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 (LDL) 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 probably responsible for some of this overlap. We have examined several technical details that may affect analytical variation. In each analysis, 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 variation of the controls by approximately 50%. Preincubation-conditions used to induce the maximum number of receptors, the concentration of fluorochrome 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-perchlorate (DiI)-LDL, labelling time, and conditions during storage of labelled cells before flow cytometry were also examined in order to reduce analytical variation. Having standardized the assay, we found among 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 suggest either the presence of some other biochemical defect or that in vivo dysfunctional receptors may be measured as normal in some patients with our assay.
Løhne
K
K
Department of Clinical Chemistry, Ullevål University Hospital, Oslo, Norway.
Urdal
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P
Leren
T P
TP
Tonstad
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S
Ose
L
L
D000328:Adult; D001055:Apolipoproteins B / Q000097:blood; D002232:Carbocyanines / Q000378:metabolism; D002784:Cholesterol / Q000097:blood; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D005456:Fluorescent Dyes / Q000378:metabolism; D006580:Genetic Carrier Screening; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000097:blood*; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D008875:Middle Aged; D011973:Receptors, LDL / Q000032:analysis*
D001055:Apolipoproteins B; D002232:Carbocyanines; D005456:Fluorescent Dyes; D011973:Receptors, LDL; C024286:3,3'-dioctadecylindocarbocyanine; D002784:Cholesterol