Hirudin versus citrate as an anticoagulant for ROTEM platelet whole blood impedance aggregometry in thrombocytopenic patients

Abstract Citrate is widely used as an anticoagulant for platelet function tests (PFTs). Due to an intrinsic inhibitory effect of citrate on platelet function, hirudin is used as an alternative. However, studies comparing the effect of these anticoagulants on rotational thromboelastometry (ROTEM) platelet whole blood impedance aggregometry in thrombocytopenic patients are scant. Cross-sectional study was done in 105 patients who entered the critical phase of Dengue hemorrhagic fever with plasma leakage and severe thrombocytopenia (<100 × 109/L). Samples were collected on two consecutive days and considered as a combined data set for analysis, out of which 200 have been included in the data analysis. Platelet count was used from routine full blood count. ROTEM platelet used TRAPTEM assay, which was performed with 3.2% sodium citrate and 525 ATU/ml hirudin anticoagulated blood. Means of all the TRAPTEM parameters were significantly higher in hirudin, compared to citrate samples (p < .05). Significantly higher overall platelet aggregation was observed in hirudinized samples with a significant mean difference (p < .05) compared to citrate in each quartile of platelet count. Higher platelet aggregation was observed with hirudin compared to citrate in ROTEM platelet whole blood impedance aggregometry in thrombocytopenic patients elaborating the importance of using hirudin anticoagulation in PFTs, particularly in patients with severe thrombocytopenia. Plain Language Summary Citrate is the most commonly used anticoagulant for coagulation studies including rotational thromboelastometry (ROTEM). Hirudin is an alternative option to be used as an anticoagulant for PFTs because of the inhibitory effect of citrate on platelet function. One study (Nissen et al. (2020)) reported higher precision and platelet aggregation with hirudinized blood of healthy individuals, over citrate using ROTEM platelet. However, none of the studies were performed in patients in actual clinical context. We evaluated the potential benefit of using hirudin anticoagulated blood over citrate in thrombocytopenic patients due to Dengue hemorrhagic fever using ROTEM platelet. We observed higher platelet aggregation with hirudin compared to citrate suggesting the importance of using hirudin anticoagulation in PFTs, particularly in patients with severe thrombocytopenia.


Introduction
Platelets play a crucial role in hemostasis. 1 The rotational thromboelastometry (ROTEM) platelet module used together with the ROTEM delta device (TEM Innovations GmbH, Munich, Germany) assesses platelet aggregation by whole blood impedance aggregometry and displays platelet aggregation graphically and numerically. 23.2% sodium citrate is the most widely used and conveniently available anticoagulant for platelet function tests (PFTs), although its chelation of ionized calcium in blood affects platelet function by the inhibition of platelet aggregation since intra-platelet calcium concentration is an important modulator of platelet function. 3Moreover, as a result of reduction in calcium levels, citrated samples have resulted in low reproducibility and sensitivity to tests associated with thrombin activation of platelets. 4he non-calcium chelating anticoagulant hirudin which was introduced subsequently, is a polypeptide present in the leech (Hirudo medicinalis) having a strong and direct antithrombin activity by inhibiting the conversion of fibrinogen to fibrin.Hence, hirudin maintains the physiological milieu of the sample. 5][8] Plasma leakage is the hallmark of the critical phase of Dengue hemorrhagic fever with thrombocytopenia (<100 × 10 9 /L) and is considered as a determining factor of mortality. 9Hence, ROTEM has been widely used in the management of the Dengue patients for the assessment of coagulopathies, [10][11][12] while the type of anticoagulant used plays a pivotal role in obtaining precise results in PFTs, particularly in patients with severe thrombocytopenia.ROTEM platelet provides three parameters; area under the aggregation curve (AUC in Ω•min), which represents overall platelet aggregation, maximum slope (MS in Ω/min), which explains the rate of aggregation and amplitude at 6 minutes run time (A6 in Ω), which reflects the extent of platelet aggregation. 13Thrombin receptor activating peptide test (TRAPTEM) in ROTEM platelet measures platelet aggregation activated by the agonist TRAP-6. 2,14As a part of a larger study, we evaluated the potential benefit of using hirudin anticoagulated whole blood samples over citrate in managing bleeding patients using ROTEM platelet.

Materials and methods
A descriptive cross-sectional study was conducted at National Hospital of Sri Lanka and National Institute of Infectious Diseases in Western Province enrolling 105 patients (>18 years) who entered critical phase of Dengue with a platelet count < 100 × 10 9 /L.Samples were collected at two consecutive days after obtaining informed and written consent from the patients: (1) within 24 hours after entering into critical phase with plasma leakage and (2) after 48 hours.At both time points, a platelet count was obtained from the routinely performed full blood count analyzed using EDTA anticoagulated blood.A sample of 1.6 mL of blood was taken using 21-gauge needle and aliquoted to a 525 ATU/ml hirudin anticoagulant tube (Sarstedt AG & Co. KG, Nümbrecht, Germany) and 1.8 mL to a 3.2% sodium citrate anticoagulant tube.Both were analyzed by ROTEM platelet TRAPTEM assay within 1 hour of blood collection.Standard reference ranges were used for three ROTEM platelet TRAPTEM parameters as published by the manufacturer. 13For citrated blood samples: A6: 15-36 Ω; MS: 5-14 Ω/min, and AUC: 61-156 Ω•min, and for hirudin blood samples: A6: 15-38 Ω, MS: 6-18 Ω/min, and AUC: 66-169 Ω•min. 13engue virus interacts with human Glycoprotein IIb/IIIa receptors and induces Dengue hemorrhagic fever.This interaction will alter the receptor causing prevention of platelets forming a plug, thereby causing bleeding.15 TRAPTEM assay assesses the glycoprotein IIb/IIIa receptor effect when platelets are activated by thrombin receptor activating peptide.Although arachidonic acid activated PFTs (e.g., ARATEM) and adenosine diphosphate-activated PFTs (e.g., ADPTEM) are available, TRAPTEM assay is widely recommended for evaluating non-drug-induced platelet dysfunction such as in trauma, sepsis, and surgical bleeding.13,[16][17][18] Furthermore, TRAPTEM provides the strongest stimulation of the platelets.Therefore, we decided to use this assay in the Dengue patient population with severe thrombocytopenia. Sice no specific antiplatelet drugs have been used in this patient population, ROTEM platelet assays designed for drug monitoring (ARATEM and ADPTEM) have not been used in this study.
Excluding the cases with missing data, a total of 200 samples obtained from both time points were analyzed as a combined data set using SPSS 23

Results and discussion
Data were normally distributed, and mean platelet count was 36.5 ± 21.7 × 10 9 /L ranging from 4 × 10 9 /L to 105 × 10 9 /L.All the patients had platelet count < 100 × 10 9 /L on the day of entering to the critical phase of dengue and only one patient had a platelet count of 105 × 10 9 /L on the following day.TRAPTEM parameters (A6, AUC and MS) exhibited a significant reduction in platelet aggregation with both anticoagulants.Comparing the means, significantly higher levels of platelet aggregation was observed in hirudin samples when compared to citrate samples.(Table I).
As a result of higher platelet aggregation and preservation of the physiological state of the sample by hirudin anticoagulation, we used ROTEM platelet aggregation parameters in hirudin whole blood samples to study the association between ROTEM platelet parameters and bleeding incidence in Dengue patients in the critical phase.TRAPTEM -AUC was able to discriminate patients with bleeding manifestations from those without, at a cutoff value of <12.5 Ω*min with a sensitivity and specificity of 73.7%, and 60.2%, respectively, which suggested the potential role for defective platelet aggregation in the pathogenesis of bleeding in the critical phase of Dengue. 19e observed that 10% (n = 20) of samples did not show any aggregation (AUC = 0) regardless of the anticoagulant used, while 6% (n = 12) aggregated only in those with citrate (AUC > 1) and 19.5% (n = 39) only with hirudin samples.Moreover, 41.5% (n = 83) showed statistically higher aggregation parameters with hirudin than citrate and 22% (n = 44) vice versa.Hence, hirudin allows for better interpretation of ROTEM platelet results in thrombocytopenic patients.Hirudin vs. citrate anticoagulant in ROTEM platelet 3 For further analysis, platelet count was divided into 4 quartile ranges: Q1: 4-19 × 10 9 /L, Q2: 20-32 × 10 9 /L, Q3: 33-46 × 10 9 /L and Q4: 47-105 × 10 9 /L.Mean of all three TRAPTEM parameters showed an increase in platelet aggregation with increase in platelet count while hirudin exhibited a higher aggregation when compared to citrate anticoagulated blood samples (Figure 1a-c).
The mean difference of overall platelet aggregation (TRAPTEM AUC) at each quartile of platelet count was significantly increased in hirudin anticoagulated samples compared to citrate (Table I).Only TRAPTEM A6 and AUC had higher median results in hirudin compared to citrate blood samples (Figure 2a-c).

Discussion
To our knowledge, this was the first study designed to evaluate the influence of hirudin and citrate anticoagulated blood in determining whole blood platelet aggregometry using the novel ROTEM platelet TRAPTEM assay in Dengue patients (n = 103) in the critical phase with varying degrees of severe thrombocytopenia (≤105 × 10 9 /L).The importance of such studies to evaluate ROTEM platelet assays in patients in a clinical setting has been suggested. 7ne published study was found on the effectiveness of hirudin and citrate anticoagulated blood on ROTEM platelet analysis, which was performed on 121 healthy individuals.However, except for TRAPTEM-MS, TRAPTEM-AUC, and TRAPTEM-A6, ADPTEM, and ARATEM showed higher precision and platelet aggregation in hirudin compared to citrate anticoagulated blood samples. 7In agreement with this study, we observed significantly higher platelet aggregation with respect to all the three TRAPTEM parameters accounting for 61% higher results in hirudin compared to citrate blood samples in thrombocytopenic patients, which prove that even at low platelet counts there is higher platelet aggregation with hirudin.However, the stability of ROTEM platelet TRAPTEM assay in citrate and hirudin samples was not assessed in the current study, which is a limitation of the study.
Though citrate is commonly used worldwide, chelation of Ca 2+ significantly affects PFT results, reducing the degree of platelet aggregation in response to different agonists. 4,20Existence of 23% higher aggregation results with citrate than hirudin while having 4% aggregation only with citrate cannot disregard the fact that thrombin too plays an important role in platelet activation and aggregation.Although, hirudin strongly and directly inhibits thrombin without affecting Ca 2+ levels and thus preserving the physiological milieu of the sample, it is unable to completely neutralize thrombin and the residual thrombin is sufficient to stimulate platelets and enhance aggregation. 20Yet, the positive aspect of hirudin was the enhanced stability and physiological milieu of hirudin than citrate in PFTs. 21hough there are certain advantages of using hirudin over citrate in PFTs, hirudin blood sampling tubes are not widely available due to the difficulty of extracting sufficient amounts of polypeptides from medicinal leeches and hence, hirudin tubes are more expensive than citrate tubes. 22However, the improved platelet function testing results may outweigh the higher costs in patients with severe thrombocytopenia, particularly when the prediction of bleeding and the decision-making for platelet transfusion in patients with severe thrombocytopenia as in Dengue can be improved. 18he potential role of Dengue virus in modifying platelet aggregation needs to be considered.Dengue is a well-known virus to change membrane characteristics, degranulation, and activation of platelets.Hence, it may alter platelet interactions with hirudin and citrate and thereby its properties of aggregation. 23t is important to consider the insensitivity of the citrated samples to provide the actual degree of platelet aggregation and its inability to maintain the physiological milieu of the sample when interpreting results obtained from ROTEM platelet as this may affect the management of bleeding patients.

Table I .
.0.0.0 (released 2015, IBM statistics for Windows version 23, IBM Corp., Armonk, NY).Standard statistical analyses were performed and mean values of hirudin and Mean difference and medians of TRAPTEM parameters in hirudin and citrate anticoagulated samples within whole range and quartiles of platelet count.werecompared using paired t-test.The graphs were developed using GraphPad Prism Software.Significance was considered when p < .05 at 95% confidence interval.Ethics approval for the study was granted by the Ethics Review Committee, Faculty of Medicine, University of Colombo (EC-19-033).