Bone Marrow Pathology

By Barbara J. Bain David M. Clark Bridget S. Wilkins

John Wiley & Sons

Copyright © 2010 John Wiley & Sons, Ltd
All right reserved.

ISBN: 978-1-4051-6825-0

Chapter One


The distribution of haemopoietic marrow

During extra-uterine life haemopoiesis is normally confined to the bone marrow, which occupies interstices within bone. An understanding of normal bone structure is necessary for interpreting bone marrow specimens. Bones are composed of cortex and medulla. The cortex is a strong layer of compact bone; the medulla is a honeycomb of cancellous bone, the interstices of which form the medullary cavity and contain the bone marrow. Bone marrow is either red marrow, containing haemopoietic cells, or yellow marrow, which is largely adipose tissue. The distribution of haemopoietic marrow is dependent on age. In the neonate virtually the entire bone marrow cavity is fully occupied by proliferating haemopoietic cells; haemopoiesis occurs even in the phalanges. As the child ages, haemopoietic marrow contracts centripetally, being replaced by fatty marrow. By early adult life haemopoietic marrow is largely confined to the skull, vertebrae, ribs, clavicles, sternum, pelvis and the proximal half of the humeri and femora; however, there is considerable variation between individuals as to the distribution of haemopoietic marrow. In response to demand, the volume of the marrow cavity occupied by haemopoietic tissue expands.

The organization of the bone marrow


The cortex and the medulla differ functionally as well as histologically. Bone may be classified in two ways. Classification may be made on the basis of the macroscopic appearance into: (i) compact or dense bone with only small interstices that are not visible macroscopically; and (ii) cancellous (or trabecular) bone with large, readily visible interstices. Bone may also be classified histologically on the basis of whether there are well-organized osteons in which a central Haversian canal is surrounded by concentric lamellae composed of parallel bundles of fibrils (lamellar bone) or, alternatively, whether the fibrils of the bone are in disorderly bundles (woven or spongy bone). The cortex is a solid layer of compact bone that gives the bone its strength. It is composed largely of lamellar bone but also contains some woven bone. The lamellar bone of the cortex consists of either well-organized Haversian systems or angular fragments of lamellar bone, which occupy the spaces between the Haversian systems; in long bones there are also inner and outer circumferential lamellae. Extending inwards from the cortex is an anastomosing network of trabeculae, which partition the medullary space. The medullary bone is trabecular or cancellous bone; it contains lamellae but the structure is less highly organized than that of the cortex. Most of the cortical bone is covered on the external surface by periosteum, which has an outer fibrous layer and an inner osteogenic layer. At articular surfaces, and more extensively in younger patients, bone fuses with cartilage rather than being covered by periosteum. The bony trabeculae and the inner surface of the cortex are lined by endosteal cells; most of these are flattened endosteal cells that can be histologically inapparent but there are some actively osteogenic cells (osteoblasts) and occasional osteoclasts, both more numerous in children. Osteocytes are found within lacunae in bony trabeculae and in cortical bone. Although osteoblasts and osteoclasts share the surface of the bone trabeculae, they originate from different stem cells. Osteoblasts, and therefore osteocytes, are of mesenchymal origin, being derived from the same stem cell as chondrocytes and probably also stromal fibroblasts. Osteoclasts, however, are derived from a haemopoietic stem cell, being formed by fusion of cells of the monocyte lineage.

The cells that give rise to bone-forming cells are designated osteoprogenitor cells; they are flattened, spindle-shaped cells that are capable of developing into either osteoblasts or chondrocytes, depending on micro-environmental factors. Osteoblasts synthesize glycosaminoglycans of the bone matrix and also the collagenous fibres that are embedded in the matrix, thus forming osteoid or non-calcified bone; subsequently mineralization occurs. Bone undergoes constant remodelling. In adult life, remodelling of the bone takes place particularly in the subcortical regions. Osteoblasts add a new layer of bone to trabeculae (apposition) while osteoclasts resorb other areas of the bone; up to 25% of the trabecular surface may be covered by osteoid. The osteoclasts, which are resorbing bone, lie in shallow hollows, known as Howship's lacunae, created by the process of resorption, while osteoblasts are seen in rows on the surface of trabecular bone or on the surface of a layer of osteoid. As new bone is laid down, osteoblasts become enclosed in bone and are converted into osteocytes. The bone that replaces osteoid is woven bone; this, in turn, is remodelled to form lamellar bone. The difference between the two can be easily appreciated by microscopy using polarized light. The organized structure of lamellar bone, with bundles of parallel fibrils running in different directions in successive lamellae, gives rise to alternating light and dark layers when viewed under polarized light. This structure is also easily seen in Giemsa- and reticulin-stained sections.

Trephine biopsy specimens from children may contain cartilage as well as bone, and endochondrial bone formation may be observed. Transition from resting cartilage to proliferating and hypertrophic cartilage can be observed, followed by a zone of calcifying cartilage, invading vessels and bone. Mature cartilage can also be seen in trephine biopsy specimens from adults.

Other connective tissue elements

Haemopoietic cells of the bone marrow are embedded in a connective tissue stroma, which occupies the intertrabecular spaces of the medulla. The stroma is composed of fat cells and a meshwork of blood vessels, branching fibroblasts, macrophages, a few myelinated and non-myelinated nerve fibres and a small amount of reticulin. Stromal cells include cells that have been designated reticulum or reticular cells. This term probably includes two cell types of different origin. Phagocytic reticulum cells are macrophages and originate from a haemopoietic progenitor. Non-phagocytic reticulum or reticular cells are closely related to fibroblasts, adventitial cells of sinusoids (see below) and probably also osteoblasts and chondrocytes. They differ from phagocytic reticulum cells in that the majority are positive for alkaline phosphatase. There is a close interaction between haemopoietic cells and their micro-environment, with each modifying the other.

The blood supply of the marrow is derived in part from a central nutrient artery, which enters long bones at mid-shaft and bifurcates into two longitudinal central arteries. Similar arteries penetrate flat and cuboidal bones. There is a supplementary blood supply from cortical capillaries, which penetrate the bone from the periosteum. Branches of the central artery give rise to arterioles and capillaries, which radiate towards the endosteum and mainly enter the bone, subsequently turning back to re-enter the marrow and open into a network of thin-walled sinusoids. Only a minority of capillaries enter the sinusoids directly without first supplying bone. The sinusoids drain into a central venous sinusoid, which accompanies the nutrient artery. Sinusoids are large, thin-walled vessels through which newly formed haemopoietic cells enter the circulation. They are often collapsed in paraffin-embedded histological sections and are therefore not readily seen. In the presence of marrow sclerosis, these vessels are often held open and are then very obvious. The walls of sinusoids consist of endothelial cells, forming a complete cover with overlapping junctions, and an incomplete basement membrane. The outer surface is clothed by adventitial cells - large, broad cells that branch into the perivascular space and therefore provide scaffolding for the haemopoietic cells, macrophages and mast cells. Adventitial cells are thought to be derived from fibroblasts; they are associated with a network of delicate extracellular fibres, which can be demonstrated with a reticulin stain. Reticulin fibres are concentrated close to the periosteum as well as around blood vessels. It is likely that both adventitial cells and fibroblasts can synthesize reticulin, which is a form of collagen. Arterioles are easily recognized both in longitudinal section (Fig. 1.7) and in cross-section. Capillaries may also be visible. Collapsed sinusoids and capillaries are better visualized with the use of an immunohistochemical stain for an endothelial cell-associated antigen.

The marrow fat content varies inversely with the quantity of haemopoietic tissue. Fat content also increases as bone is lost with increasing age. Marrow fat is physiologically different from subcutaneous fat. The fat of yellow marrow is the last fat in the body to be lost in starvation. When haemopoietic tissue is lost very rapidly it is replaced by interstitial mucin (gelatinous transformation). Subsequently this mucin is replaced by fat cells.

Haemopoietic and other cells

Haemopoietic cells lie in cords or wedges between the sinusoids. In man, normal haemopoiesis, with the exception of some thrombopoiesis at extramedullary sites, is confined to the interstitium. In pathological conditions haemopoiesis can occur within sinusoids. Mature haemopoietic cells enter the circulation by passing transcellularly, through sinusoidal endothelial cells. The detailed disposition of haemopoietic cells will be discussed below.

Bone marrow also contains lymphoid cells, small numbers of plasma cells and mast cells (see below).

Examination of the bone marrow

Bone marrow was first obtained from living patients for diagnostic purposes during the first decade of the twentieth century, but it was not until the introduction of sternal aspiration in the late 1920s that this became an important diagnostic procedure. Specimens of bone marrow for cytological and histological examination may be obtained by aspiration biopsy, by core biopsy using a trephine needle or an electric drill, by open biopsy and at autopsy. The two most important techniques, which are complementary, are aspiration biopsy and trephine biopsy.

Bone marrow aspiration causes only mild discomfort to the patient. A trephine biopsy causes moderate discomfort and, in an apprehensive patient, sedation can be useful. Intravenous midazolam, 2-10mg, is a commonly employed agent. Guidelines for safe sedation practice must be followed. In children, aspiration and trephine biopsies are often performed under general anaesthesia.

All bone marrow aspirates and needle biopsies require informed consent. Local policies should be followed as to whether written consent is required, but this is becoming customary.

Bone marrow aspiration

Aspiration biopsy is most commonly carried out on the sternum or the ilium. Aspiration from the medial surface of the tibia can yield useful diagnostic specimens up to the age of 18 months, but is mainly used in neonates in whom other sites are less suitable. Aspiration from ribs and from the spinous processes of vertebrae is also possible but is now little practised. Sternal aspiration should be carried out from the first part of the body of the sternum, at the level of the second intercostal space. Aspiration from any lower in the sternum increases the risks of the procedure. Aspiration from the ilium can be from either the anterior or the posterior iliac crest. Aspiration from the anterior iliac crest is best carried out by a lateral approach, a few centimetres below and posterior to the anterior superior iliac spine. Approach through the crest of the ilium with the needle in the direction of the main axis of the bone is also possible but is more difficult because of the hardness of the bone. Aspirates from the posterior iliac crest are usually taken from the posterior superior iliac spine. When aspiration is carried out at the same time as a trephine biopsy it is easiest to perform the two procedures from adjacent sites. This necessitates the use of the ilium. If a trephine biopsy is not being carried out there is a choice between the sternum and the iliac crest. Either is suitable in adults and older children, although great care must be exercised in carrying out sternal aspirations. In a study of 100 patients in whom both techniques were applied, sternal aspiration was found to be technically easier and to produce a suitable diagnostic specimen more frequently, although on average the procedure was more painful, both with regard to bone penetration and to the actual aspiration. Sternal aspiration is also more dangerous (see below), and is unsuitable for use in young children. Posterior iliac crest aspiration is suitable for children, infants and many neonates. Tibial aspiration is suitable for very small babies but has no advantages over iliac crest aspiration in older infants.

Bone marrow specimens yielded by aspiration are suitable for the following: preparation of wedge-spread films and films of crushed marrow fragments; study of cell markers (by flow cytometry or on films or cytospin preparations); cytogenetic study; ultrastructural examination; culture for microorganisms; culture to study haemopoietic precursors; and the preparation of histological sections of fragments. Cytogenetic analysis is most often indicated in suspected haematological neo-plasms but it also peermits rapid diagnosis of supected congenital karyotypic abnormalities such as trisomy 18; diagnosis is possible within a day, in comparison with the 3 days needed if peripheral blood lymphocytes are used.

Bone marrow aspiration may fail completely, this being referred to as a 'dry tap'. Although this can happen when bone marrow histology is normal, a dry tap usually indicates significant disease, most often metastatic cancer, chronic myeloid leukaemia, primary myelofibrosis or hairy cell leukaemia, with associated fibrosis. On other occasions only blood is obtained (a 'blood tap'); this is often also the result of bone marrow disease causing fibrosis.

Trephine biopsy of bone marrow

Trephine or needle biopsy is most easily carried out on the iliac crest, either posteriorly or anteriorly, as described above. The posterior approach appears now to be more generally preferred. If a trephine biopsy and a bone marrow aspiration are both to be carried out, they can be performed through the same skin incision but with two areas of periosteum being infiltrated with local anaesthetic and with the needle being angled in different directions. A single-needle technique in which aspiration is followed by core biopsy should not be used as the quality of the core biopsy may be inadequate. Core biopsy specimens, obtained with a trephine needle, are suitable for histological sections, touch preparations (imprints) and electron microscopy. A touch preparation is particularly important when it is not possible to obtain an aspirate since it allows cytological details to be studied. In addition, touch preparations may show more neoplastic cells than are detected in an aspirate; they may also demonstrate bone marrow infiltration when it is not detected in an aspirate, for example in hairy cell leukaemia, multiple myeloma or lymphoma. Touch preparations may be made either by touching the core of bone on a slide or rolling the core gently between two slides. Biopsy specimens can be used for cytogenetic study but aspirates are much more suitable. Frozen sections of trephine biopsy specimens are possible but they are not usually very satisfactory because of technical problems, including difficulty in cutting sections, poor adhesion of sections to glass slides during staining procedures and poor preservation of morphological detail. They are rarely used now that immunohistochemistry can be readily applied to fixed tissues. Histological sections may be prepared from fixed biopsy specimens which have either been decalcified and paraffin-embedded or have been embedded in resin without prior decalcification.


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