I Clin Pathol 1992;45:645-649 645 Occasional articles

Histology of normal haemopoiesis: 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 trephine 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 deter- mined 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 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 . of macrophages in normal marrow (unpub- Correspondence to: Occasional multinucleate 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 granulo- cytic 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 decal- cified, 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 pre- served. 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 in the cen- vessels.89 With increasing distance from the tre and progressively more mature forms trabecula or vessel, increasing numbers of towards the periphery (fig 2). Convincing metamyelocytes and mature polymorphs are evidence of a close spatial association between Histolog, oJ'noriwral haemopoiesis: Bone marrou, histol(gy I 647

such clusters and marrow sinusoids can rarely be seen in routine histological sections. Erythroid clusters are usually readily appar- ent in marrow normal trephine biopsy speci- J Clin Pathol: first published as 10.1136/jcp.45.8.645 on 1 August 1992. Downloaded from mens by virtue of the dense blue-black colour of the compact, round nuclei of mid- and late w s ...^ ^ w normoblasts in sections stained with haema- ... and . toxylin eosin. Giemsa shows staining intense cytoplasmic basophilia in proerythro- blasts and early normoblasts. Immunohis- tochemical staining of a and /3 sialoglycopro- teins (glycophorins A and C)'6 17 can also be used to demonstrate erythropoiesis (fig 2). A helpful artefact occurs in decalcified, wax embedded trephine biopsy specimens which contributes to the ease of recognition of erythroid cells. Shrinkage of the nucleus leaves > t? 5 v a clear perinuclear halo in each cell (fig 3A). This can assist in distinguishing erythroid cells (A) from lymphocytes, which have darkly staining nuclei of a similar size to those of late normoblasts. Fortunately, although perinu- clear haloes are absent from undecalcified, 6@Jgge.:.@ resin-embedded biopsy specimens, the mor- phology of red cell precursors is usually dis- tinctive in haematoxylin and eosin or Giemsa stained sections from bone marrow biopsy specimens processed in this way (fig 3B).

MEGAKARYOPOIESIS Mature megakaryocytes can easily be identi- fied in tissue sections because of their large size, voluminous cytoplasm and nuclear multi- lobulation. They are found scattered singly throughout intertrabecular spaces,9 evenly dis- tributed, except that they are usually absent from the immediate paratrabecular and peri- vascular zones occupied by early granulocytes. http://jcp.bmj.com/ As with erythroid clusters, it is often difficult to see sites of intimate association between mega- (B) karyocytes and sinusoids (fig 4). However, Figure 3 (A) Appearance of normal erythroid cells following decalcification and electron microscopic studies have shown that wax-embedding: shinkage artefact causes perinuclear halo formation. (B) Normal eythroid ceUs in an undecalcified, resin-embedded trephine biopsy specimen. Their peripheral cytoplasm of megakaryocytes pro- cytoplasmic margins are indistinct, giving the clusters a syncytia-like appearance. trudes into sinusoidal lumina and is shed into

the intravascular compartment in the form of on October 2, 2021 by guest. Protected copyright. pro-platelets which subsequently fragment to give rise to platelets.9 18 The precursors of megakaryocytes cannot always be readily identified within normal bone marrow trephine sections. Occasional, small, mononuclear forms can be found (fig 4; inset A), but their ontogenic relation to mature megakaryocytes is not known. The difficulty of recognising early megakaryocyte precursors exists because such cells are smaller than their progeny and may resemble promyelocytes morphologically. Giemsa staining can show cells in trephine biopsy sections which are probably megakaryocyte precursors: they have grainy lilac-pink cytoplasm resembling that of their mature counterparts. Some of the subtle variation in cytoplasmic coloration between haemopoietic cell lineages afforded by Giemsa staining is absent from decalcified biopsy specimens, and an alternative approach is to perform immunohistochemical staining for factor 8 related antigen or CD6 1 (platelet Figure 4 Normal megakaryocyte in close association with a marrow sinusoid (the glycoprotein Low retraction is artefactual). Inset A: small, mononuclear megakaryocyte, presumably 3a)." numbers of small, immature. Inset B: irregular bare nucleus, presumed to represent a senescent mononuclear cells, presumed to be mega- megakaryocyte. karyocyte precursors, are present which 648 wil

express these antigens. Such cells are appar- homogeneous pink cytoplasm on haematoxy ently randomly distributed throughout inter- and eosin staining and a small, dense rou trabecular spaces. nucleus. They stain metachromatically w Also normally present in small numbers are Giemsa, but are difficult to discern amc J Clin Pathol: first published as 10.1136/jcp.45.8.645 on 1 August 1992. Downloaded from scattered megakaryocyte nuclei with a convo- large numbers of developing granulocyt luted outline and condensed chromatin pat- Other metachromatic stains, such as toluid tern, apparently bare of cytoplasm (fig 4: inset blue or buffered thionine, are more useful B). These are considered to represent senes- their demonstration. Immunostaining can cent megakaryocytes. performed using the antibody AAI, react with mast cell tryptase,- which does not crc OTHER CELLS REGULARLY PRESENT IN NORMAL react with basophils. BONE MARROW Lymphocytes The distribution and morphology of lympho- Conclusion cytes within bone marrow biopsy specimens A great variety of cells, haemopoietic and n( offer no clues as to whether these cells are haemopoietic, are present within normal bc undergoing some part of their development marrow. In keeping with the highly speciali, there or are simply in transit through the functions of the tissue, there is a complex I tissue. orderly spatial organisation of cells within Small lymphocytes of B and T phenotypes Although we do not yet fully understand are present in low to moderate numbers relations between bone marrow structure a scattered throughout the intertrabecular function, knowledge in this sphere is advanc spaces. Clusters of a few lymphocytes, normal- as in vitro culture systems provide insights ii ly termed lymphoid aggregates, and larger the roles of cell-cell and cell-stroma int collections termed lymphoid nodules are also actions in haemopoiesis. Careful histologi frequently seen. The larger lymphoid nodules analysis of intact bone marrow biopsy spc may incorporate a few macrophages and small mens will also contribute to this increase vascular channels and they occasionally form knowledge of normal haemopoiesis. true follicles with germinal centres. Lymphoid For haematologists, histopathologists a aggregates and nodules only rarely lie imme- any other practitioners engaged in the di diately adjacent to trabeculae; collections of nosis ofbone marrow disorders, I hope that lymphoid cells at such sites should raise a foregoing account will be of practical use strong suspicion of lymphomatous infiltration. clarify current concepts of normal haen Criteria for distinguishing benign from malig- poietic cell organisation and to set a threshi nant lymphoid infiltrates within bone marrow against which abnormal haemopoiesis can biopsy specimens have been described in detail assessed. by Rywlin and colleagues. 9 However, even morphologically benign aggregates may be 1 Frisch B, Lewis SM, Burkhardt R, Bartl R. Introduct http://jcp.bmj.com/ associated with subsequent lymphoid malig- In: Biopsy pathology of bone and bone marrow. Lon( nancy, " and they should always be assessed Chapman and Hall, 1985:1-17. 2 Wickramasinghe SN. Organisation, blood and nerve sul cautiously. of bone marrow and some properties of non-hae: poietic cells. Human Bone Marrow. Oxford: Black Scientific Publications, 1975:60-81. Plasma cells 3 Campbell AD, Wicha MS. Extracellular matrix and These cells are usually present in small num- hematopoietic microenvironment. 7 Lab Clin bers, clustered at the adventitial of 1988;112:140-6. margins 4 Gordon MY, Riley GP, Watt SM, Greaves MF. C, on October 2, 2021 by guest. Protected copyright. small blood vessels. They are also found partmentalisation of a haematopoietic growth fa (GM-CSF) by glycosaminoglycans in the bone mar scattered singly elsewhere in the intertra- microenvironment. Nature 1987;326:403-5. becular spaces. 5 Burgio VL, Pignoloni P, Baroni CD. Immunohistolog bone marrow: a modified method of glycol-methacr3 embedding. Histopathology 1991;18:37-43. Mast cells 6 Beckstead JH, Halverson PS, Ries CA, Bainton Enzyme histochemistry and immunohistochemistry The relation between mast cells and basophil biopsy specimens of pathologic human bone mar granulocytes remains uncertain. It is believed Blood 1981;57:1088-98. 7 Dilly SA, Jagger CJ. Bone marrow stromal change- currently that both cell types arise by inde- haematological malignancies. J Clin Pathol 1990 pendent pathways from a common partially 942-6. 8 Frisch B, Bard R. Biology, structure and component committed basophil stem cell.9 Metachromatic normal bone marrow. In: Atlas of bone marrow pathoi and immunohistochemical staining of mast. Current Histopatholgy. Vol 15. London: Kluwer Acade Publishers, 1990:9-37. cells in trephine biopsy specimens shows no 9 Wickramasinghe SN. Normal Haemopoiesis: cellular c, spatial relation between developing basophils position ofnormal bone marrow. In: Wickramasinghe ed. Blood and Bone marrow. Volume 2. Systemic pathoi and mast cells, nor are cells found which 3rd Edn. Edinburgh: Churchill Livingst( exhibit transitional morphological, tinctorial, 1989:41 -72. 10 Bancroft JD, Stevens A, eds. Enzyme histochemis or immunohistochemical appearances." Diagnostic applications. In: Theory and practice of h Mast cells can be detected in about 50% of logical techniques. 3rd Edn. Edinburgh: Churchill Liv stone, 1990:406-7. trephine biopsy specimens by metachromatic 1 1 Pulford KA, ErberWN, Crick JA, et al. Use of monoch staining. Their number is generally small in antibody against human neutrophil elastase in nor and leukaemic myeloid cells. J Clin Pc normal marrow and they tend to lie imme- 1 988;41 :853-60. diately adjacent to the endosteal surfaces and 12 Dexter TM, Spooncer E. Growth and differentiation in hemopoietic system. Ann Rev Cell Biol 1987;3:423-4 at the adventitial borders ofblood vessels. They 13 WiIkins BS, Jones DB. The use of immunohistocherr are also frequently found at the periphery of techniques with fixed, decalcified, wax-embedded b marrow trephine . Haematol Rev 1989;3:149- lymphoid nodules.821 Morphologically, they 14 Van der Valk P, Mullink H, Huijgens PC, Tadema TM, are usually either spindle-shaped or oval with W, Meier CJLM. Immunohistochemistry in bone mar Histology oJ nornmal haemopoiesis: Bone nmarrow histology I 649

diagnosis. Value of a panel of monoclonal antibodies on 18 Tavassoli M, Aoki M. Localisation of megakaryocytes in the routinely processed bone marrow biopsies. Amz 7 Surg bone marrow. Blood Cells 1989;15:3-14. Pathol 1989;13:97-106. 19 Rywlin AM, Ortega RS, Dominguez CJ. Lymphoid nodules 15 Pulford KA, Rigney EM, Micklem KJ, et al. KP 1: a new of bone marrow. Blood 1974;43:389-400. monoclonal antibody that detects a monocyte/macro- 20 Faulkner-Jones BE, Howie AJ, Boughton BJ, Franklin IM. phage associated antigen in routinely processed tissue Lymphoid aggregates in bone marrow: study of eventual sections. Clin Pathol 1989;42:414-21. outcome. Clin Pathol 1988;41:768-75. J Clin Pathol: first published as 10.1136/jcp.45.8.645 on 1 August 1992. Downloaded from 16 Anstee DJ, Edwards PAW. Monoclonal antibodies to human 21 Nock ID, WiIkins BS, Jones DB. Bone marrow mast cells erythrocytes. Eur lmmunol 1982;12:228-32. revisited. Pathol 1991 ;164:352A. 17 Gatter KC, Cordell JL, Turley H, et al. The immunohisto- 22 Walls AF, Jones DB, Williams JH, Church MK, Holgate ST logical detection of platelets, megakaryocytes and throm- Immunohistochemical identification of mast cells in bi in routinely processed specimens. Histopathology formaldehyde fixed tissue using monoclonal antibodies 1988;13:257-67. specific for tryptase. 7 Pathol 1990;162:119-26. http://jcp.bmj.com/ on October 2, 2021 by guest. Protected copyright.