Detection of microchimerism by PCR is a function of amplification strategy

BACKGROUND: Suitable detection methods are needed to support larger studies of microchimerism and the allogeneic exposures that may be etiologically r elated to it. STUDY DESIGN AND METHODS: A two-tier PCR strategy for microchimerism detect ion was developed on the basis of the observation that assay sensitivity fo r the detection of microchimerism depends on the specificity with which pri mer pairs recognize sequences unique to the minor population. First, specim ens are tested to determine the host HLA class II genotype by using a locus -specific PCR strategy with low sensitivity for microchimerism. Then, a seq uence-specific PCR analysis having high sensitivity for detection of microc himerism is applied to detect and quantitate the minor population. Locus-sp ecific, group-specific, and sequence-specific amplification strategies for the detection of distinct minor WBC populations prepared ex vivo were compa red. In addition, 39 clinical samples from patients with known transfusion- associated microchimerism and 20 umbilical cord blood (CB) specimens contai ning maternal WBCs were studied. RESULTS: Locus-specific amplification detected 17 (94%) of 18 cases in whic h microchimerism was present at 10 percent, but only 1 of 51 cases with mic rochimerism II percent. Group-specific amplification detected all 63 cases with minor populations present at 10.10 percent, but only 16 of 21 cases at the 0.01 percent level. Sequence-specific amplification detected 100 perce nt of cases down to the 0.01 percent level. When applied to clinical sample s, locus-specific amplification reliably identified the major population bu t proved insensitive to low-level minor populations. CONCLUSIONS: For the detection of microchimerism, assay sensitivity is a fu nction of amplification strategy. These results suggest a simple approach t o population screening for microchimerism: the background population of WBC s is typed by a locus-specific method, while minor population(s) can then b e sought by using one or several sequence-specific amplifications.