Association between Hematological Parameters and Severity of COVID-19 disease

Introduction

In early December 2019, the world saw the beginning of an epidemic of the new coronavirus disease 2019 (COVID-19), due to the newfound virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 later evolved into a pandemic that affected billions of people worldwide. Measures taken for social distancing and the impact of the disease on socio-economic status, morbidity, and mortality have proven to be a major burden on millions of people all over the world.¹

India saw its first case in Kerala in late January 2020.² India with a total of 430422 active cases as of 16/07//2021 as per the Ministry of Health and Family Welfare, Government of India website.³ Mortality rates due to the disease have varied from 3% in China to an increase by 145% in some cities in Mexico.^4,5 India has a mortality rate of 1.33% as of 16/07/2021.³ A resurgence has been noted with JN1 as the new variant with high immune evasion properties.⁶

JN.1variant has an additional spike protein compared to its close relative BA.2.86. This could increase the infectivity of the virus and has consequently become the dominant strain in several countries.⁷ This rise of new variants also highlights the importance continuous monitoring as further variants can potentially arise with varying infectivity and virulence. Thus, simple tools for triaging and prognosticating COVID-19 are needed.

There have been several studies looking at the hematological paramaters in COVID-19. They have been used for diagnostic as well as prognostic purposes. Parameters studied include absolute neutrophil count (ANC), neutrophil lymphocyte ratio (NLR) and platelet lymphocyte ratio (PLR).⁸ Some of the abnormalities noted include anemia, leukocytosis, eosinophilia or eosinopenia and thrombocytopenia or thrombocytosis.⁹ Lymphopenia is among the most common findings.¹⁰ Neutrophilia is common especially among the severe cases, hence as a result of the above there is a high NLR.¹¹

Studies on hematological parameters of COVID-19 in our country are scarce. This study is a modest attempt to study the association of basic hematological parameters such as total leukocyte count, neutrophil-to-monocyte ratio (NMR), neutrophil to lymphocyte ratio (NLR), Systemic Inflammatory Index (SII), and platelet-to-lymphocyte ratio (PLR), which can be easily availed even in rural setups, with the severity of COVID-19, which might aid in the evaluation of patients and in detecting the progression to severe disease at the time of admission. This may even help in the judicious triaging of patients at the time of admission.

Methods

A prospective observational study was conducted by clinical examinations and lab investigation in the Department of Internal Medicine, at a tertiary care hospital, in Karnataka, India for a period of three months from 03/12/2020 to 25/03/2021.

The study was approved by our Institutional Review Board (IRB) known as Kasturba Medical College and Kasturba Hospital Institutional Ethics Committee (reference number: 740/2020). It was approved on 3^rd December, 2020 for a period of three months till 31^st March 2021. This study was conducted in accordance with the principles of the Declaration of Helsinki. Written informed consent was waved and telephonic verbal consent was taken citing restrictions during COVID-19. The same was approved by the Institutional Ethics Committee. The data collected were kept confidential.

Results

The demographic and baseline characteristics of the patients are shown in Tables 1 and 2. Among the 573 COVID-19 patients in our study, the majority were males. In the severe category, many patients in the ICU required mechanical ventilation and non-invasive ventilation. The rest were administered oxygen supplementation via a Venturi mask, non-rebreathing masks (NRBM) and nasal prongs. Comorbidities were observed in a large number of patients. Overlap of more than one chronic illness was unavoidable in some patients.

The median values of hematological parameters such as hemoglobin, total leukocyte counts, ANC, ALC, platelets, NLR, SII, NMR, and PLR were compared, and the statistically significant values are shown in Table 3. The hematological parameters were also compared between the survivors and non-survivors of the severe category, which are depicted in the same table. The mean hemoglobin values of the non-severe and severe COVID-19 categories were compared but were found to be statistically insignificant.

COVID-19 patients were categorized into two groups. The non-severe and severe categories consisted of 271 and 302 patients, respectively. Statistically significant differences were only observed in patient age, sex, and associated comorbidities. Associated comorbidities were further analyzed using multivariate logistic regression, is shown in Table 4.

Comorbidities, such as type 2 diabetes mellitus, essential hypertension, ischemic heart disease, and the abovementioned hematological parameters, which were statistically significant, were further subjected to logistic regression (Table 4). The hematological parameters between the survivors and non-survivors in the severe category of statistical significance were also subjected to logistic regression, but none of them were statistically significant. This analysis showed that type 2 diabetes mellitus, essential hypertension, NMR, and ALC were statistically significant independent predictors of COVID-19 severity while ischemic heart disease, as an independent predictor, had borderline significance.

As there was a statistically significant difference between non-severe and severe categories in the above-mentioned hematological parameters, they were further studied using Receiver Operating Characteristic (ROC) curve analyses. Regarding the observations made by ROC analyses, the following information concerning patients with COVID-19 diagnosis severity was found (Table 5 and Figure 1).

Figure 1. Receiver Operating Characteristic (ROC) curve for hematological parameters (a and b) with COVID-19 severity.

Discussion

This study was a prospective observational study that determined the mean age of the patients to be 55.82 ± 15.84 years. This conclusion aligns with earlier studies conducted in China and the USA.^13–15 However, the mean age in this study was slightly higher than that reported in an Indian cohort study conducted by Nitesh Gupta and colleagues in New Delhi.¹⁶ The prevalence of the condition was higher in men (61.6%) compared to women, which is consistent with the results reported by Fei Zhou and his colleagues in China.¹⁴ Richardson et al. conducted a study in the United States using a case series of 5700 patients. Their findings revealed that the death rate for males was consistently greater than that of females in every 10-year age interval beyond 20 years.¹⁵ A total of 85.9% of patients had comorbidities. Our study found a considerable prevalence of type 2 diabetes mellitus and essential hypertension, particularly in the severe category. Our study demonstrated a marginal level of significance indicating that ischemic heart disease can be used as a predictor of the severity of COVID-19. Nevertheless, research conducted in China and the USA revealed that patients with COVID-19 were more likely to have essential hypertension as a pre-existing condition, with additional comorbidities such as type 2 diabetes mellitus and coronary artery disease following suit.^13–15

The results of our investigation indicated a little elevation in the overall number of white blood cells and neutrophils in those with severe illness. This finding aligns with the retrospective study undertaken by Lin et al. and Rami M. Elshazli et al.^17,18 A study conducted by Agrawal et al. in Rajasthan, India, found similar results in 102 patients who had both symptomatic and asymptomatic cases of COVID.¹⁹ However, Guan and colleagues observed a higher incidence of leukopenia (33%) in the severe group compared to the non-severe group in their analysis of 1099 COVID-19 patients in China.¹³ Neutrophils, monocytes, and lymphocytes, which are diverse components of white blood cell counts, have distinct functions in the immune response within our body. Neutrophils, the predominant kind of white blood cells in the peripheral blood of humans, are now recognised as having a significant impact on safeguarding the respiratory epithelium by promoting the production of pro-inflammatory cytokines in the immediate area.²⁰ Nevertheless, it appears to have both positive and negative consequences, since excessive activation of neutrophils can lead to heightened inflammation, increased tissue damage, and hence exacerbate the disease.^20,21

The results of our investigation revealed lymphocytopenia in the severe group, accompanied by a significantly elevated median NLR (neutrophil-to-lymphocyte ratio) and PLR (platelet-to-lymphocyte ratio). W. Guan and his colleagues also observed this significant lymphocytopenia.¹³ Abhishek Agrawal and his colleagues in India observed a decrease in the average number of lymphocytes (20.58±0.87 vs 28.60±8.65), an increase in the average NLR (6.17±6.11 vs. 2.67±1.32), and an increase in the average PLR (146.29±88.90 vs. 115.47±47.88) among patients in the moderate to severely ill category, compared to asymptomatic and mildly symptomatic patients.¹⁹ The study conducted by Sha-Lin et al. in China demonstrated that an increased NLR (neutrophil-to-lymphocyte ratio) is a standalone predictive indicator for the severity of COVID-19.¹⁷ Lymphocytes are a specific type of white blood cells (WBCs) that play a crucial role in triggering adaptive immunity and the establishment of ‘self-tolerance’.^22,23

The cytokine storm typically occurs 1-2 weeks after the infection starts, when the virus moves through the circulation and primarily targets tissues with a high concentration of ACE2, the receptor for SARS-CoV-2. During this time, there is a noticeable decrease in lymphocyte count, known as lymphopenia.²⁴ Studies have demonstrated that lymphocytes in the blood express a limited quantity of ACE2 receptors on their surface. This is believed to be the reason of lymphopenia in COVID-19.²⁵ Monocytes are a distinct subset of white blood cells (WBCs) found in the human body. They can be categorised into three subtypes: classical, intermediate, and non-classical.²⁶ Recent investigations have indicated a decrease in intermediate and non-classical monocytes after SARS-CoV-2 infections.²⁷ These monocytes have the capacity to transform into activated macrophages and exhibit both anti-inflammatory and pro-inflammatory functions due to their capability to generate certain cytokines. Therefore, reducing them may disturb this equilibrium and lead to an elevation in the immunological response.²⁸

Neutrophils, lymphocytes, and monocytes have diverse functions in the immune response during various situations, such as COVID-19 disease. Therefore, it can be inferred that an elevated NMR could indicate a disturbance in the balance of the immune system cells, perhaps indicating the severity of the disease with considerable morbidity.

The Systemic Immune Inflammatory Index (SII) has demonstrated its effectiveness as an early and convenient prognostic indicator in cases of metastatic castration resistant prostate cancer (mCRPC) and metastatic renal cell carcinoma. The study also examined its significance in predicting the outcome of stomach carcinomas. It is calculated by (N×P)/L(N,P and L refer to neutrophil counts, platelet counts and lymphocyte counts respectively).^29,30 Our study focused on examining the correlation between SII and the severity of COVID-19. However, our analysis did not find any statistically significant relationship between the two variables. Fois et al. found a strong correlation between the SII and the severity of COVID-19. Nevertheless, the study found that the area under the curve (AUC) for the SII was only marginally significant, with a p-value of 0.060.³¹

NMR was found to show significant association with COVID-19 severity. The AUC also showed significance at >10.34 being the cut off value for the association of NMR with severe COVID-19 disease. A study by Rizo-Téllez SA et al showed a similar finding.³² NMR has may thus be regarded as an easy and early prognostic marker of COVID-19 severity.

The clinical implication is that, though COVID-19 is relatively under control at present compared to the initial years, patients still do present with COVID-19 and it can be fatal among those with risk factors. At present COVID-19 testing facilities are available at most centres and diagnosis is made at peripheral primary and secondary care centres. However, when it comes to management, it is imperative to have markers of severity so that, timely referral and triaging can be done to ensure a favourable outcome.

Our study has some limitations. First, this was a single-centre study with no external validation cohort. Our centre being a tertiary care centre, it was mainly the moderate-severe cases that were being managed. A good number of cases that were being managed were referred from neighbouring smaller centres of the region. For the same reason, the study findings may not be applicable to all cases of COVID-19. Secondly, the hematological parameters studied were taken at the time of admission. It remains unclear whether stepwise changes occur when the patient’s condition worsens clinically. Thirdly, with the evolution of COVID-19 and the emergence of newer variants, we are unsure of the extent to which the findings can be applicable at present.

Several markers, such as coagulation parameters, D-dimer, and CRP, were not included in this investigation, which might have offered a comprehensive understanding of the severity and underlying causes.

Conclusion

Several haematological indicators have been widely investigated for predicting the severity of COVID-19. The results of our investigation revealed a noteworthy correlation between the levels of ALC (absolute lymphocyte count), NMR (neutrophil-to-lymphocyte ratio), and the severity of COVID-19. This has enhanced the significance of our investigation, as the relationship between NMR and the severity of COVID-19 has not been investigated extensively. The utilisation of ALC and NMR at modest facilities and rural hospitals inside our nation, which possess limited resources, can effectively facilitate the triage and management of patients upon admission.