Red Cell Cadavers in Anemic Blood (Part 1) Steven Stockham, DVM, MS, DACVP (Clinical Pathology) Kansas State University Manhattan, KS

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A thorough examination of erythrocytes (RBCs) in a blood film can provide valuable information about an animal’s anemia. For those who wish to develop their microscopy skills, the following should be considered essential. • Develop techniques to make an excellent blood film that has an even distribution of cells and a good “counting window.” • Have a quality hematologic stain that can provide reproducible results; quick stains can be acceptable • Have a quality microscope that has excellent 40x- or 50x-oil and 100-x oil objectives (these objectives might cost $3000 to $5000 each) • Have excellent textbooks and atlases for the species of interest (see list below) • Have knowledge of the types of anemias that can be found and the many variations of each disorder Erythrocytes in a stained blood film are Red Cell Cadavers – they are cells that died of dehydration (air-dried) and transformed into 2-dimensional shapes that can provide clues to what they were as living cells. Every erythrocyte in a blood film is an artifact – the cell did not have that shape or appearance when it was circulating in blood. A key aspect in blood film evaluations is recognizing artifacts that tell us something about the animal versus artifacts that are distractions. During this session, we will use case information to provide a framework for the evaluation of Red Cell Cadavers. After examining pertinent microscopic fields, an audience response system will be used to assess your knowledge. The reasons for the observed poikilocytosis, anisocytosis, or other erythrocyte abnormalities will be explained. The following tables are located at the end of the document. • Table 3.6. Erythrocyte inclusions other than organisms: identifying features, clinical significance, and associated pathogenic processes • Table 3.7. Poikilocytes: identifying features, clinical significance, and pathogeneses in domestic mammals There are many books that provide images of blood cells and/or explain the significance of abnormal cells. The first listed book (by John Harvey) provides the most comprehensive set of images. The other books can also be valuable. 1. Veterinary Hematology: A Diagnostic Guide and Color Atlas: JW Harvey, 2012 2. Cowell and Tyler’s Diagnostic Cytology and Hematology of the Dog and Cat, 4th ed., A Valenciano, RL Cowell, RD Tyler, 2014 3. Veterinary Hematology Atlas of Common Domestic and Non-Domestic Species, 2nd ed, WJ Reagan, AR Irizarry Rovira, DB DeNicola, 2008 4. Fundamentals of Veterinary Clinical Pathology, 2nd ed, SL Stockham, MA Scott, 2008 5. BSAVA Manual of Canine and Feline Clinical Pathology, 3rd ed., E Villiers, J Ristic, 2016 6. Veterinary Hematology and Clinical Chemistry, MA Thrall, G Weiser, RW Allison, TW Campbell, 2012 7. Duncan & Prasse’s Veterinary Laboratory Medicine; Clinical Pathology; 5th ed., KS Latimer, 2011 8. Atlas of Veterinary Hematology: Blood and Bone Marrow of Domestic Animals, JW Harvey, 2001

1-1 RBC CVC: Blood film from anemic cat • Case: 194644 (98, 435) • Cat, domestic long hair, male(c), 6-yr-old • History: Listless, depressed, weak, and anorectic for 3 days • PE: temp 102.3 °F, heart rate 128/min, resp. rate 80/min, dehydrated (5 %), pale mucous membranes, depressed, weak, and underweight • CBC (plasma mild icterus) pTP 7.4 g/dL 6.0-8.0 WBC (corrected) 7.8 × 103/µL 5.5-19.5 ↓ Hct (s) 22 % 30-45 Seg. neut. 40 % 3.1 × 103/µL 2.5-12.5 ↓ Hct (c) 20 % 30-45 Band neut. 0 % 0.0 × 103/µL 0.0-0.3 ↓ Hgb 6.1 g/dL 9.0-15.0 Lymph. 56 % 4.4 × 103/µL 1.5-7.0 ↓ RBC 3.4 x 106/µL 5.5-10.0 Mono. 4 % 0.3 × 103/µL 0.0-0.8 ↑ MCV 59 fL 39-55 Eos. 0 % 0.0 × 103/µL 0.0-0.8 MCHC 30 g/dL 30-36 Baso. 0 % 0.0 × 103/µL 0.0-0.1 ↑ MCH 18 pg 13-17 ↑ nRBC 41 /100 WBC 0-1 Platelet clumped

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1-2 RBC CVC: Blood film from anemic cat • Case: 305729 (357, 441) • Cat, domestic long hair, female, 5-mo-old • History: Cat was presented because it was not growing well; it was considerably smaller than its littermate; no other problems were noted by the owner. To increase eating, the owner has been feeding the cat a diet consistent of chicken and vegetables (carrots, celery, & onions). • PE: temp heart rate, and resp. rate OK, not dehydrated, pale mucous membranes, underweight (2.6 lb) • CBC (plasma mild hemolysis) pTP 6.4 g/dL 6.0-8.0 ↑ WBC (corrected) 56.4 × 103/µL 5.5-19.5 ↓ Hct (s) 23 % 30-45 ↑ Seg. neut. 77 % 44.4 × 103/µL 2.5-12.5 ↓ Hct (c) 24 % 30-45 ↑ Band neut. 2 % 1.1 × 103/µL 0.0-0.3 ↓ Hgb 9.5 g/dL 9.0-15.0 Lymph. 10 % 5.6 × 103/µL 1.5-7.0 ↓ RBC 5.4 x 106/µL 5.5-10.0 ↑ Mono. 5 % 2.8 × 103/µL 0.0-0.8 MCV 44 fL 39-55 ↑ Eos. 6 % 3.4 × 103/µL 0.0-0.8 ↑ MCHC 40 g/dL 30-36 ↑ Baso. 1 % 0.6 × 103/µL 0.0-0.1 ↑ MCH 18 pg 13-17 ↑ nRBC 4 /100 WBC 0-1 Platelet clumped

1-3 RBC CVC: Blood film from anemic dog • Case: 074802 (216) • Dog, mixed breed, female (spayed), 9-yr-old • The dog was presented with a complaint of progressive lethargy and inappetence. Physical examination revealed a nonfebrile dog that had icteric mucous membranes and an enlarged spleen. • CBC (plasma icteric) pTP 7.6 g/dL 6.0-8.0 ↑ WBC 39.7 × 103/µL 6.0-17.0 ↓ Hct (s) 30 % 37-55 ↑ Seg. neut. 78 % 31.0 × 103/µL 3.0-11.5 ↓ Hct (c) 31 % 37-55 ↑ Band neut. 11 % 4.4 × 103/µL 0.0-0.3 ↓ Hgb 9.3 g/dL 12.0-18.0 ↓ Lymph. 2 % 0.8 × 103/µL 1.0-4.8 ↓ RBC 3.7 x 106/µL 5.5-8.5 ↑ Mono. 8 % 3.2 × 103/µL 0.2-1.4 ↑ MCV 85 fL 62-76 Eos. 0 % 0.0 × 103/µL 0.0-0.8 ↓ MCHC 30 g/dL 33-37 Baso. 0 % 0.0 × 103/µL 0.0-0.1 MCH 26 pg 21-26 nRBC 0 /100 WBC 0-1 Platelets × 103/µL 200-500

Chemistry profile (serum icteric) Urea nitrogen 29 mg/dL 9-33 ↓ Na+ 145 mmol/L 147–154 Creatinine 0.7 mg/dL 0.5-1.5 K+ 3.6 mmol/L 3.6-5.3 ↑ Glucose 150 mg/dL 73-113 ↑ Cl− 120 mmol/L 108–118 − Total protein 6.0 g/dL 5.4-7.5 ↓ HCO3 12 mmol/L 18–29 Albumin 3.5 g/dL 3.4-4.2 Anion gap 18 mmol/L 16-26 Globulin 2.5 g/dL 1.3-3.2 ↑ ALT 683 U/L 28-171 Ca2+, total 9.9 mg/dL 9.7-12.1 ↑ ALP 881 U/L 1-142 Phosphorus 2.5 mg/dL 2.4-6.4 ↑ CK 390 U/L 128-328 ↑ Bilirubin, total 3.1 mg/dL 0.1-0.3 Cholesterol 328 mg/dL 133-394

Urinalysis Collection Voided pH 6.5 WBC 0–2 / hpf Color None Protein Neg RBC None / hpf Transp. Clear ↑ Glucose Trace Epithelial cells Occ. Squamous / lpf USGref 1.035 Ketone Neg Casts None / lpf Heme Neg Crystals None / lpf ↑ Bilirubin +3 Bacteria None / hpf Urobilinogen 0.2

1-4 RBC CVC: Blood film from anemic dog • Case: 270417 (1467) • Dog, mixed breed, female, 1-yr-old

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• The dog was presented with a complaint of progressive weakness of 4 days duration and passing dark tarry stools 3 to 4 times per day the last 2 days. Physical examination revealed a nonfebrile dog that had pale mucous membranes, tachycardia, and mild cardiac murmur. CBC (plasma clear) ↓ pTP 4.2 g/dL 6.0-8.0 ↑ WBC 17.8 × 103/µL 6.0-17.0 ↓ Hct (s) 7 % 37-55 ↑ Seg. neut. 72 % 12.8 × 103/µL 3.0-11.5 ↓ Hct (c) 6 % 37-55 Band neut. 0 % 0.0 × 103/µL 0.0-0.3 ↓ Hgb 1.8 g/dL 12.0-18.0 Lymph. 19 % 3.4 × 103/µL 1.0-4.8 ↓ RBC 1.4 x 106/µL 5.5-8.5 ↑ Mono. 9 % 1.6 × 103/µL 0.2-1.4 ↓ MCV 43 fL 62-76 Eos. 0 % 0.0 × 103/µL 0.0-0.8 ↓ MCHC 30 g/dL 33-37 Baso. 0 % 0.0 × 103/µL 0.0-0.1 ↓ MCH 13 pg 21-26 nRBC 0 /100 WBC 0-1 ↑ Platelets 920 × 103/µL 200-500

1-5 RBC CVC: Blood film from anemic dog • Case: 299404 (518) • Dog, golden retriever, Fe(s), 7-yr-old • Two months prior to this presentation, the dog had a splenectomy for partial management of disseminated mast cell neoplasia involving spleen, lymph nodes, and skin. This presentation was part of a planned appointment to assess progress. The owner reported the dog had been eating and feeling well. Physical examination revealed a swollen left foreleg and edema in the left axillary region.

CBC (plasma clear) ↓ pTP 5.2 g/dL 6.0-8.0 WBC 16.9 × 103/µL 6.0-17.0 ↓ Hct (s) 18 % 37-55 ↑ Seg. neut. 87 % 14.7 × 103/µL 3.0-11.5 ↓ Hct (c) 17 % 37-55 Band neut. 0 % 0.0 × 103/µL 0.0-0.3 ↓ Hgb 5.7 g/dL 12.0-18.0 ↓ Lymph. 5 % 0.8 × 103/µL 1.0-4.8 ↓ RBC 2.2 x 106/µL 5.5-8.5 Mono. 8 % 1.4 × 103/µL 0.2-1.4 ↑ MCV 81 fL 62-76 Eos. 0 % 0.0 × 103/µL 0.0-0.8 MCHC 33 g/dL 33-37 Baso. 0 % 0.0 × 103/µL 0.0-0.1 ↑ MCH 26 pg 21-26 nRBC 0 /100 WBC 0-1 ↓ Platelets 100 × 103/µL 200-500

1-6 RBC CVC: Blood film from dog • Case: 275480 (326) • Dog, mixed breed, male, 10-mo-old • The dog was referred because it had intermittent diarrhea for 2 months; there was no sustained response to symptomatic treatments. Last night, the dog got into a praying position (head down between front legs, hind legs up) which suggests anterior abdominal pain. Physical examination revealed mild tachycardia, 5 % dehydration, pink mucous membranes, many fleas, and abdominal tenderness.

CBC (plasma clear) pTP 6.0 g/dL 6.0-8.0 ↑ WBC (not corrected) 24.0 × 103/µL 6.0-17.0 Hct (s) 38 % 37-55 WBC (corrected) 10.0 × 103/µL 6.0-17.0 Hct (c) 37 % 37-55 ↑ Seg. neut. 87 % 8.7 × 103/µL 3.0-11.5 Hgb 13 g/dL 12.0-18.0 Band neut. 0 % 0.0 × 103/µL 0.0-0.3 RBC 5.9 x 106/µL 5.5-8.5 ↓ Lymph. 5 % 0.5 × 103/µL 1.0-4.8 MCV 63 fL 62-76 Mono. 8 % 0.8 × 103/µL 0.2-1.4 MCHC 34 g/dL 33-37 Eos. 0 % 0.0 × 103/µL 0.0-0.8 MCH 21 pg 21-26 Baso. 0 % 0.0 × 103/µL 0.0-0.1 ↑ nRBC 140 /100 WBC 0-1 Platelets 280 × 103/µL 200-500

Tables 3.6 and 3.7 from Fundamentals of Veterinary Clinical Pathology, 2nd edition (S.L. Stockham & M.A. Scott)

Table 3.6. Erythrocyte inclusions other than organisms: identifying features, clinical significance, and associated pathogenic processes Inclusions Identifying featuresa Clinical significance Associated pathogenic processes *Basophilic stippling Fine to coarse, blue to dark purple dots Regenerative anemia (especially Young cells—persistence of 249

or specks that represent aggregated cattle), plumbism ribosomal RNA; plumbism— ribosomes dispersed in an erythrocyte’s inhibition of pyrimidine 5′- cytoplasm (Plate 4G) nucleotidase Heinz body Slightly pale, rounded, protruding Exposure to oxidants Oxidants overwhelm reductive structure that creates a membrane defect; capacity of erythrocyte; may occur as free body; stains blue with hemoglobin precipitates and NMB stain (Plate 4H and I) may bind with erythrocyte membrane Hemoglobin crystals Intensely stained, crystallized None in domestic mammals; most Occurs with hemoglobin that forms a pencil, frequent in cats (and camelids) hemoglobinopathies in people parallelogram, cube, or other polyhedron within erythrocytes (Plate 4J) *Howell-Jolly body Usually a homogeneous, dark purple– staining, round structure in erythrocytes; not associated with membrane; can be ring forms (especially in cats) (Plate 4K and L) Increased erythropoiesis, decreased Nuclear remnant that remained free splenic function in the cytoplasm after mitosis; persists in erythrocyte if the spleen does not pit it Siderotic granules Loose aggregate of fine granular Excess Fe in body; plumbism in Fe accumulates in damaged basophilic inclusions; stain blue with Fe dogs; myeloproliferative disease, mitochondria or in stains (Prussian blue) (Plate 5B) usually unknown autophagocytic vacuoles * A relatively common inclusion (Note: Basophilic stippling is more common in cattle than in dogs and cats, and it is not expected in horses.) a Appearance as seen on a Wright-stained blood film unless stated otherwise

Table 3.7. Poikilocytes: identifying features, clinical significance, and pathogeneses in domestic mammals Poikilocyte Other name Identifying features Clinical significance Pathogenesis Acanthocyte Spur cell, burr cella Spherical cell with 1–20 irregularly Hemangiosarcoma; Unknown in domestic (acantho = “spur”) spaced, membrane projections of occasionally splenic, mammals; can form from variable lengths; projections may be hepatic, and renal changes in membrane blunt spurs or clubs (Plate 5G and disorders lipids; possibly 6J) fragmentation *Codocyte (codo = Target cell, Mexican hat Central focus of Hgb that is Typical with regenerative Excess membrane “hat”) cell surrounded by a ring of pallor that anemias; also seen with relative to Hgb content; separates it from peripheral Hgb; hepatic, renal, and lipid may occur with one form of leptocyte (Plate 5H) disorders membrane lipid changes Dacryocyte — Teardrop shaped (Plate 5I Marrow diseases such as Unknown except artifacts (dacyro, dacry = and J) myelofibrosis and caused by stretching “tear”) neoplasia; also may be an during film preparation artifact Eccentrocyte Bite cell, cross-bonded Eccentric dense-staining Hgb and Overwhelming exposure Fusion of membranes (eccentro = cells, hemighost adjacent clear space or crescent to oxidants; also rare casesdamaged by oxidants “eccentric”) (Plate 5K) of G6PD or FAD deficiencies *Echinocyte Burr cella Vary from irregularly shaped cells Hyponatremic Multiple causes (see the (echino = “spiny”) (type I), to regularly spaced blunt dehydration, doxorubricin text) projections (type II), to regularly toxicosis, anionic drugs spaced pointed projections (type III) (Plate 5L) Crenated erythrocyte Crenated cells are artifacts Prolonged exposure to alkaline glass while drying Elliptocyte (See ovalocyte) — — — *Keratocyte Helmet cell Notched, flattened margin between Vasculitis, intravascular Unclear: trauma, (kerato = “horn”) two membrane projections (horns); coagulation, oxidative injury, and variant has one horn (Plate 6A) hemangiosarcoma, caval vesiculation have all been syndrome, endocarditis proposed Leptocyte — Thin cell that appears as a Fe deficiency Incomplete hemoglobin (lepto = “thin”) hypochromic cell with increased synthesis central pallor (Plate 6B)

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Poikilocyte Other name Identifying features Clinical significance Pathogenesis Ovalocyte Elliptocyte Elliptical or oval cell (Plate 6C) Protein band 4.1 Abnormal membrane (ovalo = “egg”) deficiency in dogs, mutant proteins in hereditary spectrin in a dog, form, otherwise unknown myelofibrosis, idiopathic in cats, iron deficiency Pincered cell — Button or knob joined to rest of cell PK deficiency, Unknown by a pinched area (Plate 6D) intravascular trauma Pyknocyte Irregularly contracted Spheroid erythrocyte with Overwhelming exposure Unclear; may form from (pykno = cell condensed or contracted Hgb and to oxidants; also rare caseseccentrocytes “condensed”) perhaps small tags of fragmented of G6PD or FAD membrane (Plate 6E and F) deficiencies *Schizocyte RBC fragment, Triangular, comma-shaped, small Intravascular coagulation, Same as keratocyte (schizo = “cut”) schistocyte round, or irregularly shaped piece of vasculitis, an erythrocyte (Plate 6G) hemangiosarcoma, caval syndrome, endocarditis Selenocyte — A damaged erythrocyte that is Associated with hemolytic Artifact (See the text) (seleno = “moon”) crescent-shaped and has a large anemias, fragile clear space erythrocytes *Spherocyte — Decreased central pallor, decreased Immune hemolysis, Membrane loss due to (sphero = “round”) cell diameter, increased Hgb fragmentation hemolysis, action of macrophages or staining intensity, and smooth envenomations, clostridial trauma or abnormal margins (Plate 6I and J) infections, hereditary band cytoskeleton 3 deficiency Stomatocyte — Elongated (slitlike or mouthlike) Young erythrocytes or Folding of excess (stomato = area of cytoplasmic pallor (Plate hereditary stomatocytosis membrane “mouth”) 6K) of dogs Torocyte — Punched-out, central clear space None; do not confuse with Artifact (toro = “donut that creates a donut-shaped cell hypochromia shaped”) (Plate 6L) * A relatively common poikilocyte a Classifying cells as burr cells is not recommended because the name is used for acanthocytes and echinocytes.

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Red Cell Cadavers in Anemic Blood (Part 2) Steven Stockham, DVM, MS, DACVP (Clinical Pathology) Kansas State University Manhattan, KS

2-1 RBC CVC: Blood film from anemic dog • Case: ASVCP 16-7 (2866) • Dog, mixed-breed, 5-yr-old, female • The patient presented with lethargy and hind-limb bruising. On Day 1, the dog’s TPR values were OK and it was laterally recumbent, quiet, responsive, and dehydrated and marked edema and bruising on her left hind limb with two adjacent puncture marks that oozed blood. The dog received supportive therapy for dehydration, pain control, and wound management. On Day 3 of hospitalization, the dog became progressively anemic, and clinical pathology testing was repeated (blood film from Day 3).

CBC (note: ↑ & ↓ arrows not shown for results; abnormal results in bold) D-1 D-3 D-1 D-3 Plasma Pink Pink WBC 15.7 10.8 × 103/µL 2.0-15.6 pTP 4.0 4.3 g/dL 5.9-8.2 Seg. neut. 11.4 6.2 × 103/µL 2.5-9.2 Hct (s) 40 20 % 43-62 Band neut. 0.3 2.3 × 103/µL 0.0-0.0 Hct (c) 39 20 % 43-62 Lymph. 3.1 1.2 × 103/µL 0.9-3.3 Hgb 13.5 7.3 g/dL 14.8-21.1 Mono. 0.6 0.7 × 103/µL 0.1-1.2 RBC 5.5 2.9 x 106/µL 5.9-8.6 Eos. 0.2 0.4 × 103/µL 0.0-1.9 MCV 72 68 fL 66-77 Baso. 0 0.1 × 103/µL 0.0-0.0 MCHC 34 37 g/dL 33-36 MCH 25 25 pg 21-26 nRBC 0 0 /100 WBC 0-1 Platelets 175 68 × 103/µL 150-393

Chemistry profile (serum mild hemolysis) (note: ↑ & ↓ arrows not shown; abnormal data in bold) D-1 D-3 D-1 D-3 Urea nitrogen 17 8 mg/dL 9-30 Na+ 144 140 mmol/L 147–154 Creatinine 0.7 0.3 mg/dL 0.7-1.3 K+ 3.4 3.4 mmol/L 3.6-5.3 Glucose 160 137 mg/dL 88-121 Cl− 114 113 mmol/L 108–118 − Total protein 4.3 4.2 g/dL 5.3-7.0 HCO3 21 21 mmol/L 18–29 Albumin 2.4 1.9 g/dL 2.8-3.7 Anion gap 12 9 mmol/L 16-26 Globulin 1.9 2.3 g/dL 2.1-3.8 ALT 33 23 U/L 28-171 Ca2+, total 9.0 8.2 mg/dL 9.4-10.7 ALP 108 143 U/L 1-142 Phosphorus 3.7 3.5 mg/dL 1.9-4.4 CK 2494 --- U/L 128-328 Bilirubin, total 0.2 0.4 mg/dL 0.2-0.4 Cholesterol 67 63 mg/dL 133-394

2-2 RBC CVC: Blood film from anemic dog • Case: 006320 (513) • Dog, Gordon setter, 9-yr-old • About a month ago and during diagnostic testing for inappropriate urinations, ultrasonography revealed several splenic masses. At this time, CBC results and a chemical profile results were within reference intervals. A urinalysis revealed a proteinuria. Exploratory abdominal surgery found several elevated splenic masses and the spleen was removed. The dog recovered well from the surgery. However, the current problem is weakness and icteric mucous membranes.

CBC (plasma icteric) pTP 6.9 g/dL 6.0-8.0 WBC (not corrected) 7.7 × 103/µL 6.0-17.0 ↓ Hct (s) 14 % 37-55 WBC (corrected) 6.6 × 103/µL 6.0-17.0 ↓ Hct (c) 15 % 37-55 ↓ Seg. neut. 38 % 2.5 × 103/µL 3.0-11.5 ↓ Hgb 5.1 g/dL 12.0-18.0 Band neut. 0 % 0.0 × 103/µL 0.0-0.3 ↓ RBC 2.2 x 106/µL 5.5-8.5 Lymph. 38 % 2.5 × 103/µL 1.0-4.8 MCV 68 fL 62-76 Mono. 20 % 1.3 × 103/µL 0.2-1.4 MCHC 34 g/dL 33-37 Eos. 4 % 0.2 × 103/µL 0.0-0.8 MCH 23 pg 21-26 Baso. 0 % 0.0 × 103/µL 0.0-0.1 ↑ nRBC 16 /100 WBC 0-1 Platelets 280 × 103/µL 200-500

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2-3 RBC CVC: Blood film from anemic dog Case: ASVCP 10-11 (2869) • Dog, Airedale terrier, 3-yr-old, male(c) • The patient presented to an emergency clinic because of smoke inhalation that occurred during a house fire. Adequate oxygenation was not obtained after emergency intubation and a mechanical ventilator. Continued supportive therapy included mechanical ventilation and constant rate infusions of propofol, fentanyl, ketamine, and diazepam. The laboratory data and blood film are from the 4th day of hospitalization and supportive therapy.

CBC (plasma slightly icteric) ↓ pTP 5.0 g/dL 5.7-7.2 WBC 14.0 × 103/µL 4.1-15.2 ↓ Hct (s) 32 % 36-54 ↑ Seg. neut. 87 % 11.6 × 103/µL 3.0-10.4 ↓ Hct (c) 31 % 36-54 ↑ Band neut. 5 % 0.3 × 103/µL 0.0-0.1 ↓ Hgb 10.4 g/dL 11.9-18.4 ↓ Lymph. 4 % 0.8 × 103/µL 1.0-4.6 ↓ RBC 4.4 x 106/µL 4.9-8.2 ↑ Mono. 4 % 1.3 × 103/µL 0.0-1.2 MCV 72 fL 64-75 Eos. 0 % 0.0 × 103/µL 0.0-0.5 MCHC 33.1 g/dL 33-36 Baso. 0 % 0.0 × 103/µL 0.0-0.1 MCH 24 pg 20-25 nRBC 0 /100 WBC 0-1 ↓ Platelets 105 × 103/µL 106-424

Chemistry profile (serum clear) ↑ Urea nitrogen 28 mg/dL 5-20 Na+ 155 mmol/L 141–156 ↑ Creatinine 2.9 mg/dL 0.6-1.6 K+ 5.2 mmol/L 3.8-5.5 Glucose --- mg/dL 75-120 Cl− 122 mmol/L 109–124 − ↓ Total protein 2.1 g/dL 5.1-7.1 ↓ HCO3 9 mmol/L 18–28 ↓ Albumin 1.0 g/dL 2.9-4.2 ↑ Anion gap 29 mmol/L 16-26 ↓ Globulin 1.1 g/dL 2.2-2.9 ↑ ALT 618 U/L 13-79 ↓ Ca2+, total 7.9 mg/dL 9.3-11.6 ↑ ALP 215 U/L 12-122 Phosphorus 8.1 mg/dL 3.2-8.1 ↑ CK 112,000 U/L 58-241 ↑ Bilirubin, total 2.3 mg/dL 0.1-0.4 ↑ AST 3994 U/L 12-40

2-4 RBC CVC: Blood film from anemic dog • Case: 037788 (498) • Dog mixed breed, female, 13-yr-old • The dog was referred because of anemia and an enlarged abdomen. The owner thought the dog’s abdomen had been enlarged for about a month but the dog did not appear ill. Recent problems included 2 days of vomiting and anorexia. Physical examination revealed pale mucous membranes, a distended abdomen (probably fluid-filled, and possibly an abdominal mass).

CBC (plasma icteric) pTP 6.5 g/dL 6.0-8.0 ↑ WBC 21.9 × 103/µL 6.0-17.0 ↓ Hct (s) 16 % 37-55 ↑ Seg. neut. 87 % 19.0 × 103/µL 3.0-11.5 ↓ Hct (c) 16 % 37-55 ↑ Band neut. 5 % 1.1 × 103/µL 0.0-0.3 ↓ Hgb 5.2 g/dL 12.0-18.0 ↓ Lymph. 4 % 0.9 × 103/µL 1.0-4.8 ↓ RBC 2.2 x 106/µL 5.5-8.5 Mono. 4 % 0.9 × 103/µL 0.2-1.4 MCV 71 fL 62-76 Eos. 0 % 0.0 × 103/µL 0.0-0.8 MCHC 33 g/dL 33-37 Baso. 0 % 0.0 × 103/µL 0.0-0.1 MCH 24 pg 21-26 nRBC 0 /100 WBC 0-1 ↓ Platelets 80 × 103/µL 200-500

Chemistry profile (serum icteric) ↑ Urea nitrogen 33 mg/dL 8-30 Na+ 149 mmol/L 141–156 Creatinine 0.8 mg/dL 0.5-1.5 K+ 3.9 mmol/L 3.8-5.5 Glucose 69 mg/dL 60-120 Cl− 117 mmol/L 109–124 − Total protein 6.0 g/dL 5.6-7.9 ↓ HCO3 12 mmol/L 18–28 ↓ Albumin 2.5 g/dL 3.0-4.5 Anion gap 25 mmol/L 16-26 Globulin 3.5 g/dL 1.8-4.2 ↑ ALT 984 U/L 13-79 Ca2+, total 9.1 mg/dL 8.2-12.8 ↑ ALP 197 U/L 12-122 Phosphorus 6.5 mg/dL 2.3-6.5 ↑ CK 578 U/L 58-241 ↑ Bilirubin, total 1.4 mg/dL 0.1-0.4 Cholesterol 152 mg/dL 124-335

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Urinalysis Collection Voided pH 6.5 WBC 0–3 / hpf Color None Protein Trace RBC 0-1 / hpf Transp. Hazy Glucose Neg Epithelial cells squamous / lpf USGref 1.048 Ketone Neg Casts None / lpf Heme Neg Crystals None / lpf ↑ Bilirubin +2 Bacteria None / hpf Urobilinogen 0.2

2-5 RBC CVC: Blood film from anemic dog • Case: 831139 (1791) • Dog, mixed-breed Labrador, female, 3-yr-old • The dog was presented after a sudden onset of weakness and lethargy; the owner became greatly concerned when red urine was passed. Physical examination revealed a nonfebrile dog that had pale mucous membranes.

CBC (plasma hemolyzed) pTP 7.7 g/dL 6.0-8.0 ↑ WBC (not corrected) 37.6 × 103/µL 6.0-17.0 ↓ Hct (s) 13 % 37-55 ↑ WBC (corrected) 28.7 × 103/µL 6.0-17.0 ↓ Hct (c) 14 % 37-55 ↑ Seg. neut. 59 % 16.9 × 103/µL 3.0-11.5 ↓ Hgb 4.8 g/dL 12.0-18.0 ↑ Band neut. 13 % 3.7 × 103/µL 0.0-0.3 ↓ RBC 1.9 x 106/µL 5.5-8.5 Lymph. 10 % 2.9 × 103/µL 1.0-4.8 MCV 74 fL 62-76 ↑ Mono. 15 % 4.3 × 103/µL 0.2-1.4 MCHC 34 g/dL 33-37 ↑ Eos. 3 % 0.9 × 103/µL 0.0-0.8 MCH 25 pg 21-26 Baso. 0 % 0.0 × 103/µL 0.0-0.1 nRBC 0 /100 WBC 0-1 ↓ Platelets decreased × 103/µL 200-500

2-6 RBC CVC: Blood film from anemic dog • Case: 154520 (2320) • Dog, German shepherd, male(c), 6-yr-old • The dog was presented because of lethargy and an enlarged abdomen. Physical examination revealed a nonfebrile dog that had an abdominal effusion and petechiae in oral mucous membranes.

CBC (plasma clear) pTP 6.2 g/dL 6.0-8.0 ↑ WBC 23.2 × 103/µL 6.0-17.0 ↓ Hct (s) 34 % 37-55 ↑ Seg. neut. 83 % 19.3 × 103/µL 3.0-11.5 ↓ Hct (c) 33 % 37-55 ↑ Band neut. 4 % 0.9 × 103/µL 0.0-0.3 ↓ Hgb 11.8 g/dL 12.0-18.0 Lymph. 7 % 1.6 × 103/µL 1.0-4.8 ↓ RBC 4.9 x 106/µL 5.5-8.5 Mono. 6 % 1.4 × 103/µL 0.2-1.4 MCV 66 fL 62-76 Eos. 0 % 0.0 × 103/µL 0.0-0.8 MCHC 36 g/dL 33-37 Baso. 0 % 0.0 × 103/µL 0.0-0.1 MCH 24 pg 21-26 ↑ nRBC 2 /100 WBC 0-1 ↓ Platelets 110 × 103/µL 200-500

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What are the Cells from that Bump? Steven Stockham, DVM, MS, DACVP (Clinical Pathology) Kansas State University Manhattan, KS

5-1 Cytologic biopsy CVC: fine-needle aspirate of vulvar mass • Case: 026163 (758) • Dog, mixed breed, female, 5-yr-old A 1x1 pink mass was protruding slightly from the vulvar mucosa. The owner first noticed the mass yesterday. The mass protruded into the vaginal vault; it might be extending into the submucosa. A fine-needle aspirate of the mass was collected and a smear was prepared for examination.

5-2 Cytologic biopsy CVC: fine-needle aspirate of cutaneous mass • Case: 02-7821 (754) • Dog, boxer, 1-yr-old A 1x1x1 cm, pink mass was located in the lateral skin of the right shoulder. The owner first noticed the mass a few days ago. The mass seemed to involve the dermis and epidermis and did not extend into the subcutaneous tissues. A fine-needle aspirate of the mass was collected and a smear was prepared for examination.

5-3 Cytologic biopsy CVC: fine-needle aspirate of cutaneous mass • Case: 028857 (727) • Dog, Golden retriever, male (neutered), 5-yr-old The dog was presented because of a 2x2x1 cm mass located in the lateral thoracic skin. The preparation is a smear of the sample aspirated from the mass.

5-4 Cytologic biopsy CVC: fine-needle aspirate of cutaneous mass • Case: 039973 (725) • Cat, domestic short hair The preparation is a smear of a sample aspirated from one of several small (< 1 cm) cutaneous masses.

5-5 Cytologic biopsy CVC: fine-needle aspirate of cutaneous mass • Case: 56533-98 (742) • Dog, mixed breed A smear of serosanguinous to purulent fluid was submitted; the fluid was collected from a subcutaneous swelling that had a draining tract.

5-6 Cytologic biopsy CVC: fine-needle aspirate of cutaneous mass • Case: 028729 (775) • Dog, terrier-mix, female (spayed), 14-year-old A 5x4x23 cm mass was found in the dorsal thoracic skin. The dog has several other similar masses in its thoracic and abdominal skin. The mass extends above the skin surface, the surface is ulcerated, and appears to involve dermal and possibly subcutaneous tissues. A fine-needle aspirate of the mass was collected and a smear was prepared for examination.

5-7 Cytologic biopsy CVC: fine-needle aspirate of cutaneous mass • Case: 029402 (781) • Cat, Persian, female (spayed), 19-year-old The cat was presented because of a large (about 8 cm), broad-based mass located in the area of the 3rd to 4th left mammary gland. Physical examination revealed was covered with haired skin and appeared to involve the dermis and subcutaneous tissues. A fine- needle aspirate of the mass was collected and a smear was prepared for examination.

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5-8 Cytologic biopsy CVC: fine-needle aspirate of cutaneous mass • Case: 028874 (785) • Dog, shar pei, male (neutered), 9-year-old The dog was presented because of a mass in its skin. Physical examination revealed a dermal or subcutaneous mass of the right thorax. A fine-needle aspirate of the mass was collected and a smear was prepared for examination.

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What are the Cells from this Lump? Steven Stockham, DVM, MS, DACVP (Clinical Pathology) Kansas State University Manhattan, KS

A cytologic biopsy (aka, fine needle biopsy or fine needle aspiration biopsy or “cytology”) of cutaneous and subcutaneous lesions (lumps and bumps) can result in a specific diagnosis or perhaps can better characterize a lesion. For nearly all lesions, the cytologic biopsy will not be as definitive as an incisional or excisional biopsy with a histopathological examination; but will be less expensive and yield results quicker. For some lesions (e.g., lipoma), it takes minimal expertise and diagnostic methods to arrive at a correct diagnosis; but other lesions require extensive knowledge gained through experience and excellent equipment. For those who wish to develop their cytologic biopsy skills, the following should be considered essential. • Develop techniques to obtain cytologic preparations that have monolayers of cells • Have a quality cytologic stain that can provide reproducible results; quick stains can be acceptable • Have a quality microscope that has excellent 40x- or 50x-oil and 100-x oil objectives (these objectives might cost $3000 to $5000 each) • Have excellent textbooks and atlases for the species of interest • Have knowledge of the types of lesions that can be found and the many variations of each disorder During the microscopic examination of aspirates, scrapes, imprints, or other cytologic preparations, general goals are to arrive at one of these conclusions or opinions: • Definitive diagnosis: can be achieved with a few neoplasms and some inflammatory lesions • Consistent with ______: cells populations are seen in this condition but the findings are not unique to one diagnosis; additional diagnostic efforts are needed to confirm • Suspicious of ______: findings are suggestive stated diagnoses but definitive evidence is not seen; additional diagnostic efforts are needed • Not consistent with ______: A preliminary diagnosis had been made; the findings in this sample are not likely to be found in that disorder; or, the findings do not support the preliminary diagnosis The following flowchart provides a basic guideline for the evaluation of a cytologic preparation. The concepts of the flow chart will be used during the virtual microscopy of several lesions involving the skin and subcutaneous tissues of dogs and cats.

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4-1 Cytologic biopsy CVC: Smear of fluid from subcutaneous lesion • Case: 176517 (729) • Dog, mixed breed, 3-yr-old, female (spayed) A smear of serosanguineous to purulent fluid was submitted; the fluid was collected from a subcutaneous swelling that had a draining tract.

4-2 Cytologic biopsy CVC: fine-needle aspirate of cutaneous mass • Case: 02-1975 (723) • Dog, Labrador retriever, 4-yr-old A 2x4x3 cm mass was located in the lateral skin of the left hind thigh or hip. The owner first noticed the mass a few weeks ago and it has been getting larger. The mass protruded slightly and felt like it extended into the subcutaneous tissue. A fine-needle aspirate of the mass was collected and a smear was prepared for examination.

4-3 Cytologic biopsy CVC: fine-needle aspirate of cutaneous mass • Case: 002885 (772) • Dog, Golden retriever, male, 12-yr-old The dog was presented because of a mass located on the dorsal aspect of the tail head. Physical examination revealed 2-cm, soft mass in the dermis and was covered with haired skin. A fine-needle aspirate of the rear leg mass was collected and a smear was prepared for examination.

4-4 Cytologic biopsy CVC: fine-needle aspirate of cutaneous mass • Case: 030056 (783) • Dog, basset hound, male (neutered), 7-yr-old The dog was presented because of perianal masses. Physical examination revealed a small perianal mass and possibly enlarged regional lymph node. A fine-needle aspirate of the mass was collected and a smear was prepared for examination.

4-5 Cytologic biopsy CVC: fine-needle aspirate of cutaneous mass • Case: 02-2357 (557) • Dog; breed, age, and gender not provided A smear of an aspirate obtained from a mass in the skin of a foot was submitted for evaluation.

4-6 Cytologic biopsy CVC: fine-needle aspirate of cutaneous mass • Case: 024854 (777) • Dog, schipperke, male (neutered), 15-yr-old The dog had been coughing for 2-3 weeks. During a physical exam, a mass was found in the subcutaneous tissues of the left lateral thoracic; it appeared to be firmly attached to underlying tissues. A fine-needle aspirate of the mass was collected and a smear was prepared for examination.

4-7 Cytologic biopsy L&B CVC: Imprint of moist cutaneous lesion • Case: 256285 (737) • Dog, mixed breed, male (neutered), 4-yr-old The dog was presented because of a swelling of the left flank that broke open yesterday and yellowish red material oozed out. The preparation is an imprint of the ulcerated area after superficial debris and hair were removed.

4-8 Cytologic biopsy CVC: Imprints of cutaneous mass • Case: ASVCP 1988-11 (748) • Cat, domestic short hair The cat was presented because of skin lesions. Physical examination revealed several, pea-size, cutaneous masses. One mass was excised and imprints of the mass were submitted for evaluation. .

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White Cell Cadavers of Leukocytosis Steven Stockham, DVM, MS, DACVP (Clinical Pathology) Kansas State University Manhattan, KS

Many of today’s hematologic analyzers evaluate leukocytes via flow cytometric methods and provide an automated differential count and calculated concentration of leukocyte populations. When properly calibrated for a given species (e.g., dog or cat), then the generated results are typically accurate for blood cells with relative normal leukocytes. Why, the instruments have software programs that are designed to recognize normal cells. When abnormal leukocyte are present (e.g., toxic neutrophils, reactive lymphocytes, neoplastic cells), the automated differential count will not be accurate. A thorough examination of leukocytes (WBCs) in a blood film can provide valuable information about an animal’s leukocytosis. For those who wish to develop their microscopy skills, the following should be considered essential. • Develop techniques to make an excellent blood film that has an even distribution of cells and a good “counting window.” • Have a quality hematologic stain that can provide reproducible results; quick stains can be acceptable • Have a quality microscope that has excellent 40x- or 50x-oil and 100-x oil objectives (these objectives might cost $3000 to $5000 each) • Have excellent textbooks and atlases for the species of interest (see list below) • Have knowledge of abnormal leukocytes or associated organisms that can be found and the many variations of each disorders, Leukocytes in a stained blood film are White Cell Cadavers – they are cells that died of dehydration (air-dried) and transformed into 2-dimensional shapes that can provide clues to what they were as living cells. Every leukocyte in a blood film is an artifact – the cell did not have that shape or appearance when it was circulating in blood. A key aspect in blood film evaluations is recognizing artifacts that tell us something about the animal versus artifacts that are distractions. During this session, we will use case information to provide a framework for the evaluation of White Cell Cadavers. After examining pertinent microscopic fields, an audience response system will be used to assess your knowledge. The reasons for the observed leukocytosis and leukocyte abnormalities will be explained. There are many books that provide images of blood cells and/or explain the significance of abnormal cells. The first listed book (by John Harvey) provides the most comprehensive set of images. The other books can also be valuable. 1. Veterinary Hematology: A Diagnostic Guide and Color Atlas: JW Harvey, 2012 2. Cowell and Tyler’s Diagnostic Cytology and Hematology of the Dog and Cat, 4th ed., A Valenciano, RL Cowell, RD Tyler, 2014 3. Veterinary Hematology Atlas of Common Domestic and Non-Domestic Species, 2nd ed, WJ Reagan, AR Irizarry Rovira, DB DeNicola, 2008 4. Fundamentals of Veterinary Clinical Pathology, 2nd ed, SL Stockham, MA Scott, 2008 5. BSAVA Manual of Canine and Feline Clinical Pathology, 3rd ed., E Villiers, J Ristic, 2016 6. Veterinary Hematology and Clinical Chemistry, MA Thrall, G Weiser, RW Allison, TW Campbell, 2012 7. Duncan & Prasse’s Veterinary Laboratory Medicine; Clinical Pathology; 5th ed., KS Latimer, 2011 8. Atlas of Veterinary Hematology: Blood and Bone Marrow of Domestic Animals, JW Harvey, 2001

3-1 WBC CVC: Blood film from a dog • Case: 256917 (2420) • Dog, coon hound, male, 4-yr-old The dog was presented because of lethargy, weight loss, anorexia, and dyspnea of 2 months duration. The referring veterinarian had treated the dog for pneumonia (specifics of treatment not known) Physical examination revealed labored breathing, muffled heart sounds, and distended abdomen.

CBC (plasma clear) pTP 6.7 g/dL 6.0-7.5 ↑ WBC 24.0 × 103/µL 6.0-17.0 ↓ Hct (s) 23 % 37-55 ↑ Seg. neut. 56 % 13.4 × 103/µL 3.0-11.5 ↓ Hct (c) 22 % 37-55 ↑ Band neut. 16 % 3.8 × 103/µL 0.0-0.3 ↓ Hgb 7.8 g/dL 12-18 Lymph. 18 % 4.3 × 103/µL 1.0-4.8 ↓ RBC 3.2 x 106/µL 5.5-8.5 ↑ Mono. 8 % 2.0 × 103/µL 0.2-1.4 MCV 69 fL 62-76 Eos. 2 % 0.5 × 103/µL 0.0-0.8 MCHC 35 g/dL 32-36 Baso. 0 % 0.0 × 103/µL 0.0-0.1 MCH 24 pg 19-25 259

nRBC 0 /100 WBC 0-1 Platelets clumped × 103/µL 200-500

3-2 WBC CVC: Blood film from a dog • Case: 289557 (347) • Dog, mixed breed, 8-yr-old, female(s) The dog was presented because it she had not eaten for several days and seemed lethargic. Physical examination revealed no major abnormalities except mild enlargement of mandibular, prescapular, and popliteal lymph nodes.

CBC (plasma clear) ↑ pTP 10.1 g/dL 6.0-7.5 ↑ WBC 41.7 × 103/µL 6.0-17.0 ↓ Hct (s) 36 % 37-55 Seg. neut. 12.3 % 5.1 × 103/µL 3.0-11.5 ↓ Hct (c) 36 % 37-55 Band neut. 0.0 % 0.0 × 103/µL 0.0-0.3 ↓ Hgb 11.6 g/dL 12-18 ↑ Lymph. 84.3 % 35.1 × 103/µL 1.0-4.8 ↓ RBC 5.3 x 106/µL 5.5-8.5 Mono. 1.4 % 0.6 × 103/µL 0.2-1.4 MCV 67 fL 62-76 Eos. 2.0 % 0.8 × 103/µL 0.0-0.8 MCHC 35 g/dL 32-36 Baso. 0.0 % 0.0 × 103/µL 0.0-0.1 MCH 22 pg 19-25 nRBC 0 /100 WBC 0-1 Platelets 42 × 103/µL 200-500

3-3 WBC CVC: Blood film from a dog • Case: 38501 (2319) • Dog, Irish setter, 4-yr-old, female(s) The dog was presented with a complaint of weight loss and persistent coughing. Physical examination revealed a nonfebrile, mildly dehydrated dog that had tachypnea and coarse crackles. Partial CBC results are provided.

CBC (plasma clear) pTP 7.9 g/dL 6.0-8.0 ↑ WBC 27.3 × 103/µL 6.0-17.0 Hct (s) 38 % 37-55 ↑ Seg. neut. × 103/µL 3.0-11.5 Hct (c) 38 % 37-55 ↑ Band neut. × 103/µL 0.0-0.3 Hgb 13.2 g/dL 12.0-18.0 Lymph. × 103/µL 1.0-4.8 RBC 5.6 x 106/µL 5.5-8.5 ↑ Mono. × 103/µL 0.2-1.4 MCV 68 fL 62-76 Eos. × 103/µL 0.0-0.8 MCHC 35 g/dL 33-37 Baso. × 103/µL 0.0-0.1 MCH 24 pg 21-26 nRBC 0 /100 WBC 0-1 Platelets 450 × 103/µL 200-500

3-4 WBC CVC: Blood film from a dog • Case: ASVCP 2002-16 (422) • Dog, mixed breed, female (spayed), 10-yr-old Dog was presented with the history of chronic progressive rear limb weakness, anorexia, and weight loss. Physical examination revealed depression, severe muscle atrophy of hind limbs, pain elicited with deep palpation of both femurs, and an enlarged right mandibular lymph node.

CBC (plasma slight hemolysis) pTP 6.4 g/dL 6.0-8.0 ↑ WBC 83.8 × 103/µL 6.0-17.0 ↓ Hct (s) 26 % 37-55 ↑ Seg. neut. 86 % 72.1 × 103/µL 3.0-11.5 ↓ Hct (c) 27 % 37-55 ↑ Band neut. 3 % 2.5 × 103/µL 0.0-0.3 ↓ Hgb 10.6 g/dL 12.0-18.0 Lymph. 2 % 1.7 × 103/µL 1.0-4.8 ↓ RBC 3.9 x 106/µL 5.5-8.5 ↑ Mono. 8 % 6.7 × 103/µL 0.2-1.4 MCV 70 fL 62-76 Eos. 1 % 0.8 × 103/µL 0.0-0.8 ↑ MCHC 39 g/dL 33-37 Baso. 0 % 0.0 × 103/µL 0.0-0.1 ↑ MCH 27 pg 21-26 nRBC 0 /100 WBC 0-1 Platelets 418 × 103/µL 200-500

3-5 WBC CVC: Blood film from a dog • Case: ASVCP 2006-4 (1613) • Dog, Basset hound, M(c), 4-yr-old The dog was presented after a week of inappetence. Physical examination revealed a nonfebrile, depressed, lethargic dog with pale mucous membranes and tachycardia. CBC results for blood sample collected after 2 days of IV fluids and prednisone. 260

CBC (plasma clear) ↓ pTP 5.5 g/dL 5.7-7.2 ↑ WBC 89.5 × 103/µL 4.1-15.2 ↓ Hct (s) 18 % 36-54 ↑ Seg. neut. 14 % 12.5 × 103/µL 3.0-10.4 ↓ Hct (c) 19 % 36-54 ↑ Band neut. 4 % 3.6 × 103/µL 0.0-0.1 ↓ Hgb 6.6 g/dL 11.9-18.4 Lymph. 4 % 3.6 × 103/µL 1.0-4.6 ↓ RBC 2.7 x 106/µL 4.9-8.2 ↑ Mono. 78 % 69.8 × 103/µL 0.0-1.2 MCV 71 fL 64-71 Eos. 0 % 0.0 × 103/µL 0.0-0.8 MCHC 34.5 g/dL 32.9-35.2 Baso. 0 % 0.0 × 103/µL 0.0-0.1 MCH 24 pg 21-26 nRBC 0 /100 WBC 0-1 Platelets 131 × 103/µL 106-424

3-6 WBC CVC: Blood film from dog • Case: 275449 (465) • Dog, hound-mix breed, M(c), 5-yr-old Dog was presented for the excision of a small dermal mass. The presurgical CBC results were basically within reference intervals except for microscopic findings

CBC (plasma clear) pTP 7.2 g/dL 6.0-8.0 WBC 15.0 × 103/µL 6.0-17.0 Hct (s) 45 % 37-55 Seg. neut. × 103/µL 3.0-11.5 Hct (c) 44 % 37-55 Band neut. × 103/µL 0.0-0.3 Hgb 14.8 g/dL 12.0-18.0 Lymph. × 103/µL 1.0-4.8 RBC 6.4 x 106/µL 5.5-8.5 Mono. × 103/µL 0.2-1.4 MCV 69 fL 62-76 Eos. × 103/µL 0.0-0.8 MCHC 34 g/dL 33-37 Baso. × 103/µL 0.0-0.1 MCH 23 pg 21-26 nRBC 0 /100 WBC 0-1 Platelets 280 × 103/µL 200-500

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Cases of Cavitary Effusion: What’s Filling that Space?? Steven Stockham, DVM, MS, DACVP (Clinical Pathology) Kansas State University Manhattan, KS

Pleural or peritoneal fluid analysis will be classified by evaluating the results of routine fluid analysis and virtual microscopy of digital slides. A flow chart that can be used to help establish the type of effusion is present on the first page of the previous topic (Cases of cavitary effusions: What's filling this space?).

7-1 Cavitary effusion CVC: Pleural fluid, cytocentrifuge prep. • Case: 079234 (647) (7-2 Peritoneal fluid -- next slide, same animal) • Dog, Labrador retriever, male (neutered), 8-yr-old The dog was referred because of an acute onset of a distended abdomen and a hypoproteinemia (TP = 4.2 g/dL, Alb = 2.2 g/dL). Physical examination revealed a distended abdomen due to a peritoneal effusion and muffled heart sounds. Pleural and peritoneal fluid samples were collected and submitted for analysis. Patient Patient Color, precentrifugation Blood-tinged TNCC < 1,000/µL Clarity, precentrifugation Cloudy Neutrophils % Color, postcentrifugation Colorless Monocytes/macrophages % Clarity, postcentrifugation Clear Lymphocytes % Total protein (ref) 2.9 g/dL Reactive mesothelial cells % Hct < 3 % Other %

7-2 Cavitary effusion CVC: Peritoneal fluid, cytocentrifuge prep. • Case: 079234 (651) • Dog, Labrador retriever, male (neutered), 8-yr-old • See CVC 2-1 information Patient Patient Color, precentrifugation Blood-tinged TNCC < 1,000/µL Clarity, precentrifugation Cloudy Neutrophils % Color, postcentrifugation Colorless Monocytes/macrophages % Clarity, postcentrifugation Clear Lymphocytes % Total protein (ref) 3.0 g/dL Reactive mesothelial cells % Hct < 3 % Other %

7-3 Cavitary effusion CVC: Peritoneal fluid, cytocentrifuge prep. • Case: 079660 (655) • Horse, quarter horse, male (neutered), 20-yr-old The horse was referred because of an acute colic that now is of 24-hours duration. Physical examination revealed pawing and kicking of abdomen, tachycardia, and very few gut sounds. Patient Patient Color, precentrifugation Yellow TNCC < 1,000/µL Clarity, precentrifugation Hazy Neutrophils % Color, postcentrifugation Yellow Monocytes/macrophages % Clarity, postcentrifugation Clear Lymphocytes % Total protein (ref) 1.8 g/dL Reactive mesothelial cells % Hct < 3 % Other %

7-4 Cavitary effusion CVC: Peritoneal fluid, direct smear • Case: 507605 (667) • Horse, Thoroughbred cross, male (castrated), 14-yr-old The horse was presented because of colic of 12-hr duration. The referring veterinarian reported that the horse passed a small amount of mucoid feces yesterday, rectal palpation revealed gas-distended loops of intestine, and gut sounds were absent. A small amount of peritoneal fluid was collected and submitted for analysis.

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Patient Patient Color, precentrifugation yellow TNCC 202,000/µL Clarity, precentrifugation cloudy Neutrophils % Color, postcentrifugation yellow Monocytes/macrophages % Clarity, postcentrifugation clear Lymphocytes % Total protein (ref) 5.5 g/dL Reactive mesothelial cells % Hct < 3 % Other %

7-5 Cavitary effusion CVC: Peritoneal fluid, direct smear • Case: 051233 (613) • Dog, Labrador retriever, male (neutered), 6-yr-old Owner first noticed abdominal distension about one week ago; the dog’s appetite and activity has not changed. Physical examination revealed a fluid-filled, distended abdomen and possibly a peripheral lymphadenopathy. Patient Patient Color, precentrifugation Red TNCC 7,500/µL Clarity, precentrifugation Opaque Neutrophils % Color, postcentrifugation Pink Monocytes/macrophages % Clarity, postcentrifugation Hazy Lymphocytes % Total protein (ref) 5.0 g/dL Reactive mesothelial cells % Hct 30 % Other %

7-6 Cavitary effusion CVC: Peritoneal fluid, cytocentrifuge prep. • Case: 079781 (658) • Dog, Anatolian shepherd, male (neutered), 8-yr-old The dog had intermittent episodes of diarrhea for about 2 months. About 2 weeks ago, it was dribbling urine and the referring veterinarian treated for a urinary tract infection. Urine dribbling continued up to yesterday; no urine passed in last 24 hours. Physical examination revealed a depressed dog with a distended and painful abdomen. Initial laboratory data included a mild inflammatory leukocytosis, mild hyperproteinemia, almost an erythrocytosis, azotemia (UN − 105 mg/dL, Crt 3.6 mg/dL), mild hyperphosphatemia, mild hyponatremia, almost hyperkalemia, and metabolic acidosis (HCO3 14 mmol/L) Patient Patient Color, precentrifugation Blood-tinged TNCC 2,700/µL Clarity, precentrifugation Cloudy Neutrophils % Color, postcentrifugation Pink Monocytes/macrophages % Clarity, postcentrifugation Clear Lymphocytes % Total protein (ref) 1.3 g/dL Reactive mesothelial cells % Hct < 3 % Other %

7-7 Cavitary effusion CVC: Peritoneal fluid, line prep. • Case: 08-69874 (616) • Dog, mixed breed, female, 1-yr-old A veterinarian in NE Kansas submitted pleural and peritoneal fluid from a dog. Historical or physical examination findings were not provided. Patient Patient Color, precentrifugation Blood-tinged TNCC 10,200/µL Clarity, precentrifugation Cloudy Neutrophils % Color, postcentrifugation Colorless Monocytes/macrophages % Clarity, postcentrifugation Clear Lymphocytes % Total protein (ref) 4.7 g/dL Reactive mesothelial cells % Hct < 3 % Other % Other microscopic findings: Note: A line preparation concentrates cells in the line, but also makes that area thick. Note: The analysis of pleural fluid yielded essentially the same results except the TNCC was 5,000/µL.

7-8 Cavitary effusion CVC: Pleural effusion, cytocentrifuge preparation • Case: 047859 (604)

263

• Cat, Birman, male (neutered), 16-yr-old The cat was presented because of dyspnea. The owner reported intermittent inappetence during past week. Physical examination revealed muffled heart sounds. Radiographs revealed a pleural effusion – fluid was collected for analysis. Patient Patient Color, precentrifugation Pink TNCC 8,000/µL Clarity, precentrifugation Hazy Neutrophils % Color, postcentrifugation Light yellow Monocytes/macrophages % Clarity, postcentrifugation Clear Lymphocytes % Total protein (ref) 2.6 g/dL Reactive mesothelial cells % Hct < 3 % Other %

7-9 Cavitary effusion CVC: Peritoneal fluid, cytocentrifuge prep. • Case: 080190 (663) • Dog, fox terrier, male (neutered), 6-yr-old The dog was referred because of abdominal ascites that might be due to heart failure. Physical examination revealed a grade 2-3, left- sided systolic murmur and a fluid-filled abdomen. Preliminary serum laboratory data found UN of 16 mg/dL, Crt 3.6 mg/dL), hypoproteinemia (TP 2.5 g/dL, albumin 1.2 g/dL), hypocalcemia (tCa2+ 5.7 mg/dL), mild hyponatremia (144 mmol/L), normochloremia, decreased anion gap, and urine with a specific gravity of 1.009, and negative chemistry results. Patient Patient Color, precentrifugation colorless TNCC < 1,000/µL Clarity, precentrifugation clear Neutrophils % Color, postcentrifugation colorless Monocytes/macrophages % Clarity, postcentrifugation clear Lymphocytes % Total protein (ref) 0.1 g/dL Reactive mesothelial cells % Hct < 3 % Other %

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Cases of Cavitary Effusion: What’s Filling this Space? Steven Stockham, DVM, MS, DACVP (Clinical Pathology) Kansas State University Manhattan, KS

Pleural or peritoneal fluid analysis will be classified by evaluating the results of routine fluid analysis and virtual microscopy of digital slides.

Guidelines: variations occur

Effusion

[TP] ≥ 2.0 g/dL

[TP] < 2.0 g/dL

TNCC < TNCC > 5,000/µL

• Protein-poor transudate • Uroperitoneum (early) Most exudates > 80 % neutrophils > 80 % neutrophils & macrophages Hemorrhagic (reddish) Protein-rich transudate • Early: mimics blood Infectious > 80 % neuts & macro’s Hct near blood • Bacterial • Heart failure • Later: erythrophages • Fungal • Post-sinusoidal siderophages • Protozoal congestion • Parasitic Neoplastic lymphoid Hypocellular exudates effusion • > 80 % neutrophils • Neoplastic lymphocytes Noninfectious • > 80 % neutrophils & • Neoplasms

macrophages • Foreign body

Common in FIP • Necrotic tissue Other neoplastic effusions • Bile or urine Chylous • Inflammatory cells • Creamy to white • Large atypical cells • Mostly lymphocytes (early) • Lymph, mac’s, neuts later

6-1 Cavitary effusion CVC: Peritoneal fluid; direct smear • Case: 315135 (630) • Cat, DSH, female (spayed), 8-yr-old The cat was presented because of a sudden onset of lethargy, anorexia, and more recently, vomiting. Physical examination revealed an increased rectal temperature, mild dehydration, depression, abdominal tenderness, and abdominal distension. Radiographs revealed a peritoneal effusion – fluid was collected for analysis. Patient Patient Color, precentrifugation Tan TNCC 115,000/µL Clarity, precentrifugation Cloudy Neutrophils % Color, postcentrifugation Colorless Monocytes/macrophages % Clarity, postcentrifugation Clear Lymphocytes %

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Total protein (ref) 5.1 g/dL Reactive mesothelial cells % Hct < 3 % Other %

6-2 Cavitary effusion CVC: Peritoneal fluid, direct smear • Case: 028595 (622) • Cat, DSH, male (neutered), 9-mo-old The cat was presented because of a progressive lethargy and inappetence during the past week. Physical examination revealed an increased rectal temperature, mild dehydration, and abdominal distension; the abdomen did not appear tender or painful. Radiographs revealed a peritoneal effusion – fluid was collected for analysis. Patient Patient Color, precentrifugation Yellow TNCC ** Clarity, precentrifugation Hazy Neutrophils % Color, postcentrifugation Yellow Monocytes/macrophages % Clarity, postcentrifugation Almost clear Lymphocytes % Total protein (ref) 5.1 g/dL Reactive mesothelial cells % Hct < 3 % Other % ** The viscosity of the fluid prevents accurate pipetting and thus a total nucleated cell concentration cannot be determined accurately.

6-3 Cavitary effusion CVC: direct smear (3a) and cytocentrifuge (3b) • Case: 040896 (607, 610) • Cat, DSH, female (neutered), 8-yr-old The cat was presented because it was having a hard time breathing. Physical examination revealed muffled heart sounds. Radiographs revealed a pleural effusion – fluid was collected for analysis. Patient Patient Color, precentrifugation White TNCC 3.200/µL Clarity, precentrifugation Opaque Neutrophils % Color, postcentrifugation White Monocytes/macrophages % Clarity, postcentrifugation Opaque Lymphocytes % Total protein (ref) 5.3 g/dL Reactive mesothelial cells % Hct < 3 % Other %

6-4 Cavitary effusion CVC: Pleural fluid, sediment smear. • Case: 175950 (637) • Dog, Irish setter, male (neutered), 4-yr-old The dog was presented because of difficult breathing. The owner reported intermittent inappetence for the past two weeks; also, the dog did seemed to tire easily. Physical examination revealed a lethargic dog with muffled heart sounds. Radiographs revealed a pleural effusion – fluid was collected for analysis. Patient Patient Color, precentrifugation Pale yellow TNCC 35,000/µL Clarity, precentrifugation Cloudy Neutrophils % Color, postcentrifugation None Monocytes/macrophages % Clarity, postcentrifugation Clear Lymphocytes % Total protein (ref) 3.8 g/dL Reactive mesothelial cells % Hct < 3 % Other %

6-5 Cavitary effusion CVC: Peritoneal fluid • Case: 028757 (640) • Dog, Yorkshire terrier, female (spayed), 9-yr-old The dog was presented because of an acute onset of vomiting. Physical examination revealed icteric mucous membranes and intense abdominal pain. Peritoneal fluid was collected for analysis. Patient Patient Color, precentrifugation Icteric TNCC 32,100/µL Clarity, precentrifugation Cloudy Neutrophils % Color, postcentrifugation Icteric Monocytes/macrophages % Clarity, postcentrifugation Nearly clear Lymphocytes % Total protein (ref) 4.3 g/dL Reactive mesothelial cells %

266

Hct < 3 % Other %

6-6 Cavitary effusion CVC: Peritoneal fluid • Case: 12-115895 (1727) • Dog, German shepherd, female, 8-yr-old The dog was presented because of an acute onset of vomiting. Physical examination revealed intense abdominal pain. Peritoneal fluid was collected for analysis. Patient Patient Color, precentrifugation Dark yellow TNCC Clot in sample Clarity, precentrifugation Cloudy Neutrophils % Color, postcentrifugation Dark yellow Monocytes/macrophages % Clarity, postcentrifugation Nearly clear Lymphocytes % Total protein (ref) 4.0 g/dL Reactive mesothelial cells % Hct < 3 % Other %

6-7 Cavitary effusion CVC: Peritoneal fluid, cytocentrifuge prep • Case: 075542 (620) • Dog, Cairn terrier, female (spayed), 3-yr-old The dog was presented because icterus and difficult breathing. Physical examination revealed a distended abdomen due to a peritoneal effusion. Peritoneal fluid was collected and submitted for analysis. Patient Patient Color, precentrifugation Blood-tinged TNCC < 1,000/µL Clarity, precentrifugation Hazy Neutrophils % Color, postcentrifugation Light yellow Monocytes/macrophages % Clarity, postcentrifugation Clear Lymphocytes % Total protein (ref) 0.5 g/dL Reactive mesothelial cells % Hct < 3 % Other %

6-8 Cavitary effusion CVC: Peritoneal fluid direct smear • Case: ASVCP 10-9 (885) • Dog, miniature Australian shepherd, female (spayed), 8-mo-old One week after intestinal resection, the dog was presented because of anorexia. Physical examination revealed a distended abdomen due to a peritoneal effusion. A direct smear of peritoneal fluid was prepared and submitted for evaluation (fluid was not available for analysis).

6-9 Cavitary effusion CVC: Peritoneal fluid, cytocentrifuge prep • Case: ASVCP 08-9 (1166) • Dog, Nova Scotia Duck-tolling retriever, male (neutered), 5-yr-old The dog was presented because hematemesis and melena. Physical examination revealed pale mucous membranes. Abdominal ultrasound demonstrated multiple enlarge abdominal lymph nodes and a peritoneal effusion. Peritoneal fluid was collected and submitted for analysis. Patient Patient Color, precentrifugation Light yellow TNCC 49,500/µL Clarity, precentrifugation Hazy Neutrophils % Color, postcentrifugation Light yellow Monocytes/macrophages % Clarity, postcentrifugation Clear Lymphocytes % Total protein (ref) 3.2 g/dL Reactive mesothelial cells % Hct < 3 % Other %

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Is this Lymphoma- or Just a Reactive Lymph Node? Steven Stockham, DVM, MS, DACVP (Clinical Pathology) Kansas State University Manhattan, KS

The major reason for a cytologic biopsy of lymph node aspirates is looking for the reason for an enlarged lymph node. Lymph nodes become enlarged from many diseases and typically are classified into one of the following groups.

Hyperplastic lymph node Lymph node hyperplasia is characterized by increased numbers of lymphocytes: B-lymphocytes, T-lymphocytes, or both. The proportions of different types of lymphocytes may appear normal, in which case hyperplasia is suggested by normal cell populations in association with lymphadenomegaly. There may be increases in large lymphocytes and/or plasma cells, in which case the terms reactive or reactive hyperplasia are often used in place of hyperplasia, though the nodes are enlarged because of hyperplasia. A variety of infectious and noninfectious diseases, including bacterial, viral, fungal, and neoplastic disorders, can lead to the stimulation and proliferation of lymphocytes. If there is generalized lymph node hyperplasia, a systemic illness should be considered. If only one node is hyperplastic, a disease within the drainage field of that node should be considered.

Reactive lymph node A node classified as reactive typically has increased numbers of plasma cells and/or large lymphocytes. The percentage of large lymphocytes is expected to be less than 50 % in a reactive node and is usually less than 10 %. An increase in plasma cells indicates B- lymphocyte stimulation. The causes of a reactive lymph node are essentially the same as those for lymph node hyperplasia.

Lymphadenitis Lymphadenitis is characterized by an increased number of nonlymphoid inflammatory cells in a lymph node. One inflammatory cell type might dominate (e.g., neutrophils), or there can be a mixture of inflammatory cells (e.g., neutrophils, macrophages, and eosinophils). The cause of the inflammatory state may be within the lymph node or, more commonly, in the node’s drainage field. For example, an allergic dermatitis may result in an eosinophilic lymphadenitis, or a lymph node draining a necrotic hemorrhagic lesion may have many macrophages containing cell debris and Fe pigments. Lymphadenitis is often associated with reactive (proplastic) changes, and the term reactive lymphadenitis is sometimes used to reflect both changes

Lymphoma Cytologically, lymphoma can be diagnosed when there is nearly a single population of atypical lymphocytes rather than the heterogeneous mixture of typical cell types present in normal, reactive, or inflamed lymph nodes. However, depending on the appearance of the cells, lymphoma can be an easy or difficult diagnosis cytologically. When cytologic preparations consist of single populations of large lymphocytes with prominent nucleoli, the diagnosis of lymphoma is clear. It is more difficult when the cells are of small to intermediate size or when substantial numbers of non-neoplastic cells are intermixed with neoplastic cells because of a nondiffuse form or a recent onset. In these cases, histologic examination may be necessary for a diagnosis.

Metastatic neoplasm Lymph nodes can be enlarged because of the growth of non-lymphoid neoplastic cells in the node. Metastatic cells can also be found during biopsies of lymph nodes that do not appear enlarged. Many neoplasms have the potential to spread to regional lymph nodes. Those seen more frequently in the peripheral lymph nodes included squamous cell carcinoma, mammary carcinoma or adenocarcinoma, melanoma, mast cell neoplasia, and some hemic neoplasms.

Cell populations in lymphadenopathies other than lymphoma Typical lymph nodes include popliteal, inguinal, and prescapular lymph nodes. Percentages are provided to illustrate the differences between the pathologic states. They are not firm decision limits; a true differential count is rarely completed. Normal* Hyperplasia Hyperplasia Hyperplasia Lymphadenitis*** Metastatic #1** #2 (reactive) neoplasm Lymphoid > 95 % > 95 % > 95 % > 95 % ??? Varies; depends on Small > 80 % > 80 % > 60 % > 60 % ? > 60 % how much of the Intermediate < 10 % < 10 % < 30 % < 30 % ? < 30 % LN has been Large < 5 % < 5 % < 10 % < 10 % ? < 10 % replaced by

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Plasma cells < 2 % < 2 % < 2 % > 2 % ? < 2 % neoplastic cells Neutrophils < 2 % < 2 % < 2 % < 2 % ? > 2 % Macrophages < 2 % < 2 % < 2 % < 2 % ? > 2 % Mast cells < 1 % < 1 % < 1 % < 1 % ? > 1 % Organisms ------Maybe Yes * Mandibular lymph nodes and mesenteric lymph nodes frequently have higher percentages of neutrophils, macrophages, or plasma cells ** The cell populations in this hyperplastic lymph node look like normal lymph node cells, but they came from an enlarged lymph node. *** The distribution of the cell populations vary with the severity of the inflammatory process. The aspirate may look like a normal LN with only a minor increase in neutrophil percentage. Or, the aspirate may contain very few lymphoid cells as nearly all of the cells are inflammatory cells.

Cell populations in most lymphomas* Lymphoma Lymphoma Lymphoma** (intermediate cell) (large cell) (small cell) Lymphoid > 90 % > 90 % > 90 % Small < 50 % > 10 % > 80 % Intermediate > 20 % > 30 % < 10 % Large < 10 % > 30 % < 5 % Plasma cell < 2 % < 2 % < 2 % Neutrophils < 5 % < 2 % < 2 % Macrophages < 5 % < 2 % < 2 % Mast cells < 1 % < 1 % < 1 % * Lymphoma classification based on the diameters of most of the neoplastic lymphoid cells in the sample: small cell = nuclei < 10 µm; intermediate (medium) cell = nuclei 10–15 µm; large cell = nuclei > 15 µm ** The small-cell lymphoma is difficult to recognize with certainty in an aspirate; the cell populations are similar to those of a normal lymph node or a hyperplastic lymph node. Histopathologic examination of an incised or excised lymph node is typically needed to establish the diagnosis.

8-1 Lymph node CVC: Mandibular LN aspirate • Case: 053394 (1346) • Dog, Labrador retriever, 4-yr-old Healthy dog Lymphoid % Neutrophils % Small lymphocytes % Macrophages % Intermediate lymphocytes % Mast cells % Large lymphocytes % Organisms Plasma cells %

8-2 lymph node CVC: Mandibular LN aspirate • Case: 021111 (2407) • Dog, German shepherd, 3-yr-old, female (spayed) The dog was presented because inappetence and lethargy. Physical examination revealed several mildly enlarged peripheral lymph nodes. An aspirate from the right mandibular lymph node was submitted for analysis. Lymphoid % Neutrophils % Small lymphocytes % Macrophages % Intermediate lymphocytes % Mast cells % Large lymphocytes % Organisms Plasma cell %

8-3 Lymph Node CVC: Axillary LN aspirate • Case: 079237 (548) • Dog, German shepherd, 3-yr-old, female (spayed) The dog was presented because of right, foreleg lameness. Radiographs revealed a small lytic bone lesion in the humerus. An aspirate from an enlarged axillary lymph node was submitted for analysis.

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Lymphoid % Neutrophils % Small lymphocytes % Macrophages % Intermediate lymphocytes % Mast cells % Large lymphocytes % Organisms Plasma cells %

8-4 Lymph node CVC: Prescapular LN aspirate • Case: 053708 (554) • Dog, Golden retriever, 5-yr-old, female (spayed) The dog was presented because of anorexia and lethargy. Several peripheral lymph nodes were enlarged. An aspirate from an enlarged prescapular lymph node as submitted for analysis. Lymphoid % Neutrophils % Small lymphocytes % Macrophages % Intermediate lymphocytes % Mast cells % Large lymphocytes % Organisms

8-5 Lymph node CVC: Popliteal LN aspirate • Case: 032086 (453) • Dog, Basset hound, 6-yr-old, male (neutered) The dog was presented because of polyuria and polydipsia. Initial laboratory data revealed a hypercalcemia. A slightly enlarged popliteal lymph node was aspirated and the sample was submitted for analysis. Lymphoid % Neutrophils % Small lymphocytes % Macrophages % Intermediate lymphocytes % Mast cells % Large lymphocytes % Organisms Plasma cells %

8-6 Lymph node CVC: Inguinal LN aspirate • Case: 028587 (539) • Cat, Tabbi, female (spayed), 8 years old The cat was presented because of weight loss and poor appetite. Physical examination revealed enlarged peripheral lymph node. One lymph node was aspirated and cytologic preparations were submitted for examination. Lymphoid % Neutrophils % Small lymphocytes % Macrophages % Intermediate lymphocytes % Mast cells % Large lymphocytes % Organisms Plasma cells %

8-7 Lymph node CVC: Popliteal LN aspirate • Case: 028260 (542) • Dog, Boxer, 7-yr-old, male (neutered) A cutaneous mass on the left hind leg had been removed 10 days ago. The excised mass was not submitted for histopathologic examination. When the dog was returned for suture removal, an enlarged popliteal lymph node was found. An aspirate of the lymph node was submitted for analysis. Lymphoid % Neutrophils % Small lymphocytes % Macrophages %

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Intermediate lymphocytes % Mast cells % Large lymphocytes % Organisms Plasma cells %

8-8 Lymph node CVC: Prescapular LN aspirate • Case: 028445 (537) • Dog, Cairn terrier, 2-yr-old, female The dog was presented because it was constantly scratching ears and neck. Physical examination revealed numerous fleas, red inflamed skin, and enlarged mandibular and prescapular lymph nodes. An aspirate of the lymph node was submitted for analysis. Lymphoid % Neutrophils % Small lymphocytes % Macrophages % Intermediate lymphocytes % Mast cells % Large lymphocytes % Organisms Plasma cells %

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