Studying antigen-specific T cells through a streamlined, whole blood-based extracellular approach.

Techniques currently used for the study of antigen-specific T-cell responses are either poorly informative or require a heavy workload. Consequently, many perspectives associated with the broader study of such approaches remain mostly unexplored in translational research. However, these could benefit many fields including but not limited to infectious diseases, oncology, and vaccination. Herein, the main objective of this work was to develop a standardized flow cytometry-based approach that would combine ease of use together with a relevant study of antigen-specific T-cell responses so that they could be more often included in clinical research. To this extent, a streamlined approach relying on 1/ the use of whole blood instead of peripheral blood mononuclear cells and 2/ solely based on the expression of extracellular activation-induced markers (AIMs), called whole blood AIM (WAIM), was developed and further compared to more conventional techniques such as enzyme-linked immunospot (ELISpot) and flow cytometry-based intracellular cytokine staining (ICS). Based on a cohort of 20 individuals receiving the COVID-19 mRNA vaccine and focusing on SARS-CoV-2 and cytomegalovirus (CMV)-derived antigen T-cell-specific responses, a significant level of correlation between the three techniques was found. Based on the use of whole blood and on the expression of extracellular activation-induced markers (CD154, CD137, and CD107a), the WAIM technique appears to be very simple to implement and yet allows interesting patient stratification capabilities as the chosen combination of extracellular markers exhibited higher orthogonality than cytokines that are commonly considered in ICS (IFN-γ, TNF-α, and IL-2).

CD169 and CD64 could help differentiate bacterial from CoVID-19 or other viral infections in the Emergency Department.

The identification of a bacterial, viral, or even noninfectious cause is essential in the management of febrile syndrome in the emergency department (ED), especially in epidemic contexts such as flu or CoVID-19. The aim was to assess discriminative performances of two biomarkers, CD64 on neutrophils (nCD64) and CD169 on monocytes (mCD169), using a new flow cytometry procedure, in patients presenting with fever to the ED during epidemics. Eighty five adult patients presenting with potential infection were included during the 2019 flu season in the ED of La Timone Hospital. They were divided into four diagnostic outcomes according to their clinical records: no-infection, bacterial infection, viral infection and co-infection. Seventy six patients with confirmed SARS-CoV-2 infection were also compared to 48 healthy volunteers. For the first cohort, 38 (45%) patients were diagnosed with bacterial infections, 11 (13%) with viral infections and 29 (34%) with co-infections. mCD169 was elevated in patients with viral infections, with a majority of Flu A virus or Respiratory Syncytial Virus, while nCD64 was elevated in subjects with bacterial infections, with a majority of Streptococcus pneumoniae and Escherichia coli. nCD64 and mCD169 showed 90% and 80% sensitivity, and 78% and 91% specificity, respectively, for identifying patients with bacterial or viral infections. When studied in a second cohort, mCD169 was elevated in 95% of patients with SARS-CoV-2 infections and remained at normal level in 100% of healthy volunteers. nCD64 and mCD169 have potential for accurately distinguishing bacterial and acute viral infections. Combined in an easy and rapid flow cytometry procedure, they constitute a potential improvement for infection management in the ED, and could even help for triage of patients during emerging epidemics.

Possible alternative strategies to implement basophil activation testing in multicentric studies.

The Basophil Activation Test (BAT) enables flow cytometry characterization of basophil reactivity against specific allergenic molecules. The focus now revolves around democratizing this tool, but, as blood sample stability could be challenging, after having developed a simplified approach, herein, we aimed to characterize two strategies for implementing BAT in multicentric studies: store and ship blood before or after sample processing. Fresh heparin- and EDTA-anticoagulated whole blood samples followed both BAT workflows: "collect, store, process & analyze" or "collect, process, store & analyze". Storage temperatures of 18-25 °C or 2-8 °C and preservation times from 0 to 7 days were considered. Interleukin-3 was also evaluated. With the "collect, store, process & analyze" workflow, heparin-anticoagulated blood and 18-25 °C storage were better than other conditions. While remaining possible, basophil activation exhibited a possible reactivity decay after 24 h. Under the conditions tested, interleukin-3 had no role in enhancing basophil reactivity after storage. Conversely, the "collect, process, store & analyze" workflow demonstrated that either heparin- or EDTA-anticoagulated blood can be processed and kept up to 7 days at 18-25 °C or 2-8 °C before being analyzed. Various strategies can be implemented to integrate BAT in multicentric studies. The "collect, store, process & analyze" workflow remains a simplified logistical approach, but depending on time required to ship from the clinical centers to the reference laboratories, it might not be applicable, or should be used with caution. The "collect, process, store & analyze" workflow may constitute a workflow improvement to provide significant flexibility without impact on basophil reactivity.

Leukocyte activation patterns in hospitalized children: comparing SARS-CoV-2, bacterial infections, and inflammatory pathologies.

In adults, monocytes and neutrophils play important roles in the hyperinflammatory responses characteristic of severe forms of SARS-CoV-2 infection. We assessed leukocyte activation in 55 children attending the emergency department for acute fever between March 2020 and September 2021. The following markers were analyzed by flow cytometry: CD169 and HLA-DR on monocytes, CD64 and CD16 on neutrophils, and CD38 on lymphocytes TCD8. Fifteen of the children had SARS-CoV-2 infection, 15 had bacterial infections, and 15 had inflammatory diseases. We observed overexpression of CD169 on monocytes and CD38 on T lymphocytes in all patients with a diagnosis of SARS-CoV-2, while overexpression of CD64 on neutrophils was observed with bacterial infections and inflammatory diseases. There was a decrease in the expression of HLA-DR on monocytes in the bacterial infection and inflammatory pathology groups. Leukocyte analysis identifies distinct activation patterns in children during SARS-CoV-2 infections, bacterial infections, and inflammatory diseases.

A Streamlined Strategy for Basophil Activation Testing in a Multicenter Phase III Clinical Trial.

BACKGROUND: The basophil activation test (BAT) has been limited to research settings owing to technical issues. Novel approaches using dry, ready-to-use reagents and streamlined protocols offer greater flexibility and may open opportunities for easier implementation in clinical research. OBJECTIVE: Using a streamlined basophil activation test (sBAT) strategy and the settings of the baseline study of the Epicutaneous Immunotherapy in Toddlers with Peanut Allergy (EPITOPE) trial of EPicutaneous ImmunoTherapy, we aimed to assess the feasibility of implementing BAT in a multicenter trial and to evaluate its utility in predicting the outcomes of peanut double-blind placebo-controlled food challenge (DBPCFC). METHODS: Whole blood samples were collected from subjects aged 1 to 3 years (n = 241) undergoing baseline eligibility DBPCFC in the EPITOPE study across 15 clinical sites in North America. After preparation with sBAT reagents, processed samples were analyzed in a single central laboratory within 5 days of collection and preparation. The eliciting dose (ED) at DBPCFC was determined using, Practical Allergy (PRACTALL) criteria. Using a machine learning approach that incorporated BAT-derived features, clinical characteristics, and peanut-specific immunoglobulin E, the ability to predict outcomes of interest (ED ≤ 300 mg or > 300 mg] and use of epinephrine) was assessed using data randomly split into training (n = 182) and validation (n = 59) subsets. RESULTS: The expression of basophil activation markers CD203c and CD63 correlated with ED and severity outcomes of DBPCFC. Most informative concentrations of peanut extract in the sBAT assay for these associations were 1 ng/mL and 10 ng/mL. Using machine learning to assess the ability to predict the outcomes of DBPCFC, the best models using only the BAT-derived features provided relatively high sensitivities of 0.86 and 0.85 for predicting ED and epinephrine use, respectively, whereas specificities were lower, ranging from 0.60 to 0.80. Although including specific immunoglobulin E and skin prick test data in addition to those from sBAT did not improve the ability to identify individuals most at risk for severe reactions, it did improve the ability to identify patients with an ED greater than 300 mg. CONCLUSIONS: In addition to facilitating implementation in multicenter trials, sBAT retains the potential of BAT to characterize allergic patients and confirms its potential to contribute to predicting the outcome of oral food challenges.

Promises and Remaining Challenges for Further Integration of Basophil Activation Test in Allergy-Related Research and Clinical Practice.

More than 20 years after having been initially proposed, the relevance and usefulness of basophil activation test (BAT) for the field of allergy research and testing were demonstrated on many occasions. Leveraging the fully open format of a flexible, whole blood-based functional assay, BAT has been shown to be equally important for fundamental research, clinical research, and diagnosis. Regardless of whether the focus of a study is on the characterization of the allergenic moiety, on the patient side, or on the study of the fundamental processes involved in the allergic disease or its treatment, BAT enables the gathering of very important insights. In spite of this, its full capabilities have yet to be leveraged. Various bottlenecks, including but not limited to assay logistics, robustness, flow cytometry access, and/or expertise, have indeed been limiting its development beyond experts and long-term users. Now, various initiatives, aiming at resolving these bottlenecks, have been launched. If successful, a broader use of BAT could then be contemplated. In such a situation, its more thorough integration in clinical practice has the potential to significantly change the allergic patient's journey.

A rapid, easy, and scalable whole blood monocyte CD169 assay for outpatient screening during SARS-CoV-2 outbreak, and potentially other emerging disease outbreaks.

Objective: The COVID-19 corona virus disease outbreak is globally challenging health systems and societies. Its diagnosis relies on molecular methods, with drawbacks revealed by mass screening. Upregulation of neutrophil CD64 or monocyte CD169 has been abundantly reported as markers of bacterial or acute viral infection, respectively. We evaluated the sensitivity of an easy, one-step whole blood flow cytometry assay to measure these markers within 10 min, as a potential screening test for COVID-19 patients. Methods: Patients (n = 177) with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were tested on 10 µL blood and results were compared with reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). Results: We observed 98% and 100% sensitivity in early-stage (n = 52) and asymptomatic patients (n = 9), respectively. Late-stage patients, who presented for a second control RT-qPCR, were negative for both assays in most cases. Conversely, neutrophil CD64 expression was unchanged in 75% of cases, without significant differences between groups. Conclusion: Monocyte CD169 evaluation was highly sensitive for detecting SARS-CoV-2 infection in first-presentation patients; and it returns to basal level upon infection clearance. The potential ease of fingerprick collection, minimal time-to-result, and low cost rank this biomarker measurement as a potential viral disease screening tool, including COVID-19. When the virus prevalence in the tested population is usually low (1%-10%), such an approach could increase the testing capacity 10 to 100-fold, with the same limited molecular testing resources, which could focus on confirmation purposes only.

One-step White Blood Cell Extracellular Staining Method for Flow Cytometry.

Flow cytometry is a powerful analytical technique that is increasingly used in scientific investigations and healthcare; however, it requires time-consuming, multi-step sample procedures, which limits its use to specialized laboratories. In this study, we propose a new universal one-step method in which white blood cell staining and red blood cell lysis are carried out in a single step, using a gentle lysis solution mixed with fluorescent antibody conjugates or probes in a dry or liquid format. The blood sample may be obtained from a routine venipuncture or directly from a fingerprick, allowing for near-patient analysis. This procedure enables the analysis of common white blood cell markers as well as markers related to infections or sepsis. This simpler and faster protocol may help to democratize the use of flow cytometry in the research and medical fields. Graphic abstract: One-step White Blood Cell Extracellular Staining Method for Flow Cytometry.

Direct freezing of whole blood enables analysis of leucocyte markers by flow cytometry: a proof-of-concept study.

Aim: A new one-step flow cytometry procedure has been recently demonstrated for identifying subjects with infections, but only for fresh whole blood samples. The goal of this study was to assess its applicability on frozen samples, by proposing a new method to perform the sample freezing directly and easily. Methods: Fresh blood was tested, then frozen either directly or with dimethylsulfoxide and serum. Common markers of white blood cells as well as infection-related biomarkers were tested. Results: All percentages of leucocyte subsets and levels of infection-related biomarkers were significantly correlated between frozen and fresh samples. Conclusion: The direct freezing method enables an accurate assessment of common cellular sub-populations and of levels of important infectious biomarkers via flow cytometry.

Toward Monocyte HLA-DR Bedside Monitoring: A Proof-of-Concept Study.

OBJECTIVES: Decreased expression of human leukocyte antigen-DR on monocytes (mHLA-DR) is recognized as the most appropriate marker for the monitoring of immune alterations in septic patients and critically ill subjects. Its measurement has been established for years by flow cytometry, but remains under-used due to pre-analytical constraints. The objectives of the present work were to develop a rapid and robust one-step protocol. METHODS: A novel, simplified protocol has been developed to measure mHLA-DR in whole blood using flow cytometry. It is a one-step procedure that includes red cell lysis, antibodies, and fixative reagents. It has been compared to the standardized routine protocol in two consecutive cohorts of septic shock patients (n = 37). Finally, the protocol was applied to a few subjects in point-of-care settings, by collecting capillary blood from fingerpricks. RESULTS: Strong correlation was observed between the one-step method and routine protocol in 24 patients. After testing several stabilizing agents, the procedure was further optimized by adding a low-dose formaldehyde to the stain and lyse solution. This improved method was tested in a second cohort of 13 patients, and again strongly correlated to the routine protocol. Finally, the fingerprick and venous puncture samples were shown to provide similar results. CONCLUSIONS: The present work demonstrates the feasibility of a bedside protocol for flow cytometry measurement of mHLA-DR in critically ill subjects. This helps overcome pre-analytical constraints previously identified, which have limited wider use of this biomarker in intensive care units. In addition, preliminary results from fingerprick samples are promising.

Cell Analysis from Dried Blood Spots: New Opportunities in Immunology, Hematology, and Infectious Diseases.

Blood cell analysis is a major pillar of biomedical research and healthcare. These analyses are performed in central laboratories. Rapid shipment from collection site to the central laboratories is currently needed because cells and biomarkers degrade rapidly. The dried blood spot from a fingerstick allows the preservation of cellular molecules for months but entire cells are never recovered. Here leucocyte elution is optimized from dried blood spots. Flow cytometry and mRNA expression profiling are used to analyze the recovered cells. 50-70% of the leucocytes that are dried on a polyester solid support via elution after shaking the support with buffer are recovered. While red blood cells lyse upon drying, it is found that the majority of leucocytes are preserved. Leucocytes have an altered structure that is improved by adding fixative in the elution buffer. Leucocytes are permeabilized, allowing an easy staining of all cellular compartments. Common immunophenotyping and mRNAs are preserved. The ability of a new biomarker (CD169) to discriminate between patients with and without Severe Acute Respiratory Syndrome induced by Coronavirus 2 (SARS-CoV-2) infections is also preserved. Leucocytes from blood can be dried, shipped, and/or stored for at least 1 month, then recovered for a wide variety of analyses, potentially facilitating biomedical applications worldwide.

Role of the interferons in CD64 and CD169 expressions in whole blood: Relevance in the balance between viral- or bacterial-oriented immune responses.

INTRODUCTION: CD64 expression increases on neutrophils during bacterial infections. Recently an increase in CD169 expression has been discovered on monocytes during viral infections. Generally, interferons α (IFNsα) and IFNsγ are key drivers of the infectious host immune response. The purpose of this study was to explore if a link exists between these IFNs and both biomarkers. METHODS: Whole blood samples from healthy volunteers were stimulated with either IFNs, interleukins, or infectious extracts, to mimic an infectious state. Expressions of CD64 and CD169 were assessed in these samples by multiple flow cytometry methods, over precise kinetics. RESULTS: The expression of CD64 was statistically higher in samples stimulated with IFNγ, and CD169 in those stimulated with IFNα (and all other type I IFNs). Surface expressions are directly induced by their respective IFNs via Janus kinase/signal transducer and activator of transduction pathways within 6 to 8 hours of incubation. Mixing both types of IFNs seemed to indicate that they partially inhibit each other. CONCLUSIONS: The induction of CD169 on monocytes and CD164 on neutrophils by type I and type II IFNs confirms the relevance of these markers for assessing between a viral- vs bacterial-oriented immune response.

Flow cytometry evaluation of infection-related biomarkers in febrile subjects in the emergency department.

Aim: In an Emergency Department (ED), the etiological identification of infected subjects is essential. 13 infection-related biomarkers were assessed using a new flow cytometry procedure. Materials & methods: If subjects presented with febrile symptoms at the ED, 13 biomarkers' levels, including CD64 on neutrophils (nCD64) and CD169 on monocytes (mCD169), were tested and compared with clinical records. Results: Among 50 subjects, 78% had bacterial infections and 8% had viral infections. nCD64 showed 82% sensitivity and 91% specificity for identifying subjects with bacterial infections. mCD169, HLA-ABC ratio and HLA-DR on monocytes had high values in subjects with viral infections. Conclusion: Biomarkers showed promising performances to improve the ED's infectious stratification.

A novel one-step extracellular staining for flow cytometry: Proof-of-concept on sepsis-related biomarkers.

BACKGROUND: Flow cytometry is a powerful analytical technique. However, it requires time-consuming, multi-step sample procedure. A new protocol was developed to perform extracellular staining and red blood cell lysis in one step, using dry antibodies. Common markers of white blood cells as well as sepsis biomarkers were tested as a model for modulated antigen expression. METHODS: Peripheral blood was stained using the reference and the one-step methods. Recruitment and staining of CD3-, CD4-, CD8-, CD14-, and CD15-positive cells were analyzed. Then, protocol modifications were tested for optimization. Finally, the one-step method was evaluated on subjects in septic conditions, by measuring expressions of CD64 and of HLA-DR. RESULTS: No statistical differences were observed between methods when comparing the proportions of cells. The procedure was optimized by decreasing blood volume from 100 µL to 5 µL, lysis from 1 mL to 500 µL, and time from 30 to 15 min. In the blood samples from septic subjects, an increase of CD64 on neutrophils and a decrease of HLA-DR on monocytes were observed. CONCLUSIONS: The one-step method, described here-in, enables an accurate, streamlined flow cytometry sample preparation protocol. The simplified phenotyping procedure reduces training requirements and could help overcome logistic constraints in many flow cytometry applications.

Clinical research assessment by flow cytometry of biomarkers for infectious stratification in an Emergency Department.

Aim: Management of patients with infections within the Emergency Department (ED) is challenging for practitioners, as the identification of infectious causes remains difficult with current techniques. A new combination of two biomarkers was tested with a new rapid flow cytometry technique. Materials & methods: Subjects from the ED were tested for their CD64 on neutrophils (nCD64) and CD169 on monocytes (mCD169) levels and results were compared to their clinical records. Results: Among 139 patients, 29% had confirmed bacterial infections and 5% viral infections. nCD64 and mCD169 respectively showed 88 and 86% sensitivity and 90 and 100% specificity for identifying subjects in bacterial or viral conditions. Conclusion: This point-of-care technique could allow better management of patients in the ED.