Analysis of APTT Based Clot Waveform Parameters in Various Clinical Conditions – A Study at A Tertiary Care Center

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INTRODUCTION
Clot Waveform Analysis (CWA) is an extended interrogation of the curve generated by an optical detection system during the measurement of coagulation assays such as Prothrombin Time (PT) and activated Partial Thromboplastin Time (aPTT).It detects light transmittance basd on its absorbance.This is a global hemostatic assay, that reflects the overall hemostatic factor (1).
The automated photo-optical coagulation analyzers used for the estimation of PT and APTT display the clot reaction curves along with the first and second derivative curves (first and second DCs) (2).
The height of the first DC in the APTT-CWA is used to reflect to the "thrombin burst" as a hemostatic ability.The low height of the first DC in APTT-CWA suggests a risk of bleeding.The height of the second DC in APTT-CWA is useful for detecting coagulation factor deficiency (3).Thromboelastography (TEG) also shows a different pattern of information, but is slightly expensive and time-consuming (4).
The coagulation system has a specific mechanism that includes the cascade system, thrombin burst and enhancement of clotting activation by phospholipids (PLs) (5).
The various available assays which evaluate the coagulation system these days are activated partial thromboplastin time (aPTT), PT, thromboelastography (TEG), and thrombin generation test (TGT) (6).

MATERIALS AND METHODS
It is a prospective cross-sectional study Statisctical significance was set at P<0.05.

RESULTS
A total of 145 patients were included in the study, with 20 control samples.The mean age group in the present study was 46.9 ± 20 years with male to female ratio of 2.2:1.The ranges of APTT, first, and second derivatives in controls and cases were determined (Table 1).
The     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 CWA is a global coagulation assay that evaluates the kinetics of fibrin formation during testing of aPTT or PT.Clot waveforms provide information on light transmittance during clot formation (7).Automatic optical end-point coagulation analyzers have the ability to show the clot reaction curve of the PT and APTT and reflect the "thrombin burst" with "enhancement of clotting activation by PLs (8).The plot waveform analyses the slope generated by optical detection during routine coagulation tests, such as aPTT or PT.The optical detection system generates a clot formation process with respect to the change in transmittance and absorbance of the light beam through the sample (9).Continuous measurement of the change in light transmission or absorbance during the PT and APTT assays is performed and the data are given in the form of a wave.This generated clot wave has three phases: 1) Preor increased absorbance along with formation of fibrin is seen in the clotting process which is seen as slope on waveform, 3) Post coagulation: Towards the end of coagulation, the light transmittance or absorbance stabilizes, which is seen as a linear segment on the waveform (10).TGT and CWA provide similar information, as both correlate with the rate and velocity of thrombin formation and reflect the entire process of thrombin generation.Clinical conditions such as DIC/Sepsis and prediction of bleeding risk in DIC (3), Factor VIII deficiency (3), bacterial infections (11), Covid 19 (12), patients on anticoagulant therapy exhibit different clot waveform morphology.The aim of the present study was to analyze and compare clot waveform characteristics, such as morphology and first-and second-derivative values, in cases with abnormal APTT.

with a duration of 6
months.A total of 125 patients and 20 age matched controls were included in the study.All the abnormal APTT samples were thoroughly selected through the analyzer.Patients receiving unfractionated heparin (UFH) or lowmolecular-weight heparin (LWMH) were excluded from the study.Blood samples were collected in anticoagulant tubes containing 1:9 volumes of 3.2% trisodium citrate.For obtaining platelet poor plasma: platelet-poor plasma was obtained by centrifugation of the blood samples at 3000 rpm for 15 min.The plasma is analyzed for platelet count on a cell counter, which should be less than 10000/ per microliter.PT/APTT was performed using an ACL Top 300 CTS Coagulation Analyzer.The morphology of clot waveform in all the conditions with abnormal APTT was studied.The parameters studied were the first derivative (maximum velocity of clot formation), second derivative (maximum and minimum acceleration and deceleration during clot wave formation).Statistical analysis was performed using SPS15 software.The mean and standard deviation were calculated for the APTT, First and second derivatives.Mann Whitney U test was performed and p value was determined.

4 Figure 2 .
Figure 2. The mean values of APTT and the first and second derivatives in each clinical condition were calculated and compared with those of the controls (Table2).

Figure 1 .Figure 2 .
Figure 1.Spectrum of clinical cases and various morphology patterns of clot wave morphology of the clot waveform.The precoagulation phase was entirely distorted and the first and second-derivatives were suggestive of consumptive coagulopathy (DIC).In addition, two cases of factor deficiency (Factor V and Factor VIII) showed corresponding changes in the clot wave pattern with early and late shoulders in the second derivative curves.DISCUSSION Multiple factors, such as the blood vessel wall, plasma proteins, platelets and coagulation factors are involved in the coagulation cascade.PT and aPTT are the routinely performed tests that give information regarding hemostasis.Coagulation assays are of utmost importance in this new era.(13).CWA is based on APTT and PT tests, which are global coagulation tests and is studied on the principle of optical detection system through an automated coagulation analyzer.Various ACL series are widely available and use an automated coagulation analyzer that works on the light absorbance principle and helps in studying the entire process of hemostasis depicted in the form of waves as described above (14).In the present study, clot waveforms detected hemostatic alterations and abnormal patterns in various clinical cases like Covid-19, bacterial infection, liver diseases, DIC/sepsis, hemophilia, venous thromboembolism.A rise in the incidence of thrombotic events, such as pulmonary embolism, has been observed in critical ill cases of Covid-19 with severe hypercoagulability is been seen in various studies (15).M.F Rubereto et al., (16) studied CWA in 191 patients with liver cirrhosis and found values of maximum acceleration and deceleration were lower in the cirrhotic patients as compared to the control groups which correlate with our study.Takuya et al., (12) studied the clot wave forms of APTT in 26 patients with Covid-19 and the results showed abnormal patterns of second derivative morphology (early shoulder type and late shoulder type), which were similar to those found in our study.In a study by Tan et al., (1) of 101 patients, it was concluded that patients with bacterial infections showed significantly higher CWA parameters than controls.In contrast, patients with dengue infection had significantly lower CWA parameters.This similar observation was also seen in our study.Kei et al., (3) studied the clot wave form in 211 patients of sepsis and showed first and second derivatives curves were useful in diagnosis and prediction of bleeding risks.There was a significant association between the second derivative and the disease condition in this study.A study by Dave al., (17) showed that the risk of severe bleeding in patients with Hemophilia A invariably accompanied by an aberrant clot waveform and thrombin generation test, while the Factor VIII level did not always reflect the actual bleeding severity.Similarly, we had two cases of factor deficiency in our study with abnormal clot wave formation, thus providing new insight into the potential utility of CWA in detecting hypercoagulability or risk of bleeding in various clinical conditions (17).A study by Kanouchi et al., (18) showed significant atypical peak and deceleration/acceleration ratio extension using clotting waveforms, specifically in patients with LA-positive APS.Oka et al., (19) studied the CWA for the assessment of DOAC effects and provided valuable insights into the relevance of anticoagulation to therapeutic efficacy and bleeding risk from the perspective of fibrinolysis.CWA is an extended study of the routine aPTT test that utilizes pre-existing test protocol and equipment assay.Many automated analyzers use changes in light transmittance or absorbance to measure clotting times, and these optical changes over time as clot forms are captured and presented as a clot waveform curve in the software of the analyzers (20).As more studies showing the correlation between the clot wave patterns have surfaced, the importance of full utilization of the data provided by the automation machines can be interpreted without any additional cost and turn over time (14).

Table 1 .
Range of APTT, First and Second Derivatives in Cases and Controls

Table 2 .
Mean Values of APTT, First and Second Derivative in Various Clinical Condition

Table 3 .
Association of APTT, First and Second Derivatives in Various Clinical Conditions