An effusion is the fluid that can accumulate in a body cavity due to various different pathologies. Mechanistically, effusions are caused by transudation, exudation, rupture of a blood/lymph vessel/ hollow organ, or neoplasia. Types of effusions include transudates and exudates, but also haemorrhagic effusions, chyle and uroperitoneum. The composition of the fluid gives clues as to the pathology that caused it, and the goal of fluid cytology is to describe this composition, by looking at cellularity, cell types present, protein concentration, SG and the presence of other substances like bilirubin or creatinine.
Effusions can be examined in-house or sent away to reference laboratory for evaluation. Fluid should be collected into an EDTA tube for cytological analysis, and into a plain tube for culture. Two direct smears (using the blood-smear technique) should be made from the EDTA-fluid. If the samples are to be sent away, these smears should be left unstained, packaged in plastic slide-keepers and stored at room temperature until sent. The EDTA and serum tubes should be stored at 4°C. For in-house analysis, the total nucleated cell count (TNCC) can be determined from the uncentrifuged EDTA fluid sample on an automated cell counter – in most cases the same analyzer used for measuring haematology/ CBC samples. If this is not available, an estimate of cellularity (“low” or “high”) can be made from the direct smears, with some experience. After TNCC measurement, the EDTA tube is centrifuged. Total protein and SG of the supernatant is measured with a refractometer. If the TNCC is < 30 000 cells/ µL, or cellularity is low on the direct smear, smears should be made from the sediment after centrifugation. The supernatant should be saved in case any biochemical tests need to be performed. If the fluid is very bloody, prepare a buffy coat smear to concentrate any nucleated cells of interest.
It is usual to perform a 100-cell differential count, or at least estimated the proportions of the different nucleated cell populations, when examining the smears. Note should be made of any infectious agents, foreign material, background or atypical cells. Normal pleural fluid has a protein concentration of < 25 g/L with very low numbers of macrophages and mesothelial cells. Mesothelial cells line the pleural, pericardial and peritoneal cavities and will be present in most effusions. They are large cells, present singly or in clusters, as seen in Figure 1. Mesothelial cells become hyperplastic very quickly in response to an increased volume of any type of fluid. Numbers increase, and they display many atypical features – multinucleation, mitotic figures, prominentnucleoli, coarse chromatin and nuclear moulding are not unusual (Figure 2).
They also tend to slough off in sheets when reactive. It is almost impossible to differentiate highly reactive mesothelial cells from a neoplastic population, as well as from cells originating from an adenocarcinoma. Diagnosis of a mesothelioma based on cytology alone is not considered accurate, and imaging findings should always be taken into consideration. If cytology is suspicious, fluid sediment/ pellet can be fixed in formalin and processed as a histopathology sample. Evaluation of the arrangement of the cells as well as immunohistochemical staining can then be performed.
Classification of Effusions
Readers may notice the absence of the “modified transudate” in the descriptions below. This classification is only used in veterinary, and not human medicine. The classification of transudate and exudate, based on cut-offs for TNCC, SG and protein was first introduced in a veterinary internal medicine textbook in 1968. The modified transudate appeared in 1971, in order to cover fluids that did not fit the criteria for transudate or exudate. Rather alarmingly, the veterinary classification system, using cellularity and total protein to indicate the mechanism of effusion formation, is not evidence-based and has been shown to be inaccurate. The modified transudate, in particular, is considered by many veterinary clinical pathologists to be a meaningless and unhelpful category. The classification system used here attempts to give information about the underlying aetiology for the effusion. (Regarding units for total nucleated cell counts: 1 x 109/L = 1000 /µL. The former is the SI unit, but the latter is more commonly used.)
Transudates appear when there is increased vascular hydrostatic pressure with or without loss of oncotic pressure due to hypoalbuminaemia (i.e. a disruption of Starling’s forces). Transudates are further subdivided as:
i. Low protein transudate: clear colourless effusions with low protein concentrations and low nucleated cell count (NCC):
- Dogs/ cats: TP < 25 g/L, NCC< 1500 cells/ µL
- Cell types present: mixture of non-degenerate neutrophils, lymphocytes, macrophages and reactive mesothelial cells (all in very low numbers).
- Caused by leakage of low protein fluid from the blood vessels into a body cavity, usually due to hypoalbuminaemia together an increase in hydrostatic pressure.
For transudative effusions to be present solely due to hypoalbuminaemia, it has been suggested (but not proven in animals) that the serum albumin level has to be <15 g/L. The 2 main disorders causing the formation of a low protein transudate are
- protein-losing nephropathy (protein lost through the kidney – hypoalbuminaemia – decreased oncotic pressure + abnormal regulation of blood volume – water retention – increased hydrostatic pressure)
- hepatic cirrhosis (liver cannot produce proteins – hypoalbuminaemia – decreased oncotic pressure + abnormal regulation of blood volume – water retention – increased hydrostatic pressure)
Other rarer disorders than can cause a low-protein transudate in the absence of hypoalbuminaemia are: portal vein anomalies, pulmonary hypertension. Low protein transudates are rare.ii .High protein transudate: clear to cloudy, yellow, orange or red effusions with medium to high protein concentrations and low to medium nucleated cell count (NCC):
- Dogs/ cats: TP ≥ 25 g/L, NCC< 5000 cells/ µL
- Cell types present: mixture of non-degenerate neutrophils and macrophages and reactive mesothelial cells.
Caused by increased hydrostatic pressure in the lungs or liver (sinusoidal/ post-sinusoidal) due to venous congestion. The increased hydrostatic pressure causes fluid to leak out into the pleural/ peritoneal space. The protein concentration of the fluid is not low.The two main disorders causing venous congestion in the lungs or liver, leading to a high protein transudate are:
- congestive heart failure
- chronic hepatic disease
An exudate forms due to increased vascular permeability caused by inflammation. The exudation of protein-rich fluid is accompanied by migration of inflammatory cells into the effusion:
- Dogs/ cats: TP >25 g/L, NCC >5000 cells/ µL
- Exudates are hazy to cloudy, yellow, tan, cream, orange, and may have a putrid smell if bacteria are involved.
Neutrophils are the dominant cell present in most exudates. With inflammation due to bacterial infections, degenerate neutrophils will be the predominant cells present, unless the bacterial toxin is weak or produced in small amounts. Lack of bacteria and/or organisms does not rule out an infectious cause. A septic exudate refers to the presence of intra- and extracellular bacteria, and a non-septic exudate refers to the absence of bacteria and a negative culture. The classification of an effusion as non-septic should be based on a negative culture. Non-septic exudates are caused by acute pancreatitis, necrosis associated with intracavitatory neoplasia or secondary to intracavitatory organ inflammation. Septic exudates are caused by disorders like GIT rupture or penetrating wounds (Figure 3). If acute pancreatitis is suspected to be the cause of a non-septic exudate, fluid lipase may be helpful. A DGGR-lipase activity in fluid of > 500 U/L, or twice serum lipase; or a SpecCPL of > 500 µg/L is highly specific for pancreatitis as the cause of the exudate.
Exception: FIP exudate: Caused by the FIP virus, has a very specific appearance – straw-coloured, viscous, TP > 25 g/L, NCC < 5000 cells/ µL. Cell population is a mixture of neutrophils and macrophages. (The reason for the low cellularity is that although there is a vasculitis in FIP, the focus of inflammation is inside the blood vessels and not in the body cavity – so vessels are leaky to plasma proteins, but inflammatory cells remain in the blood vessel walls)
3. Haemorrhagic Effusion
Causes include haemostatic defects, trauma or neoplasia invading blood vessel walls. The fluid is red and bloody in appearance. Dominated by red blood cells. PCV >1% All species: TP >25 g/L, NCC >2000 cells/ µL
With haemorrhage of more than 24 hours duration or chronic persistent haemorrhage, erythrophagocytosis and/or haemosiderin or haematoidin should be present. With peracute haemorrhage or iatrogenic blood contamination, platelets could be present. If both erythrophagocytosis and platelets are present, chronic persistent haemorrhage or previous haemorrhage and iatrogenic blood contamination are present. A fluid PCV of > 1% indicates that haemorrhage is contributing to the effusion. Inadvertent aspiration of the liver or spleen could also cause a cytological appearance similar to haemorrhage. The PCV of this fluid will then be higher or similar to the peripheral blood PCV. The most common causes of haemoperitoneum in dogs is splenic disease, specifically haemangiosarcoma. However, fluid cytology has a very low sensitivity for detecting cells from haemangiosarcomas in haemorrhagic fluids, due to the dilution effect of the blood and poor exfoliation of cells from these tumours. In other words, neoplastic cells from haemangiosarcoma are rarely seen in haemoperitoneum caused by this tumour. The absence of neoplastic cells does not at all rule out a haemangiosarcoma. In cats, the most common cause of haemorrhagic effusions is trauma, followed by various types of neoplasia.
Caused by a ruptured bladder. TP and NCC are variable – both very low initially (because urine has a very low protein and cell content), then increasing with time as urine is irritant to the peritoneum and causes a low-grade inflammation, so that the fluid becomes an exudate with time. There may be a urine odour. If uroperitoneum is suspected, fluid creatinine concentrations must be measured.
Two out of the following three criteria are diagnostic for uroperitoneum:
- Fluid creatinine > 2x serum creatinine
- Fluid creatinine > 4x normal serum creatinine (upper reference limit)
- Fluid K+ > 1.4 x serum K+
5. Chylus Effusion / Chyle
Chylus effusions occur due to leakage of lymphatic fluid into body cavities due to physical or functional obstructions to lymphatic ducts or to trauma (rare). These effusions therefore have characteristics similar to lymphatic fluid in the lymph ducts draining the GIT.
- They are milky and white, TP >25 g/L, NCC < 10 000 cells/ µL.
- The dominant cell population is small lymphocytes, but cytology may vary if the chylus fluid causes irritation of and inflammation is present.
6. Bile Peritoneum
Caused by a rupture in the biliary system due to gall bladder disease, choleliths or cholangitis. Starts off as a low protein transudate, but as bile is extremely irritant to the peritoneum, the protein and cellularity increase and the process becomes exudative.The fluid may have a greenish or orange tinge. Bilirubin concentrations in the fluid are >2x bilirubin in serum. Bilirubin crystals may be seen on cytology.
7. Neoplastic Effusion
Neoplasia may cause:
- obstruction to lymphatics resulting in a chylus effusion
- obstruction to blood vessels resulting in a high-protein transudate
- invasion of blood vessels resulting in a haemorrhagic effusion
- inflammation resulting in an exudate
A neoplastic effusion is identified as such when malignant cells are seen in the effusion. (Figure 4) The most common tumours associated with effusions are lymphoma and carcinomas. A cytological diagnosis of neoplasia in an effusion (excluding mesothelioma) has a high specificity and high positive predictive value. Sensitivity is however low, and a negative diagnosis does not rule out neoplasia.
8. Pericardial Effusions
In dogs, the most common causes are neoplasia (haemangiosarcoma) and idiopathic. Rarely, a pericardial effusion may be caused by left atrial rupture, coagulopathy or bacteria. Cytology usually reveals a haemorrhagic effusion with highly reactive, atypical looking mesothelial cells. Sensitivity for identifying HAS is very low. Around 75% of pericardial effusions in cats are caused by chronic heart failure (i.e. will have cellularity and protein concentration consistent with high protein transudate). However, periocardiocentesis is performed extremely rarely in cats.
Correct Sample Collection and Preparation Technique for Cytological Evaluation
Emma Hooijberg DiplECVCP
Department of Companion Animal Clinical Studies
Faculty of Veterinary Science University of Pretoria
Other than the experience of the cytopathologist evaluating the samples, one of the major factors determining the diagnostic value of cytologic specimens is the quality of the sample.
Cytology is a reliable method of obtaining a tissue diagnosis in a minimally invasive way and is most frequently used to investigate superficial cutaneous or subcutaneous masses, lymphadenomegaly and body cavity effusions. The widespread availability of ultrasonography has, however, greatly enhanced the ability to accurately sample focal lesions deep within body cavities. Cytology and histopathology remain complementary diagnostic procedures, reflecting a trade-off between the lower degree of invasiveness of sample collection and short turn-around time with cytology and the increased amount of information available due to the ability to evaluate the tissue architecture with histopathology.
With cytology, the clinician is faced with the responsibility of not only collecting an adequately representative sample, but also preparing the slides that are to be examined.
Benefits of cytology:
• Sample collection usually quick, easy and inexpensive.
• No requirement for general anaesthesia – may need light sedation in some cases.
• Less invasive collection techniques – little or no risk to the patient.
• Diagnosis within hours
• Useful for making a specific diagnosis, identifying the disease process, directing therapy, prognostication and to determine the next diagnostic step.
Fig 1: A mass is often made up of multiple”anatomical” regions . The area aspirated will affect the diagnostic quality of the sample
• Does not preserve tissue architecture; therefore less information regarding tissue of origin and behaviour of tumour (e.g. grading, degree of tissue infiltration, presence/absence of necrosis).
• Cellular yield may be very poor for certain tissues or tumours e.g. tumours of mesenchymal origin – may require concurrent histopathological examination.
• May not be representative of the lesion under investigation.(Fig 1)
SAMPLE COLLECTION AND PREPARATION
Table 1. Cytological sample collection methods and indications:
Indication for uses
Fine needle aspiration
Masses (surface or internal), lymph nodes, internal organs, fluid collection
Best method for non-invasive sampling of internal organs/masses and cutaneous/subcutaneous masses
Exudative cutaneous lesions, cytology from biopsy samples
May yield only surface cells and contamination – a problem with ulcerated tumours
Flat cutaneous lesions, cytology from poorly exfoliative biopsy specimens
Obtain some blood/serum to make cells stick to slide
Vaginal smears, fistulous tracts
Used when anatomical location is not amenable to other methods
1. Fine-needle aspiration (FNA)
The sample is collected with a 22-25 G needle and a 3-20 ml syringe. The softer the tissue is, the smaller the needle and syringe that should be used. With larger needles, tissue cores with few free cells are aspirated and blood contamination (haemodilution) is more common. Softer tissues such as lymph nodes are aspirated with a 3 ml syringe, whilst firm tissues such as fibromas and squamous cell carcinomas require a larger syringe to maintain adequate negative pressure/suction in order to collect sufficient cells. If the texture of the tissue is unknown, a 12 ml syringe should be used.
Skin preparation is similar to that required for venipuncture, i.e. an alcohol swab can be used to clean the area. In the case of ultrasound-guided aspiration, it is very important to clean the skin of any ultrasound gel before sampling, as even a very small amount of ultrasound gel contamination during aspiration could obscure cells and render a slide non-diagnostic.
There are 2 basic harvesting techniques.
• Aspiration technique
The mass is stabilized between thumb and forefinger, while the needle with syringe attached is introduced into the mass. The plunger is withdrawn two-thirds to three-fourths the syringe volume. If the animal is calm enough and the mass is large enough, the needle is redirected in the mass while negative pressure is maintained. With small masses and fractious animals, negative pressure is released, the needle redirected and negative pressure then reapplied. Negative pressure is released before the mass is exited to prevent the aspiration of surrounding tissues, the aspiration of the sample into the needle hub/syringe and excessive blood contamination.
After several areas of the mass have been sampled, negative pressure is released, the needle removed from the mass and then the needle is removed from the syringe. Air is drawn into the syringe, the needle is replaced and the sample is forced onto a clean glass slide. The material is smeared and allowed to air dry.
• Stab technique
This technique relies only on the passage of the needle through the tissue to collect enough cells, and not negative pressure. Most commonly, a needle without an attached syringe is used, but a syringe can be connected to use as a handle only. No negative pressure is applied during collection. The hub of the needle is grasped with thumb and forefinger (as if throwing a dart), the mass is stabilized and the needle is inserted into the mass. About 8–10 stabbing motions are made, staying in the same tract, but making sure that the tip of the needle stays inside the mass so that contamination by surrounding tissues is avoided.
The needle is removed from the mass; a syringe with plunger drawn back attached, and the material expelled onto a clean glass slide. It is then smeared and left to air dry. This procedure must be repeated in multiple areas of the mass. If multiple masses are sampled, always use new needles and syringes with each mass.
2. Impression smears
Impression smears (also called imprints) can be made of superficial cutaneous lesions or from tissues obtained by surgical biopsy. Ulcerative lesions must be imprinted, then cleaned with a moistened surgical swab and again imprinted. Freshly cut surfaces from biopsy samples must be blotted with absorbent paper repeatedly until dry of blood, and gently touched to the surface of a clean microscope slide, without a smearing action, as this will rupture cells.(Fig 2)
Fig 2: Impression smear being made from a blotted freshly cut edge of a biopsy sample
Scrapings could be useful for obtaining cells from firm surfaces, such as firm cutaneous lesions. Cells obtained by means of a scalpel blade are gently smeared onto a clean glass slide. This often gives many broken cells, but areas of intact cells can usually be found.
Swabs can be used to obtain cells from mucosal surfaces such as the rectum or vagina, or in areas where the other sampling methods are not practical, such as fistulous tracts. The swab must be gently rolled along the surface of a clean glass slide.
Preparation of slides
Once a sample has been collected, it needs to be transferred to a clean glass slide and spread out – the aim is to obtain a monolayer of cells. This needs to be done quickly before the material dries out or clots. There are various techniques, depending on the volume and consistency of collected material.
1. Solid tissue aspirates
• Slide over slide (“squash”) preparation
This is generally the best method for preparing slides, if done properly but does need some practice. The material collected is placed in the middle of a clean glass slide (the smear slide). A second slide (the spreader slide) is placed over the sample, perpendicular or parallel to the smear slide. The weight of the spreader slide will cause the sample to spread. Once this has happened, the spreader is gently drawn across the smear slide, without any downward pressure, as this will rupture cells. (Fig 3)The weight of the second slide is enough to cause the cells to spread. Non-fragile tissues (e.g. from carcinomas) or any thick viscous material (such as mucous strands from trans-tracheal aspirates) can be spread by this method. With some experience, and a gentle touch, this method can also be used for more fragile tissues like lymph node or spleen.
Blood smear technique
This is done in a similar manner to making a blood smear. The material is placed near the end of a smear slide. The spreader slide is placed in front of the material at a 45-degree angle, backed up until the material is touched and then moved forward. (Fig 4) The feathered edge should not be right on the edge of the slide, as the stage retaining clamps on the microscope will interfere with the oil immersion lens and the sample then cannot be viewed. Malignant cells are often found in the feathered edge. This technique works well for tissues with a fluid component, like internal organs and lymph nodes.
• “Starfish” preparation
Here the tip of a needle is used to drag the material in several different directions. It is a gentle technique that does not cause much rupture of the cells, but it does leave a thick layer of tissue fluid around the cells that may stop them from spreading to an acceptable shape and size.(Fig 5) Usually some areas that can be interpreted are found. This is not a commonly used or preferred technique (rather try a squash prep), but can be used when grainy or viscous material is aspirated.
2. Fluid samples
Examples of fluid samples are pleural or peritoneal effusions, synovial and broncho-alveolar lavages. The main considerations with fluid samples are the preservation of cell morphology during transit to the laboratory (which should happen as soon as possible after collection) and preparing smears that are sufficiently cellular.
A fluid sample must be transferred to an EDTA tube straight after sampling, which will prevent coagulation and preserve cell morphology. A sufficient amount of fluid must be placed in the EDTA tube so that protein determination by means of a refractometer is not artificially elevated by the relatively large amount of very refractive EDTA.
EDTA should preserve cell morphology overnight. Refrigeration will prolong this time. However, even if fluid is placed in an EDTA tube and kept cool, major morphological changes due to cell ageing could occur within 24 hours. It is therefore important to also make fresh smears of the fluid: use the blood smear technique, allow the smears to air-dry and send to the lab unfixed and unstained, together with the fluid in EDTA. Do not place these smears in a refrigerator, as condensation will cause lysis of cells.
If the fluid is to be evaluated in-house, and not by a referral lab, the following is done:
– A direct smear from the uncentrifuged fluid (invert the tube a few times to mix first) should always be made in order to assess cellularity.
– After this, part of the well-mixed fluid can be centrifuged for five minutes at 1000–1500 rpm in a bench top centrifuge. This will result in cellular material forming a sediment or pellet at the bottom of the tube, covered by acellular fluid, the supernatant.
– The supernatant is decanted into a clear plastic tube, leaving the sediment in the original tube (as for urinalysis). A refractometer is used to determine the total protein concentration and specific gravity of the supernatant.
– The sediment is then aspirated and smeared on to a glass slide as described previously using the blood smear technique. If the sediment is thick/too cellular, use the squash technique.
Transparent fluids are usually low in cells and are always centrifuged after a direct smear has been prepared. The sediment can be smeared by means of the concentration line technique. This is very similar to the blood smear technique, except that the spreader slide is lifted directly upwards after advancing the material about two-thirds to three-quarters of the distance required to make a smear. The smear will have a line of concentrated cells at its end, but no feathered edge.
If fluid is visibly turbid (i.e. very cellular) often only a direct smear is sufficient. The sample can also be centrifuged and the sediment smeared. If sediment is very viscous, a squash preparation can be made.
When fluid is obtained from sampling a solid lesion, drain as much fluid from the lesion as possible and prepare as described above. If a solid tissue component remains, attempt a fine needle aspiration of the lesion, and prepare as previously described. Cystic neoplasia will not exfoliate overtly neoplastic cells into the fluid, and the two types of material/methods might reveal completely different cell populations.
Staining of cytological samples
It is generally not necessary to fix or stain slides before submission to a laboratory as cytologists prefer to fix and stain smears themselves, with stains and techniques that they will adapt to the type and thickness of the smear. Sometimes special stains are required. All that is therefore required from private practitioners is to air dry the smear, and to identify and package them properly before submitting it to a laboratory. Submit the sample together with the patient history and identification and owner detail. Smears must be stained within a week after making them. Never submit slides for cytology in the same package as samples in formalin. Formalin fumes diffuse even from tightly closed containers and influence the staining properties of the cytology slides.
If the slides are to be examined in-house, they should be air-dried and can be stained with a modified Romanowsky stain like Diff-Quik.
The following measures will ensure good staining quality:
• Use only new clean slides. Samples will not spread out well on re-used slides even if they are cleaned and dried, as the surface properties of the glass will have been changed.
• Use fresh, well filtered stains to avoid precipitates and contamination with organisms or cell debris.
• Stain only completely air-dried slide, or dry slide quickly with a hair dryer.
The most common problem encountered is under-staining of slides. This will render cells and nuclei pink (while nuclei should be dark purple, with a clear demarcation of nucleus and cytoplasm). Neutrophils on the slide can be used as an internal control for staining quality. If understained, place the slide in the stains for an additional time period. If immersion oil has already been placed on the slide, wipe this off gently and place the slide in the fixative solution for a minute. This will remove the oil and also most of the stain, and the slide will need to be restained.
Identify slides by marking with pencil on the frosted end of the slide. Do not use paper labels to identify the slide – the staining process will damage/dirty paper labels or wash ink off.
• Hypertrophy: An increase in cellular size and/or functional activity in response to a stimulus.
• Hyperplasia: An increase in cellular numbers in response to a stimulus. Increased mitotic activity is a common finding. This is a reversible change. Example – generalised reactive hyperplasia of lymph nodes seen with chronic canine ehrlichiosis.
• Neoplasia: An increase in cellular growth and multiplication that is not dependent on a stimulus external to the neoplastic tissue.
• Metaplasia: A process where one mature cell type is replaced by another mature cell type. This change is seen as an adaptive replacement that is reversible e.g. chronic rhinitis.
• Dysplasia: A reversible, irregular, atypical, proliferative cellular change in response to irritation or inflammation. Can be confused with malignant changes.
• Anaplasia: A lack of differentiation of tissue cells. Usually an indicator of malignant potential.
• Dyscrasia: An increase/decrease in the numbers of one or more cell components/maturational stages of a tissue out of proportion to other cell components/maturational stages.
Additional material from the internet
1. Refer to IVD 300 Clinical Pathology notes for information regarding sample dispatch to laboratory. This video will remind you of the general principles: https://www.youtube.com/watch?v=JwcsQcQshBs&list=PLNjV05pK4JEVLl3x8_mLYXxpjfMV-Gq4P&index=9
2. This video shows the stab (“woodpecker”) and aspiration (“syringe attached”) techniques for sample collection, as well as how to make a squash preparation. https://www.youtube.com/watch?v=JTYJBNxeTH8
3. This video which shows how impression smears are made from biopsy samples: https://www.youtube.com/watch?v=hey21Z459X0&t=2s
4. This video shows hoe smear from swabs are made. https://www.youtube.com/ h?v=GsLFO8zodKk&index=3&list=PLNjV05pK4JEVLl3x8_mLYXxpjfMV-Gq4P
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