Preclinical validation of fluorescence in situ hybridization assays for clinical practice

Abstract

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; Key Words: FISH validation, metaphase FISH, interphase FISHValidation of fluorescence in situ hybridization (FISH) assays is becoming more challenging as the number of probes and applications increase, and diverse analytic strategies emerge. The Clinical Laboratory Improvement Amendments (CLIA), Food and Drug Administration (FDA), College of American Pathologists, and other accrediting agencies all require validation of new or modified FISH assays before reporting any patient results. Preclinical validation requires evaluation of the accuracy, analytic sensitivity and analytic specificity (interfering factors), normal values, precision, and reportable reference ranges of the FISH assay. [1][2][3] This publication focuses on the preclinical validation process, but validation continues into clinical practice and must be continually monitored to ensure the FISH assay works as expected and achieves the intended results. In clinical practice, validation includes proficiency testing, assessment of employee competency, instrument calibration, and correlation with clinical findings.Some regulatory agencies, such as the College of American Pathologists and the New York State Health Department, provide general standards for validation of FISH tests. 2,4 The American College of Medical Genetics (ACMG) has published guidelines to establish scoring criteria, analytic sensitivity, analytic specificity, normal cutoffs, and abnormal reference ranges. 5 The ACMG has also published a policy statement discussing the clinical considerations of FISH for prenatal screening, diagnosis of microduplication and microdeletion syndromes, and identification of acquired marker or derivative chromosomes. 6 In 1995, Schad and Dewald 7 presented an overview of quality control and quality assurance methods, and p...

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“…1c, Table 1). The frequency of the aneuploid cells in the controls was within the range reported in adult human cells in culture [46,47]. In contrast, the SWCNT-treated SAEC had a level of aneuploidy that was as high as the effect that was observed in the vanadium pentoxidetreated positive control cells (Fig.…”

Guidance for Fluorescence in Situ Hybridization Testing in Hematologic Disorders

“…The normal cutoff point would be determined by statistical analysis using a binominal distribution assessment. 10 Because the normative data are not distributed in a typical bell-shaped curve, cutoff points cannot be based on SD calculations. 10 The normal cutoff for an analysis of 200 cells can be calculated for FISH results using the Microsoft Excel ␤ inverse function, ϭ BETAINV(Confidence level, false-positive cells plus 1, number of cells analyzed).…”

“…10 Because the normative data are not distributed in a typical bell-shaped curve, cutoff points cannot be based on SD calculations. 10 The normal cutoff for an analysis of 200 cells can be calculated for FISH results using the Microsoft Excel ␤ inverse function, ϭ BETAINV(Confidence level, false-positive cells plus 1, number of cells analyzed). 10 This formula calculates a one-sided upper confidence limit for a specified percentage proportion based on an exact computation for the binomial distribution.…”

“…10 The normal cutoff for an analysis of 200 cells can be calculated for FISH results using the Microsoft Excel ␤ inverse function, ϭ BETAINV(Confidence level, false-positive cells plus 1, number of cells analyzed). 10 This formula calculates a one-sided upper confidence limit for a specified percentage proportion based on an exact computation for the binomial distribution. This can be done by examining results for 20 normal specimens for the particular sample type being validated and identifying the specimen with the greatest number of false-positive nuclei for any given signal pattern.…”

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“…All probes were validated, and their sensitivity and specificity were determined per standard procedure. 11 Specific cutoff values, confidence intervals, and control cell lines used for each probe are provided in Table 1. The automated platform, detailed biostatistical methods, and sensitivity based on dilution experiments (spiking a known number of cancer cells into a normal cell population) of the T-FISH assay have been described previously.…”