Pleural Fluid Analysis: Essential Samples and Testing Protocol
For diagnostic pleural fluid analysis, send samples in both sterile containers AND blood culture bottles (aerobic and anaerobic) for comprehensive microbiological, biochemical, and cytological evaluation. 1
Core Sample Requirements
Microbiological Samples (Priority for Infection)
- Send 5-10 mL in aerobic AND anaerobic blood culture bottles plus sterile containers for Gram stain, acid-fast bacilli (AAFB) stain, and TB culture 1, 2
- When volume is limited (2-5 mL), prioritize blood culture bottles over plain containers 1
- Blood culture bottles significantly increase diagnostic yield—36% of positive samples grow exclusively in blood culture vials, particularly anaerobes 3
- This dual-container approach is a strong recommendation by consensus for suspected pleural infection 1
Cytological Samples (Priority for Malignancy)
- Submit 25-50 mL for cytological analysis when malignancy is suspected (strong recommendation) 1, 2
- Process using both direct smear AND cell block preparation 1, 2
- If initial volume <25 mL is non-diagnostic, send larger volume on repeat (except when mesothelioma suspected, which has low cytological yield) 1
- Cytology detects only 60% of malignant effusions; negative results mandate further investigation 1, 2
Biochemical Analysis
All pleural fluid samples should be analyzed for: 1, 2
- Protein (to differentiate transudate from exudate)
- LDH (lactate dehydrogenase—clarifies borderline protein values 25-35 g/L)
- pH (measure in all non-purulent effusions when infection suspected; pH <7.2 indicates need for drainage)
Specialized Testing Based on Clinical Context
When Tuberculosis is Suspected
- Adenosine deaminase (ADA): >35-45 U/L with >50% lymphocytes suggests TB in high prevalence populations 2, 4
- Interferon-gamma (IFN-γ): 89% sensitivity, 97% specificity 2
- Tissue sampling remains strongly preferred for all suspected TB cases (69-97% sensitivity) 1, 2
- Perform differential cell count—pleural lymphocytosis requires TB and malignancy exclusion 1
When Malignancy is Suspected
- Cytology sensitivity varies by tumor type—consider direct biopsies for low-yield tumors like mesothelioma 1, 2
- Do NOT routinely use pleural fluid biomarkers for diagnosing secondary malignancy 1
- Molecular testing (EGFR/BRAF/ALK/ROS1/PD-L1) on pleural fluid can guide targeted therapies in advanced NSCLC 5
When Heart Failure is Suspected
Additional Context-Specific Tests
- Glucose: useful in differential diagnosis of exudates 6, 4
- Amylase: when pancreatic or esophageal pathology suspected 6, 4
- Hematocrit: if hemorrhagic—pleural fluid hematocrit >50% of peripheral blood confirms hemothorax 1, 2
- Pleural fluid ANA: supports diagnosis of lupus pleuritis 1
Critical Procedural Points
Sample Collection
- Use fine-bore 21G needle with 50 mL syringe 1
- Image-guided thoracentesis should ALWAYS be used to reduce complications (strong recommendation) 1
- Note appearance and odor: serous, blood-tinged, bloody, or purulent 1, 2
- If turbid/milky, centrifuge to differentiate empyema (clear supernatant) from chylothorax (turbid supernatant) 1, 2
Common Pitfall to Avoid
The 2003 BTS guidelines recommended sending samples in sterile vials AND blood culture bottles 1, but the 2023 BTS guidelines emphasize prioritizing blood culture bottles when volume is limited 1. This reflects evidence that 60% of positive samples occur in non-purulent fluid, and blood culture bottles capture organisms missed by conventional culture alone 3.
Diagnostic Algorithm Integration
- If transudate suspected clinically (heart failure, hypoalbuminemia, dialysis): treat underlying cause first 1, 2
- Perform thoracentesis with samples as outlined above 1, 2
- Classify as transudate vs. exudate using protein/LDH; apply Light's criteria if borderline (25-35 g/L) 1, 2, 6
- If exudate without diagnosis: proceed to contrast-enhanced CT thorax (with fluid present for better pleural visualization) and consider pleural biopsy 1, 2