Evaluation of clopidogrel response in healthy cats using a novel viscoelastic test and thromboelastography.

Introduction: Cats with cardiomyopathy face an increased risk of arterial thromboembolism (ATE). Although clopidogrel is frequently utilized to mitigate this risk, feline responses to this therapy exhibit variability. This study evaluated 2 viscoelastic devices, thromboelastography (TEG) and Viscoelastic Coagulation Monitor (VCM), for monitoring clopidogrel in cats in comparison to light transmission aggregometry (LTA). Methods: Twenty-eight healthy cats received clopidogrel for 7 days. Blood was collected at baseline and after treatment for analysis by TEG, VCM, and LTA. Results: On LTA, maximum amplitude, slope, and area under the curve (AUC) significantly decreased after treatment (p < 0.0001). On VCM, maximum clot firmness (MCF) significantly increased after treatment (p = 0.002). On TEG, R-time significantly prolonged (p = 0.024), while K and alpha angle significantly changed (p = 0.0002 and p = 0.0014, respectively). There was a moderate negative correlation between TEG R-time and LTA AUC (r = -0.39, p = 0.042). Eight cats were identified as non-responders to clopidogrel. Of the 8 non-responders, 6 (75%) had shortened R time after treatment. VCM appeared to be less discriminatory in identifying non-responders. Discussion: LTA remained the gold standard of monitoring clopidogrel treatment in cats. Unexpected changes on VCM and TEG were likely related to high interindividual and assay variability and increased sensitivity of feline platelets. R-time on TEG may have potential utility for point-of-care monitoring of clopidogrel response in cats.

Whole Blood Aggregometry in Mice.

Aggregometry plays a crucial role in both clinical diagnostics and research within hematology, serving as a fundamental tool for understanding platelet function and its implications in physiological and pathological processes. In research, aggregometry provides insights into platelet aggregation dynamics and aids in understanding the underlying mechanisms of hemostasis, thrombosis, and related disorders. Light transmission aggregometry (LTA) and lumi-aggregometry, as well as whole blood aggregometry, are commonly employed methods. While LTA and lumi-aggregometry allow for specific platelet function assessment under controlled conditions, whole blood aggregometry provides a more physiologically relevant approach by evaluating platelet aggregation within the context of whole blood. Although both methodologies offer unique advantages, whole blood aggregometry allows for preservation of the native cellular environment, simplicity, and potential for better clinical correlation. In a clinical setting, with human blood samples, protocols are established for both LTA and whole blood aggregometry as they are frequently used diagnostic tools. A protocol for LTA and lumi-aggregometry in murine models has been described; however, to date, there is no standardized protocol for whole blood aggregometry in murine models accessible to hematology researchers. This article aims to outline a simple, basic protocol for murine whole blood aggregometry, offering an alternative method to the commonly used LTA aggregometry in research settings. Standardizing whole blood aggregometry protocols in murine models could enhance experimental reliability and facilitate translational research efforts in hematology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Whole blood aggregometry in mice Support Protocol: Phenylhydrazine-induced anemia in wild-type mice Basic Protocol 2: Hematocrit percentage in mice.

Defining light transmission aggregometry cutoff values for clopidogrel and aspirin resistance in flow diversion treatment of intracranial aneurysms.

OBJECTIVE: Concern about thromboembolic events after flow diversion (FD) warrants dual antiplatelet therapy for 3 to 6 months. Platelet function tests are routinely performed prior to the procedure to detect clopidogrel responsiveness, as resistance is associated with CYP2C19 gene polymorphisms. This study aimed to identify optimal cutoff values in light transmission aggregometry (LTA) for clopidogrel and aspirin as predictive indicators of thromboembolic complications. METHODS: The authors conducted a retrospective analysis of aneurysms treated with FD between 2013 and 2023 at a single academic institution. Patients with LTA data for adenosine diphosphate (ADP) and arachidonic acid (ARA) were included, excluding those with aborted procedures. Receiver operating characteristic curves were plotted for ADP and ARA assays to determine optimal cutoff values. RESULTS: A total of 442 patients harboring 552 aneurysms treated in 485 procedures were selected for this analysis. Complete and near-complete aneurysm occlusion on the last radiological follow-up was achieved in 81.8% of aneurysms in a median last imaging follow-up of 13.9 months. A good functional outcome (modified Rankin Scale score ≤ 2) was achieved in 96.3% of patients on the last follow-up. Thromboembolic complications occurred in 4.9% of procedures, and intracranial hemorrhagic complications in 1.9%. For the ADP assay, a value ≥ 40% reached a sensitivity of 82.1% and a specificity of 42.9% with a positive likelihood ratio (LR) of 1.50. For the ARA assay, a value ≥ 13.5% reached a sensitivity of 82.1% and a specificity of 45.6% with a positive LR of 1.51. CONCLUSIONS: This study analyzed the largest FD-treated cohort in which optimal LTA platelet function thresholds for clopidogrel were evaluated and is the first to assess LTA values for aspirin. The authors found that values ≥ 40% for clopidogrel and ≥ 13.5% for aspirin were optimal for predicting thromboembolic complications after FD in treating aneurysms.

Platelet Reactivity in the Exacerbation of Psoriasis.

Background: Psoriasis is a chronic, inflammatory, immune-mediated disease with a specific cutaneous presentation. Increased platelet aggregation has been observed in patients with extensive psoriatic lesions. The aim of this study was to evaluate the clinical factors affecting platelet reactivity in patients with an exacerbation of psoriasis. Methods: This was a prospective, single-center, observational study, enrolling patients hospitalized for an aggravation of psoriasis. Enrolled patients underwent single platelet function testing with light transmission aggregometry on the first morning of hospitalization. Results: 120 patients were enrolled in the study. Of the compared subgroups, women had higher maximal platelet aggregation (MPA) than men (77% vs. 72%; p = 0.03), and those with BMIs < 25 kg/m2 showed higher platelet reactivity compared to subjects with BMIs ≥ 25 kg/m2 (75% vs. 73%; p = 0.02). There was a positive correlation between MPA and platelet count (r = 0.27; p < 0.01), as well as C-reactive protein concentration (r = 0.20; p = 0.03), while a negative correlation was observed with total cholesterol (r = -0.24; p = 0.01) and triglycerides (r = -0.30; p < 0.01). A two-step analysis based on multidimensional models with random effects revealed that every increase in the platelet count by 103/µL led to an increase in MPA by 0.07% (R2 = 0.07; p < 0.01), and an increase in triglycerides' concentration by 1 mg/dL was related to a reduction in MPA by 0.05% (R2 = 0.07; p < 0.01). Conclusions: The increased platelet reactivity observed in patients with psoriasis appears to be multifactorial and related to several clinical and laboratory features. Further research is warranted to put these findings into a clinical perspective.

Bleeding phenotype and hemostatic evaluation by thrombin generation in children with Noonan syndrome: A prospective study.

BACKGROUND: This study aimed to evaluate the bleeding phenotype and to conduct a comprehensive hemostatic evaluation in individuals with Noonan syndrome (NS), a dominantly inherited disorder caused by pathogenic variants in genes associated with the Ras/MAPK signaling pathway. METHODS: Children with a genetically confirmed diagnosis of NS underwent clinical evaluation, routine laboratory tests, platelet function testing, and thrombin generation (TG) assessment. RESULTS: The study included 24 children. The most frequently reported bleeding symptoms were easy bruising and epistaxis, while bleeding complications were observed in 15% of surgical procedures. Various hemostatic abnormalities were identified, including platelet dysfunction, von Willebrand disease, and clotting factor deficiencies. Abnormal platelet function was observed in 50% of the patients, and significantly lower TG parameters were found compared to controls. However, no significant correlation was observed between bleeding symptoms and TG results. CONCLUSIONS: The study suggests that the bleeding diathesis in NS is multifactorial, involving both platelet dysfunction and deficiencies of plasma coagulation factors. The potential role of TG assay as an ancillary tool for predicting bleeding tendencies in individuals with NS undergoing surgery warrants further investigation.

No impact of polymorphism in the phosphodiesterase 5A gene in Cavalier King Charles Spaniels on pimobendan-induced inhibition of platelet aggregation response.

BACKGROUND: A variant in the canine phosphodiesterase (PDE) 5A gene (PDE5A:E90K) is associated with decreased concentrations of circulating cyclic guanosine monophosphate (cGMP) and response to PDE5 inhibitor treatment. Pimobendan is a PDE inhibitor recommended for medical treatment of certain stages of myxomatous mitral valve disease (MMVD) in dogs. HYPOTHESIS: PDE5A:E90K polymorphism attenuates the inhibitory effect of pimobendan on in vitro platelet aggregation and increases basal platelet aggregation in Cavalier King Charles Spaniels (CKCS). Selected clinical variables (MMVD severity, sex, age, hematocrit, platelet count in platelet-rich plasma [PRP], and echocardiographic left ventricular fractional shortening [LV FS]) will not show an association with results. ANIMALS: Fifty-two privately owned CKCS with no or preclinical MMVD. METHODS: Using blood samples, we prospectively assessed PDE5A genotype using Sanger sequencing and adenosine diphosphate-induced platelet aggregation response (area under the curve [AUC], maximal aggregation [MaxA], and velocity [Vel]) with and without pimobendan using light transmission aggregometry. Dogs also underwent echocardiography. RESULTS: Pimobendan inhibited platelet function as measured by AUC, MaxA, and Vel at a concentration of 10 µM (P < .0001) and Vel at 0.03 µM (P < .001). PDE5A:E90K polymorphism did not influence the inhibitory effect of pimobendan or basal platelet aggregation response. CONCLUSIONS AND CLINICAL IMPORTANCE: The PDE5A:E90K polymorphism did not influence in vitro basal platelet aggregation response or the inhibitory effect of pimobendan on platelet aggregation in CKCS. Dogs with the PDE5A:E90K polymorphism did not appear to have altered platelet function or response to pimobendan treatment.

Prothrombin consumption as an indicator of hemorrhagic phenotype in mild platelet function disorders.

BACKGROUND: The bleeding risk of patients with mild platelet function disorders is difficult to assess and their phenotype remains ill-explored. AIM: This study was designed to establish a comprehensive biological phenotype of patients with mild platelet function disorders. METHODS: Twenty patients were included with persistent abnormal light transmission aggregometry (LTA). The ISTH bleeding assessment tool (ISTH-BAT) was assessed to identify laboratory analyses associated with an abnormal hemorrhagic score. RESULTS: The majority of patients had defects that might affect Gαi protein signaling pathways or minor abnormalities. No LTA nor flow cytometry parameters were associated with an above-normal hemorrhagic score. However, prothrombin consumption, which corresponds to the ratio of serum residual factor II to plasma residual factor II, was significantly higher (p = .006) in the abnormal ISTH-BAT group (mean = 14%, SD = 6) compared with the normal ISTH-BAT group (mean = 8%, SD 4). Prothrombin consumption was significantly associated with ISTH-BAT score (r = .5287, IC 95% 0.0986-0.7924, p = .0165). CONCLUSION: In this group of patients, there was an association between a pathological bleeding score and increased prothrombin consumption. This test could be used as an additional indicator of platelet function abnormality liable to be related to bleeding risk.

Automated Light Transmission Aggregometry with and without Platelet Poor Plasma Reference: A Method Comparison.

Background Light transmission aggregometry (LTA) is considered the gold standard for the evaluation of platelet function but is labor-intensive and involves numerous manual steps. Automation may contribute to standardization. Here, we evaluate the performance characteristics of a new automated instrument, Thrombomate XRA (TXRA), and compare it against a manual instrument (PAP-8). Materials and Methods Leftover blood samples from blood donors or patients were tested in parallel with identical reagents and in identical concentrations both manually using PAP-8 and automated on the TXRA. In addition to precision and method comparison, an additional evaluation was performed on the TXRA against "virtual" platelet-poor plasma (VPPP) based on artificial intelligence. The main focus was on comparing the maximum aggregation (MA%) values. Results Precision for MA% ranged from 1.4 to 4.6% on TXRA for all reagents. Normal ranges for 100 healthy blood donors on both instruments were in a similar range for all reagents, with a tendency to slightly higher values with TXRA. Most agonists resulted in normally distributed MA%. Comparing 47 patient samples on both devices showed a good correlation for both slope and MA% with some differences in individual samples with epinephrine and TRAP. Correlation between the TXRA measurement against PPP and "virtual" PPP demonstrated excellent correlation. Reaction signatures of both devices were very similar. Conclusion TXRA provides reproducible LTA results that correlate with an established manual method when tested against PPP or VPPP. Its ability to perform LTA only from platelet-rich plasma without requiring autologous PPP simplifies LTA. TXRA is an important step not only for further standardizing LTA but also for a more widespread use of this important method.

Investigation of Platelet Aggregation in Atherosclerosis.

Platelet activation and aggregation is implicated in all stages of inflammation-related atherosclerosis from the initial steps of endothelial dysfunction and plaque formation, to plaque rupture and atherothrombotic events, such as acute coronary syndrome, myocardial infarction, and ischemic incidences. Platelet aggregometry assays are the mainstream for evaluating and monitoring platelet reactivity in such conditions and for the investigation of prophylactic and therapeutic approaches. The most established methodology is light transmittance aggregometry (LTA). Here we describe the appropriate preparation of platelet suspensions from human blood and the methodology of LTA-based assays that is used for basic and clinical research for monitoring and evaluating the activities of several thrombotic mediators, as well as determining the dose efficacy and safety of several pharmaceutical and nutraceutical compounds intended for therapeutic and prophylactic interventions for atherosclerosis.

Canine Cancer Cells Activate Platelets via the Platelet P2Y12 Receptor.

In addition to their well-known functions in haemostasis, anucleated platelets have a critical role in cancer biology. Many human and non-human cancer types can directly interact with and activate platelets, promoting cancer malignancy and progression. Although naturally occurring canine neoplastic diseases mimic the biologically complex conditions of human cancers more closely than laboratory-bred mice, studies evaluating the relationship between cancer cells and platelets in dogs are scarce, and the effects of tumour cells on platelets in these animals are unknown. To evaluate whether cancer cells could activate canine platelets, we assessed the response of platelet-rich plasma to cultured canine cancer cells using light transmittance aggregometry. Similar to human and murine cancer cell research, we demonstrated that both canine osteosarcoma and mammary carcinoma cells activated canine platelets in vitro, resulting in platelet aggregation. The degree of aggregation was most pronounced at a cancer cell to platelet ratio of 1:200 for most cell lines. Mechanistic studies revealed that the platelet adenosine diphosphate (ADP) receptor P2Y12 is essential for canine platelet aggregation induced by canine cancer. ADP receptor blockage on platelets inhibited >50% of cancer cell-induced maximum platelet aggregation in all cell lines evaluated. As in other species, our results suggest that canine cancers may activate canine platelets in vivo. This mechanism is likely relevant for the biology and progression of cancer in the dog.

Screening and diagnosis of inherited platelet disorders.

Inherited platelet disorders are important conditions that often manifest with bleeding. These disorders have heterogeneous underlying pathologies. Some are syndromic disorders with non-blood phenotypic features, and others are associated with an increased predisposition to developing myelodysplasia and leukemia. Platelet disorders can present with thrombocytopenia, defects in platelet function, or both. As the underlying pathogenesis of inherited thrombocytopenias and platelet function disorders are quite diverse, their evaluation requires a thorough clinical assessment and specialized diagnostic tests, that often challenge diagnostic laboratories. At present, many of the commonly encountered, non-syndromic platelet disorders do not have a defined molecular cause. Nonetheless, significant progress has been made over the past few decades to improve the diagnostic evaluation of inherited platelet disorders, from the assessment of the bleeding history to improved standardization of light transmission aggregometry, which remains a "gold standard" test of platelet function. Some platelet disorder test findings are highly predictive of a bleeding disorder and some show association to symptoms of prolonged bleeding, surgical bleeding, and wound healing problems. Multiple assays can be required to diagnose common and rare platelet disorders, each requiring control of preanalytical, analytical, and post-analytical variables. The laboratory investigations of platelet disorders include evaluations of platelet counts, size, and morphology by light microscopy; assessments for aggregation defects; tests for dense granule deficiency; analyses of granule constituents and their release; platelet protein analysis by immunofluorescent staining or flow cytometry; tests of platelet procoagulant function; evaluations of platelet ultrastructure; high-throughput sequencing and other molecular diagnostic tests. The focus of this article is to review current methods for the diagnostic assessment of platelet function, with a focus on contemporary, best diagnostic laboratory practices, and relationships between clinical and laboratory findings.

Sex-Dependent Effect of Platelet Nitric Oxide: Production and Platelet Reactivity in Healthy Individuals.

A nitrate-rich diet has many cardiovascular benefits, but the mechanism behind this is unclear. We hypothesized that the ingestion of nitrate augments nitrate to nitrite reduction, leading to nitric oxide (NO) production, which may suppress platelet reactivity. In a randomized, double-blinded, placebo-controlled study involving healthy individuals, ingestion of nitrate augmented saliva and plasma nitrite/nitrate concentration and enhanced platelet NO production disproportionately in women compared with men. The response of elevated platelet NO in men was increased platelet reactivity and the response of markedly elevated platelet NO in women slightly inhibited platelet reactivity.

A new decade awaits sticky platelet syndrome: where are we now, how do we manage and what are the complications?

INTRODUCTION: Sticky platelet syndrome is a less known platelet function disorder with a familiar occurrence and likely genetic background. Clinically, it is characterized by an increased risk of venous and arterial thromboembolic events and obstetric placenta-mediated complications. The increased aggregation after low-dose ADP and/or epinephrine is its distinctive laboratory feature. Though described for almost 40 years, several issues regarding its etiology, involved pathomechanisms, genetic background, optimal diagnostic and treatment approach remain controversial. AREAS COVERED: The work aims to summarize published studies, the actual definition of the syndrome, and point out its drawbacks. A literature search on Medline, Embase, and archives from EHA congresses was performed (terms: 'sticky platelet syndrome' - 'platelet hyperreactivity' - 'platelet hyperaggregability'). The authors added in their unpublished data. The introductory overview of the present understanding is followed by the discussion of the pathophysiologic, diagnostic, and therapeutic problems. EXPERT OPINION: Despite the growing evidence provided by case reports and series, the lack of robust studies limits the decision-making on diagnostics and management. The diagnostic issues, particularly the standardization of light transmission aggregometry, represent the crucial problem for the broader acceptance of the syndrome.

PLAIN LANGUAGE SUMMARY Sticky platelet syndrome is aplatelet function disorder. It is associated with an increased risk of venous thromboembolism, arterial thrombosis, and obstetric placenta-mediated complications. Increased aggregation after low-dose ADP and/or epinephrine is the defining laboratory feature. Furthermore, afew studies report the familiar occurrence with possible genetic background. Several issues regarding the syndrome remain controversial: its exact etiology, genetics, optimal diagnostic, and treatment approach. These uncertainties provide ground for debate of the syndrome as aunique clinical entity. The review has two goals. Firstly, it summarizes the published studies and the actual definition of the syndrome. Secondly, it tries to point out the open pathophysiologic, diagnostic, and therapeutic problems.

The porcine abattoir blood model-Evaluation of platelet function for in-vitro hemocompatibility investigations.

BACKGROUND: The major obstacle of blood-contacting medical devices is insufficient hemocompatibility, particularly thrombogenicity and platelet activation. Pre-clinical in-vitro testing allows for the evaluation of adverse thrombogenicity-related events, but is limited, among others, by the availability and quantity of human blood donations. The use of animal blood is an accepted alternative for several tests; however, animal and particularly abattoir blood might present species-specific differences to human blood as well as elevated blood values, and pre-activated platelets due to stressed animals and non-standardized blood collection. MATERIAL & METHODS: To this end, we investigated porcine abattoir blood in comparison to human donor blood with the focus on platelet pre-activation and remaining activation potential. By means of light transmission aggregometry, aggregation kinetics of platelet rich plasma after stimulation with three different concentrations of each adenosine diphosphate (ADP) (5 µM, 10 µM, 20 µM) and collagen (2.5 µg/ml, 5 µg/ml, 10 µg/ml) were monitored. RESULTS: The activation with collagen revealed no significant differences in platelet behavior of the two species. In contrast, stimulation with ADP resulted in a lower maximum aggregation and a high disaggregation for porcine abattoir blood. The latter is a species-specific phenomenon of porcine platelets. Variations within each study cohort were comparable for human and abattoir pig. CONCLUSION: The similarities in platelet activation following collagen stimulation and the preservation of the porcine-specific reaction to ADP prove a general functionality of the abattoir blood. This finding provides a first step towards the complete validation of the porcine abattoir blood model.

Serum thromboxane B2 but not soluble P-selectin levels identify ischemic stroke patients with persistent platelet reactivity while on aspirin therapy.

BACKGROUND: Aspirin non-response due to persistent platelet reactivity has been associated with adverse vascular events. Light transmission aggregometry (LTA), the 'gold standard' for measuring the platelet response to aspirin therapy, is a cumbersome procedure and a simple and reliable alternative is required. Our aim was to explore whether serum thromboxane B2 (sTXB2) and soluble P-selectin can be used to identify patients who are at risk of increased platelet reactivity while on aspirin. METHODS AND RESULTS: We recruited 293 ischemic stroke patients, taking aspirin for more than seven days, and performed LTA to classify them. Based on therapeutic serum salicylate levels, 63 patients were excluded due to suspected non-compliance, followed by ELISA measurement of TXB2 and P-selectin in serum. Accordingly, patients were classified into 'Responders' (n = 122, 53%), 'Semi-responders' (n = 76, 33%) and 'Non-responders' (n=32, 14%) by LTA. Patients who had platelet aggregation of ≥70% with 10µM ADP and ≥20% with 0.5mM AA were defined as 'Non-responders'. In comparison with 'Responders', 'Non-responders' had 8.63-fold increased risk of secondary vascular events (p = 0.008). ROC curve analysis revealed that sTXB2, at a cut-off level of >4.15 ng/mL, could distinguish the patient group with elevated platelet reactivity with a sensitivity of 84.3% (AUC = 0.84), and was in fair agreement with the LTA-based classification of patients. Soluble P-selectin levels, on the other hand, had no discriminatory ability. CONCLUSION: We suggest sTXB2 measurement as an alternative to the LTA approach for identifying aspirin-treated ischemic stroke patients who are at risk of enhanced platelet reactivity and subsequent vascular events.

Impact of high on-treatment platelet reactivity after angioplasty in patients with critical limb ischemia.

OBJECTIVES: Dual antiplatelet therapy (DAPT) with aspirin and clopidogrel is standard of care in patients with peripheral artery disease (PAD) after percutaneous transluminal angioplasty (PTA). However, high on treatment platelet reactivity (HTPR) to DAPT is frequent and associated with major adverse limb events (MALE) in PAD patients. Nevertheless, association of MALE and HTPR in patients with critical limb ischemia (CLI) is not known. Moreover, comorbidities might confound response to antiplatelet medication further. Hence, in this trial we analyzed pharmacodynamic responses to DAPT and clinical events in CLI patients post PTA. METHODS: In this prospective single center pilot analysis, we included 71 CLI patients. Patients received DAPT after PTA. Antiplatelet effect were measured by light transmission aggregometry (LTA) and vasodilator-stimulated protein phosphorylation assay (VASP). MALE, major adverse cardiac and cerebrovascular events (MACCE) and BARC bleeding within 12 months follow-up were assessed. RESULTS: Mean age of patients was 73.37 ± 7.36 years and 47 (66.2%) were male. Overall HTPR appeared in 46 patients (64.8%). MALE and MACCE showed no differences between patients with and patients without HTPR. However, bleeding was higher in patients with sufficient pharmacodynamic response to DAPT (Bleeding - HTPR: 13.4% vs. no HTPR: 36.0%; log-rank HR: 0.32; 95% CI 0.1079 to 0.9396 p = 0.0217). This finding remained robust in multivariate analysis. CONCLUSION: HTPR to DAPT is frequent in CLI patients. However, bleeding was higher in patients with sufficient response to DAPT. Ischemic events did not differ. Hence, CLI patients might benefit from an alternative antithrombotic approach.

Platelet Aggregation Assays Do Not Reliably Diagnose Platelet Delta Granule Storage Pool Deficiency.

Background: Patients with platelet dysfunction disorders present with a variety of mucocutaneous bleeding symptoms including easy bruising, frequent epistaxis, bleeding gums upon tooth brushing and for women, heavy menstrual bleeding. Available laboratory assays to evaluate platelet function include the platelet function analyzer (PFA) and in larger centers with coagulation laboratories, light transmission platelet aggregometry (LTA) analyses. Both assays are known to have a number of limitations, especially in the diagnosis of platelet delta granule storage pool deficiency (δ-SPD). δ-SPD is an underdiagnosed condition caused by decreased numbers of platelet dense granules (DGs) and is best diagnosed by electron microscopy (EM). Patients with platelet δ-SPD have a decreased response to low levels of the agonist adenosine diphosphate (ADP) in the second wave of light transmittance with LTA or decreased ADP secretion by fluorescence lumiaggregometry. There are few reports that have evaluated patients with δ-SPD and their respective LTA results. One report published in 1987 described normal LTA assays in 23% of patients with δ-SPD; a more recent report described LTA as having the sensitivity to detect only about 52% of patients with δ-SPD. The purpose of our study was intended to review the LTA and EM results of patients suspected of having a platelet function disorder at our institution for comparison with previously published studies. Methods: Our study included 344 patients who had been evaluated by both LTA and whole mount EM. Aggregometry utilized five agonists: ADP, epinephrine, collagen, arachidonic acid, and ristocetin. DGs were enumerated in 100 whole-mounted platelets to determine a mean number of dense granules per platelet (DGs/PL). Results: Seventy-seven percent of our patients were found to have δ-SPD (264/344); 68% (179/264) of these subjects had an abnormal platelet LTA. Thirty-two percent (85/264) of our patients had normal LTA results but were found to have δ-SPD with a mean of 2.54 ± 0.15 DG/PL (normal = 4 - 6 DG/PL). Conclusion: These data confirm previous reports suggesting the utilization of LTA alone in patients with histories of unexplained bleeding may miss the diagnosis of platelet δ-SPD. It is, therefore, prudent to assess platelet DG number by EM, especially if platelet LTA assessment is normal.

Plateletworks® as a Point-of-Care Test for ASA Non-Sensitivity.

Aspirin (ASA) therapy is proven to be effective in preventing adverse cardiovascular events; however, up to 30% of patients are non-sensitive to their prescribed ASA dosage. In this pilot study, we demonstrated, for the first time, how ASA non-sensitivity can be diagnosed using Plateletworks®, a point-of-care platelet function test. Patients prescribed 81 mg of ASA were recruited in a series of two successive phases-a discovery phase and a validation phase. In the discovery phase, a total of 60 patients were recruited to establish a cut-off point (COP) for ASA non-sensitivity using Plateletworks®. Each sample was simultaneously cross-referenced with a light transmission aggregometer (LTA). Our findings demonstrated that >52% maximal platelet aggregation using Plateletworks® had a sensitivity, specificity, and likelihood ratio of 80%, 70%, and 2.67, respectively, in predicting ASA non-sensitivity. This COP was validated in a secondary cohort of 40 patients prescribed 81 mg of ASA using Plateletworks® and LTA. Our data demonstrated that our established COP had a 91% sensitivity and 69% specificity in identifying ASA non-sensitivity using Plateletworks®. In summary, Plateletworks® is a point-of-care platelet function test that can appropriately diagnose ASA non-sensitive patients with a sensitivity exceeding 80%.

Investigation of platelet function in patients with chronic kidney disease stages IV-V.

INTRODUCTION: Patients with renal failure are at increased risk of both bleeding and thrombosis. Further descriptions of laboratory investigations in these patients are required. METHODS: Investigation of 24 patients with chronic kidney disease (CKD) stages IV-V with light transmission aggregometry, platelet secretion assays and platelet nucleotide analysis. Patients were in a nonbleeding state and not on antiplatelet medication. Results were compared with our local reference range used within the clinical haematology service. RESULTS: Of the 24 patients, two had decreased responses to arachidonic acid, adenosine diphosphate, collagen, thrombin receptor activator peptide-6 and one had decreased responses to high dose ristocetin, and one had increased response to low dose ristocetin. 11 and 13 out of 24 had abnormal platelet secretion release to collagen and thrombin, respectively. Platelet nucleotide analysis in patients was normal with the exception of a reduction in ADP content in one patient and ATP/ADP ratio in one patient. CONCLUSIONS: In our collection of patients with CKD investigated for platelet function in the nonbleeding state, they generally had normal light transmission aggregometry and nucleotide analysis but around 50% had decreased platelet secretion assays. These results could be important in determining the significance of platelet function tests in patients with bleeding symptoms and renal failure. Further characterization of platelet function tests in future will help characterize haemostasis in renal failure further.

Is monitoring of antiplatelet therapy by light transmission aggregometry dependent on instrument and reagent used?

INTRODUCTION: Light transmission aggregometry (LTA), used to detect clopidogrel resistance in patients under antiplatelet therapy, is prone to multiple variables potentially influencing results and interpretation. Currently, no attention is given to type of aggregometer or reagent used. The aim of this study was to evaluate the interassay variability between two aggregometers (Chronolog700 and TA-8V), using two different ADP reagents (Chrono-Par® and Agro-Bio ADP) in patients under clopidogrel therapy. Additionally, LTA results were correlated to CYP2C19-polymorphism. METHODS: Light transmission aggregometry was performed in 20 healthy individuals and 30 patients using both aggregometers, and applying two different reagents (2.5 and 5 µmol/L). The maximum platelet aggregation (ADPmax ), the platelet aggregation at 6 minutes (ADP6min ), and the percentage of disaggregation at 6 min (ADP%disaggr ) were compared between four applied combinations. Additionally, 23 clopidogrel-resistant patients according to Chronolog700-Chrono-par® ADP reagent analysis were tested for CYP2C19*2 polymorphism. RESULTS: Comparison of the LTA of healthy individuals revealed a significant lower ADPmax , lower ADP6min , and higher ADP%disaggr with the TA-8V aggregometer compared to Chronolog700, regardless of the reagent. In contrast, LTA results in patients are depending on the reagent, with significant higher ADPmax and ADP6min and lower ADP%disaggr using Chrono-Par® compared to Agro-Bio ADP reagent. All intermediate clopidogrel metabolizers (CYP2C19*2 carriers) were correctly classified as clopidogrel resistant using Chrono-Par® , in contrast to the Agro-Bio ADP reagent. CONCLUSION: Light transmission aggregometry in clopidogrel-treated patients is mainly depending on the type of ADP reagent. Comparison of LTA with genotype reveals that the choice of instrument seems less influencing. In contrast, in the healthy individuals, differences could be attributed to the instrument.