scholarly journals Our Experiences With B Cell Clonality Analysis Using Multiplex Pcr Amplification in Paraffin-Embedded Tissue

2014 ◽  
Vol 14 (1) ◽  
pp. 5-13
Author(s):  
L. Stanek ◽  
S. Llsova ◽  
D. Tvrdlk

Abstract The clonal determination of B-cell lymphoproliferative diseases by immunoglobulin heavy chain (IgH) rearrangement by polymerase chain reaction (PCR) is widely used. In this study we report our experiences with B cell clonality analysis using multiplex PCR amplification followed by heteroduplex analysis, which was found to be essential for the efficient resolution of monoclonal bands within polyclonal backgrounds, in formalin-fixed paraffinembedded tissue.

1993 ◽  
Vol 2 (1) ◽  
pp. 42-49 ◽  
Author(s):  
Thomas J. Reed ◽  
Ann Reid ◽  
Karen Wallberg ◽  
Timothy J. O??Leary ◽  
Glauco Frizzera

1993 ◽  
Vol 2 (1) ◽  
pp. 42-49 ◽  
Author(s):  
Thomas J. Reed ◽  
Ann Reid ◽  
Karen Wallberg ◽  
Timothy J. O??Leary ◽  
Glauco Frizzera

2016 ◽  
Vol 16 (1) ◽  
pp. 17-21 ◽  
Author(s):  
M Vazan ◽  
I Kasubova ◽  
A Vanochova ◽  
P Lukac ◽  
L Plank ◽  
...  

AbstractIntroduction:A clonal population of B-cells is defined as those cells arising from the mitotic division of a single somatic cell with the same rearrangement of immunoglobulin genes. This gives rise to DNA markers for each individual lymphoid cell and its progenies and enables us to study clonality in different B-cell malignancies using multiplex polymerase chain reaction - PCR. The BIOMED-2 protocol has been implemented for clonality detection in lymphoproliferative diseases and exploits multiplex PCR reaction, subsequently analyzed by heteroduplex analysis (HDA) using polyacrylamide gel electrophoresis (PAGE). With the advent of miniaturization and automation of molecular biology methods, lab-on-chip technologies were developed and replace partially the conventional approaches. We tested device for microfluidic chip, which is used for B-cells clonality analysis, using a PCR reaction for three subregions called frameworks (FR) of the immunoglobulin heavy locus (IGH) gene.Material and Methods:For the implementation and comparison of the two methods we analyzed three unknown B-cell chronic lymphocytic leukemia (B-CLL) samples. As positive control (PK) we used one formalin-fixed, paraffin-embedded (FFPE) sample from B-CLL lymph node. The DNA was extracted from FFPE sections and multiplex PCR was used to amplify IGH gene segments. After PCR, the HDA was performed, the DNA fragments were evaluated on the PAGE and the microfluidic chip electrophoresis as well, and the results were compared.Results:Using HDA with subsequent PAGE, we were able to confirm the clonality of the positive control and the tested samples. The same results were obtained by the Bioanalyzer 2100. The microfluidic chip electrophoresis was persuasive in all tested samples.Conclusion:The implementation of microfluidic chip electrophoresis for detection of B-cell clonality by BIOMED-2 protocol on the device Agilent 2100 Bioanalyzer was successful and yielded the same results as the HDA - PAGE. Moreover, chip electrophoresis system is faster for preparation and less laborious than the conventional HDA - PAGE method.


1995 ◽  
Vol 81 (6) ◽  
pp. 405-409 ◽  
Author(s):  
Valli De Re ◽  
Salvatore De Vita ◽  
Antonino Carbone ◽  
Gianfranco Ferraccioli ◽  
Annunziata Gloghini ◽  
...  

Aims and background The detection of immunoglobulin heavy chain variable (VH)-diversity (DH)-joining (JH) region gene rearrangement by polymerase chain reaction (VDJ PCR) has been recently proposed as a rapid approach to assess B-cell clonality in lymphoproliferative disorders. The aim of the present study was to determine the efficacy of VDJ PCR in a wide spectrum of lymphoproliferative disorders previously characterized by immunohistochemistry and Southern blot (SB). Methods 83 SB-rearranged B-cell non-Hodgkin's lymphomas (NHL) of different histotype, 22 cases of SB-unrearranged classical Hodgkin's disease (HD), 18 cases of HIV-related reactive lymphadenopathy, and 4 frankly pre-lymphomatous lesions (MESA) in the course of Sjögren's syndrome were investigated by 2 different VDJ PCR protocols (FR3, FR2). Results The detection rate in NHL was 64% and 71% using the protocols FR3 and FR2, respectively. However, the overall VDJ PCR efficacy increased to 81% by combining the results of both protocols. In addition, differences in the combined, as well as in the single FR3 or FR2 protocol efficacy, were noted in the different NHL subgroups. B-cell clonality was also detected in 4/22 (18%) SB-unrearranged classical HD cases and in 2/18 (11%) reactive lymphadenopathy cases, whereas it was demonstrated in all the MESA lesions, 2 of them being SB-negative. Conclusions VDJ PCR represents a useful and rapid technique to detect B-cell clonality in NHL, although with some differences depending on the NHL histotype and the panel of primers employed. The technique may also be of value to investigate the possible progression of early B-cell clonal expansion into frankly B-cell malignancy and to contribute to the controversy about the clonal lineage origin of the putative HD malignant cells.


Sign in / Sign up

Export Citation Format

Share Document