Evidence for a graft-versus-mast-cell effect after allogeneic bone marrow transplantation

Mast cell leukemia (MCL) is a rare form of aggressive mastocytosis with a reported median survival below 6 months. Casuistic reports suggest the effectiveness of allogeneic bone marrow transplantation (BMT) for MCL. However, these reports lack clear evidence for a graft-versus-mast-cell (GvMC) effect. We prospectively investigated the GvMC at different time points after allogeneic BMT and donor-lymphocyte infusions (DLI). Samples were gathered from a patient with MCL treated with allogeneic BMT from an unrelated HLA identical donor. Parameters for detection of a GvMC effect included flow cytometrical analysis of mast cell (MC) populations in peripheral blood and BM, BM smear and histology, chimerism analysis of flow cytometrically sorted BM CD117+/CD34- MC and testing for anti-mast cell reactivity of donor lymphocytes by interferon (IFN)-gamma ELISPOT. DLIs reduced MC from 5 to 0.5%. MC chimerism analysis demonstrated a complete recipient genotype after BMT, suggesting that the persistent mastocytosis was part of residual neoplastic disease. At 3.7 years after BMT, there is some evidence for relapse. In summary, BMT and DLIs attenuated the mastocytosis from an aggressive to an indolent form and may have improved the patients' prognosis. The in vitro data of our study indicate for the first time the existence of a GvMC effect.     

Prognostic value of hematopoietic chimerism in patients with acute leukemia after allogeneic bone marrow transplantation: a prospective study

Hematopoietic chimerism as a predictive marker for the relapse of acute leukemia after allogeneic BMT was evaluated in a prospective study. Monthly assays of hematopoietic chimerism were performed from peripheral blood samples by PCR amplification of short tandem repeats or amelogenin loci. Between December 1997 and June 1999, 33 patients enrolled and 30 were evaluable (two early deaths, one lack of informative bands for chimerism evaluation). There were 14 male and 16 female patients (15 AML and 15 ALL) with a median age of 31 years (range 16-46). Mixed chimerism (MC) was observed at least once in 14 of 30 patients (47%). There was no significant difference between 14 patients who showed MC (MC group) and 16 patients who did not show MC (complete chimerism (CC) group) in terms of age, sex, disease status at BMT, donor type, and the number of bone marrow cells infused. There was no significant difference in the neutrophil and platelet engraftment rates between the two groups. After a median follow up of 10.9 months (range 4.3-22.4), five patients in the CC group and two patients in the MC group relapsed (P = 0.27). All five patients who relapsed in the CC group maintained CC up to 1 month prior to clinical relapse. Our study demonstrated that the patients who showed MC post BMT did not have higher risk of relapse of acute leukemia when compared to patients who did not show MC. Sensitive PCR-based assays for hematopoietic chimerism applied on a monthly basis after allogeneic BMT could not predict relapse of acute leukemia.     

Quantitative analysis of mixed chimerism following allogeneic bone marrow transplantation using telomere flow-FISH

Assessment of mixed chimerism is of particular interest for allogeneic bone marrow or peripheral blood stem cell transplantation with reduced-intensity conditioning in order to study contribution of donor-type and host-type lymphohematopoiesis. Because the length of telomere repeat sequences is frequently shorter in leukemic compared to normal hematopoietic cells, this telomere repeat polymorphism might be a useful marker to analyze mixed chimerism in selected patients with short telomeres. Recently, fluorescence in situ hybridization and flow cytometry (flow-FISH) have been shown to be valuable tools to analyze the mean telomere length in hematopoietic cells. Here, we demonstrate in a case study on a patient with chronic lymphocytic leukemia (CLL) that telomere flow-FISH can in principle be exploited to quantitate the amount of donor- and host-type cells for chimerism analysis based on distinct histogram distributions which reflect cell populations with different telomere length.     

Control of systemic B cell-mediated autoimmune disease by nonmyeloablative conditioning and major histocompatibility complex-mismatched allogeneic bone marrow transplantation

Systemic autoimmune disease (AID) can be controlled with conventional therapies in most patients. However, relapses are common, leading to progressive disability and premature death. Nonmyeloablative conditioning and allogeneic bone marrow transplantation (BMT) could be an effective treatment for severe AID, because of mild toxicity of the conditioning and the potential benefits of donor chimerism. We examined the effects of this treatment in experimental autoimmune arthritis. Our results demonstrate the induction of complete donor chimerism and significant suppression of disease activity. No clinical graft-versus-host disease (GVHD) was observed. The beneficial effects were most likely caused by the elimination of plasma cells producing pathogenic autoantibodies, because these antibodies disappeared rapidly after BMT. Although this type of treatment was effective in organ-specific T-cell-mediated AID, the present study provides convincing evidence that nonmyeloablative conditioning and allogeneic BMT can effectively treat severe B-cell-mediated AID with a systemic inflammatory component.     

Natural history of mixed chimerism after bone marrow transplantation with CD6-depleted allogeneic marrow: a stable equilibrium

Mixed hematopoietic chimerism (MC) is a common finding after allogeneic bone marrow transplantation (BMT), but the natural history of this phenomenon remains unclear. To understand the evolution and the implications of this finding, we performed a prospective analysis of the development of mixed chimerism in 43 patients with hematologic malignancies who received bone marrow (BM) from human leukocyte antigen (HLA)-identical sibling donors. T-cell depletion in vitro with anti-T12 (CD6) monoclonal antibody and rabbit complement was used as the only method of graft-versus-host disease (GVHD) prophylaxis. Overall, MC was identified in peripheral blood (PB) and BM in 22 of 43 (51%) patients evaluated. MC was found by restriction fragment length polymorphism (RFLP) analysis in 21 of 40 (53%) patients, by cytogenetic analysis in 6 of 29 (21%) patients, and by red blood cell phenotyping in 4 of 9 (44%) patients. RFLP studies were performed at 0.5, 1, 3, 6, 9, and 12 months post-BMT and then every 6 months, and showed a high probability of developing MC in the first 6 months after BMT followed by stabilization after 12 months. Cytogenetic analysis was less sensitive in detecting MC. Once MC was detected after BMT, the percentage of recipient cells increased very slowly over more than 3 years of follow-up, and no patient reverted to complete donor hematopoiesis (CDH). Thus, recipient and donor cells remained in a relative state of equilibrium for prolonged periods that seemed to favor recipient cells over donor cells. Patient's disease, remission status, or intensity of the transplant preparative regimen did not influence the subsequent development of mixed chimerism. Early immunologic reconstitution was the only factor that correlated with the subsequent chimeric status of the patients. The percentage and absolute number of T3 (CD3) and T4 (CD4) positive cells at day 14 after BMT were significantly higher in the patients who maintained CDH but NK cell reconstitution was similar in both groups, suggesting that early reconstitution with T cells may play a role in preventing recovery of recipient cells after BMT. GVHD was also associated with maintenance of CDH, but the probability of relapse, survival, and disease-free survival was identical in patients with MC and CDH.     

Mixed hematopoietic chimerism following bone marrow transplantation for hematologic malignancies

Twenty-nine of 172 patients (17%) who received an allogeneic bone marrow transplant (BMT) from histocompatible sibling donors for hematologic malignancies were mixed hematopoietic chimeras; ie, they had a mixture of donor and host hematopoietic or lymphohematopoietic cells at greater than or equal to 14 days after transplantation. Twenty- four of the 29 mixed chimeras (83%) have remained in continuous complete remission for up to 116 months (greater than 9 years) following BMT. Four of the 29 patients (14%) have had recurrent leukemia, and 7 of the 29 (24%) have had moderate or severe graft-v- host disease (GVHD). Twelve of these 29 patients have persisted as stable mixed chimeras for greater than or equal to 2 years after BMT, whereas other patients converted to all donor-type hematopoiesis. The incidence of mixed chimerism was independent of the pretransplant regimen, the donor or recipient age (less than 20 v greater than 20 years), remission status (first complete remission of acute leukemia and first chronic phase of chronic myelocytic leukemia v later stages of disease), and type of leukemia. Our data indicate that mixed hematopoietic chimerism is not rare after BMT for hematologic malignancies and that its presence is compatible with long-term disease- free survival. Prospective studies of mixed chimerism after BMT are warranted to achieve better understanding of its biologic importance.     

Hematopoietic donor chimerism and graft-versus-myeloma effect in relapse of multiple myeloma after allogeneic bone marrow transplantation

 A large group of patients relapsing after allogeneic bone marrow transplantation (BMT) have obtained remission after infusion of leukocytes from their original donor, suggesting a graft-versus-myeloma effect. However, side effects such as graft-versus-host disease and myelosuppression are severe, and sometimes fatal, complications of this therapeutic approach. Previously we demonstrated that patients with leukemia who lack donor hematopoiesis in relapse after BMT experience severe and lasting aplasia after infusion of donor leukocytes. In two patients – one with extramedullary and one with marrow relapse after a sex-mismatched transplantation – we analyzed hematopoietic chimerism by cell sorting and bone marrow cultures. CD34-positive cells, CD4-CD8-positive cells, committed progenitors, and LTC-IC were of donor origin, as demonstrated by two-color fluorescence in situ hybridization (FISH). Additionally, in relapse complete donor T-cell chimerism was seen. In contrast, plasma cells were of recipient origin in the patient who had a relapse in the bone marrow. Both patients were treated with infusions of donor leukocytes from their original donor. Neither patient suffered myelosuppression, and one achieved a stable complete remission. Received: February 26, 1999 / Accepted: April 14, 1999     

Detection of chimerism and early engraftment after allogeneic peripheral blood stem cell or bone marrow transplantation by short tandem repeats

Chimerism can be monitored after HLA-matched allogeneic bone marrow transplantation (BMT) or allogeneic peripheral blood stem cell transplantation (PBSCT) by detecting polymorphisms in short tandem repeats (STR). The purpose of our study was to document early complete chimerism in BMT and PBSCT recipients using STR, and to determine whether the initial WBC recovery correlated with the days required to attain complete chimerism. A total of 5 patients (2 PBSCT and 3 BMT) were followed by STR after transplantation. Peripheral blood obtained prior to transplantation was used to determine the 2 most informative STR probes for each donor/recipient pair. STR were amplified by polymerase chain reaction (PCR) with 8 commercial probes, and PCR products were visualized with silver staining. Peripheral blood was evaluated daily post-transplantation for WBC counts and to identify the presence of mixed or full chimerism by STR. The sensitivity of the STR technique varied from 0.05 to 1%, depending on the probe. Full chimerism was documented between day 9 and 14 in PBSCT recipients and on day 14 and 16 in BMT recipients. The initial rise in WBC occurred within 3 days of the onset of full chimerism, indicating that full chimerism is a more sensitive indicator of early engraftment. Periodic recipient monitoring using STR after complete chimerism identifies those patients who revert to mixed chimeras. The STR method may be useful in future studies to determine the significance of early engraftment and the clinical implications of sustained complete chimerism or mixed chimerism. © 1996 Wiley-Liss, Inc.     

Immunophenotypic and ultrastructural study in peripheral blood neutrophil granulocytes following bone marrow transplantation

Neutrophil studies after bone marrow transplantation (BMT) describe chemotactic and phagocytotic alterations and dyshaemopoiesis. Neutrophil granulocytes (NG) in peripheral blood after BMT were analysed in 28 patients. 14 patients (six receiving GM-CSF) underwent autologous BMT and 14 underwent allogeneic BMT. Immunophenotypic and electron microscopic studies were performed during post-BMT granulopoietic regeneration. Results were compared with NG from 15 healthy bone marrow donors (control group A) and from six patients receiving intensive chemotherapy before autologous BMT (control group B).
A significant increase in CD15 and a decrease in 8C7 antigen expression was observed in peripheral blood NG from BMT patients compared with controls A. MPO-7 in NG after BMT did not differ from control group A. Autologous BMT patients showed a lower percentage of NG expressing 13F6, 31D8 and CD16 (Leu 11a) than allogeneic BMT patients, and a significant decrease in 8C7 antigen expression compared with patients receiving intensive chemotherapy. Ultrastructurally, a marked decrease of azurophilic granules was observed in NG from BMT patients compared with control groups A and B. These data indicate that repopulation after BMT was made by phenotypically less mature NG with dysgranulopoietic features. Differences between autologous and allogeneic BMT patients may be partly related to GM-CSF usage. In conclusion, NG present immunophenotypic and ultrastructural changes after BMT which may be involved in abnormal NG response against bacterial infections, although further investigation is needed.     

Generation of CD8 cytolytic T cells early after autologous or allogeneic bone marrow transplantation

Longitudinal in vitro assays related to cell-mediated immunity were performed in patients following allogeneic (32) or autologous (15) bone marrow transplantation (BMT). In both groups of reconstituted patients, low CD4+/CD8+ T cell ratio and weak allogeneic mixed lymphocyte reactions were found in the first 6 months after BMT, progressively reaching values similar to controls (bone marrow donors or unrelated individuals). In contrast, a strong generation of allogeneic cytotoxic cells, assessed by the number of lytic units per 10(6) cells, was frequently found (18/38 patients tested in both groups) in the first 4 months, despite the quantitative deficit of the CD4+ subset. This in vitro differentiation was found to be independent of in vivo acute graft-versus-host disease (GVHD) and chronic GVHD in allo-transplanted patients. As also documented in autologous recipients, this observation suggests that this phenomenon could be, at least partially, related to the transplantation per se. Preliminary characterization of the effector cells indicates that they belong to the CD8+ subset and that their differentiation is interleukin-2-dependent. Experimental depletion of the CD4+ subset in normal subjects did not increase the number of lytic units in allogeneic cultures. This implies qualitative differences between BMT recipients and normal subjects, namely in CD8+ subset: i.e. that following BMT early CD8+ T cells appear to produce their own growth factor (IL-2), while in normal adult individuals, such autocrine CD8+ T cells, if present, are very rare.     

Donor multipotent mesenchymal stromal cells may engraft in pediatric patients given either cord blood or bone marrow transplantation

OBJECTIVE: Multipotent mesenchymal stromal cells (MSCs) are endowed with multilineage differentiative potential and immunomodulatory properties. It is still a matter of debate whether donor MSCs have sustained engraftment potential in host bone marrow (BM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The aim of this study was to analyze the donor/recipient origin of MSCs in children receiving allogeneic either BM or cord blood (CB) transplantation. METHODS: Thirty-seven pediatric patients undergoing allo-HSCT for either a malignant or a nonmalignant disorder were enrolled in the study; 19 received CB and 18 BM transplantation. Results were compared with those obtained in 14 adults given BM transplantation for either malignant or nonmalignant disorders. MSCs were grown from BM aspirates obtained 1-17 and 2-192 months after allo-HSCT in pediatric and adult patients, respectively. MSC samples at the third-fourth passage were phenotypically characterized. Donor/recipient origin of MSCs was assessed by amelogenin assay and microsatellite analysis. RESULTS: MSCs could be grown from 30 of 37 children; at the third-fourth passage MSCs resulted positive (> or = 98%) for CD73, CD105, CD106, CD29, CD13, CD44 and negative (< or = 1%) for CD34, CD45, CD14. Mixed chimerism with donor cells was observed in 4 BM and 5 CB transplantation recipients, respectively; full recipient chimerism was detected in the remaining children. Full recipient MSC chimerism was observed also in all assessable (12/14) adult patients. CONCLUSIONS: BM of pediatric patients might be a more favorable milieu than that of adults for sustained engraftment of transplanted MSCs. MSCs able to engraft in the host can be transferred with cryopreserved CB units.     

Mesenchymal stem cells remain of host origin even a long time after allogeneic peripheral blood stem cell or bone marrow transplantation

OBJECTIVE: Plasticity of hematopoietic stem cells (HSC) has gained major interest in stem cell research. In order to investigate whether HSC may differentiate into mesenchymal stem cells (MSC), we assessed chimerism in peripheral blood (PB), mononuclear cell fractions (MNC) of bone marrow, and MSC derived from bone marrow (BM) from 27 up to 4225 days after allogeneic transplantation. PATIENTS AND METHODS: We applied fluorescence in situ hybridization using X/Y gene probes in sex-mismatched and STR-PCR in sex-matched patients. MSC could have been generated in 27 of 55 bone marrow samples derived from 20 patients. Fifteen patients received peripheral blood stem cell transplants (PBSCT), including CD34-selected PBSCT in two. Five patients received bone marrow. RESULTS: While all patients had chimerism in PB and MNC of the BM, in all but one patient BM-derived MSC were of recipient origin. This single patient showed reproducibly MSC of donor origin in a frequency of 1% after having received a CD34-selected PBSCT. Looking at graft collections, MSCs were easily generated from BM specimens, while no MSC could be derived from PBSC samples. CONCLUSION: Even though HSC have been found to differentiate into a variety of nonhematological cell types, they usually do not differentiate into MSC after allogeneic transplantation.     

Mixed chimerism of erythro- and megakaryopoiesis following allogeneic bone marrow transplantation

Until now, studies on mixed chimerism (MCh) after allogeneic bone marrow transplantation (BMT) have predominantly focused on the B- and T-lymphocyte population, but not on distinct myeloid cell lineages like nucleated erythroid precursors and megakaryocytes. To evaluate the lineage-restricted MCh more explicitly in 10 patients with chronic myelogenous leukemia (CML), a quantitative analysis was performed on bone marrow biopsies following a sex-mismatched host/donor constellation. Techniques included immunophenotyping (antiglycophorin C, CD61) for the identification of erythro- and megakaryopoiesis and a simultaneously conducted genotyping with x- and y-chromosome-specific DNA probes. Normal bone marrow and specimens taken before BMT served as controls. Contrasting a total gender-dependent sex-typing in the latter samples in the early and late posttransplant period (up to 586 days), 3-9% erythroid precursors and about 16% megakaryocytes revealed a host-type origin. This significantly higher number of host megakaryocytes is explained by their polyploidy generating an increased probability to detect positive signals at a certain section level of the corresponding biopsies. A striking conversion of MCh to a recipient cell type was found in leukemic relapse with a more than 90% host-derived erythroid and megakaryocytic cell population in 4 patients approximately 643 days after BMT.     

Successful T cell-depleted allogeneic bone marrow transplantation in a child with recurrent multiple extramedullary plasmacytomas.

Multiple plasmacytomas with IgA paraproteinemia, a low grade lymphoid neoplasm of differentiated B cells rarely seen in the young, were encountered in a 10-year-old child. T cell-depleted allogeneic bone marrow transplantation (BMT) was performed because of progressive disease despite chemotherapy. Donor T cells were infused after transplantation in gradually increasing numbers to achieve a graft-versus-tumor effect. Complete eradication of all tumor masses was achieved following BMT with normalization of serum immunoglobulin levels. The patient remains in complete remission 4 years after BMT. Allogeneic BMT should be considered early in the course of low-grade lymphoid malignancy in the younger age group.     

Partial chimerism after T-cell-depleted allogeneic bone marrow transplantation in leukemic HLA-matched patients: a cytogenetic documentation

We evaluated serially by cytogenetics the blood and marrow chimerism of 38 leukemic recipients of HLA-matched bone marrow transplants (BMT) who were prepared by high doses of alkylating agents and fractionated total- body irradiation (2.2 Gy X 5). Donor or host mitoses were identified by examination of sex chromosomes in 32 patients or by evaluation of the polymorphism of other chromosomes after specific banding in six patients. Twenty-four patients were recipients of untreated BMT, and 14 were recipients of T-cell-depleted BMT. In the 24 patients who received untreated BMT, all showed successful engraftment, and only three had a transient mixed chimera. In the 14 recipients of T-cell-depleted BMTs, four rejected their grafts, and seven had mixed chimeras; these mixed chimeras were more frequent in blood lymphocytes than in marrow cells and could be detected up to 26 months after BMT. This high frequency of partial chimerism after T-cell-depleted BMT by comparison with a control group suggests that the donor's T cells play an important role in the eradication of host residual hematopoiesis after allogeneic BMT.     

Genetic fingerprinting in mouthwashes of patients after allogeneic bone marrow transplantation.

Detection of chimerism by PCR analysis of short tandem repeats (STR) in blood samples of patients who received allogeneic bone marrow transplantation (BMT) has proved to be an important method for early detection of relapse. The prerequisite for this type of analysis is knowledge of donor and recipient pretransplantation genotypes. In some cases, recipient cells from time points prior to BMT are not available and the pretransplant fingerprint cannot be determined. As BM recipients only alter their genotype in blood cells, we attempted to identify patient's pretransplantation genotypes after transplantation in mouthwash samples that contain easily accessible epithelial cells. Of 17 patients who had undergone BMT between one week and 45 months prior to analysis, DNA was isolated from mouthwash cell pellets or from epithelial cells obtained from mouthwashes. PCR analysis of STR loci in the von Willebrand and the tyrosine hydroxylase genes were performed. Even though the mouthwash cell pellets contained about 75% epithelial cells (presumably of recipient origin) and only about 25% leukocytes (presumably of donor origin), three of five patients showed donor genotype and only two patients exhibited chimeric DNA patterns, when cellular DNA was obtained by boiling of mouthwash cell pellets. Following phenol/chloroform extraction, eight of 10 DNA samples exhibited a chimeric pattern, while two of 10 DNAs showed only donor genotype. Of three patients, epithelial cells were attached to magnetic beads prior to DNA isolation. Even this DNA contained donor and recipient material. From our results it appears that blood cells serve as preferential DNA source in mouthwash samples and cannot be removed by epithelial cell separation.     

Long-term follow-up of recipients of allogeneic bone marrow grafts reveals no progressive telomere shortening and provides no evidence for haematopoietic stem cell exhaustion

Summary. Accelerated telomere shortening has been proposed as a possible long‐term risk of allogeneic bone marrow transplantation (allo‐BMT). In this study we monitored telomere length in white blood cells (WBC), granulocytes, and naïve and memory CD4⁺ T lymphocytes in recipients of allo‐BMT at long‐term follow‐up. Peripheral blood was collected from 10 allo‐BMT recipients and donors at a median interval of 18 years after allo‐BMT. Telomere length was determined using Southern blot analysis. Similar to results previously reported at short‐term follow‐up, a small difference in telomere length (0·1–0·3 kb) between recipients and donors was detected in WBC, granulocytes and naïve CD4⁺ T cells. Our data therefore provide no evidence for sustained telomere shortening in leucocytes, and render the possibility of long‐term haematopoietic graft failure unlikely. In addition, we observed two phenomena that may be related to involution of the thymus. First, the number of naïve CD4⁺ T cells in the blood was significantly lower in recipients (0·4 × 10⁹/l) than in donors (0·7 × 10⁹/l) (P < 0·05). Second, telomeres in memory CD4⁺ T cells from recipients were on average 0·6 kb shorter than those from donors (P = 0·01). The latter may be related to the reported rapid peripheral expansion of memory T cells immediately after transplantation.     

TCR gamma/delta positive lymphocytes after allogeneic bone marrow transplantation.

Peripheral gamma/delta+ T cells were studied in patients following allogeneic bone marrow transplantation (BMT) by indirect immunofluorescence utilizing two monoclonal antibodies (G1 and A13) able to recognize the two major subpopulations (V delta 2+ and V delta 1+, respectively) of these cells. We found that the relative percentage of 'total' (gamma/delta+ T lymphocytes) (V delta 2 + V delta 1 positive cells), and particularly of G1+ (V delta 2+) cells, in CD3+ lymphocytes was higher in transplanted patients, and especially in those presenting with acute graft-versus-host disease (aGVHD), than in normal controls. This finding was confirmed by the analysis of the V delta 2+/V delta 1+ cell ratio which was again significantly higher in patients with aGVHD as compared to controls. Similarly, the absolute number of 'total' gamma/delta+ and V delta 2+ cells was also significantly increased in patients with aGVHD. TCR gamma/delta+ T cells increased as a function of time after BMT reaching a plateau value at about day 60 post-BMT. When patients were stratified for the presence or absence of aGVHD this correlation was maintained only for patients with aGVHD. Finally, most V delta 2+ cells expressed surface T cell activation markers such as CD25 (IL-2 receptor) and DR (MHC class II) antigens. Our results suggest a possible involvement of gamma/delta+ T cells and particularly of V delta 2+ cells in the clinical and immunological events (aGVHD) occurring after allogeneic BMT.     

Patterns of hematopoietic chimerism following bone marrow transplantation for childhood acute lymphoblastic leukemia from volunteer unrelated donors

Hematopoietic chimerism was analyzed in serial bone marrow samples taken from 28 children following T-cell depleted unrelated donor bone marrow transplants (UD BMT) for acute lymphoblastic leukemia (ALL). Chimeric status was determined by polymerase chain reaction (PCR) of simple tandem repeat (STR) sequences (maximal sensitivity, 0.1%). At least two serial samples were examined in 23 patients. Of these, two had evidence of complete donor engraftment at all times and eight showed stable low level mixed chimerism (MC) (<1% recipient hematopoiesis). All 10 of these patients remain in remission with a minimum follow-up of 24 months. By contrast, 13 patients demonstrated a progressive return of recipient hematopoiesis. Five of these relapsed (4 to 9 months post BMT), one died of cytomegalovirus pneumonitis and seven remain in remission with a minimum follow-up of 24 months. Five children were excluded from serial analysis as two serial samples were not collected before either relapse (3) or graft rejection (2). We conclude that as with sibling transplants, ex vivo T depleted UD BMT in children with ALL is associated with a high incidence of MC. Stable donor engraftment and low level MC always correlated with continued remission. However, detection of a progressive return of recipient cells did not universally correlate with relapse, but highlighted those patients at greatest risk. Serial chimerism analysis by PCR of STRs provides a rapid and simple screening technique for the detection of relapse and the identification of patients with progressive MC who might benefit from detailed molecular analysis for minimal residual disease following matched volunteer UD BMT for childhood ALL.