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Post-BMT Chimerism Quantification by FISH on Routine Smears Easier, Faster and More Sensitive Than STR-PCR

, : Post-BMT Chimerism Quantification by FISH on Routine Smears Easier, Faster and More Sensitive Than STR-PCR. Blood 100(11): Abstract No 5271, November 16

Post-BMT chimerism quantification is useful for the prediction/early detection of graft failure/rejection or disease relapse, as well as to follow up response to treatment with immunosuppresion withdrawal, DLI, etc. A sensitive, quantitative, easy and rapid method is needed for the detection of residual/emerging recipient cells after BMT. We report the use of FISH to detect sex chromosomes (XY-FISH), after sex-mismatched transplants, on routine bone marrow (BM) or peripheral blood (PB) smears. The use of routine smears requires the introduction of minor modifications to the standard protocol (CEP XY, Vysis Inc.), such as pretreatment of the slides with 2xSSC at 37degreeC for 30 min, incubation with 0.001% pepsin HCl for 30 min, and longer denaturation times (5 min). High quality hybridization is obtained even after short hybridization times (4-5h), which enables the results to be obtained the same day the sample is taken. Additionally, the same protocol can be successfully performed on smears stored at room temperature for several years. Moreover, sequential FISH assays can be done with different probes in order to analyze minimal residual disease in those cells of recipient origin identified in a previous XY-FISH assay performed on the same smear. The sensitivity of the technique has been determined for different numbers of cells analyzed. Scoring only XX (female, 2 red signals) and XY (male, one red signal and one green signal) sensitivity is 1.5% when 200 nuclei are analyzed (figure used by most laboratories) and 0.6% scoring 500 nuclei, which can be achieved in less than 30 min of microscope observation. This sensitivity is considerable better than that of VNTR/STR-PCR revealed on agarose/acrilamide gel, which is 3-5% depending on the marker used. Finally, scoring 3000 cells allows to increase the sensitivity up to 0.1%, which is similar to that of expensive PCR methods using fluorescent primers and analyzing the result on automated DNA sequencing machines. Therefore, XY-FISH should be the method of choice for chimerism quantification in the sex-mismatched setting.


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