Polymerase Chain Reaction Analysis

Unlike SBA, which compares germline and rearranged alleles of the genes being analyzed, PCR amplifies rearranged alleles only because germline alleles have too great a distance between the PCR primer binding sites to allow for amplification. PCR can be performed on fresh, frozen, or paraffin-embedded tissue, as well as microdis-sected and cytology specimens, because it is not as affected by DNA fragmentation as SBA. PCR also requires much less DNA or RNA and is more rapid than SBA. Thus, PCR has versatility not achieved by SBA.

PCR amplification of genomic DNA is used for detection of AgR rearrangements as well as many BCL-associated translocations. In genes with numerous translocation partners, however, FISH is more useful than PCR. Translocations involving variable sites over an area of a chromosome too large for PCR may be amenable to RT-PCR. RT-PCR takes advantage of the splicing out of introns that occurs in the generation of mRNA. RT-PCR cannot reliably be performed on paraffin-embedded tissue because intact RNA is difficult to extract from paraffin-embedded tissues.

PCR detection of IgVH rearrangements uses V- and J-region primers and relies on the V, D, and J segments being brought into close proximity during rearrangement so that the PCR reaction can amplify across these segments. The closest V and J segments are too far apart in the germline configuration for PCR amplification to occur. IGH clonal-ity analysis by PCR uses consensus primers designed to anneal to conserved IGH V- and J-region sequences. For IGH PCR, one J-region primer will recognize all six J segments because there is a single well-conserved region among the six J regions, but there is no single V-region primer that will recognize all V segments. V regions have three more highly conserved framework regions (FR I, II, and III) and intervening highly variable sequences called complementarity-determining regions (CDR I and II). Because the FR sequences are more conserved across the different V regions, the V-region primers are designed to bind to FR sequences. Most commonly, FR III-region primers are used because the FR III region is closest to the J region in the rearranged state, thus resulting in a smaller PCR product that results in more efficient amplification. FR III- and J-region primers amplify the highly variable V-D junction (CDR III) and detect 60% to 70% of rearrange ments. The sequence of the FR III V-region primer also affects the detection rate. One interlaboratory comparison showed a difference of from 55% to 70% based on the specific sequence used for the V-region primer.39 The addition of a second PCR amplification using a FR II V-region primer with a J-region primer also increases the detection rate of the test significantly (80% to 90%).

IGH PCR amplifies any rearranged IGH allele, such that there is a background signal from the polyclonal B cells present in a specimen that may obscure the signal from a monoclonal B-cell population. Numerous strategies are used for PCR product detection, most commonly gel electrophoresis with colorimetric, fluorescent, or chemiluminescent labeling. Capillary electrophoresis (CE) with fluorescently labeled primers provides slightly enhanced sensitivity and higher throughput, with improved resolution, and is becoming the system of choice for many laboratories. Interpretation of CE results may be more objective than the standard PCR gel format for demonstration of B-cell clonality. A polyclonal B-cell population produces a smear or ladder on a gel or multiple small peaks on a CE instrument printout, while monoclonal B-cell populations produce one or two distinct bands on a gel, or one or two sharp peaks on CE (Figure 32-5). One criterion developed for determining whether a peak is monoclonal compared to the background poly-clonal peaks is that the distinct peak should be more than two to three times the height of the adjacent polyclonal peaks. Specific criteria for interpretation of IGH PCR results have not been developed, especially with regard to the number of bands allowed per reaction. Sequence analysis of IGH PCR products can be done for identification of patient-specific sequences, which may then be used to design patient-specific primers for subsequent patient-specific PCR analyses for detection of MRD following therapy.

Was this article helpful?

0 0

Post a comment