Therapeutic plasma exchange in alloimmune platelet refractoriness.

Patients with alloimmune platelet refractoriness can present complex clinical conundrums. Herein we describe a case of platelet refractoriness in the setting of combined HLA and HPA alloimmunization in a patient with acute myeloid leukemia and life-threatening bleeding. We discuss causative antibodies and compare prevailing therapeutic modalities. We highlight plasma exchange as a potentially feasible, repeatable, and personalized treatment option for patients with extensive platelet alloimmunization who require transfusion.

Evaluation of amotosalen and UVA pathogen-reduced apheresis platelets after 7-day storage.

BACKGROUND: Amotosalen/UVA pathogen-reduced platelet components (PRPCs) with storage up to 7 days are standard of care in France, Switzerland, and Austria. PRPCs provide effective hemostasis with reduced risk of transfusion-transmitted infections and transfusion-associated graft versus host disease, reduced wastage and improved availability compared with 5-day-stored PCs. This study evaluated the potency of 7-day PRPCs by in vitro characterization and in vivo pharmacokinetic analysis of autologous PCs. STUDY DESIGN AND METHODS: The in vitro characteristics of 7-day-stored apheresis PRPCs suspended in 100% plasma or 65% platelet additive solution (PAS-3)/35% plasma, thrombin generation, and in vivo radiolabeled post-transfusion recovery and survival of 7-day-stored PRPCs suspended in 100% plasma were compared with either 7-day-stored or fresh autologous conventional platelets. RESULTS: PRPCs after 7 days of storage maintained pH, platelet dose, in vitro physiologic characteristics, and thrombin generation when compared to conventional 7-day PCs. In vivo, the mean post-transfusion survival was 151.4 ± 20.1 h for 7-day PRPCs in 100% plasma (Test) versus 209.6 ± 13.9 h for the fresh autologous platelets (Control), (T-ΔC: 72.3 ± 8.8%: 95% confidence interval [CI]: 68.5, 76.1) and mean 24-h post-transfusion recovery 37.6 ± 8.4% for Test versus 56.8 ± 9.2% for Control (T-ΔC: 66.2 ± 11.2%; 95% CI: 61.3, 71.1). DISCUSSION: PRPCs collected in both 100% plasma as well as 65% PAS-3/35% plasma and stored for 7 days retained in vitro physiologic characteristics. PRPCs stored in 100% plasma for 7 days retained in vivo survival. Lower in vivo post-radiolabeled autologous platelet recovery is consistent with reported reduced count increments for allogenic transfusion.

Daily versus every other day oral iron supplementation in patients with iron deficiency anemia (DEODO): study protocol for a phase 3 multicentered, pragmatic, open-label, pilot randomized controlled trial.

BACKGROUND: Iron deficiency anemia (IDA) accounts for the majority of anemia cases across the globe and can lead to impairments in both physical and cognitive functioning. Oral iron supplementation is the first line of treatment to improve the hemoglobin level for IDA patients. However, gaps still exist in understanding the appropriate dosing regimen of oral iron. The current trial proposes to evaluate the feasibility of performing this study to examine the effectiveness and side-effect profile of oral iron once daily versus every other day. METHODS: In this open-label, pilot, feasibility, randomized controlled trial, 52 outpatients over 16 years of age with IDA (defined as hemoglobin < 12.0 g/dL in females and < 13.0 g/dL in males and ferritin < 30 mcg/L) will be enrolled across two large academic hospitals. Participants are randomized in a 1:1 ratio to receive 300 mg oral ferrous sulfate (60 mg of elemental iron) either every day or every other day for 12 weeks. Participants are excluded if they are as follows: (1) pregnant and/or currently breastfeeding, (2) have a disease history that would impair response to oral iron (e.g., thalassemia, celiac disease), (3) intolerant and/or have an allergy to oral iron or vitamin C, (4) on new anticoagulants in the past 6 months, (5) received IV iron therapy in the past 12 weeks, (6) have surgery, chemotherapy, or blood donation planned in upcoming 12 weeks, (7) a creatinine clearance < 30 mL/min, or (8) hemoglobin less than 8.0 g/dL with active bleeding. The primary outcome is feasibility to enroll 52 participants in this trial over a 2-year period to determine the effectiveness of daily versus every other day oral iron supplementation on hemoglobin at 12 weeks post-initiation and side-effect profile. DISCUSSION: The results of this trial will provide additional evidence for an appropriate dosing schedule for treating patients with IDA with oral iron supplementation. Additional knowledge will be gained on how the dosing regimen of oral iron impacts quality of life and hemoglobin repletion in IDA patients. If this trial is deemed feasible, it will inform the development and implementation of a larger multicenter definitive trial. TRIAL REGISTRATION: ClinicalTrials.gov: NCT03725384 . Registered 31 October 2018.

Novel GNE Gene Variants Associated with Severe Congenital Thrombocytopenia and Platelet Sialylation Defect.

The GNE gene encodes an enzyme that initiates and regulates the biosynthesis of N-acetylneuraminic acid, a precursor of sialic acids. GNE mutations are classically associated with Nonaka myopathy and sialuria, following an autosomal recessive and autosomal dominant inheritance pattern. Reports show that single GNE variants cause severe thrombocytopenia without muscle weakness. Using panel sequencing, we identified two novel compound heterozygous variants in GNE in a young girl with life-threatening bleedings, severe congenital thrombocytopenia, and a platelet secretion defect. Both variants are located in the nucleotide-binding site of the N-acetylmannosamin kinase domain of GNE. Lectin array showed decreased α-2,3-sialylation on platelets, consistent with loss of sialic acid synthesis and indicative of rapid platelet clearance. Hematopoietic stem cell transplantation (HSCT) normalized platelet counts. This is the first report of an HSCT in a patient with an inherited GNE defect leading to normal platelet counts.

A possible case of recipient anti-neutrophil and anti-human leukocyte antigen antibody-mediated fatal reverse transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a transfusion complication often mediated by recipient exposure to plasma from donors with human leukocyte antigen (HLA) and human neutrophil antigen (HNA) antibodies. Recipient anti-donor HLA or HNA antibodies have rarely been implicated. STUDY DESIGN AND METHODS: Herein, we describe a case of fatal TRALI mediated by recipient anti-HLA and anti-HNA antibodies. Cognate antibody-antigen match was confirmed with serologic and molecular assays. RESULTS: A 69-year-old G5P5 female with no prior transfusion history and metastatic cholangiocarcinoma with thromboembolic complications presented with heart failure and dyspnea. She was transfused 15 ml of a unit of Fya -negative red blood cells and subsequently developed acute onset dyspnea, hypoxemia, hypotension, and fever. Clinical investigations revealed bilateral infiltrates on chest X-ray and cognate recipient HLA and HNA antibodies to donor antigens. The patient died of acute respiratory failure within 24 h of transfusion. In total, the patient had Fya , HLA Class I, HNA, and human platelet antigen (HPA) alloantibodies. The 63-year-old female donor had detectable HLA class II antibodies (recipient class II genotype unavailable). CONCLUSION: The pathophysiology of TRALI has traditionally been ascribed to underlying conditions that put the recipient at risk in combination with donor biological response modifiers. This case illustrates alternative pathogenic mediators including alloantibodies to donor HLA and HNA. Additional studies to determine the contribution and frequency of recipient alloantibodies in TRALI may inform future mitigation strategies to further reduce the incidence of TRALI, particularly in female transfusion recipients.

Characterization and statistical modeling of glycosylation changes in sickle cell disease.

Sickle cell disease is an inherited genetic disorder that causes anemia, pain crises, organ infarction, and infections in 13 million people worldwide. Previous studies have revealed changes in sialic acid levels associated with red blood cell sickling and showed that stressed red blood cells bare surface-exposed clustered terminal mannose structures mediating hemolysis, but detailed glycan structures and anti-glycan antibodies in sickle cell disease remain understudied. Here, we compiled results obtained through lectin arrays, glycan arrays, and mass spectrometry to interrogate red blood cell glycoproteins and glycan-binding proteins found in the plasma of healthy individuals and patients with sickle cell disease and sickle cell trait. Lectin arrays and mass spectrometry revealed an increase in α2,6 sialylation and a decrease in α2,3 sialylation and blood group antigens displayed on red blood cells. Increased binding of proteins to immunogenic asialo and sialyl core 1, Lewis A, and Lewis Y structures was observed in plasma from patients with sickle cell disease, suggesting a heightened anti-glycan immune response. Data modeling affirmed glycan expression and plasma protein binding changes in sickle cell disease but additionally revealed further changes in ABO blood group expression. Our data provide detailed insights into glycan changes associated with sickle cell disease and refer glycans as potential therapeutic targets.

Red cell genotyping of rare blood donors: donation behaviour and data visualization.

BACKGROUND: The continual identification of rare blood among donors is critical to support national programs like the American Rare Donor Program (ARDP). Some blood centres require consent from donors to be registered with a national registry. This situation provides an opportunity to determine whether a donor's willingness to register is associated with a change in donation behaviour. METHODS: Rare donors were identified by molecular typing. The average number of donations per year was compared for each donor prior to and after receiving a consent letter. Donors were categorized as either accepting or declining the request. Non-parametric t tests compared the statistical significance within and between categories. Rare types were overlaid with consensus data to look for trends using data visualization techniques. RESULTS: A total of 270 molecularly typed rare donors received letters over 4 years. Half of the donors (132, 49%) agreed to participate in the ARDP. Overall, donation frequency increased after the letter when enrolled. Both Caucasian and non-Caucasian donors increased their donations after enrolling providing an additional 159 red blood cell units over 3 years. Declining participation did not change donation frequency. Data visualization showed that enrolled donors were more affluent, high school and college educated, and lived in their home for longer periods of time. CONCLUSION: A donor's willingness to enrol in the ARDP was associated with a post-response increase in donation frequency. New interventions to reach non-Caucasian donors may be a prerequisite to increase donation frequency and a willingness to be a rare blood donor.

A microfluidic analysis of thrombus formation in reconstituted whole blood samples comparing spray-dried plasma versus fresh frozen plasma.

BACKGROUND: Prompt resuscitation with plasma and other blood products reduces trauma-related morbidity and mortality. Standard storage and preparation techniques for frozen plasma limit its utility in the pre-hospital setting. Plasma can be dehydrated using hot air (spray-dried plasma), stored at room temperature and rehydrated quickly for use. The spray-dry process decreases high-molecular-weight multimers of von Willebrand factor compared with conventional plasma. The objective of this study was to compare platelet adhesion and thrombus formation in a microfluidic perfusion assay facilitated by spray-dried compared with frozen plasma using a non-inferiority design. STUDY DESIGN AND METHODS: Whole blood was centrifuged to obtain red cell concentrate, and a platelet pellet that was suspended in either spray-dried or frozen plasma to create recombined whole blood. Platelets were fluorescently labelled, and samples were flowed through a collagen-coated microchannel. Surface area coverage by platelets and thrombi was analysed and compared between each spray-dried and frozen plasma pair. RESULTS: Compared with whole blood samples containing frozen plasma, samples with spray-dried plasma had similar surface area coverage of platelets and thrombi after 180 s of flow. Even when diluted with von Willebrand factor-free plasma, there was no reduction thrombus formation. CONCLUSION: Spray-dried plasma is not inferior in supporting haemostasis compared with fresh frozen plasma in a paired analysis. It offers advantages with respect to portability and ease of preparation over frozen plasma in the pre-hospital setting. This study supports development of clinical studies to evaluate the efficacy and safety of spray-dried plasma in trauma patients.

Predictive modeling of complex ABO glycan phenotypes by lectin microarrays.

Serological classification of individuals as A, B, O, or AB is a mainstay of blood banking. ABO blood groups or ABH antigens, in addition to other surface glycans, act as unique red blood cell (RBC) signatures and direct immune responses. ABO subgroups present as weakened, mixed field, or unexpected reactivity with serological reagents, but specific designations remain complex. Lectins detect glycan motifs with some recognizing ABH antigens. We evaluated a 45-probe lectin microarray to rapidly analyze ABO blood groups and associated unique glycan signatures within complex biological samples on RBC surface glycoproteins. RBC membrane glycoproteins were prepared from donor RBCs, n = 20 for each blood group. ABO blood group was distinguishable by lectin array, including variations in ABH antigen expression not observed with serology. Principal component analysis highlighted broad ABO blood group clusters with unexpected high and low antigen expression and variations were confirmed with ABH antibody immunoblotting. Using a subset of lectins provided an accurate method to predict an ABO serological phenotype. Lectin microarray highlighted the importance of ABO localization on glycoproteins and glycolipids and pointed to increased glycocalyx complexity associated with the expression of A and B antigens including high mannose and branched polylactosamine. Thus, lectins identified subtle surface ABO blood group glycoprotein density variations not detected by routine serological methods. Transfusion services observe alterations in ABH expression during malignancy, and ABO incompatible solid organ transplantation is not without risk of rejection. The presented methods may identify subtle but clinically significant ABO blood group differences for transfusion and transplantation.

Complement activating ABO anti-A IgM/IgG act synergistically to cause erythrophagocytosis: implications among minor ABO incompatible transfusions.

BACKGROUND: Typically minor ABO incompatible platelet products are transfused without any incident, yet serious hemolytic transfusion reactions occur. To mitigate these events, ABO 'low titer' products are used for minor ABO incompatible transfusions. We sought to understand the role of IgM/IgG and complement activation by anti-A on extravascular hemolysis. METHODS: Samples evaluated included (i) Group O plasma from a blood donor whose apheresis platelet product resulted in an extravascular transfusion reaction, (ii) Group O plasma from 12 healthy donors with matching titers that activated complement (N = 6) or not (N = 6), and (iii) Group O sera from 10 patients with anti-A hemolysin activity. A flow cytometric monocyte erythrophagocytosis assay was developed using monocytes isolated by immunomagnetic CD14-positive selection from ACD whole blood of healthy donors. Monocytes were frozen at - 80 °C in 10% dimethyl sulfoxide/FBS and then thawed/reconstituted on the day of use. Monocytes were co-incubated with anti-A-sensitized fluorescently-labeled Group A1 + RBCs with and without fresh Group A serum as a source of complement C3, and erythrophagocytosis was analyzed by flow cytometry. The dependency of IgM/IgG anti-A and complement C3 activation for RBC erythrophagocytosis was studied. Anti-A IgG subclass specificities were examined for specific samples. RESULTS: The plasma and sera had variable direct agglutinating (IgM) and indirect (IgG) titers. None of 12 selected samples showed monocyte-dependent erythrophagocytosis with or without complement activation. The donor sample causing a hemolytic transfusion reaction and 2 of the 10 patient sera with hemolysin activity showed significant erythrophagocytosis (> 10%) only when complement C3 was activated. The single donor plasma and two sera demonstrating significant erythrophagocytosis had high IgM (≥ 128) and IgG titers (> 1024). The donor plasma anti-A was IgG1, while the patient sera were an IgG3 and an IgG1 plus IgG2. CONCLUSION: High anti-A IgM/IgG titers act synergistically to cause significant monocyte erythrophagocytosis by activating complement C3, thus engaging both Fcγ- and CR1-receptors.

Use of a cloud-based search engine of a centralized donor database to identify historical antigen-negative units in hospital inventories.

BACKGROUND: The provision of units with antigen-negative attributes is required for alloimmunized transfusion recipients and to avoid alloimmunization among patients on chronic transfusion support. Recent evidence confirms that the demand for antigen-typed units is increasing. STUDY DESIGN AND METHODS: A cloud-based search engine was designed by the blood center to find antigen-negative units. The service provided access to historical antigen information for units in hospital inventories. The hospital transfusion service was required to confirm the antigen phenotype. The results of 16 hospitals' use over 5 years were analyzed to determine trends and value of the service. The time commitment of the cloud-based query was compared to the hospital performing manual phenotyping with an outcome of at least one unit found with the desired antigen-negative attribute(s). RESULTS: Hospitals were located between 4 miles and 200 miles away from the blood center. A total of 6,081 queries were submitted over the 5 years, with an overall 50% success rate of finding at least one unit. Single antigen queries accounted for 67% of total searches, with two antigen queries and three or more antigen queries accounting for 24% and 9% of the units found, respectively. The cloud-based antigen query was most efficient for combined antigen frequencies <0.5 for two or more antigen-negative attributes. CONCLUSION: A cloud-based search engine provides hospitals with access to historical antigen information housed at the blood center. Future refinements may consider regulatory submission of a process to provide confirmed historical information through this cloud-based program.

Mass-scale red cell genotyping of blood donors: from data visualization to historical antigen labeling and donor recruitment.

Donor red cell genotyping provides efficiencies to identify "in demand" blood donors for transfusion recipients requiring antigen-negative blood. Donor red cell genotype information can be used to label units with historical types and introduced throughout the supply chain from the blood center to the hospital transfusion service and has potential to be used in recruitment strategies.

Trends in antigen-negative red blood cell distributions by racial or ethnic groups in the United States.

BACKGROUND: The overall number of red blood cell (RBC) units distributed to hospitals throughout the world and in the United States has decreased lately. This study was performed to determine if the number of antigen-negative RBC units distributed to hospitals has followed this trend. STUDY DESIGN AND METHODS: Stratified by ethnicity, data on total RBC distributions and antigen-negative RBC distributions from six large blood collectors in the United States were obtained from 2009 through 2016. An antigen-negative unit was defined as a unit with a specific RBC phenotype that had been specially ordered as such by a hospital. RESULTS: Overall, 10,103,703 RBC units were distributed by these six blood collectors; 650,516 (6.4%) were distributed as antigen-negative units. While the overall number of RBCs distributed decreased by 27.2% between 2009 and 2016, the number of antigen-negative RBC distributions increased by 39.5%. In each year, the majority of the distributed antigen-negative RBCs were donated by whites. However, antigen-negative RBC units from black or African American donors were distributed in a disproportionately high fraction compared to the overall number of RBCs distributed from these donors. Most of the one through four antigen-negative RBCs were donated by whites. However, as antigen matching became more extensive, the proportion of units distributed from black or African American donors increased such that they were the predominant donors of five or more antigen-negative units. CONCLUSION: Blood collectors will need to be aware of the trend of increasing antigen-negative distributions despite decreased overall distributions.

Targeting Myeloid-Derived Suppressor Cells in Cancer.

Myeloid derived suppressor cells (MDSC) represent only a minor fraction of circulating blood cells but play an important role in tumor formation and progression. They are a heterogeneous group of cells that influence the tumor microenvironment by depletion of amino acids, oxidative stress, decreased trafficking of antitumor effector cells, and increased regulatory T and regulatory dendritic cell responses. Investigational treatment strategies targeting MDSCs have attempted to inhibit MDSC development and expansion (stem cell factor blockade, modulate of cell signaling, and target MDSC migration and recruitment), inhibit MDSC function (nitric oxide inhibition and reactive oxygen and nitrogen species inhibition), differentiate MDSCs into more mature cells (Vitamins A and D, all-trans retinoic acid, interleukin-2, toll-like receptor 9 inhibitors, taxanes, beta-glucan particles, tumor-derived exosome inhibition, and very small size proteoliposomes), and destroy MDSCs (cytotoxic agents, ephrin A2 degradation, anti-interleukin 13, and histamine blockers). To date, there are no Food and Drug Administration approved therapies selectively targeting MDSCs, but such therapies are likely to be implemented in the future, due to the key role of MDSCs in antitumor immunity.