Clinical and Laboratory Standards Institute document MM07-A2—Fluorescence In Situ Hybridization Methods for Clinical Laboratories; Approved Guideline—Second Edition provides information to ensure appropriate and reliable use of the FISH technology. FISH may be used to detect cytogenetic aberrations that are not readily evident by standard cytogenetic banding analyses. FISH technology allows for rapid identification of deletions, duplications, amplifications, and structural abnormalities of specific genes, loci, or chromosomal DNA/RNA sequences. The regions assessed by FISH are typically larger than those studied with PCR, yet smaller than those visualized microscopically with standard cytogenetics. FISH studies have become routine in medical laboratories.

• Examples have been added to illustrate the characteristics of FISH tests. 

•  The target audience has been expanded to include all clinical laboratories, not just genetic labs. 

• Manufacturing standards are no longer included, focusing instead on testing laboratories. 

•  Greater emphasis has been placed on oncology-related FISH issues in this edition of the guideline. • Nonfluorescent detection methods are now discussed. 

• Background information on testing strategies and how FISH testing is used in a clinical setting has been expanded. 

• Detailed discussion of “measurands (analytes)” detected by FISH and their impact on test development and performance has been added.

• The sensitivity and specificity of FISH probes are distinguished from analytical sensitivity and specificity.

 • Statistical methods and their limitations for establishing normal cutoff values for detecting mosaicism and neoplastic abnormalities are discussed. 

• Issues related to formalin-fixed, paraffin-embedded samples, selected or enriched cell populations, and FISH testing in support of microarray analysis are covered.

Note that the methods and quality control (QC) approaches described in this guideline are based on current clinical applications of FISH testing and that, as new technical methods and clinical applications evolve, other QC methods may be appropriate.

This document addresses fluorescence in situ hybridization methods for medical genetic determinations, identification of chromosomal abnormalities, and gene amplification. Recommendations for probe and assay development, manufacture, qualification, verification, and validation; instrument requirements; QA; and evaluation of results are also included. The guideline is intended to facilitate the reproducible production of FISH assays and the interlaboratory comparison of results and diagnostic interpretations, as well as to ensure accuracy in diagnosis. This document is intended for use by laboratories that develop tests based on commercially manufactured and laboratory-developed FISH probes. Unlike the previous edition, this revised guideline does not specifically address issues associated with the manufacturing of FISH probes or in vitro diagnostic (IVD) devices based on FISH technology. Nevertheless, manufacturers may find value in the principles of FISH testing presented in this guideline and in better understanding how their products will be used by laboratories.