How to improve the search for an unrelated haematopoietic stem cell donor. Faster is better than more!

Many patients do not reach haematopoietic stem cell transplantation. Shortage of unrelated donors (UDs) is still seen as the main cause. However, with a worldwide UD pool containing more than 8 million donors, it is possible that other impediments are becoming more important. We analysed 549 UD searches for Dutch patients, performed between 1987 and 2000, in order to find the reasons for failure or success to reach transplantation. Between 1996 and 2000, 59% of the patients of Northwest European origin received a graft from an UD with a median time span of 4.4 months from the start of the search. In all, 11% of the patients lacked a compatible donor, while 30% became medically unfit for transplantation. This is in contrast to the patients of non-Northwest European origin for whom UD shortage is still the most important impediment; only 32% were transplanted while 50% lacked a compatible donor. We conclude that the shortage of donors is no longer the biggest constraint in unrelated stem cell transplantation for patients of Northwest European origin. It may be more effective to optimize the chance on transplantation by making the search process more efficient.

Simultaneous flow cytometric detection of antibodies against platelets, granulocytes and lymphocytes.

We present a time-saving and objective flow cytometric immunofluorescence assay for the simultaneous detection of antibodies against platelets, granulocytes or lymphocytes using a reconstituted mixture of these cell populations. Platelets, granulocytes and lymphocytes could be distinguished on the basis of their forward (FSC) and sideways (SSC) light scattering properties plotted on scales of 4 log orders. After setting FSC/SSC gates around the platelets, granulocytes and lymphocytes, the reactivity of the sera with the cell populations was determined by histogram analyses of immunofluorescence for each gate. The flow cytometric assay of reconstituted cell mixtures showed a strong, positive correlation with a reference microscopic immunofluorescence assay of separate cell suspensions. The reproducible procedures for the isolation and staining of the cells and the electronic stability of the flow cytometer permitted the use of the same gate and marker settings throughout the experiments. Consequently, the entire analysis of data stored in list mode could be performed using a keystroke, so that time consuming and subjective manual analyses were avoided.

Graft-versus-host disease following transplantation of 'one log' versus 'two log' T-lymphocyte-depleted bone marrow from HLA-identical donors.

Prevention of acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation, requires the depletion of mature T-lymphocytes from bone marrow grafts. The optimal degree of T-cell reduction is still an open question. We compared two procedures of T-cell separation in 18 consecutive recipients of genotypically HLA-matched bone marrow, who also received cyclosporin A for 6 months. The first method (A) was based on a discontinuous albumin gradient fractionation and resulted in an average T-lymphocyte content of 50 X 10(5)/kg body weight (n = 9 patients); the second method (B) was based on E-rosette sedimentation and reduced the contamination to 15 X 10(4) grafted T-lymphocytes/kg body weight on the average (n = 9 patients). Thus, approximately 90 and 99% of the original T-lymphocytes were removed from the marrow grafts respectively. Of the seven patients of the first group who were at risk of GVHD (excluding two cases of early death), five developed a minimal-to-moderately severe acute GVHD and in two cases chronic GVHD ensued. Lethal GVHD was not seen. Of group B, all recipients engrafted and none developed GVHD (0/9). The difference in the frequency of GVHD between the two groups was highly significant (P less than 0.0025). These data confirm our preclinical studies. They demonstrate that a one-log T-lymphocyte reduction of the marrow inoculum, when combined with cyclosporin A prophylaxis after major histocompatibility complex (MHC)-matched transplantation, is still associated with a considerable incidence of GVHD, whereas a two-log reduction of T-lymphocytes may provide full protection against acute GVHD.

Problems and possible solutions in finding an unrelated bone marrow donor. Results of consecutive searches for 240 Dutch patients.

To evaluate the efficiency of our protocol for finding an HLA matched unrelated bone marrow donor, search results obtained between 1990 and 1995 for 240 Dutch patients were analyzed. The percentage of patients for whom, according to information given by the registries, a fully split-HLA antigen matched donor is available, increased from 24% in 1990 to over 70% in 1995. As a result the percentage of patients transplanted rose from about 24% in 1990-1991 to 44% in 1994-1995. The median time between the start of the search and transplantation was about 6 months. The systematic use of Bone Marrow Donors Worldwide (BMDW) which comprises the HLA groups of all volunteer bone marrow donors in Europe, Israel, South Africa, North America, Canada, India, Australia and New Zealand has been essential in this context. While searching for a suitable donor several problems were encountered such as unavailability of donors (12%) and discordant typing results (8%; range < 1% to > 25%). Thus it is advisable to select several donors for a patient. For 86% of patients with at least one HLA identical donor on the serological level for HLA-A,-B,-DR,-DQ, an HLA-DRB1/3/4/5, and -DQB1 identical donor could be identified. As expected, patients with two frequent haplotypes in strong linkage disequilibrium had the best chance of obtaining an HLA matched donor. Unexpectedly, patients with only one such haplotype had an almost similar chance. It could be calculated that HLA-DR typing of HLA-A,-B identical donors was rarely cost-effective after 1992. Only 12 of the 75 transplanted patients (16%) typeable at DNA level for class II, turned out to be completely matched for HLA-A,-B,-C,-DRB1/3/4/5,-DQB1,-DPB1 and had a negative MLC test. In the group of patients transplanted with a fully matched donor and for whom a CTLp test was performed, only 7% (4/54) of the tests were negative. Search results for patients of non-European origin were dismal, with only four of 26 patients referred being transplanted. In summary, of the 240 patients for whom the Europdonor office searched for a donor, about one-third were transplanted, one-third had a potential donor but did not reach transplantation, while for the remaining one-third of patients no suitable donor could be found.

Expression of MAGE, gp100 and tyrosinase genes in uveal melanoma cell lines.

In order to determine the possible use of uveal melanoma cell lines as stimulators in immunotherapy, we evaluated the expression of the human genes for MAGE-1, -2 and -3, gp100 and tyrosinase in uveal melanoma cell lines. mRNA expression of the MAGE-1, -2 and -3, gp100 and tyrosinase genes and the HLA class I specificity were determined in five primary and three metastatic uveal melanoma cell lines. Expression of the examined genes was heterogeneous in the primary and metastatic cell lines. The cell lines OCM-1 and OMM-1 expressed MAGE-1, -2 and -3, whereas EOM-3, MEL202, 92-1 and OMM-3 were negative for these antigens. gp100 was expressed in all cell lines, and tyrosinase in all but three (EOM-29, OMM-2 and OMM-3). Except for EOM-3, the HLA-A type of all the cell lines could be determined by complement-dependent microlymphocytotoxicity assay. Since at least two melanoma-associated antigens can be found in uveal melanoma cell lines, as well as the HLA class I molecules, these cell lines may be applicable as immunogens for specific immunotherapy against metastatic uveal melanoma.

[Allogeneic bone marrow transplantation of unrelated donors; fewer complication, more donors]. / Allogene transplantaties met beenmerg van onverwante donors; minder complicaties, meer donors.

A female patient aged 37 years who suffered from chronic myeloid leukemia received an allogeneic bone marrow transplantation (BMT) from an HLA-matched unrelated donor. No life-threatening complications ensued; 2.5 years after BMT she is still in complete remission. Survival after BMT from an unrelated donor for the time being is still lower than that after BMT from a related donor, but is improving due to better prevention and treatment of opportunistic infections and better selection of registered donors by meticulous HLA matching.

Familial clustering of multiple sclerosis in a Dutch genetic isolate.

Multiple sclerosis (MS) is a complex disease with a substantial, yet poorly identified, genetic influence. We estimated the pattern of familial aggregation of MS in a recent genetically isolated population in The Netherlands. Forty-eight MS patients were identified. Their relationship was evaluated by tracing extended pedigrees, making use of municipal and church records. Of the 48 MS patients, 24 could be linked to a common ancestor in 14 generations. However, multiple relationships exist between patients and, to take these into account, we calculated inbreeding and kinship coefficients. We found that MS patients from the isolate were significantly more often related to each other and significantly more often inbred than a non-MS control group, drawn from the same isolate. There was no clustering of Type 1 diabetes and autoimmune thyroid diseases in families of MS patients from this isolate. Finally, HLA typing was performed. Although there was a trend towards a higher prevalence of the HLA DRB1*15 allele in patients compared to controls, differences did not reach significance. This study suggests familial aggregation in the genetically isolated population. The high level of inbreeding makes this population valuable for finding novel genes involved in MS.

HLA class II alleles are not a general susceptibility factor in Guillain-Barré syndrome.

OBJECTIVE: To assess whether human leukocyte antigen (HLA)-DRB1 and HLA-DQB1 alleles confer susceptibility to Guillain-Barre syndrome (GBS) or are related to specific clinical or serologic subgroups of GBS. METHODS: The HLA-DRB1 and HLA-DQB1 loci were genotyped by PCR amplification with sequence-specific primers in 164 well-documented Dutch patients with GBS and 207 healthy Dutch control subjects. Patients with GBS were divided into subgroups based on clinical features, severity of disease, antecedent infection, and anti-ganglioside antibodies. Data were compared with those of all case-control HLA studies in GBS performed previously. RESULTS: In this case-control study, HLA-DRB1 and HLA-DQB1 alleles did not differ between GBS patients and control subjects. The frequency of HLA-DRB1*01 was increased in patients who needed mechanical ventilation (odds ratio 4.2; 95% CI 1.9 to 9.6; p(c) = 0.02). Multivariate logistic regression analysis showed that this association was independent of the severity of paresis and the presence of cranial nerve involvement (all p < 0.05). There was a tendency toward an association between certain HLA alleles and several anti-ganglioside antibodies. CONCLUSIONS: Human leukocyte antigen (HLA) class II antigens are not a general susceptibility factor in Guillain-Barre syndrome (GBS). However, HLA class II alleles may be a determinant in distinct subgroups of GBS, indicating the need for further exploration in large-scale studies.

An increased MRP8/14 expression and adhesion, but a decreased migration towards proinflammatory chemokines of type 1 diabetes monocytes.

In the early development of type 1 diabetes macrophages and dendritic cells accumulate around the islets of Langerhans at sites of fibronectin expression. It is thought that these macrophages and dendritic cells are derived from blood monocytes. Previously, we showed an increased serum level of MRP8/14 in type 1 diabetes patients that induced healthy monocytes to adhere more strongly to fibronectin (FN). Here we show that MRP8/14 is expressed and produced at a higher level by type 1 diabetes monocytes, particularly after adhesion to FN, creating a positive feedback mechanism for a high fibronectin-adhesive capacity. Also adhesion to endothelial cells was increased in type 1 diabetes monocytes. Despite this increased adhesion the transendothelial migration of monocytes of type 1 diabetes patients was decreased towards the proinflammatory chemokines CCL2 and CCL3. Because non-obese diabetic (NOD) mouse monocytes show a similar defective proinflammatory migration, we argue that an impaired monocyte migration towards proinflammatory chemokines might be a hallmark of autoimmune diabetes. This hampered monocyte response to proinflammatory chemokines questions whether the early macrophage and dendritic cell accumulation in the diabetic pancreas originates from an inflammatory-driven influx of monocytes. We also show that the migration of type 1 diabetes monocytes towards the lymphoid tissue-related CCL19 was increased and correlated with an increased CCR7 surface expression on the monocytes. Because NOD mice show a high expression of these lymphoid tissue-related chemokines in the early pancreas it is more likely that the early macrophage and dendritic cell accumulation in the diabetic pancreas is related to an aberrant high expression of lymphoid tissue-related chemokines in the pancreas.

A flow cytometric platelet immunofluorescence crossmatch for predicting successful HLA matched platelet transfusions.

Platelet crossmatching may provide a useful way of selecting donors for effective platelet transfusions in patients refractory to random donor platelet concentrates due to alloimmunization. We assessed the predictive value of a flow cytometric platelet immunofluorescence crossmatch test for the outcome of HLA matched platelet transfusions in a group of alloimmunized patients. Platelet immunofluorescence (PIFT) crossmatches were performed for 104 HLA-matched platelet transfusions administered to 30 patients. A negative PIFT crossmatch correctly predicted a successful platelet transfusion (1 h post-transfusion platelet recovery >20%) in 56/75 (75%) cases. We also considered non-immunological factors that, in combination with alloimmunization, might have contributed to an unsuccessful transfusion result, i.e. fever, septicaemia, splenomegaly, disseminated intravascular coagulation and bleeding. The predictive value of a negative PIFT crossmatch was better when these non-immunological factors were absent [48/59 (81%) correct predictions] than when these factors were present [8/16 (50%) correct predictions] (P=0.01; chi-square test). The effect of ABO incompatibility between donor and recipient on the predictive value of the PIFT crossmatch was also analysed. Positive PIFT crossmatches occurred more frequently in ABO incompatible donor-recipient combinations [in 18/28 (64%) cases] than in ABO-compatible donor-recipient combinations [in 11/76 cases (14%)] (P<0.001, chi-square test). Successful platelet transfusions were observed on 53/76 (70%) occasions in ABO compatible transfusions as compared to 16/28 (57%) in ABO incompatible transfusions. This difference was not statistically significant (P=0.23; chi-square test). Consequently, a negative PIFT crossmatch appeared to be non-predictive for the transfusion outcome in cases of ABO incompatibility between donor and recipient. We conclude that the PIFT crossmatch for platelet donor selection in addition to matching for HLA antigens, is predictive for the outcome of ABO compatible transfusions in alloimmunized recipients and prediction levels are increased when non-immunological causes for platelet refractoriness are absent.

Comparison of four filters for leukocyte depletion of single donor plateletapheresis products.

Removal of leukocytes from platelet suspensions is important in preventing the development of alloimmunization to HLA antigens in repeatedly transfused patients. Several types of filters have recently become available to remove leukocytes from platelet suspensions. We have compared the results obtained with Pall PL100S, Pall PL50S, Sepacell PL-10N and Sepacell PL-5N filters. Platelets were obtained by plateletapheresis using the Haemonetics V-50 TSPP surge procedure. A total of 160 platelet suspensions were studied, 40 for each type of filter used. The median number of leukocytes pre-filtration was 321x10 6 (range: 16-4068). The median number of leukocytes post-filtration was 2x10 6 ( less than 1-521) and was dependent on the number of leukocytes present pre-filtration but not on the type of filter used. All filters tested showed approximately a 2 log 10 reduction of leukocytes. When the number of leukocytes was below 500x10 6 pre-filtration, the number of leukocytes post-filtration did not exceed 10x10 6. Overall platelet loss was 11+/-7% and was lowest with the Pall PL50S filters (7+/-7%). Mean platelet volumes post-filtration were decreased with all types of filters used.

The value of a 51Cr platelet lysis assay as crossmatch test in patients with leukaemia on platelet transfusion therapy.

Current platelet crossmatch testing still results in a significant percentage of false positive or false negative results in oncological patients during platelet support. A 51Cr platelet lysis assay was used for the detection of platelet alloantibodies. We determined the predictive value of this assay as crossmatch procedure on 28 occasions in 14 patients who received random single donor platelet transfusions. To deal with the problem of spontaneous lysis we included a panel of 10 control sera from normal individuals and applied several methods of statistical analysis to these data. It appeared that the use of only one control serum was sufficient when the percentage relative counts was used as the criterion variable, which is of advantage in the practical application of the test. The test values were retrospectively compared with the clinical transfusion response, determined as the 1 h post-transfusion platelet recovery. The 51Cr platelet lysis crossmatch showed false negative results in 1/21 cases and false positive results in 1/7 cases. These data indicate that the 51Cr platelet lysis assay adds a useful dimension to the solution of the problem of selecting compatible platelet donors. Spontaneous lysis of target platelets appeared not to be a problem in the interpretation of test results.

ABH antibodies causing platelet transfusion refractoriness.

Two alloimmunized patients with multispecific anti-HLA and high-titered ABH antibodies showed transfusion failures after ABH-mismatched HLA-identical platelet transfusions, whereas ABH-matched HLA-identical platelets showed sufficient increments. The anti-A and -B could be demonstrated on platelets by immunofluorescence tests using FITC-labeled goat anti-human IgG. These platelet antibodies could be absorbed with red cells and platelets of the appropriate ABH type. In contrast to previous reports about the influence of ABH antibodies on platelet survival, not only a proportion of the platelet population were destroyed after infusion, but almost all ABH-incompatible platelets were destroyed.

A microplate ELISA for the detection of platelet alloantibodies: comparison with the platelet immunofluorescence test.

An enzyme-linked immunoassay (ELISA) for the detection of platelet alloantibodies has been compared in detail with the platelet immunofluorescence test (PIFT). The ELISA appeared much simpler to perform than the PIFT. Both tests were comparable with regard to reproducibility and sensitivity. Alloantibodies were detected with ELISA and PIFT in 13 out of 14 patients who were refractory to random donor platelets. The value of these tests as a platelet crossmatch assay was determined in a retrospective comparison of the test results and the clinical transfusion responses expressed as the 1 h post-transfusion platelet recovery. 39/41 (95%) negative ELISA crossmatches and 30/33 (91%) negative PIFT crossmatches appeared to be associated with a successful platelet transfusion, whereas 7/10 positive ELISA crossmatches and 2/4 positive PIFT crossmatches appeared to be associated with transfusion failures. The high frequency of 'correct' negative tests indicates the importance of both assays in the prospective selection of compatible platelet donors for alloimmunized patients. However, because of its simplicity, the ELISA appears the method of choice for this purpose.