| Abstract|| |
Context: Hemophilia A is classified as mild, moderate, and severe based on Factor VIII levels (FVIII). Clot-based assays only detect initiation of thrombin generation, hence FVIII levels may not accurately predict the bleeding risk in all hemophilia patients. The entire process of thrombin generation as measured by global hemostasis tests like activated partial thromboplastin time clot waveform analysis (APTT CWA) and thrombin generation test (TGT) may reflect the actual bleeding phenotype. Aims: To assess the utility of TGT and CWA as a screening tool to identify bleeders and to evaluate the bleeding phenotype in Hemophilia A. Settings and Design: Prospective, observational study of 147 consecutive patients referred for coagulation workup. Subjects and Methods: Bleeding assessment tool was used to identify bleeders. Patients were classified as severe and nonsevere bleeders based on clinical criteria. TGT was performed by calibrated automated thrombogram, CWA by photo-optical coagulometer and factor levels by one stage clot-based assays. Statistical Analysis Used: The Kruskal-Wallis test with post-hoc analysis was done to examine the difference in CWA/TGT parameters amongst hemophilia classified by FVIII levels. Receiver operating characteristic (ROC) analysis was performed to estimate the diagnostic accuracy of CWA and TGT in discriminating between clinically severe vs nonsevere bleeders. Results: Using ROC derived cut-offs of min1, min2 and peak height of thrombin (PH), the sensitivity (min1:91.67%, min2:91.67%, PH: 97.22%, FVIII: 86.11%) and specificity (min1:100%, min2:100%, PH: 90.91%, FVIII: 90.91%) of CWA/TGT was superior to FVIII to distinguish between clinically severe vs nonsevere bleeders. Phenotypic heterogeneity of bleeding severity was identified in our study population. Clinical severity correlated with CWA/TGT parameters instead of FVIII levels. Conclusions: CWA and TGT are more effective tools than conventional factor assays to identify clinically severe bleeders and tailor prophylaxis as per bleeding phenotype.
Keywords: Clot waveform analysis, factor VIII, Hemophilia A, International Society of Thrombosis and Haemostasis-Bleeding Assessment Tool score, thrombin generation test
|How to cite this article:|
Dave RG, Geevar T, Mammen JJ, Vijayan R, Mahasampath G, Nair SC. Clinical utility of activated partial thromboplastin time clot waveform analysis and thrombin generation test in the evaluation of bleeding phenotype in Hemophilia A. Indian J Pathol Microbiol 2021;64:117-22
|How to cite this URL:|
Dave RG, Geevar T, Mammen JJ, Vijayan R, Mahasampath G, Nair SC. Clinical utility of activated partial thromboplastin time clot waveform analysis and thrombin generation test in the evaluation of bleeding phenotype in Hemophilia A. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 Jan 16];64:117-22. Available from: https://www.ijpmonline.org/text.asp?2021/64/1/117/306505
| Introduction|| |
Hemophilia A patients are conventionally classified as mild (Factor VIII >5–40%), moderate (Factor VIII 1–5%), and severe (Factor VIII <1%) based on Factor VIII (FVIII) level.
Current treatment is predominantly guided by factor VIII levels wherein severe hemophilia patients with FVIII <1 IU dL-1 are closely monitored and may be candidates suitable for early prophylaxis, whereas patients with moderate (FVIII 1–5 IU dL-1) and mild hemophilia A (FVIII >5 IU dL-1) mostly receive on-demand treatment.
However, a subset of 10 to 15% of patients with severe hemophilia A (FVIII <1%) show a milder disease phenotype with significantly reduced frequencies of spontaneous bleeding and lower requirements for factor concentrates. On the other hand, some patients with moderate hemophilia A (FVIII 1–5%) have frequent episodes of spontaneous bleeding and more frequent factor concentrate requirement.,,
Thus, FVIII clotting activity does not have an excellent predictive value to evaluate the bleeding risk in hemophilia A patients. Treatment decisions, such as starting prophylaxis, should therefore be tailored according to the bleeding pattern rather than based on the residual factor VIII activity levels.
Thrombin being the central enzyme in the coagulation cascade, there are no pathways that can bypass thrombin to form a stable clot. Therefore estimation of one's potential to generate thrombin may actually correlate closely with clinical heterogeneity of bleeding severity.
The clot based tests measure clotting time when only 5% of the total thrombin is generated and thus may not reflect the actual bleeding phenotype. More than 95% of all thrombin forms explosively in the clot after the clotting time is detected.,
Global hemostasis tests like clot waveform analysis (CWA), and thrombin generation test (TGT), reflect the entire process of thrombin generation, and thus may reflect the actual bleeding phenotype.
This study was based on the hypothesis that the total amount of thrombin generated (as reflected by the global hemostasis tests) may more accurately predict clinical severity of bleeding than the time that it takes for the initiation of thrombin formation (as reflected by clotting time-based assays).
TGT requires a costly proprietary instrument, whereas activated partial thromboplastin time (APTT) CWA can be obtained on a photo-optical coagulometer at the cost of routine APTT. Thus, APTT CWA could be a practical and cost-effective tool that may be easily used to evaluate the bleeding phenotype of Hemophilia A patients.
The purpose of this study was to evaluate the diagnostic utility of global hemostasis tests like TGT and APTT CWA in assessing bleeding phenotype of hemophilia A patients so that treatment decisions such as starting prophylaxis can be tailored according to the bleeding pattern.
| Subjects and Methods|| |
A total of 147 consecutive patients referred to our institution for complete coagulation workup were enrolled for the study. About 51 patients were diagnosed as hemophilia A, 65 patients had no hemostatic defect and were taken as controls.
Amongst the remaining patients (7 patients with Hemophilia B, 3 patients with Von Willebrand (VWD) type 1, 6 patients with VWD type 3, 4 patients with Factor V deficiency, 2 patients with Factor VII deficiency, 4 patients with Factor X deficiency, 1 patient with Factor XI deficiency and 5 patients with combined factor deficiencies) were tested but not included in view of their small sample size to be statistically significant.
Patients with platelet dysfunction and who had received any transfusions or clotting factor concentrate within the previous 2 weeks were excluded.
A detailed questionnaire based International Society of Thrombosis and Hemostasis Bleeding Assessment Tool (ISTH-BAT) score was calculated and all patients were grouped into bleeders and non-bleeders based on established cut-offs for an abnormal BAT Score of =4 for adult males, =6 for adult females and =3 for children. ISTH-BAT score was used as the reference standard to which TGT and APTT CWA parameters were compared to identify bleeders.
Hemophilia A patients (n = 51) were also grouped according to the clinical severity of bleeding into severe bleeders and nonsevere bleeders. For the purpose of this study, severe bleeders were defined as those with a history of life-threatening hemorrhages, multiple spontaneous hemorrhages such as hemarthrosis, muscle hematomas, umbilical cord, gastrointestinal, and central nervous system bleeding, patients with >2 joints involved by hemophilic arthropathy or those with Clotting factor concentrate/transfusion requirement of >5 times/year. Non-severe bleeders were defined as those who bled only after trauma or surgery, bled less frequently (about once a month) with =2 joints involved, or had less Clotting factor concentrate/transfusion requirement of <5/year or had only minor symptoms such as epistaxis and mucocutaneous bleeds.,
Blood was drawn via a 21G × 1'' [0.80 mm × 25 mm] needle directly into evacuated anticoagulant tubes containing 3.2% sodium citrate with minimal stasis.
Blood sample was centrifuged at 2500 g for 20 min and platelet-poor plasma was prepared within half an hour of venipuncture.
Factor VIII Assay was done by one stage clot based assay on the Sysmex CS2000i coagulometer. APTT clot waveform analysis was done by photo-optical automated coagulation analyzer Sysmex CS2000i using Automate as the APTT reagent. The objective data on APTT clot waveform was obtained using parameters like Min1 (the minimum value of first derivative of the APTT waveform), Min2 (the minimum value of the second derivative of the APTT waveform) and Max 2 (the maximum value of the second derivative of the APTT waveform).
Thrombin Generation test was performed by Calibrated Automated Thrombogram. Samples were run in duplicates along with a calibrator well for each sample.
To each of the 96 well round bottom microtiter plates, 20 μL of PPP trigger reagent (1 pM of recombinant relipidated tissue factor and 4 μM of phospholipids) or 20 μL of Calibrator were added along with 80 μL of respective platelet-poor plasma samples.
The plates were warmed for 10 min, followed by the automated addition of 20 μL of freshly prepared buffer fluCa substrate (2.5 mM fluorogenic substrate [Z – Gly – Gly – Arg – AMC] and Calcium chloride) to all the wells.
The concentration of thrombin generated in clotting plasma was measured by a 390 nm excitation and 460 nm of emission filter set, and comparing it to a constant known thrombin activity in parallel. The microtiter plate fluorometer used was the Fluoroskan Ascent. Fluorescence was measured in each well at 30 second intervals for 1 hour to give thrombogram. Objective parameters of TGT like lag time, endogenous thrombin potential (ETP), the peak height of thrombin (PH), time to peak (TTP) and start tail were analyzed.
The statistical analysis was done using the STATA/1C 13.1 version. In the case of the normal distribution, data were expressed as mean values with SD, while the non-normally distributed data were expressed as median with ranges. Kruskal-Wallis test along with a post-hoc analysis was done to examine the difference in APTT clot waveform parameters and thrombin generation test parameters amongst hemophilia patients classified as per FVIII levels. The Receiver operating characteristic (ROC) analysis was performed to estimate the diagnostic accuracy of waveform analysis and thrombin generation test parameters in discriminating between clinically severe and non-severe hemophilia A patients. Statistical significance was defined as P < 0.05.
| Results|| |
All the patients enrolled in the study were classified as nonbleeders and bleeders based on the ISTH-BAT score.
Hemophilia A patients were classified:
- Based on FVIII levels: Into mild (FVIII >5–40%), moderate (FVIII 1–5%), and severe (FVIII <1%) hemophilia
- Based on the clinical severity of bleeding into severe and nonsevere bleeders
- Based on ROC derived best cut-offs of CWA parameters and TGT parameters into group I (probable severe bleeders) and group II (probable nonsevere bleeders).
When Hemophilia A were characterized based on FVIII levels into mild (FVIII: C >5–40%), moderate (FVIII: C 1–5%) and severe (FVIII: C <1%), 70% patients were severe hemophilia, 14% patients were moderate hemophilia and 16% were mild hemophilia whereas when classified based on clinical severity of bleeding, 80% were severe bleeders and 20% were nonsevere bleeders.
On assessing the utility of clot waveform and TGT to distinguish bleeders vs nonbleeders, all parameters had very good sensiti?vity (min1: 92.20%, min2: 98%, peak height: 92.20%, time to peak: 94.10%) and specificity (min1: 96.90%, min2: 92.20%, peak height: 98.40%, time to peak: 87.50%) and thus can be good screening tools to distinguish non-bleeders from even mild bleeders.
When hemophilia A patients were classified as mild, moderate, or severe based on FVIII levels, there was statistically significant difference (P < 0.001) between the clot waveform parameters (min1, min2, max 2) and TGT parameters (ETP, PH, TTP, Start tail) between normal controls vs mild-to-moderate hemophilia patients and mild-to-moderate hemophilia vs severe hemophilia patients, respectively [Table 1]. However, Lag time did not show statistically significant difference. Parameters of CWA (min1, min2) and TGT (ETP, PH) were significantly different between clinically severe and non-severe bleeders with no overlap between the two groups [Figure 1].
|Table 1: Results of quantitative clot waveform analysis and TGT parameters in control group, mild.moderate and severe hemophilia A patients (classified based on FVIII level)|
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|Figure 1: Utility of CWA/TGT parameters to distinguish Clinically Severe vs Nonsevere bleeders. CWA parameters min1 and min2, and TGT parameters Endogenous thrombin potential and peak height are significantly different between clinically severe and nonsevere bleeders with no overlap between two groups|
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On applying the ROC curve to compare the discriminatory ability of CWA parameters and conventional FVIII level to identify clinically severe vs nonsevere bleeders, the discriminatory ability reflected by the AUC as well as reproducibility reflected by 95% CI was superior for clot waveform parameters compared to FVIII. Based on this ROC curve, we identified the best cut-offs of min1 (<1.33) and min 2 (<0.145) to identify clinically severe bleeders [Table 2] and [Figure 2]. On applying these cut-offs, we found that the sensitivity (min1: 91.67%, min2: 91.67%, FVIII: 86.11%), as well as specificity (min1: 100%, min2: 100%, FVIII: 90.91%) of min1 and min2, was superior to FVIII to distinguish between clinically severe vs nonsevere bleeders.
|Table 2: ROC for CWA and FVIII to discriminate between clinically severe vs nonsevere bleeders|
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|Figure 2: ROC curve for CWA parameters and FVIII to discriminate between clinically severe vs nonsevere bleeders|
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Similar ROC curve analysis for TGT parameters showed that almost all parameters had a good discriminatory ability to identify clinical severity of bleeding as reflected by AUC. The peak height of thrombin was found to be the best TGT parameter in this respect, and its best cut off (<106 nM) was found based on the ROC curve to identify clinically severe bleeders. Lag time, however, had a very low AUC, since it corresponds to conventional clotting time and thus it reflects only 5% of total thrombin generated [Table 3] and [Figure 3].
|Table 3: ROC for TGT to discriminate between clinically severe vs nonsevere bleeders|
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|Figure 3: ROC curve for TGT parameters like lag time, Time To Peak, Start Tail, ETP and PH to discriminate between clinically severe vs nonsevere bleeders shows all parameters had good discriminatory ability reflected by AUC to identify severe bleeders except for Lag time|
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Based on our identified best cut-offs of min1, min2 and peak height, we divided hemophilia patients into two groups:
- Group 1: ones with values below the cut-offs (min1 <1.33, min2 <0.145, PH <106) i.e., with poor thrombin generation and thus probable severe bleeders
- Group 2: ones with values above the cut-offs (min1 >1.33, min2 >0.145, PH >106), i.e., with good thrombin generation and thus probable nonsevere bleeders.
Our results showed that patients in group 1 had an early age of onset of symptoms, with more joints involved and more clotting factor concentrates requirement irrespective of their FVIII levels. While patients in group 2 had late onset of symptoms, with less number of joints involved and less clotting factor concentrates requirement irrespective of their FVIII levels [Table 4].
|Table 4: Clinical parameters of all hemophilia A patients divided into two groups based on cut-off values by ROC analysis, wherein Group I is Probable Severe Bleeders, Group II is Probable Nonsevere bleeders|
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There was also phenotypic heterogeneity in our study population. 15% of all hemophilia A patients had nonsevere bleeding despite FVIII <1%. All these patients had good thrombin generation reflected by min1, min2 and PH values above our calculated cutoffs (min1 >1.33, min2 >0.145, PH >106). And 8% of all hemophilia A patients had severe bleeding despite FVIII being in the range of 1-5%, all these patients had poor thrombin generation reflected by min1, min2 and PH values below calculated cut-offs (min1 <1.33, min2 <0.145, PH <106).
| Discussion|| |
While reviewing the literature, we have not found any data demonstrating phenotypic heterogeneity in hemophilia A and the utility of CWA and TGT in the Indian population.
In our study, 15% of severe hemophilia A patients had phenotypic heterogeneity with nonsevere bleeding and 8% of moderate hemophilia A patients had severe bleeding. Van den Berg et al. found heterogeneity of bleeding severity in around 10-15% of the Hemophilia A patients have which did not correlate with their Factor VIII levels. This is because clot based assays have their endpoints at the initiation of clot formation when only <5% of total thrombin is generated, whereas 95% of thrombin is generated explosively after the initiation of the clot. Thus, monitoring the entire course of thrombin generation would more closely correlate with an individual's bleeding phenotype rather than the time taken for the initiation of clot formation.
We found that when patients were classified as nonbleeders and bleeders based on the ISTH-BAT score, all the CWA and TGT parameters except the lag time had high sensitivity and specificity to distinguish nonbleeders from bleeders, and thus serve as good screening tools.
Our results demonstrated that the risk of severe bleeding in Hemophilia A patients was invariably accompanied by aberrant clot waveform and thrombin generation test parameters, while the FVIII level did not always reflect the actual bleeding severity. Dargaud et al. also found a significant correlation between severe clinical bleeding phenotype and ETP. Beltran Miranda et al. found that peak thrombin correlated better with FVIII: C level while the ETP correlated better with clinical severity.
Based on ROC curve analysis, Min1 <1.33, Min2 <0.145 and Peak Height of thrombin <106 nM were found to be the best cut-offs in our study to identify severe bleeders. Patients with parameters below these cut-offs were invariably having earlier age of onset of symptoms, more number of joints affected by hemarthrosis or more clotting factor concentrate requirement. However, these cut-offs need to be established by each lab according to the reagent-instrument combination used. Since this was a cross-sectional study, patients were not followed up and hence these cut-offs need further validation on another cohort of patients in a subsequent study.
Studies have shown that clotting time-based factor assays are limited by their sensitivity, linearity and reproducibility below 1% levels. Even in our study, the Min1 parameter in the patients with FVIII <1% ranged from 0.20 to 1.33, and Peak height ranged from 20.92 to 42.96 indicating a higher sensitivity of CWA and TGT in comparison with FVIII assays in the <1% FVIII level. Matsumoto et al. have also likewise found that CWA can distinguish between very low and absent FVIII activity in severe hemophilia A patients. Thus, the sensitivity for very low FVIII levels is another major advantage of the waveform analysis and TGT.
Moreover, APTT clot waveform parameters are available as additional information without any additional cost and at the same time while performing routine APTT using the photo-optical method. Thus, utilizing these objective clot waveform parameters to identify potential severe bleeders, in spite of having mild to moderate levels of FVIII can help identify potential candidates for FVIII prophylaxis in resource-poor settings where not all the patients can afford prophylaxis.
The superior sensitivity and discriminatory ability of the Clot waveform analysis, as well as the cost-benefit of just measuring routine APTT, make this approach a practical and promising tool for assessing coagulation in hemophilia A patients.
| Conclusion|| |
All the Parameters of global hemostasis tests like CWA and TGT could more accurately predict the bleeding severity than conventional APTT or factor VIII assay in patients with Hemophilia, except for Lag time. This is in agreement with findings of Dargaud et al. in hemophilia A and B and Al Dieri et al. in patients with rare bleeding disorders. This is because Lag time corresponds to the conventional clotting time and thus detects only the initial 5% of thrombin generated.
In conclusion, a combined approach using these global hemostasis tools can improve the prediction of bleeding phenotype and the management of patients with Hemophilia A.
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Conflicts of interest
There are no conflicts of interest.
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Rutvi G Dave
Department of Transfusion Medicine and Immunohematology, ASHA, Building 5th Floor, Christian Medical College, Vellore - 632 004, Tamil Nadu
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]