Myeloproliferative Neoplasms Notes

White Blood Cells Proliferations and Myeloid Neoplasms

White Blood Cells Normal And Reactive Proliferations

As illustrated in haematopoietic stem cells in the bone marrow differentiate to form lymphoid, and non-lymphoid (or myeloid or trilineage) stem cells. The trilineage stem cells differentiate in the bone marrow into erythroid, megakaryocytic, and granulocyte-monocyte progenitors; the last series are progenitors of mature forms of granulocyte-monocyte series which circulate in the peripheral blood.

Read And Learn More: General Pathology Notes

Thus, mature leucocytes of the peripheral blood, based on the presence or absence of granules in the cytoplasm, are of 2 types granulocytes (comprised of polymorphonuclear or PMN leucocytes, and monocytes) and nongranular leucocytes (consisting of lymphocytes).

• According to the colour and content of granules, PMN leucocytes are of 3 types neutrophils, eosinophils and basophils.
• On morphology, nongranular mature leucocytes are small and large lymphocytes but immunologically there are further subtypes as discussed in Generation of various types mature leucocytes, leucopoiesis, in the bone marrow, is under regulatory control of distinctive growth factors as discussed below.

Granulopoiesis:

Site Of Formation And Kinetics:

All forms of granulocytes are produced in the bone marrow and are termed myeloid series. Myeloid series include maturing stages: myeloblast (most primitive precursor), promyelocyte, myelocyte, metamyelocyte, band forms and segmented granulocyte (mature form). It takes about 12 days for formation of mature granulocytes from the myeloblast.

These maturing cells are further divided into:

• ‘Proliferative or mitotic pool’ comprised of myeloblast, promyelocyte and myelocyte; and
• ‘mature or post-mitotic pool’ composed of metamyelocyte, band forms and segmented granulocytes).

Normally the bone marrow contains more myeloid cells than erythroid cells in the ratio of 2:1 to 15:1 (average 3:1), the largest proportion being that of metamyelocytes, band forms and segmented neutrophils.

• The bone marrow storage compartment contains about 10-15 times the number of granulocytes found in the peripheral blood. Following their release from the bone marrow, granulocytes spend about 10 hours in circulation before they move into the tissues, where they perform their respective functions.
• The blood pool of granulocytes consists of 2 components of about equal size—the circulating pool that is included in the blood count, and the marginating pool that is not included in the blood count.
• Granulocytes spend about 4-5 days in the tissues before they are either destroyed during phagocytosis or die due to senescence.

To control the various compartments of granulocytes, a ‘feed-back system’ exists between the circulating and tissue granulocytes on one side and the marrow granulocytes on the other.

• The presence of a humoural regulatory growth factor, ‘granulopoietin’ analogous to erythropoietin and thrombopoietin, has also been identified by in vitro studies of colony-forming units (CFU) and is characterised as G-CSF (granulocyte colony-stimulating factor) and GM-CSF (granulocyte-monocyte colony-stimulating factor).
• The kinetics of monocytes is less well understood than that of other myeloid cells. Monocytes spend about 20-40 hours in circulation after which they leave the blood to enter extravascular tissues where they perform their main function of active phagocytosis.
• The extravascular lifespan of tissue macrophages which are the transformed form of blood monocytes, may vary from a few months to a few years.

Myeloid Series:

The development of myeloid cells from myeloblast takes place in the following sequence:

1. Myeloblast: The myeloblast is the earliest recognisable precursor of the granulocytes, normally comprising about 2% of the total marrow cells.

• The myeloblast varies considerably in size (10-18 µm in diameter), having a large round to oval nucleus nearly filling the cell, has fine nuclear chromatin and contains 2-5 well-defined pale nucleoli.
• The thin rim of the cytoplasm is deeply basophilic and devoid of granules. The myeloblasts of acute myeloid leukaemia may, however, show the presence of rod-like cytoplasmic inclusions called Auer’s rods which represent abnormal derivatives of primary azurophilic granules.
• The nuclei of successive stages during their development from myeloblast become progressively coarser and lose their nucleoli and the cytoplasm loses its blue colour.
• As the cells become mature lysosomal granules appear; firstly non-specific primary or azurophilic granules appear which are followed by specific or secondary granules that differentiate the neutrophils, eosinophils and basophils.

2. Promyelocyte: The promyelocyte is slightly larger than the myeloblast (12-18 µm diameter).

• It possesses a round to oval nucleus, having fine nuclear chromatin which is slightly condensed around the nuclear membrane.
• The nucleoli are present but are less prominent and fewer than those in the myeloblast. The main distinction between promyelocyte from myeloblast is in the cytoplasm which contains azurophilic (primary or non-specific) granules.

3. Myelocyte: The myelocyte is the stage in which specific or secondary granules appear in the cytoplasm, and accordingly, the cell can be identified at this stage as belonging to the neutrophilic, eosinophilic or basophilic myelocyte.

• Primary granules also persist at this stage but the formation of new primary granules stops. The nucleus of a myelocyte is eccentric, round to oval, having somewhat coarse nuclear chromatin and no visible nucleoli.
• The myeloid cells up to the myelocyte stage continue to divide and, therefore, are included in the mitotic or proliferative pool.

4. Metamyelocyte: The metamyelocyte stage is 10-18 µm in diameter and is characterised by an indented or horseshoe-shaped nucleus without nucleoli. The nuclear chromatin is dense and clumped.

The cytoplasm contains both primary and secondary granules. The metamyelocytes are best distinguished from the monocytes by the clumped nuclear chromatin while the latter have fine chromatin.

5. Band Forms: Band form is a juvenile granulocyte, 10-16 µm in diameter, characterised by further condensation of nuclear chromatin and transformation of nuclear shape into band configuration of uniform thickness.

6. Segmented Granulocytes: The mature polymorphonuclear leucocytes, namely the neutrophils, eosinophils and basophils, are described separately below.

Common surface markers for all stages of the myeloid series of cells are CD33, CD13 and CD15. However, stages from myelocytes to mature neutrophils also carry CD11b and CD14. Band forms and mature neutrophils have further CD10 and CD16.

Monocyte-Macrophage Series:

The monocyte-macrophage series of cells, though comprising a part of the myeloid series along with other granulocytic series are described separately here given different morphologic stages in their maturation.

1. Monoblast: The monoblast is the least mature of the recognisable cells of monocyte-macrophage series.

• It is very similar in appearance to myeloblast except that it has ground-glass cytoplasm with an irregular border and may show phagocytosis as indicated by the presence of engulfed red cells in the cytoplasm.
• However, differentiation from myeloblast at times may be difficult even by electron microscopy and, therefore, it is preferable to call the earliest precursor of the granulocytic series myelomonoblast.

2. Promonocyte: The promonocyte is a young monocyte, about 20 µm in diameter and possesses a large indented nucleus containing a nucleolus.

The cytoplasm is basophilic and contains no azurophilic granules but may have fine granules which are larger than those in the mature monocyte.

3. Monocyte: The mature form of monocytic series is described below, while the transformed stages of these cells in various tissues (i.e. macrophages) are a part of the RE system.

Monocyte-macrophage series having specialised function of phagocytosis secrete active products such as lysozyme, neutral proteases, acid hydrolases, components of complement, transferrin, fibronectin, nucleosides and several cytokines (TNF-α, IL-1, IL-8, IL-12, IL-18). They express lineage-specific molecules CD 14, cell surface LPS receptors etc.

Lymphopoiesis:

Sites of Formation and Kinetics:

The lymphocytes and the plasma cells are immunocompetent cells of the body. In humans, the bone marrow and the thymus are the primary lymphopoietic organs where lymphoid stem cells undergo spontaneous division independent of antigenic stimulation.

• The secondary or reactive lymphoid tissue is comprised of the lymph nodes, spleen and gut-associated lymphoid tissue (GALT). These sites actively produce lymphocytes from the germinal centres of lymphoid follicles as a response to antigenic stimulation.
• Lymphocytes pass through a series of developmental changes in the course of their evolution into lymphocyte subpopulations and subsets. It includes the migration of immature lymphocytes to other organs such as the thymus where locally-produced factors act on them.
• Functionally, the lymphocytes are divided into T, B and natural killer (NK) cells depending upon whether they are immunologically active in cell-mediated immunity (T-cells), in humoral antibody response (B-cells) or form part of the natural or innate immunity and act as a killer of some viruses (NK cells).
• In human beings, the B-cells are derived from the bone marrow stem cells, while in birds they mature in the bursa of Fabricius. After antigenic activation, B-cells proliferate and mature into plasma cells which secrete specific immunoglobulin antibodies.
• The T-cells are also produced in the bone marrow and possibly in the thymus. NK cells do not have B- or T-cell markers, nor are these cells dependent upon thymus for development. The concept of T, B and NK cells along with lymphocyte subpopulations and their functions.

Lymphoid Series:

The maturation stages in the production of lymphocytes are illustrated in are as under:

1. Lymphoblast: The lymphoblast is the earliest identifiable precursor of lymphoid cells and is a rapidly dividing cell. It is a large cell, 10-18 µm in diameter, containing a large round to oval nucleus having slightly clumped or stippled nuclear chromatin.

The nuclear membrane is denser and the number of nucleoli is fewer (1-2) as compared with those in myeloblast (2-5). The cytoplasm is scanty, basophilic and non-granular. The distinguishing morphologic features between the myeloblast and lymphoblast are summarised.

2. Prolymphocyte: This stage is an intermediate stage between the lymphoblast and mature lymphocyte. These young lymphocytes are 9-18 µm in diameter, contain round to indented nuclei with slightly stippled or coarse chromatin and may have 0-1 nucleoli.

3. Lymphocyte: The mature lymphocytes are described below.

Leucopoiesis:

• Granulopoiesis is a generation of a granular series of mature leucocytes circulating in blood (PMN neutrophils, eosinophils, basophils, monocytes) which are formed after differentiating from progenitor cells in the bone marrow under the influence of regulatory growth factor granulopoietin (G-CSF and GM-CSF).
• Myeloid precursor series of cells include myeloblasts, promyelocytes, myelocytes, metamyelocytes, band form and mature granulocytes (polymorph, eosinophil, basophil).
• Monocyte-macrophage cells are also formed from myeloid precursors, from immature monoblast or myelomonoblast.
• Mature lymphoid cells are formed from precursor cells lymphoblasts in the bone marrow. Lymphocytes undergo differentiation and maturation in the secondary lymphoid organs and thymus

Mature Leucocytes In Health And Pathologic Variation:

Normally, only mature leucocytes namely: polymorphs, lymphocytes, monocytes, eosinophils and basophils, are found in the peripheral blood. The normal range of total and differential leucocyte count (TLC and DLC expressed sequentially as P, L, M, E, B) in health in adults and children is given in Table.

White cell count tends to be higher in infants and children than in adults. It also normally undergoes a minor degree of diurnal variation with a slight rise in the afternoon. The total white cell count is normally high in pregnancy and following delivery, usually returning to normal within a week. Pathological variations in white cell values together with a brief review of their morphology and functions are considered below.

Polymorphs (Neutrophils):

Morphology: A polymorphonuclear (PMN) neutrophil, commonly called polymorph or neutrophil, is 12-15 µm in diameter. It consists of a characteristic dense nucleus, having 2-5 lobes and a pale cytoplasm containing numerous fine violet-pink granules. These lysosomal granules contain several enzymes and are of 2 types

Primary or azurophilic granules are large and coarse and appear early at the promyelocyte stage. These granules contain hydrolases, elastase, myeloperoxidase, cathepsin-G, cationic proteins, permeability-increasing proteins, and microbicidal proteins called defensins.

Secondary or specific granules are smaller and more numerous. These appear later at the myelocyte stage, are MPO-negative and contain lactoferrin, NADPH oxidase, histaminase, vitamin B₁₂ binding protein, and receptors for chemoattractants and for laminin.

The normal functions of neutrophils are as under:

1. Chemotaxis or cell mobilisation in when the cell is attracted towards bacteria or at the site of inflammation.
2. Phagocytosis in when the foreign particulate material of tiny sizes is phagocytosed by actively motile neutrophils; thus PMNs act as microphages compared to function of monocytes as macrophages.
3. Killing of the microorganism is mediated by oxygen-dependent and oxygen-independent pathways.

Pathologic Variations: Pathologic variations in neutrophils include variations in count, morphology and defective function.

Variation In Count: An increase in neutrophil count (neutrophil leucocytosis or neutrophilia) or a decrease in count (neutropenia) may occur in various diseases.

Neutrophil leucocytosis: An increase in circulating neutrophils above 7,500/µl is the commonest type of leucocytosis and occurs most commonly as a response to acute bacterial infections.

Some common causes of neutrophilia are as under:

1. Acute infections, local or generalised, especially by cocci but also by certain bacilli, fungi, spirochaetes, parasites and some viruses. For example pneumonia, cholecystitis, salpingitis, meningitis, diphtheria, plague, peritonitis, appendicitis, actinomycosis, poliomyelitis, abscesses, furuncles, carbuncles, tonsillitis, otitis media, osteomyelitis etc.
2. Other inflammations for example., tissue damage resulting from burns, operations, ischaemic necrosis (such as in MI), gout, collagen-vascular diseases, hypersensitivity reactions etc.
3. Intoxication for example., uraemia, diabetic ketosis, eclampsia, poisonings by chemicals and drugs.
4. Acute haemorrhage, internal or external.
5. Acute haemolysis.
6. Disseminated malignancies.
7. Myeloproliferative disorders for example., myeloid leukaemia, polycythaemia vera, and myeloid metaplasia.
8. Miscellaneous for example., following corticosteroid therapy, idiopathic neutrophilia.

Neutropenia: When the absolute neutrophil count falls below 2,500/µl, the patient is said to have neutropenia and is prone to develop recurrent infections.

Some common causes of neutropenia (and hence leucopenia) are as follows:

1. Certain infections for example., typhoid, paratyphoid, brucellosis, influenza, measles, viral hepatitis, malaria, kala-azar etc.

2. Overwhelming bacterial infections especially in patients with poor resistance for example., miliary tuberculosis, and septicaemia.

3. Drugs, chemicals and physical agents which induce aplasia of the bone marrow cause neutropenia, for example., antimetabolites, nitrogen mustards, benzene, and ionising radiation. Occasionally, certain drugs produce neutropenia due to individual sensitivity such as anti-inflammatory (amidopyrine, phenylbutazone), antibacterial (chloramphenicol, cotrimoxazole), anticonvulsants, antithyroids, hypoglycaemics and antihistaminics.

4. Certain haematological and other diseases for example., pernicious anaemia, aplastic anaemia, cirrhosis of the liver with splenomegaly, SLE, and Gaucher’s disease.

5. Cachexia and debility.

6. Anaphylactoid shock.

7. Certain rare hereditary, congenital or familial disorders for example., cyclic neutropenia, primary splenic neutropenia, idiopathic benign neutropenia.

Variations In Morphology:

Some of the common variations in neutrophil morphology.

These are as under:

1. Granules Heavy, dark-staining, coarse toxic granules are characteristic of bacterial infections.

2. Vacuoles In bacterial infections such as septicaemia, cytoplasmic vacuolation may develop.

3. Döhle bodies These are small, round or oval patches, 2-3 µm in size, in the cytoplasm. They are mostly seen in bacterial infections.

4. Nuclear abnormalities These include the following:

1. Sex chromatin is a normal finding in 2-3% of neutrophils in female sex. It consists of a drumstick appendage of chromatin, about 1 µm across, and attached to one of the nuclear lobes by a thin chromatin strand. Their presence in more than 20% of PMNs is indicative of female sex chromosomes.
2. A ‘shift-to-left’ is the term used for the appearance of neutrophils with a decreased number of nuclear lobes in the peripheral blood for example., the presence of band and stab forms and a few myelocytes in the peripheral blood. It is seen in severe infections, leucoerythroblastic reactions or leukaemia.
3. A ‘shift-to-right’ is the appearance of hypersegmented (more than 5 nuclear lobes) neutrophils in the peripheral blood such as in megaloblastic anaemia, uraemia, and sometimes in leukaemia.
4. Pelger-Huët anomaly is an uncommon autosomal dominant inherited disorder in which nuclei in a majority of neutrophils are distinctively bilobed (spectacle-shaped) and coarsely staining chromatin.
5. Acquired pseudo-Pelger-Huët abnormality may occur in acute infections or in MDS. However, the physiologic role of a multilobed nucleus of neutrophils is unknown and the bilobed anomaly is an innocuous condition.

Defective Functions:

The following abnormalities in neutrophil function may sometimes be found:

1. Defective chemotaxis for example., in a rare congenital abnormality called lazy-leucocyte syndrome; following corticosteroid therapy, aspirin ingestion, alcoholism, and in myeloid leukaemia.

2. Defective phagocytosis due to lack of opsonisation for example., in hypogammaglobulinaemia, hypocomplementaemia, after splenectomy, in sickle cell disease.

3. Defective killing for example., in chronic granulomatous disease, Chédiak-Higashi syndrome, and myeloid leukaemias.

Lymphocytes:

Morphology: The majority of lymphocytes in the peripheral blood are small (9-12 µm in diameter) but large lymphocytes (12-16 µm in diameter) are also found. Both small and large lymphocytes have round or slightly indented nuclei with coarsely clumped chromatin and scanty basophilic cytoplasm.

• Plasma cells are derived from B lymphocytes under the influence of appropriate stimuli. The nucleus of the plasma cell is eccentric and has a cart-wheel pattern of clumped nuclear chromatin.
• The cytoplasm is characteristically deeply basophilic with a pale perinuclear zone. Plasma cells are normally not present in peripheral blood but their pathological proliferation occurs in myelomatosis.
• Reactive lymphocytes (or Turk cells or plasmacytoid lymphocytes) are seen in certain viral infections and have sufficiently basophilic cytoplasm that they resemble plasma cells.
• Functionally, 3 types of lymphocytes possess distinct surface markers called clusters of differentiation (CD) which aid in the identification of the stage of their differentiation:

T lymphocytes i.e. thymus-dependent lymphocytes, mature in the thymus and are also known as thymocytes.

They are mainly involved in direct action on antigens and are therefore involved in cell-mediated immune (CMI) reaction by its subsets such as cytotoxic (killer) T-cells (CD3+), CD8+ T-cells, and delayed hypersensitivity reaction by CD4+ T-cells.

B lymphocytes i.e. bone marrow-dependent or bursa-equivalent lymphocytes as well as their derivatives, plasma cells, are the source of specific immunoglobulin antibodies. They are, therefore, involved in humoral immunity (HI) or circulating immune reactions.

NK cells i.e. natural killer cells are those lymphocytes which morphologically have the appearance of lymphocytes but do not possess functional features of T- or B-cells. As the name indicates they are identified with ‘natural’ or innate immunity and bring about direct ‘killing’ of microorganisms (particularly certain viruses) or lysis of foreign bodies.

Pathologic Variations: A rise in the absolute count of lymphocytes exceeding the upper limit of normal (above 4,000/µm) is termed lymphocytosis, while an absolute lymphocyte count below 1,500/µm is referred to as lymphopenia.

Lymphocytosis Some of the common causes of lymphocytosis are as under:

1. Certain acute infections for example., pertussis, infectious mononucleosis, viral hepatitis, and infectious lymphocytosis.
2. Certain chronic infections for example., brucellosis, tuberculosis, and secondary syphilis.
3. Haematopoietic disorders for example., lymphocytic leukaemias, lymphoma, heavy chain disease.
4. Relative lymphocytosis is found in viral exanthemas, convalescence from acute infections, thyrotoxicosis, and conditions causing neutropenia.

Lymphopenia Lymphopenia is uncommon and occurs in the following conditions:

1. Most acute infections.
2. Severe bone marrow failure.
3. Corticosteroid and immunosuppressive therapy.
4. Widespread irradiation.

Monocytes:

Morphology: The monocyte is the largest mature leucocyte in the peripheral blood measuring 12-20 µm in diameter.

It possesses a large, central, oval, notched indented or horseshoe-shaped nucleus which has a characteristically fine reticulated chromatin network. The cytoplasm is abundant, pale blue and contains many fine dust-like granules and vacuoles.

The main functions of monocytes are as under:

1. Phagocytosis of antigenic material or microorganisms.
2. Immunologic function as antigen-presenting cells and present the antigen to lymphocytes to deal with further.
3. As mediators of inflammation, they are involved in the release of prostaglandins, and stimulation of the liver to secrete acute-phase reactants.

Tissue macrophages of different types included in the RE system are derived from blood monocytes.

Pathologic Variations: A rise in the blood monocytes above 800/µl is termed monocytosis.

Some common causes of monocytosis are as follows:

1. Certain bacterial infections for example., tuberculosis, subacute bacterial endocarditis, syphilis.
2. Viral infections.
3. Protozoal and rickettsial infections for example., malaria, typhus, trypanosomiasis, and kala-azar.
4. Convalescence from acute infection.
5. Haematopoietic disorders for example., monocytic leukaemia, lymphomas, myeloproliferative disorders, multiple myeloma, and lipid storage disease.
6. Malignancies for example., cancer of the ovary, stomach, and breast.
7. Granulomatous diseases for example., sarcoidosis, and inflammatory bowel disease.
8. Collagen-vascular diseases.

Eosinophils:

Morphology: Eosinophils are similar to segmented neutrophils in size (12-15 µm in diameter), have coarse, deep red staining granules in the cytoplasm and have usually two nuclear lobes.

• Granules in eosinophils contain basic protein and stain more intensely for peroxidase than granules in the neutrophils.
• In addition, eosinophils also contain cell adhesion molecules, cytokines (IL-3, IL-5), and a protein that precipitates
• Charcot-Leyden crystals in lung tissues in asthmatic patients. Eosinophils are involved in reactions to foreign proteins and antigen-antibody reactions.

Pathologic Variations: An increase in the number of eosinophilic leucocytes above 400/µl is referred to as eosinophilia and below 40/µl is termed as eosinopenia.

Eosinophilia:

The causes are as under:

1. Allergic disorders for example., bronchial asthma, urticaria, angioneurotic oedema, hay fever, drug hypersensitivity.
2. Parasitic infestations for example.,. trichinosis, echinococcosis, intestinal parasitism.
3. Skin diseases for example., pemphigus, dermatitis herpetiformis, erythema multiforme.
4. Loeffler’s syndrome.
5. Pulmonary infiltration with eosinophilia (PIE) syndrome.
6. Tropical eosinophilia.
7. Haematopoietic diseases for example., CML, polycythaemia vera, pernicious anaemia, Hodgkin’s disease, following splenectomy.
8. Malignant diseases with metastases.
9. Irradiation.
10. Miscellaneous disorders for example., polyarteritis nodosa, rheumatoid arthritis, sarcoidosis.

Eosinopenia: Adrenal steroids and ACTH induce eosinopenia in men.

Basophils:

Morphology: Basophils resemble the other mature granulocytes but are distinguished by coarse, intensely basophilic granules which usually fill the cytoplasm and often overlap and obscure the nucleus.

The granules of circulating basophils (as well as their tissue counterparts as mast cells) contain heparin, histamine and 5-HT. Mast cells or basophils on degranulation are associated with histamine release.

Pathologic Variations: Basophil leucocytosis or basophilia refers to an increase in the number of basophilic leucocytes above 100/µl.

Basophilia is unusual and is found in the following conditions:

1. Chronic myeloid leukaemia
2. Polycythaemia vera
3. Myelosclerosis
4. Myxoedema
5. Ulcerative colitis
6. The following splenectomy
7. Hodgkin’s disease
8. Urticaria pigmentosa

Mature Leucocytes in Health and Pathologic Variation:

• Peripheral blood normally contains mature leucocytes expressed as total and differential cell counts which remain within normal range.
• Pathologic variation in TLC is called leucocytosis and leucopenia for abnormal rise and fall respectively. Different types of mature leucocytes have distinct causes for rise or fall in their corresponding cell type in the DLC.

Common types of variations in DLC and the most common causes are Neutrophilia (acute bacterial infections), neutropenia (drugs); lymphocytosis (viral infections), lymphopenia (acute infections); monocytosis (chronic bacterial or viral infections); eosinophilia (allergic disorders), eosinopenia (steroid therapy); and basophilia (CML).

There are morphologic changes in mature leucocytes which may be a pointer to a particular disease for example., toxic granules in neutrophils in severe bacterial infections, hypersegmented neutrophils in megaloblastic anaemia

Infectious Mononucleosis:

Infectious mononucleosis (IM) or glandular fever is a benign, self-limiting lymphoproliferative disease caused by Epstein-Barr virus (EBV), one of the herpesviruses. Infection may occur from childhood to old age but the classical acute infection is more common in teenagers and young adults.

• The infection is transmitted by person-to-person contact such as by kissing with transfer of virally-contaminated saliva. Groups of cases occur particularly in young people living together in boarding schools, colleges, camps and military institutions.
• Primary infection in childhood is generally asymptomatic, while 50% of adults develop clinical manifestations. The condition is so common that by the age of 40, most people have been infected and developed antibodies.
• It may be mentioned here that EBV is oncogenic as well and is strongly implicated in the African (endemic) Burkitt’s lymphoma and nasopharyngeal carcinoma.

Pathogenesis:

EBV, the etiologic agent for IM, is a B lymphotropic herpesvirus. The disease is characterised by fever, generalised lymphadenopathy, hepatosplenomegaly, sore throat, and appearance in the blood of atypical ‘mononucleosis cells’.

The pathogenesis of these pathologic features is outlined below:

1. In a susceptible seronegative host who lacks antibodies, the virus in the contaminated saliva invades and replicates within epithelial cells of the salivary gland and then enters B-cells in the lymphoid tissues which possess receptors for EBV. The infection spreads throughout the body via the bloodstream or by infected B cells.

2. Viraemia and death of infected B-cells cause an acute febrile illness and the appearance of specific humoral antibodies which peak about 2 weeks after the infection and persist throughout life. The appearance of antibodies marks the disappearance of the virus from the blood.

3. Though the viral agent has disappeared from the blood, the EBV-infected B cells continue to be present in the circulation as latent infection. EBV-infected B-cells undergo polyclonal activation and proliferation. These cells perform two important roles which are the characteristic diagnostic features of IM:

1. They secrete antibodies—initially IgM but later IgG class antibodies appear. IgM antibody is the heterophile anti-sheep antibody used for diagnosis of IM while IgG antibody persists for life and provides immunity against re-infection.
2. They activate CD8+T lymphocytes—also called cytotoxic T-cells (or CTL) or suppressor T cells. CD8+ T-cells bring about the killing of B-cells and are pathognomonic atypical lymphocytes seen in blood in IM.

4. The proliferation of these cells is responsible for generalised lymphadenopathy and hepatosplenomegaly.

5. The sore throat in IM may be caused by either necrosis of B-cells or due to viral replication within the salivary epithelial cells in the early stage.

Besides the involvement of EBV in the pathogenesis of IM, its role in neoplastic transformation in nasopharyngeal carcinoma, Burkitt’s lymphoma and B-cell NHL and diagrammatically depicted.

Clinical Features:

The incubation period of IM is 30-50 days in young adults, while children have a shorter incubation period. A prodromal period of 3-5 days is followed by frank clinical features lasting for 1-3 weeks, and subsequently complete recovery occurs after 2 months.

The usual clinical features are as under:

1. During the prodromal period (first 3-5 days), the symptoms are mild such as malaise, myalgia, headache and fatigue.

2. Frank’s clinical features (next 7-21 days) seen commonly are fever (90%), sore throat (80%) and bilateral cervical lymphadenopathy (95%).

• Other features are splenomegaly (50% of patients), hepatomegaly (10% of cases), transient erythematous maculopapular rash on the trunk and extremities (10%), periorbital oedema (10%) and jaundice (5%).

3. Complications Although most cases of IM run a self-limited course, complications may develop in some cases as under:

1. Neurologic manifestations in children.
2. Splenic rupture due to splenitis.
3. Upper airway obstruction due to hypertrophied adenotonsillitis.
4. Autoimmune haemolytic anaemia, cold antibody type.
5. Bacterial superinfection.
6. Rarely, myocarditis, hepatitis, pneumonia.

Laboratory Findings:

The diagnosis of IM is made by characteristic haematologic and serologic findings.

1. Haematologic findings Major abnormalities in the blood are as under:

1. TLC There is a moderate rise in total white cell count (10,000-20,000/µl) during 2nd to 3rd week after infection.
2. DLC There is an absolute lymphocytosis. The lymphocytosis is due to a rise in normal as well as atypical T lymphocytes. There is relative neutropenia.
3. Atypical T-cells Essential to the diagnosis of IM is the presence of at least 10-12% atypical T-cells (or mononucleosis cells) lying in peripheral blood lymphocytosis. The mononucleosis cells are variable in appearance and are classed as Downey type I, II and III, of which Downey type I are found most frequently. These atypical T lymphocytes are usually of the size of large lymphocytes (12-16 µm diameter). The nucleus, rather than the usual round configuration, is oval, kidney-shaped or slightly lobate due to the indentation of the nuclear membrane and contains relatively fine chromatin without nucleoli, suggesting an immature pattern but short of leukaemic features. The cytoplasm is more abundant, basophilic and finely granular and may contain vacuoles. The greatest number of atypical lymphocytes is found between the 7th to 10th day of the illness and these cells may persist in the blood for up to 2 months.
4. CD4+ and CD8+ T-cell count There is a reversal of CD4+/CD8+ T-cell ratio. There is a marked decrease in CD4+ T-cells while there is a substantial rise in CD8+ T-cells.
5. Platelets There is generally thrombocytopenia in the first 4 weeks of illness.

2. Serologic Diagnosis: The second characteristic laboratory finding is the demonstration of antibodies in the serum of infected patients.

These are as under:

1. Test for heterophile antibodies The Heterophile antibody test (Paul-Bunnell test) is used for making the diagnosis of IM. In this test, the patient’s serum is absorbed by the guinea pig kidney. Serum dilutions are prepared which are used for agglutination of red cells of sheep, horses or cows and are reported as heterophile titers of test serum.
   1. A high serum titer of 40 or more times is diagnostic of acute IM infection in symptomatic cases in the first week. Heterophile antibodies peak during the 3rd week in 80-90% of cases.
   2. The test remained positive for about 3 months after the illness started. Thus, the test has to be repeatedly performed. Similar antibody is also produced in patients suffering from serum sickness and have to be distinguished by differential absorption studies.
   3. Heterophile antibodies are not demonstrable in children under 5 years of age or in quite elderly. Currently, a more sensitive and rapid kit-based test for heterophile antibodies, mono spot, is also available.
2. EBV-specific antibody tests Specific antibodies against the viral capsid and nucleus of EBV can be demonstrated in patients who are negative for heterophile antibody test
   1. Specific antibodies against EBV capsid antigen show elevated titers in over 90% of cases during acute infection. IgM class antibody appears early and is thus most useful for diagnosis of acute infection. IgG class antibody appears later and persists throughout life; thus it does not have diagnostic value but is instead used for assessing the past exposure to EBV infection.
   2. Antibodies against EBV nuclear antigen are detected 3-6 weeks after infection and, like IgG class antibodies, persist throughout life.
   3. Antibodies to early antigens may be elevated but are less useful for making a diagnosis of IM. However, titers of these antibodies remain elevated for 3-6 months and their levels are high in cases of nasopharyngeal carcinoma and African Burkitt’s lymphoma.
   4. IgA antibodies to EBV antigen are seen in patients with nasopharyngeal carcinoma or those who are at high risk of developing this EBV-induced cancer.
3. EBV antigen detection Detection of EBV DNA or proteins can be done in blood or CSF by PCR method.

3. Liver Function Tests: In addition, abnormalities of the liver function test are found in about 90% of cases. These include elevated serum levels of transaminases (SGOT and SGPT), rise in serum alkaline phosphatase and mild elevation of serum bilirubin.

Infectious Mononucleosis:

• Infectious mononucleosis or glandular fever is a self-limited viral infection caused by the EB virus.
• In symptomatic cases, it presents with fever, soar throat, and bilateral cervical lymphadenopathy.
• The diagnosis rests on haematologic findings of leucocytosis with absolute lymphocytosis, the presence of atypical T lymphocytes in peripheral blood, and documentation of serum antibodies (heterophile antibody or Paul-Bunnel test and EBV-specific antibodies)

Leukaemoid Reactions:

• Leukaemoid reaction is defined as a reactive excessive leucocytosis in the peripheral blood resembling that of leukaemia in a subject who does not have leukaemia.
• Despite the confusing blood pictures, the clinical features of leukaemia such as splenomegaly, lymphadenopathy and haemorrhages are usually absent and the features of the underlying disorder causing the leukemoid reaction are generally obvious.
• Leukaemoid reaction may be myeloid or lymphoid; the former is much more common.

Myeloid Leukaemoid Reaction:

Causes: The majority of leukemoid reactions involve the granulocyte series. It may occur in association with a wide variety of diseases.

These are as under:

1. Infections for example., staphylococcal pneumonia, disseminated tuberculosis, meningitis, diphtheria, sepsis, endocarditis, plague, infected abortions etc.
2. Intoxication for example., eclampsia, mercury poisoning, severe burns.
3. Malignant diseases for example., multiple myeloma, myelofibrosis, Hodgkin’s disease, and bone metastases.
4. Severe haemorrhage and severe haemolysis.

Laboratory Findings:

The myeloid leukemoid reaction is characterised by the following laboratory features:

1. Leucocytosis, usually moderate, not exceeding 100,000/µl.
2. The proportion of immature cells is mild to moderate, comprised of metamyelocytes, myelocytes (5-15%), and blasts fewer than 5% i.e. the blood picture simulates somewhat with that of CML.
3. Infective cases may show toxic granulation and Döhle bodies in the cytoplasm of neutrophils.
4. Neutrophil (or Leucocyte) alkaline phosphatase (NAP or LAP) score in the cytoplasm of mature neutrophils in leukemoid reaction is characteristically high and is very useful to distinguish it from chronic myeloid leukaemia in doubtful cases.
5. Cytogenetic studies may be helpful in exceptional cases which reveal negative Philadelphia chromosome i.e. t (9; 22) or BCR-ABL fusion gene in myeloid leukemoid reaction but positive in cases of CML.
6. Additional features include anaemia, normal-to-raised platelet count, myeloid hyperplasia of the marrow and absence of infiltration by immature cells in organs and tissues. Sums up the features to distinguish myeloid leukaemoid reaction from chronic myeloid leukaemia.

Lymphoid Leukaemoid Reaction:

Causes:

Lymphoid leukemoid reaction may be found in the following conditions:

1. Infections for example., infectious mononucleosis, cytomegalovirus infection, pertussis (whooping cough), chickenpox, measles, infectious lymphocytosis, tuberculosis.
2. Malignant diseases may rarely produce lymphoid leukemoid reactions.

Laboratory Findings:

The blood picture is characterised by the following findings:

1. Leucocytosis not exceeding 100,000/µl.
2. The differential white cell count reveals mostly mature lymphocytes simulating the blood picture found in cases of CLL

Leukaemoid Reaction:

• Leukaemoid reaction is due to the reactive proliferation of leucocytes, which may be myeloid or lymphoid.
• The myeloid leukemoid reaction is more common and is due to an underlying cause for the reactive proliferation of myeloid series of cells.
• The diagnosis is made by leucocytosis with an immature myeloid series of cells in the peripheral blood (myelocytes, metamyelocytes, but blasts <5%), and raised LAP score in neutrophils.
• It requires distinction from CML—the myeloid leukemoid reaction has high LAP scores and is Philadelphia chromosome-negative.
• The lymphoid leukemoid reaction is due to marked lymphocytosis, mostly due to mature small lymphocytes. Its common causes are a viral illness or whooping cough.

Haematolymphoid Malignancies General Considerations

The most important group of leucocyte disorders are leukaemias and lymphomas which are malignant proliferation of white blood cells.

Classification Schemes:

Conventionally, leukaemias are classified into:

• Myeloid and lymphoid based on cell types predominantly involved.
• Acute and chronic based on the natural history of the disease.

Accordingly, the main types of leukaemias are:

• Acute myeloblastic leukaemia and acute lymphoblastic leukaemia (AML and ALL):
  • Acute leukaemias are characterised by a predominance of undifferentiated or earliest leucocyte precursors or leukaemic blasts of corresponding series. In general, patients with acute leukaemia have a rapidly downhill course.
• Chronic myeloid leukaemia and chronic lymphocytic leukaemias (CML and CLL):
  • Chronic leukaemias have easily recognisable late precursors of corresponding leucocyte series and they circulate in large numbers as the predominant chronic leukaemic cells. Patients with chronic leukaemia tend to have more indolent behaviour.

Besides, there are some other uncommon variants of leukaemias. The incidence of both acute and chronic leukaemias is higher in men than in women. ALL is primarily a disease of children and young adults, whereas AML occurs at all ages. CLL tends to occur in the elderly, while CML is found in middle age.

• Over the last 50 years, several classification systems have been proposed for leukaemias and lymphomas—clinicians favouring an approach based on clinical findings while pathologists have been interested in classifying them on morphologic features.
• Newer classification schemes have been based on cytochemistry, immunophenotyping, cytogenetics and molecular markers which have become available to pathologists and haematologists.
• The classification scheme proposed by the World Health Organisation (WHO) combines all tumours of haematopoietic and lymphoreticular tissues.
• The basis of the WHO classification is the cell type of the neoplasm as identified by a combined approach of clinical features and morphologic, cytogenetic and molecular characteristics, rather than the location of the neoplasm (whether in blood or tissues) because lymphohematopoietic cells are present in circulation as well as in lymphoreticular tissues.
• As per the revised WHO classification scheme (2016), neoplasms of haematopoietic and lymphoid tissues are considered a unified group and are divided into 3 broad groups.

1. Myeloid neoplasms: This group includes neoplasms of myeloid cell lineage and, therefore, includes neoplastic proliferations of red blood cells, platelets, granulocytes and monocytes.

There are 5 categories under the myeloid series of neoplasms:

1. Myeloproliferative neoplasms (MPN),
2. Myeloid/lymphoid neoplasm with eosinophilia and gene rearrangement,
3. Myelodysplastic/myeloproliferative neoplasms (MDS/MPN),
4. Myelodysplastic syndromes (MDS)
5. Acute myeloid leukaemia (AML).

2. Lymphoid neoplasms: Neoplasms of lymphoid lineage include leukaemias and lymphomas of B, T or NK cell origin. This group is divided into Hodgkin’s disease and non-Hodgkin’s lymphomas; the latter group includes several types based on maturation stages and immunophenotyping of lymphoid cells (malignancies of B-cells including plasma cell disorders, T-cells, and rarely NK cells).

3. Histiocytic neoplasms: This group is of interest mainly due to neoplastic proliferations of histiocytes in Langerhans cell histiocytosis.

• Besides the WHO classification, the FAB (French-American-British) Cooperative Group classification of lymphomas and leukaemias based on morphology and cytochemistry is also widely used.
• These as well as other classification schemes have been tabulated and discussed in appropriate sections later. But before that, the unified aetiology and pathogenesis of leukaemias and lymphomas are discussed below.

Etiology Of Haematolymphoid Neoplasms:

The exact aetiology of leukaemias and lymphomas is not known. However, many factors have been implicated:

1. Heredity:

There is evidence to suggest that there is a role of family history, occurrence in identical twins and predisposition of these malignancies in certain genetic syndromes.

1. Identical twins, There is a high concordance rate among identical twins if acute leukaemia develops in the first year of life. Hodgkin’s disease is 99 times more common in the identical twin of an affected case compared with the general population, implicating genetic origin strongly.
2. Family history Families with excessive incidence of leukaemia have been identified.
3. Genetic disease association Acute leukaemia occurs with increased frequency with a variety of congenital disorders such as Down’s, Bloom’s, Klinefelter’s and Wiskott-Aldrich’s syndromes, Fanconi’s anaemia and ataxia telangiectasia. Hodgkin’s disease has familial incidence and with certain HLA types.

2. Infections: There is evidence to suggest that certain infections, particularly viruses, are involved in the development of lymphomas and leukaemias.

1. Human T-cell leukaemia-lymphoma virus I (HTLV-I) is implicated in the aetiology of adult T-cell leukaemia-lymphoma (ATLL).
2. HTLV II for T-cell variant of hairy cell leukaemia.
3. Epstein-Barr virus (EBV) is implicated in the aetiology of Hodgkin’s disease (mixed cellularity type and nodular sclerosis type), endemic variety of Burkitt’s lymphoma, and post-transplant lymphoma.
4. HIV in diffuse large B-cell lymphoma and Burkitt’s lymphoma.
5. Hepatitis C virus (HCV) in lymphoplasmacytic lymphoma.
6. Human herpes virus 8 (HHV-8) in primary effusion lymphoma.
   Helicobacter pylori bacterial infection of gastric mucosa in MALT lymphoma of the stomach.

3. Environmental Factors:

Certain environmental factors are known to play a role in the aetiology of leukaemias and lymphomas:

1. Ionising radiation: Damage due to radiation exposure has been linked to the development of leukaemias and lymphomas.
   1. Individuals exposed to occupational radiation exposure, patients receiving radiation therapy, and Japanese survivors of the atomic bomb explosions are at higher risk of developing haematopoietic malignancies, particularly prone to development of CML, AML and ALL but not to CLL or hairy cell leukaemia.
2. Chemical carcinogens: Benzene, tobacco smoking, alcohol, use of certain hair dyes and exposure to agricultural chemicals are associated with an increased risk of development of haematopoietic malignancies.
3. Certain drugs: Long-term exposure to certain drugs such as phenytoin, alkylating agents and other chemotherapeutic agents is associated with increased incidence of leukaemias and lymphomas. Patients treated for Hodgkin’s disease can develop NHL.

4. Association With Diseases Of Immunity: Since lymphoid cells are the immune cells of the body, diseases with derangements of the immune system have a higher incidence of haematopoietic malignancies

1. Immunodeficiency diseases: Various inherited and acquired immunodeficiency diseases including AIDS and iatrogenic immunosuppression induced by chemotherapy or radiation, are associated with subsequent development of lymphomatous transformation.
2. Autoimmune disease association: A few autoimmune diseases such as Sjogren’s syndrome, nontropical sprue, rheumatoid arthritis and SLE are associated with a higher incidence of NHL.

Pathogenesis:

It needs to be emphasised that since haematopoietic cells have a rapid turnover, they are more vulnerable to chromosomal damage and cytogenetic changes under the influence of various etiologic factors listed above.

1. Genetic damage to a single clone of target cells: Leukaemias and lymphomas arise following the malignant transformation of a single clone of cells belonging to the myeloid or lymphoid series, followed by the proliferation of the transformed clone.

• The basic mechanism of malignant transformation is genetic damage to the DNA of the target white cells followed by proliferation, disrupting normal growth and differentiation.
• The heritable genetic damage may be induced by various etiologic agents listed above (for example., RNA viruses HTLV-I, EBV etc) and causes insertional mutagenesis for which oncogenes may play a role.
• The evolution of leukaemia is a multi-step process, and in many cases, acute leukaemia may develop after a pre-existing myelodysplastic or myeloproliferative disorder.

2. Chromosomal translocations: Several cytogenetic abnormalities have been detected in cases of leukaemias-lymphomas, most consistent of which are chromosomal translocations. In NHL, translocation involving antigen receptor genes, immunoglobulin genes, or overexpression of BCL-2 protein may be seen.

The most consistent chromosomal abnormality in various forms of acute and chronic leukaemias is the Philadelphia (Ph) chromosome seen in 70-90% of cases with CML, involving reciprocal translocation of parts of the long arm of chromosome 22 to the long arm of chromosome 9 i.e. t(9;22).

3. Maturation defect: In acute leukaemia, the single most prominent characteristic of the leukaemic cells is a defect in maturation beyond the myeloblast or promyelocyte level in AML, and the lymphoblast level in ALL.

It needs to be emphasised that it is the maturation defect in leukaemic blasts rather than rapid proliferation of leukaemic cells responsible for causing acute leukaemia. In fact, the generation time of leukaemic blasts is somewhat prolonged rather than shortened.

4. Myelosuppression: As the leukaemic cells accumulate in the bone marrow, there is suppression of normal haematopoietic stem cells, partly by physically replacing the normal marrow precursors, and partly by inhibiting normal haematopoiesis via cell-mediated or humoral mechanisms.

This is based on the observation that some patients with acute leukaemia have a hypocellular marrow indicating that marrow failure is not due to overcrowding by leukaemic cells alone.

Nevertheless, some normal haematopoietic stem cells do remain in the marrow which are capable of proliferating and restoring normal haematopoiesis after effective anti-leukaemic treatment.

5. Organ infiltration: The leukaemic cells proliferate primarily in the bone marrow, circulate in the blood and infiltrate into other tissues such as lymph nodes, liver, spleen, skin, viscera and the central nervous system.

6. Cytokines: The presence of reactive inflammatory cells in Hodgkin’s disease is due to the secretion of cytokines from the Reed Sternberg cells for example., IL-5 (growth factor for eosinophils), IL-13 (for autocrine stimulation of RS cells) and transforming growth factor-β (for fibrogenesis)

Haematolymphoid Malignancies General Considerations:

• The malignant proliferation of white blood cells called leukaemias and lymphomas are the most important diseases of leucocytes.
• Conventionally, leukaemias are divided into acute and chronic, and into myeloid and lymphoid; thus major types are AML, ALL, CML, and CLL.
• According to the WHO classification, neoplasms of haematopoietic cells in the blood and lymphoreticular organs are considered a unified group and are divided into neoplasms of myeloid, lymphoid and histiocytic cells.
• Although the exact aetiology of leukaemias and lymphomas is not known, a number of factors have been implicated for example., heredity, viral and certain bacterial infections, ionising radiation, chemical carcinogens, certain drugs, immunodeficiency, and autoimmune diseases.
• In general, the pathogenesis of these cancers evolves through genetic damage to a single clone of target cells, chromosomal translocations, maturation defect and myelosuppression and secretion of certain cytokines

Myeloid Neoplasms

Based on the cell line of differentiation of the pluripotent stem cell, the WHO classification divides all haematopoietic neoplasms into myeloid, lymphoid and histiocytic neoplasms.

Since myeloid trilineage stem cells further differentiate into 3 series of progenitor cells erythroid, granulocyte-monocyte, and megakaryocytic series, therefore all examples of myeloid neoplasms fall into these three categories of cell lines.

Based on this concept, myeloid neoplasms are divided into the following 5 groups:

1. Myeloproliferative neoplasms (MPN)
2. Myeloid/lymphoid neoplasms with eosinophilia and gene rearrangement
3. Myelodysplastic/myeloproliferative neoplasms (MDS/MPN)
4. Myelodysplastic syndrome (MDS)
5. Acute myeloid leukaemia (AML)

As shown, each of these groups has several subtypes. Important examples within each group are discussed here.

Myeloproliferative Neoplasms:

The myeloproliferative disorders, renamed as ‘myeloproliferative neoplasms (MPN)’ are a group of neoplastic proliferation of multipotent haematopoietic stem cells. Besides their common stem cell origin, these disorders are closely related, occasionally leading to the evolution of one entity into another during the disease.

• The WHO classification of MPN includes 9 subtypes. Classic and common examples are chronic myeloid leukaemia (CML), polycythaemia vera (PV), and essential thrombocytosis (ET), each one representing a corresponding excess of mature and immature granulocytes, red blood cells, and platelets, respectively.
• Primary myelofibrosis, another clonal disorder, is also included in the discussion here. The group as a whole has a slow and insidious onset of clinical features and indolent clinical behaviour.

Chronic Myeloid Leukaemia (CML):

Chronic Myeloid Leukaemia Definition and Pathophysiology:

By WHO definition, CML is established by the identification of the clone of a haematopoietic stem cell that possesses the balanced reciprocal translocation between chromosomes 9 and 22, forming the Philadelphia chromosome.

• The t(9;22) involves the fusion of BCR (breakpoint cluster region) gene on chromosome 22q11 with the ABL1 (named after Abelson murine leukaemia virus) gene located on chromosome 9q34.
• The fusion product so formed is termed “Ph chromosome t(9;22) (q34;q11), BCR-ABL1” which should be positive for making the diagnosis of CML.
• This identification may be done by PCR or by FISH. The underlying pathophysiologic mechanism of human CML is based on the observation that BCR-ABL1 fusion product proteins are capable of transforming haematopoietic progenitor cells in vitro and forming malignant clones.

BCR-ABL1 fusion product brings about the following functional changes:

1. ABL1 protein is activated to function as a tyrosine kinase enzyme that in turn activates other kinases which inhibits apoptosis.
2. The ability of ABL1 to act as a DNA-binding protein is altered.
3. The binding of ABL1 to actin microfilaments of the cytoskeleton is increased.

The exact mechanism of progression of CML to the blastic phase is unclear but the following mechanisms may be involved:

1. Structural alterations in tumour suppressor p53 gene.
2. Structural alterations in tumour suppressor RB gene.
3. Alterations in RAS oncogene.
4. Alterations in MYC oncogene.
5. Release of cytokine IL-1β.
6. Functional inactivation of tumour suppressor protein, phosphatase A2.

Chronic Myeloid Leukaemia Clinical Features:

Chronic myeloid (myelogenous, granulocytic) leukaemia comprises about 20% of all leukaemias and its peak incidence is seen in 3rd and 4th decades of life.

A distinctive variant of CML seen in children is called juvenile CML. Both sexes are affected equally. The onset of CML is generally insidious.

Some of the common presenting manifestations are as under:

1. Features of anaemia such as weakness, pallor, dyspnoea and tachycardia.
2. Symptoms due to hypermetabolism such as weight loss, lassitude, anorexia, and night sweats.
3. Splenomegaly is almost always present and is frequently massive. In some patients, it may be associated with acute pain due to splenic infarction.
4. Bleeding tendencies such as easy bruising, epistaxis, menorrhagia and haematomas may occur.
5. Less common features include gout, visual disturbance, neurologic manifestations and priapism.
6. Juvenile CML is more often associated with lymph node enlargement than splenomegaly. Other features are frequent infections, haemorrhagic manifestations and facial rash.

Laboratory Findings:

The diagnosis of CML is generally possible on blood picture alone. However, bone marrow, cytochemical stains and other investigations are of help.

1. Blood Picture:

The typical blood picture in a case of CML at the time of presentation shows the following features:

1. Anaemia: Anaemia is usually of moderate degree and is normocytic normochromic in type. Occasional normoblasts may be present.
2. White blood cells: Characteristically, there is marked leucocytosis (approximately 200,000/ µl or more at the time of presentation). The natural history of CML consists of 3 phases— chronic, accelerated, and blastic.
   • The chronic phase of CML begins as a myeloproliferative disorder and consists of excessive proliferation of myeloid cells of intermediate grade (i.e. myelocytes and metamyelocytes) and mature segmented neutrophils.
   • Myeloblasts usually do not exceed 10% of cells in the peripheral blood and bone marrow. An increase in the proportion of basophils up to 10% is a characteristic feature of CML. A rising basophilia is indicative of impending blastic transformation.
   • An accelerated phase of CML is also described in which there is progressively rising leucocytosis associated with thrombocytosis or thrombocytopenia and splenomegaly. The accelerated phase has an increasing degree of anaemia, blast count in blood or marrow between 10 and 20%, marrow basophils 20% or more, and platelet count falling below 1,00,000/µl.
   • Blastic phase or blast crisis in CML fulfils the definition of acute leukaemia in having blood or marrow blasts ≥20%. These blast cells may be myeloid, lymphoid, erythroid or undifferentiated and are established by morphology, cytochemistry, or immunophenotyping.
   •  Myeloid blast crisis in CML is more common and resembles AML. However, unlike AML, Auer rods are not seen in myeloblasts of CML in blast crisis.
3. Platelets: Platelet count may be normal but is raised in about half the cases.

2. Bone Marrow Examination:

Examination of marrow aspiration yields the following results:

1. Cellularity: Generally, there is hypercellularity with total or partial replacement of fat spaces by proliferating myeloid cells.
2. Myeloid cells: The myeloid cells predominate in the bone marrow with an increased myeloid erythroid ratio. The differential counts of myeloid cells in the marrow show similar findings as seen in the peripheral blood with a predominance of myelocytes.
3. Erythropoiesis: Erythropoiesis is normoblastic but there is a reduction in erythropoietic cells.
4. Megakaryocytes: Megakaryocytes are conspicuous but are usually smaller in size than normal.
5. Cytogenetics: Cytogenetic studies on blood and bone marrow cells show the characteristic chromosomal abnormality called the Philadelphia (Ph) chromosome seen in 90-95% of cases of CML. Ph chromosome is formed by reciprocal balanced translocation between part of the long arm of chromosome 22 and part of the long arm of chromosome 9{(t(9;22) (q34;q11)} forming product of fusion gene, BCR-ABL1.

3. Cytochemistry: The only significant finding on cytochemical stains is reduced scores of neutrophil alkaline phosphatase (NAP) which helps to distinguish CML from myeloid leukemoid reaction in which case NAP scores are elevated. However, NAP scores in CML return to normal with successful therapy, corticosteroid administration and infections.

4. Other Investigations:

A few other accompanying findings are seen in CML:

1. Elevated serum B₁₂ and vitamin B₁₂ binding capacity.
2. Elevated serum uric acid (hyperuricaemia).

General Principles of Treatment and Prognosis:

Insight into the molecular mechanism of CML has brought about major changes in its therapy. The approach of modern therapy in CML is targeted at the removal of all malignant clones of cells bearing BCR/ABL1 fusion protein so that the patient reverts back to prolonged non-clonal haematopoiesis i.e. molecular remission from the disease.

This is achievable by the following approaches:

1. Imatinib oral therapy: The basic principle underlying imatinib oral treatment is to competitively inhibit the ATP binding site of the ABL kinase, which in turn, inhibits signal transduction of BCR-ABL1 fusion protein.

Imatinib induces apoptosis in BCR-ABL1 positive cells and thus eliminates them. Imatinib is found more effective in newly diagnosed cases of CML. Complete hematologic remission is achieved for 18 months in 97% of cases treated with imatinib.

2. Allogenic bone marrow (stem cell) transplantation: Although this treatment modality offers a proven cure, it is complicated with mortality due to the procedure and development of posttransplant graft-versus-host disease (GVHD) and, therefore, post-transplant immunosuppressive treatment has to be continued.

3. Interferon-α: Before imatinib and allogenic transplantation, the chronic phase of CML used to be treated with interferon-α which was the drug of choice.

4. Chemotherapy: Chemotherapeutic agents are used in the treatment of CML to lower the total population of WBCs. These include the use of busulfan, cyclophosphamide (melphalan) and hydroxyurea.

5. Others: Besides the above, other forms of treatment include splenic irradiation, splenectomy and leucopheresis. The most common cause of death (in 80% of cases) in CML is disease acceleration and blastic transformation.

Polycythaemia Vera:

Definition and Pathophysiology:

Polycythaemia vera (PV) is a clonal disorder characterised by increased production of all myeloid elements resulting in pancytosis (i.e. increased red cells, granulocytes, platelets) in the absence of any recognisable cause.

• The term ‘polycythaemia vera’ or ‘polycythaemia rubra vera’ is used for primary or idiopathic polycythaemia only and is the most common of all myeloproliferative disorders.
• Secondary polycythaemia or erythrocytosis, on the other hand, may occur secondary to several causes for example.,

1. High altitude.
2. Cardiovascular disease.
3. Pulmonary disease with alveolar hypoventilation.
4. Heavy smoking.
5. Inappropriate increase in erythropoietin (renal cell carcinoma, hydronephrosis, hepatocellular carcinoma, cerebellar haemangioblastoma, massive uterine leiomyoma).
6. Sometimes relative or spurious polycythaemia may result from plasma loss such as in burns and in dehydration from vomiting or water deprivation.

None of the secondary causes of polycythaemia is associated with splenic enlargement or increased leucocytes and platelets which are typical of PV.

The exact etiology of PV is not known but about a third of cases show inconsistent and varied chromosomal abnormalities such as 20q, trisomy 8 and 9p. The major pathogenetic mechanism is a tyrosine kinase JAK2 mutation which removes the autoinhibitory control and activates the kinases.

Clinical Features:

PV is a disease of late middle life and is slightly more common in males. The disease generally runs a chronic but slowly progressive course. Clinical features are the result of hyperviscosity, hypervolaemia, hypermetabolism and decreased cerebral perfusion.

1. These are as under: Headache, vertigo, tinnitus, visual alterations, syncope or even coma.
2. Increased risk of thrombosis due to accelerated atherosclerosis.
3. Increased risk of haemorrhages due to increased blood volume and intrinsic platelet dysfunction for example., epistaxis, and peptic ulcer disease.
4. Splenomegaly produces abdominal fullness.
5. Pruritus, especially after a bath.
6. Increased risk of urate stones and gout due to hyperuricaemia.

Laboratory Findings:

PV is diagnosed by the following haematologic findings:

1. Raised haemoglobin concentration (above 17.5 g/dl in males and 15.5 g/dl in females).
2. Erythrocytosis (above 6 million/µl in males and 5.5 million/µl in females).
3. Haematocrit (PCV) is above 55% in males and 47% in females.
4. Mild to moderate leucocytosis (15,000-25,000/µl) with basophilia and raised neutrophil alkaline phosphatase scores.
5. Thrombocytosis with defective platelet function.
6. Bone marrow examination reveals erythroid hyperplasia or pan-hyperplasia.
7. Cytogenetic abnormalities such as 20q, trisomy 8 and 9p are found in 30% of cases of PV.
8. In PV, unlike secondary polycythaemia, erythropoietin levels in serum and urine are reduced.

General Principles of Treatment and Prognosis:

Since PV runs an indolent course, therapy is aimed at maintaining normal blood counts and relieving the patient of symptoms.

1. Phlebotomy (venesection) by blood-letting is done at regular intervals to reduce total blood cell mass and to induce a state of iron deficiency.
2. Anticoagulant therapy is administered in case thrombosis has occurred.
3. Chemotherapy may be indicated to induce myelosuppression.
4. Hyperuricaemia is treated with uricosuric drugs.
5. Interferon-α is associated with good results because it reduces JAK2 expression in these patients which is the underlying cytogenetic abnormality.

Patients receiving phlebotomy alone may survive for 10-12 years. About 25% of patients progress to myelofibrosis. A small proportion of patients develop secondary haematologic malignancies such as AML, non-Hodgkin’s lymphoma and multiple myeloma. The major complication and cause of death in PV is vascular thrombosis.

Essential Thrombocythaemia:

Definition and Pathophysiology:

Essential thrombocythaemia (ET), also termed essential thrombocytosis or primary (idiopathic) thrombocythaemia is a clonal disorder characterised by markedly elevated platelet count in the absence of any recognisable stimulus.

• Secondary or reactive thrombocytosis, on the other hand, occurs in response to known stimuli such as chronic infection, haemorrhage, postoperative state, chronic iron deficiency, malignancy, rheumatoid arthritis and post-splenectomy.
• ET is an uncommon disorder and represents an overproduction of platelets from megakaryocyte colonies without any added stimulus but no clonal marker is available to distinguish primary from secondary thrombocytosis.
• Though an elevated platelet count is the dominant feature, other cell lines may also be involved in the expansion of neoplastic clones.
• The underlying pathophysiologic mechanism in ET is the absence of control by thrombopoietin that regulates endomitosis in the megakaryocytes to produce platelets.
• The result is uncontrolled proliferation of not only megakaryocytes but also of the platelets. There is a probable role of heredity in ET since families with ET have been reported.

Clinical Features:

The condition has an insidious onset and is more frequent in older people. Haemorrhagic and thrombotic events are common.

These include the following:

1. Arterial or venous thrombosis.
2. Easy disability following minor trauma.
3. Spontaneous bleeding.
4. Transient ischaemic attack or frank stroke due to platelet aggregation in microvasculature of the CNS.

Laboratory Findings:

The prominent laboratory features pertain to platelets.

These include the following:

• Sustained elevation in platelet count (above 400,000 µl).
• Blood film shows many large platelets, megakaryocyte fragments and hypogranular forms.
• Consistently abnormal platelet functions, especially abnormality in platelet aggregation.
• Bone marrow examination reveals a large number of hyperdiploid megakaryocytes and a variable amount of increased fibrosis.

General Principles of Treatment and Prognosis:

ET runs a benign course and may not require any therapy. Treatment is given only if the platelet count is higher than one million. Complications of ET are the occurrence of acquired von Willebrand’s disease and bleeding but the incidence of thrombosis is not higher than matched controls.

Primary Myelofibrosis:

Definition and Pathophysiology:

Primary myelofibrosis (PMF), also called chronic idiopathic myelofibrosis or agnogenic (of unknown origin) myeloid metaplasia, and myelosclerosis, is a clonal disorder characterised by the proliferation of neoplastic stem cells at multiple sites outside the bone marrow (i.e. extramedullary haematopoiesis), especially in the liver and spleen, without an underlying etiology.

• Secondary myelofibrosis, on the other hand, develops in association with certain well-defined marrow disorders, or it is the result of the toxic action of chemical agents or irradiation.
• The exact aetiology is not known. Several chromosomal abnormalities have been reported but without a specific cytogenetic abnormality. PMF evolves from an early pre-fibrotic stage to an overt fibrotic stage.
• Fibrosis in the bone marrow is due to overproduction of transforming growth factor-β, osteosclerosis of the bone is related to osteonectin and marrow angiogenesis is due to increased production of vascular endothelial growth factor (VEGF).

Clinical Features:

The disease begins in the late middle of life and is gradual in onset. Both sexes are affected equally.

The symptomatology includes the following:

1. Anaemia with constitutional symptoms such as fatigue, weakness and anorexia.
2. Massive splenomegaly producing abdominal discomfort, pain and dyspnoea.
3. Hepatomegaly is present in half of the cases.
4. Petechial and other bleeding problems are found in about 20% of cases.
5. Less common findings are lymphadenopathy, jaundice, ascites, bone pain and hyperuricaemia.

Laboratory Findings:

1. Mild anaemia is usual except in cases where features of polycythaemia vera are coexistent.
2. Leucocytosis at the time of presentation but later there may be leucopenia.
3. Thrombocytosis initially but advanced cases show thrombocytopenia.
4. The peripheral blood smear shows bizarre red cell shapes, teardrop poikilocytes, basophilic stippling, nucleated red cells, immature leucocytes (i.e. leucoerythroblastic reaction), basophilia and giant platelet forms.
5. Bone marrow aspiration is generally unsuccessful and yields a ‘dry tap’.
6. Trephine biopsy:
   1. The prefibrotic stage shows the proliferation of megakaryocytes and atypia without reticulin fibrosis (grade 1), and myeloid hypercellularity but decreased erythropoiesis.
      
   2. The fibrotic stage shows the proliferation of megakaryocytes and atypia with reticulin fibrosis
      (grade 2-3)
7. Extramedullary haematopoiesis can be documented by liver biopsy or splenic aspiration.

General Principles of Treatment and Prognosis:

PMF does not require any specific therapy. Anaemia and ineffective erythropoiesis cannot be readily treated and do not respond to erythropoietin, androgens. Splenectomy may be necessary in some cases. In general, PMF has poorer outcomes compared with PV and ET.

Myeloproliferative neoplasms:

• These are a group of closely related clonal neoplastic disorders having a common origin from stem cells.
• CML is identified by the identification of clones of cells having reciprocal t(9;22) forming fusion gene complex BCR-ABL or Philadelphia chromosome. Clinically, CML cases have anaemia, splenomegaly and bleeding tendencies. It has a chronic phase and a more aggressive blastic phase.
• Polycythaemia vera is a clonal disorder characterised by increased production of all myeloid elements resulting in pinocytosis (i.e. increased red cells, granulocytes, platelets) in the absence of any recognisable cause.
• Essential thrombocythaemia is uncommon and has an overproduction of platelets.
• Primary myelofibrosis is characterised by bone marrow finding of proliferated and atypical megakaryocytes with variable fibrosis and the presence of extramedullary haematopoiesis

Acute Myeloid Leukaemia:

Definition And Pathophysiology:

Acute myeloid leukaemia (AML) is a heterogeneous disease characterised by infiltration of malignant myeloid cells into the blood, bone marrow and other tissues. AML is mainly a disease of adults (median age 50 years), while children and older individuals may also develop it sometimes.

• AML develops due to the inhibition of the maturation of myeloid stem cells due to mutations.
• These mutations may be induced by several etiologic factors—heredity, radiation, chemical carcinogens (tobacco smoking, rubber, plastic, paint, insecticides etc) and long-term use of anticancer drugs but viruses do not appear to have a role in the aetiology of AML.
• The defect induced by mutations causes the accumulation of precursor myeloid cells of the stage at which the myeloid maturation and differentiation are blocked.
• As seen from the revised WHO classification (2016), many more entities to the list of AML due to the availability of newer molecular biomarkers and gene expressions and these refinements in diagnosis have prognostic and therapeutic value.

Acute Myeloid Leukaemia Classification:

Currently, two main classification schemes for AML are followed:

Fab Classification: According to the revised FAB classification system, leukaemia is acute if the bone marrow consists of more than 30% blasts. Based on morphology and cytochemistry, the revised FAB classification divides AML into 8 subtypes (M0 to M7).

Who Classification:

The WHO classification for acute leukaemias differs from the revised FAB classification in the following 2 ways:

• Firstly, it places limited reliance on blast morphology and cytochemistry for making the diagnosis of a subtype of AML but instead takes into consideration clinical, cytogenetic and molecular abnormalities in different types. These features can be studied by multiparametric flow cytometry, gene expression analysis and next-generation sequencing (NGS).
• Secondly, the WHO classification for AML has revised and lowered the cutoff percentage of marrow blasts to 20% from 30% in the revised FAB classification for making the diagnosis of AML. The latest WHO classification of AML is given in Table.

Both FAB as well as WHO classifications for AML are followed in different settings depending upon the laboratory facilities available in various centres. Moreover, most of the current clinical and laboratory data in the literature are based on FAB groupings.

Hence, detailed morphologic and cytochemical features of various AML groups must be understood well.

Acute Myeloid Leukaemia Clinical Features:

AML and ALL share many clinical features and the two are difficult to distinguish on clinical features alone.

• In approximately 25% of patients with AML, a preleukaemic syndrome with anaemia and other cytopenias may be present for a few months to years before the development of overt leukaemia.
• Clinical manifestations of AML are divided into 2 groups those due to bone marrow failure, and those due to organ infiltration.

1. Due to bone marrow failure:

These are as under:

1. Anaemia produces pallor, lethargy, and dyspnoea.
2. Bleeding manifestations due to thrombocytopenia cause spontaneous bruises, petechiae, bleeding from gums and other bleeding tendencies.
3. Infections are quite common and include those of mouth, throat, skin, respiratory, perianal and other sites.
4. Fever is generally attributed to infections in acute leukaemia but sometimes no obvious source of infection can be found and may occur in the absence of infection.

2. Due To Organ Infiltration: The clinical manifestations of AML are more often due to the replacement of the marrow and other tissues by leukaemic cells.

These features are as under:

1. Pain and tenderness of bones (for example., sternal tenderness) are due to bone infarcts or subperiosteal infiltrates by leukaemic cells.
2. Lymphadenopathy and enlargement of the tonsils may occur.
3. Splenomegaly of moderate grade may occur. Splenic infarction, subcapsular haemorrhages, and rarely, splenic rupture may occur.
4. Hepatomegaly is frequently present due to leukaemic infiltration but the infiltrates usually do not interfere with the function of the liver.
5. Leukaemic infiltration of the kidney may be present and ordinarily does not interfere with its function unless secondary complications such as haemorrhage or blockage of ureter supervene.
6. Gum hypertrophy due to leukaemic infiltration of the gingivae is a frequent finding in myelomonocytic (M4) and monocytic (M5) leukaemias.
7. Chloroma or granulocytic sarcoma is a localised tumour-forming mass occurring in the skin or orbit due to local infiltration of the tissues by leukaemic cells. The tumour is greenish in appearance due to the presence of myeloperoxidase.
8. Meningeal involvement manifested by raised intracranial pressure, headache, nausea and vomiting, blurring of vision and diplopia are seen more frequently in ALL during haematologic remission. Sudden death from massive intracranial haemorrhage as a result of leucostasis may occur.
9. Other organ infiltrations include testicular swelling and mediastinal compression.

Laboratory Findings:

The diagnosis of AML is made by a combination of routine blood pictures and bone marrow examination, coupled with cytochemical stains and other special laboratory investigations.

Blood Picture:

Findings of routine haematologic investigations are as under:

1. Anaemia: Anaemia is almost always present in AML. It is generally severe, progressive and normochromic. Moderate reticulocytosis up to 5% and a few nucleated red cells may be present.

2. Thrombocytopenia: The platelet count is usually moderately to severely reduced (below 50,000/µl) but occasionally it may be normal.

• Bleeding tendencies in AML are usually correlated with the level of thrombocytopenia but most serious spontaneous haemorrhagic episodes develop in patients with fewer than 20,000/µl platelets.
• Acute promyelocytic leukaemia (M3) may be associated with a serious coagulation abnormality, disseminated intravascular coagulation (DIC).

3. White blood cells: The total WBC count ranges from subnormal to markedly elevated values. In 25% of patients, the total WBC count at presentation is reduced to 1,000-4,000/µl.

• More often, however, there is a progressive rise in white cell count which may exceed 100,000/µl in more advanced diseases.
• The majority of leucocytes in the peripheral blood blasts and there is often neutropenia due to marrow infiltration by leukaemic cells. The basic morphologic features of myeloblasts and lymphoblasts are summed up in Table.
• Typical characteristics of different forms of AML (M0 to M7) are given in Table. In some instances, the identification of blast cells is greatly aided by the company they keep i.e. by more mature and easily identifiable leucocytes in the company of blastic cells of myeloid series.
• Some ‘smear cells’ in the peripheral blood representing degenerated leucocytes may be seen.

2. Bone Marrow Examination:

An examination of bone marrow aspirate or trephine reveals the following features:

1. Cellularity: Typically, the marrow is hypercellular but sometimes a ‘blood tap’ or ‘dry tap’ occurs. A dry tap in AML may be due to pancytopenia, but sometimes even when the marrow is so packed with leukaemic cells they cannot be aspirated because the cells are adhesive and enmeshed in reticulin fibres. In such cases, trephine biopsy is indicated.

2. Leukaemic cells: The bone marrow is generally tightly packed with leukaemic blast cells. The diagnosis of the type of leukaemic cells, according to FAB classification, is generally possible with routine.

• Romanowsky stains but cytochemical stains may be employed as an adjunct to Romanowsky staining for determining the type of leukaemia.
• The essential criteria for diagnosis of AML, as per FAB classification, was the presence of at least 30% blasts in the bone marrow.
• However, as per WHO classification, these criteria have been revised and lowered to 20% blasts in the marrow for labelling and treating a case as AML.

3. Erythropoiesis: Erythropoietic cells are reduced. Dyserythropoiesis, megaloblastic features and ring sideroblasts are commonly present.

4. Megakaryocytes: They are usually reduced or absent.

5. Cytogenetics: Chromosomal analysis of dividing leukaemic cells in the marrow shows karyotypic abnormalities in 75% of cases which may have a relationship to prognosis.

• The WHO classification emphasises the categorisation of AML based on cytogenetic abnormalities. Some common mutations related to the prognosis of AML are listed in Table.

6. Immunophenotyping:

AML cells express the following biomarkers:

• Primary panel (to distinguish AML from ALL): CD13, CD33 and CD117 markers; M7 shows CD41 and CD42 positivity.
• Secondary panel (to diagnose the specific subtype of AML): CD14, CD41, CD61, CD64, lysozyme and glycophorin A.

3. Cytochemistry:

Some of the commonly employed cytochemical stains, as an aid to classify the type of AML, are as under:

1. Myeloperoxidase: Positive in immature myeloid cells containing granules and Auer rods but negative in M0 myeloblasts.
2. Sudan Black: Positive in immature cells in AML.
3. Periodic acid-Schiff (PAS): Positive in immature lymphoid cells and in erythroleukaemia (M6).
4. Non-specific esterase (NSE): Positive in monocytic series (M4 and M5).
5. Acid phosphatase: Focal positivity in leukaemic blasts in ALL and diffuse reaction in monocytic cells (M4 and M5).

4. Biochemical Investigations: These may be of some help:

1. Serum muramidase Serum levels of lysozyme (i.e. muramidase) are elevated in myelomonocytic (M4) and monocytic (M5) leukaemias.
2. Serum uric acid Because of the rapidly growing number of leukaemic cells, serum uric acid level is frequently increased. The levels are further raised after treatment with cytotoxic drugs because of increased cell breakdown.

General Principles Of Treatment And Prognosis:

The management of acute leukaemia involves the following aspects:

1. Treatment Of Anaemia And Haemorrhage: Anaemia and haemorrhage are managed by fresh blood transfusions and platelet concentrates. Patients with severe thrombocytopenia (platelet count below 20,000/µl) require regular platelet transfusions since haemorrhage is an important cause of death in these cases.

2. Treatment And Prophylaxis Of Infection: Neutropenia due to bone marrow replacement by leukaemic blasts and as a result of intensive cytotoxic therapy renders these patients highly susceptible to infection.

• The infections are predominantly bacterial but viral, fungal, and protozoal infections also occur. For prophylaxis against infection in such cases, the patient should be isolated and preferably placed in laminar airflow rooms.
• Efforts are made to reduce the gut and other commensal flora which are the usual source of infection. This is achieved by bowel sterilisation and by topical antiseptics. If these fail to achieve the desired results, systemic antibiotics and leucocyte concentrates are considered for therapy.

3. Cytotoxic Drug Therapy: The aims of cytotoxic therapy are firstly to induce remission, and secondly to continue therapy to reduce the hidden leukaemic cell population by repeated courses of therapy. Most commonly, cyclic combinations of 2, 3 and 4 drugs are given at treatment-free intervals to allow the bone marrow to recover.

The most effective treatment of AML is a combination of 3 drugs:

• Cytosine arabinoside, anthracyclines (daunorubicin, adriamycin) and 6-thioguanine. Another addition is amsacrine (mAMSA) administered with cytosine arabinoside, with or without 6-thioguanine.
• Following remission-induction therapy, various drug combinations are given intermittently for maintenance. However, promyelocytic leukaemia (M3) is treated with tretinoin orally which reduces the leukaemic cells bearing t(15;17)(q22;q21) but the development of DIC due to liberation of granules of dying cells is a problem.

4. Bone Marrow Transplantation: Bone marrow (or stem cell) transplantation from suitable allogenic or autologous donor (HLA and mixed lymphocytes culture-matched) is increasingly being used for treating young adults with AML in first remission.

• The basic principle of marrow transplantation is to reconstitute the patient’s haematopoietic system after total body irradiation and intensive chemotherapy have been given so as to kill the remaining leukaemic cells. Bone marrow transplantation has resulted in a cure in about half the cases.
• Prognosis varies with specific mutations as per the WHO classification; some common mutations in WHO type and corresponding FAB type are listed in Table. In general, the remission rate with AML is lower (50-70%) than in ALL, often takes longer to achieve remission, and disease-free intervals are shorter.

AML is the most malignant of all leukaemias; median survival with treatment is 12-18 months.

Adapted from Dohner et al: European leukaemia network recommendations. Blood 2010; 115:453.

Acute Myeloid Leukaemia:

• AML is a disease of adults characterised by infiltration of malignant myeloid cells into the blood, bone marrow and other tissues.
• According to the revised FAB classification system, the cut-off for bone marrow blasts in AML is >30%. Based on morphology and cytochemistry, FAB classification divides AML into 8 subtypes (M0 to M7).
• Revised WHO classification takes into account genetic and molecular features and lowered marrow blast count to >20% for diagnosis of AML. The WHO classification has several newer entities having prognostic and therapeutic value.
• AML is characterised by anaemia, thrombocytopenia, and leucocytosis with blasts in the blood. Bone marrow shows myeloblasts over 20% and reduced megakaryocytes.

Myelodysplastic Syndromes:

Definition And Classification:

Myelodysplastic syndromes (MDS) are a group of haematopoietic clonal neoplasms of the bone marrow characterised by ineffective haematopoiesis of different marrow elements, manifested morphologically by dysplastic haematopoietic cells and often peripheral cytopenia/s. In older literature, these conditions were termed preleukaemic syndromes.

The following three classification schemes have been described for MDS:

Conventional Classification:

Based on underlying etiologic factors, MDS is divided into primary and secondary:

1. Primary MDS is idiopathic but factors implicated in etiology are radiation exposure and benzene carcinogen.
2. Secondary (therapy-related) MDS may occur following earlier anti-cancer treatment, aplastic anaemia treated with immunosuppressive therapy, and in Fanconi’s anaemia.

Fab Classification: According to FAB (French-American-British) Cooperative Group, the marrow may contain <30% myeloblasts in MDS and this was considered as the dividing line for distinguishing cases of AML (blasts >30%) from MDS.

FAB classified MDS into the following 5 groups:

1. Refractory anaemia (RA) Blood blasts <1%, marrow blasts <5%.
2. Refractory anaemia with ringed sideroblasts (RARS) (primary acquired sideroblastic anaemia) Blood blast <1%, marrow blasts <5%; ring sideroblasts≥15%.
3. Refractory anaemia with excess blasts (RAEB) Blood blasts 5%, marrow blasts 5-20%.
4. Refractory anaemia with excess of blasts in transformation (RAEB-t) Blood blasts 5%, marrow blasts 21-30%.
5. Chronic myelomonocytic leukaemia (CMML) Blood blasts, 5%, monocytosis.

Revised Who Classification Of Mds (2016):

The WHO classification differs from FAB classification in following ways:

1. Marrow blast count for making the diagnosis of AML is taken as >20%; applying same criteria to MDS, marrow blasts should be <20% in MDS.
2. FAB category of RAEB-t (group 4 above) cases have prognosis similar to patients of AML and thus shifted from MDS to AML.
3. CMML category of FAB has been excluded from the WHO classification of MDS due to biologic behaviour of these cases and has been, instead, placed in the hybrid category of myelodysplastic/myeloproliferative neoplasm (MDS/MPN).
4. Terms ‘refractory anaemia’ and ‘refractory cytopenia’ have been removed from adult MDS. Instead, terms like single and multilineage have been used for type of MDS. However, childhood MDS cases have refractory cytopenia.
5. Morphologic criteria for diagnosis have been refined.
6. It relies on 3 parameters:
7. Degree of dysplasia (1,2 or 3 lineages),
8. Blasts percentage, and
9. Specific type/s and extent of cytopenia/s (1,2 or 3 lineages).
10. It also includes cytogenetic criteria and mutations in diagnosis of MDS.

As shown in Table 13.4, the revised WHO classification of MDS (2016) consists of following 8 categories:

1. MDS with single lineage dysplasia (MDS-SLD)
2. MDS with multilineage dysplasia (MDS-MLD)
3. MDS with ring sideroblasts (MDS-RS)
4. With single lineage dysplasia (MDS-RS-SLD)
5. With multilineage dysplasia (MDS-RS-MLD)
6. MDS with excess blasts (MDS-EB)
7. MDS with isolated del (5q)
8. MDS-unclassifiable (MDS-U)
9. Refractory cytopenia of childhood
10. Myeloid neoplasms with germ line predisposition

In addition, following comments are relevant to diagnostic criteria of MDS:

1. Threshold to define MDS is 10% dysplastic cells pertaining to any of haematopoietic cell lineage.
2. Cytopenia may pertain to 1, 2 or 3 lineages and the cut-off values are haemoglobin <10g/dl, platelet count <100,000/µl, absolute neutrophil count <1800/µl.
3. MDS-defining cytogenetic abnormality by conventional karyotyping in a cytopenic patient is isolated del (5q). Cases evolving into leukaemia more often have aneuploidy.
4. As in AML, recurrent mutations are seen in MDS; most commonly mutated genes are SF3B1 and TP53. These mutations have prognostic and therapeutic significance.
5. Although most cases of MDS (and AML) are sporadic diseases, a category of MDS in the WHO classification have predisposing syndrome or specific underlying genetic defect with germ line mutations and these cases have familial predisposition.

Clinical Features:

In general, MDS is found more frequently in older people past 6th decade of life, with slight male preponderance. Therapy-related MDS is generally not age-related and may occur about a decade after anti-cancer therapy. Clinical features are quite non-specific and MDS may be discovered during routine CBC examination done for some other cause.

At presentation, the patient may have following features:

1. Anaemia appreciated by pallor, fatigue and weakness
2. Fever
3. Weight loss
4. Sweet syndrome having neutrophilic dermatosis seen in some cases
5. Splenomegaly seen in 20% cases of MDS.

(BM, bone marrow; PB, peripheral blood). Adapted from Arber et al: The 2016 revision to the WHO classification of myeloid neoplasms and acute leukaemia. Blood 2016;127:2391-2405.

Laboratory Findings:

Various combinations of features are seen in different types of MDS. Sums up salient laboratory findings on peripheral blood, bone marrow and cytopenias in major categories of MDS.

In general, laboratory findings are as under:

Blood Findings:

There is cytopenia affecting one, two (bi-) or all the three blood cell lines (pancytopenia):

1. Anaemia Generally macrocytic or dimorphic.
2. TLC Usually normal; cases of CMML may have high TLC but these cases in WHO classification of myeloid neoplasms have been put in a separate group of myelodysplastic/myeloproliferative neoplasms and not in MDS.
3. DLC Neutrophils are hyposegmented and hypogranulated. Myeloblasts may be seen in PBF and their number correlates with marrow blasts count.
4. Platelets Thrombocytopenia with large agranular platelets.

Bone Marrow Findings:

There is constellation of findings in the marrow as under:

1. Cellularity Normal to hypercellular to hypocellular.
2. Erythroid series Dyserythropoiesis as seen by abnormally appearing nuclei and ring sideroblasts. Megaloblasts may be seen.
3. Myeloid series Hypogranular and hyposegmented myeloid precursor cells. Myeloblasts increased depending upon the type of MDS.
4. Megakaryocyte series Reduced in number and having abnormal nuclei.

General Principles Of Treatment And Prognosis:

MDS is difficult to treat and may not respond to cytotoxic chemotherapy. Broad principles of supportive care as for aplastic anaemia apply to MDS. Stem cell transplantation offers cure and longer survival.

Survival rates vary depending upon the type of MDS:

Cases of MDS-RS and 5q syndrome survive for years, while cases of MDS with excess blasts (MDS-EB-1 and MDS-EB2) and severe cytopenia with monosomy-7 have poor survival for a few months only. Even low-risk MDS develop complications more due to cytopenia than due to leukaemic transformation.

Myelodysplastic Syndromes:

• MDS are clonal neoplasms of the bone marrow characterised by ineffective haematopoiesis of different marrow elements, manifested morphologically by dysplastic haematopoietic cells and often peripheral cytopenia/s.
• As per revised WHO classification, since marrow blast count for diagnosis of AML is >20%e, cases of MDS have blast count below 20%.
• FAB category of RAEB-t cases have been included in AML in WHO classification of MDS.
• FAB category of CMML has been included in myeloproliferative disorder in WHO scheme of MDS.
• Revised WHO classification has 8 categories of MDS: 1) single lineage dysplasia (MDS SLD), 2) multilineage dysplasia (MDS-MLD), 3) with ring sideroblasts (MDS-RS), 4) with excess blasts (MDS-EB), 5) with isolated del (5q), 6) unclassifiable (MDS-U), 7) refractory cytopenia of childhood, and 8) myeloid neoplasms with germ line predisposition