Nuances

The Nuances of • The many faces of neutrophils Mastering Interpretation • Mechanisms of and • Causes of neutrophilia neutropenia Frances Moore, DVM, Diplomate ACVP • Artifactual neutrophilia and neutropenia Director of Clinical Pathology, Heska • Automated differential pitfalls

Image Noah’s Arkive Davis Thompson Foundation Image from Noah’s Arkive Davis Thompson Foundation Image from Noah’s Arkive Davis Thompson Foundation

NO EDTA

EDTA

Image from Noah’s Arkive Davis Thompson Foundation “Toxic Change”

• Response to marked stimulation (cells not toxic) • Cytoplasmic • Dohle bodies (also can be artifact from EDTA storage) • Cytoplasmic (primary)granules (purple/red) • Foamy cytoplasm (dispersed organelles)

Stem cell pool 20%

PP (proliferative pool) Myeloblasts, progran, Bone marrow Normal – 7 days from Reserve pool (RP) myeloblast to seg in . Cell division stops – 2-3 days Maturation and Storage pool metamyelocytes, 80% bands, segs

Orderly release – mature first

BLOOD Tissue Marginal pool (MP) Circulating pool (CP) Neutrophil Kinetics

• Samples of peripheral blood reflect a subset of the neutrophil population – the circulating pool of neutrophils (CNP)

Blood CNP Marginated pool MNP

Endothelium

• Neutrophils moving as fast as RBCs = CNP Circulating Pool • MP = Neutrophils moving more slowly than RBCs • Adhesion molecules • Neutrophils hesitatingly adherent • Uneven distribution of cells in blood vessels • Greater concentration of neutrophils in postcapillary venules Marginated pool = Circulating pool dog, horses, calves Marginated = 3X Circulating pool cats

Marginated pool Neutrophilia

Circulating Pool

Neutrophilia – What are the causes? Physiologic neutrophilia (Fear, excitement, etc) • Physiologic (epinephrine) • Catecholeamines (not steroids) • Steroid effect • MNP shift to CNP (TBN normal) • Inflammation • Neutrophilia and lymphocytosis • Neutrophil influx > egress • Lymphocytes < 10,000/uL usually • Particularly profound • 20 – 30 minute duration • Sequestered inflammation • Young animals • Up to 2 X URL dogs, horses, cows • Certain infectious agents • UP to 3 – 4 URL cats • Immune mediated hemolytic anemia • May relate to ischemia, thrombosis • Common in young horses and in • Paraneoplastic neutrophilia cats • Inherited conditions (LAD) • Uncommon in dogs Steroids and neutrophilia Steroid leukograms in various species

WBC Dogs Cats Horses Cows • Down regulation of adhesion molecules (decreased stickiness) • Shift from MNP to CNP Total WBC (X 103) 15 – 35 20 – 30 15 – 20 8 -18 • Increased bone marrow release • Decreased migration neutrophils into tissues Segs WRI • Neutrophilia, lymphopenia, , • Peak –4 –8 hours after steroid administration Bands WRI WRI WRI WRI • WBC return to normal 24 hrs after single injection and 2 – 3 days after Lymph cessation of long term steroid therapy • Continuous steroid therapy – Neutrophil count normalize in sev wks Monos WRI WRI WRI WRI • Common in dogs, not common in cats Eos WRI WRI WRI • Cows – stress, LDA, milk fever, ketosis, etc • Horses - exercise

Free Tethering Mechanisms of Neutrophilia circulating and slow Marginated rolling Bone Marrow Full Transmigration pool Crawling Firm Delivery Rate arrest adhesion

Circulating Pool

Tissue Extravasated neutrophil New information: 1. Neutrophil subsets Bone marrow delivery > tissue 2. Interaction with other immune consumption = neutrophilia cells: Dend C, B/T lymphs, NET endothelial cells and influence immune response Tissue Consumption Neutrophil response to inflammatory stimuli 3. Reverse migration from tissues Rate Inflammation and Sequestered Inflammation and neutrophilia Neutrophilic

• Neutrophilia with/without • Pyometra left shift • Neutrophilia can be • Toxic change (+/-) marked >125,000/uL • Monocytosis • Increases transiently after OHE • Usually lymphopenia but can be normal or slightly increased

Mesenteric abscess in a dog Images courtesy of Noah’s Arkive Davis Noah’s Arkive/Davis Thompson Fnd Thompson Foundation

Neutrophilia and Hepatozoon canis – Queensland Dog, 2018 anemia

• Asymptomatic to severe disease • IMHA • Lethargy, cachexia, fever, anemia • Rarely - marked leukocytosis (up • Extreme neutrophilia in severe to 175,000/uL) cases (up to 150,000/uL) • Primary mechanism may be • Rhipicephalus sanguineus is inflammation relating to effects main vector of ischemia and thrombosis • Dog ingests infected tick • Can be poor prognostic sign • Diagnosis: blood smear/buffy • Can be difficult to distinguish coat exam; PCR Image courtesy of Davis Thompson foundation from septic inflammation Paraneoplastic (30 – 40,000 - > 100,000/uL) Leukocyte Adhesion Deficiency • Irish Setters and Holstein cattle • G CSF production • Autosomal recessive • Metastatic Fibrosarcoma • Defect in adhesion glycoprotein • Pulmonary adenocarcinoma • Decreased neutrophil adhesion, impaired , minimal bactericidal activity • Rectal adenomatous polyp • Renal cell carcinoma • Gingivitis, oral ulcers, periodontitis, chronic pneumonia, nonhealing wounds • Splenic sarcomas • Squamous cell carcinoma • Marked neutrophilia (up to 100,000/uL) (cat) • Nonregenerative anemia • Mammary carcinoma (cat) • Polyclonal gammopathy

Noah’s Arkive; Davis Thompson Foundation

Neutropenia – how does it happen?

• Shift from CNP to MNP • Overwhelming tissue demand – neutrophils exiting blood into tissue Neutropenia faster than they can be replaced • Neutrophil destruction in the peripheral blood or marrow • Can be a mechanism in viral • Bone marrow lesions • Decreased or abnormal neutrophil production in the marrow • Can be a mechanism in viral infection • Breed associated Mechanisms of neutropenia Examples of Neutropenia Marginated pool BM • Shift from CNP to MNP • Initial reaction to endotoxin • Benign transitory neutropenia Circulating Pool • Excessive tissue demand • Acute bacterial infection (more common in cows)

Tissue Consumption Rate Noah’s Arkive/Davis Thompson Fnd

• Neutrophil destruction or sequestration • Hemophagocytic syndrome • • Hypersplenism Bone marrow disorders • Immune mediated – rare (dogs, • Decreased neutrophil production horses) • Viral diseases, estrogen toxicity, • Drug induced (antibiotics, irradiation, chemotherapy, antithyroid drugs, NSAIDS) marrow tumor infiltration, • Unknown (cats) hereditary bone marrow • Chronic idiopathic neutropenia? disorders • Increased ineffective granulopoiesis (normally 20% ineffective) • FeLV, myelodysplasia, marrow Noah’s Arkive/Davis Thompson Fnd tumor infiltration, Causes of falsely Counting Neutrophils increased WBC count

• WBC from analyzer • Incomplete lysis of RBCs • Manual differential • Can occur with Heinz bodies • Automated differential count with • Large platelets analyzer total WBC count • Primarily in cats • Verify on blood smear • • Estimate WBC count Platelet clumps • Verify Differential Count • Nucleated RBCs • Check for inclusions • RBC morphology

Causes of false low WBC counts How to ensure CBC results are valid

• Incompletely mixed blood • The blood smear is the tubes best QC • Common problem with microtainer tubes • Blood clots • Also low RBCs and platelets Manual Differential Count Automated Differential Counts

• Scan slide at low power • Leukocytes are stained with a fluorescent dye binds to DNA, RNA and • Exam feathered edge for large cells, platelet clumps, parasites cytoplasmic organelles. • Find “counting area” where RBC touch but not overlap • Laser flow cytometry separates cell populations on the basis of fluorescence and light scatter. • Count and identify 100 leukocytes • Or estimate differential to compare with analyzer • Some analyzers differentiate cells based on staining intensity, light scatter, size and lobularity • Perform RBC morphology • Cytograms display the leukocyte types in clouds • Species differences in cytogram cell “clouds”.

Automated differential count Leukocyte clouds: Advia; HT5 does not replace slide evaluation • Reduce the time spent in slide evaluation Neutrophils • Verify the differential count Monos • Differentials are not 100% reliable, especially if results are not normal

• Must verify with slide or risk misdiagnosis! Eosinophils

• Leukocyte morphology Lymphs • RBC morphology • Parasites/viral inclusions

WBCs are differentiated by size, complexity and granularity Leukocyte clouds: Sysmex, Procyte Leukocyte clouds: Sysmex, Procyte

Monos Monos

Eosinophils Eosinophils Lymphs Lymphs

Cat: Neutrophilia with a left shift Neutrophils Neutrophils reported as lymphocytosis

WBCs are differentiated by size, complexity and granularity Neutrophilia with left shift and convergence with lymphocyte cloud Can result in false report of lymphocytosis; (13% of 106 cat samples at a university hospital analyzed with Procyte; JVIM 2017; Tvedten H, et al)

Leukocyte clouds: Advia; HT5 Questions?

Remember to download the CE certificate Neutrophils in the handouts panel of Monos the webinar control panel. NOTE: CE certificate not available Eosinophils for watching the recording.

Lymphs Questions about CE? [email protected]

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