Programme

Abstract: La acreditación es la herramienta establecida a escala internacional para generar confianza sobre la correcta ejecución de las actividades de evaluación de la conformidad (ej: laboratorios clínicos), permitiendo diferenciar a los organismos que han demostrado disponer de la competencia técnica necesaria para realizarlas y que ofrecen fiabilidad en sus resultados de los que no lo han demostrado.
ENAC es el Organismo Nacional de Acreditación designado por el Gobierno en aplicación del Reglamento (CE) nº 765/2008 que reggula el funcionamiento de la acreditación en Europa.
El reglamento europeo 2017/746 sobre los productos sanitarios para diagnóstico un vitro establece una excepción en su aplicación a los productos fabricados y utilizados exclusivamente en centros sanitarios establecidos en la Unión, cuando se cumplan unas condiciones entre las que se encuentra “que el laboratorio del centro sanitario sea conforme a la norma ISO 15189 o, en su caso, a las disposiciones nacionales aplicables, incluidas las relativas a la acreditación”.
La acreditación otorgada por un organismo de acreditación reconoce la competencia técnica de un laboratorio para realizar unas actividades determinadas ya que el conjunto de sus procesos, procedimientos y recursos (personal, equipos, instalaciones) han permitido demostrar que realizan sus actividades técnicas de manera solvente.
Sin embargo, la acreditación no debe entenderse como un reconocimiento de aspectos específicos como personal, equipos o métodos/procedimientos de la entidad fuera de su contexto.
Es decir, no debe entenderse que un método/procedimiento incluido en el alcance de acreditación de un laboratorio ha obtenido un reconocimiento específico fuera de su utilización en el laboratorio que obtuvo la acreditación.
La acreditación no debe entenderse como un reconocimiento o autorización de un método de laboratorio determinado.

Abstract: The ISAC SRL Recognition Program, launched in the Fall of 2021, aims to identify and acknowledge cytometry laboratories who have put in significant efforts to adhere to best practices, with the long-term goal to generate a level of professionalism across SRLs that increases the overall quality and reproducibility of flow cytometry. In this session we will provide an overview of the Recognition Program, detailing it’s purpose, how to apply, feedback from recognized labs and data from the first two application cycles. Additionally, we will discuss the perspective of a reviewer who has partaken in judging applicants to the Recognition Program.

Background: Normalflow is a non-governmental organization (NGO) recently formed by scientists from Argentina, Colombia, Spain, Ireland and UK.

Aims: The aim to encourage and support flow cytometry laboratories in Latinamerica to obtain International Organization of Accreditation and Certification (ISO) 15189 and 9001 respectively

Methods: Normaflow sent a survey to twelve Latinamerican countries: Argentina, Bolivia, Chile, Colombia, Costa Rica, Ecuador, El Salvador, Mexico, Perú, Republica Dominicana, Uruguay and Venezuela. The survey was done through SurveyMonkey.co.uk, ten questions were sent including the type of institution and others relevant to QAS: does the laboratory have a Quality Manual and identify the pre, post and analytical, processes based on the ISO 15189 and ISO 9001, are these well documented and other QAS related questions.

Results & Conclusion: We obtained 83 responses: 27 Argentina, 2 Bolivia, 4 Chile, 7 Colombia, 3 Costa Rica, 2 Ecuador, 1 El Salvador, 24 Mexico, 9 Perú, 1 República Dominicana, 1 Uruguay and 2 Venezuela. The type of Institution was: 67,9% clinical laboratories, 26,2% State Universities, 1,2% Industry and 4,8% other. 69,5% stated that their institution had documented and implemented a QAS. 48,2% agreed that their QAS was based on standards ISO 15189 and 9001. 56,6% of laboratories had Quality Manual but 24,1% didn’t. Only 56,8% agreed that the Quality policy was following the ISO 15189 and 9001 standards. Leaders of the institutions were making progress to achieve these standards. This survey gave us a glimpse in Latinamerican's flow cytometry laboratories compliant with ISO 15189 and 9001. The conclusion from this small sample is that there is a need in Latinamerican's flow cytometry laboratories to be compliant with ISO. Normaflow is an organization with the aim to encourage and facilitate laboratories in their own language to obtain ISO accreditation offering expertise in QAS and orientation on how to obtain these with advice and practical help.

Abstract: Coeliac disease (CD) is an immune-mediated systemic disease, which is triggered by gluten ingestion in genetically susceptible individuals. Current diagnosis requires that patients are following a gluten containing diet. However, a long life gluten-free diet (GFD) constitutes the treatment of CD and many patients adopt a GFD on their own, precluding a proper diagnosis.
In 2018, our research group proposed a diagnostic test for individuals following a GFD based on detecting activated gut-homing CD8+ T cells in blood after a 3-day gluten challenge. These T cells can be detected by flow cytometry using only four markers (CD8, CD103, β7 and CD38), providing a simple method of easy implementation in clinical practice. In 2021, we showed that the suggested test provides accurate CD diagnosis with 95% specificity and 97% sensitivity. This test is very useful in diverse situations observed in daily clinical practice such as subjects following a GFD and needing review of the initial diagnosis due to absence of or incomplete original testing (serological screening and confirmatory biopsy), previous discrepant results or slow or non-responsiveness to the GFD. It appears as an alternative to the current long gluten challenge (usually >6 weeks) followed by serological and biopsy testing, which is refused by many patients worried about the clinical consequences. Additionally, it provides a standardized gluten challenge protocol consisting of 3 days supplying 10 g of gluten/day, which must be combined with two days of sample (blood) collection and analysis (before gluten challenge and 6 days later). It offers highly reproducible results for fresh analysis, but also for sample processing and analysis after 24 hours, opening the possibility of sending samples to a reference center.

Abstract: Brain tumors are the most common solid cancers in childhood, compromising 25% of the pediatric cancer diagnoses, and are the leading cause of cancer-related death in children. In particular, the prognosis of pediatric high-grade brain tumors is dismal. The combination of aggressive tumors in a delicate environment limits the therapeutic options. Studies on brain tumor biology and immune responses in adults outweigh pediatric tumor immunology knowledge. Even though many processes are similar in children and adult immunity, there are substantial age-specific developmental differences. A thorough comprehension of the pediatric brain tumor microenvironment and its influence on local and systemic immune system is essential for the translation of preclinical immune neuro-oncology research into clinical trials that improve current treatment options for these patients.
Spectral flow cytometry is an ultimate technique that offers the ability to perform complex multicolor analysis of cellular parameters. Because of its capacity for extensive panels, this method provides a comprehensive solution to investigate a substantial number of cellular characteristics in a single experiment, making it particularly useful for analyzing unique samples.
We have established a comprehensive and standardized immune monitoring platform that utilizes a combination of advanced technological solutions, such as spectral flow cytometry, to examine a wide range of immune parameters in various pediatric brain tumor entities. This platform enables us to track changes in the immune system during treatment, including the impact of surgery, radiotherapy, and chemotherapy.
Gaining a comprehensive understanding of the tumor microenvironment and immune landscape in brain malignancies is crucial to pave the path towards effective immunotherapeutic strategies in pediatric neuro-oncology.

Abstract: Common Variable Immunodeficiency (CVID) is characterized by defective antibody production and hypogammaglobulinemia with impaired differentiation of mature post-germinal-center (GC) class-switched  memory B-cells (MBC). Flow cytometry immunophenotyping has become of great relevance for its diagnosis and classification.
In this collaborative multicentric study the EuroFlow PID 8-color  Pre-GC B-cell tube, standardized sample preparation procedures (SOPs)  and innovative data analysis tools, were used to characterize the  maturation profile of pre-GC B-cells in 100 CVID patients, vs 62 age-matched healthy donors (HD).
The Pre-GC B-cell tube allowed identification within pre-GC B-cells of  three subsets of maturation associated immature B-cells and three  subpopulations of mature naïve B-lymphocytes. CVID patients showed overall reduced median absolute counts (vs HD) of the two more advanced stages of maturation in immature B cells. This was associated with an expansion of CD21- CD24- and CD21- CD24++ naïve B-cell counts above normal values. Aditionally, reduced IgMD+ and IgMD- MBC counts were found to be below normal values CVID patients, always together with severely reduced/undectatable circulating blood plasmablasts.
Comparison of the maturation pathway profile of pre-GC B cells in blood of CVID patients vs HD using EuroFlow software tools showed systematically altered patterns in CVID. These consisted of: i) a  normally-appearing maturation pathway with altered levels of expression of >1 (CD38, CD5, CD19, CD21, CD24, and/or smIgM) phenotypic marker (57/88 patients; 65%) for a total of 3 distinct CVID patient profiles (group 1: 42/88 patients, 48%; group 2: 8/88, 9%; and group 3: 7/88, 8%) and ii) CVID patients with a clearly altered pre-GC B cell maturation pathway in blood (group 4: 31/88 cases, 35%).
Our results show that maturation of pre-GC B-cells in blood of CVID is systematically altered with up to four distinctly altered maturation profiles.

Background: Several studies indicate that a group of Common Variable Immunodeficiency (CVID) patients actually suffer from late-onset combined immunodeficiency (LOCID), a different disease with a T-cell defect associated with a more aggressive clinical behavior. Some criteria based on naïve CD4+ T-cell counts have been proposed for LOCID (the DEFI and Freiburg criteria). However, these criteria have been arbitrarily established without considering age-related variations in the reference values for naïve CD4+ T cells.

Aims: Search a new age-matched criteria to better discriminate LOCID among patients with a CVID-like clinical phenotype.

Methods: In this multicentric study, 157 CVID patients were studied in parallel to 270 healthy donors using 8-colors flow cytometry based on the Euroflow standard operation procedures.

Results & Conclusion: Using the new age-matched criteria, a higher number of LOCID patients was observed (n=56) vs. the Freiburg (n=35) and DEFI (n=21) classifications. The age-based criteria was particularly efficient to identify LOCID patients, among the 18-39y (36% vs. 23% and 9%; p<0.01) and 40-59y (46% vs. 28% and 19%; p<0.001) CVID age-groups. Frequency of infections were associated with IgG and IgA serum levels at diagnosis, but presence of non-infectious complications were exclusively found among individuals with low naïve CD4+ T-cell counts, independently of the LOCID criteria. However, the age-based criteria showed a higher sensitivity for identifying of patients suffering autoimmune cytopenia (66% vs. 41% and 24%), enteropathy (60% vs. 31% and 31%) splenomegaly (60% vs. 40% and 24%), hepatomegaly (69% vs. 42% and 35%), lymphadenopathy (71% vs. 49% and 27%), granulomas (70% vs. 44% and 25%) and interstitial lung disease (59% vs. 47% and 24%) than the Freiburg and DEFI criteria, respectively. In conclusion, the new age-matched criteria to identify CVID patients with decreased naïve CD4+ T-cell counts results in a more accurate diagnosis of LOCID, associated with more severe clinical phenotypes.

Background: Pleural fluid samples are the most frequent body fluids immunophenotyped in the clinical laboratory. Pleural effusions are associated with various pathological processes. It is extremely difficult to distinguish reactive lymphocytes from hematopoietic malignancy or mesothelial cells from metastases based on cytomorphology alone, even for the most experienced Pathologist. Flow Cytometry is crucial in contributing to diagnose and monitor hematopoietic and metastatic disease in body fluids.

Aims: This study is a single-institution retrospective review of the flow cytometric analysis of pleural fluid samples from 2018 until 2022. Our goal was to identify the percentage of neoplastic samples and also the percentage of cases in which it was the first result to establish the suspicion of a diagnosis.

Methods: A screening 8-color tube (BD OneFlow™ LST) was used to discriminate reactive from abnormal lymphocytes. In case abnormal lymphocytes were identified a more extensive characterization was obtained using 8- color specific panels. Epithelial cells were identified by the positivity of CD326 (DAKO™ clone Ber-EP 4) and negativity of both CD45 and CD33.

Results & Conclusion: 513 pleural fluid samples from 432 patients were studied. The results of 302 samples (58.9%) presented no evidence of malignancy. The remaining 211 samples (41.1%) were positive for hematopoietic neoplasms (13.1%), and carcinoma (27.9%) respectively. The hematopoietic neoplasms included 2 LMA, 1 NK-NHL, 7 T-NHL and 57 B-CLPD (CLL and NHL). In 50 cases (23.7% of the positive cases) patients without prior clinical established diagnosis were newly diagnosed based on flow cytometry results. Flow cytometry for pleural fluids is of great utility for hematopoietic malignancy or carcinoma metastasis, being sometimes the only study that raises the suspicion of a diagnosis given the large number of cells analyzed, the short time of response and the accuracy to identify and classify the abnormal cells, not only at diagnosis but also for patient follow-up.

Background: Non-muscle invasive bladder cancer (NMIBC) has been treated for decades with immunotherapy with relatively good success rates. Nontheless, to date we lack a full characterisation of the microenvironment of this type of tumours. Besides, immune check-point inhibitors has entered the treatment scheme in this disease. A better understanding of the composition of these tumours could help to better patient stratification and management.

Aims: To use flow cytometry to characterise non-muscle invasive tumours in terms of subset composition and immune check-point inhibitor expression in order to find celular markers that may help stratify patients.

Methods: 98 bladder cancer patients were enrolled along three years in this study, from which we collected paired tumour and non-pathological samples. We digested freshly removed tissue samples into single cell suspensions and stained cells with two flow cytometry panels to characterise the following subsets: Urothelial/tumour cells, macrophages and M2-like macrophages, neutrophils, conventional dendritic cells type 1 and 2, T cells, NK cells, endotelial cells, mesemchymal cells and fibroblasts. Results were analysed using manual gating and unbiased computactional methods.

Results & Conclusion: We found that NMIBC tumours present all the cell subsets analysed. Compared to non-patholocal tissue, tumours display high macrophage infiltration and several signs of inflammation. Stroma infiltration and landscape varied significantly according to tumour stage. PD-L1 expression was similar between tumour and non-pathological samples in urothelial cells, but higher in the tumour microenvironment. Finally, we used an agnostic approach to find biomarkers associated to tumour grade and found that M2-like macrophages and activated fibroblasts are more abundant in high grade tumours. Our findings confirm that NMIBC tumours are infiltrated with immune and non-immune cells, and that increased PD-L1 expression comes from microenvironment cells. We also provide novel markers to help in tumour grading, of key importance for patient classification.

Any form of tissue damage will instantly recruit immune cells to control this damage and induce repair. This is valid for infectious diseases, inflammatory diseases, auto-immune diseases, and trauma as well as for many different types of medical interventions (surgery, irradiation, cytotoxic drugs, immunosuppressive treatment and immunotherapies). Obviously, dependent on the type of tissue damage, the degree and type of immune response will differ.

Thanks to many new developments in flow cytometry, cellular immune responses are stepwise better understood, particularly due to the EuroFlow-based analysis of differentiation, maturation and activation pathways with multi-color (>35-color) flowcytometry. Depending on the clinical setting, the focus of the cellular studies should be adapted to consecutive steps, such as in infection, vaccination and surgical interventions, were several mature immune subsets play consecutive roles. In contrast, specific immature subsets should be studied to understand reconstitution and regeneration during & after specific cytotoxic therapies or after stem cell transplantation. In targeted immunotherapies, such as in hemato-oncology, immune monitoring during & after therapy should be combined with minimal residual disease, regrowth of the normal targeted cells and the immune therapy itself, particularly in case of CAR T-cells.

Finally, in addition to the EuroFlow-based pathway concepts, also high levels of standardization appear to be critical to get comparable results at different sites within the same clinical protocols. This concerns, the sample processing, the choice of instruments (allowing acquisition of 25,000 to 30,000 cells per second), acquisition of 5 to 10 million cells, usage of at least 25 colors, no complex requirements for compensation settings, and comparability & stability of instruments at different sites (and overtime). This is why European collaboration should be fostered in order to obtain comparable results between many different clinical sites in international treatment protocols.