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Increased prevalence of heparin induced thrombocytopenia in COVID-19 patients

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A 59-year-old male patient referred to our hospital for COVID-19-related pneumonia with respiratory distress. Laboratory exams on admission are shown in Table 1. SARS-CoV-2 infection was confirmed by reverse-transcriptase-polymerase-chain-reaction (RT-PCR) on nasopharyngeal swab. The patient received supplemental oxygen with continuous positive airway pressure (CPAP), empirical antibiotics, hydroxychloroquine and prophylaxis for venous thromboembolism (VTE) with low-molecular-weight heparin (enoxaparin 4000 IU/day). Due to progressive severe respiratory failure, on day 5 the patient was transferred to the ICU, where invasive mechanical ventilation was initiated. Renal function worsened (creatinine serum level up to 359.87 μmol/l) and required continuous renal replacement therapy (CRRT); enoxaparin was replaced by calcium heparin at a dose of 5.000 IU/0.2 ml every 8 h. After 18 days of heparin exposure, laboratory tests showed a decline of platelet count, with a reduction of 50% by day 21 (day 15 of calcium heparin). Calculated 4 T score was 5 and HIT expert probability (HEP) score was 6. Anti-PF4-antibody testing was positive and confirmed by the high-heparin confirmatory step. Heparin was promptly stopped and replaced with fondaparinux at a dose of 1.5 mg/day with normalization of platelet count.
The clinical status was improving, when the patient experienced a cardiac arrest and computed tomographic scan of the chest showed bilateral pulmonary embolism. Fondaparinux was then replaced with argatroban. Deep vein thrombosis was excluded. Therapy with argatroban was well tolerated, without recurrence of thrombosis or adverse effects. A few days later, Candida parapsilosis was isolated from blood cultures, and the patient died of septic shock.

Case report 2
An otherwise healthy 50-year-old male patient was admitted to the ICU with SARS-CoV-2 -related bilateral pneumonia and acute respiratory distress syndrome (ARDS). He was intubated and vasopressor support was required for hemodynamic instability. Hydroxychloroquine, azithromycin and enoxaparin (6000 IU every 12 h) were initiated. Given the persistent severe hypoxia and hypercapnia, the patient received veno-venous extracorporeal membrane oxygenation (VV-ECMO) with intravenous heparin sodium. Thrombocytopenia with a 50% decrease in platelet count was observed after 16 days of heparin exposure (10 days of heparin sodium). Calculated 4 T score was 4 and HEP score was 3. Suspecting HIT, heparin was stopped and replaced by bivalirudin at standard dose with normalization of platelet count. Anti-PF4-antibodies tested positive. The improvement of the patient's medical condition enabled the discontinuation of ECMO (after 21 days) and vasopressors; VTE prophylaxis with fondaparinux (2,5 mg/ day) was initiated. Then, the patient developed urinary tract infection and Acinetobacter baumannii was isolated from bronchoalveolar lavage, but he was finally discharged home in good condition after 69 days of hospitalization.
Thrombocytopenia was a common laboratory finding among the 172 COVID-19 patients admitted to the ICU of our hospital. A platelet count below 100,000 cells/uL was found in 43 patients (25%), and a platelet count of 100,000-150,000 cells/uL was found in 42 patients (24,41%). Anti-PF4 antibody test was performed in 14 patients with suspected HIT and one case of HIT, and one of HITT were diagnosed (1.16%) while the test was negative in 11 patients and one patient presented only a weak positivity (0.41 anti PF4 Optical Density units) that suggests a very low probability of HIT (< 5%). The diagnosis of HIT relies on clinical suspicion, first assessed by probability scores, and then confirmed by laboratory tests. The 4 T score and the more recent HEP score are the most widely used pretest clinical scoring systems (6,7). When an intermediate/high pretest probability is found, it is recommended to perform laboratory tests, including the immunoassay as screening test and the functional assay as confirmatory step.
In our laboratory, we use the PF4 Enhanced® assay (Immunoglobulin G/A/M) (IMMUCOR GTI Diagnostics, Waukesha, Wisconsin, USA) as first-line test for HIT. Optical density (OD) levels reported in this study were derived from PF4 Enhanced® test only and tests were considered positive if OD > 0.40 at 410 nm. A confirmatory step using high heparin concentrations was performed.
Our patients presented an intermediate/high clinical suspicion and a positive laboratory test and both of them had high risk factors for HIT, such as sepsis and extracorporeal circulation (4).
In our past experience, from January 1st 2018 to February 20th 2020, among 2148 consecutive patients admitted to the ICU, anti-PF4 antibody test was performed in 62 patients with thrombocytopenia and only three HITT and one HIT (0.18%) were diagnosed (data obtained from ICU's medical records). This prevalence is comparable to that in previous reports (8).
An association between high risk for pulmonary embolism and COVID-19 is hypothesized by many authors (9). HITT patient's personal and familiar history was negative for thromboembolism, and although the infection may have contributed to a pro-thrombotic state, the temporal sequence of the events suggests a direct correlation of the thrombotic event with HIT.
The limitation of our data is the unavailability of the confirmatory functional assay in our laboratory. In spite of this, it is reasonable to diagnose our patients with HIT based on clinical presentation, pre-test probability, the anti PF4 antibodies OD units, and a rapid increase of platelet counts following discontinuation of heparin.
In conclusion COVID-19 is characterized by marked hypercoagulability, and heparin administration, particularly in patients with severe impairment of respiratory function, has been employed to prevent VTE. Even if HIT is a rare condition, the incidence observed in our case series is higher than that reported in critically ill patients without COVID 19, as recently observed in the literature (10). We hypothesize that the inflammatory state and the activation of the coagulation cascade could play a role in the development of anti-PF4 antibodies. Other mechanisms may also be involved, and further studies are needed.

Declaration of competing interest
None.

Acknowledgments
All authors made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. All authors participated in drafting the article and revised it critically for important intellectual content. All authors gave final approval of the version to be submitted.
The research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Thrombotic complications Site of thrombosis Bilateral pulmonary embolism None CAD denotes coronary artery disease, ICU intensive care unit, LMWH low molecular weight heparin, PF4 platelet factor 4, EGFR estimated glomerular filtration rate, INR international normalized ratio, aPTT activated partial thromboplastin time, ND not determined, and HEP heparin-induced thrombocytopenia expert probability, OD optical density.