Preservation of Human Granulocytes: II. Characteristics of Granulocytes Obtained by Counterflow Centrifugation

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Abstract
Human granulocytes were isolated from whole blood and from the buffy coat of blood by counterflow centrifugation using the Beckman JE‐6 rotor. Ninety‐one per cent of granulocytes in the human blood sample were isolated in 59 studies. The differential white blood cell count showed 97 per cent segmented neutrophils, 1 per cent lymphocytes and 2 per cent others. The ratio of polymorphonuclear white blood cells to red blood cells was 9:1 when the granulocytes were isolated from whole blood and 40:1 when the granulocytes were isolated from the buffy coat of CPD‐collected blood. The size distribution showed a single symmetrical peak. Measurements of oxygen consumption, chemotaxis, and phagocytosis in granulocytes isolated by counter‐flow centrifugation were similar to those in granulocytes isolated from blood by dextran sedimentation. The granulocytes had normal adenine and guanine di‐ and trinucleotide levels.Scanning electron microscopy demonstrated spherical granulocytes with ridge‐like profiles and leafy folded exoplasm. After fluorescein diacetate treatment, the cytoplasm of 98.5 + 0.7 per cent of fresh granulocytes was positive, while 98.5 + 1.2 per cent of the fresh granulocytes excluded ethidium bromide from their nuclei. The response of freeze‐preserved granulocytes to treatment with fluorescein diacetate and ethidium bromide showed that 75 per cent of granulocytes survived freezing and thawing in 5 per cent DMSO but were progressively unstable after incubation for two hours at 37 C.