Pleural fluid: cytology

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Overview

  • The pleura of the horse is a thin layer of connective tissue covered by a single layer of mesothelial cells.  The visceral pleura covers the lungs and the parietal pleura covers the chest wall, mediastinum and diaphragm. 
  • In the normal horse the pleural cavity is moistened by a very small amount of fluid to allow friction free movement of the surfaces during respiration.  When fluid in the pleural space exceeds this very small amount, a pleural effusion is said to exist. 
  • Factors responsible for maintenance of the normal pleural fluid are plasma oncotic pressure, capillary hydrostatic pressure, capillary permeability and lymphatic drainage.  A disturbance in any one of these factors may cause fluid to accumulate, producing a pleural effusion.
  • Pleural fluid     is a dialysate of plasma with a low cell count    and protein    .  The cytological constituents reflect pathophysiological processes in the pleural surfaces.  
  • Cell characteristics vary in septic conditions, toxic processes, chylous processes and with neoplasia.
  • Cytology enables characterization of a thoracic effusion and positive diagnosis in some cases of neoplasia.

Uses

Alone

  • Differentiate transudate from exudates.
  • Identify neoplastic cells, eg lymphosarcoma    , carcinoma, mesothelioma.
  • Identify septic effusion, eg pleuritis    .
  • The most common cause of pleural effusion in the United States is septic (bacterial) pleuropneumonia    .
  • Viral infections undoubtedly can result in a pleural effusion but these are rarely diagnosed as they are usually not clinically evident and are often self-limiting.

In combination

  • With total cell count and protein estimations to differentiate transudates from exudates and diagnose neoplastic effusions.

Differential diagnosis of pleural effusion in the horse

Result data

Normal (reference) values

  • Total nucleated cell count should be <10 x 10*9/l, cells should be non-degenerate neutrophils in the main (30-90% on differential count), with some mononuclear cells (lymphocytes 0-22%) and large mononuclear cells (monocytes, macrophages and mesothelial cells 5-66%).  
  • Assessment of cell morphology, as for peritoneal fluid    , is very important and the same criteria may be applied.

Polymorphonuclear leukocytes

  • The most common cell type encountered in pleural fluid, function of primary cellular defence against invading micro-organisms.  Morphology of PMNs provides important information on the presence or absence of sepsis and toxin-producing organisms in the pleural cavity.  
  • Two general types of PMN are recognized:
    • Non-degenerate PMNs  PMN Score Table  have tightly clumped, basophilic nuclear chromatin and may be hypersegmented due to old age.  The PMNs in peritoneal fluid do not re-enter the circulation and the nucleus eventually breaks into round, tightly clumped balls (pyknosis) within the fluid.  The presence of non-degenerate hypersegmented PMNs suggests that the fluid is not septic.  
    • Degenerate PMNs  PMN Score Table   are those that have undergone hydropic change due to bacterial toxins altering the permeability of the membrane and allowing water to diffuse into the cell.  The cells swell, show vacuolation and dissolution of the nucleus into tiny balls (karyorrhexis), or  they become fragile and may smear (karyolysis).

Mesothelial cells

  • Pleural fluid: mesothelial cells 04 - cytologyFig.1 Pleural fluid: mesothelial cells 04 - cytology
    A single layer of flattened mesothelial cells covers the visceral and parietal pleural surfaces.  Some mesothelial cells are desquamated into the pleural fluid in normal circumstances, and in their least reactive form they appear squamous and may be seen in rafts. The unstimulated mesothelial cell nucleus is round to oval and contains few visible nucleoli.   The cytoplasm is pale and evenly stippled   (Fig. 1)  .
  • Recognition of the mesothelial cell may be difficult because it can assume a variety of forms in different circumstances.  In response to an acute or sub-acute, non-septic inflammatory process, mesothelial cells proliferate and become activated, showing marked pleomorphism and intense basophilic staining.  The nucleus appears darker staining with prominent nucleoli, there is a higher nucleus:cytoplasm ratio, the cytoplasm appears denser and there may be a hairy corona around the cytoplasmic membrane. 
  • Activated mesothelial cells may show prominent nucleoli, binucleate forms and cell clusters which bud off into the pleural fluid.  In chronic effusions, mesothelial cells may show coarse chromatin, prominent nucleoli and numerous cytoplasmic vacuoles.  
  • These transformed mesothelial cells may have phagocytic properties.  The presence of transformed mesothelial cells in pleural fluid indicates that the effusive mechanism has been in operation for some time, sufficient for mesothelial proliferation and desquamation to occur.  
  • Pleural fluid: mesothelial cells 05 - cytologyFig.2 Pleural fluid: mesothelial cells 05 - cytology
    Reactive mesothelial cells can be difficult to differentiate from neoplastic cells, and specialist interpretation should be sought   (Fig. 2)  .

Mononuclear phagocytes (macrophages)

  • Macrophages are an important component of the host defence mechanism. 
  • Along with the neutrophils, macrophages are responsible for combating bacterial invasion of the body cavities.  They are helper cells to lymphocytes in immunological reactions and have an important role in the removal of damaged and old cells from the pleural cavity.  
  • Mononuclear phagocytes are commonly encountered in normal pleural fluid samles.  In unstimulated fluids, they have an appearance similar to the blood monocyte, and unstimulated mesothelial cell.  The nucleus is typically bean shaped and eccentrically placed.  In cases with acute inflammatory effusions, after the initial PMN reaction, the population of macrophages increases and macrophages containing engulfed (phagocytized) cellular elements and debris are commonly seen. 

Other cells

Eosinophils

  • Eosinophils are rarely encountered in equine pleural fluid samples.
  • Their presence has been associated with some cases of lymphosarcoma.  Aberrant parasite migration could also be implicated.

Pathologic changes

  • A pleural effusion develops when pleural fluid is produced faster than it is removed. This may be by increased transudation, producing an ascitic fluid (transudate) or by exudation.  
  • A transudate has low (normal) protein levels and cellularity.  
  • Common causes include increased capillary hydrostatic pressure, such as in congestive heart failure, and low plasma osmotic pressure as in hypoalbuminemia.  
  • An exudate results from increased pleural capillary permeability and compromised lymphatic drainage usually as a result of inflammation of the pleura.  
  • Effusions are classically grouped as follows:
    • Transudate :
      • Increased volume with low cellularity, estimate of fluid volume is needed to diagnose. 
      • Protein: &amp;lt;25 g/l (usually &amp;lt;15 g/l)    .
      • Smears have few cells.
      • Non-degenerate neutrophils (hypersegmented), normal and occasional stimulated (reactive) mesothelial cell, macrophages and small lymphocytes.
    • Modified transudates :
      • Increased volume and normal appearance, with total cell count 5-15 x 10*9/l and total protein concentration of 20-50 g/l. 
      • Non-degenerate neutrophils tend to be the predominant cell type. 
      • Modified transudates are rarely diagnosed and would be seen in cases of primary or secondary thoracic neoplasia, and cardiac failure    . 
      • Rarely a chylous effusion is seen, and this will appear opaque and white/pink with a predominance of small lymphocytes.
    • Exudate :
      • Inflammatory exudates are the most common cause of pleural effusion in horses. 
      • Exudates have greatly increased cellularity and protein content (>30 g/l).  The most common cause is aerobic and/or anaerobic bacterial infection. 
      • Exudates may be broadly classified as suppurative, neutrophil predominant (pyogranulomatous) or mixed, depending on the predominant cell types.
      • Pleural fluid: septic pleuritis - cytologyFig.3 Pleural fluid: septic pleuritis - cytology
        Nucleated cell counts in excess of 10 x 10*9/l   (Fig. 3)  .

Neoplastic cells

Neoplastic effusions

  • Pleural fluid cytology is useful in establishing a diagnosis in cases of thoracic neoplasia.  
  • Pleural fluid: thymic lymphosarcoma - cytologyFig.4 Pleural fluid: thymic lymphosarcoma - cytology
    Lymphoma is the most common neoplasm encountered, accounting for up to 73.7% of thoracic neoplasia cases. The diagnosis is usually straightforward.  The fluid which is usually a modified transudate or hemorrhagic effusion contains numerous immature lymphocytes   (Fig. 4)  . Immunophenotyping (low availability) is helpful to confirm the diagnosis.
  • Neoplastic cells are usually large with a high nuclear:cytoplasmic ratio and basophilic cytoplasm.  There is marked pleomorphism and indented nuclei are often seen.
  • Less commonly encountered tumors include mesotheliomas (abnormally large cells with highly vacuolated cytoplasm)    , metastatic carcinomas, eg renal, (large epithelial cells often in clusters)    , squamous cell carcinoma from the stomach, malignant melanomas and metastatic adenocarcinomas. These tumors are considerably less common, with metastatic spread from renal carcinoma representing 10.5% of cases and others having an incidence of 5% or less. (Figs. 5-6)

Problems in cytologic diagnosis in cases of neoplasia

  • Intrathoracic tumors may not exfoliate into the pleural fluid.  
  • Ultrasonography, radiography, and pleuroscopy may be helpful in establishing a diagnosis.
  • Tumors that obstruct lymph flow from the pleural cavity may produce a voluminous effusion characteristic of a modified transudate.

Cytopathology

Hemorrhagic effusion

  • Blood collecting within the pleural cavity is defibrinated and will not clot in a plain tube.  
  • Cytologically the smear shows marked erythrophagia or hemosiderophagia.

Errors and Artifacts

  • Neoplastic cells are difficult to distinguish from very reactive ('transformed') mesothelial cells, and care should be taken in over interpretation of dysplastic changes if there is a significant inflammatory response.
  • Erythrophagia and vacuolation of macrophages may occur in vitro if there is a delay in fixing the sample.  Nucleated cells may start to exhibit aging changes such as hypersegmentation and pyknosis, and neutrophil hyposegmentation artifact may be observed following delayed processing of EDTA anticoagulated samples.

Blood tap

  • Occasionally frank blood is obtained on attempts at pleural tap, more often there is contamination of the sample with blood at the time of sampling.  
  • The fluid has the gross and cytological appearance of blood.  Accidental blood tap at the time of sampling will result in a clot in a plain container.  
  • Platelets will be seen on the smear of a sample contaminated with blood.
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