BackgroundThe immune system is a crucial part of the body’s defense against infection and disease. However, individuals in antigen-limited environments face unique challenges that can weaken their immune systems. This systematic review aimed to investigate the impact of an exposure to an isolated, confined and extreme (ICE) environment with limited antigen diversity on human immune parameters.MethodsA systematic literature search was conducted using PubMed, Web of Science and Cochrane Library to identify relevant studies on immune system parameters in ICE environments. The studies were grouped by ICE type (space missions, microgravity simulations like bed rest studies, space simulation units like MARS500, and Antarctic research stations) to allow for clearer comparison and analysis of immune outcomes.ResultsAnalysis of 140 studies revealed considerable heterogeneity in study designs and outcomes, reflecting the complexity of immune responses across ICE environments. Nevertheless, immune dysregulation was consistently observed across environments. Space missions and Antarctic stations, in particular, showed pronounced immune changes, likely due to low antigen diversity and extreme conditions, with higher rates of infections and allergic responses suggesting increased vulnerability. Space simulation units exhibited immune changes similar to those in actual space missions, while gravity simulation studies, which focus on fluid shifts and bone loss, showed fewer immune alterations. Across environments, most immunological measures returned to baseline after isolation, indicating resilience and the potential for recovery upon re-exposure to diverse antigens.ConclusionReduced antigen diversity in ICE environments disrupts immune function, with effects often compounded by extreme conditions. Although immune resilience and recovery post-isolation are promising, the heterogeneity in current studies highlights the need for targeted research to identify specific immune vulnerabilities and to develop countermeasures. Such measures could reduce immune-related health risks for individuals in isolated settings, including astronauts, polar researchers, and vulnerable populations on Earth, such as the elderly or immunocompromised, thereby enhancing resilience in confined environments.Systematic Review Registrationhttps://www.crd.york.ac.uk/prospero/, identifier CRD42023476132.

Drug allergies (DA), particularly to anesthetics, are underexplored in Morocco. Traditional diagnostic methods such as skin tests and specific IgE assays are costly, risky, and often inaccessible. The basophil activation test (BAT) is emerging as a safer and more effective alternative, especially for diagnosing propofol allergy, an under-researched area. This study examines the role of the basophils activation test in diagnosing perioperative anaphylaxis to propofol. A 32-year-old woman experienced two anaphylactic reactions during fibroscopy after propofol administration. The basophil activation test (BAT) was performed one week later using diluted propofol, measuring basophil activation via CD63 and CD203c markers by flow cytometry. Results showed 0% basophil activation in the negative control, 85.7% in the positive control, and 19.05% at 1 mg/ml of propofol, with a strong correlation (0.973) between concentration and activation. The basophil activation test (BAT) offers a reliable diagnostic method, particularly in Morocco, where traditional tests are limited, providing a cost-effective solution for diverse allergens in low-resource settings.

Background. Routine lymphocyte counting requires quality assurance validation using quality controls (QCs) that closely resemble fresh human blood leukocytes. This study aimed to evaluate a novel artificial product designed to mimic leukocyte light scatters and marker expression. Methods. FlowCytes and TruCytes, “artificial cell mimics” (Slingshot Biosciences, Emeryville, CA, USA), were tested on CE-IVD-certified systems, namely FACSCanto, FACSLyric (BD Biosciences), Navios, and DxFlex (Beckman Coulter), using routine staining, lysing, fixation, and no-wash procedures for T, B, and NK counting. Results. FlowCytes and TruCytes provided forward and side scatter profiles comparable to human leukocytes on the FACSCanto, FACSLyric, and DxFlex systems but not on Navios despite adapting the process to be slightly different from the manufacturer’s recommendations. TruCytes demonstrated robust immunolabeling of CD3, CD4, CD8, and CD19 on the FACSCanto, FACSLyric, and DxFlex systems, with fluorescence intensities and subset distributions being similar to those usually observed in fresh human blood. However, CD16 and CD56 labeling was inconsistent and depended on the antibody clones used. Regrettably, monocyte and granulocyte mimics lacked expression of CD4, CD16, and CD14. TruCytes also displayed significantly lower concentrations of TBNK lymphocyte subsets compared to healthy human blood. Conclusions. FlowCytes and TruCytes show promises as internal quality controls for T cell and B cell immunophenotyping, but not NK cells. They are compatible with most CE-IVD cytometers, even when using lysis/fixation/no-wash routine diagnosis procedures. Further multicentric studies are warranted to assess their performance relative to existing products, such as stabilized human blood.

Drug hypersensitivity reactions to essential antibiotics like vancomycin pose significant diagnostic and therapeutic challenges, particularly in vulnerable populations such as pediatric patients with immune deficiencies. We present the case of a 7-year-old girl with primary immunodeficiency who experienced immediate hypersensitivity reactions to vancomycin, including urticaria and angioedema, managed with corticoids and antihistamines. The Basophil Activation Test (BAT) conducted two years after the last allergic episode revealed significant basophil activation across all tested vancomycin dilutions, with CD63 and CD203c expression exceeding negative and positive controls. These findings confirm vancomycin hypersensitivity and underscore the BAT's utility as a reliable in vitro diagnostic tool, especially in settings where skin testing or specific IgE assays are unavailable. This case highlights the BAT's potential for broader adoption in clinical allergy practice, particularly in resource-limited environments. It emphasizes the importance of reliable diagnostic methods for managing drug hypersensitivity in high-risk patients.

Ce travail est une synthèse de la thèse du Dr Ghazi et est basée sur une revue systématique de la littérature et une étude clinique observationelle transversalle en cours de publication. Introduction Au cours des cinq dernières décennies, la concentration en spermatozoïdes a diminué de 50 % dans la population masculine [1]. Parallèlement, l’exposition à l’aluminium a augmenté de manière significative. Une revue systématique de la littérature réalisée sur 40 articles sélectionnés à partir de 111 publications sur le sujet, nous a permis d’évaluer la reprotoxicité masculine de l’aluminium. Dans l’ensemble, elle révèle une toxicité limitée mais réelle de l’aluminium sur le système reproducteur masculin. Cette toxicité se manifeste par une altération modérée de l’épithélium séminifère entraînant une réduction limitée mais significative du nombre de spermatozoïdes dans l’éjaculat. Le mécanisme de cette toxicité pourrait être la génération d’un stress oxydatif au niveau des testicules. Par ailleurs, l’aluminium semble s’accumuler au sein de la tête du spermatozoïde [2] à proximité de l’ADN spermatique [3]. Matériel et méthodes Nous avons évalué le lien entre la concentration d’aluminium et la fragmentation de l’ADN dans le sperme de 80 patients effectuant une insémination intra-utérine. Nous avons également évalué l’exposition à l’aluminium des patients à l’aide d’un questionnaire. Résultats Aucune corrélation significative entre la quantité d’aluminium dans le sperme et la fragmentation de l’ADN des spermatozoïdes n’a été trouvée. De même, aucune différence de concentration d’aluminium dans le sperme n’a été trouvée entre les patients exposés à l’aluminium et les autres dans l’une ou l’autre des modalités d’exposition explorées, à savoir : exposition professionnelle, alimentaire, cosmétique ou médicale. Conclusion L’aluminium présent dans le sperme ne semble pas induire un taux plus élevé de fragmentation de l’ADN des spermatozoïdes. Toutefois, au vu de la littérature, l’aluminium pourrait être l’un des nombreux toxiques environnementaux contribuant à la baisse de la fertilité masculine observée dans le monde.

Rheumatoid arthritis (RA) is characterized by progressive joint destruction with subsequent serious disability. Objective biomarkers of RA course progression are lacking, which necessitates the discovery of activity indicators and predictors of the disease outcome. Musculoskeletal Ultrasound Seven-joint Score (MSUS7) is proposed as a reliable technique to evaluate radiographic RA progression. Homo sapiens-microRNA-21-5p (hsa-miR-21-5p) plays an important role during joint remodeling and the pro-inflammatory process driving RA progression. We aimed to evaluate plasma hsa-miR-21-5p as a noninvasive RA activity biomarker and to investigate if hsa-miR-21-5p is linked to MSUS7 components in the context of RA activity. This cross-sectional study included 71 RA patients classified into inactive (n = 36) and active (n = 35) groups according to the Disease Activity Score 28-joint count with ESR (DAS28-ESR). Joints were assessed by MSUS7. Gray-scale ultrasound (GSUS) and power Doppler ultrasound (PDUS) were used to rate the synovitis, tenosynovitis, and erosion in the joints. Plasma hsa-miR-21-5p expression was measured by real-time PCR. The absolute count of regulatory T cell (Treg) was calculated after Treg frequency was assessed by flow cytometry. Results: Hsa-miR-21 expression was significantly up-regulated in the active RA group with a median fold change of 51.6 in comparison to the inactive cases with a median fold change of 7.7 (p < 0.001). Hsa-miR-21-5p was positively correlated with DAS28-ESR, C reactive protein (CRP), and rheumatoid factor (r = 0.7, p < 0.001, r = 0. 0.6, p < 0.001, and r = 0.4, p = 0.002, respectively), while negatively correlated with Treg absolute count (r = -0.4, p < 0.001). Hsa-miR-21-5p levels were correlated with synovitis and tenosynovitis in GSUS (r = 0.4, p < 0.001, r = 0.3, p = 0.025, respectively) and in PDUS (r = 0.5, p < 0.001 and 0.4, p = 0.001, respectively). The hsa-miR-21-5p accurately distinguished RA activity [AUC 0.933, 94.3% sensitivity, and 86.1% specificity]. Logistic regression analysis revealed hsa-miR-21-5p as an independent predictor for RA flare (OR = 1.228, p = 0.004). Hsa-miR-21-5p was linked to synovitis and tenosynovitis components of the MSUS7. Up-regulated hsa-miR-21-5p can be utilized as a predictor for RA disease flare.

Cross reactivities are known for human leukocyte antigen inside HLA-B7 related Cross-Reactive Group (B7CREG). Some CE-IVD flow-cytometry kits use double monoclonal antibodies (mAb) to distinguish HLA-B27 and HLA-B7 but practice reveals more complexes results. This study explores the performances of this test. Analysis of 466 consecutive cases using HLA-B27 IOTest™ kit on a Navios™ cytometer from Beckman-Coulter, partially compared to their genotypes. Expected haplotypes HLA-B27-/HLA-B7- (undoubtedly HLA-B27 negative) and HLA-B27+/HLA-B7- (undoubtedly HLA-B27+) were clearly identified according to the manufacturer's instructions. On the opposite, patients strongly labeled with anti-HLA-B7 showed three different phenotypes regarding anti-HLA-B27 labeling: (1) most of the cases were poorly labeled in accordance with cross reactivity inside B7CREG (HLA-B27-/HLA-B7+ haplotype); (2) rare cases had strong B7 and B27 labeling corresponding to HLA-B27+/HLA-B7+ haplotype; (3) even less cases had strong labeling by anti-HLA-B7 but non for anti-HLA-B27, all expressing HLA-B44 and no B7CREG molecules. Surprisingly, more cases were not labeled with anti-HLA-B7 antibody but partially labeled with anti-HLA-B27 suggesting another cross reactivity out of B7CREG. mAb HLA typing suggests new, cross reactivities of anti-HLA-B27 antibody to more molecules out of B7CREG and of anti-HLA-B7 antibody but not anti-HLA-B27 to HLA-B44 molecule also out of B7CREG. HLA-B27 could surely be excluded in most samples labeled with HLA-B27, below a “grey zone” on intermediate intensity. More comparison is needed in future studies.

Introduction (contexte de la recherche) Neuromuscular blocking agents (NMBAs) are the main drugs involved during perioperative immediate hypersensitivity reaction. Identification of culprit drug and safe alternatives are challenging. Basophil activation test (BAT) has emerged as a tool to secure diagnosis. Very few authors evaluated CD203c expression in patients with NMBAs hypersensitivity reaction and questions remain regarding the threshold for positivity. Objectif To define an optimal threshold for BAT positivity, as measured by expression of the CD203c marker, determine their diagnostic value in NMBAs hypersensitivity. Méthodes 104 patients, included from March 2020 to May 2023, with clinical features suggestive of NMBA hypersensitivity and for whom skin tests (ST) and BATs for NMBAs were performed and classified into 3 groups (43 allergic patients, 49 negative patients and 12 control patients). Optimal thresholds for BAT positivity (CD203+) expressed as percentage of basophil activation (Ba%), stimulation index (SI) or Median Fluorescence Intensity (MFI) ratio were identified by ROC curve analysis. We then calculated the diagnostic performance of BATs and assessed the concordance with ST results. Résultats We calculated thresholds of 6.6% (Ba%), 3.2 (SI) and 5.5% (Ba%), 2.7 (SI) for suxamethonium (Se=74% ; Sp=100%) and rocuronium (Se=79%; Sp=100%) respectively (AUC>0.8). The optimal MFI ratio threshold was 1.1 for the 2 NMBAs. BATs demonstrated no discriminatory performance in atracurium reactions (AUC≈0.5). Global sensitivity (Se) and specificity (Sp) of BAT (all NMBAs combined) were 68% and 100% respectively. We found excellent positive predictive values (100%) combined with very high negative predictive values, ranging from 95% to 99%, for all the parameters studied. Applying the optimal thresholds expressed in Ba%, the BAT then identified sensitization to NMBAs in 5 patients with negative skin tests. Finally, we observed very good concordance between ST and BAT. Conclusions BAT, using CD203c expression, is a reliable tool for reinforcing the diagnosis of NMBAs allergy, and more specifically to suxamethonium and rocuronium. The technique can also be used to identify potential cross-reactions and safe alternatives.

Cholangiocarcinoma (CCA) is a rare form of cancer, accounting for approximately 3% of all gastrointestinal cancers and 15% of primary liver malignancies. Most patients will require a multidisciplinary approach, including palliative, radiotherapy and systemic therapy (chemotherapy or, more recently, immunotherapy). Local ablation therapy using radiofrequency ablation (RFA) has also been explored in the setting of perihiliar cholangiocarcinoma (pCCA) as an adjunctive to palliative drainage. Tumour-infiltrating lymphocytes (TILs) are an adaptive immune cell type that can travel directly to the tumoural site to suppress cancer proliferation and development. Immune checkpoint inhibitor (ICI)-based therapies that act directly on the T-cell signalling pathway have proven particularly useful in recent trials, with molecules targeting the cytotoxic T-lymphocytes associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathways showing efficacy in a wide range of indications. In this context, our study aimed to measure the potential effect of RFA on the intratumoural expression of checkpoint molecules, with an impact on modulating immunotherapy. We present here an exploratory study presenting an optimized flow cytometry protocol for tissue dissociation and cell characterization in terms of PD-1/PD-L1 and CTLA-4 expression. Our results show that RFA significantly increases the infiltration of cluster of differentiation 8(+ )(CD8(+)) cells and that the expression of PD-1 and CTLA-4 was significantly increased on cluster of differentiation 3(+) (CD3(+)) cells after RFA. RFA might promote a "switch" from an "immune-cold" to an "immune-hot" tumour microenvironment (TME) profile, with a significant impact on enabling immunotherapy for CCA patients.

PurposeIn this study, we retrospectively reviewed the use of flow cytometry (FCM) in the diagnosis of inborn errors of immunity (IEIs) at a single center in Algeria. Sharing insights into our practical experience, we present FCM based diagnostic approaches adapted to different clinical scenarios.MethodsBetween May 2017 and February 2024, pediatric and adult patients presenting with clinical features suggestive of immunodeficiency were subjected to FCM evaluation, including lymphocyte subset analysis, detection of specific surface or intracellular proteins, and functional analysis of immune cells.ResultsOver a nearly seven-year period, our laboratory diagnosed a total of 670 patients (372 (55.5%) males and 298 (44.5%) females), distributed into 70 different IEIs belonging to 9 different categories of the International Union of Immunological Societies classification. FCM was used to diagnose and categorize IEI in 514 patients (76.7%). It provided direct diagnostic insights for IEIs such as severe combined immunodeficiency, Omenn syndrome, MHC class II deficiency, familial hemophagocytic lymphohistiocytosis, and CD55 deficiency. For certain IEIs, including hyper-IgE syndrome, STAT1-gain of function, autoimmune lymphoproliferative syndrome, and activated PI3K delta syndrome, FCM offered suggestive evidence, necessitating subsequent genetic testing for confirmation. Protein expression and functional assays played a crucial role in establishing definitive diagnoses for various disorders. To setup such diagnostic assays at high and reproducible quality, high level of expertise is required; in house reference values need to be determined and the parallel testing of healthy controls is highly recommended.ConclusionFlow cytometry has emerged as a highly valuable and cost-effective tool for diagnosing and studying most IEIs, particularly in low-income countries where access to genetic testing can be limited. FCM analysis could provide direct diagnostic insights for most common IEIs, offer clues to the underlying genetic defects, and/or aid in narrowing the list of putative genes to be analyzed.

Background: Chronic lymphocytic leukemia (CLL) is characterized by a wide range of tumor-induced immune alterations. Regulatory T cells (Treg) play a central role in these immune responses. CD200 and Leukocyte- associated immunoglobulin-like receptor-1 (LAIR-1, CD305) are inhibitory markers said to be involved in Treg immune response. We aimed to analyze the expression of CD200 and LAIR-1 on leukemic cells and assess their interactions with the Treg frequency to elucidate their role in the CLL course. Subjects and methods: This study was conducted on 70 participants: 50 newly diagnosed CLL cases classified according to Rai staging system into group 1 (n = 25) patients with stages 0, I, and II, and group 2 (n = 25) of advanced patients with stages III and IV. In addition to control group (n = 20) of healthy adults. Flow cytometry was used to investigate Treg frequency in bone marrow (BM) proportional to CD4+ T cell and to assess leukemic cell expression of CD200 and LAIR-1. Also, in-silico database analysis was performed to identify study markers interactions for future personalized target therapy. Results: Comparison between CLL groups 1 and 2 revealed increased leukemic cell percentage expressing LAIR-1 (p = 0.021) in group 1. Group 2 showed significant increase in frequency of Treg in BM and leukemic cells expressing CD200. There was a strong positive correlation between frequency of Treg and leukemic cells expressing CD200 (r = 0.669, p = 0.000). On the other hand, there was a negative correlation between frequency of Treg and leukemic cell expressing LAIR-1 (r =-0.342, p = 0.015). ROC curve analysis revealed that increased frequency of leukemic cells expressing CD200 yielded sensitivity (SN) and specificity (SP) of 96 % and 84 %, respectively in detecting CLL progression, with an AUC of 0.965. Leukemic cell percentages expressing LAIR-1 yielded a lower SN (75 %), SP (72 %), with an AUC of 0.688. Conclusion: Treg frequency in BM was significantly increased in CLL advanced stages according to Rai classification. Leukemic cells CD200 and LAIR-1 expression were differently associated with Treg frequency. Increased CD200 expressions on leukemic cells can be considered a sensitive and specific biomarker in detecting CLL progression. As demonstrated by the in-silico research, CD200 blockade targeting may offer therapeutic benefits for CLL treatment through Treg suppression.

Cross reactivities are known for human leukocyte antigen inside HLA-B7 related Cross-Reactive Group (B7CREG). Some CE-IVD flow-cytometry kits use double monoclonal antibodies (mAb) to distinguish HLA-B27 and HLA-B7 but practice reveals more complexes results. This study explores the performances of this test. Analysis of 466 consecutive cases using HLA-B27 IOTest (TM) kit on a Navios (TM) cytometer from Beckman-Coulter, partially compared to their genotypes. Expected haplotypes HLA-B27-/HLA-B7- (undoubtedly HLA-B27 negative) and HLA-B27+/HLA-B7- (undoubtedly HLA-B27+) were clearly identified according to the manufacturer's instructions. On the opposite, patients strongly labeled with anti-HLA-B7 showed three different phenotypes regarding anti-HLA-B27 labeling: (1) most of the cases were poorly labeled in accordance with cross reactivity inside B7CREG (HLA-B27-/HLA-B7+ haplotype); (2) rare cases had strong B7 and B27 labeling corresponding to HLA-B27+/HLA-B7+ haplotype; (3) even less cases had strong labeling by anti-HLA-B7 but non for anti-HLA-B27, all expressing HLA-B44 and no B7CREG molecules. Surprisingly, more cases were not labeled with anti-HLA-B7 antibody but partially labeled with anti-HLA-B27 suggesting another cross reactivity out of B7CREG. mAb HLA typing suggests new, cross reactivities of anti-HLA-B27 antibody to more molecules out of B7CREG and of anti-HLA-B7 antibody but not anti-HLA-B27 to HLA-B44 molecule also out of B7CREG. HLA-B27 could surely be excluded in most samples labeled with HLA-B27, below a "grey zone" on intermediate intensity. More comparison is needed in future studies.

PurposeThe authors aim to investigate the altered monocytes subsets distribution in liver cirrhosis (LC) and subsequent hepatocellular carcinoma (HCC) in association with the expression level of plasma Homo sapiens (has)-miR-21-5p and hsa-miR-155-5p. A step toward non-protein coding (nc) RNA precision medicine based on the immune perturbation manifested as altered monocytes distribution, on top of LC and HCC.MethodsSeventy-nine patients diagnosed with chronic hepatitis C virus (CHCV) infection with LC were enrolled in the current study. Patients were sub-classified into LC group without HCC (n = 40), LC with HCC (n = 39), and 15 apparently healthy controls. Monocyte subsets frequencies were assessed by flow cytometry. Real-time quantitative PCR was used to measure plasma hsa-miR-21-5p and hsa-miR-155-5p expression.ResultsHsa-miR-21-5p correlated with intermediate monocytes (r = 0.30, p = 0.007), while hsa-miR-155-5p negatively correlated with non-classical monocytes (r = - 0.316, p = 0.005). ROC curve analysis revealed that combining intermediate monocytes frequency and hsa-miR-21 yielded sensitivity = 79.5%, specificity = 75%, and AUC = 0.84. In comparison, AFP yielded a lower sensitivity = 69% and 100% specificity with AUC = 0.85. Logistic regression analysis proved that up-regulation of intermediate monocytes frequency and hsa-miR-21-5p were independent risk factors for LC progression to HCC, after adjustment for co-founders.ConclusionMonocyte subsets differentiation in HCC was linked to hsa-miR-21-5p and hsa-miR-155-5p. Combined up-regulation of intermediate monocytes frequency and hsa-miR-21-5p expression could be considered a sensitive indicator of LC progression to HCC. Circulating intermediate monocytes and hsa-miR-21-5p were independent risk factors for HCC evolution, clinically and in silico proved.

PurposeThe gastrointestinal (GI) microbiota is a complex and dynamic ecosystem whose composition and function are influenced by many internal and external factors. Overall, the individual GI microbiota composition appears to be rather stable but can be influenced by extreme shifts in environmental exposures. To date, there is no systematic literature review that examines the effects of extreme environmental conditions, such as strict isolation and confinement, on the GI microbiota.MethodsWe conducted a systematic review to examine the effects of isolated and confined environments on the human GI microbiota. The literature search was conducted according to PRISMA criteria using PubMed, Web of Science and Cochrane Library. Relevant studies were identified based on exposure to isolated and confined environments, generally being also antigen-limited, for a minimum of 28 days and classified according to the microbiota analysis method (cultivation- or molecular based approaches) and the isolation habitat (space, space- or microgravity simulation such as MARS-500 or natural isolation such as Antarctica). Microbial shifts in abundance, alpha diversity and community structure in response to isolation were assessed.ResultsRegardless of the study habitat, inconsistent shifts in abundance of 40 different genera, mainly in the phylum Bacillota (formerly Firmicutes) were reported. Overall, the heterogeneity of studies was high. Reducing heterogeneity was neither possible by differentiating the microbiota analysis methods nor by subgrouping according to the isolation habitat. Alpha diversity evolved non-specifically, whereas the microbial community structure remained dissimilar despite partial convergence. The GI ecosystem returned to baseline levels following exposure, showing resilience irrespective of the experiment length.ConclusionAn isolated and confined environment has a considerable impact on the GI microbiota composition in terms of diversity and relative abundances of dominant taxa. However, due to a limited number of studies with rather small sample sizes, it is important to approach an in-depth conclusion with caution, and results should be considered as a preliminary trend. The risk of dysbiosis and associated diseases should be considered when planning future projects in extreme environments.Systematic review registrationhttps://www.crd.york.ac.uk/prospero/, identifier CRD42022357589.

BackgroundFully mature monocytes that express CD14, but not CD16, undergo phagocytosis within tissues, whereas non-classical monocytes, CD14-low CD16+, represent <11% of peripheral monocytes and have primary pro-inflammatory functions. Inflammation plays a major role in Covid-19 disease and adds to the inflammation caused by chronic hemodialysis. The aim of our study was to monitor monocyte subsets in five patients with end-stage kidney disease (ESKD) over a 1-year period after a mild Covid-19 infection. Five ESKD patients with a mild Covid-19 infection were monitored using CD14, CD16, CD300e, HLA-DR, CD64, and CD45 panels using a BD FACS Canto flow cytometer.ResultsCD14-low CD16+ was dramatically (p=0,001) decreased in patients during Covid-19 infection, as previously described for patients without chronic renal failure. In addition, CD14-low CD16+ monocytes remained decreased for 10 months after recovery from Covid. Intermediate monocytes increased during Covid-19 infection and decreased 10 months after infection but this subtype of monocytes retained their inflammatory activity with a significant increase in HLA-DR expression after recovery from Covid infection.ConclusionOur study shows that ESKD patients had a pro-inflammatory profile induced by Covid 19, but this status was prolonged significantly over a 10-month period. Thus, advanced renal failure treated by hemodialysis did not dramatically change the inflammatory response against to SARS Covid 2. It seems that monocytes retain their inflammatory status for many months in ESKD patients after a Covid-19 infection.

Multiple immunolabeling introduces high risks of interferences between fluorescences. As an example, in analyzing T cell clonality, we recently reported a fluorescence resonance energy transfer (FRET) effect providing an unexpected signal on B770 (PE-Cy7) detector, on the Vβ-PE positive CD3 APC-Alexa750+ T cell subsets. Here, we report another FRET effect produced by the violet laser in Vβ-FITC positive CD3-Pacific Blue (PB) T cells providing signal on V550 (Krome Orange; KrO) detector. The study was performed on fresh whole blood, labeled with anti-CD3-PB, CD8-KrO, Vbeta FITC, Vbeta PE, CD4 AA750 then fixed, treated for erythrolysis, and washed before analysis on DxFlex cytometer from Beckman Coulter. Data were analyzed using Kaluza software. Using this panel, we repeatedly observed an added CD8dim-KrO (V550) cell population on all Vβ FITC positive T cells. The unexpected green signal excited by the violet laser was still observed after removing anti-CD8-KrO (FMO) but disappeared where either anti-CD3-PB or anti-Vβ-FITC was removed. The effect was also observed with an anti-TCR gamma delta-FITC labeling, but not with another FITC labeled antibody targeting a protein out of the CD3-TCR complex. The analysis of fluorochrome spectra confirms that PB emission and FITC excitation spectra partly overlap. This observation clearly reminds users that FRET can give misleading results in case of labeling of very close markers with complementary fluorochromes. This risk has to be considered in panel design. These observations clearly highlight the potential for FRET to give misleading results in cases where very close markers are labeled with complementary fluorochromes. This risk must be considered when designing panels. To our knowledge, this is the first description of a FRET between PB and FITC as acceptor thus excited by the violet laser.

Small extracellular vesicles (EVs) are characterized by the membrane expression of CD63, CD81 and CD9 tetraspanins. Their size is inferior to 200 nm. They share the same characteristics as the native cells and are found in human fluids. Specific membrane protein biomarkers expressed on small EV are useful for the diagnosis of tumoural pathologies. Clear cell renal cell carcinoma (CCRCC) is diagnosed by imaging examinations and/or tissue biopsy. Carbonic anhydrase IX (CAIX) is a powerful biomarker of CCRCC. The detection of CAIX on small EV from the urine of patients could constitute a liquid biopsy for CCRCC. We have set up a specific protocol for the preparation, the immunostaining characterization and the transmission electron microscopy observation of small EVs isolated from the urine of CCRCC patients. The background labelling was significantly reduced. We successfully detected biomarkers on urinary small EVs from CCRCC patients. This technique could be extended with antibodies directed against other EV biomarkers for the detection and the monitoring of cancer diseases.

Serum exosomes frequently are used for liquid biopsy. Serum exosomes normally are isolated using ultracentrifugation; however, ultracentrifugation is time-consuming, labor intensive and requires a high-speed centrifuge. Many commercial kits use a precipitation-based method; however, this process can result in substantial contamination. We developed a new method to isolate pure serum exosomes. We isolated serum exosomes using precipitation, extracted them using acetone, then isolated them again by precipitation. We used transmission electron microscopy (TEM) to examine the morphology of serum exosomes. TEM indicated that our isolated exosomes were pure with typical morphology and with a size ranging from 40 to 150 nm. Flow cytometry revealed expression of exosome markers, CD63, CA81 and CD9. Our double precipitation method enables ready extraction of pure exosomes from serum. Our double precipitation method simplifies detection of serum exosomal biomarkers for diagnosis and prognosis of disease.

Early hepatocellular carcinoma (HCC) diagnosis is challenging. Moreover, for patients with alpha-fetoprotein (AFP)-negative HCC, this challenge is augmented. MicroRNAs (miRs) profiles may serve as potential HCC molecular markers. We aimed to assess plasma homo sapiens—(hsa)-miR-21-5p, hsa-miR-155-5p, hsa-miR-192-5p, and hsa-miR-199a-5p—expression levels as a panel of biomarkers for HCC in chronic hepatitis C virus (CHCV) patients with liver cirrhosis (LC), especially AFP-negative HCC cases, as a step toward non-protein coding (nc) RNA precision medicine. Subjects and methods: 79 patients enrolled with CHCV infection with LC, subclassified into an LC group without HCC (n = 40) and LC with HCC (n = 39). Real-time quantitative PCR was used to measure plasma hsa-miR-21-5p, hsa-miR-155-5p, hsa-miR-192-5p, and hsa-miR-199a-5p. Results: Plasma hsa-miR-21-5p and hsa-miR-155-5p demonstrated significant upregulation, while hsa-miR-199a-5p demonstrated significant downregulation in the HCC group (n = 39) when compared to the LC group (n = 40). hsa-miR-21-5p expression was positively correlated with serum AFP, insulin, and insulin resistance (r = 0.5, p < 0.001, r = 0.334, p = 0.01, and r = 0.303, p = 0.02, respectively). According to the ROC curves, for differentiating HCC from LC, combining AFP with each of hsa-miR-21-5p, hsa-miR-155-5p, and miR199a-5p improved the diagnostic sensitivity to 87%, 82%, and 84%, respectively, vs. 69% for AFP alone, with acceptable specificities of 77.5%, 77.5%, and 80%, respectively, and AUC = 0.89, 0.85, and 0.90, respectively vs. 0.85 for AFP alone. hsa-miR-21-5p/hsa-miR-199a-5p and hsa-miR-155-5p/hsa-miR-199a-5p ratios discriminated HCC from LC at AUC = 0.76 and 0.71, respectively, with sensitivities = 94% and 92% and specificities = 48% and 53%, respectively. Upregulation of plasma hsa-miR-21-5p was considered as an independent risk factor for HCC development [OR = 1.198(1.063–1.329), p = 0.002]. Conclusions: Combining each of hsa-miR-21-5p, hsa-miR-155-5p, and hsa-miR-199a-5p with AFP made it possible to identify HCC development in the LC patients’ cohort with higher sensitivity than using AFP alone. hsa-miR-21-5p/hsa-miR-199a-5p and hsa-miR-155-5p/hsa-miR-199a-5p ratios are potential HCC molecular markers for AFP-negative HCC patients. hsa-miR-21-5p was linked, clinically and via in silico proof, to insulin metabolism, inflammation, dyslipidemia, and tumorigenesis in the HCC patients’ group as well as for an upregulated independent risk factor for the emergence of HCC from LC in the CHCV patients.