Proudly Presents: CLINICAL PATHOLOGY

Transcription

1 Chicago Veterinary Medical Association Shaping the Future of Veterinary Medicine - Promoting the Human-Animal Bond Proudly Presents: CLINICAL PATHOLOGY With: ANNE BARGER DVM, MS, DACVP April 13, 2016

2 Chicago Veterinary Medical Association April 13, : OBTAINING HIGH QUALITY SAMPLES FOR CYTOLOGY Anne Barger, DVM, MS, DACVP University of Illinois, College of Veterinary Medicine, Urbana, IL Cytology is a critical diagnostic tool for general practice. It is easy, safe, fast, relatively inexpensive, minimally invasive and is often diagnostic. Cytology can be used to evaluate lymph nodes and masses (external or internal), and also fluids such as effusions and discharges. Results may change further diagnostic plans, treatment recommendations, or what a client chooses to do. Sample collection and preparation for cytology is simple and requires little equipment. Needles (22, 20, or 19 gauge), 6 cc syringes (although 3 cc syringes can be used for lymph nodes), clean glass slides, and occasionally a scalpel blade for scraping are all that is needed. There is no one way to obtain a sample. The most successful technique is the technique the user is most comfortable performing. This presentation will focus on the fenestration technique. The fenestration technique can be performed with a needle alone or with a syringe attached to the needle. Before obtaining the sample, make sure the slides are set out and the syringe is ready. For a single aspirate, 3-6 slides should be used. This will allow for in-house evaluation while still having the ability to send unstained slides out for evaluation. Stabilize the mass in your non-dominant hand. For the needle alone technique, direct the needle into the mass; withdraw (yet stay within skin) and push back into the mass several times rapidly. Draw several cc air into the syringe, connect it to needle and expel needle contents onto a slide (make sure the bevel is down and the contents come out near the frosted end). For the needle plus syringe procedure, connect the needle and syringe, direct the needle into the mass, draw back plunger and release several times, with pressure released withdraw the needle from mass, disconnect the needle and draw several cc air into syringe, reconnect and expel needle contents onto the slide. Slide preparation is critical for good cytology results. The samples must be smeared out quickly to prevent clotting and need to be in a thin layer for best cellular evaluation. Even if only a small dot of sample is expelled onto the slide, smearing it is important. Cells that dry in a little round droplet of tissue are often balled up and crowded and therefore cannot be appropriately evaluated. After the contents are expelled onto a slide, place a second slide onto the first cross-wise to the first slide and pick up a small amount of material. Pick up a third slide, gently place the second slide across the third slide and smear the sample by pulling the second slide away from the frosted edge of the third slide. The second slide needs to stay flat on the third slide and must be pulled gently. Now, the second slide can be used again to pick up another small bit of sample from slide 1, and can be smeared onto a 4 th slide, etc. When just a small amount of sample remains on slide 1, use slide 2 to smear it as well. Most aspirates will yield 3-6 good slides, allowing for in-house staining and evaluation of one slide while leaving several unstained for laboratory evaluation. Also, by making multiple smears, the samples are more thinly spread out which improves diagnostic ability. This is particularly helpful with very cellular aspirates. Clinical Pathology Anne Barger DVM, MS, DACVP Part 1: Page 1 of 9

3 Chicago Veterinary Medical Association April 13, 2016 Getting Started with Cytology: Techniques & Cells Anne Barger, DVM, MS, DACVP University of Illinois Clinical Pathology Cytology Techniques & Cells Part 1: Page 2 of 9

4 Chicago Veterinary Medical Association April 13, 2016 Tools of the Trade Needle Size will vary depending on the tissue aspirated Syringe 3 or 6 cc usually adequate for most samples Glass slides Frosted edge ideal for easy labeling Markers that can write on glass Sharpie s wash off!! Technique Classic fine needle aspiration Needle attached to syringe Fenestration Technique Needle attached to syringe or not Clinical Pathology Cytology Techniques & Cells Part 1: Page 3 of 9

5 Chicago Veterinary Medical Association April 13, 2016 Clinical Pathology Cytology Techniques & Cells Part 1: Page 4 of 9

6 Chicago Veterinary Medical Association April 13, 2016 Assistance from imaging Slide preparation Very important to smear the slide Especially with cellular samples, the cells need to spread out to be appropriately evaluated Slide Preparation Direct smear Similar to a blood smear Slide Preparation Squash preparation Clinical Pathology Cytology Techniques & Cells Part 1: Page 5 of 9

7 Chicago Veterinary Medical Association April 13, 2016 Clinical Pathology Cytology Techniques & Cells Part 1: Page 6 of 9

8 Chicago Veterinary Medical Association April 13, 2016 What to do with the Sample If Sending it Out, Package it Well Label it! Clinical Pathology Cytology Techniques & Cells Part 1: Page 7 of 9

9 Chicago Veterinary Medical Association April 13, 2016 Staining NMB As soon as the material is dry, the sample is ready to stain Dif Quik type stains 7 dips methanol 7 dips solution 1 15 dips solution 2 New Methylene Blue (NMB) Fast and easy but not permanent Understained sample (Diff Quik) Appropriately Stained Sample Clinical Pathology Cytology Techniques & Cells Part 1: Page 8 of 9

10 Chicago Veterinary Medical Association April 13, 2016 Mast cell tumors Limitations Stain consistently with Giemsa stains Most laboratories uses Wright Giemsa Save an unstained slide if you suspect mast cell neoplasia. Advantages Limitations Advantages and Disadvantages of Different Stains Romanowsky Papanicolaou NMB Excellent cytoplasmic detail Adequate staining of background material Easy to use Performed on air dried slides Easy identification of microorganisms Staining of cells challenging in thick samples Nuclear detail is minimal Excellent nuclear detail Useful in staining cells in thick preparations Wet fixation or rehydration of samples required Not useful for evaluation of microorganisms or background material Consistent staining of mast cell granules Nuclear detail is adequate Easy to use Use of stain on air dried smears for cytology is not permanent Conclusion Obtaining a good sample is the most critical component of the process A good sample can always be sent out or reviewed by a second pathologist Clinical Pathology Cytology Techniques & Cells Part 1: Page 9 of 9

11 Chicago Veterinary Medical Association April 13, : INTRODUCTION TO CYTOLOGY, A CASE-BASED APPROACH Anne Barger, DVM, MS, DACVP University of Illinois, College of Veterinary Medicine, Urbana, IL Cytology is the microscopic examination of cells. Specimens are obtained from masses, enlarged organs, fluidfilled cavities, urine, vaginal swabs, ear and fecal swabs among others. Probably the most common technique for obtaining a sample is fine needle aspiration (FNA). Cytology is a critical diagnostic tool for general practice. It is easy, safe, fast, relatively inexpensive, minimally invasive and is often diagnostic. Cytology can be used to evaluate lymph nodes and masses (external or internal), and also fluids such as effusions and discharges. Results may change further diagnostic plans, treatment recommendations, or what a client chooses to do. Sample collection and preparation for cytology is simple and requires little equipment. Needles (22, 20, or 19 gauge), 6 cc syringes (although 3 cc syringes can be used for lymph nodes), clean glass slides, and occasionally a scalpel blade for scraping are all that is needed. There is no one way to obtain a sample. The most successful technique is the technique the user is most comfortable performing. This presentation will focus on the fenestration technique. The fenestration technique can be performed with a needle alone or with a syringe attached to the needle. Before obtaining the sample, make sure the slides are set out and the syringe is ready. For a single aspirate, 3-6 slides should be used. This will allow for in-house evaluation while still having the ability to send unstained slides out for evaluation. Stabilize the mass in your non-dominant hand. For the needle alone technique, direct the needle into the mass; withdraw (yet stay within skin) and push back into the mass several times rapidly. Draw several cc air into the syringe, connect it to needle and expel needle contents onto a slide (make sure the bevel is down and the contents come out near the frosted end). For the needle plus syringe procedure, connect the needle and syringe, direct the needle into the mass, draw back plunger and release several times, with pressure released withdraw the needle from mass, disconnect the needle and draw several cc air into syringe, reconnect and expel needle contents onto the slide. Slide preparation is critical for good cytology results. The samples must be smeared out quickly to prevent clotting and need to be in a thin layer for best cellular evaluation. Even if only a small dot of sample is expelled onto the slide, smearing it is important. Cells that dry in a little round droplet of tissue are often balled up and crowded and therefore cannot be appropriately evaluated. After the contents are expelled onto a slide, place a second slide onto the first cross-wise to the first slide and pick up a small amount of material. Pick up a third slide, gently place the second slide across the third slide and smear the sample by pulling the second slide away from the frosted edge of the third slide. The second slide needs to stay flat on the third slide and must be pulled gently. Now, the second slide can be used again to pick up another small bit of sample from slide 1, and can be smeared onto a 4 th slide, etc. When just a small amount of sample remains on slide 1, use slide 2 to smear it as well. Most aspirates will yield 3-6 good slides, allowing for in-house staining and evaluation of one slide while leaving several unstained for laboratory evaluation. Also, by making multiple smears, the samples are more thinly spread out which improves diagnostic ability. This is particularly helpful with very cellular aspirates. Once the slide is stained it is ready for microscopic examination. Scan the slide on low power (10x) first to get an overall feel for the cellularity of the sample and then go to high power to evaluate the cell types and individual cytologic features of the cells. Inflammatory processes usually have a mixed population of cells including inflammatory and tissue cells. The inflammatory process is characterized by the cell types present. When neutrophils predominate (>85%), this is considered a suppurative response. The character of the neutrophils Clinical Pathology Anne Barger DVM, MS, DACVP Part 2: Page 1 of 17

12 Chicago Veterinary Medical Association April 13, 2016 should be closely evaluated. If there is a bacterial infection, the neutrophils will often undergo karyolysis and the nucleus will appear pale and swollen. These cells are often called degenerative neutrophils. Also the cells should be closely examined for intracytoplasmic microorganisms. A pyogranulomatous response consists of a mixture of neutrophils, epitheliod macrophages and multinucleated giant cells. This type of inflammatory response is commonly observed with fungal infections or responses to foreign material. Granulomatous inflammation consists primarily of macrophages with multinucleated giant cells and lesser numbers of other inflammatory cells. This type of inflammatory response does not always exfoliate well and may be of low cellularity. Eosinophilic inflammation is more subtle. Cytologic specimens containing greater than 12% eosinophils are considered eosinophilic inflammatory lesions. Common causes include allergic or hypersensitivity response, parasitic infestation or paraneoplastic response. Tissue cells are classified as epithelial, mesenchymal (connective tissue) or round cells. Epithelial cells are cohesive and are often observed in tight clusters. This feature can be observed on low power evaluation. The cells are generally round and the sample is often highly cellular. Mesenchymal cells do not exfoliate nearly as well as epithelial tissue. In benign lesions, it is not uncommon to see only 1-2 cells on a slide. The cells are commonly spindle shaped with wispy cytoplasmic borders. The nuclei are often oval in shape. Round cells are a specific group of neoplasms, which will be discussed in greater detail in the section on skin and subcutaneous tissue. The cells, themselves, exfoliate readily, resulting in highly cellular samples. The cells are round with a scant to moderate rim of basophilic cytoplasm. They are not cohesive but can occur in sheets which may be difficult to distinguish from epithelial cells. Once the cell type has been characterized, the next step is to determine if the process is hyperplastic or neoplastic and if it is neoplastic, is it benign or malignant. It is important to evaluate the cells for criteria of malignancy. These criteria include: prominent and multiple nucleoli, anisocytosis, anisokaryosis, multiple nuclei, mitotic figures and nuclear molding. There are others, but these are the most common. When these criteria are present it is fairly easy to identify a malignant tumor, however not all tumors are malignant and not all malignancies express these criteria. Therefore, on cytology we cannot always rule out a malignant process. The final step in cytologic evaluation is examination of the material in the background. Proteinaceous material is eosinophilic and granular and is usually observed evenly distributed throughout the sample. Granules from ruptured mast cells or melanocytes must be distinguished from bacteria. Most cytologic samples contain some level of peripheral blood contamination. With the red blood cells, come white blood cells. It is important to remember this when trying to distinguish inflammation from peripheral blood contamination. It may be necessary to do a CBC on the patient to determine the WBC. Skin and subcutaneous tissue Normal skin is predominated by mature, anucleated, keratinized squamous epithelial cells with lesser numbers of glandular and basal epithelial cells and few stromal connective tissue cells. The subcutaneous tissue is primarily composed of mature adipose tissue. Blood is almost always observed in cytology specimens. It is important to understand what cells are present in normal skin before trying to interpret abnormal tissue. Skin lesions may present as a mass, alopecic or ulcerated lesion. Subcutaneous lesions usually present as a mass. Cytology may only lead to a diagnosis of inflammation or neoplasia however there are many lesions of skin and subcutaneous tissues that have unique features, allowing for definitive diagnosis with cytology. Clinical Pathology Anne Barger DVM, MS, DACVP Part 2: Page 2 of 17

13 Chicago Veterinary Medical Association April 13, 2016 Dermal lesions (non-neoplastic and non-inflammatory) These consist primarily of cysts and hyperplastic tissue. Epidermal inclusion cysts are common masses on older dogs. Cytologically they consist of mature squamous epithelial cells and thick aggregates of keratinaceous debris. Occasionally cholesterol crystals are identified. If these cysts rupture, they elicit a tremendous inflammatory response, suppurative to pyogranulomatous. The inflammatory cells are observed surrounding the aggregates of keratinaceous material. Apocrine cysts are cytologically unremarkable. They are low cellular samples are more of a gross diagnosis confirmed with cytology. Sebaceous cysts and sebaceous hyperplasia consist of clusters of sebaceous epithelial cells. These cells are cohesive and consist of a moderate rim of cytoplasm filled with distinct cytoplasmic vacuoles. Cytologically it is difficult to differentiate sebaceous hyperplasia from adenoma. Inflammatory lesions Suppurative inflammation of the skin and subcutaneous tissue is common. Immune-mediated diseases such as pemphigus and nodular panniculitis are suppurative. The lesions are sterile and the neutrophils appear nondegenerative. Bacterial infections of the skin and subcutaneous lesions usually result in suppurative inflammation. Intracellular bacteria are fairly easy to find and the neutrophils are often degenerative. Pyogranulomatous inflammation is often caused by fungal organisms though sterile pyogranulomatous inflammation does occur. Dimorphic fungal organisms such as Blastomyces, Cryptococcus, Histoplasma and Coccidiodomycosis are more likely to cause pyogranulomatous inflammation. Granulomatous inflammation of the skin is fairly uncommon but can be observed with atypical bacterial infections such as Mycobacteria. These organisms are negative staining rods with most of the common cytologic stains. Neoplasia There are many more tumors than will be discussed in this hand-out but the most common ones will be discussed. Common epithelial tumors of the skin include squamous cell carcinoma, basal cell tumors, hair follicle tumors, sebaceous adenomas, perianal gland tumors and apocrine anal sac adenocarcinomas. Individual features of each tumor type will be discussed in more detail. It is difficult on cytology to distinguish the different mesenchymal tumors. Often a diagnosis of sarcoma or benign mesenchymal tumor is made based on the morphology of the cells. Histopathology, commonly with immunohistochemical staining, is necessary to make a more specific diagnosis. Lipomas are a mesenchymal tumor and cytologically they appear consistent with mature adipose tissue and are difficult to differentiate from underlying subcutaneous fat. Histopathology or a history of a fluctuant mass is helpful in making a definitive diagnosis. Melanomas may be characterized as mesenchymal or round cell tumors. The majority of melanomas are benign however from certain locations, such as the mouth or nail bed, malignancy is likely. Benign melanomas contain so much pigment in their cytoplasm, it is difficult to examine specific cytologic features. Malignant melanomas often have little to no pigment making it easy to identify the criteria of malignancy. Other round cell tumors include lymphoma, mast cell tumor, plasma cell tumor, histiocytoma and transmissible venereal tumors (TVT). Each of these cell types has distinct features making it fairly easy to differentiate each tumor. Mast cell tumors contain red to purple granules in their cytoplasm, TVTs have multiple, discrete, cytoplasmic vacuoles, histiocytomas have peripheral clearing of their cytoplasm whereas plasma cells have a perinuclear clearing of their cytoplasm. Occasionally, histiocytomas and plasma cell tumors look very similar and the signalment of the patient becomes very important. Histiocytomas are generally a tumor of young dogs and will often resolve on their own. Plasma cell tumors occur in middle aged to older dogs and surgical excision is often curative. Clinical Pathology Anne Barger DVM, MS, DACVP Part 2: Page 3 of 17

14 3/3/2016 A Case-Based Approach to Cytology Anne Barger VDL University of Illinois Slide Examination Cellularity Highly cellular vs. poorly cellular Can be beneficial with diagnosis Monomorphic vs. pleomorphic cell population Cellular components Inflammation vs. Neoplasia Neoplasia vs. Hyperplasia Background Blood Protein Microoroganisms Broken cells Low power evaluation Cellularity First step to evaluation of cytology Highly cellular Suppurative inflammation Epithelial and round cell neoplasms Low cellular Granulomatous inflammation Mesenchymal neoplasia Part 2: Page 4 of

15 3/3/2016 Cellularity Cellularity Low Power Evaluation Monomorphic vs. Pleomorphic Cell population Monomorphic more likely to be neoplasia Pleomorphic more likely to be inflammation Part 2: Page 5 of

16 3/3/2016 High Power Evaluation Identify cell types Tissue cells vs. inflammatory cells Identify microorganisms Evaluate material within the background Part 2: Page 6 of

17 3/3/2016 Identification of Cell Types Primarily inflammatory cells Suppurative inflammation >85% neutrophils Condition of neutrophils Look for microorganisms Pyogranulomatous inflammation Neutrophils present with multinucleated giant cells and epitheliod macrophages Look for fungal organisms or evidence of foreign material Suppurative Inflammation Pyogranulomatous Inflammation Part 2: Page 7 of

18 3/3/2016 Inflammation Granulomatous inflammation Primarily macrophages with multinucleated giant cells Low numbers of neutrophils and small mature lymphocytes Eosinophilic inflammation >12% eosinophils Parasitic, allergic or paraneoplastic Eosinophilic granuloma Eosinophilic inflammation Identification of Cell Types Origin of cell type Epithelial: Squamous, glandular, nonglandular Mesenchymal: Connective tissue Round cells: Discrete cells Hyperplasia vs. Neoplasia Consider source of aspirate Look for criteria of malignancy Part 2: Page 8 of

19 3/3/2016 Criteria of Malignancy Anisocytosis Anisokaryosis Multinucleation Prominent and/or multiple nucleoli Variation in nuclear to cytoplasmic ratio (N:C) Nuclear molding Individual Cellular Characteristics Epithelial cells Usually in clusters Distinct cell borders Round to polygonal in shape Round nucleus Individual Cellular Characteristics Mesenchymal cells Individualized cells Spindiloid in shape Indistinct to wispy cell borders Oval nucleus Exfoliate poorly Part 2: Page 9 of

20 3/3/2016 Individual Cellular Characteristics Round cells Round in shape Discrete cells Occur individually or in a sheet Generally highly cellular Examination of the Background Proteinaceous material Mucin Osteoid Amyloid Granules Ruptured mast cells or melanocytes Blood Broken cells Background Part 2: Page 10 of

21 3/3/2016 Background Cytology Cases! Case #1 4 year old, male Great Dane History of large mass near anal sac Fine needle aspiration performed Part 2: Page 11 of

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23 3/3/2016 Case #2 7 month old, female spayed, mixed breed dog History of fever and diarrhea Generalized lymphadenopathy Mild anemia and severe thrombocytopenia (<25,000/ l) Aspiration of multiple lymph nodes and rectal scrape Part 2: Page 13 of

24 3/3/2016 Case 3 4 year old, neutered male, Yorkshire Terrier History of lethargy and fever of o F Multiple lymph nodes enlarged, increased lung sounds Aspiration of multiple lymph nodes Part 2: Page 14 of

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26 3/3/2016 Case #4 8 year old mixed breed dog History of lameness left front FNA performed based on radiographic evaluation ALP Activity Part 2: Page 16 of

27 3/3/2016 Case #5 8 year old, neutered male, Shepherd mix History of decreased appetite and sensitivity around his mouth Physical examination revealed large mass in the mucosa Part 2: Page 17 of

28 Chicago Veterinary Medical Association April 13, : CYTOLOGY OF COMMON CUTANEOUS MASSES FROM DOGS AND CATS Anne Barger, DVM, MS, Diplomate ACVP University of Illinois, College of Veterinary Medicine, Urbana, IL Cutaneous masses are probably the most common structures aspirated cytologically and generally arise from one of three processes; inflammation, neoplasia and cysts. Fine needle aspiration with cytology can assist in determining what should be done next to attain a more definitive diagnosis or for diagnosis. Inflammatory masses are very common and are categorized based on the type of inflammatory cells. Suppurative inflammation is predominated by neutrophils, pyogranulomatous consists of a mixture of neutrophils, epitheliod macrophages and multinucleated giant cells and granulomatous inflammation is heavily predominated by macrophages and multinucleated giant cells. In a suppurative inflammatory response, if the cause is a bacterial infection, the neutrophils will often be degenerate and intracellular bacteria may be identified. Pyogranulomatous inflammatory responses often occurs secondary to fungal infection or a foreign body response. Blastomyces dermatitidis commonly causes multiple masses or draining tracts and elicits a pyogranulomatous response. Cysts are epithelial lined, non-neoplastic structures that are commonly seen in older dogs. Follicular cysts are often seen as dermal, raised, fluctuant masses that are filled with keratinaceous debris. Cytologically, thick aggregates of keratinaceous material and many anucleated, mature squamous epithelial cells are observed. These masses are benign though if they rupture the free keratin can elicit a severe inflammatory response. The neutrophils and macrophages are often observed surrounding the keratinaceous material. Sebaceous masses, cysts, hyperplasia and adenomas can look very similar. Sebaceous cysts are less cellular than the other two and contain thick mats of nonstaining lipid based secretory material. Low numbers of sebaceous epithelial cells may also be observed. These cells are cohesive. They have a moderate amount of basophilic cytoplasm filled with discrete cytoplasmic vacuoles. The nuclei are small and round but are often obscured by the vacuoles. Sebaceous hyperplasia and sebaceous adenomas are cytologically indistinguishable. They are highly cellular and consist of multiple clusters of uniform sebaceous epithelial cells. Grossly, sebaceous adenomas appear as pedunculated wart-like masses whereas sebaceous hyperplasia may appear as a thickened area of skin. There are many cutaneous and subcutaneous neoplasms that are diagnosed in dogs and cats but the focus of this talk is to highlight the most common tumors. Round cell tumors commonly appear as red raised hairless dermal mass. This grouping of tumors includes mast cell tumor, plasma cell tumor, lymphoma, histiocytoma and transmissible venereal tumor. Of these tumors, mast cell tumors and histiocytomas are the most common. Mast cell tumors are more common in dogs than cats. Cytologically, they are cellular and consist of a population of round cells with a moderate amount of basophilic cytoplasm, filled with metachromatic (purple) granules. These granules do not stain consistently with Diff-Quik and special staining may be necessary to clearly visualize these granules. Eosinophils often accompany mast cell tumors in dogs but do so less commonly in cats. Welldifferentiated mast cell tumors in cats usually do not contain eosinophils. Histopathologic assessment is necessary for grading in dogs but grading has not proven useful in cats. In dogs the grading is important to attempt to predict the behavior of these tumors. Histiocytomas are unique to dogs. They are benign tumors that will often resolve on their own after several weeks. Grossly they appear as a round, red, raised mass. Cytologically this tumor consists of a population of Clinical Pathology Anne Barger DVM, MS, DACVP Part 3: Page 1 of 14

29 Chicago Veterinary Medical Association April 13, 2016 round cells with a moderate amount of basophilic cytoplasm and a centrally placed nucleus. The cytoplasm often has a clearing of the basophilia at the periphery. As the tumor begins to resolve, increasing numbers of small lymphocyte (cytotoxic T lymphocytes) can be observed, sometimes outnumbering the neoplastic cells. Lipomas are a benign mesenchymal tumor and probably the most common benign tumor in dogs. Generally they are of low cellularity. Large droplets of lipid are observed on the unstained slide and often wash away during staining. Adipocytes are large cells with abundant, pale staining cytoplasm packed with lipid and a small round nucleus. Benign spindle cells can also be observed in these samples. Lipomas, cytologically, are often indistinguishable from mature subcutaneous adipose tissue. Squamous cell carcinomas (SCC) are a frequently diagnosed epithelial tumor. This tumor is the most common head and neck tumor diagnosed in cats. Samples often contain a mixture of cell types because the neoplastic squamous cells are frequently accompanied by inflammatory cells. The presence of inflammation can make diagnosis challenging because epithelium can become dysplastic secondary to inflammation. The neoplastic squamous epithelial cells are arranged in clusters with varying amounts of basophilic, occasionally keratinized cytoplasm. The nuclei are large and round with a stippled chromatin pattern and prominent nucleoli. Occasionally cells are coated in neutrophils. Grossly the masses are often ulcerated which contributes to the inflammatory component of the tumor. In conclusion, there are many benign and malignant processes that can be successfully diagnosed with cytology. The veterinarian s clinical impression is critical in the appropriate interpretation of a cytologic specimen. Neoplastic processes are more likely to have a monomorphic population of cells, inflammatory responses are more likely to be pleomorphic and be painful to the patient. If there is ever any question about the cytologic interpretation, a pathologist should be contacted and/or a biopsy performed. Suggested Reading: Raskin RE, Meyer DJ. Canine and Feline Cytology, A Color Atlas and Interpretation Guide, 2 nd ed. Saunders Elsevier, St. Louis, MO Cowell RL, Tyler RD, Meinkoth JH and DeNicola DB. Diagnostic Cytology and Hematology of the Dog and Cat, 3 rd ed. Mosby Elsevier, St. Louis, MO Barger A. Common masses in dogs and cats. Clinician s brief 7;9:9-11, Cohen M, Bohling MW, Wright JC, et al. Evaluation of sensitivity and specificity of cytologic examination:269 cases ( ). J Am Vet Med Assoc 222: , Clinical Pathology Anne Barger DVM, MS, DACVP Part 3: Page 2 of 14

30 Chicago Veterinary Medical Association April 13, 2016 Cytology of Common Cutaneous Masses Anne Barger, DVM, MS, DACVP University of Illinois Clinical Pathology - Cytology of Cutaneous Masses Part 3: Page 3 of 14

31 Chicago Veterinary Medical Association April 13, 2016 Cutaneous Masses Lumps and bumps Dermal vs. Subcutaneous Impact diagnosis Differential list 2 Inflammation Lumps and Bumps Granuloma, pyogranuloma, abscess Neoplasia and cysts Benign vs. malignant Tissue type Epithelial Mesenchymal Round cell 3 Identification of Cell Types Identification of Cell Types Primarily inflammatory cells Suppurative inflammation >85% neutrophils Condition of neutrophils Look for microorganisms Pyogranulomatous inflammation Neutrophils present with multinucleated giant cells and epitheliod macrophages Look for fungal organisms or evidence of foreign material 4 Origin of cell type Epithelial: Squamous, glandular, nonglandular Mesenchymal: Connective tissue Round cells: Discrete cells Hyperplasia vs. Neoplasia Consider source of aspirate Look for criteria of malignancy 5 Clinical Pathology - Cytology of Cutaneous Masses Part 3: Page 4 of 14

32 Chicago Veterinary Medical Association April 13, 2016 Abscess Inflammatory masses Neutrophils Degenerate Bacteria, often intra and extracellular 6 7 True mass or draining tract Foreign body Fungus Sterile Pyogranuloma 8 9 Clinical Pathology - Cytology of Cutaneous Masses Part 3: Page 5 of 14

33 Chicago Veterinary Medical Association April 13, 2016 Cytology Granuloma Macrophages Multinucleated giant cells Few neutrophils, lymphocytes, plasma cells Atypical bacterial infections Fungus Epithelial lined structures Many different types Sebaceous Follicular Apocrine Cysts Clinical Pathology - Cytology of Cutaneous Masses Part 3: Page 6 of 14

34 Chicago Veterinary Medical Association April 13, 2016 Neoplasia Round cell tumors Histiocytoma Dermal mass Most common in dogs <3 years of age Benign neoplasm, will resolve on its own Cytologically Round cell population Pale basophilic cytoplasm Centrally placed nucleus Peripheral clearing of the cytoplasm Round cell tumors Mast cell tumor Can be dermal or subcutaneous Often observed at mucocutaneous junctions Highly cellular Round cells Moderate amount of cytoplasm Filled with metachromatic granules Clinical Pathology - Cytology of Cutaneous Masses Part 3: Page 7 of 14

35 Chicago Veterinary Medical Association April 13, Round Cell Tumors Plasma Cell Tumor Common on head, face and feet Dermal mass Cellular Cells are round with eccentrically placed nuclei and perinuclear clearing Binucleation is a common feature Round Cell Tumors Plasma Cell Tumor Common on head, face and feet Dermal mass Cellular Cells are round with eccentrically placed nuclei and perinuclear clearing Binucleation is a common feature Clinical Pathology - Cytology of Cutaneous Masses Part 3: Page 8 of 14

36 Chicago Veterinary Medical Association April 13, 2016 Epithelial Tumors Sebaceous adenoma or epithelioma Dermal mass Benign Clusters of vacuolated epithelial cells Small condensed nucleus Clinical Pathology - Cytology of Cutaneous Masses Part 3: Page 9 of 14

37 Chicago Veterinary Medical Association April 13, 2016 Epithelial Neoplasia Basal Cell Tumor Common head and neck tumor of cats Recategorized for dogs as trichoblastoma Cytology Clusters of uniform epithelial cells Small and round with scant rim of basophilic cytoplasm Condensed chromatin pattern Epithelial Neoplasia Squamous Cell Carcinoma Common locations Mouth Eyes Ears Mucocutaneous junctions Clinical Pathology - Cytology of Cutaneous Masses Part 3: Page 10 of 14

38 Chicago Veterinary Medical Association April 13, 2016 Epithelial Neoplasia Squamous cell carcinoma Cellular sample commonly with secondary suppurative inflammation Clusters of epithelial cells with some differentiation to mature squamous epithelial cells Perinuclear vacuolization Nuclei are large and round with prominent nucleoli Mesenchymal Neoplasia Lipoma Benign neoplasm More common in dogs than cats Can have multiple lipomas Cytologically Low cellularity Many only see few lipid droplets Aggregates of large adipocytes Clinical Pathology - Cytology of Cutaneous Masses Part 3: Page 11 of 14

39 Chicago Veterinary Medical Association April 13, Mesenchymal Neoplasia Soft tissue sarcoma Inclusive term Hemangiopericytoma Neurofibroma Schwannoma Fibrosarcoma Behavior Usually locally invasive though higher grade Grading is only done on histopathology so cytology for diagnosis only 35 Mesenchymal Neoplasia Soft Tissue Sarcoma Samples vary greatly in cellularity Cytologically they look fairly similar Spindle shaped cells Wispy cytoplasmic borders Clinical Pathology - Cytology of Cutaneous Masses Part 3: Page 12 of 14

40 Chicago Veterinary Medical Association April 13, Mesenchymal Neoplasia Feline Vaccine Associated Sarcoma Behavior Aggressive Fast growing Locally Invasive Tumor types Fibrosarcoma Histiocytic Sarcoma Osteosarcoma Chondrosarcoma Rhabdomyosarcoma 41 Clinical Pathology - Cytology of Cutaneous Masses Part 3: Page 13 of 14

41 Chicago Veterinary Medical Association April 13, 2016 Mesenchymal Neoplasia Vaccine Associated Sarcoma Cytology Usually low cellularity Cells exhibit obvious criteria of malignancy Spindle shaped cells May have extracellular matrix May have lymphoid infiltrate or secondary inflammation Conclusions Many diagnoses can be made with cytology Often the common masses have very distinct cytologic features that can be differentiated from one another If you ever have a question about a cytology, consult a clinical pathologist Clinical Pathology - Cytology of Cutaneous Masses Part 3: Page 14 of 14

42 Chicago Veterinary Medical Association April 13, : LYMPH NODE CYTOLOGY IS ACTUALLY USEFUL Anne Barger, DVM, MS, DACVP University of Illinois, College of Veterinary Medicine, Urbana, IL Lymph node aspiration is a fairly simple diagnostic tool. Enlarged peripheral lymph nodes can be easy to aspirate in a non-sedated patient. Lymph node drain various tissues of the body and cytology can assist in diagnosing issues in draining tissues as well as primary disease of the lymph node itself. Lymph node enlargement can occur secondary to inflammation (lymphadenitis), antigenic or immune stimulation (reactive lymph node) or neoplasia, either primary (lymphoma) or metastatic. Normal lymph node Normal cytology of lymphoid tissue consists of a mixed lymphoid population predominated by small lymphocytes (>90%) with lesser numbers of large and intermediate sized lymphocytes (<10%). Low numbers of other cell types such as neutrophils, macrophages, plasma cells, mast cells, eosinophils and melanocytes should represent <1% of the cell population. Low numbers of nonstaining lipid vacuoles, red blood cells and cytoplasmic blebs often called lymphoglandular bodies may be identified. Normal lymph node histology consists of cortex, paracortex and medulla. Within the cortex there are lymphoid follicles which are composed of B cells and the follicles are surrounded by T cells. The T lymphocyte area intermingles with the paracortex which also consists of antigen presenting cells or macrophages and dendritic cells. The medulla consists of medullary cords which can contain inflammatory or lymphoid cells and endothelial lined vessels where cells can enter the node. Lymph nodes are encased in a capsul. The cells of the capsul are rarely observed in cytologic specimens but should be visualized in histopathopathology sections. Reactive Lymph Node Antigenic stimulation often results in lymphadenomegaly and the lymph nodes may be painful. The cause is nonspecific, any antigen can elicit this response in a draining lymph node. Cytologically, the sample is predominated by small lymphocytes with increased numbers of plasma cells and large and intermediate sized lymphocytes. Quantification of plasma cells consistently can be challenging. If the sample is highly cellular with a full field of cells and within every 100x field >2-3 plasma cells are visualized, the lymph node is reactive. Additionally, highly activated plasma cells, termed Mott cells can also be identified. These cells contain small discrete cytoplasmic vacuoles within their cytoplasm. These vacuoles are called Russell bodies. Low numbers of melanocytes, neutrophils, macrophages and other inflammatory cells can be identified but should not exceed 1-2% of the nucleated cell population in a purely reactive lymph node. Lymphadenitis Inflammation of the lymph node is characterized based on the cell types observed. A mixed lymphoid population is observed and is predominated by small lymphocytes with lesser numbers of large lymphocytes and plasma cells. In addition, increased numbers of inflammatory cells are identified. Because only low numbers of inflammatory cells are observed in a normal lymph node (often <1%) low numbers of inflammatory cells indicate a lymphadenitis. Suppurative lymphadenitis is diagnosed when >5% neutrophils are observed. In a highly cellular sample, a 100x field represents close to 100 cells. If, consistently, 5 or more neutrophils are observed in each 100x field a suppurative inflammatory response is diagnosed. Causes include bacterial infections, immunemediated disorders such as pemphigus and neoplasia, particularly if a tumor is large and ulcerated. Pyogranulomatous lymphadenitis consists of >5% neutrophils and macrophages with few multinucleated giant Clinical Pathology Anne Barger DVM, MS, DACVP Part 4: Page 1 of 17

43 Chicago Veterinary Medical Association April 13, 2016 cells. Fungal infections, salmon poisoning and less likely foreign body responses should be considered. Eosinophilic lymphadenitis consists of >3% eosinophils. Causes should include allergic skin disease, parasite infestation, especially fleas and paraneoplastic response (mast cell tumor). Organisms are commonly observed in lymph nodes, especially fungal yeast organisms. Neoplasia Lymphoma is one of the most common tumors diagnosed in dogs and cats. In the other processes described in lymphoid tissue, a mixed lymphoid population has been described. In diagnosing lymphoma, we are looking for more of a pure population of large, intermediate or small lymphocytes. Lymphoma is classified cytologically by cell size, large, intermediate and small. The large lymphocytes are 3-4 times the size of a red blood cell, intermediate sized lymphoma is 2-3 times an erythrocyte and small cell lymphoma which is the least common is the size of an erythrocyte. The cells should be described based on many features including cell size, nuclear size and shape, presence, number and shape of nucleoli, amount of cytoplasm and color, note if any granules are present and identify any mitotic figures. Lymphoglandular bodies or small cytoplasmic fragments are commonly observed in the background. Large and intermediate sized lymphoma are reasonable cytologic diagnoses but the diagnosis of small cell lymphoma can be quite challenging without the histopathologic architecture. Small cells are the predominate cell type in normal lymph nodes, therefore diagnosis of neoplastic small cells presents a challenge. Wedge or excisional biopsy is often necessary for definitive diagnosis. Morphologically, T and B lymphocytes can look quite similar so often immunophenotyping is necessary to classify the lymphoma more specifically. Immunocytochemistry is available at several laboratories for cytology and if immunocytochemistry is unavailable or inconclusive PCR for antigen receptor rearrangement (PARR) to determine clonality and cellular origin is also available. Grading is also often performed on cytologic specimens. This involves evaluating the cell size and mitotic index. Low grade has a low mitotic index and a small cell size, high grade has a moderate or high mitotic index and intermediate or large cell size. Metastatic neoplasia within a lymph node can be easily diagnosed with cytology. The node should be evaluated for cells that do not belong in lymphoid tissue such as epithelial cells. These cells can be easily identified because they are often seen in clusters as opposed to the lymphocytes which are individualized. Diagnosis of tumors from cells that can occur in a node in low numbers such as mast cell neoplasia and melanomas can be a little more challenging. Here we are looking for either increased numbers of cells, morphologically atypical cells or cells occurring in large groups or aggregates. When mast cells are observed in a node as part of an inflammatory response, there are often many other cell types observed including eosinophils, macrophages, neutrophils etc. In a metastatic mast cell tumor, the mast cells will often group together and the mast cells outnumber the eosinophils. It is a great idea to aspirate the draining lymph node because so much information can be gained regarding the potential behavior of the tumor. Additionally, some tumors such as nasal carcinomas can be difficult to diagnose with cytology. Identification of the cells within the lymph node makes diagnosis much more clear. Alternate Structures The most common tissues aspirated when lymph node aspiration is attempted are perinodal fat and salivary tissue. Adipose tissue consists of large aggregates of mature adipocytes. These cells are round with abundant amounts of lipid filled cytoplasm. The nuclei are small and round. Aspiration of salivary tissue consists of clusters of uniform epithelial cells. These cells often contain small discrete cytoplasmic vacuoles. The background Clinical Pathology Anne Barger DVM, MS, DACVP Part 4: Page 2 of 17

44 Chicago Veterinary Medical Association April 13, 2016 contains thick, mucinous material which often makes the cells line up in rows or windrow consistent with the viscous nature of the material. The lymph nodes are often cranial to the salivary gland. Quick examination of the slide for these cell types may be useful in getting the most from your aspirate. Suggested Reading: Raskin RE, Meyer DJ. Canine and Feline Cytology, A Color Atlas and Interpretation Guide, 2 nd ed. Saunders Elsevier, St. Louis, MO Cowell RL, Tyler RD, Meinkoth JH and DeNicola DB. Diagnostic Cytology and Hematology of the Dog and Cat, 3 rd ed. Mosby Elsevier, St. Louis, MO Avery PR, Avery AC. Molecular methods to distinguish reactive and neoplastic lymphocyte expansions and their importance in transitional neoplastic states. Vet Clin Pathol 2004;33: Burnett RC, Vernau W, Modiano JF, et al. Diagnosis of canine lymphoid neoplasia using clonal rearrangements of antigen receptor genes. Vet Pathol 2003;40: Clinical Pathology Anne Barger DVM, MS, DACVP Part 4: Page 3 of 17

45 Chicago Veterinary Medical Association April 13, 2016 Lymph Node Aspiration, it's Really Useful! Anne Barger, DVM, MS, DACVP University of Illinois Clinical Pathology - Lymph Node Aspiration Part 4: Page 4 of 17

46 Chicago Veterinary Medical Association April 13, 2016 Purpose Diagnostic Tool Neoplasia Primary or metastatic Staging and prognosis Inflammation Identification of inflammatory processes Organisms Immune stimulation Supportive evidence for localized or systemic disease 2 Normal Lymph Node Histology Encapsulated with subcapsular sinuses Cortex, paracortex, medulla Cortex Spherical follicles B cell areas» When activated a germinal center is formed Paracortex Primarily T cell areas Medulla Lymphatic and blood vessels 3 Willard-Mack CL. Toxicol Pathol Lymph Node Cytology Normal Node Primarily small lymphocytes Round cells, scant rim of basophilic cytoplasm, large nucleus <10% intermediate and large lymphocytes Equal in size or larger than a neutrophils Open chromatin pattern <1% other cell types Macrophages, neutrophils, plasma cells, mast cells, melanocytes Background structures Red blood cells Lymphoglandular bodies 5 Clinical Pathology - Lymph Node Aspiration Part 4: Page 5 of 17

47 Chicago Veterinary Medical Association April 13, 2016 Normal lymph node 6 7 Lymphoglandular Bodies 8 Lymph Node Enlargement Reactive lymph node Antigenic exposure resulting in immune stimulation Lymphadenitis Lymph node draining an inflammatory lesion Neoplasia Lymphoma Metastatic neoplasia 9 Clinical Pathology - Lymph Node Aspiration Part 4: Page 6 of 17

48 Chicago Veterinary Medical Association April 13, 2016 Reactive Lymph Node Probably the most common diagnosis Submandibular lymph nodes are often reactive Generalized lymphadenomegaly, systemic disease should be considered Tick borne diseases such as E. canis Reactive Lymph Node Immune Stimulation Lymphoid expansion and division Increased intermediate and large lymphocytes Small lymphocytes still predominate Increased (>1%) plasma cells Mott cells Still only few inflammatory cells Clinical Pathology - Lymph Node Aspiration Part 4: Page 7 of 17

49 Chicago Veterinary Medical Association April 13, 2016 Lymphadenitis Characterized based on the type of inflammatory cell Suppurative lymphadenitis >5% neutrophils Lymphoid population may appear reactive or mixed with increased large or intermediate lymphocytes Lymphadenitis Suppurative lymphadenitis Causes Bacterial infection or abscess Immune mediated disease Neoplasia Tumor is ulcerated or inflamed Suppurative lymphadenitis Clinical Pathology - Lymph Node Aspiration Part 4: Page 8 of 17

50 Chicago Veterinary Medical Association April 13, 2016 Suppurative lymphadenitis Lymphadenitis Pyogranulomatous lymphadenitis Mixed lymphoid population >5% neutrophils, macrophages and multinucleated giant cells Causes Fungal infection Foreign body response Salmon poisoning Pyogranulomatous Lymphadenitis Pyogranulomatous Lymphadenitis Clinical Pathology - Lymph Node Aspiration Part 4: Page 9 of 17

51 Chicago Veterinary Medical Association April 13, 2016 Lymphadenitis Granulomatous >5% macrophages with multinucleated giant cells Reactive lymphoid population Causes Mycobacterium Fungus Granulomatous Lymphadenitis Lymphadenitis Eosinophilic lymphadenitis >3% eosinophils Reactive lymphoid population Causes Allergic or hypersensitivity response Paraneoplastic Parasitic infestation Clinical Pathology - Lymph Node Aspiration Part 4: Page 10 of 17

52 Chicago Veterinary Medical Association April 13, 2016 Eosinophilic Lymphadenitis Neoplasia Lymphoma Primary neoplasia of the lymph node Generally the lymph node is enlarged Lymphoid population described by size Red blood cell can be used to evaluate size Pure population of lymphocytes Large Intermediate Small 28 Lymphoma Cytology Cell size based on comparison to RBC Determine the nuclear shape Also the location of the nucleus within the cell Eccentric, central etc. Identify and quantify nucleoli Also examine the shape of the nucleolus Evaluate the cytoplasm Quantity and color Describe any mitotic figures 29 Clinical Pathology - Lymph Node Aspiration Part 4: Page 11 of 17

53 Chicago Veterinary Medical Association April 13, 2016 Lymphoma Clinical Pathology - Lymph Node Aspiration Part 4: Page 12 of 17

54 Chicago Veterinary Medical Association April 13, 2016 Lymphoma Additional testing Histopathology Wedge or excisional biopsy Phenotyping: T vs. B cell lymphoma Immunocytochemistry Flow Cytometry PCR TCR and BCR gene rearrangement Excellent diagnostic test in conjunction with cytology 34 Lymphoma Clinical Pathology - Lymph Node Aspiration Part 4: Page 13 of 17

55 Chicago Veterinary Medical Association April 13, 2016 Neoplasia Metastatic Neoplasia Lymphoid population often reactive or inflammatory Identification of non lymphoid cells in the node Metastatic carcinoma Clusters of epithelial cells Metastatic mast cell neoplasia Increased numbers or aggregates of mast cells Metastatic melanoma Melanin containing cells exhibit obvious criteria of malignancy 38 Metastatic Neoplasia Clinical Pathology - Lymph Node Aspiration Part 4: Page 14 of 17

56 Chicago Veterinary Medical Association April 13, 2016 Metastatic Neoplasia 42 Identification of the residual lymphoid population Primary tumor vs. lymph node Important to aspirate the draining lymph node Tumor behavior Prognosis May change therapeutic options 43 Lymph Node Clinical Pathology - Lymph Node Aspiration Part 4: Page 15 of 17

57 Chicago Veterinary Medical Association April 13, 2016 Lymph Node Aspiration Gone Wrong Perinodal Fat The majority of the lymph nodes are embedded in deep mats of fat Unless the lymph node is significantly enlarged, it may be challenging to aspirate Lymph Node Aspiration Gone Wrong Salivary gland Neighboring structure Palpates similarly to lymph node Clusters of epithelial cells Mucin in the background Clinical Pathology - Lymph Node Aspiration Part 4: Page 16 of 17

58 Chicago Veterinary Medical Association April 13, 2016 Salivary Gland Conclusion Lymph node aspiration is an inexpensive, noninvasive diagnostic tool Important prognostic and diagnostic information can be obtained 52 Clinical Pathology - Lymph Node Aspiration Part 4: Page 17 of 17

59 Chicago Veterinary Medical Association April 13, : THE PERIPHERAL BLOOD SMEAR IS A VALUABLE COMPONENT OF THE CBC Anne Barger, DVM, MS, Diplomate ACVP University of Illinois, College of Veterinary Medicine, Urbana, IL Examination of the peripheral blood smear begins on low power. The feathered edge should be scanned for platelet clumps, large cells and microfilaria. The body of the smear should be scanned for proteinaceous background material, identification of the monolayer and again, large cells and microfilaria. A white blood cell estimate can also be performed at low power. Getting an accurate number at this level is challenging so the emphasis should be placed on whether the white blood cell count is increased, decreased or adequate. A rough estimate can be obtained by counting the cells per field and multiplying by Within the monolayer, examination of the red blood cell density and red blood cell arrangement is important. Both agglutination and rouleaux can be observed at low power and may actually be missed at higher magnification. The red blood cell density is important because a decreased density is consistent with anemia. Red blood cells should be evenly spaced from each other and almost touching. Increased space between the red blood cells indicates anemia. At higher power (40x, 50x or 100x) more specific information about red blood cells, white blood cells and platelets can be evaluated. Red blood cell morphology should be evaluated at higher power. Different morphologic changes can indicate very different disease states in the patient or even artifact. Polychromasia indicates erythrocytes which are grayish-blue in color due to the presence of RNA, ribosomes and other organelles. Reticulocytes are often polychromatophilic. All polychromatophils are reticulocytes but not all reticulocytes are polychromatophilic. Anisocytosis indicates variation in cell size and may indicate the presence of small or large cells. Reticulocytes are often larger than mature erythrocytes so they are described as macrocytes. Other causes of macrocytosis include feline leukemia virus, folic acid deficiency (uncommon in animals) and macrocytosis of toy and miniature poodles. Microcytosis or small red blood cells can be observed with several different disease processes, the most common of which is iron deficiency. Due to the impaired heme synthesis, the red blood cells undergo an additional division. Microcytosis will often precede hypochromasia. Diseases associated with iron deficiency or iron misuse include chronic blood loss, dietary deficiency in neonates and portosystemic shunts. Other causes of microcytes include hereditary microcytosis (Akita and Shiba Inu) and familial dyserythropoiesis of English Springer Spaniels 1. Hypochromasia can also be observed in patients with iron deficiency and indicates a decrease in hemoglobin content. If severe enough, this may result in a decrease in the mean corpuscular hemoglobin concentration or MCHC. Membrane abnormalities or poikilocytosis can result in various morphological abnormalities. Spherocytes are red blood cells which have lost their biconcave shape. On a blood smear, they lack central pallor and they can appear a little smaller. They are commonly associated with immune-mediated hemolytic anemias but have also been reported in dogs with spectrin deficiency 2. Acanthocytes are spiculated red cells with irregularly spaced spicules. These cells can be observed in patients with liver disease, portosystemic shunts or high-cholesterol diets. In contrast echinocytes are also spiculated red blood cells but have regularly spaced spicules. These are generally considered an artifactual change however they have been observed in dogs with glomerulonephritis, lymphoma, hemangiosarcoma and other neoplasms, immune mediated hemolytic anemia, pyruvate kinase deficiency, rattlesnake envenomation and doxorubicin toxicosis among others 3. Cats likely have echinocytes with many of these diseases as well but are specifically reported in the literature associated with chronic doxorubicin administration. Schistocytes are red blood cell fragments usually resulting from direct physical damage to the erythrocyte secondary to vascular abnormalities or turbulent blood flow. The shape of the fragments may vary from pointed or triangular to spiculated. Microangiopathic fragmentation has been described in dogs in a Clinical Pathology Anne Barger DVM, MS, DACVP Part 5: Page 1 of 24

60 Chicago Veterinary Medical Association April 13, 2016 number of different disorders including disseminated intravascular coagulation (DIC), glomerulonephritis, hemangiosarcoma, myelofibrosis, dyserythropoiesis and chronic doxorubicin toxicosis. Other poikilocytes include target cells or codocytes, which can be observed with iron deficiency, liver disease or as an artifact or dacryocytes (tear-drop shaped red blood cells) which have been reported in dogs with myelodysplastic disease and myelofibrosis. Keratocytes have one or two spicules and are thought to represent blister cells which have opened, whereas eccentrocytes have a peripheralization of their hemoglobin. The erythrocytes should also be examined for inclusions. Nucleated erythrocytes are commonly observed in anemic patients as part of the regenerative response. However in the absence of anemia or polychromasia, bone marrow and splenic disorders should be considered. Howell-Jolly bodies are nuclear remnants and appear as a small basophilic inclusion. These structures can accompany a regenerative response. Basophilic stippling are part of the regenerative response to anemia in some species such as cattle, however in cats and dogs, this observation is less common. In these species, lead toxicity should be considered especially if the patient is not anemic or has increased numbers of nucleated red blood cells. Oxidative damage can result in a number of morphologic changes to the red blood cells including the formation of Heinz bodies. Heinz bodies are small aggregates of oxidized hemoglobin which can occur as hemoglobinized projections extending from the cell or as pale areas within the cell but close to the cell membrane. Staining the sample with new methylene blue or other vital stains, stains the Heinz body pale blue, allowing them to become much more obvious. Cats are more susceptible to Heinz body formation because of their hemoglobin structure; therefore a healthy cat can have low numbers of Heinz bodies in circulation. Many toxins have been reported to cause Heinz bodies. Hemic parasites also appear as red blood cell inclusions. In dogs two forms of babesial organisms can be identified; Babesia gibsoni is a small, signet ring shaped organism with an eccentrically placed nucleus and Babesia canis is a piroplasmic organism, much larger than B. gibsoni, generally with two organisms/cell. In cats, Mycoplasma hemofelis and M. hemominutum are common extracellular parasites. These organisms appear as small cocci or chains of cocci on the erythrocyte membrane. Cytauxzoon felis is an intraerythrocytic parasite that is regionally specific and appears as a small, signet ring shaped organism, similar to B. gibsoni. A differential count of cells should be performed at high power, in the monolayer of the blood smear. The white blood cells observed in the peripheral blood include neutrophils, eosinophils, basophils, monocytes, lymphocytes and less commonly, plasma cells, mast cells and blasts. In dogs and cats, the neutrophil is the predominant white blood cell. Neutrophils are granulocytes and originate from the same precursor cell as eosinophils and basophils. Neutrophils have a multilobulated nucleus with pale, basophilic cytoplasm. During an inflammatory response, toxic changes may occur which can change the appearance of the cytoplasm. Basophilic cytoplasm, foamy cytoplasm, döhle bodies and azurophilic granules are common changes that can occur with inflammation. Additionally, if there is increased tissue demand for white blood cells, particularly neutrophils, bands and metamyelocytes or hyposegmented neutrophils may be observed in circulation. Bands have a U shaped nucleus with no indentation and metamyelocytes have a reniform shaped nucleus. Neutrophils can also have a hypersegmented nucleus. Hypersegmented neutrophils are generally thought of as older cells and can be observed in patients with chronic inflammation, myeloproliferative disorders or receiving glucocorticoids. Eosinophils are also granulocytes and have a lobulated nucleus and contain numerous eosinophilic granules. In dogs these granules are round and in cats they are rod shaped. Eosinophilia can be observed with parasitic infestation, allergic or hypersensitivity responses and marked eosinophilia (>10,000/ l) can be seen in paraneoplastic responses, hypereosinophilic syndrome, eosinophilic pulmonary granulomas, hepatozoonosis Clinical Pathology Anne Barger DVM, MS, DACVP Part 5: Page 2 of 24

61 Chicago Veterinary Medical Association April 13, 2016 and eosinophilic leukemia 4. Basophils are not commonly observed in peripheral blood but can accompany eosinophils. In cats with eosinophilia and basophilia, heartworm infestation should be considered. Basophils have a lobulated nucleus with grayish-blue cytoplasm. In dogs, few basophilic granules are observed. In cats, basophils are filled with pale, lavender granules which are difficult to visualize with Dif-Quik type stains. Lymphocytes are not granulocytes. These cells are small and round with a scant rim of basophilic cytoplasm and a round nucleus which fills the cytoplasm. Variable lymphocyte morphology can be observed in peripheral blood. Reactive lymphocytes are seen with antigenic stimulation. These cells are larger and have increased amounts of deeply basophilic cytoplasm. Observation of atypical lymphocytes is a common notation on CBCs. These cells have an increased amount of basophilic cytoplasm and are slightly larger than classic lymphocytes. The clinical significance of these lymphocytes is uncertain but if they are present in low numbers, they likely represent a reactive population. However if the patient has a significant lymphocytosis (>20,000/ l) and most or all of the lymphocytes appear atypical, a neoplastic proliferation should be considered. Monocytes are larger than lymphocytes and have a moderate rim of basophilic cytoplasm, often containing discrete cytoplasmic vacuoles and variably shaped nucleus from round to reniform to lobulated. These cells are often part of an inflammatory response but in dogs, increased monocytes can be observed as part of a stress response. Other white blood cells that can be observed in peripheral blood include mast cells, plasma cells and blasts. Mast cells are round cells with a round nucleus containing numerous metachromatic granules. These cells can be seen in peripheral blood with a number of different diseases including mast cell neoplasia, parvoviral enteritis, fibrinous pericarditis, bacterial peritonitis, aspiration pneumonia, acute pancreatic necrosis, IMHA, allergic dermatitis and gastric torsion 5. Presence of significant mastocytemia (>10,000/ l) is more consistent with a neoplastic proliferation of mast cells such as mast cell tumor, systemic mastocytosis or mast cell leukemia. Plasma cells are infrequently observed in the peripheral blood but can be observed as part of an immune response, particularly post-vaccination. Blasts in the peripheral blood are a fairly common finding especially in patients with lymphoma or other myeloproliferative or lymphoproliferative disorders disorders. Blasts are larger cells with varying amounts of basophilic cytoplasm, round nuclei with a stippled chromatin pattern and prominent nucleoli. Morphologically, we cannot consistently distinguish a large lymphocyte from myeloblast or myelomonocytic precursor 6. Immunophenotyping of these cells is essential. Acute leukemias can have a marked leukocytosis, often >100,000 cells/ l with a predominance of neoplastic cells. In patients with lymphoma, there may only low numbers of neoplastic cells in circulation, sometimes representing 1-2% of the nucleated cell population. Identification of these cells in the peripheral blood does not necessarily indicate neoplastic infiltration of the marrow however the recognition of the cells in circulation however is very important. Platelets are cytoplasmic fragments of megakaryocytes. They can be estimated on high power (100x). In the monolayer, platelets should be counted in 10 fields and the average is calculated. This value is multiplied by 15,000-20,000. The blood smear should also be evaluated at low power for platelet clumps. The presence of platelet clumps will decrease automated platelet counts as well as the estimate. Generally, platelets are smaller than red blood cells. They are a basophilic disc of cytoplasm with varying numbers of granules. However, in thrombocytopenic patients, giant platelets can be observed and indicate that the marrow is responding or attempting to respond to the thrombocytopenia. Clinical Pathology Anne Barger DVM, MS, DACVP Part 5: Page 3 of 24

62 Chicago Veterinary Medical Association April 13, 2016 In conclusion, examination of the peripheral blood smear is an essential component of a CBC. Abundant information can be gathered from the blood smear, including subtle changes such as toxic changes in the neutrophils, hemic parasites in the erythrocytes or low numbers of neoplastic cells or mast cells that may help to make a diagnosis. References 1 Allen L,et al.j Am Vet Med Assoc 1999;214: Slappendel RJ, et al. J Vet Intern Med 2005;19: Weiss DJ, et al.vet Clin Pathol 1990;19: Marchetti v, et al. Vet Clin Pathol 2005 Sep;34(3): McManus PM. J Am Vet Med Assoc 1999;215: Gauthier MJ. J Vet Intern Med 2005;19: Clinical Pathology Anne Barger DVM, MS, DACVP Part 5: Page 4 of 24

63 3/3/2016 The Peripheral Blood Smears: A Valuable Diagnostic Tool Anne Barger University of Illinois Making a peripheral blood smear Stain Air Dry No heat or alcohol fixation necessary Diff Quik Does not stain basophil granules in cats Unpredictable staining of mast cell granules Part 5: Page 5 of

64 3/3/2016 Making a Peripheral Blood Smear Components of the smear Body of the smear Includes monolayer Cell counting area All cells lay flat Feathered Edge Large cells, ruptured cells and platelet clumps Monolayer Body Feathered edge Part 5: Page 6 of

65 3/3/2016 Examining the Blood Smear Low power evaluation Scan feathered edge Platelet clumps Large cells Microfilaria Body of smear Microfilaria Large cells Background material Identify monolayer Part 5: Page 7 of

66 3/3/2016 Monolayer Arrangement Agglutination Rouleaux Density Anemia Morphology Red Blood Cells Part 5: Page 8 of

67 3/3/2016 Red Blood Cell Morphology Quantification Consistency is key! Evaluate on 100x Count abnormal cells/hpf Record as rare, occasional, 1+, 2+, 3+ or 4+ Quantification of RBC morphology Anisocytosis Dog >30 Cat >20 Polychromasia Dog >30 Cat >15 Red Blood Cell Morphology Polychromasia Reticulocytes All polychromatophils are reticulocytes but not all reticulocytes are polychromatophilic Large, basophilic cells Anisocytosis Variation in cell size Hypochromasia Decreased hemoglobin content Part 5: Page 9 of

68 3/3/2016 Red Blood Cell Morphology Spherocytes RBC that has lost its biconcave shape IMHA Part 5: Page 10 of

69 3/3/2016 Red Blood Cell Morphology Membrane Defects Acanthocytes Irregularly spaced spicules Liver disease, PSS, high-cholesterol diets Echinocyte Regularly spaced spicules Artifact, GN, lymphoma Schistocyte RBC Fragment DIC, HSA, GN, myelofibrosis Part 5: Page 11 of

70 3/3/2016 Red Blood Cell Morphology Membrane defects Less common Target cell Iron deficiency and liver disease Dacryocyte Tear-drop shaped cell Stomatocyte Keratocyte Open blister cell Result of oxidative damage Red Blood Cell Inclusions Nucleated red blood cells With polychromasia, likely part of the regenerative response Correct WBC HJ bodies Nuclear remnant Basophilic stippling Regenerative vs. lead poisoning Heinz bodies Oxidative damage to hemoglobin Can be difficult to see with Diff-quik Part 5: Page 12 of

71 3/3/2016 Red Blood Cell Inclusions Infectious organisms Distemper inclusion Dogs Babesia gibsoni Babesia canis Cats Mycoplasma hemofelis Cytauxzoon felis Part 5: Page 13 of

72 3/3/2016 B. canis Mycoplasma Hemofelis Part 5: Page 14 of

73 3/3/2016 White Blood Cells Estimate Monolayer 10x Count cells/field and multiply by 100 to x Count cells and multiply by 400 to 600 Differential count 100 to 200 cells Can multiply % and WBC to get an absolute count White Blood Cells Neutrophils Multilobulated nucleus Toxic change Basophilic cytoplasm Dohle bodies Foamy cytoplasm Azurophilic granules Part 5: Page 15 of

74 3/3/2016 White Blood Cells Hyposegmentation Bands Immature neutrophil U shaped or S shaped nuclei Uniform in diameter No area of the nucleus is less than 2/3 the diameter of any other area of the nucleus Metamyelocyte Even less mature Reniform nucleus Myelocyte Less mature still Round nucleus Part 5: Page 16 of

75 3/3/2016 White Blood Cells Neutrophils Hypersegmentation Presence of five or more nuclear lobes Indicates a well-aged cell Glucocorticoids Chronic inflammation Myeloproliferative disorders White Blood cells Eosinophils Dog: Round eosinophilic granules Cat: Rod shaped eosinophilic granules Basophils Larger than the neutrophil Dog: Few basophilic granules Cat: Abundant lavender granules Difficult to see with Diff-Quik Part 5: Page 17 of

76 3/3/2016 Part 5: Page 18 of

77 3/3/2016 White Blood Cells Lymphocyte Round cell, round nucleus Larger than red blood cell, smaller than neutrophil Reactive lymphocyte Indicates immune stimulation Larger than small lymphocyte Deeply basophilic cytoplasm Atypical lymphocyte Increased rim of cytoplasm Part 5: Page 19 of

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79 3/3/2016 White Blood Cells Monocytes Larger than the neutrophil Nuclear shape Round, reniform, band shaped or convoluted Greyish-Blue cytoplasm Part 5: Page 21 of

80 3/3/2016 White Blood Cells Other white blood cells Mast cells Round nucleus with metachromatic granules Plasma cells Eccentrically placed nucleus and prominent golgi apparatus Blasts Large cells with prominent nucleoli Part 5: Page 22 of

81 3/3/2016 White Blood Cells Inclusions Toxic changes Dohle bodies Iron Rare structures Chediak-Higashi Lysosomal storage disorders White Blood Cells Inclusions Infectious Agents Distemper inclusions Ehrlichia morulae Hepatozoon Bacteria Platelets Estimation Low power examination of blood smear Examine feathered edge for platelet clumps Monolayer 100X Count 10 fields and take average Multiply by 15,000-20,000 Part 5: Page 23 of

82 3/3/2016 Platelets Origin Cytoplasmic fragmentation of megakaryocyte Morphology Basophilic structure Anucleated Varying numbers of eosinophilic granules Conclusion Automated CBC does not complete the picture Identification of red and white blood cell morphology can provide tremendous amounts of new information Blood smear examination can also confirm information provided by an automated count Platelet count WBC count Differential count Questions??? Part 5: Page 24 of

83 Chicago Veterinary Medical Association April 13, : URINE LUCK - A THOROUGH REVIEW OF URINE SEDIMENT Anne Barger, DVM, MS, Diplomate ACVP University of Illinois, College of Veterinary Medicine, Urbana, IL Examination of Urine Urine should be examined as soon as possible. Many things can change quickly in urine, chemically and within the sediment so if it cannot be examined immediately or if it is being sent out to a laboratory, refrigeration is recommended. Every urinalysis should begin with gross examination of the urine. Color, clarity and aroma are all part of this examination. Normal urine color is yellow due to the presence of urochrome and urobilin. When urine is red, it is likely due to red blood cells or hemoglobin. Dark yellow to brown urine indicates the presence of bilirubin and reddish brown urine may be from hemoglobin or myoglobin. Normal urine should be clear, clear enough to read text. Cloudy urine indicates an active sediment which means there are cells, crystals, mucus, bacteria, casts or even sperm in the urine. Even normal urine has an aroma. Pathologic processes which can give urine a nasty odor include ammonia, protein breakdown and ketonuria. The next step to urine evaluation is determination of the concentration. The most widely used method is specific gravity. This method utilizes a refractometer and can easily be performed in most general practices. Urine specific gravity or USG is the ratio of the weight of a volume of urine to the weight of the same volume of water. It provides a general assessment of the functioning capacity of the tubules and collecting ducts. USG has a normal wide range or to in the dog and up to in the cat. It is necessary to correlate the USG value with the hydration status of the patient. We categorize urine concentration by how much it has deviated from the specific gravity of the urine when it entered the glomerulus. Hyposthenuric urine has a USG of This indicates that solute has been removed from the urine and nephron is able to dilute the urine. Isosthenuric urine has a range of This indicates that the urine has not changed since it passed through the glomerulus. This could be normal for a well hydrated patient or may suggest renal failure. Urine can become isosthenuric when 66% of the nephrons are not functioning. Dehydrated patients should have concentrated urine. If an animal is >3% dehydrated ADH should cause the renal tubules to concentrate the urine. Determination of urine chemistry is a very easy in-house test that can be performed using urine dipsticks. ph, glucose, ketones, protein, bilirubin, and blood can all be identified. Urine dipsticks can be an insensitive method for evaluation of protein, so other testing such as protein:creatinine or microprotein analysis should be performed. There are some limitations to the urine dipstick. Certain medications can cause false positives on the urine dipstick, particularly Cephalexin and Enrofloxacin can give a false positive reaction. The nitroprusside test used to identify ketones reacts with acetone and acetoac -hydroxybutyric acid which is the most common ketone in dogs and cats. There is an ELISA test method available to detect small amounts of protein in the urine which is more sensitive than the urine dipstick. Urine sediment After the urine chemistry and specific gravity are performed it s time for the examination of the urine sediment. The urine should be centrifuged for 5 minutes. The supernatant is poured off and the sediment is resuspended in a few drops of the remaining supernatant. A drop of sediment is placed on a glass slide and is covered with a Clinical Pathology Anne Barger DVM, MS, DACVP Part 6: Page 1 of 20

84 Chicago Veterinary Medical Association April 13, 2016 cover slip. The sediment is scanned at 10x (lpf) and then at high power or 40x. No oil is necessary for examination of urine sediment. If interesting cells or structures are examined in the sediment, the sample can be preserved by painting the edges of the cover slip with nail polish. Structures identified in the sediment include cells, crystals, casts, microorganisms and other structures. Several different cell types can be observed including epithelial cells (squamous, transitional and renal tubular), red blood cells and white blood cells. Squamous epithelial cells exfoliate from the distal urethra and vagina. The presence of these cells is not considered pathologic unless the morphology of the cells is unusual. Transitional epithelial cells can exfoliate from the proximal urethra, urinary bladder, ureters and renal pelvis. These cells may be observed in high numbers, even in clusters from catheterized samples. These cells do not necessarily indicate disease unless they appear morphologically abnormal. The presence of renal tubular epithelial cells in the urine indicates renal pathology. These cells are often accompanied by casts. Red and white blood cells are quantitated per high power field. >3-5 cells/hpf is considered abnormal and may indicate hemorrhage or inflammation. RBCs can be round or Crenated depending on the specific gravity. If the specific gravity is too low, the RBCs may even lyse. WBCs can lyse in alkaline urine. The presence of increased numbers of WBCs indicates an inflammatory process within the urogenital track. Casts occur when there is damage to the nephron, primarily the renal tubule. If the tubule is damaged, it secretes a protein, called Tamm-Horsfall protein. This protein fills the tubule and encompasses any material that happens to be in the tubule at that time. Casts are truly an imprint of the renal tubule and therefore should mimic the shape and the contents of the tubule. If there is inflammation or hemorrhage in the tubule white blood cell casts or red blood cell casts, respectively, may be observed in the sediment. If there is cellular debris within the tubule, granular casts will be observed. Casts are intermittently shed and therefore do not indicate severity of disease but the presence of waxy casts indicates chronicity. There are many different types of casts in addition to those already mentioned. Hyaline casts are pure protein and may be observed with glomerular disease and epithelial casts consist of renal tubular epithelial cells. The most common microorganisms observed in urine sediment are bacteria. Bacteria observed in a free catch sample must be interpreted with caution unless there are many white blood cells present. Urine is considered sterile until the miduretha so a catheterized sample or cystocentesis is preferred for collecting a sample for culture. Fungal organisms are usually considered a contaminant however Blastomyces dermatitidis and Cryptococcus neoformans have been diagnosed in urine. Formation of urine crystals is affected by a number of variables including, ph, temperature and medications given. Refrigeration can actually induce crystal formation. Presence of particular crystals can indicate very specific diseases. For instance, ammonium biurate crystals often indicate decreased hepatocellular function in most animals but may be normal for Dalmations. Tyrosine crystals can also indicate liver disease. Calcium oxylate dihydrate crystals may be normal in small animals but calcium oxylate monohydrate crystals are associated with ethylene glycol toxicity. Calcium carbonate crystals are considered normal in the horse and the rabbit. Certain medications are known to induce crystal formation such as sulfonamides, allopurinol and ampicillin. Clinical Pathology Anne Barger DVM, MS, DACVP Part 6: Page 2 of 20

85 Chicago Veterinary Medical Association April 13, 2016 It is not uncommon to identify other structures in urine sediment. Fat droplets are very common and can be challenging to differentiate from red blood cells. Plant material or pollen grains can mimic microorganisms or parasite eggs so it is important to identify these structures as contaminants. In conclusion, the urine is packed with useful information that is essential for diagnosis. It is an inexpensive, convenient test that can easily be run as an in-house test. Suggested Reading: Graff, L. A Handbook of Routine Urinalysis. 1983, JB Lippincott Co.Philadelphia, PA. Chew DJ, DiBartola SP. Interpretation of Canine and Feline Urinalysis Ralston Purina Co. Clinical Pathology Anne Barger DVM, MS, DACVP Part 6: Page 3 of 20

86 Chicago Veterinary Medical Association April 13, 2016 Urine Luck: A Thorough Review of Urine Sediment Anne Barger, DVM, MS, DACVP University of Illinois Clinical Pathology Urine Sediment Part 6: Page 4 of 20

87 Chicago Veterinary Medical Association April 13, 2016 Urine Collection Free catch/voided sample Urinary catheterization Cystocentesis Color Clarity Odor Gross Examination 2 3 Color Normal color is yellow Red urine Red blood cells or hemoglobin Dark yellow to brown Bilirubin Reddish brown Hemoglobin Clarity Normal urine should be clear Cloudy urine may indicate an active sediment Myoglobin 4 5 Clinical Pathology Urine Sediment Part 6: Page 5 of 20

88 Chicago Veterinary Medical Association April 13, 2016 Specific gravity Urine Concentration Rough assessment of functioning capacity of tubules and collecting ducts Normal, wide range Up to in the cat Urine Concentration Factors affecting specific gravity Increases of by the addition of a substrate to 1L of urine 2.7 g/dl glucose 4.0 g/dl protein 1.47 g/dl sodium 3.6 g/dl urea 7 Interpretation of Urine Concentration Hydration status of patient must be considered If an animal is >3% dehydrated, ADH should have an effect on the renal tubules to concentrate the urine Hyposthenuria Specific gravity: Isosthenuria Interpretation of Urine Concentration Remember, any specific gravity at one point in time may be normal for that patient. Hydration status, presence or absence of azotemia and clinical signs must all be considered. Specific gravity: Clinical Pathology Urine Sediment Part 6: Page 6 of 20

89 Chicago Veterinary Medical Association April 13, 2016 Urine Chemistry Measured via reagent strip ph Glucose Ketones Bilirubin Blood Protein ph H + concentration ph of urine varies with diet Carnivores: acidic Herbivores: alkalotic Abnormal ph Therapy Cystitis Glucose Normal urine is negative for glucose Renal threshold for glucose Dog: 180 mg/dl Cat: 280 mg/dl Ruminant: <100 mg/dl Glucosuria Fear primarily cat Diabetes mellitus Glucocorticoid release 12 Ketones Normal urine is negative Positive result Starvation Diabetes mellitus Bovine ketosis Ovine pregnancy toxemia High fat diet Severe vomiting and diarrhea Ketonuria present before ketonemia Ketones Acetone Acetoacetone b hydroxybutyric acid: Reagent strip is insensitive 13 Clinical Pathology Urine Sediment Part 6: Page 7 of 20

90 Chicago Veterinary Medical Association April 13, 2016 Bilirubin Unconjugated bilirubin is bound to albumin and does not pass through the glomerulus Conjugated bilirubin passes freely through the glomerulus Renal threshold for bilirubin is lower in the dog Any amount of bilirubin in the urine of cats is significant Tests detect primarily conjugated bilirubin 14 Blood Test based on pseudoperoxidase activity of erythrocytes UA strips sensitive to RBCs, hemoglobin and myoglobin Hematuria vs. Hemoglobin vs. Myoglobin Hematuria: Intact red cells in sediment, red color of urine will clear when spun Hemoglobin: hemolyzed plasma Myoglobin: colorless plasma with concurrent increase in CPK, LDH and AST 15 Blood Causes of hematuria Results from injury or disease of the urogenital tract Causes of hemoglobinuria Observed with intravascular hemolysis Lysis of red cells within the urine can also result in hemoglobinuria Vaden et al, Vet Clin Pathol 2004 showed that hematuria may not affect urine protein until macroscopic and 81% of dogs with pyuria had a normal UPC (<0.4) 16 Mechanisms of Proteinuria Damage to the glomerulus Loss of negative charge on basement membrane People, many hereditary diseases result from podocyte abnormalities Nephrin Podocin Damage to Tubules Tubulointerstitial disease prevents proximal tubules from reabsorbin low molecular weight proteins from the glomerular ultrafiltrate Over flow proteins Bence Jones proteinuria 17 Clinical Pathology Urine Sediment Part 6: Page 8 of 20

91 Chicago Veterinary Medical Association April 13, 2016 Proteinuria Definitions >300 mg/l Microalbuminuria mg/day or mg/l Dog: mg/l in urine normalized to a specific gravity of (JAVMA 2006) Proteinuria National Kidney Foundation recommends that an increase in protein excretion be used as a screening tool in patients at risk of developing renal disease (Am J Kidney Dis, 2002) Preeclampsia Diabetic nephropathy Drug toxicity Proteinuria Proteinuria Historically, 24 hour urine considered gold standard in assessment of protein excretion Variation of protein excretion throughout the day Use of creatinine can lessen this variability Constant throughout the day GFR must be stable Urine luck Protein: Creatinine can be just as useful as 24 urine (Clin Chem 2005) 20 Urine dipstick Urine protein to creatinine ratio (UPC) Urine albumin/creatinine ratio Microalbuminuria 21 Clinical Pathology Urine Sediment Part 6: Page 9 of 20

92 Chicago Veterinary Medical Association April 13, 2016 Proteinuria Whittemore et al, JAVMA 2004 Prospective study, evaluated 600 dogs Measured UAC, UPC, and semiquantitative and quantitative Evaluated 408 dogs with a negative urine dipstick 48 healthy dogs Dogs with systemic disease were more likely to have a positive microalbuminuria The semiquantitative test was the most sensitive for detection of systemic disease 4% of healthy dogs had microalbuminuria 22 Urine Sediment Cells in urine sediment Epithelial cells Red blood cells White blood cells 23 Epithelial cells Squamous epithelial cells No clinical significance Distal urethra and urogenital tract Transitional epithelial cells Proximal urethra, urinary bladder, ureter, renal pelvis No significance unless neoplastic Catheterized samples may have clusters of transitional cells Renal tubular epithelium 24 Sqames 25 Clinical Pathology Urine Sediment Part 6: Page 10 of 20

93 Chicago Veterinary Medical Association April 13, 2016 Transitional epithelial cells TCC TCC Red blood cells 28 RBCs can be round or crenated Important to differentiate from fat droplets >4 5 RBCs/hpf considered significant Important to consider how sample was taken 29 Clinical Pathology Urine Sediment Part 6: Page 11 of 20

94 Chicago Veterinary Medical Association April 13, 2016 RBCs Lipid Droplets Lipid Droplets White Blood cells WBCs are larger than RBCs and can lyse in hypotonic or alkaline urine >5 WBCs/hpf are considered significant in the absence of blood contamination Clinical Pathology Urine Sediment Part 6: Page 12 of 20

95 Chicago Veterinary Medical Association April 13, 2016 WBCs 34 Casts Formed in the distal tubules The turds of the renal tubules Types of casts Hyaline casts Granular cast Epithelial cast Waxy cast Indicate chronicity RBC cast WBC cast 35 Cast Formation Granular and waxy casts Clinical Pathology Urine Sediment Part 6: Page 13 of 20

96 Chicago Veterinary Medical Association April 13, Hyaline cast Waxy cast Clinical Pathology Urine Sediment Part 6: Page 14 of 20

97 Chicago Veterinary Medical Association April 13, 2016 Microorganisms Bacteria Sample collection very important for interpretation of bacteria If true pathogen, and not just contaminant, should see inflammation Fungi Generally considered a contaminant Parasites Stephanurus dentatus Capillaria plica Dioctophyma renale Microfilaria 42 Capillaria Capillaria Dioctophema renale Clinical Pathology Urine Sediment Part 6: Page 15 of 20

98 Chicago Veterinary Medical Association April 13, 2016 Fungal hyphae Bacteria Clinical Pathology Urine Sediment Part 6: Page 16 of 20

99 Chicago Veterinary Medical Association April 13, 2016 Crystals Crystal formation affected by: Urine ph Temperature Medications given 50 Crystals Crystals found in acidic urine Calcium oxalate Cystine Hippuric acid Sulfonamide Crystals found in alkaline urine Triple phosphate Ammonium biurate Calcium carbonate 51 Characteristics of Individual Crystals Ammonium biurate Liver disease, PSS Calcium oxalate Dihydrate Can be seen in normal urine and associated with urolithiasis Monohydrate Ethylene glycol toxicity Triple phosphate crystals One of the most common uroliths in cats Cystine Defect in amino acid transport of proximal renal tubule Calcium Carbonate Considered normal in horse and rabbit 52 Ammonium urates Clinical Pathology Urine Sediment Part 6: Page 17 of 20

100 Chicago Veterinary Medical Association April 13, 2016 Ammonium Urate Calcium Oxalate Calcium Oxalate Triple phosphate Clinical Pathology Urine Sediment Part 6: Page 18 of 20

101 Chicago Veterinary Medical Association April 13, 2016 Cystine Bilirubin Crystals Clinical Pathology Urine Sediment Part 6: Page 19 of 20

102 Chicago Veterinary Medical Association April 13, 2016 Calcium Carbonate Uric Acid Fat droplets Sperm Plant material or pollen grains Other Structures Clinical Pathology Urine Sediment Part 6: Page 20 of 20