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The article focuses on the necessity of Iodoacetamide blank compensation in FXIII functional assays, particularly in the context of inherited factor XIII deficiency, a rare bleeding disorder affecting 1 in 2,000,000 people. Topics include the tetrameric structure of plasma FXIII, the role of activated FXIII (FXIIIa) in stabilizing the fibrin network, and the life-threatening bleeding consequences associated with severe inherited FXIII deficiency, emphasizing its impact on coagulation.

Severe coronavirus disease 2019 (COVID-19) is characterized by hyperinflammation, vascular damage, and hypercoagulability. Insufficient responses of Annexin A1 (AnxA1), a pro-resolving inhibitor of neutrophil infiltration and activation, might contribute to a severe course of the disease. We longitudinally evaluated AnxA1′s role in terms of inflammation, vascular damage, and clinical outcomes in a large prospective cohort of patients with COVID-19. AnxA1 was measured at presentation and during follow-up in the sera of 220 consecutive patients who presented at our hospital during the first wave. AnxA1 was significantly higher in the moderate and severe cases of COVID-19 compared to the healthy controls. Elevated AnxA1 was associated with markers of inflammation and endothelial damage. AnxA1 was significantly higher in patients with thrombotic events and ICU admission. Multivariable logistic regression indicated baseline AnxA1 (per ten units) as a predictor of thrombotic events. Linear mixed models predicted that AnxA1 tended to increase more steeply over time in patients without adverse events, with a statistically significant rise in patients without thrombotic events. These findings might reflect an insufficient increase in AnxA1 as a response to the excessive hyperinflammation in COVID-19. Future studies should evaluate whether hyperinflammation could be reduced through the administration of human recombinant AnxA1 or Ac2-26 peptide. [ABSTRACT FROM AUTHOR]

Background: Lupus anticoagulants (LA) are detected by prolongation of clotting times for dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) screening tests. Direct oral anticoagulants (DOACs) can interfere with both screening and confirmatory tests. The present study aimed to investigate the influence of direct factor Xa inhibitors (DiXaIs) on screen, confirm and mixing tests and establish a method for differentiation from other sample types. Materials and methods: A total of 257 samples including nonanticoagulated LA positive, LA positive with DiXaIs, factor deficiency, FVIII inhibitors, warfarin and non‐APS DiXaIs were tested. APTT reagents Cephen LS/Cephen and dRVVT reagents LA1/LA2 were used, respectively, to screen/confirm the study group. Index of circulating anticoagulant (ICA) was calculated from clotting times based on the following formula as ICA screening and ICA confirmation. ICA= (1:1 Mix sample – Normal pooled plasma) / Screen patient x 100. An ICA matrix was established which suggested the presence of a DiXaI when both ICA screening and confirmation were above the cut‐off. When only ICA screening is elevated, LA is suspected. Results: Sensitivity and specificity of the ICA matrix were 52.2% and 92.8% for DiXaIs and 38.1% and 96.7% for LA in APTT, and 61.2% and 92.9% for DiXaIs and 22.2% and 88.4% for LA in dRVVT, respectively. Conclusion: The ICA matrix achieved high specificity with a lower apparent sensitivity for DiXaI samples comparatively to other devices but due only to less interferences: the matrix could contribute to differentiating DiXaIs from LA in samples where anticoagulation status is unknown. [ABSTRACT FROM AUTHOR]

Introduction: This review concerns biomarkers of blood activation and their measurement in plasma, as described in the literature up to 2019, and from our personal experience. How their measurement can be informative and useful, for research studies or for diagnostic applications, is presented and discussed. Areas covered: Biochemistry and regulation of blood clotting cascade were elucidated these past decades. Understanding the various molecular mechanisms which initiate, amplify, and propagate coagulation has allowed measuring biomarkers as early indicators of hemostasis activation. They present a high usefulness for detecting hemostasis and fibrinolysis abnormalities in the clinically silent period. Sophisticated technologies are necessary for their measurement, as biomarkers are present at very low concentrations (from <1.0 ng/ml to >1.0 µg/ml). Expert opinion: Many analytes provide useful information for pathological evolution and disease severity, but only DDimer has entered routine applications. Limitations of biomarkers concern their short half-lives, their low plasma concentration, and their rapid variations. DDimer escapes these limitations and has become the biomarker of choice for ruling out DVT and PE and has increasing applications for adjusting anticoagulation or predicting diseases. Promising emerging biomarkers concern cell releasable proteins, extracellular vesicles, activated blood cells and cell aggregates, pathological metabolites, and degradation products. [ABSTRACT FROM AUTHOR]

Although suspected conceptually in the 60 s, Protein C and Protein S activities in hemostasis were investigated and reported from the mid-80 s, followed by the discovery of Thrombomodulin, an endothelial cell membrane associated protein, playing the most important heamostatic role. These 3 proteins act in regulating thrombogenesis and protecting against thrombo-embolic events. When blood is activated, any trace of circulating thrombin is captured by Thrombomodulin in the microcirculation, making thrombin become an anticoagulant through its capacity to activate Protein C to Activated Protein C, which operates as a sentinel in blood coagulation, in the form of a complex with free Protein S, to block any new blood activation site, and more especially circulating activated Factors V and VIII. Protein S not only acts as the Activated Protein C cofactor, but also as the cofactor of Tissue Factor Pathway Inhibitor. In addition, it has some functions in the complement pathway through its binding to C4b-BP. Another capability of activated protein C is to lower fibrinolytic activity, as the Activated Protein C Inhibitor is also known as Plasminogen Activator Inhibitor 3. The Protein C-Protein S system becomes less efficient in the presence of mutated Factor V (Factor V-Leiden or other variants), which is resistant to its inactivating effect. Other pathologies linked to this system concern the development of allo- or auto-antibodies to Protein S or to thrombin, which can generate severe thrombotic complications in affected patients. Some antithrombotic drugs have originated from this regulatory system. Protein C or Protein S concentrates are used for treating deficient patients. Activated Protein C (especially in patients with sepsis) or Thrombomodulin are proposed as antithrombotic medications. Most importantly, congenital or acquired Protein C or Protein S deficiencies are associated with severe recurrent thrombotic events. From the clinical standpoint most of the patients are heterozygous, as homozygosity is almost incompatible with life in the absence of a continuous and efficient treatment. Laboratory investigation of this highly complex system involves many different specialized assays for measuring these 3 proteins' activities, their antigenic content or their genetic sequence. The Protein S in-vitro anticoagulant activity is weak and contrasts with its high antithrombotic role in-vivo, showing that diagnostic assays have not yet succeeded in reproducing all the natural mechanisms for evidencing the anticoagulant role of Protein S. This paradoxal notion is discussed and illustrated in this manuscript as well is a revisit of the major characteristics and pathophysiological functions of the Protein C-Protein S-Thrombomodulin system; the associated pathologies; and the main laboratory tools available for clinical diagnosis. In respect to future perspectives, we also focused on developing more significant and relevant assays, especially for Protein S, thanks to the understanding of its biological roles. [ABSTRACT FROM AUTHOR]

The contact system initiates the intrinsic pathway of coagulation and is started by Factor XII activation, which then activates prekallicrein to kallicrein and Factor XI to Factor XIa and, in the presence of high molecular weight kininogen, forms a "contact phase activation loop", that amplifies Factor XII activation. FXII deficiency is not associated with bleeding tendencies, but when the blood clots, the thrombus is less dense, thus favoring antithrombotic protection. Activated Factor XII inhibition emerges as an efficient target for preventing thrombo-embolic diseases without inducing a hemorrhagic risk. Activated Factor XII exhibits other activities, in that it can activate complement and provoke inflammation, contributing to innate immunity. It also stimulates fibrinolysis through uPA activation from scu-PA. Among the other components of the contact phase, Factor XI has a more important role in coagulation pathways and can directly activate FX, FVIII and FV, in a FIX independent pathway. Its deficiency is associated with a mild bleeding diathesis ("pseudo-hemophilia" or hemophilia C), with a variable incidence among kindreds. Recently, the occurrence of thrombotic events the same day following infusion of immunoglobulin concentrates has been demonstrated to be caused by the presence of trace amounts of activated Factor XI, pointing out the key role of this factor for thrombogenicity. Prekallicrein can be activated at the endothelial surface in the presence of high molecular weight kininogen, whose cleavage generates bradykinins and contributes to vessel tonicity and inflammation. The contact phase, through its activation loop, is then an important physiological system, which can initiate and regulate various biological functions and is at the crossroads of various biological activities. Many of the body's physiological functions are intimately linked between them, making the global approach of special usefulness for understanding the interactions which can result from any abnormality of one of them. New pharmaceutical drugs targeting a defined activity need to be investigated for all the possible interferences or side effects. In this article we aim to present and summarize the present understanding of contact phase system activation and regulation, its involvement in various physiological functions, and the laboratory tools for its exploration. [ABSTRACT FROM AUTHOR]

Introduction: A prolonged activated partial thromboplastin time (APTT) may be indicative of a specific or multiple factor deficiency, therapeutic anticoagulation, presence of a nonspecific factor inhibitor, or lupus anticoagulant (LA). Recently, pairing of the LA‐sensitive APTT and standard APTT reagents, Cephen LS and Cephen, respectively, has been shown to be effective in LA detection. The present study aimed to evaluate the usefulness of this reagent pair for discriminating between causes of APTT elevation and the detection of LA in conjunction with dilute Russell's viper venom time (dRVVT). Methods: Plasma samples from 50 normal and 105 non‐anticoagulated LA‐positive patients in routine dRVVT and/or dilute APTT (dAPTT) via the percent correction formula were employed. Cephen LS/Cephen and dRVVT reagents LA1/LA2 were used to screen/confirm, respectively. Thirty‐four symptomatic LA‐negative, 25 warfarinised non‐antiphospholipid syndrome, 6 coagulation inhibitors, 17 samples with hereditary elevated APTT, and 24 FVIII/IX/XI/XII and 17 FII/V/X artificial single deficiency plasmas were used. Results: Thirty‐three samples out of 105 (31%) were LA‐positive in Cephen LS/Cephen. The total percent positivity in Cephen LS/Cephen and LA1/LA2 pairs was 89.1% against samples with the routine dRVVT/dAPTT double positive. The percent corrections of Cephen LS/Cephen in the routine dAPTT/dRVVT positive group were significantly higher than those in all other groups. Conclusions: The percent correction of the APTT reagent pair showed higher values in LA‐positive samples. The combination will be useful with respect to differentiating LA from other abnormal samples and is effective in LA detection when paired with dRVVT. [ABSTRACT FROM AUTHOR]

Abstract This manuscript aims to provide a concise review on current diagnostic/ confirmatory strategies of Heparin Induced Thrombocytopenia (HIT) with the combined use of immunological / functional assays in addition to the clinical probability. Laboratory diagnosis of HIT is of primordial importance as the related complications could become rapidly severe and life-threatening and can provoke limb amputation in some cases. The first action in the presence of HIT suspicion is to withdraw heparin and to initiate an alternative anticoagulant. Whilst vitamin K antagonists are not appropriate, anticoagulant options include Fondaparinux, Sodium Danaparoid, DOACs, Argatroban, and Bivalirudin. However, if HIT is excluded, patients can benefit again from the high therapeutic and antithrombotic efficacy of this drug, which remains superior to all the substitutive anticoagulant treatments. HIT is suspected in the presence of a platelet count drop > 50% on 2 successive counts, or a platelet count < 100 G/L, and of a significant clinical probability (4 Ts score). Testing patients' plasma is required for establishing the diagnosis. Laboratory investigation involves first the immunological measurement of heparin dependent IgG antibodies (mainly targeted to Heparin-Platelet Factor 4 complexes). When positive, a functional assay for platelet activation, performed at a low and high heparin concentration, allows confirming this disease. In any case, if the immuno-assay is negative, HIT can be excluded with a high probability, and heparin can be continued (if clinical examination favors this decision). Conversely, the higher the IgG antibody concentration is (and affinity), the higher is the probability of developing HIT. The functional assay has now become for confirming the platelet activation capacity of antibodies, and therefore confirming the presence of HIT. Up to now, the gold reference method for testing antibody-dependent platelet activation is the C14-Serotonin Release Assay, available only in very few laboratories working with radio-isotopes. A simple, sensitive, and accurate flow cytometry assay becomes now available to all clinical sites, and it can be easily used for testing the capacity of heparin dependent-antibodies to activate platelets, at low heparin concentration. This technique can be performed in any laboratory equipped with a flow cytometer and can make the HIT confirmation diagnosis rapidly available, which introduces a great improvement for management of patients with HIT. We believe that an evidence–based update on this topic is timely and well warranted. [ABSTRACT FROM AUTHOR]

Background: Antithrombin (AT) is a critical regulator of coagulation, and its overall activity is typically measured using functional tests. A large number of molecular forms of AT have been identified and each individual carries multiple molecular proteoforms representing variable activities. Conventional functional tests are completely blind for these proteoforms. A method that ensures properly defined measurands for AT is therefore needed. We here assess whether mass spectrometry technology, in particular multiple reaction monitoring (MRM), is suitable for the quantification of AT and the qualitative detection of its molecular proteoforms. Methods: Plasma proteins were denatured, reduced and alkylated prior to enzymatic digestion. MRM transitions were developed towards tryptic peptides and glycopeptides using AT purified from human plasma. For each peptide, three transitions were measured, and stable isotope-labeled peptides were used for quantitation. Completeness of digestion was assessed using digestion time curves. Results: MRM transitions were developed for 19 tryptic peptides and 4 glycopeptides. Two peptides, FDTISEK and FATTFYQHLADSK, were used for quantitation, and using a calibration curve of isolated AT in 40 g/L human serum albumin, CVs below 3.5% were obtained for FDTISEK, whereas CVs below 8% were obtained for FATTFYQHLADSK. Of the 26 important AT mutations, 20 can be identified using this method, while altered glycosylation profiles can also be detected. Conclusions: We here show the feasibility of the liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) technique for the quantitation of AT and the qualitative analysis of most of its molecular proteoforms. Knowing the measurands will enable standardization of AT tests by providing in-depth information on the molecular proteoforms of AT. [ABSTRACT FROM AUTHOR]

On demand and prophylaxis usage of FVIII/ FIX concentrates for the therapeutic management of hemophilia has greatly changed quality of life, and healthy life span of affected patients. Availability of recombinant therapeutic FVIII and FIX products, and of their long-acting variants, further improves the treatment constraints, and progressively permits to hemophiliacs to have an almost normal way of life. Unlimited amounts of recombinant or engineered substitutive products become available, and open new avenues for extending the benefits of prophylaxis to all hemophiliac patients, not only in economically advanced territories, but also in emerging and developing countries, worldwide. Pharmacokinetics of injected products can be variable among treated patients, and dependent on age. In addition, patient medical status, existing diseases, and the nature of joint damages can impact protective effect of substitutive products, and risks associated to way of life and activity. Product requirements and half-life of infused products are therefore patient specific. Monitoring recoveries of injected products thus provide useful information for the most appropriate treatment adjustment. FVIII and FIX measurements in plasma of treated patients helps to establish the optimal interval between injections for each treated patient, and the overall therapeutic cost. Due to the high variability from reagent to reagent, and the different behavior from plasma derived products, clotting methods are not ideal for recombinant and long-acting products. They require to be performed only in association with a drug specific calibrator. They are not recommended for patients’ survey, due to the high variety of reagents available. Chromogenic assays (2-stage methods) offer a standard reactivity to all available FVIII or FIX products in drugs, whether the way they are obtained, or in plasma. In a subset of treated patients, inhibitory antibodies to FVIII or FIX develop and can be measured with inhibition assays (Bethesda units), or by Elisa. Unfortunately, FVIII or FIX substitutive therapies cannot be used in patients with inhibitors, and alternative clinical management is requested, such as the use of FEIBA or FVIIa for their bypassing activity. A new treatment is being introduced in the form of a bispecific antibody (Emicizumab) targeted to both FIXa and FX, and which allows activating FX by FIXa without the need for FVIII. Some chromogenic assays (Biophen FVIII), designed with human proteins, offer the possibility to measure the activity and recovery of this new drug. Chromogenic methods are then useful for establishing potency of therapeutic products or monitoring recovery and kinetics in treated patients, through plasma measurements. Availability of International Standards for FVIII and FIX, in concentrates or plasma, allows harmonization of assay results. [ABSTRACT FROM AUTHOR]

Antithrombin [AT] is the main inhibitor for activated plasma coagulation serine esterases, inhibiting thrombin, Factors Xa and IXa, but also Factors XIIa, XIa, VIIa, kallicrein, and plasmin. Its activity is highly enhanced by heparin, through binding to the pentasaccharide sequences, for inhibition of all coagulation proteases, except thrombin, which inhibition requires its additional binding to the heparin polysaccharide chain. However, AT is the major inhibitor of thrombin in the blood circulation. Congenital or acquired deficiencies of AT expose affected patients to an increased risk of developing unprovoked and recurrent thrombo-embolic diseases. Antithrombin can be measured with various laboratory techniques, by either immunological or functional methods. Earlier, a radial immunodiffusion immunoassay allowed measurement of the protein antigenic content. Functional assays are mainly designed with Anti-Thrombin or Anti-Factor Xa chromogenic methods and are useful for detecting genetic molecular mutations with decreased inhibitory activity and contributed to study the conformational changes of antithrombin and its variants, which potentially regulate the activity of this serine protease inhibitor. These assays are not equivalent in terms of diagnosing protein abnormalities, associated with increased thrombotic incidence, and they have variable performance for reflecting impaired antithrombin binding capacity for heparin, reduced progressive inhibition of serine proteases, or accelerated switch rates to the latent and less active forms. A small proportion of AT (<10%) is present in blood in the β-form, with a lower oligosaccharide content, a lower Molecular Weight, a higher binding rate to endothelial glycosaminoglycans, and a higher anticoagulant activity, hence requiring specific laboratory methods for its measurement. The β-AT form is then of critical importance for controlling blood activation by tissue injury and preventing development of thrombo-embolic diseases. This article reviews the performance characteristics of the currently available assays, and their usefulness for monitoring the use of AT concentrates in intensive care units, disseminated intravascular coagulation or severe infections, to restore the anticoagulant protective effect of heparin by supplementing the requested AT concentration. The issues of automation, harmonization and standardization are also revisited and discussed. [ABSTRACT FROM AUTHOR]

For many years, the importance of fibrinolysis has been recognized, first for its intravascular antithrombotic action, and more recently for its many extravascular activities, associated with matrix degradation and tissue remodeling. In the blood circulation system, fibrinolysis prevents thrombosis, and is associated with various biological and clinical situations: risk factors for cardio-vascular diseases in high risk clinical situations (type II diabetes, hypertension, triglycerides, high BMI, elevated glucose, etc.), probably resulting from a significant reduction of the fibrinolysis potential, and elevation of PAI-1. Noteworthy, t-PA is mainly present as an inactive complex with PAI-1, and its concentration in plasma tends to follow that of PAI-1, but in a lesser extent. Hypofibrinolysis can favor the occurrence of thrombotic events, and possibly other biological dysfunctions. Fibrinolysis activity is however difficult to evaluate as it has a delayed activity after clot formation, is initiated and regulated after fibrin generation, and conversely to clotting, its action is delayed (long lag phase) and slow, before being dramatically amplified leading to rapid clot dissolution. We have designed a new assay for evaluating the global fibrinolytic capacity (GFC) in the body. Reagents are used in association with a specific instrument, which can be connected to any computer, and dedicated software is used for analyzing clot lysis kinetics. The assay is performed in a micro-cuvette, introduced into one of the instrument wells at 37 °C, and light transmittance is continuously measured. Assayed plasma is first supplemented with a limited and constant amount of t-PA with silica and is then clotted with thrombin and calcium. Clot dissolution (measurement of turbidity change) is recorded over time using the dedicated instrument (Lysis Timer), and clot lysis kinetics are analyzed with the associated software: primary and secondary derivatives of the light transmission curve give information on kinetics and completion of clot dissolution. Total assay time is about 1 h (but in the presence of hypofibrinolysis it can be prolonged). The concentration of t-PA used for the assay has been adjusted (100 ng/ml) to obtain an optimal sensitivity to hypofibrinolysis within a short time interval, and clot dissolution occurs within about 45 min for normal individuals, with a broad range from 30 min to 60 min, with some samples presenting a clot dissolution time >60 min (hypofibrinolysis). This new assay is performed with the tested plasma intrinsic factors, especially its own fibrinogen, and only exogeneous t-PA is added. GFC is highly sensitive to PAI-1 activity, but other factors regulating fibrinolysis contribute to the clot dissolution kinetics. Freshly prepared or frozen and thawed citrated plasma can be used. The usefulness of this assay for clinical applications is under investigation. Although fibrinolysis is mainly initiated in the body upon stimulation or blood clotting, and rapidly diluted and inhibited in the circulation, evaluation of its “residual” activity in plasma is expected to reflect its global body potential. [ABSTRACT FROM AUTHOR]

Activated Protein C Resistance is mainly associated to a factor V mutation (RQ506), which induces a deficient inactivation of activated factor V by activated protein C, and is associated to an increased risk of venous and arterial thrombosis in affected individuals, caused by the prolonged activated factor V survival. Its prevalence is mainly in Caucasians (about 5%), and this mutation is absent in Africans and Asians. Presence of Factor V-Leiden is usually evidenced with clotting methods, using a two-step APTT assay performed without or with APC: prolongation of blood coagulation time is decreased if this factor is present. The R506Q Factor V-Leiden mutation is now usually characterized using molecular biology, and this technique tends to become the first intention assay for characterization of patients. Both techniques are qualitative, and allow classifying tested individuals as heterozygotes or homozygotes for the mutation, when present. A new quantitative assay for Factor V-Leiden, using a one-step clotting method, has been developed, and designed with highly purified human coagulation proteins. Clotting is triggered with human Factor Xa, in presence of calcium and phospholipids (mixture which favours APC action over clotting process). Diluted tested plasma, is supplemented with a clotting mixture containing human fibrinogen, prothrombin, and protein S at a constant concentration. APC is added, and clotting is initiated with calcium. Calibration is performed with a pool of plasmas from patients carrying the R506Q Factor V mutation, and its mixtures with normal plasma. Homozygous patients have clotting times of about <40 sec; heterozygous patients have clotting times of about 40–60 sec and normal individuals yield clotting times >70 sec. Factor V-Leiden concentration is usually >75% in homozygous patients, 30-60% in heterozygous patients and below 5% in normal. The assay is insensitive to clotting factor deficiencies (II, VII, VIII: C, IX, X), dicoumarol or heparin therapies, and has no interference with lupus anticoagulant (LA). This new assay for Factor V-Leiden can be easily used in any coagulation laboratory, is performed as a single test, and is quantitative. This assay has a high robustness, is accurate and presents a good intra- (<3%) and inter-assay (<5%) variability. It contributes solving most of the laboratory issues faced when testing factor V-Leiden. Quantitation of Factor V-L could contribute to a better assessment of thrombotic risk in affected patients, as this complication is first associated to and caused by the presence of a defined amount of FVa. [ABSTRACT FROM AUTHOR]

Anti-phospholipid syndrome is a complex and severe clinical situation, associated with symptoms such as recurrent thrombosis, arterial or venous, at any site, pregnancy loss, and other related syndromes. These clinical burdens, are highly variable from patient to patient, and are associated with biological abnormalities, such as the presence of the Lupus Anticoagulant or phospholipid dependent antibodies, confirmed on two occasions at least 12 weeks apart. From the diagnosis standpoint, both, functional (clotting) or immunological assays, are difficult to standardize and to optimize, due to the absence of reference material, or a characteristic clinical group, and international reference preparations. Large cohort studies are necessary for defining the usefulness of each assay, in terms of specificity, sensitivity, accuracy and for following-up the disease evolution. Clotting assays are based on Activated Partial Thromboplastin Time (APTT) and diluted Russell Viper Venom Time (dRVVT), performed at low and high phospholipid concentration, or on 1:1 mixtures of tested sample and a normal plasma pool. They allow evaluation of the paradoxal effects of LAs, which are pro-thrombotic in vivo, and anticoagulant in vivo. Use of synthetic phospholipids improves assay specificities and sensitivities, especially in patients treated with anticoagulants. Immunoassays can also be used for testing phospholipid dependent antibodies, first identified and measured as anti-cardiolipin antibodies, but now characterized as targeted to phospholipid cofactor proteins: mainly β2GP1 (which exposes cryptic epitopes upon binding to phospholipids), and in some cases prothrombin, and more rarely Protein S, Factor XIII, Protein Z or Annexin V. Use of optimized assays designed with well-characterized anionic phospholipids, then complexed with highly purified phospholipid cofactor protein (mainly β2GP1), offers a better link between reactivity and clinical associations, than the former assays which were empirically designed with cardiolipin. Standardization also remains complicated due to the absence of international standards and harmonized quantitation units. Validation on large cohorts of negative and positive patients remains the key approach for defining assay performance and clinical usefulness. Laboratory practice for all these methods is now greatly facilitated thanks to the use of automated instruments and dedicated software. Along with clinical criteria, laboratory assays are of great usefulness for identification and confirmation of the anti-phospholipid syndrome and they allow disease follow-up when appropriate patient management is in place. [ABSTRACT FROM AUTHOR]

Thrombotic diseases caused by cancer progression have been reported as one of the major causes of cancer associated morbidity and mortality along with cancer invasiveness and infectious complications. Moreover, anticoagulant therapy with heparin and heparin-like drugs, or vitamin K antagonists, or the Direct Oral Anticoagulants, is seeing an extended application in cancer patients and offers prolonged life expectancy to oncology patients for whom blood activation and thrombotic events have a variable incidence, depending on cancer type. Laboratory tools are highly useful for identifying patients at thrombotic risk through the measurement of blood activation markers and selecting those appropriate for anticoagulant therapy. Among the pathological markers, DDimer or Extracellular Vesicles have the highest diagnostic value in these pathological conditions. Global assays are useful for dosage adjustment, such as assessing either an induced anticoagulant effect or the measurement of drug activity. Various assays are also developed such as platelet aggregometry techniques for evaluating drug induced- aggregates or methods allowing measurement of the drug activity to its targeted coagulation factors such as: heparin to thrombin or Factor Xa; DOACs to Thrombin or Factor Xa (Dabigatran to thrombin and DiXaIs, Rivaroxaban, Apixaban, and Edoxaban, to Factor Xa). Such explorative techniques help to find the right dosage adjustment to protect patients from developing thrombosis without exposing them bleeding. It also permits exploration of unexpected drug behavior in treated patients, to check the right adherence to therapy in long-term anticoagulant protocols, and prevention of bleeding in patients with impaired renal or hepatic function. Complementary use of blood activation markers brings additional information on the curative effects of the anticoagulant therapy, and allows identification of pro-thrombotic activity in the clinically silent state. These issues are concisely addressed below. [ABSTRACT FROM AUTHOR]

Functional and genetic assays for measuring platelet microvesicles (PMVs) are presented and discussed. Functional assays concern two groups of methods: a) homogeneous assays using the cofactor activity of phospholipids (PPLs) contained in PMVs and present in assayed plasmas, and a coagulation or a thrombin generation assay (TGA) as “end points”; b) capture-based assays, in which PMVs bind to an immobilized ligand, such as Annexin V in the presence of calcium, or monoclonal antibodies (MoAbs) specific for membrane proteins. Genetic assays aim to detect micro-RNA (miRNA) present in PMVs: miRNA must be extracted from plasma, and the expression pattern can be determined by various methods such as quantitative real-time PCR, microarray or sequencing. All these technical approaches introduce new exploration tools for measuring or quantitating PMVs or their associated activities, as biomarkers for disease evolution, their diagnosis or prognosis, and for monitoring of some antithrombotic or anti-inflammatory therapies. They offer invaluable analytical tools for research, drug discovery and epidemiological studies and have a strong potential as diagnostic tests. [ABSTRACT FROM AUTHOR]

Neonates and children can develop rare bleeding disorders due to congenital/acquired coagulation Factor deficiencies, or allo-immune/autoimmune complications, or can undergo surgeries at high haemorrhagic risk. They then need specialized transfusion of blood components/products, or purified blood extracted products or recombinant proteins. Blood-derived therapies conventionally used for management of affected infants with genetic/acquired deficiencies, bleeding problems (coagulation Factor reduced or missing) or thrombotic disorders (reduced or missing anticoagulant proteins) pose some additional risks. These remedial therapies can cause tolerance when used very early in life and, sometimes needed, repeatedly. The introduction of recombinant proteins has allowed manufacturers to produce large amounts of the proteins usually present at very low concentration in blood. This has also changed the risk pattern of plasma-extracted products, especially in terms of continual reduction of viral transmission. Many efforts have been made over these past decades to reduce the risks associated with the use of all these products in terms of viral and bacterial safety, as well as immune disorders but they are not the objective of this article. Other associated side effects are the presence of undesired activities in blood products, which can produce thrombotic events or adverse reactions. The progressive introduction of blood derived products has greatly improved the prognosis and quality of life of affected patients. This concerns whole blood, but also blood cell concentrates, mainly platelets and red blood cells, plasma, while the blood extracted products are increasingly replaced by recombinant proteins. All these therapeutic products, i.e. blood extracted drugs, improve health and quality of life for hemophiliac's A or B, or patients with auto/allo-immune thrombocytopenias or with rare bleeding disorders, and those with thrombotic events occurring in childhood, which are mainly due to Protein C or Protein S deficiencies (congenital or acquired). Progress in analytical methods and biotechnology allow better control of the manufacturing processes for all blood derived or plasma extracted products and recombinant proteins, and contribute to improved manufacturing processes to minimize the occurrence of side effects. These adverse events can be due to the aging of the blood cell concentrate with release of their granule content, and generation of EVs, which can produce anaphylactic reactions and risk of thrombosis, but also to the presence of activated coagulation Factors in purified products, such as Factor Xia as recently identified in immunoglobulin concentrates. Characterization and measurement of contaminant products is of special usefulness during product preparation and for optimization of manufacturing processes for purified extracted products, but also for recombinant proteins. The pharmaceutical industry introduces these new methods for validating manufacturing processes, or for quality control assessments. The objective is first to warrant the full quality and safety of the lots produced, and assure the highest efficacy with the lowest risks when used in patients. For cell concentrates and fresh blood, storage conditions are critical and measurement of analytes such as EVs or Annexin V allows evaluation of quality of each individual transfused pouch. In addition to all the rules around viral and bacterial transmission risk, and immune tolerance, our available laboratory methods contribute to reducing the side effects of blood cell concentrates and derived plasma products, as well as those of the therapeutic recombinant proteins. [ABSTRACT FROM AUTHOR]

The key coagulation factor FVII, and its activated form FVIIa, present a major interest for their role at the initiation phase of blood coagulation, and because they can activate all blood coagulation cascade, through the extrinsic, but also the intrinsic pathway. Blood activation initiated through FVII is first presented, as it is understood nowadays. Measurement of FVII and FVIIa were of main interest for epidemiological studies, but FVIIa contribution to assay results was only deduced. The introduction of specific FVIIa assays, functional or immunoassays, allowed measuring directly FVIIa without any interference of non-activated FVII, or other coagulation factors or their activated forms. The various methods available, and their characteristics are presented, with a special focus on two assays developed by our group for FVIIa (a clotting one and a chromogenic one). The FVIIa clotting assay shows evident superiority for measuring its activity in plasma, in pathophysiological conditions. The normal range is <2.5 ng/ml, which represents less than 0.5% of the FVII protein. FVIIa is elevated in some pathological states. The chromogenic assay is of interest for assigning the potency of FVIIa concentrates, as it has a higher dynamic range. Both assays are fully automatable on laboratory instruments, and standardized in a satisfactory manner thanks to the use of the FVIIa concentrate WHO International Standard (NIBSC). The various applications and usefulness of FVIIa laboratory assays are discussed, for the measurement of therapeutic products, or for following recoveries in treated patients, including hemophiliacs with inhibitors, patients with severe bleeding risk (liver diseases, surgery, trauma, …), and lastly for measurement of its activity in therapeutic products. [ABSTRACT FROM AUTHOR]

In the past decade Direct Oral Anti-Coagulants (DOACs), targeting Thrombin or Factor Xa, have enormously facilitated the daily treatment of all relevant patients, including those requiring lifelong therapy. These DOACs have considerable advantages over the use of oral Vitamin K Antagonist (VKA) treatments, in view of having little interferences with food and other medications and also not requiring adjustment for age, gender or weight, with some well-defined exceptions. In this current What's Happening Section we focus on measurements of DiXaIs in plasma using anti-Xa assays, with the objective of providing a tribute to Professor Michel Meyer Samama, who was not only a real leader in this field but, in the past, both authors benefited from his wisdom, as a teacher who dedicated his scientific and professional life (among many other interests in hemostasis, thrombosis and fibrinolysis) to develop and promote methods and strategies for laboratory monitoring of anticoagulants. This review presents the performance characteristics of the Anti-Factor Xa assays (measuring Factor Xa inhibition by drugs), which are available for measuring Direct Factor Xa Inhibitors in plasma, and show good compliance of the results with the reference LC:MS method (which measures the mass of Direct Factor Xa Inhibitors). We also present the preparation and validation of drug specific plasma calibrators and controls which are requested for drug measurements. These assays are convenient and practical laboratory tools which can be used in any laboratory setting, and meet the requirements of regulatory bodies for making smart, quantitative, sensitive, accurate and ease of use assays for measuring DOACs when needed. The manuscript focuses mainly on the following areas of current interest: interference in coagulation assays; anti-Xa laboratory methods; development of calibrators and controls for DiXaIs; method validation and comparison with reference techniques (LC:MS); regulatory requirements and method registrations; newer clinical applications and experience on DiXaIs with Anti-Xa assays, and future perspectives. [ABSTRACT FROM AUTHOR]

We report a very high factor V inhibitor affecting the measurement of all coagulation factors besides fibrinogen, all these factors being dramatically decreased. This inhibitor could be linked to antibiotic use. The patient died of massive hemorrhage before a plasma exchange could be initiated. [ABSTRACT FROM AUTHOR]

Utility of EVs, as biomarkers of cause or consequence of various pathological complications, and prognosis of blood components' therapy in terms of safety/efficacy and their potential associated hazards, primed by EVs involvements in pro-inflammatory, immunomodulatory and activations of both pro/anti-coagulatory and others associated pathways, as well as various cellular cross talks, are highlighted as the fundamental. Today EVs are becoming the “buzz” words of the current diagnosis, development and research [DDR] strategies, with the aim of ensuring safer therapeutic approaches in the current clinical practices, also incorporating their potential in long term cost effectiveness in health care systems. The main focus of this manuscript is to review the current opinions in some fundamental areas of EVs involvements in health and diseases. Firstly, our goal is highlighting what are EVs/MVs/MPs and how are they generated in physiology, pathology or blood products; classification and significance of EVs generated in vivo; followed by consequences and physiological/pathological induced effects of EVs generation in vivo. Secondly, specific cell origin EVs and association with malignancy; focus on EVs carrying TF and annexin V as a protective protein for harmful effects of EVs, and associations with LA; and incidence of anti-annexin V antibodies are also discussed. Thirdly, utility of EVs is presented: as diagnostic tools of disease markers; prognosis and follow-up of clinical states; evaluation of therapy efficacy; quality and risk assessment of blood products; followed by the laboratory tools for exploring, characterizing and measuring EVs, and/or their associated activity, using our own experiences of capture based assays. Finally, in perspective, the upcoming low volume sampling, fast, reliable and reproducibility and friendly use laboratory tools and the standardization of measurement methods are highlighted with the beneficial effects that we are witnessing in both wound healing and tissue remodeling, with an expected blockbuster status EVs as future therapeutic directions. [ABSTRACT FROM AUTHOR]

Blood cells generate heterogeneous populations of vesicles that are delivered, as small-specialized packages of highly active cell fragments in blood circulation, having almost similar functional activities, as the mother cells. These so called extracellular vesicles are the essential part of an energy-dependent natural apoptotic process; hence their beneficial and harmful biological functions cannot be ignored. Evidence is accumulating, that cellular derived vesicles, originate from all viable cells including: megakaryocytes, platelets, red blood cells, white blood cells and endothelial cells, the highest in proportions from platelets. Shedding can also be triggered by pathological activation of inflammatory processes and activation of coagulation or complement pathways, or even by shear stress in the circulation. Structurally, so called MV/EV appear to be, sometimes inside-out and sometimes outside-in cell fragments having a bilayered phospholipid structure exposing coagulant-active phosphatidylserine, expressing various membrane receptors, and they serve as cell-to-cell shuttles for bioactive molecules such as lipids, growth factors, microRNAs, and mitochondria. Ex vivo processing of blood into its components, embodying centrifugation, processing by various apheresis procedures, leukoreduction, pathogen reduction, and finally storage in different media and different types of blood bags, also have major impacts on the generation and retention of MV content. These artificially generated small, but highly liable packages, together with the original pool of MVs collected from the donor, do exhibit differing biological activities, and are not inert elements and should be considered as a parameter of blood safety in haemovigilance programmes. Harmonization and consensus in sampling protocols, sample handling, processing, and assessment methods, in particular converting to full automation, are needed to achieve consensual interpretations. This review focuses on some of our past personal studies on the role of MV/EV focusing on characterization of platelet storage lesion and platelet therapy that shows the highest transfusion hazards [up to 25%], and loss of 25% platelet efficacy after various leukoreduction and validated platelet pathogen reduction treatments. The planned paths for the future of EV/MV involvement in immunological and viral/ non-viral transfusion hazards are also discussed. Whilst considerable advances made on the characterization of EV/MV, but disparity still exists between various surrogate markers, showing some subtle differences in the levels of MV/ EV & BRMs in platelet preparations, and the clinical outcome showing platelets derived by all current technologies are equivalents in vivo. One possible reason for such a disparity may be relatedto the fact that MVs, being the end products of apoptotic cells, have little specificity and clear rapidly from circulation [<6 h in thrombocytopoenia]. This makes their clinical usefulness rather short lived. The recent findings that pegylating smaller subsets of EV increases its circulatory life from <15 minutes to approximately about one hour is highly promising, in particular, for drug delivery on specific sides. Hence a promising clinical utility of EV/MV continues, as a journey without end, indeed. This manuscript is based mainly on the selected key readings listed below. [ABSTRACT FROM AUTHOR]

Use of Direct Oral Anticoagulants (DOACs) is continuously increasing for clinical application. The first product released was Dabigatran, which was proposed for many preventive and curative applications, especially for prevention of stroke in patients with non-valvular atrial fibrillation. Although measurement of Dabigatran Anti-IIa activity in plasma is not requested on a routine basis, in some situations its measurement is clinically useful. Especially, before an emergency surgery in treated patients, where its presence at high concentrations, which will expose the patient at an increased bleeding risk, has to be excluded. Hence, smart, specific, rapid and accurate quantitative assays are warranted as an essential required. Hemoclot™ Thrombin Inhibitors and Biophen® DTI were specifically designed for these applications, and can be used on all automated instruments with a standard range protocol for measuring concentrations at peak, or with a low range protocol for testing residual concentrations. Both functional assays have a good correlation with the reference LC-MS/MS method, and concentrations measured are similar. Performances of these assays and interferences of various substances or drugs are discussed. Some differences in variations of clotting times are observed between mechanical or optical clot detection instruments, which could be explained by the fibrin clot structure, altered by direct Factor Xa inhibitors, and more especially Rivaroxaban. Both clotting and chromogenic assays offer a safe and accurate quantitative measurement of Dabigatran in plasma in all situations where this determination is requested. In short this manuscript provides an in depth update on current opinions on laboratory aspects of measuring Dabigatran concentrations in plasma, when required. [ABSTRACT FROM AUTHOR]

Capture assays were developed and validated for measuring the global pro-coagulant activity of micro-particles (MPs, mainly originated from platelets), or specific extravascular cellular MPs (released from erythrocytes, leukocytes, monocytes, endothelial cells) as those exposing TF (MP-TF, mainly observed in patients with some cancers). Conversely to Flow Cytometry methods, these capture assays measure all coagulant activity associated with MPs, through thrombin generation (MP-Activity) or Factor Xa generation (MP-TF), and therefore they bring a complementary information, as they are more specific for the pro-coagulant activity associated with MPs. Small particles (<0.40 µ) exposing Phosphatidyl Serine (PS) exhibit a greater pro-coagulant surface than larger MPs (0.40 to >1.00 µ), those preferentially measured with flow cytometry. Activity associated with MPs is a consequence of disease but can also be a cause contributing to pathological processes and development of thrombo-embolic events. In many diseases, flow cytometry and capture assays do not totally correlate, and have different associations with disease evolution. Optimized capture based assays are presented and discussed, along with their performance characteristics and some applications. They can be performed in any technically skillful hemostasis laboratory, using a thermostated ELISA equipment, or an incubator. Dynamic ranges for MP-Activity assay is from <0.1 nM to >2.5 nM Phospholipids, expressed as Phosphatidyl Serine (PS) equivalent, in the tested dilution. For MP-TF the very sensitive bio-immunoassay reported allows measuring concentrations from <0.10 pg/ml (TF equivalent) to >5.00 pg/ml, in the assayed dilution. No measurable MP-TF was found in normals, although an important concentration was generated from whole blood treated with Lipo-Poly-Saccharides. Capture based assays are then highly useful in the laboratory setting for measuring the activities associated with pro-coagulant, or specific cellular MPs. [ABSTRACT FROM AUTHOR]

Background: Light transmission aggregometry (LTA) is considered as the gold standard for testing platelet function in the setting of both platelet disorders suspicion and response to antiplatelet therapy evaluation. LTA requires however specialized equipment, substantial blood sample volumes, is technically challenging and time-consuming. Aim: To evaluate an automated platelet aggregation method performed on a routine coagulation analyzer Sysmex CS-2000i. Methods: 46 patients presenting a bleeding syndrome and 62 patients with acute coronary syndrome receiving dual antiplatelet therapy were studied in total. Platelet aggregations were performed on CS-2000i equipped with a dedicated software and on APACT-4004 (Elitech, France) as the reference instrument. Aggregation was measured by monitoring the changes in light absorbance occurring in response to ADP 2.5, 5 and 10µM, collagen 3.3 µg/mL, epinephrin 10µM, ristocetin 1.25 mg/mL and arachidonic acid 0.5 mg/mL in platelet rich plasma (PRP). PRP were tested simultaneously on both CS-2000i and APACT-4004 devices. Platelet stirred speed were 800 rpm for both instruments. Results: Significant correlations were observed between CS-2000i and LTA after all stimulations (p< 0.001). Patients presenting a bleeding syndrome had similar aggregation profiles with both methods. A single patient presented a severe platelet disorder (Glanzmann Thrombasthenia) and its PRP showed defective aggregation in response to all agonists except ristocetin with both instruments. Finally, the inter-agreement rates for CS-2000i and APACT-4004 to detect low responders to thienopyridines or aspirine were strong (weighted kappa> 0.70). Conclusion: Platelet aggregation on the routine coagulation analyzer CS-2000i is an easily accessible, handy, reliable, standardized, and rapid tool to assess platelet function which allows to skirt most of the LTA limitations. [ABSTRACT FROM AUTHOR]

There is an increasing interest in Extracellular Vesicles released by many cells through membrane shedding. In addition to cell signaling, these particles are true messenger cargos, which can carry cell surface proteins, miRNAs and non-coding RNAs to other and distant cells. They are part of the inter-cellular crosstalk and they contribute to transferring biological messages far away from the triggering event. EVs are biomarkers of many diseases, including thrombo-embolic pathology, infections, neurological or metabolic disorders, and malignancy. Their role and significance are presented and discussed in this short review, as consequences of disease and causes of its progression. But they can also be beneficial for tissue healing or repair, and they can be prepared in vitro to be used for cell- targeted treatments. Many identification and measurement methods for EV's are sophisticated, which restricts their use to research studies, but they have, nevertheless, a high laboratory potential for diagnosis, prognosis and evolution as follow-up of many pathologies. New emerging laboratory tools offer more friendly and easy applications for characterizing EVs and testing their associated activity, especially for the procoagulant ones. [ABSTRACT FROM AUTHOR]