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Pitfalls in obtaining and interpreting bone marrow aspirates: to err is human

Barbara J Bain, Katharine Bailey
St Marys Hospital, Paddington, London, UK Correspondence to Professor Barbara J Bain, Department of Haematology, St Marys Hospital Campus of Imperial College Faculty of Medicine, St Marys Hospital, Praed Street, London W2 1NY, UK; b.bain@ic.ac.uk Accepted 22 December 2010 Published Online First 4 February 2011

ABSTRACT Pitfalls relating to bone marrow aspirates and their interpretation start even before the aspirate is obtained. There can be failure to perform an aspiration that is clinically indicated or, conversely, an aspiration may be done that is not actually necessary. Once an aspirate is obtained it may be unhelpful because it is a blood tap or very dilute, or because of the sampling error that is intrinsic to the procedure. Even if an adequate aspirate is obtained, it may be misinterpreted. Megaloblastic marrows and childrens marrows with increased haematogones or marked reactive changes are particularly prone to misinterpretation. A constant awareness of potential pitfalls and an assessment of the aspirate in the appropriate clinical context will help to reduce errors.

INTRODUCTION
Bone marrow examination is currently the gold standard investigation for diagnosing and monitoring many haematological diseases. It can also be useful for investigating various non-haematological conditions. Combining the bone marrow aspirate with a trephine biopsy enables the haematologist and pathologist to assess not only ne cytological detail but also the organisation of the bone marrow and the presence of focal abnormalities. The marrow aspirate can be rapidly and easily obtained, stained and examined, often providing a reliable diagnosis within a matter of hours. However there are pitfalls in its interpretation. A request for a bone marrow examination should be regarded as a request for a consultation, not just for the performance of a technical procedure. The haematologist should not be obtaining and subsequently interpreting the aspirate in isolation, but in relation to a detailed clinical history and with knowledge of the ndings on physical examination and of the results of other diagnostic procedures. Sometimes only an aspiration is performed and the aspirate alone gives all the information that is needed. More often a trephine biopsy is done at the same time. It is good practice, and often important for drawing the correct conclusions, to interpret the bone marrow aspirate in the light of the trephine biopsy ndings, and vice versa. Should there be any apparent inconsistency, review of both is required. Ideally the aspirate and the biopsy sections should be examined by a haematologist/haematopathologist who is trained and experienced in both elds. If this is not possible, both lms and sections should be reviewed jointly. In the UK this may be done in the context of a multidisciplinary cancer meeting, but this venue is not always suitable for a careful
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assessment of problem cases and not all patients requiring a bone marrow examination have cancer. Pitfalls in obtaining and interpreting a bone marrow aspirate commence even before the procedure is performed. We cannot emphasise too strongly the importance of the clinical features and the blood lm examination as a preliminary step. The blood lm can serve to: (i) reveal the diagnosis and thus obviate a bone marrow examination; (ii) help the assessment of whether a bone marrow examination is indicated or not; (iii) indicate the preferred site for the biopsy or sites to be avoided; and (iv) suggest a preliminary diagnosis and thus indicate what extra tests should be done on the aspirate. The pitfalls related to bone marrow aspiration are summarised in box 1. There is often little published evidence relating to these pitfalls, so we shall draw on our own unpublished experience and knowledge of misadventures as well as on what relevant literature exists.

BONE MARROW ASPIRATION IS DONE WHEN IT IS NOT NEEDED

An assessment of the history, clinical features and blood lm should mean that bone marrow aspiration is only performed when it is clinically indicated. In general, a bone marrow aspiration should not be performed on the basis of a single abnormal blood count unless other features strongly support its validity. We are aware, for example of a bone marrow aspirate that was performed to investigate pancytopenia that was actually due to a blood sample being taken from above an intravenous infusion, and of another that was done to investigate neutropenia that was also factitious, the patient having an inherited myeloperoxidase deciency that led to the neutrophils not being recognised by an automated instrument. We are aware of instances of malaria being diagnosed from a bone marrow aspirate1; clinical suspicion and careful examination of the peripheral blood should avoid this. Similarly, we are aware of two cases, one of which has been published,2 in which a bone marrow aspiration was performed for suspected autoimmune thrombocytopenic purpura, when careful examination of a blood lm subsequently revealed the MayeHegglin anomaly. It has been suggested that a bone marrow aspirate should not be used for the diagnosis of Gaucher disease since a simple blood test can conrm the diagnosis3; avoiding this unnecessary procedure requires that the possibility of this diagnosis has been considered. In the absence of randomised trials and metaanalyses, clinical guidelines can be helpful in deciding when a bone marrow examination is indicated. Autoimmune thrombocytopenic purpura
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prevalence is 4e5% in people in their 70s13 and 14% over the age of 90 years.14 It would clearly be inappropriate to consider that all these individuals require bone marrow aspiration. The UK Myeloma Forum and Nordic Myeloma Study Group have prepared a joint guideline indicating which patients should be referred to a consultant haematologist for further investigation and which patients who are referred actually require detailed investigation, including bone marrow examination.15 Following these guidelines will avoid unnecessary marrow examination.

Box 1 Pitfalls in obtaining and interpreting a bone marrow aspirate

< < < <

Bone marrow aspiration is done when it is not needed. Bone marrow aspiration is not done when it is needed. Bone marrow aspiration is done on the wrong site. The clinical context is not adequately assessed and the correct range of tests is therefore not done on the aspirate. < There is a false negative result as a consequence of a sampling error. < The aspirate is not interpreted together with the trephine biopsy sections. < The aspirate is misinterpreted.

BONE MARROW ASPIRATION IS NOT DONE WHEN IT IS NEEDED

Failure to perform a bone marrow aspiration when indicated is usually the result of inadequate assessment of clinical and peripheral blood features, but sometimes it is due to genuine diagnostic difculty with the possible benets of bone marrow examination not being apparent. There is necessarily little documentation of the effect on patient management of failing to perform a bone marrow aspiration when it was indicated. Evidence of delayed diagnosis serves as a surrogate marker. Intravascular large B-cell lymphoma is an example of a condition that is not infrequently diagnosed only at autopsy because of the lack of a clinical suspicion and yet the bone marrow is one of the tissues often involved. We have also observed extensive investigation of presumed iron deciency before an eventual bone marrow aspirate revealed that the correct diagnosis was anaemia of chronic disease subsequently found to be due to Hodgkin lymphoma,16 and misdiagnosis of thrombotic thrombocytopenic purpura, treated by plasmapheresis, before a bone marrow aspirate revealed a myelodysplastic syndrome (MDS).17

(ITP) and monoclonal gammopathy of undetermined signicance (MGUS) will be taken as examples. American Society of Hematology (ASH) guidelines recommend bone marrow aspiration at presentation in children only when atypical features are revealed by the clinical history, physical examination, blood count or blood lm.4 During follow-up, aspiration is advised if thrombocytopenia persists for more than 6e12 months or if there is no response to high dose intravenous immunoglobulin. For adults with suspected ITP, ASH guidelines recommend bone marrow aspiration only when there are atypical features, when the patient is aged over 60 years or when splenectomy is being considered.4 The validity of this approach was supported by two retrospective analyses of 665 and 836 patients, respectively, aged up to 60 years with suspected ITP whose bone marrows were examined. The British Committee for Standards in Haematology guidelines are similar but not identical to the ASH guidelines. In the case of children, the British Committee for Standards in Haematology advises aspiration not only when there are atypical features but also before corticosteroid therapy is given.7 This guidance is based on the fear that acute lymphoblastic leukaemia (ALL) will be missed. We are aware of occasional instances, including two published accounts,8 9 of patients who have inadvertently been given corticosteroids for an alternative diagnosis without the correct diagnosis of ALL having been made, but we are not aware of any well documented instance when this has occurred in a patient with suspected ITP with no atypical features. A number of large series of patients have been reviewed from this point of view. Halperin and Doyle reviewed 127 children with suspected ITP in whom a bone marrow aspirate was performed; atypical features were present in all ve patients who were found to have ALL.10 Dubansky et al retrospectively reviewed records of 2239 patients with ALL, only one of whom was found to have presented with isolated thrombocytopenia (otherwise normal blood lm, haemoglobin concentration >11 g/dl and white cell count >1.53109/l); however this child had marked hepatosplenomegaly.11 Calpin et al reviewed 484 children who had a bone marrow aspiration for ITP; three cases of leukaemia were found among 152 children with atypical features but none were found among the 332 children with no atypical features.12 The available evidence supports the position of the ASH guidelines that bone marrow aspiration is not indicated at diagnosis in children with suspected acute ITP if clinical and peripheral blood features are typical. Bone marrow aspiration is similarly not necessarily indicated in MGUS. This is a common condition as people age and it may be an incidental nding unrelated to clinical features. The
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BONE MARROW ASPIRATION IS DONE ON THE WRONG SITE

Previous tissue damage at the site of the aspiration can potentially affect the sample, so it is advisable to ascertain whether the patient has a past history of fractures or radiotherapy to the intended aspiration site prior to carrying out the procedure. Occasionally history and physical examination will indicate that aspiration should be performed from a particular site because of the presence of localised pain or bone tenderness.

THE CLINICAL CONTEXT IS NOT ADEQUATELY ASSESSED AND THE CORRECT RANGE OF TESTS IS THEREFORE NOT DONE ON THE ASPIRATE
The haematologist performing the bone marrow aspirate should have attempted to arrive at a provisional diagnosis or at least have a differential diagnosis before the procedure is performed. This will indicate when it is important to use part of the aspirate for culture (eg, for mycobacteria or leishmania), immunophenotyping or molecular or cytogenetic analysis. When the diagnostic possibilities are broad, it is useful to place some of the aspirate into preservative-free heparin and rapidly stain and examine one lm to assess whether further tests on the aspirate are indicated.

FALSE NEGATIVE RESULTS ARE OBTAINED AS A CONSEQUENCE OF A SAMPLING ERROR

Bone marrow aspiration is subject to sampling error. It may therefore fail to detect an abnormality despite there being disease in the bone marrow, or may underestimate the extent of such disease. This is because bone marrow lesions can be focal. In addition, reactive bone marrow brosis may mean that cells of interest are absent or under-represented in the aspirate. To
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some extent sampling error can be overcome by combining aspiration with trephine biopsy, which increases the chance of detecting focal lesions. Lesions that are typically focal include granulomas and inltration by lymphoma (Hodgkin or nonHodgkin), metastatic tumour or systemic mastocytosis. For example, in follicular lymphoma there may be no lymphoma cells detectable by either microscopy or immunophenotyping of the aspirate and yet there is clear paratrabecular inltration in the needle biopsy sections. Similarly, in systemic mastocytosis there may be only very infrequent mast cells in the aspirate; those present may be largely within the fragments and difcult to visualise, and yet the sections show extensive inltration. Although trephine biopsy often overcomes the problem of sampling error, this is not necessarily so, and sometimes repeated aspirations are needed. This can be true, for example, in haemophagocytic lymphohistiocytosis in which an initial nondiagnostic bone marrow aspirate does not exclude the diagnosis and should not delay therapy.18 aspirate was diagnostic in only 16.5% of patients (mainly patients with leishmaniasis) compared with the trephine biopsy specimen in 76%.25 It is our practice to always perform a trephine biopsy when obtaining an aspirate from an HIV-positive patient since often the aspirate is hypocellular and in addition the biopsy sections may reveal granulomas, lymphomatous inltration or, rarely, Kaposi sarcoma. Not only should a trephine biopsy be performed when indicated but the aspirate lms should be interpreted in conjunction with the sections or at least with an acknowledgement that these exist. The aspirate will often be reported more speedily so the nal interpretation might include a phrase such as No inltration has been detected. Trephine biopsy report to follow or The aspirate is very dilute. Await results of trephine biopsy .

THE ASPIRATE IS MISINTERPRETED

An aspirate may be misinterpreted because: (i) it is of poor technical quality; (ii) the correct stains have not been performed; (iii) features that are present are not noticed; or (iv) abnormalities are observed but their signicance is not appreciated or the ndings do not trigger the correct supplementary tests.

AN ASPIRATE IS NOT INTERPRETED IN RELATION TO A TREPHINE BIOPSY

The preliminary evaluation of the patient permits an assessment of whether a trephine biopsy is indicated in addition to an aspiration. An aspirate is superior for the assessment of cytological detail (eg, in megaloblastic anaemia and acute leukaemia, for the detection of ring sideroblasts and for assessment of granulocyte dysplasia in MDS) but only a core biopsy permits assessment of marrow architecture, bone structure and reticulin and collagen deposition. The two procedures are thus complementary. Although a trephine biopsy is usually superior for the detection of lymphoma, it should be noted that there are instances when the aspirate will demonstrate lymphomatous inltration that is not shown by trephine biopsy. In one series of 51 bone marrows of patients with non-Hodgkin lymphoma, 10% showed non-Hodgkin lymphoma in the aspirate which was not present in the trephine biopsy sections. A second trephine biopsy then conrmed these nding in those patients with a positive aspirate.19 Similarly, in neuroblastoma, combining a trephine biopsy with an aspirate will also give a higher detection rate than either alone.20 A trephine biopsy should always be performed if no marrow can be aspirated or if the aspirate appears very dilute and lacking in particles. It is also essential when the differential diagnosis includes aplastic anaemia, lymphoma, metastatic malignancy or a myeloproliferative neoplasm. We consider it is also important to perform a trephine biopsy in suspected multiple myeloma, since it is not infrequent for there to be a marked discrepancy between the numbers of plasma cells in the aspirate and in trephine biopsy sections; sometimes the number of plasma cells in the aspirate is less than 10%. A core biopsy at diagnosis is also important for comparison with post-treatment specimens when an informative aspirate may no longer be obtained. The estimated extent of bone marrow inltration is signicantly higher when based on immunohistochemistry of trephine biopsy sections than when based on aspirates, mainly because inltration may be focal and reticulin may be increased; estimates from a section also show a closer correlation with prognosis than estimates from an aspirate.21 This is not, however, a reason to discount the value of the aspirate since a number of studies have shown myeloma cell morphology to correlate with prognosis.21e24 A trephine biopsy is generally superior in the investigation of patients with fever of unknown origin, although some parasites are easier to recognise in an aspirate. In one series of patients the
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Problems relating to technical quality

Cell morphology can only be properly assessed when the cells are adequately and evenly separated, the cells are not crushed or distorted, the sample is sufciently cellular and contains adequate numbers of particles, and the sample is not clotted or partly clotted.26 A dilute specimen may be the result of taking a large volume of marrow for supplementary tests. This problem can be avoided by aspirating no more than 0.5 ml into a small syringe (eg, a tuberculin syringe) for the preparation of lms and then attaching a second larger syringe to obtain the specimen needed for ow cytometry or other tests. If a bone marrow is likely to be intensely hypercellular, for example in acute leukaemia, it can be useful to anticoagulate part of the specimen with EDTA so that it can subsequently be diluted for the preparation of thinner lms. Heparin should not be used as an anticoagulant for this purpose since it alters the staining characteristics of the cells. The aspirate must be spread and stained correctly so that the fragments and the trails behind them are stained and can be brought under the objective when the slide is placed in a secure position on the microscope stage. The aspirate should be spread towards the area where the label is to be placed; if it is spread in the opposite direction the fragments may only be under the objective when the end of the slide is in mid-air and unstable. Worse still, if an automated staining machine is used the fragments and parts of the lms adjacent to them may be unstained (gure 1). Wedge-spread lms are generally ideal for assessment of cytological detail, but it is also desirable to prepare at least one squash preparation of a fragment. This is particularly important in suspected multiple myeloma or systemic mastocytosis when the cells of interest may be trapped within the marrow particles. It is important that the lms have dried thoroughly before they are xed and stained or artefacts occur that can be confused with dysplastic changes (gure 2). Poor drying can inadvertently occur when slides are transported to the laboratory in air-tight plastic slide holders and are then immediately xed. Depending on environmental conditions in the laboratory, it can take many hours for lms to dry. Laboratories need to develop a suitable staining protocol for bone marrow specimens. Films may require exposure to the stain for considerably longer than is necessary for a peripheral blood lm.
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for examination. If a sample is found to have clotted entirely before spreading it will still be suitable for histological sections, which may permit a diagnosis. If specimens are taken into EDTA, lms should be made promptly since prolonged storage at room temperature can produce changes, for example nuclear lobulation and detached nuclear fragments, that simulate dyserythropoiesis.27 It is necessary to recognise various artefacts in order to avoid misinterpretation. Metastatic tumour cells may be fragile and therefore crushed, as may the lymphocytes of chronic lymphocytic leukaemia, but normal bone marrow cells, particularly erythroblasts, may also be crushed and may be misinterpreted as tumour cells. It is necessary to nd intact cells for a correct interpretation. Because of their size, megakaryocytes are particularly prone to a crush artefact, which may be misinterpreted as evidence of dysplasia (gure 3).

Correct stains not performed

An iron stain should be performed on the initial diagnostic marrow from every patient. Generally this should be done on a lm that includes particles, but each patient must be assessed individually. If the diagnosis suspected is acute leukaemia or MDS, it can be more important to reserve particulate lms for a Romanowsky stain. If an iron stain is not done as a normal part of the routine, it is possible to miss a diagnosis of sideroblastic anaemia. We have observed this occurrence when a blood lm was not assessed prior to performing the aspiration and the laboratory protocol did not include a routine iron stain. The use of cytochemical stains has become much less frequent in recent years. It is important that these are not neglected when their use is appropriate, for example when there is not speedy access to immunophenotyping. Appropriate cytochemistry can conrm the presence of Auer rods, aid the recognition of the variant form of acute promyelocytic leukaemia and help in making a distinction between acute monoblastic leukaemia and large cell lymphoma. The role of cytochemistry in helping the next generation of haematologists to recognise what they are seeing down the microscope should also not be forgotten.

Figure 1 Two bone marrow lms showing incorrect spreading technique (left) and correct spreading technique (right). The lms have been stained on an automated staining machine and ill-positioned fragments in the left-hand lm have not been stained. Even had they been stained, examination of them would have been difcult. Poor staining can lead to errors of interpretation, an example being that if neutrophil precursors are too pink they can be misidentied as cells of the eosinophilic lineage. If the staining is particularly poor, it can become extremely difcult to identify the cells reliably and pick up any morphological abnormality. A clotted or partly clotted sample is particularly a problem in hypercoagulable states such as acute promyelocytic leukaemia when there may be only a small part of the lm that is suitable

Features present are not noted

It is important to examine an adequate number of lms and to examine their edges and tails. Metastatic cancer cells may be

Figure 2 An erythroblast (centre) showing an apparent irregularity of the red cell membrane, which is actually an artefact due to inadequate drying prior to xation.
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Figure 3 A megakaryocyte that appears to be multinucleated; however, this apparent abnormality is due to crushing of the cell during spreading, with part of the disrupted nucleus being outside the cell.
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irregularly distributed and not present in all lms; they may be present mainly in the trails or at the edges, including the advancing edge of the lm. Occasionally at relapse of acute leukaemia or when acute transformation occurs in chronic myelogenous leukaemia, blast cells are irregularly distributed and it is necessary to examine the trails behind particles in a number of lms to detect a focal but clinically very signicant increase. Assessment of the presence or absence of iron and its quantication requires assessment of a sufcient number of particles; evaluation of a minimum of seven particles, if necessary in more than one lm, is required.28 obviously not an actual diagnosis but rather a reminder that the situation is not clear and should be kept under active review. The failure to detect tumour cells despite bone marrow inltration being present is often due to a sampling error. However it can also be due to a failure to recognise tumour cells that are present. This is particularly a problem with small cell tumours of childhood. For example, in one study of children with advanced neuroblastoma, 57 of 61 initial samples contained tumour cells that were detectable by automated immunocytochemistry on the slides.34 Of these 57 positive samples, cytology of the lms gave a 45.6% false negative rate; this was particularly likely when tumour cell numbers were low but some specimens containing up to 10% tumour cells were not recognised.34 Specialised techniques such as this, applied to lms or to the bone marrow aspirate in suspension, can circumvent this problem. The bone marrows of children can cause particular diagnostic problems because of the presence of either reactive lymphocytes or immature lymphoid precursors (haematogones). Haematogones are cytologically similar to leukaemic lymphoblasts and their presence can lead to a misdiagnosis of ALL or a misdiagnosis of relapse when there is a rebound increase of haematogones following cessation of therapy.35 Since a proportion of haematogones express terminal deoxynucleotidyl transferase (TdT) they can also be misinterpreted on ow cytometric immunophenotyping; it is important for the immunophenotyping laboratory to appreciate the spectrum of antigen expression by haematogones in contrast to the more uniform immunophenotype of leukaemic lymphoblasts to avoid making this error. The diagnosis of leishmaniasis on a bone marrow aspirate is difcult when organisms are infrequent. This may be compounded by striking reactive changes, which can include either marked dyserythropoiesis or signicant plasmacytosis. If this diagnosis is suspected, the bone marrow should be cultured for leishmania. Errors are most likely to occur in a non-endemic area, when the clinical history becomes important. For example, a patient returning to Japan from India and the USA was misdiagnosed as having lymphoma, no leishmania having been detected in the rst two of three bone marrow aspirates, which instead showed a remarkable inltration by monocytes and plasma cells.36 Similarly, three French children have been reported in whom a misdiagnosis of leishmaniasis as infectionrelated or familial haemophagocytic syndrome led to treatment with corticosteroids and etoposide, in one patient for as long as 5 months.37 Misdiagnosis as dyserythropoietic anaemia has also occurred.38 Haematologists in northern Europe need to be aware that HIV-positive patients may present with leishmaniasis even decades after exposure in the Mediterranean region. Leishmaniasis is not the only condition that can lead to a striking increase of plasma cells that can suggest MGUS or

Misinterpretation of an adequate aspirate

Bone marrow lms may be well spread, well stained and free of artefacts and yet they are misinterpreted. There are various well recognised recurrent misinterpretations that relate to the diagnostic process rather than to inadequacies of the specimen. Morphological diagnosis is an exercise in pattern recognition and yet patterns are not as specic as might be thought. Interpretation of a megaloblastic bone marrow is fraught with hazards since megaloblastosis may result from a deciency of vitamin B12 or folic acid, a haematological neoplasm or the action of a drug that interferes with DNA synthesis. Misdiagnosis of a deciency state as MDS or erythroleukaemia is a particularly serious error. Haematologists have been aware of this trap for decades and yet patients are still being started on regular transfusions or referred to leukaemia centres for treatment following misdiagnosis. The error may be compounded by immunophenotyping since megaloblastic proerythroblasts sometimes express CD34. Some aspects that can help to prevent these particular misadventures are shown in table 1. There are other non-neoplastic conditions that can lead to a misdiagnosis of MDS. This can occur in lead poisoning, arsenic poisoning, thalassaemia intermedia, congenital dyserythropoietic anaemia,29 copper deciency30 and HIV infection. The use of haemopoietic growth factors can cause granulocyte dysplasia. Agranular neutrophils and micromegakaryocytes have a high degree of specicity for a haematological neoplasm but dyserythropoiesis, even if severe, is lacking in specicity and caution must be shown when the diagnosis is based on cytopenia and erythroid dysplasia alone. Recognition of a diagnostic category of idiopathic cytopenia of undetermined signicance is useful in order to avoid drifting into the assumption that a patient has MDS because no other explanation can be found for cytopenia and dysplasia. This term was rst proposed by the International Working Group on Morphology of MDS at a meeting in Lisbon in April 2005,31 32 and was subsequently adopted in the 2008 WHO classication33 and by others. It is

Table 1 Practices that are helpful in avoiding the misdiagnosis of megaloblastic anaemia due to a vitamin deciency as myelodysplastic syndrome or erythroleukaemia
Practice to be followed Paying careful attention to the clinical history Features of relevance Is the patient a vegan? Has there been a gastrectomy or small bowel resection? Are there features suggesting pernicious anaemia or coeliac disease? Has the patient been prescribed an antifolate drug or had repeated exposure to nitrous oxide? Serum B12 levels are normal in about 5% of patients with megaloblastic anaemia due to deciency of this vitamin, and red cell folate may be normal when there is a rapid onset of deciency. Marked megaloblastosis with striking dyserythropoiesis is not specic for a deciency state. However, although giant metamyelocytes and hypersegmented neutrophils can occur in MDS, this is quite uncommon and their presence strongly suggests a vitamin deciency. A therapeutic trial of vitamin B12 and folic acid should be considered if the evidence suggesting erythroleukaemia or MDS is not absolutely conclusive.

Not placing absolute reliance on vitamin assays Paying careful attention to morphological details

Carrying out a therapeutic trial

MDS, myelodysplastic syndrome.

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multiple myeloma. Rarely, patients with infection or an autoimmune disease have 30e50% plasma cells as a reactive change; this has been observed in Sjgren syndrome,39 syphilis, relapsed acute myeloid leukaemia40 and tuberculosis in an HIV-positive patient.41 A full assessment of other clinicopathological features and assessment of the k:l ratio are needed to make the distinction. A diagnosis of Gaucher disease may be suspected in patients with numerous pseudo-Gaucher cells in the bone marrow. This difculty is best resolved by an assay of peripheral blood b glucocerebrosidase activity. Sometimes systematic errors occur in interpretation, which are resolved with increasing knowledge. This was so with the diagnosis of malignant histiocytosis. This is now known to be a rare condition, with many of the initial reports actually representing a misinterpretation of infection-associated or other reactive haemophagocytic syndromes.42 Sometimes bone marrow ndings do not trigger the right response in the observer. We are aware, for example, of a bone marrow aspirate in a child that showed marked vacuolation of haemopoietic precursors and yet this did not lead to investigation for Pearson syndrome. fault. Similarly, Lucius Annaeus Seneca (c. 4 BC e AD 65) wrote: Errare humanum est perseverare diabolicumdTo err is human; to persist is of the Devil. Keeping the possibility of error in mind means that an initial misinterpretation is more likely to be recognised and corrected.
Competing interests None. Provenance and peer review Commissioned; not externally peer reviewed.

REFERENCES
1. 2. 3. 4. 5. 6. 7. Abdulsalam AH, Sabeeh N, Bain BJ. Immature Plasmodium falciparum gametocytes in bone marrow. Am J Hematol 2010;85:943. Bain BJ, Atra A. Thrombocytopenia. Am J Hematol 2008;83:303. Beutler E, Saven A. Misuse of marrow examination in the diagnosis of Gauchers disease. Blood 1990;76:646e8. George JN, Woolf SH, Raskob GE, et al. Idiopathic thrombocytopenic purpura. A practice guideline developed by explicit methods for the American Society of Hematology. Blood 1996;88:3e40. Westerman DA, Grigg AP. The diagnosis of idiopathic thrombocytopenic purpura in adults: does bone marrow biopsy have a place? Med J Aust 1999;170:216e17. Mak YK, Yu PH, Chan CH, et al. The management of isolated thrombocytopenia in Chinese adults: does bone marrow examination have a role at presentation? Clin Lab Haematol 2000;22:355e8. British Committee for Standards in Haematology General Haematology Task Force. Guidelines for the investigation and management of idiopathic thrombocytopenic purpura in adults, children and in pregnancy. Br J Haematol 2003;120:574e96. Reid MM. Bone marrow examination before steroids in thrombocytopenic purpura or arthritis. Acta Paediatr 1992;81:1052e3. Naithani R, Kumar R, Mahapatra M, et al. Is it safe to avoid bone marrow examination in suspected ITP? Pediatr Hematol Oncol 2007;24:205e7. Halperin DS, Doyle JJ. Is bone marrow examination justied in idiopathic thrombocytopenic purpura? Am J Dis Child 1988;142:508e11. Dubansky AS, Boyett JM, Falletta J, et al. Isolated thrombocytopenia in children with acute lymphoblastic leukemia: a rare event in a Pediatric Oncology Group Study. Pediatrics1989;84:1068e71. Calpin C, Dick P, Poon A, et al. Is Bone Marrow Aspiration Needed in Acute Childhood Idiopathic Thrombocytopenic Purpura to Rule Out Leukemia? Arch Pediatr Adolesc Med 1998;152:345e7. Kyle RA, Rajkumar SV. Monoclonal gammopathy of undetermined signicance. Br J Haematol 2006;134:573e89. Crawford J, Eye MK, Cohen HJ. Evaluation of monoclonal gammopathies in the well elderly. Am J Med 1987;82:39e45. Bird J, Behrens J, Westin J, et al. UK Myeloma Forum (UKMF) and Nordic Myeloma Study Group (NMSG): guidelines for the investigation of newly detected M-proteins and the management of monoclonal gammopathy of undetermined signicance (MGUS). Br J Haematol 2009;147:22e42. Chakravorty S, Johnston R, Coulter C, et al. Teaching cases from the Royal Marsden and St Marys Hospitals, Case 26: refractory microcytic anaemia. Leuk Lymphoma 2004;45:1491e2. Samson RE, Abdalla DH, Bain BJ. Teaching cases from the Royal Marsden and St Marys Hospitals. Case 18. Severe anaemia and thrombocytopenia with red cell fragmentation. Leuk Lymphoma 1998;31:433e5. Gupta A, Tyrrell P, Valani R, et al. The role of the initial bone marrow aspirate in the diagnosis of hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2008;51:402e4. Musolino A, Guazzi A, Nizzoli R, et al. Accuracy and relative value of bone marrow aspiration in the detection of lymphoid inltration in non-Hodgkin lymphoma. Tumori 2010;96:24e7. Aronica PA, Pirrotta VT, Yunis EJ, et al. Detection of neuroblastoma in the bone marrow: biopsy versus aspiration. J Pediatr Hematol Oncol 1998;20:330e4.  Stifter S, Babarovi c E, Valkovi c T, et al. Combined evaluation of bone marrow aspirate and biopsy is superior in the prognosis of multiple myeloma. Diagn Pathol 2010;5:30. Greipp PR, Raymond NM, Kyle RA, et al. Multiple myeloma: signicance of plasmablastic morphological classication. Blood 1985;65:305e10. Carter A, Hocherman I, Linn S, et al. Prognostic signicance of plasma cell morphology in multiple myeloma. Cancer 1987;60:1060e5. Goasguen JE, Zandecki M, Mathiot C, et al. Mature plasma cells as indicator of better prognosis in multiple myeloma. New methodology for the assessment of plasma cell morphology. Leuk Res 1999;23:1133e40. Gupta R, Setia N, Arora P, et al. Hematological prole in pyrexia of unknown origin: `-vis aspiration. Hematology role of bone marrow trephine biopsy vis-a 2008;13:307e12. Riley RS, Williams D, Ross M, et al. Bone marrow aspirate and biopsy: a pathologists perspective. II. Interpretation of the bone marrow aspirate and biopsy. J Clin Lab Anal 2009;23:259e307. Wang LJ, Glasser L. Spurious dyserythropoiesis. Am J Clin Pathol 2002;117:57e9.

FUTURE PITFALLS
An interesting consideration for the future is whether there will be a time when we stop using microscopes and become reliant on digital imaging and computerised interpretation. Certainly the computer processing power now available is approaching the point where there could feasibly be the capability to scan bone marrow aspirate slides as a matter of routine.43 Would the use of digital images be an advantage, permitting collective or expert interpretation even at a distance, thus avoiding some of the interpretation pitfalls which have been discussed in this article, or if coupled with automated interpretation, would it lead to a deskilling of haematologists and thus contribute to misdiagnoses?

8. 9. 10. 11. 12. 13. 14. 15.

CONCLUSIONS
Many of the pitfalls described in this review can be avoided by full clinical assessment, careful morphological analysis of the bone marrow aspirate, appropriate use of special tests, and associated examination of a bone marrow trephine biopsy specimen. However, it is also important to be aware of ones own fallibility and to continue to question a presumptive diagnosis. Almost two millennia before Alexander Pope voiced the thought To err is human, the same characteristic of Homo sapiens had been pointed out both by the statesman and philosopher, Marcus Cicero, and by the Stoic philosopher, Seneca the Younger. Marcus Tullius Cicero (106e43 BC) wrote: Cuiusvis hominis est errare, nullius nisi insipientis in errore perseveraredAnyone can err, but only the fool persists in his
16. 17. 18. 19. 20. 21. 22.

Take-home message
23.

< Interpreting a bone marrow aspirate is a pattern-recognition

24. 25. 26. 27.

exercise that requires both appreciation of context and assessment in the light of other relevant investigations. < A bone marrow aspirate may be misleading because of sampling error or poor technical quality or may be misinterpreted, through human errors, despite a relevant abnormality being present.

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Review
28. 29. 30. 31. 32. 33. Hughes DA, Stuart-Smith SE, Bain BJ. How should stainable iron in bone marrow lms be assessed? J Clin Pathol 2004;57:1038e40. Tso A, Kumaran TO, Bain BJ. Case 41: a misdiagnosis of erythroleukemia. Leuk Lymphoma 2009;50:1e3. Gregg XT, Reddy V, Prchal JT. Copper deciency masquerading as myelodysplastic syndrome. Blood 2002;100:1493e5. Mufti G. Minimal diagnostic criteria in MDS: setting the bar. MDS Foundation Symposium The Puzzle of MDS: How Do the Pieces Fit? Annual Meeting of the American Society of Hematology, Atlanta, GA, 2005. Bain BJ. Problems in the morphological diagnosis of MDS. Satellite Symposium: An evolution in the Understanding of Myelodysplastic Syndromes. Amsterdam: European Hematology Association Congress, 2006. Brunning RD, Hasserjian RP, Porwit A, et al. Refractory cytopenia with unilineage dysplasia. In: Jaffe ES, Harris NL, Swerdlow SH, eds. World Health Organization Classication of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press, 2008:94e5. hes G, Luegmayr A, Kornmu Me ller R, et al. Detection of disseminated tumor cells in neuroblastoma: 3 log improvement in sensitivity by automatic immunouorescence plus FISH (AIPF) analysis compared with classical bone marrow cytology. Am J Pathol 2003;163:393e9. 35. 36. 37. 38. 39. 40. 41. 42. 43. Anand M, Thavaraj V. Ensuring correctness of bone marrow reports in infants: role of the pediatrician. Indian Pediatr 2005;42:834e5. Kawakami A, Fukunaga T, Usui M, et al. Visceral leishmaniasis misdiagnosed as malignant lymphoma. Intern Med 1996;35:502e6. phan JL. Hemophagocytic syndrome: a misleading Gagnaire MH, Galambrun C, Ste complication of visceral leishmaniasis in childrenda series of 12 cases. Pediatrics 2000;106:e58. Olivieri O, Gandini G, Baiocco R, et al. Visceral leishmaniasis presenting as dyserythropoiesis associated with increased i-antigenicity of erythrocytes. Haematologica 1987;72:163e5. Tanvetyanon T, Leighton JC. Severe anemia and marrow plasmacytosis as presentation of Sjo grens syndrome. Am J Hematol 2002;69:233. Torlakovic EE, Naresh KN, Brunning RD. Bone Marrow Immunohistochemistry. Chicago: ASCP Press, 2008:134. J, Thompson M. Striking bone marrow plasmacytosis in a patient Bayley J, Pavlu with sickle cell anaemia. Br J Haematol 2008;144:457. Wilson MS, Weiss LM, Gatter KC, et al. Malignant histiocytosis: a reassessment of cases previously reported in 1975 based on parafn section immunophenotyping studies. Cancer 1990;66:530e6. May M. A better lens on disease. Sci Am 2010;302:74e7.

34.

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Pitfalls in obtaining and interpreting bone marrow aspirates: to err is human

Barbara J Bain and Katharine Bailey J Clin Pathol 2011 64: 373-379 originally published online February 4, 2011

doi: 10.1136/jcp.2010.080820

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