Direct hybridization to DNA from small numbers of flow‐sorted nucleated newborn cells

Abstract

A technique is described that allows direct hybridization to the DNA of cells flow sorted onto nitrocellulose filters, which obviates an intervening DNA isolation step. The feasibility of this technique for studying small numbers of cells is demonstrated with human cord blood, which has a high proportion of nucleated cells. The cells are stained with fluorescein‐conjugated anti‐HLe‐l, a monoclonal antibody that recognizes mature leucocytes. Anti‐HLe‐l‐positive cells are all nucleated, and a controlled, precise number of them may be sorted directly onto a nitrocellulose membrane. In cord blood, a small percentage of anti‐HLe‐l‐negative cells are nucleated erythrocytes, which may also serve as a source of DNA. Studies were performed on male or female newborn cells flow sorted onto introcellulose membranes and hybridized with either a nonspecific human repeat DNA probe or a Y chromosome‐specific probe. Importantly, the sex of the newborn could be determined at the DNA level from as few as 50 sorted cord blood leucocytes or 5,000 HLe‐l‐negative cells. Since nucleated erythrocytes are common in fetal blood but rarely found in the peripheral circulation of adults, the method has potential application for the determination of fetal sex from analysis of flow‐sorted nucleated erythrocytes present in the maternal circulation during pregnancy.

Isolation of fetal DNA from nucleated erythrocytes in maternal blood.

“…Analysis and sorting were performed on a Becton Dickinson FACS-IV (19) with a Consort 40 program as described (20). The gain was standardized manually for each experiment by using identical laser power and fluorescent beads.…”

“…The DNA was transferred to Hybond-N in 20x SSC (0.10 M NaCl/0.01 M sodium citrate, pH 7) over 4 hr. Filters were baked in a vacuum oven at 80°C for 20 min. Each filter was placed in a buffer containing 5% dextran sulfate, 6x SSC, 5x Denhardt's solution (26), 5% SDS, 50%o…”