Analysis of Clotting Factors in Covid 19: A Study in indian Patients in a Tertiary Hospital

Coronavirus 2019(Covid 2019) is a global pandemic and may trigger coagulation dysfunction with extensive micro thrombosis. This study was conducted to evaluate the basic coagulation parameters in symptomatic patients with and without SARi in Covid.it was a prospective comparative cross sectional study to study the prognostic role of these markers in patients with and without SARi (severe acute respiratory illness) and survivors versus non survivors. demographic characteristics , detailed medical history and platelets, prothrombin time (PT),activated thromboplastin time (APTT), fibrinogen and d dimer was recorded and analysed in both these groups. The independent group t-test and Mann -Whitney U test was used to analyse continuous variables. RoC was plotted for significant variables to obtain area under curve. The average PT for survivors was 14.6s and non survivors was 29.4s and the difference statistically significant. The area under curve for PT was 0.751 and at a cut off value of 13s had a sensitivity of 75% and specificity of 62.5% for predicting severe Covid with SARi. The median value for aPTT for non survivors was 35.5 (iQR 32.5-42.1) and for survivors it was 31.9 (iQR 29.5-35.7) and was significant. The mean values of d dimer for patients without and with SARi was 384 and 2168 mcg/ml and the difference was statistically significant (p=0.00). The d dimer test was the single most test distinguishing survivors and non survivors with an AUC of 0.844.The levels of fibrinogen and CRP was higher in patients with severe Covid and was statistically significant (p=0.001) and (p=0.028).The platelet count was lower in patients with severe Covid but difference was not statistically significant. The basic coagulation markers have a prognostic significance in treatment of Covid atients with and without SARip.

C o r o n a v i r u s e s a r e e n v e l o p e d nonsegmented positive stranded RNA viruses belonging to the family Coronaviridae and the order Nidovirales. 4The virus spreads through direct contact of infected surfaces or through human to human contact through air borne droplet spread. 5,6he virus has a mean incubation period of 5.2 days. 7Most common symptoms include nausea, vomiting, fever, fatigue, dry cough, dyspnoea, myalgia, diarrhea, sore throat, rhinorrhea and chest pain. 8,9,10Increased mortality in COVID patients is related to severe symptoms like pneumonia, acute respiratory distress syndrome and multi organ failure. 11,12In severe cases, patients may develop acute respiratory distress syndrome (ARDS), with coagulation predominant-type coagulopathy. 13he coronavirus causing COVID-19 may trigger coagulation dysfunction because it induces abundant release of pro-inflammatory cytokines in various tissues, which can lead to systemic inflammatory response syndrome that damages the microvascular system and thereby activates the coagulation system, leading to generalised small vessel vasculitis and extensive microthrombosis1. 14,15Patients with severe COVID-19 may be at high risk of venous thromboembolism, which may be present in up to 25% of such patients. 16Risk may be exacerbated by the dehydration due to fever and diarrhea, hypotension, and prolonged bed rest characteristic of the disease, all of which are risk factors for coagulation. 17Chinese studies have revealed markedly prolonged PT/APTT and elevated D dimer in nonsurvivors in comparision to COVID 19 survivors. 13,18The coagulation parameters have not been completely reported in patients with and without SARS in Indian literature so far.Hence this research project was undertaken to ascertain the differences in coagulation parameters in symptomatic patients with and without SARI.Aims and objectives 1.To evaluate the basic coagulation parameters in symptomatic patients with and without SARI 2. To study the prognostic role of the basic coagulation parameters in both the clinical forms of disease.

MATERIALS AND METHODS
This study was a prospective comparative cross sectional study which was undertaken in COVID patients admitted to Father Muller Medical College Hospital over a period of one year from April 2020-April 2021.The Institutional Ethics Committee clearance and prior written informed consent was obtained from the study participants.
The sample size was 96 (48 in each group)

Inclusion criteria
Adult COVID patients admitted to ward and ICU of this hospital with symptoms of nausea, vomiting, fever, fatigue, dry cough, dyspnoea, myalgia, diarrhea, sore throat, rhinorrhea and chest pain.

Exclusion criteria
Asymptomatic COVID patients, pregnant COVID patients and paediatric COVID patients.
At the time of admission to the hospital, blood sample was collected for platelets, prothrombin time (PT), activated thromboplastin time (APTT), fibrinogen and D dimer.Demographic characteristics, detailed medical history and basic coagulation was recorded and was analysed.Platelet analysis was done in Beckman Coulter LH 750.PT, APTT and fibrinogen was analysed in ACL TOP 500 and ACL TOP 300.D dimer was analysed by an automated latex enhanced immunoassay on the ACL TOP.The COVID patients was divided into 2 groups according to presence or absence of severe SARI requiring ICU care The clotting factors was compared between these 2 groups of patients and also survivors and non survivors .The case definition of severe SARI was defined as those patients requiring ICU care with saturation less than 90 % on room air requiring non-invasive or invasive ventilation.

Statistical analysis
The sample size of 96 was calculated based on the standard deviation 151 of fibrinogen in admitted COVID patients and standard deviation of 83 in outpatient (in reference to previously done study), mean difference of 70, alpha error 5% for 80% power, 2 sided test, sample size required for each group is 48. 19This was calculated using Master 2 software, CMC, Vellore.
Data was analysed using unpaired t test if the results follow normal distribution or else Mann Whitney test will be used.P<0.05 is considered to be significant.Data was analysed using SPSS version 20.

Statistical analysis
Continuous variables were expressed as mean and SD or median and IQR The independent group t test was used to analyse normally distributed continuous variables, and the Mann-

RESULTS
In our study the values of the levels of all five parameters were elevated in non survivors as compared to survivors.

PT
On admission the mean level of PT in patients with and without SARI 12.8s and 21.3s.When interpreted with INR the mean values were 1.1 and 1.8.The difference in the mean values apart from being statistically significant (p=0.006) and also clinically different with moderate patients having a mean value within normal range for our laboratory(10-13s) and the severe cases well above that.Our finding is similar to Song JC 15 et al who hypothesized that these are due to liver involvement as there was significant abnormality in the liver enzymes of patients with COVID.
The average PT for survivors was 14.6 and the non survivors was 29.4s and the difference was statistically significant.Area under the curve for PT (survivors non survivor)was 0.751 and at a cut off value of 13s PT alone had a sensitivity of 75 % and a specificity of 62.5% for predicting those with severe covid.A cut off value of 16s had sensitivity to 39.6%.specificity of 95.8 % in predicting death This finding is similar to those described by Cui S et al 16 in whose study the multivariate regression analysis showed the difference in PT inpatients with SARI who survived and did not survive was not significant.

Area under the curve
The prolongation of PT is understandable.One of the mechanisms of COVID induced coagulopathy (CIC) is the over expression of tissue factor (TF) by the endothelial cells secondary to the cytokine release.Binding activation and consumption of factor VII by TF is probably the main reason for this.It can be inferred that the consumption of factor VII occurs more severely in SARI than in non SARI cases due to stronger cytokine response in them.aPTT The difference in aPTT among the moderate and sever cases was not statistically significant.This finding is similar to other studies.However the difference was significant when aPTT was survivors was compared with non survivors.(p=0.003)The median value for non survivors was 35.5 (IQR 32.5-42.1)and for survivors it was 31.9 (IQR 29.5-35.7)This finding is similar to that of study done by Huang C 11 et al.It has been suggested that CIC be categorized into three stages beginning with stage of high d-dimer and nerar normal PT and aPTT, followed by increasing PT and aPTT with high dDimer and finally with features of Classic DIC as described by International Society of Thrombosis and Hemostasis (ISTH) It is possible that the non survivors in our case had progressed to second stage as compared to our survivors .It is interesting to note that the difference in the aPTT between survivors with and without SARI was also not significant as well as between the SARI survivors and non survivors.33.9 cut off 21 severe patients and 18 moderate patients >33.9s in 11/16 non survivors(68.5%)36.25% (of survivors 29/80) D Dimer (D-DU) (non survivor vs survivor and Non Sari vs Sari).The mean values of D dimer for patients without and with SARI as 384 and 2168 mcg/ml and the difference was highly significant.The IQR of D dimer in survivors and   21 .Elevated D-dimer level is a sign of excessive coagulation activation and hyperfibrinolysis which is a significant predictor of mortality due to venous thromboembolism both of the deep veins and in the pulmonary circulation the prevalence of which ranges from 0-54% as shown in a study by Suh et al 22 The mean levels of fibrinogen values in our study in moderate and severe cases was 458 and 621 respectively and the difference was significant (p =. 001) .The CRP The levels of fibrinogen and CRP was higher in patients with severe COVID and the difference was statistically significant whereas platelet values were lower but difference was not significant

DISCUSSION
The aim of the cross-sectional study was to analyze the association between the coagulation parameters on admission with the severity and survival of adult COVID patients as thromboembolism and disseminated Intravascular Coagulation are an important cause of morbidity and mortality among COVID patients.
Early on it was discovered that the mechanism for development of DIC in patients with COVID differed from those commonly encountered during sepsis.It is believed that the release of cytokines especially the Interleukin -6(IL-6) is major contributor for development of macrovascular as well as microvascular thrombosis.The mechanism appears to be dual:1.IL -6 increases the endothelial expression of Tissue Factor, ii) Sars Co-V 2 by itself produces widespread endothelitis and also pyroptosis and apoptosis of endothelial cells.
The coagulation cascade activation that occurs I Covid can be identified by prolongation of Prothrombin Time, Activated Partial Thromboplastin Time (aPTT).The fibrinolysis that follows is represented by increased levels of D-Dimer and Fibrin Split Products (FSP).The other conventional laboratory parameters that are useful in monitoring of Covid patients is levels of In our study the values of the levels of all five parameters were elevated in non survivors as compared to survivors On admission the mean level of PT in patients with and without SARI 12.8s and 21.3s.When interpreted with INR the mean values were 1.1 and 1.8.The difference in the mean values apart from being statistically significant (p=0.006) also clinically different with moderate patients having a mean value within normal range for our laboratory (10-13s) and the severe cases well above that.Our finding is similar to Zhang et al 23 who hypothesized that these are due to liver involvement as there was significant abnormality in the liver enzymes of patients with COVID.
The levels of fibrinogen and CRP was higher in patients with severe covid and the difference was statistically significant whereas platelet values were lower but difference was not significant.
Platelet values are known to be lower in patients with severe Covid and multiple mechanisms are proposed beginning with suppression of bone marrow to increased consumption.Lower platelet counts are associated with more severe disease and are useful if monitored serially 24 .Since we did not follow up platelet counts and it may be the reason that it was not statistically significant.In our study 20 severe and 6 moderate cases had levels below 1,50,000 Both fibrinogen and CRP elevation can be explained by the fact that they are acute phase reactants.Elevation of fibrinogen could also be an indicator of prothrombotic state as shown in study done by Thachil J 25 and in conjunction with elevated CRP gives credence to the connection between inflammation and coagulation.

CONCLUSION
Basic coagulation markers may have a prognostic implication in the treatment of Covid patients with and without SARI.

Table 1 .
Group statistics

Table 2 .
Group statistics

Table 6 .
The D Dimer test was the single most important test distinguishing survivors and non Survivors with an AUC of.0.844.At values if 382 the sensitivity and specificity was 93.8 and 51, 2. A cut off of 1000 ensured a 81.3 and 81.2% specificity.At 1711 it was 56.3 and 90% respectively.Our findings is similar to Gao Y.D. et al 20 also is the most common hematological abnormality reported in COVID-19 in a study done Asakura H, Ogawa H