Contemplations on preclinical validation of fluorescence in situ hybridization probe assay for paraffin-embedded tissues in hematologic disorders.

A validation of fluorescence in situ hybridization (FISH) assays for paraffin-embedded tissues (PET) is required before clinical use. Although the FISH probe validation process for hematologic disorders has been described during recent years, none of these descriptions, to our knowledge, address the validation process for paraffin-embedded tissues in detail. We describe an applicable preclinical validation process. The following five-step validation is outlined: (1) pathologist's review of slide; (2) probe validation on metaphases to assess sensitivity and specificity; (3) a pilot study using FISH probes on PET; (4) analytical validation using normal and abnormal PET samples to establish a normal reference range or cutoff; and (5) application of the cutoffs to test samples. Although the procedure focuses on dual-color, dual-fusion FISH assays, the same steps could be used for any type of probe. We have described a preclinical validation for probes on paraffin-embedded tissue.