Flow cytometric sorting of paraffin-embedded tumor tissues considerably improves molecular genetic analysis

• Published in: American journal of clinical pathology Vol. 120; no. 3; pp. 327 - 334
• Main Authors: JORDANOVA, Ekaterina S, CORVER, Willem E, VONK, Marcel J, LEERS, Math P. G, RIEMERSMA, Sietske A, SCHUURING, Ed, KLUIN, Philip M
• Format: Journal Article
• Language: English
• Published: Chicago, IL American Society of Clinical Pathologists 01.09.2003
• Subjects: Biological and medical sciences; Chromosome Aberrations; Chromosomes, Human, Pair 6; Dissection - methods; Flow Cytometry - methods; Hematologic and hematopoietic diseases; Hematology; HLA Antigens - genetics; Humans; In Situ Hybridization, Fluorescence; Investigative techniques, diagnostic techniques (general aspects); Leukemias. Malignant lymphomas. Malignant reticulosis. Myelofibrosis; Loss of Heterozygosity; Lymphoma, B-Cell - genetics; Lymphoma, Non-Hodgkin - genetics; Medical sciences; Paraffin Embedding; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Diffuse; Human; Flow cytometry; Paraffin-embedded tissue; LOH; Immunocytochemistry; Fluorescent in situ hybridization; Malignant hemopathy; B-Lymphocyte; Large cell lymphoma; Non Hodgkin lymphoma; Anatomic pathology; Lymphoproliferative syndrome; Loss of heterozygosity; Cytogenetics; FISH; Technique; Molecular biology; HLA; Flow cytometric sorting; B-cell lymphoma
• ISSN: 0002-9173; 1943-7722
• DOI: 10.1309/HPR11R7LQ9NNCCG8

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.