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Blood Cells Indices are Determinants of the COVID-19 Outcome: A Cross-Sectional Study from Kurdistan Region-Iraq

Blood Cells Indices are Determinants of the COVID-19 Outcome: A Cross-Sectional Study ABSTRACT Introduction: The complete blood picture of patients with COVID-19 showed lymphocytopenia and neutrophilia. Changes in the circulating blood cells are served as prognostic factors of COVID-19. This study aimed to investigate the clinical importance of determining the hematological indices and ratios as diagnostic and/or prognostic markers of COVID-19. Methods: This cross-sectional observational study was performed in the West Erbil Emergency Hospital, Kurdistan region, Erbil- Iraq, between August 10 and November 19, 2020. A total of 204 patients with COVID-19 were included in this study. The hematological indices and their derived ratios were determined, C-reactive protein and the outcome events were the primary outcome measures. Results: The mean value of leukocytes is ≥ 10,000 cell/mm 3 , which characterized by neutrophilia and lymphocytopenia. The percentage of monocyte is significantly higher in patients with hypertension with and without diabetes mellitus compared with other patients. There are no significant differences between patients with and without concomitant diseases in the erythrocyte sedimentation rate and C-reactive protein. The mortality rate was 31.3% (64 out of 204). Red distribution width and neutrophil-to-lymphocyte ratio are significant discriminators of the non-survivor patients with COVID-19 (The area under the curve with 95% confidence interval: 0.618 (0.510-0.726) and 0.612 (0.505-0.718), with odd ratios of 3.02, 2.407, at cutoff values ≥13.2 % and 12.0), respectively. Conclusion: Significant high values of red distribution width and lymphocyte-to-neutrophil ratio are associated with unpleasant outcome events of COVID-19 patients, while a higher percentage of monocyte is commonly found in hypertensive patients presented with COVID-19.


INTRODUCTION
COVID-19 is a pandemic viral disease caused by a coronavirus (CoV), presented with respiratory and extrarespiratory signs and symptoms. Polymerase chain replication technology is the definite laboratory tool for the diagnosis of CoV infections. The hematological indices are also useful in the diagnosis and assessment of COVID-19. Lymphocytes and monocytes are part of immune system, which specifically determine the immune response to the foreign substances and microorganisms, while the main function of the neutrophils is protecting the humans from bacterial infections. Lymphocytopenia is commonly reported in COVID-19 patients [1,2], and other studies found that lymphocytopenia is a prognostic marker as 35-75% of patients who had lymphocytopenia did not survive [3]. Also, another study reported that patients who were admitted to the intensive care unit had a cutoff value of lymphocyte count < 0.6x10 9 /L [4].
Lymphcytopenia observed in severe COVID-19 disease is significantly characterized by a lower number of CD4+ and CD8+, and usually associated with a significant increase of Creactive protein, D-dimer and interleukins (including IL-2R, IL-6, IL-10) and tumor necrosis factor-α [5].
A small percentage of patients with severe illness showed a leukocyte count > 10,000/mm 3 which is due to a higher number of lymphocytes or neutrophils or both [1]. A significant high neutrophil count is an indication of the bacterial superinfection, cytokine storm, and hyperinflammatory state that accompanied CoV infections [6][7][8]. Moreover, the neutrophil-to-lymphocyte ratio (NLR) is significantly increased in severe COVID-19 compared with those with mild-illness [8]. The majority of patients showed significant low platelet count and linked with severe infection and hypoxia [9][10][11]. Mean platelet volume (MPV) was found to be increased in COVID-19 with unfavorable outcomes (death or venous thrombosis) compared with patients who survived without thrombosis complication [12]. Another retrospective study, including 85 COVID-19 patients, found that patients with severe pneumonia had a significant MPV-to platelet count ratio, which can be considered as an independent risk factor for severe pneumonia [13]. The rationale of this study is that the hematological indices can give a typical pattern of viral infection, specifically for CoV infections. Also, it can be applied to discriminate and predict the survivals of COVID-19 patients. This cross-sectional study aimed to investigate the clinical importance of determining the hematological indices as diagnostic and prognostic markers in a small sample of the Kurdistan population taking into consideration the concomitant diseases and the outcome events.

Design and Setting
This cross-sectional study included adult patients of both sexes hospitalized in the West Erbil Emergency Hospital, Kurdistan region, Erbil-Iraq, between August 10 and November 19, 2020. The West Erbil Emergency Hospital was established for quarantine and management of COVID-19 in the Kurdistan region, with 102 beds, thirty-four physicians, ten pharmacists, 224 nurses, specialized laboratories and radiological departments for diagnosis of CoV infections, and the facilities of artificial ventilation.
The diagnosis of COVID-19 was confirmed by polymerase chain reaction (PCR) assays on the swabs obtained from the nasopharynx. The Ethical and Scientific Board at the Ministry of the Heath in the Erbil approved this study, and exempt the need for consent.

Sample Size
The sample size was calculated using α-coefficient (type II error) = 0.05, β-coefficient (type I error) = 0.2, and power = 85%). The patients were randomly recruited from the hospital using a random numbers table according to the number of admission sheet records.

Participants
A total number of 204 patients were allocated from singlecenter (128 males and 76 females, with a mean age of 58.3 years). Current illnesses were reported in 19 (9.3%) patients with diabetes mellitus; 45 (22.1%) patients with hypertension; 80 (39.2%) patients with hypertension and diabetes mellitus; and 4 (2%) patients with blood disorders. We categorized the patients into four categories according to their outcomes (Figure 1

):
Group A: patients who recovered from illness and discharged from the hospital.
Group B: patients who were discharged from the hospital with minor clinical features (dry cough, fatigue, etc.) without any evidence of clinical and radiological investigations that indicate the presence of any complications).
Group C: Patients with clinical features of COVID-19 and required quarantine in the hospitals to avoid future complications. Some of these patients required oxygen therapy.
Group D: Patients who died (non-survivors) in the intensive care unit. Most patients admitted to the intensive care unit (ICU) required oxygen therapy and ventilator support.
Group A represented the survivors while Group D represented non-survivor patients.

Determination of Clinical Variables
On admission, samples of the blood obtained from patients under careful precautions and sent to the laboratories of the hospital to determine the hematological indices (blood samples with EDTA as an anticoagulant), and quantitative serum C-reactive protein (separated serum from a blood sample without EDTA). Neutrophil-to-lymphocyte (NLR) and platelet-to lymphocyte (PLR) ratios were simply calculated by

Statistical Analysis
The results are expressed as a number, percentage, and mean ± SE. The data were statistically analyzed using a twotailed, one-way analysis of variance (ANOVA), homogeneity test of variance (Levene's statistics), receiving operating characteristics, and calculating the risk odd ratios for continuous data, and Chi-square test for categorized data. Pvalue ≤ 0.05 is a lower significance level. SPSS-20 (IBMcompatible) was applied for statistical analysis.

Distribution of Hematological Indices and Ratios According to the Presence of Concomitant Diseases
A total number of 204 patients were included in this study. The number of male patients was non-significantly (χ 2 =0.873, P=.832) higher than the corresponding female patients, and the mean age of the patients with concomitant diseases was higher than patients without concomitant diseases ( Table 1). There is no significant difference in the red cell indices in patients with or without concomitant diseases. The red cell indices were within the normal range. White cell indices showed the mean values are ≥ 10.000 cell/mm 3 , which characterized by a higher percentage of neutrophil and a lower percentage of lymphocyte in each patient group with or without concomitant diseases. The percentage of monocyte is significantly higher in patients with hypertension with and without diabetes mellitus compared with other patients. The mean values of the blood platelet indices are within normal limits, and the platelet count was higher than the lower limit of normal platelet count (150,000/mm 3 ). Moreover, the data showed homogeneity in the variance of each patients group except the percentages of the granulocytes (monocyte, eosinophil, and basophil), which are significantly showed heterogeneity. Table 2 shows that the mortality rate was 31.3% (64 out of 204). Patients with concomitant diseases constituted the higher number of Groups C and D. Diabetic patients were more     Table 3), which is significantly less than the corresponding value of non-hypertensive non-diabetic patients (76.4±3.5% versus 81.3±1.4%). During the course of COVID-19, there are no significant differences between patients with and without concomitant diseases in the erythrocyte sedimentation rate and C-reactive protein, accounting for significantly higher values compared with normal upper limits ( Table 3).

Comparison between Category A and D Regarding Hematological Indices
There are non-significant statistical differences between patients related to the categories A and D regarding the mean values of red distribution width (RDW), platelet distribution width (PDW), MPV, NLR, and PLR. The data of these hematological indices and ratios showed homogeneity as Levene's statistic value was non-significant for each index and ratio (Table 4). Moreover, the RDW and the NLR ratio are significant discriminators of the unfavorable event (death) of patients with COVID-19 (Figure 2). The areas under the curve with 95% C.I. of the RDW and NLR are 0.618 (0.510-0.726) and 0.612 (0.505-0.718), respectively (Figure 2). The odd ratios of unfavorable (death) outcomes are 3.02, 2.407, and 2.407 at cutoff values of RDW (≥13.2), NLR (12.0), and PLR (36.8), respectively (Figure 3).

DISCUSSION
The results of this study indicate that the determination of hematological indices and ratios at the time of hospitalization can predict the outcome events of COVID-19 patients despite the presence or absence of concomitant diseases, including hypertension and/or diabetes mellitus. The characteristic hematological profile of COVID-19 is neutrophilia, lymphocytopenia, a higher monocyte percentage, and within the normal range of the blood platelet count. The results of this study are in parallel with previous studies that neutrophilia and lymphocytopenia are the characteristic features of CoV infection. Terpos et al. [14] reported that lymphocytopenia occurred after 7-14 days from the clinical presentation of the COVID-19, and considered as a prognostic factor. The causes of lymphocytopenia are due to the lysis of the lymphocyte as a result of binding the CoV to the angiotensin converting enzyme receptor -2 (ACER-2) which is expressed on the lymphocyte [15], and to the inflammatory mediators that released as a part of cytokine storm syndrome, which cause lymphocyte apoptosis [16][17][18], and atrophy of lymphoid tissue [19]. A higher number of the leucocytes (> 10,000/mm 3 ), is also a feature of COVID-19, and it may indicate superimposed secondary infection [20]. The percentage of monocyte is higher among hypertensive patients with/without type 2 diabetes mellitus. Merad and Martin [21] reported that dysregulation of the immune system as a result of hyperinflammation leads to an increase in the number of monocyte/macrophage in the bronchoalveolar fluid in severe COVID-19. This work demonstrates a significantly higher percentage of circulating monocyte in the peripheral blood, which is linked to hypertensive patients rather than to the severity of COVID-19. The explanation of this observation that peripheral monocytes are activated the vascular endothelium under the effect of excess production of IL-6 and deprivation of nitric oxide in hypertension [22]. It is well known that CoV cannot cause direct damage to the blood platelet because the platelets lack ACER-2 on their surfaces [14]. Thrombocytopenia is a feature of severe COVID-19, and it is usually noted in the non-survivors [23]. On the other side, COVID-19 patients who had a peak platelet count at the time of clinical presentation will have a worse prognosis [24]. Moreover, concomitant diseases are not the cause of the changes in the blood platelet indices of COVID-19 patients ( Table 1). The results of hematological ratios during the course of illness explore their important values to discriminate and predict the patients who may be nonsurvivors. Red distribution width significantly discriminates the non-survivor from survivor COVID-19 (Figure 2), and COVID-19 patients who had an RDW ≥ 13.2% will get a poor prognosis. This observation wasn't previously mentioned. The neutrophilto-lymphocyte ratio is significantly higher in non-survivors compared with survivor patients, which this finding agreed   with previous studies [25]. This work adds two important findings that the NLR can discriminate against the nonsurvivors from recovered patients (Figure 2), and the NLR value of ≥12.0 during the course of illness predicts the non-survivor (odds ratio: 2.407). The PLR at a cutoff value of 36.8 can predict the non-survivors of COVID-19 patients. This observation agreed with other studies that patients with a higher PLR ratio are at risk of worse prognosis [24,25]. Mean platelet volume and platelet distribution width can predict the non-survivors as their odd ratios exceeded 1.0 but they are not discriminated against the non-survivors. This study agreed with another study that observed each one femtolitre increment of the MPV will increase the mortality rate by 1.76 [26]. Higher values of serum CRP and ESR indicate that COVID-19 patients were presented with hyperinflammation, which is prescribed in a lot of studies. Also, a low mean value of blood saturated indicates that hospitalization of the patients is absolutely indicated, and a significantly low PSO2 in diabetes patients may be due to the small sample size. Limitations of the study included the variability in the duration of the disease (from the onset of clinical presentation to the hospitalization), serial measurements of complete blood count, and a small sample size of diabetic patients. The strength of this study is related to describing the cutoff values of hematological indices and ratios.
We conclude that the determination of hematological indices and ratios during the course of illness can serve as discriminators and predictors of patients who will get a poor prognosis. A significantly higher percentage of monocyte during the course of COVID-19 is a feature of hypertensive patients.
Author contributions: MS-AN provided the study concept and design, statistical analysis, data management, and wrote the manuscript. TAM provided the design, recruited the patients, and performed data management. KYM recruited the patients, and HWM made the applications of laboratory investigations. All authors critically reviewed the manuscript.