Whole-blood hypocoagulable profile correlates with a greater risk of death within 28 days in patients with severe sepsis.

BACKGROUND: Hypocoagulability and impaired platelet function have been associated with a high risk of death in sepsis. The aim of this cohort study was to determine whether sepsis-induced hypocoagulability and platelet dysfunction (assessed by ROTEM® and MULTIPLATE®, respectively) are increased in sepsis patients who died within 28 days after diagnosis compared with patients who died between 29 and 90 days after diagnosis. METHODS: Consecutive patients admitted to the intensive care unit of Padova University Hospital from March 2015 to March 2018 for severe sepsis were considered. We collected blood samples from all patients to determine ROTEM® and MULTIPLATE® parameters. Each enrolled patient underwent a 90-day follow-up and the mortality rate was recorded. RESULTS: Of 120 patients, 36 (30%) died within 28 days post-diagnosis (Group A), 23 (19%) died between days 29 and 90 post-diagnosis (Group B), and 61 (51%) were alive after 90 days (survivors). The clotting time in the ROTEM® test and clot formation time in the EXTEM test were significantly more prolonged in Group A than in B. Both groups showed a significantly higher hypocoagulability than survivors in the EXTEM test. MULTIPLATE® platelet function analysis showed that platelet function was significantly lower in Group A than in Group B. CONCLUSIONS: The present study showed that the combination of thromboelastometry and impedance aggregometry may help identifying sepsis patients at high risk of short-term death. Larger studies are warranted to corroborate our results.

Levels of circulating microparticles in septic shock and sepsis-related complications: a case-control study.

BACKGROUND: Microparticles (MP) have been largely studied as potential biomarkers in septic shock (SS) though their biological and clinical relevance is still unclear. This case-control study describes the trend of various MP subtypes during SS to evaluate their possible association with severity of illness and sepsis-related complications (disseminated intravascular coagulation [DIC] and acute kidney injury [AKI]). METHODS: Forty patients admitted to the Intensive Care Unit with SS and 40 matched healthy volunteers were recruited. AnnexinV+, E-selectin+, thrombomodulin (TM+), leukocyte-derived (CD45+, CD36+) and platelet-derived MP (PMP-expressed as PMP/platelets ratio) were measured by flow-cytometry at baseline, on day 1, 3 and 7 after diagnosis. Severity of illness was assessed by Sequential Organ Failure Assessment Score, duration of vasoactive support and mechanical ventilation. Sepsis-related complications were considered. RESULTS: Overall, septic patients showed higher levels of all MP considered compared to controls. TM+MP were significantly lower in more severe sepsis, while CD36+MP and PMP/platelets ratio were significantly increased in patients requiring longer vasoactive support and mechanical ventilation. As for sepsis-related complications, a higher PMP/platelets ratio in patients who developed DIC and increased E-selectin+MP in subjects who developed AKI were observed. PMP/platelets ratio at baseline was significantly associated with longer vasoactive support (OR=1.59 [1.05-2.42]), longer mechanical ventilation (OR=1.6 [1.06-2.42]) and DIC occurrence (OR=1.45 [1.08-1.96]). CONCLUSIONS: A global response through extra-vesiculation of endothelial cells, leukocytes and platelets during the early stages of SS was confirmed. The cellular activation was detected until day 3 after diagnosis. PMP/platelets ratio at diagnosis may be useful to evaluate SS severity and DIC occurrence.

Effect of sugammadex on coagulation as detected by rotational thromboelastometry in morbidly obese patients.

BACKGROUND: Sugammadex, which is used to reverse rocuronium-induced neuromuscular blockade, has a limited and transient effect on activated partial thromboplastin time and prothrombin time. However, no data are available on the effects of sugammadex on coagulation in morbidly obese patients, as assessed by rotational thromboelastometry (ROTEM®). METHODS: Sixty patients received sugammadex 2 mg/kg or 4 mg/kg to reverse moderate or deep rocuronium-induced neuromuscular blockade (N.=30/group) at the end of surgery under desflurane anesthesia. Arterial blood samples were collected before and 3 min and 30 min after sugammadex administration for ROTEM® analysis, including measurements of clotting time (CT), clot formation time, α angle, and maximum clot firmness in INTEM, EXTEM, and FIBTEM assays. Major and minor bleeding events were also monitored during the postoperative period. RESULTS: Sugammadex 2 and 4 mg/kg has a limited and transient (<30 min) effect on INTEM CTs of 7.7% (P=0.04) and 10.7% (P<0.0001), respectively. There were no relevant effects on other ROTEM® parameters. A multivariate analysis indicated a significant effect of total sugammadex dose (<250, 250-500, >500 mg) on the INTEM CT (P=0.002). A regression analysis showed a positive relationship between sugammadex dose and INTEM CT value at 3 min after administration (coefficient = 0.052 s; 95% CI: 0.005-0.098 s; P=0.03). No major or minor bleeding events were observed in either group during the postoperative period. CONCLUSIONS: Sugammadex produces a slight effect on coagulation in morbidly obese patients, without increasing the risk for postoperative bleeding.

Cardiopulmonary Bypass Increases Plasma Glial Fibrillary Acidic Protein Only in First Stage Palliation of Hypoplastic Left Heart Syndrome.

BACKGROUND: Univentricular congenital heart defects require open-heart surgery soon after birth, and are associated with risk of brain injury and poor neurologic outcome. METHODS: This is a prospective, observational study on children undergoing cardiac surgery. Plasma glial fibrillary acidic protein (GFAP), as an early marker of brain injury, was measured by ELISA at the end of anaesthesia induction, initiation of cardiopulmonary bypass (CPB), the end of cooling, the end of rewarming, the end of CPB, and after protamine administration. We recorded clinical and surgical parameters to assess which CPB phase and clinical parameters were associated with a GFAP increase. RESULTS: We studied 13 children less than 50 months of age: 8 underwent Norwood or Damus-Kaye-Stansel palliation (group 1) and 5 underwent Fontan procedure (group 2). A GFAP increase was only observed in group 1, with the highest median value at the end of rewarming. No quantifiable levels of GFAP were measured at pre-bypass and the start of CPB stages in all patients. End of cooling and CPB-end GFAP, GFAP maximum value, and GFAP area under the curve all correlated with the CPB time spent at a cerebral regional saturation < 45% (P = 0.021, 0.028, 0.007, 0.021, respectively). CONCLUSIONS: Children with univentricular heart defects exhibit a CPB plasma-GFAP increase only after stage 1 palliation. The maximum GFAP increase occurred at the end of rewarming. Further studies are needed to identify which clinical or surgical parameter(s) could reflect a GFAP increase during surgery for congenital heart defects, and whether GFAP levels correlate with the neurologic outcome.

Thromboelastographic evaluation of coagulative profiles in pig-to-monkey kidney xenotransplantation.

BACKGROUND: Activation of the clotting cascade is central in acute xenograft rejection (AHXR) that occurs when pig organs are transplanted into primates. The coagulopathy reported in this model is a very complex process that involves simultaneously coagulation factors, platelets and phospholipid-bearing cells (i.e., leukocytes, red blood cells, and endothelial cells). Choosing whole blood for coagulation analysis theoretically appears more favorable compared with plasma. Whole blood rotation thromboelastometry (ROTEM(®) ) is a point-of-care global coagulation analyzer able to evaluate the characteristics of clot formation and lysis by dynamic monitoring. The aim of this study was to record thromboelastographic profiles, performed by ROTEM(®) , in a series of immunosuppressed nephrectomized primates that received a life-supporting kidney. METHODS: Of the eight primates, n = 4 received a pig kidney transgenic for human decay-accelerating factor (hDAF/Gal+); n = 2, an α 1,3-galactosyltransferase gene-knockout (GT-KO) pig kidney transgenic for human CD39, CD55, CD59 and fucosyltransferase (HTF); and n = 2, a GT-KO pig kidney transgenic for hDAF. Blood samples were collected before and at least once per week after transplantation till euthanasia. Intrinsic (INTEM) and extrinsic (EXTEM) coagulation pathways and the function of fibrinogen (FIBTEM) were evaluated. Thromboelastographic parameters considered were clotting time (CT, seconds) and clot formation time (CFT, seconds) in INTEM and EXTEM and maximum clot firmness (MCF, mm) in FIBTEM. The correlations between CT in INTEM and activated partial thromboplastin time (aPTT), CT in EXTEM and PT, CFT in INTEM and EXTEM, and platelet counts and MCF in FIBTEM and fibrinogen plasma levels were also considered. RESULTS: In all animals, thromboelastographic profiles showed progressive prolongation of CT (activation of coagulative cascade) in INTEM. A close correspondence was observed between (i) the prolongation of the CFT values (propagation of clot formation), both in INTEM and EXTEM, and the decrease in platelet counts; (ii) the reduction in MCF values (clot firmness) ​​in FIBTEM and the decrease in fibrinogen plasma levels. No concordance between CT in INTEM and aPTT and between CT in EXTEM and PT was observed. CONCLUSIONS: Our study demonstrated that ROTEM(®) analyzer could be a useful and complementary tool to study the consumptive coagulopathy, either "compensated" or "non-compensated," that takes place when transgenic pig kidneys are transplanted into primates. Larger and prospective studies are needed to confirm our results and to evaluate the role of ROTEM(®) to guide the management of consumptive coagulopathy in order to prolong the survival of the transplanted organ.

Effects of long-term administration of recombinant human protein C in xenografted primates.

BACKGROUND: The role potential of recombinant human activated protein C (rhaPC), a recently developed molecule with anticoagulant and antiinflammatory properties, in prolonging survival in immunosuppressed primate recipients of porcine renal xenografts has been evaluated. METHODS: rhaPC was administered daily for 5 days (24 µg/kg/hr; group A; n = 3) or throughout the postoperative period (8-24 µg/kg/hr; group B; n = 2; or 24-48 µg/kg/hr; group C; n = 4). Animals in group D (n = 2) received rhaPC daily (24 µg/kg/hr) combined with recombinant human antithrombin (84 U/kg every 8 hr). Two animals served as control (group E). RESULTS: The results indicate that rhaPC is protective against fibrin deposition early after transplantation but does not prevent fibrin deposition and the occurrence of acute humoral xenograft rejection (AHXR) later on. Animals in the study survived between 8 and 55 days. At the dose used, rhaPC is able to prevent fibrin deposition in the graft in the first 2 weeks after xenotransplantation, except when it is administered in conjunction with antithrombin. However, rhaPC did not prevent the eventual occurrence of AHXR in primate recipients of porcine xenografts. CONCLUSIONS: In this pig to primate model, rhaPC confers a short advantage in the prevention of early perioperative xenograft damage but does not represent an effective strategy for preventing AHXR.

Reference values for thromboelastometry (ROTEM®) in cynomolgus monkeys (Macaca fascicularis).

INTRODUCTION: The imbalance in clotting homeostasis, tending towards hypercoagulation, is recognized as the real barrier to the long-term survival of porcine xenografts in pig-to-primate xenotransplatation. The present study aimed to validate in primate blood the applicability of whole blood rotation thromboelastometry, performed by ROTEM®, which evaluates the characteristics of clot formation by dynamic monitoring. MATERIALS AND METHODS: ROTEM® (Pentapharm GmbH, Munich, Germany) was used to investigate native coagulation (NATEM®), the intrinsic (INTEM®) and extrinsic (EXTEM®) pathways, the function of fibrinogen (FIBTEM®), and the presence of fibrinolysis in 40 naïve cynomolgus monkeys. Using classic validation approaches, the normal thromboelastographic profile was defined and the influence of haematocrit (Hct,%), platelet count (x10(9)/L), fibrinogen (mg/dl), and factor VIII (FVIII,%) was evaluated. RESULTS: In all four (NATEM®, INTEM®, EXTEM®, FIBTEM®) assays considered, Clotting Time (CT, sec) and Clot Formation Time (CFT, sec) were shorter in primates than humans. Moreover, α-angle (°), Maximum Clot Firmness (MCF, mm), and MaxVel (mm/min) were also higher in primates than humans. No substantial difference was observed for Hct and platelet count between the two species. On the contrary, FVIII was higher in primates than in humans whereas, interestingly enough, fibrinogen levels were lower in monkeys than in humans. CONCLUSION: ROTEM® depicts a hypercoagulable profile in primates as compared to humans. Taken together these data suggest that, with regard to coagulation, xenotransplantation in cynos may represent a much more difficult situation than xenotransplantation in humans.

Peculiar whole blood rotation thromboelastometry (Rotem) profile in 40 sideropenic anaemia patients.

The ROtation ThromboElastoMetry analyser (ROTEM, Pentapharm, Munich, Germany) is useful for studying whole blood (WB) clot formation and lysis. Reduction of haematocrit (HCT) has been reported to influence traditional thromboelastography parameters without compromising "in vitro" blood coagulation. We performed this case-control study to evaluate ROTEM profiles in sideropenic anaemia patients with different degrees of reduction of HCT levels. Forty consecutively referred patients with sideropenic anaemia were enrolled. A group of 40 healthy age and gender matched subjects acted as a control. The influence of HCT on ROTEM was assessed in the study population and in a model of artificially reconstituted blood with modified HCT values. Cases presented significantly increased levels of maximum clot firmness (MCF) as compared to controls (p < 0.001) mimicking a sort of "hypercoagulable profile". However, thrombin generation tests failed to detect an increase in thrombin generation in cases as compared to controls. A statistically significant inverse linear correlation between HCT and MCF (p < 0.0001) was found. In addition, ROTEM profiles following "in vitro" manipulation of HCT confirmed the inverse linear correlation between HCT and MCF found in the study population. In conclusion, the increased clot firmness found by ROTEM in anaemic patients is likely to be related to the method in itself rather than representing a marker of hypercoagulability "in vivo". Since ROTEM is widely used by anaesthesiologists when deciding the optimisation of products supplementation during surgery, attention should be paid in the case of anaemic patients taking depending on the peculiar thrombo-elastography profile found.