Enhanced ascertainment of microchimerism with real-time quantitative polymerase chain reaction amplification of insertion-deletion polymorphisms.
BACKGROUND
The characterization of microchimerism (MC) by gene amplification has been limited by few allogeneic markers, ascertainment bias, and assay analytic performance. To address this, a panel of 12 MC assays based on insertion-deletion (InDel) polymorphisms had been optimized.


METHODS
The InDel assays were validated with comprehensive in vitro spiking studies at the stochastic limit of detection. Their ability was also determined to ascertain MC of unknown source genotype with both theoretical and actual donor-recipient pairs, and the assays were applied to a clinical population of 73 trauma patients who received transfusions where MC was previously characterized by HLA-based assays alone.


RESULTS
In the stochastic spiking experiments, all assays were sensitive to a single copy of target DNA, and no false-positive amplification occurred among 1128 samples studied. Among 219 theoretical donor-recipient pairs, informative alleles existed for 99.5 percent with both InDel and HLA compared to 91.3 percent with HLA alone. In the clinical population, 33 cases of MC were detected (9 more cases than by HLA-DR alone) in the nonleukoreduced (non-LR) group and 8 cases (1 more case than by HLA-DR) in the LR group for the short-term follow-up. Among 27 long-term follow-up samples, 8 cases were detected overall (3 more cases than by HLA-DR alone).


CONCLUSIONS
It is concluded that an InDel-based assay panel has excellent technical performance characteristics while also allowing for ascertainment of some MC cases not detectable with HLA alone. The tandem use of both the InDel and the HLA provides a powerful tool for the enhanced ascertainment of MC.
Lee
Tzong-Hae
TH
Blood Systems Research Institute, San Francisco, California 94118, USA. tlee@bloodsystems.org
Chafets
Daniel M
DM
Reed
William
W
Wen
Li
L
Yang
Yunting
Y
Chen
Jennifer
J
Utter
Garth H
GH
Owings
John T
JT
Busch
Michael P
MP
D000328:Adult; D001782:Blood Donors; D046528:Chimerism; D005189:False Positive Reactions; D005260:Female; D005500:Follow-Up Studies; D006684:HLA-DR Antigens / Q000235:genetics*; D006801:Humans; D047589:Leukocyte Reduction Procedures; D008297:Male; D008875:Middle Aged; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D017384:Sequence Deletion; D014947:Wounds and Injuries / Q000150:complications / Q000235:genetics / Q000628:therapy
D006684:HLA-DR Antigens