The Beloved : Its Function in Health and Disease 1915 PRODUCTION AND KINETICS OF

CELLS % CELLS TIME : 1 7 - 9 Mitotic 4 Days 16

Maturation/ 22 3 – 7 Storage Band 30 Days Seg 21

Vascular: Peripheral Seg 2 6 – 12 hours 3 Marginating Pool Apoptosis and ? Tissue clearance by 0 – 3 days From: D. Myeloid Precursors Lichtman's Atlas of Hematology, 2007

Promyelocyte. Marrow film. The largest cell among the precursors, ranging from 15 to 30 μm, although as with all cell types there is a variation in size. The nucleus and cytoplasm are usually more voluminous than those structures in the myeloblast, its immediate precursor. The nucleus may be circular, rectangular, pentagonal or hexagonal and the chromatin is coarser and the nucleoli less easily discerned than in the myeloblast. The cytoplasm is bluish and a light area, the Golgi zone, is evident from which the granules are synthesized. The cytoplasm contains few to many distinct granules ranging in color from reddish to purple. The granules frequently are seen over the nucleus as well as in the cytoplasm. The granules are primary (azurophilic) granules: and their synthesis is intense during this cell stage, after which primary granule synthesis ceases and the primary granules are divided among daughter cells during mitosis and maturation to later stages and ultimately the segmented neutrophil. A band neutrophil is to the upper left of the promyelocyte.

Date of download: 7/2/2015 Copyright © 2015 McGraw-Hill Education. All rights reserved. Myelocyte, neutrophilic, early stage. Marrow film. This stage is defined by the appearance of specific (secondary) granules, in this case neutrophilic granules. The neutrophilic granule is too small to be resolved by the light microscope (unlike primary granules). Their initial appearance is appreciated by a distinctive lighter area in the paranuclear nuclear site of the Golgi zone, where they are synthesized. This is referred to as a “sunburst” or “dawn of ” and is usually very evident. This is the earliest stage of the neutrophilic myelocyte. With maturation the entire cytoplasm becomes filled with neutrophilic granules and a tan or neutral color replaces the deep blue color of the promyelocyte cytoplasm; the primary granules become less apparent. The early myelocyte is slightly smaller on average than the promyelocyte. Contains bluish cytoplasm and residual primary granules except in the area of the sunburst of neutrophilic granules. The nucleus is usually eccentric, occupies about half the area of the cell, has more condensed chromatin, and is devoid of nucleoli. A very early neutrophilic myelocyte is present in the lower left quadrant, distinguished from the promyelocyte to the upper right by the early appearance of a small clearing area (sunburst of neutrophilic granules) in the nuclear hilus (Golgi zone).

Date of download: 7/2/2015 Copyright © 2015 McGraw-Hill Education. All rights reserved. From: D. Myeloid Precursors Lichtman's Atlas of Hematology, 2007

Metamyelocyte, neutrophilic. Marrow film. The size and cytoplasmic coloration is the same as its predecessor, the late stage neutrophilic myelocyte that is a tan coloration throughout reflecting the neutral staining properties of the neutrophilic granules, not individually discernible because they are less than 0.5 μm in diameter. The singular change that determines this stage of development is the indentation of the nucleus to take on a reniform appearance.

Date of download: 7/2/2015 Copyright © 2015 McGraw-Hill Education. All rights reserved. From: D. Myeloid Precursors Lichtman's Atlas of Hematology, 2007

Band and segmented neutrophil. Marrow film. The band neutrophil has the same cytoplasmic appearance as its predecessor, the neutrophilic metamyelocyte, that is a tan coloration throughout the cytoplasm, reflecting the neutral staining properties of the neutrophilic granules, not individually discernible because they are less than 0.5 μm in diameter. The sole distinguishing feature of this state of maturation is the sausage shaped nucleus with a thickness that is nearly identical from one end of the nucleus to the other with no hint of segmentation. A segmented neutrophil sits adjacent to the band neutrophil. Note the clear segmentation with one lobe being connected to the remainder of the nucleus by a very thin strand. Note irregularities in the main body of the nucleus with minor and major constrictions and another developing segment apparent at the right end of the nucleus. The nuclear chromatin is more condensed and darker than that of the band neutrophil. A small is at the bottom of the field. In supravital preparations nuclear segmentation is a dynamic event with changing lobe numbers and areas of segmentation. Date of download: 7/2/2015 Copyright © 2015 McGraw-Hill Education. All rights reserved. Neutrophil exit from the vascular space

Kolaczkowska et al Nat. Rev 2013 13:159 &

1. Margination

Turhan et al 2002 PNAS 99(5): 3047

1. Mobilization 2. 3. Recognition (Opsonization) 4. Ingestion 5. Degranulation 6. Peroxidation 7. Killing and Digestion 8. Net formation

From JG Hirsch, J Exp Med 116:827, 1962, with permission. Infectious complications of disorders (either quantitative or functional)  Frequent and/or unusually severe bacterial, fungal - Skin, lymph nodes, lungs (portals of entry); other sites via bloodstream or tissue extension  Unusual site - e.g. liver or brain abscess  Recurrent/chronic gingivitis, aphthous ulcers  Staphylococcal common. Also Strep; Gram- negatives, unusual or opportunistic pathogens e.g. Aspergillus, Serratia, B. cepacia, Klebsiella, atypical Mtb

Neutropenia is defined as a decrease in the absolute numbers of circulating segmented neutrophils and bands in the blood. Obtaining a (CBC) with a differential count identifies this condition. The absolute neutrophil count (ANC) is determined by multiplying the total white count by the percentage of segmented and band forms. The ANC for the general population ranges between 1.5 and 8.0 x 10⁹/L.

• Definition: Reduction in the absolute neutrophil count (includes bands and segmented PMNs) below norms for age and ethnic groups in the blood circulation. • Age-related ANC: Term newborn (1 week): < 1500 Infant (1 month – 4 years): < 1000 Child, adolescent, adult: < 1500 • Ethnicity: < 800 From: H. White Cell Concentrate (Buffy Coat) Lichtman's Atlas of Hematology, 2007

Legend: Normal blood. . White cell concentrate. Note concentration of white cells. Three segmented neutrophils, two , and a , an approximately normal distribution by cell type.

Date of download: 7/2/2015 Copyright © 2015 McGraw-Hill Education. All rights reserved. Suspicion of Neutropenia

• Acute severe bacterial infections.

• History of recurrent infections.

• Prolonged or elevated temperature (> 101° F).

• Pneumonia, peritonitis, GU tract , buccal and tongue ulcers, chronic gingivitis, cellulitis, perirectal infections.

• Findings associated with a malignancy, syndrome, viral infection or drug exposure. Clinical Risk Assessment

• None: ANC of 1,000 to 1,500/µL

• Moderate: ANC of 500 to 999/µL

• Severe: ANC of 300 to 499/µL

• Very severe: ANC of < 300/µL

Clinical Risk Assessment

• Acute vs. chronic lasting more than three months (ANC < 500/µL).

• Can neutrophils be mobilized from bone marrow?

• Production vs. destruction.

Epidemiology

• Acute neutropenia occurs frequently.

• Congenital neutropenia: ~ 2 per million.

• : ~ 0.6 per million. Physical Examination of Patients with Neutropenia • Height and weight • Detailed exam of oral cavity • Skeletal abnormalities • Skin assessment for pigmentation, dystrophic nails, warts, eczema, and cellulitis • Cardiac and vascular abnormalities • Adenopathy and hepatosplenomegaly Laboratory Evaluation of Neutropenia

• CBCPD and blood smear. • If neutropenia is recurrent, repeat CBCPD 3x/week for 6 weeks. • Coombs test. • Immunoglobulin levels and lymphocyte subsets. • Antineutrophil antibodies. • Serology for viral infections if acute process. • ANA, LDH, uric acid. • Bone marrow exam and cytogenetics. Human Neutrophil Alloantigens

Previous Antigens Names Carrier Glycoproteins HNA-1a NA1 FcȣR IIIb (CD16) HNA-1b NA2 FcȣR IIIb (CD16) HNA-1c SH FcȣR IIIb (CD16) HNA-2 NB1 58-64 kDa (CD177) HNA-3 5b CTL2 (Unknown) HNA-4 HART CR3 (CD11b) HNA-5 OND LFA-1 (CD11a) Immune Neutropenia of Infancy Prevalence: 5-10 symptomatic cases/100,000

Age: 5-15 months

Infection: 80% suffered from mild infections, e.g. skin, otitis media, URI

Laboratory: ANC varies between 0 to 1500 cells/µL

Bone Marrow: Usually normal

Immunohematology: Granulocyte-specific antibodies which are reactive agents HNA-1, HNA-2, CD11b

Prognosis: Neutropenia resolves spontaneously after a median of 17 months Recovery from Neutropenia in Infancy

Positive Anti-neutrophil Negative Anti-neutrophil Antibody Antibody Patients 22 14 Gender 9 Males 6 Males 13 Females 8 Females Median Ages at Onset 1.06 0.73 (Range) (0.01 – 1.43) (0.07 – 1.24) Median ANCs Pre G-CSF x 109/L 0.167 0.255 (Range) (0.000 – 0.784) (0.041 – 0.692) Median Age at Recovery 2.98 3.99 (Range) (1.10 – 6.92) (0.81 – 9.63) Median Duration of G-CSF 1.57 2.72 (Range) (0.36 – 3.61) (0.54 – 8.03) Median G-CSF Dose in mcg/kg/day 1.75 1.77 (Range) (0.15 – 3.70) (0.51 – 4.20) Management

• Recommend vaccinations • Usually does not require G-CSF • Bone Marrow often not required • Obtain anti-neutrophil antibodies x 3 • Treat fever with antipyretics • If temperature exceeds 38.5° on antipyretics, obtain CBC, blood cultures and give broad spectrum antibiotics

Other Autoimmune Neutropenias

Associated  SLE Disorders:  Chronic active hepatitis  Infectious mononucleosis; HIV  Malignancy - lymphoma, Hodgkin's  Evan's syndrome- Coomb's + autoimmune hemolytic and/or ITP  - e.g. CVID, HyperIgM, IgA def., ALPS

Management: Treat underlying disorder G-CSF; steroids; rituximab Severe Chronic Neutropenia • Heterogeneous group of disorders of

• Associated with - decreased production of neutrophils - recurrent bacterial infections

• Severity of disease related to degree of neutropenia

Three Main Subtypes of SCN • Idiopathic

• Cyclic

• Congenital

Severe Congenital Neutropenia (SCN)

• Early childhood onset with ANC < 200

• Bone marrow shows maturation arrest at promyelocyte-myelocyte stage

• Recurrent life-threatening bacterial infections

• Associated with ELANE (ELA2) mutations

Clinical Issues in SCN

• 10% – 30% risk of evolving into MDS/AML

• HCT should be considered in G-CSF non responders to • > 20µg/kg/day or converting to MDS/AML

• Need for annual bone marrow to survey for cytogenetic changes

• Patients remain at risk for infection because of impaired neutrophil function

Classification of congenital neutropenia disorders Classification Syndrome Myelopoiesis Severe congenital neutropenia Cyclic neutropenia Ribosome /telomere dysfunction Shwachman-Diamond syndrome Dyskeratosis congenita Metabolism Reticular dysgenesis Barth syndrome Glycogen storage disease type 1b Glucose-6 phosphatase catalytic subunit 3 Granule formation and secretion Chédiak-Higashi syndrome Cohen syndrome Griscelli syndrome type II Hermansky-Pudlak type II JAGN7 Deficiency p14 deficiency VPS45 Deficiency Immune function Cartilage-hair hypoplasia GATA2 deficiency (MonoMac) Hyper-IgM syndrome Schimke immuno-osseous dysplasia TCIRG1 deficiency WHIM syndrome Wiskott- Aldrich Syndrome Cyclic Neutropenia

• Like autosomal dominant SCN, cyclic neutropenia has been linked to mutations in neutrophil elastase • ELANE (ELA2) mutations found in essentially 90% of cyclic neutropenia • NOT associated with an increased risk of AML Cyclic Neutropenia

• Dominantly inherited • Cycle of neutropenia q 21 days • Marrow during neutropenia: myelocyte arrest • Stem cell regulatory defect

Dynamics of the Absolute Monocyte Count

Congenital Neutropenia Cyclic Neutropenia 6000 6000

5000 5000

4000 4000

3000 3000

2000 2000

Peripheral Leukocyte Count (counts/µl) Count Leukocyte Peripheral 1000 1000

0 0 0 21 42 63 84 105 126 0 21 42 63 84 105 126 Days ANC ANCAMC AMC Adult Idiopathic Neutropenia

• Includes idiopathic and primary autoimmune neutropenia • Normal bone marrow and chromosome analysis • Female predominance • Management may not require G- CSF