PCR-based methodology for molecular microchimerism detection and quantification.
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.
Pujal
Josep-Maria
JM
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.org
Gallardo
David
D
D000220:Adaptation, Biological / Q000235:genetics; D000483:Alleles; D046528:Chimerism; D005260:Female; D015234:HLA-A Antigens / Q000235:genetics; D015235:HLA-B Antigens / Q000235:genetics; D006801:Humans; D008297:Male; D018895:Microsatellite Repeats; D016377:Organ Transplantation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic / Q000235:genetics
D015234:HLA-A Antigens; D015235:HLA-B Antigens