Alkaline Phosphatase (ALP)

Diagnostic reagent for determination of Alkaline Phosphatase (ALP) concentration.

Liquid. Dual Reagents (Ratio: R1/R2: 4/1). Store at +2/+8°C. For in Vitro Diagnostic Use (IVD). Do not freeze.

Ref No Package
MH-012 75 mL
MH-013 50 mL

Changes made in the instructions for use are marked as grey.

INTENDED USE the detergent effect of bile salts and enters the circulation
This test is applied for the quantitative determination of to increase enzyme activity in serum.2
ALP in human serum and plasma.
The increase in enzyme activity in extrahepatic obstruction
GENERAL INFORMATION (e.g. stones, pancreatic head cancer) can be more than
four times the upper reference limit (URL). This increase is
Alkaline phosphatase, (EC 3.1.3.1; orthophosphoric generally greater than those seen in intrahepatic
monoester phosphohydrolase [alkaline optimum]; ALP) cholestasis. The greater the obstruction, the higher the
catalyzes the alkaline hydrolysis of a wide variety of serum enzyme activity (which can reach 10 to 12 times the
naturally occurring and synthetic substrates. Some URL) and usually returns to normal within a week after
divalent ions such as Mg+2, Co+2 and Mn+2 are activators surgical removal of the obstruction. A similar increase is
of the enzyme, while Zn+2 is a metal ion constitutively also seen in patients with advanced primary liver cancer or
present in the enzyme. The correct ratio of Mg+2 to Zn+2 extensive secondary liver metastasis. ALP elevation is can
ions is necessary to avoid displacement of Mg+2 and for to be predictive of outcomes such as liver transplantation or
have optimal activity of the enzyme. Phosphate, borate, death in patients with primary biliary cholangitis (previously
oxalate and cyanide ions are inhibitors of ALP activity. known as primary biliary cirrhosis) and can increase more
Changes in Mg+2 and substrate concentrations alter the pH than twice the URL.3
optimum required for enzyme activity. ALP activity is
present in most organs of the body and is found fixed to Similar prognostic information can also be obtained from
the cell membrane via glycosylphosphatidylinositol ALP activity in primary sclerosing cholangitis. Liver
("ectoenzyme"), particularly in the mucosa of the small diseases that primarily affect parenchymal cells, such as
intestine and proximal convoluted tubules of the kidney, infectious hepatitis, typically show only moderately
bone (osteoblasts), liver and placenta. Although the exact elevated (less than threefold) or even normal serum ALP
metabolic function of the enzyme is not yet understood, activities. Increases may also be the result of a response
ALP it appears to be involved in lipid transport in the gut, to drug therapy and ALT-/ALP-based criteria remain the
the calcification process in bone and host defense through mainstay for detecting and characterizing the type of drug-
endotoxin dephosphorylation. ALP exists in multiple induced liver injury.1 In the third trimester of pregnancy, an
homodimeric forms (molecular weight ranges from 70 to increase of up to two to three times the URL is observed in
120 kDa), some of which are true isoenzymes encoded at women. This situaiton makes ALP an unreliable marker of
separate genetic loci. Bone, liver and kidney forms of ALP hepatobiliary disease in pregnancy.
it share a common primary structure encoded by the same Studies have described a benign familial increase in serum
genetic locus, but differ in their carbohydrate content. The ALP activity caused by increased intestinal ALP
ALP activity found in the serum of healthy adults is concentrations.5 Transient, well-defined increases in
primarily of liver and bone origin in a ratio of approximately serum ALP from both liver and bone forms can be seen in
1:1. Minimal amounts of intestinal ALP may also be infants and children, often reaching up to 10 times the
present in the serum, particularly in individuals with blood URL. These changes seem to reflect a reduction in the
type B or O. Increases in serum ALP activity usually removal of ALP from blood caused by transient
originate from one or both of two sources: liver and bone. modifications of enzyme glycosylation.6 Complexes
between ALP and immunoglobulins or macro-ALP are
Consequently, serum ALP measurements are particularly occasionally seen in serum and cause abnormal ALP
important in the investigation of two major conditions: values, but have no specific diagnostic value in the present
Hepatobiliary disease and bone disease associated with state of knowledge.1
increased osteoblastic activity. Serum ALP is one of the
main enzymes used in the investigation of liver disease.1
The liver induces ALP synthesis by hepatocytes in
response to any obstruction in the biliary tract. The newly
formed ectoenzyme is released from the cell membrane by

Rev: V1.1 Date: 01.2025 ALP Page 1 / 6

 Finally, loss-of-function mutations in the tissue-nonspecific
ALP gene are associated with hypophosphatasia, a rare REAGENT STABILITY AND STORAGE
inherited disorder characterized by poor bone
Reagents are stable at +2/+8˚C till the expiration date
mineralization (due to the extracellular accumulation of
stated on the label which is only for closed vials.
inorganic pyrophosphate, a natural ALP substrate and a
potent inhibitor of mineralization) and low serum ALP
Once opened vials are stable for 10 days at +2/+8°C in
activity.6
optimum conditions. On board stability is strongly related
to auto analyzers’ cooling specification and carry-over
TEST PRINCIPLE
values.
Colorimetric Measurement [in accordance with
International Federation of Clinical Chemistry and Reagent stability and storage data have been verified by
Laboratory Medicine (IFCC) reference method] using Clinical and Laboratory Standards Institute (CLSI)
EP25-A protocol.7
The IFCC reference measurement procedure uses 4-
nitrophenyl phosphate (4-NPP) as substrate and 2-amino- SAMPLE REQUIREMENTS
2-methyl-1-propanol (AMP) as phosphate acceptor buffer. Serum or Li-heparin plasma collected by standard
In accordance with the IFCC reference method, the test procedure can be used.Multiple sample freezing and
method contains 4-nitrophenyl phosphate (4-NPP) as thawing should be avoided.
substrate. Alkaline phosphatase in the sample catalyzes
the hydrolysis of colorless 4-NPP to yield colorless 4- ALP activity stability in serum and plasma:
nitrophenol and inorganic phosphate in benzenoid form. In 7 days at +20/+25°C
alkaline medium, 4-nitrophenol is converted to the yellow 7 days at +2/+8ºC
4-nitrophenoxide ion form. The enzyme reaction is 2 months at -20°C
continuously monitored by observing the rate of formation
of 4-nitrophenoxide ions at 405 nm. The rate of increase in Unit Conversion:
absorbance at this wavelength is directly proportional to
the ALP activity in the sample. U/L x 0.0167 = µkat/L

AMP was used as buffer in this assay. The type of buffer Note
present affects the rate of enzyme activity. Accordingly, • Serum or heparinized plasma, free of hemolysis, should
buffers can be classified as inert (carbonate and barbital), be used. Complexing anticoagulants -such as citrate,
inhibitory (glycine and propylamine) or activating (AMP, oxalate and EDTA must be avoided as they bind cations,
tris[hydroxymethyl]aminomethane [TRIS] and such as Mg+2 and Zn+2, which are essential cofactors for
diethanolamine). Activating buffers such as AMP buffer ALP activity. Blood transfusion (containing citrate)
used in optimal concentration increase the enzyme activity causes a transient decrease in serum ALP by a similar
two to six times over inert ones such as carbonate.1 mechanism.
• It is recommended that freshly collected serum samples
Optimized concentrations of Mg+2 and Zn+2 ions are be kept at room temperature and tested as soon as
present as cofactors to activate ALP in the sample. The possible, preferably within 4 hours after collection. In
reagent component contains the activator Mg +2 and Zn+2 sera stored at a refrigerated temperature, ALP activity
ions involved in the reaction as well as the chelating agent increases slowly (2%/d).
N-hydroxyethylenediaminetriacetic acid (HEDTA),1 which • Frozen samples should be thawed and kept at room
acts as a metal ion buffer to maintain optimal temperature for 18 to 24 hours before measurement to
concentrations of these ions. achieve full enzyme reactivation.1

REAGENT COMPONENTS CALIBRATION AND QUALITY CONTROL

2-amino-2-methyl- Calibration: Arcal Auto Calibrator should be used for this
1-propanol buffer : ≤ 0.35 M test.
pH 10.40 (30°C),
Magnesium acetate : ≤ 2 mM Arcal Auto Calibrator
Zinc sulfate : ≤ 1 mM Ref.No: VT-003
HEDTA : ≤ 2 mM
4-NPP : ≤ 16 mM Calibration stability is 10 days. Calibration stability
depends on the application characteristics and cooling
REAGENT PREPARATION capacity of the autoanalyzer used.

Reagents are ready for use.

Rev: V1.1 Date: 01.2025 ALP Page 2 / 6

 Control: Commercially available control material with any kind of pre-treatment that is between the analyte’s
established values determined by this method can be lower limit of quantitation (LLoQ) and upper limit of
used. We recommend: quantitation (ULoQ).10
The determined analytic measuring interval for ALP: 8-
1000 U/L.
Arcon N Level 1 Control- Lyophilized
Ref.No: VT-001 Detection Capability
Limit of Detection (LoD): 5 U/L
Arcon P Level 2 Control- Lyophilized
Ref.No: VT-002 Limit of Quantitation (LoQ): 8 U/L

Note: LoQ values are based on Coefficient of Variation

At least two level controls must be run once in every 24
Percentage (CV) ≤ 20%.
hours. Each laboratory should determine its own quality
control scheme and procedures. If quality control results LoD and LoQ values have been verified by using CLSI
are not within acceptable limits, calibration is required. EP17-A2:2012 protocol.11

REFERENCE INTERVALS / MEDICAL DECISION Linearity

LEVELS This method shows measurement linearity in the activities
up to 1000 U/L. Autoanaylzer’s auto-dilution system can be
Reference Range18 used if the concentrations have higher values. See device
Age Range manual for further information.
(U/L)
0 to 14 days 90 – 273 For the manual dilution procedure, dilute the sample 1:10
15 days to < 1 year 134 – 518 using 0.90% isotonic. After this process, multiply the result
1 year to < 3 years 156 – 369 of the reworked sample by the dilution factor. Do not report
3 to 5 years 144 – 327 the sample result after dilution if it is marked as lower than
6 to 10 years 153 – 367 the linear lower limit. Rerun with a suitable dilution.
11 to 15 years, Male 113 – 438
11 to 15 years, Female 64 – 359 Linearity studies data have been verified by using CLSI
16 to 21 years, Male 56 – 167 EP06-A:2003 protocol.12
16 to 29 years, Female 44 – 107
Precision
22 to 79 years, Male 50 – 116
30 to 79 years, Female 46 – 122 Running system has been developed according to 20x2x2
“The Single Site” protocol. Repeatibility and Within-
• ALP activities in serum vary with age and, in minor Laboratory Precision/Within-Device values have been
amount, with sex. obtained according to the running results.
• For women, a progressive increase in ALP activity has
been described after menopause with a reference According to the protocol in use, 2 separate runs per day
interval between 53 and 141 U/L. have been made for 20 days (no obligation for being
• Infants and peripubertal children show higher ALP consecutive days). This protocol has been applied to each
activity than healthy adults as a result of bone ALP low and high samples separately and 80 results have been
leakage from osteoblasts during bone growth. Age- obtained for each one. Statistically, the results have been
specific reference intervals are therefore crucial for the obtained using 2-factor Nested-ANOVA model.13
detection of hypophosphatasia. The decline in ALP
Repeatibility (Within Run) and Repeatibility (Day to Day)
activity to the typical adult intervals occurs on average 2
SD and CV% values of ALP have been given in the table
years earlier in females than in males.1,8
1 and 2 respectively.
Each laboratory should investigate the transferability of the
expected values to its own patient population and if Table 1. ALP Repeatibility (Within Run) results
necessary, determine its own reference range. obtained from samples in two different concentrations
Mean Concentration SD* CV% n
Reference interval has been verified by using Clinical and 83 U/L 0.59 0.71 80
Laboratory Standards Institute (CLSI) EP28-A3c protocol.9 205 U/L 0.90 0.44 80
*SD: Standard Deviation
PERFORMANCE CHARACTERISTICS
Note: This working system has been named “Within-Run
Measuring Interval
Precision” in the previous CLSI - EP05-A2 manual.14
According to CLSI EP34-ED1:2018, “Measuring Interval”
refers to the interval where the analyte concentration is
measured with intended accuracy in terms of medical and
laboratory requirements without dilution, concentrating or

Rev: V1.1 Date: 01.2025 ALP Page 3 / 6

 Table 2. ALP Repeatibility (Day to Day) results selective electrode methods) may show some negative
obtained from samples in two different concentrations effects that will cause various attempts and some
Mean Concentration SD CV% n misjudgements.16
83 U/L 2.35 2.83 80
These performance characteristics have been obtained
205 U/L 6.79 3.31 80
using an autoanalyzer. Results may vary slightly when using
Note: This working system has been named “Total
different equipment or manual procedures.
Precision” in the previous CLSI - EP05-A2 manual.14
WARNINGS AND PRECAUTIONS
Method Comparison
Correlation with a comparative method is: IVD: For in Vitro Diagnostic use only.
r= 0.999 Do not use expired reagents.
According to Passing-Bablok Fit: Reagents with two different lot numbers should not be
y= 0,95x +1,65 interchanged.
For professional use.
Interference Follow Good Laboratory Practice (GLP) guidelines.
Endogenous interferant and analyte concentrations that
CAUTION: Human source samples are processed with this
have been used in the ALP scanning tests has been
product. All human source samples must be treated as
determined according to “CLSI EP37-ED1:2018” and “CLSI
potentially infectious materials and must be handled in
EP07-ED3:2018” manuals.15,16
accordance with OSHA standards.
The total acceptable error rate, which is going to be used to Danger
detect whether the observed differential value obtained from EUH032 :Releases a very toxic gas if contacts
ALP interference scanning test is appropriate, is determined with acid.
as ±10%.17 H317 :May cause allergic skin reaction.

In ALP test results, no significant interaction has been Precaution

observed in the determined endogenous interferant and P280 :Use protective gloves / clothes / glasses
analyte concentrations or between interferants and analyte. / mask.
Interfering P264 :Wash your hands properly after using.
ALP Target Observed P272 :Contaminated work clothes should not
Substance and N*
(U/L) Recovery %
Concentration be allowed to be used outside of the
Hemoglobin workplace.
85 3 107
990 mg/dL
Bilirubin Intervention
165 3 98 P302+P352 :Wash with plenty of water and soap if it
58.5 mg/dL
Lipemia contacts with skin.
89 3 94 P333+P313 :Seek medical help if it irritates your skin
825 mg/dL
or develops rash.
* Total acceptable error rate determined as interference limit and P362+P364 :Remove contaminated clothes and
repeatability (within run) pre-detected for the related method were wash properly before using.
used for the calculations of how many times the control and test
samples prepared as a serum pool are going to be run repetitively. Disposal
In the calculations, the accepted error rate for type 1 (α error) was P501 :Dispose the vials and contents
5% and for type 2 (β error) was 10% (90% power).16 according to the local regulations.

It should be noted that endogenous interferants, as well as REFERENCES

various medicines and metabolites, anticoagulants (e.g.
Heparin, EDTA, citrate, oxalate) and preservatives (e.g. 1. Rifai, N., Chiu, R. W., & Young, I., et al., (2023) Tietz
sodium floride, iodoacetate, hydrochloride acide) such as Textbook of Laboratory Medicine (7th ed.), Chapter 32:
additives, materials that may contact with samples during Serum Enzymes, p.350.e1-36, Elsevier, St.
collection and processing (serum separator devices, sample Louis, Missouri 63043
collection containers and contents, catheters, catheter wash 2. Moss DW. Physicochemical and pathophysiological
solutions, skin disinfectants, hand cleaners and lotions, factors in the release of membrane-bound alkaline
glass washing detergents, powder gloves), dietary phosphatase from cells. Clin Chim Acta 1997;257:133–
substances known to affect some specific tests (caffeine, 40.
beta-carotene, poppy seeds, etc.), or some substances 3. Lammers WJ, van Buuren HR, Hirschfield GM, et al.
present in a sample that cause foreign proteins (heterophilic Levels of alkaline phosphatase and bilirubin are
antibodies, etc.), autoimmune response (autoantibodies, surrogate end points of outcomes of patients with
etc.), or due to malignancy (for example, interference by primary biliaty cirhosis: an international follow-up study.
paraproteins with phosphate testing and indirect ion Gastroenterology 2014;147:1338–49.

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 4. Panteghini M. Benign inherited hyperphosphatasemia of
intestinal origin: report of two cases and a brief review of Archem Sağlık Sanayi ve Tic. A.Ş. (With
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8. Ceriotti F, Panteghini M, Guerra E, Leoncini R, Cevenini
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Defining, Establishing and Verifying Reference Intervals
in the Clinical Laboratory; Approved Guideline – Third
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Establishing and Verifying an Extended Measuring
Interval Through Specimen Dilution and Spiking - First
Edition. CLSI Document EP34. Wayne, PA: CLSI; 2018.
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Evaluation of Detection Capability for Clinical Laboratory
Measurement Procedures; Approved Guideline - Second
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Evaluation of the Linearity of Quantitative Measurement
Procedures: A Statistical Approach - First Edition. CLSI
Document EP06-A. Wayne, PA: CLSI; 2003.
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Evaluation of Precision of Quantitative Measurement
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Evaluation of Precision Performance of Quantitative
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Supplemental Tables for Interference Testing in Clinical
Chemistry - First Edition. CLSI Document EP37. Wayne,
PA: CLSI; 2018.
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Interference Testing in Clinical Chemistry - Third Edition.
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of Clinical Chemistry and Molecular Diagnostics. 6th ed.
St. Louis, MO: Elsevier; 2018.

Rev: V1.1 Date: 01.2025 ALP Page 5 / 6

 SYMBOLS

In Vitro Diagnostic Medical

IVD Device

LOT Lot Number

R1 Reagent 1

R2 Reagent 2

GTIN Global Trade Item Number

REF Reference Number

GLP Good Laboratory Practice

Identifies Products to Be Used

FOR USE WITH Together

PRODUCT OF TURKEY Product of Turkey

Manufacturer

Expiration Date

Temperature Limitation

Consult Instructions for Use

Caution

Number of Tests

Rev: V1.1 Date: 01.2025 ALP Page 6 / 6