Frequency of HLA-DPB1 disparities detected by reference strand-mediated conformation analysis in HLA-A, -B, and -DRB1 matched siblings.

The role of HLA-DPB1 as transplantation antigen is controversial. The frequency and relevance of HLA-DPB1 mismatch in hematopoietic stem cell transplantation are unknown. To ascertain the rate of HLA-DBP1 mismatch in siblings that had been matched for HLA-A, -B, and -DRB1, reference strand mediated conformation analysis (RSCA) a high resolution HLA typing method was used. Locus-specific primers were used to amplify the HLA-DPB1 locus. The PCR product was then hybridized with two fluorescein-labeled references and the duplexes were analyzed after electrophoresis in a short polyacrylamide gel. Among the 113 pairs of individuals tested, six HLA-DPB1 mismatches were identified, which corresponds to a frequency of 5.31 % (95% confidence interval 3.20%-7.42 %).

Individually adjusted prophylactic donor lymphocyte infusions after CD34-selected allogeneic peripheral blood stem cell transplantation.

T cell depletion of the graft increases graft failure and relapse rate in allogeneic PBSC transplantation. Delayed lymphocyte add-back after T cell-depleted transplants might prevent these complications. We present 22 consecutive allogeneic PBSC transplants from related histocompatible donors with positive selection of CD34+ cells. Recipients received prophylactic donor lymphocyte infusions (DLI) depending on their risk of relapse and of developing GVHD. Patients were considered at high risk of relapse with AML > first CR, ALL > second CR, and CML in accelerated or blastic phase. Patients were considered at high risk of developing GVHD if older than 35 years, or with a donor sensitized through previous pregnancy or blood transfusion. Patients at high risk of relapse and low risk of GVHD were scheduled to receive three DLI. Patients at low risk of relapse and high risk of GVHD did not receive DLI. The remaining patients were scheduled to receive two DLI. The DLI were administered on days +28 (2 x 10(5)/kg), +60 (2 x 10(5)/kg) and +90 (2 x 10(6)/kg) after transplant. G-CSF mobilized peripheral stem cells from healthy donors were positively selected by an immunomagnetic method. The mean CD34+ cells and CD3+ cells infused were 4.4 x 10(6)(range 1.9-10.6) and 0.085 x 10(5) (range 0.01-0.67). Cyclosporin A was given to prevent GVHD. All the patients engrafted. Twenty-two prophylactic DLI were performed in 12 patients: seven developed acute GVHD (one case grade III-IV) and none presented pancytopenia. At a mean follow-up of 585 days (range 89-1103), 14 patients were alive in CR, one patient was alive in relapse, four patients had died of relapse and three had died of transplant-related complication. Individually adjusted prophylactic DLI at the doses we used with an escalating schedule allowed an acceptable GVHD rate and a good engraftment of donor hematopoiesis.

Disparity for the minor histocompatibility antigen HA-1 is associated with an increased risk of acute graft-versus-host disease (GvHD) but it does not affect chronic GvHD incidence, disease-free survival or overall survival after allogeneic human leucocyte antigen-identical sibling donor transplantation.

Disparity for the minor histocompatibility antigen HA-1 between patient and donor has been associated with an increased risk of acute graft-versus-host disease (GvHD) after allogeneic human leucocyte antigen (HLA)-identical sibling donor stem cell transplantation (SCT). However, no data concerning the impact of such disparity on chronic GvHD, relapse or overall survival are available. A retrospective multicentre study was performed on 215 HLA-A2-positive patients who received an HLA-identical sibling SCT, in order to determine the differences in acute and chronic GvHD incidence on the basis of the presence or absence of the HA-1 antigen mismatch. Disease-free survival and overall survival were also analysed. We detected 34 patient-donor pairs mismatched for HA-1 antigen (15.8%). Grades II-IV acute GvHD occurred in 51.6% of the HA-1-mismatched pairs compared with 37.1% of the non-mismatched. The multivariate logistic regression model showed statistical significance (P: 0.035, OR: 2.96, 95% CI: 1.07-8.14). No differences were observed between the two groups for grades III-IV acute GvHD, chronic GvHD, disease-free survival or overall survival. These results confirmed the association between HA-1 mismatch and risk of mild acute GvHD, but HA-1 mismatch was not associated with an increased incidence of chronic GvHD and did not affect relapse or overall survival.

El antígeno menor de histocompatibilidad HA-1 / The HA-1 minor histocompatibility antigen

Los antígenos menores de histocompatibilidad (mHAgs) son péptidos inmunogénicos procedentes de proteínas celulares polimórficas asociados a moléculas del complejo mayor de histocompatibilidad (MHC) de clase I o de clase II. Las disparidades en estos mHAgs entre donante y receptor de un transplante alogénico de progenitores hematopoyéticos (TPH) a partir de donante emparentado MHC idéntico están relacionadas con reacciones inmunes adversas del tipo enfermedad de injerto contra huésped (EICH) o rechazo del injerto. El mHAg HA-1 fue el primer antígeno menor no codificado por el cromosoma Y identificado por métodos celulares .Posteriores publicaciones establecieron su comportamiento inmunodominante en el contexto de TPH a partir de donante emparentado MHC idéntico. En el presente trabajo revisamos diferentes aspectos del mHAg HA-1, tales como estudios bioquímicos y genéticos, métodos de biología molecular para la asignación alélica y posibles aplicaciones clínicas (AU)

HLA-B44 subtyping in the Catalan population using reference strand mediated conformation analysis. Implications for the selection of unrelated bone marrow donors.

Cytotoxic T lymphocytes (CTLs) reactive against the disparity between HLA-B*4402 and HLA-B*4403 have been reported after unrelated donor bone marrow transplantation. These CTLs have been associated with acute graft-versus-host disease and graft rejection. This study describes the HLA-B44-subtyping in the Catalan population using reference-strand mediated conformation analysis. It has been performed on 297 unrelated HLA-B44+ cord blood units from the Barcelona Cord Blood Bank (Barcelona, Spain). We have found a predominance of HLA-B*4403 (66.04%) over HLA-B*4402 (33.02%), whereas the predominant HLA-B44 allele in Northern Europe and the United States is HLA-B*4402. This inverted proportion between HLA-B44 subtypes in Mediterranean populations compared with other Caucasian populations suggests that HLA-B44 subtyping should be performed when an HLA-B44+ unrelated donor marrow is identified.

Genomic typing of minor histocompatibility antigen HA-1 by reference strand mediated conformation analysis (RSCA).

Disparities in minor histocompatibility antigen (mHAg) HA-1 are involved in the development of acute graft-versus-host disease (GvHD) in adult recipient after HLA-identical sibling donor hematopoietic stem cell transplantation. The mHAg HA-1 is an HLA-A*0201-restricted nonapeptide, which derives from the cleavage of a protein encoded at chromosome 19. The sequence analysis of HA-1 cDNA identified two alleles, termed HA-1H and HA-1R, which differ in only two nucleotides at 3' end of exon A, at positions 500 and 504. DNA-based methods for HA-1 typing were developed in 1998, using polymerase chain reaction with sequence-specific primers (PCR-SSP) and restriction fragment length polymorphism (PCR-RFLP). Here, we report the usefulness of reference strand mediated conformation analysis (RSCA), which was developed for mutation detection and typing of polymorphic loci, to discriminate between the two HA-1 alleles. We performed genomic typing of HA-1 locus in 203 HLA-A*0201-positive samples using RSCA and we confirmed these results by PCR-SSP. The results demonstrate the high reproducibility of this method and their strong correlation with the results obtained by PCR-SSP (99%). Only two samples showed disparity between the RSCA typing and the PCR-SSP. Direct sequencing of these samples confirmed that the correct allele assignment was that obtained by the RSCA typing. Furthermore, HA-1- RSCA-based typing provides additional information about the intronic structure of both alleles. With this approach, we describe the almost constant presence (99.2%) of a 5-bp deletion at intronic position 214-218 associated to the HA-1H allele, previously unidentified. We conclude that HA-1 genomic typing by RSCA is easy to perform and that could be used as a routine typing method.

Follow-up of chimerism status after allogeneic HLA-mismatched stem cell transplantation by detection of non-shared HLA alleles.

BACKGROUND AND OBJECTIVES: Chimerism studies after allogeneic transplantation are usually performed using cytogenetic analysis, PCR-VNTR or PCR-STR. Here, we report an alternative method for following the chimerism status after an HLA-mismatched stem cell transplantation (SCT), detecting the presence of non-shared HLA alleles by reference-strand mediated conformation analysis (RSCA). DESIGN AND METHODS: We tested this new approach on allogeneic related haploidentical SCT, unrelated cord blood transplantation, and HLA-mismatched unrelated donor SCT. The quantification of the chimerism was performed by laser detection of fluorescent-labeled primers on an automated DNA sequencer. RESULTS: In all cases this technique was able to detect mixed chimeras. The technique detected above 5% of residual cells when the analysis was based on HLA-class I and above 3% for HLA-class II. This sensitivity is similar to that of the PCR-VNTR analysis. INTERPRETATION AND CONCLUSIONS: This method avoids the need to search for an informative locus (which is essential for PCR-VNTR or -STR). Moreover, we did not find the phenomenon of preferential amplification that is observed with most VNTR, thus avoiding the need for construction of standard curves to quantify mixed chimeras. We conclude that the detection of the non-shared HLA alleles by RSCA is a useful approach for chimerism follow-up after HLA-mismatched SCT.

Pathologic rupture of the spleen as the initial manifestation of acute lymphoblastic leukemia: an additional case.

Pathological rupture of the spleen (PRS) is a rare, but well known complication of some hematological malignancies. In a recent review of the literature, Giagounidis et al. Identified 136 cases of pathologic rupture of the spleen since 1861. This number gives an idea of how seldom it occurs. In this review, 34% of the cases had occurred in acute leukemias, and 13% in acute lymphoblastic leukemias. In most cases, PRS occurs on the course of the disease. PRS as initial manifestation of ALL is a very rare feature: only six cases have been reported in the literature. Before now, in our hospital, we had only seen one case of a patient suffering from chronic myelomonocytic leukemia who presented splenic rupture as the initial manifestation of this disease. We now describe the seventh case, to our knowledge, of acute lymphoblastic leukemia presenting as pathologic splenic rupture.

Hematopoietic cell transplantation using plasma and DMSO without HES, with non-programmed freezing by immersion in a methanol bath: results in 213 cases.

A simplified cryopreservation method for bone marrow (BM) and peripheral blood progenitor cells (PBPC) was utilized in hematopoietic cell transplantation of 213 patients with hematological or solid neoplasms after ablative chemotherapy (187 with peripheral blood progenitor cells and 26 with bone marrow). Cells were cryopreserved, after addition of autologous fresh plasma with DMSO, without HES, by freezing to -80 degrees C in a methanol bath and non-programmed freezer. For the patients autotransplanted with PBPC, the median period necessary for recovery of more than 0.5 x 10(9)/l granulocytes was 11 days (range 6-44), and 15 (8-204) days were required to obtain more than 20 x 10(9)/l platelets. For the patients autotransplanted with BM, the median period necessary to recover >0.5 x 10(9)/l granulocytes was 12 days (range 9-33), and 24 (12-57) days to obtain more than 20 x 10(9)/l platelets. These results support this method as being very effective in achieving high-quality cryopreservation. The procedure, which uses a non-programmed freezer, simplifies and reduces enormously the cost of the technical measures currently in use, enabling its adoption in almost any clinical oncological institution.

Haematopoietic transplantation in pulmonary alveolar proteinosis associated with chronic myelogenous leukaemia.

Pulmonary alveolar proteinosis (PAP) is a disease of unknown etiopathogenesis sometimes associated with malignant haematological disorders. The potential reversibility of the process in these cases seems to be related to recovery from the underlying disease. GM-CSF has acquired an important, potentially pathogenic role and BMT presents one therapeutic option effective in certain forms of human PAP. We present the case of a 43-year-old female patient with Ph+ CML. During pretransplantation evaluation, unexpected pulmonary infiltrates were noted in the chest X-ray, PAP being diagnosed on biopsy. In view of the progressive respiratory symptomatology and her CML being in accelerated phase, the patient underwent haematopoietic transplantation. She died on day +12 from invasive pulmonary aspergillosis before a response could be observed. Pathogenic implications in PAP and the role of haematopoietic transplantation in this disease are discussed.