Eosinophilia is typically associated with allergic disorders including drug sensitivity, skin diseases and parasitic infections. In most cases, the cause is indicated by the clinical history, which should include details of all medications and foreign travel, and by examination of the stool and urine for parasites, cysts and ova. A diagnosis of chronic eosinophilic leukaemia is made if there is dominant eosinophilia with an increase in blast cells in the blood or marrow, or cytogenetic or molecular evidence of an abnormal myeloid clone.⁵ If no other cause for eosinophilia is found it is important, because of the therapeutic implications (i.e. responsiveness to imatinib), to confirm or exclude a diagnosis of eosinophilic leukaemia related to rearrangement of PDGFRA or PDGFRB (see p. 559).⁶ The idiopathic hypereosinophilic syndrome is an unusual cause of eosinophilia in which release of the contents of eosinophil granules results in damage to the heart, lungs and other tissues. It is defined by the presence of a peripheral blood eosinophil count of 1.5 × 10⁹/l or greater for at least 6 months with resultant tissue damage. This is a diagnosis of exclusion, made only when detailed investigations exclude other possible causes of eosinophilia including systemic mastocytosis, eosinophilic leukaemia and eosinophilia associated with a phenotypically aberrant T-cell population or a neoplastic clone of T cells.

Basophilia

Basophilia as an isolated finding is unusual. However, it is a common feature of myeloproliferative neoplasms and basophils may be particularly prominent in chronic myelogenous leukaemia. In this condition, an increasing basophil count may be the first indication of accelerated phase disease.

Thrombocytosis

Thrombocytosis is often associated with infectious and inflammatory conditions such as osteomyelitis and rheumatoid arthritis. Haematological causes of thrombocytosis include chronic blood loss, red cell destruction, splenectomy and rebound following recovery from marrow suppression. Under these circumstances, a moderately increased platelet count (e.g. 400–800 × 10⁹/l) does not usually have any pathological implications. Primary thrombocythaemia is usually the result of a myeloproliferative neoplasm; rarely, it is an inherited condition. When there is isolated persistent thrombocytosis in a myeloproliferative neoplasm the diagnosis is essential thrombocythaemia (as long as a BCR–ABL1 fusion gene has been excluded). Thrombotic or haemorrhagic complications can occur but often the diagnosis is an incidental one.⁷ A significant proportion of individuals with essential thrombocythaemia have the JAK2 V617F mutation, which is associated with an increased risk of thrombosis.⁸ The criteria for this diagnosis are discussed on p. 559.

Reduced Numbers of Cells

Reductions in more than one cell line

A reduction in cell numbers occurs because of increased destruction, reduced production or increased pooling in the spleen or other organs. Reduced production of cells may be the result of aplastic anaemia, a lack of haematinics such as folate or vitamin B₁₂ or interference with normal haemopoiesis by infiltration (e.g. leukaemia, lymphoma, multiple myeloma, metastatic carcinoma – often with secondary myelofibrosis), infection (e.g. HIV infection, tuberculosis, leishmaniasis) or exposure to toxins (e.g. alcohol) or myelosuppressive drugs (e.g. hydroxycarbamide or methotrexate). Certain myeloid neoplasms, e.g. primary myelofibrosis and MDS, are characterized by cytopenias, which are at least in part the result of ineffective haemopoiesis. Cytopenia is also sometimes a feature of acute myeloid leukaemia (AML), when it is due both to ineffective haemopoiesis and to replacement of normal haemopoietic stem cells by leukaemic cells. A relatively common cause of a global reduction in circulating cells is pooling of the cells in a markedly enlarged spleen (hypersplenism), which may be secondary to conditions such as primary myelofibrosis and portal hypertension. Examination of a bone marrow aspirate and trephine biopsy specimen is often helpful in determining the cause of cytopenias for which no obvious cause is apparent

The mechanisms which result in anaemia are decreased production, reduced red cell lifespan, blood loss and splenic pooling. Anaemia is broadly divided into three types: microcytic (low MCV), macrocytic (high MCV) and normocytic (normal MCV). The choice of investigations is guided by the MCV and red cell morphology in addition to clinical features. Figures 23.1–23.3 are flow charts that provide an orderly sequence of investigations for the different types of anaemia on the basis of these indices. Examination of a blood film will usually suggest the quickest route to the diagnosis; confirmation may require the more specific tests, which are given in the text. The presence of basophilic stippling in a patient with microcytic red cells suggests thalassaemia trait or, much less often, lead poisoning. A dimorphic blood film is typical of congenital sideroblastic anaemia but is more often the result of iron deficiency responding to treatment. Pappenheimer bodies suggest that a microcytic anaemia is the result of sideroblastic erythropoiesis.

Figure 23.1 Investigation of a microcytic hypochromic anaemia. HbEP, Hb electrophoresis; HPLC, high-performance liquid chromatography.

Figure 23.2 Investigation of a macrocytic anaemia. MDS, myelodysplastic syndrome.

Figure 23.3 Investigation of a normocytic, normochromic anaemia.

Microcytic Anaemia

The most common cause of anaemia worldwide is iron deficiency, which can be suspected from a low MCV (Fig. 23.1) and the presence of hypochromic, microcytic red cells. Laboratory confirmation of iron deficiency may be based on measurements of serum ferritin, serum iron plus either total iron-binding capacity or transferrin assay, red cell protoporphyrin and staining of bone marrow aspirates for iron (see Chapter 4). Assay of soluble transferrin receptors has good sensitivity and specificity for iron deficiency and may be useful in the presence of inflammation when interpretation of ferritin levels is difficult.⁹ A diagnosis of iron deficiency must be followed by a search for the cause. This should include specific questions relating to blood loss and dietary insufficiency and may require stool examination for parasites and occult blood, endoscopic examination of the gastrointestinal tract to exclude occult malignancy and tests for coeliac disease. The differential diagnosis of iron deficiency anaemia includes anaemia of chronic disease. Clinical and laboratory features of inflammation or chronic infection may suggest this diagnosis, which is confirmed by demonstration of normal or high serum ferritin and reduced serum iron, transferrin and iron-binding capacity.

The thalassaemias also cause microcytosis, but both α and β thalassaemia trait are usually associated with an increased red blood cell count (RBC) and a normal or near-normal Hb despite a considerable reduction of the MCV and MCH. In contrast, in iron deficiency the MCV and MCH do not fall until the Hb is significantly reduced. Further investigations, such as high-performance liquid chromatography (HPLC) or haemoglobin electrophoresis supplemented by measurement of Hb A₂ and Hb F usually confirm the diagnosis of β thalassaemia trait. The diagnosis of α thalassaemia trait is more difficult; detection of infrequent Hb H inclusions is usually possible in α⁰ thalassaemia trait, but definitive diagnosis requires DNA analysis.¹⁰ A diagnosis of α⁰ thalassaemia heterozygosity can be clinically important for prediction of haemoglobin Bart’s hydrops fetalis since, if both parents have α⁰ thalassaemia, this very serious condition can occur in a fetus. DNA analysis is therefore indicated when a pregnant woman of appropriate ethnic origin has an MCH of <25 pg. Hb H inclusions may not be detected in α⁺ thalassaemia trait but this diagnosis is of less clinical importance and confirmation is thus not usually required.

Macrocytic Anaemia

A high MCV (Fig. 23.2) with oval macrocytes and hypersegmented neutrophils suggests folate or vitamin B₁₂ deficiency and is an indication for assays of these vitamins (see Chapter 10); subsequent investigations could include malabsorption studies, tests for coeliac disease and tests for intrinsic factor and gastric parietal cell antibodies. In patients with these blood film findings and normal vitamin assays, haematinic deficiency is not completely excluded and further investigation is indicated. A Schilling test permits a definitive diagnosis of pernicious anaemia but currently this test is not available due to a lack of reagents. In the absence of intrinsic factor antibodies, the diagnosis of pernicious anaemia may be presumptive. Pernicious anaemia is commonly associated with autoimmune thyroid disease and other autoimmune disorders, such as diabetes mellitus. A high MCV may also be associated with alcohol excess and liver disease or use of drugs such as hydroxycarbamide or zidovudine. Macrocytosis resulting from chronic haemolysis is associated with increased numbers of immature red cells, which appear slightly larger and bluer than normal red cells (polychromatic macrocytes) on a Romanowsky-stained peripheral blood film. Supravital staining of blood films (see p. 33) or an automated reticulocyte count can be used to confirm reticulocytosis. Untreated anaemia associated with polychromasia is likely to indicate blood loss or haemolysis. The combination of red cell fragments, thrombocytopenia and polychromasia indicates a microangiopathic haemolytic anaemia and should trigger further tests such as a platelet count, coagulation studies, assessment of renal function and a search for infection or neoplastic disease. This further assessment is urgent because these may be features of thrombotic thrombocytopenic purpura, which requires immediate treatment by plasma exchange.

Normocytic Anaemia

Normochromic, normocytic anaemia (Fig. 23.3) is frequently the result of an underlying chronic, non-haematological disease. Investigations should include screening for renal insufficiency, subclinical infections, autoimmune diseases and neoplasia. In the presence of anaemia, a lack of polychromasia, confirmed by reticulocytopenia, points toward a primary failure of erythropoiesis or lack of compensatory increased red cell production in blood loss or haemolysis. Examination of the bone marrow may be helpful in demonstrating haematological causes for a normochromic, normocytic anaemia such as aplastic anaemia or MDS.¹¹ Staining for iron may also show that there is a block in iron metabolism suggestive of anaemia associated with chronic inflammatory disease.

Leucopenia

Neutropenia

Once physiological variation, ethnicity and familial or cyclic neutropenia have been excluded (see p. 19), the non-haematological causes of isolated neutropenia to be considered include overwhelming infection, autoimmune disorders such as systemic lupus erythematosus, irradiation, drugs (particularly anticancer agents) and large granular lymphocyte leukaemia. Bone marrow examination may assist in determining whether the problem is the result of peripheral destruction (increased marrow myeloid precursors) or stem cell failure (lack of marrow myeloid precursors). Typical marrow appearances occur in drug-induced neutropenia, in which there is a relative paucity of mature neutrophils and in Kostmann’s syndrome (infant genetic agranulocytosis) where there is maturation arrest at the promyelocytic stage.

Reduced numbers of lymphocytes, monocytes, eosinophils and basophils

Lymphocytes, eosinophils and basophils may all be reduced by physical stress such as surgery, trauma and infection. Lymphopenia with neutrophilia is a common combination of haematological abnormalities in severe acute respiratory syndrome and in many other patients with acute illness or trauma. Lymphopenia, especially affecting the CD4 cells, may occur in HIV infection and renal failure. Monocytopenia (<0.2 × 10⁹/1) is typically found in hairy cell leukaemia, which is also associated with pancytopenia, typical bone marrow histology and lymphocytes with a characteristic cytology and immunophenotype.

Thrombocytopenia

Thrombocytopenia is a common isolated finding and it is important to ensure that the laboratory result reflects a true reduction in platelet count before embarking on further diagnostic tests. Frequent causes of spurious thrombocytopenia include blood clots in the sample, platelet aggregation and platelet satellitism. Platelet aggregation, which is seen on the blood film, may occur in vitro as the result of a temperature-dependent or anticoagulant-dependent autoantibody or on slides that have been made directly from a finger prick sample. True thrombocytopenia is most frequently the result of autoantibodies (autoimmune ‘idiopathic’ thrombocytopenic purpura), HIV infection, anticancer chemotherapy, other drugs (such as thiazide diuretics), alcohol excess, hypersplenism and MDS (in the elderly). The blood film should be examined for giant or hypogranular platelets, red cell fragments, May–Hegglin inclusions in leucocytes and abnormal cells that might indicate leukaemia or lymphoma. The clinical circumstances, together with blood film and bone marrow examination, usually enable the various causes of thrombocytopenia to be differentiated. An association with thrombosis, disturbed renal or hepatic function and haemolytic anaemia, should prompt investigations for other diseases such as thrombotic thrombocytopenic purpura and, in a pregnant woman, the HELLP (haemolysis + elevated liver enzymes + low platelet count) syndrome. A bone marrow examination is often carried out early in the investigation of thrombocytopenia because it is helpful in excluding conditions such as acute leukaemia, which occasionally present with isolated thrombocytopenia.

Pancytopenia

Pancytopenia (reduction in the WBC, Hb and platelet count) is most often the result of anticancer chemotherapy, HIV infection, hypersplenism and bone marrow infiltration or failure. Careful examination of a blood film is important if the reason for the pancytopenia is not apparent from the clinical history. If this does not reveal the cause, bone marrow aspiration and trephine biopsy may be needed.

Qualitative Abnormalities of Blood Cells

In health, only the most mature forms of cells appear in the peripheral blood. Cells at various stages of immaturity, such as nucleated red blood cells, polychromatic red cells, myelocytes and metamyelocytes, may be released from the bone marrow in conditions where the bone marrow is overactive (e.g. acute haemolytic states or recovery from suppression) or functionally abnormal. Their presence in the peripheral blood indicates that active haemopoiesis is taking place.

Abnormalities of All Cell Lines

The combination of anisopoikilocytosis, mild macrocytosis, hypogranular neutrophils with abnormal nuclear morphology and platelet anisocytosis, often with quantitative abnormalities, is virtually pathognomonic of MDS. These features are reflected in the bone marrow with disturbance of the normal developmental pathway and sometimes nuclear:cytoplasmic asynchrony. Cytogenetic studies are helpful for confirming the diagnosis, especially when cytological abnormalities are minor, and can also assist in determining the prognosis.

Abnormalities of Individual Cell Lines

Red cells

Congenital abnormalities of the red cell affecting the structure (e.g. spherocytosis, elliptocytosis) and content (e.g. haemoglobinopathies, enzymopathies) often produce typical morphological changes (see Chapter 5). The type of changes will guide further investigations toward analysis of structural proteins, haemoglobin electrophoresis or HPLC, or enzyme assays. Acquired red cell abnormalities may also help to indicate underlying pathology. For example, target cells may prompt investigation of liver function, whereas increased rouleaux formation may indicate the need for investigations for multiple myeloma or inflammatory conditions.

White cells

Congenital abnormalities of neutrophils are unusual, but similar morphological abnormalities (e.g. Pelger–Huët cells) may be seen in acquired conditions such as MDS. Reactive changes in lymphocytes, including basophilic, faceted cytoplasm, are typically seen in infectious mononucleosis, which can be diagnosed using an appropriate serological screening test (see p. 105) or, if this is negative, by demonstration of immunoglobulin M (IgM) antibodies to the Epstein–Barr virus. These atypical lymphocytes can sometimes be difficult to differentiate from circulating lymphoma cells. Bone marrow histology, combined with immunophenotyping studies and determination of lymphocyte clonality by demonstration of light chain restriction or by gene rearrangement studies, may be needed to reach a firm conclusion.

Platelets

Platelets that function poorly may not necessarily appear morphologically abnormal, although sometimes they are hypogranular or larger than normal. A normal platelet count with an abnormal in vitro platelet function test is characteristic of a disorder of platelet function, but some patients with abnormal platelet function are also thrombocytopenic. Hereditary disorders of platelet function are uncommon and usually present as a bleeding diathesis. When a qualitative disorder of platelets is suspected, platelets should be examined to assess size and to detect the cytological features of the grey platelet syndrome. Qualitative disorders of platelets can broadly be divided into two categories: abnormalities of the platelet membrane (e.g. Bernard–Soulier syndrome, Glanzmann’s thrombasthenia) and abnormalities of platelet secretory function (e.g. storage pool diseases). Acquired disorders of platelet function are more common than inherited disorders. Haematological conditions associated with platelet dysfunction include myeloproliferative neoplasms, MDS and dysproteinaemias (in plasma cell neoplasms). Many widely prescribed drugs, including aspirin and non-steroidal anti-inflammatory agents, interfere with platelet function. Systemic conditions, particularly chronic renal failure and cardiopulmonary bypass, are also associated with a bleeding tendency as a result of qualitative platelet defects. Most of these acquired functional defects are not associated with any abnormality in platelet appearance but in MDS and, to a lesser extent, in the myeloproliferative neoplasms, there may be hypogranular and giant platelets.

Specific tests for common haematological disorders

Common haematological disorders are outlined in the following sections with suggestions for investigations that may be helpful in confirming the diagnosis. The lists are not intended to be exhaustive because the range of tests provided locally will depend on the availability of expertise and technology. The investigations discussed are those that are likely to be available within a general haematology department.

Red Cell Disorders

Microcytic Hypochromic Anaemias

For more information, see Chapters 9 and 14.

• Measurement of serum ferritin or iron plus total iron-binding capacity or transferrin assay, red cell protoporphyrin or soluble transferrin receptors

• Bone marrow aspirate with staining for iron

• Stool examination for occult blood

• Gastrointestinal imaging and endoscopy, with biopsies if appropriate; rarely, blood loss studies with ⁵¹Cr-labelled red cells

• Tests for malabsorption

• Serological tests for coeliac disease (e.g. endomysial antibodies, tissue transglutaminase antibodies)

• Serum lead (if lead poisoning is suspected)

If thalassaemia is suspected:

• Haemoglobin electrophoresis plus Hb A₂ and Hb F measurements or HPLC

• Haemoglobin H preparation

• Family studies

• DNA analysis (when the diagnosis is clinically important).

Macrocytic Anaemias

If macrocytic, megaloblastic erythroid maturation is demonstrated, further investigations should be undertaken as described in Chapter 10. If the blood film is typical of megaloblastic anaemia, relevant assays and further investigations can often indicate the diagnosis without the need for a bone marrow aspirate. Macrocytosis may also be secondary to conditions such as alcohol excess, liver disease, MDS, hydroxycarbamide administration and hypothyroidism. Reticulocytosis from any cause can also increase the MCV.

Haemolytic Anaemias

A haemolytic process may be suspected by the presence of a falling Hb, a reticulocytosis and jaundice with an increase in unconjugated bilirubin level (see Chapters 9, 10 and 11).

White Cell Disorders

The blood film is often of critical importance in the differential diagnosis of white cell disorders though it may sometimes be normal (e.g. in some patients with lymphoma or neutrophil functional defects). Changes in white cell numbers or morphology may occur rapidly in response to local or systemic disorders. In chronic leukaemias, bone marrow aspiration may add little to the diagnosis, but the pattern of infiltration of neoplastic cells seen on trephine biopsy can have diagnostic value or prognostic significance, e.g. in lymphoma and chronic lymphocytic leukaemia.

Acute Leukaemia

• Full blood count and peripheral blood film

• Bone marrow aspirate (trephine biopsy if bone marrow aspirate is inadequate)

• Blood or marrow immunophenotyping, unless obviously myeloid, or in all patients if it is to be used for monitoring minimal residual disease (cytochemical stains can be used if immunophenotyping is not readily available)

• Cytogenetic analysis

• Molecular studies (e.g. fluorescence in situ hybridization – FISH – analysis) for detection of all patients with acute lymphoblastic leukaemia (ALL) with hyperdiploidy or ETV6–RUNX1 fusion, detection of BCR–ABL1 fusion in adults with ALL and detection of other mutations of specific oncogenes, e.g. NPM1, CEBPA and possibly FLT3 in AML.

Neutropenia

• Vitamin B₁₂ and folate assays

• Autoantibody screen including rheumatoid factor and investigations for systemic lupus erythematosus

• Serial neutrophil counts for cyclical neutropenia

• Tests for anti-neutrophil antibodies

• Bone marrow aspirate and trephine biopsy

• Flow cytometry for PNH (see aplastic anaemia above)

• Consider the need for investigation for an abnormal T-cell population.

Chronic Myelogenous Leukaemia

• Full blood count and peripheral blood film

• Bone marrow aspirate

• Cytogenetic analysis

• Molecular studies (e.g. real-time quantitative reverse transcriptase or FISH) for BCR–ABL1 transcripts

• Neutrophil alkaline phosphatase score (only if cytogenetic and molecular genetic analysis are not available).

Chronic Lymphoproliferative Disorders/Lymphadenopathy

Diagnosis may be made from various specimens including lymph nodes, bone marrow aspirates, trephine biopsy cores and peripheral blood and other fluids such as cerebrospinal fluid, ascitic fluid and pleural aspirates.¹²

• Full blood count and peripheral blood film

• Serum protein electrophoresis and immunoglobulin concentrations

• Plasma uric acid, calcium and lactate dehydrogenase (LDH)

• Serological screening for infectious mononucleosis, cytomegalovirus infection, HIV infection and toxoplasmosis (if infectious cause suspected) and human T-cell leukaemia/lymphoma virus, when clinically relevant

• Bone marrow aspirate and trephine biopsy (to demonstrate the presence and distribution of abnormal lymphocytes) and/or lymph node or other tissue biopsy

• Flow cytometry immunophenotyping or immunohistochemistry of biopsy specimens

• Cytogenetic or molecular genetic analysis including investigation for immunoglobulin heavy chain or T-cell receptor gene rearrangement if the diagnosis of lymphoma is in doubt

• Imaging (plain radiographs, ultrasonography, computed tomography scan, magnetic resonance imaging).

Myelomatosis (Plasma Cell Myeloma)

• Full blood count and peripheral blood film ¹³

• Serum protein electrophoresis, immunofixation and quantification of immunoglobulins and any paraprotein

• Urine electrophoresis and immunofixation for Bence–Jones protein (early morning urine sample and, if positive, quantification on 24 h collection)

• Serum free light chain quantification and ratio

• Serum albumin, tests of renal function, plasma uric acid, calcium, phosphate and alkaline phosphatase measurements

• β₂ microglobulin quantification

• Plasma viscosity

• Bone marrow aspirate (with cytogenetic or FISH analysis if results will influence treatment decisions, and flow cytometry immunophenotyping or DNA analysis if these analyses are to be used for monitoring minimal residual disease)

• Trephine biopsy

• Radiological skeletal survey.

Other Disorders

Myelodysplastic syndromes

• Full blood count and blood film

• Bone marrow aspirate and trephine biopsy

• Cytogenetic analysis.

Classification of haematological neoplasms

Previous classifications for haematological neoplasms have now been largely supplanted by the World Health Organization’s Classification of Tumours of Haematopoietic and Lymphoid Tissues,³ which outlines the international standards for assessment and diagnosis of haematological neoplasms. Application of the WHO criteria requires the results of history and physical examination, morphology (cytology or histology), immunophenotyping, cytogenetic analysis and, in some circumstances, molecular genetic analysis. The French–American–British (FAB) group classifications (see previous editions) continue to have a place (a) when these techniques are not all available and (b) in making a provisional morphological diagnosis, e.g. in acute leukaemia, while awaiting the results of further tests. Whichever classification is used the criteria should be strictly observed so that there is consistency between different centres and countries. To avoid confusion, FAB terminology (e.g. M1, M2) should not be applied if the WHO classification is being used. Details of the FAB classifications can be found in specialized textbooks.¹⁴

The WHO classification of haematological neoplasms has several major categories (Table 23.1)

Table 23.1 WHO classification of myeloid neoplasms and acute leukaemias ¹⁵

Myeloproliferative neoplasms (MPN)

Myeloid and lymphoid neoplasms associated with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1

Myelodysplastic/myeloproliferative neoplasms (MDS/MPN)

Myelodysplastic syndrome (MDS)

Acute myeloid leukaemia and related neoplasms

Acute leukaemias of ambiguous lineage

B lymphoblastic leukaemia/lymphoma

T lymphoblastic leukaemia/lymphoma

Classification of Acute Myeloid Leukaemia

The WHO classification categorizes cases as AML (Table 23.2) if the following criteria are met:

1. There are at least 20% of blast cells of myeloid lineage in the blood or bone marrow or

2. If the erythroid cells are at least 50% of bone marrow cells, blast cells are at least 20% of non-erythroid cells or

3. Primitive erythroid cells constitute at least 80% of bone marrow cells or

4. There is a myeloid sarcoma (granulocytic sarcoma) or

5. One of a number of specified chromosomal rearrangements is present.

Table 23.2 WHO Classification of acute myeloid leukaemia and related neoplasms ¹⁵

Therapy-related myeloid neoplasms

Acute myeloid leukaemia with recurrent genetic abnormalities

AML with t(8;21)(q22;q22); RUNX1-RUNX1T1

AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11

APL with t(15;17)(q22;q12); PML-RARA

AML with t(9;11)(p22;q23); MLLT3-MLL

AML with t(6;9)(p23;q34); DEK-NUP214

AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1

AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1

Provisional entity: AML with mutated NPM1

Provisional entity: AML with mutated CEBPA

Acute myeloid leukaemia with myelodysplasia-related changes

Acute myeloid leukaemia, not otherwise specified

AML with minimal differentiation

AML without maturation

AML with maturation

Acute myelomonocytic leukaemia

Acute monoblastic/monocytic leukaemia

Acute erythroid leukaemia

Pure erythroid leukaemia

Erythroleukaemia, erythroid/myeloid

Acute megakaryoblastic leukaemia

Acute basophilic leukaemia

Acute panmyelosis with myelofibrosis

Myeloid sarcoma

Myeloid proliferations related to Down syndrome

Transient abnormal myelopoiesis

Myeloid leukaemia associated with Down syndrome

Blastic plasmacytoid dendritic cell neoplasm

It should be noted that the WHO classification is hierarchical. If appropriate, cases are first assigned to the category of therapy-related leukaemia. Next, cases are assigned, if appropriate, to the category of AML with recurrent genetic abnormalities. Cases continue to be assigned to successive categories in the order shown in Table 23.2, with remaining cases finally being categorized as ‘AML not otherwise specified.’ This final group is further subdivided into categories resembling those of the FAB classification (but defined in quite a different manner). Blastic plasmacytoid dendritic cell neoplasm and myeloid neoplasms associated with Down syndrome are recognized as specific entities.

The WHO classification of acute leukaemia ¹⁵ lists cytogenetic abnormalities that, in combination with ≥20 blasts, indicate a diagnosis of AML with myelodysplasia-related changes; assignment to this category can also be based on a previous history of MDS or on morphological evidence of dysplasia.

Classification of the Myelodysplastic Syndromes

The WHO classification of MDS (Table 23.3) requires evidence for a myeloid neoplasm with ineffective and, generally, dysplastic haemopoiesis; blasts must be <20% in both blood and bone marrow (Table 23.4). It will be noted that cytogenetic analysis is essential for the application of the WHO classification because cases of the 5q− syndrome cannot otherwise be recognized. Like the classification of AML, this is a hierarchical classification. Therapy-related MDS is categorized with therapy-related AML. Remaining cases are then assessed as to whether they meet the criteria for the 5q− syndrome. If they do not, they are assigned to the remaining categories, depending on the number of lineages showing dysplasia, the percentage of ring sideroblasts, the presence or absence of Auer rods and the percentage of blast cells in the blood and marrow. The WHO classification differs from the FAB classification in not regarding chronic myelomonocytic leukaemia as MDS; instead it assigns it to the MDS/MPN group.

Table 23.3 WHO Classification of the myelodysplastic syndromes (MDS) (2008)

Refractory cytopenia with unilineage dysplasia

Refractory anaemia

Refractory neutropenia

Refractory thrombocytopenia

Refractory anaemia with ring sideroblasts

Refractory cytopenia with multilineage dysplasia (with or without ring sideroblasts)

Refractory anaemia with excess blasts

Myelodysplastic syndrome with isolated del(5q)

Myelodysplastic syndrome, unclassifiable

Childhood myelodysplastic syndrome

Provisional entity: refractory cytopenia of childhood

Table 23.4 WHO diagnostic criteria for the myelodysplastic syndromes (MDS)

Disease	Blood findings	BM findings
Refractory cytopenia with unilineage dysplasia (RCUD): (refractory anaemia [RA]; refractory neutropenia [RN]; refractory thrombocytopenia [RT])	Unicytopenia or bicytopenia^a No or rare blasts (<1%)^b	Unilineage dysplasia: ≥10% of the cells in one myeloid lineage <5% blasts <15% of erythroid precursors are ring sideroblasts
Refractory anaemia with ring sideroblasts (RARS)	Anaemia No blasts	≥15% of erythroid precursors are ring sideroblasts Erythroid dysplasia only <5% blasts
Refractory cytopenia with multilineage dysplasia (RCMD)	Cytopenia(s) No or rare blasts (<1%)^b No Auer rods <1 × 10⁹/l monocytes	Dysplasia in ≥10% of the cells in ≥2 myeloid lineages (neutrophil and/or erythroid precursors and/or megakaryocytes) <5% blasts in marrow No Auer rods ±15% ring sideroblasts
Refractory anaemia with excess blasts-1 (RAEB-1)	Cytopenia(s) <5% blasts^b No Auer rods <1 × 10⁹/l monocytes	Unilineage or multilineage dysplasia 5–9% blasts^b No Auer rods
Refractory anaemia with excess blasts-2 (RAEB-2)	Cytopenia(s) 5–19% blasts^c Auer rods ±^c <1 × 10⁹/l monocytes	Unilineage or multilineage dysplasia 10–19% blasts^c Auer rods ±^c
Myelodysplastic syndrome – unclassified (MDS-U)	Cytopenias ≤1% blasts^b	Unequivocal dysplasia in <10% of cells in one or more myeloid lineages when accompanied by a cytogenetic abnormality considered as presumptive evidence for a diagnosis of MDS (see Table 23.6) <5% blasts
MDS associated with isolated del(5q)	Anaemia Usually normal or increased platelet count No or rare blasts (<1%)	Normal to increased megakaryocytes with hypolobated nuclei <5% blasts Isolated del(5q) cytogenetic abnormality No Auer rods

a Bicytopenia may occasionally be observed. Cases with pancytopenia should be classified as MDS-U.

b If the marrow myeloblast percentage is <5% but there are 2–4% myeloblasts in the blood, the diagnostic classification is RAEB-1. Cases of RCUD and RCMD with 1% myeloblasts in the blood should be classified as MDS-U.

c Cases with Auer rods and <5% myeloblasts in the blood and <10% in the marrow should be classified as RAEB-2. Although the finding of 5–19% blasts in the blood is, in itself, diagnostic of RAEB-2, cases of RAEB-2 may have <5% blasts in the blood if they have Auer rods or 10–19% blasts in the marrow or both. Similarly, cases of RAEB-2 may have <10% blasts in the marrow but may be diagnosed by the other two findings, Auer rod + and/or 5–19% blasts in the blood.

From Vardiman et al (2009).¹⁵

Classification of Acute Lymphoblastic Leukaemia

The WHO classification requires that an acute leukaemia be positively shown to be lymphoid before it is categorized as ALL. This classification groups together ALL and lymphoblastic lymphoma, using the designations precursor B lymphoblastic leukaemia/lymphoblastic lymphoma and precursor T lymphoblastic leukaemia/lymphoblastic lymphoma. These designations are clearly too cumbersome to use in clinical practice and undoubtedly haematologists will continue to refer to ‘acute lymphoblastic leukaemia.’ The FAB classification of ALL is now redundant except that FAB L3 morphology, i.e. the presence of ‘blast cells’ with basophilic cytoplasm and vacuolation, is of considerable clinical significance and should be recognized. In most, but not all, of these cases the cells are immunologically mature, expressing surface membrane immunoglobulin and the condition represents a leukaemic presentation of Burkitt lymphoma. The WHO categorization of such cases as lymphoma is more appropriate than their being categorized as ALL and is clinically important because the treatment, which is urgent, differs very considerably from the treatment of ALL.

Classification of Myeloproliferative Neoplasms and Related Conditions

The WHO classification of myeloproliferative neoplasms (previously called ‘disorders’) and related conditions (Table 23.5) is increasingly taking account of cytogenetic or molecular genetic analyses.

Table 23.5 Myeloproliferative neoplasms (MPN) and related conditions ^5,^6,^15,¹⁶

Myeloproliferative neoplasms

Chronic myelogenous leukaemia

Polycythaemia vera

Essential thrombocythaemia

Primary myelofibrosis

Chronic neutrophilic leukaemia

Chronic eosinophilic leukaemia, not otherwise categorized

Mast cell disease

MPN, unclassifiable

Lymphoid and myeloid neoplasms associated with rearrangement of PDGFRA, PDGFRB or FGFR1

Lymphoid and myeloid neoplasms associated with rearrangement of PDGFRA

Myeloid neoplasms associated with rearrangement of PDGFRB

Lymphoid and myeloid neoplasms associated with rearrangement of FGFR1

WHO criteria for a diagnosis of essential thrombocythaemia are: platelet count ≥450 × 10⁹/l; megakaryocyte proliferation with large and mature megakaryocytes on examination of the bone marrow with little or no granulocyte or erythroid proliferation; not meeting WHO criteria for CML, PV, primary myelofibrosis, MDS or other myeloid neoplasm; demonstration of JAK2 V617F or other clonal marker or no evidence of reactive thrombocytosis.¹⁶

WHO criteria for a diagnosis of primary myelofibrosis are divided into major criteria (e.g. megakaryocyte proliferation and atypical megakaryocytes accompanied by reticulin and/or collagen fibrosis; demonstration of JAK2 V617F or other clonal marker; no evidence of reactive marrow fibrosis; not meeting WHO criteria for CML, PV, MDS or other myeloid neoplasm) and minor criteria (e.g. leukoerythroblastosis; increased serum LDH; anaemia; palpable splenomegaly).

WHO criteria for a diagnosis of systemic mastocytosis are highly complex.³ A trephine biopsy with a mast cell tryptase stain is often crucial in the diagnosis. Molecular analysis for a KIT mutation can also be important.

Recognition of lymphoid and myeloid neoplasms associated with rearrangement of PDGFRA, PDGFRB or FGFR1 requires cytogenetic analysis (for detection of translocations likely to indicate rearrangement of PDGFRB or FGFR1) and molecular analysis (for detection of the FIP1L1−PDGFRA fusion gene that results from a cryptic deletion at 4q12). Appropriate molecular analysis may be either FISH or reverse transcription polymerase chain reaction (RT-PCR). The diagnosis of chronic eosinophilic leukaemia, not otherwise specified, requires exclusion of rearrangement of these three specific genes.

The categorization of neoplasms with features of both myelodysplasia and myeloproliferation and their diagnostic criteria are listed in Table 23.6.

Table 23.6 Summary of the World Health Organization categories of myelodysplastic/myeloproliferative neoplasms

Category	Criteria
Chronic myelomonocytic leukaemia (CMML)	A Ph-negative, BCR–ABL1-negative disorder with monocyte count >1 × 10⁹/l
	Fewer than 20% blasts plus promonocytes in peripheral blood (PB) or bone marrow (BM)
	Either dysplasia of one or more myeloid lineages or alternative criteria met (acquired clonal cytogenetic abnormality or monocytosis persisting for at least 3 months and alternative causes of monocytosis excluded)
Atypical chronic myeloid leukaemia (aCML)	A Ph-negative, BCR–ABL1-negative disorder with leucocytosis resulting from an increase in neutrophils and their precursors, the precursors (promyelocyte to metamyelocytes) constituting a least 10% of PB white cells
	Basophils <2% of white cells
	Monocytes <10% of white cells
	Hypercellular BM with granulocytic hyperplasia and dysplasia, with or without dysplasia of other lineages
	Fewer than 20% blasts plus promonocytes in peripheral blood or bone marrow
Juvenile myelomonocytic leukaemia (JMML)	A Ph-negative, BCR–ABL1-negative disorder with monocyte count >1 × 10⁹/l
	Fewer than 20% blasts plus promonocytes in peripheral blood or bone marrow
	Plus two or more of the following
	Haemoglobin F increased for age
	Immature granulocytes in the PB
	WBC >10 × 10⁹/l
	Clonal chromosomal abnormality (monosomy 7 not excluded)
	GM-CSF hypersensitivity of myeloid precursors in vitro
Myelodysplastic/myeloproliferative neoplasm, unclassifiable	A myelodysplastic/myeloproliferative disorder in which the criteria of one of the myelodysplastic syndromes are met
	There are prominent proliferative features (e.g. a platelet count of ≥450 × 10⁹/l or a white cell count of ≥13 × 10⁹/l)
	The condition has developed de novo
	The criteria for other MDS/MPD (CMML, aCML and JMML) are not met
	There is no Philadelphia chromosome, BCR–ABL1 fusion gene, 5q−, inv(3)(q21q26) or t(3;3)(q21;q26)