Lisa Senzel

The platelet proteome.

Purpose of reviewThe 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 findingsOver 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. SummaryProteomic 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.

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

BACKGROUNDnWe sought to establish a metric for easily estimating bleeding and transfusion risks for cardiac surgery patients after antiplatelet agent use.nnnMETHODSnDeidentified 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 (nxa0=xa0276). 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).nnnRESULTSnEighty-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; Pxa0=xa0.0004) and to have high CTO or coagulation factor transfusion (OR, 2.35; Pxa0=xa0.02) than higher-PRU patients. Likewise, intermediate- and low-PRU patients had incrementally greater incidences of high CTO (OR, 1.72; Pxa0=xa0.002) and coagulation factor transfusion (OR, 2.08; Pxa0<xa0.0001) than high-PRU/no clopidogrel patients. High CTO or coagulation factor transfusion was more frequent in intermediate-PRU (OR,xa02.67; Pxa0=xa0.02) and low-PRU (OR, 5.08; Pxa0=xa0.0002) patients than in high-PRU/no clopidogrel patients.nnnCONCLUSIONSnAmong clopidogrel-treated CABG patients, preoperative platelet function testing can identify those at increased risk for postoperative bleeding and transfusion.

The role of therapeutic apheresis in the treatment of acute antibody-mediated kidney rejection.

Approximately 10% of renal transplant recipients experience acute antibody‐mediated rejection (AMR) due to alloimmunization against human leukocyte antigen (HLA) molecules and other antigens. While therapeutic apheresis is included in most treatment protocols for acute kidney allograft rejection, these protocols have been derived mainly from single center experience rather than controlled trials. This concise review focuses on the role of therapeutic apheresis in AMR treatment. Two groups have recently reported treating acute AMR using drug‐only strategies without therapeutic apheresis in particular situations, namely in clinically less severe cases or in resource‐limited situations without testing for donor specific antibodies. A randomized controlled trial, designed to test the efficacy of immunoadsorption apheresis in AMR treatment, was terminated early and suggested a benefit of apheresis. An observational study suggested efficacy of plasmapheresis in acute AMR treatment, but all patients who received plasmapheresis also received rituximab. As new therapeutic modalities are becoming available, therapeutic apheresis continues to play a role in the treatment of acute kidney allograft rejection. J. Clin. Apheresis, 2012.

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.

Undetectable HDL Cholesterol in a Patient with Flu-Like Illness

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.nnOn 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.nnView this table:nnTable 1. nLaboratory data.nnnnThe 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 <0.1%, symptoms improved greatly, and he was discharged after 3 days. Ten weeks after initial presentation, anemia and thrombocytopenia had resolved. HDL-C had returned to a value within the reference interval (52 mg/dL or 1.34 mmol/L), and ferritin, which had been high, also was within the reference interval (293 ng/mL). …

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

OBJECTIVESnDiagnosis 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.nnnMETHODSnWe 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnReplacement 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.

Contamination of Patient Blood Samples by Avian RBCs From Control Material During Automated Hematology Analysis

OBJECTIVESnAtypical 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.nnnMETHODSnCBC 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.nnnRESULTSnWe experimentally reproduced the phenomenon of contamination of a subsequent tube with avian cells from a multiply punctured reticulocyte control tube.nnnCONCLUSIONSnWe concluded that the NRBCs likely represented avian RBCs from the Retic-C Cell Control that had been introduced into the patient tubes.

Thrombotic thrombocytopenic purpura and its look-alikes: a single institution experience.

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.

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.

Platelet transcriptome and cardiovascular disease

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.