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World Health Organization states the importance of conventional cytogenetics and FISH in hematologicalmalignancy for accurate diagnosis, treatment and monitoring response to therapy. Most FISH probes,however, are Analyte- Specific reagents (not FDA approved) and thus an elaborate validation procedureprior to diagnostic use becomes essential. This study focuses on validating FISH probes by assessing theanalyte- sensitivity, specificity, accuracy, precision and determining normal reference ranges (cut-offs).Eight probes from two different manufacturers each were validated using cytogenetically normal peripheralblood (negative controls) and leukemia positive bone marrow samples (positive controls) to determine themost suitable probe for use in a diagnostic set-up. Both the controls were cytogenetically defined beforeinitiating the validation procedure. Alongside this, the probe constructs were studied to understand signalco-localization, size and intensity. Accuracy was determined by metaphase FISH, precision by standarddeviation or inter-observer variability and analyte specificity and sensitivity using standard formulae. Thecut-off or the normal reference range was derived by BETAINV function in Microsoft Excel. Based onperformance characteristics and qualitative data most relevant probes were suggested for diagnostic use.Although validation procedures may differ between test centres, it should be a mandate pre-clinical practice.A validated FISH probe surges dependability on generated reports and this study presents the mostrudimentary yet essential parameters in a FISH probe validation.

Preclinical In-House Validation of Commercially Available Fluorescence In-Situ Hybridization Probes Used in Diagnosis of Haematological Malignancies