Abstract
Blood mononuclear cells (HNC) are commonly isolated by Ficoll-Hypaque density centrifugation (FH) prior to monoclonal antibody staining and flow cytometric lymphocyte phenotype analysis. We have found that FH separation of peripheral blood is unsuitable for infants less than 4 months of age due to contamination of MNC with red blood cells (RBC's). Identically FH-prepared infant and adult samples showed RBC: lymphocyte ratios of 1.2:1 and 0.2:1 in the MNC suspension on Wright's stained cytospin preparations (n=5). Unlike adult samples, infants' RBC's could not be distinguished from lymphocytes on the basis of forward angle and 90° light scatter. Each of 8 infant samples was split into paired aliquots prepared by FH and a whole blood technique (WB) .The %T3 + was 43.5 +/−17 for FH, 76.8 +/−9 for WB, p=.0047 two sided paired t-test. Results were similar for %T11+, %T4+, %T8+, and %B1+ (Coulter) staining. Differerces are attributed to contamination in the FH-prepare lymphocyte gate with non-staining RBC's. No differencs was seen in 30 adult samples. RBC lysis should be performed on all blood samples from infants less than 4 months of age.
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Slade, H., Greenwood, J., Hudson, J. et al. SPURIOUS LYMPHOCYTE PHENOTYPES BY FLOW CYTOMETRY FROM MONONUCLEAR CELLS PREPARED BY FICOLL-HYPAQUE. Pediatr Res 21 (Suppl 4), 318 (1987). https://doi.org/10.1203/00006450-198704010-00906
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DOI: https://doi.org/10.1203/00006450-198704010-00906