Bone Marrow Pathology
Jeffery M. Klco
John L. Frater
I. NORMAL GROSS AND MICROSCOPIC ANATOMY. The bone marrow is generally considered the fourth largest organ in the human body and is composed of cells derived from a variety of lineages including stromal cells, adipocytes, lymphocytes, and hematopoietic precursors. The most frequently sampled areas are the posterior superior iliac crest and, much less frequently, the sternum and long bones. The bone marrow has an orderly microscopic anatomy. The most superficial part consists of a layer of dense cortical bone with an adjacent cover of dense fibrous periosteum. Deep into the cortex are the bony trabeculae, which consist of thin trabecular bone and the marrow cavity itself. The marrow cavity contains islands of maturing hematopoietic cells with intervening areas of fat, the latter of which increase with age.
The cellularity of the bone marrow is defined as the percentage of the marrow cavity composed of hematopoietic cells. In biopsies from the posterior iliac crest, marrow cellularity decreases with age, and is expressed by the formula: Marrow cellularity = (100 − patient age)% ± 20%. Thus, a 50-year-old individual would be expected to have a marrow cellularity of approximately (100 − 50)% ± 20%, or 30% to 70%.
Under normal circumstances, maturing myeloid and erythroid elements occupy different regions of the marrow cavity. Myeloid precursors lie adjacent to the trabecular bone, and erythroid elements form “islands” of cells between trabeculae. The ratio of myeloid to erythroid elements is roughly 2:1. Megakaryocytes are irregularly distributed throughout the bone marrow. Under normal circumstances they are not present in clusters.
It is important to be cognizant of the multidisciplinary nature of hematopathology. Diagnoses in bone marrow pathology are not generally the product of morphologic analysis of the bone marrow core biopsy or peripheral blood and bone marrow aspirate smears alone. Clinical history is extremely important in separating morphologically similar diseases and should be provided by the patient’s physicians. Also, the pertinent features of the physical examination, such as lymphadenopathy, splenomegaly, and hepatomegaly, are of importance. Other clinical laboratory information, such as complete blood counts and serum and/or urine protein electrophoresis, are often of interest. Radiographic data are of importance, particularly in the evaluation of a monoclonal protein.
II. GROSS EXAMINATION AND TISSUE SAMPLING. The same pathologist should review both the bone marrow aspirate smears and the core biopsies whenever possible to avoid ambiguities or outright contradictions. The bone marrow aspirate is generally performed before the biopsy. There are two kinds of aspirate smears: smears prepared directly from the specimen without pretreatment and smears prepared from concentrated aspirate fluid. Concentrated bone marrow aspirate smears and touch preparations prepared from the bone marrow core biopsy are particularly useful in the evaluation of specimens diluted with peripheral blood. Three to five smears are stained using the Wright-Giemsa or similar technique, one is stained for iron using the Prussian blue technique, and additional unstained smears are reserved for ancillary techniques such as fluorescence in situ hybridization (FISH), if necessary. Although examination of bone marrow cells with enzyme cytochemistry is likely to be rendered obsolete in the coming years, its use is still highly recommended by the World Health Organization (WHO) committee for the diagnosis of
acute myeloid leukemia, and high-quality aspirate smears should be reserved for this purpose when an acute myelogenous leukemia is suspected. Additional bone marrow aspirates are obtained for flow cytometric, cytogenetic, and/or molecular genetic studies. Aspirate smears are generally reviewed using high power (600× to 1000×) and are important for evaluating individual cell detail. However, because the process of aspiration disrupts cell cohesion, the relationship of the various cell types and the marrow cellularity cannot be reliably assessed. An adequate bone marrow core biopsy adds this important information.
III. DIAGNOSTIC FEATURES OF COMMON BENIGN DISEASES. The number and scope of nonneoplastic bone marrow disorders are vast. Emphasis is given to commonly encountered bone marrow diseases and conditions that may simulate neoplasia.
A. Megaloblastic anemia. It is often not necessary to perform a bone marrow biopsy in patients who present with anemia, because the most common forms of anemia (iron deficiency, megaloblastic, and anemia of chronic disease) may be diagnosed by laboratory analysis of the peripheral blood, clinical history, and response to iron, vitamin B12, and/or folate replacement. Bone marrow biopsy is performed for patients with anemia that is unexplained or therapeutically resistant.
It is important to note that megaloblastic anemia may simulate a neoplastic condition, particularly a myelodysplastic syndrome. Patients with megaloblastic anemia may present with marked cytopenias and pronounced dyspoiesis (e-Figs. 44.1 and 44.2).* Clues to discriminate this benign condition from a myelodysplastic syndrome include normal blast percentage, absence of karyotypic abnormalities, and improvement of cytopenias following administration of vitamin B12/folate in megaloblastic anemia. Also, the degree of dyspoiesis in megaloblastic anemia often exceeds that encountered in most cases of neoplastic myelodysplasia.
B. Benign causes of lymphocytosis. Numerous benign conditions may cause a transient increase in benign lymphocytes in the peripheral blood and can simulate chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), T-cell large granular lymphocytic leukemia, or other lymphoid leukemias. Stress lymphocytosis is a transient increase in morphologically normal peripheral blood lymphocytes encountered in individuals subjected to physiologic stresses, including individuals presenting to hospital emergency departments. An absolute increase in lymphocytes accompanied by “reactive” forms with increased basophilic cytoplasm and inconspicuous nucleoli may be seen in patients infected with Epstein-Barr virus (EBV, e.g., infectious mononucleosis) or, less commonly, cytomegalovirus or other viral pathogens. Interestingly, in cases of EBV infection the virus particles are present in morphologically normal lymphocytes, and the reactive lymphocytes represent cytotoxic T cells directed at the infected cells. Correlation with serum viral antibody titers is useful for arriving at the correct diagnosis and for avoiding unnecessary procedures such as bone marrow and lymph node biopsies. Many other infections are associated with lymphocytosis, including pertussis, in which the lymphocytes have characteristic clefted nuclei and thus may simulate peripheral blood involvement by a non-Hodgkin lymphoma. Persistent polyclonal B-cell hyperplasia, as its name implies, is often of longer duration than other benign forms of lymphocytes. It frequently affects women who smoke, and is associated with the human leukocyte antigen (HLA)-DR7 phenotype.
C. The granulocytic “left shift.” Other cases of leukocytosis represent a granulocytic shift to immaturity (colloquially known by the archaic term “left shift” that refers to the traditional placement of immature myeloid cell percentages to the left of neutrophils in classical peripheral blood smear reports). Because many
of these cases also demonstrate eosinophilia and/or basophilia, it is important to distinguish them from neoplastic conditions, in particular chronic myelogenous leukemia or other myeloproliferative neoplasms. Most commonly, especially in hospitalized populations, a granulocytic shift to immaturity represents an acute response to bacterial or other infections. Transient increases in mature demarginated neutrophils may also follow surgery or other physical trauma. Unusual causes of increased peripheral blood neutrophils with or without immature granulocytes include chronic idiopathic neutrophilia, hereditary neutrophilia, and leukocyte adhesion factor deficiency.
D. Benign causes of erythrocytosis. Under normal conditions, the circulating red blood cell mass is maintained at a constant level by the actions of the cytokine erythropoietin, which is produced by renal peritubular cells. An absolute increase in circulating red blood cells (polycythemia) may be primary (most commonly due to the myeloproliferative neoplasm polycythemia vera) or secondary (due to increased production of erythropoietin). Thus, in establishing a diagnosis of polycythemia vera, a number of conditions must be excluded that may cause secondary erythrocytosis, including smoking, living in a high-altitude environment, and high oxygen-affinity hemoglobins.
E. Benign causes of thrombocytosis. There are numerous causes of benign thrombocytosis (defined as a peripheral blood platelet count in excess of 450 × 109/L). Common reactive causes of peripheral thrombocytosis include childbirth, major hemorrhage, iron deficiency anemia, chronic inflammatory conditions, infection, and acute stress events.
F. Aplastic anemia. Bone marrow aplasia is usually associated with bi- or pancytopenia rather than isolated anemia, and may be identified in a variety of clinical settings. It may occur secondary to a variety of drugs, most commonly chemotherapeutic agents, benzene, alcohol, and arsenic. It may also occur secondary to exposure to radiation, viral infection (e.g., viral hepatitis, EBV), or tuberculosis. An important cause of infection-mediated isolated anemia is infection with parvovirus B19. Other causes of bone marrow hypocellularity include paroxysmal nocturnal hemoglobinuria, Fanconi anemia, dyskeratosis congenital, and other very rare inherited bone marrow failure syndromes (Int J Hematol. 2002;76, Suppl 1:207).
The common morphologic finding in bone marrow biopsies from patients with aplastic anemia is marked panhypoplasia; a notable exception is parvovirus infection in which there is selective suppression of erythroid precursors. These specimens should be carefully scrutinized for evidence of significant dyspoiesis or increased blasts because a minority of myelodysplastic syndromes and acute myeloid leukemias present with markedly hypocellular bone marrow biopsies. These specimens should also be examined for evidence of infection as evidenced by granuloma formation in the case of tuberculosis, or intranuclear inclusions in the case of viral infection. The inclusions of parvovirus are ill defined and are localized to the nuclei of proerythroblasts and can be illuminated by immunohistochemistry against viral capsid proteins.
G. Serous degeneration (serous atrophy) is a pattern of bone marrow injury most often associated with acquired immunodeficiency syndrome (AIDS) and states of chronic nutritional deficiency such as starvation, chronic alcoholism, and anorexia nervosa (Arch Pathol Lab Med. 1992;116:504). The primary morphologic finding in the bone marrow is stromal edema with associated microvesicular change. The bone marrow is hypocellular in these regions due to loss of normal hematopoietic elements. The findings may be focal, alternating with regions of relatively preserved normal hematopoiesis. It is important to recognize that the subcortical bone marrow is normally hypocellular and occasionally demonstrates edema, possibly related to trauma associated with the biopsy procedure, and thus may mimic serous degeneration.
H. Granulomas may be encountered in bone marrow core biopsies and are occasionally noted in aspirate smears. Granulomas may be quite subtle and are usually composed of admixed histiocytes, lymphocytes, and plasma cells. Some granulomas contain foci of necrosis and infiltrating neutrophils. The causes of granuloma formation in the bone marrow are similar to those in other sites; the most common etiologies are infectious, autoimmune, or idiopathic. In addition, granulomas are occasionally encountered in the bone marrow of patients with Hodgkin lymphoma, although their presence is not indicative of marrow involvement by disease. Because it is impossible to predict with certainty the etiology of bone marrow granulomas, their presence usually warrants the use of stains to aid in the identification of acid-fast bacilli or fungi. However, it should be noted that special stains are far less sensitive than microbiologic culture in the detection of most infectious agents in the bone marrow, so microbiologic analysis of fresh bone marrow tissue is recommended when an infectious etiology is considered. An important item in the morphologic differential diagnosis of granulomas is the lipogranuloma, which is generally considered to be nonpathologic and consists of a collection of histiocytes and lymphocytes surrounding an area of fat demonstrating microvesicular change.
I. Benign lymphoid aggregates are present in the bone marrow of healthy individuals and at increased incidence in elderly individuals. Because they are common, it is important to recognize the attributes of benign aggregates and distinguish them from their malignant counterparts. Benign aggregates are often well circumscribed and are small to medium sized. They are composed of an admixture of cell types including small and mature-appearing lymphocytes (typically CD3+ T cells), histiocytes, and granulocytes. They frequently contain a central small-caliber vessel. Although they occasionally abut bony trabeculae, they do not demonstrate the paratrabecular pattern of growth seen in follicular lymphoma and other non-Hodgkin lymphomas involving the marrow. In some cases, immunohistochemistry or in situ hybridization for κ and λ immunoglobulin light chains can be used to further evaluate aggregates.
J. AIDS. Since the first cases of AIDS were reported in 1981, a number of associated diseases have been identified in the bone marrow. Commonly encountered morphologic changes in the bone marrow of human immunodeficiency virus (HIV)-infected individuals include granulomas, lymphoid aggregates, plasma cell aggregates, and dysplasia in one or more hematopoietic lineages (Haemophilia. 2001;7:47). Since the development of combined drug therapy, the incidence of secondary infections has decreased. However, infectious agents are still encountered in the bone marrow of individuals infected with HIV and, because impaired inflammatory responses are a hallmark of HIV infection, it is recommended that all bone marrow biopsies from patients with HIV be examined with special stains for fungi and mycobacteria.
Lymphoid aggregates occur with increased frequency in the bone marrow of HIV-infected individuals, are sometimes large with ill-defined borders, and grow along bony trabeculae; these are all features suggestive of malignancy. They may be extremely difficult to evaluate, especially in view of the increased incidence of non-Hodgkin lymphomas in this population. Ancillary studies may be useful in the evaluation of monoclonal B-cell or phenotypically abnormal/monoclonal T-cell populations.
Dyspoiesis is a common finding in the bone marrow of patients with HIV infection and can be seen in other viral infections such as hepatitis C. The significance of this finding may be difficult if not impossible to interpret because of the increased incidence of neoplastic myelodysplasia and acute leukemias in the HIV+ population. Blasts are not typically increased in HIV-associated dysplasia, and clonal cytogenetic abnormalities are not present.
K. Hemophagocytic syndrome is a potentially deadly condition in which cytokine-stimulated benign histiocytes phagocytose other hematopoietic cells in an uncontrolled fashion. The most common causes of hemophagocytic syndrome are related to activation of benign macrophages by cytokines produced by malignant cells, and unregulated phagocytosis by macrophages following infection. Malignancy-related hemophagocytic syndrome is most commonly associated with peripheral T-cell lymphoma, although other hematopoietic and lymphoid malignancies are also rarely associated with this complication, including acute myeloid leukemias with the translocation t(16;21)(p11;q22). The most common infectious cause of hemophagocytic syndrome is EBV. Regardless of the underlying cause, hemophagocytic syndrome presents with splenomegaly, fever, and wasting. Pancytopenia, elevated liver function tests, and coagulopathy are variably present. Evaluation of the bone marrow aspirate and core biopsy reveals variable numbers of macrophages containing phagocytosed hematopoietic cells (Am J Surg Pathol. 2001;25:865).
L. Chédiak-Higashi syndrome is an autosomal recessive inherited disorder that is caused by a mutation in the CHS1-LYST gene located at chromosome 1q42. The clinical features of this syndrome are related to abnormal lysosomal trafficking and include recurrent infection, oculocutaneous albinism, neurologic disorders, and a bleeding diathesis. Granulocytes, monocytes, and lymphocytes contain abnormal large granules derived from secondary or cytotoxic granules (e-Fig. 44.3) (Platelets. 1998;9:21).
M. Mucopolysaccharidoses. Alder-Reilly anomaly is identified in the granulocytes of patients with a group of uncommon diseases characterized by X-linked or autosomal recessive transmitted defects in the enzymes involved in mucopolysaccharide metabolism (Table 44.1A) (Clin Lab Haematol. 1996;18:39). Granulocytes in the peripheral blood and bone marrow have coarse azurophilic granules superficially resembling normal primary granules. Classification of cases of mucopolysaccharidosis requires chemical and/or molecular analysis.
N. Lipid storage disorders. There are numerous genetically mediated conditions related to defects in enzymes comprising the pathway of lipid metabolism (Table 44.1B). The most commonly encountered are Gaucher disease and Niemann-Pick disease. Gaucher disease demonstrates an autosomal recessive pattern of inheritance and is caused by a defect in the enzyme β-glucocerebrosidase. Clinically, patients present with bone pain and splenomegaly related to the proliferation of morphologically abnormal histiocytes at these sites. The cytoplasm of the macrophages has a “wrinkled tissue paper” appearance which represents the accumulation of glucocerebroside in these cells (e-Fig. 44.4). Occasional cases are associated with B-lineage malignancies (including plasma cell dyscrasias) and light chain amyloidosis (J Intern Med. 1999;246:587).
Patients with Niemann-Pick disease typically present with organomegaly, neuropathy, and abnormal laboratory findings similar to those identified in Gaucher disease. The pathophysiology of Niemann-Pick disease is related to autosomal recessively inherited defects in the enzyme sphingomyelinase, with the presence of sphingomyelin in affected cells. Macrophages in this disorder have been described as “sea-blue” due to the cytoplasmic accumulation of PAS-positive material representing sphingomyelin (Ann Hematol. 2001;80:620).
The remaining lipid storage diseases, which are somewhat less common, have clinical presentations similar to those of Gaucher and Niemann-Pick diseases. Some are associated with additional findings. For example, Hermansky-Pudlak syndrome is associated with platelet storage pool deficiencies and oculocutaneous albinism (Platelets. 1998;9:21).
IV. DIAGNOSTIC FEATURES OF MALIGNANCIES
A. Myelodysplastic syndromes are clonal hematopoietic disorders characterized in most cases by peripheral cytopenias (hemoglobin <10 g/dL, absolute neutrophil
count [ANC] <1.8 × 109/L, and/or platelets <100 × 109/L) and increased bone marrow cellularity (Hematology Am Soc Hematol Educ Program. 2006;199). The entities comprising this family of diseases are summarized in Table 44.2. Diagnosis is made by assessment of the bone marrow aspirate smear for significant dyspoiesis and correlation with the clinical history, including a failure to respond to iron, vitamin B12, and folate replacement. Dyspoiesis is identified in one or more of the hematopoietic lineages. In the myeloid series, this most commonly manifests as abnormal nuclear lobation, including cells with pseudo-Pelger-Huët (hypolobate) nuclei, and cells with decreased cytoplasmic granules. Erythroid precursors demonstrate nuclear irregularities including budding, with occasional ringed sideroblasts (erythroid precursors with multiple punctate iron granules surrounding the nuclei that reflect iron abnormally trapped in mitochondria). Megakaryocytes contain multiple separate nuclei or are small with decreased nuclear lobation (e-Figs. 44.5 to 44.8). Importantly, these changes must be present in at least 10% of the cells in a given lineage to morphologically establish this diagnosis.
TABLE 44.1 Storage Diseases
A. Mucopolysaccharidoses Disease
Enzyme deficiency
Hurler syndrome (mucopolysaccharidosis type I H)
α-L-iduronidase
Scheie syndrome (mucopolysaccharidosis type I S)
α-L-iduronidase
Hurler-Scheie syndrome (mucopolysaccharidosis type I H-S)
α-L-iduronidase
Hunter syndrome (mucopolysaccharidosis type II)
Iduronidate α-sulfatase
Sanfilippo syndrome type A (mucopolysaccharidosis type III A)
Heparin N-sulfatase
Sanfilippo syndrome type B (mucopolysaccharidosis type III B)
α–N-Acetylglucosaminidase
Sanfilippo syndrome type C (mucopolysaccharidosis type III C)
α-Glucosaminide transferase
Sanfilippo syndrome type D (mucopolysaccharidosis type III D)
N-Acetylglucosamine-6-sulfatase
Morquio syndrome type A (mucopolysaccharidosis type IV A)
N-Acetylgalactosamine, 6-sulfate sulfatase
Morquio syndrome type B (mucopolysaccharidosis type IV B)
β-Galactosidase
Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI)
N-Acetylgalactosamine-4-sulfatase
Sly syndrome (mucopolysaccharidosis type VII)
β-Glucuronidase
B. Lipid storage disorders Disease
Enzyme deficiency
Substance stored
Gaucher disease
β-Glucocerebrosidase
Glucocerebroside
Niemann-Pick disease
Sphingomyelinase
Sphingomyelin
Gangliosidosis
β-galactosidase
GM1 ganglioside
Tay-Sachs disease
Hexosaminidase A
GM2 ganglioside
Sandhoff disease
Hexosaminidase A
GM2 ganglioside
Fabry disease
α-Galactosidase
Ceramide trihexalose
TABLE 44.2 Myelodysplastic Syndromes
Disease
Blood
Bone marrow
Cytogenetics (percentage of cytogenetically abnormal cases)
Outcome (median survival)
Refractory cytopenia with unilineage dysplasia
Uni- or bicytopenia
No/rare blasts
Unilineage dysplasia
<5% blasts
<15% ringed sideroblasts
<50%
˜2% progress to acute leukemia at 5 y
Refractory anemia with ringed sideroblasts
Anemia
No blasts
≥15% ringed sideroblasts
Erythroid dysplasia
<5% blasts
<10%
1-2% progress to acute leukemia (6 y)
Refractory cytopenia with multilineage dysplasia
Bi/pancytopenia
No/rare blasts
No Auer rods
<1 × 109/L monocytes
Dysplasia in ≥10% of cells in 2 or more myeloid cell lines
≥5% blasts
No Auer rods
<15% ringed sideroblasts
˜50%
˜11% progress to acute leukemia at 2 y
Refractory cytopenia with multilineage dysplasia and ringed sideroblasts
Bi/pancytopenia
No/rare blasts
No Auer rods
<1 × 109/L monocytes
Dysplasia in ≥10% of cells in 2 or more myeloid cell lines
≥ringed sideroblasts
<5% blasts
No Auer rods
Unknown; probably similar to refractory cytopenia with multilineage dysplasia
Unknown; probably similar to refractory cytopenia with multilineage dysplasia
Refractory anemia with excess blasts-1 (RAEB-1)
Cytopenias
<5% blastsa
No Auer rods
<1 × 109/L monocytes
Uni/multilineage dysplasia
5-9% blasts
No Auer rodsb
˜30-50%
˜25%
Refractory anemia with excess blasts-2 (RAEB-2)
Cytopenias
5-19% blasts ± Auer rods
<1 × 109/L monocytes
Uni/multilineage dysplasia
10-19% blasts ± Auer rods
˜30-50%
˜33%
Myelodysplastic syndrome – unclassifiable
Cytopenias
No/rare blasts
No Auer rods
Unilineage dysplasia
<5% blasts
No Auer rods
Unknown
Unknown
Myelodysplastic syndrome associated with isolated del(5q)
Anemia
Normal/increased platelet count <5% blasts
Normal/increased megakaryocytes with hypolobate nuclei
<5% blasts
Isolated del(5q) cytogenetic abnormality
No Auer rods
100% (isolated del(5q) cytogenetic abnormality; cases with additional abnormalities should not be classified under this diagnosis)
Unknown median survival—probably many years
Notable exceptions:
a A diagnosis of RAEB-1 can be established if there are 2% to 4% myeloblasts in the peripheral blood with less than 5% blasts in the bone marrow.
b Cases with Auer rods and less than 10% bone marrow myeloblasts are designated as RAEB-2. From: Swerdlow S, Campo E, Harris NL, et al., eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2008. Used with permission.
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