Direct HbA1c (Enzymatic, On-Board Lysis) Assay

Diazyme's Direct HbA1c (Enzymatic, On-Board Lysis) Assay

Assay Principle:

Diazyme Direct HbA1c Assay (Enzymatic, On-Board Lysis) test kit is intended for use in the quantitative determination of stable HbA1c in venous whole blood samples with on-board blood lysis application in a clinical laboratory. This test is not to be used to diagnose or screen for diabetes. The measurement of HbA1c concentration is for use in monitoring long-term glucose control of persons with diabetes. For in-vitro diagnostic use only.

Diazyme Direct HbA1c (Enzymatic, On-Board Lysis) Assay Kit Configuration:

Diazyme Direct HbA1c Assay (Enzymatic, On-Board Lysis) is an enzymatic test in which lysed whole blood samples are subjected to extensive protease digestion with Bacillus sp protease. The Direct HbA1c Assay (Enzymatic, On-Board Lysis) should be measured according to specific application parameters for specific chemistry analyzers. The assay kit consists of two liquid stable reagents; R1, and R2, and the Direct HbA1c (Enzymatic, On-Board Lysis) Assay calibrators and controls are packaged separately.

Assay Features:

  • NGSP certified and traceable to the Diabetes Control and Complication Trial Reference (DCCT) Method
  • Single channel assay eliminates the need for a dedicated channel for total hemoglobin measurement
  • On-board lysis allows for a faster, more efficient process
  • Fully enzymatic, no latex particle residue to cloud cuvettes
  • Virtually eliminates interference from hemoglobin variants
  • Directly measures glycated hemoglobin and is resistant to interference from post transcript modifications
  • Liquid stable reagent requires no reagent preparation, saving time and reducing sample handling
  • Cost effective test with multiple parameter options available

Assay Procedure:

Diazyme Direct HbA1c Assay (Enzymatic, On-Board Lysis) should be measured according to specific application parameters for specific chemistry analyzers. The figure below is a general example of the assay test scheme:

Assay Accuracy:

A method Comparison study was performed according to the CLSI: EP9-A2 guideline using one lot of the reagents at three different sites on three different Modular P analyzers by three different operators. A total of 374 unique K2 EDTA whole blood samples were tested. The HbA1c values were compared with that obtained by a commercial HPLC method. The comparison results are summarized in the following table.

Parameter

Linear Regression (total of 3 sites)

Deming Regression (total of 3 sites)

Slope

1.023

1.030

95% CI

1.011 – 1.035

1.018 – 1.042

Intercept

-0.090

-0.140

95% CI

-0.180 to 0.000

-0.230 to -0.050

Cor Coeff (R)

0.9937

0.9937

Sample Range

4.2 – 12.0

4.2 – 12.0

Assay Precisions:
The precision of the Diazyme Direct HbA1c Assay (Enzymatic, On-Board Lysis) was evaluated according to CLSI EP5-A2 guideline. In the study, 5 whole blood samples were tested in duplicates per run, 2 runs per day over 20 working days with three lots of reagents. The results of the within-run, between-run, between-day, between-lot, and total CV% for three lots of the reagents combined are listed in the following table (N =240).

 

Sample

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Mean (%)

4.64

5.36

7.51

9.61

11.89

Within-Run

SD

0.04

0.05

0.05

0.06

0.09

%CV

0.8%

0.9%

0.6%

0.6%

0.7%

Between-Run

SD

0.07

0.05

0.05

0.05

0.08

%CV

1.5%

0.9%

0.7%

0.5%

0.6%

Between-Day

SD

0.00

0.00

0.00

0.03

0.04

%CV

0.0%

0.0%

0.0%

0.3%

0.4%

Between-Lot

SD

0.08

0.07

0.07

0.08

0.12

%CV

1.6%

1.2%

0.9%

0.9%

1.0%

Total

SD

0.08

0.07

0.07

0.08

0.12

%CV

1.7%

1.2%

0.9%

0.9%

1.0%

 

Multi-site precision study was performed at Diazyme Laboratories and two external sites on Modular P analyzers. In this study, the same set of 5 whole blood samples were tested in duplicates per run, 2 runs per day for 5 working days with one lot of reagents at three different testing sites, by three different operators on three different Modular P analyzers. The results of the within-run, between-run, between-day, between-site, and total CV% for the three sites combined are listed in the following tables (N =60):

Sample

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Mean (%)

4.67

5.37

7.52

9.67

11.92

Within-Run

SD

0.05

0.04

0.05

0.07

0.09

%CV

1.0%

0.8%

0.7%

0.8%

0.8%

Between-Run

SD

0.04

0.05

0.06

0.11

0.09

%CV

0.8%

1.0%

0.8%

1.1%

0.8%

Between-Day

SD

0.02

0.00

0.00

0.00

0.06

%CV

0.5%

0.0%

0.0%

0.0%

0.5%

Between-Site

SD

0.07

0.06

0.07

0.12

0.14

%CV

1.4%

1.2%

0.9%

1.3%

1.2%

Total

SD

0.07

0.07

0.08

0.13

0.14

%CV

1.4%

1.2%

1.0%

1.4%

1.2%

Assay Linearity:
CLSI protocol EP6-A was used to evaluate assay linearity. Diazyme Direct HbA1c Assay (Enzymatic, On-Board Lysis) has an AMR range of 4.0% - 12.0% HbA1c.

Assay Sensitivity:
The LOB, LOD, LOQ of the Diazyme Direct HbA1c Assay (Enzymatic, On-Board Lysis) was determined according to CLSI EP17-A2. The LOB was determined to be 0.2%; the LOD was determined to be 0.5%; the LOQ was determined to be 0.8%.

Interference:
Interference was evaluated according to CLSI protocol EP7-A. The following substances showed no significant interference (<10%) up to the concentrations in the table below:

Interfering Substances

Concentration

Ascorbic Acid

12 mg/dL

Bilirubin

15 mg/dL

Bilirubin Conjugated

13 mg/dL

Hemoglobin

21 g/dL

Triglycerides

4000 mg/dL

Glucose

4000 mg/dL

Uric Acid

30 mg/dL

Urea

80 mg/dL

Acetaminophen

20 mg/dL

Acetylsalicylic Acid

65.2 mg/dL

Metformin

4 mg/dL

Ibuprofen

50 mg/dL

Glyburide

0.19 mg/dL

Total Protein

21 g/dL

Vitamin E

13.6 mg/dL

Rheumatoid Factor

375 IU/mL

Chemistry Analyzers for Diazyme Direct HbA1c Assay (Enzymatic, On-Board Lysis) including validated and proposed (pending validation) applications are listed below but are not limited to:

  • Roche Hitachi, Modular P and Cobas Series
  • Beckman AU
  • Abbott Architect

For a full list of validated and proposed (pending validation) applications please click the following link:
http://www.diazyme.com/validated-analyzer-parameters