Sequence Polymorphism Systems for Quantitative Real-Time Polymerase Chain Reaction to Characterize Hematopoietic Chimerism—High Informativity and Sensitivity As Well As Excellent Reproducibility and Precision of Measurement

Abstract

Sequence polymorphisms (SPs) can serve as genetic markers for quantitative polymerase chain reactions (qPCR) for chimerism analysis, providing a significantly higher sensitivity compared to short tandem repeat PCR. In this study, a panel of 29 selected markers was evaluated in 317 patients with leukemia and myelodysplastic syndrome, who received allogeneic stem cell transplantation. In total, 5415 posttransplantation samples were analyzed. Recipient genotype discrimination was possible in 96% with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Marker specific standard dilution series from volunteers' DNA served as standard for quantification of chimerism. Sensitivity of the method was < or =1 x 10-3 (0.1% of recipient cells) in 83.3% of the assays. By this method, it was possible to very accurately detect autologous signals in the range from 0% to 0.5% (95% confidence interval [CI] +/-0.2), from 0.5% to 1% (95% CI +/-0.4), from 1% to 2% (95% CI +/-0.6) and from 2% to 5% (95% CI +/-1.2). Reproducibility of the quantified autologous signals was independent from the amount of DNA. This is the first report on a SP-based chimerism system allowing for the performance of chimerism analyses for virtually all patients with high sensitivity, excellent reproducibility, and precision of measurement.

Validation of chimerism in pediatric recipients of allogeneic hematopoietic stem cell transplantation (HSCT) a comparison between two methods

“…10,11 In recent years, more sensitive methods for determining chimerism based on real-time PCR have been available. 17 Real-time PCR allows sensitive detection of the DNA product, ensures detection during the linear range of amplification, eliminates the need for post-PCR analysis, and incorporates specialized software to simplify data analysis, 18 such as AlleleSEQR Software.…”

“…[7][8][9] Since the 1980s, a variety of techniques that employ polymorphic markers have been established to survey chimerism status, 10 such as the following: fluorescent in situ hybridization (FISH) with XY chromosome-specific probes or polymerase chain reaction (PCR), single-nucleotide polymorphism (SNP) analyses, short tandem repeats (STR), restriction fragment length polymorphism (RFLP) and variable number tandem repeats (VNTR). [10][11][12][13] However, several limitations have been reported associated with these techniques namely low sensitivity, time-consuming, limited to sex-mismatched transplantations, high DNA requirement and limited degree of polymorphism. 11,13 For all these clinical applications, the optimal methodological approach needs to be informative, sensitive, quantitatively accurate, reproducible and cost effective.…”

“…[10][11][12][13] However, several limitations have been reported associated with these techniques namely low sensitivity, time-consuming, limited to sex-mismatched transplantations, high DNA requirement and limited degree of polymorphism. 11,13 For all these clinical applications, the optimal methodological approach needs to be informative, sensitive, quantitatively accurate, reproducible and cost effective. [13][14][15][16] In the present study, we compared and validated chimerism in pediatric recipients of post-HSCT between two methods: quantitative real-time PCR (qPCR) vs. variable number tandem repeats PCR (VNTR) to find a more accurate and efficient methodology to asses chimerism.…”

Single Nucleotide Polymorphism-Based System Improves the Applicability of Quantitative PCR for Chimerism Monitoring

“…This compares favorably with the highest level of informativity reported to date. 14 In this study we have adapted the approach of the individualized reference system used for chimerism quantification by ⌬⌬Ct. This method provides reliable quantitative data if the amplification efficiencies of both target and reference genes are similar.…”

“…Detection of 0.1% of target DNA has been reported for SNP 8,14,21 compared with 0.1% to 0.01% for bialellic short insertion/deletion polymorphisms. 6,14,22 The detection limit of our SNP-specific qPCR assay was evaluated with dilution experiments of allele-positive DNA in allele-negative DNA. A linear correlation of target DNA template fraction (r ϭ 99%) for all SNP markers was detected down to 0.1% of the test template (Figure 1).…”