Functional fibrinogen (FLEV-TEG) versus the Clauss method in an obstetric population: a comparative study.

BACKGROUND: Hemostasis is the dynamic equilibrium between coagulation and fibrinolysis. During pregnancy, the balance shifts toward a hypercoagulative state; however placental abruption and abnormal placentations may lead to rapidly evolving coagulopathy characterized by the increased activation of procoagulant pathways. These processes can result in hypofibrinogenemia, with fibrinogen levels dropping to 2 g/L or less and an associated increased risk of post-partum hemorrhage. The aim of the present study was to evaluate the concordance between two methods of functional fibrinogen measurement: the Thromboelastography (TEG) method (also known as FLEV) vs. the Clauss method. Three patient groups were considered: healthy volunteers; non-pathological pregnant patients; and pregnant patients who went on to develop postpartum hemorrhage. METHODS: A prospective observational study. Inclusion criteria were: healthy volunteer women of childbearing age, non-pathological pregnant women at term, and pregnant hemorrhagic patients subjected to elective or urgent caesarean section (CS), with blood loss exceeding 1000 mL. Exclusion criteria were age < 18 years, a history of coagulopathy, and treatment with contraceptives, anticoagulants, or antiplatelet agents. RESULTS: Bland-Altman plots showed a significant overestimation with the FLEV method in all three patient groups: bias was - 133.36 mg/dL for healthy volunteers (95% IC: - 257.84; - 8.88. Critical difference: 124.48); - 56.30 mg/dL for healthy pregnant patients (95% IC: - 225.53; 112.93. Critical difference: 169.23); and - 159.05 mg/dL for hemorrhagic pregnant patients (95% IC: - 333.24; 15.148. Critical difference: 174.19). Regression analyses detected a linear correlation between FLEV and Clauss for healthy volunteers, healthy pregnant patients, and hemorrhagic pregnant patients (R2 0.27, p value = 0.002; R2 0.31, p value = 0.001; R2 0.35, p value = 0.001, respectively). ANOVA revealed a statistically significant difference in fibrinogen concentration between all three patients groups when assayed using the Clauss method (p value < 0.001 for all the comparisons), but no statistically significant difference between the two patients groups of pregnant women when using the FLEV method. CONCLUSIONS: The FLEV method does not provide a valid alternative to the Clauss method due to the problem of fibrinogen overestimation, and for this reason it should not be recommended for the evaluation of patients with an increased risk of hypofibrinogenemia.

Thrombogenesis in Thrombophilic Pregnancy: Evaluation of Low-Molecular-Weight Heparin Prophylaxis.

The aim of this study is to investigate thrombogenesis and the hypercoagulable changes in pregnant women affected by thrombophilia who received low-molecular-weight heparin (LWMH) prophylaxis. We included 21 pregnant women affected by thrombophilia treated with LWMH and 20 nontreated normal pregnant women as the control group. The sample group of thrombophilic pregnant women included different conditions (factor V Leiden mutation, protein C deficiency, protein S deficiency, antiphospholipid antibodies syndrome, and combined defects). Three blood samples were collected during pregnancy (i.e., at 16, 20, and 24 weeks) and tested for activated partial thromboplastin time and prothrombin fragment F1 + 2 (F1 + 2); anti-FXa activity was tested only in treated thrombophilic pregnant women. F1 + 2 levels progressively increased during pregnancy in both study groups. However, the F1 + 2 increase in women exposed to heparin prophylaxis was significantly lower than that in normal pregnant women in all 3 measurements carried out during gestation (p < 0.05); a statistically significant inverse correlation between F1 + 2 levels and anti-Xa activity (R = -0.8575, p < 0.05) was observed in treated women during pregnancy. Our findings suggest that F1 + 2 in addition to anti-Xa measurement could be used to adjust LWMH prophylaxis, at least in high-risk pregnant women.

Comparison of four D-dimer assays in the context of venous thromboembolism in the emergency department.

INTRODUCTION: This observational study conducted across seven emergency care units compares the efficacy of four D-dimer detection methods, namely HemosIL D-dimer HS (HS), HemosIL D-dimer HS-500 (HS-500), VIDAS D-dimer (VIDAS), and HemosIL AcuStar D-dimer (ACUSTAR). The primary focus is on patients with a clinical suspicion of deep venous thrombosis (DVT) or pulmonary embolism (PE). METHODS: A total of 149 samples were collected from patients with suspected DVT or PE. The confirmation of DVT/PE was based on calf ultrasound or computed tomography-Angiography. Direct comparisons were made between the different detection methods, considering both their analytical performance and clinical utility. Additionally, the impact of an age-adjusted cut-off on the diagnostic accuracy of each method was assessed. RESULTS: The results revealed comparable negative predictive value, sensitivity, and specificity across the methods, with a notable exception of increased specificity for HS compared with HS-500 (50.8% vs. 41.5%, p = 0.03). Further analysis incorporating an age-adjusted cut-off demonstrated a significant improvement in specificity for HS. When using the age-adjusted cut-off, HS exhibited a substantial increase in specificity compared with HS-500 (63.1% vs. 49.2%, p = 0.004) and demonstrated significantly higher specificity compared with VIDAS (63.1% vs. 53.8%, p = 0.04). CONCLUSION: The study emphasizes the nonuniversal effect of an age-adjusted cut-off and discusses the potential necessity for different cut-off values, particularly in the case of HS-500. These findings contribute to the understanding of D-dimer detection methods in the context of DVT and PE, providing insights into their relative performances and the potential optimization through age-adjusted cut-offs.

Cooperation experience in a multicentre study to define the upper limits in a normal population for the diagnostic assessment of the functional lupus anticoagulant assays.

BACKGROUND: Phospholipid-dependent coagulation tests for lupus anticoagulant (LA) are considered an important step for the diagnosis of anti-phospholipid syndrome; however, LA laboratory detection is difficult because of many variables. Five hospital laboratories, located in a North-Italy area and using the same method for LA testing, cooperated to standardise sample treatment and analytical procedure in order to define the upper values for LA negativity. METHODS: In total, 200 normal subjects (40 for each centre) were studied for six LA functional assays, using the same procedure, reagent lot and analyser type. The first tests done were LA screen and LA confirm assays, based on diluted Russell's Viper Venom Time, with low and high phospholipid content, respectively. The second tests performed were silica clotting time screen and confirm assays, based on activated partial thromboplastin time, with low and high phospholipid content, respectively. Finally, two mixing assays were executed for both screening assays, diluting patient sample with a pool prepared with plasma collected from the study population. RESULTS: Data analysis demonstrated a difference between centres for all assays when results were expressed in seconds; the difference disappeared when results are normalised with the local mean normal value of each centre and are expressed as a normalised ratio. The study population was normally distributed; so the value corresponding to 99th percentile was used as limit value for LA negativity. Values expressed as normalised ratio, for LA and silica clotting time screenings were 1.22 and 1.23, respectively. CONCLUSIONS: The study allowed us to define a uniform approach to LA testing and evaluation for laboratories employing the same methods.

Failure of Therapeutic Anticoagulation in COVID-19 Patients With Acute Ischemic Stroke. A Retrospective Multicenter Study.

Background: Acute ischemic stroke (AIS) is a possible complication of coronavirus disease 2019 (COVID-19) infection. Although peculiar clinical features and underlying specific mechanisms of thrombogenesis have been suggested so far, there is no consensus on the appropriate vascular preventive drug regimen in patients with COVID-19. Aim and Methods: From a larger clinical series of consecutive acute ischemic strokes related to COVID-19 admitted to three cerebrovascular units in Northern Italy, herein, we describe the clinical features of a subgroup of patients in whom stroke occurred despite therapeutic anticoagulation. Results: A total of seventeen/80 AIS related to COVID-19 (21.2%) occurred in anticoagulated patients. Although no blood level was available for Direct Oral AntiCoagulant, the drug dosage was appropriate according to guidelines. Their National Institute of Health Stroke Scale (NIHSS) at admission was 12.0 (SD = 7.4) and 58.8% of them had evidence of large vessel occlusion. The case fatality rate was as high as 64.7%. Discussion and Conclusions: The occurrence of an anticoagulation failure seems to be increased in the setting of COVID-19 infection, with worse clinical outcomes if compared to non-COVID-19 related ischemic strokes. We discuss the diagnostic and therapeutic implications of such evidence, suggesting that some arterial thrombotic complications might be either resistant to or independent of the anticoagulation effect.

Early thromboelastography in acute traumatic coagulopathy: an observational study focusing on pre-hospital trauma care.

BACKGROUND: Major brain injury and uncontrolled blood loss remain the primary causes of early trauma-related mortality. One-quarter to one-third of trauma patients exhibit trauma-induced coagulopathy (TIC). Thromboelastometry (ROTEM) and thrombelastography (TEG) are valuable alternatives to standard coagulation testing, providing a more comprehensive overview of the coagulation process. PURPOSE: Evaluating thromboelastographic profile, the incidence of fibrinolysis (defined as Ly30 > 3%) in severe trauma patients, and factors influencing pathological coagulation pattern. METHODS: Prospective observational 2 years cohort study on severe trauma patients assisted by Helicopter Emergency Medical System (HEMS) and Level 1 Trauma Center, in a tertiary referral University Hospital. RESULTS: Eighty three patients were enrolled, mean NISS (new injury severity score) 36 (± 13). Mean R value decreased from 7.25 (± 2.6) to 6.19 (± 2.5) min (p < 0.03); 48 (60%) patients had a reduction in R from T0 to T1. In NISS 25-40 and NISS > 40 groups, changes in R value increased their significance (p = 0.04 and p < 0.03, respectively). Pathological TEG was found in 71 (88.8%) patients at T0 and 74 (92.5%) at T1. Hypercoagulation was present in 57 (71.3%) patients at T0, and in 66(82.5%) at T1. 9 (11.3%) patients had hyperfibrinolysis at T0, 7 (8.8%) patients at T1. Prevalence of StO2 < 75% at T0 was greater in patients whose TEG worsened (7 patients, 46.7%) against whose TEG remained stable or improved (8 patients, 17.4%) from T0 to T1 (p = 0.02). 48 (57.8%) patients received < 1000 mL of fluids, while 35 (42.2%) received ≥ 1000 mL. The first group had fewer patients with hypercoagulation (20, 41.6%) than the second (6, 17.6%) at T1 (p < 0.03). No differences were found for same TEG pattern at T0, nor other TEG pattern. CONCLUSION: Our population is representative of a non-hemorrhagic severe injury subgroup. Almost all of our trauma population had coagulation abnormalities immediately after the trauma; pro-coagulant changes were the most represented regardless of the severity of injury. NISS appears to affect only R parameter on TEG. Hyperfibrinolysis has been found in a low percentage of patients. Hypoperfusion parameters do not help to identify patients with ongoing coagulation impairment. Small volume resuscitation and mild hypotermia does not affect coagulation, at least in the early post-traumatic phase.

Contribution and Effectiveness of Laboratory Testing in the Diagnostic Assessment of Juvenile Ischemic Stroke and Transient Ischemic Attack.

Introduction Strokes in young people require an extensive diagnostic workup to detect their possible several etiopathogenetic mechanisms. There is no consensus indicating what and when it should be tested. The clinical benefit and cost-effectiveness ratio of laboratory tests is unclear as well. Methods In one series of 104 consecutive juvenile ischemic stroke patients, under 45 years old, admitted between January 1, 2012, and December 31, 2017, we considered a wide panel of laboratory biomarkers exploring both the patient's basal status and specific risk factors for thrombotic disorders. To combine conventional and unconventional risk factors, structural defects, and other stroke-related diseases, we defined four categories of etiologic probability. We then studied the contribution of laboratory testing in changing the rate of "definite or probable stroke etiology" and the "proportion of patients with at least one additional risk factor" for stroke. Results The mere clinical assessment clarified stroke etiopathogenesis in 31% of cases. Abnormal values of the panel of biomarkers we considered were found in 30.1% of young ischemic strokes, while 11.5% of patients had unclear or borderline values. The benefit of laboratory assessment consisted of a relevant 14% gain in patients with a "definite or probable stroke etiology." Conclusion Several areas of uncertainty are still pending and herein discussed, such as the low re-testing rate during follow-up and the neglect of some relevant biomarkers. However, our results support the importance of laboratory testing in this setting. An improvement of diagnostic protocols in juvenile ischemic stroke would even increase their effectiveness, and this is still an unsolved issue in the field of cerebrovascular diseases. The same age limit, conventionally considered for juvenile stroke, could be better defined according to the effectiveness of both laboratory and clinical assessment in identifying unconventional stroke risk factors.

Retinal vein occlusion: evaluation of "classic" and "emerging" risk factors and treatment.

Retinal vein occlusion (RVO) is the second most common retinal vein disease and an important cause of blindness and visual morbidity. Systemic risk factors are commonly associated with RVO, while unclear it is the role of the thrombophilic and coagulation disorders. To evaluate "classic" and "emerging" risk factors, and to establish a good treatment for RVO. Fifty patients, 31 males and 19 females, with RVO were selected for our study. RVO patients were divided into two groups: those with central retinal vein occlusion (CRVO) and those with branch retinal vein occlusion (BRVO). All patients were subjected to an anamnestic investigation and were tested for thrombophilia, coagulation disorders and hyperlipidemia. Treatment and prophylaxis were evaluated. We have named "classic" the systemic risk factors associated with RVO and "emerging" those risk factors, haemostasis related, not clearly associated with RVO. RVO occurs more commonly in patients aged over 50. "Emerging" risk factors were more frequent in CRVO, "classic" in BRVO. Hyperhomocysteinemia is the most common "emerging" risk factor related to RVO. 71.4% of tested patients had hypercholesterolemia. Treatment with LMWH would appear to be safe and effective, but the small number of patients considered not allow us a definitive evaluation of its efficacy. Although our study has shown the correlation between RVO and the "emerging" risk factors, more studies are necessary to better know the real role of thrombophilic and coagulation disorders in this disease and to determine a specific protocol for the treatment and prophylaxis of RVO.

ADMA as a possible marker of endothelial damage. A study in young asymptomatic patients with cerebral small vessel disease.

Sporadic small vessel disease (SVD) has high prevalence in aging population and stroke patients, but also in younger asymptomatic subjects. In this last group it can represents a prelude to stroke and cognitive impairment. Still nowadays, its pathogenesis is unclear. 35 consecutive patients with SVD at brain MRI and 35 age- and sex-matched controls, between January 2016 and February 2018, underwent an extended screening for thrombophilia, autoimmunity and evaluated levels of blood markers of inflammation and endothelial activation. Asymmetric DiMethyl Arginine (ADMA) levels proved higher in patients (70.44 ± 36.25 ng/ml vs. 46.58 ± 30.67 ng/ml; p = 0.004), also after controlling for confounding factors. ADMA levels showed positive correlation with Fazekas score (r = 0.304; p = 0.01). ROC curve analysis showed a moderate accuracy in discriminating patients and controls (AUC = 0.70; CI 0.57-0.82; p = 0.004): a cut-off of 46 ng/ml is associated with 80% sensitivity, but limited (54%) specificity. Higher ADMA levels characterize selected subjects with sporadic SVD, asymptomatic for vascular diseases and without latent inflammatory conditions or coagulopathy. This reinforces the hypothesis of the key role of endothelial dysfunction in SVD. Further studies should explore the cause-effect relationship between ADMA pathway and SVD.

A collaborative study by the Working Group on Hemostasis and Thrombosis of the Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC) on the interference of haemolysis on five routine blood coagulation tests by evaluation of 269 paired haemolysed/non-haemolysed samples.

INTRODUCTION: Haemolysis is the leading cause of sample rejection in laboratory haemostasis. Most studies focused on artificially haemolysed samples. The aim of this study was a prospective assessment of spontaneous haemolysis on haemostasis tests, by comparing results of haemolysed (H) versus new, non-haemolysed (NH) specimens, collected within 4hrs. As new coagulometers can identify interfering substances, visual assessment of haemolysis was also compared with instrumental haemolysis index and stratified in subclasses. MATERIALS AND METHODS: Two hundred and sixty nine paired samples were collected and analysed using ACL TOP750-CTS (Instrumentation Laboratory, Bedford, USA), for prothrombin time (PT), activated partial thromboplastin time (aPTT), D-Dimer (DD), fibrinogen (Fib) and antithrombin (AT). Bias between H and NH was calculated and compared with the respective critical difference (CD). RESULTS: Mean bias was - 0.1 s for PT (P = 0.057), - 1.1 s for aPTT (P < 0.001), 1025 ng/mL for DD (P < 0.001), - 0.04 g/L for Fib (P = 0.258) and 1.4% for AT (P = 0.013). Bias exceeding the CD varied according to the method, with larger differences for aPTT (36.1%) and DD (17.1%) and < 8% for PT, Fib and AT. No correlation emerged between free haemoglobin values and difference in haemostasis tests in H and NH samples for any tests. Moderate/severe haemolysis involved > 95% of samples. The agreement between visual assessment and instrumental evaluation of haemolysis was 0.62. CONCLUSION: Spurious haemolysis deeply influences aPTT and DD, and to a lesser extent AT and Fib. Prothrombin time seems only slightly influenced, suggesting that PT can be accepted also in haemolysed samples. Although a good inter-observer correlation of haemolysis evaluation was found, the instrumental assessment of haemolysis seems recommendable.

Exploring the plasmatic platelet-activating factor acetylhydrolase activity in patients with anti-phospholipid antibodies.

PURPOSE: To explore the role of plasmatic platelet-activating factor acetylhydrolase (PAF-AH), a marker of cardiovascular risk, in patients with anti-phospholipid antibodies (aPL). METHODS: PAF-AH activity was assessed in a series of 167 unselected patients screened for aPL in a context of thrombotic events, risk of thrombosis or obstetric complications and in 77 blood donors. RESULTS: 116/167 patients showed positive results for at least one aPL among IgG/IgM anti-prothrombin/phosphatidylserine (aPS/PT), anti-cardiolipin (aCL), anti-beta2-glycoprotein I (aß2GPI) or lupus anticoagulant (LAC), while 51/167 patients resulted aPL-negative. LAC+ patients disclosed higher PAF-AH than LAC-negative (22.1 ± 6.4 nmol/min/ml vs. 19.5 ± 4.1 nmol/min/ml; p = 0.0032), and aPL-negative patients (p = 0.03). Patients presenting positive IgG aß2GPI disclosed higher PAF-AH than patients with only IgM aß2GPI-positive antibodies (23.1 ± 7.2 nmol/min/ml vs. 20.1 ± 5.3 nmol/min/ml; p = 0.035), as well as than patients showing only isolated LAC, aCL or aPS/PT (16.9 ± 3.8 nmol/min/ml; p = 0.003). CONCLUSIONS: PAF-AH plasmatic activity is particularly up-regulated in LAC+ and in aß2GPI IgG+ patients, possibly representing an alternative prognostic biomarker for the therapeutic management of APS patients.

Lupus anticoagulant: a multicenter study for a standardized and harmonized reporting.

Laboratory assessment of Lupus anticoagulant (LAC) is very challenging because of inter and intralaboratory variability, which makes it difficult to standardize and harmonize results expression. Five hospital laboratories in North-eastern Italy shared their efforts and their experience in a cross-laboratory study, conducting the diagnostic process as homogeneously as possible and providing a better interpretation for LAC positivity. Hundred normal samples from healthy subjects (20 from each center) were processed to confirm negative upper limits and calculate positivity cutoffs of LAC integrated assays, that is dilute Russell's viper venom time (dRVVT) and silica clotting time (SCT). Moreover, 311 samples previously diagnosed by the laboratories as positive for LAC were analyzed to characterize different positivity levels for each assay. As far as the analysis of healthy subjects is concerned, negative upper limits are set at 1.17 and 1.19 for dRVVT and SCT screen ratio, respectively. Positivity cutoffs are set at 1.20 for dRVVT and 1.23 for SCT, expressed as Test Ratio calculated on screen and confirm integrated tests. Positive results for each integrated assay are subsequently divided into three subgroups: weak, moderate and strong; the results obtained are presented as a score proposal that can provide LAC interpretation. The combined use of both dRVVT and SCT assays and the definition of different positivity levels may lead to clearer, more objective LAC reporting. An interpretative table for LAC-proposed score provides LAC-positive results and it is now adopted by all centers involved in the study.

Age dependency for coagulation parameters in paediatric populations. Results of a multicentre study aimed at defining the age-specific reference ranges.

Understanding of developmental haemostasis is critical to ensure optimal prevention, diagnosis, and treatment of haemorrhagic and thrombotic diseases in children. As coagulation test results are known to be dependent on the reagents/analysers used, it is recommended for each laboratory to define the age-dependent reference ranges by using its own technical condition. That study was carried out in seven centers to establish age-specific reference ranges using the same reagents and analyser. Plasma samples were obtained from 1437 paediatric patients from the following age groups: 15 days-4 weeks (n=36), 1-5 months (n=320), 6-12 months (n=176), 1-5 years (n=507), 6-10 years (n=132) and 11-17 years (n=262). Indication of coagulation testing was pre-operative screening for non-acute diseases in most cases. PT values were similar in the different age groups to those in adults, whereas longer aPTTs were demonstrated in the younger children. Plasma levels of all clotting factors, except for FV, were significantly decreased (p<0.0001) in the youngest children, adult values being usually reached before the end of the first year. The same applied to antithrombin, protein C/S, and plasminogen. In contrast, FVIII and VWF levels were elevated in the youngest children and returned to adult values within six months. The same applied to D-dimer levels, which were found elevated, particularly until six months of life, until puberty. These data suggest that most coagulation test results are highly dependent on age, mainly during the first year of life, and that age-specific reference ranges must be used to ensure proper evaluation of coagulation in children.

The introduction of anti-phosphatidylserine/prothrombin autoantibodies in the laboratory diagnostic process of anti-phospholipid antibody syndrome: 6 months of observation.

PURPOSE: To evaluate the impact of the introduction of the anti-phosphatidylserine/prothrombin autoantibodies (aPS/PT) in the laboratory diagnostic process of anti-phospholipid antibody syndrome (APS). METHODS: Four hundred and twenty-one patients (71.5 % females; 53 ± 15 years) presenting a medical prescription for aPS/PT antibodies were consecutively enrolled in the study from March 2013 to August 2013. During the same period, aPS/PT were additionally investigated in a selected series of 62 patients characterized by difficult lupus anticoagulant (LA) tests interpretation and in a retrospective series of 52 LA positive cases with available data about anti-prothrombin (aPT) antibodies. The aPS/PT antibodies, as well as the anti-cardiolipin (aCL), the anti-ß2 glycoprotein I (aß2GPI) and the aPT antibodies were analyzed by ELISA. LA was tested according to the recommended criteria, performing both the screen and the confirm steps. RESULTS: Overall, aPS/PT IgM positive (>30 U/ml) and/or IgG frankly positive (>40 U/ml) antibodies were found in 49/421 (11.6 %) cases. Among the LA positive patients, we found 56.1 % aPS/PT positive versus 31.7 % aCL and/or aß2GPI positive cases, with limited (17.1 %) simultaneous positivity. The PS/PT complex resulted the newly recognized specificity in about 27 % of patients recruited from the subset with difficult LA test interpretation. Compared to aPT antibodies, the aPS/PT antibodies displayed a much higher sensitivity (55.8 versus 15.4 %) in LA positive patients. CONCLUSIONS: The introduction of aPS/PT antibodies in the diagnostic process of APS is highly recommended, since they disclose a notable diagnostic performance and a high correlation with LA activity, such that they can be a viable alternative.

WITHDRAWN: Retinal vein occlusion: Risk factors evaluation, treatment and prophylaxis.

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Frequency of factor V, prothrombin and methylenetetrahydrofolate reductase gene variants in preeclampsia.

BACKGROUND: The association between thrombophilic variants (Leiden mutation of the factor V gene, G20210A mutation of the prothrombin gene and C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene) with preeclampsia was investigated in a north-eastern Italian population. METHODS: Fifty-eight preeclamptic (PE) women and 74 normal pregnancies were evaluated. Genotypes were determined by polymerase chain reaction. RESULTS: The frequency of heterozygous carriers of the factor V Leiden was similar between PE women (5.2%) compared to the control subjects (4.1%; p 0.76). Also the frequencies of G20210A and C677T mutations were similar between PE and control subjects. CONCLUSIONS: In this population, we found no difference in the prevalence of genetic risk factors for thrombosis in women with preeclampsia compared with control subjects.