| Titre : | Implémentation de la méthode flowfish en routine pour l’analyse de la taille des télomères |
| Auteurs : | Romain KOUHAÏL, Auteur ; J.-P. Defour, Promoteur |
| Type de document : | Travail de fin d'études |
| Editeur : | Bruxelles : Institut Paul Lambin, 2020 |
| Langues: | Français |
| Index. décimale : | TFE - Biologie médicale |
| Mots-clés: | telomeres ; PNA ; FlowFISH ; Hybridation ; PCR ; flow cytometry ; chromosomes |
| Résumé : |
Telomeres are significative parts of the chromosomes. Their main function is to preserve the extremities of the chromosomes, avoiding any cellular’s senescence. Researchers pointed a correlation between several pathologies and the decrease of the telomere’s lenght, as bone marrow deficiency, idiopathic pulmonary fibrosis or heavy emphysema and cancers.
To detect them, several technics were tested, as qPCR, Southern Blot or QFISH. But despite how advanced those technics are, results carried on being very differents for a same patient because of how difficult and changing the telomeres are (depending on the person). However, a new technic made its proofs, it is called FlowFISH. This technic uses a PNA probe to mark the chromosomes contained in the White cells. This DNA is analyzed by Flow Cytometry, which will give the fluorescence results (being proportionals to the lenght of the telomeres, and normalized by MESF beads). This technic shows a lot of positive aspects. The main purpose of this work will be to implemente the FlowFish in the routine of the laboratory of Saint-Luc. To succed in this project, a lot of parameters had to be taken in count. Firstly, we had to work on its own protocol. For this, we had to adapt the protocol of « Nature Procols » written by Peter Landsorp's team in 2007, to make it specific to Saint-Luc'slab. Despite how complete this procol was, we had to adapt it to the automates, material and reagents/products of the laboratory. Furthermore, FlowFISH uses several internal’s controls, as a flow test (used to verify the calibration of the cytometer), but also bovine thymocytes. We had to work with our own thymocytes that we preleved ourselves. The bovine thymocytes had shorter telomere’s lenght than our partners of De Duve Institute. That means we had to work to find a correction's factor, This factor allowed us to create the Saint-Luc curves based on the patient’s results of Anabelle Decottignies from De Duve Institute. After, we had to adapt several steps of the protocol to make it faster in the laboratory’s routine, as the Red Globules lysis. We had to compare same samples used with differents lysis to determine the most practiceful one, it recquiered a lot of stats and work. Finaly, The FlowFish is a technic which can be easyly made in a daily’slab, but it also recquiers time. The main challenge will be to introduce the technicians to this méthod. Indeed, they will have to practice and manipulate in the same time than the other analysis. As a conclusion, FlowFISH was a challenge on several points, from the technic's dicussions to the teaching of the FlowFISH to the technicians. But we arrived to make it work in the lab, which is an accomplishment on several points. However, the technicians still have to learn how to practice, using a protocol which works, but remains in process of rewriting. |
| Accès : | Identifiez-vous avant d'accéder au document électronique |
| Disponible en ligne : | Oui |
| Lieu du stage : | Laboratoire d’hématologie - Cliniques universitaires Saint-Luc |
| Département du TFE : | Biologie médicale |
Documents numériques (1)
 TFE biologie médicale Adobe Acrobat PDF |
