The Role of Ischemia Modified Albumin in Patients With COVID-19

The purpose of the study is to recognize the diagnostic and/or prognostic value of IMA, as It reflects the degree of ischemia regardless of the affected organ.

Our sample, which will be taken from the Pulmonology/Covid-19 Department and the Outpatient Clinic of the Pulmonology Department of University Hospital of Larissa, will be divided into two groups. The first group will be the study group, which will include patients with confirmed COVID-19 infection, while the second group will be the control group, which will include healthy volunteers. From the study population will be collected demographics, medical history, medication, symptoms, vital points, arterial blood gases, viral load from RT-PCR for SARS-COV2 and findings from imaging and laboratory assessment. On a daily basis, during their treatment, their vital points, their laboratory tests and the presence of possible complications will be recorded.

Expected results are: 1) Comparison of IMA levels between COVID-19 patients and healthy volunteers, 2) The IMA contribution, during their admission to the hospital, to the prediction of the risk of deterioration and severe respiratory failure, 3) The increase of the predictive accuracy of SuPAR as a risk stratification biomarker, after its combination with IMA, 4) The estimation of IMA on the 10th day of illness in patients with severe respiratory failure, 5) The possibility of predicting with greater accuracy the probability of admission to the ICU, by measuring the IMA on the 10th day of illness compared to the IMA of admission.

Endothelial activation and dysfunction are associated with COVID-19 severity. Epidemiological studies suggest that severe cases or deaths due to COVID-19 frequently present with underlying comorbidities, such as advanced age, hypertension, diabetes, and cardiovascular diseases. Endothelial dysfunction, with the subsequent use of the complement, the hypercoagulable state and production of thrombin appears to be the common denominator of varieties of clinical signs and symptoms of COVID-19, which are directly associated with thromboembolic events. Necropsy findings in patients with COVID-19 have shown catastrophic microvascular injury, with pulmonary endotheliitis, thrombosis and angiogenesis being distinct pathophysiological features of the lungs in patients with COVID-19 compared with H1N1 patients and uninfected controls.

Venous thromboembolic disease, which can manifest as Deep Vein Thrombosis (DVT) or Pulmonary Embolism (PE), often occurs in patients with severe COVID-19, despite prophylactic anticoagulation. PE has been found in almost 20% of patients, despite having received anticoagulant treatment. In another study, necropsy was performed in 12 patients with COVID-19, of whom were receiving anticoagulants. DVT was detected in 7 (58%) patients, although none of them had a clinical suspicion. PE was the leading cause of death in 4 patients. Venous thrombosis occurs in particularly high rates (14-81%) in critically ill COVID-19 patients admitted to Intensive Care Units (ICU). In fact, PE has been recorded as the most common thrombotic complication in these patients with a frequency of up to 81%. In hospitalized patients with COVID-19, venous thrombosis occurs less frequently (3-21%), while for outpatients there are insufficient data. In addition to DVT, cases of acute ischemia of the upper or lower extremity with a potential need for surgery have also been reported as a less common arterial thrombotic complication in COVID-19.

In addition, acute myocardial ischemia is the most commonly reported cardiovascular complication of COVID-19. The prevalence of myocardial ischemia varies from 7% to 28%. Studies have correlated the increase in troponin in hospitalized patients with a more severe clinical course and worse prognosis.

Ischemic stroke is another common extrapulmonary thromboembolic complication of COVID-19. There are reports of ischemic stroke as the first symptom of infection (appearing especially as a middle cerebral artery thrombosis, even in young patients) and as a complication during hospitalization. In retrospective observational studies, the prevalence of ischemic stroke in this population ranges from 1.3% to 46%. The most likely mechanism of strokes during COVID-19 is considered to be subsequent hypercoagulability and vascular endothelial dysfunction through activation of inflammatory cytokines.

Human serum albumin is a peptide, which consists of 585 amino acids bonded in a specific sequence in humans. The ischemia modified albumin (IMA) molecule was identified in early 2000s, when it was observed that hypoxic-induced ischemia resulted in a modification in the circulating albumin molecule. Under ischemic conditions, the N-terminus of albumin is differentiated, possibly as a result of hypoxia, oxidation, free radicals and energy-dependent changes in membranes. This modification at the N-terminus of albumin can be indirectly evaluated. When cobalt (in vitro) is added to an ischemic sample, normal albumin molecules will bind to it, leaving a small percentage free, while IMA cannot bind to the added cobalt due to its modification at its binding site. Elevated IMA percentages result in more unbound cobalt, which can be detected by the addition of a chromatographic agent, such as dithiothreitol, and evaluated photospectrometrically. The increase of IMA is inversely related to the amount of cobalt, which causes an increase in the colour product. This is the basis of the Albumin Cobalt - Binding test, ACB test.

IMA has been studied as an indicator of cardiac ischemia but has also been found increased in patients with endothelial dysfunction, infections and sepsis. It also increases in patients with cerebral ischemia, pulmonary embolism, deep vein thrombosis, heart failure, intestinal ischemia etc. IMA values have a normal distribution in the control population and are not related to age and gender. IMA, which is the only ischemia biomarker that has reached the level of clinical evaluation, increases within a few minutes of the onset of ischemia regardless of the affected organ-tissue.

SuPAR (Soluble urokinase Plasminogen Activating Receptor) is the soluble form of uPAR, a protein that is primarily expressed in cells of the immune system, including neutrophils, activated T-lymphocytes, and macrophages. The uPAR binds to the cell membrane with a glycosylphosphatidyl-inositol (GPI). When uPAR is released from the cell membrane, it becomes soluble (SuPAR) and can be measured as a stable protein in various biological fluids, such as plasma, urine and cerebrospinal fluid. SuPAR levels in healthy people are quite stable in the blood and urine. Elevated levels of SuPAR reflect the activation of the immune system and have been evaluated as an indicator of inflammation and organ damage in a few diseases, including COVID-19. Studies on the concentration of SuPAR in 57 patients with COVID-19 pneumonia, showed that SuPAR concentrations were higher in patients who eventually developed severe respiratory failure and required mechanical ventilation, compared to patients who did not develop severe respiratory failure. More specifically, concentrations greater than or equal to 6 ng/ml had a sensitivity of 85.7% to predict the adverse outcome even 12 days before happening. SuPAR appears to be an important risk stratification tool in patients with COVID-19, as it can early identify patients who will develop severe respiratory failure or that need mechanical ventilation.

Since this is the first study of IMA in patients with COVID-19, the primary aim of the study is to investigate whether IMA levels at admission or at day 10 from symptoms onset (peak of symptoms) will be higher in patients with COVID-19 pneumonia compared to healthy individuals. The secondary aim is to assess whether IMA can predict severe respiratory failure and deterioration risk and whether combination with SuPAR will increase its accuracy.

Participants: 2 study groups will be included:

i) 128 patients with confirmed COVID-19 disease (study group, group 1) ii) 64 healthy volunteers without any of the exclusion criteria (control group, group 2) Patients from the study group will be divided into two subgroups, those with severe respiratory failure (PaO2/FiO2 \<150mmHg) and those without severe respiratory failure (PaO2/FiO2 \>150mmHg) Furthermore, they will be divided into those with or without deterioration risk defined as need for high flow nasal cannula, mechanical ventilation, ICU admission or death.

Type of study: This will be a prospective observational study that will be carried out at the Pulmonology/Covid-19 Department of the University General Hospital of Larissa.

Power analysis: For a statistical power of 90% with a probability of α-error of 0.05 and Group 1/Group 2 ratio 2:1, it was estimated that 64 people are needed in the control group (group 2) and 128 in the study group (group 1).

Interventions: Blood samples will be collected at admission for measurement of IMA and SuPAR levels and at day 10 from symptom onset (peak of symptoms) for IMA level.

Blood sample collection: Samples will be collected in blood collection tubes containing K2EDTA as anticoagulant and serum separation gel tubes for SuPAR and IMA assays respectively. The samples will be centrifuged (at 3000 x g for 10 minutes) within the first 3 hours of sampling. Then, both plasma and serum samples will be placed in Eppendorf tubes and stored at -80oC.

IMA and SuPAR measurements: IMA levels will be determined according to the principles of the ACB assay, using the commercially available "Ischemia Modified Albumin Assay Kit" method (Abbexa LTD, Cambridge, UK), on the Architect c8000 automatic analytical system (Abbott, USA). SuPAR levels will be determined by the suPARnostic TurbiLatex quantitative turbidimetric immunoassay (ViroGates A/S, Denmark), also on the Architect c8000 automatic analytical system.

Monitoring/Recording: Demographics (age, sex, nationality), date of admission, comorbidities, medication, symptoms, vital signs (heart rate, temperature, blood pressure, respiratory rate, arterial blood oxygen saturation and PaO2/FiO2 ratio), arterial blood gases (PaO2, PaCO2, pH, HCO3), lactic acid, viral load from RT-PCR for SARS-COV2 and the findings from the imaging and laboratory test (hemoglobin, hematocrit, white blood cells, neutrophils, lymphocytes, platelets, PT, INR, aPTT, fibrinogen, d-dimers, CRP, ferritin, LDH, troponin, urea, creatinine, AST, ALT, ALP, CPK, potassium, sodium, calcium, total protein, albumin, total bilirubin) will be recorded on admission.

Vital signs (heart rate, temperature, blood pressure, respiratory rate, arterial blood oxygen saturation and PaO2/FiO2 ratio), laboratory findings (as mentioned above) and the presence of any complications (pneumonia, ARDS, pulmonary embolism, stroke, acute renal failure, multiorgan failure, arrhythmias, shock, pseudomembranous colitis, etc.) will be recorded daily.

Expected results: i) IMA levels are expected to be higher in patients with COVID-19 compared to healthy volunteers ii) IMA levels at admission are expected to be higher in patients that will develop severe respiratory failure or with deterioration risk iii) IMA levels at day 10 from symptom onset are expected to be higher in patients with severe respiratory failure and predict deterioration risk with a greater accuracy iv) SuPAR levels are expected to be higher in patients with COVID-19 compared to healthy volunteers v) SuPAR levels at admission are expected to be higher in patients that will develop severe respiratory failure or with deterioration risk vi) Combination of IMA and SuPAR, is expected to increase the predictive accuracy of SuPAR

To our knowledge, this will be the first study of Ischemia Modified Albumin in patients with COVID-19. Prognosis is a key driver of clinical decision-making. If the correlation between IMA levels and poor prognosis is proved, it will become a useful tool for the risk stratification of COVID-19 patients. Furthermore, the combination of an ischemia biomarker, such as IMA, with an inflammatory biomarker, like SuPAR might be the key to this approach.

Eligibility Criteria:
Inclusion Criteria:

* \> 18 years old
* Positive nasopharyngeal test for SARS-CoV-2 confirmed by RT-PCR

Exclusion Criteria:

* Age \< 18 years old
* SARS-CoV-2 infection not confirmed by RT PCR
* No consent for participation in the study
* Acute ischemic disease prior to SARS-CoV-2 (trauma, mesenteric ischemia, stroke, liver disease, venous thromboembolic disease, acute coronary syndrome in the last 3 months, etc.)
* Pregnancy
* Immunosuppression
* Albumin \< 2gr/dl or \> 5.5gr/dl