title: Technical Report: Triple-Colour Staining Flow Cytometry for Co-Distribution of Thrombospondin Receptor (CD36), Ribonucleic Acid (RNA) and Fetal Haemoglobin (HbF) in Sickle Red Blood Cells.
abstract: Red blood cells (RBCs) from sickle cell patients (SS) express thrombospondin receptor (CD36), contain ribonucleic acid (RNA, recognised as reticulocytes) and fetal haemoglobin (HbF, defined as F cells) in a higher proportion than RBCs from healthy individuals. The co-distribution of CD36, RNA and HbF on the same RBCs has not been demonstrated due to a lack of detection methods. A triple-colour staining flow cytometry for the co-distribution of CD36, RNA and HbF was developed. The method can simultaneously determine CD36-expressing RBCs (CD36 cells), RNA-bearing RBCs (reticulocytes), HbF-bearing RBCs (F cells), CD36-expressing reticulocytes (CD36 reticulocytes), CD36-expressing-F cells (CD36-F cells), HbF-bearing reticulocytes (F reticulocytes) and CD36-expressing-F reticulocjrtes (CD36-F reticulocytes). Mouse monoclonal antibody against CD36 (MoAb-CD36), antibodagainst mouse-immunoglobulin conjugated to biotin (Ab-Molg-Bi), streptavidin conjugated to rhodamine phycoerythrin (StA-RFE), MoAb against HbF conjugated to Tri-Colour® (MoAb-HbF-TC), Thiazole orange (TO), Glutaraldehyde and Triton X-100 were used. The procedure takes approximately 7 hours. The numbers of CD36 cells, reticulocytes and F cells obtaining from single and triple staining were well correlated and not significantly different. Intra- and inter-assay coefficient of variation percents (%CVs) of the triple-colour staining were less than 10 and 15% respectively. EDTA blood samples stored at 4°C for less than 3 days are suitable. The method trial was then employed on blood samples from SS and healthy individuals. The method is reproducible, objective and applicable for determination of co-distribution of other membrane and intracellular markers in RBCs.
authors.affiliation: a Department of Haematology , University College London Medical School , London , UK.|d Department of Clinical Microscopy, Faculty of Associated Medical Sciences , Chiang Mai University , Thailand.
authors.forename: Y
authors.initials: Y
authors.lastname: Mundee
authors.affiliation: b William Beaumont Hospital , Royal Oak , MI , USA.
authors.forename: N C
authors.initials: NC
authors.lastname: Bigelow
authors.affiliation: c Maine Medical Center Research Institute , South Portland , ME , USA.
authors.forename: B H
authors.initials: BH
authors.lastname: Davis
authors.affiliation: a Department of Haematology , University College London Medical School , London , UK.
authors.forename: J B
authors.initials: JB
authors.lastname: Porter
keywords: CD36; F cells; Flow cytometry; HbF; reticulocytes; sickle cell disease