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)

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¡ 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)

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¡ 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)

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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

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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 ALDER-REILLY ANOMALY

¡ Seen in patients with mucopolysaccharidoses

¡ Morphologic changes similar to Chédiak-Higashi syndrome ¡ Historically known as - Hunter syndrome, Hurler syndrome, Morquio syndrome, Sanfilippo syndrome, & Scheie syndrome ¡ X-linked or autosomal recessive

¡ Typically adults with underlying myeloid disorders ¡ May be seen in association with acute myeloid leukemia, including acute promyelocytic leukemia or ¡ Predominately affects granulocytes myelodysplastic syndromes ¡ Variably present in monocytes

¡ Abnormal large granules within a subset of myeloid blasts ¡ Large numbers of coarse, azurophilic granules in cytoplasm ¡ May be misidentified as toxic granulation

¡ May see vacuolated/abnormally granulated lymphocytes

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CONGENITAL ANOMALIES MAY-HEGGLIN ANOMALY

¡ Group of diseases with mutations in the myosin heavy chain-9 (MYH9) gene on chromosome 22 ¡ May-Hegglin, Fechtner, Sebastian and Epstein syndromes ¡ Autosomal dominant giant-platelet disorders INFECTIOUS ETIOLOGIES ¡ Most patients are asymptomatic

¡ Triad of thrombocytopenia, giant platelets and leukocyte inclusions (granulocytes and monocytes) ¡ Absence of toxic granulation

¡ Large blue cytoplasmic inclusions that resemble Döhle bodies

¡ Döhle-like bodies are paracrystalline arrays of ribosomes and RNA ¡ Larger than true Döhle bodies (2-4 µm)

INFECTIOUS ETIOLOGIES INFECTIOUS ETIOLOGIES ANAPLASMA EHRLICHIA

¡ Human Granulocytic Anaplasmosis (HGA) ¡ Human Monocytic Ehrlichiosis (HME) ¡ Fever and septic shock like symptoms ¡ Tick borne disease Ehrlichia chaffeensis and E. ewingii - lone star tick (Ambylomma americanum) ¡ Tick borne disease - blacklegged tick (Ixodes scapularis) and western blacklegged tick (Ixodes pacificus) ¡ Bacteria Ehrlichia chaffeensis, E. ewingii, or E. muris eauclairensis in the United States ¡ Eastern United States and west coast ¡ The majority of reported cases are due to infection by E. chaffeensis ¡ Bacterium Anaplasma phagocytophilum ¡ Southcentral and eastern United States

¡ Clinical and laboratory findings are similar to those seen with Anaplasmosis ¡ Nonspecific laboratory features ¡ Typically more serious than HGA, often with hospitalization, especially if immunocompromised ¡ Leukopenia, thrombocytopenia, elevated serum transaminases

¡ Location of morulae in Ehrlichiosis ¡ Cluster of purple dots within phagosomes of neutrophils ¡ E. chaffeensis most commonly infects monocytes ¡ So-called elementary bodies of Anaplasma resemble a mulberry, known as a morulae ¡ E. ewingii more commonly infects granulocytes

INFECTIOUS ETIOLOGIES ¡ 59 yo, M status post kidney transplant on immunosuppressive therapy HISTOPLASMA

¡ Histoplasmosis ¡ Fungal infection - Histoplasma capsulatum var. capsulatum

¡ Survives in soil containing bird and bat droppings ¡ Central and eastern United States – especially the Ohio and Mississippi River valleys

¡ Rare in the peripheral blood and bone marrow

¡ Typically immunocompromised and HIV patients

¡ Histoplasma antigen detection in urine and/or serum is the most widely used and most sensitive method for diagnosis

¡ Yeast forms are phagocytized by neutrophils, monocytes and macrophages Photos by K. Haberichter (Giemsa, 1000x) ¡ Ye a s t f or ms ( 2 -5 µm) with basophilic crescent or ring shaped nucleus, often with a pericellular halo ¡ May see extracellular forms

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INFECTIOUS ETIOLOGIES INFECTIOUS ETIOLOGIES CRYPTOCOCCUS TALAROMYCES MARNEFFEI

¡ Fungal infection ¡ Cryptococcus neoformans ¡ Opportunistic dimorphic fungal infection, related to Aspergillus

¡ Found in soil, decaying wood, tree hollows, and bird droppings ¡ Previously named Penicillium marneffei ¡ Worldwide distribution ¡ Immunocompromised individuals from Southeast Asia, southern China, or eastern India ¡ Immunosuppressed patients (HIV and status post organ transplants) ¡ Rare case reports disease in non-immunocompromised patients ¡ May be associated with disseminated intravascular coagulation (DIC)

¡ Symptoms include skin lesions, fever, weight loss, cough, lymphadenopathy ¡ Cultures are the gold standard for diagnosis ¡ May develop symptoms weeks to years after exposure

¡ Typically detected in body fluids and tissue sections ¡ Morphologically similar to Histoplasma ¡ Grocott-Gomori's methenamine silver stain (GMS) ¡ Contain a characteristic central transverse septum ¡ Rare in the bone marrow and peripheral blood ¡ Have one or two small red to purple nuclei ¡ May be engulfed by neutrophils and monocytes

REFERENCES

¡ “Anaplasmosis.” Centers for Disease Control and Prevention, 11 Jan 2019, https://www.cdc.gov/anaplasmosis/.

¡ “C. neoformans Infection.” Centers for Disease Control and Prevention, 9 Oct 2018, https://www.cdc.gov/fungal/diseases/cryptococcosis-neoformans/index.html.

¡ Courville EL, Crisman S, Linden MA, and Yohe S. Green Neutrophilic Inclusions are Frequently Associated With Liver Injury and May Portend Short-Term Mortality in Critically Ill Patients. Lab Med. 2017 Feb;48(1):18-23.

¡ “Ehrlichiosis.” Centers for Disease Control and Prevention, 17 Jan 2019, https://www.cdc.gov/ehrlichiosis/. ¡ Gorup T, Cohen AT, Sybenga AB, and Rappaport ES. Significance of green granules in neutrophils and monocytes. Proc (Bayl Univ Med Cent). 2017 Dec 29;31(1):94-96. THANK YOU! ¡ Haberichter K and Crisan D. Green Neutrophilic Inclusions and Acute Hepatic Failure: Clinical Significance and Brief Review of the Literature. Annals of Clinical & Laboratory Science, 2017; 47(1):58-61.

¡ Harris VN, Malysz J and Smith MD. Green neutrophilic inclusions in liver disease. J Clin Pathol, 2009; 62: 853-854.

¡ “Histoplasmosis.” Centers for Disease Control and Prevention, 13 Aug 2018, https://www.cdc.gov/fungal/diseases/histoplasmosis/index.html.

¡ Hodgson TO, Ruskova A, Shugg CJ, McCallum VJ, and Morison IM. Green neutrophil and monocyte inclusions – time to acknowledge and report. British Journal of Hematology, 2015;170: 229-235.

¡ Jazaerly T, Gabali AM. Green neutrophilic inclusions could be a sign of impending death! Blood, 2014;123(5): 614.

¡ Kahwash E and Gewirtz AS. Howell-Jolly Body-Like Inclusions in Neutrophils. Arch of Pathol Lab Med, 2003;127:1389-1390.

¡ Kjeldsberg, Carl R., and Sherrie L. Perkins. Practical Diagnosis of Hematologic Disorders. ASCP Press, 2010.

¡ O’Malley DP and His E. “Non-neoplastic Morphologic Abnormalities of White Blood Cells and Macrophages.” Hematopathology, edited by Eric D. Hsi. Elsevier/Saunders, 2012,pp. 99-110. ¡ Nayal B, Veena, Niveditha S, Chethan M. Detection of cryptococcosis in peripheral blood smear: A case report. Int J Appl Basic Med Res. 2011;1(2):116-7.

¡ Qureshi A and Akhtar N. Bilirubin inclusions in neonatal neutrophils. Blood. 2017;130(13):1601.

¡ Srinivas U, Kar R, Saxena R, Prasad PH. Cryptococcal neoformans profiles in peripheral blood neutrophils: An unusual presentation. Indian J Pathol Microbiol 2008;51:296-7.

¡ “Talaromycosis (formerly Penicilliosis).” Centers for Disease Control and Prevention, 26 Sept 2017, https://www.cdc.gov/fungal/diseases/other/talaromycosis.html.

¡ Yang JA and Gabali AB. Green neutrophilic inclusions: current understanding and review of the literature. Current Opinion in Hematology. 2018 Jan;25(1):3-6.

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