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Platelet‐mapping assay for monitoring antiplatelet therapy during mechanical circulatory support in children: A retrospective observational study

Abstract Essentials Thromboelastography (TEG)‐platelet mapping (PM) assay is used for antiplatelet‐drug monitoring. The TEG‐PM was evaluated for dypiridamole and aspirin monitoring in 4 BH‐implanted children. Some TEG‐PM tracings were atypical leading to wrong results. The TEG‐PM has to be improved for antiplatelet‐drug monitoring during ventricular assistance. Introduction The complex hemostatic changes associated with Berlin Heart (BH) implantation in children require a challenging antithrombotic treatment. The aim of this retrospective analysis was to evaluate the thromboelastography (TEG)‐platelet mapping (PM) assay to monitor antiplatelet therapy in children implanted with a BH. Methods TEG‐PM was performed in 4 BH‐implanted patients receiving dipyridamole and aspirin, and 9 healthy volunteers. Patients’ antiplatelet therapy was adjusted to TEG‐PM results. Light transmission aggregometry (LTA) was also available for 2 of these patients. Results Between 2009 and 2014, 4 BH‐implanted patients received a dual antiplatelet therapy monitored by TEG‐PM. In 2 patients, 18 of 34 tracings were atypical, because the maximum amplitude due to fibrin never stabilized, which made difficult antiplatelet therapy adjustment as recommended by BH's guidelines. To overcome this difficulty, TEG‐PM and LTA were next performed in parallel. However, both methods led to different decisions to adjust antiplatelet therapy in 57% of the cases. In order to better understand this atypical tracing, TEG‐PM was also performed in 9 volunteers and surprisingly 3 of them had the same atypical tracing. This atypical tracing was corrected by adding apyrase, suggesting that adenosine diphosphate (ADP) participates to spontaneous platelet activation in heparinized samples. In addition, we evidenced a high variability in the responses of TEG‐PM with ADP in volunteers. Conclusions Antiplatelet therapy monitoring in BH‐implanted children remains challenging, as TEG‐PM is sensitive to several preanalytical and analytical conditions.

Ventricular assist devices (VADs) are essential for the management of patients with terminal heart failure or cardiomyopathy, awaiting heart transplantation or myocardial recovery. [1][2][3][4] In 1992, with the introduction of miniaturized pumps for clinical use, the Berlin Heart (BH) device became the first pulsatile VAD available for small children. 5,6 Mechanical assistance in pediatrics has considerably evolved over a decade, providing excellent results, even for small children. 7 However, device and/or systemic thrombosis and hemorrhagic events remain the major causes of mortality and morbidity with complication rates (mainly neurologic) ranging from 20% to 60%. 2,[7][8][9][10][11] The hemostatic changes associated with BH implantation are complex and require a challenging antithrombotic treatment. 11 Thus, an antithrombotic protocol combining anticoagulant and antiplatelet molecules is recommended. 5 Unfractionated heparin (UFH) is started soon after VAD implantation and is relayed by vitamin K antagonists or low-molecular-weight heparin once the patient's condition is fully stabilized. Within 2 to 10 days after VAD implantation, platelet inhibitors (dipyridamole or clopidogrel and aspirin) are introduced. The bitherapy is continued until the VAD is removed.
Determining hemostatic function in these patients is essential, but standard coagulation tests performed in plasma are not designed to evaluate global hemostatic function. Thus, BH's guidelines recommend using whole blood thromboelastography (TEG) and (TEG)-platelet mapping-(PM) to monitor anticoagulation and platelet inhibition in children with a BH. 5,12,13 TEG-PM is designed to specifically evaluate platelet inhibition secondary to antiplatelet therapy. Although this assay has been shown to correlate with light transmission aggregometry (LTA) and to have a predictive value of bleeding risks in patients undergoing percutaneous coronary intervention 14,15 , some concerns with the design of TEG-PM assay have been recently raised. 16,17 The aim of this retrospective analysis was to evaluate the TEG-PM assay to monitor antiplatelet therapy in children implanted with a BH.  7 Inlet and outlet cannulae and artificial devices were monitored daily for micro thrombi which could potentially migrate to the brain.

Patients and healthy volunteers
Blood samples were obtained from the French Blood Bank Institute (Etablissement Français du Sang, Paris, France N°13/CABANEL/008) from nine healthy volunteers after they had given their written informed consent and after it had been verified that they had no increased bleeding tendency and had not taken medication that could decrease platelet function.

Hemostasis monitoring
Blood from patients and healthy volunteers was collected in Vacutainer tubes (BD Vacutainer System, Paymouth, UK) containing 0.109 mol L −1 sodium citrate or 15 U mL −1 heparin. The blood was drawn from the patients' arterial line following the recommendations of avoiding heparin contamination and sample dilution 19 or from the antecubital vein of healthy volunteers. To avoid platelet activation, blood specimens were transported to the laboratory by hand delivery (the pneumatic tube system was never used). 20,21 Blood was analysed within 30 minutes after sampling.

TEG-PM
This technique was performed according to the manufacturer's instructions using the platelet-mapping kit (Haemonetics, Braintree, MA, USA). 22 For the standard TEG reaction, kaolin-treated citrated blood (360 μL) was transferred into a prewarmed cup containing heparinase and calcium chloride (20 μL) to measure thrombin-induced plateletfibrin clot-strength. Under these conditions, maximum amplitude (MA) is dependent on fibrin and platelet contributions (MA thrombin ).
To eliminate the thrombin effect on platelet activation, heparinized blood (340 μL) is used and activated by reptilase in the presence of factor XIIIa (activator F) (10 μL), enabling the strength of the fibrin clot (MA fibrin ) to be specifically measured. The addition of platelet agonists (10 μL), either 1 mmol L −1 AA or 2 μmol L −1 ADP to activator F ensures determination of the strength of the platelet-fibrin clot (MA ADP or MA AA ).
The difference between MA ADP or AA and MA thrombin is considered as platelet inhibition (%) and the results are expressed as the percentage of
• The TEG-PM was evaluated for dypiridamole and aspirin monitoring in 4 BH-implanted children.
• Some TEG-PM tracings were atypical leading to wrong results.
• The TEG-PM has to be improved for antiplatelet-drug monitoring during ventricular assistance. The BH's guidelines recommend starting platelet inhibitors according to some of these parameters, namely when MA thrombin is >56 mm, G ADP >6 dynes cm −2 and AA platelet inhibition <70%. The dipyridamole dose should be adjusted to the G ADP value: for G ADP 6 to 10 dynes cm −2 , no dose change is required; otherwise the dose must be adjusted as described in Table 1. The aspirin dose must be adjusted to the percentage of platelet inhibition in the presence of AA: when inhibition is 70-95%, no dose change is required; otherwise the dose must be adjusted (Table 1).
Platelet-rich plasma was incubated 2 minutes at 37°C and was then stirred at 122 g for 2 minutes before adding saline, ADP or AA (5 μmol L −1 and 1 mmol L −1 , respectively). The platelet response was recorded during 5 minutes. Platelet aggregation was quantified as the percentage of maximal optical change.
In the last version of the BH's guidelines, when ADP-induced platelet aggregation is <50%, no dose change of dipyridamole is required; when AA-induced platelet aggregation is <30%, no dose change of aspirin is required.

TEG-PM in healthy volunteers
TEG-PM assay was also performed in healthy volunteers using activator F with standard ADP and AA concentrations (ie, 2 μmol L −1 and 1 mmol L −1 , respectively) or 10 μmol L −1 ADP.
In addition, control tests were run with activator F alone (without any platelet activators) after incubating blood with 5 U mL

Characteristics of patients
Demographic and clinical patients' characteristics are presented in Table 2. Six patients were affected by dilated cardiomyopathy with end-stage heart failure and were assisted with a bi-VAD; one patient had myocarditis-induced dilated cardiomyopathy and was supported by a left VAD. The median duration of the support was 54 6-107 days.
Among patients with bi-VAD, 4 were successfully transplanted and 2 died. The patient assisted by a left BH was successfully explanted. T A B L E 1 Antiplatelet therapymonitoring with TEG-PM according to the Berlin Heart manufacturer's plateletinhibition protocol

Anticoagulation and platelet inhibition after BH implantation
Conventional tests were monitored at least daily (not shown). UFH was started 24-48 hours after BH implantation. The heparin dose was adjusted to achieve a target anti-Xa range of 0.35-0.5 IU mL −1 . For 6 patients, heparin was maintained during the whole assistance period.
Antithrombin was monitored daily and human antithrombin (Aclotine) administration was adjusted to achieve a target value above 70%.
The combined administration of heparin and antithrombin resulted in consistent therapeutic anticoagulation for all patients. No significant bleeding event was observed. Vitamin K antagonist (warfarin) was used in only one patient (patient 3) with an initial dose of 0.2 mg kg −1 d −1 after 40 days of assistance; the dose was adjusted to maintain the INR between 2.7 and 3.5. Patients who were without bleeding and hemodynamically stable (patients 3, 5, 6, and 7) received dual antiplatelet therapy (Table 3). Aspirin was given between 6 and 9 days postimplantation when TEG-PM platelet inhibition with AA decreased below 70% according to BH's guidelines. On the contrary, dipyridamole was started between 5 and 47 days postimplantation to limit the risk of thrombosis, although the 6 dynes cm −2 threshold for G ADP indicated in BH's guidelines was reached for none of these patients.

Platelet inhibition monitoring
In the 4 patients receiving antiplatelet therapy (Table 3)   The monitoring of children receiving dual antiplatelet therapy was also done with LTA for the last 2 patients (6 and 7, Table 2). For pa-

TEG-PM in healthy volunteers
To in ADP-induced platelet aggregation was previously reported, suggesting that 2 μmol L −1 ADP could be insufficient to reach full platelet activation in some subjects. 24 We thus compared the effect of ADP

DISCUSSION
This retrospective analysis underlines the difficulties of monitoring antiplatelet therapy with TEG-PM in children implanted with pediatric BH.
An antithrombotic protocol including platelet inhibitors is essential during mechanical circulatory support to prevent thromboembolic complications. 5,25 Anticoagulant monitoring is relatively well-standardized, albeit sometimes more difficult in children than in adults. 26 In contrast, antiplatelet-drug monitoring remains an issue.
In general, bridged patients have suppressed platelet and coagulation functions during the acute phase after device implantation, with more frequent bleeding. This first phase is usually followed by a hypercoagulable phase during which anticoagulation and platelet inhibitors are required. These complex and clinically unpredictable hemostatic changes explain why BH recommends using the TEG-PM assay, which is presented as a convenient tool to provide an analogous profile of platelet function over time that can accurately monitor platelet antagonists in whole blood. 14,27,28 BH provides algorithms specifying the dipyridamole and aspirin dose changes according to G ADP and the percentage of platelet inhibition in the presence of AA, respectively.
However, no correlative studies on TEG-PM results and patient outcomes have been reported. Moreover, some publications highlighted the weaknesses of TEG-PM. 16,22,29,30 Four out of the 7 patients implanted with BH in our series were treated by antiplatelet therapy after BH implantation. These results are explained by the interindividual variability in ADPinduced platelet aggregation, as previously shown in a study on 98 healthy volunteers, among whom 2 phenotypic groups of subjects with high or low responsiveness to 2 μmol L −1 ADP could be identified. 24 However, the percentage of ADP platelet inhibition, also decreasing, remains high in healthy subjects free of antiplatelet therapy when 10 μmol L −1 ADP was used.
Our study has several limitations. The implantation of BH in children, although steadily increasing, is still rare and our study is limited by its small sample size and heterogeneity of patients. Moreover, healthy volunteers were not run in parallel of patients, and blood collection was not standardized between patients and healthy volunteers.
Thus, no comparison is allowed between patients and volunteers, but this was not the aim of this study.
Results of TEG-PM in healthy volunteers improved our understanding of patients' results by showing that spontaneous platelet activation involving ADP occurs in heparinized blood samples. This compromises TEG-PM's ability to specifically reflect platelet inhibition secondary to antiplatelet therapy with ADP inhibitors. Our conclusion is consistent with a study on 40 healthy volunteers and volunteers taking daily antiplatelet therapy that compared 5 platelet-function tests, and concluded that TEG-PM is the least suited to monitor the effects of antiplatelet agents. 30 Unfortunately, due to the necessary blood volume, such a study comparing different platelet-function test is not possible in pediatrics.

CONCLUSION
Antiplatelet drug management by monitoring TEG parameters is difficult in VAD-bearing children and requires individual decision-making according to each patient's clinical status. Given the broad variability of TEG-PM results, its sensitivity to several preanalytical and analytical conditions, and the lack of validation of its use in clinical settings, we believe that TEG-PM has to be improved for antiplatelet-drug monitoring during ventricular assistance.

AUTHOR CONTRIBUTIONS
Planned and designed the study: DL, CBL. Conducted the experi-