Analytical Validation of Familial Hypercholesterolemia Biomarkers in Dried Blood Spots.

Michael Lasarev,Amy Peterson,Vanessa Horner,Kristin Campbell,Amy E Wiberley-Bradford,Patrice K Held

doi:10.3390/ijns8010014

Abstract

Heterozygous familial hypercholesterolemia (HeFH) is a common, treatable genetic disorder characterized by premature atherosclerosis and cardiovascular disease, yet the majority of affected individuals remain undiagnosed. Newborn screening could play a role in identification of at-risk individuals and provide an opportunity for early intervention, prior to the onset of symptoms. The objective of this study was to develop and validate assays for quantification of candidate HeFH biomarkers in dried blood spots (DBS). Commercially available enzyme assay kits for quantification of serum total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C) were modified for high-throughput analysis of DBS. Apolipoprotein B (ApoB) concentrations in DBS were measured using an immunoassay with modifications from published studies. All three assays were validated according to the College of American Pathologists guidelines for clinical laboratories. The performance of TC, LDL-C, and ApoB assays was assessed by precision, recovery, limit of quantification (LOQ) and linearity. Precision studies yielded coefficients of variation (CV) of less than 15%, with recovery greater than 75% for all three assays. The determined LOQ and linearity were comparable to serum-based assays. In a direct comparison between serum and DBS concentrations, positive correlations were demonstrated for TC, LDL-C, and ApoB. Additionally, the initial evaluation of the three biomarker concentrations within the unaffected population was similar to values obtained in previous published studies. This study reports on methods for quantification of TC, LDL-C, and ApoB in DBS. Assay validation results were within acceptable limits for newborn screening. This is an important first step toward the identification of newborns with HeFH.

Highlights

Heterozygous familial hypercholesterolemia (HeFH) is the most common, potentially fatal genetic disease in humans, with an incidence of about 1 in 200–300 individuals across all ethnic groups [1]
HeFH is characterized by markedly increased concentrations of low-density lipoprotein-cholesterol (LDL-C) that are present from birth, causing early and aggressive atherosclerotic plaque formation
Method precision for total cholesterol (TC), LDL-C, and Apolipoprotein B (ApoB) was assessed through analysis of three quality control materials: endogenous levels, and two spiked control specimens

Summary

Introduction

Heterozygous familial hypercholesterolemia (HeFH) is the most common, potentially fatal genetic disease in humans, with an incidence of about 1 in 200–300 individuals across all ethnic groups [1]. HeFH is characterized by markedly increased concentrations of low-density lipoprotein-cholesterol (LDL-C) that are present from birth, causing early and aggressive atherosclerotic plaque formation. This predisposes affected individuals to early onset of atherosclerotic cardiovascular disease [1–4]. Individuals may be affected with the homozygous form of familial hypercholesterolemia (HoFH). HoFH affects an estimated 1 in 160,000 to 400,000 individuals and is characterized by even higher levels of LDL-C, ranging from 500 to 1000 mg/dL. Affected individuals rarely live past 30 years old unless heroic measures are undertaken, including treatment with multiple lipid-lowering medications, LDL-C apheresis, and/or organ transplant [11,12]

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