Glycated Hemoglobin

Latex Turbidimetry (HbA1c)

PRODUCT CODE 3. Allow to stand for 5 minutes or until complete lysis is evident.
TL005 Hemolysates may be stored up to 10 days at 2-8°C.
INTENDED USE PROCEDURES
Quantitative determination of glycated hemoglobin (HbA1c) in human 1. Assay conditions:
blood IVD. Wavelength: 660 nm (600 - 660)
CLINICAL SIGNIFICANCE Temperature: 37ºC
Throughout the circulatory life of the red cell, Hemoglobin A1c is formed Cuvette ligth path: 1 cm
continuously by the adduction of glucose to the N-terminal of the 2. Adjust the instrument to zero with distilled water.
hemoglobin beta chain. This process, which is non-enzymatic, reflects the 3. Pipette into a cuvette: (Note 2)
average exposure of hemoglobin to glucose over an extended period. In a R1 (µl) 360
classical study, Trivelli et al1 showed Hemoglobin AB1c in diabetic
subjects to be elevated 2-3 fold over the levels found in normal individuals. Calibrator or Sample (µl) 10
Several investigators have recommended that Hemoglobin A1c serve as an 4. Mix and incubate 5 minutes.
indicator of metabolic control of the diabetic, since Hemoglobin A1c levels 5. Pippete into the cuvette:
approach normal values for diabetics in metabolic control.2,3,4 R2 (µl) 120
Hemoglobin A1c has been defined operationally as the “fast fraction”
hemoglobins (Hb1A, A1B, A1c) that elute first during column 6. Mix and read the absorbance after 5 minutes (A) of the R2 addition.
chromatography with cation-exchange resins. The non-glycosylated
hemoglobin, which consists of the bulk of the hemoglobin has been CALCULATIONS
designated HbA0. The present procedure utilizes an antigen and antibody HbA1c concentration (%): Plot (A) obtained against the HbA1c
reaction to directly determine the concentration of the HbAB1c.
concentration of each calibrator (1 to 4 Level). HbA1c percentage in the
PRINCIPLE sample is calculated by interpolation of its absorbance (A) in the
This method utilizes the interaction of antigen and antibody to directly calibration curve.
determine the HbA1c in whole blood. Total hemoglobin and HbAB1c have
the same unspecific absorption rate to latex particles. When mouse QUALITY CONTROL
antihuman HbAB1c monoclonal antibody is added (R2), latex HbA1c- HbA1c Control is recommended to monitor the performance of manual and
mouse anti human HbAB1c antibody complex is formed. Agglutination is automated assay procedures. Controls require hemolysis pretreatment after
formed when goat anti-mouse IgG polyclonal antibody interacts with the being reconstituted. Each laboratory should establish its own Quality
monoclonal antibody. The amount of agglutination is proportional to the
amount of HbA1c absorbed on to the surface of latex particles. The amount Control scheme and corrective actions if controls do not meet the
of agglutination is measured as absorbance. The HbAB1c value is obtained acceptable tolerances
from a calibration curve.
REFERENCE VALUES:
REAGENTS Recommended Values: less than 6% for a non-diabetic, less than 7% for
(R1) Latex 0,13%, Buffer, stabilizer. glycemic control of a person with diabetes. Each laboratory should
(R2) Mouse anti-human HbA1c monoclonal establish its own expected values. In using Hemoglobin A1c to monitor
antibody 0,05mg/ml, goat anti-mouse IgG diabetic patients, results should be interpreted individually. That is, the
polyclonal antibody 0,08mg/dl, Buffer, patient should be monitored against him or herself. There is a (3–4) week
stabilizers time lag before Hemoglobin A1c reflects changes in blood glucose level
(R3) Water and stabilizers
(Hemolysis Reagent) PERFORMANCE CHARACTERISTICS
1. Measuring range: From detection limit of 2% to linearity limit of 16%.
PREPARATION 2. Precision:
R1, R2 and R3 are ready to use. Mix gently before use. Intra-assay (n=20)

STORAGE AND STABILITY Mean (%) 5.95 12.15

All the components of the kit are stable until the expiration date on the SD 0.19 0.18
label when stored tightly closed at 2-8ºC and contaminations are prevented
CV (%) 3.20 1.47
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 Inter-assay (n=20)
and R2 are stable for at least one month after opening stored at 2-8°C. Mean (%) 5.97 12.21
Reagent deterioration: Alterations in the physical appearance of the
SD 0.14 0.15
reagents or values of control materials outside of the manufacturer’s
acceptable range may be an indication of reagent instability CV (%) 2.31 1.24
3. Sensitivity: 1% = 0,056 (A)
PRECAUTIONS 4. Accuracy:
All human specimens should be regarded as potentially biohazardous. Results obtained using Bioresearch reagents (y) did not show
Therefore, universal precautions should be used in specimen handling differences when compared with another commercial reagent (x). The
(gloves, lab garments, avoid aerosol production, etc.)
results obtained using 40 samples were the following: Correlation
SPECIMEN AND SAMPLE PREPARATION coefficient (r)2 0,995 Regression equation: y= 0,989x -0,047 The
Special preparation of the patient is unnecessary. Fasting specimens are not results of the performance characteristics depend on the analyzer used.
required. No special additives or preservatives other than anticoagulants are 5. INTERFERENCES
required. Collect venous blood with EDTA using aseptic technique. HbA1c 1. Bilirubin to 50 mg/dL, ascorbic acid to 50 mg/dL, triglycerides to 2000
in whole blood collected with EDTA is stable for one week at 2-8°C. mg/dL, carbamylated Hb to 7,5 mmol/L and acetylated Hb to 5,0 mmol/L
do not interfere in this assay.
To determine HbA1c, a hemolysate must be prepared for each sample: 2. It has been reported that results may be inconsistent in patients who have
1.Dispense 1 mL Hemolysis Reagent into tubes labeled: Calibrator, the following conditions: opiate addiction, lead-poisoning, alcoholism,
Control, Patients, etc. Note: Plastic or glass tubes of appropriate size are ingest large doses of aspirin.
acceptable. 3. It has been reported that elevated levels of HbF may lead to
2. Place 20 µL of well mixed whole blood into the appropriately labeled underestimation of HA1c and, that uremia does not interfere with HbAB1c
determination by immunoassay.10 It has been reported that labile
lyse reagent tube. Mix. intermediates (Schiff base) are not detected and therefore, do not interfere

Bio Research For Medical Diagnostics

MDSS GmbH
Muslim Al Attar Street,P.O.Box:1235, Doc.No.: IFU-TL-005
Schiffgraben 41
Amman-11953,Jordan Rev.: 01
30175 Hannover, Germany Page 1 of 2
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 Glycated Hemoglobin
Latex Turbidimetry (HbA1c)

with HbAB1c determination by immunoassay.

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.

*NOTES
1. In order to avoid contamination, it is recommended to use disposable
material.
2. Use clean disposable pipette for its dispensation.

SYMBOL ON LABELS

REFRENCES
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)

Bio Research For Medical Diagnostics

MDSS GmbH
Muslim Al Attar Street,P.O.Box:1235, Doc.No.: IFU-TL-005
Schiffgraben 41
Amman-11953,Jordan Rev.: 01
30175 Hannover, Germany Page 2 of 2
Tel:+962 64892525, Fax: +962 64892526,
www.bioresearch.com.jo