Evaluation of hemolysis effect on hemoglobin measurement by capillary electrophoresis

Abstract Background Hemoglobin (Hb) is a hemeprotein with two linked pairs of globin chains. Each chain is connected to a heme residue in its center. Hemoglobinopathies are divided into quantitative and qualitative defects in globin synthesis. Hemolysis is a pre-analytical problem that reduces quality of sample for measurement of many analytes. Methods Blood samples from 311 female and 189 male subjects with normal and abnormal Hb electrophoresis patterns were studied. Three milliliters of whole blood was obtained from all subjects and transferred into EDTA tubes. To analyze hemolysis, 1.5 ml of blood from each tube was aliquoted and frozen, and the remaining blood was stored at 4 °C for 24 h. Hemoglobin was measured in both hemolyzed and non-hemolyzed samples by capillary electrophoresis. Results and discussion Data was analyzed with linear regression. The results were linear for the lower limit of detection of 9, 0.5, and 0.1% up to at least 99.5, 6.6, and 99.4% for HbA, HbA2, and HbF, respectively. Method comparison demonstrated good agreement between non-hemolyzed and hemolyzed conditions for hemoglobin measurement. Conclusion Use of hemolyzed samples had no effected on hemoglobin measurements

Evaluation of 25-OH vitamin D by high performance liquid chromatography: validation and comparison with electrochemiluminescence

Abstract Objectives The aim of the present study was to develop a robust and easy to use high performance liquid chromatography (HPLC) to analyze 25(OH)D3 in human serum. Background Vitamin D is a fat-soluble steroid hormone precursor that is mainly produced in the skin by exposure to sunlight. It is also supplied in the diet and plays a pivotal role in calcium homeostasis and skeletal metabolism throughout life. Methods To assess its analytical performance, we used the RECIPE HPLC Complete Kit and an HPLC-UV instrument. Our HPLC results were compared with a validated electrochemiluminescence method. Results The method was linear for the lower limit of quantification from 3 ng/l up to at least 200 ng/l for 25(OH)D3, with the following equation for the regression line: y = 0.172 X + 2.45 (R 2 = 0.989). Intra-assay precision was determined by extracting and quantifying 10 serum replicates from one patient. The mean was 37.875 ng/ml, the standard deviation was 0.22, and the coefficient of variation was 0.58%. Comparisons of results demonstrated good agreement between HPLC and ECL methods (R 2 = 0.883). Conclusion The HPLC assay demonstrates excellent linearity, acceptable accuracy and precision, and good agreement with a validated ECL method. The simple sample preparation and ease of use make it practical for the routine clinical laboratory