BONE MARROW PATHOLOGY

BARBARA J. BAINMBBS, FRACP, FRCPathReader in Diagnostic HaematologySt Mary’s Hospital Campus of Imperial College School of Medicine, Londonand Honorary Consultant HaematologistSt Mary’s Hospital, London

DAVID M. CLARKMD, MRCP, FRCPathConsultant HistopathologistUnited Lincolnshire Hospitals NHS TrustHonorary Consultant HistopathologistUniversity Hospital, Nottinghamand Special Lecturer in PathologyUniversity of Nottingham

IRVIN A. LAMPERTMB, ChB, DCP, FRCPathConsultant HistopathologistEaling Hospital, LondonHonorary Senior Lecturer, Hammersmith Hospital Campus of Imperial College School of Medicineand Honorary Consultant HistopathologistHammersmith Hospital, London

BRIDGET S. WILKINSDM, PhD, FRCPathConsultant HistopathologistNewcastle upon Tyne Hospitals NHS Trustand Honorary Senior LecturerUniversity of Newcastle

THIRD EDITION

BONE MARROW PATHOLOGY

This book is dedicated to Professor David A.G. Galton

BONE MARROW PATHOLOGY

BARBARA J. BAINMBBS, FRACP, FRCPathReader in Diagnostic HaematologySt Mary’s Hospital Campus of Imperial College School of Medicine, Londonand Honorary Consultant HaematologistSt Mary’s Hospital, London

DAVID M. CLARKMD, MRCP, FRCPathConsultant HistopathologistUnited Lincolnshire Hospitals NHS TrustHonorary Consultant HistopathologistUniversity Hospital, Nottinghamand Special Lecturer in PathologyUniversity of Nottingham

IRVIN A. LAMPERTMB, ChB, DCP, FRCPathConsultant HistopathologistEaling Hospital, LondonHonorary Senior Lecturer, Hammersmith Hospital Campus of Imperial College School of Medicineand Honorary Consultant HistopathologistHammersmith Hospital, London

BRIDGET S. WILKINSDM, PhD, FRCPathConsultant HistopathologistNewcastle upon Tyne Hospitals NHS Trustand Honorary Senior LecturerUniversity of Newcastle

THIRD EDITION

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CONTENTS

Preface to the third edition, vi

Acknowledgements, vii

Abbreviations, viii

1 The normal bone marrow, 1

2 Special techniques applicable to bone marrow diagnosis, 51

3 Infection and reactive changes, 90

4 Acute myeloid leukaemia, the myelodysplastic syndromes and histiocytic neoplasms, 141

5 Chronic myeloproliferative and myeloproliferative/myelodysplastic disorders, 191

6 Lymphoproliferative disorders, 231

7 Multiple myeloma and related disorders, 332

8 Disorders of erythropoiesis, granulopoiesis and thrombopoiesis, 360

9 Miscellaneous disorders, 391

10 Metastatic tumours, 430

11 Diseases of bone, 462

Appendix, 474

Index, 483

v

In this book we have set out to provide a practicalguide to bone marrow diagnosis, based on an inte-grated assessment of peripheral blood and bonemarrow aspirate films, trephine biopsy sections andvarious supplementary investigations. We believethat a trephine biopsy specimen should not be ex-amined and interpreted in isolation. We have there-fore discussed the clinical context of bone marrowdiagnosis and have given equal weight to cytolog-ical and histological features. Since bone marrowdiagnosis is no longer based on morphological fea-tures alone we have also, in this edition, discussedin considerable detail the role of immunopheno-typic, cytogenetic and molecular genetic analysis.

We have dealt very fully with haematological disorders for which bone marrow examination iscommonly performed. However, we have alsosought to be as comprehensive as possible, includ-

ing whatever information is available on uncom-mon and rare disorders so that the book will serveas a useful reference source. When possible, wehave illustrated rare as well as common conditionsand have cited the relevant scientific literatureextensively.

In this edition we have introduced a new sectionentitled ‘Problems and pitfalls’ which we believewill be of value. We have also expanded the descrip-tion of technical methods. We hope that haemato-logists and histopathologists will continue to findBone Marrow Pathology a useful aid in their day-to-day practice and that trainees in these disciplineswill find it indispensable.

BJB, DMC, IAL, BSW

December 2000

PREFACE TO THE THIRD EDITION

vi

We should like to thank our many friends and colleagues in North America, Europe, Africa, Asiaand Australia who have provided illustrations orhave permitted us to photograph microscopic slidesfrom their personal collections. They are individu-ally acknowledged in the legends of specific figures.In addition we should like to thank our technical andmedical colleagues in St Mary’s Hospital, Hammer-smith Hospital, Nottingham University Hospitals

and Southampton University Hospitals for the directand indirect help they have given us.

This book is dedicated to Professor EmeritusDavid Galton who taught us and countless otherhaematologists and histopathologists for a greatmany years. Those who have worked with him haveadmired him both for his diagnostic skills and for his mindfulness of the individual patient. It is withmuch pleasure that we make this dedication to him.

ACKNOWLEDGEMENTS

vii

aCML atypical CMLAIDS acquired immune deficiency syndromeAILD angio-immunoblastic

lymphadenopathy (withdysproteinaemia)

ALCL anaplastic large cell lymphomaALIP abnormal localization of immature

precursorsALL acute lymphoblastic leukaemiaAML acute myeloid leukaemiaATLL adult T-cell leukaemia/lymphomaATRA all-trans retinoic acidBCL2 B-cell leukaemia/lymphoma 2BCR breakpoint cluster regionBFU-E erythroid burst-forming unitBM bone marrowB-PLL B-lineage prolymphocytic leukaemiaCD cluster of differentiationCDA congenital dyserythropoietic anaemiaCFU colony-forming unitCFU-GM granulocyte–monocyte CFUCFU-Meg megakaryocyte CFUCGL chronic granulocytic leukaemiaCHAD cold haemagglutinin diseaseCLL chronic lymphocytic leukaemiaCML chronic myeloid leukaemiaCMML chronic myelomonocytic leukaemiaCMV cytomegalovirusCT computerized tomographyCyIg cytoplasmic immunoglobulinDIC disseminated intravascular coagulationDNA deoxyribonucleic acidEBER Epstein–Barr early RNAEBNA Epstein–Barr nuclear antigenEBV Epstein–Barr virusEDTA ethylene diamine tetra-acetic acidEGIL European Group for the Immunological

Classification of Leukaemia

EMA epithelial membrane antigenESR erythrocyte sedimentation rateET essential thrombocythaemiaFAB French–American–British (co-operative

group)FICTION fluorescent immunophenotyping and

interphase cytogeneticsFISH fluorescence in situ hybridizationG-CSF granulocyte colony-stimulating factorGM-CSF granulocyte–macrophage colony-

stimulating factorGMS Grocott’s methenamine silver (stain)GPI glycosyl phosphatidylinositolGVHD graft-versus-host diseaseH&E haematoxylin and eosin (stain)Hempas hereditary erythroid multinuclearity

with positive acidified serum testHHV human herpesvirus HIV human immunodeficiency virusHLA human leucocyte antigenHTLV-I human T-cell lymphotropic virus IIg immunoglobulinIGH immunoglobulin heavy chain (gene)IL interleukinISH in situ hybridizationJMML juvenile myelomonocytic leukaemiaLDH lactate dehydrogenaseLE lupus erythematosusLGL large granular lymphocyteLGLL large granular lymphocyte leukaemiaLTC-IC long-term culture-initiating cellMALT mucosa-associated lymphoid tissueMBR major breakpoint regionMcAb monoclonal antibodyMCR minor cluster regionMDS myelodysplastic syndromesM:E myeloid : erythroid ratioMGG May–Grünwald–Giemsa (stain)

ABBREVIATIONS

viii

ABBREVIATIONS ix

MGUS monoclonal gammopathy ofundetermined significance

MIC morphologic, immunologic, cytogenetic(classification)

MIC-M morphological, immunological,cytogenetic, molecular genetic(classification)

MPD myeloproliferative disorderMPO myeloperoxidasemRNA messenger RNANEC non-erythroid cellsNHL non-Hodgkin’s lymphomaNK natural killerPAS periodic acid–Schiff (stain)PB peripheral bloodPCR polymerase chain reactionPh Philadelphia chromosomePLL prolymphocytic leukaemiaPNET primitive neuro-ectodermal tumour PNH paroxysmal nocturnal haemoglobinuriaPOEMS polyneuropathy, organomegaly,

endocrinopathy, M-protein, skinchanges (syndrome)

PRV polycythaemia rubra veraRA refractory anaemia

RAEB refractory anaemia with excess of blastsRAEB-T refractory anaemia with excess of blasts

in transformationRARS refractory anaemia with ring

sideroblastsREAL Revised European–American

Lymphoma (classification)RNA ribonucleic acid RT-PCR reverse transcriptase polymerase chain

reactionSBB Sudan black B (stain)SD standard deviationSKY spectral karyotypingSLVL splenic lymphoma with villous

lymphocytesSmIg surface membrane immunoglobulinTAR thrombocytopenia-absent radii

(syndrome)TCR T-cell receptorTdT terminal deoxynucleotidyl transferaseT-PLL T-lineage prolymphocytic leukaemiaTRAP tartrate-resistant acid phosphataseWBC white blood cell (count)WHO World Health OrganizationZN Ziehl–Neelsen

may also be classified histologically on the basis ofwhether there are well-organized osteons in whicha central Haversian canal is surrounded by concen-tric lamellae composed of parallel bundles of fibrils(lamellar bone) (Fig. 1.1) or, alternatively, whetherthe fibrils of the bone are in disorderly bundles(woven bone) (Fig. 1.2).

The cortex is a solid layer of compact bone whichgives the bone its strength. It is composed largely oflamellar bone but also contains some woven bone.The lamellar bone of the cortex consists of eitherwell-organized Haversian systems or angular frag-ments of lamellar bone which occupy the spacesbetween the Haversian systems; in long bones thereare also inner and outer circumferential lamellae.Extending inwards from the cortex is an anasto-mosing network of trabeculae which partition themedullary space (Fig. 1.3). The medullary bone istrabecular or cancellous bone; it contains lamellaebut the structure is less highly organized than thatof the cortex. Most of the cortical bone is covered onthe external surface by periosteum which has anouter fibrous layer and an inner osteogenic layer. Atarticular surfaces, and more extensively in youngerpatients, bone fuses with cartilage rather than beingcovered by periosteum. The bony trabeculae andthe inner surface of the cortex are lined by end-osteal cells including osteogenic cells (osteoblasts)and osteoclasts. Osteocytes are found within lacunaein bony trabeculae and in cortical bone. Althoughosteoblasts and osteoclasts share the surface of thebone trabeculae, they originate from different stemcells. Osteoblasts, and therefore osteocytes, are ofmesenchymal origin, being derived from the samestem cell as chondrocytes and probably also stromalfibroblasts. Osteoclasts, however, are derived from ahaemopoietic stem cell, being formed by fusion ofcells of the monocyte lineage.

ONE

THE NORMAL BONE MARROW

The distribution of haemopoieticmarrow

During extra-uterine life haemopoiesis is normallyconfined to the bone marrow. Bones are composedof cortex and medulla. The cortex is a strong layer of compact bone; the medulla is a honeycomb ofcancellous bone, the interstices of which form the medullary cavity and contain the bone mar-row. Bone marrow is either red marrow contain-ing haemopoietic cells or yellow marrow which is largely adipose tissue. The distribution ofhaemopoietic marrow is dependent on age. In theneonate virtually the entire bone marrow cavity isfully occupied by proliferating haemopoietic cells;haemopoiesis occurs even in the phalanges. As thechild ages, haemopoietic marrow contracts cen-tripetally, being replaced by fatty marrow. By earlyadult life haemopoietic marrow is largely confined tothe skull, vertebrae, ribs, clavicles, sternum, pelvisand the proximal half of the humeri and femora;however, there is considerable variation betweenindividuals as to the distribution of haemopoieticmarrow [1]. In response to demand, the volume ofthe marrow cavity occupied by haemopoietic tissueexpands.

The organization of the bone marrow

Bone

The cortex and the medulla differ functionally aswell as histologically. Bone may be classified in twoways. Classification may be made on the basis of themacroscopic appearance into: (i) compact or densebone with only small interstices that are not visiblemacroscopically; and (ii) cancellous (or trabecular)bone with large, readily visible interstices. Bone

1

The cells that give rise to bone-forming cells aredesignated osteoprogenitor cells; they are flattened,spindle-shaped cells which are capable of develop-ing into either osteoblasts or chondrocytes, depend-ing on micro-environmental factors. Osteoblastssynthesize glycosaminoglycans of the bone matrixand also the collagenous fibres which are embeddedin the matrix, thus forming osteoid or non-calcifiedbone; subsequently mineralization occurs. Boneundergoes constant remodelling. In adult life,remodelling of the bone takes place particularly in

the subcortical regions. Osteoblasts add a new layerof bone to trabeculae (apposition), while osteoclastsresorb other areas of the bone; up to 25% of the trabecular surface may be covered by osteoid. Theosteoclasts which are resorbing bone lie in shallowhollows, known as Howship’s lacunae, created bythe process of resorption, while osteoblasts are seenin rows on the surface of trabecular bone or on thesurface of a layer of osteoid. As new bone is laiddown, osteoblasts become immured in bone and areconverted into osteocytes. The bone that replaces

2 CHAPTER ONE

Fig. 1.1 BM trephine biopsysection showing normal bonestructure; the trabeculae arecomposed of lamellar bone.Paraffin-embedded, reticulin stain×188.

Fig. 1.2 BM trephine biopsysection showing woven bone (pale pink; without lamellae) in a hypocellular but otherwiseunremarkable bone marrow.Paraffin-embedded, H&E ×188.

THE NORMAL BONE MARROW 3

osteoid is woven bone which, in turn, is remodelledto form lamellar bone. The difference between thetwo can be easily appreciated by microscopy usingpolarized light. The organized fibrillar structure oflamellar bone, with bundles of parallel fibrils run-ning in different directions in successive lamellae,gives rise to alternating light and dark layers whenviewed under polarized light.

Trephine biopsies from children may contain cartilage as well as bone and endochondrial boneformation may be observed (Figs 1.4 and 1.5).

Transition from resting cartilage to proliferating andhypertrophic cartilage can be observed, followed bya zone of calcifying cartilage, invading vessels andbone. Mature cartilage can also be seen in trephinebiopsy specimens from adults (Fig. 1.6).

Other connective tissue elements

The haemopoietic cells of the bone marrow areembedded in a connective tissue stroma whichoccupies the intertrabecular spaces of the medulla.

Fig. 1.3 BM trephine biopsysection showing normal bonestructure; there are anastomosingbony trabeculae. Paraffin-embedded, H&E ×48.

Fig. 1.4 BM trephine biopsysection from a child showingendochondrial ossification in an island of cartilage. Paraffin-embedded, H&E ×188.

Fig. 1.5 BM trephine biopsysection from a child showingendochondrial ossification; a bonyspicule with a core of cartilage islined by osteoblasts. Paraffin-embedded, Giemsa ×376.

The stroma is composed of fat cells and a meshworkof blood vessels, branching fibroblasts, macrophages,some myelinated and non-myelinated nerve fibresand a small amount of reticulin. Stromal cells in-clude cells that have been designated reticulum orreticular cells. This term probably includes two celltypes of different origin. Phagocytic reticulum cellsare macrophages and originate from a haemopoieticprogenitor. Non-phagocytic reticulum or reticularcells are closely related to fibroblasts, adventitialcells of sinusoids (see below) and probably alsoosteoblasts and chondrocytes. They differ fromphagocytic reticulum cells in that the majority are

positive for alkaline phosphatase. There is closeinteraction between haemopoietic cells and theirmicro-environment, with each modifying the other.

The blood supply of the marrow is derived in partfrom the central nutrient artery which enters longbones at mid-shaft and bifurcates into two longi-tudinal central arteries [2]. Similar arteries penetrateflat and cuboidal bones. There is a supplementaryblood supply from cortical capillaries which pene-trate the bone from the periosteum. The branches of the central artery give rise to arterioles and capil-laries which radiate towards the endosteum andmainly enter the bone, subsequently turning back

4 CHAPTER ONE

Fig. 1.6 BM trephine biopsyspecimen from an adult showingcartilage adjacent to the cortex. By contrast with childhoodappearances, a well-defined layerof cortical bone separates thiscartilage from the bone marrow.Cartilage cells are dispersed singlyor in small groups and are notaligned into columns, as they are in childhood. Paraffin-embedded,H&E ×188.

THE NORMAL BONE MARROW 5

to re-enter the marrow and open into a network of thin-walled sinusoids [2]. Only a minority of capillaries enter the sinusoids directly without firstsupplying bone. The sinusoids drain into the centralvenous sinusoid which accompanies the nutrientartery. Sinusoids are large, thin-walled vesselsthrough which newly formed haemopoietic cellsenter the circulation. They are often collapsed inhistological sections and are therefore not readilyseen. In the presence of marrow sclerosis, these ves-sels are often held open and are then very obvious.The walls of sinusoids consist of endothelial cells,forming a complete cover with overlapping junc-tions, and an incomplete basement membrane. Theouter surface is clothed by adventitial cellsalarge,broad cells which branch into the perivascular spaceand therefore provide scaffolding for the haemopoi-etic cells, macrophages and mast cells. Adventitialcells are thought to be derived from fibroblasts; theyare associated with a network of delicate extracellu-lar fibres which can be stained with a reticulin stain.Reticulin fibres are concentrated close to the perios-teum as well as around blood vessels. It is likely thatboth adventitial cells and fibroblasts can synthesizereticulin [3], which is a form of collagen.

The marrow fat content varies inversely with the quantity of haemopoietic tissue. Fat contentalso increases as bone is lost with increasing age.Marrow fat is physiologically different from subcu-taneous fat. The fat of yellow marrow is the last fat in the body to be lost in starvation. Whenhaemopoietic tissue is lost very rapidly, it is replacedinitially by interstitial mucin. Subsequently thismucin is replaced by fat cells.

Haemopoietic and other cells

Haemopoietic cells lie in cords or wedges betweenthe sinusoids. In man normal haemopoiesis, withthe exception of some thrombopoiesis at extra-medullary sites, is confined to the interstitium. In pathological conditions haemopoiesis can occurwithin sinusoids. Mature haemopoietic cells enterthe circulation by passing transcellularly, throughsinusoidal endothelial cells [2]. The detailed dis-position of haemopoietic cells will be discussedbelow.

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

Examination of the bone marrow

Bone marrow was first obtained from living patientsfor diagnostic purposes during the first decade ofthe last century but it was not until the introductionof sternal aspiration in the late 1920s that this be-came an important diagnostic procedure. Specimensof bone marrow for cytological and histologicalexamination may be obtained by aspiration biopsy,by core biopsy using a trephine needle or an electricdrill, by open biopsy and at autopsy. The two mostimportant techniques, which are complementary,are aspiration biopsy and trephine biopsy.

Bone marrow aspiration causes only mild dis-comfort to the patient. A trephine biopsy causesmoderate discomfort and, in an apprehensive patient,sedation can be useful. In children, aspiration andtrephine biopsies are often performed under gen-eral anaesthesia.

Bone marrow aspiration

Aspiration biopsy is most commonly carried out on the sternum or the ilium. Aspiration from themedial surface of the tibia can yield useful diag-nostic specimens up to the age of 18 months but ismainly used in neonates in whom other sites areless suitable. Aspiration from ribs and from thespinous processes of vertebrae is also possible but isnow little practised. Sternal aspiration should becarried out from the first part of the body of the ster-num, at the level of the second intercostal space.Aspiration from any lower in the sternum increasesthe risks of the procedure. Aspiration from the iliumcan be from either the anterior or the posterior iliaccrest. Aspiration from the anterior iliac crest is bestcarried out by a lateral approach, a few centimetresbelow and posterior to the anterior superior iliacspine. Approach through the crest of the ilium withthe needle in the direction of the main axis of thebone is also possible but is more difficult because of the hardness of the bone. Aspirates from the pos-terior iliac crest are usually taken from the posteriorsuperior iliac spine. When aspiration is carried outat the same time as a trephine biopsy, it is easiest toperform the two procedures from adjacent sites.This necessitates the use of the ilium. If a trephinebiopsy is not being carried out, there is a choicebetween the sternum and the iliac crest. Either is

suitable in adults and older children, although greatcare must be exercised in carrying out sternal aspi-rations. In a study of 100 patients in whom bothtechniques were applied, sternal aspiration wasfound to be technically easier and to produce a suit-able diagnostic specimen more frequently, althoughon average the procedure was more painful, bothwith regard to bone penetration and to the actualaspiration [4]. Sternal aspiration is also more dan-gerous. Although deaths are very rare, at least 20have been reported and we are aware of three fur-ther fatalities, not reported in the scientific liter-ature; deaths have been mainly consequent on laceration of vessels or laceration of the heart withpericardial tamponade. Sternal aspiration is unsuit-able for use in young children. Posterior iliac crestaspiration is suitable for children, infants and manyneonates. Tibial aspiration is suitable for very smallbabies but has no advantages over iliac crest aspira-tion in older infants.

Bone marrow specimens yielded by aspiration aresuitable for the following: (i) preparation of wedge-spread films and films of crushed marrow frag-ments; (ii) study of cell markers (by flow cytometryor on films or cytospin preparations); (iii) cytoge-netic study; (iv) ultrastructural examination; (v)culture for micro-organisms; and (vi) culture tostudy haemopoietic precursors and preparation ofhistological sections of fragments. Cytogenetic ana-lysis is most often indicated in suspected haemato-logical neoplasms but it also permits rapid diagnosisof suspected congenital karyotypic abnormalitiessuch as trisomy 18; diagnosis is possible within aday, in comparison with the 3 days needed if periph-eral blood lymphocytes are used.

Bone marrow aspiration may fail completely, thisbeing referred to as a ‘dry tap’. Although this canhappen when bone marrow histology is normal, adry tap usually indicates significant disease, mostoften metastatic cancer, chronic myeloid leukaemia,idiopathic myelofibrosis or hairy cell leukaemia [5],with associated fibrosis.

Trephine biopsy of bone marrow

Trephine or needle biopsy is most easily carried outon the iliac crest, either posteriorly or anteriorly, asdescribed above. The posterior approach appearsnow to be more generally preferred. If a trephine

biopsy and a bone marrow aspiration are both to becarried out, they can be performed through thesame skin incision but with two areas of periosteumbeing infiltrated with local anaesthetic and with theneedle being angled in different directions. Corebiopsy specimens, obtained with a trephine needle,are suitable for histological sections, touch prepara-tions (imprints) and electron microscopy. A touchpreparation is particularly important when it is notpossible to obtain an aspirate since it allows cytolog-ical details to be studied. In addition, touch prepa-rations may show more neoplastic cells than aredetected in an aspirate and may detect bone marrow infiltration when it is not detected on anaspirate, for example in hairy cell leukaemia, mul-tiple myeloma or lymphoma [6]. Touch preparationsmay be made either by touching the core of bone ona slide or rolling the core gently between two slides.Biopsy specimens can be used for cytogenetic studybut aspirates are much more suitable. Frozen sec-tions of trephine biopsy specimens are possible andallow the application of a wide range of immuno-logical markers. However, they are not usually verysatisfactory because of technical problems, includ-ing difficulty in cutting sections, poor adhesion of sections to glass slides during staining proceduresand poor preservation of morphological detail. His-tological sections may be prepared from fixed biopsyspecimens which have either been decalcified andparaffin-embedded or have been embedded in plastic without prior decalcification.

Processing of trephine biopsies

The two principal methods of preparation of fixedtrephine biopsy specimens have advantages anddisadvantages. Problems are created because of thedifficulty of cutting tissue composed of hard boneand soft, easily torn bone marrow. Alternative approaches are to decalcify the specimen or toembed it in a substance that makes the bone mar-row almost as hard as the bone. Decalcification canbe achieved with weak organic acids, e.g. formicacid and acetic acid, or by chelation, e.g. with ethy-lene diamine tetra-acetic acid (EDTA). Decalcifica-tion and paraffin-embedding lead to considerableshrinkage and loss of cellular detail. Nuclear stainingmay become blurred. Some cytochemical activity is lost; for example chloro-acetate esterase activity is

6 CHAPTER ONE

THE NORMAL BONE MARROW 7

lost when acid decalcification is used. Because sec-tions are thicker, cellular detail is harder to appreci-ate. However, immunological techniques are muchmore readily applicable to paraffin-embedded thanto plastic-embedded specimens and, with carefultechniques, very good histochemical results can beobtained. Plastic-embedding techniques are moreexpensive and, for laboratories which are processingonly small numbers of trephine biopsy specimens,are technically more difficult. However, there is no shrinkage, preservation of cellular detail is excel-lent and the thinness of the sections means that fine cytological detail can be readily appreciated.Some enzyme activities, for example chloro-acetateesterase, are retained. Although immunologicaltechniques can be applied, excessive backgroundstaining is often a problem. Plastics with differingqualities are available for embedding. Methyl metha-crylate requires lengthy processing and is thereforenot very suitable for routine diagnostic laboratories.Glycol methacrylate is more satisfactory; however,when cellularity is low, sections tend to tear and, inthis circumstance, a small amount of decalcificationmay be useful. Methods which we have found satis-factory are given in the Appendix.

Relative advantages of aspiration and corebiopsy

Bone marrow aspiration and trephine biopsy eachhave advantages and limitations. The two proceduresshould therefore be regarded as complementary.Bone marrow aspirates are unequalled for demon-stration of fine cytological detail. They permit awider range of cytochemical stains and immunolog-ical markers than is possible with histological sec-tions. Aspiration is particularly useful, and may wellbe performed alone, when investigating patientswith suspected iron deficiency anaemia, anaemia ofchronic disease, megaloblastic anaemia and acuteleukaemia. Trephine biopsy is essential for diagnosiswhen a ‘dry tap’ or ‘blood tap’ occurs as a con-sequence of the marrow being fibrotic or very denselycellular. Only a biopsy allows a complete assess-ment of marrow architecture and of the pattern of distribution of any abnormal infiltrate. This technique is particularly useful in investigating sus-pected aplastic or hypoplastic anaemia, lymphoma,metastatic carcinoma, myeloproliferative disorders

and diseases of the bones. We have also foundtrephine biopsy generally much more useful thanbone marrow aspiration when investigating patientswith the advanced stages of HIV infection in whomhypocellular, non-diagnostic aspirates are common.It should not be forgotten, however, that trephinebiopsy undoubtedly causes more pain to the patientthan does aspiration.

Complications of bone marrow aspiration and tre-phine biopsy are very rare. Cardiac and great vessellaceration has been mentioned above. Otherwise,haemorrhage is uncommon but, when proceduresare carried out on patients with a haemostaticdefect, prolonged firm pressure is necessary afterwardsto ensure that bleeding has stopped. Haemorrhageis also occasionally a problem when a biopsy is car-ried out on bone with an abnormal vasculature, forexample in Paget’s disease. Severe retroperitonealhaemorrhage has also been observed in patientswith osteoporosis and in several patients with nor-mal bones but with haematological disorders.Damage to the lateral cutaneous nerve of the thighoccurs rarely and is suggestive of poor technique.Pneumothoraces have occurred following sternalaspiration and sternomanubrial separation has been observed in one patient. In patients withosteosclerosis, needles may break. Infection is a rarecomplication.

Other techniques

It is occasionally necessary to obtain a bone marrowspecimen by open biopsy under a general anaes-thetic. This is usually only required when a specificlesion has been demonstrated, by radiology or bonescanning, at a relatively inaccessible site.

At autopsy, specimens of bone marrow for histo-logical examination are most readily obtained fromthe sternum and the vertebral bodies, although anybone containing red marrow can be used. Unlessthe autopsy is performed soon after death, the cyto-logical detail is often poor.

Cellularity

Bone marrow cellularity can be assessed most ac-curately in histological sections (Fig. 1.7), althoughassessment can also be made on aspirated bonemarrow fragments in wedge-spread films (Fig. 1.8).

Specimens which are suitable for histological assess-ment of cellularity are: (i) aspirated fragments; (ii)needle or open biopsy specimens; and (iii) autopsyspecimens. The cellularity of the bone marrow inhealth depends on the age of the subject and the sitefrom which the marrow specimen was obtained. It is also influenced by technical factors, sincedecalcification and paraffin-embedding lead tosome shrinkage of tissue in comparison with plastic-embedded specimens; estimates of cellularity basedon the former are approximately 5% lower thanestimates based on the latter [7].

The cellularity of histological sections can beassessed most accurately by computerized imageanalysis or, alternatively, by point-counting usingan eyepiece with a graticule; the process is knownas histomorphometry. Results of the two proceduresshow a fairly close correlation [7,8]. Cellularity canalso be assessed subjectively. Such estimates are lessreproducible and may lead to some under-estimationof cellularity but show a reasonable correlation withhistomorphometric methods; in one study the meancellularity was 78% by histomorphometry (point-counting) and 65% by visual estimation, with the

8 CHAPTER ONE

Fig. 1.7 Section of normal BM:normal distribution of all threehaemopoietic lineages; note themegakaryocyte adjacent to asinusoid. Plastic-embedded, H&E ×188.

Fig. 1.8 Aspirate of normal BM:fragment showing normalcellularity. MGG ×377.

800

Age (years)

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10 20 30 40 50 60 70

THE NORMAL BONE MARROW 9

correlation between the two methods being 0.78[7]. Bone marrow cellularity is expressed as the per-centage of a section that is occupied by haemopoi-etic tissue. However, the denominator may vary.The cellularity of sections of fragments is expressedin terms of haemopoietic tissue as a percentage ofthe total of haemopoietic and adipose tissue. In thecase of a trephine biopsy, however, the cellularitymay be expressed either as a percentage of theentire biopsy (including bone) [9] or as a percentageof the marrow cavity [7,10]. There are advantagesin the latter approach, in which the area occupiedby bone is excluded from the calculation, since thepercentages obtained are then directly comparablewith measurements made on histological sectionsof aspirated fragments or estimates made from frag-ments in bone marrow films.

The bone marrow of neonates is extremely cellu-lar, negligible fat cells being present. Cellularitydecreases fairly steadily with age, with an acceler-ated rate of decline above the age of 70 years (Figs 1.9 and 1.10) [9–13]. The decreasing percent-age of the marrow cavity occupied by haemopoietictissue is a consequence both of a true decline in theamount of haemopoietic tissue and of a loss of bonesubstance with age requiring adipose tissue toexpand to fill the larger marrow cavity. In subjectswith osteoporosis this effect can be so great thateven young persons who are haematologically normal may have as little as 20% of their marrowcavity occupied by haemopoietic cells [11]. Inhaematologically normal subjects without bone disease, typical reported rates of decline in averagemarrow cellularity (expressed as a percentage ofhaemopoietic cells plus adipose cells) are: (i) from64% in the second decade to 29% in the eighthdecade in the iliac crest [10]; (ii) from 85% at age 20 to 40% at age 60, also in the iliac crest [11]; and(iii) from 66% at age 20 to 30% at age 80 in the sternum [13].

Bone marrow cellularity is determined also bythe bone on which the assessment is made. Study ofthe two tissues by the same techniques has shownthat the cellularity of lumbar vertebrae is, on aver-age, about 10% more than the cellularity of the iliaccrest [9]. Vertebrae are also more cellular than thesternum. Because of the considerable dependenceof the assessment of cellularity on methods of pro-cessing and counting, it is much more difficult to

make generalizations when different tissues havenot been assessed by the same techniques. Bennikeet al. [4], in comparing the two sites in 100 subjects,considered the sternum to be on average somewhatmore cellular than the iliac crest. However, compar-ison of the results of histomorphometric studies by different groups found that, comparing a singlestudy of the sternum with four studies of the iliaccrest, the sternum was generally less cellular [9–13].It should be noted that the lowest estimates of iliaccrest cellularity are from a study using decalcified,paraffin-embedded bone marrow specimens [10],while the highest estimates are from a study usingnon-decalcified plastic-embedded specimens [9].Some studies have been conducted on biopsy speci-mens [13] and others on specimens obtained atautopsy [9,10,12]. Because of such technical con-siderations it is difficult to make any generalizations

Fig. 1.9 Mean and 95% range of cellularity at variousages of anterior iliac crest bone marrow which has beendecalcified and paraffin-embedded. Cellularity isexpressed as a percentage of the bone marrow cavity.Calculated from Hartsock et al. [10].

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about normal bone marrow cellularity. However, itis possible to say that, except in extreme old age, cel-lularity of less than 20% indicates hypoplasia and,except in those less than 20 years of age, cellularityof more than 80% is likely to indicate hyperplasia.

In making a subjective assessment of the cellular-ity of films prepared from aspirates, the cellularityof fragments is of more importance than the cellu-larity of trails, although occasionally the presence ofquite cellular trails despite hypocellular fragmentssuggests that the marrow cellularity is adequate. Anaverage fragment cellularity between 25 and 75% isusually taken to indicate normality, except at theextremes of age.

Because of the variability of cellularity from oneintertrabecular space to the next, it is not possible to assess marrow cellularity if few fragments areaspirated or if a biopsy core is of inadequate size. In particular, a small biopsy sample containing only asmall amount of subcortical marrow does not allowassessment of cellularity since this area is often oflow cellularity, particularly in the elderly. A biopsyspecimen containing at least five or six intertra-becular spaces is desirable, not only for an adequateassessment of cellularity but to give a reasonableprobability of detecting focal bone marrow lesions.This requires a core of 20–30 mm in length. Smallerspecimens may fail to show a focal lesion that is present in the bone marrow (Fig. 1.11).

10 CHAPTER ONE

Fig. 1.10 Mean value of bone marrow cellularity atvarious ages expressed as percentage of bone marrowcavity: (a) iliac crest, autopsy, not decalcified (recalculatedfrom Frisch et al. [9]); (b) iliac crest, autopsy, notdecalcified [12]; (c) sternum, biopsy, not decalcified [13];(d) ilium, autopsy, decalcified [10].

Fig. 1.11 A section of a trephinebiopsy specimen of adequate sizefrom a patient with Hodgkin’sdisease showing only a small areaof infiltration at one end of thespecimen, illustrating how a smallbiopsy may miss focal lesions.Paraffin-embedded, H&E ×24. (By courtesy of Dr K Maclennan,Leeds.)

THE NORMAL BONE MARROW 11

Haemopoietic cells

A multipotent stem cell gives rise to all types ofmyeloid cell: (i) erythrocytes and their precursors;(ii) granulocytes and their precursors; (iii) macro-phages, monocytes and their precursors; (iv) mastcells; and (v) megakaryocytes and their precursors(Fig. 1.12). It should be mentioned that the term‘myeloid’ can be used with two rather differentmeanings. It is used to indicate all cells derived fromthe common myeloid stem cell and also to indicateonly the granulocytic and monocytic lineages, as inthe expression ‘myeloid:erythroid ratio’ (M:E). It is usually evident from the context which sense isintended but it is important to avoid ambiguity inusing this term. The common myeloid stem cell andstem cells committed to the specific myeloid lineagescannot be identified morphologically but it is likelythat they are cells of similar size and appearance to a lymphocyte. The various myeloid lineages differ

both morphologically and in their disposition in thebone marrow. The normal bone marrow contains,in addition to myeloid cells, smaller numbers oflymphoid cells (including plasma cells) and the stro-mal cells which have been discussed above.

Erythropoiesis

Cytology

Precursors of erythrocytes are designated erythro-blasts. The term normoblast can also be used but has a narrower meaning; ‘erythroblast’ includes allrecognizable erythroid precursors, whereas ‘nor-moblast’ is applicable only when erythropoiesis isnormoblastic. There are at least five generations oferythroblast between the morphologically unrecog-nizable erythroid stem cell and the erythrocyte.Erythroblasts develop in close proximity to a macro-phage, the cytoplasmic processes of which extend

Fig. 1.12 A semi-diagrammatic representation of granulopoiesis and erythropoiesis. Cell division occurs up to themyelocyte and intermediate erythroblast stages.

between and around individual erythroblasts. Several generations of erythroblast are associatedwith one macrophage, the whole cluster of cellsbeing known as an erythroblastic island [14]. Intact erythroblastic islands are sometimes seen inbone marrow films (Fig. 1.13). Erythroblasts areconventionally divided, on morphological grounds,into four categoriesaproerythroblasts and early, intermediate and late erythroblasts. An alternativeterminology is: proerythroblast, basophilic erythro-blast, early polychromatophilic erythroblast and latepolychromatophilic erythroblast. The term orthochro-matic erythroblast is better avoided since the mostmature erythroblasts are only orthochromatic (thatis acidophilic, with the same staining characteristicsas mature red cells) when erythropoiesis is abnormal.

Proerythroblasts (Fig. 1.14) are large round cellswith a diameter of 12–20 µm and a large roundnucleus. The cytoplasm is deeply basophilic with a pale perinuclear zone, attributable to the Golgiapparatus, sometimes being apparent. The nucleushas a finely granular or stippled appearance andcontains several nucleoli.

Early erythroblasts (Fig. 1.15) are smaller thanproerythroblasts and more numerous. The nucleo-cytoplasmic ratio is somewhat lower. They havestrongly basophilic cytoplasm and a granular orstippled chromatin pattern without visible nucleoli.A perinuclear halo, which is less strongly basophilicthan the rest of the cytoplasm, may be apparent.

Intermediate erythroblasts (Figs 1.14 and 1.15)

are smaller again, with a lower nucleocytoplasmicratio than that of the early erythroblast, less baso-philic cytoplasm and moderate clumping of thechromatin. They are more numerous than earlyerythroblasts.

Late erythroblasts (Figs 1.14 and 1.15) are smaller and more numerous than intermediate erythroblasts. They are only slightly larger thanmature red cells. Their nucleocytoplasmic ratio islower than that of the intermediate erythroblastand the chromatin is more clumped. The cytoplasmis only weakly basophilic and in addition has a pinktinge due to the increased amount of haemoglobin.Because of the resultant pinky-blue colour the cellis described as polychromatophilic.

Late erythroblasts extrude their nuclei to formpolychromatophilic erythrocytes, which are slightlylarger than mature erythrocytes. These cells can beidentified by a specific stain as reticulocytes; whenhaemopoiesis is normal they spend about 2 days oftheir 3-day life span in the bone marrow.

Small numbers of normal erythroblasts showatypical morphological features such as irregularnuclei, binuclearity and cytoplasmic bridging be-tween adjacent erythroblasts [15].

Histology

Erythroblastic islands (Figs 1.16 and 1.17) are recog-nizable as distinctive clusters of cells in which oneor more concentric circles of erythroblasts closely

12 CHAPTER ONE

Fig. 1.13 BM aspirate: an erythroidisland. MGG ×940.

Fig. 1.15 Aspirate of normal BM:early, intermediate and lateerythroblasts and a lymphocyte.MGG ×940.

Fig. 1.14 Aspirate of normal BM: a proerythroblast, intermediateerythroblast, four lateerythroblasts, a myelocyte, largeand small lymphocytes and aneutrophil. MGG ×940.

Fig. 1.16 Section of normal BM: an erythroid island (centre).Plastic-embedded, H&E ×377.

THE NORMAL BONE MARROW 13

surround a macrophage. The erythroblasts whichare closer to the macrophage are less mature thanthe peripheral ones. The central macrophage sendsout extensive slender processes which envelop each erythroblast. The macrophage phagocytosesdefective erythroblasts and extruded erythroblastnuclei; nuclear and cellular debris may therefore be recognized in the cytoplasm and a Perls’ stain(see page 54) may demonstrate the presence ofhaemosiderin. Erythropoiesis occurs relatively closeto marrow sinusoids although it is probable that, as

in the rat [16], only a minority of erythroblasticislands actually abut on sinusoids.

Early erythroblasts (Fig. 1.18) are large cells; theyhave relatively little cytoplasm and large nucleiwith dispersed chromatin and multiple, small, irreg-ular or linear nucleoli often abutting on the nuclearmembrane. The nuclei are rounder than those ofmyeloblasts but, in contrast to the nuclei of earlyerythroid cells in bone marrow aspirates of healthysubjects, in histological sections some appear ovoidor slightly irregular. More mature erythroid cells

14 CHAPTER ONE

Fig. 1.17 Section of normal BM: an erythroid island containingintermediate and late erythroblastsand a haemosiderin-ladenmacrophage; a Golgi zone is seen in some of the intermediateerythroblasts. Plastic-embedded,H&E ×970.

Fig. 1.18 Section of normal BM: an erythroid island containing earlyand intermediate erythroblasts.Plastic-embedded, Giemsa ×940.

THE NORMAL BONE MARROW 15

have condensed nuclear chromatin and cytoplasmwhich is less basophilic. The chromatin in the erythroblast nuclei is evenly distributed and, aschromatin condensation occurs, an even, regularpattern is retained.

There are four features which are useful in distin-guishing erythroid precursors in the marrow fromother cells: (i) in normal bone marrow they occur indistinctive erythroblastic islands containing severalgenerations of cells of varying size and maturity; (ii)erythroblasts adhere tightly to one another; (iii)

their nuclei are round; and (iv) in late erythroblaststhe chromatin is condensed in a regular mannerwhereas nuclei of small lymphocytes show coarseclumping. With a Giemsa stain (Fig. 1.19), theintense cytoplasmic basophilia with a small, neg-atively staining Golgi zone adjacent to the nucleus is also distinctive. In paraffin-embedded specimens(Fig. 1.20), artefactual shrinking of cytoplasm oflater erythroblasts can be useful in distinguishingthem from lymphocytes. Shrinkage artefact isabsent in plastic-embedded sections, in which the

Fig. 1.19 Section of normal BM:erythroid island containing threeearly, one intermediate andnumerous late erythroblasts; notethe cytoplasmic basophilia of earlyerythroblasts. Plastic-embedded,Giemsa ×940.

Fig. 1.20 Section of normal BM:erythroid island showingintermediate and late erythroblastswith haloes surrounding the nuclei.Paraffin-embedded, H&E ×940.

identification of erythroid cells is aided by their syn-cytial appearance (Fig. 1.21).

When the bone marrow is regenerating rapidly,erythroid islands may be composed of cells, all ofwhich are at the same stage of maturation. Thisresults in some islands consisting only of immatureelements. A similar pattern is sometimes seen when erythropoiesis is abnormal, for example inmyelodysplasia, in which the intramedullary deathof erythroblasts is a major mechanism.

The identification of abnormal erythroblasts can be more difficult than the identification of theirnormal equivalents, for example, if well-organizederythroblastic islands are not present or if they containonly immature cells. When there is any difficulty in recognizing erythroid precursors, their identitycan be confirmed by cytochemical or immunocyto-chemical staining (see page 67).

Granulopoiesis

Cytology

There are at least four generations of cells betweenthe morphologically unrecognizable committedgranulocyte–monocyte precursor and the maturegranulocyte but cell division does not necessarilyoccur at the same point as maturation from one

stage to another. The first recognizable granulo-poietic cell is the myeloblast (Figs 1.22 and 1.23). It is similar in size to the proerythroblast, about 12–20 µm. It is more irregular in shape than a proery-throblast and its cytoplasm is moderately rather thanstrongly basophilic. The chromatin pattern is diffuseand there are several nucleoli. Myeloblasts are gen-erally defined as being cells that lack granules but,in the context of the abnormal myelopoiesis ofacute myeloid leukaemia and the myelodysplasticsyndromes, primitive cells with granules may alsobe accepted as myeloblasts. Myeloblasts are capableof cell division and mature to promyelocytes.

Promyelocytes (Fig. 1.23) have a nucleolated,slightly indented nucleus, a Golgi zone and primaryor azurophilic granules which are reddish-purplewith a Romanowsky stain. Promyelocytes are largerthan myeloblasts, usually 15–25 µm, and theircytoplasm is often more strongly basophilic. By lightmicroscopy, promyelocytes of the three granulo-cytic lineages cannot be distinguished but, by ultrastructural examination, the distinction can bemade. Promyelocytes are capable of cell divisionand mature to myelocytes.

Myelocytes (Fig. 1.23) are smaller than promye-locytes and are quite variable in sizeafrom 10 to 20 µm. Their nuclei show partial chromatin con-densation and lack nucleoli. Their cytoplasm is less

16 CHAPTER ONE

Fig. 1.21 Syncytial appearance oferythroblasts in an erythroid islandin sections from a resin-embeddedtrephine biopsy specimen. Plastic-embedded, H&E ×592.

THE NORMAL BONE MARROW 17

basophilic than that of promyelocytes and specificneutrophilic, eosinophilic and basophilic granulescan now be discerned, staining lilac, orange–red andpurple, respectively. Eosinophil myelocytes mayalso contain some granules that take up basic dyesand stain purple; these differ ultrastructurally fromthe granules of the basophil lineage and are bestdesignated pro-eosinophilic granules. There areprobably normally at least two generations of mye-locytes so that at least some cells of this category are

capable of cell division. Late myelocytes mature tometamyelocytes which are 10–12 µm in diameterand have a markedly indented or U-shaped nucleus(Fig. 1.23). The metamyelocyte is not capable of cell division but matures to a band form which has a ribbon-shaped nucleus. The band cell, in turn,matures to a polymorphonuclear granulocyte witha segmented nucleus and specific neutrophilic,eosinophilic or basophilic granules. The bone mar-row is a major reservoir for mature neutrophils.

Fig. 1.22 Aspirate of normal BM: a myeloblast, three neutrophils andtwo monocytes; the myeloblast hasa high nucleocytoplasmic ratio, adiffuse chromatin pattern and anucleolus. MGG ×940.

Fig. 1.23 Aspirate of normal BM: a myeloblast and a promyelocyte(centre), a myelocyte (lower right),a metamyelocyte, band forms, aneutrophil and a late erythroblast;the promyelocyte is larger than themyeloblast and is showing somechromatin condensation but withpersisting nucleoli, well-developedcytoplasmic granulation and aGolgi zone. MGG ×940.

Histology

Myeloblasts (Fig. 1.24) are the earliest granulocyteprecursors identifiable histologically; they are pre-sent in small numbers and are most frequentlyfound adjacent to the bone marrow trabecular sur-faces or to arterioles. They are fairly large cells withround to oval nuclei and one to five relatively smallnucleoli. There is no chromatin clumping. Theyhave relatively little cytoplasm. They are readily dis-tinguished from lymphoid cells by the absence ofchromatin clumping and the presence of nucleoli.Myeloblasts are far outnumbered in normal mar-

rows by the promyelocytes (Figs 1.24 and 1.25) andmyelocytes (Fig. 1.25) which are recognized, atleast in plastic-embedded sections, by their granu-larity. Primary and neutrophilic granules may beseen as faintly eosinophilic granules in good qualityhaematoxylin and eosin (H&E) stains on sectionscut from plastic-embedded specimens, but they arebest seen with a Giemsa stain. Granules of cells ofeosinophil lineage are large, refractile and morestrongly eosinophilic. They are therefore easily re-cognized on both H&E and Giemsa stains of eitherparaffin- or plastic-embedded biopsies. Basophil gran-ules are water-soluble and, since trephine biopsy

18 CHAPTER ONE

Fig. 1.24 Section of normal BM:myeloblasts and promyelocytesadjacent to a bony trabecula.Plastic-embedded, H&E ×970.

Fig. 1.25 Section of normal BM:promyelocytes, myelocytes andmaturing neutrophils andeosinophils adjacent to a sinusoid.Plastic-embedded, H&E ×970.