Guidelines for the diagnosis and management of adult aplastic anaemia: A British Society for Haematology Guideline.

Pancytopenia with hypocellular bone marrow is the hallmark of aplastic anaemia (AA) and the diagnosis is confirmed after careful evaluation, following exclusion of alternate diagnosis including hypoplastic myelodysplastic syndromes. Emerging use of molecular cyto-genomics is helpful in delineating immune mediated AA from inherited bone marrow failures (IBMF). Camitta criteria is used to assess disease severity, which along with age and availability of human leucocyte antigen compatible donor are determinants for therapeutic decisions. Supportive care with blood and platelet transfusion support, along with anti-microbial prophylaxis and prompt management of opportunistic infections remain key throughout the disease course. The standard first-line treatment for newly diagnosed acquired severe/very severe AA patients is horse anti-thymocyte globulin and ciclosporin-based immunosuppressive therapy (IST) with eltrombopag or allogeneic haemopoietic stem cell transplant (HSCT) from a matched sibling donor. Unrelated donor HSCT in adults should be considered after lack of response to IST, and up front for young adults with severe infections and a readily available matched unrelated donor. Management of IBMF, AA in pregnancy and in elderly require special attention. In view of the rarity of AA and complexity of management, appropriate discussion in multidisciplinary meetings and involvement of expert centres is strongly recommended to improve patient outcomes.

D-dimer Release From Livers During Ex Situ Normothermic Perfusion and After In Situ Normothermic Regional Perfusion: Evidence for Occult Fibrin Burden Associated With Adverse Transplant Outcomes and Cholangiopathy.

BACKGROUND: Deceased donor livers are prone to biliary complications, which may necessitate retransplantation, and we, and others, have suggested that these complications are because of peribiliary vascular fibrin microthrombi. We sought to determine the prevalence and consequence of occult fibrin within deceased donor livers undergoing normothermic ex situ perfusion (NESLiP) and evaluate a role for fibrinolysis. METHODS: D-dimer concentrations, products of fibrin degradation, were assayed in the perfusate of 163 livers taken after 2 h of NESLiP, including 91 that were transplanted. These were related to posttransplant outcomes. Five different fibrinolytic protocols during NESLiP using alteplase were evaluated, and the transplant outcomes of these alteplase-treated livers were reviewed. RESULTS: Perfusate D-dimer concentrations were lowest in livers recovered using in situ normothermic regional perfusion and highest in alteplase-treated livers. D-dimer release from donation after brain death livers was significantly correlated with the duration of cold ischemia. In non-alteplase-treated livers, Cox proportional hazards regression analysis showed that D-dimer levels were associated with transplant survival ( P = 0.005). Treatment with alteplase and fresh frozen plasma during NESLiP was associated with significantly more D-dimer release into the perfusate and was not associated with excess bleeding postimplantation; 8 of the 9 treated livers were free of cholangiopathy, whereas the ninth had a proximal duct stricture. CONCLUSIONS: Fibrin is present in many livers during cold storage and is associated with poor posttransplant outcomes. The amount of D-dimer released after fibrinolytic treatment indicates a significant occult fibrin burden and suggests that fibrinolytic therapy during NESLiP may be a promising therapeutic intervention.

Transplant-associated thrombotic microangiopathy and immune haematological complications following intestine-containing organ transplantation: experience from over 100 consecutive cases.

Descriptions of passenger lymphocyte syndrome (PLS), immune cytopenias and transplant-associated thrombotic microangiopathy (TA-TMA) after intestine-containing transplants remain scarce. We describe our centre's experience of these complications from 2007 to 2019. Ninety-six patients received 103 transplants. PLS occurred in 9 (9%) patients (median 12 days post-transplant); all due to ABO antibodies. There were 31 minor ABO mismatch transplants. No patient required change in immunosuppression. Immune cytopenias (excluding PLS) occurred in six patients at an incidence of 1·7/100 patient years; three immune haemolysis, one immune thrombocytopenia, one acquired Glanzmann's and one immune neutropenia; 50% occurred with other cytopenias. All cases eventually responded to treatment, with a median of four treatments (range 1-8) and 5/6 were treated with rituximab. One patient with immune haemolysis required bortezomib. Complications were common in patients with immune cytopenias; 4/6 with infection needing intravenous antibiotics and 3/6 with venous thromboembolism. In 3/6 cases, a secondary cause for the immune cytopenia was evident. Switching from tacrolimus to ciclosporin was not necessary. There were five cases of transplant-associated thrombotic microangiopathy (TA-TMA; 1·5/100 patient years) requiring calcineurin inhibitor withdrawal; two cases associated with acute rejection. Two cases were managed with plasma exchange, one with plasma infusions and one with eculizumab. Further research in this patient group is required.

The use of third-party packed red blood cells during ex situ normothermic machine perfusion of organs for transplantation: Underappreciated complexities?

Ex situ normothermic machine perfusion (NMP) is being used increasingly in the assessment of higher risk deceased donor organs and to facilitate prolonged organ storage. Third-party packed red blood cells (pRBCs) are often used as an oxygen carrier in the perfusate of ex situ NMP. Despite the increasing interest in NMP, comparatively little attention has been paid to the appropriate selection of pRBCs. This includes the choice of ABO blood group and Rhesus D status, the need for special requirements for selected recipients, and the necessity for traceability of blood components. Flushing organs with cold preservation solution after NMP removes the overwhelming majority of third-party allogeneic pRBCs, but residual pRBCs within the organ may have biologically relevant effects following implantation as they enter the recipient's circulation. This review considers these issues, and suggests that national transplant and blood transfusion agencies work together to develop a co-ordinated approach within each country. This is especially important given the possibility of organ re-allocation between centers after ex situ NMP, and the ongoing development of organ perfusion hubs.

The effect of variation in donor platelet function on transfusion outcome: a semirandomized controlled trial.

The effect of variation in platelet function in platelet donors on patient outcome following platelet transfusion is unknown. This trial assessed the hypothesis that platelets collected from donors with highly responsive platelets to agonists in vitro assessed by flow cytometry (high-responder donors) are cleared more quickly from the circulation than those from low-responder donors, resulting in lower platelet count increments following transfusion. This parallel group, semirandomized double-blinded trial was conducted in a single center in the United Kingdom. Eligible patients were those 16 or older with thrombocytopenia secondary to bone marrow failure, requiring prophylactic platelet transfusion. Patients were randomly assigned to receive a platelet donation from a high- or low-responder donor when both were available, or when only 1 type of platelet was available, patients received that. Participants, investigators, and those assessing outcomes were masked to group assignment. The primary end point was the platelet count increment 10 to 90 minutes following transfusion. Analysis was by intention to treat. Fifty-one patients were assigned to receive platelets from low-responder donors, and 49 from high-responder donors (47 of which were randomized and 53 nonrandomized). There was no significant difference in platelet count increment 10 to 90 minutes following transfusion in patients receiving platelets from high-responder (mean, 21.0 × 109/L; 95% confidence interval [CI], 4.9-37.2) or low-responder (mean, 23.3 × 109/L; 95% CI, 7.8-38.9) donors (mean difference, 2.3; 95% CI, -1.1 to 5.7; P = .18). These results support the current policy of not selecting platelet donors on the basis of platelet function for prophylactic platelet transfusion.

Development and validation of a comprehensive genomic diagnostic tool for myeloid malignancies.

The diagnosis of hematologic malignancies relies on multidisciplinary workflows involving morphology, flow cytometry, cytogenetic, and molecular genetic analyses. Advances in cancer genomics have identified numerous recurrent mutations with clear prognostic and/or therapeutic significance to different cancers. In myeloid malignancies, there is a clinical imperative to test for such mutations in mainstream diagnosis; however, progress toward this has been slow and piecemeal. Here we describe Karyogene, an integrated targeted resequencing/analytical platform that detects nucleotide substitutions, insertions/deletions, chromosomal translocations, copy number abnormalities, and zygosity changes in a single assay. We validate the approach against 62 acute myeloid leukemia, 50 myelodysplastic syndrome, and 40 blood DNA samples from individuals without evidence of clonal blood disorders. We demonstrate robust detection of sequence changes in 49 genes, including difficult-to-detect mutations such as FLT3 internal-tandem and mixed-lineage leukemia (MLL) partial-tandem duplications, and clinically significant chromosomal rearrangements including MLL translocations to known and unknown partners, identifying the novel fusion gene MLL-DIAPH2 in the process. Additionally, we identify most significant chromosomal gains and losses, and several copy neutral loss-of-heterozygosity mutations at a genome-wide level, including previously unreported changes such as homozygosity for DNMT3A R882 mutations. Karyogene represents a dependable genomic diagnosis platform for translational research and for the clinical management of myeloid malignancies, which can be readily adapted for use in other cancers.

Prospective study of rabbit antithymocyte globulin and cyclosporine for aplastic anemia from the EBMT Severe Aplastic Anaemia Working Party.

Rabbit antithymocyte globulin (rATG; thymoglobulin, Genzyme) in combination with cyclosporine, as first-line immunosuppressive therapy, was evaluated prospectively in a multicenter, European, phase 2 pilot study, in 35 patients with aplastic anemia. Results were compared with 105 age- and disease severity-matched patients from the European Blood and Marrow Transplant registry, treated with horse ATG (hATG; lymphoglobulin) and cyclosporine. The primary end point was response at 6 months. At 3 months, no patients had achieved a complete response to rATG. Partial response occurred in 11 (34%). At 6 months, complete response rate was 3% and partial response rate 37%. There were 10 deaths after rATG (28.5%) and 1 after subsequent HSCT. Infections were the main cause of death in 9 of 10 patients. The best response rate was 60% for rATG and 67% for hATG. For rATG, overall survival at 2 years was 68%, compared with 86% for hATG (P = .009). Transplant-free survival was 52% for rATG and 76% for hATG (P = .002). On multivariate analysis, rATG (hazard ratio = 3.9, P = .003) and age more than 37 years (hazard ratio = 4.7, P = .0008) were independent adverse risk factors for survival. This study was registered at www.clinicaltrials.gov as NCT00471848.

Quiescent (5-fluorouracil-resistant) aplastic anemia hematopoietic cells in vitro.

OBJECTIVE: Bone marrow from aplastic anemia (AA) patients shows reduced numbers in long-term culture (LTC)-initiating cell (LTC-IC) assays. The LTC-IC assay is based on assumptions of the culture kinetics of normal hematopoietic stem cells (HSC), which are not necessarily justified in a disease state. We therefore undertook a detailed examination of the kinetics of quiescent HSC from AA patients in LTC. METHODS: Colony formation by quiescent HSC in LTC was tested by pretreating control (n=6) and AA bone marrow (n=7) with 5-fluorouracil. Secondly, we manipulated normal samples to inoculate cultures with proportions of CD34+ cells similar to those from AA samples. We obtained enough CD34+ cells to reconstitute one AA sample to "normal" levels. RESULTS: Patient cells showed altered kinetics with rapid proliferation and premature termination of LTC. In vivo, decreased numbers of HSC may induce rapid proliferation and differentiation; a similar phenomenon could explain the observations in culture. We therefore manipulated normal samples to contain a proportion of CD34+ HSC similar to that in AA samples. Although absolute numbers of secondary colonies in LTC were reduced, the kinetics of culture were not altered. However, when AA CD34+ HSC were reconstituted to "normal" levels, the cultures still demonstrated early termination. CONCLUSIONS: The kinetics of LTC are not affected by CD34+ HSC number. However, quiescent HSC derived from patients with AA have qualitative differences from normal cells, as reflected by distinct kinetics in long-term culture. This has implications for the interpretation of the LTC-IC assay with AA samples.

In vitro effects of interferon-gamma and tumor necrosis factor-alpha on CD34+ bone marrow progenitor cells from aplastic anemia patients and normal donors.

Acquired aplastic anemia is characterized by loss or dysfunction of hematopoietic stem and progenitor cells. The proinflammatory cytokines Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) may be responsible for the immune-mediated pathology observed in some patients. The CD34+ population of bone marrow mononuclear cells contains primitive cells responsible for hemopoiesis. We investigated the response of CD34+ cells from aplastic anemia patients to a combination of IFN-gamma and TNF-alpha, and compared them to cells from normal volunteer donors. This was to determine whether aplastic CD34+ cells are more sensitive than normal cells to IFN-gamma/TNF-alpha-mediated effects, and whether cytokine-induced CD95 expression can explain the high levels of apoptosis observed in CD34+ cells from aplastic patients. CD34+38- cells were most affected by overnight incubation with these cytokines, their proportion and numbers being reduced in both normal donors and patients. There was no evidence for increased apoptosis, suggesting that this effect may be due to differentiation. IFN-gamma/TNF-alpha induced upregulation of CD95 on both normal and aplastic CD34+ cells, although the basal level of CD95 expression was increased in aplastic cells. However, CD95 induction did not make cells from normal donors or aplastic anemia patients susceptible to induction of apoptosis by agonistic anti-CD95 antibodies, soluble CD95 ligand, or membrane-bound CD95L. In vivo CD95L is required for CD95 induced apoptosis. No forms of this protein were detectable in lymphocytes from aplastic patients. We conclude that increased apoptosis in aplastic CD34+ cells is not due to increased sensitivity to IFN-gamma/TNF-alpha. We further show that normal and aplastic CD34+ cells are resistant to CD95 apoptosis, even in the presence of mCD95L.

Direct binding of antithymoctye globulin to haemopoietic progenitor cells in aplastic anaemia.

Antithymocyte globulin (ATG) is widely used in the treatment of aplastic anaemia (AA) and a response occurs in 60-80% of patients. However, its exact mechanism of action in the treatment of AA has yet to be determined. Previously, we have shown that ATG increases colony growth from purified bone marrow CD34+ cells of AA patients in vitro, and decreases stem cell apoptosis and the expression of soluble Fas receptor after ATG therapy in vivo. The aim of this study was to further examine the association of ATG with AA haemopoietic progenitor cells. We describe here that ATG bound directly to CD34+ cells. Forty-six patients and 20 normal control subjects were studied. ATG bound to CD34+ cells in normal control subjects (mean 90.38%) as determined by flow cytometry. The mean percentage of CD34+ cells binding to ATG was 59.90% in untreated aplastic patients, 83.24% in partial responders, 58.3% in non-responders and 62.73% in relapsed patients. In completely recovered patients, ATG binding was indistinguishable from control subjects. The functionality of AA patients' haemopoietic progenitor cells was assessed using colony assays. These results demonstrate the direct binding of ATG to CD34+ cells and suggest that differences in its binding to AA CD34+ cells could reflect functional differences in the haemopoietic stem cell compartment throughout the disease process.