Abstract
Purpose: :
Currently, several genetic/molecular tests are available to patients for estimating risk associated with age-related macular degeneration (AMD) progression based on the detection of genetic variants ranging from 1 to 6 loci per test. Saliva, blood, and buccal swabs in equal frequency serve as the matrix type for these tests. We have developed a new genetic laboratory developed test (LDT) capable of genotyping 13 polymorphic sites that strongly associate with risk of AMD progression to the wet form. Here we investigate the clinical appropriateness of buccal swabs and EDTA whole blood specimens as the sample matrix for this test.
Methods: :
Buccal swabs and EDTA whole blood specimens were obtained under informed consent from healthy donors. Processing to DNA from blood and buccal samples utilized a variety of methodologies, both manual and automated. Following PCR amplification, single base extension allowed for detection of genotypes via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In addition to DNA yields, genotyping success, genotyping coverage, rate of detection primer extension, and signal-to-noise ratio were the metrics monitored to assess the AMD test performance.
Results: :
A comparison of blood and buccal samples from the same donor (n=100) demonstrated 100% agreement in genotype consensus and no difference in AMD test analytical metrics. Buccal swab collections (Isohelix SK-1 kit) did not demonstrate any significant differences in DNA yield when collections were self- versus professionally-administered (p=0.508); taken from left versus right cheek (p=0.161); underwent DNA extraction using Isohelix versus Qiagen procedures and reagents (p=0.230). However, statistically significant differences in yields were observed when buccal specimens underwent automated DNA extraction using a magnetic-particle based (10.55ng/uL) versus semi-automated silica membrane based (79.7ng/uL) technologies. When hemoglobin, bacterial DNA, starch, or ethanol was added to buccal collections, no effect was observed on downstream AMD performance metrics. The limit of detection for the AMD test was at approximately 53 genomic copies, 99% CI.
Conclusions: :
DNA extracted from buccal swabs and whole blood proved to be a suitable matrix for the AMD genetic test. A wide range of DNA yields could be accommodated by the test and no discernible effect on analytical test performance was observed by the inclusion of interferents common to the oral cavity.
Keywords: age-related macular degeneration • clinical laboratory testing • detection