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Practical Hematology Practical Hematology

Leukocytosis 1. 101 2. Loss Anemia Wendy Blount, DVM 3. Hemolysis 4. Non-Regenerative Anemia 5. Transfusion Medicine 6. Polycythemia 7. 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 Basophils Hypersegmented Look at the blood smear for every CBC with More than 5 nuclear lobes abnormalities – esp if Histograms don’t match No automated Diff identifies band cells Prolonged Many fail to identify basophils

Leukocytosis Mike Overton McKinney TX

1 Leukocytosis Leukocytosis

Anaplasma phagocytophilum

Leukocytosis Leukocytosis

Band neutrophil monocyte

Leukocytosis Leukocytosis

Segmented neutrophil RBC - Basophilic stippling

2 Leukocytosis Leukocytosis

Giant platelet polychromatophil

Leukocytosis Leukocytosis

Lymphocyte basophil

Leukocytosis Leukocytosis

RBC – distemper inclusions RBC – Howell Jolly Bodies

3 Leukocytosis Leukocytosis

Eosinophil monocyte

Leukocytosis Leukocytosis

Histoplasma spp Mast cell

Leukocytosis Leukocytosis

basophil

4 Leukocytosis Leukocytosis

Mast cell Segmented neutrophil

Agnes Rupley Leukocytosis College Station TX

basophil

Leukocytosis Leukocytosis

monocyte nRBC Counted in the WBC, not the RBC

5 Leukocytosis Leukocytosis

toxic band neutrophil eosinophil Dohle bodies

Leukocytosis Leukocytosis

monocyte Segmented neutrophil

Leukocytosis Leukocytosis

monocyte Hepatozoon spp gamont

6 Leukocytosis Leukocytosis

Lymphocyte band eosinophil

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

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

DDx Necrosis • Left shift indicates acute, intense inflammation • • >1000/ul bands/nonsegs = left shift • Pansteatitis (necrosis of fat) • 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 • • Metamyelocytes • Myelocytes • promyelocytes • Peripheral myeloblasts often indicate leukemia

Left Shift Toxic Neutrophils • Left shift indicates acute, intense inflammation DDx toxic neutrophils - toxemia • >1000/ul bands = left shift • Sepsis – pyometra, parvovirus, pneumonia • 300-1000/ul = mild left shift • Viral infection – feline URI • Rarely Pelger-Huet Anomaly or granulocytic • Toxic disease – acute renal failure leukemia • Necrosis – pancreatitis, neoplasia, etc. • Immune mediated disease – IMHA • immature unsegmented neutrophils indicates a • Massive systemic inflammation – DIC, SIRS, more intense inflammation • Metamyelocytes • Myelocytes Toxic neutrophils appear in cats with disease • promyelocytes that is relatively less severe than that causing • Peripheral myeloblasts often indicate toxic neutrophils in dogs leukemia

8 Chronic Inflammation Prognosis for Neutrophilia • indicates inflammatory process is at least 10 days old • Poor prognostic indicators • Progressive degenerative left shift • Elevated globulins also indicate chronicity • More nonsegs than segs • Left shift rarely seen with chronicity • Most common cause is sepsis • WBC can be normal with significant chronic • WBC > 60-75,000/ul correlated with inflammation increased risk of sudden death in dogs • Other clues: • Extremely high mature neutrophilia • Recurring • “Leukemoid response” • Marked toxic changes in the neutrophils • Increased rouleau formation • Graded 1+ to 4+ • and other secondary immune mediated • Severe persistent lymphopenia disease can develop with time • Sustained stress on the body Normal leukogram does not rule out significant • Magnitude of feline neutrophilic response infection or inflammation is less than canine

Peanut Peanut 9 year old female Chihuahua Exam • Has not been feeling well for • Temp 102.8, P-165, R-panting about a week • Eyes are red • Decreased appetite – still eating some • QAR, well hydrated • No vomiting or diarrhea, no coughing • Tenses a little on abdominal palpation • Current on preventative health • Never goes outside – uses puppy pads Diagnostics • Saw your associate a few days ago, and she • CBC, chemistries – normal is not any better on Clavamox • CBC – neutrophils 28K/ul • Chemistries – no abnormalities • UA – bacteriuria, pyuria • Dilated loops of bowel seen on ultrasound

Peanut Peanut Abdominal X-rays Abdominal Ultrasound

9 Peanut Peanut Abdominal Ultrasound Diagnosis • Pyometra

Surgery

Peanut DDx Leukemoid Response Lessons from Peanut • Internal abscess • Normal CBC does not rule out • Pyometra, Bacterial pyometra • Discospondylitis • Always ask if the animal is spayed • Pyothorax, septic peritonitis • Closed pyometra can present many ways • Pancreatic/hepatic abscess • Red eyes • Neutrophil count often will continue to • Non-specific signs accelerate for at least one week after resolving abscess • PU-PD • IMHA • High, normal or low white count • Neoplasia • sepsis • Hepatozoon canis • CLAD of Irish Setters – Canine Leukocyte Adhesion protein Deficiency

Stress/ Response Epinephrine/Exercise Response • Mild to moderate neutrophilia • Mild to moderate neutrophilia • WBC move from marginalized to circulating • <40,000/ul in the dog • Lymphocytosis (6-15,000/ul) • <30,000/ul in the cat • Especially cats • Lymphopenia • Increased HCT • (overlaps with normal) • Splenic contraction • Monocytosis (>2,500/ul) in dogs • Near death experience accompanied by • Mature neutrophilia marked hyperglycemia • Increased hypersegmented segs • “right shift” Marked Neutrophilia >50,000/ul in the dog, • Onset within 4-13 hours >30,000 in the cat • Resolves within 24 hours Severe Neutrophilia >100,000/ul in the dog, >50,000 in the cat

10 Work-Up for Occult Infection Work-Up for Occult Infection

First Tier Tests: Second Tier Tests: • CBC • Thoracic and abdominal x-rays • General health profile • GlobalFAST®, Abdominal ultrasound • Electrolytes and venous blood gases • Including FNA of liver, spleen and enlarged • Urinalysis and urine culture lymph nodes • FeLV/FIV test in cats, • Heartworm test in dogs Look especially hard for infection if: • Toxic neutrophils • Degenerative left shift • Pronounced rouleaux

Work-Up for Occult Infection Work-Up for Occult Infection

Third Tier Tests Third Tier Tests • Echocardiogram if murmur • CPK if muscle pain • “to & fro” murmur at L heart base, bounding pulses • Muscle biopsies or PCR if Hepatozoon suspected or • when febrile increased CPK • use ARD (antimicrobial removal device) if • Lung FNA, and/or BAL for respiratory signs on antibiotics • If indicated by ultrasound - • 2 samples several hours apart • Gallbladder FNA for bile culture • Collect aseptically • Pyelocentesis for urine culture • CSF tap and/or spinal rads if neck/back pain • Bone marrow sampling or CNS deficits • Especially if cytopenias, markedly high cell • Joint taps if joint swelling or shifting lameness counts, or uncharacterized atypical cells • Looking for latent FeLV, Histoplasma, other rare , occult neoplasia, etc.

Work-Up for Occult Infection Trip Third Tier Tests • Infectious Disease Panels, according to systemic signs • Respiratory PCR Panel (canine, feline) Signalment • GI Pathogen panel (canine, feline) • 2 year old castrated male border collie • Tick PCR panel Chief Complaint/History • Babesia Panel • Productive Cough, weight loss for 2 months • Hepatozoon panel • Fungal antigen tests or panels • Breathing hard for a 2 days • Etc. • Energy good; did well in agility 4 days ago • Owner thinks has had lifelong PU-PD • Has wanted to be in AC this summer – unlike last summer when he enjoyed being outside

11 Trip Trip

Exam Exam • T 102.2, P 168, R 42, CRT 3 sec • 3 murmurs: • BCS 2.5 1. PMI left base • BP 100 – To-and-fro murmur 3/6 – aortic stenosis in systole, regurg in diastole • Bounding pulses, notable in dorsal pedal artery 2. PMI left apex, but heard all over chest • Precordial – exaggerated left apical heave – Holosystolic murmur 3/6 • Lung sounds clear – Mitral regurgitation due to LHF 3. PMI Carotid artery – 2/6 ejection murmur – aortic stenosis

Cd TripPh Trip

Differential Diagnoses • VetBLUE® - ULR 3+ to infinity Ph and Cd, 1-3 Cr and • Aortic endocarditis Md • SAS with aortic regurgitation • Mitral regurgitation (endocarditis?) CBC – segs 38K/ul Md Cr Panel/lytes - normal • VetBLUE®

Trip Trip

EKG EKG • Normal sinus rhythm for 10 minutes • Normal sinus rhythm for 10 minutes Thoracic Radiographs • Interstitial pattern caudal lung fields • Vertebral heart score 10.5 • Enlarged cranial pulmonary lobar vein • Mildly enlarged left atrium • Rapid onset left congestive heart failure

12 Trip - Echo Trip - Echo

Trip - Echo Trip - Echo

Diagnosis • Aortic endocarditis

Therapeutic Plan • Elected euthanasia due to poor prognosis

Trip DDx Monocytosis

• Necrosis • Infection that attracts mononuclear cells • viral (especially FIP) • Fungal • Mycobacterial • L-form, mycoplasma, Ureaplasma • Parasitic • Tissue foreign body (Radlinksy et al) • Neoplasia • Immune mediated inflammation • Corticosteroids (lymphopenia, eosinopenia)

13 DDx Lymphocytosis • Activated lymphocytes – normal response • aka reactive lymphocytes, immunocytes, variant • Stress/epinephrine response lymphocytes • Chronic infection (activated lymphocytes) • Large, immunostimulated lymphocytes • Viremia • Dark blue cytoplasm with perinuclear clear zone (making IgM & IgG) • Ehrlichia spp., Anaplasma spp. • Irregular, scalloped or cleaved nuclei • Toxoplasma gondii • Present with lymphoid hyperplasia • Immune mediated disease (esp feline IMHA) • Immunoblasts – marked inflammation • Recent vaccination • aka blast transformed lymphocytes • Lighter, more lacy chromatin in large nucleus • Lymphoid neoplasia • Prominent nucleoli or nucleolar rings • Addison’s Disease (5-10%) • Atypical lymphocytes – usually malignant • Characteristics of malignancy • basophilic cytoplasm, heterochromic nuclei • Large and atypical nucleolus • Immature granular chromatin • Uniform population

Holly Harris Activated lymphs Atypical cells College Station TX

Immunoblast

ALL

CLL

Joshua CGL

Lymphocytosis

• Puppies and kittens have higher • >5,000/ul – hypereosinophilia lymphocyte counts • Strong eosinophilic response • And more immunoblasts • Eosinophils of sight hounds stain poorly – look like segs • Adult dogs >2 years – lymphs <1000/ul • Rottweiler and GSD have higher • 8-24 month puppies – 1000-1500/ul reference range • 3-6 month puppies – 1,500-2,000/ul

14 DDx Eosinophilia DDx Eosinophilia

• Infection • Mast Cell Tumor • Parasitic infection of soft tissues • Other neoplasia • Insect bites, parasitic enteritis • Lymphoma • Fungal • Mucinous carcinoma • Viral – FeLV • fibrosarcoma • Streptococcus, Staphylococcus spp. • Eosinophilic leukemia (rare) • / • Canine estrus • Immune mediated disease • Hypereosinophilic syndrome - Rottweilers, cats • Eosinophilic – huskies, malamutes, Cavalier King Charles spaniels • Eosinophilic meningioencephalitis - Rottweilers • PIE, eosinophilic pneumonia – huskies, malamutes

Rosey Rosey Grier Hypereosinophilic Syndrome (HES) Grier Hypereosinophilic Syndrome (HES)

• Primarily a disease of cats • Abdominal masses are possible • Persistent eosinophilia (25-30,000/ul) • Eventually causes organ failure and death • Organ infiltration with eosinophils • Difficult to distinguish from eosinophilic • Bone marrow, Spleen, Liver leukemia (EL) • Lymph nodes (often mesenteric) • May be two forms of the same disease • Gut • More immature eos in circulation with EL • skin • Treatment • Clinical Signs • No known effective treatment • Diarrhea, vomiting • Anorexia, weight loss • Corticosteroids – immunosuppressive • Intermittent, recurring fever • Hydroxyurea • Pruritus, • Alpha interferon • Gleevec® (imatinib) has been used in people (Palladia®??)

DDx Leukemia

Basophils can be difficult to identify • Malignant blood cells (usually blasts – Mistaken for monocytes or eos atypical cells) in circulation • Parasites • Or >20-30% malignant blood cells (usually • Allergy blasts) in the marrow • Mast Cell Tumor • Often accompanied by cytopenias in other cell lines • Lipemia • Clinical signs • Basophilic leukemia (very rare) • Hepatosplenomegaly • Lymphadenopathy (FNA can be diagnostic) • Fever, weight loss if acute • Symptoms of cytopenias

15 Leukemia Leukemia • Cell lines of leukemia • Types of leukemia • Acute undifferentiated (stem cell) • Aleukemic leukemia – no cancer cells in • Erythroleukemia (RBC) circulation; just in bone marrow • Myelomonocytic (monos & grans) • Subleukemic leukemia – small amounts • Granulocytic - Neutrophilic, of cancer cells in circulation Eosinophilic, Basophilic • Leukemic leukemia – many cancer cells • Monocytic in circulation • Megakaryocytic • Maturity of leukemia • Lymphoproliferative Leukemia • Acute leukemia – proliferation of blasts, • Lymphoblastic (ALL) tends to be more severe • Lymphocytic (CLL) • Chronic leukemia – proliferation of more • Plasma Cells (multiple myeloma) mature blood cells, tends to be less severe and more treatable • Mast Cells

Leukemia Leukemia

• Pre leukemia (Myelodysplasia) • Lymphoproliferative Disease • Bone marrow dysplasia, with maturation • Arises from the lymph nodes or other arrest organs with reticuloendothelial function • Usually presents as cytopenia of the • Spleen, liver, skin affected cell line • , bronchial-associated • Causes lymphoid tissue, GALT • FeLV • Very little lymphoid production in marrow • folate deficiency (B12 in giant schnauzers) • Myeloproliferative Disease • Drug and toxin exposure • Arises from the bone marrow or other • Sometimes responds to treatment with organs that can undergo EMH and cell line stimulators (Epogen®/ProCrit®, Neupogen®) $$$ • Spleen • Multiple CBCs over time to monitor for • Rarely liver, lymph nodes, etc. leukemic leukemia or cytopenias

Leukemia Pippin

• CGL – chronic leukemia (rare) • 4 month old female snowshoe – 4.2 lbs • No atypical cells as seen with acute • Had 2 generalized seizures this week Exam, neurologic exam – normal • Bands sometimes seen (left shift) CBC – vacuolated lymphocytes • Lymphadenopathy Panel – SAP 436, ALT 383 • LN cytology can be diagnostic – EMH Tx – start Zonisamide 15 mg PO SID with very rare myeloblasts • Liver and spleen may be similar • Oddly, bone marrow aspiration is often not diagnostic – resembles myeloid hyperplasia • Need bone marrow biopsy • Dogs are often not sick

16 Pippin Pippin

• Over the next 30 days Added phenobarbital – 5 mg PO BID • Seizures become more frequent • Still having 5-10 seizures per week • 2-3 per week Phenobarbital level – 35 ug/ml Zonisamide level – 15 ug/ml Added prednisone 5 mg PO SID Titrated zonisamide dose up until level 38 Seizures eventually became uncontrollable ug/ml (target 10-14 ug/ml) and Pippin was euthanized at 6 months old • Having 5-10 seizures per week. Necropsy – lysosomal storage disease Toxoplasma paired sera – IgM, IgG negative FeLV neg, FIV neg CSF tap – mononuclear cells with vacuoles, increased microprotein eye exam – central corneal precipitates, fundic exam normal

Lysosomal Storage Summary

• Congenital enzyme defects cause PowerPoints - Leukocytosis accumulations of metabolites in cells • .pptx • Mucopolysaccharidosis (MPS) & many • .pdfs – 1 and 6 slides per page others • Progressive CNS disease Articles • Seizures eventually become • ACVIM - Ehrlichiosis uncontrollable • Some affected children are controlled with ketogenic diet (Lorenzo’s Oil) • And/or musculoskeletal disease (dwarfism) • And/or corneal dystrophy • Accumulations can be seen on manual diff CBC

Acknowledgements

Chapter 2: The , Bone Marrow Examination, and Blood Banking • Douglass Weiss and Harold Tvedten • Small Animal Clinical Diagnosis by Laboratory Methods, eds Michael D Willard and Harold Tvedten, 5th Ed 2012

Chapter 4: Leukocyte Disorders • Harold Tvedten and Rose Raskin • Small Animal Clinical Diagnosis by Laboratory Methods, eds Michael D Willard and Harold Tvedten, 5th Ed 2012

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