Laser beam microdissection in the diagnosis of cutaneous B-cell lymphoma

Abstract

Interpretation of molecular analyses of cutaneous lymphoid infiltrates may be difficult because a heterogeneous group of cells in usually present within the neoplasms. Extraction of DNA from tissue sections does not provide exact information about which cell population has been analysed. We present a laser microscope system that allows selective molecular analysis of single cells or small groups of cells in cases of cutaneous lymphoma. An ultraviolet (UV)-laser microscope system (PALM, Wolfratshausen, Germany) was used to isolate particular populations of cells from a routinely processed specimen of a cutaneous follicular lymphoid proliferation. Using the UV-laser beam, a circle was cut around a target germinal centre in order to separate it from neighbouring tissues and to isolate a pure population of germinal centre cells. Isolated cells were scraped off with a micromanipulator and placed in a proteinase-K solution. DNA was extracted and amplified by the polymerase chain reaction (PCR) technique. Analysis of immunoglobulin JH gene rearrangement showed a distinct monoclonal band. In a second phase, using the same procedure in the same specimen, mantle zone cells around a germinal centre and single interfollicular B lymphocytes were isolated for PCR analysis of immunoglobulin JH gene rearrangement. In this population of cells, no clonality could be detected. This new technique allows the selective elimination of undesired cells and tissue from cutaneous neoplasms. By destruction of unwanted tissues with laser-beam energy a contamination-free sample is obtained. Analysis of isolated cells in our case demonstrated a clonal rearrangement derived from germinal centre cells and not from other B lymphocytes in the specimen, confirming the diagnosis of cutaneous follicle centre lymphoma. The method described has exciting implications for dermatology and dermatopathology, allowing precise correlation of morphological features with findings by molecular genetics.