A procedure is described for making leucocyte preparations from blood samples fixed ex vivo. Briefly, blood is treated with buffered formaldehyde and the erythrocytes and platelets removed subsequently by lysis and differential centrifugation. The fixed leucocyte preparations can then be processed or fixed further for various types of microscopy as required. In stained preparations examined by light microscopy, the different leucocytes, i.e. lymphocytes, polymorphonuclear leucocytes (PMN) and monocytes, can readily be identified by their characteristic nuclear morphology, and polarized PMN can easily be recognized by their non-spherical shape. Experiments with chemotaxins in vitro indicated that the procedure could be used to determine whether polarized PMN circulate in the blood of normal individuals and patients with inflammatory disease. A study of the blood from eleven normal subjects showed that 84.0 +/- 10.8% (mean +/- 2 SEM) of circulating PMN were spherical, while the majority of the remainder (14.3 +/- 10.5%) were only 'slightly' polarized.
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