What does a FISH (Fluorescence In Situ Hybridization) lab result indicate and what conditions does it apply to?

FISH Lab Results: Methodology, Interpretation, and Clinical Applications

FISH (Fluorescence In Situ Hybridization) is a molecular cytogenetic technique that uses fluorescent DNA probes to detect and localize specific genetic sequences on chromosomes, providing critical diagnostic and prognostic information for genetic disorders, cancers, and prenatal conditions.

What FISH Detects

FISH identifies chromosomal abnormalities by visualizing specific DNA sequences through fluorescent signals under microscopy. The test reports should clearly state whether results are normal/negative or abnormal/positive, though terms like "inconclusive," "equivocal," or "borderline" may be used when results are ambiguous 1.

Key Detectable Abnormalities:

• Microdeletions and microduplications - submicroscopic chromosomal losses or gains (e.g., DiGeorge syndrome, Prader-Willi syndrome where deletions account for ~70% of cases) 1
• Aneuploidies - abnormal chromosome numbers, particularly trisomies 13,18,21, and sex chromosome abnormalities 2
• Gene amplifications - increased copy numbers of oncogenes (e.g., HER2 in breast cancer, MYCN in neuroblastoma) 1, 3
• Gene rearrangements - translocations and fusions (e.g., BCR/ABL1 in chronic myeloid leukemia, IGH rearrangements in lymphomas) 1, 4
• Marker chromosomes - identification of origin and content of supernumerary chromosomes 1

How FISH Results Are Reported

Every FISH report must identify the specific probe(s) used (gene or locus symbol), probe manufacturer, and number of cells examined 1. For microarray follow-up studies, the linear genomic position with corresponding genome build must be referenced 1.

Essential Report Components:

• Nomenclature: Results described using the International System for Human Cytogenetic Nomenclature, with separate nomenclature strings for multiple simultaneous assays 1
• Documentation: Normal results require images of two representative cells; abnormal results require images of at least two cells for each abnormal signal pattern 1
• FDA Disclaimer: For tests using Analyte Specific Reagents (ASRs), a mandatory statement must appear: "This test was developed and its performance characteristics determined by [laboratory name] as required by CLIA '88 regulations. It has not been cleared or approved for specific uses by the U.S. Food and Drug Administration" 1

Analysis Standards:

• Metaphase analysis: Examines chromosomes during cell division, suitable for detecting microdeletions but may miss microduplications unless interphase nuclei are also analyzed 1
• Interphase analysis: Examines non-dividing cells, essential for microduplication detection and when metaphase cells are unavailable 1
• Mosaic conditions: Require examination of 30 metaphase cells plus at least 50 interphase nuclei when suspected 1

Clinical Applications by Category

Birth Defects and Developmental Disorders

FISH serves as either a primary diagnostic test when clinical suspicion for specific microdeletion syndromes is high, or as a follow-up to routine cytogenetics when phenotypes are less specific 1.

• Detection rates vary by syndrome: 70% for Prader-Willi/Angelman syndromes versus 11-12% for Rubinstein-Taybi syndrome 1
• Clinical utility depends on whether deletions are frequent mutational mechanisms for the suspected condition 1
• Common pitfall: Probes may target the smallest region of overlap rather than the critical gene, potentially missing very small deletions or point mutations 1

Prenatal Diagnosis

In advanced maternal age patients (>35 years), FISH using probes for chromosomes X, Y, 13,18, and 21 detects approximately 80% of clinically significant abnormalities 1.

• Overall detection rate across all prenatal indications: 65-70% of significant cytogenetic abnormalities 1
• Concordance with cytogenetic results exceeds 99.5% in informative samples 2
• Critical limitation: FISH detects only the specific chromosomes tested and does not substitute for complete karyotype analysis 1
• Uninformative results occur in up to 20% of cases, particularly in abnormal specimens, requiring classical cytogenetic follow-up 2

Cancer Diagnostics

FISH provides prognostic information and clarifies diagnoses in hematologic malignancies and solid tumors 4, 3.

Hematologic Applications:

• Chronic lymphocytic leukemia (CLL): Panel detects 13q deletion (most frequent), trisomy 12, 11q deletion, and 17p deletion in 72.9% of cases 4
• IGH rearrangements identify cases requiring reclassification (e.g., mantle cell lymphoma with CCND1/IGH fusion) 4
• Interpretation caveat: Borderline-positive results in newly diagnosed CML should not occur with BCR/ABL1 testing; such results warrant careful clinical correlation 1

Solid Tumor Applications:

• MYCN amplification in neuroblastoma: Detectable in paraffin-embedded tissue, showing amplification heterogeneity and whether amplification occurs as double-minute chromosomes or homogeneously staining regions 3
• Gene amplification thresholds: Disease-specific (HER2 ratio >2.2 in breast cancer versus MYCN ratio >4 in neuroblastoma) 1
• When amplification is detected and dividing cells are available, conventional chromosome analysis should be recommended to detect homogeneously staining regions or double minutes 1

Microarray Confirmation

FISH confirms or visualizes copy number variants identified by microarray, but requires specific validation considerations 1.

• Parental FISH should be performed by the same laboratory that conducted the initial microarray and proband FISH evaluation 1
• Critical requirement: Without probe signal pattern confirmation in the proband, subtle abnormalities like small duplications cannot be adequately interpreted in parental samples 1
• Very small tandem duplications (<500 kb) may yield closely spaced signals unresolvable by interphase FISH, requiring alternative confirmation methods 1

Important Limitations and Pitfalls

Technical Limitations:

• Metaphase FISH provides information only about the specific probe locus tested and does not substitute for complete karyotypic analysis 1
• Whole chromosome paints may not hybridize uniformly across small derivative chromosome regions 1
• Microdeletion testing performed only on metaphase cells cannot exclude microduplications 1
• Fixation variables (type, age of paraffin blocks) may negatively impact hybridization efficiency in archived specimens 1

Interpretive Cautions:

• Borderline results require extreme caution and clinical context: Cut-off values are estimates of the upper limit for abnormal patterns in normal populations 1
• Low-level positive results are unexpected for common microduplication syndromes due to large duplication size and absence of expected mosaicism 1
• Normal FISH results with probes not targeting the critical gene should include disclaimers about potential false negatives from very small deletions or point mutations 1
• Parental testing caveat: Identical FISH abnormalities in parent and proband strongly suggest but do not prove identical copy number states; further modifications or undetected mosaicism remain possible 1

Quality Control Requirements:

• For probes with limited clinical experience, concurrent normal controls must be run with patient material 1
• Databases for interpreting mosaicism must be established for each probe set; without this, test sensitivity for detecting mosaicism is unknown 1
• Rare individuals with small numbers of repeated sequence targets may yield false-negative results with repeated sequence probes 1