Hemoglobin A1c Assay Kit （Fructosyl Oxidase Assay）

Hemoglobin A1c (HbA1c) has been regarded as the gold standard for measuring diabetes control by American Diabetes Association (ADA). It is a good indicator of average blood glucose level over 2 to 3 months. The HbA1c kit is used for in vitro test for the quantitative determination of HbA1c concentration in human whole blood on photometric systems.

Principle:

Under the action of protease, the n-terminal of β chain in HbA1c is cut off and glycosylated dipeptides are released. In the first reaction, Hb concentration can be obtained by measuring the absorbance of 480 nm. In the second reaction, fructosyl peptide oxidase (FPOX) acts on glycosylated dipeptides to generate hydrogen peroxide which can react with chromogenic agents to generate a absorbance at 660nm in the presence of peroxidase, then the concentration of HbA1c can be obtained by measuring the absorbance of 660nm. According to the obtained HbA1c concentration and Hb concentration, the percentage of HbA1c(HbA1c%) can be calculated.

Reagents:

sample treatment

Take 25 μL whole blood sample or red blood cells (centrifuged from whole blood sample, 2000rpm, 5min) into 500 μL sample solution. Mix the sample and then measure it on the automatic chemistry analyzer.

Method

1. Reagent preparation：Liquid reagent can be used when opened.

2. Measurement：

⑴Two items measured separately

1 Detection of hemoglobin

ABS Ⅰ ： Difference of absorbance between 480 nm and 800nm, and difference of absorbance between 660 nm and 800nm.

ABS Ⅱ ： Difference of absorbance between 660nm and 800nm.

Notes：

The apparatus must meet the following conditions:

①Different test items can correspond to the same reagent site.

② The absorbance at different wavelengths of the same reaction cup can be measured simultaneously

Calculation

The results of HbA1c depend on the concentration of hemoglobin and glycated hemoglobin, can be expressed directly by HbA1c%. In order to match the standardized value of HbA1c recommended by different organizations, the inter-project calculation formula of automatic analyzer should be used:

1. Standard method of Japan diabetes association: HbA1c=96.3*HbA1c(μmol/L)/Hb(μmol/L)+1.62

2. Method of National glycosylated hemoglobin standardization program/method of diabetes control and complications study (NGSP/DCCT):

HbA1c=91.5*HbA1c(μmol/L)/Hb(μmol/L)+2.15

3. Method recommended by IFCC: HbA1c=HbA1c(μmol/L)/Hb(μmol/L)*100

Storage and stability

12 months from manufacturation when stored at 2-8℃ in the dark environment and sealed package. Once opened, the reagents are stable for 28 days when refrigerated on the analyzer or refrigerator. Contamination of the reagents must be avoided. Do not freeze the reagents.

Reference Interval

≤6.5% Determination method: No less than 100 whole blood samples of normal population were selected in clinical trials and measured by automatic chemistry analyzer. The measured values were processed by statistical methods and the reference interval was calculated. It is recommended that each laboratory establishes its own reference interval !

Limitations

No obvious interference is observed when bilirubin ≤ 15mg/dL, Intrilipid ≤ 200 mg/dL, ascorbic acid ≤ 50 mg/dL or uric acid ≤ 5 mg/dL in the sample, and the result is calculated comparing to the concentration in the untreatedsample.

Interpretation

Human error, sample processing and deviation of analytical instruments can all affect the results. When individual results deviate too far from the expected value, re-determination is required.

Performance index

1. Reagent blank absorbance: When using pure water as sample, the blank absorbance of reagents is no more than0.100.

2. Repeat-ability and lot-to-lot variation: CV shall notexceed 5%; relative range R between batches shall notexceed 10%.

3. Accuracy: test the quality control for 3 times, the relative deviation between the mean value and the target value shall no more than ±10%.
   
   4. Linear range:

   (1) HB: [90.0, 300.0] μmol/L, the linear correlation coefficient R ≥ 0.990, and the linear relative deviation shall not exceed ± 10%.

   (2) HbA1c: [3.00, 16.00] μmol/L, the linear correlation coefficient R ≥ 0.990;

   [3.00, 5.00] μmol/L, the absolute deviation of linearity shall not exceed ± 0.50 μmol/L； (5.00, 16.00] μmol/L, the relative deviation of linearity shall not exceed ± 10%.

   Sensitivity

   1. Hb: the absorbance difference (∆A) of 100 μmol/L hemoglobin sample shall be 0.100 ~0.300.
   2. HbA1c: the absorbance difference (∆A) of 10 μmol/L HbA1c sample shall be 0.050 ~0.100.

   Sensitivity

   1. Hb: the absorbance difference (∆A) of 100 μmol/L hemoglobin sample shall be 0.100 ~0.300.

   Attention

   Reagents with different batch numbers cannot be mixed. Please calibrate again when changing the reagent batch number!

   References

   [1] Liu Limin,Hood Stefanie,Wang Yuping,Bezverkov Robert,Dou Chao,Datta Abhijit,Yuan Chong. Direct enzymatic assay for %HbA1c in human whole blood samples.[J]. Clinical Biochemistry, 2008, 41: 576-583. [2] Teodoro Morrison Tracy, Janssen Marcel J W et,al. Evaluation of a next generation direct whole blood enzymatic assay for hemoglobin A1c on the ARCHITECT c8000 chemistry system. [J]. Clinical chemistry and laboratory medicine, 2015, 531. [3] Kozo Hirokawa,Kazuhiko Shimoji,Naoki Kajiyama. An enzymatic method for the determination of hemoglobinA1C [J]. Biotechnology Letters, 2005, 27: 963-968.

In vitro test for the quantitative determination of HbA1c concentration in human whole blood on photometric systems. HbA1c is a product of hemoglobin (Hb) that produces a slow and continuous non-enzymatic glycation reaction under high blood glucose. Glucose modifies hemoglobin specifically in its n-terminal valine residue to form glycated hemoglobin. Under normal physiological conditions, the production of non-enzymatic glycosylation reaction products is positively proportional to the concentration of reactants. Since hemoglobin concentration remains relatively stable, glycosylation levels are mainly dependent on glucose concentration and are also related to the length of hemoglobin and glucose exposure. Therefore, HbA1c is a good indicator to the average blood glucose level of patients for the last 2~3 months.