Interpretation of Canine Leukocyte Responses

Interpreting a leukogram can be a challenging task. One has to interpret both the presence of as well as the degree of the abnormality. Evaluating the leukogram, including a total leukocyte count, a differential leukocyte count, absolute numbers of specific leukocytes and examination of morphology on a blood smear, can help identify abnormalities that may suggest specific diseases such as a viral or bacterial infection or even a neoplastic process.

1. Interpretation of Canine Leukocyte Responses Gaonkar Pankaj Prakash. MVSc II year [email protected] Department of Veterinary Pathology, Nagpur Veterinary College, Maharashtra Animal & Fishery Sciences University, Nagpur, India. Masters Seminar – VPP 691

2. Introduction Neutrophilia and Neutropenia Eosinophilia and Eosinopenia Basophilia and Basopenia Lymphocytosis and Lymphopenia Monocytosis and Monocytopenia Leukogram Differentiation Pattern Approach towards Interpretation of leukogram results. INDEX

3. INTRODUCTION Total white blood cell (WBC) count - 5,000 to 14,100 cells/ μL of blood. Neutrophil -commonly encountered leukocyte. Lymphocytes - less numerous than neutrophils. Monocytes and eosinophils - less frequently. Basophils are seen rarely. The interpretation of result , while taking into account the basic factors that influence alteration in : Total number of leukocytes. Proportion of different types of leukocytes. Morphological alteration

4. Neutrophil Eosinophil Basophil Monocyte Lymphocyte

5. Frequent causes of neutrophilia: physiologic leukocytosis corticosteroid- induced leukocytosis inflammation Other causes of neutrophilia: haemolysis hemorrhage genetic defects in leukocyte adhesion molecules immune- mediated diseases Several benign and malignant neoplastic conditions cause paraneoplastic syndromes that can result in neutrophilia Thrombosis, infarction, burns, and uremia NEUTROPHILIA It is defined as > 12,000 neutrophils/ μL of blood, is a frequent observation in canine blood films

6. Mechanism: Shift from marginating to circulating pool Increased release from bone marrow Increased production by bone marrow Delayed apoptosis Decreased migration into tissue Ref- A.J.ROSENFELD , S.M.DIAL. Clinical pathology for the veterinary team. 1 st edition. Wiley-Blackwell publishing,2010 Normal neutrophil pool Normal neutrophil pool

7. Stress neutrophilia Excitement neutrophilia (physiologic) Inflammatory Neutrophilia Ref- A.J.ROSENFELD , S.M.DIAL. Clinical pathology for the veterinary team. 1 st edition. Wiley-Blackwell publishing,2010

8. Inflammation is the most frequent cause of neutrophilia !!! The intensity of the underlying disease process has a direct effect on the magnitude of the total neutrophil response. Localized purulent lesions , like abscesses, induce greater neutrophilia than do more generalized diseases, such as septicemia . Neutrophilia with a left shift is the hallmark of acute inflammatory reactions . The degree of the left shift is considered a direct indication of severity of disease. Left shifts of great magnitude may occur in dogs that have pleuritis , peritonitis, pyoderma , or pyometra . Release of myelocytes and metamyeolcytes from bone marrow is related to the increased tissue demand in purulent inflammation. In some diseases, inflammation is too mild to induce a left shift. Left shifts may be mild or nonexistent in seborrheic dermatitits , catarrhal enteritis, or hemorrhagic cystitis, conditions in which tissue demand for neutrophils is minimal.

9. NEUTROPENIA Neutropenia is decreased numbers of circulating segmented neutrophils. Mechanisms:

10. Morphologic Alterations in Neutrophils

11. Fig. Hypersegmented neutrophil from a dog. Wright’s stain. Nuclear Hypersegmentation Characteristic changes- Neutrophils with > 5 nuclear lobes are seldom observed in the blood of dogs. .....Continued Morphologic Alterations in Neutrophils

12. Nuclear Hyposegmentation Associated with- left shift of inflammation or infection Fig. Late-band and metamyelocyte neutrophils from a dog that had inflammation. Wright’s stain .....Continued Morphologic Alterations in Neutrophils

13. Pelger - Huët Anomaly: Etiology - Autosomal dominant trait (hereditary disorder of granulocyte development) Fig. Neutrophil from dog with Pelger-Huët anomaly. Notice coarse mature chromatin pattern and hyopsegmented nucleus. Wright’s stain. .....Continued Morphologic Alterations in Neutrophils

14. .....Continued Morphologic Alterations in Neutrophils Ethylenediaminetetraacetic acid (EDTA) induced Artifact: Cause-When whole blood is collected in EDTA and allowed to incubate at room temperature Cytoplasmic basophilia and foamy vacuolation (i.e. signs of toxic change) are not observed as EDTA induced artifactual change .

15. Happens in-vitro if EDTA anticoagulated blood is allowed to stand. Fig. Neutrophil aggregation

16. Cytoplasmic vacuolation- .....Continued Morphologic Alterations in Neutrophils Fig. Toxic vacuolization. Toxic Changes: A set of disease - induced morphologic alterations in neutrophils, including cytoplasmic vacuolation , cytoplasmic basophilia , Döhle bodies , or prominently stained primary granules ( toxic granulation ) is referred to as a toxic change. .

17. Döhle bodies- Angular, blue - to - gray, cytoplasmic inclusions Remnants of rough endoplasmic reticulum. Toxic Granulation- (B & D) Increased permeability of primary granule membranes to Romanowsky stains.

18. Infectious Agents : Neutrophil with negatively stained intracellular bacillus (arrow) in blood smear from a dog that has mycobacteriosis, Wright’ s stain. .....Continued Morphologic Alterations in Neutrophils

19. .....Continued Morphologic Alterations in Neutrophils (Infectious Agent ) Canine distemper virus inclusion bodies : Intracytoplasmic inclusions are round to irregularly shaped, homogeneous, magenta to gray blue structures. Fig. Magenta- colored canine distemper virus inclusion in a neutrophil, Wright’ s stain .

20. Hepatozoon canis and Hepatozoon americanum gametocytes: Fig. Two segmented neutrophils with intracytoplasmic , clear to ice-blue H. canis gametocytes. Wright’s stain. .....Continued Morphologic Alterations in Neutrophils (Infectious Agent )

21. Histoplasma capsulatum They have a small round, purple nucleus , and can occur singly or in clusters within the cytoplasm. Fig . Segmented neutrophil with intracytoplasmic Histoplasma capsulatum . Wright’s stain .....Continued Morphologic Alterations in Neutrophils

22. Ehrlichia canis morulae : The Ehrlichia morulae vary from magenta to blue- gray in color and resemble a mulberry. Fig . Three neutrophils containing morulae of Ehrlichia ewingii , Wright’s stain. .....Continued Morphologic Alterations in Neutrophils

23. Hemosiderin granules: The brown granules , that stain positive for iron. Multiple granules can be observed in some cells. The mechanism of their occurrence in neutrophils is uncertain. Fig. Segmented neutrophil with hemosiderin crystal from a dog that had immune- mediated hemolytic anemia . Wright’ s stain. .....Continued Morphologic Alterations in Neutrophils

24. Mucopolysaccharidosisis type VI ( arylsulfatase B deficiency ) and type VII : Autosomal recessive pattern Neutrophils and lymphocytes - large, pink to purple staining inclusions ( Alder- Reilly bodies ) in the cytoplasm in routine Romanowsky - stained blood films (Fig.). The granules represent intermediate products that accumulate in cells caused by the arylsulfatase B (type VI) or β - glucuronidase deficiency (type VII). Fig. Segmented neutrophil with Alder - Reilly bodies from a dog that had mucopolysaccharidosis type VII. Wright’ s stain. .....Continued Morphologic Alterations in Neutrophils

25. Causes / Mechanism of Eosinophilia : Parasitism : Eosinophil bind to parasite opsonized with IgG , IgE , complement. Release of MBP,EPO Toxic to parasite Inflammation or local hypersensitivity: MBP Degranulation of mast cell and basophil . Eosinophil release histaminase Neutralize histamine Paraneoplastic response : Release of cytokines by the neoplastic cells that promote eosinophil production. (e.g. Interleukin-5). EOSINOPHILIA Eosinophilia , defined as > 1,300 eosinophils/ μ L of blood.

26. Eosinopenia is a decreased numbers of circulating eosinophils. It may be difficult to document because the eosinophil normal range may go down to zero . True eosinopenia is difficult to document and is of limited clinical significance . Eosinopenia is most commonly attributed to increased corticosteroids (stress leukogram). Etiology- emotional or physical stress, hyperadrenocorticism ( Cushing ’s syndrome ). High dose of corticosteroid for long term It is hypothesized that corticosteroids inhibit histamine release , neutralize circulating histamine, and initiate release of cytokines that mediate alterations in eosinophil distribution. Eosinopenia

27. BASOPHILIA & BASOPENIA Basophils : Poorly lobulated nucleus- a twisted ribbon . blue - gray cytoplasm metachromatic granules -scattered , round, varied size Count: <2 % of the differential leukocyte count or an absolute count of 0– 140 basophils / μ L. Basophilia: Defined as a increase in circulating numbers of basophils ( > 140 basophils / μL of blood), is a rare event. Parasite - Dirofilaria immitis , oslerus larvae . Basopenia: Since most healthy animals have basophil counts of 0/ μL , therefore basopenia is not a relevant finding.

28. Causes of Monocytosis: Stress response : Inflammation   Recovery from acute marrow injury : E.g. secondary to chemotherapeutic agents. Paraneoplastic response : e.g. lymphoma cytokine secretion (GMSF). Monocytic/ monoblastic leukemia:   A variant of acute or chronic myeloid leukemia. Monocytopenia may be difficult to document because monocyte reference intervals often go down to zero, and has no clinical significance . Monocytosis & Monocytopenia Monocytosis is defined by numbers that exceed 1,400 monocytes / μL .

29. LYMPHOCYTOSIS Lymphocytosis : > 2,900 lymphocytes/ μ L of blood. Cause : Physiologic: Excitement - ( epinephrine response ) Vaccination Antigenic stimulation:   Chronic Infection/inflammation : (long standing exposure) Ehrlichia canis   result in proliferation of lymphocytes and expansion of the blood lymphocyte pool. Trypanosomiasis,leishmaniasis and brucellosis also may cause lymphocytosis by a similar mechanism.

30. Hypoadrenocorticism (Addison ’ s disease) : Reported to cause lymphocytosis in 11– 20% of affected dogs. The lack of lymphopenia in severely stressed dog provides supportive evidence for glucocorticoid deficiency. Fig. Lymphocytosis and Eosinophilia in dog with hypoadrenocorticism .

31. Lymphoid neoplasia : Malignant lymphoma , acute or chronic lymphocytic leukemia, and thymoma have been associated with lymphocytosis The highest lymphocyte counts usually are associated with chronic lymphocytic leukemia . This can occur in an animal of any age, but should be considered the main differential diagnosis in an older animal with an unexplained lymphocytosis.

32. LYMPHOPENIA Lymphopenia , defined as < 400 lymphocytes/ μ L blood. Causes : Stress leukogram : This is due to endogenous or exogenous corticosteroids . It is due to a shift of lymphocytes from the circulating to other pools (such as decreased efflux from lymph nodes or retention in bone marrow). Acute infection : By means of stress - induced corticosteroid release and redistribution of lymphocytes. canine distemper virus and canine parvovirus , cause lymphocyte destruction, atrophy of lymphoid tissues, and depletion of lymphocyte subpopulations. Loss of lymphocytes:   Chylothorax Loss of lymphocyte - rich fluid into the intestinal lumen. (ulcerative enteritis , granulomatous enteritis ).

33. Occlusion of the flow of lymph: Disseminated granulomatous infl ammation , Neoplasia Autosomal recessive severe combined immunodeficiency: Jack Russell terrier puppies - marked lymphopenia and decreases serum immunoglobulins hypoplasia of all lymphoid tissues. Basset hounds that have combined immunodeficiency have lymphopenia. Recurrent infection.

34. Morphologic Alterations in Lymphocytes Reactive Lymphocytes: Antigentically stimulated lymphoid cells that are seen in the blood of dogs. Morphology : Deeply basophilic cytoplasm . Vary in size, nuclear chromatin clumping , and number of nucleoli visualized. Fig . Reactive lymphocyte in a canine blood smear. Wright’s stain

35. Granular lymphocytes : They have a few distinct azurophilic granules that tend to cluster at the nuclear margin or indentation. Fig . Large granular lymphocytes in a canine blood smear. Wright’ s stain

36. Lymphoblasts lymphoma and lymphoblastic leukemia . Morphology- large nucleus with vesicular chromatin and prominent nucleoli . The cytoplasm is abundant and has a deep blue hue. Fig. Lymphoblast in blood smear of a dog that has malignant lymphoma. Notice the prominent nucleoli. Wright’s stain.

37. Acute myeloid leukemia Large number of blasts multiple cytopenias Acute lymphoid leukemia Large number of blasts small to intermediate cells( Lymohocyte ) multiple cytopenia LEUKEMIA Lymphoma   Variable number of lymphoma cells   Typically no or mild cytopenias Clinical Examination , phenotypic testing (e.g. Flowcytometry ) are required for confirmative diagnosis. Chronic leukemia Rarely Blasts Involved cell type increased Mild cytopenias

38. Acute Myeloid leukemia Lymphoid leukemia Chronic leukemia of granular lymphocytes Acute lymphoblastic lymphoma

39. Fig. Intermediate "blasts” and toxic neutrophil. Fine chromatin.(Wright's stain, 1000x). (Acute Myeloid Leukemia)

40. Eosinophilia Neutrophilia Lymphocytosis Basophilia Monocytosis Neutropenia Eosinopenia Lymphopenia Physiological Leukocytosis Inflammatory Leukocytosis Stress Leukocytosis

41. Leukogram patterns Differentiation Ref- www.eclinpath.com(Cornell Uni.)

42. Approach towards interpreting WBC results.

43. supported by a lymphopenia and/or eosinopenia . A neutrophilia, without a left shift, with or without a lymphopenia or eosinopenia  could be due to inflammation or corticosteroids, so you would need to evaluate other findings (clinical and laboratory) for evidence of inflammation. Is there evidence of endogenous corticosteroid release (“stress”) or is the animal on corticosteroid treatment? Are there abnormal cells in the blood or an unexplained lymphocytosis ?  If yes, then you may have a leukemia (acute, chronic or the leukemia phase of lymphoma).

44. Is there an inflammatory leukogram? Supported by – Left shift  (immature neutrophils in the circulation) . Toxic change  in neutrophils (which are usually found together, but not always). If there is an inflammatory leukogram, how severe is it?   severity of the left shift (degenerative generally supports a severe inflammatory response). degree of toxicity in neutrophils (marked toxic change in neutrophils would support severe inflammation). If there is inflammation, is the bone marrow responding?  Neutrophilia and mature neutrophils out number immature neutrophils , this would support a bone marrow response.

45. If there is inflammation, who is winning ? The animal or the inflammation? This can be difficult to determine from a single hemogram . A degenerative left shift , in the face of a Neutropenia or normal neutrophil count. Thus depletion of marrow stores and failure of the bone marrow to keep up with the demand from the tissue The inflammation is winning...!!! Neutrophilia and more immature than mature neutrophils Marrow has had time to respond Inflammation is worsening Here, we are not sure who is winning. Continued…

46. So how do we tell who is winning ? When in doubt, always do serial hemograms – every 24 hours (or less if really needed)! Continued from….If there is inflammation , who is winning? The animal or the inflammation?

47. Conclusion Interpretation of Leukogram Valuable Diagnostic Tool Treatment Prognosis

48. References Available on request. ([email protected])

49. THANK YOU