CHIMERISM ANALYSIS BY LINEAGE-SPECIFIC FLUORESCENT POLYMERASE CHAIN REACTION IN SECONDARY GRAFT FAILURE AFTER ALLOGENEIC STEM CELL TRANSPLANTATION

Background. Chimerism analysis is essential in understanding the etiology of graft failure occurring after allogeneic stem cell transplantation. The detection of marrow and/or blood host cells suggests graft rejection, relapse of the underlying disease, or a state of stable mixed chimerism. However, complete donor chimerism may be observed in some cases. Our objective was to characterize, by a sensitive process of chimerism analysis, six cases of graft failure occurring after transplant. Methods. Six cases of secondary graft failure, in which previous analysis had shown complete donor chimerism by standard polymerase chain reaction amplification of variable number of tandem repeats, were studied. In order to detect a minority population of recipient cells, we increased the sensitivity of the process by using fluorescent polymerase chain reaction and analyzing the origin of T, B, and natural killer lymphocytes at the time of graft failure. Results. The complete donor origin of mononuclear cells and lymphocytic populations was confirmed with this method in five of six patients. In the remaining patient, diagnosis of graft failure was clarified by the detection of a previously undetected mixed chimerism, compatible with graft rejection. In the other five patients, graft rejection was thereby excluded and graft failure could be related to viral infection or to graft-versus-host disease. Conclusion. Our sensitive process of fluorescent lineage-specific chimerism analysis may help in distinguishing between graft rejection and other mechanisms of graft failure, which is essential for deciding appropriate therapy. Secondary graft failure, the occurrence of pancytopenia after initial engraftment of an allogeneic transplant, is often a diagnostic problem. Recipients of T cell-depleted marrow may be particularly at risk for graft failure (1, 2), but it may be seen in recipients of unmodified, HLA-identical or nonidentical, marrow (3, 4). Immunologically mediated graft rejection, secondary lack of sustained marrow engraftment, and relapse of the underlying disease are the main diagnoses to be considered in this situation. Chimerism analysis is necessary to distinguish among these possibilities, if relapse is not apparent in marrow morphology. In some cases, the detection of residual host cells by chimerism analysis may represent a small number of malignant host cells not evident on marrow slides (5, 6). In other cases, the detection of residual host cells suggests either graft rejection (7) or a state of long-term stable mixed chimerism (8). To distinguish between these possibilities, the degree and evolution of mixed chimerism, determined by serial analysis over time, may be helpful. In contrast, the persistence of complete donor chimerism favors secondary lack of engraftment, as a result of infection (especially cytomegalovirus), the toxic effect of drugs, or graft-versus-host disease (GVHD) (9, 10). The sensitivity of detection of recipient cells is the essential parameter of chimerism analysis in this setting (11). A minority population of recipient cells could be undetected against a background of donor cells if an insensitive process is used. The suitable process of chimerism analysis must be applicable to the small number of cells that can be collected from hypocellular blood or marrow at the time of graft failure. Our objective was to study cases of secondary graft failure with a sensitive method of chimerism analysis. We retrospectively studied six patients, in whom previous analysis had shown complete donor chimerism using standard polymerase chain reaction (PCR) amplification of variable number of tandem repeat (VNTR) sequences. In order to detect a minority population of recipient cells, we sought to increase the sensitivity of the process by using fluorescent primers and automated GeneScan analysis of PCR products. In five patients, we applied this method separately to lymphocytic T-, B-, and natural killer (NK) cell subpopulations, sorted at the time of graft failure. We also examined the potential etiologies of graft failure and the impact of therapy on achieving subsequent engraftment.