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Digital archive of theses discussed at the University of Pisa

 

Thesis etd-02282014-091716


Thesis type
Tesi di dottorato di ricerca
Author
OTTAVIANO, VIRGINIA
URN
etd-02282014-091716
Thesis title
Development of a new flow cytometric method to study chronic lymphocytic leukemia.
Academic discipline
MED/15
Course of study
SCIENZE BIOLOGICHE E MOLECOLARI
Supervisors
tutor Carulli, Giovanni
tutor Prof. Petrini, Mario
Keywords
  • chronic lymphocytic leukemia.
  • cytometric
Graduation session start date
11/03/2014
Availability
Full
Summary
Background: B-cell chronic lymphocytic leukaemia (B-CLL) is a B-cell neoplasm that in many patients shows an indolent course and survive for a prolonged period without therapy, but in others, CLL progresses rapidly and they die of the disease. Because of the difficulty in identifying such patients at diagnosis, only patients with progressive or symptomatic disease currently are recommended for therapy. Although easily characterized by diagnostic markers, the evaluation of an individual CLL patient’s prognosis remains a problematic issue. .
Methods: We have performed a simple six colours flow cytometry assay for ZAP-70 in peripheral blood of 5 healthy donors and 20 B-CLL patients. Samples were incubated with the following monoclonal antibodies: anti CD3-PerCP, anti CD5 FITC anti CD38-APC, anti CD45 APC Cy-7, anti CD19 PE Cy-7, treated with the Intrasure kit and finally stained with the anti-human/mouse ZAP-70-PE conjugate. Samples were acquired on a FACSCanto flow cytometry and analyzed with FacsDiva software (Becton-Dickinson). Quantification of CLLU1 expression in CD19+ cells of donors and buffy coat of patients was performed with the use of CLLU-1 ProfileQuant Kit (Ipsogen) by RQ-PCR.
Conclusion: About ZAP-70, the ratio between B-MFI/ T-MFI could be used to discriminate positive and negative patients instead of T-cells residual method. About CLLU-1 expression we have analyzed 5 healthy donors and 20 patients, donors showed very low levels of expression of the gene while CLL patients showed variable levels of expression.
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