Evaluation of Imprecision for Analysis of Short Tandem Repeats by Use of Mixed Blood Cells in Variable Concentrations

Abstract

Monitoring of chimerism after allogeneic stem cell transplantation is important for the early diagnosis of graft failure or disease relapse. Two main approaches used for monitoring chimerism are fluorescence in situ hybridization and PCR of short tandem repeats (STRs) expressing high degrees of polymorphism (1). Although both methods are useful, the STR assay has been increasingly used because fluorescence in situ hybridization can be used only in cases with specific genetic aberrations or a sex-mismatched donor (2)(3)(4). The STR assay, which produces quantitative results within 1 day, uses fluorescence-labeled primers and a capillary electrophoresis system (5)(6). However, the precision of the assay’s performance with respect to the chimeric stages of hematopoietic cells has not been fully investigated. We therefore aimed to evaluate the assay imprecision (CV) for seven STRs, D7S820, D8S1179, D16S539, D18S51, D21S11, TH01, and TPOX, to determine whether the precision changes according to the degree of chimerism. We used cell mixtures at various concentrations to simulate hematopoietic chimerism, and we also determined the detection limit. In an initial screening to find adequate samples for evaluation, we obtained peripheral blood specimens from 96 volunteer donors. Genomic DNA was isolated by use of Wizard Genomic DNA purification reagents (Promega) and was assayed for allele determination for seven different STRs. PCRs were set up in a final volume of 25 μL containing 10× buffer, 200 μM deoxynucleotide triphosphates, 5 pmol of each primer labeled with a fluorescence dye, 1 U of Taq polymerase (Roche), and template DNA. PCR was carried out in a GeneAmp PCR System 9600 (Applied Biosystems), and PCR products were analyzed by capillary electrophoresis on an ABI 3100 (Applied Biosystems) with performance-optimized polymer 4 (POP-4), a 47-cm capillary, and GA buffer plus EDTA. We mixed 1 μL …