Peripheral blood cell anomalies in COVID-19 patients in the United Arab Emirates: A single-centered study

Introduction In this study, we aimed at exploring the morphologic and quantitative abnormalities in the peripheral blood counts of coronavirus disease 2019 (COVID-19) patients. Methods A cohort of 131 COVID-19 patients was recruited at University Hospital Sharjah (UHS), UAE. Their peripheral blood smears were examined for morphological evaluation. Also, their clinical laboratory investigations and radiological findings were retrieved from the medical records. Our cohort consisted of 63 males and 68 females with an age of 63.6 ± 18.6 years. Results The presence of atypical lymphocytes was observed in around 80% of the recruited COVID-19 patients. Further, monocytes with toxic cytoplasmic vacuoles were identified in 55% of the cases. Neutrophil-associated changes, including pseudo-Pelger-Huët, bands, and long nuclear endoplasm, were reported in around 25–35% of the patients. RBCs associated changes such as microcytic and hypochromic RBCs, as well as targetoid, dacrocytes, ovalocytes, echinocytes/burr cells, and schistocytes, were described. According to disease severity, RBCs chromicity was found to be significantly different between stable and critical patients. COVID-19 patients with CO-RADS 5 showed a similar change in RBCs as well as a decrease in the neutrophils with hypogranular cytoplasm. Conclusion Peripheral blood smear assessment in COVID-19 patients could provide information about the disease state and pulmonary involvement.

It was found that COVID-19 pathogenesis was associated with an activation of the immune system and subsequent immune dysregulation (6). A major clinical feature of COVID-19 was neutrophilia with concomitant lymphopenia, that were linked to the severity of the disease (7). Despite the known quantitative abnormalities in the peripheral blood, little is known about the morphologic changes in circulating blood cells in COVID-19 (8). Such changes could aid in the diagnosis of COVID-19 and management decisions in COVID-19 patients. The reported abnormalities in peripheral blood smears include a range of atypical lymphocytes, acquired Pelger-Huët anomaly, and fetus-shaped neutrophils (9). Furthermore, abnormal platelets and red blood cells (RBCs) morphology were also reported in peripheral blood of COVID-19 infected patients, thus inducing coagulopathies and malfunction of oxygen carrying capacity (10). In this study, we aim to explore the morphologic and quantitative abnormalities in the peripheral blood counts of COVID-19 patients recruited to a single center in the UAE.

Subjects and methods
This is a retrospective study conducted on 131 COVID-19 patients that were recruited at University Hospital Sharjah (UHS), UAE. Our cohort was composed of 63 males and 68 females, aged 63.6 ± 18.6 years (mean ± SD). The cases were diagnosed based on a positive nasopharyngeal swab result using reverse transcriptase-polymerase chain reaction (RT-PCR). Out of 270 patients that were admitted from July 2020 to July 2021, 131 COVID-19 patients were selected as they were not previously vaccinated for SARS-CoV-2. The study was approved by the Ethics and Research Committee of UHS (UHS-HERC-035-03052020).
Peripheral blood samples were collected from COVID-19 patients in EDTA sterile vacutainers, after which peripheral blood smears were prepared, and laboratory investigations were performed. These tests included complete and differential blood such as platelets, white blood cells (WBCs), neutrophils, lymphocytes, monocytes count, oxygen saturation and hemoglobin that were done using Sysmex XN 20 Hematology Analyzer (Sysmex, Germany). Also, prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (aPTT), and D-dimer were measured using STA Compact Max 3 (Stago, France). Moreover, lactate dehydrogenase (LDH) and C-reactive protein (CRP) were measured using Atellica R CH 930 Analyzer (Siemens Healthineers, Germany), while procalcitonin and ferritin were measured Atellica R IM 1300 Analyzer (Siemens Healthineers, Germany). All these tests are summarized in Table 1. Blood films were prepared and stained using the Leishman stain standard protocol. The smears were examined under the light microscope (Olympus BX43, Japan) by a hematopathologist for morphological evaluation, and images were captured using the digital camera (Olympus SC50, Japan). The evaluation was done blindly in terms of laboratory investigations. Radiological evaluation was done using chest X-ray and high-resolution computed tomography (CT) scans, followed by an assessment using the COVID-19 Reporting and Data System (CO-RADS) as a standardized assessment of pulmonary involvement of COVID-19 (11). The CO-RADS classification is described in Table 2.
Values represent mean ± SD for the continuous variables, or percentage relative to the total number of patients in each group for the categorical variables. Statistical analysis was performed using GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA). Chi-square test was used for the comparison between the categorical variables. P-value < 0.05 was considered statistically significant.

Results
Our cohort was composed of 131 patients that were proven to be COVID-19 positive by RT-PCR of the nasopharyngeal  swabs. Their quantitative hematologic abnormalities were documented along with the microscopic examination of the peripheral smears to include various anomalies such as changes in WBCs, RBCs, and platelets. The most common reported morphologic finding was the presence of atypical lymphocytes in around 80% of the COVID-19 patients ( Figure 1A). This was followed by the presence of monocytes with toxic cytoplasmic vacuoles in 55% of the cases. As shown in Figure 1B, activated monocytes were observed showing prominent cytoplasmic vacuolization and few granules. Also, the nuclei were large, having fine chromatin with nuclear blebbing.
Regarding platelet counts, the mean number was within the normal range in COVID-19 patients, which aligns with the coagulation parameters (PT, INR, and aPTT) that were almost in their normal ranges. Around half of the COVID-19 patients presented with normocytic RBCs along with anisocytosis and hyperchromicity ( Table 4). Other common RBCs associated changes included erythrocytopenia and microcytic RBCs as well as targetoid, dacrocytes, ovalocytes, echinocytes/burr cells, and schistocytes (Figures 3A-E).
The COVID-19 patients were classified into different groups according to their CO-RADS score. Almost 77% of the patients fell into the CO-RADS 5 category (Figure 4), indicating pulmonary involvement and a high probability of COVID-19 infection before confirmatory tests by qRT-PCR ( Table 5).
In order to search for a relation between morphological changes in peripheral blood cells and disease severity, COVID-19 patients were classified into stable (patients not requiring ICU admission, n = 48) and critical (ICU admitted patients, n = 83) groups. No statistical significance was found between the two groups except for RBCs changes and platelets' thrombocytosis ( Table 6). There was a significant increase in the percentage of patients showing normocytic (p = 0.0301), normochromic (p = 0.0246), with a significant decrease in the patients' microcytic (p = 0.0109), and hypochromic RBCs (p = 0.0158) TABLE 4 Changes in the platelets and red blood cells (RBCs) count and their associated morphological anomalies.
Since gender plays a critical role in the COVID-19 pathogenesis (11, 12), it was interesting to explore if there is any difference in the peripheral blood anomalies between males (n = 63) and females (n = 68). There was a statistical significance in the chromicity of RBCs, where males showed a higher significant percentage of normochromic (p = 0.0093), with a concomitant decrease in the percentage of hypochromic RBCs (p = 0.0054, Table 8).

Discussion
This study highlights the quantitative and morphological changes in the peripheral blood cells of COVID-19 patients. To our knowledge, this is the first study to report these changes in the UAE, which has taken extraordinary precautionary measures to restrict the spread of COVID-19 and guarantee the safety of citizens. Furthermore, this study explored if there is an association between disease severity and peripheral blood anomalies.
The most common morphological anomalies in our cohort were atypical lymphocytes, large monocytes with vacuoles, and hypogranular neutrophil cytoplasm of the peripheral blood smears. Despite the small number of reports on the peripheral morphological anomalies associated with COVID-19 infection, our findings go in line with a study by Zhang et al. where large monocytes with vacuoles were observed in peripheral smears of COVID-19 patients (13). In addition, our observed morphological anomalies were consistent with the findings by Zini et al. that reported various peripheral morphological blood changes, specifically in the neutrophils (14). Further, the presence of atypical lymphocytes was highly found to be similar to the previous reports (15)(16)(17). In addition, granulocytes and particularly neutrophils showed a pseudo-Pelger-Huët anomaly affecting 25% of the recruited COVID-19 patients, both stable and critical cases, unlike the observed findings by Ahnach et al. (18). Similarly, another type of neutrophil-associated anomaly was the presence of hypogranular cytoplasm in more than 30% of the patients (18).
Despite the advances in COVID-19 research, little information about the morphological changes in peripheral blood smears of infected individuals and their association with patients' clinical outcomes is still unknown. Thus, we were interested in correlating the difference in these peripheral anomalies in ICU-admitted and stable COVID-19 patients. An increase in the percentage of patients showing neutrophilia, neutrophils with pseudo-Pelger-Huët, bands, long nuclear endoplasm, and fetus shaped nucleus, as well as large vacuolated monocytes, was recognized in the critical/ICU admitted group; however, such changes did not reach statistical significance. An increase in the percentage of aforementioned anomalies was also observed in COVID-19 patients with a CO-RADS score 5 compared to others. On the other hand, upon the classification of COVID-19 patients according to the severity of the disease or CO-RADS, there was a decrease in the number of patients showing hypogranular neutrophilic cytoplasm, which could be a sign of pulmonary deterioration. On the contrary, a study by Gabr et al. reported that the abundance of peripheral morphological abnormalities was significantly associated with unfavorable clinical outcomes in COVID-19 patients (17).
A plethora of abnormalities associated with RBCs was previously described in COVID-19 patients (19,20). Morphological changes in COVID-19 patients were detected, along with a comparison between stable and critical cases. There was a significant decrease in the percentage of COVID-19 patients in the critical group with microcytic and hypochromic RBCs, along with a significant increase in the percentage of patients with normocytic and normochromic RBCs. A similar pattern was observed in the classification of COVID-19 according to CO-RADS. Additionally, there was a concomitant decrease in hemoglobin concentration in stable COVID-19 patients (11.4 ± 2.3) compared to critical (12.2 ± 2.0) cases. Hence, this could be attributed to other factors such as ferritin

Conclusion
To our knowledge, this is the first study in the UAE describing the morphological changes in the peripheral blood smears of COVID-19 patients and their association with disease severity. Peripheral blood smear assessment at the time of diagnosis in COVID-19 patients could provide information about the disease state and pulmonary involvement. One of the limitations of this study is the lack of information about the recruited patients' mortality and the lack of peripheral smears of healthy controls for comparative investigation. Also, another limitation of this study is the lack of functional analysis of the aforementioned peripheral blood cells (including oxygencarrying capacity of RBCs, ROS generation by leukocytes and immunoglobulin production by lymphocytes, phagocytosis capacity of macrophages/monocytes and leukocytes), that should be explored in future studies to further understand their role in the fight against SARS-CoV-2. Additionally, future studies could explore the association between antibody titers against SARS-CoV-2 proteins and hematological abnormalities that will aid in understanding the effect of infection and vaccinations on various blood cells.

Data availability statement
The original contributions presented in this study are included in this article/supplementary material, further inquiries can be directed to the corresponding authors.

Ethics statement
The studies involving human participants were reviewed and approved by the Ethics and Research Committee of University Hospital Sharjah in June 2020 (UHS-HERC-035-03052020). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

Author contributions
NE and IT designed the study, analyzed, interpreted the results, and wrote the original draft. FB interpreted the slides of the peripheral blood smears. RG assessed the radiological imaging of the patients. DZ, AAl, NA, AH, FA, AE, AAh, and SA recruited and collected the data of the patients. RB designed the study. FB, RG, and RB reviewed the manuscript. All authors read and approved the final manuscript.