EXAMINATION OF PLEURAL FLUID

The main purpose of testing is to ascertain their transudative or exudative nature and to find a causative organism if an infective process is indicated. The scheme of examination is almost the same as for CSF except that the determination of specific gravity is important in these fluids while the determination of chloride can be omitted. The most reliable test for differentiating between a transudate and an exudate is the simultaneous analysis of pleural fluid and serum for total protein and lactic dehydrogenase levels. A transudate is an effusion in which the ratio of serous fluid total protein to serum protein is less than 0.5, while the corresponding LD ratio is less than 0.6. If the fluid is labelled as transudate, no other tests are required, but if it is exudate then gram staining, cultures and counter-immuno-electrophoresis is indicated. A cytologic examination and biopsy may be indicated in a case of suspected malignancy.
SPECIMEN COLLECTION
The Medical Officer collects specimens in the ward under aseptic conditions. Fluid is collected in 3-4 sterile containers as for CSF. It is good to take a separate specimen in EDTA for the purpose of taking a cell count.

ROUTINE EXAMINATION
Appearance: Note the amount, colour and transparency. Normal fluid is straw-coloured and clear without coagulum or pellicle.
Specific Gravity: Determine specific gravity either by a refractometer or by using copper sulphate solutions of known specific gravity. Normal specific gravity is less than 1.016.
Cell Count: The procedure used is the same as for CSF. Normally these fluids contain 0-8 cells per mm3 and these are lymphocytes and mesothelial cells.
Preparation of Smears for staining is exactly as for CSF.
Table1: Differences between Transudate and Exudate

Transudate
Exudate

Appearance
Clear
Cloudy or turbid

Colour
Watery or straw
Turbid to purulent or bloody

Specific gravity
<1.016 ≥1.016 Cell count <1X109/L Lymphocytes and mesothelial cells >1X109/L Neutrophils early but mononuclear cells later

RBC
Absent
Often present

Clot formation
None
Usual

Glucose
Same as serum
Same as serum or reduced (>50% of serum level)

Total proteins
<20 g/L (<50% serum level) ≥20 g/L Rivalta Test Negative or faint Positive LD <60% of serum activity >60% of serum activity.

Fluid total protein to serum total protein ratio
<0.5 >0.5

Fluid LD to serum LD ratio
<0.6 >0.6

Estimation of Proteins: This method is the same as for CSF. However, as the protein content of these fluids is higher than that of CSF, these should be diluted prior to making protein estimations. Dilution depends upon specific gravity. If the specific gravity is high, then further dilution should be made. Results are then multiplied with the dilution factor accounted for, accordingly.
Estimation of Globulins: A qualitative test is usually performed. The test performed on serous fluids is the Rivalta Test. The required reagent is prepared by adding one drop of glacial acetic acid to 100 ml of distilled water in a conical flask. To this are added 1-2 drops of centrifuged supernatant fluid. Normal fluids do not produce any cloud in the reagent. Transudate produces a faint cloud, while a distinct cloud appears if the fluid is an exudate.
Estimation of Glucose is important. Glucose levels in pleural fluid below 3.5 mmol/L (60 mg/100 ml); or 2.3 mmol/L (40 mg/100 ml) less than the simultaneous plasma glucose level is considered ‘decreased’. The decreased value of glucose in exudates may be seen in bacterial infections, especially when the exudate is purulent, rheumatoid arthritis, malignant pleuritis and tuberculous pleuritis.
α-Amylase: Pleural effusion may be the first sign of pancreatic disease. α-Amylase activity should be measured in all unexplained effusions. α-Amylase activity is considered elevated when the level in the fluid is 1.5 to 2.0 times the simultaneous serum level. Pleural fluid α-amylase activity may be increased in a variety of conditions, including acute and chronic pancreatitis, pancreatic pseudocyst, oesophageal rupture and, rarely, primary or metastatic carcinoma of the lung.
Creatine kinase: Isoenzyme BB is high in pleural and pericardial fluids in the case of adenocarcinoma of the prostate gland. This enzyme is also high in adenocarcinoma and anaplastic carcinoma of the lung.
pH of normal pleural fluid is 7.64.pH. <7.30 is associated with empyema, malignant disorders, collagen disorders, tuberculosis, oesophageal rupture, or haemothorax. A pleural fluid with pH 7.3-7.4 usually indicates a benign condition. A pH of <6.0 is highly suggestive of oesophageal rupture. The pH<7.1 of pericardial fluid is associated with connective tissue diseases and bacterial infection. A pH of 7.2-7.4 is associated with neoplasms, idiopathic disorders and tuberculosis or uraemic pericarditis. A pH>7.4 is associated with post-cardiotomy states and hypothyroidism.
Staining: If a fluid is an exudate and an infective process is suspected, then cultures must be done. The third container, which was set aside, is used for this purpose. Gram and acid-fast staining are fundamental to any examination of fluids.
Culture: In fungal disease, an appropriate culture is usually necessary.
Agglutination techniques for the identification of certain bacterial antigens (S. pneumoniae) can be done on the fluid.
Tumour Markers: The determination of tumour markers in pleural fluid is sometimes helpful in the diagnosis of certain malignancies. These are done if the presence of malignant cells is suspected. The test is positive in cases of adenocarcinoma of the lung, carcinoma of the breast and ovary.
In addition to the tests mentioned above, a few additional tests may also be required:

Test for Viscosity
Aspirate the fluid in a pipette and then release. If a falling drop draws into a band of 5cm or longer, the viscosity is normal. If the length of the band is less than 5cm, viscosity is decreased.
Test for Mucin (Hyaluronic Acid)
To 5 ml of 1:5 diluted fluid add 0.14 ml 7N acetic acid (408 ml glacial acetic acid in 1 litre distilled water). Stir with a glass rod, examine immediately and after 2 hours. A tight ropy mass is termed good. A softer, shreddy precipitate is termed; fair and a poor precipitate shows shreds of mucin in turbid solution. The latter two indicate reduced hyaluronic acid content.
Wet Preparation for Crystals and Inclusions
A drop of fluid is placed on a clean slide and covered lightly with a cover slip. The preparation is then examined under a microscope with the condenser lowered down. Needle-like crystals of urates are seen in gouty arthritis. In rheumatoid arthritis small, multiple, dark inclusions, are seen in polymorphs. These are immunoglobulins with RA factor activity.
Table 2: Work up of pleural effusion
Pleural fluid protein to serum protein ratio
<0.5 No further tests required Pleural fluid LD to serum LD ratio <0.6 Pleural fluid protein to serum protein ratio >0.5
Gram stain, culture, total WBC and differential counts, cytology, pH, glucose, α-amylase, tumour markers pleural biopsy

Pleural fluid LD to serum LD ratio
>0.6

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