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Soluble fibrin (SF) in blood consists of monomers lacking both fibrinopeptides A with a minor population in multimeric clusters. It is a substantial component of isolated fibrinogen (fg), which spontaneously self-assembles into protofibrils progressing to fibers at sub-physiologic temperatures, a process enhanced by adsorption to hydrophobic and some metal surfaces. Comparisons of SF-rich (FR) and SF-depleted (FD) fg isolates disclosed distinct molecular imprints of each via an adsorption/desorption procedure using gold surfaced silica microplates. Accelerated plasminogen activator-induced lysis and decreased stiffness (G′) of thrombin-induced FR fg clots were revealed by thomboelastography. Erythrocyte sedimentation (ESR) in afibrinogenemic plasma (Hematocrit 25–33%) was accelerated by FR fg nearly threefold that of FD fg. Stained smears disclosed frequent rouleaux formations and fibers linking stacked erythrocytes in contrast to no rouleaux by FD fg. Rouleaux formations were more pronounced at 4 °C than at ambient temperatures and at fiber-membrane contacts displayed irregular, knobby membrane contours. One of several FR fg isolates also displayed incomplete fiber networks in cell-free areas. What is more, pre-mixing FR fg with each of three monoclonal IgG anti-fg antibodies at 1.5 mol/mol fg, that inhibited fibrin polymerization, prevented rouleaux formation save occasional 2–4 erythrocyte aggregates. We conclude that spontaneously generated SF fibers bound to erythrocytes forming intercellular links culminating in rouleaux formation and ensuing ESR acceleration which in clinical settings reflects hypercoagulability. Also, the results can explain the reported fg binding to erythrocytes via ligands such as CD47, stable in vivo RBC aggregates in capillaries, and red areas of pathologic thrombi.

The recent FDA approval of the first CAR immunotherapy marks a watershed moment in the advancement toward a cure for cancer. CD19 CAR treatment for B cell acute lymphocytic leukemia has achieved unprecedented remission rates. However, despite success in treating previously relapsed and refractory patients, CD19 CAR faces similar challenges as traditional chemotherapy, in that malignancy can adapt and overcome treatment. The emergence of both antigen positive and negative blasts after CAR treatment represents a need to bolster current CAR approaches. Here, we report on the anti-tumor activity of a CAR T cell possessing 2 discrete scFv domains against the leukemic antigens CD19 and CD123. We determined that the resulting compound CAR (cCAR) T cell possesses consistent, potent, and directed cytotoxicity against each target antigen population both in vitro and in vivo. Our findings indicate that targeting CD19 and CD123 on B-ALL cells may be an effective strategy for augmenting the response against leukemic blasts and reducing rates of relapse.

Chimeric antigen receptor (CAR) T cells directed against CD19 for B-cell malignancies represent the first form of gene-transfer therapy to gain FDA approval. CARs are synthetic receptors that reprogram T cells to target cell-surface antigens following the transfer of genes encoding engineered T-cell receptors. CAR T-cell therapy combines the specificity of an antibody with the cytotoxic and memory functions of T cells. Once infused, each CAR T cell can kill many tumor cells. This chapter discusses the principles of synthetic immunity, efficacy against various tumor types, role of apheresis in manufacturing the product, toxic effects, tumor antigen escape, other challenges, and potential future applications of CAR T-cell therapy. Although autologous CAR T-cell therapies have immense potential, the cost and complexity of autologous T-cell therapies remain problematic. Emerging techniques may provide an opportunity to generate off-the-shelf T cells that combine favorable attributes, including antigen specificity, lack of alloreactivity, histocompatibility, and enhanced functional properties.

Current clinical outcomes using chimeric-antigen receptors (CARs) against multiple myeloma show promise in the eradication of bulk disease. However, these anti-BCMA (CD269) CARs observe relapse as a common phenomenon after treatment due to the reemergence of either antigen-positive or -negative cells. Hence, the development of improvements in CAR design to target antigen loss and increase effector cell persistency represents a critical need. Here, we report on the anti-tumor activity of a CAR T-cell possessing two complete and independent CAR receptors against the multiple myeloma antigens BCMA and CS1. We determined that the resulting compound CAR (cCAR) T-cell possesses consistent, potent and directed cytotoxicity against each target antigen population. Using multiple mouse models of myeloma and mixed cell populations, we are further able to show superior in vivo survival by directed cytotoxicity against multiple populations compared to a single-expressing CAR T-cell. These findings indicate that compound targeting of BCMA and CS1 on myeloma cells can potentially be an effective strategy for augmenting the response against myeloma bulk disease and for initiation of broader coverage CAR therapy.

Objectives To review the use of laboratory tests for antiplatelet agents to determine escalation of antiplatelet therapy and for emergent reversal of P2Y12 inhibitors. Methods A case scenario and review of cardiovascular and neurointerventional literature are described. Results In cardiovascular disease patients, large randomized trials failed to demonstrate superiority of tailored antiplatelet regimens using the VerifyNow P2Y12 assay, where earlier studies had shown promise. Platelet transfusions restored platelet function measured by vasodilator-stimulated phosphoprotein, light transmission aggregometry, or thromboelastography but not VerifyNow P2Y12, with the most restoration for clopidogrel and the least for ticagrelor. Conclusions Current evidence does not support changing antiplatelet therapy based on the results of platelet function monitoring tests. For emergent reversal of P2Y12 inhibitors, test method can affect platelet dosing recommendations, as different methods may give different results.

The recent Food and Drug Administration approval of the use of L-glutamine for the reduction of pain episodes in patients with sickle cell disease (SCD)2 highlights the importance of familiar amino acids in new therapeutics. For example, lysine analogs are used as antifibrinolytics to stop bleeds, whereas an arginine analog is used as an antithrombotic agent. What is the mechanism of action of L-glutamine in SCD? L-glutamine has a favorable impact on the ratio of reduced to total NAD and redox potential in sickle red blood cells (RBCs) exposed to oxidative stress (1). RBCs from individuals with SCD who were treated with L-glutamine showed reduced sickling on the peripheral blood smear and were less sickled and less adherent to vascular cells than RBCs from untreated individuals (2). Oral L-glutamine also reduced endothelial adhesion of sickle RBCs to human umbilical vein endothelial cells (3). Improved redox biology might make sickle RBCs less adhesive. Both tranexamic acid and e-aminocaproic acid, which are analogs of lysine, competitively block the binding of fibrinogen and fibrin to their lysine binding sites on plasminogen, thus reducing the rate of fibrinolysis. Aprotinin, a serine protease inhibitor, has been removed from the market due …

Objectives: Atypical nucleated RBCs (NRBCs) found on several patient blood smears between 2010 and 2012 were noted to resemble avian RBCs. NRBCs are not normally found in the circulation beyond the neonatal period and may indicate hematologic disease, malignancy in the bone marrow, or other severe conditions. Our blood smears with unusual NRBCs did not contain other abnormalities that typically accompany NRBCs, such as immature cells or dysplastic granulocytes. To investigate this anomaly, we considered possibilities such as contaminated collection tubes and instrument problems. The Retic-C Cell Control used with the LH 750 Hematology Analyzer contains a mixture of human and avian RBCs. Methods: CBC count with differential tests were performed on blanks and routine laboratory samples run immediately after the Retic-C Cell Control on the LH 750 and LH 780 analyzers to recreate the conditions that might cause spillage into the next tube. Results: We experimentally reproduced the phenomenon of contamination of a subsequent tube with avian cells from a multiply punctured reticulocyte control tube. Conclusions: We concluded that the NRBCs likely represented avian RBCs from the Retic-C Cell Control that had been introduced into the patient tubes.

Objectives: Diagnosis of heparin-induced thrombocytopenia (HIT) is complicated by a high false-positive rate for the screening enzyme immunoassay (EIA) and limited availability of confirmatory platelet activation assays such as serotonin release assay (SRA). We evaluate the impact of a massive transfusion on EIA and SRA testing and emphasize that the timing of the confirmatory sample is important. Methods: We present a case in which separate samples for HIT testing were collected before and after a major bleed requiring massive transfusion. We also discuss a recent study in which HIT serum samples were diluted in vitro and in vivo. Results: The EIA was strongly positive, but SRA was negative, leading to suspicion of a false-positive EIA result. However, SRA performed on the initial EIA specimen was strongly positive. A second EIA, drawn after a massive transfusion, was negative. Conclusions: Replacement of several blood volumes diluted the HIT antibodies below the limit of detection. Confirmatory testing for HIT antibodies should be done on the specimen that initially tested positive.

An 85-year-old man presented to his family physician with complaints of generalized weakness, fatigue, loss of appetite, excessive sleeping, and mild laryngitis. He reported that his temperature taken orally at home that morning was 102.6 °F. These symptoms arose during the previous week, before which he had been healthy, exercising vigorously every day. His past medical history included type 2 diabetes, hypercholesterolemia, and Paget disease of bone, and medications included metformin and pravastatin. One year earlier he had a tick bite followed by appearance of a bullseye rash and took a course of doxycycline. He was not symptomatic after that and was not known to have any additional tick bites. He lived in Long Island, New York, and did not have a history of travel or sick contacts. On physical exam the patient was alert but somewhat confused, with shaking chills. Cardiac and respiratory exam findings were normal. Laboratory testing included examination of a peripheral blood smear, which revealed intraerythrocytic ring forms, consistent with Babesia species, in 2.7% of the cells. The patient also had a lipid panel drawn (presumably because it was a visit to his family physician who ordered this as a routine) and was found to have an undetectable concentration of HDL cholesterol (HDL-C).2 Other laboratory data are summarized in Table 1. View this table: Table 1. Laboratory data. The patient was diagnosed with babesiosis and admitted to the hospital, where he was treated with atovaquone, azithromycin, and doxycycline. Follow-up molecular testing confirmed infection with Babesia microti. Parasitemia declined to

At presentation, variant or "look-alike" conditions can resemble TTP. We reviewed charts of 26 consecutive patients treated for presumed TTP. Of 15 classic TTP patients, 11 were tested for ADAMTS13; all showed severe deficiency, and inhibitor levels correlated with probability of relapse. The variant TMA group consisted of 8 patients who had active clinical disorders which overlapped with TTP. Variant TMA patients had higher creatinine and worse prognosis than classic TTP patients. "Look-alike" disorders included ITP with intravascular hemolysis following administration of WinRho™, and human granulocytic anaplasmosis. These conditions had not been previously described as TTP look-alikes.

Objective: Band neutrophil counts are helpful for the detection of neonatal sepsis. Our goal was to optimize our laboratory process to detect elevated band neutrophils whenever present, without performing unnecessary manual differential counts. Methods: We retrospectively tracked consecutive complete blood count with differential (CBCD) orders from neonatal intensive care unit (NICU) specimens according to the level of manual review. We identified specimens for which manual differential counts were performed due to telephone request and categorized them according to band percentage. Results: Our laboratory routinely performed manual scans on 96% of NICU specimens, but only 87% of specimens required manual differential counts. Abnormal band counts were present in 5 of 70 specimens for which manual differential counts were requested because of clinically suspected sepsis. We elected to perform universal manual scans on all NICU specimens. Conclusion: The revised laboratory process could be useful for other hospitals treating neonatal patients at risk of sepsis.

The proteome is the pool of proteins expressed at a given time and circumstance. The word 'proteomics' summarizes several technologies for visualization, quantitation and identification of these proteins. Recent advances in these techniques are helping to elucidate platelet processes which are relevant to bleeding and clotting disorders, transfusion medicine and regulation of angiogenesis.Over 1100 platelet proteins have been identified using proteomic techniques. Various subproteomes have been characterized, including platelet releasates (the 'secretome'), alpha and dense granules, membrane and cytoskeletal proteins, platelet-derived microparticles, and the platelet 'phosphoproteome'. Proteomic data about platelets have become increasingly available in integrated databases.Proteomic experiments in resting and activated platelets have identified novel signaling pathways and secreted proteins which may represent therapeutic targets, as well as potential cancer biomarkers.

BACKGROUND: Decisions for when to select, and when to discontinue, antigen-negative blood in hematopoietic progenitor cell transplantation (HPCT) recipients with red blood cell (RBC) antibodies can be confusing. In HPCT performed for sickle cell anemia patients who require extremely rare antigen-negative blood, the balance of caution and practicality is further complicated. CASE REPORTS: Four sickle cell anemia patients with current or historic RBC antibodies underwent allogeneic HPC transplantation. One required extremely rare (group O D–, hrB–) blood. None of the antibodies caused significant hemolysis after transplant. In the case requiring rare blood, antigen-negative blood was requested after donor RBC engraftment because of incomplete donor white blood cell (WBC) chimerism. CONCLUSIONS: RBC antibodies derived from a recipient of allogeneic HPCT rarely cause significant hemolysis, in contrast to the more severe picture sometimes seen with donor-derived antibodies. When donor WBC chimerism is delayed past the time of donor RBC engraftment, there can be concern for the possibility of future recipient-type antibody production. Even 100 percent donor lymphocyte chimerism is no guarantee of total host plasma cell ablation. Immunoglobulin allotyping, when informative, can suggest chimerism for several years. Recipient-type blood, when extremely rare, may not be available for that duration.

The T-domain of diphtheria toxin, which extends from residue 202 to 378, causes the translocation of the catalytic A fragment (residues 1-201) across endosomal membranes and also forms ion-conducting channels in planar phospholipid bilayers. The carboxy-terminal 57-amino acid segment (residues 322-378) in the T-domain is all that is required to form these channels, but its ability to do so is greatly augmented by the portion of the T-domain upstream from this. Here we show that in association with channel formation by the T-domain, its hydrophilic 63-amino acid NH2-terminal region (residues 202-264) as well as the entire catalytic A fragment (residues 1-201) cross the lipid bilayer. The phenomenon that enabled us to demonstrate this was the rapid closure of channels at cis negative voltages when a histidine tag was placed at various positions in the NH2-terminal region of the T-domain or in the A fragment; the inhibition of this effect by trans nickel established that the histidine tag was present on the trans side of the membrane. Thus, all of the machinery necessary to translocate the A fragment across membranes is built into the 114 residues at the carboxy-terminal end of the T-domain (residues 265-378), without the requirement of any proteins in the plasma membrane (e.g., toxin receptor) or of any other cellular components.

When diphtheria toxin encounters a low pH environment, the channel-forming T domain undergoes a poorly understood conformational change that allows for both its own membrane insertion and the translocation of the toxin's catalytic domain across the membrane. From the crystallographic structure of the water-soluble form of diphtheria toxin, a “double dagger” model was proposed in which two transmembrane helical hairpins, TH5-7 and TH8-9, anchor the T domain in the membrane. In this paper, we report the topography of the T domain in the open channel state. This topography was derived from experiments in which either a hexahistidine (H6) tag or biotin moiety was attached at residues that were mutated to cysteines. From the sign of the voltage gating induced by the H6 tag and the accessibility of the biotinylated residues to streptavidin added to the cis or trans side of the membrane, we determined which segments of the T domain are on the cis or trans side of the membrane and, consequently, which segments span the membrane. We find that there are three membrane-spanning segments. Two of them are in the channel-forming piece of the T domain, near its carboxy terminal end, and correspond to one of the proposed “daggers,” TH8-9. The other membrane-spanning segment roughly corresponds to only TH5 of the TH5-7 dagger, with the rest of that region lying on or near the cis surface. We also find that, in association with channel formation, the amino terminal third of the T domain, a hydrophilic stretch of ∼70 residues, is translocated across the membrane to the trans side.

The T domain of diphtheria toxin, which extends from residue 202 to 378, causes the translocation of the catalytic A fragment (residues 1–201) across endosomal membranes and also forms ion-conducting channels in planar phospholipid bilayers. The carboxy terminal 57-amino acid segment (322–378) in the T domain is all that is required to form these channels, but its ability to do so is greatly augmented by the portion of the T domain upstream from this. In this work, we show that in association with channel formation by the T domain, its NH2 terminus, as well as some or all of the adjacent hydrophilic 63 amino acid segment, cross the lipid bilayer. The phenomenon that enabled us to demonstrate that the NH2-terminal region of the T domain was translocated across the membrane was the rapid closure of channels at cis negative voltages when the T domain contained a histidine tag at its NH2 terminus. The inhibition of this effect by trans nickel, and by trans streptavidin when the histidine tag sequence was biotinylated, clearly established that the histidine tag was present on the trans side of the membrane. Furthermore, the inhibition of rapid channel closure by trans trypsin, combined with mutagenesis to localize the trypsin site, indicated that some portion of the 63 amino acid NH2-terminal segment of the T domain was also translocated to the trans side of the membrane. If the NH2 terminus was forced to remain on the cis side, by streptavidin binding to the biotinylated histidine tag sequence, channel formation was severely disrupted. Thus, normal channel formation by the T domain requires that its NH2 terminus be translocated across the membrane from the cis to the trans side, even though the NH2 terminus is >100 residues removed from the channel-forming part of the molecule.

HSCs are rare cells that have the unique ability to self-renew and differentiate into cells of all hematopoietic lineages. The lack of donors and current inability to rapidly and efficiently expand HSCs are roadblocks in the development of successful cell therapies. Thus, the challenge of ex vivo human HSC expansion remains a fertile and critically important area of investigation. Here, we show that either SALL4A- or SALL4B-transduced human HSCs obtained from the mobilized peripheral blood are capable of rapid and efficient expansion ex vivo by >10 000-fold for both CD34+/CD38− and CD34+/CD38+ cells in the presence of appropriate cytokines. We found that these cells retained hematopoietic precursor cell immunophenotypes and morphology as well as normal in vitro or vivo potential for differentiation. The SALL4-mediated expansion was associated with enhanced stem cell engraftment and long-term repopulation capacity in vivo. Also, we demonstrated that constitutive expression of SALL4 inhibited granulocytic differentiation and permitted expansion of undifferentiated cells in 32D myeloid progenitors. Furthermore, a TAT-SALL4B fusion rapidly expanded CD34+ cells, and it is thus feasible to translate this study into the clinical setting. Our findings provide a new avenue for investigating mechanisms of stem cell self-renewal and achieving clinically significant expansion of human HSCs.

Platelet hyper-reactivity is likely to play a role in cardiovascular disease, but there are no standardized tests to evaluate platelet responsiveness. A ‘platelet chip’ (a synthetic oligonucleotide microarray representing all platelet-restricted genes) is under development as a tool for high-throughput characterization of platelet-based bleeding and clotting disorders. In future, platelet gene profiling may be used to improve thrombohemorrhagic risk assessment and to guide antiplatelet therapy for patients at risk of cardiovascular disease.

The transcriptome is the mRNA pool found within a cell. Transcriptomic discovery approaches include microarray-based technologies as well as sequencing-based technologies. Transcriptomic experiments provide dynamic information about gene expression at the tissue level. The proteome is the pool of proteins expressed at a given time and circumstance. The word PROTEOMICS summarizes several technologies for visualization, quantitation, and identification of these proteins. Protein separation can be accomplished by two-dimensional electrophoresis, use of protein chips with an affinity matrix, or by a variety of advanced chromatographic methods. Mass spectrometry is used to identify the proteins in conjunction with protein sequence databases. Recent proteomic experiments in resting and activated platelets have identified novel signaling pathways and secreted proteins. Platelet transcriptomic studies in essential thrombocythemia, atherosclerotic disease, sickle cell disease, and an inherited platelet defect are reviewed. Transcript profiling has the potential to distinguish molecular signatures in normal and diseased platelets and to classify prothrombotic patient phenotypes to tailor their therapy.

The T domain of diphtheria toxin is known to participate in the pH-dependent translocation of the catalytic C domain of the toxin across the endosomal membrane, but how it does so, and whether cellular proteins are also required for this process, remain unknown. Here, we report results showing that the T domain alone is capable of translocating the entire C domain across model, planar phospholipid bilayers in the absence of other proteins. The T domain therefore contains the entire molecular machinery for mediating transfer of the catalytic domain of diphtheria toxin across membranes.

1. What is this patient’s red blood cell (RBC) antigen phenotype? 2. What is unusual about this patient’s RBC antigen phenotype? 3. What antibody is present in this patient’s serum, and what is its significance? 4. Is this patient an immunologic “non-responder,” since she never made an anti-k antibody despite 5 pregnancies and deliveries? 5. Should this patient receive k-negative blood even though she has not thus far made anti-k?

BackgroundWe sought to establish a metric for easily estimating bleeding and transfusion risks for cardiac surgery patients after antiplatelet agent use.MethodsDeidentified records of patients who underwent coronary artery bypass grafting (CABG) at our institution (January 2010-June 2011) were searched for patients without identified risk factors for excessive bleeding who underwent documented P2Y12 testing after clopidogrel administration (n = 276). Clinical outcomes were analyzed according to whether preoperative platelet function was higher (platelet reactivity units [PRUs], ≥237) or lower (PRU, 290, or no clopidogrel), intermediate (200-290), or low (437 mL) in 62 (42%) of the clopidogrel-treated patients. Lower-PRU patients were more likely to receive coagulation factors (odds ratio [OR], 2.82; P = .0004) and to have high CTO or coagulation factor transfusion (OR, 2.35; P = .02) than higher-PRU patients. Likewise, intermediate- and low-PRU patients had incrementally greater incidences of high CTO (OR, 1.72; P = .002) and coagulation factor transfusion (OR, 2.08; P

[Figure][1] A 96-year-old woman with a past medical history of hypertension, medication noncompliance, and lack of medical follow-up was brought to the emergency room with altered mental status. She was to found to have malignant hypertension with concomitant acute pontine hemorrhage. Her

Annexin A5 (A5) is a potent anticoagulant protein that crystallizes over phospholipid surfaces, shielding them from availability for coagulation enzyme reactions. The antiphospholipid antibody (aPL) syndrome (APS) is an autoimmune thrombophilia that is marked by the presence of antibodies against phospholipid-binding proteins and the paradoxical lupus anticoagulant phenomenon. aPL antibodies can promote coagulation by interfering with the crystallization of A5, thereby increasing the exposure of blood to thrombogenic anionic phospholipids. Since a flow cytometric assay for aPL measuring A5 binding to frozen thawed platelets was previously reported, we investigated whether phosphatidylserine-bound polystyrene beads could provide a robust platform for the assay. Beads were incubated with sera from patients with the aPL syndrome and from non-aPL controls. Fluorescence-tagged A5 was added and samples were analyzed by flow cytometry. Similarly treated beads were also used in coagulation assays to determine whether A5 anticoagulant function was also inhibited. Control sera permitted fluorescent labeling of nearly all beads. In contrast, sera from aPL syndrome patients produced a major population of unlabeled beads, sometimes accompanied by a minor population of labeled beads. 7 of 13 confirmed aPL syndrome patients, and 7 of 12 anticardiolipin antibody positive patients, demonstrated reduced A5 binding in this assay, while 10 of 10 healthy blood donor controls did not. Dilution of the aPL sera resulted in progressive increase of A5 binding. Reduction of A5 binding appeared to correlate with reduced A5 anticoagulant activity (r=0.42, n=33, p=.01). This assay shows promise for investigating the effects of aPL antibodies on A5 binding and for the clinical diagnosis of the aPL syndrome. Download : Download high-res image (186KB) Download : Download full-size image Figure Download : Download high-res image (184KB) Download : Download full-size image Figure

Hematopoietic stem cells (HSCs) are widely used in transplantation therapy to treat a variety of blood diseases. The success of hematopoietic recovery is of high importance and closely related to the patient’s morbidity and mortality after Hematopoietic stem cell transplantation (HSCT). We have previously shown that SALL4 is a potent stimulator for the expansion of human hematopoietic stem/progenitor cells in vitro. In these studies, we demonstrated that systemic administration with TAT-SALL4B resulted in expediting auto-reconstitution and inducing a 30-fold expansion of endogenous HSCs/HPCs in mice exposed to a high dose of irradiation. Most importantly, TAT-SALL4B treatment markedly prevented death in mice receiving lethal irradiation. Our studies also showed that TAT-SALL4B treatment was able to enhance both the short-term and long-term engraftment of human cord blood (CB) cells in NOD/SCID mice and the mechanism was likely related to the in vivo expansion of donor cells in a recipient. This robust expansion was required for the association of SALL4B with DNA methyltransferase complex, an epigenetic regulator critical in maintaining HSC pools and in normal lineage progression. Our results may provide a useful strategy to enhance hematopoietic recovery and reconstitution in cord blood transplantation with a recombinant TAT-SALL4B fusion protein.