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Luigi del Vecchio. CEINGE-Università di Napoli. Nápoles (Italia)

Luigi del Vecchio. CEINGE-Università di Napoli. Nápoles (Italia) Image-in-flow cytometry in leukemia studies. Background.

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Luigi del Vecchio. CEINGE-Università di Napoli. Nápoles (Italia)

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  1. Luigi del Vecchio. CEINGE-Università di Napoli. Nápoles (Italia) Image-in-flow cytometry in leukemia studies

  2. Background • To date, the strength of onco-hematological flow cytometry is based upon the identification of complex and specific phenotypic mosaics thanks to the increasing poly- or hyper-chromaticability of the technique. • Thus, • CD34+/CD33+/MPO+/CD7+/CD117+ is an immunophe- notype strongly indicating an acute leukemia of myeloid origin, while CD19+/CD5+/CD20+/CD23+/SIg+ profile suggest the presence of a low grade non-Hodgkin lymphoma.

  3. Immunolocalization • Another concept is rapidly emerging in diagnostic flow cytometry: the specific detection of intracellular localization of a given protein. • The concept is based upon a series of observations mainly produced by Brunangelo Falini in Perugia, who demonstrated that several molecules tend to change their pattern of localization within the neoplastic cells, thus offering the possibility of identifying a hematopoietic tumor, by localizing a molecule susceptible of changes in its sub-cellular distribution.

  4. Examples of immunolocalization PML localization within nuclei of acute promyelocytic leukemia (APL) cells is abnormal (micro-speckled) as compared to normal cells (macro-speckled). The aberrant NPM1 cytoplasmic localization within an acute myeloblastic leukemia cell can be a surrogate of NPM1 mutation. The differential localization (nuclear, cytoplasmic or both) of the anaplastic lymphoma kinase (ALK) in anaplastic large-cell lymphoma carrying chromosomal translocations that cause the (ALK) gene to fuse with a variety of gene partners allows the sub-classification of this kind of neoplasm.

  5. IMAGESTREAM David A. Basiji is the inventor of the ImageStream system and founder of Amnis Corporation. Prior to founding Amnis, Dr. Basiji worked at the University of Washington, under the guidance of Ger van den Engh, the inventor of MoFlo and InFlux.

  6. Internal side Imagestream is able to analyze not only the entire cell, but also the thousands pixels forming the structure of the cell image External side

  7. Internalization Internalization of zymosan (green) by murine RAW cells (orange) identified by immunophenotyping. Phagocytosis is measured as the percentage of cells with internalized zymosan at 15, 30 and 60 minutes at 37 degrees C.

  8. MHC class II upregulation and colocalization with Fas in experimental models of immune-mediated bone marrow failure Co-expression and co-localization Erie, 2012

  9. Neutrophils

  10. + Morphology Immunophenotype I Level Suspect of APL ATRA Immuno- localization of PML RT-PCR Karyotype FISH II Level

  11. non APL APL • PML nuclear bodies are multimolecular complexes that contain apart from PML, other proteins such asi Daxx, CBP and BLM • The t(15;17) modifies the architecture of such nuclear domains • There is a change of pattern: from normal to “microgranular”

  12. non-APL APL

  13. HL-60 (normal PML)

  14. NB-4 (PML/RARa)

  15. HL-60 Modulation = (max pixel – min pixel)/(max pixel + min pixel). The bigger the range in the per-cell PML stains, the higher the modulation scores.

  16. NB-4 Modulation = (max pixel – min pixel)/(max pixel + min pixel). The bigger the range in the per-cell PML stains, the higher the modulation scores.

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