Clot waveform analysis: Where do we stand in 2017?

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1 Received: 28 April 2017 Accepted: 11 July 2017 DOI: /ijlh REVIEW Clot waveform analysis: Where do we stand in 2017? P. O. Sevenet F. Depasse Diagnostica Stago S.A.S, Asnières sur Seine, France Correspondence Pierre-Olivier Sevenet, Diagnostica Stago, Clinical Development, Asnières-sur-Seine, France. Abstract Analysis of the optical waveform generated during global coagulation assays, such as activated partial thromboplastin time and prothrombin time, can provide much precious information on the global coagulation state of the plasma sample tested, in addition to a single clotting time. Many studies have been published concerning patient diagnosis and management in haemophilia A, and in the early diagnosis and prognosis of disseminated intravascular coagulation and sepsis. However, many other works have also been published on further potential clinical applications such as lupus anticoagulant diagnosis and anticoagulant monitoring. Altogether, these publications have demonstrated the ability for clot waveform analysis (CWA) to improve patient management, especially as this tool is inexpensive, rapid and readily available on coagulation analysers with optical detection systems. By an extensive review of the literature related to studies performed on CWA, this publication aims at providing a review of current knowledge in this specific field, ranging from research data to potential clinical applications and future trends. KEYWORDS activated partial thromboplastin time, biphasic waveform, clot waveform, disseminated intravascular coagulation, haemophilia, transmittance 1 INTRODUCTION Coagulation is a highly complex physiological mechanism that involves multiple plasma proteins, cellular components and the vessel wall. Increasing coagulation and fibrinolysis knowledge has led to major improvements in clinical practice decisions, resulting in optimized and safer patient management in both haemorrhagic and thrombotic settings. Among the numerous laboratory assays exploring haemostasis, global functional assays have been generating more and more interest over time. By analysing an end- point of the coagulation cascade (eg thrombin or fibrin clot), they combine, within a single assay, the analysis of a complete process and allow the detection of several coagulation abnormalities. The most widely known global coagulation assays are the thrombin generation test (TGT) and the viscoelastometrics assays (such as ROTEM or TEG ). Through the evaluation of the kinetic of fibrin formation during tests such as activated partial thromboplastin time (aptt) or prothrombin time (PT), clot waveform analysis (CWA) is also considered as a global coagulation assay. Both quantitative and qualitative CWA parameters have been shown to be associated with pathophysiological processes and have a potential use in clinical applications. Among them, the clinical phenotype of severe haemophilia A (HA) patients could very accurately be predicted by CWA. 1,2 The management of haemophilia treatments and bypass therapy in HA patients with inhibitors is also of great interest. 3,4 Additionally, it has been shown that CWA can help in the early diagnosis and prognosis of sepsis and disseminated intravascular coagulation (DIC). 5,6 This review has been written using an exhaustive analysis of original articles in English, present in the MEDLINE/PubMed database, about CWA. Fifty- six publications concerning the use of CWA were found using the search fields: (waveform[all fields] AND (clot[all fields] OR activated partial thromboplastin time[all fields])) AND (analysis[all fields] OR biphasic[all fields] OR haemophilia[all fields] OR disseminated intravascular coagulation[all fields]) and activated partial thromboplastin time[all fields] AND (reaction curve[all fields] OR optical profil[all fields] OR transmittance[all fields] OR first derivative[all Int J Lab Hem. 2017;39: wileyonlinelibrary.com/journal/ijlh 2017 John Wiley & Sons Ltd 561

2 562 fields] OR second derivative[all fields]). After exclusion of noneligible papers (reviews, editorials), 44 original articles were included for analysis. 2 WHAT IS CLOT WAVEFORM ANALYSIS? Clot waveform analysis corresponds to the extended study of the slope generated by an optical detection system during routine coagulation assays, such as aptt or PT. The optical detection system recognizes the clot formation process by measuring changes in transmittance, or absorbance, of a light beam through the analysed sample. Transmittance or absorbance is continuously recorded over time. The qualitative examination of the clot formation curve and the multiple quantitative parameters provided by CWA can give valuable information in addition to clotting time obtained by routine assays. CWA is mainly based on the aptt assay; however, PT or modified assays have also been used in various studies. The resultant curves obtained are called waveform because of their sigmoidal profile (Figure 1). 7 The coagulation process can be divided into 3 distinct periods, the precoagulation phase, the coagulation phase and the postcoagulation phase. These 3 phases can be characterized by parameters defining the time interval, the rate, the slope and the magnitude of the signal variation during the reaction. Multiple quantitative parameters can be recorded in addition to clotting time. While the transmittance (or absorbance) curve indicates the appearance of the fibrin clot in the sample, the first derivative of the curve reflects the velocity of the clot formation. The maximum absolute value of the first derivative is thus considered as the maximum velocity. The second derivative represents the acceleration of clot formation. The maximum acceleration and maximum deceleration (steepest point of the deceleration slope) are 2 parameters of interest, frequently reported in the literature. Denomination of these parameters varies depending on whether the recorded signal is transmittance or absorbance. In the case of transmittance, which is the most widely reported detection system in the literature, maximum velocity is called Min1, maximum acceleration is called Min2, and maximum deceleration is called Max2. The normal transmittance waveform pattern is illustrated in Figure 1. The pattern of the absorbance detection system is the reverse of the transmittance pattern. Hence, maximum velocity, maximum acceleration and maximum deceleration are called Max1, Max2 and Min2, respectively. To aid comprehension, transmittance nomenclature will be used in this review. Besides quantitative parameters reflecting fibrin formation kinetics, CWA is able to identify other potential reactions of interest in clinical practice such as detection of the CRP- VLDL complexes. The formation of these complexes results in the biphasic waveform pattern (BWP). 6 This is of special relevance in the prediction of impending DIC as we will see further in this article. To carry out CWA, an automated coagulation analyser that uses an optical- based system for clot formation detection is required. The analyser is usually equipped with software that automatically computerizes absorption or transmittance raw data and translates them into CWA parameters. Alternatively, raw data can also be exported and processed by external software. The standardization of CWA methods was proposed during the Scientific and Standardization Committee (SSC) sessions of the International Society of Haemostasis and Thrombosis (ISTH) in 2013 by Shima et al 8 They formulated the minimal requirements for performing CWA, which are the use of colourless and clear reagents, a system able to detect sensitive changes in the opacity of the sample, and an adjusted aptt to ensure a normal clotting time between 30 and 40 seconds. FIGURE 1 Representation of a normal clot waveform transmittance pattern, with first and second derivatives. Min1 corresponds to the maximum velocity of the clotting process, Min2 corresponds to the maximum acceleration, and Max2 corresponds to the maximum deceleration

3 3 POTENTIAL CLINICAL INDICATIONS 3.1 Haemophilia The first experiments with CWA in haemophilia were performed in 1997 by Braun et al, who suggested the potential expanded interpretation of aptt and PT assays using optical transmittance data. 7 Then, Shima et al also demonstrated the promising correlation between small amount of factor VIII (FVIII) in the sample and coinciding variation of CWA parameters. 1 They observed significant qualitative differences in aptt clot waveform curves across 36 patients with severe HA (ie patients with FVIII one- stage clotting assay result <1.0 IU/dL). Spiked plasma with increasing concentrations of FVIII in the very low values impacted clotting time and maximum acceleration (Min2 in the study) in a dose- dependent manner. The Min2 parameter appeared to be very well correlated (r = 0.922) with FVIII clotting assay (FVIII:C) and furthermore had the advantage of being more sensitive than FVIII:C in the very low values (<1.0 IU/dL with FVIII activity clotting assay). Matsumoto et al extended these assumptions with the testing of aptt CWA on FVIII and factor IX (FIX)- deficient plasmas spiked with their respective factor. 2 They showed a good predictable dose- response of CWA parameters (clotting time, maximum velocity and maximum acceleration) with low amounts of FVIII and FIX. In addition, they showed that CWA correlated well with TGT across the whole range of concentrations for FVIII (0-100 IU/dL) and in the moderate to high concentrations ( IU/dL) for FIX. According to their observations, CWA seems to be very accurate, even more than FVIII:C clotting assay, for detecting low values (<1.0 IU/dL) of FVIII. The concept that CWA could be a valuable tool for severe haemophiliac patients has emerged following the repeated observation that some patients categorized as severe haemophiliacs, based on FVIII:C assay, exhibit only a moderate bleeding phenotype. Similarly, patients with moderate HA according to the FVIII:C assay can present with a severe bleeding phenotype characterized by frequent episodes of spontaneous bleeding. 9,10 CWA, through its good correlation with the FVIII:C assay and its increased sensitivity in the lower values, appears to be a useful prognosis assay for the segregation of patients according to their bleeding phenotype. The clinical relevance of CWA has been assessed by Nair et al in a cohort of severe, moderate and mild HA patients. 11 They demonstrated that the median value of maximum acceleration (Max2) could discriminate firstly haemophiliac patients from healthy patients, and secondly between moderate HA and severe HA. However, as did Shima, 1 they showed that Max2 presents more variability in regard to FVIII:C in severe HA subject samples (ie with FVIII:C<1.0 IU/dL) than in spiked samples for the same range of FVIII:C level. This could suggest that FVIII is not the only determinant for clot formation acceleration in clinical samples and that factors other than FVIII could be implicated in the expression of bleeding tendency in HA patients. Based on this assumption, and the fact that it is a global coagulation test, CWA reflects the balance between different procoagulant and anticoagulant factors present in the plasma sample more effectively, and thus predicts the bleeding tendency for haemophiliac patients better than the determination of FVIII:C alone. This 563 is also supported by the publication of Yada and colleagues, who investigated a specific variant (Arg1781His of F8 gene) associated with moderate clinical phenotype in severe haemophilic patients. CWA, as well as thrombin generation and thromboelastography, shows that results from these severe patients are more comparable to plasma at 5 IU/dL of FVIII:C than <1.0 IU/dL. Even though the mechanism remains only partially understood, it would appear that binding affinity between FVIII and FX is higher in Arg1781His patients than in normal FVIII patients. 12 The absolute maximum values of the first and second derivatives of the aptt curve (Min1 and Min2, respectively) are the most widely used parameters for CWA in the evaluation of low levels of factor VIII and factor IX. However, other sets of parameters can be used for the expression of CWA modifications, as shown in the study led by Milos. 13 They expressed, with a parameter called Delta, the steepness of the aptt curve during the coagulation phase. They then showed that Delta is significantly different between severe and nonsevere HA patients and that this parameter could be used to discriminate between those 2 groups of patients with a remarkable sensitivity and specificity (ROC analysis: area under the curve = 0.978, P <.001; sensitivity = 97.3%; specificity = 93.2%). Moreover, they showed that the occurrence of severe clinical events (age of first bleed, number of annual joint bleeds and number of joints with haemophiliac arthroplasty) is better correlated with the Delta parameter than with FVIII activity assays (either one- stage clotting assay or chromogenic assay). This confirms the potential of aptt CWA in the prediction of bleeding phenotypes regardless of CWA parameters used to express the results. Another concept of CWA has been made by Tokunaga et al as they observed different profiles in the second- derivative curves of the aptt assay: normal pattern, a shoulder type curve and a biphasic type curve with a double peak. The presence of the double peak was associated with severity of intrinsic pathway factor deficiency. In addition, in acquired von Willebrand disease patients, the biphasic pattern is normalized following DDAVP infusions which increase von Willebrand factor levels. 14 Siegemund et al made a new approach to CWA in On the assumption, as in TGT, that the derivative of a substrate concentration curve over time represents the activity of the substrate s enzyme, they considered that the first derivative of the aptt assay curve represents thrombin activity, the second derivative represents prothrombinase activity, and finally, the third derivative represents tenase activity. 15 They correlated the maximum absolute value of the first derivative with the activity of thrombin calculated with a Michaelis- Menten mathematical model and found an excellent correlation (r 2 = 0.95). In addition, this study is the first to present the third derivative as the best parameter correlated with low concentrations of FVIII. In view of the interest of these parameters in the evaluation of severe haemophilia A and B, the implementation into clinical practice as an aid for determining the bleeding risk should be considered. It could be even more valuable for laboratories which do not have capabilities for measuring FVIII:C or FIX:C. However, aforementioned studies were conducted on small series of patients only. Conducting larger and prospective studies including reliable collection of clinical

4 564 data should be considered to confirm the potential of clot waveform in haemophilia. 3.2 Replacement therapy management In the course of haemophiliac patient management, development of antibodies targeting FVIII or FIX is the most important complication in replacement therapies. Haemophiliac patients with high responding inhibitors are nowadays treated for bleeding prevention with bypassing therapies, usually human recombinant activated factor VII (rfviia) or activated prothrombin complex concentrates (apcc). Optimization of bypassing therapies in these particular patients remains challenging. Neither practical guidelines for optimal therapy nor haemostasis monitoring has been adequately defined. Furthermore, the optimal dosage to obtain sufficient efficacy with the lowest risk of thrombotic events varies across patients. At present, standard one- stage clotting factor assays do not always adequately reflect haemostasis in the presence of an inhibitor, and thus, are not relevant enough for efficient monitoring. Assays evaluating global clotting function, such as automated thromboelastometry, TGT and CWA, may be useful for evaluating and monitoring haemophiliac patients with inhibitors Following observations on the correlation between CWA and very low values of FVIII:C levels, it was proposed by Shima and colleagues that this technique could provide a very useful method for monitoring HA patients with inhibitors. They showed a clear dose- response relationship between the improvement in clot waveform parameters and the ex vivo added amount of rfviia. Furthermore, they highlighted the benefits of this assay as it could be run as a routine coagulation assay. They also demonstrated that CWA results are independent of pre- analytical variables such as in vitro coagulation activation. 18 More recently, Haku et al explored the performances of CWA in bypassing therapy monitoring, for both rfviia and apcc. They assessed different triggers for coagulation and found that the best results, after in vivo infusion of bypassing therapy, were yielded when aptt is activated with a mix of low doses of tissue factor and ellagic acid. 4 This study confirms the promising potential of CWA in bypassing therapy monitoring, even with a modified aptt trigger reagent. Furthermore, they were the first to monitor the real- time clinical effectiveness of replacement therapy using CWA, in a small number of patients. Using clot waveform, Kasuda et al assessed the effectiveness of immune tolerance induction in patients with high responding inhibitors. They observed that, in patients with inhibitors, CWA based on the aptt assay more effectively reflects clinical improvement after factor concentrates infusion than the FVIII:C assay. 19 Clot waveform analysis is also used to assist in the development of new haemostatic drugs, by the evaluation of coagulation parameters. Hence, Shirathata assessed, in a phase 1 trial, the potential effect on coagulation and thrombin generation of a new bypassing drug in haemophilia, MC710 (KAKETSUKEN; Kumamoto, Japan). By observing maximum velocity and maximum acceleration, they found that MC710 had a greater bypassing activity than FEIBA or rfviia. 20 Furthermore, CWA was used by Yada et al to evaluate the effects of different alloantibodies against different epitopes of FVIII. This helped to understand the variability of responses to FVIII replacement therapy in patients with HA with inhibitors. 21 It can thus be seen that CWA is a very promising tool for the evaluation of the effects of haemostatic drugs, either for research and development purposes by pharmaceutical companies or in clinical use for the follow- up of patients haemostasis recovery. 3.3 Disseminated intravascular coagulation and sepsis Disseminated intravascular coagulation is an acquired disorder of the coagulation and fibrinolytic system, secondary to defined groups of severe diseases such as sepsis or trauma. 22 It was observed by Downey et al in 1997 that a patient in which DIC has been diagnosed could present an abnormal and characteristically distinct transmittance waveform appearance on the aptt assay. 23 They noticed that the curve can show an immediate and progressive decrease in light transmittance after initiation of the aptt assay, that is in the precoagulation phase, while the normal profile is characterized by a 100% light transmittance prior to the clot formation. This modified appearance was designated as the BWP (Figure 2). The presence of BWP was shown to be independent of the aptt clotting time, aptt activator reagent, decreased or increased coagulation factors and is not influenced by anticoagulant therapy such as heparin. 24,25 In a second study led by the same author, the correlation between CWA and DIC diagnosis was assessed in a prospective cohort of 747 patients. The study showed that the sensitivity and specificity of BWP for DIC diagnosis was 97.6% (95% confidence interval [CI]: 85.6%- 99.9%) and 98% (95% CI: 96.6%- 98.9%), respectively. 5 Toh and colleagues demonstrated that the addition of Ca 2+ in the test sample induces the rapid formation of a precipitate that contains C- reactive protein (CRP) and very low- density lipoprotein (VLDL). The calculated concentration of CRP- VLDL complexes is correlated with turbidity changes in the sample, thus confirming the assumption. 6 Furthermore, VLDL complexed to CRP has been shown to induce a procoagulant effect through enhancement of the prothrombinase complex, by modification of the anionic phospholipid surface. 26 The same group prospectively assessed CWA in 1,187 patients admitted in the ICU over a 24- month period and concluded that CWA predicts DIC outcome more effectively than D- dimer, CRP or VLDL measurements alone. 6 The results also showed that the appearance of BWP preceded, by an average of 18 hours (range 2-47 hours), the time of DIC diagnosis (using Japanese Ministry of Health and Welfare JMHW criteria for DIC diagnosis). Furthermore, the quantitation of the degree of abnormality could provide an index for the prediction of the clinical response of DIC to therapy. 27 In this cohort, the overall death rate was found to be 44% for patients who are positive for BWP whereas it was only 25% for patients with a normal aptt waveform. 28 Another prospective study over 217 consecutive ICU patients demonstrated that the presence of the BWP has a sensitivity of 88% and a specificity of 97% for diagnosis of DIC, as compared to a blind diagnosis based on experts opinion. 29

5 565 FIGURE 2 The biphasic waveform pattern could be seen in patients with disseminated intravascular coagulation (DIC) (waveform in dotted line) In non- ICU hospitalized patients, the BWP showed a moderate sensitivity of 59.2% or 47.9% for the diagnosis of DIC by ISTH or JMHW criteria, respectively. However, specificity was high (95.4% for both scores) and the presence of the BWP was significantly associated with DIC (P <.0001) with odds ratios of 29.9 and 19.0 for ISTH and JMHW criteria, respectively. 30 In addition to DIC condition, BWP has been examined among sepsis- affected patients. Several studies have shown a positive correlation between the presence of BWP and the early development of sepsis in patients admitted to ICU. 28,31-33 They showed a modest sensitivity, while the specificity is higher. BWP has thus been proposed to evaluate a high- risk ICU population, principally in sepsis- affected patients. Given the elevated negative predictive value (NPV) determined in the studies conducted by Toh (NPV: 91.0%) and Downey (NPV: 90.6%), the presence of BWP in aptt waveform could be used as a marker of sepsis and for ruling- out sepsis in ICU patients when the result is normal. In cardiopulmonary bypass surgery population, a publication of Delannoy et al demonstrated, in a cohort of 32 patients, that the presence of a BWP discriminated between sepsis and nonseptic systemic inflammatory response syndrome (SIRS) with a sensitivity of 100% and a specificity of 93% (ROC analysis, area under the curve: ± 0.039; P <.01). 34 This finding reinforces what Chopin et al described in a ICU population of 187 consecutive patients: the biphasic aptt waveform was able to discriminate between patients with severe sepsis and sepsis shock from patients with SRIS and nonsevere sepsis with a sensitivity of 90% (95% CI: 82%- 94%) and a NPV of 92% (95% CI: 87%- 96%). 35 It has also been reported in a non- ICU population that the presence of aptt BWP is related to the poorest clinical outcomes (infection prevalence, overall mortality). 36 Consecutively, enrolled patients who showed positive aptt BWP also had positive microbial culture in 67% of cases, whereas only 13% of patients without BWP had positive microbial culture. 32 CWA has been assessed in meningococcal sepsis- affected children. Results showed that the presence of BWP is associated with the poorest outcomes (prolonged hospital stay) and that this convenient assay could facilitate a prompt diagnosis, in combination with procalcitonin measurement. 37 The combination with procalcitonin levels was notably assessed by Zarariah et al and has demonstrated that, at optimized thresholds, this combination showed enhanced sensitivity of 79% (95% CI: 64%- 90%), specificity of 96% (95% CI: 93%- 98%) and negative predictive value of 96% (95% CI: 94%- 98%). 38 Altogether, the cumulative evidences confirm the considerable capacity of CWA in predicting DIC or sepsis. However, this parameter is not actually used in a clinical setting, although the sensitivity has been described as elevated (up to 98%). 28,29 This may be due to the actual limited number of laboratories providing CWA and to the lake of large prospective confirmatory studies. However, it could be of great interest to implement it in different scores, as the test is inexpensive, easy to perform and has shown high performances for both DIC and sepsis Other potential indications Since the introduction of the assay into clinical studies, CWA has been assessed to detect procoagulant or prohaemorrhagic patients status in a variety of disorders. Lupus anticoagulants (LA) were notably studied with CWA by Su et al 40 Investigation of patients with antiphospholipid antibodies (APA) was performed using both aptt and PT waveforms. They showed that the slope of the precoagulation phase of PT assay (called slope 1) presents an abnormal result (a difference of more than 2 SD from a healthy group) in 61.5% of patients with APA. In contrast, 5.1% of patients without APA but treated by warfarin, and no healthy patients (untreated by warfarin) present an abnormal slope 1. However, these results were highly reagent- dependent as it has only been observed with the Simplastin reagent, and no significant decrease in slope 1 was observed with other thromboplastin reagents. Concerning aptt assay, none of the extracted parameters consistently distinguished patients with APA from other groups of patients, even aptt clotting time due to its weak specificity. In other studies, abnormal aptt waveform

6 566 has been observed in 25% of patients with APA, 41 but the authors did not determine any aptt waveform parameter that would allow the segregation of APA- positive patients from DIC patients, as the curve profiles are too similar. 23 A recent study observed that absolute Min1 value of aptt assay is decreased in patients with both positive LA and clinical antiphospholipid syndrome (2.2 ± 0.5; mean value ± SD), and in patients with LA but without antiphospholipid syndrome (1.6 ± 0.2), compared to normal patients (3.1 ± 0.1). Furthermore, Min1 for patients with mild HA or acquired HA is significantly decreased (1.3 ± 0.2 and 0.9 ± 0.5, respectively) compared to the aforementioned groups. 42 However, even if differences are significant, the distribution of Min1 values overlaps, and single CWA testing could not distinguish between patients in the different groups. An atypical pattern for Min1 and Min2 curves has also been observed in patients with LA, but none in normal patients, using silica- based aptt reagent. 43 This observation is however highly reagent- dependent as ellagic acid reagent produces a similar atypical pattern in normal patients and in LA- positive patients. Ultimately, many different observations have been made in patients with LA, and further studies are required to confirm these observations and add proof of the interest of CWA in LA laboratory diagnosis. Most of the time, aptt- based CWA assay is used. However, CWA could also be valuable with a PT assay. Matsumoto and colleagues used PT- derived CWA in a study intended to show the prediction potential of the bleeding phenotype in patients with acquired anti- factor V inhibitors. 44 They found discriminating differences in the clot time, maximum velocity (Min1) and maximum acceleration (Min2) between patients with life- threatening bleeding and asymptomatic patients. CWA, in conjunction with TGT, was notably used to understand the mechanisms underlying the variations in the phenotypes of patients with this condition. Fibrinogen assessment has also been investigated. The difference between the plateau phase of precoagulation and postcoagulation for both aptt and PT assays correlates well with fibrinogen levels, independently of clotting times and plasma quality (haemolysis, icterus and lipaemia). Despite the positive results, this method of fibrin measurement is not innovative and will not replace present conventional fibrinogen assays such as the Clauss method, as performances (precision and accuracy) are clearly superior. Nonetheless, these studies show the direct correlation between fibrinogen concentration and the impact on CWA parameters. 45,46 Clot waveform analysis has been used by Shima s team to demonstrate the increased procoagulant state in chronic spontaneous urticaria 47 and for the exploration, in association with other global coagulation assays, of the paradoxical procoagulant effect induced by plasmin DISCUSSION CWA is based on the recording of an optical signal over time and is intended to reflect the whole process of clot formation. Whether initiated by a contact activator (silica, kaolin, polyphenols, ellagic acid) or by tissue factor, the waveform pattern reflects the combined activity of all factors implicated in the clot formation process. In this respect, CWA can be considered as a global haemostasis assay. Other global haemostasis assays are thrombin generation and thromboelastometry. They differ greatly from CWA in their operating principles. In TEG /ROTEM, viscoelastic properties of the fibrin clot are measured whereas thrombin formation rate is detected by mean of a fluorescent marker in TGT. 49,50 The TEG /ROTEM devices evaluate the kinetics and strength of the fibrin clot over the time and can evaluate both prothrombotic and prohaemorrhagic conditions. The most used clinical application is the management of bleeding during surgery or invasive procedures. This approach is mentioned in different guidelines for tailoring transfusion strategy during surgery. 51,52 In addition, the point- of- care format and the ease- of- use of these devices allow its implementation in the operating room. Further applications are however possible as numerous trigger reagents could be used (extrinsic pathway assessment, heparin levels, fibrinogen measurement and resistance to lysis). Thrombin generation could also assess both thrombotic states and bleeding tendencies. Several applications are now entering the scope of clinical practice. The management of haemophiliac patients has been actively assessed with thrombin generation. Some studies have been performed jointly with CWA for this purpose. 1,2,18,42 The 2 approaches are very sensitive in low FVIII:C values in HA patients. Nevertheless, both still require clinical validation in larger clinical trials. Clot waveform analysis provides similar information to TGT as it correlates with the rate and velocity of thrombin formation, and reflects the whole process of thrombin generation over time. This contrasts with routine coagulation assays in which clotting time only reflects coagulation initiation. In severe haemophilia or in the presence of a specific factor inhibitor, the rate of thrombin is decreased 53 and this results in statistically significant variations in CWA parameters. 2,19 Increased thrombin generation is observed in thrombophilic states; however, no study correlating CWA parameters and thrombophilic states have been published as yet. Clot waveform analysis is a global assay which presents many advantages. Based on widely used and very simple assays such as PT and aptt, it appears to be easily carried out on widely available automated analysers with optical detection systems, using routine reagents, and is not associated with additional costs for laboratories. In contrast to CWA, thromboelastometric assays and TGT require specific equipment. Furthermore, CWA could be run as a regular routine assay on the same sample as PT and aptt (0.109 or mol/l trisodium citrate blood collection tubes), and with a fast turnaround time compared to other global assays. As clotting time in aptt assay occurs when only 5% of thrombin is generated, CWA enables the exploration of the coagulation process with more information than the single clotting time. Throughout studies on DIC, it has been shown that the presence of an abnormal pattern associated with DIC is not influenced by pre- analytical conditions nor by the presence of an anticoagulant. Furthermore, clotting time has no impact on the presence of the biphasic pattern. However, CWA does have some limitations. As in PT and aptt assays, the use of whole blood or platelet- rich plasma is not possible.

7 567 Using an optical measurement method, interferences colouring plasma such as haemolysis, lipaemia and icterus can impact CWA results. These interferences depend on the wavelength assigned for optical detection. At present, no study has been run to assess the minimal levels of these interferences to perform CWA in optimal conditions nor the choice of optimal wavelength for clot detection. Many questions remain open on the potential clinical use of CWA in daily practice. Scientific subcommittees of ISTH proposed standardization using a sensitive optical detection system, colourless and nonopaque reagent and an adjusted normal clotting time for aptt assay. 8 However, many experimental factors such as aptt reagents, types of activator and phospholipids preparations could impact CWA results. As yet no study nor correlation has been published on the variability between reagents or analysers on CWA results. The choice of wavelength for detection would seem to be crucial to the sensitivity of the assay, but it is not dealt with in the SSC communication. Furthermore, different curve smoothing algorithms or curve evaluation modes exist and may be a source of variability between analyser results. 54 For it to become a clinically validated tool, further studies and calibration methodologies should be developed. However, in contrast with other global assays, CWA appears to be the easiest assay to standardize because of the easy-to-perform protocol, and convenient and very wellknown assays involved. Despite the accumulated data suggesting benefits in various clinical situations and the many attempts to democratize it, CWA is still only investigated by a small number of research teams and seems to be under- recognized by physicians and pathologists. The number of original articles present in the literature is relatively low. Between 1997 and 2016, only 56 original articles and reviews are referenced in MEDLINE/PubMed, compared to more than 6000 and more than 4000 when using keywords thrombin generation and thrombelastography, respectively. Shown to be a very promising tool, CWA should be extensively studied in many different clinical conditions, especially as more and more coagulation analysers provide optical detection system and possess integrated software for waveform analysis. Furthers physiopathological conditions, such as hypercoagulable states, inflammation, anticoagulant monitoring and risk prediction, have to be extensively evaluated, to integrate this rapid, inexpensive and already available tool into clinical practice. 5 CONCLUSION It is now generally accepted that CWA has huge potential and could provide much more information on the haemostasis system than clotting time alone. The most widely used assays are aptt, and to a lesser extent PT or combined tissue factor plus contact phase activator triggered assays. Efforts have been made to standardize the assay; however, further recommendations are still needed for an extensive clinical application. At present, HA management and DIC diagnosis and prognosis are the 2 main clinical fields in which CWA is designed to deliver improvements to patient management. Nevertheless, as studies are limited to small cohorts, extensive prospective clinical trials are mandatory to make the leap to clinical application. REFERENCES 1. Shima M, Matsumoto T, Fukuda K, et al. The utility of activated partial thromboplastin time (aptt) clot waveform analysis in the investigation of hemophilia A patients with very low levels of factor VIII activity (FVIII:C). Thromb Haemost. 2002;87: Matsumoto T, Shima M, Takeyama M, et al. 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Int J Lab Hematol. 2010;32: How to cite this article: Sevenet PO, Depasse F. Clot waveform analysis: Where do we stand in 2017?. Int J Lab Hem. 2017;39: