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Approved
Prepared by Reviewed by
by

Preparation Approval
Review Date
Date Date

Standard Operating Procedure for Detection of HbA1c

1 Purpose
This test is an enzyme-linked immunosorbent assay (ELISA) for the determination of
HbA1c in anticoagulated whole blood. It is of great reference value for the diagnosis,
observation of the changes of diseases and the treatment of diseases.

2 Method& Reaction Principle

2.1 Method
Enzymatic Assay Method
2.2 Reaction Principle
In the first reaction, the concentration of hemoglobin is measured at absorbance of fixed
wavelength, and simultaneously the fructosyl dipeptides are generated from the N-terminus
amino groups of the beta-chain of HbA1c by the reaction of protease. In the second reaction, the
reaction of Fructosyl peptide oxidase (FPOX) with fructosyl dipeptides, generated hydroperoxide
allows 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino) phenothiazine sodium salt to
develop a color in the presence of peroxidase. The change in absorbance is measured for HbA1c
determination. The combined assay results for hemoglobin and HbA1c are used by the system to
calculate and express HbA1c%.

3 Performance characteristics
3.1 Linearity Range and Analytical Specificity

Instrument
BS series chemistry analyzers
Performance Analytical Specificity (mg/dL) (the influence of the
Precision (%) following concentrations of interferents on the results
Chemistry Linearity should not exceed ±10%)
Range
Ascorbic
Within-run Batch-to-batch Glucose Bilirubin Lipemia
acid
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3%～
HbA1c ≤5.0 ≤10.0 30 1000 50 2000
16%
3.2 Precision

Precision performance using the CLSI Approved Guideline EP5-A2 to assay serum control

appears in the table below. U: %

TYPE OF LEVEL II LEVEL III

IMPRECISION Mean SD CV% Mean SD CV%

WITHIN-RUN 0.141 3.194 0.133 1.434

BETWEEN-RUN 0.085 1.923 0.092 0.982

4.43 9.32
BETWEEN-DAY 0.031 0.705 0.023 0.245

WITHIN-DEVICE 0.168 3.794 0.164 1.755

3.3 Comparison

A comparison between Mindray System (Mindray BS series analyzers/Mindray HbA1c

Reagent) (y) and Hitachi/SEKISUI System (Hitachi /SEKISUI HbA1c) (x) using 40 samples

gave following correlation(%):y=0.9975x-0.0455，R2=0.9925,

Details of the comparison experiments are available on request.

4 Specimen collection and preparation

K2-EDTA, K3-EDTA, NaF-EDTA, lithium heparin and sodium heparin whole blood are
suitable for samples.
1) Use the suitable tubes or collection containers and follow the instruction of the
manufacturer, avoid effect of the materials of the tubes or other collection containers.
2) Do not centrifuge samples.
On-board lysis
1) Mix all specimens thoroughly by low speed vortexing or gently inverting 10 times prior
to loading onto the Mindray BS series System.
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2) The proportion of whole blood sample and Pretreatment Solution is 10ul:200ul.
Note: Warning: For the anticoagulation tubes, the height of total sample in the tube
should be into the range of 10mm-55mm. If out of the range, adjust to the height of
sample or change to the manual lysis.
Manual lysis
1) Mix all specimens thoroughly by low speed vortexing or gently inverting 10 times.
2) Get 10 μL whole blood into a sample cup or an Eppendorf microfuge tube. The whole
blood is easily adsorbed to the sample probe and pipette, which may cause exceed the
target volume.
3) Add 200 μL of Pretreatment Solution into test tube.
4) Close the test tube and lysis the blood by inverting 10 times or shaking for 10s on the
vortex mixer.
5) The hemolysate can be used as working samples after 4 minutes.
6) The volume of whole blood and Pretreatment Solution can be amplified in equal
proportion if necessary.
7) Specimens should be tested as soon as possible after pre-analytical treatment.

Whole blood Stability: 8 hours at 15-25°C

7 days at 2-8°C
30 days at (-25)-(-15) °C
Hemolysate Stability :8 hours at 15-25℃ 24 hours at 2–8℃

5 Test Requirements
5.1 Instrument: Mindray BS series biochemical analyzer.
5.2 Reagent: The original reagent is used. Do not mix different batches of reagent kit. The
unopened reagent kit is stored in the 2℃~8℃ for a period of eighteen months. After being
opened, reagent should be kept away from light and stable for 28 days at 2℃~8℃. Reagent
Do not freeze. The opened reagent should be used up as soon as possible. The expired
reagent cannot be used.
5.3 Environmental requirements (Operating environment requirements of the detection system):
Temperature: 15℃~30℃;
Humidity: 35 %RH~85 %RH, without condensation.
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5.4 Safety control: Take necessary precautions when using the monitor. For example, wear
protective suit and gloves. reagent Do not swallow the monitor and avoid contact with the
skin and mucous membrane. reagent and the corresponding calibrators and quality control
products have been tested and confirmed to be negative for HbsAg, HIV and TP antibody of
Treponema pallidum. For caution, please treat them as infectious dangerous articles during
use. Pay full attention to them during operation. Dispose of all samples and waste in
accordance with national and local laws and regulations.

6 Calibration
6.1 Calibrator: Use the manufacturer's matching calibrator (the calibrator is dissolved in 1mL
double distilled water or deionized water respectively, and there is no need to process the
sample. The calibrator should be taken immediately, and used immediately after sampling.
Do not put the used sample back into the calibrator bottle.)
1) Take out the 2~8℃ storage environment, prepare a proper amount of double
distilled water or deionized water, and balance the water to room temperature;
2) Tap the cap gently to ensure that the lyophilized sample is scattered at the
bottom of the bottle. Carefully open the cap and rubber stopper to avoid loss of
the content.
3) Weigh 1 mL of distilled water or deionized water, inject it into the bottle slowly,
cover the rubber stopper, and invert the bottle gently for several seconds. Keep
the bottle away from light for 30 minutes.
4) Gently invert the vial to ensure that the contents are completely dissolved to
avoid foam.
5) After redissolution, select appropriate centrifugal tubes or sample cups for
dispensing and marking;
6) Refer to the Standard Operating Procedure for Mindray Biochemistry Analyzer
and use the reference values in the calibrator reference value table to set the
calibration values and calibrate the analyzer.
Unopened calibrators are stored at 2~8 ℃ for 18 months in a dark place. After
reconstitution, keep the solution in a dark place under 2~8℃ conditions. The solution can be
stable for 15 days.
Calibrator type:
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S1: Mindray calibrators.
S2: Mindray calibrators.
6.2 Calibration method: It is recommended to use the calibrator in kits for two-point calibration.
For details, see《Standard Operating Procedure for Mindray Biochemistry Analyzer》.
6.3 Calibration period and requirements (including but not limited to the following):
1) When it is used for the first time;
2) Replace the reagent lot number.
3) When replacing key parts;
4) When the indoor QC is out of control.
6.4 Traceability: See the traceability file provided by the manufacturer.

7 Procedure
Prepare the analyzer/reagent preparation → sample ID/data entry → QC → analysis QC
results → sample analysis → analysis results → abnormal result review → test results
review → instrument maintenance and use registration → shutdown of the instrument.
For details about the operations, see 《 Standard Operating Procedure of Mindray
Biochemistry Analyzer》. Analyze and test the collected samples in time. STAT samples shall be
tested first, and the results shall be reported within 30 minutes generally. The report time can be
extended for the samples to be retested. Non-STAT samples should be completed within 2 hours
after blood sampling. When reviewing test results, special attention should be paid to evaluating
their compliance with available patient-related clinical information.

8 Quality Control
8.1 Control type: The corresponding commercial control provided by the manufacturer.
8.2 QC measure
8.2.1 QC
Run controls before running samples.
1) Take out the 2~8℃ storage environment, prepare a proper amount of double
distilled water or deionized water, and balance the water to room temperature;
2) Tap the cap gently to ensure that the lyophilized sample is scattered at the bottom of
the bottle. Carefully open the cap and rubber stopper to avoid loss of the content.
3) Weigh 1 mL of distilled water or deionized water, inject it into the bottle slowly,
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cover the rubber stopper, and turn the bottle over for several seconds to keep it
away from light for 30 minutes.
4) Gently invert the vial to ensure that the contents are completely dissolved to avoid
foam.
5) After redissolution, select appropriate centrifugal tubes or sample cups for
dispensing, marking and use (the unopened control can be stored in a dark place
under 2~8℃ conditions for 18 months. After uncapping and redissolution, keep the
solution in a dark place under 2~8℃ conditions. The solution can be stable for 15
days.
6) For details about QC test, see the Standard Operating Procedure of Mindray
Biochemistry Analyzer.
The test results of the control shall be within the reference range; otherwise, the test system
shall be checked according to the quality control procedure, such as the control position, validity
period, storage method, calibration process, performance and status of the instrument, etc.
Record the condition of the instrument on the day of quality control.

9 Principle of Result Calculation Program

Sample Reagent

Reagent color
development
The absorbance principle
changes.
Analysis method

Response

Calibrator
Response
Sample
concentration

10 Calculation
The analyzer calculates the HbA1c concentration of each sample automatically after
calibration.

HbA1c% is the most common form in the reports. Report results of hemoglobin A1c in
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mmol/mol (IFCC) or percent (NGSP):

Protocol 1 according to IFCC: HbA1c (mmol/mol)= HbA1c/Hb×1000

Protocol 2 according to NGSP/DCCT: HbA1c(%)= 91.5×HbA1c /Hb +2.15

11 Reference range

Whole blood

According to IFCC 20.20-42.06 mmol/mol

According to NGSP/DCCT 4.0%-6.0%

12 Reportable interval of test results

If the value of sample exceeds 300 μmol/L(Hb) or 39 μmol/L(HbA1c), the sample should
be diluted with Pretreatment Solution (e.g. 1+1) and rerun; the result of Hb and HbA1c should be
multiplied by 2. No conversion factor is required for the mmol/mol HbA1c (IFCC) and %
HbA1c (DCCT/NGSP) result.

13 Warning or Critical Value

For critical values of biochemistry analysis, see 《 Standard Operating Procedure for
Reporting Critical Values in the Biochemistry Laboratory》. In case of a critical value, follow this
SOP.

14 Laboratory clinical interpretation

1) HbA1c is related to the life span of erythrocytes and the average blood glucose level. It
is an ideal indicator for evaluating the long-term blood glucose control of diabetic
patients. It reflects the average blood glucose level in the past 2-3 months and is not
affected by daily blood glucose fluctuation.
2) Closely related to the occurrence of microvascular and macrovascular complications.
The risk of diabetic retinopathy, nephropathy, neuropathy, and cardiovascular events
increases with the increase of HbA1c level.
3) HbA1c has a good predictive ability for the occurrence of diabetes. In 2010, the
American Diabetes Association (ADA) published the diagnosis and treatment of
diabetes as one of the diagnostic criteria for diabetes.
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15 References
1) Prepared by the Japanese Diabetes Society: Diabetes Treatment Guidelines 2006~2007,
p.9 Wen guangtang (2006)
2) National Committee for the Standardization of Glycohemoglobin, Diabetes
37855(1994)
3) Junge W, Wilke B, Halabi A et al. Determination of reference intervals in adults for
hemoglobin A1c (HbA1c). Poster presentation 18th International Diabetes Federation
Congress, Paris, 2003

16 Related Documents
1) Standard Operating Procedure of Mindray Biochemistry Analyzer;
2) Standard Operating Procedure for Laboratory Critical Value Reporting