Methodological Aspects of Flow Cytometry to Measure Low-Density Lipoprotein Receptor Expression on Peripheral Monocytes

Conflicting reports question the value of low-density lipoprotein (LDL) cholesterol receptor expression assays by flow cytometry to diagnose individual cases of heterozygous familial hypercholesterolemia (FH). We used genetically proven index patients with FH to assess an optimized LDL receptor expression assay on mononuclear cells. Assay conditions (preincubation period, staining procedure, mode of data calculation) were investigated for their impact on test reproducibility and plausibility in 20 patients with clinically diagnosed FH and 6 control subjects. This approach was then validated in 4 genetically characterized index patients. Good discrimination of controls and patients was observed after 4 and 7 days but not terol, resulting in hypercholesterolemia, premature atherosclerosis, and coronary heart disease. Unlike sporadic hypercholesterolemia, FH is associated with such high coronary heart disease risk that drug treatment is always necessary.1,2 More than 300 different mutations have been identified, which can be diagnosed by molecular techniques. Since no routine test of LDL receptor expression in vivo is presently available, the LDL receptor is assessed ex vivo by activity assays. The labor-intensive analysis of LDL binding, uptake, and internalization from patientderived skin fibroblasts is widely accepted for research use. However, no routine assay has been established. The measurement of LDL receptor activity can be performed on peripheral blood mononuclear cells using flow cytometry and fluorescent dye-conjugated LDLreceptor specific antibodies. Conflicting reports question the value of these methods to diagnose individual cases of heterozygous FH. Several investigators noted that LDLreceptor studies are possible for homozygous FH, but result in a large overlap between patients with heterozygous FH and normal subjects.3-5 In this respect, it is also of concern that studies relied on clinically diagnosed rather than genetically typed cases of heterozygous FH to assess the value of LDL-receptor assays. We assessed assay conditions such as preincubation period, staining procedure, and mode of data calculation, and investigated their impact on test reproducibility and plausibility in control subjects and clinically diagnosed cases of heterozygous FH. Based on these experiments we assessed patients with characterized mutation of the LDL receptor gene. Our results indicate that standardization of cell preincubation time and repeated testing significantly improves methods currently used to distinguish healthy subjects from patients with heterozygous FH.