STANDARDIZATION OF A FLOW CYTOMETRIC METHOD FOR MEASUREMENT OF LOW-DENSITY-LIPOPROTEIN RECEPTOR ACTIVITY ON BLOOD MONONUCLEAR-CELLS

Flow cytometric methods for measurement of low-density lipoprotein (LD L) receptor activity on peripheral blood mononuclear cells (PBMC) may be used to identify patients with familial hypercholesterolemia (FH). However, cellular LDL receptor activities measured in FH heterozygotes may overlap with those of healthy subjects. Analytical variation is p robably responsible for some of this overlap, We have examined several technical details that may affect analytical variation. In each analy sis, we included one standard and two control cell preparations. These were cells isolated from three donors and stored in aliquots at -135 degrees C. Use of standard cells reduced between-series analytical var iation of the controls by approximately 50%. Preincubation conditions used to induce the maximum number of receptors, the concentration of f luorochrome 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine-pe rchlorate (DiI)-LDL, labelling time, and conditions during storage of labelled cells before now cytometry were also examined in order to red uce analytical variation. Having standardized the assay, we found amon g 20 healthy subjects a median receptor activity of 100% vs. 51% among 26 patients who fulfilled clinical criteria for FH. However, four of the patients showed distinctly normal receptor activities, which may s uggest either the presence of some other biochemical defect or that in vivo dysfunctional receptors may be measured as normal in some patien ts with our assay. (C) 1995 Wiley-Liss, Inc.