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Clin Chem Lab Med 2011;49(9):1439–1446
2011 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/CCLM.2011.621

IFCC primary reference procedures for the measurement of

catalytic activity concentrations of enzymes at 37 8C. Part 9:
Reference procedure for the measurement of catalytic
concentration of alkaline phosphatase
International Federation of Clinical Chemistry and Laboratory
Medicine (IFCC) Scientific Division, Committee on Reference
Systems of Enzymes (C-RSE)1)

Gerhard Schumann1, Rainer Klauke1, Francesca trations of enzymes at 37 8C and the certification of reference
Canalias2, Steffen Bossert-Reuther3, Paul F.H. preparations. Other parts deal with: Part 1. The concept of
Franck4, F.-Javier Gella5, Poul J. Jørgensen6, reference procedures for the measurement of catalytic activ-
Dongchon Kang7, Jean-Marc Lessinger8, Mauro ity concentrations of enzymes; Part 2. Reference procedure
Panteghini9 and Ferruccio Ceriotti10,* for the measurement of catalytic concentration of creatine
1
kinase; Part 3. Reference procedure for the measurement of
Medizinische Hochschule Hannover, Institut für Klinische
catalytic concentration of lactate dehydrogenase; Part 4. Ref-
Chemie, Hannover, Germany
2
erence procedure for the measurement of catalytic concen-
Departament de Bioquı́mica i Biologia Molecular,
tration of alanine aminotransferase; Part 5. Reference
Laboratori de Referència d’Enzimologia Clı́nica,
procedure for the measurement of catalytic concentration of
Universitat Autònoma de Barcelona, Cerdanyola del Vallès,
aspartate aminotransferase; Part 6. Reference procedure for
Spain
3
the measurement of catalytic concentration of g-glutamyl-
Roche Diagnostics GmbH, Research and Development
transferase; Part 7. Certification of four reference materials
Department, Mannheim, Germany
4
for the determination of enzymatic activity of g-glutamyl-
Klinisch Chemisch en Hematologisch Laboratorium,
transferase, lactate dehydrogenase, alanine aminotransferase
HagaZiekenhuis, Den Haag, The Netherlands
5
and creatine kinase at 37 8C; Part 8. Reference procedure for
BioSystems S.A., Barcelona, Spain
6
the measurement of catalytic concentration of a-amylase.
Department of Clinical Chemistry, Kolding Hospital,
The procedure described here is derived from the previously
Kolding, Denmark
7 described 30 8C IFCC reference method. Differences are
Department of Clinical Chemistry and Laboratory
tabulated and commented on in Appendix 1.
Medicine, Kyushu University Graduate School of Medical
Sciences, Fukuoka, Japan
8 Keywords: alkaline phosphatase; IFCC reference procedure;
Hôpitaux Universitaires de Strasbourg, Laboratoire de
reference intervals.
Biochimie Générale et Spécialisée, Strasbourg, France
9
Centro Interdipartimentale per la Riferibilità Metrologica
in Medicina di Laboratorio (CIRME), Università degli
Studi, Milano, Italy
10
Diagnostica e Ricerca San Raffaele, Scientific Institute Introduction
H. San Raffaele, Milan, Italy
The catalytic concentration of alkaline phosphatase (ALP)
(Orthophosphoric-Monoester Phosphohydrolase, Alkaline
Abstract
Optimum, EC 3.1.3.1) in serum represents the activity of
This paper is the ninth in a series dealing with reference multiple forms of the enzyme. More than 17 isoforms of
procedures for the measurement of catalytic activity concen- ALP are detectable by use of an isoelectric focusing tech-
nique (1). The catalytic concentration of ALP in serum of
1)
Members: F. Ceriotti (IT), J. Gella (ES), D. Kang (JP), J.M. Lessinger healthy adults originates from the liver and from bone in
(FR), R. Reja (US), G. Schumannb (DE), F. Canaliasc (SP), P.J.
Jørgensenc (DK); amember since 2011, bmember until 2009, cmember similar proportions. ALP from the small intestine contributes
until 2010. to approximately 10 % of total ALP in healthy individuals.
*Corresponding author: Ferruccio Ceriotti, Diagnostica e Ricerca If not physiologically induced by growth of bone or in preg-
San Raffaele, via Olgettina 60, 20132 Milan, Italy nancy, increase of ALP in serum occurs as a consequence of
Phone: q39-2-2643 2282, Fax: q39-2-2643 2640,
E-mail: [email protected] disease of the liver and/or bone.
Received April 11, 2011; accepted April 11, 2011; The determination of the catalytic concentration of ALP
previously published online June 25, 2011 in serum depends strongly on the chosen measurement para-

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1440 Schumann et al.: IFCC reference procedures: part 9

meters. Among these, the choice of the buffer is of great Table 1 Concentrations in the final complete reaction mixture for
importance. In 1983 an expert group of the International Fed- the measurement of ALP.
eration of Clinical Chemistry (IFCC) provided a proposal for
2-Amino-2-methyl-1-propanol 750 mmol/L
a standardized measurement procedure for ALP (2, 3). The
pH (37 8C) 10.20"0.05a
reaction principle was based on the use of 2-amino-2-methyl- 4-Nitrophenyl phosphate 16 mmol/L
1-propanol (AMP) and 4-nitrophenyl phosphate (NPP); the Zinc sulfate 1 mmol/L
measurement temperature was 30 8C. Even though this pro- Magnesium acetate 2 mmol/L
cedure for ALP was never endorsed as official IFCC rec- HEDTA 2 mmol/L
ommendation by the IFCC member societies, many Volume fraction of sample 0.0196 (1:51)
commercial test kits for ALP currently use the AMP buffer. a
Maximum allowable expanded uncertainty (95 % probability).
This paper describes the IFCC primary reference meas-
urement procedure for ALP, which is based on the previous
IFCC work (2, 3). The change of the measurement temper-
ature from 30 8C to 37 8C necessitated a re-evaluation of the confirmed if the expanded uncertainty of the calibration
measurement conditions. The results of this re-evaluation (95 % probability) is equal to or smaller than the maximum
with very detailed definition of the measurement parameters allowable expanded uncertainty, and the result of the cali-
relevant for high-level standardization are described. This bration does not differ significantly (pF0.05) from the target
paper is the ninth in a series dealing with reference proce- value. This is valid if
dures for the measurement of catalytic activity concentra-
tions of enzymes at 37 8C and the certification of reference
preparations (4–11).
The reference measurement procedure for ALP will be of
Z valuetargetIvaluecalibration
y(Umax2HUcal2) Z
F1

use in a reference measurement system for the certification

of calibrators and control materials, thus providing the basis
to allow the clinical laboratories to produce traceable meas- where valuetarget is the target value prescribed in the IFCC
urement results and to establish definitive reference intervals document; valuecalibration is the result determined with the cali-
(12, 13). In particular, here we provide only preliminary ref- bration procedure; Umax is the maximum allowable expanded
erence intervals for adult subjects; ALP reference intervals uncertainty prescribed in the IFCC document; and Ucal is the
undergo important changes dependent on age and gender and maximum expanded uncertainty of the result of the
further work is needed to obtain the full range of reference calibration procedure.
intervals.

Reagents
Reaction principle

ALP catalyzes the hydrolysis of NPP, forming phosphate and 1. 2-Amino-2-methyl-1-propanol (C4H11NO), Mrs89.14
free 4-nitrophenol; under alkaline conditions, 4-nitrophenol 2. 4-Nitrophenyl phosphate, disodium salt, hexahydrate
is converted to the 4-nitrophenoxide ion. AMP and H2O are (C6H4NNa2O6PØ6H2O), Mrs371.14
used as phosphate-acceptors: 3. Magnesium acetate wMg(C2H3O2)2Ø4H2Ox, Mrs214.46
4. Zinc sulfate (ZnSO4Ø7H2O), Mrs287.54
5. N-(2-Hydroxyethyl)ethylenediamine-N,N9,N9-triacetic
1) 4-Nitrophenyl phosphateqH2O ALP
——— ) 4-Nitrophenoxide acid (HEDTA), trisodium salt (C10H15N2Na3O7), Mrs
qPhosphate 344.20 (anhydrous)
2) 4-Nitrophenyl phosphateqAMP ALP
———) 4-Nitrophenoxide 6. Hydrochloric acid (HCl), Mrs36.46, 25 %
qAMP-Phosphate 7. Hydrochloric acid (HCl), Mrs36.46, 2 mol/L
8. Sodium chloride (NaCl), Mrs58.44

Specimens
Table 2 Measurement conditions for the measurement of ALP.
Calibration materials, control specimens and human sera.
Temperature 37.0 8C"0.1 8Ca
Wavelength 405 nm"1 nma
Measurement conditions Band width F2 nm
Light path 10.00 mm"0.01 mma
Concentrations in the final reaction mixture and the meas- Incubation time 60 s
urement conditions are listed in Tables 1 and 2. Delay time 90 s
Note: The compliances with the prescribed maximum Measurement interval 120 s
allowable expanded uncertainties (95 % probability) of the Readings (measurement points) G6
a
values for temperature, pH, light path and wavelength are Maximum allowable expanded uncertainty (95 % probability).

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Note: AMP may contain inhibitors for ALP. The manu- gent chemical employed is less (e.g., yz %), the amount
facturers should declare that the buffer substance is suitable equivalent to the given mass is calculated by the use of a
for the investigation of ALP. factor:
Note: The content of water in commercially available
HEDTA differs. The percentage of water in the used lot shall Fcontents100/yz
be documented in a certificate of analysis of the manufac-
turers and shall be taken into account by the preparation of
Highly purified water shall be used for the preparation of
the metal ion buffer.
the reagent solutions. Guidelines describing the preparation
Note: The NPP reagent must meet the following criteria
and testing of reagent water are published elsewhere (15).
(declared in manufacturers’ certificate of analysis or con-
The expanded (ks2) combined uncertainty (normally dis-
firmed by investigations of the user):
tributed) of each weighing procedure (including the uncer-
• Enzymatic conversion of NPP to 4-nitrophenol should tainty of the purity of the substance) shall be F1.5 %.
result in a hydrolysis of )0.98,
• The molar absorption coefficient of NPP at 311 nm in Solution 1
sodium hydroxide, 10 mmol/L at 25 8C, should be
986.7 m2/mol"7.6 m2/mol, 0.878 g (25.50 mmol/L) HEDTA; 0.367 g (12.75 mmol/L)
• The mol fraction of 4-nitrophenol to NPP must be zinc sulfate; 0.547 g (25.50 mmol/L) magnesium acetate.
-0.0003, and • Dissolve the HEDTA in approximately 70 mL water.
• The mol fraction of inorganic phosphate to NPP must be • Add the zinc sulfate to the solution.
-0.01.

Detailed procedures for checking these conditions are given Table 3 Dependence of the pH of the reaction solution upon
by Bowers et al. (14). temperature.
Reagents of the highest purity must be used. If a chemical
is suspected of containing impurities affecting the catalytic Temp, pH Temp, pH Temp, pH
activity of the analyte further investigations must be per- 8C 8C 8C
formed: e.g., comparisons with products from different man- 15.00 10.906 23.50 10.619 32.00 10.352
ufacturers and different lots. 15.25 10.897 23.75 10.611 32.25 10.344
It is recommended to use reagents which have already 15.50 10.889 24.00 10.603 32.50 10.337
been tested and approved in comparisons. 15.75 10.880 24.25 10.595 32.75 10.329
16.00 10.871 24.50 10.587 33.00 10.321
16.25 10.862 24.75 10.579 33.25 10.314
16.50 10.854 25.00 10.571 33.50 10.306
Charts for the adjustment and control of pH 16.75 10.845 25.25 10.563 33.75 10.298
17.00 10.836 25.50 10.555 34.00 10.291
17.25 10.828 25.75 10.547 34.25 10.283
Procedure for the adjustment of pH at temperatures 17.50 10.819 26.00 10.539 34.50 10.276
diverging from 37 8C 17.75 10.811 26.25 10.531 34.75 10.268
18.00 10.802 26.50 10.523 35.00 10.260
Both the thermometer and the pH electrode are suspended in 18.25 10.794 26.75 10.515 35.25 10.253
the mixed solution simultaneously. The stirred solution is 18.50 10.785 27.00 10.507 35.50 10.245
then titrated to the pH listed in the chart for the actually 18.75 10.777 27.25 10.500 35.75 10.238
19.00 10.768 27.50 10.492 36.00 10.230
measured temperature (Table 3). The speed of agitation
19.25 10.760 27.75 10.484 36.25 10.223
should be the same during the calibration and the control of 19.50 10.751 28.00 10.476 36.50 10.215
the pH-meter and the adjustment of the pH of the reagent 19.75 10.743 28.25 10.468 36.75 10.208
solutions. The pH electrode should be positioned in the cen- 20.00 10.735 28.50 10.460 37.00 10.200
ter of the stirred solution. 20.25 10.726 28.75 10.453 37.25 10.192
The possibility that the temperature can change during the 20.50 10.718 29.00 10.445 37.50 10.185
titration must be taken into account. For this reason, the tem- 20.75 10.710 29.25 10.437 37.75 10.177
perature in the proximity of the target value should be con- 21.00 10.701 29.50 10.429 38.00 10.170
trolled again and the target pH has to be corrected if 21.25 10.693 29.75 10.421 38.25 10.162
necessary. The same applies for the adjustment of the tem- 21.50 10.685 30.00 10.414 38.50 10.155
21.75 10.677 30.25 10.406 38.75 10.147
perature compensation of the pH meter.
22.00 10.668 30.50 10.398 39.00 10.140
22.25 10.660 30.75 10.390 39.25 10.132
Preparation of solutions 22.50 10.652 31.00 10.383 39.50 10.125
22.75 10.644 31.25 10.375 39.75 10.117
The given mass of the compounds for the preparation of 23.00 10.636 31.50 10.367 40.00 10.110
solutions refers to 100 % content. If the content of the rea- 23.25 10.628 31.75 10.360

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1442 Schumann et al.: IFCC reference procedures: part 9

• Add the magnesium acetate only when the zinc sulfate is The reagent blank shows a non-linear kinetic. The accept-
completely dissolved. able blank rate is positive and -3.0=10 –5 s –1
• Dissolve the magnesium acetate in the solution. (-0.0018 min –1).
• Transfer to a 100 mL volumetric flask. Note: Due to the non-linearity of the kinetic, the reagent
• Equilibrate volumetric flask and water to 20 8C. blanks have a lower reproducibility. It is advisable to perform
• Fill water up to the calibration mark of the volumetric at least three replicates and use the mean for further
flask (20 8C). calculations.
Stability at 2–8 8C: 3 months.

Reaction solution
Sample blank rate

8.52 g (956.3 mmol/L) 2-Amino-2-methyl-1-propanol. For the determination of the sample blank rate, the start rea-
gent solution is replaced by 9 g/L (154 mmol/L) sodium
• Dissolve in approximately 70 mL water. chloride solution. The measurement procedure is then carried
• Adjust pH (37 8C) 10.3 – pH (37 8C) 10.5 with 25 % out as described above.
hydrochloric acid. Note: The sample blank rate is determined and docu-
• Add 10 mL solution 1. mented, but not taken into account for calculation of the
• Adjust pH (37 8C) 10.2 with hydrochloric acid (2 mol/L). catalytic concentration of ALP in control sera and calibra-
• Transfer to a 100 mL volumetric flask. tors. In case of the value of the sample blank rate exceeding
• Equilibrate volumetric flask and water to 20 8C. 1 % of total ALP, a warning that the respective material is
• Fill water up to the calibration mark of the volumetric not appropriate for calibration should be issued.
flask (20 8C). Note: The reagent blank rate for the sample blank rate is
Stability at 2–8 8C: 3 months. determined by replacing the start reagent solution and the
sample by 9 g/L (154 mmol/L) sodium chloride solution.
Start reagent solution Note: Effects of the matrix of the sample on the indicator
reaction have not been considered due to the omission of
0.757 g (81.6 mmol/L) 4-Nitrophenyl phosphate, disodium NPP from the reaction mixture.
salt, hexahydrate.
• Dissolve in approximately 15 mL water.
• Transfer to a 25 mL volumetric flask. Upper limit of the measurement range
• Equilibrate volumetric flask and water to 20 8C.
• Fill water up to the calibration mark of the volumetric If the change of absorbance exceeds 0.0042 s –1 (0.25 min –1)
flask (20 8C). in the measurement interval, an analytical portion of the
sample must be diluted with 9 g/L (154 mmol/L) sodium
Stability at 2–8 8C: 1 week. chloride solution and the measurement procedure must be
repeated with the diluted specimen (16). The obtained value
must then be multiplied by the corresponding factor of the
Measurement procedure dilution.
Note: The following rules for the dilution of samples are
Degas carefully the amount of reaction solution required for recommended for improved standardization.
the experiment. The degassing can be performed by using a The sample shall be diluted if the interval of the combined
vacuum oven with a temperature set at 35 8C or warming expanded uncertainty and the upper limit of the measurement
the solution up to approximately 35 8C following by storage range are overlapping. In this case pre-dilution of 10 volume
of it for 1 h in a vacuum desiccator while stirring with a parts of the sample with one volume part of 9 g/L
magnetic stirrer. If the degassed solution is not used within (154 mmol/L) sodium chloride solution is recommended.
the day, degas it again just before use. Adequate dilution shall be performed if the interval of the
Equilibrate only an adequate volume of start reagent solu- combined expanded uncertainty is completely located above
tion at 37 8C in preparation for the measurement procedure.
The remaining volume of the start reagent solution should
be stored at 2–8 8C. Table 4 Analytical system for the measurement of ALP.
Pipette the volumes as listed in Table 4 one after another 2.000 mL reaction solution
into the cuvette. Equilibrate to 37.0 8C
0.050 mL sample
Mix thoroughly and incubate for 60 s. At the end of the
Reagent blank rate incubation time, the temperature of the solution in the cuvette
shall have reached 37.0 8C
To determine the reagent blank rate, the specimen is replaced 0.500 mL start reagent solution
by 9 g/L (154 mmol/L) sodium chloride solution. The meas- Mix thoroughly, wait 90 s and monitor time and absorbance for
urement procedure is then carried out as described above. an additional 120 s

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the upper limit of the measurement range. The change of Appendix I

absorbance of the reaction mixture containing the pre-diluted
sample shall be in a range from 0.0034 s –1 (0.20 min –1) to Changes in the IFCC reference procedure for
0.0038 s –1 (0.23 min –1) measurements at 37 8C compared with the reference
method for measurements at 30 8C as described in
the original IFCC document

Sources of error The primary reference procedure is derived from the draft of
the IFCC reference method (2, 3), which provides optimized
conditions for the measurement of catalytic concentration of
• The temporal conversion rate is not linear for all samples. ALP at 30 8C.
The non-linearity depends on the matrix of the material The measurement temperature of 37 8C instead of 30 8C
and the catalytic concentration of ALP. Deviations from requires only minor changes of certain measurement para-
the described delay time and measurement time can lead meters to retain the optimum measurement conditions. The
to non-commutability of the material and changed modifications are listed and commented on in this appendix.
method. Furthermore, if in comparison to the 30 8C reference method
• The optimum pH in some control materials and calibra- a more accurate specification has become necessary for
tors differs from the described pH of the reference pro- improving the standardization of the measurements, it is also
cedure. This leads to an increased sensitivity to the described here in Table 5.
uncertainty of the pH adjustment.
• Absorbed carbon dioxide from the air changes the pH of
the reaction solution. Therefore, the container of reaction Appendix II
solution shall be closed tightly. The pH in solution 1 shall
be controlled at least weekly. Determination of preliminary reference intervals
• The catalytic concentration of ALP in some control mate-
rials increases during the storage. Such a material is not Sample collection was performed in four different European
suitable as a calibrator or as control material. centres: two sites in Milan (Italy) (Ospedale L. Sacco and
• A thorough mixing of the final complete reaction mixture Ospedale S. Raffaele), one in Nancy (France) (Centre Med-
is necessary due to the high buffer concentration. Other- icine Preventive) and one in Bursa (Turkey) (Uludag Uni-
wise the formation of streaks leads to a poor precision of versity Medical School). The serum was collected using a
the measurement results. BD Vacutainer system (BD Becton, Dickinson and Compa-
ny) into plastic tubes with serum separator (SSTTM II). The
samples were allowed to clot at room temperature and cen-
trifuged (2000 g) within 1 h. Serum was separated and ali-
quoted within 4 h from blood collection. The aliquots were
Calculation frozen at –80 8C. The frozen aliquots were sent in dry ice
to the reference laboratory in Hannover where all the anal-
The temporal change of absorbance (s –1) is calculated with yses were performed. The reference analyses were performed
analysis of regression (method of the least squares). After on a Konelab 30i instrument (Thermo Fisher Scientific). The
subtraction of the reagent blank rate the corrected change of reagents were self-prepared according to the prescriptions in
absorbance is multiplied with the factor, Fs2729 (measure- this document. The measurement parameters and the meas-
ment at 405 nm, ´405s1869 m2/mol). urement design were configured as closely related as tech-
Note: The use of the molar absorption coefficient nically feasible. The calibration was performed by use of a
´405s1869 m2/mol is recommended by IFCC and IRMM. set of pooled sera with assigned target values for ALP
The catalytic concentration of ALP is calculated in obtained by measurements using the manually performed
mkat/L. DA/DtALP is the change of absorbance after correc- primary IFCC reference measurement procedure.
tion of the reagent blank rate (in s –1); bALP is the catalytic Subjects in Italy and Turkey were specifically enrolled for
concentration of ALP (mkat/L); and bALPs2729 DA/DtALP. the reference interval experiment and they gave informed
The catalytic concentration in mkat/L can be converted to consent and replied to an ad hoc questionnaire; the samples
U/L by multiplication by the factor fs60. from France were left over samples from those collected for
a health screening program. All of the samples were obtained
from individuals in the fasting state. In addition to ALP, the
Preliminary reference values following tests were performed locally in all the samples:
aspartate aminotransferase (AST), alanine aminotransferase
(ALT), g-glutamyltransferase (GGT), glucose and creatinine;
• Females (18–49 years): 0.55–1.64 mkat/L; 33–98 U/L. in addition, total calcium and inorganic phosphate were
• Males (G20 years): 0.72–1.92 mkat/L; 43–115 U/L. measured in the Hannover reference laboratory.
The ages of the enrolled subjects spanned from 5 to
Details about the definition of the preliminary reference 87 years, but the number of subjects in the younger and older
values are given in Appendix II. age ranges was insufficient to provide reliable reference

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1444 Schumann et al.: IFCC reference procedures: part 9

Table 5 Comparison of the IFCC methods for the measurement temperatures of 30 8C and 37 8C.

37 8C IFCC reference procedure 30 8C reference method Comment

Specimen of investigation
Calibration material, control Human sera The reference procedure will be used primarily for the measurement of
specimens and human sera control samples and calibration materials.
Uncertainty of the measurement temperature adjustment
Target uncertainty F0.1 8C (ks2) Bias: less "0.05 8C High quality spectro-photometer with devices for temperature
Imprecision: less "0.1 8C adjustment and control provide an uncertainty (ks2) of the
temperature F0.1 8C.
Incubation time
60 s No incubation time 60 s are necessary for mixing and closing the lid of the spectrometer.
Loss of temperature can be compensated in this time.
Delay time
90 s No information about The reagent blank rate is non-linear after the addition of the start reagent
the delay time solution. The non-linearity decreases with increasing delay time.
Measurement time
120 s Up to 300 s The reaction rate is not linear and the non-linearity proportionally
increases with the time and the catalytic concentration of ALP. Shorter
measurement time implies less non-linearity.
Concentration of AMP in the final complete reaction mixture
750 mmol/L 350 mmol/L 750 mmol/L AMP is the optimum buffer concentration. The pH value
of the buffer solution decreases faster with lower buffer concentrations.
The buffer solution is more stable with the increased AMP
concentration.
Start of the reaction
Solution R 2000 mL Solution R 2500 mL The reaction is started with serum in the 30 8C method and with
Serum 50 mL Serum 50 mL substrate in the 37 8C procedure.
Start solution 500 mL
Temperature of the start reagent solution before use
Start with substrate: the start Start with serum: no The use of start reagent solution with ambient temperature decreases the
reagent solution should be temperature equilibration temperature in the cuvette.
37 8C before use of the serum is described
Collection of data
Number of readings G6 Monitoring of the Modern spectrophotometers employ digital data processing. A number
increase in absorbance of readings G6 should ensure a sufficient precision of the measurement
results. Devices for a continuous monitoring are no longer in use.
Determination of the slope (time vs. absorbance)
Regression analysis of the No information A well-defined statistical method is necessary to ensure the
method of least squares reproducibility of the calculation of the slope.
Reference values
See Table 7 No reference values were The reference values for women and men were investigated separately.
given
Unit of catalytic enzymatic concentration
mkat/L and U/L mkat/L U/L is commonly used in clinical laboratory, but mkat/L is
based on the SI system.
Sample blank rate
Determined, but not taken Subtraction Usually, sample blank rates are not subtracted in routine procedures.
into account Therefore, assigned values in calibrators and control materials are
only useful for routine methods, if they include the sample blank
rate value.

intervals. Thus, results are presented only for adult pre-men- To exclude the possibility that any of the reference indi-
opausal females and adult males. The origin of the partici- viduals were in late puberty, only individuals who were
pants and their age group distributions are shown in Table 6. 18 years or older if females and 20 years or older if males
The population sizes from the different sites were similar, were included in the calculation. The decrease in ALP cat-
but the Turkish group had a larger number of young subjects. alytic activity to typical adult ranges is known to differ from

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Schumann et al.: IFCC reference procedures: part 9 1445

Table 6 Age and origin of the enrolled subjects.

Age, Italy France Turkey Total

years (Milan) (Nancy) (Bursa)
Females 18–30 32 32 56 120
31–40 19 21 28 68
41–49 34 27 8 69
Total 85 80 92 257
Males 20–30 14 19 56 89
31–40 20 25 31 76
41–50 30 19 8 57
51–60 13 18 0 31
)60 7 6 0 13
Total 84 87 95 266

Table 7 ALP reference intervals.

Females, age 18–49 years Males, age G20 years

Number of subjects 257 266
2.58 percentile (90 % CI) 0.55 mkat/L (0.48–0.59) 0.72 mkat/L (0.65–0.76)
33 U/L (28.7–35.4) 43 U/L (39.2–45.8)
97.58 percentile (90 % CI) 1.64 mkat/L (1.50–1.73) 1.92 mkat/L (1.84–2.03)
98 U/L (89.8–103.6) 115 U/L (110.1–121.6)

subject to subject and occurs on average 2 years earlier in across the three sites were less pronounced than those of the
females than in males. women. The application of the Lahti algorithm indicated
Moreover, as for females a progressive increase of both only marginal differences.
lower and upper reference limit following the menopause is The reference limits, calculated with the non-parametric
described (17), we excluded females older than 49 years. As approach, are reported in Table 7.
for males no age-related increase in ALP has been described,
all the data, including those from elderly people, were
included in the calculation. Acknowledgments

Reference subject samples and additional medical laboratory infor-

Exclusion criteria
mation was provided by Yesim Ozarda, Uludag University Medical
School, Department of Biochemistry and Clinical Biochemistry,
Participants were excluded from the study if suffering for
Bursa, Turkey through support of the Uludag University Research
diabetes (glucose )7.0 mmol/L), renal impairment wcreati-
Fund (Grant UAP(T)-2009/10), Joseph Henny, Laboratoire de
nine )115 mmol/L if females, 133 mmol/L if malesx, liver Biologie Clinique, Centre de Médecine Préventive, Vandoeuvre-lès-
damage (AST, ALT or GGT )100 U/L), total calcium and Nancy, France, and Cristina Valente, Laboratorio Analisi Chimico-
inorganic phosphate outside the reference intervals (2.15– Cliniche, Azienda Ospedaliera Luigi Sacco, Milano, Italy. Elena
2.60 mmol/L and 0.80–1.50 mmol/L, respectively). Guerra (Diagnostica e Ricerca S. Raffaele, Milano, Italy) and Rolf
Nagel (Diagnostics GmbH, Research and Development Department,
ALP results Mannheim, Germany) contributed with their suggestions and tech-
nical assistance. James Boyd, Josep M. Queraltó and Shen Zyiu,
The results of adult males and adult pre-menopausal females members of the IFCC Committee Reference Intervals and Decision
were compared using the approach proposed by Lahti et al. Limits (C-RIDL), contributed with fruitful discussion and sugges-
(18, 19) and partitioning was suggested, so the two groups tions. The German Society for Clinical Chemistry and Laboratory
were considered separately. Medicine (DGKL) has granted the work of the accredited reference
laboratory DKD-K-20602.
Results for women from the three collection sites were
compared using the Lahti et al. (18, 19) statistical method
and the Turkish group was found to be significantly different
from the French group, whereas difference with the Italian Conflict of interest statement
group was only marginal; the Italian and French groups were
Authors’ conflict of interest disclosure: The authors stated that
significantly different only at the lower reference limit. there are no conflicts of interest regarding the publication of this
Hence, statistically the three groups were different; however, article.
because the Turkish group was significantly younger and the Research funding: None declared.
lower reference limit is clinically less relevant, all the data Employment or leadership: None declared.
were merged. The differences among the results from men Honorarium: None declared.

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1446 Schumann et al.: IFCC reference procedures: part 9

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