A Strategy for Multiple Immunophenotyping by Image Cytometry: Model Studies Using Latex Microbeads Labeled with Seven Streptavidin-Bound Fluorochromes

Multiple immunophenotyping is aimed at identifying several cell populations in a single labeling procedure by their ability to bind combinations of specific labeled antibodies. The present work demonstrates the simultaneous discrimination by using image cytometry of aminomethylcoumarin acetate (AMCA), Lucifer yellow (LY), fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), PE-Texas red tandem (Red613), peridinin-chlorophyll protein (PerCP), and allophycocyanin (APC), which were all bound to latex beads as streptavidin-conjugated fluorochromes. This has been the result of a step-by-step optimization of the several factors affecting the sensitivity and specificity of multiple immunofluorescence analysis. First, 14 streptavidin-conjugated fluorochromes were evaluated by using spectrofluorometry. A primary selection was then made of ten spectrally separable dyes that could be evaluated by using image cytometry. These dyes were bound to latex particles, and specific filter combinations were assembled to minimize crosstalk between fluorophores while preserving sufficient fluorescence intensity and counting statistics. Potential probe associations were then assessed by measuring the emissions of all fluorochromes that were detected by each filter combination. The resulting crosstalk matrix served as the basic tool both for final selection of the optimal filter combination and for dye set (composed, in this case, of the seven fluorochromes described above) and for mathematical correction of residual spectral overlap. Next, an image cytometry system was adapted to collect seven images of matched brightness with the selected combination of excitation/emission filters and dichroic mirrors. Finally, seven-parameter synthetic images were generated by digital image processing

Hemophilia : a disease on the move

peer reviewedOver the last hundred years, the treatment of hemophilia has evolved considerably. To date, its principle is still to prevent the occurrence of hemorrhages by regular intravenous injections of factor VIII or IX concentrate. It allows to reach a life expectancy similar to the general population. The quality of life is constantly improving despite the constraint imposed by the modality and frequency of injections. The main complication remains the development of antibodies that inhibit the administered factors. Concentrates of long-acting factors are now available allowing to limit for example the frequency of injections. A bispecific monoclonal antibody reproducing the action of factor VIII and injectable subcutaneously has recently become available to hemophilia A patients, with the advantage of being effective even in the presence of inhibitors. Other non-substitute products are being studied offering interesting leads. Finally, gene therapy shows promising results, giving hope for access to this therapeutic option in a relatively near future. These advances are, however, a challenge for clinical laboratories, which must adapt their measurement techniques to ensure optimal monitoring. The future is on its way for hemophilia. Treatment remains expensive but it is worth the price.Depuis un siècle, le traitement de l’hémophilie a considérablement évolué. À ce jour, son principe est toujours de prévenir la survenue d’hémorragies par injections intraveineuses régulières de concentré de facteur VIII ou IX. Il permet d’atteindre une espérance de vie similaire à la population générale. La qualité de vie est en constante amélioration, malgré la contrainte imposée par les modalités et la fréquence des injections. La complication principale reste le développement d’anticorps inhibant les facteurs administrés. Des concentrés de facteurs à action prolongée sont, maintenant, disponibles et permettent, notamment, de limiter la fréquence des injections. Un anticorps monoclonal bispécifique reproduisant l’action du facteur VIII et injectable par voie sous-cutanée est, depuis peu, à la disposition des patients hémophiles A, avec l’avantage d’être efficace même en présence d’inhibiteurs. D’autres produits non substitutifs sont à l’étude offrant des pistes intéressantes. Enfin, la thérapie génique montre des résultats prometteurs, laissant espérer un accès à cette option thérapeutique dans un futur relativement proche. Ces avancées sont cependant un défi pour les laboratoires d’analyse qui doivent adapter leurs techniques de mesure pour assurer un suivi optimal. Le futur est en marche pour l’hémophilie. Le traitement reste coûteux, mais il en vaut le prix

Continuous platelet infusion in patient refractory to platelet transfusion : a retrospective study.

editorial reviewedPlatelet transfusion is commonly used for the prevention of bleeding in central thrombocytopenia. It is estimated that 7-34 % of chronically transfused patients become progressively refractory until they no longer have an increase in post-transfusion platelet count. In this case, and in the presence of bleeding, a therapeutic option is continuous transfusion (CT) of platelet concentrates (PC). We performed a retrospective study of patients at the University Hospital of Liege who received a CT between 01/01/2019 and 31/12/2020. We explored the etiology, immune or non-immune, of the refractory state and analyzed the clinical and biological evolution after TC. In our cohort, 13 patients received CT during the study period; for 8 of them, the refractory state was explained by non-immune causes. The mean platelet count increased during CT and in 61 % of cases, an improvement in bleeding symptomatology was obtained.La transfusion plaquettaire est couramment utilisée pour la prévention des saignements en cas de thrombopénie centrale. On estime que 7 à 34 % des patients transfusés chroniquement deviennent progressivement réfractaires jusqu’à ne plus présenter d’augmentation de la numération plaquettaire post-transfusionnelle. Dans ce cas, et en présence de saignements, une option thérapeutique correspond à la transfusion continue (TC) de concentrés plaquettaires (CP). Nous avons réalisé une étude rétrospective sur les patients du CHU de Liège ayant reçu une TC entre le 01/01/2019 et le 31/12/2020. Nous avons exploré l’étiologie, immune ou non immune, de l’état réfractaire et analysé l’évolution clinique et biologique après TC. Dans notre cohorte, 13 patients ont bénéficié de TC durant la période étudiée; pour 8 d’entre eux, l’état réfractaire s’expliquait par des causes non immunes. La numération plaquettaire moyenne a augmenté durant la TC et, dans 61 % des cas, une amélioration de la symptomatologie hémorragique a été obtenue

How to explore… Inherited platelet disorders

peer reviewedInherited platelet disorders (IPD) include a set of rare diseases whose diagnosis is often difficult because it requires the use of complex biological assays in specialized centers. They are probably under-diagnosed. Clinicians should consider an IPD when facing a chronic thrombocytopenia resistant to intravenous immunoglobulins (IVIG) and steroids together with a family history of thrombocytopenia. A syndromic thrombocytopenia will be suspected by the family survey and specific clinical signs. The confirmation of the diagnosis will then require the use of specialized biological assays such as platelet aggregation, flow cytometry, electron microscopy, platelet secretion assays, karyotype and molecular biology.Les thrombopénies et thrombopathies constitutionnelles constituent un ensemble de pathologies rares dont le diagnostic est souvent difficile car il nécessite le recours à des analyses biologiques souvent réservées à des centres spécialisés. Elles sont probablement sous-diagnostiquées. Le clinicien devra les envisager devant une thrombopénie chronique ne répondant pas aux immunoglobulines intraveineuses et aux corticoïdes et la présence d’antécédents familiaux. Une thrombopénie syndromique sera suspectée en fonction des éléments de l’anamnèse familiale et de signes cliniques spécifiques. La confirmation du diagnostic nécessitera la réalisation d’examens biologiques spécialisés (agrégation plaquettaire, cytométrie en flux, microscopie électronique, tests de sécrétion, caryotype et biologie moléculaire)

Monoclonal B-cell lymphocytosis: asymptomatic condition or pretumoral stage

peer reviewe

Machine learning identification of specific changes in myeloid cell phenotype during bloodstream infections

The early identification of bacteremia is critical for ensuring appropriate treatment of nosocomial infections in intensive care unit (ICU) patients. The aim of this study was to use flow cytometric data of myeloid cells as a biomarker of bloodstream infection (BSI). An eight-color antibody panel was used to identify seven monocyte and two dendritic cell subsets. In the learning cohort, immunophenotyping was applied to (1) control subjects, (2) postoperative heart surgery patients, as a model of noninfectious inflammatory responses, and (3) blood culture-positive patients. Of the complex changes in the myeloid cell phenotype, a decrease in myeloid and plasmacytoid dendritic cell numbers, increase in CD14(+)CD16(+) inflammatory monocyte numbers, and upregulation of neutrophils CD64 and CD123 expression were prominent in BSI patients. An extreme gradient boosting (XGBoost) algorithm called the “infection detection and ranging score” (iDAR), ranging from 0 to 100, was developed to identify infection-specific changes in 101 phenotypic variables related to neutrophils, monocytes and dendritic cells. The tenfold cross-validation achieved an area under the receiver operating characteristic (AUROC) of 0.988 (95% CI 0.985–1) for the detection of bacteremic patients. In an out-of-sample, in-house validation, iDAR achieved an AUROC of 0.85 (95% CI 0.71–0.98) in differentiating localized from bloodstream infection and 0.95 (95% CI 0.89–1) in discriminating infected from noninfected ICU patients. In conclusion, a machine learning approach was used to translate the changes in myeloid cell phenotype in response to infection into a score that could identify bacteremia with high specificity in ICU patients

Curcuma longa and Boswellia serrata Extracts Modulate Different and Complementary Pathways on Human Chondrocytes In Vitro: Deciphering of a Transcriptomic Study

peer reviewedObjectives:Curcuma longa (CL) and Boswellia serrata (BS) extracts are used to relieve osteoarthritis symptoms. The aim of this in vitro study was to investigate their mechanisms of action at therapeutic plasmatic concentrations on primary human osteoarthritic (OA) chondrocytes.Methods: BS (10–50 μg/ml) and CL (0.4–2 μg/ml corresponding to 1–5 µM of curcumin) were evaluated separately or in combination on primary chondrocytes isolated from 17 OA patients and cultured in alginate beads. Ten patients were used for RNA-sequencing analysis. Proteomic confirmation was performed either by immunoassays in the culture supernatant or by flow cytometry for cell surface markers after 72 h of treatment.Results: Significant gene expression modifications were already observed after 6 h of treatment at the highest dose of CL (2 μg/ml) while BS was significantly effective only after 24 h of treatment irrespective of the concentration tested. The most over-expressed genes by CL were anti-oxidative, detoxifying, and cytoprotective genes involved in the Nrf2 pathway. Down-regulated genes were principally pro-inflammatory cytokines and chemokines. Inversely, BS anti-oxidant/detoxifying activities were related to the activation of Nrf1 and PPARα pathways. BS anti-inflammatory effects were associated with the increase in GDF15, decrease in cholesterol cell intake and fatty acid metabolism-involved genes, and down-regulation of Toll-like receptors (TLRs) activation. Similar to CL, BS down-regulated ADAMTS1, 5, and MMP3, 13 genes expression. The combination of both CL and BS was significantly more effective than CL or BS alone on many genes such as IL-6, CCL2, ADAMTS1, and 5.Conclusion: BS and CL have anti-oxidative, anti-inflammatory, and anti-catabolic activities, suggesting a protective effect of these extracts on cartilage. Even if they share some mechanism of action, the two extracts act mainly on distinct pathways, and with different time courses, justifying their association to treat osteoarthritis