When diagnostics meets translational research: detection of hemoglobin fractions in cellular lysates from in vitro erythroid cultures by Capillarys 2 Flex Piercing analyzer (Sebia)

摘要

Detection of hemoglobin (Hb) variants represents an important issue for diagnosis and adequate treatment of hemoglobinopathies. The Capillarys 2 Flex Piercing analyzer (Capillarys) by Sebia is routinely used in our clinical laboratories to detect Hb variants in peripheral blood (PB). This automated method separates Hb fractions by capillary electrophoresis, giving a spectrophotometric measure of their relative proportion. The scientific research in the field of hemoglobinopathies needs robust procedures to evaluate the efficacy of experimental therapies, as gene therapy. We investigated for the first time the feasibility to use Capillarys on cellular lysates from in vitro erythroid cultures. Because total Hb concentration in erythroid lysates is up to 20-fold lower than in hemolysates from PB, we analyzed diluted blood samples, thanks to the manual mode included in the Capillarys setting. We compared analytical precision, accuracy, sensitivity, and specificity of this procedure to the automatic method, routinely used in diagnostics. For instance, adult Hb intra- and interassay precision were estimated as coefficient of variation 0.2% and 0.3%, respectively. The manual mode is less robust for detection of fractions <3% and the lower level of sensitivity is 2 g/L of total Hb. Specificity of manual and automatic settings was equivalent. We confirmed the performance of the method by analyzing erythroid lysates from thalassemic patients' cultures. Our study demonstrated that the Capillarys 2 Flex Piercing manual method is comparable to the automatic one. The analysis is very robust at low Hb concentrations, as in erythroid cultures from patients affected by hemoglobinopathies, representing a useful tool also in translational research.