Enrichment by preferential mitosis of fetal lymphocytes from a maternal blood sample

A method for selecting and culturing fetal lymphocytes from a maternal blood sample, comprising the steps of:

• (1) adding a reagent solution comprising sodium chloride, ammonium chloride, and carbonic anhydrase inhibitor to obtain a cell suspension;
• (2) adding a bicarbonate solution selected from the group of ammonium bicarbonate and sodium bicarbonate to the cell suspension and reacting the cell suspension with the bicarbonate solution for about four minutes in order to lyse erythrocytes;
• (3) adding a sodium chloride shock solution to the reacted cell suspension;
• (4) centrifuging the reacted cell suspension;
• (5) sequentially treating a cell pellet obtained from step (4) with
• i) the reagent solution of step (1);
• ii) ammonium bicarbonate solution;
• and
• iii) a sodium chloride shock solution in an amount sufficient to condition fetal lymphocytes to undergo mitotic division, thereby yielding a treated cell suspension;
• (6) centrifuging the treated cell suspension;
• (7) washing a cell pellet obtained from step (6) in a phosphate buffered saline wash solution;
• and
• (8) culturing fetal lymphocytes present in the washed cell pellet obtained from step (7) in the presence of a sufficient concentration of a mitotic stimulant to induce mitotic division of the fetal lymphocytes and allowing the lymphocytes to divide.

A method for enriching the fetal lymphocyte population relative to other cells in a maternal blood sample, comprising the steps of:

• (a) lysing erythrocytes in the maternal blood sample by adding a solution comprising ammonium chloride, ammonium bicarbonate and carbonic anhydrase inhibitor in an amount sufficient to preferentially lyse erythrocytes in the blood sample;
• (b) adding a hypertonic salt solution to the remaining cells in the blood sample which comprises the fetal lymphocytes in an amount sufficient to cause the cells to be subjected to hypertonic conditions;
• (c) adding a mitogenic stimulant to the remaining cells in an amount sufficient to cause the fetal lymphocytes to divide more rapidly than the maternal lymphocytes and;
• (d) allowing the lymphocytes to divide.