Scand. J. Immunot. 31, 289-296. 1990 A New Technique Using an Aggregating Antibody Against Glycophorin-A for Purging Ficoll-Paque-Separated Leucocytes of Contaminating Erythroid Lineage Cells J. HELDRUP Department of Paediatrics, University Hospital, University of Lund, Sweden Heldrup, J.A. New Technique Using an Aggregating Antibody Against Glycophorin-A for Purging FicoU Paque-Separated Leucocytes of Contaminating Erythroid Lineage Cells. Scand. J. Immunol. 31, 2ii9 296, 1990 Ficoll-separated leucocytes used for phenotyping may be contaminated with crythroblasts or erythrocytes, which can eause problems in cytofluorography, especially if erythroid lineage eells outnumber leucocytes in the preparation. Diflerent means have been deseribed for removing erythroid cells from leucocyte preparations, such as BD-FACS lysing solution, hypotonie shock and ammonium chloride, but none of these is entirely satisfactory. In my hands all ihese techniques damaged the leucocytes to a greater or lesser extent and did not adequately eliminate the erythroblasts, sometimes not even the reiiculocytcs and erythrocytes. To obtain pure leucocyte suspensions, I developed ;i new technique based on an aggregating antibody {a-Gly-A, 73.1/8F7, from BioCarb, Sweden) directed against glyeophorin-A which is fotind on the cell surface of erythroid lineage cells. Agglutinated erythroid eells could be removed by filtration or low-speed eentrifugation. With the new technique there was no loss or damage of the leucocytes. Je.<iper Hetdrup. Department of Pediatrics. Unirer^ity Hospital. University of Lund. S- 221H5 Lund. Sweden Immunological phenotyping of leucocytes is now used as a diagnostic tool in patients suffering from immunodeficiency, lymphoblastic leukae- mia, lymphoma and autoimmune disease. As erythroblasts have nearly the same density as leucocytes, they cannot be separated from the latter by gradient centrifugation on Fieoll Paque solution [19]. High concentrations of erythroid lineage cells {ELC) are often observed in prep- arations of Ficoll-Paque-separated celis (FSC) from bone marrow aspirates, especially when the patients have undergone chemotherapy and the bone marrow is in a regenerative phase. This is also the case in FSC from peripheral blood from neonates and the patients with autoimmune haemolytic anaemia. The effect of ELC contami- nation on immunological phenotyping with cytofluorography has not been systemically stu- died, although several means of removing ELC have been developed, using such substances as Saponin, Triton X-100, Cetavlon, hypotonie shock and ammonium chloride [I. 4. 5. 7, 9, 15, 17, 18, 20]. Neither their efficacy nor their effect on the viability and recovery of leucocytes has been adequately documented. In the work reported here, I studied some of the these meth- ods systematically and compared them with a new technique developed in our laboratory, using an antibody against glycophorin-A, a glycopro- tein found on the cell surface of ELC [10. 12, 14, 21]. MATERIALS AND METHODS Leucocyte preparation and staining Leucocytes were obtained from heparinized cord blood from healthy full-term newborns and from peripheral venous biood from healthy adults. The low- density leucocytes (i.e. with a density less than i .077 g/ cm') were separated from whole blood hy dilutmg the blood with an equal amount of Hanks' balanced salt solution (HBSS) followed by Ficoll-Paque gradient- 289 J