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Case report In this article, we wanted to present our case in which we detected SMZL during examining for defects in coagulation tests and correlated the PT and aPTT elevation with the development of inhibitors against coagulation factors related to this disease. The PT and aPTT values of the patient diagnosed with MZL did not improve in the mixing test, and no other etiology was found. With the second course of chemotherapy, the patient's values improved.

Background & Aims Prophylactic administration of platelets and fresh frozen plasma (FFP) has been recommended in patients with cirrhosis with low platelets and/or prolonged international normalized ratio (INR) without scientific evidence to support this practice. In this analysis, we evaluated the use of prophylactic administration of blood products in outpatients with cirrhosis undergoing endoscopic band ligation (EBL). Methods This is a multicenter retrospective analysis of consecutive EBL procedures in patients with cirrhosis at 4 hospitals in Spain from 01/2010-01/2017. FFP and/or platelet transfusion were given at the discretion of the physician if INR was >1.5 and/or platelet count, Graphical abstract, Highlights • Multicenter analysis of prophylactic administration of blood products in 536 outpatients with cirrhosis undergoing EBL. • The prophylactic transfusion protocol was only followed in 16% and 28% of procedures with high INR and/or low platelets, respectively. • Post EBL-bleeding occurred in 26 patients – 4.8% of patients and in 2.2% of procedures. • Patients that bled had higher Child-Pugh and MELD scores compared to those that did not bleed. • There was no clear relationship between post-EBL bleeding and the baseline INR/platelet count before the procedure.

Background Recent reports of thrombotic events after SARS-Cov-2 vaccination raised concern. However, modifications of the most common coagulation parameters after vaccination are unknown. Aims We measured parameters of coagulation including (i) basic coagulation tests, (ii) procedures aimed to assess the ex-vivo potential capacity to generate thrombin and (iii) in vivo thrombin activity. We also assessed anti-platelet factor 4 (aPF4) with two methods. Design Laboratory measurements were performed for a cohort of subjects (n = 30) before (baseline) and after (7 and 21days after first dose, and 14days after second dose) SARS-Cov-2 vaccination. Results All subjects received the Pfizer-BioNTech vaccine, and none developed symptomatic thrombotic events during the study period. None of the parameters showed clinically relevant variations at different time-points before and after vaccination. Only platelet count showed a slight increase, and F1.2 and the thrombin generation parameters ETP and ETP-TM ratio, showed a small decline, at the last time-point after vaccination when compared to baseline. aPF4 was negative in all the subjects, except two, who were positive (one with the chemiluminescent and the other with the ELISA assay). Conclusions The study shows no modifications of the coagulation parameters nor the presence of biochemical signs of coagulation activation following the administration of the Pfizer-BioNTech vaccine.

This data article accompanies the manuscript entitled: “Prothrombotic Disturbances of hemostasis of Patients with Severe COVID-19: a Prospective Longitudinal Observational Cohort Study” submitted to Thrombosis Research by the same authors. We report temporal changes of plasma levels of an extended set of laboratory parameters during the ICU stay of the 21 COVID-19 patients included in the monocentre cohort: CRP, platelet count, prothrombin time; Clauss fibrinogen and clotting factors II, V and VIII levels, D-dimers, antithrombin activity, protein C, free protein S, total and free tissue factor pathway inhibitor, PAI-1 levels, von Willebrand factor antigen and activity, ADAMTS-13 (plasma levels); and of two integrative tests of coagulation (thrombin generation with ST Genesia) and fibrinolysis (global fibrinolytic capacity - GFC). Regarding hemostasis, we used double-centrifuged frozen citrated plasma prospectively collected after daily performance of usual coagulation tests. Demographic and clinical characteristics of patients and thrombotic and hemorrhagic complications were also collected from patient's electronic medical reports.

Thrombosis is a complex disorder involving interaction between many different types of genes with environment factors. Modification in genes coding for coagulation and regulatory proteins of hemostasis are important risk factors for thrombosis. Historically, the measurement of a clot endpoint has been the basis of coagulation testing mainly focused on bleeding disorders. Thrombosis diagnosis has slowly increased with most modern automated coagulation instruments utilizing optic, immunologic, and chromogenic methods. However, molecular diagnostics has added another dimension for the evaluation of clotting and bleeding disorders. DNA-based tests are commonly available for the detection of the factor V Leiden mutation, the prothrombin 20120A mutation, and the methyltetrahydrofolate reductase mutation.

The purpose of this review is to describe structure and function of the multiple proteins of the coagulation system and their subcomponent domains. Coagulation is the process by which flowing liquid blood plasma is converted to a soft, viscous gel entrapping the cellular components of blood including red cells and platelets and thereby preventing extravasation of blood. This process is triggered by the minimal proteolysis of plasma fibrinogen. This transforms the latter to sticky fibrin monomers which polymerize into a network. The proteolysis of fibrinogen is a function of the trypsin-like enzyme termed thrombin. Thrombin in turn is activated by a cascade of trypsin-like enzymes that we term coagulation factors. In this review we examine the mechanics of the coagulation cascade with a view to the structure-function relationships of the proteins. We also note that two of the factors have no trypsin like protease domain but are essential cofactors or catalysts for the proteases. This review does not discuss the major role of platelets except to highlight their membrane function with respect to the factors. Coagulation testing is a major part of routine diagnostic clinical pathology. Testing is performed on specimens from individuals either with bleeding or with thrombotic disorders and those on anticoagulant medications. We examine the basic in-vitro laboratory coagulation tests and review the literature comparing the in vitro and in vivo processes. In vitro clinical testing typically utilizes plasma specimens and non-physiological or supraphysiological activators. Because the review focuses on coagulation factor structure, a brief overview of the evolutionary origins of the coagulation system is included.

Background and objectives: Major burn surgery causes large hemorrhage and coagulation dysfunction. Treatment algorithms guided by ROTEM® and factor VIIa reduce the need for blood products, but there is no evidence regarding factor XIII. Factor XIII deficiency changes clot stability and decreases wound healing. This study evaluates the efficacy and safety of factor XIII correction and its repercussion on transfusion requirements in burn surgery. Methods: Randomized retrospective study with 40 patients undergoing surgery at the Burn Unit, allocated into Group A those with factor XIII assessment (n = 20), and Group B, those without assessment (n = 20). Erythrocyte transfusion was guided by a hemoglobin trigger of 10 g.dL−1 and the other blood products by routine coagulation and ROTEM® tests. Analysis of blood product consumption included units of erythrocytes, fresh frozen plasma, platelets, and fibrinogen. The coagulation biomarker analysis compared the pre- and post-operative values. Results and conclusions: Group A (with factor XIII study) and Group B had identical total body surface area burned. All patients in Group A had a preoperative factor XIII deficiency, whose correction significantly reduced units of erythrocyte concentrate transfusion (1.95 vs. 4.05, p = 0.001). Pre- and post-operative coagulation biomarkers were similar between groups, revealing that routine coagulation tests did not identify factor XIII deficiency. There were no recorded thromboembolic events. Correction of factor XIII deficiency in burn surgery proved to be safe and effective for reducing perioperative transfusion of erythrocyte units. Resumo: Justificativa e objetivos: A cirurgia no grande queimado causa hemorragia de grande porte e disfunção da coagulação. Os algoritmos de tratamento guiados por ROTEM® e fator VIIa reduzem as necessidades de hemoderivados, mas falta evidência em relação ao fator XIII. A deficiência do fator XIII altera a estabilidade do coágulo e diminui a cicatrização. Este estudo avalia a eficácia e a segurança da correção do fator XIII e sua repercussão nas necessidades transfusionais na cirurgia do queimado. Métodos: Estudo retrospectivo randomizado de 40 doentes submetidos à cirurgia na Unidade de Queimados alocados em grupo A com estudo do fator XIII (n = 20) e grupo B sem estudo (n = 20). A transfusão eritrocitária foi guiada por gatilho de hemoglobina de 10 g.dL−1 e os outros hemoderivados por testes de coagulação de rotina e ROTEM®. A análise do consumo de hemoderivados incluiu unidades de eritrócitos, plasma fresco congelado, plaquetas e fibrinogênio. A análise dos biomarcadores da coagulação comparou os valores pré e pós-operatórios. Resultados e conclusões: O grupo A (com estudo de fator XIII) e o grupo B apresentaram área de superfície corporal total queimada idêntica. Todos os doentes do grupo A revelaram déficit pré-operatório de fator XIII, cuja correção reduziu significativamente a transfusão de unidades de concentrado eritrocitário (1,95 vs. 4,05, p = 0,001). Os biomarcadores de coagulação pré e pós-operatórios foram semelhantes entre os grupos, revelaram que os testes de coagulação de rotina não identificam o déficit de fator XIII. Sem eventos tromboembólicos registrados. A correção do fator XIII na cirurgia do queimado revelou-se segura e eficaz na redução da transfusão perioperatória de unidades de eritrócitos. Keywords: Intensive care, Burned, Surgery, Coagulation and hemostasis, Factor XIII, Palavras-chave: Cuidados intensivos, Queimados, Cirurgia, Coagulação e hemostase, Fator XIII

Introduction Orthotopic liver transplantation (OLT) can be associated with significant bleeding requiring multiple blood product transfusions. Rotational thromboelastometry (ROTEM) is a point-of-care device that has been used to monitor coagulation during OLT. Whether it reduces blood loss/transfusions during OLT remains controversial. Materials and Methods We aim to compare ROTEM with conventional coagulation tests (aPTT, PT, INR, platelet count, fibrinogen) to guide transfusion of platelets, cryoprecip-itate, and fresh frozen plasma (FFP) during OLT over 3 years. Thirty-four patients who had transfusions guided by ROTEM were compared to 34 controls who received transfusions guided by conventional coagulation tests (CCT). Intraoperative blood loss, type/ amount of blood products transfused, and direct costs were compared between the two groups. Results The ROTEM group had significantly less intra-operative blood loss (2.0 vs. 3.0 L, p = 0.04) and fresh frozen plasma (FFP) transfusion (4 units vs. 6.5 units, p = 0.015) compared to the CCT group (2.0L vs. 3.0L, p = 0.04). However, total number of patients transfused cryoprecipitate was increased in ROTEM (n = 25;73%) as compared to CCT (n = 19; 56%), p = 0.033. The direct cost of blood products plus testing was reduced in the ROTEM group ($113,142.89 vs. $127,814.77). Conclusion. In conclusion implementation of a ROTEM-guided transfusion algorithm resulted in a reduction in intra-operative blood loss, FFP transfusion and a decrease in direct cost during OLT. ROTEM is a useful and safe point of care device in OLT setting.

In Wilson disease, a regular clinical, biological, and radiological follow-up is essential to ensure good efficiency and tolerance of treatment and observance. Ongoing monitoring should continue throughout life. It must be enhanced in the adolescents, when the follow-up is transferring in adult care and during pregnancy. The monitoring is quite different according to the phase of the disease. Indeed, the major difficulties are the risk of paradoxical worsening of neurological symptoms during the initial phase and the adherence to the treatment during the maintenance phase. The follow-up must also be adapted to the severity of the hepatic and neurological features. So, the regular evaluation is often carried out by a multidisciplinary team of specialists: pediatrician, hepatologist, neurologist, psychiatrist, dermatologist, physiotherapist, speech therapist, psychologist, dietician, and social worker. Biologists have also to be associated to these follow-up: blood cell count, liver function and coagulation tests, proteinuria, blood and urinary copper monitoring, etc. are essential to confirm the efficiency and the observance of treatment and to detect complications of treatment or overtreatment. Red flags concerning weak treatment compliance or copper deficiency need to be known.

The coagulopathy of liver disease is often multifactorial, and symptoms may be minimal for the degree of coagulopathy until the liver disease is very severe. Decreases in production of coagulation factors and associated disturbances in biliary function cause abnormalities that lead to both prothrombotic and hemorrhagic diatheses. Platelet counts are decreased due to decreased thrombopoiesis from the lack of thrombopoietin as well as increased destruction from hypersplenism. The pathologies that cause the hepatopathy often have an effect on hemostasis: alcohol, warfarin, autoimmune disorders, and hepatitis all have direct coagulopathic consequences. In this chapter, we hope to help the reader understand the intricate balance between clotting and bleeding in liver disease and to interpret the results of both routine and specialized coagulation testing in the assessment of the patient with liver disease that will result in optimal management for these complex patients.

In this chapter, errors in coagulation testing that are known to significantly affect test results have been addressed. The sources of these errors can be found in both pre-analytical and analytical phases of testing. Knowing the sources of errors is essential for trouble-shooting unexpected laboratory results that are not correlated with clinical information. There are also pre-analytical artifacts present in patient samples that cause rejection of samples by the laboratory. Similarly, interferences in analytical testing may produce unexpected results in coagulation testing. Knowing these artifacts and interferences should help clinicians to work with laboratory personnel to prevent future incidences or to pursue alternative methods of testing.

Major orthopaedic surgery is associated with an increased risk of venous thromboembolism. Direct oral anticoagulants (DOACs) are recommended as thromboprophylactic agents after orthopaedic surgery. Although routine monitoring of DOACs in general is not required, measuring DOAC concentration may be necessary in clinical settings. The effects of DOACs on routine coagulation assays in spiked material are studied extensively, however, few data are available on DOAC concentration in patients after major orthopaedic surgery. We measured trough and peak DOAC concentrations with UPLC-MS/MS and routine coagulation tests in a prospective study including 40 patients receiving thromboprophylactic treatment with dabigatran 220 mg od and 40 patients receiving rivaroxaban 10 mg od after major orthopaedic surgery. For rivaroxaban, the median trough concentration with UPLC-MS/MS was 17.1 ng/mL and median peak concentration was 149 ng/mL. The anti-Xa assay displayed a good correlation, but a positive bias in comparison to the reference method. Furthermore, trough levels were mostly below the LOD of the anti-Xa assay. For dabigatran, the median trough concentration with UPLC-MS/MS was 12.1 ng/mL, and median peak level was 80.8 ng/mL. A positive bias was found when results from coagulation assays were compared to UPLC-MS/MS data. However, the addition of glucuronidated metabolites to dabigatran concentration UPLC-MS/MS data generally resolved most of this bias. Age was found to have a significant influence on dabigatran pharmacokinetics, irrespective of kidney function, whereas no effect of age was found during rivaroxaban treatment. In both treatment groups, female subjects displayed faster pharmacokinetics in comparison to male subjects, although not reaching significance. We conclude that UPLC-MS/MS is the method of choice to measure trough concentrations of DOACs in patients after orthopaedic surgery. Current coagulation assays are not suited for this purpose. We found large heterogeneity in both peak and trough concentrations of DOACs, and showed that pharmacokinetics of novel oral anticoagulants may be influenced by age and gender. Whether patients with high or low trough concentrations are at increased risk for bleeding or thromboembolic events respectively remains to be established.

A 24-year-old man presented with impending ulceration of a large thigh swelling which appeared after minor trauma. Imaging revealed a large well-encapsulated lesion with no vascularity. He was diagnosed to have a pseudotumor and underwent successful excision of the mass under blood and cryoprecipitate cover. The unusual presentation was suspected to be secondary to a transient drug-induced factor XIII deficiency because the result of the final coagulation study was normal.

Background Postpartum haemorrhage (PPH) may be exacerbated by haemostatic failure. Based on data from trauma studies, empirical infusions of fresh frozen plasma (FFP) are often given during severe PPH if coagulation tests are unavailable. This study observed a cohort of women with moderate/severe PPH in whom FFP infusion was guided by the use of viscoelastometric point-of-care testing (VEPOCT) and clinical assessment. Methods Women were enrolled into this observational study when blood loss was measured or suspected to be about 1000 mL. If Fibtem A5 remained >15 mm, or bleeding stopped, FFP was withheld. If Fibtem A5 was ≤15 mm and bleeding ongoing women were randomised into an interventional study as previously reported. Clinical and laboratory outcomes were recorded. Results The study recruited 605 women and 98% had FFP withheld. The median (25th-75th centile) total blood loss was 1500 (1300-2000) mL with 300 (50-545) mL occurring after enrolment. Total blood loss was >2500 mL in 40/605 (6.6%) women. RBCs were transfused in 141/605 (23.3%) cases and 11 (1.8%) received ≥4 units. At least one invasive procedure was performed in 283/605 (46.8%) women. Level 3 care was required for 10/605 (1.7%) women. No women developed clinically significant haemostatic impairment. Conclusions A restrictive use of FFP guided by clinical assessment of bleeding and VE-POCT is feasible and did not result in clinically significant haemostatic impairment. Studies should compare the clinical and cost effectiveness of empirical FFP infusions, according to current guidelines, with a targeted use of FFP based on VE-POCT.

Background and aims Conventional coagulation tests (CCTs) in patients with cirrhosis only assess procoagulant factors and are poor predictors of bleeding risk. In spite of this knowledge, they are routinely used prior to invasive procedures, and attempts are made to correct these abnormalities before invasive procedures. These practices are not supported by the evidence and are harmful to the patients. Methods This prospective study included 150 patients of cirrhosis undergoing invasive procedures. CCTs [bleeding time (BT), clotting time (CT), international normalized ratio (INR), activated partial thromboplastin time (aPTT) and platelet count], thromboplastin generation time (TGT) and thromboelastography (TEG) were done in all patients, and they were observed for post procedural bleeding. None of the patients received prophylactic transfusion before the procedure. Results BT, CT and TGT were normal in all 150 patients. One of 150 patients developed clinically significant post procedural bleeding. No statistically significant association was seen among INR, aPTT, platelet count and Child class with bleeding. TEG values (R time and MA value) were normal in 61% and 75% patients respectively in spite of abnormal CCTs in most of them. Comparison of abnormal TEG values (R time and MA value) with INR and platelet count, respectively, in patients with no bleeding showed a statistically significant lower percentage of abnormal values of R time and MA value compared to INR and platelet count. Conclusion Abnormal CCTs in patients of cirrhosis do not predict bleeding risk. TEG may be useful in patients undergoing invasive procedures to assess bleeding risk and prevents erroneous prophylactic transfusions.

Antiphospholipid syndrome (APS) is a clinical condition characterized by recurrent thrombosis and obstetric-related adverse events associated with the persistent presence of antiphospholipid antibodies (aPL). The aPL family is a heterogeneous group of autoantibodies with different antigenic specificities. The dominant antigenic targets for aPL in APS are phospholipid-binding plasma proteins such as β2glycoprotein I (β2GPI) and prothrombin. In the laboratory, aPLs can be detected by a diverse range of tests. There is not a unique or gold-standard test for the diagnosis of APS. The aPL tests can be broadly categorized into two groups: solid-phase assays such as anticardiolipin antibodies, anti-β2GPI antibodies, antiprothrombin antibodies, or phosphatidylserine-dependent antiprothrombin antibodies, and functional assays characterized by their ability to prolong phospholipid-dependent coagulation tests, known as lupus anticoagulant. In this chapter, we detail the currently available laboratory assays for aPL detection, and discuss the value of each aPL as laboratory marker for APS.

The monitoring of bleeding and clotting risk is a critical component of patient care, as either excessive bleeding or abnormal clotting can have obvious and serious consequences for health and well-being and is related to many other co-morbidities. Classical hemostasis management has been around the monitoring of the time it takes activated blood samples to clot. Such techniques have moved from central laboratory tests to point-of-care technologies, allowing more effective management and treatment. However, such techniques are limited in the information they provide on clotting or bleeding status. There is a big move toward novel approaches which purport to be more representative of an individual's clotting and bleeding risk. These techniques are already being used in, and developed for, point-of-care applications.

This chapter addresses the preoperative assessment of surgical patients in terms of relevant history, including medication history and examination, as well as preoperative testing in order to assess risk of bleeding as well as take steps to minimize bleeding during the intraoperative and postoperative periods. Among preoperative testing, coagulation-based testing, blood bank testing and tests for liver and renal function are also discussed. Appropriate steps, such as holding various medications prior to surgery and administration of various blood products and other pharmacotherapeutic agents, are also addressed.

Hematology tests such as complete blood count and peripheral blood smear analysis are commonly ordered test in clinical laboratory ,and sources of errors in such tests could be preanalytical, analytical, or postanalytical. Errors related to sample collection, transport, and storage must be avoided to get accurate results in hematology testing. The amount and concentration of dipotassium ethylenediaminetetraacetic acid (EDTA) in the collection tube require that blood should be collected up to a specific mark on the tube. If too little blood is collected, dilution of the sample can become an issue with alteration of parameters. Relative excess EDTA in such cases also affects the morphology of blood cells. Transport of specimen should ensure that high temperatures are avoided. Red cell fragmentation is a feature of excess heat. Falsely high white blood cell (WBC) counts are more common than falsely low WBC counts. In certain situations, hematology analyzers are known to provide a falsely low platelet count when the true platelet count is adequate. Other common sources of errors in hematology and coagulation tests are also discussed.

The Liver is one of the organs involved in the multiorgan failure that occurs in sickle cell disease, the pathophsiology of liver disease in this condition is complex because of the interrelated multifactorial causes. Liver dysfunction was assessed in both paediatric and adult sickle cell disease patients in the steady state. The transaminases and alkaline phosphatase were analysed by automation while coagulation studies were done manually. The mean (range) of Alanine transaminase (ALT), Aspartate transaminase (AST) and alkaline phosphatase (ALP) were 23.0 (2-77) IU, 48.5 (15-120) IU, 227.5 (37-1200) IU respectively. ALT and AST levels were less than 100 IU in over 95% of the patients. The gender or age of the patients did not significantly affect the level of these three enzymes. There was close association between the liver size and elevation of the liver enzymes except for alkaline phosphatase (ALT = .017, AST = .009, ALP = .056). Twenty-five percent of the patients had normal enzymes while 13% had derangement of the three enzymes, 19%, 50% and 74% had abnormal ALT, AST and ALP respectively. Only 22% and 5% had deranged PT and APTT respectively. In conclusion minimal elevation of the tramsaminases which is not gender or age dependent were observed in steady state sickle cell disease, higher levels of alkaline phosphatase may be due to associated vasoocclussive crises involving the bones rather than a pathology of the liver.

Acute kidney injury (AKI) occurs in over half of patients with acute liver failure. Despite prolonged prothrombin times and thrombocytopenia, continuous renal replacement therapy circuits frequently develop clots during patient treatment. Here we assessed factors contributing to this by measuring coagulation parameters (standard coagulation tests, pro- and anticoagulant factors, thromboelastography, and thrombin generation) in 20 consecutive patients with acute liver failure; mean age 42 years. Within 48 h, 10 had developed stage 3 AKI and 9 required continuous renal replacement therapy, of whom 2 had frequent circuit clots. The patients with stage 3 AKI were found to have significantly lower platelet counts and levels of factor V and the natural anticoagulants antithrombin, Protein C and Protein S, but increased extrinsic pathway activation and von Willebrand factor levels. Tissue factor levels were greater in those with stage 3 AKI, as was microparticle activity. Although patients with acute liver failure and advanced AKI requiring continuous renal replacement therapy have an even more marked thrombocytopenia and more prolonged extrinsic pathway activation, this was not associated with increased bleeding. Thus, more frequent circuit clots during continuous renal replacement therapy appear to be due to a combination of increased tissue factor and microparticle release, endothelial activation, and reduction in natural anticoagulants., peer-reviewed

This chapter addresses errors in hematology and coagulation testing that are known to significantly affect test results. The sources of these errors can be found in both pre-analytical and analytical phases of testing. Knowing the sources of errors is essential for troubleshooting unexpected laboratory results that are not correlated with clinical information. There are also pre-analytical artifacts present in patients’ samples that cause rejection of samples by the laboratory. Similarly, interferences in analytical testing may produce unexpected results in hematology and coagulation testing. Knowing these artifacts and interferences should help clinicians to work with laboratory personnel to prevent future incidences or to pursue alternative methods of testing.

Publisher Summary This chapter presents an introduction to coagulation testing. The clinical laboratory analysis of in vitro coagulation function is fairly a complicated task and coagulation testings are further compromised by the necessity of highly artificial testing environment. The advances and ongoing innovations greatly improve laboratory analysis of coagulation medicine and careful attention must be paid to rapidly evolving literature and stringent criteria must be applied to the diagnostic validity of new and existing methodologies. An analysis of proteins involved in coagulation is described including measurement of its activity, measurement of the protein and measurement of gene sequence. Other factors involved in coagulation testing include analyzing the cellular biology of coagulation, analysis of therapeutic interventions and integration of coagulation laboratories with patient care. The landscape of coagulation medicine is further complicated by the fact that evidence-based data are lacking for many assays of coagulation, especially given the rapid pace of development of new systems or modification of existing tests.

The hematologist may be consulted to see a patient with bleeding in order to make a diagnosis, suggest therapy, and advise regarding follow-up. While a large variety of coagulation tests are available, none are perfect. Nevertheless, the astute clinician usually can produce a differential diagnosis. However, many hemorrhaging patients have no abnormal test results, while other patients with several abnormal coagulation tests may not be hemorrhaging, or their hemorrhaging is not a consequence of the test results. The value of the physical examination, medical history of the patient and their family, examination of the peripheral blood smear, and results of a modest battery of tests will typically reveal a path to pursue and secure the appropriate diagnosis. As any correct treatment depends on a correct diagnosis, this craft is important to learn.

This chapter discusses the qualitative disorders of coagulation. Endothelial cells secrete substances that repel platelets, initiate coagulation, promote platelet adhesion, platelet aggregation, and fibrin dissolution, catalyze the inhibition of thrombin, and inhibit the initiation of fibrin dissolution. The fibrinolytic system provides a mechanism for removal of physiologically deposited fibrin and the clotlysis is brought about by the action of plasmin on fibrin. In addition to the physiologic role of fibrinolysis, other inhibitors also play critical roles in the control of hemostasis. The participation of platelets is a fundamental component of the physiologic process of coagulation in hemostasis. Platelet interactions in coagulation are initiated by adhesion to areas of vascular injury. The plasma coagulation factors are produced in the liver and factor VIII is produced by endothelial cells. The evaluation of a patient for a hemostatic defect can be done by: detailed history, complete physical examination, and laboratory evaluation. Coagulation factor screen tests: prothrombin partial thromboplastin time assays. The acquired coagulation factor disorders are vitamin K deficiency, hepatic dysfunction, and disseminated intravascular coagulation. Isolated coagulation factor disorders are hemophilia A and B. The genetics, prevalence, coagulation studies, and symptoms of inherited coagulation factor disorders are described.

Lupus anticoagulants (LACs) are immunoglobulins that interfere with in vitro phospholipid-dependent coagulation tests. Initially recognized in the context of systemic lupus erythematosus (SLE), LACs are now known to occur frequently in patients with other disorders, as well as in individuals with no apparent underlying disease. It is now recognized that LACs and anti-cardiolipin antibodies (ACAs) are separate entities, but both are members of a diverse group of immunoglobulins that bind to phospholipid-bound proteins including, but not limited to, β2-glycoprotein-I (β2GPI) and prothrombin. LACs and ACAs are both associated with a high risk of arterial and venous thromboembolicevents, although the mechanism of this thrombotic risk is still unknown. In this chapter the term “anti-phospholipid antibody (APL)” is used to denote the presence of a LAC and/or ACA. The chapter discusses the diagnosis of LACs, the possible thrombotic mechanisms associated with APLs, and the treatment of thrombosis in patients with APLs.

Publisher Summary This chapter focuses on lupus anticoagulants (LAs), which are immunoglobulins (usually IgG, IgM, or a mixture of both) that interfere with in vitro phospholipid-dependent coagulation tests, such as prothrombin times (PT), activated partial thromboplastin time (APTT), Russell viper venom time (RVVT), and Textarin time. They may be encountered in various clinical settings including autoimmune diseases and non-autoimmune conditions, malignancies, certain medications, and infectious diseases. They are detected serendipitously as a result of routine coagulation testing, and their prevalence in various clinical conditions is widely variable. The high degree of variation seen between laboratories may be due to the choice of test systems. The wide disparity of LA detection may reflect a variety of variables including patient selection, test sensitivities, and response to treatment. The identification of LA in patients is important since its presence indicates a greater risk of thromboembolic complications. In addition to the association of LA/anticardiolipin (aCL) antibodies with venous/arterial thromboembolic events, other clinical complications include recurrent spontaneous abortion, transverse myelopathy, and nonbacterial thrombotic endocarditis. Drug-induced LA has been associated with a various medications including hydralazine, chlorpromazine, quinidine, and procainamide. The heterogeneity of LAs explains the multiple laboratory assay systems necessary to detect these inhibitors.

This chapter focuses on two topics: the animal models used for evaluating the pathobiology of venous thrombosis and their usefulness, reliability, and limitations; and the techniques employed to generate and evaluate experimental data. Many laboratories have applied animal models and techniques to better define two broad areas in coagulation biology: heparin anticoagulation and its reversal with protamine sulfate and protamine-like agents; and the inflammatory response associated with venous thrombosis. Protamine sulfate reversal of standard unfractionated heparin and low-molecular-weight heparin (LMWH) anticoagulation may cause adverse side effects such as decreased mean arterial blood pressure (MAP), decreased cardiac output (CO), decreased oxygen consumption ( V O2 ), and thrombocytopenia. Due to the unpredictable toxic effects of protamine sulfate, there is a need to develop nontoxic protamine variants that can effectively reverse the anticoagulant effects of heparin and its LMWH fragments. In the investigation of polycationic compounds, it is necessary to develop an animal model to evaluate the hemodynamic and physiologic effects of these compounds used alone and in combination with heparin and LMWH. The dog ( Canis familiaris ) has proven to be an excellent animal model to evaluate hemodynamic and hematologic toxicity of anticoagulation reversal and antithrombotic agents. The hemodynamic effects of heparin, LMWH, and protamine reversal in dogs are very similar to those in humans, but magnified on a scale that allows thorough evaluation. Additionally, the same coagulation tests used in humans may be used in dogs.

Publisher Summary This chapter discusses current understanding of coagulation and fibrinolytic systems, their clinical impact, and variables affecting the laboratory assessment of thrombotic and bleeding disorders. It begins with the introduction about coagulation and fibrinolysis. Then goes on to discuss basic concepts of coagulation—namely, role of platelets, activation of the coagulation cascade, contribution of leukocytes to coagulation, and inhibitors of coagulation. Next the chapter has discussed about basic concepts of fibrinolysis—namely, fibrinolysis activators and fibrinolysis inhibitors. Anticoagulant theory is also discussed in the chapter wherein conventional heparin, low-molecular-weight heparins, oral anticoagulant therapy, thrombin inhibitors, and platelet inhibitors are described. The chapter has also explored clinical aspects and discussed about molecular defects, laboratory assessment of hemostatic activation, antiphospholipid antibodies, and the study of platelet function by flow cytometry. The chapter then turns to the discussion of nonanalytical variable affecting the laboratory evaluation of hemostasis—namely, preanalytical variables affecting global coagulation tests, minimizing in vitro platelet activation and preanalytical variables affecting analysis of fibrinolysis. Strategy for the systematic examination of thrombophilia is also discussed in the chapter. Finally the chapter closes with the conclusion stating that our concepts of coagulation and fibrinolysis are still evolving. The increasing use of molecular methods in the laboratory has uncovered new mutations and has facilitated the understanding of the genetic basis of hemostatic defects.

The existing techniques to evaluate hemostasis in clinical laboratories are not sensitive enough to detect hypercoagulable and mild hypocoagulable states. Under different experimental conditions, the thrombin generation test may meet these requirements. This technique evaluates the overall balance between procoagulant and anticoagulant forces and has provided new insights in our understanding of the coagulation cascade, as well as of the diagnosis of hypocoagulability and hypercoagulability conditions. Thrombin generated in the thrombin generation test can be quantified as platelet-rich or platelet-poor plasma using the calibrated automated thrombogram method, which monitors the cleavage of a fluorogenic substrate that is simultaneously compared to the known thrombin activity in a non-clotting plasma sample. The calibrated automated thrombogram method is an open system, in which different antibodies, proteins, enzymes and peptides can be introduced to answer specific questions regarding hemostatic processes. The thrombin generation test has great clinical potential, such as in monitoring patients taking anticoagulants and antiplatelet drugs, screening for genetic or acquired thrombotic disorders, and evaluating bleeding risk control in patients with hemophilia using bypass agents or replacement therapy. Different to conventional coagulation tests, the thrombin generation test can be used for an overall evaluation of hemostasis, the results of which can then be used to evaluate specific characteristics of hemostasis, such as prothrombin time, activated partial thromboplastin time, and levels of fibrinogen and other coagulation factors. The introduction of this method will contribute to a better understanding and evaluation of overall hemostatic processes; however, this method still requires standardization and clinical validation.

This chapter presents the tests for blood coagulation. To study patients thought to be suffering from blood clotting defects, various tests may be carried out. The simplest of these are the platelet count, bleeding time, whole blood clotting time, and Quick's one-stage prothrombin time. Normal results in these four tests do not exclude hemorrhagic disorder. Further tests are necessary before the patient can be said to have a normal clotting mechanism. Capillary blood can be used for the platelet count test. The chapter also explains Duke's method and Ivy's method for bleeding time. Quick's one-stage prothrombin time technique is so-called because it was designed to measure prothrombin on the basis of Morawitz's Classical Theory of Coagulation .

Publisher Summary This chapter focuses on coagulation studies on thoracic duct lymph in hypovolemic shock dogs. Progressive interstitial edema following increased microvascular permeability is a central feature in the disseminated intravascular coagulation. The chapter describes a study in which 50 ml blood per minute was taken from 20 healthy adult Basset dogs under pentobarbital narcosis from the right femoral artery up to an arterial mean blood pressure of 40 mm Hg, with simultaneously measuring of arterial and central venous blood pressure and monitoring of EGG. Thoracic duct lymph is normally known to have coagulation properties, but the coagulation activity is significantly lower than that of plasma. Endogenous coagulation factors are a good tool to study the relationship between molecular size and transcapillary escape. The increase activities of coagulation factors in lymph might arise from an increased transcapillary filtration, from mobilization of factors out of extravascular pools by a washout and extravascular partial activation of some factors, and from a decreased peripheral lymph flow.

Publisher Summary This chapter discusses the equipment and general requirements for the coagulation laboratory. Most coagulation studies are performed at constant temperatures and water baths supplied with a thermostat accurate enough to maintain a water temperature of 37±1°C are widely used. Glass or plastic straight-walled containers are preferable to round containers because they allow observation and magnification without removing tubes. A good light source placed at the side or the bottom of the container is helpful. The prerequisite for reproducable and quantitative results in blood coagulation tests is a clean venipuncture. Minimal contamination with tissue thromboplastin or contact of the blood with human skin or dirty glassware are factors that invariably produce serious errors. Most routine assays are performed in buffer systems covering the physiologic pH range of the blood. The use of compounds capable of binding ionized calcium is indispensable in coagulation work. Trisodium citrate, sodium oxalate, and disodium ethylenediamine tetracetate are all highly effective. Cationic exchange resins such as Dowex 50 or Amberlite IRC-50 can be used to remove ionized calcium from blood.