Inter-laboratory comparison of different rapid methods for the detection of bacterial contamination in platelet concentrates

Background Bacterial contamination of platelet concentrates still represents a major risk in transfusion medicine, and a variety of screening methods have been available to improve the safety of PCs. In the present study, the analytical quality of three different rapid screening methods (BactiFlow flow cytometry, Pan Genera Detection Assay, 23S rRNA RT‐PCR) was evaluated in an inter‐laboratory comparison in three different German blood services. Methods Samples were inoculated with different bacteria [Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli (two strains), Klebsiella pneumoniae (two strains), Enterobacter aerogenes (one strain), Serratia marcescens (one strain)] at different counts (4·5 × 10³–4·5 × 10⁸ CFU/ml) alternating with negative samples in one transfusion facility. Samples were blinded with a random order for each screening method, shipped to partners and analysed immediately after receipt with different rapid screening methods. Results The inter‐laboratory comparison revealed that the BactiFlow assay and 23S rRNA RT‐PCR‐screening detected all samples correctly (positive: 12/12, negative: 8/8). The Pan Genera Detection Assay test detected only four of the positive samples. Four of the non‐detected positive samples were below the assay’s detection limit. Another four inoculated samples with comparatively high bacteria counts were detected false negative (E. coli (two strains): 9·87 × 10⁵ and 2·10 × 10⁷ CFU/ml, respectively, K. pneumoniae: 4·79 × 10⁶ CFU/ml, S. aureus: 6·03 × 10⁵ CFU/ml). All rapid screening methods revealed no false‐positive results. Conclusions Both BactiFlow and 23S rRNA RT‐PCR demonstrated a high sensitivity to detecting bacterial contamination in PCs. The Pan Genera Detection Assay had some shortcomings regarding sensitivity, especially for the detection of Gram‐negative strains.     

Prospective observational cohort study of the association between thromboelastometry,coagulation and platelet parameters and bleeding in patients with haematological malignancies‐ The ATHENA study

Previous studies have shown that total platelet count (TPC) inadequately predicts bleeding in thrombocytopenic patients with haematological malignancies. This prospective cohort study evaluated whether rotational thromboelastometry (ROTEM), coagulation or other platelet parameters were more strongly associated with bleeding than TPC. Adults treated at two UK haematology centres for haematological malignancy were enrolled if they had thrombocytopenia (TPC ≤ 50 × 10⁹/l) at beginning of, or during treatment (International Standard Randomized Controlled Trial Number 81226121). TPC and bleeding symptoms were recorded daily for up to 30 d or until platelet count recovery, hospital discharge or death. Blood samples were tested thrice weekly using ROTEM, Platelet Function Analyser (PFA)‐100^®, coagulation and platelet cytometry assays. Bleeding symptoms and TPC from 49/50 enrolled participants who completed the study were recorded on 754/760 study days. Mean platelet volume and PFA‐100^® closure times were frequently inestimatable because of thrombocytopenia. TPC, absolute immature platelet number (AIPN) and ROTEM maximum clot firmness were significantly associated with bleeding on the day after blood sampling. Only AIPN was associated with bleeding after adjustment of test results for TPC (Odds Ratio 0·52, 95% confidence interval 0·28–0·97; P = 0·038). In a predictive model, AIPN was superior to TPC for predicting bleeding. This study indicates that AIPN may be more clinically useful than TPC at predicting bleeding.     

Romiplostim monotherapy in thrombocytopenic patients with myelodysplastic syndromes: long‐term safety and efficacy

Romiplostim can improve platelet counts in about 50% of patients with low‐ or intermediate 1‐risk (lower risk) myelodysplastic syndromes (MDS) and thrombocytopenia, but its long‐term toxicity and efficacy are not known. This open‐label extension study evaluated the long‐term safety and efficacy of romiplostim in 60 patients with lower risk MDS and platelet counts ≤50 × 10⁹/l. The primary endpoint was adverse event (AE) incidence. Secondary endpoints were efficacy parameters, including bleeding events and platelet response. Median (range) treatment time in the extension study and the median observation times thereafter were 25 (2–181) and 57 (11–209) weeks, respectively. Treatment‐related AEs and serious AEs were reported in 14/60 (23%) and 4/60 (7%) patients, respectively. Progression to acute myeloid leukaemia (AML) occurred in two patients after 44 and 46 weeks. Patients (n = 34, 57%) with a platelet response were further evaluated for length of response. Median (range) response duration was 33 (7–174) weeks; 28/34 (82%) patients had a continuous response. Five of 34 patients (15%) had grade ≥3 bleeding events; three when the platelet count was >50 × 10⁹/l. There were no new safety concerns and the rate of progression to AML was low; response to romiplostim was maintained for most patients.     

Establishment of the first international repository for transfusion-relevant bacteria reference strains: ISBT working party transfusion-transmitted infectious diseases (WP-TTID), subgroup on bacteria

Background Bacterial contamination of platelet concentrates (PCs) still remains a significant problem in transfusion with potential important clinical consequences, including death. The International Society of Blood Transfusion Working Party on Transfusion‐Transmitted Infectious Diseases, Subgroup on Bacteria, organised an international study on Transfusion‐Relevant Bacteria References to be used as a tool for development, validation and comparison of both bacterial screening and pathogen reduction methods. Material and Methods Four Bacteria References (Staphylococcus epidermidis PEI‐B‐06, Streptococcus pyogenes PEI‐B‐20, Klebsiella pneumoniae PEI‐B‐08 and Escherichia coli PEI‐B‐19) were selected regarding their ability to proliferate to high counts in PCs and distributed anonymised to 14 laboratories in 10 countries for identification, enumeration and bacterial proliferation in PCs after low spiking (0·3 and 0·03 CFU/ml), to simulate contamination occurring during blood donation. Results Bacteria References were correctly identified in 98% of all 52 identifications. S. pyogenes and E. coli grew in PCs in 11 out of 12 laboratories, and K. pneumoniae and S. epidermidis replicated in all participating laboratories. The results of bacterial counts were very consistent between laboratories: the 95% confidence intervals were for S. epidermidis: 1·19–1·32 × 10⁷ CFU/ml, S. pyogenes: 0·58–0·69 × 10⁷ CFU/ml, K. pneumoniae: 18·71–20·26 × 10⁷ CFU/ml and E. coli: 1·78–2·10 × 10⁷ CFU/ml. Conclusion The study was undertaken as a proof of principle with the aim to demonstrate (i) the quality, stability and suitability of the bacterial strains for low‐titre spiking of blood components, (ii) the property of donor‐independent proliferation in PCs, and (iii) their suitability for worldwide shipping of deep frozen, blinded pathogenic bacteria. These aims were successfully fulfilled. The WHO Expert Committee Biological Standardisation has approved the adoption of these four bacteria strains as the first Repository for Transfusion‐Relevant Bacteria Reference Strains and, additionally, endorsed as a project the addition of six further bacteria strain preparations suitable for control of platelet contamination as the next step of enlargement of the repository.     

The PFA‐100® does not predict delta‐granule platelet storage pool deficiencies

Summary.  There are no published reports investigating the ability of the platelet function analyzer (PFA‐100^®) to detect the presence of delta‐granule platelet storage pool deficiencies (δ‐PSPD), a common mild bleeding disorder. Prior studies of the PFA‐100^® and congenital platelet disorders have been limited by small numbers of patients with a variety of disorders. We examined PFA‐100^® results in a large paediatric patient population diagnosed specifically with δ‐PSPD, and determined the relationship between PFA‐100^® and platelet electron microscopy (the gold standard for diagnosis). This study is a retrospective review of patients <19 years of age diagnosed with δ‐PSPD at Nationwide Children’s Hospital from 2008 to 2010. To examine the correlation between PFA‐100^® and average number of granules per platelet we used Spearman’s Rho as a non‐parametric measure of dependence. A total of 105 patients diagnosed with δ‐PSPD were included, of which 99 patients underwent PFA‐100^® testing. Of those tested 46% had at least one abnormal closure time, whereas 16% had abnormal results for both cartridges. We found no statistical correlation between C‐EPI closure time and average number of granules per platelet (ρ= ?0.0095, P‐value = 0.9328), nor between C‐ADP closure time and the average number of granules (ρ = 0.0315, P‐value = 0.7798). The PFA‐100^®, a widely used screening test for suspected bleeding disorders, did not correlate with presence or severity of δ‐PSPD as determined by platelet electron microscopy. When evaluating patients with suspected bleeding disorders, PFA‐100^® alone cannot be used to rule out the presence of a δ‐PSPD.     

A multi‐centre,single‐arm,open‐label study evaluating the safety and efficacy of fixed dose rituximab in patients with refractory,relapsed or chronic idiopathic thrombocytopenic purpura (R‐ITP1000 study)

The efficacy of a fixed‐dose rituximab schedule was prospectively explored in primary/acute refractory, relapsed or chronic (platelet count >10 × 10⁹/l and ≤50 × 10⁹/l) idiopathic thrombocytopenic purpura (ITP). Patients received two doses of rituximab (1000 mg) on days 1 and 15 and were followed‐up on weeks 1–8, 12, 26, 39 and 52. A total of 122 patients were included in the safety population; efficacy was analysed in 108 patients. Overall response rate (ORR) at week 8, defined as the proportion of patients achieving complete response (CR; platelet count >150 × 10⁹/l) or partial response (PR; platelet count >50 × 10⁹/l) was 44%. Therapeutic response, defined as achieving a response at week 8, with at least a minor response (MR; platelet count >30 × 10⁹/l), sustained up to weeks 26 and 52 and accompanied by a reduction in ITP medications, was achieved in 44% (week 26) and 35% (week 52) of patients, respectively. Treatment was well tolerated with no safety concerns. While this study failed to meet its primary endpoint of an ORR of 50%, the efficacy of two fixed doses of rituximab appear to provide similar efficacy to the standard 375 mg/m² four‐dose schedule in relapsed/chronic ITP.     

Long‐term tolerability,immunogenicity and efficacy of Nuwiq® (human‐cl rhFVIII) in children with severe haemophilia A

Introduction

Nuwiq^® (human‐cl rhFVIII, simoctocog alfa) is a 4th generation recombinant human FVIII, without chemical modification or fusion with any other protein, produced in a human cell line.

Aim/Methods

This study (GENA‐13) was an extension of the GENA‐03 study in which previously treated children aged 2‐12 years with severe haemophilia A received Nuwiq^® prophylaxis for ≥6 months. GENA‐13 examined long‐term tolerability, immunogenicity and efficacy of Nuwiq^® prophylaxis in children.

Results

Of 59 patients enrolled in GENA‐03, 49 continued Nuwiq^® prophylaxis in GENA‐13 for a median (range) of 30.0 (9.5‐52.0) months. No patient withdrew due to drug‐related adverse events or developed inhibitors. Only 2 of 20 518 infusions were associated with possibly related adverse events (dyspnoea, fever). The estimated annualized bleeding rate (ABR) was 0.67 (95% CI: 0.44, 1.02) for spontaneous and 2.88 (95% CI: 1.86, 4.46) for all bleeds. Younger children (2‐5 years) had lower ABRs than children aged 6‐12 years. Annualized bleeding rates were reduced in GENA‐13 vs GENA‐03, especially for spontaneous bleeds in younger children (71% reduction; ABR ratio 0.29 [95% CI: 0.11, 0.74]). Nuwiq^® efficacy was rated as excellent/good in the treatment of 83.0% of 305 evaluated breakthrough bleeds. Surgical prophylaxis with Nuwiq^® was rated as excellent for all 17 assessed procedures.

Conclusion

Long‐term treatment with Nuwiq^® for the prevention of bleeds in children with severe haemophilia A was well tolerated, effective and reduced spontaneous bleeding by up to 70% compared with GENA‐03.     

Nitric oxide‐releasing microparticles as a potent antimicrobial therapeutic against chronic rhinosinusitis bacterial isolates

Background

Bacteria, particularly in the biofilm state, may be implicated in the pathogenesis of chronic rhinosinusitis (CRS) and enhance antibiotic resistance. Nitric oxide (NO) is a gaseous immunomodulator with antimicrobial activity and a short half‐life, complicating achievement of therapeutic concentrations. We hypothesized that a novel microparticle‐based delivery platform, which allows for adjustable release of NO, could exhibit potent antibacterial effects.

Methods

Porous organosilica microparticles (SNO‐MP) containing nitrosylated thiol groups were formulated. Dissociation of the nitrosothiol groups generates NO at body temperature. The susceptibility of bacterial isolates from CRS patients to SNO‐MP was evaluated through a colony forming unit (CFU) assay. Serial dilutions of SNO‐MP in triplicate were incubated with isolates in suspension for 6 hours followed by plating on tryptic soy agar and overnight incubation followed by CFU quantification. Statistical analysis was performed with SPSS using one‐way analysis of variance with Bonferroni correction.

Results

SNO‐MP displayed antibacterial activity against gram‐positive (methicillin‐resistant and ‐sensitive Staphylococcus aureus) and gram‐negative (Pseudomonas aeruginosa, Enterobacter aerogenes, and Proteus mirabilis) isolates. SNO‐MP induced dose‐dependent reductions in CFU across all strains. Compared with controls and blank nanoparticles, SNO‐MP (10 mg/mL) induced a 99.99%‐100% reduction in CFU across all isolates, equivalent to a 5–9 log kill (p < 0.005). There was no statistically significant difference in CFU concentration between controls and blank microparticles.

Conclusion

SNO‐MP demonstrates potent bactericidal effect against antibiotic‐resistant CRS bacterial strains.

     

A fibrinogen concentrate Haemocomplettan® (Riastap®) or a Factor XIII concentrate Fibrogammin® combined with a mini dose of tranexamic acid can reverse the fibrin instability to fibrinolysis induced by thrombin‐ or FXa‐inhibitor

To assess whether Haemocomplettan^® (fibrinogen concentrate) or Fibrogammin^® (Factor XIII concentrate) can be used to manage bleeding complications of antithrombotic treatment, we examined a normal plasma pool spiked with AR‐H067637 (thrombin inhibitor) or rivaroxaban (activated factor X‐inhibitor), to which one of the concentrates was added. Fibrin network permeability (Ks), images of Scanning Electron Microscopy (SEM) and Clot Lysis Time (CLT) were examined. Both inhibitors increased the Ks levels, which could be fully or partly reversed by Haemocomplettan^® or Fibrogammin^® respectively. However, these modified clots with tightened network remained non‐resistant to fibrinolysis, shown as unaffected CLT. Tranexamic acid at a very low concentration (0·4 mg/ml) aided the two concentrates to stabilize the clots, where the prolongation of CLT was more pronounced for a lower dose than a higher dose of Haemocomplettan^® while Fibrogammin^® brought the greatest delay to CLT out of all additions. These observations were partly supported by SEM images, displaying alterations of fibrin fibre arrangement known to influence fibirinolysis. The in vitro data suggest that Haemocomplettan^® or Fibrogammin^® given in combination with a mini dose of tranexamic acid may slow down the natural clearance of fibrin clot by plasmin and thus prevent patients from haemorrhagic complications during antithrombotic therapy.     

Field evaluation of GeneXpert® (Cepheid) HCV performance for RNA quantification in a genotype 1 and 6 predominant patient population in Cambodia

GeneXpert^® (Cepheid) is the only WHO prequalified platform for hepatitis C virus (HCV) nucleic acid amplification testing that is suitable for point‐of‐care use in resource‐limited contexts. However, its application is constrained by the lack of evidence on genotype 6 (GT6) HCV. We evaluated its field performance among a patient population in Cambodia predominantly infected with GT6. Between August and September 2017, we tested plasma samples obtained from consenting patients at Médecins Sans Frontières’ HCV clinic at Preah Kossamak Hospital for HCV viral load (VL) using GeneXpert^® and compared its results to those obtained using COBAS^® AmpliPrep/Cobas^® TaqMan^® HCV Quantitative Test, v2.0 (Roche) at the Institut Pasteur du Cambodge. Among 769 patients, 77% of the seropositive patients (n = 454/590) had detectable and quantifiable VL using Roche and 43% (n = 195/454) were GT6. The sensitivity and specificity of GeneXpert^® against Roche were 100% (95% CI 99.2, 100.0) and 98.5% (95% CI 94.8, 99.8). The mean VL difference was ?0.01 (95% CI ?0.05, 0.02) log₁₀ IU/mL for 454 samples quantifiable on Roche and ?0.07 (95% CI ?0.12, ?0.02) log₁₀ IU/mL for GT6 (n = 195). The limit of agreement (LOA) was ?0.76 to 0.73 log₁₀ IU/mL for all GTs and ?0.76 to 0.62 log₁₀ IU/mL for GT6. Twenty‐nine GeneXpert^® results were outside the LOA. Frequency of error and the median turnaround time (TAT) for GeneXpert^® were 1% and 0 days (4 days using Roche). We demonstrated that the GeneXpert^® HCV assay has good sensitivity, specificity, quantitative agreement, and TAT in a real‐world, resource‐limited clinical setting among GT6 HCV patients.     

Evaluation of the von Willebrand factor antigen (vWF‐Ag) assay using an immuno‐turbidimetric method (STA Liatest® vWF) automated on the MDA®180 coagulometer

The standard enzyme‐linked immunosorbent assay (ELISA) test for von Willebrand factor antigen (vWF‐Ag), though sensitive and specific, does not deliver the flexibility to handle single sample assessments economically or provide rapid, emergency testing capability. The present study examined the performance characteristics of an immuno‐turbidimetric assay kit (STA Liatest^®) modified for automation on the MDA^®180 coagulation analyser using a supplied protocol. One hundred and sixteen patient samples were assessed by both the standard and the modified method. The correlation coefficient was 0.98 across the range of values 1–487 IU/dl. Above 200 IU/dl, where specimen dilution was indicated, there was greater diversity (r = 0.86) between the techniques. Plotting the difference between methods against the mean of both showed excellent agreement between methods below 100 IU/dl vWF. The intra‐assay and interassay coefficients of variation were less than 3% at both low and normal range levels. The MDA^®180 automated vWF assay merits consideration as an alternative to ELISA testing that provides random access and result availability within 30 min.     

Presenting D‐dimer and early symptom severity are independent predictors for post‐thrombotic syndrome following a first deep vein thrombosis

Post‐thrombotic syndrome (PTS) is the most common complication of deep vein thrombosis (DVT). Current preventative strategies are limited to the daily wear of graduated compression stockings (GCS). The aim of this study was to evaluate early predictors of PTS. One hundred and twenty‐two consecutive patients with a first DVT were prospectively recruited from diagnosis and followed for up to 6 months post‐end of anticoagulation. D‐dimer was measured in 107 participants at presentation and Villalta scale was evaluated in 70 participants at a median of 2 weeks following diagnosis. PTS developed in 51·6% of participants. GCS were obtained by 78·1% of participants, with 33·7% reporting daily wear at the end of follow‐up. Mean early Villalta scale was significantly higher in those with PTS (8·1 ± 3·7) compared to those without (2·6 ± 2·7, P < 0·001). Median D‐dimer was significantly higher in those with PTS [3260 ng/ml, interquartile range (IQR) 820–8000 ng/ml] compared to those without (1540 ng/ml, IQR 810–2520 ng/ml, P < 0·001). The adjusted odds ratio for every one point increase in early Villalta scale was 1·78 [95% confidence interval (CI), 1·19–2·64; P = 0·005] and for D‐dimer >1910 ng/ml it was 2·71 (95% CI, 1·05–7·03; P = 0·04). These markers could enable targeted counselling regarding GCS for those at high risk of PTS.     

Chemomobilization with high‐dose etoposide and G‐CSF results in effective and safe stem cell collection in heavily pretreated lymphoma patients: report from a single institution study and review

The optimal mobilization strategy prior to autologous stem cell transplantation (auto‐SCT) for patients with lymphoma is yet to be determined. We reviewed our institutional experience using chemomobilization with high‐dose (HD) etoposide (1.6 g/m²) and G‐CSF (300 μg/day) in 79 patients with lymphoma. The majority (76%) had received at least two prior regimens of chemotherapy, and 12 (15.2%) patients had previously failed to mobilize following HD cyclophosphamide or DHAP or ICE with G‐CSF. HD etoposide and G‐CSF chemomobilization resulted in successful collection (>2 × 10⁶ CD34+ cells/kg) in 82.3% of patients within a median 2 (1–6) apheresis days. Patients had stem cells collected between days +8 and +15, with a median +12 day. Median total CD34+ cells/kg collected was 5.95 × 10⁶ (0.1–36.8). Seventy‐one percent of patients yielded >2 × 10⁶ CD34+ cells/kg in ≤2 d of apheresis and were defined as good mobilizers. While median CD34+ cells/kg collected for good mobilizers was 7.6 × 10⁶, it was 2.6 × 10⁶ for poor mobilizers (P < 0.001). This regimen was safe with a low rate of febrile neutropenia (7.6%) and acceptable rates of RBC (40.5%) and platelet transfusions (22.8%). Hematopoietic recovery after auto‐SCT was achieved on expected time. Therapy‐related myelodysplastic syndrome/acute myeloid leukemia occurred in only one patient (1.3%) with in a median follow‐up of 16 months after chemomobilization. We conclude that HD etoposide and G‐CSF chemomobilization appear to result in effective, tolerable, and safe stem cell collection in the majority of heavily pretreated lymphoma patients.     

Development and validation of a model to predict platelet response to romiplostim in patients with lower‐risk myelodysplastic syndromes

Low endogenous erythropoietin levels and limited red blood cell transfusion history can predict response to erythropoiesis‐stimulating agents in anaemic patients with myelodysplastic syndromes (MDS). The relationship between endogenous thrombopoietin (THPO) levels and platelet response to romiplostim is unknown. Variables including baseline endogenous THPO levels, transfusion needs, and platelet response were analysed in a randomized trial of 250 thrombocytopenic, lower‐risk MDS patients (International Prognostic Scoring System low/intermediate‐1). A predictive scoring system was developed based on log–likelihood ratios and logistic coefficients. Patients with HI–P (haematological improvement – platelets) responses had lower mean baseline THPO levels (P = 0·0497) and were more likely to have <6 platelet units transfused in the past year (P = 0·0027), as did patients with platelet responses ≥50% of weeks on romiplostim (P = 0·001 and P = 0·0037, respectively). A model for predicting response to romiplostim was developed and validated in a separate MDS cohort (N = 72). Patients in low‐, intermediate‐, and high‐response groups had response rates of 17·4%, 29·6%, and 50·7%, respectively, for HI‐P, and 17·4%, 33·8%, and 65·2%, respectively, for ≥50% response. For thrombocytopenic patients with lower‐risk MDS, lower baseline THPO levels (<500 pg/ml) and limited platelet transfusion history predicted a greater likelihood of a subsequent platelet response to romiplostim.     

Low‐dose anti‐CD20 veltuzumab given intravenously or subcutaneously is active in relapsed immune thrombocytopenia: a phase I study

Low doses of the humanized anti‐CD20 monoclonal antibody, veltuzumab, were evaluated in 41 patients with immune thrombocytopenia (ITP), including 9 with ITP ≤1 year duration previously treated with steroids and/or immunoglobulins, and 32 with ITP >1 year and additional prior therapies. They received two doses of 80–320 mg veltuzumab 2 weeks apart, initially by intravenous (IV) infusion (N = 7), or later by subcutaneous (SC) injections (N = 34), with only one Grade 3 infusion reaction and no other safety issues. Thirty‐eight response‐assessable patients had 21 (55%) objective responses (platelet count ≥30 × 10⁹/l and ≥2 × baseline), including 11 (29%) complete responses (CRs) (platelet count ≥100 × 10⁹/l). Responses (including CRs) occurred with both IV and SC administration, at all veltuzumab dose levels, and regardless of ITP duration. Responders with ITP ≤1 year had a longer median time to relapse (14·4 months) than those with ITP >1 year (5·8 months). Three patients have maintained a response for up to 4·3 years. SC injections resulted in delayed and lower peak serum levels of veltuzumab, but B‐cell depletion occurred after first administration even at the lowest doses. Eight patients, including 6 responders, developed anti‐veltuzumab antibodies following treatment (human anti‐veltuzumab antibody, 19·5%). Low‐dose SC veltuzumab appears convenient, well‐tolerated, and with promising clinical activity in relapsed ITP.( Clinicaltrials.gov identifier: NCT00547066.)     

Treatment of Epstein Barr virus‐induced haemophagocytic lymphohistiocytosis with rituximab‐containing chemo‐immunotherapeutic regimens

Haemophagocytic lymphohistiocytosis (HLH) is a life threatening complication of Epstein–Barr virus (EBV) infection. The anti‐CD20 antibody rituximab depletes B cells, leading to improved outcomes for patients with EBV‐associated B‐lymphoproliferative disorders. To gather data on the use of rituximab in EBV‐HLH, we performed a retrospective investigation involving 42 EBV‐HLH patients who had received treatment with rituximab‐containing regimens. On average, patients received 3 rituximab infusions (range 1–10) at a median dose of 375 mg/m². In all patients, rituximab was administered with other HLH‐directed medications, including steroids, etoposide and/or ciclosporin. Rituximab‐containing regimens appeared well tolerated and improved clinical status in 43% of patients. Examination of laboratory data obtained prior to and within 2–4 weeks after the first rituximab dose revealed significant reductions in EBV load (median load pre‐rituximab: 114 200 copies/ml, median post‐rituximab: 225 copies/ml, P = 0·0001) and serum ferritin levels (median ferritin pre‐rituximab: 4260 μg/l, median post‐rituximab: 1149 μg/l, P = 0·001). Thus, when combined with conventional HLH‐directed therapies, rituximab improves symptoms, reduces viral load and diminishes inflammation. These data support the incorporation of rituximab into future prospective clinical trials for patients with EBV‐HLH.     

Efficacy and safety of romiplostim in refractory aplastic anaemia: a Phase II/III,multicentre, open‐label study

A previous dose‐finding study has suggested that romiplostim is effective in patients with refractory aplastic anaemia (AA) and 10 µg/kg once weekly was recommended as a starting dose. In this Phase II/III, multicentre, open‐label study, romiplostim was administered subcutaneously at a fixed dose of 10 µg/kg once weekly for 4 weeks (weeks 1–4) followed by weekly doses (5, 10, 15 and 20 µg/kg) titrated by platelet response for up to 52 weeks (weeks 5–52). A total of 31 patients with AA who were refractory to immunosuppressive therapy (IST) and thrombocytopenia (platelet count of ≤30 × 10⁹/l) were enrolled. The primary efficacy endpoint of the proportion of patients achieving any haematological (platelet, neutrophil and erythrocyte) response at week 27 was 84% [95% confidence interval (CI) 66–95%]. Trilineage response was 39% (95% CI 22–58%) at week 53. The most common treatment‐related adverse events (AEs) were headache and muscle spasms (each 13%). All AEs were mild or moderate except for three patients with Grade 3 hepatic AEs; no AEs necessitated romiplostim discontinuation. Two patients developed cytogenetic abnormalities, of whom one returned to normal karyotype at last follow‐up. High‐dose romiplostim is effective and well tolerated in the treatment of patients with AA refractory to IST.     

Circulating myeloid‐derived suppressor cells correlate with clinical outcome in Hodgkin Lymphoma patients treated up‐front with a risk‐adapted strategy

In the attempt to find a peripheral blood biological marker that could mirror the dysregulated microenvironment of Hodgkin Lymphoma (HL), we analysed the amount of myeloid‐derived suppressor cells (MDSC), including the three main sub‐types (monocytic, granulocytic and CD34 + fraction). The absolute MDSC count was investigated in 60 consecutive newly diagnosed HL patients and correlated with clinical variables at diagnosis and outcome. Patients received standard‐of‐care chemotherapy with the exception of interim fluorodeoxyglucose positron emission tomography (PET‐2)‐positive patients, who were switched early to a salvage regimen. All MDSC subsets were increased in HL patients compared to normal subjects (P < 0·0001) and were higher in non‐responders. However, a strong prognostic significance was limited to immature (CD34⁺) MDSC. A cut‐off level of 0·0045 × 10⁹/l for CD34⁺MDSC resulted in 89% (95% confidence interval [CI] 52–99%) sensitivity and 92% (95% CI 81–98%) specificity. The positive predictive value to predict progression‐free survival was 0·90 for PET‐2 and 0·98 for CD34⁺MDSC count; the negative predictive value was 0·57 for PET‐2 and 0·73 for CD34⁺MDSC. PFS was significantly shorter in patients with more than 0·0045 × 10⁹ CD34⁺MDSC cells/l at diagnosis and/or PET‐2 positivity (P < 0·0001). In conclusion, all circulating MDSC subsets are increased in HL; CD34⁺MDSC predict short PFS, similarly to PET‐2 but with the advantage of being available at diagnosis.     

Evaluation of two,commercial, multi‐dye,nucleic acid amplification technology tests,for HBV/HCV/HIV‐1/HIV‐2 and B19V/HAV,for screening blood and plasma for further manufacture

Background The cobas^® TaqScreen MPX Test, version 2.0, a multiplex, multi‐dye nucleic acid amplification technology (NAT) test from Roche was evaluated by two European Blood Banks, the German Red Cross Blood Donor Service, Frankfurt, Germany and Centro de Hemoterapia y Hemodonación de Castilla y León, Valladolid, Spain. In addition, the cobas^® TaqScreen DPX Test was evaluated for the simultaneous detection and quantitation of parvovirus B19 and the detection of hepatitis A virus (HAV). Study Design and Methods The performances of the two tests were evaluated regarding the analytical sensitivity, the reproducibility of the tests using samples containing low concentrations of each virus and cross‐contamination using samples containing high titres of virus. Results The analytical sensitivity of the MPX Test, version 2.0, obtained by the German Red Cross Blood Donor Service was 1·1, 3·9 and 43·3 IU/ml for HBV, HCV and HIV‐1, respectively. The comparable analytical sensitivity at Centro de Hemoterapia y Hemodonación de Castilla y León was 3·5, 17·6 and 50·6 IU/ml for HBV, HCV and HIV‐1, respectively. The analytical sensitivity of the DPX test determined by the German Red Cross Blood Donor Service was 0·6 and 3·8 IU/ml for HAV and B19. Conclusion These multiplex and multi‐dye blood screening assays represent a flexible NAT screening system for mini‐pools between 6 and 96 samples per pool and fulfil all requirements of the European Pharmacopoeia for HCV and B19V testing of plasma for fractionation. The inclusion of a new multi‐dye technology means discriminatory assays are no longer required for either test thus improving workflow, turn‐around time and minimize the risk of obtaining a reactive result for which the virus cannot be identified.     

The PFA‐100®: a potential rapid screening tool for the assessment of platelet dysfunction

The PFA‐100^® is a device that simulates high shear dependent platelet function in vitro and thus is particularly useful for screening for von Willebrand's disease (VWD). The aim of this study was to assess the overall potential of the PFA‐100^® as a primary clinical screening tool using the wide spectrum of clinical samples assessed for platelet function within our institution. The PFA‐100^® test was performed using both collagen/ADP (CADP) and collagen/epinephrine (CEPI) cartridges on samples from 337 patients with a wide variety of haemostatic defects. One hundred and eighty‐two patients were defined as having normal platelet function based on classical laboratory tests and von Willebrand factor levels. The overall clinical sensitivity of the PFA‐100^® for platelet abnormalities (including VWD) was 81% for CADP and 86% for CEPI. The overall specificity was found to be 82% for CADP and 80% for CEPI. When utilizing both cartridges in combination (with both results either higher or lower than the upper cutoff of the normal ranges), the overall false positive and false negative rates were 12% and 6%, respectively. The PFA‐100^® proved to be sensitive in detecting classical defects by giving prolonged closure times in samples from patients with major platelet function defects (e.g. von Willebrand's disease, Glanzmann's thrombasthenia and Bernard Soulier syndrome). However, there were a small number of false negative results (6%) obtained with various milder platelet defects (e.g. Hermansky Pudlak syndrome, storage pool and release defects, type I VWD and macrothrombocytopenia). The PFA‐100^® test provides a useful rapid screening tool and should increase the efficiency and reduce the cost of the routine diagnosis of platelet dysfunction.