Minimizing red blood cell contamination while isolating mononuclear cells from whole blood: the next step for the treatment of severe hemolytic disease of the fetus/newborn

Objective We examined whether mononuclear cell purification of human blood could be done while minimizing contamination with RhD-positive red blood cells to treat hemolytic disease of the fetus/newborn that was caused by rhesus disease. Study design Whole blood from 16 individuals who tested rhesus positive was diluted and centrifuged over a Ficoll gradient. The cell pellet was incubated with red blood cell lysis buffer, divided into three samples, and analyzed for cell count, mononuclear cell yield, and RhD-positive red cell contamination by flow cytometry. Results Mean RhD-positive red cell contamination was 0.24% (range, 0%-1.9%). The average yield of mononuclear cells was 11.5% (range, 1.8%-23.6%). Through regression analysis, 34 to180 mL of paternal whole blood would be necessary to achieve an antigen load that is sufficient for an HLA antibody response. Conclusion Purification of human blood is possible to produce reasonable mononuclear cell yields with minimal rhesus activity, which makes paternal leukocyte therapy a plausible treatment for severe rhesus alloimmunization.