Anti-PF4 testing for vaccine-induced immune thrombocytopenia and thrombosis (VITT): Results from a NEQAS, ECAT and SSC collaborative exercise in 385 centers worldwide
Manuscript handled by: Robert Gosselin
Final decision: Robert Gosselin, 11 March 2022
Abstract
Background
Vaccine-induced immune thrombocytopenia and thrombosis (VITT) following the administration of the AstraZeneca (AZ) ChAdOx1 nCOV-19 vaccine is a well recognized clinical phenomenon. The associated clinical and laboratory features have included thrombosis at unusual sites, thrombocytopenia, raised D-dimer levels and positivity for immunoglobulin G (IgG) anti-platelet factor 4 (PF4) antibodies.
Objectives
A collaborative external quality assessment (EQA) exercise was carried out by distributing five lyophilized samples from subjects with VITT and one from a healthy subject to 500 centers performing heparin-induced thrombocytopenia (HIT) testing.
Methods
Participating centers employed their locally validated testing methods for HIT assays, with some participants additionally reporting results for VITT modified assays.
Results
A total of 385 centers returned results for anti-PF4 immunoassay and functional assays. The ELISA assays used in the detection of anti-PF4 antibodies for the samples distributed had superior sensitivities compared with both the functional assays and the non-ELISA methods.
Conclusion
ELISA-based methods to detect anti PF4 antibodies have a greater sensitivity in confirmation of VITT compared with functional assays regardless of whether such functional assays were modified to be specific for VITT. Rapid immunoassays should not be employed to detect VITT antibodies.
Essentials
- Comprehensive review of regular heparin-induced thrombocytopenia (HIT) assays employed for vaccine-induced immune thrombocytopenia and thrombosis (VITT) diagnosis.
- Calculate the sensitivities for anti-platelet factor 4 (PF4) immunoassays and functional assays for samples with a clinical diagnosis of VITT.
- Calculate the specificity for a non-VITT healthy subject sample distributed as part of the exercise.
- Compile assay list of methods employed in the diagnosis and management of VITT.
1 INTRODUCTION
Vaccine-induced immune thrombocytopenia and thrombosis (VITT) following the administration of AstraZeneca (AZ) ChAdOx1 vaccine was first reported in Spring 2021 across Europe.1-3 The associated clinical and laboratory features have included thrombosis often at unusual sites, thrombocytopenia, raised D-dimer levels plus positive anti-platelet factor 4 (PF4) antibodies.1-4 The information available with respect to the sensitivity and specificity of the different anti-PF4 assays employed by laboratories for HIT when used in the diagnosis of VITT have included two laboratories performing multiple testing5, 6 or an interlaboratory comparison using clinically positive VITT samples,7 or an external quality assessment (EQA) program distributing VITT samples.8 This exercise was designed as a multicenter investigation of a wide range of methodologies in international usage. The prime objective was to aid the diagnosis of VITT following the roll-out of AZ COVID-19 vaccination across the globe.
2 METHODS
Table 1 gives clinical details for the six samples, showing prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), D-dimer, and platelet count.
| Sample details | Clinical diagnosis | PT (s) | APTT (s) | FIB g/L | D-dimer ng/ml | Platelet count X109/L | Anticoagulation, which? |
|---|---|---|---|---|---|---|---|
| VITT 01 received IVIG and given Argatroban prior to first PEX | VITT | 13 | 24.8 | 1.5 | 29,284 | 2 | Argatroban |
| VITT 02 received IVIG and given Argatroban prior to second PEX | VITT | 12.7 | 26.4 | 1.5 | >50,000 | 16 | Argatroban |
| VITT 03 received IVIG and given Argatroban prior to fourth PEX | VITT | 12.0 | 24.6 | 1.8 | 41,596 | 6 | Argatroban |
| VITT 04 received IVIG and given no anticoagulation prior to PEX | VITT | 12.4 | 27 | 0.97 | 24,070 | 9 | None |
| VITT 05 received IVIG and given Fondaparinux prior to PEX | VITT | 14.0 | 37 | 2.9 | 4500 | 121 | Fondaparinux |
| VITT 06 contains LMWH at approx. 0.8 IU/ml | No clinical suspicion of VITT | NT | NT | NT | NT | NA | Low molecular weight heparin |
- Abbreviations: APTT, activated partial thromboplastin time; FIB, fibrinogen; IVIG, intravenous Ig; LMWH, low molecular weight heparin; NA, not applicable; NT, not tested; PEX, plasma exchange; PT, prothrombin time; VITT, vaccine-induced immune thrombocytopenia and thrombosis.
Plasma from patients with a clinical diagnosis of VITT was collected following written informed consent as part of a plasma exchange (PEX) process. Samples VITT 01, VITT 02, and VITT 03 were from one patient at the first, second, and fourth round of PEX. Samples VITT 04 and VITT 05 were from two separate patients at their first round of PEX.
The PEX plasma samples collected from the patients were in sufficient volumes to allow for the large scale EQA exercise. The PEX plasma for samples VITT 01–05 were lyophilized as previously described9 before dispatching to participants as lyophilized samples. Lyophilized plasma which had been spiked with LMWH (VITT 06) from an unvaccinated healthy subject was also distributed as part of this exercise.
The lyophilized aliquots were distributed at ambient temperatures to participants within the UK NEQAS Blood Coagulation distribution database, those registered for the ECAT HIT EQA program, and to the laboratories of members of the International Society on Thrombosis and Hemostasis Scientific and Standardization Committee (ISTH SSC) for Platelet Immunology. Samples were distributed to a total of 500 centers.
Laboratories were invited to perform their regular HIT assays for suspected VITT on all samples and return results for data analysis. A survey monkey link was provided to capture the raw results plus interpretations as well as details of the methodologies employed by the participating centers. Centers were invited to perform as many different assays as they employ at their testing sites to further edify the laboratory community on assay sensitivities and specificities. A further round of questioning with regards to the functional assays was undertaken to elucidate the nature of assay modifications for suspected VITT. The participants employed a range of PF4 concentrations including 5, 10, 12.5, 20, 25, and 50 μg/ml for the VITT modified assay. Both low molecular weight heparin and unfractionated heparin at concentrations of 0.1, 0.2, 0.35, 0.5, 1, 10, 48, and 100 IU/ml were used by participants with the VITT modified assay at their centers. Data were analyzed in a Microsoft Excel spreadsheet.
3 RESULTS
Results were returned from 385 centers with the methods employed by the participants shown in Table 2. A total of 105 centers returned more than one set of results for the samples distributed. The sensitivity and specificity of the ELISA methods, VITT modified functional methods, and non-ELISA methods as a whole have been calculated and are shown in Table 3. The sensitivity % was calculated by dividing the total number of positive interpretations by the total number of all the interpretations for the samples VITT 01–05. The specificity % was calculated for VITT 06 by dividing the number of negative interpretations by the total number of all interpretations for sample VITT 06. The Immucor ELISA method was the ELISA assay most widely used by participants and the results are illustrated in Figure 1. Optical densities (ODs) returned by participants are graphically illustrated to show the drop in antibody titer. The median OD for these three samples from one patient did fall over the course of their therapy with intravenous immunoglobulin (IVIG) and plasma exchange (PEX) (Figure 1). The data for the other ELISA assays for the three samples from the same patient show a similar trend over time.
| Assay method | Number of results returned |
|---|---|
| ELISA: Aeskulisa | 6 |
| ELISA: Stago Asserachrom | 28 |
| ELISA: Hyphen | 43 |
| ELISA: Immucor Lifecodes | 69 |
| Non-ELISA: BioRad Diamed | 72 |
| Non-ELISA: Biotec Melinia Quickline & Stago STic | 99 |
| Non-ELISA: Werfen Acustar Chemiluminescence | 66 |
| Non-ELISA: Werfen Latex Immunoassay | 53 |
| Functional: flow cytometry | 10 |
| Functional: heparin induced platelet aggregation | 8 |
| Functional: multi electrode aggregometry | 7 |
| Functional: platelet ATP release | 1 |
| Functional: Platelet Aggregation | 17 |
| Functional: serotonin release | 6 |
- Abbreviation: VITT, vaccine-induced immune thrombocytopenia and thrombosis.
| Method | VITT 01 sensitivity % | VITT 02 sensitivity % | VITT 03 sensitivity % | VITT 04 sensitivity % | VITT 05 sensitivity % | VITT 06 specificity % |
|---|---|---|---|---|---|---|
| AeskulisaELISAn = 6 | 100 | 100 | 67 | 100 | 50 | 100 |
| AsserachromIgG ELISAn = 17 | 100 | 100 | 82 | 100 | 88 | 100 |
| Asserachrom IgGAM ELISAn = 6 | 67 | 50 | 33 | 100 | 33 | 100 |
| HyphenIgG ELISAn = 41 | 100 | 100 | 76 | 98 | 71 | 100 |
| HyphenIgGAM ELISAn = 2 | 100 | 100 | 100 | 100 | 100 | 100 |
| ImmucorIgG ELISAn = 59 | 100 | 100 | 97 | 100 | 98 | 100 |
| ImmucorIgGAM ELISAn = 9 | 100 | 100 | 100 | 100 | 100 | 100 |
| BioRad Diamednon ELISAn = 72 | 67 | 49 | 7 | 69 | 40 | 99 |
| Biotec Melinia Quickline & Stago STic non ELISAn = 99 | 6 | 3 | 4 | 0 | 1 | 100 |
| Acustar Chemiluminescencenon ELISA n = 66 | 2 | 0 | 0 | 0 | 0 | 98 |
| Latex Immunoassaynon ELISAn = 53 | 2 | 2 | 0 | 0 | 0 | 85 |
| Heparin-induced platelet aggregation assayFunctionaln = 6 | 33 | 33 | 17 | 50 | 17 | 83 |
| Flow cytometry assay Functionaln = 10 | 70 | 40 | 40 | 90 | 40 | 90 |
| Multiplate assayFunctionaln = 7 | 57 | 57 | 43 | 57 | 29 | 100 |
| Platelet aggregationFunctionaln = 15 | 27 | 20 | 14 | 53 | 21 | 100 |
| Serotonin release assay Functionaln = 2 | 50 | 0 | 0 | 50 | 0 | 100 |
| Platelet aggregationATP release assayFunctional n = 1 | 0 | 0 | 0 | 0 | 0 | 100 |
| VITT modifiedFunctional assaysn = 9 | 78 | 67 | 78 | 100 | 67 | 100 |
- Abbreviation: VITT, vaccine-induced immune thrombocytopenia and thrombosis.
Interpretations of the functional assays returned by participants were split into two groups: modified assays for VITT and functional HIT assays not modified to be specific for VITT. The VITT modified functional assays have a greater sensitivity to clinically positive VITT samples, distributed as part of the exercise, than functional assays that have not been modified (Table 3).
4 DISCUSSION
The samples distributed for this exercise and the returns and data presented here were to inform a larger community of clinical laboratories across the NEQAS, ECAT, and SSC networks of the performance, sensitivity, and specificity of the locally available anti-PF4 laboratory assays when used to help with patients with diagnosis and management of suspected or confirmed VITT.
The non-ELISA methods showed poor sensitivity to the VITT samples distributed as part of this exercise as previously reported5-8, 10 (Table 3).These non-ELISA RAPID/POC methods included: Werfen Acustar HIT assay, Werfen LIA, Stago STIC, and Biotec Milenia Quickline are were not sufficiently sensitive for the detection of anti-PF4 antibodies in VITT clinical cases. Therefore they should not be employed to detect VITT antibodies.
The VITT modified functional assays had a greater sensitivity to the clinically positive VITT samples than the functional HIT assays that have not been modified to be more specific for VITT. The modifications were to serotonin release assays (n = 4), heparin-induced platelet aggregation assays (n = 3), and platelet aggregation assays (n = 2).
With this in mind, it may help if testing algorithms used by clinical teams to select laboratory assays recommend that a VITT-modified functional assay is requested to confirm clinical VITT in a patient. However, modified assays were still not 100% sensitive, which may well be due to the IVIG therapy11 that the three patients were having for treatment of VITT.
The sensitivity of the ELISA methods to the VITT samples distributed in the exercise is superior to that of the functional assays, regardless of whether the functional assays are modified or non-modified.
The exercise included serial PEX samples from one patient. The ODs of these samples decreased over the course of their therapy with IVIG and PEX, with the ELISA assays used by participants. However, the ODs returned by participants using the Immucor IgG assay were above the cut-off for 99.4% of results (178/179) for the samples VITT 01, VITT 02, and VITT 03. Therefore, we can conclude that for the plasma from three patients with VITT, the ELISA assays had the greatest sensitivity compared with the functional assays regardless of whether they have been modified or not. The ISTH guidelines for VITT recommend first-line testing with an ELISA anti-PF4 assay and confirmation with a functional assay. There are no recommendations to employ non-ELISA RAPID anti-PF4 assays.12 Our data suggest that further information on the type of functional assay and any modifications required should be detailed if recommending use of a functional assay in the VITT setting.
AUTHOR CONTRIBUTIONS
Steve Kitchen, Ian Jennings, Piet Meijer, Chris Reilly-Stitt, and Isobel Walker devised the study. Ian Jennings and Chris Reilly-Stitt performed the data analysis. Mike Makris and Marie Scully provided clinical details and revised the manuscript. Chris Reilly-Stitt wrote the first draft of the manuscript. Ian Jennings, Steve Kitchen, Mike Makris, Piet Meijer, Moniek de Maat, Marie Scully, Tamam Bakchoul, and Isobel D Walker contributed to the review and revision of the manuscript.
CONFLICT OF INTEREST
All authors, including Chris Reilly-Stitt, Ian Jennings, Steve Kitchen, Mike Makris, Piet Meijer, Moniek de Maat, Marie Scully, Tamam Bakchoul, and Isobel D Walker declare no relevant conflicts of interest.
