Disorders of Granulocyte Number & Function

1. Blood-Hematopoiesis-Lymphatics 2013 Disorders of Granulocyte Number & Function William F. Kern, MD Director, Laboratory Hematology william-kern@ouhsc.edu

2. Downloading any of the photographs, images or diagrams from this presentation for any purpose other than studying for the BHL course is prohibited.

3. Neutrophils • Bone marrow storage compartment • Blood compartments: • Circulating pool: ~50% • Marginating pool: ~50% • Lifespan in blood: A few hours • Extravascular pool: Size unknown • Lifespan: A few days

4. Neutrophils: Requirements for Function • Adherence to endothelium • Passage through endothelium into tissues (“diapedesis”) • Chemotaxis • Phagocytosis • Microbial killing Deficiencies can occur at any of these steps

5. From: Hoffman’s Hematology: Basic Principles & Practice

6. Neutrophils: Adherence to Endothelium • Regulated by cell adhesion molecules (CAMs) • Types of CAMs: • Selectins and their ligands • Integrins • Immunoglobulin superfamily: • Intercellular adhesion molecule family (ICAMs) Not for memorization

7. Cell Adhesion Molecules (CAMS):Integrins • Heterodimer: a and b subunits • b2-Integrin family important in leukocyte function (“leukocyte integrins”) • Common b subunit (b2; CD18) • Different a subunits: • CD11a/CD18: LFA-1 • CD11b/CD18: Receptor for C3bi • CD11c/CD18 Remember the concept – not the specific CD numbers!

8. Neutrophil-Endothelium Adhesion • “Stickiness” is variable for both PMNs and endothelium: • Upregulation of expression of CAMS on cell surfaces • Some transformed to more active form • Inflammation or thrombosis increases adhesion

9. Neutrophil-Endothelium Adhesion • Initial attachment mediated by selectins: • Relatively weak • Leukocytes “roll” along surface • Secondary attachment mediated by integrins: • Stronger • Leads to passage through endothelium For reference only

10. Chemotaxis • Neutrophils move along chemical gradients • Chemotactic agents: • Bacterial products: Small peptides • Complement components: C5a • Chemokines: IL-8 • Platelet activating factor (PAF) • Leukotriene (LBT4) • Others Remember the concept – not the specific chemotactic factors

11. Phagocytosis • Phagocytosis stimulated by opsonins on surface of microorganisms • Important opsonins: • Immunoglobulins: IgG, polymeric IgA • Complement: C3b • Specific receptors on neutrophil surface for Fc portion of immunoglobulins and complement components

12. Microbial Killing Mechanisms • Oxygen dependent: “Respiratory burst” • Generation of superoxide, H2O2, hydroxyl radical and hypochlorous acid (HOCl) • Nitric oxide pathway: Nitric oxide synthetase: • Reactive nitrogen intermediates • Non-oxidative mechanisms: • Antimicrobial compounds in PMN granules • “Defensins”: Cathepsin G, others

13. Neutrophil Respiratory Burst OH• Fe (non-enzymatic) NADPH Oxidase* NADP+ + 2 O2- + H+ NADPH + 2 O2 Superoxide Dismutase H2O2 Myeloperoxidase * Also called “respiratory burst oxidase” HOCl Hypochlorous acid

14. Neutrophil Respiratory Burst • NADPH generated from NADP+ via hexose monophosphate shunt: • Glucose-6-phosphate dehydrogenase (G6PD) = first enzyme • Glutathione also required A minorityof people with severe G6PD deficiency have impaired neutrophil function, with increased susceptibility to infection; most do not.

15. NADPH Oxidase • Key enzyme in respiratory burst • Complex enzyme with multiple subunits • Tightly regulated • Cytochrome b558: catalytic & redox center • gp91-phox: Gene on X chromosome: CYBB • Genes for other subunits on autosomal chromosomes

16. NADPH Oxidase Percentages indicate frequency of involvement in deficiency of NADPH oxidase Hoffman Hematology: Basic Principles & Practice

17. Inherited Disorders of Neutrophil Function • Chronic granulomatous disease (CGD) • Myeloperoxidase deficiency • G6PD deficiency • Leukocyte adhesion deficiency (I & II) • Specific granule deficiency • Chédiak-Higashi disease • Others

18. Chronic Granulomatous Disease • Deficiency of NADPH oxidase • Absence of respiratory burst with neutrophil stimulation • Inability to produce H2O2 and hypochlorous acid • Heterogeneous: Varied genetic basis, clinical manifestations & severity • Uncommon

19. Chronic Granulomatous Disease • Majority of cases: X-linked inheritance • Mutation of CYBB gene for gp91-phox • May be associated with McLeod phenotype (absence of Kell antigens on RBCs), Duchenne’s muscular dystrophy, retinitis pigmentosa • Other cases: Autosomal recessive My impression is that the “board” exams love questions about patterns of inheritance and linkage of different diseases….

20. Chronic Granulomatous Disease • Recurrent bacterial & fungal infections • Granulomatous inflammation: Histiocytes & lymphocytes • Severe cases (majority): Onset of infections during first year of life • Milder cases: May not present until adolescence

21. Chronic Granulomatous Disease • Catalase-positive organisms cannot be killed by neutrophils: • Neutralize any H2O2 that they produce • Catalase-negative organisms can be handled: • Obligingly produce H2O2 for neutrophils • H2O2 converted to hypochlorous acid by myeloperoxidase

22. Staph. aureus Gram-negative enterics Serratia marcesans Burkholderia cepacia Pseudomonas sp. Aspergillus sp. Pneumonitis Lymphadenitis Cutaneous infections Hepatic abscesses Osteomyelitis Perirectal abscesses Septicemia Pyelonephritis/UTI’s Chronic Granulomatous Disease Organisms: Infections: For reference, not for memorization.

23. Chronic Granulomatous Disease (CGD) For reference, not for memorization.

24. Chronic Granulomatous Disease (CGD) For reference, not for memorization.

25. CGD: Diagnosis • History of recurrent infections • ± Family history of recurrent infections in males • Histology: Granulomatous inflammation • Nitroblue tetrazolium test (NBT test)* • Flow cytometry: Reduction of dihydroxyrhodamine 123 to rhodamine 123 *NBT sounds like a good “board” question to me….

26. CGD: Treatment • Antibiotics for infections • Aggressive surgical drainage of abscesses • Prophylactic trimethoprim-sulfamethoxazole ± dicloxacillin • g-Interferon helpful in some cases: • Mechanism unknown

27. Myeloperoxidase (MPO) Deficiency • Most common inherited disorder of phagocytes: • Complete deficiency in ~1:4,000; partial deficiency in 1:2,000 • Autosomal recessive inheritance • Majority of patients asymptomatic: • No increase in bacterial infections • Disseminated fungal infections in minority of patients, most commonly diabetics

28. Neutrophil Respiratory Burst OH• Fe (non-enzymatic) NADPH Oxidase* NADP+ + 2 O2- + H+ NADPH + 2 O2 Superoxide Dismutase H2O2 Myeloperoxidase * Also called “respiratory burst oxidase” HOCl Hypochlorous acid

29. Myeloperoxidase (MPO) Deficiency • Unable to generate hypochlorous acid (HOCl) • Respiratory burst is prolonged & intensified • Bacterial killing occurs, but delayed • PMNs unable to kill Candida or Aspergillus in vitro: • Fungal infections uncommon in vivo

30. G6PD Deficiency & Neutrophil Dysfunction • NADPH for NADPH oxidase generated by hexose monophosphate shunt • G6PD = first enzyme • Neutrophil dysfunction may occur with severe G6PD deficiency: • Normal PMN function in most cases

31. Leukocyte Adhesion Deficiency (LAD I) • Disorder of leukocyte adhesion & chemotaxis • Rare • Deficiency of b2-integrins on surface of PMNs: • b2-integrins involved in tight adherence to and passage through endothelium • Inability to synthesize common b2 subunit (CD18)

32. Leukocyte Adhesion Deficiency (LAD I) • Autosomal recessive inheritance • PMNs unable to adhere tightly to endothelium • Adherence, chemotaxis & complement-mediated phagocytosis all deficient • Clinical manifestations heterogeneous; variable severity: • Severe cases: Death in early childhood • Moderate: Can survive into 20’s-30’s For reference only

33. Leukocyte Adhesion Deficiency (LAD I): Diagnosis • Diagnosis by flow cytometry: Deficiency of CD18 and/or CD11b on resting & stimulated PMNs For reference only

34. Disorders with Morphologic Abnormalities in Neutrophils • Chédiak-Higashi syndrome • Alder-Reilly anomaly • May-Hegglin anomaly • Pelger-Huët anomaly The only one of these I am going to discuss is Chédiak-Higashi syndrome

35. Chédiak-Higashi Syndrome • Rare • Autosomal recessive inheritance • Includes: • Partial oculo-cutaneous albinism • Abnormal large lysosomal granules in PMNs & other cells • Neutropenia & defective neutrophil function • Recurrent infections • ± Other congenital anomalies

36. Chédiak-Higashi Syndrome

37. Chédiak-Higashi Syndrome • Believed due to abnormal fusion & function of granule membranes • Mutation in CHS1 gene • Defects in PMN & lymphocyte function • Recurrent infections: Pyoderma, otitis media, gingivitis, septicemia

38. Chédiak-Higashi Syndrome:Accelerated Phase • May terminate as lymphoma-like syndrome • First or second decade • Infiltration of nodes, liver, spleen, marrow • May be related to inability to handle EBV infection • Often fatal

39. Acquired Neutrophil Function Defects:Defects in Chemotaxis • Autoimmune diseases: SLE, RA, polymyositis • Diabetes mellitus • Sarcoidosis • Leprosy • Hodgkin lymphoma • Severe malnutrition Acquired defects are more common than inherited defects

40. Decreased Phagocytosis due to Impaired Opsonization • Multiple myeloma • Acquired hypogammaglobulinemia • Hereditary agammaglobulinemia • Asplenism • Complement deficiency states Key: Deficiency of immunoglobulins or complement

41. Neutrophilia • Definition: Generally >7,000 neutrophils/mL in adults • Number differs between labs • Number varies in children: • Upper normal limit generally lower • Varies by age OUMC: Normal upper limit for segmented neutrophils in adults = 8,600/mL

42. Neutrophilia: Causes • Acute stress or physical exertion • Acute infection • Inflammation due to other causes • Tissue necrosis • Acute hemorrhage • Non-hematologic malignancies • Medications: • Lithium, corticosteroids, epinephrine • Idiopathic chronic neutrophilia

43. “Shift Neutrophilia” • Demargination of marginated granulocyte pool • No increase in actual intravascular granulocyte number • No increase in proportion of “bands” • Rapid but transient: ~20-30 minutes • Causes: Acute stress, exercise, epinephrine, corticosteroids

44. “Leukemoid Reaction” • Marked increase in leukocytes which is not due to acute or chronic leukemia • Reactive process - not neoplastic • Usually defined as >50,000/mL • Usually consists predominantly of mature cells: • May contain occasional immature cells Main concern: Reactive leukocytosis vs. leukemia

45. Leukemoid Reactions: Causes • Infections: • Pneumonitis, meningitis, diphtheria, TB • Severe stress: • Intoxications, eclampsia, severe burns • Malignant disease, especially with bone metastases • Multiple myeloma, Hodgkin lymphoma • Severe hemorrhage or acute hemolysis • Cytokine therapy (G-CSF)

46. Reactive Leukocytosis: Distinction from Leukemia • Clinical situation • Predominance of mature cells • WBC usually <50,000/mL • No anemia, thrombocytopenia or thrombocytosis • No lymphadenopathy or splenomegaly • Distinction from chronic myelogenous leukemia (CML) may be difficult

47. Granulocytic Leukemoid Reaction: Distinction from CML • Predominance of mature cells: • Blasts, promyelocytes usually absent • No splenomegaly • Absence of BCR/ABL rearrangement, Philadelphia chromosome [t(9;22)] or other cytogenetic abnormality • Leukocyte alkaline phosphatase (LAP) score

48. Granulocytic Leukemoid Reaction vs. CML

49. Neutropenia • Definition: • ANC <1,500/mL: Caucasians* • ANC <1,200/mL: African-Americans • Calculation of absolute neutrophil count: • ANC = WBC x (%Segs + %Bands) x 0.01 • Multiplication by 0.01 corrects for percent (Segs + Bands) * ANC = Absolute neutrophil count Lower limit of reference range varies for different labs Lower limit of ANC also varies with age

50. Neutropenia & Risk of Infection • Depends on severity and duration of neutropenia: • Also bone marrow neutrophil stores • Definition of severity: • Mild: 1000-1,500/mL • Moderate: 500-1,000/mL • Severe: <500/mL • Severe infections not common until ANC <500/mL • Brief periods usually well tolerated