PCR-Based Methodology for Molecular Microchimerism Detection and Quantification

This Article Figures Only Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Pujal, J.-M. Articles by Gallardo, D. Search for Related Content PubMed PubMed Citation Social Bookmarking What’s this?

Josep-Maria Pujal1 and
David Gallardo

Translational Research Laboratory, Institut Català d’Oncologia, Hospital Duran i Reynals, L’Hospitalet de Llobregat, Barcelona, Spain

¹ Translational Research Laboratory, Institut Català d’Oncologia, Hospital Duran i Reynals, Avda Gran Via s/n, Km 2.7, 08907 L’Hospitalet de Llobregat, Barcelona, Spain. E-mail: jmpujal{at}idibell.org

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 1:10⁵ and 1:10⁶ 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 1:10⁴ DPRC for HLA-DRB1 PCR-SSP. The clinical application of these techniques in solid organ transplanted recipients showed microchimerism levels ranging from 1:10⁴ to 1:10⁶ DPRC after kidney or heart transplantation, and 1 log higher (1:10³ to 1:10⁶ DPRC) after liver transplantation. Inconclusion, the standardization of molecular microchimerismdetection techniques will allow for comparable interpretationof results in microchimerism detection for diagnostic or researchstudies.

Keywords: microchimerism, real-time PCR, detection, quantification

This study was partially financed by a Fundació MaratóTV3 2000 grant. The author (JMP) was the recipient of a grantFundació Crèdit Andorrà 2002–2005.

CiteULike    Complore    Connotea    Delicious    Digg    Facebook    Reddit    Technorati    Twitter    What’s this?