Bone Marrow Biopsy Evaluation for Mast Cell DisordersBy Daniel Bustamante, MD and Tracy I. George, MDAuthor Contact:Daniel Bustamante, MDDepartment of Pathology, University of New MexicoTracy I. George, MDProfessor of PathologyUniversity of UtahExecutive Director, Clinical Trials and PharmaDx, ARUP LaboratoriesTracy.firstname.lastname@example.org IntroductionMastocytosis is a neoplastic process characterized by an increase of clonal mast cells in one or more organs in the body. The location and extent of involvement in the body is important for further characterization of the disease process. Systemic mastocytosis typically involves the bone marrow and a bone marrow biopsy is therefore crucial for complete diagnostic and prognostic determination.Bone Marrow BiopsyA bone marrow biopsy is a minimally invasive surgical procedure that can be performed in a hospital or outpatient facility. Local anesthesia is often used to reduce pain and discomfort associated with the procedure. The procedure typically consists of two steps: the aspirate and the biopsy. The aspirate is usually performed first and involves insertion of a needle into the marrow cavity, typically at the iliac crest. A syringe is used to aspirate the cellular component of the marrow (Figure 1A). Aspirate smears are then prepared at the bedside from the aspirate fluid.After collecting the aspirate sample, the bone marrow biopsy is performed using a trephine surgical instrument to obtain a solid piece of bone marrow. The size of the biopsy varies; a specimen between 1.5 to 3.0 cm is optimal for complete diagnostic interpretation. The biopsy specimen is touched to the surface of a few slides at the bedside to create touch preparations. The biopsy specimen is then sent to the laboratory for preparation, which consists of fixation of tissue, decalcification, processing, sectioning, and staining (Figure 1B and 1C).Figure 1AFigure 1BFigure 1CFigure 1. Bone marrow biopsy1A. Bone marrow aspirate in syringe. This fluid contains bone marrow hematopoietic cells, including red blood cells. The aspirate can be expelled drop by drop onto a glass slide, smeared, allowed to air dry, and stained.1B. Sectioning of bone marrow clot. The remaining bone marrow aspirate is expelled into a dish and allowed to clot. It is then fixed in a fixative, processed overnight, and embedded in paraffin wax. A plastic cassette containing the paraffin block of clotted bone marrow aspirate is placed in a microtome for cutting thin sections. These sections can be placed on glass slides, stained, and examined under the microscope.1C. Core biopsy specimen embedded in paraffin. The core of bone is fixed in fixative, decalcified to make the bone soft, processed overnight, and embedded in paraffin wax. This core biopsy specimen can then be sliced into thin sections that are placed on glass slides, stained, and examined under the microscope.Diagnostic Components of the Bone Marrow Pathology ReportPeripheral blood is not collected during the bone marrow procedure, although a sample should be collected before or soon after the procedure to prepare a peripheral blood smear slide. For complete interpretation, the pathologist concurrently reviews the peripheral blood smear and the bone marrow specimen. The cells that circulate in peripheral blood most often originate within the bone marrow; thus, the peripheral blood serves as a “window” into the bone marrow and can help confirm certain diagnoses. A complete blood cell count should be reviewed along with the peripheral blood smear.The bone marrow aspirate can be rapidly interpreted following the procedure. Once the sample is placed on a slide, the cells are stained with a Romanowsky stain. This preparation of the bone marrow specimen allows pathologists to conduct a cytologic (cellular) assessment of the individual cellular components that make up the patient’s bone marrow. Spicules are cellular-heavy areas on the slide and are required for optimal representation and interpretation of the bone marrow study.The bone marrow clot section is prepared in the laboratory. Any residual marrow aspirate sample that was not used in making aspirate slides is sent to the laboratory for processing. The sample is spun down and concentrated, and ultimately paraffin embedded. Thin slices of the paraffin embedded sample are placed on slides and stained with hematoxylin-eosin (“clot section”). This preparation offers an alternative view of the aspirate specimen. It should be noted that the overall architecture of the bone marrow is often disrupted during the aspiration procedure. General structure, cellularity, and localization of cellular components often cannot be evaluated on aspirate and clot preparations.Bone marrow touch preparations are cellular representatives of the components of the core biopsy specimen. As with aspirate preparations, these can be rapidly interpreted following the procedure. The sample is air-dried and stained with a Romanowsky stain to show individual cellular components.The bone marrow biopsy technique gives rise to a physical core of bone and bone marrow. The biopsy is prepared in a manner similar to the clot section specimen. The biopsy is unique as it is the only specimen that shows cellular architecture and cellularity and is therefore, often essential. Bone marrow hematopoietic cells are arranged in a particular pattern within the marrow space. Cells that are displaced, infiltrated, or arranged in an abnormal fashion, may indicate a worrisome process developing within the bone marrow. Bone trabeculae are also assessed on the marrow biopsy specimen and can also give clues regarding an underlying reactive or neoplastic process.A pathologist reviews all of the above-mentioned diagnostic components of the bone marrow biopsy in conjunction with a thorough clinical history, laboratory studies, and ancillary studies. Laboratory studies include a complete blood count: white blood cell count (WBC) with differential counts (percentage of neutrophils, lymphocytes, monocytes, eosinophils, basophils, and other cell types); red blood cell count (RBC); associated RBC measurements and calculations such as hemoglobin, hematocrit, red cell distribution width (RDW), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC); and platelet count (PLT). Ancillary studies such as flow cytometry, cytogenetics, fluorescence in situ hybridization (FISH), and molecular genetic studies may be included in the bone marrow pathology report. These studies are often reported on separately and are only briefly discussed in the bone marrow pathology report. At some institutions, an integrated report is compiled to incorporate results of all related investigations.Mast Cell-Specific Bone Marrow FindingsMast cells are part of the innate immune system. They are a normal cellular component of the bone marrow but are not very numerous in normal bone marrow. Normal mast cells have a characteristic microscopic morphology with a central round nucleus and abundant granular cytoplasm (Figure 2A). When mast cell disease involves the bone marrow this is called systemic mastocytosis. Involvement of the bone marrow is often distinct, as mast cells manifest with unique histologic findings. Neoplastic mast cells often aggregate, and appear spindled and elongated, or immature or bilobed, rather than isolated, round, and granular (Figure 2B-2E). They can aggregate along the bony trabeculae or along the vasculature in the biopsy specimen. Neoplastic mast cells can also form characteristic “target” lesions in the marrow that are often a core of lymphocytes surrounded by mast cells. These lesions are more commonly found in indolent systemic mastocytosis. Fibrosis and associated thickening of the adjacent bony trabeculae (osteosclerosis) are often associated with mast cell neoplasms.Ancillary Studies on Bone Marrow SpecimensConcurrent ancillary studies are almost always performed with the bone marrow biopsy and are often essential for complete diagnosis. Different stains may be performed on the specimens obtained from the bone marrow biopsy. Usually a select few are performed based on differential diagnoses identified by histology and clinical history. For mast cell disease in particular, the number and microscopic appearance of mast cells are analyzed by CD117 and/or tryptase immunohistochemical stains. Because mast cell diseases in the bone marrow are associated with fibrosis, reticulin and trichrome stains are used to assess the extent of fibrosis. Abnormal or neoplastic mast cells can stain for markers that are not normally expressed by mast cells. These include CD25, CD2, and CD30 (Figure 2F). It is critical that the appropriate stains be utilized to make an accurate diagnosis of SM based on the WHO criteria (Table 2).Flow cytometry is an essential diagnostic tool performed on the bone marrow aspirate specimen. It is a laboratory technique that can help determine the lineage of a cell line and identify markers expressed by cells. In mast cell disease, flow cytometry can help quantify the number of mast cells present and identify the markers that they express. This can help differentiate neoplastic from normal mast cells.Conventional cytogenetic studies can identify a neoplastic/clonal process and can be of significant diagnostic and prognostic value. Cytogenetic studies can be utilized to evaluate the chromosomal complement in a neoplastic process. Different cytogenetic abnormalities can have different prognostic implications. Fluorescence in situ hybridization (FISH) is a technique that uses probes to evaluate particular areas of interest in the cellular genome. Different probes are used to identify translocations, deletions, rearrangements, and gains or losses of genetic material. There are many molecular studies that can provide diagnostic and prognostic information on a variety of neoplasms. The majority of the assays utilize DNA or RNA that is extracted from cells of interest. In mast cell disease, a KIT mutation can often be identified; this has significant diagnostic value in that it supports the presence of a mast cell neoplasm.The Diagnosis of MastocytosisThe World Health Organization (WHO) Classification of Tumours of Haematopoietic and Lymphoid Tissues outlines diagnostic criteria for the classification of mastocytosis. Cutaneous mastocytosis is defined as skin involvement by mast cells without involvement of other organs, including the bone marrow. Systemic mastocytosis (SM) typically involves the bone marrow and spleen, but can involve other organs including the skin.The diagnosis of SM requires the identification of one major and one minor criteria, or three minor criteria (Table 1). Variants of SM are indolent systemic mastocytosis or advanced mastocytosis, including SM with an associated clonal hematologic non-mast cell disease (SM-AHNMD); aggressive systemic mastocytosis; and mast cell leukemia. Patients with SM-AHNMD have both SM and an associated clonal hematological non-mast cell disorder. This is typically a myeloid disorder such as myelodysplastic syndrome, chronic myelomonocytic leukemia, or acute myeloid leukemia, although others have been described. Patients with aggressive SM have “C” findings (Table 2) that reflect organ dysfunction due to the presence of mast cells in bone marrow, liver, spleen, and/or the gastrointestinal tract. Mast cell leukemia is unique in that mast cells make up at least 20% of all nucleated cells in bone marrow aspirate smears. Smoldering systemic mastocytosis is a variant of indolent systemic mastocytosis. These patients lack “C” findings, but have two or more “B” findings (Table 2). The classification of mast cell disease therefore requires careful clinical, laboratory, and histopathologic investigation and correlation.Figure 2AFigure 2BFigure 2CFigure 2DFigure 2EFigure 2FFigure 2. Mast cell-specific bone marrow findings.2A. Normal mast cell with round, tightly packed granules filling the cytoplasm and obscuring the oval to round nucleus. Wright Giemsa stain, 100X.2B. Normal mast cell with round shape, slightly oval nuclei, and abundant lightly eosinophilic cytoplasm. On H&E stain as shown, the cytoplasmic granules are not readily visible. H&E, 60X.2C. Neoplastic spindle-shaped mast cell with an elongated cytoplasmic projection. The cell is enlarged and the cytoplasm is hypogranular compared to normal mast cells. Wright Giemsa stain, 100X.2D. Neoplastic mast cell: promastocyte. Two promastocytes are present in the center of the image next to each other. Unlike normal mast cells, these are indented but can sometimes be bilobed, and the cytoplasm is hypogranular. The mast cells are round in shape, but larger than their normal counterparts. Wright Giemsa stain, 100X.2E. Bone marrow biopsy showing a cluster of spindle-shaped mast cells surrounding a blood vessel. The mast cells show elongated nuclei and abundant, lightly pink to clear cytoplasm, and are admixed with eosinophils. H&E stain, 40X.2F. This bone marrow biopsy from a 61 year old man with indolent systemic mastocytosis shows a large mast cell aggregate surrounding a blood vessel. The brown stain is an antibody directed against CD25, the interleukin 2 receptor, which highlights neoplastic but not normal mast cells. The compact cluster of mast cells has many round cells, but toward the edges of the aggregate they appear spindle shaped. CD25 immunohistochemical stain, 20X.Table 1. World Health Organization 2008 Criteria for Systemic Mastocytosis3Major:Dense, multifocal mast cell aggregates in bone marrow or other non-cutaneous organsMinor:More than 25% of total mast cells show atypical morphologyThe presence of an activation mutation at KIT codon 816Aberrant mast cell phenotype (expression of CD25 and/or CD2)Elevated total serum tryptase above 20 ng/mL**This criterion is not valid if there is an associated hematological disorder, as these other disorders can express tryptase.Table 2. “B” and “C” Findings (World Health Organization 2008 Criteria for Variants of Systemic Mastocytosis)3“B” Findings:Bone marrow biopsy showing >30% infiltration by mast cells (focal, dense aggregates) and/or total serum tryptase level > 200 ng/mLSigns of dysplasia or myeloproliferation in non-mast cell lineage(s), but insufficient criteria for definitive diagnosis of hematopoietic neoplasm (AHNMD*), with normal or only slightly abnormal blood countsHepatomegaly without impairment of liver function, and/or palpable splenomegaly without hypersplenism, and/or lymphadenopathy on palpation or imaging“C” Findings:Bone marrow dysfunction manifested by one or more cytopenias (ANC <1.0×109/L, Hb < 10 g/dL, or platelets <100×109/L), but no obvious non-mast cell hematopoietic malignancyPalpable hepatomegaly with impairment of liver function, ascites, and/or portal hypertensionSkeletal involvement with large osteolytic lesions and/or severe osteoporosis with pathological fracturesPalpable splenomegaly with hypersplenism (splenic dysfunction)Malabsorption with weight loss due to gastrointestinal mast cell infiltrates*Associated clonal hematologic non-mast cell disease (AHNMD)Edited byDenyse Nanan, MDValerie M. 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