title: Fluorescence flow cytometry of human leukocytes in the detection of LDL receptor defects in the differential diagnosis of hypercholesterolemia.
abstract: A flow-cytometric method with fluorescence-labeled monoclonal antibodies (MABs) against the low density lipoprotein (LDL) receptor (C7A MAB) or 3,3'-dioctadecylindocarbocyanin-iodide (DiI) LDL has been developed that allows the quantification of LDL receptors on leukocytes and the identification of patients with familial hypercholesterolemia (FH) within 48 hours. Leukocytes were isolated from 10 mL anticoagulated blood by density gradient centrifugation. To induce maximal expression of LDL receptors, mononuclear cells were preincubated with either phytohemagglutinine (PHA) or lipoprotein-deficient serum (LPDS). LPDS-treated monocytes provided a more homogeneous cell population with regard to LDL receptor activity than did the PHA-treated lymphocytes; they also provided a greater discrimination between the fluorescence of the receptor probes and cellular autofluorescence. The C7A MAB was able to compete for DiI LDL binding by about 40%. In competition with unlabeled LDL, DiI LDL revealed linear binding, indicating an affinity similar to native LDL. The binding characteristics of DiI LDL were also similar to 125I-LDL binding. LDL isolated from familial defective apolipoprotein B-100 was not able to compete for DiI LDL binding on monocytes, whereas native LDL reduced it by about 80%. In monocytes from FH heterozygous patients, the cellular mean fluorescence using either C7A MAB or DiI LDL at 4 degrees C was 30% to 70%; in FH homozygotes, cellular mean fluorescence was less than 20% of that in monocytes from normal individuals. In patients with familial defective apolipoprotein B-100 antibody binding was normal, but one patient's own LDL failed to compete with normal DiI LDL for 4 degrees C binding on U937 test monocytes. Patient monocytes having internalization defects showed normal 4 degrees C DiI LDL binding, but at 20 degrees C cell-associated fluorescence was reduced by about 40%. In our study 384 hypercholesterolemic patients (preselected according to serum cholesterol levels, clinical symptoms, and family history) were analyzed for LDL receptor expression using the C7A MAB-based assay. In 71.8% of the patients with cholesterol levels higher than 300 mg/dL, an LDL receptor deficiency was observed. Apolipoprotein E isoforms and lipoprotein[a] were found to be independent from the LDL receptor status. In some patients with high cholesterol levels but normal LDL receptor expression with the C7A MAB assay, LDL receptor defects could be diagnosed when either reduced binding or internalization of DiI LDL or familial defective apolipoprotein B-100 was detected.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Institut für Klinische Chemie und Laboratoriumsmedizin, Universität Regensburg, FRG.
authors.forename: G
authors.initials: G
authors.lastname: Schmitz
authors.forename: T
authors.initials: T
authors.lastname: Brüning
authors.forename: E
authors.initials: E
authors.lastname: Kovacs
authors.forename: S
authors.initials: S
authors.lastname: Barlage
mesh_terms: D000328:Adult; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001666:Binding Sites, Antibody; D002232:Carbocyanines; D002469:Cell Separation; D003937:Diagnosis, Differential; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D006937:Hypercholesterolemia / Q000175:diagnosis*; D007963:Leukocytes, Mononuclear / Q000648:ultrastructure*; D008297:Male; D009000:Monocytes / Q000378:metabolism; D011973:Receptors, LDL / Q000276:immunology / Q000502:physiology*
chemical_list: D000911:Antibodies, Monoclonal; D002232:Carbocyanines; D011973:Receptors, LDL