Quantitative determination of glycated hemoglobin (HbAB1Bc) in
human blood
IVD
Store 2 - 8ºC.
PRINCIPLE OF THE METHOD
This method utilizes the interaction of antigen and antibody to directly determine the
HbA1c in whole blood. Total hemoglobin and HbAB1c have the same unspecific absorption
rate to latex particles. When mouse antihuman HbAB1c monoclonal antibody is added
(R2), latex-HbA1c-mouse anti human HbAB1c antibody complex is formed. Agglutination is
formed when goat anti-mouse IgG polyclonal antibody interacts with the monoclonal
antibody. The amount of agglutination is proportional to the amount of HbA1c absorbed
on to the surface of latex particles. The amount of agglutination is measured as
absorbance. The HbAB1c value is obtained from a calibration curve.
CLINICAL SIGNIFICANCE P
Throughout the circulatory life of the red cell, Hemoglobin A1c is formed continuously by
the adduction of glucose to the N-terminal of the hemoglobin beta chain. This process,
which is non-enzymatic, reflects the average exposure of hemoglobin to glucose over an
extended period. In a classical study, Trivelli et al1
showed Hemoglobin AB1c in diabetic
subjects to be elevated 2-3 fold over the levels found in normal individuals. Several
investigators have recommended that Hemoglobin A1c serve as an indicator of metabolic
control of the diabetic, since Hemoglobin A1c levels approach normal values for diabetics
in metabolic control.2,3,4
Hemoglobin A1c has been defined operationally as the “fast fraction” hemoglobins (Hb1A,
A1B, A1c) that elute first during column chromatography with cation-exchange resins. The
non-glycosylated hemoglobin, which consists of the bulk of the hemoglobin has been
designated HbA0. The present procedure utilizes an antigen and antibody reaction to
directly determine the concentration of the HbAB1c.
PRECAUTIONS
All human specimens should be regarded as potentially biohazardous. Therefore,
universal precautions should be used in specimen handling (gloves, lab garments, avoid
aerosol production, etc.).
PREPARATION
R1, R2 and R3 are ready to use. Mix gently before use.
STORAGE AND STABILITY
All the components of the kit are stable until the expiration date on the label when stored
tightly closed at 2-8ºC and contaminations are prevented during their use. Reagents
should not be left inside the analyzer after use, they must be stored refrigerated at 2-8ºC.
Latex may sediment. Mix reagents gently before use. Do not use reagents over the
expiration date.
R1 and R2 are stable for at least one month after opening stored at 2-8°C.
Reagent deterioration: Alterations in the physical appearance of the reagents or values
of control materials outside of the manufacturer’s acceptable range may be an indication
of reagent instability.
SAMPLES
Special preparation of the patient is unnecessary. Fasting specimens are not required.
No special additives or preservatives other than anticoagulants are required. Collect
venous blood with EDTA using aseptic technique. HbAB1c in whole blood collected with
EDTA is stable for one week at 2-8°C5
.
To determine HbA1c, a hemolysate must be prepared for each sample:
1. Dispense 0,5 mL Hemolysis Reagent into tubes labeled: Calibrator, Control,
Patients, etc. Note: Plastic or glass tubes of appropriate size are acceptable.
2. Place 10 µL of well mixed whole blood into the appropriately labeled lyse reagent
tube. Mix.
3. Allow to stand for 5 minutes or until complete lysis is evident. Hemolysates may be
stored up to 10 days at 2-8°C.
QUALITY CONTROL
HbAB1c Control (ref: 43106) is recommended to monitor the performance of manual and
automated assay procedures. Controls require hemolysis pretreatment after being
reconstituted.
Each laboratory should establish its own Quality Control scheme and corrective actions
if controls do not meet the acceptable tolerances.
REFERENCE VALUES11
Recommended Values: less than 6% for a non-diabetic, less than 7% for glycemic control
of a person with diabetes.
Each laboratory should establish its own expected values. In using Hemoglobin A1c to
monitor diabetic patients, results should be interpreted individually. That is, the patient
should be monitored against him or herself. There is a 3-4 weeks time lag before
Hemoglobin A1c reflects changes in blood glucose level.
NOTES
1. In order to avoid contamination, it is recommended to use disposable material.
2. Use clean disposable pipette for its dispensation.
3. SPINREACT has instruction sheets for several automatic analyzers.
INTERFERENCES
1. Bilirubin to 50 mg/dL, ascorbic acid to 50 mg/dL, triglycerides to 2000 mg/dL,
carbamylated Hb to 7,5 mmol/L and acetylated Hb to 5,0 mmol/L do not interfere in
this assay.
2. It has been reported that results may be inconsistent in patients who have the
following conditions: opiate addiction, lead-poisoning, alcoholism, ingest large
doses of aspirin.6, 7, 8, 9
3. It has been reported that elevated levels of HbF may lead to underestimation of
HA1c and, that uremia does not interfere with HbAB1c determination by
immunoassay.10 It has been reported that labile intermediates (Schiff base) are not
detected and therefore, do not interfere with HbAB1c determination by immunoassay.5
4. It has been determined that Hemoglobin variants HbA2, HbC and HbS do not
interfere with this method.
5. Other very rare variants of hemoglobin (e.g. HbE) have not been assessed.
BIBLIOGRAPHY
1. Trivelli, L.A., Ranney, H.M., and Lai, H.T., New Eng. J. Med. 284,353 (1971).
2. Gonen, B., and Rubenstein, A.H., Diabetologia 15, 1 (1978).
3. Gabbay, K.H., Hasty, K., Breslow, J.L., Ellison, R.C., Bunn, H.F., and Gallop, P.M.,
J. Clin. Endocrinol. Metab. 44, 859 (1977).
4. Bates, H.M., Lab. Mang., Vol 16 (Jan. 1978).
5. Tietz, N.W., Textbook of Clinical Chemistry, Philadelphia, W.B. Saunders
Company, p.794-795 (1999).
6. Ceriello, A., et al, Diabetologia 22, p. 379 (1982).
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).
9. Nathan, D.M., et al, Clin. Chem. 29, pp. 466-469 (1983).
10. Engbaek, F., et al, Clin. Chem. 35, pp. 93-97 (1989).
11. American Diabetes Association: Clinical Practice Recommendations (Position
Statement). Diabetes Care 24 (Suppl. 1): S33-S55, (2001).
