TIPS ON TECHNIQUES PCR Amplification of DNA Obtained From Archived Hematoxylin-Eosin– and Giemsa-Stained Breast Cancer Aspirates Fernando Carlos Schmitt, M.D., Ph.D., M.I.A.C.,* Raquel Soares, B.S., Luis Cirnes, C.T., and Raquel Seruca, M.D., Ph.D. The study of DNA abnormalities in fine-needle aspiration (FNA) specimens for breast cancer could be helpful in improving the capacity of diagnosis and specially to obtain prognostic or predictive information. The aim of the present study was to verify whether it is possible to perform molecular analysis in slides of breast cancer FNA specimens, previously stained by hematoxylin- eosin (H&E) and Giemsa, stored at least for 3 years. For this purpose, 10 cases of FNA obtained from breast cancer patients diagnosed between 1993 and 1994 in our institute were used. Five cytologic smears were alcohol-fixed and stained with H&E. The other five were air-fixed and Giemsa stained. DNA was isolated from the cytologic smears and amplified by using radioactive polymerase chain reaction (PCR) aimed at BAT-26 marker. Our results demonstrate that archived stained smears prepared for cytologic examinations can be used for molecular analyses by using a PCR amplification method. DNA could be isolated and PCR amplified independently of the prior fixation and staining procedures. So, we conclude that the application of molecular biology techniques to the existing archival smears may become a valuable tool to detect genetic changes in samples from breast cancer aspirates. Diagn. Cytopathol. 1998;19:395–397.  1998 Wiley-Liss, Inc. Key Words: cytologic techniques; breast carcinoma; molecular genetics Fine-needle aspiration (FNA) cytology is a simple and accurate method that is a standard of care in the evaluation of breast lesions. Furthermore, FNA material can also be used for some special studies such as immunohistochemis- try 1 and cytometry and in situ hybridization techniques. 2 One of the limitations of FNA is the possibility of obtaining only a small number of representative cells for interpreta- tion, particularly in small mammographically detected le- sions. Polymerase chain reaction (PCR) amplification is a technique that enables the detection of DNA changes, even when a smear contains a small number of cells. The study of DNA abnormalities in FNA specimens for breast cancer could be helpful in improving the capacity of diagnosis and especially to obtain prognostic or predictive information. The possibility of performing molecular studies in microdissected, previously archived and stained material will provide several advantages such as improvement of morphologic concomitant analysis and the use of the same aspirated material for different analysis and retrospective studies. The ability to study DNA obtained from archived cytologic specimens had been described in Papanicolaou- stained cervical smears for detection of the human papilloma virus L1 region. 3 The aim of the present study was to verify whether it is possible to perform molecular analysis of slides of breast cancer FNA specimens, previously stained by H&E and Giemsa, stored at least for 3 years. Materials and Methods The study was carried out on 10 cases of FNA specimens obtained from patients age 37–70 yr old with primary breast carcinomas diagnosed between 1993 and 1994 at our institute. Fine-Needle Aspiration Percutaneous FNA biopsies were performed with a 0.6-mm (23-gauge) diameter needle by using the procedure de- scribed by Zajicek. 4 The aspirated material was expelled on Institute of Molecular Pathology and Immunology - IPATIMUP, Medical School of the University of Porto, Portugal Grant sponsor: Junta Nacional de Investigac ¸a ˜o Cientı ´fica e Tecnolo ´ gica - JNICT - Portugal; Grant number: PRAXIS XXI/BCC/6445/95; Grant number: PRAXIS/PSAU/C/SAU/12/96. *Correspondence to: Dr. Fernando C. Schmitt, IPATIMUP, Rua Roberto Frias s/n°,4200-Porto-Portugal. E-mail: fernando.schmitt @ipatimup.pt Received 20 November 1997; Accepted 7 July 1998  1998 WILEY-LISS, INC. Diagnostic Cytopathology, Vol 19, No 5 395