Flow cytometric sorting of paraffin-embedded tumor tissues considerably improves molecular genetic analysis.
The characterization of genetic aberrations in paraffin-embedded tumor material is impaired by contaminating normal cells. In the present study on the genetic causes of loss of HLA expression in diffuse large B-cell lymphoma (DLBCL), we compared the efficacy of microdissection with flow cytometric sorting of tumor cells. Single-cell suspensions from paraffin-embedded material of 5 DLBCL cases were stained for CD79a and DNA content (propidium iodide). Fluorescent in situ hybridization (FISH) using HLA class II and chromosome 6 centromeric probes and loss of heterozygosity (LOH) analysis with 5 HLA-specific microsatellite markers were performed on microdissected and flow cytometry-sorted fractions. FISH confirmed considerable enrichment of the samples after flow cytometric sorting and disclosed tumor heterogeneity in 4 cases. Moreover, lymphomas with a so-called zebra LOH pattern in the microdissected material showed unambiguous LOH after flow cytometric sorting, revealing in 1 case a biologically relevant hemizygous deletion in the HLA region.
D002869:Chromosome Aberrations; D002896:Chromosomes, Human, Pair 6; D004210:Dissection / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D006680:HLA Antigens / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D019656:Loss of Heterozygosity; D016393:Lymphoma, B-Cell / Q000235:genetics*; D008228:Lymphoma, Non-Hodgkin / Q000235:genetics*; D016612:Paraffin Embedding
D006680:HLA Antigens