Sop of Hemoglobins Determination.

a. PURPOSE:

The Bio-Rad D-10 Dual Program is intended for the percent determination of hemoglobins
A2, F, and A1c, and for the detection of abnormal hemoglobins in human whole blood using
ion-exchange high-performance liquid chromatography (HPLC).Hemoglobins A2 and F – A
frequently occurring thalassemia, beta-thalassemia (β-thalassemia), is commonly found in the
heterozygous state as β-thalassemia minor or β-thalassemia trait. Adult blood contains
primarily hemoglobin A (HbA), a small percentage of hemoglobin A2 (HbA2), and trace amounts
of fetal hemoglobin (HbF). Carriers of β-thalassemia typically have HbA2 levels of 4–9% and HbF
levels of 1–5%. The D-10 Dual Program HbA2/F/A1c assay can be used for β-thalassemia
screening by quantitation of HbA2 and HbF. The most commonly occurring hemoglobin variants
include hemoglobins S, E, C, and D.10 Presumptive identification of these hemoglobin variants
is made using retention time windows, such as an "S-Window" and "C-Window." Final
determination of specific variants eluting in the windows is left to the educated judgment of the
user. For the positive confirmation of any particular hemoglobin variant, alternative separation
methods are required.

b.PRINCIPLE OF TEST:

The D-10 Dual Program is based on chromatographic separation of the analytes by ion-
exchange high-performance liquid chromatography (HPLC). The samples are automatically
diluted on the D-10 and injected into the analytical cartridge. The D-10 delivers a programmed
buffer gradient of increasing ionic strength to the cartridge, where the hemoglobins are
separated based on their ionic interactions with the cartridge material. The separated
hemoglobins then pass through the flow cell of the filter photometer, where changes in the
absorbance at 415 nm are measured. The D-10 software performs reduction of raw data
collected from each analysis. Two-level calibration is used for quantitation of the HbA2/F/A1c
values. A sample report and a chromatogram are generated for each sample. The A2, F, and A1c
peaks are shaded. The A1c area is calculated using an exponentially modified Gaussian (EMG)
algorithm that excludes the labile A1c and carbamylated peak areas from the A1c peak area.
The D-10 Dual Program is for use only with the Bio-Rad D-10 Hemoglobin Testing System.

d.SAMPLES:

Specimen Type:

The whole blood specimens should be collected in a vacuum collection tube containing EDTA.
Specimen should be collected with Precaution considering any materials of human origin as
infectious and handle them using typical biosafety procedures.
Sample stability:

Whole blood specimens may be stored up to 4 days at 2–8 °C or 1 day at room temperature (15–30 °C).

Sample requirement:

Minimum sample volume required is 2.0 ml in Lavender capped tube containing K3 EDTA is preferred
for automated processing. Volumes lower than 2.0 ml have to be pre diluted before processing.

Sample rejection:

Haemolysed , inadequate sample volume, unlabeled sample.

Specimen Preparation

1. Allow sample tubes to reach room temperature (15–30 °C) before performing the assay. No
sample preparation is required. Mixing the tubes prior to loading is not necessary. The sample
tubes are loaded into the D-10 sample rack and placed in the D-10. Ensure that the sample
barcodes are facing towards the back of the instrument. Use special rack inserts for 12, 13, and
14 mm diameter tubes. Remove all inserts for 16 mm diameter tubes. Tubes with a height of 75
mm to 100 mm are acceptable for use.

2. If the sample is in an abnormal size/type tube, or if there is less than 2.0 mL of sample in the
tube, then the sample must be prediluted. Before pipetting, thoroughly mix the sample by
gently inverting the tube. To predilute, pipet 1.5 mL of Wash/Diluent Solution into a labeled 1.5
mL vial, followed by 5 μL of the whole blood sample. Cap the sample vial and mix thoroughly.
Use a sample vial adapter for 1.5 mL vials.

e. PATIENT PREPARATION:

Random sample can be collected. Pre transfusion sample is preferred to rule out existing blood
disorder which in most case is masked by the donor samples.

f. TYPE OF CONTAINER:

Blood sample should be collected in lavender capped tube containing K3 EDTA.

g. EQUIPMENTS AND REAGENT REQUIREMENT:

EQUIPMENTS:

BIO-RAD D-10 Dual Program HPLC and Rack loader.

REAGENTS:
Elution Buffer 1. Two bottles containing 2000 mL of a Bis-Tris/Phosphate buffer, pH 6.0.
Contains <0.05% sodium azide as a preservative.

Elution Buffer 2. One bottle containing 1000 mL of a Bis-Tris/Phosphate buffer, pH 6.7.

Contains <0.05% sodium azide as a preservative.
Wash /Diluent Solution. One bottle containing 1600 mL of deionized water with <0.05%
sodium azide as a preservative.

HbA1c Calibrator/Diluent Set. One set consisting of 3 vials of Calibrator Level 1, 3 vials of
Calibrator Level 2, and 1 bottle of Calibrator Diluent. The calibrator vials contain lyophilized
human red blood cell hemolysate with gentamicin, tobramycin, and EDTA as preservatives.
Reconstituted volume is 7 mL per vial. Calibrator Diluent contains 100 mL deionized water
with <0.05% sodium azide as a preservative.

HbA2/F/A1c Calibrator/Diluent Set. One set consisting of 3 vials of Calibrator Level 1,

3 vials of Calibrator Level 2, and 1 bottle of Calibrator Diluent. The calibrator vials contain
lyophilized human red blood cell hemolysate with gentamicin, tobramycin, and EDTA as
preservatives. Reconstituted volume is 7 mL per vial. Calibrator Diluent contains 100 mL
deionized water with <0.05% sodium azide as a preservative.

Analytical Cartridge. One cation exchange cartridge, 4.0 mm ID x 30 mm.

Whole Blood Primer. Four vials of lyophilized human red blood cell hemolysate with
gentamicin, tobramycin, and EDTA as preservatives. Reconstituted volume is 1.0 mL per
vial.

working solutions
Elution Buffers and Wash/Diluent Solution
1. Allow the Elution Buffers and Wash/Diluent Solution to reach room temperature (15–30 °C)
before performing the assay. Mix each bottle by gently inverting prior to installation.

2. The Elution Buffers and Wash/Diluent Solution will be stable until the expiration date when
stored unopened at 15–30 °C. After opening the bottles, these reagents are stable for 30 days
when stored at 15–30 °C.

3. With a new reorder pack, install one bottle of each reagent and follow the procedure for
Installing a New Reorder Pack Lot in the Procedure section. After 200 A1c or 100 A2/F/A1c
injections, install a fresh bottleof Elution Buffer 1. Reset the volume in the LOT INFO/Buffer 1
screen after installing this reagent.

NOTE: When using the optional D-10 Rack Loader, the two bottles of Elution Buffer 1 are
installed simultaneously. Manually resetting the volume is not required.
4. The Wash/Diluent Solution is interchangeable between Reorder Pack lots.

Whole Blood Primer

Use fresh aliquots of Whole Blood Primer when installing a new cartridge.
1. The Whole Blood Primer will be stable until the expiration date when stored unopened at 2–
8 °C.
2. Prepare the Whole Blood Primer by adding 1 mL of deionized water to the vial.
3. Allow to stand for 10–15 minutes at 15–30 °C.
4. Swirl gently to dissolve and ensure complete mixing.
5. Write the reconstitution date on the label. The reconstituted Whole Blood Primer is stable
for 1 day when
stored at 2–8 °C.
6. The Whole Blood Primer is interchangeable between lots.

HbA1c Calibrators
Reconstitute and store the HbA1c Calibrators as directed in the Dual Program HbA1c
Calibrator/Diluent Set Insert.

HbA2/F/A1c Calibrators
Reconstitute and store the HbA2/F/A1c Calibrators as directed in the Dual Program HbA2/F/A1c
Calibrator/
Diluent Set Insert.

Extracted Standards
This HPLC method does not use extracted standards.

Controls

• Bio-Rad Lyphochek Diabetes Controls and Hemoglobin A2 Controls must be diluted 1:300
prior to analysis. To achieve this each vial is reconstituted with 500µ of distilled water.
Pipette 1.5 mL of Wash/Diluent Solution into a labeled 1.5 mL vial, followed by 5 μL of the
reconstituted control.
Cap each control vial and mix thoroughly.

INDICATIONS OF INSTABILITY OR DETERIORATION OF REAGENTS

• If reagents were frozen during shipment, mix each bottle by gently inverting before installing
on
instrument.
• Do not use any reagents which have any indications of discoloration, cloudiness or
precipitation.
• Do not use any reagents that show any signs of leakage.
• Do not use the calibrator or whole blood primer if the pellet is brown or the vial is broken. If
the lyophilized material contains insoluble matter, discard the material and reconstitute a new
vial.
Reagent handling :

Ready for use.

Pipetting parameters

Diluent (H2O)
R1 75 µL 11 µL
Sample 27.5 µL
SR 17 µL 10 µL
Total volume 140.5 µL

STORAGE AND STABILITY:

Shelf life at 2-8 °C See expiration date on cobas c pack label

On-board in use at 10-15 °C 4 weeks

h.SAFTEY:

Exercise the normal precautions required for handling all laboratory reagents.

To avoid the possible build-up of azidecompounds,flush waste -pipes with water after the disposal of
undiluted reagent.

Dispose all waste material in accordance with BMW guidelines.

The materials used as a calibrator or quality control should be handled as infectious and universal
precautions to be followed.

i.CALIBRATION:

CALIBRATOR REQUIRED:

Lyophilized serum : Calibrator f.a.s. ref. 10759350 190

Use deionized water as zero calibrator.

CALIBRATION FREQUENCY:

AS per manufacture’s instruction recalibration lot /lot recovery 30days or whenthe following occurs:

 Change in reagent lot or significant shift in control values.

 Major preventive maintenance was performed on the analyzer or a critical part was
replaced.
However daily or bottle to bottle calibration may be done as per the discretion of the lab manager.

CALIBRATOR PREPARATION:

CALIBRATOR STABILITY:

TRACEABILITY:

This method has been standardized against the IFCC procedure (2011).

j. TESTING PROCEDURE:

K.QUALITY CONTROL:

INTERNAL CONTROL;

EXTERNAL CONTROL:

l. INTERFERENCE AND LIMITATIONS:

Substance Limits
Icterus No significant interference up to an I index of 42 for conjugated bilirubin and 60
for unconjugated bilirubin (approximate conjugated bilirubin concentration:
718 µmol/L or 42 mg/dL; approximate unconjugated bilirubin concentration:
1026 µmol/L or 60 mg/dL)
Hemolysis No significant interference up to an H index of 250 (approximate hemoglobin
concentration: 155 µmol/L or 250 mg/dL)
Drugs No interference was found at therapeutic concentrations using common drug
panels

m. CALCULATING RESULTS TO INCLUDE MEASURMENT UNCERTANITY:

n. BIOLOGICAL TEST INTERVALS:

Adults:

Males (n = 221) -40-129 U/L (0.67-2.15 µkat/L)

Females (n = 229)35-104 U/L (0.58-1.74 µkat/L)

Children

Male
0 - 14 days 83-248 U/L (1.39-4.14 µkat/L)

15 days - < 1 year 122-469 U/L (2.04-7.83 µkat/L)

1 - < 10 years 142-335 U/L (2.37-5.59 µkat/L)

10 - < 13 years 129-417 U/L (2.15-6.96 µkat/L)

13 - < 15 years 116-468 U/L (1.94-7.82 µkat/L)

15 - < 17 years 82-331 U/L (1.37-5.53 µkat/L)

17 - < 19 years 55-149 U/L (0.92-2.49 µkat/L)

Female

0- 14 days 83-248 U/L (1.39-4.14 µkat/L)

15 days - < 1 year 122-469 U/L (2.04-7.83 µkat/L)

1 - < 10 years 142-335 U/L (2.37-5.59 µkat/L)

10 - < 13 years 129-417 U/L (2.15-6.96 µkat/L)

13 - < 15 years 57-254 U/L (0.95-4.24 µkat/L)

15 - < 17 years 50-117 U/L (0.84-1.95 µkat/L)

17 - < 19 years 45-87 U/L (0.75-1.45 µkat/L)

o.REPORTABLE RANGE:3.0-1200 U/L (0.05-20 µkat/L)

p.DETERMINING RESULTS WHEN THE RESULT IS NOT WITHIN MEASURABLE RANGE:

LINEARITY:The test is linear within a concentration range of 3.0-1200 U/L (0.05-20 µkat/L for serum or
plasma

Limits of detection (LOD) verified in the lab:3.0-1200 U/L (0.05-20 µkat/L

POTENTIAL SOURCE OF VARIATION:

In very rare cases, gammopathy, in particular type IgM (Waldenström’s macroglobulinemia), may
cause unreliable results.11 For diagnostic purposes, the results should always be assessed in
conjunction with the patient’s medical history, clinical examination and other findings.

C.PERFORMANCE CHARECTERISTICS:

Linearity:

Stated Linearity:

Test is linear within a Measuring range of3.0-1200 U/L (0.05-20 µkat/L) for serum and plasma.
Conversion factor: U/L × 0.0167 = µkat/L

Determine samples having higher activities via the rerun function. Dilution of samples via the rerun
function is a 1:5 dilution. Results from samples diluted using the rerun function are automatically
multiplied by a factor

Precision:
PERFORMANCE CHARACTERISTICS
Precision
The precision of the D-10 Dual Program was evaluated based on the NCCLS EP5-T2 guideline (for the Short Program) and EP5-A guideline (for
the Extended Program), "Evaluation of Precision Performance of Clinical Chemistry Devices." In these studies, 40 runs were performed over 20
working days. In each run, aliquots of low and high specimens were analyzed in duplicate. The results of the precision study are summarized in

Tables 2–5.
Low Patient High Patient Low Patient High Patient
Mean (% HbA1c) 5.7 9.4 Mean (% HbA1c) 5.9 13.1
Within Run (%CV) 0.8 0.5 Within Run (%CV) 0.8 0.3
Between Day (%CV) 0.7 1.0 Between Day (%CV) 1.4 0.8
Between Run (%CV) 0.5 0.5 Between Run (%CV) 0.7 0.3
Total Precision (%CV) 1.2 1.2 Total Precision (%CV) 1.8 0.9

Table 2: Precision results for HbA1c, Short Program Table 3: Precision results for HbA1c, Extended Program
Patient State HbA2 Level HbF Level
Heterozygous β-thalassemia 4–9% 1–5%
Homozygous β-thalassemia
Normal or
Increased
80–100%
Heterozygous HPFH* <1.5% 10–20%
Homozygous HPFH* Absent 100%

Table 1: HbA2/F Reference Intervals 9

* Hereditary Persistence of Fetal Hemoglobin

Low Patient High Patient Low Patient High Patient

Mean (% HbA2) 2.2 5.4 Mean (% HbF) 2.1 8.7
Within Run (%CV) 4.5 1.7 Within Run (%CV) 2.2 1.4
Between Day (%CV) 3.4 2.7 Between Day (%CV) 1.8 1.3
Between Run (%CV) 0.0 0.0 Between Run (%CV) 1.7 0.0
Total Precision (%CV) 5.3 3.1 Total Precision (%CV) 3.3 2.0
Table 4: Precision results for HbA2, Extended Program Table 5: Precision results for HbF, Extended Program

Accuracy
Hemoglobin A1c
Hemoglobin A1c results obtained
from the D-10 Dual Short and
Extended Programs were
compared to each other. The
comparison study was performed
on 40 patient samples analyzed in
duplicate. See Figure 1.
slope = 1.0663
intercept = −0.5416
R2 = 0.9953
D-10 Dual Extended vs Short Program
4
6
8
10
12
14
4 6 8 10 12 14
D-10 Dual Short Program %HbA1c
D-10 Dual Extended Program %HbA1c
Figure 1: Correlation: D-10 Dual Extended Program (HbA1c) vs D-10 Dual
Short Program (HbA1c)
D-10™ Dual Program
L20012304 Instruction Manual 15
Hemoglobin A1c
Hemoglobin A1c results obtained from the
D-10 Dual Short and Extended Programs
were compared to values obtained on the
VARIANT™ II HbA1c Program ( 270-
2101). The comparison study was
performed on 40 patient samples
analyzed in duplicate. See Figures 2 and
3.
slope = 0.9743
intercept = 0.3078
R2 = 0.9945
slope = 0.9906
intercept = 0.431
R2 = 0.9843
D-10 Dual vs VARIANT II HbA1C
4
6
8
10
12
14
4 6 8 10 12 14
VARIANT II %HbA1C
D-10 Dual Extended Program %HbA1c

Figure 3: Correlation: D-10 Dual Extended Program (HbA1c) vs

VARIANT II HbA1c Program ( 270-2101)
Hemoglobin A2
Hemoglobin A2 results obtained from
the D-10 Dual Extended Program were
compared to values obtained on the
VARIANT II β-thalassemia Short
Program. The comparison study was
performed on 40 patient samples
analyzed in duplicate. See Figure 4 for
results.
slope = 1.0898
intercept = −0.2407
R2 = 0.9832
D-10 HbA1c vs VARIANT II HbA 1c
VARIANT II %HbA 1c
D-10 %HbA1c

Figure 2: Correlation: D-10 Dual Short Program (HbA1c) vs

VARIANT II HbA1c Program ( 270-2101)
D-10 Dual Program vs VARIANT II β-thalassemia Short Program (HbA 2)
0
1
2
3
4
5
6
7
8
9
10
0 1 2 3 4 5 6 7 8 9 10
VARIANT II %HbA 2
D-10 Dual Program %HbA2
Figure 4: Correlation: D-10 Dual Extended Program (HbA2) vs
VARIANT II β-thalassemia Short Program
16 Instruction Manual L20012304
Hemoglobin F
Hemoglobin F results from the D-10
Dual Extended Program were
compared to values obtained on the
VARIANT II β-thalassemia Short
Program. The comparison study was
performed on 40 patient samples
analyzed in duplicate. See Figure 5 for
results.
slope = 0.9497
intercept = −0.1785
R2 = 0.9959

Linearity/Recovery
To demonstrate linearity on the D-10 Dual Program throughout the reportable range, low and high specimens
were derived as follows:
HbA1c (Short Program)
Low: Whole blood from a normal patient sample was supplemented with Bio-Rad Lyphochek
Hemoglobin A1c Linearity Set Level 1 to yield relative HbA1c level of 3.8%.
High: Whole blood from a diabetic patient sample was supplemented with Bio-Rad Lyphochek
Hemoglobin A1c Linearity Set Level 4 to yield relative HbA1c level of 18.5%.
HbA1c (Extended Program)
Low: Whole blood from a normal patient sample was supplemented with Bio-Rad Lyphochek
Hemoglobin A1c Linearity Set Level 1 to yield relative HbA1c level of 3.7%.
High: Whole blood from a diabetic patient sample was supplemented with Bio-Rad Lyphochek
Hemoglobin A1c Linearity Set Level 4 to yield relative HbA1c level of 18.4%.
HbA2
Low: Whole blood with a HbA2 level of 1.5%.
High: Normal whole blood spiked with purified HbA2 to a level of 11.4%.
HbF
Low: Normal whole blood with HbF level of 0.8%.
High: Normal whole blood spiked with an elevated HbF sample to a level of 16.5%.
The high specimen was diluted with the low specimen in varying ratios to yield specific relative analyte
percentages (theoretical percent). These diluted samples were analyzed with the D-10 Dual Program
(observed percent). Percent recovery was determined by dividing the observed percent by the theoretical
percent and multiplying the result by 100. Results from the Linearity Study are shown in Tables 6–9.
D-10 Dual Program vs VARIANT II β-thalassemia Short Program (HbF)
-2
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12 14 16
VARIANT II %HbF
D-10 Dual Program %HbF
Figure 5: Correlation: D-10 Dual Extended Program (HbF) vs
VARIANT II β-thalassemia Short Program
D-10™ Dual Program
L20012304 Instruction Manual 17
% Contribution
Theoretical % HbA1c Observed % HbA1c % Recovery
Low High
100 0 3.8 3.8 100
80 20 6.6 6.6 100
67 33 8.6 8.5 98.8
50 50 11.0 11.0 100
33 67 13.5 13.2 97.8
20 80 15.4 15.2 98.7
0 100 18.5 18.5 100
Table 6: Results of Study on Linearity and Recovery for HbA1c, Short Program
% Contribution
Theoretical % HbA1c Observed % HbA1c % Recovery
Low High
100 0 3.7 3.7 100
80 20 7.1 6.7 94.4
67 33 9.2 8.8 95.7
50 50 11.7 11.4 97.4
33 67 14.1 13.7 97.2
20 80 15.9 15.7 98.7
0 100 18.4 18.4 100
Table 7: Results of Study on Linearity and Recovery for HbA1c, Extended Program
% Contribution
Theoretical % HbA2 Observed % HbA2 % Recovery
Low High
100 0 1.5 1.5 100
80 20 3.5 3.3 94.3
67 33 4.9 4.7 95.9
50 50 6.5 6.4 98.5
33 67 8.2 8.1 98.8
20 80 9.5 9.6 101.1
0 100 11.4 11.4 100
Table 8: Results of Study on Linearity and Recovery for HbA2, Extended Program
18 Instruction Manual L20012304
% Contribution
Theoretical % HbF Observed % HbF % Recovery
Low High
100 0 0.8 0.8 100
80 20 4.0 3.7 92.5
67 33 6.1 6.0 98.4
50 50 8.7 8.8 101.1
33 67 11.3 11.4 100.9
20 80 13.4 13.6 101.5
0 100 16.5 16.5 100
Table 9: Results of Study on Linearity and Recovery for HbF, Extended Program
Interfering Substances
• Icterus, as indicated by bilirubin concentrations up to 20 mg/dL, does not interfere with the percent
determination of HbA2, HbF, or HbA1c.
• Lipemia, as indicated by triglyceride concentrations up to 5680 mg/dL, does not interfere with the
percent determination of HbA2, HbF, or HbA1c.
• Hemoglobin F concentrations up to 10% do not interfere with the percent determination of HbA1c.
• Labile A1c (LA1c/CHb-1) concentrations up to 4% do not interfere with the percent determination of
HbA1c.
• Labile A1c (LA1c/CHb-1) concentrations up to 2.6% do not interfere with the percent determination of
HbF.
• Carbamylated hemoglobin (LA1c/CHb-2) concentrations up to 3.5% do not interfere with the percent
determination of HbA1c.
SAMPLE REPORT FORMAT
Several of the following sample report examples include both IFCC (mmol/mol) and NGSP (%) units for
HbA1c. Beginning with D-10 software version 3.60, your lab's preferred reporting units for standardized HbA1c
results (i.e., NGSP only or NGSP and IFCC) can be configured in the D-10 Service Software by a Bio-Rad
representative.
NOTE: The sample report examples with the HbA1c result in % only were generated using D-10 software version
3.50.
D-10™ Dual Program
L20012304 Instruction Manual 19
SAMPLE REPORT FORMAT
Patient report
Bio-Rad DATE: 09/23/2010
D-10 TIME: 09:16 AM
S/N: #DA3G222606 Software version: 3.57
Sample ID: RACKB3-3-23-22-9-2010
Injection date 09/22/2010 11:45 AM
Injection #: 23 Method: HbA1c
Rack #: B3 Rack position: 3
Peak table - ID: RACKB3-3-23-22-9-2010
Peak R.time Height Area Area %
Unknown 0.14 8245 9082 0.3
A1a 0.20 6676 17769 0.5
A1b 0.28 8455 40358 1.2
F 0.44 5438 30425 0.9
LA1c/CHb-1 0.61 6180 46070 1.4
A1c 0.78 12575 125339 5.1
P3 1.36 32535 149519 4.5
A0 1.45 734587 2909750 87.4
Total Area: 3328311
Concentration: % mmol/mol
A1c 5.1 32
Figure 6: Non-Diabetic (Normal) Sample, Short Program
20 Instruction Manual L20012304

Precision was determined using human samples and controls in an internal protocol with repeatability
(n = 21) and intermediate precision (1 aliquot per run, 1 run per day, 10 days).

The following results were obtained:

Repeatability Mean U/L (µkat/L) SD U/L (µkat/L) CV %

Precinorm U 83.7 (1.40) 0.6 (0.01) 0.7
Precipath U 226 (3.77) 1 (0.02) 0.4
Human serum 1 84.4 (1.41) 0.3 (0.01) 0.4
Human serum 2 217 (3.62) 1 (0.02) 0.4

Intermediate precision Mean U/L (µkat/L) SD U/L (µkat/L) CV %

Precinorm U 84.0 (1.40) 1.8 (0.03) 2.1
Precipath U 227 (3.80) 4 (0.07) 1.7
Human serum 1 85.7 (1.43) 1.7 (0.03) 2.0
Human serum 2 218 (3.64) 5 (0.08) 2.2

Method comparison:

{ALP values for human serum and plasma samples obtained on a COBAS INTEGRA 400 analyzer with the
COBAS INTEGRA ALP IFCC Gen.2 (ALP2L) reagent and the AP2LP application (y) were compared to
commercially available ALP assay.

Result of linear regression analysis were as follows:

R= n=

This has been verified by the lab}

ALP values for human serum and plasma samples obtained on a COBAS INTEGRA 700 analyzer with the
COBAS INTEGRA ALP IFCC Gen.2 (ALP2L) reagent and the AP2LP application (y) were compared to those
determined using the same reagent but the ALP2L application on a COBAS INTEGRA 700 analyzer (x),
and to those determined using the previous reagent using the ALP6P application on a
COBAS INTEGRA 700 analyzer (x).

COBAS INTEGRA (ALP2L) Sample size (n) = 54

Passing/Bablok14 Linear regression

y = 1.003x - 0.195 U/L y = 0.999x + 2.11 U/L

τ = 0.976 r = 1.00 SD (md 95) = 9.19 Sy.x = 4.06

The sample activities were between 40 and 1010 U/L (0.668 and 16.9 µkat/L).

COBAS INTEGRA (ALP6P) Sample size (n) = 67 Passing/Bablok14 Linear regression y = 0.991x + 0.808 U/L
y = 0.997x - 1.35 U/L τ = 0.976 r = 1.00 SD (md 95) = 7.16 Sy.x = 3.39

The sample activities were between 39 and 862 U/L (0.651 and 15.8 µkat/L)

INTERPRETATION:

Alkaline phosphatase in serum consists of four structural genotypes: the liver-bone-kidney type, the
intestinal type, the placental type and the variant from the germ cells. It occurs in osteoblasts,
hepatocytes, leukocytes, the kidneys, spleen, placenta, prostate and the small intestine. The liver-
bonekidney type is particularly important.

LOW:

HIGH:

A rise in the alkaline phosphatase occurs with all forms of cholestasis, particularly with obstructive
jaundice. It is also elevated in diseases of the skeletal system, such as Paget’s disease,
hyperparathyroidism, rickets and osteomalacia, as well as with fractures and malignant tumors. A
considerable rise in the alkaline phosphatase activity is sometimes seen in children and juveniles. It is
caused by increased osteoblast activity following accelerated bone growth.

REFERENCE: