Anti-CD9 Monoclonal antibody, clone LND9

Introduction In the context of neuroblastoma (NB), the screening for bone marrow (BM) metastasis is a recurrent issue for hematology laboratory routine practice. Detection of low tumor burden using light microscopy is often difficult. In this regard, our objective was to evaluate the performance of multiparametric flow cytometry (FC) for detecting NB metastatic cells in BM. Methods We applied a new FC multiparametric panel allowing the analysis of the co-expression of 5 surface markers: GD2 (disialoganglioside 2), CD9, CD56, CD81, and CD90, on CD45-negative BM cell populations, and compared results with BM biopsy immunohistochemistry, which is the reference method. Results In spike-in tests, the multiparametric FC successfully detected NB cells mixed in peripheral blood mononuclear cells to a level of 0.01%. FC analysis was performed on 45 sets of BM aspirates sampled from 21 children, either at diagnosis or during follow-up. Combining multiparametric FC with light microscopy improved NB metastasis detection, with a higher sensitivity (76.9% vs 61.5%) and a higher specificity (94.4% vs 77.8%) as compared to light microscopy alone. At the time of diagnosis, multiparametric FC detected NB metastatic cells in all cases. Conclusion These results illustrate the performance of multiparametric FC analysis to detect metastatic BM infiltration of NB. This is of particular interest in an emergency context, since when combined with light microscopy, it enhances the detection of metastatic invasion within a short timeframe, allowing an adapted and rapid clinical management.

Objective To identify whether factors toxic to oligodendrocytes (OLs), released by B cells from patients with MS, are found in extracellular microvesicles enriched in exosomes. Methods Conditioned medium (Sup) was obtained from cultures of blood B cells of patients with MS and normal controls (NCs). Exosome-enriched (Ex-En) fractions were prepared by solvent precipitation from Sup containing bovine serum and from serum-free Sup by ultracentrifugation (UC) or immunoprecipitation (IP) with antibodies to CD9. Ex-En fractions were diluted 1:4 with OL culture medium and screened for toxic effects on cultured rat OLs as measured by trypan blue uptake. Proteomic analysis was performed on Sup fractions. Results MS B cell-derived Ex-En fractions prepared from Sup by solvent extraction, UC, or IP induced OL death, whereas corresponding Ex-En fractions from NC showed little toxicity. Proteomic analysis of Sup demonstrated enrichment of proteins characteristic of exosomes from both NC and MS B-cell Sup. Ontology enrichment analysis suggested differences in the types and cargo of exosomes from MS Sup compared with NC, with proteins related to cell surface, extracellular plasma membrane, and gliogenesis enriched in MS. Conclusions Much of the in vitro toxicity of Sup from B cells of patients with relapsing-remitting MS is found in Ex-En fractions, as confirmed by 3 methods. Proteomic analysis of B-cell Sup indicates multiple differences between MS and NC.