3.3 SARS-CoV-2 induces cell death and morphological changes in leukocytes of COVID-19 patients.
We analyzed cell death levels by annexin-V and propidium iodide reactivity as a phenotypic marker for apoptosis and necrosis. Single annexin-V expression shows early apoptosis, while the presence of annexin-V and propidium iodide results in late apoptosis, and only staining with propidium iodide establishes necrosis.
According to cell death (Fig. 4), an increase in both early apoptosis (243.1%, Fig. 4A) and late apoptosis (466.7%, Fig. 4B) was observed in the viremia-COVID-19 group, respect to control; in contrast, the MODS-COVID-19 group, showed a greater increase in late apoptosis (1363%, p<0.001) compared to the early apoptosis (147.6%, p<0.01), was presented reflecting rapid changes in cellular damage processes. Also, the MODS-COVID-19 negative group showed an increase in early and late apoptosis processes (130% and 496%, respectively) compared to the control group.
Finally, in the necrotic death process (Fig. 4C) it is observed that the viremia-COVID-19 group presents higher levels (3.8 folds more, p<0.01) than the control; while the MODS-COVID-19 group presented lower levels compared to the viremia group, but higher than the control (1.6 folds higher, p<0.05). Regarding the negative MODS COVID group, there was also a slight increase in necrosis (0.5 folds), although it was not significant compared to the control.
In regard to morphological changes in leukocytes are show in Fig. 5. It is observed that infection by SARS-CoV-2 induces modifications in the morphology of leukocytes, generating the presence of cellular structures that indicate changes in their functions. In Figure 5A, Band neutrophils with coarsely clumped chromatin and cytotoxic granulation, observed in greater quantity in the MODS stage. 5B and 5C, Hypersegmented neutrophils with vacuoles, in addition, loss of membrane integrity is observed, these cells were found in the sepsis stage. 5D, Band neutrophils with cytotoxic granulation and vacuoles, observed at all stages. 5E, monocytes with both cytoplasmic and nuclear digestive vacuoles are observed, indicating increased phagocytic activity. These monocytes were observed in greater quantity in COVID-19 patients in MODS stage and in smaller quantity in the sepsis stage; 5F, vacuolated neutrophils, these neutrophils present vacuoles in the cytoplasm and were observed in all stages of the disease. 5G: Leukocytes with membrane rupture. In the MODS-COVID-19 stage, leukocytes with a completely ruptured membrane were observed, indicating death from cell lysis. in 5H: Hypersegmented neutrophils with cytotoxic granulation are observed, granulations indicate a systemic inflammatory process in addition to a viral or bacterial infectious process, granulations were observed in all stages of the disease, however hypersegmentation was observed at the stage of MODS. 5I, reactive lymphocytes (hyperbasophilic) are observed, which presents a scarce cytoplasm in addition to a marked basophilia, smaller in size as a mature lymphocyte and observed at all stages of SARS-CoV-2 infection.