Platelets Count During Circulatory Assistance: Involvement in the Changes of Oxygenation Membrane

Background To study the evolution of platelet count during patient treatment with Extra-Corporeal Membrane Oxygenation (ECMO) and the causes of Membrane Oxygenation (MO) exchange. Assessment of the morbidity and mortality rate was our secondary objective. Methods A single center retrospective study was conducted from January 2014 to December 2015. One-hundred and thirty-nine MO exchanges were studied over 73 patients who received 66 MO exchange. Alterations in biological parameters were compared before and after each MO exchange, to study the device-related platelet count evolution.


Introduction
Over the last 47 years, Extra-Corporeal Membrane Oxygenation (ECMO) and Extra-Corporeal Life Support (ECLS) have emerged as techniques providing both cardiac and respiratory support. 1 According to Extracorporeal Life Support Organization (ELSO), ECLS stand for Veno-Arterial ECMO (VA-ECMO) and ECMO for Veno-Venous ECMO (VV-ECMO). 2any deaths occur in the rst 3 months after the discontinuation of ECMO.On the other hand, those who survive the initial critical period after ECMO seem to have a longer life expectancy, especially if treated for an infectious disease. 3Membrane Oxygenation (MO) complication events during ECMO have decreased in recent decades due to improved coatings and biocompatible materials. 4,5chnical complications remain critical during ECMO therapy representing 30% of all ECMO treatments.Forty-ve percent of system exchanges had to be conducted urgently, whereas the remaining percentage could be performed electively.Maintaining a platelet count of greater than 200.000 /µL, results in decreased overall bleeding complications, without increasing morbidity or mortality. 6 10% of cases there were unpredictable mechanical complications and in 5% a suspected infection of the device which was a rare complication requiring a system exchange. 7Eighty ve percent of technical complications caused by clot formation in the MO affect the gas transfer and device-related coagulation disorder.The ECMO pump can cause hemolysis.Controlling the gas exchange capability and the pressure drop across the MO, as well as monitoring of coagulation and hemolysis parameters over time, allow earlier identi cation of these complications ultimately reducing emergency exchanges. 7ny complex factors interact to control hemostasis; the platelet number and function has a signi cant impact on the development of primary hemostasis.Platelets are consumed and damaged by the arti cial circuit and the MO.The use of systemic anticoagulation and the onset of circuit-related thrombocytopenia contribute to the increased incidence of hemorrhagic complications during ECMO, signi cantly affecting the average platelet count, administration of platelets and bleeding complications. 6 the platelet count is less than 20,000 /µL spontaneous bleeding can occur.The usual practice is to transfuse platelets (adherence to the MO) to keep the count greater than 80,000/µL.Even though the platelet count is over 80,000/µL, platelet functions may be impaired. 2

Materials And Methods
The present study was conducted at the university hospital of Clermont-Ferrand, Department of Cardiovascular surgery.A non-randomized retrospective study was done from January 2014 to December 2015.The population of study was 87 patients, treated with ECMO by using the Quadrox D (Maquet®, Rastatt, Germany) circuit.Fifty eight patients had VA-ECMO and 29 VV-ECMO; MO exchange was performed 66 times.Fourteen patients who died under ECMO in less than 48 hours were excluded.
Among the patients who bene ted from multiple MO exchanges, we studied only the rst four MOs (First MO built-in ECMO and the next three MO exchanges), knowing that the maximum exchange number was 6 times.The total number of MOs studied was 139 ( gure 1).
All MO exchanges were performed by highly trained team including Cardiac surgeon, ECMO perfusionist and practitioner nurse.
Two scores have been used to predict the survival rate developed by ELSO.The Respiratory Extracorporeal membrane oxygenation Survival Prediction score (RESP) for adult patients undergoing VV-ECMO for respiratory failure and second score is the Survival After Veno-arterial ECMO score (SAVE) which is designed for adult patients undergoing VA-ECMO for refractory cardiogenic shock.
In order to study the evolution of platelet count and its normalization, we compared two-day platelet count results before MO exchange and two days after it.In addition, we continued to evaluate the platelet count ve days after ECMO weaning.
We evaluated the association between MO exchanges and alterations in biological laboratory parameter.The exchange procedures were associated with a full examination on daily basis of the circuit, hemolysis, coagulation, and hemostasis parameters.These factors could predict the need for a MO exchange.This retrospective analysis compared consecutive platelet count on admission and during the total duration of MO to study the device-related platelet count evolutions.According to our ECMO perprocedural protocol for the anticoagulation, we gave unfractionated heparin: bolus 50-100 unit/Kg in the time of cannulation, then continuous infusion (no standard dose; 20-70 u/Kg/h), then Activating Clotting Time (ACT) measured hourly (more frequently if needed), ACT: 1.5 time's normal level.Platelet transfusion was given prophylactically in patients with a platelet count lower than 80,000 /µL or therapeutically in active bleeding, in patient known case of thrombocytopenia or platelet function defect.

Statistical analysis
Statistical analysis was performed using Stata software, version 13 (StataCorp, College Station, TX, US).
All tests were two-sided with a Type I error set at 0.05.Continuous data were expressed as means ± standard deviations (SD) or as medians with interquartile range [IQR] according to statistical distribution and categorical parameters as frequencies and associated percentages.Comparisons according to patient status (dead or alive) were realized for continuous data by Student t-test or Mann-Whitney test when assumptions of t-test were not met.The normality was studied with the Shapiro-Wilk test and the homoscedasticity with the Fisher-Snedecor test.Concerning categorical data, Chi-squared or Fisher's exact tests were performed.For longitudinal data (platelet count), statistical analysis was conducted with mixed models to take into account the repeated measurements per subject ("subject" random effect) and the time covariate.These models were also adjusted on platelet transfusions.The normality of models' residuals was studied.
The procedure of MO exchanges and even the number of MO did not affect the mortality rate, but did in multiple RBC and platelet transfused patients (p value 0.03, 0.02 respectively).The average transfusions rate was: platelet 0.7 ± 1.5 units, red blood cell (RBC) 4.5 ± 6.6 units and fresh frozen plasma (FFP) 1.2 ± 2.7 units.In table 2, ECMO and transfusion are described in details.
The incidence of major complications of both the patient and the circuit included renal failure in 26% then tamponade in 20%.The table 3 depicts the incidence of major patients and circuit complications.
The median duration of ECMO was 7 days (IQR: 4 to 11).The mean duration of MO was 5.8 ± 3.2 days, the minimum was less than one day and the maximum duration was 19 days.
We found a signi cant drop in the mean platelet count (p<0.001) from the initiation of ECMO to day 2, with an additional signi cant decrease on day 3 (112.5 ± 56.1 x10 9 /L) (p=0.001).The delta of platelet count pre ECMO and day 1 post ECMO was 58.0 ± 19 x10 9 /L.
In relation to MO exchange, a signi cant decrease in average platelet count (p<0.001) was observed between the rst and second exchange in comparison with the baseline.Table 4, demonstrates the mean platelet count variations relating to exchange in MO.Subsequently, we observed a stable decreased platelet count after the third MO exchange.On the other hand, a normalization of platelet count occurred ve days after the weaning of ECMO.In gure 2, Pattern of average platelet count along with MO exchange was reported.

Discussion
This retrospective analysis in 73 patients compared consecutive platelet count on admission and during the total duration of the ECMO.It aimed to study the device-related platelet count disorders.
The most common technical complication of ECMO is clot formation. 8Clot formation in MOs can occur despite adequate anticoagulation that compromises gas transfer to the extent where MO exchange is necessary. 9Many studies and publications, both domestic and international, have detailed this phenomenon.Researchers hypothesize that it is the activation of platelets that subsequently may cause the deposition of brin, enhancing thrombus formation.
According to ELSO, thrombocytopenia is common in ECMO.This correlates with our ndings.Circulating platelets adhere to plastic surfaces and undergo a release reaction that attracts other platelets. 2moval of the MO resulted in a reduction of D-Dimer levels, delaying increases in brinogen concentration and platelet count over the next ve days. 7binson et al. reported a mean decrease of 26% in the platelet count after the rst 15 minutes of the initiation of ECMO, with an additional mean decrease of 16% by the end of one hour (p < 0.05).The resolution of platelet aggregation abnormalities and normalization of platelet count occurred 8 hours after the weaning of ECMO. 10 This corresponds to our rst result, regarding the signi cant mean decrease in the platelet count from the baseline platelet count found on day 2 after the initiation of ECMO (p<0.001), with an additional signi cant mean decrease on day 3 (p 0.001).Furthermore, we observed a signi cant decrease in average platelet count (p<0.001) between the rst and second MO exchange in comparison with the baseline.
Then a steady decrease in platelet count was witnessed after the third MO exchange.However, a resolution of platelet count occurred ve days after the discontinuation of ECMO.
Using anticoagulation to avoid the formation of clots in the ECMO circuit is mandatory whereas it's important to balance the patient's risk of bleeding.Clots are very dark non-moving areas on the MO surfaces.Large clots on MO require the exchange to obtain the maximum bene t from this membrane.
No intervention is necessary unless the white thrombi are greater than 5 mm or growing. 2 Broi et al. 7 resume the causes of system exchanges into acute (mechanical failure, acute clot formation) and elective system exchanges (progressive clot formation and worsening of gas transfer, suspected infection and device-related coagulation disorder).This corroborates the ndings of our study in which the incidence of MO exchange was due to the clot formation of MO, thrombocytopenia, transformation from VA-ECMO to VV-ECMO, failure of ECMO weaning, technical problems with the machine, and mechanical failure of the blood pump.No MOs were exchanged due to the suspicion of infection.Dornia et al., 11 reported thrombotic clot formation in MO device after 8 days from the onset, and had an average duration of 5.9 ± 3.1 days.
In our experience, bleeding is the most common complication during ECMO because of systemic anticoagulation, thrombocytopenia, and thrombocytopathy.Furthermore, a platelet transfusion was ordered for a patient on ECMO if the platelet count falls below 100,000/µL to prevent generalized hemorrhage.Consequently, platelet transfusion carries risks as well as bene ts.Infections due to bacteria or fungal are the most commonly reported complications of platelet transfusions. 12lood products transfusion was routinely administered during ECMO.Particularly, MO exchange permitted a clear reduction in platelets and RBC transfusion, yet a less evident of FFP.We hypothesised that RBC and platelets are directly affected by the MO but the FFP need is dependent of the systemic in ammatory response induced by the pump itself.
Hemodynamic management can be particularly challenging.The management of bleeding begins with returning the coagulation status to normal as much as possible.Hemorrhagic complications can occur in up to 35% during ECMO by affecting the primary hemostasis. 6ult ECMO patients with lower Hb require more daily RBC and FFP.According to Ang et al., on multivariate analysis, average daily transfusion of RBC increased with hemoglobin < 7•5 g/dl.An average daily platelet transfusion > 3 units was also associated with increased ECMO duration (p = 0•024). 13 our study, the average platelet count was 195.8 ± 84.3x10 9 /L, with an average 0.5 ± 1.4 platelet transfusion associated with high mortality rate (p=0.02).Even with the average number of RBC transfusions at 3.4 ± 5.5, we found the same correlation (p=0.03).
Stallion et al. suggest maintaining a platelet count of greater than 200.000/µL while on ECMO, as it results in overall decreased bleeding complications without increasing morbidity or mortality. 6 the other hand, if the platelet count is less than 20,000 /µL spontaneous bleeding can occur.The usual practice is to transfuse platelets (adherence to the MO) to keep the count greater than 80,000 /µL.Even though the platelet count is over 80,000 /µL, platelet functions may be impaired. 2ture studies are needed to validate our results, particularly to investigate how ECMO survivors do in terms of cardio-pulmonary function, cognitive function, and quality of life.

Conclusion
There is a signi cant decrease in platelets count mainly on day 2 that indicates platelets transfusions in order to prevent complications of hemostasis.A daily examination of the circuit, hemolysis, coagulation, and hemostasis parameters can predict the need for MO exchange to get the maximum bene ts of this treatment.

Figure 1 Total
Figure 1

Table 2 :
Description of ECMO and transfusion.

Table 3 :
Incidence of complications of patient and circuit.Membrane Oxygenation, SD: Standard Deviations, RBC: Red blood cell, FFP: Fresh frozen plasma, 2DB: 2 days before MO exchange, 2DA: after MO exchange.P-value 1 : Consicutive comparision of platelets count between every 2 consicutive MO exchanges.