Prolonged Clot Formation Time Research Articles

Tocilizumab reduces hypercoagulation in COVID-19 – Perspectives from the coagulation and immunomodulation Covid assessment (Coag-ImmCovA) clinical trial

BackgroundDespite medical interventions, COVID-19 continues to persist at pandemic proportions. A hypercoagulation state was rapidly observed in the severely ill, and the incidence of thromboembolic events remains elevated. Interleukin inhibitors have demonstrated positive effects on the hyperactivation of the immune system in COVID-19, with the interleukin-6 inhibitor tocilizumab showing promising results in reducing mortality. Nevertheless, the impact of interleukin inhibitors on the coagulation system remains incompletely understood. MethodsIn this clinical trial conducted in Stockholm, Sweden, interleukin inhibitors, namely anakinra (ANA) or tocilizumab (TOCI), were randomly administered in addition to standard care (SC) to hospitalized patients with COVID-19. A control group received only SC. The primary outcome sought to measure effects on global hemostasis, as indicated by changes in functional coagulation tests, specifically Rotational Thromboelastometry (ROTEM) or Overall Hemostatic Potential (OHP), visualized through scanning electron microscopy images. Secondary outcomes included effects on conventional coagulation laboratory tests. ResultsThe study enrolled 74 patients who were randomized to receive either ANA or TOCI in addition to SC, or SC alone. In the TOCI group, ROTEM variables exhibited less hypercoagulation after 29 days compared with ANA or SC treatment groups, characterized by prolonged clot formation time and decreased clot firmness. OHP decreased, but there were no significant differences among the three treatment groups. Plasma fibrinogen levels, initially elevated, decreased significantly in TOCI recipients over time. ConclusionTocilizumab treatment demonstrated a significant reduction of hypercoagulation in hospitalized COVID-19 patients, by improvements in both global coagulation tests and conventional laboratory tests, in comparison with anakinra or SC alone. This finding underscores the significance of tocilizumab as a viable treatment option in severe COVID-19 cases, with the potential to decrease thrombosis incidence.

Diagnostic value of clot formation parameters determined by rotational thromboelastometry in 63 patients with congenital dysfibrinogenemia.

Rotational thromboelastometry (ROTEM) is a global hemostasis assay. The diagnosis added value of ROTEM in congenital dysfibrinogenemia remains to be established. The aim of this study was to analyze clot formation by ROTEM in a cohort of dysfibrinogenemic patients and to establish correlations with genotype, clinical features, and coagulation parameters. The study included genetically confirmed congenital dysfibrinogenemia cases (n = 63) and healthy controls ( n = 50). EXTEM, INTEM, FIBTEM tests were used to measure ROTEM parameters, that is, clotting time (CT), clot formation time (CFT), maximal clot firmness (MCF) and amplitude 10 min after CT (A10). The ISTH bleeding assessment tool was used to determine bleeding episodes. CT (INTEM) was statistically significantly shorter in congenital dysfibrinogenemia patients compared to controls while CFT (EXTEM) was prolonged. Patients's MCF in EXTEM, INTEM, and FIBTEM were similar to controls while A10 (FIBTEM) was statistically significantly lower. Fibrinogen activity was positively correlated with fibrinogen antigen, A10 and MCF in all three assays. Bleeding phenotypes were observed in 23 (36.5%) patients. Only CFT in EXTEM and CT in INTEM were statistically different in patients with bleeding phenotype versus controls. Carriers of the FGA mutation p.Arg35His had a CT (EXTEM) slightly prolonged and a reduced A10 (FIBTEM) compared to controls. Some ROTEM parameters were able to distinguish congenital dysfibrinogenemia patients from controls, and patients with a bleeding phenotype. Prolonged CFT in EXTEM were associated with congenital dysfibrinogenemia and bleeding phenotype. Bleeding episodes in most patients were generally mild and prevalence of thrombosis was very low.

Cryopreserved platelets and amotosalen-treated plasma in an experimental clot formation set-up.

Amotosalen treatment of plasma and cryopreservation of platelets affect the quality and potentially the interplay between platelets and coagulation factors. We set up an experimental clot formation study to test the hypothesis that amotosalen treatment of plasma affects the interaction with different platelet preparations. Pooled plasma units (n=16) were subjected to coagulation tests before and after pathogen inactivation with amotosalen treatment (PI) and aliquots were frozen at -80°C. Fresh and cryopreserved platelets were analyzed for phenotypic and activity markers. Finally, coagulation properties of different combinations of platelets and plasma, before and after PI, were analyzed by viscoelastography (ROTEM). PI of plasma reduced the concentration of several coagulation factors (p<0.01). Cryopreservation altered phenotypic expression and reduced the platelets' ability to respond to agonists (p<0.0001). The interplay between all plasma derivatives and cryopreserved platelets resulted in shortened coagulation time (p<0.0001) but prolonged clot formation time and reduced clot strength (p<0.0001) as compared to the interaction between fresh platelets with different plasma variants. PI of the plasma does not seem to have a major impact on coagulation time, clot formation time or clot strength. Our data show that the reduced concentration of coagulation factors after PI treatment of plasma are negligible measured by viscoelastography, with fresh and cryopreserved platelets in this experimental clot formation setup, and that platelets play a more pronounced role. Cryopreserved platelets are more activated and result in reduced clot stability.

Thromboelastometry assessment of hemostatic properties in various murine models with coagulopathy and the effect of factor VIII therapeutics

BackgroundRotational thromboelastometry (ROTEM) has been commonly used to assess the viscoelastic properties of the blood clotting process in the clinic for patients with a hemostatic or prothrombotic disorder. ObjectiveTo evaluate the capability of ROTEM in assessing hemostatic properties in whole blood from various mouse models with genetic bleeding or clotting disease and the effect of factor VIII (FVIII) therapeutics in FVIIInull mice. MethodsMice with a genetic deficiency in either a coagulation factor or a platelet glycoprotein were used in this study. The properties of platelet‐ or plasma‐FVIII were also assessed. Citrated blood from mice was recalcified and used for ROTEM analysis. ResultsWe found that blood collected from the vena cava could generate reliable results from ROTEM analysis, but not blood collected from the tail vein, retro‐orbital plexus, or submandibular vein. Age and sex did not significantly affect the hemostatic properties determined by ROTEM analysis. Clotting time (CT) and clot formation time (CFT) were significantly prolonged in FVIIInull (5‐ and 9‐fold, respectively) and FIXnull (4‐ and 5.7‐fold, respectively) mice compared to wild‐type (WT)‐C57BL/6J mice. Platelet glycoprotein (GP)IIIanull mice had significantly prolonged CFT (8.4‐fold) compared to WT‐C57BL/6J mice. CT and CFT in factor V (FV) Leiden mice were significantly shortened with an increased α‐angle compared to WT‐C57BL/6J mice. Using ROTEM analysis, we showed that FVIII expressed in platelets or infused into whole blood restored hemostasis of FVIIInull mice in a dose‐dependent manner. ConclusionROTEM is a reliable and sensitive assay for assessing therapeutics on hemostatic properties in mouse models with a bleeding or clotting disorder.

Evaluation of Global Coagulation Assays in Normal and Thrombocytopenic Populations

IntroductionQuantitative platelet count is not always a true reflection of an individual's bleeding risk with thrombotic complications previously reported in thrombocytopenic patients. A better laboratory assessment of bleeding risk in these patients is required. Global coagulation assays such as thromboelastography (TEG), thrombin generation with calibrated automated thrombogram (CAT) and fibrin generation assessment with overall haemostatic potential (OHP) assay may provide more complete assessment of an individual's coagulation profile. We aim to evaluate how global coagulation assays differ between thrombocytopenic patients and normal controls.MethodsBlood samples were collected from thrombocytopenic patients (platelet count <150x109/L). The samples were processed for baseline investigations and global coagulation assays. Citrated whole blood was analysed for thromboelastography using the TEG® 5000 analyser and additional citrated blood samples were double spun at -2500G to obtain platelet-poor plasma (PPP) and stored at -80°C within 2 hours of sample collection. The stored PPP were then used for later testing with CAT and OHP, using standard manufacturer guidelines. The results were compared to our previously collected healthy normal control population (n=90).ResultsFifty-eight samples (30 males, 28 females) with mean age of 57.5 years were collected. This included 34 patients with chemotherapy or malignancy-related thrombocytopenia and 24 participants with immune thrombocytopenia or other secondary causes of thrombocytopenia. In comparison to the normal control population (average platelet count 245 x 109/L), thrombocytopenic participants (average platelet count 79 x 109/L, p<0.001) had reduced clot lysis (LY30, 0.0% vs 0.6%; p<0.001) without significant difference in other TEG® parameters including comparable clot strength (maximum amplitude, 59.3 vs 57.8 mm; p=0.38). Thrombin generation using CAT showed reduced endogenous thrombin potential (1252.2 vs 1353.0 nM.min; p=0.040) while fibrin generation showed no difference in the OHP parameters (p>0.05).On sub-analysis, participants with marked thrombocytopenia (platelet count 0 - 50 x 109/L; n=13) had significantly more hypocoagulable TEG® parameters including prolonged clot formation time (K-time, 3.4 vs 2.3 min; p=0.002) as well as reduced α-angle (41.2° vs 57.9°; p=0.001), clot strength (47.2 vs 57.8 nM.min; p=0.002) and clot lysis (0.0% vs 0.6%; p<0.001). Interestingly, the CAT parameters generally did not meet significance, possibly due to small numbers in this subgroup, apart from prolonged lag time (4.0 vs 3.1 min; p=0.018) although there was a trend towards lower endogenous thrombin potential (1184.0 vs 1353.0; p=0.056). Conversely, participants with platelet count 101 - 150 x 109/L appeared more hypercoagulable on TEG® with shorter clot formation time (1.7 vs 2.3 min; p<0.001) and increased clot strength (66.4 vs 57.8 mm; p<0.001) compared to normal controls. CAT parameters were not significantly different in this subgroup. Fibrin generation was relatively preserved in both sub-groups as well. When comparing the 2 groups of thrombocytopenic patients (ITP vs chemotherapy/malignancy) patients, there was no difference in all the parameters of the three global coagulation assays apart from reduced clot lysis in the latter group (p=0.018)ConclusionGlobal coagulation assays, particularly thromboelastography is useful in measuring the differences in coagulation in thrombocytopenic patients. Marked thrombocytopenia (<50 x 109/L) showed hypocoagulable parameters, which validate current clinical practice of managing this population as high bleeding risk. In contrast, thrombocytopenic patients with platelet counts between 101 - 150 x 109/L were found to be hypercoagulable, providing a possible explanation of overcompensation, which may lead to some thrombocytopenic patients experiencing thrombotic events. Clot lysis was consistently markedly reduced in thrombocytopenic population, suggesting again, possible compensatory mechanisms to reduce bleeding risk. CAT and OHP assay using platelet-poor plasma, on the other hand, does not appear to be sensitive in differentiating the thrombocytopenic and normal healthy populations. Larger prospective studies will be undertaken to confirm these findings. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mesenchymal stromal cell delivery via an ex vivo bioreactor preclinical test system attenuates clot formation for intravascular application

While mesenchymal stromal cells are an appealing therapeutic option for a range of clinical applications, their potential to induce clotting when used systemically remains a safety concern, particularly in hypercoagulable conditions, such as in patients with severe COVID‐19, trauma, or cancers. Here, we tested a novel preclinical approach aimed at improving the safety of mesenchymal stromal cell (MSC) systemic administration by use of a bioreactor. In this system, MSCs are seeded on the exterior of a hollow‐fiber filter, sequestering them behind a hemocompatible semipermeable membrane with defined pore‐size and permeability to allow for a molecularly defined cross talk between the therapeutic cells and the whole blood environment, including blood cells and signaling molecules. The potential for these bioreactor MSCs to induce clots in coagulable plasma was compared against directly injected “free” MSCs, a model of systemic administration. Our results showed that restricting MSCs exposure to plasma via a bioreactor extends the time necessary for clot formation to occur when compared with “free” MSCs. Measurement of cell surface data indicates the presence of known clot inducing factors, namely tissue factor and phosphatidylserine. Results also showed that recovering cells and flushing the bioreactor prior to use further prolonged clot formation time. Furthermore, application of this technology in two in vivo models did not require additional heparin in fully anticoagulated experimental animals to maintain target activated clotting time levels relative to heparin anticoagulated controls. Taken together the clinical use of bioreactor housed MSCs could offer a novel method to control systemic MSC exposure and prolong clot formation time.

Clinical and pathophysiologic aspects of ECMO-associated hemorrhagic complications.

Extracorporeal membrane oxygenation (ECMO) is increasingly used to treat severe cases of acute respiratory or cardiac failure. Hemorrhagic complications represent one of the most common complications during ECMO, and can be life threatening. The purpose of this study was to elucidate pathophysiological mechanisms of ECMO-associated hemorrhagic complications and their impact on standard and viscoelastic coagulation tests. The study cohort included 27 patients treated with VV-ECMO or VA-ECMO. Hemostasis was evaluated using standard coagulation tests and viscoelastic parameters investigated with rotational thromboelastometry. Anticoagulation and hemorrhagic complications were analyzed for up to seven days depending on ECMO duration. Hemorrhagic complications developed in 16 (59%) patients. There were 102 discrete hemorrhagic episodes among 116 24-hour-intervals, of which 27% were considered to be clinically significant. The highest number of ECMO-associated hemorrhages occurred on the 2nd and 3rd day of treatment. Respiratory tract bleeding was the most common hemorrhagic complication, occurring in 62% of the 24-hour intervals. All 24-hours-intervals were divided into two groups: “with bleeding” and “without bleeding”. The probability of hemorrhage was significantly associated with abnormalities of four parameters: increased international normalized ratio (INR, sensitivity 71%, specificity 94%), increased prothrombin time (PT, sensitivity 90%, specificity 72%), decreased intrinsic pathway maximal clot firmness (MCFin, sensitivity 76%, specificity 89%), and increased extrinsic pathway clot formation time (CFTex, sensitivity 77%, specificity 87%). In conclusions, early ECMO-associated hemorrhagic complications are related to one traditional and two novel viscoelastic coagulation abnormalities: PT/INR elevation, reduced maximum clot firmness due to intrinsic pathway dysfunction (MCFin), and prolonged clot formation time due to extrinsic pathway dysfunction (CFTex). When managing hemostasis during ECMO, derangements in PT/INR, MCFin and CFTex should be focused on.

Post-TBI splenectomy may exacerbate coagulopathy and platelet activation in a murine model

IntroductionTraumatic brain injury (TBI) induces acute hypocoagulability, subacute hypercoagulability, and persistently elevated risk for thromboembolic events. Splenectomy is associated with increased mortality in patients with moderate or severe TBI. We hypothesized that the adverse effects of splenectomy in TBI patients may be secondary to the exacerbation of pathologic coagulation and platelet activation changes. MethodsAn established murine weight-drop TBI model was utilized and a splenectomy was performed immediately following TBI. Sham as well as TBI and splenectomy alone mice were used as injury controls. Mice were sacrificed for blood draws at 1, 6, and 24 h, as well as 7 days post-TBI. Viscoelastic coagulation parameters were assessed by rotational thromboelastometry (ROTEM) and platelet activation was measured by expression of P-selectin via flow cytometry analysis of platelet rich plasma samples. ResultsAt 6 h following injury, TBI/splenectomy demonstrated hypocoagulability with prolonged clot formation time (CFT) compared to TBI alone. By 24 h following injury, TBI/splenectomy and splenectomy mice were hypercoagulable with a shorter CFT, a higher alpha angle, and increased MCF, despite a lower percentage of platelet contribution to clot compared to TBI alone. However, only the TBI/splenectomy continued to display this hypercoagulable state at 7 days. While TBI/splenectomy had greater P-selectin expression at 1-h post-injury, TBI alone had significantly greater P-selectin expression at 24 h post-injury compared to TBI/splenectomy. Interestingly, P-selectin expression remained elevated only in TBI/splenectomy at 7 days post-injury. ConclusionSplenectomy following TBI exacerbates changes in the post-injury coagulation state. The combination of TBI and splenectomy induces an acute hypocoagulable state that could contribute to an increase in intracranial bleeding. Subacutely, the addition of splenectomy to TBI exacerbates post-injury hypercoagulability and induces persistent platelet activation. These polytrauma effects on coagulation may contribute to the increased mortality observed in patients with combined brain and splenic injuries.

Effect of Albumin in Combination With Mannitol on Whole-blood Coagulation In Vitro Assessed by Thromboelastometry

Albumin and mannitol may interfere with hemostasis, but their coinfluence is unclear. We aimed to determine the effects of albumin alone and in combination with mannitol or Ringer acetate (RAC) on hemostasis in crossover in vitro study. From citrated fresh whole blood withdrawn from 10 volunteers, we prepared 2.5, 5, 10, 15, and 20 vol% dilutions of 4% albumin (Alb group). Each sample was thereafter diluted by 15% mannitol (Alb/Man group) or RAC (Alb/RAC group) at a ratio of 9:1. Using thromboelastometry, FibTEM (fibrinogen ROTEM) and ExTEM (extrinsic ROTEM) tests were performed. A 20 vol%, but not 2.5 to 15 vol% dilution of albumin caused a prolonged clot formation time, α-angle decrease, and maximum clot firmness (MCF) weakening compared with undiluted sample (P<0.05). Clot formation time prolonged more in Alb5/Man than in Alb5 and Alb5/RAC dilution (P<0.05). In Alb2.5/Man, Alb10/Man, and Alb15/Man, dilution α-angle was lower than in corresponding Alb/RAC and Alb-group dilutions (P<0.05). In ExTEM, MCF decreased similarly in every dilution of Alb/Man and Alb/RAC compared with Alb group (P<0.05). In FibTEM, MCF decreased more in Alb10/Man than in Alb10/RAC dilution (P<0.05). In up to 15 vol% dilutions, albumin alone did not impair hemostasis in vitro, but in combination with mannitol or RAC coagulation was disturbed similarly at most concentrations. There was some significant additional effect with mannitol at certain concentrations. Our results indicate that coadministration of mannitol and albumin needs further study in vivo.

The effects of protamine sulfate on clot formation time and clot strength thromboelastography variables for canine blood samples

To determine the effects of protamine sulfate on clot formation time and clot strength thromboelastography variables for canine whole blood samples. Blood samples obtained from 11 healthy dogs. Blood samples were collected from jugular veins of dogs into syringes with 3.2% sodium citrate (blood to citrate ratio, 9:1). Blood samples were divided into aliquots, and protamine sulfate was added to various concentrations (0 [control], 22, 44, and 66 μg/mL). Prepared samples were activated with kaolin (n = 8) or not activated (8), CaCl₂ was added, and thromboelastography was performed. Reaction time (R), clot formation time (K), rate of clot formation (α angle), and maximum amplitude (MA) were measured. For kaolin-activated and nonactivated blood samples, protamine (66 μg/mL) significantly increased R and K and decreased α angle and MA, compared with values for control samples. Also, protamine (44 μg/mL) decreased MA in nonactivated blood samples and increased K and decreased α angle in kaolin-activated samples, compared with values for control samples. Results indicated protamine prolonged clot formation time and decreased overall clot strength in a dose-dependent manner; such effects may contribute to a hypocoagulable state in dogs. Kaolin-activated and nonactivated blood samples were appropriate for measurement of the effects of protamine on coagulation. Administration of protamine to reverse the effects of heparin should be performed with caution.

Antithrombotic properties of rafigrelide: a phase 1, open-label, non-randomised, single-sequence, crossover study

Platelets play a central role in atherothrombotic events. We investigated the effect of a novel platelet-lowering agent, rafigrelide, on thrombus formation and characteristics. In this phase 1, open-label, non-randomised, single-sequence, crossover study, healthy male volunteers received rafigrelide for 14 days (Period 1). Following a ≥6-week washout period, they then received rafigrelide + acetylsalicylic acid (ASA) for 14 days (Period 2). Thrombus formation was assessed ex vivo using the Badimon perfusion chamber, and thrombus characteristics were assessed using thromboelastography. A total of 15 volunteers were enrolled in the study and were assigned to Panel A or Panel B, which had different schedules of assessments. In Panel A, after treatment with rafigrelide alone (Period 1), mean (± standard deviation) platelet count was reduced from 283(±17)× 10⁹/l at Day 1, to 125(±47)× 10⁹/l at Day14 (n=6) and thrombus area reduced under high and low shear conditions. Reductions in thrombus area under high shear conditions correlated with reductions in platelet count (r²=0.11, p=0.022; n=12). Rafigrelide treatment prolonged clot formation time and reduced clot strength. The addition of ASA to rafigrelide (Period 2) had no additional effect on platelet count or thrombus area under high or low shear conditions. Similar results were seen in Panel B for all parameters. The most common adverse events (≥3 participants per period) were thrombocytopenia and headache. While confirming the platelet-lowering effects of rafigrelide, this early phase study also indicates that rafigrelide has antithrombotic properties under both high and low shear conditions.

Rotational thromboelastometry for diagnosing sudden hyperfibrinolysis immediately after cardiopulmonary bypass during cardiac surgery

Rotational thromboelastometry for diagnosing sudden hyperfibrinolysis immediately after cardiopulmonary bypass during cardiac surgery

Impact of cell salvage during cardiac surgery on the thrombelastomeric coagulation profile: a pilot study

Intraoperative cell salvage of the cardiopulmonary bypass residual volume can dilute platelets and coagulation factors. This is a report of a randomised control trial of 20 patients undergoing coronary bypass surgery. Residual cardiopulmonary bypass volume was processed and transfused after surgery in the cell salvage group and the residual volume was transfused unprocessed in the control group. The coagulation profile was measured using the Rotem(®) thrombelastometry system. Mean (SD) maximum clot firmness after surgery was 52.8 (5.4) mm in the cell salvage group compared to 57.2 (5.0) mm in the control group (p=0.04). Clot formation time was prolonged after surgery by 39 (27) s in the cell saver group compared to 19 (17) s in the control group (p=0.045). Platelet count was reduced after surgery by 96 (32) x 10(9).L(-1) in the cell saver group and 70 (19) x 10(9).L(-1) in the control group (p=0.03). Blood volume in the chest drains 4 hours after surgery was similar in both groups. There was a strong association between clot formation time after surgery and blood loss (R = 0.68, p=0.001). The increase in blood loss was 4.1 ml for every one-second increase in clot formation time (95% CI 1.9 - 6.4, p=0.001). Cell salvage of the residual cardiopulmonary bypass volume reduced platelet numbers and prolonged clot formation time and maximum clot firmness was less in this group.

Thromboelastometry to Guide Recombinant Activated Factor VII Therapy for Postoperative Refractory Intracranial Bleeding

Thromboelastometry to Guide Recombinant Activated Factor VII Therapy for Postoperative Refractory Intracranial Bleeding

Hyperfibrinolysis After Major Trauma: Differential Diagnosis of Lysis Patterns and Prognostic Value of Thrombelastometry

The aim of this study was to diagnose hyperfibrinolysis (HF) and its pattern using thrombelastometry and to correlate the diagnosis with mortality. Furthermore, routine laboratory based and the rotational thrombelastometry analyzer (ROTEM)-derived variables were also correlated with survival. Severe trauma patients showing HF in ROTEM were consecutively enrolled in the study. Three different HF patterns were compared: fulminant breakdown within 30 minutes, intermediate HF of 30 to 60 minutes, and late HF after 60 minutes. Injury severity score (ISS), hemodynamics, hemoglobin, hematocrit, platelet count (PC), fibrinogen, and ROTEM variables at admission were analyzed. The observed mortality was compared with the predicted trauma and injury severity score mortality. Thirty-three patients were diagnosed with HF. The mean ISS was 47 +/- 14. Fulminant, intermediate, or late HF (n = 11 each group) resulted in 100%, 91%, or 73% mortality, respectively, with the best prognosis for late HF (p = 0.0031). The actual overall mortality of HF (88%) exceeded the predicted trauma and injury severity score mortality (70%) (p = 0.039). Lower PC (123 +/- 53 vs. 193 +/- 91; p = 0.034), ROTEM prolonged clot formation time [CFT, 359 (140/632) vs. 82 (14/190); p = 0.042], and lower platelet contribution to maximum clot firmness [MCF(EXTEM) - MCF(FIBTEM), 34 (20/40) vs. 46 (40/53); p = 0.026] were associated with increased mortality. ROTEM-based diagnosis of HF predicted outcome. Further independent predictors of death were combination of HF with hemorrhagic shock, low PC, and prolonged CFT in ROTEM. ROTEM-based point of care testing in the emergency room is thus able to identify prognostic factors such as prolonged CFT and low platelet contribution to clot firmness (MCF(EX) - MCF(FIB)) earlier than standard laboratory-based monitoring.

Venous air embolism induces both platelet dysfunction and thrombocytopenia

In vitro, air bubbles can induce platelet activation and platelet to air bubble binding. We therefore tested in vivo the hypothesis that venous air embolism (VAE) induces (1) platelet dysfunction and (2) thrombocytopenia. Adult swine (60.8+/-3.9 kg; n=8) were anaesthetized, mechanically ventilated, and placed in a semi-upright position. Air boli (0.5-80 ml) were injected randomly via an ear vein, and arterial blood was sampled after cumulative air dosages of 0, 80, 160, and 240 ml. Coagulation was assessed by impedance aggregometry, rotational thrombelastometry, whole blood count, plasmatic coagulation variables, and fibrinogen, d-dimer, protein C, and antithrombin plasma concentrations, respectively. VAE induced a 47% decrease in platelet count (303 vs. 160 nl(-1); P<0.001) over the dose range assessed, with haematocrit being unaltered. Furthermore, VAE-impaired platelet aggregation induced by adenosine diphosphate, arachidonic acid, collagen, and the thromboxan analogue U46619 over the dose range assessed independent of thrombocytopenia. (P<0.05 vs. baseline). In contrast, rotational thrombelastometry alone was quite insensitive in detecting VAE-induced coagulation changes, showing only at near lethal air dosages a prolonged clot formation time following activation with tissue factor, contact activator, and during spontaneous coagulation (P<0.05 vs. baseline). VAE induces both a dose-dependent decrease in platelet count and a marked decrease in platelet aggregation, independent of thrombocytopenia (P<0.05 vs. baseline).

Thromboelastometry in Patients Receiving Platelet Transfusion after Chemotherapy.

Prophylactic platelet transfusion is frequently administered to reduce the risk of hemorrhage in patients undergoing chemotherapy for leukemia or cancer. A platelet count of 10 or 20x109 L−1 is often used as an occasion for platelet transfusion. In clinical studies, the bleeding risk was similar using either threshold (Rebulla, NEJM 1997; 337:1870, Wand, Blood 1998; 91:3601). Moreover, serious bleeding events were not related to the patients' platelet count. Thromboelastometry with ROTEM® Whole-Blood Coagulation Analyzer (Pentapharm, Munich, Germany) is a method that may provide a better estimate of the risk of hemorrhage because it also depends on platelet function and plasma coagulation. We therefore performed a pilot study in 13 adult patients receiving prophylactic platelet transfusion after chemotherapy for leukemia or lymphoma. ROTEM® was performed using an ellagic acid based activating reagent (in-TEM®, Pentapharm). Clotting profiles were evaluated using the following thromboelastometric measures: clotting time (CT), clot formation time (CFT), and maximum clot firmness (MCF). The median platelet count before transfusion was 9x109 L−1 (range 1 to 20). CT was within the normal range in all patients (median 168 s, range 125–212, reference range 100–240). In contrast, a prolonged CFT (median 139 s, range 51–4526, reference range 30–110) and reduced MCF (median 34 mm, range 19–54, reference range 50–72) was recorded. Correlation between platelet count and CFT (R=−0.41, Spearman) or MCF (R=+0.48) was weak and not statistically significant. 15 min after platelet transfusion, there was an increase in platelet count (median 39x109 L−1, range 23–48) and a substantial improvement of CFT (median 99 s, range 44–332) and MCF (median 49 mm, range 40–64). Changes in thromboelastometric measures were due to platelet transfusion as the addition of cytochalasin D (fib-TEM®, Pentapharm) resulted in identical profiles before and after transfusion. Changes in platelet count correlated with changes in MCF (R=+0.73, P<0.01), but not CFT (R=−0.40, P=0.17). Comparing patients who had a maximum of one mild bleeding symptom (n=9) with patients who experienced one severe or at least two mild bleeding symptoms (n=4), there was no difference in platelet counts (median 10 vs. 9x109 L−1, p=0.77). In contrast, there was a trend towards a shorter CFT (median 111 vs. 388 s, P=0.09) and higher MCF (median 39 vs. 29.5 mm, P=0.09) in the group of non-bleeding patients. In summary, ROTEM® seems to be a sensitive method to detect hemostatic changes in patients with severe thrombocytopenia receiving platelet transfusion and to identify thrombocytopenic patients with an increased bleeding risk. Therefore, further studies to evaluate ROTEM® as a means to estimate the bleeding risk in thrombocytopenic patients are warranted.

Gerinnungsbeurteilung in unkomplizierten und hypertensiven Schwangerschaften mittels der Thrombelastometrie - Einsatz des ROTEM® in der Geburtshilfe

Purpose of the Study: Thrombelastography provides an overall assessment of hemostatic function through the analysis of the viscoelastic properties of whole blood and is useful in managing perioperative coagulopathies. Our study investigated the use of an enhanced method, thrombelastometry with specific coagulation activators (ROTEM®), in obstetrics. We looked for differences between non-pregnant and pregnant women and coagulation changes in hypertensive pregnancies. Patients and Methods: Citrate plasma was collected from 18 healthy non-pregnant pre-menopausal women, 50 women in the 3rd trimester of uncomplicated pregnancies, 14 women with preeclampsia and 8 women suffering from HELLP syndrome. Thrombelastometry was performed with ROTEM® (Pentapharm GmbH, Munich, Germany), using specific activators to evaluate the extrinsic and intrinsic systems. Coagulation time (CT), clot formation time (CFT) and maximal clot firmness (MCF) were determined. Results: Pregnant women showed a significant decrease of CT and CFT in the extrinsic system and an increase of MCF in both systems compared to non-pregnant women. In the preeclamptic group, a tendency toward further coagulation activation with decreased levels of CT and CFT could be observed. Patients with HELLP syndrome showed a significantly prolonged CFT (127s and 102 s) and a decreased MCF (55 mm and 57 mm) in both the extrinsic and the intrinsic system compared with healthy (CFT: 74 s and 60 s; MCF: 66 mm and 68 mm) and preeclamptic (CFT: 65 s and 56 s; MCF: 66 mm and 69 mm) pregnant women. There was a highly significant correlation between CFT and MCF, respectively, and the platelet count. Conclusion: ROTEM® provides a quick, reproducible assessment of hemostatic functions and can be used as a bedside test. We confirmed hypercoagulability during pregnancy. Our results suggest that ROTEM® may be a tool to monitor coagulation changes during hypertensive pregnancies and to detect changes of coagulation activation including the phase of factor consumption with risk of hemorrhagia. Furthermore, in cases of thrombopenia it will be possible to determine the actual state of coagulation before a necessary caesarean section.