Direct Enzymatic HbA1c Methods

How many HbA1c testing methods are available to clinical laboratories?

There are 3 major HbA1c testing methods currently available to clinical laboratories. They are:

  1. Chromatography based HPLC assay
  2. Antibody based immunoassay
  3. Enzyme based enzymatic assay

How do these methods work?

1. Chromatographic method:

The chromatographic assay uses an HPLC instrument and ion exchange or affi nity column to separate HbA1c molecules from other hemoglobin molecules. The HbA1c content is calculated based on the ratio of HbA1c peak area to the total hemoglobin peak areas as shown in the figure below:

2. Latex enhanced Immunoassay method:

The latex enhanced immunoassay for HbA1c is based on interactions between antigen molecules (HbA1c) and HbA1c specifi c antibodies coated on latex beads. This cross-link reaction results in changes in the solution turbidity which is proportional to the amount of the antigen in the samples as depicted in the scheme below:

3. Enzymatic HbA1c assay method:

Recent innovation has yielded a Direct Enzymatic HbA1c AssayTM which uses a single channel test and reports %HbA1c values directly, without the need for a separate THb test or a calculation step.

Assay Principle

Oxidizing agents in the lysis buffer react with the whole blood sample to eliminate low molecular weight and high molecular weight signal interfering substances. After lysis, the whole blood samples are subjected to extensive proteolytic digestion. This process releases amino acids, including glycated valines, from the hemoglobin beta chains. The Direct Enzymatic HbA1c Assay™ glycated valines serve as substrates for a specific recombinant fructosyl valine oxidase (FVO) enzyme. The recombinant FVO specifically cleaves N-terminal valines and produces hydrogen peroxide in the presence of selective agents. This, in turn, is measured using a horseradish peroxidase (POD) catalyzed reaction and a suitable chromagen. The signal produced in the reaction is used to directly report the %HbA1c of the sample using a suitable linear calibration curve expressed in %HbA1c. The Direct Enzymatic HbA1c Assay principle is depicted in Figure 1 below.


(Figure 1)

Assay Advantages The Direct Enzymatic HbA1c Assay™ has all the advantages of both the HPLC and immunoassays methods in the areas of accuracy, specificity, applicability to chemistry analyzers and yet is cost effective, simpler and has less interferences. The direct enzymatic HbA1c test uses two ready-to-use liquid stable reagents. Since it does not require a separate measurement of total hemoglobin content in the samples, the Direct Enzymatic HbA1c Assay™ only needs a single channel to perform the test on chemistry analyzers in comparison with some immunoassays that require a separate measurement of total hemoglobin and need two channels for the test on chemistry analyzers as shown in the Figure 2 below.


(Figure 2)

The Direct Enzymatic HbA1c Assay™ procedure is simple and straight forward. After addition of Reagent R1, sample and Reagent R2, the result of %HbA1c will be reported within 2 min as shown in the Figure 3 below.


(Figure3)

In addition, the reagents do not contain latex particles, and hence do not coat analyzer cuvettes and lines. Most importantly, enzymatic HbA1c assays have the highest specificity among all HbA1c assays as shown in the Tables 1 and 2.

Modification Methods Affected Methods Unaffected
Carbamyl Hb HPLC, up to 300% errors Enzymatic HbA1c assay Immunoassay
Labile fraction Some HPLC and some Immunoassays Enzymatic HbA1c assay
Table 1. Effect of Chemically Modified Hemoglobin Variants

Variant Methods Affected Methods Unaffected
Hb C Immunoassay, HPLC assay, up to 40% errors Enzymatic HbA1c assay
HbS Immunoassay, HPLC assay, up to 40% errors Enzymatic HbA1c assay (<10% error
Hb Graz Immunoassay, HPLC assay, up to 30% errors Enzymatic HbA1c assay
Hb Sherwood Forest Immunoassay, HPLC assay, up to 30% errors Enzymatic HbA1c assay
Hb D Immunoassay, HPLC assay, up to 30% errors Enzymatic HbA1c assay
Hb O padova Immunoassay, HPLC assay, up to 30% errors Enzymatic HbA1c assay
Table 2. Effects of Genetic Hemoglobin Variants

As shown in Tables 1 and 2, enzymatic HbA1c assays are not interfered by either chemical or genetically modified hemoglobin variants. Therefore, enzymatic Hb1c tests are reliable tests, and it does not report false results regardless of the patient's hemoglobin variant types. In summary, the Direct Enzymatic HbA1c Assay™ offers the following advantages over HPLC and Immunoassays:

  • Two reagents, liquid stable
  • No need for total hemoglobin measurement
  • Single channel on analyzers Faster, simpler and more cost effective
  • No interferences from hemoglobin variants
  • On-board blood lysis possible
  • Applicable to most analyzers
  • Excellent correlation with HPLC and immunoassays

Performance Characteristics

Below is a brief summary of the performance characteristics of the Direct Enzymatic HbA1c Assay™.

Assay Precision

  Level 1 (% HbA1c) Level 2 (% HbA1c)
N 20 20
Mean 5.7% 10.3%
SD 0.06 0.07
CV% 1.0 0.7
Table 3. CV% of Intra Precision

  Level 1 (% HbA1c) Level 2 (% HbA1c)
N 80 80
Mean 5.7% 10.3%
SD 0.10 0.18
CV% 1.8 1.8
Table 4. CV% of Inter Precision

Correlation with HPLC and Immunoassays:

The Direct Enzymatic HbA1c Assay™ has been compared with Tosoh's HPLC assay and Roche's immunoassay with 100 of random patient blood samples. As shown in the figures below, the HbA1c results obtained by Direct Enzymatic HbA1c assay exhibited excellent correlation with those obtained by HPLC method and immunoassay method with correlation coefficient r2 values ranging from 0.96-0.98 (Figures 4 and 5).


(Figure 4)


(Figure 5)

The Direct Enzymatic HbA1c Assay™ has an assay linearity range from 4 to 16% of HbA1c as shown in Table 5 and Figure 6.

Expected %HbA1c Recovered %HbA1c
4.0% 3.9%
7.0% 7.2%
10.0% 10.2%
13.0% 13.2%
16.0% 16.2%
21.9% 21.6%
Table 5. Assay Linearity of Direct Enzymatic HbA1c Assay™


(Figure 6)

Interferences

Direct Enzymatic HbA1c testing is not adversely affected by HbS, C and E variants in samples. For this study, variants in samples were confirmed by Primus HPLC analysis at the Diabetes Diagnostic lab (University of Missouri). These frozen samples were tested with Direct Enzymatic HbA1c Assay™ and results were compared with those obtained by HPLC and immunoassay methods as shown in Figures 7 and 8.


(Figure 7)

 


(Figure 8)

However, several samples were identified to be incorrectly value reported by the TOSOH HPLC method. These samples (shown in table below) were accurately tested with the Direct Enzymatic HbA1c Assay™ reagents and by the Roche Tinaquant II assay reagents.

Sample TOSOH Test Roche Tinaquant II Direct Enzymatic
10924315 13.4% 6.4% 6.9%
10923294 10.8% 6.4% 6.5%

In summary, the Direct Enzymatic HbA1c Assay™ is not only simple in assay procedure but also more accurate in assay results. Therefore, the Direct Enzymatic HbA1c Assay™ is a better assay over the immunoassay and the HPLC assay, and is highly recommended for use in all clinical laboratories. Application parameters for most of chemistry analyzers are available.

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