Flow cytometric analysis of DNA content differences in blood samples obtained by leucoconcentration

Abstract

The leucoconcentration technique allows rapid obtainment of cellular suspensions from total blood or bone marrow for flow cytometric analysis. The technique is based on picric acid in ethyl alcohol fixation and saponin red cell lysis, followed by mithramycin staining for DNA. It gives a good resolution of DNA distributions that allow detection of slight variations in DNA content. These results were obtained with cellular suspensions differing only in one X or Y chromosome (male, female, Klinefelter and Turner syndromes).In these studies the ratio of the DNA content of X and Y chromosomes agrees with the chromosomal mass ratio already reported by other authors, but the "absolute values" are 10-fold more compared to these same works. Our conclusion is that leucoconcentration technique followed by DNA staining with mithramycin increases the difference in the dye's penetration and binding between X and Y chromosomes.Key terms: Internal standard, leucoconcentration, mithramycin, chromosome X and Y, DNA distribution, flow cytometryIn a previous work, we described use of the leucoconcentration technique for flow cytometric analysis of hematopoietic cells (13). Besides its rapidity, this technique has the advantage of conserving all nucleated cells, even abnormal or rare cells, because it uses saponin red cell lysis.The conditions of DNA staining with mithramycin have been adjusted to the leucoconcentration technique. The histogram resolution (CV=2.2%, (1.2-3.9), n = 10) led us to use this technique to measure small DNA content differences. To confirm this application, we quantified the smallest DNA content difference easily identifiable between two cell populations, such as the difference between X and Y chromosomes. MATERIALS AND METHODSFlow cytometric analysis was performed on peripheral blood samples from 20 healthy subjects (10 men, 10 women), and 2 patients with karyotypic abnormality (one Klinefelter (47,XXY) and one Turner (45,XO) syndromes). Peripheral blood samples (5 ml) were collected by a method previously described (13). The following reagents (Merck, Darmstadt, East Germany) were used: 1) solution A-sodium chloride 60 g; sodium citrate, 40 g; polyvinylpirrolidone, 100 g; formol, 75 ml; picric acid 1% in 95% ethyl alcohol, 75 ml; distilled water qsp, 1,000 ml; at pH 7.2; 2) solution B-solution A, 15 ml; distilled water, 85 ml; at pH 7.2; 3) lysis solution-water, 50 ml; saponin, 1 g; 95% alcohol, 50 ml; formol, 2 ml; 4) phosphate buffer saline solutin (PBS) pH 7.4,O.l M.Peripheral blood samples were collected into a 20-ml syringe containing 10 ml of solution B without any anticoagulant. Lysis of erythrocytes in peripheral blood was achieved by admixture of lysis solution drop by drop with a gentle shaking until translucency was obtained. Immediately after red cell lysis, 10 ml of solution B were added. Then the cell suspensions were centrifuged at 700g for 15 min at room temperature. The supernatant was discarded. The cell pellets were carefully resuspended, with a Pasteur-pipette, in a few microliters of P...