Evaluation of hematological parameters as an indicator of disease severity in Covid‐19 patients: Pakistan's experience

Abstract Background The severity of COVID‐19 could be evaluated by examining several blood parameters mainly white blood cell (WBC) count, granulocytes, platelet, and novel hemocytometric markers neutrophils to lymphocyte ratio (NLR), platelet‐to‐lymphocyte (PLR), and lymphocyte to monocyte ratio (LMR). The current study was conducted to investigate alteration in blood parameters and their association with the severity and mortality of COVID‐19 patients. Methodology An observational cross‐sectional study was conducted retrospectively, a total of 101 COVID‐19 positive patients were examined: 52 were mild, 24 were moderate, 09 were severe, and 16 were critically diseased patients. We also recorded 16 deaths associated with the critical group. The overall mean age observed in our study was 48.94 years, where the mean age for critical individuals was 62.12 ± 14.35 years. Results A significant association between the disease severity and elevation in blood parameters were observed. The WBC's and granulocyte count were significantly increased (p value <0.001) while the mean platelet count (165.0 × 109/L) and red blood cell volume distribution width (RDW) were decreased in the critical group (57.86%) compared to mild group's patients (177.3%) (p = 0.83). The lymphocytes count was decreased in critical patients (1.40 × 109/L) compared to mild patients (1.92 × 109/L) (p = 0.28). A significant association was observed in platelet‐lymphocyte ratio (p < 0.001), Neutrophil‐Lymphocyte ratio (p = <0.001), and Lymphocyte‐Monocyte ratio (0.011). Conclusion These blood parameters could be used as a suitable biomarker for the prognosis and severity of COVID‐19. Evaluating novel hemograms NLR, PLR, and LMR can aid clinicians to identify potentially severe cases at early stages, initiate effective management in time, and conduct early triage which may reduce the overall mortality of COVID‐19 patients.


| INTRODUC TI ON
The coronavirus disease 2019  caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) was first described in the late December 2019 in Wuhan, China. 1,2 The COVID-19 is a high contagious disease and spread around the globe within a short time, and the world health organization (WHO) has declared it a pandemic on March 12, 2020. 3 COVID-19 has varied clinical manifestations, ranging from mild flu-like symptoms and asymptomatic phase to life-threatening acute respiratory distress syndrome (ARDs), and organ failure that may lead to death. 4 The initial symptoms of COVID-19 are shortness of breath, cough, fatigue, fever, dyspnea, myalgia, and muscle pain.
However, they could progress to acute respiratory distress syndrome (ARDS), multi-organ dysfunction, shock, and metabolic acidosis when the condition becomes worsened. 5  disease. 8 Different respiratory factors such as oxygen saturation rate, lesion progression in pulmonary rate, and respiratory rate were the key factors considered during the classification of the severity levels of COVID-19. 9 Patients with severe COVID-19, mostly critical individuals, usually have dysfunction complications of other organs, such as shock, septic, disseminated intravascular coagulation (DIC), and heart failure. In critical cases, some thrombotic complications such as strokes, venous thromboembolism, and ischemic limbs have been reported. 10 Except for clinical symptoms and pulmonary computed tomography (CT) findings, most confirmed COVID-19 patients revealed laboratory fluctuations in different serological parameters, including renal and liver function tests, coagulation parameters, and inflammatory, biochemical and hemocytometric parameters. 9,11,12 To show the prognosis and hyperinflammation state, a combination of laboratory tests has been evaluated. The combination of the various test includes platelet-to-lymphocyte (PLR) and neutrophils to lymphocyte ratio (NLR). COVID-19 leads to variation in the hematological parameters, including lymphocytes, white blood cells, platelets, neutrophils, etc. [12][13][14] These variations are different from case to case and level of the disease severity. Lymphopenia has been previously reported in about 35%-85% of patients and was the most common blood count abnormality. [10][11][12] Moreover, knowledge about the risk of infections along with other comorbid conditions would provide valuable insights on risk stratification and making a clinical decision in severe COVID-19 patients. 15 According to different studies, NLR in severe patients was raised as compared to those with mild or moderate disease. 4,16 Further, Liu et al 16 found out in hospitalized patients that NLR serves as an independent risk factor for mortality. CBCs are the most appropriate and potent laboratory examination. The main objective of this research was to evaluate and review the variations among CBC level of COVID-19 patients with the disease severity level and how the CBC level changes after the onset of disease to identify the stage and disease's key indicator to provide information for the diagnosis and treatment basis for health professionals.
Moreover, this study aimed to explore novel inflammatory markers NLR, PLR as a valuable marker in predicting severity and outcome of COVID-19 infection as they were previously reported in different infections and inflammatory conditions. 17 2 | MATERIAL S AND ME THODS

| Study design
A retrospective cross-sectional study was conducted from May 2020 to September 2020 in the Pathology Department of Sahiwal Medical College, Sahiwal.

| Patients
Our study participants include n = 101 patients who were tested

| Exclusion and inclusion criteria
All those individuals who were tested positive for the SARS-CoV-2 according to the WHO and CDC guidelines for the detection and diagnosis of COVID-19 were included in this study. Individuals below 18 years of age and those with missing data were excluded.

| Neutrophil-to-lymphocyte ratio
Neutrophil-to-lymphocyte ratio is determined by dividing the relative percentage of neutrophils by lymphocytes. In normal individuals, it should be <3 but a ratio of >3 signifies acute infection, and a ratio of more than nine reveals sepsis. But when it comes to the cut-off value, there is a lot of diversity of NLR in populations, with some studies suggesting a cut-off value of 4. 18 The NLR of our patients was also analyzed as shown in Table 3 and Figure  Interestingly, the study shows that systolic blood pressure and NLR were independently associated with sHT. Hence, NLR at admission can accurately predict sHT in patients with AIS undergoing revascularization. 22 It can also as a diagnostic tool in inflammatory bowel disease. 23 This ratio has also been considered as a determinant of outcomes of various malignancies and coronary arterial diseases as well as patients suffering from recurrent ulcerative colitis. All these conditions are associated with overt inflammation as in COVID-19.

| Platelet-to-lymphocyte ratio
Absolute platelet counts (×10 9 cells/µl) hence divided by absolute lymphocyte count (×10 9 cells/µl), gives this ratio and it usually 50-150 but it also has a great diversity amongst people. 7 In this study, PLR levels seem to be independently associated with an unfavorable outcome and death after three months. PLR is also identified as a diagnostic tool in inflammatory bowel syndrome. 23 Similarly, Mean Platelet volume to lymphocyte ratio (MPVLR) was also found to be associated with frailty in type 2 diabetic individuals. 23

| Statistical analysis
Different statistical tools were used for the analysis of data.  Comorbidities were also mentioned for also each group and a statistically significant difference was observed between mortality and disease severity as shown in Table 2.
The red cells distribution width was significantly decreased Chi-square test was applied and p < 0.05 was considered as significant at 95% confidence of interval.

F I G U R E 3
Platelets count (10 9 cells per L) in various diseased groups. Chi-square test was applied and p < 0.05 was considered as significant at 95% confidence of interval.

F I G U R E 4
Association of platelet count (10 9 cells per L) with gender. Chi-square test was applied and p < 0.05 was considered as significant at 95% confidence of interval.  Table 3. A significant increase was also observed in NLR in various disease groups. The highest value of NLR was recorded in critically ill patients (6.87) followed by severe, moderate, and mild disease groups. A significant association (p value = <0.001) was observed in various disease groups as shown in Table 3. Similarly, a decrease in the LMR was observed in the critical disease group patients compared to other groups. The lowest value of LMR was recorded for critically ill patients (4.37). A significant association (p value = 0.011) was observed in the critical disease group as compared to other groups. ROC curves reveal the relative diagnostic performance of hematological parameters and age, forecasting the severity of disease in patients with SARS-CoV-2 infection, WBC, white blood cell; NLR, neutrophil-to-lymphocyte ratio; PLR, platelet-to-lymphocyte ratio; LMR, lymphocyte-to-monocyte ratio as shown in Figure 1.

| DISCUSS ION
The second wave of COVID-19 has been started and cases are steadily increasing around the globe. There is a need to assess the disease severity and mortality risk associated with COVID-19 in the current pandemic for the optimal management of the patients. 29 Several inflammatory and hematological parameters are thought to associate with the severity of COVID-19. 18,24 The current study was aimed to examine the different blood parameters in the confirmed positive COVID-19 patients. We analyzed several blood parameters and their association with the severity of the COVID-19 disease. In this study, we classified the patients into different categories based on disease severity. The patients were categorized into mild, moderate, severe, and critical groups. Among the total 101 patients, 52 were mild, 24 were moderate, 09 were severe and 16 were critical diseased patients. We recorded 16 deaths that were associated with the critical group.
Among the previously reported mortality predictors for COVID-19, the increasing age was an important factor and associated with the poor outcomes. 12,25 The mean age for deceased individuals in our study was 62.12 ± 14.35 years which was greater than any other diseased group (represented in Table 4). The statistical analysis represented a significant association between the increased age and mortality due to COVID-19. There was no association between the severity of COVID-19 and gender in our study.
Similar to our study, Hypertension, senility, diabetes, heart disease, COPD, and asthma were reported in different studies as predictors for severe COVID-19. 30,31 A statistically significant difference was shown between comorbid conditions and mortality as shown in Table 2. A significant increase in the WBCs level in the critical and diseased individuals was observed. It has been previously reported that the WBCs count increased with the severity of the COVID-19 disease. 26 We observed a decrease in the lymphocytes in severe and critical patients. Based on our observation, it could be speculated that the lymphocytes count depletion is directly associated with the COVID-19 disease severity and the survival rate of the disease could be linked with the ability of T lymphocytes which are essential for the destruction of infected viral particles. 32 Our observation supports the previous investigations which documented differential diagnostic criteria for COVID-19 patients based on the increased WBC count along with lymphopenia. 33 Thrombocytopenia was increased with the severity of the disease and the lowest value of platelets was recorded for critical patients. Our findings are consistent with the previous studies in which thrombocytopenia was associated with severely diseased individuals and dead patients. Therefore, thrombocytopenia could be used as a useful indicator for disease progression. 33 The mechanism of F I G U R E 5 Granulocytes level (10 9 cells per L) in various diseased groups. Chi-square test was applied and p < 0.05 was considered as significant at 95% confidence interval. The critically diseased individuals represented the highest level of granulocytes as compared to other diseased groups Examination of the platelet could be a suitable biomarker for recognition of coagulopathy and its severity.
The lymphocytes functions testing has been suggested to assess the severity of COVID-19 disease. 4 We observed the decreased lymphocytes count and increased granulocytes in the critical diseased individuals which could be attributed to increased inflammation and suppression of the immune system caused by

| Limitations of the study
The present study has several limitations, particularly the very small sample size. Therefore, exact implication of our findings on a large scale population could not be a good practice. Importantly, this is the first study from Pakistan to the best of our knowledge and our findings could be very helpful for policy makers in the current pandemic. The cases of COVID-19 are surging as the second wave has been started globally. We, therefore, believe that the current study could provide more important literature while dealing with the cases of COVID-19 in the overburdened hospitals. 44,45 All the subjects in our study were Pakistani nationals and therefore our findings could hardly be inferred to other ethnic groups.
Further, we only reported hospitalized patients in our study. Nonhospitalized individuals, and their clinical characteristics were not studied. Further exploration of the topic is urgently needed to provide optimal management to the patients during the COVID-19 pandemic.

| CON CLUS ION
Several blood parameters need to be continuously screened in the current pandemic to assess the severity and mortality of COVID-19. Thes parameters such as lymphocytes, white blood cells, and platelet count could be used as suitable biomarkers for prognosis of COVID-19 disease. The elevation in the mentioned parameters can be easily used for severity and mortality analysis of COVID-19 as routine blood tests are easy and readily available. The topic needs to be explored further for the early target of treatment options and optimal management of the COVID-19 patients during the pandemic. Evaluating novel hemograms NLR, PLR, and LMR can aid clinicians to identify potentially severe cases at early stages, initiate effective management in time, and conduct early triage which may reduce the overall mortality of COVID-19 patients.

CO N FLI C T O F I NTE R E S T
The author has no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm that the data supporting the findings of this study are available within the article.