Selection of Primers and Probes for Clonality Tests

Each laboratory should individually evaluate which assays are most suitable to answer the diagnostic questions that arise in its patient population, and which set of probes and primers are most appropriate. A recent multicenter study16 reports that laboratories routinely use two to five different restriction enzymes for SBA analysis of the TCRB chain gene. The majority of laboratories that participated in this study (9 of 13) used three enzymes, as recommended by CLSI.12 Ten of 13 participating laboratories also used only a single hybridization probe, whereas the remaining three laboratories in the study used two probes. The use of the additional second probe may be a valid alternative to the use of a larger number of enzymes when the available tissue is minimal. Whereas each additional enzyme digest requires additional tissue, the same Southern blot can be stripped and evaluated with additional probes without the need for more sample. In such cases, however, hybridized material must be removed efficiently without significant loss of genomic DNA from the membrane.

As mentioned previously,the false-negative rate of TCRG PCR can be significantly reduced if primers directed against all of the V and J regions are used. Such an approach at a single institution was recently reported to detect 95% of clonal T-cell proliferations by PCR analysis.

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