The problem

 

There are a number of proteins that regulate blood clotting so that under normal circumstances, when we need our blood to clot, they prevent the clot from getting too big and eventually ‘switch off’ some of the biochemical reactions. If a patient has a hereditary deficiency (reduction) of one of these regulators they are said to have a type of disorder called a thrombophilia, which increases their risk of having a thrombosis. There are different ways of testing for deficiencies of these proteins, and we sometimes have to do multiple tests to find them.

Having major surgery increases your risk of thrombosis so patients are often treated with drugs called anticoagulants to slow down clotting mechanisms. Interestingly, one of those anticoagulants, called heparin, works by binding to antithrombin, one of the patient’s regulatory proteins, which then changes its shape and becomes a powerful anticoagulant. However, a sub-type of antithrombin deficiency is due to reduced ability to bind to heparin, so these patients cannot be treated with heparin or require massive doses for it to work. Doing an assay that identifies the type of antithrombin defect, and why heparin isn’t working, can directly affect patient treatment.

 

The science

 

Antithrombin is so-called because it mainly regulates a pivotal enzyme of blood clotting biochemistry, thrombin, which if left alone can cause uncontrolled clotting. If a person has a deficiency of antithrombin, it can predispose them to developing thrombosis because of loss of regulation. There are two main types of antithrombin deficiency. In Type 1, patients make the same antithrombin molecules as you and I, meaning it functions properly, but they don’t make enough, so it is a quantitative deficiency. In Type 2, patients make normal amounts of antithrombin but it doesn’t function properly and are qualitative deficiencies. As well binding to heparin, antithrombin has to bind to the enzymes that it switches off, so the assay for measuring function harnesses both of these properties.

In the assay, the patient’s antithrombin is first reacted with heparin to increase the speed with which it works in the next step of the assay where it binds to one of the enzymes it switches off, either thrombin or factor Xa. Any leftover enzyme is reacted with a substance that produces a coloured product because of the reaction, and the analytical equipment reads the intensity of the colour. The more antithrombin the patient has in their plasma, there is less enzyme left over so the colour intensity is low, so a patient with a deficiency will have more enzyme left over and the colour intensity is higher. The degree of colour intensity is converted to a concentration by comparing it to the results from a plasma whose antithrombin level is already known, called a standard plasma.

 

What lab assays aren’t detecting

 

Patients with Type 2 antithrombin deficiency have a defect in their ability to bind either to clotting enzymes or heparin, and rarely, both. A research publication by PL Harper et al in 1991 revealed that if the incubation step with heparin is allowed to go on too long, the ability of the antithrombin from a patient with a heparin binding defect is exaggerated and the result reported to the doctor will not be low enough, or could even be normal, leading to a missed diagnosis.

Consequently, most diagnostic laboratories will have their analytical equipment programmed for a short incubation time with heparin before the enzyme is then added to the reaction. These assays will generate a result indicating reduced levels of antithrombin whether it is due to a Type 1 or 2 deficiency but not give more detailed information about the sub-type. For many patients this won’t make any difference to their treatment. Interestingly, most patients with heparin binding defect mutations of antithrombin have a much lower prevalence of thrombotic events compared to patients with other sub-types.  Nonetheless, their identification is relevant to patients who do experience clinical events, co-inheritance with another thrombophilia as their thrombosis risk increases, and in cases where standard doses of heparin do not achieve the required degree of anticoagulation. However, differentiation between type II subtypes is not commonly undertaken in clinical practice as the assays are not widely available, largely because they are technically demanding and time consuming.

 

The concept for a new assay

 

Consultant Scientist for the Haemostasis & Thrombosis centre at St. Thomas’ Hospital in London, Dr Gary Moore, said, “I considered that if you do the usual assay with the short incubation and do it deliberately again with a long incubation period, the result will be stable in non-heparin binding defects however long you incubate it but there will be a discrepancy if there is a heparin binding defect.”  Gary came up with the idea of quantifying the discrepancy by generating a ratio of the antithrombin result from the short incubation time and prolonged incubation time, which he termed the heparin-antithrombin binding ratio (HAB).

Gary’s concept needed to be tested in practice and he designed an experimental protocol which his colleague Naomi de Jager undertook for her MSc degree, which she passed with distinction, plus some subsequent work so that it could be submitted for publication in an international, peer reviewed specialist medical journal. Their colleague Jacqueline Cutler also contributed by supplying results from genetic studies which proved the presence or absence of heparin binding defects in the patients that were studied. The study was published in the journal Thrombosis Research.

 

Why it’s important

 

The chances of developing a thrombosis are about 1 in 1000 per year, although certain circumstances can greatly increase the risk, such as cancer, major surgery and immobility. Young people are less likely to develop thrombosis, although patients with a thrombophilia may develop a thrombosis in their late teens or early twenties. When this happens, laboratory scientists can look for a pre-disposition for thrombosis. Gary’s department conducts these tests on about 5000 patients a year.

Performing a HAB-ratio assay in response to an initial finding of a reduced antithrombin activity assay result will increase the specificity of the diagnosis. Although the risk of thrombosis is lower in antithrombin deficient patients with heparin binding defects, identifying that defect in a symptomatic patient may lead to counselling of a patient’s affected family members that they may be at relatively low risk for thrombosis.

 

Conclusions

 

The assay doesn’t always give a positive result as some mutations of antithrombin completely remove the ability to bind to heparin, so it cannot be exaggerated upon prolonged incubation, but a positive result is diagnostically conclusive. Naomi has done even more hard work to transfer the assay to existing equipment and it will soon be included in the diagnostic testing repertoire for diagnostic testing at Guy’s & St Thomas’ NHS Foundation Trust’s.

The acid test for any newly described and published test is whether it works in the hands of others. Dr René Mulder, a researcher well known for his work in this area, recently published a study in the British Journal of Haematology which included HAB-ratio testing, successfully identifying all his patients who had heparin binding defects in their antithrombin.

 

Bibliography

Harper PL, Daly M, Price J, Edgar PF, Carrell RW. Screening for heparin binding variants of antithrombin. J Clin Pathol 1991; 44: 477-479

Moore GW, de Jager N, Cutler JA. Development of a novel, rapid assay for detection of heparin-binding defect antithrombin deficiencies: the heparin-antithrombin binding (HAB) ratio. Thrombosis Research 2015;135:161-166

Mulder R, Croles FN, Mulder AB, Huntington JA, Meijer K, Lukens MV. SERPINC1 gene mutations in antithrombin deficiency. Br J Haematol 2017 Mar 20. doi: 10.1111/bjh.14658. [Epub ahead of print]