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Severe combined immunodeficiencies (SCID) are commonly fatal early in life. Adequate diagnosis and rapid institution of treatment, such as allogeneic stem cell transplantation (SCT), is essential. Several studies demonstrated that reconstitution of B-cell function after SCT is better in B-positive SCID than in B-negative SCID. We demonstrate that B-cell reconstitution in a B-negative SCID patient due to an Artemis mutation required the elimination of the autologous precursor-B-cells in bone marrow, probably to create physical space in the precursor-B-cell niches. Apparently, occupation of the precursor-B-cell niches is a potential dominant factor influencing repopulation of a functional B-cell compartment in B-negative SCID.

To understand the nature of long-term Th immune responses, we investigated in the present study the TCRBV gene repertoire of CD4(+) T cells specific for the recall antigen tetanus toxoid (TT) in recipients of an allogeneic bone marrow transplantation (allo-BMT) at several time points after transplantation and in their BM donors. We observed that the TCR repertoire of TT-specific CD4(+) Th cells was heterogeneous, and differed between allo-BMT recipients and their respective donors. Some individuals, however, used similar TCR-complementarity-determining region (CDR) 3 motifs that could reflect recognition of and selection by similar promiscuous epitopes of TT. Longitudinal analysis of this TT-specific T cell response revealed that T cells with completely identical TCR were present at several time points after the first analysis in allo-BMT recipients, most probably reflecting long-term stability of at least part of the antigen-specific TCR repertoire. Similar stability of the TT-specific TCR repertoire in time was also noted in the allo-BMT donors. These observations reveal that within a given individual the dominant antigen-specific T cell clones persist in time in an otherwise diverse TT-specific CD4(+) T cell immune response.

To study the impact of an MHC class II-negative environment on T cell immune reconstitution, we have analyzed the phenotypical and functional characteristics of FACS-sorted cultured CD4(+) and CD8(+) T cells in two Bare Lymphocyte Syndrome (BLS) patients before and after allo-BMT. A similar analysis was performed in two MHC class II expressing pediatric leukemia patients after treatment with an allo-BMT who were included in our study as control. It was observed that CD4(+) T cells displayed cytolytic alloreactivity in both BLS patients prior to and within the first year after allo-BMT, whereas such cells were absent at a later time-point, in the donors and pediatric leukemia controls. In addition, reduced MHC class II expression was observed in CD8(+) T cells of both recipients early after allo-BMT, irrespective of the T cell chimerism pattern. Lack of endogenous MHC class II expression in BLS patients, therefore, results in aberrant T cell selection within the first year after allo-BMT, analogous to T cell selection before transplantation. These T cell selection processes seem to be normalized at a later time point after allo-BMT probably due to migration and integration of graft-derived MHC class II-positive antigen presenting cells to sites of T cell selection.

We prospectively studied the reconstitution of lymphocyte subpopulations in a group of 22 children, who survived disease-free at least 6 months after allogeneic BMT for a haematological malignancy. Absolute counts of total lymphocytes, B lymphocytes, T lymphocytes, and CD4+ helper T lymphocytes reached the 5th percentile (p5) of age-matched reference values within 6 months after BMT in 15, 17, 7 and 2 patients, respectively. In particular, CD4+ helper T lymphocyte reconstitution was very slow. Unexpectedly, CMV reactivation had a profound positive influence upon the number of CD4+ helper T lymphocytes in the children. In five patients, absolute B lymphocyte counts above the 95th percentile were reached from 6 months after BMT onwards, mimicking normal ontogeny. Unlike normal ontogeny, the percentages of helper T lymphocytes expressing the 'naive' CD45RA isoform were low and those expressing the 'memory' CD45RO isoform were high in the first 3 months after BMT, as described before. Thereafter, the CD45RA:CD45RO ratio slowly normalised. Also, CD7 expression was absent on up to 90% of T lymphocytes in the first months after BMT, and on a steadily decreasing percentage thereafter, as recently described in adults. However, the absolute counts of CD45RO+/CD4+ and CD7-/CD4+ helper T lymphocytes did not change significantly. So, we found no evidence of peripheral expansion of previously primed donor-derived 'memory' T lymphocytes during the follow-up period which spanned 1-18 months after BMT. The absolute counts of 'naive' CD45RA+ helper T lymphocytes did not show a faster increase after BMT than in adults, despite the presumed presence of a non-involuted thymus in children. Bone Marrow Transplantation (2000) 25, 267-275.

To evaluate the role of T-cell selection in the thymus and/or periphery in T-cell immune reconstitution after allogeneic bone marrow transplantation (allo-BMT), we have analyzed the overall and antigen-specific T-cell repertoires in pediatric allo-BMT recipients treated for leukemia. We observed a lack of overall T-cell receptor (TCR) diversity in the repopulating T cells at 3 months after allo-BMT, as was deduced from complementarity determining region 3 (CDR3) size distribution patterns displaying reduced complexity. This was noted particularly in recipients of a T-cell-depleted (TCD) graft and, to a lesser extent, also in recipients of unmanipulated grafts. At 1 year after allo-BMT, normalization was observed of TCR CDR3 size complexity in almost all recipients. Analysis of the antigen-specific T-cell repertoire at 1 year after BMT showed that the T cells responding to tetanus toxoid (TT) differed in TCR gene segment usage and in amino acid composition of the CDR3 region when comparing the recipient with the donor. Moreover, the TT-specific TCR repertoire was found to be stable within a given allo-BMT recipient, because TT-specific T cells with completely identical TCRs were found at 3 consecutive years after transplantation. These observations suggest an important role for T-cell selection processes in the complete restoration of the T-cell immune repertoire in children after allo-BMT.

To study the effects of major histocompatibility complex (MHC) class II expression on T-cell development, we have investigated T-cell immune reconstitution in two MHC class II-deficiency patients after allogeneic bone marrow transplantation (allo-BMT). Our study showed that the induction of MHC class II antigen expression on BM graft-derived T cells in these allo-BMT recipients was hampered upon T-cell activation. This reduction was most striking in the CD8(+) T-cell subset. Furthermore, the peripheral T-cell receptor (TCR) repertoire in these graft-derived MHC class II-expressing CD4(+) and in the CD8(+) T-cell fractions was found to be restricted on the basis of TCR complementarity determining region 3 (CDR3) size profiles. Interestingly, the T-cell immune response to tetanus toxoid (TT) was found to be comparable to that of the donor. However, when comparing recipient-derived TT-specific T cells with donor-derived T cells, differences were observed in TCR gene segment usage and in the hydropathicity index of the CDR3 regions. Together, these results reveal the impact of an environment lacking endogenous MHC class II on the development of the T-cell immune repertoire after allo-BMT.

To evaluate the long-term reconstitution of the T cell immune repertoire in recipients of an allogeneic Bone Marrow Transplantation (allo-BMT), we have analyzed the T cell receptor (TCR) repertoire in the periphery and the T cell response against tetanus toxoid in two T- B+ Severe Combined Immunodeficiency Disease (SCID) patients more than 11 years after HLA haplo-identical allo-BMT. Our studies demonstrate that in the periphery of allo-BMT recipients, on the basis of TCR V-gene segment usage, the T cell immune repertoire long after allo-BMT is diverse, as is that of the donor. However, when donor and allo-BMT recipient were compared, differences were noted in the TCR Complementarity Determining Region 3 (CDR3) size distributions and in the T cell response against tetanus toxoid. In particular, the tetanus toxoid specific T cell clones differed in their use of HLA restriction elements, and expressed different T cell receptors. Moreover, we have uncovered donor-type tetanus toxoid specific T cell clones which were established from allo-BMT recipient derived peripheral blood lymphocytes and were found to be restricted by the non-shared recipient allele. This observation suggests a role for recipient-mediated T cell selection processes, in the thymus or at extra-thymic sites.

A powerful approach to documenting engraftment after allogeneic BMT is the quantification of the degree of chimaerism in distinct haematopoietic cell lineages. This cannot be achieved by the recently developed, quantitative, modifications of PCR amplification of highly polymorphic DNA markers, unless this technique is applied to separated cell populations. Here, we report the development of a new method, in which cells are simultaneously characterized by enzymatic immunophenotyping and identified for their origin by two-colour fluorescence in situ hybridization with X and Y chromosome-specific DNA probes (XY-FISH/immunostaining). The method enables the rapid, reliable and quantitative analysis of chimaerism within distinct cell lineages after sex-mismatched BMT, without the requirement for cell separation techniques. This is illustrated by investigation of the pattern of chimaerism in patients receiving a sex-mismatched BMT for the treatment of primary immunodeficiencies. The results obtained with the quantitative XY-FISH/immuno staining method show a good correlation with the data generated by the semi-quantitative analysis of PCR amplified minisatellites in FACS-sorted cell fractions. In addition, XY-FISH/immunostaining was successfully applied to detect materno-fetal engraftment of T cells in a SCID patient.

B cell lymphoproliferative disorders (BLPD) are relatively frequent after genotypically non-HLA-identical BMT. We performed univariate analysis to study which BMT-related variables were associated with an increased risk of developing BLPD. Sixty-five recipients of other than genotypically HLA-identical BM grafts were included in the study. Seventy-seven recipients of genotypically HLA-identical BM grafts served as a comparison group. BLPD occurred in nine of 65 children after non-HLA-identical BMT (14%) and in none of the 77 children after HLA-identical BMT (0%). In all cases, BLPD was proven to be EBV-related. Our data suggest that the combined use of Campath 1G and anti-LFA1 was associated with an increased risk of developing BLPD, particularly children who had received a T cell-depleted BM graft, using albumen density gradient sedimentation followed by E-rosetting, and who were conditioned with Ara-C, CY and TBL. In addition, T cell numbers below 50/microliters at 1 month and below 100/microliters at 2 months after BMT, respectively, were associated with an increased risk of developing BLPD. Longitudinal determination of T cell numbers after non-HLA-identical BMT is a simple method for identifying patients at risk of developing BLPD. In addition to monitoring levels of circulating EBV-infected lymphocytes, monitoring T cell numbers may allow early intervention to prevent progression of BLPD.

After allogeneic BMT, transient homogeneous Ig components (H-Ig) can be detected in the sera of most graft recipients. So far, data on the antigen-specificity and therefore the function of these H-Ig are not available. Such information may be important for our understanding of the underlying mechanisms that are responsible for these excessive clonal B cell expansions, and it may help to delineate the functional antibody repertoire after BMT. In the present study, sera of 98 paediatric BM graft recipients were investigated for the potential presence of H-Ig of IgG isotype (H-IgG) with specificity towards a panel of antigens, including vaccine and herpes virus antigens, auto-antigens and allo-antigens. The vast majority of H-IgG in sera of BM graft recipients were unreactive when tested for this panel of antigens. However, in four cases, antigen-specificity of H-IgG to tetanus toxoid could be demonstrated after vaccination with that antigen. An explanation for the negative findings may be either that a restricted antibody production had been elicited by other non-tested antigens, eg substances of colonizing and translocating bacteria or of food antigens, or that the H-IgG components may have anti-idiotype or anti-'self' specificity.

Allogeneic BMT using a non-genotypically HLA-identical donor may be curative for children suffering from lethal haematological diseases, who lack a genotypically HLA-identical donor. Unfortunately, graft failures are often seen, especially after T cell depletion of the graft. We studied whether untimely decreased counts of leucocytes and reticulocytes in peripheral blood might predict graft failure at an early stage. Fifty-five recipients of a non-genotypically HLA-identical BM graft were included in the study; data from these children were compared with those of 77 recipients of a genotypically HLA-identical BM graft. Time-related reference values of peripheral blood leucocyte and reticulocyte counts were established in graft recipients with proven donor-origin recovery after BMT. Graft failure after nonHLA-identical BMT was observed in 16 out of 55 children (29%) and after HLA-identical BMT in one out of 77 (1.3%). With respect to early graft failure, the predictive value of granulocyte numbers falling below the lower limit of the reference values and a rapid decline of reticulocyte numbers after their appearance in peripheral blood was 100% (95% confidence intervals of 83-100% and of 80-100%, respectively). Early immunosuppressive intervention was applied in six patients and was successful in three of them.

Pediatric recipients (n = 25) of an allogeneic bone marrow (BM) graft were selected on the basis of informative IgG allotype (Gm) differences between the BM donor and the recipient. To investigate the kinetics of the appearance of IgG of donor origin and the disappearance of IgG of recipient origin, G1m and G2m allotype levels were quantified in sera obtained at regular intervals between 3 months and 5 years after BM transplantation (BMT). For this quantification, a dot immunobinding assay (DIBA) has been developed. In 19 of 22 informative recipients, the Gm allotype distribution had reached the range of values expected on the basis of the Gm phenotype of the donor within 6 months after BMT. Remarkably, IgG of recipient origin persisted in 15 of 18 informative recipients until last follow up, ie, for several years after BMT. In addition to the origin of total IgG production, the origin of homogeneous IgG components (H-IgG) appearing after BMT was investigated. H-IgG of donor origin could be detected as early as 3 weeks after BMT, but also H-IgG of recipient origin were present in 8 of 13 informative recipients for a period of up to 1 year after BMT. We conclude that host-type IgG-producing cells were not eradicated by the (myeloablative) conditioning regimen and persisted in a high number of graft recipients. It is our hypothesis that lack of graft-versus-host disease (GVHD) in the majority of these recipients results in the persistence of IgG-producing cells of host origin. These observations may be relevant for the evaluation of patients who received allogeneic BMT for the treatment of multiple myeloma.

After bone marrow transplantation (BMT), a prolonged dysregulation of humoral immunity can be observed. In the present study, we investigated whether this is reflected in an abnormal production of specific antibodies (Ab) to the T-cell-dependent recall antigen tetanus-toxoid (TT). The study group consisted of children receiving transplants of an unmodified allogeneic graft and of adults receiving either a T-cell-depleted allogeneic or an unmodified autologous BM graft. Findings were compared with those in healthy controls. In pediatric graft recipients, who were routinely revaccinated early after BMT, the Ab response was quantitatively superior to that in adult graft recipients who did not receive early revaccination. In the majority of graft recipients, the time period after vaccination required to reach the peak level of antibodies was prolonged and the number of responding TT-specific B-cell clones was markedly decreased in comparison with controls. In controls, a low frequency of dominant B-cell clones may produce low quantities of homogeneous Ab components (H-Ab) against a heterogeneous background. However, in BM graft recipients, "overshooting" of Ab production by separate B-cell clones was observed, resulting in the development of H-Ab at a relatively high concentration. These abnormalities were present up to 10 years after BMT, irrespective of either the age of the recipient, the modulation of the graft, or the vaccination schedule used. It is hypothesized that the dysregulated Ab production is the consequence of activation of a restricted number of resting memory B cells, present in germinal centers, repopulating gradually after BMT. Our data show that routine revaccination early after BMT improves the humoral immune response. However, because of a clonally dysregulated Ab production, long-lasting qualitative defects may be present even after normalization of Ab titers.

We report the outcome of allogeneic bone marrow transplantation (BMT) as treatment for severe combined immunodeficiency disease (SCID) in 31 patients grafted from 1968 until 1992. The patients received a graft from an HLA-identical related (n = 10), an HLA-haplo-identical related (n = 19), or a closely HLA-matched unrelated (n = 2) donor that resulted in the long-term survival of 6 of 10, 9 of 19, and 0 of 2 children, respectively. Major complications included failure of engraftment and early death caused by respiratory failure. The chimerism pattern and immunologic reconstitution were evaluated in 15 children who survived more than 1 year with sustained engraftment. The pattern of engraftment was investigated within flow-sorted peripheral blood (PB) T- and B-lymphoid, natural killer (NK), and myelomonocytic cell populations using the amplification of variable number of tandem repeats by the polymerase chain reaction. The immunologic reconstitution was assessed by various in vitro and in vivo parameters. Although the number of PB T cells and the in vitro T-cell proliferative response was in the lower region of normal in the majority of cases and even subnormal in some, in all cases donor T-cell engraftment and reconstitution of T-cell immunity was observed. Residual host-type T cells (1% to 5%) were detected in eight cases at multiple occasions. All children showed normal serum IgM and IgG subclass levels and produced specific IgG antibodies after vaccination, irrespective of donor B-cell engraftment. However, three HLA haplo-identical graft recipients with host-type B lymphoid and myeloid cells have a persistent selective IgA deficiency. NK cells were either of donor, host, or mixed origin. Donor NK cell engraftment restored defective in vitro NK cell function of the recipient. We conclude that determination of lineage-specific engraftment patterns provides valuable information for the understanding of the immunologic reconstitution after allogeneic BMT for SCID.

We investigated the chimerism pattern within flow-sorted peripheral blood- or bone marrow-derived cell populations after allogeneic bone marrow transplantation (BMT) for the treatment of leukemia in children. This study was performed to define the identity of persistent host-type cells, to identify prognostic variables for the persistence of host-type hematopoiesis, and to determine the prognostic significance of the chimerism pattern on the duration of the leukemia-free interval, the overall survival, and the leukemia-free survival. The patients received either HLA-identical non-T-cell-depleted (n = 46) or HLA nonidentical T-cell-depleted (n = 7) BMT. In the peripheral blood, the children showed either stable mixed chimerism (SMC; ie, persistent host-type hematopoiesis; n = 14), (transient) mixed T-lymphoid chimerism (MTLC; n = 9), or complete chimerism (CC; n = 30). In the bone marrow, only donor-type cells were found in children with either CC (n = 8) or MTLC (n = 2), and a mixture of donor- and recipient-type cells was found in children with SMC (n = 7). The persistence of host-type hematopoiesis (SMC) was significantly related to a lower age of the recipient, the type of conditioning regimen, a lower total body irradiation dose, T-cell depletion of the bone marrow graft, and the use of cyclosporine A for acute graft-versus-host disease prophylaxis. No significant differences were found between patients with (SMC) or without (CC/MTLC) persistent host-type hematopoiesis with respect to the duration of the leukemia-free interval, the overall survival, or the leukemia-free survival. We conclude that ablation of host-type hematopoiesis is not compulsory for long-term leukemia-free survival after allogeneic BMT for various hematologic malignancies.

Bone marrow graft recipients suffer profound immunodeficiency during at least 3 months after transplantation. B-cell reconstitution following allogeneic bone marrow transplantation (BMT) in children was studied longitudinally by quantification of Ig (sub)class levels in serum and by investigation of numbers and characteristics of homogeneous Ig components (H-Ig); ie, monoclonal gammopathies (MG). For the latter purpose, a sensitive immunoblotting technique capable of detecting H-Ig of a concentration as low as 0.5 microgram/mL was used. Sera of 40 children grafted for a variety of diseases were investigated and followed up for 5 years. It was found that Ig (sub)classes reached normal levels from 3 months after BMT onward. The sequential increase of the different Ig isotypes was in accordance with that seen in normal ontogeny. This was especially clear following BMT for severe congenital immunodeficiency. H-Ig appeared from as early as 6 weeks after BMT in increasing numbers, beginning within IgM, IgG3, and IgG1, and afterward within other isotypes. After an initial increase of serum Ig levels, "overshooting" occurred accompanied by high frequency of H-Ig. H-Ig were still present at 5 years after BMT, when Ig levels normalized. Our data indicate that B-cell reconstitution after allogeneic BMT recapitulates normal ontogeny but in a clonally dysregulated fashion; that is, with overexpression of some clones and underexpression of others.

We performed polymerase chain reaction-variable number of tandem repeats analysis of flow-sorted peripheral blood T-, B-, natural killer-, and myeloid cell populations (van Leeuwen et al, Br J Haematol 79:218, 1991) in 32 children following allogeneic bone marrow transplantation (BMT) for leukemia to evaluate the relationship between mixed lymphoid chimerism and leukemia relapse. Five patients showed a stable mixed chimerism pattern characterized by the presence of both recipient as well as donor type cells in all cell populations up to 1 year posttransplantation. Five others showed transient mixed chimerism in the T-lymphoid cell lineage. In one patient, host T cells persisted until leukemia relapse. The remaining 21 patients showed a complete chimerism throughout the period of investigation. Twenty-five of these patients were classified according to the presence (n = 10) or absence (n = 15) of recipient type T cells. Statistical analysis did not show significant differences in the distribution of a number of clinical variables between the two groups, nor in the actuarial survival (P = .11) and leukemia-free interval (P = .97). Therefore, these results suggest that persistence of recipient type T lymphoid cells after allogeneic BMT for hematologic malignancies is not correlated with leukemia relapse. In addition, we observed that persistence of host cells within the original leukemia cell lineage and at the correct maturational stage was predictive for leukemia relapse in one case.

The in vitro colony formation of hematopoietic progenitor cells of bone marrow samples, taken before and early after allogeneic bone marrow transplantation (BMT), was investigated prospectively. In order to circumvent culture-related and sample-related variations, a serum-free recombinant growth factor-replenished culture system was developed using T cell- and monocyte-depleted bone marrow samples. Samples of healthy bone marrow donors were used to validate the technique. The standardized culturing technique gave reproducible results, with numbers of colonies above those in conventional conditioned-medium technique. Colony formation in vitro of myelomonocytic precursor cells was found decreased in graft recipients, also after addition of growth factors, in comparison with healthy donors. The growth-promoting effect of the combination of IL-3 + GM-CSF was superior to that of either growth factor alone or conditioned medium. No effect was observed of T lymphocytes and monocytes on in vitro colony formation after bone marrow transplantation, probably as a result of functional impairment of these cells at that period after transplantation.

A rapid recovery of specific humoral immunity in the recipient of an allogeneic bone marrow transplantation (BMT) can be observed after immunization of the donor before graft sampling. This has been attributed to transfer of specific immunity from donor to recipient. However, to maintain the concept of transfer the origin of the antibody producing cells in the recipient after BMT must be demonstrated. To this end, donor-recipient pairs with differences in Gm-allotypes were selected and immunized before BMT with the neo-antigen Helix pomatia haemocyanin (HPH) according to three immunization protocols. Additionally, the recipients were immunized at day 42 after BMT. Serum samples were weekly obtained from the recipients in the first 100 d after BMT. The origin of HPH-specific antibody producing cells was assessed by two approaches: (1) determination of the Gm-allotypes of anti-HPH antibodies within a distinct IgG subclass, (2) analysis of anti-HPH antibody spectrotypes by isoelectric focusing combined with immunoblotting. The results obtained with these two approaches show concordance in most instances and led to the conclusion that the antibody producing cells are of donor origin.

Transfer of specific immunity was investigated in a group of 28 paediatric and adult leukaemia patients during the first 100 d after allogeneic bone marrow transplantation (BMT). These patients and/or their donors were immunized 7-13 d before transplantation with the recall antigen tetanus toxoid (TT) and the neo-antigen Helix pomatia haemocyanin (HPH). The recipients were booster immunized with both antigens at day 42 after transplantation. Transfer of a primary IgM and IgG response to HPH was successful in most paediatric and adult patients, but transfer of a secondary response to TT was established in only a few paediatric recipients. After booster immunization at day 42 most paediatric recipients responded with a rise in serum antibody titre to HPH as opposed to only two of 18 adult recipients. This incapability of the adult recipients to mount a secondary immune response may be related to their conditioning regimen which included Campath-IG in vivo. The results from this study indicate that transfer of immunity against recall- and neo-antigens is possible. However, the establishment of long-term memory may be affected by the regimen used to condition the graft recipient.

Growth characteristics of stromal cells, assessed as adherent cells in long-term bone marrow cultures, and of hematopoietic progenitor cells, prior to and shortly after allogeneic bone marrow transplantation (BMT), were investigated, more specifically with regard to their possible correlations. The main constituent cells of the bone marrow stroma, that is, endothelial cells, reticular cells/fibroblasts, and monocytes/macrophages, showed an as yet inexplicable increased growth in samples taken from recipients prior to BMT, as compared with the growth in samples from their healthy donors and those taken after BMT. In the third week after BMT the in vitro outgrowth of hematopoietic precursors was severely depressed, but cell numbers in the adherent layer were normal. No relationship between in vitro growth of hematopoietic precursor cells and stromal cells was observed at that time. Probably the precursor cells growing in vitro are committed progenitor cells, relatively independent of stromal influences. In the eight week after grafting, endothelial cell outgrowth in vitro was highly correlated with granulocyte-macrophage colony-forming unit (CFU-GM) colony formation and to a lesser extent with mixed-lineage colony-forming unit (CFU-Mix) colony formation. This may indicate the reappearance of cytokine-mediated influences or the reappearance of a direct interaction, for example, by cell-cell contact between stromal cells and hematopoietic progenitor cells at that time.

The amplification of Variable Number of Tandem Repeats (VNTR) by the polymerase chain reaction (PCR) was used to determine the extent of chimaerism in flow sorted lymphoid and myeloid cell populations following allogeneic bone marrow transplantation (BMT). Pre-BMT screening with a set of five VNTR revealed that at least one marker was maximally informative in 95% of donor-recipient pairs. Mixing reconstruction experiments indicated that detection of 1-5% of the minor cell population in a sample of 5 x 10(3) nucleated cells is feasible. Flow sorted post-transplant peripheral blood B- and T-lymphocyte, natural killer and monocyte cell populations were subjected to PCR-VNTR marker analysis. It was shown that this procedure can be used for the early detection of engraftment and the identification of mixed chimaerism in various haematopoietic cell lineages in patients with leukaemia or severe combined immune deficiency, treated with allogeneic BMT.