What investigations should be sent on a pleural fluid sample suspected of malignancy?

Pleural Fluid Investigations for Suspected Malignancy

Send 25-50 mL of pleural fluid for cytological analysis with both direct smear and cell block preparation as the primary diagnostic test for suspected malignant pleural effusion. 1

Essential Investigations

Cytological Analysis (Primary Test)

• Submit 25-50 mL of pleural fluid for cytological examination—this is the optimal volume range that maximizes diagnostic sensitivity 1, 2
• Process samples using both direct smear and cell block preparation to increase diagnostic yield 1
• If less than 25 mL is obtained, send it anyway but recognize the significantly reduced sensitivity for detecting malignancy 1
• Volumes greater than 50 mL do not improve sensitivity further 2

The British Thoracic Society (2023) provides strong consensus-based recommendations for these volumes, and research confirms that 25-50 mL represents the plateau phase for diagnostic accuracy 1, 2. Smaller volumes (10 mL) perform significantly worse, with one study showing statistical superiority for volumes ≥150 mL when only direct smear is used, but 60 mL is adequate when both smear and cell block are prepared 3.

Immunocytochemistry (Essential Adjunct)

• Order immunocytochemical staining when malignant cells are identified to distinguish tumor type and primary site 1, 4
• Key markers for adenocarcinoma: CEA, B72.3, Leu-M1, BerEP4, MOC-31 1, 4
• Key markers for mesothelioma: Calretinin, cytokeratin 5/6, D2-40, WT-1 1, 4
• Epithelial membrane antigen (EMA) confirms epithelial malignancy 1

Immunocytochemistry is particularly critical because cytology alone has variable sensitivity depending on tumor type—highest for adenocarcinoma (60-70%), but much lower for mesothelioma, squamous cell carcinoma, lymphoma, and sarcoma 1, 5.

Biomarkers NOT Recommended

• Do not routinely order pleural fluid biomarkers for diagnosing secondary pleural malignancy—they lack diagnostic utility 1
• Do not order serum biomarkers for diagnosing pleural malignancy 1

Additional Investigations Based on Clinical Context

If Initial Cytology is Negative

• Consider a second pleural fluid sample if the first is non-diagnostic and adequate volume (≥25 mL) can be obtained 1
• This yields an additional 25-28% diagnostic rate, though most diagnoses (65%) come from the first specimen 1, 4
• Do not repeat thoracentesis if mesothelioma is suspected, as cytology has particularly low sensitivity for this tumor type—proceed directly to pleural biopsy 1, 5

Microbiological Testing (If Infection Possible)

• Send 5-10 mL in aerobic and anaerobic blood culture bottles if pleural infection cannot be excluded 1
• Also send fluid in plain sterile containers for routine culture 1
• If volume is limited, prioritize 2-5 mL to blood culture bottles over plain containers 1

Other Pleural Fluid Tests to Consider

• Adenosine deaminase (ADA) or interferon-gamma if tuberculous effusion is in the differential (particularly in high-prevalence populations) 1
• Antinuclear antibody (ANA) if lupus pleuritis is suspected 1

Procedural Considerations

• Always use image-guided thoracentesis (ultrasound preferred) to reduce complication risk 1, 6
• Blind thoracentesis should never be performed 6
• Limit fluid removal to 1-1.5 L per session to avoid re-expansion pulmonary edema 6

Critical Pitfalls to Avoid

• Do not rely on negative cytology alone—sensitivity is only approximately 60% overall, and a negative result should prompt consideration of pleural biopsy (thoracoscopic or image-guided) 1
• Do not send inadequate volumes (<25 mL) unless unavoidable—this substantially reduces diagnostic sensitivity 1, 2
• Do not order pleural fluid NT-proBNP routinely—serum NT-proBNP is equally useful and more cost-effective 1
• Consider tumor-specific diagnostic strategies—for suspected mesothelioma or lymphoma, proceed directly to tissue biopsy rather than repeated thoracenteses given the low cytological yield 1, 5