The Dysregulation of the Monocyte-Dendritic Cell Interplay Is Associated with In-Hospital Mortality in COVID-19 Pneumonia.

Background: The monocyte-phagocyte system (MPS), including monocytes/macrophages and dendritic cells (DCs), plays a key role in anti-viral immunity. We aimed to analyze the prognostic value of the MPS components on in-hospital mortality in a cohort of 58 patients (M/F; mean age ± SD years) with COVID-19 pneumonia and 22 age- and sex-matched healthy controls. Methods: We measured frequencies and absolute numbers of peripheral blood CD169+ monocytes, conventional CD1c+ and CD141+ (namely cDC2 and cDC1), and plasmacytoid CD303+ DCs by means of multi-parametric flow cytometry. A gene profile analysis of 770 immune-inflammatory-related human genes and 20 SARS-CoV-2 genes was also performed. Results: Median frequencies and absolute counts of CD169-expressing monocytes were significantly higher in COVID-19 patients than in controls (p 0.04 and p 0.01, respectively). Conversely, percentages and absolute numbers of all DC subsets were markedly depleted in patients (p < 0.0001). COVID-19 cases with absolute counts of CD169+ monocytes above the median value of 114.68/µL had significantly higher in-hospital mortality (HR 4.96; 95% CI: 1.42-17.27; p = 0.02). Interleukin (IL)-6 concentrations were significantly increased in COVID-19 patients (p < 0.0001 vs. controls), and negatively correlated with the absolute counts of circulating CD1c+ cDC2 (r = -0.29, p = 0.034) and CD303+ pDC (r = -0.29, p = 0.036) subsets. Viral genes were upregulated in patients with worse outcomes along with inflammatory mediators such as interleukin (IL)-1 beta, tumor necrosis-α (TNF-α) and the anticoagulant protein (PROS1). Conversely, surviving patients had upregulated genes related to inflammatory and anti-viral-related pathways along with the T cell membrane molecule CD4. Conclusions: Our results suggest that the dysregulated interplay between the different components of the MPS along with the imbalance between viral gene expression and host anti-viral immunity negatively impacts COVID-19 outcomes. Although the clinical scenario of COVID-19 has changed over time, a deepening of its pathogenesis remains a priority in clinical and experimental research.

New Insights into the Identification of Metabolites and Cytokines Predictive of Outcome for Patients with Severe SARS-CoV-2 Infection Showed Similarity with Cancer.

SARS-CoV-2 infection is characterized by several clinical manifestations, ranging from the absence of symptoms to severe forms that necessitate intensive care treatment. It is known that the patients with the highest rate of mortality develop increased levels of proinflammatory cytokines, called the "cytokine storm", which is similar to inflammatory processes that occur in cancer. Additionally, SARS-CoV-2 infection induces modifications in host metabolism leading to metabolic reprogramming, which is closely linked to metabolic changes in cancer. A better understanding of the correlation between perturbed metabolism and inflammatory responses is necessary. We evaluated untargeted plasma metabolomics and cytokine profiling via 1H-NMR (proton nuclear magnetic resonance) and multiplex Luminex assay, respectively, in a training set of a limited number of patients with severe SARS-CoV-2 infection classified on the basis of their outcome. Univariate analysis and Kaplan-Meier curves related to hospitalization time showed that lower levels of several metabolites and cytokines/growth factors, correlated with a good outcome in these patients and these data were confirmed in a validation set of patients with similar characteristics. However, after the multivariate analysis, only the growth factor HGF, lactate and phenylalanine retained a significant prediction of survival. Finally, the combined analysis of lactate and phenylalanine levels correctly predicted the outcome of 83.3% of patients in both the training and the validation set. We highlighted that the cytokines and metabolites involved in COVID-19 patients' poor outcomes are similar to those responsible for cancer development and progression, suggesting the possibility of targeting them by repurposing anticancer drugs as a therapeutic strategy against severe SARS-CoV-2 infection.

Immune profiling of SARS-CoV-2 epitopes in asymptomatic and symptomatic pediatric and adult patients.

BACKGROUND: The infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has unpredictable manifestations of coronavirus disease (COVID-19) and variable clinical course with some patients being asymptomatic whereas others experiencing severe respiratory distress, or even death. We aimed to evaluate the immunoglobulin G (IgG) response towards linear peptides on a peptide array containing sequences from SARS-CoV-2, Middle East respiratory syndrome-related coronavirus (MERS) and common-cold coronaviruses 229E, OC43, NL63 and HKU1 antigens, in order to identify immunological indicators of disease outcome in SARS-CoV-2 infected patients. METHODS: We included in the study 79 subjects, comprising 19 pediatric and 30 adult SARS-CoV-2 infected patients with increasing disease severity, from mild to critical illness, and 30 uninfected subjects who were vaccinated with one dose of SARS-CoV-2 spike mRNA BNT162b2 vaccine. Serum samples were analyzed by a peptide microarray containing 5828 overlapping 15-mer synthetic peptides corresponding to the full SARS-CoV-2 proteome and selected linear epitopes of spike (S), envelope (E) and membrane (M) glycoproteins as well as nucleoprotein (N) of MERS, SARS and coronaviruses 229E, OC43, NL63 and HKU1 (isolates 1, 2 and 5). RESULTS: All patients exhibited high IgG reactivity against the central region and C-terminus peptides of both SARS-CoV-2 N and S proteins. Setting the threshold value for serum reactivity above 25,000 units, 100% and 81% of patients with severe disease, 36% and 29% of subjects with mild symptoms, and 8% and 17% of children younger than 8-years reacted against N and S proteins, respectively. Overall, the total number of peptides in the SARS-CoV-2 proteome targeted by serum samples was much higher in children compared to adults. Notably, we revealed a differential antibody response to SARS-CoV-2 peptides of M protein between adults, mainly reacting against the C-terminus epitopes, and children, who were highly responsive to the N-terminus of M protein. In addition, IgG signals against NS7B, NS8 and ORF10 peptides were found elevated mainly among adults with mild (63%) symptoms. Antibodies towards S and N proteins of other coronaviruses (MERS, 229E, OC43, NL63 and HKU1) were detected in all groups without a significant correlation with SARS-CoV-2 antibody levels. CONCLUSIONS: Overall, our results showed that antibodies elicited by specific linear epitopes of SARS-CoV-2 proteome are age dependent and related to COVID-19 clinical severity. Cross-reaction of antibodies to epitopes of other human coronaviruses was evident in all patients with distinct profiles between children and adult patients. Several SARS-CoV-2 peptides identified in this study are of particular interest for the development of vaccines and diagnostic tests to predict the clinical outcome of SARS-CoV-2 infection.

Cytokine signature and COVID-19 prediction models in the two waves of pandemics.

In Europe, multiple waves of infections with SARS-CoV-2 (COVID-19) have been observed. Here, we have investigated whether common patterns of cytokines could be detected in individuals with mild and severe forms of COVID-19 in two pandemic waves, and whether machine learning approach could be useful to identify the best predictors. An increasing trend of multiple cytokines was observed in patients with mild or severe/critical symptoms of COVID-19, compared with healthy volunteers. Linear Discriminant Analysis (LDA) clearly recognized the three groups based on cytokine patterns. Classification and Regression Tree (CART) further indicated that IL-6 discriminated controls and COVID-19 patients, whilst IL-8 defined disease severity. During the second wave of pandemics, a less intense cytokine storm was observed, as compared with the first. IL-6 was the most robust predictor of infection and discriminated moderate COVID-19 patients from healthy controls, regardless of epidemic peak curve. Thus, serum cytokine patterns provide biomarkers useful for COVID-19 diagnosis and prognosis. Further definition of individual cytokines may allow to envision novel therapeutic options and pave the way to set up innovative diagnostic tools.

Clinical Outcome Prediction in COVID-19 Patients by Lymphocyte Subsets Analysis and Monocytes' iTNF-α Expression.

In December 2019, a novel coronavirus, "SARS-CoV-2", was recognized as the cause of coronavirus disease 2019 (COVID-19). Several studies have explored the changes and the role of inflammatory cells and cytokines in the immunopathogenesis of the disease, but until today, the results have been controversial. Based on these premises, we conducted a retrospective assessment of monocyte intracellular TNF-α expression (iTNF-α) and on the frequencies of lymphocyte sub-populations in twenty-five patients with moderate/severe COVID-19. We found lymphopenia in all COVID-19 infected subjects compared to healthy subjects. On initial observation, in patients with favorable outcomes, we detected a high absolute eosinophil count and a high CD4+/CD8+ T lymphocytes ratio, while in the Exitus Group, we observed high neutrophil and CD8+ T lymphocyte counts. During infection, in patients with favorable outcomes, we observed a rise in the lymphocyte count, in the monocyte and in Treg lymphocyte counts, and in the CD4+ and in CD8+ T lymphocytes count but a reduction in the CD4+/CD8+ T lymphocyte ratio. Instead, in the Exitus Group, we observed a reduction in the Treg lymphocyte counts and a decrease in iTNF-α expression. Our preliminary findings point to a modulation of the different cellular mediators of the immune system, which probably play a key role in the outcomes of COVID-19.

An observational study of a cohort of citizens receiving the AZD1222 vaccine against SARS-CoV-2.

In this retrospective study, a cohort of 67 subjects vaccinated with AZD1222 was retrospectively observed. Consistently with published findings, no serious adverse event was reported, and all adverse events reported (fever, muscle ache and/or pain in the site of injection) had resolved by day 8. Of note, some citizens were prescribed low-dose aspirin and even heparin for thrombosis prevention. We also found variations in laboratory test results (full blood count and chemistry) on day 1 compared with day 8. Physicians should be aware that no prevention therapy for thrombosis is currently recommended, given the very low incidence of this side effect. Additional studies are warranted to interpret our findings.

Mindray BC-6800 haematological analyser: 3D-DIFF scattergram usefulness in infectious mononucleosis diagnosis.

INTRODUCTION: 3D-DIFF scattergram of the Mindray BC-6800 haematological analyser shows morphological abnormalities and lymphocyte cluster splitting related to the presence of reactive lymphocytes. This study aims to assess whether these cytographic changes are useful in detecting both activated and apoptotic lymphocytes, leading to an improvement in the laboratory diagnostic process of infectious mononucleosis. METHODS: Two hundred three samples with modified shape and doubled lymphocyte cluster of DIFF scattergram (study group) were divided into two different subgroups: with and, respectively, without serological evidence of ongoing IM. Activated and apoptotic cells in peripheral blood were counted by light microscopy or gating in the instrumental dot plots. Values of apoptotic cells counted by microscopy were compared with those resulting from gating. RESULTS: Samples with both shape change and doubled lymphocyte cluster had serological profiles according to the diagnosis of ongoing infectious mononucleosis. Blood smears review was positive for reactive lymphocytes in all 112 samples (100%). An underestimation of apoptotic cell count by light microscopy compared with the gating in the instrumental scatterplot was also observed (96 out of 112, 85.7%). CONCLUSION: The additional lymphocyte cluster was significantly associated with activated and apoptotic lymphocytes in samples with serology suggesting ongoing infectious mononucleosis. Considering the significance of clue for infectious mononucleosis assigned to the apoptotic lymphocytes, a specific flag such as "apoptotic cells?" could be associate with the related cluster. Such a flag could be used for dedicated rules for smears review, thus increasing infectious mononucleosis detection in laboratories that do not usually practise instrumental cytograms observation.

The diagnostic use of ADVIA 2120i Siemens and an "APL criteria" can help to reduce the rate of early death in the APL.

INTRODUCTION: Acute promyelocytic leukemia (APL) is a type of acute myeloid leukemia (AML) with a life-threatening coagulopathy. Once it is suspected, ATRA should be started. Appreciation of APL details is critical, but an experienced hematopathologist may not be available. We developed an algorithm, based on the parameters generated by automated blood cell counter ADVIA 2120i Siemens that can aid the diagnosis of APL. METHODS: All parameters in the algorithm were selected on the bases of the pathophysiology of the APL and the analyzer's technology. We used c1 cutoff: PLT < 150 × 103 ; % Mono<10; % LUC<4; % hyperchromic cells > 2; %saturated cells ≥ 1; % blasts > 4. Satisfying at least five of six cutoffs, we obtained an "APL criteria". It was tested on 247.209 raw-data from routine and emergency samples and on 124 raw-data from APL. We performed a multiparametric analyses and obtained definitive cutoffs (c2). It was validated on a new group of 51.002 raw-data from routine and emergency. Finally, it was tested on AML and ALL, MDS, MPN, oncologic samples, and hemolytic and megaloblastic anemia. RESULTS: The algorithm provided a high sensitivity (86.30%) and high specificity (99.83%) in APL with high or normal number of WBC and satisfactory results in APL with cytopenia (80%). The specificity was very high also in groups of hematological and nonhematological diseases. It can be used as an "APL criteria" to alert pathologists of the possible presence of APL. It together with instrumental and classical morphology may allow to reduce the time of diagnosis with further reduction in early death.

Short preheating at 41°C leads to a red blood cells count comparable to that in RET channel of Sysmex analysers in samples showing cold agglutination.

AIMS: The presence of cold agglutinin in blood samples can cause a spontaneous agglutination of red blood cells (RBCs) when low temperature occurs. This phenomenon causes a spurious lowering of RBC count on the automated haematological analysers that are detected by incongruous values (≥370 g/L) of the mean cellular haemoglobi concentration (MCHC). A preheating at 37°C can remove the RBC agglutination generally resulting in a reliable count. It has been reported that the same result can be reached by using the optical reticulocyte (RET) channel of Sysmex analysers where the RBC count is not influenced by the presence of cold agglutinin. This study aims to evaluate these data in a larger population, with regard to environmental conditions on Sysmex analysers. We have also evaluated the influence of different thermal pretreatments on the RBC count. METHODS: This study was performed on 96 remnants of peripheral blood samples (48 with MCHC in normal range and 48 with MCHC>370 g/L) which have been analysed in different preanalytical conditions on the Sysmex analysers. RESULTS: A preheating of samples at 41°C for 1 min leads to a reversibility of the cold agglutination comparable to the one observed in the RET channel and yields better results compared with 37°C for 2 hours. CONCLUSIONS: None of described procedures assure the complete cold agglutination reversibility in every case. Consequently, since the haematological analysers not yet provide reliable parameters to confirm the complete resolution of agglutination, further verification of RBC count accuracy needs to be performed.

Automatic wedge smears preparation may cause traumatic morphological changes in peripheral blood cells.

In recent years, several automated analysers that prepare and stain blood smears have been introduced in clinical laboratories. Despite the use of instrumental settings based on physical characteristic of individual samples, traumatic injuries of neutrophil and lymphocytes can be observed. Some samples present a very high percentage of damaged cells, allowing the speculation that a cellular susceptibility may enhance mechanical traumatism. These artefacts can puzzle morphological evaluation in both traditional and digitised microscopy; in addition, unskilled operators can be misled.

A prolonged microscopic observation improves detection of underpopulated cells in peripheral blood smears.

We evaluated an extended time in the microscopic review in samples in which the potential clinical information could be increased with respect to those that could be achieved with the usual laboratory methodologies. We used samples containing nucleated red blood cells in a small amount and cytopenic samples. For these purposes for each peripheral blood smear, the timing of eye-count differential was increased up to 20 min, regardless of the final number of cells which could be counted. In addition, an automated system for digital analysis of peripheral blood smears was employed and the number of cells counted was brought up to 1000 leukocytes. In both manual and automatic light microscopy extended observation, we obtained more diagnostic information in respect to those with routine or standard methods. Both automated and manual increase systems of the timing for microscopic review are useful tools to find diagnostic information that otherwise would be lost using normal and standard procedures. So, these methods should be used especially when there is a higher pre-test probability for discovery of pathological cells.

Detection of Apoptotic Lymphocytes Through Sysmex XN-1000 As a Diagnostic Marker for Mononucleosis Syndrome.

BACKGROUND: The infectious mononucleosis (IM) includes clue elements, apoptotic and atypical lymphocytes. In IM, the evaluation of dot plot provided by Sysmex XN-1000 analyzer revealed a stretched lymphocytic cluster, white cell differential channel (WDF), on cytogram. METHODS: In this study, we analyzed 698 samples that include 39 IM, 76 chronic lymphoproliferative disorders, 25 nonclonal lymphocytosis, and 40 healthy donors. Five hundred eighteen samples with other diseases or interference were evaluated. The algorithm was validated on 40,000 files that were received from internal database of Sysmex-Dasit. RESULTS: The analysis of flow cytometry standard (FCS) files in WDF channel and presumed apoptotic lymphocytes counts on side scatter/forward scatter (SSC/FSC) and SSC/SFL (where SFL is side fluorescence) dot plot revealed excellent correlation among apoptotic cells on peripheral blood smear (R(2)  = 0.79 and 0.75). There was a variation of positional parameters in lymphocyte clusters WX, WY, and WZ. If WX-SSC > 500 and WY-SFL > 1,000 and WZ-FSC > 700, specificity equals to 99% and sensitivity equals to 100%. If nucleated red blood cell (NRBC) <0.03 × 10(3) /µl, specificity equals to 100%. In received files, positives were 1% adding the simultaneous presence of a percentage of events in the two gates relating to the apoptotic lymphocytes of 1.2% of WBC. CONCLUSION: On Sysmex XN-1000, dot-plot observation allowed immediate detection of IM. Meanwhile, an algorithm based on the parameters on these plots can be calculated with excellent performance.