CD4+ T cells proliferation assay to analyze Mo-MDSCs suppressive function.

Among myeloid regulatory cells (MRCs), some particular subsets termed myeloid-derived suppressor cells (MDSCs) have been described. They are suppressor myeloid cells characterized by their ability to regulate innate and adaptive immune responses and known to accumulate in the context of chronic diseases and cancer. The lack of specific markers makes their classification difficult and requires functional studies to distinguish them from other myeloid cells. In this sense, the in vitro analysis of the proliferation of T lymphocytes cultured with MDSCs provides information about the regulatory function of these cells. Here, we provide a detailed protocol to assess the ability of human Mo-MDSCs to suppress T cell proliferation in vitro after obtaining Mo-MDSCs and CD4+T cell from peripheral blood.

Plasma Phosphorylated Tau 231 Increases at One-Year Intervals in Cognitively Unimpaired Subjects.

Background: Plasma biomarkers of Alzheimer's disease (AD) constitute a non-invasive tool for diagnosing and classifying subjects. They change even in preclinical stages, but it is necessary to understand their properties so they can be helpful in a clinical context. Objective: With this work we want to study the evolution of p-tau231 plasma levels in the preclinical stages of AD and its relationship with both cognitive and imaging parameters. Methods: We evaluated plasma phosphorylated (p)-tau231 levels in 146 cognitively unimpaired subjects in sequential visits. We performed a Linear Mixed-effects Model to analyze their rate of change. We also correlated their baseline levels with cognitive tests and structural and functional image values. ATN status was defined based on cerebrospinal fluid biomarkers. Results: Plasma p-tau231 showed a significant rate of change over time. It correlated negatively with memory tests only in amyloid-positive subjects. No significant correlations were found with any imaging measures. Conclusions: Increases in plasma p-tau231 can be detected at one-year intervals in cognitively healthy subjects. It could constitute a sensitive marker for detecting early signs of neuronal network impairment by amyloid.

Where Should I Draw the Line: PET-Driven, Data-Driven, or Manufacturer Cut-Off?

Background: The optimal cut-off for Alzheimer's disease (AD) CSF biomarkers remains controversial. Objective: To analyze the performance of cut-off points standardized by three methods: one that optimized the agreement between 11C-Pittsburgh compound B PET (a-PET) and CSF biomarkers (Aß1-42, pTau, tTau, and Aß1-42/Aß1-40 ratio) in our population, called PET-driven; an unbiased cut-off using data from a healthy research cohort, called data-driven, and that provided by the manufacturer. We also compare changes in ATN classification. Methods: CSF biomarkers measured by the LUMIPULSE G600II platform and qualitative visualization of amyloid positron emission tomography (a-PET) were performed in all the patients. We established a cut-off for each single biomarker and Aß1-42/Aß1-40 ratio that optimized their agreement with a-PET using ROC curves. Sensitivity, Specificity, and Overall Percent of Agreement are assessed using a-PET or clinical diagnosis as gold standard for every cut-off. Also, we established a data-driven cut-off from our cognitively unimpaired cohort. We then analyzed changes in ATN classification. Results: One hundred and ten patients were recruited. Sixty-six (60%) were a-PET positive. PET-driven cut-offs were: pTau > 57, tTau > 362.62, Aß1-42/Aß1-40 < 0.069. For a single biomarker, pTau showed the highest accuracy (AUC 0.926). New PET-driven cut-offs classified patients similarly to manufacturer cut-offs (only two patients changed). However, 20 patients (18%) changed when data-driven cut-offs were used. Conclusions: We established our sample's best CSF biomarkers cut-offs using a-PET as the gold standard. These cut-offs categorize better symptomatic subjects than data-driven in ATN classification, but they are very similar to the manufacturer's.

Influence of Physiological Variables and Comorbidities on Plasma Aß40, Aß42, and p-tau181 Levels in Cognitively Unimpaired Individuals.

Plasma biomarkers for Alzheimer's disease (AD) are a promising tool that may help in early diagnosis. However, their levels may be influenced by physiological parameters and comorbidities that should be considered before they can be used at the population level. For this purpose, we assessed the influences of different comorbidities on AD plasma markers in 208 cognitively unimpaired subjects. We analyzed both plasma and cerebrospinal fluid levels of Aß40, Aß42, and p-tau181 using the fully automated Lumipulse platform. The relationships between the different plasma markers and physiological variables were studied using linear regression models. The mean differences in plasma markers according to comorbidity groups were also studied. The glomerular filtration rate showed an influence on plasma Aß40 and Aß42 levels but not on the Aß42/Aß40 ratio. The amyloid ratio was significantly lower in diabetic and hypertensive subjects, and the mean p-tau181 levels were higher in hypertensive subjects. The glomerular filtration rate may have an inverse relationship on plasma Aß40 and Aß42 levels but not on the amyloid ratio, suggesting that the latter is a more stable marker to use in the general population. Cardiovascular risk factors might have a long-term effect on the amyloid ratio and plasma levels of p-tau181.

ANCA detection with solid phase chemiluminescence assay: diagnostic and severity association in vasculitis.

ANCA-associated vasculitis (AAV) comprises a group of necrotizing vasculitis that mainly affects small- and medium-sized vessels. Serum anti-neutrophil cytoplasmic antibodies (ANCA), mainly anti-myeloperoxidase (anti-MPO) and anti-proteinase 3 (anti-PR3), levels may correlate to severity, prognosis, and recurrence of the disease. A retrospective analysis of 101 patients with MPO-positive and 54 PR3-positive vasculitis was performed, using laboratory established cut-off value, measured by chemiluminescence. Furthermore, data of renal disease and pulmonary involvement were collected at vasculitis diagnosis, as well as the progress, requiring dialysis, transplant, or mortality. For anti-MPO antibodies with a diagnosis of vasculitis (n = 77), an area under the curve (AUC) was calculated (AUC = 0.8084), and a cut-off point of 41.5 IU/ml was determined. There were significant differences in anti-MPO levels between patients with renal or pulmonary dysfunction (n = 65) versus those without them (n = 36) (p = 0.0003), and a cut-off threshold of 60 IU/ml was established. For anti-PR3 antibodies with a diagnosis of vasculitis (n = 44), an area under the curve (AUC) was calculated (AUC = 0.7318), and a cut-off point of 20.5 IU/ml was determined. Significant differences in anti-PR3 levels were observed between those patients with renal or pulmonary dysfunction (n = 30) and those without them (n = 24) (p = 0.0048), and a cut-off threshold of 41.5 IU/ml was established. No significant differences between those patients who had a worse disease progression and those who did not were found for anti-MPO and anti-PR3. Anti-MPO and anti-PR3 levels at the moment of vasculitis diagnosis are related with disease severity but not with disease outcome or vasculitis recurrence.

Total error in lymphocyte subpopulations by flow cytometry-based in state of the art using Spanish EQAS data.

OBJECTIVES: Flow cytometry analyses of lymphocyte subpopulations (T, B, NK) are crucial for enhancing clinical algorithms and research workflows. Estimating the total error (TE) values for the percentage and absolute number of lymphocyte subpopulations using the state-of-the-art (SOTA) approach with real data from an external proficiency testing (EPT) scheme was performed. A comparison with previously published Biological Variability (BV)-based specifications was carried out. METHODS: A total of 44,998 results from 86 laboratories over 10 years were analysed and divided into two five-year periods (2012-2016) and (2017-2021). Data come from the IC-1 Lymphocytes scheme of the Spanish External Quality Assurance System (EQAS) GECLID Program. This quantitative scheme includes percentages and absolute numbers of CD3+, CD3+CD4+, CD3+CD8+, CD19+, and CD3-CD56+CD16+ NK cells. The percentage of TE was calculated as: |reported value - robust mean|*100/robust mean for each laboratory and parameter. The cut-off for TE is set at 80 % best results of the laboratories. RESULTS: A significant reduction in the SOTA-based TE for all lymphocyte subpopulations in 2017-2021 was observed compared to 2012-2016. The SOTA-based TE fulfils the minimum BV-based TE for percentages of lymphocyte subpopulations. The parameter with the best analytical performance calculated with SOTA (2017-2021 period)-based TE was the percentage of CD3+ (TE=3.65 %). CONCLUSIONS: The values of SOTA-based specifications from external quality assurance program data are consistent and can be used to develop technical specifications. The technological improvement, quality commitment, standardization, and training, reduce TE. An update of TE every five years is therefore recommended. TE assessment in lymphocyte subsets is a helpful and reliable tool to improve laboratory performance and data-based decision-making trust.

The Cantabria Cohort, a protocol for a population-based cohort in northern Spain.

Cantabria Cohort stems from a research and action initiative lead by researchers from Valdecilla Research Institute (IDIVAL), Marqués de Valdecilla University Hospital and University of Cantabria, supported by the regional Goverment. Its aim is to identify and follow up a cohort that would provide information to improve the understanding of the etiology and prognosis of different acute and chronic diseases. The Cantabria Cohort will recruit between 40,000-50,000 residents aged 40-69 years at baseline, representing 10-20% of the target population. Currently, more than 30,000 volunteers have been enrolled. All participants will be invited for a re-assessment every three years, while the overall duration is planned for twenty years. The repeated collection of biomaterials combined with broad information from participant questionnaires, medical examinations, actual health system records and other secondary public data sources is a major strength of its design, which will make it possible to address biological pathways of disease development, identify new factors involved in health and disease, design new strategies for disease prevention, and advance precision medicine. It is conceived to allow access to a large number of researchers worldwide to boost collaboration and medical research.

Recommendations for the use of anti-dsDNA autoantibodies in the diagnosis and follow-up of systemic lupus erythematosus - A proposal from an expert panel.

Anti-dsDNA autoantibodies are listed as one of the classification criteria for systemic lupus erythematosus (SLE) and are relatively effective indicators for monitoring disease activity and treatment response. Therefore, clinicians rely on them to diagnose and adjust medication and treatment strategies for SLE patients. However, the use of anti-dsDNA antibodies is not free from controversy. Part of this controversy stems from the fact that anti-dsDNA antibodies are found in several disorders, besides SLE. In addition to this, anti-dsDNA antibodies are a heterogeneous group of antibodies, and their determination still lacks proper standardization. Moreover, anti-dsDNA testing specificity and diagnostic performance change depending on the population under study. These and other issues result in inconsistency and encumber the clinical use of anti-dsDNA antibodies. A panel of medical laboratory and clinical experts on SLE discussed such issues based on their clinical experience in a first meeting, establishing a series of recommendations. The proceedings of this first meeting, plus an exhaustive review of the literature, were used to compose a paper draft. The panel subsequently discussed and refined this draft in a second meeting, the result of which is this paper. This document is relevant to clinical laboratories as it guides to improving diagnosis and monitoring of SLE. Simultaneously, it will help laboratories compile more informative reports, not limited to a mere number. It is also relevant to clinical doctors who wish to better understand laboratory methods so that they can do a more efficient, better-aimed laboratory test ordering.

No evidence of association between inherited thrombophilia and increased risk of liver fibrosis.

BACKGROUND: Preliminary evidence suggests that inherited hypercoagulable disorders can lead to an increased risk of significant liver fibrosis. OBJECTIVE: We aimed to investigate the prevalence of significant fibrosis in patients with inherited thrombophilia, assessed by using liver stiffness (LS), and to compare this prevalence to that found in a large population-based cohort from the same region. METHODS: This was a single-center, cross-sectional study. A complete laboratory analysis for liver disease, LS by transient elastography and an abdominal ultrasound were performed in patients with inherited thrombophilia diagnosed between May 2013-February 2017. These patients were propensity score matched (ratio 1:4) with a population-based cohort from the same region (PREVHEP-ETHON study; NCT02749864; N = 5988). RESULTS: Of 241 patients with inherited thrombophilia, eight patients (3.3%) had significant fibrosis (LS ≥8 kPa). All of them had risk factors for liver disease and met diagnostic criteria for different liver diseases. After matching 221 patients with thrombophilia with 884 patients of the PREVHEP-ETHON cohort, the prevalence of significant fibrosis was similar between both cohorts (1.8% vs. 3.6%, p = 0.488). Multivariate analysis showed that age and liver disease risk factors, but not belonging to the thrombophilia cohort, were associated with the presence of significant fibrosis. The magnitude of the increased risk of significant fibrosis in patients with risk factors for liver disease was also similar in both cohorts. CONCLUSIONS: Our findings do not provide evidence supporting an association between inherited thrombophilia and an increased risk of significant liver fibrosis, independent of the presence of liver-related causes of fibrosis.

Prevalence and sociodemographic correlates of antinuclear antibody testing by indirect immunofluorescence or solid-phase assays in a Spanish population: the Camargo Cohort.

Autoantibodies are the hallmark of autoimmunity, and specifically, antinuclear antibodies (ANA) are one of the most relevant antibodies present in systemic autoimmune diseases (AID). In the present study, we evaluate the relationship between ANA and sociodemographic and biobehavioral factors in a population with a low pre-test probability for systemic AID. ANA were determined in serum samples at baseline visit from 2997 participants from the Camargo Cohort using indirect immunofluorescence assay, and two solid phase assays (SPA), addressable laser bead immunoassay, and fluorescence enzyme immunoassay. Sociodemographic and biobehavioral features of the subjects were obtained at baseline visit using a structured questionnaire. The prevalence of ANA positive results was significantly higher when indirect immunofluorescence assay was used as screening method in comparison with SPAs, being higher in females, older subjects, and those with higher C-reactive protein levels. Considering biobehavioral features, the prevalence was higher in those individuals with a sedentary lifestyle, and in ex- and non-alcohol users. Moreover, considering the relevance of the antibody load using ANA Screen, the prevalence of the antibody load also increased with age, especially in females. In conclusion, the prevalence of ANA varies depending on sociodemographic and biobehavioral features of the subjects, which could be relevant specifically in a population with a low pre-test probability for systemic AIDs.

Age-related hearing loss is not linked to cerebrospinal fluid levels of ß-amyloid or p-tau181.

INTRODUCTION: As Hearing loss and dementia affect people with the same profile, several epidemiological studies have evaluated their relationship. However, the link between age-related hearing loss and Alzheimer's disease is still unclear. METHODS: We selected subjects with no history of exposure to loud noises, blasts, head trauma with hearing loss, or sudden sensorineural hearing loss from a cohort intended to study preclinical phases of Alzheimer's disease. Participants are volunteers over 55 years without cognitive impairment. We correlated the results of an objective auditory evaluation with brain amyloid and p-tau181 levels and with the outcomes of a comprehensive neuropsychological assessment. RESULTS: Fifty-five subjects at different stages of the Alzheimer's disease continuum were evaluated. There were no statistically significant correlations between amyloid-ß and p-tau levels and any of the objective auditory measures. A weak but significant correlation was found between amyloid-ß values and the Hearing Handicap Inventory for the Elderly. The neuropsychological domains more correlated to hearing loss were executive function and processing speed. DISCUSSION: Age-related hearing loss is not linked to any pathological markers of Alzheimer's disease nor to neuropsychological domains typically affected in this disease. The Hearing Handicap Inventory for the Elderly has an important component of subjectivity and further studies are needed to explore its relationship with amyloid-ß levels.

Accuracy of plasma Aß40, Aß42, and p-tau181 to detect CSF Alzheimer's pathological changes in cognitively unimpaired subjects using the Lumipulse automated platform.

BACKGROUND: The arrival of new disease-modifying treatments for Alzheimer's disease (AD) requires the identification of subjects at risk in a simple, inexpensive, and non-invasive way. With tools allowing an adequate screening, it would be possible to optimize the use of these treatments. Plasma markers of AD are very promising, but it is necessary to prove that alterations in their levels are related to alterations in gold standard markers such as cerebrospinal fluid or PET imaging. With this research, we want to evaluate the performance of plasma Aß40, Aß42, and p-tau181 to detect the pathological changes in CSF using the automated Lumipulse platform. METHODS: Both plasma and CSF Aß40, Aß42, and p-tau181 have been evaluated in a group of 208 cognitively unimpaired subjects with a 30.3% of ApoE4 carriers. We have correlated plasma and CSF values of each biomarker. Then, we have also assessed the differences in plasma marker values according to amyloid status (A - / +), AD status (considering AD + subjects to those A + plus Tau +), and ATN group defined by CSF. Finally, ROC curves have been performed, and the area under the curve has been measured using amyloid status and AD status as an outcome and different combinations of plasma markers as predictors. RESULTS: Aß42, amyloid ratio, p-tau181, and p-tau181/Aß42 ratio correlated significantly between plasma and CSF. For these markers, the levels were significantly different in the A + / - , AD + / - , and ATN groups. Amyloid ratio predicts amyloid and AD pathology in CSF with an AUC of 0.89. CONCLUSIONS: Plasma biomarkers of AD using the automated Lumipulse platform show good diagnostic performance in detecting Alzheimer's pathology in cognitively unimpaired subjects.

DQA1 Eplet Mismatch Load As an Independent Risk Factor of CLAD After Lung Transplantation.

Lung transplantation remains the treatment of choice for end-stage lung diseases, and recipient selection is currently based on clinical urgency, ABO compatibility, and donor size. The risk of allosensitization is classically based on HLA mismatch, but eplet mismatch load is increasingly seen to be important in long-term outcomes in solid organ transplantation. Chronic lung allograft dysfunction (CLAD) is relatively common and relevant, affecting almost 50% of patients 5 y after transplantation and being the first cause of death from the first year after transplantation. The overall class-II eplet mismatch load has been associated with CLAD development. Methods: Based on clinical data, 240 lung transplant recipients were eligible for CLAD, and HLA and eplet mismatch was analyzed using the HLAMatchmaker 3.1 software. Results: A total of 92 (38.3%) lung transplant recipients developed CLAD. The time free-of-CLAD was significantly decreased in patients with presence of DQA1 eplet mismatches (P = 0.015). Furthermore, when other previously described CLAD risk factors were studied in a multivariate analysis, the presence of DQA1 eplet mismatches was found to be independently associated with the early onset of CLAD. Conclusions: The concept of epitope load has arisen as a new tool to better define donor-recipient immunologic compatibility. The presence of DQA1 eplet mismatches potentially would increase the likelihood of developing CLAD.

CD64 expression on neutrophils as a potential biomarker for bacterial infection in ascitic fluid of cirrhotic patients.

BACKGROUND: CD64 expression on neutrophils surface (CD64N) by flow cytometry has been validated as a rapid biomarker for bacterial infections in both peripheral blood and other biological fluids. Ascites is a common complication in cirrhotic patients that a variety of factors can cause, including bacterial infections. Manual counting of polymorphonuclear (PMN) cells in ascitic fluid and microbiologic culture are essential for its diagnosis. We aimed to validate the determination of CD64N by flow cytometry in ascitic fluid and assess its potential usefulness in the rapid identification of bacterial infections. MATERIALS AND METHODS: A prospective unicentre study was conducted. Flow cytometry was used to analyse the expression of CD64N in 77 ascitic fluid samples from the initial paracentesis of 60 cirrhotic patients in different admission episodes from November 2021 to December 2022. RESULTS: Seventeen samples were diagnosed with bacterial infection based on a positive microbiologic culture or by PMN count (>250 PMN/mm3 in ascitic fluid). The median of CD64N MFI was significantly increased in the bacterial infection group (3690.5 MFI [1635.23-6521.18] vs. 1105.9 MFI [737.3-2048.2], p < 0.001). The CD64 MFI ratio of granulocytes to lymphocytes was elevated in the bacterial infection group (13.06 [6.38-24.58] vs. 5.01 [3.38-7.36], p < 0.001). A CD64N ratio higher than 9.9 identified those patients with bacterial infection with 70.6 and 86.7% sensitivity and specificity, with an area under the curve (AUC) of 79.4%. CONCLUSION: The CD64N determined by flow cytometry on ascitic fluid could help quickly identify bacterial infections in ascites patients, allowing early antibiotic treatment.

Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAP.

OBJECTIVES: Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA). METHODS: A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group "Autoimmunity Testing"; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP). RESULTS: In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations. CONCLUSIONS: These recommendations are an important step to achieve high quality ANA testing.

Comparison of ANA testing by indirect immunofluorescence or solid-phase assays in a low pre-test probability population for systemic autoimmune disease: the Camargo Cohort.

OBJECTIVES: Autoantibodies and, specifically antinuclear antibodies (ANA), are the hallmark of systemic autoimmune diseases (AID). In the last decades, there has been great technical development to detect these autoantibodies along with an increased request for this test by clinicians, while the overall pre-test probability has decreased. In this study, we compare the diagnostic performance of three different methods for ANA screening (indirect immunofluorescence [IIF], addressable laser bead immunoassay [ALBIA], and fluorescence enzyme immunoassay [FEIA]). METHODS: Serum samples at baseline visit from 2,997 participants from the Camargo Cohort, a population with an overall low pre-test probability for systemic AID, were analyzed with the three methods. Participants have a minimum follow-up of 10 years and the development of autoimmune diseases was collected from clinical records. RESULTS: The highest frequency of positive ANA was observed by IIF assay. However, ALBIA showed high sensitivity for AID. Likewise, solid phase assays (SPA) presented higher specificity than IIF for AID. ANA prevalence with any method was significantly higher in females and overall increased with age. Triple positivity for ANA was significantly related to the presence of anti-dsDNA-SSA/Ro60, Ro52, SSB/La, RNP, Scl-70, and centromere-specificities. No association was found for anti-Sm - RNP68, or ribosomal P - specificities. Noteworthy, triple positivity for ANA screening was associated with diagnosis of systemic AID both at baseline visit and follow-up. CONCLUSIONS: ANA detection by IIF may be better when the pre-test probability is high, whereas SPA techniques are more useful in populations with an overall low pre-test probability for systemic AID.

Development of SARS-CoV-2 specific IgG and IgA antibodies in serum and milk with different SARS-COV-2 vaccines in lactating women.

BACKGROUND: Our main objective was to determine the evolution of IgG and IgA antibodies directed against SARS-CoV-2 protein S in the blood of lactating women and in breast milk. METHODS: A cohort of 110 uninfected and vaccinated breastfeeding women was followed-up for 6 months at the Marqués de Valdecilla University Hospital, Spain, in 2020. An additional group of 23 breastfeeding mothers who had no previously documented infection and had not been vaccinated against SARS-CoV-2 were included as a control group. The antibodies in blood and breast milk and their evolution at 6 months post-vaccination were analysed. RESULTS: One hundred ten breastfeeding mothers were included; 70 women (63.6%) were vaccinated with two doses of BNT162b2, 20 women (18.2%) received two doses of mRNA-1273, and 20 women (18.2%) received a single dose of ChAdOx1-S. No evidence of differences between concentrations of antibodies was found according to the type of vaccine, with the exception of serum IgA antibodies, which was higher in women vaccinated with mRNA-1273: mean [95%CI]: 0.05 AU/mL [0.03,0.06] with mRNA-1273, 0.02 AU/mL [0.01,0.03] with BNT162b2 and 0.01 AU/mL [0.00,0.03] with ChAdOx1-S, ANOVA p value = 0.03. The lack of difference between vaccines was also found when anti-S1 specific IgG in serum and breast milk were measured. CONCLUSIONS: In lactating women vaccinated against COVID-19, anti-SARS-CoV-2 antibodies can be detected in both serum and breastmilk 6 months after receiving the second dose, although their concentrations decreased when compared with concentrations reached immediately after vaccination.

A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants.

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.