Skip to main content

Anti-PF4 and Serotonin Release Assay (SRA) for Diagnosing Heparin-induced Thrombocytopenia/Thrombosis (HIT/HITT)

Test code(s) 414, 14627, 14874, 16284

The anti-PF4 (ie, heparin-induced platelet antibody) test is an immunologic, enzyme-linked immunosorbent assay (ELISA) that detects IgG antibodies against the platelet factor 4 (PF4)/heparin complex. The assay uses heparin bound to protamine sulfate, and PF4 is supplemented through a platelet lysate.

The SRA test is a functional assay that measures heparin-dependent platelet activation. Patient serum is incubated with donor platelets containing radiolabeled serotonin and different concentrations of heparin. Antibody present in the patient serum will bind and activate donor platelets, releasing radiolabeled serotonin from the platelet granules. A positive SRA is expected to show >20% release of the radiolabeled serotonin when mixed with patient serum and low-dose heparin. In addition, the high-dose heparin must reduce the % release obtained with low-dose heparin by at least 50% in order to demonstrate that the platelet activation is heparin dependent.

The SRA is considered a better predictor of thrombosis than the anti-PF4 assay.

OD is the optical density; it is an indication of antibody strength. Antibody present in the patient sample binds to the heparin-PF4 coated wells, leading to a color-producing reaction. A higher antibody concentration leads to more color production and a higher OD reading. The positive cutoff is ≥0.300 OD, and high-titer antibodies may yield values up to 3.500 OD. 

The anti-PF4 test has a sensitivity of >95% and a specificity ranging from 50% to 89%. However, the higher the OD, the greater the probability is for clinical HIT/HITT. Similarly, the specificity increases when the OD is >1.000.1

The SRA test has a sensitivity of 88% to 100% and a specificity of 89% to 100% for HIT.2

The combined sensitivity of the anti-PF4 and the SRA is about 99%.3

HIT/HITT is a diagnosis that requires both clinical judgment and laboratory corroboration, ie, a clinical, pathologic diagnosis.

Thrombocytopenia in a hospitalized patient is very common and may be due to a vast number of causes (eg, consumption, dilutional effect from fluids or transfusions, drugs, sepsis, splenic sequestration). For patients exposed to heparin, HIT/HITT is an important consideration, given the life-threatening potential. Applying a standardized and objective approach to the clinical assessment can help determine the pretest probability of HIT/HITT.4 The most common and widely accepted scoring model is called the 4T’s (Thrombocytopenia, Timing of onset, Thrombosis, oTher cause).5

Another diagnostic challenge is that antibodies may be identified in patients with little or no clinical symptomatology.

No, testing for suspected HIT is not needed in this situation. HIT can be excluded based on a low pretest probability score (4T’s score of 0-3).6

A published algorithm that incorporates the clinical assessment suggests that patients with a moderate pretest probability and the laboratory results above are “indeterminate” for HIT/HITT.4

This scenario may represent a true antibody response, but one that lacks sufficient titer or avidity to activate platelets in vitro. The fact that the antibodies are measurable only with the anti-PF4 ELISA may also be indicative of the assay’s greater sensitivity. While this explanation has a good scientific foundation, some patients may still have clinically significant antibodies, thereby the rationale for the “indeterminate for HIT/HITT diagnosis.” False-positive anti-PF4 ELISA results have also been attributed to cross-reactivity to antiphospholipid antibodies.7

Quest Diagnostics offers both tests individually, together as a panel, and as a reflex (a positive heparin-induced platelet antibody reflexes to SRA).

A cautionary note when ordering the panel or reflex—if the result interface (connection from Quest Diagnostics to your computer system) does not accept partial results, the anti-PF4 ELISA results will not be received until the SRA is reported. Confirming the status of your interface with an IT specialist is recommended. Alternatively, physicians may view all results irrespective of this issue using the Care 360 product. 

Yes. Only IgG antibodies activate platelets, thus IgM and IgA classes are of minor clinical relevance.8,9 Furthermore, the British Society for Haematology recommends that only the IgG isotype be tested.10

Quest Diagnostics does not perform the heparin neutralization procedure, sometimes referred to as a confirmatory step. It is our experience that virtually every positive sample is neutralized, and therefore we concluded that it offered no significant insight beyond that of the OD value. A retrospective study also reported that some patients with high ELISA OD values were misclassified by a confirmed negative result despite presence of clinical HIT/HITT.11

Given the short half-life (30 minutes to 2 hours) of unfractionated heparin (UFH), waiting a minimum of 2 hours is suggested. 

A SRA result is considered indeterminate when there is a >20% release of radiolabeled serotonin in the patient serum/low dose heparin mixture and an absence of inhibition with high-dose UFH. There may be other nonheparin-dependent antibodies present (ie, Class I HLA antibodies or platelet receptor specific antibodies) that may cause platelet activation.

A low-positive SRA is repeated using platelets from a different donor to demonstrate reproducibility. At this point, specific conclusions about a low-positive result cannot be made. It is recommended that the result be considered in the context of all the data at hand, specifically the clinical assessment and anti-PF4 result.


  1. Zwicker JI, Uhl L, Huang WY, et al. Thrombosis and ELISA optical density values in hospitalized patients with heparin-induced thrombocytopenia. J Thromb Haemost. 2004;2(12):2133-2137. doi:10.1111/j.1538-7836.2004.01039.x
  2.  Ortel TL. Heparin-induced thrombocytopenia: when a low platelet count is a mandate for anticoagulation. Hematology Am Soc Hematol Educ Program. 2009;2009(1):225-232. doi:10.1182/asheducation-2009.1.225
  3. Linkins LA, Dans AL, Moores LK, et al. Treatment and prevention of heparin-induced thrombocytopenia: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians evidence-based clinical practice guidelines. Chest. 2012;141(2)(Suppl):e495S-e530S. doi:10.1378/chest.11-2303
  4. Arepally GM, Ortel TL. Clinical practice: heparin-induced thrombocytopenia. N Engl J Med. 2006;355(8):809-817. doi:10.1056/NEJMcp052967
  5. Warkentin TE, Aird WC, Rand JH. Platelet-endothelial interactions: sepsis, HIT, and antiphospholipid syndrome. Hematology Am Soc Hematol Educ Program. 2003;2003(1):497–519. doi:10.1182/asheducation-2003.1.497
  6. Cuker A, Gimotty PA, Crowther MA, et al. Predictive value of the 4Ts scoring system for heparin-induced thrombocytopenia: a systematic review and meta-analysis. Blood. 2012;120(20):4160-4167. doi:10.1182/blood-2012-07-443051
  7. Pausner R, Greinacher A, Selleng K, et al. False-positive tests for heparin-induced thrombocytopenia in patients with antiphospholipid syndrome and systemic lupus erythematosus. J Thromb Haemost. 2009;7(7):1070-1074. doi:10.1111/j.1538-7836.2009.03335.x
  8. Warkentin TE. How I diagnose and manage HIT. Hematology Am Soc Hematol Educ Program. 2011;2011(1):143-149. doi:10.1182/asheducation-2011.1.143
  9. Greinacher A, Juhl D, Strobel U, et al. Heparin-induced thrombocytopenia: a prospective study on the incidence, platelet-activating capacity and clinical significance of antiplatelet factor 4/heparin antibodies of the IgG, IgM, and IgA classes. J Thromb Haemost. 2007;5(8):1666-1673. doi:10.1111/j.1538-7836.2007.02617.x
  10. Watson H, Davidson S, Keeling D. Guidelines on the diagnosis and management of heparin-induced thrombocytopenia: second edition. Br J Haemotol. 2012;159(5):528-540. doi:10.1111/bjh.12059
  11. Whitlatch NL, Perry SL, Ortel TL. Anti-heparin/platelet factor 4 antibody optical density values and the confirmatory procedure in the diagnosis of heparin-induced thrombocytopenia. Thromb Haemost. 2008;100(4):678-684. doi:10.1160/th08-02-0118


This FAQ is provided for informational purposes only and is not intended as medical advice. A physician’s test selection and interpretation, diagnosis, and patient management decisions should be based on the physician’s education, clinical expertise, and assessment of the patient.

Document FAQS.06 Version: 3

Version 3 effective 02/20/2023 to present

Version 2 effective 12/16/2016 to 02/20/2023
Version 1 effective 09/17/2013 to 12/16/2016
Version 0 effective 12/06/2011 to 09/16/2013