TRUEchemie HbA1c Test Kit

(Immunoturbidimetric Assay)
for the direct quantitative determination of Hemoglobin A1c (HbA1c) in whole blood 2

INTENDED USE WARNINGS AND PRECAUTIONS

The TRUEchemie HbA1c liquid reagent test kit is used for the direct quantitative 1. For in vitro diagnostic use.
determination of percent HbA1c (hemoglobin fraction) in human whole blood. 2. Specimens should be considered infectious and handled appropriately.
3. Avoid ingestion. DO NOT PIPETTE BY MOUTH.
INTRODUCTION 4.Volume of reagents and samples can be adjusted as per the instrument but the ratio of the
Normal levels of glucose produce a normal amount of glycated hemoglobin. As the average reagent be the same
5. If the reagents became turbid or the absorbance of blank reagent is higher than 1.000, it
amount of plasma glucose increases, the fraction of glycated hemoglobin increases in a
means that the reagent is invalid and you should discard it.
predictable way. The test is limited to a three-month average because the lifespan of a red 6. The disposal of the residues has to be done as per local legal regulations.
blood cell is four months (120 days). However, since RBCs do not all undergo lysis at the
MATERIALS REQUIRED BUT NOT PROVIDED
same time, HbA1c is taken as a limited measure of 3 months. In diabetes mellitus, higher
amounts of glycated hemoglobin, indicating poorer control of blood glucose levels, have been 1. Pipettes to accurately measure required volumes.
2. Test tubes/rack
associated with cardiovascular disease, nephropathy, neuropathy, and retinopathy. 3. Timer
4. 37 °C heating block or water bath
PRINCIPLE
5. Photometer capable of accurately measuring absorbance at 340 nm
The percentage of HbA1c in whole blood can be directly determined by utilizing the interaction
of antigen and antibody. Total hemoglobin and HbA1c have the same unspecific absorption TEST PROCEDURE FOR PREPARATION OF CALIBRATION CURVE
rate to Latex particles. When we add mouse antihuman HbA1c monoclonal antibody
(containing Multiple subunits), “Latex--HbA1c--mouse antihuman HbA1c monoclonal Wavelength : 660 nm
Temperature : 37 °C
antibody--HbA1c--Latex” complex is formed. The amount of this complex is proportional to
Prewarm the Reagent to reaction temperature.
the amount of HbA1c absorbed on to the surface of latex particles. The amount of
agglutination is measured as absorbance. The HbA1c value is obtained from a calibration
curve. Reagent Cal -1 Cal -2 Cal -3 Cal -4 Cal -5
HbA1c Reagent-1 (R1) (ml) 0.300 0.300 0.300 0.300 0.300
PACK SIZE Calibrator (ml) 0.008 0.008 0.008 0.008 0.008
Kit Size 20 ml (1x20 ml) 40 ml (2x20 ml) 20 ml (1x20 ml) 40 ml (2x20 ml) 5 x 1 ml Mix well and incubate for 5 min at 37 ◦C
Cat No. ADX941 ADX942 ADX943 ADX944 ADX945 HbA1c Reagent-2 (R2) (ml) 0.100 0.100 0.100 0.100 0.100
Kit Contents
1) HbA1C Reagent-1 (R1) 1 x 15 ml 1 x 30 ml 1 x 15 ml 1 x 30 ml -- Mix well, incubate 5 min. at 37 °C and read the absorbance of blank, calibrators at 660 nm.
2) HbA1C Reagent 2 (R2) 1 x 5 ml 1 x 10 ml 1 x 5 ml 1 x 10 ml --
3) HbA1C Reagent-3 (R3) 1 x 25 ml 1 x 50 ml 1 x 25 ml 1 x 50 ml --
Calculations :
4) HbA1C Calibrators 5 x 1 ml 5 x 1 ml -- -- 5 x 1 ml

Δ O.D of Calibrator = O.D Calibrator-O.D Blank.

REAGENTS COMPOSITION Plot the Δ O.D of each calibrator versus assigned concentration (HbA1c%) on a non-linear
graph.

Reagent 1 (R1) Latex Reagent: Latex 0.13%, Buffer, stabilizer Sodium azide (0.95 g/L)
TEST PROCEDURE FOR PREPARATION OF SPECIMEN
Reagent 2 (R2) HbA1c Antibody: Buffer, Mouse anti-human HbA1c monoclonal antibody:
0.05 mg/mL, goat anti-mouse IgG polyclonal antibody 0.08 mg/dL., Stabilizers Wavelength : 660 nm
Temperature : 37 °C
Reagent 3 (R3) Hemolysing reagent: Water and stabilizers. Prewarm the Reagent to reaction temperature.

STORAGE AND STABILITY Blank (ml) Sample (ml)

HbA1c Reagent-1 (R1) 0.300 0.300
All the kit compounds are stable until the expiry date stated on the label. Do not use reagents
Sample -- 0.008
over the expiration date. Store the Bottle tightly closed, protected from light and prevent
contaminations during the use. Distilled Water 0.008 --
Calibrator -- --
Calibrators: Reconstituted calibrators are stable for 20 days at 2-8℃. Mix well and incubate for 5 min at 37 ◦C
HbA1c Reagent-2 (R2) 0.100 0.100
Mix well, incubate 5 min. at 37 ◦C and read the absorbance of blank, samples at 660 nm.
SAMPLE STORAGE and STABILITY
Calculations:
Whole Blood samples and Hemolysed samples (lysate) are stable for 5 days at 2-8℃.
Δ O.D of Sample = O.D Sample - O.D Blank HbA1c % in the sample is calculated by
SAMPLE PREPARATION interpolation of OD of sample on the calibration curve.

1. Only below samples were tested and found suitable for use:
The TRUEchemie HbA1c assay is traceable to the International Federation of Clinical
Dipotassium EDTA(K2-EDTA) Chemistry (IFCC) reference standard.
Lithium Heparin(Li-Heparin) The default result unit for the assay is % HbA1c. For alternative units, manual calculations
can be used according to below equations:
2. Do not use samples that are heat-inactivated, pooled or with obvious microbial
contamination.
NGSP % HbA1c to IFCC mmol/mol: ［%HbA1c x 10.93］-23.50
3. Fresh/Non-Frozen samples: Do not centrifuge fresh/Non-frozen samples. Samples must IFCC mmol/mol to NGSP %HbA1c: mmol/mol x 0.09148］+2.152
be mixed thoroughly prior to use.
HbA1c results are calculated from a spline data reduction method to generate a calibration
4. Frozen samples: a. Thaw the samples for a minimum of 30 minutes. b. Mix thawed curve.
samples thoroughly by inverting the vials until they are visibly homogeneous. c. To ensure QUALITY CONTROLS
consistency in results, frozen and thawed samples must be transferred to a centrifuge tube
and centrifuge at >10,000 RCF (Relative Centrifuge Force) for 5 minutes before testing. Control Sera are recommended to monitor the performance of manual and automated assay
5.HEMOLYSATE PREPARATION: procedures. Each laboratory should establish its own Quality Control scheme and corrective
To determine the level of HbA1c, a hemolysate must be prepared for each sample: actions if controls do not meet the acceptable tolerances.

a. Dispense 0.500 ml (500 µl) of Hemolyzing Solution (HbA1c Reagent -3 (R3)) into a tube. NORMAL VALUES
b. Add 0.010 ml (10 µl) of well mixed whole blood sample.
c. Mix thoroughly by gentle vertexing for 30 seconds HbA1c Value Glycaemic Goal
d. Stand for 30 minutes at room temperature until complete lysis. < 8 % HbA1c Less Stringent
e. Take 0.008 ml (8 µl) of above lysate for testing. < 7 % HbA1c General (Non-Pregnant Adults)
< 6.5 % HbA1c More Stringent
CALIBRATOR PREPARATION
It is strongly recommended that glycemic goals are individualized following current
1. Reconstitute the calibrator with 1ml DI water. professional society recommendations
2. Mix thoroughly by gentle vertexing for 30 seconds. AUTOMATED PROCEDURE
3. Stand for 30 minutes at room temperature and protect from light. Meanwhile, shake and
flip over the brown bottle gently to ensure the content is fully dissolved. Avoid air bubbles. Appropriate Program sheet is available for different analyzers upon request.
4.Take 0.008 ml (8 µl) of reconstituted calibrator for testing.
5. Reconstituted calibrators are stable for 20 days at 2-8 ℃ and 30 days at -20 ℃.
 TRUEchemie HemoglobinA1c (HbA1c) Test Kit
8
(Immunoturbidimetric Assay)
2
for the direct quantitative determination of Hemoglobin A1c (HbA1c) in whole blood 2 2

CALIBRATION

When using TRUEchemie HbA1c calibrators to do the multi-point calibration, the calibration
cycle is 14 days. Re-calibration is required when changing reagent batch. It is recommended
to use the normal & pathological quality control as internal quality control. Corrective action
shall be done if controls do not recover within the acceptable tolerance.

LIMITATIONS

Linearity: upto 18.0 %

Sensitivity: 2%

INTERFERENCES

The assay is not affected by the following interfering substances at the indicated
concentrations:
Bilirubin up to 688 µ mol/L no interference effect
Triglyceride up to 11.3 mmol/L no interference effect
Ascorbic acid up to 0.5 g/L no interference effect

SYSTEMS PARAMETERS

Mode : Endpoint
Factor : --
Primary Wave length : 660 nm
Linearity : 2-18 %
Units : %
Flow cell Temp : 37 °C
Blank : Reagent
Calibration type : Multi Standard Calibration
Reagent volume : 0.300 ml (R1) + 0.100 ml (R2)
Sample volume : 0.008 ml
Incubation : 5 + 5 min.
Low Normal : 3%
High Normal : 5.8 %

REFERENCES

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)

Consult instructions for use Catalogue number

Use-by date
For in vitro diagnostic Batch code
use only Do not use if
8 package is
Temperature limit 2-8 °C Date of manufacture
2 damaged
Keep dry
Keep away from sunlight Manufacturer

Effective date: 23.07.2018 Rev. B

English version

+91-44-22541131

See instructions for

use
For in vitro
diagnostic use only

Use by

Store between 2-8°C

Manufacturer

Date of manufacture