Impedance Aggregometry Research Articles

Association of whole blood microRNA expression with platelet function and turnover in patients with coronary artery disease

IntroductionThe risk of recurrent cardiovascular events in patients with coronary artery disease (CAD) is determined by multiple factors including platelet function and turnover. MicroRNAs (miRs) may regulate both platelet function and turnover. We aimed to identify candidate miRs associating with platelet function and turnover in a cohort of stable CAD patients. Furthermore, we retrieved information on binding targets of the candidate miRs to obtain a more comprehensive biological insight into miR regulation of platelet function and turnover. MethodsBased on existing literature and a pilot study, we identified nine candidate miRs. Subsequently, we investigated the expression of the candidate miRs in whole blood and their relation to platelet function and turnover in 749 CAD patients. Platelet function was analysed using impedance aggregometry, optical aggregometry and serum thromboxane B2 measurements. Platelet turnover markers (immature platelet count, immature platelet fraction and mean platelet volume) were measured using monochromatic automated flow cytometry. ResultsExpression of miR-93-5p, miR-126-3p, miR-150-5p, miR-423-3p and miR-1180-3p showed negative correlations with platelet function (p-values from <0.0001 to 0.0006, rho from −0.13 to −0.36). In addition, expression of miR-423-3p showed negative correlation with platelet turnover markers (p-values from 0.001 to 0.004, rho from −0.11 to −0.12). ConclusionsWe identified several novel miRs that may regulate platelet function and turnover, thereby contributing to the increased risk of recurrent cardiovascular events in CAD patients.

Impact of high platelet turnover on the platelet transcriptome: Results from platelet RNA-sequencing in patients with sepsis.

Sepsis is associated with high platelet turnover and elevated levels of immature platelets. Changes in the platelet transcriptome and the specific impact of immature platelets on the platelet transcriptome remain unclear. Thus, this study sought to address whether and how elevated levels of immature platelets affect the platelet transcriptome in patients with sepsis. Blood samples were obtained from patients with sepsis requiring vasopressor therapy (n = 8) and from a control group of patients with stable coronary artery disease and otherwise similar demographic characteristics (n = 8). Immature platelet fraction (IPF) was determined on a Sysmex XE 2100 analyser and platelet function was tested by impedance aggregometry. RNA from leukocyte-depleted platelets was used for transcriptome analysis by Next Generation Sequencing integrating the use of unique molecular identifiers. IPF (median [interquartile range]) was significantly elevated in sepsis patients (6.4 [5.3-8.7] % vs. 3.6 [2.6-4.6] %, p = 0.005). Platelet function testing revealed no differences in adenosine diphosphate- or thrombin receptor activating peptide-induced platelet aggregation between control and sepsis patients. Putative circular RNA transcripts were decreased in platelets from septic patients. Leukocyte contamination defined by CD45 abundance levels in RNA-sequencing was absent in both groups. Principal component analysis of transcripts showed only partial overlap of clustering with IPF levels. RNA sequencing showed up-regulation of 524 and down-regulation of 118 genes in platelets from sepsis patients compared to controls. Upregulated genes were mostly related to catabolic processes and protein translation. Comparison to published platelet transcriptomes showed a large overlap of changes observed in sepsis and COVID-19 but not with reticulated platelets from healthy donors. Patients with sepsis appear to have a less degraded platelet transcriptome as indicated by increased levels of immature platelets and decreased levels of putative circular RNA transcripts. The present data suggests that increased protein translation is a characteristic mechanism of systemic inflammation.

A Comparative Assessment Study of Known Small-molecule GPVI Modulators.

The GPVI platelet receptor was recently validated as a safe antiplatelet target for the treatment of thrombosis using several peptidic modulators. In contrast, few weakly potent small-molecule GPVI antagonists have been reported. Those that have been published often lack evidence for target engagement, and their biological efficacy cannot be compared because of the natural donor variability associated with the assays implemented. Herein, we present the first side-by-side assessment of the reported GPVI small-molecule modulators. We have characterized their functional activities on platelet activation and aggregation using flow cytometry as well as light transmission and electrical impedance aggregometry. We also utilized microscale thermophoresis (MST) and saturation transfer difference (STD) NMR to validate GPVI binding and have used this along with molecular modeling to suggest potential binding interactions. We conclude that of the compounds examined, losartan and compound 5 are currently the most viable GPVI modulators.

High on-treatment platelet reactivity to aspirin in patients after myocardial infarction

Aspirin (ASA) is widely used as an antiplatelet therapeutic drug in secondary prevention. Last years brought many reports on ASA resistance or high on-treatment platelet reactivity (HTPR) to aspirin.This study is a post-hoc prospective analysis with 30 patients evaluated during follow up on average of 6.3 years after hospitalization from myocardial infarction. The examined population was divided into two subgroups according to the response to ASA. In order to estimate the function of blood platelets and their responsiveness to acetylsalicylic acid therapy, ASPI-test was used. The measurements were performed by the method of whole blood impedance aggregometry. During long-term follow up significantly higher percentage of high platelet reactivity was observed, compared with previous visits (p = 0.00001). Considering clinical endpoints of the research that were connected with coronary disease, no differences were obtained.The frequency of HTPR to aspirin in this study was higher than data reported in literature among subjects with CV diseases. In long-term observation the highest percentage of ASA non-responders was reported (58.6%). HTPR to aspirin did not affect the presence of the clinical endpoints for the study.

Quantification of platelet function - a comparative study of venous and arterial blood using a novel flow cytometry protocol

Studies of platelet function in surgical patients often involve both arterial and venous sampling. Possible effects of different sampling sites could be important, but have not been thoroughly investigated. We aimed to compare platelet function in arterial and venous blood samples using a novel flow cytometry protocol and impedance aggregometry. Arterial and venous blood was collected before anesthesia in 10 patients undergoing cardiac surgery of which nine was treated with acetylsalicylic acid until the day before surgery. Flow cytometry included simultaneous analysis of phosphatidylserine exposure, active conformation of the fibrinogen receptor (PAC-1 binding), α-granule and lysosomal release (P-selectin and LAMP-1 exposure) and mitochondrial membrane integrity. Platelets were activated with ADP or peptides activating thrombin receptors (PAR1-AP/PAR4-AP) or collagen receptor GPVI (CRP-XL). Leukocyte-platelet conjugates and P-selectin exposure were evaluated immediately in fixated samples. For impedance aggregometry (Multiplate®), ADP, arachidonic acid, collagen and PAR1-AP (TRAP) were used as activators. Using impedance aggregometry and in 27 out of 37 parameters studied with flow cytometry there was no significant difference between venous and arterial blood sampling. Arterial blood showed more PAC-1 positive platelets when activated with PAR1-AP or PAR4-AP and venous blood showed more monocyte-platelet and neutrophil-platelet conjugates and higher phosphatidylserine exposure with CRP-XL alone and combined with PAR1-AP or PAR4-AP. We found no differences using impedance aggregometry. In conclusion, testing of platelet function by flow cytometry and impedance aggregometry gave comparable results for most of the studied parameters in venous and arterial samples. Flow cytometry identified differences in PAC-1 binding when activated with PAR1-AP, exposure of phosphatidyl serine and monocyte/neutrophil-platelet conjugates, which might reflect differences in blood sampling technique or in flow conditions in this patient cohort with coronary artery disease. These differences might be considered when comparing data from different sample sites, but caution should be exercised if a different protocol is used or another patient group is studied.

Effect of lactic acid addition to equine whole blood on platelet aggregation measured by impedance aggregometry

Acidemia in sick or injured horses is often due to lactic acid accumulation. Alterations in platelet function and hemostasis are among numerous deleterious effects caused by decreased physiologic pH. We aimed to evaluate the effect of hyperlactatemia and resultant acidemia on platelet aggregation in equine whole blood using impedance aggregometry. Platelet aggregation was measured using the Multiplate analyzer in whole blood from 34 healthy horses at baseline and after in vitro addition of lactic acid to adjust the pH. Platelet aggregation of each sample was quantified by the area under the curve measurement reported by the Multiplate system. The association between platelet aggregation and pH was analyzed using a linear mixed-effects model. The association of baseline platelet aggregation with hematocrits (Hcts), white blood cell (WBC) counts, and platelet counts was evaluated using Pearson's correlations. There was a significant association between acidemia and decreased platelet aggregation. No significant correlations were detected between platelet aggregation and Hct, WBC count, or platelet count. Platelet aggregation measured in healthy horses using the Multiplate analyzer showed substantial variation between animals. Acidemia caused by the addition of lactic acid to equine whole blood was associated with a mild though statistically significant decrease in platelet aggregation. In conjunction with other factors, this change may contribute to morbidity-related disorders of hemostasis, although its precise clinical relevance is uncertain.

Multiplate platelet aggregometry in dogs undergoing laparoscopic liver biopsy for diagnosis of chronic hepatopathy.

To assess platelet function via the Multiplate analyser in dogs undergoing laparoscopic liver biopsy for diagnosis of chronic hepatopathy. Twenty-seven client-owned dogs were prospectively enrolled. Before laparoscopic liver biopsy, whole blood impedance platelet aggregometry via the Multiplate analyser was performed. Buccal mucosal bleeding time was performed in 23 of 27 dogs. Tissue factor-activated thromboelastography was also performed, in addition to plasma-based coagulation testing. Descriptive statistics were calculated and the prevalence of platelet function abnormalities and results of other biochemical and coagulation testing were reported. Seventeen (63%) of 27 dogs had evidence of decreased platelet function as assessed by aggregometry, with all 17 dogs having decreased responsiveness to adenosine diphosphate, and 11 of 17 dogs demonstrating decreased responsiveness to arachidonic acid. Based on maximum amplitude, most dogs were classified as normocoagulable on thromboelastography (15/25; 60%). Other frequent coagulation abnormalities included increased D-dimers (20/27;74%), thrombocytopenia (11/27; 41%), hypofibrinogenemia (4/27; 15%), and decreased antithrombin (4/27; 15%). Decreased platelet function as assessed by whole blood impedance aggregometry was common in dogs with chronic liver disease. Further study is necessary to determine whether this finding is repeatable or indicative of increased bleeding risk.

Whole blood platelet impedance aggregometry with the ROTEM platelet device: comparison of 2 anticoagulants and storage times for the establishment of canine reference intervals.

The ROTEM platelet device, a point-of-care whole blood platelet impedance aggregometer, is an add-on to the rotational thromboelastometry ROTEM delta device. The latter has been validated in dogs. We examined whether canine whole blood is suited for analysis with the ROTEM platelet device using adenosine-5'-diphosphate (ADP) and arachidonic acid (ARA) as agonists for platelet activation, and if there are significant differences between sample storage times and anticoagulants used. Subsequently, we determined canine reference intervals (RIs) for the ROTEM platelet device for ADP and ARA. In a pilot study, we examined whole blood from 7 dogs after 15-min and 60-min storage of lithium-heparinized samples and 40-min and 80-min storage of hirudinized samples. Statistical analysis showed no significant differences between ROTEM platelet device results for both ADP and ARA in lithium-heparin and hirudin anticoagulated canine whole blood. Lithium-heparinized blood samples analyzed after 15-min storage had the lowest coefficient of variation. RIs were determined for heparinized whole blood samples from 49 dogs after 15 min of storage.

Increased symmetric dimethyl-arginine is a predictor factor of decreased platelet reactivity and increased bleeding risk in patients with acute coronary syndrome

Abstract Background One of the promising biomarkers in CVD are asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), which are products of L-arginine methylation and are both involved in endothelial dysfunction. ADMA, SDMA and L-homoarginine, have emerged as biomarkers linked to cardiovascular outcomes [1]. Purpose To investigate the association of SDMA with platelet reactivity and bleeding risk in patients with acute coronary syndrome (ACS) treated with potent P2Y12 inhibitors prasugrel and ticagrelor. Methods Our prospective observational study enrolled 292 patients with ACS undergoing percuteneus coronary intervention [2]. Plasma concentrations of SDMA were measured during the hospitalization for ACS. Impedance aggregometry was used. The primary study endpoint was the concentration of metabolites and platelet reactivity. The primary clinical outcome endpoint was the incidence of Thrombolysis in Myocardial Infarction (TIMI) bleeding events (major, minor and minimal). The efficacy endpoint was the composite of major adverse cardiac events (MACE: stent thrombosis, myocardial infarction, stroke and cardiac death). Results There was an inverse correlation between SDMA serum levels and platelet reactivity (r=−0.25; p&amp;lt;0.000). The ADP+PGE1-induced platelet reactivity was 33% lower among patients with the highest SDMA quartile (4th) as compared to those with the 1–3rd SDMA quartile (8 [0–29] vs 12 [0–126] U; p&amp;lt;0.001). The AA-induced platelet reactivity was 56% lower among patients with the highest SDMA quartile (4th) as compared to those with the 1–3rd SDMA quartile (4 [0–48] vs 9 [0–133]; p&amp;lt;0.001). In a multivariate model, the highest SDMA (4th) quartile was found to be an independent predictor of the lowest ADP+PGE1 and AA induced platelet aggregation (OR: 2.666, 95% CI [1.184–5.999], p=0.018). Conclusions Our study shows that high plasma concentration of SDMA, but not ADMA, is independently associated with low platelet reactivity to ADP and AA and is associated with major and minor bleeding events in patients with ACS on potent antiplatelet therapies. Therefore, SDMA might have a potential to be further evaluated as a blood biomarker for individualization of duration and potency of antiplatelet therapies in an ACS population at high risk of bleeding complications. Acknowledgment I-COMET research team Funding Acknowledgement Type of funding sources: None. Figure 1Table 1

Long-term post-discharge coagulation abnormalities in patients with moderate to severe COVID-19

IntroductionCoagulopathy plays a significant role in COVID-19 pathogenesis. Benefit from anticoagulation is well established in hospitalized patients. But since there is a lack of data on coagulopathy resolution: there is no consensus in guidelines if extended anticoagulation is required.PurposeThe purpose of our work was to analyze coagulation abnormalities at 2 to 5 months after moderate to severe COVID-19.MethodsCOVID-19 reconvalescents (CR), discharged from our hospital, were called for follow-up at 2–3 (CR1 group, 21 patients) or 5–6 (CR2 group, 26 patients) months after discharge. All CR were not on the anticoagulation therapy by that time. In addition to clinical examination and standard lab tests, we performed an FMD-test to analyze endothelial function, impedance aggregometry to analyze platelet aggregation, and a thrombodynamics test to assess thrombogenesis and fibrinolysis. The control group was recruited before the pandemic started.ResultsAll CR were free from thrombotic complications after discharge from the hospital.Endothelial function was not significantly impaired in CR compared with control, and was still in the normal range (7,07, IQR (3,36; 11,56) vs. 7,87 (5,42; 13,45)).Platelet aggregation was significantly lower in CR1 than in the control group in ADP-induced mode (37, IQR (19; 47) vs. 46, IQR (41; 50), p=0,02) and didn't differ in other groups and other modes (Asa, TRAP-induced).Thrombodynamics tests revealed suppression of the clot formation process in both CR1 and CR2 compared with control. There were decreased clot growth rates (μm/min) (CR1/CR2: 27,1, IQR (26,1; 29,2)/27,6, IQR (26,4; 30,0) vs. 32,2, IQR (30,0; 35,1), both p<0,001); decreased clot size (μm) (CR1/CR2: 1099, IQR (1069; 1194)/1199, IQR (1058; 1221) vs. 1304, IQR (1164; 1380), both p<0,001), and decreased optical density (arb units) (CR1/CR2: 21'607, IQR (20'363; 24'545)/22'741, IQR (21'344; 25'961) vs. 26'556, IQR (24'672; 29'387, p<0,001 and p=0,09 respectively.Fibrinolysis was enhanced in CR groups compared with control (lysis progression was significantly higher for CR2 only, CR1/CR2: 2,9, IQR (2,5; 3,8)/3,8, IQR (2,6; 5,4) vs. 2,5, IQR (1,1; 3,4) %/min, p=0,087 and p=0,007 respectively; expected clot lysis time was shorter in both CR1 and CR2: 36,5, IQR (29,8; 44,2)/31,7, IQR (24,3; 42,7) vs. 65,9. IQR (36,3; 95,5) min, p=0,019 and p=0,016 respectively).There was no statistical difference in clot formation and in fibrinolysis between CR1 and CR2.ConclusionIn the deferred period (2–5 months) of COVID-19 the fibrinolysis process remains still active whereas the process of clot formation is mostly suppressed. Endothelial function assessed via the FMD test is within the normal range in the post COVID period.FUNDunding AcknowledgementType of funding sources: None.

The use of impedance aggregometry to evaluate platelet function after the administration of DDAVP in healthy dogs treated with aspirin or clopidogrel.

To evaluate the effect of 1-Desamino-8-d-arginine vasopressin (DDAVP; desmopressin acetate) on platelet aggregation in healthy dogs receiving aspirin or clopidogrel. 7 healthy staff-owned dogs. In this randomized double-blinded crossover study, impedance aggregometry was performed on samples of lithium-heparinized whole blood samples from dogs before (T0) treatment with aspirin (1 mg/kg, PO, q 24 h for 4 days; ASP group) or clopidogrel (1 mg/kg, PO, q 24 h for 4 days; CLP group) and then before (T1) and after (T2) treatment with DDAVP (0.3 μg/kg, IV, once). There was a 14-day washout period before the crossover component. Aggregometry was performed with 4 different assays, each of which involved a different agonist reagent to stimulate platelet function: ADP, thrombin receptor activating peptide-6, arachidonic acid, or collagen type 1. Median results for platelet aggregometry with agonist reagents ADP, arachidonic acid, or thrombin receptor activating peptide-6 significantly decreased between T0 and T1 for the CLP group; however, no meaningful difference in platelet aggregation was detected in the ASP group. Results for platelet aggregometry did not differ substantially between T1 and T2 regardless of treatment group or assay. Findings suggested that administration of DDAVP may have no effect on platelet aggregation (measured with platelet aggregometry) in healthy dogs treated with clopidogrel. Because no inhibition of platelet aggregation was detected for dogs in the ASP group, no conclusion could be made regarding the effects of DDAVP administered to dogs treated with aspirin.

Platelet reactivity is higher in e-cigarette vaping as compared to traditional smoking

IntroductionVaping emerges as alternative to standard tobacco smoking. However, there is evidence for critical cardiovascular, gastrointestinal and respiratory side effects. Nevertheless, long-term vaping effects on thrombocyte reactivity have not been investigated. Therefore, we investigated the influence of vaping on thrombocyte reactivity in comparison to standard smoking and non-smoking. MethodsPlatelet function was measured by Multiplate Impedance Aggregometry as area under the curve (AUC). Smoking habits and characteristics were assessed by questionnaire. Results were analyzed using inverse probability of treatment weighting (IPTW) and conventional t-tests to test for robustness. ResultsAfter IPTW adjustment, participants in all groups were balanced by age, gender, body height and weight. Collagen-induced aggregation was higher in vapers compared to non-smokers (non-smokers 52.55 ± 23.97 vs. vapers 66.63 ± 18.96 AUC, p = 0.002) and to smokers (vapers vs. smokers 49.50 ± 26.05 AUC, p < 0.0001). ADP-induced aggregation in vapers was higher compared to non-smokers (non-smokers 33.16 ± 16.61 vs. vapers 45.27 ± 18.67 AUC, p = 0.001) and was numerically increased compared to smokers (vapers vs. smokers 40.09 ± 19.80 AUC, p = 0.08). These findings remained robust in t-test analysis. ConclusionThis study provides first evidence that vaping leads to enhanced platelet reactivity compared to standard smoking and non-smoking. This suggests health effects of vaping might be more severe than previously assumed. Whether this effect translates to clinical outcome with a higher incidence of major cardiovascular events, should be evaluated in large-scaled clinical studies.

Pre-operative platelet reactivity is a strong, independent predictor of bleeding complications after branched endovascular thoracoabdominal aortic aneurysm repair

Endovascular aortic repair (EVAR) an alternative to open surgical repair of thoracoabdominal aortic aneurysm (TAAA). The effect of EVAR on platelet reactivity is unknown. We prospectively determined the effect of branched EVAR (bEVAR) on platelet reactivity in patients with TAAA, and evaluated the predictive value of preoperative platelet reactivity for post-operative bleeding in 50 consecutive patients undergoing elective bEVAR (mean age 70.9 ± 5.7 years, 66% male). Blood samples were collected within 24 hours before bEVAR, after bEVAR and at hospital discharge. Platelet reactivity was assessed with impedance aggregometry using ASPI, ADP and TRAP tests. Platelet reactivity decreased within 24 hours after bEVAR compared to the measurement before bEVAR in all tests (p ≤ 0.04), with a further decrease in hospital discharge in the ADP test (p = .004). Twenty-three patients experienced post-operative bleeding complications (transfusion ≥2 red blood cell [RBC] units). Preoperative platelet reactivity below the cutoff value of 30 AUC units predicted post-operative bleeding with 78% sensitivity and 59% specificity (p = .045). In the multivariable analysis, platelet reactivity was the only independent predictor of postoperative bleeding (OR 6.507, 95% CI 1.227–34.506, p = .028). We conclude that platelet reactivity decreases following bEVAR of TAAA and is a strong and independent predictor for postoperative bleeding complications.

Effects of pentoxifylline on canine platelet aggregation.

BackgroundPentoxifylline can decrease platelet function in humans, but the anti‐platelet effects of pentoxifylline in dogs is unknown. The addition of a luciferin–luciferase reagent during platelet aggregometry can induce a dose‐dependent potentiation of platelet aggregation.ObjectiveTo determine if exposure to pentoxifylline, without the addition of a luciferin–luciferase reagent during aggregometry, causes canine platelet dysfunction. Our hypotheses were that pentoxifylline would inhibit platelet function, and that the addition of a luciferin–luciferase reagent would obscure detection of pentoxifylline‐induced platelet dysfunction as measured via aggregometry.MethodsSeven healthy Walker hound dogs. Platelet‐rich plasma (PRP) and whole blood were treated for 30 minutes with pentoxifylline: 0 (control), 1 and 2 μg/mL. The platelet aggregation was determined using optical (maximum amplitude) and impedance (ohms) aggregometry using collagen as the agonists, with and without a luciferin–luciferase reagent. Four samples were analysed per concentration and the results were averaged.ResultsBased on optical aggregometry, there was no difference (p = 0.964) in the mean maximum amplitude at any pentoxifylline concentration, with and without the luciferin–luciferase reagent. During impedance aggregometry, the addition of a luciferin–luciferase reagent was associated with significantly (p < 0.001) greater platelet aggregation in response to a collagen agonist, regardless of the presence or absence of pentoxifylline.ConclusionsPentoxifylline does not exert an in vitro anti‐platelet effect on canine platelet aggregation when collagen is used as an agonist, but it is unknown if long‐term oral drug administration will inhibit platelet aggregation. The addition of a luciferin–luciferase reagent during platelet aggregometry can artificially enhance canine platelet aggregation.

High Levels IPF as Possible Predictor Heparin-Induced Thrombocytopenia

Background: Heparin-Induced Thrombocytopenia (HIT) represents a serious complication of heparin treatment. IgG antibodies binding Platelet Factor 4 (PF4) and heparin trigger the clinical manifestations of HIT. A 4T score is used to stratify the selection of patients suitable for examination. However, the selection of suitable patients remains at the discretion of the clinician, who is confronted with determining the cause of thrombocytopenia. The inclusion of the evaluation of the Immature platelet fraction result seems to be a suitable complement to the stratification of patients because we do not climb elevated IPF values when consuming platelets due to their immunization. Materials and Methods: In a group of 432 thrombocytopenic samples IPF was detected and analyzed in 45 patients with suspected HIT, a 4T score was determined; IPF and HIT functional tests were examined. IPF was determined by oxazine fluorescent dyeing structures of nucleic acid-containing platelets and fluorescence detection on a Sysmex XN 1000 analyser. To determine HIT, impedance aggregometry using the Multiplate® analyser (MEA) as heparin-induced aggregation techniques. The MEA method uses sensitization of donor platelets with patient plasma in the presence of heparin at a concentration of 0.5IU/mL. Results: From the results of the test, it is evident that 10 patients from our group of 45 examined showed positivity of HIT, which is a significant number due to the proven occurrence of HIT in patients treated with LMWH and showing thrombocytopenia. If we evaluate these 10 patients in terms of IPF value, it is evident that 6 of them have an increased value of IPF &gt;10%, which is a 33% positive predictive value and 4 have IPF &gt;30%, when the positive predictive value is even 100%. Conclusions: Diagnosis of HIT remains a complicated clinical laboratory issue. However, new diagnostic options provide considerable potential for solving this problem. The implementation of IPF assays helps us in the diagnosis of HIT on two levels. On the one hand, it provides us with information on platelet consumption in hospitalized patients and thus draws our attention to HIT as one of the options for congestive thrombocytopenia, unless, of course, disseminated intravascular coagulation or thrombotic microangiopathy. Secondly, its implementation will increase the predictive value of the 4T score in patients at medium risk, which is, however, the vast majority indicated for HIT examination.

Platelet aggregation in whole blood is not impaired by a platelet‐sparing leukoreduction filter and instead depends upon the presence of leukocytes

Recent studies characterizing in vitro hemostatic properties of whole blood (WB) leukoreduced (LR) with a platelet-sparing filter have described subtle, if any, changes to viscoelastic clotting; however, reductions in platelet (PLT) content and impedance aggregometry (IA) responses have been noted. The effects of filtration of WB (i.e., filter-contact effects, reduction in platelet and leukocyte count) have not been rigorously investigated as to their individual impacts on platelet IA responses. WB units from healthy donors were collected and characterized to assess the effects of platelet-sparing leukoreduction (LR) upon the in vitro hemostatic measures of platelet IA and thromboelastometry. Further characterization of platelet IA responses was carried out in WB samples to delineate the effects of platelet count and leukocyte presence/absence upon the response. WB filtration reduced the platelet count and IA responses but had no impact on viscoelastic clotting measures in fresh WB. Experiments revealed that IA responses have a linear correlation with platelet count in both apheresis platelets and WB and that passage of platelets through the WB-LR filter has no impact upon the strength of this response. Further experiments in LR WB showed that addition of autologous leukocytes back to the platelets fully restored the platelet aggregation response to pre-filtration levels. WB filtration results in platelet count reduction and leukocyte removal; however, platelet IA is not degraded by passage through the filter. Apparent declines in platelet IA responses can be fully attributed to the reduction in platelet count and the removal of leukocytes.

Coagulopathy As a Dose-Limiting Toxicity in Systemic Administration of Mesenchymal Stem Cells (MSCs): Relative Safety of Bone Marrow Versus Adipose Derived MSCs

Introduction: Systemic administration of mesenchymal stem cells (MSCs) is an option that allows MSCs to interact with circulatory immune cells, or to deliver MSCs into multiple traumatic sites that are not reachable through regional delivery. The number of MSCs per injection also needs to be determined in order to achieve the maximal beneficial effect, and optimize the ratio of MSCs to target cells. However, MSCs express tissue factor, which is known to activate coagulation, which could potentially cause inappropriate clot formation, counteracting the beneficial effect of MSCs on tissue regeneration and immunomodulation at the site of injury. In this study, we sought to determine the maximum dose of MSCs that can safely be used for systemic administration by measuring their impact on coagulation in rats.Method: Bone marrow and adipose derived MSCs (BMSC and AMSC) were isolated from bone (femur and tibia) and visceral fat tissue, respectively, in normal young Sprague Dawley (SD) rats. Both BMSCs and AMSCs were cultured and passaged using Dulbecco's Modified Eagle's Medium (DMEM) with 20% fetal bovine serum. MSCs at passage 2-5 were labeled with green fluorescent chloromethyl derivatives of fluorescein diacetate (CMFDA), resuspended in normal saline with anticoagulant (Citrate Phosphate Dextrose (CPD)), and infused into the SD rats through the femoral vein at doses of 2.5, 5, 10, 20, and 40 million/kg under anesthesia. Citrated whole blood was collected at the base line, immediately after infusion, 1 hour, and 3 hours after infusion of MSCs. The prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen were measured by hemostasis analyzer (ST4); coagulation properties and platelet aggregation (response to ADP (Adenosine Diphosphate) and collagen) were measured by hemostasis analyzer, rotational thromboelastometry (ROTEM) and impedance aggregometry (Multiplate), respectively. The MSCs in circulation were measured by flow cytometry. The MSCs in tissue were determined by histology.Results: At the dose of 20 million/kg, rats survived with BMSCs, but died with AMSCs (n=3 per group). Two of three rats survived with infusion of BMSCs at 40 million/kg. The platelet counts were significantly reduced at 1hr after MSC infusion (%change to baseline: AMSCs: -4±0; -24±3; -39±8 % at 2.5, 5 and 10 million/kg, respectively; BMSCs: -3±0, -4±3, -18±4, -19±6, -63±5 % at 2.5, 5, 10, 20 and 40 million/kg, respectively). Both PT and aPTT were significantly prolonged (PT: 35±4(AMSC) vs. 9±3(BMSC)% increased; aPTT 81±6(AMSC) vs. 17±3(BMSC)% increased), and fibrinogen levels significantly declined (-39±8(AMSC) vs. -18±4(BMSC)% decreased) after 10 million/kg AMSC or BMSC infusion. In comparison to the baseline, clotting time (CT) was significantly prolonged (AMSCs: 124±14 (vs. 68±6); BMSCs: 82±8 (vs. 57±4) seconds), and maximum clot firmness (MCF) was significantly reduced (AMSC: 39±3 (vs. 58±2) mm); BMSCs: 38±7 (vs. 62±4) mm) at 1hr after infusion of AMSCs at 10 million/kg or BMSCs at 20 million/kg respectively. Platelet aggregation (area under the curve (mm2)) significantly declined compared to the baseline after infusion of 10 million/kg AMSCs (ADP: 10±4 (vs. 75±7.0); Collagen: 4±4 (vs. 74±5)) or 20 million/kg BMSCs (ADP: 20±15 (vs. 89±12); Collagen: 36±18 (vs.102±10)). There were few circulating AMSCs or BMSCs found after 1 and 3 hours after infusion. The MSCs were found mostly in the lung, as determined by histology.Conclusion: AMSCs and BMSCs are not maintained in the circulation after systemic administration. However, infusion of MSCs has a significant impact on the hemostatic system, as shown by prolongation of PT, aPTT, and CT; and a decrease in fibrinogen levels, platelet counts, platelet function, and clot firmness. These data suggest that systemic administration of MSCs has a potential risk of causing consumptive coagulopathy, which may be initiated by the tissue factor expression of MSCs. This study also suggests that BMSCs have less impact than AMSCs on hemostasis, and BMSCs at the dose of 5 million/kg or less has the least risk of causing coagulopathy. Future studies are necessary to investigate the appropriate methods of down-regulating the procoagulant properties of MSCs prior to systemic administration in order to minimize the potential risk of coagulopathy. DisclosuresNo relevant conflicts of interest to declare.

Absence of hypercoagulability after nCoV-19 vaccination: An observational pilot study

BackgroundIt is still unknown whether COVID-19 vaccines induce a prothrombotic state or increase the hypercoagulable condition in subjects with a predisposition to thrombosis. ObjectivesWe evaluated the coagulation profile in a series of healthy subjects who received the first dose of the BNT162b2 or the ChAdOx1 vaccines and assessed whether hypercoagulability developed. Patients/methodsVolunteers among the staff of the University of Padua or health care professionals in the Padua University Hospital who had received either the ChAdOx1 or BNT162b2 vaccine in the previous 10 ± 2 days were eligible. A cohort of unvaccinated volunteers among family members of the University staff acted as control group. Global coagulation monitoring was assessed by whole blood rotational thromboelastometry, whole blood impedance aggregometry and thrombin generation. Platelet count was also obtained. ResultsOne hundred and ninety subjects were enrolled: 101 (53.2%) received the ChAdOx1 vaccine and 89 (46.8%) the BNT162b2 vaccine. Twenty-eight non-vaccinated subjects acted as controls. Thromboelastometry parameters were all comparable among groups. Thrombin receptor activating peptide (TRAP)-, ADP- and ASPI-induced platelet aggregation were similar among groups, as well as platelet count. Endogenous thrombin potential (ETP) was comparable among groups. The results were confirmed after controlling for age, gender and hormonal. Considering women taking combined oral contraceptives or thrombophilia carriers, no differences were detected in thromboelastometry or thrombin generation parameters between subjects who received ChAdOx1 vs. BNT162b2 vaccines. ConclusionsNo significant activation of fibrinogen-driven coagulation, plasma thrombin generation or clinically meaningful platelet aggregation after ChAdOx1 or BNT162b2 vaccination was observed.

Novel Predictors of Future Vascular Events in Post-stroke Patients-A Pilot Study.

Introduction: A modified platelet function test (mPFT) was recently found to be superior compared to impedance aggregometry for selection of post-stroke patients with high on-treatment platelet reactivity (HTPR). We aimed to explore some peripheral blood cell characteristics as predictors of recurrent ischemic episodes. The predictive value of mPFT was also assessed in a cohort followed up to 36 months regarding recurrent ischemic vascular events.Methods: As a novelty, not only whole blood (WB), but after 1-h gravity sedimentation the separated upper (UB) and lower half blood (LB) samples were analyzed including neutrophil antisedimentation rate (NAR) in 52 post-stroke patients taking clopidogrel. Area under the curve (AUC, AUCupper and AUClower, respectively) was separately measured by Multiplate in the WB, UB and LB samples to characterize ex vivo platelet aggregation in the presence of ADP. Next, the occurrence of vascular events (stroke, acute coronary syndrome, ACS) were evaluated during 36-month follow-up.Results: A total of 11 vascular events (stroke n = 5, ACS n = 6) occurred during the follow-up period. The AUCupper was significantly higher in patients with recurrent stroke compared to those with uneventful follow-up (p = 0.03). The AUCupper with a cut-off value ≥70 based on the mPFT, was able to predict all stroke events (p = 0.01), while the total vascular events were independently predicted by NAR with a sensitivity of 82% and specificity of 88%.Conclusions: A combination of NAR reflecting the inflammatory state and AUCupper indicating HTPR may provide a better prediction of recurrent ischemic events suggesting a better selection of patients at risk, thus providing an individually tailored vascular therapy.

Electrical impedance vs. light transmission aggregometry: Testing platelet reactivity to antiplatelet drugs using the MICELI POC impedance aggregometer as compared to a commercial predecessor

BackgroundPatients' responses to antiplatelet therapy significantly vary, with individuals showing high residual platelet reactivity associated with thrombosis. To personalize thrombosis management, platelet function testing has been suggested as a promising tool able to monitor the antithrombotic effect of antiplatelet agents in real-time. We have prototyped the MICELI, a miniature and easy-to-use electrical impedance aggregometer (EIA), measuring platelet aggregation in whole blood. Here, we tested the capability of the MICELI aggregometer to quantify platelet reactivity on antiplatelet agents, as compared with conventional light-transmission aggregometry (LTA). MethodsPlatelet aggregation in ACD-anticoagulated whole blood and platelet-rich plasma of healthy donors (n = 30) was evaluated. The effect of clopidogrel, ticagrelor, cangrelor, cilostazol, and tirofiban on ADP-induced aggregation was tested, while aspirin was evaluated with arachidonic acid and collagen. Platelet aggregation was recorded using the MICELI or BioData PAP-8E (Bio/Data Corp.) aggregometers. ResultsThe MICELI aggregometer detected an adequate and comparable dose-dependent decrease of platelet aggregation in response to increments of drugs' concentrations, as compared to LTA (the inter-device R2 = 0.79–0.93). Platelet aggregation in platelet-rich plasma recorded by LTA showed higher sensitivity to antiplatelet agents, but it couldn't distinguish between different drug doses as indicated by saturation of the aggregatory response. ConclusionPlatelet aggregation in whole blood as recorded by EIA represents a better model system for evaluation of platelet reactivity as compared with platelet aggregation in platelet-rich plasma as recorded by LTA, since EIA takes into consideration the modulatory effect of other blood cells on platelet hemostatic function and pharmacodynamics of antiplatelet drugs in vivo. As such, the MICELI impedance aggregometer could be potentially employed for the point-of-care monitoring of platelet function in patients on-treatment for personalized tailoring of their antiplatelet regimen.