Title: Pilot study to evaluate hypercoagulation and inflammation using rotational thromboelastometry and calprotectin in COVID-19 patients.

Introduction: Abnormal coagulation and inflammation are hallmarks of SARs-COV-19. Stratifying affected patients on admission to hospital may help identify those who are risk of developing severe disease early on. ROTEM is a point of care test that can be used to measure abnormal coagulation and calprotectin is a measure of inflammation. Aim: Assess if ROTEM can measure hypercoagulability on admission and identify those who will develop severe disease early on. Assess if calprotectin can measure inflammation and if there is a correlation with ROTEM and calprotectin. Methods: COVID-19 patients were recruited on admission and ROTEM testing was undertaken daily for a period of 7 days. Additionally inflammatory marker calprotectin was also tested. Results : 33 patients were recruited to the study out of which 13 were admitted to ITU and 20 were treated on the ward. ROTEM detected a hypercoagulable state on admission but did not stratify between those admitted to a ward or escalated to ITU. Calprotectin levels were raised but there was no statistical difference (p=0.73) between groups. Significant correlations were observed between FIBA5 (p<0.00), FIBCFT (p<0.00), FIBMCF (p<0.00) and INMCF (p<0.00) and calprotectin. Conclusion: COVID19 patients were hypercoagulable in admission. The correlations between ROTEM and calprotectin underline the interactions between inflammation and coagulation.


Introduction
Patients infected with the coronavirus SARS-CoV-2 leading to the coronavirus disease 2019  become symptomatic after an average incubation of 5.2 days (1). Severely affected patients develop shortness of breath at a median of 8 days from illness onset, with acute respiratory distress syndrome, pneumonia developing at day 9 and admission to Intensive Therapy Unit (ITU) at day 10.5 (2).
There is extensive cross talk between inflammation and coagulation systems in response to invasion by pathogens (4)(5)(6). Reflecting this, several studies have reported abnormalities in laboratory markers of coagulation and fibrinolysis in COVID-19 patients (7)(8)(9)(10)(11). Retrospective data indicates that there is significantly more derangement in coagulation parameters (namely prothrombin time (PT) and D-Dimer) at the point of admission in patients who don't survive, versus those who do (12). These changes in individual coagulation parameters point to a disruption in haemostasis, but do not provide any guidance as to the biological effect of the changes. Traditional coagulation tests provide a snapshot of a particular aspect of coagulation in cell depleted plasma, but do not provide an assessment of overall haemostasis. Rotational thromboelastometry (ROTEM ® ) provides a composite assessment of the dynamic process of clot initiation, thrombin generation and whole blood clot formation which is arguably more representative of physiological processes (13).
A number of studies have used ROTEM ® to assess haemostatic status and demonstrate a prothrombotic phenotype in patients admitted to ITU (14)(15)(16). Furthermore, studies have also shown that ROTEM ® can be potentially used as a predictor for thrombosis and disease severity on admission (17)(18)(19)(20).
In this study we investigated if COVID-19 patients demonstrated a prothrombotic phenotype as measured by ROTEM Sigma and if it could be used as a predictor of disease severity on admission to hospital. Correlations between ROTEM and inflammatory marker calprotectin were also undertaken to understand its role in contributing to hypercoagulation in COVID-19 patients.

Materials and Methods
in line with the Declaration of Helsinki, ethical approval was obtained from the Cornwall and Plymouth Research Ethics Committee. IRAS No: 284755. As part of the ethical approval where patients had severe disease and were not able to consent, assent was obtained from an independent medical practitioner who was not involved in the direct care of the patient. Patients who were admitted to hospital with a suspected/known diagnosis of COVID-19 and over the age of 18 were included in the study. Patients who had a negative COVID-19 test after recruitment were subsequently excluded from analysis. Any patients with a previous history of coagulation disorders including venous . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 27, 2022. ; https://doi.org/10.1101/2022.05.27.22275701 doi: medRxiv preprint thromboembolisn within 6 months and those on anticoagulant therapy excluding low molecular weight heparin thromboprophylaxis were excluded from the study. Admitted patients either went to a normal ward or to ITU depending on clinical severity. Patients with mild to moderate respiratory failure were administered oxygen via nasal cannula and venturi mask upto a maximum of 15 L/minute, if this was insufficient continuous positive airway pressure (CPAP) was administered. Those with severe respiratory failure were treated in ITU with invasive ventilation support. On admission patients were treated with a single prophylactic dose of enoxaparin in ED and then transferred to either a ward or ITU. Anticoagulation treatment was given twice daily (6am/6pm) and categorised based on weight.
Enoxaparin 40mg if <100kg, 60mg if 100-150Kg and 80mg if >150kg. All patients were also given a stat dose of vitamin K IV with the first dose of enoxaparin. This was given as testing at the start of the pandemic had shown several patients had low protein C or S on admission.
Bloods were taken on admission and everyday for the first 7 days of hospital admission. The follow-up bloods were taken approximately 3 hours after anticoagulant treatment. Rotational thromboelastometry was tested within 4 hours of taking the blood and the plasma aliquots were frozen for batch testing of calprotectin.

Rotational thromboelastometry
ROTEM Sigma (Werfen UK) is a viscoelastic point of care test with a fully automated system containing a sample handler and cartridge that uses whole blood to measure coagulation (21). All tests were analysed for 60 minutes and within 4 hours of sample collection. Reference ranges were obtained from healthy volunteer data (n=26) previously collected.
Data from three ROTEM channels are presented, FIBTEM, INTEM and EXTEM. FIBTEM uses cytochalasin D to inhibit platelet activity and provide a clot tracing that reflects the presence of fibrinogen. It provides information on fibrin formation and polymerisation without platelet contribution (22). In EXTEM tissue factor is used to initiate the extrinsic clotting cascade. The INTEM test uses ellagic acid to initiate clotting via the intrinsic pathway. The EXTEM and INTEM tests provide information on the extrinsic and intrinsic pathways respectively. The viscoelastic characteristics of the clot are measured from clot formation to lysis and include clotting time (CT), clot formation time (CFT), maximum clot firmess (MCF), A5-A30 (measure of clot firmess from 5-30 minutes) and maximum lysis (ML) which is the degree of fibrinolysis relative to MCF achieved during measurement and is reported as percentage of clot firmness lost (23). A shortened CFT with an increased MCF is indicative of a hypercoagulable state.
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Calprotectin
Circulating calprotectin levels were measured in plasma using a chemiluminescent immunoassay (QUANTA Flash, Werfen, UK) according to manufacturer's instructions. The chemiluminescent immunoassay utilises a predefined lot specific master curve that is stored in the reagent cartridge barcode.

Statistics
Demographics of the patient population are presented as frequencies and percentages. All continuous variables are presented with medians and interquartile ranges (IQR). Mann-Whitney U test was used to test whether there were significant differences between patients admitted to ITU vs ward for ROTEM and calprotectin. We included individuals in the ITU cohort if they were admitted to the high dependency unit (HDU) or intensive treatment unit (ITU) at any point during admission (n=13). The ward cohort represents the remaining individuals in the study (n=20). Spearman's rank correlation coefficients were calculated to assess associations between variables. A Bonferroni correction was used to evaluate significance with both tests. A p-value below 0.05 at a 95% confidence interval was considered significant. Analysis was performed in Anaconda 3 with Python 3.8.8.

Results
Blood was obtained from 47 patients admitted to Hampshire Hospitals from October 2020-April 2021. 14 patients were excluded as they tested negative for COVID-19, 33 who tested positive were . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)    Table 3.
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(which was not certified by peer review)
The copyright holder for this preprint this version posted May 27, 2022. ; https://doi.org/10.1101/2022.05.27.22275701 doi: medRxiv preprint Figure 1: Calprotectin trend between ITU patients and those on the ward for admission through to week 2. Normal range for calprotectin is <1.99µg/ml.  . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted May 27, 2022 admitted to internal medicine wards and ICU. They found that ICU patients had a significantly higher FIBMCF compared to those admitted to the internal medicine wards (19). Lastly, ROTEM parameters were also measured in COVID-19 patients with varying severities of pneumonia; a hypercoagulable ROTEM pattern due to shortened EXCT, higher than normal EXMCF and FIBMCF and shortened EXCFT, high clot strength and hypofibrinolysis in advanced disease and patients with high levels of IL-6 were observed (20). It is likely that we did not see a difference between the two groups because of the small numbers of patients in our ITU cohort.
Calprotectin levels have been shown to be elevated in COVID-19 patients (24)(25)(26). In our study we found that calprotectin levels were raised overall, and patients admitted to ITU although not statistically significant had higher calprotectin levels compared to those admitted to a ward. Various studies have reported that raised calprotectin levels in COVID-19 patients. A marked difference in the serum calprotectin levels between COVID-19 survivors and non-survivors (24). Another study demonstrated that reduced frequency of non-classical monocytes along with raised serum calprotectin levels has the potential to identify patients who will develop severe COVID-19 (27). A review of calprotectin in COVID-19 provided evidence suggesting that calprotectin could be useful in assessing disease severity (28). Similar to our ROTEM results, it is likely we did not see a statistical difference between the two groups because of the small numbers in the ITU cohort.
We are the first to report significant correlations between FIBA5, FIBCFT and FIBMCF and calprotectin. It is known that fibrinogen is an acute phase reactant which is increased during an inflammatory response (29). In COVID-19 patients increased fibrinogen levels have been associated with excessive inflammation (30). We suggest that the significant correlation we have observed between fibrinogen and calprotectin is because of fibrinogen's role in inflammation. Calprotectin also correlated with INMCF which measures the intrinsic pathway of coagulation. FVIII and VWF which are part of the intrinsic pathway of coagulation are also linked to inflammation (31)(32)(33) and are known to be raised in COVID-19 patients (33)(34)(35)(36)(37). Similar to fibrinogen, the link that FVIII and vWF have to inflammation could be the reason for the significant correlation calprotectin and INMCF. These . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted May 27, 2022. ; https://doi.org/10.1101/2022.05.27.22275701 doi: medRxiv preprint interesting findings further highlights the role of inflammation in COVID-19 and its impact on coagulation which is measurable using ROTEM and should be investigated further. In addition to undertaking ROTEM testing in COVID-19 patients, consideration should be given to measuring calprotectin levels. ROTEM is a point of care test with quick turnaround times that gives an overview of coagulation. Calprotectin has the potential to be used as a marker of inflammation in these patients.
In summary, ROTEM detected a hypercoagulable state in COVID-19 patients but could not stratify severity of disease on admission in this pilot study. Significant correlations were observed between ROTEM and calprotectin demonstrating the synergy that exists between inflammation and coagulation.

Limitations
No definite conclusions can be drawn from this pilot study because of the small sample size. The study consisted mainly of Caucasian patients and as coagulation status and predisposition to the development of coagulopathies varies between race and ethnicity (38) this limits the generalisability of our study. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

Appendix 1
Comparison of ITU vs ward for the various tests over time.          . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 27, 2022. ; https://doi.org/10.1101/2022.05.27.22275701 doi: medRxiv preprint