Routine Coagulation Analyses Research Articles

Von Willebrand factor and the thrombophilia of severe COVID-19: in situ evidence from autopsies

BackgroundCOVID-19 is accompanied by a hypercoagulable state and characterized by microvascular and macrovascular thrombotic complications. In plasma samples from patients with COVID-19, von Willebrand factor (VWF) levels are highly elevated and predictive of adverse outcomes, especially mortality. Yet, VWF is usually not included in routine coagulation analyses, and histologic evidence of its involvement in thrombus formation is lacking. ObjectivesTo determine whether VWF, an acute-phase protein, is a bystander, ie, a biomarker of endothelial dysfunction, or a causal factor in the pathogenesis of COVID-19. MethodsWe compared autopsy samples from 28 patients with lethal COVID-19 to those from matched controls and systematically assessed for VWF and platelets by immunohistochemistry. The control group comprised 24 lungs, 23 lymph nodes, and 9 hearts and did not differ significantly from the COVID-19 group in age, sex, body mass index (BMI), blood group, or anticoagulant use. ResultsIn lungs, assessed for platelets by immunohistochemistry for CD42b, microthrombi were more frequent in patients with COVID-19 (10/28 [36%] vs 2/24 [8%]; P = .02). A completely normal pattern of VWF was rare in both groups. Accentuated endothelial staining was found in controls, while VWF-rich thrombi were only found in patients with COVID-19 (11/28 [39%] vs 0/24 [0%], respectively; P < .01), as were NETosis thrombi enriched with VWF (7/28 [25%] vs 0/24 [0%], respectively; P < .01). Forty-six percent of the patients with COVID-19 had VWF-rich thrombi, NETosis thrombi, or both. Trends were also seen in pulmonary draining lymph nodes (7/20 [35%] vs 4/24 [17%]; P = .147), where the overall presence of VWF was very high. ConclusionWe provide in situ evidence of VWF-rich thrombi, likely attributable to COVID-19, and suggest that VWF may be a therapeutic target in severe COVID-19.

Comparison of the Two Coagulation Techniques: Thrombolyzer XRM and Diagon Coag XL.

The aim of this study was to evaluate the performance characteristics of the Thrombolyzer XRM against the Diagon Coag XL during the exchange of the coagulation analyzer. The Partial Thromboplastin Time (PTT) and prothrombin time (PT) measurements were performed by using the Thrombolyzer XRM and Diagon CoagXL analyzers. The precision, accuracy, method comparison, and reference range verification studies were performed on the Thrombolyzer XRM based on Clinical Laboratory Standards Institute guidelines. The precision study was performed with normal and pathologic controls for the PT and PTT tests, all co-efficients of variation (%) were found ≤ 2.5%. In the accuracy study, both analyzers displayed less than 6.3% and 10.8% bias for PT and PTT tests, respectively. The method comparison study demonstrated good agreement for the Bland-Altman plots for PTT and PT with a bias of -2.0 and -3.3 between each analyzer, respectively. The Passing and Bablok analysis showed no significant differences for PTT and PT between each analyzer (p = 0.65 and p = 0.33, respectively). However, there was a proportional bias for PT with a slope of 1.40 (95% CI 1.2 - 1.8). The manufacturer ranges were acceptable as a result of the reference range verification study. The routine coagulation analysis can be performed on the Thrombolyzer XRM with satisfactory precision and the obtained minor differences can be eliminated in future standardization studies.

Impaired secretion of platelet granules in patients with Duchenne muscular dystrophy – results of a prospective diagnostic study

A tendency to bleed during scoliosis surgery has been reported repeatedly in Duchenne muscular dystrophy (DMD) and diagnostic studies show a prolonged bleeding time. The pathophysiological background is still not fully understood. The short dystrophin isoform dp71 is expressed in platelets and mediates contractile properties. We performed a bicentric, non-blinded, prospective diagnostic study in 53 patients with confirmed DMD. Extensive laboratory analyses included platelet aggregometry and platelet flow cytometry, as well as routine coagulation analyses. Results of laboratory diagnostics were correlated with clinical data. Patients were subgrouped and analyzed according to ambulatory status and cardiac involvement. Platelet aggregation was reduced after stimulation with ADP (adenosine triphosphate) [60%; reference range 66–84%]. In addition, in the DMD cohort the expression of platelet activation markers CD62 and CD63 (flow cytometry analyses) was significantly lower than in healthy controls, most prominent in non-ambulatory patients with cardiac involvement. There was no clear association with the location of the underlying mutations in the dystrophin gene. No further abnormalities were identified regarding primary or secondary hemostasis. This study shows that platelets of patients with DMD have decreased expression of CD62 and CD63 which are markers for platelet granule release. This may indicate that patients with DMD have an impaired platelet granule secretion which may explain to some extent the increased bleeding, especially in mucocutaneous areas and perioperatively.

Validation of a New Small-Volume Sodium Citrate Collection Tube for Coagulation Testing in Critically Ill Patients with Coagulopathy.

Blood loss due to phlebotomy leads to hospital-acquired anemia and more frequent blood transfusions that may be associated with increased risk of morbidity and mortality in critically ill patients. Multiple blood conservation strategies have been proposed in the context of patient blood management to minimize blood loss. Here, we evaluated a new small-volume sodium citrate collection tube for coagulation testing in critically ill patients. In 46 critically adult ill patients admitted to an interdisciplinary intensive care unit, we prospectively compared small-volume (1.8 mL) sodium citrate tubes with the conventional (3 mL) sodium citrate tubes. The main inclusion criterium was a proven coagulopathy (Quick < 60% and/or aPTT > 40 second) due to anticoagulation therapy or perioperative coagulopathy. In total, 92 coagulation analyses were obtained. Linear correlation analysis detected a positive relationship for 7 coagulation parameters (Prothrombin Time, r = 0.987; INR, r = 0.985; activated Partial Thromboplastin Time, r = 0.967; Thrombin Clotting Time, r = 0.969; Fibrinogen, r = 0.986; Antithrombin, r = 0.988; DDimer, r = 0.969). Bland-Altman analyses revealed an absolute mean of differences of almost zero. Ninety-five percent of data were within two standard deviations of the mean difference suggesting interchangeability. As systematic deviations between measured parameters of the two tubes were very unlikely, test results of small-volume (1.8 mL) sodium citrate tubes were equal to conventional (3 mL) sodium citrate tubes and can be considered interchangeable. Small-volume sodium citrate tubes reduced unnecessary diagnostic-related blood loss by about 40% and, therefore, should be the new standard of care for routine coagulation analysis in critically ill patients.

Autovalidation and automation of the postanalytical phase of routine hematology and coagulation analyses in a university hospital laboratory.

The need to satisfy high-throughput demands for laboratory tests continues to be a challenge. Therefore, we aimed to automate postanalytical phase in hematology and coagulation laboratory by autovalidation of complete blood count (CBC) and routine coagulation test results (prothrombin time [PT], international normalized ratio [PT-INR], activated partial thromboplastin time [APTT], fibrinogen, antithrombin activity [AT] and thrombin time [TT]). Work efficacy and turnaround time (TAT) before and after implementation of automated solutions will be compared. Ordering panels tailored to specific patient populations were implemented. Rerun and reflex testing rules were set in the respective analyzers' software (Coulter DxH Connectivity 1601, Beckman Coulter, FL, USA; AutoAssistant, Siemens Healthcare Diagnostics, Germany), and sample status information was transferred into the laboratory information system. To evaluate if the automation improved TAT and efficacy, data from manually verified results in September and October of 2015 were compared with the corresponding period in 2016 when autovalidation was implemented. Autovalidation rates of 63% for CBC and 65% for routine coagulation test results were achieved. At the TAT of 120 min, the percentage of reported results increased substantially for all analyzed tests, being above 90% for CBC, PT, PT-INR and fibrinogen and 89% for APTT. This output was achieved with three laboratory technicians less compared with the period when the postanalytical phase was not automated. Automation allowed optimized laboratory workflow for specific patient populations, thereby ensuring standardized results reporting. Autovalidation of test results proved to be an efficient tool for improvement of laboratory work efficacy and TAT.

Correlation of complement depletion and the severity of sepsis in children

Objective To analyze the relationship between complement key component C3, C4 and the severity of sepsis in children, in order to explore the role of complement activation in the progression of sepsis and provide a reference for diagnosis and treatment of severe sepsis. Methods Four hundred and twenty-four children diagnosed as sepsis from December 2012 to December 2015 in Children′s Hospital of Nanjing Medical University were enrolled in this study, among whom 347 children with sepsis were eligible for the following research including 169 cases of common sepsis and 178 cases of severe sepsis. Blood specimens were collected in 24 hours after their admission into pediatric intensive care unit(PICU) for the analysis of lymphocyte subsets, humoral immunity, blood routine analysis, coagulation, liver and renal function analysis. General information was collected by consulting their medical records, laboratory analysis and clinical treatment. The relationship between complement C3, C4 and the severity of sepsis was analyzed, and the correlation between C3 and coagulation, liver, renal, myocardium damage was also studied. Logistic regression was used to analyze the relationship between C3 and the progression to severe sepsis and multiple organ dysfunction syndrome(MODS), while Cox regression was used for survival analysis. Results Natural killer(NK) cell percentage was lower in severe sepsis group than that in common sepsis group[6.6%(3.7%, 10.7%) vs. 8.5%(4.7%, 13.3%), Z=2.635, P=0.008], while C3 decreased in severe sepsis group compared with common sepsis group [0.653(0.462, 0.985) g/L vs. 0.991(0.678, 1.265) g/L, Z=5.684, P<0.001], and C4 decreased in severe sepsis group compared with common sepsis group [0.160(0.102, 0.244) g/L vs. 0.190(0.121, 0.265) g/L, Z=2.513, P=0.012]. The proportion of severe pneumonia was higher in severe sepsis group than that in co-mmon sepsis group (34.3% vs. 19.5%, χ2=9.540, P=0.002), and liver function damage proportion was increased in severe sepsis group than that in common sepsis group (48.3% vs. 16.0%, χ2=41.28, P<0.001), and the duration of PICU treatment was longer in severe sepsis group than that in common sepsis group[10.7(6.5, 17.4) d vs. 7.5(4.0, 12.4) d, Z=-4.039, P<0.001]. C3 was significantly decreased in children with single organ dysfunction, multiple organ dysfunction and death group compared with common sepsis group (K=33.04, P=0.001), and the median of each group decreased with the severity of sepsis, but C4 had no difference among 4 groups(K=7.36, P=0.061). C3 was positively correlated with coagulation marker platelet (ρ=0.31, P<0.001) and fibrinogen (ρ=0.53, P<0.001), but negatively correlated with international normalized ratio (INR) (ρ=-0.39, P<0.001) and activated partial thromboplastin time (ρ=-0.34, P<0.001). C3 was also negatively correlated with liver damage marker alanine transaminase (ρ=-0.30, P<0.001) and total bilirubin (ρ=-0.28, P<0.001), and had a negative correlation with renal function marker creatinine (ρ=-0.24, P<0.001) and myocardial damage marker creatine kinase-MB (ρ=-0.27, P<0.001). The depletion of C3 was a risk factor of severe sepsis(OR=3.45, P<0.001) and MODS(OR=3.03, P=0.005) after being adjusted for confounding factors by using Logistic regression. In stratification analysis, C3 depletion was still a risk factor of severe sepsis(OR=2.78, P=0.019) and MODS(OR=3.57, P=0.015) among children less than 1 year old, and was also a risk factor of severe sepsis(OR=4.76, P=0.008) among children more than 1 year old as well. In children without liver function damage, C3 depletion was still a risk factor of severe sepsis(OR=4.17, P=0.002) and MODS(OR=9.09, P=0.002). Cox regression showed that C3 depletion was a hazard in 28-day mortality(HR=3.57, P=0.026) in children with sepsis. Conclusion The decrease of C3 is correlated with coagulation dysfunction and organ damage marker, while C3 depletion was a risk factor of severe sepsis, MODS and 28-day mortality, and could be a potential prognostic marker of children with sepsis. Key words: Sepsis; Severe sepsis; Complement C3; Complement C4

Coagulative safety of epidural catheters after major upper gastrointestinal surgery: advanced and routine coagulation analysis in 38 patients.

BackgroundThe risk of spinal haematoma in patients receiving epidural catheters is estimated using routine coagulation tests, but guidelines are inconsistent in their recommendations on what to do when results indicate slight hypocoagulation. Postoperative patients are prone to thrombosis, and thromboelastometry has previously shown hypercoagulation in this setting. We aimed to better understand perioperative haemostasis by comparing results from routine and advanced tests, hypothesizing that patients undergoing major upper gastrointestinal surgery would be deficient in vitamin K-dependent coagulation factors because of malnutrition, or hypocoagulative because of accumulation of low molecular weight heparin (LMWH).MethodsThirty-eight patients receiving epidural analgesia for major upper gastrointestinal surgery were included. We took blood at the time of preoperative epidural catheterization and at catheter withdrawal. Prothrombin time-international normalized ratio (PT-INR), activated partial thromboplastin time (aPTT) and platelet count (Plc) were analysed, and also albumin, proteins induced by vitamin K absence (PIVKA-II), rotational thromboelastometry (ROTEM®), multiple electrode aggregometry (Multiplate®) and activities of factors II, VII, IX, X, XI, XII and XIII.ResultsPostoperative coagulation was characterized by thrombocytosis and hyperfibrinogenaemia. Mean PT-INR increased significantly from 1.0 ± 0.1 to 1.2 ± 0.2 and mean aPTT increased significantly from 27 ± 3 to 30 ± 4 s. Activity of vitamin K-dependent factors did not decrease significantly: FIX and FX activity increased. FXII and FXIII decreased significantly. Mean Plc increased from 213 ± 153 × 106/L while all mean ROTEM-MCFs (maximal clot firmnesses) especially FIBTEM-MCF increased significantly to above the reference interval. All mean ROTEM® clotting times were within their reference intervals both before and after surgery. ROTEM® (HEPTEM minus INTEM) results were spread around 0. There were significant correlations between routine tests and the expected coagulation factors, but not any of the viscoelastic parameters or PIVKA-II. Multiplate® area under curve and EXTEM-MCF correlated significantly to Plc as did EXTEM-MCF to fibrinogen, FIX, FX and FXIII; and FIBTEM-MCF to Plc, FII, FXI and FXIII.ConclusionsThe increase in PT-INR may be caused by decreased postoperative FVII while the elevated aPTT may be caused by low FXII. The mild postoperative hypocoagulation indicated by routine tests is not consistent with thromboelastometry. The relevance of ROTEM® and Multiplate® in the context of moderately increased routine tests remains unclear.Trial registration number is not applicable since this is not a clinical trial.Electronic supplementary materialThe online version of this article (doi:10.1186/s13741-016-0053-0) contains supplementary material, which is available to authorized users.

Dabigatran for thromboprophylaxis after robotic assisted laparoscopic prostatectomy: Retrospective analysis of safety profile and effect on blood coagulation

Objective. The aims of this study were to analyse the efficacy and safety of orally administered dabigatran in prostate cancer patients who have undergone robotic assisted laparoscopic prostatectomy (RALP) and to analyse the effects of RALP on selected markers of coagulation activity. Material and methods. Data were collected prospectively on the first 400 patients who underwent RALP at Helsinki University Central Hospital between 2009 and 2011. As thromboprophylaxis, intermittent compression devices were used in association with anaesthesia. Dalteparin sodium at 2500 IU was administered on the preoperative evening and at 5000 IU daily until discharge. Then patients were prescribed 220 mg dabigatran etexilate once daily for the next 15 days. Bleeding and thromboembolic complications were recorded. A set of routine coagulation analyses was undertaken in 61 patients preoperatively, on the first, second and eight postoperative days. Results. One patient with obesity- and cancer-related risk factors developed venous thromboembolism 18 days after the operation. Nine patients (2.3%) had postoperative blood loss or bleeds, eight patients required blood transfusions and three underwent reoperation before dabigatran administration. Increased fibrinogen, factor VIII, d-dimer and thrombocytosis were observed postoperatively, reflecting coagulation activity. Conclusions. RALP activates coagulation, and thromboprophylaxis for high-risk patients even after minimally invasive surgery may be beneficial. Orally administered dabigatran after discharge from hospital appears safe for venous thromboembolism prophylaxis after RALP.

Reference ranges for thromboelastography (TEG®) and traditional coagulation tests in term parturients undergoing caesarean section under spinal anaesthesia*

There has been little published work defining 'normal' thromboelastography (TEG(®) ) values in healthy parturients, and few large studies defining reference ranges for traditional coagulation tests in this patient group. Our aim was to establish peri-operative reference ranges for TEG and for standard laboratory coagulation tests in our pregnant population. Fifty healthy term parturients presenting for elective caesarean section under spinal anaesthesia had blood samples taken pre-operatively, on arrival in the recovery room and, in a subset of 33 women, 4 h after routine thromboprophylaxis with enoxaparin 40 mg. All three samples had TEG analysis, the first and second having standard laboratory coagulation tests in addition. Reference ranges for our pregnant population were established, demonstrating a hypercoagulable state in term parturients and a significant effect of enoxaparin. The standard coagulation reference ranges were within 98% of the local non-pregnant ranges. These reference ranges provide a useful comparator for peri-operative TEG and routine coagulation analysis in term parturients.

Effect of Pneumatic Tube Delivery System Rate and Distance on Hemolysis of Blood Specimens

We evaluated the effects of pneumatic tube system (PTS) transport rates and distances on routine hematology and coagulation analysis. PTS effects on centrifuged blood samples were also examined. The study was completed at Dicle University Hospital, which has the longest pneumatic tube system in Turkey. Blood samples were collected at three different locations within the hospital and an emergency department, and delivered to the central laboratory by the PTS or a human carrier. Samples were transported at different rates and over varying distances. Each specimen's potassium (K) and lactic dehydrogenase (LDH) levels, in both the serum and plasma, were tracked to monitor hemolysis. Measurements of LDH and K were obtained using heparin or citrate. A positive correlation was observed between distance and hemolysis in serum samples transported at 4.2 m/sec, and at 3.1 m/sec for more than 2200 m (r = 0.774 and r = 0.766, respectively). Distance and hemolysis were also correlated in non-centrifuged samples (r = 0.871). The alterations in plasma LDH and K levels at different rates and PTS lengths were not statistically significant. The rate of hemolysis in PTS transported samples, dependent on PTS length and rate, may seriously affect routine tests of non-centrifuged samples.

Robust, routine haematology reference ranges for healthy adults

Accurate, reliable laboratory reference ranges are essential for effective clinical evaluation and monitoring. We present robust reference ranges established for haematology, coagulation and haematinic parameters using the Sysmex XE 2100, CA 1500 and Beckman-Coulter Access analysers. Blood samples were taken from 250 healthy laboratory personnel and routine haematology, coagulation and haematinic parameter analysis performed. Our data represent findings from an extensive study to establish reference ranges in healthy adults.

Perioperative autotransfusion and functional coagulation analysis in total hip replacement

Functional coagulation analyses like Sonoclot and thromboelastography have not been evaluated during perioperative autotransfusion. We have prospectively studied three different transfusion regimes in 45 patients undergoing total hip arthroplasty. Blood losses were replaced either with heterologous erythrocyte concentrate (group I), intra- and postoperative autotransfusion of blood salvaged with cellsaver technique (group II) or predonated autologous erythrocyte concentrates together with salvaged blood (group III). Routine and functional coagulation analyses with a Sonoclot were performed preoperatively, 6 hours postoperatively (6 h), day 1-5 and 10. An early postoperative hypo- and late postoperative hypercoagulative phase could be detected with Sonoclot signs of platelet function and fibrin deposition in all groups. Sonoclot coagulation analyses better correlated to both blood loss and dextran dosage than APTT and platelet count in the routine coagulation analyses. Functional coagulation analysis has a potential use in individualizing plasmasubstitution and thromboprophylaxis regimes during autotransfusion in THR.

Global and Molecular Hemostatic Markers in Acute Myeloid Leukemia

Patients with acute myeloid leukemia have multiple hemostatic and thrombotic complications, which may or may not result from disseminated intravascular coagulation. Previous studies incorporating routine coagulation analyses failed to detect any clinically useful information in most of these patients. In this study, the first comprehensive evaluation of the various aspects of the hemostatic system in a population of patients with acute myeloid leukemia was performed. Eighteen patients (23-71 years of age) were studied at either diagnosis or relapse. Hemostatic studies were performed at onset and on days 3, 7, and 30 after initiation of therapy. The bone marrow blast counts ranged from 8% to 98%; prothrombin time and activated partial thromboplastin time showed only minor prolongations in a few of these patients. However, in all patients measurement of platelet-associated markers revealed elevated platelet factor 4 and thromboxane B2 and normal 6-keto-prostaglandin F1 alpha levels. Fibrinolytic markers showed an increase in D-dimer and tissue plasminogen activator and a decrease in alpha 2-antiplasmin levels. Plasminogen, plasminogen activator inhibitor, and fibrinogen levels were normal. Coagulation markers demonstrated a decrease in protein C and antithrombin III levels and an elevation of the thrombin-antithrombin complex. The pretreatment values for all hemostatic markers studied were similar to the values obtained on days 3, 7, and 30 during treatment. This investigation demonstrated a subclinical activation of the components of the hemostatic system possibly leading to a hypercoagulable state. Although only six patients (33%) experienced hemorrhagic complications, the risk of bleeding and/or thrombosis was strongly evident in all patients. The significance of finding abnormal levels of specific molecular markers of hemostasis will be established in the future application of such markers in clinical evaluations of leukemic patients known to be at risk for coagulation disorders.