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PCR-based methodology for molecular microchimerism detection and quantification.
Exp Biol Med (Maywood). 2008 Sep; 233(9):1161-70.EB

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.

Authors+Show Affiliations

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. jmpujal@idibell.orgNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

18535170

Citation

Pujal, Josep-Maria, and David Gallardo. "PCR-based Methodology for Molecular Microchimerism Detection and Quantification." Experimental Biology and Medicine (Maywood, N.J.), vol. 233, no. 9, 2008, pp. 1161-70.
Pujal JM, Gallardo D. PCR-based methodology for molecular microchimerism detection and quantification. Exp Biol Med (Maywood). 2008;233(9):1161-70.
Pujal, J. M., & Gallardo, D. (2008). PCR-based methodology for molecular microchimerism detection and quantification. Experimental Biology and Medicine (Maywood, N.J.), 233(9), 1161-70. https://doi.org/10.3181/0802-RM-35
Pujal JM, Gallardo D. PCR-based Methodology for Molecular Microchimerism Detection and Quantification. Exp Biol Med (Maywood). 2008;233(9):1161-70. PubMed PMID: 18535170.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - PCR-based methodology for molecular microchimerism detection and quantification. AU - Pujal,Josep-Maria, AU - Gallardo,David, Y1 - 2008/06/05/ PY - 2008/6/7/pubmed PY - 2008/10/1/medline PY - 2008/6/7/entrez SP - 1161 EP - 70 JF - Experimental biology and medicine (Maywood, N.J.) JO - Exp Biol Med (Maywood) VL - 233 IS - 9 N2 - 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. SN - 1535-3702 UR - https://www.unboundmedicine.com/medline/citation/18535170/PCR_based_methodology_for_molecular_microchimerism_detection_and_quantification_ L2 - https://journals.sagepub.com/doi/10.3181/0802-RM-35?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -