Flow cytometric method for simultaneous assay of foetal haemoglobin containing red cells, reticulocytes and foetal haemoglobin containing reticulocytes.

摘要

Level of foetal haemoglobin (HbF) containing red cells (F cells) is a parameter for monitoring sickle cell anaemia (SS) patients undergoing treatment with HbF modulating drugs (including hydroxyurea (HU)). One convenient technique for F cell assay is flow cytometry. A flow cytometric method for the simultaneous assay of F cells, reticulocytes and HbF-containing reticulocytes (F reticulocytes) is described in this paper. These three parameters can be obtained within 2 h using double colour staining flow cytometry. Glutaraldehyde fixation, Triton X-100 permeabilization, monoclonal antibody to HbF Tri-colour conjugate (MoAb-HbF-TC; deep-red fluorescence) immuno-staining and thiazole orange (TO; green fluorescence) are employed. The red cell gate was set on forward scatter (FSC) and logarithmic side scatter (logSSC) for 50 000 cells on the flow cytometer. Fluorescent signals were acquired from fluorescent channel 1 (FL1; green) and (FL4; deep-red). Coefficient of variation percent (%CVs) of intra- and inter-assay were less than 9% and 15%, respectively. EDTA, citrate, heparin and CTAD anticoagulants are all suitable; the samples can be stored at 4 degrees C for up to 6 days. The method is a simple, rapid, convenient, reproducible and useful way of determining F cell, reticulocyte and F reticulocyte levels in sickle cell and thalassaemic patients.