Analysis of Body Cavity Fluids

1. Analysis of Body Cavity Fluids Lab 8

2. Indications and Sampling Indications: - Identifies the type of fluid present: transudate, exudate, neoplastic or other effusion and may identify the cause of fluid accumulation Sampling: - Sterile preparation of site - Use a fine needle (21- 23 G) - Avoid movement or causing pain during sampling - Split sample into EDTA & plain sterile tubes - Process as soon as possible - Monitor the animal

3. Tests Applied Four basic tests are applied: • Appearance of fluid • Protein content • Nucleated cell count (NCC) • Examination of a direct and/or sediment smear to identify cell type Additional tests such as biochemistry may be used in certain clinical situations, e.g. urea or creatinine, if uroabdomen (from bladder rupture) is suspected.

4. Specimen Management for Smears - Mix sample well - Make a direct smear - Centrifuge & smear the deposit (sediment smears) - Air-dry rapidly & stain Special centrifuges (cytocentrifuges) yield better smears A standard centrifuge may be used at a slow speed for a short period (<1000 rpm)

5. Procedure to get a smear “Wedge” method Flat-slide method A drop of the fluid is placed on a cleaned glass slide A smear can be made by the “wedge” method used for making blood smears Alternatively, a 2nd slide may be superimposed on the first, and the two are drawn smoothly apart to make two thin smears.

6. Examination of sediment smears • Blood stains e.g. Diff-Quik or Giemsa usually used • The smear is scanned at low power, to locate cells and cell clusters • NORMAL FINDINGS: Nucleated cells (NC) are usually a mixed population of neutrophils, macrophages and round cells (lymphocytes and monocytes), occasionally with mesothelial cells. • Cells present are noted, most common cells first, giving approximation of cell numbers

7. Classification of Body Cavity Fluids Analysis of a body cavity fluid should allow classification as: • Transudate • Modified transudate • Exudate • Neoplastic effusions • Others: Haemorrhagic effusion Uroperitoneum Chylous effusion

8. A B Transudate - Appearance; Clear, watery - Protein; <20 g/l - NCC; <1 x109/l - Cell type; Few RBC - Small mixed nucleated cells: - Neutrophils up to 60%, - Lymphocytes, monocytes, MΦ, reactive mesothelial cells - Occurs in health, but volume too small for collection except in the horse - Common pathological cause is marked hypoproteinemia

9. Reactive Mesothelial Cells Seen singly & in clumps in all body cavity fluids. Occur in increased numbers, and may become dysplastic when there is irritation or inflammation present.

10. Reactive Mesothelial Cells • Mesothelial cells, if dysplastic, may appear neoplastic, with deeply basophilic cytoplasm, variable nuclear to cytoplasmic ratio. They maybe multi- nucleated, with clumped chromatin & prominent nucleoli. However, an apparent fringe of pink villi on some cells can help to identify them as mesothelial cells. • Reactive mesothelial cells are seen mainly in chronic effusions in dogs and cattle, rarely in horses and cats.

11. Modified Transudate - Appearance; Clear/Turbid serosanguinous - Protein;5 – 35 g/l - NCC; <5 x109/μl - Cell type; Moderate mixed nucleated cell population - Occurs; with increased venous hydrostatic pressure, e.g. heart failure, liver fibrosis

12. Exudate - Appearance; Turbid Bloody/Purulent - Protein; >30 g/l - NCC; >5 x109/l - Cell Type; Many RBC, Nucleated cells are mostly neutrophils, they may be karyolysed, and bacteria may be present - Occur in inflammation, infection & necrosis

13. 1. 2. 3. Exudate 1. Non septic peritonitis in a horse 2. Septic peritonitis in a cow 3. Septic pleuritis in a cat caused by Nocardia spp

14. Neoplastic Effusions - Appearance; May be bloody and/or turbid - Protein; Often > 35 g/l - NCC; Often 5-25 x109/l - Smear; RBC Mixed nucleated cell population; neoplastic cells may be seen - Occurs; due to exfoliation of the neoplasm Since many neoplasms have an irritant or “foreign body” effect producing inflammation or necrosis, neoplastic effusions can have characteristics of an exudate, eg raised protein content and nucleated cell counts. Neoplastic cells may be seen if the neoplasm exfoliates heavily, otherwise they may not.

15. Haemorrhagic Effusion • Appearance; blood-like, but does not clot • Protein; variable, often >50 g/l • PCV; variable, often high (>0.25 l/l) • NCC; variable, similar to blood (>5-15 x109/l) • Smear; no platelets, mixed nucleated & mesothelial cells, erythrophagia & possibly haemosiderin and haematoidin crystals • Occur; with trauma, ruptured neoplasm or coagulopathy

16. Haemorrhagic Effusion • Macrophages which have phagocytosed RBC can processed Hb producing haemosiderin (green-bluish black granules) or hematoidin (brown rhomboid) crytals, which will indicate a longer standing effusion. • The appearance and results vary with the duration of the haemorrhage - immediately after it occurs, results are similar to those of a blood sample.

17. Uroperitoneum • Appearance; usually clear, but also can be serosanguinous. It may be an odour of urine especially if heated • Protein, NCC and Cell Type; similar to those of transudates. It is differentiated by the finding of higher concentrations of creatinine in the effusion than in blood from the same animal. • Occurs; with bladder rupture or urinary tract obstruction

18. Uroperitoneum • Creatinine is more reliable in differentiating uroperitoneum than urea as it is slower to diffuse from the peritoneal cavity into blood. • A crude test is to mix 1 drop of the fluid with a drop of nitric acid on a glass slide, heat gently over a bunsen flame and examine by microscope: hexagonal crystals indicate uroperitoneum.

19. Chyle • Appearance; Milky, white or pink • Protein; 10-30 g/l NCC; 5 - 20 x109/l • Smear; Cells vary with age of lesion- mostly mature lymphocytes - Occurs; with trauma –rupture- of the thoracic duct, also heart failure, neoplasia or any other causes of increased hydrostatic pressure in lymphatics, producing leakage of chyle (lymph and lipids) into the body cavity Findings that helps confirm lipid is present are: - supernatant turbidity/milkiness after centrifugation - staining with sudan allows to visualise fat droplets

20. Characteristics of some benign lesions

21. Types of Benign Lesions • Inflammation • Acute • Sterile • Septic • Eosinophilic • Chronic • Haematoma • Lipoma • Cyst • Sialocoele

22. Acute inflammation • >70% of nucleated cells are neutrophils. • The rest may be mononuclear cells (monocytes, macrophages, lymphocytes, plasma cells). • Types: • - Non-septic or sterile inflammation;neutrophils predominate but arewell-preserved • - Septic inflammation, • neutrophils are degenerate (karyolysed) & bacteria are often present • - Eosinophilic inflammation; • The cell content may be mixed, but eosinophils predominate

23. Acute Sterile Inflammation Intact neutrophils predominate, no micro-organisms seen Mainly well-preserved neutrophils, with a few macrophages and lymphocytes in a firm rapidly-growing submaxillary mass in a dog. Examples of causes of sterile inflammation can be insect bite or immune-mediated polyarthritis.

24. Acute Septic Inflammation Karyolysed neutrophils predominate, bacteria are present Karyolysed and pyknotic neutrophils & many bacteria in an aspirate from a swollen joint in a calf

25. Acute Septic Inflammation 1 2 Streptococcal infection with reactive lymphocytes & karyolysed cells in post-castration discharge in a horse karyolysed neutrophils

26. Acute eosinophilic inflammation A mixed cell content, mainly eosinophils in an aspirate smear from an acute swelling on the head of a dog (presumed to be due to an insect sting)

27. Chronic Inflammation The number of mononuclear cells (monocytes, MΦ, lymphocytes, plasma cells) is increased and may be >50% of nucleated cells. Red blood cells (RBCs), macrophages, neutrophils and lymphocytes in smears from a chronic skin lesion in a dog

28. Example of Chronic Inflammation There are many neutrophils, and reactive macrophages and lymphocytes in this smear from a chronic skin lesion in a horse.

29. Example of Chronic Inflammation: Granulomatous Inflammation This is characterised by the presence of multinucleated giant cells Granulomatous inflammation is chronic inflammation from persistent irritation and infection with particular organisms, e.g. staphylococci and fungi, and characterized by the presence of multinucleated giant cells (Langhans type cell). RBC and a multinucleated giant cell in a granulomatous pedal mass in a dog

30. Haematoma - Findings depend on age of lesion - Fluid blood-like, dark, does not clot - Cell counts/protein content may be similar to those in blood - Smear looks like blood, but no platelets may be present, and macrophages with erythrophagia and hematoidin crystals can appear Fluid swelling on the head of a pup. Was a haematoma, but could have been hydrocephalus or an abscess

31. LIPOMA Clinically, they are soft, smooth, slow-growing, non- nodular painless masses occurring particularly in dogs Aspirate is clear, greasy …and stains+ with Sudan III

32. Lipoma - Aspirate smears are of low cellularity, with a few clumps of benign connective tissue cells & adipocytes

33. Cysts Cysts vary widely in aetiology and therefore in the contents of an aspirate smear. They may be acellular and watery, or filled with necrotic material, or with a proteinaceous secreta. Aspirates from a crusty skin lesion (an epidermal cyst) on the back of a dog; contains a cholesterol crystal and disrupted cells and cell debris

34. Sialocoele - Firm painless fluid swelling of acute onset in the submaxillary space - Smears contain many RBC, few WBC, clouds of pink amorphous material (mucin) and many macrophages