SUB MODULE 5:

GRANULOCYTES DISORDERS

TEACHER GUIDANCE
Contributors :
Budiman, dr., SpPK(K) Budi Darmawan M, dr., SpPD-KHOM Dr. Endang Sri Wahyuni, dr., MS Setyohadi, drg., MS Djoko Heri Hermanto, dr., SpPD Susanto Nugroho, dr., SpA

FACULTY OF MEDICINE UNIVERSITY OF BRAWIJAYA MALANG 2008

SUB MODULE 5: GRANULOCYTE DISORDERS

I. DESCRIPTION Definition and Overview The white blood cells (leukocytes) may be divided into two broad group: the phagocytes and the immunocytes. Granulocytes, which include three types of cell: neutrophils (polymorphs), eosinophils and basophils, together with monocytes comprise the phagocytes. The function of phagocytes and immunocytes in protecting the body against infection is closely connected with two soluble protein systems of the body, immunoglobulins and complement. These proteins, which may also be involved in blood cell destruction in a number of diseases. Granulopoiesis, the function of granulocytes, and granulocytes disorders are discussed in this sub module. Area of competence : 3rd of the Doctor Competencies Standard from Indonesian Medical Council To apply the concept and principles of granulopoiesis, the morphology and the function of granulocytes, granulocytes disorders, and also clinical approach to diagnose these. 1. Student can describe granulopoiesis 2. Student can describe the morphology and the function of granulocytes 3. Student can describe causes of granulocytes disorders 4. Student can do the clinical approach to diagnose causes of granulocytes disorders Active learning with modul task, group discussion, expert lecture, and skill development (history taking) Classroom, worksheet, computer, LCD and screen Active learning with modul task 3x50 minutes; group discussion 2x50 minutes; expert lecture 1x50 minutes; and skill development (history taking) overall 3x50 minutes. 1. Budiman, dr., SpPK(K) 2. Budi Darmawan M, dr., SpPD-KHOM 3. Dr. Endang Sri Wahyuni, dr., MS 4. Setyohadi, drg., MS 5. Djoko Heri Hermanto, dr., SpPD 6. Susanto Nugroho, dr., SpA Middle exams at the end of modul programmes with multiple choice questions, and final exams at the end of semester with OSCE. 1. Hoffbrand AV, Pettit JE, Moss PAH. The White Cells 1: Granulocytes, Monocytes and their Benign Disorders. In: Essential Haematology. 4th ed. London: Blackwell Science. 2001. 2. Holland SM, Gallin JI. Disorders of Granulocytes and Monocytes. In: Fauci AS, Kasper DL, Longo DL et al, eds. Harrisons Principles of Internal Medicine. 17th ed. New York: The McGraw-Hill Companies. 2008.

Competency Area Competency Component Clinical Competence

Learning Methode Equipment Time Lecturer

Evaluation Suggested Refferences

II. LECTURE CONTENTS OVERVIEW The white blood cells (leukocytes) may be divided into two broad group: the phagocytes and the immunocytes. Granulocytes, which include three types of cell: neutrophils (polymorphs), eosinophils and basophils, together with monocytes comprise the phagocytes. The function of phagocytes and immunocytes in protecting the body against infection is closely connected with two soluble protein systems of the body, immunoglobulins and complement. These proteins, which may also be involved in blood cell destruction in a number of diseases. Granulopoiesis, the function of granulocytes, and granulocytes disorders are discussed in this sub module. GRANULOCYTES Neutrophils (polymorph) This cell has a characteristic dense nucleus consisting of between two and five lobes, and a pale cytoplasm with an irregular outline containing many fine pink-blue (azurophilic) or grey-blue granules (Fig.1.a). The granules are divided into primary, which appear at the promyelocyte stage, and secondary (specific) which appear at the myelocyte stage and predominate in the mature neutrophil. Both types of granule are lysosomal in origin; the primary contains myeloperoxidase, acid phosphatase and other acid hydrolases, the secondary contains collagenase, lactoferrin and lysozyme. The lifespan of neutrophils in the blood is only about 10 hours. Neutrophil precursors These do not normally appear in normal peripheral blood but are present in the marrow. The earliest recognizable precursor is the myeloblast, a cell of variable size which has a large nucleus with fine chromatin and usually two to five nucleoli. The cytoplasm is basophilic and no cytoplasmic granules are present. The normal bone marrow contains up to 4% of myeloblasts. Myeloblasts give rise by cell division to promyelocytes which are slightly larger cells and have developed primary granules in the cytoplasm. These cells then produce myelocytes which have specific or secondary granules. The nuclear chromatin is now more condensed and nucleoli are not visible. Separate myelocytes of the neutrophil, eosinophil, and basophil series can be identified. The myelocytes give rise by cell division to metamyelocytes, non-dividing cells, which have an idented or horseshoe-shaped nucleus and a cytoplasm filled with primary and secondary granules. Neutrophil forms between the metamyelocyte and fully mature neutrophil are termed band, stab or juvenile. These cells may occur in normal peripheral blood. They do

not contain the clear fine filamentous distinction between nuclear lobes which is seen in mature neutrophils. Monocytes These are usually larger than other peripheral blood leucocytes and possess a large central oval or idented nucleus with clumped chromatin (Fig.1.b). The abundant cytoplasm stains blue and contains many fine vacuoles, giving a ground-glass appearance. Cytoplasmic granules are also often present. The monocyte precursors in the marrow (monoblasts and promonoblasts) are difficult to distinguish from myeloblasts and monocytes.

(a)

(b)

(c)

(d)

Fig.1. White blood cells (leucocytes): (a) neutrophil (polymorph); (b) monocyte; (c) eosinophil; (d) basophil

Eosinophils These cells are similar to neutrophils, except that the cytoplasmic granules are coarser and more deeply red staining and there are rarely more than three nuclear lobes (Fig.1.c). Eosinophil myelocytes can be recognized but earlier stages are indistinguishable from neutrophil precursors. The blood transit time for eosinophils is longer than for neutrophils. They enter inflammatory exudates and have a special role in allergic responses, defence against parasites and removal of fibrin formed during inflammation. Basophils These are only occasionally seen in normal peripheral blood. They have many dark cytoplasmic granules which overlie the nucleus and contain heparin and histamine (Fig.1.d). In

the tissues they become mast cells. They have immunoglobulin E (IgE) attachment sites and their degranulation is associated with histamine release. GRANULOPOIESIS Granulopoiesis (or granulocytopoiesis) is hematopoiesis of granulocytes. The blood granulocytes and monocytes are formed in the bone marrow from a common precursor cell (Fig.2). In the granulopoietic series progenitor cells, myeloblasts, promyelocytes and myelocytes form a proliferative or mitotic pool of cells while the metamyelocytes, band and segmented granulocytes, make up a postmitotic maturation compartment (Fig.3). Large numbers of band and segmented neutrophils are held in the marrow as a reserve pool or storage compartment. The bone marrow normally contains more myeloid cells than erythroid cell in the ratio of 2:1 to 12:1, the largest proportion being neutrophils and metamyelocytes. In the stable or normal state, the bone marrow storage compartment contains 10-15 times the number of granulocytes found in the peripheral blood. Following their release from the bone marrow, granulocytes spend only 6-10h in the circulation before moving into the tissues where they perform their phagocytic function. In the bloodstream there are two pools usually of about equal size the circulating pool (included in the blood count) and the marginating pool (not included in the blood count). It has been estimated that they spend on average 4-5 days in the tissues before they are destroyed during defensive action or as the result of senescence.

Fig.2. Diagrammatic representation of the bone marrow pluripotent stem cell and the cell lines that arise from it

Control of granulopoiesis: myeloid growth factors

The granulocytes series arises from bone marrow progenitor cells which are increasingly specialized. Many growth factors are involved in this maturation process including interleukin-1 (IL-1), IL-3, IL-5 (for eosinophils), IL-6, IL-11, granulocyte-macrophag colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF) and monocyte CSF (M-CSF) (Fig.4). The growth factors stimulate proliferation and differentiation and also affect the function of the mature cells on which they act (e.g. phagocytosis, superoxide generation and cytotoxicity in the case of neutrophils; phagocytosis, cytotoxicity and production of other cytokines by monocytes). Increased granulocyte and monocytes production in response to an infection is induced by increased production of growth factors from stromal cells and T lymphocytes, stimulated by endotoxin, IL-1 or tumour necrosis factor (TNF).

Fig.3. Neutrophil kinetics

THE FUNCTION OF GRANULOCYTES The normal function of neutrophils and monocytes may be divided into three phases: 1. Chemotaxis (cell mobilization and migration) The phagocyte is attracted to bacteria or the site of inflammation by chemotactic substances released from damaged tissues or by complement components and also by the interaction of leucocyte adhesion molecules with ligands on the damaged tissues. 2. Phagocytosis The foreign material (bacteria, fungi, etc.) or dead or damaged cells of the host are phagocytosed (Fig.5). Recognition of a foreign particle is aided by opsonization with immunoglobulin or complement because both neutrophils and monocytes have Fc and C3b receptors. Opsonization of normal body cells (e.g. red cells or platelets) also makes them liable to destruction by macrophages of the reticuloendothelial system, as in autoimmune

hemolysis, idiopathic (autoimmune) thrombocytopenic purpura or many of the drug-induced cytopenias.

Fig.4. A diagram of the role of growth factors in normal hemopoiesis.

Macrophages have a central role in antigen presentation processing and presenting foreign antigens on human leucocyte antigen (HLA) molecules to the immune system. They are also secrete a large number of growth factors which regulate inflammation and immune responses.

Fig.5. Phagocytosis and bacterial destruction

Chemokines are chemotactic cytokines of which there are two main classes CXC ( ) chemokines, small (8-10.000 MW) pro-inflammatory cytokines which mainly act on

neutrophils, and CC () chemokines such as macrophage inflammatory protein (MIP)-1 and RANTES which act on monocytes, basophils, eosinophils and natural killer (NK) cells. Chemokines may be produced constitutively and control lymphocyte traffic under physiological conditions; inflammatory chemokines are induced or up-regulated by inflammatory stimuli. They bind to and activate cells via chemokine receptors and play an important part in recruiting appropriate cells to the sites of inflammation. Chemokine receptors have been identified as coreceptors for human immunodeficiency virus (HIV) entry into cells. 3. Killing and digestion This occurs by oxygen-dependent and oxygen-independent pathways. In the oxygendependent reactions, superoxide (O2-), hydrogen peroxide (H2O2) and other activated oxygen (O2) species, are generated from O2 and reduced nicotinamide adenine dinucleotide phosphate (NADPH). In neutrophils, H2O2 reacts with myeloperoxidase and intracellular halide to kill bacteria; activated oxygen may also be involves a fall in pH within phagocytic vacuoles into which lysosomal anzymes are released. An additional factor, lactoferrin an iron-binding protein present in neutrophil granules is bacteriostatic by depriving bacteria of iron (Fig.5). Defects of phagocytic cell function 1. Chemotaxis These defects occur in rare congenital abnormalities (e.g. lazy leucocyte syndrome) and in more common acquired abnormalities either of the environment, e.g. corticosteroid therapy, or of the leucocytes themselves, e.g. in acute or chronic myeloid leukemia, myelodysplasia and the myeloproliferative syndromes. 2. Phagocytosis These defects usually arise because of a lack of opsonization which may be caused by congenital or acquired causes of hypogammaglobulinemia or lack of complement components. 3. Killing and digestion This abnormality is clearly illustrated by the rare X-linked or autosomal recessive chronic granulomatousdisease which results from abnormal leucocyte oxidative metabolism. There is an abnormality affecting different elements of the respiratory burst oxidase or its activating mechanism. The patients have reccuring infections, usually bacterial but sometimes fungal, which present in infancy or early childhood in most cases. Other rare congenital abnormalities may also result in defects of bacterial killing e.g. myeloperoxidase deficiency and the Chediak-Higashi syndrome. Acute or chronic myeloid

leukemia and myelodysplastic syndromes may also be associated with defective killing of ingested microorganisms. LEUCOCYTOSIS AND MONOCYTOSIS Neutrophil leucocytosis An increase in circulating neutrophils to levels greater than 7.5x10 9/l is one of the most frequently observed blood count changes. The causes of neutrophil leucocytosis are given in Table 1. Neutrophil leucocytosis is sometimes accompanied by fever as a result of the release of leucocyte pyrogens. Other characteristic features of reactive neutrophilia may include: a. A shift to the left in the peripheral blood differential white cell count, i.e. an increase in the number of band forms and the occasional presence of more primitive cells such as metamyelocytes and myelocytes b. The presence of cytoplasmic toxic granulation and Doehle bodies c. An elevated neutrophil alkaline phosphatase (NAP) score For this the strength of the staining of each of 100 neutrophils is scored between 0 and 4. The maximum score is therefore 400; a normal score is between 20-100.
Table 1. Causes of neutrophil leucocytosis Bacterial infection (especially pyogenic bacterial, localized or generalized) Inflammation and tissue necrosis, e.g. myositis, vasculitis, cardiac infarct, trauma Metabolic disorders, e.g. uremia, eclampsia, acidosis, gout Neoplasms of all types, e.g. carcinoma, lymphoma, melanoma Acute hemorrhagic of hemolysis Corticosteroid therapy (inhibits margination) Myeloproliferative disease, e.g. chronic myeloid leukemia, polycythemia vera, myelosclerosis Treatment with myeloid growth factors, e.g. G-CSF, GM-CSF

The leukemoid reaction The leukemoid reaction is a reactive and excessive leucocytosis usually characterized by the presence of immature cells (e.g. myeloblasts, promyelocytes and myelocytes) in the peripheral blood. Occasionally lymphocytic reactions occur. Associated disorders include severe or chronic infections, severe hemolysis or metastatic cancer. Leukemoid reactions are often particularly marked in children. Granulocyte changes such as toxic granulation and Doehle bodie and a high NAP score help to differentiate the leukemoid reaction from chronic myeloid leukemia (in which the NAP score is low). Eosinophilic leucocytosis (eosinophilia)

The causes of an increase in blood eosinophils above 0.4x10 9/l are listed in Table 2. Sometimes no underlying cause is found and if the eosinophil count is elevated (>1.5x10 9/l) for over 6 months and associated with tissue damage then the hypereosinophilic syndrome is diagnosed.
Table 2. Causes of eosinophilia Allergic diseases, especially hypersensitivity of the atopic type, e.g. brochial asthma, hay fever, urticaria and food sensitivity Parasitic diseases, e.g. amoebiasis, hookworm, ascariasis, tapeworm infestation, filariasis, schistosomiasis and trichinosis Recovery from acute infection Certain skin diseases e.g psoariasis, pemphigus and dermatitis herpetiformis Pulmonary eosinophilia and the hypereosinophilic syndrome Drug sensitivity Polyarteritis nodosa Hidgkins disease and some other tumours Metastatic malignancy with tumour necrosis Eosinophilic leukemia (rare) Treatment with GM-CSF

Basophilic leucocytosis (basophilia) An increase in blood basophils above 0.1x109/l is uncommon. The usual cause is a myeloproliferative disorder such as chronic myeloid leukemia or polycythemia vera. Reactive basophil increases are sometimes seen in myxoedema, during smallpox or chickenpox infection, and in ulcerative colitis. Monocytosis A rise in blood monocyte count above 0.8x10 9/l is infrequent. The conditions listed in Table 3 may be responsible.
Table 3. Causes of monocytosis Chronic bacterial infections: tuberculosis, brucellosis, bacterial endocarditis, typhoid Protozoan infections Chronic neutropenia Hodgkins disease and other malignancies Myelodysplasia (especially chronic myelomonocytic leukemia) Treatment with GM-CSF or M-CSF

NEUTROPENIA The lower limit of the normal neutrophil count is 2.5x109/l. When the absolute neutrophil level falls below 0.5x109/l the patient is likely to have recurrent infections and when the count

falls to less than 0.2x109/l the risks are very serioud, particularly if there is also a functional defect. Neutropenia may be selective or part of a general pancytopenia (Tabel 4).
Table 4. Causes of neutropenia Selective neutropenia Congenital Kostmanns syndrome Acquired Drug induced Anti-inflammatory drugs (aminopyrine, phenylbutazone) Antibacterial drugs (chloramphenicol, co-trimoxazole, sulfaslazine, imipenem) Anticonculsants (phenytoin, carbamazepine) Antithyroids (carbimazole) Hypoglicemics (tolbutamide) Phenothiazines (chlorpromazine, thioridazine) Psychotropics and antidepressants (clozapine, mianserin, imipramine) Bark derivatives, e.g. paclitaxil Miscellaneous (glod, penicillamine, mepacrine, amodiaquine, ticlopidine, furosemide, etc) Benign (racial or familial) Cyclical Immune Autoimmune Systemic lupus erythematosus Feltys syndrome Hypersensitivity and anphylaxis Large granular lymphocytic leukemia Infections Viral, e.g. hepatitis, influenza, HIV Fulminant bacterial infection, e.g. typhoid, military tuberculosis Part of general pancytopenia Bone marrow failure Splenomegaly

a. Congenital neutropenia Kostmanns syndrome is an autosomal recessive disease presenting in the first year of life with life-threatening infections. Most cases are due to mutation of the gene coding for neutrophil elastase. G-CSF produces a clinical response although marrow fibrosis and acute myeloid leukemia may supervene.

b. Drug-induced neutropenia A large number of drugs have been implicated (Table 4) and may induce neutropenia either by direct toxicity or immune-mediated damage. c. Cyclical neutropenia This is a rare syndrome with 3-4 weeks periodicity. Severe but temporary neutropenia occurs. Monocytes tend to rise as the neutrophils fall. Mutation of the gene for neutrophil elastase underlies some cases. d. Autoimmune neutropenia In some cases of chronic neutropenia an autoimmune mechanism can be demonstrated. The antibody may be directed against one of the neutrophil-specific antigens (NA, NB, etc.). e. Idiopathic benign neutropenia An increase in the marginating fraction of blood neutrophils and a corresponding reduction in the circulating fraction is one cause of benign neutropenia. Clinical features Severe neutropenia is particularly associated with infections of the mouth and throat. Painful and often intractable ulceration may occur at these sites (Fig.6), on the skin or the anus. Septicemia rapidly supervenes. Organisms carried as commensals by normal individuals, such as Staphylococcus epidermidis or Gram-negative organisms in the bowel, may become pathogens. Diagnosis Bone marrow examination is useful in determining the level of damage in granulopoiesis, i.e. whether there is reduction in early precursors or whether there is reduction only of circulating and marrow neutrophils with late precursors remaining in the marrow. Marrow aspiration and trephine biopsy may also provide evidence of leukemia, myelodysplasia or other infiltration.

Fig.6. Ulceration of the tongue in severe neutropenia

Algorithms of leucocytosis, eosinophilia, and neutropenia (see Fig.7, 8 and 9)

III. MODUL TASK 1. Describe in brief three types of granulocytes (neutrophils, eosinophils, basophils)

and monocytes ! 2. Describe in brief the normal function of neutrophils and monocytes into three phases ! 3. Describe in brief defects of phagocytic cell function !
IV. SUGGESTED READINGS 3. Hoffbrand AV, Pettit JE, Moss PAH. The White Cells 1: Granulocytes, Monocytes and their Benign Disorders. In:Essential Haematology. 4th ed. London: Blackwell Science. 2001. 4. Holland SM, Gallin JI. Disorders of Granulocytes and Monocytes. In: Fauci AS, Kasper DL, Longo DL et al, eds. Harrisons Principles of Internal Medicine. 17th ed. New York: The McGraw-Hill Companies. 2008.