WMDA guidelines for subsequent donations following initial BM or PBSCs.

Unrelated donor SCT activity is increasing, and in 5-10% of cases a subsequent donation of stem cells or donor lymphocytes may be requested. Second donations of stem cells are not associated with an increased chance of donor complications, but the yield of CD34+ cells may be lower in some donors. It is acceptable practice for any registry to request subsequent donations and it is recommended that donors should be counselled about this possibility before their first donation. Guidance is provided on the requirements for further medical assessment, the procedures used to agree requests, frequency and timing of donation and timing and duration of donor follow up.

Transportation of cellular therapy products: report of a survey by the cellular therapies team of the Biomedical Excellence for Safer Transfusion (BEST) collaborative.

BACKGROUND AND OBJECTIVES: Most cell therapy products (CTP) are infused or processed shortly after collection but in some cases this may be delayed for up to 48 h. A number of variables such as temperature and cell concentration are of critical importance for the integrity of CTP during this time. MATERIALS AND METHODS: We conducted a survey of cellular therapy laboratories to ascertain current practices for CTP transportation. RESULTS: There were 194 respondents of whom 90% shipped or received CTP--84% allogeneic, 71% autologous and 62% therapeutic cells. Processing facilities shipped or received the following products--hematopoietic progenitor cells (HPC), Marrow 73%; HPC, Apheresis 90%; HPC, Cord Blood 54% and others 14%. Other CTP included donor lymphocytes, mesenchymal stem cells (MSC), natural killer cells, buffy coat neutrophils and virus-specific cytotoxic T lymphocytes (CTL). More than 70% of respondents believed that it was acceptable for CTP to be held for up to 2 h without checking the temperature or cell density and a similar proportion agreed that putting products in containers to control parameters such as temperature within this time period was unnecessary. The majority of centres shipped or received between 1 and 10 CTP annually and 66% received products taking more than 2 h to ship. Of these, 82% specified the conditions for temperature in transit whilst 57% monitored temperature in transit and 74% of these used a data logger. The temperature range most commonly specified was 18-24 degrees C. The majority of processing facilities did not request an adjustment to the cell density even for products taking more than 2 h to reach their facility. More than 90% of respondents tested HPC for CD34(+) cells, viability and sterility; 40-48% performed colony-forming unit-granulocyte macrophage (CFU-GM) analysis. Only viability was thought by > 50% of respondents to be impacted by temperature, cell density and other parameters. CONCLUSION: Understanding current practice will help in the design of future studies for CTP storage and transportation.

Early emergence of PNH-like T cells after allogeneic stem cell transplants utilising CAMPATH-1H for T cell depletion.

CAMPATH-1H (C-1H) is widely used in vivo and / or in vitro for T cell depletion in hematopoietic SCT. This humanised monoclonal antibody is specific for CD52, a marker coexpressed on the majority of human lymphocytes with CD48 and other glycosylphosphatidyl-inositol (GPI) anchored proteins. We detected CD52 / CD48 dual expression on >99% of CD3(+) lymphocytes from normal individuals and all 15 post-SCT patients whose transplants did not utilise C-1H. By contrast, 23 / 26 patients with transplants involving C-1H (in vivo, in vitro or both) exhibited populations lacking CD52 expression that accounted for 49.7% (4.2-86.2%) of the CD3+ lymphocytes (median and range) in samples evaluated at a median of 2 months post-SCT. Most CD52- cells also lacked CD48 expression. These GPI- T cells were of either donor or mixed donor / recipient origin. They were predominant in the early months after SCT at times of profound lymphopenia and inversely correlated with the recovery of the absolute lymphocyte count (r= - 0.663, P<0.0001). The presence of CD52- cells has been correlated previously with clinical outcome after CAMPATH therapy for both malignant and nonmalignant diseases.

High peripheral blood progenitor cell counts enable autologous backup before stem cell transplantation for malignant infantile osteopetrosis.

Autosomal recessive osteopetrosis (OP) is a rare, lethal disorder in which osteoclasts are absent or nonfunctional, resulting in a bone marrow cavity insufficient to support hematopoiesis. Because osteoclasts are derived from hematopoietic precursors, allogeneic hematopoietic cell transplantation can cure the bony manifestations of the disorder. However, high rates of graft failure have been observed in this population. It is not possible to harvest bone marrow from these patients for reinfusion should graft failure be observed. We report that 8 of 10 patients with OP had high numbers of circulating CD34(+) cells (3% +/- 0.9%). This increased proportion of peripheral CD34(+) cells made it possible to harvest 2 x 10(6) CD34(+) cells per kilogram with a total volume of blood ranging from 8.3 to 83.7 mL (1.3-11.6 mL/kg). In addition, colony-forming assays documented significantly more colony-forming unit-granulocyte-macrophage and burst-forming unit-erythroid in the blood of osteopetrotic patients compared with controls; the numbers of colony-forming units approximated those found in control marrow. We conclude that OP patients with high levels of circulating CD34(+) are candidates for peripheral blood autologous harvest by limited exchange transfusion. These cells are then available for reinfusion should graft failure be observed in patients for whom retransplantation is impractical.

Influence of cytomegalovirus (CMV) sero-positivity on CMV infection, lymphocyte recovery and non-CMV infections following T-cell-depleted allogeneic stem cell transplantation: a comparison between two T-cell depletion regimens.

We compared the incidence and outcome of preemptively treated cytomegalovirus (CMV) infection, lymphocyte recovery and non-CMV infections between two different TCD modalities, one employing CD34+ selection and T-cell add-back (TCAB), preceded by Campath-1H in vivo (CD34+/TCAB group, n=29), and the other using grafts incubated with Campath-1H in vitro (Campath-1H in vitro group, n=32). The probabilities of CMV reactivation and recurrence were 67 and 83.6% in the CD34+/TCAB group and 42.9 and 20% in the Campath-1H group (P=0.07 and 0.02). Donor sero-positivity reduced CMV reactivation in the Campath-1H group, but not in the CD34+/TCAB group. The durations of positive PCR/antigenemia positivity and antiviral therapy were also significantly longer in the CD34+/TCAB group. However, only two patients developed CMV disease in each group. The mean absolute lymphocyte counts (x 10(9)/l) at 30 days (0.27 vs 0.4, P=0.03) and 100 days (0.77 vs 1.4, P=0.01) were significantly lower in the CD34+/TCAB group along with a higher incidence of non-CMV infections in CMV at-risk patients, but not in the CMV low-risk group. These findings suggest that the modality of TCD should be tailored according to the CMV risk status, and CMV sero-positive patients should receive a less extensively T-cell-depleted graft and a CMV sero-positive graft if possible.

Leukaemic stem cells.

All haemopoietic cell lineages arise from multipotential self-renewing stem cells that give rise to committed progenitor cells. These progenitor cells subsequently differentiate into more lineage-committed cells with a restricted range of plasticity. A hierarchical order is considered to exist, where lineage commitment and differentiation are thought to be irreversible. As cells differentiate, they gradually lose the ability to self-renew. The most primitive haemopoietic progenitor cells have the ability to reconstitute long-term haemopoiesis in myeloablated recipients. However, as cells differentiate, there is an orchestrated silencing of some genes and activation of others, resulting in lineage commitment and generally a reduction in proliferative ability. Here, we discuss potential differences between normal and leukaemic stem cells, some of which may have therapeutic implications.

Early lymphocyte recovery is an important determinant of outcome following allogeneic transplantation with CD34+ selected graft and limited T-cell addback.

We evaluated the outcome of 29 patients (age 22-60 years), who received a CD34+selected related (n=16) or unrelated graft (n=13) with limited T-cell addback (TCAB) (median 5.9 x 10(4)/kg) following full-intensity conditioning for haematological malignancies. In all, 16 patients (55%) had either advanced disease or previous transplants. The cumulative incidences of grade 2-4 acute GVHD were 15.4 and 19.2% and that for chronic extensive GVHD were 35 and 37% in related and unrelated graft recipients, respectively. The strongest predictor of nonrelapse mortality and overall survival was the absolute lymphocyte count (ALC) at 30 days; patients with ALC<0.35 x 10(9)/l having an NRM and OS of 59.2 and 24.7%, compared to 10 and 90% in those with a higher ALC. Patients with acute leukaemia had poorer survival and this was associated with a lower ALC as well. Thus, TCAB with a CD34+ selected graft resulted in a comparable outcome in both older and younger patients, but the survival was strongly influenced by early lymphocyte recovery.

An optimised biphasic culture system for the generation of functional dendritic cells from patients with acute lymphoblastic leukaemia at presentation and in clinical remission.

We have tested the hypothesis that functional dendritic cells (DC) may be generated from patients with acute lymphoblastic leukaemia (ALL). We evaluated the production of DC from blast cells taken at presentation from nine children with ALL. Blast cells were expanded in serum-free medium supplemented with Flt3L, G-CSF, GM-CSF, IL-3, IL-6 and SCF for 7 days and subsequently stimulated with Flt3L, GM-CSF and TGF-beta for a further 14 days, with the addition of TNF-alpha for the final 48 h of culture. Cultured cells had the morphological appearance of DC and expressed the DC-associated antigens CD1A (range 2-87%) and CD83 (15-44%). Expression of the co-stimulatory molecules CD80 and CD86 was increased and the majority of these cells retained their expression of CD34 (73+/-4%) and HLA-DR (79+/-5%). Seven of the nine ALL had a leukaemia-specific abnormality and DC generated from five of these seven cases were derived from the leukaemic clone. Leukaemic DC derived from four HLA-A*02-positive ALL pulsed with CMV-associated peptides could induce significant proliferation of peptide-specific CD8+ T cells. This specificity was verified using tetrameric complexes of HLA class l/antigenic peptide. DC could also be generated from cells taken at times of complete remission of ALL and from normal controls using these culture conditions. These findings show that functional DC can be generated both from ALL blasts and from patients in remission; these might be utilised in future for immunotherapeutic strategies in the treatment of ALL.

The clinical features, risk factors and outcome of thrombotic thrombocytopenic purpura occurring after bone marrow transplantation.

In this study, we retrospectively analysed the clinical features, risk factors and outcome of 22 patients with thrombotic thrombocytopenic purpura (TTP) occurring after allogeneic stem cell transplantation. All but two of these patients received stem cells from unrelated donors (UDs), two-thirds were female, three-quarters were adults and leukaemia was the major reason for transplant. The incidence of TTP was 20 out of 332 patients (6%) with UD transplants and two out of 104 recipients (2%) of matched sibling allografts (P = 0.16). In order to ascertain basic demographic risk factors for the development of TTP, we compared the 22 patients with 434 patients who did not develop TTP. Compared with patients who did not develop TTP, patients with TTP were nearly three times older (P < 0.001) and were more than twice as likely to be female (P = 0.001). Because > 90% of patients were recipients of UD marrow, we then compared the 20 UD-bone marrow transplantation (BMT) patients with 60 randomly selected UD-BMT patients who did not develop TTP. On univariate analysis, age and female gender were again significant risk factors, as was grade II-IV acute graft-versus-host disease (GvHD) (P = 0.002), and there was a trend towards an association with chronic GvHD (P = 0.083). However, after logistic regression analysis, only age and sex remained significant (P < 0.001 and 0.009 respectively). We report an 86% mortality with only three survivors out of 22 patients, and one of these remains thrombocytopenic and red cell transfusion dependent, possibly in part because of graft hypoplasia. Six out of 17 patients responded to plasmapheresis, but the majority of them ultimately succumbed because of TTP, often in association with GvHD or fungal infection.

T cell-depleted unrelated donor bone marrow transplantation for acute myeloid leukemia.

The outcome for 39 patients with acute myeloid leukemia (AML) in remission who had CAMPATH 1M T cell-depleted unrelated donor bone marrow transplantations (BMTs) is described. Conditioning was mainly with cyclophosphamide (120 mg/kg) and total body irradiation (TBI) (14.4 Gy), but 5 patients received busulfan in place of TBI and 200 mg/kg cyclophosphamide. All patients received cyclosporin, and short-course methotrexate was given to recipients of mismatched grafts. The patient population was predominantly pediatric (median age, 10 years), but one third of the patients was aged 15 years or above. Twenty-five patients were in second complete remission (CR2), and 14 had high-risk CR1 disease (primarily failed remission induction or antecedent myelodysplastic syndrome, often with complex cytogenetic abnormalities). Both recipient and donor were cytomegalovirus seronegative in 15 of 37 cases (38%); 51% of patients were matched for HLA class I and II. Grade II to IV acute graft-versus-host disease (GVHD) occurred in 24% of patients; chronic GVHD occurred in 5 of 31 evaluable patients (16%), 4 extensive and 1 limited. Relapse occurred in 5 cases (13%); 1 of these 5 patients survives, 24 months after a second unrelated donor transplantation. Two of these relapses were associated with secondary graft failure (incidence rate, 5%). All patients engrafted primarily. Severe viral infection was the major transplant-associated complication, with 12 episodes in 9 patients, 5 of them lethal. Twenty-five patients survive at a median follow-up of 44 months (range, 2-102 months), with estimated actuarial overall and disease-free survival rates at 44 months of 61% (SE 8%) and 57% (SE 8%), respectively. Nineteen patients are more than 2 years post-BMT and may be cured. The functional status of long-term survivors is excellent, with 19 of 21 patients who survive 6 months or more in full-time employment or full-time students. These encouraging results suggest that in patients lacking a sibling donor, unrelated donor BMT for AML in remission achieves survival figures as good as or better than those reported on patients with autologous stem cell transplantation, and that T-cell depletion of grafts is associated with a low relapse rate and an excellent functional status. However, only a randomized study comparing unrelated donor BMT and auto-grafting will resolve which of these treatment strategies is better for patients with AML.

Differential leucocyte subpopulation analysis of leucodepleted red cell products.

Donor leucocytes are responsible for many adverse transfusion effects. Clinical reactions may be attributed to specific leucocyte subsets. In this study leucocyte subpopulations were identified and quantified pre- and post-leucodepletion by integral filtration using novel Optipac(R) configurations incorporating either WBF-1 (Pall Medsep) or RS2000 (Asahi) whole blood filters. Leucocytes were analysed by flow cytometry using direct, four-colour, membrane immunofluorescence with monoclonal antibodies specific for CD 3, 14, 16, 19 and 45. Filtration reduced the leucocyte load by 3-4 log10, consistently giving products with < 2 cells microL-1. Subset distributions were also affected with the proportion of neutrophils and monocytes increased and the lymphocyte/monocyte ratio inverted. These effects were independent of the preprocessing hold conditions, filter used and buffy coat (BC) removal. All filtered red cell products contained 75-80% neutrophils, 16-20% monocytes and 2-7% lymphocytes. Results presented here demonstrate that whole blood filtration, and BC removal, significantly reduce the content and substantially alter the subpopulation distribution of the donor leucocytes remaining in leucodepleted red cell products.

Impact on T-cell depletion and CD34+ cell recovery using humanised CD52 monoclonal antibody (CAMPATH-1H) in BM and PSBC collections; comparison with CAMPATH-1M and CAMPATH-1G.

BACKGROUND: Ex vivo T-cell depletion of allogeneic BM (BM) grafts can effectively reduce graft versus host disease (GvHD) and may also apply to transplantation of allogeneic peripheral blood stem cell (PBSC) transplants. METHODS: Here we have evaluated T-cell depletion and progenitor cell recovery by antibody-mediated cells lysis using three CD52 monoclonal antibodies (MAbs) at different concentrations and cell densities. RESULTS: CAMPATH-1M was superior to CAMPATH-1H for T-cell depletion of BM samples. Treatment with CAMPATH-1M resulted in up to 2.55 log depletion of CD3+ cells, with recoveries of >or=45% CD34+ cells, >or=67% CFU-GM and >or=65% BFU-E. CAMPATH-1H treatment resulted in up to 1.64 log depletion of CD3+ cells and similar recoveries of CD34+ cells, CFU-GM and BFU-E as seen with CAMPATH-1M. Depletion of CD19+ cells was similar to that observed for CD3+ cells while natural killer (NK) cells were relatively spared compared with the T and B cell populations. Log depletions of T cells from PBSC, as determined by immunofluorescence studies and limiting dilution analyses, were similar using CAMPATH-1M, -1H, and -1G. There were also no differences in the depletion of CD19+ cells or NK cells using the three MAbs. Similar results were obtained for recoveries of CD34+ cells, CFU-GM and BFU-E using all three MAbs, although the recovery of CD34+ cells using the highest concentration of MAbs was significantly greater in CAMPATH-1H treated samples. Increasing the number of PBSC treated with CAMPATH-1H and -1M had no effect on the log depletion of T, B or NK cells and there were no major differences in the log depletions achieved with CAMPATH-1H or -1M. Likewise, the higher PBSC density had no effect on the recoveries of CD34+ cells or committed progenitors and once again CAMPATH-1H gave similar recoveries to those obtained using CAMPATH-1M. DISCUSSION: Although CAMPATH-1M resulted in greater ex vivo T-cell depletion of BM than CAMPATH-1H, in all other respects, the humanised CAMPATH-1H was just as effective as CAMPATH-1M and -1G.

Outcome and clinical course of 100 patients with adenovirus infection following bone marrow transplantation.

We conducted a retrospective review of the clinical features and outcome of adenovirus infection in 572 consecutive patients transplanted in a single centre over a 10 year period. One hundred patients (17%) had a total of 105 episodes of adenovirus infection diagnosed at a median of 18 days post transplant (range 2-150 days). The incidence was higher in children than adults (21% vs 9%, P < 0.001) and in unrelated donor vs matched sibling donor transplants (26% vs 9%, P < 0.001). Diarrhoea and fever were the most common presenting features. Reflecting these symptoms, the most common site of isolation was the stool. Serotypes 1, 2 and 7 were the most frequently seen (total of 41/68 or 60% of evaluable cases). In six patients (6%) adenovirus infection was the direct cause of death occurring at a median of 72 days post transplant (range 18-365 days). Five of these six patients had pulmonary involvement and four had associated graft-versus-host disease (GVHD). Three further patients were considered to have severe adenoviral disease (total incidence 9%). Isolation of virus from multiple sites correlated with a poor outcome (P < 0.001). Comorbid viral infection was common in this group with 50% of all patients having other viruses isolated (predominantly polyoma virus and cytomegalovirus). We conclude that adenovirus is commonly isolated after bone marrow transplant and is a cause of significant morbidity but was a rare cause of mortality (6/572 = 1%) in our patient group as a whole. The relative infrequency of severe infection will make it difficult for the transplant physician to decide which patients should receive experimental antiviral drugs such as ribavirin and cidofovir or immunomodulatory therapy with donor white cell infusions.

Children with acute lymphoblastic leukemia who receive T-cell-depleted HLA mismatched marrow allografts from unrelated donors have an increased incidence of primary graft failure but a similar overall transplant outcome.

Disparity for HLA in unrelated donor bone marrow transplantation (BMT) increases the risk of graft rejection and graft-versus-host disease (GVHD) and may compromise transplant outcome. We have compared the outcome of matched and mismatched transplants from unrelated donors in 137 children with acute lymphoblastic leukemia (ALL). Their disease status was complete remission (CR)-1, 24 patients; CR-2, 88 patients; CR-3, 18 patients; CR-4, 2 patients; and relapse, 5 patients. CAMPATH monoclonal antibodies were used for T-cell depletion and cyclosporin A was given to 134 children together with short-course methotrexate in 43, mainly when there was HLA disparity. Fifty-two donor/recipient pairs were HLA-mismatched, 41 at HLA-A and -B and 11 at HLA-DR and -DQ loci. Overall graft failure was increased in recipients of marrow mismatched at either HLA-A, -B, -DR, or -DQ (15.7% v 4.8%; P =.057) mainly because there was a higher proportion of children with primary graft failure (11. 8% v 1.2%; P =.012). The presence of an HLA-C locus mismatch did not independently increase the likelihood of graft failure. There was no significant difference in the incidence of acute GVHD >/= grade 2 between the matched and mismatched groups (P =.849). For patients in CR-2, the risk of relapse post-BMT was significantly lower if leukemic relapse occurred off-treatment (P =.005). The Kaplan-Meier overall and leukemia-free survival (LFS) estimates for recipients of matched and mismatched BMT, respectively, at 36 months were 49% versus 42% (P =.380) and 45% versus 40% (P =.654). Although HLA mismatching results in an increased occurrence of primary graft failure with T-cell-depleted allografts, it allows more donors to be identified rapidly for children with ALL without compromising overall transplant outcome.

Prevention of transfusion-transmitted cytomegalovirus infection.

Cytomegalovirus (CMV) is a double-stranded DNA virus which can be transmitted by blood transfusion. Its seroprevalence in adults ranges from 40% to 100% depending on geographical and socioeconomic conditions. Seropositive individuals have latent CMV infection with viral DNA present in peripheral blood leucocytes. CMV can be associated with considerable morbidity and mortality in susceptible individuals, e.g. CMV-seronegative bone marrow allograft patients. Evidence, from a number of reports, suggests that provision of leucodepleted blood components may be as effective as the use of components from CMV-seronegative donors in preventing CMV infection and disease. This is relevant in the UK because Blood Transfusion Services are implementing universal leucodepletion of cellular blood components to minimize the theoretical risk of transmission of new variant Creutzfeldt-Jakob disease. This review examines data on the biology of CMV, discusses options for testing and summarizes the impact of CMV-seronegative and leucodepleted blood components on transfusion-transmitted CMV.

Evaluation of plasma and red cells obtained after leucocyte depletion of whole blood.

We evaluated whole blood integral filtration to produce leucocyte-depleted red cells and plasma by using the WBF1 whole blood filter (Pall Medsep). Whole blood units were filtered after either warm (2-4 h at room temperature) or cold (12-24 h at 4 degrees C) holds. Filtered and control units were processed using either a bottom-and-top or top-top method. Red cells were tested weekly for 6 weeks, and plasma 3 monthly for 12 months. All filtered red-cell packs contained < 5 x 10(6) leucocytes/unit with 71 of 72 containing < 1 x 10(6) leucocytes/unit. No clinically significant differences in red-cell storage parameters were seen, although haemolysis was less and pO2/pCO2 values were better maintained in filtered units. Plasma units contained < 2.5 x 10(3) leucocytes/unit with no significant loss of factor VIII except in the warm hold units processed by the top-top method. There was no evidence of complement or coagulation activation with significant removal of preformed C3a in cold hold units. Plasma storage parameters were maintained at control levels for 12 months.

Infections in adults undergoing unrelated donor bone marrow transplantation.

This study retrospectively reviews infections over a 7-year period in 60 consecutive adults (median age 25 years) undergoing their first unrelated donor bone marrow transplant (UD-BMT). T-cell depletion was employed in 93%. More than half the patients had one or more severe, potentially life-threatening, infections. There was a high incidence of invasive fungal infections (Aspergillus 17, Candida four), despite the use of itraconazole or amphotericin prophylaxis. Ten Aspergillus infections occurred beyond 100 d. Two patients (11%) with invasive aspergillosis survived. Clustering of infections was noted, with invasive fungal infections significantly associated with bacteraemias (OR 3.73, P=0.06) and multiple viral infections (OR 4.25, P=0.05). There were 21 severe viral infections in 16 patients, with CMV disease occurring in four patients only; viral pneumonitis was predominantly due to 'community respiratory' viruses. Most early bacteraemias (68%) were due to Gram-positive organisms. The majority of episodes of Gram-negative sepsis were caused by non-fastidious non-fermentative bacteria, such as Pseudomonas spp. and Acinetobacter spp., historically regarded as organisms of low pathogenicity. In patients with successful engraftment and minimal graft-versus-host disease, late infections suggestive of continued immune dysfunction (shingles, recurrent lower respiratory infections, Salmonella enteritis and extensive warts) were common.