Cancer Genetics and Cytogenetics 123 (2000) 102–108 0165-4608/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S0165-4608(00)00315-0 Standardization criteria for the detection of BCR/ABL fusion in interphase nuclei of chronic myelogenous leukemia patients by fluoresc in situ hybridization Ninette Cohen a , Ilya Novikov a , Izhar Hardan a , Arif Esa b , Frida Brok-Simoni a , Ninette Amariglio a, *, Gideon Rechavi a , Isaac Ben-Bassat a , Luba Trakhtenbrot a a The Institute of Hematology, The Chaim Sheba Medical Center, Tel-Hashomer, Sackler School of Medicine, Tel Aviv University, Tel b Kirchhoff Institute for Physics, University of Heidelberg, Heidelberg, Germany Received 26 April 2000; accepted 28 June 2000 Abstract Fluorescence in situ hybridization (FISH), as a new clinical test, is not presently standardized. For practical reasons, each laboratory must build its own criteria. In this work, we present our stan- dardization criteria for clinical practice, which include not only the methods for cell fixation, spec- imen preparation, and hybridization conditions, but mainly the definition of false-positive range and the scoring criteria of microscopic analysis. These include signal assessment, difference be- tween individual microscopists, evaluation of specimen homogeneity, and the minimum number of scored nuclei required for a clinically reliable result. For this purpose, we analyzed by FISH 24 healthy volunteer donors, 31 patients affected by non-chronic myelogenous leukemia (CML) he- matological malignancies, 47 CML patients at diagnosis, and 82 CML patients during treatment for the BCR/ABL fusion. In this article, we present several quality control and assurance methods that can be useful in providing standardization of the FISH technique. © 2000 Elsevier Science Inc. All rights reserved. 1. Introduction Fluorescence in situ hybridization (FISH) is a relatively new and valuable tool for the analysis of rearranged chro- mosomes in human malignancies in general and in chronic myelogenous leukemia (CML) in particular [1–5]. Fluores- cence in situ hybridization permits visualization of BCR/ ABL fusion in both interphase and metaphase cells and can be readily used as an adjunct to conventional cytogenetic methods. The precise evaluation of the proportion of the BCR/ABL -positive cells by interphase FISH is of great sig- nificance for diagnosis and monitoring therapy in CML pa- tients [6–9]. However, FISH, as a new clinical test, is not yet standardized [4, 10], and for practical reasons, each lab- oratory must build its own criteria. We present several quality control and assurance meth- ods that can be useful in providing standardization of the FISH technique. The standardization for clinical practice in- cludes not only the methods for cell fixation, specimen preparation, hybridization conditions, and the definition of false-positive range, but also the scoring criteria of micr scopic analysis. These include signal assessment and number of scored nuclei required for a clinically reliable sult. In addition, we added experiments to establish the dardization: comparison of results between two indepen investigators, comparison between results of interphase FISH and G-banded mitoses, determination of range BCR/ABL fusion in each group, and false-positive rates. For this purpose, we analyzed by FISH 24 healthy vol teer donors, 31 patients affected by non-CML hematolog cal malignancies, 47 CML patients at diagnosis, and CML patients during treatment for BCR/ABL fusion. 2. Materials and methods 2.1. Patients Twenty-four healthy volunteer donors, 31 non-CML hemato-oncological patients, 47 CML patients at diagno and 82 CML patients during treatment were examined b for the detection of BCR/ABL fusion. Normal peripheral blood (PB) cells were obtained from the healthy donors. The P and/or bone marrow cells (BM) were collected from CM patients and non-CML hematological patients. Periph * Corresponding author.Tel.: 1 972-3-5302128; fax: 1 972-3- 5302377. E-mail address : ncohen@post.tau.ac.il (N. Cohen).