Flow cytometry vs conventional methods for the evaluation of anti-PF4/heparin antibodies: a single center study

Abstract

Aim: Heparin-induced thrombocytopenia (HIT) is a rare, life-threatening, immune-mediated adverse effect of heparin administration. This study compares frequently used laboratory assays in terms of their effectiveness in HIT diagnosis. Materials & methods: Fifty patients with suspected HIT were tested by gel immunoassay and solid phase PF4/heparin antibody ELISA. On positive results, platelet activation markers P-selectin and Annexin V were assayed using flow cytometry. Results: Thirty/50 patients were negative for both immunoassays. Flow cytometry was performed in the 20 immunoassay positive patients. Platelet activation was observed in 7/20 in the presence of low heparin concentration (0.2 IU/ml). Conclusion: The results are in accordance with the currently available literature and flow cytometry seems a promising alternative in HIT laboratory investigation.

Article highlights

Background

• Heparin-induced thrombocytopenia (HIT) is a clinical-laboratory syndrome; therefore its diagnosis is based on both clinical and laboratory data.

• Two types of laboratory tests are used to diagnose HIT: immunoassays that detect antibodies against the PF4/heparin complex, and functional assays that detect platelet activation triggered by the formation of heparin-induced antibodies.

• The use of platelet-specific antibodies in flow cytometry is essential in order to avoid the implication of cells other than platelets. An accurate determination of both activated and non-activated platelets is achieved by using antibodies that bind either all platelets or the activated platelets only.

• P-selectin and Annexin V are reliable markers of platelet activation, with the P-selectin, being the most widespread marker to assess platelet activation by flow cytometry.

Results

• The study was conducted on a sample of 50 patients. The majority of them were inpatients (34 ICU and nine surgical), while seven were outpatients from neighboring hospitals. All patients had received heparin and were referred to laboratory, presenting with thrombocytopenia and clinical suspicion of HIT.

• Thirty/50 patients were negative for both immunoassays. Flow cytometry was performed in the 20 immunoassay positive patients. Platelet activation was observed in 7/20 in the presence of low heparin concentration (0.2 IU/ml).

Discussion

• The two immunoassays performed in the study showed comparable sensitivity, specificity and negative predictive value.

• The application of flow cytometry as a functional method for the laboratory diagnosis of HIT and the selection of Annexin V and P-selectin as platelet activation markers, constitute a promising method for the verification of the immunological techniques.

Keywords: :

• Annexin V
• anti-PF4/heparin antibodies
• ELISA
• flow cytometry
• heparin-induced thrombocytopenia
• P-selectin
• PaGIA
• platelet activation

Acknowledgments

The study was conducted in the context of post-graduate program “Thrombosis – Bleeding – Transfusion Medicine” of National and Kapodistrian University of Athens.

Author contributions

All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

Financial disclosure

The study was conducted in 2021–2022 in the context of the post-graduate program “Thrombosis-Bleeding-Transfusion Medicine” organized by the School of Medicine, of National and Kapodistrian University of Athens, Greece and it was partly funded by the National and Kapodistrian University of Athens. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Disclosure statement

The authors report there are no competing interests to declare.

Funding

The study was partly funded by the National and Kapodistrian University of Athens.

Ethical conduct of research

The study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki. Informed consent was obtained from all individuals participated in the study.

Additional information

Funding

The study was conducted in 2021–2022 in the context of the post-graduate program “Thrombosis-Bleeding-Transfusion Medicine” organized by the School of Medicine, of National and Kapodistrian University of Athens, Greece and it was partly funded by the National and Kapodistrian University of Athens.