title: When diagnostics meets translational research: detection of hemoglobin fractions in cellular lysates from in vitro erythroid cultures by Capillarys 2 Flex Piercing analyzer (Sebia).
abstract: 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.
authors.affiliation: San Raffaele-Telethon Institute for Gene Therapy (TIGET), Division of Regenerative Medicine, Stem Cells and Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy; University of Rome "Tor Vergata", Rome, Italy.
authors.forename: Annamaria
authors.initials: A
authors.lastname: Aprile
authors.affiliation: Service of Laboratory Medicine, San Raffaele Hospital, Milan, Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Passerini
authors.affiliation: Università di Milano, Ca Granda Foundation IRCCS, Milan, Italy.
authors.forename: Maria Domenica
authors.initials: MD
authors.lastname: Cappellini
authors.affiliation: Hematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Sarah
authors.initials: S
authors.lastname: Marktel
authors.affiliation: Hematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy; Immunohematology and Transfusion Medicine Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Ciceri
authors.affiliation: San Raffaele-Telethon Institute for Gene Therapy (TIGET), Division of Regenerative Medicine, Stem Cells and Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele University, Milan, Italy.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Ferrari
authors.affiliation: Service of Laboratory Medicine, San Raffaele Hospital, Milan, Italy. Electronic address: ceriotti.ferruccio@hsr.it.
authors.forename: Ferruccio
authors.initials: F
authors.lastname: Ceriotti
mesh_terms: D002478:Cells, Cultured; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D004912:Erythrocytes / Q000737:chemistry* / Q000166:cytology; D006454:Hemoglobins / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D057230:Limit of Detection; D015203:Reproducibility of Results; D057170:Translational Research, Biomedical
chemical_list: D006454:Hemoglobins