Special Techniques Applicable to Bone Marrow Diagnosis
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TWO SPECIAL TECHNIQUES APPLICABLE TO BONE MARROW DIAGNOSIS Peripheral blood samples, bone marrow aspirates and niques that may be applied to trephine biopsy trephine biopsy specimens are suitable for many sections include: (i) a wider range of cytochemical diagnostic investigations, in addition to routine stains; (ii) immunohistochemistry; (iii) cytogenetic microscopy of Romanowsky-stained blood and bone and molecular genetic analysis; and (iv) ultrastruc- marrow films and haematoxylin and eosin-stained tural examination. histological sections. Some of these techniques, for example Perls’ stain to demonstrate haemosiderin Cytochemical and histochemical stains in a bone marrow aspirate, are so often useful that they are performed routinely, whereas other tech- Cytochemical stains on bone marrow niques are applied selectively. This chapter will deal aspirates predominantly with special techniques that are applicable to bone marrow aspirates and trephine Perls’ stain for iron biopsy sections but reference will be made to the peripheral blood where this is the more appropriate A Perls’ or Prussian blue stain (Figs 2.1 and 2.2) tissue for study. demonstrates haemosiderin in bone marrow macro- Bone marrow aspirate films are stained routinely phages and within erythroblasts. Consequently, it with a Romanowsky stain such as a May– allows assessment of both the amount of iron in Grünwald–Giemsa (MGG) or a Wright–Giemsa stain. reticulo-endothelial stores and the availability of Other diagnostic procedures that may be of use iron to developing erythroblasts. in individual cases include: (i) cytochemistry; (ii) Assessment of storage iron requires that an ade- immunophenotyping (by immunocytochemistry or quate number of fragments are obtained. A bone flow cytometry); (iii) cytogenetic and molecular marrow film or squash will contain both intracellu- genetic analysis; (iv) ultrastructural examination; lar and extracellular iron, the latter being derived (v) culture for micro-organisms; and (vi) culture for from crushed macrophages. It is usual to base assessment of haemopoietic progenitor cells. assessment of iron stores mainly on intracellular iron In most countries, histological sections cut from since iron stains are prone to artefactual deposits bone marrow trephine biopsies are stained rou- and it can be difficult to distinguish between extra- tinely with H&E. Most laboratories also use silver cellular iron and artefact. Iron stores may be stains routinely to demonstrate reticulin and some assessed as normal, decreased or increased, or may employ, in addition, a Giemsa stain, a Perls’ stain be graded as 1+ to 6+ as shown in Table 2.1, grades or both. We recommend the routine use of H&E, of 1+ to 3+ being considered normal. Alternatively, Giemsa and reticulin stains. Giemsa staining per- iron stores may be graded as 1+ to 4+ [3,4]. mits the easy identification of mast cells, facilitates Examination of a Perls’ stain of a bone marrow recognition of plasma cells and helps in making film allows adequate assessment of erythroblast a distinction between early erythroid cells and iron as long as a thinly spread area of the film is myeloblasts. If a Giemsa stain is not performed examined with optimal illumination. A proportion routinely, then it is important that it is used when- of normal erythroblasts have a few (one to five) fine ever necessary for these indications. Other tech- iron-containing granules randomly distributed in 51 52 CHAPTER TWO Fig. 2.1 Aspirate of normal BM: bluish-black iron in macrophages in a fragment. Perls’ stain ×377. Fig. 2.2 Aspirate of normal BM: a fragment with no stainable iron. Perls’ stain ×377. the cytoplasm (Fig. 2.3). Such erythroblasts are de- Table 2.1 Grading of bone marrow storage iron [1,2]. signated sideroblasts. In haematologically normal subjects with adequate iron stores, 20–50% of 0 No stainable iron bone marrow erythroblasts are sideroblasts [5–7]. 1+ Small iron particles just visible in reticulum cells using an oil objective Examination of an iron stain allows detection not 2+ Small, sparse iron particles in reticulum cells, only of an increased or decreased proportion of visible at lower power sideroblasts but also of abnormal sideroblasts. The 3+ Numerous small particles in reticulum cells latter include those in which siderotic granules 4+ Larger particles with a tendency to aggregate are merely increased in size and number and those into clumps in which granules are also distributed abnormally 5+ Dense, large clumps within the cytoplasm, being sited in a ring around 6+ Very large clumps and extracellular iron the nucleus rather than randomly (ring sideroblasts). SPECIAL TECHNIQUES 53 Fig. 2.3 BM aspirate from a healthy volunteer showing a normal sideroblast. Perls’ stain ×960. In certain pathological conditions, plasma cells performed on bone marrow films since there are contain haemosiderin inclusions which are irregular usually only small numbers of immature cells in the in shape and relatively large. On an MGG stain they peripheral blood. are greenish-black (Fig. 2.4a). Their nature is con- The techniques recommended for diagnosis and firmed by a Perls’ stain (Fig. 2.4b). Haemosiderin classification of acute leukaemia are either myelo- inclusions in plasma cells are observed mainly in peroxidase or Sudan black B staining, to identify iron overload (for example in haemochromatosis cells showing granulocytic differentiation, plus a and transfusional siderosis) and in chronic alco- non-specific esterase or combined esterase stain, holism [8]. to identify cells showing monocytic differentiation. Enzyme cytochemistry for either α-naphthyl butyrate esterase or α-naphthyl acetate esterase is suitable as Problems and pitfalls a ‘non-specific’ esterase staining method for the iden- Since iron is distributed irregularly within bone tification of monocytic differentiation. In a combined marrow macrophages, it is necessary to assess a esterase stain either of these methods is combined number of fragments before concluding that storage with demonstration of naphthol AS-D chloro- iron is absent or reduced. If necessary, a Perls’ stain acetate esterase (chloro-acetate esterase), the latter can be performed on more than one bone marrow to show granulocytic differentiation. The applica- film. Stain deposit on the slide must be distin- tion of these stains will be discussed in Chapter 4. guished from haemosiderin. Careful examination Other cytochemical stains which are occasionally will show that it is not related to cells and is often in used include toluidine blue to demonstrate the another plane of focus. metachromatic granules in basophils and mast cells and staining of cells of mast cell lineage for ε- aminocaproate. When immunophenotyping is avail- Other cytochemical stains able, periodic acid–Schiff (PAS) and acid phosphatase Cytochemical stains are employed mainly in the stains are redundant in the investigation of acute investigation of acute leukaemia and the myelodys- leukaemias, although differences can be observed plastic syndromes (MDS). In acute leukaemia there between different types of acute lymphoblastic may be numerous blast cells in the peripheral leukaemia (ALL) and acute myeloid leukaemia blood and it is then useful to perform cytochemical (AML) [9,10]. Either a Sudan black B or a myelo- stains on blood and bone marrow in parallel. Cyto- peroxidase stain should also be used in cases of chemical investigation of suspected MDS should be suspected MDS to facilitate detection of Auer rods. 54 CHAPTER TWO (a) Fig. 2.4 BM aspirate from a patient with chronic alcoholism showing haemosiderin in plasma cells. (b) (a) MGG ×960. (b) Perls’ stain ×960. Cytochemistry now has little place in the inves- presence of iron when none has been detected in tigation of lymphoproliferative disorders. However, an aspirate. Haemosiderin can often be detected, the demonstration of tartrate-resistant acid phos- particularly when it is increased, as golden brown phatase activity is still of value in the diagnosis of refractile pigment in an unstained or H&E-stained hairy cell leukaemia, particularly when a large section (Fig. 2.5). In a Giemsa-stained section it panel of appropriate immunophenotyping reagents is greenish-blue (Fig. 2.6). An iron stain (Fig. 2.7) is not available. can be successfully carried out using either plastic- embedded or paraffin-embedded biopsy specimens. However, plastic-embedded specimens give more reli- Histochemical stains on trephine biopsy able results. Decalcification of a paraffin-embedded sections specimen, whether by acid decalcification or by chelation, leads to some leaching out of iron. Perls’ stain for haemosiderin Plastic-embedded samples are also superior for the Because of the irregular distribution of iron within detection of ring sideroblasts or other abnormal bone marrow macrophages, a biopsy may show the sideroblasts. These can sometimes also be detected SPECIAL TECHNIQUES 55 Fig. 2.5 Section of BM showing haemosiderin within macrophages in an HIV-positive patient with iron overload. Paraffin-embedded, H&E ×376. Fig. 2.6 Section of BM trephine biopsy specimen showing haemosiderin in stromal macrophages demonstrated by Giemsa staining. A distinctive yellow–green colour is obtained that is easily visible against background haemopoietic cell staining. Paraffin-embedded, Giemsa ×940. Fig. 2.7 Section of normal BM: macrophage containing iron. Plastic-embedded, Perls’ stain ×940. 56 CHAPTER TWO Fig. 2.8 Section of trephine biopsy specimen from a child with iron overload associated with