However this protocol uses large quantity of formamide (toxic), and is preferably avoided.
References
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2. Henegariu O, Bray-Ward P, Ward DC (2000) Custom fl uorescent-nucleotide synthesis as an alternative method for nucleic acid labeling.
Nat Biotechnol 18:345–348
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Thomas S.K. Wan (ed.), Cancer Cytogenetics: Methods and Protocols, Methods in Molecular Biology, vol. 1541, DOI 10.1007/978-1-4939-6703-2_10, © Springer Science+Business Media LLC 2017
Chapter 10
Fluorescence In Situ Hybridization Probe Validation for Clinical Use
Jun Gu , Janice L. Smith , and Patricia K. Dowling
Abstract
In this chapter, we provide a systematic overview of the published guidelines and validation procedures for fl uorescence in situ hybridization (FISH) probes for clinical diagnostic use. FISH probes—which are clas- sifi ed as molecular probes or analyte-specifi c reagents (ASRs)—have been extensively used in vitro for both clinical diagnosis and research. Most commercially available FISH probes in the United States are strictly regulated by the U.S. Food and Drug Administration (FDA), the Centers for Disease Control and Prevention (CDC), the Centers for Medicare & Medicaid Services (CMS) the Clinical Laboratory Improvement Amendments (CLIA), and the College of American Pathologists (CAP). Although home- brewed FISH probes—defi ned as probes made in-house or acquired from a source that does not supply them to other laboratories—are not regulated by these agencies, they too must undergo the same indi- vidual validation process prior to clinical use as their commercial counterparts. Validation of a FISH probe involves initial validation and ongoing verifi cation of the test system. Initial validation includes assessment of a probe’s technical specifi cations, establishment of its standard operational procedure (SOP), determi- nation of its clinical sensitivity and specifi city, development of its cutoff, baseline, and normal reference ranges, gathering of analytics, confi rmation of its applicability to a specifi c research or clinical setting, test- ing of samples with or without the abnormalities that the probe is meant to detect, staff training, and report building. Ongoing verifi cation of the test system involves testing additional normal and abnormal samples using the same method employed during the initial validation of the probe.
Key words Validation , Fluorescence in situ hybridization , FISH , Probe , Sensitivity , Specifi city , Cutoff value , Normal range , BETAINV function
1 Introduction
Accredited cytogenetic and molecular genetics laboratories use fl uo- rescence in situ hybridization (FISH) probes extensively to make clinical diagnoses, and under United States law, these laboratories must validate their FISH probes before use. The U.S. Food and Drug Administration (FDA) regulates the manufacture of commer- cial FISH probes, while the Centers for Medicare & Medicaid Services (CMS) and the Centers for Disease Control and Prevention (CDC) regulate the testing methodology through their Clinical
Laboratory Improvement Amendments (CLIA). Thus, it is man- dated in the United States that all FISH probes must be validated before clinical use in an accredited clinical diagnostic laboratory.
Guidelines for FISH probe validation have been published by the American College of Medical Genetics (ACMG) and the College of American Pathologists (CAP). The Clinical and Laboratory Standards Institute (CLSI) has produced similar guidelines (International Organization for Standardization (ISO) 15189). Currently, the responsibility of FISH probe validation rests with each accredited laboratory, and the details of the validation procedures adapted by these laboratories can vary. The major steps for FISH test validation include assessment of a probe’s technical specifi cations, establishment of its standard operational procedure (SOP), determination of its clinical sensitivity and specifi city, development of its cutoff, baseline, and normal reference ranges, gathering of analytics, confi rmation of its applicability to a specifi c research or clinical setting, testing of sam- ples with or without the abnormalities that the probe is meant to detect, staff training, and report building. Documentation of the vali- dation should be maintained for laboratory accreditation and reac- creditation purposes. Based on available guidelines and regulations, this chapter provides a systematic overview of FISH probes and their validation for use in clinical diagnosis.
Home-brew FISH probes are not currently regulated by the FDA, although the FDA requires the inclusion of the following dis- claimer on all reports of in-house tests using them: “ This test was developed and its performance characteristics determined by [Laboratory Name]. It has not been cleared or approved by the U.S. Food and Drug Administration .” [ 1 ]. In contrast, the FDA does control the manufacture of molecular probes (called analyte- specifi c reagents or ASRs), which are classifi ed as Class I, II, or III according to the level of oversight necessary to expect that they are reasonably effective and safe. While the majority of the probes in use are Class I—meaning that they are not required to undergo premarket approval—those in Class II or III, including FISH in vitro diagnostic devices, must be authorized by the FDA before they can be marketed in the United States. According to the CLIA’88, FISH probes that the FDA approves as Class II or III devices must only be used by clinical laboratories qualifi ed to per- form high-complexity testing. Such laboratories must document that the assay’s performance characteristics are the same as or bet- ter than those stated by the probe manufacturer in the package insert. Clinical laboratories must also maintain documentation of subsequent biannual calibrations of the test system. The ACMG, the CAP, and the CLSI have also produced guidelines for FISH probe validation. The ACMG requires laboratories to validate FISH probes by establishing their clinical sensitivity and specifi city, cutoff values, baseline reference ranges, and normal ranges.
The organization published its fi rst position statement on the use 1.1 Guidelines
of interphase FISH in prenatal diagnosis in 1993 [ 2 ]. The fi rst description of validation methods for FISH tests was made by Schad in 1995 [ 3 ]. The preclinical validation of FISH recom- mended by Wiktor et al. was widely accepted [ 4 ]. The ACMG has also published guidelines relevant to FISH probe validation in con- stitutional and oncologic clinical cytogenetic analysis [ 5 – 8 ]. The CAP emphasizes the documentation of FISH probe validation in CYG.42700 of its Cytogenetic Checklist, and in CYG.42900 indi- cates that written procedures for establishing normal cutoff values and records from cutoff-value studies should be maintained for interphase FISH tests. In CYG.43250, CAP requires that there be a system in place to ensure FISH probe colocalization , which can be accomplished by using metaphase cells in an interphase cell analysis or by including an internal or external target that could give a positive signal for each hybridization. In CYG.48399, CAP provides guidelines for FISH validation in formalin-fi xed, paraffi n- embedded ( FFPE ) tissues. The National Committee on Clinical Laboratory Standards (NCCLS) published its fi rst FISH validation guidelines in 2004 [ 9 ] and, after being renamed as the Clinical and Laboratory Standards Institute, published guideline ISO 15189 for probe validation. In August 2013, CLSI also made recommenda- tions for FISH probe and test validation in an updated version of regulation MM07-A2 for clinical laboratory FISH methods [ 10 ], and this new version of MM07-A2 fully agreed with the FISH rec- ommendations from the ACMG. Table 1 summarizes the guidelines produced by the agencies that regulate FISH test validation.
Figure 1 gives an overview of the work fl ow for FISH test valida- tion. Steps 1 and 2 are preclinical evaluations, step 3 is the major part of the clinical evaluation, and step 4 deals with the post- validation work required before a test can be put into clinical use.
2 Assessment of FISH Probe Technical Specifi cations
Commonly used FISH probes include chromosome-painting probes, repetitive-sequence probes, and locus-specifi c probes.
Locus-specifi c probes include those with dual-color/ single-fusion , dual-color/ extra-signal , dual-color/ dual-fusion , triple-color/
dual-fusion, and dual-color/break-apart designs. The use of control probes with each type of FISH probe design is essential to reduce the number of false-positive and false-negative results.
A probe’s slide hybridization adequacy should be evaluated before determining its analytic sensitivity and specifi city. Slide hybridization adequacy is determined by assessing the probe’s signal intensity, background, and localization validation on metaphase cells. The specimen’s target viewing area should be located using low- power objective lens with a DAPI fi lter and should have a minimum of 1.2 Work Flow Chart
2.1 Types of FISH Probes
2.2 Hybridization Adequacy
Table 1 FISH test validation and verifi cation guidelines FD A (1996) ACMG (2009) CLSI (2013) C AP (2015) Code of Federal Regulations, Title 21 (21 CFR) 21 CFR §809 Regulates in vitro diagnostic products for human use 21 CFR §809.30(e) ASR disclaimer 21 CFR §809.30(f) Order restriction for in-house tests Standards and Guidelines for Clinical Genetics Laboratories, Section E (Clinical Cytogenetics) E9.1.1 Types of molecular probes E9.2.1.1 Validation of unique sequence FISH probes not approved of Class II or III ASR kits E9.4 Target tissues E9.7.3.1 ASR disclaimer E10.1.7 Interphase FISH validation E10.3.1 Database collection E10.3.2 Reportable reference ranges E11.2 Multitarget FISH probe validation International Organization for Standardization (ISO) Quality Document 15189, Molecular Methods (MM) MM07-A2-7 Production of new FISH probes MM07-A2-8.3 Test sensitivity and specifi city MM07-A2-8.4.1 The hybridization optimization process MM07-A2-8.4.3 Establishing normal cutoff values MM07-A2-8.4.4 FISH performed on selected cells or cell populations subjected to enrichment procedures MM07-A2-8.4.5 FISH on FFPE MM07-A2-8.6 Controls Cytogenetics Standards (CYG) CYG.42700 FISH probe validation CYG.42900 Interphase FISH cutoff values CYG.43000 FISH scoring CYG.43200 FISH contr CYG.43250 FISH probe intended target CYG.43600 ASR disclaimer CYG.48399 HER2 (ERBB) assay validation
50 interphase nuclei in order to pass the initial hybridization ade- quacy assessment. The specimen is classifi ed as uninformative if it fails the hybridization adequacy assessment. Generally, hybrid- ization effi ciency should be 98 % or higher for chromosome enu- meration probes.
The goal of signal colocalization is to demonstrate that the test probe only hybridizes to the intended target, i.e., without cross- hybridization . A 60× or 100× objective lens with corresponding fi lters should be used to check anticipated signal patterns on metaphase cells from fi ve normal male individuals. Although ACMG recommends evaluating a minimum of fi ve metaphases to confi rm localization [ 8 ], autosomal locus validation typically requires 20 metaphases, and sex chromosome locus validation typically requires 40 [ 10 ]. To determine chromosomal localiza- tion, standard chromosome analysis should be performed on the sample on which the FISH probe will be used. Either karyotyp- ing (using inverted DAPI images from a computer imaging sys- tem) (Fig. 2 ) or sequential staining (FISH on G-banded slides ) may be employed [ 11 ].
2.3 Signal Colocalization
HYBRIDIZATION ADEQUACY CROSS- HYBRIDIZATION OR CONTAMINATION
SIGNAL INTENSITY REPRODUCIBILITY
SIGNAL COLOCALIZATION PROBE SENSITIVITY SIGNAL PATTERNS SCREENING PROBE SPECIFICITY
INCLUSION AND EXCLUSION OF
SIGNAL PATTERNS ANALYTICAL SENSITIVITY ESTABLISHMENT OF QUANTITATION
CRITERIA ANALYTICAL SPECIFICITY
DISTRIBUTION OF NORMAL VS.
PATIENT SAMPLES
SCREENING WITH KNOWN POSITIVE/
NEGATIVE SAMPLES CUTOFF VALUES
CLINICAL SENSITIVITY REFERENCE RANGES CLINICAL SPECIFICITY
TECHNOLOGIST TRAINING REPORT BUILIDING STEP ONE
(PRECLINICAL EVALUATION):
ASSESSMENT OF PROBE TECHNICAL SPECIFICATIONS
STEP TWO (PRECLINICAL EVALUATION):
ESTABLISHMENT OF FISH ASSAY
STANDARD OPERATIONAL
PROCEDURE
STEP THREE (CLINICAL EVALUATION):
CLINICAL VALIDATION OF
FISH ASSAY
STEP FOUR (POST- VALIDATION):
IMPLEMENTATION OF FISH ASSAY
Fig. 1 FISH probe validation fl owchart
The main steps of sequential G-band FISH are as follows: