Rodak's Hematology: Clinical Principles and Applications, 5th Ed.

CHAPTER 17. Bone marrow examination

George A. Fritsma*


Bone Marrow Anatomy and Architecture

Indications for Bone Marrow Examination

Bone Marrow Specimen Collection Sites

Bone Marrow Aspiration and Biopsy


Core Biopsy


Patient Care

Managing the Bone Marrow Specimen

Direct Aspirate Smears

Anticoagulated Aspirate Smears

Crush Smears

Imprints (Touch Preparations)

Concentrate (Buffy Coat) Smears

Histologic Sections (Cell Block)

Marrow Smear Dyes

Examining Bone Marrow Aspirate or Imprint

Low-Power (100×) Examination

High-Power (500×) Examination

Prussian Blue Iron Stain Examination

Examining the Bone Marrow Core Biopsy Specimen

Definitive Bone Marrow Studies

Bone Marrow Examination Reports


After completion of this chapter, the reader will be able to:

1. Diagram bone marrow architecture and locate hematopoietic tissue.

2. List indications for bone marrow examinations.

3. Specify sites for bone marrow aspirate and biopsy.

4. Assemble supplies for performing and assisting in bone marrow specimen collection.

5. Assist the physician with bone marrow sample preparation subsequent to collection.

6. List the information gained from bone marrow aspirates and biopsy specimens.

7. Perform a bone marrow aspirate smear and core biopsy specimen examination.

8. List the normal hematopoietic and stromal cells of the bone marrow and their anticipated distribution.

9. Perform a bone marrow differential count and compute the myeloid-to-erythroid ratio.

10. Characterize features of hematopoietic and metastatic tumor cells.

11. Prepare specimens for and assist in performing specialized confirmatory bone marrow studies.

12. Prepare a systematic written bone marrow examination report.


After studying the material in this chapter, the reader should be able to respond to the following case study:

A patient came for treatment complaining of weakness, fatigue, and malaise. Complete blood count results were as follows:

HGB concentration: 7.5 gm/dL

Segmented neutrophils: 21 × 109/L (70%)

HCT: 21%

Immature neutrophils: 6 × 109/L (20%)

RBC count: 2.5 × 1012/L

Basophils: 1.5 × 109/L (5%)

WBC count: 30 × 109/L

Eosinophils: 0.3 × 109/L (1%)

Platelet count: 540 × 109/L


Bone marrow was hypercellular with 90% myeloid precursors and 10% erythroid precursors. There were 15 megakaryocytes per 10× microscopic objective field.

1. What bone marrow finding provides information on blood cell production?

2. What is the myeloid-to-erythroid ratio in this patient, and what does it indicate?

3. What megakaryocyte distribution is normally seen in a bone marrow aspirate?

Bone marrow anatomy and architecture

In adults, bone marrow accounts for 3.4% to 5.9% of body weight, contributes 1600 to 3700 g or a volume of 30 to 50 mL/kg, and produces roughly 6 billion blood cells per kilogram per day in a process calledhematopoiesis.1 At birth, nearly all the bones contain red hematopoietic marrow (Chapter 7). In the fifth to seventh year, adipocytes (fat cells) begin to replace red marrow in the long bones of the hands, feet, legs, and arms, producing yellow marrow, and by late adolescence hematopoietic marrow is limited to the lower skull, vertebrae, shoulder, pelvic girdle, ribs, and sternum (Figure 7-2). Although the percentage of bony space devoted to hematopoiesis is considerably reduced, the overall volume remains constant as the individual matures.2 Yellow marrow reverts to hematopoiesis, increasing red marrow volume, in conditions such as chronic blood loss or hemolytic anemia that raise demand.

The arrangement of red marrow and its relationship to the central venous sinus are illustrated in Figure 7-3. Hematopoietic tissue is enmeshed in spongy trabeculae (bony tissue) surrounding a network of sinuses that originate at the endosteum (vascular layer just within the bone) and terminate in collecting venules.3 Adipocytes occupy approximately 50% of red hematopoietic marrow space in a 30- to 70-year-old adult, and fatty metamorphosis increases approximately 10% per decade after age 70.4

Indications for bone marrow examination

Because the procedure is invasive, the decision to collect and examine a bone marrow specimen requires clinical judgment and the application of inclusion criteria. With the development of cytogenetic chromosome studies, flow cytometry, immunohistochemistry, and molecular diagnostics, peripheral blood may often provide information historically available only from bone marrow, reducing the demand for marrow specimens. On the other hand, these advanced techniques also augment bone marrow–based diagnosis and thus potentially raise the demand for bone marrow examinations in assessment of conditions not previously diagnosed through bone marrow examination.

summarizes indications for bone marrow examination.Table 17-15 Bone marrow examinations may be used to diagnose and stage hematologic and nonhematologic neoplasia, to determine the cause of cytopenias, and to confirm or exclude metabolic or infectious conditions suspected on the basis of clinical symptoms and peripheral blood findings.6

TABLE 17-1

Indications for Bone Marrow Examination



Neoplasia diagnosis

Acute leukemias 

Myeloproliferative neoplasms such as chronic leukemias, myelofibrosis 

Myelodysplastic neoplasms such as refractory anemia 

Lymphoproliferative disorders such as acute lymphoblastic leukemia 

Immunoglobulin disorders such as plasma cell myeloma, macroglobulinemia 

Metastatic tumors

Neoplasia diagnosis and staging

Hodgkin and non-Hodgkin lymphoma

Marrow failure: cytopenias

Hypoplastic or aplastic anemia 

Pure red cell aplasia 

Idiosyncratic drug-induced marrow suppression 

Myelodysplastic syndromes such as refractory anemia 

Marrow necrosis secondary to tumor 

Marrow necrosis secondary to severe infection such as parvovirus B19 infection 

Immune versus amegakaryocytic thrombocytopenia 

Sickle cell crisis 

Differentiation of megaloblastic, iron deficiency, sideroblastic, hemolytic, and blood loss anemia 

Estimation of storage iron to assess for iron deficiency 

Infiltrative processes or fibrosis

Metabolic disorders

Gaucher disease 

Mast cell disease


Granulomatous disease 

Miliary tuberculosis 

Fungal infections 

Hemophagocytic syndromes

Monitoring of treatment

After chemotherapy or radiation therapy to assess minimal residual disease 

After stem cell transplantation to assess engraftment

Each bone marrow procedure is ordered after thorough consideration of clinical and laboratory information. For instance, bone marrow examination is most likely unnecessary in anemia when the cause is apparent from red blood cell (RBC) indices, serum iron and ferritin levels, or vitamin B12 and folate levels. Multilineage abnormalities, circulating blasts in adults, and unexpected pancytopenia usually prompt marrow examination. Bone marrow puncture is prohibited in patients with coagulopathies such as hemophilia or vitamin K deficiency, although thrombocytopenia (low platelet count) is not an absolute contraindication. Special precautions such as bridging therapy may be necessary to prevent uncontrolled bleeding when a bone marrow procedure is performed on a patient receiving antithrombotic therapy, for instance, Coumadin or heparin.

Bone marrow specimen collection sites

Bone marrow specimen collection is a collaboration between a medical laboratory scientist and a skilled specialty physician, often a pathologist or hematologist.7 Prior to bone marrow collection, a medical laboratory practitioner or phlebotomist collects peripheral blood for a complete blood count with blood film examination. During bone marrow collection, the laboratory scientist assists the physician by managing the specimens and producing initial preparations for examination.

Red marrow is gelatinous and amenable to sampling. Most bone marrow specimens consist of an aspirate (obtained by bone marrow aspiration) and a core biopsy specimen (obtained by trephine biopsy), both examined with light microscopy using 100× and 500× magnification. The aspirate is examined to identify the types and proportions of hematologic cells and to look for morphologic variance. The core biopsy specimen demonstrates bone marrow architecture: the spatial relationship of hematologic cells to fat, connective tissue, and bony stroma. The core biopsy specimen is also used to estimate cellularity.

The core biopsy specimen is particularly important for evaluating diseases that characteristically produce focal lesions, rather than diffuse involvement of the marrow. Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, metastatic tumors, amyloid, and granulomas produce predominantly focal lesions. Granulomas, or granulomatous lesions, are cell accumulations that contain Langerhans cells—large, activated granular macrophages that look like epithelial cells. Granulomas signal chronic infection. The biopsy specimen also allows for morphologic evaluation of bony spicules, which may reveal changes associated with hyperparathyroidism or Paget disease.8

Bone marrow collection sites include the following:

• Posterior superior iliac crest (spine) of the pelvis (Figure 17-1). In both adults and children, this site provides adequate red marrow that is isolated from anatomical structures that are subject to injury. This site is used for both aspiration and core biopsy.

• Anterior superior iliac crest (spine) of the pelvis. This site has the same advantages as the posterior superior iliac crest, but the cortical bone is thicker. This site may be preferred for a patient who can only lie supine.

• Sternum, below the angle of Lewis at the second intercostal space. In adults, the sternum provides ample material for aspiration but is only 1 cm thick and cannot be used for core biopsy. It is possible for the physician to accidentally transfix the sternum and enter the pericardium within, damaging the heart or great vessels.

• Anterior medial surface of the tibia in children younger than age 2. This site may be used only for aspiration.

• Spinous process of the vertebrae, ribs, or other red marrow–containing bones. These locations are available but are rarely used unless one is the site of a suspicious lesion discovered on a radiograph.


FIGURE 17-1 The posterior superior iliac crest is the favored site for obtaining the bone marrow aspirate and core biopsy specimen because it provides ample marrow and is isolated from structures that could be damaged by accidental puncture. Source: (Courtesy Indiana Pathology Images, Indianapolis, IN.)

Adverse outcomes are seen in less than 0.05% of marrow collections. Infections and reactions to anesthetics may occur, but the most common side effect is hemorrhage associated with platelet function disorder or thrombocytopenia.

Bone marrow aspiration and biopsy


Less than 24 hours prior to bone marrow collection, the medical laboratory scientist or phlebotomist collects venous peripheral blood for a complete blood count and blood film examination using a standard collection procedure. Peripheral blood collection is often accomplished immediately before bone marrow specimen collection. The peripheral blood specimen is seldom collected after bone marrow collection to avoid stress-related white blood cell (WBC) count elevation.

Most institutions purchase or assemble disposable sterile bone marrow specimen collection trays that provide the following:

• Surgical gloves.

• Shaving equipment.

• Antiseptic solution and alcohol pads.

• Drape material.

• Local anesthetic injection, usually 1% lidocaine, not to exceed 20 mL per patient.

• No. 11 scalpel blade for skin incision.

• Disposable Jamshidi biopsy needle (Care Fusion, McGaw Park, IL; Figure 17-2) or Westerman-Jensen needle (Becton, Dickinson and Company, Franklin Lakes, NJ; Figure 17-3). Both provide an obturator, core biopsy tool, and stylet. A Snarecoil biopsy needle also is available (Kendall Company, Mansfield, MA). The Snarecoil has a coil mechanism at the needle tip that allows for capture of the bone marrow specimen without needle redirection (Figure 17-4).

• Disposable 14- to 18-gauge aspiration needle with obturator. Alternatively, the University of Illinois aspiration needle may be used for sternal puncture. The University of Illinois needle provides a flange that prevents penetration of the sternum to the pericardium.

• Microscope slides or coverslips washed in 70% ethanol.

• Petri dishes or shallow circular watch glasses.

• Vials or test tubes with closures.

• Wintrobe hematocrit tubes.

• Anticoagulant liquid tripotassium ethylenediaminetetraacetic acid (K3EDTA).

• Zenker fixative: potassium dichromate, mercuric chloride, sodium sulfate, and glacial acetic acid; B5 fixative: aqueous mercuric chloride and sodium acetate, or 10% neutral formalin. Because Zenker fixative and B5 contain toxic mercury, controlled disposal is required.

• Gauze dressings.


FIGURE 17-2 Disposable sterile Jamshidi bone marrow biopsy and aspiration needle. The outer puncture cannula is advanced to the medulla with the obturator in place to prevent bone coring. The physician removes the obturator and slides the core biopsy needle through the cannula and into the medulla with the expulsion stylus removed. The core biopsy needle is removed from the puncture needle with the specimen in place. The specimen is expelled using the stylus. Source: (Courtesy Care Fusion, McGaw Park, IL.)


FIGURE 17-3 Bone marrow biopsy technique using the Westerman-Jensen needle.


FIGURE 17-4 Snarecoil bone marrow biopsy needle. The coil mechanism resides within the biopsy needle as illustrated in the magnified image. The coil is turned to draw the marrow specimen into the needle. Source: (Courtesy Tyco Healthcare/Kendall, Mansfield, MA.)

The patient is asked to lie supine, prone, or in the right or left lateral decubitus position (lying on the right or left side). With attention to standard precautions, the skin is shaved if necessary, disinfected, and draped. The physician infiltrates the skin, dermis, and subcutaneous tissue with a local anesthetic solution, such as 1% or 2% lidocaine or procaine, through a 25-gauge needle, producing a 0.5- to 1.0-cm papule (bubble). The 25-gauge needle is replaced with a 21-gauge needle, which is inserted through the papule to the periosteum (bone surface). With the point of the needle on the periosteum, the physician injects approximately 2 mL of anesthetic over a dime-sized area while rotating the needle, then withdraws the anesthesia needle. Next, the physician makes a 3-mm skin incision over the puncture site with a No. 11 scalpel blade to prevent skin coring during insertion of the needle.

Core biopsy

The biopsy specimen is usually collected first, because aspiration may destroy marrow architecture. After the incision is made, the physician inserts a Jamshidi outer cannula with the obturator in place 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 physician removes the obturator, inserts the biopsy needle through the cannula and slowly advances the needle 2 to 3 cm with continuous reciprocating rotation along the long axis. The physician changes the needle angle slightly to separate the core cylinder specimen from its marrow cavity attachments, and withdraws the biopsy needle and cannula 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 onto the slide. Using sterile forceps, the laboratory 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 physician removes the obturator, inserts the cutting blades through the cannula, and advances the blades into the medullary cavity. The cutting blades are pressed into the medullary bone, with the outer cannula held firmly in a stationary position. The physician slowly withdraws the blades so that the cannula entraps the tissue, then withdraws the entire unit. 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.


In a separate location from the biopsy, the physician inserts a 14- to 18-gauge aspiration needle such as the University of Illinois needle, with obturator, through the skin and cortex of the bone. The obturator is removed, and a 10- to 20-mL syringe is attached. The physician withdraws the plunger to create negative pressure and aspirates 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 physician detaches the syringe and passes it immediately to the laboratory scientist, who expels the material onto a series of clean and sterile microscopic slides or coverslips. The physician may attach a second syringe to aspirate an additional specimen for cytogenetic analysis, molecular diagnosis, 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 physician removes the needle and syringe, applies pressure, and begins the procedure at a new site. If the marrow is fibrotic, acellular, or packed with leukemic cells, the first and second aspiration may be unsuccessful, known as a dry tap. In this case, a biopsy is necessary, and the laboratory scientist may observe cell morphology using a slide imprint, or touch preparation.

Patient care

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

Managing the bone marrow specimen

Direct aspirate smears

The medical laboratory scientist receives the aspirate syringe from the physician at the bedside and immediately transfers drops of the marrow specimen onto six to eight ethanol-washed microscope slides. Marrow clots rapidly, so good organization is essential. Using spreader slides, the scientist spreads the drop into a wedge-shaped smear 12 to 34 the length of the slide, similar to a peripheral blood film. Bony spicules 0.5 to 1.0 mm in diameter and larger fat globules follow behind the spreader and become deposited on the slide. In the direct smear preparation the scientist avoids crushing the spicules. The scientist may lightly fan the smears to promote rapid drying in an effort to preserve cell morphology.

In the syringe, the specimen consists of peripheral blood with suspended light-colored bony spicules and fat globules. The scientist evaluates the syringe blood for spicules: more spicules mean a specimen with more cells to identify and categorize. If the specimen has few fat globules or spicules, the scientist may alert the physician to collect an additional specimen.

Anticoagulated aspirate smears

Anticoagulated specimens are a more leisurely alternative to direct aspirate smears. The scientist expresses the aspirate from the syringe into a vial containing K3EDTA and subsequently pipettes the anticoagulated aspirate to clean glass slides, spreading the aspirate using the same approach as in direct smear preparation. All anticoagulants distort cell morphology, but 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. The scientists places additional glass slides directly over the specimens at right angles and presses gently to crush the spicules. The slides are separated laterally to create two rectangular smears, which the scientist may fan 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 (Chapter 16).

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. Imprints are valuable when the specimen has clotted or there is a dry tap: the cell morphology may closely replicate aspirate morphology, although few spicules are transferred.

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. The scientist transfers approximately 1.5 mL of K3EDTA-anticoagulated marrow specimen 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 the scientist has prepared aspirate smears and has distributed aliquots of marrow 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 subsequently centrifuged, and the pellet is decalcified and wrapped in an embedding bag or lens paper and placed in a paraffin-embedding cassette. A histotechnologist sections the embedded specimen, applies hematoxylin and eosin (H& E) dye, and examines the section.

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 (Chapter 20). Further, a number of cytochemical dyes may be used for cell identification or differentiation (Table 17-2).

TABLE 17-2

Cytochemical Dyes Used to Identify Bone Marrow Cells and Maturation Stages

Cytochemical Dye


Myeloperoxidase (MPO)

Detects myelocytic cells by staining cytoplasmic granular contents

Sudan black B (SBB)

Detects myelocytic cells by staining cytoplasmic granular contents

Periodic acid–Schiff (PAS)

Detects lymphocytic cells and certain abnormal erythrocytic cells by staining of cytoplasmic glycogen


Distinguish myelocytic from monocytic maturation stages (several esterase substrates)

Tartrate-resistant acid phosphatase (TRAP)

Detects tartrate-resistant acid phosphatase granules in hairy cell leukemia

Examining bone marrow aspirate or imprint

describes the uses of low- and high-power objectives in examining bone marrow aspirate direct smears or imprints. Box 17-1

BOX 17-1

Bone Marrow Aspirate Microscopic Smear Examination: Low and High Power

Low power: 10x objective (100x total magnification)

• Assess peripheral blood dilution

• Find bony spicules and areas of clear cell morphology

• Observe fat-to-marrow ratio, estimate cellularity

• Search for tumor cells in clusters

• Examine and estimate megakaryocytes

High power: 50x and 100x objective (500x and 1000x total magnification)

• Observe myelocytic and erythrocytic maturation

• Distinguish abnormal distribution of cells or cell maturation stages

• Perform differential count on 300 to 1000 cells

• Compute myeloid-to-erythroid ratio

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 linked 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 observation by residents, fellows, medical laboratory students, and attending staff. The microscopist locates the bony spicules, aggregations of bone and hematopoietic cells, which stain dark blue (Figure 17-5). In imprints, spicules are sparse or absent, and the search is for hematopoietic cells in the absence of spicules. Within these areas the microscopist selects intact and nearly contiguous nucleated cells for examination, avoiding areas of distorted morphology or areas diluted with sinusoidal blood.


FIGURE 17-5 Bone marrow aspirate specimen illustrating intact, contiguous cells and adipocytes. This is a good site for morphologic evaluation and cell counting (Wright stain, 100×).

Near the spicules, cellularity is estimated by observing the proportion of hematopoietic cells to adipocytes (clear fat areas).9 For anterior or posterior iliac crest marrow, 50% cellularity is normal for patients aged 30 to 70 years. In childhood, cellularity is 80%, and after age 70, cellularity becomes 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 peripheral 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 from each other 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 17-6). Megakaryocytes are the largest cells in the bone marrow, 30 to 50 μm in diameter, with multilobed nuclei (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 and are 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.


FIGURE 17-6 Bone marrow aspirate smear showing megakaryocyte with budding platelets at the plasma membrane (Wright stain, 1000×). Megakaryocytes are counted at 100× magnification, but if there is abnormal morphology, cells are examined at 500× or 1000×.

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 17-7 through 17-10) and erythrocytic (rubricytic, normoblastic; Figure 17-11) series should be present, along with eosinophils, basophils, lymphocytes, plasma cells, monocytes, and histiocytes. Chapters 7, 8, and 12 provide detailed cell and cell maturation stage descriptions. Table 17-3 names all normal marrow cells and provides their expected percentages.


FIGURE 17-7 Bone marrow aspirate smear. Myelocytic stages include a myeloblast (MyBl), promyelocyte (ProMy), and myelocyte (Myel). The lymphocyte (Lymph) diameter illustrates its size relative to the myelocytic stages. The source of the lymphocyte is sinus blood (Wright stain, 1000×).


FIGURE 17-8 Bone marrow aspirate smear. Myelocytic stages include a myeloblast (MyBl), promyelocytes (ProMy), a myelocyte (Myel), and metamyelocyte (Meta). One orthochromic normoblast (OrthoN) and one lymphocyte (Lymph) are present (Wright stain, 1000×).


FIGURE 17-9 Bone marrow aspirate smear. Myelocytic stages include myelocytes (Myel), a metamyelocyte (Meta), and neutrophilic bands (Wright stain, 1000×).


FIGURE 17-10 Bone marrow aspirate smear illustrating neutrophilic bands and segmented neutrophils (SEGs) (Wright stain, 1000×).


FIGURE 17-11 Bone marrow aspirate smear showing an island of erythrocytic precursors with polychromatophilic and orthochromic normoblasts (Wright stain, 1000×).

TABLE 17-3

Anticipated Distribution of Cells and Cell Maturation Stages in Aspirates or Imprints

Cell or Cell Maturation Stage


Cell or Cell Maturation Stage








Basophilic normoblasts/prorubricytes




Polychromatophilic normoblasts/rubricytes




Orthochromic normoblasts/metarubricytes


Neutrophilic bands




Segmented neutrophils


Plasma cells


Eosinophils and eosinophilic precursors




Basophils and mast cells





2–10 visible per low-power field

Myeloid-to-erythroid ratio

1.5:1–3.3 : 1

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 17-12). Osteoblasts are responsible for bone formation and remodeling, and they derive from endosteal (inner lining) cells. Osteoblasts resemble plasma cells with eccentric round to oval nuclei and abundant blue, mottled cytoplasm, but they 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.


FIGURE 17-12 Bone marrow aspirate smear showing a cluster of osteoblasts that superficially resemble plasma cells. Osteoblasts have round to oval eccentric nuclei and mottled blue cytoplasm that is devoid of secretory granules. They may have a clear area within the cytoplasm but lack the well-defined central Golgi complex of the plasma cell (Wright stain, 1000×).

Osteoclasts are nearly the diameter of megakaryocytes, but their multiple, evenly spaced nuclei distinguish them from multilobed megakaryocyte nuclei (). 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. Figure 17-13


FIGURE 17-13 Bone marrow core biopsy section from a patient with hyperparathyroidism. The large multinucleated cell near the endosteal surface is an osteoclast, a cell that reabsorbs bone. The spindle-shaped cells are fibroblasts (hematoxylin and eosin stain, 500×).

Adipocytes, endothelial cells that line blood vessels, and fibroblast-like reticular cells complete the bone marrow stroma (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. 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 leach 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. Figure 17-14


FIGURE 17-14 Prussian blue dye on bone marrow aspirate smears (500×). A, Normal iron stores. B, Absence of iron stores. C, Increased iron stores.

Examining the bone marrow core biopsy specimen

The standard dye for the core biopsy specimen is H& E. Other dyes and their purposes are listed in . Bone marrow core biopsy specimen and imprint (touch preparation) examinations are essential when the aspiration procedure yields a dry tap, which may be the result of hypoplastic or aplastic anemia, fibrosis, or tight packing of the marrow cavity with leukemic cells. The key advantage of the core biopsy specimen is preservation of bone marrow architecture so that cells, tumor clusters (Table 17-4Figure 17-15), and maturation stages may be examined relative to stromal elements. The disadvantage is that individual hematopoietic cell morphology is obscured.


FIGURE 17-15 Bone marrow aspirate smear showing a cluster or syncytia of tumor cells. Nuclei are irregular and hyperchromatic, and cytoplasm is vacuolated. Cytoplasmic margins are poorly delineated (Wright stain, 500×).

TABLE 17-4

Dyes Used in Examination of Bone Marrow Core Biopsy Specimens



Hematoxylin and eosin (H& E)

Evaluate cellularity and hematopoietic cell distribution, locate abnormal cell clusters.

Prussian blue (ferric ferricyanide) iron stain

Evaluate iron stores for deficiency or excess iron. Decalcification may remove iron from fixed specimens; thus ethylenediaminetetraacetic acid chelation or the aspirate smear is preferred for iron store estimation.

Reticulin and trichrome dyes

Examine for marrow fibrosis.

Acid-fast stains

Examine for acid-fast bacilli, fungi, or bacteria in granulomatous disease.

Gram stain

Examine for acid-fast bacilli, fungi, or bacteria in granulomatous disease.

Immunohistochemical dyes

Establish the identity of malignant cells with dye-tagged monoclonal antibodies specific for tumor surface markers

Wright or Wright-Giemsa dyes

Observe hematopoietic cell structure. Cell identification is less accurate in a biopsy specimen than in an aspirate smear.

The microscopist first examines the core biopsy specimen preparation using the 10× objective (100× total magnification) to assess cellularity. Because the sample is larger, the core biopsy specimen provides a more accurate estimate of cellularity than the aspirate. The microscopist compares cellular areas with the clear-appearing adipocytes, using a method identical to that employed in examination of aspirate smears to assess cellularity. All fields are examined because cells distribute unevenly. Examples of hypocellular and hypercellular core biopsy sections are provided for comparison with normocellular marrow in Figure 17-16.


FIGURE 17-16 A, Representative core biopsy section showing normal cellularity, approximately 50% fat and 50% hematopoietic cells (hematoxylin and eosin, 50×). B, Hypocellular core biopsy specimen with only fat and connective tissue cells from a patient with aplastic anemia (hematoxylin and eosin stain, 100×). C, Hypercellular core biopsy specimen from a patient with chronic myelogenous leukemia. There is virtually 100% cellularity with no fat visible (hematoxylin and eosin stain, 100×). Source: (Bcourtesy Dennis P. O’Malley, MD, director, Immunohistochemistry Laboratory, Indiana University School of Medicine, Indianapolis, IN.)

Megakaryocytes are easily recognized by their outsized diameter and even distribution throughout the biopsy. They exhibit the characteristic lobulated nucleus, although nuclei of the more mature megakaryocytes are smaller and more darkly stained in H& E preparations than on a Wright-stained aspirate. Their cytoplasm varies from light pink in younger cells to dark pink in older cells (). Owing to the greater sample volume, microscopists assess megakaryocyte numbers more accurately by examining a core biopsy section than an aspirate smear. Normally there are 2 to 10 megakaryocytes per 10× field, the same as in an aspirate smear or imprint. Figure 17-17


FIGURE 17-17 Core biopsy section containing many large lobulated megakaryocytes and increased blasts (Giemsa stain, 400×).

Using the 50× oil immersion objective, the microscopist next observes cell distribution relative to bone marrow stroma. For instance, in people older than age 70, normal lymphocytes may form small aggregates in nonparatrabecular regions, whereas malignant lymphoma cell clusters are often paratrabecular. In addition, normal lymphocytes remain as discrete cells, whereas lymphoma cells are pleomorphic and syncytial.

If no aspirate or imprint smears could be prepared, the core biopsy specimen may be stained using Wright, Giemsa, or Wright-Giemsa dyes to make limited observations of cellular morphology. In Wright- or Giemsa-stained biopsy sections, myeloblasts and promyelocytes have oval or round nuclei with cytoplasm that stains blue (). Neutrophilic myelocytes and metamyelocytes have light pink cytoplasm. Mature segmented neutrophils (SEGs) and neutrophilic bands (BANDs) are recognized by their smaller diameter and darkly stained C-shaped nuclei (BANDs) or nuclear segments (SEGs). The cytoplasm of BANDs and SEGs may be light pink or may seem unstained (Figure 17-18Figure 17-19).


FIGURE 17-18 Core biopsy section infiltrated by blasts with blue cytoplasm and a few myelocytes with pink cytoplasm (Giemsa, 400×).


FIGURE 17-19 Core biopsy section showing myelocytes, metamyelocytes, bands, segmented neutrophils, and bright red-orange eosinophils (Giemsa stain, 400×).

The cytoplasm of eosinophils stains red or orange, which makes them the most brightly stained cells of the marrow. Basophils cannot be recognized on marrow biopsy specimens fixed with Zenker glacial acetic acid solution.

The microscopist may find it difficult to differentiate myelocytic cells from erythrocytic cells in biopsy specimens other than to observe that the latter tend to cluster with more mature normoblasts and often surround histiocytes. Polychromatophilic and orthochromic normoblasts, the two most common erythrocytic maturation stages, have centrally placed, round nuclei that stain intensely (). Their cytoplasm is not appreciably stained, but the plasma membrane margin is clearly discerned, which gives the cells of these stages a “fried egg” appearance. Because erythrocytic cells have a tendency to cluster in small groups, they are more easily recognized using the 10× objective, although their individual morphology cannot be seen. Figure 17-20


FIGURE 17-20 Erythrocytic island in a core biopsy section. Late-stage normoblasts often have a “fried egg” appearance (Giemsa stain, 400×).

Lymphocytes are among the most difficult cells to recognize in the core biopsy specimen, unless they occur in clusters. Mature lymphocytes exhibit speckled nuclear chromatin in a small, round nucleus, along with a scant amount of blue cytoplasm (). Immature lymphocytes (prolymphocytes) have larger round or lobulated nuclei but still only a small rim of blue cytoplasm. Figure 17-21


FIGURE 17-21 Core biopsy section showing small mature lymphocytes. A few are immature with larger nuclei containing a single prominent nucleolus (Giemsa, 400×).

Plasma cells are difficult to distinguish from myelocytes in H& E-stained sections but are recognized using Wright-Giemsa dye as cells with eccentric dark nuclei and blue cytoplasm and a prominent pale central Golgi apparatus (Figure 17-22). Characteristically, plasma cells are located adjacent to blood vessels.


FIGURE 17-22 Plasma cells in a core biopsy section. Nuclei are eccentric, cytoplasm is blue with a prominent central unstained Golgi apparatus (Giemsa stain, 400×).

Definitive bone marrow studies

Although in many cases the aspirate smear and biopsy specimen are diagnostic, additional studies may be needed. Such studies and their applications are given in . These studies require additional bone marrow volume and specialized specimen collection. Information on Prussian blue iron stains and cytochemistry was provided earlier. Each study is described in the chapter referenced in Table 17-5Table 17-5.

TABLE 17-5

Definitive Studies Performed on Selected Bone Marrow Specimens

Bone Marrow Study




Iron stain

Identification of iron deficiency, iron overload

Fresh marrow aspirate


Cytochemical studies

Diagnosis of leukemias and lymphomas

Fresh marrow aspirate

29, 33, 35, 36

Cytogenetic studies

Diagnosis of acute leukemias via deletions, translocations, and polysomy; remission studies

1 mL marrow in heparin


Molecular studies

Polymerase chain reaction for diagnostic point mutations; minimal residual disease studies

1 mL marrow in EDTA


Fluorescence in situ hybridization

Staining for diagnostic mutations; minimal residual disease studies

Fresh marrow aspirate


Flow cytometry

Immunophenotyping, usually of malignant hematopoietic cells, clonality; minimal residual disease studies

1 mL marrow in heparin, EDTA, or ACD


ACD, Acid-citrate dextrose; EDTA, ethylenediaminetetraacetic acid.

Bone marrow examination reports

The components of a bone marrow report should be generated systematically and are given in . An example of a bone marrow examination report is provided in Table 17-6Figure 17-23.


FIGURE 17-23 Example of a bone marrow examination report, including reference intervals, peripheral blood complete blood count results, marrow differential, and narrative. PB,Peripheral blood; BMA, bone marrow aspirate; BMBX, bone marrow biopsy specimen; DX, diagnosis.

TABLE 17-6

Components of a Bone Marrow Examination Report



Patient history

Patient identity and age, narration of symptoms, physical findings, findings in kindred, treatment

Complete blood count (CBC)

Peripheral blood CBC collected no more than 24 hours before the bone marrow puncture, includes hemogram and peripheral blood film examination


Hypocellular, normocellular, or hypercellular classification based on ratio of hematopoietic cells to adipocytes


Estimate using 10× objective (100× magnification), compare with reference interval and comment on morphology


Narrative characterizing the maturation of the myelocytic and erythrocytic (normoblastic, rubricytic) series

Additional hematologic cells

Narrative describing numbers and morphology of eosinophils, basophils, mast cells, lymphocytes, plasma cells, monocytes, and histiocytes if appropriate, with reference intervals

Stromal cells

Narrative describing numbers and morphology of osteoblasts, osteoclasts, bony trabeculae, fibroblasts, adipocytes, and endothelial cells; appearance of sinuses; presence of amyloid, granulomas, fibrosis, necrosis

Differential count

Numbers of all cells and cell stages observed after performing a differential count on 300 to 1000 cells and comparing results with reference intervals

Myeloid-to-erythroid ratio

Computed from nucleated hematologic cells less lymphocytes, plasma cells, monocytes, and histiocytes

Iron stores

Categorization of findings as increased, normal, or decreased iron stores

Diagnostic narrative

Summary of the recorded findings and additional laboratory chemical, microbiologic, and immunoassay tests


• Adult hematopoietic tissue is located in the flat bones and the ends of the long bones. Hematopoiesis occurs within the spongy trabeculae of the bone adjacent to vascular sinuses.

• Bone marrow collection is a safe but invasive procedure performed by a pathologist or hematologist in collaboration with a medical laboratory scientist to obtain specimens used to diagnose hematologic and systemic disease and to monitor treatment.

• The necessity for a bone marrow examination should be evaluated in light of all clinical and laboratory information. In anemias for which the cause is apparent from the RBC indices, a bone marrow examination is not required. Examples of indications for bone marrow examination include multilineage abnormalities in the peripheral blood, pancytopenia, circulating blasts, and staging of lymphomas and carcinomas.

• A peripheral blood specimen is collected for a complete blood count no more than 24 hours before the bone marrow is collected, and the results of the CBC are reported with the bone marrow examination results.

• Bone marrow may be collected from the posterior or anterior iliac crest or sternum using sterile disposable biopsy and aspiration needles and cannulas. The site and equipment depend on how old the patient is and whether both an aspirate and a biopsy specimen are desired.

• The medical laboratory scientist receives the bone marrow specimen and prepares aspirate smears, crush preparations, imprints, anticoagulated bone marrow smears, and fixed biopsy sections, and specimens for confirmatory studies.

• The medical laboratory scientist and pathologist collaborate with residents, fellows, attending physicians, and medical laboratory science students to stain and review bone marrow aspirate smears, biopsy sections, and confirmatory procedure results.

• Confirmatory procedures include cytochemistry, cytogenetics, and immunophenotyping by flow cytometry; fluorescence in situ hybridization; and molecular diagnostics.

• The medical laboratory scientist and pathologist determine cellularity and megakaryocyte distribution, then perform a differential count of 300 to 1000 bone marrow hematopoietic cells and compute the M:E ratio, comparing the results with reference intervals.

• The pathologist characterizes features of hematopoietic disease, metastatic tumor cells, and abnormalities of the bone marrow stroma and prepares a systematic written bone marrow examination report including a diagnostic narrative.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

1. Where is most hematopoietic tissue found in adults?

a. Liver

b. Lungs

c. Spleen

d. Long bones

2. What is the preferred bone marrow collection site in adults?

a. Second intercostal space on the sternum

b. Anterior or posterior iliac crest

c. Any of the thoracic vertebrae

d. Anterior head of the femur

3. The aspirate should be examined under low power to assess all of the following except:

a. Cellularity

b. Megakaryocyte numbers

c. Morphology of abnormal cells

d. Presence of tumor cell clusters

4. What is the normal M:E ratio range in adults?

a. 1.5:1 to 3.3:1

b. 5.1:1 to 6.2:1

c. 8.6:1 to 10.2:1

d. 10:1 to 12:1

5. Which are the most common erythrocytic stages found in normal marrow?

a. Pronormoblasts

b. Pronormoblasts and basophilic normoblasts

c. Basophilic and polychromatophilic normoblasts

d. Polychromatophilic and orthochromic normoblasts

6. What cells, occasionally seen in bone marrow biopsy specimens, are responsible for the formation of bone?

a. Macrophages

b. Plasma cells

c. Osteoblasts

d. Osteoclasts

7. What is the largest hematopoietic cell found in a normal bone marrow aspirate?

a. Osteoblast

b. Myeloblast

c. Pronormoblast

d. Megakaryocyte

8. Which of the following is not an indication for a bone marrow examination?

a. Pancytopenia (reduced numbers of RBCs, WBCs, and platelets in the peripheral blood)

b. Anemia with RBC indices corresponding to low serum iron and low ferritin levels

c. Detection of blasts in the peripheral blood

d. Need for staging of Hodgkin lymphoma

9. In a bone marrow biopsy specimen, the RBC precursors were estimated to account for 40% of the cells in the marrow, and the other 60% were granulocyte precursors. What is the M:E ratio?

a. 4:6

b. 1.5:1

c. 1:1.5

d. 3:1

10. On a bone marrow core biopsy sample, several large cells with multiple nuclei were noted. They were located close to the endosteum, and their nuclei were evenly spaced throughout the cell. What are these cells?

a. Megakaryocytes

b. Osteoclasts

c. Adipocytes

d. Fibroblasts

11. The advantage of a core biopsy bone marrow sample over an aspirate is that the core biopsy specimen:

a. Can be acquired by a less invasive collection technique

b. Permits assessment of the architecture and cellular arrangement

c. Retains the staining qualities of basophils owing to the use of Zenker fixative

d. Is better for the assessment of bone marrow iron stores with Prussian blue stain


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7.  Ryan D. H. Examination of the marrow. In: Kaushansky K, Lichtman M, Beutler E, et al. Williams Hematology. 8th ed. New York : McGraw Hill 2010.

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*The author thanks Lynne Shaw, MT (ASCP), director of the University of Alabama at Birmingham Hospital Laboratory of Bone Marrow Pathology, for substantive contribution to this chapter.