Jenica L. Harrison, PhD, MT (ASCP)
Heparin is frequently given to hospitalized patients to treat and prevent the formation of venous thromboembolism. Although heparin serves as a widely used and efficacious anticoagulant, its administration can lead to adverse outcomes in some patients. One adverse outcome of heparin use is the development of heparin-induced thrombocytopenia (HIT). HIT is an adverse prothrombotic condition caused by antibodies that are elicited against complexes of platelet factor 4 (PF4) and heparin. (Ahmed et al,2007) During HIT, heparin from an exogenous source, binds to PF4 in vivo, leading to the untoward formation of immunogenic PF4/heparin complexes. Moreover, the binding of anti-PF4/heparin antibodies to PF4/heparin complexes can result in 1) adverse thrombosis via platelet activation and/or 2) thrombocytopenia (i.e., decreased platelet count). (Chahal et al, 2017) The diagnosis of HIT dependent on both the patient’s clinical presentation in concert with the results of the clinical laboratory test. When evaluating a patient for the presence of clinically relevant HIT antibodies (i.e., anti-PF4/heparin antibodies), the 4T score in concert with specific screening and confirmatory clinical laboratory tests for anti-PF4/heparin antibodies are utilized. Currently, ELISAs are widely utilized in clinical laboratories as a screening assay to assess for the presence of antibodies elicited to the PF4/heparin complex which has the potential to cause HIT-associated thrombocytopenia.
The objective of this study was to test and implement an ELISA protocol that detects IgG/A/M antibodies that bind PF4 /heparin complexes. At the present time, the Department of Medical Laboratory Sciences at (VCU) does not have a laboratory exercise in which students have hands-on experience with a specific screening assay that assesses for HIT-associated antibodies. This optimized protocol can then be used as an important teaching tool for future Medical Laboratory Science (MLS) students to demonstrate the benefits and practical use of advanced hemostasis testing.
In this study, the Asserachrom® Heparin-PF4-Induced Antibodies ELISA (Diagnostica Stago Inc; Parsippany, NJ). was used in concert with the Thermo Scientific™ Varioskan™ LUX multimode microplate reader (ThermoFisher Scientific Inc; Waltham, MA) to assess for the presence of IgG/A/M antibodies elicited to the PF4/heparin complex which have the potential to cause HIT associated thrombocytopenia. ELISA protocol was evaluated in the presence of assay controls, provided by Diagnostica Stago, Inc. (Parsippany, NJ), and was tested along with spiked and unspiked human serum/plasma in order to determine the suitability of the ELISA kit. Spiked samples were created using 200 µL of either human serum or plasma and 200 µL of the Reference standard.
Pertaining to the controls the Plate Acceptance Criteria from the Stago Asserachrom® HPIA kit indicated that: 1) Reference Standard O.D. = 1.73 ± 0.35 = Pass; 2) Positive control O.D. = 1.20 ± 0.30 = Pass. Pertaining to the samples (note: this is reflected in the Control O.D./RS O.D. (%) column of the data table) 3) Sample O.D. values must be >23.0% the magnitude of the O.D. value obtained for the RS within the same run to be considered Positive. The interpretation can be made that the plate controls passed the acceptance criteria provided by the manufacturer. According to the manufacture, insert, it recommended that samples measured at an absorbance of 450 nm would need an absorbance value greater than 23.0% of the absorbance value obtained by reagent 6 and 7b in order to be considered positive. Spiked and unspiked human serum and plasma samples were run in duplicate and when compared to the positive and negative control requirements they both resulted in a negative interpretation.
The results show that the Asserachrom® Heparin-PF4-Induced Antibodies ELISA kit can be employed as a screening assay to detect the presence of IgG, IgA, and IgM class anti-PF4/heparin complex antibodies in human plasma and serum when used in conjunction with the Thermo Scientific™ Varioskan™ LUX multimode microplate reader. Using these protocols in the MLS student laboratory at VCU has the potential to enhance students’ knowledge and understanding of hematological processes and advance testing prior to entering their clinical rotations. Lastly, this project served to help the undergraduate student researcher gain a deeper understanding of advanced clinical laboratory assays for the assessment of abnormal primary hemostasis, but also allowed them the opportunity to learn how to develop and test a scientific protocol that will help the student researcher in the future research endeavors.