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Received: 28 April 2017    Accepted: 11 July 2017

DOI: 10.1111/ijlh.12724

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.
Email: pierreolivier.sevenet@stago.com
Abstract
Analysis of the optical waveform generated during global coagulation assays, such as
activated partial thromboplastin time and prothrombin time, can provide much pre-
cious information on the global coagulation state of the plasma sample tested, in addi-
tion 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 anti-
coagulant diagnosis and anticoagulant monitoring. Altogether, these publications have
demonstrated the ability for clot waveform analysis (CWA) to improve patient man-
agement, especially as this tool is inexpensive, rapid and readily available on coagula-
tion 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 set-
tings. Among the numerous laboratory assays exploring haemostasis,
global functional assays have been generating more and more inter-
est 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 co-
agulation abnormalities. The most widely known global coagulation
assays are the thrombin generation test (TGT) and the viscoelastomet-
rics assays (such as ROTEM® or TEG®). Through the evaluation of the
kinetic of fibrin formation during tests such as activated partial throm-
boplastin 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 phe-
notype 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 in-
terest.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 orig-
inal 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 (anal-
ysis[all fields] OR biphasic[all fields] OR haemophilia[all fields] OR dis-
seminated intravascular coagulation[all fields])” and “activated partial
thromboplastin time[all fields] AND (reaction curve[all fields] OR op-
tical profil[all fields] OR transmittance[all fields] OR first derivative[all

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fields] OR second derivative[all fields]).” After exclusion of noneligi-
ble 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 co-
agulation 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 sig-
nal 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 rep-
resents 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 trans-
mittance, 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 maxi-
mum 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 comput-
erizes 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 al8 They formulated the minimal requirements for per-
forming 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.
F I G U R E   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
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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 interpre-
tation 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 vari-
1
ation of CWA parameters. They observed significant qualitative dif-
ferences 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 (1.0-­100 IU/dL) for FIX. According to their ob-
servations, 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 haemo-
philiac 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 accord-
ing 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 se-
vere 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 ten-
dency 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 haemo-
philiac patients better than the determination of FVIII:C alone. This
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is also supported by the publication of Yada and colleagues, who in-
vestigated 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 re-
sults 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 re-
mains 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 be-
tween those 2 groups of patients with a remarkable sensitivity and
specificity (ROC analysis: area under the curve = 0.978, P < .001; sen-
sitivity = 97.3%; specificity = 93.2%). Moreover, they showed that the
occurrence of severe clinical events (age of first bleed, number of an-
nual 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 phe-
notypes 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 asso-
ciated 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 2014. 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 (r2 = 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 ca-
pabilities 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
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564       SEVENET and DEPASSE
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 bypass-
ing therapies, usually human recombinant activated factor VII (rFVIIa)
or activated prothrombin complex concentrates (aPCC). Optimization
of bypassing therapies in these particular patients remains challeng-
ing. 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 throm-
botic 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.16-18
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 monitor-
ing 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
18
pre-­analytical variables such as in vitro coagulation activation.
More recently, Haku et al explored the performances of CWA in
bypassing therapy monitoring, for both rFVIIa and aPCC. They as-
sessed different triggers for coagulation and found that the best re-
sults, after in vivo infusion of bypassing therapy, were yielded when
aPTT is activated with a mix of low doses of tissue factor and ellagic
4
acid. This study confirms the promising potential of CWA in bypass-
ing therapy monitoring, even with a modified aPTT trigger reagent.
Furthermore, they were the first to monitor the real-­time clinical ef-
fectiveness 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 inhib-
itors. 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 param-
eters. 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 ther-
apy in patients with HA with inhibitors.21
It can thus be seen that CWA is a very promising tool for the eval-
uation of the effects of haemostatic drugs, either for research and de-
velopment 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 characteristi-
cally 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 modi-
fied 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 be-
tween CWA and DIC diagnosis was assessed in a prospective co-
hort 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%), re-
spectively.5 Toh and colleagues demonstrated that the addition of
Ca2+ in the test sample induces the rapid formation of a precipi-
tate 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 clini-
cal 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 wave-
form.28 Another prospective study over 217 consecutive ICU pa-
tients 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
SEVENET and DEPASSE
F I G U R E   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 cor-
relation between the presence of BWP and the early development of
sepsis in patients admitted to ICU.28,31-33 They showed a modest sen-
sitivity, 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) deter-
mined 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 sys-
temic inflammatory response syndrome (SIRS) with a sensitivity of
100% and a specificity of 93% (ROC analysis, area under the curve:
0.948 ± 0.039; P < .01).34 This finding reinforces what Chopin et al de-
scribed 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 pres-
ence of aPTT BWP is related to the poorest clinical outcomes (infec-
tion 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 pos-
itive 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)
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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 ca-
pacity 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.39
3.4 | 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
al40 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 pa-
tients with APA from other groups of patients, even aPTT clotting time
due to its weak specificity. In other studies, abnormal aPTT waveform
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566       SEVENET and DEPASSE
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 pa-
tients with mild HA or acquired HA is significantly decreased (1.3 ± 0.2
42
and 0.9 ± 0.5, respectively) compared to the aforementioned groups.
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 nor-
mal 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 observa-
tions 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 po-
tential 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, inde-
pendently of clotting times and plasma quality (haemolysis, icterus and
lipaemia). Despite the positive results, this method of fibrin measure-
ment is not innovative and will not replace present conventional fibrin-
ogen 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 im-
pact on CWA parameters.45,46
Clot waveform analysis has been used by Shima’s team to demon-
strate the increased procoagulant state in chronic spontaneous ur-
ticaria47 and for the exploration, in association with other global
coagulation assays, of the paradoxical procoagulant effect induced by
plasmin.48
4 |  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 thromboe-
lastometry. They differ greatly from CWA in their operating princi-
ples. 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 op-
erating room. Further applications are however possible as numerous
trigger reagents could be used (extrinsic pathway assessment, hepa-
rin levels, fibrinogen measurement and resistance to lysis). Thrombin
generation could also assess both thrombotic states and bleeding ten-
dencies. 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 per-
formed 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 re-
flects the whole process of thrombin generation over time. This con-
trasts 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 decreased53 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 ad-
vantages. 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 equip-
ment. Furthermore, CWA could be run as a regular routine assay on
the same sample as PT and aPTT (0.109 or 0.105 mol/L trisodium ci-
trate blood collection tubes), and with a fast turnaround time com-
pared 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 stud-
ies on DIC, it has been shown that the presence of an abnormal pat-
tern 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.
SEVENET and DEPASSE
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 standard-
ization using a sensitive optical detection system, colourless and non-
opaque 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 wave-
length 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 be-
cause of the easy-to-perform protocol, and convenient and very well-­
known assays involved.
Despite the accumulated data suggesting benefits in various clin-
ical 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 “thrombelas-
tography,” 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 physio-
pathological 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 clot-
ting 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
|
      567
studies are limited to small cohorts, extensive prospective clinical tri-
als are mandatory to make the leap to clinical application.
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How to cite this article: Sevenet PO, Depasse F. Clot
waveform analysis: Where do we stand in 2017?. Int J Lab
Hem. 2017;39:561–568. https://doi.org/10.1111/ijlh.12724