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Pathological effects of SARS-CoV-2 on hematological and immunological cells: Alterations in count, morphology, and function

The novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 outbreak, spread rapidly and infected more than 140 million people with more than three million victims worldwide. The SARS-CoV-2 causes destructive changes in the immunological and hematological system of the host. These alterations appear to play a critical role in disease pathology and the emerging of clinical manifestations. In this review, we aimed to discuss the effect of COVID-19 on the count, function and morphology of immune and blood cells and the role of these changes in the pathophysiology of the disease. Knowledge of these changes may help with better management and treatment of COVID-19 patients.


Background
In December 2019, the novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-COV-2) emerged from Wuhan, China and rapidly spread over more than 200 countries. Eventually, the World Health Organization (WHO) officially declared Coronavirus Disease-2019 (COVID-19) a pandemic on March 11, 2020 [1,2]. SARS-CoV-2 is highly contagious and it has so far infected more than 140 million patients with more than three million victims across the world [3]. COVID-19 mostly involves the lower respiratory tract causing respiratory symptoms ranging from mild respiratory illness to acute respiratory distress syndrome (ARDS) [4]. The most common reported symptoms of COVID-19 patients are fever, cough, dyspnea, myalgia or fatigue, and sputum production [5].
Infection with SARS-CoV-2 causes crucial hematological and immunological changes in patients, which are involved in the pathophysiology of the disease [6,7]. The immune system has an essential role in the control and eradication of viral infections. However, an insufficient or over-responsive immune system may cause serious adverse conditions [8,9]. Inflammatory cells like macrophages are recruited in almost all viral infections and release cytotoxic cytokines, lipid mediators, metalloproteinases, and reactive oxygen species that induce tissue damage [9]. In severe cases of COVID-19, elevated levels of cytokines and chemokines (IL1α/β, IP-10, MCP-1) promote hyper inflammation in the lungs and underlies ARDS. Furthermore, postmortem analysis has shown infiltration of inflammatory cells such as macrophages and lymphocytes in the lung tissue of COVID-19 patients [5,10,11]. Several studies have found a decreased number of CD8 + and NK cells and functional exhaustion of these cells due to overexpression of inhibitory markers like NKG2A and PD-1 [12][13][14].
The most common hematological changes in covid-19 patients include lymphopenia [15,16], neutrophilia [17,18], eosinopenia [19,20], mild thrombocytopenia [15], and thrombocytosis with a lower frequency. The leukocyte count varies between different cases and may be normal, reduced [15,20,21], or increased [3,22]. In some studies, the lymphocyte count is proposed as an indicator for classification of disease severity besides pulmonary imaging as the main test for classification of disease type [23,24]. The platelet count is another indicator of disease severity and prognosis and can reflect the pathophysiological alterations in COVID-19 patients in the early stages [25]. A combination of thrombocytopenia, prolonged prothrombin time (PT) and increased D-dimer is indicative of disseminated intravascular coagulation (DIC) in COVID-19 patients. In addition, deposition of fibrin and thrombin in the pulmonary microvasculature is a factor that contributes to ARDS and coagulopathy in patients dying from COVID-19 [26,27].
Daily global reports warn that the number of patients is rising sharply. Moreover, a lack of information about different aspects of the disease is still a serious obstacle despite all efforts [28]. Hence, the study of the pathophysiology of disease and the effect of the virus on the immune cells and other blood cells, besides improving our knowledge about the disease, can also help to choose effective treatment strategies.
In this comprehensive study, we reviewed the destructive effects of COVID-19 on the quality and quantity of immune and blood cells and discussed the role of these changes in the pathophysiology of the disease.

Alteration in count
While many studies have reported findings related to the NK cell function in COVID-19, the exact role of these cells in defense against SARS-CoV-2 and the pathophysiology of the disease is not defined. In SARS-CoV-2 infected patients, the frequency of NK cells in the blood is decreased [13,[29][30][31][32], or increased [33], or does not show any significant changes [34]. A similar NK cell reduction was reported in SARS-CoV-1 infection previously [35]. After recovery, the NK cell count returns to normal levels in severe COVID-19 patients [33,36]. In addition, the NK cell frequency has the potential to predict COVID-19 severity. The NK cell frequency is lower in patients with ARDS and pneumonia compared to those with a mild phenotype of the disease, which suggests an indirect link between the NK cells count and disease severity in COVID-19 patients [12]. It is still unclear whether NK cell reduction is because of cell death resulting from SARS-CoV-2 or infiltration of inflammatory NK cells into infected organs (particularly the lung) [37]. However, it is unlikely that SARS-CoV-2 induces cell death in NK cells similar to what is observed in other viral infections like influenza [38] because NK cells lack the entry receptor, angiotensin-converting enzyme 2 (ACE-2) [39]. In a study, analysis of the bronchoalveolar lavage fluid (BLAF) of ARDS patients showed no CD16 +CD57 + mature NK cells in the lung tissue, suggesting that the reduction of circulatory NK cells is not a consequence of the migration of these cells into infected lungs [12]. On the contrary, the single cell landscape of bronchoalveolar immune cells in two recent studies reported an increased frequency of NK cells in BALF of COVID-19 patients [40,41]. Therefore, more studies are needed to determine the etiology of NK cell reduction in COVID-19 patients.

Alteration in cell surface markers and function
While several studies have investigated the potential responses of NK cells during COVID-19, there are still many debates in this regard. In a cohort study by Demaria et al. [12], the frequency of NK group 2 member A (NKG2A) expressing NK cells was lower in ARDS patients compared to healthy controls while the NKG2A density on the surface of NK cells was up-regulated. Likewise, other regulatory molecules such as programmed cell death protein-1 (PD-1), CD244 (also known as 2B4), and CD39 were upregulated in COVID-19 patients. More importantly, the levels of cytotoxic effector molecules including granzyme A and perforin decreased in the patients [12,32]. Besides, isolated NK cells from BALF of patients had higher expression of NKG2A, CD39, and PD-1 molecules, even higher than those observed in NK cells derived from blood samples. Interestingly, incubation of NK cells isolated from ARDS patients with monalizumab, an anti-NKG2A monoclonal antibody, unleashed their cytotoxic functions and killing ability [12]. In a study comparing mild and severe forms of COVID-19 patients, Zheng et al. [31] found a significant reduction in the frequency of NK and CD8 + T cells in severe COVID-19 patients. In addition, they reported higher expression levels of inhibitory receptor NKG2A in NK and CD8 + T cells in the severe form of the disease. The expression level of CD107a (a degranulation marker of NK cell) [29] and production of IFN-γ and TNF-α are lower in severe COVID-19 patients [31]. Accordingly, it is hypothesized that NK and CD8 + T cells have an exhausted phenotype in severe COVID-19 patients, which might be due to NKG2A overexpression (Table 1) [31]. To evaluate the possible role of SARS-CoV-2 spike protein (SP) in altering the NK cell function, an in vitro study explored NK cell responses towards lung epithelial cells transfected with spike protein. The results showed that the spike protein might induce chemo-attraction and IFN-γ production of NK cells [42]. Furthermore, CD107a degranulation marker decreased in SP1 transfected cell culture. Further analysis exhibited that the HLA-E/NKG2A/CD94 interaction may be a possible mechanism for the reduction in NK cell cytotoxicity functions [42]. These studies emphasize the exhausted phenotype of NK cells with over-expression of inhibitory molecules and describe NKG2A as a potential cause for this exhaustion during COVID-19 infection. In addition, the data suggests that the exhausted phenotype of NK cells can be a prognostic factor for a more severe phenotype of COVID-19.
In the disease course, a dynamic alteration is seen in the lymphocyte count. According to a recent study, during the disease progression, the lowest level of B cells and CD4+ T cells is seen in the second week after the onset of symptoms, whereas the CD8+ T cells show the lowest level in the third week after the onset of symptoms [58]. In addition, in the recovery phase, the T cell population is larger compared to the acute phase of infection [58] but a sustained lymphopenia with decreased CD3+, CD4+, and CD8+ T cells is found in more severe cases of COVID-19 [46]. After treatment and in the convalescent phase of the disease, the lymphocyte count gradually returns to the range of COVID-19 mild cases [46,64].

B lymphocytes and SARS-CoV-2 infection
The data on B-lymphocytes is still conflicting and needs further investigation. A study on the total B cells and its different subsets indicated that the frequency and the absolute number of total B cells did not change considerably in COVID-19 patients relative to healthy controls [65]. The results of this study showed that transitional B cells and antibody secreting cells (plasmablast/plasma cell) increased in COVID-19 patients whereas the memory B cells reduced significantly indicating a positive correlation with disease severity (Table 1) [65]. Other studies also reported that the number or proportion of plasma cells increased in COVID-19 patients [52,[65][66][67].

T lymphocyte subset and SARS-CoV-2 infection
A decrease in both CD4+ and CD8+ T cells is reported in COVID-19 patients with different disease severities, which is more significant in severe and critical patients [43,45,51,52,54,57,58,60]. Histological analysis of deceased COVID-19 patients showed that the CD4+ and CD8+ T cells decreased in the spleen and lymph nodes of these patients [68].

CD4+ T cells.
The absolute count of Th1, Th2, Th17, Th22, and Treg is reduced in COVID-19 patients [45]. The Th subset differentiation and Th1/Th2 coordination can determine the COVID-19 severity and outcome [44]; for instance, it has been observed that a high percentage of exhausted Th2 cells is associated with a high rate of mortality in COVID-19 patients (Table 1) [44]. Furthermore, the memory T cells also decrease in COVID-19 patients, which can be one of the reasons why infected individuals are not immune to the virus after infection [52].
Th1 cells play a vital role in anti-viral response and induce proliferation and activation of cytotoxic T cell, which leads to removal virusinfected cells [45]. On the other hand, Th2 cytokines are reported to be high in COVID-19 patients [45,51,61]; these cytokines have a suppressive effect on inflammatory Th1 response and induce antibody response [44,45].
Reduction of Treg cells [52,54] and exhaustion of CD4+ and CD8+ T cells in COVID-19 [52,54,68] patients exacerbate the inflammatory status and cause more damage to the lung [46,54]. On the other hand, intense inflammatory cytokines may promote lymphopenia and lymphocyte exhaustion [52] creating a damage cycle all together. A possible reason for Treg cell reduction is direct infection with SARS-CoV-2 or migration of these cells to the lungs [52]. In addition, a decrease in IL-2 level is seen in bronchoalveolar lavage of severe COVID-19 patients, which may lead to Treg apoptosis [52].

CD8+ T cells.
One study found that only CD8+ T cells reduced in asymptomatic COVID-19 patients and the other subsets of lymphocytes in these patients were similar to healthy individuals [58]. It may indicate that CD8+ T cells are more susceptible to SARS-CoV-2 infection [48,51,52,55,63] and their reduction in the early phase of infection may predict the need for ICU admission [69]. Interestingly, a study found that the baseline levels of ACE2 and TMPRSS2 in the lung tissue were negatively related to the level of CD8+ T cells that reside in the lungs before infection [70]. This finding may indicate why some people are more susceptible to more severe infections with the SARS-CoV-2 [70]   since the presence of cytotoxic cells in the lungs can play an important role in rapid containment of the infection in the early stages of the virus infection [70]. Moreover, this study revealed that the baseline level of chemokines attracting CD8+ T cells to the lung had a negative correlation with ACE2 and TMPRSS2 expression levels, which, in return, exacerbates the lack of cytotoxic cells [70]. However, it has been observed that the level of these chemokines increases following the SARS-CoV-2 infection resulting in the migration of CD8+ T cells and other cytotoxic cells to the lungs [70].

Alteration in cell surface markers and function
Although ACE2 is not expressed on lymphocytes, CD147 may act as an alternative receptor for SARS-CoV2 entry, which is expressed on B cells as well as CD4+ and CD8+ T cells [51]. In addition, CD26 (DPP4) is also suggested to be an entry receptor for SARS-CoV-2 on T cells (but does not exist on B cells) [51]. However, the exact role of these receptors is not yet proven warranting further research [51].

CD4+ T cells
The percentage of CD4+ T cells that express HLA-DR and CD38 is also elevated in severe disease, which indicates the activation of these cells [45,56,68]. Increased expression of HLA-DR may be due to a high level of inflammatory cytokines in these patients and therefore it does not indicate specific activation of these cells (Table 1) [45]. A recent study found that the expression of OX40 and 4-1BB increased on CD4+ and CD8+ T cells of COVID-19 patients, respectively. Both markers indicate the active phenotype of these cells and increase more in severe cases [56]. The expression of PD-1 and TIM-3 as exhaustion markers on T lymphocytes increases in severe COVID-19 patients, which is associated with a poor prognosis [51,52,56,68,71].
It has been reported that SARS-CoV-2 specific CD4+ T cells cannot efficiently generate am interferon response, specifically in ICU patients [71]. Additionally, it has been shown that in critical patients admitted to the ICU, the neutralizing antibody titer is not concordant with the level of CD4+ T lymphocytes indicating discordance between B cells, as major participants in the humoral response, and CD4+ T cells [71].

CD8+ T cells
Generally, most studies have reported a hyperactive status in CD4+ and CD8+ T cells [51,63,68,69]. A recent study found that the expression of CD38 and HLA-DR on CD8+ T cells reduced following SARS-CoV-2 infection, which is possibly associated with Th1 dysfunction [45]. However, on the opposite side, it has been revealed that the cytotoxicity of these cells is increased in COVID-19 patients for compensating their count reduction [47,52,69]. In this regard, some studies reported increased expression of HLA-DR, CD38, GrA, GrB, and perforin on CD8+ T cell in SARS-CoV-2 infected patients [47,66,72].

Alteration in count
Neutrophils, as the innate immune cells, are the first cells that are recruited in inflammations. They have important roles in defense against COVID-19 [73]. Neutrophils are recruited to the lungs of COVID-19 patients, and neutrophilia acts as a prognostic factor of ARDS in these patients [22,[74][75][76][77].

Alteration in cell surface markers and function
BALF analysis of COVID-19 patients has shown increased levels of some chemokines such as CXCL2 and CXCL8 leading to recruitment of PMNs to lungs. Although neutrophils are important in defense against viruses, neutrophilia can cause lung injury and ARDS [17,[93][94][95][96][97][98][99]. A study reported an increase in the expression of some genes such as Cathepsin G, IFNγR1, ATP6AP2, CD36 that induce neutrophil apoptosis and downregulation of the TGFB gene in macaques model of COVID-19 infection [78].
Degranulation of neutrophils and release of some proteolytic enzymes such as elastase can cause more injury to lung capillaries and result in edema formation [100]. The level of calprotectin, as a neutrophil activation marker, is correlated with the severity of COVID-19 disease. In addition, it has positive correlations with C-reactive protein, ferritin, lactate dehydrogenase, absolute neutrophil count, and platelet count. Therefore, calprotectin, as an inflammatory marker, can activate neutrophils and lead to their degranulation and phagocytosis. Furthermore, cross-talk between neutrophils and platelets and calprotectin can cause artery and vein thrombosis in COVID-19 [101]. A study revealed strong associations between markers of hyperactive neutrophils (calprotectin and cell-free DNA) and D-dimer [102]. Neutrophils and platelets interact closely [103] via P-selectin and its ligand (PSGL-1) [104,105]. Platelets induce the release neutrophil extracellular traps (NET) from neutrophils in viral infections [106,107]. Neutrophils and platelets are entrapped in the fibrin meshwork in alveolar capillaries of SARS-COV-2 patients. these neutrophils are destructed to some extent and form NETs [77,89,102,108,109].

NETosis during COVID-19 infection
NET is composed of chromatin fibers and citrullinated histones, some special enzymes such as neutrophil elastase, myeloperoxidase, and cathepsin G [110]. The NETosis process can act as a double-edged sword; although it is an essential mechanism for virus entrapment, excessive NET formation increases inflammation, autoimmunity, vascular disease, and thrombosis [102,[111][112][113][114]. COVID-19 patients may develop thromboembolism and arterial thrombosis [95,115]. NETs are associated with the severity of COVID-19 disease and can trigger macrophages to produce more IL-1β precursors [116]. An active form of IL-1β can help with NET formation in vivo and in vitro [117,118]. The NET-IL1β loop is activated in severe COVID-19. An enhanced production of NETs and IL1β can accelerate respiratory decompensation, formation of micro thrombi, and aberrant immune responses. Importantly, IL1β induces IL6 [119], and IL6 is considered as a promising target for COVID-19 treatment [31,120]. NETs can activate the coagulation cascade by acting as a scaffold for VWF and tissue factor (TF) and also by activating the contact pathway. NETs also has an inhibitory effect on anticoagulant pathway such as protein C and tissue factor pathway inhibitors (TFPI) Fig. 1 [111,121]. Some studies found evidence for NETosis in COVID-19 patients, such as aggregation of neutrophils and platelets in the blood, presence of cell-free DNA and MPO-DNA complex [102,109,112,[121][122][123][124][125]. Elevated levels of soluble platelet derived factors that trigger NETosis, including PF4 and RANTES can be observed as the prognosis of the severity of the disease in these patients Table 2 [121]. One study found that the branch length of the NETs released by neutrophils was larger in COVID-19 patients compared to healthy controls [112]. NETs are positively correlated with ARDS [121], multi-organ failure and mortality [122] but NET formation normalizes during convalescence after COVID-19 [121]. NET levels are also significantly higher in the tracheal aspirate fluid than in plasma samples in COVID-19 patients [121]. NET is related to the complement system, and inhibition of C3 and C5 can reduce NET formation [126,127]. Therefore, targeting some parts of the NET formation, thrombosis, or complement system can be a good therapeutic approach for reducing the mortality of COVID-19 patients [128].

Eosinophil alterations in COVID-19 patients
Eosinophils account for 1-3% of circulating leukocytes and have some roles in antiviral responses [129]. Some studies found that more than half the patients admitted with COVID-19 had eosinopenia (defined as an absolute eosinophil count<0.02 * 10 9 cells/L) [83,95,130,131]. This eosinopenia is correlated with the severity of COVID-19 disease [129,132,133]. A low eosinophil count will normalize over time ( Table 2) [83]. No eosinophil enrichment has been found in the pulmonary tissue of patients with COVID-19 in postmortem analyses [134]. Type 2 immune response, including type 2 cytokines (IL-4, IL-13, etc.) and accumulation of eosinophils, might provide potential protective effects against COVID-19 [135]. However, there is controversy over the protective or exacerbating role of eosinophils during SARS-CoV-2 infection [129].

Monocytes/macrophages
Monocytes and macrophages are the main cells of the mononuclear phagocyte system (MPS) that play an important role in both innate and adaptive immune systems [136]. The presence of these two cell populations in all phases of SARS-CoV-2 infection can act as a double-edged sword that can ameliorate infection or exacerbate it [136]. They are the main players in the innate anti-viral immunity. They can also trigger systemic inflammation, pro-coagulant syndrome and cytokine release syndrome (CRS) leading to ARDS and multi organ failure in COVID-19 patients [136][137][138][139][140]. Monocytes and tissue macrophages express a broad set of membrane receptors and secretory elements that mediate their role in both anti-viral and inflammatory responses [136]; the balance between these two responses and the contribution of other immunity mechanisms can determine the severity and outcome of SARS-CoV-2 infection [136].

Alteration in count and morphology
The absolute number of total monocytes and their frequencies are not affected by SARS-CoV-2 virus [141][142][143][144][145]; however, some studies found a decrease in the number of monocytes in critical patients [146,147] and an increase in mild cases [147]. Based on previous studies, it can be concluded that monocytes increase their antiviral response in the early stages, which may indicate a good prognosis. However, in the severe stages of the disease, a decrease occurs in monocytes, suggesting the destructive effects of the virus on monocytes [146]. In patients with SARS-CoV-2 infection, the monocyte distribution width (MDW) shows a higher value compared to healthy individuals. Moreover, among infected individuals, those requiring ICU admission have significantly higher Fig. 1. Role of neutrophils in hypercoagulable state of COVID-19 patients. After the entrance of the SARS-CoV2 virus into the respiratory system, it enters the epithelial cells through ACE2 and causes some damages. This virus travels to the alveoli of the lungs and innate immune cells are recruited. Monocytes activate by identifying PAMPs and DAMPs and neutrophils are recruited and cytokine storm occurs by producing some inflammatory cytokines such as IL-6, IL-1b, TNF-α. Therefore, infiltration of neutrophils occurs, which produce NETs. The NETosis process has some connections with macrophages in the tissue. NETs are composed of MPO, NE, H3, H4, LL37, etc. LL37 activates the p2x7r channel on macrophages and then the efflux of K + ions occur. Hence, NLRP3 inflammasome activates and macrophages produce some cytokines such as IL-1b and IL-18 that can help neutrophils to produce more NETs. On the other hand, the neutrophils that produce NETs can link to platelets in the blood through PSGL1 and p-selectin on the platelets and activate them. These activated platelets release poly p and PDI. PDI, as an enzyme, causes the release of TF micro-vesicles from activated monocytes. Not only this TF activates the extrinsic pathway of coagulation but also adheres to the NETs. VWF adheres to these NETs too, and they can form a scaffold for thrombosis happening. Also, the intrinsic pathway can be activated by activating factor XII on this scaffold. NE extraction of neutrophils inhibits some inhibitory factors of the coagulation system such as protein C, thrombomodulin, and TFPI. Finally, all of the above cause thrombosis in the veins or arteries of COVID19 patients.
MDW values relative to patients with mild symptoms [148]. Therefore, MDW can be considered a prognostic parameter along with other laboratory parameters for predicting unflavored outcomes [148].

Alteration in morphology
Large monocytes with vacuolated cytoplasm are seen specifically in COVID-19 patients [145]. Meanwhile, the results of several studies have confirmed a change in the frequency of different subsets of monocytes in COVID-19 patients [143]; A study showed that the non-classical (NC) monocytes could determine the severity of the disease; NC monocytes below 4% indicate more severe disease and the recovery phase also occurs when the NC monocytes start to increase [144]. Two studies reported a mixed phenotype expressing both M1 and M2 macrophage markers in this specific population (Table 3) [142,145]. Nonetheless, there is a profound contradiction in the results of studies on each cell population, and more research is needed to draw a definitive conclusion about the changes in each monocyte subset.

Alteration in cell surface markers and function
Comparison of COVID-19 patients with healthy individuals shows a significant reduction in HLA-DR expression on monocytes of COVID-19 patients, which has a direct relationship with the severity of symptoms [92,143,147,149]. The high amounts of IL-6 and CRP in COVID-19 patients can downregulate the HLA-DR expression on classical monocytes [143,144]. Impaired HLA-DR expression leads to less antigen presentation and lower anti-viral response by T cells [143,149]. On the other hand, the expression of some functional markers such as CD64 and CD11b increases on monocytes, which is independent of COVID-19 severity [143]. Moreover, one study reported an increase in CD38  expression on monocytes in critical patients, which indicates the enhanced inflammatory status of monocytes [147]. A recent study found reduced CD4 expression on monocytes in severe COVID-19 patients [146]. CD40 expression as an activation marker in blood monocytes has a propensity to decrease in all monocyte subtypes in critically ill patients; however, this reduction is not associated with IL-6 and other inflammatory elements (Table 3) [138]. Monocytes, macrophages (particularly M1), and dendritic cells have a transporting role for SARS-CoV-2 as they bind to the virus by their lectin-binding receptors such as CD169 and carry it to local lymph nodes. Through this transportation, the virus gains access to the lower respiratory tract and even other tissues through the bloodstream [136,149,150]. When SARS-CoV-2 disseminates in the body and reaches other organs, resident tissue macrophages evoke an inflammatory response and cause some dysfunction in the related organ [136]. During the early phase of infection with SARS-CoV-2, overexpression of CD169 on monocytes has been observed; however, its expression decreases in the severe phase of the disease (Table 3) [151]. Under inflammatory conditions, it has been shown that mononuclear phagocytes can activate platelets and produce procoagulant factors such as tissue factors [136,137], factor 5, and factor 13, which indicates their role in hyper-coagulation and generation of thrombo-embolism (Fig. 2) [136]. Furthermore, a recent study reported an increase in monocyte-platelet aggregation (MPA) in COVID-19 patients compared to healthy individuals and a positive relationship between MPA and the severity of disease [152]. These MPAs can be one of the underlying mechanisms for increased risk of thrombotic complications in COVID-19 patients [152].
On the opposite side of the pathological processes that are driven by these cells, the recovery process also depends on macrophages in COVID-19 infection [136]. M2-macrophages can reduce inflammation by producing some lipid metabolites such as resolvins, protectins, and maresins [136]. Besides, secretion of elastase, collagenase, TGFβ, and IGF-1 and blotting out the necrotic cells is a cooperative mechanism done by macrophages resulting in recovery [136]. It has been shown that the number and function of monocytes are restored in the course of COVID-19 remission [147].

Alteration in count
Thrombocytopenia is a common occurrence in COVID-19 patients [158,[161][162][163][164]. The platelet count shows a decreasing trend throughout the disease progression proportional to the severity of the COVID-19 disease [25,158,[161][162][163][164][165][166]. However, it gradually begins to increase after admission in survivors [25,166]. Several retrospective studies showed a more severe reduction in the platelet count in non-survivor COVID-19 patients compared to survivors [25,167]. A low platelet count at the onset of infection can indicate a poor prognosis and a higher chance of mortality [25,166]. A decrease in the platelet count in COVID-19 patients may be associated with several factors including hyper activation of platelets and platelet consumption [25,155,162,168,169]. However, some studies found that the platelet count increased or did not change in COVID-19 patients [153,[156][157][158]166,170]. Elevated levels of IL-6 and other inflammatory cytokines as characteristic inflammatory conditions in COVID-19 patients may promote platelet biogenesis through increasing thrombopoietin production by hepatocytes [153,157,158,170].
An increase in the mean platelet volume (MPV) is directly related to the severity of COVID-19 disease and the rate of platelet activation [155,162,166,168]. However, a prospective study of asymptomatic children that tested positive for SARS-CoV-2 infection found a high MPV even without any clinical signs (Table 4) [154]. A possible underlying

Fig. 2. Interaction between Monocytes-Platelets-T cells in COVID-19 patients.
In COVID-19 patients, the relationship between platelets, monocytes, and T cells exacerbates the cytokine storm, enhances coagulopathies, and diminished the antiviral response of T lymphocytes. As shown figure, the monocyte-platelet interaction through Mac-1/αIIbβ3 and PSGL-1/CD62P leads to the release of TF, FV, and FXIII from platelets. On the other hand, activated platelets can affect the T cell responses by altering some CD4+ T cell functions and Antigen presentation to CD8+ T cells. Moreover, excessive secretion of GM-CSF by T cells exacerbates cytokine release and TF production by monocytes and differentiates them toward M1 pro-inflammatory macrophages. A decrease in the HLA-DR expression on monocytes results in more attenuation of the T cell response due to a decrease in antigen presentation. mechanism for a high MPV in COVID-19 patients may be an increase in the release of young platelet in response to platelet destruction after infection and cytokines such as IL-6 [154,157]. A study reported that MPV/platelet count ratio (MPR) had a correlation with COVID-19 severity as the patients with elevated MPR were more prone to severe pneumonia and were at a high risk of death [157].

Alteration in morphology and coagulation parameters
A short report showed the presence of macro thrombocytes and platelet aggregation in peripheral blood smears of ICU COVID-19 patients while such a manifestation was not observed in stable non-ICU patients [170]. Macro thrombocytes indicate the increased turnover of platelets [168,170] leading to PDW elevation [168]. An autopsy examination showed an increased number of megakaryocytes in the lungs and bone marrow of dead COVID-19 patients [159]. The morphology of BM megakaryocyte indicates the active platelet biogenesis. Moreover, electron microscopy examination of these cells has shown the presence of small amounts of virions in megakaryocytes [159].
Severe thrombocytopenia causes some abnormalities in the results of PT, APTT, INR, D-dimer, and FDP measurement in COVID-19 patients [158,[161][162][163]. The levels of D-dimer [158,161,166,[171][172][173] and fibrinogen [166,172,174] and the fibrinogen-albumin ratio (FAR) [171] are higher in severe COVID-19 patients and the platelet count and albumin level were lower in these patients compared to non-severe patients [25,169,171]. It has been reported that a high level of fibrinogen in COVID-19 patients can make platelets more active and intensify coagulopathies [162,174].

Platelet and map kinase pathway
An in vitro study showed direct infection of platelets by SARS-CoV-2 through ACE2 and TMPRSS2 expression on platelets [162] while other studies did not report such a finding [156,169,175]. The effect of SARS-CoV-2 on platelets is mediated by activation of the MAPK pathway and production of thromboxane A2, which has been shown to increase in COVID-19 patients [156,162,174]. Activation of the MAPK pathway rearranges the platelet cytoskeleton, which subsequently results in the reinforcement of platelet secretory profile, clot retraction, and platelet aggregation [156,162]. Enhanced platelet-leukocyte aggregates are seen in COVID-19 patients [153,156,162,176] and makes platelets respond to IL-6 by assembling the IL-6R subunits and subsequently activates them significantly [175].

Platelet and hyper activation
Hyper activation of platelets following the SARS-CoV-2 infection is reported by several studies [153,156,157,169]. It can occur in several ways including TLR response to DAMP released from damaged tissues and stimulation of complement receptors on platelets leading to thrombosis and platelet depletion [163,175]. Moreover, Fc receptor activation on platelets may be involved in antibody-mediated platelet count reduction in COVID-19 patients [163,175]. There are reports of the increased expression of αIIbβ3 integrin [153,162] and CD62P (P-selectin) on the platelets of COVID-19 patients, indicating the active state of these cells (Table 4) [156,162,174,177]. TF release by damaged endothelial cells together with decreased levels of nitric oxide and prostacyclin are the possible causative mechanisms of this hyper activation [169]. CD62P (P-selectin) expression as a marker of platelet activation increases in COVID-19 patients, especially in severe ones [153,156,157,162,169,174]. Enhancement of CD62P expression results in augmentation of its interaction with PSGL-1 on leukocytes, which causes platelet-leukocyte aggregation [153,156,162,169]. Furthermore, SARS-CoV-2 and its spike protein can trigger the secretion of factor V, factor XIII, PF4, IL-1β, IL-8, and TNFα from platelets (Table 4) [162]. The PF4 level is significantly elevated in COVID-19 patients and is correlated with disease severity [162]. It has been found that spike protein (S1, not S2) can increase αIIbβ3 integrin activation and CD62P expression on platelets in the presence or absence of agonist stimulation [153,162].

Platelets and other blood cells
The platelet-monocyte and platelet-neutrophil interactions play an auxiliary role in tissue factor secretion, micro-thrombosis formation, and ARDS in COVID-19 [175]. The interrelation between platelets and neutrophils can promote the formation of neutrophil extracellular traps (NETs) [153,158,174], resulting in platelet activation and thrombotic complications in COVID-19 patients [158]. In addition, the platelet-monocyte aggregation has been shown to increase in COVID-19 patients, particularly in severe patients, and is associated with the mortality rate [169,174]. In vitro and in vivo observations indicate that adhesion of platelets to monocytes induces TF expression on monocytes [174,178], which is mediated by CD62P adhesion molecule and αIIbβ3 integrin and subsequently contributes to hyper-coagulation in COVID-19 patients [174]. Aggregation of platelets and T cells is increased in COVID-19 patients, which may affect the T cell inflammatory and immune response [156]. In addition, the platelet-to-lymphocyte ratio is higher in patients with SARS-CoV-2 infection compared to non-infected individuals [179].

Platelets and endothelial cells
Activation of endothelial cells, monocytes, granulocytes, and platelets after viral infection may lead to the secretion of pro-coagulant macrovesicles (MVs) containing phosphatidyl serine (PS) and TF, which plays a role in triggering the coagulation cascade [169]. It has been shown that TF-containing MVs are more abundant in the plasma of COVID-19 patients compared to healthy individuals [169]. According to a prospective study of COVID-19 patients, platelets, monocytes, and granulocytes that express tissue factor are higher in COVID-19 patients compared to healthy subjects [169]. In COVID-19, ACE2 expression on endothelial cells causes some damage to these cells, which in turn leads to an increased expression of VonWillebrand factor and a hyper coagulation state in the patients [158,163,175]. Destructed endothelial cells increase platelet adhesion and activation through collagen-GPIV interaction [175] and cause platelet aggregation in the lungs [25,167,168]. On the other hand, due to lung damage, the function of lung megakaryocytes may be impaired and they may no longer be able to produce platelets efficiently [25,155,158,[164][165][166]170]. Moreover, megakaryocyte secretions, which contain cytokines, may cause further injury to the lungs [170]. Adverse immune mechanisms such as autoantibodies against platelets and immune complexes may result in platelet loss [25,158].

Alteration in count and morphology
COVID-19 as a viral infection causes some changes in erythrocytes and their hematological markers. One study found that red blood cell counts were lower in COVID-19 patients compared to normal individuals. It is suggested that inhibition of hematopoiesis by this virus in the bone marrow of patients causes anemia [180]. Anisocytosis, some changes in the lipid and protein membrane of circulating RBCs, erythrophagocytosis, and hemophagocytosis by macrophages engulfed with both erythroblasts and mature erythrocytes are reported in COVID-19 patients [181]. Polychromasia and basophilic stippling can be seen in the blood film of patients, probably as a consequence of an increased reticulocyte count [182]. Huge rouleaux formations and auto agglutination are reported in the blood film too [182]. Spherocytes and schistocytes are also observed, probably as a result of membrane damage induced by fibrin strands occurring in vascular thrombosis with microangiopathy [183]. Another common observation is the presence of high percentages of stomatocytes, knizocytes, and cup-shaped erythrocytes in the blood smear of COVID-19 patients. These morphological changes are associated with COVID-19-related anemia [182,184]. One study reported a few mushroom-shaped cells in the blood film [185]. Red blood cell distribution width (RDW) is increased in severe COVID-19 patients so it can be used as a significant predictor and prognostic factor for the severity and kidney damage of COVID-19 patients [181]. The hemoglobin (Hb) concentration tends to decrease during COVID-19. Therefore, a mild to severe anemia may ensue [186]. There are reports of coated RBCs with some complement proteins such as C3b/iC3b/C3dg. C4d is also seen on the RBCs of COVID-19 patients [187].

Conclusions
In conclusion, the SARS-CoV-2 infection can change immunological and hematological cells in different aspects. These changes appear to play a critical role in disease pathology and the emerging of clinical manifestations. Infection with SARS-CoV-2 contributes to lymphopenia, neutrophilia, eosinopenia, and mild thrombocytopenia. Severe thrombocytopenia in COVID-19 patients causes abnormalities in the results of PT, APTT, INR, D-dimer, and FDP measurements. Hyperactivation of platelets and development of DIC are observed in COVID-19 patients. Furthermore, SARS-CoV-2 and its spike protein can trigger the secretion of factor V, factor XIII, PF4, IL-1β, IL-8, and TNFα from platelets. Accordingly, SARS-CoV-2 can induce coagulopathy and thrombosis in infected patients. COVID19 as a viral infection causes some changes in erythrocytes and its related hematological markers. Anisocytosis, echinocytosis, spherocytosis, schistocytes, stomatocytes, knizocytes, polychromasia, basophilic stippling, and huge rouleaux formation may appear in the blood film of patients. In addition, the hemoglobin (Hb) concentration tends to decrease during COVID19 disease, therefore, mild to severe anemia may occur in the patients. The virus can cause attenuated immune responses or uncontrolled inflammatory responses, which lead to improper defense against the virus and damage to the host respectively. Neutrophils, as first responders to inflammation, can trigger inflammation and lung injury by production of proteolytic enzymes, ROS, and NET. Furthermore, inflammatory cells like monocytes and macrophages are associated with systemic inflammation, procoagulant syndrome, and cytokine release syndrome, which can result in ARDS condition and multi-organ failure in COVID-19 patients. Large monocytes (high MDW) with vacuolated cytoplasm are another feature of SARS-CoV-2 infection. The presence of DC-SIGN, L-SIGN, and CD147 along with ACE2 expression on DCs makes these cells a vulnerable target for SARS-CoV-2 entry and infection. HLA-DR and co-stimulatory molecules such as CD80 and CD86 are downregulated in DCs following infection with SARS-CoV-2 virus and can subsequently affect the T cell responses and even cause their apoptosis. The absolute count of CD4+ and CD8+ T cells is lower in severe patients. The effect of SARS-CoV-2 on lymphocyte function is a controversial subject and further studies are needed. Lymphocyte morphology is also affected by SARS-CoV-2 and atypical lymphocytes with plasmacytoid features are common in the blood smear of COVID-19 patients. The SARS-CoV-2 can turn normal NK and CD8+ T cells, which are important to eliminate the virus, to exhausted phenotype, which makes the patients prone to other viral infections. Considering these changes may help to manage and treat COVID-19 patients better.

Author contributions
EA: Originated the study, Drafted the manuscript. AO: final approval of the version to be published. ZB: Made substantial contributions to the conception. EZ: revised article for important intellectual content. All authors have approved the submitted version and have agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.

Funding
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Consent for publication
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CRediT authorship contribution statement
Ehsan Ahmadi: Originated the study, acquired data, Drafted the manuscript. Azadeh Omidkhoda: Made substantial contributions to the conception and substantively revised manuscript. Zahra Bagherpour: Acquired data and Design some part of the work. Elmira Zarei: Design some part of the work. All authors have approved the submitted version and have agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.

Conflict of interest statement
The authors have no conflicts of interest to declare.

Data Availability
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