Pleural fluid analysis: Laboratory Investigation of Pleural fluid

Introduction

Pleural fluid analysis is a procedure that focuses on examination of a sample of fluid that has collected in the pleural cavity. Pleural cavity is a potential space between chest wall and lung lined by a layer of flattened epithelium called as mesothelium. It has a small amount of fluid (less than 10 ml in each pleural cavity) which is an ultra-filtrate of plasma.

The function of this fluid is to lubricate the two opposing parietal and visceral pleural layers. Pleural fluid is being formed continuously depending on hydrostatic pressure, plasma colloid oncotic pressure, and permeability of capillaries in parietal pleura. Resorption of pleural fluid happens through lymphatics and venules in visceral pleura.

The contents of pleural fluid is similar to that of plasma, except that it has lower protein level (less than 1.5 gm/dl). Normal pleural fluid is clear and straw or pale yellow in color.

Build up of excess fluid in pleural cavity is termed as pleural effusion. Presence of pleural effusion is usually confirmed by lateral decubitus chest X-ray or ultrasonography. Radiography can also provide information about the cause of effusion e.g. bilateral effusion with enlarged heart suggests congestive cardiac failure, radiological signs of pneumonia suggest parapneumonic effusion.

Causes of Pleural Effusion

There are various causes of pleural effusion.
Common causes include;

Congestive cardiac failure,
Bacterial pneumonia,
Tuberculosis,
Cirrhosis with ascites, and cancer.

The Cause of pleural effusion may be evident clinically such as;

lymphoma or carcinoma (enlargement of lymph nodes, liver or spleen),and
pulmonary embolism (deep vein thrombosis of lower limbs, hemoptysis, pleuritic type of chest pain, and dyspnea out of proportion to amount of fluid accumulated).
congestive cardiac failure (raised jugular venous pressure, edema of feet),
cirrhosis of liver (ascites, other signs of portal hypertension),

The fluid build up may be either a transudate or an exudate.

Transudates come from non-inflammatory conditions and are due to rise in capillary hydrostatic pressure (e.g. congestive cardiac failure, portal hypertension) or low oncotic pressure of plasma (e.g. hypoproteinemia).

Exudates come from inflammatory conditions and are due to rise in capillary permeability (e.g. infections) or obstruction of pleural lymphatics(e.g. malignancy). Further testing of transudates is not required, while it is necessary to analyze exudates further.

Below is a Comparative features of transudates and exudates

Specimen collection for pleural fluid analysis

Sample collection for pleural fluid analysis is achieved by inserting a needle in the chest wall. The procedure is termed as thoracentesis. It may be either diagnostic or therapeutic. Diagnostic thoracentesis is carried out to know the cause of pleural effusion. Conditions that can be definitely diagnosed are:

Chylothorax,
Hemothorax,
Rheumatoid pleurisy
Lupus pleurisy.
Empyema,
Malignancy,
Tuberculous effusion,
Fungal effusion,
Esophageal rupture,

It is recommended to perform diagnostic thoracentesis when pleural effusion is clinically significant (thus more than 10 mm thick on lateral decubitus chest X-ray or ultrasonography) and if cause is not obvious. Therapeutic thoracentesis is performed either to relieve respiratory insufficiency because of massive pleural effusion or to introduce antineoplastic drugs in pleural space following withdrawal of pleural fluid. A qualified and experienced physician does the procedure.

It is contraindicated in:

Mechanical ventilation,
A small volume of fluid,
Unstable angina, and
Cardiac arrhythmias.
Non-cooperative patients,
Coagulation disorders,
Respiratory insufficiency,
Infection at the site of chest wall puncture,

Complications of the procedure include

Introduction of infection,
Pneumothorax,
Haemothorax,
Air embolism,
Injury to the spleen or liver,and
Pulmonary edema if large amount of pleural fluid is rapidly removed.

For diagnostic studies, pleural fluid is obtained in heparinised tubes to prevent clotting. For cell counts, sample is collected in EDTA anticoagulant. For microbiologic studies, sterile sample tubes are necessary; for culture, aliquots of sample are best inoculated in blood culture bottles at the bedside. In suspected malignancy, maximum amount of fluid should be submitted to
enhance the cellular yield

Procedure for Pleural fluid analysis

This consists of:

Appearance
Chemical examination
Cell counts
Microbiological examination
Cytological examination.

1. Physical Appearance:

Transudates are clear, pale yellow, or straw-colored and do not clot on standing.
Exudates are opaque or turbid because of increased proteins, leukocytes, or presence of malignant cells, and often clot on standing, due to high fibrinogen content.
Frank pus with putrid odor indicates empyema.
Straw-colored transudative fluid happens in congestive cardiac failure, pulmonary embolism, and cirrhosis of liver.
Thick exudative fluid happens in pneumonia and cancer.
Bloody or hemorrhagic fluid indicates traumatic tap, pulmonary infarction, or malignancy. Hemorrhage in pleural space is referred to as hemothorax. A traumatic tap progressively becomes less bloodstained during removal of pleural fluid.
Buildup of a milky or chylous pleural fluid is referred to as chylothorax. True chylous effusion results from obstruction of lymphatic duct (due to injury, carcinoma, or lymphoma). A characteristic feature of chylous effusion is triglyceride level more than 110 mg/dl. Microscopy of such fluid shows lymphocytosis and fat droplets that stain with Sudan III. Following centrifugation of a chylous fluid, a layer of creamy chylomicrons forms at the top. A pseudochylous effusion results from long-standing chronic pleural effusion through breakdown of cellular lipids (tuberculous or rheumatoid effusion). It has a bright, shiny appearance, and microscopic examination shows mixed cellularity and cholesterol crystals. Triglyceride level is not more than 50 mg/dl and chylomicrons are absent.
A foul smelling pleural fluid indicates a possible anerobic infection, while urinous (ammoniacal) odor indicates possible urinothorax.
In mesothelioma, pleural fluid is viscous.

2. Chemical examination:

For Chemical examination in pleural fluid analysis, the old criteria for differentiation of exudates and transudates (like protein level, specific gravity, cell count, and clots in sample), significant cases are misclassified. Therefore, in pleural fluid, differentiation of exudates from transudates is based on following criteria (called Light’s criteria):

Pleural fluid protein to serum protein ratio greater than 0.5
Pleural fluid LDH to serum LDH ratio greater 0.6
Pleural fluid LDH above two thirds of upper limit of normal for serum LDH.

Exudates have at least one of the above criteria. if all the three criteria are present, this best differentiates an exudate from a transudate. Transudates have none of these criteria.
If the fluid is an exudate, further investigations are indicated like;

Cell counts,
Glucose estimation,
Cytological examination,
Test for tuberculosis,
Gram smear,
Culture.

In case of transudates, diagnostic considerations include;

Congestive cardiac failure,
Cirrhosis,
Pulmonary embolism.
Low glucose levels are got in empyema, rheumatoid pleurisy, tuberculous pleurisy, and malignant effusion.
There is a direct correlation between lactate dehydrogenase levels in pleural fluid with degree of pleural inflammation.

3. Cell counts:

For cell counts in pleural fluid analysis, a total leukocyte count must routinely be obtained on all fluids. In transudates, leukocytes are less than 1000/ml and are mostly small, mature lymphocytes.
Predominance of neutrophils (greater than 50%) indicates an acute process (e.g. parapneumonic effusion, pulmonary infarct), while predominance of lymphocytes indicates a chronic process (e.g. tuberculosis, rheumatoid pleurisy, malignancy).

Very high leukocyte count (greater 50,000/ml) with predominance of neutrophils suggests parapneumonic effusion (usually empyema). Presence of many small, mature lymphocytes with only a few or no mesothelial cells is suggestive of tuberculosis. Blood-tinged pleural fluid is not diagnostic of any condition and can occur in a transudate or an exudate.

Grossly hemorrhagic pleural fluids have red cells more than 100,000/ml and are seen in pulmonary infarction, malignancy, or traumatic tap. Traumatic bloody effusion is suggested by gradual clearing with aspiration, clotting of fluid after some time, and lack of hemosiderin-laden macrophages.

4. Microbiological examination:

For Microbiological pleural fluid analysis Gram’s smear and culture must be carried out on exudates, especially on:

purulent,
bloodstained,
cloudy samples.

The likelihood of isolation of organisms is increased if pleural fluid is inoculated in blood culture bottles at the bedside. Ziehl-Neelsen stained smear is positive in less than 20% of tuberculous pleural effusions and culture in less than 40% of cases. If tuberculosis is suspected and culture is negative, polymerase chain reaction for mycobacterial DNA or increased level of adenosine deaminase (released in pleural fluid from activated lymphocytes) may establish the diagnosis.

5. Cytological examination:

For cytological pleural fluid analysis, pleural fluid is centrifuged, smears are prepared from the sediment, and are stained with Papanicolaou stain. In older age, metastatic malignancy is responsible for majority of pleural effusions. The common malignancies include;

bronchogenic carcinoma,
carcinoma of breast,
lymphoma.

Pleural effusion results from lymphatic obstruction by tumor cells. Typically, effusion is blood tinged or hemorrhagic. Examination of three separately obtained pleural fluid samples increases sensitivity of detection of cancer cells to 80%.

6. Other tests:

For investigation of pulmonary embolism, D-dimer test (a breakdown product of fibrin) should be done on peripheral blood sample.
Flow cytometric analysis of pleural fluid sample must be done if lymphoma is suspected.
In parapneumonic effusion, if pleural fluid pH is less than 7.20, drainage is indicated; if pH is greater than 7.30, complete resolution will happen with medical treatment.
In malignant pleural effusion, low pH indicates poor prognosis and poor response to tetracycline pleurodesis.
A pleural fluid rheumatoid factor of atleast 1:320 is suggestive of rheumatoid pleurisy. Presence of LE cells in pleural fluid makes diagnosis of lupus nephritis very certain.
Elevated pleural fluid amylase occur in pancreatitis and esophageal rupture.
Pleural fluid triglyceride of atleast 110 mg/dl indicates chylous effusion

Special studies on pleural fluid to determine the cause of pleural effusion

Pancreatic disease, esophageal disease: Amylase
Malignancy: Carcinoembryonic antigen
Chylothorax: Triglycerides
Tuberculous pleural effusion: Adenosine deaminase, gamma interferon, polymerase chain reaction
Rheumatoid effusion: Rheumatoid factor
Lupus pleuritis: Antinuclear antibodies

Pleural Biopsy

Percutaneous needle biopsy of the parietal pleura is indicated in patients with exudative pleural effusion pleural fluid analysis does not yield a specific diagnosis. It is done especially if tuberculosis or malignancy is suspected. For better results, thoracoscopy (direct visualization of pleural surface by introducing a thoracoscope through the chest wall into the pleural
cavity previously filled with air) can be useful in localizing the site of biopsy.

For investigation of tuberculosis, pleural biopsy is more sensitive than pleural fluid analysis and culture. In malignancy, improved techniques in pleural fluid cytology have increased the diagnostic yield to 80%; combination of pleural fluid cytology and pleural biopsy can increase the detection rate further.

For pleural biopsy, special needles like Abrams, Cope and tru-cut are available. Needle is inserted with its cutting chamber closed into the pleural effusion. It is recommended to obtain at least three biopsy samples (from 3, 6, and 9 ‘O’ clock positions of cutting chamber) for histology and culture.

Chances of hemothorax and pneumothorax are higher as compared to thoracentesis. Spread of malignant cells can occur along the needle track.

Thoracoscopy:

In thoracoscopy, an endoscope is inserted into the pleural cavity for direct visualization of pleural surfaces. It is indicated if pleural fluid analysis is non-diagnostic in a case of pleural effusion and to localize the site of biopsy.

More pleural space is created by removing pleural fluid and replacing it with an equal amount of atmospheric air. Thoracoscope is inserted through a skin incision via a trocar into the pleural cavity. It is especially helpful for diagnosis of metastatic cancer and mesothelioma. Appearance of pleura is also diagnostic in tuberculosis and rheumatoid pleurisy. The procedure can be combined with aspiration of pleural fluid, pleural biopsy, and pleurodesis.

Thorascopy is better than blind pleural biopsy for diagnosis of malignancy. Thoracoscopy is contraindicated if pleural space is obliterated and in the presence of coagulopathy and severe cardiac disease.