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Objective: Coating of autologous red blood cells (RBCs) with polyclonal antibodies to Rh(D) antigen may result in an immunomodulation and improved outcome in Rh(D) positive women with recurrent pregnancy loss (RPL). Design: Retrospective data analysis. Setting: Rh(D) positive women with a history of RPL and ineffective treatment with low molecular weight heparin (LMWH) and/or aspirin Methods: Anti-D (300 µg) was given subcutaneously to 60 women either prior to pregnancy and/or two times within 12 weeks of gestation. Main outcome measures: Births of healthy child/children. Results: Treatment with Anti-D resulted in successful pregnancies in 67% of all cases. The remaining women had only aborts (23%) or did not become pregnant (10%). None of the treated women has developed anaemia due to this treatment or any other significant adverse reaction. The rate of successful pregnancies does not appear to be influenced by the administration of: Anti-D prior to pregnancy, age, thrombophilia or previous alive births. Conclusion: The improved outcome following the administration of Anti-D in women with RPL might be explained by immune modulations induced by different immune reactions including polarization of decidual macrophages. The results obtained in this study clearly indicate that Anti-D is safe and highly effective in treatment of Rh(D) positive women with RPL. However, further studies are required to support our results and to find out the optimal dose and timing of Anti-D administration. Funding: There was no funding included. Key words: abort, Anti-D, aspirin, low molecular weight heparin, new treatment, recurrent pregnancy loss

Objectives To assess the efficacy and safety of intravenous immunoglobulin (IVIG) 10% (Panzyga® ), a novel human normal IVIG 10%, in patients with chronic immune thrombocytopenia (ITP). Background First-line treatment options in ITP include IVIGs. Methods In this prospective, open-label, non-controlled, multicentre, phase III study, patients received a daily dose of IVIG 10% (1 g kg-1 body weight) for two consecutive days. The primary end point was clinical response rate; secondary end points included alternate response definitions, time to response, response duration, platelet counts, regression of bleeding and safety. Results Forty patients were enrolled (57·5% male, mean age 36·7 years); the full analysis set comprised 36 patients. A clinical response was seen for 29 of 36 patients (80·6%). Median time to response and response duration was 2 days and 14 days, respectively. IVIG 10% was well tolerated at a maximum infusion rate of 8 mg (kg min)-1 in all but one patient; adverse events were mainly mild to moderate in severity, and the most frequent was headache (42·5%). Conclusion IVIG 10% is well tolerated even at a high infusion speed and induces a rapid platelet count increase, thus decreasing the bleeding rate and the severity of bleeding events. Trial registry ClinicalTrials.gov record: NCT01349790.

Introduction: Uncrossmatched ABO-compatible red blood cells (RBCs) are generally recommended in patients with life-threatening massive bleeding. There is little data regarding RBC transfusion when patients are transfused against clinically significant alloantibodies because compatible RBCs are not immediately available. Methods/Patients: All patients reviewed in this study (n = 6,109) required emergency blood transfusion and were treated at the Charité – Universitätsmedizin Berlin between 2001 and 2015. Primary uncrossmatched O Rh(D)-positive or -negative RBC units were immediately transfused prior to complete regulatory serological testing including determination of ABO group, Rhesus antigens, antibody screening, and crossmatching. Results: Without any significant change in the protocol of emergency transfusion of RBCs, a total of 63,373 RBC units were transfused in 6,109 patients. Antibody screening was positive in 413 patients (6.8%), and 19 of these patients received RBC units against clinically significant alloantibodies. None of these patients appeared to have developed significant hemolysis, and only one patient with anti-D seems to have developed signs of insignificant hemolysis following the transfusion of three Rh(D)-positive units. One patient who had anti-Jka received unselected units and did not develop a hemolytic transfusion reaction. Conclusion: Transfusion of uncrossmatched ABO-compatible RBCs against alloantibodies is highly safe in patients with life-threatening hemorrhage.

Patients with persistent/chronic immune thrombocytopenia (cITP) have low platelet counts, increased risk of bleeding and bruising, and often suffer from reduced health‐related quality of life (HRQoL). cITP treatments may either improve HRQoL by increasing platelet counts or decrease it because of side effects. The open‐label EXTEND study (June 2006 to July 2015) evaluated long‐term safety, tolerability, and efficacy of eltrombopag (an oral thrombopoietin‐receptor‐agonist) in adults with cITP who completed a previous eltrombopag ITP trial. The final results of EXTEND were published and used to assess changes in patient‐reported HRQoL over time and association between HRQoL and platelet response. Four validated HRQoL instruments were administered: SF‐36v2 including physical component summary (PCS) and Mental Component Summary; Motivation and Energy Inventory Short Form (MEI‐SF); Fatigue Subscale of FACIT (FACIT‐Fatigue); and FACT‐Thrombocytopenia Subscale Six‐Item Extract (FACT‐Th6). For the 302 patients enrolled, median duration of eltrombopag treatment was 2.37 years. All 4 HRQoL instruments demonstrated positive mean changes from baseline over time adjusted for patient baseline characteristics and rescue therapy use, and had positive association with platelet response (platelet count ≥30 × 109/L; ≥50 × 109/L; and ≥50 × 109/L and >2 times baseline). Improvements from baseline started within 3 months and persisted through 5 years of treatment for FACIT‐Fatigue and FACT‐Th6 (P

Background: Intravenous immunoglobulin (IVIG) IQYMUNE® is a highly purified 10% IVIG that was assessed using the new stringent definition of response described in the revised guideline on the clinical investigation of IVIG. The efficacy and the safety of IQYMUNE® were investigated in adult patients with chronic primary immune thrombocytopenia (ITP). Methods: In this phase III multinational, multicentre, prospective, uncontrolled, open-label, single-arm study, adult patients with a baseline platelet count < 30 × 10 9 /L were treated with IVIG 10% at a dose of 2 g/kg body weight administered over 2 consecutive days. The primary endpoint was Response over the study period and was defined according to the recent and most stringent European Medicines Agency guidelines (platelet count ≥ 30 × 10 9 /L and a ≥ 2-fold increase from baseline, no new bleeding, and no concomitant treatment with drugs that affect platelet count and/or induce bleeding cessation). Results: Thirty-eight patients were enrolled; 73 infusions were administered (38 on Day 1 and 35 on Day 2). Response was reached by 24 patients corresponding to 63.2% of patients in the full analysis set (95% CI: 46.0; 78.2) and 68.6% of patients in the per-protocol set (95% CI: 50.7; 83.1). The median time to Response was 1 day. The median duration of Response was 13.5 days. Reasons for non-response were failure to reach the required platelet count (n = 12), a new bleeding event (n = 1), and forbidden medication use (n = 1). Among the 23 patients with a baseline platelet count ≤ 20 × 10 9 /L, 19 patients (82.6%) achieved a platelet count ≥ 50 × 10 9 /L at least once before Day 5 (previous European Medicines Agency definition of response). Treatment was well tolerated even in patients with a high flow rate (≥ 6 mL/kg/h in 40% of patients). Headache (34.2%), pyrexia (15.8%), and creatinine renal clearance decrease, including one case of decrease in glomerular filtration rate (10.5%) were the most frequently reported drug-related adverse events. Conclusions: Administration of IQYMUNE® for 2 consecutive days at a dose of 2 g/kg was safe and efficacious. These results support the treatment of adult patients with chronic ITP with IQYMUNE®. J Hematol. 2018;7(3):87-95 doi: https://doi.org/10.14740/jh385w

Immune thrombocytopenic purpura (ITP) is an idiopathic bleeding disorder. B cell activating factor (BAFF) and 'A proliferation-inducing ligand' (APRIL) have regulatory effects on B and T cells and may represent relevant factors in the pathogenesis of ITP. Serum levels and gene expression were investigated in ITP patients. Both BAFF and APRIL serum levels were significantly elevated in active ITP. However, gene expression analysis revealed both factors to have a tendency toward downregulation. Glucocorticoid treatment significantly reduced BAFF but not APRIL serum levels, which may be mediated by differences in transcription factor binding sites. The glucocorticoid receptor binding site is present in the BAFF promotor region, but not in the APRIL promotor region. Prednisolone in combination with vitamin D3 may be effective in reducing APRIL serum levels. In conclusion, glucocorticoid treatment exerts different regulatory effects on both BAFF and APRIL, whereas antioxidant supplementation may also be beneficial in reducing serum levels.

Objective Haemolysis and anaemia related to autoimmune haemolytic anaemia (AIHA) of warm type (wAIHA) and of cold type (cAIHA) are believed to be solely due to antibody and/or complement-mediated destruction and clearance of red blood cells (RBCs). There is evidence that RBCs of affected patients may also undergo eryptosis, the suicidal death of RBCs. Method RBCs from 24 patients with wAIHA, 7 patients with chronic cAIHA, and one patient with AIHA of mixed-type were analysed for exposed phosphatidylserine (PS) by treatment with phycoerythrin (PE)-labelled Annexin V and cell-associated fluorescence was measured using a MACSQuant flow cytometer. Results PS exposing RBCs were detected in 7 of 13 patients with clinically significant wAIHA. Haemolysis was mostly related to IgM or IgA autoantibodies (aab) in those patients. In contrast, PS exposure in 11 wAIHA patients in complete remission was comparable to that in healthy blood donors. All patients with chronic cAIHA and the patient with AIHA of mixed-type showed haemolytic activity and high numbers of PS exposing RBCs. Patients with decompensated AIHA appear to respond to treatment with erythropoietin, which is a known inhibitor of eryptosis. Conclusion Eryptosis may frequently occur in AIHA related to IgM or IgA aab. Inhibition of eryptosis may represent a new therapeutic option in the treatment of AIHA. This article is protected by copyright. All rights reserved.

The thrombopoietin receptor agonists (TPO-RAs), romiplostim and eltrombopag, stimulate megakaryopoiesis and thereby increase platelet counts. Both drugs are increasingly used in the treatment of immune thrombocytopenic purpura (ITP). To assess the effect of TPO-RAs on trilineage haematopoiesis, colony-forming cell (CFC) assays were performed on peripheral blood mononuclear cells of 8 healthy donors and 52 ITP patients. Additionally, we revaluated the regular and complete blood counts (CBCs) performed during romiplostim therapy in 45 patients and the CBCs performed in 9 patients during eltrombopag therapy. The clonogenic capacity of PBMCs was significantly increased in patients treated with TPO-RAs compared with healthy donors and untreated patients [BFU-E, 69 ± 47; CFU-GM, 61 ± 48; CFU-GEMM, 16 ± 11; CFU-total, 145 ± 94; P values

BACKGROUND Patients treated with intravenous immunoglobulins (IVIG) rarely experience symptomatic hemolysis. Although anti-A and anti-B isoagglutinins from the product are involved in most cases, the actual mechanisms triggering hemolysis are unclear. STUDY DESIGN AND METHODS A prospective, open-label, multicenter, single-arm clinical trial in 57 patients with immune thrombocytopenia treated with IVIG (Privigen, CSL Behring) was conducted. RESULTS Twenty-one patients received one infusion (1 g/kg) and 36 received two infusions (2 × 1 g/kg) of IVIG. After a study duration of more than 2 years, no cases of clinically significant hemolysis as defined in the protocol were identified. Data of patients with mild hematologic and biochemical changes were analyzed in more detail. Twelve cases (10/23 patients with blood group A1 and 2/11 patients with blood group B, all having received 2 g/kg IVIG) were adjudicated as mild hemolysis (median hemoglobin [Hb] decrease, −3.0 g/dL); Hb decreases were transient, with partial or full recovery achieved by last visit. Eighteen patients (31.6%), all with non-O blood group, of whom 16 (88.9%) received 2 g/kg IVIG, fulfilled post hoc criteria for hemolytic laboratory reactions. Red blood cell (RBC) eluates of all direct antiglobulin test–positive samples were negative for non-ABO blood group antibodies. Blood groups A and B antigen density on RBCs appeared to be a risk factor for hemolytic laboratory reactions. Platelet response to treatment was observed in 42 patients (74%); eight of 12 patients with complete response had blood group A1. CONCLUSION Isoagglutinins are involved in clinically nonsignificant hemolysis after treatment with IVIG, but individual susceptibility varies greatly.

Purpose Immune-mediated heparin-induced thrombocytopenia (HIT type II, HIT) is a potentially serious adverse drug reaction characterized by an increased risk of venous and arterial thrombosis. This study aimed to identify risk factors associated with the development of these complications. Methods Our study cohort included patients with HIT assembled in our pharmacovigilance center by reports from 51 collaborating hospitals in Berlin, Germany. To identify risk factors for thromboembolic complications, patients with thromboembolic events (cases) were compared to those without thromboembolic events (controls) in a case-control design. We applied univariable and multivariable logistic regression analysis to estimate odds ratios (OR) and corresponding 95% confidence intervals (CI) for potential risk factors of thromboembolic complications. Results Our cohort comprised 209 HIT patients. Of those, 53 developed thromboembolic complications. Most HIT patients received heparin for medical indications (42.1%) or in the context of cardiovascular surgery (40.2%). Of the 78 thromboembolic complications, 49 (63%) and 29 (37%) were observed in the venous and arterial vascular bed, respectively. The main locations were deep vein thrombosis (39.7%), pulmonary embolism (16.7%), and limb artery thrombosis (16.7%). In multivariable analysis, immobilization prior to HIT (OR 4.6, 95% CI 1.2-18.0; P = .026) and higher platelet counts before initiation of heparin therapy (OR 1.004, 95% CI 1.000-1.008; P = .046) were independently associated with the occurrence of thromboembolic events. Conclusions Immobilization and a high platelet count (with a low effect size) are additional risk factors of thromboembolic complications in the course of HIT.

BACKGROUND Routine quantification of platelet-derived extracellular vesicles (PL-EVs) may be useful in the quality control (QC) of platelet concentrates (PCs). The aim of this multicenter study was to establish and validate a consensus protocol for the standardized PL-EV quantification using conventional flow cytometers. STUDY DESIGN AMD METHODS Eighty-six PCs were investigated in five blood transfusion centers (A-E) on Days 0 and 5. The centers used different apheresis instruments: Trima Accel (n = 56) and/or Amicus (n = 30). PCs were prepared using standard methods (sd-PCs; n = 73; A-D) or with pathogen inactivation (PI [PI-PCs]; n = 13; E). Platelet (PLT) count was determined using conventional hematology analyzers. PLT degranulation (P-selectin expression in response to thrombin receptor PAR1 activation) and PL-EVs were analyzed by flow cytometry. RESULTS During storage, PLT count remained stable in 58 PCs (A, C, E), whereas a decrease was observed in 12 PCs (B). PLT degranulation declined in all PCs (p < 0.001) and PL-EVs increased in 74 PCs (A, C-E; p < 0.001). Certain donor variables (e.g., plasma cholesterol, immature PLT fraction) were associated with lower PL-EVs. In Trima-produced PCs, PL-EVs were significantly lower (D) and PLT degranulation was superior compared to PCs prepared with the Amicus (A, D). PL-EVs were 10-fold lower in PI-PCs, compared to sd-PCs. However, similar QC trends were demonstrated for both PC groups during storage. CONCLUSION PL-EV analysis in a QC program of PCs was successfully performed with results comparable among the different centers. PLT degranulation and vesiculation were primarily affected by preparation techniques.

Immune thrombocytopenic purpura (ITP) is one of the best characterized autoimmune diseases. Autoantibodies (AABs) against platelet antigens are considered as the diagnostic hallmark of ITP, but are detectable in only 50% of patients. We designed and applied a novel proteomic approach termed Mass Spectroscopy-based Antibody-Mediated Identification of Autoantigens (MS-AMIDA) for platelet antigens. Patients were separated into patients with classical AABs [ITP(+)] and patients without AABs [ITP(−)]. Altogether, 181 potential AAGs were found in ITP(+) and 135 AAGs in ITP(−), with 34 and 23 AAGs reproducibly found in two runs of MS-AMIDA. After subtracting identifiers from the controls, 57 AAGs in ITP(+) and 29 AAGs in ITP(+) remained, with 16 AAGs commonly found in ITP(+) and ITP(−) patients. Label-free quantification (LFQ) revealed 15 potential AAGs that are quantitatively stronger in ITP. Dot blot validation was performed on hexokinase 1 (HK1), E1 pyruvate dehydrogenase (E1-PDH), coagulation factor XIII, filamin A (FLNA), non-muscle myosin 9. Eleven patients were found to have anti-HK1 AABs, one patient had anti-E1-PDH AABs, and two patients had anti-FLNA AABs. Most antigens were of intracellular origin with significant association with actin-cytoskeleton and regulation of programmed cell death. In conclusion, novel AAGs for ITP were identified using MS-AMIDA.

Platelet storage lesions (PSLs) occur during platelet concentrate (PC) storage. Adverse transfusion reactions (ATRs) have been demonstrated to be more frequent in older PCs and removal of the supernatant prior to transfusion reduces their occurrence. Proteomic profiling of PC supernatants was thus performed to identify proteins associated with PSLs and ATRs. Twenty-four PCs were investigated daily from day 0 to day 9 for platelet pre-activation (PPA), platelet-derived extracellular vesicles (PEVs), and platelet function. Using antibody microarrays, 673 extracellular proteins were analysed in PC supernatants on days 0, 3, 5, 7, and 9. During 5 days of storage, PPA and PEVs continuously increased ( P P = 0.1751) and decreased thereafter. Comparison of all time points to day 0 revealed the identification of 136 proteins that were significantly changed in abundance during storage, of which 72 were expressed by platelets. Network analysis identified these proteins to be predominantly associated with exosomes ( P = 4.61 × 10 − 8 , n = 45 genes) and two clusters with distinct functions were found with one being associated with haemostasis and the other with RNA binding. These findings may provide an explanation for ATRs. Significance Changes in platelet concentrate (PC) supernatants during storage have been so far only poorly addressed and high abundant proteins burden the identification of quantitative changes in the secretome. We applied a high-throughput antibody microarray allowing for the sensitive quantification of 673 extracellular factors. PCs account for the highest number of adverse transfusion reactions (ATRs). ATRs have been demonstrated to be more frequent in older PCs and removal of the supernatant prior to transfusion reduces their occurrence. Comprehensive interpretation of the changing proteins in the secretome during platelet storage under blood banking conditions may help to identify mechanisms leading to the occurrence of adverse transfusion reactions.

Background: Dependent on the absence or presence of associated diseases, autoimmune thrombocytopenia (ITP) can be classified as primary or secondary form. The manifestation of the associated diseases is not temporally defined and may occur during observation. Thus the question which disease is the primary one remains unanswered. Methods: All 386 patients included in this study were treated by a single primary physician between 1996 and 2015 at the Charité Berlin and met current ITP criteria. Medical records and investigations were reviewed to assess diseases associated with ITP. Results: Initially, the vast majority of patients presented with primary ITP (isolated disease). Based on our findings, ITP was found to be associated with other abnormalities in most cases. These abnormalities included: positive direct antiglobulin test in 49 of 386 tested patients (13%), affections of the thyroid gland in 41 of 386 tested patients (11%), infections in 30 (8%), solid malignancies in 20 (5%) and hematological malignancies in 10 patients (3%), as well as many other miscellaneous diseases. Moreover, of 160 patients who did not receive prior intravenous immunoglobulin treatment, 40 (25%) showed antibody deficiency. Conclusion: In conclusion, the incidence of ‘true' ITP as a primary disease is less common than has yet been suggested. Additionally, there is evidence that ITP itself predispose affected subjects toward development of other diseases.

Background The aim of this study was to determine retrospectively the efficacy of combined therapy using erythropoietin (EPO) and erythrocytapheresis (EA) in patients with hereditary hemochromatosis (HH) who did not tolerate phlebotomy. Patients and Methods Twenty patients (age range, 43–74 years) with genetically confirmed HH had received low-dose EPO (4,000 IU) in accordance to the patient's hemoglobin levels between each EA session. Laboratory parameters including hemoglobin, ferritin, transferrin, and iron were measured at regular intervals. Results Anemia did not occur in a single patient and no serious side effects were observed. Combined treatment with EPO and EA was well tolerated, and all 18 patients who suffered from fatigue prior to therapy recovered. Median ferritin values were 678.5 ng/L before treatment and 145 ng/L after treatment. Conclusion EA in combination with EPO is safe and effective in treating patients with HH. Prospective studies comparing this therapeutic option to phlebotomy are warranted. J. Clin. Apheresis, 2016. © 2016 Wiley Periodicals, Inc.

BACKGROUND: Hypertension is one of the leading global risks for cardiovascular events worldwide. There is preliminary evidence that regular blood donation may be beneficial. STUDY DESIGN AND METHODS: Unselected blood donors were included in this observational study. Blood pressure (BP) was measured before and after blood donation, with participants donating between one and four occasions in a 1-year study period. RESULTS: In this study, 292 donors were enrolled. At baseline, 146 had elevated BP (>140/90 mmHg). In hypertensives, after four blood donations, systolic and diastolic blood pressure (SBP and DBP, respectively) decreased from a mean of 155.9 613.0 to 143.7 615.0 mmHg and from 91.4 69.2 to 84.5 69.3 mmHg, respectively (each p

Background: Alloimmunization to red blood cells (RBCs) may result in fetal anemia prior to 20 weeks gestation. The question as to whether early commencement of antenatal treatment with high-dose intravenous immunoglobulins (IVIG) may prevent or at least delay the development of fetal anemia in the presence of alloantibodies to RBCs is highly relevant. Patients and Results: Here we describe a patient with high-titer anti-K and two other severely affected pregnant women with a history of recurrent pregnancy loss due to high-titer anti-D or anti-D plus anti-C. Early commencement of treatment with IVIG (1 g/kg/week) resulted in prevention of intrauterine transfusion (IUT) in the former two cases, and in a significant delay of development of fetal anemia in the remaining case (26 weeks gestation). Conclusion: Based on our findings and of previously published cases, early initiation of treatment of severely alloimmunized women with IVIG (1 g/kg/week) could potentially improve the outcome of fetuses at risk.

Hamatopoetische Stammzelltransplantationen fuhren gelegentlich zur Entwicklung einer schweren autoimmunhamolytischen Anamie (AIHA) vom Warmetyp. Die „konventionelle“ immunsuppressive Therapie dieser Form der AIHA ist haufig ineffektiv und mit schweren Nebenwirkungen verbunden. Die Hamolyse kann in Einzelfallen durch die Transfusion von Erythrozytenkonzentraten (EKs) verstarkt werden. Bei drohender hypoxischer Anamie infolge einer AIHA sollten dem Patienten EK-Transfusionen dennoch nicht vorenthalten werden. Im nachfolgenden Fallbericht stellen wir ein Transfusionsstufenschema vor, das bei einer Patientin mit schwerer akuter Post-Transplant-AIHA vertraglich war, wahrend die Patientin auf Standardtransfusionen mit ausgepragter Hamolyse reagierte.

Until now, treatment of primary autoimmune hemolytic anemia of the warm type (wAIHA) is primarily based on immunosuppression. However, many patients do not respond adequately to treatment, and treated patients may develop severe side effects due to uncontrolled, mixed and/or long-lasting immunosuppression. Unfortunately, the newly used therapeutic monoclonal antibodies are unspecific and remain frequently ineffective. Thus, development of a specific therapy for AIHA is necessary. The ideal therapy would be the identification and elimination of the causative origin of autoimmunization and/or the correction or reprogramming of the dysregulated immune components. Blood transfusion is the most rapidly effective measure for patients who develop or may develop hypoxic anemia. Although some effort has been made to guide physicians on how to adequately treat patients with AIHA, a number of individual aspects should be considered prior to treatment. Based on my serological and clinical experience and the analysis of evidence-based studies, we remain far from any optimized therapeutic measures for all AIHA patients. Today, the old standard therapy using controlled steroid administration, with or without azathioprine or cyclophosphamide, is, when complemented with erythropoiesis-stimulating agents, still the most effective therapy in wAIHA. Rituximab or other monoclonal antibodies may be used instead of splenectomy in therapy-refractory patients.

Background: Pseudothrombocytopenia (PTCP) is a well-known phenomenon. However, confusion may occur due to unusual characteristics. Case Reports: Two patients with autoimmune thrombocytopenia (ITP) and long-lasting PTCP are described. Initially, only the diagnosis of ITP was confirmed. During observation, discrepancies were recognized between clinical findings and platelet counts. Re-examination resulted in the additional diagnosis of EDTA-dependent PTCP. Subsequently, the latter diagnosis was changed to citrate-dependent PTCP in both cases. Interestingly, PTCP was observed to change again and became recognizable in citrate or heparin, and only during the first 20-30 min following phlebotomy in EDTA specimens. Conclusion: The incidence of concomitant ITP with PTCP might be higher than previously reported, and PTCP may have variable dynamics and characteristics.

Background and Objectives Pharmacokinetics, safety and tolerability of escalating infusion rates of BT090, a 10% intravenous immunoglobulin (IVIg), were studied in patients with primary immunodeficiency disease. Materials and Methods In Part A, patients (n = 30) received 3 infusions of BT090 at their pretrial dose and dosing interval; the infusion rate of BT090 was increased from 0·3 to 1·4 to 2·0 ml/kg/h for each infusion in each patient initially at 30-min intervals. Pharmacokinetics was evaluated at the 3rd infusion (n = 24). At the 4th infusion, infusion rates were to be gradually escalated from 0·3 to 1·4 to 4·0 to a maximum of 8·0 ml/kg/h initially at 30-min intervals to establish the maximum tolerated infusion rate per patient. Results The pharmacokinetic characteristics and safety profile of BT090 were comparable with those of other IVIgs, including Intratect®. Escalation of infusion rates was well tolerated, allowing identification of individual patient's maximum tolerated infusion rate. At subsequent infusions, all patients tolerated their individually defined maximum infusion rate: 17 patients (68·0%) tolerated infusion rates of 6·0 or 8·0 ml/kg/h and four patients (16%) had maximum tolerated infusion rates of

Background: Drug-induced immune haemolytic anaemia (DIHA) is difficult to diagnose, and its true incidence remains obscure. Here, we present cases of DIHA identified at our institute over the last two decades. Methods: Serological tests were performed according to standard procedures. Detection of drug-dependent antibodies was performed in the presence and absence of the relevant drug and/or their ex vivo antigens. Results: Over the last 20 years, 73 patients have been identified with DIHA in our institute, which was related to 15 different drugs. The most common single drugs identified were diclofenac (n = 23), piperacillin (n = 13), ceftriaxone (n = 12) and oxaliplatin (n = 10). As far as data were available, haemolysis was acute in all patients, and signs of intravascular haemolysis were present in 90% of the cases. Haemolysis resulted in death in 17 patients (23%). The remaining patients recovered, but haemolysis was complicated by transitory renal and/or liver failure or shock in 11 patients. Upon initial evaluation, the antibody screening test was positive in 36 cases. A positive direct antiglobulin test (DAT) at least with anti-C3d was found in 65 cases, with anti-IgG only in 6 cases, and with anti-IgA only in 1 case. Conclusion: DIHA is a rare but potentially life-threatening disorder that should be considered if a patient develops haemolysis under drug treatment. The main serological finding is a positive DAT, primarily with anti-C3d.

Autoimmune haemolytic anaemias (AIHAs) are well-characterized disorders. They can be differentiated from one another and from other non-immune haemolytic anaemias by clinical, laboratory and serological testing. However, several misleading clinical presentations and/or serological findings may result in misinterpretation, delay and/or misdiagnosis. Such failures are avoidable by adequate clinical and serological experience of the responsible physicians and serologists or, at least, by an optimised bidirectional communication. As long as this has not been achieved, unpleasant failures are to be expected. A true diagnosis of AIHA can neither be verified by clinical nor serological findings alone. Thus, a collective clinical and serological picture remains obligatory for fulfilling the criteria of optimal diagnosis and therapy. Ultimately, the majority of pioneer scientific and practical work in this field stems from scientists who were simultaneously involved in both the clinic and serology.

Background: The phenomena of co-incidence of transfusion-induced allo- and autoantibodies, blockage and/or loss of red blood cell (RBC) antigens are conspicuous and may result in confusion and misdiagnosis. Case Report: A 67-year-old female was transferred to the intensive care unit due to hemolysis which developed 2 days following transfusion of three Rh(D)-negative RBC units in the presence of strongly reactive autoantibodies. Standard serological testing and genotyping were performed. Upon arrival, the patient was typed as Ccddee. Her hemolysis was decompensated, and an immediate blood transfusion was required. In addition, direct and indirect antiglobulin tests (DAT and IAT) as well as the eluate were strongly positive. Emergency transfusion of Rh(D)-negative RBCs resulted in increased hemolysis and renal failure. An exhaustive testing revealed anti-D, anti-c, CCddee phenotype and CCD.ee genotype. Three units of cryopreserved CCddee RBCs were transfused, and the patient's condition immediately improved. The discrepancy between Rh-D phenotyping and genotyping was likely caused by masking of the D-epitopes by the autoantibodies. In fact, further enquiry revealed that the patient had been phenotyped as Rh(D)-positive 6 months ago and had been transfused at that time following hip surgery. Conclusion: The phenomena of transfusion-induced autoantibodies, masked alloantibodies, antigen blockage and/or loss are rare but important features which should be considered in patients presenting with autoimmune hemolytic anemia and/or hemolytic transfusion reactions.

SUMMARY Objective The objective of this study was to elucidate whether complement activation occurs during the storage of RBCs in newly formulated PAGGS-M storage medium. Background The reason for red blood cell (RBC) storage lesions is not yet fully understood. The contribution of complement to RBC storage lesion has not been extensively characterised. Study design and methods We investigated the surface expression of CD35, CD55, CD59 and CD47, as well as deposition of C3d, using flow cytometry over a storage period of up to 42 days on a weekly basis. C3d and the immunoglobulins IgG, IgM and IgA were additionally investigated via the direct antiglobulin test (DAT). The effect of contact with homologous serum for 30 min at 37 °C was also performed for C3d and CD35 and is subsequently termed as a ‘transfusion simulation (TS)’. Results A weak but significant increase of C3d was observed prior to TS (anova P = 0·0103), whereas a stronger increase from 74·0 ± 12·4 to 101·2 ± 9·7 was observed post-TS (anova; P

Background and objectives The clinical significance of immune thrombocytopenia (ITP) is mainly reflected by bleeding and/or bleeding risks, which, in some cases, cannot be adequately controlled by standard therapy. Tranexamic acid (TA) is increasingly used in preventing and reducing bleeding in several medical settings. There is little information on whether TA may also be useful in the management of ITP. Materials and methods Twelve patients with ITP were treated with TA (0·5–3 g/day) due to recognizable bleeding. Ten of the 12 patients were under regular treatment for ITP. The remaining two patients did not require additional therapy. Results Cessation or, at least, significant improvement of bleeding was achieved shortly after the initiation of TA in all cases. TA was well tolerated and discontinued after cessation of bleeding. Conclusions We recommend the use of TA in ITP patients with bleeding and/or an increased bleeding risk. Ultimately, cessation of bleeding plays a key role in the management of such affected patients. However, future studies are required to optimize dose and administration routes (intravenous or oral).

In phase 2/3 trials, eltrombopag treatment of 6 months or less in patients with chronic/persistent immune thrombocytopenia (ITP) increased platelet counts and reduced bleeding. The open-label EXTEND study evaluated long-term safety and efficacy of eltrombopag in adults with ITP who had completed a previous eltrombopag study. For the 302 patients enrolled, median duration of eltrombopag treatment was 2.37 years (2 days-8.76 years). Median platelet counts increased to 50 × 109/L or more by week 2 and were sustained throughout the treatment period. Overall, 259 patients (85.8%) achieved a response (platelet count ≥50 × 109/L at least once in the absence of rescue), and 133 (52%) of 257 patients achieved a continuous response of 25 weeks or longer. Responses in patients with platelet counts lower than 15 × 109/L, more previous therapies, and/or splenectomy were somewhat lower. Thirty-four (34%) of 101 patients receiving concomitant ITP medication discontinued 1 or more medication. In patients with assessments, bleeding symptoms (World Health Organization grades 1-4) decreased from 57% at baseline to 16% at 1 year. Forty-one patients (14%) withdrew because of adverse events. Hepatobiliary adverse events (n = 7), cataracts (n = 4), deep vein thrombosis (n = 3), cerebral infarction (n = 2), headache (n = 2), and myelofibrosis (n = 2) occurred in more than 1 patient; the remaining adverse events occurred only once. Rates of thromboembolic events (6%) and hepatobiliary adverse events (15%) did not increase with treatment duration past 1 year. EXTEND demonstrated that long-term use of eltrombopag was effective in maintaining platelet counts of 50 × 109/L or more and reducing bleeding in most patients with ITP of more than 6 months' duration. Important adverse events (eg, thrombosis, hepatobiliary, and bone marrow fibrosis) were infrequent. (ClinicalTrials.gov:NCT00351468).

Introduction: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by autoantibody production against platelets, increased platelet destruction, and, in some cases, impaired thrombopoiesis. The majority of affected patients have significant bleeding risks due to low platelet counts and require treatment. The etiology of ITP is an immunological labyrinth. Currently available treatment options are usually not only nonspecific, but are also associated with some risks.Areas covered: Several useful drugs for the treatment of ITP are currently available. Furthermore, ongoing trials with new drugs and preclinical development of additional drugs may help to improve and determine their value.Expert opinion: ITP is a heterogeneous complex requiring individualized treatment. None of the available drugs are specific, nor are they invariably safe and effective. Thus, the need for specific therapy is evident.

Background We describe a patient with a high-titer warm immunoglobulin (Ig)A autoantibody resulting in death due to hemagglutination rather than to hemolysis. Case Report A 47-year-old male patient presented with an intriguing pronounced vascular erythema of the skin. A livedo reticularis associated with cold agglutinin of high thermal amplitude was suspected. The patient's condition unexpectedly and abruptly deteriorated resulting in death 3 days after admission. Study Design and Methods Conventional serologic procedures and immunochemical methods were used. Results Serologic and immunochemical examinations revealed a warm IgA autoantibody of high titer with anti-Band 3 specificity. Although the patient presented with severe anemia, only mild signs of hemolysis were observed, with no evidence of complement activation. The autopsy revealed an enormous accumulation of agglutinated red blood cells in liver and spleen and a B-cell lymphoma and cerebral edema. Thus, the patient's death was largely caused by hypoxia related to hemagglutination rather than to hemolysis and/or anemia per se. Conclusion Strongly hemagglutinating antibodies may not only cause immune hemolysis but also hypoxia due to intravascular hemagglutination.

Background: In general, patients with significant anti-Ig-A do not tolerate intravenous (i.v.) administration of normal blood products. Here, we present our experiences in the induction of immune tolerance (IIT) and long-term treatment in a series of such patients affected in such a way. The question whether blood components from IgA-deficient donors are required will be discussed. Methods: Ten adult patients (4 females and 6 males; age ranging from 36 to 75 years) with anti-IgA were included in this study. All patients required long-term treatment with blood components. One patient had IgA deficiency and paroxysmal nocturnal hemoglobinuria (PNH), and all other patients had common variable immunodeficiency (CVID). The particle gel immunoassay was used for the detection of anti-IgA. Immune tolerance to IgA was induced by controlled subcutaneous (s.c.) and/or i.v. infusions of IgG preparations. Results: Prior to IIT, anti-IgA was detectable in plasma samples of all patients and significantly diminished or abolished by controlled s.c. and/or i.v. infusions of IgG. Multiple transfusions with normal blood components could be repeatedly performed with the patient suffering from PNH without any complications. As long as i.v. IgG (IVIgG) infusions were consequently administered as individually required (intervals 2-8 weeks), none of the patients developed reactions during observation (up to 10 years). However, interruption of treatment and re-exposure to IVIgG resulted in adverse reactions. Conclusion: Patients with significant anti-IgA can be safely desensitized and tolerate long-term IgG substitutions independent of the IgA concentration of the used blood component.

Immune hemolytic anemia (IHA) is a rare complication of drug administration. However, its true incidence remains obscure, as there are a number of factors that may lead to misdiagnosis. The clinical and serologic pictures are variable, and there is a great deal of unawareness that certain drugs can cause IHA. Furthermore, serologic results can be easily misinterpreted, resulting in a wrong diagnosis. Immunohematology 2014;30:80–84.

Drug-induced agranulocytosis (DIAG) is a rare but serious adverse drug reaction. The Berlin Case-Control Surveillance Study (FAKOS) aimed to identify pharmaceuticals with an increased risk for this condition.Adult patients with acute non-chemotherapy-induced agranulocytosis, developed in hospital or in the outpatient setting, were ascertained by active surveillance in all 51 Berlin hospitals between the years 2000 and 2010. Applying the criteria of the World Health Organization, a standardized drug causality assessment was conducted for each agranulocytosis patient to determine possible drug aetiology. Drug risks were quantified in a case-control design with unconditional logistic regression analysis.Sixty-three out of 88 validated cases of agranulocytosis were identified as being at least probably drug-related. Drug causality assessment resulted in 36 pharmaceuticals with a certain or probable relationship to agranulocytosis. Drugs involved in ≥ 3 cases with a probable or certain causality were metamizole (dipyrone) (N = 10), clozapine (N = 6), sulfasalazine (N = 5), thiamazole (N = 5), and carbamazepine (N = 3). In case-control analysis, six drugs were identified with significant odds ratios for DIAG. The highest odds ratios were observed for clozapine, sulfasalazine, and thiamazole.Our findings are generally in agreement with those of earlier case-control studies. The spectrum of drugs causing acute agranulocytosis has not changed considerably over recent years, despite many newly marketed drugs. Evidence for induction of agranulocytosis by some new pharmaceuticals is supported.

To understand more specific abnormalities of humoral autoimmunity, we studied 31 spleens from immune thrombocytopenia (ITP) patients and 36 control spleens. Detailed analysis identified at least 2 different splenic structures accommodating proliferating B cells, classic germinal centers (GCs), and proliferative lymphoid nodules (PLNs). PLNs were characterized by proliferating Ki67+ B cells close to follicular dendritic cells (FDCs) and lacked polarization into dark and light zones. As opposed to cells in GCs, proliferating B cells in PLN lacked expression of Bcl6. In both PLNs and GCs of ITP spleens, the density of T cells was significantly reduced. Both T follicular helper cells (TFH) and regulatory T cells were reduced within PLNs of ITP spleens suggesting a defect of tolerance related to a loss of T-cell control. Within PLNs of ITP, but not controls, abundant platelet glycoprotein (GP) IIb/IIIa autoantigens was found in IgM containing immune complexes tightly bound to FDCs and closely approximated to proliferating B cells. GPIV was found less often, but not in the same PLNs as GPIIb/IIIa. Autoantigens were not found in the GCs of ITP or controls indicating that PLNs are the sites of autoantigen stimulation in ITP potentially related to a lack of control by T cells and/or the present autoantigen.

Patients treated with intravenous immunoglobulins (IVIG) rarely experience symptomatic hemolysis. Although anti-A and anti-B isoagglutinins from the product are involved in most cases, the actual mechanisms triggering hemolysis are unclear.A prospective, open-label, multicenter, single-arm clinical trial in 57 patients with immune thrombocytopenia treated with IVIG (Privigen, CSL Behring) was conducted.Twenty-one patients received one infusion (1 g/kg) and 36 received two infusions (2 × 1 g/kg) of IVIG. After a study duration of more than 2 years, no cases of clinically significant hemolysis as defined in the protocol were identified. Data of patients with mild hematologic and biochemical changes were analyzed in more detail. Twelve cases (10/23 patients with blood group A1 and 2/11 patients with blood group B, all having received 2 g/kg IVIG) were adjudicated as mild hemolysis (median hemoglobin [Hb] decrease, -3.0 g/dL); Hb decreases were transient, with partial or full recovery achieved by last visit. Eighteen patients (31.6%), all with non-O blood group, of whom 16 (88.9%) received 2 g/kg IVIG, fulfilled post hoc criteria for hemolytic laboratory reactions. Red blood cell (RBC) eluates of all direct antiglobulin test-positive samples were negative for non-ABO blood group antibodies. Blood groups A and B antigen density on RBCs appeared to be a risk factor for hemolytic laboratory reactions. Platelet response to treatment was observed in 42 patients (74%); eight of 12 patients with complete response had blood group A1.Isoagglutinins are involved in clinically nonsignificant hemolysis after treatment with IVIG, but individual susceptibility varies greatly.

There is evidence that the thrombopoietin-receptor agonists romiplostim and eltrombopag may have different therapeutic values and adverse reaction profiles. Here we present new data and provide a review of all studies dealing with switching between these two drugs.A total of 89 patients (38 males and 51 females, aged between 14-87 years) were treated with eltrombopag and/or romiplostim between 2007 and 2016 at our institution. Eltrombopag was switched to romiplostim or vice versa in 32 patients. In addition, all published data concerning patients treated sequentially with different thrombopoietin-receptor agonists were identified via a computer-assisted search and summarised in this article.Thirty-two of 89 patients treated with a thrombopoietin-receptor agonist in our institution were given both eltrombopag and romiplostim sequentially. Therapy was switched to the alternate thrombopoietin-receptor agonist 36 times, due to inefficacy (n=21), adverse reactions (n=14), and a patient's preference (n=1). In addition, data from 126 patients who have been treated with both agonists have been published by other groups. In total eltrombopag was replaced by romiplostim in 56 cases due to poor or no response or to adverse reactions. Forty-five patients responded to treatment with romiplostim, 11 patients shared cross-resistance and nine had adverse reactions to romiplostim. In contrast, romiplostim was replaced by eltrombopag in 106 cases. Seventy-eight patients responded to eltrombopag, 27 shared cross-resistance and 19 had adverse reactions to eltrombopag.Eltrombopag and romiplostim often share bidirectional cross-resistance and/or adverse reactions. Both drugs appear to cause more adverse reactions than have been previously reported.

The increased risk of subsequent primary malignancies (SPM) in survivors of adult-onset Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) remains a challenging clinical problem worldwide. The German cancer registry database, pooled from 14 federal states, was used to calculate the standardized incidence ratio (SIR) and excess absolute risk (EAR) of SPM in 128 587 patients registered with first primary HL/NHL between 1990 and 2012. Conversely, SIRs were also calculated for a subsequent HL/NHL following other first cancers. The risk of developing SPM was significantly increased over twofold for HL survivors (SIR = 2.14, EAR = 51.87 cases/10 000 person-years) and 1.5-fold for NHL survivors (SIR = 1.48, EAR = 55.23) compared with the general German population. For solid cancers, SIRs were significantly elevated (1.6- and 1.4-fold; respectively) and were highest (threefold) in patients below 30 years of age upon initial diagnosis. Overall, SIRs were consistently elevated for lip/oral cavity, colon/rectum, lung, skin melanoma, breast, kidney and thyroid. Significantly increased SIRs for oesophagus, stomach, liver, pancreas, testis, prostate, and brain/central nervous system were observed following NHL only. For certain SPM, SIRs remained significantly elevated more than 10 years following HL/NHL diagnosis. Positive reciprocal associations were demonstrated between HL/NHL and several solid cancers mentioned above; for some, common aetiological mechanisms seem plausible.

Background and Objectives The clinical significance of autoimmune thrombocytopenia (ITP) is reflected by bleeding and/or an increased bleeding risk due to low platelet counts. In most cases, treatment with high-dose (1–2 g/kg body weight) intravenous immunoglobulin has been demonstrated to result in an increase in platelet counts after one day of treatment. Until now, there is little information on the true beginning of therapy effect in patients treated with high-dose intravenous immunoglobulin. In this study, we focused on the kinetic of platelet counts and cessation of bleeding within the first 24 h of treatment. Materials and methods Nineteen patients with chronic ITP were treated with high-dose intravenous immunoglobulin due to clinically relevant bleeding and/or for increased bleeding risk. Results Although the response was variable in treated patients, cessation of bleeding was observed within 12 h in all such affected patients (n = 7), even in patients with platelet counts that were not increased (n = 3). Furthermore, platelet counts were observed to increase already within 1 h in 10 (53%) patients and within 8 h in 12 (63%) patients. Conclusions From a clinical perspective, the onset of therapeutic effects of intravenous immunoglobulin in patients with ITP may occur at an earlier stage and be superior to that previously expected. Failure to measure an increase in platelets in the circulation of ‘non-responders’ may be explained by an increased consumption of platelets due to recognizable or unrecognizable bleeding in such affected patients.

Routine quantification of platelet-derived extracellular vesicles (PL-EVs) may be useful in the quality control (QC) of platelet concentrates (PCs). The aim of this multicenter study was to establish and validate a consensus protocol for the standardized PL-EV quantification using conventional flow cytometers.Eighty-six PCs were investigated in five blood transfusion centers (A-E) on Days 0 and 5. The centers used different apheresis instruments: Trima Accel (n = 56) and/or Amicus (n = 30). PCs were prepared using standard methods (sd-PCs; n = 73; A-D) or with pathogen inactivation (PI [PI-PCs]; n = 13; E). Platelet (PLT) count was determined using conventional hematology analyzers. PLT degranulation (P-selectin expression in response to thrombin receptor PAR1 activation) and PL-EVs were analyzed by flow cytometry.During storage, PLT count remained stable in 58 PCs (A, C, E), whereas a decrease was observed in 12 PCs (B). PLT degranulation declined in all PCs (p0.001) and PL-EVs increased in 74 PCs (A, C-E; p0.001). Certain donor variables (e.g., plasma cholesterol, immature PLT fraction) were associated with lower PL-EVs. In Trima-produced PCs, PL-EVs were significantly lower (D) and PLT degranulation was superior compared to PCs prepared with the Amicus (A, D). PL-EVs were 10-fold lower in PI-PCs, compared to sd-PCs. However, similar QC trends were demonstrated for both PC groups during storage.PL-EV analysis in a QC program of PCs was successfully performed with results comparable among the different centers. PLT degranulation and vesiculation were primarily affected by preparation techniques.