Erythrocyte and leucocyte cytochemistry

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Chapter 15 Erythrocyte and leucocyte cytochemistry

Barbara J. Bain, David Swirsky

Erythrocyte cytochemistry

Siderocytes and Sideroblasts

Siderocytes are red cells containing granules of non-haem iron. They were originally described by Grüneberg ¹ in small numbers in the blood of normal rat, mouse and human embryos and in large numbers in mice with a congenital anaemia. The granules are formed of a water-insoluble complex of ferric iron, lipid, protein and carbohydrate. This siderotic material (or haemosiderin) reacts with potassium ferrocyanide to form a blue compound, ferriferrocyanide; this reaction is the basis of a positive Prussian-blue (Perls’) reaction. The material also stains with Romanowsky dyes and then appears as basophilic granules, which have been referred to as ‘Pappenheimer bodies’ (Fig. 15.1).² By contrast, ferritin, which is a water-soluble non-haem compound of iron with the protein apoferritin, is not detectable by Perls’ reaction. Ferritin is normally present in all cells in the body, whereas, in health, haemosiderin is mainly found in macrophages in the bone marrow, liver (Kupffer cells) and spleen. When the body is overloaded with iron, as in haemochromatosis or transfusional haemosiderosis, excess iron is also found in other tissues.

Figure 15.1 Siderotic granules and Pappenheimer bodies. Photomicrographs of erythrocytes in a peripheral blood film from a splenectomized patient with a myelodysplastic syndrome (refractory anaemia with ring sideroblasts) showing Pappenheimer bodies: May–Grünwald–Giemsa (A); and siderotic granules: Perls’ reaction (B).

Iron is transported in plasma attached to a β globulin, transferrin, and is taken up selectively by the bone marrow, where the iron–transferrin complex binds to transferrin receptors on the surface of the erythroblast; the iron is released from transferrin and enters the cell. Most of the iron is rapidly converted to haem, synthesis being partly in the cytosol and partly in the mitochondria. The non-haem residue is in the form of ferritin. Degradation of the ferritin turns some of it into haemosiderin, which can be visualized under the light microscope as golden-yellow refractile particles in phagocytic cells. When stained by Perls’ reaction, haemosiderin is blue.

In health, siderotic granules can normally be seen, in preparations stained by Perls’ reaction, in the cytoplasm of many of the erythroblasts of human bone marrow and in marrow reticulocytes.³ However, they are not normally seen in human peripheral blood red cells. After splenectomy, siderocytes can always be found in the peripheral blood, often in large numbers. The reason for this is probably because reticulocytes, after delivery from the marrow, are normally sequestered for a time in the spleen and there they complete haem synthesis, utilizing, for this purpose, the iron stored in their cytoplasm within the siderotic granules. After splenectomy, this stage of reticulocyte maturation has to take place in the bloodstream, with the result that, even in an otherwise healthy person, a small percentage of siderocytes can then be found in the peripheral blood. The spleen is also probably able to remove large siderotic granules – as may be found in disease – from red cells by a process of pitting,⁴ and in its absence such granules persist in the red cells throughout their lifespan.

Method of Staining Siderotic Granules

Air dry films of peripheral blood or bone marrow and fix with methanol for 10–20 min. When they are dry, place the slides in a solution of 10 g/l potassium ferrocyanide in 0.1 mol/l HCl made by mixing equal volumes of 47 mmol/l (20 g/l) potassium ferrocyanide and 0.2 mol/l HCl immediately before use.

Leave the slides in the solution for about 10 min at about 20°C. Wash well in running tap water for 20 min, rinse thoroughly in distilled water and then counterstain with 1 g/l aqueous neutral red or eosin for 10–15 s. Care must be taken to avoid contamination by iron that may have been present on the slides or in staining dishes. Prepare the glassware by soaking in 3 mol/l HCl before washing (see p. 623). For quality control, a positive bone marrow film should always be stained together with the test films.

Prussian-blue staining can be applied to films that have previously been stained by Romanowsky dyes, even after years of storage. It is advisable to let the films stand in methanol overnight to remove most of the Romanowsky stain. The film should be checked before carrying out Perls’ reaction to ensure that there is no residual blue staining that could obscure Prussian-blue staining. Sundberg and Bromann described a technique whereby films were stained first by a Romanowsky dye (Wright’s stain) and then overstained by the acid-ferrocyanide method.⁵ This can give beautiful pictures, but the small blue-stained iron-containing granules tend to be masked in young erythroblasts by the general basophilia of the cell cytoplasm. Hayhoe and Quaglino described a method for combined periodic acid–Schiff (PAS) and iron staining.⁶ This may be helpful in the investigation of abnormal erythropoiesis in which the erythroblasts give a positive PAS reaction (see p. 343). A rapid method has been described for demonstrating siderotic granules by staining with 1% bromochlorphenol blue for 1 min.⁷ Iron-containing granules stain dark purple.

Significance of siderocytes

Siderocytes contain one or two (rarely many) small, unevenly distributed iron-containing granules that stain a Prussian-blue colour. There are normally a few very small scattered siderotic granules in about 40% of late erythroblasts.³ They stain faintly and may be difficult to see by light microscopy. The percentage of erythroblasts recognizable as sideroblasts is increased in haemolytic anaemias and megaloblastic anaemias and in haemochromatosis and haemosiderosis, in proportion to the degree of saturation of transferrin (i.e. to the amount of iron available). A disproportionate increase in the percentage of erythroblasts that are sideroblasts occurs when the synthesis of haemoglobin is impaired, in which case the siderotic granules are both more numerous and larger than normal (Fig. 15.2). When there is a defect in haem synthesis, the granules are deposited in mitochondria and frequently appear to be arranged in a collar around the nucleus (Fig. 15.3) giving the ‘ring sideroblasts’ characteristic of sideroblastic anaemias. In contrast, the distribution of the granules within the cell tends to be mainly normal in conditions in which globin synthesis alone is affected (e.g. in thalassaemia) or when there is iron overload.

Figure 15.2 Pathological sideroblasts. Thalassaemia. There is massive accumulation of iron-containing granules in normoblasts and phagocytic cells. Perls’ reaction.

Figure 15.3 Pathological sideroblasts. Sideroblastic anaemias. Accumulation of iron-containing granules in normoblasts, arranged characteristically around the nucleus. (A) Hereditary type; (B) myelodysplastic syndrome. Perls’ reaction.

There are several types of sideroblastic anaemia. These include the congenital (hereditary) type, pyridoxine (vitamin B₆) deficiency (rarely), sideroblastic anaemia caused by B₆ antagonists (e.g. drugs used in antituberculosis therapy) and secondary sideroblastic anaemia in alcoholism and lead poisoning. The presence of ring sideroblasts is a defining feature of refractory anaemia with ring sideroblasts ⁸ and refractory cytopenia with multilineage dysplasia and ring sideroblasts, two of the World Health Organization (WHO) categories of myelodysplastic syndrome (MDS). They may also occur in other categories of MDS. Ring sideroblasts are not uncommon in other haematological neoplasms, including primary myelofibrosis and acute myeloid leukaemia (AML), particularly erythroleukaemia and the WHO categories of therapy-related AML and AML with multilineage myelodysplasia. Ring sideroblasts have been defined as erythroblasts with at least five siderotic granules surrounding at least one-third of the nucleus.^9,¹⁰

In sideroblastic anaemia as a feature of a haematological neoplasm, erythroblasts at all stages of maturity may be loaded with siderotic granules, whereas in the secondary sideroblastic anaemias and in the hereditary types, the more mature cells seem most affected.

In addition to the siderotic granules within erythroblasts, haemosiderin can normally be seen in marrow films as accumulations of small granules, lying free or in macrophages in marrow fragments.¹¹ The amount of haemosiderin will be markedly increased in patients with increased iron stores, whereas haemosiderin is absent in iron deficiency anaemia (Fig. 15.4). In practice, staining to demonstrate iron stores in marrow fragments and siderotic granules in erythroblasts is a simple and valuable diagnostic procedure and should be applied as a routine to marrow films from the initial bone marrow aspirate of each patient.

Figure 15.4 Prussian-blue staining (Perls’ reaction) on aspirated bone marrow particles to demonstrate iron stores. (A) Normal, (B) absent, (C) increased and (D) grossly increased.

In chronic infections and in other examples of anaemia of chronic disease, the iron stores may be increased, with much siderotic material in macrophages but little or none visible in erythroblasts. Markedly excessive iron in macrophages is also a feature of thalassaemia intermedia and major and some dyserythropoietic anaemias. Conversely, absence of iron is diagnostic of iron deficiency or iron depletion (the latter term indicating the state in which storage iron is absent but anaemia is not yet evident). One study has shown that to establish the absence of stainable iron, at least seven particles must be examined, if necessary using more than one slide for this purpose.¹² There is no cytochemical method of demonstrating ferritin; methods of assay are described in Chapter 9.

Haemoglobin Derivatives

Heinz Bodies in Red Cells

Heinz, in 1890, was the first to describe in detail inclusions in red cells developing as the result of the action of acetylphenylhydrazine on the blood.¹³ It is now known that Heinz bodies can be produced by the action on red cells of a wide range of aromatic nitro- and amino-compounds, as well as by inorganic oxidizing agents such as potassium chlorate. They also occur when one or other of the globin chains of haemoglobin is unstable. In man, the finding of Heinz bodies is a sign of either chemical poisoning, drug toxicity, glucose-6-phosphate dehydrogenase (G6PD) deficiency or the presence of an unstable haemoglobin (e.g. Hb Köln). When of chemical or drug origin, Heinz bodies are likely to be visible in red cells only if the patient has been splenectomized previously or when large doses of the chemical or drug have been taken. When they are due to an unstable haemoglobin, they are rarely visible in freshly withdrawn red cells except after splenectomy. They may nevertheless develop in vitro in the blood of patients who have not been splenectomized if the specimen is incubated for 24–48 h.¹⁴ Heinz bodies are a late sign of oxidative damage and represent an end-product of the degradation of haemoglobin. Reviews dealing with Heinz bodies include those by Jacob¹⁵ and by White.¹⁶

Demonstration of Heinz Bodies

Unstained preparations

Heinz bodies may be seen as refractile objects in dry, unstained films, if the illumination is reduced by lowering the microscope condenser. They also can be seen by dark-ground illumination or phase-contrast microscopy. However, it is preferable to look for them in stained preparations (see below). In size they vary from 1 to 3 μm. One or more may be present in a single cell. They are usually close to the cell membrane and may cause a protrusion of the membrane; in wet preparations, they may move around within the cells in a slow Brownian movement.

The degradation product of an unstable haemoglobin (e.g. Hb Köln) exhibits green fluorescence when excited by blue light at 370 nm in a fluorescence microscope.¹⁷

Stained preparations

Dissolve approximately 0.5 g of methyl violet in 100 ml of 9 g/l NaCl and filter. Add 1 volume of blood (in any anticoagulant) to 4 volumes of the methyl violet solution and allow the suspension to stand for about 10 min at room temperature. Then prepare films and allow them to dry or view the suspension of cells between slide and coverglass. The Heinz bodies stain an intense purple (Fig. 15.5).

Figure 15.5 Glucose-6-phosphate dehydrogenase deficiency. Many of the cells contain large Heinz bodies. Stained supravitally by methyl violet.

(Courtesy of Mr David Roper.)

Heinz bodies also stain with other basic dyes. Brilliant green stains them well and none of the stain is taken up by the remainder of the red cell.¹⁸ Rhodanile blue (5 g/l solution in 10 g/l NaCl) stains them rapidly¹⁹ (i.e. within 2 min), at which time reticulocytes are only weakly stained. Compared with methyl violet, Heinz bodies stain less intensely with brilliant cresyl blue or New methylene blue. Nevertheless, they may be readily seen as pale blue bodies in a well-stained reticulocyte preparation, if the preparation is not counterstained.

If permanent preparations are required, fix the vitally stained films by exposure to formalin vapour for 5–10 min. Then counterstain the fixed films with 1 g/l eosin or neutral red, after thoroughly washing in water. If films are fixed in methanol, Heinz bodies are decolourized.

In β thalassaemia major, methyl violet staining of the bone marrow will demonstrate precipitated α chains. These appear as large irregular inclusions in late normoblasts, usually single and closely adhering to the nucleus. If such patients are splenectomized, inclusions are also found in reticulocytes and mature red blood cells, in the circulation as well as in the bone marrow.

Demonstration of Haemoglobin H Inclusions

Patients with α thalassaemia, who form haemoglobin H (β₄), have red cells in which multiple blue-green spherical inclusions develop on exposure to brilliant cresyl blue or New methylene blue as in reticulocyte preparations ²⁰ (Fig. 15.6). This is mainly a feature of haemoglobin H disease, but small numbers of similar cells may be seen in α thalassaemia trait, particularly, but not only, in α° thalassaemia heterozygosity.

Figure 15.6 Denaturation of haemoglobin H by brilliant cresyl blue. The round bodies consist of precipitated Hb H.

Method

Mix together in a small tube, as for staining reticulocytes (see p. 33), equal volumes of fresh blood or blood collected into ethylenediaminetetra-acetic acid (EDTA) and 10 g/l brilliant cresyl blue or 20 g/l New methylene blue in iso-osmotic phosphate buffer pH 7.4. Leave the preparation at 37°C for 3 h and make films at intervals during this time. Allow the films to dry and examine them without counter-staining. Haemoglobin H precipitates as multiple pale-staining greenish-blue, almost spherical, bodies of varying size (Fig. 15.7), which can be clearly differentiated from the darker-staining reticulofilamentous material of reticulocytes (Fig. 15.8).

Figure 15.7 Haemoglobin H disease. Almost every erythrocyte is affected.

Figure 15.8 Film of blood from patient with hereditary spherocytosis. There is an increased number of reticulocytes. Stained supravitally by brilliant cresyl blue.

The number of cells containing inclusions varies according to the type of α thalassaemia. In α° thalassaemia heterozygosity only 0.01–1.0% of the red cells contain inclusions, but this finding can provide a significant clue to diagnosis. In haemoglobin H disease (e.g. resulting from α° thalassaemia/α⁺ thalassaemia compound heterozygosity, − −/−α), as a rule at least 10% of the cells develop inclusions and, in some cases, the percentage is considerably greater. Haemoglobin H inclusions are also detectable in patients with acquired haemoglobin H disease as a feature of a myelodysplastic syndrome.

It should be noted that a haemoglobin H preparation is not recommended when precise diagnosis of the type of α thalassaemia trait is required (e.g. in antenatal diagnosis). DNA analysis is then indicated (see p. 147).

Carboxyhaemoglobin and Methaemoglobin

Carboxyhaemoglobin- and methaemoglobin-containing cells can be demonstrated cytochemically. These methods are described by Kleihauer and Betke.²¹ They have little practical value in modern practice.

Fetal Haemoglobin

An acid-elution cytochemical method that was introduced by Kleihauer et al.²² is a sensitive procedure to identify individual cells containing haemoglobin F even when few are present. Their detection in the maternal circulation has provided valuable information on the pathogenesis of haemolytic disease of the newborn.

The identification of cells containing haemoglobin F depends on the fact that they resist acid elution to a greater extent than do normal cells; thus, in the technique described in the following, they appear as isolated, darkly stained cells among a background of palely staining ghost cells. The occasional cells that stain to an intermediate degree are less easy to evaluate; some may be reticulocytes because these also resist acid elution to some extent. The following method, in which elution is carried out at pH 1.5, is recommended.²³

Reagents

Fixative. 80% ethanol

Elution solution. Solution A: 7.5 g/l haematoxylin in 90% ethanol. Solution B: FeCl₃, 24 g; 2.5 mol/l HCl, 20 ml; doubly distilled water to 1 litre.

For use, mix well 5 volumes of A and 1 volume of B. The pH is approximately 1.5. The solution can be used for about 4 weeks; if a precipitate forms, the solution should be filtered.

Counterstain. 1 g/l aqueous erythrosin or 2.5 g/l aqueous eosin.

Method

Prepare fresh air-dried films. Immediately after drying, fix the films for 5 min in 80% ethanol in a Coplin jar. Then rinse the slides rapidly in water and stand them vertically on blotting paper for about 10 min to dry. Next, place the slides for 20 s in a Coplin jar containing the elution solution. Then wash the slides thoroughly in water and finally place them in the counterstain for 2 min. Rinse in tap water and allow them to dry in the air. Fetal cells stain red and adult ghost cells stain pale pink (Fig. 15.9). Films prepared (a) from a mixture of cord blood and adult blood and (b) from normal adult blood should be stained alongside the test films as positive and negative controls, respectively.

Figure 15.9 Cytochemical demonstration of fetal haemoglobin. Acid elution method. The preparation consists of a mixture of cord and normal adult blood. The darkly staining cells are fetal cells.

A number of modifications of the Kleihauer method have been proposed. In one, New methylene blue is incorporated in the buffer solution, the reaction time is prolonged and buffer is used for washing the films.²⁴ The advantage of this technique is that reticulocytes stain blue, whereas cells containing haemoglobin F stain pink.

An immunofluorescent staining method has been developed based on the use of a specific antibody against haemoglobin F, which does not react with haemoglobin A.²⁵ By using a double-labelling procedure with rhodamine-labelled antibody against γ globin and a fluorescein-labelled antibody against β globin, it is possible to detect the presence of haemoglobin F and haemoglobin A in the same cell.²⁶

Haemoglobin S and Other Haemoglobin Variants

Immunodiffusion with specific antibodies has been used for the identification of haemoglobin S, haemoglobin A₂ and haemoglobin F in red cells.^27,²⁸ An alternative method is by detection of cells after labelling the cells with fluorescein isothiocyanate (FITC).²⁷ By a double-labelling method similar to that described earlier, it is possible to identify haemoglobin S as well as another haemoglobin in individual cells.

Leucocyte cytochemistry

Leucocyte cytochemistry encompasses the techniques used to identify diagnostically useful enzymes or other substances in the cytoplasm of haemopoietic cells. These techniques are particularly useful for the characterization of immature cells in AML and the identification of maturation abnormalities in the myelodysplastic syndromes and myeloproliferative neoplasms. There are many variations in the staining techniques, as discussed in the recommendations of an Expert Panel of the International Committee (now Council) for Standardization in Haematology.^29,³⁰ Detailed reference works discussing the theoretical and practical aspects of cytochemistry are available.³¹ The use of cytochemistry to characterize lymphoproliferative disorders has been largely superseded by immunological techniques (see Chapter 16). The results of cytochemical tests should always be interpreted in relation to Romanowsky stains and immunological techniques. Control blood or marrow slides should always be stained in parallel to ensure the quality of the staining. The principal uses of cytochemistry are as follows:

1. To characterize the blast cells in acute leukaemia as myeloid (leading to a diagnosis of AML unless there is also evidence of lymphoid differentiation)

2. To demonstrate myeloperoxidase or non-specific esterase activity and thus contribute to a diagnosis of mixed-phenotype acute leukaemia, according to the criteria of the 2008 WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues ³²

3. To identify granulocytic and monocytic components in AML

4. To identify unusual lineages occasionally involved in clonal myeloid disorders (e.g. basophils and mast cells)

5. To detect cytoplasmic abnormalities and enzyme deficiencies in myeloid disorders (e.g. myeloperoxidase-deficient neutrophils in myelodysplasia or acute leukaemia, neutrophil alkaline phosphatase-deficient neutrophils in chronic myelogenous leukaemia, CML)

6. To identify Auer rods in MDS (and thus classify a case as refractory anaemia with excess of blasts II in the WHO classification)

7. To confirm a diagnosis of hairy cell leukaemia.

It is particularly important that cytochemistry is not neglected in under-resourced countries when immunophenotyping is not readily available.

Myeloperoxidase

Myeloperoxidase (MPO) is located in the primary and secondary granules of neutrophils and their precursors, in eosinophil granules and in the azurophilic granules of monocytes. The MPO in eosinophil granules is cyanide resistant, whereas that in neutrophils and monocytes is cyanide sensitive. MPO splits H₂O₂ and in the presence of a chromogenic electron donor forms an insoluble reaction product. Various benzidine substitutes have been used, of which 3,3′-diaminobenzidine (DAB) is the preferred chromogen.^33,³⁴ The reaction product is stable, insoluble and non-diffusible. Staining can be enhanced by immersing the slides in copper sulphate or nitrate, but this is generally not required in normal diagnostic practice. Alternative non-benzidine-based techniques use 4-chloro-1-naphthol (4CN)³⁵ or 3-amino-9-ethylcarbazole.³⁶ The former gives very crisp staining but is soluble in some mounting media and immersion oil; the latter shows some diffusibility and does not stain as strongly as DAB.

Method with 3,3′-Diaminobenzidine

Results and interpretation

The reaction product is brown and granular (Fig. 15.10A). Red cells and erythroid precursors show diffuse brown cytoplasmic staining. The most primitive myeloblasts are negative, with granular positivity appearing progressively as they mature toward the promyelocyte stage. The positivity may be localized to the Golgi region. Promyelocytes and myelocytes are the most strongly staining cells in the granulocyte series, with positive (primary) granules packing the cytoplasm. Metamyelocytes and neutrophils have progressively fewer positive (secondary) granules. Eosinophil granules stain strongly and the large specific eosinophil granules are easily distinguished from neutrophil granules. Eosinophil granule peroxidase is distinct biochemically and immunologically from neutrophil peroxidase. Monoblasts and monocytes may be negative or positive. When positive, the granules are smaller than in neutrophils and diffusely scattered throughout the cytoplasm. MPO activity is present in basophil granules but is not demonstrable in mature basophils by the DAB reaction described earlier.

Figure 15.10 (A) Myeloperoxidase (MPO) and (B) Sudan Black B (SBB). Bone marrow in acute myeloid leukaemia (French–American–British [FAB] M1). Myeloperoxidase staining shows Auer rods and cytoplasmic granular staining, whereas with SBB localized positive reaction in the blast cells is more definite and Auer rods are prominent.

Pathological variations

Some individuals have congenital deficiency of neutrophil MPO. All stages of the neutrophil lineage, from the myeloblast onward, are negative. In these individuals, the eosinophils stain normally. Other individuals have an MPO deficiency confined to eosinophils or monocytes. Dysplastic neutrophils may be MPO negative. Auer rods stain well with DAB and are seen more frequently on MPO staining than on Romanowsky-stained films.

Sudan Black B

Sudan Black B (SBB) is a lipophilic dye that binds irreversibly to an undefined granule component in granulocytes, eosinophils and some monocytes. It cannot be extracted from the stained granules by organic dye solvents and gives comparable information to that of MPO staining.³⁷ The currently used staining solution is essentially that described by Sheehan and Storey.³⁸

Reagents

Fixative. Vapour from 40% formaldehyde solution

Stain. SBB (Sigma S-2380) 0.3 g in 100 ml absolute ethanol

Phenol buffer. Dissolve 16 g crystalline phenol in 30 ml absolute ethanol. Add to 100 ml distilled water in which 0.3 g Na₂HPO₄.12H₂O has been dissolved

Working stain solution. Add 40 ml buffer to 60 ml SBB solution

Counterstain. May–Grünwald–Giemsa or Leishman stain (see p. 61).

Method

1. Fix air-dried smears in formalin vapour as follows. Place a small square of filter paper in the bottom of a Coplin jar. Add 2 drops of 40% formalin, put on the lid and leave for 15 min to allow vaporization. Place the slides in the Coplin jar and replace the lid. After 5–10 min, remove the slides and stand on end for 15 min to ‘air wash’.

2. Immerse the slides in the working stain solution for 1 h in a Coplin jar with a lid on.

3. Transfer slides to a staining rack and immediately flood with 70% alcohol. After 30 s, tip the 70% alcohol off and flood again for 30 s. Repeat three times in total.

4. Rinse in gently running tap water and air dry.

5. Counterstain without further fixation with Leishman stain or May–Grünwald–Giemsa.

Technical Considerations

Buffered formal acetone fixation for 30 s is a satisfactory alternative to formalin vapour. The working stain solution should be replaced after 4 weeks. Bone marrow smears with fatty particles containing lipid-soluble SBB benefit from a 5-s swirl in xylene followed by rinsing in running tap water and air drying prior to counterstaining. The Romanowsky counterstain gives excellent cytological detail of all cells present.

Results and Interpretation

The reaction product is black and granular. The results are essentially similar to those seen with MPO staining, both in normal and leukaemic cells (Fig. 15.10B). MPO-negative neutrophils are also SBB negative. The only notable difference is in eosinophil granules, which have a clear core when stained with SBB. Rare cases (1–2%) of acute lymphoblastic leukaemia (ALL) show non-granular smudgy positivity not seen with MPO staining.³⁹ Basophils are generally not positive but may show bright red/purple metachromatic staining of the granules.

Neutrophil Alkaline Phosphatase

Alkaline phosphatase activity is found predominantly in mature neutrophils, with some activity in metamyelocytes. Although demonstrated as a granular reaction product in the cytoplasm, enzyme activity is associated with a poorly characterized intracytoplasmic membranous component distinct from primary or secondary granules.⁴⁰ Other leucocytes are generally negative, but rare cases of lymphoid malignancies show cytochemically demonstrable activity.⁴¹ Bone marrow macrophages are positive. Early methods of demonstrating alkaline phosphatase relied on the use of glycerophosphate or other phosphomonoesters as the substrate at alkaline pH, with a final black reaction product of lead sulphide.⁴² Azo-dye techniques are simpler, giving equally good results. These methods use substituted naphthols as the substrate and it is the liberated naphthol rather than phosphate that is used to combine with the azo-dye to give the final reaction product.⁴³^–⁴⁵

Reagents

Fixative. 4% formalin methanol. Add 10 ml 40% formalin to 90 ml methanol. Keep at −20°C or in the freezer compartment of a refrigerator. Discard after 2 weeks

Substrate. Naphthol AS phosphate (Sigma N-5625). Store in freezer

Buffer. 0.2 mol/l Tris buffer, pH 9.0 (see p. 623)

Stock substrate solution. Dissolve 30 mg naphthol AS phosphate in 0.5 ml N,N-dimethylformamide (Sigma D-4551). Add 100 ml 0.2 mol/l Tris buffer, pH 9.1. Store in a refrigerator at 2–4°C. The solution is stable for several months

Coupling azo-dye. Fast Blue BB salt (Sigma F-0250). Store in freezer

Counterstain. Neutral red, 0.02% aqueous solution.

Method

1. Fix freshly made air-dried blood films for 30 s in cold 4% formalin methanol.

2. Rinse with tap water and air dry.

3. Prepare working substrate solution by allowing 40 ml of stock substrate solution to warm to room temperature. Add 24 mg of Fast Blue BB and mix thoroughly until dissolved. Incubate slides for 15 min.

4. Wash in tap water and air dry.

5. Counterstain for 3 min in 0.02% aqueous neutral red, rinse briefly and air dry.

Technical considerations

N,N-dimethylformamide may dissolve some types of plastic; therefore a glass tube should be used to dissolve the substrate. Blood films should be made soon after blood collection, preferably within 30 min because neutrophil alkaline phosphatase (NAP) activity decreases rapidly in EDTA-anticoagulated blood. Once spread, the blood film should be stained within 6 h. A control film with a predictably high score, e.g. from a patient with reactive neutrophilia or from a pregnant woman, should be processed together with the patient film. The technical aspects of blood film preparation and the effects of fixation on NAP activity are discussed by Kaplow.⁴⁶

Results and Interpretation

The reaction product is blue and granular. The intensity of reaction product in neutrophils varies from negative to strongly positive, with coarse granules filling the cytoplasm and overlying the nucleus (Fig. 15.11). An overall score is obtained by assessing the stain intensity in 100 consecutive neutrophils, with each neutrophil scored on a scale of 1–4 as follows:

0 – Negative, no granules

1 – Occasional granules scattered in the cytoplasm

2 – Moderate numbers of granules

3 – Numerous granules

4 – Heavy positivity with numerous coarse granules crowding the cytoplasm, frequently overlying the nucleus.

Figure 15.11 Neutrophil alkaline phosphatase. A strongly positive (4+) and moderately positive (3+ and 2+) intensity of reaction are shown.

The overall possible score will range between 0 and 400 (assessed on 100 neutrophils). Reported normal ranges show some variations, owing possibly in part to variations in scoring criteria and methodology: 13–160 (mean 61);⁴⁶ 14–100 (mean 46);⁴⁷ 37–98 (mean 68);⁴⁸ 11–134 (mean 48).⁴⁹ A normal range should therefore be established in each laboratory.

In normal individuals, it is rare to find any neutrophils with a score of 3 and a score of 4 should not be present. There is some physiological variation in NAP scores. Newborn babies, children and pregnant women have high scores and premenopausal women have, on average, scores one-third higher than those of men.⁴⁰ In pathological states, the most significant diagnostic use of the NAP score is in CML. In the chronic phase of the disease, the score is almost invariably low, usually zero. Transient increases may occur with inter-current infection. In myeloid blast transformation or accelerated phase, the score rises. Low scores are also commonly found in paroxysmal nocturnal haemoglobinuria (PNH) and the very rare condition of hereditary hypophosphatasia. There are many causes of a raised NAP score, notably in the neutrophilia of infection, polycythaemia vera, leukaemoid reactions and Hodgkin lymphoma. In aplastic anaemia, the NAP score is high, but it falls if PNH supervenes. With the greater use of cytogenetic and molecular genetic techniques to confirm the diagnosis of CML, the NAP score is rarely needed.

Acid Phosphatase Reaction, including Tartrate-Resistant Acid Phosphatase Reaction

Cytochemically demonstrable acid phosphatase is ubiquitous in haemopoietic cells. The staining intensity of different cell types is somewhat variable according to the method used. Its main diagnostic use is in the diagnosis of T-cell ALL and hairy cell leukaemia.^50,⁵¹ These diseases are more reliably diagnosed and characterized by immunophenotyping when this is available (see Chapter 16). The unmodified acid phosphatase stain is now largely redundant but the tartrate-resistant acid phosphatase stain is still useful for confirmation of the diagnosis of hairy cell leukaemia when immunophenotyping is not available. The pararosaniline method given in the following section, modified from Goldberg and Barka,⁵² is recommended for demonstrating positivity in T lymphoid cells. Use of Fast Garnet GBC as coupler may be preferred for the demonstration of tartrate-resistant acid phosphatase activity.^29,⁵¹

Reagents

Fixative. Methanol, 10 ml; acetone, 60 ml; water, 30 ml; and citric acid, 0.63 g. Adjust to pH 5.4 with 1 mol/l NaOH before use

Buffer pH 5.0. Sodium acetate trihydrate, 19.5 g; sodium barbiturate, 29.5 g; water to 1 litre (Michaeli’s veronal acetate buffer)

Substrate solution. 25 mg naphthol AS-BI phosphate (Sigma N-2125) dissolved in 2.5 ml N,N-dimethylformamide

Sodium nitrite. 4% NaNO₂ aqueous solution

Coupling reagent

1. Stock pararosaniline. Dissolve 1 g pararosaniline (Sigma P-7632) in 25 ml warm 2 mol/l HCl. Filter when cool. Store at room temperature in the dark. Stable for 2 months

2. 4% sodium nitrite solution. Dissolve 200 mg sodium nitrite in 5 ml distilled water. Stable for 1 week at 4–10°C

3. Hexazotized pararosaniline. Mix equal volumes of pararosaniline and 4% sodium nitrite together 2 min before use.

Counterstain. 1% Aqueous methyl green or aqueous haematoxylin

Tartaric acid L(+) (Sigma T-1807)

Working solution A. Mix 92.5 ml of buffer with 2.5 ml of substrate solution. Add 32.5 ml of distilled water and then add 4 ml of hexazotized pararosaniline. Mix well and adjust pH to 5.0 using 1 mol/l NaOH

Working solution B. Add 375 mg of crystalline L(+)-tartaric acid to 50 ml of working solution A; the final concentration is then 50 mmol/l.

Method

1. Air dry films for several hours (24 h if possible).

2. Fix for 10 min in methanol/acetone/citric acid, rinse in tap water and air dry.

3. Incubate for 1 h at 37°C in working solutions A (acid phosphatase reaction) or incubate two films in working solutions A and B, respectively (tartrate-resistant acid phosphatase reaction).

4. Rinse in tap water and air dry.

5. Counterstain in 1% aqueous methyl green or aqueous haematoxylin for 5 min.

6. Rinse in tap water and mount wet in warmed glycerin jelly.

Results and Interpretation

The reaction product is red with a mixture of granular and diffuse positivity (Fig. 15.12). In T cells, acid phosphatase is an early differentiation feature. Almost all acute and chronic T-lineage leukaemias show strong activity. In T-lineage ALL, the activity is usually highly localized (polar). Granulocytes are strongly positive. Monocytes, eosinophils and platelets show variable positivity. In the bone marrow, macrophages, plasma cells and megakaryocytes are strongly positive.

Figure 15.12 Acid phosphatase. T-cell acute leukaemia with intense localized staining.

In hairy cell leukaemia the majority of leukaemic cells react equally positively in the presence and absence of tartaric acid (Fig. 15.13).

Figure 15.13 Acid phosphatase. Hairy cell leukaemia: acid phosphatase (A) and tartrate-resistant acid phosphatase (B).

When immunophenotyping is available, the unmodified acid phosphatase reaction is redundant. If a full range of appropriate monoclonal antibodies is available, the tartrate-resistant acid phosphatase reaction is also redundant.

Periodic Acid–Schiff Reaction

Periodic acid specifically oxidizes 1–2 glycol groups to produce stable dialdehydes. These dialdehydes give a red reaction product when exposed to Schiff’s reagent (leucobasic fuchsin). Positive reactions occur with carbohydrates, principally glycogen, but also monosaccharides, polysaccharides, glycoproteins, mucoproteins, phosphorylated sugars, inositol derivatives and cerebrosides.⁵³ Glycogen can be distinguished from other positively reacting substances by its sensitivity to diastase digestion. In haemopoietic cells, the main source of positive reactions is glycogen.

Reagents

Fixative. Methanol

1% periodic acid. HIO₄.2H₂O, 10 g/l in distilled water

Schiff’s reagent. Dissolve 5 g basic fuchsin in 500 ml of hot distilled water. Filter when cool. Saturate with SO₂ gas by bubbling for 1–12 h in a fume cupboard. Shake vigorously with 2 g activated charcoal for 1 min in a conical flask in a fume cupboard and filter immediately through a large Whatman No. 1 filter into a dark bottle. The reagent is stable for 6 months at room temperature, stored in the dark

Counterstain. Aqueous haematoxylin.

Technical Considerations

Formalin vapour (5 min), formalin/ethanol (10 ml 40% formalin/90 ml ethanol) (10 min) and buffered formal acetone (45 s) are satisfactory alternative fixatives. Previously fixed, iron-stained or Romanowsky-stained films can be overstained with the PAS reaction satisfactorily. Romanowsky-stained smears can be partly decolourized by soaking in methanol for 1 h prior to step 4. The intensity of the reaction product depends on the quality of the Schiff’s reagent. Normal neutrophils should always stain intensely red and deterioration of the Schiff’s reagent can be detected by examination of control normal films. Some methods recommend rinsing in a dilute sodium metabisulphite HCl solution (‘SO₂ water’) after step 6, but this is not necessary with good-quality Schiff’s reagent.

Results and Interpretation

The reaction product is red, with intensity ranging from pink to bright red (Figs 15.14, 15.15). Cytoplasmic positivity may be diffuse or granular. Granulocyte precursors show diffuse weak positivity, with neutrophils showing intense confluent granular positivity. Eosinophil granules are negative, with diffuse cytoplasmic positivity. Basophils may be negative but often show large irregular blocks of positive material not related to the granules. Monocytes and their precursors show variable diffuse positivity with superimposed fine granules, often at the periphery of the cytoplasm. Normal erythroid precursors and red cells are negative. Megakaryocytes and platelets show variable, usually intense, diffuse positivity with superimposed fine granules, coarse granules and large blocks. Granular positivity with negative background cytoplasm is found in 10–40% of peripheral lymphocytes, with no detectable differences between T and B cells.^54,⁵⁵ Lymphoblasts show variable PAS-positive cytoplasmic granules or blocks on a clear background; it is block positivity on a clear background that is most characteristic of lymphoblasts rather than myeloblasts. When immunophenotyping is available, the PAS reaction is redundant for the diagnosis of ALL. It can still be useful in AML and MDS to identify abnormal erythroblasts and dysplastic megakaryocytes and to demonstrate the cytoplasmic blush that helps to confirm a diagnosis of acute promyelocytic leukaemia (Fig. 15.15).

Figure 15.14 Periodic acid–Schiff stain. (A) Dysplastic micromegakaryocytes with diffuse cytoplasmic staining and some coarse granules; (B) dyserythropoiesis with diffuse staining in a trinucleate normoblast and coarse granular and diffuse staining in a proerythroblast; and (C) acute lymphoblastic leukaemia with blasts showing block positivity.

Figure 15.15 Acute promyelocytic leukaemia. (A) May–Grünwald–Giemsa stain shows hypergranular promyelocytes with scattered Auer rods; (B) Sudan black B with strongly stained cytoplasm; (C) periodic acid–Schiff staining shows diffuse cytoplasmic blush; and (D) chloroacetate esterase gives strong cytoplasmic staining.

Esterases

Leucocyte esterases are a group of enzymes that hydrolyse acyl or chloroacyl esters of α-naphthol or naphthol AS. Li et al.⁵⁶ identified nine esterase isoenzymes using polyacrylamide gel electrophoresis of leucocyte extracts from normal and pathological cells. The gels were stained in parallel with cell smears. The isoenzymes fell into two groups: bands 1, 2, 7, 8 and 9 corresponded to the ‘specific’ esterase of neutrophils, staining specifically with naphthol AS-D chloroacetate esterase (chloroacetate esterase, CAE), whereas bands 3, 4, 5 and 6 corresponded to ‘non-specific’ esterase (NSE), staining with α-naphthyl acetate esterase (ANAE) and α-naphthyl butyrate esterase (butyrate esterase, ANBE). Band 4 was best demonstrated by ANBE and band 5 by ANAE. The NSEs are inhibited by sodium fluoride (NaF). Naphthol AS acetate and naphthol AS-D acetate react with both specific and non-specific esterases, but only the reaction with the NSEs is inhibited by NaF. The methods using parallel slides with and without NaF are not generally used anymore because it is usually more informative to perform a combination of chloroacetate esterase and one of the ‘non-specific’ esterase stains on a single slide. The combined methods have the advantage of demonstrating pathological double staining of individual cells. All the esterase stains can be performed using a variety of coupling reagents, each of which gives a different coloured reaction product. The methods outlined as follows have been chosen for their simplicity and reliability.

Naphthol AS-D Chloroacetate Esterase

Reagents

Fixative. Buffered formal acetone (see p. 623)

Buffer. 66 mmol/l phosphate buffer, pH 7.4 (see p. 622)

Naphthol AS-D chloroacetate substrate solution.²⁹ Dissolve 0.1 g of naphthol AS-D chloracetate (Sigma N-0758) in 40 ml N,N-dimethyl-formamide (Sigma D-4254). Keep refrigerated

Working substrate solution. Add 2 ml of naphthol AS-D chloroacetate stock solution to 38 ml of 66 mmol/l phosphate buffer, pH 7.4. Mix well. Add 0.4 ml of freshly prepared hexazotized New fuchsin. Mix well

Coupling reagent

1. Hexazotized New fuchsin. Dissolve 4 g of New fuchsin in 100 ml of 2N HCl.

2. Sodium nitrite solution 0.3 mol/l. Dissolve 2.1 g of sodium nitrite (NaNO₂) in 100 ml of water.

3. Immediately prior to using, add 0.2 ml of the hexazotized New fuchsin to 0.4 ml sodium nitrite, mix well and leave for 1 min before adding to substrate solution

Counterstain. Aqueous haematoxylin.

Method

1. Fix air-dried smears in cold buffered formal acetone for 30 s.

2. Rinse in gently running tap water and air dry.

3. Immerse the slides in the working substrate solution in a Coplin jar for 5–10 min.

4. Rinse in running tap water and air dry.

5. Counterstain in aqueous haematoxylin for 1 min.

6. ‘Blue’ in running tap water for 1 min and air dry.

Technical considerations

The CAE stain is robust and reliable. A satisfactory alternative to New fuchsin is 40 mg of Fast blue BB, but it requires thorough vigorous mixing with the substrate solution. The incubation time is important because most haemopoietic cells show some scattered granular staining if the incubation is prolonged. Hydrolysis of the substrate is rapid, with staining virtually complete within 3–5 min.

Results and interpretation

The reaction product is bright red (Fig. 15.15D). It is confined to cells of the neutrophil series and mast cells. Cytoplasmic CAE activity appears as myeloblasts mature to promyelocytes. Positivity in myeloblasts is rare, but promyelocytes and myelocytes stain strongly, with the reaction product filling the cytoplasm. More mature cells stain strongly but less intensely. It is therefore useful as a marker of cytoplasmic maturation in myeloid leukaemias. In acute promyelocytic leukaemia, the cells show heavy cytoplasmic staining. The characteristic multiple Auer rods stain positively, often with a hollow core. It is rare to see CAE-positive Auer rods in other forms of AML except in cases with the t(8;21) translocation.⁵⁷

α-Naphthyl Butyrate Esterase

Reagents

Fixative. Buffered formal acetone

Buffer. 100 mmol/l phosphate buffer (Sorensen’s), pH 8.0

Substrate stock solution. α-Naphthyl butyrate (Sigma N-8125) 100 μl in 5 ml acetone. The solution should be stored at −20°C and is stable for at least 2 months

Coupling reagent. Fast Garnet GBC (Sigma F-8761) 15 mg

Counterstain. Aqueous haematoxylin.

Method

1. Fix air-dried smears in buffered formal acetone for 30 s. Rinse in gently running tap water and air dry.

2. Add the Fast Garnet GBC to 50 ml buffer and mix well.

3. Add 0.5 ml of the α-naphthyl butyrate/acetone solution and mix well.

4. Pour the incubation medium into a Coplin jar containing the fixed slides and incubate for 20–40 min.

5. Rinse thoroughly by running tap water into the Coplin jar until clear.

6. Air dry and counterstain in aqueous haematoxylin for 1–5 min.

Technical considerations

The reaction product is soluble in immersion oil and synthetic mounting media. If slides are to be looked at repeatedly, they should be mounted in an aqueous mounting medium (e.g. Apathy’s gum-arabic mountant or glycerin/gelatin). There may be batch-to-batch variation of the Fast Garnet GBC. Staining can be controlled by removing the control slide from the incubation medium after 20 min and examining it wet under a low-power (e.g. ×20) objective, returning it to the incubation medium while still wet. When the monocytes show as dark brown, staining is complete. Hexazotized pararosaniline is an alternative coupling reagent, which gives an insoluble brown reaction product and is suitable for mounting in synthetic mounting media.⁵⁶

Results and interpretation

The reaction product is brown and granular (Fig. 15.16). The majority of monocytes (>80%) stain strongly, the remainder showing some weak staining. Negative monocytes are rare. Neutrophils, eosinophils, basophils and platelets are negative. B lymphocytes are negative and T lymphocytes are more variably stained. In the bone marrow, monocytes, monocyte precursors and macrophages stain strongly. α-Naphthyl butyrate is more specific for identifying a monocytic component in AML than α-naphthyl acetate (see later).

Figure 15.16 Non-specific esterase. Positive (brown) reaction in acute monocytic leukaemia (FAB M5).

α-Naphthyl Acetate Esterase

Reagents

Fixative. Buffered formal acetone

Buffer. 66 mmol/l phosphate buffer, pH 6.3

Substrate solution. Dissolve 100 mg α-naphthyl acetate (Sigma N-8505) in 5 ml ethylene monomethyl ether. Store at 4–10°C

Coupling reagent

1. Stock pararosaniline. Dissolve 1 g pararosaniline (Sigma P-7632) in 25 ml warm 2 mol/l HCl. Filter when cool. Store at room temperature in the dark. Stable for 2 months.

2. 4% sodium nitrite solution. Dissolve 200 mg sodium nitrite in 5 ml distilled water. This solution is stable for 1 week at 4–10°C.

3. Hexazotized pararosaniline. Mix equal volumes of pararosaniline and 4% sodium nitrite together 1 min before use.

Incubation medium. Add 2 ml of the α-naphthyl acetate solution to 38 ml of the 66 mmol/l phosphate buffer, pH 6.3, and mix well. Add 0.4 ml of freshly prepared hexazotized pararosaniline and mix well

Counterstain. Aqueous haematoxylin.

Method

1. Fix air-dried smears in cold buffered formal acetone for 30 s.

2. Rinse in running tap water and air dry.

3. Immerse the slides for 30–60 min in the incubation medium in a Coplin jar.

4. Rinse in gently running tap water in the Coplin jar until clear and air dry.

5. Counterstain in aqueous haematoxylin for 2–5 min.

Technical considerations

Fast Blue BB 80 mg can be substituted as a coupling reagent. This gives a dark green/brown granular reaction product, which is soluble in mounting media and immersion oil. The haematoxylin staining time should be adjusted to give clear nuclear detail without overstaining to obscure nucleoli and chromatin texture.

Results and interpretation

The reaction product is diffuse red/brown in colour. Normal and leukaemic monocytes stain strongly. Normal granulocytes are negative, but in MDS or AML may give positive reactions of varying intensity. Megakaryocytes stain strongly and leukaemic megakaryoblasts may show focal or diffuse positivity. Most T lymphocytes and some T lymphoblasts show focal ‘dot-like’ positivity, but immunophenotyping has superseded cytochemistry for identifying and subcategorizing T cells. Leukaemic erythroblasts may show focal or diffuse positivity.

Sequential Combined Esterase Stain Using ANAE and CAE

Reagents

As earlier for ANAE and CAE stains.

Method

1. Follow the method and steps 1–4 listed earlier for α-naphthyl acetate esterase stain, rinse in tap water and air dry.

2. Without further fixation, prepare the naphthol AS-D chloroacetate incubation medium as explained previously, substituting 10 mg Fast Blue BB (Sigma F-0250) for hexazotized New fuchsin and incubate for 10 min.

3. Rinse in tap water and counterstain with aqueous haematoxylin for 1–3 min.

Technical considerations

Fast Blue BB is relatively insoluble and the chloroacetate incubation medium should be mixed vigorously before use.

Results and interpretation

The ANAE gives a brown reaction product and the CAE gives a granular bright blue product (Fig. 15.17). Staining patterns are identical to those seen with the two stains used separately. The double-staining technique avoids the need to compare results from separate slides and reveals aberrant staining patterns. In myelomonocytic leukaemias, cells staining with both esterases may be present. In MDS and AML with dysplastic granulocytes, double staining of individual cells may be present. This may be helpful when a diagnosis of MDS is not otherwise certain, but the same abnormal pattern may be seen in non-clonal dysplastic states such as megaloblastic anaemia.

Figure 15.17 Combined esterase stain. Acute myelomonocytic leukaemia with almost equal numbers of chloroacetate esterase (blue) and non-specific esterase (brown) positive cells.

Single Incubation Double Esterase (Naphthol AS-D Chloroacetate and α-Naphthyl Butyrate)⁵⁸

Technical considerations

Steps 4 and 5 should be carried out rapidly. Staining can be extended to 30 min if necessary to ensure maximal ANBE staining, but at longer incubation times some nonspecific granular CAE staining may occur.

Results and interpretation

The CAE reaction product is bright blue (granulocytes); the ANBE product is dark green/brown (monocytes). ANBE does not stain megakaryocytes or T cells as strongly as α-naphthyl acetate. Lam et al. suggest the use of hexazotized pararosaniline as coupling reagent in a single incubation combined esterase, which gives contrasting bright red and brown reaction products.⁵⁹

In AML, the stain is useful for identifying monocytic and granulocytic components.

Toluidine Blue Stain

Toluidine blue staining is useful for the enumeration of basophils and mast cells. It binds strongly to the granules in these cells and is particularly useful in pathological states in which the cells may not be easily identifiable on Romanowsky stains. In AML and in CML and other myeloproliferative neoplasms, basophils may be dysplastic and poorly granular, as may the mast cells in systemic mastocytosis.

Reagents

Toluidine blue 1% w/v in methanol. Add 1 g of toluidine blue (BDH 34077) to 100 ml methanol and mix for 24 h on a roller or with a magnetic flea. The stain is stable indefinitely at room temperature. Keep tightly stoppered.

Method

1. Place air-dried smears on a staining rack and flood with the toluidine blue solution.

2. Incubate for 5–10 min.

3. Rinse briefly in gently running tap water until clear and air dry.

Results and Interpretation

The granules of basophils and mast cells stain a bright red/purple and are discrete and distinct (Fig. 15.18). Nuclei stain blue and cells with abundant RNA may show a blue tint to the cytoplasm. Although toluidine blue is said to be specific for these granules, with >10 min incubation, the primary granules of promyelocytes are stained red/purple. However, these are smaller and finer than the mast cell or basophil granules and easily distinguished.

Figure 15.18 Toluidine blue. Chronic myelogenous leukaemia in accelerated phase. There are five strongly positive basophils.

Cytochemical Reactions and Leukaemia Classification

Myelodysplastic Syndromes and Acute Myeloid Leukaemia

MDS is an acquired clonal preleukaemic bone marrow disorder characterized largely by a cellular or hypercellular marrow, peripheral cytopenias and variable morphological abnormalities of the haemopoietic cells. The classification system proposed by the French-American-British (FAB) cooperative group in 1982 ⁶⁰ was widely used for many years but is now being superseded by the WHO classification (see Chapter 23). A Perls’ reaction for haemosiderin is essential for the demonstration of ring sideroblasts. Other cytochemical evidence of dysplasia includes double staining of cells with chloroacetate and ANAE, the presence of SBB- or MPO-negative neutrophils and the presence of Auer rods (identified with SBB and MPO).

In AML, cytochemistry is helpful in defining monocytic cells (ANAE and ANBE), identifying Auer rods and demonstrating dysplasia (as mentioned earlier).

Acute Lymphoblastic Leukaemia

The modern diagnosis and classification of ALL is by cytology, followed by immunophenotyping (see Chapter 16). If immunophenotyping is not available, cytochemistry remains important. It should be noted that, on Romanowsky staining, lymphoblasts may rarely contain fine azurophilic granules. However, Auer rods are never seen and MPO and CAE are negative, whether or not fine granules are present. Occasionally granules give a weak reaction with SBB. Although not lineage specific, the pattern of any PAS positivity may be helpful.³¹ In ALL, 95% of cases show positive blocks or granules of bright red PAS-positive material. This may be present in very few blasts (<1%) or the majority. The critical difference from granular or block positivity in other leukaemic cells is the glass-clear background cytoplasm in lymphoblasts. Myeloblasts, monoblasts, leukaemic erythroblasts and megakaryoblasts all show some degree of diffuse cytoplasmic positivity and occasionally block positivity is seen. Acid phosphatase staining is more likely to give focal positivity in T-lineage than B-lineage acute leukaemias, but the difference is not clear enough to be of diagnostic certainty and focal positivity is sometimes also seen in the erythroblasts of erythroleukaemia. Esterase staining is generally unhelpful; some T-cell cases show focal positivity with ANAE, but this is not specific.

Chronic Myeloproliferative Neoplasms

Although low NAP scores are typical in chronic phase CML and high scores are usually found in other myeloproliferative neoplasms, the finding of a high NAP score is too non-specific to be of diagnostic help.

Chronic Lymphoproliferative Disorders

Chronic lymphoproliferative disorders are now characterized by immunophenotyping (Chapter 16). The reactions for acid hydrolases (acid phosphatase, ANAE, β-glucuronidase and β-glucosaminidase) show focal positivity in most T-cell disorders but are negative in B-cell disorders. The tartrate-resistant acid phosphatase reaction for hairy cell leukaemia is the only cytochemical stain that is sufficiently specific to still be regarded as diagnostically useful (in the absence of immunophenotyping).

References

1 Grüneberg H. Siderocytes: a new kind of erythrocyte. Nature (London). 1941;148:114-115.

2 Pappenheimer A.M., Thompson K.P., Parker D.D., et al. Anaemia associated with unidentified erythrocytic inclusions after splenectomy. Q J Med. 1945;14:75-100.

3 Kaplan E., Zuelzer W.W., Mouriquand C. Sideroblasts: a study of stainable nonhemoglobin iron in marrow normoblasts. Blood. 1954;9:203-213.

4 Crosby W.H. Siderocytes and the spleen. Blood. 1957;12:165-170.

5 Sundberg R.D., Bromann H. The application of the Prussian blue stain to previously stained films of blood and bone marrow. Blood. 1955;10:160-166.

6 Hayhoe F.G., Quaglino D. Refractory sideroblastic anaemia and erythraemic myelosis: possible relationship and cytochemical observations. Br J Haematol. 1960;6:381-387.

7 Kass L., Eickholt M.M. Rapid detection of ringed sideroblasts with bromchlorphenol blue. Am J Clin Pathol. 1978;70:738-740.

8 Bennett J.M. Classification of the myelodysplastic syndromes. Clin Haematol. 1986;15:909-923.

9 Mufti G.J., Bennett J.M., Goasgen J., et al. Diagnosis and classification of MDS: International Working Group on Morphology of MDS (IWGM-MDS) consensus proposals for the definition and enumeration of myeloblasts and ring sideroblasts. Haematologica. 2008;93:1712-1717.

10 Hasserjian R.P., Gatterman N., Bennett J.M., et al. Refractory anaemia with ring sideroblasts. In: Swerdlow S.H., Campo E., Harris N.L., et al, editors. World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2008:96-97.

11 Rath C.E., Finch C.A. Sternal marrow hemosiderin: a method for the determination of available iron stores in man. J Lab Clin Med. 1948;33:81-86.