Flow cytometry with a monoclonal antibody to the low density lipoprotein receptor compared with gene mutation detection in diagnosis of heterozygous familial hypercholesterolemia

Abstract

We used a fluorescence flow cytometry assay with a monoclonal low density lipoprotein (LDL) receptor-specific antibody to detect LDL receptor expression on blood T lymphocytes and monocytes. We prepared peripheral blood mononuclear cells from patients with genetically verified LDL receptor-defective (Trp66-Gly mutation, n = 17) or receptor-negative (Trp23-stop mutation, n = 17) heterozygous familial hypercholesterolemia (FH) and from healthy individuals (n = 24). The cells were stimulated to express the maximum amount of LDL receptor by preincubation in lipoprotein-deficient medium. A dual-labeling technique allowed flow cytometric analysis of LDL receptor expression on cells identified by fluorescently conjugated surface marker antibodies. Knowing the LDL receptor gene mutation of the FH patients allowed us to compare the diagnostic capability of this functional assay with the DNA diagnosis and to validate the assay with molecular genetics instead of clinical indices of heterozygous FH. T lymphocytes expressed more LDL receptors and gave better diagnostic results than monocytes, and cells from patients with either the Trp66-Gly or the Trp23-stop mutation had variable but significantly reduced LDL receptor expression. The data indicate that this fluorescence flow cytometry assay is unsuitable for diagnosis of individual cases of heterozygous FH but that it may be useful for functionally characterizing mutations in the LDL receptor gene.

The complex of the insect LDL receptor homolog, lipophorin receptor, LpR, and its lipoprotein ligand does not dissociate under endosomal conditions

“…Together, our data indicate that the complex of LpR and HDLp remains intact during its intracellular itinerary, which is in complete agreement with the occurrence of ligand recycling [14,[16][17][18], and may provide a vital determinant of the ligand-recycling capacity of LpR. In several studies, flow cytometry has been used to quantify lipoprotein binding and uptake [29,[53][54][55][56][57][58][59]. In most cases, the experiments were performed on blood cells.…”

Flow cytometric assessment of LDL receptor activity in peripheral blood mononuclear cells compared to gene mutation detection in diagnosis of heterozygous familial hypercholesterolemia

“…A complete functional characterization of mutations in the LDL receptor gene, therefore, requires measurements of LDL receptor expression, LDL binding, and LDL internalization. We have recently reported our experience with a fluorescence flow cytometry (FFC) assay using a monoclonal LDL receptor specific antibody to determine LDL receptor expression on stimulated peripheral blood mononuclear cells (27). In the present study, we report our experience with an FFC assay using DiI-LDL to measure LDL binding and internalization on stimulated peripheral blood mononuclear cells.…”

“…Blood was cooled to 20°C and diluted with Hank's buffered saline solution. Peripheral blood mononuclear cells were prepared under sterile conditions using a modified version of the method of Böyum (5) and preincubated for 46 h at 37°C in a humidified carbon dioxide incubator as described previously (27).…”

Functional characterization of two low density lipoprotein receptor gene mutations by fluorescence flow cytometric assessment of receptor activity in stimulated human T‐lymphocytes

“…Measurements of LDL receptor activity were performed using a two-color FFC assay with stimulated peripheral blood T-lymphocytes from patients with heterozygous FH. We have previously validated the FFC assay in evaluating LDL receptor activity in heterozygous FH patients (10,11). The assay uses monoclonal LDL receptor specific antibody (C7) to determine LDL receptor expression and fluorescently conjugated LDL (DiI-LDL) to measure binding and internalization of LDL.…”

“…The procedures of this investigation have been described in detail in previous reports (10,11) and are briefly summarized here.…”