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Regular Manuscript

PCR-based methodology for molecular microchimerism detection and quantification

¹ Hospital Duran i Reynals - Institut Catala d'Oncologia

^* To whom correspondence should be addressed. E-mail: jmpujal{at}idibell.org.

	Abstract

Background: Peripheral blood microchimerism after pregnancy or solid organ transplantation has been widely studied but a consensus on its detection has not yet been adopted. Objectives: To establish a panel of reproducible molecular PCR-based methods for detection and quantification of foreign cells in an individual. Methods: We analysed length polymorphisms generated by STR and VNTR markers. HLA-A and -B polymorphisms were detected by RSCA. Class II polymorphisms on HLA-DRB1 locus were analysed both by classical PCR-SSP and by quantitative PCR (Q-PCR). Also, 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 lowest detectable microchimerism level, when present. Results: We could detect microchimerism in more than 96% and more than 86% of cases at levels as low as 1:10e5 and 1:10e6 donor per recipient cells (DPRC) respectively, using Q-PCR for SRY or for non-shared HLA-DRB1 alleles. These techniques allowed as low as 1 genome-equivalent cell detection. Lower levels (nanochimerism) could be detected but not quantified due to technique limitations. On the other hand, classical PCR methods allowed detection down to 1:10e4 DPRC for HLA-DRB1 SSP-PCR. The clinical application of these techniques in solid organ transplanted recipients showed microchimerism levels ranging from 1:10e4 to 1:10e6 DPRC after kidney or heart transplantation, and 1 log higher (1:10e3 to 1:10e6 DPRC) after liver transplantation. Conclusion: The standardization of molecular microchimerism detection techniques will allow for comparable interpretation of results in microchimerism detection for diagnostic or research studies.

Key Words: Microchimerism, Transplantation, Macrochimerism, Quantification