Disorders of Erythropoiesis, Granulopoiesis and Thrombopoiesis

Peripheral blood

The peripheral blood shows initially a normocytic,normochromic anaemia and later, when thedeficiency is more severe, a hypochromic, micro-cytic anaemia. Red cells also show anisocytosis,anisochromasia and poikilocytosis, particularly thepresence of elliptocytes. Occasional patients showthrombocytosis, thrombocytopenia or the presenceof occasional hypersegmented neutrophils.

Bone marrow cytology

Bone marrow cellularity is mildly increased as aresult of a moderate degree of erythroid hyperpla-sia. Erythropoiesis is micronormoblastic with ery-throblasts being smaller than normal with scanty orragged cytoplasm or with cytoplasmic vacuolation(Fig. 8.1). There is a minor degree of dyserythro-poiesis. An iron stain shows siderotic granules to beseverely reduced or absent and there is a completeor virtual absence of the iron within macrophageswhich usually constitutes the body’s iron stores (seeFig. 2.1). Since iron is irregularly distributed in themarrow, a number of bone marrow fragments mustbe available for the performance of an iron stainbefore it can be concluded that storage iron is lack-ing. In iron deficiency, the bone marrow sometimesshows occasional giant metamyelocytes but granu-lopoiesis and thrombopoiesis are otherwise usuallynormal. Individuals whose bone marrow lacks storage iron but in whom erythropoiesis is normalshould be regarded as iron depleted rather than asiron deficient; a significant proportion of healthywomen fall into this group.

EIGHT

DISORDERS OF ERYTHROPOIESIS,GRANULOPOIESIS ANDTHROMBOPOIESIS

In this chapter we shall discuss non-neoplastichaematological disorders, both congenital andacquired, which affect predominantly a single lineageaeither erythroid, granulocytic or mega-karyocytic. For a more detailed discussion of theperipheral blood features, the reader is referred toBlood Cells: A Practical Guide [1]. In the majority ofthese conditions diagnosis is based on peripheralblood and bone marrow aspirate features and onsupplementary tests. In general a trephine biopsy isof little importance and is not often performed. Thechanges consequent on infection have been dis-cussed in Chapter 3 and will therefore not be dealtwith in this chapter.

Iron deficiency anaemia

Iron deficiency anaemia results from inadequateiron intake, increased loss of iron from the body or acombination of the two. Peripheral blood features,supplemented by biochemical assays, are oftensufficient for a definitive diagnosis. In more com-plicated cases bone marrow aspiration permits adefinitive diagnosis. Trephine biopsy is of little im-portance and, if iron is leached out during decalci-fication, histological sections can be misleading.

Useful biochemical tests in the diagnosis or irondeficiency include estimations of serum ferritin andserum iron concentration, the latter only if com-bined with an estimate of either transferrin concen-tration or serum total iron binding capacity. Serumferritin and serum iron concentrations are reduced,whereas serum transferrin concentration and totaliron binding capacity are increased. The concentra-tion of soluble serum transferrin receptors is alsoincreased but this test is not very specific for irondeficiency since concentration is also increased ifthere is increased erythropoiesis.

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ERYTHROPOIESIS, GRANULOPOIESIS, THROMBOPOIESIS 361

Bone marrow histology

Trephine biopsy sections show mild hypercellular-ity, erythroid hyperplasia and absent iron stores.Megakaryocytes are sometimes increased (Fig. 8.2).

Problems and pitfalls

An iron stain performed on a plastic-embedded,non-decalcified trephine biopsy section permitsreliable assessment of iron stores. However, itshould be noted that the decalcification needed for

paraffin-embedded biopsy specimens leads toleaching out of some or all of the iron. It is thereforepossible to exclude a diagnosis of iron deficiency ifstainable iron is present but it is not possible to statethat iron stores are absent or reduced. A diagnosis ofiron deficiency therefore cannot be made from abiopsy specimen that has been decalcified.

Sideroblastic anaemia

Sideroblastic anaemia as a feature of myelodysplas-tic syndromes (MDS)arefractory anaemia with ring

Fig. 8.1 BM aspirate, irondeficiency anaemia, showingerythroblasts with poorlyhaemoglobinized vacuolatedcytoplasm. MGG ×940.

Fig. 8.2 BM trephine biopsysection, iron deficiency anaemia.Paraffin-embedded, H&E ×376.

362 CHAPTER EIGHT

sideroblasts or primary acquired sideroblasticanaemiaahas been discussed in Chapter 4. Sidero-blastic anaemia may also be inherited or may be secondary to exogenous agents such as alcohol,chloramphenicol or certain drugs used in the treatment of tuberculosis. Congenital (inherited)sideroblastic anaemia occurs predominantly but not exclusively in males, most cases being caused by defects in synthesis of 5-amino-laevulinic acid.Other cases, in either males or females, are a fea-ture of Pearson’s syndrome (see page 407) or of thiamine-responsive megaloblastic anaemia (seepage 371). Sideroblastic anaemia is most readily diag-nosed from a bone marrow aspirate but diagnosis is also possible from sections of plastic-embeddedtrephine biopsy specimens.

Peripheral blood

Congenital and secondary sideroblastic anaemiasare associated with microcytosis and hypochromia(Fig. 8.3), in contrast to the macrocytosis which isusual when sideroblastic erythropoiesis is a featureof MDS. In some patients the peripheral blood filmis dimorphic with a mixture of hypochromic micro-cytes and normochromic normocytes. Congenitalsideroblastic anaemia varies in severity from mod-erate to severe. Secondary sideroblastic anaemia isof mild to moderate severity. In families in whichmales have sideroblastic anaemia, females may showa small population of hypochromic microcytes.


The bone marrow shows mild hypercellularity andmild erythroid hyperplasia. A proportion of the erythroblasts show micronormoblastic maturationand defective haemoglobinization with ragged orvacuolated cytoplasm (Fig. 8.4). An iron stainshows the presence of abnormal sideroblasts includ-ing frequent ring sideroblasts (Fig. 8.5). Iron storesare usually increased. Plasma cells may containhaemosiderin (see Fig. 2.8).


Trephine biopsy sections show some degree of erythroid hyperplasia. Increased storage iron andring sideroblasts are detectable in plastic-embeddedsections but not in sections of decalcified paraffin-embedded biopsy specimens. Plasma cells may con-tain haemosiderin (see Fig. 2.8). A trephine biopsyis not indicated in the investigation of suspectedcongenital sideroblastic anaemia but is useful ifacquired sideroblastic anaemia, particularly as a feature of MDS, is suspected.


Making a distinction between congenital andacquired sideroblastic anaemias and between prim-ary and secondary sideroblastic anaemias is notalways possible from the peripheral blood and bone

Fig. 8.3 PB film from a boy withcongenital sideroblastic anaemia,showing many hypochromic andmicrocytic cells; there is moderatepoikilocytosis and one cellcontaining multiple Pappenheimerbodies. MGG ×940.


marrow features alone. In some cases a family history, drug history and supplementary tests areneeded.

A diagnosis of sideroblastic anaemia cannot bemade from decalcified trephine biopsy specimens.

Thalassaemia trait and thalassaemiaintermedia

The various thalassaemic disorders, including tha-lassaemia trait, are most readily diagnosed fromperipheral blood features but it is necessary for

haematologists and pathologists to be aware of thebone marrow features to avoid misdiagnosis as otherconditions. Bone marrow aspiration and trephinebiopsy are not of any importance in the diagnosis.

Thalassaemia trait is the term used to describe anasymptomatic condition usually consequent ondysfunction of one of the two β genes or lack of oneor two of the four α genes. The term ‘thalassaemiaintermedia’ denotes a symptomatic condition, moresevere than thalassaemia trait, but in which bloodtransfusion is not generally necessary; the geneticbasis is diverse.

Fig. 8.4 BM aspirate from a boywith congenital sideroblasticanaemia (same case as in Fig. 8.3),showing granulocyte precursorsand five erythroblasts, one of which has a very severe defect inhaemoglobinization. MGG ×940.

Fig. 8.5 BM aspirate from a boywith congenital sideroblasticanaemia (same case as in Fig. 8.3),showing abnormal sideroblasts,including one ring sideroblast.Perls’ stain ×940.

364 CHAPTER EIGHT

Diagnosis of β thalassaemia trait is based on a typical blood count and blood film together withdemonstration of an elevated percentage ofhaemoglobin A2. There may or may not be an ele-vated percentage of haemoglobin F. The diagnosisof β thalassaemia intermedia is made on the basis ofclinical and haematological features, haemoglobinelectrophoresis and DNA analysis. A presumptivediagnosis of α thalassaemia trait is made when there is microcytosis that is not explained by othermore readily diagnosable conditions such as irondeficiency anaemia or β thalassaemia trait. Adefinitive diagnosis of α thalassaemia trait requiresDNA analysis, most cases being caused by deletionof one or more of the α genes.

Peripheral blood

In β thalassaemia trait, and in cases of α thalas-saemia trait in which two of the four α genes arelacking, the peripheral blood shows microcytosisand sometimes a degree of hypochromia. Some, butnot all, cases of β thalassaemia trait also havebasophilic stippling and moderate poikilocytosis,including the presence of target cells. In cases of αthalassaemia trait in which only one of the four αgenes is lacking, the haematological abnormalitiesare much less and the diagnosis may not be sus-pected. In β thalassaemia intermedia, the haemato-logical features are intermediate between those ofthalassaemia trait and thalassaemia major.


In thalassaemia trait, the bone marrow aspirateshows moderate erythroid hyperplasia. Erythro-poiesis is micronormoblastic and there is moderatedyserythropoiesis including nuclear lobulation andnuclei of irregular shape (Fig. 8.6). An iron stainshows increased siderotic granulation and occa-sional ring sideroblasts. Storage iron is commonlyincreased. In thalassaemia intermedia, erythroidhyperplasia and dyserythropoiesis are marked andstorage iron is increased.


Trephine biopsy sections show erythroid hyper-plasia and dyserythropoiesis.


Misdiagnosis of β thalassaemia intermedia as MDScan occur if the possibility of thalassaemia is notconsidered and if it is not appreciated that dysplasticfeatures are confined to the erythroid lineage.

Thalassaemia major

Thalassaemia major indicates a transfusion-depen-dent thalassaemic condition, usually consequent onhomozygosity or compound heterozygosity for βthalassaemia.

Fig. 8.6 BM aspirate, βthalassaemia trait, showingerythroid hyperplasia anddyserythropoiesis. There is a binucleated early erythroblast and the late erythroblasts are smalland have irregular or lobulatednuclei. MGG ×940.


Peripheral blood

The peripheral blood shows striking hypochromia,microcytosis, anisocytosis and poikilocytosis. Baso-philic stippling, Pappenheimer bodies and dysplas-tic circulating erythroblasts are also present. If the patient has been transfused, the blood film isdimorphic.


The bone marrow shows very marked erythroidhyperplasia, severe erythroid dysplasia and poorhaemoglobinization (Fig. 8.7). Some erythro-blasts contain cytoplasmic inclusions, seen with difficulty in MGG-stained films, which represent precipitated α chains. There is an increase inmacrophages which contain degenerating ery-throblasts, cellular debris and haemosiderin. Insome patients the increased cell turnover leads tothe formation of pseudo-Gaucher cells and sea-blue histiocytes (see pages 416 and 420). An ironstain shows numerous abnormal sideroblasts andsmall numbers of ring sideroblasts. Storage iron isconsiderably increased. Plasma cells may containhaemosiderin.


Bone marrow sections show marked erythroidhyperplasia with disappearance of fat spaces.

Dyserythropoiesis is also very marked and ironstores are increased. Pseudo-Gaucher cells and sea-blue histiocytes may be present. Plasma cells andthe endothelial cells lining sinusoids may containhaemosiderin.

Haemoglobin H disease

Haemoglobin H disease is a thalassaemic conditionresulting from the lack of three of the four α genesor from a functionally similar defect. There is also a decreased red cell life span. Diagnosis rests onperipheral blood features and the results of hae-moglobin electrophoresis; bone marrow examina-tion contributes little. Haemoglobin electrophoresisshows a small percentage of haemoglobin H andhaemoglobin H inclusions are seen within red cellsthat have been exposed to a suitable supravital dye.Occasionally, haemoglobin H disease is an acquiredcondition, occurring as a feature of MDS.

Peripheral blood

The peripheral blood shows marked hypochromia,microcytosis, anisocytosis and poikilocytosis. Be-cause of the haemolytic component, there is also poly-chromasia and the reticulocyte count is elevated.


The bone marrow is hypercellular with marked ery-

Fig. 8.7 BM aspirate, βthalassaemia major, showingerythroid hyperplasia anddyserythropoiesis. Several cellscontain cytoplasmic inclusions,composed of precipitated α chains.MGG ×940.

366 CHAPTER EIGHT

throid hyperplasia, defective haemoglobinizationand some dyserythropoietic features (Fig. 8.8).


Bone marrow sections show hypercellularity due toerythroid hyperplasia.


It is important to distinguish acquired haemoglobinH disease from the much more common inheritedcondition. This is possible by examination of cells ofother haemopoietic lineages.

Haemolytic anaemias

Haemolytic anaemia may be inherited or acquired.Aetiological factors, pathogenetic mechanisms andmorphological features are very varied [1]. Exam-ination of the peripheral blood is of great impor-tance in the diagnosis but examination of the bonemarrow adds little, except in detecting complicatingmegaloblastic anaemia or pure red cell aplasia or,occasionally, an associated lymphoma.

Peripheral blood

Haemolytic anaemias have in common poly-chromasia and an increased reticulocyte count.Macrocytosis is usual in those patients in whomhaemolysis is chronic and severe. Other morpho-

logical features are very variable, depending on theprecise nature of the condition [1].


The bone marrow is hypercellular as a consequenceof erythroid hyperplasia (Fig. 8.9). The degree of hyperplasia reflects the extent to which the redcell life span is shortened. In some patients, fat cellsare totally lost. Haemopoiesis is often macronor-moblastic, i.e. the erythroblasts are increased in sizebut have nuclear and cytoplasmic characteristicssimilar to those of normoblasts. Some cases ofhaemolytic anaemia have quite marked dysery-thropoiesis. This may occur transiently in auto-immune haemolytic anaemia [2] and has also beenobserved in haemolytic anaemia associated with the familial auto-immune lymphoproliferative dis-order caused by FAS gene mutations [3]. Dysery-thropoiesis is often very striking when severehaemolytic anaemia occurs in a neonate, e.g. inhaemolytic disease of the newborn. A specificdyserythropoietic feature is associated with hae-molytic anaemia due to haemoglobin C disease; normoblasts have irregular nuclear membranes(Fig. 8.10). Macronormoblastic erythropoiesisshould be distinguished from mildly megaloblasticerythropoiesis which may occur in the haemolyticanaemias when there is complicating folic aciddeficiency. When haemolysis is extravascular, bonemarrow macrophages are increased and containcellular debris. Iron stores are commonly increased,

Fig. 8.8 BM aspirate, haemoglobinH disease, showing markederythroid hyperplasia withmicronormoblastic maturation.MGG ×376.


except when there is severe intravascular haemo-lysis with consequent loss of iron from the body.Siderotic granulation is somewhat increased.


The bone marrow is hypercellular with erythroidhyperplasia (Fig. 8.11) and a variable degree ofdyserythropoiesis. The number of erythroid islandsis increased and the central macrophage is large andprominent, often staining a dirty greenish colourwith a Giemsa stain because of the presence of

increased haemosiderin. An iron stain confirmsincreased storage iron.


Misinterpretation of erythroid hyperplasia with avariable degree of dyserythropoiesis, which is a con-sequence of haemolytic anaemia, is possible if aperipheral blood film is not examined as part of theassessment of a bone marrow aspirate and trephinebiopsy. This may result in a failure to considerhaemolysis as a diagnostic possibility.

Fig. 8.9 BM aspirate, auto-immune haemolytic anaemia,showing an erythroid islandcomposed of erythroblasts clusteredaround a debris-laden macrophage.MGG ×940.

Fig. 8.10 BM aspirate,haemoglobin C disease, showingerythroid hyperplasia and anirregular nuclear outline which ischaracteristic of this condition.MGG ×940.

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Congenital dyserythropoietic anaemia

The congenital dyserythropoietic anaemias (CDAs)are a diverse group of inherited conditions [4,5], allof which are characterized by anaemia resul-ting from dysplastic and ineffective erythropoiesis.Splenomegaly and expansion of the bone marrowcavity are common. Three major types of CDA havebeen recognized but a considerable number of casesnot conforming to these categories have also beendescribed. Both peripheral blood and bone marrow

aspirate features are important in making the diag-nosis. In type II CDA, demonstration of a positiveacidified serum lysis test, using a number of normalsera to exclude false-negative results, is required forconfirmation. Trephine biopsy is not important indiagnosis.

Peripheral blood

Specific morphological features vary, depending onthe category of CDA (Table 8.1). All are character-

Fig. 8.11 BM trephine biopsysection, auto-immune haemolyticanaemia, showing erythroidhyperplasia. Paraffin-embedded,H&E ×97.

Table 8.1 Genetic, peripheral blood and bone marrow features of the congenital dyserythropoietic anaemias.

Inheritance

Peripheral blood

Bone marrow

* Hereditary erythroid multinuclearity with positive acidified serum test.

Type III

Autosomal dominant

Mild anaemia, macrocytosis,marked anisocytosis andpoikilocytosis, basophilicstippling

Hyperplastic, megaloblastic,giant erythroblasts withsingle nuclei or marked multinuclearity—up to adozen nuclei per cell,karyorrhexis

Type II (Hempas)*

Autosomal recessive

Mild to severe anaemia,normocytic red cells, moderateanisocytosis and poikilocytosis,including teardrop poikilocytes,variable anisochromasia,irregularly contracted cells,basophilic stippling

Hyperplastic, normoblastic,marked binuclearity andmultinuclearity

Type I

Autosomal recessive

Mild to moderate anaemia,macrocytosis, marked anisocytosisand poikilocytosis includingteardrop poikilocytes, basophilicstippling

Hyperplastic, megaloblastic,moderate binuclearity andinternuclear chromatin bridges,nuclear budding and karyorrhexis


ized by anisocytosis and by poikilocytosis (Fig. 8.12)which often includes the presence of fragments andirregularly contracted cells. Basophilic stippling iscommon. In all categories, the reticulocyte count isnot elevated appropriately for the degree of anaemia.


Bone marrow features characteristic of the differentcategories of CDA are summarized in Table 8.1 andillustrated in Figs 8.13–8.15. In all types there iserythroid hyperplasia and dyserythropoiesis. Intype II CDA the increase in cell turnover is such that

pseudo-Gaucher cells may be present. Iron storesare commonly increased. Ultrastructural examina-tion of bone marrow cells is diagnostically useful,showing a ‘Swiss cheese’ appearance of the nucleusin CDA type I, a double membrane parallel to thecell membrane in CDA type II and a variety ofdefects in CDA type III [5].


Examination of trephine biopsy or bone marrowclot sections confirms erythroid hyperplasia anddyserythropoiesis (Fig. 8.16).

Fig. 8.12 PB film, congenitaldyserythropoietic anaemia, type I,showing macrocytosis, markedanisocytosis and poikilocytosis.MGG ×940.

Fig. 8.13 BM aspirate, congenitaldyserythropoietic anaemia, type I,showing erythroid hyperplasia and dyserythropoietic featuresincluding two pairs of erythroblastsjoined by cytoplasmic and nuclearbridges, respectively. The cell (top right) with two nuclei joinedtogether over a considerabledistance is typical of this type of CDA. MGG ×940.

Fig. 8.14 BM aspirate, congenitaldyserythropoietic anaemia, type II, showing one binucleateerythroblast and one erythroblastwith a multilobulated nucleus.MGG ×940.

Fig. 8.15 BM aspirate, congenitaldyserythropoietic anaemia, type III,showing giant, multinucleatederythroblasts. MGG ×940.(By courtesy of Professor SNWickramasinghe, London.)

Fig. 8.16 BM clot section,congenital dyserythropoieticanaemia, type I, showing markederythroid hyperplasia with largenumbers of immature erythroidprecursors and markeddyserythropoiesis. The chromatinpattern is very abnormal. Paraffin-embedded, H&E ×390.

370 CHAPTER EIGHT



Some cases of CDA present quite late in life.Misdiagnosis as MDS may occur if the possibility of CDA is not considered and if due consideration is not given to the fact that the abnormalities areessentially confined to the erythroid lineage.

Megaloblastic anaemia

Megaloblastic anaemia is usually consequent on adeficiency of vitamin B12 or folic acid. Less often, itis attributable to administration of a drug that inter-feres with DNA synthesis or, rarely, to a congenitalmetabolic defect. Some patients with acute myeloidleukaemia (AML) or MDS also have megaloblasticerythropoiesis. The presence of megaloblasticanaemia can usually be suspected from examina-tion of the peripheral blood and, if features aretotally typical, bone marrow aspiration is often notdone. The ready availability of accurate assays forvitamin B12 and folic acid has lessened the im-portance of bone marrow examination. However, iftypical peripheral blood features of megaloblasticerythropoiesis are lacking or if atypical features arepresent, bone marrow aspiration should be per-formed. Further tests indicated in patients withmegaloblastic anaemia are assays of serum vitaminB12 and red cell folate followed, when appropriate,by tests for auto-antibodies and a Schilling test. If pernicious anaemia is suspected, tests for par-ietal cell and intrinsic factor antibodies are indi-cated; the former is more sensitive but less specificthan the latter. If coeliac disease is suspected as acause of malabsorption of folic acid or vitamin B12,tests indicated include those for relevant auto-antibodies (antireticulin and antiendomysium anti-bodies), antigliadin antibodies and a small bowelbiopsy.

Peripheral blood

In most cases there is a macrocytic anaemia, withoval macrocytes being particularly characteristic.The mildest cases have macrocytosis withoutanaemia. Some degree of anisocytosis and poikilo-cytosis is usual and, when anaemia is severe, thereare striking morphological abnormalities includingthe presence of teardrop poikilocytes, fragments,basophilic stippling and occasional Howell–Jolly

bodies and circulating megaloblasts. Hyperseg-mented neutrophils are usually present; they arehighly suggestive of megaloblastic erythropoiesisalthough not pathognomonic. They persist for aweek or more after commencement of vitamin B12

or folic acid therapy. There may also be increasednumbers of macropolycytes (tetraploid neutrophils)but this feature is less strongly associated withmegaloblastic erythropoiesis. In severe megaloblas-tic anaemia, leucopenia and thrombocytopenia alsooccur.


The bone marrow is hypercellular, often markedlyso. Erythropoiesis is hyperplastic and is character-ized by the presence of megaloblasts (Fig. 8.17).These are large cells with a chromatin pattern moreprimitive than is appropriate for the degree of mat-uration of the cytoplasm. Late megaloblasts may be fully haemoglobinized and lack any cytoplasmicbasophilia. They may therefore be described asorthochromatic, a term which is not really appro-priate in describing normal erythropoiesis, in whichthe most mature erythroblasts are polychromatic.Erythropoiesis is ineffective so that early erythroidcells are over-represented in comparison withmature cells; macrophages are increased and con-tain defective red cell precursors and cellular debris. An iron stain shows abnormally prominentsiderotic granules and sometimes occasional ringsideroblasts. Storage iron is usually increased.Plasma cells may contain iron. The mitotic count isincreased and examination of cells in metaphasemay show that chromosomes are unusually long.

Granulopoiesis is also hyperplastic although lessso than erythropoiesis. Giant metamyelocytes areusually present (Fig. 8.17). They are twice to threetimes the size of a normal metamyelocyte and oftenhave nuclei of unusual shapes, e.g. E- or Z-shapedrather than U-shaped. Myelocytes and promyelo-cytes are also increased in size but this abnormalityis less obvious and less distinctive than the abnor-mality of metamyelocytes. When megaloblastic fea-tures in erythroblasts are partly or largely maskedby co-existing iron deficiency, the detection of giantmetamyelocytes is diagnostically important.

Megakaryocytes are hypersegmented and havemore finely stippled chromatin than normalmegakaryocytes.

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There is a variable hypercellularity with loss of fatcells. In some cases this can be so severe that it mayresemble the ‘packed marrow’ appearance seen inacute leukaemia on examination at low power.There is erythroid hyperplasia with predominanceof immature precursors (Figs 8.18 and 8.19). Theearly erythroid cells have large, round-to-ovalnuclei with one or more basophilic nucleoli whichoften appear to have rather irregular margins and

often abut on the nuclear membrane (Fig. 8.19);there is usually a moderate amount of intenselybasophilic cytoplasm. Small Golgi zones may beseen. The later erythroid cells show asynchrony of nuclear and cytoplasmic maturation with cellshaving immature nuclei but haemoglobinized cyto-plasm. Granulocytic precursors are increased butmay appear relatively inconspicuous in the presenceof profound erythroid hyperplasia. Giant metamyel-ocytes are usually easily seen (Fig. 8.19). Megakary-ocyte numbers may be normal or decreased.

Fig. 8.18 BM trephine biopsysection, megaloblastic anaemia,showing marked erythroidhyperplasia with numerous early,intermediate and late megaloblasts.Giant metamyelocytes are alsopresent. Paraffin-embedded, H&E ×390.

Fig. 8.17 BM aspirate,megaloblastic anaemia, showinghyperplastic megaloblasticerythropoiesis and a giantmetamyelocyte. MGG ×940.



It is critically important that a bone marrow aspiratein severe megaloblastic anaemia is not interpretedas AML. The likelihood of these errors has probablyincreased in recent years as haematologists havehad less experience in interpreting bone marrowsfrom patients with straightforward megaloblasticanaemia. An appearance of ‘maturation arrest’ andgross dyserythropoiesis may suggest acute leu-kaemia but these are also features of severe mega-loblastosis. Confusion with M6 AML should notoccur since the bone marrow in megaloblasticanaemia does not have any increase in myeloblasts.However, confusion may occur with M6 variantAML in which the primitive cells present are all erythroid (see page 154). It is important that thediagnosis of megaloblastic anaemia is always con-sidered in any such patient. Hypersegmented neu-trophils and giant metamyelocytes should be soughtsince they are not a feature of AML. If there is any real doubt as to the correct diagnosis, a trial ofvitamin B12 and folic acid therapy should be given.

Examination of a trephine biopsy specimen israrely useful in the diagnosis of megaloblasticanaemia but it is important for pathologists to beable to recognize the typical histological features sothat misdiagnosis, particularly as acute leukaemia,does not occur. Megaloblastic change in biopsy

sections may be mistaken for acute leukaemia if thebiopsy is reported without referring to the bloodfilm and marrow aspirate findings and if the possi-bility of megaloblastic anaemia is not considered.Less often, there may have been failure to obtain anaspirate or the presence of immature cells in theperipheral blood in a patient with complicating in-fection may have given rise to the clinical suspicionof leukaemia; in these circumstances misdiagnosisof leukaemia is more likely [6].

Erythroid islands composed of early megalo-blasts are also sometimes mistaken for clusters of carcinoma cells or for ‘abnormal localization ofimmature precursors’ in MDS (see Fig. 4.41). Ifthere is any real doubt as to their nature, immuno-histochemistry can be used.

Anaemia of chronic disease

The anaemia of chronic disease is characterized by a normocytic, normochromic anaemia or, when more severe, by a hypochromic, microcyticanaemia. Such anaemia is secondary to infection,inflammation or malignancy. Diagnosis is usuallybased on peripheral blood features and biochemicalassays. Serum iron and transferrin are reducedwhereas serum ferritin is normal or increased.Serum transferrin receptor concentration tends tobe normal. A bone marrow aspirate is sometimes

Fig. 8.19 BM trephine biopsysection, megaloblastic anaemia,showing several early megaloblastswith prominent, often elongatednucleoli which frequently abut onthe nuclear membrane. Numerouslate megaloblasts are also seen.Plastic-embedded, H&E ×970.

necessary to confirm or exclude co-existing irondeficiency in a patient who has features of anaemiaof chronic disease. A bone marrow biopsy does notusually give diagnostically useful information.

Peripheral blood

In addition to the possible occurrence of hypo-chromia and microcytosis, the peripheral bloodusually shows increased rouleaux formation andsometimes increased background staining due to areactive increase in various serum proteins. Theerythrocyte sedimentation rate is increased.


The bone marrow is usually of normal cellularity.Erythropoiesis may show no specific abnormality ormay be micronormoblastic with defective haemo-globinization. An iron stain shows storage iron to be increased, often markedly so when the condi-tion is very chronic. Erythroblasts show reduced or absent siderotic granulation. The bone marrowoften shows non-specific inflammatory changesincluding increased plasma cells, mast cells andmacrophages.


Sections of bone marrow trephine biopsy cores usually show normal cellularity. There may beincreased lymphoid nodules, plasma cells, mast cellsand macrophages. An iron stain shows increasedstorage iron.


An iron stain may be falsely negative if a trephinebiopsy specimen has been decalcified, leading to a mistaken assumption that the patient has irondeficiency anaemia.

Sickle cell anaemia and other sickling disorders

Sickle cell anaemia denotes the disease resultingfrom homozygosity for the βS genes and the consequent replacement of haemoglobin A byhaemoglobin S. Diagnosis of sickle cell anaemia isdependent on peripheral blood features and

haemoglobin electrophoresis or an equivalent tech-nique. Haemoglobin S comprises almost all the totalhaemoglobin, haemoglobin A being absent. Bonemarrow aspiration is usually only indicated todetect suspected complications such as megalo-blastic anaemia, pure red cell aplasia or bone mar-row necrosis. Trephine biopsy is not often indicated.The clinical features consequent on sickling of red blood cells can also be found in various com-pound heterozygous states, such as sickle cell/haemoglobin C disease and sickle cell/β thalas-saemia trait.

Peripheral blood

The peripheral blood shows anaemia, usually with a haemoglobin concentration of 6–10 g/dl. Thereare variable numbers of sickle cells and, in addition,target cells and polychromasia; nucleated red bloodcells may be present. After the age of 6 months, features of hyposplenism start to appear, par-ticularly Howell–Jolly bodies and Pappenheimerbodies. The neutrophil count may be increased, particularly during episodes of sickling. The bloodfilm in compound heterozygous states is often similar to that of sickle cell anaemia, althoughpatients with sickle cell/haemoglobin C disease mayhave occasional cells containing haemoglobin Ccrystals and those with sickle cell/β thalassaemiahave microcytosis.


The bone marrow aspirate shows hypercellularitydue to erythroid hyperplasia. Iron stores are oftenincreased and sickle cells are usually present. Some-times they are much more elongated than is usualfor sickle cells in the circulating blood. When thereare complicating conditions, such as megaloblasticanaemia, pure red cell aplasia or bone marrownecrosis, the appropriate morphological featuresare superimposed on those of the underlying disease. Bone marrow macrophages may containoccasional or numerous sickle cells (Fig. 8.20).Macrophages and various storage cells are some-times increased (Figs 8.20 and 8.21) as a con-sequence of increased cell turnover and episodes of bone marrow infarction. In sickle cell/β thalas-saemia, erythropoiesis is hyperplastic and micro-normoblastic (Fig. 8.22).

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Fig. 8.20 BM aspirate, sickle cell anaemia, showing a foamymacrophage and a macrophagecontaining a sickled cell. MGG×940. (By courtesy of ProfessorSally Davies, London.)

Fig. 8.21 BM aspirate, sickle cellanaemia, showing erythroidhyperplasia, a partially sickled cell and a sea-blue histiocyte. MGG ×940.

Fig. 8.22 BM aspirate, sickle cell/β thalassaemia compoundheterozygosity, showing erythroidhyperplasia and erythroblasts withscanty cytoplasm and defectivehaemoglobinization; several sicklecells are present. MGG ×940.


376 CHAPTER EIGHT


Trephine biopsy sections show hypercellularity dueto erythroid hyperplasia. During episodes of sick-ling, sickle cells may be seen within bone marrowsinusoids (Fig. 8.23). As for bone marrow aspiratefilms, sickle cells in bone marrow sections may bemuch more elongated than the typical sickle cellsthat are seen in blood films (Fig. 8.24). Infarctedbone and bone marrow may be present in patientswho are experiencing a sickling crisis, and foamymacrophages and small fibrotic scars may mark thesites of previous bone marrow infarction.


It should be noted that at autopsy sickle cells may bepresent in histological sections of bone marrow, notonly in patients with sickle cell disease but also inthose with sickle cell trait.

Pure red cell aplasia (includingBlackfan–Diamond syndrome)

Pure red cell aplasia has been defined as severeanaemia with the reticulocyte count being less than1% and mature erythroblasts in a normocellular

Fig. 8.23 BM trephine biopsysection, sickle cell anaemia,showing sinusoids distended byirreversibly sickled erythrocytes.Paraffin-embedded, H&E ×377.

Fig. 8.24 Section of clotted BMaspirate showing macrophagesstuffed with sickle cells and severalextremely long sickled cells.Paraffin-embedded, H&E ×940.


bone marrow being less than 0.5% [7]. Pure red cellaplasia may be either constitutional or acquired andeither acute or chronic. Constitutional pure red cell aplasia, also known as the Blackfan–Diamondsyndrome, is a chronic condition which usuallybecomes manifest during the first year of life. Itappears to be a trilineage disorder, consequent onan inherited stem cell defect, rather than a purelyerythroid disorder. It shows some responsiveness tocorticosteroids. Inheritance is usually autosomaldominant, with variable penetrance, but some casesare autosomal recessive or occur sporadically. Asignificant proportion of cases are due to a mutationin the gene encoding ribosomal protein S19 at19q13 [8]. A small percentage of patients with the Blackfan–Diamond syndrome subsequentlydevelop bone marrow aplasia [9], myelodysplasia[9] or AML. Infants, usually but not always over 1year of age, may also suffer from acute pure red cellaplasia designated transient erythroblastopenia ofchildhood [10,11]; in this condition the aplasia,which is consequent on infection by human her-pesvirus 6 [12], lasts only a matter of months anddoes not require specific treatment. In older chil-dren and adults, the most commonly recognizedcause of acute aplasia is parvovirus infection; theaplasia is usually of brief duration and thereforecauses symptomatic anaemia only in subjects with apre-existing red cell defect associated with a short-ened red cell life span. In adults, chronic aplasia iscommonly immunological in origin and may beassociated with a thymoma or with auto-immunedisease. Rare cases are associated with pregnancy[13]. Pure red cell aplasia can also be a complicationof T-cell granular lymphocytic leukaemia [14]; thelymphoproliferative disorder may be occult. In oneseries, almost 20% of cases of pure red cell aplasiawere attributed to large granular lymphocyteleukaemia (LGLL) [15]. Chronic parvovirus B19infection can also cause chronic pure red cell aplasiaparticularly, but not exclusively, in patients withevident causes of immune deficiency such as con-genital immune deficiency, HIV infection or theprior administration of immunosuppressive drugs[16]. Cases have occurred following renal and bonemarrow transplantation, following chemotherapyfor solid tumours [17] and Waldenström’s macro-globulinaemia [18], and during maintenance treat-ment for acute lymphoblastic leukaemia (ALL).

Marked erythroid hypoplasia may also be a featureof protein-calorie deprivation (kwashiorkor), beinduced by hypothermia [19], occur as part of ahypersensitivity reaction to a drug, or be the dom-inant feature of MDS.

Peripheral blood

The peripheral blood shows no specific abnormal-ity. There is a complete absence of polychromaticcells and the reticulocyte count is zero or virtuallyzero. Associated features differ according to thecause of the red cell aplasia. Macrocytosis is usual inthe Blackfan–Diamond syndrome and the red cellshave some characteristics similar to those of fetalred cells; occasionally, there is mild neutropenia andthe platelet count may be somewhat elevated [10].In transient erythroblastopenia of childhood, the red cells are of normal size and lack fetal character-istics; neutropenia, which may be moderately severe,occurs in about a quarter of cases and thrombocy-tosis in about a third [10,11]. Since symptomaticanaemia following parvovirus-induced aplasia islargely confined to patients with an underlying redcell defect, the blood film usually shows features ofan associated disease, most often hereditary sphero-cytosis or sickle cell anaemia. In such cases theabsence of polychromasia, despite marked anaemia,is diagnostically important and should lead to areticulocyte count being performed. Neutrophil andplatelet counts are only occasionally reduced inpatients with parvovirus-induced red cell aplasia.Patients with red cell aplasia associated with thy-moma or with auto-immune disease sometimesalso have neutropenia or thrombocytopenia. Inpatients with red cell aplasia as the dominant feature of MDS it is sometimes possible to detectdysplastic features in cells of other lineages.


Bone marrow cellularity is usually somewhat re-duced. There is a striking reduction of maturing ery-throid cells. Proerythroblasts are present in normalnumbers and sometimes they may be increased(Fig. 8.25). Other lineages are usually normal. InBlackfan–Diamond syndrome (Fig. 8.26) there arescattered proerythroblasts and sometimes minimalevidence of maturation; haematogones and lym-

Fig. 8.27 BM aspirate, chronicpure red cell aplasia caused byparvovirus B19 infection in an HIV-positive child. MGG ×940.

Fig. 8.25 BM aspirate, chronicidiopathic pure red cell aplasia,showing increased proerythroblastsand a lack of maturingerythroblasts. MGG ×940.

Fig. 8.26 BM aspirate,Blackfan–Diamond syndrome,showing a single intermediateerythroblast but no maturing cells.MGG ×940. (By courtesy of Dr R Brunning, Minneapolis.)

378 CHAPTER EIGHT


Fig. 8.28 BM trephine biopsysection, pure red cell aplasia,showing absence of erythroblasticislands and late erythroblasts; onlyoccasional early and intermediateerythroblasts are present. Paraffin-embedded, H&E ×390.

Fig. 8.29 BM trephine biopsysection, Blackfan–Diamondsyndrome, showing prominentproerythroblasts and earlyerythroblasts but very fewmaturing erythroid cells (same case as in Fig. 8.26). H&E ×940.(By courtesy of Dr R Brunning,Minneapolis.)

phocytes may be increased [20]. In transient ery-throblastopenia of childhood, granulopoiesis maybe left shifted and there may be an infiltrate ofimmature lymphocytes [12]. In parvovirus-inducedaplasia, giant proerythroblasts with prominentnucleoli are often noted (Fig. 8.27). Iron stores arecommonly increased since the iron normally in erythroid cells has been deposited in the stores.


The overall bone marrow cellularity is somewhatreduced. There is a striking lack of erythroid islandsand of maturing erythroblasts (Figs 8.28 and 8.29).

Large proerythroblasts with strongly basophiliccytoplasm are readily apparent. Occasionally, thereis a striking increase in proerythroblasts (Fig. 8.30).In parvovirus infection, the giant proerythroblastsmay show intranuclear eosinophilic degenera-tion with peripheral condensation of chromatin(Fig. 8.31). In immunocompetent patients the bonemarrow is hypercellular and megakaryocytes areincreased [20]. Immunohistochemistry can be usedto show parvovirus antigens.


Since red cell aplasia may be the major manifestation

380 CHAPTER EIGHT

of MDS, it is important to examine other lineagescarefully for dysplastic features.

Occasionally, patients with pure red cell aplasiashow a striking increase of proerythroblasts whichcan cause diagnostic confusion (see Fig. 8.30).

Congenital neutropenia

Severe congenital neutropenia (Kostmann’s syn-drome) is a heterogeneous group of disorders witheither autosomal dominant or autosomal recessiveinheritance. Isolated congenital neutropenia mayalso be mild with a benign clinical course. Con-

genital neutropenia may be cyclical with variationover a period of 3 weeks or more from very low to normal or above normal levels. Congenital neu-tropenia also occurs as a feature of other congenitalsyndromes.

Peripheral blood

In Kostmann’s syndrome, the peripheral bloodshows severe neutropenia and often monocytosis,eosinophilia and the effects of chronic or recurrentinfection such as anaemia and increased rouleauxformation.

Fig. 8.30 BM trephine biopsysection, chronic pure red cellaplasia, probably auto-immune innature, showing a striking increaseof proerythroblasts. H&E ×940. (Bycourtesy of Dr Haley, Vancouver.)

Fig. 8.31 BM trephine biopsysection in chronic parvovirus B19-induced pure red cell aplasia in an HIV-positive child (same case as in Fig. 8.27), showingseveral apoptotic cells and aproerythroblast with aneosinophilic intranuclear inclusion.Paraffin-embedded, H&E ×960.


Bone marrow cytology and histology

Most cases of Kostmann’s syndrome show an arrest at the promyelocyte stage of differentiation(Fig. 8.32). Haematogones may be increased. Somecases show a severe reduction of all granulopoieticcells with residual cells sometimes being mor-phologically atypical. The latter pattern may be predictive of failure of response to granulocytecolony-stimulating factor (G-CSF) therapy [21]. Anassociation between Kostmann’s syndrome andosteoporosis has been observed [22].

In Schwachman’s syndrome (congenital neutro-penia with exocrine pancreatic insufficiency), thebone marrow may show apparent maturation arrest[23]. In cyclical neutropenia there is myeloid hypo-plasia during the neutropenic phase but, when theneutrophil count is normal, the bone marrowappears normal. In neutropenia associated withCohen’s syndrome bone marrow examinationshows left-shifted granulopoiesis [24].

Agranulocytosis

Agranulocytosis is an acute, severe, reversible lackof circulating neutrophils consequent on an idio-syncratic reaction to a drug or chemical. At leastsome cases result from the development of anti-bodies against the causative drug with destructionof neutrophils being caused by the interaction of theantibody and the drug. However, some cases may

result from abnormal metabolism of a drug so thattoxic levels develop when normal doses are admin-istered. Clinical features are due to sepsis con-sequent on the neutropenia.

Peripheral blood

The neutrophil count is greatly reduced, usually to less than 0.5 × 109/l. Residual neutrophils may be morphologically normal but often they showtoxic changes consequent on superimposed sepsis.During recovery there is an outpouring of imma-ture granulocytes into the peripheral blood, consti-tuting a leukaemoid reaction.


The bone marrow aspirate shows a marked reduc-tion in mature neutrophils. Sometimes myelocytesare also greatly reduced. The degree of granulocytecompartment depletion is predictive of speed ofrecovery; if promyelocytes and myelocytes are pres-ent, recovery usually occurs in 4–7 days, withoutadministration of growth factors, whereas if pro-myelocytes and myelocytes are absent recoverytakes 14 days or more [25]. In severe cases withsuperimposed sepsis the majority of cells of granu-locytic lineage may be promyelocytes with veryheavy granulation. This appearance has been con-fused with hypergranular promyelocytic leukae-mia. Useful points allowing differentiation of the

Fig. 8.32 BM aspirate inKostmann’s syndrome showingneutrophil maturation apparentlyarrested at the promyelocyte stage;cells of eosinophil lineage areincreased. MGG ×940.

382 CHAPTER EIGHT

two conditions are the prominent Golgi zone in thepromyelocytes of agranulocytosis and the absenceof Auer rods and giant granules.


Bone marrow sections show a lack of mature granulocytes and, often, superimposed changes dueto infection.

Auto-immune neutropenia

Auto-immune neutropenia may occur as an iso-lated phenomenon or be one manifestation of anauto-immune disease such as systemic lupus ery-thematosus. It may also occur in association withthymoma and as a complication of T-cell granularlymphocytic leukaemia (with or without associ-ated rheumatoid arthritis). Neutropenia associatedwith T-cell granular lymphocytic leukaemia may becyclical [15].

Peripheral blood

There is a reduction in neutrophils but those pres-ent are cytologically normal.


Granulopoiesis appears normal or hyperplastic with

a reduced proportion of mature neutrophils. Anuncommon observation is phagocytosis of neu-trophils by bone marrow macrophages (Fig. 8.33)[26]. In agranulocytosis associated with thymoma,the bone marrow may show either apparent arrestat the promyelocytic stage or a total absence ofmyelopoiesis [27].

Idiopathic hypereosinophilic syndrome

The idiopathic hypereosinophilic syndrome is acondition of unknown aetiology characterized bysustained hypereosinophilia and damage to tissues,usually including the heart and central nervous sys-tem, by eosinophil products. The clinical featuresare due to this tissue damage. The idiopathic hyper-eosinophilic syndrome has been arbitrarily definedas requiring the eosinophil count to be greater than1.5 × 109/l for greater than 6 months and for tissuedamage to have occurred [28]. Diagnosis of theidiopathic hypereosinophilic syndrome is mainlydependent on peripheral blood and clinical fea-tures and on the exclusion of other diagnoses. Abone marrow aspirate and trephine biopsy are ofimportance in excluding eosinophilic leukaemiaand lymphoma, the latter being an important cause of reactive eosinophilia. Some cases of idiopathichypereosinophilic syndrome represent a myelo-proliferative disorder (MPD) and subsequentlytransform to AML despite initially displaying no

Fig. 8.33 BM aspirate in auto-immune neutropenia showingneutrophil shadows withinmacrophages. MGG ×940.


specific evidence of the nature of the underlyingdisorder. Some cases of otherwise unexplainedeosinophilia result from cytokine secretion by aber-rant, sometimes monoclonal, T cells [29].

Peripheral blood

The eosinophil count is considerably elevated andeosinophils usually show some degree of hypogran-ularity and cytoplasmic vacuolation; completelyagranular eosinophils are sometimes present.Eosinophil nuclei may be non-segmented or hyper-segmented or occasionally ring-shaped. Neutrophils

may show heavy granulation. In contrast to eosino-philic leukaemia, there are usually only occasionalif any granulocyte precursors in the peripheralblood. There may be a mild anaemia and thrombo-cytopenia with red cells showing anisocytosis andpoikilocytosis. Nucleated red cells are sometimespresent.


The bone marrow shows an increase of eosinophilsand their precursors (Fig. 8.34). Some eosinophilmyelocytes show granules with basophilic staining

Fig. 8.34 BM aspirate, idiopathichypereosinophilic syndrome,showing eosinophil hyperplasia;note partial degranulation ofeosinophils. MGG ×940.

Fig. 8.35 BM trephine biopsyspecimen, idiopathichypereosinophilic syndrome,showing marked granulocytichyperplasia and increased numbersof immature eosinophil precursors.Plastic-embedded, H&E ×390.

384 CHAPTER EIGHT

Fig. 8.36 Chediak–Higashisyndrome. (a, b) BM aspirates,showing giant granules andvacuolation of granulocyteprecursors: (a) MGG; (b) Sudanblack B ×960.

(a)

(b)

characteristics but this feature is much less strikingthan in AML of M4Eo type (see page 150). There isno increase in blast cells. Macrophages may containCharcot–Leyden crystals [30].


Eosinophils and their precursors are increased (Fig.8.35). It is important to exclude marrow infiltrationby lymphoma since this may be easily overlooked.However, it should be noted that benign lymphoidaggregates may be associated with the idiopathichypereosinophilic syndrome [31].


The idiopathic hypereosinophilic syndrome is adiagnosis of exclusion. The bone marrow aspirateshould be examined for any increase of blast cells,indicative of a diagnosis of eosinophilic leukaemia.Bone marrow aspirate films and trephine biopsysections should be examined carefully for featuresof systemic mastocytosis, which occasionally pre-sents with striking eosinophilia. Bone marrow cyto-genetic analysis is indicated since demonstration ofa clonal abnormality means that the condition is not‘idiopathic’ but represents a chronic eosinophilic

(c)

Fig. 8.36 Chediak–Higashisyndrome. (c, d) BM trephinebiopsy sections: (c) H&E stainshowing giant granules; (d)Dominicini stain showing giantgranules and vacuolation. Paraffin-embedded ×960. (By courtesy of Dr CJ McCallum, Kirkaldy.) (d)

leukaemia [32,33]. Immunophenotyping of periph-eral blood lymphocytes is indicated and, when anaberrant population is found, T-cell receptor geneanalysis can be used to identify a monoclonal T-cellproliferation. If such a clonal population is demon-strated, the eosinophilia should be regarded as sec-ondary to a T-cell neoplasm rather than idiopathic.

Despite thorough investigation, some patientswith apparently idiopathic hypereosinophilic syndrome can be recognized as having chroniceosinophilic leukaemia only in retrospect whenthey subsequently develop a granulocytic sarcomaor AML.

Chediak–Higashi syndrome

The Chediak–Higashi syndrome is a fatal inheritedcondition characterized by a defect in formation oflysosomes in multiple cell lineages. Patients sufferfrom albinism, neurological abnormalities and re-current infections. Haematological abnormalitiesare most apparent in the granulocyte series althoughanaemia and thrombocytopenia also occur.

Peripheral blood

All granulocyte lineages show striking abnormalities.


386 CHAPTER EIGHT

Granules are very large and also have abnormalstaining characteristics. Lymphocytes and monocytesmay also have abnormally prominent granules.With disease progression, there is development ofanaemia, neutropenia and thrombocytopenia.


Granulocyte precursors as well as mature granulo-cytes show giant granules with unusual stainingcharacteristics (Fig. 8.36a,b). Sometimes there isalso vacuolation. A secondary haemophagocyticsyndrome may occur; it is likely to be con-sequent on immune deficiency and superimposedinfection.


Giant granules may be apparent in granulocyte precursors (Fig. 8.36c,d) but, in general, detection is easier in bone marrow aspirate films. Markedhaemophagocytosis may be seen during the termi-nal phase (see page 119).

Congenital thrombocytopenias

Congenital thrombocytopenia may be inherited ormay be secondary to intra-uterine infection, muta-gen exposure or platelet destruction by maternalanti-platelet antibodies.

Peripheral blood

Morphological features are dependent on whichspecific defect is responsible for thrombocytopenia[1]. In inherited thrombocytopenia, the plateletsmay be of normal size, increased in size as inBernard–Soulier syndrome or decreased in size as in the Wiskott–Aldrich syndrome. In the grey plate-let syndrome they are increased in size and lacknormal azurophilic granules. In the May–Hegglinanomaly, and in several other rare inheriteddefects, thrombocytopenia and giant platelets areassociated with weakly basophilic cytoplasmic inclu-sions in neutrophils. When thrombocytopenia issecondary to intra-uterine platelet destruction ordamage to megakaryocytes, the platelets are usuallynormal in size and morphology.

Other lineages are generally normal but infantswith the thrombocytopenia-absent radii (TAR) syn-drome have been noted to be prone to leukaemoidreactions.


In inherited thrombocytopenia, megakaryocytesmay be present in normal numbers, as in Bernard–Soulier syndrome, or may be severely reduced innumber, as in constitutional amegakaryocytic throm-bocytopenia. In the TAR syndrome megakaryocytesare greatly reduced in number and are small with

Fig. 8.37 BM aspirate, auto-immune thrombocytopenicpurpura, showing fivemegakaryocytes of varying size and ploidy levels. MGG ×377.


poorly lobulated nuclei; eosinophilia is common. Inthe Jacobsen syndrome, associated with a constitu-tional deletion of the long arm of chromosome 11,there are increased numbers of micromegakary-ocytes [34]. In other rare syndromes characterizedby familial thrombocytopenia, megakaryocyte num-bers are variously increased, normal or decreasedand megakaryocyte size may likewise be increased,normal or decreased [35].

When thrombocytopenia results from intra-uterine damage to megakaryocytes, these cells areusually reduced in number. When platelets havebeen destroyed by exposure to maternal anti-platelet antibodies, megakaryocytes are present innormal or increased numbers.


A bone marrow biopsy is not often needed in determining the cause of congenital thrombocy-topenia but it can be useful in permitting an accurate assessment of megakaryocyte numbersand morphology. In the grey platelet syndromethere may be associated myelofibrosis, probablyresulting from intramedullary release by megakary-ocytes of granular contents capable of stimulatingfibroblasts.

Acquired thrombocytopenias

Isolated acquired thrombocytopenia is commonlydue to peripheral destruction of platelets, caused byanti-platelet antibodies, drug-dependent antibodiesor immune complexes; the latter may attach toplatelets both in auto-immune diseases and duringor after viral infections, including infection by HIV.Thrombocytopenia may also be consequent onplatelet consumption, as in thrombotic thrombocy-topenic purpura or in disseminated intravascularcoagulation. Less often, acquired thrombocytopeniaresults from megakaryocytic hypoplasia, such asthat induced by thiazide diuretics, or a failure ofmegakaryocytes to produce platelets, as in somepatients with MDS who present with isolatedthrombocytopenia. Antibody-mediated amega-karyocytic thrombocytopenia is a rare cause [36].Auto-immune thrombocytopenia and, rarely,amegakaryocytic thrombocytopenic purpura maybe associated with LGLL [15].

Peripheral blood

When thrombocytopenia is caused by a sustainedincrease in the peripheral destruction or consump-tion of platelets, there is usually an increase inplatelet size with some giant platelets being present.When thrombocytopenia is due to failure of pro-duction, as in sepsis or during chemotherapy, theplatelets are small. When thrombocytopenia is con-sequent on MDS, platelets often show increasedvariation in size and hypogranular or agranularforms may be present.


When thrombocytopenia resulting from peripheraldestruction or consumption of platelets has devel-oped acutely, the bone marrow may show no rele-vant abnormality, megakaryocytes being present innormal numbers. With sustained thrombocytope-nia, there is an increase in megakaryocyte numbers(Fig. 8.37) and a reduction in average size. There isoften very little morphological evidence of plateletproduction despite the increased platelet turnoverwhich can be demonstrated by isotopic studies.

When thrombocytopenia results from ineffectivethrombopoiesis, for example in MDS, megakary-ocytes may be present in normal or increased numbers and may show dysplastic features. Inacquired megakaryocytic hypoplasia, for exampleas an adverse drug effect, megakaryocytes are usually morphologically normal although reducedin number. Antibody-mediated amegakaryocyticthrombocytopenia may be cyclical. In these cases,megakaryocyte numbers are reduced and mega-karyocytes are small when the platelet count isfalling. When the count is rising, they are normal or increased in number and cytologically normal[36].


Trephine biopsy is not usually necessary in the in-vestigation of suspected immune thrombocytopeniabut is useful in confirming megakaryocytic hypo-plasia and in investigating suspected myelodysplasia.

In idiopathic (auto-immune) thrombocytopenia,the bone marrow is normocellular with increasednumbers of megakaryocytes (Fig. 8.38). Mean

guidelines, however, do not regard a bone marrowexamination as necessary if there are no atypicalclinical or haematological features.

Familial thrombocytosis

Familial thrombocytosis has been reported in atleast 17 individuals in eight families [39]. Inheri-tance appears to be autosomal dominant. In some,but not all, families the cause appears to be a muta-tion in the thrombopoietin gene. A minority of individuals have had splenomegaly.

Peripheral blood

The blood film and count show thrombocytosis asan isolated abnormality. Platelet morphology hassometimes been reported as abnormal [39].

Bone marrow cytology and histology

Megakaryocytes are increased in number and havesometimes been considered to be cytologicallyabnormal [39]. Bone marrow cellularity is some-times increased.


Use of the term ‘essential thrombocythaemia’ todescribe familial thrombocytosis is not recom-

388 CHAPTER EIGHT

megakaryocyte diameter is decreased. There isincreased variation in size so that, although smallmegakaryocytes predominate, there are also in-creased numbers of giant forms. There is no abnor-mal localization of megakaryocytes and clusters are not usually seen [37]. In cyclical, antibody-mediated amegakaryocytic thrombocytopenia, mega-karyocyte numbers are reduced when the plateletcount is falling and normal when it is rising [36].


The differential diagnosis of isolated thrombocy-topenia in children includes ALL. Leukaemia isunlikely if the haemoglobin concentration andwhite cell count are normal. However, a small butsignificant proportion of children in whom a pre-sumptive diagnosis of auto-immune or post-viralthrombocytopenia is made turn out to have ALL.This has led to controversy as to whether a bonemarrow aspirate is required in children with iso-lated thrombocytopenia [38]. There is concern thatadministration of corticosteroids without a pre-treatment bone marrow examination may lead toinadvertent suboptimal treatment of undiagnosedALL. For this reason, UK guidelines suggest that bonemarrow aspiration should be performed before corticosteroid therapy is given, whereas this is notconsidered essential prior to high-dose immuno-globulin therapy or if no treatment is required. USA

Fig. 8.38 BM trephine biopsyspecimen, auto-immunethrombocytopenic purpura,showing increased megakaryocyteswhich are not clustered and arenormally sited; two are adjacent to sinusoids. Paraffin-embedded,H&E ×375. (By courtesy of Dr S Wright, London.)


mended since the condition clearly differs from theMPD that is usually intended by this term. MPD israre in children with a significant proportion ofcases of primary thrombocythaemia being found tobe familial. Investigation of parents and siblings istherefore indicated when persistent unexplainedthrombocytosis is found in a child or adolescent.

Reactive thrombocytosis

The platelet count may increase in response toinfection, inflammation and malignant disease. Inreactive thrombocytosis it is uncommon for theplatelet count to exceed 1000 × 109/l.

Peripheral blood

In contrast to MPD, there is no increase in plateletsize when thrombocytosis is reactive. The blood film may show other reactive changes includingleucocytosis and neutrophilia but the presence ofbasophilia suggests MPD.


The bone marrow aspirate shows increased num-bers of megakaryocytes of normal morphology.


Megakaryocyte numbers are increased. The averagemegakaryocyte diameter is increased in comparisonwith normal and there is increased variation in size.There is no clustering or abnormality of distribution[37].


The differential diagnosis of reactive thrombocyto-sis includes hyposplenism and essential thrombo-cythaemia. Changes of hyposplenism shouldtherefore be sought in the blood film. An increasedbasophil count, increased reticulin deposition in thebone marrow and clustering of megakaryocytesfavour a diagnosis of essential thrombocythaemia.

References1 Bain BJ. Blood Cells: A Practical Guide,3rd edn. Blackwell

Science, Oxford, 2002.

2 Bodnar A, Rosenthal NS and Yomtovian R (1996)Severe hemolytic anemia with marrow dyserythro-poiesis. Am J Clin Pathol, 105, 509.

3 Bader-Meunier B, Rieux-Laucat F, Croisille L, Yvart J,Mielot F, Dommergues JP et al. (2000) Dyserythro-poiesis associated with a Fas-deficient condition inchildhood. Br J Haematol, 108, 300–304.

4 Wickramasinghe SNW (1997) Dyserythropoiesis andcongenital dyserythropoietic anaemias. Br J Haematol,98, 785–797.

5 Wickramasinghe SN (1998) Congenital dyserythropoi-etic anaemia: clinical features, haematological morpho-logy and new biochemical data. Blood Rev, 12, 178–200.

6 Dokal IS, Cox TC and Galton DAG (1990) Vitamin B12and folate deficiency presenting as leukaemia. BMJ,300, 1263–1264.

7 Maung ZT, Norden J, Middleton PG, Jack FR andChandler JE (1994) Pure red cell aplasia: further evi-dence of a T cell clonal disorder. Br J Haematol, 87,189–192.

8 Ramenghi U, Garelli E, Valtolina S, Campagnoli MF, Timeuus F, Crescenzio N et al. (1999)Diamond–Blackfan anaemia in the Italian population.Br J Haematol, 104, 841–848.

9 Giri N, Kang E, Tisdale JF, Follman D, Rivera M,Schwartz GN et al. (2000) Clinical and laboratory evi-dence for a trilineage haematopoietic defect in patientswith refractory Diamond-Blackfan anaemia. Br J Hae-matol, 108, 167–175.

10 Glader BE (1987) Diagnosis and management of redcell aplasia in children. Hematol Oncol Clin North Am, 1,431–447.

11 Foot ABM, Potter MN, Ropner JE, Wallington TB andOakhill A (1990) Transient erythroblastopenia ofchildhood with CD10, TdT, and cytoplasmic µ lympho-cyte positivity in bone marrow. J Clin Pathol, 43,857–859.

12 Penchansky L and Jordan JA (1997) Transient ery-throblastopenia of childhood associated with humanherpesvirus type 6, variant B. Am J Clin Pathol, 108,127–132.

13 Fleming AF (1999) Pregnancy and aplastic anaemia.Br J Haematol, 105, 313–320.

14 Kwong YL and Wong KF (1998) Association of purered cell aplasia with large granular lymphocyte leu-kaemia. J Clin Pathol, 51, 672–675.

15 Lamy T and Loughran TP (1999) Current concepts:large granular lymphocyte leukemia. Blood Rev, 13,230–240.

16 Frickhofen N, Chen ZJ, Young NS, Cohen BJ, HeimpelH and Abkowitz JL (1994) Parvovirus B19 as a cause ofacquired chronic pure red cell aplasia. Br J Haematol,87, 818–824.

17 Shaw PJ, Eden T and Cohen BJ (1993) Parvovirus as acause of chronic anemia in rhabdomyosarcoma.Cancer, 72, 945–949.

29 Simon HU, Plötz SG, Dummer R and Blaser K (1999)Abnormal clones of interleukin-5-producing T cells in idiopathic eosinophilia. N Engl J Med, 341, 1112–1120.

30 Brunning RD and McKenna RW. Atlas of TumorPathology, 3rd Series, Fascicle 9, Tumors of the bonemarrow. Armed Forces Institute of Pathology,Washington, 1993.

31 Metz J, McGrath KM, Savoia HF, Begley CG andChetty R (1993) T cell lymphoid aggregates in bonemarrow in idiopathic hypereosinophilic syndrome. JClin Pathol, 46, 955–958.

32 Bain BJ (1996) Eosinophilic leukaemias and the idio-pathic hypereosinophilic syndrome. Br J Haematol, 95,2–9.

33 Bain BJ (1999) Eosinophiliaaidiopathic or not? N EnglJ Med, 341, 1141–1143.

34 Gangarossa S, Schiliró G, Mattina T, Scardilli S, MollicaF and Cavallari V (1996) Dysmegakaryopoietic throm-bocytopenia in patients with distal chromosome 11qdeletion. Blood, 87, 4915–4916.

35 Bellucci S (1997) Megakaryocytes and inheritedthrombocytopenias. Baillières Clin Haematol, 10,149–162.

36 Zent CS, Ratajczak J, Ratajczak MZ, Anastasi J,Hoffman PC and Gewirtz AM (1999) Relationshipbetween megakaryocyte mass and serum thrombopoi-etin levels as revealed by a case of cyclic amegakary-ocytic thrombocytopenic purpura. Br J Haematol, 105,452–458.

37 Thiele J and Fischer R (1991) Megakaryocytopoiesis inhaematological disorders: diagnostic features of bonemarrow biopsies. Virchows Arch (A), 418, 87–97.

38 Bolton-Maggs PHB (1998) The management of acutechildhood immune thrombocytopenic purpuraaacontroversy revisited. CME Bull Haematol, 1, 79–81.

39 Dror Y and Blanchette VS (1999) Essential thrombo-cythaemia in children. Br J Haematol, 107, 691–698.

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18 Karmochkine M, Oksenhendler E, Leruez-Ville M,Jaccard A, Morinet F and Herson S (1995) Persistentparvovirus B19 infection and pure red cell aplasia inWaldenström’s macroglobulinemia: successful treat-ment with high dose intravenous immunoglobulin.Am J Hematol, 50, 227–228.

19 O’Brien H, Amess JAL and Mollin DL (1982) Recur-rent thrombocytopenia, erythroid hypoplasia andsideroblastic anaemia associated with hypothermia. BrJ Haematol, 51, 451–456.

20 Koduri PR (1998) Novel cytomorphology of the giantproerythroblasts of parvovirus B19 infection. Am JHematol, 58, 95–99.

21 Ryan M, Will AM, Testa N, Hayworth C andDarbyshire PJ (1995) Severe congenital neutropeniaunresponsive to G-CSF. Br J Haematol, 91, 43–45.

22 Simon M, Lengfelder E, Reiter S and Hehlmann R(1996) Osteoporosis in severe congenital neutropenia:inherent in the disease or a sequel of G-CSF treat-ment? [letter] Am J Hematol, 52, 127.

23 Van den Tweel JG (1997) Preleukaemic disorders inchildren: hereditary disorders and myelodysplasticsyndrome. Current Diagn Pathol, 4, 45–50.

24 Kivitie-Kallio S, Rajantie J, Juvonen E and Norio R(1997) Granulocytopenia in Cohen syndrome. Br JHaematol, 98, 308–311.

25 Patton WN (1993) Use of colony stimulating factors forthe treatment of drug-induced agranulocytosis. Br JHaematol, 84, 182–186.

26 Shimizu H, Sawada K, Katano N, Sasaki K, Kawai Sand Fujimoto T (1990) Intramedullary neutrophil pha-gocytosis by histiocytes in autoimmune neutropenia ofinfancy. Acta Haematol, 84, 201–203.

27 Yip D, Rasko JEJ, Lee C, Kronenberg H and O’Neill B(1996) Thymoma and agranulocytosis: two case re-ports and literature review. Br J Haematol, 95, 52–56.

28 Chusid MJ, Dale DC, West BC and Wolff SM (1975)The hypereosinophilic syndrome. Medicine, 54, 1–27.

Disorders of Erythropoiesis, Granulopoiesis and Thrombopoiesis

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