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International audience; Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin therapy. It is due to the synthesis of antibodies most often directed against platelet factor 4 (FP4) modified by heparin (H). HIT is manifested by a platelet count fall, associated with a high risk of venous or arterial thrombosis. The diagnosis of HIT is based on the assessment of clinical probability (4Ts score or change in platelet count after cardiac surgery) and the demonstration of heparin-modified anti-FP4 antibodies (FP4/H). If the immunological tests are positive, functional tests should be performed. In case of suspicion of HIT, it is necessary to urgently stop heparin therapy, to perform a doppler ultrasound of the lower limbs, and to prescribe an alternative anticoagulation agent at a curative dose. Currently, danaparoid sodium and argatroban are authorized. The diagnosis and management of HIT remain complex and requires multidisciplinary collaboration. (C) 2021 Societe Nationale Francaise de Medecine Interne (SNFMI).

In response to the COVID-19 pandemic, vaccines for SARS-CoV-2 were developed, tested, and introduced at a remarkable speed. Although the vaccine introduction had a major impact on the evolution of COVID-19, some potential rare side-effects of the vaccines were observed. Within a short period, three scientific groups from Norway, Germany, and the UK reported cerebral venous sinus thrombosis with thrombocytopenia and anti-platelet factor 4 (anti-PF4) antibodies in individuals following AstraZeneca-Oxford vaccination and named this new syndrome vaccine-induced immune thrombotic thrombocytopenia (VITT). This syndrome was subsequently reported in individuals who received Johnson & Johnson vaccination. In this Viewpoint, we discuss the epidemiology, pathophysiology, and optimal diagnostic and therapeutic management of VITT. Presentation of an individual with possible VITT should raise prompt testing for anti-PF4 antibodies and initiation of treatment targeting autoimmune processes with intravenous immunoglobulin and prothrombotic processes with non-heparin anticoagulation.

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

In late February 2021, the first cases of vaccine-induced thrombosis and thrombocytopenia (VITT) were observed in patients after immunization with the ChAdOx1 nCoV-19 adenoviral vector vaccine (Oxford, AstraZeneca) against SARS-CoV-2.1 This prothrombotic response is presumed to be mediated by anti-platelet factor 4 (PF4) antibodies that activate platelets and induce activation and extracellular traps formation of neutrophils.2 These antibodies can also be seen in autoimmune heparin-induced thrombocytopenia, but all the described cases had no previous exposure to heparin.

Objectives Recent years have brought new developments in interventional chronic pain management, namely regenerative orthopedics utilizing platelet-rich plasma (PRP) as well as further evolution of pulsed radiofrequency neuromodulation (PRF). Both methods have been used separately. Here, we investigated whether PRF may potentiate the activation of platelets in PRP samples when both these techniques are combined together in in vitro conditions. Materials and methods Studies were performed on concentrated PRP samples (PRPs) obtained from acid citrate dextrose-treated blood taken from 11 healthy volunteers. PRPs were divided into four groups: 1) nonactivated PRP; 2) thrombin-activated PRP as a positive control for maximal platelets activation; 3) PRF-treated PRP exposed for 20 min to PRF energy generated by neurotherm radio frequency generator at 500 kHz, with a voltage of 40 V and maximal temperature of 42°C; and 4) a combination of groups 2 and 3. Results PRF-induced platelet activation measured by platelet factor 4 (PF4) and ATP release from PRPs was significantly higher compared to nonactivated PRPs, and similar to PF4 and ATP release from thrombin-activated PRPs. Thrombin activation did not potentiate PF4 release in PRF samples and even reduced ATP level. Additionally, PRF neither induced any platelet membrane damage measured by lactic dehydrogenase release from PRP nor modified any platelets viability or metabolism measured by MTT. Conclusions We confirmed that PRF may activate PRP without additional platelet activators. So, a combination of both methods PRF and PRP application may provide a more effective opportunity for tissue regeneration in dentistry, surgery, dermatology, or in orthopedics.

INTRODUCTION The development of anti-platelet factor 4 (PF4) antibodies is linked to a rare thrombotic complication described now as vaccine-induced immune thrombotic thrombocytopenia (VITT). This clinical syndrome with thrombosis and thrombocytopenia was reported after exposure to the Oxford-AstraZeneca COVID-19 vaccine, ChAdOx1 nCoV-19 vaccine (AZD1222), and Ad26.COV2.S vaccine (Janssen/Johnson & Johnson). In the absence of the clinical features, the incidence of positive anti-PF4 antibodies in asymptomatic individuals post-vaccination is unclear. METHODS The aim of this study was to evaluate the development of anti-PF4 antibodies in asymptomatic individuals 14-21 days after receiving the first dose of ChAdOx1 nCoV-19 vaccine (AZD1222) and BNT162b2 vaccine. Prospectively, we collected serum from individuals before and after ChAdOx1 nCoV-19 vaccine and BNT162b2 vaccine and measured anti-PF4 antibodies using the Asserachrom HPIA IgG ELISA (Stago, Asnieres, France). RESULTS We detected positive anti-PF4 antibodies in 5 of 94 asymptomatic individuals post-vaccine with a rate of 5.3% with low titers (OD 0.3-0.7). Four of 5 individuals who tested positive after the vaccine had also positive anti-PF4 antibodies before the vaccine, which indicates that a majority of the positive results are due to preexisting anti-PF4 antibodies. We did not find a relation between the development of anti-PF4 antibodies and the immune response to the vaccine, status of prior COVID-19 infection, and baseline characteristics of participants. None of the participants developed thrombosis nor thrombocytopenia. CONCLUSION Our results provide new evidence to guide the diagnostic algorithm of suspected cases of VITT. In the absence of thrombosis and thrombocytopenia, there is a low utility of testing for anti-PF4 antibodies.

We report a case of vaccine-induced immune thrombotic thrombocytopenia (VITT) in a young man diagnosed 13 days after Ad26.COV2.S COVID-19 (Johnson & Johnson/Janssen) vaccination. He presented to us with 5 days of progressive left leg pain, thrombocytopenia, hypofibrinogenemia, and markedly elevated d-dimers, but without radiographically demonstrable thrombosis. Despite negative imaging, we initiated treatment of presumptive VITT given the striking clinical picture that included the timing of his recent adenovirus-based COVID-19 vaccine, leg symptoms, marked thrombocytopenia, and consumptive coagulopathy. He received intravenous immune globulin, prednisone, and argatroban and was discharged 7 days later much improved. His positive platelet factor 4 enzyme-linked immunosorbent assay antibody test returned after treatment was initiated. To our knowledge, this is the first reported case of VITT following Ad26.COV2.S vaccination presenting without radiographically demonstrable thrombosis. Our patient highlights the importance of knowing vaccine status and initiating treatment as soon as possible in the right clinical setting, even in the absence of radiographic evidence of thrombus. Early VITT recognition and treatment provide an opportunity to prevent serious thrombotic complications.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a life-threatening complication described after administration of recombinant adenoviral vector encoding the spike protein antigen of Severe Acute Respiratory Syndrome CoronaVirus-2. The syndrome is characterized by platelet consumption and thrombosis. High levels of antibodies to platelet factor 4 (PF4)–polyanion complexes were identified in many patients with VITT by enzyme-linked immunosorbent assay (ELISA). A 64-year-old woman presented with thrombocytopenia, right renal vein thrombosis with renal infarction, right intra-right atrium and intra-right ventricle thrombosis and pulmonary embolism after ChAdOx1-S vaccine administration. ELISA for antibodies to PF4–polyanion complexes tested positive, while functional tests were not. Thrombocytopenia was refractory to intravenous immunoglobulins and corticosteroids. Eltrombopag was introduced and platelet gradually rose to normal values. VITT is a novel complication yet to be understood. The clinical case we reported highlights the difficulties in the management of this disorder and discloses a new potential therapy in refractory conditions.

BACKGROUND: Cases of ChAdOx1 nCoV-19 (AstraZeneca) vaccinated patients with thrombocytopenia, elevated D-dimer, and elevated platelet factor 4 (PF4) antibody levels with- and without thrombosis have been reported. No recommendations regarding the duration of anticoagulation have been made, because data on the long-term course beyond the first weeks is lacking. OBJECTIVE: To report on the treatment, medical course, and longitudinal follow-up of laboratory parameters in patients with vaccine-induced prothrombotic immune thrombocytopenia (VIPIT). PATIENTS: We followed VIPIT patients with- (n = 3) and without (n = 3) venous thromboembolism fulfilling the aforementioned laboratory criteria. RESULTS: Elevated D-dimer (median: 35.10 µg/ml, range: 17.80-52.70), thrombocytopenia (42 G/l, 20-101), and strong positivity in the platelet factor 4 (PF4)/heparin-enzyme-immunoassay (2.42 optical density [OD], 2.06-3.13; reference range

A series of cases with rare thromboembolic incidents including cerebral sinus vein thrombosis (some of them fatal) and concomitant thrombocytopenia occurring shortly after vaccination with the coronavirus disease 2019 (COVID-19) vaccine AZD1222 (Vaxzevria) have caused significant concern and led to its temporary suspension in many countries. Immediate laboratory efforts in four of these patients have identified a tentative pathomechanism underlying this syndrome termed initially vaccine-induced prothrombotic immune thrombocytopenia (VIPIT) and renamed recently vaccine-induced immune thrombotic thrombocytopenia (VITT). It encompasses the presence of platelet-activating antibodies to platelet factor-4/heparin complexes, possibly emulated by polyanionic constituents of AZD1222, and thus resembles heparin-induced thrombocytopenia (HIT). Because these immune complexes bind and activate platelets via Fcγ receptor IIA (FcγRIIA), high-dose intravenous immunoglobulin G has been suggested for treatment of VITT in addition to non-heparin anticoagulants. Here we propose inhibitors of Bruton tyrosine kinase (Btk) approved for B cell malignancies (e.g., ibrutinib) as another therapeutic option in VITT, as they are expected to pleiotropically target multiple pathways downstream of FcγRIIA-mediated Btk activation, for example, as demonstrated for the effective inhibition of platelet aggregation, dense granule secretion, P-selectin expression and platelet-neutrophil aggregate formation stimulated by FcγRIIA cross-linking. Moreover, C-type lectin-like receptor CLEC-2- and GPIb-mediated platelet activation, the interactions and activation of monocytes and the release of neutrophil extracellular traps, as encountered in HIT, could be attenuated by Btk inhibitors. As a paradigm for emergency repurposing of approved drugs in COVID-19, off-label use of Btk inhibitors in a low-dose range not affecting haemostatic functions could thus be considered a sufficiently safe option to treat VITT.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a newly described hematologic disorder, which presents as acute thrombocytopenia and thrombosis after administration of the ChAdOx1 nCov-19 (AstraZeneca) and Ad26.COV2.S (Johnson & Johnson) adenovirus-based vaccines against COVID-19. Due to positive assays for antibodies against platelet factor 4 (PF4), VITT is managed similarly to autoimmune heparin-induced thrombocytopenia (HIT) with intravenous immunoglobulin (IVIG) and non-heparin anticoagulation. We describe a case of VITT in a 50-year-old man with antecedent alcoholic cirrhosis who presented with platelets of 7 × 103 /μL and portal vein thrombosis 21 days following administration of the Ad26.COV2.S COVID-19 vaccine. The patient developed progressive thrombosis and persistent severe thrombocytopenia despite IVIG, rituximab and high-dose steroids and had persistent anti-PF4 antibodies over 30 days after his initial presentation. As such, delayed therapeutic plasma exchange (TPE) was pursued on day 32 of admission as salvage therapy, with a sustained improvement in his platelet count. Our case serves as proof-of-concept of the efficacy of TPE in VITT.

We hypothesize that thrombosis with thrombocytopenia syndrome recently described after administration of adenovirus‐vectored vaccines for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) occurs as a result of the unique properties of the adenovirus vectors, which can have widespread biodistribution throughout the body. The antigen is delivered to megakaryocyte cells, which act as part of the primary immune system and distribute the antigen within progeny platelets, also a key component of the immune system. The interaction of the antigen induces preformed antiplatelet factor 4 (PF4) antibodies to bind to PF4–heparan sulfate complexes in the absence of exogenous heparin, at sites where the heparan sulfate concentration in the vascular glycocalyx is optimal for complex formation, causing thrombosis and thrombocytopenia as observed clinically. This hypothesis is testable in cell culture and animal models, and potentially in vivo, and if proven correct has significant implications for vaccine development and our understanding of the links between the coagulation and immune systems., We hypothesize that thrombosis with thrombocytopenia syndrome recently described after administration of adenovirus‐vectored vaccines for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) occurs as a result of the unique properties of the adenovirus vectors, which can have widespread biodistribution throughout the body. The antigen is delivered to megakaryocyte cells, which act as part of the primary immune system and distribute the antigen within progeny platelets, also a key component of the immune system. The interaction of the antigen induces preformed antiplatelet factor 4 (PF4) antibodies to bind to PF4–heparan sulfate complexes in the absence of exogenous heparin, at sites where the heparan sulfate concentration in the vascular glycocalyx is optimal for complex formation, causing thrombosis and thrombocytopenia as observed clinically.

Thrombocytosis has been feared as a source of thrombotic complications during the conduct of cardiopulmonary bypass (CPB) for patients undergoing cardiac procedures. We present a patient urgently requiring repair/replacement of three heart valves that had preexisting myelofibrosis with thrombocytosis (platelet count of 800,000 per µl) and neutrophilia (40,000 per µl). Despite achieving an activated clotting time > 500 s with heparin and antithrombin concentrate administration prior to CPB, the pump oxygenator and reservoir demonstrated significant clot just prior to restoration of the patient’s circulation. The patient subsequently suffered a severe protamine reaction that was successfully managed. A review of the literature of similar patients and the relevant cellular and biochemical mechanisms in this setting are presented, with potential therapeutic approaches to prevent such complications noted.

Blood component transfusion is often used as the primary therapy as it is still considered safe. Platelet Concentrate (PC) transfusion plays a critical role in preventing bleeding in patients with severe thrombocytopenia. Allergic reactions are the most frequent transfusion reactions after PC administration. Regulated on Activation Normal T-Cell Expressed and Secreted (RANTES) and Platelet Factor 4 (PF4) cytokines released by platelets during PC storage are responsible for allergic reactions after transfusion. The purpose of this study was to analyze changes in RANTES and PF4 levels during PC storage. This study was an observational analytical research with a time series design carried out at the Clinical Pathology Laboratory and Blood Bank of the Dr. Soetomo Hospital, Surabaya, from June to July 2019. RANTES and PF4 levels in 27 bags derived from Platelet Rich Plasma (PRP) on storage for day 1, day 3, and day five were measured using the ELISA sandwich method. Subject same variant test or Friedman test was used for statistical analysis. The results showed no significant differences in RANTES and PF4 levels based on the storage duration of PCs on days 1, 3, and 5, with p=0.717, and p=0.614, respectively. There was no difference in the storage of PCs from day 1 to day five, and there was no effect on allergic reactions after PC transfusion.

Herein, we report a case of a new-onset Raynaud’s phenomenon (RP), which occurred in an otherwise healthy 31-year-old Caucasian woman, who lacked any known risk factors and associations with possible causes for secondary RP. However, 2 weeks prior to the development of RP, the patient had received her first injection of the COVID-19 vaccine containing ChAdOx1-SARS-COV-2. The patient presented with well-demarcated, white-pale, cold areas involving the middle fingers of both hands and the ring finger of the right hand, which were triggered by exposure to cold environment and accompanied by a sensation of numbness. Infrared thermography revealed notable temperature differences of up to 10.9°C between affected and nonaffected fingers. Coagulation and immunological parameters, including cryoglobulins and pathological autoantibodies, were within the normal range and antibodies to the heparin/platelet factor 4 complex not detectable. It remains unclear if the development of RP in our patient is causally related to antecedent COVID-19 vaccination; however, the temporal connection to the vaccination, the complete absence of RP in her past medical history, and the lack of any risk factors and triggers raise the suspicion of a yet unknown association with the vaccine. Whether a clear association between the development of RP and COVID-19 vaccination exists or whether RP represents a bystander effect needs to be awaited in case observational reports on RP accumulate. Given the steadily rising numbers of people receiving COVID-19 vaccinations, physicians may remain alert to still unrecognized side effects.

BACKGROUND: A substantial proportion of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe/critical coronavirus disease 2019 (COVID-19) characterized by acute respiratory distress syndrome (ARDS) with thrombosis. OBJECTIVES: We tested the hypothesis that SARS-CoV-2--induced upregulation of tissue factor (TF) expression may be responsible for thrombus formation in COVID-19. METHODS: We compared autopsy lung tissues from 11 patients with COVID-19--associated ARDS with samples from 6 patients with ARDS from other causes (non-COVID-19 ARDS) and 11 normal control lungs. RESULTS: Dual RNA in situ hybridization for SARS-CoV-2 and TF identified sporadic clustered SARS-CoV-2 with prominent co-localization of SARS-CoV-2 and TF RNA. TF expression was 2-fold higher in COVID-19 than in non-COVID-19 ARDS lungs (P = .017) and correlated with the intensity of SARS-CoV-2 staining (R2 = .36, P = .04). By immunofluorescence, TF protein expression was 2.1-fold higher in COVID-19 versus non-COVID-19 ARDS lungs (P = .0048) and 11-fold (P

Supplemental Digital Content is available in the text., In the spring of 2021, reports of rare and unusual venous thrombosis in association with the ChAdOx1 and Ad26.COV2.S adenovirus-based coronavirus vaccines led to a brief suspension of their use by several countries. Thromboses in the cerebral and splanchnic veins among patients vaccinated in the preceding 4 weeks were described in 17 patients out of 7.98 million recipients of the Ad26.COV2.S vaccine (with 3 fatalities related to cerebral vein thrombosis) and 169 cases of cerebral vein thrombosis among 35 million ChAdOx1 recipients. Events were associated with thrombocytopenia and anti-PF4 (antibodies directed against platelet factor 4), leading to the designation vaccine-induced immune thrombotic thrombocytopenia. Unlike the related heparin-induced thrombotic thrombocytopenia, with an estimated incidence of

Background Vaccine-induced immune thrombocytopenia and thrombosis (VITT) following the administration of the AstraZeneca (AZ) ChAdOx1 nCOV-19 vaccine has recently been reported. The associated clinical and laboratory features have included thrombosis at unusual sites, thrombocytopenia, and raised D-dimers with positivity for IgG anti-platelet factor 4 (PF4) antibodies. Objectives A UK National External Quality Control Assessment Scheme external quality control exercise was carried out by distributing liquid and lyophilized samples from a subject with VITT, a pool of samples from subjects with classical heparin-induced thrombocytopenia (HIT), and a non-VITT/non-HIT case to 85 centers performing HIT testing. Methods Participating centers employed their locally validated testing methods for HIT assays. Results The lyophilized and liquid samples were found to be commutable for the ELISA assays used in the detection of anti-PF4 antibodies. The Aeskulisa, Stago, Hyphen, and LIFECODES anti-PF4 ELISA assays successfully detected the VITT antibody, whereas the Acustar HIT, Werfen LIA, and the Stago STIC assays did not. Conclusion It is important that clinical and laboratory teams are aware of the limitations of some anti-PF4 assays when using them to aid diagnosis of VITT syndrome.

Vaccine-induced immune thrombosis and thrombocytopenia (VITT) syndrome is a new entity, characterised by severe thrombocytopenia and multiple sites of thrombosis1 . The presumed mechanism is driven by anti-platelet factor 4 (PF4) antibodies causing platelet activation1 . Donors with VITT syndrome have a particular relevance for liver transplantation (LT), due to the associated risk of passenger lymphocyte syndrome, and a predisposition for porto-mesenteric or hepatic vein thrombosis2 .

Shortly after the worldwide initiation of vaccination against SARS-CoV-2, concerns emerged about a possible link between vaccination, severe thrombocytopenia, and the development of atypical venous thrombosis. Concerns were primarily about AstraZeneca (ChAdOx1 nCov-19), later Johnson & Johnson (Ad26.COV2.S), but cases of acute immune thrombocytopenic purpura (ITP) and bleeding without thrombosis and also atypical venous thrombosis after exposure to the messenger RNA-based vaccines produced by Pfizer-BioNTech and Moderna have been reported. Examination of the circumstances of these complications revealed that this is a similar mechanism to heparin-induced thrombocytopenia (HIT), a prothrombotic thrombocytopenic hypercoagulable disorder with venous and arterial thrombosis. HIT is caused by platelet-activating IgG antibodies directed against an antigen that is a macromolecular complex consisting of platelet factor 4 (PF4) and heparin. Naming this new entity vaccine-induced immune thrombotic thrombocytopenia (VITT) was suggested to avoid confusion with HIT. Patients had high levels of antibodies to the immune complex formed by PF 4 and the polyanionic component of the vaccine (double-stranded DNA). In patients with thrombosis at any vascular site after vaccination, accompanied by absolute or relative thrombocytopenia and systemic manifestations, HIT Ig ELISA assay to detect antibodies against PF4 and platelet-activating functional tests may be used for VITT recognition and differentiation from venous thromboembolic disease. Immune globulin impedes antibody-mediated platelet clearance and down-regulate platelet activation by immune complexes, as in HIT. It is prudent to choose from among the nonheparin antithrombotic agents – direct oral F.Xa inhibitors, direct thrombin inhibitors and indirect F.Xa inhibitors for the treatment of thrombosis. © 2021, SOLEN s.r.o.. All rights reserved.

Diabetic nephropathy (DN) is a common complication of diabetes. The Notch pathway plays an important role in podocyte injury and decreases the expression of podocyte-specific proteins in DN. However, the exact mechanisms are still elusive. The purpose of this study was to investigate the effect of the Notch pathway on podocyte injury by regulating Kruppel-like factor 4 (KLF4) in high glucose (HG)-induced podocytes. The expression of the Notch pathway and KLF4 was regulated in HG-induced podocytes. Western blotting was used to detect the protein expression of Notch1, Notch intracellular domain 1 (NICD1), KLF4, nephrin, and integrin β1. The mRNA levels of Notch1, KLF4, nephrin, and integrin β1 were determined by real-time polymerase chain reaction. HG increased the expression of Notch1 and NICD1 in podocytes and inhibited the expression of KLF4 (p

Background Consistent with fulminant endothelial cell activation, elevated plasma von Willebrand factor (VWF) antigen levels have been reported in patients with COVID‐19. The multimeric size and function of VWF are normally regulated through A Disintegrin And Metalloprotease with ThrombSpondin Motif type 1 motif, member 13 (ADAMTS‐13)‐‐mediated proteolysis. Objectives This study investigated the hypothesis that ADAMTS‐13 regulation of VWF multimer distribution may be impaired in severe acute respiratory syndrome‐coronavirus‐2 (SARS‐CoV‐2) infection contributing to the observed microvascular thrombosis. Patients and Methods Patients with COVID‐19 (n = 23) were recruited from the Beaumont Hospital Intensive Care Unit (ICU) in Dublin. Plasma VWF antigen, multimer distribution, ADAMTS‐13 activity, and known inhibitors thereof were assessed. Results We observed markedly increased VWF collagen‐binding activity in patients with severe COVID‐19 compared to controls (median 509.1 versus 94.3 IU/dl). Conversely, plasma ADAMTS‐13 activity was significantly reduced (median 68.2 IU/dl). In keeping with an increase in VWF:ADAMTS‐13 ratio, abnormalities in VWF multimer distribution were common in patients with COVID‐19, with reductions in high molecular weight VWF multimers. Terminal sialylation regulates VWF susceptibility to proteolysis by ADAMTS‐13 and other proteases. We observed that both N‐ and O‐linked sialylation were altered in severe COVID‐19. Furthermore, plasma levels of the ADAMTS‐13 inhibitors interleukin‐6, thrombospondin‐1, and platelet factor 4 were significantly elevated. Conclusions These findings support the hypothesis that SARS‐CoV‐2 is associated with profound quantitative and qualitative increases in plasma VWF levels, and a multifactorial down‐regulation in ADAMTS‐13 function. Further studies will be required to determine whether therapeutic interventions to correct ADAMTS‐13‐VWF multimer dysfunction may be useful in COVID‐microvascular thrombosis and angiopathy.

To describe the prevalence of heparin-induced thrombocytopenia (HIT) in patients on extracorporeal membrane oxygenation (ECMO) and to explore ways to improve the diagnostic accuracy of the HIT enzyme-linked immunosorbent assay (ELISA). Retrospective review of all patients needing ECMO between September 2011 and September 2020 who underwent evaluation for HIT while on ECMO. The diagnosis of HIT required a confirmatory serotonin release assay (SRA). Various break points for the optical density (OD) that defines a positive HIT ELISA were examined to estimate their utility as screening tests for HIT. Patient outcomes served as a secondary endpoint. Among 417 ECMO patients, 162 (38.8%) had a HIT ELISA. Of these, 114 (70.4%) had a subsequent SRA. Although the HIT ELISA was positive at an OD ≥ 0.4 in 1/3rd of subjects, only 15 subjects met criteria for HIT by SRA. Hence, the prevalence of HIT equaled 3.6%. At an OD ≥ 0.4 the ELISA had both poor specificity (71.7%) and accuracy (74.6%). Changing the definition of the ELISA to an OD ≥ 1.2 improved both specificity and accuracy with only a limited impact on sensitivity. Nearly 60% of those with HIT developing during ECMO died. HIT is infrequent in persons requiring ECMO. However, HIT remains associated with substantial mortality. The HIT ELISA as currently implemented performs poorly as a screening test and likely results in the unnecessary overuse of alternatives to unfractionated heparin. Altering the definition of a positive OD improves the HIT ELISA’s accuracy.

Background Anti-heparin/platelet factor 4 (PF4) antibodies may trigger severe thrombotic complications in hemodialysis (HD) patients. Tetrameric PF4 has a high affinity for extracellular DNA, which is a key component of neutrophil extracellular traps (NETs); therefore, the interactions between anti-heparin/PF4 antibodies and NETs can contribute to prothrombotic events. Methods Anti-heparin/PF4 antibody levels were measured by enzyme-linked immunosorbent assay; an optical density > 1.8 was regarded as clinically significant. In incident HD patients, we additionally measured serum nucleosome levels as representative markers of NETs, and the contributions of anti-heparin/PF4 and increased serum nucleosome levels to the primary functional patency loss of vascular access was assessed. Results The frequency of anti-heparin/PF4 antibodies was significantly higher in incident HD patients compared to prevalent HD patients (23.6% vs. 7.7%). Serum nucleosome levels, as well as the white blood cell counts, neutrophil counts, and high-sensitivity C-reactive protein levels, were significantly higher in anti-heparin/PF4 antibody-positive patients compared to the control. Platelet counts tended to be lower in the patients with anti-heparin/PF4 of >1.8 than in the controls. Relative risk calculations showed that the presence of anti-heparin/PF4 antibodies increased the risk of primary functional patency failure by 4.28-fold, and this risk increased further with higher nucleosome levels. Furthermore, in the anti-heparin/PF4 antibody-positive group, the time to first vascular intervention was much shorter, and the risk of repeated intervention was higher, compared to the controls. Conclusion In incident HD patients, the presence of anti-heparin/PF4 antibodies was associated with increased NET formation; this could be a strong predictor of vascular access complications.

Heparin-induced thrombocytopenia (HIT) is characterized clinically by thrombocytopenia, hypercoagulability, and increased thrombosis risk, and serologically by platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies. Heparin-"induced" acknowledges that HIT is usually triggered by a proximate immunizing exposure to heparin. However, certain non-heparin medications (pentosan polysulfate, hypersulfated chondroitin sulfate, fondaparinux) can trigger "HIT". Further, naturally-occurring polyanions (bacterial lipopolysaccharide, DNA/RNA) can interact with PF4 to recapitulate HIT antigens. Indeed, immunologic presensitization to naturally-occurring polyanions could explain why HIT more closely resembles a secondary, rather than a primary, immune response. In 2008 it was first reported that a HIT-mimicking disorder can occur without any preceding exposure to heparin or polyanionic medications. Termed "spontaneous HIT syndrome", two subtypes are recognized: (a) surgical (post-orthopedic, especially post-total knee arthroplasty, and (b) medical (usually post-infectious). Recently, COVID-19 adenoviral vector vaccination has been associated with a thrombotic thrombocytopenic disorder associated with positive PF4-dependent enzyme-immunoassays and serum-induced platelet activation that is maximal when PF4 is added. Vaccine-induced immune thrombotic thrombocytopenia (VITT) features unusual thromboses (cerebral venous thrombosis, splanchnic vein thrombosis) similar to those seen in spontaneous HIT syndrome. The emerging concept is that classic HIT reflects platelet-activating anti-PF4/heparin antibodies whereas spontaneous HIT syndrome and other atypical "autoimmune HIT" presentations (delayed-onset HIT, persisting HIT, heparin "flush" HIT) reflect heparin-independent platelet-activating anti-PF4 antibodies-although the precise relationships between PF4 epitope targets and the clinical syndromes remain to be determined. Treatment of spontaneous HIT syndrome includes non-heparin anticoagulation (direct oral Xa inhibitors favored over direct thrombin inhibitors) and high-dose immunoglobulin.

Contains fulltext : 238007.pdf (Publisher’s version ) (Closed access) IMPORTANCE: Cases of cerebral venous sinus thrombosis in combination with thrombocytopenia have recently been reported within 4 to 28 days of vaccination with the ChAdOx1 nCov-19 (AstraZeneca/Oxford) and Ad.26.COV2.S (Janssen/Johnson & Johnson) COVID-19 vaccines. An immune-mediated response associated with platelet factor 4/heparin antibodies has been proposed as the underlying pathomechanism. OBJECTIVE: To determine the frequencies of admission thrombocytopenia, heparin-induced thrombocytopenia, and presence of platelet factor 4/heparin antibodies in patients diagnosed with cerebral venous sinus thrombosis prior to the COVID-19 pandemic. DESIGN, SETTING, AND PARTICIPANTS: This was a descriptive analysis of a retrospective sample of consecutive patients diagnosed with cerebral venous sinus thrombosis between January 1987 and March 2018 from 7 hospitals participating in the International Cerebral Venous Sinus Thrombosis Consortium from Finland, the Netherlands, Switzerland, Sweden, Mexico, Iran, and Costa Rica. Of 952 patients, 865 with available baseline platelet count were included. In a subset of 93 patients, frozen plasma samples collected during a previous study between September 2009 and February 2016 were analyzed for the presence of platelet factor 4/heparin antibodies. EXPOSURES: Diagnosis of cerebral venous sinus thrombosis. MAIN OUTCOMES AND MEASURES: Frequencies of admission thrombocytopenia (platelet count 0.4, in a subset of patients with previously collected plasma samples). RESULTS: Of 865 patients (median age, 40 years [interquartile range, 29-53 years], 70% women), 73 (8.4%; 95% CI, 6.8%-10.5%) had thrombocytopenia, which was mild (100-149 ×103/μL) in 52 (6.0%), moderate (50-99 ×103/μL) in 17 (2.0%), and severe (

Differentiation therapy is a less toxic but still a very effective treatment for a subset of acute myeloid leukaemia (AML) cases. With the goal to identify novel compounds that can effectively and safely induce the terminal differentiation of non-acute promyelocytic leukaemia (APL) AML cells, we performed a chemical screening and identified albendazole (ABZ), a widely used anti-helminthic drug, as a promising lead compound that can differentiate non-APL AML cells by stimulating the Krüppel-like factor 4-dihydropyrimidinase-like 2A (KLF4-DPYSL2A) differentiation axis to the monocytes. Our in vitro and in vivo findings demonstrate that ABZ is an attractive candidate drug as a novel differentiation chemotherapy for patients with non-APL AML.

Cases of unusual thrombosis and thrombocytopenia after administration of the ChAdOx1 nCoV-19 vaccine (AstraZeneca) have been reported. The term vaccine-induced prothrombotic immune thrombocytopenia (VIPIT) was coined to reflect this new phenomenon. In vitro experiments with VIPIT patient sera indicated that high-dose intravenous immunoglobulins (IVIG) competitively inhibit the platelet-activating properties of ChAdOx1 nCoV-19 vaccine induced antibodies. Here, we report a case of a 62-year-old woman who had received this vaccine and developed VIPIT. She visited the emergency ward because of petechiae and hematomas. In the laboratory work-up, thrombocytopenia, low fibrinogen, elevated D-dimer, and positivity in the platelet factor 4/heparin-enzyme-immunoassay were present. Signs and symptoms of thrombosis were absent. Upon immediate therapy with non-heparin anticoagulation, high-dose IVIG, and prednisolone, laboratory parameters steadily improved and the patient was discharged from hospital without thrombotic complications. We conclude that early initiation of VIPIT treatment results in a swift response without thrombotic complications.

Background Coronavirus disease 2019 vaccine ChAdOx1 nCov-19 may rarely lead to vaccine-induced thrombotic thrombocytopenia (VITT). Antibody-mediated, platelet factor 4 (PF4)-dependent platelet activation appears to resemble a key mechanism in VITT, partially comparable to heparin-induced thrombocytopenia. The use of PF4/heparin immunoassays has been proposed as part of a diagnostic approach, but their sensitivity has not been established. Methods Sera from 12 well-defined VITT patients were first studied by two different laboratories in functional assays. Sera where then used for an interlaboratory comparison, in which five different PF4/heparin immunoassays were used by four laboratories. Results Results for functional testing were highly concordant. VITT antibodies were also reliably detected by PF4/heparin enzyme-linked immunosorbent assays (ELISAs) (92–100%). In contrast, only 25% of VITT antibodies were reactive in a particle gel immunoassay (PaGIA), and 8% in a lateral flow assay (LFA). An automated chemiluminescence immunoassay (CLIA) was negative for all sera tested (0%). Conclusion It seems feasible to establish functional antibody testing for the confirmation of VITT. For the initial screening of suspected VITT cases, PaGIA, LFA, and CLIA are useless when applied as single tests. Only ELISA-based PF4/heparin immunoassays are sensitive enough to be incorporated in the diagnostic workup. However, a combination of a positive ELISA and a negative CLIA may be useful to identify VITT antibodies in the absence of confirmatory functional assays.

Advanced SARS-CoV-2 infections not uncommonly associate with the occurrence of silent or manifest thrombotic events which may be found as focal or systemic disease. Given the potential complexity of COVID-19 illnesses, a multifactorial causation is likely, but several studies have focused on infection-induced coagulopathy. Procoagulant states are commonly found in association with the finding of antiphospholipid antibodies. The correlation of the latter with thrombosis and/or clinical severity remains controversial. Although measures of antiphospholipid antibodies most commonly include assessments for lupus anticoagulant, anticardiolipin, and anti-ß2-glycoprotein-I antibodies, lesser common antibodies have been detected, and there remains speculation that other yet undiscovered autoimmune thrombotic events may yet be found. The recent discovery of post-vaccination thromboses associated with platelet factor 4 antibody has created another level of concern. The pathogenesis of antiphospholipid antibodies and their role in COVID-19-related thrombosis deserves further attention. The multifactorial nature of thrombosis associated with both infection and vaccination should continue to be studied as new events unfold. Even if a cause-and-effect relationship is variable at best, such dedicated research is likely to generate other valuable insights that are applicable to medicine generally.

Background Several cases of unusual thrombotic events and thrombocytopenia have developed after vaccination with the recombinant adenoviral vector encoding the spike protein antigen of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (ChAdOx1 nCov-19, AstraZeneca). More data were needed on the pathogenesis of this unusual clotting disorder. Methods We assessed the clinical and laboratory features of 11 patients in Germany and Austria in whom thrombosis or thrombocytopenia had developed after vaccination with ChAdOx1 nCov-19. We used a standard enzyme-linked immunosorbent assay to detect platelet factor 4 (PF4)–heparin antibodies and a modified (PF4-enhanced) platelet-activation test to detect platelet-activating antibodies under various reaction conditions. Included in this testing were samples from patients who had blood samples referred for investigation of vaccine-associated thrombotic events, with 28 testing positive on a screening PF4–heparin immunoassay. Results Of the 11 original patients, 9 were women, with a median age of 36 years (range, 22 to 49). Beginning 5 to 16 days after vaccination, the patients presented with one or more thrombotic events, with the exception of 1 patient, who presented with fatal intracranial hemorrhage. Of the patients with one or more thrombotic events, 9 had cerebral venous thrombosis, 3 had splanchnic-vein thrombosis, 3 had pulmonary embolism, and 4 had other thromboses; of these patients, 6 died. Five patients had disseminated intravascular coagulation. None of the patients had received heparin before symptom onset. All 28 patients who tested positive for antibodies against PF4–heparin tested positive on the platelet-activation assay in the presence of PF4 independent of heparin. Platelet activation was inhibited by high levels of heparin, Fc receptor–blocking monoclonal antibody, and immune globulin (10 mg per milliliter). Additional studies with PF4 or PF4–heparin affinity purified antibodies in 2 patients confirmed PF4-dependent platelet activation. Conclusions Vaccination with ChAdOx1 nCov-19 can result in the rare development of immune thrombotic thrombocytopenia mediated by platelet-activating antibodies against PF4, which clinically mimics autoimmune heparin-induced thrombocytopenia. (Funded by the German Research Foundation.)

Intracerebral hemorrhage (ICH) is the most devastating subtype of stroke with high morbidity and mortality. The previous study has confirmed the therapeutic effect of Baihui (DU20)-penetrating-Qubin (GB7) acupuncture on ICH, while the related mechanism is left to be revealed. The aim of this study was to investigate the relevant mechanisms. ICH rat models were established utilizing the autologous blood injection method and the beneficial effect was found after DU20-penetrating-GB7 acupuncture along with decreased miR-34a-5p levels in the perihemorrhagic penumbra. Inversely, upregulating miR-34a-5p expression inhibited microglia M2 polarization while accelerated M1 polarization through targeting Kruppel-like factor 4 (Klf4), and thereby diminished the protective effect of DU20-penetrating-GB7 acupuncture on ICH. The results suggested the therapeutic effect of DU20-penetrating-GB7 acupuncture on ICH might be attributed to its modulation on microglia polarization through miR-34a-5p/Klf4 signaling.

Vaccine-induced thrombosis and thrombocytopenia (VITT) may follow immunisation with the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2. Autoantibodies to platelet factor 4 (PF4) may mediate VITT through antibody-dependent platelet activation, though the underlying etiology is uncertain. Anti-PF4 antibodies are also seen in heparin-induced thrombocytopenia, though most cases of VITT do not have prior heparin exposure. More than 20 million people in the United Kingdom (UK) have received the ChAdOx1 nCoV-19 vaccine.

COVID‐19 (coronavirus disease 2019) represents a prothrombotic disorder, and there have been several reports of platelet factor 4/heparin antibodies being present in COVID‐19‐infected patients. This has thus been identified in some publications as representing a high incidence of heparin‐induced thrombocytopenia (HIT), whereas in others, findings have been tempered by general lack of functional reactivity using confirmation assays of serotonin release assay (SRA) or heparin‐induced platelet aggregation (HIPA). Moreover, in at least two publications, data are provided suggesting that antibodies can arise in heparin naïve patients or that platelet activation may not be heparin‐dependent. From this literature, we would conclude that platelet factor 4/heparin antibodies can be observed in COVID‐19‐infected patients, and they may occur at higher incidence than in historical non‐COVID‐19‐infected cohorts. However, the situation is complex, since not all platelet factor 4/heparin antibodies may lead to platelet activation, and not all identified antibodies are heparin‐dependent, such that they do not necessarily reflect “true” HIT. Most recently, a “HIT‐like” syndrome has reported in patients who have been vaccinated against COVID‐19. Accordingly, much more is yet to be learnt about the insidious disease that COVID‐19 represents, including autoimmune outcomes in affected patients.

Vaccination using the adenoviral vector COVID-19 vaccine ChAdOx1 nCoV-19 (AstraZeneca) has been associated with rare vaccine-induced immune thrombotic thrombocytopenia (VITT). Affected patients test strongly positive in platelet factor 4 (PF4)/polyanion enzyme immunoassays (EIAs), and serum-induced platelet activation is maximal in the presence of PF4. We determined the frequency of anti-PF4/polyanion antibodies in healthy vaccinees and assessed whether PF4/polyanion EIA+ sera exhibit platelet-activating properties after vaccination with ChAdOx1 nCoV-19 (n = 138) or BNT162b2 (BioNTech/Pfizer; n = 143). In total, 19 of 281 participants tested positive for anti-PF4/polyanion antibodies postvaccination (All: 6.8% [95% confidence interval (CI), 4.4-10.3]; BNT162b2: 5.6% [95% CI, 2.9-10.7]; ChAdOx1 nCoV-19: 8.0% [95% CI, 4.5% to 13.7%]). Optical densities were mostly low (between 0.5 and 1.0 units; reference range

Vaccination is crucial in combatting the severe acute respiratory syndrome coronavirus 2 pandemic. The rare complication of thrombocytopenia and thrombotic complications at unusual sites after ChAdOx1 nCov-19 vaccination is caused by platelet-activating antibodies directed against platelet factor 4 (PF4). We present a widely applicable whole-blood standard flow cytometric assay to identify the pathogenic antibodies associated with vaccine-induced immune-mediated thrombotic thrombocytopenia (VITT) after ChAdOx1 nCov-19 vaccination. This assay will enable rapid diagnosis by many laboratories. This trial was registered at www.clinicaltrials.gov as #NCT04370119.

We describe the first Danish case of presumed inflammatory and thrombotic response to vaccination with an adenoviral (ChAdOx1) vector based COVID-19 vaccine (AZD1222). The case describes a 60-year old woman, who was admitted with intractable abdominal pain seven days after receiving the vaccine. Computer Tomography (CT) of the abdomen revealed bilateral adrenal hemorrhages. On the following day she developed a massive right sided ischemic stroke and Magnetic Resonance Imaging (MRI) angiography showed occlusion of the right internal carotid artery. The ischemic area was deemed too large to offer reperfusion therapy. During admission, blood tests showed a remarkable drop in platelet counts from 118,000 to 5,000 per µL and a substantial increase in D-dimer. The patient died on the 6th day of hospitalization. Blood test revealed platelet factor 4 (PF-4) reactive antibodies, imitating what is seen in heparin-induced thrombocytopenia. This may be a novel immune mediated response to the vaccine.

Background: Hyperfibrinolysis and pro/anti-inflammatory imbalance usually occur in the early stage of severe burns. Soluble urokinase-type plasminogen activator receptor (suPAR) is involved in fibrinolysis and inflammation. To date, the levels of circulating suPAR in non-survivors with severe burns remain unknown. This study aimed to investigate the early association between circulating suPAR levels and biomarkers of fibrinolysis, pro/anti-inflammatory, and prognosis. Methods: Sixty-four consecutive Chinese patients with severe burns and 26 healthy volunteers were enrolled in a prospective observational cohort. Clinical characteristics and laboratory data were collected prospectively. Blood samples were collected at 48 h post-burn, and suPAR and biomarkers of pro/anti-inflammatory and fibrinolysis were detected by enzyme-linked immunosorbent assays. Important indicators between non-survivors and survivors were compared. Linear regression analysis was performed to screen variables associated with suPAR. Logistic regression analysis and receiver operating characteristic curve (ROC) analysis were performed to evaluate the prognostic value of suPAR. Result: Compared with the control group, the circulating suPAR levels in the survivors (P

Background Functional platelet activation assays, such as the serotonin release assay (SRA), are the gold standard for the diagnosis of heparin-induced thrombocytopenia (HIT). Recently, platelet activation assays using added platelet factor 4 (PF4) have been described and suggest improved sensitivity. Direct comparisons of these assays have not been performed. Objective We compare the performance characteristics of three PF4-enhanced platelet activation assays, the PF4/heparin-SRA (PF4/hep-SRA), the PF4-SRA, and the P-selectin expression assay (PEA), at a single reference laboratory. Methods Serum samples from two cohorts of patients were used. The referral cohort (n = 84) included samples that had previously undergone routine diagnostic testing for HIT and tested positive or negative using the SRA. The clinical cohort (n = 101) consisted of samples from patients with clinically confirmed HIT whose serum contained platelet-activating antibodies. We simultaneously tested all samples in PF4-enhanced SRA-based assays (PF4/hep-SRA, PF4-SRA) and the flow cytometry-based PEA. Results In the referral cohort, the three PF4-enhanced assays identified all samples that were previously determined to be positive in the SRA. However, specificity of the PF4/hep-SRA was 96.6%, the PF4-SRA was 84.7%, and the PEA was 67.8%. In the clinical cohort of samples, all SRA-based assays displayed high performance characteristics (>92.1% sensitivity, >98.4% specificity). Sensitivity and specificity of the PEA was the lowest, 65.8% and 63.5%, respectively; but improved to 92.1% and 96.8% using preselected platelet donors. Conclusions All PF4-enhanced assays demonstrated good performance characteristics when platelet donors were preselected. Further comparisons across multiple laboratories should be conducted for consensus on optimal HIT diagnostic testing.

Background The mechanism by which immune cells regulate metastasis is unclear. Understanding the role of immune cells in metastasis will guide the development of treatments improving patient survival. Methods We used syngeneic orthotopic mouse tumour models (wild-type, NOD/scid and Nude), employed knockout (CD8 and CD4) models and administered CXCL4. Tumours and lungs were analysed for cancer cells by bioluminescence, and circulating tumour cells were isolated from blood. Immunohistochemistry on the mouse tumours was performed to confirm cell type, and on a tissue microarray with 180 TNBCs for human relevance. TCGA data from over 10,000 patients were analysed as well. Results We reveal that intratumoral immune infiltration differs between metastatic and non-metastatic tumours. The non-metastatic tumours harbour high levels of CD8+ T cells and low levels of platelets, which is reverse in metastatic tumours. During tumour progression, platelets and CXCL4 induce differentiation of monocytes into myeloid-derived suppressor cells (MDSCs), which inhibit CD8+ T-cell function. TCGA pan-cancer data confirmed that CD8lowPlatelethigh patients have a significantly lower survival probability compared to CD8highPlateletlow. Conclusions CD8+ T cells inhibit metastasis. When the balance between CD8+ T cells and platelets is disrupted, platelets produce CXCL4, which induces MDSCs thereby inhibiting the CD8+ T-cell function.

Adenoviral vector vaccines are effective against SARS-CoV-2 but have been associated with a rare side effect termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Here, we discuss our understanding of how vaccine-induced antibodies to platelet factor 4 (PF4) form immune complexes that activate platelets and trigger the thrombotic events seen in VITT., Here, John Kelton and colleagues provide an overview of vaccine-induced immune thrombotic thrombocytopenia (VITT), a very rare complication that has been observed following vaccination with adenoviral vector-based COVID-19 vaccines.

Currently, blood donors in Ghana are not screened for malaria parasites. Therefore, this study assessed platelet thrombogenicity in blood donors infected asymptomatically with Plasmodium falciparum and the relationship between tumour necrosis factor alpha (TNF-α), 8-iso-prostaglandin F2α oxidative stress biomarker (8-iso-PG2α), C-reactive protein (hs-CRP) and D-dimer, and platelet thrombogenes levels. Haematology analyser was used to enumerate platelet count and platelet indices in 80 P. falciparum infected blood donors and 160 matched non-infected controls. Replicate serum levels of von Willebrand Factor (vWF), platelet factor 4 (PF4), P-selectin thrombogenic factors as well as TNF-α and 8-iso-PG2α were determined using enzyme immuno-assay while high sensitive hs-CRP and D-dimer concentrations were determined by fluorescent immunoassay. The geometric mean of parasite density in malaria infected donors was 1784 parasites/µL (505-2478 parasites/µL). This led to significant increase in the mean levels of 8-iso-PG2α, hs-CRP, TNF-α and D-dimer. However, PF4, P-selectin were significantly lower in infected donors while vWF levels did not differ significantly among the groups even though lower levels were observed in the infected donors. Significant direct relationship existed between both P-selectin and PF4 and platelet count, and plateletcrit and platelet large cell ratio whereas these thrombogenic factors varied inversely to 8-iso-PG2α, TNF-α and hs-CRP. Relative thrombocytopaenia was associated with significant reduction in P-selectin and platelet factor 4 levels together with increased 8-iso-PG2α, hs-CRP, TNF-α and D-dimer levels. Taken together, it is recommended that all P. falciparum infected blood donors should be deferred. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12288-020-01390-w) contains supplementary material, which is available to authorized users.

OBJECTIVE The aim: This study aims to compare the lateral and fourth ventricles of CPs using hepatocyte nuclear factor 4 alpha (HNF4α), metabolism marker, to evaluate the functional activity of this tissue in two regions. PATIENTS AND METHODS Materials and methods: Ten adult male albino rats were used to study the histological features of the CPs and to study the functional activity by quantitative immunohistochemical labelling with HNF4α marker. RESULTS Results: The CP of the fourth ventricle had more functional activity than the CP of the lateral ventricle. A quantitative assessment of HNF4α using Aperio ImageScope Software Analysis showed that the lateral ventricle CP mean positivity equalled 0.264 ± 0.083 pixel/micron² while the fourth ventricle CP has mean positivity 0.297 ± 0.043 pixel/micron². The immunohistochemical expression of marker in the fourth ventricle CP was significantly (p ≤ 0.05) higher than those in the lateral ventricle (P ≤ 0.05). CONCLUSION Conclusions: Immunohistochemical detection of metabolism marker went along with findings of other histological and biochemical studies to define the CP as a highly dynamic structure with regional variations forming a continuum of one entity tissue capable of functional adaptation according to body needs.

IgG-specific and polyspecific PF4-dependent enzyme-immunoassays (EIAs) have exceptionally high sensitivity (≥99%) for diagnosis of heparin-induced thrombocytopenia (HIT), a drug reaction caused by platelet-activating antibodies detectable by serotonin-release assay (SRA). The IgG-specific EIAs are recommended for screening, as their high sensitivity is accompanied by relatively high specificity vis-à-vis polyspecific EIAs. We investigated the frequency of SRA-positive/EIA-negative (SRA+/EIA-) HIT, prompted by referral to our reference HIT laboratory of serial blood samples from a patient ("index case") with false-negative IgG-specific EIAs. Despite initial clinical suspicion for HIT, repeat negative IgG-specific EIAs prompted heparin resumption, which triggered recurrent thrombocytopenia and near-fatal cardiac arrest, indicating likely post-heparin HIT-associated anaphylactoid reaction. Further investigations revealed a strong-positive SRA, whether performed with heparin alone, PF4 alone, or PF4/heparin, with inhibition by Fc receptor-blocking monoclonal antibody (indicating IgG-mediated platelet activation); however, five different IgG-specific immunoassays yielded primarily negative (or weak-positive) results. To investigate the frequency of SRA+/EIA- HIT, we reviewed the laboratory and clinical features of patients with this serological profile during a 6-year period in which our reference laboratory investigated for HIT using both SRA and IgG-specific EIA. Although ~0.2% of 8546 patients had an SRA+/EIA- profile, further review of 15 such cases indicated clerical/laboratory misclassification or false-positive SRA in all, with no SRA+/EIA- HIT case identified. We conclude that while SRA+/EIA- HIT is possible-as shown by our index case-this clinical picture is exceptionally uncommon. Moreover, the requirement for a positive EIA is a useful quality control maneuver that reduces risk of reporting a false-positive SRA result.

Objective: Combination of ‘4Ts’ clinical scoring system and Particle gel immunoassay to determine the frequency of heparin-induced thrombocytopenia during heparin treatment in cardiac surgery patients. Study Design: Prospective observational study. Place and Duration of Study: This study was conducted in the Pathology Department, Army Medical College incollaboration with Armed Forced Institute of Cardiology and Armed Forced Institute of Transfusion, Rawalpindi,from Jan 2019 to Dec 2019. Methodology: A total of 115 suspected cases of heparin-induced thrombocytopenia irrespective of age and gender were included in the study. A clinical scoring system the ‘4Ts’ was used for the classification of patients based on probability into three groups such as low, intermediate, and high probability groups. For the detection of antibodies against heparin/platelet factor 4 complexes, the Particle gel immunoassay was used. Results: There were 39 (33.9%) females and 76 (66.1%) males, the age range of 20 to 86 ± 12.9 years. Among the low probability group, there was no positive result, 2 (2.3%) patients showed positive serological evidence in the intermediate probability group. In the high probability group, 4 (23.5%) patients showed positive results with the chosen assay. Conclusion: Heparin-induced thrombocytopenia was found an overall 5.2% of patients undergoing cardiacsurgery receiving unfractionated heparin. A combination of ‘4Ts’ pretest clinical scoring systems followed byPaGIA constitutes a simple strategy to screen for heparin-induced thrombocytopenia in suspected patients. This will avoid serious complications if the detection is not delayed.

There are currently no effective substitutes for high intensity therapy with unfractionated heparin (UFH) for cardiovascular procedures based on its rapid onset of action, ease of monitoring and reversibility. The continued use of UFH in these and other settings requires vigilance for its most serious non-hemorrhagic complication, heparin induced thrombocytopenia (HIT). HIT is an immune prothrombotic disorder caused by antibodies that recognize complexes between platelet factor 4 (PF4) and polyanions such as heparin (H). The pathogenicity of anti-PF4/H antibodies is likely due to the formation of immune complexes that initiate intense procoagulant responses by vascular and hematopoietic cells that lead to the generation of platelet microparticles, monocyte and endothelial cell procoagulant activity, and neutrophil extracellular traps (NETs), among other outcomes. The development of anti-PF4/H antibodies after exposure to UFH greatly exceeds the incidence of clinical disease, but the biochemical features that distinguish pathogenic from non-pathogenic antibodies have not been identified. Diagnosis relies on pretest clinical probability, screening for anti-PF4/H antibodies and documentation of their platelet activating capacity. However, both clinical algorithms and test modalities have limited predictive values making diagnosis and management challenging. Given the unacceptable rates of recurrent thromboembolism and bleeding associated with current therapies, there is an unmet need for novel rational non-anticoagulant therapeutics based on the pathogenesis of HIT. We will review recent developments in our understanding of the pathogenesis of HIT and its implications for future approaches to diagnosis and management.

Krüppel-like factor 4 (KLF4) is known to preserve vascular homeostasis. In the present study, we sought to correlate serum KLF4 levels with arterial aneurysm size and their clinical presentation. We also explored the association between serum KLF4 levels and the severity of extracranial carotid and peripheral arterial disease.Patients undergoing surgery for various forms of atheromatosis (ATH group) or for arterial aneurysm repair (AA group) were eligible for inclusion. KLF4 levels were measured via enzyme-linked immunosorbent assay.Patients in the atheromatic and aneurysmal groups had significantly higher serum KLF4 levels compared with controls. Patients with permanent end-organ damage (ATH3) had higher serum KLF4 (6.96 ± 0.75 pg/mL) compared with patients with asymptomatic internal carotid stenosis70% or claudication (ATH1) (2.76 ± 0.68 pg/mL; mean difference [MD], -4.20; 95% confidence interval [95% CI], -5.35 to -3.04; P 0.01) and those with transient ischemic attack or rest pain (ATH2) (4.47 ± 1.08 pg/mL; MD, -2.48; 95% CI, -3.76 to -1.21). Furthermore, patients with an asymptomatic aneurysm of a diameter 250-300% of that of the normal artery (AA1, 5.01 ± 1.08 pg/mL) had considerably lower serum KLF4 compared with those suffering from either a symptomatic aneurysm or an asymptomatic aneurysm of a diameter350% of that of normal artery (AA3, 6.63 ± 1.92 pg/mL; MD, -2.61; 95% CI, -5.04 to -0.18; P 0.01).Serum KLF4 levels are significantly increased in patients with end-organ damage related to atheromatosis as well as those with extensive aneurysmal disease.