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Platelet Indices and Platelet to Lymphocyte Ratio (PLR) as Markers for Predicting COVID-19 Infection Severity

Background Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (or coronavirus disease 2019; COVID-19) has caused a large number of infections across the globe. Numerous markers are being used to predict the severity of infection. This study was undertaken to assess the utility of platelet count, mean platelet volume (MPV), platelet distribution width (PDW), and platelet lymphocyte ratio (PLR) as markers of severity and mortality among patients with COVID-19 infection. Methodology This is a retrospective study conducted in a tertiary care center in India from April 2021 to June 2021. Patients admitted with COVID-19 infection were included in the study. Based on the severity, patients were categorized into the mild and severe (moderate severity included) groups. Platelet count, MPV, PDW, and PLR done at admission were studied and correlated with the disease severity and mortality. Statistics The independent t-test was used to compare the variables. The receiver operating characteristic (ROC) curve was done to identify the cut-off value. Statistical analysis was performed using SPSS 18 software (SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago: SPSS Inc). Results One hundred patients admitted with COVID-19 infection were studied. 51 patients had a mild and 49 had a severe infection. The mean PLR was 141.40 among patients with mild illness and 252.6 with severe infection (P<0.001). The mean PLR among survivors was 104.4 (SD-23.56) and among nonsurvivors was 302.78 (SD-34.5) (P<0.001). There was no statistically significant difference between the two groups with respect to platelet count, MPV, and PDW. Conclusion PLR was found to be a reliable marker of severity and mortality among patients with COVID-19 illness.


Introduction
Novel severe acute respiratory syndrome coronavirus 2 (or coronavirus disease 2019; COVID-19) has caused a large number of infections and mortalities worldwide. A dysregulated immune response and cytokine storm is the underlying mechanism that determines the adverse outcome [1]. Numerous inflammatory markers can predict severity and mortality among patients with COVID-19. Platelet count, mean platelet volume (MPV), platelet distribution width (PDW), and platelet lymphocyte ratio (PLR) have been studied as markers of severity and mortality among patients with COVID-19 [2]. PLR is a marker of inflammation, which is economical and available in most clinical settings. PLR is a ratio between the absolute platelet count and absolute lymphocyte count. PLR has been used as a marker of inflammation in cardiovascular diseases and autoimmune diseases [2,3]. Platelet count and its variations are associated with an increased risk of severe disease and mortality in patients with COVID-19 and are a clinical indicator of worsening illness during hospitalization [4]. High PLR has been demonstrated as a good marker of severity and patients having elevated PLR had a longer duration of hospitalization. In COVID-19, the decrease in lymphocyte count is more marked than the decrease in platelet count. This explains the increase in PLR in COVID-19 infection [5]. MPV and PDW can be used as auxiliary tests in predicting mortality among patients with COVID-19 in a few studies. There is a paucity of such studies done in India. Hence, this study was undertaken to assess the utility of platelet count, MPV, PDW, and PLR as early markers of severity among admitted patients having COVID-19 infection in a tertiary care center in South India.

Materials And Methods
This is a retrospective study conducted in a tertiary care hospital in South India. The study was done after obtaining approval from the Institutional Ethics Committee of Ramaiah Medical College (MSRMC/EC/SP-07/04-2021). All procedures were carried out as per the Declaration of Helsinki 2013. Consecutive patients being admitted to the hospital suffering from COVID-19 infection were included in the study. A total of 100 adult patients (aged more than 18 years) were included in the study. COVID-19 infection was confirmed using reverse transcriptase polymerase chain reaction (RT-PCR COVID-19) on a throat swab. Patients suffering from any hematological diseases like leukemia, lymphoma, myelodysplastic syndrome, or idiopathic thrombocytopenic purpura, patients on chemotherapy, and patients on antiplatelet drugs were excluded, as these can alter the platelet count and platelet indices. As per Indian Council of Medical Research guidelines, patients with mild COVID-19 illness were those having symptoms of upper respiratory tract infection and/or fever without shortness of breath, moderate COVID-19 illness includes patients with a respiratory rate more than 24 per minute or oxygen saturation less than 93% on ambient air, and severe illness are those patients whose saturation is less than 90% on ambient air or respiratory rate more than 30 per minute [6]. For the purpose of the study and analysis, these patients were categorized into two groups, mild illness and severe illness (which included patients with moderate and severe illness). The data regarding these patients were extracted from the files and electronic health records. Care was taken not to collect or disclose the patients' personal information. The clinical profile and laboratory data were studied. Complete blood counts, platelet count, MPV, PDW, and PLR, which were done at the time of admission were collected. The high-resolution computed tomography (HRCT) of the chest of the patients was also studied. The HRCT was reported by a radiologist. The correlation of platelet count, PLR, MPV, and PDW at admission with the severity and mortality of the illness was studied. Hemoglobin estimation was done using sodium lauryl sulfate detection, total leucocyte count estimation by a Sysmex device (Sysmex Corporation, Kobe, Japan), peripheral smear examination, C-reactive protein (CRP), and D-dimer measurement was done by immune-turbidometry.

Sample size with justification
A study conducted by Qu et al. has observed that the mean platelet to lymphocyte ratio among non-severe patients was 262.27 +/-97.78 and among severe was 627.27 +/-523.64, which was found to be statistically significant [4]. The present study expected similar results with 80% power, 95% confidence level, and an effect size of 0.64. The study required 98 patients; 49 in each group.

Statistical methods
Descriptive statistics of platelet count, lymphocyte count, PLR, and platelet indices were analyzed in both groups and summarized in terms of mean with standard deviation. The independent t-test was used to compare mean platelet count, MPV, PDW, and PLR between the two groups. The receiver operator characteristic (ROC) curve was constructed to identify the cut-off value, and the area under the curve (AUC) and the sensitivity and specificity of hematological parameters in predicting severity and mortality were analyzed. Correlation between parameters was done using Pearson's correlation coefficient. Statistical analysis was performed using SPSS 18 (SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago: SPSS Inc.).

Discussion
The above study was conducted to study the utility of platelet count, MPV, PDW, and PLR as markers of severity of COVID-19 illness. The results of the above study indicate that PLR at admission was higher among the patients having severe COVID-19 illness as compared to the patients having a mild illness. PLR and NLR values had a significant positive correlation with CT severity scores. It was also observed that PLR at admission was found to be significantly elevated among patients who succumbed to COVID-19 infection as compared to patients who survived. The above study did not find any statistical difference in platelet count, mean platelet volume, and PDW among mild and severe illness.
The findings of the present study were similar to the study done by Simadibrata DM et al., which showed that the patients with severe COVID-19 illness had higher PLR levels on admission (SMD 0.68; 95%CI 0.43-0.93; I2 =58%) [7].
Similar findings were observed in a study conducted by Rong Qu et al. which showed that values of PLR done at the highest platelet count were an independent factor that inversely influenced the outcome in patients with severe COVID-19 illness [4].
In the present study, PLR and NLR had a positive correlation with CT severity scores, which was statistically significant. These findings were concordant with the study conducted by Man A et al., which demonstrated that both NLR and PLR had a positive correlation with the radiological severity of COVID-19 illness as per CT severity scores. In this study, NLR was 2.90 and PLR was 186, having a good specificity and positive correlation with the CT severity score (0.89, p = 0.001; 0.92, p<0.001, respectively) [8].
Similarly, the study conducted by Eslamijouybari  Sarkar et al. retrieved 32 studies of COVID-19 patients for outcomes. It was seen that critically ill patients and nonsurvivors had higher PLR levels on admission in comparison to survivors and non-severe patients [14].
As per the results of the study conducted by Giuseppe Lippi et al., patients having severe COVID infections have significantly low platelet count [5]. However, in the present study, it was seen that patients with severe COVID had slightly low platelet count as compared with patients having a mild illness. But the difference was not statistically significant.
As per the study conducted by Seyit M et al., the CRP (p = 0.0001), lactate dehydrogenase (LDH) (p=0.038), PLR (p=0.0001), and NLR (p=0.001) were significantly higher in patients with a positive SARS-CoV-2 PCR test result [15]. Similar findings were also noted in the current study.
PLR is a ratio between the absolute platelet count and the absolute lymphocyte count. In COVID-19 illness, the decrease in lymphocyte count is more marked than the decrease in the platelet count. This explains the increase in PLR in COVID-19 infection. SARS-CoV-2 infection can induce pyroptosis in lymphocytes through NLRP3 inflammasome activation. This mechanism leads to lymphopenia of COVID-19 illness. Cytokine IL-6 utilizes lymphocytes and can further reduce lymphocyte counts. Thus, an increase in PLR can be associated with severe COVID-19 illness [16].
The limitation of the study was that the sample size was small. Long-term follow-up and prospective studies will be required to understand the utility of these markers in COVID-19 infection.

Conclusions
This study demonstrates that PLR can be used as a reliable marker of severity and mortality among patients with COVID-19 infection. PLR has a positive correlation with CT severity score. PLR is easily available as an economical marker and can be utilized in resource-limited settings. MPV, PDW, and platelet count were not found to be good markers of the severity of COVID-19 infections.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Ramaiah Medical College issued approval MSRMC/EC/SP-07/04-2021. The study was conducted after obtaining clearance from the Institutional Ethics Committee of Ramaiah Medical College, number (MSRMC/EC/SP-07/04-2021). Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: The study received academic grants from Rajiv Gandhi University of Health Sciences, Bangalore. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.