Key words : Blood coagulation, Clot formation, Fibrinolysis

Introduction

Liver transplant recipients can have associated coagulopathies and can develop serious intravascular thrombosis in their portal venous and hepatic artery systems.^1,2 Changes in platelet counts and function, as well as coagulation factors and inhibitors, can result in bleeding and thrombosis. Platelets play a critical role in the initiation of in?ammation, angiogenesis, ischemia/reperfusion injury, and thrombosis.^3,4

Splenectomy during living related donor liver transplant is performed by surgeons to reduce portal flow and to avoid small-for-size liver graft syndrome.⁵ Splenectomy increases platelet survival time because of removal of the major site of platelet elimination, which subsequently results in thrombocytosis.⁶ Routine laboratory coagulation tests are not sufficient to monitor platelet hyperactivity in the perioperative settings.^3,7 Standard rotational thromboelastometry (ROTEM-delta; Tem Innovations) cannot detect platelet dysfunction, apart from its contribution to maximum clot firmness (MCF).^8-12

Platelets are activated by different pathways, including the arachidonic acid, adenosine diphosphate, and thrombin pathways. The use of ROTEM-platelet is an additional module, designed to work with the ROTEM-delta device. It is based on impedance aggregometry that allows measurement of platelet aggregation in whole blood samples and provides a more specific analysis of function by also analyzing the various platelet activation pathways. ROTEM-platelet can identify various antiplatelet drug effects.¹³ In this study, our primary aim was to monitor perioperative platelet function among living donor liver transplant recipients and to study the effects of splenectomy on platelet function. The secondary aim was to report any thrombotic events and to investigate the antiplatelet effects of oral acetyl salicylic acid.

Materials and Methods

The study was approved by the local ethics and research committee of the Faculty of Medicine (7/2018ANET8, July 1, 2018) at Menoufia University, Egypt.

We conducted a prospective observational cohort study to assess platelet function among liver transplant recipients with and without splenectomy. We included 40 adult recipients with end-stage liver disease (20 recipients with splenectomy, 20 recipients without splenectomy). All recipients were scheduled for elective living related donor liver transplant. The 20 recipients scheduled for splenectomy were identified based on their perioperative clinical and investigational data. Hepatitis C-related end-stage liver disease is known to be the main indication for living related liver transplant in Egypt, with a rate of 89.8% as reported by Yosry and colleagues.¹⁴ Exclusion criteria included recipients on preoperative oral anticoagulants or antiplatelet drugs, recipients with Budd-Chiari syndrome, and recipients who used any drugs causing pancytopenia.

We collected the following preoperative data: recipient age, sex, body mass index, Modified End-Stage Liver Disease score, and present and past history of any medical illness. We analyzed the following perioperative laboratory tests: protein C and protein S levels, antithrombin III, factor V Leiden mutation, lupus anticoagulant, homocysteine immunoglobulin (IgG/IgM), and antiphospholipid antibodies. Furthermore, prothrombin time, inter-national normalized ratio, activated partial throm-boplastin time, and complete blood count, including platelet count, were assessed.

Analysis

ROTEM-delta assays assessed both the intrinsic (INTEM pathway) and extrinsic (EXTEM pathway) coagulation pathways of clot formation and fibrinolysis. Assessment of fibrin contribution to clot firmness was made with tissue factor activation and platelet inhibition (FIBTEM) test. The following ROTEM-delta variables were reported for each blood sample: clotting time (in seconds), amplitude of clot firmness 10 minutes after clotting (in mm), and MCF (in mm). Clotting time was the time from start of measurement until clot firmness of 2 mm was reached (reference range for INTEM clotting time: 100-240 s; reference range for EXTEM clotting time: 38-79 s). Maximum clot firmness is the clot firmness during the measurement by platelet aggregation, polymerized fibrin, and crosslinking by factor XIII (normal reference range of EXTEM and INTEM MCF of 50-72 mm). The FIBTEM MCF reflects fibrin contribution to clot firmness without platelet contribution (normal reference range of FIBTEM MCF of 8-24 mm).¹⁵

Single-use reagents were used with ROTEM-delta. Blood samples were immediately mixed with 3.2% citrate sodium solution (9 NC; Becton, Dickinson, and Co). Analyses with ROTEM-delta was at 37 °C.

Platelet activation with arachidonic acid (ARATEM), adenosine diphosphate (ADPTEM), and thrombin receptor-activating peptide 6 (TRAPTEM) pathways were used with ROTEM-platelet. The area under the aggregation curve (AUC) was assessed for each whole blood sample. In accordance with the manufacturer’s manual, ROTROL N and P were used for quality control to identify potential problems with the ROTEM device, reagents, consumables (tips, cups), or user technique.^16-20 ROTEM-platelet is a whole blood impedance aggregometry module that uses single-use reagents and is used to detect the effect of antiplatelet drugs such as cyclo-oxygenase inhibitors, ADP receptor inhibitors, and protease-activated receptor inhibitors. The module ROTEM-platelet can be used to diagnose platelet dysfunction and can help to predict bleeding and thrombotic or ischemic complications.

Other conventional coagulation tests were performed, including activated partial thromboplastin, prothrombin time, international normalized ratio, and plasma fibrinogen concentration (Clauss method). These factors were measured semi-automatically in citrated platelet-poor plasma by fibrin timer (Siemens-Dade Behring Healthcare Diagnostics).²¹ Platelet count was measured in EDTA blood with a Coulter counter (Beckman Coulter Diagnostics).^13,22-24

Perioperative ROTEM-delta and ROTEM-platelet assays and standard coagulation tests were assessed during living donor liver transplant at dedicated time points (preoperatively, 10 minutes after hepatectomy [anhepatic phase], 5 minutes after reperfusion, and on postoperative days 1, 3, 7, 14, and 21). Recipients on acetylsalicylic acid, clopidogrel, or antiplatelet drugs during the 10 days preceding surgery were excluded.

Packed red blood cells were transfused to keep hematocrit above 25%. Procedures for ROTEM were utilized to guide intraoperative transfusion of blood products as described by Gorlinger.¹⁸ Perioperative fluid regimen consisted of Ringer acetate solutions (6 mL/kg/h) and 5% albumin guided by the corrected flow time of transesophageal Doppler (Cardio QP; Deltex Medical) according to Sinclair and colleagues.²⁵

Monitoring and general anesthesia were induced and maintained according to the standards in our transplant center.²⁶ Intraoperative body (model 750-Bair Hugger temperature management unit; Arizant Healthcare) and by warming all infused fluids. The same team of surgeons was present for all surgeries. All recipients underwent the piggyback technique. All recipients underwent portal vein anastomosis first, followed by hepatic artery bile duct reconstruction, with no veno-venous bypass or temporary portocaval shunts. Recipients were admitted to the intensive care unit after surgery for sedation and mechanical ventilation. The aim was to extubate all recipients as soon as liver graft function was shown and systemic and hepatic hemodynamics data were adequate.

Intravenous unfractionated heparin was admi-nistered postoperatively for 2 days (60-180 U/kg/day) guided by conventional coagulation tests and ROTEM parameters, followed by subcutaneous low-molecular-weight heparin (40 mg/24 h), which commenced on postoperative day 3. Oral acetyl salicylic acid (150 mg/12 h) initiated only when platelet count increased to >50 × 103/?L.

We collected recipient outcomes and 3-month mortality rates. We also reviewed blood and platelet transfusion requirements, duration of surgery, and length of stays in the intensive care unit and hospital. Recipients scheduled for splenectomy during the transplant procedure received immunization before surgery.

Statistical analyses

We calculated the minimal sample size in accordance with a previous study from Song and colleagues who aimed to evaluate whether EXTEM, INTEM, and FIBTEM correlated with platelet count and fibrinogen concentration in hypocoagulable patients undergoing living donor liver transplant.¹⁰ Song and colleagues demonstrated that ROTEM, as an early variable for clot firmness, was effective in detecting critically low platelet and fibrinogen and thus could be reliable to guide transfusion therapy in hypocoagulable patients undergoing transplant.¹⁰ The findings of Song and colleagues resulted in a minimal required sample size of 20 patients per group (number of groups = 2, with 40 total patients).¹⁷ Sample size did not need to be increased to control for attrition (withdrawal) bias.18 We used a power of 90% and level of significance of 95% (? = 0.05). We used G Power version 3.1.9.2 to calculate sample size.^27-29 We used SPSS version 21 (Statistical Package for Social Science) to collect and analyze data. We presented results as numerical or categorical data. We used the Kolmogorov-Smirnov test to examine data with normal distribution. We presented results of parametric tests as mean and standard deviation and results of nonparametric tests as median and interquartile range, as appropriate.

Results

Forty-two recipients with end-stage liver disease scheduled for elective living related donor liver transplant were enrolled in the study. We excluded 2 recipients (1 because of anatomic concerns with donor liver size during surgery and 1 because of lack of ROTEM reagents). Our study groups included 20 consecutive recipients with splenectomy and 20 consecutive recipients without splenectomy.

Demographics of transplant recipients are listed in Table 1. Graft-to-body weight ratio was lower in the splenectomy group (0.82 ± 0.12) compared with the nonsplenectomy group (1.03 ± 0.07; P <.001). Operation time was longer with splenectomy then without (11.70 ± 1.75 vs 10.50 ± 1.43 h; P = .009); however, no differences in warm (58.25 ± 35.88 vs 46.35 ± 4.53; P = .76) or cold ischemia times (43.15 ± 5.92 vs 46.55 ± 5.36; P = .17) were shown in groups with versus without splenectomy. The preoperative blood levels of protein C and antithrombin III were low but were not different between groups with and without splenectomy. In groups with versus without splenectomy, protein S activity was low (<60 U/dL) in 8 (40%) versus 9 (45%; P = .749) recipients, and factor V Leiden mutation was positive in 2 (10%) versus 4 recipients (15%), respectively. Methylenetetrahydrofolate reductase gene mutation and anticardiolipin IgM antibodies were negative in all recipients.

Platelet count and function were low preo-peratively because of end-stage liver disease, but levels gradually increased during postoperative time until full recovery after 2 weeks (Table 2).

Splenectomy greatly enhanced recovery of platelet count and function (Figure 1). Oral acetyl salicylic acid reduced platelet function, as represented by ARATEM AUC on postoperative days 7 and 21. However, acetyl salicylic acid exerted no effect on ADAPTEM and TRAPTEM (Figures 2--4and Table 2).

Oral acetyl salicylic acid was required earlier among 8 recipients in the splenectomy group on postoperative day 3 (8/20) compared with no patients in the nonsplenectomy group (P = .006). On postoperative day 7, 14 of 20 patients in the splenectomy group required acetyl salicylic acid compared with only 10 of 20 patients in the nonsplenectomy group (P = .19). Two weeks after transplant, all recipients (20/20) required acetyl salicylic acid.

In the splenectomy group, platelet count correlated significantly with AUC ARATEM, ADPTEM, and TRAPTEM (? = 0.24, ? = 0.58, ? = 0.49, respectively; n = 160; P < .001). In the group without splenectomy, platelet count correlated with ADPTEM and TRAPTEM (? = 0.58, ? = 0.59; P < .001), respectively, but did not correlate with ARATEM (? = 0.06; P = .224). Platelet counts in both groups correlated with EXTEM MCF (? = 0.56; n = 320, P < .001) and INTEM MCF (? = 0.61; n = 320, P < .001). No platelets were transfused perioperatively. Intraoperative transfusion of packed red blood cells was comparable between groups. Six recipients (30%) with splenectomy versus 10 recipients (50%) without splenectomy required only 2 units of packed red blood cells (P = .13). No units of cryoprecipitate were needed in 17 recipients (85%) with splenectomy versus 12 recipients (60%) without splenectomy (P = .145). Patients with splenectomy required significantly less fresh frozen plasma transfusion, with 16 recipients with splenectomy (80%) versus 8 recipients without splenectomy (40%) who required no plasma transfusion (P =.016).

One recipient had portal vein thrombosis and another recipient had hepatic artery thrombosis, both on postoperative day 13 and in the splenectomy group, but with no signs of hypercoagulation or thrombocytosis. The 2 recipients died on posto-perative day 22 and day 60 from graft failure and sepsis. No recipient in the group without splenectomy presented with hepatic or portal vein thrombosis. Three-month mortality was not different between groups. Three recipients (15%) died on postoperative days 22, 24, and 60 in the splenectomy group compared with 2 recipients (10%) on postoperative days 30 and 35 in the nonsplenectomy group (P = 1.00).

Discussion

Despite improvements in liver transplant surgical and anesthetic techniques, coagulopathy remains one of the important factors that contribute to morbidity and mortality. De Pietri and colleagues reported that portal hypertension, hypervolemia, and infections were important predicting factors for coagulopathy among liver recipients.³⁰ Hemostasis could shift between hypo- and hypercoagulability in any liver transplant recipient, as demonstrated by Kamel and colleagues in their prospective observational cohort study.31 Results of our study helped to illuminate several important points. One is the perioperative utilization of ROTEM-delta and whole blood impedance aggregometry with ROTEM-platelet as point of care devices for monitoring the platelet contribution to clot firmness. In addition, the ROTEM-platelet device was able to monitor platelet function and to assess the efficacy of antiplatelet effects of acetyl salicylic acid.

The low platelet number and function observed in this study before patients had liver transplant surgery was because of several reasons. One is the effect of end-stage liver disease on platelet pro-duction and elimination.^32-34 Another is platelet sequestration in the spleen (hypersplenism) and the presence of portal hypertension, both of which can lead to thrombocytopenia among patients with liver disease, as demonstrated by Gangireddy and colleagues and Massoud and colleagues.^32,33 The low level of thrombopoietin and the subsequent reduced platelet production, in the presence of antiplatelet antibodies, can lead to thrombocytopenia. Finally, folate deficiency, chronic low-grade disseminated intravascular coagulopathy, and direct viral sup-pression of platelet production may also contribute to thrombocytopenia.^32,33 Eyraud and colleagues also referred in their prospective observational study to the defect in platelet function as a result of low glycoprotein IIb/IIIa and P-selectin levels among patients with liver disease. Both were restored on posttransplant day 3.³⁴

The low preoperative platelet number and function continued in our study patients intraoperatively and during the immediate postoperative days. After 2 weeks, platelet number and function fully recovered, as shown by standard laboratory tests and ROTEM results. This delay in recovery could be because of the residual effects of intraoperative bleeding, hemodilution, immunological reactions, and platelet sequestration that are associated with transplant surgery.

Porte and colleagues and Stricker and colleagues previously attributed the reduction in platelet number and function to the activation of the fibrinolytic system. Activation results in an increase in tissue plasminogen activator during the anhepatic phase and after reperfusion of the liver graft. This increase contributes to graft injury and substantial activation and consumption of platelets.^35,36

Juttner and colleagues also reported a decrease in platelet aggregation after liver graft reperfusion and during the immediate postoperative phase. The investigators associated this observation to the low platelet glycoprotein IIb/IIa receptors and P selectin, as mentioned previously by Eyraud and colleagues.^34,37

Conversely, Tripodi and colleagues studied platelet function among patients with liver cirrhosis and demonstrated that platelet capacity maintained thrombin generation despite thrombocytopenia.³⁸ The investigators concluded that platelet function might not be changed significantly as thought before. A compensatory mechanism with platelet dys-function and bleeding disorders in patients with cirrhosis was noted. This mechanism depends on the compensatory elevation of von Willebrand factor and factor VIII, produced and liberated from endothelial cells, which can reach 10-fold the plasma level of healthy controls. At the same time, the von Willebrand factor-cleaving enzyme produced in the liver is significantly decreased in patients with chronic liver disease and may also contribute to this effect.³⁸

Pereboom and colleagues used flow cytometry to investigate platelet function and observed stability of platelet function during and after liver transplant despite thrombocytopenia.³⁹ The investigators found no proteolysis of platelet receptors, which may have been as a result of improvements in organ preser-vation and the policy of keeping cold ischemia times as short as possible.

In our study, platelet count and function were not different on postoperative day 1 and postoperative day 3 compared with findings after liver graft reperfusion. A possible cause for this persistently low platelet count and function during the 3 days posttransplant could be because of sequestration of the platelet in the liver graft and less likely because of heparin-induced thrombocytopenia.

Bachmann and colleagues reported a low incidence of heparin-induced thrombocytopenia in their single-center study. Of 205 liver graft recipients, only 1.95% had elevated levels of heparin-associated antibody type II in blood.⁴⁰ Monitoring levels of heparin-associated antibodies and correlating levels with platelet count and function may be beneficial in the future.

In our study, transplant recipients required 2 weeks to recover platelet function and plasma fibrinogen concentration. Surprisingly, recovery in a few recipients exceeded the normal reference range. Thus, future studies should investigate whether extreme thrombocytosis or platelet hyperactivity and/or hyperfibrinogenemia can affect the blood flow to the transplanted liver graft. At 2 weeks after liver transplant, patients are usually on surgical wards and monitored with only daily standard coagulation tests. The value of the routine laboratory coagulation tests (prothrombin time, activated partial thromboplastin time, and platelet count) are questionable in the perioperative setting because of their inability to adequately reflect the complex changes in hemostasis among liver transplant recipients.⁴¹

In 2008, the introduction of ROTEM-delta and ROTEM-platelet to our center led to an increase in the interest to monitor coagulation and platelet function perioperatively. Recipients included in our study were monitored for 3 weeks postoperatively and not only during the immediate perioperative duration; however, our study was limited to 40 recipients. We identified 2 recipients with portal vein and hepatic artery thrombosis in the splenectomy group; however, both recipients demonstrated no hypercoagulation signs.

Several studies have analyzed the role of ROTEM as a point-of-care device for monitoring perioperative bleeding in liver transplant recipients,^42-44 but few have utilized the device as a tool to detect hyper-coagulability and monitor platelet function for a longer duration posttransplant.

In our study, we showed that platelet count and function, as tested by the ARATEM, ADPTEM, and TRAPTEM pathways, were only normalized between postoperative day 14 and 21 but not before. This slow recovery of platelet count and function were also reported by Peck-Radosavljevic and colleagues.45 The investigators studied throm-bopoietin serum levels and markers of platelet production from bone marrow (reticulated platelets) before and serum levels increased significantly on the first day after transplant preceding the increase in reticulated platelets by day 3. Peripheral platelet count recovered after day 5 and normalized within 14 days, with increase in platelet production by bone marrow.

The decrease in thrombopoietin production among patients with liver cirrhosis is an important etiologic factor that can lead to thrombocytopenia in patients with chronic liver disease. Lisman and colleagues also reported that the decrease in thrombopoietin was rapidly reversed on the first day after transplant.⁴⁶ The slow and gradual increase of thrombopoietin in the blood of recipients after liver transplant and after removal of the diseased liver could explain this delayed and the slow increase in platelet count and functions until postoperative day 14.

A limitation of our study was not measuring results with the Coombs test and blood viscosity in the presence of a 50% of recipients being diagnosed with hepatitis C. The standard coagulation tests can be affected to a certain extent by viscosity, but the effects of viscosity on the viscoelastic tests measured by ROTEM devices should be minimal. There is no direct link between blood or plasma viscosity and viscoelastic testing results, since viscosity testing requires flow conditions, and, in viscoelastic testing devices such as ROTEM, there is only an oscillation of pin or cup by 5.75 degrees. No relation between ROTEM results and Coombs test could be found in searched scientific databases. Viscosity, if present, will only have an effect on standard coagulation tests and not ROTEM parameters.

Conclusions

End-stage liver disease lowers platelet function and count. Two weeks were required for platelet function and count to fully recover in patients after liver transplant. Splenectomy significantly increased platelet count and function on postoperative day 3 but not before. Platelet count and function in the postoperative period should be monitored to avoid individual excessive increases, particularly after 2 weeks, to avoid any possible thromboembolic events. A few recipients in our study demonstrated extreme platelet hyperactivity. The inhibitory effect of acetyl salicylic acid on platelet functions was only expressed by ROTEM ARATEM, whereas the other platelet activation pathways (ADPTEM and TRAPTEM) were not affected.

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