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Clinical Infection & Immunity

Original Article

Volume 8, Number 1, March 2023, pages 13-23

Mortality Prognostic Hematological Parameters in COVID-19 Patients

In late December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolved in Wuhan, China causing coronavirus disease 2019 (COVID-19) [1]. COVID-19 soon emerged to be a global pandemic affecting more than 500 million people worldwide until now. SARS-CoV-2 is the seventh member of coronavirus infecting humans and causes severe lower respiratory tract infections through binding to angiotensin-converting enzyme 2 (ACE-2) receptors [1, 2]. Although the primary clinical manifestations of COVID-19 are respiratory tract infection and pneumonia, other systemic illness disorders are common, such as cardiovascular, neurological, and gastrointestinal complications [3-5]. Additionally, SARS-CoV-2 was shown to have significant implications on hematopoietic system, such as blood cells and coagulation disorders [6, 7]. For instance, lymphopenia is being used as a cardinal laboratory marker with a prognostic value of COVID-19 [6].

Trends in leukocyte count, lymphocyte percentage, and platelet count have received attention in characterizing the severity of the condition and prediction of the clinical outcomes [7, 8]. An association between the grade of lymphopenia and prognosis of the disease was also noted [9]. New emerging parameters such as neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR) were described as possible prognostic factors of disease severity and comparison of treatment strategies [7, 9]. Many studies were performed to clarify the crucial role of SARS-CoV-2 in acute respiratory illness as well as develop new viral detection platforms [10, 11], yet data are still lacking on the clinical characteristics of the infections caused by the virus, and specifically the general trends of hematological and coagulation parameters during the course of the infection.

This study was conducted following approval of the Institutional Review Board. This was a retrospective study focusing on the hematological parameters of laboratory-confirmed cases of COVID-19 in Waterbury Hospital, Waterbury, CT, USA, from March 2020 to August 2020. Study subjects were hospitalized adult patients with oxygen saturation below 94% without oxygen supplementation on presentation, and laboratory confirmation of SARS-CoV-2 infection. Laboratory confirmation of SARS-CoV-2 was performed with the use of real-time reverse-transcriptase-polymerase chain reaction (RT-PCR) assay of a nasopharyngeal swab specimen. All patients had demographic and clinical data collected (age, gender, and comorbidities including hypertension, diabetes, chronic lung disease (chronic obstructive lung disease or asthma), and chronic kidney disease). Daily laboratory values including complete blood count with differential were reported. Primary outcome was in-hospital mortality and secondary outcomes were intensive care unit (ICU) level of care, mechanical ventilation, and hospital length of stay (LOS). Severe disease was defined as patients requiring higher level of care or admission to ICU, the need for mechanical ventilation and in-hospital mortality. Leukocyte count was documented on admission and on discharge. Leukopenia was defined as white blood cell (WBC) count less than 4,000/mm³. Leukocytosis was defined as WBC count more than 10,000/mm³. Absolute lymphocyte count was documented on admission. Nadir levels of lymphocytes were documented as well. Lymphopenia was defined as patients with levels less than 1,000/mm³. NLR was calculated as absolute neutrophil count divided by absolute lymphocyte count. PLR was calculated as absolute platelet count divided by absolute lymphocyte count. Platelet counts were reported as thrombocytopenia if the number < 100,000/mm³. All collected parameters were correlated to primary and secondary endpoints to assess correlation. P values less than 0.05 were considered statistically significant. The study was conducted in compliance with the ethical standards of the responsible institution on human subjects as well as with the Helsinki Declaration

For the patient characteristics, the categorical variables were made as percentages using Chi-square test while the continuous variables were described mean using descriptive analysis.

Both univariate and multivariate logistic regression analyses were conducted, and odds ratio (OR) were calculated between the hematologic markers and the primary and secondary outcome with a confidence interval of 95% (95% CI) and alpha value of 0.05. No imputation was used for missing data.

For the univariate logistic regression, the hematologic markers were analyzed using 95% CI and statistical significance of 0.05.

For the multivariate logistic regression analysis, the hematologic markers were analyzed to determine the rate of COVID-19 related death, ICU admission and mechanical ventilation requirements while adjusting with for prespecified baseline covariates such as diabetes mellitus status, hypertension, lung disease (asthma and chronic obstructive pulmonary disease (COPD)), pre-existing cancer and end-stage renal disease to determine the risk factors of death among critically ill patients.

The descriptive analysis, univariate and multivariate logistic regression analyses were performed using SAS software, version 9.4.

During the period of data collection, a total of 276 patients were enrolled in the study and included in data analysis. The mean age of the study population was 63 years. There were slightly more males than females in the study. Nearly half of the patients had hypertension and about a third had diabetes mellitus. The mean values of the hematologic markers (WBC, lymphocytes, platelet count, platelet/lymphocyte ratio and neutrophil/lymphocyte ratio) can be reviewed in Table 1.

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Table 1. Patient Characteristics

Seventy-three of the 276 patients were admitted to ICU with 58 required mechanical ventilation. About one-fifth (n = 59) of the study population died in the hospital. The average length of hospitalization was 9.4 days (Table 1).

The primary outcome event defined as in-hospital mortality from COVID-19 occurred in 58 patients out of a total of 276 patients (Table 1).

The univariate unadjusted association was analyzed between the hematologic markers and the primary outcome event. Leukocytosis showed statistically significant association with death from COVID-19 infection (OR of 7.67; 95% CI: 3.277 - 17.970; P < 0.0001). Moderate and severe neutrophil/lymphocyte ratio were found to be associated with death from COVID-19 infection: (OR of 3.691; 95% CI: 1.636 - 8.326; P < 0.0017) and (OR of 12.819; 95% CI: 5.285 - 31.090; P < 0.0001), respectively. In addition, the third and fourth quartiles of platelet/lymphocyte ratio were also found to be associated with death from COVID with OR of 2.417 (95% CI: 1.033 - 5.654; P < 0.0419) and OR of 2.715 (95% CI: 1.171 - 6.293; P < 0.0199) (Tables 2, 3).

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Table 2. Univariate Unadjusted Association Analysis Between the Hematologic Markers and the Primary Outcome Event (In-Hospital Mortality)

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Table 3. The Association With In-Hospital Mortality Using the Multivariate Analysis (After Adjusting for Possible Confounding Factors)

While adjusting for the various confounding variables, the hematologic markers were analyzed against the primary outcome events. In the same manner, the variables that were associated using the unadjusted analysis were associated with the primary outcome using the multivariate analysis except for platelet/lymphocyte ratio.

Leukocytosis was associated with death from COVID (OR of 6.894; 95% CI: 2.871 - 16.552; P < 0.0001). Moderate and severe neutrophil/lymphocyte ratio had OR of 3.488 (95% CI: 1.468 - 8.291; P < 0.0047) and OR of 15.489 (95% CI: 5.786 - 41.462; P < 0.0001), respectively. All the quartiles of platelet/lymphocyte ratio were found not to be associated with death from COVID after adjustment (Tables 2, 3).

The secondary outcomes are ICU admission and mechanical ventilation requirements.

The results of unadjusted univariate association between the hematologic markers and the secondary outcome events showed that: leukocytosis, leukopenia, thrombocytopenia, platelet/lymphocyte ratio and neutrophil/lymphocyte ratio were all found to be associated with the patient being admitted to the ICU.

Leukocytosis was associated with increased ICU admission (OR of 6.555; 95% CI: 3.111 - 13.809; P < 0.0001). Leukopenia was also found to be associated with increased ICU admission with OR of 3.392 (95% CI: 1.638 - 7.022; P < 0.0010) as well as thrombocytopenia with OR of 2.067 (95% CI: 1.046 - 4.084; P < 0.0367).

Mild, moderate and severe neutrophil/lymphocyte ratio were found to be associated with increased ICU admission as well, OR of 3.627 (95% CI: 1.363 - 9.656; P < 0.0099), OR of 9.250 (95% CI: 4.042 - 21.170; P < 0.0001) and OR of 19.733 (95% CI: 7.733 - 50.355; P < 0.0001), respectively. The third and fourth quartiles of platelet/lymphocyte ratio were also found to be associated with higher ICU admission with OR of 3.894 (95% CI: 1.527 - 9.929; P < 0.044) and OR of 8.971(95% CI: 3.598 - 22.364; P < 0.0001), respectively (Tables 4, 5).

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Table 4. The Association Between Hematological Parameters and ICU Admission Using Univariate Association Analysis

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Table 5. The Association With ICU Admission Using the Multivariate Analysis

While adjusting for the confounding variables in the multivariate analysis, the hematologic markers were analyzed. The correlation between the previous hematologic markers and ICU admission was also statistically significant except thrombocytopenia.

Leukocytosis was associated with increased ICU admission (OR of 6.453, 95% CI: 2.929 - 14.217; P < 0.0001). Mild, moderate, and severe neutrophil/lymphocyte ratio were also associated with higher ICU admissions: OR of 3.344 (95% CI: 1.202 - 9.306, P < 0.0208), OR of 8.329 (95% CI: 3.483 - 19.917; P < 0.0001) and OR of 20.225 (95% CI: 7.403 - 55.255; P < 0.0001), respectively. Also, the third and fourth quartiles of platelet/lymphocyte ratio were found to be associated with increased ICU admissions: OR of 3.223 (95% CI: 1.220 - 8.517; P < 0.0182) and OR of 8.022 (95% CI: 3.107 - 20.711; P < 0.0001) (Tables 4, 5).

Leukocytosis, platelet/lymphocyte ratio and neutrophil/lymphocyte ratio were found to be associated with higher mechanical ventilation rates.

Leukocytosis was found to be associated with increased mechanical ventilation (OR of 5.072; 95% CI: 2.325 - 11.063; P < 0.0001). Moderate and severe neutrophil/lymphocyte ratio were found to be associated with higher ventilation rates: OR of 3.292 (95% CI: 1.517 - 7.144; P < 0.0026) and OR of 7.055 (95% CI: 3.006 - 16.556; P < 0.0001), respectively. The third and fourth quartiles of platelet/lymphocyte ratio were also found to be associated with OR of 3.877 (95% CI: 1.331 - 11.294; P < 0.0130) and OR of 6.720 (95% CI: 2.383 - 18.949; P < 0.0003), respectively (Tables 6, 7).

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Table 6. The Association Between Hematological Parameters and Mechanical Ventilation Using Univariate Association Analysis

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Table 7. The Association With Mechanical Ventilation Using the Multivariate Analysis

While adjusting for the confounding variables in the multivariate analysis, the hematologic markers were analyzed. All the previous hematologic markers were also statistically significant.

Leukocytosis was found to be associated with higher need for mechanical ventilation (OR of 5.159; 95% CI: 2.265 - 11.754; P < 0.0001). Moderate and severe neutrophil/lymphocyte ratio were found to be associated with higher need for mechanical ventilation: OR of 2.906 (95% CI: 1.274 - 6.629; P < 0.0112) and OR of 7.269 (95% CI: 2.875 - 18.378; P< 0.0001), respectively. Also, the third and fourth quartiles of platelet/lymphocyte ratio were found to be associated with increased mechanical ventilation: OR of 3.474 (95% CI: 1.156 - 10.439; P < 0.0266) and OR of 5.899 (95% CI: 2.037 - 17.079; P < 0.0011) (Tables 6, 7).

Inflammation plays a significant role in the pathogenesis of COVID-19 [11, 12]. Neutrophils are the major component of leukocytes that activate response to an infectious insult, and they are among the first innate leukocytes recruited to the site of infection; their primary function is phagocytosis, and they also mediate tissue damage and induce apoptosis of virally infected cells. They also stimulate B lymphocyte to initiate humoral immunity. COVID-19 leads to severe inflammatory response and cytokine storm leading to higher levels of interleukin-6, interleukin-8, tumor necrosis factor (TNF)-α, interferon gamma, and granulocyte colony stimulating factor These factors activate neutrophils leading to proliferation and migration of neutrophils to viral-infected sites. Because neutrophils have a short half-life, neutrophilia usually indicates an acute inflammatory reaction [13, 14]. High levels of neutrophil counts may lead to tissue damage and cytotoxicity [12, 13].

The human immune response triggered by viral infection mainly relies on lymphocytes. Lymphopenia has been reported in up to 40% of COVID-19 cases [15]. There are several postulated hypotheses that might explain lymphocytopenia in those patients. SARS-CoV-2 has the ability to directly infect T cells through ACE receptor, the portal entry protein into the cells resulting in decreased levels of CD3⁺, CD4⁺, CD8⁺ T lymphocytes and increased regulatory T cells [11, 15]. Studies showed levels of total T cells, and CD4⁺ and CD8⁺ T cells were significantly lower in critical patients [15] and leukocytopenia was noted to be more common in patients with severe disease. The inflammatory cytokine storm is likely playing a key role in the observed decline of lymphocyte counts. Pro-inflammatory cytokines such as TNF-α and interleukin-6 can activate the neutrophils and damage the lymphocytes [11]. They have a cytotoxic effect on lymphocytes leading to apoptosis. Another theory is that COVID-19 can lead to exhaustion of T cells as T cells were observed to have higher level of programmed cell death. Lymphopenia has been found to be an indication of immunosuppression in sepsis and may be a predictor of mortality in patients who developed acute respiratory distress syndrome (ARDS) [15].

NLR is defined as the ratio of absolute neutrophil count to absolute lymphocyte count. As NLR represents the balance between the two cell subsets, neutrophilia and lymphopenia leading to high NLR values can be indicative of severe inflammatory process [13]. NLR is not a novel parameter, and it has been shown to be a prognostic factor in various diseases, such as solid tumor, cardiovascular disease, COPD, and systemic lupus erythematosus (SLE) [13]. For example, Yu et al found that NLR was significantly elevated in 212 patients with SLE compared to 201 healthy controls and was positively correlated with erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) reflecting its correlation with SLE disease activity [16].

NLR can be classified according to severity to mild (6 - 8), moderate (9 - 18) and severe (> 18). Our study showed that higher levels of NLR are associated with worse outcomes thus can be a useful prognostic tool in COVID-19. Our results match previously published data [3, 17, 18].

Platelets are important immune cells in the human body, which are produced by mature megakaryocytes in the bone marrow. Platelets play a vital role in blood coagulation, angiogenesis, immune response, and inflammation [19]. Thrombocytopenia is commonly observed in COVID-19 patients. Possible mechanisms include the ability of SARS-CoV-2 to directly invade hematopoietic cells or bone marrow stromal cells. Hypercoagulability causing platelet aggregation and thrombogenesis can further reduce platelet count [19, 20].

PLR is defined as the ratio of absolute platelet count to absolute lymphocyte count, so changes in both platelet count and lymphocyte counts can affect PLR. Although thrombocytopenia is commonly reported in COVID-19, higher PLR values have been associated with more severe disease as reported in our study, which could be explained by the fact that the degree of lymphopenia is much higher than the degree of thrombocytopenia leading to high PLR noticed in the severe forms of COVID-19. PLR has also been shown as an inflammatory indicator in some diseases including malignancies, diabetes, and infections including viral and bacterial [21]. Some clinical trials have demonstrated the relationship between PLR and autoimmune disease. Uslu et al showed a high PLR was associated with severe disease activity in rheumatoid arthritis [22]. Although different studies have investigated the correlation between PLR and the disease severity, reports are still contradictory. Some studies have reported that the increase in PLR was correlated with the poor prognosis of COVID-19. Qu et al noticed that the larger ΔPLR, the more severe the cytokine storm, and the worse the prognosis [7, 21]; however other studies found PLR was not associated with the disease severity and no significant relationship was found between PLR and disease severity [23]. Our study showed a statistically significant correlation between PLR and disease severity.

Most studies reported an association between the severity of lymphopenia with higher mortality rates [24]. Although our study noted an association between lymphopenia and ICU mortality, the association between lymphopenia and mortality was not statistically significant in our study.

There are some limitations that need to be acknowledged in our study. First, this represents a single-center retrospective observational study. Second, the possible confounding effect of corticosteroids and other medications that can affect blood cell count, can subsequently affect NLR and PLR parameters and complicate interpretation. Systemic steroids were used for severe and critical COVID-19 infection in September 2020 after the preliminary report of the RECOVERY trial [25]. Our study included patients till August 2020, this should be taken into consideration for patients on corticosteroids. Changes in PLR can be shifted in those who suffer from possible autoimmune or neoplastic comorbidities.

The study results reflect the early phase of the pandemic, which could be different from other variants as delta and omicron. More studies are needed to compare effect of different variants of COVID-19 on hematological parameters. Finally, single measurements of laboratory parameters do not reflect their dynamic change through the course of the disease. However, the sample size was large, and the results match most previously published data.

NLR and PLR are simple biomarkers that reflect the presence of systemic inflammation. Both markers were found to be associated with ICU admission, need for mechanical ventilation, and in-hospital mortality. Lymphopenia was noted to be associated with a higher risk for ICU admission but an association with mechanical ventilation or mortality was not statistically significant. NLR and PLR can be used to assess severity of COVID-19 infection and predict possible worse outcomes, thus guiding appropriate triage, monitoring, and possible earlier intervention.

Acknowledgments

None to declare.

Financial Disclosure

None to declare.

Conflict of Interest

None to declare.

Informed Consent

This is a retrospective study that did not include any direct interaction with the patients. All patients’ identifiers were removed. Informed consent was not required.

Author Contributions

Sara Soliman developed the original idea, collected and reviewed data for all patients, reviewed literature data, prepared the manuscript, and provided additional review. Jamie Hundaniele collected and reviewed data. Akintayo Akinyele did the statistical analysis for the project and added tables. Medhat Ghaly contributed to data collection and manuscript revision for important intellectual content.

Data Availability

The authors declare that data supporting the findings of this study are available within the article.

Abbreviations

NLR: neutrophil-lymphocyte ratio; PLR: platelet-lymphocyte ratio; RT-PCR: real-time reverse-transcriptase-polymerase chain reaction; ICU: intensive care unit; ACE: angiotensin-converting enzyme; ARDS: acute respiratory distress syndrome

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