Journalc( ImmunologicalMethods', 72 (1984) 349-353 349 Elsevier JIM03162 Enumeration of Human Lymphocyte Subsets by Monoclonal Antibodies and Flow Cytometry: a Comparative Study Using Whole Blood or Mononuclear Cells Separated by Density Gradient Centrifugation Paolo De Paoli, Michele Reitano, Sandro Battistin, Carla Castiglia and Gianfranco Santini Sert,izio di Microbiolog 'a-Immunologia, Ospedale Cit,ile USL l 1. 33170 Pordenone. Italy (Received 16 November 1983, accepted 24 April 1984) Enumeration of lymphocyte subpopulations by combined use of flow cytomet~ and monoclonal antibodies is influenced by the separation method used. T or B lymphocyte antigen frequencies do not differ in samples of whole blood or after separation on Ficoll-Paque or Percoll, but there is a significant increase of Leu7 + Leull + lymphocytes showing strong natural killer activity at the sample-separation medium interface. At the bottom of the tubes a selective loss of OKT8 ~. Leu7 +, Leull cells is found; at this level cytotoxicity is very_ low. Our data suggest that Leu7 + cells could be subdivided into 2 subpopulations differing in reactivity with the monoclonal antibody to Leull, natural killer activity and density. Differences observed in the percentages of Leu7 +, Leull ' lymphocytcs between whole blood and separated cells could be due to an enrichment produced by centrifugation or to the presence in whole blood of a factor interfering with antibody binding. Key words: monoclonal antibodies - lvmpho~ivtes - whole blood - density gradient centrifugation Introduction Enumeration of T, B and natural killer cells in peripheral blood is important in assessing the immunological status in leukaemias and lymphomas (Ritz et al., 1980, 1981; Kung et al., 1981; Schroff et al., 1982), infectious diseases (Reinherz et al., 1980; Bach et al., 1981; Carney et al., 1981; De Paoli et al., 1983b), immuno- deficiencies (Fisher et al., 1981; Phan Dinh Tuy et al., 1981; Pandolfi et al., 1982) and in other pathological conditions (Morimoto et al., 1980; Cosimi et al., 1981a, b). The use of monoclonal antibodies combined with flow cytometry requires small volumes of blood and allows a very rapid enumeration of lymphocyte subsets. We used this procedure on both whole blood (Hoffman et al., 1980) and cells separated 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.