Core Biopsy

The biopsy specimen is usually collected first, because aspiration may destroy marrow architecture. After the incision is made, the Jamshidi outer cannula with the obturator in place is inserted through the skin and cortex of the bone. The obturator prevents coring of skin or bone. Reciprocating rotation promotes the forward advancement of the cannula until the resistance weakens, which indicates penetration through the cortex to the medullary cavity of the bone. The obturator is removed and the biopsy needle is inserted through the cannula and advanced slowly 2 to 3 cm with continued reciprocating rotation along the long axis. The needle angle is changed slightly to separate the core cylinder specimen from its marrow cavity attachments, and the biopsy needle and cannula are withdrawn from the bone, taking the core cylinder with them. The core cylinder is 1 to 1.5 cm long and 1 to 2 mm in diameter, and weighs about 150 mg. The biopsy needle is placed over an ethanol-cleaned slide and the stylus is pushed through to dislodge the core cylinder. Using sterile forceps, the scientist prepares imprints (touch preparations) and transfers the core cylinder to the chosen fixative, Zenker, B5, or formalin.

When the Westerman-Jensen needle is used, the obturator is removed, the cutting blades are inserted through the cannula, and the blades are advanced into the medullary cavity. The cutting blades are pressed into the medullary bone, with the outer cannula held firmly in a stationary position. The blades are withdrawn so that the cannula entraps the tissue, and the entire unit is withdrawn. The core cylinder is removed by inserting the probe through the cutting tip and extruding the specimen through the hub of the needle to the selected slide and fixative-containing receptacles.

Aspiration

In a separate location from the biopsy, a 14- to 18-gauge aspiration needle such as the University of Illinois needle, with obturator, is inserted through the skin and cortex of the bone. The obturator is removed and a 10- to 20-mL syringe is attached. The plunger is withdrawn to create negative pressure and aspirate 1.0 to 1.5 mL of marrow into the syringe. Collecting more than 1.5 mL dilutes the hematopoietic marrow with sinusoidal (peripheral) blood. The syringe is detached and is passed immediately to the scientist, who expels the material onto a series of clean and sterile microscopic slides or coverslips. A second syringe may be attached and additional specimen aspirated for cytogenetic analysis, molecular diagnosis (polymerase chain reaction testing), or immunophenotyping using flow cytometry. The needle is then withdrawn, and pressure is applied to the wound.

If no marrow is obtained, the physician returns the obturator to the needle, advances the needle, attaches a fresh syringe, and tries again. The syringe and needle are retracted slightly and the process is repeated. If this attempt is unsuccessful, the needle and syringe are removed, pressure is applied, and the procedure is begun at a new site. If the marrow is fibrotic, acellular, or packed with leukemic cells, the aspiration may be unsuccessful, known as a dry tap. In this case, a biopsy is necessary, and cell morphology may be observed using a slide imprint, or touch preparation.

Patient Care

Subsequent to bone marrow biopsy or aspiration, a pressure dressing is applied, and the patient is required to remain in the same position for 60 minutes to prevent bleeding.

Anticoagulated Aspirate Smears

Anticoagulated specimens are a more leisurely alternative to direct aspirate smears. The aspirate is expressed from the syringe into a vial of K₃EDTA and subsequently pipetted to clean glass slides and spread using the same approach as in direct aspirate smear preparation. All anticoagulants distort cell morphology; however, K₃EDTA generates the least distortion.

Crush Smears

To prepare crush smears, the medical laboratory scientist expels a portion of the aspirate to a Petri dish or watch glass covered with a few milliliters of K₃EDTA solution and spreads the aspirate over the surface with a sterile applicator. Individual bony spicules are transferred using applicators, forceps, or micropipettes (preferred) to several ethanol-washed glass slides. Additional glass slides are placed directly over the specimens at right angles and pressed gently to crush the spicules. The slides are separated laterally to create two rectangular smears, which may be fanned to encourage rapid drying.

Some scientists prefer to transfer aspirate directly to the slide, subsequently tilting the slide to drain off peripheral blood while retaining spicules. Once drained, the spicules are then crushed with a second slide as described earlier.

The scientist may add one drop of 22% albumin to the EDTA solution, particularly if the specimen is suspected to contain prolymphocytes or lymphoblasts, which tend to rupture. The albumin reduces the occurrence of “smudge” or “basket” cells often seen in lymphoid marrow lesions.

The crush preparation procedure may also be performed using ethanol-washed coverslips in place of slides. The coverslip method demands adroit manipulation but may yield better morphologic information, because the smaller coverslips generate less cell rupture during separation. Use of glass slides offers the opportunity for automated staining, whereas coverslip preparations must first be affixed smear side up to slides and then stained manually (see Chapter 15).

Imprints (Touch Preparations)

Core biopsy specimens and clotted marrow may be held in forceps and repeatedly touched to a washed glass slide or coverslip, so that cells attach and rapidly dry. The scientist lifts directly upward to prevent cell distortion. The cell morphology of imprint preparations may imitate aspirate morphology, although few spicules are transferred, and imprints are valuable when the specimen has clotted or there is a dry tap.

Concentrate (Buffy Coat) Smears

Buffy coat smears are useful when there are sparse nucleated cells in the direct marrow smear or when the number of nucleated cells is anticipated to be small, as in aplastic anemia. Approximately 1.5 mL of K₃EDTA-anticoagulated marrow specimen is transferred to a narrow-bore glass or plastic tube such as a Wintrobe hematocrit tube. The tube is centrifuged at 2500 g for 10 minutes and examined for four layers.

The top layer is yellowish fat and normally occupies 1% to 3% of the column. The second layer, plasma, varies in volume depending on the amount of peripheral blood in the specimen. The third layer consists of nucleated cells and is called the myeloid-erythroid (ME) layer. The ME layer is normally 5% to 8% of the total column. The bottom layer is RBCs, and its volume, like that of the plasma layer, depends on the amount of peripheral blood present. The scientist records the ratio of the fat and ME layers using millimeter gradations on the tube.

Once the column is examined, the scientist aspirates a portion of the ME layer with a portion of plasma and transfers the suspension to a Petri dish or watch glass. Marrow smears are subsequently prepared using the crush smear technique.

The concentrated buffy coat smear compensates for hypocellular marrow and allows for examination of large numbers of nucleated cells without interference from fat or RBCs. On the other hand, cell distribution is distorted by the procedure. Therefore, the scientist does not estimate numbers of different cell types or maturation stages on a buffy coat smear.

Histologic Sections (Cell Block)

After aspirate smears are prepared and aliquots of marrow have been distributed for cytogenetic, molecular, and immunophenotypic studies, the remaining core biopsy specimen, spicules, or clotted specimen is submitted for histologic examination. The specimen is suspended in 10% formalin, Zenker glacial acetic acid, or B5 fixative for approximately 2 hours.

The fixed specimen is centrifuged, and the pellet is decalcified and wrapped in an embedding bag or lens paper and placed in a paraffin embedding cassette. The embedded specimen is sectioned and dyed with hematoxylin and eosin (H&E) dye and examined.

Marrow Smear Dyes

Marrow aspirate smears are stained with Wright or Wright-Giemsa dyes using the same protocols as for peripheral blood film staining. Some laboratory managers increase staining time to compensate for the relative thickness of marrow smears compared with peripheral blood films.

Marrow aspirate smears and core biopsy specimens may also be stained using a ferric ferricyanide (Prussian blue) solution to detect and estimate marrow storage iron or iron metabolism abnormalities (see Chapter 19). Further, a number of cytochemical dyes are used for cell identification or differentiation (Table 16-2 and Chapter 30).

Low-Power (100×) Examination

Once the bone marrow aspirate direct smear or imprint is prepared and stained, the scientist or pathologist begins the microscopic examination using the low-power (10×) dry lens, which, when coupled with 10× oculars, provides a total 100× magnification. Most bone marrow examinations are performed using a teaching report format that employs projection or multiheaded microscopes to allow viewing by residents, fellows, medical laboratory science students, and attending staff. The microscopist locates the bony spicules, aggregations of bone and hematopoietic marrow, which stain dark blue (Figure 16-5). In imprints, spicules are sparse or absent, and the search is for hematopoietic cells instead. Within these areas, intact and nearly contiguous nucleated cells are selected for examination, whereas areas showing distorted morphology or dilution with sinusoidal blood are avoided.

Near the spicules, cellularity is estimated by observing the proportion of hematopoietic cells to adipocytes (clear fat areas).⁹ For iliac crest marrow, 50% cellularity is normal for patients aged 30 to 70 years. In childhood, cellularity is 80%, and after age 70, cellularity is reduced. For those older than age 70, a rule of thumb is to subtract patient age from 100% and add ±10%. Thus for a 75-year-old, the anticipated cellularity is 15% to 35%. By comparing with the age-related normal cellularity values, the microscopist classifies the observed area as hypocellular, normocellular, or hypercellular. If a core biopsy specimen was collected it provides a more accurate estimate of cellularity than an aspirate smear, because in aspirates there is always some dilution of hematopoietic tissue with sinus blood. In the absence of leukemia, lymphocytes should total fewer than 30% of nucleated cells; if more are present, the marrow specimen has been substantially diluted and should not be used to estimate cellularity.¹⁰

Using the 10× objective the microscopist searches for abnormal, often molded, cell clusters (syncytia) of metastatic tumor cells or lymphoblasts. Tumor cell nuclei often stain darkly (hyperchromatic), and vacuoles are seen in the cytoplasm. Tumor cell clusters are often found near the edges of the smear.

Although myelocytic cells and erythrocytic cells are best examined using 500× magnification, they may be more easily distinguished using the 10× objective. The erythrocytic maturation stages stain more intensely and their margins are more sharply defined, features more easily distinguished at lower magnification.

The microscopist evaluates megakaryocytes using low power (Figure 16-6). Megakaryocytes are the largest cells in the bone marrow, 30 to 50 µm in diameter, with multilobed nuclei (see Chapter 13). Although in special circumstances microscopists may differentiate three megakaryocyte maturation stages—megakaryoblast, promegakaryocyte, and megakaryocyte (MK-I to MK-III)—a total megakaryocyte estimate is generally satisfactory. In a well-prepared aspirate or biopsy specimen, the microscopist observes 2 to 10 megakaryocytes per low-power field. Deviations yield important information reported as decreased or increased megakaryocytes. Bone marrow megakaryocyte estimates are essential to the evaluation of peripheral blood thrombocytopenia and thrombocytosis; for instance, in immune thrombocytopenia, marrow megakaryocytes proliferate markedly.

Abnormal megakaryocytes may be small, lack granularity, or have poorly lobulated or hyperlobulated nuclei. Indications of abnormality may be visible using low power; however, conclusive descriptions require 500× or even 1000× total magnification.

High-Power (500×) Examination

Having located a suitable examination area, the microscopist places a drop of immersion oil on the specimen and switches to the 50× objective, providing 500× total magnification. All of the nucleated cells are reviewed for morphology and normal maturation. Besides megakaryocytes, cells of the myelocytic (Figures 16-7 through 16-10) and erythrocytic (rubricytic, normoblastic, Figure 16-11) series should be present, along with eosinophils, basophils, lymphocytes, plasma cells, monocytes, and histiocytes. See Chapters 7, 8, and 12 for detailed cell and stage descriptions. Table 16-3 names all normal marrow cells and provides their expected percentages.

The microscopist searches for maturation gaps, misdistribution of maturation stages, and abnormal morphology. Although the specimen is customarily reviewed using the 50× oil immersion objective, the 100× oil immersion objective is frequently employed to detect small but significant morphologic abnormalities in the nuclei and cytoplasm of suspect cells.

Many laboratory directors require a differential count of 300 to 1000 nucleated cells. These seemingly large totals are rapidly reached in a well-prepared bone marrow smear at 500× magnification and compensate statistically for the anticipated uneven distribution of spicules and hematopoietic cells. The microscopist counts cells and maturation stages surrounding several spicules to maximize the opportunity for detecting disease-related cells. Some laboratory directors eschew the differential in favor of a thorough examination of the smear.

Many microscopists choose not to differentiate the four nucleated erythrocytic maturation stages, and others may combine three of the four—basophilic, polychromatophilic, and orthochromic normoblasts—in a single total, counting only pronormoblasts separately. In normal marrow, most erythrocytic precursors are either polychromatophilic or orthochromic normoblasts, and differentiation yields little additional information. On the other hand, differentiation may be helpful in megaloblastic, iron deficiency or refractory anemias.

The microscopist may infrequently find osteoblasts and osteoclasts (Figure 16-12). Osteoblasts are responsible for bone formation and remodeling, and derive from endosteal (inner lining) cells. Osteoblasts resemble plasma cells with eccentric round to oval nuclei and abundant blue, mottled cytoplasm, but lack the prominent Golgi apparatus characteristic of plasma cells. Osteoblasts are usually found in clusters resembling myeloma cell clusters. Their presence in marrow aspirates and core biopsy specimens is incidental; they do not signal disease, but they may create confusion.

Osteoclasts are nearly the diameter of megakaryocytes, but their multiple, evenly spaced nuclei distinguish them from multilobed megakaryocyte nuclei (Figure 16-13). Osteoclasts appear to derive from myeloid progenitor cells and are responsible for bone resorption, acting in concert with osteoblasts. Osteoclasts are recognized more often in core biopsy specimens than in aspirates.

Adipocytes, endothelial cells that line blood vessels, and fibroblast-like reticular cells complete the bone marrow stroma (see Chapter 7). Stromal cells and their extracellular matrix provide the suitable microenvironment for the maturation and proliferation of hematopoietic cells, but are seldom examined for diagnosis of hematologic or systemic disease.

Finally, Langerhans cells, giant cells with “palisade” nuclei found in granulomas, signal chronic inflammation.

Once the differential is completed, the myeloid-to-erythroid (M : E) ratio is computed from the total of myeloid to the total of nucleated erythroid cell stages. Excluded from the M : E ratio are lymphocytes, plasma cells, monocytes, histiocytes, nonnucleated erythrocytes, and nonhematopoietic stromal cells.

Prussian Blue Iron Stain Examination

A Prussian blue (ferric ferricyanide) iron stain is commonly used on the aspirate smear. Figure 16-14 illustrates normal iron, absence of iron, and increased iron stores in aspirate smears. The iron stain may be used for core biopsy specimens, but decalcifying agents used to soften the biopsy specimen during processing may leech iron, which gives a false impression of decreased or absent iron stores. For this reason, the aspirate is favored for the iron stain if sufficient spicules are present.