Evaluation of the Analytical Performance of Five Assays in Measurement of Hemoglobin A1c in Iran

Diabetes mellitus, being one of the most prevalent endocrine disorders, is a major public health concern both in developed and developing countries. The prevalence of diabetes was estimated 12.9% among the population older than 20 in the United States based on a survey carried from 2005 to 2006 (National Health and Nutrition Examination Survey) (1). The prevalence of diabetes is also high in Iran and it is estimated that 7.7% of the adults aged 25 to 64 years old suffer from diabetes, half of them arguably being undiagnosed (2). In another study the prevalence rates of diabetes and prediabetes were 7.04% and 8.58%, respectively (3). Measurement of Hemoglobin A1c (HbA1c) blood levels is considered as a reliable indicator of glycemic control in diabetic patients since 1977 (4). Hemoglobin A1c measurement is currently considered as the main method for monitoring long-term glycemic control in both type 1 and type 2 diabetic patients. Moreover, it is demonstrated that HbA1c blood levels also can predict the risk of developing diabetes micro- and macro-vascular complications (5). Approximately, 100 different HbA1c assays have been invented and are currently in use in different countries. The instruments utilize different technologies, which range from low-throughput research laboratory systems to high-throughput automated systems invented exclusively for the measurement of blood HbA1c levels. Therefore, the HbA1c results reported for a same blood sample can considerably differ unless they are standardized according to a common reference system (6). In response to this need, National Glycohemoglobin Standardization Program (NGSP), which is partly sponsored by the American Diabetes Association, was established in 1996 with the primary goal of standardization of HbA1c assays. Based on the NGSP requirements, manufacturers of A1C testing devices are awarded a certificate of traceability to the Diabetes Control and Complications Trial (DCCT) reference method provided that their assay methods are precise and accurate enough to pass the stringent test administered which needs to be renewed on a yearly basis (7, 8). Notwithstanding the relative success of NGSP, it is reported that the presence of inter method variability among NGSP-certified methods could still decrease the clinical usefulness of HbA1c testing. This can cause even more dramatic results in mismanagement of diabetic patients when different methods are used interchangeably, which may be quite inevitable when patients change health providers and diagnostic laboratories (9).

This study aimed to investigate the compatibility of various HbA1c assays used in Iran, both NGSP-certified and non-NGSP certified.

This cross-sectional study was conducted in diabetes and metabolic clinic of endocrine and metabolic institute of Tehran University of Medical Sciences from October 2013 to December 2013. A total of 45 fresh blood samples from diabetic type 2 patients with different HbA1c levels (4.8 - 12.7%) were collected in sterile tubes containing K₂ Ethylene Diamine Tetraacetic Acid (EDTA). Patients with any kind of hemoglobinopathy were excluded. No further inclusion/exclusion criteria were used. The study was approved by the ethics committee of endocrinology and metabolism research institute and a written informed consent was obtained from all participants.

The samples were tested by five devices within the same day otherwise refrigerated up to two days. The HbA1c assays, which were evaluated in this study included:

1. TOSOH G8 Ion exchange (Tosoh Bioscience, Japan), High performance liquid chromatography (HPLC)

2. NycoCard Reader II (Axis-Shield, Norway), Boronate affinity

3. CERA STAT 2000 (Ceragem Medisys Inc, Korea), Boronate affinity

4. DS5 (Drew Scientific inc, France), Ion exchange chromatography

5. Biosystems (BioSystems S.A, Spain), Ion exchange chromatographic (manual)

6. Pars Azmoon (Pars azmoon, Iran), Immunoturbidometry

TOSOH G8, NycoCard Reader II and CERA STAT 2000 are NGSP-certified assays. TOSOH G8 was considered as the Standard Reference Method (SRM) and comparability of HbA1c results was evaluated by the analysis of TOSOH G8 results.

The Clinical Laboratory Standardization Institute (CLSI) EP 15-A2 and EP-9 A2 protocols were used to investigate assay imprecision and compare the results of the methods (10, 11). Microsoft Excel 2007 and SPSS version 15 packages (SPSS, Chicago, IL, USA) were used to analyze the data. Total Error (TE) was calculated as: bias ± 1.96 SD of differences.

Total imprecision of TOSOH G8, NycoCard Reader II, CERA STAT 2000, DS5 , Biosystems and Pars Azmoon in different levels of HbA1c were less than 0.5%, 3.1% , 3% , 3.3%, 3.4% and 2.3%, respectively. A significant difference between SRM and the mean of each test method was evident (Table 1). The results were classified into three groups (less than 6.5%, 6.5% - 8% and more than 8% of HbA1c) and in each group, the means of the assays, partitioned bias, SD of difference and TE (percent) were calculated (Table 2).

The UK Prospective Diabetes Study results have demonstrated that the risk of retinopathy, nephropathy, and potentially neuropathy is significantly reduced when blood sugar levels in type 2 diabetes are kept strictly low by means of tight glycemic control, which is defined by a median HbA1c of 7.0% in contrast with the standard glycemic control with a median HbA1c of 7.9%. It was found that with the tight glycemic control monitored by continuous measurement of blood HbA1c levels; the overall microvascular complication rate was decreased by 25% (12). Therefore, considering the severity of diabetes complications, in diabetes control, low imprecision (CV of 1.9%) of analytical methods is considered critical (13). In our study, imprecision of all evaluated assays (except TOSOH G8) was higher than 1.9%. Although a strong correlation was observed in comparison study (r: 0.86 to 0.96), the mean of different methods were significantly different from that of the reference method (P value < 0.01). Moreover, in a great majority of them, a negative bias was observed, which was not dependent to concentration. Besides, calculated TEs for all assays ranged from 9.65% to 28.68%, which were remarkably higher in comparison with optimal ranges (NGSP and CAP criteria are less than 7% and 6%, respectively) (14, 15). Furthermore, our findings demonstrated that the results reported by NGSP-certified and non-NGSP certified devices can differ significantly when the same sample is assayed. The findings of our study demonstrated that the differences between results reported by different HbA1c assays in Iran are too large to be acceptable when used interchangeably and this can lead to confusing clinical interpretations. Moreover, our results indicated that the routine assays used in laboratories in Iran show different degrees of negative bias. According to the NGSP criteria as described before, the calculated bias is quite significant. The extent of bias among the evaluated methods was large and highly suggestive that unacceptable intermethod differences can most commonly be observed when various HbA1c assays are utilized for the analysis of the same sample. In conclusion, it seems that national programs are needed to standardize HbA1c assays. In the current situation, for better monitoring of diabetic patients using the same laboratory for HbA1c measurement is recommended. To shed more light on this issue and its potential to distort clinical decisions in diabetes care, further studies with larger sample sizes are warranted.