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Authors: Carissimo et al. 

Journal/ Pre-Print:bioRxiv 

NB: Supplementary tables/figures not available at time of review 

Tags: Clinical, Diagnostics, Immunology/Immunity, Inflammation 

Research Highlights

  1. Acute Covid-19 patients (n=54) had decreased absolute numbers of CD8+ T-cells and VD2 γδ T-cells in the circulation compared to healthy controls, but these cells showed an activation phenotype. 

  1. Acute Covid-19 patients (n=54) had increased absolute numbers of immature neutrophils in the circulation compared to healthy controls or recovered patients, even though the number of total neutrophils was not different. 

  1. In this cohort the ratio of immature neutrophils to VD2 γδ T-cellor to CD8+ T-cells in the blood was a diagnostic marker for pneumonia and hypoxia. 

Summary

Carissimo et al. revealed increased immature neutrophil counts and decreased CD8+ T-cells and VD2 γd T-cells counts in the blood of patients with acute COVID-19 (n=54) compared to recovered patients (n=28) and healthy controls (n=19) using in-depth flow-cytometry analysis (total 44 markers). The immature neutrophil counts correlated with IL-6 and IP-10 levels in the blood. Decreased eosinophils, basophils, DCs, and NK-cells in acute stages was also shown.  

Samples were stratified into no pneumonia, pneumonia only, pneumonia and hypoxia, or pneumonia and hypoxia requiring ICU admission. ROC analysis revealed that the count ratio of immature neutrophils to CD8+ or VD2 T-cells correlates to disease severity.  

Impact for SARS-CoV2/COVID19 research efforts 

Understand the immune response to  SARS-CoV2/COVID19  

Develop diagnostic tools for SARS-CoV2/COVID19 
 

Study Type 

  • Patient Case study54 patients with laboratory-confirmed SARS-CoV-2 infection were recruited at the National Centre for Infectious Diseases (NCID), Singapore from end March to 90 mid-May 2020. 

Strengths and limitations of the paper 

Novelty: Extensive phenotyping of whole blood samples with 3 large FACS panels with a useful focus on a large number of leukocyte subsets and their activation status including neutrophils and  γδ T-cells. While most studies examine percentages of different leukocyte types in the blood, this study also quantified absolute numbers, which is more informative.  

Standing in the field:In line with previous publications CD8+, CD4+, MAIT, VD1 and VD2 T-cells showed a gradual reduction in the peripheral blood with increasing disease severity. In contrast with other studies total circulating neutrophils showed no significant change but only in the immature neutrophils compartment. The presence of immature neutrophils in the blood of Covid-19 patients has been described multiple times before. 

Appropriate statistics: YesAlthough sample size was based on sample availability, and restrictions due to cross-sectional methodology of the study rather than longitudinal. Not clear which ANOVA test was used – both Brown-Forsythe and Welch were mentioned. 

Viral model used:SARS-CoV-2 infected patients 

Translatability:The immature neutrophil/VD2 T-cell ratio may associate with disease severity 

Main limitations:  

> The main limitation is that this is a cross-sectional study that cannot claim to identify any prognostic information. The study is rather an associative assessment of the immune phenotype that correlates with acute infection at the time of sampling.  

> There is a significant flaw in performing the analysis and then using the same patients to assess predictive value (Figure 5B). A separate independent test set of patients should have been used to determine predictive value.  

Potential confounders (e.g. patient age, gender, treatment, comorbidities) were not taken into account when calculating the correlation between cell count ratios and disease severity. 

> Overall a small sample number for each group particularly when they are subsequently subdivided into the various pneumonia/severity groups 

> The recovered samples were not paired with the acute COVID-10 patient samples (not the same patients) 

> Large degree of heterogeneity between patients within the acute COVID-19 group in terms of clinical outcome but unable to assess this further due as supplementary data not available 

Similarly, it is unknown if the different patient groups were well-matched. In demographics 

> Not clear whether the more severe patients were no treatment that may interfere with immune phenotype (e.g. steroids) 

A number of figures report only the mean of each group and don’t report on spread of the data e.g. by SD (Fig. 1B/D/E, 2C, 4A). 

> The number of paired plasma samples was rather low (19 out of 54 patients), therefore the fit of the correlation between IL-6/IP-10 and immature neutrophils is poor.