HbA1c Direct with Calibrator

Intended Use
For the quantitative determination of Hemoglobin A1c (HbA1c) in human
blood. The determination of HbA1c is most commonly performed for the
evaluation of glycemic control in diabetes mellitus. HbA1c values provide an
indication of glucose levels over the preceding 4-8 weeks. A higher HbA1c
value indicates poorer glycemic control. For in vitro diagnostic use only.
Storage and Stability
Summary and Explanation of Test 1. All reagents are stable to the expiration date stated on the labels. Do
Throughout the circulatory life of the red cell, Hemoglobin A1c is formed not use the reagents past their expiration date.
continuously by the adduction of glucose to the N-terminal of the 2. Hemoglobin A1c in whole blood collected with EDTA is stable for one
hemoglobin beta chain. This process, which is non-enzymatic, reflects the week at 2-8°C.5
average exposure of hemoglobin to glucose over an extended period. In a GENERAL SYSTEM PARAMETER
classical study, Trivelli et al1 showed Hemoglobin A1c in diabetic subjects to
Mode of reaction End point
be elevated 2-3 fold over the levels found in normal individuals. Several
Slope of reaction Increasing
investigators have recommended that Hemoglobin A1c serve as an indicator
Wavelength 660nm (600-660nm)
of metabolic control of the diabetic, since Hemoglobin A1c levels approach
Temperature 37°C
normal values for diabetics in metabolic control.2, 3, 4
Calibrator Concentration Refer Kit Insert
Hemoglobin A1c has been defined operationally as the "Fast Fraction"
Linearity 16%
hemoglobins (HbA1a, A1b, A1c) that elute first during column
Blank Reagent blank
chromatography with cation-exchage resins. The non-glycosylated
Sample volume 10 µL
hemoglobin which consists of the bulk of the hemoglobin has been
Reagent 1 Volume 360 µL
designated HbA0. The present procedure utilizes a antigen and antibody
Reagent 2 Volume 120 µL
reaction to directly determine the concentration of the HbA1c.
Light Path 1 cm
Principle PROCEDURE
This method utilizes the interaction of antigen and antibody to directly
determine the HbA1c in whole blood. Total hemoglobin and HbA1c have the Blank Calibrator Sample/control
same unspecific absorption rate to latex particles. When mouse antihuman Latex Reagent 360 µL 360 µL 360 µL
HbA1c monoclonal antibody is added (R2), latex-HbA1c-mouse antihuman
HbA1c antibody complex is formed. Agglutination is formed when goat anti- Hemolysate (Calibrator) - 10 µL -
mouse lgG polyclonal antibody interacts with the monoclonal antibody. The Hemolysate (sample) - - 10 µL
amount of agglutination is proportional to the amount of HbA1c absorbed on Mix & Incubate for 5 min at 37°C.
to the surface of latex particles. The amount of agglutination is measured as Antibody Reagent 120 µL 120 µL 120 µL
absorbance. The HbA1c value is obtained from a calibration curve.
Mix and incubate for 5 min at 37°C and read absorbance(A) at 660 nm.
Contents Calculation
Reagent 1 : Latex Reagent
Reagent 2 : Antibody Reagent. Calibration curve
Reagent 3 : Hemolysis Reagent Calculate the Abs of calibrators = Abs calibrator – Abs Blank.
Reagent 4 : Calibrator Plot the Abs of each calibrator verus assigned concentration (HbA1c
[4 Level Lyophilized 4x0.5ml] %) on a linear graph paper. HbA1c results according to NGSP for the
Reagent Storage samples and controls are determined using the prepared calibration
Store all reagents refrigerated at 2-8°C. curve.
An example curve is illustrated below.
Reagents Preparation
2.0
R1, R2 and Hemolysis reagents are supplied as ready to use liquids, Mix
gently before use. x
A.
1.0 x
Calibrator : Reconstitute the Calibrator with 0.5ml distilled water. Stable for b
30 days at 2-8°C. Do Not Freeze.
S. x
0.5 x
Reagent Deterioration 0
Alterations in the physical appearance of the reagents or values of control 0 4 8 12 16 20
materials outside of the manufacture's acceptable range may be an HbA1c (%)

indication of reagent instability. Calculate

Abs of sample i.e. abs sample - abs blank
Specimen Collection and Preparation HbA1c % in the sample is calculated by interpolation of Abs of sample on
Special preparation of the patient is unnecessary. Fasting specimens are the calibration curve. For calculation of results according to IFCC, use
not required. No special additives or preservatives other than anticoagulants IFCC calibrator values or use following equation.
are required. Collect venous blood with EDTA using aseptic technique. All NGSP = (0.915 x IFCC) + 2.15
human specimens should be regarded as potentially biohazardous.
Therefore, universal precautions should be used in specimen handling Interferences & Limitations
(gloves, lab garments, avoid aerosol production, etc.). 1. Bilrubin to 50mg/dL, ascorbic acid to 50mg/dl, triglycerides to 2000
mg/dl, Carbamylated Hb to 7.5 mmol/L and acetylated Hb to 5.0
To determine HbA1c, a hemolysate must be prepared for each sample : mmol/L do not interfere in this assay.
1. Dispense 1ml Hemolysis Reagent into tubes labeled : Calibrator, 2. It has been reported that results may be inconsistent in patients who
Samples etc. Note : Plastic or glass tubes of appropriate size are have the following conditions : opiate addiction, lead-poisoning,
acceptable. alcoholism, ingest large doses of aspirin.6, 7, 8, 9
2. Place 20ul of well mixed whole blood into the appropriately labeled lyse 3. It has been reported that elevated levels of HbF may lead to
reagent tube, Mix. underestimation of HbA1c.10 Also, it has been reported that labile
3. Allow to stand for 5 minutes or until complete lysis is evident. intermediates (Schiff base) are not detected and do not interfere with
Hemolysates may be stored up to 10 days at 2-8°C. HbA1c determination by immunoassay.5
4. It has been determined that Hemoglobin varients HbA2, HbC and
Instruments HbS do not interfere with this method.
Refer to specific instrument application for suggested settings. 5. Other very rare variants of hemoglobin (e.g. HbE) have not been
assessed.
Precautions 6. Patient specimens should always be assayed using a calibration
1. This reagent is for in vitro diagnostic use only. curve.
2. Not for internal or external use in humans or animals.
Expected Values11
Recommended values: less than 6% for a non-diabetic,less than 7% for 4. Sensitivity : Sensitivity was investigated by reading the change
glycemic control of a person with diabetes. in absorbance at 660 mm for a saline sample and a whole
blood sample with a known concentration. Ten replicates of
Each laboratory should establish its own expected values. In using each sample were performed. The results of this investigation
hemoglobin A1c to monitor diabetic patients ,results should be indicated that, on the analyzer used (Hitachi 717), the HbA1c
interpreted individually. That is, the patient should be monitored against reagent showed little or no drift on the zero sample. Under the
him or herself. There is a 3-4 week time lag before Hemoglobin A1c reaction conditions described, a 0.073 absorbance change is
reflects changes in blood glucose level. approximately equivalent to 1.0% HbA1c.

Linearity Reference :
This Procedure linear up to 16% (NGSP)
1. Trivelli, L.A. Ranney, H.M. and Lai, H.T., New Eng. J. Med.
Performance 284,353 (1971).
1. Linearity : The Hemoglobin A1c assay range is 2.0% - 16.0% 2. Gonen , B., and Rubenstein, A.H.,Diabetologia 15,1(1978).
2. Comparison : A study using 40 human specimens between this 3. Gabbay K.H., Hasty K., Breslow J.L., Ellision R.C., Bunn H.F.
Hemoglobin A1c procedure and an automated HPLC procedure and Gallop P.M., J. Clin, Endocrinol Metab. 44, 859 (1977).
(Tosoh) yielded a correlation coefficient of 0.988 and a linear 4. Bates,H.M., Lab. Mang.,Vol 16,(jan.1978).
regression equation of y = 1.050x - 0.481. (syx = 0.332) 5. Titez, N.W.,Textbook of Clinical Chemistry, Philadelphia, W.B.
3. Precision : Saunders Companay, p.794-795 (1999).
Within Run : The within run precision was established by assaying 6. Ceriello,A., et al, Diabetologia, 22, p.379 (1982).
two blood samples following NCCLS protocol EP5 on a Hitachi 917. 7. Little, R.R.,et al, clin. chem. 32,pp. 358-360 (1986).
8. Fluckiger, R., et al, New eng.J.med. 304 pp. 823-827(1981).
Level Mean Std. Dev. % C.V. 9. Nathan, D.M.et al,Clin Chem. 29,pp.466 -469(1983) .
Low 5.48 0.078 1.43 10. Engbaek,F., et al, Clin. chem.35 pp.93-97 (1989).
High 10.28 0.176 1.72 11. american Diabetes Association: Clinical practice
recommendations (Position statement). Diabetes care 24
Day to Day : The between day precision was established by (Suppl. 1):S33-S55,(2001).
assaying two blood samples following NCCLS protocol EP5 on a
Hitachi 917. Code No. Pack Size Reagent 1 Reagent 2 Reagent 3 Reagent 4

Level Mean Std. Dev. % C.V. T07 40ml 1x30ml 1x10ml 1x125ml 4x0.5ml
Low 5.48 0.152 2.77
High 10.28 0.275 2.68

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ISO 9001:2015 ISO 13485:2003

ASSIGNED VALUES ACCORDING TO NGSP (%)

REFERENCE

INSTRUMENT CALIBRATOR 1(%) CALIBRATOR 2(%) CALIBRATOR 3(%) CALIBRATOR 4(%)

Vchem Plus/
Turbicon Plus/ 5.3 8.3 11.4 15.5
DS 140/DS 302

COBAS MIRA 5.2 8.2 11.6 15.5

ENVOY 500 4.7 8.2 11.2 15.2

HITACHI 717 5.2 8.3 11.6 15.5

HITACHI 917 5.0 8.1 11.3 15.4
BECKMAN AU 5.3 8.3 11.4 15.5
MINDRAY BS-200 5.6 8.3 12.0 15.5