key: cord-1000892-1n6jcl2j
authors: Favaloro, Emmanuel J.; Henry, Brandon Michael; Lippi, Giuseppe
title: The complicated relationships of heparin‐induced thrombocytopenia and platelet factor 4 antibodies with COVID‐19
date: 2021-05-17
journal: Int J Lab Hematol
DOI: 10.1111/ijlh.13582
sha: 32ba7711040d7cbffdff73a39ee21399dd900d64
doc_id: 1000892
cord_uid: 1n6jcl2j

COVID‐19 (coronavirus disease 2019) represents a prothrombotic disorder, and there have been several reports of platelet factor 4/heparin antibodies being present in COVID‐19‐infected patients. This has thus been identified in some publications as representing a high incidence of heparin‐induced thrombocytopenia (HIT), whereas in others, findings have been tempered by general lack of functional reactivity using confirmation assays of serotonin release assay (SRA) or heparin‐induced platelet aggregation (HIPA). Moreover, in at least two publications, data are provided suggesting that antibodies can arise in heparin naïve patients or that platelet activation may not be heparin‐dependent. From this literature, we would conclude that platelet factor 4/heparin antibodies can be observed in COVID‐19‐infected patients, and they may occur at higher incidence than in historical non‐COVID‐19‐infected cohorts. However, the situation is complex, since not all platelet factor 4/heparin antibodies may lead to platelet activation, and not all identified antibodies are heparin‐dependent, such that they do not necessarily reflect “true” HIT. Most recently, a “HIT‐like” syndrome has reported in patients who have been vaccinated against COVID‐19. Accordingly, much more is yet to be learnt about the insidious disease that COVID‐19 represents, including autoimmune outcomes in affected patients.

COVID-19 (coronavirus disease 2019) is a recognized global pandemic caused by infection with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). This infectious disease is thought to have originated in Wuhan, China, in late 2019, and at time of writing has infected over 141 million people and caused over 3 million deaths. 1 Severe COVID-19 reflects primarily a prothrombotic disorder, with thrombosis appearing in various forms. [2] [3] [4] For example, a recent meta-analysis has indicated a venous thromboembolism (VTE) rate, including deep vein thrombosis (DVT) and pulmonary thrombosis (PE), of close to 30% in patients with severe COVID-19. 5 Acute myocardial ischaemia (infarction) and cerebrovascular accidents may also develop in as many as 8% and 3% of COVID-19-infected patients needing intensive care, 6 whilst systemic coagulopathy and disseminated intravascular coagulation (DIC) may occur in as many as 7% of such patients. 7 Evidence of microthrombosis in multiple organs including lungs, kidneys and liver, also occurs, although only identifiable on autopsy in patients who have died due to COVID-19. [8] [9] [10] [11] Indeed, COVID-19 appears to affect all facets of haemostasis, including primary haemostasis (ie platelets, von Willebrand factor, endothelium), secondary haemostasis and fibrinolysis. [12] [13] [14] [15] In addition, thromboses may arise from disturbances in immune response, creating cytokine disturbance (so-called "cytokine storm"), according to immunothrombosis/endotheliitis type mechanisms. 2 Perhaps unsurprisingly, then, a series of autoimmune events have also been reported in patients with COVID-19, including for example the presence of antiphospholipid antibodies potentially associated with antiphospholipid syndrome. 16 Of relevance to the current narrative review is that there have been several reports of platelet factor 4/heparin (PF4/H) antibodies being present in COVID-19infected patients. Accordingly, we critically appraise this literature to answer the question: is heparin-induced thrombocytopenia (HIT) a feature of COVID-19?

This is a narrative review. The PubMed database (https://pubmed.ncbi.

nlm.nih.gov) was searched as required for both background information on PF4/H antibodies and HIT as well as specific papers related to COVID-19. For the latter, we primarily used the search term (heparin AND (antibodies OR thrombocytopenia OR thrombocytopaenia OR HIT)) AND (COVID OR SARS)). An initial search performed on 24

March 2021 was later updated to be current as of 1 April 2021. Of 102 separate articles identified by this specific search, we then excluded general reviews and commentaries (ie not presenting original data), and papers otherwise found to be irrelevant to the topic.

PF4/H antibodies develop in some individuals after exposure to heparin, primarily unfractionated heparin (UFH), but in some cases also to low molecular weight heparin (LMWH). 17 These antibodies can be detected immunologically via a wide range of assays, including ELISA (enzyme-linked immunosorbent assays), LIA (latex immunoassay), CLIA (chemiluminescence immunoassays) and particle/lateral flow-based methods. 18, 19 There is some considerable inter-assay variability regarding the detection of these PF4/H antibodies. For example, ELISA assays tend to detect a proportionally greater number of PF4/H antibodies than does CLIA. 20 Irrespective, the presence of these PF4/H antibodies in themselves does not identify pathological HIT, which also requires resultant activation of platelets. 21 This represents a functional event detected by functional assays, such as SRA (serotonin release assay) and HIPA (heparin-induced platelet aggregation). 22, 23 Only a small proportion of detected PF4/H antibodies will cause platelet activation, and thus "positivity" in the functional assays, and therefore, the anticipated pathological consequence of thrombosis. 21, 24 There is also an association between the level of detected PF4/H antibodies and the likelihood of "positivity" in a functional assay. For example, an ELISA OD >1.0 or a CLIA value >10 U/mL may be characterized as more likely associated with functional assay positivity. 18, 20, 21, 24 However, it is generally accepted that a functional assay is required to confirm pathological HIT in a patient with immunologically detected PF4/H antibodies.

It may also be important to note some nomenclature issues and arising confusion in the literature. The development of PF4/H antibodies is variously identified by authors as "immunological HIT," or just HIT. A transient heparin-induced nonimmune reduction in platelet count is sometimes called "HIT type I." The secondary confirmation by antibody-mediated platelet activation causing pathological HIT is variously called "HIT Type II," "HITT" (ie HIT with thrombosis) or else may also just be referred to as HIT. In this review, we will refer to the first and last of these entities as (immunologically detected) PF4/H antibodies and pathological HIT, respectively. Where it is not clear from the literature whether pathological HIT has been appropriately defined, we will use the term "HIT" to denote this uncertainty.

The background risk of developing PF4/H antibodies and subsequent pathological HIT depends on the clinical setting. The overall reported incidence of "HIT" in patients exposed to heparin varies from 0.2% to up to 5%. 25, 26 Interestingly, "HIT" is relatively rare in intensive care unit (ICU) populations, despite high use of heparin, with estimates approximating 0.5%. 27 The risk of HIT is higher amongst surgical compared with medical patients. 28 Amongst surgical patients, post-cardiac surgery patients may have a higher risk of developing PF4/H antibodies than do post-orthopaedic surgical patients, but the development of pathological HIT was historically potentially more likely after orthopaedic surgery when UFH was used. 29 Patients with major trauma are at higher risk of being PF4/H antibody positive and also developing pathological HIT as compared to those with minor trauma. 30 Pathological HIT is very rare in obstetric or paediatric patients. The risk of developing "HIT" is also higher amongst women than men, 31 and in patients on UFH vs patients receiving LMWH. 17, 32 The incidence of PF4/H antibodies and pathological HIT with ECMO (extracorporeal membrane oxygenation) is also not insignificant, although accurate numbers are difficult to determine. However, Sokolovic et al 33 reported that in a cohort of adult ECMO patients, 8/96 (8.3%) were positive for "HIT" (positive SRA or PF4/H antibodies by ELISA with optical density [OD] >1) and 7/9 had documented thrombotic events (HITT or pathological HIT) based on predefined criteria (thus, "HIT" and "HITT" incidence in the study group were 8.3% and 7.3%, respectively). Vayne et al, 34 in a single-centre prospective trial, reported on 57 adult patients who were supported by ECMO for at least 5 days. HIT was suspected in two patients (ie 3.5%) with ECMO circuit dysfunction and unexpected platelet count decrease after day 5. High levels of PF4-specific IgG were detected in both patients, and HIT was confirmed by SRA. Additional data on such patients have been reported in published reviews. 35,36

The arising literature from our specific literature search is listed and summarized in Table 1 . [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] Most published reports were individual case reports, or small case series. The information specifically provided by each publication varied considerably. Of interest, not all studies performed laboratory testing for PF4/H antibodiessome "diagnosed" "HIT" based on clinical grounds, such as the 4T score (4Ts; being evidence of Thrombocytopenia, and additional information about the Timing of platelet count fall, the presence of Thrombosis or other sequelae and Other causes for thrombocytopenia). Alternatively, some publications did not report 4Ts data, and instead "diagnosed" "HIT" on the basis of positive PF4/H antibody tests, only sometimes confirmed by additional testing using functional assays such as SRA or HIPA. Not all publications reported on the anticoagulant used ahead of the "HIT" "diagnosis." Where anticoagulant use was identified, this typically being heparin, it was not always specified if this was UFH or LMWH. Sometimes, anticoagulant use was changed for patients according to clinical need; for example, LMWH use initiated as standard prophylaxis for prevention of thrombosis in COVID-19 might be changed to UFH for use in ECMO, with this being a common treatment in severe COVID-19.

Not all publications identified the day of "HIT" diagnosis in regard to hospital day (HD) or day post-heparin initiation, or subsequent to progression of thrombocytopenia. When laboratory testing was performed for PF4/H antibodies, methodologies were not always identified, or test results were not always reported.

One could hypothesis that the reported incidence of COVID-19associated anti-PF4/H antibodies or HIT would be highly biased due to the type of reported study. In particular, case reports and small cases series, comprising the current literature (Table 1) , would be biased simply due to patient selection. Thus, authors are more likely to publish positive rather than negative findings. Second, researchers may actively look for anti-PF4/H antibodies or HIT in select COVID-19-infected patient cohorts, such as those with clinical suspicion, either due to thrombocytopenia or 4Ts. It can be noted that (mild) thrombocytopenia is common in COVID-19. 15 Third, there may be anticoagulation bias, where, given that UFH is more often reported to be associated with "HIT" than LMWH, studies may be limited to investigation only of UFH-treated COVID-19-infected patients. In total, in such studies, a relatively high incidence of anti-PF4/H antibodies or "HIT" may be identified, as might be anticipated, irrespective of the presence of COVID-19. This is important to note in any evaluation of anti-PF4/H antibodies or "HIT" in COVID-19.

Details provided in Table 1 are further summarized in Table 2 or negative (68.4%) by functional assay. It can be noted that this distribution follows the anticipated pattern of hospitalized patients with COVID-19, being predominantly male, and above the age of 55 years. 2

As noted earlier, the COVID-19 literature is sometimes unclear regarding whether UFH or LMWH was the anticoagulant in use prior to development of anti-PF4/H antibodies or "HIT," or the time course of their development. However, based on the information gleaned from the literature, UFH (55.7%) and LMWH (44.3%) seemed "similarly" involved in development of anti-PF4/H antibodies or "HIT."

Note here, however, that selection bias is particularly problematic, as some studies only evaluated cases under UFH therapy. The number of cases according to functional assay positivity vs negativity is numerically low, but similar "equality" was seen for LMWH vs UFH in the separated cohorts (Table 2 ).

Again, accepting that selection bias would play a part in findings, the median 4Ts for cases identified in Table 1 was 5, with an IQR of 4-6 ( Table 2) . Interestingly, the median value for those positive in functional assays was 6, and higher than the median (ie 4) for those with negative functional assay findings. Although this would be as expected, study numbers are low overall, and the difference was not statistically significant ( Figure 1) . Also, the 4Ts is noted to be less useful in ICU cohorts, and many of the "HIT" assessed population derived from ICU.

Again, cognizant of study bias, the median (IQR) for the whole cohort for ELIZA OD was 0.67 (0.47-1.20), with higher median in functionally positive patients (1.02) than negative patients (0.59). However, the difference was not statistically significant ( Figure 1) . Similarly, using CLIA assays, the median (IQR) for the whole cohort was 1.9

(1.9-18.5) U/mL, with higher median in functionally positive than negative patients (21 vs 0.35). Again, this fits expectations, although study numbers are low. Here, however, the difference was statistically significant (Figure 1 ).

It is not possible to be entirely sure of the true incidence of pathological HIT in COVID-19, given selection bias noted above TA B L E 1 Summary data for case reports and case series related to heparin (and non-heparin)-induced thrombocytopenia in COVID-19 a 

The literature on "HIT" in COVID-19 is conflicting. Some researchers find a high level of "HIT" in COVID-19-infected patients, but these findings need to be tempered by methodological flaws, where patients were either not fully worked up, or else key details were missing. It needs to be noted that in non-COVID-19 literature, many patients may be found to have PF4/H antibodies, but most of these will not develop pathological HIT. 56 Moreover, a high level of PF4/H antibodies (especially as detected by ELISA) will not be positive by a functional assay such as SRA. 20, 24 In contrast, some researchers reported a low level of "HIT" in did not appear to be a trigger for the event as patients were heparin naïve.

Thus, the overall situation in regards to identification of anti--PF4/H antibodies or "HIT" is complex. Indeed, the situation has recently become even more complex, given a recent publication reporting a rare phenomenon in 9 patients recently vaccinated against COVID-19, primarily leading to venous thrombotic episodes (especially cerebral venous thrombosis, CVT) accompanied by relevant thrombocytopenia (ie 39 ± 30×10 9 /L) 4-16 days after receiving the vaccine. 57 Interestingly, the German researchers indicated a mechanism similar to HIT, with the serum of 4 such patients referred for investigation of platelet-activating antibodies directed against PF4/H. All test results revealed strong positivity for "PF4/-H antibodies" using an immunoassay (ELISA) and also activated platelets using a platelet activation assay, but these events appeared to occur independently of heparin, and reactivity against PF4 (without heparin) was later also shown. Although a causal link between COVID-19 vaccination (especially with AstraZeneca AZD1222) and CVT has yet to be clarified, these patients (mostly young women in the German study) appeared to develop "PF4 anti- Janssen). 61 In the UK series, the phenomenon was more generally observed in both men and women, and in both "young" and "old." 60 The events are unquestionably rare, but physicians should be advised about its possible development after COVID-19 vaccination. 62

Taking all this information into consideration, we need to accept that PF4/H antibody development does represent a feature of COVID-19, at least in some patients. However, this does not always indicate pathological HIT for most. Also, there appears to be a question over the role of heparin in some of the cases reported in the literature, and instead, a "HIT-like" event may be occurring in some patients without heparin exposure. This may be linked to theoretical concepts around PF4/H mimicry in infectious disease, although additional confounders may exist in relation to COVID-19. The binding of SARS-CoV-2 spike protein to platelet surface receptor(s), leading to platelet activation, is another potential mechanism that deserves further scrutiny in the pathogenesis of thrombosis in COVID-19. 63 However, whether pathological HIT truly develops in a high proportion of COVID-19-infected patients remains unresolved, and more needs to be done to fully investigate the phenomena of PF4/H antibody development, and arising pathophysiology in COVID-19. We encourage further studies on the development of HIT or PF4(/H) antibodies, either following heparin exposure or not. These studies need to document all aspects of patient exposure to heparin (or not), the type of heparin, the time course for antibody development or arising pathology, and the methods employed for laboratory testing, including any employed modifications (eg heparin inhibition).

The opinions expressed in this review are those of the authors and do not necessarily reflect the opinions of their respective employers, NSW Health Pathology, The Heart Institute, Cincinnati Children's Hospital Medical Center and the University of Verona.

The authors have no competing interests. 

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