The influence of sample freezing at – 80 °C for 2–12 weeks on glycated haemoglobin (HbA1c) concentration assayed by HPLC method on Bio-Rad D-10® auto analyzer

Introduction The aim of the study was to evaluate the effect of a single freeze/thaw cycle on HbA1c concentrations measured by commercially available HPLC method. Materials and methods Study included 128 whole blood samples collected from diabetic patients (N = 60) and healthy volunteers (N = 68). HbA1c concentrations were measured in fresh blood samples. Then samples were frozen at - 80 °C for up to 12 weeks. HbA1c was assayed by ion-exchange HPLC method on Bio-Rad D-10® analyzer. Variables were compared using Wilcoxon and ANOVA Kruskal-Wallis tests. Bias between HbA1c measured in fresh and frozen samples was calculated. The comparability of HbA1c concentrations was assessed by Bland-Altman plot. Results Median (IQR) HbA1c concentration was 45.3 (36.6–61.2) mmol/mol for fresh and 45.3 (36.6–60.6) mmol/mol for frozen/thawed samples. No significant difference in HbA1c concentrations was found comparing fresh and frozen/thawed samples (P = 0.070) in the whole group, as well as in healthy and diabetic subjects. The median calculated bias between fresh and frozen/thawed samples was 0% in whole group and healthy subjects, and 1.19% in diabetic patients. No significant difference was found between the biases according to baseline HbA1c values (P = 0.150). The Bland-Altman plot analysis showed a positive bias of 0.4% (95% CI: - 2.8 - 3.7%), which indicates high compliance between HbA1c values and no relevant influence of sample freezing on clinical significance of HbA1c measurement. Conclusions Storage for up to 12 weeks at – 80 °C with a single freeze/thaw cycle does not affect HbA1c concentrations measured with HPLC method on Bio-Rad D-10® analyzer.


Introduction
Glycated haemoglobin (HbA 1c ) is a product of the non-enzymatic binding of glucose to N-terminal valine residues of haemoglobin's β chains. It is considered a "gold standard" for monitoring diabetes, however current guidelines of the American Diabetes Association (ADA) recommend its use as a diagnostic tool for diabetes (1). HbA 1c concentration is strongly correlated with the development of long-term complications (micro-and macroangiopathy) in both Type 1 and Type 2 diabetes (2,3).
Changes in the structure and chemical-physical properties of haemoglobin have become the starting point for the development of various methods for its quantification. Many in vitro diagnostic manufacturers developed automatic analyzers based on enzymatic, immunological and separation methods for HbA 1c assay. According to the standardization programme of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), determination of HbA 1c concentration should be performed using high performance liquid chromatography (HPLC) combined with capillary electrophoresis or mass-spectrometry (4). Autoanalyzers and methods used in clinical practice are obliged to obtain IFCC certification.
Effect of sample freezing on HbA 1c assayed by HPLC Glycated haemoglobin concentration is stable in blood during short-term storage, which is its main advantage over glucose (5). Usually, it is measured in fresh samples immediately after blood collection or in samples stored at 4 °C up to 1-2 weeks. Bio-Rad D-10 ® Hemoglobin A1c Program (Bio-Rad Laboratories, CA, USA) is based on ion-exchange HPLC method which is certified by IFCC. According to the manufacturer, whole blood samples are stable up to 7 days when stored at 2-8 °C or 3 days at room temperature (15-30 °C). Sample freezing is not recommended (6).
Several studies evaluated repeatability of HbA 1c concentration on different instruments after freezing the blood samples which might be useful if prolonged storage is required (i.e. for research or evaluation of new methods). For prolonged storage (several months or years) recommended storage temperatures are -70 °C or lower (7)(8)(9). HbA 1c concentration is stable at 4 °C usually for 2-3 weeks, while at room temperature relevant changes in HbA 1c concentration are observed within 1-3 days (8,9).
The aim of this study was to evaluate the effect of a 2-12 weeks storage period and single freeze/ thaw cycle on HbA 1c concentrations measured by HPLC on Bio-Rad D-10 ® autoanalyzer in clinically healthy and diabetic individuals. Most studies evaluating the influence of sample storage on HbA 1c concentration were performed using very small numbers of analysed samples. For example, in studies mentioned above only 3 and 5 whole blood samples were analyzed, which can considerably limit their clinical importance (8,9). Moreover, many studies do not distinguish results obtained in healthy and diabetic subjects. In this study, we made an attempt to compare indicators of the analytical quality and recommended bias related to clinical significance of HbA 1c measurements.

Blood sampling
From each subject, one sample of fasting venous blood was drawn in the morning (7.00-9.00 am) using sterile 2.0 mL plastic tubes Vacutainer ® with potassium -EDTA (cat. no. 367841; Becton Dickinson, NJ, USA). Samples were delivered to the laboratory and HbA 1c concentrations were measured immediately after collection in fresh blood samples. Several samples (approximately [16][17][18]) that could not be tested immediately after collection (for example, blood was drawn on Sunday) were stored at 2-8 °C for no longer than 24 hours. Samples were then transferred into sterile, tightly closed polypropylen tubes (cat. no. 20-1203-1; Medlab-Products, Raszyn, Poland) suitable for freezing up to -90 °C to avoid peptide degradation and potential interference with the material of blood collection tubes, and frozen at -80 °C. Samples were stored for up to 12 weeks.
Samples were divided according to the three storage time intervals: 2-5 weeks (N = 40), 6-9 weeks (N = 45) and 10-12 weeks (N = 43). This division was used to obtain comparable size groups and similar number of diabetic and healthy subjects matched by age, sex and baseline HbA 1c concentration in each storage time interval.
Samples were thawed in the refrigerator, brought to room temperature and thoroughly mixed on a roller mixer (MX-T6-S, Medlab-Products, Raszyn, Poland) prior to the assay.

Methods
HbA 1c was assayed by ion-exchange high performance liquid chromatography (HPLC) method certified by National Glycohemoglobin Standardization Program (NGSP) and IFCC on Bio-Rad D-10 ® (Bio-Rad Laboratories, CA, USA) autoanalyzer with reportable range 18 -179 mmol/mol (3.8-18.5%) and total precision CV = 1.16%. The instrument automatically dilutes whole blood samples and injects them into the analytical cartridge. In the next step, different fractions of haemoglobin are separated using buffer gradient of increasing ionic strength. Each type of haemoglobin is measured by spectrophotometry at 415 nm.
The quality control of this assay was performed every day during the study using Bio-Rad Lyphochek ® Diabetes Control with the following reference values: level 1 (low) 5.4%-42.1 mmol/ mol) and level 2 (high) 9.6%; ranged 8.4-10.8% (81.4 mmol/mol; ranged 68.3-94.5 mmol/mol). The intra-assay variation calculated from 10 assays of the same sample (in duplicates) was 0.87% and the inter-assay variation calculated from 10 runs on duplicate samples was 1.47%. The intra-and inter-assay variations were performed using quality control material at level 1.

Statistical analysis
Statistical analysis was performed using Statistica 12 To evaluate the significance of changes in HbA 1c concentrations after sample freezing, the repeatability was calculated. Repeatability was considered as absolute difference between HbA 1c concentration measured in fresh blood samples and in frozen/thawed samples. A difference of 5 mmol/mol (0.5%) in HbA 1c concentration between samples was considered clinically significant.
Bias between HbA 1c in fresh and frozen samples was calculated using the following formula: Bias (%) = [(HbA 1c fresh -HbA 1c frozen) / HbA 1c fresh] x 100%. According to the recent findings from "Guideline-Driven Medical Decision Limits" bias less than 1.0% is recommended to obtain clinically useful HbA 1c results (10). Repeatability of HbA 1c concentration after single freeze/thaw cycle is presented in Figure 1. No changes in HbA 1c concentration was found in 48.4% of subjects (N = 62), while in 41.4% cases (N = 53) the obtained results were lower (mean 1.4 mmol/mol) and in 10.1% cases (N = 13) the results were higher (mean 2.3 mmol/mol) than baseline HbA 1c values, respectively. However, the observed differences in HbA 1c concentrations between fresh and frozen/thawed samples were not statistically significant (P = 0.110).

Median of
Samples were divided according to storage time: 2-5, 6-9 and 10-12 weeks, respectively (Table 1). No significant differences in HbA 1c concentration were found comparing frozen/thawed samples stored in different time intervals when compared to fresh blood samples.

Percentage of results (%)
The median calculated bias between fresh and frozen samples was 0% in whole group and in healthy subjects (interquartile range (IQR): 0-1.64%) and 1.19% (IQR: 0-2.15%) in diabetic patients, respectively. No significant difference was found between the biases according to the baseline HbA 1c values (P = 0.150; Figure 3).

Discussion
We demonstrated good stability of HbA 1c measured in whole blood samples stored for  (12). Control material was stored up to 7 days in 2-8 °C and alternatively frozen in 10 µL aliquots at -80 °C. The coefficient of variation (CV) obtained by standard procedure were 2.33% and 2.51% for level 1 and 2, respectively; while the CV obtained by alternative procedure were significantly lower: 1.93% (P < 0.001) and 0.99% (P = 0.003). Moreover, control materials freezing reduced costs of quality control by 80%. Optimization of quality control and cost saving may be important especially in small-scale laboratories, where HbA 1c measurements are not performed every day.
Although freezing seems to be an effective way to store blood samples for HPLC methods, some au-  thors consider that long-term storage may increase HbA 1c concentration assayed by mass spectrometry methods. D'Alessandro et al. observed relative percentage increase in HbA 1c concentration in samples frozen for 28-42 days compared with samples stored up to 14 days, however the authors did not clearly state the freezing temperature (13). This effect was explained by use of additive solutions, such as SAGM (saline-adenine-glucose-mannitol) for blood banking which results in prolonged exposure of erythrocytes to high glucose concentrations. However, their study group consisted of only 10 healthy volunteers, thus the results should be interpreted with caution. The influence of SAGM on HbA1c concentrations should be verified in samples obtained from patients with diabetes or elderly subjects, who have increased rate of glycation.
Recent studies suggest that repeatability of HbA 1c measurements depends on baseline HbA 1c concentration in fresh blood samples. In a study by Liotta et al., including 237 subjects with type 2 diabetes, HbA 1c levels measured by ion-exchange HPLC method were compared in fresh samples and samples stored for 1 year at -80 °C (14). HbA 1c concentrations were significantly decreased after freezing and refreezing in patients with lower (< 6.5%), increased (6.5-8.0%) and very high (> 8.0%) baseline concentrations. Moreover, bias was significantly higher in subjects with HbA 1c ≥ 6.5% compared with those with HbA 1c < 6.5%, and increasing bias values were observed in subsequent concentration intervals of baseline HbA 1c . However, our results did not confirm this tendency in both diabetic and healthy subjects.
In the Atherosclerosis Risk in Communities (ARIC) study HbA 1c concentration in 14 069 whole blood samples was measured by HPLC method after storage for 14 and 18 years at -70 °C. The correlation between measurements at the two time points was high; however a slight positive bias of 0.29% was found when comparing samples stored for 18 years to those stored for 14 years (15).
For HbA 1c measurement used in particular as a diagnostic tool for diabetes diagnosis, evaluation of diabetes risk in general population or establishing therapeutic goals in diabetic patients the corresponding values of analytical quality indicators must be maintained. According to generally accepted rule in clinical practice, difference of 5 mmol/mol (0.5%) in HbA 1c concentration between patients samples is considered as a significant change in glycemic control (16,17). When comparing this criterion with the repeatability observed in our study, it can be concluded that single freeze/ thaw cycle does not affect the clinical significance of HbA 1c measurement. However, the evaluation of clinical significance of HbA 1c concentrations should also consider the analytical bias of assay used. A recent study by Hyltoft Petersen and Klee, which appraises the influence of analytical bias and imprecision on the number of false positive results, recommends bias less than 1.0% to obtain clinically useful HbA 1c results (10). In our study a slight positive bias of 0.4% between HbA 1c values in fresh and frozen/thawed samples was found in Bland-Altman plot analysis. Thus, high compliance of results obtained in both diabetic and healthy individuals was demonstrated, with no significant effect on HbA 1c measured from samples stored at -80 °C. Several studies indicate different bias of HbA 1c concentration assayed by comparable methods. For example, study performed in Denmark proposed a more restrictive bias (i.e. less than 0.3%), which provides acceptable accuracy for the detection of significant changes in glycemic control and risk prediction of diabetic complications (18).
Despite potentially interesting findings, we should emphasize the important limitations of this study. First, study consisted of relatively small number of subjects and storage time was relatively short, therefore results require confirmation in extended population-based study. Moreover, the time between blood collection, HbA 1c assay and samples freezing should be unified. Almost all samples were delivered to the laboratory, measured and frozen immediately after collection; however, several of them were stored at 4 °C and assayed not later than within 24 hours, which may possibly induce some variation in HbA 1c concentration. However, it is worth emphasizing that our study included significantly greater number of analyzed sam-ples, when compared to previously published research. Moreover, it is one of the few studies which evaluated the stability of HbA 1c concentration during freezing in both healthy and diabetic subjects.
Obtained results indicate that sample storage at -80 °C does not affect the clinical significance of HbA 1c measurement on HPLC autoanalyzer, therefore this method of storage might be crucial for maintaining analytical quality of assay, as well as for its cost-effectiveness.
In conclusion, whole blood samples storage at -80 °C up to 12 weeks and a single freeze/thaw cycle do not affect the concentration of HbA 1c measured by HPLC method on Bio-Rad D-10 ® analyzer in healthy and diabetic subjects. However, due to relatively short storage period studied, results should be confirmed by measuring HbA 1c levels in longterm stored samples.

Potential conflict of interest
None declared.