title: Molecular studies on single cells harvested by micromanipulation from archival tissue sections previously stained by immunohistochemistry or nonisotopic in situ hybridization.
abstract: Molecular analysis of isolated single cells is a powerful tool for analyzing heterogeneity within a population of cells and for clarifying issues of cell origin and clonality. Current techniques are limited by the availability of suitable fresh tissue. To broaden the applicability of molecular techniques at single-cell level, we have developed an approach that uses routinely processed archival tissue. Immunoglobulin heavy chain (IgH) gene rearrangement was analyzed in large tumor cells from four cases of diffuse large cell B-non-Hodgkin's lymphoma and in small reactive T and B lymphocytes from three cases of lymphocytic predominance Hodgkin's disease. One case of Epstein-Barr virus (EBV)-encoded RNA (EBER)-positive angiocentric pulmonary T-cell lymphoma was assayed for the presence of the BamHI-W multiple-copy fragment of the EBV genome. T- and B-lymphoid cells were immunostained with anti-CD3 and CD20, respectively. The tissue sections from the EBER-positive T-cell lymphoma were stained by nonisotopic in situ hybridization. Single cells were mobilized after proteolytic treatment under an inverted microscope using a hydraulic micromanipulator at a magnification of 400 x. Isolated cells were aspirated into a micropipette fixed to a second micromanipulator and transferred into a PCR tube. The IgH complementarity determining region (CDR)3 was successfully amplified in 17 of 52 (33%) small B-lymphocytes from lymphocytic predominance Hodgkin's disease using a previously reported semi-nested PCR method, and the products from each case differed in size as expected of a polyclonal population. None of the 49 small T lymphocytes demonstrated any amplifiable IgH CDR3 products, indicating no significant cellular contamination. The IgH CDR3 sequence analysis of the PCR products indicated a clonal relationship among harvested cells. In the T-cell lymphoma case, the harvested EBER-positive cells were amplifiable for the multiple-copy fragment BamHI-W of the EBV genome. Our study indicates that single-cell analysis can be performed on paraffin-embedded archival tissue after being subjected to immunoperoxidase and in situ hybridization procedures.
authors.affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68198-3135, USA.
authors.forename: F
authors.initials: F
authors.lastname: d'Amore
authors.forename: J A
authors.initials: JA
authors.lastname: Stribley
authors.forename: T
authors.initials: T
authors.lastname: Ohno
authors.forename: G
authors.initials: G
authors.lastname: Wu
authors.forename: R S
authors.initials: RS
authors.lastname: Wickert
authors.forename: J
authors.initials: J
authors.lastname: Delabie
authors.forename: S H
authors.initials: SH
authors.lastname: Hinrichs
authors.forename: W C
authors.initials: WC
authors.lastname: Chan
mesh_terms: D018951:Antigens, CD20; D001402:B-Lymphocytes / Q000166:cytology; D001483:Base Sequence; D017252:CD3 Complex / Q000032:analysis; D004854:Herpesvirus 4, Human / Q000235:genetics; D006689:Hodgkin Disease / Q000235:genetics; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008223:Lymphoma / Q000235:genetics; D008846:Micromanipulation / Q000379:methods*; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000032:analysis; D013601:T-Lymphocytes / Q000166:cytology
chemical_list: D018951:Antigens, CD20; D017252:CD3 Complex; D007143:Immunoglobulin Heavy Chains; D016693:Receptors, Antigen, T-Cell, alpha-beta