ABSTRACT
We have evaluated the conservation prior to HbA(1c) determination of three whole blood samples stored at -80 degrees C, -20 degrees C, 4 degrees C and 20 degrees C, for a maximal duration of one year. HbA(1c) was measured by an ion-exchange high performance liquid chromatography (HPLC) method (Variant II). We have analyzed the HbA(1c) value and the quality of the chromatographic separation for each sample. Storage of whole blood samples at -80 degrees C is good for at least one year. Storage at 4 degrees C is correct for two weeks, without major sample degradation. A more important and earlier degradation occurs at -20 degrees C. The conservation at 20 degrees C (room temperature) is very short. In conclusion, the temperatures of 4 and -80 degrees C are of interest for whole blood storage before HbA(1c) measurement, respectively for short and long term conservations. The temperatures of 20 and -20 degrees C are not recommended.
Subject(s)
Blood Preservation/methods , Chromatography, High Pressure Liquid/methods , Glycated Hemoglobin/analysis , Drug Stability , Humans , Reproducibility of Results , Temperature , Time FactorsABSTRACT
HbA(1c) represents a key parameter in the follow-up of glycemic balance in diabetic patients. It may be assayed by different methods, among which high-pressure liquid chromatography (HPLC). We have evaluated a new method available on HPLC Variant II analyzer (BioRad) equipped with the new kit 270-2101 NU. Chromatographic separation is improved, allowing a better identification of peaks. Intra- and inter-assay coefficients of variation are respectively lower than 1.1% and 1.8%. Linearity is excellent from 3.2% to more than 18%. The correlation with the previous method (kit 270-2101) is good: y (% HbA(1c) new kit) = 0.944x (% HbA(1c) previous kit) + 0.299, r(2) = 0.995. There is no inter-sample contamination. This method is less sensitive to interferences frequently found in practice (labile glycated hemoglobin, carbamylated haemoglobin) than the previous one. Validation is possible in more circumstances when an abnormal hemoglobin is present (especially in case of hemoglobin D or E). As the control of analytic quality is a major element for validation and clinical use of HbA(1c) results, the characteristics of this new method make it a well-suited tool for daily laboratory practice.
Subject(s)
Blood Glucose/metabolism , Diabetes Mellitus/blood , Glycated Hemoglobin/metabolism , Anticoagulants/therapeutic use , Bilirubin/blood , Chromatography, High Pressure Liquid , Glycated Hemoglobin/drug effects , Glycated Hemoglobin/isolation & purification , Hemoglobin E/metabolism , Hemoglobin J/metabolism , Hemoglobins, Abnormal/metabolism , Humans , Regression Analysis , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
HbA(1c) assay by high pressure liquid chromatography remains submitted to interferences, among which that of labile HbA(1c) in 1 to 2% of samples. We have evaluated the interference of labile HbA(1c) on HbA(1c) assay using Variant II analyzer (Biorad), by in vitro formation of labile glycated haemoglobin and by evaluation of two protocols of elimination of labile HbA(1c) (wash and incubation of red blood cells in saline solution, or incubation in the wash/dilution solution of the analyzer). Levels of labile HbA(1c) higher than 4.5 % lead to underestimation of HbA(1c). The different protocols tested proved efficient and were adapted to routine conditions. The fastest method is the incubation of red blood cells in the wash/dilution solution for at least two hours, or more if labile fraction is unusually high.
