Towards standardization of clot waveform analysis and recommendations for its clinical applications

Abstract

Automated coagulation analyzers can provide a wealth of information in addition to that provided by conventional clotting tests such as the prothrombin time (PT) and the activated partial thromboplastin time (APTT), which are often considered of limited use for clinical purposes 1.Braun P.J. Givens T.B. Stead A.G. Beck L.R. Gooch S.A. Swan R.J. Fischer T.J. Properties of optical data from activated partial thromboplastin time and prothrombin time assays.Thromb Haemost. 1997; 78: 1079-87Crossref PubMed Scopus (54) Google Scholar. One particular type of analysis, the clot waveform, which was originally described using the Multichannel Discrete Analyzer (MDA series; Organon, Technika, Durham, NC, USA), defines changes in light transmittance that occur during the process of clot formation. A number of recent reports have described the use of this type of automated clotting instrument for clot waveform analysis (CWA), and there appears to be significant advantages in using this assay for the assessment of global coagulation function. In this communication, we propose standardization of methods for the CWA using currently available clotting analyzers and overview the potential clinical applications. Changes in light transmittance or absorbance are determined by continuous measurements during the APTT and are designated the clot waveform (CW) (Fig. 1). This clotting process is categorized into three parts: the pre‐coagulation, coagulation and post‐coagulation phases. Pre‐coagulation is described as the first segment of the trace, from the beginning of the signal to the onset of coagulation. After the onset of coagulation, light transmittance is decreased or absorbance is increased by the formation of fibrin, and this is defined by a slope in the waveform. At the end of coagulation, light transmittance or absorbance tends to stabilize and is characterized again by a linear segment. If fibrinolysis is enhanced due to acquired or congenital abnormalities of hemostasis, light transmittance may increase or absorbance may decrease again in the post‐coagulation phase. There are two types of clotting machines for CWA. One utilizes a system to detect transmittance during the APTT clotting reaction, and is represented by the MDA‐II or CS series. In this type, transmittance is decreased after initiation of clotting (Fig. 1A). The other type monitors the absorbance, and is represented by the ACL series. In this type, 0% absorbance defines the pre‐coagulation phase, and the absorbance increases after the initiation of clotting (Fig. 1B). Other analyzers having similar features should also be able to provide CWA data easily, particularly if manufacturers incorporate the relevant software. Even if an automated CWA is not available, there are several analyzers that are able to provide adequate raw data (Table S1). CWA is possible using such analyzers by statistical evaluation of this raw data of transmittance or absorbance (Fig. S1). Plasma should be prepared from fresh citrated whole blood as for the standard APTT assay. Pooled plasma from normal individuals or commercial normal plasma is available as reference plasma. Colorless APTT reagents, without opacity, are recommended to detect sensitive changes in transmittance or absorbance, and for subsequent precise measurements of the various parameters. Although any APTT reagent that fulfills this criteria should be usable for CWA, among the reagents tested so far (Table S1), Thrombocheck APTT‐SLA/0.02M CaCl2 is suitable for MDA‐II and CS series and HemosIL SynthASil for ACL‐Top series. Other instrument and reagent combinations should also be possible to use but these need to be tested. APTT reagents used for the detection of anti‐phospholipid antibodies are not recommended for the CWA because their sensitivity for assessing low clotting function is not sufficiently high. The APTT for CWA is performed in a similar manner to that for the standard APTT assay. The first derivative of the transmittance reflects coagulation velocity, and the second derivative reflects coagulation acceleration. Clotting time (CT), maximum coagulation velocity (Min1), maximum coagulation acceleration (Min2) and maximum coagulation deceleration (Max2) are common basic parameters. Among these measurements, Min2 has been reported to be correlated with clotting function in hemophilia 2.Shima M. Matsumoto T. Fukuda K. Kubota Y. Tanaka I. Nishiya K. Giles A.R. Yoshioka A. 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: 436-41Crossref PubMed Scopus (83) Google Scholar. Initial evaluation of clotting function by CWA is undertaken by qualitative assessment of the CW pattern. In particular, two characteristic CW patterns are observed in various coagulation abnormalities compared with normal reference plasma (Fig. S2). In normal plasma, the pre‐coagulation phase is short and the slope, reflecting the coagulation phase, is steep. In factor (F) XII, X, IX, VIII, V and II deficiencies, the pre‐coagulation phase is prolonged but the changes in slope are different 1.Braun P.J. Givens T.B. Stead A.G. Beck L.R. Gooch S.A. Swan R.J. Fischer T.J. Properties of optical data from activated partial thromboplastin time and prothrombin time assays.Thromb Haemost. 1997; 78: 1079-87Crossref PubMed Scopus (54) Google Scholar. Changes in slope are more evident in FVIII and FIX deficiencies than in other deficiencies. Thus, qualitative analysis of CW may have diagnostic value in various clinical settings of impaired clotting function. While assays of FVIII:C and FIX:C are most important for the clinical management of hemostasis in patients with hemophilia, CWA provides a potentially widely available platform for assessment of global hemostasis in these patients 3.Matsumoto T. Shima M. Takeyama M. Yoshida K. Tanaka I. Sakurai Y. Giles A.R. Yoshioka A. The measurement of low levels of factor VIII or factor IX in hemophilia A and hemophilia B plasma by clot waveform analysis and thrombin generation assay.J Thromb Haemost. 2006; 4: 377-84Crossref PubMed Scopus (70) Google Scholar (Figs S3 and S4). This assay could then also provide a novel method not only for diagnosis and correlations with the bleeding phenotype but also for monitoring of hemostasis in cases of replacement therapy for serious hemorrhage or surgery. Furthermore, the aPTT CWA is also useful for assessing very low levels of FVIII or FIX, for example less than 1 IU dL−1. Studies in a number of patients with severe HA diagnosed by conventional clotting assays, demonstrated that CWA patterns differed from patient to patient. The APTT clotting time was prolonged in all patients with severe HA, but there was variation in the slope 2.Shima M. Matsumoto T. Fukuda K. Kubota Y. Tanaka I. Nishiya K. Giles A.R. Yoshioka A. 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: 436-41Crossref PubMed Scopus (83) Google Scholar. Using mixtures of severe HA plasma and exogenous FVIII ranging from zero to 1.0 IU dL−1, the slope and the APTT clot time and the min2 appeared to change in a dose‐dependent manner. Similarly, in further studies of 36 patients with severe HA, significant correlations between min2 and very low levels of FVIII:C were confirmed 4.Shima M. Matsumoto T. Ogiwara K. New assays for monitoring haemophilia treatment.Haemophilia. 2008; 14: 83-92Crossref PubMed Scopus (55) Google Scholar. These results indicated that in some patients, the presence of trace amounts of FVIII mediated higher coagulation acceleration, characterized by the steeper slope, although it was possible that factors other than FVIII:C alone may have influenced clotting kinetics reflected in the waveform profile. Nevertheless, the data suggested that CWA could discriminate between different levels of FVIII:C in this critical category of severe HA, defined as having < 1.0 IU dL−1 FVIII:C by conventional assays (Figs S3 and S4). The evidence suggests that CWA can provide more specific data on global hemostasis in such patients, which could correlate better with the clinical phenotype. Some HA patients, classified as severe on the basis of standard coagulation assays, exhibit milder clinical symptoms. It appeared possible, therefore, that CWA might provide valuable data for evaluating in vivo clotting function in various types of hemophilia A. To investigate this possibility, severe hemophilia A patients based on < 1 IU dL−1 of FVIII:C were divided into clinically severe and non‐ severe groups 4.Shima M. Matsumoto T. Ogiwara K. New assays for monitoring haemophilia treatment.Haemophilia. 2008; 14: 83-92Crossref PubMed Scopus (55) Google Scholar. Clinically severe patients were characterized by the presence of spontaneous bleeding episodes at the age of < 1 year, the onset of joint or muscular bleeding before the age of 3 years old, or the presence of severe bleeding such as intracranial bleeding or refractory oral bleeding. The differences between the severe and the non‐severe phenotype were significant for four CW parameters: clot time, maximal coagulation velocity (Min1), maximal coagulation acceleration (Min2) and maximal coagulation deceleration (Max2), These results strongly suggested, therefore, that CW parameters reflect clinical severity (Fig. S5). The hemostatic benefits of various agents used for bypassing therapy, including activated prothrombin complex concentrates (APCC) and recombinant factor VIIa (rFVIIa), can be monitored by CWA 4.Shima M. Matsumoto T. Ogiwara K. New assays for monitoring haemophilia treatment.Haemophilia. 2008; 14: 83-92Crossref PubMed Scopus (55) Google Scholar, 5.Shima M. Understanding the hemostatic effects of recombinant factor VIIa by clot waveform analysis.Semin Hematol. 2004; 41: 125-31Crossref PubMed Scopus (33) Google Scholar. In addition, CWA was also utilized effectively in a recent clinical phase 1 study for the assessment of a new bypassing agent based on mixtures of plasma‐derived FVIIa and X 6.Shirahata A. Fukutake K. Mimaya J. Takamatsu J. Shima M. Hanabusa H. Takedani H. Takashima Y. Matsushita T. Tawa A. Higasa S. Takata N. Sakai M. Kawakami K. Ohashi Y. Saito H. Results of clot waveform analysis and thrombin generation test for a plasmin‐derived factor VIIa and X mixture (MC710) in haemophilia patients with inhibitors‐Phase I trial: 2nd report‐.Haemophilia. 2013; 19: 330-7Crossref PubMed Scopus (16) Google Scholar. In two hemophilia A patients with high responding inhibitors, CWA demonstrated improved hemostasis. Moreover, CWA was shown to reflect the prophylactic effect of regular infusions of FVIII during immune tolerance induction therapy (ITI) 7.Kasuda S. Tanaka I. Shima M. Matsumoto T. Sakurai Y. Nishiya K. Giles A.R. Yoshioka A. Effectiveness of factor VIII infusions in haemophilia A patients with high responding inhibitors.Haemophilia. 2004; 10: 341-6Crossref PubMed Scopus (16) Google Scholar. The findings confirmed that CWA is very sensitive to low levels of clotting factors, and suggested that the technique could also be useful for monitoring therapy using FVIII or FIX concentrates in patients with inhibitor. FVIII:C levels do not reflect clinical severity in many cases of acquired hemophilia A, and it may be difficult to determine clotting function precisely in these patients. CWA illustrates severely impaired patterns in these cases, however, characterized by a remarkably prolonged pre‐coagulation phase and low values for maximum coagulation velocity and acceleration 8.Matsumoto T. Nogami K. Ogiwara K. Shima M. A putative inhibitory mechanism in the tenase complex responsible for loss of coagulation function in acquired haemophilia A patients with anti‐C2 autoantibodies.Thromb Haemost. 2012; 107: 288-301Crossref PubMed Google Scholar. Assessment of clotting function by aPTT CWA, in addition to the measurement of FVIII activity, can be useful, therefore, to confirm decisions on hemostatic treatment and the monitoring of bypass therapy in these complicated clinical circumstances. There are several advantages to the use of CWA. The method has broad utility as a simple global test of hemostasis and is capable of providing sensitive, quantitative parameters as well as qualitative waveform patterns. Furthermore, CWA can be usefully applied in various difficult clinical settings. Not all current coagulation analyzers can be used for CWA, however, although the number of appropriate analyzers is increasing. Finally, the CWA is based on APTT‐based coagulation mechanisms using an ‘intrinsic’ trigger. A modified CWA using trace amounts of tissue factor may extend the application of this technique. Among the global hemostasis tests, CWA is perhaps the simplest to establish and standardize. It therefore needs to be tested more widely using standardized methods in different clinical situations to decide its place in the assessment of hemostasis and its disorders. M. Shima chaired the working party, performed the research, analyzed the data and wrote the manuscript. J. Thachil and S.C. Nair performed research and collected data. A. Srivastava supervised the study. M. Shima is supported for APTT reagents from Sysmex. Download .jpg (.59 MB) Help with files Figure S1. Data sheet of the transmittance and presentation by waveform. Download .jpg (.17 MB) Help with files Figure S2. APTT clot waveforms of various clotting factor deficiencies. Download .jpg (.16 MB) Help with files Figure S3. Dose‐dependent waveform changes in plasma containing various concentrations of FVIII. Download .jpg (.31 MB) Help with files Figure S4. Waveform changes in hemophilia A with various levels of FVIII. Download .jpg (.05 MB) Help with files Figure S5. CWA parameters and clinical severity of severe hemophilia A. Download .xls (.02 MB) Help with xls files Table S1. Coagulation analyzers and APTT reagents for clot waveform analysis.