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Evidence links osteoporosis and cardiovascular disease but the cellular and molecular mechanisms are unclear. Here we identify skeleton-secreted platelet-derived growth factor-BB (PDGF-BB) as a key mediator of arterial stiffening in response to aging and metabolic stress. Aged mice and those fed high-fat diet (HFD), relative to young mice and those fed normal chow food diet, respectively, had higher serum PDGF-BB and developed bone loss and arterial stiffening. Bone/bone marrow preosteoclasts in aged mice and HFD mice secrete an excessive amount of PDGF-BB, contributing to the elevated PDGF-BB in blood circulation. Conditioned medium prepared from preosteoclasts stimulated proliferation and migration of the vascular smooth muscle cells. Conditional transgenic mice, in which PDGF-BB is overexpressed in preosteoclasts, had 3-fold higher serum PDGF-BB concentration and developed simultaneous bone loss and arterial stiffening spontaneously at a young age. Conversely, in conditional knockout mice, in which PDGF-BB is deleted selectively in preosteoclasts, HFD did not affect serum PDGF-BB concentration; as a result, HFD-induced bone loss and arterial stiffening were attenuated. These studies confirm that preosteoclasts are a main source of excessive PDGF-BB in blood circulation during aging and metabolic stress and establish the role of skeleton-derived PDGF-BB as an important mediator of vascular stiffening.

Tissue transglutaminase (TG2), a multifunctional protein of the transglutaminase family, has putative transamidation-independent functions in aging-associated vascular stiffening and dysfunction. Developing preclinical models will be critical to fully understand the physiologic relevance of TG2’s transamidation-independent activity and to identify the specific function of TG2 for therapeutic targeting. Therefore, in this study, we harnessed CRISPR-Cas9 gene editing technology to introduce a mutation at cysteine 277 in the active site of the mouse Tgm2 gene. Heterozygous and homozygous Tgm2-C277S mice were phenotypically normal and were born at the expected Mendelian frequency. TG2 protein was ubiquitously expressed in the Tgm2-C277S mice at levels similar to those of wild-type (WT) mice. In the Tgm2-C277S mice, TG2 transglutaminase function was successfully obliterated, but the transamidation-independent functions ascribed to GTP, fibronectin, and integrin binding were preserved. In vitro, a remodeling stimulus led to the significant loss of vascular compliance in WT mice, but not in the Tgm2-C277S or TG2−/− mice. Vascular stiffness increased with age in WT mice, as measured by pulse-wave velocity and tensile testing. Tgm2-C277S mice were protected from age-associated vascular stiffening, and TG2 knockout yielded further protection. Together, these studies show that TG2 contributes significantly to overall vascular modulus and vasoreactivity independent of its transamidation function, but that transamidation activity is a significant cause of vascular matrix stiffening during aging. Finally, the Tgm2-C277S mice can be used for in vivo studies to explore the transamidation-independent roles of TG2 in physiology and pathophysiology.

Rurioctocog alfa pegol prophylaxis targeting factor VIII (FVIII) troughs ≥1% has shown to be efficacious with an acceptable safety profile in people with hemophilia A (PwHA). The PROPEL trial compared safety and efficacy of 2 target FVIII troughs in PwHA aged 12 to 65 years, with severe disease, annualized bleeding rate ≥2, and previous FVIII treatment. PwHA were randomized to 12 months’ pharmacokinetic (PK)-guided rurioctocog alfa pegol prophylaxis targeting FVIII troughs of 1% to 3% (reference arm) or 8% to 12% (elevated arm); first 6 months was treatment-adjustment period. The primary endpoint was absence of bleeds during the second 6 months, analyzed using multiple imputations (full analysis set [FAS]). In the 1% to 3% and 8% to 12% arms, respectively, point estimates (95% confidence interval) of proportions of PwHA with zero total bleeds were 42% (29% to 55%) and 62% (49% to 75%) in FAS (N = 115; P = .055) and 40% (27% to 55%) and 67% (52% to 81%) in per-protocol analysis set (N = 95; P = .015). Dosing frequency and consumption varied in each arm. Adverse events (AEs) occurred in 70/115 (60.9%) PwHA; serious AEs in 7/115 (6%) PwHA, including 1 treatment-related in 8% to 12% arm (transient anti–FVIII inhibitor). There were no deaths, serious thrombotic events, or AE-related discontinuations. PK-guided prophylaxis was achievable and efficacious in both arms. No new safety signals were observed in the 8% to 12% arm. These results demonstrate elevated FVIII troughs can increase the proportion of PwHA with zero bleeds and emphasize the importance of personalized treatment. This trial was registered at www.clinicaltrials.gov as #NCT02585960.

BACKGROUND Acoustic cell separation uses ultrasound waves to separate cells from plasma to perform plasmapheresis. Although the fundamental process has been studied for decades, no acoustic cell separation has been studied in a disposable plastic format suitable for clinical applications. STUDY DESIGN AND METHODS We developed a disposable, rectangular, polystyrene microchannel acoustic cell-separation system and applied acoustic energy relevant for plasmapheresis. Fresh blood from healthy volunteers was exposed in vitro to acoustic energy in an open microfluidic circuit with and without ultrasound applied. Blood was tested for perturbations in red blood cells, platelets, and coagulation using clinical assays. RESULTS Red blood cell and platelet size parameters as well as coagulation activation all were within 3% of baseline values. P-selectin expression on platelets increased by an average of 10.9% relative to baseline. Hemolysis increased with flow through the microfluidic channel, but percentage hemolysis remained below 0.3%. CONCLUSION Blood parameters in a single-pass, microfluidic acoustic cell-separation apparatus remained within normal limits. In vivo animal studies that model continuous separation in a physiologic environment are warranted.

Background Babesiosis, a tickborne zoonotic disease caused by intraerythrocytic protozoa of the genus babesia, is characterized by nonimmune hemolytic anemia that resolves with antimicrobial treatment and clearance of parasitemia. The development of warm-antibody autoimmune hemolytic anemia (also known as warm autoimmune hemolytic anemia [WAHA]) in patients with babesiosis has not previously been well described. Methods After the observation of sporadic cases of WAHA that occurred after treatment of patients for babesiosis, we conducted a retrospective cohort study of all the patients with babesiosis who were cared for at our center from January 2009 through June 2016. Data on covariates of interest were extracted from the medical records, including any hematologic complications that occurred within 3 months after the diagnosis and treatment of babesiosis. Results A total of 86 patients received a diagnosis of babesiosis during the 7.5-year study period; 18 of these patients were asplenic. WAHA developed in 6 patients 2 to 4 weeks after the diagnosis of babesiosis, by which time all the patients had had clinical and laboratory responses to antimicrobial treatment of babesiosis, including clearance of Babesia microti parasitemia. All 6 patients were asplenic (P

DISC-0974 is a humanized monoclonal anti-HJV antibody with high binding affinity for human, rat and cynomolgus monkey HJV (100 pM, 240 pM and 110 pM Kd, respectively) [Kovac 2016]. DISC-0974 inhibits the interaction between HJV and the key hepcidin regulatory ligands, bone morphogenetic proteins (BMPs), leading to decreased SMAD phosphorylation (SMAD-P) and reduced hepcidin expression [Kovak 2016]. Elevated hepcidin is a hallmark of and plays a critical role in the pathogenesis of anemia of inflammation (and other forms of anemia) by reducing transferrin saturation and erythroid iron availability, leading to failure of erythropoiesis and decreased hemoglobin synthesis. DISC-0974 is being developed to reduce hepcidin synthesis and treat anemia in these patients. A critical early signal of therapeutic effect is the effect of DISC-0974 on transferrin saturation (TSAT). TSAT has therefore been used as the primary pharmacodynamic marker in a series of non-clinical studies to determine the relationship between drug exposure and pharmacological activity. In non-human primates (NHP) DISC-0974 elicits a dose-dependent reduction in hepcidin levels leading to increases in serum iron and consequently TSAT. PK/PD responses were examined over 60 days for a single 6 mg/kg intravenous dose of DISC-0974 in three male NHPs and in repeat dose studies occurring at 14 day intervals administered 3 times (vehicle, 0.6 and 3 mg/kg) or two times (60 mg/kg). In these studies, the serum iron response saturated in an exposure-dependent manner over the 0.6-60 mg/kg dose range. The onset of iron elevation lagged compared to the DISC-0974 maximal plasma concentrations by 1- 4 days post dose and persisted beyond what could be predicted by DISC-0974 plasma concentration alone. The observed PD responses were well-described by a delayed response PK/PD model of serum iron which was fit globally to all studies. We will present single and multiple dose simulations showing that low monthly doses of DISC-0974 are sufficient to elicit a biologically significant and controlled PD modulation (serum iron, TSAT, hepcidin) over a sustained period-of-time (Figure 1). DISC-0974 is under development for the treatment of anemia of inflammation associated with high hepcidin. Disclosures Blaustein: Disc Medicine:Current Employment, Current equity holder in private company.King:Disc Medicine:Current Employment, Current equity holder in private company.Nguyen:Disc Medicine:Current Employment, Current equity holder in private company.Savage:Disc Medicine:Current Employment, Current equity holder in private company.Beconi:Disc Medicine:Current Employment, Current equity holder in private company.

Background . The majority of hematopoietic stem cell (HSC) transplants are performed using peripheral blood mobilized with granulocyte-colony stimulating factor (G-CSF) given over 5 days. The goal of a successful transplant is to reliably mobilize optimal numbers of HSCs necessary for rapid and consistent multilineage engraftment. Infusion of mobilized allogeneic grafts results in significant acute and chronic graft-versus-host disease (GvHD) in up to 80% of allogeneic transplant recipients. A reliable and rapid method to mobilize HSC-rich grafts with reduced GvHD potential would be clinically meaningful. In a Phase 1 study of normal volunteers, MGTA-145 (GroβT), a CXCR2 agonist, when combined with plerixafor, a CXCR4 inhibitor, robustly and rapidly mobilized sufficient HSCs for a safe transplant after only a single day of dosing and apheresis/collection. Here, we phenotypically and functionally profile these mobilized grafts obtained from human volunteers and show that MGTA-145 + plerixafor mobilizes grafts with >10-fold higher engraftment potential (as measured by SCID-repopulating units in NSG mice), a marked reduction in xenogeneic GvHD, and enhanced overall survival compared to G-CSF or plerixafor alone grafts. Results . In healthy donors, a peak of 40 CD34+ cells/μL were mobilized with MGTA-145 + plerixafor (n=12 donors). 11 of 12 (92%) of these donors mobilized >20 CD34+ cells/μL with single day dosing compared to only 8 of 14 (57%) achieving the same CD34+ cell target treated with plerixafor alone. Eight donors were mobilized with a single dose of MGTA-145 + plerixafor and apheresed on the same day. A median of 4x106 (1.5-7.0x106) CD34+ cells/kg were obtained (n=8 donors) from a median 20 (13-20) L collection. 35.8 (18.5-40.9)% of these cells were CD90+CD45RA-, a CD34+ subset enriched for HSCs, compared to only 6.9 (5.3-9.0)% with G-CSF (p To assess engraftment, we transplanted mobilized peripheral blood cells from healthy donors after a 5-day regimen of G-CSF or a single dose of plerixafor alone or MGTA-145 + plerixafor at limit dilution into sublethally irradiated primary and secondary NSG mouse recipients (n=3 cell doses, n=7-8 mice/group). Multilineage human engraftment was measured by flow cytometry 16 weeks post-transplant and SCID-repopulating cell (SRC) number was calculated (Figure 1A). MGTA-145 + plerixafor mobilized grafts (n=4 donors) led to a 23-fold increase in engraftment compared to G-CSF mobilized grafts (p Immune cell subsets (B, T, and NK cells and cell subsets) mobilized by MGTA-145 + plerixafor were similar to those mobilized by plerixafor alone. While CD3+ T-cell numbers were comparable between MGTA-145 + plerixafor and plerixafor alone, MGTA-145 + plerixafor mobilized 0.2 (0.0-0.6) x108/kg CD8+ T-cells, constituting 1.8 (0.5-4.8)% of the graft, a number and proportion significantly lower than that mobilized by either G-CSF or plerixafor alone. To determine the effect of the mobilization regimen on xenogeneic GvHD, we developed a xenograft GvHD model in NSG mice where 6x106 PBMCs from various graft sources were infused into sublethally-irradiated animals (n=3-6 donors per graft source). Notably, MGTA-145 + plerixafor mobilized grafts resulted in significantly less GvHD than G-CSF (p Conclusions . These data demonstrate that MGTA-145 + plerixafor is a rapid, reliable, and G-CSF free method to obtain high numbers of HSCs with durable engraftment potential and a graft with highly immunosuppressive properties. These data suggest that MGTA-145 + plerixafor is an effective single-day mobilization/collection regimen for both autologous and allogeneic stem cell transplantation resulting in enhanced engraftment and reduced GvHD in this xenograft model. Disclosures Goncalves: Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Hyzy:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hammond:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Falahee:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Howell:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Pinkas:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Schmelmer:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hoggatt:Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company. Scadden:Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Devine:Magenta Therapeutics: Consultancy. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Savage:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Davis:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Hepcidin is known as the master regulator of systemic iron homeostasis with reduction in synthesis leading to the development of iron overload. Hepcidin gene expression is negatively modulated by matriptase-2 (MT-2), a liver-specific type II transmembrane serine protease. MT-2 cleaves hemojuvelin (HJV), leading to the extracellular release of soluble HJV fragments and suppression of hepcidin expression. Loss-of-function of MT-2 leads to increased hepcidin expression, as has been established by human genetics (Finberg et al., 2008) and genetic mouse models (Du et al., 2008). Therefore, inhibition of MT-2 represents a potential therapeutic strategy for diseases caused by inappropriately low hepcidin leading to iron overload or where therapeutic iron restriction may be used to control excessive erythrocytosis. Here we describe the characteristics of DISC-A, a potent (low nM Ki) small molecule MT-2 inhibitor for treatment of low hepcidin disorders, with a favorable pharmacokinetics profile in rats (Clp 6.4 ml/min/kg, and t ½ 4.6 hr) and monkeys (Clp 8.1 ml/min/kg, and t ½ 2.8 hr) and drug-like properties. DISC-A inhibits proteolytic activity of MT-2 expressed on the surface of transfected HEK293 cells and prevents shedding of MT-2 from the membrane (autocleavage). In addition, in MT-2 and HJV co-transfected HEK293A cells, DISC-A shows a dose dependent inhibition of HJV cleavage. The efficacy of DISC-A was evaluated in a rat model of low hepcidin. In this model, when Sprague-Dawley rats who are fed a standard iron diet (45 ppm) reach 8 - 9 weeks of age, they are administered erythropoietin (EPO) at 30 IU/animal/day for 4-consecutive days, before dosing with DISC-A. Under the conditions of the model, the increased erythropoiesis leads to increased iron utilization and consequently suppressed hepcidin levels. We determine hepcidin changes by measuring the changes in the expression of liver HAMP (the gene that encodes hepcidin) mRNA expression. Circulating soluble HJV is assayed as a direct measure of MT-2 activity. In this model, a single subcutaneous administration of DISC-A at 20 mg/kg resulted in a 50% reduction in soluble HJV, 14-fold increase in liver HAMP expression and >50% reduction in serum iron and transferrin saturation (TSAT) at 2, 4, 6, and 8 hours. The pharmacokinetics/pharmacodynamics response was robust. In summary, we have identified DISC-A, a novel, potent inhibitor of MT-2. We have demonstrated that DISC-A inhibits MT-2 proteolytic activity, prevents cleavage of HJV, and modulates hepcidin gene expression and iron homeostasis in vitro and in vivo. The favorable pharmacokinetics suggest compounds from these chemical series have the potential for clinical therapeutic benefit. Disclosures Hong: Disc Medicine: Current Employment, Current equity holder in private company. Babu:Aurigene Discovery Technologies: Current Employment. Blaustein:Disc Medicine: Current Employment, Current equity holder in private company. Nguyen:Disc Medicine: Current Employment, Current equity holder in private company. Rao:Aurigene Discovery Technologies: Current Employment. Savage:Disc Medicine: Current Employment, Current equity holder in private company. MacDonald:Disc Medicine: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Beconi:Disc Medicine: Current Employment, Current equity holder in private company. Venkatraman:Disc Medicine: Current Employment, Current equity holder in private company.

Background:Autologous haematopoietic stem cell (HSC) transplantation is a recommended therapeutic option for selected patients with autoimmune diseases. G-CSF mobilisation of HSCs requires 4-7 days of injections that are associated with significant side effects and potential for severe complications including disease flares (e.g., scleroderma and multiple sclerosis). MGTA-145 is a biologic that activates CXCR2 on neutrophils, and with plerixafor rapidly mobilises HSCs in mice and non-human primates. The combination promises to be a same-day, G-CSF-free mobilisation regimen.Objectives:To evaluate the safety, tolerability, and mobilisation efficacy of MGTA-145 monotherapy and combination therapy with plerixafor in healthy volunteers.Methods:This healthy volunteer phase 1 study consisted of 4 parts- Part A: single-agent MGTA-145 or placebo; Part B: MGTA-145 or placebo given immediately or 2 hours after plerixafor; Part C: MGTA-145 or placebo given 2 hours after plerixafor on 2 consecutive days; Part D: MGTA-145 given 2 hours after plerixafor, just prior to apheresis cell collection.Results:Monotherapy of MGTA-145 mobilised CD34+ cells within minutes and peaked within 1 hour post MGTA-145 (median 11 CD34+ cells/µL, a 7-fold increase vs baseline). White blood cells and neutrophils followed a similar pattern. Importantly, markers of neutrophil activation were relatively unchanged (≤2-fold vs baseline).MGTA-145 combined with plerixafor increased CD34+ cell mobilisation, whether given simultaneously or 2h after plerixafor (Fig. 1A). Mobilisation was highly enriched for CD34+CD90+CD45RA- HSCs, which tracked closely with the total CD34 count. At the 0.03 mg/kg dose with 2h stagger, median peak CD34+ peripheral blood mobilisation was ≥40 cells/µL in Part B. On a second consecutive day of dosing, MGTA-145 + plerixafor mobilises HSCs to levels comparable to day 1. Initial data from the ongoing Part D show that sufficient numbers of cells (median 4.3 x 10^6 CD34+ cells/kg) for transplant were collected in a single day.. Preliminary data from NSG mouse transplant studies of those mobilised HSCs in part D show higher engraftment rates of MGTA-145 + plerixafor mobilised HSCs, compared to G-CSF-mobilised HSCs.Figure.Peripheral blood mobilisation after plerixafor + 0.03 mg/kg MGTA-145 in healthy subjects with simultaneous and 2h stagger dosing after plerixafor. Dotted line: previously reported CD34+ counts with plerixafor alone mobilisation (Chenet al,Blood Advances. 2018).MGTA-145 monotherapy was well tolerated with no significant adverse events (AEs). Grade 1, transient lower back pain that dissipated within minutes was reported. The combination of MGTA-145 with plerixafor was well tolerated, with some subjects experiencing grade 1/2 gastrointestinal AEs commonly observed with plerixafor and one grade 2 back pain with MGTA-145 at 0.075 mg/kg that resolved within minutes.Conclusion:MGTA-145 monotherapy was well-tolerated and induced rapid mobilisation of significant numbers of HSCs. CD34+ cell mobilisation with MGTA-145 + plerixafor was immediate and superior to plerixafor alone. These data suggest that the combination can enable the collection of sufficient HSCs for transplant in one day without the need for G-CSF. Further development as a first line mobilisation product is warranted in autoimmune diseases, gene therapy and haematologic malignancies.Table.Single-day Mobilisation and Apheresis Cell Yields in Part DSubjectTotal CD34+ Yield (x106 cells)CD34+/kg (x106 cells)CD90+ (%)8013194.139%8073224.441%8175005.326%821 (*completed only 13L of planned 20L collection)2392.719%Median3214.333%Disclosure of Interests:John Dipersio Shareholder of: Magenta, Consultant of: Cellworks, Tioma, Rivervest, Bioline, Asterias, Amphivena and Bluebird, Celgene, Incyte, NeoImuneTech, Macrogenics, Steven Devine: None declared, Jonathan Hoggatt Shareholder of: Magenta, Grant/research support from: Magenta, Consultant of: Magenta, David Scadden Shareholder of: Magenta, Consultant of: Magenta, Haley Howell Shareholder of: Magenta, Employee of: Magenta, Veit Schmelmer Shareholder of: Magenta, Employee of: Magenta, Jason Neale Shareholder of: Magenta, Employee of: Magenta, Tony Boitano Shareholder of: Magenta, Employee of: Magenta, Michael Cooke Shareholder of: Magenta, Employee of: Magenta, Dwight Morrow Shareholder of: Magenta, Employee of: Magenta, Glen Raffel Shareholder of: Magenta, Employee of: Magenta, Will Savage Shareholder of: Magenta, Employee of: Magenta, Kevin Goncalves Shareholder of: Magenta, Employee of: Magenta, Pat Falahee Shareholder of: Magenta, Employee of: Magenta, John Davis Shareholder of: Magenta, Employee of: Magenta

Chronic graft-versus-host disease (cGVHD) affects >50% of hematopoietic stem cell transplant patients. Extracorporeal photopheresis (ECP), an immunomodulatory therapy, provides clinical benefit in steroid-refractory (SR) cGVHD, possibly via regulatory T (Treg) and natural killer (NK) cell expansion. We demonstrated that low-dose interleukin-2 (IL2) led to clinical improvement in SR-cGVHD and stimulated preferential Treg and NK-cell expansion with minimal effect on conventional T (Tcon) cells. We evaluated the effect of ECP (weeks 1-16) plus IL2 (1 × 106 IU/m2, weeks 9-16) in 25 adult patients with SR-cGVHD in a prospective phase 2 trial. Objective responses occurred in 29% and 62% of evaluable patients at weeks 8 (ECP alone) and 16 (ECP plus IL2), respectively. Eight weeks of ECP alone was associated with a marked decline in CD4+ Tcon (P = .03) and CD8+ T cells (P = .0002), with minimal change in Treg cells, Treg:Tcon cell ratio, or NK cells. Adding IL2 induced an increase in Treg cells (P < .05 at weeks 9-16 vs week 8), Treg:Tcon cell ratio (P < .0001 at weeks 9-16 vs week 8), and NK cells (P < .05 at weeks 9-16 vs week 8). Patients responding to ECP alone had significantly fewer CD4+ Tcon and CD8+ T cells at baseline compared with patients who responded after IL2 addition and patients who did not respond; neither Treg nor NK cells were associated with response to ECP alone. Altogether, ECP plus IL2 is safe and effective in patients with SR-cGVHD. ECP and IL2 have distinct immunologic effects, suggesting different therapeutic mechanisms of action. This trial was registered at www.clinicaltrials.gov as #NCT02340676.

Allergic transfusion reactions are the most common adverse reaction to blood products, particularly platelets and plasma. Reactions most commonly are mild, occur within 2 hours of transfusion, and manifest as localized urticaria and pruritus; however, more severe reactions and anaphylaxis can occur. Selective protein deficiencies, e.g. IgA deficiency, underlie the pathophysiology of some severe allergic reactions, but in most cases the cause is unknown. Plasma reduction of cellular blood products decreases the incidence of allergic reactions. Diphenhydramine, the most commonly used premedication for allergic transfusion reactions, likely does not prevent allergic transfusion reactions from occurring but does alleviate symptoms once they occur.

In diseases characterized by chronic inflammation, hepcidin levels are often elevated causing reduced transferrin-bound iron, reduced erythroid iron availability, diminished erythropoiesis and anemia despite the presence of adequate iron stores. Hemojuvelin (HJV) is a key selective regulator of hepcidin production in humans; patients with homozygous loss-of-function in the gene coding for HJV exhibit dramatically reduced hepcidin levels and a severe iron overload syndrome, juvenile hemochromatosis Type 2A. DISC-0974 is a humanized monoclonal anti-HJV antibody with high binding affinity for human, rat and cynomolgus monkey HJV (100 pM, 240 pM and 110 pM Kd, respectively) [Kovac 2016]. DISC-0974 inhibits the interaction between HJV and the key hepcidin regulatory ligands, bone morphogenetic proteins (BMPs), leading to decreased SMAD phosphorylation (SMAD-P) and reduced hepcidin expression [Kovak 2016]. The multiple-dose pharmacokinetics and pharmacodynamics (PK/PD) of DISC-0974 was first evaluated in healthy NHPs in which regulation of iron metabolism is primarily controlled by BMP/SMAD regulation of hepcidin synthesis. Dose levels were selected to modulate the pharmacodynamic response (transferrin saturation [TSAT]) but to be either below saturation (0.6 mg/kg), or to saturate the TSAT response (3 and 60 mg/kg). As expected from the mechanism of action, hepcidin concentrations decreased following DISC-0974 administration with return to baseline that was exposure dependent. The decrease in hepcidin was associated with increased TSAT, which DISC-0974 modulated in a dose-dependent manner. At 0.6 mg/kg, the TSAT response was maintained below saturation (~65%) after the first dose; saturation increased following subsequent doses, consistent with higher DISC-0974 serum concentrations. At the 3.0 and 60 mg/kg doses TSAT saturation (>85%) was achieved within 5 days post first dose and remained elevated through the end of the study, also consistent with the serum concentrations of DISC-0974. In vivo efficacy of DISC-0974 was evaluated in an NHP model of inflammation-induced (IL-6) high-hepcidin to establish whether inhibition of BMP/SMAD signaling could effectively control hepcidin increases generated by inflammatory signals. In this study, monkeys were challenged with 6×104 IU IL-6/kg on Day 1 and on Day 4. Groups of N=3 received 0 (vehicle), 0.6 or 6 mg/kg DISC-0974. On Day 11 all groups received a second challenge of 6×104 IU IL-6/kg. TSAT, hepcidin-25 and DISC-0974 concentrations were determined. DISC-0974 was effective in preventing IL-6-induced hepcidin increase in a dose-dependent manner in this model (Figure 1). In summary, in healthy NHPs, administration of DISC-0974 causes reduction in hepcidin levels leading to release of stored iron from macrophages of the reticuloendothelial system, making iron available for erythropoiesis as demonstrated by the dose-dependent modulation of TSAT. DISC-0974 is also effective in preventing hepcidin increases caused by IL-6, which is generally regarded at the classical cytokine stimulator of hepcidin synthesis. Given the similarity of the mechanism of hepcidin regulation between NHPs and humans, DISC-0974 is anticipated to provide a similar effect in the treatment of anemia of inflammation in humans by lowering cytokine-induced increases in hepcidin and improving iron availability for erythropoiesis. Figure Disclosures MacDonald: Disc Medicine: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Blaustein:Disc Medicine: Current Employment, Current equity holder in private company. Nguyen:Disc Medicine: Current Employment, Current equity holder in private company. King:Disc Medicine: Current Employment, Current equity holder in private company. Hong:Disc Medicine: Current Employment, Current equity holder in private company. Savage:Disc Medicine: Current Employment, Current equity holder in private company. Venkatraman:Disc Medicine: Current Employment, Current equity holder in private company. Beconi:Disc Medicine: Current Employment, Current equity holder in private company.

ABO incompatibility of red blood cells leads to brisk complement-mediated lysis, particularly in the setting of red cell transfusion. The ABO blood group is the most clinically significant blood group because of preformed immunoglobulin M (IgM) and IgG antibodies to ABO blood group antigens (isohemagglutinins) in everyone except group AB individuals. In addition to transfusion, ABO incompatibility can cause hemolysis in hematopoietic and solid organ transplantation, hemolytic disease of the newborn, and intravenous immunoglobulin infusion. It is important to prevent ABO incompatibility when possible and to anticipate complications when ABO incompatibility is unavoidable.

BACKGROUND: Red blood cell (RBC) transfusions remain essential in the treatment of patients with sickle cell disease (SCD) and -thalassemia. Alloimmunization, a well-documented complication of transfusion, increases the risk of delayed hemolytic transfusion reactions, complicates crossmatching and identifying compatible units, and delays provision of transfusions. Guidance is required to optimize the RBC product administered to these patients. STUDY DESIGN AND METHODS: An international, multidisciplinary team conducted a systematic review and developed, following the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) methodology, recommendations to assist treating physicians and transfusion specialists in their decision to select RBCs for these patients. RESULTS: Eighteen studies (17 clinical studies and one cost-effectiveness study) were included in the systematic review. The overall quality of the studies was very low. In total, 3696 patients were included: 1680 with -thalassemia and 2016 with SCD. CONCLUSION: The panel recommends that ABO D CcEe K-matched RBCs are selected for individuals with SCD and -thalassemia, even in the absence of alloantibodies, to reduce the risk of alloimmunization. In patients with SCD and -thalassemia who have developed clinically significant alloantibodies, selection of RBCs antigen negative to the alloantibody is recommended, if feasible. In these patients, selection of more extended phenotype-matched RBCs will likely reduce the risk of further alloimmunization. However, given the limited availability of extended phenotype-matched units, attention should be given to ensure that a delay in transfusion does not adversely affect patient care.

Background Transfusion-related characteristics have been hypothesized to cause allergic transfusion reactions (ATRs) but they have not been thoroughly studied. The primary objective of this study is to evaluate the associations of infusion rate, infusion volume, ABO mismatching, component age, and pretransfusion medication with the incidence and severity of ATRs. A secondary objective is to compare the risk of these attributes relative to the previously reported risk factor for aeroallergen sensitization in transfusion recipients, as measured by an aeroallergen-specific immunoglobulin (Ig)E antibody screen. Study Design and Methods Clinical and transfusion-related data were collected on subjects with reported ATRs and uneventful (control) apheresis platelet (PLT) transfusions over a combined 21-month period at two academic medical centers. Control transfusions were selected as the next uneventful transfusion after an ATR was reported. Logistic regression, Mann-Whitney, and t tests were used to assess associations with ATRs. Previously reported aeroallergen-specific IgE screening data were incorporated into a multivariable logistic regression. Results A total of 143 ATRs and 61 control transfusions were evaluated among 168 subjects, ages 2 to 86 years. Infusion rate, infusion volume, ABO mismatching, component age, and pretransfusion medication showed no significant association with ATRs (p > 0.05). Neither infusion rate nor infusion volume increased the risk of anaphylaxis versus mucocutaneous-only ATRs. Aeroallergen sensitization has previously been associated with ATRs. After transfusion-related covariates were controlled for, aeroallergen sensitization remained significantly associated with ATRs (odds ratio, 2.68; 95% confidence interval, 1.26-5.69). Conclusions Transfusion- and component-specific attributes are not associated with ATRs. An allergic predisposition in transfusion recipients is associated most strongly with ATR risk.

Background Thirty percent of chronically transfused patients with sickle cell disease (SCD) become alloimmunized. Antigen-matching reduces alloimmunization. Matching may be prospective (for all patients) or based on history (only for patients with an alloimmunization history). We assessed the clinical and financial value of a potential assay to identify at-risk patients to guide antigen-matching.

Background The decision to transfuse red blood cells in surgical patients should be based on multiple clinical variables, rather than on isolated hemoglobin (Hb) measurements alone. An important but often unrecognized clinical variable is the postoperative downward drift in Hb concentration (Hb drift), but the etiology, predictors, and time course of Hb drift are not well understood. Study Design and Methods Data were retrospectively collected for patients who did not receive postoperative transfusion. Initially, 11 common surgical procedures from one institution (n = 3179) were compared to assess clinical predictors of Hb drift. Data were analyzed in detail for two procedures associated with a large Hb drift (Whipple [n = 82] and lumbar spinal fusion [n = 74]), to determine the clinical predictors and temporal pattern of postoperative Hb drift. Results Surgical procedures with greater intraoperative intravenous (IV) fluid and blood requirements had greater postoperative Hb drift. The maximum Hb drifts after the Whipple and spinal fusion procedures were −2.5 ± 1.1 g/dL (occurring on Day 4, p

Background: The maximum surgical blood order schedule (MSBOS) is used to determine preoperative blood orders for specific surgical procedures. Because the list was developed in the late 1970s, many new surgical procedures have been introduced and others improved upon, making the original MSBOS obsolete. The authors describe methods to create an updated, institution-specific MSBOS to guide preoperative blood ordering. Methods: Blood utilization data for 53,526 patients undergoing 1,632 different surgical procedures were gathered from an anesthesia information management system. A novel algorithm based on previously defined criteria was used to create an MSBOS for each surgical specialty. The economic implications were calculated based on the number of blood orders placed, but not indicated, according to the MSBOS. Results: Among 27,825 surgical cases that did not require preoperative blood orders as determined by the MSBOS, 9,099 (32.7%) had a type and screen, and 2,643 (9.5%) had a crossmatch ordered. Of 4,644 cases determined to require only a type and screen, 1,509 (32.5%) had a type and crossmatch ordered. By using the MSBOS to eliminate unnecessary blood orders, the authors calculated a potential reduction in hospital charges and actual costs of $211,448 and $43,135 per year, respectively, or $8.89 and $1.81 per surgical patient, respectively. Conclusions: An institution-specific MSBOS can be created, using blood utilization data extracted from an anesthesia information management system along with our proposed algorithm. Using these methods to optimize the process of preoperative blood ordering can potentially improve operating room efficiency, increase patient safety, and decrease costs.

Background Sickle cell disease is associated with extensive health care utilization; estimated lifetime costs exceed $460,000 per patient. Approximately 30% of chronically transfused sickle cell patients become alloimmunized to red blood cell antigens, but these patients cannot be identified a priori. Prospective antigen matching can prevent alloimmunization, but is costly and may not benefit most patients. Study design and methods A Markov-based model was constructed to compare the health and financial implications of four alternative antigen-matching strategies for chronically transfused sickle cell patients. The strategies varied by the group of patients receiving matched blood (all patients prophylactically or only patients with a history of alloimmunization [history-based]), and by the extent of antigen matching (limited to C, E, and K, or extended to 11 antigens). Direct medical costs and alloimmunization events were assessed over 10- and 20-year periods, for a hypothetical cohort of initially transfusion-naive patients and for a dynamic population. Results Within a hypothetical cohort of initially transfusion-naive patients, implementing prophylactic limited matching for all chronically transfused patients instead of history-based limited matching is expected to cost an additional $765.56 million over 10 years, but result in 2072 fewer alloimmunization events. Within the same cohort, implementing prospective extensive matching is expected to cost $1.86 billion more than history-based extensive matching, but result in 2424 fewer alloimmunization events. Averting a single alloimmunization event using prospective matching would cost $369,482 to $769,284. Among a dynamic population over 10 years, prospective limited matching is expected to cost $358.34 million more than history-based limited matching. Conclusions While prospective matching for all transfused patients would reduce alloimmunization, this strategy requires considerable expenditure.

Silent cerebral infarction (SCI) is a prevalent problem in sickle cell anaemia (SCA, defined herein as HbSS or HbSB0 thalassaemia); by age six years, 27% of children with SCA have had a SCI (Kwiatkowski et al, 2009) and by age 14 years, 37% (Bernaudin et al, 2011). Children with SCI are at greater risk for overt stroke, recurrent or progressive SCI, and impaired cognitive function than the general SCA population. SCI is presumed, but not proven to represent infarctive injury. The mechanism underlying SCI in SCA is not fully understood, but is probably multifactorial. Anaemia and relative high blood pressure, endothelial cell activation, hypercoagulopathy, alterations in vasomotor tone secondary to decreased nitric oxide bioavailabilty, reperfusion injury, intravascular haemolysis and the impaired haemodynamics and resultant increased blood flow to the brain that result from anaemia have been postulated to contribute to the pathogenesis of infarction in SCA patients (DeBaun et al, 2012; Kato et al, 2007). Thrombospondin 1 (THBS1, TSP1) and L-selectin have been implicated in sickle cell-related complications, including vaso-occlusive crisis and stroke, but their role in SCI has not been evaluated. We tested the hypothesis that children with SCA and SCI would have altered plasma THSB1 and L-selectin levels when compared to children with SCA and no SCI. We used commercially available immunoassays to measure plasma THSB1 and L-selectin levels in 116 children with SCA (n=65 with SCI, n=51 without SCI, one sample per patient). These steady state plasma samples were obtained from the Silent Infarct Transfusion (SIT) Trial Biologic Repository at the Johns Hopkins University. This was a convenience sample, selected from 524 available undiluted plasma samples from patients who were enrolled in the trial between February 2007 and May 2009 and had plasma samples that were processed per protocol (Casella et al, 2010). Baseline characteristics were similar between the SCI and non-SCI groups; no differences in mean Hb, white blood cell or platelet counts were observed. Children with SCI had higher median THSB1 values than the non-SCI controls (8.4 versus 6.2 μg/ml for SCI and non-SCI, respectively, P=0.03; Fig 1A). Compared to without SCI, 14% (n=9/65) of children with SCI had THSB1 values above the 95th percentile for non-SCI THSB1 values (Fig 1B). L-selectin was significantly higher in children with SCI when compared to non-SCI controls (median values of 1.46 versus 1.35 μg/ml for SCI and non-SCI controls, respectively, P=0.03; Fig 2A). Fifteen percent (10/65) of children with SCI had L-selectin values above the 95th percentile for non-SCI values (Fig 2B). Baseline oxygen saturation, a reported risk factor for a number of sickle-related complications, correlated inversely with THSB1 in children with SCA (r=-0.4, P

Background A necessary component of an effective blood management program is the accurate and comprehensive collection and analysis of blood utilization data. This study describes innovative methods for analyzing and presenting data for red blood cell (RBC) utilization that compare hemoglobin (Hb) transfusion triggers and targets to those representing the restrictive transfusion strategy advocated by previous large outcome studies. Study Design and Methods From one institution, blood utilization data for 134,456 patients, 23,559 of whom were transfused with RBCs, were analyzed. Hb triggers and targets for transfused patients were plotted and graphically compared to the trigger and target ranges from previously published randomized clinical trials. Results Nine hospital services with the highest transfusion rates were selected for analysis. The service with the highest Hb trigger and target was further analyzed by comparing transfusion thresholds for individual providers. Differences among services and among individual providers for mean Hb transfusion triggers and targets were significant (up to 1.5 g/dL, p

Background Allergic transfusion reactions (ATRs) are among the most common complications of transfusion. Storage in platelet additive solution (PAS) has been shown to reduce ATRs from apheresis platelets (APs). This study evaluated the cost-effectiveness of using PAS storage as an alternative method to reduce ATRs. Study Design and Methods A Markov-based decision tree was constructed to compare ATR rates and associated costs expected from current practice and from alternative strategies of using APs stored in PAS. The potential use of pretransfusion medication was also incorporated. Using a hospital perspective and including direct medical expenses only (US$2012), Monte Carlo microsimulations were run to evaluate outcomes under a base-case analysis. One-way and probabilistic sensitivity analyses were used to assess outcome uncertainty. Results Under base-case variables, using APs stored in PAS for all patients as an initial transfusion protocol is expected to avert ATRs and associated costs, compared to current practice. Using PAS for all patients along with pretransfusion medication would be cost-saving only when the additional cost of PAS is below $9.14. If PAS storage could eliminate pretransfusion medication use, it is expected to result in cost savings when the additional unit cost of PAS is under $11.90. At a PAS cost of $15, averting one ATR would cost $701.95. Using PAS storage only in response to recurring mild ATRs is associated with cost savings under all costs of PAS evaluated. Conclusions Using PAS storage for all AP transfusions to prevent ATRs may be financially and clinically beneficial, compared to current practice.

Acoustic cell separation uses ultrasound waves to separate cells from plasma to perform plasmapheresis. Although the fundamental process has been studied for decades, no acoustic cell separation has been studied in a disposable plastic format suitable for clinical applications.We developed a disposable, rectangular, polystyrene microchannel acoustic cell-separation system and applied acoustic energy relevant for plasmapheresis. Fresh blood from healthy volunteers was exposed in vitro to acoustic energy in an open microfluidic circuit with and without ultrasound applied. Blood was tested for perturbations in red blood cells, platelets, and coagulation using clinical assays.Red blood cell and platelet size parameters as well as coagulation activation all were within 3% of baseline values. P-selectin expression on platelets increased by an average of 10.9% relative to baseline. Hemolysis increased with flow through the microfluidic channel, but percentage hemolysis remained below 0.3%.Blood parameters in a single-pass, microfluidic acoustic cell-separation apparatus remained within normal limits. In vivo animal studies that model continuous separation in a physiologic environment are warranted.

Transfusion reactions are common occurrences, and clinicians who order or transfuse blood components need to be able to recognize adverse sequelae of transfusion. The differential diagnosis of any untoward clinical event should always consider adverse sequelae of transfusion, even when transfusion occurred weeks earlier. There is no pathognomonic sign or symptom that differentiates a transfusion reaction from other potential medical problems, so vigilance is required during and after transfusion when a patient presents with a change in clinical status. This review covers the presentation, mechanisms, and management of transfusion reactions that are commonly encountered, and those that can be life-threatening.

Allergic transfusion reactions (ATRs) are a spectrum of hypersensitivity reactions that are the most common adverse reaction to platelets and plasma, occurring in up to 2% of transfusions. Despite the ubiquity of these reactions, little is known about their mechanism. In a small subset of severe reactions, specific antibody has been implicated as causal, although this mechanism does not explain all ATRs. Evidence suggests that donor, product, and recipient factors are involved, and it is possible that many ATRs are multifactorial. Further understanding of the mechanisms of ATRs is necessary so that rationally designed and cost-effective prevention measures can be developed.

Age-group analyses were conducted of patients in the prophylactic platelet dose trial (PLADO), which evaluated the relation between platelet dose per transfusion and bleeding. Hospitalized patients with treatment-induced hypoproliferative thrombocytopenia were randomly assigned to 1 of 3 platelet doses: 1.1 × 1011, 2.2 × 1011, or 4.4 × 1011 platelets/m2 per transfusion, given for morning counts of ≤ 10 000 platelets/μL. Daily hemostatic assessments were performed. The primary end point (percentage of patients who developed grade 2 or higher World Health Organization bleeding) was evaluated in 198 children (0-18 years) and 1044 adults. Although platelet dose did not predict bleeding for any age group, children overall had a significantly higher risk of grade 2 or higher bleeding than adults (86%, 88%, 77% vs 67% of patients aged 0-5 years, 6-12 years, 13-18 years, vs adults, respectively) and more days with grade 2 or higher bleeding (median, 3 days in each pediatric group vs 1 day in adults; P < .001). The effect of age on bleeding differed by disease treatment category and was most pronounced among autologous transplant recipients. Pediatric subjects were at higher risk of bleeding over a wide range of platelet counts, indicating that their excess bleeding risk may be because of factors other than platelet counts. This trial was registered at www.clinicaltrials.gov as #NCT00128713.

Therapies that disrupt or repair blood-brain barrier integrity can result in major changes in MRI images even when the tumor volume remains constant. Thus, a reliable blood-based tumor biomarker could significantly improve clinical care and research studies in these patients. This study was performed to assess plasma concentrations of glial fibrillary acidic protein (GFAP) in patients with high- and low-grade gliomas before and after debulking surgery. Pre-operative plasma was collected from 33 patients with radiation- and chemotherapy-naïve gliomas. Additional plasma was collected 24-48 h post-operatively from 23 of these patients. Plasma GFAP (pGFAP) concentrations were determined using an electrochemiluminescent immunoassay and were analyzed as a function of tumor grade, tumor GFAP expression, the integrity of the blood-brain barrier, and post-operative status. Detectable pGFAP levels (≥ 0.04 ng/mL) were found pre-operatively in 52 % of patients and post-operatively in 96 %. Detectable pGFAP was more common in patients with WHO grade IV (100 %) than WHO grade III (56 %) or WHO grade II gliomas (20 %). No patient with undetectable GFAP had WHO grade IV glioma. Higher pGFAP concentrations were also associated with contrast enhancement but not related to tumor GFAP expression. GFAP is commonly detected in the plasma of patients with high-grade gliomas. pGFAP levels rise rather than fall following debulking surgery which is probably a result of surgical trauma. GFAP remains a potentially informative plasma biomarker for gliomas. Longitudinal studies are required to correlate pGFAP levels with patient outcomes.

BACKGROUND: The overall risk of hemolytic transfusion reactions (HTRs) from plasma (minor)-incompatible platelet (PLT) transfusions and the role of a critical anti-A or anti-B titer in predicting and preventing these reactions has not been clearly established. STUDY DESIGN AND METHODS: We evaluated all apheresis PLT (AP) transfusions for 3 months. Using the gel titer method, we determined the anti-A and/or the anti-B immunoglobulin (Ig)G titer for all incompatible APs. Reported febrile transfusion reactions and HTRs were recorded; transfusions were not prospectively evaluated by the study team. A posttransfusion direct antiglobulin test (DAT) and eluate were performed after a reported febrile or hemolytic reaction for patients who received plasma-incompatible APs. RESULTS: A total of 647 of 4288 AP transfusions (15.1%) were plasma incompatible. Group O APs (n = 278) had significantly higher anti-A and anti-B titers than group A or B APs (p

Background—Concentrating and washing apheresis platelets (APs) substantially reduce the number of allergic transfusion reactions likely due to removal of plasma. However, these processes may damage platelets. This study evaluated whether concentrating or washing APs decrease the Corrected Count Increment (CCI). Study Design and Methods—This retrospective study evaluated individuals who initially received unmanipulated APs and subsequently received concentrated and/or washed APs at a large university hospital between 1998 and 2009. Concentrated units were prepared by reducing the plasma volume of APs by a goal of >67%. Washed units were prepared by washing the APs with 1L normal saline. The CCI (plt × m 2 /uL) for all transfusions was calculated. Hypothesis testing was performed with Student’s t-tests for continuous variables and chi-square tests for dichotomous variables. Results—We evaluated 121 individuals; 46 patients who received unmanipulated, concentrated and then washed APs, 59 patients who received unmanipulated and then concentrated APs; and 16 patients who received unmanipulated and then washed APs. Patient demographics were similar among the three groups. The mean CCI for unmanipulated AP transfusions at 0–2 hours post transfusion were significantly higher than concentrated and washed platelet transfusions (p

To determine whether, in children, plasma glial fibrillary acidic protein is associated with brain injury during extracorporeal membrane oxygenation and with mortality.Prospective, observational study.Pediatric intensive care unit in an urban tertiary care academic center.Neonatal and pediatric patients on extracorporeal membrane oxygenation (n = 22).Serial blood sampling for glial fibrillary acidic protein measurements.Prospective patients age 1 day to 18 yrs who required extracorporeal membrane oxygenation from April 2008 to August 2009 were studied. Glial fibrillary acidic protein was measured using an electrochemiluminescent immunoassay developed at Johns Hopkins. Control samples were collected from 99 healthy children (0.5-16 yrs) and 59 neonatal intensive care unit infants without neurologic injury. In controls, the median glial fibrillary acidic protein concentration was 0.055 ng/mL (interquartile range, 0-0.092 ng/mL) and the 95th percentile of glial fibrillary acidic protein was 0.436 ng/mL. In patients on extracorporeal membrane oxygenation, plasma glial fibrillary acidic protein was measured at 6, 12, and every 24 hrs after cannulation. We enrolled 22 children who underwent extracorporeal membrane oxygenation. Median age was 7 days (interquartile range, 2 days to 9 yrs), and primary extracorporeal membrane oxygenation indication was: cardiac failure, six of 22 (27.3%); respiratory failure, 12 of 22 (54.5%); extracorporeal cardiopulmonary resuscitation, three of 22 (13.6%); and sepsis, one of 22 (4.6%). Seven of 22 (32%) patients developed acute neurologic injury (intracranial hemorrhage, brain death, or cerebral edema diagnosed by imaging). Fifteen of 22 (68%) survived to hospital discharge. In the extracorporeal membrane oxygenation group, peak glial fibrillary acidic protein levels were higher in children with brain injury than those without (median, 5.9 vs. 0.09 ng/mL, p = .04) and in nonsurvivors compared with survivors to discharge (median, 5.9 vs. 0.09 ng/mL, p = .04). The odds ratio for brain injury for glial fibrillary acidic protein0.436 ng/mL vs. normal was 11.5 (95% confidence interval, 1.3-98.3) and the odds ratio for mortality was 13.6 (95% confidence interval, 1.7-108.5).High glial fibrillary acidic protein during extracorporeal membrane oxygenation is significantly associated with acute brain injury and death. Brain injury biomarkers may aid in outcome prediction and neurologic monitoring of patients on extracorporeal membrane oxygenation to improve outcomes and benchmark new therapies.

BACKGROUND: The mechanisms that underlie allergic transfusion reactions (ATRs) are not well characterized, but likely involve recipient, donor, and product factors. To assess product factors associated with ATRs, we investigated candidate mediators in apheresis platelet (PLT) products associated with ATRs and controls. STUDY DESIGN AND METHODS: Using bead-based and standard enzyme-linked immunosorbent assays, we tested supernatants from 20 consecutive apheresis PLT transfusions associated with ATRs and 30 control products for concentrations of mediators in three categories: acute inflammatory mediators, direct agonists of basophils and mast cells, and growth and/or priming factors of basophils and mast cells. RESULTS: Median concentrations of the direct allergic agonists C5a, brain-derived neurotrophic factor (BDNF), and CCL5 (RANTES) were 16.6, 41.8, and 13.9% higher, respectively, in the supernatant of apheresis PLT products that were most strongly associated with ATRs (p 0.2 for all associations). CONCLUSION: The allergic agonists C5a, BDNF, and CCL5 may be mediators of ATRs in apheresis PLT products. Acute inflammatory proteins and basophil and/or mast cell growth and priming factors do not appear to be associated with apheresis PLT products that cause ATRs.

Objective Investigate the natural history of PNH clones in patients with acquired aplastic anemia (AA).

BACKGROUND: The biologic mechanisms of allergic transfusion reactions (ATRs) are largely unknown. We sought to compare the atopic predisposition of platelet (PLT) recipients who experienced an ATR to nonreactive control recipients. STUDY DESIGN AND METHODS: We identified 37 consecutive apheresis PLT recipients who experienced an ATR and 26 matched controls. Total immunoglobulin (Ig)E and aero- and food allergen–specific IgE were quantified in plasma by a blood test for allergies (ImmunoCAP: Phadiatop and Fx5). IgE testing of apheresis PLT supernatants was also performed. RESULTS: Pruritus and urticaria were manifest in 91.9 and 83.8% of all ATRs, with more severe respiratory symptoms and angioedema occurring in less than 15% of cases. No subject had anaphylaxis. Sex, age, and primary diagnosis were balanced between the two groups. Total and aeroallergen-specific IgE was higher among subjects experiencing an ATR in comparison to control subjects (median total IgE, 55.5 kU/L vs. 8.3 kU/L, p = 0.002; and median aeroallergen-specific IgE, 0.57 kUa/L vs. 0.36 kUa/L, p = 0.046). IgE antibody levels in apheresis products associated with ATRs were similar to control products (p > 0.1 for all IgE tests). CONCLUSION: Recipient atopic predisposition, as defined by IgE sensitization, is a risk factor associated with ATRs.

BACKGROUND: The incidence of allergic transfusion reactions (ATRs) ranges from 1% to 3% of all transfusions, and they are difficult to prevent. This study evaluated whether removing plasma from apheresis platelets (APs) or red blood cells (RBCs) by concentrating or washing transfusion products can decrease the incidence of ATRs. STUDY DESIGN AND METHODS: A retrospective cohort study of 179 individuals who received unmanipulated and subsequently concentrated and/or washed APs was conducted. Poisson regression with generalized estimating equations was used to estimate the incident rate ratios and 95% confidence intervals (CIs) of ATRs. RESULTS: The incidence of ATRs to unmanipulated APs was 5.5% (306 ATRs/5575 AP units). The incidence decreased to 1.7% (135 ATRs/4327 AP units) when individuals received concentrated APs (73% reduction; 95% CI, 65%-79%) and 0.5% (21 ATRs/4082 AP units) when individuals received washed APs (95% reduction; 95% CI, 91%-97%). Of the 39 individuals who received unmanipulated RBCs and subsequently washed RBCs, the incidence of ATRs decreased from 2.7% (33 ATRs/1236 RBC units) to 0.3% (2 ATRs/733 RBC units; 89.4% reduction; 95% CI, 55.5%-97.5%). The median number of AP transfusions to first ATR was six (interquartile range [IQR], 2-19) for unmanipulated APs and increased to 13 (IQR, 4-32) for concentrated APs and 40 (IQR, 29-73.5) for washed APs. CONCLUSIONS: Concentrating APs and washing APs and RBCs substantially reduces ATRs, suggesting that the plasma component of APs and RBCs has an essential role in the etiology of ATRs.

BACKGROUND: Mechanisms of allergic transfusion reactions (ATRs) are not well understood. The aim of this study was to distinguish recipient-, donor-, and product-specific factors associated with ATRs. STUDY DESIGN AND METHODS: We conducted a retrospective cohort study of apheresis platelet (AP) products transfused from April 2000 through March 2010. The concordance rate of ATRs when split AP products were transfused to at least two individuals was compared to the overall ATR rate among all AP products. Per-person ATR rates also were compared to the overall ATR rate. RESULTS: We observed 1616 ATRs among 93,737 transfusions, for an overall incidence of 1.72% (95% confidence interval [CI], 1.64%-1.81%). Of the 1616 ATRs, 630 occurred when split AP products were transfused to at least two recipients. Of these 630 AP products, ATRs were observed in at least two different recipients of the same AP collection only 6 of 630 times, for a concordant incidence of 0.95% (95% CI, 0.35%-2.06%), which is similar to the overall ATR rate (p = 0.17). On an individual level, 30.0% of recipients had ATR rates of more than 5%, and these 30.0% accounted for 62.1% of ATRs. Donors of AP products associated with concordant ATRs donated AP products that had an ATR rate of 5.8% (95% CI, 3.1%-9.7%), which is higher than the overall ATR rate (p

To the Editor Allergic transfusion reactions (ATRs) occur in up to 3% of platelet transfusions1, are under-reported, and range in severity from mild itching and hives to fatal anaphylaxis. Clinical data have broadly defined factors that may be involved, such as a pathogenic role for plasma. Retrospective data of plasma reduction maneuvers to prevent ATRs to blood products are confounded by selection bias: patients always receive unmanipulated platelets before being switched to volume reduced or washed platelets following ATRs. An individual's risk of ATRs might decrease with increasing numbers of platelet transfusions, independent of platelet product manipulation. Thus, plasma reduction may temporally coincide with, but not cause, a reduction in ATR risk2. To address the question of whether ATR risk varies with increasing platelet transfusion number, we analyzed severe urticarial reaction data from the Trial to Reduce Alloimmunization to Platelets (TRAP)3. There were 31 severe urticarial reactions reported in 24 subjects in the TRAP study (n=8,770 transfusions). Details about minor allergic reactions or the criteria for defining a severe urticarial reaction were not recorded in the study. Anaphylaxis was an evaluable outcome, but anaphylaxis was not reported after any platelet transfusion. However, three severe urticarial reactions were accompanied by dyspnea or bronchospasm. These reactions meet current criteria for likely anaphylaxis4. The figure shows the frequency of severe urticarial reactions by transfusion number. The risk for a severe urticarial reaction is not constant and is highest among the first 5 transfusions: 18 severe urticarial reactions in transfusions 1-5 (n=2889) vs. 13 severe urticarial reactions in the remaining transfusions (n=5881; P=0.006, Fisher exact test). We conducted a logistic regression analysis using a clustered sandwich estimator to account for subjects receiving different total numbers of transfusions. The odds ratio for a severe urticarial reaction for every additional platelet transfusion is 0.91 (95% CI 0.85-0.98, P=0.007). Five percent of platelets in TRAP were volume reduced. When these transfusions are excluded from the analysis, the findings do not materially change (OR 0.92; 95% CI 0.86-0.98). Figure The rate of severe urticarial reactions to platelets in the Trial to Reduce Alloimmunization to Platelets decreases over time It is possible that subjects with an increased ATR predisposition received fewer platelet transfusions than subjects with no predisposition to ATRs, and this would bias the results. Indeed, the 24 subjects who experienced a severe urticarial reaction had a median (range) of 7 (1-32) platelet transfusions during the study period vs. 9 (1-92) for the subjects who did not have a severe reaction (P

Biomarkers have enormous potential to improve patient care by establishing tests of diagnosis, prognosis, and treatment effects. Successfully translating a biomarker from discovery to clinical application demands high-quality discovery research and high-quality clinical studies for biomarker validation; however, there are additional challenges that face biomarker research in pediatrics. There are also additional characteristics of pediatric medicine that make biomarker research especially needed. This review focuses on the fundamentals of biomarkers, the additional considerations needed for applying biomarker research to children, and recommendations for advancing pediatric biomarker research.

Purpose: Hemoglobin (Hb) depletion with nickel affinity chromatography has been shown to increase the number of proteins identified in proteomic studies of erythrocytes, but limited data exist on the application of this technique in depletion of Hb from plasma or serum required for clinical biomarker studies. The aim of this study was to explore the potential of using nickel-beads for Hb depletion of plasma. Experimental design: Nickel–nitrilotriacetic acid (Ni–NTA) affinity chromatography was used to deplete Hb from hemolyzed plasma samples obtained from children with sickle cell disease (SCD, n=7) and normal human plasma (n=4). Ni–NTA-bound proteins were analyzed by one-dimensional GE, followed by in-gel digestion for characterization using an LTQ-Orbitrap hybrid mass spectrometer. In addition, the loss of two non-Hb-related plasma proteins, thrombospondin1 and L-selectin, by Ni–NTA was determined by ELISA (SCD n=6, non-SCD controls n=2). Results: Ni–NTA resulted in an average 60% decrease in plasma protein concentration, which was not hemolysis dependent. Specifically, Hb (7 peptides) and the top three proteins, α-2-macroglobulin (75 peptides), apolipoprotein B-100 (73 peptides), and albumin (42 peptides) were Ni–NTA bound. In addition, using an ELISA assay two non-Hb-associated plasma proteins thrombospondin1 and L-selectin were decreased by Ni-NTA. Conclusions and clinical relevance: Hb depletion with Ni–NTA is effective for Hb removal but is not specific. There is a potential for deleterious depletion of potential biomarkers that may limit the applicability of this method. Consideration of alternate methods of Hb depletion for clinical proteomics may be warranted.

Seventeen patients transplanted with hematopoietic cells to correct severe T lymphocyte immunodeficiency resulting from complete DiGeorge anomaly were identified worldwide, and retrospective data were obtained using a questionnaire-based survey. Patients were treated at a median age of 5 months (range, 2-53 months) between 1995 and 2006. Bone marrow was used in 11 procedures in 9 cases: 6 from matched unrelated donors, 4 from human leukocyte antigen (HLA)-identical siblings, and one haploidentical parent with T-cell depletion. Unmobilized peripheral blood was used in 8 cases: 5 from HLA-identical siblings, one from a matched unrelated donor, one from an HLA-identical parent, and one unrelated matched cord blood. Conditioning was used in 5 patients and graft-versus-host disease prophylaxis in 11 patients. Significant graft-versus-host disease occurred in 9 patients, becoming chronic in 3. Median length of follow-up was 13 months, with transplantation from HLA-matched sibling showing the best results. Median survival among deceased patients (10 patients) was 7 months after transplantation (range, 2-18 months). The overall survival rate was 41%, with a median follow-up of 5.8 years (range, 4-11.5 years). Among survivors, median CD3 and CD4 counts were 806 (range, 644-1224) and 348 (range, 225-782) cells/mm3, respectively, CD4+/CD45RA+ cells remained very low, whereas mitogen responses were normalized.

Over the last decade, translational science has come into the focus of academic medicine, and significant intellectual and financial efforts have been made to initiate a multitude of bench-to-bedside projects. The quest for suitable biomarkers that will significantly change clinical practice has become one of the biggest challenges in translational medicine. Quantitative measurement of proteins is a critical step in biomarker discovery. Assessing a large number of potential protein biomarkers in a statistically significant number of samples and controls still constitutes a major technical hurdle. Multiplexed analysis offers significant advantages regarding time, reagent cost, sample requirements and the amount of data that can be generated. The two contemporary approaches in multiplexed and quantitative biomarker validation, antibody-based immunoassays and MS-based multiple (or selected) reaction monitoring, are based on different assay principles and instrument requirements. Both approaches have their own advantages and disadvantages and therefore have complementary roles in the multi-staged biomarker verification and validation process. In this review, we discuss quantitative immunoassay and multiple reaction monitoring/selected reaction monitoring assay principles and development. We also discuss choosing an appropriate platform, judging the performance of assays, obtaining reliable, quantitative results for translational research and clinical applications in the biomarker field.

BACKGROUND: Transfusion-associated bacterial sepsis is a significant risk of morbidity and mortality related to platelet (PLT) transfusions. Previously the rate of septic PLT transfusion reactions (SPTRs) to single-donor PLTs (SDPs) in our hospital was determined to be 1 in 15,098 (6.6 per 100,000; 95% confidence interval [CI], 0.17-36.9 per 100,000) transfusions. The goal of this study was to determine if bacterial testing of SDPs reduced the rate of SPTRs in our hospital. STUDY DESIGN AND METHODS: An automated microbial detection system was implemented by our blood supplier in February 2004. A retrospective examination of the number of SPTRs that have occurred to SDPs at our hospital since that time was performed, using the same criteria used before bacterial screening. Transfusions over a 3.5-year period were examined. Clinical and laboratory data were gathered and correlated from transfusion reaction files and three independent computer systems. RESULTS: From March 1, 2004, through August 31, 2007, there were 49,625 transfusions of SDPs with 1096 transfusion reactions reported. Only one reaction detected the same organism in two of three sites, meeting our criteria for a SPTR. The rate of SPTRs in SDPs was identified as 1 in 49,625 (2.0 per 100,000; 95% CI, 0.05-11.2 per 100,000). This represents a 69.7% reduction in the incidence of SPTRs (p = 0.41). CONCLUSION: With the implementation of bacterial testing, a decrease in the rate of SPTRs to SDPs from 6.6 per 100,000 to 2.0 per 100,000 transfusions was observed. Although not significant, these findings suggest a trend.

Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon acquired hemolytic anemia that manifests with abdominal pain, esophageal spasm, fatigue, and thrombosis. The hallmark of PNH at the cellular level is a deficiency in cell surface glycosylphosphatidylinositol anchored proteins; this deficiency on erythrocytes leads to intravascular hemolysis. Free hemoglobin from hemolysis leads to circulating nitric oxide depletion and is responsible for many of the clinical manifestations of PNH, including fatigue, erectile dysfunction, esophageal spasm, and thrombosis. The recently FDA approved complement inhibitor eculizumab has been shown to decrease hemolysis, decrease erythrocyte transfusion requirements, and improve quality of life for PNH patients.