Search

Introduction: Thrombin generation assays (TGAs) assess the overall functionality of the hemostatic system and thereby provide a reflection of the hemostatic capacity of patients with disorders in this system. Currently, four (semi-)automated TGA platforms are available: the Calibrated Automated Thrombogram, Nijmegen Hemostasis Assay, ST Genesia and Ceveron s100. In this study, we compared their performance for detecting patients with congenital single coagulation factor deficiencies., Materials and Methods: Pooled patient samples, healthy control samples and normal pooled plasma were tested on all four platforms, using the available reagents that vary in tissue factor and phospholipid concentrations. The TGA parameters selected for analysis were peak height and thrombin potential. Results were normalized by using the calculated mean of healthy controls and a correction for between-run variation. Outcomes were presented as relative values, with the mean of healthy controls standardized to 100 %., Results: Across all platforms and reagents used, thrombin potentials and peak heights of samples with coagulation factor deficiencies were lower than those of healthy controls. Reagents designed for bleeding tendencies yielded the lowest values on all platforms (relative median peak height 19-32 %, relative median thrombin potential 19-45 %). Samples representing more severe coagulation factor deficiencies generally exhibited lower relative peak heights and thrombin potentials., Conclusions: Thrombin generation assays prove effective in differentiating single coagulation factor deficient samples from healthy controls, with modest discrepancies observed between the platforms. Reagents designed for assessing bleeding tendencies, featuring the lowest tissue factor and phospholipid concentrations, emerged as the most suitable option for detecting coagulation factor deficiencies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: There are significant differences in the activated partial thromboplastin time (APTT) critical values reported in different studies, most of which does not make recommendations for any specific clear detection systems. The International Council for Standardization in Hematology (ICSH) recommends that APTT critical values be established based on the reagent type, coagulation factor sensitivity and heparin response. The objective of this study was to establish APTT critical values by using different reagents and based on single coagulation factor deficiencies., Methods: The APTT values were determined in commercial endogenous coagulation factor-deficient plasma at concentrations of 1 IU/dL, 2 IU/dL, 5 IU/dL, 10 IU/dL, 20 IU/dL, and 30 IU/dL by using four assay systems. The retrospective collection of data from patients who lacked factor VIII (FVIII), FIX, or FXI alone was performed. Receiver operating characteristic (ROC) curves were constructed to assess the diagnostic accuracy of APTT for identifying patients with an endogenous coagulation factor activity < 5 IU/dL., Results: The APTT values in the plasma samples with the same concentrations of endogenous coagulation factors were significantly different among the four assay systems (P < 0.001). The suggested critical values of APTT were 40.0 s for Sysmex CS5100 (Actin FSL), 58.0 s for Sysmex CS5100 (Actin), 51.8 s for STA-R Evolution (STA-PTTA), and 64.8 s for ACL TOP 700 (HemosIL SynthasIL). On the basis of the ROC curve, the optimal threshold values for APTT (STA-PTTA) were 55.8 s in patients with a simple deficiency of FVIII (sensitivity = 100%, specificity = 85.7%, area under the ROC curve (AUC) = 0.982), 54.3 s in patients with a simple deficiency of FIX (sensitivity = 100%, specificity = 92.9%, AUC = 0.986), and 71.7 s in patients with a simple deficiency of FXI (sensitivity = 100%, specificity = 94.1%, AUC = 0.992), which were closer (difference of 0.6-2.5 s) to the cutoff points for commercial plasma at equal factor levels., Conclusions: APTT critical values need to be established for different reagents based on the presence of a single coagulation factor deficiency., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Rattlesnakes are a diverse clade of pit vipers (snake family Viperidae, subfamily Crotalinae) that consists of numerous medically significant species. We used validated in vitro assays measuring venom-induced clotting time and strength of any clots formed in human plasma and fibrinogen to assess the coagulotoxic activity of the four medically relevant Mexican rattlesnake species Crotalus culminatus, C. mictlantecuhtli, C. molossus , and C. tzabcan . We report the first evidence of true procoagulant activity by Neotropical rattlesnake venom in Crotalus culminatus . This species presented a strong ontogenetic coagulotoxicity dichotomy: neonates were strongly procoagulant via Factor X activation, whereas adults were pseudo-procoagulant in that they converted fibrinogen into weak, unstable fibrin clots that rapidly broke down, thereby likely contributing to net anticoagulation through fibrinogen depletion. The other species did not activate clotting factors or display an ontogenetic dichotomy, but depleted fibrinogen levels by cleaving fibrinogen either in a destructive (non-clotting) manner or via a pseudo-procoagulant mechanism. We also assessed the neutralization of these venoms by available antivenom and enzyme-inhibitors to provide knowledge for the design of evidence-based treatment strategies for envenomated patients. One of the most frequently used Mexican antivenoms (Bioclon Antivipmyn®) failed to neutralize the potent procoagulant toxic action of neonate C. culminatus venom, highlighting limitations in snakebite treatment for this species. However, the metalloprotease inhibitor Prinomastat substantially thwarted the procoagulant venom activity, while 2,3-dimercapto-1-propanesulfonic acid (DMPS) was much less effective. These results confirm that venom-induced Factor X activation (a procoagulant action) is driven by metalloproteases, while also suggesting Prinomastat as a more promising potential adjunct treatment than DMPS for this species (with the caveat that in vivo studies are necessary to confirm this potential clinical use). Conversely, the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) inhibited the direct fibrinogen cleaving actions of C. mictlantecuhtli venom, thereby revealing that the pseudo-procoagulant action is driven by kallikrein-type serine proteases. Thus, this differential ontogenetic variation in coagulotoxicity patterns poses intriguing questions. Our results underscore the need for further research into Mexican rattlesnake venom activity, and also highlights potential limitations of current antivenom treatments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Seneci, Zdenek, Chowdhury, Rodrigues, Neri-Castro, Bénard-Valle, Alagón and Fry.)

Introduction: Rare coagulation factor deficiencies are less-known disorders with variable effects on the patient's life. Management of such patients is a challenge due to the paucity of evidence-based data, more so when patients with these rare disorders encounter a more rare, related condition, like inhibitor development or thrombosis., Area Covered: A comprehensive literature search related to RCFDs and management was performed in PubMed in order to discuss therapeutic options and challenges, prophylaxis, management of minor and major surgeries, obstetric and gynecological complications, inhibitor development, and thrombosis., Expert Opinion: Although significant changes have occurred in the management of RCFDs in recent years, more evidence-based studies besides expert opinion are needed for optimal management.

Background: Coagulopathy is common in multitrauma patients and repletion of procoagulant factor deficiency with fresh frozen plasma (FFP) improves hemostasis. Optimal kaolin-thromboelastography thresholds for FFP transfusion in trauma patients have not been well established., Study Design: Adult trauma patients with an Injury Severity Score ≥15 were included in this retrospective observational cohort study. The primary end point was area under the receiver operating characteristic curve (AUROC) for reaction time (R-time) to detect procoagulant factor deficiency, as reflected by an elevated international normalized ratio (INR) or aPTT. Test characteristics for the optimal R-time threshold calculated in our study were compared against thresholds recommended by the American College of Surgeons for FFP transfusion., Results: Six hundred and ninety-four pairs of thromboelastography and conventional coagulation tests were performed in 550 patients, with 144 patients having additional pairs of tests after the first hour. The R-time was able to detect procoagulant factor deficiency (INR ≥1.5 AUROC 0.80; 95% CI, 0.75 to 0.85; aPTT ≥40 seconds AUROC 0.85; 95% 0.80 to 0.89) and severe procoagulant factor deficiency (INR ≥2.0 AUROC 0.82; 95% CI, 0.73 to 0.99; aPTT ≥60 seconds AUROC 0.89; 95% CI, 0.81 to 0.98) with good accuracy. Optimal thresholds to maximize sensitivity and specificity were 3.9 minutes for detection of INR ≥1.5, 4.1 minutes for detection of aPTT ≥40 seconds, 4.3 minutes for detection of INR ≥2.0, and 4.3 for detection of aPTT ≥60 seconds. Currently recommended R-time thresholds for FFP transfusion had 100% specificity for detecting procoagulant factor deficiency, but low sensitivity (3% to 7%)., Conclusions: R-time can detect procoagulant factor deficiency in multitrauma patients with good accuracy, but currently recommended R-time thresholds are highly specific and not sensitive. Use of low-sensitivity thresholds might result in undertreatment of many patients with procoagulant factor deficiency., (Copyright © 2020 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: Congenital disorders of glycosylation are rare inherited diseases affecting many different proteins. The lack of glycosylation notably affects the hemostatic system and leads to deficiencies of both procoagulant and anticoagulant factors., Objective: To assess the hemostatic balance in patients with multiple coagulation disorders by using a thrombin generation assay., Method: We performed conventional coagulation assays and a thrombin generation assay on samples from patients with congenital disorder of glycosylation. The thrombin generation assay was performed before and after activation of the protein C system by the addition of soluble thrombomodulin., Results: A total of 35 patients were included: 71% and 57% had low antithrombin and factor XI levels, respectively. Protein C and protein S levels were abnormally low in 29% and 26% of the patients, respectively, whereas only 11% displayed low factor IX levels. Under baseline conditions, the thrombin generation assay revealed a significantly higher endogenous thrombin potential and thrombin peak in patients, relative to controls. After spiking with thrombomodulin, we observed impaired involvement of the protein C system. Hence, 54% of patients displayed a hypercoagulant phenotype in vitro. All the patients with a history of stroke-like episodes or thrombosis displayed this hypercoagulant phenotype., Conclusion: A thrombin generation assay revealed a hypercoagulant in vitro phenotype under baseline condition; this was accentuated by impaired involvement of the protein C system. This procoagulant phenotype may thus reflect the risk of severe vascular complications. Further research will have to determine whether the thrombin generation assay is predictive of vascular events., (© 2019 French National Institute of Health and Medical Research. © 2019 International Society on Thrombosis and Haemostasis.)

Direct oral anticoagulants (DOACs) are small molecule inhibitors of the coagulation proteases thrombin and factor Xa that demonstrate comparable efficacy to warfarin for several common indications, while causing less serious bleeding. However, because their targets are required for the normal host-response to bleeding (hemostasis), DOACs are associated with therapy-induced bleeding that limits their use in certain patient populations and clinical situations. The plasma contact factors (factor XII, factor XI, and prekallikrein) initiate blood coagulation in the activated partial thromboplastin time assay. While serving limited roles in hemostasis, pre-clinical and epidemiologic data indicate that these proteins contribute to pathologic coagulation. It is anticipated that drugs targeting the contact factors will reduce risk of thrombosis with minimal impact on hemostasis. Here, we discuss the biochemistry of contact activation, the contributions of contact factors in thrombosis, and novel antithrombotic agents targeting contact factors that are undergoing pre-clinical and early clinical testing., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Congenital factor deficiencies (CFDs) refer to inherited deficiency of coagulation factors in the blood. A total of 481 patients with CFDs, who were diagnosed and followed at our Pediatric Hematology and Oncology Clinic between 1990 and 2015, were retrospectively evaluated. Of the 481 cases, 134 (27.8%) were hemophilia A, 38 (7.9%) were hemophilia B, 57 (11.8%) were von Willebrand disease (vWD), and 252 (52.3%) were rare bleeding disorders (RBDs). The median age of the patients at the time of diagnosis and at the time of the study was 4.1 years (range: 2 months to 20.4 years) and 13.4 years (range: 7 months to 31.3 years), respectively. The median duration of the follow-up time was 6.8 years (range: 2.5 months to 24.8 years). One hundred nineteen (47.2%) of 252 patients with RBDs were asymptomatic, 49 (41.1%) of whom diagnosed by family histories, 65 (54.6%) through preoperative laboratory studies, and 5 (4.2%) after prolonged bleeding during surgeries. Consanguinity rate for the RBDs was 47.2%. Prophylactic treatment was initiated in 80 patients, 58 of whom were hemophilia A, 7 were hemophilia B, 13 were RBDs, and 2 were vWD. Significant advances have been achieved during the past 2 decades in the treatment of patients with CFDs, particularly in patients with hemophilias. The rarity and clinical heterogeneity of RBDs lead to significant diagnostic challenges and improper management. In this regard, multinational collaborative efforts are needed with the hope that can improve the management of patients with RBDs.

Background: Protein C is a vitamin K dependent plasma zymogen. It inhibits clotting by inhibiting clotting by inactivating factor V and factor VIII. Protein C activation pathway involves three steps: (i) Activation of protein C; (ii) Inhibition of coagulation through inactivating factor V and VIII by activated protein C and (iii) Inhibition of activated protein C by plasma protease inhibitors specific for this enzyme. Proteinases converting the zymogen protein C (PC) of vertebrates into activated PC have been detected in several snake venoms. Most PC activators have been purified from venom of snake species belonging to the genera of the Agkistrodon complex. Unlike the physiological thrombin-catalyzed PC activation reaction which requires thrombomodulin as a cofactor, most snake venom activators directly convert the zymogen PC into the catalytically active form which can easily be determined by means of coagulation or chromogenic substrate techniques., Conclusion: The fast-acting PC activator Protac® from Agkistrodon contortrix contortrix (Southern copperhead snake) venom has found a broad application in diagnostic practice for the determination of disorders in the PC pathway. Recently, screening assays for the PC pathway have been introduced, based on the observation that the PC pathway is probably the most important physiological barrier against thrombosis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Rare diseases are defined as life-threatening or chronically debilitating diseases with a prevalence of less than one per 2000 according to the European Union or one per 1250 according to the USA. Congenital rare bleeding disorders RBD are reported in most populations, with incidence varying from 1 in 5000 (Hemophilia A), 1:30,000 (Hemophilia B) to much rarer (1:500,000 for FVII deficiency, 1-3 million for Prothrombin or FXIII deficiency). Acquired Hemophilia A is also a rare bleeding disorder with estimated frequency of 1 in million. Most RBDs are inherited as autosomal recessive (AR); however, heterozygous carriers with varying degrees of corresponding factor deficiency may render an unpredictable propensity for bleeding. In patients with bleeding symptoms, laboratory assessment and especially molecular techniques currently enable accurate diagnosis and may provide tools for prenatal and family counseling. Currently hemostasis control is mainly based upon replacement of the missing coagulation factors (unless presence of inhibitors renders it impossible), however future gene therapy and disruptive, non-replacement alternatives may be promising for patients with RBD., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mixing tests in coagulation studies allow the laboratory to correctly differentiate factor deficiency from inhibitors as the cause of a prolonged clotting time. This is important in terms of subsequent laboratory follow-up and clinical patient management. In this chapter, a suggested method of performing mixing tests is given, with procedures applying two different formulas for interpretation, the percent correction and the index of circulating anticoagulant.

The activated partial thromboplastin time (APTT) is a useful global assay for the assessment of the contact factor pathway of hemostasis and its inhibitors. The test is usually performed on fully automated analyzers using commercially prepared reagents. The three main clinical areas of interest are detection of factor deficiencies, detection of lupus anticoagulants and in the monitoring of therapy with unfractionated heparin. Methods are described here for assessing APTT reagents for their sensitivity to clotting time prolongation in each of these areas of interest.

Rare bleeding disorders (RBDs) are a heterogeneous group of coagulation disorders characterized by fibrinogen, prothrombin, factors V, VII, X, XI, or XIII (FV, FVII, FX, FXI, or FXIII, respectively), and the combined factor V + VIII and vitamin K-dependent proteins deficiencies, representing roughly 5% of all bleeding disorders. They are usually transmitted as autosomal, recessive disorders, and the prevalence of the severe forms could range from 1 case in 500 000 for FVII up to 1 in 2-3 million for FXIII in the general population. Patients affected with RBDs may present a wide range of clinical symptoms, varying from mucocutaneous bleeding, common to all types of RBDs to the most life-threatening symptoms such as central nervous system and gastrointestinal bleeding. Treatment of these disorders is mainly based on the replacement of the deficient factor, using specific plasma-derived or recombinant products. In countries where these facilities are not available, bleedings could be managed using cryoprecipitate, fresh frozen plasma (FFP), or virus-inactivated plasma. Minor bleedings could be managed using antifibrinolytic agents. Recently, 2 novel drugs, recombinant FXIIIA and a plasma-derived FX, have been added to the list of available specific hemostatic factors; only prothrombin and FV deficiencies still remain without a specific product. Novel no-replacement therapies, such as monoclonal antibody anti-tissue factor pathway inhibitor, RNA interference, and a bispecific antibody that is an FVIIIa mimetic, enhancing thrombin generation through different mechanisms, were developed for patients with hemophilia and may in the future be a good therapeutic option also in RBDs., (© 2016 by The American Society of Hematology. All rights reserved.)

Introduction: There are several clinical settings and patient conditions especially in intensive care units, emergency departments, and operating theaters, where the coagulation status of a patient must be known immediately and point-of-care (POC) systems are beneficial due to low time to result., Methods: This noninterventional, single-blinded, multicenter study with prospectively collected whole blood samples was performed to evaluate the diagnostic accuracy of the CoaguChek PT Test (POC PT) and CoaguChek aPTT Test (POC aPTT) compared to standard laboratory testing in patients with suspected deficiencies of coagulation factors., Results: In total, 390 subjects were included. Both POC PT and POC aPTT showed concordance with the laboratory PT and aPTT. Lot-to-lot variation was below 2% both for POC PT and for POC aPTT. The mean relative difference of capillary blood compared to venous blood was 0.2 % with POC PT and 8.4% with POC aPTT. The coefficients of variation for repeatability of POC PT using whole blood were found to be between 2% and 3.6%., Conclusion: Our findings suggest reliable quantitative results with this POC system to support on-site decision-making for patients with suspected deficiencies of coagulation factors in acute and intensive care., (© 2016 John Wiley & Sons Ltd.)

Introduction: Activated partial thromboplastin time (aPTT) is a routine clotting assay that is widely used to globally screen for coagulation abnormalities. It is commonly admitted that a prolonged test result, may trigger the need for specific assays to be performed, particularly factor measurement. However, the sensitivity of aPTT reagents to deficiencies of clotting factors varies., Methods: We evaluated, according to the recommendation of the CLSI H47-A2 guideline, the responsiveness to single factor levels of five aPTT reagents by using factor-deficient plasmas spiked with a calibration plasma to produce individual factor activities ranging from <1 to ~100 Unit (U)/dL. Test results were expressed as the sample-to-control ratio, the latter was defined as the clotting time obtained in the calibration plasma containing ~100 U/dL factor activity. The factor activity producing a prolongation of aPTT above the upper limit of its specific normal range (in ratio) was assigned as the factor responsiveness in U/dL to that reagent., Results: Responsiveness ranged from 34 to 47 U/dL to, Fviii: C, from 18 to 57 U/dL to FIX, from 38 to 52 U/dL to FXI, from 29 to 50 U/dL to FXII, from 40 and 59 U/dL to FV, from 7.5 to 49 U/dL to FX, and from 9.1 to 10.5 U/dL to FII., Conclusions: These results suggest that the sensitivity of the tested aPTT reagents to single factor deficiency is highly variable. Moreover, for one given aPTT reagent, its sensitivity was very different depending on the deficient factor. This must be considered when analyzing clinical materials., (© 2016 John Wiley & Sons Ltd.)

Objectives: An isolated prolongation of activated partial thromboplastin time (aPTT) found in paediatric pre-operative screening could be due to bleeding or non-bleeding aetiologies. The aim of our study was to evaluate clinical benefits of an additional ActinFS and/or a mixing aPTT study to identify a bleeding-related factor deficiency (BRFD)., Methods: Over a 4-yr period, isolated prolongation of aPTT with PlatelinLS was detected in 308 paediatric patients and confirmed in 161 cases by a 2nd sample. ActinFS, a mixing study, FVIII, FIX, FXI, FXII and lupus anticoagulant (LA) were performed. Three different aPTT ratios between PlatelinLS and ActinFS were analysed., Results: We found 17 BRFD, 31 FXII deficiencies and 64 positive LA. A prolonged ActinFS had a significant association with BRFD (P < 0.0001) while a corrected mixing study did not. The direct aPTT ratio had a significant relationship with positive LA (P < 0.05), and with BRFD (P < 0.0001). Using this ratio, the sensitivity of BRFD's and LA's detection could be increased, respectively, to 82% from 59% using ActinFS alone, and to 86% from 55% using mixing study., Conclusions: Applying this direct aPTT ratio allows to quickly and reliably identify both BRFD and LA sequential to an isolated prolongation of aPTT., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Pseudoxanthoma elasticum (PXE)-like syndrome is characterized by the association of PXE and cutis laxa (CL) features with a deficiency of vitamin K-dependent clotting factors. It was first described in 1971 and was identified as a distinct genetic entity in 2007 with analysis of the GGCX (γ-glutamyl carboxylase) gene, which is involved in congenital deficiency in vitamin K-dependent clotting factors. Here we report a new case of this extremely rare syndrome., Patients and Methods: A 23-year-old female patient was seen for the emergence of loose and redundant skin following extensive weight loss. She also presented a deficiency of vitamin K-dependent clotting factors. Physical examination revealed excessive, leathery skin folds in the axillary and neck regions. A skin biopsy revealed polymorphous and fragmented elastic fibers in the reticular dermis. These were mineralized, as was demonstrated by Von Kossa staining. The clinical features of CL associated with the histopathological features of PXE and vitamin K-dependent clotting factor deficiency led us to a diagnosis of PXE-like syndrome. A molecular study of the GGCX gene showed compound heterozygosity., Discussion: The GGCX gene is usually responsible for PXE-like syndrome. GGCX encodes a γ-glutamyl carboxylase necessary for activation of gla-proteins. Gla-proteins are involved both in coagulation factors in the liver and in the prevention of ectopic mineralization of soft tissues. Uncarboxylated forms of gla-proteins in fibroblast would thus enable mineralization and fragmentation of elastic fibers., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Objective: Mild bleeding symptoms are commonly seen in the general population. The aim of this study was to determine the final clinical and laboratory features of children referred for a first evaluation with a suspected bleeding disorder in the pediatric outpatient clinic of İstanbul University., Materials and Methods: The medical records of 26,737 outpatients who were admitted to the Division of Ambulatory Pediatrics between 31 October 2011 and 31 October 2012 were evaluated retrospectively. Ninety-nine patients were initially diagnosed as having probable bleeding disorders and were followed up. The symptoms of bleeding in addition to coagulation tests were analyzed., Results: Of the 99 patients, 52 (52.5%) were male and 47 were female, and the mean age of the entire study group was 9.1±4.1 years (minimum-maximum: 2-18 years). Major bleeding symptoms were epistaxis in 36 patients (36.4%), easy bruising in 32 (32.3%), and menorrhagia in 6 (6.1%). After initial tests ordered by the pediatrician, 36 of 99 patients (36.4%) were diagnosed as having bleeding disorders that included von Willebrand disease in 12 (12.1%), hemophilia A or B in 9 (9.1%), and other rare factor deficiencies in 9 (9.1%). Six patients (6.1%) were found to have combined deficiencies. Seven of 36 patients had a family history of bleeding., Conclusion: Among the patients referred for bleeding disorders, 36.4% were diagnosed with a bleeding disorder with the help of primary screening tests ordered in the outpatient clinic.

Introduction: Prothrombin time (PT) and activated partial thromboplastin time (APTT) sensitivity for detecting isolated factor deficiencies varies with different reagents and coagulometers. The Clinical and Laboratory Standards Institute (CLSI) H47A2 guideline proposed a method to calculate these sensitivities, but some inconsistency has been reported. This study aimed to calculate factor sensitivities using CLSI guideline and to compare them with those obtained from single factor-deficient patients' data., Methods: Different mixtures of normal pooled and deficient plasmas were prepared (<1IU/dL to 100 IU/dL) according to the CLSI H47A2 guideline. PT with rabbit brain (RB) and human recombinant (HR) thromboplastins, APTT and factors' activities were measured in an ACL TOP coagulometer. Sensitivities (maximum factor concentration that produces PT or APTT values out of the reference range) were calculated from mixtures and from patients with single-factor deficiencies: 17 factor FV, 36 FVII, 19 FX, 39 FVIII, 15 FIX 15 FXI and 24 FXII., Results: PT sensitivity with RB was as follows: FV 38 and 59, FVII 35 and 58, FX 56 and 64 IU/dL; PT sensitivity with HR was as follows: FV 39 and 45, FVII 51 and 50, FX 33 and 61 IU/dL; and APTT sensitivity was as follows: FV 39 and 45, FX 32 and 38, FVIII 47 and 60, FIX 35 and 44, FXI 33 and 43, FXII 37 and 46 IU/dL, respectively., Conclusions: Reagent-coagulometer combination has adequate sensitivities to factor deficiencies according to guideline recommendations (>30 IU/dL). These should not be considered as actual sensitivities because those obtained by analysing patients' plasmas with single-factor deficiencies were higher for most factors and could induce misinterpretation of the basic coagulation test results., (© 2015 John Wiley & Sons Ltd.)

Background: Rare factor deficiencies (RFDs) are autosomal recessively inherited coagulation factor deficiencies encountered at a frequency of between one in 500, 000 and one in two million., Materials and Methods: One hundred and ninety-two patients, diagnosed as having RFD, followed and treated in our clinic between 1990 and 2013 were retrospectively evaluated in this study., Results: From the 192 patients, 142 had FVII, 15 had FX, 14 had FXI, 10 had fibrinogen, six had FV, two had FXIII, two had FV + FVIII and one had FII deficiency. One hundred and thirty of the cases were boys and 62 were girls. The age range was 2 weeks to 24 years and the ages at the admission were between 2 weeks and 16 years. The rate of consanguinity was 49.4%. Eighty-eight of our patients were asymptomatic (45.8%) and 104 were symptomatic (54.2%). Asymptomatic patients were diagnosed by family histories (39.8%), preoperative laboratory studies (54.6%) and operational bleeding (5.6%). Sixty-eight of our symptomatic patients displayed grade II (65.4%) and 36 displayed grade III bleeding symptoms (34.6%). First bleeding regions were skin (33%), nose (28%), central nervous system (CNS) (15.5%), oral cavity (10.5%), soft tissue (6%), joint (3%), urinary system (2%) and gastrointestinal system (GIS) (2%), respectively. The bleeding prevalence rates of our symptomatic patients are listed as epistaxis 62.5%, skin bleedings 53%, oral cavity bleeding 28.8%, haematomas 18.3%, CNS bleedings 17.3%, haemarthrosis 14.4%, GIS bleedings 3.8%, menorrhagia 2.9%, haematuria 1.9%, bleeding because of operations 1.9% and iliopsoas bleedings 1.9%. CNS bleedings (41%) take the first place among the serious bleedings of our cases, followed by haemarthrosis (36.4%), GIS bleedings (18.1%) and iliopsoas bleedings (4.5%). Prophylaxy was applied to nine patients (five patients with FVII, two patients with fibrinogen and one each with FV and FX deficiency)., Conclusions: The characteristics of clinical presentations, first bleeding attacks, bleeding prevalence and severe bleedings as well as prophylactic approaches are discussed in this article.

This is a retrospective study on six multiple myeloma patients with upfront coagulopathy and bleeding. A detailed description and analysis of clinical characteristics, coagulation factor deficiencies, treatments and outcome of those six multiple myeloma patients are presented. All six patients presented with significant bleeding. One patient was detected with single factor X deficiency and another with single factor VII (FVII) deficiency, whereas four other patients had complex factor deficiencies. The time from symptom presentation to diagnosis ranged from 3 to 10 months. After correct diagnosis and coagulation factor supplementation, those patients were treated with bortezomib/adriamycin/dexamethasone (PAD) or melphalan/dexamethasone/thalidomide (MTD) regimen. It took 29-71 days (median time 46 days) to completely correct coagulation factor deficiencies since the start of therapy for multiple myeloma. Multiple myeloma patients with acquired bleeding disorders may present with large, deep and multiple sites of haematoma or other types of significant bleeding, which may affect bone marrow examination in some of the cases. Patients may be easily misdiagnosed. The routine examinations of erythrocyte sedimentation rate, serum immunoglobulins and blood urine light chain are the key to diagnosis, hence requiring the treating physician to think broadly and look for traits suggesting myeloma as the underlying cause.

Thrombin is a key enzyme of the coagulation system, having both pro- and anticoagulant functions. Thus, the generation of thrombin is one of the most important steps in coagulation. Global haemostasis assay, the so-called thrombin generation test is appropriate for its assessment. Since thrombin generation is sensible for both pro- and anticoagulant processes it can be applied for the general characterisation of the risk of thrombosis and bleeding, too. Clinical studies confirmed augmented thrombin generation in patients with high risk of venous or arterial thrombosis. Anticoagulant therapy (also novel oral anticoagulant treatment) can be monitored by thrombin generation. In case of haemophilia thrombin generation assays reflect bleeding severity. It is applicable for monitoring of both conventional haemophilia treatment and inhibitor-bypassing therapy, which is needed when inhibitors develop in patients. Standardization of thrombin generation methods and determination of cut off values are required before its application in clinical practice.

Objective: To explore the diagnostic value of protein induced by vitamin K absence or antagonist -II(PIVKA-II) in non-infant with acquired deficiency of vitamin K-dependent coagulation factors(ADVKCF)., Methods: PIVKA-II levels were measured by ELISA in 50 patients with ADVKCF on day 0, 3, 7 after vitamin K treatment. Prothrombin time(PT), APTT, FII: C, FVII: C, FIX: C, and FX: C were analyzed simultaneously. Twenty healthy subjects were enrolled as controls., Results: The average level of PIVKA-II in ADVKCF group was (3.83 ± 1.40)µg/L, while (1.30 ± 0.54) µg/L in the control group (P < 0.05). The PIVKA-II levels on day 0 and 3 did not show significant difference [(3.83 ± 1.40) µg/L vs (3.79 ± 0.66) µg/L, P > 0.05], but decreasing significantly on day 7 compared to the control group(P < 0.05). The PIVKA-II level was (3.78 ± 1.30) µg/L in patients receiving plasma transfusion, while (3.91 ± 1.49)µg/L in no-plasma-transfusion group (P > 0.05). Coagulation factors II, VII, IX and X activity which decreased significantly before treatment returned to normal range after one week use of vitamin K, leading to complete correction of prolonged APTT and PT (>100 seconds)., Conclusions: The PIVKA-II level in ADVKCF patients is significantly higher than that of healthy subjects within one week treatment of vitamin K, which is not influenced by plasma transfusion. This study suggests that PIVKA-II is a more sensitive parameter than APTT, PT and the activity of coagulation factor, which could be a valuable factor in the early diagnosis of ADVKCF.

Rare bleeding disorders (RBDs) comprise the inherited deficiencies of coagulation factors such as fibrinogen, factor (F)II, FV, FV + FVIII, FVII, FX, FXI, and FXIII, and are usually transmitted as autosomal recessive disorders. RBDs are characterized by a wide variety of symptoms from mild to severe; however, due to their rarity, only little information is available on the adequate management of patients affected with these deficiencies. Moreover, the limitations of laboratory assays and the lack of a definitive consensus concerning their classification have prevented adoption of optimal approaches to their individual management. To overcome these limitations, new strategies are therefore necessary, such as the establishment of global collaborations and networks among treatment centers, as well as increasing support provided by public health organizations., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Background: The outcomes of colorectal cancer are determined by host factors, including systemic inflammation. The purpose of this study was to evaluate the prognostic significance of fibrinogen and inflammation-based scores, as markers of the inflammatory response, in colon cancer., Methods: We retrospectively reviewed the medical records of patients with nonmetastatic colon cancer who underwent curative resection between January 2005 and December 2007. Fibrinogen, albumin, C-reactive protein, neutrophil, lymphocyte, and platelet counts were measured at the time of diagnosis. Correlations between preoperative plasma fibrinogen levels and clinicopathologic characteristics were analyzed. Univariate and multivariate survival analyses were performed to identify factors associated with disease-free and overall survival., Results: A total of 624 patients who underwent curative resection for colon cancer were eligible for this study. Mean preoperative plasma fibrinogen levels were 325.24±88.19 mg/dl. Higher preoperative plasma fibrinogen levels were associated with sex (male), old age, poorly/mucinous differentiated tumor, advanced tumor stage, elevated carcinoembryonic antigen (CEA) levels, higher modified Glasgow Prognostic Score, and higher neutrophil:lymphocyte and platelet:lymphocyte ratios. In multivariate analysis, elevated plasma fibrinogen level [disease-free survival: hazard ratio (HR) 1.999, 95% confidence interval (95% CI) 1.081-3.695, P=.027; overall survival: HR 3.138, 95% CI 1.077-9.139, P=.036], advanced tumor stage, and higher CEA levels were independently associated with worse disease-free survival and overall survival. None of the inflammation-based scores were significantly associated with survival., Conclusions: Fibrinogen as one of inflammatory markers may be considered a possible prognostic marker in colon cancer.

The investigation of inherited bleeding disorders with routine tests of hemostasis will yield clear diagnostic information in the majority of subjects with an unequivocal history of bleeding and especially in those where the phenotypic severity is severe and where an obvious family history of bleeding is present. Nevertheless, a significant minority of subjects with obvious bleeding symptoms will remain without a definite diagnosis after extensive hemostatic testing. With these facts in mind, the role of molecular testing for inherited disorders of hemostasis now includes the following: confirmation of a phenotypic diagnosis through targeted genetic analysis, the distinction of bleeding phenocopies by molecular analysis, and provision of genetic testing as the investigation of choice in situations such as prenatal diagnosis and detection of the carrier state for inherited bleeding traits. In addition, molecular testing can sometimes be used to provide supplementary knowledge that can be used to enhance clinical care. Finally, the utility of genome-wide approaches to identify novel genetic associations may provide new information to explain the cause of bleeding in the population of bleeders without established diagnoses., (© 2013 Blackwell Publishing Ltd.)

The Q analyzer is a recently launched fully automated photo-optical analyzer equipped with primary tube cap-piercing and capable of clotting, chromogenic, and immunoturbidometric tests. The purpose of the present study was to evaluate the performance characteristics of the Q analyzer with reagents from the instrument manufacturer. We assessed precision and throughput when performing coagulation screening tests, prothrombin time (PT)/international normalized ratio (INR), activated partial thromboplastin time (APTT), and fibrinogen assay by Clauss assay. We compared results with established reagent instrument combinations in widespread use. Precision of PT/INR and APTT was acceptable as indicated by total precision of around 3%. The time to first result was 3  min for an INR and 5  min for PT/APTT. The system produced 115 completed samples per hour when processing only INRs and 60 samples (120 results) per hour for PT/APTT combined. The sensitivity of the DG-APTT Synth/Q method to mild deficiency of factor VIII (FVIII), IX, and XI was excellent (as indicated by APTTs being prolonged above the upper limit of the reference range). The Q analyzer was associated with high precision, acceptable throughput, and good reliability. When used in combination with DG-PT reagent and manufacturer's instrument-specific international sensitivity index, the INRs obtained were accurate. The Q analyzer with DG-APTT Synth reagent demonstrated good sensitivity to isolated mild deficiency of FVIII, IX, and XI and had the advantage of relative insensitivity to mild FXII deficiency. Taken together, our data indicate that the Q hemostasis analyzer was suitable for routine use in combination with the reagents evaluated.

Purpose of Review: By definition, rare factor deficiencies have a prevalence of less than 200,000 in the US population, or an incidence of less than one in 2000 in Europe. The very small numbers of patients with rare disorders present challenges in diagnosis, evaluation of bleeding risk and treatment. Use of new assays, full genome sequencing, and global clotting assays will significantly improve diagnosis of patients with rare bleeding disorders., Recent Findings: In addition to new assays available for monitoring patients, new therapy, both recombinant and plasma derived, is now available. Registries and clinical trials have demonstrated decreased bleeding and improved outcomes when patients are treated with these agents. Expanding international registries have been initiated to correlate genotype and bleeding phenotype in conjunction with global assays., Summary: Ongoing research continues to expand our understanding of the pathophysiology of rare factor deficiencies. This work complements medical practice to incorporate early diagnosis and new treatment options for patients, resulting in safer and less sensitizing regimens and much improved clinical outcomes.

Children presenting with bleeding symptoms pose a diagnostic challenge. The evaluation should include a comprehensive medical and bleeding history, detailed family history, a physical examination, and selected laboratory tests. Evaluation of the bleeding history should include an objective quantitation of the bleeding symptoms, followed by objective quantitation of the family members' bleeding symptoms. If a bleeding disorder is suspected, based on personal and/or family bleeding history, specific laboratory testing is needed for attaining a diagnosis. The screening tests for coagulation factor deficiencies are considered reliable, routinely available, and easy to perform. However, for primary hemostatic defects, the various screening tests have not yet proven to be reliable and thus referral for a specialized hematology consultation and specific laboratory testing are needed. Perhaps a Bayes theorem approach, combining the personal and family bleeding history with the results of a screening test for a primary hemostatic defect would prove to be useful in the identification of those children who will most benefit from specific laboratory testing., (© Thieme Medical Publishers.)

Inherited antithrombin deficiency is estimated to carry a 50% risk of a venous thrombotic complication during each pregnancy and puerperium. We present a case of a female with heterozygous type I antithrombin deficiency who presented with a central nervous system transverse sinus thrombosis in the third trimester of pregnancy despite the use of therapeutic doses of low molecular weight heparin, as venous thromboembolic prophylaxis, since conception. A successful pregnancy outcome was achieved with the combined use of therapeutic anticoagulation and regular plasma-derived antithrombin concentrate infusions to normalize her antithrombin levels. This case lends further debate to the issue of whether antithrombin concentrate, in addition to anticoagulation, should be routinely administered for venous thromboembolic prophylaxis during pregnancy and the puerperium to women with inherited antithrombin deficiency. This point may become more relevant as further experience is gained with the use of recombinant human antithrombin., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

Congenital dysfibrinogenemia is a rare disease characterised by inherited abnormality in the fibrinogen molecule, resulting in functional defects. Two patients, a 26-year-old woman and a 61-year-old man, both with history of thrombotic events, had abnormal coagulation test results. DNA sequencing showed the heterozygous gamma Y363N mutation (Fibrinogen Praha III) and the heterozygous Aalpha N106D mutation (Fibrinogen Plzen), respectively. Fibrin polymerisation, after addition of either thrombin or reptilase, showed remarkably delayed polymerisation in both cases. Fibrinolysis experiments showed slower tPA initiated lysis of clots. SDS-PAGE did not show any difference between normal and Praha III and Plzen fibrinogens. Both mutations had a significant effect on platelet aggregation. In the presence of either ADP or TRAP, both mutations caused the decrease of platelet aggregation. SEM revealed abnormal clot morphology, with a large number of free ends and narrower fibres of both fibrin Praha III and Plzen. Praha III mutation was situated in the polymerisation pocket "a". The replacement of the bulky aromatic side chain of tyrosine by the polar uncharged small side chain of asparagine may lead to a conformational change, possibly altering the conformation of the polymerisation pocket. The Plzen mutation is situated in the coiled-coil connector and this replacement of polar uncharged asparagine residue by polar acidic aspartate changes the alpha-helical conformation of the coiled-coil connector; and may destabilise hydrogen bonds in its neighborhood. Although both mutations are situated in different regions of the molecule, both mutations have a very similar effect on fibrinogen functions and both are connected with thromboses.

Congenital combined deficiency of the vitamin K-dependent coagulation factors is a rare bleeding disorder caused by either a defect in the gamma-glutamyl carboxylase or the vitamin K epoxide reductase enzyme complex. The diagnosis should be considered when vitamin-K dependent factor activities are decreased and liver dysfunction, vitamin K deficiency, and factitious coumarin ingestion have been excluded. We report a case of VKCFD in a child resulting from compound heterozygosity for two novel splice site mutations of the gamma-glutamyl carboxylase gene. Oral vitamin K supplementation resulted in partial resolution of proteins and complete resolution of bleeding., (Copyright 2009 Wiley-Liss, Inc.)

Whether severe coagulation factor deficiency can cause adverse pregnancy outcomes or recurrent fetal loss is not definitely known. We report here on five women with severe deficiency of coagulation factors (two factor X, one factor XI, one factor VII and one von Willebrand factor) who presented with history of unexplained fetal loss or with adverse pregnancy outcome. Detailed investigations of thrombophilia showed that four patients were positive for antiphospholipid antibodies, one of whom was also homozygous for the plasminogen-activator inhibitor-1 4G/4G polymorphism, and the fifth patient was deficient for protein C. Despite the concomitant presence of both coagulation factor defect and thrombophilia, fetal loss may be attributed to factor defect that in reality is a red herring, with underlying thrombophilia not being evaluated.

Background and Objectives: Mucocutaneous bleeding (MCB) is the main expression of inherited disorders of primary hemostasis. However, the relative prevalence of these disorders, their clinical differential diagnosis, and the proportion of patients with MCB of unknown cause (BUC) after an initial comprehensive laboratory testing are unknown., Design and Methods: We studied prospectively 280 consecutive patients with MCB and 299 matched controls, using strict inclusion and exclusion criteria. A single physician recorded the clinical data in a bleeding score and estimated the severity of bleeding in clinical categories. Laboratory criteria for the diagnosis of von Willebrand's disease (VWD) and platelet function defects were established from reference values derived from controls., Results: Fifty patients (17.9%) had VWD (type 1VWD=45, type 2=5). Platelet function defects and mild clotting factor deficiencies were found in 65 (23.2%) and 11 (3.9%) patients, respectively. Thirteen (11.5%) patients had combined defects. The remaining 167(59.6%) patients had BUC, with prolonged bleeding time in 18.6% as their only abnormality. All these disorders, including BUC, were clinically undistinguishable. Moreover, no relationship was found between the severity of bleeding and VWF/platelet function variables., Interpretation and Conclusions: The diagnostic efficacy of a first laboratory testing in patients with hereditary MCB is 40.4%. Most patients have a disease(s) of high prevalence but unknown pathogenesis. Concurrent bleeding disorders in the same patient are frequent. Our results support the proposal that low plasma VWF levels, but also platelet function defects, should be considered risk factors rather than unequivocal causes of hemorrhages.

Normal prothrombin time (PT) and partial thromboplastin time (PTT) are recommended for administration of recombinant tissue-plasminogen activator (rt-PA) in stroke, but waiting for results can delay use. We examined the charts of 365 stroke patients to assess predetermined risk factors associated with elevated PT/PTT. Elevated PT/PTT can be predicted in patients taking warfarin or heparin/heparinoid or on hemodialysis, according to emergency department triage, with 100% sensitivity and 94.7% specificity. These results could be applied to rt-PA candidates and reduce potential delays.

Amyloid diseases can be associated with potentially life-threatening hemorrhage. Pathogenetic factors contributing to the abnormal bleeding tendency in this setting are heterogeneous and depend on the type of amyloidosis and pattern of organ involvement. In patients with light-chain (AL) amyloidosis, acquired hemostatic abnormalities, including coagulation factor deficiencies, hyperfibrinolysis, and platelet dysfunction, can be regarded as the most important pathogenetic factors. In patients with other types of amyloidosis, acquired hemostatic defects are rare, and amyloid deposition has also been reported to be the main cause of abnormal bleeding manifestations. Amyloid angiopathy with increased fragility of blood vessels and impaired vasoconstriction may promote bleeding in this setting. Rupture of solid organs caused by amyloid deposition also was reported. Whereas therapeutic options in bleeding caused by local amyloid deposition are restricted to supportive measures and, in severe cases, surgery, acquired hemostatic defects may be treated according to the causative mechanism. In this review, we focus on bleeding risks in patients with amyloid diseases. Current concepts with regard to pathophysiology, diagnosis, and treatment are summarized and discussed.

Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder with laboratory findings similar to those for congenital von Willebrand disease (VWD). Unlike the congenital disease, AVWS usually occurs in individuals with no personal or family history of bleeding. The prevalence of AVWS in the general population is unknown because data from large prospective studies of this syndrome are not available. Although AVWS is particularly frequent in lymphoproliferative or myeloproliferative disorders, it can also be associated with solid tumors, immunologic and cardiovascular disorders, and other miscellaneous conditions. Diagnosis of AVWS is based on assays measuring the activity of von Willebrand factor (VWF). This tends to be abnormally low, but factor VIII (FVIII) coagulant activity can sometimes be normal. FVIII/VWF inhibiting activity is found in only a minority of cases. Bleeding episodes in patients with AVWS are mostly of the mucocutaneous type and can be managed with desmopressin, plasma-derived FVIII/VWF concentrates, and intravenous immunoglobulin (IVIg). Recombinant activated factor VII can be useful in patients unresponsive to standard therapy. An updated version of the International Registry on AVWS, recently available online, will provide more information on this rare, but underdiagnosed and misdiagnosed, disorder.

The sensitivity of commercial prothrombin time (PT) tests was assessed based on a dilution series of equine pooled plasma (EPP) (experiment 1) and on 40 equine plasma samples with reduced activity of coagulation factors II, V, VII and X (experiment 2). Two different PT reagents (reagent 1, human placental thromboplastin; reagent 2, recombinant human tissue factor) were used according to the manufacturers' instructions (standard test, PT([ST])) and compared to a modified test procedure (modified test, PT([MT])) using sample dilution and fibrinogen addition. In all samples, sensitivity was lower (P<0.01) when using PT([ST]) with reagent 2 (0.20) than when using either PT([ST]) with reagent 1 (0.65) or PT([MT]) with both reagents (reagent 1, 0.60-0.75, reagent 2, 0.58-0.70, depending on sample dilution). The highest sensitivity was found for PT([MT]) when using a 1:20 sample dilution. In those samples in which at least one coagulation factor activity was decreased (by 20%; n=18), the sensitivity of PT([ST]) with reagent 2 (0.33) was found to be inadequate, in contrast to all other test procedures (0.83-0.94). This low sensitivity corresponded to shorter time intervals between different coagulation activity levels prepared by EPP dilution. The results indicate that adequate sensitivity of PT measurements in equine plasma can be achieved using a standard test procedure as long as a suitable reagent is used.

The Italian Association of Haemophilia Centres reviewed and finally approved in November 2004 the new Italian Guidelines for the diagnosis and treatment of patients with clotting factor inhibitors. The recommendations have been based on the identification of levels of clinical evidence derived from the systematic review carried out in 2003 by the School of Health and Related Research, the University of Sheffield, UK, and further integrated by clinical studies published from 2003 to 2004. The Italian guidelines consist of six major domains concerning inhibitor definition, epidemiology, risk factors, diagnosis, inhibitor eradication, management of bleeding episodes, in patients with congenital and acquired coagulation disorders, with 121 statements, 59 synthesis and 54 recommendations. We report here recommendations and open issues concerning the diagnosis and monitoring of inhibitors, inhibitor eradication and the management of bleeding in patients with haemophilia A and B.