Occasional Articles Histology of Normal Haemopoiesis: Bone Marrow

I Clin Pathol 1992;45:645-649 645 Occasional articles Histology of normal haemopoiesis: Bone marrow J Clin Pathol: first published as 10.1136/jcp.45.8.645 on 1 August 1992. Downloaded from histology I B S Wilkins Introduction trabecular spaces,2 but little is known about The bone marrow trephine biopsy specimen the dynamics of blood flow through human holds an unusual position among pathological bone marrow. Arterioles and venules tend to lie specimens. Having been obtained in most towards the centres of intertrabecular spaces. cases by a haematologist, the biopsy specimen They are usually seen in only a small propor- is processed in a histopathology laboratory and tion of intertrabecular spaces in any one biopsy is then reported either by a haematologist or a specimen, suggesting that each may supply or histopathologist, depending on local custom. drain a number of such spaces. Individual practitioners may acquire great skill The trabeculae, arterioles, and venules form in the interpretation of trephine biopsy speci- the structural framework around which mens, but others in both branches ofpathology granulopoiesis develops. Erythropoiesis and are sometimes perplexed when faced with megakaryopoiesis occur in apposition to the tissue appearances for which other experience fine, branching sinusoids. within their individual disciplines may not have prepared them fully. Ideally, interpretation MARROW STROMA should be a collaborative procedure, combin- The trabecular and vascular architecture of ing the clinical and cytological knowledge of medullary bone provides the basic framework, the haematologist with the histopathologist's nutritional supply, and waste removal system skills in analysis ofnormal and abnormal tissue for haemopoiesis, but specialised support of structure. the self-renewal and differentiation of haemo- An important starting point for all con- poietic precursors is provided by the bone cerned is to have a good appreciation of the marrow stroma. This stroma consists of a normal appearances of haemopoiesis as repre- heterogeneous mixture of adipocytes, fibro- sented in a trephine biopsy specimen, against blasts, and macrophage-like cells, together which abnormal changes can then be assessed. with a complex extracellular matrix of reticu- http://jcp.bmj.com/ The purpose of this article is to describe these lin, binding proteins, including fibronectin and normal appearances and to indicate briefly haemonectin, and proteoglycans.3 In culture some uses and limitations ofvarious processing systems specific haemopoietic microenviron- and staining techniques in bone marrow biopsy ments-for example, for erythropoiesis or specimen interpretation. granulopoiesis-can be shown to be determined by the nature of the stromal cells and the binding of growth factors to restricted on October 2, 2021 by guest. Protected copyright. General structure of bone marrow matrix sites.4 How this correlates with the It is impossible to describe the haemopoietic spatial distribution of haemopoiesis within tissue of bone marrow without at least a brief intact marrow in vivo is not yet understood. reference to the underlying bony structure. A Despite this evidence of its important con- trephine biopsy specimen may have a quantity tribution to normal haemopoiesis, bone mar- of dense, cortical bone (and even extraneous row stroma appears disappointingly bland on elements such as cartilage or tendon) at its conventional histological examination. Adipo- outer end, but for the most part consists of cytes are readily apparent and small numbers trabecular bone-a meshwork of delicate bony of scattered fibroblasts can usually be dis- plates and strands within which the haemo- cerned. Reconisable macrophages are remark- poietic tissue and its stroma are suspended.' ably few: those which contain storage iron can The precise distribution of bony trabeculae, in be demonstrated by Perls's stain, but they are the iliac crest as at other sites, is determined not numerous in normal marrow and only primarily by mechanical aspects of bone func- occasionally can they be seen to lie in close spaces of size and proximity to developing erythrocytes. Immu- University tion, but the varying shape Department of which lie between adjacent trabeculae com- nostaining of paraffin wax embedded trephine Pathology, prise the basic structural units of haemo- biopsy sections using macrophage reactive Southampton General monoclonal antibodies such as Mac387 (anti- Hospital, Tremona poiesis. Road, Southampton The trabecular surfaces are covered by a calgranulin; Dako UK Ltd) and KP1 (CD68; S09 4XY layer of endosteal cells, usually inconspicuous Dako UK Ltd) also shows only small numbers B S Wilkins but sometimes recognisable as osteoblasts. of macrophages in normal marrow (unpub- Correspondence to: Occasional multinucleate osteoclasts are also a lished observation). Dr B S Wilkins normal at trabecular margins. A sinu- Spindle cells expressing alkaline phospha- Accepted for publication finding 20 December 1991 soidal network arborises throughout the inter- tase, butyrate esterase, or fibroblast associated 646 Wilkins seen. This radial orientation of granulopoiesis can be appreciated easily in reactive granulocytic hyperplasia or chronic granulocytic leu- A~~~~~~~~~~~~, kaemia but is not always obvious in normal marrow, in which the zone of promyelocytes J Clin Pathol: first published as 10.1136/jcp.45.8.645 on 1 August 1992. Downloaded from ~~~~. and myelocytes may only be one or two cells thick. In undecalcified resin-embedded biopsy specimens the immature granulopoietic zone can be highlighted by Leder's stain for chlor- oacetate esterase,'0 which tends to react more strongly with promyelocytes and myelocytes than with later granulocytes. Unfortunately, this visually impressive staining technique can- not be performed successfully with biopsy Figure i Normal paratrabecular zone of promyelocytes specimens decalcified by formic or acetic acids, and myelocytes. Streptavidin-biotin complex as the enzyme activity is lost. However, a very immunoperoxidase technique using the monoclonal antibody NP57, reactive with neutrophil elastase. similar demonstration of the distribution of early granulocytes can be achieved in decalcified, wax-embedded biopsy specimens by immunohistochemical staining for neutrophil elastase" (fig 1). In general, using routine stains, little granu- ~~~ ~ ~ ~ ~ ~ ~ ~ ~ A larity can be appreciated in granulocytes of trephine biopsy specimens which have under- gone acid decalcification, with the exception of eosinophils, whose granules are well preserved. Occasional promyeloctes and myelocytes, scattered singly or in small clusters, occur at sites distant from trabeculae and vessels, and it is therefore not necessarily abnormal to see immature granulocytes in the centres of inter- Figure 2 Normal clusters of proerythroblasts and normoblasts. Streptavidin-biotin complex trabecular spaces. immunoperoxidase technique using the monoclonal antibody Ret4Of,ff-sialoglycoproteinreactive with MONOPOIESIS (glycophorin C). The close association of granulopoiesis and monopoiesis in bone marrow culture systems in vitro'2 suggests that within intact marrow http://jcp.bmj.com/ antigens have been shown in frozen or resin monocyte precursors should be found in a embedded sections5` and in cultured marrow similar distribution to that of granulocytes. stroma, but the precise functions of these cells Monocytes, however, are difficult to recognise remain uncertain. in trephine biopsy sections and little is known about the spatial organisation of their develop- BONE MARROW CELLULARITY ing precursors. No staining technique is cur- This term is usually used with reference to the rently widely available for reliable and specific on October 2, 2021 by guest. Protected copyright. haemopoietic cell content of a bone marrow demonstration of monocytes in fixed trephine biopsy specimen relative to its adipocyte con- biopsy specimens. Butyrate esterase enzyme tent. Biopsy specimens are generally con- activity does not survive routine processing, sidered to be of normal cellularity if 40-60% of although it may be preserved by some resin- the non-bony tissue is composed of haemo- embedding protocols.6 Most immunohisto- poietic cells. Cellularity varies widely with age chemical agents which will react in decalcified, and site, however, so that in a neonate normal wax embedded trephine biopsy specimens for cellularity may be 100% and in an elderly demonstration of monocytes (Mac387, CD 15, patient it may be 20%.8 Haematologists and C68) also react strongly with granulo- pathologists who regularly assess trephine cytes,'3-' which predominate in normal mar- biopsy specimens will also be well aware that row and tend to obscure any monocytes intertrabecular spaces immediately beneath present. the bony cortex are frequently hypocellular, especially in the elderly, and may be unrepre- ERYTHROPOIESIS sentative of the cellularity deeper in the biopsy Erythrocytes develop in small clusters which specimen. are dispersed throughout the intertrabecular spaces9 but which tend not to encroach on the GRANULOPOIESIS paratrabecular and perivascular zones of early The earliest recogznisable gyranulop)oietic pre- granulocytes. In a trephine biopsy section cursors, the promyelocytes and myelocytes, lie these clusters measure about 5-10 cells in adacent to the surfaces of bony trabeculae (fig diameter. Erythroid clusters have a radial 1) and the adventitial aspect of small blood organisation, with proerythroblasts

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