Microfluidic System-Based Quantitative Analysis of Platelet Function through Speckle Size Measurement.

Platelets play essential roles in the formation of blood clots by clumping with coagulation factors at the site of vascular injury to stop bleeding; therefore, a reduction in the platelet number or disorder in their function causes bleeding risk. In our research, we developed a method to assess platelet aggregation using an optical approach within a microfluidic chip's channel by evaluating the size of laser speckles. These speckles, associated with slowed blood flow in the microfluidic channel, had a baseline size of 28.54 ± 0.72 µm in whole blood. Removing platelets from the sample led to a notable decrease in speckle size to 27.04 ± 1.23 µm. Moreover, the addition of an ADP-containing agonist, which activates platelets, resulted in an increased speckle size of 32.89 ± 1.69 µm. This finding may provide a simple optical method via microfluidics that could be utilized to assess platelet functionality in diagnosing bleeding disorders and potentially in monitoring therapies that target platelets.

Platelet function testing: Update on determinant variables and permissive windows using a platelet-count-based device.

While there are various aspects of platelet biology that can be studied in the lab (i.e. adhesion, degranulation, integrin activation), the master test for platelet function is that which gives a measure of the platelet aggregation capacity upon stimulation with an agonist. Platelet function testing is necessary for the diagnosis of platelet disorders and the monitoring of patients receiving anti-platelet treatments. Furthermore, it becomes relevant in the quality control of platelet concentrates for transfusion purposes, especially considering the global concern about long term storage, other forms of storage (i.e. cryopreservation, lyophilization), and the impact of Pathogen Reduction Treatments (PRTs) on platelet performance upon transfusion. However, it has been acknowledged as technically difficult and demanding, since a fine platelet function test must be carried out under specific conditions. Still, there might be occasions that preclude the platelet function testing abiding to the gold standard requirements, thus, leaving us with the necessity to redefine which variables may condition or limit the analysis of platelet function testing. In the present manuscript, we test different variables (such as the anticoagulant used or the time elapsed since extraction) and the possibility to reconstitute blood prior to platelet function analysis. This study aims to provide windows of action at the diagnostics lab, especially when not all of the recommended procedures and conditions can be followed: for example, when a sample is sent from a long distance, when there is a limitation on blood extraction volume or when certain parameters (platelet count) preclude reliable test results.

Evaluation of an automated platelet aggregation method for detection of congenital or acquired platelet function defects.

BACKGROUND: Light transmission aggregometry (LTA) is the most widely used laboratory method for an initial screening of patients with a suspected platelet function defect (PFD), and its use has also been proposed for assessing the efficacy of antiplatelet treatment (APT). An automated LTA method has been developed by Sysmex (Kobe, Japan) on a routine coagulation analyzer (CS-2400), together with a new research parameter called PAL (platelet aggregation level) to evaluate patients on APT. MATERIALS AND METHODS: We evaluated the performance of CS-2400 compared to a stand-alone lumi-dual-aggregometer device in the diagnosis of PFD and in assessing the efficacy of APT. For these purposes, the study population was represented by a cohort of 23 patients with a previous diagnosis of PFD and a cohort of 28 patients on APT. RESULTS: Compared to healthy volunteers, patients with PFD showed a statistically significant reduction (p<0.05) in the maximal %light transmission, irrespective of the agonist used, both with the CS-2400 and the lumi-dual-aggregometer. As regards PFD patients, CS-2400 was effective in identifying the more severe defects, with a good sensibility and specificity, but less effective in identifying milder forms of PFD, such as platelet secretion defects. Patients on APT showed a statistically significant (p=0.001) reduced median %light transmission and PAL scores compared to healthy controls. DISCUSSION: Thanks to this LTA technology, CS-2400, a routine coagulation analyzer widely available in routine laboratories, could prove useful for initial assessment of patients with a suspected PFD. Moreover, the PAL scores were a fairly accurate reflection of the platelet response to APT.

Quantification and optimization of clot retraction in washed human platelets by Sonoclot coagulation analysis.

INTRODUCTION: Clot retraction is a pivotal process for haemostasis, where platelets develop a contractile force in fibrin meshwork and lead to the increased rigidity of clot. The pathophysiological alteration in contractile forces generated by the platelet-fibrin meshwork can lead to haemostatic disorders. Regardless of its utter significance, clot retraction remains a limited understood process owing to lack of quantification methodology. Sonoclot analysis is a point-of-care technique used in clinical laboratories for whole blood analysis that provides in vitro qualitative as well as quantitative assessment of coagulation process from initial fibrin formation to clot retraction. METHODS: Human washed platelets were isolated by differential centrifugation method and analysed via optical imaging, microscopy and Sonoclot analysis using 1-2 × 108 /mL of washed platelets, 1 U/mL of thrombin, 1 mg/mL of fibrinogen and 1 mM of calcium chloride. RESULTS: In this study, we demonstrate the novelty of this instrument in the quantitative evaluation of clot retraction in washed platelets and attempted to optimize the reference range of Sonoclot parameters including ACT - 87.3 ± 20.997, CR - 16.23 ± 3.538 and PF - 3.57 ± 0.629, (n = 10). DISCUSSION: Sonoclot analysis provides a simple and quantitative method to better understand in vitro clot retraction and its modulation by retraction components including platelet count, fibrinogen and platelet-fibrin interaction compared with existing conventional methods. Sonoclot may prove to be a valuable tool in thrombus biology research to understand fundamental basis of blood clot retraction.

Microelectromechanical System Measurement of Platelet Contraction: Direct Interrogation of Myosin Light Chain Phosphorylation.

Myosin Light Chain (MLC) regulates platelet contraction through its phosphorylation by Myosin Light Chain Kinase (MLCK) or dephosphorylation by Myosin Light Chain Phosphatase (MLCP). The correlation between platelet contraction force and levels of MLC phosphorylation is unknown. We investigate the relationship between platelet contraction force and MLC phosphorylation using a novel microelectromechanical (MEMS) based clot contraction sensor (CCS). The MLCK and MLCP pair were interrogated by inhibitors and activators of platelet function. The CCS was fabricated from silicon using photolithography techniques and force was validated over a range of deflection for different chip spring constants. The force of platelet contraction measured by the clot contraction sensor (CCS) was compared to the degree of MLC phosphorylation by Western Blotting (WB) and ELISA. Stimulators of MLC phosphorylation produced higher contraction force, higher phosphorylated MLC signal in ELISA and higher intensity bands in WB. Inhibitors of MLC phosphorylation produced the opposite. Contraction force is linearly related to levels of phosphorylated MLC. Direct measurements of clot contractile force are possible using a MEMS sensor platform and correlate linearly with the degree of MLC phosphorylation during coagulation. Measured force represents the mechanical output of the actin/myosin motor in platelets regulated by myosin light chain phosphorylation.

Point-of-care platelet function tests: relevance to arterial thrombosis and opportunities for improvement.

Studies using whole blood platelet aggregometry as a laboratory research tool, provided important insights into the mechanism and modulators of platelet aggregation. Subsequently, a number of point-of-care (POC) platelet function tests (PFTs) were developed for clinical use, based on the concept that an individual's thrombotic profile could be assessed in vitro by assessing the response to stimulation of platelet aggregation by specific, usually solo agonists such as adenosine diphosphate (ADP), collagen and thrombin. However, adjusting antiplatelet medication in order to improve the results of such POC PFTs has not translated into a meaningful reduction in cardiovascular events, which may be attributable to important differences between the POC PFT techniques and in vivo conditions, including patient-to-patient variability. Important limitations of most tests include the use of citrate-anticoagulated blood. Citrate directly and irreversibly diminishes platelet function and even after recalcification, it may result in altered platelet aggregation in response to ADP, epinephrine or collagen, and interfere with thrombin generation from activated platelets. Furthermore, most tests do not employ flowing blood and therefore do not assess the effect of high shear forces on platelets that initiate, propagate and stabilize arterial thrombi. Finally, the effect of endogenous thrombolysis, due to fibrinolysis and dislodgement, which ultimately determines the outcome of a thrombotic stimulus, is mostly not assessed. In order to accurately reflect an individual's predisposition to arterial thrombosis, future tests of thrombotic status which overcome these limitations should be used, to improve cardiovascular risk prediction and to guide pharmacotherapy.

Standardization and harmonization in hematology: Instrument alignment, quality control materials, and commutability issue.

INTRODUCTION: In the hub and spoke laboratory network, the number of hematology analyzers (HAs) within each core center has increased, and the control of HAs alignment is becoming necessary requirement to ensure analytical quality. In this scenario, HA alignment can be assessed by analyzing the same control material used for internal quality control on multiple HAs, assuming its commutability. The aim of the study was to verify the applicability of a protocol for the alignment of HAs based on control material rather than on fresh whole-blood samples. METHODS: The alignment of five HAs was evaluated for red (RBC, Hb, MCV, RET), white (WBC, NE, LY, MO, EO, BA, IG), and platelet (PLT) series parameters, following a protocol by SIBioC, using human sample (HS) and quality control material (QC), after the verification of commutability, according to the IFCC protocol. Maximum bias was derived from biological variation data. RESULTS: A complete alignment between instruments was confirmed for the majority of the parameters investigated both for HS and QC material. Partial misalignments or inconcludent results were instead evident for MCV, MO, EO, BA, and IG. Interestingly, QC material was found to be not commutable for LY, MO, and BA. CONCLUSION: The alignment of hematologic analyzers for main cell population parameters may be verified with both QC and HS, displaying consistent results and interpretation. The evaluation for some white series parameters (EO, BA, and IG) is critical, and particular attention must be paid to the values of the material used for the alignment.

Correlation Between the VerifyNow P2Y12 Assay and the Newly Developed APAL System in Neuroendovascular Patients.

OBJECTIVE: Light transmission aggregometry (LTA) is the gold standard method for assessing platelet function. Recently, a new parameter called adenosine diphosphate (ADP)-induced platelet aggregation level (APAL) was developed to aid interpretation of LTA results. APAL is a score calculated based on platelet aggregation patterns upon exposure to 1 µM and 10 µM ADP and is determined using an automated coagulation analyzer. We compared APAL and VerifyNow P2Y12 assay for neuroendovascular patients. METHODS: 42 patients who have received antiplatelet therapy were studied. Platelet function tests were performed on CS-2400 for APAL and VerifyNow P2Y12 assay was used for P2Y12 reaction unit (PRU) and % inhibition. RESULTS: Moderate correlations were observed between APAL and PRU (r=0.64, p<0.001) and between APAL and % inhibition (r=-0.74, p<0.001). The optimal threshold for APAL was 8.2 for PRU (threshold=240) and 8.1 for % inhibition (threshold=26%). The percentage of agreement between the above thresholds was 90.9% between PRU and APAL and 77.3% between % inhibition and APAL. CONCLUSIONS: The APAL system exhibits moderate correlation with PRU and % inhibition. APAL testing is a good choice for a clinical laboratory already in possession of Sysmex CS series analyzers. In this setting, APAL testing can significantly decrease the cost of platelet function testing for patients on antiplatelet therapy.

Multicenter evaluation of analytical performances of platelet counts and platelet parameters: Carryover, precision, and stability.

INTRODUCTION: The correctness of the results of automated platelet analysis is still highly debated. The aim of this multicenter study, conducted according to international guidelines, was to verify the analytical performance of nine different types of hematology analyzers (HAs) in the automated platelet analysis. METHODS: Four hundred eighty-six peripheral blood samples (PB), collected in K3 EDTA tubes, were analyzed by ABX Pentra, ADVIA2120i, BC-6800, BC-6800 Plus, Cell-DYN Sapphire, DxH800, XE-2100, XE-5000, XN-20 with PLT-F App. Within-run imprecision and between-run imprecision were carried out using PB and material control, respectively. The carryover, low limit of quantification (LoQ), and the PB stability were evaluated. RESULTS: The carryover was absent for all HAs. The LoQ of PLT ranged between 2.0 (Cell-Dyn Sapphire) and 25.0 × 109 /L (ADVIA 2120i), while immature platelet fraction (IPF) ranged between 1.0 (XN-20) and 12.0 × 109 /L (XE-5000). The imprecision (%CV) increases as the platelet count decreases. No HAs showed desirable CVAPS for PLT counts less than 50.0 × 109 /L, with the exception of Cell-DYN Sapphire (CV 3.0% with PLT-O mean value of 26.7 × 109 /L), XN-20 (CV 2.4% with PLT-F mean value of 21.5 × 109 /L), and BC-6800 Plus (CV 1.9% with PLT-O mean value of 26.5 × 109 /L). The sample stability ranged between under two hours for MPV by ADVIA2120i and 8 hours for other PLT parameters and HAs. CONCLUSION: The findings of this study may provide useful information regarding carryover, precision, and stability of platelet counts and parameters, especially in thrombocytopenic samples. Moreover, the stability of sample for platelet analysis is conditioned by the HA and by temperature and storage time.

Platelet-Derived Thrombogenicity Measured by Total Thrombus-Formation Analysis System in Patients With ST-Segment Elevation Myocardial Infarction Undergoing Primary Percutaneous Coronary Intervention.

BACKGROUND: Prompt and potent antiplatelet effects are important aspects of management of ST-elevation myocardial infarction (STEMI) patients undergoing primary percutaneous coronary intervention (PPCI). We evaluated the association between platelet-derived thrombogenicity during PPCI and enzymatic infarct size in STEMI patients.Methods and Results:Platelet-derived thrombogenicity was assessed in 127 STEMI patients undergoing PPCI by: (1) the area under the flow-pressure curve for the PL-chip (PL18-AUC10) using the total thrombus-formation analysis system (T-TAS); and (2) P2Y12reaction units (PRU) using the VerifyNow system. Patients were divided into 2 groups (High and Low) based on median PL18-AUC10during PPCI. PRU levels during PPCI were suboptimal in both the High and Low PL18-AUC10groups (median [interquartile range] 266 [231-311] vs. 272 [217-317], respectively; P=0.95). The percentage of final Thrombolysis in Myocardial Infarction (TIMI) 3 flow was lower in the High PL18-AUC10group (75% vs. 90%; P=0.021), whereas corrected TIMI frame count (31.3±2.5 vs. 21.0±2.6; P=0.005) and the incidence of slow-flow/no-reflow phenomenon (31% vs. 11%, P=0.0055) were higher. The area under the curve for creatine kinase (AUCCK) was greater in the High PL18-AUC10group (95,231±7,275 IU/L h vs. 62,239±7,333 IU/L h; P=0.0018). Multivariate regression analysis identified high PL18-AUC10during PPCI (ß=0.29, P=0.0006) and poor initial TIMI flow (ß=0.37, P<0.0001) as independent determinants of AUCCK. CONCLUSIONS: T-TAS-based high platelet-derived thrombogenicity during PPCI was associated with enzymatic infarct size in patients with STEMI.

Monitoring Antiplatelet Aggregation In Vivo and In Vitro by Microtiter Plate Method.

BACKGROUND: The current light transmission aggregation method is a recognized conventional method for platelet function evaluation, but it is time-consuming and poor in parallelism and cannot simultaneously monitor multiple inducers at multiple levels. The microtiter plate method has been established because of the high-throughput characteristic, but it needs more practical applications. OBJECTIVES: To evaluate the microtiter plate method by using aspirin and clopidogrel in vivo and in vitro. METHODS: In vitro, the platelet aggregations inhibited by aspirin (0.3, 1, 3, 10, 30, 90 µM) and clopidogrel (1, 3, 10, 30, 100, 300 µM) were evaluated with the presence of arachidonic acid (AA) and adenosine diphosphate (ADP) agonists. Using the combination index (CI), the effect of the combination of aspirin and clopidogrel on platelet aggregation was evaluated. In vivo, New Zealand rabbits (n = 18) were randomly divided into 3 groups, aspirin group (5 mg/kg, intragastrical gavage [i.g.]), clopidogrel group (14 mg/kg at the first day, followed by 4 mg/kg, i.g.), and the combination of these two drugs, administered (i.g.) continuously for 7 days. Then, the blood was collected to measure platelet aggregation. RESULTS: Different concentrations of AA (12.5, 25, 50, 100 µM) and ADP (1.25, 2.5, 5, 10 µM) could promote platelet aggregation in concentration-dependent manner, and the most stable induction concentrations of AA and ADP were 50 and 5 µM. In vitro, with the above optimized detection system, aspirin and clopidogrel alone or in combination had concentration-dependent antiplatelet aggregation. The combination of aspirin and clopidogrel also showed synergistic inhibition effect within the concentration range studied. In vivo, aspirin and clopidogrel alone or in combination inhibited platelet aggregation induced by multiple concentrations of AA and ADP agonists, and the combined inhibition was more significant during the administration than aspirin or clopidogrel alone. CONCLUSIONS: The improved microtiter plate method combining the use of multiple levels of multiple agonists avoids the variation of the effective inducer concentrations due to individual different response of platelets to agonists. It may be a potential approach in the detection of platelet aggregation.

The MICELI (MICrofluidic, ELectrical, Impedance): Prototyping a Point-of-Care Impedance Platelet Aggregometer.

As key cellular elements of hemostasis, platelets represent a primary target for thrombosis and bleeding management. Currently, therapeutic manipulations of platelet function (antithrombotic drugs) and count (platelet transfusion) are performed with limited or no real-time monitoring of the desired outcome at the point-of-care. To address the need, we have designed and fabricated an easy-to-use, accurate, and portable impedance aggregometer called "MICELI" (MICrofluidic, ELectrical, Impedance). It improves on current platelet aggregation technology by decreasing footprint, assay complexity, and time to obtain results. The current study aimed to optimize the MICELI protocol; validate sensitivity to aggregation agonists and key blood parameters, i.e., platelet count and hematocrit; and verify the MICELI operational performance as compared to commercial impedance aggregometry. We demonstrated that the MICELI aggregometer could detect platelet aggregation in 250 µL of whole blood or platelet-rich plasma, stimulated by ADP, TRAP-6, collagen, epinephrine, and calcium ionophore. Using hirudin as blood anticoagulant allowed higher aggregation values. Aggregation values obtained by the MICELI strongly correlated with platelet count and were not affected by hematocrit. The operational performance comparison of the MICELI and the Multiplate® Analyzer demonstrated strong correlation and similar interdonor distribution of aggregation values obtained between these devices. With the proven reliability of the data obtained by the MICELI aggregometer, it can be further translated into a point-of-care diagnostic device aimed at monitoring platelet function in order to guide pharmacological hemostasis management and platelet transfusions.

Comparison of three common whole blood platelet function tests for in vitro P2Y12 induced platelet inhibition.

In the context of interventional cardiology, platelet function testing may identify patients treated with P2Y12-inhibitors at an increased risk of mortality, thrombosis and bleeding. Several whole blood point-of-care platelet function analyzers are available; however, inter-device differences have not been examined systematically. To compare three platelet function tests under standardized in vitro conditions. Healthy volunteer (n = 10) blood samples were spiked with increasing concentrations of ticagrelor (0-7500 ng/mL) and/or ASA (0-3280 ng/mL), measured on three platelet function analyzers (TEG®6s, Multiplate®, and VerifyNow®) and respective Effective Concentration (EC) levels EC10, EC50 and EC90 were calculated. Repeatability was assessed in a separate group of pooled blood samples (n = 10) spiked with ticagrelor at EC10, EC50 and EC90. ASA had no impact on ADP-activated channels for all three devices. TEG®6s was able to distinguish (p ≤ 0.05) between all ticagrelor EC zones; VerifyNow® and Multiplate® were able to distinguish between three and two zones, respectively. Multiplate® showed the largest window between EC10 and EC90 (19-9153 ng/mL), followed by TEG®6s (144-2589 ng/mL), and VerifyNow® (191-1100 ng/mL). Drug effect models distribution of disagreements were identified for TEG®6s (5.0%), VerifyNow® (8.3%), and Multiplate® (13.3%). TEG®6s showed the smallest average coefficient of variation between EC conditions (5.1%), followed by Multiplate® (14.1%), and VerifyNow® (17.7%). Linear models could be generated between TEG®6s and Multiplate®, but not VerifyNow®. Significant differences were found between whole blood point-of-care platelet function analyzers and the clinical impact of these differences needs to be further investigated.

Platelet Activity Measured by VerifyNow® Aspirin Sensitivity Test Identifies Coronary Artery Bypass Surgery Patients at Increased Risk for Postoperative Bleeding and Transfusion.

BACKGROUND: Identifying predictors of bleeding in patients before coronary artery bypass grafting surgery is important, given the complications of bleeding and finite supply of blood. Patient response to aspirin is heterogeneous and can be evaluated using point-of-care platelet function tests. We postulated that patients who hyper-respond to aspirin given preoperatively, as identified by VerifyNow® Aspirin assay (Accumetrics, Inc., San Diego, CA, USA), are at increased risk of bleeding and transfusion. METHODS: This prospective pilot study examined response to aspirin in patients undergoing coronary artery bypass grafting surgery (n = 61) from 2009 to 2013. Patients with aspirin reaction unit (ARU) values in the lower 50th percentile as identified by VerifyNow® assays were defined as aspirin hyper-responders. The proportion of patients transfused and the median adjusted indexed drop in haemoglobin were compared between aspirin hyper-responders and non-hyper-responders. Logistic regression was performed to determine factors associated with increased risk of transfusion. RESULTS: Seventy per cent (70%) of aspirin hyper-responders were transfused perioperatively compared with 39% of patients who did not hyper-respond, (OR 3.694, 95% CI 1.275-10.706, p = 0.014). VerifyNow® Aspirin hyper-responders had a greater median adjusted indexed drop in haemoglobin compared to non-hyper-responders (34.1 g/L versus 26.6 g/L respectively, p = 0.032). Multivariate analysis also showed VerifyNow® Aspirin hyper-response to be an independent predictor of transfusion (p = 0.016). Other variables such as age, gender, body mass index, renal insufficiency, and cross clamp and bypass times were not predictors of postoperative bleeding in this pilot cohort. CONCLUSIONS: VerifyNow® Aspirin is able to preoperatively identify aspirin hyper-responders at an increased risk of bleeding and subsequent transfusion in the context of coronary artery bypass graft surgery.

Whole blood platelet aggregation determined by the ROTEM platelet equipment; reference intervals and stability.

Point of care testing of residual effect of antiplatelet therapy in trauma patients or during major surgery may result in improved clinical management of significant bleeding. We included 121 healthy individuals (57 females and 64 males, aged 22-65 years) in order to establish reference intervals for platelet aggregation induced by adenosine diphosphate (ADPTEM, 10 µM), arachidonic acid (ARATEM, 0.42 mM) and thrombin activating peptide (TRAPTEM, 36 µM) employing the ROTEM platelet module. Further, the impact of citrate (3.2%) and hirudin (>15 µg/ml) as anticoagulants was evaluated. Finally, we investigated assay stability (15, 30, 60, and 120 min after blood sampling) (n = 8) and between-day variation (n = 5). We report reference intervals for 121 healthy individuals and reference intervals by gender. We observed significantly higher platelet aggregation in females than in males (all P-values < 0.05). No correlation between age and platelet aggregation was observed, except for the parameter TRAPTEM amplitude (A6), in which a decline in A6 was observed with increasing age (P = 0.03). We observed significantly lower levels of platelet aggregation in citrate tubes than in hirudin tubes (all P-values < 0.05), except from TRAPTEM maximum slope, where no significant difference was observed (P = 0.40).The stability was acceptable (≤20% deviation) for up to 120 min for ARATEM in citrate tubes, and up to 60 min for the ADPTEM and TRAPTEM assays in citrate tubes. In hirudin tubes we found ADPTEM and ARATEM assays to be stable for 60 min, while the stability of TRAPTEM in hirudin tubes was found to be stable for 30 min. Using citrate tubes, the between-day variation (mean coefficient of variation, CV) was 19-20% for ADPTEM, 19-26% for TRAPTEM, and 10% for ARATEM, whereas the mean CV was 11-13% for all three assays in hirudin tubes.In conclusion, we established combined and gender-specific reference intervals for three platelet aggregation assays in both citrate- and hirudin tubes. In citrate tubes, the stability of the ROTEM platelet assays was 60-120 min, while the stability in hirudin tubes was 30-60 min. The between-day variation was lowest for samples obtained in hirudin tubes.

Whole blood platelet aggregation kinetics under cardiopulmonary bypass: A pilot study.

Assessing the platelets' functional status during surgery on cardiopulmonary bypass is challenging. This study used multiple electrode impedance aggregometry (Multiplate®) to create a timeline of platelet aggregation changes as induced by cardiopulmonary bypass in antiplatelet-naive patients undergoing elective surgery for mitral valve regurgitation. We performed six consecutive measurements (T1: pre-operatively, T2: after heparinization, T3: 3 min after establishment of cardiopulmonary bypass, T4: immediately after administration of cardioplegia, T5: 5 min after administration of cardioplegia, and T6: 45 min after administration of cardioplegia). Platelet aggregation was determined after stimulation with 3.2-µg/mL collagen, 6.4-µM adenosine diphosphate, and 32-µM thrombin receptor activating peptide. Five patients were included (age: 64 ± 10 years, one female). We observed a decrease in hematocrit levels by -17.1% ± 3.7% (T1 vs T6) with a drop after establishment of cardiopulmonary bypass (T2 vs T3) and slightly decreasing platelet counts by -6.2% ± 7.7% (T1 vs T6). Immediately after establishment of cardiopulmonary bypass (T2 vs T3), we observed reduced platelet aggregation responses for stimulation with adenosine diphosphate (-19.7% ± 12.8%) and thrombin receptor activating peptide (-19.3% ± 6.3%). Interestingly, we found augmented platelet aggregation for all stimuli 45 min after administration of cardioplegia (T5 vs T6) with the strongest increase for collagen (+83.4% ± 42.8%; adenosine diphosphate: +39.0% ± 37.2%; thrombin receptor activating peptide: +34.5% ± 18.5%). Thus, after an initial drop due to hemodilution upon establishment of cardiopulmonary bypass, platelet reactivity increased over time which was not outweighed by decreasing platelet counts due to mechanical platelet destruction and absorption. These findings have implications for rational transfusion, peri-operative antiplatelet therapy, and for the management of patients on other extracorporeal support, such as extracorporeal life support or extracorporeal membrane oxygenation.

Evaluation of the Newly Developed Adenosine Diphosphate-Induced Platelet Aggregation Level System in Aggregometer on Automated Coagulation Analyzer.

BACKGROUND: Light transmission aggregometry (LTA) is the gold standard for platelet function assessment. The automated coagulation analyzer from Sysmex that performs LTA offers the advantage of being a walk-away technology. Recently, a new parameter "ADP-induced platelet aggregation level (APAL)" was developed to support the interpretation of results. APAL is calculated as a score from 0.0 to 10.0 based on platelet aggregation patterns with 1 and 10 µM adenosine diphosphate (ADP). Here, the basic performance of the newly developed APAL system and comparison with the maximum aggregation rate of ADP (ADP-MA) was evaluated. METHODS: The within-run precision was calculated by conducting five replicate analyses of the platelet-rich plasma (PRP) from healthy volunteers and 0.05 µM of cangrelor-spiked PRP. Cangrelor is a P2Y12 inhibitor that does not require liver CYP activation. The reference interval was calculated from the results of 67 healthy volunteers. The effect of the antiplatelet P2Y12 agent was evaluated using several concentrations of cangrelor. A comparative study was performed using 103 PRP samples with different levels of aggregation. Each test was analyzed with both APAL and ADP-MA. RESULTS: The percentage coefficient of variation in within-run precision was within 7% for APAL and 10 µM ADP-MA. Reference interval of APAL and 10 µM ADP-MA was 7.1 - 10.0 and 80.0 - 99.2%, respectively. APAL signifi-cantly decreased with the addition of 0.02 µM cangrelor, while 10 µM ADP-MA was barely affected. A significant correlation was observed between APAL and 10 µM ADP-MA (r = 0.94; p < 0.0001). CONCLUSIONS: The newly developed APAL system exhibited an acceptable performance. APAL score showed a good correlation with ADP-MA and was adequate to detect the weak effect of P2Y12 inhibitors. APAL is a new platelet aggregation scoring system with the potential to monitor the effects of P2Y12 inhibitor over a wide range.