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J Med Biochem 2022; 41 (3): 263– 432 ©2022 by SMBS, Belgrade
Contents Sadràj
REVIEW PAPER/REVIJSKI RAD Bara’ah Khaleel, Al-Motassem Yousef, Mazhar Salim

AL-Zoubi, Muhammad AL-Ulemat, Ahmad A. Masadeh,
Xiaoping Yang, Yuanyuan Yu, Yong Wang, Wen Jiang,
Ali Abuhaliema, Khalid M AL-Batayneh, Bahaa Al-Trad
Wenqing Jiang, Bin Yin
IMPACT OF GENETIC POLYMORPHISMS AT THE
GENETIC POLYMORPHISM OF MATRIX
PROMOTER AREA OF IL-10 GENE ON TACROLIMUS
METALLOPROTEINASE 9 AND SUSCEPTIBILITY TO
LEVEL IN JORDANIAN RENAL TRANSPLANTATION
CHRONIC OBSTRUCTIVE PULMONARY DISEASE:
RECIPIENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
A META-ANALYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Osman Oğuz, Huriye Serin, Fatma Sinem Hocaoglu
ORIGINAL PAPER/ORIGINALNI NAU^NI RAD ALKALINE PHOSPHATASE INTERFERENCE IN
Kürşad Ramazan Zor, İsmail Sarı, Gamze Yıldırım Biçer, IMMUNO-ENZYMATIC ASSAYS . . . . . . . . . . . . . . . . . 335
İnayet Güntürk, Erkut Küçük, Serpil Erşan, Gönül Şeyda
Seydel XiaoZe Li, LiHong Wang, ZeRong Yao, FangYing Ruan,
EVALUATION OF OXIDATIVE STRESS, ZhiPeng Hu, WenXia Song
3-NITROTYROSINE, AND HMGB-1 LEVELS IN PATIENTS CLINICAL EVALUATION OF NON-INVASIVE PRENATAL
WITH WET TYPE AGE-RELATED MACULAR SCREENING IN 32,394 PREGNANCIES FROM
DEGENERATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 CHANGZHI MATERNAL AND CHILD HEALTH CARE
HOSPITAL OF SHANXI CHINA. . . . . . . . . . . . . . . . . . 341
Miron Sopi}, Ana Nini}, Barbara Ostanek, Dragana Bojanin,
Chunbao Xie, Jianbo Zhang, Jiangrong Luo, Meiling Jian,
Tatjana Milenkovi}, Jelena Munjas, Marija Mihajlovi}, Jelena
Taiqiang Zhao, Jiaqiang Wang, Linxi Jiang, Chao Dai,
Veki}, Janja Marc, Vesna Spasojevi}-Kalimanovska
Yao Wei, Li Jiang, Yi Shi
DOWNREGULATION OF MAPK/MAK/MRK
FOCUS-PDCA CAN EFFECTIVELY OPTIMIZE
OVERLAPPING KINASE 1 IN PERIPHERAL BLOOD
THE CRITICAL VALUE OF TEST ITEMS . . . . . . . . . . . 347
MONONUCLEAR CELLS OF PEDIATRIC PATIENTS
WITH TYPE 1 DIABETES MELLITUS . . . . . . . . . . . . . 282
Binghua Yin, Bing Dong, Xiaohui Guo, Can Wang,
Huazhi Huo
Haixi Yan, Shuaishuai Chen, Yang Qiong, Linling Cai
GABPA PROTECTS AGAINST GASTRIC CANCER
PREOPERATIVE PREALBUMIN-TO-FIBRINOGEN
DETERIORATION VIA NEGATIVELY REGULATING
RATIO TO PREDICT SURVIVAL OUTCOMES IN
GPX1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
HEPATOCELLULAR CARCINOMA PATIENTS AFTER
HEPATIC RESECTION . . . . . . . . . . . . . . . . . . . . . . . . . 290
Mirjana Stojkovic, Biljana Nedeljkovic-Beleslin, Milorad Tesic, XXII SRPSKI KONGRES MEDICINSKE I
Zoran Bukumiric, Jasmina Ciric, Milos Stojanovic, Marija LABORATORIJSKE MEDICINE
Miletic, Ana Djordjevic-Dikic, Vojislav Giga, Branko Beleslin, sa me|unarodnim uče{ćem
Milos Zarkovic XXII SERBIAN CONGRESS OF MEDICAL
SPECIFIC IMPACT OF CARDIOVASCULAR RISK FACTORS BIOCHEMISTRY AND LABORATORY MEDICINE
ON CORONARY MICROCIRCULATION IN PATIENTS with international participation
WITH SUBCLINICAL HYPOTHYROIDISM . . . . . . . . . . 299 16th BELGRADE SYMPOSIUM FOR
BALKAN REGION . . . . . . . . . . . . . . . . . . . . . . . . . . 363
Mingxing Chen, Simeng Qin, Sitao Yang, Huaping Chen, PLENARY SESSIONS . . . . . . . . . . . . . . . . . . . . . . . 363
Liuyi Lu, Xue Qin POSTER SESSIONS . . . . . . . . . . . . . . . . . . . . . . . . 395
PERFORMANCE EVALUATION BETWEEN TWO
AUTOMATED BIOCHEMICAL ANALYZER SYSTEMS: TECHNICAL REPORTS
ROCHE COBAS 8000 AND MINDRAY BS2000M . . . . 306 OBAVE[TENJA
Neda Milinkovi}, Milica Zekovi}, Margarita Dodevska, PROGRAM NAU^NIH, STRU^NIH SKUPOVA
Briìta \or|evi}, Branimir Radosavljevi}, Svetlana Ignjatovi}, I EDUKATIVNIH SEMINARA . . . . . . . . . . . . . . . . . . . 413
Nevena Ivanovi}
MAGNESIUM SUPPLEMENTATION AND IRON STATUS INSTRUCTIONS FOR AUTHORS . . . . . . . . . . . . . . . . 421
AMONG FEMALE STUDENTS: THE INTERVENTION
STUDY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
Volume: 41 Belgrade, July – September 2022 Issue: 3

J Med Biochem 2022; 41 (3) DOI: 10.5937/jomb0-34155
UDK 577.1 : 61 ISSN 1452-8258
J Med Biochem 41: 263 –274, 2022 Review paper

Revijski rad
GENETIC POLYMORPHISM OF MATRIX METALLOPROTEINASE 9

AND SUSCEPTIBILITY TO CHRONIC OBSTRUCTIVE PULMONARY DISEASE:
A META-ANALYSIS
GENETSKI POLIMORFIZAM MATRIKS METALOPROTEINAZE 9 I OSETLJIVOST
NA OPSTRUKTIVNU BOLEST PLU]A: META ANALIZA
Xiaoping Yang1#, Yuanyuan Yu2#, Yong Wang1, Wen Jiang1, Wenqing Jiang1, Bin Yin1*
1Department
2 of Respiratory and Critical Care (Lung disease) Center,
Qingdao Hospital of Traditional Chinese Medicine (Haici Hospital), Qingdao, China
2Department of Anesthesiology, Qingdao Hospital of Traditional Chinese Medicine (Haici Hospital),
Qingdao, China
Summary Kratak sadràj

Background: To systematically analyze the influence of Uvod: Sistematska analiza uticaja genetskih polimorfizama
genetic polymorphisms of matrix metalloproteinase 9 matriks metaloproteinaze 9 (MMP9) na osetljivost hroni~ne
(MMP9) on susceptibility to chronic obstructive pulmonary opstruktivne bolesti plu}a (HOBP).
disease (COPD). Metode: Relevantna literatura koja izve{tava o MMP9 i pod-
Methods: Relevant literatures reporting MMP9 and suscep- lo`nosti HOBP u bazama podataka PubMed, Web of Scince,
tibility to COPD in PubMed, Web of Science, VIP, Wanfang VIP, Wanfang i CNKI pretraìvana je kori{}enjem klju~nih re~i
and CNKI databases were searched using the key words »matriks metaloproteinaze 9/MMP9, COPD/hroni~na
»matrix metalloproteinases 9/MMP9, COPD/chronic opstruktivna bolest plu}a«. Podaci iz kvalifikovane literature
obstructive pulmonary disease«. Data of eligible literatures
su ekstrahovani i analizirani za odnos {anse (OR) i odgovara-
were extracted and analyzed for the odds ratio (OR) and
ju}i 95% CI.
corresponding 95% CI.
Results: A total of 16 independent studies reporting Rezultati: Ukupno je uklju~eno i analizirano 16 nezavisnih
MMP9-1562C/T and COPD patients were enrolled and studija koje su izve{tavale o pacijentima sa MMP9-1562C/T
analyzed. None of the genetic models revealed the rela- i HOBP. Nijedan od genetskih modela nije otkrio vezu
tionship between MMP9-1562C/T and susceptibility to izme|u MMP9-1562C/T i osetljivosti na HOBP. Analize pod-
COPD. Subgroup analyses identified lower risk of COPD in grupa identifikovale su niì rizik od HOBP kod kineske popu-
Chinese population carrying the TT genotype for theMMP- lacije koja nosi TT genotip za MMP-9 rs3918242 u odnosu
9 rs3918242 relative to those carrying CT and CC geno- na one koji nose CT i CC genotipove (P=0,03, OR=0,67,
types (P=0.03, OR=0.67, 95% CI=0.46–0.97). 95% CI=0,46–0,97).
Conclusions: Chinese population carrying the TT genotype Zaklju~ak: Kineska populacija koja nosi TT genotip za MMP-
for the MMP-9 rs3918242 present lower susceptibility to 9 rs3918242 predstavlja manju osetljivost na HOBP u odno-
COPD relative to those carrying CT and CC genotypes. su na one sa CT i CC genotipovima.
Keywords: MMP9, polymorphism, COPD, meta-analysis Klju~ne re~i: MMP9, polimorfizam, HOBP, meta-analiza
Address for correspondence:

Bin Yin, MM. Department 2 of Respiratory and Critical Care
(Lung disease) Center, Qingdao Hospital of Traditional Chinese
Medicine (Haici Hospital), 4 Renmin Road, Shibei District,
Qingdao, Shandong 266033, China
Tel: 860532-83776223 # Xiaoping Yang and Yuanyuan Yu contributed equally to this
e-mail: qd0532ybª163.com work
264 Yang et al.: Genetic polymorphism of matrix metalloproteinase 9 and susceptibility to COPD
Introduction Exclusive criteria were as follows: 1) Repeated

literatures; 2) Literatures lacked valid raw data; 3)
Chronic obstructive pulmonary disease (COPD) Reviews, comments, animal experiments, researches
is a worldwide disease affecting approximately 3 mil- on mechanism and case reports;4) The latest studies
lion people. It is estimated that COPD will be the third or those with a larger sample size were selected if
leading cause of death by 2020 (1). As a chronic air- data overlapping; 5) Unpublished data.
way inflammatory disease, COPD is characterized by
incomplete reversible airflow limitation, inflammatory Flow diagram of literature searching was depict-
cell infiltration, excessive mucus secretion, and airway ed in Figure 1.
remodeling (2). The precise molecular mechanism
underlying the pathogenesis of COPD remains
unclear. At present, it is generally believed that several Data extraction
risk factors are directly related to the pathogenesis of Data were independently extracted and ana-
COPD, including host and environmental factors (3). lyzed by two researchers, and the third one was
Among environmental factors, smoking, exposure to responsible for solving any disagreement. Extracted
chemicals, indoor and outdoor air pollution are risk data included: 1) Baseline data of literatures, includ-
factors for COPD (4). Host factors of COPD include ing publication origin, first author, year or publication,
antitrypsin-1, excessive deposition of extracellular and etc.; 2) Basic characteristics of subjects, including
matrix (ECM), corticosteroids, inflammatory stimuli, sample size, research country, genotype number and
and metabolic imbalances (5, 6). distribution, HWE in control group and etc.
Matrix metalloproteinases (MMPs) are members
of the metformin group and they are capable of
degrading ECMs and regulating extracellular signal- Statistical analysis
ing networks (7). MMPs are important in COPD. They
Heterogeneity test was conducted by calculating
degrade matrix proteins (elastin, collagen) during the
odds ratio (OR) and the corresponding 95% CI with
disease progression (8). In the past decade, abundant
the I2 test and the Q test. The pooled OR in studies
researches have been conducted to analyze the rela-
lacking the heterogeneity was calculated by the fix-
tionship between single nucleotide polymorphisms
effects model. Otherwise, a random-effects model
(SNPs) of MMPs and COPD risk in some populations
was used. Sensitivity analysis was performed by
(9 –12). However, the conclusions were controversial.
removing one study each time and analyzing the
Some reports demonstrated the certain influence of
remaining in a combination way. The HWE of control
MMPs on the occurrence of COPD (13–18), while
genotype distribution was evaluated using the 2 test
others did not (9, 12, 19, 20). These conflicting find-
and P<0.05 considered as inequivalent. Publication
ings may be explained by limited sample size, false
bias was evaluated by depicting funnel plots and
positive results, and publication bias. In this paper, we
quantified by Egger’s test. Data analyses were carried
performed a comprehensive meta-analysis to assess
out using RevMan 5.3 and STATA12.0.
the influence of MMP polymorphisms on COPD.
Materials and Methods

Search strategy of literatures
Relevant literatures reporting the relationship
between polymorphisms of MMP9-1562C/T and sus-
ceptibility to COPD in PubMed, Web of Science, VIP,
Wanfang and CNKI databases were searched using
the key words »matrix metalloproteinases 9/MMP9,
COPD/chronic obstructive pulmonary disease«.
There were no limitations on published languages.
Citations in each literature were manually reviewed.
Inclusive and exclusive criteria

Inclusive criteria were as follows:1) Case-control
studies conducted in humans; 2) Literatures published
complete data or raw data that could calculate the
genotype distribution; 3) COPD patients underwent
diagnosis of pulmonary function index; 4) Literatures
were conducted on the influence of polymorphisms of
MMP9-1562C/T on susceptibility to COPD. Figure 1 Flow diagram of the publication selection process.
J Med Biochem 2022; 41 (3) 265
Results literatures were excluded after the first-round screen-

ing. Subsequently, 14 literatures on mechanisms, 6
Baseline characteristics of eligible literatures reviews, 6 literatures reporting other diseases, 2 liter-
Initially, 157 literatures in PubMed, 151 in Web atures without complete data and 2 reporting other
of Science, 1 in CNKI, 77 in VIP and 15 in Wanfang mutant sites were excluded. Finally, 16 literatures
database were searched out, with a total of 395 li- were included in this study (Figure 1).
teratures. A total of 62 replicates and 287 irrelevant
Table I Main characteristics of studies included in the meta-analysis.

Author Year Country Journal name/ Genotyping SNP loci (PHWE) Sample size Control Sample
publication methods
origin
Zhou 2004 China Chinese Medical PCR- rs3918242 100 (male=98, 100 (male=99, Whole
Journal sequence (pHWE=0.92) female=) female=1) blood
Isao Ito 2005 Japan Am J Respir Crit PCR-RFLP rs3918242 84 (male=81, 85 (male=69,
Care Med (pHWE=0.41) female=3) female=16)
Zhang 2005 China Chin J Epidemiol PCR-RFLP rs3918242 147 (male=135, 120 Whole
Rongbao (pHWE=0.09) female=12) (male=110, blood
female=10)
Han 2006 Asian Chin J Tuberc PCR-RFLP rs3918242 60 52 Whole
Respir Dis (pHWE=0.48) blood
Testaigzi 2006 Caucasian Int J Chron PCR-RFLP rs3918242 123 262 Whole
Obstruct Pulmon (pHWE=0.39) blood
Dis
Korytina 2008 Russia Russian Journal PCR-RFLP rs3918242 318 319 Whole
of Genetics (pHWE=0.53) blood
Shih-Lung 2009 Taiwan Biochem Genet PCR-RFLP rs3918242 184 (male=152, 212 Whole
Cheng (China) (pHWE=0.23) female=32) (male=182, blood
female=30)
H. Schirmer 2009 Brazil Genetics and PCR rs3918242 89 97 Whole
Molecular (pHWE=0.60) blood
Research
Shih-Yup 2010 Korean Basic Science PCR-sequence rs3918242 301 333 Whole
Lee Investigations (pHWE=0.376) blood
Hua 2010 China Int J Respi PCR-RFLP rs3918242 180 (male=142, 180 Whole
(pHWE=0.04) female=38) (male=130, blood
female=50)
Korytina 2012 Russia Molecular PCR-RFLP rs3918242 391 514 Whole
Biology (pHWE=0.67) blood
Sarra Bchir 2015 Tunisia Mol Diagn Ther PCR-RFLP rs3918242 138 (male=122, 216 Whole
(pHWE=0.02) female=16) (male=155, blood
female=61)
Marja 2016 Serbia Environmental rs3918242 86 100 Whole
Stankovic and Molecular (pHWE=0.28) blood
Mutagenesis.
PCR-RFLP
Marja 2017 Serbia JOURNAL PCR-RFLP rs3918242 122 100 Whole
Stankovic OF CHRONIC (pHWE=0.28) blood
OBSTRUCTIVE
PULMONARY
DISEASE
Tan Jie 2017 China Journal PCR-RFLP rs3918242 186 (male=92, 219 Whole
Of Inner (pHWE<0.001) female=294) (male=105, blood
Mongolia female=112)
Medical Universit
Lwona 2018 Poland BioMed Research PCR-RFLP rs3918242 335 309 Whole
Gilowska International (pHWE=0.33) (male=87, (male=229, blood
female=248) female=80)
SNP=Single nucleotide polymorphism; HWE = Hardy-Weinberg equilibrium; pHWE=p-value of Hardy-Weinberg Equilibrium test in
controls for each locus; PCR = polymerase chain reaction
Baseline characteristics of eligible literatures Meta-analysis

were listed in Table I. Briefly, 16 case-control studies
A total of 2011 COPD patients and 2249
were published from 2004–2018, including 13 stud-
healthy controls were enrolled. The influence of
ies published in English-language scientific journals
MMP9 (-1562) C/T on susceptibility to COPD was
and 3 in Chinese-language scientific journals.
assessed using different genetic models. No relation-
Genotyping methods were conducted using poly-
ship was found between the CC vs.TT genotype of
merase chain reaction (PCR), PCR-RFLP and PCR-
MMP9 rs391842 and susceptibility to COPD in the
sequence. Identification of single nucleotide polymor-
allele model (P=0.41, OR=1.12, 95% CI=0.86-
phisms (SNPs) was conducted by extracting blood
1.47) (Figure 2 A-C). The other three genetic models
samples of subjects.
obtained the same conclusion, including the domi-
In the 16 eligible literatures, 5 analyzed Chinese nant model (CC vs. CT+TT, P=0.13, OR=0.82,
population, 1 analyzed Japanese population, 2 ana- 95% CI=0.63–1.06), recessive model (TT vs.
lyzed Russian population, 1 analyzed Brazilian popu- CC+CT, P=0.87, OR=0.97, 95% CI=0.65–1.43)
lation, 1 analyzed Korean population, 1 analyzed and over-dominant model (CT vs. CC+TT, P=0.51,
Tunisian population, 2 analyzed Serbian population, 1 OR=1.13, 95% CI=0.79–1.61).
analyzed Poland population, 1 analyzed Asian popu-
Subgroup analyses were performed based on
lation and 1 analyzed Caucasian population. Sample
the ethnic populations, involving Asian population (8
size of each literature was 60-391.
literatures), European population (3 literatures),
Caucasian population (3 literatures) and African pop-
ulation (2 literatures). The random-effects model was
utilized owing to the different degrees of hetero-
Figure 2A Forest map of the relationship between the SNP of MMP-9 rs3918242 and susceptibility to COPD.
J Med Biochem 2022; 41 (3) 267
Figure 2B Forest map of the relationship between the SNP of MMP-9 rs3918242 and susceptibility to COPD.
Figure 2C Forest map of the relationship between the SNP of MMP-9 rs3918242 and susceptibility to COPD.
geneity (I2>50%, P<0.05). The data showed no Heterogeneity and sensitivity analysis
relationship between MMP9 polymorphisms and
Significant heterogeneity was identified in the
COPD risk under the different genetic models
dominant model, over-dominant model and allele
(P>0.05) (Figure 3 C-D).
model analyzing the relationship between MMP9
Subsequently, we individually analyzed the (-1562) C/T and susceptibility to COPD (all
relationship between MMP9 polymorphisms and P<0.001). No remarkable changes in I2 and P values
COPD in Chinese population, involving 5 literatures were observed after removing a single study. In addi-
(15, 18, 21–23). Except for the recessive model (TT tion, sensitivity analysis was not altered by removing
vs. CC&CT) analyzed by the fix-effects model any study each time (data not shown).
(P=0.13, I2=46%), the remaining were assessed
In the subgroup analyses based on different eth-
using the random-effects model (I2>50%, P<0.05)
nic populations, all genetic models showed the results
(Figure 4). Our data showed that Chinese popula-
of I2>50% and P<0.05. We did not find any changes
tion carrying the TT genotype for the MMP-9
in I2 and P values after removing a single study.
rs3918242 was closely related to susceptibility to
Sensitivity analysis was not influenced by removing a
COPD relative to those carrying CT and CC geno-
single study (data not shown).
types (P=0.03, OR=0.67, 95% CI=0.46 – 0.97).
Such a difference was not observed in the dominant
model (CC vs. CT&TT), over-dominant model (CT
vs. CC&TT) and allele model (C Allele vs. T Allele)
(P>0.05) (Figure 4).
J Med Biochem 2022; 41 (3) 269
Figure 3A, B Subgroup analyses of the relationship between the SNP of MMP-9 rs3918242 and susceptibility to COPD in dif-
ferent regions and different pairs of comparisons.
Figure 3C, D Subgroup analyses of the relationship between the SNP of MMP-9 rs3918242 and susceptibility to COPD in dif-
ferent regions and different pairs of comparisons.
J Med Biochem 2022; 41 (3) 271
Figure 4 Subgroup analyses of the relationship between the SNP of MMP-9 rs3918242 and susceptibility to COPD in Chinese
population and different pairs of comparisons.
Publication bias susceptibility to COPD in the three genetic models

except for the allele model (CC vs. CT+TT, P=0.325;
A wide range of search strategies was carried
TT vs. CC+CT, P=0.541; CT vs. CC&TT, P=0.553;
out to minimize potential publication biases. After
C allele vs. T allele, P=0.017) (Figure 5).
quantification using Egger’s test, the data showed no
publication biases between MMP9 (-1562) C/T and
Figure 5 Subgroup analyses of the relationship between the SNP of MMP-9 rs3918242 and susceptibility to COPD in Chinese
population and different pairs of comparisons.
Discussion activity owing to the deletion of the transcriptional

repressor binding site (34).
MMPs are a class of zinc-dependent endopepti-
dases that degrade major protein components of the So far, studies focusing on the correlation
ECM. They participate in development- and inflam- between MMP9 -1562 C/T polymorphism and COPD
mation-related tissue remodeling and repair (7). are relatively rare and uncertain. Studies with a small
MMP-9 (gelatinase B) can degrade ECM proteins, sample size lack the statistical power and often lead
such as type IV collagen and gelatin (24). In addition, to contradictory conclusions. Meta-analysis provides
it exerts a vital role in airway inflammation and convincing evidences by calculating data extracted
remodeling (25, 26). MMP-9 protects ventilator- from multiple studies. In this paper, we obtained the
induced lung injury by reducing infiltration of alveolar conclusion that MMP9 -1562 C/T polymorphism was
neutrophils (27). not associated with susceptibility toped in different
putative genetic models. Subgroup analyses showed
COPD is a common respiratory disease charac- that Chinese population carrying the TT genotype for
terized by airflow limitation. The pathogenesis of the MMP-9 rs3918242 are risky of COPD relative to
COPD is complex, involving inflammatory response, those carrying CT and CC genotypes.
oxidant-antioxidant imbalance, and MMPs-induced
proteolysis of the alveolar wall. MMP9, one of the Inconsistent with our results, some studies have
most widely studied MMPs, decomposes most of the demonstrated that the MMP9 -1562 C>T polymor-
components of ECM by degrading structural proteins, phism indeed influences COPD risk. Zhou et al. (35)
such as collagen and elastin (28). Many studies have illustrated that the TT genotype of MMP9 -1562 C/T
reported the involvement of MMP9 in the develop- polymorphism is a genetic risk factor for severe
ment of lung diseases (29). MMP9 polymorphism is COPD. Korytina et al. (36) have indicated the corre-
identified to increase the susceptibility to respiratory lation between the TT genotype of MMP9 -1562 C/T
diseases (30–33). Multiple SNPs of MMP9 have been polymorphism and COPD severity. Similarly, a study
discovered. Among them, C/T mutation on MMP9 conducted in Russia showed a significant difference
(-1562) rs3918242 results in the increased promoter in the frequency distribution of MMP9 -1562 C>T
J Med Biochem 2022; 41 (3) 273
among COPD patients with different severity levels ty to COPD relative to those carrying CT and CC
(37). genotypes.
Some shortcomings in this study should be Acknowledgements. No.
pointed out. First of all, many complex factors were
not adjusted, such as gender, age, and smoking his- Financial Disclosure. This study was supported
tory. Secondly, some studies (16, 20, 23) had small by the Key Project of Qingdao 2020 Traditional
sample sizes and did not have enough capacity to Chinese Medicine Scientific Research Plan (No:
detect the risk of COPD. Thirdly, the lack of raw data 2020-zyz002) and Shandong Traditional Chinese
limited the further analysis of the potential interac- Medicine Science and Technology Project (No:
tions between genetic risks and environmental factors 2020M108).
in COPD. Studies with large sample sizes in a multi-
center hospital are required for further validation.
Conflict of interest statement
Conclusions All the authors declare that they have no conflict
of interest in this work.
Chinese population carrying the TT genotype
for the MMP-9 rs3918242 present lower susceptibili-
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Received: September 28, 2021

Accepted: December 14, 2021
UDK 577.1 : 61 ISSN 1452-8258
J Med Biochem 41: 275 –281, 2022 Original paper

Originalni nau~ni rad
EVALUATION OF OXIDATIVE STRESS, 3-NITROTYROSINE, AND HMGB-1

LEVELS IN PATIENTS WITH WET TYPE AGE-RELATED MACULAR DEGENERATION
PROCENA OKSIDATIVNOG STRESA, NIVOA 3-NITROTIROZINA I HMBG-1
KOD PACIJENATA SA VLA@NOM VRSTOM MAKULARNE DEGENERACIJE
Kürşad Ramazan Zor1, İsmail Sarı2, Gamze Yıldırım Biçer1, İnayet Güntürk3,
Erkut Küçük1, Serpil Erşan2, Gönül Şeyda Seydel4
1Niğde Ömer Halisdemir University School of Medicine Department of Ophthalmology, Bor Yolu, Niğde, Turkey
2Niğde Ömer Halisdemir University School of Medicine Department of Biochemistry, Bor Yolu, Niğde, Turkey
3Niğde Ömer Halisdemir University, Healthcare Services,
Zübeyde Hanım Health Services Vocational High School, Bor Yolu, Niğde, Turkey
4Niğde Ömer Halisdemir University School of Medicine Department of Medical Biochemistry,
Bor Yolu, Niğde, Turkey

Background: This study aims to compare serum HMGB-1, Uvod: Ova studija ima za cilj da uporedi serumske nivoe
3-nitrotyrosine (3-NT), TAS, TOS, and OSI levels in Wet- HMBG-1, 3-nitrotirozina (3-NT), TAS, TOS i OSI kod pacije-
type Age-Related Macular Degeneration (wAMD) patients nata sa vla`nom starosnom makularnom degeneracijom
and healthy controls to determine the correlation of these (vAMD) i zdravih kontrola kako bi se utvrdila korelacija ovih
parameters with each other. parametri me|usobno.
Methods: Thirty patients with Wet-type Age-Related Metode: Trideset pacijenata sa vla`nom starosnom maku-
Macular Degeneration (wAMD) and 27 healthy adults, as larnom degeneracijom (wAMD) i 27 zdravih odraslih osoba
controls were enrolled in the study. We determined the TAS
su uklju~ene u studiju. Odre|ivali smo nivoe TAS i TOS u
and TOS levels in serum samples of both groups using
uzorcima seruma obe grupe pomo}u komercijalnih komple-
commercial kits on a microplate reader. Serum HMGB-1
and 3-NT levels were measured with the enzyme-linked ta na ~ita~u mikroplo~a. Serumski nivoi HMBG-1 i 3-NT
immunosorbent assay method. mereni su pomo}u enzimsko-imunolo ke analize.
Results: HMGB-1 levels were significantly higher in the Rezultati: Nivoi HMBG-1 bili su zna~ajno ve}i u grupi paci-
patient group (137.51 pg/mL, p=0.001), while there was jenata (137,51 pg/mL, p = 0,001), dok nije bilo razlike
no difference between the two groups in serum 3-NT levels izme|u dve grupe u serumskim nivoima 3-NT (p = 0,428).
(p = 0.428). A statistically significant difference found in Statisti~ki zna~ajna razlika prona|ena u nivoima TOS i OSI
the levels of TOS and OSI (p = 0.001 and p = 0.045, (p = 0,001 i p = 0,045, respektivno) izme|u pacijenata i
respectively) between the patients and controls, however, kontrola, me|utim, nije prime}ena zna~ajna razlika izme|u
no significant difference was observed between the groups grupa u pogledu nivoa TAS (p = 0,228).
in terms of TAS levels (p = 0.228). Zaklju~ak: Oksidativni stres i nivo HMBG-1 su pove}ani kod
Conclusions: Oxidative stress and HMGB-1 levels were pacijenata sa wAMD-om, a poja~ani oksidativni stres moè
increased in wAMD patients and enhanced oxidative stress biti povezan sa pove}anom nekrozom tkiva i upalom. Stoga

Kürşad Ramazan Zor, MD,
Address: Niğde Ömer Halisdemir University School of
Medicine Department of Ophthalmology
BOR YOLU/NIGDE
e-mail: kursadzorªhotmail.com
Fax: +90 388 212 14 11
Phone number: +90 388 232 22 20
276 Zor et al.: Oxidative stress, 3-NT and HMGB-1 levels in patients with wAMD
may be associated with increased tissue necrosis and bi primenu antioksidativnog tretmana pored rutinske terapije
inflammation. Thus administration of antioxidant treatment trebalo razmotriti wAMD-u.
in addition to routine therapy should be considered in
wAMD. Klju~ne re~i: HMBG-1, 3-nitrotirozin, TAS, TOS, OSI,
mokri tip AMD
Keywords: HMGB-1, 3-Nitrotyrosine, TAS, TOS, OSI,
wet type AMD
Introduction In this case, 3-NT levels in AMD patients can provide

information on pathogenesis and damage.
Age-related macular degeneration (AMD) is one
of the most common causes of central vision loss in The formation and removal of free radicals in
the elderly (1). As a result of abnormalities in the the living system occur in perfect balance, and the
Bruch’s membrane, retinal pigment epithelium (RPE), organism is not affected by these reactive molecules
photoreceptor, and choroid complex, AMD can often if this sensitive condition stabilizes the oxidative
result in regional atrophy and/or neovascularization. balance. If one of the steps to achieve this balance is
For this reason, the disease is basically divided into interrupted, it causes oxidative stress that can lead to
two subgroups: wet and dry type (2). cell damage. Therefore, while oxidative stress is so
important in AMD pathogenesis, plasma oxidant and
AMD, is associated with various pathological antioxidant levels also gain importance. The total
factors such as chronic oxidative stress, decrease in effects of all antioxidants in serum and other body
autophagy, and chronic inflammation (3). Although fluids are measured according to the total antioxidant
several local and systemic inflammatory molecules status (TAS), and the total effects of oxidants are
are recommended as markers for AMD, a specific determined by the total oxidant status (TOS). In
and reliable marker has not been established. addition, a global oxidative stress index (OSI)
High Mobility Group Box-1 (HMGB-1), a non- reflecting both oxidative and antioxidant responses is
histone protein, is found in large amounts in the informative (10).
nucleus and plays a role in DNA transcription, The relationship of AMD, whose inflammatory
replication, and repair (4). HMGB-1 is a newly base has been demonstrated in many studies, with
described inflammatory molecule that is a mediator HMGB-1, an inflammation-related molecule, has not
of endotoxin shock and is elevated in the blood of been previously examined and we aimed to
septic patients (5). The expression of HMGB-1 contribute to the literature on this matter. We
increases significantly in dry-type AMD and also measured 3-NT levels which is a marker of nitrosative
increases the aging rate of RPE cells (6, 7). In stress and hasn’t studied in patients with AMD; and
addition, the application of recombinant HMGB-1 TOS, TAS, and OSI levels which is an indicator of
initiates a pro-inflammatory response in human oxidative stress that has been shown to be associated
endothelial cells (5). Considering the importance of with AMD before, in serum of wAMD patients.
these molecules and inflammatory status in AMD
pathogenesis, it is understood that HMGB-1 is
important in the prognosis and pathogenesis of this
Material and Methods
disease. However, apart from a few limited studies,
which are all based on cell culture, there are no Study population
studies investigating the role and level of serum
The study was approved by the Noninvasive
HMGB-1 in macular diseases in humans.
Ethics Committee of Niğde Ömer Halisdemir
Recent research has provided a lot of evidence University (The Decision Number: 2020/52) and
on the relationship between inflammation drusen and written consent was obtained from each patient
oxidative stress in the development of wet type AMD before taking the first blood samples. The study was
(wAMD). During inflammation, nitric oxide NO plays conducted in accordance with the principles of the
an important role and it may be released in high Helsinki Declaration. 30 patients with wAMD and 27
amounts with stimulation. Higher levels of NO in healthy controls constituted the study group of our
plasma have been reported in AMD (8). NO is an research. All individuals included in the study groups
important physiological regulator but it can form were over 50 years old. Patients with other eye
other reactive intermediates such as nitrite (NO2), pathologies, such as glaucoma, uveitis, who had
peroxynitrite (ONOO), which can change the undergone intraocular surgery, smoking, and
structure of tissues. Peroxynitrite changes the systemic disease were not included in the study. The
structure of molecules by adding a nitro group to the diagnosis of wAMD was carried out by examination,
phenolic ring of tyrosine in proteins or in free form. fundus photographs, optical coherence tomography,
Thus, 3-nitrotyrosine (3-NT) is a specific marker for and fundus fluorescein angiography. The control
oxidative damage to proteins and NO production (9). group of our study consisted of individuals without the
J Med Biochem 2022; 41 (3) 277
systemic disease who had a complete healthy eye Table I Demographic data of patients and the control group.
examination. Patient Group Control Group
p
(N=30) (N=27)
Mean Age
Blood sample collection 71.13±9.37 66.51±8.09 0.053
(year±SD)
Five mL of venous blood samples were taken Sex
into sterile biochemistry tubes with gel from the 13/17 10/17 0.138
(Female/Male)
patient and control groups. Then serum samples
SD, standard deviation; * p < 0.05
were obtained by centrifuging at 1600xg for ten min.
Sera samples were kept at -80 °C until analysis day.
Table II HMGB-1, 3-NT, TAS, TOS, and OSI levels in
patients with wAMD and the control group.
Determination of Serum HMGB-1 and 3- Contor Group Patient Group
p
Nitrotyrosine Levels (N=27) (N=30)
HMGB-1 and 3-Nitrotyrosine levels were HMGB-1 95.93 137.51

0.001*
(pg/mL) (92.90–114.63) (126.47–166.17)
measured with ELISA kits (Cusabio; CSB-E08223h
and Yehua; YHB0033Hu-96, respectively) according 3-NT (nmol/L) 900.33±197.47 951.53±236.62 0.428
to the instructions of the manufacturers.
TAS (mmol
1.73 (0.73–2.96) 2.00 (1.06–2.89) 0.228
Trolox Equiv. /L)
Measurement of TAS and TOS levels in the TOS (mmol
7.93 (6.99–9.18) 17.03 (7.11–30.79) 0.001*
serum samples H2O2 Equiv. /L)
OSI (%) 0.56 (0.25–1.13) 0.79 (0.56–1.42) 0.045*
TOS and TAS levels were measured using
commercially available kits (Rel Assay, Mega Tıp, HMGB-1, TAS, TOS, and OSI levels were expressed as the medi-
Gaziantep, Turkey) by Erel’s colorimetric method. The an (min-max), whereas 3-NT levels as the mean ± standard
results were expressed mmol Trolox equivalents/L for deviation. HMGB-1, High Mobility Group Box-1; 3-NT, 3-nitroty-
TAS; mmol H2O2 equiv/L for TOS (11, 12). rosine; TAS, Total antioxidant status; TOS, Total oxidant status;
OSI, Oxidative stress index. * p < 0.05.
Determination of oxidative stress index 66.51 ± 8.09 years in the controls. There was no
OSI was calculated through the following significant difference between the groups in terms of
formula: gender (p = 0.138) and age (p=0.053).
OSI =TOS (mmol Trolox equivalent/L)/TAS Table II summarizes the statistical comparison of
(mmol H2O2 equivalent /L) X100 HMGB-1, 3-NT, TAS, TOS, and OSI levels deter-
mined in the sera samples of the study groups.
Median levels of HMGB-1 were 137.51 pg/mL in
Statistical analysis wAMD patients and 95.93 pg/mL in controls. The
median 3-NT levels were 951.53 nmol/L in the
Statistical analysis was made with IBM SPSS patient group and 900.33 nmol/L in the control
Statistics version 23 software. The normal distribution group. The levels of HMGB-1 were meaningfully
of the variables was assessed with the Shapiro–Wilks higher in the patient group than in the control group
test. Statistical data were stated as mean ± standard while there was no difference between the two groups
deviation (⎯x ± SD), or median and inter-quartile in terms of 3-NT levels. Furtheremore, while there
range for normally and non-normally distributed was no significant difference between TAS values,
variables, respectively. Comparisons between groups TOS and OSI levels were significantly higher in the
for continuous variables were performed using the patients then the control (Table II). Lastly, we found a
Student t-test (normal distribution) or the Mann- significant correlation coefficient between the OSI
Whitney U test (non-normal distribution). Data were and HMGB-1 levels measured in the patient group (r2
considered statistically significant at a value of =0.6; p=0.0001).
p<0.05.
Discussion
Results
The retina is one of the tissues with the highest
As can be seen in Table I, there were 13 females oxygen consumption in humans (13). Indeed, Hanus
and 17 males in the patient group while 10 females et al. (14) reported that RPE cells undergo necrosis
and 17 males in the control group. The mean age due to increased oxidative stress. As it is known, ROS
was 71.13 ± 9.37 years in the patient group, and attacks the cells, especially the lipids in the
membrane, DNA, RNA, and proteins by disrupting prevent the progression of RPE cell aging. We found
their functions. Such a situation may increase the significantly higher HMGB-1 levels in wAMD patient
necrosis of RPE cells in wAMD patients. It has been sera (137.51 pg/mL) compared to the control group
suggested that RPE cell injury and death caused by (95.93 pg/mL) in our study and to our knowledge this
oxidative stress promote the release of damage- was the first study in the literature to examine the
associated molecular pattern molecules (DAMP) due HMGB-1 level in the serum of wAMD patients
to intracellular and extracellular damage. DAMP can (p=0.001).
stimulate immune and inflammatory responses and
Nitrotyrosine is an indicator of the production of
lead to AMD progression (15). Consistent with these
reactive nitrogen species and this end product causes
findings, we found that levels of TOS and OSI were
damage to cellular components. Therefore, tyrosine
higher in wAMD patients in our study (p <0.05).
nitration has been recently investigated in the patho-
HMGB-1 is a large DAMP molecule that genesis of diseases (29). In a study by Thomson (30),
releases from the nucleus during necrosis and is 3-nitrotyrosine levels were found to be associated
secreted into the extracellular matrix and induces with higher cardiovascular risk. Bandookwala et al.
inflammation (16). It has been reported that HMGB- (31) reported that the change in the nitrotyrosine
1 is secreted from also necrotic RPE cells and induces profile occurred in the presymptomatic stage of the
inflammation (17). HMGB-1 is a non-histone DNA neurodegenerative diseases and this could be used
binding protein that was first described by Goodwin for early diagnosis. Qian et al. (32) found that
and Johns in 1973 (18). It plays an important role in nitrotyrosine level was associated with mortality in
DNA transcription, replication, and repair also patients with acute kidney injury. Khan et al. (33)
contributes to the collection of nuclear proteins. In reported that serum 3-NT has increased in SLE
the cytoplasm, it functions as a signal regulator and patients and preventing nitrosative damage could be
takes a role in the inflammatory cascade, and acts as new treatment methods for some diseases.
a pro-inflammatory cytokine in the extracellular Nitrosative stress also attracts attention in the field of
environment (19). HMGB-1 serum levels, which have ophthalmology. It was shown that the formation of
an important role in mediating inflammation, have nitrotyrosine significantly increases in the diabetic
been shown to be high in various diseases (20). retina and is an inflammatory element in the
Furthermore, HMGB1plays an important regulatory development of diabetic retinopathy (29). Wang et al.
role in angiogenesis and it affects many (34) reported that 3-nitrotyrosine levels in blood
angiogenesis-related conditions such as proliferative serum were higher in type 2 diabetic patients than in
diabetic retinopathy, cancer, and wound healing via healthy individuals. In RP, nitrosative stress has been
p53. Thus, HMGB1 is a promising therapeutic target reported to be effective in cone photoreceptor deaths
in many malignancies such as prostate and colon (35). In another study, it has been reported that
cancer, and epidermal tumors (21–24). retinal nitrosative stress, plays an important role in
retinal ganglion cell loss in glaucoma (36). In a study
HMGB-1, which is intertwined with inflam-
in the tissue culture, it was reported that the non-
mation and oxidative stress, also attracts attention in
enzymatic nitration of the RPE basement membrane
the field of ophthalmology. Sakamoto et al. (25)
may have significant harmful effects on the RPE
showed that HMGB-1 induced cell death of retinal
function and they found that the accumulation of 3-
ganglion cells with increased oxidative stress in the rat
nitrotyrosine increased with the age of the patient on
retina. Chang et al. (26) reported that the release of
the human Bruch membrane (8). Wang et al. (37)
HMGB-1 peptides caused by hypoxia-regulated the
reported that nitrite-induced changes in the normal
production of angiofibrogenic factors in RPE cells,
basement membrane can mimic the harmful effects
thereby contributing to the pathogenesis of hypoxia-
of the aging Bruch membrane on RPE function. In
associated diabetic retinopathy. Watanabe et al. (27)
line with these findings, in our study, in which we
reported that the extracellular HMGB-1 contributes to
planned to evaluate the importance of 3-nitrotyrosine
ocular inflammation in autoimmune uveoretinitis.
in AMD pathogenesis, we found that the level of
Murakami et al. (28) investigated the vitreous of reti-
serum 3-nitrotyrosine did not show a statistically
nitis pigmentosa (RP) patients and found a significant
significant difference between the two groups
release of extracellular HMGB-1 associated with
(p=0.428). Although our findings indicate that
necrotic cell death and they reported that HMGB-1
nitrosative stress is not significantly effective in
could be a new therapeutic target in RP. In a study on
patients with AMD, different results can be obtained
pluripotent stem cells from healthy individuals, Sun et
in large-scale studies conducted with study groups
al. (7) showed that HMGB-1 caused RPE cell aging.
consisting of more individuals, thus we consider that
They also reported that HMGB-1 upregulated
additional studies are needed.
Caveolin-1, which is also associated with RPE cell
aging. With these results, they demonstrated that Free radicals are reactive molecules and these
HMGB-1 may be associated with RPE cell aging and molecules damage cell components, and oxidative
may play a role in AMD pathogenesis, thus stress occurs (38). The formation and removal of free
suggesting that it is a potential therapeutic target to radicals in the body occur in perfect balance,
J Med Biochem 2022; 41 (3) 279
imbalance between these two processes causes and normal TAS values that we have found indicate
oxidative stress that can cause cell damage in the increased oxidative stress and a sufficient antioxidant
living systems. Oxidative stress plays an important role system in wAMD patients. Furthermore, increased
in the pathogenesis of many diseases (39). Icel et al. HMGB-1 levels in patients with wAMD and
(40) reported that TOS level was found to be determined the significant correlation between OSI
significantly higher while TAS was found to be low in and this parameter may suggest that increased tissue
the study of diabetic rats. Oruç et al. (41) found that necrosis and inflammation in these patients are
the TOS value was high while the TAS value was associated with OSI. However, further studies are
found low in humorous aqueous of pseudoexfoliation needed to examine the effects of OSI and
patients, and they reported that normal these levels inflammation on the pathogenesis of wAMD and their
may slow the progression of pseudoexfoliation relationship in this disease.
syndrome. In a study conducted by Altını ık et al. (42)
in patients with retinal vein occlusion, it was found Authors’ contributions: All authors have read the
that the oxidative stress index in humor aqueous was final manuscript within their respective areas of
high, while TAS values were low. Recently, the expertise and participated sufficiently in the study to
relationship between AMD and oxidative stress has take responsibility for it and accept its conclusions.
drawn attention in a study (13, 14). Elbay et al. (43)
investigated wAMD patients and reported that serum
TOS levels increased and TAS values decreased in Declarations
wAMD patients. In the literature, there is no other Funding: Not applicable
study examining TOS and TAS values in wAMD
patients. In our study, it was found that TOS levels
increased and there is no difference between TAS Research involving Human Participants
values. We believe that our study is the second study and/or Animals
on TOS and TAS values in AMD patients. As a result
of high TOS values, OSI was found significantly This article does not contain any studies with
higher in the patient group. human participants or animals performed by any of
the authors.
Conclusion
We have found significantly higher OSI in
wAMD patients and thus oxidative stress may have All the authors declare that they have no conflict
important roles in AMD pathogenesis. The high TOS of interest in this work.
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Received: April 12, 2021

Accepted: August 19, 2021
UDK 577.1 : 61 ISSN 1452-8258
J Med Biochem 41: 282–289, 2022 Original paper

DOWNREGULATION OF MAPK/MAK/MRK OVERLAPPING KINASE 1

IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF PEDIATRIC PATIENTS
WITH TYPE 1 DIABETES MELLITUS
NISHODNA REGULACIJA MAPK/MAK/MRK PREKLAPAJU]E KINAZE U MONONUKLEARNIM
]ELIJAMA PERIFERNE KRVI PEDIJATRIJSKIH PACIJENATA SA TIP1 DIIABETES MELLITUS-OM
Miron Sopi}1, Ana Nini}1, Barbara Ostanek2, Dragana Bojanin3, Tatjana Milenkovi}4, Jelena Munjas1,
Marija Mihajlovi}1, Jelena Veki}1, Janja Marc2, Vesna Spasojevi}-Kalimanovska1
1Department of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Serbia
2Department of clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Slovenia
3Mother and Child Health Care Institute of Serbia »Dr Vukan ûpi}«, Biochemical Laboratory, Belgrade, Serbia
4Mother and Child Health Care Institute of Serbia »Dr Vukan ûpi}«, Department of Endocrinology,
Belgrade, Serbia

Background: Type 1 diabetes mellitus (T1DM) is one of the Uvod: Dijabetes melitus tip 1 (T1DM) jedno je od naj~e{}ih
most common endocrine diseases in children. T-cell autore- endokrinih oboljenja kod dece. Autoreaktivnost T-}elija
activity toward b-cells is controlled by significant changes in prema b-}elijama kontroli{e se zna~ajnim promenama u
metabolism of T cells. Mammalian target of rapamycin metabolizmu T }elija. Cilj delovanja rapamicina kod sisara je
(mTOR) is an important intracellular regulator of metabolism vaàn unutar}elijski regulator metabolizma i rasta }elija.
and cell growth. MAPK/MAK/MRK overlapping kinase 1 MAPK/MAK/MRK preklapaju}e kinaze koja se preklapa
(MOK1) is one of the less known regulators of mTOR. We (MOK1) jedan je od manje poznatih regulatora mTOR-a. Cilj
sought to investigate if MOK1 and mTOR mRNA levels in istraìvanja je bio da se ispita da li su nivoi iRNK MOK1 i
peripheral blood mononuclear cells (PBMCs) of T1DM pedi- mTOR u mononuklearnim }elijama periferne krvi razli~iti
atric patients are different compared to healthy subjects. kod pedijatrijskih pacijenata sa T1DM u odnosu na zdrave
Methods: This study included 172 adolescents with T1DM ispitanike.
and 36 healthy adolescent volunteers designated for control Metode: Ovo istraìvanje je obuhvatilo 172 adolescenta sa
group (CG). MOK1 and mTOR mRNA levels were deter- T1DM i 36 zdravih adolescenata dobrovoljaca koji su ~inili
mined in PBMCs by qPCR. kontrolnu grupu (CG). Nivoi MOK1 i mTOR mRNA odre-
Results: T1DM patients have significant downregulation of |eni su u PBMC-ima pomo}u qPCR-a.
MOK1 mRNA levels in PBMCs compared CG (P=0.018), Rezultati: Pacijenti sa T1DM imali su zna~ajno niè nivoe
while there was no significant difference in mTOR mRNA iRNK MOK1 u PBMC u odnosu na CG, dok razlike u nivoima
levels (P=0.891). Furthermore, in T1DM patients, MOK1 iRNK mTOR nisu bile zna~ajne (P = 0,891). [tavi{e, kod
significantly correlated with age, triglycerides and mTOR, pacijenata sa T1DM, MOK1 je zna~ajno korelirao sa godina-
while mTOR correlated significantly with BMI and systolic ma, trigliceridima i mTOR, dok je mTOR zna~ajno korelirao
blood pressure. Overweight T1DM subjects had significantly sa BMI i sistolnim krvnim pritiskom. Ispitanici sa prekomer-
lower MOK1 (P=0.034) and mTOR (P=0.017) mRNA nom teìnom T1DM imali su zna~ajno niè nivoe iRNK
levels, together with significantly higher levels of systolic MOK1 (P = 0,034) i mTOR (P = 0,017), zajedno sa
blood pressure (P<0.001), total cholesterol (P=0.001), zna~ajno ve}im nivoima sistolnog krvnog pritiska (P
LDL-cholesterol (P=0.001) and CRP (P<0.001). Multi- <0,001), ukupnog holesterola (P = 0,001), LDL-holesterola
variate analysis showed that MOK1 was independently (P = 0,001) i CRP (P <0,001). Multivarijantna analiza je
negatively associated with T1DM when adjusted for sex, age, pokazala da je MOK1 nezavisno negativno povezan sa
List of abbreviations: MOK1, MAPK/MAK/MRK overlapping

Address for correspondence: kinase1; mTOR, Mammalian target of rapamycin; mTORC1,
Jelena Munjas Mammalian target of rapamycin complex 1; NLRP3, NLR family
Vojvode Stepe 450 pyrin domain containing 3; PBMC, Peripheral blood mono-
+381 11 3951 284 nuclear cell; T1DM, Type 1 diabetes mellitus; T2DM, Type 2 dia-
jelena.munjasªpharmacy.bg.ac.rs betes mellitus.
J Med Biochem 2022; 41 (3) 283
HDL-C and CRP (OR=0.417 (95%CI: 0.175–0.997), T1DM kada je prilago|en polu, starosti, HDL-C i CRP (OR
p=0.049). = 0,417 (95%CI: 0,175–0,997), p = 0,049).
Conclusions: Our study demonstrated for the first time that Zaklju~ak: Na{a studija je prva koja je pokazala da je T1DM
T1DM is associated with MOK1 downregulation. In addition, udruèn sa nishodnom regulacijom MOK1. Pored toga,
downregulation of both mTOR and MOK1 gene expressions snièna regulacija ekspresije gena mTOR i MOK1 bila je
was associated with cardiovascular risk factors in overweight povezana sa kardiovaskularnim faktorima rizika kod pacije
T1DM patients. nata sa T1DM sa prekomernom teìnom.
Keywords: type 1 diabetes mellitus, Mammalian target of Klju~ne re~i: dijabetes melitus tip 1, meta rapamicina kod
rapamycin, MAPK/MAK/MRK overlapping kinase 1 sisara, MAPK/MAK/MRK kinaza 1 koja se preklapa
Introduction downstream regulators. MOK1 overexpression has

been mostly associated with different types of cancer,
Type 1 diabetes mellitus (T1DM) is a chronic and this overexpression might be caused by hypo-
disease characterized by dysfunction of pancreatic methylation of its promoter (10). In a recent transcrip-
islet b-cells and concomitant insulin deficiency and tome-wide twin study, Huang et al. (11) showed that
hyperglycemia. It is considered as one of the most upregulation of MOK1 in peripheral blood mononu-
common endocrine and metabolic diseases in chil- clear cells (PBMC) is associated with hypertension.
dren, and over 500,000 children are currently living Principal component analysis of proteins has also
with this condition worldwide (1, 2). Although revealed MOK1 as relevant to type 2 diabetes melli-
observed loss of pancreatic islet b-cell’s function is tus (12).
currently poorly understood, it is evident that autore-
active T lymphocytes play a critical pathological role Considering the fact that 70–90% of PBMCs are
in this process (1, 3). Early stages of T1DM are asso- T lymphocytes (12), as well as their important role in
ciated with B cells mediated activation of CD4+ and pathogenesis of T1DM, the main aim of this study
CD8+ T cells that lead to selective loss of b-cells (1, was to analyze for the first time whether gene expres-
3). The analysis of Langerhans islets in post mortem sion levels of MOK1 and mTOR in PBMCs are
samples obtained close to T1DM diagnosis showed changed in pediatric patients with T1DM. In order to
rare cellular infiltrates dominated by CD4+ and CD8+ get a deeper insight into association of these two
T lymphocytes (4). It seems that T cell autoreactivity kinases with other factors tied with T1DM, we sought
in b-cells autoimmunity is controlled by significant to investigate if their mRNA levels are related to dif-
changes in metabolic pathways (5). Previous studies ferent demographic, clinical and laboratory character-
have shown that these metabolic shifts are highly istics of T1DM patients.
related to T cells activation, proliferation and differen-
tiation into different subsets, the generation of mem-
ory T cells, and the capacity to respond to recall anti- Materials and Methods
gen in the long term (5). Metabolic changes in T cells This study included 172 adolescents with T1DM
are even considered as targets for therapy in preclini- (84 males and 88 females) and 36 healthy adolescent
cal models of autoimmunity and transplantation (5). volunteers (6 males and 30 females) without family
Mammalian target of rapamycin (mTOR) is consid- history of T1DM designated for control group (CG).
ered to be an important intracellular regulator of Recruitment of subjects was done in The Mother and
metabolism and cell growth (5, 6). This serine/threo- Child Health Care Institute of Serbia »Dr Vukan
nine kinase, which is a part of phosphoinositide-3- ûpi}«, Belgrade, Serbia, during regular follow-up in
kinase (PI3K)-AKT pathway, is related to functional the outpatient clinic. Criteria from Serbian national
regulation of T cells, B cells, neutrophils, macro- guidelines of good clinical practice for diagnosis and
phages, dendritic cells, mast cells, and natural killer treatment of diabetes mellitus were used for diagnosis
cells (4). In addition, mTOR seems to be an impor- of diabetes (13, 14). Tanner scale was used for assess-
tant player in cytotoxic T cell proteome shaping (7). ment of puberty stages. The groups were matched by
mTOR signaling dysfunction has been associated pubertal stage and body mass index (BMI).
with type 2 diabetes, cancer, neurodegeneration and
ageing (6). One of the less known regulators of All T1DM patients were on insulin therapy. 162
mTOR is MAPK/MAK/MRK overlapping kinase1 patients were on the intensive insulin therapy regime
(also known as renal tumour antigen-1, and serine/ that included multiple daily insulin injections, and 10
threonine kinase 30) (MOK1). It was suggested that patients were treated with continues subcutaneous
insulin infusion through insulin pump. The patients
MOK1 negatively regulates cilium length of renal
were not on any antihypertensive or lipid-lowering
epithelial cells by inhibition of mTOR complex 1
therapy, and there was no clinical nor laboratory evi-
(mTORC1) signaling (8). MOK1 was firstly identified
dence of any diabetic complications.
in renal carcinoma cells as an antigen recognized by
autologous cytolytic T cells (9). So far, relatively little This study was conducted according to guide-
is known about MOK1 functions or its upstream and lines laid down in the Declaration of Helsinki and
284 Sopi} et al.: Downregulation of MOK1 in T1DM
approved by the Ethics Committees of University of Inc., Chicago, USA). Normality of data distribution
Belgrade-Faculty of Pharmacy and Mother and Child was tested with Shapiro-Wilk test. Data did not follow
Health Care Institute of Serbia »Dr Vukan ûpi}«. All normal distribution and were presented as median
the subjects and their parents were thoroughly with interquartile range. Comparisons between the
informed about all aspects of the study, and written tested groups were done by Mann-Whitney test.
inform consent was obtained. Categorical data were given as absolute frequencies
and compared by Chi-square test for contingency
Whole blood was obtained from all subjects tables. Associations between clinical data were tested
after 12 hours fasting period. Immediately after plas- by Spearman’s bivariate correlation analysis.
ma separation, PBMCs were isolated using density
gradient (Ficoll-Paque® PLUS gradient-gel), added Multivariate binary regression analysis was con-
to TrizolTM (Invitrogen Life Technologies, Foster City, ducted to determine possible independent associa-
USA) and stored at -80 °C. tion of MOK1 mRNAs and T1DM using data signifi-
cantly different between tested groups and data
Glucose, total cholesterol, HDL-cholesterol, and which correlate significantly with MOK1 mRNA levels
triglycerides levels were measured in serum using as covariates (sex, age, HDL-C and CRP). Statistically
routine laboratory methods. Friedewald formula was significant p-value was less than 0.05.
used to calculate LDL-cholesterol. HbA1c level was
determined by competitive turbidimetric inhibition
immunoassay. C-reactive protein (CRP) was meas- Results
ured using immunoturbidimetric method. All the
analyses were performed on Roche/Hitachi c501 Demographic, clinical and laboratory character-
automated analyzer (Roche, Mannheim, Germany). istics of T1DM and healthy controls are presented in
the Table I. The T1DM group had significantly higher
Protocols for RNA isolation, reverse transcription percentage of males (P<0.001) and was significantly
and real-time PCR were described elsewhere (15). In younger compared to CG (P=0.003). Glucose,
brief, total RNA was isolated using TRIZOLTM-chloro- HbA1c, CRP and HDL-C levels were significantly
form extraction with modified protocol (16). RNA higher in T1DM patients (P<0.01, P<0.001 and
concentration and contamination for proteins and P=0.009, P=0.001 respectively). In addition, there
organic solvents was assessed using UV analysis at was no significant difference in systolic blood pres-
260 nm, 280 nm and 230 nm respectively. Integrity sure, total cholesterol, LDL-C and triglycerides levels
of isolated RNA was checked using electrophoresis between observed groups.
on 1% agarose gel.
Normalized MOK1 mRNA levels were signifi-
Reverse transcription was performed on the cantly downregulated in T1DM patients compared to
7500 Real-Time PCR System (Applied Biosystems, CG (P=0.018) (Figure 1A), while there were no sig-
Foster City, CA, USA). nificant differences in normalized mTOR mRNA lev-
MOK1 gene expression levels were measured by els between observed groups (P=0.891) (Figure 1B).
quantitative PCR on 7500 Real-Time PCR System MOK1 gene expression levels were not significantly
(Applied Biosystems, Foster City, CA, USA) using Taq- different between males in females in CG (P=0.865),
Man® 5’-nuclease gene expression assays (Applied nor in T1DM group (P=0.709). Correlation analysis
Biosystems, Foster City, CA, USA) for MOK1 in T1DM group revealed significant negative correla-
(Hs00179504_m1) and HOT FIREPol® DNA Poly- tion of normalized MOK1 mRNA levels with age, and
merase (Solis BioDyne, Tartu, Estonia). MOK1 mRNA triglycerides and positive association with normalized
levels were normalized to beta-actin (Hs01060665_g1) mTOR mRNA levels, as well as negative association
as a housekeeping gene. of normalized mTOR mRNA levels with BMI per-
centiles and systolic blood pressure. (Table II). In addi-
MTOR gene expression levels were measured by tion, we have observed negative trend between BMI
quantitative PCR on Lightcycler II (Roche diagnostics) percentiles and MOK1 mRNA levels (P=0.091)
using specific primers (F: 5 -AGGCCGCATTGTCTC-
TATCAA-3 ; R: 5 -GCAGTAAATGCAGGTAGTCATC- In order to further explore association between
CA-3 ) and 5x HOT FIREPol® EvaGreen® qPCR MOK1 and mTOR gene expression with BMI, we
Supermix (Solis BioDyne, Tartu, Estonia). MTOR have divided T1DM patients according to 85th per-
mRNA levels were normalized to GAPDH (F: 5 - centile BMI. Namely, subjects with BMI larger then
TGCACCACCAACTGCTTAGC-3 ; R: 5 -GGCATG- 85th percentile according to age and gender were
GACTGTGGTCATGAG-3 ) as housekeeping gene. considered as overweight. Interestingly, we have
observed significant downregulation of MOK1
(P=0.034) and mTOR (P=0.017) in overweight sub-
jects, together with significantly higher levels of sys-
Statistical Analysis
tolic blood pressure (P<0.001), total cholesterol
Statistical analysis was performed using a statis- (P=0.001), LDL-cholesterol (P=0.001) and CRP
tical program IBM® SPSS® Statistics version 22 (SPSS (P<0.001). Overweight subjects also had higher
J Med Biochem 2022; 41 (3) 285
Table I Demographic, clinical and laboratory characteristics of CG and T1DM patients.
Parameter CG T1DM P
Sex (male/female)a 6/30 84/88 <0.001
Age (years)b 17 (13–18) (12–16) 0.003
Tanner stage 0.088
Tanner I 3 28
Tanner II 1 13
Tanner III 3 27
Tanner IV 3 26
Tanner V 26 81
Diabetes duration (years)b / 7 (5-8)
BMI percentilesb 54.4 (33.6 –78.4) 56.4 (33.4–78.1) 0.831

Systolic blood pressure (mm
110 (94–120) 110 (100 –115) 0.678
Hg) b
Glucose (mmol/L)b 4.79 (4.66–5.13) 10.20 (7.72–13.85) <0.001
HbA1c (%) 5.2 (4.8–5.0) 7.6 (7.0–8.7) <0.001
Total cholesterol (mmol/L)b 4.01 (3.56– 4.46) 3.98 (3.54– 4.62) 0.600
HDL-cholesterol (mmol/L)b 1.49 (1.26–1.64) 1.63 (1.42–1.88) 0.001
LDL-cholesterol (mmol/L)b 2.21 (1.87–2.51) 2.02 (1.68 –2.50) 0.246
Triglycerides (mmol/L)b 0.81 (0.62–1.03) 0.71 (0.58–0.93) 0.224
CRP (mg/L)b 0.30 (0.20–0.50) 0.70 (0.30 –1.70) 0.009
a – categorical variables (compared with Chi-square test); b – data are presented as median and interquartile range (compared with Mann-
Whitney test)
Figure 1 Normalized mRNA levels of MOK1 (A) and mTOR (B) in CG and T1DM patients.
TableI II Spearman correlation analysis of MOK1 and mTOR mRNA levels with demographic, clinical and laboratory param-
eters in T1DM patients.
Normalized Normalized
Parameter
MOK1 mRNA r / P mTOR mRNA r / P
Age (years) -0.168 / 0.027 -0.086 / 0.259
BMI percentiles -0.117 / 0.091 -0.190 / 0.006
Systolic blood pressure (mm Hg) -0.134 / 0.068 -0.173 / 0.019
Glucose (mmol/L) -0.080 / 0.302 -0.039 / 0.613
Total cholesterol (mmol/L) 0.005 / 0.945 0.091 / 0.233
HDL-cholesterol (mmol/L) 0.101 / 0.192 0.024 / 0.758
LDL-cholesterol (mmol/L) -0.016 / 0.841 0.093 / 0.229
Triglycerides (mmol/L) -0.159 / 0.037 0.010 / 0.892
CRP (mg/L) -0.066 / 0.397 -0.145 / 0.062
HbA1c (%) 0.051 / 0.502 0.122 /0.111
Normalized MOK1 mRNA / 0.350 /<0.001
Normalized mTOR mRNA 0.350 /<0.001 /
Table III Demographic, clinical and laboratory characteristics of CG and T1DM patients.
Parameter BMI < 85 percentile BMI 85 percentile P
Sex (male/female)a 69/71 13/14 0.914
Age (years)b 14 (12–17) (13–16) 0.641
Systolic blood pressure (mm Hg)b 106 (100–110) 120 (110–120) <0.001
Glucose (mmol/L)b 10.19 (7.74–14.16) 11.59 (7.33–14.04) 0.838
HbA1c (%) 7.6 (6.9–8.6) 7.8 (7.4–9.0) 0.231
Total cholesterol (mmol/L)b 3.91 (3.48-4.56) 4.53 (4.14–5.15) 0.001
HDL-cholesterol (mmol/L)b 1.64 (1.42–1.89) 1.63 (1.46–1.93) 0.793
LDL-cholesterol (mmol/L)b 1.91 (1.62–2.36) 2.48 (2.04–3.08) 0.001
Triglycerides (mmol/L)b 0.69 (0.57– 0.93) 0.79 (0.67–1.16) 0.052
CRP (mg/L) b 0.60 (0.30 –1.20) 1.70 (0.90–3.10) <0.001
Normalized MOK1 mRNA b 0.962 (0.709–1.319) 0.805 (0.654–1.019) 0.034
Normalized mTOR mRNA b 1.023 (0.842–1.245) 0.840 (0.769–1.039) 0.017
a – categorical variables (compared with Chi-square test); b - data are presented as median and interquartile range (compared with Mann-
Whitney test)
J Med Biochem 2022; 41 (3) 287
triglycerides levels, but this was of borderline signifi- population, especially in T1DM patients. It is charac-
cance (P=0.052) (Table III). terized by proatherogenic lipid profile and increased
systemic inflammation (19). In our study, T1DM
Binary logistic regression analysis was used to
patients above the threshold for overweight children
test associations of MOK1 with the presence of
(85th percentile BMI according to age and gender),
T1DM. Univariate analysis revealed significant nega-
along with higher levels of systolic blood pressure,
tive association between MOK1 mRNA and T1DM
total cholesterol, LDL-cholesterol, triglycerides and
(OR=0.449 (95%CI: 0.224–0.897), p=0.023). In
CRP, had significantly lower levels of MOK1 and
multivariate analysis, when MOK1 was adjusted for
mTOR gene expressions. Namely, the hormonal
sex, age, HDL-C and CRP, it demonstrated significant
changes of normal puberty cause a transient physio-
independent negative association with T1DM
logic state of insulin resistance. This insulin resistance
(OR=0.417 (95%CI: 0.175–0.997), p=0.049).
is markedly exaggerated in adolescents T1DM, espe-
cially in obese ones, leading to defects in both the
plasma glucose and lipid-lowering effects of insulin
Discussion
(20). In addition, peripheral insulin resistance, both in
This study, for the first time, demonstrated that well controlled and poorly controlled T1DM, is reflect-
MOK1 gene expression levels were downregulated in ed by impaired insulin suppression of fatty tissue
PBMCs of patients with T1DM compared to healthy lipolysis and lowering of plasma free fatty acids and
controls, whereas there was no significant difference glycerol levels (20, 21), followed by increased trigly-
in gene expression level of mTOR between groups. In cerides levels, which contribute to cardiovascular risk
addition, MOK1 mRNA showed positive correlation (19, 20). Therefore, it is not surprising that over-
with mTOR mRNA, and negative correlation with weight T1DM patients in our study showed higher
age, and TG, while mTOR correlated negatively with levels of proatherogenic lipids (total cholesterol, LDL-
systolic blood pressure and BMI percentiles. cholesterol, triglycerides) compared to non-over -
weight ones. Along with that, MOK1 was negatively
Although MOK1 was identified 20 years ago as correlated with serum triglycerides, and downregulat-
an antigen in renal carcinoma cells that can bind to ed in overweight T1DM group, suggesting potential
autologous cytolytic T cells, not much is known about link to higher risk of cardiovascular disease develop-
its role in cell signaling (9). Since its discovery, MOK1 ment in T1DM patients.
overexpression was observed in numerous types of
cancer cells (10). Quite surprisingly, changes in Furthermore, mTORC1 represents a regulator
MOK1 expression levels in PBMCs along with 12 of cellular nutrient and energy status through stimu-
other genes were associated with hypertension in lation of protein, lipid and nucleotide synthesis (22,
transcriptome-wide twin study (11). Our study is the 23). It has been found that attenuation of mTOR sig-
first to report that downregulation of MOK1 in naling in the form of protein complex mTORC1 is
PBMCs is associated with T1DM. This association was associated with increased lipolysis in adipose tissue,
independent of sex, age, HDL-C and CRP levels. The as well as with enhanced autophagy in adipocytes of
effects of observed MOK1 downregulation on func- obese patients with T2DM (24). In addition, it was
tions of PBMCs in T1DM are currently not known. suggested that the relation between mTORC1 activity
One study conducted in cell models for amyotrophic and insulin resistance follows a U-shaped curve,
lateral sclerosis suggested that MOK1 inhibition where too little or too much mTORC1 activity has a
might lead to inflammatory response. Namely, this negative impact on systemic metabolism (25). In that
study showed that aggregates of pathological protein way, downregulation of mTOR seen in overweight
TDP-43 bind to MOK1, disrupting its phosphorylation T1DM could further aggravate processes leading to
status supposedly leading to NLRP3/caspase-1 peripheral insulin resistance. Taken all together, our
inflammasome activation and secretion of IL-1b and findings imply that downregulation of both MOK1
IL-18 (17). NLRP3/caspase-1 inflammasome activa- and mTOR genes, together with proatherogenic lipid
tion stimulates secretion of IL-1b and IL-18 (17, 18) profile and increased SBP, could contribute to
and increases systemic inflammation, while IL-1 alone increased risk of atherosclerosis development and
is a major regulator of T-cell proliferation and function cardiovascular complications seen in these patients.
leading to polarization of T cells towards proinflam- However, our conclusions are drawn from different
matory immunity (18). Therefore we can presume studies that explored function of MOK1 and mTOR in
that the observed downregulation of MOK1 in various cell types. Our findings are limited to PBMCs,
PBMCs could lead to increased proinflammatory and require further functional studies to back up our
activity of T cells and contribute to the overall proin- premises.
flammatory scenery seen in patients with long stand-
ing T1DM.
Limitations
Obesity is considered as a major risk factor for
atherosclerosis development and progression and Our study has several limitations. Firstly, this
subsequent cardiovascular complications in general study was performed on relatively small number of
healthy participants. Considering that the participants Conclusion

were pediatric population, we had to take into
Our study demonstrated for the first time that
account the underlying ethical rationale. Next, the MOK1 downregulation is associated with T1DM,
percentage of females was significantly higher in CG which could lead to increased proinflammatory state
compared to T1DM group. However, mRNA levels of in T1DM. In addition, downregulation of both mTOR
MOK1 and mTOR were not significantly different and MOK1 gene expressions was associated with car-
between males in females in CG, nor in T1DM group, diovascular risk factors in overweight T1DM patients.
suggesting that uneven sex distribution between
groups did not significantly influence our conclusions. Acknowledgments. This work was supported by
a grant from the Ministry of Education, Science and
Thirdly, CG was significantly older in comparison to
Technological Development, Republic of Serbia
T1DM group. Even though MOK1 exhibited a signif-
(Project No. 175035), and CEEPUS project (CIII-SI-
icant negative correlation with age, we find that this 0611-08-1819) Novel diagnostic and therapeutic
correlation has no significant effect on our conclu- approaches to complex genetic disorders. The
sions. Namely, our study demonstrated negative asso- Authors would like to thank students Nikola Tomi},
ciation of MOK1 levels and age, and since our CG is Olivera Borojev and PhD Klemen Kodri~ for help in
older we can presume that this age difference didn’t laboratory work and primers optimization.
affect the observed difference in MOK1 mRNA levels.
Moreover, multivariate binary logistic regression
showed that MOK1 is associated with T1DM inde- Conflict of interest statement
pendently of sex, age, HDL-C and CRP levels.
All the authors declare that they have no conflict
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Received: July 20, 2021

Accepted: October 18, 2021
UDK 577.1 : 61 ISSN 1452-8258

PREOPERATIVE PREALBUMIN-TO-FIBRINOGEN RATIO TO PREDICT

SURVIVAL OUTCOMES IN HEPATOCELLULAR CARCINOMA PATIENTS
AFTER HEPATIC RESECTION
PREOPERATIVNI ODNOS PREALBUMIN-FIBRINOGEN ZA PREDVI\ANJE PRE@IVLJAVANJA
KOD PACIJENATA SA HEPATOCELULARNIM KARCINOMOM NAKON HEPATEKTOMIJE
Haixi Yan1,2, Shuaishuai Chen2, Yang Qiong2, Linling Cai2

1Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang Province, China
2Department of Clinical Laboratory, Taizhou Hospital of Zhejiang Province affiliated to Wenzhou
Medical University, Linhai 317000, Zhejiang Province, China

Background: This study aimed to evaluate the clinical appli- Uvod: Cilj ove studije je bio da proceni klini~ku primenu
cation of the preoperative prealbumin-to-fibrinogen ratio preoperativnog odnosa prealbumin-fibrinogena (PFR) u
(PFR) in the clinical diagnosis of hepatocellular carcinoma klini~koj dijagnozi pacijenata sa hepatocelularnim karci-
(HCC) patients and its prognostic value. nomom (HCC) i njegovu prognosti~ku vrednost.
Methods: The clinical and laboratory data of 269 HCC Metode: Sprovedena je retrospektivna analiza klini~kih i
patients undergoing surgical treatment from January 2012 laboratorijskih podataka 269 pacijenata sa HCC koji su bili
to January 2017 in Taizhou Hospital were retrospectively na hirur{kom le~enju od januara 2012. do januara 2017.
analysed. The Cox regression model was used to analyse u Taizhou bolnici. Za analizu korelacije izme|u PFR-a i
the correlation between the PFR and other clinicopatholog- drugih klini~ko-patolo{kih faktora u ukupnom preìvljava-
ical factors in overall survival (OS) and disease-free survival nju (OS) i preìvljavanju bez bolesti (DFS) kori{}en je
(DFS). Koksov regresioni model.
Results: Cox regression analysis showed that the PFR (haz- Rezultati: Koksova regresiona analiza je pokazala da je PFR
ard ratio (HR)=2.123; 95% confidence interval (95% CI), (indeks rizika (HR) = 2,123; 95% interval pouzdanosti
1.271–3.547; P=0.004) was an independent risk factor (95% CI), 1,271–3,547; P = 0,004) bio nezavisni faktor
affecting the OS of HCC patients. Furthermore, a nomo- rizika koji je uticao na ukupno preìvljavanje pacijenata sa
gram was built based on these risk factors. The C-index for HCC. Dodatno, na osnovu ovih faktora rizika je ura|en
the OS nomogram was 0.715. nomogram. C-indeks za nomogram ukupnog preìvljavanja
Conclusions: Nomograms based on the PFR can be recom- je bio 0,715.
mended as the correct and actual model to evaluate the Zaklju~ak: Nomogrami zasnovani na PFR-u se mogu pre-
prognosis of patients with HCC. poru~iti kao ispravan i stvarni model za procenu prognoze
kod pacijenata sa HCC.
Keywords: hepatocellular carcinoma, prealbumin-to-fib-
rinogen ratio, survival analysis, nomogram Klju~ne re~i: hepatocelularni karcinom, odnos prealbu-
mina i fibrinogena, analiza preìvljavanja, nomogram

Linling Cai
Department of Clinical Laboratory, Taizhou Hospital of Zhejiang
Province affiliated to Wenzhou Medical University, 150 Ximen
Street of Linhai City, Linhai 317000, Zhejiang Province, China
e-mail:cllªenzemed.com
J Med Biochem 2022; 41 (3) 291
Introduction differentiation degree were obtained through patho-

logical examination. The intraoperative lymph nodes
Hepatocellular carcinoma (HCC) is a globally
were examined to determine the lymph node metas-
prevalent malignant tumour with a 5-year survival rate
tasis in each study group. Preoperative blood exami-
(<5%) (1). HCC is mainly diagnosed by imaging
nation data was exhaustive. Furthermore, the exclu-
examinations, serological markers (alpha-fetoprotein,
sion criteria were (a) patients with other benign liver
a-L-fucosidase, abnormal prothrombin, and so on),
tumours (e.g., haemangiomas and hepatic adeno-
and liver histopathological diagnoses. Moreover,
mas) and (b) patients with infections or other inflam-
China has the highest incidence of liver cancer.
matory diseases. Finally, 269 patients were included.
According to informal statistics, the number of
Chinese people who die of liver cancer each year is
approximately 110,000, accounting for 45% of the
Treatment and follow-up
world’s liver cancer deaths (2).
All patients underwent a 1-year regular follow-
Fibrinogen is an important acute-phase protein.
up visit. The follow-up deadline was April 22, 2017.
Recent studies have found that fibrinogenaemia is
The recurrence and survival times were all recorded
strongly related to various tumours’ occurrence,
in months, from surgery to recurrence or death.
development, and prognosis. These tumours mainly
According to the follow-up data criteria, tumour
include renal cell carcinoma, lung cancer, ovarian
recurrence or metastasis was the DFS endpoint.
cancer, and hepatocellular carcinoma (3–6).
Furthermore, the overall survival (OS) endpoint was
Furthermore, prealbumin levels can often be used to
death.
assess a patient’s nutritional status. Consequently,
prealbumin levels can reflect postoperative effects
such as cervical cancer, metastatic renal cell carcino-
Statistical analysis
ma, and non-small-cell lung cancer (7–9).
Furthermore, a new prognostic indicator has been The Statistical Package for the Social Sciences,
reported, which is a combination of prealbumin and version 22.0, and X-tile, version 3.6.1, were used for
fibrinogen. Several articles have shown that prealbu- analysis. The optimal PFR cut-off value was calculat-
min and fibrinogen (PFR) ratio has good prognostic ed using the X-tile plot. The X″ test was used to anal-
significance in acute pancreatitis (10). yse the differences between the categorical variables.
Moreover, the Cox proportional hazards model was
Therefore, the trend of early detection and early
used for univariate and multivariate survival analyses
intervention is crucial for HCC treatment and mortal-
to determine the risk factors for prognosis. Finally, the
ity reduction. Finding new HCC biomarkers to predict
nomogram for prediction value was established using
the prognosis of HCC patients after surgery is urgent.
R software. Consequently, model accuracy was evalu-
Thus, this study aimed to use nomograms to analyse
ated by Harrell’s concordance index (c-index), cali-
the relationship of the preoperative PFR in the prog-
bration plots, decision curves, and clinical impact
nosis of HCC patients to determine its prognostic
curves. P values ≤ 0.05 were considered statistically
value in HCC.
significant.

Results
Patients
Analysis and calculation of the PFR optimal cut-
This study recruited 367 HCC patients admitted off value
to the Department of Hepatobiliary Surgery of
This study group enrolled 269 patients. Taking
Taizhou Hospital of Zhejiang Province, Taizhou,
postoperative mortality of the patients as the end-
China, from January 2012 to January 2017. The
point, 0.8 was the optimal PFR cut-off value calculat-
inclusion criteria were as follows: (a) complete clinical
ed by the X-tile plot (Figure 1).
and laboratory data (current medical history, previous
medical history, family history, personal history, phys-
ical examination, and complete pathological data);
Patient characteristics
(b) abdominal computed tomography or ultrasound
to exclude liver abscess, liver-occupying lesions, and The patients’ baseline and clinicopathological
other diseases; (c) the use of required pathological characteristics stratified by the PFR are described in
data to exclude secondary liver cancer; (d) postoper- Table I. Furthermore, the table shows that the patients
ative TNM clinical-pathological stages I, II, and III; were divided into two groups for further analysis
and (e) exhaustive whole blood cell analysis, bio- (PFR<0.8 and PFR 0.8). Moreover, the PFR was
chemical analysis, and examinations. The preopera- associated with the Barcelona Clinic Liver Cancer
tive clinical diagnosis was clear, and the tumour loca- (BCLC) stage, Child-Pugh score, complications (hep-
tion, size, number, pathological stage, and atic rupture, portal hypertension, and intraoperative
292 Yan et al.:
Figure 1 X-tile OS analyses were performed using patient data to determine the optimal PFR cut-off values. The optimal cut-
off values are shown in the histograms of the entire cohort (left panels), and Kaplan–Meier plots are displayed (right panels).
The P values were determined by using the cut-off values defined in the training sets and applying them to the validation sets.
A. For OS, the optimal cut-off value of the PFR was 0.8. B. For DFS, the optimal cut-off value of the PFR was 0.7.
ascites), survival, tumour length, and red cell distribu- Prognostic value of the PFR
tion width. The average age of the 269 patients was
Univariate analysis was carried out using the Cox
57 years, with 219 (81.4%) males and 50 (18.6%)
regression model. Consequently, the clinicopatholog-
females. Moreover, patients with stages A and B–C,
ical parameters predicting OS and DFS were further
using the BCLC staging system, accounted for 55.8%
studied. In the univariate analysis, the BCLC stage,
and 44.2% of the total patients, respectively.
nerve invasion, vascular invasion, T stage, M stage,
However, patients with grades A and B accounted for
tumour size, number of tumours and PFR were signif-
89.6% and 10.4% of the total patients’ Child-Pugh
icantly related to OS (p<0.05). Furthermore, the
scores, respectively. Patients with complications of
BCLC stage, portal hypertension, vascular invasion, T
liver rupture bleeding, portal hypertension, and hep-
stage, M stage, tumour size, number of tumours
atic encephalopathy accounted for 7.8%, 6.3%, and
and PFR were associated with DFS (p<0.05). For the
0.7% of the total patients, respectively. Consequently,
multivariate Cox regression OS model, the PFR
173 patients survived (64.3%). Of the total patients,
(hazard ratio (HR)=2.123; 95% confidence interval
167 (62.1%) and 102 (37.9%) were diagnosed with
(95% CI), 1.271–3.547; P=0.004), vascular invasion
tumours <5 and 5 cm, respectively.
(HR=2.272; 95% CI, 1.032–5.003; P=0.041), M
stage (HR =8.095; 95% CI, 3.518–18.627;
J Med Biochem 2022; 41 (3) 293
Table I Comparison of baseline clinicopathological characteristics based on PFR.

Cases PFR
P
No. (%) <0.8 ≥0.8
≤60 164 (61%) 25 139
Age (years) 0.312
>60 105 (39%) 21 84
Male 219 (81.4%) 35 184
Gender 0.308
Female 50 (18.6%) 11 39
No 140 (52%) 25 115
Smoking 0.731
Yes 129 (48%) 21 108
No 196 (72.9%) 37 159
Drinking 0.205
Yes 73 (27.1%) 9 64
A 150 (55.8%) 18 132
BCLC stage 0.013
B, C 119 (44.2%) 28 91
A 241(89.6%) 31 210
Child-Pugh score 0.000
B 28 (10.4%) 15 13
Liver rupture No 248 (92.2%) 33 215
0.000
bleeding Yes 21 (7.8%) 13 8
No 252 (93.7%) 37 215
Portal hypertension 0.000
Yes 17 (6.3%) 9 8
No 267 (99.3%) 45 222
Hepatic encephalopathy 0.215
Yes 2 (0.7%) 1 1
No 146 (54.3%) 21 125
Recrudescence 0.197
Yes 123 (45.7%) 25 98
Survival 173 (64.3%) 23 150
Survival situation 0.026
Mortality 96 (35.7%) 23 73
No 69 (25.4%) 7 62
Cirrhosis 0.075
Yes 200 (74.3%) 39 161
Liver capsule No 220 (81.8%) 37 183
0.795
invasion Yes 49 (18.2%) 9 40
No 247 (91.8%) 39 208
Liver margin 0.056
Yes 22 (8.2%) 7 15
No 259 (96.3%) 45 214
Nerve invasion 0.543
Yes 10 (3.7%) 1 9
No 201 (74.7%) 30 171
Vascular invasion 0.103
Yes 68 (25.3%) 16 52
T0–T1 157 (58.4%) 24 133
T stage 0.350
T2–T4 112 (41.6%) 22 90
N0 264 (98.1%) 45 219
N stage 0.862
N1 5 (1.9%) 1 4
M0 263 (97.8%) 45 218
M stage 0.977
M1 6 (2.2%) 1 5
<5 cm 167 (62.1%) 20 147
Tumour size (cm) 0.004
5 cm 102 (37.9%) 26 76
Number of 1 209 (77.7%) 38 171
0.379
tumours 2 60 (22.3%) 8 52
Hepatitis B No 47 (17.5%) 43 4
0.097
infection Yes 222 (82.5%) 181 41
300 × 109/L 259 (96.3%) 45 214
PLT 0.543
>300 × 109/L 10 (3.7%) 1 9
20 mg/L 116 (43.1%) 15 101
AFP 0.114
>20 mg/L 153 (56.9) 31 122
≤5 ng/mL 232 (86.2%) 41 191
CEA 0.533
>5 ng/mL 37 (13.8%) 5 32
294 Yan et al.:
Table II Univariate and multivariate survival analyses of OS and DFS in HCC patients.
OS DFS
Univariate analysis Multivariate analysis Univariate analysis Multivariate
P P P P
HR (95% CI) HR (95% CI) HR (95% CI) HR (95% CI)analysis
Age (years) 0.489 0.389 0.937 0.525
≤60 1.000 1.000 1.000 1.000
>60 1.157 (0.766 –1.749) 1.010 0.985 (0.680–1.427) 1.133 (0.772–1.663)
Gender 0.948 0.796 0.985 0.991
Male 1.000 1.000 1.000 1.000
Female 0.982 (0.574–1.682) 0.927 (0.520–1.651) 0.995 (0.621–1.595) 0.997 (0.614–1.621)
Smoking 0.545 0.424
No 1.000 1.000
Yes 1.132 (0.758–1.690) 1.157 (0.809–1.653)
Drinking 0.536 0.386
No 1.000 1.000
Yes 1.146 (0.744–1.766) 1.189 (0.804–1.757)
BCLC stage 0.000 0.421 0.001 0.852
A 1.000 1.000 1.000 1.000
B, C 2.171 (1.445–3.263) 0.766 (0.401–1.466) 1.800 (1.257–2.579) 0.945 (0.523–1.709)
Child’s score 0.055 0.123
A 1.000 1.000
B 1.742 (0.988–3.073) 1.532 (0.891–2.635)
Liver rupture bleeding 0.083 0.238
No 1.000 1.000
Yes 1.746 (0.930–3.278) 1.433 (0.789–2.604)
Portal hypertension 0.068 0.002 0.062
No 1.000 1.000 1.000
Yes 1.842 (0.955–3.551) 2.462 (1.378–4.397) 1.870 (0.970–3.604)
Hepatic encephalopa- 0.547 0.727
No 1.000 1.000
Yes 1.833 (0.255–13.188) 1.421 (0.198–10.193)
Cirrhosis 0.699 0.403
No 1.000 1.000
Yes 1.100 (0.678–1.787) 1.203 (0.780–1.858)
Liver capsule invasion 0.328 0.881
No 1.000 1.000
Yes 1.270 (0.787–2.048) 1.035 (0.661–1.621)
Liver margin 0.085 0.234
No 1.000 1.000
Yes 1.782 (0.923–3.439) 1.459 (0.784–2.717)
Nerve invasion 0.034 0.330 0.168
No 1.000 1.000 1.000
Yes 2.456 (1.071–5.633) 1.596 (0.623–4.086) 1.787 (0.783–4.078)
Vascular invasion 0.000 0.041 0.001 0.151
No 1.000 1.000 1.000 1.000
Yes 2.472 (1.619–3.774) 2.272 (1.032–5.003) 1.938 (1.315–2.856) 1.709 (0.823–3.550)
T stage 0.000 0.938 0.001 0.778
T1 1.000 1.000 1.000 1.000
T2, T3, T4 2.111 (1.412–3.157) 0.966 (0.408–2.286) 1.823 (1.276–2.603) 0.894 (0.411–1.947)
N stage 0.876 0.623
N0 1.000 1.000
N1 1.119 (0.272–4.598) 0.698 (0.167–2.925)
M stage 0.000 0.000 0.000 0.000
M0 1.000 1.000 1.000 1.000
M1 13.114 (5.515–31.184) 8.095 (3.518–18.627) 8.556 (3.679–19.897) 5.015 (2.253–11.163)
Tumour length(cm) 0.000 0.007 0.001 0.073
<5 cm 1.000 1.000 1.000 1.000
≥5 cm 2.320 (1.551–3.471) 2.188 (1.240–3.859) 1.868 (1.306–2.671) 1.611 (0.957–2.711)
Number of tumours 0.025 0.224 0.004 0.103
1 1.000 1.000 1.000 1.000
≥2 1.658 (1.067–2.577) 1.532 (0.770–3.048) 1.770 (1.197–2.617) 1.714 (0.896–3.278)
Hepatitis B infection 0.630 0.674
No 1.000 1.000
Yes 0.816 (0.357–1.867) 0.849 (0.395–1.822)
PFR 0.001 0.004 0.007 0.092
≥0.8 1.000 1.000 1.000 1.000
<0.8 2.261 (1.411–3.624) 2.123 (1.271–3.547) 1.829 (1.175–2.848) 1.525 (0.934–2.491)
J Med Biochem 2022; 41 (3) 295
Figure 2 Kaplan–Meier curves for OS according to the PFR in each subgroup and the total HCC patients. A. total HCC
patients, B. vascular invasion-negative subgroup, C. vascular invasion-positive subgroup, D. solitary tumour subgroup, and E.
multiple tumours subgroup.
P=0.000), and tumour size (HR=2.188; 95% CI, (<0.8) were associated with short OS (Figure 2A).
1.240–3.859; P=0.007) were verified to be indepen- Based on vascular invasion and the number of
dent prognostic factors in patients with HCC (Table tumours, a separate subgroup analysis was also con-
II). ducted to investigate the significance of the PFR for
the prognosis of HCC patients. Moreover, short OS
was found in patients with low PFR in the solitary and
Survival PFR analysis multiple tumour subgroups (P<0.001 and p<0.001,
respectively) and the vascular invasion-negative sub-
This study used Kaplan-Meier analysis to deter- group (P<0.001) but not in the vascular invasion-
mine the prognostic value of the PFR. Low PFR levels positive subgroup (p=0.596; Figure 2B, 2C, 2D, 2E).
296 Yan et al.:
Figure 3 The PFR was an independent predictive factor for disease progression in HCC patients. A. Nomogram predicting the
OS of HCC patients. B. 1-year OS calibration plot. C. 3-year OS calibration plot. D.5-year OS calibration plot.
Figure 4 Decision curve for the disease progression of HCC patients. A. Nomogram, red line; BCLC stage, blue line. TMN
stage, purple line. The abscissa of this graph is the threshold probability, and the ordinates are the net benefit. B. Clinical
Impact Curve.
J Med Biochem 2022; 41 (3) 297
Development and validation of nomograms for thesised by cancer cells (26, 27). Meanwhile, highly
predicting OS in HCC patients concentrated fibrinogen induces epithelial-mesenchy-
mal transition, which increases cancer cell invasion
The nomograms can be explained by adding up and metastasis using a cell line model by increasing
the number of points assigned to each variable at the vimentin expression and decreasing E-cadherin
top of the scale. At the bottom of the scale, the total expression (28).
score translates into predicting the patient’s 5-year
probability of mortality. A nomogram, based on inde- Therefore, in theory, prealbumin and fibrinogen
pendent risk factors (the PFR, vascular invasion, M are two valuable markers for monitoring HCC pro-
stage, tumour size), was established to predict OS in gression. Furthermore, this study showed that the
HCC patients (Figure 3A). The C-index for the OS PFR, an inflammatory marker, is a potential prognos-
nomogram was 0.715 (95% CI, 0.662–0.768). tic factor for HCC patients, combining the two factors
Moreover, the calibration curves by internal validation of HCC patients’ nutritional status and inflammatory
demonstrated good agreement between the predicted response status and having the advantages of low
and actual probability of 1-, 3- and 5-year OS (Figure cost and convenience, rapidity, and easy detection.
3B, 3C, 3D). The decision curve analysis found that the This study tried to explain the prognostic value of
nomogram model that included the PFR had better net the PFR for HCC patients. X-tile was used to calculate
benefits than the model that included the BCLC and the optimal PFR cut-off value of 0.8. Moreover, many
TNM stages to identify OS for HCC patients (Figure 4). studies previously used the receiver operating charac-
teristic (ROC) curve to select the cut-off value.
However, most of the ROC curves included only the
Discussion event outcomes and experimental indicators but did
Malnutrition and fibrinogen abnormalities are not include important factors for cancer prognosis. The
common in cancer patients and have major effects on X-tile software precisely includes the time worth choos-
their quality of life, treatment outcomes, and progno- ing as the cut-off value. Thus, the cut-off value of this
sis (11, 12). This study indicates that a low preopera- article may be more accurate. Furthermore, this study
tive PFR (<0.8) is an independent risk factor for OS suggests that the PFR is associated with OS and DFS in
in HCC patients. HCC patients in univariate analysis, and the PFR is
related to the patient’s OS (HR=2.123; 95% CI,
Chronic infections, including HCC, cause >15% 1.271–3.547; P=0.004) and has nothing to do with
of malignancies worldwide (13). Studies have shown DFS in multivariate analysis. The risk of mortality of
that systemic inflammatory responses boost angio- HCC patients with a low PFR is significantly increased.
genesis and tumour invasion by upregulating Moreover, the nomogram was used to establish the
cytokines (14–16). This shows that the inflammatory prognostic model of liver cancer, and the accuracy of
response plays a critical role in tumorigenesis and the nomogram was proven by calibration, decision, and
tumour development. Furthermore, the two functions clinical influence curves. The nomogram shows that
of prealbumin may be related to the occurrence and the PFR has an important predictive value.
prognosis of tumours. The first is that inflammation is
associated with decreased prealbumin levels in sever- Although the PFR can predict OS in HCC patients,
al studies (17, 18). Moreover, prealbumin levels may there are some limitations in this study. First, this single-
be affected in other ways during inflammation centre retrospective study may have selection bias as it
because cytokines (e.g., IL-6, IL-1, and TNF-a) can only included HCC patients undergoing surgical resec-
downregulate synthesis (19) and increase vascular tion. Moreover, this study does not represent HCC
permeability (20). The second is that prealbumin can patients who refuse surgery for different reasons.
respond to the nutritional status of the reaction body. Second, verification queues are lacking to verify whether
Serum prealbumin has a shorter half-life than albu- the findings of this study are commonly used. Therefore,
min and is synthesised by hepatocytes. However, the results of this study need to be further verified in for-
synthesis rapidly declines when hepatocytes are dam- ward-looking and large-scale cooperative research.
aged. Furthermore, tumours cause protein malab-
sorption in the body and can also cause prealbumin
levels to decline. Studies have recently shown that Conclusion
hypoproteinaemia is a poor prognostic indicator of In conclusion, the results of this study suggest
oesophageal and colorectal cancers (21, 22). that the PFR (<0.8) is a prognostic indicator of OS in
Fibrinogen is the most acutely reactive plasma HCC patients. Thus, a PFR-containing nomogram
protein (1). It plays an important role in activating the can be used as a more practical model for evaluating
coagulation cascade (23). Moreover, fibrinogen is OS in HCC patients.
also the key factor in regulating the inflammatory cas-
cade through the interaction of ligand-receptor Conflict of interest statement
mechanisms involving immune cells (e.g., monocytes
and microvasculature) (2, 24, 25). Some studies have All the authors declare that they have no conflict
indicated that fibrinogen can be endogenously syn- of interest in this work.
298 Yan et al.:
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Accepted: October 12, 2021
J Med Biochem 2022; 41 (3) DOI 10.5937/jomb0-34545
UDK 577.1 : 61 ISSN 1452-8258

SPECIFIC IMPACT OF CARDIOVASCULAR RISK FACTORS ON CORONARY

MICROCIRCULATION IN PATIENTS WITH SUBCLINICAL HYPOTHYROIDISM
SPECIFIÂN UTICAJ KARDIOVASKULARNIH FAKTORA RIZIKA NA KORONARNU
MIKROCIRKULACIJU U PACIJENATA SA SUBKLINI^KOM HIPOTIREOZOM
Mirjana Stojkovic1,2, Biljana Nedeljkovic-Beleslin1,2, Milorad Tesic1,3, Zoran Bukumiric1,4,

Jasmina Ciric1,2, Milos Stojanovic1,2, Marija Miletic1,2, Ana Djordjevic-Dikic1,3, Vojislav Giga1,3,
Branko Beleslin1,3, Milos Zarkovic1,2
1Facultyof Medicine, University of Belgrade, Belgrade, Serbia
2Clinic of Endocrinology, Diabetes and Metabolic Disease, University Clinical Centre of Serbia, Belgrade, Serbia
3Clinic for Cardiology, University Clinical Centre of Serbia, Belgrade, Serbia
4Instuitute of Medical Statistics and Informatics, Faculty of Medicine, University of Belgrade, Belgrade, Serbia

Background: Although thyroid hormones have significant Uvod: Poznato je da tiroidni hormoni imaju zna~ajan efekat
effect on cardiovascular system, the impact of subtle thy- na kardiovaskularni sistem, ali i dalje ostaje da se utvrdi uti-
roid dysfunction such as subclinical hypothyroidism (SCH) caj suptilnih promena na nivou tiroidne osovine kao {to je
remains to be determined. We investigated coronary flow subklini~ka hipotireoza (SHT). Ispitivali smo koronarnu re-
reserve (CFR) in patients with subclinical hypothyroidism. zervu protoka (KRP) kod pacijenata sa subklini~kim hipo-
Methods: Thirty two subjects with SCH and eighteen con- tireoidizmom.
trol subjects with normal serum thyroid hormones and thy- Metode: Trideset dva ispitanika sa subklini~kom hipotireo-
roid-stimulating hormone (TSH) levels were included in the zom i osamnaest kontrolnih ispitanika sa urednim tiroidnim
study. TSH, free thyroxine, free triiodothyronine, glucose, hormonskim statusom su bili uklju~eni u studiju. Mereni su
insulin, HbA1c, cholesterol, triglyceride and plasma levels TSH, fT4, fT3, glukoza, insulin, HbA1c, holesterol, tri-
of C-reactive protein were measured. Coronary diastolic gliceridi i CRP. Koronarne dijastolne brzine protoka u levoj
peak flow velocities in left anterior descending coronary prednjoj silaznoj koronarnoj arteriji merene su na po~etku i
artery were measured at baseline and after adenosine infu- nakon infuzije adenozina. Koronarna rezerva protoka je
sion. CFR was calculated as the ratio of hyperemic to base- izra~unata kao odnos hiperemijske i osnovne dijastolne
line diastolic peak velocity. brzine protoka.
Results: CFR values were not significantly different between Rezultati: Vrednosti koronarne rezerve protoka se nisu
the two groups (SCH 2.76±0.35 vs controls 2.76±0.42). zna~ajno razlikovale izme|u dve grupe (SHT 2,76 ± 0,35
There was a significant correlation of CFR with waist to hip u odnosu na kontrole 2,76 ± 0,42). Postojala je zna~ajna
ratio, hypertension, smoking habits, markers of glucose sta- korelacija KRP sa odnosom struka i kukova, hipertenzijom,
tus (glucose level, HbA1c, insulin level, HOMA IR), choles- navikama pu{enja, markerima glikoregulacije (nivo glu-
terol, LDL-cholesterol and triglyceride levels in SCH group, koze, HbA1c, nivo insulina, HOMA IR), holesterolom, LDL-
whereas only cholesterol level showed significant correla- holesterolom i nivoom triglicerida u SHT grupi, dok je
tion with CFR in controls. There was no correlation samo nivo holesterola pokazao zna~ajnu korelaciju sa KRP
between CFR and thyroid hormones. u kontrolnoj grupi. Nije bilo korelacije izme|u KRP i hor-
mona {titaste `lezde.

Mirjana Stojkovi}
Clinic of Endocrinology, Diabetes and Metabolic Disease,
University Clinical Centre of Serbia, Dr Subotica 13,
11000 Belgrade, Serbia
phone: +381-69-3755-440 List of abbreviations: SCH, subclinical hypothyroidism; CFR,
fax: +381-11-2685-357 coronary flow reserve; LAD, left anterior descending coronary
e-mail: mirjana.stojkovicªgmail.com artery; DPFV, diastolic peak flow velocity
300 Stojkovi} et al.: Coronary risk in subclinical hypothyroidism
Conclusions: We concluded that there is a different impact Zaklju~ak: Zaklju~ili smo da postoji druga~iji uticaj kardio-
of cardiovascular risk factors on CFR in SCH patients com- vaskularnih faktora rizika na koronarnu rezervu protoka kod
pared to healthy control and that these two groups behave pacijenata sa subklini~kom hipotireozom u pore|enju sa
differently in the same circumstances under the same risk zdravom kontrolom i da se ove dve grupe pona{aju razli~ito
factors. The basis for this difference could be that the u istim okolnostima, pod istim faktorima rizika. Osnova za
altered thyroid axis »set point« changes the sensitivity of the ovu razliku mogla bi biti da promenjen »set point« tiroidne
microvasculature in patients with SCH to known risk fac- osovine menja osetljivost mikrovaskulature kod pacijenata
tors. sa SHT na poznate faktore rizika.
Keywords: cardiovascular risk factors, coronary flow Klju~ne re~i: kardiovaskularni faktori rizika, koronarna
reserve, subclinical hypothyroidism, thyroid rezerva protoka, subklini~ka hipotireoza, {titasta `lezda
Introduction years). SCH was diagnosed on the basis of persistent

TSH increase with free thyroid hormones level within
Subclinical hypothyroidism (SCH) is defined as
the referent range. Patients were included in the study
mild elevation of thyroid-stimulating hormone (TSH)
only if they had stable SCH which was demonstrated
in the presence of normal free thyroxine (fT4) and
by repeated thyroid hormone profile after minimum
free triiodothyronine (fT3) levels (1). It is well known
four weeks. The institutional ethics committee
that overt hypothyroidism has negative impact on car-
approved the study protocol, and all participants
diovascular function (2, 3). The clinical importance of
subclinical hypothyroidism in cardiovascular disease signed informed consent to the study.
and mortality is still controversial, because of the The exclusion criteria for SCH group as well as
inconsistent results (4–6), on the impact of SCH on for the control group were history of coronary artery
cardiovascular function. Several studies including disease, valvular or congenital heart disease, cardiac
meta-analyses have suggested that there is an associ- rhythm abnormalities, diabetes mellitus, systemic,
ation between SCH and cardiovascular diseases (2, 3, hepatic or renal diseases. Controls had a normal thy-
7–10), and that SCH is an independent risk factor for roid hormonal status.
atherosclerosis and myocardial infarction in elderly
women (11). Razvi et al. (12) in their meta-analysis All blood samples were collected between
postulated that SCH is associated with increased car- 08.00 and 09.00 h in the morning after overnight
diovascular morbidity and mortality only in younger fast. Serum lipid levels (total cholesterol, high-density
subjects. On the other hand, in the last few years lipoprotein cholesterol, triglyceride), HbA1c and fast-
there were several studies that did not show relation ing glucose levels were measured using spectropho-
between SCH and cardiovascular disease or cardio- tometry commercial kits on an automatic analyzer
vascular and all-cause mortality (5, 13, 14). Likewise, c501 (Roche Diagnostics, GmbH, Mannheim,
treatment of subclinical hypothyroidism in older per- Germany). C-reactive protein values were analyzed
sons did not show any clinical benefit (15). by Immunoturbidimetric assay for the in vitro quanti-
tative determination on a Cobas c501 analyzer
Coronary flow velocity reserve (CFR) is defined (Roche Diagnostics, GmbH, Mannheim, Germany),
as the ratio of hyperemic coronary blood flow velocity using the latex-enhanced immunoturbidimetric assay.
to baseline and reflects functional integrity of coro- Low density lipoprotein cholesterol was calculated by
nary microcirculation. It has been shown that reduced Friedewald’s formula. The serum TSH, fT4, fT3,
CFR is an early manifestation of atherosclerosis and TPOAb and insulin levels were measured using a
coronary artery disease (16). CFR measured by electrochemiluminescence immunoassay (ECLIA) on
transthoracic Doppler echocardiography (TTDE) has the Roche Cobas e601 automated analyzer (Roche
an excellent correlation with CFR measured by Diagnostics, Mannheim, Germany) and using a
positron emission tomography, which has been vali- chemiluminescent microparticle immunoassay
dated as a gold standard for noninvasive CFR meas-
(CMIA) on an Alinity instrument (Abbott Diagnostics,
urement (17).
Wiesbaden, Germany). Normal range for TSH was
The present study was designed to investigate 0.27– 4.2 mIU/L, for fT3 was 3.1–6.8 pmol/L, for
the impact on persistent SCH on the value of CFR, as fT4 12–22 pmol/L and for TPOAb 0–34 IU/mL).
assessed by transthoracic Doppler echocardiography, Body mass index (BMI), waist-to-hip ratio (WHR) and
and consequently microcirculatory function. HOMA IR were also calculated, using standard for-
mulas. Systolic and diastolic blood pressures (BP)
were measured on the right arm of subjects in an
Materials and Methods upright sitting position after at least 5 min of rest
using a sphygmomanometer.
The study group consisted of 32 patients with
newly diagnosed persistent SCH (31 female, one CFR was performed using the Acuson Sequoia
male; mean age 52.6±14.8 years), and 18 healthy C 256 (Siemens Medical Solutions, Mountain View,
controls (17 female, one male; mean age 50.1±15.4 CA, USA) with 4-MHz transducer. With the patient
J Med Biochem 2022; 41 (3) 301
positioned in the left lateral decubitus, coronary flow Statistical analysis

was searched for in the mid/distal portion of the left
Results were presented as mean ± standard
anterior descending (LAD) coronary artery with the
deviation, frequency (percent) and median (range) in
transducer placed at the cardiac apex or one inter-
case of not normal distribution of data. Chi-square
costal space higher in order to obtain modified, three-
test was used to test differences between nominal
chamber view. Color Doppler imaging was performed
data (frequencies). For parametric data independent
by decreasing the Nyquist limit to 1624 cm/s. With a
samples t-test was used to test differences between
sample volume 3–5 mm wide and positioned on the
groups. For numeric data with non-normal distribu-
LAD color flow signal in diastole, pulsed Doppler trac-
tion and ordinal data Mann-Whitney U test was used.
ings of peak flow velocities were recorded. After
Chi-square test or Fisher’s exact test were used to test
acquiring Doppler tracings in baseline conditions,
differences between nominal data (frequencies).
under continuous echocardiographic monitoring,
Correlation between the CFR for LAD as dependent
adenosine 140 mg/kg/min was administrated over 2
variable and potential predictors was analyzed by lin-
min and peak diastolic coronary flow velocities were
ear regression. All p-values less than 0.05 were con-
obtained during maximal hyperemia. Three optimal
sidered significant.
flow profiles at rest and during hyperemia were
obtained and results were averaged. CFR was calcu-
lated as the ratio of hyperemic to baseline diastolic
flow velocities. Preserved CFR was defined as 2.0. Results
All patients abstained from caffeine-containing drinks Age, gender, BMI, glucose, insulin levels, HOMA
for at least 12 hours before the tests. IR, cholesterol, HDL, LDL, levels, systolic and diastolic
blood pressure, smoking habits were similar in SCH
group and in controls. Triglyceride levels were higher
in SCH group, whereas CRP also showed borderline
higher values in SCH group. The fT3/fT4 ratio was
Table I Patients characteristic.
SCH group
Control group(n=18) P
(n=32)
Age 52.6±14.8 50.1±15,4 0.509
Male/female 1/31 1/17 0.595
BMI 26.6±5.1 24.2±3.0 0.069
WHR 0.84±0.07 0.82±0,06 0.620
Hypertension (%) 40.6% 38.9% 0.904
Systolic BP (mmHg) 120.3±11.6 119.4±10.1 0.792
Diastolic BP (mmHg) 75.8±7.8 73.3±7.3 0.283
Smokers (%) 21.9% 22.2% 0.923
Glycose (mmol/L) 5.5±0.7 5.4±0.6 0.708
Insulin (mIU/L) 8.5 (1.4–23.5) 6.8 (1.4 –16.1) 0.983
HbA1c (%) 5.7±0.4 5.5±0.3 0.212
HOMA IR 1.8 (0.3–6.7) 1.6 (0.3-3.6) 0.861
Cholesterol (mmol/L) 5.60±1.02 5.63±1.08 0.914
HDL (mmol/L) 1.48±0.32 1.67±0.53 0.113
LDL (mmol/L) 3.47±0.85 3.46±0.70 0.986
Triglyceride (mmol/L) 1.30 (0.48–3.66) 0.97 (0.52–2.19) 0.044
CRP (nmol/L) 1.3(0.3–9.6) 0.7(0.1–2.1) 0.051
fT4 (mgl/L) 11.8±1.6 12.6±1.7 0.082
fT3 (pmol/L) 4.03±0.42 4.01±0.42 0.881
fT3/fT4 0.35±0.05 0.31±0.04 0.015
TSH (mIU/L) 7.70 (4.60–15.35) 2.08 (0.51–4.14) <0.001
TPOAb (IU/mL) 248.5 (4–7413.5) 14.4(0.3–793.3) <0.001
BMI – body mass index; WHR – waist to hip ratio
Table II Coronary flow velocity values in SCH and CFR (2.0), but with a wide range of scatterplot data
controls. (Table II).
SCH group Control group By univariate linear regression analysis with CFR
P for LAD as dependent variable, CFR was inversely
(n =32) (n=18)
associated with the age and total cholesterol values in
Baseline DPVF controls, whereas in SCH, CFR was related to the
0.27±0.04 0.26±0.06 0.402
of LAD (cm/s) age, hypertension, smoking, total and LDL choles-
terol, triglycerides, and glucose metabolism deteriora-
Hyperemic tion, and waist-hip ratio, implicating specific contrib-
DPVF of LAD 0.75±0.18 0.70±0.17 0.379 utory effect of cardiovascular risk factors on CFR in
(cm/s)
patients with SCH (Table III). There was no associa-
CFR 2.76±0.35 2.76±0.42 0.999
tion between TSH level and CFR nor between fT4
level and CFR in both control and SCH group.
DPVF – diastolic peak flow velocity; CFR – coronary flow reserve;
LAD – left arterior descending coronary artery
Discussion
Table III Univariate linear regression with CFR for We have shown that in patients with SCH,
LAD as dependent variable. microcirculatory function as assessed by 2D Doppler
echocardiography derived CRF is generally preserved
SCH group Control group with wide scatter of data and without significant dif-
Variable
B p B p ferences to patients with normal thyroid function.
Age -0.012 0.003 -0.019 0.002 However, it seems that in patients with SCH, in com-
parison to normal thyroid function, the value of CFR
BMI -0.013 0.282 -0.018 0.610
is more dependent on traditional cardiovascular risk
WHR -2.090 0.013 -1.897 0.307 factors including hypertension, smoking, high choles-
Hypertension 0.249 0.045 0.362 0.075 terol, and glucose metabolism deterioration. CFR by
Systolic Doppler echocardiography, over last 10 years has
-0.010 0.066 -0.013 0.192 been shown to be highly reproducible, efficacious
tension
Dyastolic and feasible noninvasive to assess microcirculatory
-0.010 0.231 -0.013 0.362 dysfunction in different clinical scenarios affecting
tension
Smokers -0.256 0.037 0.033 0.877
coronary microcirculation (18, 19).
Glucose -0.249 0.006 -0.311 0.063 Based on a large number of studies and meta
HbA1c -0.357 0.021 -0.547 0.168 analyses conducted in the last twenty years, it is clear
that SCH leads to a somewhat increased risk for car-
Insulin -0.023 0.024 -0.013 0.629 diovascular disease, cardiovascular mortality and
HOMA. IR -0.102 0.009 -0.116 0.326 overall mortality (2, 3, 6, 9, 11, 12, 20, 21), but the
Cholesterol -0.165 0.005 -0.179 0.056 pathophysiologic mechanisms involved in this phe-
HDL -0.077 0.701 -0.334 0.082 nomenon are still to be defined.
LDL -0.160 0.028 -0.166 0.153 Since subclinical hypothyroidism is a laboratory
Triglyceride -0.195 0.020 0.146 0.496 finding, the diagnosis of SCH should be made with
caution. Different physiological conditions as well as
CRP -0.008 0.798 -0.043 0.815
other diseases can change the pituitary-thyroid axis,
fT4 0.038 0.337 -0.016 0.810 i.e. lead to a transient increase in TSH. There is also
fT3 0.057 0.693 0.340 0.216 an increase in TSH with age, and this increase does
fT3/fT4 -0.790 0.496 4.873 0.150 not lead to increased cardiovascular mortality CVD
(22). One way to overcome such doubts is to prove
TSH 0.009 0.698 0.121 0.169
persistently elevated TSH over a period of time, and
TPOAb <0.001 0.802 0.001 0.124 as Hashimoto’s thyroiditis is the most common cause
of both overt and subclinical hypothyroidism (1), find-
significantly higher in SCH group, as well as titer of ing of elevated TPOAb could reinforce the diagnosis
thyroid peroxidase (TPOAb) autoantibodies (Table I). of SCH (23). In our study the SCH group showed a
significantly higher titer of TPOAb compared to the
Baseline diastolic peak flow velocity (DPFV) of control group, thus confirming the existence of an
LAD was similar between the groups, as well as autoimmune process in the thyroid gland in SCH
hyperemic DPFV. Accordingly, there was no statisti- group. We also confirmed persistently higher TSH val-
cally significant difference in CFR for LAD between ues which, after initial elevated values, were con-
the SCH group and the control group, and all the val- firmed by TSH re-determination. There was a signifi-
ues in both groups were above the preserved limit of cant increase in the fT3/fT4 ratio in the SCH group
J Med Biochem 2022; 41 (3) 303
in our study, which we know to represent the adaptive (2.03 ± 0.13 vs 2.54 ± 0.18) but the study was con-
mechanism of the thyroid axis due to increased activ- ducted on only ten patients with SCH (38).
ity of deiodinase 2 (D2), which mediates T4 to T3
Biondi et al. (39) also showed a significant dif-
conversion, as well as due to higher TSH-induced
ference in CFR between the SCH and control group
increase of T3 synthesis and secretion from the thy-
(SCH 20, control 15), but they induced endothelium-
roid gland (24).
dependent vasodilation and hyperaemia using a cold
Of all anthropometric and biochemical parame- pressor test as an inducer ( SCH 1.4 ± 0.2 vs. con-
ters, only C-reactive protein (CRP) and triglyceride trols 1.9 ± 0.3 p< 0.0001) (39).
level were significantly higher in the SCH group than
It is challenging to explain about the significant
in the controls. This agrees with studies that have
differences between our and previous results in CFR
shown similar results (25, 26). CRP is known to be a
values, but few points should be emphasized regard-
strong independent risk factor for cardiovascular
ing our study population, methodology and results.
events (27), not only among those with stable and
Our study population was older (SCH 52.6 ± 14, 8;
unstable angina (28) but also among individuals with
controls 50.1 ± 15, 4) than study populations in
no current evidence of cardiovascular disease (29).
Oflaz et al. (37) (SCH 45 ± 2; controls 48 ± 2 years),
Triglyceride level was also higher in SCH group in two
Baycan et al. (36) (41.4 ± 9.5; controls 41.3 ± 9.4
large observational studies (30, 31), and it is well
years) and Biondi et al. (39) (SCH38.4 ± 12.1; con-
known that elevated plasma triglyceride level is an
trols 41.4 ± 14.5 years), and since CRF is significant-
independent risk factor for cardiovascular disease
ly negatively correlated with age, it is possible that the
(32). The values of cholesterol, its fractions and gly-
subtle vascular changes that might be detected in
cose related parameters (basal glucose level, insulin,
SCH are outweighed by changes due to aging.
HOMA IR) did not differ significantly between groups,
Further, in the study by Biondi et al. (39) CFR was
which was also shown in some of the published
measured after induction of endothelium-dependent
papers (30, 33), but there are also papers that show
vasodilatation, while in the remaining tree studies,
a significant difference between groups in relation to
including our study, endothelium-independent vaso-
these parameters (34, 35).
dilatation was induced. And third, all of these studies
Only four studies have previously evaluated CFR were performed on a relatively small number of sub-
in middle-aged patients with SCH (36–39). The two jects.
of them used dipyridamole (36) and adenosine (37)
If we look at the dependence of CFR for LAD in
as a stressor, and both adenosine and dipyridamole
our study, it is expected that in both groups there is a
induce a hyperaemic stimulus that relaxes vascular
significant dependence of CFR on the age of the sub-
smooth muscle cells in mostly endothel-indepen-
jects. However, apart from age, there is only a signif-
dent way. In the third study, conducted by the Oflaz
icant dependence of CFR for LAD on total cholesterol
et al. (38) CFR for LAD was evaluated before and
in the control group. It is interesting, however, that in
after the introduction of levothyroxine replacement
the SCH group, the dependence of CFR for LAD on
therapy. The fourth study evaluated endothelial-medi-
several anthropometric and metabolic parameters
ated CRF in SCH subjects using cold pressor test to
(WHR, HTA, smoking, glycemia, HbA1c, basal
induce endothelium-dependent vasodilation (39).
insulin, HOMA IR, cholesterol, LDL, triglycerides) was
Importantly, in comparison to previous studies our
obtained. These results suggest that individuals with
study did not find significant deterioration of CFR in
subclinical hypothyroidism are more sensitive to cer-
SCH patients (36, 37, 39).
tain metabolic, proatherogenic parameters, and this
In particular, Baycan et al. (36) in 50 SCH finding could be one of the explanations for the
patients and 30 controls (hyperaemia was induced by increased morbidity and mortality from cardiovascular
dipyridamole), showed no significant difference in disease in patients with SCH, which has been shown
anthropometric and biochemical parameters between in several studies and meta-analyzes (2, 3, 9, 10).
the groups (BMI, lipids, CRP), but a significant dete- Since CRP is shown to be higher in patients with SCH
rioration of CFR due to blunted hyperaemic response compared to healthy controls, low but prolonged
in SCH group (2.38 ± 0.44 vs. 2.98 ± 0.47, chronic inflammation could be the basis for greater
p<0.0001) (36). sensitivity of the microvasculature in patients with
SCH to other known cardiovascular risk factors and
Oflaz et al. (37) with a smaller group of subjects
mechanism linking SCH and CVD, i.e. that SCH facil-
(18 SCH, 24 controls) and adenosine as a stimulus of
itate the effect of traditional risk factors on microvas-
hyperaemia-endothelium independent vasodilation
cular function.
obtained similar results for CFR (SCH 1.97 ± 0.09 vs.
controls 2.58 ± 0.08.The same authors evaluated
CFR for LAD before and after the introduction of
Study limitations
levothyroxine replacement therapy and showed that
there was a significant increase in CFR for LAD in Our study reflects a single-center experience
SCH group after six month levothyroxine substitution with a relatively small number of participants. Second,
the cross-sectional design of our study limits its ability ent impact of cardiovascular risk factors on CFR for
to establish causality between SCH and CFR, and LAD suggests that these two groups behave different-
long-term effects of SCH on microcirculatory and car- ly in the same circumstances under the same risk fac-
diovascular function. tors. The basis for this difference could be that the
altered »set point« of the thyroid axis changes the
sensitivity of the microvasculature in patients with
Conclusion SCH to known risk factors, making them more sus-
ceptible for low prolonged chronic inflammation.
Our study has shown that people with subclini-
Further investigations on a larger number of partici-
cal hypothyroidism have a higher risk of chronic
pants are needed to address in depth the relation
inflammation, which plays an important role in the
between SCH, CFR, chronic inflammation and car-
development of atherogenesis and thus an increased
diovascular risk factors.
risk of developing CHD. We also showed that in
patients with SCH several known risk factors for
atherogenesis have a significant impact on CFR for
LAD which is not the case in the control group.
Although we did not find a significant difference All the authors declare that they have no conflict
between groups in relation to CFR for LAD, the differ- of interest in this work.
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Received: October 29, 2021

Accepted: November 20, 2021
UDK 577.1 : 61 ISSN 1452-8258

PERFORMANCE EVALUATION BETWEEN TWO AUTOMATED BIOCHEMICAL

ANALYZER SYSTEMS: ROCHE COBAS 8000 AND MINDRAY BS2000M
PROCENA PERFORMANSI DVA AUTOMATIZOVANA SISTEMA BIOHEMIJSKIH
ANALIZATORA: ROCHE COBAS 8000 I MINDRAY BS2000M
Mingxing Chen1†, Simeng Qin1†, Sitao Yang1, Huaping Chen1, Liuyi Lu1, Xue Qin1,2
1Department of Clinical Laboratory, First Affiliated Hospital of Guangxi Medical University, Nanning, China
2Key Laboratory of Early Prevention and Treatment for Regional High-Incidence-Tumor,
Guangxi Medical University, Ministry of Education, Nanning, Guangxi, P.R. China

Background: The values of biomarkers play a central role in Uvod: Vrednosti biomarkera igraju centralnu ulogu u rutin-
routine clinical decision-making. Whereas the performance skom klini~kom dono{enju odluka. S druge strane, perfor-
of different automated chemical analyzers remains unclear. manse razli~itih automatizovanih hemijskih analizatora
To determine the performance of different platforms, we ostaju nejasne. Kako bismo utvrdili performanse razli~itih
compared the consistency and accuracy between Roche platformi, izvr{eno je upore|ivanje konzistentnosti i ta~nosti
Cobas 8000 and Mindray BS2000M. izme|u analizatora Roche Cobas 8000 i Mindray
Methods: A total of 1869 remaining serum samples were BS2000M.
collected. CK, LDH-1, RBP, Cys-C, IgA, IgM, and IgG were Metode: Prikupljeno je ukupno 1869 preostalih uzoraka
assessed using paired t-test, Passing-Bablok regression
seruma. Vrednosti CK, LDH-1, RBP, Cys-C, IgA, IgM i IgG
analysis, and Bland-Altman analysis according to CLSI EP5-
su odre|ene kori{cenjem uparenog t-testa, Passing-Bablok
A3.
Results: There were significant differences in the average regresije i Bland-Altmanove analize prema CLSI EP5-A3.
bias of all items between the two machines (P<0.001). Rezultati: Pokazale su se zna~ajne razlike u prose~noj pris-
Because the 95% confidence interval of intercept A includ- trasnosti svih stavki izme|u dve ma{ine (P < 0,001). Po{to
ed 0, CK, LDH-1, Cys-C and IgG did not show systematic je interval poverenja od 95% preseka A uklju~ivao 0, CK,
error in Passing-Bablok regression analysis. The confidence LDH-1, Cys-C i IgG nisu pokazali sistematsku gre{ku u
interval of 95% of the slope B in IgM contained 1, and Passing-Bablok regresionoj analizi. Kod intervala poverenja
there was no difference in the two measurements in IgM. od 95% nagiba B u IgM koji je sadrào 1, nije bilo razlike u
Except for IgA, the r values and correlation coefficient of all dva merenja u IgM. Osim za IgA, r vrednosti i koeficijent
items were higher than 0.91, which showed that the corre- korelacije kod svih stavki su bili ve}i od 0,91, {to je poka-
lation and consistency were good. The Bland-Altman zalo dobru korelaciju i konzistentnost. Bland-Altmanova
analysis showed that two instruments had more than 95% analiza je pokazala da su dva instrumenta imala vi{e od 95
of the points apart from CK, LDH-1, and IgA. procentna poena osim za CK, LDH-1 i IgA.
List of abbreviations: CLSI, Clinical and Laboratory Standards

Institute; CK, creatine kinase; AMI, acute myocardial infarc-
tion; LDH-1, lactate dehydrogenase-1; RBP, retinol-binding
Address for correspondence: protein; Cys-C, Cystatin-C; IgA, immunoglobulin A; IgM,
immunoglobulin M; IgG, immunoglobulin G; CSF, cere-
Xue Qin
brospinal fluid; NABL, the National Laboratory Accreditation
Department of Clinical Laboratory,
Board; CI, confidence interval; CV, coefficient of variation;
First Affiliated Hospital of Guangxi Medical University
LOA, the limit of agreement; Min, minimum; Max, maximum;
No. 6 Shuangyong Road, Nanning, Guangxi, China
CC, correlation coefficient.
Fax: +86-0771-865353342
e-mail: qinxue919ª126.com
†These authors contributed equally to this work and should
be considered as co-first authors.
J Med Biochem 2022; 41 (3) 307
Conclusions: It can be considered that the two instruments Zaklju~ak: Moè se re}i da dva instrumenta imaju dobru
have good correlation and consistency in CK, LDH-1, RBP, korelaciju i konzistentnost u pogledu CK, LDH-1, RBP, Cys-C,
Cys-C, IgM, and IgG, and the two instruments are inter- IgM i IgG, te se mogu koristiti bez razlike.
changeable and can replace each other.
Klju~ne re~i: analiti~ke tehnike i oprema, validacija,
Keywords: Analytical techniques and equipment, valida- Roche Cobas 8000, Mindray BS2000M, pore|enje
tion, Roche Cobas 8000, Mindray BS2000M, comparison
Introduction all know, automated chemistry analyzers can detect

organ functions and all kinds of metabolites. These
The biomarkers in clinical laboratories have published studies have indicated excellent perform-
played a key role in medical decisions for patients’ ance in the precision and accuracy of automated bio-
diagnosis, treatment, and prognosis (1–4). There- chemical analyzers. Mindray BS2000M, a new gener-
fore, the results tested by diagnostic machines must ation automated chemistry analyzer, is a high test, less
be more precise, accurate, sensitive, and specific. reaction volume, and multi-wavelengths system. None
Technological advances have greatly improved the of the studies have focused on the performance and
development of laboratory medicine and met the evaluation of Mindray BS2000M. Moreover, none of
growing demands in routine biochemistry analysis, these studies have defined the performance at low,
such as high throughput analysis of multi-parameters, normal, and high concentration. Therefore, we
leading to the increasing use of new analyzers. designed the present study and paid attention to the
Implementing an automation analyzer in laboratory differences in myocardial enzyme, kidney function,
diagnostics provides advantages and convenience for and immunoglobulin in Roche and Mindray. As far as
requiring a high degree of value with precision and we know, we are first to compare the two machines’
accuracy (5). However, the detected results of the performance. For myocardial enzymes, such as crea-
same samples by different machines are sometimes tine kinase (CK), which appears very early after the
inconsistent. For example, some common clinical attack of acute myocardial infarction (AMI), its sensi-
chemistry analytes have shown that comparable prob- tivity reaches 98% in the diagnosis after the onset of
lems still exhibited unacceptable or suboptimal bias the disease of AMI. Moreover, a previous study
compared to the true value (6). Imprecise or incorrect revealed that patients with high CK had a worse prog-
results might lead to immeasurably serious conse- nosis (16). Another enzyme, lactate dehydrogenase-1
quences for patients, clinicians, and even the entire (LDH-1), owing to being increased in blood 510
health care system. Hence, emphasis should be hours after AMI, was also treated as an early biomark-
placed on examining the standardized protocol (7). er of AMI (16). As mentioned, many studies have
However, it remains controversial whether the shown the discrepancies of common kidney biomark-
values of the different equipment in an identical med- ers, for instance, creatinine, blood urea nitrogen, and
ical laboratory from the same specimen may be uric acid, between different measurements. We
inconsistent and not the same according to the stan- focused on other markers that were more sensitive
dardized operating procedures. It is high time that the and specific. Retinol binding protein (RBP), which can
emphasis is placed on creating different reference remain stable in acid urine and quickly appear after an
intervals for different machines. Recently, most previ- early renal proximal injury, is considered to be a reli-
ous studies have compared automated hematology able and sensitive parameter for kidney injury (17).
analyzers about Beckman Coulter, Sysmex, and Cystatin-C (Cys-C) improves the risk classification of
Mindray (8 –11), automated hemostasis analyzer patients with chronic kidney disease, death, cardiovas-
between Sysmex and Atellica (12), automated bacte- cular disease (3), and end-stage renal disease (18). As
rial identification, and drug sensitivity analyzer the effector molecules of the adaptive humoral
between GENECUBE and Vitek (13). However, some immune system, high or low levels of immuno-
research has studied other different automated globulins cause an allergic reaction or immunodefi-
chemistry analyzers, such as Abbott, Roche, Beckman ciency diseases. Since immunoglobulin A (IgA) can
Coulter, and Hitachi (6, 14, 15). limit antigen access to host tissues, it was referred to
as the mucosal barrier in immune exclusion and shed
Nikolac Gabej et al. (14) recently focused on light on the importance of regulating food allergen
three parameters about hemoglobin and bilirubin sensitization (19). At the same time, the patients with
intralipid in Abbott, Roche, and Beckman Coulter. Crohn’s disease or ulcerative colitis also showed that
Having included 21 items, Lim et al. (6) emphasized serum IgA in blood was elevated (20). While for com-
liver and kidney function and blood lipid. Though mon variable immunodeficiency and primary immun-
Leitner-Ferenc et al. (1) study underlined the reference odeficiency diseases, the level of IgA of patients may
intervals on the Roche Cobas 8000 platform based on be deficient (21). Immunoglobulin M (IgM), involved
the Clinical and Laboratory Standards Institute (CLSI), in both immune protection and immunoregulatory
their study only focused on gender difference. As we functions, is treated as the first line of humoral defense
308 Chen et al.: Evaluated performance of Roche Cobas 8000 and Mindray BS2000M
against pathogens (22). Reducing IgM might increase Table I Characteristics of the compared methods between
the risk of infection, exacerbate autoimmunity as well Cobas 8000 and Mindray BS2000M.
as atherosclerosis (23). High immunoglobulin G (IgG) Mindray BS2000M
helps to diagnose autoimmune hepatitis (24) and IgG Parameter Cobas 8000 method
method
in cerebrospinal fluid (CSF), which is useful for the Creatine phosphate
diagnosis of multiple sclerosis (15). In addition to the CK Colorimetry
substrate method
mean bias in the two instruments, test characteristics Lactic acid substrate
related to consistency and correlation in two measure- LDH-1 Rate method
method
ments were investigated.
Latex immunotur- Latex enhanced
RBP
Measurement of laboratory analytical errors falls bidimetry immunoturbidimetry
into two main categories, systematic error and ran- Latex immunotur- Latex enhanced
Cys-C
dom error. Systematic errors are predictable problems bidimetry immunoturbidimetry
that influence observations consistently in one direc- IgA Immunoturbidimetry Immunoturbidimetry
tion, while random errors are more unpredictable.
Sources that contribute to uncertainty may include IgM Immunoturbidimetry Immunoturbidimetry
samples, calibrators, reference materials, input quan-
IgG Immunoturbidimetry Immunoturbidimetry
tities, equipment, and environmental conditions.
Materials and Methods Quality control

Samples All reagents, quality control products, and cali-
A total of 1869 remaining serum samples were bration products were original reagents that matched
collected from outpatients and inpatients at the with the machine. The instrument was calibrated
according to the manufacturer’s guidelines using cal-
Second Affiliated Hospital of Guangxi Medical
ibration samples provided by the manufacturer. High,
University from July 2019 to October 2019 for diag-
normal, and low control samples were run every day
nostic accuracy. All samples were tested within 2
to monitor the system’s performance according to the
hours after centrifugation of 4000 g for 5 minutes.
National Laboratory Accreditation Board (NABL)
Specimens that could not be tested immediately were guideline and CLSI EP5-A3 (25). To evaluate the
refrigerated at 4 after centrifugation, and tests were quality of our results from two machines, two levels of
completed within 24 hours. Samples must be thawed control in seven parameters were detected every
to room temperature and mixed thoroughly after time, including Lot 32419602 and 32419602 in CK
refrigeration. After being tested on Cobas 8000 c702 and LDH-1, Lot 1293uN, and 983uE in RBP and
(Roche, Basel, Switzerland), those serum samples Cys-C, and Lot 48902 and 48903 in IgA, IgM, and
were immediately tested on Mindray BS2000M IgG. The coefficient of variation of quality control in
(Mindray Bio-Medical Electronics Co., Ltd, Shenzhen, all parameters was less than 10% which means that
China) to guarantee the consistency of time and the the results of quality control were in control. There
accuracy of results. Those samples were categorized was nothing unusual in control, which demonstrates
as being of abnormally high, abnormally low, or nor- that the quality of controls was acceptable. Then the
mal value. This study was approved by the Ethics serum samples were tested according to the manu-
Committee of the Second Affiliated Hospital of facturer’s instruction and strictly followed standard
Guangxi Medical University. operating procedure.
Reagents Statistical analysis

All of the procedures were carried out according All statistical analyses were performed using
to the manufacturer’s protocols. In brief, CK tested by SPSS 20.0 (SPSS Inc., Chicago, IL, USA) and
Cobas 8000, and Mindray BS2000M used colorime- MedCalc v18.2.1 (Ostend, Belgium). The paired t-
try and phosphocreatine substrate method, respec- test was used to compare the mean bias of results in
tively. LDH-1tested by Cobas and Mindray used the two instruments. Bland-Altman plot (26, 27) was
rate method and lactic acid substrate method, respec- used to evaluate the consistency of the two machines.
tively. Latex immunoturbidimetry by using Cobas- Passing-Bablok regression analysis (28) was used to
reagents was used for RBP, Cys-C analysis, while they evaluate the regression equation and the correlation
were examined by latex enhanced immunoturbidime- of the two instruments. If the 95% confidence interval
try in Mindray. For IgA, IgM, and IgG, all were detect- (CI) of intercept A does not contain 0, there are sys-
ed by immunoturbidimetry in two automatic analyz- tematic errors in the two instruments. The slope B
ers. All methods in seven parameters are summarized was used to measure the difference in the ratio
in Table I. between the two instruments. The 95% CI for slope B
J Med Biochem 2022; 41 (3) 309
did not include 1, which means that there are a few The comparison between seven items of two instru-
differences between the two methods. The Cusum ments was carried out using Passing and Bablok
test for linearity was used to test the applicability of regression analysis and Bland-Altman plots. The
the Passing-Bablok regression. If P<0.05, it indicates results of this statistical analysis are shown in Table III
that there is no linear relationship between the two and Table IV. A high correlation was obtained for
apparatuses, so this method is not applicable. When analysis compared with two instruments for most
the correlation coefficient r is lower than 0.4, the cor- parameters in all results but not subgroups in six
relation degree is low. If r is more than 0.4 but lower items (r ranging from 0.904 to 0.995) except for IgA
than 0.7, the correlation degree is moderate. If r is (r=0.857) by Spearman rank correlation analysis.
higher than 0.7, the correlation degree is high. All However, the high level of IgA (>4.53 g/L) between
comparison with P-value <0.05 was considered sta- the two instruments showed little correlation
tistically significant. (r=0.089). Moreover, there was a high correlation
between 7 parameters in the two machines according
to correlation coefficient (CC) results. All CC of items
Results were more than 0.7, whether the items had low, mod-
erate, and high values, except when IgA was more
Descriptive analysis of different methods than 4.53 g/L (CC: 0.605, 95%CI 0.426–0.738)
As shown in Table II, the IgG in Cobas 8000 had (Table III). All correlations were statistically significant
a minimum CV value of 2.64%, while CK in Cobas (P<0.001).
8000 reached 7.10%. However, all CVs of the param-
On the Bland-Altman plot, the average bias in
eter in the two instruments were lower than 10%. The
Cys-C, IgA, and IgM was close to zero (0.520, 0.189,
paired t-test was performed, and the results revealed
and 0.046, respectively), while the average bias of CK
a statistically significant difference in all items (both
and LDH-1 in the two machines were -11.938 and
P<0.001). All methods of different items between
12.180, respectively (Table III). In particular, the com-
the two platforms were summarized in Table I.
parison of Cobas 8000 and Mindray data showed a
significant negative bias for CK while the bias was
positive for LDH-1 and RBP (Figure 1). In addition,
Comparison methods three-sevenths of two instruments had more than
Based on the clinical significance of these 95% of the points within the 95% consistency limit
parameters level, serum samples were divided into (RBP 96.4%, IgM 95.6%, and IgG 95.0%) in Bland-
two levels (low and high level) and three levels (low, Altman analysis, meeting the consistency require-
normal, and high level). Three of seven items (LDH- ments. The remaining four items were also more than
1, RBP, and Cys-C) and four of seven items (CK, IgA, 90% (data not shown). The absolute value of the dif-
IgM, and IgG) were divided into either two or three ference between the two machines was less than 10%
levels according to the clinical reference range. All which demonstrates that the difference is clinically
subgroups of these parameters are shown in Table III. acceptable.
Table II Evaluation of the imprecision between the two measurements.

Parameter (units) Cases Instrument Min Max Mean CV (%) P
Cobas 8000 11 1026 147.5 7.10
CK (U/L) 241 <0.001
Mindray BS2000M 25 1000 159.44 6.99
Cobas 8000 25 572 97.48 4.79
LDH -1 (U/L) 261 <0.001
Mindray BS2000M 10 500 85.3 4.83
Cobas 8000 7.9 157.5 44.19 3.47
RBP (mg/L) 275 <0.001
Mindray BS2000M 10 150 38.63 4.14
Cobas 8000 0 10 1.49 5.17
Cys-C (mg/L) 272 <0.001
Mindray BS2000M 0.4 6.34 0.971 5.84
Cobas 8000 0.06 8.61 2.8654 3.72
IgA (g/L) 311 <0.001
Mindray BS2000M 0.4 6.06 2.6767 3.40
Cobas 8000 0.056 4.02 1.2744 4.71
IgM (g/L) 271 0.001
Mindray BS2000M 0.35 3.6 1.3206 4.43
Cobas 8000 1.99 36 12.8547 2.64
IgG (g/L) 238 <0.001
Mindray BS2000M 1.03 27.6 10.501 2.81
Min: minimum, Max: maximum, CV: coefficient of variation
Table III Spearman rank correlation, Bland-Altman plot analysis and correlation coefficient in the two systems.
Parameter Group N r Average bias (95%CI) LOA CC (95%CI) P
All 241 0.995 -11.938 (-13.968 to -9.907) (-43.303 to 19.428) 0.997 (0.997 to 0.998) <0.001
7–40 (U/L) 65 0.807 -3.846 (-4.636 to -3.056) (-10.095 to 2.402) 0.955 (0.927 to 0.972) <0.001
CK
40–300 (U/L) 127 0.994 -7.937 (-9.175 to -6.699) (-21.756 to 5.882) 0.997 (0.995 to 0.998) <0.001
>300 (U/L) 49 0.975 -33.041 (-39.782 to -26.300) (-79.040 to 12.958) 0.933 (0.884 to 0.962) <0.001
All 261 0.956 12.180 (10.053 to 14.307) (-22.020 to 46.380) 0.977 (0.971 to 0.982) <0.001
LDH-1 <90 (U/L) 125 0.910 7.288 (6.190 to 8.386) (-4.873 to 19.449) 0.933 (0.905 to 0.952) <0.001
90 (U/L) 136 0.934 16.677 (12.852 to 20.501) (-27.522 to 60.874) 0.898 (0.860 to0.927) <0.001
All 275 0.910 5.579 (4.635 to 6.524) (-10.019 to 21.178) 0.980 (0.974 to 0.984) <0.001
RBP <70 (U/L) 218 0.913 5.845 (5.299 to 6.391) (-2.167 to 13.856) 0.962 (0.950 to 0.970) <0.001
70 (U/L) 57 0.459 4.563 (0.4200 to 8.706) (-26.042 to 35.169) 0.886 (0.814 to 0.932) <0.001
All 272 0.981 0.520 (0.475 to 0.564) (-0.208 to 1.246) 0.954 (0.942 to 0.964) <0.001
Cys-C <1.03 (mg/L) 119 0.618 0.313 (0.300 to 0.327) (0.168 to 0.459) 0.770 (0.686 to 0.835) <0.001
1.03 (mg/L) 153 0.981 0.679 (0.611 to 0.748) (-0.157 to 1.516) 0.963 (0.950 to 0.973) <0.001
All 311 0.851 0.189 (0.107 to 0.271) (-1.252 to 1.630) 0.935 (0.919 to 0.948) <0.001
<0.82 (g/L) 57 0.145 -0.220 (-0.344 to -0.095) (-1.139 to 0.700) 0.839 (0.740 to 0.902) <0.001
IgA
0.82–4.53 188 0.957 0.070 (0.041 to 0.099) (-0.328 to 0.468) 0.983 (0.977 to 0.987) <0.001
>4.53 (g/L) 67 0.089 0.867 (0.560 to 1.174) (-1.601 to 3.334) 0.605 (0.426 to 0.738) <0.001
All 271 0.950 0.046 (-0.073 to -0.020) (-0.481 to 0.389) 0.981 (0.976 to 0.985) <0.001
<0.46 (g/L) 68 0.436 -0.058 (-0.082 to -0.034) (-0.255 to 0.139) 0.829 (0.736 to 0.891) <0.001
IgM
0.46–3.04 174 0.912 -0.071 (-0.097 to -0.044) (-0.421 to 0.279) 0.962 (0.950 to 0.972) <0.001
>3.04 (g/L) 29 0.251 0.129 (-0.049 to 0.307) (-0.787 to 1.045) 0.695 (0.441 to 0.846) <0.001
All 238 0.904 2.354 (2.139 to 2.569) (-0.945 to 5.652) 0.947 (0.932 to 0.958) <0.001
<7.51 (g/L) 46 0.737 0.793 (0.569 to 1.018) (-0.688 to 2.274) 0.861 (0.761 to 0.921) <0.001
IgG
7.51–15.60 127 0.751 2.343 (2.139 to 2.546) (0.0712 to 4.615) 0.860 (0.807 to 0.900) <0.001
>15.60 (g/L) 65 0.739 3.479 (2.960 to 3.999) (-0.633 to 7.592) 0.820 (0.720 to 0.886) <0.001
LOA: limit of agreement, CC: correlation coefficient, CI: confidence intervals
Figure 1 Spearman rank correlation of evaluated parameter between the two machines (A) CK, (B) LDH-1 (C) RBP, (D)
Cys-C, (E) IgA, (F) IgM, (G) IgG.
J Med Biochem 2022; 41 (3) 311
Table IV A Passing-Bablok regression analysis for the two analyzers.
Parameter Regression equation Intercept A (95%CI) Slope B (95%CI)

CK y = 0.769 + 1.077 x 0.769 (-0.027 to 1.333) 1.077 (1.067 to 1.087)
LDH-1 y = -0.206 + 0.8504 x -0.206 (-1.579 to 0.775) 0.851 (0.838 to 0.868)
RBP y = -4.351 + 0.947 x -4.351 (-4.960 to -3.705) 0.947 (0.928 to 0.966)
Cys-C y = -0.023 + 0.653 x -0.023 (-0.050 to 0.003) 0.653 (0.628 to 0.678)
IgA y = 0.092 + 0.925 x 0.092 (0.066 to 0.118) 0.925 (0.914 to 0.936)
IgM y = 0.044 + 0.995 x 0.044 (0.029 to 0.056) 0.995 (0.981 to 1.011)
IgG y = -0.238 + 0.840 x -0.238 (-0.600 to 0.115) 0.840 (0.808 to 0.871)
CI: confidence intervals
Figure 2 Bland-Altman plots of the difference of the evaluated parameter between the two machines (A) CK, (B) LDH-1 (C)
RBP, (D) Cys-C, (E) IgA, (F) IgM, (G) IgG.
According to Passing and Bablok regression cept A was very close to zero. Only a relatively high
analysis, 95% CI for the intercept A of the regression intercept A can be found in RBP (intercept A: -4.351,
equation for CK, LDH-1, Cys-C, and IgG includes 0, 95% CI -4.960 to -3.705). Except for IgM, the 95%
and there is no systematic error between the two CI of the slope B contains 1 (0.9806–1.0105), anoth-
instruments. For IgA and IgM, the 95% CI for inter- er the slope B of CK, RBP, and IgA were almost equal
Figure 3 Passing-Bablok regression of the difference of the evaluated parameter between the two machines (A) CK, (B) LDH-
1 (C) RBP, (D) Cys-C, (E) IgA, (F) IgM, (G) IgG.
to 1 (1.077, 0.947, and 0.925, respectively). For Abbott, Beckman Coulter, Roche Cobas, and Mind-
LDH-1, Cys-C, and IgG, slope B did not contain 1 ray. Different detection systems using different meth-
(0.851, 0.653, and 0.840, respectively), which ods will produce different results for different samples
shows some proportional differences between the two on different detected platforms, and this difference
instruments (Table IV). Therefore, it can be consid- may affect routine clinical decision-making. Hence,
ered that the results of the two pieces of equipment when utilizing different analyzers to disclose the same
are consistent, and the two devices are interchange- items, the instrument needs to be contrasted with
able. guaranteeing the consistency and conformity of the
detected results. Numerable studies have focused on
comparing biomarkers in Abbott, Hitachi, and Roche
Discussion (6, 14). As previously described in the literature,
these clinical chemistry assays are accurate and reli-
The availability of rapid and automated methods able and are readily applicable on various platforms.
regarded as a major breakthrough in the laboratory Some newly launched and advanced chemical and
can decrease the labor force and increase consistency immune analyzers remain uncertain. This study
and repeatability. Indeed, in addition to improving the aimed to compare basic biochemistry parameters
clinical effectiveness, the new generation of automat- between Roche Cobas 8000 and Mindray BS2000M.
ed analyzers increases laboratory efficiency by reduc-
ing working time and costs associated with the optical To our knowledge, this is the first large study
validation of the results. At present, the most regularly using two automated chemistry platforms Roche
used chemistry platforms in the laboratory are Cobas 8000 and Mindray BS2000M, to assess the
J Med Biochem 2022; 41 (3) 313
equivalence of common organ function parameters. value of parameters exceeds upper detection limits,
A total of 1869 samples were screened in our study. one of the common solutions in regular work of labo-
The ultimate objective was to evaluate whether the ratories to solve high-level samples is for an operator
detected values in different analyzers were identical to dilute the sample by adding low level serum or
and therefore interchangeable when informing clini- matrix (31). A previous study also demonstrated that
cians’ decisions in diagnosis, treatment, and progno- substrate depletion plays a key role in causing nega-
sis. All items in the two platforms were appraised tive results. The enzyme linearity extension function in
according to CLSI protocols (7, 25). BS-2000M2 can effectively solve the risk of false-neg-
ative results for high-level samples (32). Hence, to
In the assessment of linearity, the r for all ana- avoid unnecessary misleading and misconceptions,
lytes at all levels was more than 0.9 except for IgA the sample from one patient should not be detected
(r=0.851). All items within the clinical reference separately on different methods of different systems
range showed excellent linearity. However, the linear- in the same laboratory. One should not use sample
ity of RBP, Cys-C, IgA, and IgM at a low or high level internal quality control rule if it is necessary to use a
was verified outside the range as claimed by the man- different sample to verify or review the values of
ufacturer. Regarding the correlation of parameters in parameter. Moreover, it is wise and advisable for dif-
two systems, we found that the correlation of all ana- ferent laboratories to establish reference ranges and
lytes at all levels was highly relevant (CC> 0.95, dilute high levels samples beyond upper limitation.
P<0.001). However, the CC of IgA and IgM at a high
level showed a low correlation (0.605 and 0695, This study has mentioned limitations. One of
respectively). According to the regression equation them was that the performance of our study only
between Roche Cobas 8000 and Mindray BS2000M, compared with two analyzers (Roche Cobas 8000
CK, IgA, and IgM performance were excellent in our and Mindray BS2000M) and did not include more
study, which did not show a statistically significant clinical chemistry platforms, such as Abbott and
proportional error or constant error. On the contrary, Hitachi. Due to the small volume of samples, there
RBP in the two instruments displayed a significant was no possibility of repeating the analysis with every
constant error (intercept A=-4.351), and Cys-C showed analyzer once more. Another disadvantage was that
obviously proportional error (slope B=0.653). There the samples included in our study contained all kinds
remained a small proportional error in LDH-1 (slope of patients and healthy people. Further study on the
B=0.851) and IgG (slope B=0.840). performance of biochemical or immune items by var-
ious analyzers in a more significant number of cases
In Bland-Altman’s plot, Cys-C, IgA, IgM, RBP, and multicenter should be performed to validate the
and IgG showed a low average bias (0.520, 0.189, findings of this study. Based on the data in our study,
0.046, 5.579, and 2.354, respectively), and their we can conclude that the analytical performances of
mean bias in the former three almost closed to 0. RBP, Cys-C, IgA, IgM, and IgG are excellent, while CK
While for CK and LDH-1, the mean differences were and LDH-1 need to be improved to decrease or
higher (-11.938 and 12.180, respectively) and the remove the systematic error as much as possible.
same as the limit of agreement (LOA), proportionally
increasing with the growing levels (CK: -43.303 to Taken together and to the best of our knowl-
19.428, LDH-1: -22.020 to 46.380). For instance, edge, this is the first study to describe the perform-
their average bias showed significant differences in ance characteristics of the Roche Cobas 8000 and
CK (-3.846 to -33.041) and LDH-1 (7.288 to Mindray BS2000M systems. The two platforms have
16.677), compared with the Cys-C, IgA, and IgM. We good correlation and bias for detecting CK, LDH-1,
suggest three possible explanations for why the aver- RBP, Cys-C, IgM, and IgG analytes. They have a high
age bias of CK and LDH-1 was so wide. One possible method agreement in CK, LDH-1, IgA, IgM, and IgG.
reason is that the two platforms use the different In summary, Cobas and Mindray clinical chemistry
detection methods for CK (colorimetry vs. creatine assays are reliable and precise, and applicable to dif-
phosphate substrate method) and LDH-1 (rate ferent analytic platforms.
method vs. lactic acid substrate method). The study
of He et al. demonstrated that the coefficient of vari- Acknowledgments
ation of Cys-C showed a significant difference
(P=0.016), very low pass rates, and widespread dis- This research was funded by Key Laboratory of
tributions (from 3.63% to 6.74%) in internal quality Early Prevention and Treatment for Regional High-
control of laboratories using different systems from Incidence-Tumor, Guangxi Medical University,
2014 to 2017 in China (29). Meanwhile, Han et al. Ministry of Education (GKE2019-05, GKE-
(30) study also showed that LDH-1 should be ZZ202132).
improved their precision and accuracy at the same
time after being evaluated sigma index, further sup-
porting our investigation. Another factor caused by
the significant mean difference was that the detection All the authors declare that they have no conflict
limits of different platforms are different. If the true of interest in this work.
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UDK 577.1 : 61 ISSN 1452-8258

MAGNESIUM SUPPLEMENTATION AND IRON STATUS AMONG

FEMALE STUDENTS: THE INTERVENTION STUDY
SUPLEMENTACIJA MAGNEZIJUMA I STATUS GVO@\A KOD STUDENTKINJA: STUDIJA INTERVENCIJE
Neda Milinkovi}1, Milica Zekovi}2, Margarita Dodevska3, Briìta \or|evi}4,

Branimir Radosavljevi}5, Svetlana Ignjatovi}1,6, Nevena Ivanovi}4
1University of Belgrade-Faculty of Pharmacy, Department of Medical Biochemistry
2Center of Excellence in Research, Nutrition and Metabolism, Institute for Medical Research,
National Institute of the Republic of Serbia, University of Belgrade
3Institute of Public Health of Serbia »Dr Milan Jovanovi} Batut«, Center for Hygiene and Human Ecology
4University of Belgrade-Faculty of Pharmacy, Department of Bromatology
5Institute of Chemistry in Medicine, University of Belgrade-Faculty of Medicine
6Center for Medical Biochemistry, University Clinical Center of Serbia

Background: Literature data indicate the benefit of magne- Uvod: Literaturni podaci ukazuju na benefit suplementacije
sium (Mg) supplementation. The aim of this study was to magnezijumom (Mg). Cilj ove studije bio je da se ispita uticaj
examine the effect of short-term Mg supplementation on kratkotrajne suplementacije Mg na status gvo`|a kod zdravih
iron status in healthy female participants. èna.
Methods: One hundred healthy female students of the Metode: Sto zdravih studentkinja Univerzitet u Beogradu –
University of Belgrade - Faculty of Pharmacy participated the Farmaceutskog fakulteta je u~estvovalo u istraìvanju tokom
study during eleven intervention days. Students ingested Mg jedanaest dana intervencije. Studenti su uzimali preparate
preparations with the same dose of the active substance. The Mg sa istom dozom aktivne supstance. U serumu je
analysis included the measurement of serum iron, unsaturat- odre|ivano gvo`|e, nezasi}en kapacitet vezivanja gvo`|a
ed iron binding capacity (UIBC), total iron binding capacity (UIBC), ukupan kapacitet vezivanja gvo`|a (TIBC), ukupan
(TIBC), total Mg (tMg), ionized Mg (iMg), complete blood Mg (tMg), jonizovni Mg (iMg), kompletna krvna slika, met-,
count, met-, carboxy- and oxy-haemoglobin (metHgb, karboksi- ioksi- hemoglobin (metHgb, COHgb, O2Hgb).
COHgb, O2Hgb). Transferrin concentrations and percentage Transferin i saturacija transferina (SAT) su izra~unati ru~no.
of transferrin saturation (SAT) were calculated manually. The Povezanost analiziranih biohemijskih parametara je ispitana
association among the analyzed biochemical parameters was pomo}u polinomalne regresije. Za procenu me|uzavisnosti
examined using polynomial regression. A principal compo- izme|u analiziranih parametara kori{}ena je analiza glavnih
nent analysis (PCA) was used for the evaluation of interde- komponenti (PCA).
pendence between the analyzed parameters. Rezultati: Utvr|en je statisti~ki zna~ajan trend promene
Results: A statistically significant trend for change in O2Hgb O2Hgb (%) po tertilima koncentracija iMg (P = 0,029).
(%) by tertiles of iMg concentrations was found (P = 0.029). Ukupan Mg je dostigao zna~ajnu pozitivnu korelaciju sa SAT
Serum tMg reached significant positive correlation with the pri koncentracijama ve}im od 0,9 mmol/L, nakon 11 dana
SAT at concentration levels greater than 0.9 mmol/L, after intervencije (R2 = 0,116). Jonizovani Mg u koncentraciji
11 days of intervention (R2=0.116). Ionized Mg in a con- ve}oj od 0,6 mmol/L pozitivno korelira sa SAT i gvo`|em (R2
centration higher than 0.6 mmol/L is positively correlated = 0,214; 0,199, redom). PCA analizom je pokazana varija-
with SAT and serum Fe (R2=0.214; 0.199, respectively). bilnost od 64,7% za dve ose nakon 11 dana.

Neda Milinkovi}
Vojvode Stepe 450, 11121 Beograd, Srbija
064-422-3685
e-mail: nedanªpharmacy.bg.ac.rs
J Med Biochem 2022; 41 (3) 317
PCA revealed variability of 64.7% for two axes after 11 days. Zaklju~ak: Suplementacija Mg dovodi do pobolj{anja
Conclusions: Mg supplementation leads to an improvement odre|enih parametara statusa gvo`|a ~ak i kod pojedinaca
in the certain iron status parameters even in individuals with sa optimalnim nivoima ovih parametara. Me|utim treba biti
optimal levels of these indices. However, caution should be oprezan pri suplementaciji Mg, a dodatno je neophodno i
exercised when supplementing Mg, and laboratory monitor- laboratorijsko pra}enje ovih interakcija.
ing of the interaction is required.
Klju~ne re~i: magnezijum, suplementacija, status gvo`|a,
Keywords: magnesium, supplementation, iron status, ène, studenti
female, students
Introduction ing associated diseases, when the recommended

daily intake cannot be provided solely by the diet (1–
Magnesium (Mg) plays an important role in
3, 13). According to the recently conducted survey,
many physiological functions. As a cofactor for over
Mg supplements were among ten most popular
600 enzymes and an activator of an additional 200
dietary supplements used in adult population (17).
enzymes, Mg is involved in almost all major biochem-
Magnesium supplementation led to improved anemia
ical and metabolic processes in the body. Magnesium
in thalassemic mice and improved erythrocyte mem-
has an important role in macronutrient and energy
brane transport abnormalities in patients with sickle
metabolism, neuromuscular function, bone develop-
cell disease (18, 19), while in athletes, Mg supple-
ment, cell proliferation and signalling pathways (1, 2).
mentation increased erythrocyte count and hae-
Magnesium is an essential micronutrient, and there-
moglobin levels (20). Moreover, according to the Shi
fore it must be supplied regularly via food sources to
reach the recommended intake and prevent deficien- et al. (16) Mg supplementation may be a safer alter-
cy. Insufficient dietary intake of Mg is one of the most native than iron (Fe) supplementation in the preven-
important causes of hypomagnesaemia (3). Recom- tion of anemia.
mendations for Mg intake for adults in the United Although previously published data suggest the
States (Recommended Daily Allowances, RDA) are beneficial effects of increased Mg intake on Fe status
320 mg/day for women and 420 mg/day for men among anaemic individuals, research on this issue is
and in Europe 300 mg/day for women and 350 lacking for the non-anaemic population (15, 16). It is
mg/day for men (Dietary Reference Values, DRVs) (4, generally accepted that the female population is more
5). Although Mg is widely distributed in plant and ani- prone to Fe deficiency anemia and more vulnerable
mal foods as well as in beverages, literature data indi- on Fe status than men (21, 22). Therefore, it is
cate that the dietary intake of Mg is below the recom- important to ensure stable Fe status, particularly in
mended levels in a large percentage of European and the reproductive period of life. The aim of this study
US populations where there is a higher prevalence of was to examine the effect of short-term Mg supple-
the Western dietary pattern (6–9). There is also evi- mentation in doses of 375 mg corresponding to the
dence that many young women in various European 100% of Mg Nutritive Referent Value NRV on Fe sta-
countries and in the US fail to achieve these recom- tus in healthy female participants.
mended intakes (2, 10). Furthermore, epidemiologi-
cal studies have shown that people who follow a
Western-style diet, characterized by a high intake of Materials and Methods
processed foods, have an inadequate intake of several
micronutrients, with dietary Mg intake of less than Ethics statement
30–50% of RDA (11, 12). This study was conducted following the guide-
In recent decades, insufficient Mg intake and lines laid down in the Declaration of Helsinki and the
consequent hypomagnesaemia have been associated study protocols were approved by the Ethics
with several cardiovascular conditions, including Commission of the University of Belgrade - Faculty of
hypertension, an increased risk of glucose intoler- Pharmacy, Belgrade, Serbia (approval number:
ance, metabolic syndrome, and type 2 diabetes (1–3, 188/2, 2020). All subjects went through verbal and
13). Moreover, a meta-analysis of eight prospective written consent processes.
cohort studies has reported a significant inverse asso-
ciation between Mg intake and risk of type 2 diabetes
in a dose-response manner (14). In addition, Mg defi- Study design and subjects
ciency is closely related to the development of
One hundred healthy female students of the
anaemia (15, 16).
University of Belgrade – Faculty of Pharmacy agreed
There is accumulating research regarding the to participate in this study. Eligible students were
importance and modalities for achieving adequate approached in the Faculty setting. Recruitment
Mg intake. Magnesium supplementation may be a brochure contained detailed information regarding
good strategy for preventing deficiency and prevent- the purpose of the study, procedures involved as well
318 Milinkovi} et al.: Magnesium supplementation and iron status
as rights and expectations of the potential partici- i.e., Dietary Reference Values (DRVs) were applied
pants. The main inclusion criteria were: age between for micronutrient adequacy evaluation (26).
18 and 30 years, body mass index (BMI) between
18.5 and 29.9 kg/m2 and willingness to maintain
regular dietary habits throughout the study. The study Biochemical assessment
did not include students who had altered Fe status
Serum biochemical parameters were analyzed
(primarily based on complete blood count analysis),
before the initiation of the intervention, at baseline
who had taken Mg supplements over the previous
(t0), on the fifth day (t1) and the eleventh day (t2) of
three months, or students who had confirmed chronic
the intervention period. Blood samples were collected
kidney and/or gastrointestinal tract disease. Finally,
by professional phlebotomists via venipuncture
after applying the exclusion criteria, 46 respondents
between 7:00 am and 9:00 am. All studied partici-
remained in the analytical sample.
pants were donated blood samples after the 12h of
Anthropometric parameters were measured at overnight fast. Standard operating procedures for
the beginning of the study. Height was measured to blood collection and sample preparation were fol-
the nearest 0.1 cm (Perspective Enterprises, lowed (27). A closed venipuncture system, Beckton
Kalamazoo, MI, USA). Body weight and body fat per- Dickinson (BD) 22 Standard Wire Gauge (SWG), a
centage were determined using the bioimpedance reusable adapter and vacutainers were used.
method (BC-418MA, Tanita, USA). Body Mass Index Vacutainers with clot activator (BD Vacutainer® SST™
(BMI) was calculated as weight (kg) / height2 (m2). Tubes) were used to obtain serum samples. Serum
samples were used for measurement of serum Fe,
During eleven intervention days, students unsaturated iron binding capacity (UIBC), total iron
ingested Mg preparations (citrate, oxide and carbon- binding capacity (TIBC) and total Mg (tMg).
ate) with the same dose of the active substance (375 Transferrin concentrations and percentage of transfer-
mg/day). The subjects were randomly assigned to rin saturation (SAT) were calculated according to the
three groups according to the form of the Mg prepa- formula proposed by Dacie et al. (28). Complete
ration. blood count was determined using the whole blood
samples collected in a tube with liquid anticoagulant
(ethylenediaminetetraacetic acid, EDTA) (BD
Dietary intake assessment Vacutainer® EDTA Tubes). For the analyses of met-,
carboxy- and oxy-haemoglobin (metHgb, COHgb,
Dietary intake data was collected via partici- O2Hgb) and ionized Mg (iMg), lithium-heparin spray-
pants’ subjective retrospective reports in two study coated tube (BD Vacutainer® Heparin Tubes) were
points. Participants were administered 24h dietary used. Serum Fe, UIBC, TIBC and tMg concentrations
recalls on two consecutive days before the initiation of were measured using the spectrophotometric method
the supplementation (t0) and after 11 days of using with commercial reagents, on Olympus AU400 bio-
the provided Mg supplements, within the follow-up chemical analyzer (Beckman Coulter, Inc., California,
assessment (t2). Dietary information and relevant USA). Complete blood count was measured in whole
contextual data were obtained over the course of blood samples using electrical impedance on Coulter
face-to-face structured interviews led by a trained Ac•T diff Hematology Analyzer (Beckman Coulter,
researcher. In order to improve the accuracy of the Inc., California, USA). Total haemoglobin (tHgb) con-
report, enhance participants’ memory, and assure the centrations were measured by spectrophotometry on
provision of detailed descriptions of consumed items Coulter Ac•T diff Hematology Analyzer. MetHgb,
multiple-pass methodological approach was applied. COHgb, O2Hgb and iMg were determined approxi-
To assist respondents in portion size quantification mately up to 60 min after collection using Stat Profile
interviewers used validated, 135-item Food Atlas fea- Prime Plus Critical Care blood gas analyzer (Nova
turing coloured photographs of increasing portion Biomedical, USA). The precision and accuracy of the
sizes for a selection of Balkan region-specific simple methods were verified using commercial control sam-
foods and composite dishes (23). Additionally, sub- ples for all the listed parameters.
jects reported quantities of foods consumed based on
standardized household measures, natural units and
labelling information for packaged products. Diet Statistical analysis
Assess & Plan – original software-based platform for The normality of the data distribution was anal-
comprehensive nutritional assessment was applied in ysed using the One-sample Kolmogorov-Smirnov
questionnaire processing (24). Subsequent conver- test. Normally distributed data were presented as
sion of food consumption information into energy mean and standard deviation (SD). Non-normally dis-
and nutrient intake estimates was performed accord- tributed data were presented as a median and
ing to data compiled in National Serbian Food interquartile range (IQR), by subtracting the first from
Composition Database (25). Age and gender-adjust- the third quartile of the distribution. For data that
ed nutritional recommendations proposed by EFSA were not normally distributed, values were log-trans-
J Med Biochem 2022; 41 (3) 319
formed before analysis. Linear regression analysis was Table I Baseline participant characteristics (N=46).
performed to evaluate the relationship between inves-
Parameters Mean±SDa Median (IQR)b
tigated parameters. The association among serum Fe
and Mg (as tMg, iMg and iMg/tMg) with certain Age, years 23 (2)
(some) biochemical parameters was presented as BMI, kg/m2 21.8 (2.8)
polynomial regression. A principal component analy-
Total body fat, % 25.49±4.85
sis (PCA) was used for the evaluation of interdepen-
dence between serum Fe and Mg (as tMg, iMg and Systolic pressure (mmHg) 113.8±10.6
iMg/tMg) and biochemical parameters (UIBC, TIBC, Diastolic pressure (mmHg) 80.3±8.6
Transferrin, tHgb, hematocrit, SAT and Fe). We have
WBC, 109/L 7.29±1.13
considered p-value < 0.05 as statistically significant.
All statistical analyses were performed using IBM Lymphocytes, % 33.5±5.7
SPSS version 24 (SPSS Inc., USA). Monocytes, % 6.0±1.6
Granulocytes, % 60.4±5.9
Results Lymphocytes, # 2.4±0.5

Monocytes, # 0.4 (0.1)
Baseline participants’ characteristics are pre-
sented in Table I. Participants were on average 23 Granulocytes, # 4.4±0.9
years old, with a BMI of 21.8 kg/m2. Erythrocyte RBC, 1012/L 4.51±0.28
indices and biochemical parameters indicated a tHgb, g/L 140 (4)
favourable Fe status. All presented parameters were
within the reference values routinely used in the lab- Hct, L/L 0.439±0.029
oratory, and recommended by the test manufacturer. MCV, fL 89.6±4.2
Given that the aim of this study was to examine the MCH, pg 29.9±1.7
effect of the applied dose of magnesium of 375 mg,
in which magnesium is most often found in dietary MCHC, g/L 334±7
supplements on the Serbian market (dose that meets RDW, % 14.5±1.7
100% nutritional reference value for Mg), data was iMg, mmol/L 0.59±0.032
further analysed without partition of subjects into sub
tMg, mmol/L 0.89±0.054
cohorts in relation to different types of supplemented
magnesium (citrate, oxide and carbonate). Fe, mol/L 14.89±5.65
Estimated daily energy and nutrient intakes are UIBC, mol/L 51.8±13.8
presented in Table II. Based on the average of dietary TIBC, mol/L 66.7±12.5
recalls for two consecutive days at baseline evaluation SAT, % 23.9±9.8
(t0) and after 11 days (t2) of using provided dietary
supplements, the results obtained indicate that food Transferrin, g/L 2.6 (0.7)
intake did not change over time. MetHgb, % 0.4 (0.15)
Estimated intake levels of eight food groups and COHgb, % 3.66±2.02
their corresponding contributions to daily iron intake O2Hgb, % 40.6±19.7
based on repeated 24 h dietary recalls are presented aMean±SD, the standard deviation for normal distribution
in Table III. Dominant Fe dietary sources were grains bMedian (IQR); IQR, interquartile range (quartile3-quartile1)
and cereal products (28.6%), meat and meat prod- for
not normally distribution
ucts (22.3%) and vegetables and vegetable products WBC, white blood cells (Leucocytes); RBC, red blood cells; tHgb,
(15.7%). haemoglobin; Hct, hematocrit; MCV, mean cell volume; MCH,
A statistically significant trend for change in mean haemoglobin concentration; MCHC, amount of haemo-
O2Hgb (%) by tertiles of whole blood iMg concentra- globin per unit volume in a single red blood cell; Fe, iron; UIBC,
unsaturated iron-binding capacity; TIBC, total iron-binding
tions was found. With the increase of iMg, the per- capacity; SAT, total transferrin saturation
centage of O2Hgb decreases. Additionally, we found
a significant increase in changes of SAT (%) by quar-
tile until the third quartile of tMg values. Interestingly,
was no statistically significant correlation between
in the fourth quartile of serum tMg values, a signifi-
serum tMg and SAT (Figure 1A). Ionized Mg in a
cant decrease in SAT (%) was observed.
concentration higher than 0.6 mmol/L is positively
Based on polynomial regression analyses, serum correlated with SAT and serum Fe (R2=0.214; 0.199,
tMg reached significant positive correlation with the respectively; Figure 2B) after supplementation, which
SAT at concentration levels greater than 0.9 mmol/L, was not the case before the supplementation, because
after 11 days of supplementary intervention at the mentioned concentration the serum Fe
(R2=0.116; Figure 1B). Before the intervention, there decreased slightly (Figure 2A).
Table II Daily energy and nutrient intakes among study participants assessed by the average of dietary recalls for two consecutive
days at baseline evaluation (t0) and after 11 days (t2) of using provided dietary supplements.
Intervention Group (N=46)

Energy/Nutrients t0 t2 P
Energy (kcal) 1733.6±550.6 1804.9±485.6 0.501
Carbohydrates (TEI%) 29.9±9.8 28.6±7.8 0.516
Proteins (%TEI) 10.5±3.9 11.7±3.3 0.066

Fats (%TEI) 29.0±13.1 31.9±13.1 0.283
Fe (mg) 7.8±3.3 8.6±3.0 0.177
Mg (mg) 236.2±85.1 230.8±74.1 0.741
Zn (mg) 7.3±3.3 8.4±3.2 0.052
Folic acid (g) 189.2±96.6 210.9±78.2 0.269
Vitamin B12 (g) 2.5±1.3 2.6±1.2 0.053
Vitamin C (mg) 61.5±7.3 68.9±6.0 0.631
%TEI, percentage of total energy intake; p < 0.05 – statistically significant difference between t0 and t2 within the same intervention
group.
aMean±SD, the standard deviation for normal distribution
Table III Daily intake levels presented as the median levels and 5th and 95th percentiles of eight food groups and their corre-
sponding contributions to daily iron intake based on repeated 24 h dietary recalls among study participants.
The average contribution

Intake of the food group
Food groups to the total iron intake
(mg/day)
(10.52 mg/day)
5th 95th Iron intake

Median %
percentile percentile (mg/day)
Milk and dairy products 0.18 0.01 0.44 2.25 0.24
Eggs and egg products 0.63 0.15 3.34 8.01 0.84
Meat and meat products 1.64 0.38 5.92 22.28 2.34
Grains and cereal products 2.28 0.71 5.60 28.57 3.00
Nuts and seeds 0.42 0.03 2.24 6.94 0.73
Vegetables and vegetable products 1.29 0.14 4.03 15.67 1.65
Fruit and fruit products 0.51 0.04 1.54 4.84 0.51
Sugar and confectionary products 0.21 0.01 1.95 4.37 0.46
Table IV Associations between estimated O2Hgb and tertiles of whole blood ionized Mg at the eleventh day.
Whole blood ionized Mg on the eleventh day

Biochemical
parameter T1 (N=17) T2 (N=16) T3 (N=13)
P for trend
<0.59 0.60–0.63 0.64–0.68
49.9 41.3 27.3
O2Hgb (%) 0.029
(38.7– 61.1) (29.4 –53.2) (20.9–33.7)
p < 0.05 – a statistically significant difference for trend
T, tertil; O2Hgb, oxy-haemoglobin; N, number of participants
J Med Biochem 2022; 41 (3) 321
Figure 1 Interdependence between serum total magnesium (tMg) with serum iron (Fe),unsaturated iron binding capacity
(UIBC), total iron binding capacity (TIBC) and transferrin saturation (SAT) at the beginning (A) and after 11 days (B) of sup-
plementary intervention.
PCA was applied to integrate results of bio- two axes participated in total variability with 55.8%
chemical parameters, to discover the possible correla- (F1: 34.6% and F2: 21.2%), and after 11 days, the
tions among measured parameters, and to classify axes participated in total variability with 64.7% (F1:
the parameters in a factor plane. PCA is a factor 46.7% and F2: 18.0%). According to the PCA,
model in which the factors are based on summarizing Transferrin, TIBC, UIBC, SAT, iMg/tMg and iMg cor-
the total variance. The first two factors should corre- related mainly with the first axis - factor (0.952;
spond to a high % of the variance to ensure that the 0.952; 0.940; -0.607; -0.579 and -0.520, respec-
maps based on the first two factors are a good quality tively), while serum Fe, tHgb, SAT, Mg; hematocrit;
projection of the initial multi-dimensional table. At iMg/tMg, and MetHgb were mainly connected to the
the beginning of the experiment, PCA revealed that second axis - factor (0.758; 0.721; 0.588; 0.442;
Figure 2 Interdependence between serum ionized magnesium (iMg) with serum iron (Fe), hematocrit (Hct), transferrin and
transferrin saturation (SAT) at the beginning (A) and after 11 days (B) of supplementary intervention.
0.317; -0.554; -0.510; respectively), Figure 4A. In -0.890, -0.890, 0.890, 0.822, 0.777, and 0.698,
the first factor (F1) there is a strong positive correla- respectively), whereas F2 was determined with
tion between TIBC, UIBC and Transferrin, while SAT, iMg/tMg; iMg and serum Mg (0.981; 0.720 and -
iMg/tMg and iMg are negatively correlated. The 0.616; respectively), Figure 4B. The first factor (F1) is
second factor (F2) is positively correlated with serum positively correlated with SAT; serum Fe; tHgb and
Fe, tHgb, SAT, serum Mg; hematocrit, and negatively hematocrit, and negatively with UIBC, TIBC and
with iMg/tMg, and MetHgb. After 11 days of the Transferrin, while second factor (F2) is positively
study, F1 was determined with UIBC, TIBC, Trans- correlated with iMg/tMg and iMg, and negatively with
ferrin; SAT; serum Fe; tHgb and hematocrit (-0.982, serum tMg.
J Med Biochem 2022; 41 (3) 323
Figure 3 Principal Component Analysis for serum iron (Fe) and serum magnesum (Mg) (as total (tMg), ionized (iMg) and
iMg/Mg) and biochemical parameters (unsaturated iron binding capacity (UIBC), total iron binding capacity (TIBC), transferrin,
transferrin saturation (SAT), total haemoglobin (tHgb), MetHgb and hematocrit (Hct)) content at the beginning (A, t0) and
after 11 days (B, t2) of supplementary intervention.
Discussion adapts to the additional intake of magnesium, calci-

To the best of our knowledge this is the first um and phosphorus in the period from 7 to 10 days
study examining the short-term effects of Mg supple- and that the mineral balance is achieved in the period
mentation in daily doses corresponding to 100% NRV of a few days after supplementation intake (29). After
on Fe status in young healthy women. Intervention 11 days of supplementation in a dose corresponding
study was conducted among young female subjects to Mg DRV (i.e. 375 mg), direct association was
with an aim to explore the effects of short-term Mg found between the serum tMg concentration and
supplementation on indices of iron status. Namely, SAT. Furthermore, whole blood iMg correlated posi-
the literature data indicate that the human body tively with SAT and serum Fe. These observations
were also confirmed by polynomial regression. These using the PCA approach, before and after Mg supple-
finding suggest that Mg supplementation and mentation. Given that the diet did not change over
increased Mg levels, both serum tMg and blood iMg, intervention period intake of Mg and Fe were not con-
might exert beneficial impact on obtaining favorable sidered in PCA. At the beginning of the study, before
Fe status among young female population. the initiation of the supplementation, there was a
strong positive correlation between UIBC, TIBC and
In this sample, the average iron intake was sig-
transferrin, but after 11 days of supplementation we
nificantelly bellow the DRI and DRV for women, i.e.,
found a strong negative correlation among the same
18 mg/day and 16 mg/day, respectively (4, 5). These
analyzed parameters. Analyses revealed that even in a
findings are consistent with the results of other stud-
short period of intervention there is a noticeable
ies which reported dietary iron intake in women of
effect of Mg supplementation on Fe status para-
reproductive age in Europe (30, 31). Regardless the
meters (serum Fe, tHgb, hematocrit). PCA analysis
suboptimal dietary intake of Fe, biomarkers of Fe sta-
revealed a positive correlation between serum Fe and
tus were within the normal range. This can be
SAT after 11 days of supplementation. Reddy et al.
explained by the following facts: iron metabolism was
have explained similiar associations in Fe status
primarily regulated at the level of absorption, and the
parameters but in patients with functional anemia in
examined population did not have gastrointestinal
chronic kidney disease (37). Anemia could be present
disorders. Also, the amount of iron that enters the
as a latent condition, mostly in young women who are
body from food is regulated by the body’s need for
in the reproductive period. In order to optimize Fe
iron. Other studies also indicate that a significant pro-
status it is important to monitor biochemical
portion of the UK population has Fe intake below rec-
parameters and routinely examine relevant markers
ommendations and a low prevalence of poor Fe sta-
(38).
tus (32–34). This might be because there are
important uncertainties in the DRVs for Fe intake Previously published data suggest an interaction
which may be too high, particularly for girls and between the resorption of divalent cations such as Mg
women of reproductive age. It is recommended that and Fe. Namely, the deficiency of one divalent cation
the DRVs for iron should be reviewed when more in the intestine can lead to increased resorption of
data becomes available. Good quality dose-response other divalent cations (39). In an animal model, it has
data are required to enable a reassessment of the been shown that Fe deficiency can lead to increase in
DRVs for iron. Knowledge of the systemic regulation intestinal absorption of Mg, calcium and phosphorus
and mediation of iron homeostasis should be applied since the same receptor may be involved in the
to characterize better the responses to increased and resorption of these chemically similar cations (40).
reduced systemic needs for Fe and development, or Low dietary intake of Mg in rats has been shown to
better validation, of existing markers used to assess increase Fe resorption (41). Furthermore, in vitro
the adequacy of Fe status in populations and individ- studies have demonstrated that Mg salts can nega-
uals. The main food sources of Fe among participants tively affect absorption of Fe by raising the pH value
in our study were grains and cereal products with con- as the availability of Fe salts in the intestinal tract is
tribution of more than a quarter of total dietary intake pH dependent (42). Moreover, certain Mg salts can
of this nutrient followed by meat and meat products absorb Fe and thus interact with its absorption (42).
and vegetables. These three food groups together
On the contrary, there are studies linking Mg
contributed to 65% of estimated iron intake in the
intake and the risk of developing anemia (15, 16, 42–
participants. These observations are in accordance
44). In our study, we have demonstrated that short
with previously published data for European popula-
term supplementation with Mg and increased level of
tion (35, 36). Furthermore, dietary intake assessment
serum tMg and iMg could have benefical effects on %
revealed that Mg intake among female student popu-
SAT and serum Fe. Magnesium is a cofactor of a
lation were also below the recommended level
large number of enzymes, with an important role in
although mean baseline tMg concentration was ade-
the synthesis of hemoglobin. Accordingly, Mg defi-
quate.
ciency can interrupt hemoglobin synthesis and ery-
There is a lack of literature data regarding the throcyte energy metabolism and result in anemia. In
effect of increased Mg intake on Fe status in young addition, Mg deficiency has been reported to be asso-
women in the reproductive period. This issue needs to ciated with an inflammatory process, which could
be addressed taking into account that Mg supple- lead to anemia (45). Therefore, the question arises as
ments are one of the most popular dietary supple- to whether Mg supplementation is required in persons
ments used in adult population (17) and the fact that who are not deficient in this trace element, and leaves
anemia is most prevalent among females of child- space to the future studies to examine longitudinal
bearing age (36). effects of Mg supplementation on Fe status indices.
In this study, we tried to explore the connection The key limitation of the present study is missing
between serum Fe and serum Mg (as tMg, iMg and data regarding the leukocyte, thrombocyte, and ery-
iMg/Mg) and other biochemical indices of Fe status throcyte counts as well as ferritin level, after the inter-
J Med Biochem 2022; 41 (3) 325
vention period although changes in these parametres the analyzed parameters was achieved. However, cau-
couldn’t be expected during short intervention period. tion should be exercised when supplementing Mg,
Additionally, acknowledge that calculated transferrin and laboratory monitoring of the interaction is
concentrations provide limited information. required. Further research is warranted regarding the
Furthermore, the duration of the Mg supplementation possible impact of the forms of Mg preparations that
may have been short to explore the dynamical exist on the market, and whether they equally affect
changes of the supplements’ effects on biomarkers of biochemical changes of iron status in healthy young
Fe status. Similar studies are of interest in the male people as well as in specific target groups of patients.
population, as well.
Acknowledgements. This work was supported by the
Ministry of Education, Science and Technological
Conclusion Development of Serbia on the basis of contract
No.175036 and No.451-03-68/2020-14/200161.
This study results indicate that Mg supplementa-
tion leads to an improvement in the certain iron status
parameters even in individuals with optimal levels of Conflict of interest statement
these indices. Additionally, the analyzed parameters
were significantly correlated, and after the interven- All the authors declare that they have no conflict
tion period, a significant positive association among of interest in this work.
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UDK 577.1 : 61 ISSN 1452-8258

IMPACT OF GENETIC POLYMORPHISMS AT THE PROMOTER AREA

OF IL-10 GENE ON TACROLIMUS LEVEL IN JORDANIAN RENAL
TRANSPLANTATION RECIPIENTS
UTICAJ GENETSKIH POLIMORFIZAMA U OBLASTI PROMOTERA GENA IL-10 NA NIVO
TAKROLIMUSA U JORDANSKIH PACIJENATA SA TRANSPLANTACIJOM BUBREGA
Bara’ah Khaleel1, Al-Motassem Yousef 2, Mazhar Salim AL-Zoubi3, Muhammad AL-Ulemat4,

Ahmad A. Masadeh4, Ali Abuhaliema2, Khalid M AL-Batayneh1, Bahaa Al-Trad1
1Department of Biological Sciences, Faculty
of Science, Yarmouk University, Irbid, Jordan
2Department of Biopharmaceutics and Clinical Pharmacy, School of Pharmacy,
The University of Jordan, Amman, Jordan
3Department of Basic Medical Sciences, Faculty of Medicine, Yarmouk University, Irbid, Jordan
4Pharmacy Department, Royal Medical Services, Amman, Jordan

Background: Tacrolimus is a widely used immunosuppres- Uvod: Takrolimus je {iroko kori{}en imunosupresiv koji
sant that prevents solid organ transplant rejection. The spre~ava odbacivanje presa|enog ~vrstog organa. Farmako-
pharmacokinetics of Tacrolimus show considerable varia- kinetika takrolimusa pokazuje zna~ajnu varijabilnost inter-
bility. Interleukin-10 (IL-10), in the host’s immune response leukin-10 (IL-10), u imunolo{kom odgovoru doma}ina
after transplantation, contributes to the variable CYP3A- nakon transplantacije, doprinosi promenljivoj dispoziciji leka
dependent drug disposition of Tacrolimus. In the current zavisne od CIP3A kod takrolimusa. U ovoj studiji, cilj nam je
study, we aim to evaluate the impact of single nucleotide da procenimo uticaj polimorfizama pojedina~nih nukleotida
polymorphisms (SNP) in the promoter region of IL-10 on (SNP) u promotorskom regionu IL-10 na zahteve za dozom
Tacrolimus dose requirements and the Dose Adjusted takrolimusa i koncentraciju prilago|enu dozi (DAC) takro-
Concentration (DAC) of Tacrolimus among kidney trans- limusa me|u primaocima transplantacije bubrega.
plantation recipients. Metode: Nivoi takrolimusa u krvi su mereni kori{}enjem imu-
Methods: Blood levels of Tacrolimus were measured using noeseja mikro~estica enzima (MEIA) tokom {est meseci
Microparticle Enzyme Immunoassay (MEIA) for six months nakon transplantacije. Analiza genotipizacije je izvedena
post-transplantation. Genotyping analysis was utilized using kori{}enjem specifi~ne lan~ane reakcije polimeraze (PCR)
specific Polymerase Chain Reaction (PCR) followed by pra}ene metodama sekvenciranja za 98 jordanskih prima-
sequencing methods for 98 Jordanian kidney transplant laca transplantiranih bubrega.
recipients. Rezultati: U~estalosti genotipizacije IL-10 (-592) su
Results: Genotyping frequencies of IL-10 (-592) were (CC/CA/AA: 38, 46,7, 15,2%); IL-10 (-819) su (CC/CT/TT:
(CC/CA/AA: 38, 46.7, 15.2%); IL-10 (-819) were 40,4, 44,1, 15,1%); i IL-10 (-1082) su (AA/AG/GG: 42,6,

Al-Motassem Yousef, PhD
Professor of Pharmacology and Therapeutics,
Vice Dean for Quality and Accreditation,
Dept. Biopharmaceutics and Clinical Pharmacy,
College of Pharmacy, University of Jordan, Amman 11942, Jordan
Mobile: +962-777-486-930
Office: +962-6-5355000 ext 23357
Phone #:00962-777-486930 List of abbreviations: ESRD, End-stage renal disease; SNPs,
Fax #: 00962-6-5339649 Single-nucleotide polymorphisms; SD, Standard deviation;
e-mail: ayousefªju.edu.jo BSA, Body surface area; DAC, Dose-adjusted concentration
328 Khaleel et al.: IL-10 polymorphism and tacrolimus level in renal transplantation
(CC/CT/TT: 40.4, 44.1, 15.1%); and IL-10 (-1082) were 44,7, 12,8%). Uticaj IL-10 (-1082) na takrolimus DAC za-
(AA/AG/GG: 42.6, 44.7, 12.8%). The impact of IL-10 visio je od pola. Mu{karci koji su nosili najmanje jedan alel A
(-1082) on Tacrolimus DAC was gender dependent. Men imali su zna~ajno niì DAC od mu{karaca koji su nosili
carrying at least one A allele had significantly lower DAC genotipizaciju GG samo u prvom mesecu nakon transplan-
than men carrying GG genotyping only in the first month tacije 88,2±32,1 naspram 117,5±22,5 ng/mL po
post-transplantation 88.2±32.1 vs. 117.5±22.5 ng/mL mg/kg/dan, p=0,04.
per mg/kg/day, p=0.04. Zaklju~ak: Na{a trenutna studija je pokazala da interakcija
Conclusions: Our current study showed that the interaction izme|u pola i IL-10-1082 uti~e na takrolimus DAC kod jor-
between gender and IL-10 -1082 affects Tacrolimus DAC danskih primalaca transplantacije bubrega tokom prvog
in Jordanian kidney transplant recipients during the first- meseca nakon transplantacije.
month post-transplantation.
Klju~ne re~i: IL-10 genetski polimorfizam, transplantacija
Keywords: IL-10 genetic polymorphism, kidney trans- bubrega, farmakokinetika, takrolimus
plantation, pharmacokinetics, tacrolimus
Introduction ing prospects in Tacrolimus dose individualization

(13).
End-stage renal disease (ESRD) is the final stage
of kidney failure, characterized by a decreased The IL-10 promoter area is highly polymorphic;
glomerular filtration rate and increased urinary albu- many studies have been conducted showing varia-
min excretion (1). Kidney transplantation is consid- tions in IL-10 expression linked to promoter area poly-
ered the most effective treatment of End-stage renal morphisms. Five SNPs tagging the promoter area of
disease (ESRD) when compared to dialysis (2, 3). In IL-10 have been widely studied and they are (-3575),
1954, Murray, and Merrill (4) performed the first suc- (- 2763), (-1082), (-819), and (-592) (14).
cessful kidney transplant operation; it was made pos-
sible because the donor and recipient were monozy- IL-10 production level showed that the GG
gotic identical twins. The first kidney transplantation genotype -1082 is higher versus (AA and AG) geno-
in the Arab world was performed in Jordan in 1972, types, independently of the polymorphisms at posi-
the kidney was obtained from a deceased donor (5). tions -819 and -592, and also associated with higher
serum concentration (15 –17). Furthermore, in vivo
Transplantation patients during their post-opera- study showed that higher IL-10 decreases CYP3A
tive phase run a great risk of developing major life- activity, which is involved in the metabolism of
threatening complications which include: (1) cardio- tacrolimus among renal transplantation recipients
vascular diseases most likely caused by calcification of (18, 19).
vessels and left ventricular hypertrophy associated
originally with ESRD; (2) delayed graft function which Our current study aimed to investigate the asso-
is defined as the use of dialysis due to poor kidney ciation between the dose required to reach the target
function in the first week of graft life; (3) infection due level of Tacrolimus and genetic variations in renal
to the high level of immunosuppressants given to the transplantation recipients through the study of IL-10
patient, but the improved use of antimicrobials and (-592, rs1800872, A/C); IL-10 (–819, rs1800871,
antimicrobial regimens has decreased infection sever- T/C) and IL-10 (–1082, rs1800896, A/G). The SNPs
ity; (4) graft rejection (6–9). were selected due to the reported relationship with IL-
10 production and level (15, 20).
To solve these complications, immunosuppres-
sants are essential for successful organ transplan-
tation as they suppress rejection and inhibit the Materials and Methods
autoimmune process, however, they also lead to
undesired consequences such as immunodeficiency, Patients and ethical approval
infection or malignancy, and non-immune toxicity Ninety-eight adult renal transplant recipients,
(10). Tacrolimus is a fermentation product of Strepto- who had received a renal graft between 2009 and
myces and belongs to the family of calcineurin in- 2011 from Jordanian Royal Medical Services, were
hibitors. It is a widely used immunosuppressive drug included in the study. The inclusion criteria were
for preventing solid-organ transplant rejection (11). patients with a newly transplanted kidney and who
But its usage is complicated due to its narrow thera- were on a Tacrolimus-based immunosuppressive
peutic index and considerable inter-and intra-individ- maintenance therapy starting immediately following
ual pharmacokinetic variability (12).
transplantation. Tacrolimus was given in two equally
Many single-nucleotide polymorphisms (SNPs) divided doses. All patients treated with Tacrolimus
have been studied concerning the pharmacokinetics used the capsule formulation Prograf® (Fujisawa,
of Tacrolimus, especially CYP3A4, CYP3A5, and Munich, Germany). Patients who received medica-
ABCB-1; as their alleles have been involved in the tions known to interact with Tacrolimus were excluded
metabolism of calcineurin inhibitors showing promis- from the study.
J Med Biochem 2022; 41 (3) 329
Table I Primers, PCR conditions of genotyping analysis for IL-10 -1082A/G, IL-10 -592A/C and IL-10 -819T/C.
Allele Positiona Primers PCR conditions
Forward primer 5’ GGCTTCCTACAGTACAGGCG 3’ Denaturing 95 °C for 1 min

IL-10 -1082A/G rs1800896
Annealing 60 °C for 1 min
Reverse Primer 5’ GGTAGAGCAACACTCCTCGC 3’
Extension 72 °C for 1 min
35 cycles
rs1800872 Forward primer 5’GATGAATACCCAAGACTTCTCCT 3’
IL-10 –592A/CIL-10 Size of PCR product
–819T/C 447 bp and 783 bp
rs1800871 Reverse Primer 5’ CCTTCCCCAGGTAGAGCAACAC 3’
This study was approved by local Research an ABI Prism Genetic Analyzer system 3130xl
Ethics Committees of Jordanian Royal Medical (Applied Biosystems, Waltham, MA, USA).
Services (IRB: TF1/3/ethics obtained on June 27th,
2016); and has been performed following the ethical
standards laid down in the 2000 Declaration of Statistical analysis
Helsinki as well as the Declaration of Istanbul 2008.
Written informed consent was obtained from all par- Data were coded and entered into Statistical
ticipants. Details that might disclose the identity of Packages for Social Sciences (SPSS version 20.0,
the subjects in the study were omitted. 2012). Data were summarized as counts and per-
centages for categorical data and as means and stan-
dard deviation (SD) for continuous data. A data set
was tested for normality of distribution using the
Tacrolimus blood level measurements
Shapiro-Wilk test. Homogeneity of variance was
Blood samples were collected before the admin- assessed by Levene’s test. Comparison between cate-
istration of the morning dose of Tacrolimus for the gorical data was conducted using the Fisher exact test
determination of trough blood concentrations of the or Chi-square test, when appropriate. Comparison
drug. The trough concentration was measured in between continuous data was performed utilizing
whole blood by IMx Tacrolimus II assay which utilizes independent t-test; ANOVA, Mann-Whitney or
MEIA in the Abbott IMx system (Tacrolimus II; Abbott Kruskal Wallis, based on which was most appropriate.
Laboratories, IL. USA). This measurement was per- A p-value of 0.05 or less was considered statistically
formed in the laboratory of Jordanian Royal Medical significant.
Services, and the dose-adjusted concentration was
calculated by dividing the pre-dose concentration by
the corresponding 24-hour dose in milligram Results
Tacrolimus per kilogram body weight.
Ninety-eight kidney transplant recipients met
our inclusion criteria. The age, weight and gender of
Genomic DNA isolation and genotype analysis donors and recipients are comparable. Demographic
data of recipients and corresponding donors are sum-
Genomic DNA was isolated from 300 mL EDTA- marized in Table II. The most common cause of
treated whole blood using a Commercial kit (Wizard chronic kidney disease among our patients was hyper-
genomic DNA purification kit, Promega,WI, USA). tensive nephropathy (49%). Other identifiable causes
The procedure was carried according to the kit man- of chronic kidney disease included glomerulonephritis
ufacturer’s recommendation. (8.2%), chronic pyelonephritis (6.1%), diabetic
Genotyping analysis for detection of 3 SNPs of
IL-10s was performed for all patients by using specific
Table II Demographic data of Jordanian kidney transplant
PCR primers. Table I describes primers used and PCR recipients and corresponding donors.
conditions. PCR was performed in a total volume of
25 mL using 100 ng of genomic DNA with 1.5 mL of Parameter Donors Recipients p
10 mmol/L of each primer and 12.5 mL of 2X
KAPA2G Fast ReadyMix PCR Kit (Kappa Biosystems, Gender N Male 53 (54.1%) 60 (61.2%) 0.38
USA). PCR amplifications were performed in PTC- (%) Female 45 (45.9%) 38 (38.8%)
100 Peltier Thermal Cycler (MJ Research, MA, USA).
Age, years, mean (±SD) 34.1±8.9 35.6±9.6 0.26
PCR reaction products were sequenced using
Big Dye Terminator version 3.1 kit (Applied Bio- Weight, kg, mean (±SD) 70.9±16.4 72.1±17.4 0.62
systems, Waltham, MA, USA). Samples were run on Chi-Squared with one degree of freedom
Table III Medical history data for Jordanian kidney trans- Table IV Genotype frequencies of Jordanian kidney trans-
plant recipients. plant recipients.
Parameter N (98) Allele frequency
Genotype N (%) X2 P
%
Glomerulonephritis 8 (8.2%)
IL-10 CC 35 (38) Minor 61 0.01 0.99
Chronic pyelonephritis 6 (6.1%)
(-592,
Causes of Diabetic nephropathy 4 (4.1%) CA 43 (46.7) Major 36
rs1800872,
chronic C/A) AA 14 (15.2)
Hypertensive nephropathy 48 (49.0%)
kidney disease,
N (%) Polycystic kidney disease 4 (4.1%) IL-10 CC 38 (40.9) Minor 37 0.1 0.96
Undetermined 8 (8.2%) (–819,
CT 41 (44.1) Major 63
rs1800871,
Others 20 (20.4%) C/T) TT 14 (15.1)
Transplantation First 95 (96.9%)
events IL-10 AA 40 (42.6) Minor 34 0.06 0.97
N (%) Second 3 (3.1%) (–1082,
AG 42 (44.7) Major 66
Relative, living 91 (92.9%) rs1800896,
Type of donors A/G)
N (%) GG 12 (12.8)
Non relative, living 7 (7.1%)
Immunosuppressant use N: number of recipients. c2: Chi-Squared with one degree of
freedom.
Prednisolone N (%) 95 (96.9%)
Total daily dose
11.8±6
( mean±SD), mg
Azathioprine N (%) 6 (6.1%)
Total daily dose
75 ± 27.4
(mean±SD), mg
Mycophenolate N (%) 89 (90.0%)
Total daily dose
1393.3±491.2
(mean±SD), mg
N: number of recipients. SD: standard deviation.
nephropathy (4.1%), and polycystic kidney disease

(4.1%). Ninety-seven percent of patients underwent
the transplantation operation for the first time, with
the majority of them receivingthe graft from a living
relative (93%). The medical history of kidney trans- Figure 1 Effect of gender-genotype at IL-10 (-592,
plant recipients is summarized in Table III. rs1800872, C/A) on Tacrolimus concentration level (ng/mL)
of Jordanian kidney transplant recipients in the first month
post transplantation.
Genotypes and alleles frequencies Y-axis is Tacrolimus concentration level (ng/mL), X-axis is the
genotypes at IL-10 -592. CC: Homozygous ancestral genotype,
Among the 98 kidney transplant recipients, AA: Homozygous variant genotype and CA: Heterozygote vari-
some cases were not genotyped due to unsuccessful ant genotype.
PCR results. The allele frequencies of the 3 SNPs in
all patients were in accordance with the Hardy-
Weinberg Equilibrium equation (P>0.05). Genotype of IL-10. All mentioned parameters did not differ sig-
frequencies of patients are summarized in Table IV. nificantly among IL-10 (–819, rs1800871, T/C) and
IL-10 (–1082, rs1800896, A/G) during the first six
months post-transplantation as shown in Supple-
Effect of recipients genotypes on Tacrolimus mentary Tables (Table I and Table III). However, we
pharmacokinetics parameters found that patients carrying AA at IL-10 (-592,
Daily dose (mg/day), concentration level rs1800872, A/C) had significantly a higher tacro-
(ng/mL), weight-adjusted daily dose (mg/kg/day), limus concentration level than those patients carrying
body surface area (BSA) adjusted dose (mg/m2) and AC or CC genotypes in the first month, post-trans-
dose-adjusted concentration (DAC) (ng/mL per plantation (AA: 20.1±4.95 ng/mL; AC: 14.58±4.4
mg/kg/day) of Tacrolimus were compared among ng/mL; and CC: 13.86±4.1 ng/mL, p = 0.01). This
recipients with different allelic statuses of three SNPs difference in Tacrolimus concentration level disap-
J Med Biochem 2022; 41 (3) 331
peared after the first month as shown in Figure 1 and

Supplementary Tables (Table II).
Effect of gender-genotypes interaction of

recipients on Tacrolimus pharmacokinetics
parameters
Recipients were grouped according to their gen-
der (male vs female) then categorized into two sub-
groups according to the presence of at least one
ancestral allele versus the homozygous variant geno-
type (-592; CC and CA vs AA), (-819; CC and CT vs
TT) and (-1082; AA and AG vs GG). All mentioned
parameters did not differ significantly amongIL-10
(-592, rs1800872, A/C); IL-10 (–819, rs1800871,
T/C) during the first six months post-transplantation Figure 2 Effect of gender-genotype interaction at IL-10
(as shown in Supplementary Tables, Table V, VI). (–1082, rs1800896, A/G) on Tacrolimus dose adjusted
However, we found that patients carrying GG concentration (ng/mL per mg/kg/day) of Jordanian kidney
genotype atIL-10 (–1082, rs1800896, A/G) versus transplant recipients in the first month post transplantation.
patients carrying at least one A allele (AA or AG) Y-axis is dose adjusted concentration (ng/mL per mg/kg/day),
X-axis is the genotypes at IL-10 –1082. AA: Homozygote ances-
show differences. Men carrying at least one A allele tral genotype, GG: Homozygous variant genotype and AG:
had significantly lower Tacrolimus adjusted concen- Homozygous variant genotype.
tration than men carrying GG genotype in the first-
month post-transplantation. This reduction in DAC,
however, disappeared after the first month
88.2±32.1 vs. 117.5±22.5 ng/mL per mg/kg/day, Discussion
p=0.04. On the other hand, in women, non of men- This study examined the contribution of IL-10 (-
tioned parameters differed significantly between dif- 592, rs1800872, A/C); IL-10 (–819, rs1800871,
ferent IL-10 -1082 A>G genotype groups during the T/C) and IL-10 (–1082, rs1800896, A/G)polymor-
first six months post-transplantation as shown in phisms in Jordanian renal transplant recipients to
Figure 2 and Supplementary Tables (Table IV). Tacrolimuspharmacokinetics parameters within the
first six months post-transplantation. The clinical use
of Tacrolimus is complicated by its narrow therapeutic
range and highly variable pharmacokinetics among
individuals. Some patients do not reach target con-
Table V Haplotype Distribution of IL-10 –592, –819 and – centrations using the recommended initial doses of
1082 among renal transplantation recipients one month post Tacrolimus, and therefore, have an increased risk of
transplantation. inadequate immunosuppression and subsequent
acute rejection during the early period following
<= median >median organ transplantation (21). The association of the IL-
Haplotype Total (%) P
N=27 (%) N=23 (%)
10 gene SNPs with Tacrolimus dose requirements has
ATA 31.6 7(27.3) 8(36.7) 0.99 been recognized as a genetic base for the observed
inter-individual differences in pharmacokinetics (22).
CCG 29.9 6(24.8) 8(35.9) 0.99
Our current study aimed to determine whether
CCA 27.6 11(41.1) 3(11.7) 0.04 the genotype of IL-10 could explain variability in phar-
macokinetic parameters of Tacrolimus in kidney recip-
ACA 3.6 1(1.8) 1(5.7) 0.94 ients during the proposed period. We hypothesized
that the recipient’s polymorphisms of IL-10 are asso-
ACG 3.0 0(1.0) 1(5.4) ciated with changes in Tacrolimuspharmacokinetics
CTA 2.1 1(2.0) 1(2.3) 0.94 parameters during the early period post-transplanta-
tion.
CTG 2.1 1(2.0) 1(2.3) 0.94
In our current study, IL-10 alleles frequencies
N: number of recipients. C (first): the ancestral allele of IL-10
were found to be as follows; the A allele: 65% and the
–592. C (middle): the ancestral allele of IL-10 –819. A (last) G allele: 35%. this is consistent with data from previ-
the ancestral allele of IL-10–1082. A (first): the variant allele ously published research on such frequencies among
of IL-10 –592. T (middle): the variant allele of IL-10 –819. G Caucasians (A allele: 58.5–65.6%, G allele: 34–
(last): the variant allele of IL-10–1082. 41.5%) (23, 24).
Turner and Williams (15) found that following CYP3A catalytic activity (18). Hence, a lower tacro-
stimulation, IL-10 production was measured by ELISA limus dose is required to reach a significantly higher
showed that the GG genotype -1082 is significantly Tacrolimus dose-adjusted concentration. This is evi-
higher compared to the AA and AG genotypes. This dent during the early phase after transplantation.
correlation was independent of the polymorphisms at
positions -819 and -592. Later, studies found that the Multiple studies have demonstrated linkage dis-
G allele at position -1082 is the most important equilibrium between the polymorphism at position
genetic factor in the regulation of constitutive IL-10 -1082 in the IL-10 promoter area and other SNPs in
mRNA level, and is associatedwith a greater serum the same area including SNPs at positions -819 and
concentration (16, 17). Furthermore, an important -592, suggesting that the functional effects may be
relationship was noted between IL-10 and cyto- haplotype-dependent (28–30).
chrome P450 activity through an in vivo study that The current study shows that Tacrolimus adjust-
showed IL-10 to significantly decrease CYP3A activity ed concentration is sex-genotype-dependent in
(P ≤ 0.02) (18). Interestingly, a previous study con- Jordanian kidney transplant recipients during the first-
ducted among Jordanian kidney transplant recipients month post-transplantation at IL-10 -1082 A>G.
revealed a correlation between genetic variations in This effect was observed in the first-month post-trans-
both CYP3A4 and CYP3A5 enzymes and tacrolimus plantation in male patients carrying at least one A
blood levels among renal transplant recipients (19). allele who showed significantly lower DAC than male
In a previous study of liver transplant recipients, patients carrying the GG genotype. This reduction in
significantly higher Tacrolimus dose-adjusted concen- DAC disappeared after the first month. On the other
trations were measured in patients carrying -1082 AA hand, non of the mentioned parameters differed sig-
versus those carrying GG and GA during an interme- nificantly between different IL-10 -1082 A>G geno-
diate value within the first three weeks after transplan- type groups during the first six months post-transplant
tation (22). On the other hand, a Chinese study in the female patients.
demonstrated the impact of IL-10 gene polymor-
phism on Tacrolimus dosage requirement in 53 liver
transplant recipients and found no statistically signifi- Limitations
cant differences in Tacrolimus dose-adjusted concen-
The number of studied patients was small due to
tration among recipients. The same study revealed a
the long follow-up period of 6 months per patient. As
significantly higher Tacrolimus dose-adjusted concen-
well there were cases where data was missing due to
tration in recipients with donors with the -1082 AA
genotype than those whose donors with IL-10 -1082 the difficulty in interviewing patients, or loss of con-
GA genotype (25). tact with patients. Because of the small sample size,
we couldn’t detect rare mutations and their frequency
In a later study including 240 renal transplant impact on tacrolimus pharmacokinetic parameters.
recipients, IL-10 (-1082) variants did not show a sig- However, it should be noted that our sample size is
nificant relationship between Tacrolimus metabolism similar to other previously published studies that were
and -1082 genotypes within the first four weeks fol- close to (240) or even smaller (53) than the present
lowing transplantation (26). The current study did not study (22, 25, 26).
find a significant relationship between studied IL-10
SNPs among kidney transplant recipients and Acknowledgment. This research was supported
Tacrolimus pharmacokinetics parameters. Remarkably, by unconditional support by Yarmouk University
gender analysis revealed that males carrying at least (15/2017) and the University of Jordan/Deanship of
one A allele at IL-10 (-1082) had significantly lower Academic Research (469/2017). We gratefully thank
Tacrolimus dose-adjusted concentration than males the local research ethics committee of Royal Medical
carrying GG genotype in the first-month post-trans- Services and all the patients who agreed to partici-
plantation. We divided the patients according to their pate.
gender due to the differences in liver and renal func-
tion between males and females (27).
Our results can be explained by hypothesizing
that the -1082 GG allele is associated with increased All the authors declare that they have no conflict
IL-10 production (15–17), which leads to decreased of interest in this work.
J Med Biochem 2022; 41 (3) 333
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UDK 577.1 : 61 ISSN 1452-8258

ALKALINE PHOSPHATASE INTERFERENCE IN IMMUNO-ENZYMATIC ASSAYS

INTERFERENCIJA ALKALNE FOSFATAZE U IMUNO-ENZIMSKIM ANALIZAMA
Osman Oğuz1, Huriye Serin2, Fatma Sinem Hocaoglu3
1Department of Medical Biochemistry, Istanbul Education and Research Hospital, Istanbul, Turkey
2Department of Medical Biochemistry, Istanbul Education and Research Hospital, Istanbul, Turkey
3
Department of Clinical Biochemistry, Düzen Lab, Istanbul,Turkey

Background: Alkaline phosphatase (ALP) enzymes are Uvod: Enzimi alkalne fosfataze (ALP) se {iroko koriste kao
widely used as signal amplifiers in immunoenzymatic meth- poja~iva~i signala u imunoenzimskim metodama. Stanja
ods. Conditions that cause ALP elevations, such as bone or koja izazivaju povi{enje ALP, kao {to su bolesti kostiju ili
liver diseases, can cause interference in immunoenzymatic jetre, mogu izazvati smetnje u imunoenzimskim meto-
methods. We aimed to examine ALP’s effect on immu- dama. Na{ cilj je bio da ispitamo efekat ALP-a na
noenzymatic assay by adding isolated pure ALP to the pre- imunoenzimski test dodavanjem izolovanog ~istog ALP-a u
pared serum pool. pripremljenu grupu uzoraka seruma.
Methods: We prepared a serum pool and divided it into 4 Metode: Pripremili smo ve}i broj uzoraka seruma i podelili
groups. By adding isolated pure ALP at different concentra-
ih u 4 grupe. Dodavanjem izolovanog ~istog ALP u
tions to each group, we obtained sample groups containing
razli~itim koncentracijama svakoj grupi, dobili smo grupe
ALP enzyme at concentrations of 85 U/L, 340 U/L, 870
U/L, and 1570 U/L. 20-repetition of bhCG, ferritin, FT4, uzoraka koje sadrè ALP enzim u koncentracijama od 85
TSH, troponin I, and Vit B12 tests were performed in each U/L, 340 U/L, 870 U/L, i 1570 U/L. Ura|eno je 20 po-
group. The coefficient of variation, bias, and total error was navljanja u svakoj grupi testova za bhCG, feritin, FT4, TSH,
calculated. All groups were compared by using the troponin I, i Vit B12. Izra~unati su koeficijent varijacije, pris-
Friedman test for paired samples. trasnost i ukupna gre{ka. Sve grupe su upore|ene
Results: After ALP addition, the calculated total error val- kori{}enjem Fridmanovog testa za uparene uzorke.
ues of FT4, bhCG and troponin I tests were above the Rezultati: Nakon dodavanja ALP, izra~unate ukupne vred-
acceptable error limits. There were statistically significant nosti gre{ke testova FT4, bhCG i troponina I su bile iznad
differences in bhCG, FT4, troponin I, and Vit B12 tests prihvatljivih granica gre{ke. Postojale su statisti~ki zna~ajne
compared to the baseline ALP level (P<0.0125). razlike u testovima za bhCG, FT4, troponin I i Vit B12 u
Conclusions: Isolated ALP elevations can be a source of pore|enju sa osnovnim nivoom ALP (P<0,0125).
interference for immunoenzymatic methods. Zaklju~ak: Izolovana povi{enja ALP mogu biti razlog smet-
nji za sprovo|enje imunoenzimskih metoda
Keywords: Alkaline phosphatase, ALP, bias, immunoen-
zymatic, total error Klju~ne re~i: alkalna fosfataza, ALP, pristrasnost, imuno-
enzimski, ukupna gre{ka
Address for correspondence: List of abbreviations: ALP, Alkaline phosphatase; bhCG, beta
Osman Oğuz human chorionic gonadotropin; FT4, Free thyroxine; TSH, thy-
Department of Medical Biochemistry, Istanbul Education and roid-stimulating hormone; Vit B12, Cobalamin; TE, Total Error;
Research Hospital, Istanbul, Turkey CV, coefficient of variations; MEIA, microparticle immunoassay;
e-mail: osmanoguzzªgmail.com FIA, fluorometric immunoassay.
336 Oğuz et al.: Alkaline phosphatase interference in immuno-enzymatic assays
Introduction was reported that while the endogenous ALP had an

interfering effect on immunochemical methods in the
Alkaline phosphatases (ALP; orthophosphate
previous automated systems, this effect is prevented
mono-ester phosphohydrolase (alkaline optimum) EC
through the increased washing processes in the
3.1.3.1) are homodimeric and glycoprotein enzymes
renewed automated systems (4, 7).
in the hydrolase group with a molecular weight of 86
kilodaltons. These groups of enzymes are commonly In our laboratory, we use UniCelDxl 800 and
found in nature in both eukaryotes and most prokary- Access II (Beckman Coulter, Brea, CA) auto analyzers
otes. Each monomer is encoded by multiple genes to that measure by immunoenzymatic method and use
contain five cysteine residues, two zinc atoms and one ALP conjugates as signal amplifiers. Our purpose is to
magnesium atom, which are vitally important for its evaluate the interference caused by the ALP elevation
catalytic function. on these systems through beta human chorionic
gonadotropin (bhCG), ferritin, free thyroxine (FT4),
ALP enables the detachment of phosphate
thyroid-stimulating hormone (TSH), troponin I and
groups from a variety of molecules, including
nucleotides, proteins and alkaloids, in alkaline pH Cobalamin (Vit B12) tests.
environments (1).
ALP mainly functions as bound by hydrophobic Materials and Methods
glucosyl-phosphatidylinositol to the cell membrane
(2). It is mostly found in the canalicular membrane of We created a serum pool with 20 patients’ sera
hepatocytes and bile duct epithelium lumen, bone who have previously consulted our laboratory in
osteoblasts, brush border membrane of the intestinal January 2020 and whose ALP, bhCG, ferritin, FT4,
mucosa, placenta, proximal kidney tubules, and TSH, troponin I, and Vit B12 tests were found to be
breast tissue during lactation. A healthy human within the reference range.
serum contains four different ALP isoenzymes under The prepared serum pool was divided into four
normal conditions. These are Intestinal ALP, Placental groups. Commercially prepared isolated ALP (Toyoba
ALP, Germ cell ALP and tissue nonspecific alkaline Enzymes, Osaka, Japan), with an activity of
phosphatase. The difference between the isoenzymes 30,000,000 U/L, was added at different doses to the
stem from the sialic acid found in them at varying groups. Commercial ALP enzyme is in transparent liq-
rates, and also, the protein amount of the placental uid form, has grade 2 activity (30,000 U/mL or more)
isoenzyme is different (2, 3). and contains Adenosine deaminase and Phospho-
Serum ALP measurement plays a vital role in the diesterase. First of all, due to the enormous enzyme
diagnosis of hepatobiliary and bone diseases associ- activity, isolated ALP was diluted to a working stock by
ated with an increased osteoblastic activity. adding 2 mL of commercial ALP to 998 mL of distilled
Intrahepatic and extrahepatic cholestasis, space- water. We prepared a stock solution immediately
occupying lesions in the liver, metastasis and infiltra- before the assay. We added 5 mL, 10 mL and 20 mL of
tive liver diseases cause an increase in serum ALP lev- this stock solution, respectively, to the serum pools of
els (4). Apart from the liver diseases, serum ALP 8 mL each. We obtained four groups, one being our
levels elevate as a result of Paget’s disease (associat- reference group without ALP addition and the others
ed with increased osteoblastic activity), osteomalacia containing ALP at concentrations of 340 U/L, 870
and rickets associated with vitamin D deficiency, pri- U/L and 1590 U/L after adding isolated ALP.
mary and secondary hyperparathyroidism, and during
the healing process of bone fractures. Likewise, pla-
centa-induced ALP enzyme elevation is seen in the Biochemical analysis
third trimester of pregnancy and placental or germ The ALP was measured spectrophotometrically
cell malign diseases (4). with a Beckman Coulter AU 5800 (Beckman Coulter,
ALP is used to provide signal amplification by Brea, CA) auto-analyzer. Ferritin, FT4, TSH and Vit
conjugating it with antibodies in immunoenzymatic B12 tests were measured immunoenzymatically with
methods (4 – 6). ALP is frequently used in immuno- an UniCelDxl 800 (Beckman Coulter, Brea, CA) auto-
chemical methods along with Horseradish Peroxidase analyzer and bhCG and troponin I test with an Access
due to its substrate diversity, cheapness and accessi- II (Beckman Coulter, Brea, CA) auto-analyzer. All
ble possibility. Once conjugated with antibodies, anti- assays are two-site sandwich immunoassay using
gens and streptavidin, these enzymes increase the enzyme-conjugated antibodies with direct chemilumi-
test sensitivity owing to their low background effect, nometric technology. In all groups, each test was per-
linear reaction rate, and extended incubation time. formed with 20 repetitions. All measurements were
Using ALP as a signal amplifier provides an enhanced completed on the same day and within-run.
and longer-lasting lumination obtained at the end of
the reaction. Thus, the target analyte can be assayed
in lesser concentrations and much broader linearity. It
J Med Biochem 2022; 41 (3) 337
Statistical analysis Bonferroni correction to determine the adjusted sig-

nificance level as p<0.0125.
In the evaluation of the effect of the ALP
enzyme on the immunoenzymatic tests, Total error
(TE), Bias and coefficient of variations (CV) were cal-
Results
culated by 20 repetitions of each group.
CV, Bias, and TE values of each analyte are
TE was calculated by the following formula:
reported as separate groups in Table I. ALP interfer-
TE = │Bias % │+ 2CV ence was assessed by calculating CV, bias, and TE for
each analyte and comparing it to allowable total error
TE = total error
(9). Results for each sample pool are summarized in
CV = coefficient of variation. Table I. Figure 1 shows the TE values of bhCG,
Ferritin, FT4, troponin I, TSH and Vit B12 between
Bias was calculated as follows (8): the groups, while Figure 2 shows the calculated CV’s.
C2 -C1 After adding ALP, in bhCG, calculated TE values
Bias % =
( C1 ) x 100 at ALP concentrations of 340 U/L and 1590 U/L
were found to be above the acceptable error limits. In
the FT4 test, the calculated TE value at ALP concen-
C1 = mean value of reference group and C2 = trations of 340 U/L was found to be above the
mean value of groups added with ALP acceptable error limits. Calculated TE values were
found to be above the acceptable error limits in all
We applied the Friedman test to show the differ- groups for the troponin I test. We observed that the
ence between groups using Med Calc (MedCalc calculated CV values for the bhCG test increased with
Software Mariakerke, Belgium) software. We used

ALP (85 U/L) ALP (340 U/L) ALP (870 U/L) ALP (1590 U/L)
Mean Mean Mean
Test Name Mean (CV) Bias % TE Bias % TE Bias % TE aTE
(CV) (CV) (CV)
0.77 0.72 0.84
bHCG, IU/L 0.68 (3.29) 13.23 20.71 5.88 14.88 23.5 40.76 17.00
(3.74) (4.50) (8.63)
105.46 104.42 104.62
Ferritin, pmol/L 107.57 (2.55) -1.96 10.00 -2.92 8.56 -2.73 10.85 13.50
(4.02) (2.82) (4.06)
11.58 11.19 10.94
FT4, pmol/L 10.94 (4.88) 5.88 14.28 2.35 9.81 0.05 7.57 13.00
(4.2) (3.73) (3.76)
15.19 16.20 15.29
Troponin I, ng/L 19.78 (3.74) -23.2 35.34 -18.02 25.02 -22.6 33.18 20.00
(6.07) (3.5) (5.29)
2.46 2.44 2.40
TSH, mIU/L 2.43 (2.70) 6.99 10.83 0.40 4.10 -1.23 6.01 13.50
(1.92) (1.85) (2.39)
169.69 166.74 166.74
vitB12, pmol/L 160.10 (4.60) 5.99 15.43 4.14 13.74 4.14 11.82 25.00
(4.72) (4.80) (3.84)
ALP, alkaline phosphatase; aTE, allowable total error; CV, coefficient of variations; FT4, free thyroxine; TE, total error; TSH, thy-
roid-stimulating hormone; Vit B12, cobalamin; The values marked as bold indicate TE values that exceed the upper limit that rec-
ommended by Rili-BAEK (9).
Table II P-values of statistical association analysis by Friedman test with Bonferroni correction for comparison of ALP effects on
each test. P<0.0125 is considered statistically significant.
ALP (340 U/L) ALP (870 U/L) ALP (1570 U/L)
bHCG, IU/L P<0.0125 P<0.0125 P<0.0125
Ferritin, pmol/L p>0.0125 p>0.0125 p>0.0125
ALP (85 U/L) FT4, pmol/L P<0.0125 p>0.0125 p>0.0125
Troponin I, ng/L P<0.0125 P<0.0125 p>0.0125
TSH, mIU/L p>0.0125 p>0.0125 p>0.0125
vitB12, pmol/L P<0.0125 P<0.0125 P<0.0125
Figure 1 Total error distributions of βhCG, Ferritin, FT4, Troponin I, TSH and Vit B12 tests at different ALP concentrations.
Groups containing different ALP concentrations are shown on the x-axis and total error values on the y-axis.
Figure 2 Coefficient of variations of each test at different ALP concentrations. Groups containing different ALP concentrations
are shown on the x-axis, and the calculated coefficient of variations on the y-axis.
J Med Biochem 2022; 41 (3) 339
increasing ALP concentrations. In the troponin I test, nents of the system are pipetting, washing, and
calculated CV’s at ALP concentrations of 340 U/L checking the luminometer. If system updates and
and 1590 U/L were found to be higher than the weekly maintenance are skipped, and the washing
group without ALP added. performance of the autoanalyzer is not working effi-
ciently, high ALP values may cause interference.
Table II shows the p values obtained by compar-
ing the groups with different ALP concentrations with In a way similar to our study, Dasguptaet et al.
the group containing baseline ALP. We observed that (11) evaluated ALP interference on troponin I
there were statistically significant differences in all assayed by microparticle immunoassay (MEIA) and
groups for bhCG and Vit B12, in the concentration of fluorometric immunoassay (FIA). They did not
ALP 340 U/L for FT4, and in concentrations of 340 observe ALP interference in the MEIA method, while
and 870 U/L for troponin I when compared to the they observed that interference increased with the
baseline ALP level (P<0.0125). There were no statis- elevation of ALP enzyme concentrations in the FIA
tically significant differences in ferritin and TSH method (11). Similarly, Butch et al. (12) demonstrat-
among the groups. ed the interference of endogenous ALP on troponin I
measurement by the FIA method. They evaluated and
reported that the reason for this interference may be
Discussion related to the washing performance of the system
they use; they contacted the device manufacturers
In our study, ALP interference was observed in and reported that the interference was reduced by
immunoenzymatic assays for bhCG, FT4, troponin I improving the washing steps (12).
and Vit B12 tests. We observed that TE and CV values
increased after ALP addition, especially in troponin I Similar to our study, Marinheiro et al. (13) com-
and bhCG tests. Likewise, Sofronescu et al. (10) pared two troponin I methods using ALP as conjugate
observed that their patient who used ALP enzyme (Beckman Coulter Access AccuTnI+3®) and acridini-
externally for treatment had low Total testosterone um as conjugate (Abbott Architect STAT high sensi-
levels than usual. They decided to measure total tive TnI®). They reported that troponin I was falsely
testosterone by liquid chromatography-mass spec- higher in the method which uses ALP. Interestingly,
trometry for comparison and found the result in the like in our case, the ALP level was normal in their case
normal range. They observed negative interference report. They concluded that endogenous ALP may
as we observed in the troponin I test. They concluded interfere with the assay by interacting with micropar-
that ALP could potentially interact and cause interfer- ticles even if it is in the normal reference range (13).
ence after binding the antibody (10). There are some While preparing the study plan, we had to deter-
similarities between their study and ours. They used mine the final ALP concentration that we would
the same auto-analyzer and manufacturer’s kit as us. reach. The linearity upper limit of the system that we
But they calculated neither TE nor CV. At the end of used (Beckman AU5800) was set at 1500 U/L for the
their study, they mentioned inadequate washing of ALP test. Nargis et al. (14) analyzed patients with per-
the unbound analyte could also lead to false results sistent ALP elevations in their study. They have report-
(10). Similarly, Herman et al. (5) reported that bhCG ed that in the population they studied, ALP values
and troponin I measurements were found to be incor- were above 3000 U/L in only 3% of the patient group
rectly high as a result of improper washing steps of (14). So, we decided to stay within the values that we
samples containing elevated ALP. Herman et al. (5) might encounter clinically in the daily workflow.
reported that this effect was seen especially above Although we could elevate the ALP levels to much
ALP> 1000 U/L concentrations. higher levels by adding ALP externally, we preferred
We observed that ALP addition could cause to keep our upper limit within the linearity limits of the
interference on bhCG, FT4, troponin I and Vit B12 assay as we did not want to exceed the general
tests. While Herman et al. (5) found an erroneous patient population.
elevation in both of the tests after the addition of ALP, The interference effect of the presence of het-
we found an erroneous elevation in the bhCG test erophilic antibodies on the test for immunoenzymatic
and an erroneously low reading in the troponin I test. methods is reported as a generally erroneous test
We used Access II auto-analyzer for these tests, while result (15). One of the limitations of our study is that
Herman et al. (5) used DXI-800 auto-analyzer. we did not examine the samples used in the serum
Although Access II and DXi-800 use the same kits pool for the presence of heterophilic antibodies. If
and calibrators and are manufactured by the same some of the selected samples had contained het-
manufacturer, they are systems whose operation per- erophilic antibody would have possibly affected our
formances are different from each other. These sys- entire pool. To avoid this, we could have used het-
tems perform the washing process in three cycles by erophilic antibody inhibitors. However, since we did
using special washing solutions and eliminate any not want to create a different interference source by
unbound molecules from the medium by creating a using a heterophilic antibody inhibiting tube, we
magnetic field using a magnet. Three critical compo- ignored the presence of heterophilic antibodies.
Other studies investigating ALP interference are troponin I could be affected by high ALP levels. It
often presented as case reports and are not statistical- would be beneficial to repeat the bhCG and troponin
ly strong. They tried to understand and show the I tests with DXI-800. There is a need for repeating
interference through a patient. Our study design was the study with samples free of any heterophilic anti-
structured very well, and we compared our results to bodies and with samples containing higher rates of
the reported guideline.
ALP.
Based on the findings obtained from our study,
we determined that elevated ALP caused interference
on bhCG, FT4, troponin I and Vit B12 tests, but it did Conflict of interest statement
not cause a significant interference on Ferritin and
TSH tests. Especially in terms of misdiagnosing All the authors declare that they have no conflict
myocardial infarction, it should be considered that of interest in this work.
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Received: September 10, 2021

UDK 577.1 : 61 ISSN 1452-8258

CLINICAL EVALUATION OF NON-INVASIVE PRENATAL SCREENING IN 32,394

PREGNANCIES FROM CHANGZHI MATERNAL AND CHILD HEALTH CARE
HOSPITAL OF SHANXI CHINA
KLINI^KA PROCENA NE-INVAZIVNOG SKRININGA U 32.394 TRUDNICE
U CHANGZHI PORODILI[TU I DEÎJOJ BOLNICI U SHANXI U KINI
XiaoZe Li, LiHong Wang, ZeRong Yao, FangYing Ruan, ZhiPeng Hu, WenXia Song
Department of Medical Genetic, Changzhi Maternal and Child Health Care Hospital Affiliated
Hospital of Changzhi Medical College, Changzhi City, Shanxi Province, 046000, China

Background: Non-invasive prenatal screening (NIPS) is a Uvod: Neinvazivni prenatalni skrining (NIPS) je veoma
highly sensitive and specific screening test to detect fetal osetljiv i specifi~an skrining test za otkrivanje fetalnih hromo-
chromosomal abnormalities. The primary objective of this zomskih abnormalnosti. Primarni cilj ove studije bio je da se
study was to evaluate the NIPS as an effective method for proceni NIPS kao efikasan metod za prenatalno otkrivanje
prenatal detection of aneuploidies in both high-risk and aneuploidije u trudno}ama visokog i niskog rizika.
low-risk pregnancies. Metode: U trenutnoj studiji, NIPS smo uradili u 32.394 trud-
Methods: In current study, we performed NIPS in 32,394 no}e, od kojih su rezultati bili dostupni u 32.361 (99,9%)
pregnancies, out of which results were available in 32,361 trudno}a. Illumina sekvenciranje je izvr{eno za NIPS skrining.
(99.9%) of them. Illumina sequencing was performed for Z test hipoteze je kori{}en za klasifikaciju fetalne autozomne
NIPS screening. Hypothesis Z test was used to classify fetal aneuploidije T21, T18 i T13. Kariotipizacija je ura|ena da bi
autosomal aneuploidy of T21, T18, and T13. Karyotyping se utvrdili pravi negativni i istinski pozitivni rezultati NIPS.
was performed to determine the true negative and true Rezultati: Me|u 32.361 potvr|enim uzorkom, 164 slu~aja
positive NIPS results. je imalo pozitivne rezultate, a 32.197 slu~ajeva je imalo ne-
Results: Among the 32,361 confirmed samples, 164 cases gativne rezultate. Od ovih pozitivnih slu~ajeva, 116 slu~ajeva
had positive results and 32197 cases had negative results. je bilo trizomija 21,34 slu~aja trizomija 18 i 14 slu~ajeva tri-
Of these positive cases, 116 cases were trisomy 21, 34 zomija 13. Nisu prona|eni la`no negativni rezultati u ovoj
cases were trisomy 18 and 14 cases were trisomy 13. No kohorti. Ukupna osetljivost i specifi~nost bile su 100% i
false negative results were found in this cohort. The overall 99,91%, respektivno. Nije bilo zna~ajne razlike u perfor-
sensitivity and specificity were 100% and 99.91%, respec- mansama testa izme|u 7.316 visokorizi~nih i 25.045 trud-
tively. There was no significant difference in test perform- no}a sa niskim rizikom, (osetljivost, 100% naspram 100%
ance between the 7,316 high-risk and 25,045 low-risk (P>0,05); specifi~nost, 99,96% naspram 99,95% (P >
pregnancies, (sensitivity, 100% vs 100% (P>0.05); speci- 0,05)). Faktori koji su doprineli la`no pozitivnim rezultatima
ficity, 99.96% vs 99.95% (P > 0.05)). Factors contributing uklju~ivali su varijante broja kopija fetusa (CNV), fetalni
to false-positive results included fetal copy number variants mozaicizam i tipi~no stvaranje Z rezultata izme|u 3 i 4.
(CNVs), fetal mosaicism and typically producing Z scores [tavi{e, analizirali smo sekvenciranje celog genoma NIPS da
between 3 and 4. Moreover, we analyzed NIPS whole- bismo istraìli povezanost polimorfizama jednog nukleotida

WenXia Song, Department of Medical Genetic,
ChangZhi Maternal and Child Health Care Hospital Affiliated
Hospital of ChangZhi Medical College, 48 Weiyuanmen Middle
Road, Luzhou District, ChangZhi City, ShanXi Province,
046000, China
e-mail: songwenxia.loveª163.com
342 Li et al.: NIPS in 32,394 pregnancies from of Shanxi China
genome sequencing to investigate the Single Nucleotide (SNP) sa odgovorom na lek. ili rizik od bolesti. U pore|enju
Polymorphisms (SNPs) associations with drug response or sa podacima o genomu isto~ne Azije od 1000 g, rezultati su
risk of disease. As compare to the 1000g East Asian otkrili zna~ajnu razliku u 7.285.418 varijanti SNP-a kod trud-
genome data, the results revealed a significant difference nica u Shanki-u, uklju~uju}i 19.293 zabeleène varijante
in 7,285,418 SNPs variants of Shanxi pregnant women klinvara i 7.266.125 zabeleènih ne-clinvara.
including 19,293 clinvar recorded variants and 7,266,125 Zaklju~ak: Na{i nalazi su pokazali da je NIPS bio efikasan
non-clinvar recorded. test koji se moè primeniti kao rutinski skrining za fetalne tri-
Conclusions: Our findings showed that NIPS was an effec- somije u prenatalnom okruènju. Pored toga, ova studija
tive assay that may be applied as routine screening for fetal tako|e pruà ta~nu procenu zna~ajnih razlika u 7.285.418
trisomies in the prenatal setting. In addition, this study also varijanti SNP-a kod trudnica u Shanki-u koje su ranije bile
provides an accurate assessment of significant differences nedostupne klini~arima u populaciji Shanki-a.
in 7,285,418 SNPs variants in Shanxi pregnant women
that were previously unavailable to clinicians in Shanxi pop- Klju~ne re~i: NIPS, trizomija, la`no pozitivan, istinski po-
ulation. zitivan, Z skor, SNP varijante
Keywords: NIPS, trisomy, false positive, true positive, Z

score, SNPs variants
Introduction high-risk and low-risk pregnancies. Moreover, we also

investigated the SNPs induced risk of disease based
China has the largest number of birth defects in on 32,394 samples obtained from the Changzhi
the world, with about 900,000 new cases of birth Maternal and Child Health Care Hospital, and SNPs
defects every year (1), including about 240,000 induced variation in drug response.
cases of chromosomal abnormalities, most of which
cannot be cured till today (2). In 1997, Lo et al. (3)
discovered the presence of fetal cell free DNA in
maternal peripheral (also named cfDNA). The cfDNA
in maternal plasma grows in concentration with the Study design
gestational age, and it also harbors genomic informa-
This study was approved by the institutional
tion of the fetus. When a fetus has an abnormal num-
review board of the Changzhi Maternal and Child
ber of chromosomes (aneuploidy), the cfDNA ratio
Health Care Hospital. This was a prospective, large-
regarding that chromosome will be altered (4).
scale, blinded cohort study conducted from 11
However, the concentration of cfDNA in the maternal
October 2016 to 26 June 2019. Only the patients
plasma circulation may vary widely (5, 6), ranging
with written consent were included in this study. The
from 4% to over 30% (7). Thus, advanced technolo-
inclusion criteria for participants were: pregnant
gies such as digital polymerase chain reaction or mas-
women, at least 18 years old, and a gestational age
sively parallel sequencing have been used to study
of 11 to 30 weeks. The women who were excluded
cfDNA in maternal blood, differentiate fetal DNA
included those: (i) who had recent stem cell therapy
from maternal DNA, and detect fetal chromosomal
or immune therapy; (ii) who had cancer diagnosed;
abnormalities (8). These discoveries made non-inva-
(iii) who had known chromosome abnormalities or
sive prenatal screening (NIPS) of fetal aneuploidies a
whose partners had known aneuploidy.
clinical reality and led to a new era of non-invasive
prenatal screening with high sensitivity and specifici-
ty in multiple clinical centers (9, 10). NIPS had been
NIPS screening
commercialized in China since May 2010, and the
clinicians showed strong interest and attempted to Five milliliter peripheral blood of pregnant
adopt the technology for detection of fetal aneuploi- women was collected by EDTA anticoagulant tube
dies (11). Find Gene NIPS is one of the earliest NIPS and delivered to the laboratory within 6 hr. of collec-
assays developed in China. In November 2019, based tion. The plasma was isolated from the peripheral
on retrospective clinical trials conducted in multiple blood by two-step centrifugation including at 1,600
clinical centers, FindGene NIPS got approval from × g for 10 min and then at 16,000 ×g for 10 min at
the National Medical Products Administration for 4 °C. After that the samples were frozen and delivered
screening fetal trisomies (T) 21, 18, and 13 in China. to Shanghai Findgene clinical laboratories (Shanghai,
Moreover, sufficiently large NIPS samples also indi- China) at which plasma was prepared for library con-
cate population genetics databases such as SNPs and struction, quality control and pooling. The plasma
allele frequency, genomic locations, and functional DNA was extracted from 600 mL plasma of each
annotations. Single Nucleotide Polymorphisms (SNPs) sample using Circulating Nucleic Acid Kit from
are known to contribute to variation in various dis- Findgene (Chengdu Findgene Medical Equipment
eases and drugs responses. The primary objective of Co., Ltd). The detailed general technical procedure
this study was to evaluate the NIPS as an effective was described previously (12). The prepared library
method for prenatal detection of aneuploidies in both was quantified using real time PCR, 96 barcoded
J Med Biochem 2022; 41 (3) 343
libraries which were titrated and evenly pooled and Statistical analysis
sequenced with 36-cycles single-end multiplex
Statistical analysis between the different groups
sequencing strategy on an Illumina Next Seq. 500
was performed using a chi square test or Fisher’s
platform (Illumina, USA) (13). exact test, and P values of <0.05 were considered
Hypothesis Z test was used to classify fetal auto- statistically significant.
somal aneuploidy of T21, T18, and T13. An
approach integrating dynamic GC reference library
(14) and LOESS (locally weighted scatterplot smooth- Results
ing) regression (15) was applied to correct the GC NIPS failure rate
bias before Z test. Integrating maternal copy number
variant (CNV) and fetal fraction correction (2) were Between 11 October 2016 and 26 June 2019,
applied to correct the Z score after Z test. The a total of 32,394 maternal blood samples were
sequences of each sample were mapped to the refer- obtained for NIPS screening at Findgene from the
ence genome of human and Z scores were calculated Changzhi Maternal and Child Health Care Hospital in
for each chromosome (16, 17). Z  3 would repre- Shanxi, China. Out of these samples, 33 cases yield-
sent a chromosomal aneuploidy and -3.0 < Z < 3.0 ed no NIPS results including 24 cases of haemolysis
would represent a chromosomal euploid. Fetal frac- and 9 cases of cancellation. The rate of NIPS failure
tion of male fetuses was calculated based on the Y was 0.1% (33/32394).
chromosome fraction. Re-sequencing, retesting and
resampling was applied when samples did not meet
quality control (QC) standards. Re-sequencing was Demographic characteristics of pregnant women
performed on samples with insufficient unique reads undergoing non-invasive prenatal screening
only. Common reasons for retested sample included (NIPS) for aneuploidies
insufficient cfDNA content after library preparation The demographic characteristics of the remaining
quantification and unsuitable GC content to the refer- 32,361 samples are shown in Table I. Data from a total
ence or Z score chaos (Z  3 or Z  -3) for more than of 32,361 cases were included in this study, which
five chromosomes. Samples with hemolysis, gesta- mainly consisted of women from Shanxi province,
tional age of less than 12 weeks, insufficient fetal China. The mean maternal age was 31 years, with
fraction, and repeating Z score chaos would be
resampled. Only one chance of resampling was
allowed. Table I Demographic characteristics of pregnant women
undergoing NIPS for aneuploidies between 11 October 2016
and 26 June 2019.
Clinical follow-up Maternal age
Patients with negative NIPS screening results Mean age (year) 31
were advised for regular prenatal care; genetic coun- Advanced maternal age > = 35 7,316 (22.8%)
seling was provided if routine ultrasound examination Pregnancies with the age < 35 25,045 (77.2%)
showed abnormalities. Karyotyping was performed
for all NIPS screening samples. On the basis of kary- 18–20 year 89
otyping a negative NIPS result was considered as a 20–24 year 4,012
true negative if the prenatal or neonatal karyotyping 25–29 year 12,260
results were normal, or if the neonate looked pheno-
30–34 year 8,684
typically normal. By contrast, a negative NIPS result
was defined as a false negative if the prenatal or 35– 40 (year) 6,272
neonatal showed aneuploidy in karyotyping, or if the >40 (year) 1,044
newborn was phenotypically abnormal. Pregnant In Vitro Fertilization sample 549
women with positive NIPS results were recommended
Twin samples 875
for confirmatory invasive prenatal diagnosis like
amniocentesis. The positive NIPS result was defined Single pregnant sample 31,486
as true positive upon karyotyping invasive confirma- Gestational age at blood sampling
tion or clinical follow-up results. The false positive was Median (week) 18.5
defined as high risk trisomy report in a case that was
subsequently shown to be without aneuploidy upon Range (week) 11–30
invasive diagnostic confirmation or clinical physical 11 to 15 weeks (n, %) 1,060 (3.3%)
examination. Patients without confirmatory diagnostic 16 to 20 weeks (n, %) 25,651 (79.2%)
results were excluded from calculation of test param-
20 to 25 weeks (n, %) 4,819 (15%)
eters (sensitivity, specificity, positive perspective value
(PPV), and negative perspective value (NPV)). 26-30 weeks (n, %) 831 (2.5%)
Table II Efficiency of NIPS for T21/T18/T13. Table III Performance of non-invasive prenatal screening
(NIPS) in high-risk pregnancies and low-risk pregnancies.
Trisomies TP FP FN Sensitivity Specificity PPV NPV
Category Verified TP FP Sensitivity Specificity
T21 65 17 0 100% 99.95% 79.27% 100% T13 (<35yrs) 3 2 1 100% 99.99%
T18 (<35yrs) 13 10 3 100% 99.99%
T18 17 8 0 100% 99.98% 68% 100%
T21 (<35yrs) 56 44 13 100% 99.96%
T13 4 4 0 100% 99.99% 50% 100% T13 (>=35yrs) 5 2 3 100% 99.99%
T18 (>=35yrs) 11 7 4 100% 99.99%
Total 86 29 0 100% 99.91% 74.78% 100%
T21 (>=35yrs) 25 21 5 100% 99.98%
TP = true positive, FP = false positive, NIPS = noninvasive pre- Low-risk
natal screening, PPV = positive predictive value, NPV= Negative pregnancies 73 56 17 100% 99.95%
predictive value. (<35yrs)
High-risk
22.8% maternal age  35 years upon delivery. In this pregnancies 41 29 12 100% 99.96%
cohort, 31,486 cases were single pregnant, 875 cases (>=35yrs)
were twins and 549 cases were in-vitro fertilization TP = true positive, FP = false positive, NIPS = noninvasive pre-
(IVF). The gestational age (GA) ranges from 11 to natal screening, T13 = trisomy 13, T18 = trisomy 18, T21 =
30 weeks, with a mean GA of 18 weeks. trisomy 21.
Z score and gray zone

Efficiency of NIPS for T21/T18/T13
Out of 29 false positive cases, 20 cases (69%)
Among the 32,361 confirmed cases who had Z score with 3 < Z < 4 and 9 cases (31%) had
obtained NIPS results, 164 were positive and Z score > 4. In contrast, 85 cases got true positive
32,197cases were negative (Table I). Pregnancies results of NIPT, 80 (95%) cases got Z > 4, and only
with the NIPS positive results were recommended for 5 cases (5%) cases got Z score ranging from 3 to 4.
confirmatory invasive prenatal diagnosis using chro- These results indicated that Z score between 3 and 4
mosomal microarray analysis. After informed con- represented a gray zone where most false positive
sent, only 114 cases agreed to go through the confir- cases resided.
matory invasive prenatal diagnosis while, 50 cases
declined for further diagnosis. Table II shows the pre-
natal diagnosis results. The NIPS sensitive, specificity, Follow-up investigation of test negative cases
positive predictive values and negative predictive val- At the time of writing, all pregnant women
ues were 100%, 99.91%, 74.78%, 100% respectively. included in this series had given birth. To date, among
32,197 NIPS negative cases, no abnormalities were
reported through our feedback mechanism.
Efficiency of NIPS in high-risk pregnancies and
low-risk pregnancies
In present study, the women whose age  35 Characteristics of single-nucleotide polymor-
(high-risk pregnancies) years old were 7,316 cases. phisms (SNPs) in the Shanxi pregnant genome
Among 7,316 advanced maternal age women, 29 As compared to 1000 g Eastern Asian popula-
cases were the NIPS positive and 12 cases were NIPS tion there were significant difference alleles frequen-
false positive results of T21/T18/T13 (Table II). Of all cies in 7,285,418 SNPs variants (p<0.05) (Supple-
25,045 cases of the women whose age < 35 (low mentary 1). Out of these SNPs variants, 19, 293 SNPs
risk pregnancies), there were 56 cases with NIPS pos- variants were recorded by clinivarhttps://www.ncbi.
itive results and 17 cases with false positive results. nlm.nih.gov/clinvar/including 17,167 benign variants,
False negative cases were not found after at least 10 67 pathogenic variants, 5 affects variants, 82 associat-
months of follow-up. There was no significant differed variants, 408 not provided/other variants, 8 protec-
ence in test performance between the 7,316 high- tive variants, 85 risk factor variants, 977 uncertain sig-
risk and 25,045 low-risk subjects (sensitivity, 100% nificance variants, 387 conflicting interpretation of
vs. 100% (P >0.05); specificity, 99.96% vs. 99.95% pathogenicity, and 107 drug response variants.
(P > 0.05)). These results suggested that the applica-
tion of NIPS could significantly reduce the cost of
invasive prenatal diagnosis, which had a positive yield Discussion
of only 0.56% (41/7,316) in high-risk group and
0.29% (73/25,045) in low-risk group. In the past few years, NIPS has been widely used
as a powerful screening tool to detect the chromoso-
mal aneuploidies such as T21, T18, and T13 (18).
J Med Biochem 2022; 41 (3) 345
Table IV The characteristics of Z score in the false and true other studies, ranging from 1.6 to 3.2 % (23, 28).
positive cases. This was because this study included a large number
Z score T21 T18 T13 Total of low-risk pregnancies (25,046 cases (77.3%)) for
False
NIPS with a mean maternal age of 31 years. Out of
3<Z<4 14 3/7 3/4 20/29 (69%) the 7,316 advanced maternally aged women, there
positive
(n=29) Z>4 4 4/7 1/4 9/29 (31%) were only 29 patients (0.39%) showing positive
True results; Among 25,046 low-risk cases (<35 years),
3<Z<4 4 1 0 5/85 (5%)
positive there were 55 cases (0.22%) showing positive results.
(n=85) Z>4 60 16 4 80/85 (95%) Although NIPS was often recommended for maternal
T13 = trisomy 13, T18 = trisomy 18, T21 = trisomy 21. ages above 35, we demonstrated its efficacy in low-
risk pregnancies. It could significantly reduce the cost
This prospective study was performed to evaluate the of invasive prenatal diagnosis, many of which would
efficiency of NIPS in the Changzhi Maternal and Child be unnecessary. In fact, in this study, application of
Health Care Hospital. The results indicated a signifi- NIPS significantly reduced the rate of invasive prena-
cant difference in 7,285,418 SNPs variants of Shanxi tal diagnosis to 0.56% (41/7316) in high-risk group
pregnant women including 19,293 clinvar and and 0.29% (73/25046) in low-risk group (Table III).
7,266,125 nonclinvar recorded variants. Our data Surprisingly, this study found 44/64 cases (69%) of
also showed that the sensitivity, specificity, PPV and Down syndrome coming from the low-risk pregnan-
NPV were 100%, 99.91%, 74.56% and 100% cies (Table III), this highlighted the value of NIPS in
respectively, which were very competitive compared this often-overlooked subgroup.
to earlier reports (19–21). When compared to studies
in high-risk pregnancies, our results were comparable
to those of Qi et al. (22) and Hu et al. (23), showing Conclusion
that the efficacy of NIPS screening was consistent
from low-risk to high-risk pregnancies. In conclusion, this study showed that NIPS was an
effective method for prenatal detection of aneuploidies
In parallel, false positives were analyzed in (29). It showed comparable results when applied to
detail. Some possible reasons of false positives in both high-risk and low-risk pregnancies, and was able
NIPS had been reported in literature, including low Z to provide valuable information for more cost-effective
scores, fetal pathogenic CNVs and placental utilization of invasive diagnostic methods. Although our
mosaicism. Bianchi et al. (24) demonstrated that a
study provides an authentication of NIPS as effective
Z-score between 2.5 and 4 should be considered as
method to detect the prenatal detection of aneuploi-
a borderline value (24), and false-positives were likely
dies on the basis of being highly sensitivity and speci-
to occur at borderline Z scores (25). Indeed, this
study indicated that 20 NIPS positive cases (80%) ficity with lower false-positive rates, but still there are
with Z score from 3 to 4 were later confirmed to be some limitations to NIPS and our study. Although NIPS
false positives (Table IV). The algorithm of NIPS is Z has high sensitivity and specificity, the overall specificity
test with the standard normal distribution. If the true and sensitivity is not uniform for all chromosomes
negative is judged to be negative by Z 3 (99.87%), because of the variation in GC content of sequences.
the probability of false positive would be 0.13%. Another limitation is that selected patients for NIPS
Theoretically, the probability of false positives of may not reflect a general obstetrical population. Also,
0.13% can be accepted. We also developed an algo- our study didn’t include the abnormalities determined
rithm to exclude the effect of maternal CNV and using ultrasound in comparison to NIPS. Further
refined the Z-score that can determine fetal aneu- studies are needed to overcome these issues to
ploidy. However, biological factors such as fetal path- narrower the gap between diagnosis and treatment.
ogenic CNV played an important role in causing the
false positives (26). This study indicated that 2 cases
of false positives were related to fetal pathogenic Data Availability
CNV, including a case T13 for 1450KB amplifica- The data used to support the findings of this
tion at 13q12.12 and a case T21 for amplification at study are available from the corresponding author
21B, 21I points. Fetal mosaicism had also been upon request.
reported to cause false results (27). Moreover, our
data also showed that 2 cases of false positive were Funding Statement. This research received no
fetal mosaicism by invasive diagnostic confirmation. external funding.
These factors must therefore be taken into account Acknowledgment. The authors thank all collab-
when interpreting NIPS results, and post-test genetic orating medical centers, patients and their families.
counseling should be provided to pregnant women
following recommendations, such as those of the
National Society of Genetic Counselors’ statement. Conflict of interest statement
In present study, the percentage of positive NIPS All the authors declare that they have no conflict
was 0.51%, which was lower than those reported in of interest in this work.
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UDK 577.1 : 61 ISSN 1452-8258
FOCUS-PDCA CAN EFFECTIVELY OPTIMIZE

THE CRITICAL VALUE OF TEST ITEMS
FOCUS-PDCA MO@E EFIKASNO DA OPTIMIZUJE KRITI^NU VREDNOST ISPITIVANJA
Chunbao Xie1, Jianbo Zhang1, Jiangrong Luo2, Meiling Jian3, Taiqiang Zhao3,
Jiaqiang Wang3, Linxi Jiang4, Chao Dai4, Yao Wei4, Li Jiang3, Yi Shi4
1Departmentof Laboratory Medicine, Sichuan Provincial People’s Hospital,
University of Electronic Science and Technology of China, Chengdu 611731, China
2Department of Operation Management, Sichuan Provincial People’s Hospital,
University of Electronic Science and Technology of China, Chengdu 610072, China
3Department of Laboratory Medicine, Sichuan Academy of Medical Sciences
& Sichuan Provincial People’s Hospital, Chengdu 610072, China
4Sichuan Provincial Key Laboratory for Human Disease Gene Study and Institute of Laboratory Medicine,
Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China,
Chengdu 610072, China

Background: To optimize the critical value of test items Uvod: Svrha rada je optimizacija kriti~ne vrednosti ispitivanja
using FOCUS-PDCA (find, organize, clarify, understand, pomo}u FOCUS-PDCA (prona}i, organizovati, razjasniti,
select, plan, do, check and act), and to set the personalized razumeti, izabrati, planirati, uraditi, proveriti i delovati), i
critical value of the test for different departments. postaviti personalizovanu kriti~nu vrednost testa za razli~ita
Methods: We searched for literature reporting on the criti- odeljenja.
cal value and FOCUS-PDCA published over recent 5 years Metode: Traìli smo literaturu koja izve{tava o kriti~noj vred-
in order to understand the significance and status quo of nosti i FOCUS-PDCA objavljenoj u poslednjih 5 godina da
critical value and FOCUS-PDCA. We also collected and bismo razumeli zna~aj i status kriti~ne vrednosti i FOCUS-
analyzed the critical value data of hospital tests performed PDCA. Tako|e smo prikupili i analizirali podatke kriti~ne
in Sichuan province hospitals in 2019, which were later vrednosti bolni~kih testova obavljenih u bolnicama provincije
compared to data from 2020 to determine the FOCUS- Se~uan 2019. godine, koji su kasnije upore|eni sa poda-
PDCA cycle. cima iz 2020. da bismo odredili ciklus FOCUS-PDCA.
Results: The proportion of critical values in the whole hos- Rezultati: Udeo kriti~nih vrednosti u celoj bolnici smanjen je
pital decreased from 3.5% before optimization to 2.5% to sa 3,5% pre optimizacije na 2,5% do 3% nakon optimizacije.
3% after optimization. The critical values of ICU, hematol- Kriti~ne vrednosti odeljenja intenzivne intenzivne nege,
ogy, nephrology, urology, and neonatal departments after hematologije, nefrologije, urologije i neonatalnog odeljenja
optimization significantly decreased compared with those posle optimizacije su zna~ajno smanjene u odnosu na one
before optimization, while the critical values of cardiac sur- pre optimizacije, dok kriti~ne vrednosti kardiohirurgije,

Yi Shi
e-mail: shiyi1614ª126.com Research funding: This work was supported by the National
Li Jiang Natural Science Foundation of China (81870683,
e-mail: jianglijykªmed.uestc.edu.cn 82121003), the Department of Science and Technology of
Address: 32, West Section 2, 1st Ring Road, Chengdu, Sichuan Province (2020JDTD0028), the CAMS Innovation
Sichuan 610072, China Fund for Medical Sciences (2019-12M-5-032).
348 Xie et al.: Optimize the critical value
gery, emergency ICU, cardiology, and neurosurgery ICU urgentne intenzivne nege, kardiologije i neurohirurgije ICU
showed no significant difference before and after optimiza- nisu pokazale zna~ajnu razliku pre i posle optimizacije.
tion. Contrary, the critical values of the infection depart- Naprotiv, kriti~ne vrednosti infektivnog odeljenja nakon opti-
ment after optimization significantly increased before optimizacije su zna~ajno porasle pre optimizacije.
mization. Zaklju~ak: FOCUS-PDCA moè efikasno optimizovati
Conclusions: FOCUS-PDCA can effectively optimize the kriti~nu vrednost ispitivanja, {to je korisno za racionalno
critical value of test items, which is beneficial for rational kori{}enje medicinskih resursa.
utilization of medical resources.
Klju~ne re~i: kriti~na vrednost, optimizacija, FOCUS-
Keywords: critical value, optimization, FOCUS-PDCA PDCA
Introduction Methods
The term critical value was first proposed by Literature Research
Lundberg in 1972 (1), referring to the laboratory test
We searched for literature reporting on the criti-
value that is life-threatening to a patient without time-
cal value and FOCUS-PDCA published over recent 5
ly clinical intervention (2). The item with critical value
years in order to understand the significance and sta-
is called critical value item, while the critical value
tus quo of critical value and FOCUS-PDCA. Then, we
threshold or critical value boundary is called critical
collected and analyzed the critical value data of hos-
value reporting limit, which refers to the analyte-spe-
pital tests performed in Sichuan province hospitals in
cific set limits that define a test result as a »critical
2019, which was later compared to data from
value (3). The concept of critical values was
2020to determine the FOCUS-PDCA cycle.
endorsed by many countries including China. For
example, in 2012 China developed a »medical labo-
ratory quality and criteria for recognition, »which
Find Improvement Items
requires that the critical value of clinical laboratory
represents a standardized reporting system (4); yet, at The following data were then analyzed: clinical
present, no unified critical value of the project and the laboratory specimen, critical value of specimen. After
threshold value has been proposed (5). the critical value ratio of the whole hospital and all
departments in each month was analyzed, the critical
Optimizing the critical value reporting process
value ratio of some departments was too high.
and improving the critical value reporting rate and
timely rate of critical value reporting have been Taking the optimization of critical value as the
explored worldwide. PDCA is a popular iterative goal of this improvement project, we expected the
methodology that can fix a problem or improve a project target to be »SMART«, i.e., the target belongs
process and reduce the failure rate of critical value in to the specific field of »critical value management«.
the laboratory department (6–7). The four processes The proportion of critical value can be used to meas-
of the PDCA cycle (Plan-Do-Check-Act) are not com- ure the target situation.
pleted once after running and are carried out repeat-
edly. Some researchers have applied the PDCA cycle
method to test critical value management, effectively Organize Improvement Team
reducing the return time of critical value manage-
An improvement group, which was set up
ment and medical intervention and improving the
according to the optimized critical value, included
critical value registration rate and the qualified rate of
those affected by the excessively high proportion of
registration (8).
critical value and those who will be affected by the
FOCUS-PDCA is a novel management mode of critical value reform into the group.
continuous quality improvement proposed by
American hospital organizations based on the PDCA
cycle. It creatively combines FOCUS and continuous Clarify the Current Process
cycle improvement (PDCA) and produces a manage-
According to the current test critical value ver-
ment improvement mode (9–12). The characteristics
sion, when the LIS system detected the critical value,
of FOCUS-PDCA are big ring with small ring, step
the test ends are automatically sent to the doctor, and
rise, and scientific statistics (13), which are more
the test staff informs the department and registers the
widely used in patient care, drug management, and
value within the effective time. The critical value items
medical record management.
and threshold values for the whole hospital are the
This study aimed to optimize the critical value of same versions and include: blood biochemistry proj-
the test by FOCUS-PDCA and set the personalized ect, blood gas project, coagulation project, and blood
critical value of the test for different departments. routine project.
J Med Biochem 2022; 41 (3) 349
Understand Analyze the Root Cause At the beginning of the revision of the critical value
version, some staff were not familiar with the new crit-
We hypothesized three fundamental reasons
ical value; thus, it was necessary to further adjust the
that could lead to a high proportion of critical values:
personalized critical value for some departments.
(1) laboratory staff did not know how to optimize crit-
ical values on the new system; (2) there were no rules The improvement team collected the critical
and regulations on the regular optimization of critical value specimen information and total specimen infor-
values, and there was little communication between mation of the whole hospital from May 2020 to
clinical departments and clinical laboratory depart- March 2021.The proportion of critical value in each
ments on critical values; (3) there was no personal- month after optimized critical value in the whole hos-
ized critical value, and medical staff adopted different pital and all clinical departments was then counted
clinical treatment methods for patients in different and compared with the data in 2019. Next, a table
departments. was created to observe the difference in the propor-
tion of critical values before and after optimization.
Select the Improvement Plan

Act
We selected the problem points that needed to
be improved and set the personalized critical value. After this critical value optimization, the hospital
The improvement team reviewed relevant literature in formed a »new version of critical value with personal-
the Medical Department and clinical laboratory in ized critical value«, and the clinical laboratory applied
early February 2020 and selected the way of »clinical continuous improvement measures of critical value,
critical value communication meeting« to establish including the improvement team to evaluate the hos-
personalized critical value in clinical departments. pital’s critical value periodically every year.
PDCA (Plan-Do-Check-Act) Results

Plan Changes of critical value before and after
It was necessary to improve team planning criti- optimization (monthly data)
cal value communication to implement specific mat- Data from January to March 2019 (before opti-
ters. The project improvement team leader held a mization) and 2021 (after optimization) were ana-
critical value clinical communication meeting in the lyzed and compared. The data from January to March
medical department conference room (February 2020 were actually data from January to March 2021
2020) regarding the critical value items and reporting in order to exclude the impact of the epidemic in
limits. The improvement team members then sum- 2020. Data from May to December 2019 and 2020
marized the critical value communication and were analyzed and compared. As shown in Figure 1,
informed the medical department (within one week the proportion of critical values in each month of the
after the meeting). The new critical values were hospital decreased from 2.7%–4.1% before optimiza-
released to the whole hospital after being confirmed tion to 2.6% –3.1% after optimization.
by the medical department (March 2020). Soon after
that, clinical laboratory and clinical departments held
department meetings, respectively, to inform all the Proportion change of critical values in
staff regarding the new critical value. departments before and after optimization
The proportion of critical value in each month
Do before and after the optimization of critical value in
each department was counted. The proportion of crit-
From February to March 2020, the improve- ical value in some departments decreased significant-
ment team successfully implemented the action as ly, as shown in Table I and Table II. The critical values
planned. In April 2020, the whole hospital began to of ICU, hematology, nephrology, urology, and neona-
apply the new critical value. After that, the clinical lab- tal departments after optimization decreased signifi-
oratory regularly communicated the critical value to cantly compared with those before optimization,
the clinical department. while the critical values of cardiac surgery, emergency
ICU, cardiology, and neurosurgery ICU showed no
significant difference before and after optimization.
Check Contrary, the critical values of the infection depart-
ment after optimization significantly increased before
It was necessary to periodically check whether
optimization.
the LIS system missed the critical value and the under-
standing degree of laboratory staff to the current crit- The proportion of critical values after ICU optimization
ical value version, and to find the following problems: was only half of that before optimization. The data from
4.50%
4.00%
Critical Value Percentage
3.50%
3.00%
2.50%
2.00%
1.50%
1.00%
0.50%
0.00%
Jan Feb Mar May Jun Jul Aug Sept Oct Nov Dec
2019 3.94 4.01 3.72 3.46 3.70 2.72 3.64 3.40 3.42 3.27 3.48
2020 3.02 2.90 2.74 2.60 2.95 2.92 2.95 2.79 3.10 2.64 2.87
Figure 1 Proportion of critical values of the hospital during 2019 (before optimization) and 2020 (after optimization).
Table I Change of critical value rate before and after optimization of critical value in each department.
Hematology Nephrology Organ transplantation

ICU New pediatric
Month department department center
2019 2020 2019 2020 2019 2020 2019 2020 2019 2020
Jan 13.15% 5.54% 15.75% 2.51% 7.65% 3.34% 4.64% 1.77% 7.67% 5.04%
Feb 11.69% 4.89% 16.75% 1.85% 7.89% 3.79% 5.64% 1.73% 8.52% 6.28%
Mar 12.17% 4.53% 19.76% 2.35% 5.93% 3.31% 7.03% 2.21% 7.80% 5.69%
May 12.46% 4.28% 19.65% 2.43% 5.84% 4.08% 6.16% 1.53% 6.91% 7.61%
Jun 12.24% 4.98% 18.94% 3.04% 7.29% 4.12% 4.87% 2.33% 10.98% 8.35%
Jul 11.87% 5.80% 17.50% 3.15% 7.00% 2.93% 2.90% 1.98% 11.83% 8.33%
Aug 13.68% 6.14% 21.19% 2.13% 6.47% 2.98% 7.79% 2.26% 10.23% 6.05%
Sept 12.83% 4.21% 15.74% 2.70% 7.05% 3.96% 5.67% 3.02% 9.85% 6.35%
Oct 11.31% 3.60% 16.96% 3.59% 7.42% 4.08% 4.68% 4.63% 10.59% 6.92%
Nov 11.48% 4.01% 16.66% 2.20% 5.98% 3.65% 6.60% 2.68% 13.29% 8.11%
Dec 13.65% 3.66% 16.71% 2.97% 6.28% 3.41% 7.33% 1.05% 8.89% 7.30%
January to March 2020 were actually the data from January ent, with the lowest value being 2.90% in July and the highest
to March 2021, as shown in Figure 2. The proportion of crit- value being 7.79% in August. However, the optimized data
ical value after optimization in the Department of for 2020 and 2021 showed a small difference from month to
Hematology was less than 1/3 of that before optimization month that was stable at about 2%. October was an excep-
(Figure 3), while the proportion of critical value after opti- tion, with the critical value ratio reaching 4.63% (Figure 5).
mization in the Department of Nephrology was about 1/2 to
Different from the above departments, the proportion
2/3 of that before optimization (Figure 4).
of critical values in the Infection Department after optimiza-
The proportion of critical value between the adjacent tion could be as low as 1.5 times and as high as 5 times
months of external urology in 2019 was significantly differ- before optimization (Figure 6).
J Med Biochem 2022; 41 (3) 351
Table II Change of critical value rate before and after optimization of critical value in each department.
Infectious Cardiology Neurosurgical

Cardiac surgery Emergency ICU
Month department department ICU
2019 2020 2019 2020 2019 2020 2019 2020 2019 2020
Jan 2.15% 0.90% 11.27% 10.42% 0.91% 4.99% 5.04% 6.76% 6.20% 4.59%
Feb 1.64% 0.64% 11.02% 10.02% 1.44% 4.33% 5.01% 6.42% 5.16% 3.68%
Mar 2.41% 1.86% 9.31% 11.36% 1.83% 4.46% 4.80% 5.46% 3.89% 4.54%
May 2.44% 2.10% 9.20% 7.25% 0.86% 4.93% 4.70% 4.21% 3.86% 2.84%
Jun 2.60% 1.87% 8.03% 9.76% 1.29% 4.81% 4.89% 5.36% 4.99% 6.02%
Jul 2.05% 1.55% 7.97% 10.46% 1.88% 3.89% 4.61% 5.23% 4.32% 4.43%
Aug 1.92% 2.44% 8.11% 8.71% 1.76% 3.23% 5.31% 5.85% 3.15% 4.96%
Sept 2.31% 2.74% 7.57% 9.22% 1.85% 3.21% 5.22% 6.34% 3.11% 4.58%
Oct 2.61% 2.04% 8.06% 8.32% 1.37% 5.85% 5.25% 6.82% 4.09% 4.10%
Nov 1.54% 1.28% 8.34% 8.52% 1.54% 5.36% 5.06% 5.79% 4.92% 2.70%
Dec 1.63% 1.87% 10.23% 10.00% 1.36% 4.79% 5.23% 6.29% 3.32% 2.14%
2019 2020 2019 2020

16.00% 25.00%
14.00%
20.00%
12.00%
10.00% 15.00%
8.00%
6.00% 10.00%
4.00%
5.00%
2.00%
0.00%
0.00%
Jan Feb Mar May Jun Jul Aug Sept Oct Nov Dec Jan Feb Mar May Jun Jul Aug Sept Oct Nov Dec
Figure 2 Proportion of critical values of ICU test items Figure 3 Proportion of critical value before (2019) and after
before and after optimization. (2020) optimization in Department of Hematology.
2019 2020 2019 2020

9.00% 9.00%
8.00% 8.00%
7.00% 7.00%
6.00% 6.00%
5.00% 5.00%
4.00% 4.00%
3.00% 3.00%
2.00% 2.00%
1.00% 1.00%
0.00% 0.00%
Jan Feb Mar May Jun Jul Aug Sept Oct Nov Dec Jan Feb Mar May Jun Jul Aug Sept Oct Nov Dec
Figure 4 Proportion of critical value before (2019) and after Figure 5 Proportion of critical value before (2019) and after
(2020) optimization in Department of Nephrology. (2020) optimization in Organ Transplantation Center.
2019 2020
improve processes. It is mainly used in drug management,
patient care, and medical record management, while it is
7.00%
rarely applied for critical value optimization management.
6.00% Some researchers applied FOCUS-PDCA to solve drug man-
5.00% agement problems after investigating the procurement, allo-
cation, and use of essential national drugs in the Affiliated
4.00% Hospital of Nantong University (26). Also, the application of
3.00% FOCUS-PDCA significantly reduced the dispensing error rate
in pharmacies (27). Moreover, some studies applied the
2.00% FOCUS-PDCA cycle method to effectively reduce the inci-
1.00% dence of drug proximity error (28).
0.00% In terms of patient care, FOCUS-PDCA has been used

Jan Feb Mar May Jun Jul Aug Sept Oct Nov Dec to effectively solve the difficulties in moisture management of
maintenance hemodialysis patients during dialysis (29).
Figure 6 Proportion of critical value before (2019) and after Some researchers also applied FOCUS-PDCA to treat trauma
(2020) optimization in Infection Department. patients, effectively improving the treatment rate of patients
(30).
Discussion Furthermore, FOCUS-PDCA can reduce the incidence

of unreasonable medical orders of parenteral nutrition (31).
»Critical value« result is an abnormal laboratory test In addition, the application of this model effectively improves
value that is life-threatening to a patient and is reported by the completion rate of the first page of inpatient medical
laboratory staff based on preset critical limits. It also refers to records (32). This circulation can also improve the overall
closely related disease outcomes of test results or to the management of blood (33) and help medical institutions
national major communicable diseases (14, 15). achieve significant process improvement (10).
In China, the concept of critical values has been Other researchers adopted FOCUS-PDCA during the
defined in the Patient Safety Objectives (2014–2015) issued epidemic to improve the capacity of hospitals dealing with
by China Hospital Association in 2014 (16) and Medical COVID-19 outbreaks (34–36). Previous studies have also
Quality Control Indicators for Clinical Laboratory Profes- suggested that FOCUS-PDCA effectively improves the con-
sionals issued by National Health and Family Planning version rate of intravenous drug preparations and reduces
Commission in 2015. However, standardized optimization of medical costs (10).
critical values remains a challenge. For example, the effective
critical value of auxiliary departments is not consistent with FOCUS-PDCA has been rarely applied for critical value
that of clinical identification. Different departments treat the optimization management. The aim of this study was to opti-
same critical value items differently, and the selection of crit- mize the critical value of test items using FOCUS-PDCA (find,
ical value items and the determination of critical value limits organize, clarify, understand, select, plan, do, check and act)
have not been standardized at home and abroad. So far, only and to set the personalized critical value of the test for differ-
a few studies have reported on critical value optimization ent departments. Our improvement team collected the total
(17). number of clinical specimens and critical value specimen
information of each department in Sichuan Provincial
AT present, PDCA is the most common method used People’s Hospital in January 2020 and analyzed the change
for critical value optimization. PDCA cycle was first proposed of critical value proportion of each department in the whole
by Hughart, then perfected and popularized by Dr. Deming in year and each month in 2019. The new version of the critical
1950 (18, 19). Many researchers have applied the PDCA value was implemented in April 2020. The critical value and
cycle to improve management quality (20–22). However, total specimen data from May 2020 to March 2021 were
PDCA is mainly used to establish critical value reporting pro- collected in April 2021 and then compared to data from
cedures or improve the timely rate and qualified rate of criti- 2019. In 2020–2021, the hospital’s critical values (each
cal value in medicine, while it is rarely used to optimize critical
month) were 2.5%–3%, while in 2019, the proportion was
value testing projects. Some researchers used the PDCA
3.5%, indicating a significant decrease in the value.
cycle to improve the registration rate, registration pass rate,
and rescue success rate (23), while others used it to analyze In some representative departments, such as ICU,
the critical value management of hospital clinical laboratory, hematology, nephrology, external urology, and neonatology,
improve existing problems, and finally improve the standard- critical values decreased significantly after optimization. A
ization of critical value management of hospital clinical labo- particular decrease has been observed in the ICU and the
ratory (24). After applying PDCA to manage critical value, department of hematology (ICU: 12% before optimization to
laboratory staff’s working attitude, test quality, safety aware- about 4% after optimization; department of hematology from
ness, and operation standardization score have been 15%  22% to 1.5%  3.6%).
improved (25).
The optimization of critical value items and their
FOCUS-PDCA is relatively a new approach developed threshold values is not to reduce the proportion of critical
by Hospital Corporation of America (HCA) and used to value, but to set the critical value according to specimen
J Med Biochem 2022; 41 (3) 353
items, item values, and department personalization. For FOCUS-PDCA. The clinical laboratory has effectively opti-
example, no significant change in critical values was found in mized the critical value items and their boundary values,
the cardiac surgery and emergency ICU department before screened out the critical value that can truly reflect the critical
and after optimization. Interestingly, the critical value in the state of patients in each clinical department, and established
infection department increased after optimization (less than continuous improvement measures for the critical value.
2% before and 3%-6% after optimization).Because the infec-
tion department has narrowed the range of critical coagula-
tion values. Conflict of interest statement
To sum up, a new version of critical value with person- All the authors declare that they have no conflict
alized critical value has been formed in the hospital using of interest in this work.
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Received: November 17, 2021

Accepted: January 03, 2022
UDK 577.1 : 61 ISSN 1452-8258

GABPA PROTECTS AGAINST GASTRIC CANCER DETERIORATION

VIA NEGATIVELY REGULATING GPX1
GABPA [TITI OD POGOR[ANJA KARCINOMA @ELUCA NEGATIVNIM REGULISANJEM GPX1
Binghua Yin1#, Bing Dong2#, Xiaohui Guo3, Can Wang3, Huazhi Huo3*
1CT Room, Handan Central Hospital, Handan, China
2Department of Gastroenterology, Handan Central Hospital, Handan, China
3Department of General Surgery, Handan Central Hospital, Handan, China

Background: To explore the anti-cancer role of GABPA in Uvod: Cilj je bio da se istraì antikarcenogena uloga
the progression of gastric cancer (GC), and the underlying GABPA u progresiji raka èluca (GC) i osnovni mehanizam.
mechanism. Metode: Kvantitativna lan~ana reakcija polimeraze u
Methods: Quantitative real-time polymerase chain reaction realnom vremenu (kRT-PCR) je sprovedena da bi se otkrio
(qRT-PCR) was conducted to detect the expression pattern obrazac ekspresije GABPA u 45 parova GC i netumorskih
of GABPA in 45 pairs of GC and non-tumoral tissues. The tkiva. Analiziran je odnos izme|u ekspresije GABPA i
relationship between GABPA expression and clinic patho- klini~kopatolo{kih pokazatelja pacijenata sa GC. U }elijama
logical indicators of GC patients was analyzed. In AGS and AGS i SGC-7901 koje prekomerno eksprimiraju GABPA,
SGC-7901 cells overexpressing GABPA, their migratory njihova migraciona sposobnost je odre|ena testom
ability was determined by trans well and wound healing transvella i zarastanja rana. Interakcija izme|u GABPA i
assay. The interaction between GABPA and its downstream njegovog nizvodnog ciljanog GPKS1 istraèna je testom sa
target GPX1 was explored by dual-luciferase reporter assay, dvostrukom luciferazom, a njihova sinergisti~ka regulacija
and their synergistical regulation on GC cell migration was migracije GC }elija je kona~no razja{njena.
finally elucidated. Rezultati: GABPA je smanjena u GC tkivima u pore|enju sa
Results: GABPA was downregulated in GC tissues in com- normalnim. Nizak nivo GABPA predvi|a visoku incidencu
parison to normal ones. Low level of GABPA predicted high limfnih i udaljenih metastaza u GC. Prekomerna ekspresija
incidences of lymphatic and distant metastasis in GC. GABPA je blokirala AGS i SGC-7901 }elije da migriraju.
Overexpression of GABPA blocked AGS and SGC-7901 GABPA bi mogao da cilja GPKS1 preko predvi|enog mesta
cells to migrate. GABPA could target GPX1 via the predict- vezivanja. GPKS1 je bio poja~an u klini~kim uzorcima GC i
ed binding site. GPX1 was upregulated in clinical samples imao je negativnu korelaciju sa nivoom GABPA. Efekat
of GC, and negatively correlated to GABPA level. The anti- GABPA protiv raka na GC oslanjao se na u~e{}e GPKS1.
cancer effect of GABPA on GC relied on the involvement of
GPX1.
List of Abbreviations: Gastric cancer (GC); GA-binding protein alpha

(GABPA); telomerase reverse transcriptase (TERT); Helicobacter
Pylori (HP); gastric mucosal epithelial cell line (GES-1); American
Address for correspondence: Type Culture Collection (ATCC); Dulbecco’s Modified Eagle’s
Medium (DMEM); fetal bovine serum (FBS); ethylenediaminete-
Huazhi Huo, MM. Department of General Surgery, Handan
traacetic acid (EDTA); phosphate-buffered saline (PBS);
Central Hospital, 59 Congtai North Road, Handan 056001,
Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR);
China
complementary deoxyribose nucleic acids (cDNAs); Glyceraldehyde
Tel: 86013283406185
3-phosphate dehydrogenase (GAPDH); radio immunoprecipitation
e-mail:huohuazhi123456ªsina.com
assay (RIPA); bicinchoninic acid (BCA); sodium dodecyl sulphate-
polyacrylamide gel electrophoresis (SDS-PAGE); polyvinylidene
#Binghua Yin and Bing Dong contributed equally to this work difluoride (PVDF); glutathione peroxides (GPXs).
356 Yin et al.: GABPA is an anti-cancer gene in gastric cancer progression
Conclusions: GABPA is downregulated in GC samples, Zaklju~ak: GABPA je smanjena u GC uzorcima, {to se

which can be utilized to predict GC metastasis. Serving as moè koristiti za predvi|anje GC metastaza. Sluè}i kao
a tumor suppressor, GABPA blocks GC cells to migrate by supresor tumora, GABPA blokira GC }elije da migriraju
targeting GPX1. ciljaju}i GPKS1.
Keywords: GABPA, GPX1, gastric cancer, migration Klju~ne re~i: GABPA, GPKS1, rak èluca, migracija
Introduction my and stored in liquid nitrogen. Follow-up of each

GC patient through telephone and outpatient review
Gastric cancer (GC) is the number two killer of was conducted after discharge, including physical
cancer death (1, 2). Recently, a growing number of conditions, clinical symptoms and signs, and imaging
therapeutic strategies for GC have been developed and examinations. In addition, the patients with other
improved, especially novel targeted therapy. However, malignancies; mental disease; myocardial infarction;
the 5-year survival of metastatic GC is not ideal (3, 4). heart failure or other chronic diseases, or those previ-
The study results demonstrated that GC cells are prone ously exposed to radioactive rays were excluded. This
to metastasize through lymph nodes (4). A large num- investigation was approved by the research Ethics
ber of GC patients are in the progressive stage at the Committee of Handan Central Hospital and complied
first time of clinical diagnosis, thus losing the best sur- with the Helsinki Declaration. Informed consent was
gical opportunity (4-6). Therefore, to clarify the mech- obtained from patients.
anism of GC metastasis and actively explore new bio-
markers are urgently to be solved (7–9).
GA-binding protein alpha (GABPA) encodes one Cell Lines and Reagents
of the three GA-binding protein transcription factor GC cell lines (AGS, BGC-823, SGC-7901) and
subunits, serving as a DNA-binding subunit. Since this the human gastric mucosal epithelial cell line (GES-1)
subunit has a common feature to that encoding the were provided by American Type Culture Collection
nuclear respiratory factor 2 gene, it may participate in (ATCC) (Manassas, VA, USA). Cells were cultivated in
the activation of cytochrome oxidase expression and Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco,
nuclear control of mitochondrial function (10, 11). Rockville, MD, USA) containing 10% fetal bovine
GABPA also has a similar structure to that of the tran- serum (FBS) (Gibco, Rockville, MD, USA), 100 U/mL
scription factor E4TF1. Hence, it is involved in the penicillin and 100 mg/mL streptomycin. Cell passage
expression of the adenovirus E4 gene (12). Current was conducted at 90% confluence using 1×tyrpsin
evidences have proven the interaction between containing EDTA (ethylenediaminetetraacetic acid).
GABPA and several important transcription factors (i.e.
HCFC1, SP1 and SP3), and its vital function in affect-
ing pathways of interactions at neuromuscular junc- Transfection
tion and mitochondrial gene expression (13). In mul- Transfection plasmids were synthesized by
tiple types of tumor cells, GABPA can selectively bind GenePharma (Shanghai, China). Cells were cultured to
to the ETSdomain of the mutated telomerase reverse 30–50% density in a 6-well plate, and transfected using
transcriptase (TERT) promoter and activate its tran- Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).
scription (14, 15). Previous studies have already After 48 h cell transfection, cells were collected for ver-
reported the involvement of GABPA in the progression ifying transfection efficacy and functional experiments.
of bladder cancer and breast cancer (16, 17). Its
potential influence in GC, however, is unclear.
Transwell Migration Assay
This study aims to elucidate the role of GABPA
in the malignant progression of GC and the molecular Cell suspension was prepared at 5×105
mechanism, which provides a novel target for individ- cells/mL. 200 mL of suspension and 700 mL of medi-
ualized therapy. um containing 20% FBS was respectively added on
the top and bottom of a transwell insert and cultured
for 48 h. Migratory cells on the bottom were induced
Materials and Methods with methanol for 15 min, 0.2% crystal violet for 20
min and captured using a microscope. Five random
GC Samples fields per sample were selected for capturing and
Forty-five GC patients with surgical resection in counting migratory cells.
our hospital were retrospectively analyzed, and these
patients did not have preoperative anti-cancer treat-
Wound Healing Assay
ment, previous infection of Helicobacter Pylori (HP)
was all positive as well as pathologically was con- Cell suspension in serum-free medium was pre-
firmed as GC. Invasive GC tissues and adjacent nor- pared at 5×105 /mL and implanted in 6-well plates.
mal tissues were harvested during subtotal gastrecto- Cells were cultivated to 90% density, followed by cre-
J Med Biochem 2022; 41 (3) 357
ating an artificial scratch using a sterilized pipette tip. separated by sodium dodecyl sulphate-polyacry-
Cells were washed in phosphate-buffered saline (PBS) lamide gel electrophoresis (SDS-PAGE) and loaded
for 2-3 times and cultured in the medium containing on polyvinylidene difluoride (PVDF) membrane
1% FBS. 24 hours later, wound closure percentage (Millipore, Billerica, MA, USA). The membrane was
was calculated. cut into small pieces according to the molecular size
and blocked in 5% skim milk for 2 h. They were incu-
bated with primary and secondary antibodies, fol-
Quantitative Real-Time Polymerase Chain lowed by band exposure and grey value analyses.
Reaction (qRT-PCR)
Cells were lysed using TRIzol reagent (Invitrogen,
Dual-Luciferase Reporter Assay
Carlsbad, CA, USA) for isolating RNAs. Qualified RNAs
were reversely transcribed into complementary deoxyri- Wild-type and mutant-type GABPA vectors were
bose nucleic acids (cDNAs) using AMV reverse tran- synthesized based on bioinformatics screening on the
scription kit (TaKaRa, Otsu, Shiga, Japan), followed by binding site to GPX1. They were co-transfected in
qRT-PCR using SYBR®Premix Ex Taq™ (TaKaRa, Otsu, HEK293T cells with either pcDNA-NC or pcDNA-
Shiga, Japan). Glyceraldehyde 3-phosphate dehydro- GPX1 for 48 h. Luciferase activity was finally meas-
genase (GAPDH) was the internal reference. Each ured in a standard method (Promega, Madison, WI,
sample was performed in triplicate, and relative level USA).
was calculated by 2-DDCt. Primer sequences were as fol-
Statistical Analysis
lows. GABPA: Forward: 5’-GGAGGAAGTGGAGGGA-
CTGA-3’, reverse: 5’-GCTTACACATTCAGCTGGCG-3’; GraphPad Prism 5 V5.01 (La Jolla, CA, USA)
GPX1: Forward: 5’-TATCGAGAATGTGGCGTCCC-3’, was used for statistical analyses and data were
reverse: 5’-TCTTGGCGT TCTCCTGATGC-3’; GAPDH: expressed as mean ± standard deviation. Differences
forward: 5’-CCTGGCACCCAGCACAAT-3’, reverse: 5’- between groups were compared by the t-test. The
TGCCGTAGGTGTCCCTTTG-3’. relationship between GABPA expression and clinico-
pathological indicators of GC patients was analyzed
by Chi-square test. P<0.05 was considered as statis-
Western Blot tically significant.
Cells were lysed in radio immunoprecipitation
assay (RIPA) (Beyotime, Shanghai, China) on ice for
Results
15 min, and the mixture was centrifuged at
14000×g, 4 for 15 min. The concentration of cellu- GABPA Was Lowly Expressed in GC
lar protein was determined by bicinchoninic acid
(BCA) method (Beyotime, Shanghai, China). Protein Forty-five cases of GC and paired adjacent normal
samples with the adjusted same concentration were tissues were collected in our center. It is shown that
GABPA was downregulated in GC tissues (Figure 1A).
Table I Association of GABPA expression with clinicopathologic characteristics of gastric cancer.

GABPA expression
Parameters Number of cases P-value
High (n=23) Low (n=22)
Age (years) 0.181
<60 23 14 9
≥60 22 9 13
Gender 0.295
Male 22 13 9
Female 23 10 13
T stage 0.182
T1-T2 25 15 10
T3-T4 20 8 12
Lymph node metastasis 0.023
No 28 18 10
Yes 17 5 12
Distance metastasis 0.011
No 27 18 9
Yes 18 5 13
J Med Biochem 2022; 41 (3) 359
Figure 3 3GABPA was bound to GPX1. (A) Protein level of GPX1 in AGS and SGC-7901 cells overexpressing GABPA; (B)
Differential levels of GPX1 in GC and adjacent normal tissues; (C) A negative correlation between mRNA levels of GPX1 and
GABPA in GC tissues; (D) GPX1 levels in GC cell lines; (E) Transfection efficacy of pcDNA-GPX1; (F) GABPA level in AGS and
SGC-7901 cells overexpressing GPX1; (G) Binding relationship between GABPA and GPX1. *P< 0.05, ***P< 0.001.
Figure 4 GABPA and GPX1 synergistically regulated GC migration. (A) GPX1 level in AGS and SGC-7901 cells co-overexpress-
ing GABPA and GPX1; (B) GABPA level in AGS and SGC-7901 cells co-overexpressing GABPA and GPX1; (C) Migration in AGS
and SGC-7901 cells co-overexpressing GABPA and GPX1 (magnification 20×); (D) Wound closure in AGS and SGC-7901 cells
co-overexpressing GABPA and GPX1 (magnification 20×). *P< 0.05, **P< 0.01.
J Med Biochem 2022; 41 (3) 361
In addition, GABPA was lowly expressed in GC cells in Discussion

comparison to the human gastric mucosal epithelial
Globally, GC is a severe cancer that has high
cells (Figure 1B). It is speculated that GABPA may be a
incidence and mortality, and is also one of the diges-
tumor suppressor gene involved in GC. We subse-
tive system in our country (1–3). Epidemiological
quently analyzed clinicopathological indicators of GC
investigations have proposed that the carcinogenesis
patients based on their GABPA levels. It is demonstrat-
of GC involves environmental, biological, genetic and
ed that GABPA was closely linked to the incidences of
epigenetic factors (4, 5). Microarray analysis is an
lymphatic and distant metastasis in GC patients (Table
effective technology to identify differentially
I). As expected, GC cases with lymphatic or distant
expressed genes in the profile of cancer. These abnor-
metastasis expressed lower level of GABPA than non-
mally activated or inactivated genes are potential
metastatic ones (Figure 1C).
molecular biomarkers for screening or predicting the
progression of human cancers (6–8).
Knockdown of GABPA Blocked GC to Migrate Previous study reported that GABPA could inhibit
the metastasis of papillary thyroid carcinoma through
To explore the biological functions of GABPA in regulating DICER1 (18). In addition, another study
GC, pcDNA-GABPA was generated and transfection revealed that it served as a novel biomarker for the
of pcDNA-GABPA effectively upregulated GABPA in prognosis of hepatocellular carcinoma since GABPA
AGS and SGC-7901 cells (Figure 2A). Later, overex- was able to block the migration of cancer cells by reg-
pression of GABPA was identified to reduce migratory ulating E-cadherin (19). However, the function of
cell number and wound closure percentage in GC GABPA in HCC is not clear. Our findings showed that
cells, suggesting the attenuated migratory potential GABPA was downregulated in GC tissues, compared to
(Figure 2B, 2C). adjacent normal ones. Low level of GABPA predicted
high incidences of lymphatic and distant metastasis in
GC patients. Thus, it is speculated that GABPA could
GABPA Was Bound to GPX1 be a tumor suppressor involved in the progression of
Using online bioinformatics tools, it is predicted GC. Later, a series of functional experiments identified
that GABPA may bind to GPX1. Western blot analysis that overexpressing GABPA, the migratory potential
showed that protein level of GPX1 was markedly was markedly inhibited in AGS and SGC-7901 cells.
downregulated in AGS and SGC-7901 cells overex- We thereafter verified that GPX1 was the down-
pressing GABPA (Figure 3A). GPX1 was detected to stream target binding GABPA by miRDB starbase.
be highly expressed in GC tissues and cell lines GPXs (glutathione peroxides) are vital antioxidant
(Figure 3B, 3D). It was negatively correlated to mRNA enzymes in the body, which are responsible for elimi-
level of GABPA in clinical samples of GC (Figure 3C). nating ROS (20). GPX1 is the most common antiox-
Subsequently, transfection efficacy of pcDNA-GPX1 idant enzyme in the GPX family. It is widely present in
was examined (Figure 3E). Overexpression of GPX1 human cells, and has antioxidant and detoxifying
downregulated GABPA in GC cells, further supporting effects (21, 22). Protein level of GPX1 was found to
their negative correlation (Figure 3F). Dual-luciferase be downregulated by overexpression of GABPA in GC
reporter assay revealed a decline of luciferase activity cell lines. In addition, we also found that GPX1 was
in the wild-type GABPA vector after overexpression of upregulated and negatively correlated to GABPA level
GPX1. Nevertheless, GPX1 could not affect in GC tissues. Furthermore, the rescue experiments
luciferase activity in the mutant-type one (Figure 3G). identified the migration ability of GPX1 to reverse the
As a result, we have proven that GPX1 could be tar- regulatory effect of GABPA on GC cell lines. Taken
geted by GABPA through the predicted binding site. together, GABPA was a tumor suppressor that inhibit-
ed migratory potential in GC through negatively reg-
ulating GPX1, which could be utilized in the clinical
GABPA and GPX1 Synergistically Regulated GC targeted therapy of GC.
Migration
To further uncover the synergistical regulation of
Conclusions
GABPA and GPX1 on GC, we co-transfected pcDNA-
GABPA and pcDNA-GPX1 in AGS and SGC-7901 GABPA is downregulated in GC samples, which
cells. Compared with those overexpressing GABPA, can be utilized to predict GC metastasis. Serving as a
both GPX1 and GABPA levels were much higher in tumor suppressor, GABPA blocks GC cells to migrate
AGS and SGC-7901 cells co-overexpressing GABPA by targeting GPX1.
and GPX1 (Figure 4A, 4B). Co-overexpression of
GABPA and GPX1 enhanced migratory potential in
GC cells with solely overexpression of GABPA (Figure Conflict of interest statement
4C, 4D).
All the authors declare that they have no conflict
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Received: December 18, 2021

Accepted: January 05, 2022
XXII srpski kongres medicinske
i laboratorijske medicine
sa me|unarodnim u~e{}em
XXII Serbian Congress

of Medical Biochemistry
and Laboratory Medicine
with international participation
&
16th Belgrade Symposium
for Balkan Region
Plenarne sekcije / Plenary Sessions

Apstrakti / Abstracts
364
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Opening lecture
LABORATORY MEDICINE IN THE ERA OF COVID-19:

LESSONS FOR THE FUTURE
Mario Plebani
Dept of Laboratory Medicine University Hospital – Padova, University of Padova, Padova, Italy
The lockdown due to the coronavirus disease 2019 (COVID-19), a major healthcare challenge, is a worldwide
threat to public health, social stability, and economic development. The pandemic has affected all aspects of soci-
ety, dramatically changing our day-to-day lives and habits. It has also changed clinical practice, including practices
of clinical laboratories. After two years, it is time to rethink what has happened, and is still happening, in order to
learn lessons for the future of laboratory medicine and its professionals. The first issue is the increased visibility of
the central role of clinical laboratories in modern healthcare. Before the pandemic, several documents, papers
and initiatives emphasized the importance of laboratory testing in numerous clinical pathways, but the pandemic
further raised awareness of the essential contribution made by clinical laboratories to diagnostic reasoning and
the management of cases of suspected or confirmed SARS-CoV-2 infection. These include etiological diagnosis,
patient monitoring, and epidemiological surveillance. Further evidence of the importance of laboratory medicine
is being gained thanks to serological testing for SARS-CoV-2 antibodies in vaccine(s) clinical trials, in properly
monitoring vaccinated subjects (eventually with different vaccines and different clinical histories), and in better
understanding the effects of virus variants from both diagnostic and clinical viewpoints. The second lesson is that
»speed must never compromise quality, and a marriage between accuracy, reliability and quickness should be
assured«. A thrid lesson is that we have to »assure and monitor quality in all phases of the testing process and
measure clinical and economical outcomes to provide evidence of the effectiveness of laboratory services. The
IFCC Model of Quality Indicators (MQI) is a valuable tool for achieving this goal«. A fourth lesson is that laboratory
professionals have to evaluate all well-developed and promising technologies, validate and deploy them according
to established guidelines and recommendations focusing on patient needs. And we have to integrate different
diagnostic approaches in a clear and reliable report so that the information is conducive to diagnostic accuracy,
effective therapy and the best possible clinical outcome. However, the most important lesson is to move slinical
laboratories out of the silo, avoid isolation and integrate laboratory testing in diagnostic and clinical pathways that
effectively prevent disease, and provide early diagnosis, valuable monitoring, personalized therapy and epidemio-
logical surveillance. The ultimate goal is, in fact, effectiveness, not just efficiency.
J Med Biochem 2022; 41 (3) 365
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Plenary Lecture
LABORATORY MANAGEMENT
IN THE NEW NORMAL
Dunja Rogi}
Clinical Institute of Laboratory Diagnostics

Clinical Hospital Center Zagreb, Croatia
Laboratory leadership is a skill mostly learned by experience and intuition (often referred to as emotional
inteligence). The art of leadership is akin to the art of living – we learn as we go along. Preanalytical, analytical
and postanalytical processes need to be constantly monitored and optimized, with no visible disruptions in the
service. It is often presumed that the most prominent sign of a smoothly operated laboratory are happily oblivious
customers who never give a laboratory a second thought, which means that results are always delivered as
requested, in an efficient and timely manner. This notion might be true in a fee for service oriented laboratory,
but not neccessarily in the one which aspires to provide an added value. An ”added value” laboratory is the one
where not all requests are uniformely received as unquestionable orders to be fullfilled, where results are not only
numbers but may and should contain meaningful and informative comments and where laboratory people bear
names and faces known to their users.This presentation therefore deals with all the additional chalenges faced by
hospital laboratory leaders during the two years of the Covid 19 pandemic. All the hospital laboratories needed
to promptly adapt to the sudden unprecedented demands, not only related to SARS CoV-2 diagnostic, but also
to all the other challenges connected to swift and often chaotic hospital workflow and case mix changes. Staff
routines were adapted to accomodate the highest priority – no disruption of laboratory services due to within lab-
oratory infection spread. Preanalytical workflow suddenly became crucial in terms of timely and safe specimen
delivery while minimising human contact, both from emergency departements and intensive care units dealing
with covid patients. Any process changes which were not carefully talked through have resulted in serious TAT
delays, as will be discussed in detail with practical examples. New tests (SARS CoV2 PCR and antibodies, IL-6)
needed to be quickly added to the existing STAT routine. But, above all, throughout this period laboratory tech-
nicians and other employees had to feel safe and cared for. As in any emergency situation, people’s individual
vulnerabilities became visible and needed immediate attention. All this had to be done by the laboratory leader,
who simultaneously held an 24 hours’ open hotline with hospital administration and their various questions and
demands. Finally, the laboratory remains as usual the only voice of reason to be heard regarding excessive unnec-
cessary testing conected with Covid and suspected post Covid sydromes.
366
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J Med Biochem 41: 366 –393, 2022 Plenarne sekcije

Plenary sessions
MEETING THE LEADERSHIP CHALLENGE OF DISRUPTIVE INNOVATION

Gilbert Wieringa
Department of Biomedical Sciences, University of Manchester, UK
The 20th century digital revolution has seen the introduction of faster, innovative and easier to use technologies
that have taken laboratory medicine services closer to patients in primary and community care. For the 21st cen-
tury artificial intelligence driven algorithms are increasingly supporting evidence-based decision making that is
reducing the need for expert human resource and opening opportunities for global, information technology
providers to disrupt conventional ways of working at the point of care. A new leadership challenge emerges for
specialists in laboratory medicine. Perhaps no longer laboratory-based specialists will extend their knowledge,
skills and competence to a) guiding appropriate services for local environments based on clinical need, b) ensur-
ing technology solutions are cost-effective, safe and reliable, c) developing the business acumen to market, nego-
tiate and manage change d) getting a better understanding of imaging technologies, genomics, and health infor-
mation science (data mining and health economics) that also drive the changing landscape. In providing examples
of the new ways of working this talk will also emphasise the potential to exploit specialist leadership to ensure
effective use of resource across the diagnostics and information technology industries, service commissioners,
academia and policy related healthcare organisations.
ADDED VALUE BY INTERPRETATIVE COMMENTING
Wytze P. Oosterhuis
Zuyderland Medical Center, Sittard/Heerlen, The Netherlands

EFLM Working Group »Patient Focused Laboratory Medicine«
Consultation by adding interpretative comments to reports has long been recognized in laboratory medicine as
one of the activities that can support physicians and help to improve patient treatment outcomes. Interpretation
of laboratory test results might in some cases considerably be supported when additional tests are performed on
the available samples. This activity was named reflective testing-where the reflection is done by the laboratory spe-
cialist - and that can improve the diagnostic efficiencyconsiderably. Both the need, clinical value and appreciation
by stakeholders of these forms of consultation have been proven by a diversity of studies. Both general practition-
ers and medical specialists have been shown to value interpretative commenting. Other forms of consultation are
emerging: reporting of laboratory results to patients is becoming the rule. Most patients have little understanding
of these results, and consultation of patients could add a new dimension to the service of the laboratory. These
developments have been recognized by the European Federation of Clinical Chemistry and Laboratory Medicine,
which has established the working group on Patient Focused Laboratory Medicine for work on the matter.
Providing proper interpretative comments is, however, labor intensive. Harmonization is necessary to maintain
quality between individual specialists. In present-day high-volume laboratories, there are few options on how to
generate high-quality, patient-specific comments for all the relevant results without overwhelming the laboratory
specialists. Automation and application of expert systems could be to only solution.
J Med Biochem 2022; 41 (3) 367
SMARTPHONE APPLICATIONS USING LABORATORY MEDICINE DATA

– RELIABILITY AND BENCHMARKING
Sneàna Jovi~i}
Center for Medical Biochemistry, Clinical Center of Serbia

Department of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Serbia
ELFM Working Group "Patient Focused Laboratory Medicine"
Thanks to their accessibility, many of smartphone mobile applications (apps) are used for delivering health inter-
ventions to clinicians and patients. However, the burning issue is the quality of health related apps and how to
evaluate it. The content of most of them is not officially regulated, unless they function as part of a medical device.
Mobile App Rating Scale (MARS) is a multidimensional tool for classifying and rating the quality of mobile health
apps. Its quality criteria consider engagement, functionality, aesthetics, and information quality of the app content.
The project of EFLM Patient Focused Laboratory Medicine Working Group was to analyze the number and quality
of smartphone apps available on the market using in any way laboratory medicine data. Seven categories were
distinguished: 1) apps that offer medical advice about symptoms and health queries with the possibility to upload
laboratory test results, which can be seen, stored and shared; 2) reference ranges of selected analysis with basic
information about the causes of increase or decrease designed for patients; 3) quick reference for laboratory tests
for medical students and doctors; 4) apps for monitoring the state of user’s health through a wide range of health
parameters, including glucose and/or cholesterol as laboratory data, 5) apps that provide access to patients’ lab-
oratory results to physicians; 6) apps that enable patients to access their laboratory test results directly from the
diagnostic center; and 7) electronic health records apps that include laboratory test results. MARS score values
revealed the poorest performance and quality of the apps intended for patients, with significant issues of security
of personal information used by the apps, and the questionable affiliation of developers, without referencing the
source of information cited. The working group also analyzed the users’ (i.e. patients’) opinion on selected apps,
which pointed out the trustworthiness, the adequate style of presenting information the graphics, and the appear-
ance of the app as the key issues of the app quality.
368
THE PROFESSIONAL DEVELOPMENT OF LABORATORY MEDICINE

PROFESSIONALS IN/THROUGH EFLM COUNTRIES: WHAT TOOLS AND
OPPORTUNITIES DO WE HAVE?
Evgenija Hom{ak
EFLM Professional Committee, Chair
University Clinical Centre Maribor, Slovenia
The International Federation of Clinical Chemistry and Laboratory medicine (IFCC), and the European Federation
of Clinical Chemistry and Laboratory Medicine (EFLM) are two essential professional organizations in a field of
Clinical Chemistry and Laboratory Medicine (CCLM), that join and linking together Professional National societies
(NSs) and their members through Europe and all over the world. In a scope of both organizations, professionals
as NSs representatives are actively involved through different roles and issues in various professional working
groups and committees. EFLM has several essential Committees: for Education and Training (C-ET),
Communication (C-C), Quality and Regulation (C-QR), Science (C-S) and Professional Committee (C-P). C-P is
responsible for several important issues, to represent the professional interests of specialists in laboratory medicine
across Europe. One of the most important is the effort to achieve recognition of professional qualifications under
European Union (EU) legislation based on the principles of free movement of professionals within Europe.
According to the »EU Directive 2013/55/EU on recognition of professional Qualifications«, this effort could be
achieved with the harmonization of our profession on EFLM level through Common Training Framework (CTF)
and confirmed exit qualifications. This process/approach is started almost 20 years ago, with the established
European Communities Confederation of Clinical Chemistry and Laboratory Medicine (EC4) who put the base for
rules and minimal criteria for harmonization of our diverse education system through EU Countries. Since 2016
EC4 has been transferred to the EFLM C-P. Through these years, several (5) versions of the Syllabus for post-
graduate training in CCLM was prepared, which present the education/training program, with important areas of
knowledge and essential competencies for our profession. It represents the cornerstone for later established
Equivalents of standards (EoS) for European Specialist in Laboratory Medicine. According to determined criteria,
first EoS have been delivered to the Countries whose education program/system for specialization post-graduate
training fulfil and include all important parts of these established rules for EoS: education/training duration,
program (polyvalent), final exam/exit qualification.
Equivalence of Standards in Education and Training (EoS)

• Minimum 9 years (ideally 10): years academic (4 or 5 years) and specialist training (5 years);
• Education and training to standards set in the EFLM syllabus version 5;
• A Master’s degree in Medicine, Pharmacy or Science;
• An EFLM Profession Committee recognised ‘Equivalence of Standards’ exit qualification;
• Evidence of participation in continuous professional development (CPD).
It requires curriculum content to include:
• General chemistry of at least 35%;
• General chemistry plus haematology of at least 65%;
• Flexibility as to the remaining 35%, including general chemistry, haematology, microbiology, and genetics and
IVF in a proportion consistent with the requirements in the country of destination.
For the recognition and legislation of our profession, on the EU level through Directive, it is essential to obtain
EoS of post-graduate education and training and confirmation of CTF on government level at least in 33% of EU
Member States. Once the recognition and legislation of our profession would be accepted there is an EU
requirement for all Member States to implement it. This is important especially for non-medical specialists since
the specialists who are medical doctors have been already recognised on EU level. Now we have already 15
countries with achieved/confirmed EoS and the number still growing. To support the Profession Committee in its
strategy to achieve recognition of Specialists in Laboratory Medicine, EFLM has established the EFLM Register
of European Specialists in Laboratory Medicine (EuSpLM). It was first established in 1997 by EC4, the merger of
EC4 with FESCC culminated in 2016 with the transfer of the Register to EFLM. Through the standards it sets and
the code of conduct it expects from its registrants, the Register has identified a cohort of nearly 3000 individuals
with unique knowledge, skills and competencies for leading/delivering high-quality laboratory medicine services.
EFLM members in this register who already fulfil and have confirmed exit qualification according to the EoS
criteria obtain the common title EuSpLM. However, the goal of our efforts and for our profession is to achieve EoS
in all EFLM countries and raising the level of professional knowledge and skills in all field of Laboratory Medicine.
To achieve this goal, EFLM through activities of their professional units have developed several tools and
J Med Biochem 2022; 41 (3) 369
opportunities. Education Committee through important efforts and work of their working groups (WG) offer
several possibilities for additional education and training. WG: Congresses and Postgraduate Education has
launched post-graduate courses, that NSs can apply for them, to offer their members additional knowledge in two
fields: Biostatistics, How to write a good professional article. Another very important tool is EFLMLabX project of
exchanging practical knowledge and skills through EFLM web-platform (https://eflmlabx.eflm.eu/en), where
potential users can search and apply for practice/visiting/research in different laboratories/institutions through
EFLM countries and get direct contacts. WG: Distance Education and e-Learning offer several different webinars
on different topics and recordings of conferences/congresses. Lately, under Task Group: EFLM Syllabus Course
it has been launched first webinars also on Syllabus topics, which could be an important additional tool for the
trainees/specialists, for their professional development. Science Committee with several and different WGs
provide evidence and recommendations for harmonization of knowledge and practice of our profession on
different field of LM. In 2019 EFLM has, next to EuSpLM Register, established EFLM-Academy. Its memberships
offer/bring a lot of benefits not only to already registered EuSpLM but also to all other (non-EuSpLM) members
who want to be a part of »big EFLM family« and show the interest for Laboratory Medicine (from non- EFLM
countries, other profession (medical doctors, nurses), engineers on field of LM..).
The important benefits of EFLM-Academy are:

• Free on-line subscription to CCLM, the official EFLM journal;
• Unlimited access to all documents (laboratory standards) of the CLSI (Clinical and Laboratory Standards
Institute) database;
• Regular e-mail notifications of all EFLM activities, programmes and opportunities;
• Eligibility to apply for EFLM travel grants (subordinated to application’s criteria of each specific EFLM initiati-
ve)
• Reduced registration fee to all EFLM conferences and courses
• Free access to EFLM webinars
• Enrollment in the EuSpLM Register for those who meet the Educational and Training EFLM Equivalence of
Standards (subordinated to the evaluation of the requested documentation by the EFLM Profession
Committee).
• With all the activities, efforts, opportunities and benefits that come and grow with the enthusiastic work of
experts in EFLM WGs and committees and across EFLM countries, we help to create the tools for expanding
our knowledge of Laboratory Medicine. By using these tools and opportunities, we also rising the strength of
our profession for present and for the future.
References:
1. The European Federation of Clinical Chemistry and Laboratory Medicine syllabus for postgraduate education
and training for Specialists in Laboratory Medicine: version 5 – 2018. Jassam N, Lake J, Dabrowska M,
Queralto J, Rizos D, Lichtinghagen R, et al. Clin Chem Lab Med doi.org/10.1515/cclm-2018-0344.
2. Directive 2013/55/EU of the European Parliament and of the Council of 20 November 2013 amending
Directive 2005/36/EC on the recognition of professional qualifications. Official Journal of the European Union,
28.12.2013, L 354/132
3. Our profession now has a European name: Specialist in Laboratory Medicine. Zerah S, McMurray J, Horvath
AR. Biochem Med (Zagreb). 2012; 22: 272–3.
4. The European Register of Specialists in Clinical Chemistry and Laboratory Medicine: Guide to the Register,
Version 3-2010. McMurray J, Zerah S, Hallworth M, Schuff-Werner P., Haushofer A, Szekeres T, et al. Clin
Chem Lab Med 2010; 48: 999–10.
5. The European Register of Specialists in Clinical Chemistry and Laboratory Medicine: Code of Conduct, Version
2 – 2008. McMurray J, Zerah S, Hallworth M, Koeller U, Blaton V, Tzatchev K, et al. Clin Chem Lab Med 2009;
47: 372–5.
6. EFLM project »Exchange of practical knowledge and skills in Laboratory Medicine« Homsak E. eJIFCC 2018;
29: 191–5.
7. Continuing professional development crediting system for specialists in Laboratory Medicine within 28 EFLM
National Societies. Topic E, Beletic A, Zima T. Biochem Med (Zagreb) 2013; 23: 332–41.
370
ACCREDITATION OF LABORATORIES
Dunja Rogi}
Department of Laboratory Diagnostics, University Hospital Center Zagreb
Ki{pati}eva 12, Zagreb, Croatia
An actual contribution of accreditation certificate to day to day laboratory routine is not easily measurable.
Does it represent an objective, independent confirmation of the quality of laboratory practice and reliability of lab-
oratory test reports, or is it a mere formality to be fulfilled and carefully followed? How does accreditation con-
tribute towards minimizing error occurences and risks? In the past, the quality of laboratory practice was tradition-
ally and exclusively demonstrated by means of external quality assessment (EQA) results. When considering
eligibility of a laboratory for participation in clinical studies, EQA certificates are even today still the most often
required and sufficient proof of quality.Increasingly, however, this approach does not allways suffice as EQA covers
only some (mostly analytical) aspects of laboratory practice.Therefore, in 2003 the first version of the international
standard for implementation of the quality management system in medical laboratories was adopted and pub-
lished, i.e. ISO 15189: Medical laboratories – Requirements for quality and competence. A new and somewhat
altered and updated version of this standard was published in 2012.This specific standard consists of a number
of regulations and requirements to be met by an accredited laboratory, and is intended for all medical laboratories
that perform biological, microbiological, immunological, chemical, immunohematological, hematological, bio-
physical, cytological, pathological and other examinations of human material. ISO 15189 standard was adopted
as a national standard in the Republic of Croatia in 2006, and so far 13 medical diagnostic laboratories have been
accredited (domains of work: medical biochemistry, microbiology and transfusion). Accreditation of laboratories
in Croatia (and of one laboratory in Slovenia) has been carried out by the Croatian Accreditation Agency, an inde-
pendent and non-profit public institution founded according to the Croatian Governmentdecree based on the
Accreditation Act. Accreditation of Croatian laboratories is voluntary and for the time being it does not confer any
particular privileges to accredited as compared to unaccredited laboratories in public healthcare system. The only
advantage worth mentioning is a comparatively simpler acceptance of laboratories for participation in clinical stud-
ies, however laboratories with acceptable EQA results are included without problems as well. Accreditation of
medical biochemistry laboratories according to ISO 15189 standard requires continuous monitoring, surveillance
and improvements of all laboratory processes (preanalytical, analytical, postanalytical), active interpretation of lab-
oratory test results, and establishment of full laboratory users’ trust in the quality of reported results. It should be
stated that the aim of an accredited laboratory is not only to issue accurate results based on physician’s request,
but also to participate in correct test selection and in interpretation of results, to respect patients’ rights to privacy
and to focus its attention on patient safety and on laboratory practice according to ethical principles. All these
requirements are put in place in order to elevate the role of the laboratory from mere anonymus service towards
diagnostic partnership. A question whether a fully accredited medical laboratory achieves this goal, remains to be
answered.
J Med Biochem 2022; 41 (3) 371
IMPORTANCE OF LABORATORY GUIDELINES FOR

MEDICAL LABORATORY PRACTICAL WORK
Nata{a Bogavac Stanojevi}

University of Belgrade – Faculty of Pharmacy, Department of Medical Biochemistry,
Belgrade, Serbia
Despite recent progress towards laboratory test standardization and harmonization, there are still huge problems
that have to be addressed. Since in clinical guidelines only the laboratory parameters are defined, in different
countries are useddiverse laboratory methods for the same diagnoses. In order to improve the quality of treatment
and achieve standardization in treatment along with using of resources properly, there is a need for formulation
of common laboratory guidelines for European countries and beyond. On this way, problems which can be found
in developing laboratory guidelines on national or hospital level can be avoided and update of the new scientific
advances would be faster. One of the first steps towards laboratory guidelines development is creation of guide-
line’s frame that incorporate essential information related preanalytical, analytical and postanalytical process, clin-
ical benefit and cost-effectiveness data. The most guidelines enclose data related to sample collection, biological
variations, sample type, transport and storage of the sample. For most methods, analytical information (reference
material, total error, bias, inaccuracies and interferences) is also known. From the postanalytical information, mea-
surement units, reference interval, maximum allowed TAT is available. Future laboratory guidelines should focus
on laboratory tests that may influence the decision-making process, treatment optimization, disease prediction
and improvement of patient outcome while also be cost – effective. Laboratory results are critical to ensuring the
treatment of most patients. A number of locally accepted laboratory guidelines remain too vague with respect to
new scientific information and optimal analytical approaches. In order to develop a laboratory guideline, many
obstacles need to be overcome. It should consider different patients’ needs and reimbursement systemsin differ-
ent countries. Also, laboratory guidelines should be synchronized with clinical guidelines. Guidelines should be
translated into national languages and be accepted by the most European countries.
MANAGING THINGS AND LEADING PEOPLE
Katerina Tosheska-Trajkovska
Head of Department of Medical and Experimental Biochemistry, Medical Faculty, University
Ss.»Kiril and Metodij«, 1000 Skopje,
Republic of North Macedonia
What is a Leadership?
It is the process through which leaders influence the values, behavior and attitude of others. Leadership
qualities can either be innate or also can be acquired. A Leader is someone who shows a direction, influences,
motivates and inspires. A Leader is a person who can bring constructive change. Core values of a Leader are moral
courage, integrity, decisiveness and assertiveness. Good Leader has to have: knowledge and skills, sense of prior-
ity, focus, vision, judgment, charisma, trust and emotional intelligence. A Leader motivates the team members in
any situation, if they perform well or in case of failure. A good Leader applies the following approaches to lead:
strategic approach, human assets approach, expertise approach, unbox approach, change approach. A Leader
does the right thing at the right time, in right place. Manager does things right. A Manager is someone who plans,
organizes and allocates resources, controls and solves problems. Manager administers while Leader innovates.
Manager focuses on system processes, a Leader focuses on people; Manager relies on control, a Leader inspire
trust; Manager has a short range view; a Leader has a long-range perspective. A Leader knows the way, goes the
way and shows the way. All Managers are not Leaders but all Leaders can be Managers.
372
PRIMENA TEHNIKA KONTROLE IMPLEMENTATION OF

KVALITETA RADA U REALNOM PATIENT-BASED REAL TIME
VREMENU KOJE KORISTE QUALITY CONTROL
REZULTATE PACIJENATA U TECHNIQUES IN MEDICAL
MEDICINSKIM LABORATORIJAMA LABORATORIES
Svetlana Ignjatovi} Svetlana Ignjatovi}
Katedra za medicinsku biohemiju, Department of Medical Biochemistry,

Laboratorija za medicinsko biohemijske analize, Laboratory for medical biochemical analysis,
Univerzitet u Beogradu, Farmaceutski fakultet i University of Belgrade, Faculty of Pharmacy and
Centar za medicinsku biohemiju, Centre for Medical Biochemistry,
Klini~ki centar Srbije, Beogard, Srbija Clinical Centre of Serbia, Belgrade, Serbia
Tehnike kontrole kvaliteta u realnom vremenu Patient-Based Real Time Quality Control
(Patient-Based Real Time Quality Control, PBRTQC) (PBRTQC) techniques in medical laboratories use
koje koriste rezultate pacijenata u medicinskim labo- parameters calculated from patient samples in real
ratorijama koriste izra~unate parametre iz uzoraka time as a form of quality control (QC). The use of
pacijenata u realnom vremenu kao oblik kontrole patients samples have been widely used in haematol-
kvaliteta (QC). Upotreba uzoraka pacijenata {iroko se ogy as QC tool for over forty years. In medical labo-
koristi u hematologiji kao QC alat vec ~etrdeset god- ratories, even patient-based QC techniques have
ina. U medicinskim laboratorijama, mada se QC been described for more than fifty years, the concept
tehnike koje koriste rezultate pacijenata opisane pre were seen as interesting, however, because of practi-
vi{e od pedeset godina, ovaj koncept se smatra zan- cal issues it has not been widely utilized. Patient
imljivim, ali zbog prakti~nih problema nije {iroko based QC strategies such as the delta check, average
kori{cen. Strategije QC koje koriste rezultate pacije- of normal (AON), moving average (MA) and average
nata, kao {to su delta provere (delta check), prose~na of delta (AOD) are becoming more commonplace in
vrednost normala (average of normals, AON), medical laboratories because of availability of labora-
pokretni prosek (moving average, MA) i prose~na tory information system (LIS)/middleware programs.
delta vrednost (average of delta, AOD) su sve ~e{}e There are many advantages of PBRTQC in compari-
zasupljeni u medicinskim laboratorijama zbog dos- son with »conventional« QC strategies (internal qual-
tupnosti laboratorijskog informacionog sistema (LIS)/ ity control, IQC) and there is growing concerns that
middleware. U pore|enju sa »konvencionalnim« QC IQC alone is not sufficient to rapidly detect analytical
strategijama (interna kontrola kvaliteta, IQC), error. Traditional QC materials may be non-com-
PBRTQC ima vi{e prednosti i postoji sve ve}a zabrin- mutable, some IQC materials state only so-called
utost da IQC nije dovoljna za brzo otkrivanje assay targets that are specific to anlyzers instead of
analiti~ke gre{ke. Tradicionalni QC materijali nisu the true analyte concentration. Today analyzers are
komutabilni, neki IQC materijali navode samo takoz- more reliable and medical laboratories run fewer QC
vane »ciljne« vrednosti koje su specifi~ne za anliza- samples which increases the number of patients sam-
tore umesto prave koncentracije analita. Danas su ples reported before systematic error or bias event is
analizatori pouzdaniji i medicinske laboratorije detected by a subsequent QC failure. The AON and
odre|uju manji broj QC uzoraka {to povecava broj MA techniques continuously monitor assay perfor-
rezultata iz uzoraka pacijenata koji se izve{tavaju pre mance in medical laboratories. The mean or median
nego {to se otkrije sistematska gre{ka ili »odstupan- for groups of patient results is tracked over sequential
je« (bias) se otkrije tek naknadnim neodgovaraju}im time intervals can be used in a statistical QC process.
rezultatom QC. AON i MA tehnike kontinuirano prate An AoN or MA approach is more of risk based
performanse odre|ivanja u medicinskim laboratorija- approach using the patient population characteristics
ma. Srednja vrednost ili medijana za grupe rezultata to detect a shift in the measured population mean.
pacijenata koje se prate u odre|enim vremenskim PBRTQC is next generation medical laboratory QC,
intervalima mogu da se koriste u statisti~koj QC. AoN but is not as simple to implement as conventional
ili MA pristup je vi{e zasnovan na riziku koji koristi QC. There are some requirements of LIS/middleware
karakteristike populacije pacijenta da otkrije pomer- that are crucial for adoption of PBRTQC. The essen-
anje u izmerenoj prose~noj vrednosti za populaciju. U tial features of LIS/middleware for successful implan-
medicinskim laboratorijama PBRTQC predstavlja QC tation and operational application of PBRTQC are:
nove generacije, ali njena implemntacija nije jednos- data capture and storage, data extraction, analysis,
tavna kao {to je to za »konvencionalnu« QC. Postoje visualization, exploration and transformation, statisti-
odre|eni zahtevi za LIS/middlware-a koji su klju~ni za cal analysis and testing environment, live application
primenu PBRTQC. Potrebne su slede}e neophodne and reporting. Also, a software has to have some
karakteristike LIS/middlware softvera za uspe{nu additional features: advanced data visualization, for-
J Med Biochem 2022; 41 (3) 373
implantaciju i operativnu primenu PBRTQC-a: priku- mal statistical analysis and possibility to incorporate
pljanje i ~uvanje podataka, ekstrakcija podataka, internal quality data. Patient-based QC programs are
analiza, vizualizacija, istraìvanje i transformacija, currently widely used and PBRTQC is in rapid growth
statisti~ka analiza i okruènje za testiranje, aplikacija phase. However, there is only limited software sup-
u realnom vremenu i izve{tavanje. Tako|e, softver port, and to implement a PBRTQC program requires
mora da poseduje neke dodatne karakteristike: na- considerable time and statistical skills. PBRTQC tech-
prednu vizualizaciju podataka, formalnu statisti~ku niques cannot totally replace traditional IQC. For
analizu i mogucnost da se inkorporiraju podaci unu- high volume assays they are superior to conventional
tra{nje kontrole kvaliteta rada. QC programi koji QC, but for small batch type assays, conventional QC
koriste rezultate pacijenata trenutno se {iroko koriste has a place. A combination of techniques QC will
i primena PBRTQC je u fazi brzog rasta. Me|utim, provide the best protection against erroneous results
softverska podr{ka je ograni~ena, a za sprovo|enje being reported.
programa PBRTQC potrebno je dosta vremena i sta-
tisti~kih ve{tina. PBRTQC tehnike ne mogu u pot-
punosti da zamene tradicionalnu IQC. One su
superiornije u odnosu na klasi~nu QC za veliki broj
odre|ivanja, ali za odre|ivanja u malim serijama,
konvencionalna QC je pogodnija. Kombinacija tehni-
ka QC omogu}ava najbolju za{titu protiv pogre{nih
rezultata koji se izve{tavaju.
DA LI SE MOVING AVERAGE CAN MOVING AVERAGE

PROCEDURE MOGU KORISTITI PROCEDURES BE USED
KAO KONTINUIRANA KONTROLA AS A CONTINUOUS QUALITY
KVALITETA RADA U MEDICINSKIM CONTROL IN MEDICAL
LABORATORIJAMA? LABORATORIES?
Vera Luki} Vera Luki}
Odeljenje za laboratorijska ispitivanja, Laboratory Department,

Zavod za zdravstvenu za{titu radnika Railway Health Care Institute,
»@eleznice Srbije«, Beograd Belgrade, Serbia
Tradicionalno se kontrola kvaliteta analiti~kog Traditionally, the analytical quality control in

rada u medicinskim laboratorijama sprovodi anal- medical laboratories is conducted by analysing com-
iziranjem komercijalno dostupnih kontrolnih uzoraka mercially available control materials in certain time
u odre|enim vremenskim intervalima, kao i u~e{}em intervals, as well as by participating in external quality
u spolja{njim programima kontrole kvaliteta. Nedo- control programs. The disadvantages of traditional
staci tradicionalne kontrole su intermitentnost i ne- control are intermittency and non-commutability.
komutabilnost. Zbog toga se javlja potreba za Therefore, there is a need to introduce additional
uvo|enjem dodatnih kontrolnih mehanizama koji bi control tools that can overcome these shortcomings
mogli prevazi}i ove nedostatke i obezbediti konti- and ensure continuous control of the analytical pro-
nuirani nadzor nad analiti~kim procesom. U tu svrhu cess. In this light, the idea of quality control based on
u savremenoj laboratorijskoj praksi se razmatra ideja patient results is being considered in modern labora-
kontrole kvaliteta zasnovane na rezultatima pacijena- tory practice. One of possible methods of using
ta. Jedan od mogu}ih na~ina kori{}enja rezultata patient results for control purposes is the moving
pacijenata u svrhu kontrole kvaliteta analiti~kog rada average (MA). MA involves calculating the average
jeste pokretni prosek (eng. moving average, MA). value from the obtained set of patient results and fur-
MA podrazumeva izra~unavanje prose~ne vrednosti ther using that value for the purpose of continuous
iz dobijenog seta rezultata pacijenata i dalje kori{- quality control. It is called «moving« because the MA
}enje te vrednosti u kontrolne svrhe. Naziva se value is recalculated every time a new result is
pokretnim, jer se vr{i rekalkulacija MA vrednosti svaki received, that is, the data is continuously updated
put kada se primi novi rezultat, odnosno podaci se and evaluated as patient samples are analysed. The
kontinuirano aùriraju i evaluiraju, kako se uzorci most commonly used algorithms for calculating MA
pacijenata analiziraju. Naj~e{}e kori{}eni algoritmi za values are: simple MA, exponentially weighted MA
izra~unavanje MA vrednosti su: prosti MA, eksponen- and Bull’s algorithm. Although the concept of MA
cijalno ponderisani MA i Bulov algoritam. Iako je kon- has been known for decades, it has never been wide-
374
cept MA poznat ve} decenijama, on nikada nije u{ao ly adopted in medical laboratories for many reasons.
u {iru primenu u medicinskim laboratorijama iz vi{e One is the complexity of defining optimal MA proce-
razloga. Jedan je sloènost definisanja optimalnih dures that are specific to each test and each labora-
MA procedura koje su specifi~ne za svaki test i svaku tory and therefore cannot be generalized or down-
laboratoriju i stoga ne mogu biti generalizovane niti loaded from another source, but require individual
preuzete iz nekog drugog izvora, ve} zahtevaju poje- selection, optimization, and validation. Another rea-
dina~ni izbor, optimizaciju i validaciju. Drugi razlog son limiting the use of MA procedures is the lack of
koji ograni~ava primenu MA procedura jeste nepo- insight in the ability of selected MA procedures to
znavanje detalja sposobnosti odabrane MA proce- detect clinically significant bias, especially their ability
dure da otkriva klini~ki zna~ajan bias, pri ~emu se to detect the occurrence of bias for less frequently
posebno name}e pitanje da li je ovim procedurama required tests and in laboratories with a small daily
mogu}e otkriti pojavu bias-a za re|e zahtevane number of samples and tests performed. In recent
testove i u laboratorijama koje imaju mali dnevni broj years, there has been a renewed interest of
uzoraka i ura|enih testova. Poslednjih godina ponovo researchers in this topic, with new suggestions for
raste interesovanje istraìva~a za ovu temu, sa novim ways in which MA procedures could be optimized for
predlozima za na~ine na koje bi se MA procedure routine laboratory use.
mogle optimizovati za rutinsku upotrebu.
KORI[]ENJE REZULTATA THE USE OF PATIENT RESULTS

PACIJENATA ZA IZRAÛNAVANJE FOR CALCULATION OF
REFERENTNIH VREDNOSTI REFERENCE INTERVALS
Neda Milinkovi}1, Svetlana Ignjatovi}1,2 Neda Milinkovi}1, Svetlana Ignjatovi}1,2

1Katedra za medicinsku biohemiju, Farmaceutski 1Department of Medical Biochemistry, Faculty of
fakultet, Univerzitet u Beogradu, Beograd, Srbija Pharmacy, University of Belgrade, Belgrade, Serbia
2Centar za Medicinsku biohemiju, Klini~ki centar 2Center for Medical Biochemistry,
Srbije, Beograd, Srbija Clinical Center of Serbia, Belgrade, Serbia
U cilju interpretacije rezultata u medicinsko bio- In order to interpret the results in the medical
hemijskim laboratorijama, naj~e{}e se kvantitativni biochemical laboratories, the quantitative result is
rezultat posmatra u odnosu na referentni interval most often viewed in relation to the reference interval
(RI), koji predstavlja fiksni procenat referentne (RI), which represents a fixed percentage of the ref-
populacije u intervalu koje opisuju donje i gornje erence population at the interval described by the
referentne granice. Preporu~eno odre|ivanje RI ili lower and upper reference limits. Recommended RI
direktno odre|ivanje, podrazumeva predhodan oda- determination, or direct determination, involves pre-
bir referentne populacije po ta~no definisanim krite- selecting a reference population according to well-
rijumima, uzorkovanje biolo{kog materijala i analizi- defined criteria, sampling biological material and
ranje uzoraka. Alternativni pristup odre|ivanju RI ili analyzing samples. An alternative approach to RI
indirektno odre|ivanje podrazumeva kori{}enje veli- determination, or indirect determination, involves the
kog broja postoje}ih, odra|enih rezultata iz uzoraka use of a large number of existing, extracted results
koji se sakupljaju u rutinske svrhe, iz kojih se bio- from samples collected for routine purposes, from
hemijski parametri odre|uju u svrhu skrininga, po- which biochemical parameters are determined for
stavljanja dijagnoze ili pra}enja, i koji se ~uvaju u screening, diagnosis or monitoring purposes, and
bazama laboratorijskih informacionih sistema. Veliki stored in the laboratory information system databas-
broj objavljenih radova ukazuje na prednost indirekt- es. The large number of published papers points to
no odre|enih RI uz uslov pravilne selekcije »nezdrav- the advantage of indirectly determined RIs with the
ih« osoba, kao i razlikovanja hospitalizovanih od condition of proper selection of “unhealthy” persons,
ambulantnih pacijenata, ali i kori{}enje sloènih sta- as well as separation of hospitalized from outpatients,
tisti~kih algoritama za dobijanje krajnjeg RI. Trenutne as well as the use of complex statistical algorithms to
smernice i vodi~i ne podràvaju indirektni metod kao obtain the ultimate RI. Current guidelines and docu-
primarni zbog ~injenice da ve}ina podataka mo`da ments do not support the indirect method as primary,
ne poti~e od zdravih osoba. Tako|e, statisti~ka ana- due to the fact that most data may not come from
liza koja se koristi za obradu velikog broja podataka healthy individuals. Also, the statistical analysis used
je primarno namenjena za direktno odre|ivanje RI, tj. to process large amounts of data is primarily intend-
analizu preporu~enih 120 rezultata referentne popu- ed to directly determine RI, i.e. analysis of the recom-
J Med Biochem 2022; 41 (3) 375
lacije. Me|utim, postoje posebni staisti~ki programi i mended 120 results of the reference population.
tehnike kojima je omogu}ena pravilna statisti~ka However, there are special statistical programs and
analiza velikog broja podataka za indirektno odre- techniques that allow proper statistical analysis of a
|ivanje RI. Preporuka da svaka laboratorija defini{e large amount of data for indirect RI determination.
sopstvene RI, a da to bude ekonomi~no i minimalno The recommendation that each laboratory defines its
kompleksno, daje prednost indirektnom odre|ivanju own RIs, while being cost-effective and minimally
RI, u smislu kori{}enja postoje}ih baza podataka. complex, favors indirectly determining RIs, in terms
Tako|e, nijedan RI nije apsolutno ta~an i predstavlja of using existing databases. Also, no RI is absolutely
samo procenu. Budu}a ispitivanja bi trebalo da re- correct and is only an estimate. Future trials should
guli{u eti~ke aspekte indirektnog odre|ivanja, kao i regulate the ethical aspects of indirect determination
pravilnu verifikaciju na ovaj na~in definisanih RI. as well as the proper verification of RIs defined in this
way.
NOVI BIOMARKERI AKUTNOG NEW BIOMARKERS

O[TE]ENJA BUBREGA OF ACUTE KIDNEY INJURY
Du{an Paripovi}1,2 Du{an Paripovi}1,2

1Medicinski fakultet, Univerzitet u Beogradu 1Medical School, University of Belgrade, Belgrade,
2Odeljenje nefrologije, Univerzitetska de~ja klinika, 2Department of Nephrology, University Children’s
Beograd Hospital, Belgrade
Akutno o{te}enje bubrega (AOB) je ~esto i po Acute kidney injury (AKI) is a common and life-
ìvot opasno stanje. AOB se u detinjstvu naj~e{ce threatening condition. AKI in childhood is most com-
javlja tokom prve godine ìvota. Deca sa epizodom mon during the first year of life. Infants with an
AOB imaju povecan rizik od razvoja hroni~ne bolesti episode of AKI have increased risk of developing
bubrega. AOB je reverzibilno ako se prepozna u ranoj chronic kidney disease. AKI is reversible when recog-
fazi i ako se brzo preduzmu terapijske mere. nized early and promptly treated. Serum creatinine as
Kreatinin u serumu kao tradicionalni biomarker koji traditional biomarker used to define and grade AKI is
se koristi za definisanje i ocenjivanje stepena AOB not sensitive enough. Furthermore, it takes some
nije dovoljno senzitivan biomarker. Naime, potrebno time after kidney injury or decrease in urine output
je neko vreme nakon o{te}enja bubrega odnosno until serum creatinine level rises, so creatinine is a
smanjenja diureze, da bi se nivo kreatinina u serumu late marker of AKI. In addition, serum creatinine
povisio, pa je kreatinin kasni marker AOB. Pored depends on age, gender, muscular mass and medica-
toga, kreatinin u serumu zavisi od uzrasta, pola, tion. Better biomarkers than creatinine should be
mi{icne mase i upotrebe lekova. Potrebni su nam more sensitive and specific, allowing faster diagnosis
dakle bolji biomarkeri od kreatinina, koji bi trebali biti of AKI, and use of appropriate clinical intervention
senzitivniji i specifi~niji, omogucujuci brù dijagnozu that may stop or reverse AKI. Limitations of serum
AOB i upotrebu odgovarajuce klini~ke intervencije creatinine stimulated research that developed bio-
koja moè zaustaviti ili preokrenuti AOB. Ograni~enja markers of damage in AKI including cystatin C, neu-
serumskog kreatinina podstakla su istraìvanja koja trophil gelatinase–associated lipocalin, kidney injury
su razvila biomarkere AOB, uklju~ujuci cistatin C, molecule 1, interleukin-18, liver type fatty acid–bind-
lipokalin povezan sa èlatinazom neutrofila, molekul ing protein, tissue inhibitor of metalloproteinase-2,
o{te}enja bubrega 1, interleukin-18, protein koji veè and IGF-binding protein 7. In comparison to cardiol-
masne kiseline u jetri, inhibitor tkiva metalopro- ogy, clinical use of novel biomarkers in nephrology
teinaza-2 i protein 7-vezujuci protein. U pore|enju sa has been limited. Although intensive research activity
kardiologijom, klini~ka upotreba novih biomarkera u identified several new AKI biomarkers further investi-
nefrologiji je ograni~ena. Iako je intenzivna istraì- gation is needed to define their clinical role. Factors
va~ka aktivnost otkrila nekoliko novih biomarkera influencing use of AKI biomarkers include price,
AOB, potrebna su dodatna istraìvanja kako bi se availability at the local lab or point of care, and sim-
odredila njihova klini~ka uloga. înioci koji uti~u na ple use. Application of innovative learning and artifi-
upotrebu biomarkera AOB uklju~uju cenu, dostup- cial intelligence may allow faster detection and earlier
nost u lokalnoj laboratoriji ili na mestu le~enja i jed- treatment of AKI.
nostavnu upotrebu. Primena inovativnog u~enja i
ve{ta~ke inteligencije moè omoguciti brè otkrivanje
376
ZAKASNELI PUBERTET – DELAYED PUBERTY –

IZAZOVI U DIJAGNOSTICI DIAGNOSTIC CHALLENGES
Rade Vukovi}1,2, Tatjana Milenkovi}1, Rade Vukovi}1,2, Tatjana Milenkovi}1,

Katarina Mitrovi}1, Sla|ana Todorovi}1, Katarina Mitrovi}1, Sla|ana Todorovi}1,
Sanja Pani} Zari}1 Sanja Pani} Zari}1
1Institut za zdravstvenu za{titu majke i deteta Srbije 1Motherand Child Healthcare Institute of Serbia
»Dr Vukan ûpi}« »Dr Vukan ûpi}«
2Medicinski fakultet, Univerzitet u Beogradu 2School of Medicine, University of Belgrade
Zakasneli pubertet se vi|a kod pribli`no 5% Delayed puberty can be observed in approxi-
adolescenata oba pola, a u diferencijalnoj dijagnozi, mately 5% of the adolescent population, with major-
pored konstitucionalnog zakasnelog puberteta kao ity of the affected youth having a benign variant of
naj~e{}eg uzroka, na drugom mestu se nalazi hipog- pubertal development - constitutional delay of puber-
onadotropni hipogonadizam. Veliki broj uro|enih i ty, with hypogonadotropic hypogonadism being the
ste~enih uzroka mogu dovesti do hipogonado- most frequent differential diagnosis. Multitude of
tropnog hipogonadizma, koji tako|e moè nastati i congenital and acquired etiologies can lead to hypo-
usled intenzivnih sportskih treninga, poreme}aja u gonadotropic hypogonadism, including the develop-
ishrani ili u sklopu klini~ke slike hroni~nih sistemskih ment of »functional« hypogonadotropic hypogo-
bolesti. Razlikovanje izolovanog hipogonadotropnog nadism due to secondary causes, such as vigorous
hipogonadizma od konstitucionalnog zakasnelog exercise, eating disorders or systemic chronic illness-
puberteta predstavlja veliki dijagnosti~ki izazov u pu- es. Distinguishing between constitutional delay of
bertetskom uzrastu i zasniva se na klini~kom nadzoru, puberty and isolated hypogonadotropic hypogo-
izuzev kada postoje jasne udruène fenotipske odlike nadism remains a major clinical challenge during
hipogonadotropnog hipogonadizma, poput anosmije adolescence, and is mainly based on watchful wait-
ili hiposmije u sklopu Kallmannovog sindroma. Po- ing, unless specific features suggestive of hypogo-
slednjih godina sve zna~ajniju ulogu u diferenciranju nadotropic hypogonadism are present, such as anos-
konstitucionalnog zakasnelog puberteta u odnosu na mia or hyposmia in Kallmann syndrome. During the
hipogonadotropni hipogonadizam ima odre|ivanje recent years, inhibin B levels are proving more useful
koncentracija inhibina B, a sa sve dostupnijim genet- in distinguishing between constitutional delay of
skim analizama metode molekularne dijagnostike puberty and isolated hypogonadotropic hypogo-
dobijaju sve ve}u ulogu ne samo u istraìva~kom, ve} nadism, and with the genetic analyses becoming
i u klini~kom kontekstu. U terapijskom pogledu, bez more available, molecular diagnostics are becoming
obzira da li se radi o izolovanom hipogonadotropnom increasingly important in both research and clinical
hipogonadizmu ili konstitucionalnom zakasnelom practice. Regarding treatment approach, whether the
pubertetu, kod de~aka koji imaju psiholo{ke tegobe aetiology is constitutional delay of puberty or hypog-
zbog ka{njenja ili zastoja u pubertetskom razvoju onadotropic hypogonadism, in boys with psychoso-
treba razmotriti kratkoro~nu primenu depo preparata cial complaints resulting from delayed or arrested
testosterona. Kod najve}eg broja de~aka sa hipogo- pubertal development, short-term treatment with
nadotropnim hipogonadizmom je mogu}a dalja testosterone should be considered. In most of the
indukcija pubertetskog razvoja i fertiliteta, uklju~uju}i boys with hypogonadotropic hypogonadism, com-
razvoj i porast testisa uz uspostavljanje spermato- plete pubertal development including the testicular
geneze u periodu adolescencije upotrebom huma- growth and spermatogenesis during adolescence can
nog horionskog gonadotropina i rekombinantnog be acquired by the use of human chorionic gonado-
folikulo-stimuli{u}eg hormona. tropin and follicle-stimulating hormone subcuta-
neous injections.
J Med Biochem 2022; 41 (3) 377
NOVI MARKERI SEPSE NEW MARKERS FOR SEPSIS

U NEONATOLOGIJI IN NEONATOLOGY
Ana \or|evi}-Vuji~i} Ana \or|evi}-Vuji~i}
Institut za neonatologiju, Beograd Institute of Neonatology, Belgrade
Morbiditet i mortalitet u neonatalnoj sepsi su Morbidity and mortality in neonatal sepsis are
zna~ajni uprkos kontinuiranom napretku u neona- significant, regardless of the continuous advance-
tologiji i izboru novih generacija antibiotika. Pre- ment in neonatology and emerging new-generation
terminska novoro|en~ad veoma niske telesne mase antibiotics. Preterm infants of very low weight are
su posebno osetljiva zbog imunske nezrelosti, te{kog particularly sensitive due to immune immaturity, seri-
op{teg stanja i ~este potrebe za primenom invazivnih ous general condition and frequent necessary appli-
procedura u toku le~enja. Kod ovih pacijenata je cation of invasive procedures in the course of treat-
pove}an rizik od razvoja sepse, antibiotske toksi~nosti ment. The risks of development of sepsis, antibiotic
i lo{eg ishoda le~enja. Hemokultura je »zlatni stan- toxicity and poor treatment outcomes are increased
dard« u dijagnostici bakterijske sepse, ali na rezultate in these patients. Blood culture is considered the gold
treba ~ekati 24–48 sati. Rezultati mogu biti la`no standard for diagnosis of bacterial sepsis, but the
negativni u slu~aju postojanja pneumonije ili menin- results are available after 24–48 hours. Additionally,
gitisa. Tradicionalni laboratorijski pokazatelji sepse they can be false-negative in the case of pneumonia
pokazuju nedostatke, kao {to je {irok opseg referent- or meningitis. Traditional laboratory indices of sepsis
nih vrednosti za hematolo{ke testove, zbog ~ega su have certain shortcomings, such as wide reference
nekad te{ki za interpretaciju, ili spadaju u kasne intervals for haematological tests, which make them
markere sepse, kao {to je C-reaktivni protein. Razvoj difficult to interpret, or belong to late sepsis markers,
novih tehnologija je omogu}io bolje upoznavanje such as C-reactive protein. The development of new
neonatalnog imuniteta i odgovora na infekciju kao i technologies has enabled better understanding of
otkrivanje novih biomarkera koji bi mogli da pobolj- neonatal immunity and response to infection, as well
{aju rano otkrivanje infekcije i pravovremeno zapo- as the discovery of new biomarkers which could
~injanje terapije. Pored biomarkera koji su ve} u improve early detection of infection and timely initia-
upotrebi, kao {to su C-reaktivni protein i prokalci- tion of therapy. In addition to biomarkers already in
tonin, u fazi ispitivanja ili u po~etnim fazama primene use, such as C-reactive protein and procalcitonin,
su presepsin, neki citokini, serumski amiloid A, lipo- presepsin, some cytokines, serum amyloid A, lipo-
polisaharid-vezuju}i protein i povr{inski leukocitni polysaccharide-binding protein and surface leukocyte
antigeni. Za novoro|en~e bi bila ve}a {teta da infek- antigens are in the phase of investigation or initial
cija nije dijagnostikovana i le~ena, nego la`no dijag- application phases. It would be more harmful for the
nostikovana i nepotrebno le~ena. Zato je va`nija oso- newborn if the infection was not diagnosed and treat-
bina dijagnosti~kog testa za neonatalnu infekciju ed, than to have false diagnosis and unnecessary
visoka osetljivost i negativna prediktivna vrednost treatment. Therefore, a more important feature of
blizu 100%, nego visoka specifi~nost. Mnogi autori the diagnostic test for neonatal infection is the high
smatraju da kombinacija serumskih markera infekcije sensitivity and negative predictive value near 100%,
pokazuje bolju dijagnosti~ku specifi~nost i osetljivost than the high specificity. Many authors consider that
nego pojedina~ni markeri. Kad je u pitanju neonatal- the combination of serum markers of infection shows
na populacija pri izboru biomarkera sepse se vodi better diagnostic specificity and sensitivity than indi-
ra~una o fiziolo{kim varijacijama njihovih koncen- vidual markers. When it comes to the selection of
tracija u prvim danima ìvota, kao i o vrsti i zapremini sepsis biomarkers in neonatal population, physiolog-
uzorka koji se koristi za analizu. ical variations in their levels in the first days of life and
the types and volume of the sample for analysis have
to be taken into account.
378
ULOGA LABORATORIJE U THE ROLE OF LABORATORY

DIJAGNOSTICI I PRA]ENJU IN DIAGNOSIS AND MONITORING
KOMORBIDITETA TIP 1 OF CO-MORBIDITIES IN TYPE 1
DIJABETES MELITUSA DIABETES MELLITUS
Dragana Bojanin1, Jelena Veki}2, Tatjana Dragana Bojanin1, Jelena Veki}2, Tatjana
Milenkovi}1, Vesna Spasojevi}-Kalimanovska2 Milenkovi}1, Vesna Spasojevi}-Kalimanovska2
1Institut
za zdravstvenu za{titu majke i deteta 1Mother and Child Healthcare Institute of Serbia
»Dr Vukan ûpi}«, Beograd »Dr Vukan ûpi}«, Belgrade
2Katedra za medicinsku biohemiju, 2Department of Medical Biochemistry,
Univerzitet u Beogradu – Farmaceutski fakultet University of Belgrade – Faculty of Pharmacy
Deca i adolescenti, oboleli od tip 1 dijabetes Children and adolescents with type 1 diabetes
melitusa (T1DM), imaju pove}an rizik za razvoj jedne mellitus are at increased risk for developing one or
ili vi{e pridruènih autoimunskih bolesti. Autoimunski more associated autoimmune diseases. Autoimmune
tireoiditis i celija~na bolest imaju najve}u prevalencu, thyroiditis and celiac disease have the highest pre-
a slede autoimunske bolesti vezivnih tkiva, gastroin- valence, followed by autoimmune diseases of con-
testinalnog sistema, koè i primarna adrenalna insu- nective tissue, gastrointestinal system and skin, and
ficijencija. Laboratorijski skrining na funkciju tiroidne primary adrenal insufficiency. Therefore, laboratory
`lezde i celija~nu bolest je neophodan pri postavlja- screening for thyroid function and celiac disease is
nju dijagnoze T1DM i kasnije u redovnim intervali- necessary at the diagnosis of T1DM, and later, at
ma, u cilju ranog otkrivanja i le~enja bolesti. Prema regular intervals, in order to early detect and treat the
preporukama, skrining na autoimunski tireoiditis disease. According to the recommendations, screen-
uklju~uje odre|ivanje tireostimuliraju}eg hormona i ing for autoimmune thyroiditis involves determination
antitela na tiroidnu peroksidazu. Kod asimptomatskih of thyroid stimulating hormone and antithyroid per-
pacijenata, skrining se ponavlja svake druge godine oxidase antibodies. In asymptomatic patients, screen-
posle dijagnostikovanja dijabetesa, odnosno ~e{}e u ing is repeated every second year after diabetes is
prisustvu karakteristi~nih simptoma bolesti. Odre|i- being diagnosed, or more frequently in the presence
vanje antitela na tkivnu transglutaminazu (anti-tTG of characteristic symptoms of the disease. Deter-
IgA i/ili anti-tTG IgG) je primarno u dijagnostici celi- mination of tissue transglutaminase antibodies (tTG
ja~ne bolesti kod asimptomatskih pacijenata sa IgA and/or tTG IgG) is primary in the diagnosis of
T1DM. Laboratorijski skrining se ponavlja svake celiac disease in asymptomatic patients with T1DM.
druge, odnosno svake pete godine, posle dijagno- Laboratory screening is repeated every second or
stikovanja dijabetesa. Frekventnost analiziranja zavisi every fifth year after diagnosis of diabetes. The fre-
od ispoljenih simptoma, uzrasta i genetske predis- quency of analysis depends on clinical symptoms,
pozicije pacijenta. Preporu~uje se i skrining na deficit age and the genetic predisposition of the patient.
vitamina D, posebno kod dece sa pridruènom celi- Screening for vitamin D deficiency is also recom-
ja~nom bole{}u ili promenama na koì. Autoimunske mended, especially in children with coexisting celiac
bolesti udruène sa T1DM predstavljaju dodatni rizik disease or skin disorders. Autoimmune diseases asso-
za mikro- i makrovaskularne komplikacije. Hroni~na ciated with T1DM pose an additional risk for micro-
inflamacija, koja je pratilac autoimunskih poreme}aja vascular and macrovascular complications. Chronic
i inflamacija u aterosklerozi imaju sli~ne karakte- inflammation, that accompanies autoimmune disor-
ristike, pri ~emu patofiziolo{ki ~inioci karakteristi~ni ders and inflammation in atherosclerosis have similar
za autoimunsku bolest mogu da ispolje nezavisno ili characteristics. Pathophysiological factors related to
sinergisti~ko dejstvo na razvoj ateroskleroze i pove- autoimmune disease, can have independent or syn-
}anje kardiovaskularnog rizika. Laboratorijska evalu- ergistic effect on the development of atherosclerosis
acija potencijalnog proaterogenog efekta autoimun- and increase cardiovascular risk. A laboratory evalu-
skog tireoiditisa i celija~ne bolesti, kao udruènih ation of the potential proatherogenic effect of auto-
autoimunskih bolesti, mogla bi da identifikuje dija- immune thyroiditis and celiac disease, as associated
beti~are sa pove}anim kardiovaskularnim rizikom u autoimmune diseases, could identify diabetics at
detinjstvu i adolescenciji. Odre|ivanje markera infla- increased cardiovascular risk in childhood and ado-
macije, indeksa ateroskleroze uz standardni lipidni lescence. Determination of inflammatory markers,
profil i brzine izlu~ivanja albumina, ukazalo bi na atherosclerosis indexes, standard lipids profile and
eventualni disbalans u proaterogenim i antiateroge- albumin excretory rate could indicate possible imbal-
nim komponentama kod obolelih ance between proatherogenic and antiatherogenic
components in patients.
J Med Biochem 2022; 41 (3) 379
LABORATORIJSKA DIJAGNOSTIKA LABORATORY DIAGNOSIS

ALERGIJA KOD DECE OF ALLERGIES IN CHILDREN
Iva Perovi} Blagojevi}1, Sneàna Radi}2, Iva Perovi} Blagojevi}1, Sneàna Radi}2,
Dragana Begovi}1 Dragana Begovi}1
1Slu`ba 1Department of Laboratory Diagnostic,

za laboratorijsku dijagnostiku,
KBC »Dr Dragi{a Mi{ovi} – Clinical Hospital Center »Dr Dragi{a Mi{ovi} –
Dedinje«, Beograd Dedinje«, Belgrade
2Bolnica za de~je plu}ne bolesti i TBC, 2Children Hospital for Pulmonary Diseases
KBC »Dr Dragi{a Mi{ovi} – and Tuberculosis, Clinical Hospital Center

Dedinje«, Beograd »Dr Dragi{a Mi{ovi} – Dedinje«, Belgrade
Alergijska reakcija predstavlja neo~ekivan i ne- An allergic reaction is an unexpected and inad-
adekvatan odgovor imunolo{kog sistema na razli~ite equate response of the immune system to various
faktore (alergene) iz spolja{nje sredine. Naj~e{}e se environmental factors (allergens). It is most com-
ispoljava kao reakcija rane preosetljivosti (tip I) i monly manifested as a hypersensitivity reaction (type
posredovana je alergen-specifi~nim IgE antitelima. I) and is mediated by allergen-specific IgE antibodies.
Alergijske bolesti su danas naj~e{}a hroni~na obo- Today, allergies are the most common chronic dis-
ljenja kod dece i odraslih, posebno u razvijenim zem- eases in children and adults, especially in developed
ljama. Procenjeno je da 20% svetske populacije bo- countries. It is estimated that 20% of the world’s pop-
luje od neke vrste alergije. Za razliku od drugih ulation has some type of allergy. Unlike other chronic
hroni~nih bolesti, alergijske bolesti po~inju jo{ u naj- diseases, allergies begin in the earliest childhood,
ranijem detinjstvu, a prema mi{ljenju nekih autora even prenatal according to some authors. Food aller-
~ak i prenatalno. Alergije na nutritivne alergene se gy affects 2–8% of children, most often infants and
sre}u kod 2–8% dece i to naj~e{}e u uzrastu odoj~eta children under three years of age. Results of a major
i malog deteta. Sa druge strane, rezultati velike Inter- International Study of Asthma and Allergies in
nacionalne studije astme i alergija kod dece (ISAAC) Children (ISAAC) have shown that the highest preva-
pokazali su da je najve}a prevalenca simptoma astme lence of asthma is determined in preschool and
kod dece pred{kolskog i {kolskog uzrasta. Laborato- school-age children. Laboratory diagnosis of allergies
rijska dijagnostika alergija uklju~uje ~itav niz testova includes a battery of tests that should verify an aller-
koji se koriste da potvrde alergijsku reakciju, odrede gic reaction, determine the type/mechanism of reac-
tip/mehanizam reakcije (posredovana imunoglobuli- tion (mediated by immunoglobulins or cellular medi-
nima ili }elijama), identifikuju pokreta~a/uzro~nika ators), identify triggers of allergic reaction and follow
senzibilizacije (alergen), i za pra}enje uspe{nosti ter- up therapy. Assessment of the type/mechanism of
apije. Odre|ivanje tipa/mehanizma alergijske reakci- allergic reaction involves measuring of total serum
je uklju~uje merenje koncentracije ukupnog serum- IgE concentration (screening for atopy, as well as dif-
skog IgE (skrining za atopiju, kao i razlikovanje ferentiation of atopic disease-allergy from non-atopic
atopijskih-alergija od neatopijskih bolesti-intoleranci- disease-intolerance), the eosinophilic granulocyte
ja), broja eozinofilnih granulocita u cirkulaciji, perifer- count in the blood and nasal swab (assessment of
nom razmazu krvi i razmazu brisa nosa (pomo} u current allergen exposure and asthma phenotyping),
proceni trenutne izloènosti alergenu i fenotipizaciji basophilic granulocyte count (additional parameter in
astme), te bazofilnih granulocita (dodatni parameter assessment of allergic disease) and the eosinophil
u proceni alergijske bolesti) i serumske koncentracije cationic protein – ECP concentration (eosinophil acti-
eozinofilnog katjonskog proteina – ECP (marker akti- vation parameter, for monitoring the level of inflam-
vacije eozinofila, pogodan za pra}enje stepena in- mation in asthma and the therapy). The trigger of the
flamacije kod astmati~ara i efikasnosti terapije). allergic reaction is determined indirectly by measur-
Uzro~nik alergijske reakcije se odre|uje posredno ing the allergen-specific IgE concentration. Different
merenjem serumske koncentracije alergen-specifi~- immunoassays are in use: semi-quantitative (immuno-
nog IgE. U tu svrhu se koriste razli~ite imunohemijske blot method with panels containing food/inhalative
metode: semikvantitativne (imunoblot metoda, pa- allergens) and quantitative methods (immunoassays
neli koji sadrè nutritivne/inhalatorne alergene) i characterized by high diagnostic specificity and sen-
kvantitativne metode (imunohemijske metode koje sitivity). Determination of allergen-specific IgE in
odlikuje velika dijagnosti~ka specifi~nost i osetljivost). serum is also used when skin tests or provocation
Odre|ivanje alergen-specifi~nog IgE u serumu se tests cannot be administered, as well as to monitor
koristi i kada se ne mogu primeniti ko`ni testovi the effectiveness of therapy. Although laboratory test-
(atopijski dermatitis) ili testovi provokacije (opasnost ing of allergies is necessary for the diagnosis of aller-
od anafilakse – nutritivni alergeni), kao i za pra}enje gic diseases, there are limitations that affect the
efikasnosti terapije. Iako je laboratorijsko ispitivanje accuracy of the test results. Preanalytical problems
380
alergija neophodno za postavljanje dijagnoze alergij- include time of blood collection for the measurement
skih bolesti, postoje ograni~enja koja uti~u na ta~nost of total IgE, specific IgE, ECP and eosinophilic gran-
rezultata primenjenih testova. Preanaliti~ke gre{ke ulocyte count (seasonal allergies, insect sting aller-
uklju~uju vreme uzorkovanja krvi za odre|ivanje gies, drug allergies, influence of coagulation time as
ukupnog IgE, specifi~nog IgE, ECP i broja eozinofil- well as temperature on ECP levels). Analytical prob-
nih granulocita (sezonske alergije, alergije na lekove lems are related to the use of standardized immuno-
i otrove insekata, uticaj brzine koagulacije i tempera- assays for the determination of total and specific IgE.
ture na koncentraciju ECP). Analiti~ki problemi Post-analytical problems are related to the correlation
odnose se na primenu standardizovanih imunohemij- between in vitro test results with the in vivo test
skih metoda za odre|ivanje ukupnog i specifi~nog results (skin tests, provocation tests), as well as with
IgE. Post-analiti~ki problemi odnose se na korelaciju clinical data. This correlation is most important for
izme|u rezultata in vitro testova sa rezultatima in vivo the diagnosis and control of allergic disease.
testova (ko`ni testovi, provokacijski testovi) kao i sa
klini~kim podacima. Pomenuta korelacija je najva`-
nija za dijagnozu i kontrolu alergijske bolesti.
ZNAÂJ BIOHEMIJSKIH MARKERA BIOCHEMICAL PARAMETERS

U PROCENI RIZIKA ZA RAZVOJ IN PREGNANCY COMPLICATIONS
KOMPLIKACIJA U TRUDNO]I RISK ASSESSMENT
Daniela Ardali} Daniela Ardali}
Ginekolo{ko-aku{erska klinika Gynecology and Obstetrics Clinic

»Narodni front«, Beograd, Srbija »Narodni Front«, Belgrade, Serbia
Razvoj komplikacija u trudno}i, kao {to su pre- Pregnancy complications development as

eklampsija, gestacijski dijabetes, prevremeni poro|aj, preeclampsia, gestational diabetes, preterm birth,
intrauterini zastoj u razvoju ploda, ukazuje na sve foetal intrauterine growth restriction, indicates neces-
ve}u potrebu za {to jasnijim definisanjem potencijal- sity of definition and identification of specific early
nih biomarkera ~ijim bi odre|ivanjem u ranom prvom pregnancy biomarkers for pregnancy complication
trimestru trudno}e bilo mogu}e predvideti rizi~an tok prediction. Those biomarkers would be used in order
Time se omogu}avaju pravovremena klini~ka ispiti- to preventing or treating these complications more
vanja i intervencije kod visoko rizi~nih trudno}a, u appropriately. Nowadays, combinations of several
cilju prevencije ili adekvatnijeg tretmana komplikaci- biomarkers are generally used to predict certain com-
je. Danas se u cilju predikcije odre|enih komplikacija plications of pregnancy, thus achieving greater diag-
trudno}e uglavnom koriste kombinacije nekoliko bio- nostic accuracy. In preeclampsia, combinations of
markera ~ime se postiè ve}a dijagnosti~ka ta~nost. placental growth factor (PLGF), soluble fms-like tyro-
Kada je preeklampsija u pitanju, koriste se kombi- sine kinase-1 (sFlt-1) and vascular endothelial growth
nacije placentalnog faktora rasta (PLGF), tirozin sol- factor (VEGF) are used, followed by PAPP-A and plas-
ubilnog proteina (sFlt-1) i vaskularnog endotelnog ma protein 13 (PP13) and other proteins of placental
faktora rasta (VEGF), zatim PAPP-a i plazma protein origin, including inhibin A and activin A. Risk assess-
13 (PP13) i drugi proteini placentalnog porekla kao ment for preterm birth and intrauterine growth
{to je inhibin A i aktivin A. Procena rizika za razvoj restriction, beside mentioned tests, use free beta
prevremenog poro|aja i intrauterinig zastoja u rastu, human chorionic gonadotropin (free b-HCG) and
pored navedenih testova koriste jo{ i free beta human metalloprotease (ADAM12). An aneuploidy risk
chorionic gonadotropin (free b-HCG) i metalopro- assessment also includes early first trimester screen-
teazu (ADAM12). Procena rizika za razvoj aneuploidi- ing for PAPP-A and free b-hCG. These biochemical
ja uklju~uje tako|e screening ranog prvog trimestra parameters are combined with ultrasound parame-
kojim se odre|uje PAPP-a i free b-hCG. Navedeni ters as well as risk factors parameters associated with
biohemijski parametri kombinuju se sa ultrazvu~nim mother (age, BMI, smoking, etc.), which are pro-
parametrima kao i karakteristikama i faktorima rizika cessed by an adequate software for risk assessment.
majke (godine starosti, teìna, pu{enje i dr.), {to se The aim of further investigations will be addressed to
obra|uje adekvatnim sofwaer-om za procenu rizika.U development of some new combinations of biomark-
narednom periodu trebalo bi i dalje unapre|ivati ers with associated software that would be integrate
J Med Biochem 2022; 41 (3) 381
kombinacije biomarkera uz prate}e sofwear-e koji bi screening to evaluate the risks for the development of
objedinili i kvalitetan screening za procenu rizika za these complications, as well as screening for aneu-
razvoj navedenih komplikacija, kao i screening za ploidies. The results of our studies have identified the
aneuploidije. Rezultati na{ih studija su izdvojili atero- atherogenic index of plasma (AIP) and lipid peroxida-
geni indeks palzme (AIP) i markere lipidne peroksi- tion markers as potential markers for pregnancy com-
dacije kao potencijalne markere predikcije komp- plications prediction (preeclampsia, gestational dia-
likacija u trudno}i (preeklampsija, gestacijski dijabet, betes, IUGR).
IUGR).
DIJAGNOSTI^KA TA^NOST TESTS OF OVARIAN

TESTOVA ZA PROCENU RESERVE – DIAGNOSTIC
OVARIJALNE REZERVE ACCURACY
Aleksandra Stefanovi} Aleksandra Stefanovi}
Katedra za medicinsku biohemiju, Farmaceutski Department of Medical Biochemistry, Faculty of

Ovarijalna rezerva podrazumeva veli~inu, kao i Ovarian reserve is a term that is refers to imply
kvalitet ovarijalnog pula ène odre|ene ìvotne dobi. the capacity of the ovary to produce quality egg cells.
Ona predstavlja funkcionalni kapacitet jajnika, It is a functional capacity of the ovaries that is their
odnosno njihovu biolo{ku starost i mogu}nost jajnika biological age. Ovarian reserve is a very complex
da proizvedu kvalitetne jajne }elije. Radi se o veoma clinical parameter that is primarily associated with the
kompleksnom klini~kom parametru koji je pre svega woman’s age, but also with different genetic and
uslovljen godinama starosti ène, ali i razli~itim ge- environmental factors. The respectable test for the
netskim, kao i fakorima okruènja. Idealni test za pro- ovarian reserve evaluation should be highly sensitive,
cenu ovarijalne rezerve trebao bi da bude visoko repeatable, with no variations between menstrual
osetljiv, ponovljiv, bez varijacija u rezultatima izme|u cycles and highly specific, to avoid false positives, as
ciklusa, kao i visoko specifi~an, ~ime bi se izbegli well as false negative results. Ovarian reserve tests
la`no pozitivni, kao i la`no negativni rezultati. Testovi are based on specific hormonal analyses and ultra-
su bazirani na specifi~nim hormonskim analizama i sound examinations. According to current expert’s
ultrazvu~nim pregledima. Broj antralnih folikula i opinions, antral follicles count (AFC) and the concen-
koncentracija anti-Mullerian (AMH) hormona se tration of anti-Mullerian (AMH) hormone provides
prema ve}ini autora smatraju testovima koji pokazuju more sensitive and specific results than other bio-
ve}u specifi~nost i osetljivost u odnosu na ostale biochemical tests, basal follicle stimulating hormone
hemijske testove, bazalnu koncentraciju folikostimuli- (FSH) concentration, estradiol concentration and
raju}eg hormona (FSH), koncentraciju estradiola, dynamic clomiphene citrate test. Most of these tests
kao i u odnosu na dinami~ki test sa klomifen cit- are complementary and combined. However, analy-
ratom. Ve}ina ovih testova se me|usobno dopunjuje ses have shown that the use of multiple different tests
i kombinuje, me|utim analize su pokazale da upotre- for ovarian reserve evaluation does not significantly
ba vi{e razli~itih testova za ispitivanje ovarijalne rez- improve diagnostic accuracy. The results vary
erve ne dovodi do zna~ajnog pobolj{anja dijagno- depending on the cut-off test used and the tests out-
sti~ke ta~nosti. Rezultati se razlikuju u zavisnosti od come (usually the response to ovarian stimulation in
upotrebljenih grani~nih (cut-off) vrednosti testova, the process of in vitro fertilization). Test results sug-
kao i u zavisnosti od toga koji ishod testa se prati gesting a declining ovarian reserve might be helpful
(naj~e{}e je to odgovor na stimulaciju jajnika u proin pregnancy planning. However, these results are
cesu vantelesne oplodnje). Rezultati testova koji not reliable
ukazuju na oslabljenu ovarijalnu rezervu mogu pruìti
èni informacije zna~ajne za pravovremenu interven-
ciju i ranije planiranje trudno}e, ali ne ukazuju i na
nemogu}nost za~e}a.
382
ULOGA POLNIH STEROIDA THE ROLE OF SEXUAL STEROIDS

KOD @ENA I MU[KARACA IN WOMEN AND MEN OLDER
POSLE 50. GODINE THAN FIFTY YEARS OF AGE
Svetlana Vujovi} Svetlana Vujovi}
Medicinski fakultet, Univerzitet u Beogradu, Faculty of Medicine, University of Belgrade,

Klinika za endokrinologiju, dijabetes i Clinic of Endocrinology, Diabetes and
bolesti metabolizma Klini~kog centra Srbije, Diseases of Metabolism, Clinical Center of Serbia,
Beograd, Srbija Belgrade, Serbia
Menopauza predstavlja period u zivotu ène koji Menopause represents a period in woman’s life
se javlja godinu dana posle poslednje menstruacije i starting one year after the last menstruation and con-
traje do kraja ìvota. Involutivni hopoandrogenizam u tinuing all life. Involutive hypoandrogenism in male is
mu{karaca odlikuje se padom testosterona i pojavom characterized by insufficient testosterone and typical
tipi~nih simptoma i znakova. Insuficijencija estradiola symptoms and signs. Estradiol insufficiency in
u èna i testosterona u mu{karaca dovodi do valun- women and testosterone insufficiency in male are
ga, promena raspolozenja, depresije, nervoze, lo{e characterized by hot flushes, changes in psychic sta-
koncentracije, nesanice, kardiovaskulnih bolesti, tus, depression, irritability, lack of concentration,
osteoporoze, metaboli~kog sindroma, dijabetesa i insomnia, cardiovascular diseases, osteoporosis,
zna~ajno smanjuje kvaliteta ìvota i dovodi do ve}eg metabolic syndrome, diabetes, significantly decreas-
mortaliteta. Naj~e{}i uzrok smrti svih ljudi su kardio- ing quality of life and increasing mortality rate.
vaskulne bolesti. Upravo pad gonadnih steroida Cardiovascular diseases are the most frequent cause
dovodi do dislipidemije, vazokontrikcije, promene u of all deaths. Gonadal steroid insufficiency induce
simpatktikusnom sistemu, hipertenzije, aritmija, dislipidaemia, vasocontriction, changes in symphati-
uslovljavaju}i infarkt miokarda ili sr~anu insuficijenci- cus system, hypertension, arrhythmias, leading to
ju. Najve}i denzitet receptora za testosterone je u myocardial infarction and heart insufficiency. In the
miokardu. Dve tre}ine teìne mozga ~ine krvni myocard the greatest density of gonadal steroid
sudovi, a na njima su prisutni receptori za polne receptors are found. Two thirds of brain weight are
steroide. Takodje, receptori su prisutni i u kar- blood vessels and gonadal steroid receptors are pre-
diomiocitima. Sa padom polnih steroida poja~ava se sent on them. As well, the same receptors are pre-
inflamacija preko interleukina , citokina i brojnih fak- sent on cardiomyocytes. Gonadal steroid insufficien-
tora inflamacije. Adaptivni mehanizmi slabe, a stre- cy increase inflammation by inducing cytokines and
sori dovode do sloma adaptacije i brojnih bolesti koje other inflammatory factors. Adaptive mechanism are
predstavljaju stresogeno stanje. Osteoporoza je izaz- becoming more fragile, stressores brake adaptation
vana nedovoljno{}u gonadnih steroida. Pove}ava se and induce diseases, as a stress status. Low levels of
broj fraktura. Seksualnost se zna~ajno menja odliku- gonadal steroid induce osteoporosis. Number of frac-
ju}i se smanjenom seksualnom èljom kod èna i tures are increasing. Sexuality changes are character-
impotencijom kod mu{karaca. U cilju prevencije ized by typical hypoactive sexual desire in women and
bolesti neophodno je oko 50. godine uraditi sistem- erectile dysfunction in male. In order to do a preven-
atski pregled koji obuhvata odredjivanje hormonskog tion complete examination are needed about the age
statusa, internisti~ki nalaz, nalaz ginekologa/urologa, of 50 years of age. Hormonal analysis, internal
osteodenzitometriju, ultrasonografske preglede. examination, gynecological/urological examination,
Posle obavljene dijagnostike i isklju~ivanja apsolutnih osteodensitometry, ultrasonography, mammography
kontraindikacija neophodno je uvesti supstitcionu ter- are needed. After excluding absolute contraindica-
apiju svih insuficijentnih hormona. Na taj na~in se tions hormone replacement therapy of all insufficient
spre~avaju brojne bolesti, invaliditeti, smanjuju mor- hormones are needed. In such an approach many
biditet, mortalitet i pobolj{ava kvalitet ìvota. diseases can be prevented, morbidity and mortality
rate reduced and better quality of life obtained.
J Med Biochem 2022; 41 (3) 383
METABOLIZAM GVO@\A IRON METABOLISM AND

I ISHOD TRUDNO]E PREGNANCY OUTCOME
Danica ]uji} Danica ]uji}
Laboratorija za biologiju reprodukcije, Laboratory for Biology of Reproduction,

Institut za primenu nuklearne energije – INEP, Institute for the Application of Nuclear Energy
Univerzitet u Beogradu, Beograd, Srbija – INEP, University of Belgrade, Belgrade, Serbia
Poreme}aj homeostaze gvo`|a se ~esto javlja u Dysregulation of iron homeostasis is commonly

trudno}i, kada su potrebe za gvo`|em pove}ane. seen during pregnancy, when iron needs are
Nivo peptidnog hormona hepcidina, koji je klju~ni increased. In non-complicated pregnancy, levels of
regulator metabolizma gvo`|a, opada tokom trud- peptide hormone hepcidin, a key regulator of iron
no}e, ~ime se omogu}avaju optimalna apsorpcija i homeostasis, decline through the pregnancy course,
bioraspoloìvost gvo`|a. Nedostatak gvo`|a je naj- in order to ensure optimal iron absorption and
~e{}i tip nutritivne deficijencije i uglavnom se mani- bioavailability. Iron deficiency, the most common
festuje anemijom. Anemija usled deficijencije gvo`|a nutritional deficiency, is often manifested by
je ~esta kod èna u reproduktivnom periodu: izme|u anaemia. Iron deficiency anaemia is frequent in
30 i 70% èna nema dovoljno gvo`|a u depoima pre women of reproductive age: 30-70% of women have
za~e}a, dok je oko polovine trudnica anemi~no. insufficient iron reserves before conception and
Fetus dobija gvo`|e isklju~ivo iz krvi majke, te svaki around 50% of pregnant women are anaemic.
poreme}aj homeostaze gvo`|a kod majke ima Foetus is completely dependent on mother‘s serum
posledice i po fetus. Nedostatak gvo`|a povezan je iron, so any disturbance of maternal iron homeosta-
sa pove}anim rizikom za prevremeni poro|aj, pore- sis, reflects on the developing foetus. Iron deficiency
me}ajima u rastu i razvi}u fetusa, pove}anim rizikom is related to increased risk for preterm birth, inade-
ka nastanku infekcija, anemijom, ali i komplikacijama quate foetal growth and development, anaemia,
u odraslom dobu. Primena suplemenata gvo`|a je increased risk for infection, but also to complications
uobi~ajena u praksi, ali je veoma va`no utvrditi da li in adult life. Iron supplementation is common in rou-
je zaista potrebna, proceniti odnos koristi i eventual- tine practice, but it is important to assess is it really
nih rizika, odnosno pravilno utvrditi dozu koja se pri- necessary, to estimate potential benefits and possible
menjuje. Iako je neophodno za pravilno funkcioni- risks and to optimise dosage. While iron is essential
sanje organizma, vi{ak gvo`|a moè biti {tetan. for optimal organism functioning, its excess might be
Pove}an sadràj gvo`|a doveden je u vezu sa pore- deleterious. Higher body iron content is linked to
me}ajima u metabolizmu glukoze i mogao bi imati impairment of glucose metabolism and might be
veze sa razvojem gestacijskog dijabetes melitusa. involved in development of gestational diabetes mel-
Povi{ene vrednosti gvo`|a u serumu majke imaju litus. Elevated iron levels were reported in preeclamp-
veze sa preeklampsijom (PE), stanjem koje je speci- sia (PE), pregnancy specific condition, related to high
fi~no za trudno}u, a ima za posledicu visok mor- mortality and morbidity of both mother and the new-
biditet i mortalitet i majke i novoro|en~eta. Slobodni born. The free radicals, released in the presence of
radikali, nastali usled vi{ka gvo`|a, mogu indukovati iron excess, might lead to oxidative stress and
oksidativni stres, strukturno i funkcionalno o{te}enje endothelial damage and dysfunction, contributing to
endotela, {to moè doprineti razvoju PE. U odnosu the PE development. Higher hepcidin concentrations
na zdrave trudnice, pove}ane vrednosti hepcidina su in maternal serum, compared to healthy controls, are
izmerene tokom infekcije, kao i kod gojaznih trudni- seen in infection and obesity and might adversely
ca, {to moè imati negativan uticaj na dostupnost affect the iron bioavailability. One of the topics
gvo`|a. Deo istraìvanja Laboratorije za biologiju addressed by the Laboratory for Biology of
reprodukcije usmeren je na ispitivanje uloge metabo- Reproduction is impact of iron metabolism on preg-
lizma gvo`|a na ishod trudno}e, kao i na eventualni nancy outcome, and the potential role of maternal
zna~aj hepcidina kao biomarkera u komplikacijama hepcidin as biomarker of pregnancy-related compli-
vezanim za trudno}u. cations.
384
FARMAKOGENETIKA U ONKOLOGIJI CANCER PHARMACOGENETICS

– MOĆNO ORU\E – A MIGHTY PRECISION
PRECIZNE MEDICINE MEDICINE TOOL
Milena âvi} Milena âvi}
Laboratorija za molekularnu genetiku Laboratory for Molecular Genetics

Odeljenje za eksperimentalnu onkologiju Department of Experimental Oncology
Institut za onkologiju i radiologiju Srbije Institute of Oncology and Radiology of Serbia
Moderna farmakogenetika je u potpunosti Modern pharmacogenetics has completely

transformisala le~enje onkolo{kih pacijenata omo- transformed oncological patient care enabling the
gu}avaju}i preciznu upotrebu molekularno-ciljanih precise use of molecularly targeted therapies in
terapija u odabranim pacijentima i u specifi~nom selected patients and in a specific evolution point of
trenutku evolucije njihovih tumora. Centralizovani far- their tumors. A centralized pharmacogenetics Service
makogenomski Servis je oformljen naInstitutu za was formed at the Institute for Oncology and
onkologiju i radiologiju Srbije sa ciljem da se omo- Radiology of Serbia (IORS) with the purpose of pro-
gu}i personalizovani molekularni pristup svakom srp- viding a personalized molecular approach to each
skom onkolo{kom pacijentu. Testiranje pojedina~nih Serbian cancer patient. Single-gene testing was initi-
gena je zapo~eto 2008. godine i pro{ireno na NGS ated in 2008 and expanded to NGS panels and liquid
panele i testiranje te~nih biopsija 2016. godine. Tre- biopsy testing in 2016. Currently, metastatic colorec-
nutno, u Servisu se uspe{no obavljaju analize tal cancer samples are successfully tested for the
KRAS/NRAS mutacija u metastatskom karcinomu presence of KRAS/NRAS mutations (sensitivity to
kolorektuma (osetljivost na anti-EGFR monoklonska anti-EGFR monoclonal antibodies), as well as
antitela), primarnih i ste~enih EGFR mutacija u uzna- advanced lung adenocarcinoma patients for the pres-
predovalom adenokarcinomu plu}a iz FFPE i te~nih ence of primary and acquired EGFR mutations from
biopsija (osetljivost na prvu i tre}u generaciju tirozin FFPE biopsies and/or liquid biopsy (sensitivity to first
kinaznih inhibitora), BRAF mutacija u metastatskom and third generation tyrosine kinase inhibitors),
melanomu (osetljivost na tirozin kinazne inhibitore) i metastatic melanoma patients for the presence of
BRCA1/2 mutacija u karcinomu ovarijuma (oset- BRAF mutations (sensitivity to tyrosine kinase inhi-
ljivost na PARP inhibitore). Budu}i planovi uklju~uju bitors), and ovarian cancer patients for the presence
uvo|enje prediktivnog NGS testiranja za imunoter- of somatic BRCA1/2 mutations (sensitivity to PARP
apiju odre|ivanjem nivoa mutacionog optere}enja, inhibitors). Future plans include the introduction of
mikrosatelitske nestabilnosti i deficijencije mismatch immunotherapy predictive NGS tests for the level of
mehanizma popravke DNK, kao idetekciju klini~ki tumor mutational burden, microsatellite instability
zna~ajnih onkogenih geneti~kih rearan`mana kao {to and mismatch repair deficiency, and also for clinically
su ALK, NTRK, RET, ROS1 fuzije i sl. Prate}a far- actionable oncogenic gene rearrangements as ALK,
makogeneti~ka dijagnostika je najvi{epomogla paci- NTRK, RET, ROS1 fusions etc. Patients with onco-
jentima ~iji tumori imaju onkogene vodi~-promene genically driven cancers benefit strongly from this
kada se uzmu uobzir preìvljavanje i kvalitet ìvota. companion diagnostic approach when both survival
Dalji razvoj eksperimentalnih tehnika i bioinforma- and quality of life are taken into account. Further
ti~kih analiza doprine}e boljoj nezi onkolo{kih pacije- development of experimental techniques and bioin-
nata i smanjenju tro{kova le~enja. formatics data analyses will improve overall cancer
patient care management and lower treatment costs.
J Med Biochem 2022; 41 (3) 385
BIOMARKERI PRO[IRENOG ADVANCED LIPID STATUS

LIPIDNOG STATUSA U BIOMARKERS IN
KOLOREKTALNOM KARCINOMU COLORECTAL CANCER
Aleksandra Zeljkovi}1, Sandra Vladimirov1, Aleksandra Zeljkovi}1, Sandra Vladimirov1,
Marija Mihajlovi}1, Tamara Gojkovi}1, Marija Mihajlovi}1, Tamara Gojkovi}1,
Jelena Veki}1, Aleksandra Stefanovi}1, Jelena Veki}1, Aleksandra Stefanovi}1,
Dejan Zeljkovi}2, Bratislav Trifunovi}2, Dejan Zeljkovi}2, Bratislav Trifunovi}2,
Vesna Spasojevi}-Kalimanovska1 Vesna Spasojevi}-Kalimanovska1
1Katedra za medicinsku biohemiju, Univerzitet u 1Department of Medical Biochemistry, University of
Beogradu – Farmaceutski fakultet, Beograd, Srbija Belgrade – Faculty of Pharmacy, Belgrade, Serbia
2Klinika za op{tu hirurgiju, 2Clinic for General Surgery,
Vojnomedicinska akademija, Beograd, Srbija Military Medical Academy, Belgrade, Serbia
Kolorektalni karcinom se svrstava me|u ma- Colorectal cancer is among the most prevalent
ligne bolesti sa najve}om u~estalo{}u u savremenom malignant diseases worldwide. Therefore, numerous
svetu, te su brojna biomedicinska istraìvanja posve- biomedical researches are focused to identification
}ena otkrivanju i evaluaciji prediktivnih i dijagno- and evaluation of predictive and diagnostic markers
sti~kih biomarkera za ovo oboljenje. S obzirom da of this disease. Given the fact that colorectal cancer
bolest ima kompleksnu etiopatogenezu, koja uklju- has complex aetiology, which includes a wide spec-
~uje {irok spektar metaboli~kih poreme}aja, para- trum of metabolic disturbances, lipid status parame-
metri lipidnog statusa bi mogli imati zna~ajnu ulogu ters might have a role in its prediction and diagnosis.
u dijagnostici i predikciji nastanka kolorektalnog Routine determination of serum lipid parameters in
karcinoma. Rutinsko odre|ivanje serumskih lipidnih these patients usually reveals a typical profile, charac-
parametara kod ovih pacijenata naj~e{}e pokazuje terized by decreased levels of total cholesterol,
tipi~an profil koji se karakteri{e sniènim koncentraci- triglycerides, low density lipoprotein (LDL) – choles-
jama ukupnog holesterola, triglicerida, te holesterola terol and high density lipoprotein (HDL) – choles-
sadrànog u ~esticama lipoproteina niske (LDL) i terol. Such findings could be explained by the
visoke gustine (HDL). Ovakav nalaz se obja{njava cachexia – anorexia syndrome, which is frequently
stanjem kaheksije i anoreksije, ali i pove}anim pre- seen in these subjects. However, decreased choles-
uzimanjem holesterola iz cirkulacije u maligno izme- terol concentration might as well develop as a conse-
njene }elije. Osim toga, opse`na ispitivanja markera quence of its increased uptake by malignant cells.
pro{irenog lipidnog statusa u kolorektalnom karcino- Moreover, detailed investigations of advanced lipid
mu ukazala su na prisustvo specifi~nih promena status parameters in colorectal cancer pointed
metabolizma holesterola i lipoproteinskih ~estica. U towards characteristic changes in cholesterol and
na{im istraìvanjima u ovoj oblasti izdvojio se niz lipoprotein metabolism. Our research in this area
parametara sa potencijalno zna~ajnim prediktivnim ili revealed a range of parameters with possibly signifi-
dijagnosti~kim kapacitetom u koje se ubrajaju: mar- cant predictive and diagnostic capacity, including
keri sinteze i apsorpcije holesterola, markeri metabo- markers of cholesterol synthesis and absorption,
lizma HDL ~estica i pojedini metaboliti vitamina D. markers of HDL particles metabolism and several
Ipak, pojedina~ni lipidni parametri u pravilu ne zado- vitamin D metabolites. Yet, single lipid parameters
voljavaju sve kriterijume koji se podrazumevaju za usually do not meet the criteria for reliable and effi-
pouzdane i efikasne biomarkere. U tom smislu, pred- cient biomarkers of colorectal cancer. Therefore, a
loèn je »multimarkerski pristup«, odnosno formira- novel multimarker approach is proposed, which com-
nje adekvatnih kombinacija individualnih biomarkera, prises clustering and simultaneous determination of
~ijim bi se odre|ivanjem unapredila postoje}a dijag- several individual biomarkers. It is considered that
nostika i predvi|anje nastanka bolesti. Ovakav pristup such approach might significantly improve diagnos-
u analizi parametara pro{irenog lipidnog statusa tics and prediction of various diseases. Namely, deter-
otvara brojne mogu}nosti za definisanje panela lako mination of selected lipid status parameters, within
dostupnih analita, ~ijom bi se primenom mogla po- carefully designed diagnostic panels, could enhance
bolj{ati kako dijagnostika, tako i skrining. Osim toga, both diagnosis and screening of colorectal cancer. In
integrativni »multimarkerski pristup« u istraìvanjima addition, integrative multimarker approach in
ukazuje na kriti~ne ta~ke lipidnog metabolizma biomedical investigations could shed light on critical
zna~ajne za nastanak kolorektalnog karcinoma, {to points of lipid metabolism during cancerogenesis,
unapre|uje razumevanje samog patofiziolo{kog thereby enhancing the understanding of its patho-
procesa i omogu}ava bolju prevenciju nastanka ove physiological basis and consequently, improving the
bolesti. prevention of this disease.
386
GALEKTINI KAO BIOMARKERI: GALECTINS AS BIOMARKERS:

POTENCIJAL I PERSPEKTIVE POTENTIAL AND PERSPECTIVES
@anka Boji}-Trbojevi}, Danica ]uji}, @anka Boji}-Trbojevi}, Danica ]uji},

Ljiljana Vi}ovac Ljiljana Vi}ovac
Laboratorija za biologiju reprodukcije, Laboratory for Biology of Reproduction,

Institut za primenu nuklearne energije – INEP, Institute for the Application of Nuclear Energy,
Univerzitet u Beogradu, Srbija University of Belgrade, Serbia
Galektini pripadaju {iroko rasprostranjenoj Galectins are members of a widely distributed

familiji proteina koju karakteri{e prisustvo o~uvanog protein family defined by specificity for b-galactoside
domena (engl. carbohydrate recognition domain – containing glycans and presence of conserved carbo-
CRD) odgovornog za vezivanje glikana koji sadrè hydrate recognition domain (CRD). They can be
b-galaktozidne strukture. U }eliji mogu biti prisutni u found in the nucleus, cytoplasm, at the cell surface
jedru, citoplazmi, na povr{ini }elije kao i u van}elij- as well as in the extracellular matrix. Galectins can
skom matriksu. Galektini mogu delovati unutar }elije act inside the cell mainly through protein-protein
kroz interakcije sa drugim proteinima (protein-pro- interactions and outside the cell displaying lectin
tein) i izvan }elije, pokazuju}i lektinsku aktivnost. Na activity, thereby regulating and modulating cellular
taj na~in u~estvuju u regulaciji i modulaciji }elijskih events in biological processes. In line with this multi-
doga|aja i biolo{kih procesa. U skladu sa u~e{}em u functionality, altered expression and/or function of
raznovrsnim biolo{kim funkcijama, promenjena galectins has often been associated with various
ekspresija i/ili funkcija se ~esto povezuje sa razli~itim pathologies. An increasing number of studies have
patolo{kim stanjima. Veliki broj studija identifikovao identified members of this protein family as relevant
je ~lanove ove familije proteina kao relevanne biomarkers in cancer, heart, renal and liver disease,
biomarkere u karcinomima, bolestima srca, bubrega, as well as in infections. To date, detected changes in
jetre i infekcijama. Promene u ekspresiji galektina galectin expression may aid in diagnosis of various
mogu pomo}i u dijagnozi razli~itih bolesti, mogu se diseases, can be linked to patient outcome, and
povezati sa ishodom le~enja i terapijskim pristupi- galectin-based therapeutic approaches. Increasing
ma.Veliki broj studija je ispitivao promene galektina- number of studies are mainly focused on galectin-1
1 i galektina-3 u tkivnoj ekspresiji ili u cirkulaciji kod and galectin-3 in different diseases, whether
razli~itih bolesti. Ve}ina istraìvanja je izvedena na expressed in tissue or present in circulation. The
uzorcima pacijenata obolelih od karcinoma i ~esto je majority were performed on cancer patients, often
ukazivala na postojanje veze izme|u uo~enih prome- showing correlation between changes in circulating
na u cirkulaciji i tkivnoj ekspresiji galektina. Tako|e, galectin and altered tissue expression. Accumulated
akumulirani podaci ukazuju na sve ve}i zna~aj otkri- data also indicates increasing importance of galectin
vanja i odre|ivanja galektina kod koronarnih bolesti, detection and measurement in coronary diseases,
reproduktivnih poreme}aja, bubre`ne insuficijencije i reproductive disorders, renal failure and others.
drugih oboljenja. Uprkos nekim odstupanjima, posto- Despite some discrepancies, there is ample evidence
ji dovoljno dokaza koji pokazuju zna~aj galektina kao showing significance of galectins as biomarkers.
biomarkera. Me|utim, ispitivanje galektina kod However, screening for galectin family members in
raznih bolesti je ukazalo na potrebu razvijanja adek- various diseases has pointed out a need for additional
vatnih metoda koje bi osigurale pobolj{anu osetljivost studies in order to develop adequate galectin detec-
i ta~nost kod detekcije galektina, konsenzus izme|u tion methods insuring improved sensitivity and accu-
laboratorija i uspostavljanje referentnog opsega. Dalji racy, enabling consensus between laboratories and
napredak na ovom polju zahteva integrativni i sistem- establishment of normal reference range. Further
atski pristup, kojim bi se dodatno potkrepio klini~ki progress in the field requires integrative and system-
zna~aj odre|ivanja galektina. atic approach in support of galectin determination
clinical utility.
J Med Biochem 2022; 41 (3) 387
IZAZOVI U LABORATORIJSKOJ CHALLENGES IN LABORATORY

DIJAGNOSTICI BOLESTI DIAGNOSTICS OF THYROID
TIROIDNE @LEZDE DISORDERS
Bosa Mirjani}-Azari} Bosa Mirjani}-Azari}
Katedra za patolo{ku fiziologiju, Medicinski fakultet, Department of Pathophysiology, Faculty of Medicine,

Univerzitet u Banjoj Luci, Republika Srpska University of Banja Luka, Republic of Srpska
Laboratorijski testovi imaju klju~nu ulogu u po- Laboratory testing plays a key role in the diag-
stavljanju dijagnoze i le~enju bolesti tiroidne `lezde. nosis and treatment of thyroid disease. Today,
Imunohemijske metode su danas metoda izbora za immunochemical methods are methods of choice for
odre|ivanje koncentracije hormona u krvi, zahvalju- determining the level of hormones in the blood, due
ju}i potpunoj automatizaciji, kratkom vremenu obra- to complete automatization, short processing time,
de i visokoj specifi~nosti i osetljivosti prema velikom and high specificity and sensitivity to a large panel of
panelu heterogenih molekula. Pri svakodnevnom heterogeneous molecules. When using these, seem-
kori{}enju ovih, naizgled jednostavnih, testova javlja- ingly simple tests on a daily basis, numerous interfer-
ju se brojne interferencije koje ometaju dobijanje ences occur, interfering with the obtaining accurate
ta~nih rezultata, te zahtevaju veliko poznavanje inter- results and requiring a high level of knowledge in
ferencija od strane biohemi~ara, kako bi se broj order to minimize inaccuracy. Accurate testing results
neta~nih rezultata sveo na minimum. Ta~ni rezultati are essential for successful diagnosis and successful
testiranja su neophodni za uspe{nu dijagnostiku i treatment of patients. In addition, there are other key
uspe{no le~enje pacijenata. Pored obezbe|ivanja challenges that arise in laboratory endocrinology.
ta~nih rezultata postoje i drugi klju~ni izazovi koji se Today, it is certainly the greatest challenge to stan-
javljaju u laboratorijskoj endokrinologiji, a svakako dardize and harmonize immunochemical assays,
danas su najve}i izazovi standardizacija i harmo- which is still uncompleted task, regardless the enor-
nizacija imunohemijskih testova koje, bez obzira na mous efforts. For successful diagnosis and treatment
ogromne napore koji se ulaù, nisu jo{ uvek potpune. of patients, it is necessary to determine reference val-
Za uspe{nu dijagnostiku i uspe{no le~enje pacijenata ues for thyroid hormones, which is rarely done in our
neophodno je izrada referentnih vrednosti za hor- country. Instead, we use the reference values
mone {titne `lezde u sopstvenoj populaciji, koja se obtained by the reagents manufacturers. It is also
uglavnom ne sprovodi u na{em okruènju, nego se necessary to reconsider the levels of TSH which are
koriste referentne vrednosti po preporuci proizvo|a~a significant for clinical decisions in case of hypothy-
reagenasa. Tako|e, potrebno je analizirati i nivoe roidism and which strongly depend on the method
TSH koje se koriste kao granice klini~kih odluka za used by the laboratory to measure TSH. Finally, when
hipotireozu, a koje su ovisne izme|u ostalog, o meto- interpreting the results, blood sampling time for TSH
di koju laboratorija koristi za merenje TSH. I na kraju, analysis should be considered, because TSH levels
pri interpretaciji rezultata moralo bi se uzeti u obzir i are the highest during sleep and the lowest in the late
vreme uzimanja krvi za analizu TSH, imaju}i u vidu afternoon. All of the above mentioned, without
da je nivo TSH najvi{i u vreme sna, a najniì u kasnim enough understanding and enough attention of bio-
poslepodnevnim satima. Sve gore pomenuto, bez chemists, could lead to the wrong assessment of thy-
dovoljnog razumevanja i dovoljne pa`nje biohemi- roid functional condition, with substantial conse-
~ara, moglo bi dovesti do pogre{ne procene quences for the patient.
funkcionalnog stanja {titne `lezde sa velikim
posledicama za pacijenta.
388
DOKTORI NAUKA U PHD IN IVD INDUSTRY

IVD INDUSTRIJI – OÊKIVANJA – EXPECTATIONS AND
I MOGU]NOSTI POSSIBILITIES
Gordana Dmitra{inovi} Gordana Dmitra{inovi}
Makler d.o.o., Slu`ba za stru~nu podr{ku Makler d.o.o.o., Customer Support Department,
korisnicima, Beograd Srbija Belgrade, Serbia
Poslednjih nekoliko decenija zapaà se zna~ajan Over the last few decades there have been
rast i razvoj u oblasti industrije koja se bavi medicinskim noticed considerable rise and development in med-
sredstvima. Prema definiciji, in-vitro dijagnosti~ka ical devices industrial segment. According to defini-
sredstva (IVD) obuhvataju neinvazivne testove koji se tion, in-vitro diagnostics (IVD) are non-invasive tests
izvode na razli~itom biolo{kom materijalu sa ciljem performed on biological samples used for diagnose,
postavljanja dijagnoze, radi skrininga pacijenata ili screening or therapy monitoring. IVD industry offers
pra}enja terapije. IVD industrija nudi {irok opseg wide range of different solutions, from the simplest
razli~itih re{enja po~ev{i od onih najjednostavnijih ones like the point-of-care tests for glucose monitor-
poput point-of-care testova za pra}enje nivoa gluko- ing, over the analyzers intended for routine clinical
ze, preko ure|aja namenjenih za rutinski rad klini~kih laboratories, up to the sophisticated solutions for
laboratorija do sofisticiranih tehnologija za molekular- molecular testing like the real-time PCR technology.
na testiranja poput real-time PCR tehnologije. Para- Together with the intensive development of this
lelno sa intenzivnim razvojem ove industrijske grane industry area, there is a growing need for profession-
raste i potreba za stru~nim kadrovima unutar kom- als within the companies. There is a special demand
panija. Poseban akcenat se stavlja upravo na visoko- for highly qualified employees who could offer ade-
kvalifikovano stru~no osoblje koje moè pruìti adek- quate professional support to clinical laboratories and
vatnu stru~nu podr{ku klini~kim laboratorijama i help clinicians to choose and implement appropriate
pomo}i klini~arima u odabiru i primeni adekvatnih assays in order to get timely and accurate diagnose.
testova sa ciljem postavljanja brze i ta~ne dijagnoze. Therefore, it is of the great importance to give the
Stoga je veoma va`no dati prave smernice doktoran- right guidelines to PhD students about employment
tima o mogu}nostima koje im se otvaraju u ovom possibilities in this segment after graduation, as well
segmentu, po zavr{enom doktoratu, i pruìti informa- to inform them how skills and knowledge, that they
cije na koji na~in znanja i ve{tine ste~ene tokom have already gained during studies, can be recog-
doktorskih studija mogu biti prepoznate od strane nized by employers and implemented into their
budu}ih poslodavaca i implementirane u svako- everyday work routine. Not only can they work in
dnevni rad. Ne samo da mogu da rade u sektoru za research and development department, but they can
istraìvanje i razvoj, ve} mogu konkurisati za pozicije also apply to positions in scientific marketing and sci-
u okviru nau~nog marketinga i stru~ne podr{ke. Rad entific support. The work in both multinational and
u IVD kompanijama, bilo da su u pitanju multina- local IVD companies can put in front of the young
cionalne kompanije ili kompanije lokalnog tipa, pred people great challenges they have not faced before.
mlade ljude postavlja izazove sa kojima se nisu susre- Parallel to the professional knowledge for the area
tali ranije. Pored stru~nog znanja iz oblasti za koje su they have been educated for, it demands certain
se {kolovali, zahteva se posedovanje dodatnih additional skills. Some of them, like project and time
ve{tina. Neke od njih, poput upravljanja projektima i management, are skills PhDs have already acquired.
vremenom, su ve{tine kojima su doktori nauka ve} Except them, the skills that are of special value for
ovladali. Osim njih posebno zna~ajne za rad u IVD the work in IVD industry are communication skills,
industriji su komunikacione ve{tine va`ne za ko- which are necessary for effective communication
munikaciju kako sa krajnjim korisnicima tako i sa both with end-users and management, financial and
menad`mentom, sposobnost upravljanja finansijama budget management skills, as well marketing skills
i budètima, kao i sposobnost primene marketin{kih necessary for appropriate and successful positioning
principa za pravilno i uspe{no pozicioniranje proizvo- of the final product at the market.
da na trì{tu.
J Med Biochem 2022; 41 (3) 389
DOKTORSKE STUDIJE KAO PHD AS A COMPREHENSIVE

SVEOBUHVATNA NADOGRADNJA UPGRADE FOR
ZA USPE[AN RAZVOJ KARIJERE SUCCESSFUL CARRIER
U IN VITRO DIJAGNOSTICI DEVELOPMENT IN IVD
Tijana Krnjeta Jani}ijevi} Tijana Krnjeta Jani}ijevi}
Roche Diagnostics International Ltd. Roche Diagnostics International Ltd.

Forrenstrasse 2, 6343 Rotkreuz, Switzerland Forrenstrasse 2, 6343 Rotkreuz, Switzerland
Globalni strate{ki prioriteti u in vitro dijagnostici Global IVD strategic priorities are transforma-
su transformacija zdravstvene za{tite na globalnom tion of global healthcare and improvement of out-
nivou i bolji rezultati kroz inovacije u efikasnosti testi- comes through innovation in testing efficiency, med-
ranja, klini~koj vrednosti i digitalnim re{enjima. ical value and digital insights. Innovation in medical
Inovacije u klini~koj vrednosti podrazumevaju razvoj value considers development of medically differenti-
klini~kih re{enja, koja se odnose na medicinske ated solutions, which address unmet needs and pro-
potrebe koje jo{ uvek nisu re{ene i koja obezbe|uju vide a real and superior benefit for patients. Medically
pravi benefit za pacijenta. Inovacije u klini~kim differentiated solutions (either new biomarkers, new
re{enjima, bilo da su novi biomarkeri, nove indikacije indications of existing biomarkers or clinical algo-
postoje}ih biomarkera ili digitalna re{enja, ostvaruju rithms) bring the innovation with breakthrough clini-
se kroz klinicke studije. U ulozi globalnog menadèra cal studies. In the role of Global Medical Affairs
za medicinske poslove, kandidat mora da bude spo- Manager, candidate needs to be able to select, com-
soban da prepozna, kombinuje, analizira i interpretira bine, analyze and interpret the data from different
razli~ite grupe klini~kih podataka, uklju~uju}i po- data sets, including randomized clinical trials as well
datke iz randomizovanih klini~kih studija kao i iz as real-world evidence (RWE), electronic medical
opservacionih studija, elektronskih kartona, i novijih records, and novel sources of data, such as genomics
tipova podataka, kao sto su genomika, u kombinaciji in combination with innovative ways of mining and
sa inovativnim na~inima interpretacije tih podataka. interpreting that data. PhD background is essential
Doktorat je esencijalan za uspe{no kreiranje novih for the generation of new data as well as analysis and
podataka kao i za analizu interpretaciju ve} posto- interpretation of already existing data. It allows per-
je}ih. On omogu}ava odgovaraju}e implementiranje son to implement properly medical knowledge
medicinskog znanja kao i znanja iz translacionog together with knowledge in translational sciences and
istraìvanja, epidemiologije i biostatistike. Doktorat bi most importantly biostatistics and epidemiology. PhD
dodatno trebalo da omogu}i kandidatu fokus na ino- should furthermore enable candidate to focus on
vativan na~in kreiranja novih klini~kih dokaza (mak- innovative evidence generation (leverage on RWE
simalno iskoristiti opservacione studije, korisiti alter- generation, use of other robust, credible evidence
nativne robusne kredibilne izvore preko digitalnih available on digital channels for publications) and
kanala za publikacije) i fokus na transformaciju transformation of medical engagement for proper
medicinskog angaòvanja za odgovaraju}e prenosen- dissemination of medical information (global
je klini~kih informacija (globalni pristup medicinskom approach to medical stakeholder engagement with
angaòvanju sa sofisticiranom kvalitetnom funkcijom, robust medical excellence function, rigorous analyti-
rigorozni analiti~ki pristup u identifikaciji i prioritizaciji cal approach for identification and prioritization of
klju~nih uticajnih osoba na globalnom nivou i priori- the key opinion leaders globally and coordination
tizacija izme|u dràva, pravilna upotreba digitalnih across countries, proper use of digital tools, e-con-
sredstava, saradnja sa nezavisnim edukativnim plat- gresses and other innovative methods, collaboration
formama i digitalnim programima). Menadèr medi- with independent medical education platforms and
cinskih poslova sa doktoratom bi trebalo da ima jasno digital programs). Medical Affairs Manager with PhD
razumevanje potreba korisnika I da bude u stanju da should have clear understanding of stakeholder
generi{e i dalje promovi{e solidan sadràj koji kreira. needs and be able to generate and disseminate
To moè biti pokreta~ka snaga jedinstvenog pristupa strong value story to support it. It can be the driving
kompanije u IVD u predstavljanju vrednosti svojim force behind IVD Company’s one unified collabora-
korisnicima. tive approach to delivering value to its stakeholders.
390
GENSKA EKSPRESIJA GENE EXPRESSION OF

ADIPONEKTINSKIH RECEPTORA ADIPONECTIN RECEPTORS
KOD PACIJENATA SA IN PATIENTS WITH COLORECTAL
KOLOREKTALNIM KARCINOMOM CANCER
Marija Mihajlovi}, Ana Nini}, Miron Sopi}, Marija Mihajlovi}, Ana Nini}, Miron Sopi},
Vesna Spasojevi}-Kalimanovska, Vesna Spasojevi}-Kalimanovska,
Aleksandra Zeljkovi} Aleksandra Zeljkovi}
Katedra za medicinsku biohemiju, Farmaceutski Department of Medical Biochemistry, Faculty of

fakultet, Univerzitet u Beogradu, Srbija Pharmacy, University of Belgrade, Serbia
Neadekvatan imuni odgovor je prepoznat kao Immune irregularity is recognized as one of the
jedan od faktora koji doprinose sloènoj etiopato- factors that contribute to complex etiopathogenesis
genezi kolorektalnog karcinoma (CRC). Nova istra- of colorectal cancer (CRC). Novel studies imply sig-
ìvanja ukazuju na zna~aj razli~itih adipocitokina u nificance of different adipocytokines in immune
imunolo{kom odgovoru. Adiponektin se ovde po- response. Adiponectin can be singled out, consider-
sebno izdvaja, s obzirom na njegovu ulogu u pla- ing its role in macrophages’ plasticity and polariza-
sti~nosti i polarizaciji makrofaga, pri ~emu istovre- tion, while also influencing tumor necrosis factor
meno uti~e i na nivo cirkuli{u}eg faktora nekroze alpha (TNF alpha) circulating levels. The aim of our
tumora alfa (TNF-alfa). Cilj na{e studije je bio ispiti- study was to investigate messenger ribonucleic acid
vanje nivoa informacione ribonukleinske kiseline (mRNA) levels of TNF alpha and adiponectin’s
(iRNK), TNF-alfa i adiponektinskih receptora 1 i 2 receptors 1 and 2 (adipor 1 and adipor 2), in periph-
(adipor 1 i 2) u mononuklearnim }elijama periferne eral blood mononuclear cells (PBMCs). Additionally,
krvi (M]PK). Pored toga smo ispitali i njihovu pove- we explored their association with lipids as indicators
zanost sa lipidima kao pokazateljima naru{ene of impaired metabolic control. This study included
metaboli~ke kontrole.U istraìvanje su bile uklju~ene two cohorts: CRC patients (N=73) and control group
dve grupe: pacijenti sa CRC-om (N= 73) i kontrolna (CG) (N=80). Polymerase chain reaction (PCR) was
grupa (KG) (N = 80). Za procenu relativnih nivoa employed for evaluation of relative mRNA expression
ekspresije iRNK upotrebljena je lan~ana reakcija levels, while beta actin was used as a constitutively
polimeraze (PCR), dok je beta aktin kori{}en kao expressed gene for normalization of data. Lipid status
konstitutivno eksprimiran gen za normalizaciju parameters were obtained by using commercially
podataka. Parametri lipidnog statusa odre|eni su available enzymatic methods on automated analyzer
kori{}enjem komercijalno dostupnih enzimskih meto- ILAB 300+. Normalized adipor1 and TNF alpha
da na automatskom analizatoru ILAB 300+. mRNA levels were reduced in CRC (p<0.001;
Normalizovani nivoi adipor 1 i TNF-alfa iRNK su bili p<0.050; respectively), while adipor 2 mRNA didn’t
snièni u CRC (p <0,001; p<0,050), dok se nivo differ between our two groups (p=0.442). Significant
adipor 2 iRNK-a nije zna~ajno razlikovao izme|u positive correlation was observed between TNF alpha
grupa (p = 0,442). Uo~ena je zna~ajna pozitivna mRNA and high density lipoprotein cholesterol
korelacija izme|u TNF-alfa iRNK i koncentracije (HDL-C) in CRC (r = 0.242; p<0.05), while HDL-
holesterola lipoproteina visoke gustine (HDL-H) u C levels negatively correlated with adipor 1 mRNA (r
CRC (r = 0,242; p <0, 05), dok je koncentracija = -0.262; p<0.05). Furthermore, adipor 1 mRNA
HDL-H negativno korelirala sa adipor 1 iRNK (r = - positively correlated with adipor 2 in CRC, as well as
0,262; p <0, 05). Osim toga, nivo adipor 1 iRNK je in CG (r = 0.268; p<0.05, r = 0.498; p<0.001;
pozitivno korelirao sa adipor 2, kako u CRC tako i u respectively). In CG total cholesterol correlated
KG (r = 0,268; p <0, 05, r = 0,498; p <0,001). negatively with TNF alpha and adipor1 mRNA (r =
U KG ukupni holesterol je negativno korelirao sa -0.228; p<0.05; r = -0.230; p<0.05, respective-
TNF-alfa i sa adipor 1 iRNK (r = -0.228; p <0, 05; ly). Our results suggest that disrupted metabolic con-
r = -0,230; p<0,05). Na{i rezultati ukazuju da je trol is associated with complex genetic dysregulation
naru{ena metaboli~ka kontrola povezana sa slo- of immunity. The observed relationship could repre-
ènom genetskom deregulacijom imuniteta. Dobijeni sent important novel information for cancer research,
rezultati mogli bi predstavljati novu, va`nu informa- which could be especially significant for more individ-
ciju u istraìvanjima karcinoma, koja bi mogla biti ualized approach in patients’ diagnosis and progno-
posebno zna~ajna u razvijanju individualizovanog sis. Therefore our results warrant future studies.
pristupa u dijagnozi i prognozi bolesti. Stoga dobijeni
rezultati mogu predstavljati temelj za budu}a
istraìvanja.
J Med Biochem 2022; 41 (3) 391
MARKERI HOMEOSTAZE CHOLESTEROL HOMEOSTASIS

HOLESTEROLA U VISOKORIZI^NOJ MARKERS IN HIGH-RISK
TRUDNO]I PREGNANCY
Tamara Antoni}1, Daniela Ardali}2, Tamara Antoni}1, Daniela Ardali}2,

Sandra Vladimirov1, Gorica Banjac2, Sandra Vladimirov1, Gorica Banjac2,
Petar Cabunac2, Aleksandra Zeljkovi}1, Petar Cabunac2, Aleksandra Zeljkovi}1,
Nata{a Karadòv-Orli}2, Nata{a Karadòv-Orli}2,
Vesna Spasojevi}-Kalimanovska1, Vesna Spasojevi}-Kalimanovska1,
@eljko Mikovi}2, Aleksandra Stefanovi} @eljko Mikovi}2, Aleksandra Stefanovi}
1Katedra za medicinsku biohemiju, Farmaceutski 1Department od Medical Biochemistry, Faculty of
2Ginekolo{ko – aku{erska klinika Narodni front, 2Gynecology and Obstetrics Clinic Narodni Front,
Dislipidemija koja se razvija kod èna sa pre- Dyslipidemia observed in women with pre-
eklampsijom (PEK) po nekim autorima se smatra eclampsia (PEC) is considered to be a significant fac-
zna~ajnim ~iniocem razvoja endotelne disfunkcije i tor in the development of endothelial dys-function
ne-potpune invazije trofoblasta koje se nalaze u and incomplete trophoblast invasion that underlie the
osnovi razvoja ove komplikacije trudno}e. Molekularni development of PEC. Molecular mechanisms of this
mehanizmi nastanka ove dislipidemije nisu u pot- dyslipidemia have not been fully elucidated so our
punosti razja{njeni, tako da je cilj na{e studije bio da study aimed to investigate changes in non-choles-
ispitamo promjene u koncentraciji ne-holesterolskih terol sterols (NCSs), cholesterol synthesis and
sterola (NHS), surogat markera sinteze i apsorpcije absorption surrogate markers, and to define the
holesterola, i da defini{emo metaboli~ki profil holes- cholesterol metabolic profile in a high-risk pregnancy.
terola kod èna sa visoko-rizi~nom trudno}om. Ninety pregnant women who were at risk of develop-
Devedeset trudnica koje su na osnovu vaè}ih pre- ing PEC based on valid recommendations were
poruka bile u riziku da razviju PEK uklju~eno je u included in the study and monitored longitudinally.
studiju i pra}eno longitudinalno u 4 ta~ke tokom trud- Twenty pregnant women developed PEC. Blood sam-
no}e. Dvadeset trudnica je razvilo PEK. Uzorci krvi su ples were taken once in each trimester and before
uzimani jednom u svakom trimestru i ne-posredno delivery. Circulating profiles of NCSs were deter-
prije poro|aja. Te~nom hromatografijom sa tandem mined by liquid chromatography with tandem mass
masenom spektrometrijom (LC-MS/MS) odre|ena je spectrometry (LC-MS/MS). In a high-risk group a
koncentracija NHS. U grupi sa visokim rizikom od 2. significant increase in desmosterol (p<0.001), 7-
trimestra je uo~en zna~ajan porast dezmosterola de-hydrocholesterol (p<0.05) and lathosterol
(p<0,001), 7-dehidroholesterola (p<0,05) i latos- (p<0.001), i.e. cholesterol synthesis markers, were
terola (p<0,001), tj. markera sinteze holesterola, a observed from the 2nd trimester. In PEC complicated
vrijednosti su ostale visoke do kraja trudno}e. Kod pregnancy, a significant increase was observed only
trudno}e komplikovane PEK-om zna~ajan porast je for lathosterol (p<0.05) from the 3rd trimester. First
uo~en samo za latosterol (p<0.05) i to od 3. trimes- trimester latosterol was higher in the PEC compared
tra. Latosterol u 1. trimestru je bio vi{i u grupi èna sa to the high-risk group (p<0.05), indicating choles-
PEK-om u pore|enju sa grupom sa visokim rizikom terol synthesis was higher from the onset of PEC
(p<0,05), ukazuju}i da je sinteza holesterola bila complicated pregnancy. A significant decrease in β-
pove}ana od samog po~etka trudno}e komplikovane sitosterol, cholesterol absorption marker, was
PEK-om. Zna~ajan pad b-sitosterola, markera apsorp- observed only in the high-risk group before delivery
cije holesterola, je uo~en samo u grupi sa visokim (p<0.05). First trimester β-sito-sterol was lower in
rizikom u ta~ki prije poro|aja (p<0,05). b-sitosterol u the PEC group compared to the high-risk group
1. trimestru je bio niì u grupi sa PEK-om u pore|enju (p<0.05). However, a positive corre-lation between
sa grupom sa visokim rizikom (p<0,05). Me|utim, desmosterol and β-sitosterol in the PEC group in the
pozitivna korelacija izme|u dezmosterola i b-sitostero- 1st trimester (ρ=0.459, p<0.05), implied choles-
la u 1. trimestru u grupi sa PEK-om (r=0,459; terol absorption was increased in presence of
p<0,05) ukazuje da je a-psorpcija holesterola bila increased cholesterol synthesis, i.e. cholesterol
povi{ena u prisustvu povi{ene sinteze holesterola, tj. homeostasis was disrupted early in PEC complicated
da je homeostaza holesterola bila naru{ena rano u pregnancy. In conclusion, the cholesterol metabolic
trudno}i komplikovanoj PEK-om. Dakle, dobijeni profile obtained suggests increased cholesterol syn-
metaboli~ki profil holesterola sugeri{e da je sinteza thesis and impaired cholesterol homeostasis in
holesterola povi{ena, a homeostaza holesterola women with PEC as early as the first trimester.
naru{ena ve} u prvom trimestru kod èna sa PEK-om.
392
LONGITUDINALNE PROMENE LONGITUDINAL CHANGES OF HIGH

LIPOPROTEINSKIH ÊSTICA VISOKE DENSITY LIPOPROTEIN PARTICLES
GUSTINE I ENZIMA PARAOKSONAZE AND PARAOXONASE 1 ENZYME
1 TOKOM RIZI^NE TRUDNO]E DURING RISK PREGNANCY
Gorica Markovi}1, Daniela Ardali}1, Gorica Markovi}1, Daniela Ardali}1,
Tamara Gojkovi}2, Marija Mihajlovi}2, Tamara Gojkovi}2, Marija Mihajlovi}2,
Jasmina Ivani{evi}2, Jelena Jana}2, Jasmina Ivani{evi}2, Jelena Jana}2,
Aleksandra Zeljkovi}2, Jelena Veki}2, Aleksandra Zeljkovi}2, Jelena Veki}2,
Petar Cabunac1, Nata{a Karadòv-Orli}1, Petar Cabunac1, Nata{a Karadòv-Orli}1,
Vesna Spasojevi}-Kalimanovska2, Vesna Spasojevi}-Kalimanovska2,
@eljko Mikovi}1, Aleksandra Stefanovi}2 @eljko Mikovi}1, Aleksandra Stefanovi}2
1Ginekolo{ko aku{erska klinika 1Obstetrics
and Gyneacology Clinic
»Narodni Front«, Beograd, Srbija »Narodni Front, Belgrade, Serbia
2Katedra za Medicinsku biohemiju, Farmaceutski 2Department of Medical Biochemistry, Faculty of
fakultet, Univerzitet u Beogradu, Srbija Pharmacy, University of Belgrade, Serbia
Preeklampsija (PE) je komplikacija trudno}e Preeclampsia (PE) is a pregnancy complication,

koja se karakteri{e de novo razvojem hipertenzije i characterized by de novo development of hyperten-
proteinurije posle 20-te nedelje gestacije kao i sion and proteinuria after 20th weeks of gestation
prestankom svih simptoma do 6-te nedelje nakon and disappearance of all symptoms by the 6th week
poro|aja. Incidenca PE je od 3% do 7% i predstavlja postpartum. The incidence of PE ranges from 3% to
jedan od glavnih uzro~nika smrtnosti kako fetusa 7% and is one of the leading causes of mortality for
tako i majke tokom trudno}e i poro|aja. Osnovni both the fetus and the mother during pregnancy and
patogenetski mehanizam nastanka PE je neadekvat- childbirth. The pathogenetic mechanism of PE for-
no vaskularno remodelovanje koje je neophodno za mation is inadequate vascular remodeling, which is
adekvatnu perfuziju placente i razvoj fetusa. necessary for adequate placental perfusion and fetal
Posledi~no dolazi do neadekvatne perfuzije placente, development. Consequently, inadequate placental
oksidativnog stresa, inflmacije i disfunkcije maj~inog perfusion, oxidative stress, inflammation and dys-
endotela. U PE kod trudnica se razvija dislipidemija, function of the maternal endothelium occur.
pri ~emu lipoproteinske ~estice visoke gustine (HDL) Dyslipidemia develops in PE, with high density
gube svoja antiaterogena i antioksidativna svojstva, lipoprotein (HDL) particles losing their antiathero-
doprinose}i progresiji endotelne disfunkcije. Cilj ovog genic and antioxidant properties, progressing
rada bio je pra}enje raspodele subfrakcija HDL ~esti- endothelial dysfunction. The aim of this study was to
ca i antioksidativnog kapaciteta ovih ~estica preko monitor the distribution of HDL particle subfractions
aktivnosti enzima paraoksonaze 1 (PON1), trudnica and antioxidant capacity of these particles via the
koje su u visokom riziku da razviju PE. Studija je activity of the enzyme paraoxonase1 (PON1), preg-
uklju~ila 91 trudnicu sa jednim ili vi{e faktora rizika za nant women at risk of developing PE. The study
nastanak PE. Krv je uzorkovana u ~etiri ta~ke, od included 91 pregnant women with one or more risk
prvog do tre}eg trimestra (T1, T2, T3), kao i u 37-oj factors for PE. Blood was sampled sequentially in four
nedelji gestacije pre poro|aja (T4). Aktivnost PON1 points, from the first to third trimesters (T1, T2, T3)
je odre|ena preko brzine razgradnje supstrata as well as at the 37th week of gestation before birth
paraoksona, dok je razdvajanje HDL subfrakcija (T4). PON1 activity was determined by the rate of
vrseno metodom vertikalne elektroforeze na poliakril- degradation of the paraoxonase substrate, while the
amidnom gradijent gelu. Rezultati su pokazali da je separation of HDL subfractions was performed by
relativni udeo HDL 2b subfrakcije statisti~ki zna~ajno vertical electrophoresis on a polyacrylamide gradient
ve}i u T2 u odnosu na T1 kod obe grupe ispitanica, gel. The results showed that relative proportion of
trudnica koje su razvile PE i kod trudnica koje su bile HDL2b subfractions was significantly higher in T2
u riziku da razviju PE (p<0.05). Relativni udeo velikih compared to T1 in both groups of pregnant women
HDL 2a ~estica se smanjivao tokom trudno}e, sa who developed PE as well as in high-risk group
zna~ajnom razlikom u T2 u odnosu na T1 (p<0.05). (p<0.05). The relative proportion of large HDL2a
Tako|e, relativni udeo HDL 3a ~estica je bio manji u particles was lower in T2 compared to T1 (p<0.05).
T2 u odnosu na T1 (p<0.001). Aktivnost enzima Also, relative proportion of HDL3a particles was
PON1 od po~etka trudno}e kre}e da raste i statisti~ki lower in T2 compered to T1 (p<0.05). The activity
je zna~ajno ve}a u T2 u odnosu na T1 kada posma- of PON1 enzyme from the beginning of pregnancy
tramo sve ispitanice zajedno (p<0.05), s tim da trud- starts to grow and it is significantly higher in T2 com-
nice koje su razvile PE, u startu imaju znatno ve}u pered to T1 when we look at all subject together, but
J Med Biochem 2022; 41 (3) 393
aktivnost ovog enzima kroz T1, T2 i T4 ta~ku u odno- pregnant women who developed PE, at start having
su na trudnice koje su bile u visokom riziku od razvoja significantly higher activity of this enzyme trough T1,
PE (p<0.05). Na osnovu rezultata ove studije moè T2 and T4 point compered to pregnant women who
se zaklju~iti da su trudno}e komplikovane PE pra}ene were in high risk. Based on the results of this study, it
kvalitativnim i kvantitativnim promenama HDL ~e- can be concluded that the pregnancies complicated
stica. with PE are accompanied by qualitative and quantita-
tive changes in HDL particles.
GAMA-GLUTAMIL TRANSFERAZA GAMMA-GLUTAMYL TRANSFERASE

KAO MARKER EKSTRAĆELIJSKIH AS A MARKER OF EXTRACELLULAR
VEZIKULA U SEMENOJ PLAZMI VESICLES IN HUMAN SEMINAL
ČOVEKA PLASMA
Tamara Jankovi} Tamara Jankovi}
Institut za primenu nuklearne energije, INEP, Institute for the Application of Nuclear Energy, INEP,
Univerzitet u Beogradu, Beograd, Srbija University of Belgrade, Serbia
Ekstra}elijske vezikule (EV) su membranske strukture Extracellular vesicles (EVs) are nano-sized mem-
nano veli~ine, koje se sastoje od proteina, lipida i branous structures, carrying diverse cargoes
nukleinskih kiselina. Njihov sloèn sastav u velikoj including proteins, lipids and nucleic acids. Their
meri odslikava }eliju od koje vode poreklo, a dodatno complex composition largely reflects the cells which
moè biti promenjen u zavisnosti od fiziolo{kih i they originated from and can be changed in
patolo{kih procesa. Upotreba EV u klini~koj dijag- physiological and pathological processes. With the
nostici kao oru|a »te~ne biopsije« je, stoga, sve vi{e emerging interest in the use of EVs for clinical and
u porastu. Na{e istraìvanje se bavi EV, poznatim kao diagnostic purposes, its application as a ’liquid
prostazomi, koje su izolovane iz semene plazme biopsy’ tool have exponentially growed. Our research
mu{karaca sa normospermijom i oligospermijom. deals with EVs, known as prostasomes, isolated from
Uporedna analiza molekulskih svojstava povr{ine human seminal plasma of normozoo- and oligo-
prostazoma sa aspekta glikanskog sastava, bila je zoospermic men. Comparative examination of mole-
usmerena na mogu}e razlike u manozilovanim i sija- cular properties of prostasomal surface, exemplified
linizovanim glikoproteinima. Posebna pa`nja je bila by glycan compositions as possible distinction factor,
posve}ena pra}enju membranskog glikoproteina, was focused on mannosylated and sialylated
gama-glutamil transferaze (GGT; EC 2.3.2.2.). U glycoproteins. Specifically, membranous glycoprotein
oblasti reproduktivne fiziologije, GGT se, do sada, gamma-glutamyl transferase (GGT; EC 2.3.2.2.) was
nije ispitivala kao marker prostazoma. Sticanjem monitored. So far, GGT was not studied as a prosta-
uvida u obrasce distribucije GGT na razli~itim supo- somal marker in relation to reproductive physiology.
pulacijama ili domenima EV, moè se pove}ati njen Getting insight into distribution patterns of GGT on
biomarkerski potencijal u rutinskoj laboratorijskoj different EVs subpopulation or domains can add new
dijagnostici. Intaktni ili solubilizovani prostazomi, value to its common use as a biomarker by raising its
izolovani diferencijalnim centrifugiranjem i gel filtra- laboratory diagnostic potential. Intact or detergent-
cijom, okarakterisani su pomo}u elektronske mikro- treated prostasomes, isolated by differential centrifu-
skopije, afinitetne hromatografije, odre|ivanjem gation and gel filtration, were characterized by elec-
aktivnosti GGT i blota kori{}enjem lektina konkavali- tron microscopy, affinity chromatography, GGT
na A, lektina iz p{eni~nih klica i antitela na tetra- activity and blotting using concanavalin A, wheat
spanine. Dobijeni rezultati su pokazali da distribucija germ lectin and antibodies to tetraspanins. The
GGT, generalno, prati distribuciju CD63 i N-glikana. results obtained indicated that GGT distribution gen-
U odnosu na ko-distribuciju ostalih ispitivanih erally overlapped distribution of CD63 and N-gly-
membranskih glikoproteina, molekulski obrasci po- cans. In relation to co-distribution of individual
vezani sa GGT su odraàvali razlike u prostazomima membrane glycoproteins studied, distinct GGT-asso-
mu{karaca sa normospermijom i oligospermijom. ciated molecular patterns were found to reflect
Dobijeni rezultati su otkrili GGT na EVs kao analit i differences in prostasomes from normozoo- and
referentni parametar. oligozoospermic men. Consequently, GGT on EVs as
an analyte and new reference parameter emerged.
XXII srpski kongres medicinske
i laboratorijske medicine
sa me|unarodnim u~e{}em
XXII Serbian Congress

of Medical Biochemistry
and Laboratory Medicine
with international participation
Posterske sekcije / Poster Sessions

Apstrakti / Abstracts
396
UDK 577.1 : 61 ISSN 1452-8258
J Med Biochem 41: 396 –411, 2022 Poster sessions

Posterske sekcije
P001 P001
PRESENTATION OF THE RESULTS PREZENTACIJA REZULTATA
OF PREGNANCY CHROMOSOMAL HROMOZOMSKIH ABNORMALNOSTI
ABNORMALITIES IN PHO CLINICAL TRUDNO]E U PHO KLINI^KOJ
HOSPITAL BITOLA FOR 2020 YEAR BOLNICI BITOLJA ZA 2020. GOD.
Biljana Ilkovska1, Bisera Kotevska Trifunova2 Biljana Ilkovska1, Bisera Kotevska Trifunova2
1Department of Medical Biochemistry, PHO Clinical 1Odeljenje za medicinsku biohemiju, PHO Klini~ka
Hospital Dr. Trifun Panovski, Bitola, Macedonia bolnica Dr Trifun Panovski, Bitolj, Makedonija
2Department of Dermato-venerology, Tokuda 2Odeljenje za dermato-venerologiju, bolnica Tokuda,
Hospital, Sofia, Bulgaria Sofija, Bugarska
Genetic screening for chromosomopaty is performed Genetski skrining za hromozomopatiju se sprovodi u

in the first trimester of pregnancy by determining prvom tromese~ju trudno}e odre|ivanjem fetalne
fetal nuchal translucency and pregnancy associated nuhalne translucencije i plazma proteina A povezane
plasma protein A and free human chorionic sa trudno}om i slobodnog humanog horionskog
gonadotropin hormone in maternal serum. This gonadotropina hormona u serumu majke. Studija je
study was performed in 2020 year in Clinical ura|ena 2020. godine u Klini~koj bolnici Bitola.
Hospital Bitola. A total of 503 pregnant women were Ukupno 503 trudnice su pregledane tokom prvog
screened during the first trimester. The serum was trimestra. Serum je odvojen i izmereni su proteini
separated and pregnancy associated plasma protein- plazme-A povezani sa trudno}om i slobodni beta
A and free beta human chorionic gonadotrophin hor- humani horionski gonadotropin hormon. Ultrazvu~ni
mone were measured. The ultrasound scan included pregled je uklju~ivao potpuni strukturalni pregled i
a full structural survey, and nuchal translucency. Risks nuhalnu translucenciju. Rizici za hromozomske
for chromosomal abnormalities were calculated abnormalnosti su izra~unati kori{}enjem softvera
using the software Prisca - mathematical model Prisca – matemati~ki model koji daje individualne
which gives individual risks for trisomy 21, 18 and rizike za trizomiju 21, 18 i 13. Skrining je sproveden
13.Screening was carried out in 503 pregnancies. u 503 trudno}e. Srednja starost majke bila je 22,98
Median maternal age was 22,98 ±0.37 years ±0,37 godina (raspon: 16 do 42 godine). Me|u
(range: 16 to 42 years). Among the 503 pregnant ukupno 503 trudnice, 63 (13%) fetusa je imalo
women overall, 63 (13%) fetuses had an estimated procenjeni rizik za trizomiju 21 i trizomiju 13/18. Od
risk for trisomy 21 and trisomy 13/18. Of the 440 440 (87%) slu~ajeva hromozomska abnormalnost
(87%) cases chromosomal abnormality was not nije prona|ena. Od najve}e va`nosti za trudnicu i
found.Of utmost importance for pregnant woman dru{tvo je skrining na hromozomske abnormalnosti u
and for the society is screened for chromosomal trudno}i i procenjen rizik od Daunovog sindroma,
abnormalities in pregnancy and assessed the risk of Edvardovog sindroma i Pataiovog sindroma. Ovim
Down syndrome, Edward syndrome and Patay. With skriningom }emo spre~iti njihovu pojavu i umanjiti
this screening we are going to prevent their psihi~ku i fizi~ku patnju roditelja i dru{tva, posebno u
occurrence and we’ll reduce the psychological and dana{njem savremenom dru{tvu, gde postoje
physical suffering of parents and society, especially in najrazvijenije tehnologije u industriji i prevencija je
today’s modern society, where there are the most zaista mogu}a!
developed technologies in the industry and
prevention is really possible!
J Med Biochem 2022; 41 (3) 397
P002 P002
THE INFLUENCE OF OBESITY UTICAJ GOJAZNOSTI NA
TO INFLAMMATORY AND INFLAMATORNE I ANTIOKSIDANTNE
ANTIOXIDATIVE MARKERS IN MARKERE KOD STUDENATA
UNIVERSITY STUDENTS WITH SA POVE]ANIM
INCREASED CARDIOVASCULAR KARDIOVASKULARNIM
RISK RIZIKOM
Emina ôlak1, Dragana Pap2, Ljubinka Nikoli}3, Emina ôlak1, Dragana Pap2, Ljubinka Nikoli}3,
Vesna Dimitrijevi} Sre}kovi}4 Vesna Dimitrijevi} Sre}kovi}4
1Institute of Medical Biochemistry, 1Centar za medicinsku biohemiju Univerzitetskog
University Clinical Center of Serbia, Belgrade, Serbia klini~kog centra Srbije, Beograd, Srbija
2Students Health Protection Institute, 2Zavod za zdravstvenu za{titu studenata, odeljenje
Department of Laboratory Diagnostics, Novi Sad laboratorijske dijagnostike, Novi Sad
3Department for Hematology and Transfusion 3Klinika za ginekologiju i aku{erstvo Univerzitetskog
laboratory, Clinic for Gynecology and Obstetrics, klini~kog centra Srbije, odeljenje hematolo{ke i
University Clinical Center of Serbia, Belgrade, Serbia, transfuziolo{ke laboratorije, Beograd, Srbija
4Clinic for Endocrinology, Diabetes and Metabolic 4Klinika za endokrinologiju, dijabetes i bolesti
disorders, University Clinical Center of Serbia and metabolizma Univerzitetskog klini~kog centra Srbije
School of Medicine, University of Belgrade, i Medicinski fakultet Univerziteta u
Belgrade, Serbia Beogradu, Srbija
The aim of this study was to assess the oxidative Cilj ovog rada je bio da se utvrdi status oksidativnog
stress status through the values of inflammatory stresa kroz vrednosti inflamatornih (CRP) i antioksi-
(CRP) and antioxidative parameters (SOD, GPx, GR dantnih parametara (SOD, GPx, GR i TAS), zajedno
and TAS), together with cardiovascular risk factors sa faktorima rizika za kardiovaskularne bolesti i
and anthropometric parameters in a group of obese antropometrijskim parametrima u grupi gojaznih stu-
University students. Study included 238 students denata. U ovoj studiji je bilo uklju~eno 238 studenata
(126 men and 112 women), with a mean age of (126 mu{karac i 112 èna), prose~ne starosti
22.32±1.85 years. According to the body mass 22,32±1,85 godina. U odnosu na indeks telesne
index (BMI) lower and higher than 25 kg/m2 and mase (BMI) manjeg ili ve}eg od 25 kg/m2 i obima
waist circumference (WC) of less and more than struka (WC) ve}eg ili manjed od 94 cm (za mu{-
94cm (80cm for females) the whole group of 238 karce), odnosno 80 cm (za ène), celokupna grupa
students was divided into 2 subgroups: the group at od 238 studenata je podeljena na 2 podgrupe: grupa
increased risk for CVD (n=164) and the group at sa pove}anim kardiovaskularnim rizikom (n=164) i
lower risk for CVD (n=74). Reduced SOD and GPx grupa sa snièm kardiovaskularnim rizikom (n=74).
and increased GR and TAS, inflammatory and Zna~ajno sniène vrednosti SOD-a i GPx-a a pove-
lipoprotein parameters were obtained in the high risk }ane vrednosti GR i TAS-a zajedno sa inflamatornim
group compared to the controls (p<0.05). Positive (CRP) i lipoproteinskim parametrima su dobijene u
association of CRP, TAS and GR and negative grupi gojaznih studenata u odnosu na kontrolnu
association of GPx and HDL-cholesterol with grupu (p<0,05). Pozitivna asocijacija je dobijena za
cardiovascular risk were found in obese students. CRP, TAS i GR, a negativna za GPx i HDL-holesterol
According to the ROC analysis, GR>44.8 U/L, sa faktorima rizika za kardiovaskulane bolesti u grupi
TAS>1.35 mmol/L, CRP>1.71 mg/L and HDL-cho- gojaznih studenata. ROC analiza je pokazala da su
lesterol<1.13 mmol/L had sufficient predictive abili- GR>44,8 U/L, TAS>1,35 mmol/L, CRP>1,71
ty for cardiovascular disease in obese students. mg/L and HDL-cholesterol<1,13 mmol/L zna~ajni
Significant association of antioxidant defense param- prediktori kardiovaskularnih bolesti kod gojaznih stu-
eters, anthropometric, lipid and inflammatory mark- denata. Zna~ajna asocijacija koja je dobijena izme|u
ers with increased cardiovascular risk suggest that parametara oksidativnog stresa, inflmacije, antro-
screening of these parameters is necessary and high- pometrijskih i lipoproteinskih parametara, ukazuje na
ly recommended. to da je screening ovih gojaznih ososba zaista potre-
ban i preporu~ljiv.
398
P003 P004
ABNORMALITIES IN LABORATORY LIPID PROFILE IN GERIATRIC
PARAMETERS IN PATIENTS WITH PATIENTS WITH VASCULAR
COVID-19 – CASE STUDY DEMENTIA AND
ALZHEIMER DISEASE
Roberto Cvetkovski1, Snezhana Volcheska2,
Svetlana Cekovska3 Jordan Petrov
1University Clinic for Infectious Diseases PHI Specialized Hospital for Geriatric and Palliative
and Febrile Conditions, Skopje, Macedonia Medicine »13 November« Skopje, North Macedonia
2Institute of Medical and Experimental Biochemistry,
Skopje, Macedonia
3Institute of Medical and Experimental Biochemistry, Lipids are part of the dry mass of the brain and are
Skopje, Macedonia associated with healthy and pathologic functions of
the brain. It is found that most common genetic risk
Objective: COVID-19 (CoronaVIrus Disease 2019) is factor of Alzheimer disease is ApoE e4 variant, lipids
a respiratory and multiple organ disease caused by are also involved in blood-brain barrier function and
SARS-CoV-2. Virus member of the Coronaviridae. processing of Amyloid precursor protein, inflamma-
Immunocompromised patients, older people and tion, and energy balance. Most common types of
people with chronic medical conditions/underlying dementia in our facility are Alzheimer and Vascular
conditions are at a higher risk of developing severe dementia. For this study we analyze lipid profile of 67
form. The objective of this paper is to present the patients, 37 patients with Alzheimer disease and 30
Abnormalities in laboratory parameters in survived patients with vascular dementia. 15 patients were
and in non-survived patients. male or 5 with AD and 10 with VD, and 52 female
Material and methods: Average values of several patients or 32 with AD and 20 with VD. Age range of
serum biomarkers are shown in this paper: CRP, patients was from 68 to 98 years. Serum samples
LDH, CK, ALT, AST, Urea, Creatinine, Total Bilirubin, were collected and we analyzed samples on Cobas
Total proteins, Albumin, differential blood counts for Integra 400 automated clinical chemistry analyzer for
20 hospitalized patients: 10 recovered and 10 non- total cholesterol, triglycerides, and high and low den-
survivors. Biochemical analysis and blood tests were sity lipoprotein. Results in two groups have lower lev-
performed several times in different time intervals els for cholesterol, triglycerides and LDL, also was
depending on the clinical course of the patients. found that patients with Alzheimer disease have
Results: Average results in recovered patients: lower triglyceride levels from those with vascular
Complete blood count: Hbg 132 g/L; Er 4.533×10 dementia. Mean median for triglycerides in AD group
3 /mL; Leuk 7,1×10 3 /mL; Tromb 245×10 3 /mL; was 1.12 mmol/L IQR=0.5 different from those with
Hct 0,39%; Neutr 0,58%; Limf 0,30%; Mono 0,10%; VD 1.41 mmol/L IQR=1.0. High density lipoprotein
Eoz 0,02%; Biochemical parameters: TBIL 9 mmol/L; was significantly lower in patients with VD Mean
UREA 4,2 mmol/L; CREA 50 mmol/L; GLUC 6 Median 0.913 mmol/L IQR=0.6 and normal in AD
mmol/L; ALT 58 IU/L; AST 45 IU/L; LDH 273 IU/L; patients 1.21 mmol/L IQR=0.3. From those results
CK 41 IU/L; TP 68 g/L; Alb 36 g/L; CRP 22 mg/L. we can propose that high triglyceride levels are char-
Average results in patients with fatal outcome: acteristic for vascular dementia and must consider
Complete blood count: Hbg 127 g/L; Er 4.422× the link between Stable levels of high density lipopro-
103/mL; Leuk 12,7×103 /mL; Tromb 256×103 /mL; tein and Alzheimer Disease.
Hct 0,38%; Neutr 0,84%; Limf 0,11%; Mono 0,05%;
Eoz 0,01%. Biochemical parameters: TBIL 10 Alzheimer disease Vascular Dementia
mmol/L; UREA 18,0 mmol/L; CREA 171 mmol/L;
Cholesterol (4.1) (3.79)
GLUC 8,5 mmol/L; ALT 79 IU/L; AST 84 IU/L; LDH
847 IU/L; CK 506 IU/L; TP 64 g/L; Alb 32 g/L; CRP Triglycerides (1.12) IQR=0.5 (1.41) IQR=1
220 mg/L.
Conclusion: No significant changes/abnormalities were HDL (1.21) IQR=0.3 (0.913) IQR=0.6
noticed in the blood count of recovered patients;
serum biomarkers ALT, LDH, CRP were slightly ele-
vated. In non-survived patients significant laboratory
abnormalities were noticed; neutrophilia with lym-
phopenia, multiple elevated levels of LDH (4x), CK
(3x) and CRP (20x).
Key words: COVID-19, blood count test, biochemical
parameters.
J Med Biochem 2022; 41 (3) 399
P005 P005
PORE\ENJE KONCENTACIJA COMPARISON OF CALCIUM AND
KALCIJUMA I MAGNEZIJUMA MAGNESIUM CONCENTRATIONS
ODRE\ENO U UZORCIMA DETERMINED IN HEPARINIZED
HEPARINIZIRANE PUNE KRVI WHOLE BLOOD AND PLASMA
I PLAZME SAMPLES
Neda Milinkovi}1, Nevena Ivanovi}2, Neda Milinkovi}1, Nevena Ivanovi}2,

Marija Sari} Matutinovi}1, Ksenija Veli~kovi}3, Marija Sari} Matutinovi}1, Ksenija Veli~kovi}3,
Briìta \or|evi}2, Branimir Radosavljevi}4, Briìta \or|evi}2, Branimir Radosavljevi}4,
Sneàna Jovi~i}1,5, Svetlana Ignjatovi}1,5 Sneàna Jovi~i}1,5, Svetlana Ignjatovi}1,5
1Katedra za medicinsku biohemiju, Univerzitet u 1Department of Medical Biochemistry, University
Beogradu-Farmaceutski Fakultet, Beograd, Srbija of Belgrade – Faculty of Pharmacy, Belgrade, Serbia
2Katedra za Bromatologiju, Univerzitet u Beogradu 2Department of Bromatology, University of
– Farmaceutski Fakultet, Beograd, Srbija Belgrade – Faculty of Pharmacy, VBelgrade, Serbia
3Katedra za biologiju }elije i tkiva, Biolo{ki fakultet 3Department of Cell and Tissue Biology, Faculty of
Univerziteta u Beogradu, Beograd, Srbija Biology, University of Belgrade, Belgrade, Serbia
4Institut za hemiju u medicini, Medicinski fakultet 4Institute of Chemistry in Medicine, Faculty of
Univerziteta u Beogradu, Beograd, Srbija Medicine, University of Belgrade, Belgrade, Serbia
5Centar za medicinsku biohemiju, Univerzitetski 5Center for Medical Biochemistry, Clinical Center
klini~ki centar Srbije, Beograd, Srbija of Serbia, Belgrade, Serbia
Kalcijum (Ca) i magnezijum (Mg) su minerali od Calcium (Ca) and magnesium (Mg) are minerals of
velikog zna~aja za regulaciju mnogih procesa u orga- great importance for the regulation of many process-
nizmu. Tradicionalno se koncentracije ukupnog Ca es in the body. Traditionally, the concentrations of
(uCa) i ukupnog Mg (uMg) odre|uju u serumu, {to je total calcium (tCa) and total magnesium (tMg) are
i naj~e{}i tip uzorka u medicinsko biohemijskoj labo- determined in serum, which is the most common
ratoriji. Jonizovani oblici ovih minerala (jCa i jMg) se type of sample in the medical biochemical laboratory.
odre|uju u uzorcima pune krvi. Iako je serum Ionized forms of these minerals (iCa and iMg) are
naj~e{}i i osnovni uzorak za odre|ivanje ve}ine bio- determined in whole blood samples. Although serum
hemijskih analita i dalje postoji kontinuirana nau~na is the most common and basic sample for determin-
debata o tome koji tip uzorka moè biti uzorak izbora. ing most biochemical analytes, there is still an ongo-
Cilj ovog ispitivanja je da se analiziraju parametri sta- ing scientific debate about which type of sample can
tusa Ca i Mg u uzorcima pune krvi i heparinizirane be the sample of choice. The aim of this study was to
plazme. Istraìvanjem je obuhva}eno 87 uzoraka analyze the parameters of Ca and Mg status in whole
populacije zdravih studenata prose~ne starosti od 23 blood and heparinized plasma samples. The study
godine. Ukupne koncentracije Ca i Mg odre|ene su included 87 samples of the population of healthy stu-
na biohemijskom analizatoru Olympus AU400 dents with an average age of 23 years. Total Ca and
(Beckman Coulter Diagnostics, Hamburg, Nema~ka) Mg concentrations were determined on an Olympus
u uzorcima heparinizirane plazme. Koncentracije jCa AU400 biochemical analyzer (Beckman Coulter
i jMg izmerene su na Stat Profile Prime Critical Care Diagnostics, Hamburg, Germany) in heparinized
Analyzer (New Biomedical Corporation, Waltham, plasma samples. Concentrations of iCa and iMg were
MA, SAD) iz heparinizirane pune krvi. Indeksi measured on a Stat Profile Prime Critical Care
uMg/uCa i jMg/jCa su izra~unati ra~unski. Izmerene Analyzer (New Biomedical Corporation, Waltham,
koncentracije ispitivanih parametara, izuzev uCa, MA, USA) from heparinized whole blood. Also,
pratile su normalnu raspodelu podataka (P > 0,05). tMg/tCa and iMg/iCa indices were calculated. The
Dobijena je statisti~ki zna~ajna korelaciju izme|u uCa indices tMg/tCa and iMg/iCa were calculated. The
vs jCa i uMg vs jMg (rho = 0,307; P = 0,004 i rho measured concentrations of the examined parame-
= 0,281; P = 0,008, redom). Utvr|eno je i da ters, except for tCa, followed the normal distribution
izme|u vrednosti uMg vs jMg/jCa i jMg vs uMg/uCa of data (P> 0.05). A statistically significant correla-
postoji jaka pozitivna korelacija (rho = 0,286; P = tion was obtained between tCa vs iCa and tMg vs iMg
0,007 i rho = 0,267; P = 0,013, redom). Tako|e je (rho = 0.307; P = 0.004 and rho = 0.281; P =
utvr|ena zna~ajna pozitivna korelacija izme|u indek- 0.008, respectively). It was also found that there is a
sa (rho = 0,312; P = 0,003). Me|utim, kona~no strong positive correlation between the values of tMg
slaganje izme|u ispitivanih parametara nije utvr|eno. vs iMg/iCa and iMg vs tMg/tCa (rho = 0.286; P =
Iako rezultati ovog ispitivanja ukazuju da postoji 0.007 and rho = 0.267; P = 0.013, respectively). A
400
zna~ajna korelacija izme|u parametara statusa Ca i significant positive correlation was also found
Mg izmereno u razli~itom tipu uzoraka, potrebne su between the indices (rho = 0.312; P = 0.003).
budu}e prospektivne, dobro kontrolisane studije, i na However, the final agreement between the examined
specifi~nim populacijama ispitanika, kako bi se parameters was not determined. Although the results
potvrdila zna~ajna povezanost i slaganje i mogu}a of this study indicate that there is a significant corre-
predikcija vrednosti izme|u ukupnih i jonizovanih lation between Ca and Mg status parameters meas-
oblika ovih minerala. ured in different sample types, future prospective,
well-controlled studies, and examination on specific
patient populations, are needed to confirm signifi-
cant correlation and agreement and possible predic-
tion of values between total and ionized forms of
these minerals.
P006 P006
UTICAJ DUŽINE DIJALIZIRANJA INFLUENCE OF DIALYSIS
NA PARAMETRE MINERALNO LENGTH ON PARAMETERS OF
KO[TANOG METABOLIZMA KOD MINERAL BONE METABOLISM
BOLESNIKA NA HRONIČNOJ IN PATIENTS ON CHRONIC
HEMODIJALIZI HEMODIALYSIS
Aleksandra Stefanovi} Tomi}1, Biljana Dap~evi}2 Aleksandra Stefanovi} Tomi}1, Biljana Dap~evi}2
1Centar za laboratorijsku dijagnostiku 1Centerfor laboratory diagnostics,
JZU Dom zdravlja Herceg-Novi Primary Healthcare Center Herceg-Novi
2Odjeljenje za hemodijalizu, JZU Dom zdravlja 2Hemodialisys Department, Primary Healthcare
Herceg-Novi, Crna Gora Center Herceg-Novi, Montenegro
Hroni~na bubre`na insuficijencija (HBI) je progre- Chronic renal failure (HBI) is a progressive and irre-
sivno i ireverzibilno o{te}enje bubrega uz smanjenje versible damage to the kidneys with a decrease in the
broja funkcionalnih nefrona koje dovodi do potupnog number of functional nephrons, which leads to a
gubitka bubre`ne funkcije i potrebe za lije~enjem complete loss of kidney function and the need for
hemodijalizom. Jedna od komplikacija koja se javlja u hemodialysis treatment. One of the complications
sklopu HBI je i poreme}aj mineralno-ko{tanog that occurs within HBI is a disorder of mineral-bone
metabolizma {to vremenom dovodi do ko{tane metabolism, which eventually leads to bone disease.
bolesti. Cilj istraìvanja je bio procjena uticaja duìne The aim of the study was to assess the influence of
dijaliznog staà na biohemijske parametre mineralno the length of dialysis on the biochemical parameters
ko{tanog metabolizma kod pacijenata na hemodijal- of mineral and bone metabolism in patients on
izi. Istraìvanje je obuhvatilo 35 pacijenata prosje~ne hemodialysis. The study included 35 patients with an
starosti 62.94±14.85 godina, podjeljenih u 3 grupe average age of 62.94 ± 14.85 years, divided into 3
u odnosu na duìnu dijaliznog staà (I grupa-do 5 groups according to the length of dialysis (I group-up
godina, II grupa-5 do 10 godina; III grupa-preko 10 to 5 years, II group-5 to 10 years; III group-over 10
godina). Na{im istraìvanjem smo pokazali da su vri- years). Our research has shown that phosphorus val-
jednosti fosfora povi{ene kod gotovo svih pacijenata ues are elevated in almost all patients in all groups.
u svim grupama. Vrijednosti kalcijuma su pribli`no Calcium values are approximately the same in all
iste u svim grupama. Vrijednosti PTH su niè kod groups. PTH values were lower in patients in group I
pacijenata u I grupi u donosu na pacijente u II i III. compared to patients in groups II and III.
Dok su vrijednosti ALP ne{to viso~ije u I u odnosu na
II i III grupu.
J Med Biochem 2022; 41 (3) 401
P007 P007
STATUS VITAMINA D KOD VITAMIN D STATUS IN COVID-19
BOLESNIKA SA KOVID-19 I PATIENTS AND IT’S INFLUENCE
UTICAJ NA TEŽINU BOLESTI ON DISEASE SEVERITY
Marina Chubrinoska1, Verica Jakjimoska2 Marina Chubrinoska1, Verica Jakjimoska2
1,2Centralna biohemiska laboratorija, 1,2CentralBiochemical Laboratory,
Gradska op{ta bolnica »8 Septembar« – City General Hospital »8th September« –
Skoplje, Severna Makedonija Skopje, North Macedonia
Vitamin D je uklju~en u modulaciju uro|enog i Vitamin D is involve in the modulation of the innate
ste~enog imunog sistema, proizvodnju antimikrobnih and acquired immune system and also in the
peptida, kao i u ekspresiji gena uklju~enih u production of antimicrobial peptides, as well as in the
intracelularno uni{tavanje patogena. Nizak nivo 25- expression of genes involved in the intracellular
OHD u serumu se ~esto nalazi kod starijih osoba ili destruction of pathogens. Low serum 25OHD levels
kod onih sa hroni~nim stanjima, koji su tako|e are frequently found in elderly individuals or in those
prijavljeni kao lo{i prognosti~ki faktori za COVID-19. with chronic conditions, which have also been
Smanjenje ACE2 od strane SARS-CoV-2 dovodi do reported as poor prognostic factors for COVID-19.
disregulacije sistema renin-angiotenzin, {to doprinosi The downregulation of ACE2 by SARS-CoV-2 leads
»oluji citokina« koja prethodi sindromu akutnog to a dysregulation of the renin–angiotensin system,
respiratornog distresa karakteristi~nom za te{ki oblik which contributes to the »cytokine storm« that
COVID-19. U tom smislu, vitamin D moè inhibirati precedes the acute respiratory distress syndrome
proizvodnju proinflamatornih citokina u ljudskim characteristic of the severe form of COVID19. In this
monocitima/makrofagima, a hroni~ni nedostatak sense, vitamin D can inhibit proinflammatory cyto-
vitamina D moè izazvati aktivaciju RAS, {to dovodi kine production in human monocytes /macrophages,
do proizvodnje fibroznih faktora i, prema tome, o{te- and chronic vitamin D deficiency may induce RAS
}enja plu}a. S obzirom na razlike u teìni i fatalnosti activation, leading to the production of fibrotic
COVID-19 u svetu, va`no je razumeti razloge koji factors and, therefore, lung damage. Considering the
stoje iza toga. Pobolj{anje imuniteta kroz bolju differences in the severity and fatality of COVID-19 in
ishranu moè biti zna~ajan faktor. Ova studija je the globe, it is important to understand the reasons
procenila korelaciju koncentracija vitamina D sa behind it. Improvement of immunity through better
slu~ajevima COVID-19 i njegov uticaj na teìnu i nutrition might be a considerable factor. This study
smrtnost od COVID-19. U studiju su uklju~ena 83 evaluated the correlation of vitamin D concentrations
pacijenta (55,2% mu{karaca, prose~ne starosti 57 with COVID-19 cases and its impact on the severity
godina i 45,8% èna prose~ne starosti 56 godina) sa and mortality of COVID-19. Included in the study
potvr|enom COVID-19 pneumonijom, dijagnosti- were 83 patients (55.2% men, mean age was 57
kovan i le~en, izme|u 1. juna i 12. avgusta 2020. years and 45.8% women mean age 56 years) with
godine u Gradskoj op{toj bolnici »8. Septembra«- confirmed COVID-19 pneoumonia, diagnosed and
Skoplje. U celoj studiji prime}en je veoma nizak nivo treated , between 1 June and 12 August 2020 in City
vitamina D kod oba pola. Medijan VitD je bio General Hospital »8th of September«-Skopje. In the
zna~ajno niì kod èna (28,01 nmol/L) u odnosu na entire study, very low vitamin D levels are observed in
podgrupu mu{karaca (43,96 nmol/L). Uo~eno je da both genders. Median VitD level was significantly
kod èna, iako je manji procenat hospitalizovanih od lower in the female (28.01 nmol/L) versus the male
COVID-19, one imaju ve}u stopu mortaliteta koja subgroup (43.96 nmol/L). It has been noticed that in
iznosi 18,42%, u pore|enju sa mu{karcima kod kojih women, althought the percentage of hospitalized
iako imamo ve}i procenat hospitalizovanih, mortalitet from COVID-19 is lower, they have a higher mortality
je manji i iznosi 8,9 % . Tako|e, duìna hospitalizacije rate which is 18.42%, compared to men where
kod èna je duà, 19 dana, u odnosu na mu{karce althought we have a higher percentage of
koja iznosi 15,5 dana. Ukratko, ova opservaciona hospitalized, mortality is lower and is 8.9%. Also the
studija me|u pacijentima sa COVID-19 koji su do- length of hospitalization among women is longer, 19
ìveli definitivan ishod, pokazuje povezanost izme|u days, compared to men which is 15.5 days. In
VitD statusa i teìne i mortaliteta od COVID-19. summary, this observational study among patients
with COVID-19 who have experienced a definite
outcome, shows an association between VitD status
and severity of and mortality from COVID-19.
402
P008 P009
NEUTRALIZING ANTIBODIES (NABS) EVALUATION OF ANTI-SARS-COV-2
AFTER IMMUNIZATION WITH ANTIBODY RESPONSES IN
GAM-COVID-VAC VACCINE MACEDONIAN HEALTHCARE
IN A SAMPLE OF HEALTHCARE WORKERS: AN INTERIM ANALYSIS
WORKERS
Hristina Ampova, Melda Emin,
Melda Emin, Hristina Ampova, Elena Petrusevska-Stanojevska, Irena Kostovska,
Elena Petrusevska-Stanojevska, Irena Kostovska, Sonja Topuzovska, Jasna Bogdanska,
Sonja Topuzovska, Jasna Bogdanska, Katerina Tosheska-Trajkovska
Institute of Medical and Experimental Biochemistry,
Institute of Medical and Experimental Biochemistry, Medical Faculty, University »Ss Cyril and Methodius«,
Medical Faculty, University »Ss Cyril and Methodius«, Skopje, 50 Divizija No 6, 1000 Skopje
Skopje, 50 Divizija No 6, 1000 Skopje
Amidst the COVID-19 pandemic, healthcare workers
Background: The process of conferring immunity are exposed to an anticipated higher risk of infection
after vaccination for any virus can be best estimated with SARS-CoV-2 considering their work environ-
by determining the levels of specific neutralizing anti- ment, than other members of society. Antibody test-
bodies (NAbs). The specific immune response can ing and seroprevalence of COVID-19 antibodies can
diminish over the course of few months, leading to be a beneficial tool for comprehension of the inci-
uncertainty about how long the patients are protect- dence of disease exposure in this population. This
ed. The aim of the present study is to evaluate the study aims to examine and evaluate the level of
presence of NAbs after vaccination with the Gam- SARS-CoV-2 antibodies among HCW in North
COVID-Vac vaccine (viral vector vaccine). Macedonia during the period from 28/05/2020 to
Material and Methods: The sample consisted of 131 20/08/2020. A total of 2334 HCW have been tested
healthcare workers (HCW), out of which 66 patients for SARS-Cov-2 IgM and IgG antibody assays, using
were female, while the median age of the sample the Chemiluminescent (CLIA) method on the
was 36 years (IQR 32-42). At enrollment, patients Maglumi 800 platform. Out of the 2334 HCW test-
provided baseline data and information about previ- ed, 1676 (71.87%) were women and 656 (28.13%)
ous SARS-CoV-2 infections. Patients were examined were men. The age range was between 19 and 70
6 months after 2nd dose of vaccine for the presence years old with the mean age being 45.23. A total of
of NAbs. The blood samples were analyzed by the 195 HCWs tested positive for either IgM or IgG anti-
CLIA method with the MAGLUMI 800 analyzer. SARS-CoV-2 serum antibodies. Of them, 167 in-
Results: From the whole sample, 38 patients report- dividuals tested positive for IgM antibodies and 54
ed previous known SARS-CoV-2 infection. Six tested positive for IgG antibodies. The cumulative in-
months after the vaccination, all patients with previ- cidence during the period from 28/05/2020 to
ous infection achieved NAbs above the threshold 20/08/2020 of anti-SARS-CoV-2 antibody response
value of 0.3 , while from the other group only two in HCWs was estimated at 8.355% (95% CI =
patients had NAbs levels below 0.3. The median 7.279–9.57%). HCWs represent a population predis-
value of the NAbs in the whole sample was 1.56 posed to getting infected with COVID-19. We report
(IQR 0.42 – 5.73), while patients with previous a relatively low seroprevalence rate in the tested
SARS-CoV-2 infection had median value of 6.45 group among HCW, in the set period, which can be
(IQR 4.16 – 9.03), reaching striking difference when due either to an early test request by the participants
compared to patients without previous infection (p < or increased perception of risk and proper preventa-
0.001). tive behavior.
Conclusion: The immunization with the Gam- Keywords: SARS-CoV-2, health care workers, anti
COVID-Vac produced NAbs titer above the threshold SARS CoV-2 IgM/IgG antibodies, CLIA, COVID-19
value in 98.5% of the participants, six months after
the second dose. Participants with previous docu-
mented infection had substantially higher titer of
NAbs, leaving room for further exploration on the
best practice for immunization in this group of partic-
ipants.
Keywords: MAGLUMI 800; neutralizing antibodies;
SARS-CoV-2; Gam-COVID-Vac; immunization; vac-
cine
J Med Biochem 2022; 41 (3) 403
P010 P010
VITAMIN D STATUS KOD VITAMIN D STATUS IN OVERWEIGHT
PREDGOJAZNE I GOJAZNE AND OBESE SCHOOLCHILDREN
[KOLSKE DJECE U PODGORICI IN PODGORICA
Marina Jak{i}1, Milica Martinovi}2, Marina Jak{i}1, Milica Martinovi}2,

Mirjana Nedovi}-Vukovi}3 Mirjana Nedovi}-Vukovi}3
1Klini~kicentar Crne Gore, Institut za bolesti djece, 1Clinical Center of Montenegro, Institute for
Odjeljenje za laboratorijsku dijagnostiku, Children’s Diseases, Center for Laboratory
Podgorica, Crna Gora Diagnostics, Podgorica, Montenegro
2Medicinski fakultet, Univerzitet Crne Gore, 2University of Montenegro, Medical Faculty,
Katedra za patolo{ku fiziologiju i laboratorijsku Department of Pathophysiology and Laboratory
medicinu, Podgorica, Crna Gora Medicine, Podgorica, Montenegro
3Institut za javno zdravlje Crne Gore, 3Institute for Public Health of Montenegro, Center
Centar za evidenciju podataka i istraìvanja u oblasti for Data Evidence and Research in Public Health,
javnog zdravlja, Podgorica, Crna Gora Podgorica, Montenegro
Brojne studije sugeri{u udruènost gojaznosti i Numerous studies suggest an association between
nedostatka vitamina D kod djece i odraslih. Kao obesity and vitamin D deficiency in children and
obja{njenja se ~esto navode poja~ana akumulacija i adults. Increased accumulation and altered vitamin D
izmijenjen metabolizam vitamina D u hipertrofi~nom metabolism in hypertrophic adipose tissue are often
adipoznom tkivu. Cilj: ispitivanje serumskih vrijednos- cited as explanations. Objective: to examine the
ti vitamina D kod predgojazne i gojazne djece {kol- serum levels of vitamin D in overweight and obese
skog uzrasta u odnosu na njihove normalno uhra- school aged children in relation to their normal
njene vr{njake. Istraìvanje je obuhvatilo 202 {kolske weight peers. The survey included 202 schoolchild-
djece uzrasta 7–15 godina (63,9% dje~aka, 36,1% ren aged 7–15 (63.9% boys, 36.1% girls) from
djevoj~ica) iz Podgorice, Crna Gora. U~esnici su podi- Podgorica, Montenegro. Participants were divided
jeljeni u 3 grupe prema nutritivnom statusu (kriteriju- into 3 groups according to nutritional status
mi International Obesity Task Force): normalno uhra- (International Obesity Task Force criteria): normal
njeni (42,1%), predgojazni (40,6%) i gojazni weight (42.1%), overweight (40.6%) and obese
(17.3%). Antropometrijska mjerenja obuhvatila su (17.3%). Anthropometric measurements performed:
tjelesnu masu i visinu, indeks tjelesne mase (BMI, body weight and height, body mass index (BMI,
kg/m2), i obim struka. Ukupna koli~ina tjelesne masti kg/m2), waist circumference. Total body fat was
odre|ivana je metodom bioelektri~ne impedance determined by bioelectrical impedance device (Tanita
(Tanita BC-418, Tokio, Japan). Vrijednost 25(OH) vit- BC-418, Tokyo, Japan). The value of 25 (OH) vita-
amina D (nmol/L) odre|ivana je iz seruma kod 176 min D (nmol/L) was determined from the serum of
djece (imunohemija, Cobas 6000, Roche, Manhajm, 176 children (immunochemistry, Cobas 6000,
Njema~ka). Deficijencijom su smatrane vrijednosti Roche, Mannheim, Germany). Vitamin D values 50
vitamina D 50 nmol/L. Medijana vrijednosti vitam- nmol/L were considered deficient*. The median
ina D za normalno uhranjenu djecu iznosila je 77,2 value of vitamin D for normal weight children was
(interkvartilni rang (IR) 67,70–95,10), za predgo- 77.2 (interquartile range (IR) 67.70–95.10), over-
jaznu 70,1 (IR 56,00–86,60) i gojaznu 69,6 (59,30– weight 70.1 (IR 56.00–86.60) and obese 69.6
85,87); ova razlika je bila grani~no statisti~ki (59.30–85.87), this difference was borderline statis-
zna~ajna (p < 0,046). U grupi gojaznih, vrijednost tically significant (p < 0.046). In the obese group,
vitamina D je negativno korelirala sa vrijedno{}u the value of vitamin D was negatively correlated with
obima struka (r = - 0,403). Deficijencija vitamina D the value of waist circumference (r = - 0.403).
je utvr|ena kod 4,3% normalno uhranjene, 16,0% Vitamin D deficiency was found in 4.3% of normal
predgojazne i 12,5% gojazne djece. Nije bilo sta- weight, 16.0% of overweight and 12.5% of obese
tisti~ki zna~ajne razlike u u~estalosti deficijencije vita- children. There was no statistically significant differ-
mina D u odnosu na nutritivni status kod ispitivane ence in the frequency of vitamin D deficiency in rela-
djece (c2 = 5,185, p = 0,075). Tako|e, nije utvr- tion to the nutritional status of the examined children
|ena statisti~ki zna~ajna korelacija izme|u ukupne (c2 = 5.185, p = 0.075). In addition, no statistically
koli~ine tjelesne masti kod predgojaznih i gojaznih, i significant correlation was found between the total
vrijednosti vitamina D (r = 0,133. r = - 264). U za- body fat in overweight and obese and the value of
klju~ku, vrijednost vitamina D bila je nià u serumu vitamin D (r = 0.133. r = - 264). In conclusion, the
predgojaznih i gojaznih u odnosu na normalno uhra- value of vitamin D was lower in the serum of over-
njenu djecu i negativno je korelirala sa centralnom weight and obese compared to normal weight chil-
gojazno{}u u grupi gojazne djece. Ipak, primjenom dren, and negatively correlated with central obesity in
404
datog kriterijuma za deficijenciju vitamina D, nije the group of obese children. Nevertheless, by apply-
utvr|ena razlika u u~estalosti deficijencije vitamina D ing the given criterion for vitamin D deficiency, no dif-
izme|u ispitivanih grupa. ference was found in the frequency of vitamin D defi-
ciency between the examined groups.
*Holick MF et al. (2011). Evaluation, Treatment, and
Prevention of Vitamin D Deficiency, JCEM; *Holick MF et al. (2011). Evaluation, Treatment,
https://www.endocrine.org/clinical-practice-guide- and Prevention of Vitamin D Deficiency, JCEM;
lines/vitamin-d-deficiency https://www.endocrine.org/clinical-practice-guide-
lines/vitamin-d-deficiency
P011 P011
CASE REPORT: SOLITARY PRIKAZ SLUČAJA: SOLITARNI
MASTOCYTOMA DIAGNOSED MASTOCITOM USPE[NO
SUCCESSFULLY WITH DIJAGNOSTIKOVAN
MEASUREMENT OF SERUM MERENJEM NIVOA TRIPTAZE
LEVELS OF TRYPTASE U SERUMU
Ljubica Adji Andov1, Lidija Petrovska2 Ljubica Adji Andov1, Lidija Petrovska2
1DiagnosticMedicalbiochemistry 1Dijagnosti~ka medicinskobiohemijska laboratorija
Laboratory Ramus, Skopje Ramus, Skoplje
2Clinical Hospital, Department of 2Klini~ka bolnica, Odeljenje za dermatovenerologiju,
Dermatovenerology, Shtip, Severna Makedonija [tip
Solitary mastocytoma, a rare dermatological entity Solitarni mastocitom, retki dermatolo{ki entitet, ~ini
accounts for 10 –15% of cutaneous mastocytosis. 10–15% ko`ne mastocitoze. Serumska triptaza,
One of the most important blood tests in the field of jedan od najva`nijih testova krvi u oblasti alergije,
allergy, mast cell tryptase has numerous diagnostic ima brojne dijagnosti~ke upotrebe, osobito u anafi-
uses, particularly for anaphylactic reactions and for lakti~ke reakcije i u dijagnosticiranju mastocitoze.
the diagnosis of mastocytosis. Hypertryptasemia (ele- Hipertriptazemija (povi{eni nivoi triptaze u serumu)
vated serum tryptase levels) is present in multiple dis- prisutna je kod vi{estrukih poreme}aja poput sis-
orders like systemic mastocytosis, hematological temske mastocitoze, hematolo{kih maligniteta i
malignancies, and chronic kidney disease. We repre- hroni~ne bolesti bubrega. Predstavljamo redak slu~aj
sent a rare case of solitary bullous mastocytoma pre- solitarnog buloznog mastocitoma prisutan pri ro-
senting at birth, diagnosed successfully with subse- |enju, uspe{no dijagnostikovan uzastopnim meren-
quent measurement of serum tryptase levels thus jem nivoa triptaze u serumu, ~ime se izbegava biop-
avoiding biopsy of the patient. sija pacijenta.
J Med Biochem 2022; 41 (3) 405
P012 P012
OKSIDATIVNI STRES I STATUS OXIDATIVE STRESS AND
VITAMINA D KOD COVID-19 VITAMIN D STATUS IN
PACIJENATA COVID PATIENTS
Jovana Panti}1, Irena Premovi}1, Jovana Panti}1, Irena Premovi}1,

Olivera Mirkovi}1, Marina Roksandi} Olivera Mirkovi}1, Marina Roksandi}
Milenkovi}2, Dejan Dimi}2, Nemanja Dimi}3, Milenkovi}2, Dejan Dimi}2, Nemanja Dimi}3,
Ljiljana Timotijevi}2, Danica ]uji}4, Ljiljana Timotijevi}2, Danica ]uji}4,
Azra Guzonji}1, Jelena Veki}1, Azra Guzonji}1, Jelena Veki}1,
Nata{a Bogavac-Stanojevi}1, Nata{a Bogavac-Stanojevi}1,
Jelena Kotur-Stevuljevi}1 Jelena Kotur-Stevuljevi}1
1Katedra za medicinsku biohemiju, Farmaceutski 1Department of Medical Biochemistry, Faculty of
2Gradski zavod za plu}ne bolesti i tuberkulozu, 2City Institute for Lung Diseases and Tuberculosis,
3KBC Dr Dragi{a Mi{ovi} Beograd, Srbija, 3KBC Dr Dragi{a Mi{ovi} Belgrade, Serbia,
Medicinski fakultet Univerzitet u Beogadu Faculty of Medicine, University of Belgrade
4Institut za primenu nuklearne energije, INEP, 4Institute for the Application of Nuclear Energy,
Zemun, Beograd INEP, Zemun, Belgrade
Koronavirusi pripadaju porodici Coronaviridae koji Coronaviruses belong to the family Coronaviridae
spadaju u jednolan~ane RNK viruse; SARS-CoV-2 je which are single – chain RNA viruses; SARS-CoV-2
prvi put identifikovan u gradu Vuhanu u Kini 2019. was for the first time identified in the city Wuhan in
godine nakon {to je izazvao pandemiju COVID-19. China 2019th causing a pandemic COVID-19. The
Zna~ajnu ulogu u razvoju i progresiji ove infekcije important role in the development and progression of
ima i razvoj oksidativnog stresa kao pratioca infla- infection also has the development of oxidative stress
macije, posebno u komplikovanijim oblicima ove as an inflammation consequence, especially in com-
bolesti. Ova studija je sprovedena s ciljem da ispita plicated forms of this disease. This study was per-
promene parametara oksidativnog stresa kod formed with the aim of testing the oxidative stress
COVID-19 pacijenata. Parametri oksidativnog stresa parameters changes in COVID-19 patients. Oxidative
su odre|ivani u serumu 31 pacijenta, sa lak{im stress parameters were determined in the serum of
oblikom bolesti, le~enih ambulantno, u tri ta~ke 31 patients, with a milder form of the disease, treat-
(postavljanje dijagnoze, kontrola nakon 14 i nakon ed on an outpatient basis, at three time-points (diag-
21 dana). Parametri redoks statusa koji su odre|ivani nosis, control after 14 and after 21 days). The redox
u ove tri ta~ke obuhvataju prooksidanse (totalni status parameters determined at these three points
oksidativni status (TOS), superoksidni anjon radikal include prooxidants (total oxidative status TOS);
(O2.-), prooksidantno -antioksidantni balans (PAB) superoxide anion radical (O2.-); prooxidant – antiox-
kao i produkte njihovog delovanja: uznapredovali idant balance (PAB); as well as products of their activ-
produkti oksidacije proteina (AOPP), malondialdehid ity: advanced oxidation protein products (AOPP),
(MDA), ishemijom modifikovan albumin (IMA)) i malondialdehyde (MDA), ischemia – modified albu-
antioksidanse: totalni antioksidativni status (TAS), min (IMA) and antioxidants: total antioxidant status
aktivnost enzima superoksid-dismutaze (SOD) i (TAS), superoxide dismutase (SOD) and paraoxonase
paraoksonaze 1 (PON1), odnos totalnog antioksida- 1 (PON1) activity, ratio of total antioxidant and total
tivnog i totalnog oksidativnog statusa (TAS/TOS), oxidative status (TAS/TOS), total sulfhydryl groups
ukupne sulfhidrilne grupe (SHG). Svi prooksidansi i (SHG). All prooxidants and markers of their activity
markeri njihovog delovanja su pokazali zna~ajan pad showed a significant decrease during the 14 days of
tokom 14 dana trajanja studije, dok su antioksidansi the study, while antioxidants at the same time
istovremeno pokazali zna~ajan rast {to je ukazivalo showed a significant increase, which indicated the
na oporavak pacijenata. U prvoj ta~ki ispitivanja recovery of patients. In the first point of the examina-
odre|ene su i vrednosti vitamina D kako bi se ispitala tion, the values of vitamin D were determined in
veza deficijencije vitamina D sa porastom oksida- order to examine the connection between vitamin D
tivnog stresa. Prema vrednostima vitamina D, paci- deficiency and the increase in oxidative stress.
jenti su podeljeni u tri grupe: 50 ng/mL, 51–70 According to the values of vitamin D, patients were
ng/mL, 71 ng/mL. Prime}eno je da pacijenti sa divided into three groups: 50 ng/mL, 51–70
vi{im vrednostima vitamina D imaju i zna~ajno ve}i ng/mL, 71 ng/mL. It was noticed that patients with
406
ukupni broj leukocita i vi{e vrednosti O2.-, dok su higher values of vitamin D had a significantly higher
vrednosti TAS/TOS odnosa i PAB niè (p<0,05). total number of leukocytes and higher values of O2.;
Vitamin D uti~e na funkciju imunskog sistema, sin- while the values of TAS/TOS ratio and PAB were sig-
teti{u ga T i B limfociti, {to omogu}ava njegovo auto- nificantly lower (p<0.05). Vitamin D affects the func-
krino delovanje. Deficijencija vitamina D povezana je tion of the immune system, it is synthesized by T and
sa razvojem teìh oblika ove bolesti. Pored anti- B lymphocytes, which enables its autocrine action.
virusne, antibiotske, kortikosteroidne, antikoagu- Vitamin D deficiency is associated with the develop-
lantne terapije u COVID-19 i terapija antioksidansi- ment of more severe forms of this disease. Besides
ma, mineralima i vitaminom D je bila deo protokola antiviral, antibiotic, corticosteroid, anticoagulant ther-
le~enja ovih pacijenata, ~iju opravdanost potvr|uju apy in COVID-19 also therapy with antioxidants, min-
rezultati na{e studije. erals and vitamin D were the part of the treatment
protocol of these patients, the justification of which is
confirmed by the results of our study.
P013 P013
EVALUACIJA TITARA EVALUTATION OF ANTI-SARS-COV-2
ANTI-SARS-COV-2 S1-RBD IGG S1-RBD IGG ANTIBODY TITERS
ANTITJELA KOD PRELEŽANIH I IN RECOVERED AND VACCINATED
VAKCINISANIH ISPITANIKA PARTICIPANTS
Suad Me{i}1, Violeta Filip~e2, Nada Minovska2, Suad Me{i}1, Violeta Filip~e2, Nada Minovska2,
Tatjana Baevska Vu~kovi}1, Eleonora Lazarova1, Tatjana Baevska Vu~kovi}1, Eleonora Lazarova1,
Gjurgjica Delenikova2, Tijana Argirova2, Gjurgjica Delenikova2, Tijana Argirova2,
Sofija Cacanoska2 Sofija Cacanoska2
1PZU Nikob Medical, Skoplje, Severna Makedonija 1PZU Nikob Medical, Skopje, North Macedonia
2PZU Nikob Lab, Skoplje, Severna Makedonija 2PZU Nikob Lab, Skopje, North Macedonia
Cilj na{e studije bio je uporediti titre anti-RBD anti- The aim of our study was to compare the titers of
tjela kod preleànih, vakcinisanih i vakcinisanih anti-RBD antibodies in COVID-19-recovered individ-
ispitanika koji su preleàli COVID-19. Na{a studija je uals, vaccinated and in vaccinated individuals with
uklju~ila 206 ispitanika; 73 preleànih, 71 vakcini- past-COVID-19. Our study included 206 partici-
sanih i 62 vakcinisanih sa preleànim COVID-19. pants; 73 recovered, 71 vaccinated and 62 vaccinat-
Titar anti-SARS-CoV-2 S1-RBD IgG antitjela mjeren ed with past COVID-19. The titers of nti-SARS-CoV-
je u serumu ispitanika SARS-CoV-2 IgG Quant 2 S1-RBD IgG antibodies were measured in the sera
testom (metoda CMIA) na integrisanom imuno- of participants using the SARS-CoV-2 IgG Quant test
biohemijskom analizatoru Abbott Architect ci4100. (CMIA method) on the integrated Abbott Architect
Preleàni ispitanici su imali najniì titar antitjela. ci4100 analyser. Recovered participants had the low-
BNT162b2 stvara vi{i titar antitjela u odnosu na est titters. BNT162b2 elicited higher titters com-
preleàne (p=0,001). BBIBP-CorV stvara niì titar pared to recovered participants (p=0.001). BBIBP-
antitjela u odnosu na preleàne (p=0,004), ali CorV elicited lower titters compared to recovered
znatno vi{i titar ukoliko su ispitanici i preleàli participants (p=0.004), but higher titters if they also
COVID-19 (p=0,009). Ispitanici, vakcinisani koji su i had past COVID-19 (p=0.009). Participants, vacci-
preleàli, imali su najvi{i titar antitjela (p<0,001). nated and with past COVID-19 had the highest titters
Titar antitjela pozitivno je korelirao sa godinama kod (p<0.001). Antibody titters correlated positively with
preleànih (r=0,059), negativno kod vakcinisanih age in the recovered group (r=0.059), negatively in
(r=-0,146), i pozitivno kod preleànih i vakcinisanih the vaccinated group (r=-0.146), and positively in
(r=0,146). U odnosu na pol, u na{oj studiji nije se the vaccinated with past COVID-19 group
pokazala statisti~ki signifikantna razlika u titarima (r=0.146). There was no statistically significant dif-
antitjela. Zaklju~ujemo da preleàni ispitanici vakci- ference in the titters based on gender. We conclude
nisani protiv SARS-CoV-2 virusa imaju znatno vi{i titar that COVID-19-recovered individuals that have been
u odnosu na sve druge grupe, nezavisno od primljene vaccinated against SARS-CoV-2 have significantly
vakcine. higher titers compared to all other groups regardless
of the vaccine received.
J Med Biochem 2022; 41 (3) 407
P014 P014
KONCENTRACIJA HEPCIDINA KOD HEPCIDIN CONCENTRATION AMONG
ŽENA SA IZOSTALIM ABORTUSOM WOMEN WITH MISSED ABORTION
Ana ]uk1, Ivanka Mikuli}1, Ivona Cvetkovi}1, Ana ]uk1, Ivanka Mikuli}1, Ivona Cvetkovi}1,
Nikolina Penava2, Ante Pu{i}1, Kristina Ljubi}1, Nikolina Penava2, Ante Pu{i}1, Kristina Ljubi}1,
Vinka Mikuli}1 Vinka Mikuli}1
1Zavod za laboratorijsku dijagnostiku, 1Department of Laboratory Diagnostic, University

Sveu~ili{na klini~ka bolnica Mostar, BiH Clinical Hospital Mostar, Bosnia and Herzegovina
2Klinika za ginekologiju i opstetriciju, 2Department of Gynaecology and Obstetrics,
Sveu~ili{na klini~ka bolnica Mostar, BiH University Clinical Hospital Mostar, Bosnia and
Herzegovina
Iako je poznat zna~aj gvo`|a za zdravlje majke, rast i Although it is well known how important iron is for
razvoj placente, embriona i fetusa, jo{ uvek postoje maternal health, growth and development of the
mnoge nejasno}e u razumevanju regulacije gvo`|a placenta, embryo and fetus, there are still many
tokom (ab)normalne trudno}e. Nije poznato da li ambiguities in the regulation of iron during
izostali poba~aj uzrokuje poreme}aj homeostaze (ab)normal pregnancy. It is unknown whether missed
gvo`|a ili obrnuto, da li poreme}ena homeostaza abortion causes disorder of iron homeostasis or vice
gvo`|a pre za~e}a moè doprineti poba~aju. Postoji versa, whether disturbed iron homeostasis may
11–22% rizika od poba~aja do 20. nedelje trudno}e, contribute to spontaneous abortion before its onset.
pri ~emu je rizik od poba~aja najve}i do 14.nedelje. There is 11–22% risk of miscarriage up to the 20th
Izostali poba~aj je abortus kod kojeg postoji week of pregnancy, with the risk ofmiscarriage being
asimptomatska ili »odloèna«smrt embriona ili fetusa highest up to the 14th week. Missed abortion is an
bez dovoljnih kontrakcija materice za izbacivanje abortion inwhich there is asymptomatic or »delayed«
fetusa. Hepcidin je peptidni hormon i glavni je death of the embryo or fetus without sufficient
regulator gvo`|a koji kontroli{e apsorpciju i uterine contractions to expel the fetus. Hepcidin is a
distribuciju gvo`|a u tkivu. Odre|ivanje koncentracije peptide hormone and isa major regulator of iron that
hepcidina u serumu u normalnoj ranoj trudno}i i controls the absorption and distribution of iron in
ranom izostalom poba~aju je prvi korak ka boljem tissue.Determining serum hepcidinconcentrations in
razumevanju homeostaze gvo`|a u ovimuslovima. U normal early pregnancy and early missed miscarriage
ovu studiju su uklju~ene ukupno 72 ispitanice, od is the first steptoward a better understanding of iron
~ega 36 èna u kontrolnoj grupi (normalna trud- homeostasis in these conditions. A total of 72
no}a) i 36 èna u studijskoj grupi (izostali spontani respodents were included in this study: 36 women
poba~aj), do 14. nedelje gestacije. Za ovo istra- in the control group (normal pregnancy) and 36
ìvanje pribavljena je saglasnost Eti~kog povjerenstva women in the study group (missed abortion), up to
Sveu~ili{ne klini~ke bolnice (SKB) Mostar, a svi the 14th week of gestation. The consent of the Ethics
ispitanici su prije uzimanja krvi potpisali informirani Committee of the University Clinical Hospital (UCH)
pristanak. Uzorak krvi ispitanika je uzet nakon ultra- Mostar was obtained for infomed consent before
zvu~no potvr|enog izostalog poba~aja ili normalnog blood sampling.The bloog sample of the respodents
toka trudno}e. Kriterijumi isklju~enja za obe grupe: was sampled after ultrasound-confirmed of missed
krvarenje, anemija, hipertenzija, dijabetes, endokrine abortion or the normal course of pregnancy.
abnormalnosti, ultrazvu~no vidljive abnormalnosti Exclusion criteria for bothgroups: bleeding, anemia,
fetusa, disfunkcija {titne `lezde, infekcija urinarnog hypertension, diabetes, endocrine abnormalities,
trakta, malformacije materice, onkolo{ke bolesti, ultrasound visible fetal abnormalities, thyroid dys-
ste~ena i nasledna trombofilija, autoimune bolesti, function, urinary tract infection, uterine malfor -
ve{ta~ka oplodnja, suplementacija. Uzorci seruma su mations, oncological diseases, acquired and
dobijeni nakon koagulacije i centrifugiranja pune krvi hereditary thrombophilia, autoimmune diseases,
bez antikoagulansa u trajanju od 10 minuta na 3500 artificial insemination, iron supplementation. Serum
rpm i ~uvani na -80 °C do analize. Koncentracija samples were obtained after coagulation and
hepcidina je odre|ena imunohemijskom ELISA centrifugation of whole blood without anticoagulant
metodom (engl. Enzyme Linked Immunosorbent for 10 minutes at 3500 rpm and stored at -80 °C until
Assay). Statisti~ka analiza (MedCalc verzija 20.027) analysis. Hepcidin concentration was determined by
pokazala je da je koncentracija hepcidina statisti~ki immunochemical ELISA (Enzyme Linked Immuno-
zna~ajno ve}a od koncentracije hepcidina kod èna sorbent Assay). Statistical analysis (MedCalc version
sa normalnom trudno}om (P < 0,05). Srednja kon- 20.027) showed that the concentration of hepcidin
centracija hepcidina kod èna sa normalnom was statistically significantly higher than the
408
trudno}om bila je 3,4895 (1,3825–5,1580) ng/mL, concentration of hepcidin in women with normal
a srednja koncentracija hepcidina kod èna sa pregnancy (P < 0.05). The median hepcidin con-
odloènim poba~ajem bila je 5,0130 (3,2880– centration in women with normal pregnancies was
7,8265) ng/mL. Iako je poreme}ena homeostaza 3.4895 (1.3825–5.1580) ng/mL, and the median
gvo`|a uklju~ena u mnoga patolo{ka stanja i bolesti, hepcidin concentration in women with delayed
malo se zna o ulozi poreme}ene homeostaze gvo`|a miscarriage was 5.0130 (3.2880–7.8265) ng/mL.
u abnormalnim trudno}ama. U~injeni su brojni Although disturbed iron homeostasis is involved in
poku{aji da se odrede prognosti~ki markeri koji bi many pathological conditions and diseases, there are
ukazivali na pojavu poba~aja u ranoj trudno}i kod limited information available about the role of
èna. Ovde smo prona{li poreme}enu homeostazu disturbed iron homeostasis inabnormal pregnancies.
gvo`|a kod èna sa izostalim poba~ajem, o ~emu Numerous attempts have been made to determine
svedo~e povi{ene koncentracije hepcidina u serumu. prognostic markers to indicate the occurrence of
Regulatorni mehanizmi homeostaze gvo`|a u ovim miscarriage during early pregnancy inwomen. In this
uslovima zahtevaju dalja istraìvanja. research, we found disturbed iron homeostasis in
women with missed miscarriage, which is proven by
elevated serum hepcidin concentrations. The
regulatory mechanisms of iron homeostasis in these
conditions require further research.
P015 P016
THE POTENTIAL OF SALIVARY PCOS AND ISULIN RESISTANCE
PROTEOME IN LABORATORY
ANALYSIS OF SJOGREN’S Danche Kolarovska, Marija Hristovska,
SYNDROME Hristijan Trpchevski
Marija Hiljadnikova-Bajro, Angela Simovska Hospital Plodnost Bitola, North Macedonia
Polycystic ovary syndrome (PCOS) is a hormonal dis-

Faculty of Pharmacy, Ss. Cyril and Methodius order common among women of reproductive age.
University in Skopje, Skopje, Women with PCOS may have infrequent or pro-
Republic of North Macedonia longed menstrual periods or excess male hormone
(androgen) levels. Due to the recent research that
Detection of pathological processes at an early stage PCOS is probably caused by insulin resistance, we
can significantly affect the clinical course and outcome are beginning to check insulin in blood in patients
of the disease, and hence the choice of appropriate who have at least 2 of 3 features irregular periods,
therapeutic intervention. In order to achieve this and excess androgen (high levels of »male« hormones in
avoid invasive sampling procedures like surgical biopsy y our body) and polycystic ovaries. We examine hor-
and repeated phlebotomies which introduce additional mones in blood in 86 patients between ages 21 to
stress and safety issues in patients, research is greatly 43 years old on t hird day of menstrual cycle, oppo-
focused on identifying alternative samples and novel site to a control group of 30 patients in same condi-
biomarkers for advanced laboratory analysis. Since tions: LH, FSH, testosterone, DHEAS and insulin with
many oral but also systemic pathological processes are automates the immunoassay reactions utilizing elec-
being reflected in the salivary composition, it is getting trochemiluminescence (ECL). LH/FSH ratio was
increasing attention as an alternative sample to blood, 2,6:1 with mean values in LH 14,65 mIU/mL+/ 4,2;
FSH 5,6 mIU/mL +/ 1,9; the levels of testosteron
especially for some population groups like children,
77,5 ng/dL,+/ 23,8; DHEAS 450,7 mg/dL +/
adolescents, geriatric or psychiatric patients, where 203,0 and insulin 24,75 mU/mL +/ 10,7. Many
blood sampling is often compromised by insufficient women with PCOS 68,2% have higher levels of
cooperation from the patients or individual factors insulin which had not been diagnosed. There are sta
related to the patiens’ health. Sjögren’s syndrome tistic significant p< 0.01 in levels in testosterone,
(SS) is an autoimmune disease with an insidious onset, DHEAS and insulin. PCOS cannot be diagnosed by
variable course and clinical presentation, so its symptoms alone. PCOS is a very complicated
diagnosis is usually established about 6 years after the endocrine disorder. Blood tests to measure hormone
initiation of the disease and based on the detection levels, an US of reproductive organs and genes per-
and quantification of circulating autoantibodies such sonal and family histories should be completed
as: anti-Ro/SSA, anti-La/SSB, anti-muscarinic receptor before a PCOS diagnosis is confirmed. 68% of
antibodies, anti-nuclear antibodies and rheumatoid women with PCOS have insulin resistance which
factor. Apart from molecular diagnostics and increases risk for type 2 diabetes.
J Med Biochem 2022; 41 (3) 409
nanotechnology, several new approaches emerge for P017

detecting even subtle alterations of the salivary VERIFICATION OF VIDAS 3®
constituents, like proteins which are employed in the
proteomic analysis. Numerous proteomic studies SARS-CO-V-2 IGG II TEST USING
involving sophisticated methodology like LC-MS/MS ENZYME LINKED FLUORESCENT
have identified aberrant expression of specific proteins ASSAY (ELFA) TECHNIQUE
in saliva of SS patients, making them potential markers
of the disease and indicators of progression. Beside Elena Petrushevska Stanojevska,
cytokines, many other proteins involved in the Hristina Ampova, Melda Emin, Irena Kostovska,
inflammatory process are upregulated vs proteins Sonja Topuzovska, Jasna Bogdanska,
associated with the salivary production which are
downregulated. Further to changes in salivary
concentrations of MMP9, Complement factor B,
Azurocydin Kallikrein, many other proteins including Institute of Medical and Experimental Biochemistry,
proteases like Trypsin, Catepsin, and Myeloblastin, Medical Faculty, University »Ss Cyril and Methodius«,
inhibitors of Cystein proteases, Calreticulin, Protein Skopje, 50 Divizija No 6, 1000 Skopje
29, -amylase precursor of carbonanhydrase VI, -2
microglobulin, enolase etc. have shown aberrant COVID-19 is unfortunately still present and continues
expression in saliva from SS patients and a recent to spread worldwide. The use of serological tests is
study has even proposed the combination of serum intended to determine if individuals have developed
anti-SSA/Ro and upregulated salivary TRIM29, as an a humoral immune response to SARS-CoV-2. The
optimal marker with high diagnostic accuracy for fast verification of the SARS-CoV-2 IgG II test was con-
and noninvasive diagnosis of SS. But, in patients with ducted, as part of the laboratory’s quality control pro-
SS, efficient saliva collection is difficult because saliva cedure before introducing a new test in the scope of
secretion is significantly reduced. Therefore, when our lab. The precision of the test VIDAS 3® SARS-
examining the scientific potential of this diagnostic CoV-2 IgG II was performed following the CLSI
sample, stimulated saliva is mainly used. Additionally, EP05-A3 recommendations. Two panels of human
the great heterogeneity of the results among various pool serum samples (HPSs) representing negative
studies is greatly attributed to variations in the (N) and positive (P) level indexes (i) were performed
preanalytical procedures as well as the analytical to evaluate the variability of the assay within and
methodology itself. Hence, further studies with larger between day. The tests were performed into two-lev-
cohorts applying consensual study protocols are els, over five days, each panel in triplicate. According
needed to validate the currently proposed and identify to the manufacturer the acceptable level of precision
new potential markers in saliva for diagnosis, is fixed at 20% CV (and in our case corresponds to
monitoring of SS or be used as targets in drug design 7.27% and 12.31% for P and N HPSs, respectively.
in accordance with the precise medicine initiatives. Moreover, the estimated within-run precision of our
lab was 5.98% which is less than the claimed one
(6.10%) with SD index of 0.293 (claimed- 0.50).
N/A data for N HPSs to compare. The daily variance
was estimated as 1.27% and 0.51% for P and N
HPSs, respectively. The findings have demonstrated
that imprecision and repeatability are in compliance
with the manufacture claims, therefore the test is
suitable for use. The obtained data represent a pre-
requisite for appropriate utilization of immune
response to RBD/spike viral protein.
Keywords: SARS CoV-2 IgG II, quality control, ELFA
410
P018 P018
SERUMSKI NIVOI IL-1b I IL-1RA KOD SERUM LEVELS OF IL-1b AND IL-1RA
PACIJENATA SA GREJVSOVOM IN GRAVES’ ORBITOPATHY
ORBITOPATIJOM PATIENTS
Sari} Matutinovi} M1, Nedeljkovi} Beleslin B2,3, Sari} Matutinovi} M1, Nedeljkovi} Beleslin B2,3,
]iri} J2,3, @arkovi} M2,3, Ignjatovi} S1,4 ]iri} J2,3, @arkovi} M2,3, Ignjatovi} S1,4
1Univerzitet u Beogradu-Farmaceutski fakultet, 1Universityof Belgrade, Faculty of Pharmacy,
2Univerzitetski klini~ki centar Srbije, 2Clinic for Endocrinology, Diabetes and Metabolic
Klinika za endokrinologiju, Disorders, University Clinical Center of Serbia,
dijabetes i bolesti metabolizma, Beograd, Srbija Belgrade, Serbia
3Univerzitet u Beogradu – Medicinski fakultet, 3University of Belgrade, Medical Faculty,
4Univerzitetski klini~ki centar Srbije, Centar za 4Center for Medical Biochemistry, University
medicinsku biohemiju, Beograd, Srbija Clinical Center of Serbia, Belgrade, Serbia
Grejvsova orbitopatija (GO) predstavlja inflamatorni Graves’ orbitopathy (GO) is an inflammatory disorder
poreme}aj orbite koju karakteri{e specifi~an lokalni of the orbit characterized by a specific local cytokine
citokinski milje. Naru{avanjem ravnoteè izme|u milieu. By disrupting the balance between inter-
interleukina- b (IL-1b) i njegovog prirodnog anatago- leukin-b (IL-1b) and its natural antagonist, the inter-
niste, antagoniste receptora interleukina-1 (IL-1RA) leukin-1 receptor antagonist (IL-1RA), the natural
gubi se prirodni regulatorni mehanizam, ~ime se regulatory mechanism is lost, thus creating a pro-
stvara proinflamatorni fenotip orbitalnih fibroblasta inflammatory phenotype of orbital fibroblasts that
koji koordini{e dalje procese u osnovi ove bolesti. Cilj coordinates further processes underlying this disease.
nau~nog istraìvanja je bio da se analizira povezanost Aim of the study was to analyze the association of IL-
IL-1b i IL-1RA sa klini~kim oblikom GO. Istraìvanjem 1b and IL-1RA with the clinical form of GO. A total
je obuhva}eno 65 pacijenata sa klini~ki prisutnom of 65 consecutive patients presented with GO were
GO, le~enih na Klinici za endokrinologiju, dijabetes i enrolled in the study. All patients were regularly treat-
bolesti metabolizma, Univerzitetskog klini~kog centra ed at the Clinic for Endocrinology, Diabetes and
Srbije. Pacijenti su klasifikovani prema aktivnosti i Metabolic Diseases, University Clinical Center of
teìni GO, kao pacijenti sa aktivnim ili neaktivnim, Serbia. Patients were classified according to the activ-
odnosno blagim ili umerenim do te{kim oblikom GO. ity and severity of GO, as patients with active or inac-
IL-1 b i IL-1RA su analizirani u uzorcima seruma tive, and mild or moderate to severe form of GO. IL-
pacijenata, primenom komercijalnih enzimskih imu- 1 b and IL-1RA were analyzed in patient sera, using
nohemijskih testova (ELISA), prema preporukama commercial enzyme-linked immunosorbent assays
proizvo|a~a. Serumska koncentracija IL-1b bila je (ELISA), according to the manufacturer’s instruc-
statisti~ki zna~ajno vi{a, a IL-1RA grani~no nià u tions. Significantly higher IL-1b and marginally lower
grupi pacijenata sa aktivnom GO u odnosu na paci- IL-1RA serum concentration was observed in patients
jente sa neaktivnom GO (4,86 (4,25–5,66) pg/mL i with active GO, compared to patients with inactive
3,83 (2,96–4,83) pg/mL, p = 0,027; 487 (285– GO (4.86 (4.25–5.66) pg/mL vs. 3.83 (2.96–4.83)
694) pg/mL i 618 (359–812) pg/mL, p = 0,059, za pg/mL, p = 0.027; 487 (285–694) pg/mL vs. 618
IL-1b i IL-1RA, redom). Dodatno je uo~ena i pozitiv- (359–812) pg/mL, p = 0.059, for IL-1b and IL-1RA,
na korelacija IL-1b sa vrednostima skora klini~ke respectively). Additionally, IL-1b concentration posi-
aktivnosti (r = 0,261, p = 0,036). Teìna GO nije tively correlated with the clinical activity score (r =
bila zna~ajno povezana sa vrednostima IL-1b i IL-1RA 0.261, p = 0.036). There was no significant associ-
u ispitivanom uzorku pacijenata. Rezultati ovog istra- ation between IL-1b and IL-1RA values, and the
ìvanja ukazuju na mogu}nost upotrebe IL-1b kao severity of GO in the analyzed patient sample. Results
zna~ajnog biomarkera aktivnosti i klini~kog toka GO. of this study indicate the possibility of using IL-1b as
Kombinovana primena IL-1b i IL-1RA, zajedno sa a significant biomarker of the activity and the clinical
tradicionalnim parametrima, mogla bi unaprediti la- course of GO. The combined application of IL-1b
boratorijsku dijagnostiku ove kompleksne patologije. and IL-1RA, along with traditional parameters, could
substantially improve the laboratory diagnosis of this
complex pathology.
J Med Biochem 2022; 41 (3) 411
P019 P019
OKSIDATIVNO-STRESNI STATUS OXIDATIVE-STRESS STATUS
KOD PACIJENATA NA HEMODIJALIZI OF HEMODIALYSIS PATIENTS
Nina Miti}1, Jasmina Ivani{evi}1, Nina Miti}1, Jasmina Ivani{evi}1,
Tamara Milo{evi}2, Azra Guzonji}1, Tamara Milo{evi}2, Azra Guzonji}1,
Sanja Vuj~i}1, Jelena Kotur-Stevuljevi}1, Sanja Vuj~i}1, Jelena Kotur-Stevuljevi}1,
Sneàna Pe{i}3, Radomir Naumovi}3 Sneàna Pe{i}3, Radomir Naumovi}3
1Katedra za medicinsku biohemiju, Farmaceutski 1Department of Medical Biochemistry,
fakultet, Univerzitet u Beogradu, Srbija Faculty of Pharmacy, University of Belgrade, Serbia
2Medicinsko-biohemijska laboratorija, KBC 2Medical-biochemistry laboratory,
»Zvezdara«, Beograd, Srbija CHC »Zvezdara«, Belgrade, Serbia
3Bolnica za nefrologiju i metaboli~ke bolesti 3Hospital for nephrology and metabolic disorders
»Prof. dr Vasilije Jovanovi}«, KBC »Zvezdara«, »Prof. dr Vasilije Jovanovi}«, CHC »Zvezdara«,
Poslednji stadijum bolesti bubrega karakteri{e se The last stage of kidney disease is characterized by a
brzinom glomerularne filtracije manjom od 15 glomerular filtration rate less than 15 mL/min/
mL/min/1,73 m2 zbog ~ega su potrebni tretmani koji 1.73 m2. As a consequence, treatments such as
zamenjuju renalnu funkciju poput hemodijalize (HD) hemodialysis (HD) or kidney transplantation are
ili transplantacije bubrega. Dvogodi{nji nivo rizika od necessary to substitute for the lost function. The two-
smrtnog ishoda (engl. mortality risk score) se zasniva year mortality risk score is based on multiple
na vi{e laboratorijskih, klini~kih i parametara HD. laboratory, clinical and HD parameters. Patients on
Zbog starosti i pojave drugih komorbiditeta, pacijenti HD treatment are susceptible to oxidative stress due
na HD su osetljiviji na oksidativni stres. Pove}an to aging and other comorbidities. Increased oxidative
oksidativni stres moè da doprinese i pove}anom stress can also contribute to an increased risk of
riziku od mortaliteta. Cilj na{e studije je bio da se mortality. The aim of our study was to determine the
odrede parametri oksidativno-stresnog statusa pre i parameters of oxidative-stress status before and after
posle HD i 6 meseci nakon tretmana, kao i promena HD and 6 months after treatment, as well as the
ovih parametara u zavisnosti od nivoa rizika od changes of these parameters depending on the levels
smrtnog ishoda. U studiji je u~estvovalo 130 pacije- of risk of death. The data from 130 patients on HD
nata na hemodijalizi. Parametri oksidativno-stresnog treatment were collected in this study. The
statusa: uznapredovali produkti oksidacije proteina parameters of oxidative-stress status as: advanced
(AOPP), prooksidativni-antioksidativni balans (PAB), oxidation protein products (AOPP), prooxidant
superoksidni anjon (O2.-), malondialdehid (MDA), antioxidant balance (PAB), superoxide anion (O2.-),
ishemijom modifikovan albumin (IMA), superoksid- malondyaldehide (MDA), ischemia modified albumin
dizmutaza (SOD) i sulfhidrilne (SH) grupe su odre eni (IMA), superoxide-dismutase (SOD), and sulfhydryl
spektrofotometrijskim metodama na ILAB 300+ (SH) groups were determined using spectro-
analizatoru. Vrednosti parametara O2.- (P<0,05), photometric methods on ILAB 300+ analyzer. The
IMA (P<0,01), AOPP, SOD, SH gr, PAB i MDA results of O2.- (P<0.05), IMA (P<0.01), AOPP,
(P<0,001) 6 meseci nakon tretmana su bile zna- SOD, SH gr, PAB and MDA (P<0.001), six months
~ajno razli~ite u pore enju sa vrednostima dobijenim after treatment, were significantly different compared
pre i/ili posle HD. Umereni i najvi{i nivo rizika od to the values obtained before and/or after HD. The
smrtnog ishoda se karakteri{u zna~ajno razli~itim moderate and the highest mortality risk levels were
koncentracijama SH grupa, PAB-a (P<0,05) i O2.- characterized by significantly different concentrations
(P<0,001), u odnosu na grupu pacijenata sa of SH groups, PAB (P<0.05) and O2.- (P<0.001)
najmanjim rizikom. Dobijeni rezultati pokazuju da bi compared to the group of patients with the lowest
hemodijaliza mogla zna~ajno da uti~e na oksida- risk. The obtained results show that hemodialysis
tivno-stresni status pri ~emu je i rizik od smrtnog could significantly affect the oxidative-stress status
ishoda povezan sa razli~itim nivoima oksidativnog and the risk of death is associated with different levels
stresa. of oxidative stress.
J Med Biochem 2022; 41 (3) 413
UDK 577.1 : 61 ISSN 1452-8258
J Med Biochem 41: 413, 2022 Technical reports

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tion into account when deciding how to use their describing their journal’s review process, such as num-
review. Generally speaking, people with a direct finan- ber of manuscripts submitted, acceptance rate, and
cial interest in the results of the manuscript should average times from manuscript submission to reject
not be reviewers. Editors should avoid using only letter to authors and, for accepted manuscript, time to
author-recommended peer reviewers to review a publication.
427
STATISTICAL GUIDELINES Graphs showing data of comparable magnitude should

be of similar size and design. All individual points should
be displayed where possible by displacing overlapping
These guidelines are designed to help authors prepare points. Error bars showing the standard error of the
statistical data for publication and are not a substitute mean (SEM) or interquartile range, as appropriate, can
for the detailed guidance required to design a study or be used to aid the interpretation of data.
perform a statistical analysis. Each section of a scientific The results of significance tests such as Student’s and
paper is addressed separately. chi-squared should be presented with descriptive statis-
tics, degrees of freedom (if appropriate) and probability
P. The validity of any assumptions should be checked
Summary
(e.g. conventional t-tests assume a normal distribution
The number and source of data must be stated and con- and equal variance for each set of data). For 2 × 2 con-
clusions which have a statistical basis must be substanti- tingency table analysis by the chi-squared test the conti-
ated by inclusion of pertinent descriptive statistics [mean nuity correction must be applied, and for small expected
or median, standard deviation (SD) or interquartile range, frequencies Fisher’s Exact Test used.
percentage coefficient of variation (%CV), 95% confiden-
ce limits, regression equations, etc.]. P values should be reported in full in 1 or 2 significant
figures. Describing P values as > 0.05 or NS (not signi-
ficant) should be avoided. If the results are highly signi-
Methods ficant and the calculated P value from the computer is
e.g. 0.000, then the use of P < 0.0005 is acceptable.
Experimental design, subject selection and randomiza- Confidence intervals should be stated, particularly for
tion procedures should be described and analytical pre- non-significant results.
cision quoted when appropriate. The hypotheses to be
tested by a statistical procedure must be stated and The conventional use of statistical significance is P ≤
where appropriate power calculations for the sample 0.005. If a different significance level needs to be used,
size used should be given (it is recommended that the then the reasons for this must be clearly stated in the sta-
power is <80%). In case-control studies, clearly define tistical method section.
how cases and controls were selected and what match-
ing has taken place.
Statistical tests should be described but need not be re- Discussion
ferenced unless they are unusual or are applied in a Statistical significance should not be equated to impor-
non-standard way. Computer software used should be tance and P values should not be compared between
referenced. different statistical tests. Association should not be inter-
If the paper is reporting the results of a diagnostic trial preted as causation without additional evidence.
read the STARD statement (1) and for a clinical trial
read the CONSORT statement (2) to improve the quali-
ty of your report. Problem Areas
Multiple comparisons can produce spurious and mislea-
ding significance values. The primary hypothesis should
Results
always be clearly stated, and associations detected by ret-
Unnecessary precision, particularly in tables, should be rospective analysis should be interpreted with caution.
avoided. Rounded figures are easier to compare and Whenever possible a single overall statistical test should
extra decimal places are rarely important. Descriptive be applied first e.g. ANOVA. If this is not significant, then
statistics require an additional digit to those used for the multiple comparisons must not be applied. If it is signi-
raw data. Percentages should not be expressed to more ficant then some form of multiple range test can be
than one decimal place and not be used at all for small applied. If a single overall test is not possible, then multi-
samples. ple comparisons must use a Bonferroni type significance
Normally distributed data should be described using a level.
mean, SD and/or %CV and expressed as »mean (SD)« With paired data the differences between individual pairs
not »mean ± SD«. When data are not normally distribu- of data and the variability of the differences are more
ted, following demonstration by tests such as the Sha- important than the individual values. Graphical repre-
piro-Wilk test (3), then medians and interquartile ranges sentation should also show the difference between indi-
should be used in place of mean and SD. Skewed data vidual pairs, e.g. by plotted lines joining the paired data
can often be normalized by logarithmic transformation points.
or a power transformation. The statistical analysis and
calculation of summary statistics should be carried out Standard regression analysis requires data points to be
on the transformed data and the summary statistics independent (repeated measurements are not indepen-
transformed back to the original scale for presentation. dent). The independent variable should be measure-
If a logarithmic scale is used, then graphs should display ments without significant error, e.g. age or time, and the
non-transformed data on a logarithmic scale. points should be evenly distributed over the range and
428 Statistical guidelines
have no outliers (this can be easily examined with a scat- References

ter plot). These requirements are rarely satisfied with
1. Bossuyt PM, Reitsma JB, Bruns DE, et al., for the STARD
biological data.
Group. Towards complete and accurate reporting of stu-
Method comparison using regression and correlation dies of diagnostic accuracy: the STARD initiative. Ann
coefficients is inappropriate and should be performed Clin Biochem 2003; 40: 357– 63.
using Altman and Bland difference plots (4). If a stan-
2. Moher D, Schultz KF, Altman DG, for the CONSORT
dard scatter plot and regression line are thought to be
Group. The CONSORT statement: revised recommen-
useful they can be given along with the Altman – Bland
dations for improving the quality of reports of parallel-
plot. Remember, if two methods are supposed to be
group randomization trials. Lancet 2001; 357: 1191–4.
measuring the same thing, then it is extremely likely they
will be correlated so that a statistical tool correlation not 3. Altman DG. Practical Statistics for Medical Research.
tell you anything new. London: Chapman & Hall, 1991: 132–12.
If you are carrying out complicated statistical analyses, 4. Bland JM, Altman DG. Statistical methods for assessing
e.g. multivariate analysis, ROC analysis etc., then it is re- agreement between two methods of clinical measure-
commended that you seek advice from a statistician. ment. Lancet 1986; 1 (8476): 307–10.
429
UDK 577.1 : 61 ISSN 1452-8258
Guidelines for authors
STATEMENT ON HUMAN AND ANIMAL RIGHTS

Journal of Medical Biochemistry1
The Journal of Medical Biochemistry adheres to the Uniform Requirements for Manuscripts Submitted to
Biomedical Journals: Writing and Editing for Biomedical Publication1. All articles under consideration that experi-
ment on human subjects and animals in research are required to have institutional review board approval in
accord with ethical standards set forth in the Uniform Requirements for Manuscripts Submitted to Biomedical
Journals at http://www.icmje.org/ethical_6protection.html.
The authors should provide the written informed consent that study was approved by the Institutional Ethics
Committee and was carried out in accordance with The Code of Ethics of the World Medical Association for exper-
iments involving humans.
1
International Committee of Medical Journal Editors. Uniform Requirements for Manuscripts Submitted to Biomedical Journals: Writing and
Editing for Biomedical Publication. October 2007. Available at: http://www.icmje.org/index.html. Accessed December 17, 2009.
430
UDK 577.1 : 61 ISSN 1452-8258
Guidelines for authors
CONFLICT OF INTEREST STATEMENT
It is the policy of the Society of Medical Biochemists of Serbia (SMBS) who is publisher of Journal of Medical
Biochemistry that individuals who submit or review manuscripts for publication disclose any proprietary, financial,
professional or other personal interests that may influence positions presented in, or the review of, the manuscript.
As a condition for publication and/or participation in the reviewing process, the SMBS requires that
Corresponding Author on the behalf of all other authors and reviewer complete the following:
I, , declare that I or other authors

have no proprietary, financial, professional or other personal interest of any nature or kind in any product, service
and/or company that could be construed as influencing the position presented in, or the review of, the manu-
script entitled,
except for the following:
Corresponding Author or Reviewer Date

CIP - Katalogizacija u publikaciji
Narodna biblioteka Srbije, Beograd
JOURNAL of Medical Biochemistry : official Journal of the Society of Medical Biochemists of

Serbia / Editor-in-Chief Nada Majki}-Singh. - [tampano izd. - Vol. 41, No. 3 (2022) - Belgrade
(Vojislava Ili}a 94B/7) : Society of Medical Biochemists of Serbia, 2022 - ([Belgrade] : Sprint). -
28 cm
Tromese~no. - Je nastavak: Jugoslovenska medicinska biohemija = ISSN 0354-3447. - Drugo
izdanje na drugom medijumu: Journal of Medical Biochemistry (Online) = ISSN 1452-8266
ISSN 1452-8258 = Journal of Medical Biochemstry ([tampano izd.)
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JOURNAL OF MEDICAL BIOCHEMISTRY

The Journal of Medical Biochemistry

Committee on Publication Ethics

Journal of Medical Biochemistry

Publisher of electronic Journal issues

Journal of Medical Biochemistry is published by financial support

Grafi~ko ure|enje [tampa

Danijela Mijailovi} SPRINT

REVIEW PAPER/REVIJSKI RAD Bara’ah Khaleel, Al-Motassem Yousef, Mazhar Salim

Volume: 41 Belgrade, July – September 2022 Issue: 3

UDK 577.1 : 61 ISSN 1452-8258

J Med Biochem 41: 263 –274, 2022 Review paper

GENETIC POLYMORPHISM OF MATRIX METALLOPROTEINASE 9

Summary Kratak sadr`aj

Address for correspondence:

Introduction Exclusive criteria were as follows: 1) Repeated

Materials and Methods

Inclusive and exclusive criteria

Results literatures were excluded after the first-round screen-

Table I Main characteristics of studies included in the meta-analysis.

Baseline characteristics of eligible literatures Meta-analysis

Publication bias susceptibility to COPD in the three genetic models

Discussion activity owing to the deletion of the transcriptional

Received: September 28, 2021

UDK 577.1 : 61 ISSN 1452-8258

J Med Biochem 41: 275 –281, 2022 Original paper

EVALUATION OF OXIDATIVE STRESS, 3-NITROTYROSINE, AND HMGB-1

Summary Kratak sadr`aj

Address for correspondence:

Introduction In this case, 3-NT levels in AMD patients can provide

HMGB-1 and 3-Nitrotyrosine levels were HMGB-1 95.93 137.51

Received: April 12, 2021

UDK 577.1 : 61 ISSN 1452-8258

J Med Biochem 41: 282–289, 2022 Original paper

DOWNREGULATION OF MAPK/MAK/MRK OVERLAPPING KINASE 1

Summary Kratak sadr`aj

List of abbreviations: MOK1, MAPK/MAK/MRK overlapping

Introduction downstream regulators. MOK1 overexpression has

Table I Demographic, clinical and laboratory characteristics of CG and T1DM patients.

Sex (male/female)a 6/30 84/88 <0.001

Age (years)b 17 (13–18) (12–16) 0.003

Tanner stage 0.088

Diabetes duration (years)b / 7 (5-8)

BMI percentilesb 54.4 (33.6 –78.4) 56.4 (33.4–78.1) 0.831

HbA1c (%) 5.2 (4.8–5.0) 7.6 (7.0–8.7) <0.001

HDL-cholesterol (mmol/L)b 1.49 (1.26–1.64) 1.63 (1.42–1.88) 0.001

LDL-cholesterol (mmol/L)b 2.21 (1.87–2.51) 2.02 (1.68 –2.50) 0.246

Triglycerides (mmol/L)b 0.81 (0.62–1.03) 0.71 (0.58–0.93) 0.224

CRP (mg/L)b 0.30 (0.20–0.50) 0.70 (0.30 –1.70) 0.009

Age (years) -0.168 / 0.027 -0.086 / 0.259

BMI percentiles -0.117 / 0.091 -0.190 / 0.006

Systolic blood pressure (mm Hg) -0.134 / 0.068 -0.173 / 0.019

Glucose (mmol/L) -0.080 / 0.302 -0.039 / 0.613

Total cholesterol (mmol/L) 0.005 / 0.945 0.091 / 0.233

HDL-cholesterol (mmol/L) 0.101 / 0.192 0.024 / 0.758

LDL-cholesterol (mmol/L) -0.016 / 0.841 0.093 / 0.229

Triglycerides (mmol/L) -0.159 / 0.037 0.010 / 0.892

CRP (mg/L) -0.066 / 0.397 -0.145 / 0.062

HbA1c (%) 0.051 / 0.502 0.122 /0.111