Comparison of Viscoelastic Testing by Rotational Torsion and Harmonic Resonance Methods.

OBJECTIVES: To compare the performance of the TEG 5000 and TEG 6S Global Hemostasis cartridge. METHODS: We reviewed validation data of the TEG 5000 and TEG 6S Global Hemostasis cartridge. The specimens were analyzed in parallel according to the manufacturer's operating instructions. RESULTS: Fifty-four healthy donors and 13 donors with known hemostatic abnormalities were included. The correlations between instrument types were only moderate-the Spearman rank correlations were 0.55, 0.62, 0.64, and 0.72, respectively, for CK R, K, angle, and maximum amplitude (MA) parameters. Using the manufacturer's device-specific reference ranges to classify results as normal/abnormal, there was weak agreement in the qualitative interpretation of all parameters (Cohen's κ for agreement for CK R, K, angle, and MA was 0.418, 0.154, -0.083, and 0.127, respectively). This could lead to discordant transfusion decisions. CONCLUSIONS: These findings indicate that the TEG 5000 and TEG 6S may not be used interchangeably.

Functional Assessment of Platelet Dense Granule ATP Release.

OBJECTIVES: This study was undertaken to explore the feasibility of assessing platelet dense granule release in response to platelet stimuli, using less than 1 mL of whole blood (WB). METHODS: Optimization of the luciferin-luciferase (LL) assay for ATP release, together with additional modifications, was applied to 1:10 diluted WB. RESULTS: LL assay optimization using nonstirred 1:10 diluted WB resulted in dense granule ATP release in response to thrombin receptor-activating peptide (TRAP) of similar magnitude to that observed using stirred platelet-rich plasma. Stirring of the 1:10 diluted WB restored collagen-induced dense granule secretion. Addition of lyophilized, formalin-fixed platelets, together with stirring, restored dense granule secretion responsiveness to ADP. TRAP, ADP, and collagen all stimulated ATP release in 1:10 diluted WB under the optimized conditions of this study at levels close to those observed using platelet-rich plasma. Blood sample reconstitution experiments offer hope that this assay may prove robust down to WB platelet counts as low as 50 × 103/µL. CONCLUSIONS: Platelet dense granule release in response to a number of classic stimuli, including ADP, was accomplished from less than 1 mL WB with minimal specimen processing, using widely available reagents and instrumentation.

The Impact of COVID-19 Disease on Platelets and Coagulation.

Coronavirus disease 2019 (COVID-19) causes a spectrum of disease; some patients develop a severe proinflammatory state which can be associated with a unique coagulopathy and procoagulant endothelial phenotype. Initially, COVID-19 infection produces a prominent elevation of fibrinogen and D-dimer/fibrin(ogen) degradation products. This is associated with systemic hypercoagulability and frequent venous thromboembolic events. The degree of D-dimer elevation positively correlates with mortality in COVID-19 patients. COVID-19 also leads to arterial thrombotic events (including strokes and ischemic limbs) as well as microvascular thrombotic disorders (as frequently documented at autopsy in the pulmonary vascular beds). COVID-19 patients often have mild thrombocytopenia and appear to have increased platelet consumption, together with a corresponding increase in platelet production. Disseminated intravascular coagulopathy (DIC) and severe bleeding events are uncommon in COVID-19 patients. Here, we review the current state of knowledge of COVID-19 and hemostasis.

A Descriptive and Quantitative Immunohistochemical Study Demonstrating a Spectrum of Platelet Recruitment Patterns Across Pulmonary Infections Including COVID-19.

OBJECTIVES: Pulmonary platelet deposition and microangiopathy are increasingly recognized components of coronavirus disease 2019 (COVID-19) infection. Thrombosis is a known component of sepsis and disseminated intravascular coagulation. We sought to compare the level of platelet deposition in the pulmonary vasculature in cases of confirmed COVID-19 infection to other lung injuries and infections. METHODS: Immunohistochemistry was performed on 27 autopsy cases and 2 surgical pathology cases targeting CD61. Multiple cases of normal lung, diffuse alveolar damage, COVID-19, influenza, and bacterial and fungal infections, as well as one case of pulmonary emboli, were included. The levels of CD61 staining were compared quantitatively in the autopsy cases, and patterns of staining were described. RESULTS: Nearly all specimens exhibited an increase in CD61 staining relative to control lung tissue. The area of CD61 staining in COVID-19 infection was higher than influenza but still comparable to many other infectious diseases. Cases of aspiration pneumonia, Staphylococcus aureus infection, and blastomycosis exhibited the highest levels of CD61 staining. CONCLUSIONS: Platelet deposition is a phenomenon common to many pulmonary insults. A spectrum of staining patterns was observed, suggestive of pathogen-specific mechanisms of platelet deposition. Further study into the mechanisms driving platelet deposition in pulmonary injuries and infections is warranted.

Improved outcomes after radiotherapy for prostate cancer: Anticoagulation, antiplatelet therapy, and platelet count as key factors in disease progression.

BACKGROUND: Several studies have suggested that antiplatelet (AP) or anticoagulant (AC) therapy may improve outcome in men with prostate cancer. We evaluated the effects of AP/AC therapy and tested the hypothesis that platelet count may also be associated with outcomes. METHODS: A total of 482 patients received primary radiotherapy (median dose 72 Gy) for nonmetastatic prostate cancer; 49% received androgen deprivation therapy. NCCN risk was low/intermediate/high risk in 39%/39%/22%. AP/AC therapy and platelet counts were analyzed with respect to freedom from biochemical failure (FFBF, nadir+2), distant metastasis (FFDM), and cause specific survival (CSS). RESULTS: After a median follow-up of 103 months, 10-year FFBF, FFDM, and CSS were 77%, 92%, and 96%, respectively. The 10-year cumulative incidence of BF and DM (with death as a competing event) was 19% and 7.0%, respectively. The 32% of men on AP/AC therapy had a lower incidence of 10-year BF (P = .016) and a trend toward a lower incidence of DM (P = .084) and CSS (P = .091). In the entire cohort, lowest platelet quartile (platelet count <187) was associated with higher 10-year BF (31% vs 16%, P = .0042) but not DM (9.4% vs 5.2%, P = .22) nor CSS (P = .76) compared with those patients with platelet count ≥187. AP/AC therapy was associated with a larger absolute reduction in BF for men with lowest platelet quartile (10-year BF of 21% vs 38%, P = .092) vs platelet ≥187 (10-year BF of 10% vs 18%, P = .053). Lowest platelet quartile remained associated with higher BF and DM on multivariable analysis controlling for risk category, WBC, and Hg. CONCLUSION: AP/AC was associated with improved FFBF. Low platelet count was associated with inferior FFBF and FFDM after prostate radiotherapy. This association was tempered when antiplatelet and anticoagulant therapy was administered.

Development of a Nonradioactive Platelet Serotonin Uptake and Release Assay by Micro-Liquid Chromatography Tandem Mass Spectrometry Using Minimal Blood Volume.

OBJECTIVES: Analysis of platelet functional responses to stimuli is presently quite limited with respect to measurement of dense granule secretion. We sought to develop a nonradioactive assay of stimulated serotonin release using liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS: Citrated whole blood (200 µL) was incubated with deuterated serotonin (d45-HT). Following uptake by platelets, blood was diluted 10-fold and aliquots were incubated with platelet stimuli. Following stimulation, blood was further diluted, centrifuged, and supernatant was assayed for released d45-HT by micro-LC-MS/MS. RESULTS: This study demonstrated a broad linear range of 50 to 2,000 pg/mL d45-HT, with a total precision of less than 15.0% coefficient of variation at all quality control levels and a limit of quantitation of 50 pg/mL. CONCLUSIONS: Quantification of d45-HT by micro-LC-MS/MS assay offers a highly sensitive, nonradioactive methodology for quantitating platelet serotonin uptake and dense granule secretion, requiring only small volumes of patient blood.

Green Machine: A Card Game Introducing Students to Systems Thinking in Green Chemistry by Strategizing the Creation of a Recycling Plant.

Green Machine is a competitive strategy card game facilitating a systems thinking approach to learning recycling processes and green chemistry in accordance with the United Nations Sustainable Development Goals. Players compete to be the first to be able to launch their recycling plant by collecting a series of playing cards. Players must use interpersonal skills to consider the interconnected systems while showing an appreciation for commercial awareness and versatility, as dynamic problem solving (reflecting real-world scenarios) is required to play the game successfully. The card game was implemented with 19 U.K. graduate students and 29 U.S. second-year undergraduate students. Survey feedback showed that Green Machine was an innovative resource that was enjoyable to play and engaged students in learning recycling processes through systems thinking. On the basis of pre- and post-test questions to evaluate learning gain, Green Machine is a helpful resource to introduce students not only to green chemistry and sustainability but also to taking a systems thinking approach to learning.

Role of Sphingomyelin in Alphaherpesvirus Entry.

Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that causes disease in cattle populations worldwide. Sphingomyelin (SM) is the most abundant sphingolipid in the mammalian cell membrane, where it preferentially associates with cholesterol to form lipid raft domains. SM is a substrate for the lysosome-resident enzyme acid sphingomyelinase, which plays a role in cell membrane repair following injury. Treatment of cells with noncytotoxic concentrations of Staphylococcus aureus-derived sphingomyelinase successfully reduced cell surface-exposed sphingomyelin but did not significantly inhibit BoHV-1 entry and infection, as measured by the beta-galactosidase reporter assay. Interestingly, entry of the porcine alphaherpesvirus pseudorabies virus (PRV) was inhibited by sphingomyelin-depletion of cells. Treatment of BoHV-1 particles with sphingomyelinase inhibited viral entry activity, suggesting that viral SM plays a role in BoHV-1 entry, while cellular SM does not. Treatment of cells with noncytotoxic concentrations of the functional inhibitors of host acid sphingomyelinase, imipramine and amitriptyline, which induce degradation of the cellular enzyme, did not significantly inhibit BoHV-1 entry. In contrast, inhibition of cellular acid sphingomyelinase inhibited PRV entry. Entry of the human alphaherpesvirus herpes simplex virus 1 (HSV-1) was independent of both host SM and acid sphingomyelinase, in a manner similar to BoHV-1. Together, the results suggest that among the alphaherpesviruses, there is variability in entry requirements for cellular sphingomyelin and acid sphingomyelinase activity.IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is an ubiquitous pathogen affecting cattle populations worldwide. Infection can result in complicated, polymicrobial infections due to the immunosuppressive properties of the virus. Available vaccines limit disease severity and spread but do not prevent infection. The financial and animal welfare ramifications of BoHV-1 are significant. In order to develop more effective prevention and treatment regimens, a more complete understanding of the initial steps in viral infection is necessary. We recently identified a low pH endocytosis pathway for BoHV-1. Here, we examine the role of cellular factors responsible for membrane integrity and repair in alphaherpesviral entry. This study allows comparisons of the BoHV-1 entry pathway with those of other alphaherpesviruses (pseudorabies virus [PRV] and herpes simplex virus 1 [HSV-1]). Lastly, this is the first report of sphingomyelin and lysosomal sphingomyelinase playing a role in the entry of a herpesvirus. The results may lead to the development of more effective prevention and treatment regimens.

Low-pH Endocytic Entry of the Porcine Alphaherpesvirus Pseudorabies Virus.

The alphaherpesvirus pseudorabies virus (PRV) is the causative agent of pseudorabies, a disease of great economic and welfare importance in swine. Other alphaherpesviruses, including herpes simplex virus (HSV), utilize low-pH-mediated endocytosis to enter a subset of cell types. We investigated whether PRV used this entry pathway in multiple laboratory model cell lines. Inhibition of receptor-mediated endocytosis by treatment with hypertonic medium prevented PRV entry. PRV entry into several cell lines, including porcine kidney (PK15) cells and African green monkey kidney (Vero) cells, was inhibited by noncytotoxic concentrations of the lysosomotropic agents ammonium chloride and monensin, which block the acidification of endosomes. Inactivation of virions by acid pretreatment is a hallmark of viruses that utilize a low-pH-mediated entry pathway. Exposure of PRV virions to pH 5.0 in the absence of host cell membranes reduced entry into PK15 and Vero cells by >80%. Together, these findings suggest that endocytosis followed by fusion with host membranes triggered by low endosomal pH is an important route of entry for PRV.IMPORTANCE PRV is a pathogen of great economic and animal welfare importance in many parts of the world. PRV causes neurological, respiratory, and reproductive disorders, often resulting in mortality of young and immunocompromised animals. Mortality, decreased production, and trade restrictions result in significant financial losses for the agricultural industry. Understanding the molecular mechanisms utilized by PRV to enter host cells is an important step in identifying novel strategies to prevent infection and spread. A thorough understanding of these mechanisms will contribute to a broader understanding of alphaherpesvirus entry. Here, we demonstrate PRV entry into multiple model cell lines via a low-pH endocytosis pathway. Together, these results provide a framework for elucidating the early events of the PRV replicative cycle.

Intracranial hemorrhage as initial manifestation of plasma cell myeloma: A case report.

This is the case of a previously healthy 48 year-old male whom presented with mild confusion, low-grade headache, and left sided weakness. Computed tomography of the head revealed a large acute right frontal lobe intracranial hemorrhage (ICH) and intraventricular extension, with normal vascular imaging. Initial laboratory testing was inconsequential. The patient required emergent evacuation, with pathology revealing only elements of a hematoma. Further laboratory testing and bone marrow biopsy results confirmed the diagnosis of plasma cell myeloma. Other systemic signs/symptoms of this disease were notably absent. This report provides the first description of an ICH as the presenting manifestation of plasma cell myeloma (PCM; multiple myeloma).

Acquired factor XI deficiency and therapeutic plasma exchange.

Congenital factor XI (FXI) deficiency is associated with a variable bleeding phenotype. Recent reports have documented the use of therapeutic plasma exchange to rapidly and isovolumetrically increase FXI levels before invasive procedures in patients with congenital FXI deficiency. We report a case of acquired FXI deficiency in a pregnant woman with lupus. We proved that the inhibitor was an IgG, therefore potentially capable of crossing the placenta. While immune suppression eliminated detectable circulating inhibitor, the woman's FXI remained quite low. A multi-disciplinary team was formed and therapeutic plasma exchange with 100% plasma replacement was performed when the patient went into labor, to acutely raise her FXI level and remove any potential non-neutralizing inhibitor. The mother had a controllable level of bleeding during post-TPE cesarean section; the baby had no bleeding and the baby's FXI levels were not overtly abnormal. Therapeutic plasma exchange in acquired FXI deficiency (or other acquired hemophilias) can both acutely isovolumetrically raise factor levels and remove any circulating inhibitor.

Therapeutic plasma exchange as part of multimodal treatment of acquired hemophilia in a patient with concurrent acute intracerebral bleed and pulmonary embolism.

BACKGROUND: Autoantibodies against Factor VIII (FVIII) define the rare but life-threatening bleeding disorder acquired hemophilia A (AHA). Correction of FVIII deficiency and eradication of the factor inhibitor are the ultimate therapeutic goals in this disorder. Bypassing agents such as recombinant factor VIIa (rFVIIa) or FVIII inhibitor bypassing agent are often used to control coagulopathy before the inhibitor is eradicated. Bypassing agents carry a risk of thrombosis, however. CASE REPORT: We report a patient with newly diagnosed AHA and thalamic bleed who additionally had active atrial fibrillation and developed a segmental pulmonary embolism, limiting tolerable rFVIIa dosage. This patient with very-high-risk brain bleed and concurrent thrombosis on bypass agent represented a significant management dilemma for which we successfully utilized therapeutic plasma exchange (TPE) to reduce the inhibitor titer. RESULTS: FVIII inhibitor was undetectable and FVIII level was above the lower limit of normal within 12.5 days from starting TPE. While the patient ultimately died 24 days from admission for reasons unrelated to bleeding, his intracerebral hemorrhage was unchanged in size and no other bleeding morbidity was observed. CONCLUSION: This patient achieved eradication of FVIII inhibitor and did not have bleed expansion while receiving multimodal therapy including corticosteroids, rituximab, and TPE. We discuss the periprocedural risks of TPE in an acquired hemophilia patient and our multiteam management of that risk.

Estrogens antagonize RUNX2-mediated osteoblast-driven osteoclastogenesis through regulating RANKL membrane association.

In addition to its thoroughly investigated role in bone formation, the osteoblast master transcription factor RUNX2 also promotes osteoclastogenesis and bone resorption. Here we demonstrate that 17ß-estradiol (E2), strongly inhibits RUNX2-mediated osteoblast-driven osteoclastogenesis in co-cultures. Towards deciphering the underlying mechanism, we induced premature expression of RUNX2 in primary murine pre-osteoblasts, which resulted in robust differentiation of co-cultured splenocytes into mature osteoclasts. This was attributable to RUNX2-mediated increase in RANKL secretion, determined by ELISA, as well as to RUNX2-mediated increase in RANKL association with the osteoblast membrane, demonstrated using confocal fluorescence microscopy. The increased association with the osteoblast membrane was recapitulated by transiently expressed GFP-RANKL. E2 abolished the RUNX2-mediated increase in membrane-associated RANKL and GFP-RANKL, as well as the concomitant osteoclastogenesis. RUNX2-mediated RANKL cellular redistribution was attributable in part to a decrease in Opg expression, but E2 did not influence Opg expression either in the presence or absence of RUNX2. Diminution of RUNX2-mediated osteoclastogenesis by E2 occurred regardless of whether the pre-osteoclasts were derived from wild type or estrogen receptor alpha (ERα)-knockout mice, suggesting that activated ERα inhibited osteoblast-driven osteoclastogenesis by acting in osteoblasts, possibly targeting RUNX2. Indeed, microarray analysis demonstrated global attenuation of the RUNX2 response by E2, including abrogation of Pstpip2 expression, which likely plays a critical role in membrane trafficking. Finally, the selective ER modulators (SERMs) tamoxifen and raloxifene mimicked E2 in abrogating the stimulatory effect of osteoblastic RUNX2 on osteoclast differentiation in the co-culture assay. Thus, E2 antagonizes RUNX2-mediated RANKL trafficking and subsequent osteoclastogenesis. Targeting RUNX2 and/or downstream mechanisms that regulate RANKL trafficking may lead to the development of improved SERMs and possibly non-hormonal therapeutic approaches to high turnover bone disease.

Investigation of the cause of death in a gene-therapy trial.

We present a case of disseminated histoplasmosis, complicated by retroperitoneal bleeding and leading to death, in a patient who was receiving systemic immunosuppressive therapy for rheumatoid arthritis and who was enrolled in a gene-therapy trial. This trial was designed to evaluate intraarticular delivery of a tumor necrosis factor alpha (TNF-alpha) antagonist, through an adeno-associated virus (AAV) type 2 delivery system, for inflammatory arthritis. The patient's receipt of concurrent anti-TNF-alpha therapy and other immunosuppressive therapy while she was living in an area where histoplasmosis was endemic was thought to be the most likely explanation for the infection; the evidence presented suggests that this fatal infection was unlikely to have been related to exposure to the agent administered in the gene-therapy trial. This case reinforces the importance of considering infectious complications, such as those from endemic mycoses, in patients receiving treatment with a TNF-alpha antagonist and the importance of having a well-designed monitoring plan when subjects in a research study become ill. (ClinicalTrials.gov number, NCT00126724.)

Glycoprotein analysis for the diagnostic evaluation of platelet disorders.

Platelet glycoproteins subserve a wide variety of critical functions in blood platelets. Congenital deficiencies or functional abnormalities in platelet glycoproteins may produce serious bleeding disorders such as Glanzmann thrombasthenia or Bernard-Soulier syndrome. Other hematologic disorders, such as Fanconi anemia and various myelodysplastic syndromes, may also be associated with abnormalities in platelet glycoproteins. Additionally, several acquired disorders involving the major platelet glycoproteins are increasingly being recognized. The large number of techniques, now available to characterize platelet glycoprotein disorders, reflect the many advances in biochemistry, molecular analysis, flow cytometry, and, most recently, proteomics. The application of platelet glycoprotein analysis to a wide range of clinical disorders is reviewed in this article.

Resident training in laboratory hematology.

The authors of this article formulate a strategy to guide residents and faculty in laboratory hematology. The authors build upon the recommendations of the Academy of Clinical Laboratory Physicians and Scientists (ACLPS) published earlier as well as draw from their own experiences, in discussing principles that should be considered when implementing a program that effectively trains residents to be competent pathologists in the various settings that they may encounter once they complete training.

Presence of direct thrombin inhibitors can affect the results and interpretation of lupus anticoagulant testing.

In vitro experiments were conducted to determine whether the direct thrombin inhibitors argatroban and lepirudin can interfere with the results of lupus anticoagulant (LA) testing. Concentration-response curves were generated to calculate the concentration of anticoagulant that prolongs the activated partial thromboplastin time (aPTT) to 75 seconds (2.5 times the baseline average). Corresponding concentrations of anticoagulant were added to plasma samples before running dilute Russell viper venom tests (DRVVTs) and LA-sensitive aPTTs (PTT-LAs). Because the DRVVT test system contains an antiheparin agent, DRVVT results were not prolonged in the presence of heparin. With argatroban added to normal plasma samples, neither the DRVVT percent correction of ratio nor the DRVVT test/confirm ratio were elevated, but when added to LA-positive plasma, some false-negative results were observed. Lepirudin increased the DRVVT percent correction of ratio and the DRVVT test/confirm ratio into a range that could lead to false-positive identifications of LAs. In sharp contrast to the DRVVT test system, distinction between LA-positive and LA-negative plasma samples was maintained and possibly even enhanced in the platelet neutralization procedure correction phase of the PTT-LA test system.

Alterations in the intrinsic properties of the GPIbalpha-VWF tether bond define the kinetics of the platelet-type von Willebrand disease mutation, Gly233Val.

Platelet-type von Willebrand disease (PTVWD) is a bleeding disorder in which an increase of function mutation in glycoprotein Ibalpha (GPIbalpha), with respect to binding of von Willebrand factor (VWF), results in a loss of circulating high molecular weight VWF multimers together with a mild-moderate thrombocytopenia. To better ascertain the specific perturbations in adhesion associated with this disease state, we performed a detailed analysis of the kinetic and mechanical properties of tether bonds formed between PT-VWD platelets and the A1-domain of VWF. Results indicate that the GPIbalpha mutation, Gly233Val, promotes and stabilizes platelet adhesion to VWF at shear rates that do not support binding between the native receptor-ligand pair due to enhanced formation and increased longevity of the mutant tether bond (k0 off values for mutant versus native complex of 0.67 +/- 0.11 s-1 and 3.45 +/- 0.37 s-1, respectively). By contrast, the sensitivity of this interaction to an applied force, a measure of bond strength, was similar to the wild-type (WT) receptor. Although the observed alterations in the intrinsic properties of the GPIbalpha-VWF tether bond are comparable to those reported for the type 2B VWD, distinct molecular mechanisms may be responsible for these function-enhancing bleeding disorders, as interactions between the mutant receptor and mutant ligand resulted in a greater stability in platelet adhesion. We speculate that the enhanced cellular on-rate together with the prolongation in the lifetime of the mutant receptor-ligand bond contributes to platelet aggregation in circulating blood by permitting the formation of multiple GPIbalpha-VWF-A1 interactions.