Normal bone marrow – generalities and function

The term ‘bone marrow’ (BM) refers to the tissue occupying the cavities under the cortex within the honeycomb of trabecular bone. Normal marrow is either red, consisting of the hematopoietic tissue, or yellow, composed mainly of fat cells (adipose tissue). In children most bones contain hematopoietic marrow, almost to the exclusion of fat cells. In the adult, red marrow is found in the skull, sternum, scapulae, vertebrae, ribs, pelvic bones and the proximal ends of the long bones (e.g. femora and humeri). The hematopoietic marrow produces the mature blood cells, which have a finite life span and must be constantly replaced (see Chapter 2). The weight of the total BM is 1600–3700 g. The BM is composed of:

• the parenchyme: the hemopoietic cells including precursors and mature cells of the erythroid, myeloid and megakaryocytic lineages, i.e. various stages of red cells, white cells, megakaryocytes, lymphocytes, plasma cells and mast cells

• the stroma: fat cells, histiocytes/macrophages, fibroblasts, blood vessels and intercellular matrix.

The BM can be assessed in aspirated material and in biopsied tissue. This chapter will describe the histology of BM in the trephine biopsy.

Bone marrow structure

The constituents of the normal BM are closely packed within a hard bony ‘container’. Hemopoiesis occurs in the intertrabecular space within marrow cavities. The bony trabeculae (cancellous bone) are lined by endosteum, osteoblasts and osteoclasts. The stromal elements form an extensive, closely woven network in which the hematopoietic precursors are embedded, attached in various ways and to different components by the adhesive proteins and by other cells, such as the central macrophages in the erythroid islands. Hematopoietic precursors receive their nutrients, vitamins, hormones, regulatory factors, cytokines and modulators through the extracellular matrix (ECM), which also contributes to the regulation of the cell cycle, cellular differentiation and apoptosis. The blood supply to the BM consists of two systems: periosteal arteries, which give off branches to the BM after they penetrate the bone, and nutrient arteries. Blood drains from the BM cavity through central veins. The BM receives approximately 2–4% of cardiac output. The microvasculature of the BM comprises a network of sinusoids. Hemopoiesis only occurs in the interstital space between these sinusoids, thereby ensuring that hemopoietic progenitors are located close to the blood supply. Normal BM contains a network of fine branching reticulin fibers between parenchymal cells, which provide the extracellular matrix for the BM. There is a higher concentration of thicker fibers around arterioles and near the endosteum. The BM also has a nerve supply.

Bone marrow trephine biopsy

The process of obtaining a bone marrow trephine biopsy (BMTB) originates in the ancient procedure of trepanning.¹ Prior to the advent of BMTB needles, clot preparations of aspirated marrow were prepared for diagnostic purposes. BM biopsies were obtained only as a means of diagnosis if marrow was inaspirable, a ‘dry tap’. The modification of needles by Jamshidi in the 1970s revolutionized the process of obtaining intact cores of bone and bone marrow for examination, primarily from the pelvis. The most common site biopsed is the posterior superior iliac crest. Other sites which can be biopsied include the anterior superior iliac crest, tibia, and vertebrae. Biopsy of the sternum is contraindicated due to the significant morbidity and mortality associated with this practice. The optimal length of a BMTB is 2–3 cm; shorter biopsies may not be representative and may not detect diseases that have a focal or patchy pattern of BM involvement.²

Processing and stains

A number of methods are available for fixation, decalcification and embedding of the BMTB ² (Table 3.1). For optimal evaluation, sections are cut at a thickness of 1–3 µm. Conventional stains are hematoxylin and eosin (H&E) (Fig. 3.1A) and Giemsa (Fig. 3.1B) stains. Giemsa staining generally gives better discrimination of cell types based on cytoplasmic staining characteristics, that is:

Table 3.1 The bone marrow trephine biopsy: processing techniques

Fig. 3.1 (A, B) Normal bone marrow biopsy showing normal marrow cellularity and architecture. (A) Hematoxylin & Eosin stain. × 200. (B) Giemsa stain. × 400.

1. myeloid/eosinophil granules: red

2. mast cell granules: purplish red

3. erythroid precursor cytoplasm: blue

4. plasma cell cytoplasm: purplish blue

5. perinuclear Golgi complex (hof) also seen as a pale area.

Other stains commonly used include the Perls stain for iron, silver stains for reticulin and Masson’s trichrome stain or Martius scarlet blue stain for collagen. Immunohistochemistry (IHC) using monoclonal or polyclonal antibodies, and in situ hybridization using nucleic acid probes can be used to identify specific cell types and genetic aberrations; these are discussed in sections below.³

Marrow cellularity

The BMTB is particularly useful for the assessment of marrow cellularity. This is the relative amount of BM cells to adipocytes, which is assessed subjectively and should be interpreted in the context of the age of the patient. The terms normocellular (normal for age), hypercellular (increased cellularity for age) and hypocellular (reduced cellularity for age) are used. Cellularity reduces with increasing age (Table 3.2).^4,⁵ In practice, the formula (cellularity = 100 − patient age) can be applied for adults; however, it does not correlate with cellularity at the extremes of the age range. The intertrabecular spaces adjacent to the marrow cortex tend to be hypocellular and should not be assessed when determining overall BM cellularity.

Table 3.2 Cellularity ranges for various age groups

Age	Cellularity
Newborn to 3 months	80–100%
Childhood	60–80%
20–40 years	60–70%
40–70 years	40–50%
>70 years	30–40%

Marrow architecture

The BMTB enables the assessment of bone marrow architecture, the distribution of cellular elements and the bone and stromal cells. The outermost elements of the biopsy are composed of collagenous periosteal connective tissue, followed by a zone of cartilage or cortical bone (depending on the age of the patient). After this the bone breaks up into a meshwork of trabeculae, between which are the intertrabecular spaces. Hemopoietic cells are present within these intertrabecular spaces and are supported by fat cells, stromal cells, histiocytes extracellular matrix and blood vessels (Fig. 3.2). The hemopoietic cells are located within the intertrabecular spaces. The intertrabecular areas can be divided into three zones which contain different hemopoietic cell types (Fig. 3.3):

Fig. 3.2 (A, B) Low power view of a bone marrow biopsy. (A) Periosteal connective tissue is seen external to cortical bone. (B) Bone trabeculae with the intertrabecular spaces (one marked – arrow) containing hemopoietic cells and fat cells. × 40.

Fig. 3.3 Organization of the bone marrow: zones and distribution of various cell types. × 100.

1. Endosteal or paratrabecular zone: immediately adjacent to the trabecular bone and composed predominantly of myeloid precursor cells

2. Intermediate zone: contains erythroid colonies and maturing myeloid cells

3. Central zone: in the center of the intertrabecular space. In addition to erythroid cells and maturing myeloid cells, this contains sinusoids and megakaryocytes.

Small arteries and arterioles are often seen in the intermediate and central zones; these may be surrounded by cuffs of immature myeloid cells around them.

Hemopoiesis

The process of formation of blood elements from hemopoietic stem cells has been described in detail in Chapter 2. The BMTB enables the visualization of the spatial localization of the individual cell lineages during their development. Hemopoietic progenitors are present in cords, islands or clusters. Fully mature erythroid and granulocytic cells and platelets migrate through the sinusoidal endothelial cells to enter the bloodstream.