Reactive versus neoplastic bone marrow: problems and pitfalls.
Abstract: Examination of the bone marrow poses several unique challenges to the pathologist: it is a semisolid organ without helpful gross correlation, it exists in a dynamic state with the peripheral blood and other organs of the lymphohemopoietic system, and the diagnosis of diseases affecting bone marrow often depends heavily on ancillary special studies. The bone marrow examination ideally encompasses review of the bone marrow biopsy histology (with or without additional nondecalcified clot preparation material), bone marrow aspirate smear cytology, and the peripheral blood smear; optimal procurement and processing of these samples is critical in ensuring that a maximal level of diagnostic information can be extracted. The pathologist must be aware of the clinical context of the bone marrow and the results of ancillary tests, whether these are ordered by the pathologist or the clinician. A combination of excellent diagnostic samples, appropriate ancillary tests, and knowledge of the clinical context provides the best background to distinguish between the common reactive and neoplastic processes that involve the bone marrow and to avoid diagnostic pitfalls in making these distinctions.
Subject: Tumors (Diagnosis)
Blood (Medical examination)
Medical research
Medicine, Experimental
Antimitotic agents
Antineoplastic agents
Author: Hasserjian, Robert P.
Pub Date: 04/01/2008
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2008 College of American Pathologists ISSN: 1543-2165
Issue: Date: April, 2008 Source Volume: 132 Source Issue: 4
Topic: Canadian Subject Form: Tumours
Product: Product Code: 8000428 Blood Test Procedures; 8000200 Medical Research; 9105220 Health Research Programs; 8000240 Epilepsy & Muscle Disease R&D; 2834140 Anticancer Drugs; 2834146 Chemotherapeutic Drugs NAICS Code: 6215 Medical and Diagnostic Laboratories; 54171 Research and Development in the Physical, Engineering, and Life Sciences; 92312 Administration of Public Health Programs; 325412 Pharmaceutical Preparation Manufacturing SIC Code: 2834 Pharmaceutical preparations
Accession Number: 230151943
Full Text: (Arch Pathol Lab Med. 2008;132:587-594)

DIAGNOSTIC SAMPLES USED TO EVALUATE THE BONE MARROW

The core biopsy histology and aspirate smear cytology are complementary to one another both in terms of sample type (core tissue sample vs liquid aspirate of intertrabecular marrow material) and presentation of the microscopic pathology (histologic sections vs air-dried Wright-Giemsa-stained cytology). Both methods have strengths and weaknesses and should be reviewed in concert. If the same diagnostician does not review both biopsy and aspirate, it is critical that the individuals reviewing the aspirate and biopsy communicate and come to a consensus diagnosis rather than issuing possibly contradictory or noncommittal reports.

In addition, ancillary studies are often concurrently performed on the marrow sample at the discretion of the pathologist and/or submitting clinician. Common ancillary studies relevant to bone marrow diagnosis are listed in Table 1. These tests complement the bone marrow morphologic examination; it is critical that the pathologist reviewing the morphology and the various laboratories conducting the ancillary studies are in close communication. Such communication ensures maximal diagnostic power and accuracy, as the likelihood of achieving a specific (and cost-effective) diagnosis is increased if the pathologist and the ancillary laboratories are aware of the diagnostic considerations of one another. Ultimately, the pathologist must be able to orchestrate and synthesize the various morphologic and ancillary data into a clinically meaningful diagnosis, or at least a useful differential diagnosis.

TECHNICAL CONSIDERATIONS

Technical quality of the bone marrow sample frequently compounds difficulties in interpretation. The core biopsy may be of inadequate length, may consist predominantly of cortical bone, or may exhibit obscuring crush artifact. While the aspirate smear preparations give the pathologist a "second chance" when the core biopsy is poor, the aspirate may be inadequate due to a poor aspiration technique or a fibrotic marrow space (so-called dry tap). The latter is a more serious problem than an inadequate core biopsy, since many critical ancillary studies require unfixed smear slides or living cells in suspension. It is recommended that if a dry or poor aspirate is obtained, touch imprints should be made from the fresh, unfixed core biopsy. These touch preparation slides can be used for Wright-Giemsa staining as well as for ancillary studies, such as fluorescent in situ hybridization. If cytogenetics and/or flow cytometry are critical in a dry tap scenario, a second core biopsy may be obtained and placed in sterile saline. Using sterile needles or a mechanical disaggregation system (such as Medimachine; BD Biosciences, San Jose, Calif), viable marrow cells in suspension can be released from fibrotic tissue samples.

Even if an adequate biopsy is obtained, evaluating subtle histologic findings requires excellent fixation, processing, and sectioning of the tissue. While neutral-buffered formalin is an adequate fixative, superior nuclear detail is often produced with fixatives containing metals, such as Zenker, B-5, aceto-zinc formalin, and B-plus (BBC Biochemical, Stanwood, Wash). (1) Unlike Zenker and B-5, B-plus fixative and aceto-zinc formalin do not contain mercury, thereby circumventing problems of hazardous waste generation and providing better preservation of DNA for molecular studies. (1,2) Decalcifying agents include, in order of decreasing speed of action and decreasing adverse effects on morphology, immunohistochemistry and DNA quality for fluorescent in situ hybridization and molecular studies: strong acids (hydrochloric, nitric, sulfuric), buffered acids (formic, acetic), and calcium chelators. (3-5) Proprietary decalcifying agents, such as RDO (APB Engineering Products, Plainfield, Ill) and RapidCal (BBC Biochemical) may also be used. Processing and embedding bone marrow biopsies in plastic resins produces superior sections, but this procedure is time consuming and labor intensive and is not widely used.

Small particles of marrow are often present in blood clot submitted along with the marrow core and are usually prevalent in clots prepared from residue from the marrow aspirate syringe. These clots should be fixed and processed routinely, as they do not need to be decalcified; they often produce superior morphology to the core biopsy. At Massachusetts General Hospital, we fix bone marrow core biopsies for a minimum of 4 hours in B-plus and decalcify for 1 hour in RapidCal Immuno (BBC Biochemical), which provides adequate fixation and good antigen preservation for immunohistochemistry.

Thin sectioning of paraffin-embedded sections is at least as important as good fixation in producing an interpretable slide: all bone marrow sections are cut at a thickness of 2 [micro]m at Massachusetts General Hospital. In addition to a hematoxylin-eosin section, a well-prepared Giemsa stain is useful in differentiating myeloid and erythroid elements and in identifying plasma cells and mast cells. Iron stain may be performed on the biopsy section or clot, but it is less sensitive than an iron stain on an aspirate smear, as some iron is lost during tissue processing. (6) Reticulin silver stain on the core biopsy is essential in evaluating myeloproliferative diseases and may be helpful in detecting general or focal marrow abnormalities, which can nonspecifically cause an increase in reticulin fibers (see below). Reticulin grading systems include three and four grade systems, which are reproducible if the stain is technically adequate (Table 2). (7,8)

THE CLINICAL CONTEXT IN BONE MARROW EXAMINATION

The pathologist should be aware of important clinical patient information, such as complete blood count results and the presence or absence of splenomegaly and lymphadenopathy. In evaluating samples taken after therapy, the pathologist should be aware of the details of the disease history, type of therapy, and timing of the biopsy in relation to the disease course and therapy administration. Various types of therapies may influence the appearance of both neoplastic and normal cell populations, confounding distinction between residual disease and reactive normal cells.

Knowledge of the clinical reason for bone marrow sampling is important in practical bone marrow diagnosis. The clinical scenario in which the bone marrow biopsy is performed influences the choice of ancillary studies and also places the interpretation of morphologic findings in a context; this context may allow a more specific diagnosis to be made or may help the pathologist in avoiding an erroneous diagnosis suggested by morphology. Table 3 illustrates the clinical context for bone marrow sampling performed during 1-year periods at 2 institutions. This Table illustrates that a large proportion of bone marrow biopsies are performed to follow-up or stage known disease (particularly at tertiary/referral institutions). These types of scenarios present quite a different set of problems for the pathologist than bone marrow examinations performed to evaluate a newly presenting, unknown disease. While Table 3 provides the clinical settings of bone marrow examination at 2 example institutions, these will, of course, vary in different pathology practices. Each indication sets the stage for a typical differential diagnosis and will suggest appropriate ancillary studies. Ordering of ancillary studies inappropriate for the context is costly and may generate confusing and misleading results, which may complicate an otherwise routine diagnosis. However, the pathologist must still retain an open mind and realize that diseases may present atypically with unexpected clinical findings; he or she must be prepared to alter the ancillary testing profile in cases with unexpected morphologic findings.

COMMON SITUATIONS THAT CHALLENGE THE PATHOLOGIST TO DETERMINE A PRIMARY NEOPLASTIC VERSUS REACTIVE PROCESSES

Anemia

Unexplained anemia is a common indication for bone marrow examination (and is the most common indication at many institutions). Generally, the anemia is chronic in duration and the hematologist has typically already excluded iron, vitamin [B.sub.12], and folate deficiencies as well as possible effects of a drug or known infection, although this cannot always be assumed. The degree and duration of anemias that elicit a bone marrow examination vary among different hematologists. As mentioned above, examination of the bone marrow in this context should include knowledge of the complete blood count results and examination of the peripheral smear.

Typically, the main clinical suspicion for bone marrow biopsy in anemia workup is a myelodysplastic syndrome (MDS). In this setting, MDS should be considered a diagnosis of exclusion: striking morphologic dysplasia may be seen in a variety of reactive conditions, such as effects of alcohol, drug or toxin, folate, or vitamin [B.sub.12] deficiency. (9,10) Morphologic dysplasia may also occur in inherited conditions, such as hemoglobinopathies (Figure 1, A through D). (9) In general, the pathologist should strive to find an "excuse" for the dysplasia and should be particularly cautious of making a diagnosis of MDS when only unilineage dysplasia is present. Unlike MDS, which usually exhibits architectural disorganization of hemopoiesis, reactive marrow processes typically maintain relative segregation of erythropoietic and myelopoietic "islands" (Figure 1, E and F). A cytogenetic abnormality characteristic of MDS confirms the diagnosis, but cytogenetic abnormalities are only seen in about 50% of MDS cases. (11) An increase in blasts in the context of morphologic dysplasia and prolonged anemia in the absence of growth factor therapy is also a very strong indicator of MDS. Other less definitive features that increase the suspicion of MDS in the anemic patient are macrocytosis (if folate and vitamin B12 deficiency have been excluded), other cytopenias, and the presence of ringed sideroblasts. Flow cytometric identification of aberrant antigen expression can also be helpful in raising the suspicion of MDS. (12,13) However, unless the diagnosis of MDS is clear cut, it is most prudent to raise the possibility of MDS without rendering a definitive diagnosis: clinical follow-up will often eventually confirm a diagnosis of "true" MDS by establishing the chronicity of the abnormality and by allowing time for exclusion of possible reactive/reversible causes.

Aside from MDS, other neoplasms presenting with anemia include lymphoma and multiple myeloma. Hairy cell leukemia is an important consideration, as the interstitial infiltration pattern may be subtle in early stages of the disease, and hairy cells may be rare in the peripheral blood and the often poor or dry aspirate. CD20 and/or DBA.44 immunostaining of the bone marrow core is very helpful in these situations (Figure 2, A and B). (14) It is important to keep in mind that marrow lymphomas (particularly hairy cell leukemia and also multiple myeloma) can elicit a "sympathetic" morphologic dysplasia in the hematopoietic elements and may lead to misdiagnosis as MDS. (15,16) Helpful information to avoid this pitfall includes correlation with clinical features (splenomegaly, for example, is uncommon in MDS), flow cytometry, and the typical limitation of such reactive morphologic dysplasia to the erythroid lineage.

Common reactive causes of anemia are often diagnosed clinically by the hematologist through a detailed history, physical examination,

and laboratory tests, and these patients do not undergo bone marrow biopsy. Evolving aplastic anemia or paroxysmal nocturnal hemoglobinuria may present with progressive anemia; the latter can be excluded by peripheral blood immunophenotyping for the glycosylphosphatidylinositol-anchored proteins CD55 and CD59. (17) Rare disorders, such as Gaucher and other storage diseases, bone marrow involvement by sarcoidosis, and systemic mastocytosis, may also present with anemia or pancytopenia. (18) Anemia and pancytopenia in infants present a different differential diagnosis; primary MDS is less likely, whereas aplastic anemia, hereditary bone marrow failure syndromes, and hemophagocytic syndromes must be considered. (19,20) Unfortunately, no specific diagnosis can be rendered in many bone marrow biopsies performed to evaluate anemia. Such cases likely represent a mixture of anemia of chronic disease, examples of reactive disorders described above that were not diagnosed prior to the bone marrow biopsy, or early cases of MDS in which morphologic changes are insufficiently well developed to warrant a definitive diagnosis.

[FIGURE 1 OMITTED]

Thrombocytopenia

By far the most common cause for thrombocytopenia eliciting a bone marrow examination is idiopathic thrombocytopenic purpura, which is mediated by antiplatelet autoantibodies. Isolated thrombocytopenia is less commonly a presenting manifestation of a myelodysplastic syndrome, acute leukemia, or a bone marrow failure syndrome. (21) The pathologist's role in evaluating the bone marrow in this setting is in establishing a normal or increased number of megakaryocytes and in excluding dysplastic changes diagnostic of MDS; however, isolated thrombocytopenia with minimal morphologic dysplasia may occur in indolent forms of MDS, particularly in those with a del(20q) cytogenetic abnormality. (22)

[FIGURE 2 OMITTED]

Distinction between idiopathic thrombocytopenic purpura and other causes of platelet consumption (splenic sequestration, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura) cannot be made on the basis of the bone marrow findings alone. Decreased or absent megakaryocytes in the setting of thrombocytopenia suggest suppression of megakaryopoiesis, usually due to a drug or toxin. It is important to note that in rare cases of idiopathic thrombocytopenic purpura there may be antibodies directed against megakaryocytes, with resulting decrease or absence of bone marrow megakaryocytes. (23)

Leukopenia or Pancytopenia

In the absence of any comorbid condition, pancytopenia is often an indicator of a primary bone marrow disease. In particular, bone marrow sampling is often done for isolated leukopenia or pancytopenia in patients with autoimmune diseases, HIV infection, or other chronic diseases. In these situations, the underlying disease itself may be the cause of the cytopenias and may produce an abnormal-appearing bone marrow.

Special Scenario: Cytopenias in HIV-infected Patients.--Disseminated infections, such as histoplasma, leishmania, and mycobacteria, may manifest in the bone marrow of HIV-infected patients with cytopenias.24 Pancytopenic presentation due to primary bone marrow nonHodgkin or Hodgkin lymphoma is also not uncommon in the setting of HIV infection. In the absence of a specific identifiable infectious agent or neoplasm, cytopenias may be due to autoimmune destruction or direct bone marrow effects due to the virus itself as well as the effects of antiviral drugs. (24) Various abnormalities described in the bone marrow of HIV-infected patients are listed in Table 4 and are illustrated in Figure 2, C and D. In particular, morphologic dysplasia may be present and may mimic a primary myelodysplastic syndrome. (25,26)

Special Scenario: Cytopenias in Autoimmune Diseases.--Systemic lupus erythematosus and other autoimmune diseases may cause marked pancytopenia. Histologic changes in the bone marrow include serous atrophy/ gelatinous transformation, varying degrees of reticulin fibrosis, lymphocytosis, plasmacytosis, and morphologic dysplasia. (27-29) A diagnosis of a primary myelodysplastic syndrome should be made with utmost caution in the setting of known autoimmune disease (or HIV infection).

Special Scenario: Bone Marrow Granulomas.--Granulomas occurring in the bone marrow raise the possibility of mycobacterial or fungal infection, systemic granulomatous diseases, or a paraneoplastic phenomenon. However, bone marrow granulomas may also be idiopathic and occur in the absence of any underlying disease. (27,30)

Elevated Peripheral Counts

Analogous to making a primary diagnosis of MDS, myeloproliferative disease (MPD) as a cause for peripheral counts should be considered a diagnosis of exclusion. Marked reactive thrombocytosis may occur in iron deficiency, infection, and as a paraneoplastic phenomenon. Bone marrow examination is helpful in these cases, because the megakaryocytes in MPDs are almost always morphologically abnormal, in contrast to the increased but morphologically normal megakaryocytes in reactive thrombocytosis. (31) Identifying a V617F mutation in the JAK2 gene by molecular study on peripheral blood and/ or bone marrow is very helpful in confirming a diagnosis of MPD; this mutation is present in almost all cases of polycythemia vera and in about 50% of cases of essential thrombocythemia and chronic idiopathic myelofibrosis. (32-35) However, distinction among these entities continues to rely on the clinical and laboratory features as well as bone marrow histologic features. (31)

Bone marrow examination is becoming less important in distinguishing chronic myeloid leukemia (CML) from reactive leukocytosis and other MPDs, as cytogenetics and/or molecular testing of peripheral blood can confirm this diagnosis. However, bone marrow examination is important in establishing the phase of disease (chronic, accelerated, or blastic), which depends on the blast count in bone marrow as well as peripheral blood. (36)

Special Scenario: Increased Reticulin Fibrosis (Myelofibrosis).--An important issue often encountered in bone marrow samples taken for increased peripheral counts (as well as some taken for cytopenias) is the presence of myelofibrosis. As well as complicating assessment by causing a dry tap and often poor, crushed core biopsy morphology, myelofibrosis presents a broad differential diagnosis. Causes of significantly increased reticulin (with the formation of a reticulin fiber meshwork and thick or bundled fibers) are listed in Table 5. The term myelofibrosis merely connotes increased reticulin fibers with many possible causes. Diagnosis of the specific MPD entity chronic idiopathic myelofibrosis/primary myelofibrosis requires the typical morphologic and clinical features of this disease and the exclusion of other causes of myelofibrosis. Especially important to exclude is CML, which may present with a myelofibrosis-like picture. (37) As CML has a unique clinical behavior (inevitable progression to blast crisis without appropriate therapy) and specific, highly effective targeted therapies (imatinib mesylate and related tyrosine kinase inhibitors), CML should always be excluded by molecular and/or cytogenetic testing in any fibrotic marrow of unknown cause.

Lymphoid Aggregates

Benign lymphoid aggregates may occur in the bone marrow in a variety of reactive conditions and are also more likely to be found in older individuals. (30,38) Conditions associated with the presence of benign lymphoid aggregates include autoimmune diseases and HIV infection, as well as myeloid neoplasms, such as MDS and myeloproliferative diseases; in the latter cases, the problem may arise whether the aggregates are reacting to a myeloid neoplasm or whether there is reactive marrow "dysplasia" to lymphoma involving bone marrow (see above)! In contrast to neoplastic lymphoid aggregates, reactive aggregates tend to be few in number, are nonparatrabecular (although many lymphomas may have exclusively nonparatrabecular aggregates), are small with good circumscription and minimal infiltration of surrounding hemopoietic marrow, have a polymorphous lymphoid population, and may contain reactive germinal centers. However, there is considerable morphologic overlap between reactive and neoplastic lymphoid aggregates. Unfortunately, immunohistochemistry is often not helpful, as both reactive and neoplastic lymphoid aggregates may have a variable admixture of B and T cells. Certainly, a vast excess of aggregated B cells or phenotypic aberrancy, such as abnormal coexpression of CD5 or CD43 in B cells, helps in confirming a neoplastic lymphoid population. Immunohistochemistry and/or in situ hybridization for [kappa] and [lambda] light chains can also be helpful in establishing a clonal B-cell population in lymphomas with a plasmacytic component (lymphoplasmacytic lymphoma and marginal zone lymphomas).

Flow cytometry is a critical adjunct to morphology in establishing lymphoid aggregates as neoplastic or reactive. Immunophenotypic demonstration of a clonal B-cell population or markedly aberrant T-cell population can confirm lymphoma involving bone marrow, but it may not provide definitive classification. Conversely, failure to identify an abnormal lymphoid population by flow cytometry is reassuring, but it does not necessarily exclude marrow lymphoma. Typical pitfalls producing a false-negative flow cytometry result in a bone marrow involved by lymphoma include sampling artifact due to hemodilution, inadequate sampling of paratrabecular aggregates due to associated reticulin fibrosis, and preferential loss of lymphoma cells; plasma cells and large cell neoplasms are particularly vulnerable to the latter effect. Many lymphomas, such as Hodgkin lymphomas and some T-cell lymphomas, do not have a diagnostically abnormal immunophenotype.

In the situation of morphologically suspicious lymphoid aggregates with negative flow cytometry (or no available flow cytometry) and ambiguous immunohistochemistry, the pathologist should step back and consider the whole picture: Is there an appropriate clinical context for reactive aggregates, such as HIV infection or autoimmune disease? Is there lymphadenopathy or organomegaly? Is there peripheral lymphocytosis? Do the aggregates occupy sufficient marrow space to explain any cytopenias? Biopsy of any clinically involved extramedullary site is the best way to accurately diagnose and classify most lymphomas. If this proves to be impractical or difficult, flow cytometry on peripheral blood may yield a diagnosis even if there is no lymphocytosis; most lymphomas that primarily involve the bone marrow manifest some degree of peripheral blood involvement. Finally, a fresh bone marrow biopsy may be obtained to include flow cytometry and molecular studies to demonstrate clonality. Communication with the clinician is important in determining the appropriate handling of these situations.

Paraprotein

A paraprotein, discovered either incidentally or during the workup of anemia or other symptoms, frequently elicits a bone marrow examination. Bone marrow examination may also be done as a follow-up response to therapy for known multiple myeloma, although this can also be done by assessing paraprotein levels. In either scenario, obtaining an accurate plasma cell count is critical. Fortunately, sensitive and specific plasma cell markers, such as CD138 and CD38, are available for immunohistochemistry on paraffin sections, which allow for a reasonably accurate plasma cell count in tissue sections to correlate with the plasma cell count in the aspirate smear. (39,40) While plasma cells are rare in normal bone marrow, they may increase to striking levels in reactive disorders such as infection and autoimmune disease that may also have associated paraproteins. (27) The plasma cell pattern is helpful in these situations, as plasma cells in reactive conditions usually occur around blood vessels and in small clusters rather than large aggregates or sheets away from vascular structures (Figure 2, E and F). (41) Nevertheless, unless the plasma cells are overtly malignant, it is recommended that plasma cell clonality be proven by light chain immunohistochemistry and/or flow cytometry immunophenotyping in all new diagnoses of multiple myeloma.

It is important that the diagnosis rendered fulfill the diagnostic criteria of the World Health Organization Classification: a plasma cell percentage of 10% fulfills only a minor criterion for the diagnosis of plasma cell myeloma, and additional features are required for diagnosis. (42) It is reasonable to render a diagnosis of "plasma cell neoplasm" in such cases if the full clinical information allowing a diagnosis of myeloma is not available. New or untreated cases with less than 10% bone marrow plasma cells, even if they are shown to be monotypic, must be placed into the category of monoclonal gammopathy of undermined significance. (42)

Non-Hodgkin lymphomas (including T-cell lymphomas) may present with paraproteins. An immunoglobulin M (IgM) paraprotein is almost always indicative of lymphoma rather than myeloma, but it may be present in any type of B-cell lymphoma. Although a high IgM paraprotein usually correlates with lymphoplasmacytic lymphoma, classification of lymphomas in bone marrow samples should rest on morphology, immunophenotype, and genetic features as well as the clinical context. (43)

Special Scenario: Bone Marrow Sampling in Amyloidosis Patients.--In the setting of systemic amyloidosis, bone marrow examination should focus on finding any monotypic plasma cell population, regardless of whether the numbers fulfill criteria for plasma cell myeloma. (44) Identifying any clonal plasma cell population establishes a diagnosis of systemic amyloid light chain amyloidosis, with significant treatment implications. Amyloid light chain amyloidosis patients often have very small numbers of clonal plasma cells; immunohistochemistry and/or in situ hybridization for light chains is recommended to disclose these subtle populations (which may produce highly significant amounts of amyloid protein), even if plasma cells do not appear to be increased on biopsy and aspirate morphologic review. (45)

References

(1.) Naresh KN, Lampert I, Hasserjian R, et al. Optimal processing of bone marrow trephine biopsy: the Hammersmith Protocol. J Clin Pathol. 2006;59:903-911.

(2.) Bonds LA, Barnes P, Foucar K, Sever CE. Acetic acid-zinc-formalin: a safe alternative to B-5 fixative. Am J Clin Pathol. 2005;124:205-211.

(3.) Mullink H, Henzen-Logmans SC, Tadema TM, Mol JJ, Meijer CJ. Influence of fixation and decalcification on the immunohistochemical staining of cell-specific markers in paraffin-embedded human bone biopsies. J Histochem Cytochem. 1985;33:1103-1109.

(4.) Wickham CL, Sarsfield P, Joyner MV, Jones DB, Ellard S, Wilkins B. Formic acid decalcification of bone marrow trephines degrades DNA: alternative use of EDTA allows the amplification and sequencing of relatively long PCR products. Mol Pathol. 2000;53:336.

(5.) Brown RS, Edwards J, Bartlett JW, Jones C, Dogan A. Routine acid decalcification of bone marrow samples can preserve DNA for FISH and CGH studies in metastatic prostate cancer. J Histochem Cytochem. 2002;50:113-115.

(6.) DePalma L. The effect of decalcification and choice of fixative on histiocytic iron in bone marrow core biopsies. Biotech Histochem. 1996;71:57-60.

(7.) Bauermeister DE. Quantitation of bone marrow reticulin--a normal range. Am J Clin Pathol. 1971;56:24-31.

(8.) Thiele J, Kvasnicka HM, Facchetti F, Franco V, van der Walt J, Orazi A. European consensus on grading bone marrow fibrosis and assessment of cellularity. Haematologica. 2005;90:1128-1132.

(9.) Wickramasinghe SN, Bain BJ. Pathology of the marrow: general considerations. In: Wickramasinghe SN, McCullogh J, eds. Blood and Bone Marrow Pathology. London, England: Elsevier Science Limited; 2003:87-128.

(10.) Krause JR. The bone marrow in nutritional deficiencies. Hematol Oncol Clin North Am. 1988;2:557-566.

(11.) Fenaux P. Chromosome and molecular abnormalities in myelodysplastic syndromes. Int J Hematol. 2001;73:429-437.

(12.) Kussick SJ, Fromm JR, Rossini A, et al. Four-color flow cytometry shows strong concordance with bone marrow morphology and cytogenetics in the evaluation for myelodysplasia. Am J Clin Pathol. 2005;124:170-181.

(13.) Wells DA, Benesch M, Loken MR, et al. Myeloid and monocyticdyspoiesis as determined by flow cytometric scoring in myelodysplastic syndrome correlates with the IPSS and with outcome after hematopoietic stem cell transplantation. Blood. 2003;102:394-403.

(14.) Hasserjian RP, Pinkus GS. DBA.44: an effective marker for detetion of hairy cell leukemia in bone marrow biopsies. Appl Immunohistochem. 1994;2:197-204.

(15.) Pittaluga S, Verhoef G, Maes A, Boogaerts MA, De Wolf-Peeters C. Bone marrow trephines: findings in patients with hairy cell leukaemia before and after treatment. Histopathology. 1994;25:129-135.

(16.) Castello A, Coci A, Magrini U. Paraneoplastic marrow alterations in patients with cancer. Haematologica. 1992;77:392-397.

(17.) Kwong YL, Lee CP, Chan TK, Chan LC. Flow cytometric measurement of glycosylphosphatidyl-inositol-linked surface proteins on blood cells of patients with paroxysmal nocturnal hemoglobinuria. Am J Clin Pathol. 1994;102:30-35.

(18.) Chang KL, Gaal KK, Huang Q, Weiss LM. Histiocytic lesions involving the bone marrow. Semin Diagn Pathol. 2003;20:226-236.

(19.) Sills RH. Indications for bone marrow examination. PediatrRev. 1995;16: 226-228.

(20.) McKenna RW. Myelodysplasia and myeloproliferative disorders in children. Am J Clin Pathol. 2004;122(suppl):S58-S69.

(21.) Sashida G, Takaku TI, Shoji N, et al. Clinico-hematologic features of myelodysplastic syndrome presenting as isolated thrombocytopenia: an entity with a relatively favorable prognosis. Leuk Lymphoma. 2003;44:653-658.

(22.) Gupta R, Johari V, Hasserjian R. Myelodysplastic syndrome with isolated del(20q): a distinct clinicopathologic entity? Mod Pathol. 2006;19:229A.

(23.) Katsumata Y, Suzuki T, Kuwana M, et al. Anti-c-Mpl (thrombopoietin receptor) autoantibody-induced amegakaryocytic thrombocytopenia in a patient with systemic sclerosis. Arthritis Rheum. 2003;48:1647-1651.

(24.) Evans RH, Scadden DT. Haematological aspects ofHIVinfection. Baillieres Best Pract Res Clin Haematol. 2000;13:215-230.

(25.) Thiele J, Zirbes TK, Bertsch HP, Titius BR, Lorenzen J, Fischer R. AIDS-related bone marrow lesions--myelodysplastic features or predominant inflammatory-reactive changes (HIV-myelopathy)? A comparative morphometric study by immunohistochemistry with special emphasis on apoptosis and PCNA-labeling. Anal Cell Pathol. 1996;11:141-157.

(26.) Karcher DS, Frost AR. The bone marrow in human immunodeficiency virus (HIV)-related disease: morphology and clinical correlation. Am J Clin Pathol. 1991;95:63-71.

(27.) Diebold J, Molina T, Camilleri-Broet S, Le Tourneau A, Audouin J. Bone marrow manifestations of infections and systemic diseases observed in bone marrow trephine biopsy review. Histopathology. 2000;37:199-211.

(28.) Ng MH, Li EK, Feng CS. Gelatinous transformation of bone marrow in systemic lupus erythematosus. JRheumatol. 1989;16:989-992.

(29.) Paquette RL, Meshkinpour A, Rosen PJ. Autoimmune myelofibrosis: a steroid-responsive cause of bone marrow fibrosis associated with systemic lupus erythematosus. Medicine (Baltimore). 1994;73:145-152.

(30.) Brunning RD, McKenna RW. Lesions simulating lymphomas. In: Tumors of the Bone Marrow. Washington, DC: Armed Forces Institute of Pathology; 1994: 409-455. Atlas of Tumor Pathology; 3rd series, fascicle 9.

(31.) Thiele J, Kvasnicka HM, Orazi A. Bone marrow histopathology in myeloproliferative disorders--current diagnostic approach. Semin Hematol. 2005;42: 184-195.

(32.) Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005;365:1054-1061.

(33.) James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005;434: 1144-1148.

(34.) Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005;352:1779-1790.

(35.) Levine RL, Wadleigh M, Cools J, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell. 2005;7:387-397.

(36.) Vardiman JW, Imber M, Pierre R, Brunning RD,Thiele J, Flandrin G. Chronic myelogenous leukemia. In: Jaffe ES, Harris NL, Stein H, Vardiman JW, eds. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2001:20-26. World Health Organization Classification of Tumours.

(37.) Kimura A, Katoh O, Hyodo H, Kuramoto A, Satow Y. Platelet derived growth factor expression, myelofibrosis and chronic myelogenous leukemia. Leuk Lymphoma. 1 995;1 8:237-242.

(38.) Rywlin AM, Ortega RS, Dominguez CJ. Lymphoid nodules of bone marrow: normal and abnormal. Blood. 1974;43:389-400.

(39.) Ng AP, Wei A, Bhurani D, Chapple P, Feleppa F, Juneja S. The sensitivity of CD138 immunostaining of bone marrow trephine specimens for quantifying marrow involvement in MGUS and myeloma, including samples with a low percentage of plasma cells. Haematologica. 2006;91:972-975.

(40.) Thiry A, Delvenne P, Fontaine MA, Boniver J. Comparison of bone marrow sections, smears and immunohistological staining for immunoglobulin light chains in the diagnosis of benign and malignant plasma cell proliferations. Histopathology. 1993;22:423-428.

(41.) Kass L, Kapadia IH. Perivascular plasmacytosis: a light-microscopic and immunohistochemical study of 93 bone marrow biopsies. Acta Haematol. 2001; 105:57-63.

(42.) Grogan TM, Camp BV, Kyle RA, Muller-Hermelink HK, Harris NL. Plasma cell neoplasms. In: Jaffe ES, Harris NL, Stein H, Vardiman JW, eds. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2001:142-156. World Health Organization Classification of Tumours.

(43.) Arber DA, George TI. Bone marrow biopsy involvement by non-Hodgkin's lymphoma: frequency of lymphoma types, patterns, blood involvement, and discordance with other sites in 450 specimens. Am J Surg Pathol. 2005;29:1549 1557.

(44.) Swan N, Skinner M, O'Hara CJ. Bone marrow core biopsy specimens in AL (primary) amyloidosis: a morphologic and immunohistochemical study of 100 cases. Am J Clin Pathol. 2003;120:610-616.

(45.) Hasserjian RP, Goodman HJ, Lachmann HJ, Muzikansky A, Hawkins PN. Bone marrow findings correlate with clinical outcome in systemic AL amyloidosis patients. Histopathology. 2007;50:567-573.

Robert P. Hasserjian, MD

Accepted for publication October 17, 2007.

From the Department of Pathology, Massachusetts General Hospital, Boston.

The author has no relevant financial interest in the products or companies described in this article.

Presented in part at the 28th Annual Course, Current Concepts in Surgical Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, November 2006.

Reprints: Robert P. Hasserjian, MD, Department of Pathology, Massachusetts General Hospital, 55 Fruit St, Boston, MA 02115 (e-mail: rhasserjian@partners.org).
Table 1. Common Ancillary Studies That Complement Bone Marrow
Morphologic Examination*

1. Cytogenetics on aspirated bone marrow or peripheral blood
2. FISH studies performed on aspirated bone marrow or touch
preparations
3. Molecular studies (typically PCR or RT-PCR) for antigen receptor
gene rearrangements and/or to detect specific translocations
4. Immunophenotyping of aspirated bone marrow or peripheral blood
cells by flow cytometry
5. Immunohistochemistry on paraffin sections
6. Enzyme cytochemistry on marrow aspirate or peripheral smear slides

* FISH indicates fluorescence in situ hybridization; PCR, polymerase
chain reaction; and RT-PCR, reverse transcriptase-polymerase chain
reaction.

Table 2. Reticulin Grading Systems

Bauermeister system *

No increase     Cell outlines visible, but no black fibers
Grade 1         Fine black fibers with few crossings
Grade 2         Fine black fibers with numerous crossings forming a
                 meshwork
Grade 3         Black fiber meshwork with some thick coarse fibers
Grade 4         Meshwork of thick coarse fibers (trichrome stain
                 typically positive)

European consensus system([dagger])

Grade 0         Scattered linear reticulin with no intersections
                 (crossovers)
Grade 1         Loose network of reticulin with many intersections,
                 especially in perivascular areas
Grade 2         Diffuse and dense increase in reticulin with extensive
                 intersections, occasionally with only focal bundles
                 of collagen and/or focal osteosclerosis
Grade 3         Diffuse and dense increase in reticulin with extensive
                 intersections with coarse bundles of collagen, often
                 associated with significant osteosclerosis.

* Modified with permission from Bauermeister.7 Copyright 1971 American
Society for Clinical Pathology.

([dagger]) Modified with permission from Haematologica
(http://www.haematologica.org; accessed October 3, 2007) from Thiele
et al. (8)

Table 3. Clinical Indications for Bone Marrow
Sampling Performed at 2 Institutions, Including Both
Adult and Pediatric Patients

                                            Percentage of All Cases

                                                         Institution 2
Clinical Indication                    Institution 1 *    ([dagger])

Primary evaluation for disease
  Anemia                                     28               14
  Paraprotein                                19                8
  Thrombocytosis and/or leukocytosis          9                2
  Thrombocytopenia                            5                3
  New diagnosis of leukemia                   4                4
  Leukopenia or pancytopenia                  1                3
  Other indications                           1                4
  Total Performed as Primary
    Evaluation of Disease                    67               38

Follow-up of known disease
  Staging or follow-up of lymphoma           22               26
  Follow-up for leukemia on
    treatment                                10               30
  Other follow-up                             1                7
  Total Performed as Follow-up
    of Known Disease                         33               63

* Institution 1 is Baystate Medical Center, Springfield, Mass, an
institution with predominance of primary hematology practice.

([dagger]) Institution 2 is Massachusetts General Hospital, Boston,
an institution with a predominance of tertiary/referral hematology
practice.

Table 4. Bone Marrow Abnormalities in Human
Immunodeficiency Virus-Infected Patients *

1. Hypercellularity or hypocellularity
2. Morphologic dysplasia (especially in megakaryocytes, which
   appear as "bare nuclei")
3. Plasmacytosis
4. Lymphoid aggregates
5. Reticulin fibrosis
6. Serous atrophy (gelatinous transformation)

* Adapted from Karcher and Frost. (26) Copyright 1991 American Society
for Clinical Pathology.

Table 5. Causes of Increased Bone Marrow Reticulin *

1. Chronic idiopathic myelofibrosis
2. Other MPD (including CML), MDS, or MDS/MPD
3. Acute leukemias (especially, but not exclusively, megakaryoblastic)
4. Lymphomas (especially hairy cell leukemia)
5. Metastatic neoplasms
6. Infections (especially HIV)
7. Lupus and other autoimmune diseases

* MPD indicates myeloproliferative disease; CML, chronic myeloid
leukemia; MDS, myelodysplastic syndrome; and MDS/MPD, myelodysplastic
/myeloproliferative disease.
Gale Copyright: Copyright 2008 Gale, Cengage Learning. All rights reserved.