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.

Unrelated donor bone marrow transplantation for children and adolescents with Philadelphia-positive acute lymphoblastic leukemia.

PURPOSE: Few patients with Philadelphia-positive acute lymphoblastic leukemia (Ph-positive ALL) have been cured by chemotherapy alone. Registry figures show that 38% of patients who have a matched-sibling bone marrow transplant (BMT) are disease-free 2 years after transplant, but the majority of patients lack a sibling donor. Most modern ALL protocols recommend unrelated donor (UD) BMT for patients with Ph-positive ALL in first complete remission (CR1), but the outcome of this is unknown. PATIENTS AND METHODS: We report the results of 15 children and adolescents who had a T-cell depleted UD-BMT for Ph-positive ALL. Thirteen of 15 had been previously treated on United Kingdom ALL protocols. Nine were in CR1 and six had more advanced disease. Eleven donor recipient pairs were matched at HLA-A, HLA-B, HLA-DR, and HLA-DQ, and four were mismatched at one or two HLA loci. RESULTS: The incidence of greater than grade I acute and chronic graft-versus-host disease (GVHD) was low (13% and 8%, respectively). Six patients have relapsed and seven patients survive at a median of 21 months post-BMT; six of seven are disease free. All seven survivors are in full-time education or work. The 2-year overall and disease-free survivals are 44% +/- 13% and 37% +/- 13% (+/- SE). None of four patients who had mismatched donors survived, but seven of 11 matched recipients survive (P < .05). CONCLUSION: UD-BMT can produce prolonged disease-free survival in young patients with Ph-positive ALL who otherwise would have an extremely poor outlook.

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.

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.

Ultraviolet irradiation for the prevention of graft-versus-host disease and graft rejection in bone marrow transplantation.

This review describes an approach to the prevention of graft-versus-host disease (GVHD) and graft rejection following allogeneic BMT that differs from conventional methods. Ultraviolet (UV) irradiation inhibits the proliferative responses of lymphoid cells to mitogens and alloantigens by inactivation of T lymphocytes and dendritic cells, and in animal models this can prevent both GVHD and graft rejection. It is important that the marrow repopulating capacity of haemopoietic stem cells is not damaged by the irradiation process. We have found that polymorphic microsatellite markers are a sensitive way of assessing the impact of UV irradiation on chimerism after BMT in rodents.

Graft-versus-host disease in children receiving HLA-identical allogeneic bone marrow transplants with a low adjusted T lymphocyte dose.

Fourteen children with high risk leukaemia received allogeneic bone marrow transplants from HLA-identical MLC-compatible sibling donors. All bone marrows were T cell depleted and a T cell addback was prepared from the donor's peripheral blood so that the mean total number of CD3+ cells given was 2.6 (1.0-4.1) x 10(5)/kg recipient body weight. This was administered as a short infusion prior to the bone marrow. The children were conditioned with 1440 cGy fractionated total body irradiation and cyclophosphamide 120 mg/kg and were not given cyclosporin A or methotrexate. All patients engrafted and none showed late graft rejection. Acute graft-versus-host disease (GVHD) developed in nine of 14 children and required treatment with steroids. Two children with grade IV GVHD and one with grade I acute GVHD who subsequently developed severe chronic GVHD died. There have been two relapses (both fatal) and one death from cytomegalovirus pneumonitis. Survival is currently 57% (8/14) with a mean follow-up of 548 days (range 384-810). A high incidence of GVHD which was fatal in three patients can occur despite infusion of low T cell numbers in the absence of post-graft immunosuppression.

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.

A laboratory comparison of T cell depletion by CD34+ cell immunoaffinity selection and in vitro Campath-1M treatment: clinical implications for bone marrow transplantation and donor leukocyte therapy.

Donor leukocyte infusions (DLI) have been used effectively to induce remission in patients who relapse after BMT. Using CD34+ cell immunoaffinity enrichment, donor T cells may be captured in the unadsorbed (residual) fraction and we assessed this as a potential source of functional T cells for post-BMT immunotherapy. We extended our study to compare CD34+ cell selection and antibody-mediated cell lysis using Campath-1M and measured T cell-depletion, CD34+ cell recovery and relative progenitor proliferative potential. The recovery of CD3+ cells (responsive to IL-2 or PHA) in the unadsorbed fraction was 84+/-12% (mean+/-s.d.) using a laboratory scale CD34+ cell selection process (CEPRATE LC). The immunoselected (CD34+ cell enriched) product contained 55+/-12% of the starting CD34+ cells (purity, 75+/-6%) with recoveries of 44+/-12% and 42+/-13% for CFU-GM and BFU-E respectively. T cell depletion was 99.8+/-0.2% (FACS) and the frequency of clonable T cells estimated at 1:640 (limiting dilution assay). In comparison, Campath-1M-treated marrow samples gave recoveries of CD34+ cells, CFU-GM and BFU-E of 50+/-7%, 78+/-20% and 79+/-18%, respectively. The frequency of clonable T cells was 1:2700 despite an estimated T cell depletion of 98.4+/-1.9%. Data obtained from four BM harvests processed on the clinical grade CEPRATE SC system was comparable in every respect to the laboratory scale system. The yield of 1259 +/- 222 x 10(6) CD3+ cells in the unadsorbed fraction would allow for multiple graded incremental T cell aliquots for DLI for patients with acute leukaemia.

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.

Second allogeneic bone marrow transplants from unrelated donors for graft failure following initial unrelated donor bone marrow transplantation.

Graft failure is a common and severe complication of unrelated donor bone marrow transplantation (UD-BMT). However, there are few reports of a second UD-BMT in this setting. We describe 12 patients with graft failure (five primary, seven secondary) who had a second transplant, five from their original donor and seven from a different donor. Their median age was 9 years. Two patients died before day 10 of regimen-related toxicity. Nine of 10 evaluable patients engrafted in a median of 17 days. Secondary graft failure was seen in one patient. Transplant-related morbidity was significant. Six of nine developed acute GHVD, there were five severe infections and five patients developed Bearman grade 3 or 4 extramedullary toxicity. Overall, five patients survive at a median of 38 months after the second BMT and two are in continuous complete remission. Second transplants from unrelated donors for graft failure can result in prolonged survival.

Influence of ultraviolet-B irradiation on engraftment, graft-versus-host disease and graft-versus-leukemia effect in a rat model for allogeneic bone marrow transplantation.

Ultraviolet-B (UVB) irradiation is known to inhibit lymphocyte activity and consequently to reduce the incidence of graft-versus-host disease (GVHD) in experimental models for allogeneic bone marrow transplantation (BMT). GVHD is frequently associated with morbidity and mortality, but also with the beneficial graft-versus-leukemia (GVL) effect, demonstrated by a reduction in the incidence of leukemia relapse. In this study, we investigated whether UVB treatment of allogeneic T cells could prevent GVHD while sparing the beneficial GVL effect following allogeneic BMT in the Brown Norway myelocytic leukemia (BNML) rat model analogous to human acute myelocytic leukemia (AML). The dose of UVB required to abolish lethal GVHD in the rat allogeneic BMT model (WAG/Rij donors into BN recipients) was 4000 J/m2. However, this UVB dose simultaneously abrogated all GVL activity mediated by the T cells in the graft, while the radio-protective capacity of rat BM cells was strongly reduced. The number of allogeneic BM cells required to protect lethally irradiated BN rats was increased 50 to 100-fold. It is concluded that UVB acts as a non-selective form of T cell inactivation, and that UVB pretreatment of an allogeneic marrow graft is unlikely to be useful clinically as a preventive measure for GVHD, since other means of reduction of the number of functional T cells are less damaging to bone marrow stem cells.

Double T cell depletion of bone marrow using sequential positive and negative cell immunoaffinity or CD34+ cell selection followed by Campath-1M; effect on CD34+ cells and progenitor cell recoveries.

An advantage of CD34+ cell selection over antibody purging is that a component allograft is produced comprising a stem cell enriched and an unadsorbed fraction, the latter containing T cells which may be used for post-transplant immunotherapy. Initial reports with PBSC allografts suggested that T cell depletion (TCD) by CD34+ cell selection and post-graft cyclosporin A +/- methotrexate was insufficient prophylaxis against acute GVHD. We compared sequential TCD (of a CD34+ cell-selected fraction) using a second (CD2) immunoaffinity step or Campath-1M monoclonal antibody and complement. Since a high stem cell 'dose' enhances engraftment across HLA barriers and improves overall post-transplant outcome, the recovery of CD34+ cells and progenitors were assessed. Sequential positive (CD34+) and negative (CD2+) immunoaffinity selection resulted in a 3.4 log depletion of T cells as compared to a 4.05 log depletion when CD34+ cell selection was followed by Campath-1M treatment. Recoveries of CD34+ cells, CFU-GM and BFU-E following double depletion using CD34+ cell selection plus CD2+ cell depletion were 28, 25 and 17% as compared to 20, 18 and 16% when CD34+ cells were treated with Campath-1M. The unadsorbed fraction contained 85% of the original T cells, from which donor leukocyte infusions in the range of 10(5) to 10(7) CD3+ cells per kg body weight of the recipient were harvested. Despite the advantages of component allografts, the loss of stem/progenitor cells may restrict sequential TCD steps unless single BM harvests are supplemented and/or replaced with mobilised PBSCs.

Transplantation of peripheral blood progenitor cells from unrelated donors.

Six patients with high risk haematological malignancies received peripheral blood progenitor cells (PBPC) from unrelated donors. Four patients received PBPC as primary treatment and 2 following graft failure. Five donors were HLA-A, -B and -DR identical and one had a one antigen mismatch. PBPC were mobilised by treatment with G-CSF for 4-6 days. The patients received a range of 3.4 to 11.4 x 10(8) mononuclear cells/kg and 1.0 to 15.0 x 10(6) CD34 positive cells/kg. Four patients were given Campath 1G and 2 ATG prior to transplantation. The patient with one antigen mismatch received in vitro T-cell depleted PBPC using Campath 1M. All received cyclosporin and 5 in addition methotrexate. All recipients were given G-CSF and all engrafted. The patients developed no or mild acute GVHD. Two patients had limited chronic GVHD of the skin. The recipient of the mismatched graft died from extensive chronic GVHD. Three patients have had a relapse and two are alive and free of leukaemia.

Immune reconstitution after BMT in children.

Serial assessment of peripheral blood T and B cell recovery and serum immunoglobulins was performed in 19 children for the first year following BMT and compared with normal values established from healthy children. Immunophenotypic analysis on bone marrow was performed in selected cases by Southern blotting of the immunoglobulin heavy chain (IgH) gene. We found no significant differences between T cell-replete or depleted allogeneic bone marrow transplants. Lymphocyte numbers were low until 9 months post-BMT. T cell numbers (CD2, CD3, CD5) were also low until 12 months but B cell numbers (CD19) became normal at 3 months. Both CD4+ and CD8+ T cell subsets were low post-BMT with depression of CD4+ greater and more prolonged than that of CD8+. No overshoot of CD8+ was seen. The principal effect of GVHD or its treatment was further depression of CD4+ cells but with no increase in CD8+; recovery of B cells was also delayed. Recovery of IgG was slow with only six of 11 children reaching an age-adjusted normal level by 1 year, whereas there was more rapid recovery of IgM and IgA. Several children had an increase in lymphocytes of immature appearance in their bone marrow at varying times post-BMT with increased cells of phenotype CD19+, CD10+, HLA-DR+ and TdT+. In each case Southern blotting showed a germline pattern of the IgH indicating a polyclonal early B cell regenerative population.

Successful second unrelated donor BMT in a child with juvenile chronic myeloid leukaemia: documentation of chimaerism using the polymerase chain reaction.

A 3-year old child with juvenile chronic myeloid leukaemia received a T cell-depleted BMT from a male unrelated donor. There was early graft failure associated with increasing splenomegaly and hypersplenism. Splenectomy was performed 53 days post-transplant and was followed by autologous marrow recovery with return of leukaemia. A second unrelated donor BMT was performed 9 months later using T cell-replete marrow from a similarly matched female donor. Grade 2 GVHD involving the skin and gut responded to treatment with steroids. Chimaerism was assessed using Y-specific polymerase chain reaction (PCR) and microsatellites. Samples taken at the time of splenectomy showed no donor marrow engraftment but there was significant engraftment in the spleen. Following the second transplant, donor-type haematopoiesis was documented using a panel of microsatellite probes. The patient remains well 6 months after transplant. Splenectomy should be considered prior to transplant in patients with significant splenomegaly and hypersplenism. Partial chimaerism in the spleen, but not bone marrow, post-BMT, has not previously been documented. PCR technology is a useful and highly sensitive way to assess chimaerism post-BMT and is informative in sex-matched cases, whilst the small amount of material required is advantageous in paediatric patients.

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.

PCR amplification of short tandem repeat sequences allows serial studies of chimaerism/engraftment following BMT in rodents.

Animal models of bone marrow transplantation (BMT) allow evaluation of new experimental treatment strategies. One potential strategy involves the treatment of donor marrow with ultra-violet B light to allow transplantation across histocompatibility boundaries without an increase in graft rejection or graft-versus-host disease. A major requirement for a new experimental protocol, particularly if it involves manipulation of the donor marrow, is that the manipulated marrow gives rise to long-term multilineage engraftment. DNA based methodologies are now routinely used by many centres to evaluate engraftment and degree of chimaerism post-BMT in humans. We report the adaptation of this methodology to the serial study of engraftment in rodents. Conditions have been defined which allow analysis of serial tail vein samples using PCR of short tandem repeat sequences (STR-PCR). These markers have been used to evaluate the contribution of ultraviolet B treated marrow to engraftment following BMT in rodents without compromising the health of the animals under study. Chimaerism data from sequential tail vein samples and bone marrow from selected sacrificed animals showed excellent correlation, thus confirming the validity of this approach in analysing haemopoietic tissue. Thus the use of this assay may facilitate experimental studies in animal BMT.

The outcome of 26 patients with respiratory syncytial virus infection following allogeneic stem cell transplantation.

Respiratory syncytial virus (RSV) is known to cause acute lung injury in the immunocompromised host, especially recipients of bone marrow allografts. Specific prognostic factors for the development of severe life-threatening disease remain to be identified as does the optimum treatment of established disease. Over a 5-year period the incidence and outcome of RSV in BMT recipients was analysed retrospectively. Prognostic factors assessed included type of transplant, engraftment status at the time of infection, the presence of lower respiratory tract disease, viral genotype and treatment received. During the study period, 26 of 336 (6.3%) allogeneic stem-cell recipients were identified as having RSV. Five patients (19.2%) died as a direct result of RSV. One patient died secondary to an intracranial bleed with concomitant RSV. There were four patients with graft failure (two primary and two secondary) attributable to the presence of RSV, two of whom subsequently died of infections related to prolonged myelosuppression. The presence of lower respiratory tract infection and a poor overall outcome was the only statistically significant association. Unrelated donor transplants and AML as the underlying disease appeared to be associated with a poorer outcome. Engraftment status, viral genotype and RSV treatment received did not correlate with outcome. We conclude that future studies are required to identify early sensitive and reproducible prognostic factors of RSV in the immunocompromised host. The roles of intravenous and nebulised ribavirin need to be clarified by prospective controlled trials.

Lack of significant effect of therapeutic propranolol on measurable platelet function in healthy subjects.

Propranolol, a non-selective beta blocker, was administered orally in therapeutic doses. The effects of a single dose (160 mg) and one week's treatment (80 mg twice a day) on platelet function were compared in healthy young subjects. There were no significant changes in circulating platelet aggregates, template bleeding time, platelet factor 3 availability and thromboxane beta 2 (TX beta 2) generation. Platelet aggregation responses as assessed by angle of slope and maximal percentage aggregation (all agonists) and lag phase (collagen) showed no changes of biological importance, although minor changes reaching significance were observed with some agonists. These findings suggest that propranolol does not significantly affect platelet function when taken at doses commonly encountered in clinical practice.

Acquired factor XI inhibitor in chronic lymphocytic leukaemia.

A 71 year old man with chronic lymphocytic leukaemia (CLL) experienced excessive bleeding following transurethral resection of the prostate. Investigations showed a prolonged kaolin cephalin clotting time (KCCT) with low concentrations of factor XI. The prolonged KCCT was largely corrected by mixing with normal plasma but this correction was lost on incubation, confirming the presence of an inhibitor. He was treated with pulsed methylprednisolone and chlorambucil which resulted in the resolution of the bleeding problem and the loss of detectable circulating inhibitor.