PCR-Based Methodology for Molecular Microchimerism Detection and Quantification

Abstract

Peripheral blood microchimerism after pregnancy or solid organ transplantation has been widely studied, but a consensus on its detection has not yet been adopted. The objective of this study was to establish a panel of reproducible molecular polymerase chain reaction (PCR)-based methods for detection and quantification of foreign cells in an individual. We analyzed length polymorphisms generated by short tandem repeat (STR) and variable number tandem repeat (VNTR) markers. Human leukocyte antigen (HLA)-A and -B polymorphisms were detected by reference strand conformation analysis (RSCA). Class II polymorphisms on HLA-DRB1 locus were analyzed both by classical PCR-sequence-specific primers (SSP) and by quantitative PCR (Q-PCR). Also, sex-determining region-y gene (SRY) gene allowed specific male donor discrimination and quantification by Q-PCR in female recipients. Binomial statistical distribution analysis was used for each molecular technique to determine the number of PCR replicates of each sample. This analysis allowed the detection of the lowest detectable microchimerism level, when present. We could detect microchimerism in more than 96% and more than 86% of cases at levels as low as 1:10(5) and 1:10(6) donor per recipient cells (DPRC), respectively, using Q-PCR for SRY or for nonshared HLA-DRB1 alleles. These techniques allowed as low as 1 genome-equivalent cell detection. Lower levels (nanochimerism) could be detected but not quantified because of technique limitations. However, classical PCR methods allowed detection down to 1:10(4) DPRC for HLA-DRB1 PCR-SSP. The clinical application of these techniques in solid organ transplanted recipients showed microchimerism levels ranging from 1:10(4) to 1:10(6) DPRC after kidney or heart transplantation, and 1 log higher (1:10(3) to 1:10(6) DPRC) after liver transplantation. In conclusion, the standardization of molecular microchimerism detection techniques will allow for comparable interpretation of results in microchimerism detection for diagnostic or research studies.

HLA-targeted flow cytometric sorting of blood cells allows separation of pure and viable microchimeric cell populations

“…[25][26][27][28] The detection of Y-chromosome-localized genes by qPCR offers a reproducible and sensitive technique, 2,7,9,13,26 but it is restricted to sex-mismatched combinations only. HLAs represent a broader applicable target for the detection of microchimerism 8,25,26,29 because they are highly polymorphic 30 and thus frequently mismatched between 2 persons. The oligonucleotide primers that are suitable for PCR-based HLA typing of patients who undergo transplantation can be applied to detect microchimerism.…”

Naturally acquired microchimerism

“…126 Multicopy genes localized on the Y-chromosome represent suitable targets for reproducible and sensitive analyses by qPCR. 29,57,117,127,128 Its application is, however, restricted to sex-mismatched combinations, whereby male DNA targets are targeted in a female host. DYZ1 primers target a Y-chromosome selective sequence that is found 8 times in total in the DAZ2, DAZ3, and DAZ4 genes.…”

“…[116][117][118][119] Increased sensitivity may be achieved when the preliminary PCR is followed by second-round PCR with sequence-specific primers that target the first amplicon. [120][121][122][123] However, as we (M.M., B.M.K.-M., J.J.M.D., M.E.…”

“…HLA represents a broad applicable target for microchimerism detection, 116,117,129,130 as they are highly polymorphic 131 and thus frequently mismatched between two individuals, irrespective whether there is a gender mismatch or not. Over the past years, several research groups, including those from Lee Nelson and Nathalie Lambert (Seattle WA) and from Diana Bianchi (Boston MA), have been developing HLA-allele specific qPCR assays.…”

Comparison of different blood sample processing methods for sensitive detection of low level chimerism byRHDreal-time PCR assay

“…[8][9][10][11][12] So far, RHD qPCR has been used primarily to detect RHD sequences in cell-free fetal DNA, present in maternal plasma during pregnancy to predict the D blood group status of the fetus, without invasive procedures to obtain fetal genetic material [13][14][15] and for determination of (paternal) RHD zygosity. 16 concentrations from 900 to 50 nM and 250 to 50 nM, respectively.…”