Preclinical validation of fluorescence in situ hybridization assays for clinical practice

Anne E. Wiktor, Daniel L. Van Dyke, Peggy J. Stupca, Rhett P. Ketterling, Erik C. Thorland, Brandon M. Shearer, Stephanie R. Fink, Kimberly J. Stockero, Jason R. Majorowicz, Gordon W. Dewald

Research output: Contribution to journalArticlepeer-review

73 Scopus citations


Purpose: Validation of fluorescence in situ hybridization assays is required before using them in clinical practice. Yet, there are few published examples that describe the validation process, leading to inconsistent and sometimes inadequate validation practices. The purpose of this article is to describe a broadly applicable preclinical validation process. Methods: Validation is performed using four consecutive experiments. The Familiarization experiment tests probe performance on metaphase cells to measure analytic sensitivity and specificity for normal blood specimens. The Pilot Study tests a variety of normal and abnormal specimens, using the intended tissue type, to set a preliminary normal cutoff and establish the analytic sensitivity. The Clinical Evaluation experiment tests these parameters in a series of normal and abnormal specimens to simulate clinical practice, establish the normal cutoff and abnormal reference ranges, and finalize the standard operating procedure. The Precision experiment measures the reproducibility of the new assay over 10 consecutive working days. To illustrate documentation and analysis of data with this process, the results for a new assay to detect fusion of IGH and BCL3 associated with t(14;19)(q32; q13.3) in lymphoproliferative disorders are provided in this report. Results: These four experiments determine the analytic sensitivity and specificity, normal values, precision, and reportable reference ranges for validation of the new test. Conclusion: This report describes a method for preclinical validation of fluorescence in situ hybridization studies of metaphase cells and interphase nuclei using commercial or home brew probes.

Original languageEnglish (US)
Pages (from-to)16-23
Number of pages8
JournalGenetics in Medicine
Issue number1
StatePublished - Jan 1 2006


  • FISH validation
  • Interphase FISH
  • Metaphase FISH

ASJC Scopus subject areas

  • Genetics(clinical)


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