Indications for bone marrow examination
Bone marrow examination, including both aspiration and biopsy sampling, can be performed on virtually any patient. However, patients with coagulation deficiencies or profound thrombocytopenia may experience prolonged bleeding, which cannot be controlled by pressure bandages. In these rare cases, specific treatment (e.g., platelet transfusion) may be indicated. Indications for performing bone marrow examination are summarized in Table 1.1. In the vast majority of cases, both a bone marrow aspiration and biopsy should be performed. Bone marrow aspiration and bone marrow biopsy are complementary (Bain, 2001a, 2001b). Bone marrow aspiration provides excellent cytologic detail; however, marrow architecture cannot be assessed. Bone marrow core biopsy allows for an accurate analysis of architecture; however, cytologic details may be lost. Table 1.2 shows the accepted indications for performing a bone marrow biopsy. This includes cases with inadequate or failed aspiration, need for accurate assessment of cellularity, cases in which the presence of focal lesions (e.g., granulomatous disease or metastatic carcinoma) is suspected, suspected bone marrow fibrosis, need to study bone marrow architecture, need to study bone structure, bone marrow stroma, or assessment of bone marrow vascularity. In general, patients with hypocellular marrows or bone marrow fibrosis are likely to need a trephine biopsy for adequate assessment. In such patients, an aspirate would probably be inadequate or even impossible. Unexplained pancytopenia and unexplained leukoerythroblastic blood pictures are further indications for a biopsy, because they are likely to indicate the presence of bone marrow metastatic disease or fibrosis.
Lymphoproliferative disorders frequently involve the peripheral blood and bone marrow, and bone marrow studies may be performed for primary diagnosis or as a staging procedure in patients with lymphoproliferative disorders. A primary diagnosis with accurate classification can often be made on bone marrow samples alone, if a combined morphologic and immunophenotypic approach is used. The addition of molecular or cytogenetic studies can resolve some of the low percentage of cases that are equivocal after morphologic and immunophenotypic analysis.
Many of the low-grade B-cell lymphomas will involve the bone marrow, but the precise morphologic classification of these diseases is complicated by the fact that characteristic architectural patterns seen in lymph nodes involved by these diseases are not present in the bone marrow. Despite this, many of the low-grade B-cell lymphomas have characteristic immunophenotypes that allow for proper classification. Flow cytometric immunophenotyping of involved peripheral blood or bone marrow aspirate material allows for evaluation of the largest number of antigens, as well as confirming aberrant co-expression of antigens that are characteristic of certain disease types. However, paraffin section immunophenotyping, which can be performed on core or clot biopsy material, can also be used successfully to detect many antigens of interest in these cases. The characteristic immunophenotypic features of the various small B-cell lymphoid proliferations are summarized in Table 11.1.
Molecular genetic or cytogenetic studies may also be useful in selected cases to confirm the presence of a clonal population, when the differential diagnosis is between reactive and neoplastic lymphoid proliferations (Arber, 2000).
Myelodysplastic syndromes (MDS) are a clinically heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis associated with dysplastic changes in one or more marrow lineages together with progressive cytopenias. MDS may occur as primary diseases, or may follow toxic exposures or therapy (Jaffe et al., 2001). Primary MDS is more often seen in elderly patients and frequently progresses to marrow failure, or evolves to acute leukemia, usually myeloid. The overall rate of transformation to acute leukemia depends on the subtype of MDS, ranging from 10% to over 60%. As a rule, the degree of trilineage dysplasia and percentage of blast cells correlate with the aggressiveness of the disease. It should be stressed that dysplastic features of hematopoietic elements is not unique to MDS and may occur in many reactive conditions, such as inflammatory states, HIV infection, endocrine dysfunctions, autoimmune disorders, and may also be associated with certain medications.
MDS have been traditionally subdivided according to the French–American–British (FAB) system (1982) into five major categories, primarily based on the percentage of blast cells in the peripheral blood and bone marrow (Table 7.1), the presence of ringed sideroblasts in the marrow, and the absolute count of monocytes in peripheral blood (Bennett et al., 1982). These subgroups show different rates of progression to acute myeloid leukemia (AML) and overall survival. In particular, refractory anemia and refractory anemia with ringed sideroblasts are associated with much longer median survival and a lower incidence of progression to acute leukemia than the other three FAB subtypes and can be considered “low-grade” MDS.
Granulomas in bone marrow
Although aggregates of histiocytes may be present on aspirate smears, granulomas are best identified with bone marrow trephine and clot biopsy materials (Bodem et al., 1983; Bhargava & Farhi, 1988; Vilalta-Castel et al., 1988; Foucar, 2001; Chang et al., 2003). Granulomas in the bone marrow should be approached in a similar fashion to those in other sites.
There are generally two types of granulomas encountered in the marrow. The lipogranuloma is a collection of histiocytes surrounding adipose tissue and is usually not associated with disease (Rywlin & Ortega, 1972) (Fig. 3.1). Epithelioid granulomas without associated adipose tissue may have admixed lymphocytes, plasma cells, neutrophils, or eosinophils and may have associated necrosis (Fig. 3.2). Mycobacteria and fungal organisms should be excluded by special stains in all cases with epithelioid granulomas, and fresh bone marrow aspirate material should be submitted for culture in all patients being evaluated for infectious diseases.
The most common causes of infectious granulomas are summarized in Table 3.1. Patients with immunodeficiency syndromes may have a disseminated atypical mycobacterial infection caused by Mycobacterium avium–intracellulare (MAI) even when well-formed granulomas are not present. In such cases, special stains for organisms are indicated when any increase in histiocytes is noted. MAI may also be associated with the presence of “pseudo-Gaucher” macrophages in which the intracellular organisms mimic the characteristic cytoplasmic striations of Gaucher cells. MAI shows a characteristic periodic acid-Schiff (PAS) positivity, while in tuberculosis the organisms are usually PAS-negative (Fig. 3.3).
A decrease in bone marrow cellularity for a patient's age may be related to a variety of causes. Artifactual hypocellularity due to sampling of only subcortical marrow should not be misinterpreted, and an accurate estimate of marrow cellularity cannot be made on small biopsy specimens that contain only the subcortical marrow space. True hypocellularity may be due to a decrease in all marrow cell lines or decreases in only selected cell lines.
Aplastic anemia (AA) may be acquired or constitutional (congenital), and represents a decrease in granulocytic, erythroid, and megakaryocytic cells (Guinan, 1997; Young, 1999). The principal conditions associated with the development of acquired and inherited aplastic anemia are summarized in Table 4.1.
Diagnostic criteria for severe aplastic anemia are (1) a bone marrow of less than 25% of normal age-related cellularity values, and (2) two of the following three peripheral blood findings: a neutrophil count of less than 0.5 × 109/L, a platelet count of less than 20 × 109/L, anemia with a corrected reticulocyte count of less than 1%. A grading system for AA patients is summarized in Table 4.2.
The peripheral blood demonstrates pancytopenia without obvious abnormalities of the circulating cells (Camitta et al., 1979). Aspirate smears usually show small particles containing histiocytes, mast cells, lymphocytes, and plasma cells with no or rare normal hematopoietic cells. The biopsy specimens in these patients are variably hypocellular (Fig. 4.1).
Bone marrow biopsies performed to evaluate for metastatic disease are among the most common samples received. While advances in radiologic techniques have reduced the number of such bone marrow examinations, this method is still valuable for selected patients. Determination of the tumor type is usually not difficult in patients with a known primary tumor. However, unexpected malignancies may be identified, for example during an anemia work-up (Wong et al., 1993), or bone marrow biopsy may be performed prior to a primary tumor biopsy. For example, a bone marrow biopsy may be performed as the initial procedure in a child with an abdominal mass. The ability to diagnose a tumor based on a bone marrow examination may reduce the need for further procedures or alter the approach of a second procedure. In the setting of an unknown primary, immunohistochemical studies are often essential for characterization. Even with these studies, clinical correlation is often needed to confirm the primary diagnosis. The most common non-hematologic malignancies to involve the bone marrow are prostate, breast, and lung carcinomas, neuroblastoma, Ewing sarcoma/peripheral neuroectodermal tumor (PNET), rhabdomyosarcoma, and malignant melanoma (Anner & Drewinki, 1977; Papac, 1994).
Peripheral blood and bone marrow aspirate
Peripheral blood involvement by metastatic tumor, termed carcinocythemia, is extremely uncommon and usually represents a late event with short survival (Gallivan & Lokich, 1984) (Fig. 13.1). Other, non-specific abnormalities of the blood are more common in patients with metastatic carcinoma. These usually manifest as anemia, leukoerythroblastosis, leukocytosis, and microangiopathic hemolytic anemia.
A variety of therapy regimens and toxin exposures can cause bone marrow changes. Post-therapy evaluation of the marrow may be useful to evaluate for residual disease, to assess the degree of marrow ablation, or to look for signs of marrow recovery. While proper marrow evaluation after therapy in individual patients requires knowledge of the type of prior therapy and original disease, some changes after therapy are common to all cases and vary primarily by the degree of marrow ablation.
General marrow changes after myeloablative therapy
There are many similarities in the marrow findings following high-dose chemotherapy or combined chemotherapy and radiation, as is often used in preparation for hematopoietic stem cell transplantation, and even after toxin or drug injuries to the marrow (Sale & Buckner 1988; van den Berg et al., 1989, 1990; Michelson et al., 1993; Wilkins et al., 1993). Common bone marrow changes after myeloablative therapy are summarized in Table 14.1. In the first week after the most severe types of injuries, the marrow shows complete aplasia with a complete or near-complete absence of normal hematopoietic elements and marrow fat. There is marked edema with dilated marrow sinuses, intramedullary hemorrhages, and scattered stromal cells, histiocytes, lymphocytes, and plasma cells. The histiocytes may contain cellular remnants, and fibrinoid necrosis may be prominent. Zonal areas of tumor necrosis may also be present, although myeloablative therapy is often given in the absence of prior marrow disease.
Acute leukemia is a proliferation of immature bone marrow-derived cells (blasts) that may also involve peripheral blood or solid organs. The percentage of bone marrow blast cells required for a diagnosis of acute leukemia has traditionally been set arbitrarily at 30% or more. However, more recently proposed classification systems have lowered the blast cell count to 20% for many leukemia types, and do not require any minimum blast cell percentage when certain morphologic and cytogenetic features are present.
The traditional classification of acute leukemia used criteria proposed by the French–American–British Cooperative Group (FAB) (Table 8.1), using the 30% bone marrow blast cell cutoff (Bennett et al., 1976, 1985a). This classification system originally distinguished different leukemia types by morphologic features and cytochemical studies, particularly myeloperoxidase (or Sudan black B) and non-specific esterase staining. It was revised to include leukemia types that could only be accurately identified with the addition of immunophenotyping or electron microscopic studies (Bennett et al., 1985b, 1991). Although the FAB classification failed to distinguish immunophenotypic groups of acute lymphoblastic leukemias, did not recognize the significance of myelodysplastic changes in acute myeloid leukemias or cytogenetic abnormalities in either leukemia type, and resulted in some subcategories of little clinical significance, this system provided very clear guidelines for classification. In addition, some distinct leukemia subtypes, particularly acute promyelocytic leukemia and acute myeloid leukemia with abnormal eosinophils, were found to correlate with specific cytogenetic aberrations and had unique clinical features, and those remain in recently proposed classification systems.
The category of myelodysplastic/myeloproliferative disorders (MDS/MPD) includes malignant hematopoietic proliferations which, at the time of their initial presentation, display features of both myelodysplastic syndromes and myeloproliferative disorders (Oscier, 1997; Jaffe et al., 2001). Cytopenias and dysplastic changes of any cell line may be seen, similar to the myelodysplastic syndromes. Elevated white blood cell counts, hypercellular marrows with fibrosis, and organomegaly, features more commonly associated with myeloproliferative disorders, may also be present. The presence of fibrosis alone in cases that are otherwise typical of myelodysplasia should not be placed in this category. The three best-defined mixed myeloproliferative and myelodysplastic syndromes are atypical chronic myeloid leukemia (atypical CML), chronic myelomonocytic leukemia (CMML), and juvenile myelomonocytic leukemia (JMML). Features that help in differentiating the chronic phase of CML from atypical CML and CMML are listed in Table 10.1.
Atypical chronic myeloid leukemia
Atypical CML (Bennett et al., 1994) is a Philadelphia chromosome-negative and BCR/ABL-negative proliferative disorder that affects elderly patients, with an apparent male predominance. Its incidence is <2 cases for every 100 cases of t(9;22), BCR/ABL-positive CML (Jaffe et al., 2001). Patients have some features of CML including splenomegaly, an elevated white blood cell count of predominantly granulocytic cells, and moderate anemia. The major characteristic which distinguishes atypical CML is dysgranulopoiesis, which is often severe. Moreover, atypical CML may have an initial presentation more typical of myelodysplasia with a low white blood cell count and normal to decreased platelet counts (Oscier, 1996).
It is often easiest to evaluate a bone marrow specimen by comparing it to what would be expected in the normal bone marrow (Brown & Gatter, 1993; Bain, 1996). The initial evaluation on low magnification includes the assessment of sample adequacy and marrow cellularity. The latter is usually based on the biopsy. Estimates of cellularity on aspirate material have been described (Fong, 1979) but may be unreliable in variably cellular marrows (Gruppo et al., 1997). The normal cellularity varies with age (Table 2.1), and evaluation of cellularity must always be made in the context of the patient's age (Hartsock et al., 1965) (Fig. 2.1). The marrow is approximately 100% cellular during the first three months of life, 80% cellular in children through age 10 years; it then slowly declines in cellularity until age 30 years, when it remains about 50% cellular. The usually accepted range of cellularity in normal adults is 40–70% (Hartsock et al., 1965; Gulati et al., 1988; Bain, 1996; Friebert et al., 1998; Naeim, 1998). The marrow cellularity declines again in elderly patients to about 30% at 70 years. Because of the variation in cellularity by age, the report should clearly indicate whether the stated cellularity in a given specimen is normocellular, hypocellular, or hypercellular.
Estimates of cellularity may be inappropriately lowered by several factors. Subcortical bone marrow is normally hypocellular, and the first three subcortical trabecular spaces are usually ignored in the cellularity estimate (Fig. 2.2).
Fibrosis of the bone marrow is caused by a reactive (non-clonal) proliferation of fibroblasts which may occur in association with a variety of neoplastic and non-neoplastic conditions (McCarthy, 1985). Fibrosis is usually of the reticulin type, as detected by silver stains, in the early stages, but it may progress to a collagen fibrosis that is detectable by trichrome stains (Fig. 6.1). Normally, reticulin staining is minimal but increases slightly with age (Beckman et al., 1990). Extensive marrow fibrosis is typically associated with a leukoerythroblastic reaction in the peripheral blood that is characterized by the presence of immature granulocytes (usually myelocytes and metamyelocytes), late-stage erythroblasts, and teardrop-shaped red blood cells and enlarged platelets. Diffuse fibrosis usually results in the inability to aspirate the bone marrow. Bone marrow fibrosis is common in chronic myeloproliferative disorders, and extensive fibrosis with peripheral blood leukoerythroblastosis is typical of chronic idiopathic myelofibrosis with myeloid metaplasia. Patchy areas of fibrosis are also seen with bone marrow involvement by mast cell disease (Horny et al., 1985), which may accompany other hematologic malignancies at diagnosis or relapse. Many other neoplasms involving the marrow, including some acute leukemias, malignant lymphomas, and metastatic tumors, result in focal or diffuse marrow fibrosis (Table 6.1).
Marrow fibrosis may also be associated with non-neoplastic conditions, especially inflammatory diseases, reparative changes, or metabolic disorders. Among these reactive cases, a particularly severe degree of fibrosis can be seen in patients with autoimmune conditions (Bass et al., 2001) (Fig. 6.2).
Non-neoplastic hyperplasias of one or more bone marrow cell lineages are often related to changes occurring outside the marrow and must be correlated with peripheral blood findings, other appropriate laboratory data, and, above all, clinical information. Patients recovering from toxic insults, including chemotherapy and radiation therapy, may demonstrate a transient bone marrow hyperplasia. In a similar fashion, destruction of particular hematopoietic elements outside the marrow, as in some autoimmune diseases, often results in hyperplasia of the corresponding cell lineage in the marrow. Because the bone marrow changes may represent a reaction to events elsewhere in the body, the bone marrow specimen alone is often not diagnostic of the patient's underlying disease process.
Erythroid hyperplasias represent a response to peripheral red blood cell loss or destruction or are related to ineffective erythropoiesis, as seen in some chronic anemias. The causes of erythroid hyperplasias are best addressed in combination with the evaluation of red blood cell features in the peripheral blood (Walters & Abelson, 1996; Peterson & Cornacchia, 1999) (Fig. 5.1). In general, ancillary laboratory testing is needed to precisely characterize the cause of the erythroid hyperplasia. Dyserythropoiesis, particularly mild irregularities of the nuclear contours of erythroid precursors, is common in cases of florid erythroid hyperplasia and should not be over-interpreted as evidence of myelodysplasia.
Erythroid hyperplasias associated with normocytic anemia
Hemorrhage, hemolytic anemia, intrinsic bone marrow disease (including aplastic anemia and malignant neoplasms), and anemia of chronic disease are the most common causes of erythroid hyperplasia associated with normocytic anemia in patients with no history of a toxic insult, chemotherapy, or hemoglobinopathy.
Illustrated Pathology of the Bone Marrow is designed to help pathologists and pathologists in training answer the practical diagnostic questions that arise during routine analysis of bone marrow core biopsy specimens. Although emphasis has been placed on the histologic interpretation of the bone marrow biopsy, an attempt has been made to integrate histologic findings with clinical and laboratory features and peripheral blood and bone marrow aspiration morphology. In recent years, integration between morphology, immunophenotype, genetic features, and clinical features has been increasingly used to distinguish between distinct clinical entities. This integrated multiparametric approach forms the basis for the WHO classification of tumors of hematopoietic and lymphoid tissue. As a consequence, morphology, immunophenotype, genetics, and clinical features are integrated throughout the book in an effort to summarize the current best practice of bone marrow interpretation. The illustrative case material in this book has been gathered from several institutions, including Indiana University School of Medicine in Indianapolis, Indiana; the College of Physicians and Surgeons of Columbia University, New York, New York; the City of Hope National Medical Center, Duarte, California; and Stanford University, Stanford, California. A systematic, analytical approach to interpretation of pathological changes is used throughout the book, which will enable pathologists with varying backgrounds and experience to feel confident in their assessment of bone marrow specimens during their routine everyday analysis.
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Immunosecretory and plasma cell disorders cover a broad spectrum of clinical and pathologic entities. Some of the processes, such as monoclonal gammopathy of undetermined significance (MGUS), have relatively indolent behavior, while others, such as plasma cell leukemia, are associated with very poor prognosis and high mortality. This group of disorders includes lymphoplasmacytic lymphoma (LPL), a neoplastic entity which overlaps both with B-cell lymphoma and with the immunosecretory disorders group.
Benign plasma cells and reactive plasmacytosis
Plasma cells are a normal component of adult bone marrows. They typically represent about 0–1% of the overall cellularity seen in bone marrow aspirate smears (Foucar, 2001). Levels above 5% are considered abnormal in immunologically unstimulated marrows. In normal bone marrow biopsies, plasma cells are most typically located in perivascular locations.
In Wright–Giemsa-stained preparations, plasma cells have a distinctive, light to dark blue cytoplasm with an eccentrically placed nucleus. The nuclear chromatin is quite dense, and in appropriately thin histologic sections is classically described as “clockface chromatin.” Adjacent to the nucleus is a clearing in the cytoplasm, referred to as a hof, which represents the Golgi apparatus of the cell.
The cytologic features of benign and malignant plasma cells can overlap (Fig. 12.1). Inclusions which may be seen in plasma cells include Russell bodies, which are globular eosinophilic collections of immunoglobulin in the cytoplasm. Dutcher bodies are not true nuclear inclusions but rather cytoplasmic inclusions that overlie the nucleus. Dutcher bodies are only rarely seen in benign proliferations of plasma cells.
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