Whole Blood Platelet Research Articles

Comprehensive analysis of L-PRF exudate components and their impact on whole blood platelets.

This study assessed the cellular composition and effects of leukocyte-platelet-rich fibrin (L-PRF) exudate on whole blood platelets from healthy volunteers. Key objectives included evaluating leukocyte subpopulations, platelet activation markers, platelet-leukocyte interactions and quantifying inflammatory cytokines within the L-PRF exudate. L-PRF was obtained from 20 healthy donors. Flow cytometry methodologies were used to assess intracellular calcium kinetics and activated GPIIbIIIa, and P-selectin expression. Leukocyte subpopulations and platelet-leukocyte interactions were characterized using monoclonal antibodies. Inflammatory cytokines (IL-8, IL-1β, IL-6, IL-10, TNF, IL-12p70) within L-PRF exudate were quantified using a cytometric bead array. The expression of activated GPIIbIIIa, and P-selectin exhibited a significant increase (p < 0.001) when L-PRF exudate was added to platelets of whole blood. Regarding intracellular Ca2+ mobilization, the L-PRF exudate elicited significant responses (p < 0.001). L-PRF exudate contained different leukocytes populations, being TCD4 + the most representative of T cells. It was possible to stablish a profile of cytokines produced by the L-PRF exudate, with human IL-8 cytokine exhibiting the highest average (16.90 pg/mL). Despite the study limitations, the research yielded important insights: 1- L-PRF exudate can stimulate platelet activation, essential in healing, tissue inflammation and remodeling. 2-The presence of leukocyte subpopulations within L-PRF exudate reflexes its complexity and potential to enhance immune responses. 3-The analysis of inflammatory cytokines within L-PRF exudate revealed its immunomodulatory potential. These findings are valuable evidences for understanding the potential role of L-PRF exudate in regenerative dentistry and medicine, offering innovative therapeutic strategies. This research highlights crucial aspects that could significantly influence the clinical use of L-PRF exudate in the oral cavity. The findings support the application of L-PRF exudate in both surgical and regenerative dentistry, facilitating the development of innovative therapeutic strategies to enhance patient outcomes.

The differential formation and composition of leukocyte-platelet aggregates induced by various cellular stimulants

BackgroundLeukocyte-platelet aggregates comprise a pathogenic link between hemostasis and immunity, but the prerequisites and mechanisms of their formation remain not understood. AimsTo quantify the formation, composition, and morphology of leukocyte-platelet aggregates in vitro under the influence of various cellular activators. MethodsPhorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS), thrombin receptor-activating peptide (TRAP-6), and adenosine diphosphate (ADP) were used as cellular activators. Flow cytometry was utilized to identify and quantify aggregates in whole human blood and platelet-rich plasma. Cell types and cellular aggregates were identified using fluorescently labeled antibodies against the appropriate cellular markers, and cell activation was assessed by the expression of appropriate surface markers. For confocal fluorescent microscopy, cell membranes and nuclei were labeled. Neutrophil-platelet aggregates were studied using scanning electron microscopy. ResultsIn the presence of PMA, ADP or TRAP-6, about 17–38 % of neutrophils and 61–77 % of monocytes formed aggregates with platelets in whole blood, whereas LPS did not induce platelet aggregation with either neutrophils or monocytes due the inability to activate platelets. Similar results were obtained when isolated neutrophils were added to platelet-rich plasma. All the cell types involved in the heterotypic aggregation expressed molecular markers of activation. Fluorescent and electron microscopy of the aggregates showed that the predominant platelet/leukocyte ratios were 1:1 and 2:1. ConclusionsFormation of leukocyte-platelet aggregates depends on the nature of the cellular activator and the spectrum of its cell-activating ability. An indispensable condition for formation of leukocyte-platelet aggregates is activation of all cell types including platelets, which is the restrictive step.

Photodynamic inactivation of antibiotic-resistant bacteria in whole blood using riboflavin photodynamic method.

Treating bacteremia caused by antibiotic-resistant bacteria is a global concern. Antibacterial photodynamic inactivation is a promising strategy to combat it. However, it's challenging to achieve the inactivation of antibiotic-resistant bacteria in whole blood because of its opacity and complexity. We investigated a riboflavin photodynamic method to effectively inactivate antibiotic-resistant bacteria in whole blood. Four strains of antibiotic-resistant bacteria were isolated, identified, and cultured in this research: methicillin-resistant Staphylococcus aureus (MRSA), pan-drug-resistant Acinetobacter baumannii (PDRAB), ESBLs-producing Escherichia coli (EPEC) and pan-drug-resistant Klebsiella pneumoniae (PDRKP). To simulate bacteremia, antibiotic-resistant bacteria was added into whole blood. Whole blood was treated using riboflavin photodynamic method with ultraviolet irradiation (308 nm and 365 nm). The ultraviolet irradiation dose was divided into 18 J/cm2, 36 J/cm2, and 54 J/cm2. Microbial count of antibiotic-resistant bacteria in whole blood was used for evaluating inactivation effectiveness. The roles of red blood cells, lymphocytes, coagulation factors, and platelets in whole blood were assessed. In results, inactivation effectiveness increased as the ultraviolet dose increased from 18 J/cm2 to 54 J/cm2. At the dose of 18 J/cm2, inactivation effectiveness of four antibiotic-resistant bacteria were more than 80%, while only 67% of MRSA. The antibacterial effect was enhanced by the combination of riboflavin photodynamic treatment and antibiotic. The red blood cell function was susceptible to ultraviolet dose. At the dose of 18 J/cm2, hemolysis rate was less than 0.8% and there was no change in levels of ATP and 2,3-DPG. At the same dose, the proliferation, cell killing, and cytokine secretion activities of lymphocytes decreased 20-70%; Factor V and Factor VIII activities decreased 50%; Fibrinogen and platelet function loss significantly but reparable. Consequently, we speculated that riboflavin photodynamic method with a ultraviolet dose of 18 J/cm2 was effective in inactivating four antibiotic-resistant bacteria in whole blood while whole blood function was preserved. We also provided a novel extracorporeal circulation phototherapy mode for treating bacteremia caused by antibiotic-resistant bacteria.

Assessment of hemostasis in hyperthyroid and euthyroid cats using two viscoelastic assays and platelet aggregometry.

Hyperthyroidism in humans is associated with a hypercoagulable state and an increased risk of thromboembolism. To evaluate hemostatic variables in hyperthyroid and euthyroid cats with the hypothesis that hyperthyroid cats will have evidence of altered hemostasis consistent with a potential hypercoagulable state. Client-owned hyperthyroid (n = 16) and euthyroid (n = 15) cats over 8 years of age. Prospective observational study. Hyperthyroid and euthyroid cats were enrolled. Rotational thromboelastometry (ROTEM), whole-blood platelet impedance aggregometry (WBPIA) and a point-of-care viscoelastic coagulation monitor (VCM-Vet) were performed immediately after minimally traumatic venipuncture under sedation. Hyperthyroid cats had significantly higher values for variables as assessed by VCM-Vet: A10 (34 [17-47] vs 25 [17-38], P = .003); A20 (39.5 [23-55] vs 31 [21-45], P = .003); and MCF (41 [24-58] vs 35 [22-49], P = .03). Hyperthyroid cats had significantly different values versus the euthyroid cohort as assessed by different ROTEM channels: increased A10, INTEM (61.5 [39-75] vs 54 [23-66], P = .007) and FIBTEM (18 [10-35] vs 13 [2-27], P = .01); increased A20, INTEM (68 [45-78] vs 61 [30-70], P = .006) and FIBTEM (17 [10-34] vs 11 [2-25], P = .002); increased MCF, EXTEM (72 [65-81] vs 69 [34-78], P = .04), INTEM (70 [45-85] vs 62 [35-71], P = .01) and FIBTEM (18 [13-37] vs 14 [3-27], P = .02); increased alpha angle, EXTEM (80 [68-85] vs 76 [41-84], P = .01); shortened CT, EXTEM (52.5 [29-73] vs 60 [52-92], P = .003) and FIBTEM (52.5 [16-75] vs 65 [53-165], P = .001); and decreased ML, FIBTEM (20 [1-36] vs 33 [19-59], P <.001). No significant differences were found with WBPIA. The hyperthyroid cats in this study had evidence of altered hemostasis as assessed by 2 viscoelastic methodologies, and characterized by increased clot amplitude, firmness, and faster coagulation times vs euthyroid controls.

Convolutional neuronal network for identifying single-cell-platelet-platelet-aggregates in human whole blood using imaging flow cytometry.

Imaging flow cytometry is an attractive method to investigate individual cells by optical properties. However, imaging flow cytometry applications with clinical relevance are scarce so far. Platelet aggregation naturally occurs during blood coagulation to form a clot. However, aberrant platelet aggregation is associated with cardiovascular disease under steady-state conditions in the blood. Several types of so-called antiplatelet drugs are frequently described to reduce the risk of stroke or cardiovascular diseases. However, an efficient monitoring method is missing to identify the presence and frequency of platelet-platelet aggregates in whole blood on a single cell level. In this work, we employed imaging flow cytometry to identify fluorescently labeled platelets in whole blood with a conditional gating strategy. Images were post-processed and aligned. A convolutional neural network was designed to identify platelet-platelet aggregates of two, three, and more than three platelets, and results were validated against various data set properties. In addition, the neural network excluded erythrocyte-platelet aggregates from the results. Based on the results, a parameter for detecting platelet-platelet aggregates, the weighted platelet aggregation, was developed. If employed on a broad scale with proband and patient samples, our method could aid in building a future diagnostic marker for cardiovascular disease and monitoring parameters to optimize drug prescriptions in such patient groups.

PSIV-28 Evaluation of Possible Interactions Between Platelets and Neutrophil in Cow Blood

Abstract Neutrophils have a central role in the inflammatory response and fight infection. Platelets are essential for hemostasis and thrombosis and fighting vascular damage. Studies in man show that the interaction between these cells can modulate their function. We aimed to assess the effect of possible relationships between the platelet count in whole blood on neutrophil activation. Blood samples (50 mL) from five clinically healthy Holstein Friesian cows at the NC A&amp;T Dairy research unit were collected. Differential white blood cell counts were conducted using a VetScan (Zoetis). Neutrophils were isolated through differential centrifugation and hypotonic lysis of red blood cells. Duplicate samples of neutrophils were maintained in phosphate-buffered saline for 30 minutes at 37 °C. The total and viable neutrophil count was evaluated using TC20 (Bio-Rad). Samples were centrifuged. The supernatant was used to determine the concentration of total secreted protein using the Bicinchoninic acid assay (Pierce). Enumeration of bovine blood platelets and white blood cells was accomplished using the Vetscan study found that there was no significant difference observed in the total cell count or percent viability in neutrophils between the five cows (P &amp;gt; 0.05). However, protein concentration showed a significant change between animals. Correlations between total platelet count, neutrophil count, viability, and protein secretion were evaluated. No correlation was found between neutrophil count, protein secretion in isolated neutrophils and concentration of platelets in whole cow blood. The number of circulating platelets was not related to neutrophil viability or protein secretion. This approach can be used in further investigation using imaging and is needed to evaluate the effect and functional consequences of interactions between platelets and neutrophils in cow blood.

Effects of Antiplatelet Drugs on Platelet-Dependent Coagulation Reactions.

Activated platelets are involved in blood coagulation by exposing phosphatidylserine (PS), which serves as a substrate for assembling coagulation complexes. Platelets accelerate fibrin formation and thrombin generation, two final reactions of the coagulation cascade. We investigated the effects of antiplatelet drugs on platelet impact in these reactions and platelet ability to expose PS. Washed human platelets were incubated with acetylsalicylic acid (ASA), ticagrelor, ASA in combination with ticagrelor, ruciromab (glycoprotein IIb-IIIa antagonist), or prostaglandin E1 (PGE1). Platelets were not activated or activated by collagen and sedimented in multiwell plates, and plasma was added after supernatant removal. Fibrin formation (clotting) was monitored in a recalcification assay by light absorbance and thrombin generation in a fluorogenic test. PS exposure was assessed by annexin V staining using flow cytometry. Ticagrelor (alone and in combination with ASA), ruciromab, and PGE1, but not ASA, prolonged the lag phase and decreased the maximum rate of plasma clotting and decreased the peak and maximum rate of thrombin generation. Inhibition was observed when platelets were not treated with exogenous agonists (activation by endogenous thrombin) and pretreated with collagen. Ticagrelor (alone and in combination with ASA), ruciromab, and PGE1, but not ASA, decreased PS exposure on washed platelets activated by thrombin and by thrombin + collagen. PS exposure on activated platelets in whole blood was lower in patients with acute coronary syndrome receiving ticagrelor + ASA in comparison with donors free of medications. These results indicate that antiplatelet drugs are able to suppress platelet coagulation activity not only in vitro but also after administration to patients.

Association of ADP-Induced Whole-Blood Platelet Aggregation with Serum Low-Density Lipoprotein Cholesterol in Patients with Coronary Artery Disease When Receiving Maintenance Ticagrelor-Based Dual Antiplatelet Therapy.

The degree of platelet inhibition in patients undergoing dual antiplatelet therapy (DAPT) affects cardiovascular outcomes after acute coronary syndromes (ACS) and/or percutaneous coronary intervention. Our aim was to search for correlates of residual ex vivo platelet reactivity and circulating soluble P-selectin (sP-selectin), an index of in vivo platelet activation, in patients being treated by DAPT with ticagrelor. Adenosine diphosphate (ADP)-induced platelet aggregability (by multiple electrode aggregometry) and plasma sP-selectin were estimated in 62 stable post-ACS subjects (46 men and 16 women; mean age: 64 ± 10 years; 30 with type 2 diabetes (T2DM)) undergoing maintenance DAPT with ticagrelor and aspirin. These patients did not exhibit heart failure or other relevant coexistent diseases except for properly controlled T2DM, mild renal insufficiency, and hypertension. We also assessed this in 64 subjects on clopidogrel-based DAPT matched for age, sex, and T2DM status. ADP-induced platelet aggregation was below the optimal levels (190-460 arbitrary units (AU) * min) in most patients receiving ticagrelor-based DAPT, especially in those with below-median (<1.9 mmol/L) serum concentrations of low-density lipoprotein cholesterol (LDL-c) (128 ± 61 vs. 167 ± 73 AU * min for below-median and above-median LDL-c, respectively, p = 0.025). In contrast, platelet reactivity did not differ by LDL-c on clopidogrel-based DAPT (246 ± 101 vs. 268 ± 108 AU * min for below-median and above-median LDL-c, respectively, p > 0.4). Plasma sP-selectin was found to be unrelated to serum LDL-c when receiving DAPT with ticagrelor (p > 0.4) or clopidogrel (p > 0.8). In conclusion, our preliminary observational study suggests the association of lower residual ex vivo platelet aggregability with better LDL-c control in patients undergoing ticagrelor-based maintenance DAPT, which does not appear to be reflected by plasma sP-selectin. Whether the serum LDL-c level should be considered among the factors affecting the degree of platelet inhibition for those treated with ticagrelor-based DAPT needs to be investigated in larger studies.

Effects of radiation exposure and storage on the energy metabolome of platelets in whole blood.

Exposure to radiation through battlefield use of nuclear weapons, terrorist attacks or accidents at nuclear power plants is a current concern for the military. Beyond the risk of exposure to personnel is the intentional or accidental irradiation of our blood banking supply system. It is unknown how large doses of ionizing radiation affect storage of blood and blood products, including platelets. The major function of platelets is clot formation which includes aggregation, shape change, vesicle release, and fibrinogen attachment; these tasks require a significant amount of energy. Here, we determine whether the ionizing radiation effects the energy metabolome of platelets in storage. Fresh whole blood from healthy volunteers was subjected to 0, 25, or 75Gy of X-irradiation, and stored at 4°C. Platelets were isolated from stored WB at 0, 1, 7, 14, and 21 days of storage. Krebs cycle intermediates, nicotinamide adenine dinucleotides, and the tri-, di, and mono- phosphorylated versions of adenosine and guanosine were extracted and measured by tandem mass spectroscopy. Irradiation at either 25Gy or 75Gy had no significant effect on the amount of any metabolite measured compared to control (0Gy). However, there was a significant fall over time in storage for most of the metabolites measured. These data show that irradiation at high doses has no effect on the concentration of the energy metabolome of platelets derived from whole blood stored in 4°C for up to 21 days and suggests that platelets can maintain their metabolome even after radiation exposure.

Laboratory rationale for dose reduction anticoagulant for thrombosis complicing treatment patients with hemoblastosis on the background of chemioinduced thrombocytopenia

Purpose of the study: To substantiate the minimum hemostatic threshold for platelet count, which regulates the anticoagulant therapy of thrombosis against the background of induced thrombocytopenia in children with malignant neoplasms. Material and research methods: 23 patients (group 1) with hemorrhagic syndrome caused by thrombocytopenia (less than 50∙109/l) were examined. The control group consisted of 21 patients (group 2) with a similar diagnosis, platelet count less than 150∙109/l, who did not have bleeding. All examined patients with hemoblastoses had signs of febrile neutropenia. Additionally, under the conditions of a bench experiment, dilutions of platelet donor plasma were prepared with a platelet content of 5.0×109/l, 10.0×109/l, 20.0×109/l, 30.0×109/l, 40.0×109/L, 50.0×109/L, 60.0×109/L, 70.0×109/L, 80.0×109/L, 90.0×109/L, 100.0×109 /l, 200.0×109/l. Platelets were counted in peripheral blood for each sample using an XN-3000 hematology analyzer (manufactured by Sysmex GmbH, Japan) by the impedance method using original reagents (SysmexGmbH). The endogenous thrombin potential (ETP) in patients' platelet plasma was determined by the Hemker method on a Fluoroskanascent fluoroscan manufactured by Thermo Electron Corporation (Maastricht, Netherlands) using reagent kits from Thrombinoscop eBV. Research results: Between the content of platelets in whole blood and EPT of platelet plasma of patients with hemoblastoses, a relationship was revealed, which is reflected by the regression equation: y = 15.1356 + (0.0745∙ x), where y is the content of platelets in the blood (109/l), and x - EPT nM/l∙min in venous blood plasma. The minimum threshold value of EPT of platelet plasma, which provides hemostasis, 250 nM/l∙min, corresponded to the minimum platelet content of 30.0∙109/l in the blood of FN patients. Decrease in the content of platelets in donor plasma less than 20×109/l led to a decrease in EPT less than 250 nM/l•min. Between the EPT generated in the platelet donor plasma and the content of platelets in the donor plasma in the range (100.0 -20.0) ×109/l, a linear relationship was revealed. When the content of platelets was more than 100.0×109/l, generated by platelet plasma, EPT increased, regardless of the increase in the content of platelets in the plasma under study. Conclusion: in case of thrombosis against the background of chemo-induced thrombocytopenia in the range (100.0 -20.0)×109/l, the patient should receive LMWH at a dose reduced in proportion to the content of platelets in the blood.

In vitro analysis of anti-HPA-1a dependent platelet phagocytosis and its inhibition using a new whole blood phagocytosis assay (WHOPPA).

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a serious bleeding condition mostly caused by the reaction between maternal anti-HPA-1a antibodies and fetal platelets. This reaction leads to Fc-dependent platelet phagocytosis. Although several serological methods have been developed to identify maternal antibodies, a reliable laboratory parameter as a prognostic tool for FNAIT severity is still lacking. In this study, we developed whole blood platelet phagocytosis assay (WHOPPA), a flow cytometry-based phagocytosis assay that uses a pH-sensitive fluorescent dye (pHrodo-SE) to analyze anti-HPA-1a-dependent platelet phagocytosis in whole blood. WHOPPA revealed a high phagocytosis rate for the anti-HPA-1a opsonized platelets by monocytes but not by neutrophils. Analysis of different monocyte populations showed that all monocyte subsets, including classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes, were able to engulf opsonized platelets. A unique monocyte subset, termed shifted monocytes (CD14+CD16-), showed the highest phagocytosis rate and was detected after platelet engulfment. FcγR inhibition tests revealed that except for FcγRIIa, FcγRI and FcγRIII on monocytes were responsible for the phagocytosis of anti-HPA-1a opsonized platelets. Analysis of anti-HPA-1a antibodies from FNAIT cases (n = 7) showed the phagocytosis of HPA-1aa but not of HPA-1bb platelets by monocytes. The phagocytosis rate was highly correlated with bound antibodies measured by flow cytometry (p < 0001; r = 0.9214) and MAIPA assay (p < 0.001; r = 0.7692). The phagocytosis rates were equal for type I and II anti-HPA-1a antibodies recognizing the plexin-semaphoring-integrin (PSI) domain and PSI/epidermal growth factor 1 domain of β3 integrin, respectively. By contrast, type III anti-HPA-1a antibodies reacting with αvβ3 integrin did not induce platelet phagocytosis. Furthermore, effector-silenced mAbs against HPA-1a inhibited the phagocytosis of anti-HPA-1a opsonized platelets. In conclusion, WHOPPA is a reliable in vitro platelet phagocytosis assay that mimics the phagocytosis of anti-HPA-1a opsonized platelets in whole blood. This assay allows to prove platelet phagocytosis ex vivo and evaluate the inhibitory capacity of different inhibitors as therapeutically strategies for the prevention of fetal thrombocytopenia in FNAIT in the future.

Modulation of ultralarge immune complexes in heparin–induced thrombocytopenia

BackgroundHeparin–induced thrombocytopenia (HIT) is a serious thrombotic disorder caused by ultralarge immune complexes (ULICs) containing platelet factor 4 (PF4) and heparin that form the HIT antigen, together with a subset of anti–PF4 antibodies. ULICs initiate prothrombotic responses by engaging Fcγ receptors on platelets, neutrophils, and monocytes. Contemporary anti–thrombotic therapy for HIT is neither entirely safe nor entirely successful and acts downstream of ULIC formation and Fcγ receptor–initiated generation of thrombin. ObjectivesTo determine whether HIT antigen and ULIC formation and stability could be modified favorably by inhibiting PF4-heparin interactions with fondaparinux, together with blocking formation of PF4 tetramers using a humanized monoclonal anti–PF4 antibody (hRTO). MethodsResults: The combination of fondaparinux and hRTO inhibited HIT antigen formation, promoted antigen dissociation, inhibited ULIC formation, and promoted ULIC disassembly at concentrations below the effective concentration of either alone and blocked Fcγ receptor–dependent induction of factor Xa activity by monocytic THP1 cells and activation of human platelets in whole blood. Combined with hRTO, fondaparinux inhibited HIT antigen and immune complex formation and activation through Fcγ receptors at concentrations at or below those used clinically to inhibit FXa coagulant activity. ConclusionsHIT antigen and immune complexes are dynamic and amenable to modulation. Fondaparinux can be converted from an anticoagulant that acts at a downstream amplification step into a rationale, disease-specific intervention that blocks ULIC formation. Interventions that prevent ULIC formation and stability might increase the efficacy, permit use of lower doses, shorten the duration of antithrombotic therapy, and help prevent this serious thrombotic disorder.

Testing the Impact of Vibration on Changes in the Number of Erythrocytes and Platelet Counts in Whole Blood

Blood or plasma is transferred from the unit car to the Blood Donor Unit using a conventional cooler box without any tools to maintain the effect of vibration which is very possible for a decrease in blood quality. Examination carried out to determine the quality of blood through routine hematological examination by counting the number of erythrocytes and platelets. The purpose of this study was to determine the impact of vibration on the number of red blood cells and thrombocytes. The research design was a pre and post test with control group design. The sample size for each group was nine tubes of blood. Data was processed by one way ANOVA and tukeys HSD test. Results There was a significant difference in the number of erythrocytes between the group that was vibrated at a frequency of 11 HZ compared to the group that was vibrated at a frequency of 0 HZ and 5 HZ with p values 0.00 and 0.01, respectively. There was a significant difference in the number of platelets between the groups that were vibrated with a frequency of 11 HZ compared to the groups that were vibrated with a frequency of 0 HZ and 5 HZ with a p value of 0.00. It was concluded that vibrations exceeding 10 Hz could decrease the number of erythrocytes and platelets in whole blood

Testing the Impact of Vibration on Changes in the Number of Erythrocytes and Platelet Counts in Whole Blood

Blood or plasma is transferred from the unit car to the Blood Donor Unit using a conventional cooler box without any tools to maintain the effect of vibration which is very possible for a decrease in blood quality. Examination carried out to determine the quality of blood through routine hematological examination by counting the number of erythrocytes and platelets. The purpose of this study was to determine the impact of vibration on the number of red blood cells and thrombocytes. The research design was a pre and post test with control group design. The sample size for each group was nine tubes of blood. Data was processed by one way ANOVA and tukeys HSD test. Results There was a significant difference in the number of erythrocytes between the group that was vibrated at a frequency of 11 HZ compared to the group that was vibrated at a frequency of 0 HZ and 5 HZ with p values 0.00 and 0.01, respectively. There was a significant difference in the number of platelets between the groups that were vibrated with a frequency of 11 HZ compared to the groups that were vibrated with a frequency of 0 HZ and 5 HZ with a p value of 0.00. It was concluded that vibrations exceeding 10 Hz could decrease the number of erythrocytes and platelets in whole blood

Modulation of Ultralarge Immune Complexes in Heparin-Induced Thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is serious prothrombotic disorder in which the target antigen, composed of tetramers of platelet factor 4 (PF4) and heparin or cellular glycosaminoglycans (GAGs), forms ultralarge immune complexes (ULICs) with a subset of anti-PF4 antibodies. ULICs initiate prothrombotic responses in large part by engaging Fcγ receptors on platelets, neutrophils, and monocytes. Contemporary anti-thrombotic therapy for HIT is neither entirely safe nor entirely successful, and acts downstream of ULIC formation and Fcγ receptor-initiated generation of thrombin. Therefore, we asked whether HIT antigen and ULIC formation and stability could be modified to attenuate cellular activation by inhibiting PF4-heparin interactions with fondaparinux in conjunction with disrupting PF4 tetramers with a humanized form of a monoclonal anti-PF4 monomer antibody (hRTO, Figure 1). Using dynamic light scattering to measure HIT antigen (PF4 10 µg/ml and heparin 0.2U/ml) and ULIC (PF4 10 µg/ml, heparin 0.2U/ml, KKO 35µg/ml) size, we found that either fondaparinux (FPX 5-20U/ml) or hRTO (400 - 1000 µg/ml) inhibited formation of HIT antigen and ULIC, and promoted HIT antigen and ULIC disassembly. Successively higher concentrations of hRTO were required as PF4 formed large antigenic complexes and then ULICs, likely because the bonds between PF4 and UFH have been strengthened and access to inhibitors lessened (Figure 1A). Combination of fondaparinux and hRTO was effective below the concentration of either alone (FPX 2.5U/ml + hRTO 330 µg/ml). Importantly, fondaparinux and hRTO blocked Fcγ receptor-dependent induction of factor Xa activity by monocytic THP1 cells and activation of human platelets in whole blood (Figure 1B). When combined with hRTO, fondaparinux inhibited HIT antigen and immune complex formation at concentrations at or below those used clinically to inhibit FXa coagulant activity. Even lower concentrations of inhibitors (FPX < 0.1-0.5 U/ml + hRTO 50 µg/ml) were required to block cell activation than were required to act on ULICs in solution, likely because the affinity of PF4 for GAGs is less than for UFH. These studies show that HIT antigen and immune complexes are dynamic and amenable to modulation. Fondaparinux can be converted from an anticoagulant that acts at a downstream amplification step into a rational, disease-specific intervention that blocks ULIC formation when used in combination with hRTO. Our studies demonstrate that interventions that prevent ULIC formation and stability might increase the efficacy, permit use of lower doses, and shorten the duration of anti-thrombotic therapy and help prevent this serious thrombotic disorder. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Оцінка ефективності різних режимів подвійного центрифугування для отримання чистої збагаченої тромбоцитами плазми

The purpose - to determine the optimal parameters of double centrifugation in the preparation of pure platelet-rich plasma with a maximum reduction of other blood cellular elements. Materials and methods. The study included 30 apparently healthy individuals aged 18 to 60 years who underwent venous blood sampling. The first centrifugation of all blood samples was performed in the 160 g × 10 min mode. The second centrifugation was performed in 12 different modes: 190 g × 10 min, 190 g × 15 min, 220 g × 10 min, 220 g × 15 min, 250 g × 10 min, 250 g × 15 min, 290 g × 10 min, 290 g × 15 min, 320 g × 10 min, 320 g × 15 min, 360 g × 10 min, 360 g × 15 min. The number of erythrocytes, leukocytes, platelets (in whole blood, after the first and second centrifugation), platelet capture efficiency, platelet enrichment factor, erythrocytes and leukocytes reduction efficiency, concentration of major growth factors in plasma after the second centrifugation were determined. Results. In whole blood, the concentration of erythrocytes was 4.455±0.301 × 1012/l, leukocytes - 6.485±0.501 × 109/l, platelets - 281.5±22.7 × 109/l. After the first centrifugation, the volume of the obtained plasma containing platelets was 4.51±0.25 ml, the number of platelets was 482.1±47.4 × 109/l, with platelet reduction rates of 14.39±1.1%, their preservation - 85.6±1.1% and enrichment factor - 1.71±0.1; the number of erythrocytes was 0.104±0.006 × 1012/l, leukocytes - 0.205±0.025 × 109/l, and their reduction efficiency - 98.83±0.09% and 98.42±0.14%, respectively. After the second centrifugation in different modes, the 250 g × 15 min mode turned out to be the most effective, which made it possible to achieve a platelet enrichment factor of about 4.93 with a concentration of platelets in the final product of 1386.7±116.5 × 109/l, their capture efficiency of 82.1±1.1% and erythrocytes and leukocytes reduction efficiency of 98.86±0.08% and 98.45±0.14%, respectively. Conclusions. In case of double-spin centrifugation, the sequential combination of 160 g × 10 min and 250 g × 15 min regimens is most effective for the preparation of pure platelet-rich plasma. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of all participating institutions. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors.

Characterization of Two Anti-P-Selectin Monoclonal Antibodies (mAbs): Crizanlizumab Shows Comparable or Stronger Effects Versus Inclacumab across Cell Adhesion Assays in Vitro

Background: Vaso-occlusive crises (VOCs) are the hallmark of sickle cell disease (SCD) and are associated with significant morbidity and mortality. Crizanlizumab, a first-in-class humanized anti-P-selectin IgG2 mAb, is approved in >40 countries to reduce/prevent VOCs in SCD patients aged ≥16 yrs. Inclacumab, a fully human anti-P-selectin IgG4 mAb, is in clinical development for SCD. In vitro data suggest that inclacumab may show stronger affinity to P-selectin and greater maximal inhibition of cell-cell interactions vs crizanlizumab (Geng et al ASH 2020).Aim: We investigated in blinded experiments whether crizanlizumab and inclacumab can be differentiated in terms of P-selectin binding and inhibition of P-selectin-mediated cell-cell interactions, via 5 different in vitro assays, including testing of blood samples from healthy volunteers and SCD patients.Methods: For both mAbs, we assessed: P-selectin affinity (surface plasmon resonance [SPR]/Biacore assay); inhibition of adhesion of P-selectin-expressing cells to P-selectin glycoprotein ligand-1 (PSGL-1; cell adhesion bioassay); inhibition of platelet aggregation in native blood from healthy volunteers (whole blood impedance aggregometry [WBA] and flow cytometry-based platelet-leukocyte aggregate [PLA] assays); and inhibition of adhesion of SCD patient whole blood or isolated leukocytes to P-selectin-coated substrate under physiologically relevant shear rates in microfluidic systems (flow adhesion bioassays).Results: The SPR/Biacore assay indicated that both mAbs recognize P-selectin with binding affinities of the same order of magnitude, although there was a trend towards higher binding affinity with inclacumab (mean equilibrium dissociation constant [K D ± standard deviation]: 12.4 ± 1.0 nM [crizanlizumab] vs 6.7 ± 0.7 nM [inclacumab]). However, the function-related cell adhesion bioassay revealed a significant difference between the mAbs in their ability to inhibit adhesion of P-selectin-expressing cells to PSGL-1. Crizanlizumab showed stronger inhibition than inclacumab (potency vs crizanlizumab reference standard: 98.0% for the crizanlizumab control sample [95% CI 85.2‒112.7%] and 26.2% for the inclacumab sample [95% CI 20.7%‒32.6%]).Native whole blood samples for WBA and PLA assays were provided by 12 and 10 healthy volunteers, respectively. Blocking of platelet (WBA assay) or platelet-leukocyte aggregation (PLA assay) in whole blood was comparable for crizanlizumab and inclacumab, with a clear trend towards stronger inhibition by crizanlizumab. In the WBA assay, the half maximal inhibitory concentration (IC 50) was twice as high with inclacumab (10.36 μg/mL) as crizanlizumab (5.11 μg/mL); similar data were seen in the PLA assay (Figure 1; IC 50: 4.11 vs 2.81 μg/mL for inclacumab vs crizanlizumab).Blood samples were provided by 9 SCD patients for the flow adhesion bioassays. Dose-dependent inhibition of adhesion of whole blood and isolated leukocytes to P-selectin was seen with both crizanlizumab and inclacumab, with no difference observed between them (Figure 2). A mAb concentration of 10 μg/mL resulted in statistically significant inhibition of adhesion by 60‒75%; further increasing the mAb concentration did not result in stronger inhibition of adhesion beyond this threshold. In relation to the clinically approved crizanlizumab dose (5.0 mg/kg), a concentration of 100 μg/mL corresponds with the maximum plasma concentration (C max) and ~10-15 μg/mL corresponds with the trough concentration (C trough).Conclusions: While the SPR/Biacore data suggested higher P-selectin binding affinity for inclacumab compared with crizanlizumab, there was a stronger inhibition of P-selectin-mediated cell adhesion with crizanlizumab vs inclacumab in other well-characterized functional in vitro assays. In microfluidic flow adhesion bioassays, blockage of cell adhesion from SCD whole blood and leukocytes was comparable for both mAbs. In summary, these data indicate that comparable or stronger blockage of cell adhesion with crizanlizumab vs inclacumab does not require superior P-selectin binding affinity. The data from healthy volunteers will be complemented by data from SCD patients for the WBA and PLA assays. Ultimately, clinical data are required to evaluate potential differences in the profiles and efficacy of crizanlizumab and inclacumab as treatments for SCD patients. [Display omitted] DisclosuresRubic-Schneider: Novartis Pharma AG: Current Employment. Sundd: CSL Behring Inc: Research Funding; Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ledieu: Novartis Pharma AG: Current Employment, Current holder of stock options in a privately-held company. Garcia: Novartis Pharma AG: Current Employment. Hehlen: Novartis Pharma AG: Current Employment. Burnet-Merlin: Light Chain Bioscience - Novimmune SA: Current Employment; Novartis Pharma AG: Ended employment in the past 24 months. Cochin de Billy: Novartis Pharma AG: Current Employment. Greutmann: Novartis Pharma AG: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Krӧner: Novartis Pharma AG: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: WO2021087050A1. Verneret: Novartis Pharma AG: Current Employment, Current holder of stock options in a privately-held company. Bruederle: Novartis Pharma AG: Current Employment. Gao: Functional Fluidics: Current Employment. Dajee: Novartis: Current Employment.

Heritable platelet disorders: an enigma even guidelines can't unravel.

Heritable platelet disorders: an enigma even guidelines can't unravel.

Why PRP works only on certain patients with tennis elbow? Is PDGFB gene a key for PRP therapy effectiveness? A prospective cohort study

BackgroundThere is variability in individual response to platelet-rich plasma (PRP) therapy in tennis elbow treatment. Genetic variation, especially within genes encoding growth factors may influence the observed inter-individual differences. The purpose of this study was to identify polymorphic variants of the platelet-derived growth factor beta polypeptide gene (PDGFB) that determine an improved individual response to PRP therapy in tennis elbow patients.MethodsThis prospective cohort study was designed in accordance with STROBE and MIBO guidelines. A cohort of 107 patients (132 elbows, 25 bilateral) was studied, including 65 females (77 elbows) and 42 males (55 elbows), aged 24–64 years (median 46.00 ± 5.50), with lateral elbow tendinopathy treated with autologous PRP injection. The effectiveness of PRP therapy was recorded in all subjects at 2, 4, 8, 12, 24 and 52 weeks after PRP injection using the Visual Analog Scale (VAS), quick version of Disabilities of the Arm, Shoulder and Hand score (QDASH) and Patient-Rated Tennis Elbow Evaluation (PRTEE). In order to determine the PDGFB variants with the best response to PRP therapy, patient reported outcome measures were compared between individual genotypes within studied polymorphic variants (rs2285099, rs2285097, rs2247128, rs5757572, rs1800817 and rs7289325). The influence of single nucleotide polymorphisms on blood and PRP parameters, including the concentration of PDGF-AB and PDGF-BB proteins was also analyzed.ResultsOur analysis identified genetic variants of the PDGFB gene that lead to a better response to PRP therapy. The TT (rs2285099) and CC (rs2285097) homozygotes had higher concentration of platelets in whole blood than carriers of other genotypes (p = 0.018) and showed significantly (p < 0.05) lower values of VAS (weeks 2–12), QDASH and PRTEE (weeks 2–24). The rs2285099 and rs2285097 variants formed strong haplotype block (r2 = 98, D’=100). The AA homozygotes (rs2247128) had significantly lower values of VAS (weeks 4–52), QDASH and PRTEE (weeks 8, 12).ConclusionsPDGFB gene’s polymorphisms increase the effectiveness of PRP therapy in tennis elbow treatment. Genotyping two polymorphisms of the PDGFB gene, namely rs2285099 (or rs2285097) and rs2247128 may be a helpful diagnostic tool while assessing patients for PRP therapy and modifying the therapy to improve its effectiveness.

Polydiacetylene Liposome Microarray toward Facile Measurement of Platelet Activation in Whole Blood.

The necessity of a simple measurement of platelet activation has been increasing in clinical medicine to regulate the proper dose of the antiplatelet drugs for patients having clinical outcomes in acute situations such as angina pectoris, stroke, or peripheral vascular disease or procedures involving angioplasty or coronary thrombolysis. We developed a self-signaling polydiacetylene (PDA) liposome microarray to detect activated platelets from whole blood samples in a single step. A specific antibody, 9F9 antibody, to platelet-bound fibrinogen was selected and conjugated to the PDA liposome microarray to quantify the fibrinogen-bound platelets. The developed PDA liposome-9F9 microarray generated an intense fluorescence signal when activated platelets in whole blood were introduced and also successfully distinguished the reduced platelet activation in the presence of Tirofiban, a model antiplatelet drug. The results of this single-step benchtop assay incorporates simple, sensitive, and rapid attributes that can detect the extent of platelet activation prior to needed clinical procedures.