What are the recommendations for handling and analyzing a lung biopsy sample for immunohistochemistry in a patient with suspected lung disease?

Lung Biopsy Immunohistochemistry: Handling and Analysis Recommendations

Specimen Handling Protocol

Lung biopsy specimens must be divided immediately upon receipt into multiple portions for different analytical techniques to maximize diagnostic yield. 1

Required Specimen Divisions

• Formalin fixation for routine histopathology and immunohistochemistry 1
• Microbiologic culture to exclude infectious etiologies 1
• Frozen tissue for possible immunofluorescence or other special studies 1
• Glutaraldehyde fixation for electron microscopy 1

Critical Timing Considerations

• Ischemia time (warm and cold) must be minimized and kept under 30 minutes between tissue acquisition and fixation 1
• Environmental temperature significantly impacts tissue degradation during this delay period 1
• Formalin fixation should occur for 6-48 hours before paraffin embedding, with duration dependent on tissue volume 1
• This fixation window preserves adequate DNA quality and most immunohistochemistry-detectable antigens 1

Tissue Adequacy Requirements

Minimum Biopsy Recommendations

• At least 4 EBUS/endoscopic ultrasound needle aspiration passes per target lesion 1
• At least 2 percutaneous core needle biopsies using 18-20 gauge needles; consider 3-6 core biopsies to maximize tissue volume 1
• For bronchoscopic biopsies, consider an additional 5 forceps biopsies or 2 cryobiopsies beyond standard sampling 1

Specimen Processing

• Manual microdissection is mandatory before molecular analysis to ensure only tumor-containing tissue is analyzed 1
• Small biopsy samples with limited tumor cells may only permit diagnosis and classification but compromise additional molecular testing 1
• For small tumors in resection specimens, complete embedding is recommended 1

Immunohistochemistry Panel Selection

Primary Lung Cancer Classification

For non-small cell lung cancer subtyping, a minimum immunohistochemical panel must include TTF-1, p63, CK5/6, and surfactant markers to differentiate adenocarcinoma from squamous cell carcinoma. 2, 3

• TTF-1 (thyroid transcription factor 1) is the most useful relatively lung-specific marker for adenocarcinoma 4, 3
• Napsin A complements TTF-1 in defining lung primary adenocarcinoma 4
• p63 and CK5/6 identify squamous cell carcinoma 2, 3
• 85% of non-small cell lung cancer cases display binary staining (TTF-1 positive/p63 negative for adenocarcinoma, or vice versa for squamous) 2

Critical Diagnostic Scenarios

• Immunohistochemistry is mandatory even when morphology suggests a specific tumor subtype, as diagnosis can change in a small proportion of cases after IHC 2
• Small cell carcinoma requires neuroendocrine markers for distinction from non-small cell carcinoma, particularly in artifact-limited small biopsies 4, 3
• For distinguishing primary lung tumors from metastases, use TTF-1 combined with site-specific markers (CK7, CK20, CDX2 for GI, ER/PR for breast, PSA for prostate) 4, 5

Molecular Testing Integration

Predictive Biomarker Assessment

• ALK, ROS1, and EGFR should be assessed by immunohistochemistry as initial screening, with molecular confirmation as needed 3
• PD-L1 (CD274) immunohistochemistry is required for immunotherapy eligibility determination 3
• BRAF V600E mutation testing via immunohistochemistry or molecular methods should be performed when histiocytic disorders are suspected 6

Tissue Prioritization

• When tissue is limited, prioritize immunohistochemistry for classification first, then molecular testing for treatment-relevant mutations 1, 2
• The favor adenocarcinoma immunoprofile correlates with higher EGFR positivity (25%) and ALK positivity (7%) compared to favor squamous (11% and 0% respectively) 7

Quality Assurance Requirements

Preanalytical Standards

• Vacuum preservation of resection material at 4°C in sealed plastic bags should be considered for standardization 1
• Buffered formalin remains the standard fixative; alternative fixatives (HOPE, FineFix, alcohol) have not proven superior 1
• Record ischemia time as it impacts subsequent analyses 1

Diagnostic Accuracy Benchmarks

• Sensitivity for malignancy should exceed 85-90% in lesions over 2 cm 1
• Sample adequacy should exceed 90% 1
• False positive rate must remain below 1% 1
• All testing should be accompanied by external quality assurance programs 1

Common Pitfalls to Avoid

• Never rely solely on H&E morphology for histiocytic lung diseases; immunohistochemistry is mandatory as histiocytes appear similar across multiple diseases 6
• Do not assume all immunohistochemical results are definitive—approximately 5% of cases with available second specimens show diagnostic discordance 2
• Avoid performing immunohistochemistry on inadequately fixed tissue (fixation <6 hours or >48 hours) as antigen preservation may be compromised 1
• Do not proceed with molecular testing without first confirming adequate tumor content through microdissection and pathologist review 1