Morphological Changes in Blood Cells as Indicators for Disease Progression in COVID 19

A novel highly pathogenic human corona virus (COVID19) has been recently recognised in Wuhan, China as the cause of corona disease outbreak. It has rapidly spread from China to various countries across the world evolving as a pandemic. In our study we have categorized the covid positive patients into mild, moderate and severe based on the clinical criteria suggested by WHO. The coagulation parameters of the patients were analysed and documented. A peripheral smear was made for every patient and the morphological changes in blood cells were documented. The peripheral smear ndings were then correlated with the disease stage and coagulation parameters. There were signicant differences in the total WBC count and the differential WBC count between stages 1 &2 and stages 1 & 3 (p<0.005). Leucocytosis, neutrophilia and toxic changes in neutrophils were seen in severe stage of the disease and in covid coagulopathy suggesting these are important indicators of disease severity. Schistocytes an important nding in any other coagulopathy was not present in covid associated coagulopathy. Activated lymphocytes was found to be the most common morphological presentation seen in all covid patients irrespective of the disease stage whereas plasmacytoid lymphocytes was an important nding in severe stage disease. Monocyte cytoplasmic vacuoles, large/giant platelets were other morphological ndings observed but these ndings did not have any signicant correlation with disease stage. Since follow up smears of the same patient were not analysed during disease progression and also post recovery, additional research in this eld will provide further insights.


Introduction
A novel highly pathogenic human corona virus (COVID19) has been recently recognized in Wuhan, China as the cause of corona disease outbreak. It has rapidly spread from China to various countries across the world evolving as a pandemic. The pandemic has an alarming morbidity and mortality occurring as a result of acute respiratory distress syndrome [1]. Covid 19,emerged in Wuhan China in 2019. The disease is caused by coronavirus which belongs to a group of RNA viruses that causes disease in mammals and birds. It belongs to the family coronaviridae in the order Nidovirales. They are positively charged RNA virus with the largest genome [2].
The mode of spread of this virus is by droplet infection and close contact [3]. The pathogenesis of Covid 19 seems to be a little complicated. The virus enters through the respiratory tract and attaches to the epithelial cells, the macrophages and the dendritic cells causing their activation. Their activation results in the activation of innate immune system and release of a variety of cytokines and chemokines.This leads to a dysregulated immune response activation resulting in a prothrombotic state [3].The prothrombotic states results in formation of microclots along the microvasculature. D dimer, a degradation product of brin (FDP) is increased in the peripheral blood of the Covid positive patients supporting the thrombotic state [4].Thus as a result the coagulation system and the hemostasis is deranged leading to abnormalities in the coagulation studies [5][6][7]. Viral-induced morphologic changes in the peripheral blood cells are well characterized in certain infections and can direct diagnostic workup to ensure timely therapeutic intervention. Our study represents a systematic analysis of peripheral smears of COVID positive patients. The patients were divided into mild, moderate and severe based on the clinical presentation as suggested by WHO [10,11]. The various coagulation parameters of the patients like PT, APTT, D dimer and platelet count were estimated and documented simultaneously. Peripheral smears of the patients stained with Leishman stain were analysed to look for morphological changes. The peripheral smear ndings were then correlated with the clinical stage of the disease and coagulation parameters. An analysis was done to identify whether the morphological changes in peripheral smears can be used as a parameter to indicate disease progression.

Study design and population
The study was planned as a prospective, multicenter study. A total of thousand Covid positive patients admitted in the tertiary care centre during the study period were taken for the study.

Inclusion criteria
All Covid RT PCR positive patients were taken into the study irrespective of the age, sex and pre existing health status.

Exclusion criteria
RT PCR negative patients were excluded from the study irrespective of the CT chest ndings.

Data collection
The patients were classi ed as mild, moderate & severe based on the clinical criteria suggested by the WHO and the ndings were documented.
The coagulation parameters like prothrombin time(PT), activated partial thromboplastin time (aPTT), Ddimer of the patients were analysed in blood collected in blue top vacutainers (3.2% sodium citrate). The preanalytical procedures were strictly adhered to and the samples were run within two hours of collection in IL-ACL top semi-automated coagulation analyser. The values were documented simultaneously.
The EDTA sample of the Covid patients were used to prepare the peripheral smears. The smears were stained using Leishman stain following the standard operating protocol. The stained smears were then analysed by a trained pathologist.
Morphological changes in various blood cells were analysed and documented. Morphological changes in neutrophils included toxic changes and shift to the left. Toxic change is de ned as presence of coarse purple cytoplasmic granules and cytoplasmic vacuoles. Morphological changes in lymphocytes were divided into two groups, activated monocytoid lymphocytes and plasmacytoid lymphocytes. Monocytoid lymphocytes have abundant vacuolated cytoplasm with a large sometimes lobated nucleus and cytoplasmic vacuoles resembling a monocyte. Plasmacytoid lymphocytes are cells with dark basophilic cytoplasm , round peripherally placed nucleus condensed chromatin and sometimes with a perinuclear hoff resembling a plasma cell. Morphological changes in monocytes like cytoplasmic vacuolations were documented. Presence of large/giant platelets were looked for and documented. Giant platelets are those platelets which are almost the size of RBC where as large platelets are somewhat smaller in size when compared to RBC's. Other parameters like blood hemoglobin level, WBC count(total count & differential count)and the platelet count were also documented.

De nitions
The WHO criteria for clinical classi cation of Covid 19 positive patients.

Mild :
Symptomatic patients meeting the case de nition for COVID-19 without evidence of viral pneumonia or hypoxia Moderate: Clinical signs of pneumonia (fever, cough, dyspnoea, fast breathing) but no signs of severe pneumonia, including SpO2 ≥ 90% on room air Severe: With clinical signs of pneumonia (fever, cough, dyspnoea, fast breathing) plus one of the following: respiratory rate > 30 breaths/min; severe respiratory distress; or SpO2 < 90% on room air

DATA ANALYSIS
The peripheral smear ndings documented were then correlated with the coagulation parameters and the disease severity. The data was analyzed using SPSS software. Descriptive statistics were used to summarize data. Categorical data were presented as number-percentages, and numerical data were presented as median, minimum, and maximum. Roc analysis was performed to nd a cut-off point for differential morphological pattern between mild, moderate and severe disease stage.

Results
Thousand patients of Covid 19 admitted during the study period were taken into study. The patients were classi ed as mild(stage 1) moderate(stage 2)& severe (stage 3) based on the clinical criteria suggested by WHO. In the 1000 patients 79% belonged to mild category, 16% belonged to moderate and 5% belonged to severe category.

Comparison of Peripheral blood counts and Clinical Stage
The clinical stage of the patient was correlated with total WBC count and differential count. The variation of total WBC count , differential neutrophil count, differential lymphocyte count and differential monocyte count between stages 1&2 and stages 1&3 was found to be signi cant (p<0.005). Whereas the correlation between other groups were not signi cant(p>0.005).
The mean blood count values in different clinical stage of the disease is described in Table 1. An increase in mean WBC count, differential neutrophil count and a decrease in differential lymphocyte count was seen in stage 3 disease.
Totally 12% of cases (out of 1000 cases) showed leucocytosis. 8% cases in stage 1(out of 789 cases), 27% of cases in stage 2(out of 163 cases) and 32 % of cases in stage 3(out of 48 cases) showed leucocytosis. Thus emphasizing the fact that an increase in WBC count was seen in the course of disease progression.
35% of cases (out of 1000 cases) showed neutrophilia. 25% cases in stage 1(out of 789 cases), 72% cases in stage 2 (out of 163 cases) & 89% (out of 48 cases) cases in stage 3 showed neutrophilia. Thus an increase in neutrophil count serves as a signi cant factor in determining disease progression.
14.7% of cases (out of 1000 cases) showed thrombocytopenia. There was no signi cant difference in platelet count between groups.

Comparison of Peripheral smear morphological changes and Clinical Stage
The morphological changes of the blood cells and their presentations in various disease stages are discussed in Table 2. From the ndings it has been observed that presence of neutrophilia with toxic changes were more prevalent in stage 3 disease thus emphasizing the fact that an increase in neutrophil count and occurrence of toxic changes in neutrophils is an important indicator for disease progression[ Fig. 1]. Activated lymphocytes and monocytoid lymphocytes were common in all stages signifying the fact that this is a common morphological parameter seen in all covid patients irrespective of the stage of the disease [Fig. 2]. Whereas occurrence of plasmacytoid lymphocytes was more common in stage 3 disease emphasizing the fact this morphological nding can also be a factor indicating disease progression [Fig. 2].
Monocyte cytoplasmic vacuolation and giant platelets were other morphological ndings observed in the peripheral smear but there was no signi cant relationship between these changes and the disease stage [ Fig. 3  3.5% of cases in the study presented with prolonged PT. 60% of these cases belonged to stage 2 and stage 3 and 40% belonged to stage 1. The peripheral smear ndings of the patients with prolonged PT are discussed in Table 2 (ii) Prolonged APTT & peripheral smear ndings 3.3% of cases in the study presented with prolonged APTT. 55% of these cases belonged to stage 2 and stage 3 and 45% belonged to stage 1. The peripheral smear ndings of the patients with prolonged APTT are discussed in Table 2 (ii) Elevated D dimer & peripheral smear ndings 19.5% of cases in the study presented with elevated D dimer values. 55% of these cases belonged to stage 2 and stage 3 and 45% belonged to stage 1. The peripheral smear ndings of the patients with elevated D dimer values are discussed in Table 4 (iv) Prolonged PT, APTT , elevated D dimer values & peripheral smear ndings 1% of covid patients in our study presented with derangement of all coagulation parameters( PT, APTT & D dimer). The peripheral smear ndings of these patients are described in table 5 The peripheral smear ndings of patients with deranged coagulation parameters ( prolonged PT , APTT & elevated Dimer) showed neutrophilia, toxic changes in neutrophils & activated lymphocytes as the most common presentation. Therefore presence or development of these morphological parameters in covid positive patients may suggest that the patient is progressing into a state of coagulopathy.

Discussion
Our study demonstrates signi cant numerical and morphological changes in the blood cells in Covid positive patients.It shows that there is signi cant variation in these parameters between different stages of the diseases.
Our study showed signi cant variation in WBC counts between stages 1 &2 and between stages 1&3. This was in correlation with the study by Irene S Pakos et al who described an increase in WBC count as a signi cant factor to suggest disease progression [12]. Similarly an increase in neutrophil count and associated lymphopenia were noted in severe stage of the disease which also was in correlation with the study by Chen H et al [13].
There was signi cant changes in the morphology of WBC seen in the peripheral smear. The most common morphological change observed in our study is activated lymphocytes which was seen in all Covid 19 patients irrespective of the disease stage, this was in correlation with a study by Florian Luke et al [14]. Yue Ping Lee et al reports lower incidence of plasmacytoid lymphocytes when compared to monocytoid lymphocytes which was in correlation with our study [15]. However our study showed increased incidence of plasmacytoid lymphocytes in stage 3 disease which was also in correlation with a study by Chun Tsu lee [9].
The neutrophils also showed signi cant morphological changes. The presence of toxic changes in the cytoplasm of neutrophils was found in higher percentage in stage 3 disease, which was in correlation with the study by Olga Pozdnyalova et al [16]. However Pozdnyalova et al described a higher percentage of shift to left of neutrophils in stage 3 disease whereas in our study the percentage of occurrence was almost equal in all stages. Pseudo Pelger-Huet anomaly and hypogranulation were described as a common morphological change in neutrophils in a study by Ilhami Berber et al but in our study these changes were found only in insigni cant numbers [17].
The morphological changes observed in monocytes in our study were cytoplasmic vacuolations and granulations. These changes were also described by Amindersingh et al [1]. However in our study there was no correlation between the monocyte morphology and disease stage but a study by Ilhami Berber et al described the occurrence of monocyte cytoplasmic vacuoles in higher percentage in severe disease.
Thrombocytopenia was observed in 14.5% of cases in our study but there was no signi cant correlation between platelet count and disease stage. A study by Julie Brogaard Larsen et al described mild thrombocytopenia as a common presentation in stage 3 disease [18]. Large /giant platelets were found in 50% of patients in our study which was in correlation with a study by Maryame Abnach et al [19].
Only limited studies have compared coagulation parameters with the peripheral smear ndings. A study by Charles et al suggested toxic changes in neutrophils as the common morphological ndings in patients with Covid associated coagulopathy [20].This was in correlation with our study which showed neutrophils with toxic changes and also activated lymphocytes as the most common presentation.
The most common nding, presence of schistocytes (fragmented RBC's) seen in any other coagulopathy was almost absent in Covid coagulopathy. The patients with deranged coagulation parameters in our study also did not show any signi cant association with presence of schistocytes [ Fig.5].
This study has some limitations. Even though the study population was high (1000 patients) the percentage of patients in stage 3 severe disease was comparatively low (5%). Second, follow up smears of the same patient was not analysed during disease progression and also post recovery. So any occurrence or disappearance of the preexsisting morphological changes were not studied.     Schistocytes and microsherocytes in a patient with covid associated coagulopathy