[Microcytofluorometrical measurement of DNA content in immunologically identified micromegakaryocytes of human bone marrow].

Abstract

We have developed a method to microfluorometrically determine the amount of DNA in immunologically identified micromegakaryocytes on bone marrow smears. Bone marrow smears were fixed with acetone-formalin buffer and immunostained with a monoclonal anti-GPIIb/IIIa antibody, followed by FITC conjugated anti-mouse IgG. After the smears were re-fixed with methanol, DAPI (4', 6-diamidino-2-phenylindole) staining was performed. Using an automatic Digital-Microfluorometer (Olympus MMSP-FR-II), megakaryocytes on the smears were identifies by the GPIIb/IIIa immunofluorescence and, after changing the barrier filters, their nuclear DNA content was measured by the intensity of DAPI fluorescence, which is proportional to the amount of DNA. Using this method, we found that the DNA histogram of the megakaryocytes from a patient with myelodysplastic syndrome showed a shift to small ploidy compared with normal controls. This method may be valuable in the measurement of the megakaryocyte DNA content.