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Cytology cases Kathleen Tennant BVetMed Cert SAM Cert VC FRCPath MRCVS Clinical Lead Diagnostic Laboratories Langford Veterinary Services Aims • • • • Case based Mixture of levels Some recurring problems Practice ‘screening’ slides First, the set-up • A major reason many people won’t screen their cytology is because they can’t see down the microscope properly.... Which of these would apply to you? 1. Microscope’s always covered in oil 2. Forgotten what I’m supposed to do with the condenser 3. Our Diff-Quik’s scummy and won’t stain right 4. All of the above 5. None of the above Quick fixes Microscope’s always covered in oil Clean it! Ethanol, methanol, not acetone. You can take the lenses off to make it easier but be careful not to get anything inside it... Forgotten what I’m supposed to do with the condenser Up – just below the stage, if it will move, usually open Our Diff-Quik’s scummy and won’t stain right You can scrape the scum off the last one with filter paper – use small narrow upright jars so you are more willing to throw it away Which of the following can not be successfully used as immersion oil? 1. Liquid paraffin 2. A mix of two different commercial immersion oils 3. Perfume grade cedar oil 4. Coverslipping glue Immersion oils • You cannot mix different oil types – each has its own refractive index and a combination will be impossible – clean the oils off both the lens and the slide to fix it • Coverslipping glue works suprisingly well for approximately 20 seconds. Takes about 2 hours to mend the microscope (sorry, Nikon technician....) Which of these is not a ‘cytology sin’? 1. Putting thick samples in a slide holder before they are dry 2. Putting the slides in the same bag as the histopath sample 3. Staining a slide with Diff-quik for a look and then sending it to an external lab 4. Staining a slide with Diff-quik then not including it with the rest of the slides to go out Cytology ‘sins’ • Slides going into holders stay wet in the humid environment for a long time and this can affect morphology • Even a small amount of formalin affects the way the cells stain and drastically cuts down detail • External labs prefer to have all slides – the one that you have stained may be the only one with material on it Case 1 • 5 year old Great Dane from a rescue centre with cough, pyrexia and anorexia • Radiographs show widely distributed alveolar pattern • Tracheal wash taken as initially unresponsive to antibiotics • Gelatinous material retrieved – direct smears made Direct smear, 100x lens oil immersion – what is this? Is this/are these....? 1. 2. 3. 4. 5. 6. Bacteria Stain precipitate Necrosis Inflammation KY gel Mucus KY/ ultrasound gel • Pink to deeply purple staining, irregular flecked material • Stain precipitate looks very similar but (hopefully) should not be this thick Mucus Case 1 • Same dog, second sample (one reason it’s good to screen a sample before waking up – can hair dry them if in a hurry, pre- and post staining) Tracheal wash # 2 Is this... 1. 2. 3. 4. Normal Neutrophilic inflammation Septic Mast cell metastasis Normal tracheal wash • When screening your samples, consider what the normal cell population in that area should be • Airways have regular columnar epithelial cells – sometimes the cilia are visible • Goblet cells (mucus producing) look very similar to mast cells Tracheal wash # 2 Same dog, BAL left Closer up What’s your diagnosis? 1. 2. 3. 4. Fungal pneumonia Gram positive bacterial pneumonia Gram negative bacterial pneumonia Mixed bacterial pneumonia Same dog, BAL left, neutrophils Septic neutrophilic inflammation • The demonstration of organisms INSIDE neutrophils is needed to confirm sepsis • Use neutrophil size to compare organisms to – bacteria rarely > 1/20th neutrophil diameter • Don’t forget that Diff-Quik tells you nothing about the Gram characteristics of bacteria... • Neutrophils will often degenerate in the presence of bacterial toxins, or irritants such as pancreatic fluid, bile or urine Non – degenerate/ degenerate Aspirate from a nodule on a 2 y.o. DSH’s head What’s your diagnosis? 1. 2. 3. 4. 5. Fungal infection Gram positive bacterial infection Gram negative bacterial infection Mycobacterial infection Mast cell tumour Aspirate from a nodule on a 2 y.o. DSH’s head • The cell here is a macrophage • The white (non- staining) linear stripes within it are Mycobacteria • Macrophages are very good at ‘presenting’ the cause of some infections – keep an eye out for what’s inside them Peritoneal fluid from an ascitic dog • The gross appearance of the fluid is serosanguinous • The nucleated cell is a macrophage When did the bleeding occur? 1. Current/ iatrogenic 2. Minutes to hours ago 3. Last week Peritoneal fluid from an ascitic dog • There are no platelets present, which makes current/ iatrogenic bleeding less likely (not impossible) • The macrophage has had time to phagocytose a red cell – minutes to hours • Might expect a week old bleed to have cleared, or macrophages to contain haem pigment breakdown products Recent haemorrhage - cytology Long standing haemorrhage - cytology Longer standing haemorrhage Tracheal wash from a 7 y.o. Coughing Yorkshire Terrier Is this... 1. Normal 2. Squamous cell carcinoma 3. Oropharyngeal contamination and neutrophilic inflammation 4. Septic neutrophilic inflammation Oropharyngeal contamination • Oropharynx contains nucleated squamous cells • The ‘striped’ organisms are Simonsiella, which are particular to the oropharynx • Neutrophilic inflammation also present – but interpreting any culture results will be difficult Pollen and mucus • Other ‘interlopers’ in airway washes include plant, food and pollen material • Anything green on a stained slide has to be self pigmented and is often of plant origin Nasal flush from a 3y.o. GSD Is this... 1. 2. 3. 4. Bacterial infection Yeast/fungal infection Plant contamination Nematode infection Yeast infection • Yeast are generally larger than bacteria and often have a nonstaining capsule • Look for septae • Aspergillus usually large and may have long hyphae Aspergillus Yeast from laboratory cactus! BAL from a 4y.o. Coughing cat Similar BAL from a coughing dog Is this... 1. 2. 3. 4. Eosinophilic inflammation Basophilic inflammation Neutrophilic inflammation Normal Eosinophils • Round granules in dogs, elongated granules (more difficult to see) in cats • Look for potential causes • Pulmonary infiltrate with eosinophils in dogs, ‘asthma’ in cats Angiostrongylus • Note the size of the organism versus the dots in the background, which are neutrophils • Neutrophilic inflammation can be associated with this as much as eosinophils