Clinical Chemistry 60:3 Pediatric Clinical Chemistry

518–529 (2014)

Pediatric Within-Day Biological Variation and Quality

Specifications for 38 Biochemical Markers in the
CALIPER Cohort
Dana Bailey,1,2,3† Victoria Bevilacqua,1,2† David A. Colantonio,1,2 Maria D. Pasic,1,2,4 Nandita Perumal,5
Man Khun Chan,1 and Khosrow Adeli1,2*

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BACKGROUND: Studies of biological variation provide insight into pediatric physiology, are of use for refer-
insight into the physiological changes that occur within ence change value calculations, clarify the appropriate-
and between study participants. Values obtained from ness of reference interval use, and aid in the develop-
such investigations are important for patient monitor- ment of quality management strategies specific to
ing and for establishing quality specifications. In this pediatric laboratories.
study we evaluated the short-term biological variation © 2013 American Association for Clinical Chemistry
of 38 chemistry, lipid, enzyme, and protein analytes in
a pediatric population, assessed the effect of age parti-
tions on interindividual variation, and compared the
Biological variation is an important factor to be con-
findings to adult values.
sidered when interpreting laboratory test results in a
METHODS: Four plasma samples each were obtained clinical setting. Studies of biological variation provide
within 8 h from 29 healthy children (45% males), age insight into the physiological changes that occur within
4 –18 years. Samples were stored at ⫺80 °C and analyzed and between individuals for a given analyte. Although
in 3 batches, with samples from 9 –10 study participants several studies have explored biological variation in
per batch. Within-person and between-person biological adult populations, few studies have examined these at-
variation values were established using nested ANOVA tributes in pediatric samples. This information is cru-
after exclusion of outliers by use of the Tukey outlier test. cial in result interpretation for 3 key reasons. First,
Analytical quality specifications were established with the within- and between-person biological variation can
Fraser method. be used to establish reference change values (RCV),4
which provide the information required to determine if a
RESULTS: Biological variation coefficients and analytical
change in concentration of a specific analyte qualifies as
goals were established for 38 analytes. Age partitioning
clinically significant (1 ). Establishing the usual amounts
was required for 6 analytes. Biological variation char-
of observed analyte fluctuation by use of within- and
acteristics of 14 assays (37%) were distinct from adult
between-person biological variation is central to long-
values found in the Westgard database on biological
term monitoring and follow-up of children. It is also an
variation. Biological variation characteristics were es-
especially important consideration for analytes with cycli-
tablished for 2 previously unreported analytes, uncon-
cal rhythms for which the time of collection may affect the
jugated bilirubin and soluble transferrin receptor.
expected reference interval (2 ).
CONCLUSIONS: This study is the first to examine biolog- The second reason why this information is impor-
ical variation and to establish analytical quality specifi- tant is that data information on biological variation can
cations on the basis of biological variation for common be used to establish quality specifications (2 ) such as
assays in a pediatric population. These results provide bias, precision, and total allowable error (TE). These

1
CALIPER program, Department of Pediatric Laboratory Medicine, The Hospital Received August 8, 2013; accepted December 3, 2013.
for Sick Children, Toronto, Ontario, Canada; 2 Department of Laboratory Med- Previously published online at DOI: 10.1373/clinchem.2013.214312
icine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; 3 cur- 4
Nonstandard abbreviations: RCV, reference change value; TE, total allowable
rent address: Gamma-Dynacare Medical Laboratories, London, Ontario, Canada; error; CVI, within-subject/intraindividual biological CV; CVG, between-subject/
4
current address: Department of Laboratory Medicine, St. Joseph’s Health interindividual biological CV; CALIPER, Canadian Laboratory Initiative on Pae-
Centre, Toronto, Ontario, Canada; 5 Department of Pediatrics, The Hospital for diatric Reference Intervals; A1AT, ␣-1 antitrypsin; AGP, ␣-1 acid glycoprotein;
Sick Children, Toronto, Ontario, Canada. C3, complement component 3; CRP, C-reactive protein; STfR, soluble transferrin
*
Address correspondence to this author at: Clinical Biochemistry, The Hospital for receptor; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, as-
Sick Children, University of Toronto, Toronto, Ontario, M5G 1X8 Canada. Fax partate aminotransferase; CK, creatine kinase; GGT, ␥ glutamyl transferase;
416-813- 6257; e-mail [email protected]. HDL-C, HDL cholesterol; LDH, lactate dehydrogenase; CVdd, between-day CV;
†
Dana Bailey and Victoria Bevilacqua contributed equally to the work, and both CVA, total CV; II, index of individuality.
should be considered as first authors.

518
Pediatric Biological Variation for 38 Biochemical Markers

specifications can have important implications for re- Laboratory Initiative on Paediatric Reference Inter-
sult interpretation. For example, in adult populations, vals (CALIPER) outreach program (7 ). The study
changes in bias can affect how many low-risk individ- was completed at The Hospital for Sick Children in
uals are diagnosed with diabetes (3 ) or how many in- Toronto, Canada, with institutional ethics board ap-
dividuals with cholesterol values within reference in- proval. Before participation, the health of each child
tervals are further investigated or sent for treatment was confirmed via interview and by health and life-
(4 ). Although the same misclassifications are likely style questionnaires, with specific exclusion criteria as

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present in pediatric cohorts, data are lacking. described (7 ). Children were instructed to fast over-
Although there are several ways to establish quality night for at least 8 h before the study. A total of 30
specifications, a consensus statement states that the use of children ages 4 –18 years were recruited; 1 child was
biological variation is the preferred method (5 ). Accord- removed from the study owing to an inability to obtain
ing to the hierarchy, a model based on biological variation sufficient samples at all time points. The demographics
is second only to one in which quality specifications are of the children have been previously reported (8 ). For
determined on the basis of the effect of analytical perfor- each participant, blood was drawn at 4 time points,
mance on specific clinical decision-making. However, the with a mean time period of approximately 2.5 h be-
latter approach can be incredibly time-consuming and tween each collection. Sample collection was per-
difficult (5 ) and therefore, biological variation represents formed after an overnight fast, midmorning after
both the most rigorous and the most efficient means by breakfast, within 2 h after lunch, and late afternoon.
which to establish quality specifications. After collection into serum separator tubes, all samples
Finally, biological variation can provide important were processed by the clinical chemistry laboratory and
insight into the usefulness of reference intervals for dif- stored at ⫺80 °C until batch testing.
ferent laboratory tests. Specifically, biological variation
data can be used to generate the index of individuality— ANALYTICAL PROCESSING
the ratio of within- to between-individual variation for a To avoid multiple freeze–thaw cycles and to minimize
given analyte. Although a high index of individuality analytical variability, all 38 assays were run on all sam-
(⬎1.4) suggests that the use of reference intervals ples from each child within the same day. Assays were
would be appropriate, a low index of individuality completed in batches on the Cobas Integra 400 (Roche)
(⬍0.6) suggests that other tools such as RCVs should [␣-1 antitrypsin (A1AT), ␣-1 acid glycoprotein (AGP),
also be taken into consideration (6 ). complement component 3 (C3), C4, ceruloplasmin,
Clearly, the production of data on biological vari- C-reactive protein (CRP), haptoglobin, IgG, IgA, IgM,
ation can generate a wealth of information not only soluble transferrin receptor (STfR), transferrin] and
regarding the physiological changes that occur within the VITROS 5,1 FS chemistry systems analyzer (Ortho
and between individuals, but also on what quality spec- Clinical Diagnostics) [albumin, alkaline phosphatase
ifications are appropriate for a given test and how and (ALP), alanine aminotransferase (ALT), amylase, as-
when to use reference intervals. Although such data are partate aminotransferase (AST), unconjugated biliru-
evidently valuable, there is a clear lack of information on bin, calcium, cholesterol, creatine kinase (CK), chlo-
biological variation in pediatric populations. As such, in ride, CO2, creatinine, iron, ␥ glutamyl transferase
this study we aimed to evaluate the short-term (8 h) bio- (GGT), glucose, HDL cholesterol (HDL-C), potas-
logical variation of 38 chemistry, lipid, enzyme, and pro- sium, lactate dehydrogenase (LDH), magnesium, so-
tein analytes in a pediatric population of 29 healthy chil- dium, phosphate, total bilirubin, total protein, triglyc-
dren age 4 –18 years. The within- and between-person eride, uric acid, urea] over a 3-day period, with samples
biological variation (CVI and CVG) for these analytes from 9 –10 individuals run per day. All assays were per-
were estimated and the effects of age-specific partitions on formed according to the manufacturers’ recommenda-
between-person variation were assessed. Differences in tions. Before use, both analyzers were calibrated using
biological variation between adult and pediatric popula- manufacturer-provided reagents and calibrators. QC
tions were also evaluated. RCVs and indices of individu- materials and 2 patient samples of known analyte con-
ality were generated for each of the 38 analytes and, finally, centrations were run with each batch. Analytical spec-
the estimates of biological variation were used to generate ifications for the 38 analytes tested have already been
analytical goals. published (7 ).

Materials and Methods STATISTICAL ANALYSIS

SPSS Statistical Software (version 21, IBM), EP Evalu-
STUDY PARTICIPANTS ator (version 9), and GraphPad Prism (version 4.0)
Healthy community children were recruited to take were used for data analysis. Data from males and fe-
part in this 1-day (9:30 –17:30) study via the Canadian males were analyzed together. All data followed a

Clinical Chemistry 60:3 (2014) 519

gaussian distribution (8 ). Outliers were eliminated by identify significantly different group means or vari-
use of the Tukey method, which defines an outlier as ances, respectively. Reference intervals were not
1.5 interquartile ranges below the 25th percentile or available for AGP, A1AT, bilirubin (unconjugated),
above the 75th percentile (9 ). ceruloplasmin, CK, chloride, glucose, potassium, so-
Within- and between-person biological variation dium, and STfR (7 ).
were calculated using GraphPad Prism and STATA and Adult values for CVI and CVG were obtained from
expressed as CVs, CVI and CVG, respectively. CVG was the Westgard Biological Variation database (12 ). This

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estimated using a nested ANOVA. CVI was estimated database is an updated compilation of multiple papers
by averaging the CV for each child, defined as the examining biological variation in adult populations
square root of the variance in results divided by the (13 ). The weighted-mean CVG across pediatric age
mean concentration for that individual, across all indi- partitions was used for the pediatric cohort. Pediatric
viduals. In contrast to estimates of CVI, for which the to adult CVI, CVG, RCV, and total allowable error (TE)
overall mean is used [e.g., (10 )]. This approach is more ratios ⬍0.5 or ⬎1.5 were arbitrarily defined as being
appropriate for situations in which the overall mean is significantly different.
not representative of the mean for each individual.
Both within- and between-person biological variation Results
contain components of biological and analytical varia-
tion, which were minimized through experimental de- A total of 29 children ages 4 –18 years (13 male, 16
sign and removed mathematically by subtracting the female) were recruited to participate in this 1-day (8 h)
between-day CV (CVdd) or the total CV (CVA), respec- study. Before participation, the health of each child was
tively. Analytical CV was calculated using QC material ensured through an interview as well as health and life-
run concurrently with the collected samples. The index style questionnaires. Participants were instructed to
of individuality (II), RCV, and analytical goals includ- fast for at least 8 h before the study. Each child was
ing imprecision, bias, and total allowable error, were sampled at 4 time points with approximately 2.5 h be-
calculated as follows: tween each sampling, yielding 116 data points for each
analyte; a total of 38 analytes were examined, yielding
II ⫽ CVI/CVG (note that the CVA was not included in 4408 analytical results.
calculating II) (11 ); To correctly estimate CVG, it was necessary to par-
RCV ⫽ z(2)1/2(CVA2 ⫹ CVI2)1/2; z ⫽ 1.96 for P ⬍ 0.05 tition children by age for 6 of the 38 analytes, specifi-
for a bidirectional change; cally ALP, AST, creatinine, LDH, phosphate, and uric
acid (Fig. 1, panel A). Suitable age partitions for this
Analytical imprecision:
cohort were derived from the CALIPER database, as
Optimal ⫽ 0.25 ⫻ CVI, recently described (14 ). To determine whether age par-
Desirable ⫽ 0.50 ⫻ CVI, titioning was necessary to minimize CVG, children
Minimal ⫽ 0.75 ⫻ CVI; were partitioned by age, and the mean concentration
Analytical bias: and variance of each age group was compared statisti-
Optimal ⫽ 0.125 ⫻ (CVI2 ⫹ CVG 2 1/2
) , cally, as described in Methods. Partitioning by age did
not significantly affect CVG for HDL-C, CRP, albumin,
Desirable ⫽ 0.25 ⫻ (CVI ⫹ CVG) ,
2 2 1/2
total bilirubin, and IgG (Fig. 1B).
Minimal ⫽ 0.375 ⫻ (CVI2 ⫹ CVG 2 1/2
) ; Table 1 lists the CVA, between-run CV, and CVdd
Total allowable error (TAE) ⫽ (1.65 ⫻ imprecision) ⫹ CVs, the CVI and CVG, the II, and the RCV for each of
bias. the 38 analytes, as partitioned by age. Only 1 analyte,
AST, had an index of individuality that exceeded 1.4.
When statistically indicated, the population of The majority of analytes examined (albumin, ALP,
children was partitioned according to age on the basis AGP, A1AT, ALT, amylase, AST, C3, C4, ceruloplas-
of previously determined CALIPER reference interval min, cholesterol, CK, chloride, CRP, iron, GGT, hap-
age partitions (7 ). To determine whether CALIPER- toglobin, HDL-C, IgA, IgG, IgM, LDH, STfR, total pro-
determined age partitions were appropriate for this tein, transferrin, uric acid, and urea) showed marked
substudy population, a Student t-test and F-test were individuality, with II values of ⬍0.6.
performed on analytes containing 2 age partitions (uric When comparing the pediatric cohort and adult
acid, HDL-C, CRP, urea, ALT, haptoglobin, IgG), populations, CVI and CVG components of 24 of 38
whereas a 1-way ANOVA and Bartlett test were per- analytes (63%) were found to be consistent with adult
formed on analytes containing 3 or more age parti- values published in the Westgard database (Table 2)
tions (total bilirubin, CO2, creatinine, phosphate, (12 ). Four analytes showed marked differences, arbi-
ALP, AST, LDH, albumin, total protein, and IgA) to trarily defined as a more than 50% reduction or more

520 Clinical Chemistry 60:3 (2014)

Pediatric Biological Variation for 38 Biochemical Markers

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Fig. 1. Relationship of within- and between-person biological variation with age in a pediatric cohort.
Numbers on the y axis refer to the study participant identification numbers, with children sorted by ascending age. The range
of values for each child is indicated by the box-and-whisker plot. (A), Representative analytes for which within- and/or
between-person biological variation change with age in a pediatric cohort. Dark grey shading indicates that the age partitions
determined by prior CALIPER reference interval studies [Colantonio et al. (7 )] significantly affect estimates of CVI and/or CVG.
(B), Representative analytes for which within- and/or between-person biological variation do not change significantly with age
in a pediatric cohort. Light grey shading indicates that the age partitions determined by prior CALIPER reference interval studies
[Colantonio et al. (7 )] did not significantly affect estimates of CVI and/or CVG.

than 150% increase, in both within- and between- adult populations for AGP, AST, CK, CRP, GGT, and
person variation between the pediatric and adult pop- STfR, whereas it was increased for ceruloplasmin and
ulations. Specifically, CRP values in the pediatric co- glucose.
hort demonstrated reduced CVI and increased CVG To provide a guide for analytical quality specifica-
compared to adult values, GGT showed reduced CVI tions for pediatric testing based on biological variation,
and CVG, and ceruloplasmin and glucose showed in- we calculated optimal, desirable, and minimal analyti-
creased CVI and CVG (Table 2). Additionally, 10 ana- cal goals for imprecision, bias, and total allowable error
lytes showed marked differences in either CVI or CVG. (Table 3) and compared them with adult specifications
Specifically, AGP, AST, cholesterol, CK, HDL, IgG, and (Table 2). The total allowable error based on biological
STfR had reduced CVI, sodium had reduced CVG, and variation characteristics was reduced by more than half
iron and transferrin had increased CVG. Interestingly, of that allowed for adult populations for CK (14.3% vs
for iron, the smaller CVI and larger CVG values seen in 30.3%, pediatric vs adult, respectively) and STfR (6.8%
the pediatric population resulted in a smaller II (0.38 vs vs 17.4%); it was increased to ⬎150% of that allowed
1.14) (12 ). As a consequence of the differences ob- for an adult population for ceruloplasmin (15.1% vs
served in CVI, the pediatric RCV was lower than that of 7.9%) and glucose (13.1% vs 7.2%).

Clinical Chemistry 60:3 (2014) 521

 Table 1. Biological variation characteristics in a pediatric cohort.

Analytical variation Biological variation

Analyte n Age, years Mean CVr r , % CVdd, % CVA, % CVI, % CVG, % II RCV, %

Albumin, g/dL 29 1 to ⬍19 4.5 1.5 0.2 1.5 2.3 4.7 0.5 7.5
ALP, U/L 5 1 to ⬍10 241.1 1.9 1.0 2.0 5.6 27.2 0.1 16.4

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7 10 to ⬍13 219.0 24.0 0.1
3 13 to ⬍15 215.8 27.2 0.1
4 15 to ⬍17 151.4 60.9 0.1
10 17 to ⬍19 86.5 18.7 0.2
AGP, mg/dL 28 1 to ⬍19 80.0 0.9 0.7 1.1 3.7 20.5 0.2 10.8
A1AT, mg/dL 28 1 to ⬍19 120.0 1.9 0.0 1.9 5.0 13.0 0.4 14.7
ALT, U/L 29 1 to ⬍19 16.4 4.8 5.1 7.0 15.6 27.7 0.6 47.4
Amylase, U/L 29 1 to ⬍19 67.0 2.9 1.6 3.3 5.1 24.9 0.2 16.7
AST, U/L 2 1 to ⬍7 35.5 1.7 0.8 1.9 4.7 0.6 8.4 13.9
6 7 to ⬍12 28.5 21.5 0.2
21 12 to ⬍19 22.9 23.9 0.2
Bilirubin (total), mg/dL 29 1 to ⬍19 0.3 1.7 2.0 2.6 28.1 38.2 0.7 78.3
Bilirubin (unconjugated), 28 1 to ⬍19 0.2 1.7 2.6 3.1 51.1 57.0 0.9 141.8
mg/dL
C3, mg/dL 29 1 to ⬍19 120.0 0.9 2.5 2.7 4.8 12.1 0.4 15.2
C4, mg/dL 24 1 to ⬍19 20.0 2.6 2.1 3.4 5.5 28.1 0.2 18.0
Calcium, mg/dL 29 1 to ⬍19 10.0 1.0 0.9 1.3 1.6 2.5 0.7 5.7
Ceruloplasmin, mg/dL 28 1 to ⬍19 19.6 3.8 1.3 4.1 11.3 20.3 0.6 33.4
Chloride, mmol/L 29 1 to ⬍19 105.9 0.2 0.5 0.6 0.8 1.5 0.6 2.8
Cholesterol, mg/dL 29 1 to ⬍19 166.0 0.9 1.1 1.5 2.4 15.7 0.2 7.9
CO2, mmol/L 29 1 to ⬍19 24.6 2.8 2.3 3.7 3.4 5.3 0.6 13.8
CK, U/L 28 1 to ⬍19 89.5 1.7 4.3 4.7 4.1 43.4 0.1 17.2
Creatinine, mg/dL 2 2 to ⬍5 0.3 1.1 0.7 1.3 4.2 4.1 1.1 12.3
5 5 to ⬍12 0.5 23.3 0.2
8 12 to ⬍15 0.6 11.0 0.4
14 15 to ⬍19 0.8 15.7 0.3
CRP, mg/L 27 1 to ⬍19 0.9 1.6 2.9 3.3 19.3 125.4 0.2 54.1
GGT, U/L 28 1 to ⬍19 18.0 0.9 0.5 1.1 2.7 18.7 0.1 8.0
Glucose, mg/dL 29 1 to ⬍19 90.0 1.2 0.0 1.2 11.4 9.1 1.3 31.8
Haptoglobin, mg/dL 29 1 to ⬍19 90.0 1.2 1.3 1.7 10.7 50.8 0.2 30.1
HDL-C, mg/dL 25 1 to ⬍19 54.0 2.2 3.4 4.1 2.9 22.2 0.1 13.9
IgA, mg/dL 29 1 to ⬍19 150.0 0.6 1.3 1.5 4.4 42.1 0.1 12.9
IgG, mg/dL 29 1 to ⬍19 1110.0 2.4 1.1 2.6 1.1 14.7 0.1 7.8
IgM, mg/dL 28 1 to ⬍19 120.0 0.6 1.9 2.0 4.0 37.3 0.1 12.3
Iron, ␮g/dL 29 1 to ⬍19 83.2 2.3 4.2 4.7 14.6 38.9 0.4 42.5
LDH, U/L 5 1 to ⬍10 652.9 2.0 0.9 2.2 4.7 12.5 0.4 14.4
10 10 to ⬍15 528.4 18.1 0.3
14 15 to ⬍19 420.2 13.0 0.4
Magnesium, mg/dL 29 1 to ⬍19 1.9 1.7 0.0 1.7 2.7 4.5 0.6 8.8
Continued on page 523

522 Clinical Chemistry 60:3 (2014)

Pediatric Biological Variation for 38 Biochemical Markers

Table 1. Biological variation characteristics in a pediatric cohort. (Continued from page 522)

Analytical variation Biological variation

Analyte n Age, years Mean CVr r , % CVdd, % CVA, % CVI, % CVG, % II RCV, %

Phosphate, mg/dL 2 1 to ⬍5 5.3 1.1 0.0 1.1 6.0 7.0 0.9 16.9
9 5 to ⬍13 4.6 8.0 0.8

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8 13 to ⬍16 4.6 10.4 0.6
10 16 to ⬍19 4.0 5.1 1.2
Potassium, mmol/L 29 1 to ⬍19 4.4 0.6 1.5 1.6 4.6 5.3 0.9 13.5
Sodium, mmol/L 29 1 to ⬍19 142.7 0.5 0.6 0.8 0.4 0.4 0.9 2.4
STfR, mg/dL 27 1 to ⬍19 350.0 3.2 0.0 3.2 1.4 22.4 0.1 9.6
Total protein, g/dL 29 1 to ⬍19 8.0 1.5 0.8 1.7 1.7 4.6 0.4 6.7
Transferrin, mg/dL 28 1 to ⬍19 2.2 1.9 0.0 1.9 3.0 10.8 0.3 9.8
Triglycerides, mg/dL 29 1 to ⬍19 97.3 1.0 0.4 1.1 27.0 24.8 1.1 74.9
Urea, mg/dL 29 1 to ⬍19 14.6 1.3 0.7 1.5 7.5 22.1 0.3 21.3
Uric acid, mg/dL 7 1 to ⬍12 3.3 1.1 0.7 1.3 6.8 25.1 0.3 19.2
22 12 to ⬍19 4.6 23.0 0.3

Discussion for adults. A reduced CVI was observed for 9 analytes,

but this may have resulted from the short-term nature
Biological variation and its application in serial results of this study. Previous studies have determined that
monitoring and analytical goal establishment has been measurements made 24 h apart had smaller CVI values
widely examined in adult populations. However, there than those taken at intervals of 4 days or longer (17 ).
is no literature available exploring biological variation Additionally, an increased CVG for pediatric vs adult
in a pediatric population. As a consequence, pediatric populations was observed for 5 analytes, which may
laboratories have been obliged to adopt RCV and ana- indicate a need for further age and/or sex partitioning,
lytical quality goals based on adult cohorts. In the ab- as previously demonstrated by reference interval stud-
sence of data on biological variation, the clinical pedi- ies (18 ). Only 4 analytes, CRP, GGT, ceruloplasmin,
atric laboratory has been unable to provide a complete and glucose, showed marked differences in both
picture of the dynamic changes expected for analytes in within- and between-person variation in the pediatric
the pediatric population. This is especially crucial given vs the adult populations (Table 2).
the many changes associated with childhood and teen- CRP presented with a reduced CVI (19.3% vs
age development. Previous CALIPER studies have 42.2%) and a larger CVG (125.4% vs 76.3%) in the
highlighted the substantial differences between pediat- pediatric population. This increase in CVG largely re-
ric and adult reference intervals (7, 15, 16 ) and the ef- sulted from 5 children with median CRP concentra-
fects of fasting and sampling time in a pediatric cohort tions greater than approximately 1.5 mg/L (Fig. 2). Al-
(8 ). However, addressing this gap in information rele- though the reason for the increase in CRP in these
vant to the pediatric population presents particular individuals remains unknown, the findings are consis-
challenges, including the acquisition of samples and tent with NHANES (National Health and Nutrition
the necessary introduction of age partitioning to pro- Examination Survey) data for 10- to 15-year-old chil-
vide an accurate estimate of CVG. By examining the dren, in which 15% of children had CRP values be-
short-term biological variation of 38 chemistry ana- tween 0.9 and 2.7 mg/L and 10% had values ⬎2.7 mg/L
lytes in a pediatric cohort, we have made the first steps (19 ).
to fill this gap in pediatric laboratory medicine. The age GGT demonstrated a reduced CVI and CVG (Fig.
partitions established in previous CALIPER studies 2). Because increases in GGT in adults are known to be
were tested in this analysis and it was determined that nonspecific, with modest increases noted in conjunc-
age partitioning was required for 6 of the 38 analytes tion with various common metabolic risk factors such
examined, specifically, ALP, AST, creatinine, LDH, as increased blood pressure and decreased HDL-C
phosphate, and uric acid (Fig. 1A). (20 ), the tighter biological control of GGT observed in
The CVI and CVG estimates in this pediatric pop- children may be partially explained by an absence of
ulation were found to be largely consistent with those these subclinical conditions.

Clinical Chemistry 60:3 (2014) 523

 Table 2. Comparison between pediatric and adult biological variation characteristics.

Pediatric Adulta

Analyte N Age, years CVI, % CVG, %b RCV, % TE, %c CVI, % CVG, % RCV, % TE, %c

Albumin 29 1 to ⬍19 2.3 4.7 7.5 3.2 3.1 4.2 9.5 3.9
ALP 29 1 to ⬍19 5.6 28.1 16.4 11.8 6.4 24.8 18.2 11.7

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AGP 28 1 to ⬍19 3.7d 20.5 10.8d 8.3 11.3 24.9 31.5 16.2
A1AT 28 1 to ⬍19 5.0 13.0 14.7 7.6 5.9 16.3 16.8 9.2
ALT 29 1 to ⬍19 15.6 27.7 47.4 20.8 18.0 42.0 53.5 26.3
Amylase 29 1 to ⬍19 5.1 24.9 16.7 10.6 8.7 28.3 25.8 14.6
AST 29 1 to ⬍19 4.7d 21.8 13.9d 9.5 11.9 17.9 33.4 15.2
Bilirubin (total) 29 1 to ⬍19 28.1 38.2 78.3 35.0 23.8 39.0 66.4 31.1
Bilirubin (unconjugated) 28 1 to ⬍19 51.1 57.0 141.8 61.3
C3 29 1 to ⬍19 4.8 12.1 15.2 7.2 5.2 15.6 16.2 8.4
C4 24 1 to ⬍19 5.5 28.1 18.0 11.7 8.9 33.4 26.4 16.0
Calcium 29 1 to ⬍19 1.6 2.5 5.7 2.1 1.9 2.8 6.4 2.4
Ceruloplasmin 28 1 to ⬍19 11.3e 20.3e 33.4e 15.1e 5.8 11.1 19.7 7.9
Chloride 29 1 to ⬍19 0.8 1.5 2.8 1.1 1.2 1.5 3.7 1.5
Cholesterol 29 1 to ⬍19 2.4d 15.7 7.9 6.0 5.4 15.2 15.5 8.5
CO2 29 1 to ⬍19 3.4 5.3 13.8 4.4 4.8 5.3 16.8 5.7
CK 28 1 to ⬍19 4.1d 43.4 17.2d 14.3d 22.8 40.0 64.5 30.3
Creatinine 29 2 to ⬍19 4.2 14.9 12.3 7.3 6.0 14.7 17.0 8.9
CRP 27 1 to ⬍19 19.3d 125.4e 54.1d 47.6 42.2 76.3 117.3 56.6
GGT 28 1 to ⬍19 2.7d 18.7d 8.0d 7.0d 13.8 41.0 38.4 22.2
Glucose 29 1 to ⬍19 11.4e 9.1e 31.8e 13.1e 6.1 6.1 17.1 7.2
Haptoglobin 29 1 to ⬍19 10.7 50.8 30.1 21.8 20.4 36.4 56.7 27.3
HDL-C 25 1 to ⬍19 2.9d 22.2 13.9 8.0 7.1 19.7 22.7 11.1
IgA 29 1 to ⬍19 4.4 42.1 12.9 14.2 5.4 35.9 15.5 13.5
IgG 29 1 to ⬍19 1.1e 14.7 7.8 4.6 4.5 16.5 14.4 8.0
IgM 28 1 to ⬍19 4.0 37.3 12.3 12.7 5.9 47.3 17.3 16.8
Iron 29 1 to ⬍19 14.6 38.9e 42.5 22.4 26.5 23.2 74.6 30.7
LDH 29 1 to ⬍19 4.7 14.7 14.4 7.7 8.6 14.7 24.6 11.4
Magnesium 29 1 to ⬍19 2.7 4.5 8.8 3.5 3.6 6.4 10.7 4.8
Phosphate 29 1 to ⬍19 6.0 7.6 16.9 7.4 8.5 9.4 23.7 10.2
Potassium 29 1 to ⬍19 4.6 5.3 13.5 5.5 4.8 5.6 14.0 5.8
Sodium 29 1 to ⬍19 0.4 0.4d 2.4 0.5 0.7 1.0 2.9 0.9
STfR 27 1 to ⬍19 1.4d 22.4 9.6d 6.8d 13.6 20.8 38.4 17.4
Total protein 29 1 to ⬍19 1.7 4.6 6.7 2.6 2.7 4.0 8.8 3.4
Transferrin 28 1 to ⬍19 3.0 10.8e 9.8 5.3 3.0 4.3 9.1 3.8
Triglycerides 29 1 to ⬍19 27.0 24.8 74.9 31.4 20.9 37.2 58.0 27.9
Urea 29 1 to ⬍19 7.5 22.1 21.3 12.0 12.3 18.3 34.3 15.7
Uric acid 29 1 to ⬍19 6.8 23.5 19.2 11.7 9.0 17.6 25.2 12.4
a
Westgard website available: http://www.westgard.com/biodatabase1.htm.
b
Weighted CVG.
c
Desirable TE ⫽ 1.65 (0.5 ⫻ CVI) ⫹ [0.25 ⫻ ⻫(CVI2 ⫹ CVG2)].
d
Reduced variation compared to adult cohorts (pediatric CV/adult CV ⬍0.5).
e
Increased variation compared to adult cohorts (pediatric CV/adult CV ⬎1.5).

524 Clinical Chemistry 60:3 (2014)

Pediatric Biological Variation for 38 Biochemical Markers

Table 3. Analytical goals for pediatric testing based on short-term biological variation.

Analytical goals

Imprecision, CV, % Bias, % TE, %

Analyte Age, Years Optimal Desirable Minimal Optimal Desirable Minimal Optimal Desirable Minimal

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Albumin 1 to ⬍19 0.6 1.1 1.7 0.6 1.3 1.9 1.6 3.2 4.8
ALP 1 to ⬍10 1.4 2.8 4.2 3.5 6.9 10.4 5.8 11.5 17.3
10 to ⬍13 3.1 6.2 9.2
13 to ⬍15 3.5 7.0 10.4
15 to ⬍17 7.6 15.3 22.9
17 to ⬍19 2.4 4.9 7.3
AGP 1 to ⬍19 0.9 1.9 2.8 2.6 5.2 7.8 4.1 8.3 12.4
A1AT 1 to ⬍19 1.2 2.5 3.7 1.7 3.5 5.2 3.8 7.6 11.3
ALT 1 to ⬍19 3.9 7.8 11.7 4.0 8.0 11.9 10.4 20.8 31.2
Amylase 1 to ⬍19 1.3 2.5 3.8 3.2 6.3 9.5 5.3 10.5 15.8
AST 1 to ⬍7 1.2 2.3 3.5 0.6 1.2 1.8 2.5 5.0 7.5
7 to ⬍12 2.7 5.5 8.2
12 to ⬍19 3.0 6.1 9.1
Bilirubin (total) 1 to ⬍19 7.0 14.1 21.1 5.9 11.9 17.8 17.5 35.1 52.6
Bilirubin (unconjugated) 1 to ⬍19 12.8 25.6 38.3 9.6 19.1 28.7 30.7 61.3 92.0
C3 1 to ⬍19 1.2 2.4 3.6 1.6 3.2 4.9 3.6 7.2 10.8
C4 1 to ⬍19 1.4 2.8 4.2 3.6 7.2 10.8 5.9 11.7 17.6
Calcium 1 to ⬍19 0.4 0.8 1.2 0.4 0.7 1.1 1.0 2.1 3.1
Ceruloplasmin 1 to ⬍19 2.8 5.7 8.5 2.9 5.8 8.7 7.6 15.2 22.8
Chloride 1 to ⬍19 0.2 0.4 0.6 0.2 0.4 0.6 0.5 1.1 1.6
Cholesterol 1 to ⬍19 0.6 1.2 1.8 2.0 4.0 6.0 3.0 6.0 8.9
CO2 1 to ⬍19 0.8 1.7 2.5 0.8 1.6 2.3 2.2 4.3 6.5
CK 1 to ⬍19 1.0 2.0 3.0 5.4 10.9 16.3 7.1 14.2 21.4
Creatinine 2 to ⬍5 1.1 2.1 3.2 0.7 1.5 2.2 2.5 5.0 7.5
5 to ⬍12 3.0 5.9 8.9
12 to ⬍15 1.5 3.0 4.4
15 to ⬍19 2.0 4.1 6.1
CRP 1 to ⬍19 4.8 9.6 14.4 15.9 31.7 47.6 23.8 47.6 71.4
GGT 1 to ⬍19 0.7 1.3 2.0 2.4 4.7 7.1 3.5 6.9 10.4
Glucose 1 to ⬍19 2.9 5.7 8.6 1.8 3.7 5.5 6.5 13.1 19.6
Haptoglobin 1 to ⬍19 2.7 5.4 8.0 6.5 13.0 19.5 10.9 21.8 32.7
HDL-C 1 to ⬍19 0.7 1.4 2.2 2.8 5.6 8.4 4.0 8.0 11.9
IgA 1 to ⬍19 1.1 2.2 3.3 5.3 10.6 15.9 7.1 14.2 21.3
IgG 1 to ⬍19 0.3 0.6 0.8 1.8 3.7 5.5 2.3 4.6 6.9
IgM 1 to ⬍19 1.0 2.0 3 4.7 9.4 14.1 6.3 12.7 19.0
Iron 1 to ⬍19 3.6 7.3 10.9 5.2 10.4 15.6 11.2 22.4 33.7
LDH 1 to ⬍10 1.2 2.4 3.5 1.7 3.3 5.0 3.6 7.2 10.8
10 to ⬍15 2.3 4.7 7.0
15 to ⬍19 1.7 3.5 5.2
Magnesium 1 to ⬍19 0.7 1.3 2.0 0.7 1.3 2.0 1.8 3.5 5.3
Continued on page 526

Clinical Chemistry 60:3 (2014) 525

 Table 3. Analytical goals for pediatric testing based on short-term biological variation. (Continued from
page 525)

Analytical goals

Imprecision, CV, % Bias, % TE, %

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Analyte Age, Years Optimal Desirable Minimal Optimal Desirable Minimal Optimal Desirable Minimal

Phosphate 1 to ⬍19 1.5 3.0 4.5 1.1 2.3 3.4 3.6 7.2 10.9
5 to ⬍13 1.3 2.5 3.8
13 to ⬍16 1.5 3.0 4.5
16 to ⬍19 1.0 2.0 2.9
Potassium 1 to ⬍19 1.1 2.3 3.4 0.9 1.7 2.6 2.8 5.5 8.3
Sodium 1 to ⬍19 0.1 0.2 0.3 0.1 0.1 0.2 0.2 0.4 0.7
STfR 1 to ⬍19 0.3 0.7 1.0 2.8 5.6 8.4 3.4 6.7 10.1
Total protein 1 to ⬍19 0.4 0.9 1.3 0.6 1.2 1.8 1.3 2.6 4.0
Transferrin 1 to ⬍19 0.7 1.5 2.2 1.4 2.8 4.2 2.6 5.3 7.9
Triglycerides 1 to ⬍19 6.8 13.5 20.3 4.6 9.2 13.7 15.7 31.5 47.2
Urea, 1 to ⬍19 1.9 3.8 5.6 2.9 5.8 8.7 6.0 12.0 18.1
Uric acid 1 to ⬍12 1.7 3.4 5.1 3.2 6.5 9.7 6.1 12.1 18.2
12 to ⬍19 3.0 6.0 9.0

Interestingly, glucose presented with increases in The need for properly established analytical goals,
both CVI and CVG relative to adult populations (Fig. and the consequences that result from a lack thereof,
2). These increases are believed to be due to the more have been well documented (3, 4, 6, 24 ). As such, it
prominent effect of fasting on glucose homeostasis in was necessary to determine whether any differences ex-
children. In comparison to adults, children are more ist in the laboratory test quality specifications required
prone to decreases in plasma glucose and increases in in the pediatric population compared with those in the
ketone body production [reviewed in (21 )], thereby adult population. Our analysis revealed that 4 analytes
widening the range of observed glucose concentra- showed discernible differences in TE between the adult
tions. Indeed, 7 pediatric study participants had fasting and pediatric populations: CK and STfR in the pediat-
glucose concentrations ⱕ4.0 mmol/L or 72 mg/dL, ric population had a decreased TE compared with the
with a nonfasting upper limit of 8.0 mmol/L or 144 adult population, whereas ceruloplasmin and glucose
mg/dL. had an increased TE (Table 2). It should be noted that
Due to a decrease in CVI (14.6% vs 26.5%) and a interpretation of these results should take into consid-
modest increase in CVG (38.9% vs 23.2%), the II of eration the clinical context and the downstream effect
iron was reduced from 1.14 to 0.38 (adult vs pediatric) of clinical misclassification. For example, as a conse-
(Fig. 2). Consistent with the reduction in CVI, it has quence of the increase in both components of biologi-
previously been shown that infants and young children cal variation, the calculated TE for glucose increased
lack the diurnal variation of serum iron due to the ab- from 7.2% to 13.1%. However, we argue that the ob-
sence of a sustained period of sleep (22 ). Furthermore, servation of increased susceptibility to hypoglycemia in
the discrepancy in CVG may be explained, in part, by pediatric individuals suggests the need for accurate and
the fact that iron deficiency is common in the pediatric precise glucose measurements in this population, par-
population, with females aged 12–19 years at especially ticularly at low concentrations.
high risk for anemia (23 ). The observation that the In terms of the II, it has been argued that analytes
CVG for a related analyte, transferrin, was also mark- with II values of ⬍0.6 show a high degree of individu-
edly increased in the pediatric population (10.8% vs ality and, therefore, the RCV as opposed to a reference
4.3%) suggests substantial variations in pediatric indi- interval should be used to assess the patient. On the
viduals with respect to iron status. Future long-term other hand, analytes with II values of ⬎1.4 show very
studies will be needed to explore whether or not this little individuality and, therefore, the use of reference
observation persists when the duration of the sampling intervals is deemed to be appropriate (6 ). Analysis of
period is increased. the samples obtained from this pediatric population

526 Clinical Chemistry 60:3 (2014)

Pediatric Biological Variation for 38 Biochemical Markers

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Fig. 2. Within- and between-person biological variation characteristics unique to pediatric samples.
Estimates of CVG for glucose, GGT, CRP, and iron are indicated by the top horizontal line and vertical dashed lines. Numbers
on the y axis refer to study participant identification numbers, with individuals sorted by ascending age. The range of values
for each child is indicated by the box-and-whisker plot.

revealed that only 1 analyte, AST, had an II value that concentration that would clearly fall outside of the es-
exceeded 1.4, whereas 27 of the 38 analytes examined tablished reference interval. For example, bilirubin has
had an II value below 0.6. Of particular interest, the an II value of 0.74, which falls only slightly above the
reduction of II for iron to 0.38 suggests further inves- 0.6 cutoff, showing a fair degree of individuality. Ref-
tigation of the utility of an iron reference interval for erence intervals derived from a sample of healthy chil-
clinical decision-making in a pediatric population. dren indicate that total bilirubin for children of ages
Although it may be appropriate to further examine birth to 14 days should fall between 0.19 and 16.60
the usefulness of population-based reference intervals mg/dL, and for children ages 15 days to 1 year, total
in cases in which the II falls below 0.6, it is also impor- bilirubin should fall between 0.05 and 0.68 mg/dL (7 ).
tant to consider the clinical context in which the ana- Cases of kernicterus have rarely been reported in neo-
lyte is likely to be used (25 ). In many cases, a patholog- nates with bilirubin concentrations of ⬍25 mg/dL and
ical state would result in a dramatic increase in analyte are not reported in neonates with bilirubin peak con-

Clinical Chemistry 60:3 (2014) 527

centrations of ⬍20 mg/dL (26 ). These clinically rele- mates of CVI are likely smaller than what would be
vant values far exceed the reference intervals estab- obtained from a study of longer duration. Lastly, given
lished, and it is unlikely that, despite the small II value the short-term nature of this study, preanalytical sam-
for bilirubin, a clinical diagnosis based on bilirubin pling variation may contribute significantly to the vari-
concentrations would be adversely affected by compar- ation observed. Future long-term studies will be
ison with a population-based reference interval. There- needed to confirm the findings described herein.
fore, it is important that measures like II, as well as In conclusion, this is the first study to analyze the

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clinical context, be taken into account when interpret- components of biological variation in a healthy pediat-
ing results and determining a course of action for the ric cohort. We have derived estimates of RCVs and
patient in question. quality indicators for precision, bias, and total allow-
Finally, it is important to note certain limitations able error. Future studies are needed to refine our esti-
of our study. First, previous CALIPER studies have de- mates of the components of biological variation in pe-
termined that, for the creation of reference intervals, diatric populations, to extend our analysis of short-
certain analytes (e.g., creatinine, albumin, and ALT) term biological variation to longer time periods, and to
show sex differences and, therefore, must be parti- increase the sample size investigated.
tioned accordingly (7, 16, 27 ). However, due to sample
size limitations, we were unable to assess the appropri-
ateness of partitioning CVI and CVG by sex. Larger-
scale studies will be needed to determine the role of sex Author Contributions: All authors confirmed they have contributed to
in biological variation for these analytes. Second, ow- the intellectual content of this paper and have met the following 3 re-
ing to the sample size of our study and the need to run quirements: (a) significant contributions to the conception and design,
samples in batches over several days, our protocol in- acquisition of data, or analysis and interpretation of data; (b) drafting
troduced components of between-run analytical im- or revising the article for intellectual content; and (c) final approval of
the published article.
precision into estimates of CVG which were removed
mathematically. Third, owing to the challenges associ- Authors’ Disclosures or Potential Conflicts of Interest: Upon man-
ated with obtaining pediatric samples, this study was uscript submission, all authors completed the author disclosure form.
Disclosures and/or potential conflicts of interest:
restricted to a single day. Therefore, calculations such
as analytical quality values or RCV might be relevant Employment or Leadership: None declared.
only for repeat samples collected within the same time- Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
frame. Fourth, in comparing the pediatric estimates of
Honoraria: None declared.
CVI to those of adult populations, it was assumed that Research Funding: K. Adeli, CIHR.
the within-person biological variation in a single day Expert Testimony: None declared.
would be representative of long-term variation. Al- Patents: None declared.
though we attempted to account for some effects of Role of Sponsor: The funding organizations played no role in the
diurnal variation and fed/fasting-related variation by design of study, choice of enrolled participants, review and interpre-
sampling in a fasting and postprandial state, the esti- tation of data, or preparation or approval of manuscript.

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