clinical

• Cytology allows evaluation of cellular details but

  -          Limitation of a small number of cells and relative loss of architecture.

  -          An easy technique, which requires simple equipment

  -          Minimally invasive.

  ð  therefore less expensive and has a fast turnaround time.
• Cytology gives valuable information, but it is not always possible to make a definitive diagnosis on cytological smears alone.

  -          Histology provides the advantage of tissue architecture and margins,

  -          Allows re-cuts for special stains or immunohistochemistry.

  However histology is more expensive, is more invasive and the animal needs to be under anaesthesia, requires specialized equipment, and has a longer turnaround time.
• Fine needle aspirate (FNA) – suitable for cutaneous masses, internal masses and organs. A needle is inserted into the lesion and then either a suction technique (suction applied as needle redirected several times within the lesion, suction is released then the needle is removed) or non-suction technique (insert the needle to the lesion, redirect then remove)

  -          Non suction techniques cause less cell damage and haemorrhage so are ideal for delicate tissues but the number of cells obtained may be less
• methods available for making cytology smears:

  Fine needle aspirate

  Swabs – suitable for tissues which aren’t easy to reach such as the vagina. Use a moistened cotton swab ROLL down a slide (not swipe)

  Impression smears – external lesions and excised tissues. Yield fewer cells than scrapings and there is a risk of only detecting surface inflammation. Tissues must be fresh NOT FIXED

  Scrapings – suitable for the same samples as impression smears but collect more cells. A scalpel blade, blunt spatula or brush can be used. Cells are smeared onto a slide then the squash technique is used
• In cytology Fluids are first assessed for their gross appearance.

  -          Body fluids such as body cavity effusions, CSF and joint fluid can be assessed for protein and total cell count before cytology.

  -          Cystic fluids and samples obtained using saline (e.g. BAL/TTW where saline is injected then re-extracted, traumatic catheterization and prostate wash) can be sent for normal cytology
• fluid sample preparation Technique used depends of cellularity and viscosity of the sample plus the presence of particulate material.

  You can make direct smears (which will also be useful to estimate cellularity) and you can concentrate samples by spinning them, removing the supernatant, and re suspending. Include blood smear technique, line smear technique and squash technique
• ·       Blood smear technique

  -          A drop of sample is placed at one end of the slide. A spreader slide is placed in front of the drop at a 45 degree angle, slid back to make contact then slid smoothly forward to form a ruffled edge
• ·       Line smear technique

  -          Similar to blood smear technique except a line of concentrated sample is made instead of a ruffled edge by lifting the spreader slide straight up after spreading. This works best for low cellular samples.
• ·       Squash technique

  Similarly to in an FNA, this is best used for viscous samples such as joint fluid, flocculent (puffy) material or aggregates. Press sample between two slides and submit the clearest one.
• Slides must be labelled with the patient identity and source of material IN PENCIL. The details of what has been sampled and clinical details should also be written on the submission form.

  The main stains used in practice are diff-quik or rapi-diff for general cytology and haematology. They do not stain all cells well, particularly mast cells. Laboratories use their own stains.
• How to evaluate cytology smears:

  1.      Examine with the naked eye for location and quantity of material

  2.      Use low power to look over the smear for large structures such as particulate artefacts, large clusters of cells and parasites

  3.      Move to a higher power to identify areas of interest and other structures

  4.      Drop oil onto the slide and move to oil immersion lens x100.

  Look at all areas of the smear! When examining cytological preparations, try to determine:

  • The type of cell(s) present

  • The nature of the process(es) present

  • The degree of abnormality
• cytological features of inflammation may be:

  -          Neutrophilic: associated with bacteria (especially if neutrophils appear degenerate), autoimmune disorders and acute inflammation

  -          Macrophages: generally associated with chronic inflammation, foreign bodies, fungal or protozoal infections.

  -          Lymphocytes and plasma cells, together with macrophages, can be associated with a more chronic inflammation or viral infections.

  Eosinophilic inflammation are associated with hypersensitivity, parasites, and neoplastic conditions such as MCT.
• On cytology, in response to tissue injury you may see Haemorrhage, fluid, fibrosis, necrotic material and inflammation. There are likely to be macrophages, possibly with dark haemosiderin granules or erythrophagocytosis. Plump elongated mesenchymal cells (fibroblasts) are expected as they form fibrous scar tissue.

  Necrotic material create a blue/ grey smudgey background  
• Upon cytology a cyst will contain fluid and FEW cells. The fluid is usually low in protein and often with reactive macrophages predominating.  There may be haemorrhage with RBCs, erythrophagocytosis (engulfed RBCs in the cytoplasm of macrophages) and blue/black haemosiderin granules (processed haemoglobin from previously phagocytosed RBCs) in macrophage cytoplasm.
• Epithelial cells will appear as cohesive groups of cells which exfoliate well. May also exfoliate as papillary structures, rows and occasionally acinar structures. May appear round, oval, columnar or polygonal with defined cell borders.
• Mesenchymal cells are mainly connective tissue cells e.g. fibroblasts, osteoblasts. Cells do not exfoliate well so cellular samples are often low and individualised. Cytoplasmic borders are indistinct

  -          Perivascular appearance: cells accumulate around a vascular structure

  -          Storiform: intersecting streams of cells with some stroma e.g. sarcomas
• Round cells have high cellularity but exfoliate individually. Have defined cell borders. Lymphocytes, transmissible venereal tumour (TVT), mast cells, plasma cells, histiocytes (remember acronym L.Y.M.P.H.)
• On cytology benign neoplasia may resemble the normal cells of that type, or appear mildly pleomorphic (different shape)

  Malignant neoplasia may have anisocytosis (different cell size but nucleus consistent), anisokaryosis (enlarged nucleus), display immature features/ abnormal nucleoli, be multinucleate, have abnormal mitosis or demonstrate nuclear moulding
• EDTA (Ethylenediamine tetra acetic acid) tubes must be used for haemotology which preserves call morphology as it does not cause clotting.

  We do not use heparin except for some reptile and bird species as it activates anticoagulation processes in the blood.
• erythrocyte parameters can be interpreted by Numerical data

  -          microhaematocrit a.k.a. PCV; buffy coat thickness, check plasma colour, total solids using refractometer

  -          haematology analyser; haematocrit, total RBC count, Hg concentration, mean cell volume, mean cell haemoglobin concentration (MCHC), reticulocyte count (demonstrates regeneration capacity.
• erythrocyte parameters can be determined by Smear examinations

  -          The terms anisocytosis (variation in size) and poikilocytosis (variation in shape) are useful for describing RBC morphology
• An increase in RBCs is erythrocytosis. May be due to hypoxia and some tumours, or can be physiologic e.g. in racehorses and sighthounds

   

  A decrease in RBCs is anaemia. May be regenerative or non-regenerative
• Regenerative anaemia: may be haemorrhagic (loss of RBCs) e.g. due to flea anaemia., or haemolytic where RBCs are lysed.

   

  Non-regenerative anaemia: Where there is no evidence of regeneration i.e. no reticulocytes seen. May be pre-regenerative as it takes 3-5 days for reticulocytes to appear in circulation. May be a secondary response to inflammatory disease (common but mild), chronic renal disease, iron deficiency or bone marrow disease.

   
• Reticulocytes are immature RBCs in their final step before maturity. They don’t have nuclei but they have clumps of cytoplasmic RNA, seen when stained with methylene blue. Those with a lot of reticulin can be seen in normal blood films as slightly purple polychromatophils.

  In dogs and cats they appear in increased numbers in regenerative anaemia; in large animals it is rare to see them in the circulation, even if the anaemia is regenerative.
• Erythrocyte maturation:

  Rubriblast -> prorubricyte -> early rubricyte -> late rubricyte -> metarubricyte (final stage with a nucleus) -> polychromatophils ->  erythrocyte
• mean corpuscular volume is the average volume of erythrocytes present. Based on a normal reference interval, cells can be:

  Normocytic: size is within reference interval for the species

  Macrocytic: size is increased. This could be due to regeneration (younger RBCs are larger than mature ones), genetic characteristics, FeLV, or artefacts

  Microcytic: size is decreased. Can be due to iron deficiency, a portosystemic shunt or liver disease, age, genetic characteristics or marked fragmentation.
• Mean corpuscular haemoglobin concentration is the average concentration of RBC haemoglobin on a weight per volume basis.

  Calculated by (Hb/haematocrit) x100.

  RBCs can either be normochromic or hypochromic (poor Hb content)

  Causes of hypochromia include regenerative response (young erythrocytes have a slightly lower Hb content) and iron deficiency.

  Increased MCHC is always the result of artefact (e.g. Heinz bodies, haemolysis, lipaemia, methaemoglobinemia) as there is no physiologic mechanism for forming too much Hb in a RBC.
• • Regenerative anaemia – macrocytic hypochromic (small animals) (big pale cells)

  • Non-regenerative anaemia – normocytic normochromic (normal size, normal colour)

  • Iron deficiency anaemia – microcytic hypochromic (small, pale cells)
• Rouleaux: ‘coin stack’ of erythrocytes. Can be normal at low levels in cats, horses and pigs but in other species, negative charges prevent stacking. Hyperproteinaemia from inflammation or a tumour can cause rouleaux.

  Agglutination: immune mediated clumping of erythrocytes due to anti-erythrocyte antibodies.

   

  Can be distinguished from eachother by performing a saline distribution test, rouleaux will disperse, agglutination won’t
• Echinocytes are Crenated RBCs with EVENLY arranged sharp spikes around the periphery. Usually an artefact due to slow drying of the slide. Very common
• Poikilocytes are abnormally shaped RBCs
• Acanthocyte: Uneven, rounded spikes or irregular fragments caused by damage to RBCs in the circulation due to turbular blood flow, DIC, hemangiosarcoma and abnormalities of lipid metabolism
• Heinz bodies: small ‘buttons’ attached to RBCs which indicate oxidative damage. Can be present in low numbers in healthy cats. Oxidised Hb doesn’t carry oxygen so can lead to hypoxia.
• Howell-Jolly bodies: Nuclear remnants of RBCs which may increase due to RBC turnover, defective production or defective splenic removal
• Codocytes: aka target cells. Seen with regeneration, lipid imbalances or liver disease
• Spherocyte: small, round densely staining RBCs without central pallor.  seen in immune-mediated haemolytic anaemia where macrophages break down the membrane, making them globular.
• how to interpret leukocyte parameters:

  Examination includes total WBC count and a differential count on a blood smear (both are essential)

  • In dogs and cats, use differentials in terms of absolute figures (% of each WBC type x total WBC count) rather than percentages. This is because they have a large storage pool

  • In horses, ruminants and rabbits, significant changes in percentage can occur without a significant increase in the total count, so examine percentage and absolute figures. This is because they have a small storage pool.
• Neutrophilia occurs in inflammation but also in

  -          Excitement (catecholamine mediated stress) with a splenic squeeze where the marginated pool joins the circulating pool

  -          corticosteroid mediated reactions (chronic stress and Cushing’s)

  -          paraneoplastic conditions, leukaemia and inherited neutrophil disfunctions
• Neutropenia occurs in overwhelming infections, endotoxemia and decreased bone marrow production. There may also be immunde-mediated destruction
• toxic changes to neutrophils may appear as:

  Dohle bodies- blue/grey inclusions in the cytoplasm

  Cytoplasmic basophilia- blue cytoplasm

  Vacuolation- foamy cytoplasm

  Granulation

   

  The nucleus may also appear doughnut shaped