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Introduction

Hello. My name is Dr. Rajiv Pruthi. I am a professor of Medicine in the Mayo Clinic College of Medicine, a co-director in the Special Coagulation Laboratory, and director of the Hemophilia Center at Mayo Clinic in Rochester, Minnesota. I will be presenting on heparin induced thrombocytopenia, and approach diagnosis and the role of the functional serotonin release assay. 

Disclosures

I have no relevant disclosures for this presentation.

Objectives

At the end of this presentation, the audience will understand the clinical presentation and pathophysiology of heparin induced thrombocytopenia, also known as HIT; know the HIT diagnostic algorithm, including the clinical scoring system; recall the role and limitations of immunoassays for HIT; and apply the results of the serotonin release assay, also known as SRA, to patient management. 

HIT background

Briefly, HIT is an important clinical pathologic syndrome. HIT represents the most common drug-induced immune-mediated thrombocytopenia. The incidence of HIT is common, and occurs in up to 3% of people who get exposed to therapeutic doses of unfractionated heparin. It can be difficult to confirm the diagnosis, and therefore the management of HIT may be fairly difficult and complex. It is the most important drug-induced thrombocytopenia due to the high thrombosis risk, with up to 50% of patients with HIT experiencing thrombosis.

Case example

We will start with a case example. A 73-year-old male was hospitalized for community-acquired pneumonia. In addition to receiving antibiotic therapy, he also received subcutaneous unfractionated heparin for deep vein thrombosis prophylaxis. 

This slide shows the clinical course of his thrombocytopenia. On admission, he had a normal platelet count, and on about day five of heparin therapy the platelet count started drifting down and eventually reached a nadir on day seven of heparin therapy. At this point, the clinicians suspected HIT, discontinued heparin, and ordered the anti-heparin platelet factor IV antibody immunoassay, which turned out to be strongly positive, and he was diagnosed as having isolated heparin induced thrombocytopenia.

Isolated HIT is not a benign condition. Different studies demonstrate the incidence of thrombosis associated with HIT as ranging between 18% to 52%, hence the importance of making an early and accurate diagnosis of HIT so that optimal therapy can be provided in order to result in a good patient outcome. 

HIT diagnostic algorithm

HIT should be suspected in patients on heparin therapy who develop thrombocytopenia. It can occur in those receiving both low molecular weight heparin and unfractionated heparin, but the incidence is higher in those on unfractionated heparin. In addition, HIT occurs in those on prophylactic and therapeutic doses of heparin. The highest risk appears to be in patients receiving heparin boluses during cardiac bypass procedures. Perhaps the second most common situation is orthopedic surgery, such as joint arthroplasty. Less commonly, HIT can occur in patients undergoing general surgery, and rarely in those receiving thromboprophylaxis during hospitalization for medical indications and during pregnancy.

HIT recognition

Recognition of HIT relies on selected clinical features. This consists of assessing the degree and timing of onset of thrombocytopenia, presence or absence of thrombosis, which can be venous, or arterial, or both. In addition, it is important to exclude other potential causes of thrombocytopenia. Assessing these clinical findings led to the development of a scoring system known as the 4Ts scoring system, which is very commonly used to assess the likelihood of HIT.

HIT pretest probability scoring

Based on the timing of onset of thrombocytopenia, the degree of thrombocytopenia, and presence or absence of thrombosis, and exclusion of other potential explanations for the thrombocytopenia, a 4Ts score is generated that provides a likelihood of the patient having HIT. As shown, a low score of three or less suggests a low probability of HIT, whereas a score of six or higher suggests a high probability of HIT. The final score is used in the calculation of a negative and positive predictive value for HIT that assists in decision-making process on the need for empiric change in anticoagulation therapy and laboratory testing. 

Suspect heparin induced thrombocytopenia (HIT)

The first step in the diagnostic process for suspected HIT is to calculate the 4Ts score. 

Predictive value of 4TS

A very well written systematic review and meta-analysis of the literature concluded that the negative predictive value for a low probability 4Ts score was 99.8%. However, the positive predictive value for a non-low probability, in other words, a moderate or high probability, was between 14% to 64%. This data helps guide in the next steps. 

Suspect HIT

A low clinical pretest probability has a negative predictive value of 99.8%. Therefore, the current guideline recommendation is to avoid laboratory testing, and if a non-heparin anticoagulant like a direct thrombin inhibitor was initiated, it can be discontinued and the heparin may be continued or resumed.

Since a non-low probability 4Ts score, which is also known as moderate or high probability, does not have a very high positive predictive value, in this situation, heparin induced thrombocytopenia testing should be ordered. The most commonly available and widely ordered assay is an immunoassay. Pending results of testing, however, heparin should be discontinued and a non-heparin anticoagulant such as a direct thrombin inhibitor should be initiated.

In this setting of an intermediate or high probability of HIT, where an immunoassay result has been ordered, and once the result is available and is negative, this provides a very high negative predictive value for HIT. At this stage, the non-heparin anticoagulant can be discontinued and heparin can be continued or resumed if it was discontinued. A positive immunoassay result suggests presence of antibodies directed against heparin platelet factor IV complexes. The next step is to ask the question, whether these antibodies are platelet activating, or functional antibodies that are implicated in the pathogenesis of HIT.

HIT laboratory assays

Performance characteristics of immunoassays and functional assays are shown in this table. Of the available functional assays, heparin induced platelet aggregation assay, also known as the HIPA, has been around for a long time. However, the current gold standard functional assay is the serotonin release assay, also known as the SRA, which is based on either radioactive or non-radioactive methodologies.

EIA-GTI

The relationship between a positive immunoassay test result and a positive SRA is shown in this study. A positive cutoff is at an optical density of 0.4. As you can see, all patients with an optical density of 0.4 or less were negative for the SRA assay. However, as the optical density increased, there was a higher proportion of patients with the positive SRA. Close to 90% of patients with an optical density greater than or equal to 2 had a positive SRA.

Mayo Clinic SRA method

In the serotonin release assay implemented at Mayo Clinic, fresh platelets are obtained from donors who are known to be reactive in this assay. Platelets undergo standard washing process, which is fairly labor intensive. The platelets are then incubated with the patient's serum, which contains the heparin platelet factor IV antibodies, and two different concentrations of heparin: a low concentration and a high concentration. If the antibodies are platelet activating, platelet activation occurs, which releases platelet contents. Serotonin is one of the platelet contents, which is measured by liquid chromatography tandem mass spectrometry, also known as LCMS.

Interpretation

Interpretation of the Mayo Clinic serotonin release assay is based on a cutoff of 20% serotonin release. Less than 20% serotonin release is a negative test result; however, greater than 20% serotonin release, which is inhibited by high dose heparin, is a positive test result. On occasion, there will be patients with antibodies that release serotonin at greater than 20%, however, this release is not inhibited by heparin. In other words, at a high heparin level, serotonin release is still greater than 20%. This is categorized as an indeterminate test result, and not classical for heparin platelet factor IV platelet activating antibodies.

Correlation of H/PF4 (IGG) ELISA with SRA positive test result

Here is shown the correlation of the Mayo Clinic serotonin release assay with optical density of the heparin platelet factor IV IgG ELISA assay. As you can see, the large majority of patients with a high optical density, above 1.5, had a positive serotonin release assay test result. We will talk a little more detail about the one patient who had a negative test result. On the other hand, the large majority of patients with a negative heparin platelet factor IV ELISA had a negative serotonin assay test result. In our experience, there was a variable degree of positivity of the SRA, depending on the optical density between 0.4 to 1.5 of the heparin platelet factor IV ELISA.

Positive ELISA and negative SRA: N=1

The one patient who had a positive ELISA with a very high optical density, but a negative SRA assay test result, had a 4Ts score that was moderate. This patient developed post chemotherapy thrombocytopenia, and was receiving unfractionated heparin for deep vein thrombosis prophylaxis. The Mayo SRA and the send out test, which is radioactive methodology, were both negative. This case represents an example of a non-platelet activating heparin platelet factor IV antibody.

HIT ELISA negative and SRA indeterminate

The one indeterminate SRA test result in the group of patients with a negative heparin platelet factor IV IgG test result was a patient with a new diagnosis of acute myeloid leukemia. The patient was receiving unfractionated heparin DVT prophylaxis, and the 4Ts score was low. Both the Mayo SRA and the mail out radioactive methodology based SRA were indeterminate. The heparin was discontinued due to the induction chemotherapy related thrombocytopenia, and there was no evidence of thrombosis.

What about SRA negative HIT?

What about the rare presentation of patients who have HIT but are SRA negative? There are reports of such patients who have a high clinical suspicion of HIT and a positive HIT immunoassay test result, but a negative SRA. These are confined to case reports and small case series. Therefore, clinical evaluation is critical in the diagnosis and management of such patients.

Conclusions

In summary, heparin induced thrombocytopenia is a complex clinical pathologic syndrome. Initial evaluation of patients with thrombocytopenia who are on heparin or have recently been exposed to heparin should consist of a thorough clinical evaluation, assessment of a pretest clinical probability known as the 4Ts scoring system, and to follow the guidelines in follow-up testing and decisions on anticoagulation management. The likelihood of HIT will influence the decision to interrupt heparin, to initiate non-heparin anticoagulation, and whether or not to perform laboratory testing. A positive HIT immunoassay test result should prompt confirmation of the pathogenicity of the antibody in HIT. Functional assay, which is a serotonin release assay, is currently the gold standard for detection of platelet activating antibodies. Having said that, it should be kept in mind that there are rare cases of SRA negative HIT, and cases of a negative ELISA based HIT test result, but a positive serotonin release assay. But these are extremely rare.

Questions?

Contact us: mcleducation@mayo.edu

Image credit: Getty images