4/8/19 THE SIGNIFICANCE OF VARIOUS DISCLOSURES GRANULOCYTIC INCLUSIONS ¡ No relevant financial interests to disclose. KRISTLE HABERICHTER, DO, FCAP GRAND TRAVERSE PATHOLOGY, PLLC OBJECTIVES GRANULOCYTES ¡ Innate immune system ¡ Travel to sites of infection, recognize and phagocytose pathogens ¡ Recognize common and uncommon granulocytic inclusions, including those associated with certain ¡ Utilize numerous cytotoxic mechanisms to kill pathogens inherited disorders and infectious etiologies ¡ Granulopoiesis occurs in the bone marrow ¡ Sufficient stem cells, adequate microenvironment, and regulatory factors ¡ Identify newly described green neutrophilic inclusions ¡ Granulocyte colony stimulating factor (G-CSF) → Granulocytes ¡ Monocyte colony stimulating factor (M-CSF) → Monocytes ¡ Understand the clinical significance and implications of various inclusions ¡ Granulocyte-monocytes colony stimulating factor (GM-CSF) → Granulocytes & Monocytes ¡ 1-3 weeks for complete granulopoiesis to occur ¡ Neutrophils only circulate for a few hours before migrating to the tissues Photo by K. Haberichter (Giemsa, 1000x) GRANULOCYTES INCLUSION CATEGORIES ¡ Primary granules → Myeloperoxidase Reactive/Acquired Changes Congenital Abnormalities Infectious Etiologies ¡ “Late” myeloblasts and promyelocytes ¡ To x ic G r a n u la t io n ¡ Chédiak-Higashi Syndrome ¡ Anaplasma ¡ Secondary granules → Leukocyte alkaline phosphatase ¡ Döhle Bodies ¡ Alder-Reilly Anomaly ¡ Ehrlichia ¡ Myelocytes, metamyelocytes, band and segmented neutrophils ¡ Cytokine Effect ¡ May-Hegglin Anomaly ¡ Histoplasma ¡ Neutrophils ¡ Howell-Jolly Like-Body ¡ Cryptococcus ¡ Round to oval of variable size (10-18 µm) ¡ Green Neutrophilic Inclusions ¡ Talaromyces marneffei ¡ Multiple segmented nuclear lobes (2-5 lobes) connected by a thin ¡ Bilirubin Crystals filament ¡ Clumped nuclear chromatin without nucleoli ¡ Abundant pale pink-staining cytoplasm filled with many lilac colored fine granules (secondary granules) Photos by K. Haberichter (Giemsa, 1000x) 1 4/8/19 ¡ 72 yo, M with history of chronic lymphocytic leukemia ¡ Presented to ED with sepsis and pneumonia ¡ WBC: 82.7 K/µL, PMNs: 16.2 K/µL, Lymphs: 66.5 K/µL REACTIVE/ACQUIRED CHANGES Photos by K. Haberichter (Giemsa, 1000x) REACTIVE/ACQUIRED CHANGES REACTIVE/ACQUIRED CHANGES TOXIC CHANGES TOXIC CHANGES ¡ Toxic changes = excessive granulation/toxic granulation, toxic vacuolation and Döhle bodies ¡ May be seen in association together or in any combination of the above ¡ Associated with infections and inflammation ¡ Toxic granulation due to abnormal accelerated maturation from the marrow space into the circulation ¡ Infectious etiologies, particularly bacterial septicemia ¡ Burns, trauma, acute alcoholism, pregnancy and uremia ¡ Persistence of deep blue-purple color of primary/azurophilic granules ¡ Larger in size than normal granules ¡ Leukocytosis due to absolute neutrophilia ¡ Left shift to myelocytes and metamyelocytes ¡ Typically seen in neutrophils and band forms ¡ WBC usually < 30 bil/L and rarely >50 bil/L Photo by K. Haberichter (Giemsa, 1000x) ¡ May also be associated with thrombocytosis, eosinophilia, or monocytosis ¡ Thrombocytopenia may be present with sepsis ¡ 25 yo, F with no significant past medical history REACTIVE/ACQUIRED CHANGES ¡ 20 weeks pregnant DÖHLE BODIES ¡ WBC: 10.5 K/µL, PMNs: 7.5 K/µL, Lymphs: 2.0 K/µL, Monos: 1.0 K/µL ¡ Typically seen in association with other toxic changes ¡ Toxic granulation and vacuolation ¡ May be seen in numerous medical conditions including: ¡ May-Hegglin Anomaly, pregnancy, myelodysplasia and post chemotherapy ¡ Pale blue or blue-gray inclusions (1-3 µm) of variable shape ¡ Aggregates of denatured ribosomes and remnants of RNA from the rough endoplasmic reticulum ¡ Cytoplasm of neutrophils and their precursors ¡ May also be seen in eosinophils and monocytes ¡ Frequently found on the inner side of the cell wall Photos by K. Haberichter (Giemsa, 1000x) 2 4/8/19 ¡ 3 yo, M with no known medical history REACTIVE/ACQUIRED CHANGES ¡ WBC: 55.4 K/µL, PMNs: 19.4 K/µL with left shift CYTOKINE EFFECT ¡ Granulocytic colony stimulating factor (G-CSF) ¡ Neupogen, Granix, or Zarxio (Filgrastim) ¡ Granulocytic-macrophage colony stimulating factor (GM-CSF) ¡ Leukine (Sargramostim) ¡ Why are cytokines administered? ¡ Used to increase the amount of neutrophils in circulation in neutropenic patients ¡ Patients undergoing stem cell mobilization procedures ¡ Stem cell transplants or stem cell donations Photos by K. Haberichter (Giemsa, 1000x) REACTIVE/ACQUIRED CHANGES CYTOKINE EFFECT Barr Body Howell-Jolly Body-Like Karyorrhexis ¡ Peripheral blood changes include: ¡ Bone marrow changes include: ¡ “Toxic” granulation ¡ Increased granulation, similar to the peripheral blood changes ¡ Granulocytic left shift with a transient ¡ Increased myeloid:erythroid ratio increase in circulating blasts ¡ Binucleated promyelocytes and ¡ Döhle bodies myelocytes ¡ Vacuolation ¡ Giant myeloid precursors ¡ Variable nuclear-cytoplasmic dyssynchrony ¡ Abnormalities in nuclear segmentation Photos by K. Haberichter (Giemsa, 1000x) ¡ 48 yo, F with no known medical history REACTIVE/ACQUIRED CHANGES ¡ Presented to ED with septic shock secondary to MRSA pneumonia HOWELL-JOLLY BODY-LIKE INCLUSIONS ¡ WBC: 31.2 bil/L, PMNs: 25.9 bil/L, ALT: 890 U/L, AST: 3,387 U/L ¡ HIV/AIDS patients ¡ Antiviral therapy with nucleoside analogs ¡ Patients on immunosuppressive therapy (i.e. organ transplant patients or chemotherapy) ¡ May also be seen in the minority of granulocytes in myelodysplastic syndromes ¡ Peripheral blood – Neutrophils ¡ Bone Marrow – Segmented and band neutrophils, metamyelocytes and myelocytes ¡ Round nuclear fragments, separated from the remaining nucleus ¡ Intracytoplasmic inclusions of DNA material ¡ Dark purple color of nuclear material ¡ Differential diagnosis includes infectious etiologies and Chédiak-Higashi Syndrome Photos by K. Haberichter (Giemsa, 1000x) 3 4/8/19 REACTIVE/ACQUIRED CHANGES REACTIVE/ACQUIRED CHANGES GREEN NEUTROPHILIC INCLUSIONS GREEN NEUTROPHILIC INCLUSIONS ¡ 43 cases have been reported in the literature since 2009 ¡ Newly described neutrophil cytoplasmic inclusion ¡ Rarely described in monocytes ¡ Associated with numerous medical conditions ¡ Predominately seen in patients with acute liver failure and septic shock ¡ Vibrant bright-green inclusions ¡ Irregularly shaped and coarse in appearance ¡ The majority of patients reported in the literature have elevated liver transaminases (ALT and AST) and lactic acid ¡ May be single or multiple inclusions per cell levels ¡ Typically involve the minority of neutrophils on peripheral smears ¡ A subset of patients have been noted to die within 72 hours after identification of these inclusions Photo by K. Haberichter (Giemsa, 1000x) ¡ Termed the “Green neutrophilic inclusions of death” REACTIVE/ACQUIRED CHANGES REACTIVE/ACQUIRED CHANGES GREEN NEUTROPHILIC INCLUSIONS GREEN NEUTROPHILIC INCLUSIONS ¡ Postmortem liver sections with extensive centrilobular necrosis and hepatocellular lipofuscin ¡ Exact etiology is unclear ¡ Once thought to be derived from biliverdin, lipofusion-like substance or similar lysosomal degradation product phagocytized following liver injury ¡ Newer research favors the inclusions are due to neutrophils phagocytizing a lipofusion-like substance following ischemic liver injury ¡ Special stains have been performed for further classification and reported as negative ¡ Bilirubin, Prussian blue iron, Myeloperoxidase, Periodic acid Schiff (PAS), Warthin-Starry, Gomori methenamine silver, and Gram stains ¡ Oil Red O and long Zeihl-Neelsen stains have been reported as positive PAS-D (500x magnification) Giemsa (500x magnification) Photos by K. Haberichter REACTIVE/ACQUIRED CHANGES REACTIVE/ACQUIRED CHANGES GREEN NEUTROPHILIC INCLUSIONS BILIRUBIN CRYSTALS ¡ Neonates and children with severe jaundice ¡ Why are these inclusions important to start recognizing and reporting? ¡ Known to be associated with a poor prognosis ¡ Unconjugated hyperbilirubinemia associated with septicemia more than hemolytic disease of the newborn ¡ Findings should be communicated to the clinical team ¡ Better recognition of this new entity will allow us to further evaluate the true incidence of patients ¡ Often associated with a poor outcome who develop these characteristic inclusions ¡ Allow us to further investigate the underlying etiology ¡ Refractile golden color, rhomboid to rectangular cytoplasmic crystals within segmented and band neutrophils ¡ May be extracellular ¡ Only seen in samples collected in EDTA ¡ The crystals can be confirmed as unconjugated bilirubin by the indirect Diazo reaction 4 4/8/19 CONGENITAL ANOMALIES CHÉDIAK-HIGASHI SYNDROME ¡ Autosomal recessive ¡ CHS1 (LYST) mutation, located on chromosome 1 CONGENITAL ANOMALIES ¡ Abnormal endosomal-lysosomal fusion ¡ Recurrent severe pyogenic infections ¡ Immunodeficiency, oculocutaneous albinism, bleeding tendency, and neurologic abnormalities ¡ Patients often die in childhood ¡ Often associated with neutropenia and thrombocytopenia ¡ Due to ineffective hematopoiesis CONGENITAL ANOMALIES ¡ 19 yo, M with no known medical history CHÉDIAK-HIGASHI SYNDROME ¡ WBC: 37.3 K/µL, PMNs: 3.8 K/µL, Blasts 23.0 K/µL (62%) ¡ Giant, abnormal granules in granulocytes, monocytes and lymphocytes ¡ Fusion of normal secondary granules - neutrophils, eosinophils and basophils ¡ Fusion of cytotoxic granules - monocytes and lymphocytes Photos by K. Haberichter (Giemsa, 1000x) CONGENITAL ANOMALIES CHÉDIAK-HIGASHI-LIKE CRYSTALS