Application Guide - QIAxcel Advanced

56 QIAxcel Advanced Application Guide 10/2016 Reliable and fast detection of B-cell clonality in lymphoproliferations Jean Benhattar Institute of Pathology, Centre Hospitalier Universitaire Vaudois (CHUV) and University of Lausanne, Lausanne, Switzerland DNA fragments amplified using multiplex PCR that was developed to detect immunoglobulin and T-cell receptor gene rearrangements were resolved using the QIAxcel® system. Accurate size determination enabled identification of the type of B-cell clonality in lymphoproliferation. Introduction Lymphoma is a cancer of the lymphatic or the immune system (1). The World Health Organization (WHO) lists 43  different forms of lymphoma, including well-known Hodgkin’s and several non-Hodgkin lymphomas (2). Research on lymphoid malignancies relies on histomorphological, cytomorphological, and immunohistological methods and evaluations. The vast majority of lymphomas (>98%) exhibit rearrangements in immunoglobulin (Ig) and/or T-cell receptor (TCR) genes. Because of the clonal origin, all cells of a lymphoma exhibit identical gene rearrangements, and, therefore, monoclonality in lymphoid tissue suggests a malignant disease. Polyclonality, in contrast, indicates normal B-cell maturation (3). Thus, clonal gene rearrangement assessments are of great importance for understanding lymphoid malignancies. In the BIOMED-2 European collaborative project, multiplex PCR assays were developed and standardized to study such rearrangements. By detecting rearrangements in the in IGH and IGK genes using multiplex PCR, it is possible to identify virtually all B-cell proliferations of clonal origin (3). Here, we describe the use of the QIAxcel system for fast and reliable capillary electrophoresis analysis to identify the clonality of B-cells in lymphoproliferative tissues. Materials and methods Detection of B-cell clonality was performed using BIOMED-2 primer sets for IGH (IGH-FR3 and IGH-FR2) and IGK (IGK-A and IGK-B) gene rearrangements (3). DNA was extracted from archival formalin-fixed, paraffin-embedded tissue sections. PCR amplification (40 cycles) was performed in duplicate with 10–100  ng of extracted DNA in a total volume of 20 µl. PCR products were denatured at high temperature (5  minutes at 95°C) before being subjected to rapid renaturation at low temperature (1 hour at 4°C) to enhance the formation of multiple heteroduplexes. Homoduplexes exhibit the same migration rate during electrophoretic separation, whereas, heteroduplexes exhibit different migration rates, allowing monoclonal lymphoproliferations to be distinguished from polyclonal lymphoproliferations.

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