Degree Of Platelet Aggregation Research Articles

Predicting the development of delayed cerebral ischemia in patients with ruptured arterial aneurysms

Introduction. Vasospasm and delayed cerebral ischemia (DCI), which develop after rupture of cerebral arterial aneurysms and their surgical treatment, are serious complications that worsen the clinical outcome of the disease. The ability to predict the risk of developing these complications is an urgent task allowing to reduce neurological deficits and improve the quality of the patients’ life after aneurysm rupture. Aim – to develop a prognostic model for the development of DCI in patients with ruptured cerebral arterial aneurysms based on the study of a complex of clinical manifestations and a number of laboratory parameters. Material and methods. We examined 91 patients with ruptured arterial aneurysms and the development of subarachnoid hemorrhage in the acute period, identifying the main group with the development of DCI (n=67) and the comparison group without DCI (n=24) in the pre- and postoperative periods. Clinical neurological and neuroimaging studies have been carried out. We studied indicators of blood oxygen transport function, platelet aggregation, coagulogram, concentration of nitrates/nitrites (NOx), angiotensin-converting enzyme (ACE) in venous blood in the pre- and postoperative periods. Results. Based on the logistic regression equation, we determined that predictors influencing the probability of DCI development after aneurysm rupture and clipping include a score on the Glasgow Coma Scale (p=0.001), the p50 value is the partial pressure of oxygen in the blood at which hemoglobin is oxygen saturated by 50% (p=0.001), ACE concentration (p=0.013), NOx/ACE index (p=0.001) and the degree of platelet aggregation according to Traptest (p=0.005). The threshold value of the result of the logistic regression equation was 0.58 with a diagnostic sensitivity of 85.4% and specificity of 69.3%, the area under the ROC of the AUC curve was 0.88±0.015 (p<0.001), which indicates the high quality of the constructed model. Conclusion. The developed predictive model of the probability of DCI development in patients with ruptured arterial cerebral aneurysms with a diagnostic efficiency of 88% allows timely identifying patients for whom it is advisable to begin neuroprotective therapy before surgical treatment.

Evaluating the impact of transient shear stress on platelet activation, adhesion, and aggregation with microfluidic chip technique.

When nonphysiological stenosis occurs, the transient high shear stress formed in vessels increases the risk of thrombosis and is a potential factor for cardiovascular diseases. But the platelet adhesion and aggregation behavior at nonphysiological post-stenosis and its affecting factors are not fully understood yet. In this experiment, platelet aggregation on collagen and fibrinogen at different shear stresses and different hematocrits were observed by microfluidic technology. Platelet activation (P-selectin, glycoprotein IIb/IIIa) and monocyte-platelet aggregate (MPA) levels under different shear stresses were analyzed by flow cytometry. On fibrinogen, platelets aggregate more at higher shear stress conditions. While on collagen, it becomes more difficult for platelets to form stable aggregation at higher shear stress conditions. If platelets adhere initially at low shear stress, stable platelet aggregation can be formed at subsequent high shear stress. Moreover, when the shear stress increases, platelet activity markers (P-selectin, glycoprotein IIb/IIIa and MPAs) increase significantly. Hematocrit affects the degree of platelet aggregation, and the influence of hematocrit is obvious at high shear stress. Transient high shear stress (46 ms) can effectively activate platelets. Platelet aggregation behavior was different for coated fibrinogen and collagen protein. Stable platelet adhesion at post-stenosis is more dependent on fibrinogen and platelet aggregation is stable on both fibrinogen and collagen. Hematocrit can significantly affect the formation of platelet aggregation.

Effect of pathological high shear exposure time on platelet activation and aggregation.

Circulating platelets are sometimes exposed to high shear rate environments due to vascular stenosis, and the effect of transiently elevated pathological high shear rates on platelet activation and aggregation function has not been clarified. The aim of this study was to investigate the effect of pathological high shear rate (8302s-1) exposure time (3.16-25.3 ms) on platelet activation and aggregation function. In addition, by adding active ingredients of antiplatelet drugs such as ASA (an active ingredient of aspirin), Ticagrelor, Tirofiban and GP1BA (platelet membrane protein GPIb inhibitor) in vitro, we studied TXA2, P2Y12-ADP, GPIIb/IIIa-fibrinogen and GPIb /IX/V-vWF receptor pathways to determine platelet activation function mediated by pathological high shear rate. In this study, we designed a set of microfluidic chips with stenosis lengths of 0.5 mm, 1 mm, 2 mm, 3 mm, and 4 mm, all with 80% stenosis, to generate pathological high shear forces that can act at different times. The whole blood flowing through the microchannels was collected by perfusion of sodium citrate anticoagulated whole blood at a physiological arterial shear rate (1500 s-1), and the expression levels of platelet surface activation markers (P-selectin and GP IIb/IIIa) and the degree of platelet aggregation were analyzed by flow cytometry; platelet aggregation patterns were observed by microscopic examination of blood smears. The results showed that shearing significantly increased platelet activation and aggregation levels compared to un-sheared whole blood, and the activation and aggregation levels increased with increasing duration of pathological high shear rate. In vitro inhibition studies showed that ASA barely inhibited the expression of P-selectin and PAC-1 on the platelet surface; Ticagrelor effectively inhibited the expression of both P-selectin and PAC-1; Tirofiban significantly inhibited the expression of PAC-1 on the platelet surface and slightly inhibited the expression of P-selectin; GP1BA significantly inhibited the expression of both. Our results suggest that transient pathological high shear rate (8302s-1) exposure can induce platelet activation in a time-dependent manner; however, the mechanism is more complex and may be due to the following reasons: transient elevated pathological high shear rate activates platelets through the GPIb/IX/V-vWF receptor pathway, and after platelet activation, its surface membrane protein GPIIb/IIIa receptors activate platelets through fibrinogen to form platelet-platelet aggregates, and further activation of active substances such as ADP and TXA2 released by platelet alpha particles, which contribute to the formation of irreversible platelet aggregation.

ГЕМОРЕОЛОГИЯ И ГЕЛИОГЕОФИЗИЧЕСКАЯ ВОЗМУЩЕННОСТЬ: СОВРЕМЕННОЕ СОСТОЯНИЕ ВОПРОСА (ОБЗОР)

There are quite a lot of scientific works that reveal some aspects of the heliogeophysical factors’s influence on the state of the rheological properties of human blood. Nevertheless, the study of the mechanisms of such influence remains open, which is associated with the difficulty of reproducing laboratory experiments, the heterogeneity and short duration of the analyzed time series of medical and biological data, as well as the interdisciplinarity of this area. The aim of this review is to analyze current research results and new original approaches to studying this issue. The results of studies published in the PubMed, Cyberleninka and eLibrary databases for the period from 1990-2021 were devoted to the study of the influence of heliogeophysical disturbances on the state of human hemorheology, and also this data was analyzed. The analysis of the literature showed a connection between heliogeophysical disturbance and the development of hemocoagulation disorders of humans, a tendency to an increase in the degree of platelet aggregation and an acceleration of the onset of thrombosis, a deterioration in the state of the microvasculature, which is most pronounced among people with diseases of the cardiovascular system. In patients with coronary artery disease, the same type of reactions was revealed, expressed by an increase in coagulation, there were heart rhythm disorders and the appearance of episodes of myocardial ischemia.

Determination of Antibody Activity by Platelet Aggregation.

Platelets play an important role in hemostasis by forming clots and stopping bleeding. In immune thrombotic conditions, platelets and leukocytes are aberrantly activated by pathogenic antibodies resulting in platelet aggregates and NETosis, leading to thrombosis and thrombocytopenia. A simple assay that assesses platelet function and antibody activity is light transmission aggregometry. This assay can be used to determine antibody activity in patients with disorders such as heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombotic thrombocytopenia (VITT). Briefly, for detection of pathogenic antibody, platelet-rich plasma (PRP) is treated with a specific agent (e.g., patient sera or purified patient antibodies) with constant stirring. Upon activation, platelets undergo a shape change and adhere to each other forming aggregates. This causes a reduction in opacity allowing more light to pass through PRP. Light transmission through the cuvette is proportional to the degree of platelet aggregation and is measured by the photocell over time. The advantage of this protocol is that it is a simple, reliable assay that can be applied to assess antibody activity in thrombotic conditions. Light transmission aggregometry does not require the use of radioactive reagents and is technically less demanding compared with 14C-serotonin release assay, another common assay for detecting antibody activity. Key features • This protocol can be used to assess platelet function and to detect platelet activating antibodies in diseases such as HIT and VITT. • Does not require radioactive reagents, requires an aggregometer; based on the light transmission aggregometry protocol, adapted for detection of VITT and other platelet-activating antibodies. • Two positive controls are required for reliable detection of antibodies in diseases such as HIT/VITT, namely a weak HIT/VITT antibody and a physiological agonist. • For detection of HIT/VITT antibodies, it is essential to use donors known to have platelets reactive to these antibodies to avoid false negative results.

The amino acid content in the daily diet of seniors negatively correlates with the degree of platelet aggregation in a sex- and agonist-specific manner.

Aging is a significant risk factor for the development of thrombotic diseases, dependent on blood platelet reactivity. However, the risk of thrombosis also appears to be significantly modulated by dietary nutrient content.The aim of the current study was to assess the relationship between the amount of amino acids present in the daily diet (not supplemented) and the reactivity of blood platelets to arachidonate, collagen and ADP in 246 women and men aged 60–65 years.Platelet reactivity was tested using whole blood impedance aggregometry. Amino acid intake was assessed with a 24-hour Recall Questionnaire and calculated with Dieta 5.0 software.Older subjects receiving higher amounts of all essential amino acids with their daily diet exhibit significantly lower platelet responsiveness to AA-, COL- and ADP in a sex-specific manner: dietary amino acid content was more closely associated with AA- and, to some extent, ADP-induced platelet reactivity in women, and with COL-induced platelet aggregability in men. Therefore, dietary amino acid content may be a novel factor responsible for attenuating platelet reactivity in a sex- and agonist-specific manner.

Platelet aggregation under the conditions of vortex flow in vitro in patients with chronic heart failure

Aim. To compare the effect of increased concentrations of aggregation inducers (five-fold addition) under standard conditions and under the conditions of vortex flow in vitro on platelet aggregation in patients with chronic heart failure (CHF).Materials and methods. The study included 28 patients. The activity of platelet aggregation in platelet-rich plasma (PRP) was evaluated according to light transmission curves (%) and the average size of aggregates (relative units (rel. units)). The aggregation inducer was added once at 10 seconds of the study (standard procedure) and five times at 10 seconds, 1, 2, 3, and 4 minutes of the study with a constant stirring rate of 800 rpm. The same parameters were evaluated under the conditions of vortex flow, which was created by changing the stirring rate of the PRP from 800 rpm to 0 rpm and again to 800 rpm by pressing the centrifugation button on the analyzer.Results. In the course of the study, the size of the aggregates increased in patients with CHF only under the conditions of vortex flow. When a collagen aggregation inducer was added both at the concentration of 2 mmol / l and 10 mmol / l, platelet aggregation parameters increased under the conditions of vortex flow. During the study of epinephrine-induced platelet aggregation in patients with CHF, an increase in the aggregation parameters was revealed, both at five-fold addition of the inducer and under the conditions of vortex flow compared with the standard method.Conclusion. The proposed methodological approaches to creating the conditions for vortex flow in vitro and to five-fold addition of epinephrine showed an increase in the size of the aggregates and the degree of platelet aggregation. Collagen-induced aggregation under the conditions of vortex flow revealed 7 (25%) patients with high residual platelet reactivity (HRPR), and epinephrine-induced aggregation detected 15 (54%) patients with HRPR.

Safety, pharmacokinetics and pharmacodynamics of an original glycoprotein IIb/IIIa inhibitor in healthy volunteers: results of the clinical trial

Aim. To study the tolerability, safety, pharmacokinetics (PK) and pharmacodynamics of single intravenous infusions of Angipur in healthy male volunteers.Material and methods. The Phase I trial included 20 healthy male volunteers (mean age, 30,8±7,7 years; mean body weight, 77,4±12,1 kg). Angipur (0,02% concentrate for solution for infusion) was administered to every subject in single doses 0,015, 0,05, 0,09 mg/kg for 3 consecutive days. Volunteers were divided in 6 groups (1, 1, 3, 5, 5, 5); every following group was recruited only after the previous one finished the study. The following were assessed: rate and severity of adverse events (AEs), key PK parameters of Angipur and its antiplatelet activity by impedance aggregometry.Results. No moderate or severe AEs, as well as no serious AEs were reported according to obtained data of clinical and laboratory monitoring of healthy subjects. Totally 6 mild AEs were registered in 4 subjects. Four AEs (mild hematological deviations and episode of nose bleed) were classified as possibly related to study drug and 1 AE (positive fecal occult blood test) — probably related. Key PK parameters of Angipur in single intravenous doses 0,015, 0,05 и 0,09 mg/kg were determined as follows: Cmax — 12,44±4,689, 46,10±14,295, 92,48±33,896 ng/ml; Vd — 304,01±55,300, 299,67±64,244, 252,96±47,790 l; T1/2 — 6,72±1,290, 6,84±2,341, 6,06±2,287 h; Cl — 32,19±6,919, 32,29±8,357, 31,55±10,113 l/h, respectively. Dose proportionality (linear PK) for parameters Cmax, AUC0-t and AUC0-∞ was established. Dose-dependent reduction of ADP-induced platelet aggregation degree and area under curve was revealed at period of 15 min to 2-4 h after Angipur infusion in doses 0,05 and 0,09 mg/kg.Conclusion. Results of phase I clinical trial demonstrated good tolerability of single intravenous infusions of Angipur (0,015, 0,05 и 0,09 mg/kg) in healthy subjects. We determined key PK parameters and indicated dose-dependent antiplatelet activity of Angipur.

VISCOSE AND THROMBOCYTIC-AGGREGATION DISORDERS ON PATIENTS WITH MYOCARDIAL INFARCTION WITH STABLE ELEMENTATION OF ST SEGMENT

Relevance. Clinical studies indicate a significant role in the pathogenesis of acute myocardial infarction, the functional state of platelet aggregation and blood viscosity, which determine the nature of blood flow in the vessels at the level of macro– and microcirculation.
 Objective: the aim of the study was to analyze changes in viscosity and platelet-aggregation disorders under the influence of different tactical, diagnostic and interventional approaches in patients with myocardial infarction and stable elevation of the ST segment in the presence of multivascular coronary artery disease.
 Materials and methods. Patient inclusion criteria for the study: MI with persistent ST-segment elevation; multi-vascular atherosclerotic coronary artery disease; angiographically significant stenosis of the coronary vessels more than 50-70%, at least in 2 projections; functionally significant stenosis of the coronary arteries, according to the fractional blood flow reserve (FBFR) (<0.80). 102 patients were divided into 4 groups of the same type. The groups used 4 different tactical and treatment-diagnostic approaches. Group 1 (n = 25) - occlusion of an infarct-dependent vessel was detected using coronary angiography and its immediate stenting was performed; FBFR was established on days 2-3, and stenosis of infarction-independent arteries was proved; using optical coherence tomography (OCT), the stability, size, length of atheroma was assessed and delayed stenting of these vessels was performed. Group 2 (n = 26) - stenting of the infarct-dependent artery was performed; FBFR was established on days 2-3, but without OCT, followed by stenting of infarct-independent arteries. Group 3 (n = 25) - carried out simultaneous stenting of infarct-dependent and infarct-independent vessels after coronary angiography. Group 4 (n = 26) - only stenting of the infarct-dependent vessel was performed. All patients received standard two-component antiplatelet therapy (acetylsalicylic acid 75-150 mg/day and clopidogrel 75 mg/day) and received 20 mg of atorvastatin and 10 mg of ezetimibe. The results obtained in patients were compared with those results obtained in 30 practically healthy people of the same age and gender. The number of platelets and the concentration of fibrinogen were determined in each subject. Using a viscometer and a laser aggregometer the degree of platelet adhesion, the time of platelet adhesion, the degree of platelet aggregation, and blood viscosity were assessed. All indicators were studied three times - on the first day of myocardial infarction, after 3-4 weeks, and after a year. 
 Results. Hyperviscous and hypercoagulable syndromes were established in patients with MI with persistent ST-segment elevation. They manifested themselves in the first weeks by a compensatory increase in the number of platelets, blood viscosity, fibrinogen levels, and an increase in platelet aggregation. Delayed stenting of infarct-independent arteries used in group 1 contributed to a lower amplitude of fluctuations of all laboratory parameters in comparison with other tactical and therapeutic approaches.
 Conclusions. It is possible to optimize the state of viscosity and the processes of adhesion/aggregation of platelets due to the systemic stepwise improvement of arterial blood supply. Traditional tactical and therapeutic-diagnostic principles of management of such category of patients do not fundamentally improve the state of viscous and coagulation-aggregation parameters even when using double antiplatelet therapy.

ПОКАЗАТЕЛИ КОАГУЛЯЦИОННОГО РАВНОВЕСИЯ И АГРЕГАЦИИ ТРОМБОЦИТОВ У ПАЦИЕНТОВ С ИНФЕКЦИЕЙ COVID-19

Objective. To determine changes in coagulation balance and platelet aggregation in patient during the treatment of COVID-19 infection. Methods. A pilot non-randomized prospective clinical study of coagulation balance and platelet aggregation in patients admitted to the intensive care unit with acute respiratory distress syndrome and the diagnosis of COVID-19 (n=50) was performed. Out of 50 patients, 19 patients died, 31 patients were transferred to the therapeutic department. The study of indicators of coagulation balance and platelet aggregation was carried out once in 1-3 days starting from the patient’s admission to the hospital using coagulation analyzer ACL 10000 (Instrumentation Laboratory, USA) and platelet aggregation analyzer AP 2110 (ZAO “SOLAR”, Republic of Belarus). Results. In 45 (90%) patients with COVID-19, there is a significant increase of von Willebrand factor activity 350 (244.5; 480) %. There were no statistically significant differences in the level of von Willebrand factor activity among the deceased and surviving patients: 450.0 (338.8; 530.5) % in deceased patients and 342.0 (188.8; 480.0) % in survivors. At von Willebrand factor activity level of up to 250%, the mortality rate was 8.3%, at a level of 250-400% - 31.3%, at a level of more than 400% - 59.1%. Significantly above the normal range in most patients were fibrinogen (above normal in 68% of patients, 4.63 (3.49; 5.87) g/L) and D-dimers (above normal in 88% of patients, 0.73 (0,31; 1.4) μg/ml). Antithrombin III was below normal in 56% of patients (82 (67.1; 97.2) %). The degree of platelet aggregation has a strong direct correlation with the von Willebrand factor level: with an ADP inducer 0.3 μg/ml (R=0.71, р=0.003); ADP 0.6 μg/ml (R=0.74, р=0.0001); ADP 1.25 μg/ml (R=0.53, р=0.01). Conclusion. Analysis and evaluation of coagulation balance and platelet aggregation should be an integral part in the treatment of patients with COVID-19 infection. What this paper adds For the first time, the indicators of coagulation balance and platelet aggregation have been assessed in the treatment of COVID-19 infection. It has been found that 90% of patients had the increasedlevels ofvon Willebrand factor(VWF) - 350 (244.5; 480)%. In addition to an increase of von Willebrand factor activity, 56% of patients show a reduction of antithrombin III levels, and 88% and 68% of patients have an increasing D-dimers and fibrinogen, respectively. Analysis and assessment of coagulation balance and platelet aggregation indicators should be integral components of the treatment of patients with COVID-19 infection.

High residual platelet aggregation in patients with coronary artery disease: a new methodological approach to detection

Aim. To develop a new methodological approach to assessment of collagen-induced platelet aggregation in patients with coronary artery disease (CAD) and to determine the quality of various methods for detecting high residual platelet reactivity (HRPR) to predict the risk of myocardial perfusion disturbance. Materials and methods. 36 patients (10 men and 26 women) aged 41–83 years and having stable CAD were examined. All patients had been undergoing continuous antiaggregation therapy for 6 months. We evaluated platelet aggregation using a laser analyzer with collagen as an aggregation inducer by the standard method 1 and our own patented method 2. The degree of platelet aggregation (%) and the size of aggregates in relative units (r.u.) in platelet-rich plasma were estimated. Myocardial perfusion scintigraphy with 99mTc-methoxy-isobutylisonitrile was performed according to a two-day stress-rest protocol. The summed stress score (SSS) values were used for analysis. SSS < 4 was regarded as normal myocardial perfusion. Results . The degree of platelet aggregation according to method 1 was 12 (5; 64)%, the aggregate size was 3 (2; 7) r.u. The degree of platelet aggregation according to method 2 was 44 (13; 78)%, and the aggregate size was 5 (4; 8) r.u. Method 2 allowed to diagnose the presence of myocardial ischemia with an aggregation degree ≥ 44.9% with sensitivity of 84% and specificity of 92% (area under the curve (AUC) = 0.89; p < 0.0001; odds ratio (OR) 2.18; 95% confidence interval (CI) 0.57–0.98) and an increase in aggregate size ≥ 4.80 r.u. with sensitivity of 84% and specificity of 84% (AUC = 0.95; p < 0.00001; OR 5.83; 95% CI 0.72–0.99). Conclusion. In patients with CAD, detection of high rates of collagen- induced platelet aggregation using the patented technique is associated with the risk of impaired myocardial perfusion. The developed new methodological approach to detection of HRPR allowed to determine high risk of atherothrombotic complications in additional 22% of the examined patients.

ANALYSIS OF THE STATE OF PLATELET AGGREGATION IN PREGNANT WOMEN WITH THROMBOPHILIA AND BURDENED OBSTETRIC HISTORY

The analysis of platelet aggregation in pregnant women with thrombophilia and burdened obstetric history was performed. It has been determined that the development of adverse effects of pregnancy is significantly influenced by conditions such as placental abruption, fetal growth retardation, preeclampsia and fetal death, which are provoked by an abnormal vascular network of the placenta and hemostasis disorders caused by various thromboembolic disorders due to thrombophilia. Therefore, in recent years, the problem of thrombophilia has attracted much attention as a risk factor for pregnancy complications caused by various thromboembolic disorders, which are responsible for preeclampsia, fetoplacental insufficiency, fetal growth retardation and fetal loss, and others. Given the above, the aim of the work was to analyze the state of platelet aggregation of pregnant women with thrombophilia and burdened obstetric history. To achieve this goal, a study of pregnant women with thrombophilia and burdened obstetric history was conducted: 137 pregnant women, which were divided into two study groups – the main group, which included 101 pregnant women with a burdened obstetric history and thrombophilia, and the control group, which included 36 pregnant women with a burdened obstetric history without thrombophilia. The analysis of platelet aggregation of pregnant women with thrombophilia and burdened obstetric history was performed. It was found that the degree of platelet aggregation with the addition of an inducer of adenosine diphosphate at a concentration of 0.0625 prevailed in the control group as compared with pregnant women with thrombophilia (26.3 [24.3; 28.4] % and 21,4 [14,6; 31,1] % in contrast to the aggregation time and its speed (which were lower in the controls (58.0 [54.0; 72.0] s and 22.9 [20.4; 24.9] min) as compared with the main group (71.0 [48.0; 530.5] s) (and 26.5 [14.5; 38.3] min)). It was found that at a concentration of adenosine diphosphate of 0.125, all indicators of platelet aggregation prevailed in the main group (degree of aggregation (35.4 [25.6; 52.5] %; U = 1236.0; p = 0.004), its time (115.0 [47.0; 324.0] s; U = 1623.5; p = 0.341) and speed (45.2 [32.1; 57.5] min; U = 968.5; p < 0.001) compared to the controls (30.1 [26.7; 31.2] %, 84,0 [78.0; 103.5] s and 26.4 [30.4; 35.8] min). It was determined that the concentration of adenosine diphosphate of 0.250 caused an increase in the aggregation time in the control group as compared to the main group (225.5 [196.5; 269.3] s and 181.0 [57.0; 347.0] s; U = 1554.5; p = 0,198) in contrast to the degree [43,3 [39,6; 48.8] % and 51.1 [35.4; 63.8] %; U = 1417.0; p = 0.050) and aggregation rates (57.8 [42.8; 67.0] min and 40.1 [34.4; 47.9] min; U = 786.5; p < 0.001). It was also found that the maximum concentration of adenosine diphosphate (0.500) caused a significant (U = 11178.0; p = 0.002) increase in the aggregation time in the controls (329.0 [269.5; 390.0] s) compared to the main group (211.0 [72.5; 381.0] s) in contrast to the degree and rate of aggregation [51.9 [50.1; 54.3] %; U = 1606.5; p = 0.300 and 42.7 (38.8; 49.4] min; U = 923.5; p < 0.001 and 55.1 [38.6; 69.0] % and 63.6 [44.7; 72.6] min).

How Much Does the Platelet Aggregate Influence the Total Leukocyte Count? Comparison Between Manual and Automated Impedance Methods in Domestic Cats

Background: Automated hematology analyzers have been developed to optimize the time between analyses and have promising precision and accuracy. Complete blood count (CBC) is often requested as part of veterinary clinical examination. Automated analyzers are often used to determine CBCs, since processing as well as container-related errors may occur owing to variable sizes, aggregates, white or red blood cell fragments, and effects of EDTA on cell morphology. Platelet aggregates frequently occur in felines, with studies reporting a prevalence of approximately 71%. The aim of the present study was to evaluate the influence of exercise aggregates on the global white blood cell count of domestic cats using automated hematological counters with the impedance method.Materials, Methods & Results: Blood samples of 140 cats, irrespective of age, sex, and breed, were collected into EDTA-containing tubes. The samples were obtained via routine clinical examinations at the Veterinary Hospital of the Federal Rural University of Rio de Janeiro (UFRRJ) and processed at the Veterinary Parasitology Experimental Chemotherapy Laboratory (LQEPV), belonging to the same institution. All the samples were processed on the Sysmex pocH-100iV Diff automated hematology apparatus according to the manufacturer's recommendations. Leukocyte counts were also manually determined using a duplicate Neubauer chamber. Standard dilutions were prepared immediately after the automated analysis. To identify the occurrence of platelet aggregates, a blood smear was made and visualized under a brightfield microscope at a magnification of 10× and scored 0 to 3 (G1, G2, G3, and G4) based on the aggregation intensity. In case of changes, the groups were subdivided according to the intensity of occurrence. Of the 140 samples analyzed, 76.4% (107/140) showed some degree of platelet aggregation. The maximum variation in leukocyte counts determined by the automatic and the manual technique in G1 was 2,500 cells. In G2, it was possible to identify a variation of 6,500 nucleated cells, whereas in G3, this value was 7,100 cells. In G4, where platelet aggregation was intense, the variation between counts was up to 15,000 nucleated cells. A significant difference of variation in total white blood cell count between manual and automated methods was observed when compared to animals that did not show any degree of platelet aggregation (P < 0.05). Of the total samples, 23.57% (33/140) comprised G1, 24.28% (34/140) G2, 22.14% (31/140) G3, and 30% (42/140) G4. Of the 140 samples analyzed, 107 showed aggregates, pseudo-thrombocytopenia, and changes in the total number of leukocytes.Discussion: Samples with higher platelet aggregate formation showed greater interference in global leukometry when analyzed using the hematological counter. White blood cell counts determined by automated analyzers should be interpreted with caution and compared to manual counts when there is significant platelet aggregation in the sample. The findings reinforce the importance of reconfirming the results obtained using an automated equipment in order to avoid misinterpretations that may influence diagnosis and therapy. It is essential to re-check the values obtained from an automated equipment with traditional methods in order to minimize possible errors generated by the equipment, since such errors may affect the clinical diagnosis and subsequently, the therapeutic approach chosen.

Effect of perioperative infusion therapy on the functional state of the hemostatic system in patients with concomitant coronary heart disease

Background. The choice of the infusion therapy regimen in the perioperative period remains a complex and controversial issue of modern anesthesiology. This is especially true for elderly patients with concomitant cardiovascular diseases, primarily coronary heart disease (CHD). Excessive fluid restriction in the perioperative period during the intervention can contribute to the development of arterial hypotension and hypoperfusion of vital organs. At the same time, excessive fluid intake in these patients is dangerous in terms of developing complications such as decompensation of heart activity, ischemic myocardial damage. There are many factors of the perioperative period that affect the processes of fluid metabolism in the body, the state of hemodynamics and other vital functions. Among them, the most significant factors are operational stress, features of the underlying disease and surgical intervention, the influence of anesthetics, the functional state of the cardiovascular system, kidneys, etc. One of the insufficiently considered factors that may influence the choice of infusion therapy, in our opinion, is the functional state of the hemostatic system in the preoperative period.
 Objective. To investigate the effect of perioperative infusion therapy on the functional state of the hemostatic system in patients with concomitant CHD.
 Materials and methods. A total of 92 patients who underwent abdominal surgery under combined general anesthesia with a ventilator were examined. The average age of patients was 61±12 years; risk on the ASA scale – II-III; risk of cardiac complications on the RCRI – 1-3; risk of thrombosis on the Caprini scale – 6.5±0.1. The functional state of platelets was assessed using the platelet aggregation analyzer AR 2110 (Belarus); the state of plasma hemostasis was assessed using standard coagulogram indicators.
 Results and discussion. When studying platelet aggregation in the initial state, significant fluctuations in the studied parameters were found from significant hypoaggregation to significant platelet hyperaggregation. For further analysis and differential correction, patients were divided into three groups depending on the degree of platelet aggregation. Group 1 included 22 patients with established hypoaggregation, 2nd group – 38 patients with established normal platelet aggregation, and 3rd group – 32 patients with platelet hyperaggregation. The coagulogram in the majority of patients in the initial state characterized normocoagulation or a tendency to hypercoagulation. Correction of changes in primary hemostasis was performed using infusion therapy, depending on the initial data of platelet aggregation. In the group with greegreece platelets was conducted infusion therapy with the liberal type – 5-10 ml/kg/h for intraoperative stage and 20-25 ml/kg/day after surgery; in the group with hoareau for restrictive type an average of 3-5 ml/kg/h for intraoperative stage and 20-25 ml/kg/day after surgery; in the group with normoergic the relatively restrictive type that was 5-7 ml/kg/h intraoperatively; 25 ml/kg/day after surgery. For specific correction of platelet-vascular hemostasis, etamzilate 12.5 % 4.0 ml was used in group 1 patients before surgery and later 4.0 ml three times a day; in group 3 patients, pentoxifylline 2 % 5.0 ml twice a day. Thromboprophylaxis with low-molecular-weight heparins in the perioperative period was performed in all patients according to current recommendations. As a result of this approach to the correction of established disorders of platelet-vascular hemostasis, a clear trend towards normalization of the studied parameters was established already at the intraoperative stage, this trend persisted a day after the operation. Thus, the indicators of platelet aggregation in group 1 patients at the intraoperative and early postoperative stages were 68.2 (59.5; 78.1) and 63.6 (60; 72.6); in group 3 patients – 79.7 (75.3; 94.2) and 74.6 (59.2; 83.4), respectively.
 Conclusions. Individualized infusion and pharmacological therapy allows correction of disorders of platelet-vascular hemostasis in patients with concomitant CHD, which may be useful for reducing the risk of thrombotic complications.

Assessment of platelet function utilizing viscoelastic testing.

Assessment of platelet function utilizing viscoelastic testing.

Эндотелиопротекторные свойства ATACL и катехин гидрата в условиях истощающих нагрузок крыс

Цель исследования - изучение эндотелиопротекторных свойств ATACL и катехин гидрата в сравнении с известным лекарственным препаратом Сулодексид в условиях истощающих нагрузок крыс. Методика. Истощающие нагрузки воспроизводили методом Порсолта - принудительного плавания животных с нагрузкой до полного истощения и борьбы за жизнь. Эксперименты выполнены на 50 крысах-самцах Wistar (200-220 г), разделенных на 5 групп (n=10). Вазодилатирующую функцию эндотелия сосудов головного мозга оценивали методом допплерографии с использованием программного комплекса «Minimax Doppler», производства компании «Минимакс» (Санкт-Петербург, Россия). Результаты. Показано, что на фоне истощающих нагрузок наблюдалось значительное ухудшение вазодилатирующей и антитромботической функций эндотелия мозговых сосудов, что проявлялось снижением реакции сосудов в ответ на введение анализаторов (АЦХ и L- NAME), развитием «феномена L-аргининового парадокса», повышением как степени, так и агрегационной способности (скорости) тромбоцитов. Исследуемые соединения ATACL и катехин гидрат способствовали коррекции эндотелиальной дисфункции, что выражалось в статистически значимом увеличении скорости кровотока при внутривенном введении АЦХ у животных групп, получавших ATACL на 26,1%, катехин гидрат - на 24,5%, относительно исходной скорости кровотока. ATACL (4-гидрокси-3,5-ди-трет-бутилкоричнаякислота) оказывал выраженное влияние на антитромботическую функцию, при этом статистически значимо снижалась степень агрегации тромбоцитов на 99,3% и скорости агрегации - на 136,6%, относительно аналогичных показателей группы крыс негативного контроля. Заключение. Соединение ATACL представляет интерес для дальнейшего изучения в качестве актопротекторного средства, обладающего эндотелиопротекторной активностью. Aim. To study possible endothelium-protective properties of catechin hydrate and ATACL in comparison with a well-known drug Sulodexide in exhaustive physical exercise of rats. Methods. Exhaustive exercise was produced by forced swimming of rats with a load until exhaustion and struggle for life. Experiments were performed on 50 Wistar male rats (200-220 g) divided into 5 groups (n=10 in each group). The endothelial vasodilatory function of cerebral blood vessels was evaluated with the Doppler method using Minimax Doppler equipment and software (Minimax, St. Petersburg, Russia). Results. Exhaustive exercise induced a significant impairment of vasodilatory and antithrombotic endothelial functions, which was evident as decreased response of cerebral blood vessels to acetylcholine (ACH) and L-NAME, development of the L-arginine paradox, and increases in both platelet aggregation degree and rate. ATACL (4-hydroxy-3,5-di-tert-butyl cinnamic acid) and catechin hydrate provided correction of the endothelial dysfunction, which was evident from increased blood flow velocity in response to intravenous administration of ACH by 26.1% in the ATACL group (p<0.05) and by 24.5% in the catechin hydrate group (p<0.05) compared to the baseline blood flow velocity. ATACL exerted a pronounced effect on the endothelial antithrombotic function evident as decreases in aggregation degree by 99.3% (p<0.05) and aggregation rate by 136.6% (p<0.05) compared to the group of negative control. Conclusion. ATACL is of interest for further studies as an actoprotector with endothelium-protective activity.

Prevalence and Features of Aspirin Resistance in Patients with Coronary Artery Disease

Purpose. To determine the frequency of development of aspirin resistance in patients with coronary heart disease and to identify risk factors for the development of resistance to acetylsalicylic acid (ASA) in patients with coronary heart disease. Material and Methods: 96 patients were enrolled in this study with stable forms of ischemic heart disease who received a standard dose of aspirin 75 mg/day for a long time. Patients were divided into 4 groups according to the prescription of ASA. Group 1 consisted of patients who did not receive aspirin, the second group consisted of patients taking aspirin up to 1 year, the third group consisted of patients taking aspirin from 1 year to 5 years, and the fourth group included patients taking aspirin for more than 5 years. An addition, according to the results of determining aspirin resistance, the patients were divided into 2 groups. Platelet aggregation was measured using a laser analyzer: adenosine diphosphate (ADP) with 1.0 and 5.0 μmol was used. Resistance criterion: ADP ≥72% at 5.0 μmol. Results: 17.7% of patients who took aspirin in a standard dose of 75 mg/day were resistant to aspirin, and a third of them took aspirin for more than 5 years. According to the results of a study of platelet aggregation activity, the average degree of platelet aggregation with 5.0 μmol of ADP was 82.4% in patients with no reaction to aspirin. An inadequate response to aspirin was reliably often observed in women and the elderly (58.8% and 70.5%). When analyzing the results of routine laboratory studies in patients with aspirin resistance, there was a tendency to higher levels of cholesterol and glucose (p˂0.05). Possible risk factors for the development of ASA resistance are hypercholesterolemia and hyperglycemia, since aspirin resistance was more common in patients with diabetes (13 out of 17) and obesity (11 out of 17).Conclusion: High rates of ADP-induced platelet aggregation were obtained in ¼ patients with coronary heart disease. A

Experimental estimation of the effects of exogenous carbon monoxide on blood cells

The aim of the study was to investigate the effect of the carbon monoxide (CO) donor on the Ca 2+ -activated potassium permeability of the erythrocyte membrane and platelet aggregation ability. Materials and methods. Healthy volunteers ( n = 27) and patients with chronic coronary heart disease (CHD) ( n = 32) of both sexes were examined. The material of the study was packed red blood cells and platelet-rich plasma obtained from patient’s venous blood. The change of Ca 2+ -dependent potassium conductivity of the erythrocyte membrane was evaluated by potentiometric method, and the platelet aggregation was studied by turbidimetric method. Carbon monoxide releasing molecule-2 ( CORM-2) was used as a CO donor. The amplitude of A23187- and redox-induced hyperpolarization response (HR) of erythrocytes, and the rate and degree of platelet aggregation were estimated. Results. It was shown that the addition of CORM-2 (10 and 100 μM) in the erythrocyte suspension caused a dose-dependent decrease in the amplitude of A23187- and redox-dependent HR in healthy donors, as well as in patients with chronic CHD. The maximum decrease was observed in the presence of 100 μM CORM-2. The effect of CORM-2 at concentrations of 10 and 100 μM on collagen-induced platelet aggregation led to a decrease in the degree and rate of aggregation in healthy donors. The maximum effect was shown at 100 μM of CO donor. However, such an unambiguous effect of CORM-2 on the aggregation parameters in patients with CHD was not observed. Conclusion . The results suggest that CO has a significant effect on the ion transport function of the erythrocyte membrane and platelet aggregation activity of both healthy donors and patients with CHD.

Relation of Functional Activity of Platelets to Prognosis of Unfavorable Cardiovascular Events in Patients with Acute Coronary Syndrome. Results of a Registry Study

to assess relation ofhigh functional activity ofplatelets to prognosis ofunfavorable cardiovascular events in patients with Acute Coronary Syndrome (ACS). The study was based on the data of a single center ACS registry conducted in the Central Clinical Hospital of the Presidential Affairs Department of Russian Federation. Of 529 included patients in 425 without contraindications to double antiplatelet therapy we carried out analysis of dependence of 30 days level of unfavorable events on parameters of functional activity of platelets. High on-treatment platelet reactivity (HTPR) was found to be associated with 3.5 increase of mortality in the group of patients with high cardiovascular risk. Logistic model of prognosis of unfavorable events based on multifactorial analysis of data from patients with measured platelet aggregation included chronic kidney disease, type of myocardial infarction, and degree ofplatelet aggregation >45%. C -statistic was equal to 0.77. We also present in this paper discussion of problems related to studying approaches to individualization of anti-aggregation therapy in real clinical practice and problems of organization ofsimilar studies. The study showed that patients with ACS increased platelet aggregation, as well as chronic kidney disease and type 2 MI are associated with a 30 day prognosis of adverse events.

Коррекция нарушений показателей свертывающей системы крови крыс, вызванных церебральной ишемией, соединением PIR-10

Нарушения антитромботической функции эндотелия сосудов вызывали моделированием окклюзии левой средней мозговой артерии у крыс-самцов линии Вистар. Первая группа — ложнооперированные особи, вторая — крысы негативного контроля. Животным третьей группе вводили препарат сравнения сулодексид (30 ЛЕ, единицы высвобождения липопротеинлипазы), четвертой — исследуемое соединение PIR-10 (50 мг/кг). Все изучаемые вещества вводили внутрибрюшинно сразу после операции и ежедневно однократно в течение 3 сут. Соединение PIR-10 в равной степени, в сравнении с сулодексидом, способствовало уменьшению АДФ-индуцированной агрегации тромбоцитов (степень на 53,63 % (p < 0,05), скорость на 62,94 % (p < 0,05)). Кроме того, данное соединение препятствовало гиперкоагуляционному сдвигу по параметрам: активированное частичное тромбопластиновое время, протромбиновое время, тромбиновое время — на 117,62 % (p < 0,05), 57,55 % (p < 0,05), 92,06 % (p < 0,05), соответственно, и вызывало уменьшение концентрации фибриногена в плазме крови на 42,39 % (p < 0,05) в сравнении с животными, не получавшими фармакологические вещества, соединение PIR-10 предотвращало нарушения антитромботической функции эндотелия в условиях церебральной ишемии крыс, не уступая препарату сравнения сулодексиду.