Practical

1. 101 2. Blood Loss Anemia 3. Hemolysis 4. Non-Regenerative Anemia Practical Hematology 5. Transfusion Medicine 6. Polycythemia 7. Disease Wendy Blount, DVM 8. Coagulopathy 9. Central IV Lines 10.Leukophilia 11. 12.Splenic Disease

Leukocytosis Leukocytosis

Total WBC x %cell = absolute Use WBC percentages only to calculate absolutes Count: - Bands Lymphocytes Eosinophils Basophils Hypersegmented Look at the blood smear for every CBC with More than 5 nuclear lobes abnormalities No automated Diff identifies band cells Prolonged inflammation Many fail to identify basophils

Leukocytosis Leukocytosis

lymphocyte Anaplasma phagocytophilum

1 Leukocytosis Leukocytosis

Band neutrophil

Leukocytosis Leukocytosis

monocyte Segmented neutrophil

Leukocytosis Leukocytosis

RBC - Basophilic stippling Giant platelet

2 Leukocytosis Leukocytosis

polychromatophil Lymphocyte

Leukocytosis Leukocytosis

basophil RBC – distemper inclusions

Leukocytosis Leukocytosis

RBC – Howell Jolly Bodies Eosinophil

3 Leukocytosis Leukocytosis

monocyte Histoplasma spp

Leukocytosis Leukocytosis

Mast cell eosinophil

Leukocytosis Leukocytosis

basophil Mast cell

4 Leukocytosis Leukocytosis

Segmented neutrophil basophil

Leukocytosis Leukocytosis

monocyte nRBC Counted in the WBC, not the RBC

Leukocytosis Leukocytosis

toxic band neutrophil eosinophil Dohle bodies

5 Leukocytosis Leukocytosis

monocyte Segmented neutrophil

Leukocytosis Leukocytosis

monocyte Hepatozoon spp gamont

Leukocytosis Leukocytosis

Lymphocyte band eosinophil

6 Leukocytosis Leukocytosis

Segmented neutrophil Activated lymphocyte

Leukocytosis Leukocytosis

Segs vs. Bands vs. Monocytes • Nucleus shape • 1 indentation indicates seg, bands have no dents • Bands parallel sides, segs & monos don’t • Segs thinner nucleus, bands fatter, monos thin to round nucleus • Seg nucleus longer than band • Nucleus color – segs darkest, monos lightest • Chromatin pattern – segs most clumps Ehrlichia morulae (heterochromatin), then bands, monos lacy • Cytoplasm color – segs/bands light, monos lavender

Neutrophilia DDx • Two “pools” of neutrophils in circulation • Infection • TBNP - Total Blood Neutrophil Pool • Neutrophil lifespan in circulation - 10 hours • Sterile inflammation • Circulating pool – 50% K9, 25% feline • Necrosis • In blood flow • Stress/corticosteroids • sampled for CBC • Exercise/epinephrine • Marginated pool – 50% K9, 75% feline • Attached to endothelial cells • Neutrophilic leukemia • Ready to mobilize into tissue by • Neoplasia (rare) • Or back into circulation by stress or strenuous exercise (physiologic leukocytosis) • CGL • Especially in lung and spleen • Also GI and urinary tracts in health • Other tissues on demand, to form pus

7 DDx Necrosis Left Shift • Left shift indicates acute, intense inflammation • Pancreatitis • >1000/ul bands/nonsegs = left shift • Pansteatitis • 300-1000/ul = mild left shift • Immune mediated disease • Rarely Pelger-Huet Anomaly or granulocytic • Caustic substances leukemia • Venomous bites and stings • immature hyposegmented (round nuclei) • Neoplasia neutrophils indicates a more intense inflammation • Ischemia • Metamyelocytes • Myelocytes • promyelocytes • Peripheral often indicate leukemia

Left Shift Toxic Neutrophils • Left shift indicates acute, intense inflammation Changes in the cytoplasm (any or all) • >1000/ul bands = left shift • Dohle Bodies & Toxic granulation • 300-1000/ul = mild left shift • Cytoplasmic • Rarely Pelger-Huet Anomaly or granulocytic • Cytoplasmic vacuolation leukemia • Rarely giant toxic neutrophils Indicate severe disease and worse prognosis • immature unsegmented neutrophils indicates a more intense inflammation • Metamyelocytes • Myelocytes • promyelocytes • Peripheral myeloblasts often indicate leukemia

Toxic Neutrophils Chronic Inflammation DDx toxic neutrophils - toxemia • indicates inflammatory process is at • – pyometra, parvovirus, pneumonia least 10 days old • Viral infection – feline URI • Elevated globulins also indicate chronicity • Toxic disease – acute renal failure • Left shift rarely seen • Necrosis – pancreatitis, neoplasia, etc. • WBC can be normal with significant chronic • Immune mediated disease – IMHA inflammation • Massive systemic inflammation – DIC, SIRS, • Other clues: peritonitis • Recurring fever • Increased rouleau formation Toxic neutrophils appear in cats with disease • Vasculitis and other secondary immune mediated that is relatively less severe than that causing disease can develop with time toxic neutrophils in dogs Normal leukogram does not rule out significant infection or inflammation

8 Prognosis for Neutrophilia DDx Increased nRBC

• Poor prognostic indicators • Heat Stroke • Progressive degenerative left shift • More nonsegs than segs • Sepsis • Most common cause is sepsis • WBC > 60,000/ul correlated with increased • Bone Marrow Disease risk of sudden death in dogs • Splenic Disease • Extremely high mature neutrophilia • “Leukemoid response” • Post Splenectomy • Marked toxic changes in the neutrophils • Graded 1+ to 4+ • Regenerative anemia • Severe persistent lymphopenia • Iron deficiency anemia • Sustained stress on the body • Magnitude of feline neutrophilic response is less than canine

DDx Leukemoid Response Stress/Corticosteroid Response

• Internal abscess • Mild to moderate neutrophilia • Pyometra, Bacterial prostatitis • <40,000/ul in the dog • Discospondylitis • <30,000/ul in the cat • Pyothorax, septic peritonitis • Lymphopenia • Pancreatic/hepatic abscess • Eosinopenia (overlaps with normal) • Neutrophil count often will continue to • Monocytosis (>2,500/ul) in dogs accelerate for at least one week after resolving abscess • Mature neutrophilia • IMHA ( Ralph ) • Increased hypersegmented segs • Neoplasia • “right shift” • Hepatozoon canis • Onset within 4-13 hours • CLAD of Irish Setters – Canine Leukocyte • Resolves within 24 hours Adhesion protein Deficiency

Epinephrine/Exercise Response Work-Up for Occult Infection

• FeLV/FIV test in cats • Mild to moderate neutrophilia • Heartworm test in dogs • WBC move from marginalized to circulating • CBC • Lymphocytosis (6-15,000/ul) • General health profile • Especially cats • Electrolytes and venous blood gases • Increased HCT • Thoracic and abdominal x-rays • Splenic contraction • GlobalFAST®, Abdominal ultrasound • Urinalysis and urine culture Marked Neutrophilia >50,000/ul in the dog, >30,000 in the cat • Look especially hard for infection if: • Toxic neutrophils Severe Neutrophilia >100,000/ul in the dog, • Degenerative left shift >50,000 in the cat • Pronounced rouleaux

9 Work-Up for Occult Infection DDx Monocytosis

• Echocardiogram if murmur • Chronic infection >10 days • “to and fro” murmur at left heart base • Necrosis • bounding pulses • Infection • Blood culture when febrile • viral (especially FIP) • use ARD (antimicrobial removal device) if • Fungal on antibiotics • Mycobacterial • 2 samples several hours apart • L-form, mycoplasma, Ureaplasma • Collect aseptically • Parasitic • CSF tap if neck/back pain or CNS deficits • Tissue foreign body • Joint taps if joint swelling or shifting lameness • Neoplasia • CPK if muscle pain • Immune mediated inflammation • Muscle biopsies or PCR if Hepatozoon • Corticosteroids (lymphopenia, eosinopenia) suspected or increased CPK

DDx Lymphocytosis Lymphocytosis

• Stress/epinephrine response • Chronic infection (activated lymphocytes) • Viremia • Ehrlichia spp., Anaplasma spp. • Toxoplasma gondii • Immune mediated disease (esp feline IMHA) • Recent vaccination • Lymphoid neoplasia • Addison’s Disease (5-10%) Lymphocyte

Lymphocytosis Activated lymphs Atypical cells

Immunoblast

ALL

CLL

Activated lymphocyte

CGL

10 Lymphocytosis Lymphocytosis • Activated lymphocytes – normal response • aka reactive lymphocytes, immunocytes, variant • Puppies and kittens have higher lymphocytes lymphocyte counts • Large, immunostimulated lymphocytes • And more immunoblasts • Dark blue cytoplasm with perinuclear clear zone (making IgM & IgG) • Irregular, scalloped or cleaved nuclei • Adult dogs >2 years – lymphs <1000/ul • Present with lymphoid hyperplasia • Immunoblasts – marked inflammation • 8-24 month puppies – 1000-1500/ul • aka blast transformed lymphocytes • 3-6 month puppies – 1,500-2,000/ul • Lighter, more lacy chromatin in large nucleus • Prominent nucleoli or nucleolar rings • Atypical lymphocytes – usually malignant • Characteristics of malignancy • basophilic cytoplasm, heterochromic nuclei • Large and atypical nucleolus • Immature granular chromatin • Uniform population

Eosinophilia DDx • >5,000/ul – hypereosinophilia • Infection • Strong eosinophilic response • Parasitic infection of soft tissues • Insect bites, parasitic enteritis • Eosinophils of sight hounds stain • Fungal poorly – look like segs • Viral – FeLV • Rottweiler and GSD have higher • Streptococcus, Staphylococcus spp. reference range • Allergy/asthma • Immune mediated disease • Hypereosinophilic syndrome - Rottweilers, cats • Eosinophilic granuloma – huskies, malamutes, Cavalier King Charles spaniels • Eosinophilic meningioencephalitis - Rottweilers • PIE, eosinophilic pneumonia – huskies, malamutes

DDx Eosinophilia DDx Basophilia

• Mast Cell Tumor Basophils can be difficult to identify • Other neoplasia Mistaken for monocytes or eos • Lymphoma • Parasites • Mucinous carcinoma • fibrosarcoma • Allergy • Eosinophilic leukemia (rare) • Mast Cell Tumor • Canine estrus • Lipemia • Basophilic leukemia (very rare)

11 Leukemia Leukemia • Cell lines of leukemia • Malignant blood cells (usually blasts – atypical cells) in circulation • Acute undifferentiated (stem cell) • Or >20-30% malignant blood cells (usually • Erythroleukemia (RBC) blasts) in the marrow • Myelomonocytic (monos & grans) • Often accompanied by cytopenias in other • Granulocytic - Neutrophilic, cell lines Eosinophilic, Basophilic • Clinical signs • Monocytic • Hepatosplenomegaly • Megakaryocytic • Lymphadenopathy (FNA can be • Lymphoproliferative Leukemia diagnostic) • Lymphoblastic (ALL) • Fever, weight loss if acute • Lymphocytic (CLL) • Symptoms of cytopenias • Plasma Cells (multiple myeloma) • Mast Cells

Leukemia Leukemia

• Types of leukemia • Pre leukemia (Myelodysplasia) • Aleukemic leukemia – no cancer cells in • Bone marrow dysplasia, with maturation circulation; just in bone marrow arrest • Subleukemic leukemia – small amounts • Usually presents as cytopenia of the of cancer cells in circulation affected cell line • Leukemic leukemia – many cancer cells • Causes in circulation • FeLV • Maturity of leukemia • folate deficiency (B12 in giant schnauzers) • Acute leukemia – proliferation of blasts, • Drug and toxin exposure tends to be more severe • Sometimes responds to treatment with • Chronic leukemia – proliferation of more prednisone and cell line stimulators mature blood cells, tends to be less (Epogen®/ProCrit®, Neupogen®) severe • Multiple CBCs over time to monitor for leukemic leukemia

Leukemia Leukemia

• Lymphoproliferative Disease • CGL – chronic leukemia (rare) • Arises from the lymph nodes or other • No atypical cells as seen with acute organs with reticuloendothelial function leukemias • Spleen, liver, skin • Bands sometimes seen (left shift) • Thymus, bronchial-associated • Lymphadenopathy lymphoid tissue, GALT • LN cytology can be diagnostic – EMH • Very little lymphoid production in marrow with very rare myeloblasts • Myeloproliferative Disease • Liver and spleen may be similar • Arises from the bone marrow or other • Oddly, bone marrow aspiration is often not organs that can undergo EMH diagnostic – resembles myeloid hyperplasia • Spleen • Need bone marrow biopsy • Rarely other organs • Dogs are often not sick

12 Lysosomal Storage Diseases Acknowledgements

• Congenital enzyme defects cause Chapter 2: The , Bone accumulations of metabolites in cells Marrow Examination, and Blood Banking • Mucopolysaccharidosis (MPS) & many • Douglass Weiss and Harold Tvedten others • Small Animal Clinical Diagnosis by Laboratory • Progressive CNS disease Methods, eds Michael D Willard and Harold Tvedten, 5th Ed 2012 • Seizures eventually become uncontrollable Chapter 4: Leukocyte Disorders • Some affected children are controlled • Harold Tvedten and Rose Raskin with ketogenic diet • Small Animal Clinical Diagnosis by Laboratory • And/or musculoskeletal disease (dwarfism) Methods, eds Michael D Willard and Harold • And/or corneal dystrophy Tvedten, 5th Ed 2012 • Accumulations can be seen on manual diff CBC

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