Britta Höchsmann

Bacterial contamination of platelet concentrates: results of a prospective multicenter study comparing pooled whole blood-derived platelets and apheresis platelets.

BACKGROUND: The GERMS Group initiated a prospective multicenter study to assess prevalence and nature of bacterial contamination of pooled buffy‐coat platelet concentrates (PPCs) and apheresis platelet concentrates (APCs) by routine screening with a bacterial culture system.

A randomized controlled study in patients with newly diagnosed severe aplastic anemia receiving antithymocyte globulin (ATG), cyclosporine, with or without G-CSF: a study of the SAA Working Party of the European Group for Blood and Marrow Transplantation.

We evaluated the role of granulocyte colony-stimulating factor (G-CSF) in patients with severe aplastic anemia (SAA) treated with antithymocyte globulin (ATG) and cyclosporine (CSA). Between January 2002 and July 2008, 192 patients with newly diagnosed SAA not eligible for transplantation were entered into this multicenter, randomized study to receive ATG/CSA with or without G-CSF. Overall survival (OS) at 6 years was 76% ± 4%, and event-free survival (EFS) was 42% ± 4%. No difference in OS/EFS was seen between patients randomly assigned to receive or not to receive G-CSF, neither for the entire cohort nor in subgroups stratified by age and disease severity. Patients treated with G-CSF had fewer infectious episodes (24%) and hospitalization days (82%) compared with patients without G-CSF (36%; P = .006; 87%; P = .0003). In a post hoc analysis of patients receiving G-CSF, the lack of a neutrophil response by day 30 was associated with significantly lower response rate (56% vs 81%; P = .048) and survival (65% vs 87%; P = .031). G-CSF added to standard ATG and CSA reduces the rate of early infectious episodes and days of hospitalization in very SAA patients and might allow early identification of nonresponders but has no effect on OS, EFS, remission, relapse rates, and mortality. This study was registered at www.clinicaltrials.gov as NCT01163942.

Eculizumab in Pregnant Patients with Paroxysmal Nocturnal Hemoglobinuria

BACKGROUND Eculizumab, a humanized monoclonal antibody against complement protein C5 that inhibits terminal complement activation, has been shown to prevent complications of paroxysmal nocturnal hemoglobinuria (PNH) and improve quality of life and overall survival, but data on the use of eculizumab in women during pregnancy are scarce. METHODS We designed a questionnaire to solicit data on pregnancies in women with PNH and sent it to the members of the International PNH Interest Group and to the physicians participating in the International PNH Registry. We assessed the safety and efficacy of eculizumab in pregnant patients with PNH by examining the birth and developmental records of the children born and adverse events in the mothers. RESULTS Of the 94 questionnaires that were sent out, 75 were returned, representing a response rate of 80%. Data on 75 pregnancies in 61 women with PNH were evaluated. There were no maternal deaths and three fetal deaths (4%). Six miscarriages (8%) occurred during the first trimester. Requirements for transfusion of red cells increased during pregnancy, from a mean of 0.14 units per month in the 6 months before pregnancy to 0.92 units per month during pregnancy. Platelet transfusions were given in 16 pregnancies. In 54% of pregnancies that progressed past the first trimester, the dose or the frequency of use of eculizumab had to be increased. Low-molecular-weight heparin was used in 88% of the pregnancies. Ten hemorrhagic events and 2 thrombotic events were documented; both thrombotic events occurred during the postpartum period. A total of 22 births (29%) were premature. Twenty cord-blood samples were examined for the presence of eculizumab; the drug was detected in 7 of the samples. A total of 25 babies were breast-fed, and in 10 of these cases, breast milk was examined for the presence of eculizumab; the drug was not detected in any of the 10 breast-milk samples. CONCLUSIONS Eculizumab provided benefit for women with PNH during pregnancy, as evidenced by a high rate of fetal survival and a low rate of maternal complications. (ClinicalTrials.gov number, NCT01374360.).

Screening of platelet concentrates for bacterial contamination: spectrum of bacteria detected, proportionof transfused units, and clinical follow-up

Screening of platelet concentrates (PCs) for bacterial contamination with cultivation methods is carried out as a routine procedure in some countries. The aim is to prevent the transfusion of contaminated PCs. The German Evaluation of Regular Monitoring Study Group conducted a prospective multicenter study on 52,243 PCs to investigate the prevalence of bacteria (BacT/ALERT, bioMerieux). This study describes the detected bacterial spectrum, the proportion of PCs with a positive test result that had been transfused, and the results of the clinical follow-up. One hundred thirteen (67%) of 169 potentially or confirmed positive units had already been transfused at the time of the first positive signal. The transfusion of units contaminated by Staphylococcus aureus, Serratia marcescens, and 73% of the units contaminated with Staphylococcus epidermidis, Staphylococcus capitis, or Staphylococcus saccharolyticus was prevented. In contrast, 85% of units with Propionibacterium acnes were transfused. A clonal relationship of the isolates from the pooled PCs and from the associated red blood cell concentrates was found in all investigated cases. The follow-up revealed six febrile reactions to culture-positive PCs not classified as transfusion reaction (TRs) by treating physicians. This demonstrates the importance of hemovigilance. Serious septic reactions due to Klebsiella pneumoniae in two units of one apheresis PC that had tested false-negative were reported; one had a fatal outcome. Culture systems reduce the risk of transfusion of contaminated PCs but cannot guarantee sterility. Physicians must be aware of bacterial contamination of PCs as a potential cause of TRs and must report all adverse events.

Similar outcome of upfront-unrelated and matched sibling stem cell transplantation in idiopathic paediatric aplastic anaemia. A study on behalf of the UK Paediatric BMT Working Party, Paediatric Diseases Working Party and Severe Aplastic Anaemia Working Party of EBMT

We explored the feasibility of unrelated donor haematopoietic stem cell transplant (HSCT) upfront without prior immunosuppressive therapy (IST) in paediatric idiopathic severe aplastic anaemia (SAA). This cohort was then compared to matched historical controls who had undergone first‐line therapy with a matched sibling/family donor (MSD) HSCT (n = 87) or IST with horse antithymocyte globulin and ciclosporin (n = 58) or second‐line therapy with unrelated donor HSCT post‐failed IST (n = 24). The 2‐year overall survival in the upfront cohort was 96 ± 4% compared to 91 ± 3% in the MSD controls (P = 0·30) and 94 ± 3% in the IST controls (P = 0·68) and 74 ± 9% in the unrelated donor HSCT post‐IST failure controls (P = 0·02).The 2‐year event‐free survival in the upfront cohort was 92 ± 5% compared to 87 ± 4% in MSD controls (P = 0·37), 40 ± 7% in IST controls (P = 0·0001) and 74 ± 9% in the unrelated donor HSCT post‐IST failure controls (n = 24) (P = 0·02). Outcomes for upfront‐unrelated donor HSCT in paediatric idiopathic SAA were similar to MSD HSCT and superior to IST and unrelated donor HSCT post‐IST failure. Front‐line therapy with matched unrelated donor HSCT is a novel treatment approach and could be considered as first‐line therapy in selected paediatric patients who lack a MSD.

A case of paroxysmal nocturnal hemoglobinuria caused by a germline mutation and a somatic mutation in PIGT

To ascertain the genetic basis of a paroxysmal nocturnal hemoglobinuria (PNH) case without somatic mutations in PIGA, we performed deep next-generation sequencing on all exons of known genes of the glycosylphosphatidylinositol (GPI) anchor synthesis pathway. We identified a heterozygous germline splice site mutation in PIGT and a somatic 8-MB deletion in granulocytes affecting the other copy of PIGT. PIGA is essential for GPI anchor synthesis, whereas PIGT is essential for attachment of the preassembled GPI anchor to proteins. Although a single mutation event in the X-chromosomal gene PIGA is known to cause GPI-anchored protein deficiency, 2 such hits are required in the autosomal gene PIGT. Our data indicate that PNH can occur even in the presence of fully assembled GPI if its transfer to proteins is defective in hematopoietic stem cells.

Targeted Therapy with Eculizumab for Inherited CD59 Deficiency

Patients with CD59 deficiency have progressive neurologic dysfunction and episodes of hemolytic anemia. Eculizumab, a complement inhibitor, was administered to reverse hemolytic anemia in a patient. After the anemia improved, neurologic improvement was also noted.

Incomplete inhibition by eculizumab: mechanistic evidence for residual C5 activity during strong complement activation

Eculizumab inhibits the terminal, lytic pathway of complement by blocking the activation of the complement protein C5 and shows remarkable clinical benefits in certain complement-mediated diseases. However, several reports suggest that activation of C5 is not always completely suppressed in patients even under excess of eculizumab over C5, indicating that residual C5 activity may derogate the drugs therapeutic benefit under certain conditions. By using eculizumab and the tick-derived C5 inhibitor coversin, we determined conditions ex vivo in which C5 inhibition is incomplete. The degree of such residual lytic activity depended on the strength of the complement activator and the resulting surface density of the complement activation product C3b, which autoamplifies via the alternative pathway (AP) amplification loop. We show that at high C3b densities required for binding and activation of C5, both inhibitors reduce but do not abolish this interaction. The decrease of C5 binding to C3b clusters in the presence of C5 inhibitors correlated with the levels of residual hemolysis. However, by employing different C5 inhibitors simultaneously, residual hemolytic activity could be abolished. The importance of AP-produced C3b clusters for C5 activation in the presence of eculizumab was corroborated by the finding that residual hemolysis after forceful activation of the classical pathway could be reduced by blocking the AP. By providing insights into C5 activation and inhibition, our study delivers the rationale for the clinically observed phenomenon of residual terminal pathway activity under eculizumab treatment with important implications for anti-C5 therapy in general.

Drugs that inhibit complement

The complement system is an important part of the innate immune system. Complement plays a crucial role in the pathophysiology of many disorders. Despite the pivotal role of the complement system, an approved targeted inhibitor of a complement factor became available only recently. Eculizumab is a humanized monoclonal antibody that inhibits complement factor C5. It is a targeted, disease modifying, treatment of paroxysmal nocturnal hemoglobinuria (PNH). It was approved be the US FDA and the European Commission in 2007. In this review we will update the experience with eculizumab in PNH and discuss potential use of eculizumab in other disorders (e.g. cold agglutinin disease; atypical HUS) and new approaches to complement inhibition with drugs other than eculizumab.

Should irradiated blood products be given routinely to all patients with aplastic anaemia undergoing immunosuppressive therapy with antithymocyte globulin (ATG)? A survey from the European Group for Blood and Marrow Transplantation Severe Aplastic Anaemia Working Party

In view of the lack of guidance on the use of irradiated blood products (IBP) in patients with aplastic anaemia (AA) receiving immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and ciclosporin, we conducted the first survey from European and USA centres to determine current practice on this issue. The routine use of pre-storage leucocyte-depleted blood products has reduced the risk of allosensitization in haematological malignancies, although data is lacking in AA [The Trial to reduce Alloimmunization to Platelets study group, 1997; Marsh et al, 2009]. Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare but invariably fatal complication following transfusion of viable lymphocyte-containing blood products. It is abolished by the use of IBP in at-risk patients. For haematological patients, the routine use of IBP is recommended for all autologous and allogeneic haemopoietic stem cell tranplantation (HSCT) recipients, patients with congenital immune deficiency, Hodgkin lymphoma, and patients receiving purine analogues. In Germany, Non-Hodgkin lymphoma is also an indication for IBP [British Committee for Standards in Haematology (BCSH) Blood Transfusion Task Force, 1996; Williamson et al, 2007; Agbaht et al, 2007; The Board of the German Medical Association on the recommendation of the Scientific Advisory Board 2009]. Treatments with either ATG or Alemtuzumab (anti-CD52 monoclonal antibody) are also under consideration as an indication by the BCSH (J. Treleaven, Royal Marsden Hosptial, Sutton, UK, personal communication, 2009). The rationales for considering giving only IBP to patients with AA are (1) to prevent TA-GVHD during ATG treatment and (2) to help reduce allo-sensitization, which occurs frequently in AA patients (Killick et al, 1997; Laundy et al, 2004). Animal data show that irradiation of all red cell and platelet transfusions before HSCT reduces the risk of sensitization to minor histocompatibility antigens and the risk of graft rejection after dog leucocyte antigen-identical marrow grafts [Bean et al, 1994]. It is likely, but unproven, that the routine clinical use of leucocyte-depleted blood products has helped to reduce this risk. Leucodepletion should also reduce the chances of a recipient developing TA-GVHD. There were 13 cases of TA-GVHD reported to Serious Hazards of Blood Transfusion (SHOT) in the UK between 1996 and 2005, and of these, 11 occurred after transfusion of non-leucocyte depleted products and 2 after transfusion of leucocyte-depleted products; there have been no reported cases since 2001 [Williamson et al, 2007]. We were not aware of any published cases of TA-GVHD following ATG treatment for haematological disorders. We therefore performed an email survey of 12 European Group for Blood and Marrow Transplantation (EBMT) centres and two USA centres in 2008. Questionnaires were sent to lead representatives who were also representatives on the EBMT Severe Aplastic Anaemia Working Party (EBMT SAAWP) from the following countries: UK, Switzerland, Germany, Italy, Sweden, Netherlands, Italy and France. Two large centres in the USA were also included, The National Institutes of Health and Medical College of Wisconsin, on account of their specialist interest in AA. Both adult and paediatric centres were included in the survey. The questionnaire comprised the following questions: (1) Do you routinely give only IBP after ATG? (2) If so, how long for? (3) Which blood products do you irradiate? (4) What is/are your reason(s) for giving IBP? (5) Have you ever seen a case of TA-GVHD after ATG? (6) Do you have the same policy for children and (7) Any other comments to add? The results are summarized in Figure 1. Twelve of 14 centres (85%) routinely only give IBP after ATG. Of these, 6 of 12 centres indicated that they give IBP to all AA patients regardless of treatment. The reason for not giving IBP at 2 centres was lack of published evidence. There was no consensus on how long to continue IBP after IST. Two of 12 centres gave IBP indefinitely, 5 until patients became transfusion independent, two for 6 months, two for 12 months and one until the lymphocyte count was > 1.5 × 109/1. All of the 12 centres gave IBP to avoid TA-GVHD after ATG. Two centres had a universal IBP policy to avoid TA-GVHD for all haematology/oncology patients, to avoid possible errors of not giving IBP to patients known to be at risk. Only three centres indicated that reduction in allosensitization was another indication for giving IBP. Seven of 10 centres irradiated both red cell and platelet transfusions, and three irradiated red cells, platelets and fresh frozen plasma. Eight centres managed both children and adults with AA, and all reported the same policy for children and adults. A patient with severe AA was reported to have developed probable TA-GVHD 20 years ago after treatment with ATG. A further case was reported in a liver transplant recipient more than 10 years ago. No further details on these two cases of TA-GVHD were available. Figure 1 Results of survey on the use of irradiated blood products in aplastic anaemia patients treated with immunosuppressive therapy. The risk of TA-GVHD post-ATG is not known but appears to be low. This may reflect irradiation practices, and the use of leucodepleted blood products, or it may be truly low. However, horse ATG (Lymphoglobuline) was routinely used as treatment for AA, but was withdrawn from the market in 2007. Rabbit ATG is more immunosuppressive than horse ATG. It causes a more prolonged lymphopenia, higher peak Epstein-Barr virus (EBV) viral levels post-ATG for AA than horse ATG [Scheinberg et al, 2007], more EBV post-transplant lymphoproliferative disease after HSCT [Scheinberg et al, 2007]. Therefore, there is concern that rabbit ATG might increase the risk of TA-GVHD compared with horse ATG. Therefore, based on background information we have on the beneficial effect of IBP, and on the results of this survey, we propose that patients with AA should receive IBP during and after ATG treatment. This policy should probably be continued for at least as long as patients are receiving IST, such as ciclosporin.