MOLECULAR STUDIES ON SINGLE CELLS HARVESTED BY MICROMANIPULATION FROMARCHIVAL TISSUE-SECTIONS PREVIOUSLY STAINED BY IMMUNOHISTOCHEMISTRY OR NONISOTOPIC IN-SITU HYBRIDIZATION

Molecular analysis of isolated single cells is a powerful tool for ana lyzing heterogeneity within a population of cells and for clarifying i ssues of cell origin and clonality. Current techniques are limited by the availability of suitable fresh tissue. To broaden the applicabilit y of molecular techniques at single-cell level, we have developed an a pproach 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 pr edominance Hodgkin's disease. One case of Epstein-Barr virus (EBV)-enc oded RNA (EBER)-positive angiocentric pulmonary T-cell lymphoma was as sayed for the presence of the BamHI-W multiple-copy fragment of the EB V 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 ce lls were mobilized after proteolytic treatment under an inverted micro scope using a hydraulic micromanipulator at a magnification of 400 x. Isolated cells were aspirated into a micropipette fixed to a second mi cromanipulator and transferred into a PCR tube. The IgH complementarit y 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 I gH CDR3 sequence was amplifiable in 9 of 26 (37%) tumor cells in the f our cases of B-non-Hodgkin's lymphoma. In case four, sequence analysis of the PCR products indicated a clonal relationship among harvested c ells. In the T-cell lymphoma case, the harvested EBER-positive cells w ere amplifiable for the multiple-copy fragment BamHI-W of the EBV geno me. Our study indicates that single-cell analysis can be performed on paraffin-embedded archival tissue after being subjected to immunoperox idase and in situ hybridization procedures.