Because bone marrow clots faster than peripheral blood, films should be made from the aspirated material without delay at the bedside. The remainder of the material may then be delivered into a bottle containing an appropriate amount of ethylenediaminetetra-acetic acid (EDTA) anticoagulant and used later to make more films. Preservative-free heparin should be used rather than EDTA if immunophenotyping or cytogenetic studies are needed. Some material can be preserved in fixative rather than anticoagulant for preparation of histological sections (see p. 133). Fix some of the films in absolute methanol as soon as they are thoroughly dry for subsequent staining by a Romanowsky method or Perls’ stain for iron. Appropriately fixed films are also suitable for cytochemical staining (Chapter 15). If there has been a ‘dry tap’, insert the stilette into the needle and push any material in the lumen of the needle onto a slide and spread it; in lymphomas and carcinomas, especially, sufficient material may be obtained using this approach to allow a diagnosis.⁹ Squash preparations of marrow fragments can be a useful supplement to bone marrow films. A drop of aspirated marrow is placed in the centre of a slide and, unless the aspirate is very cellular, the fragments are concentrated by removing the more dilute part of the aspirate with a plastic pipette. A second slide is then placed on top of the first and the fragments are crushed by rotating one slide on the other. Both squashes are then fixed and stained. Bone marrow aspirates in adults can be performed from the ilium, the sternum or the spinous processes; the latter site is rarely used and the procedure is described in the 10th edition of this book.

Puncture of the Ilium

The usual sites for puncture in adults are the posterior and, less commonly, the anterior iliac spine. If serial punctures are being performed, a different site should be selected for each to avoid aspirating marrow that has been diluted by intramedullary haemorrhage resulting from previous punctures. The posterior iliac spine overlies a large marrow-containing area and relatively large volumes of marrow can be aspirated from this site. Posterior iliac puncture can be carried out with the patient lying sideways, as for a lumbar puncture, or prone. The anterior superior iliac spine may be easier to locate in individuals who are very obese and the bone overlying it is said to be thinner than that of the iliac crest.

Puncture of the Sternum

The specific risks of sternal marrow aspiration were discussed earlier (see p. 124). Puncture of the sternum must be performed with care to avoid pushing the aspiration needle through the bone. The usual site for puncture is the manubrium or the first or second parts of the body of the sternum. The manubrium is formed of rather denser bone than the body of the sternum and, in elderly subjects at least, it tends to contain more fatty marrow than is found elsewhere in the sternum. The thickness of the cortex here varies from 0.2 mm to 5.0 mm, so it may be difficult to be certain that the needle point has reached the cavity of the bone.

The site for puncture of the manubrium should be about 1 cm above the sterno-manubrial angle and slightly to one side of the midline; if the body of the bone is to be punctured, this should be done opposite the second intercostal space slightly to one side of the midline. It is essential to use a needle with a guard that cannot slip, such as a Klima type. After piercing the skin and subcutaneous tissues, when the needle point reaches the periosteum, adjust the guard on the needle to allow it to penetrate about 5 mm further. If the guard cannot be advanced to this extent, it is not safe to proceed. Push the needle with a boring motion into the cavity of the bone. It is usually easy to appreciate when the cavity of the bone has been entered. Aspiration is then carried out as described earlier.

Comparison of Different Sites for Marrow Puncture

There is considerable variation in the composition of cellular marrow withdrawn from adjacent or different sites. Aspiration from only one site may give misleading information; this is particularly true in aplastic anaemia since the marrow may be affected patchily.¹⁰ In general, however, the overall cellularity, the haemopoietic maturation pathways and the balance between erythropoiesis and leucopoiesis are similar at all sites. In practice, it is an advantage to have a choice of several sites for puncture, particularly when puncture at one site results in a ‘dry tap’ or when only peripheral blood is withdrawn. Aspiration at a different site may yield cellular marrow or strengthen suspicion of a widespread change affecting the bone marrow, such as fibrosis or hypoplasia. In aplastic anaemia, several punctures or, much to be preferred, a trephine biopsy may be necessary to arrive at the diagnosis.

Aspiration of the bone marrow in children

Iliac puncture, particularly in the region of the posterior spine, is usually the method of choice in children. Occasionally, in an older child who is obese, the posterior iliac spine cannot be felt. In this case, a satisfactory sample usually can be obtained from the anterior ilium. In small babies, marrow can be withdrawn from the medial aspect of the upper end of the tibia just below the level of the tibial tubercle. This site should be used with caution because it is vulnerable to fractures and laceration of the adjacent major blood vessels. In older children, the tibial cortical bone is usually too dense and the marrow within is normally less active. It must be remembered that sternal puncture in children should be avoided because the bone is thin and the marrow cavities are small.

Marrow puncture needles

Needles should be stout and made of hard stainless steel, about 7–8 cm in length, with a well-fitting stilette and they must be provided with an adjustable guard. With reusable needles, the point of the needle and the edge of the bevel must be kept well sharpened. The most common reusable needles are the Salah and Klima needles (Fig. 7.2). A slightly larger needle with a T-bar handle at the proximal end was developed by Islam (Fig. 7.3); it provides a better grip, is more manoeuvrable and is more successful for biopsies of excessively hard (e.g. osteosclerotic) or soft (e.g. profoundly osteoporotic) bone.¹¹ A modified version of the Islam needle has multiple holes in the distal portion of the shaft in addition to the opening at the tip to overcome sampling error when the marrow is not uniformly involved in a pathological lesion. Several types of disposable bone marrow aspiration and trephine biopsy needles are now available; their design is similar to the traditional reusable needles (Fig. 7.4). The increasing use of disposable needles by haematologists is based on considerations of safety for patient and operator.

Figure 7.2 Marrow puncture needles. Salah (A) and Klima (B).

Figure 7.3 Islam’s bone marrow aspiration needle. The dome-shaped handle and T-bar are intended to provide stability and control during operation.

Figure 7.4 Disposable bone marrow needles. For trephine biopsy (A) and aspiration (B).

Processing of aspirated bone marrow

There is little advantage in aspirating more than 0.3 ml of marrow fluid from a single site for morphological examination because this increases peripheral blood dilution. If large amounts of marrow are needed for several tests, such as immunophenotyping, cytogenetic analysis and molecular studies, the syringe can be detached from the aspiration needle and the stilette can be replaced, leaving the aspiration needle in the bone. After the marrow smears have been prepared, the same or another syringe can be attached to the needle and another 5–10 ml of marrow can be aspirated.

It is good practice to obtain a sample of peripheral blood from the patient at the same time as the bone marrow so that films from both specimens can be examined and stored together. This can be done simply by preparing some films from blood obtained from a finger prick after completing the bone marrow sampling or by venepuncture so that a full blood count can be obtained. The blood film should be permanently stored with the bone marrow films.

Preparing Films from Bone Marrow Aspirates

Make films, 3–5 cm in length, of the aspirated marrow using a smooth-edged glass spreader of not more than 2 cm in width (Fig. 7.5). The marrow fragments are dragged behind the spreader and leave a trail of cells behind them. Spreading should be towards the area to which the label is to be applied to avoid having particles dragged to the tip of the slide, where it is difficult to examine them. If there are insufficient fragments, they can be concentrated. This is not usually necessary for marrows that are very cellular such as in acute and chronic myelogenous leukaemia and megaloblastic anaemia. Concentration of marrow can be achieved by delivering single drops of aspirate onto slides about 1 cm from one end. Most of the blood is quickly sucked off from the edge of the drop with the marrow syringe or a fine plastic pipette. The irregularly shaped marrow fragments tend to be left behind on the slide and can be lifted off with the spreader; films can then be prepared as explained earlier.

Figure 7.5 Film of aspirated bone marrow. The marrow particles are easily visible, mostly at the tail of the film (×1.5). Note that the bone marrow has been spread towards the label. This ensures that the area that is optimal for examination is easily positioned beneath the objective when the slide is placed on the stage.

After thorough drying, fix the films of bone marrow and stain them with Romanowsky dyes, as for peripheral blood films. However, a longer fixation time (at least 20 min in methanol) is essential for high-quality staining. If a film needs to be stained urgently, fix and stain one film only and permit the others to dry thoroughly. This avoids having all films showing artefacts caused by fixation of slides before thorough drying has been achieved. Films can also be stained by Perls’ method (see p. 334) to demonstrate the presence or absence of iron.

The preparation can be considered satisfactory only when marrow particles and free marrow cells can be seen in stained films. It is in the cellular trails that differential counts should be made, commencing from the marrow fragment and working back toward the head of the film; in this way, smaller numbers of cells from the peripheral blood are included in a differential count.

When the aspirated marrow is put into an anticoagulant in a tube (e.g. EDTA) care should be taken that appropriate amounts are used for the volume of marrow to be anticoagulated. When films of marrow containing a gross excess of anticoagulant are spread (as when a few drops of marrow are added to a tube containing sufficient EDTA to prevent the clotting of 5 ml of blood), masses of pink-staining amorphous material may be seen and some of the erythroblasts and reticulocytes may clump together.

Concentration of Bone Marrow by Centrifugation

Centrifugation can be used to concentrate the marrow cells and to assess the relative proportions of marrow cells, peripheral blood and fat in aspirated material. While concentration of poorly cellular samples is useful, especially when an abnormal cell is present in small numbers,¹² it is unnecessary when the aspirated material is of average or increased cellularity. Volumetric data, too, are of little value in individual patients because of the wide range of values encountered even in health. Methods for separation of marrow cells are described on p. 65.

Preparation of Films of Post-mortem Bone Marrow

Films made of bone marrow obtained at post mortem are seldom satisfactory. If satisfactory results are to be achieved, the procedure must be carried out as soon after death as possible. When the marrow is spread in the ordinary way, the majority of the cells tend to break up and appear as smears. The rate and pattern of cellular autolysis during the first 15 h after death has been studied and the differences between the changes of post-mortem autolysis and those that occur in life as a result of blood diseases have been defined.¹³ Blood cells are much better preserved if a small piece of marrow is suspended in 1–2 ml of 5% bovine albumin (1 volume 30% albumin, 5 volumes 9 g/l NaCl). The suspension is then centrifuged and the deposited marrow cells are resuspended in a volume of supernatant approximately equal to, or slightly less than, that of the deposit. Films are made of this suspension in the usual way.

Examination of aspirated bone marrow

Principles of Marrow Aspirate Examination

Aspirated bone marrow material spread on glass slides yields individual separate bone marrow cells that have not been subject to prior processing or been cut to form sections. The specimen may therefore be examined using an oil-immersion lens to allow excellent assessment of cytological detail. Marrow aspiration therefore has particular value where recognition of individual cells or abnormal cytological features is paramount or where individual cells need to be recognized, classified and counted. Examples where bone marrow aspiration is of major value include the cytological assessment of abnormal cell maturation of bone marrow cells or the cytological classification and numerical assessment of acute leukaemia (Fig. 7.6). Aspirated bone marrow cells are also well suited to further examination by cytogenetic, molecular or flow cytometric methods. However, the aspiration process generally disrupts structural elements within the marrow, preventing assessment of the structural relationships of normal or abnormal elements within marrow, and loose structural features such as lymphoid aggregates or granulomata are often not detected. The bone marrow aspirate may also be misleading if the cells of interest do not ‘spill’ from marrow particles and therefore do not appear on the slides. This problem is most readily apparent when marrow is subject to fibrotic processes; for example, the reticulin fibrosis of hairy cell leukaemia or the collagenous fibrosis of established myelofibrosis. An aspirate is also frequently insufficient when infiltrating cells form cohesive structures. Clumps of cells from carcinoma may on occasion be seen in aspirates, but are usually better revealed by biopsy.

Figure 7.6 (A) Aspirated cells from a case of megaloblastic anaemia. (B) Marrow aspirate spread of acute myeloid leukaemia arising from myelodysplastic syndrome. The films each illustrate very abnormal cells (megaloblastic or dysplastic, respectively) within the mature or maturing compartments. These cells are best recognized by their characteristic abnormal cytology.

Quantitative Cell Counts on Aspirated Bone Marrow

A number of values for the cell content of aspirated normal bone marrow have been given in the literature.^14,¹⁵ The percentage of marrow in the sternum of healthy adults that is cellular rather than fatty is 48–79%. However, quantification of the cell content of aspirated marrow is not reliable in view of the tendency of the marrow to be aspirated in the form of particles of varying size as well as free cells and the uncontrollable factor of dilution with peripheral blood, which according to some authors may amount to 40–100% in 0.25–0.5 ml bone marrow samples.

Quantitative cell counts on aspirated marrow are therefore difficult to interpret. For practical purposes, the degree of marrow cellularity can be assessed within broad limits as increased, normal or reduced by inspection of a stained film containing marrow particles. As a rough guide, if less than 25% of the particle is occupied by haemopoietic cells, it is hypocellular and if more than 75–80% is occupied, it is hypercellular. Less subjective quantitative measurement can be obtained by ‘point counting’ of sections; a normal range of 30–80% has been reported in the anterior iliac spine.

Physiological variation in the cell content has to be taken into account. The cellularity of the marrow is affected by age. In adults, a smaller proportion of the marrow cavity is occupied by haemopoietic marrow than in children and the proportion of fat cells to cellular marrow is increased. In one study, by means of point counting of sections from the iliac crest, the range of cellularity in children younger than 10 years was reported as 59–95% with a mean of 79%; at 30 years, the mean was 50% and at 70 years it was 30% with a range of 11–47%. The decrease in cellularity in elderly subjects is even more marked in the manubrium sterni. The marrow undergoes slight to moderate hyperplasia in pregnancy.

Differential Cell Counts on Aspirated Bone Marrow

For general purposes, it is not usually necessary to document the proportion of every stage of each cell type on the marrow slide. A 200–500-cell differential using the categories erythroid, myeloid, lymphoid and plasma cells is generally adequate providing that a systematic scheme for examining the morphology of these, and all other, cells is also used. In some conditions, such as acute and chronic myelogenous leukaemia and myelodysplastic syndrome, detailed differential counts are important because precise counts are essential for diagnosis and the results may indicate prognosis and affect treatment. Occasionally, it may be important to specifically count one cell type (e.g. blasts in acute leukaemia for assessing response to chemotherapy). Follow-up bone marrows should always be compared with previous bone marrow films to assess the course of a disease or the effect of treatment.

Sources of Error and Physiological Variations

Because of the naturally variegated pattern of the bone marrow, the irregular distribution of the marrow cells when spread in films and the variable amount of dilution with blood, differential cell counts on marrow aspirated from normal subjects vary widely. Aspirating only a small volume and counting cells in the trails left behind marrow particles as they are spread on the slide minimizes the dilutional effect of blood. When there is an increase in associated reticulin, some cell types may resist aspiration or remain embedded in marrow fragments and will therefore be under-represented in the differential count. Megakaryocytes in particular are irregularly distributed and tend to be carried to the tail of the film. The chance aspiration of a lymphoid follicle would result in an abnormally high percentage of lymphocytes.¹⁶

Ideally, differential counts should be performed on sectioned material, but difficulties in identification make this impractical. Methacrylate embedding offers a better opportunity for correctly identifying cells. The incidence of the various cell types is usually expressed as percentages. The normal values for cell differentials in bone marrow (Table 7.1) can only be taken as an approximate guide.^14,¹⁵ The cellular composition of the bone marrow varies between normal infants, children and young adults. Variation is marked in the first year, particularly in the first month. The percentage of erythroblasts decreases from birth and at 2–3 weeks they constitute only about 10% of the nucleated cells. Myeloid cells (granulocyte precursors) increase during the first 2 weeks of life, following which a sharp decrease occurs at about the 3rd week; however, by the end of the 1st month about 60% of the cells are myeloid. Lymphocytes constitute up to 40% of the nucleated cells in the marrow of small infants; the mean value at 2 years is approximately 20%, falling to about 15% during the rest of childhood. The percentage of plasma cells is especially low from infancy up to the age of 5 years.

Table 7.1 Normal ranges for differential counts on aspirated bone marrow

The hyperplasia that occurs in pregnancy affects both erythropoiesis and granulopoiesis, the latter proportionately less, although with some increase in the relative proportion of immature cells. The hyperplasia is maximal in the 3rd trimester; a return to normal begins in the puerperium but is not completed until at least 6 weeks’ postpartum.

Cellular Ratios

Ratios based on a count of 200–500 cells can provide useful qualitative information. The myeloid:erythroid ratio has been widely used and is the ratio of neutrophil and neutrophil precursor cells to erythroid precursors. The inclusion of monocytes, eosinophils and basophils is controversial but in practice makes little difference to the overall ratio, which varies from 2:1 to 4:1. As an alternative, the leuco-erythrogenetic ratio can be calculated. For this, mature cells are excluded; the normal ratio has been reported as 0.56–2.67:1. The myeloid:lymphoid ratio varies widely, 1–17:1 and the lymphoid:erythroid ratio has a similarly wide variation, 0.2–4.0:1.

Reporting bone marrow aspirate films

A systematic examination of the marrow aspirate, combined with knowledge of the clinical context, provides the best chance of arriving at a diagnosis.³ Choose several of the best-spread stained films that contain easily visible marrow particles. Several particles should then be examined with a low-power (×10) objective to estimate whether the marrow is hypocellular, normocellular or hypercellular. Megakaryocytes and clumps of non-haemopoietic cells (e.g. metastatic carcinoma cells) should be looked for at this stage of the examination; they are most often found toward the tail of the film.

Select for detailed examination – still using the ×10 objective – a highly cellular area of the film where the nucleated cells are well stained and well spread. Areas such as these can usually be found toward the tails of films behind marrow particles. The cells in these cellular areas should then be examined with a higher-power (e.g. ×40) objective and, subsequently, if necessary, with the ×100 oil-immersion objective. It is important always to examine marrows in a systematic fashion because it is easy to overlook subtle abnormalities. A suggested scheme for this is outlined below.

Systematic Scheme for Examining Bone Marrow Aspirate Films

Low Power (×10)

Determine cellularity by examining several particles.

Identify megakaryocytes and note morphology and maturation sequence (higher power may be needed for smaller immature megakaryocytes and micromegakaryocytes).

Look for clumps of abnormal cells that could indicate infiltration by metastatic tumour (higher power may be needed to examine content and morphology of clumps).

Identify macrophages and examine at higher power for evidence of haemophagocytosis, malaria pigment and bacterial or fungal organisms that may be present in the cytoplasm.

Higher Power (×40, ×100 Oil-Immersion)

Identify all stages of maturation of myeloid and erythroid cells. This is usually easiest to achieve by starting with mature red cells and working backward to the most immature cells. Repeat the process for the myeloid series starting with mature neutrophils. Maturation abnormalities, such as giant proerythroblasts or evidence of dysfunctional maturation, including nuclear–cytoplasmic asynchrony, will suggest specific diagnoses such as parvovirus B19 infection, myelodysplastic syndrome or megaloblastic anaemia, respectively. Changes in the proportion of primitive to mature myeloid cells may reflect response to treatment in leukaemia or recovery from agranulocytosis. The actual percentage of blast cells is of significance in the differentiation of myelodysplastic syndromes from acute leukaemia, in determining prognosis in myelodysplastic syndrome and in assessing whether chronic myelogenous leukaemia is in chronic, accelerated or acute phase.

Determine the myeloid:erythroid ratio. Whereas a lack of myeloid cells may be obvious without performing a formal differential count, it is easy to overlook an increase in erythroid cells, which might suggest blood loss or peripheral destruction.

Perform a differential count using the categories erythroid, myeloid, lymphoid, plasma cell and ‘others’, simultaneously noting any morphological abnormalities. The normal lymphocyte percentage in the marrow is 5–20%; moderate increases to 30–40%, which may indicate a significant disorder such as lymphoma, are not likely to be identified simply by rapidly surveying the slide. Plasma cells should be less than 2%; in plasma cell dyscrasias, they may be increased, occur in clumps or have an abnormal morphological appearance.

Look for areas of bone marrow necrosis.¹⁷ In necrotic areas, the cells stain irregularly, with blurred outlines, cytoplasmic shrinkage and nuclear pyknosis. Bone marrow necrosis may occur in sickle cell disease; it also occurs occasionally in lymphomas, acute lymphoblastic and chronic lymphocytic leukaemia, myeloproliferative neoplasms and metastatic carcinoma, as well as in septicaemia, tuberculosis and anorexia nervosa.¹⁸ In patients with anorexia nervosa or cachexia, there may be gelatinous transformation of the ground substance of the marrow.¹⁸

Assess the iron content of macrophages and look for iron granules in erythroid cells on a slide stained with a Perls’ stain. At least seven particles should be examined to optimally assess a bone marrow aspirate for iron stores.¹⁹ If fewer particles are available, a diagnosis of iron deficiency can only be tentative. In sideroblastic anaemia, the granules incompletely encircle the nucleus. Abnormal patterns of iron staining may also be seen in dyserythropoietic anaemias such as the thalassaemias.

Reporting Results

It is helpful to report bone marrow films on a printed form on which the report and conclusion can be set out in an ordered fashion (Fig. 7.7).³ Where a computerized reporting system is in use, it is useful to have a template with headings to ensure that the marrow reports are systematic and consistent. A list of the various descriptive comments that may be used can be provided in coded form to facilitate data entry. Report summaries should be intelligible to clinicians who are not haematology specialists.

Figure 7.7 Example of report form for bone marrow films.

Preparation of sections of aspirated bone marrow fragments

If it is not possible to obtain a trephine biopsy (see below), the small fragments obtained by marrow aspiration can be fixed, stained and examined to contribute to diagnosis. Such samples are useful for assessing cellularity and for detecting granulomas and tumour cells. If sections are required, it is convenient to let residual aspirated marrow clot within a plastic syringe and tease the sample out into the fixative once it has clotted.

Percutaneous trephine biopsy of the bone marrow

Like marrow aspirations, trephine biopsy may be carried out in the inpatient ward or in outpatient departments. The posterior iliac spine is the usual site, although the anterior iliac spine can also be used. The posterior iliac spine is said to provide samples that are longer and larger and the aspiration is less uncomfortable for the patient.²⁰

The trephine specimen is obtained by inserting the biopsy needle into the bone, using a to-and-fro rotation and then rocking the needle gently from side to side to detach a core of tissue that can be extracted within the needle. The main problems with this method are that the specimen may be crushed, thereby distorting the architecture, and it can be difficult to detach the core of bone from inside the marrow space. Trephine biopsy needles, both reusable and disposable, have been specifically designed to overcome these problems. The Jamshidi needle has a tapering end to reduce crush artefact (Fig. 7.8) and the Islam trephine has a core-securing device (Fig. 7.9). If larger specimens are needed, trephine needles that have bores of 4–5 mm may be used. Other needles occasionally used for trephine biopsy specimens are a 2 mm bore ‘microtrephine’ needle and a Vim-Silverman needle. However, compared with other needles, these yield smaller specimens of marrow that are prone to fracturing.

Figure 7.8 Jamshidi trephine needle for bone marrow biopsy.

Figure 7.9 Islam trephine needle for bone marrow biopsy. The distal cutting edge is shaped to hold the core secure during extraction of the material.

For the investigation of thrombocytopenia and neutropenia in neonates, sections of aspirated bone marrow can be obtained that allow assessment of marrow cellularity and architecture.²¹ A 19G, half-inch Osgood needle (Cadence Inc, New York) is introduced 2 cm below the tibial tuberosity. The trocar is removed and the hollow needle is advanced by twisting 2–3 mm into the marrow space. A syringe is used to apply suction to the needle until marrow appears; then the needle and syringe are withdrawn. The marrow clot is gently dislodged with the tip of a needle and placed into fixative. The specimen is processed as if it were an adult biopsy except that decalcification is not required.

Principles Behind Marrow Trephine Biopsy Examination

Bone marrow trephine biopsy provides a core of tissue, which is fixed and embedded to yield a histological specimen in which the structural relationships between cells, bone and bone marrow stroma are preserved. The nature of the specimen allows cellular distributions to be recognized: for example, the tendency for precursor myeloid cells to mature close to bony trabeculae or the normal development of erythroid cells in the form of small islands (Fig. 7.10). Bone marrow biopsy also delineates the abnormal distribution of cells that is characteristic of particular pathological conditions. For example, the paratrabecular collection of abnormal cells that is typical of follicular lymphoma or the clustered megakaryocytes seen in certain myeloproliferative neoplasms (Fig. 7.11). In addition, since a trephinebiopsy specimen is a core of tissue, all cells present within the specimen will be represented irrespective of fibrosis or cohesion. Cell types within a bone marrow biopsy may be recognized by cytological characteristics, but that identification is reinforced by their distribution within the biopsy. Cellular recognition on trephine biopsy sections is frequently supplemented by immunostaining in which monoclonal or polyclonal antibodies are used (with cytochemical reactions) to detect a range of lineage, maturational or cell-type restricted markers. Such markers allow confirmation of cell lineage or of maturation stage within a particular lineage (Fig. 7.12). Markers may also highlight abnormal distribution of cells: for example, markers of B-cell lineage may highlight infrequent paratrabecular aggregates of cells in cases of follicular lymphoma. Specific cell markers may highlight infrequent abnormal cells that represent residual or recurrent disease (e.g. CD72 (DBA.44) can be used to highlight neoplastic cells in hairy cell leukaemia). Finally, since trephine biopsy tissue is fixed and preserved, additional tests may be performed some time after the specimen was obtained.

Figure 7.10 Areas of bone marrow trephine biopsy stained with H&E showing an erythroid island (upper left), maturing myeloid lineage cells (lower left) and paratrabecular area with early precursor cells (right).

Figure 7.11 Bone marrow trephine biopsy showing increased and abnormally clustered megakaryocytes.

Figure 7.12 Immunostaining of marrow infiltrated by plasma cells highlighting the abnormal cells.

Imprints from Bone Marrow Trephine Biopsy Specimens

Whenever a trephine biopsy is obtained, imprints can be taken before the specimen is transferred into fixative. This is particularly useful if the bone marrow aspirate is inadequate. The bony core is gently dabbed or rolled across the slide, which is then fixed and stained as for bone marrow smears (see p. 61). This allows immediate examination of cells that fall out of the specimen onto the slide and may provide a diagnosis several days before the trephine biopsy specimen has been processed.

Processing of Bone Marrow Trephine Biopsy Specimens

The specimen should be fixed in 10% formal saline, buffered to pH 7.0, for 12–48 h prior to decalcifying, dehydrating and embedding in paraffin wax by the usual histological procedures. Cell shrinkage and distortion from the decalcification process may obscure cellular detail. These disadvantages can be overcome by methyl methacrylate (‘plastic’) embedding (Fig. 7.13). Details of the preparation of sections of bone marrow biopsies can be found in Bain et al.²²

Figure 7.13 Photomicrograph of section of normal bone marrow. Iliac crest biopsy. Methacrylate embedding. Stained by May–Grünwald–Giemsa.

Staining of Sections of Bone Marrow Trephine Biopsy Specimens

Bone marrow sections should be routinely stained with haematoxylin and eosin (H&E) and a silver impregnation method for reticulin. Sections can also be stained for iron by Perls’ reaction. H&E staining is excellent for demonstrating the cellularity (Fig. 7.14) and pattern of the marrow and for revealing pathological changes such as fibrosis or the presence of granulomata or carcinoma cells. Haemopoietic cells may be more easily identified in a Romanowsky-stained preparation. Both paraffin- and plastic-embedded specimens are suitable for immunohistochemistry.

Figure 7.14 Photomicrographs of sections of bone marrow. Iliac crest bone marrow illustrating the range of cellularity. (A) hypocellular; (B,C) normal cellularity; (D) hypercellular.

Silver impregnation stains the glycoprotein matrix, which is associated with connective tissue. The bone marrow always contains a small amount of this material, which is referred to as ‘reticulin’ and is an early form of collagen. The normal reticulin content of iliac bone marrow is shown in Figure 7.15A. An increase in marrow reticulin appears as an increase in the number, thickness and length of fibres (Fig. 7.15B). Increased reticulin deposition can occur in myeloproliferative neoplasms, particularly those associated with proliferation of megakaryocytes and in lymphoproliferative disorders, secondary carcinoma with marrow infiltration, osseous disorders such as hyperparathyroidism and Paget’s disease and inflammatory reactions.²³ In primary myelofibrosis, a more ‘mature’ form of collagen is present, which, unlike reticulin, is visible on H&E staining (Fig. 7.16).