Non-malignant haematology research in the UK: looking forward to new opportunities.

Over the last few years there has been rapid and radical change in the way clinical research in the UK is funded and supported within the NHS. This has resulted from restructuring and major new investment in research infrastructure, co-ordinated through Clinical Local Research Networks (CLRNs) and equivalent organisations in the devolved nations. CLRNs have resources to support local researchers undertake studies that have been adopted on to the national research portfolio. For example, CLRNs can help with gaining local approvals or provide research nurses to recruit patients, undertake study procedures and perform data entry. CLRNs can establish Local Speciality Groups in a number of areas of medicine, including nonmalignant haematology. These new networks offer non-malignant haematology access to significant new resources and a major opportunity to support clinical research for the benefit of our patients.

Targeted sequencing to identify novel genetic risk factors for deep vein thrombosis: a study of 734 genes.

Essentials Deep vein thrombosis (DVT) has a large unknown genetic component. We sequenced coding areas of 734 hemostasis-related genes in 899 DVT patients and 599 controls. Variants in F5, FGA-FGG, CYP4V2-KLKB1-F11, and ABO were associated with DVT risk. Associations in KLKB1 and F5 suggest a more complex genetic architecture than previously thought. SUMMARY: Background Although several genetic risk factors for deep vein thrombosis (DVT) are known, almost all related to hemostasis, a large genetic component remains unexplained. Objectives To identify novel genetic determinants by using targeted DNA sequencing. Patients/Methods We included 899 DVT patients and 599 controls from three case-control studies (DVT-Milan, Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis [MEGA], and the Thrombophilia, Hypercoagulability and Environmental Risks in Venous Thromboembolism [THE-VTE] study) for sequencing of the coding regions of 734 genes involved in hemostasis or related pathways. We performed single-variant association tests for common variants (minor allele frequency [MAF] ≥ 1%) and gene-based tests for rare variants (MAF ≤ 1%), accounting for multiple testing by use of the false discovery rate (FDR). Results Sixty-two of 3617 common variants were associated with DVT risk (FDR < 0.10). Most of these mapped to F5,ABO,FGA-FGG, and CYP4V2-KLKB1-F11. The lead variant at F5 was rs6672595 (odds ratio [OR] 1.58, 95% confidence interval [CI] 1.29-1.92), in moderate linkage with the known variant rs4524. Reciprocal conditional analyses suggested that intronic variation might drive this association. We also observed a secondary association at the F11 region: missense KLKB1 variant rs3733402 remained associated conditional on known variants rs2039614 and rs2289252 (OR 1.36, 95% CI 1.10-1.69). Two novel variant associations were observed, in CBS and MASP1, but these were not replicated in the meta-analysis data from the International Network against Thrombosis (INVENT) consortium. There was no support for a burden of rare variants contributing to DVT risk (FDR > 0.2). Conclusions We confirmed associations between DVT and common variants in F5,ABO,FGA-FGG, and CYP4V2-KLKB1-F11, and observed secondary signals in F5 and CYP4V2-KLKB1-F11 that warrant replication and fine-mapping in larger studies.

Discovery and characterization of an antibody directed against exosite I of thrombin.

UNLABELLED: ESSENTIALS: An IgA paraprotein with anti-thrombin activity was not associated with a severe bleeding phenotype. This observation challenges the paradigm that anticoagulant therapy necessarily increases bleeding risk. Characterization of the antibody showed that it specifically binds to thrombin exosite I. A therapeutic drug with the properties of this antibody might be an antithrombotic that doesn't cause bleeding. BACKGROUND: We report the case of a 54-year-old female who presented with a traumatic subdural hemorrhage. Coagulation tests were markedly prolonged due to the presence of an anti-thrombin IgA paraprotein at 3 g L(-1) . The patient made a complete recovery and has had no abnormal bleeding during a 7-year follow-up, despite the persistence of the paraprotein. OBJECTIVES: To determine how the paraprotein prolonged clotting tests by defining its target and its epitope. METHODS: The paraprotein was purified and added to normal pooled plasma for in vitro clotting assays. Binding studies were conducted to determine the affinity of the IgA for thrombin. The Fab was isolated and crystallized with thrombin. RESULTS: The purified IgA was sufficient to confer the patient's in vitro coagulation profile in normal pooled plasma, and was found to bind specifically and with high affinity to thrombin. A crystal structure of the Fab fragment in complex with thrombin revealed an exosite I interaction involving CDRH3 of the antibody. CONCLUSIONS: Although the patient originally presented with a subdural bleed, the hematoma resolved without intervention, and no other bleeding event occurred during the subsequent 7 years. During this period, the patient's IgA paraprotein levels have remained constant at 3 g L(-1) , suggesting that the presence of a high-affinity, exosite I-directed antibody is consistent with normal hemostasis. A therapeutic derivative of this antibody might therefore permit antithrombotic dose escalation without an associated increase in the risk of bleeding.

Identification and removal of a promiscuous CD4+ T cell epitope from the C1 domain of factor VIII.

BACKGROUND: The development of inhibitors in hemophiliacs is a severe complication of factor VIII (FVIII) replacement therapy and is a process driven by FVIII specific T helper cells. OBJECTIVES: To finely map T cell epitopes within the whole FVIII protein in order to investigate the possibility of engineering FVIII variants with reduced propensity for inhibitor development. PATIENTS AND METHODS: T cell lines were generated from five patients with severe hemophilia who had developed inhibitors, and were screened for T cell proliferation against pools of overlapping peptides spanning the entire B domain deleted (BDD) FVIII sequence. Positive peptide pools were decoded by screening individual peptides against the T cell lines. Positive peptides, and mutants thereof, were tested for their ability to bind major histocompatibility complex (MHC) Class II and stimulate T cell proliferation in a panel of healthy donors. The activities of the corresponding mutant proteins were assessed via chromogenic assay. RESULTS: One peptide, spanning FVIII amino acids 2098-2112, elicited a vigorous response from one hemophiliac donor, induced strong T cell responses in the panel of healthy donors and bound to a number of HLA-DR alleles. Mutations were made in this peptide that removed its ability to stimulate T cells of healthy donors and to bind to MHC Class II while retaining full activity when incorporated into a mutant BDD-FVIII protein. CONCLUSIONS: Fine T cell epitope mapping of the entire FVIII protein is feasible, although challenging, and this knowledge may be used to create FVIII variants which potentially have reduced immunogenicity.

The risk of a first and a recurrent venous thrombosis associated with an elevated D-dimer level and an elevated thrombin potential: results of the THE-VTE study.

BACKGROUND: D-dimer and thrombin generation have been associated with the risk of recurrent venous thrombosis. However, for both measurements, different assays are available, and in vitro thrombin generation may be affected by the problem of contact activation during blood sampling. OBJECTIVES: To determine the association between hypercoagulability and first and recurrent thrombosis by the use of different D-dimer and thrombin generation assays, to assess whether the addition of corn trypsin inhibitor (CTI) prior to blood sampling to inhibit contact activation improved the association between thrombin generation and thrombosis risk, and to calculate the DASH score with two different D-dimer assays. METHODS: A case-control study (626 patients and 361 controls) with subsequent follow-up of the cases was performed (2987 patient-years after stopping of anticoagulant therapy). Blood was drawn 2-3 months after discontinuation of anticoagulation for the first event in citrate tubes with and without CTI. RESULTS/CONCLUSIONS: An elevated D-dimer level and elevated thrombin generation were associated with an increased risk of a first event regardless of the assay used (odds ratios: 1.8-3.4). An elevated D-dimer level but not elevated thrombin generation was associated with the risk of recurrence. Patients with elevated D-dimer levels had a more than two-fold increased recurrence rate (Vidas - hazard ratio [HR] 2.3, 95% confidence interval [CI] 1.4-3.8; HemosIL - HR 2.4, 95% CI 1.5-3.9; Thrombinoscope and Technoclone assay - HR 1.3). Elimination of contact factor activation did not improve the predictive value of thrombin generation. In patients with unprovoked first events, the DASH score had a similar predictive value for deep vein thrombosis and pulmonary embolism, both when calculated with Vidas D-dimer and when calculated with HemosIL D-dimer.

Predictive value of von Willebrand factor to ristocetin cofactor ratio and thrombin-antithrombin complex levels for hepatic vessel thrombosis and graft rejection after liver transplantation.

Acute cellular rejection and hepatic vessel thrombosis are significant postoperative complications of liver transplantation. The study investigated changes in endothelial cell-related hemostatic proteins in the peripheral circulation of patients after liver transplantation, and assays for hemostatic parameters were compared with data from routine hematologic and biochemical investigations, together with clinical information. Of the 12 patients, 8 underwent acute rejection episodes. No significant differences in any hemostatic parameter measured were seen between rejection and nonrejection groups, with the exception of the platelet count, which increased after treatment of the rejection episode. Two of the 12 patients suffered fatal hepatic vessel thrombosis during the study. A number of significant differences were found between these patients and those with no thrombotic complications, most notably and increase in the von Willebrand factor antigen to ristocetin cofactor ratio and thrombin-antithrombin complex generation. These changes occurred before clinical detection of thrombosis. Thus, measurement of these parameters may be of predictive value in the diagnosis and monitoring of post-transplant thrombosis.

Veno-occlusive disease of the liver complicating bone marrow transplantation.

Veno-occlusive disease (VOD) of the liver is a major complication of bone marrow transplantation. The clinical features are those of intrahepatic portal hypertension and while the reported frequency varies considerably, severe disease is readily identifiable and is associated with a high mortality. The single most important risk factor for the development of VOD is an elevated ALT prior to transplantation. The incidence of VOD may be reduced by prophylactic administration of anticoagulant or antiplatelet drugs and established disease can be treated by thrombolytic therapy with tissue plasminogen activator. These observations suggest that endothelial damage and thrombin generation are fundamental to the disease process. Protection of the endothelium from toxic damage and inhibition of thrombin generation are potential targets for preventing VOD.

Veno-occlusive disease of the liver complicating ABMT successfully treated with recombinant tissue plasminogen activator (rt-PA).

A 45-year-old man underwent autologous bone marrow transplantation for relapsed myeloma. The procedure was complicated by the development of veno-occlusive disease (VOD) of the liver. Thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA) resulted in rapid resolution of hepatic encephalopathy and ascites with improvement in liver function. The use of rt-PA in the management of VOD appears effective and should be evaluated in further patients.

An assessment of the efficacy of antimicrobial prophylaxis in bone marrow autografts.

Fifty-three patients undergoing autologous bone marrow transplantation received antimicrobial prophylaxis with ciprofloxacin with or without erythromycin and low dose intravenous amphotericin B. Eight patients remained afebrile throughout the neutropenic period. All other patients had one or more febrile episodes. The median time to fever after the onset of neutropenia was 7 days. There were no gram-negative organisms isolated from blood cultures during any of these episodes whereas gram-positive organisms were isolated in 28. There was one death in this series associated with sepsis. The use of low-dose prophylactic parenteral amphotericin did not prevent the subsequent successful use of full dose amphotericin for antibiotic-resistant fever. Ciprofloxacin effectively prevents gram-negative sepsis. The addition of erythromycin does little to prevent gram-positive sepsis. The use of regimens with agents with activity against gram-positive organisms is appropriate initial treatment of all febrile neutropenic episodes.

High-dose carmustine, etoposide and melphalan ('BEM') with autologous stem cell transplantation: a dose-toxicity study.

We have investigated the toxicity of dose-escalation of BCNU, etoposide and melphalan ('BEM') chemotherapy with autologous stem cell transplantation in patients with haematological malignancies. Seventy-two patients with haematological malignancies were treated with BCNU (600 mg/m2, 450 mg/m2 or 300 mg/m2), etoposide 2 g/m2 and melphalan 140 mg/m2 followed by autologous bone marrow transplantation (ABMT), n = 51, or autologous peripheral blood progenitor cell transplantation (APBPCT), n = 21. Liver and pulmonary function was monitored pretransplant and at regular intervals post-transplant. Mucositis was graded daily during in-patient stay. There was a significantly higher incidence of symptomatic pulmonary toxicity in the patients who received BCNU at 600 mg/m2 than in the other two groups, and there was a significant increase in the incidence of asymptomatic decrease in carbon monoxide (KCO) in the patients who received BCNU 450 mg/m2. There was no significant difference between the three groups in the incidence and severity of mucositis or in the incidence of transiently abnormal liver function. We conclude that etoposide at 2 g/m2 can be used without unacceptable mucositis. BCNU at 600 mg/m2 is associated with an unacceptably high incidence of lung toxicity, but at 450 mg/m2 there is minimal symptomatic lung toxicity.

Measurement of reticulated platelets following peripheral blood progenitor cell and bone marrow transplantation: implications for marrow reconstitution and the use of thrombopoietin.

Neutrophil and platelet engraftment times are significantly shorter in patients undergoing PBPCT compared with ABMT. The explanation for this is unclear. The reticulated platelet percentage (RP%) has been established as a measure of bone marrow platelet production. Using this measurement we have followed thrombopoiesis over the transplant period in 10 patients undergoing PBPCT, eight ABMT and four alloBMT. Neutrophil and platelet engraftment times were significantly shorter in patients undergoing PBPCT than either ABMT or alloBMT. The RP% fell to a nadir in parallel with the platelet count in all patients following conditioning therapy consistent with an aplastic state and rose before platelet recovery as young platelets were released. The peak rise in the RP% was significantly greater and occurred earlier in PBPCT compared with BMT. The total number of reticulated platelets released during the time of engraftment was significantly greater in PBPCT than BMT. The potential role of the RP% in the timing of thrombopoietin therapy is explored. Finally the diagnostic use of the RP% in post-transplant thrombocytopenia is illustrated by a case in which a persistently high RP% accurately predicted a consumptive aetiology.

Peripheral blood stem cell transplantation after high-dose therapy in patients with malignant lymphoma: a retrospective comparison with autologous bone marrow transplantation.

We report the results of peripheral blood progenitor cell (PBPC) harvesting in 22 patients with lymphoma who underwent leucapheresis after cells were mobilised using 3 g/m2 cyclophosphamide and G-CSF. In 19 patients, the total CFU-GM collected was greater than 7.5 x 10(4)/kg. These patients underwent successful autologous PBPC transplantation. This group of patients was compared to a historical group of 24 patients with lymphoma who underwent ABMT with the same conditioning chemotherapy. The time to engraftment of neutrophils to 0.5 x 10(9)/l was significantly reduced (median 11 days vs 19 days, P < 0.0001) and consequently in-patient stay was reduced (median 21 days vs 28 days, P < 0.001). Blood product support (median 3 vs 4 units blood, P = 0.02; median 15 vs 40 units platelets, P = 0.005) and use of TPN (median 0 days vs 8 days, P < 0.001) were reduced. We estimate a saving of approximately pounds 2370 per patient using PBPC for autologous transplantation compared to bone marrow progenitor cells. This saving is significant (P < 0.001).

Autografting for multiple myeloma: a 5-year experience at a single institution.

Over a 5-year period, we have performed 33 autologous bone marrow or PBPC transplantations for multiple myeloma. Nine patients were in complete remission and 24 in partial remission at time of transplantation. Conditioning regimens were BEM (BCNU, etoposide and melphalan) in 29, busulphan and cyclophosphamide in three and melphalan alone in one. Two patients (6%), died within 3 months of transplant-related mortality, seven (21.3%), died of disease progression at a median follow-up of 11 months (range 4-24). Twenty-four patients (72.7%) are alive at a median follow-up of 15 months (range 4-61). Of nine patients transplanted in CR, four have relapsed and are alive and five remain in CR. Of 24 patients transplanted in PR, nine have died, six remain in PR, eight achieved CR and one has progressive disease. The overall median progression-free survival (PFS) is 31 months (95% CI = 20-42). For patients transplanted in PR the median PFS is 24 months (95% CI = 22-26), the median PFS for patients transplanted in CR has not yet been reached. The median PFS for patients achieving CR pre- or post-transplantation was better than for patients neither achieving CR pre- nor post-transplantation (P = 0.05). The median PFS was also significantly improved for patients requiring only primary therapy, compared to patients needing second-line therapy to achieve CR or stable PR prior to transplantation (31 vs 11 months, P = 0.02).

High-dose therapy and autologous stem cell rescue for poor risk and refractory lymphoma: a single centre experience of 123 patients.

Over an 8-year period we autografted 123 patient with poor-risk lymphoma. Sixty-three patients had Hodgkin's disease (HD) and 60 non-Hodgkin's lymphoma (NHL). Of the patients with HD, 45 had responsive and 18 resistant disease prior to high-dose therapy. Fifty-three patients with NHL had responsive and seven had resistant disease at the time of transplantation. Seventy-seven patients received autologous bone marrow (BM) rescue, 39 autologous peripheral blood progenitor cell (PBPC) rescue, and seven combined BM and PBPC rescue. High-dose chemotherapy was BEM in 67, BEAM in 39, TBI and cyclophosphamide or etoposide or BCNU in 10, etoposide/mitozantrone in six and etoposide/melphalan in one. There was eight (6.5%) deaths due to treatment-related toxicity, within the first 100 days post-transplantation. Of the patients with HD 41 (65%) are alive at a median follow-up of 39 months (range 2-94). Thirty-three (52%) patients remain in CR. The median DFS of the 63 patients with HD is 34 months (95% CI 7-61). The median DFS for patients transplanted with responsive disease was significantly better than for those transplanted with refractory disease (61 vs 21 months P < 0001). Thirty-five (58%) of the patients with NHL are alive, and 20 (33%) remain in CR. The median DFS for patients transplanted with responsive and refractory disease was 11 months (95% CI 3-19) and 4 months (95% CI 0-9; P = NS) respectively. The median DFS for patients transplanted with HD was significantly better than for patients transplanted with NHL (34 vs 8 months, P < 0.002). In both groups there was no significant difference in DFS in patients receiving one, two, three or more lines of therapy prior to transplantation. In summary, in patients with poor-risk lymphoma who have responsive disease high-dose therapy may result in durable CRs. Conversely, only a small proportion of patients with HD or NHL with resistant disease achieve CR after autologous stem cell rescue.

Peripheral blood progenitor cell transplantation: a single centre experience comparing two mobilisation regimens in 67 patients.

Between June 1991 and January 1995 we performed 67 peripheral blood progenitor cell transplants (PBPCT). Ten patients (group 1) were mobilised with 7 gm/m2 of cyclophosphamide followed by daily G-CSF injections (5 micrograms/kg, subcutaneously). When the white cell count reached 1 x 10(9)/1 they were leukapheresed for 5 days. After stem cell infusion they received G-CSF (10 micrograms/kg/day) until the neutrophil count reached 1.5 x 10(9)/1. Fifty-six patients had PBPCs mobilised with 3 gm/m2 of cyclophosphamide followed by daily subcutaneous G-CSF (5 micrograms/kg) and PBPCs were harvested on 2 consecutive days, when the white cell count rose to 4 x 10(9)/1. After stem cell infusion this group did not receive G-CSF. In 47 of the 56 patients (group 2) adequate MNC (> or = 4 x 10(8)/kg) and/or CFU-GM (> or = 10 x 10(4)/kg) were obtained. Insufficient MNC and/or CFU-GM were obtained in 10 patients. They were therefore transplanted using a combination of bone marrow and peripheral blood progenitor cells (group 3). Overall 64 patients successfully engrafted. Median days to neutrophils > or = 0.5 x 10(9)/1 were 9 (range 8-13), 12 (range 8-25) and 11 (range 9-16) and to platelets > or = 50 x 10(9)/1 were 11 (range 9-23), 13 (range 9-90) and 16 (range 13-99) in groups 1, 2 and 3 respectively. Patients in group 1 had a faster neutrophil recovery than patients in group 2 (P = 0.0002). The three patients who failed to engraft all received a combination of autologous peripheral blood and bone marrow cells.

Heparin induced thrombocytopenia thrombosis (HIT/T) syndrome: diagnosis and treatment.

Heparin induced thrombocytopenia thrombosis (HIT/T) is associated with a high morbidity and mortality. Diagnosis is essentially clinical and negative results of laboratory assays do not exclude the diagnosis. Treatment involves stopping all heparin immediately and giving an alternative thrombin inhibitor. The adoption of low molecular weight heparins is one reason for the reduced incidence of this disease in recent years.

Fatal pulmonary embolism in hospitalised medical patients.

This study aimed to determine the frequency of fatal pulmonary emboli in hospitalised medical patients by a retrospective necropsy review and prospective non-interventional patient follow up study. The main outcome measure, necropsy proven fatal pulmonary embolism, was determined from 400 consecutive necropsy records and 200 consecutive medical inpatient episodes. Fatal pulmonary embolism was recorded in 29 of 400 necropsies; 17 were medical patients. Thirty one of 200 consecutive medical patients died. Fourteen necropsies were performed and revealed pulmonary embolism as the cause of death in five patients. The incidence of necropsy proven fatal pulmonary embolism was therefore 2.5% (95% confidence intervals 0.8% to 5.7%). Therefore, one in 40 medical patients had pulmonary embolism recorded as the cause of death at necropsy. As the necropsy rate was only 45% the incidence of fatal pulmonary embolism may be greater. There is, therefore, a need to perform more large prospective studies to confirm the incidence of fatal pulmonary embolism in medical patients and to identify risk factors and effective antithrombotic prophylaxis.

Circulating high molecular weight IgG fibronectin complexes in myeloproliferative disorders.

The plasma of patients with myeloproliferative diseases was examined by polyethylene glycol (PEG) precipitation, analytical ultracentrifugation, and immunoaffinity chromatography for the presence of high molecular weight complexes of IgG and fibronectin. Abnormal circulating high molecular weight material was identified by ultracentrifugation in all patients. This was precipitated by PEG and was shown by exclusion chromatography to contain IgG in a high molecular weight form. Examination of plasma by immunoaffinity chromatography supported previous evidence for complex formation between IgG and fibronectin. These findings are further evidence that abnormal high molecular weight IgG complexes are a prominent feature of myeloproliferative disorders and implicate IgG fibronectin complex formation.

Antibiotic resistant fever associated with herpes simplex virus infection in neutropenic patients with haematological malignancy.

The incidence of mucocutaneous herpes simplex virus infection confirmed by culture and occurring during febrile neutropenic episodes was determined in 43 patients with haematological malignancy. The outcome of 72 episodes of neutropenic fever was determined and correlated with the presence or absence of herpes simplex virus (HSV) infection. Twenty four patients had mucocutaneous HSV infection during at least one episode. In 24 episodes in which HSV was isolated only 12.5% of fevers responded to antibiotics and 75% of fevers were otherwise unexplained. Conversely, in 48 episodes of neutropenic fever in which HSV was not isolated 67% of fevers responded to antibiotics and only 8.3% were unexplained. The difference in incidence of antibiotic resistant fever in the two groups was significant. There was, therefore, a strong association between mucocutaneous HSV infection and antibiotic resistant fever in immunosuppressed neutropenic patients. As most HSV infections are the result of virus reactivation, establishing the HSV serological state of patients would identify those at risk of infection and hence those in whom the prophylactic use of acyclovir would be indicated.

Composition of immune complexes and their relation to plasma fibronectin in chronic myeloproliferative disorders.

High concentrations of circulating immune complexes were detected by polyethylene glycol precipitation in 11 of 20 patients with myelofibrosis secondary to chronic myeloproliferative disease. Circulating immune complexes showed a positive correlation with plasma IgG concentrations both in patients and controls. Covariance analysis of the two groups showed significantly increased polyethylene glycol precipitable IgG in patients when adjusted for plasma IgG concentrations, indicating that the patients had significantly increased concentrations of complexed IgG. The immune complexes contained IgG, C3, and fibronectin and were inversely correlated with plasma fibronectin concentrations, suggesting that this major non-specific opsonin is important for the normal clearance of immune complexes. Therapeutic plasmapheresis efficiently removed circulating complexes and produced an increase in plasma fibronectin. This suggests that plasmapheresis may be useful for controlling immune complex mediated complications of these disorders.