| Abstract|| |
Background: In the laboratory, factor VIII can be measured by three different methodologies, such as one-stage clotting assay, two-stage clotting assay, and chromogenic assay. These assays differ in ease of use, variety of reagents available, sensitivity to mild hemophilia A, and interference from lupus anticoagulants (LACs). Certain factor VIII gene mutations can cause discrepancy in results between one-stage activated partial thromboplastin time (APTT) and chromogenic assays. Materials and Methods: The coagulometer for factor VIII assay is Sysmex CS-5100. All data were expressed as mean ± standard deviation (SD). Results: A total of 135 cases were studied. Of these, 100 cases were of mild hemophilia A diagnosed by molecular genetics and, 15 cases were positive for LAC, which were confirmed by dilute Russell Viper venom test. Clot-based one-stage APTT assay showed 65% sensitivity and 80% specificity in diagnosing mild hemophilia A cases and out of 15 LAC cases, it showed false positivity in five cases. Chromogenic assay showed 85% sensitivity and 90% specificity in diagnosing mild hemophilia cases and was 100% specific in excluding LAC cases. Conclusions: One-stage APTT assay is the most commonly used test for determining factor VIII levels but chromogenic assay are considered as the gold standard and recommended as the reference method by European Pharmacopoeia and ISTH subcommittee. Mild hemophilia A patients with missense mutations show discrepancy between the one-stage clot-based APTT assay and chromogenic assays for determination of factor VIII level and this can lead to misdiagnosis or misclassification of mild hemophilia A. Therefore, it is recommended that both the assays should be used in the evaluation of mild hemophilia cases.
Keywords: Activated partial thromboplastin time, chromogenic assay, factor VIII, hemophilia A
|How to cite this article:|
Baig MA, Swamy K B. Comparative analysis of chromogenic vs clot.based one stage APTT assay for determination of factor VIII level. Indian J Pathol Microbiol 2021;64:123-7
|How to cite this URL:|
Baig MA, Swamy K B. Comparative analysis of chromogenic vs clot.based one stage APTT assay for determination of factor VIII level. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 May 10];64:123-7. Available from: https://www.ijpmonline.org/text.asp?2021/64/1/123/306541
| Introduction|| |
Hemophilia A is the most common hereditary clotting factor deficiency with a prevalence of 30–100 cases per million population. The inheritance is sex-linked but up to one-third of patients have no family history and result from recent mutation. [Figure 1] depicts summary of normal coagulation cascade pathway.
The factor VIII gene is situated near the tip of the long arm of the X chromosome (Xq2.8 region). It consists of 26 exons that encode a 2351 amino acid precursor polypeptide. FVIII protein consists of three homologous A domains, two homologous C domains, and the unique B domain, which are arranged in the order A1-A2-B-A3-C1-C2 from the amino terminus to the carboxyl-terminal end. Approximately half of the patients have missense or frameshift mutations or deletions in the factor VIII gene. In others, a characteristic ' Flip-tip ' inversion is seen in which the factor VIII gene is broken by inversion at the end of the X chromosome.
In mild to moderately severe hemophilia A, missense mutations within the exons coding for the three A domains or the two C domains account for most of the mutations detected [Table 1].
An accurate measure of factor VIII activity is necessary to make a correct diagnosis of hemophilia and classify the severity of the disease. FVIII activity can be measured by one- stage clot-based activated partial thromboplastin time (APTT) assay, two-stage clot assay (complex, rarely performed), and chromogenic assay.
Certain factor VIII gene mutations can cause discrepant results between one-stage APTT and chromogenic assays. A total of 16% of mild hemophilic patients show normal factor VIII level by clot-based one-stage APTT assay, which can be correctly detected by the chromogenic assay.
Based on the type of mutation and loci of the chromosome involved [Table 2], either chromogenic assay gives higher factor VIII concentration than one-stage APTT assay or vice versa.
Previous studies show that chromogenic assays are more useful than the clot-based one-stage APTT assay as the former does not show interference due the presence of lupus anticoagulants (LACs) and gives more precise factor VIII level. However, the number of studies conducted on this interesting topic is less and this has prompted me to undertake the present study.
The main objectives of this study are to
- Compare the sensitivity and specificity of chromogenic vs clot-based one-stage APTT assay in detecting factor VIII level
- Study the effect of LACs on the assays
| Materials and Methods|| |
This is a prospective study of 1-year duration (February 2018 – January 2019) carried out in a tertiary care referral Hemophilia center and Medical college in India.
The coagulometer used for inhibitor study is Sysmex CS-5100 and APTT reagent used is Pathromtin SL provided by Siemens Healthcare Diagnostics Products, Germany.
Mild hemophilia cases and LAC cases were included.
Severe hemophilia cases and patients on direct-acting oral anticoagulants (DOACs) were excluded.
Interpretation of Mixing tests is done by Rosners index or changes% correction
(<58% is inhibitor and < 70% is correction
- Perform APTTs on serial dilutions of standard reference plasma (diluted using factor deficient plasma) and plot the results to create the best fit line
- Then perform APTTs on serial dilutions of the patient's plasma
- Based on a log-linear calibration curve (X-axis serial dilutions, Y-axis APTT)
- This should produce the best fit line parallel to the reference best fit, from which the patient's factor level can be read [Figure 2].
Causes of non-parallel lines
- You made a mistake in plotting the lines
- An inhibitor is present, with increasing dilutions the inhibitor is diluted out
- You cannot dilute anything – if the factor level is <1% the clotting times will be grossly prolonged and not change with dilution.
Interference with the one-stage Assay
- Antiphospholipid antibodies, recombinant FVIII, and pre-activation of FVIII.
Chromogenic factor VIII assay,
- Pooled patient plasma + Reagent Cocktail (IXa, X, Thrombin, Phospholipid, Calcium)
- Incubate at 37°C to produce Xa
- Add chromogenic substrate and incubate
- Color change occurs on the cleavage of the substrate by Xa —<Calculate Xa concentration.
- Step 1 - produce FXa (incubation step)
- Step 2 – measure FXa via clotting time
- Since the ability to produce FXa is proportional to the amount of FVIII present, the FVIII activity is derived from FXa activity using a standard/calibration curve.
The strategy employed for molecular detection of mild hemophilia cases with missense mutation, deletions, inversions was by amplification of the FVIII gene by polymerase chain reaction (PCR) followed by detection of mutations by various screening methods or/and DNA sequencing and linkage analysis.,
| Results|| |
A total of 135 cases were studied. Of these, 100 cases were of mild hemophilia A diagnosed by molecular genetics and 15 cases were positive for LAC, which were confirmed by dilute Russell Viper venom test. A total of 20 healthy cases served as control.
Clot-based one-stage APTT assay showed 65% sensitivity and 80% specificity in diagnosing mild hemophilia cases and out of 15 LAC cases, it showed false positivity in five cases [Table 3].
|Table 3: Comparison of one stage APTT assay vs Chromogenic assay for FVIII detection|
Click here to view
The chromogenic assay showed 85% sensitivity and 90% specificity in diagnosing mild hemophilia cases and was 100% specific in excluding LAC cases.
The above results demonstrate that chromogenic assays are not interfered by LAC and are better than one-stage APTT assay in detecting factor VIII.
| Discussion|| |
Factor VIII can be measured by three different methodologies in the laboratory: one-stage clotting assay, two-stage clotting assay, and chromogenic assay. These assays differ in ease of use, variety of reagents available, sensitivity to mild hemophilia A, and interference from LACs.
One-stage APTT assay is the most commonly used test for determining factor VIII levels as it is simple, less expensive, and fully automated but recent studies have demonstrated significant problems with the measurement of correct FVIII due to tremendous variability of the APTT reagents used. The chromogenic FVIII assay is currently considered the gold standard for quantitation of factor VIII levels in both hemophilia A patients and as part of screening for thrombophilia.
Few genetic mutations in factor VIII gene show discrepancy in results between one-stage and chromogenic FVIII assays, in mild hemophilia A patients (20–30% cases). There are 2 distinct types of discrepancies. The commonest form of discrepancy gives a <2 times higher one-stage FVIII: C than chromogenic assays. The former test can even give a result in the normal range missing mild hemophilia. The reverse discrepancy gives <2 times higher chromogenic FVIII: C than one stage. The bleeding phenotype usually correlates with the chromogenic assay.,
Results of the present study show that clot-based one-stage APTT assay gives 65% sensitivity and 80% specificity in diagnosing mild hemophilia cases and out of 15 LAC cases, it showed false positivity in five cases. The chromogenic assay showed 85% sensitivity and 90% specificity in diagnosing mild hemophilia cases and was 100% specific in excluding LAC cases.
The above results demonstrate that chromogenic assays are not interfered by LAC and are better than one-stage APTT assay in detecting factor VIII.
The discrepancy in one-stage APTT assay and chromogenic assays were also reported in other studies, which are in comparison with the present study and our results very well correlate with the majority of these studies.
In a study of 133 patients with hemophilia by Parquet-Gernez et al. in 1988, the authors found a clear discrepancy between the procoagulant activity levels obtained with the one- and two-stage assays in 11/73 (15%) patients with mild or moderate hemophilia.
In 2013, Bowyer et al. carried outa study of the incidence of assay discrepancy in 84 patients with mild hemophilia A and found assay discrepancy in 31% of the patients: 12% with lower activity in the two-stage assay and 19% with lower activity in the one-stage assay.
In 2008, Cid et al. carried out a study of 163 patients with mild hemophilia A and found discrepancies in 20% of the patients, most of whom had higher levels of FVIII: C with one-stage assay.
In 2009, Poulsen et al. carried out a study of 109 patients with mild hemophilia A. They reported that an assay discrepancy pattern was found quite frequently among their mild hemophilia A families with 36% showing a lower two-stage assay compared to the one-stage assay. However, when they selected a cut-off level for the FVIII: C chromogenic/FVIII: C clot ratios at 0.7, 0.6, and 0.5, respectively, they found that 38 (72%), 27 (51%), and 19 (36%) of families, respectively, displayed this assay discrepancy.
In 1994, Duncan et al. carried out a study of 95 patients with hemophilia A. They found that results were equivalent in all 21 patients with severe hemophilia (16 families) and in 45 of the patients with mild or moderate hemophilia (18 families). However, the results were discrepant (FVIII: C by the one-stage assay was two- to seven-fold higher than that by the two-stage assay) in the other 29 (39%) patients with mild or moderate hemophilia (12 other families). They concluded that in some families with hemophilia A, the gene defect leads to a discrepancy between the one-stage and two-stage assay results and may be more widespread than previously recognized.
LAC interferes with one-stage clot-based APTT assays and gives false decreased FVIII levels whereas chromogenic assays give correct results because it includes initial high dilution at which the assay is performed, the decreased phospholipid dependency of the reaction, and the bypass of contact activation and prothrombinase complex in the chromogenic assay.
The chromogenic assays use high dilution of plasma making it insensitive to LAC, low-molecular-weight heparins (LMWHs), and lepirudin all of which may result in major interference with one-stage clot-based APTT assay. It is however sensitive to DOACs.
| Conclusions|| |
Mild hemophilia A patients show discrepancy between the one-stage clot-based APTT assay and chromogenic assays for the determination of factor VIII level. Approximately 16% of patients with mild hemophilia A have normal factor VIII level when measured by one-stage clot-based APTT assay.
So if only the one-stage assay is used, some patients will have normal FVIII levels and not be diagnosed as having hemophilia or be considered to have a milder bleeding risk, which is not the case. Other patients who have normal FVIII activity will be diagnosed as hemophilia A. This can lead to misdiagnosis or misclassification of mild hemophilia A. So it is recommended that both the assays should be used in evaluating mild hemophilia cases.
Chromogenic assays measure correct factor VIII levels and do not show interference with LAC, so it has been recommended as the reference and gold standard method by European Pharmacopoeia and ISTH subcommittee.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Mirza Asif Baig
Lincoln University College, Selangor
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]