What tests can be performed on a suspicious tissue specimen for pathological evaluation?

Pathological Testing for Suspicious Tissue

A suspicious tissue specimen should undergo histopathological examination with H&E staining as the foundational test, supplemented by immunohistochemistry (IHC) for diagnostic clarification, and molecular testing (FISH, chromosomal microarray analysis, or next-generation sequencing) when specific genetic alterations will guide treatment decisions.

Core Pathological Testing Algorithm

Primary Evaluation: Histopathology

The initial and most critical test is histopathological examination using hematoxylin and eosin (H&E) staining 1. This provides:

• Tissue architecture assessment
• Cell morphology evaluation
• Differentiation between normal and malignant cells
• Identification of areas with adequate tumor cellularity for further testing

A pathologist must review H&E-stained sections before any advanced testing to delineate tumor regions and ensure adequate specimen quality 1.

Secondary Testing: Immunohistochemistry (IHC)

IHC should be performed on formalin-fixed paraffin-embedded (FFPE) tissue to:

• Confirm cell lineage and tumor type
• Identify specific protein markers
• Guide selection of targeted molecular tests
• Delineate tumor boundaries when morphology alone is insufficient 1

Advanced Molecular Testing

The choice of molecular test depends on the tumor type and clinical context 1:

Fluorescence In Situ Hybridization (FISH)

Use FISH when specific gene rearrangements or amplifications are suspected:

• Acceptable specimens: FFPE tissue (most common), fresh-frozen tissue, touch preparations, cultured cells, cytospin preparations 1
• Critical requirement: Pathologist must mark tumor regions on H&E slides before FISH scoring, as malignant cells cannot be reliably distinguished using only DAPI counterstain 1
• Avoid: Tissues fixed in Bouin's or B5 fixative, or decalcified with strong acids 1

Chromosomal Microarray Analysis (CMA)

CMA is the primary method for tumors where copy number alterations drive management (neuroblastoma, Wilms tumor, most CNS tumors) 1:

• Detects DNA loss, gain, amplification, and copy-neutral loss of heterozygosity
• Acceptable specimens: Fresh tumor tissue, FFPE tissue, frozen tumor, cultured cells 1
• Requires pathologist review to identify high tumor cellularity areas for DNA extraction 1

Next-Generation Sequencing (NGS)

For comprehensive mutational profiling when multiple targetable alterations are possible:

• Success rate of 85.9% on cytological preparations 2
• Can be performed on limited material including small biopsies and cytology specimens 3, 2
• Requires adequate tumor content; pathologist marks tumor areas before DNA extraction 2

Specimen Handling Considerations

Tissue Preservation

• Immediate fixation: Place tissue in 10% buffered formalin within minutes of excision 4
• Optimal fixation time: Approximately 16 hours for formalin 4
• FFPE tissue: Standard for most pathological testing; sections mounted on positively charged slides 1, 4

Sample Adequacy

• Multiple levels: Obtain 6 initial levels on small biopsies, particularly when evaluating for dysplasia or when initial impression shows atypia 5
• Tumor dissection: Essential in heterogeneous tumors with necrosis or prominent stroma to ensure DNA is tumor-derived 1
• Reserve material: Request several unstained sections for potential repeat studies 1

Common Pitfalls to Avoid

1. Inadequate tissue sampling: Small biopsies may require additional levels; 21.4% of cases show diagnostic changes with deeper sections 5

2. Improper fixation: Using incompatible fixatives (Bouin's, B5) or strong acid decalcification renders tissue unsuitable for FISH 1

3. Molecular testing without histopathology: Multiplex molecular tests are highly sensitive but detect commensal and contaminating organisms; interpretation requires histopathologic correlation 6

4. Insufficient tumor content: Mark tumor areas before molecular testing; extraneous results are common without proper tissue selection 6, 2

5. Delayed processing: For postmortem or delayed specimens, samples remain adequate up to 3 days with refrigeration 6

Testing Selection Based on Clinical Context

• Suspected solid tumor with targetable mutations: NGS on FFPE tissue 3, 7
• Suspected gene amplification (e.g., HER2): FISH on FFPE with pathologist-marked tumor regions 1
• Pediatric tumors (neuroblastoma, Wilms, CNS): CMA as primary method 1
• Limited material (cytology, small biopsy): Fine-needle aspiration shows highest sensitivity (75.0%) for diagnosis; can proceed to molecular testing if adequate 8, 7