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FU Berlin
Digitale Dissertation

Sabine Helmes :
Analysis of Hodgkin cells in B cell chronic lymphatic leukemia by single cell PCR and experiments on the establishment of the single cell PCR on cells isolated from formalin-fixed paraffin-embedded biopsies
Analyse von Hodgkin-Zellen in B-Zell chronischer lymphatischer Leukämie mittels Einzelzell-PCR und Untersuchungen zur Etablierung der Einzelzell-PCR von Zellen aus Formalin-fixierten, in Paraffin eingebetteten Biopsaten

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Abstract

The occurence of HRS cells in cases of human CLL is a rare phenomenon. Until recently, investigations on the origin of these HRS cells were restricted on morphological description and immunohistochemical analysis. In this thesis, the first molecular analysia of Ig gene rearrangements of a case of B-CLL with HRS cells on the level of individual cells by single cell PCR is presented. The analysis aimed at a more detailed understanding of the possible relationship between Hodgkin´s disease and NHL.
The sequences of the Ig gene rearrangements of the NHL B cells were identical, indicating clonality of these cells. An identical pattern was obtained for the HRS cells. Comparing the sequences from the HRS cells and the B cells of the NHL, a difference of on base was observed. Thus, the assumption that the two cell populations are closely related is valid.

Due to the spareseness of important cases as studied in this thesis as a fresh tissue biopsy, such single cell investigations are limited. A higher number of comparable cases are conserved as formalin-fixed and paraffin-embedded samples in different institutes interested in hematology. For that reason, we wanted to transfer the single cell PCR technique established on fresh tissue to the analysis of formalin-fixed and paraffin-embedded cells. Since DNA is degraded by formalin, primers were established that bind to sequences in the FR2 and FR3 of the V genes in order to generate PCR products shorter than those obtained with primers binding to sequences in the FR1 of the genes in the Ig heavy chain locus.

We could show that generally the detection of gene rearrangements in single cells isolated from paraffin tissue is possible. However, compared to the efficiency obtained using fresh tissue, the observed efficiency of 10% was low.

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More Information:

Online available: http://www.diss.fu-berlin.de/1998/52/indexe.html
Language of PhDThesis: german
Keywords: Hodgkin´s-disease; b-lymphocytes; polymerase-chain-reaction
DNB-Sachgruppe: 34 Veterinärmedizin
Date of disputation: 20-Jun-1997
PhDThesis from: Fachbereich Veterinärmedizin, Freie Universität Berlin
First Referee: Univ.-Prof. Dr. G. Monreal
Second Referee: Univ.-Prof. Dr. M.-L. Hansmann / Köln
Date created:14-Dec-2000
Date available:07-Feb-2001

 

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