An improved method for removal of red cell-bound immunoglobulin using chloroquine solution

In some patients with autoimmune hemolytic anemia or bemolytic disease of the newborn, the red cells are so heavily coated with immunoglobulin that phenotyping cannot be carried out unless the antibody is removed without destroying the red cell antigens. Studies were performed initially to determine the optimum conditions for removal of immunoglobulin from red blood cells (RBCs) using chloroquine. Group O, R1r RBCs were coated with serial dilutions of anti-D; aliquots were incubated in chloroquine diphosphate (CDP) solution (200 g/L, pH 5.0) at 18 degrees C, 25 degrees C, 30 degrees C, and 37 degrees C, and test- ed by the antiglobulin technique at intervals of 30 minutes for up to 2 hours, the results being expressed as titration scores. These studies showed that antibody removal was much more efficient at 30 degrees C and 37 degrees C than at 18 degrees C or 25 degrees C. A further series of experiments was then carried out to assess the effect of chloroquine on red cell antigenicity. A 5 percent suspension of RBCs heterozygous for c, D, and E antigens, and for Kell, Duffy, and Kidd antigens, was incubated at 30 degrees C and at 37 degrees C in chloroquine solution. Aliquots were removed at 30-minute intervals for up to 2 hours, tested with serial dilutions of the appropriate antisera, and titration scores obtained. The antigens were well preserved after two hours of chloroquine treatment at 30 degrees C. However, when treatment was performed at 37 degrees C, aitigenicity had markedly deteriorated by 60 minutes, although the antigens were still reasonably well preserved (except for Jkb) at 30 minutes. It is therefore recommended that treatment with chloroquine solution prior to typing RBCs heavily coated with antibody should be carried out for 90 minutes at 30 degrees C or for not more than 30 minutes at 37 degrees C.