Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1471-1477
Nested Polymerase Chain Reaction With Sequence-Specific
Primers Typing for HLA-A, -B, and -C Alleles: Detection
of Microchimerism in DR-Matched Individuals
Anthony S. Carter,
Lucia Cerundolo,
Mike Bunce,
Dicken D.H. Koo,
Kenneth I. Welsh,
Peter J. Morris, and
Susan V. Fuggle
From the Nuffield Department of Surgery, University of Oxford, John
Radcliffe Hospital, Oxford, UK.
It is widely accepted that donor leukocytes survive within the
recipient periphery after blood transfusion or solid organ transplantation. The significance of this microchimerism remains unclear, partially because of the insecurity of assays used to detect
the donor-derived material. The techniques used to detect donor-derived
DNA within recipient peripheral blood rely largely on major
histocompatibility complex class II polymorphism. We and others have
shown that the sensitivity of polymerase chain reaction with
sequence-specific primers (PCR-SSP) typing for HLA class
II alleles can be increased 100-fold by the addition of a primary
amplification step (nested PCR-SSP). We have now extended this
technique to encompass typing for HLA class I alleles, thereby adding
flexibility to microchimerism testing by enabling testing of recipients
HLA-DR matched with their donors. However, the high level of
sensitivity achieved with the technique (1:100,000) leads to a
concomitant decrease in the specificity that results in the amplification of unexpected products, a phenomenon we encountered in
the development of our nested PCR-SSP typing system for HLA class II
alleles. We describe here how it is possible to compensate for these
anomalies by including multiple testing of a pretransfusion sample that
acts as a specificity control, establishing a rigorous baseline for
subsequent analysis.
