Thrombin Generation in Cardiac Versus Noncardiac Surgical Cohorts.

BACKGROUND: Bleeding can be a significant problem after cardiac surgery. As a result, venous thromboembolism (VTE) or anticoagulation or both following mechanical valve implantation are often delayed in these patients. The calibrated automated thrombin (CAT) generation assay has become the gold standard to evaluate thrombin generation, a critical step in clot formation independent of other hemostatic processes (eg, platelet activation, fibrin cross-linking, and fibrinolysis), and is increasingly used to examine thrombotic and hemorrhagic outcomes. No study has currently used this assay to compare the thrombin generation profiles of cardiac surgical patients to noncardiac surgical patients. We hypothesize that noncardiac patients may be less prone to postoperative changes in thrombin generation. METHODS: A prospective, observational, cohort study was undertaken using blood samples from 50 cardiac and 50 noncardiac surgical patients preoperatively, immediately postoperatively, and on postoperative days 1 to 4. Platelet-poor plasma samples were obtained from patients preoperatively, on arrival to the postanesthesia care unit (PACU) or intensive care unit (ICU), and daily on postoperative days 1 to 4 if patients remained inpatient. Samples were evaluated for CAT measurements. Patient and surgical procedure characteristics were obtained from the electronic medical record. RESULTS: The primary outcome variable, median endogenous thrombin potential (ETP), measured in nanomolar × minutes (nM × min), was decreased 100% in cardiac surgical versus 2% in noncardiac patients (P < .001). All parameters of thrombin generation were similarly depressed. Cardiac (versus noncardiac) surgical type was associated with -76.5% difference of percent change in ETP on multivariable regression analysis (95% confidence interval [CI], -87.4 to -65.5; P value <.001). CONCLUSIONS: Cardiac surgical patients exhibit a profound decrease in thrombin generation postoperatively compared with noncardiac surgical patients evaluated by this study. Hemodilution and coagulation factor depletion likely contribute to this decreased thrombin generation after cardiac surgery.

Targeting Two Antigens Associated with B-ALL with CD19-CD123 Compound Car T Cell Therapy.

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.

Laboratory Monitoring of Platelet P2Y12 Receptor Inhibitors and Reversal of Antiplatelet Agents.

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.

Negative Heparin-Induced Thrombocytopenia Test Result After Massive Transfusion: Believe It or Not?

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.

Enhancing bone marrow regeneration by SALL4 protein.

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.

Platelet activity measured by a rapid turnaround assay identifies coronary artery bypass grafting patients at increased risk for bleeding and transfusion complications after clopidogrel administration.

BACKGROUND: We sought to establish a metric for easily estimating bleeding and transfusion risks for cardiac surgery patients after antiplatelet agent use. METHODS: Deidentified 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, <237) and according to preoperative PRU cutoffs: high (>290, or no clopidogrel), intermediate (200-290), or low (<200). RESULTS: Eighty-five patients (57%) received allogeneic blood products at 24 hours or less postoperatively: 33 (22%) received fresh frozen plasma, and 57 (38%) received platelets. The median 12-hour chest tube output (CTO) was 350 mL (interquartile range, 260-490 mL); CTO was "high" (>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 < .0001) than high-PRU/no clopidogrel patients. High CTO or coagulation factor transfusion was more frequent in intermediate-PRU (OR, 2.67; P = .02) and low-PRU (OR, 5.08; P = .0002) patients than in high-PRU/no clopidogrel patients. CONCLUSIONS: Among clopidogrel-treated CABG patients, preoperative platelet function testing can identify those at increased risk for postoperative bleeding and transfusion.

Contamination of patient blood samples by avian RBCs from control material during automated hematology analysis.

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.

SALL4 is a robust stimulator for the expansion of hematopoietic stem cells.

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.

The platelet proteome.

PURPOSE OF REVIEW: 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. RECENT FINDINGS: 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. SUMMARY: 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.

Genomic and proteomic applications in diagnosis of platelet disorders and classification.

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.

Transfusion policy: when to stop the use of extremely rare blood for an allogeneic hematopoietic progenitor cell transplant recipient with a history of red cell alloimmunization.

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-, hr(B)-) 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.