Bone Marrow

27 Bone Marrow Jamie L. Haddad, Sarah C. Roode, and Carol B. Grindem

Bone marrow is the main hematopoietic organ in the body, and bone marrow examination is a valuable tool in the identification and characterization of many hematopoietic and hematological disorders. Bone marrow is located throughout the flat and long bones of the body and is composed of hematopoietic cell populations and associated microenvironmental elements that support hematopoiesis. Basic understanding of the normal hematopoietic tissue components and familiarity with hematopoietic disorders are necessary for accurate and thorough bone marrow evaluation and interpretation. Because of the complex nature of bone marrow assessment, referral to a pathologist for review is often necessary. Knowledge of normal and potentially abnormal findings in bone marrow is crucial to comprehension of the pathology report and for correlation of the results with the accompanying clinical and clinicopathological data.1-3 This chapter will outline the approach to the cytological and histological evaluations of normal and abnormal bone marrow samples. The discussion will highlight key elements in this assessment, including utility of aspiration cytology versus core biopsy of bone marrow; sample collection and submission guidelines; sample quality effects; marrow cellularity evaluation; lineage assessment of erythroid, myeloid, and megakaryocytic components; and other cellular and stromal elements in bone marrow. Common infectious disorders and hematopoietic and nonhematopoietic neoplasia in bone marrow will also be discussed.

INDICATIONS AND CONTRAINDICATIONS Bone marrow evaluation is commonly performed in patients with abnormalities in peripheral blood and has several specific indications (Box 27.1). Bone marrow assessment is most helpful in patients with unexplained or persistent cytopenias, such as neutropenia, thrombocytopenia, and/or nonregenerative or poorly regenerative anemia, or in those with immature, atypical, or dysplastic cells in circulation. Bone marrow evaluation is also indicated with unexpected or inappropriate cellular responses in peripheral blood, such as increased nucleated red blood cells (nRBCs) without reticulocytosis; unexplained persistent leukocytosis, erythrocytosis, or thrombocytosis; or other hematological abnormalities that cannot be explained by patient history, physical examination findings, and peripheral blood smear evaluation. Additional indications include staging of neoplasia that commonly involves bone marrow; investigation of lytic bone lesions; workup for serum chemistry abnormalities, including hyperglobulinemia or hypercalcemia; monitoring of treatments, such as chemotherapy; investigation for systemic infectious diseases; or assessment of iron stores.4-9 Bone marrow collection is typically a safe procedure with minimal complications and is generally no more of a risk than the restraint,

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sedation, and/or anesthesia required for the collection procedure. Hemorrhage is rare, and infection is unlikely when using proper sampling techniques and precautions.4 The main contraindication for bone marrow sampling is therefore evaluation being unnecessary for further characterization of a disease process. Examples of this type of unnecessary sampling include (1) an explainable hematological abnormality, such as an appropriate neutrophilia in response to inflammation; (2) unconfirmed cytopenia, such as spurious thrombocytopenia caused by traumatic venipuncture; (3) situations where bone marrow assessment would not reliably differentiate between disease states, such as between chronic myeloid leukemia and an inflammatory leukemoid reaction; or (4) cases where cytopenia is acute and could be in a pre-regenerative phase, such as acute anemia without reticulocytosis when there has not been sufficient time for an appropriate bone marrow response to develop. 

BOX 27.1  Indications for Bone Marrow

Evaluation

Abnormal CBC Findings • Unexplained or persistent cytopenias (neutropenia, nonregenerative or poorly regenerative anemia, and/or thrombocytopenia) • Immature, atypical or dysplastic cells in circulation • Unexpected or inappropriate cellular responses in peripheral blood (i.e. increased nRBCs without reticulocytosis) • Unexplained persistent leukocytosis, thrombocytosis, or erythrocytosis • Other unexplained hematological abnormalities  Historical, Physical Examination, or Diagnostic Imaging Abnormalities • Staging of neoplasia (lymphoma, mast cell tumor, histiocytic neoplasia) • Monitoring of treatment (chemotherapy, treatment with other drugs) • Lytic bone lesions (multiple myeloma, metastatic neoplasia, infectious disease) • Investigation for systemic infectious disease (histoplasmosis, leishmaniasis, mycobacteriosis) • Fever of unknown origin  Serum Chemistry Abnormalities • Hyperproteinemia/hyperglobulinemia (lymphoid or plasma cell neoplasia, tickborne/rickettsial disease, systemic inflammatory conditions) • Hypercalcemia (multiple myeloma, lymphoma, fungal disease, other bone neoplasia)  Assessment of Iron Status • Suspected iron deficiency • Differentiation of causes of anemia (chronic inflammation versus chronic blood loss)

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TABLE 27.1  Aspiration Cytology versus Core Biopsy Aspiration Cytology Advantages Best for cell morphology (including evidence of dysplasia)

Core Biopsy Advantages Tissue architecture preserved (including cell distribution and microanatomical location)

More precise differential count, M:E ratio, maturation assessment

Best estimate of cellularity (including megakaryocyte numbers)

Quick and easy sample collection/preparation

Focal lesions (metastatic foci, early/occult neoplasia, granulomas)

Best for small etiological agents Disadvantages

Less accurate cellularity assessment

Stromal changes (bony abnormalities, myelofibrosis) Disadvantages

Less distinct cell morphology

May not be representative if uneven cell distribution

Differential count and calculated M:E ratio more difficult

Less able to capture necrosis, myelofibrosis, bony remodeling

Requires laboratory for sample processing and histopathological evaluation

M:E, myeloid to erythroid ratio.

ASPIRATION CYTOLOGY VERSUS CORE BIOPSY Bone marrow evaluation is most thorough and accurate when both cytological and histological assessments are performed together, because each modality has unique qualities to contribute to this analysis (Table 27.1). Bone marrow aspiration cytology is more commonly pursued compared with core biopsy and histopathology; however, it is recommended that both types of analysis be performed concurrently in all cases, whenever possible. Cytology of bone marrow is generally preferred for individual cell identification and morphological characterization, including assessment for maturation and evidence of dysplasia, as well as for investigation for small etiological agents, including hemoparasites. Aspiration cytology samples are quick and simple to obtain with minimal equipment needed. The quality of the sample can be assessed at the time of collection to allow for additional sampling, if necessary, either via repeated aspiration or the addition of core biopsy. Disadvantages of aspiration cytology include the risk of inaccurate representation of marrow cellularity; inadequate reflection of stromal changes within bone marrow, as with myelofibrosis or bone remodeling; and the risk of missing focal or predominantly paratrabecular lesions, as with foci of metastatic neoplasia. Core biopsy with histopathology is the preferred modality for the most accurate assessment of bone marrow cellularity, particularly in cases with suspected hypocellular marrow. Core biopsy with histopathology is also preferred for evaluation of megakaryocyte density; myelofibrosis and other stromal/ vascular or bony changes, including edema, hemorrhage, necrosis, fibrin, and inflammation; occult neoplasia; and focal lesions, such as metastatic neoplasia or granulomatous inflammation. If core biopsy is not initially performed but sampling for cytology results in repeated low yield “dry taps,” core biopsy with histopathology is strongly recommended to assess whether there may be hypocellular marrow, densely packed hypercellular marrow, or myelofibrosis as an explanation for the poor cytological yield. A unique feature of core biopsy with histopathological evaluation of bone marrow is the preservation of tissue architecture, which allows for assessment of the microanatomical location of the cells present (Figs. 27.1 and 27.2).1-3 This contrasts with cytological assessment, which more commonly represents the interstitial tissue components within the marrow and does not allow for distinct assessment of the specific localization of the hematopoietic compartments. Anatomically, the marrow environment consists of dense lamellar cortical bone along the bone surfaces with interior trabeculae of cancellous bone and an intertrabecular meshwork of thin-walled capillary–venous sinuses with accompanying extracellular matrix. It is within this intertrabecular

Fig. 27.1 Schematic of bone marrow microanatomy. Bone marrow comprises hematopoietic elements, including erythroid, myeloid, and megakaryocytic lineage cells, as well as a supportive network of bony trabeculae (red), vascular sinuses and stromal tissue. Early myeloid cells are paratrabecular (adjacent to bone), whereas later stage myeloid cells with their more lobulated nuclei are interstitial (central). Erythroid cells are adjacent to sinuses and may be arranged in erythropoietic islands around a central macrophage (green). Megakaryocytes (pink) are also adjacent to sinuses to allow for platelet release into the bloodstream. Plasma cells (dark blue) and mast cells are often perivascular, and small lymphocytes (yellow) are dispersed in the interstitium. With remodeling of the bone, osteoblasts (pale blue) and osteoclasts (orange) may line the bony trabeculae. (Drawing by Cari Grindem-Corbett.)

meshwork that the hematopoietic elements reside. Immature granulocytes generally are distributed along the paratrabecular zone within the marrow with maturing granulocytes located more centrally within the interstitium. Megakaryocytes and erythropoietic islands (composed of erythroid precursor cells and supportive macrophages) are located adjacent to the sinuses within the interstitial regions. Resident plasma cells and mast cells are generally perivascular in their orientation, and lymphocytes are dispersed within the interstitium or can be in perivascular aggregates. 

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SAMPLE COLLECTION AND PREPARATION

Sample Site

Bone marrow aspiration and core biopsy sites and techniques have been reviewed in the literature and will only briefly be described here.4,10-27 Common issues with sample collection, preparation, and quality are listed in Table 27.2.

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Proper bone marrow collection and sample preparation are necessary to maximize the diagnostic yield of bone marrow sampling. Once the decision to sample bone marrow has been made and a method has been chosen (aspiration for cytology versus core biopsy for histopathology versus a combination of both), a sample site must be selected (Fig. 27.3). The most commonly used sites for both aspiration cytology and core biopsy with histopathology are the proximal humerus in dogs and cats and the trochanteric fossa of the proximal femur in cats. Additionally, the iliac crest is frequently used in large dogs and occasionally used in cats. For cytological evaluation only, sternebrae or rib sites can also be considered. Caution must be exercised to avoid puncturing the thoracic cavity with use of these sites.15,16 Considerations regarding sample site selection, including advantages and disadvantages, can be found in Table 27.3. When considering the location for sample collection, it should be noted that in young animals, active hematopoiesis occurs throughout the flat and long bones. As growth ceases with maturity, the central/diaphyseal areas of the long bones transition to fatty tissue, with ongoing hematopoiesis being more concentrated in the metaphyseal areas of the long bones and the flat bones. 

Aspiration Versus Core Biopsy

Fig. 27.2 Histological architecture of bone marrow. Microanatomical localization of the hematopoietic elements is evident with histopathology. The marrow spaces are lined by bony trabeculae (top right corner), and the medullary cavity contains adipocytes (large clear spaces) and hematopoietic cells. As noted schematically in Fig. 27.1, early myeloid cells are paratrabecular (arrow adjacent to bone), and later stage myeloid cells are interstitial (central arrows denote groups of granulocytes with lobulated nuclei). Megakaryocytes (denoted by “*”) are adjacent to sinusoids, and erythroid cells are also adjacent to sinusoids (arrowheads denote groups of erythroid cells with dark bulleted nuclei) (hematoxylin and eosin [H&E] stain, original magnification 400×).

Whether performing aspiration for a cytological sample or core biopsy for histopathological sample, the overall approach is similar, and thus similar equipment is needed (Box 27.2). All equipment should be assembled and readily available for immediate use before starting the collection procedure. Sedation or anesthesia is often needed to ensure patient compliance. If the patient has an extremely calm demeanor or is critically ill, a local anesthetic without sedation may be sufficient for sample collection. For either cytology or biopsy, the sampling site is generally clipped and prepared with aseptic/sterile technique, and local anesthetic (2% lidocaine) is injected into the skin, subcutis, and periosteum. A small stab incision is made into the skin with a #11 scalpel blade. The incision can be made just adjacent to the biopsy site to avoid direct connection between the skin surface and the underlying bone tissue, and this may help prevent infection.

TABLE 27.2  Sample Collection and Quality Issues With Aspiration Cytology and Core Biopsy

Samples

Aspiration Cytology No sample obtained

Poor sample yield/quality

Needle plugged with skin or bone

Core Biopsy No sample obtained

Needle not seated in marrow cavity

Needle not seated in marrow cavity

Core not cut/retrieved from marrow cavity

Myelofibrosis or hypercellular marrow (“dry tap”)

Aspiration needle used, rather than core biopsy needle

Hemodilution

Poor sample yield/quality

Sample too short (not deep enough in marrow cavity or not adequately cut/severed before removing needle)

Hypocellular marrow

Sample damaged during collection (crushed while obtaining sample from bone, while removing core from needle, or while making touch imprints)

Bevel of needle lodged against cortical bone

Sample taken from prior aspiration site with disruption of medullary tissue by the cytological collection procedure

Sampling difficult site with small needle (e.g., sternum or rib)

Sample appears hypocellular due to sampling of only subcortical area (naturally hypocellular)

Good sample obtained but Sample too thick/not well spread Good sample obtained but Laboratory processing issues (chatter from microtome if not cannot be evaluated well unable to evaluate well decalcified sufficiently, over-decalcification, cut too thick) microscopically microscopically Cells ruptured during aggressive squash Lost during processing (small sample not placed in cassette) preparation Formalin exposure Understained sample

Fig. 27.3  Bone marrow sample collection sites Left: Proximal humerus. This is a good bone marrow collection site for dogs and cats. Middle: Iliac crest and proximal femur. In large dogs, a dorsal approach to the iliac crest is a good sample site. In small dogs and cats, a transilial approach can be considered (see right image), or the trochanteric fossa of the proximal femur is a good option. Right: For small dogs and cats, the lateral approach to the wing of the ilium (transilial) is a good location for sample collection. (Reprinted with permission from Grindem CB. Bone marrow biopsy and evaluation. Vet Clin Small Anim. 1989;19[4]:673–674.)

TABLE 27.3  Bone Marrow Sample Site Considerations Site

Patient Positioning

Landmarks for Sampling

Proximal humerus Best for: • Large dogs • Small dogs • Cats Considerations: • Avoid the articular cartilage • Young growing animals should not be sampled at this location because of proximity of growth plate

Considerations for Use

Lateral recumbency

• L ocate the greater tubercle by palpation. • Flex the shoulder and stabilize the limb. • Insert the biopsy needle into the flat area just distal to the greater tubercle and advance caudomedially along the long axis of the bone.

Iliac crest

Sternal recumbency preferred; • Palpate the greatest prominence of the iliac crest. can consider sitting, stand- • Stabilize the ilium by placing a finger on either side ing, or lateral recumbency of the wing. • Insert the biopsy needle parallel to the ilium, and direct it ventromedially keeping it parallel to the long axis of the wing of the ilium. • Alternate approach: transilial13

Best for: • Large dogs Considerations: • May not be accessible in obese animals • Transilial approach is useful in cats, small dogs, and obese dogs

• L ocate the greater trochanter of the proximal femur by palpation. • Stabilize the femur by grasping the stifle; slight internal rotation of the stifle may enhance exposure of the fossa. • Insert the biopsy needle medial to the trochanter with the long axis of the needle parallel to the long axis of the femur.

Trochanteric fossa Best for: of the femur • Small dogs • Cats Considerations: • May not be accessible in larger, well-muscled, or obese patients • Cortical bone may be too dense in older patients • Avoid the sciatic nerve located medial and caudal to the greater trochanter

Lateral recumbency

Sternebrae

Sternal recumbency preferred, • Locate the first sternebra, and stabilize with one can consider sitting or hand. standing or lateral recum • Insert a 1-inch 20-gauge needle with attached bency for 2–4 sternebrae 3-cc syringe into the cortex of the first sternebra and advance carefully until firmly embedded, then aspirate.15 • Alternate approach: 2–4 sternebrae14

Considerations: • Danger of penetrating thoracic cavity • Aspiration only, not core biopsy • May be more feasible in elderly or debilitated patients • May be performed with only light sedation

Continued

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TABLE 27.3  Bone Marrow Sample Site Considerations—cont’d Site

Considerations for Use

Patient Positioning

Rib

Considerations: Lateral recumbency • Danger of penetrating thoracic cavity • Aspiration only, not core biopsy • May be more feasible in elderly or debilitated patients • May be performed with only light sedation • May not yield representative sample, especially in older dogs with little active hematopoiesis

BOX 27.2  Bone Marrow Sampling

Equipment

• S urgical preparation supplies (gloves, scrub kit), local anesthetic (2% lidocaine) and sedative, scalpel blade (#11) • 15- to 18-gauge, 1- to 2-inch bone marrow needles (Rosenthal, Illinois sternal, or Jamshidi) for aspiration cytology, and 11- to 15-gauge Jamshidi needles for core biopsy • Alternative recent option: intraosseous infusion system needles and bone injection guns (EZ-O and OnControl, Vidacare Corp)13,21-23 • 10- to 12-mL syringes • 2.5%–3% ethylenediaminetetraacetic acid (EDTA) solution and EDTA tubes • To make EDTA solution, add 0.35 mL sterile isotonic saline to 7-mL EDTA tube to produce a 2.5%–3% EDTA solution (2.5% if EDTA tube contains liquid and 3% if tube contains powder) • Microscope slides, coverslips, and pencil to label slides at frosted edge • Clean Petri dish (or watch glass) and microhematocrit tubes (optional)

For aspiration sampling, a 15- to 18-gauge Jamshidi, Rosenthal, or Illinois sternal needle (preflushed with ethylenediaminetetraacetic acid [EDTA], if desired) is inserted into the stab incision with the stylet locked in place. The needle is advanced into the appropriate area of the bone (see Fig. 27.3) with a twisting/rotating motion (alternating clockwise and counterclockwise) until the needle and stylet are firmly seated in the bone. A slight decrease in resistance may be encountered upon entry into the medullary cavity. The stylet is removed and a 10- to 12-mL syringe containing a small amount (0.3 mL) of 2.5% to 3% EDTA is attached to the needle. Strong negative pressure is applied to the syringe, pulling back two-thirds to threefourths the volume of the syringe in multiple quick successive pulls, until red marrow fluid is seen at the hub of the needle. As soon as bone marrow sample starts to enter the syringe, the negative pressure is released to avoid subsequent hemodilution. Approximately 0.2 to 0.4 mL of bone marrow fluid in the syringe is usually sufficient to prepare several smears. The needle and syringe are then withdrawn from the bone to prepare the sample. Direct pressure to the skin will aid in hemostasis, and the skin can then be sutured, if needed. The next steps in the preparation of the marrow sample are described below in the section “Sample Preparation and Staining.” If marrow is not obtained, the procedure can be repeated. The needle can be repositioned at the same site by either advancing or retracting slightly or angling medially or laterally. Alternatively, the needle can be fully redirected through a different site on the same bone, or a new anatomical sampling site can be selected. Causes of aspiration failure may include poor technique, occlusion of the needle with skin or bone tissue, marrow fibrosis, hypoplasia, or a densely packed hypercellular marrow.

Landmarks for Sampling • P alpate the 10th rib and costochondral junction. • Stabilize the 10th rib. • Advance a 1-inch 22-gauge needle with attached 3-cc syringe dorsally into the medullary cavity just above the costochondral junction keeping needle parallel to the rib, and gently aspirate.16

Core biopsy with histopathology can be performed instead of aspiration cytology, although, ideally, both sampling techniques should be performed concurrently. The core biopsy sample is preferably taken from an adjacent site slightly different from that of the aspiration sample, for example, by reangling the needle so that the aspiration procedure does not damage the area of the bone to be sampled for biopsy.10,12,28 Jamshidi bone marrow needles (11- to 15-gauge, most often 13-gauge) are utilized for core biopsy sampling, and the placement of the needle is the same as described for cytological sampling. The needle is similarly embedded in the bone via a rotating/twisting motion of the needle, but for core biopsy sampling, the stylet is removed just after the needle is initially seated into the bone. Then, the needle is advanced at least 3 mm and to up to 1 to 2 cm deeper into bone to fully access the medullary cavity and cut a diagnostic quality sample. The needle is rotated in place completely (360 degrees) multiple times to sever the core biopsy sample from the sample site, and the needle is then removed from the bone. The sample is removed from the needle by inserting the probe into the narrow end of the needle and pushing the marrow retrograde out through the wider end. An impression smear can be made before formalin fixation of the biopsy sample via gentle rolling of the core on a glass slide, taking great care not to crush or damage the sample in the process. The core sample is then placed in 10% neutral-buffered formalin for submission to the laboratory.24 Samples can be placed in a cassette, with or without sponge inserts, to ensure that the sample is retained and not lost during processing. Formalin-fixed samples and cytological preparations should not be shipped in the same package to avoid artifact from the formalin fumes, which can affect the cytological sample staining quality (Fig. 27.4). 

Sample Preparation and Staining Once the cytological sample is obtained and is within the syringe, the marrow material will clot very quickly (within 30 seconds) if anticoagulant is not utilized. Therefore non-anticoagulated marrow samples need to be placed on glass slides immediately. Even with EDTA, the sample should be prepared right away. This can be either via direct application of the material from the syringe onto the slides or via expulsion of the material into a Petri dish or watch glass containing EDTA solution. Bone marrow spicules can then be identified in the Petri dish and transferred to the slides with a microhematocrit tube or pipette. The spicules within a Petri dish are clear to slightly opaque, light-gray, and irregularly shaped. Once the material is on the slides, the slides are tilted 45 to 70 degrees to allow the blood to drip off the slide while the bone marrow flecks remain adhered. The sample is then spread with a squash technique or, less commonly, a smear technique, as with a blood film (Fig. 27.5). The squash technique is best performed by gently placing a second glass slide onto the sample, orienting it 90 degrees to the original slide, and then smoothly separating the slides.

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Fig. 27.4  Effects of exposure to formalin fumes on a cytological preparation. A bone cytology sample was shipped in the same container as a sealed biopsy specimen jar containing formalin. The exposure to formalin fumes, even through a sealed jar, alters the staining characteristics of the cytological sample. Formalin fumes impart a blue-green hazy quality to the cytological sample. Note the blue-green color of the red blood cells, which are typically pink to red with Wright-Giemsa stain. Smudging of the cellular features also occurs, obscuring accurate assessment of cell morphology and cellular characterization (Wright-Giemsa stain, original magnification 500×). (Courtesy Dr. Andrea Siegel.)

A

B

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Fig. 27.6 Macroscopic appearance of two cytological preparations. Top: The very low number and small size of the marrow flecks (tiny blue specks denoted by the arrow) suggests a hypocellular marrow sample. Alternatively, this may be a poor-quality, low-diagnostic-yield sample. The number of unit particles on a slide may be more reflective of the sample adequacy than the actual marrow cellularity. Bottom: The darkblue flecks of marrow represent large unit particles (indicated by the arrow). This suggests a good-quality/high-yield sample, likely from a hypercellular marrow. Note the deep-blue staining quality to the marrow flecks, consistent with a well-stained preparation.

Similarly, a coverslip can be used instead of a second spreader slide for samples with very fragile cells. If flecks are not identified, the sample can be centrifuged in a small tube and additional squash preparations made from the buffy coat layer. Once the smears are prepared and correctly labeled with pencil on the frosted edge of the slides, they are stained with a typical Romanowsky-type stain (Wright-Giemsa or Diff-Quik). Because bone marrow smears are thick, additional staining time is required, typically at least twice the length of time in each buffer and stain as would be used for a blood smear. Slides should not be blotted dry but, instead, air-dried to allow for full development of the stain color in the cells. If slides are understained, they can be restained to enhance dye penetration into the cells. Properly stained marrow has dark blue-purple spicules macroscopically (Fig. 27.6). If slides are to be submitted to a laboratory, one slide can be stained before submission to check for sample quality and then the rest submitted unstained, along with a current complete blood count (CBC) and blood smear. Any remaining bone marrow fluid can also be submitted in EDTA. 

Necropsy/Postmortem Sampling

C

D

Fig. 27.5 Squash technique for bone marrow cytology samples. (A) A marrow fleck, collected from the Petri dish containing the sample, is placed on a glass microscope slide. (B) A microscope slide or a coverslip (as pictured) is placed over the fleck at a 45-degree angle to the slide. This spreads the fleck and accompanying fluid. (C) The coverslip or spreader slide is slid horizontally and smoothly off the glass slide. (D) Both the original glass microscope slide preparation and the coverslip or spreader slide preparation can be used for microscopic evaluation. However, a coverslip preparation is usually hard to handle during staining and is often discarded.

Samples can still be obtained from deceased patients for both cytological and histological evaluation. For cytology in particular, to ensure sufficient preservation of cellular morphology, samples are ideally obtained within minutes (less than 30 minutes) from the time of death to prevent introduction of autolysis and cellular degradation. This cellular degeneration can lead to misidentification of cell types and prevents accurate assessment of marrow with cytology. For histological assessment, a longer postmortem interval of several hours or even days may still preserve enough architecture to evaluate the sample, although if there is a delay in sample collection for histopathology, refrigeration of the body (not freezing) can help slow autolysis and preserve sample integrity. Postmortem sample sites typically include the metaphyseal region of the long bones, most commonly the femur. The diaphyseal region should be avoided because this is predominantly fatty tissue and may not accurately reflect hematopoietic activity. For cytological evaluation, if the sample is collected immediately after death, aspiration and smear preparation can be performed as previously described. Otherwise, a paintbrush or a gentle

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BOX 27.3  Special Stains and Advanced

BOX 27.4  Systematic Approach to Bone

Special Stains Cellular Identification (Core Biopsy) • Giemsa (highlights erythroid cells deeper blue, highlights mast cell granules) • PAS (highlights granularity in myeloid cell cytoplasm, as well as cytoplasm of plasma cells and megakaryocytes) 

1. Sample quality (an adequate yield, diagnostic sample) 2. Hematopoietic cellularity (relative to patient age and complete blood count [CBC] findings) 3. Iron stores (in dogs; normally absent in cats) 4. Myeloid-to-erythroid (M:E) ratio and differential count (interpreted relative to CBC findings, patient age, and marrow cellularity) 5. Assessment of each lineage (erythroid, myeloid, and megakaryocytic) for numbers, maturation, and morphology 6. Other cell types (lymphocytes, plasma cells, mast cells, macrophages/histiocytes [including phagocytic activity]) 7. Stromal components (myelofibrosis, necrosis, bone changes) 8. Etiological agents (if inflammation or necrosis present)

Diagnostics in Bone Marrow Evaluation

Infectious Agent Investigation (Cytology or Core Biopsy) • PAS, GMS (fungal organisms) • Acid fast, Fites-Faraco (Mycobacterium spp.) • Gram (bacteria)  Substances in Marrow (Core Biopsy) • Iron (Perl’s iron, Prussian blue)—can also be performed on cytology slides • Myelofibrosis (reticulin, Masson’s trichrome) • Serous atrophy of fat (Alcian blue)  Advanced Diagnostics Cytochemical Evaluation for Subtyping Leukemia (Cytology) • Peroxidase, Sudan black B, chloroacetate esterase, nonspecific esterases, acid phosphatase  Flow Cytometry (Liquid Bone Marrow Sample) • CD34; lymphoid, myeloid, histiocytic, and megakaryocytic markers  PARR (PCR for Antigen Receptor Rearrangement; Cytology or Core Biopsy) • Assesses for clonality within a lymphoid population to aid in confirmation of lymphoid neoplasia  Immunocytochemistry/Immunohistochemistry • Aids in tumor identification (cytokeratin for carcinoma, lymphoid markers for lymphoma, Mum1 for plasma cell neoplasia, etc.) GMS, Gomori methenamine silver; PAS, periodic acid Schiff; PCR, polymerase chain reaction.

rolling technique (using a needle to roll the sample along the slide) can be used to apply a postmortem sample to a glass slide. For histological evaluation, a wedge of tissue can be collected to fix in formalin for routine processing. Marrow tissue collected postmortem for histological assessment can be placed in a cassette to keep the sample together during fixation and aid in sample processing at the laboratory. Before enclosure in a cassette, a small portion of the soft part of the marrow can be placed directly in formalin to observe whether the tissue sinks, as with a cellular marrow, or floats, as with a fatty marrow. 

Sample Submission to the Laboratory A combination of stained and unstained air-dried bone marrow cytology preparations should be submitted in break-proof containers. Include patient information, current CBC and blood smear, and any additional liquid bone marrow in EDTA. Unstained slides will then either be routinely stained at the laboratory or retained for potential special staining or advanced diagnostic testing, if warranted (Box 27.3). Bone marrow core biopsies should be mailed separately from cytology slides, even if the formalin jar containing the core biopsies is well sealed, because the formalin fumes can still escape and alter the cellular features on the cytological preparations (see Fig. 27.4). 

Marrow Evaluation

OVERALL APPROACH TO BONE MARROW EVALUATION Whether utilizing aspiration cytology, core biopsy with histopathology of bone marrow, or both modalities concurrently, the approach to bone marrow evaluation is similar (Box 27.4). Accurate interpretation and complete methodical assessment of marrow changes require a current CBC and blood smear assessment, ideally collected simultaneously with the bone marrow sample or within 24 hours. Additional information should include patient history (illnesses, drug administration or other therapies, travel history, diet, transfusion history, chronicity of CBC abnormalities); physical examination findings (mass lesions, organomegaly, petechiae, lymphadenopathy); additional bloodwork (chemistry or urinalysis abnormalities, testing for tickborne disease, Coomb test results); and diagnostic imaging results (hepatosplenomegaly, lung lesions, bone lesions). Bone marrow evaluation encompasses both low- and highmagnification assessments. Features assessed at low magnification include sample quality, marrow cellularity, iron stores, and megakaryocyte numbers. Low-magnification assessment is also used to identify areas of the sample with an abnormal or distinct appearance, such as with metastatic neoplasia or focal cell aggregates, and to identify ideal areas to subsequently examine at high magnification. Components assessed at high magnification include specific cell morphology and lineage identification, maturation evaluation, and examination for etiological agents. With cytology, the most accurate high-magnification assessment requires thin areas that contain a monolayer of intact cells with adequate staining and relatively little hemodilution. These areas are commonly identified directly adjacent to unit particles or between particles. For the most representative assessment of overall marrow findings versus a regional or focal change, evaluation should include assessment of multiple areas on multiple slides. With core biopsy, the anatomical location of the cells can aid in interpretation as to the appropriateness and nature of the population. Noting immature mitotically active cells in a paratrabecular location is an appropriate reaction for development of early myeloid precursors, but a similar immature mitotically active cell population in the interstitial area would be cause for concern about a neoplastic proliferation. Special stains can also be utilized with core biopsies to aid in classification of hematopoietic cellular elements, such as Giemsa to highlight erythroid cells and periodic acid–Schiff (PAS) to highlight granulocytes, megakaryocytes, and plasma cells. Special stains can also highlight stromal elements (reticulin, trichrome) or infectious agents (PAS, Gomori methenamine silver [GMS], acid fast, Gram stains) (see Box 27.3).

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475

Caution in assessment of bone marrow findings is necessary to avoid overinterpreting the changes. Bone marrow findings need to be interpreted in light of sample quality and cellularity, serial CBC results with attention to chronicity of hematological abnormalities, and patient information. It is important to understand that a single bone marrow sample captures only a “snapshot in time,” reflecting a single moment in a constantly changing and evolving hematopoietic picture. 

SAMPLE QUALITY Adequate sample quality is necessary for accurate assessment of bone marrow cytology or core biopsy samples.1 The most important factor with regard to sample quality is to avoid overinterpretation of a poor quality or inadequate sample. Artifacts within the sample or inadequate yield of cells or tissue can lead to inaccurate interpretation (see Box 27.2). For cytology, abundant hemodilution can affect accuracy of cellularity assessment and may lead to a disproportionate percentage of erythroid lineage cells or, if there is a peripheral neutrophilia, a disproportionate component of late stage myeloid cells. A “dry tap” sample with very little yield of unit particles can be misinterpreted as a hypocellular sample. Areas too thick for evaluation cytologically can be very difficult to accurately assess for the myeloid-to-erythroid (M:E) ratio and the morphological features of the cells present. Improperly stained samples can lead to inaccurate assessment of cell morphology and, in some cases, can lead to the impression of an increased component of immature blast cells because nucleoli are often more apparent in understained samples. A more aggressive squash preparation technique can lead to excessive cell rupture, obscuring identification of the cells present. Importantly, exposure of a cytological sample to formalin fumes, even through a tightly sealed biopsy specimen jar, can impart a blue-green hazy staining quality to the sample. This can obscure accurate assessment of cell morphology and characterization (see Fig. 27.4). Therefore biopsy samples should not be shipped in the same box or container as cytological specimens. On core biopsy with histopathology, a large amount of bone dust or crush artifact resulting from difficult sample collection, aggressive handling of the sample, or performing biopsy in a previous aspiration site can lead to an inconclusive result or can falsely mimic the appearance of myelofibrosis (Fig. 27.7). The medullary spaces in the subcortical zone (the first 2–3 trabeculae deep) are naturally hypocellular compared with the deeper medullary tissue, and therefore a shallow core biopsy or a sample taken parallel, rather than more perpendicular, to the cortical bone can lead to a falsely hypocellular appearance to the marrow (Fig. 27.8). 

CELLULARITY Cellularity of a marrow sample can be partially or initially assessed at the time of sample collection and then subsequently confirmed microscopically. At the time of collection for cytology, the sample may be of little yield without much fat (suggesting possible fibrosis), mostly fat without clear flecks of marrow (suggesting a hypocellular, fatty sample), or contain many flecks of marrow tissue (suggesting a normal to hypercellular sample). At the time of collection for core biopsy, red-gray coloration of the tissue suggests normal to hypercellular marrow, whereas white or yellow coloration of the sample suggests hypocellular, fibrotic or fatty marrow, or marked white blood cell (WBC) proliferation, as with lymphoma or leukemia. With a

Fig. 27.7 Bone dust and crush artifact in a core biopsy. Core biopsy sample of low diagnostic yield as a result of abundant bone dust and crushed bony and medullary tissue (blue-purple to pink smudged material). A small amount of yellow-brown iron pigment is identifiable, but hematopoietic cells are not intact to evaluate. The streaming pink material should not be mistaken for myelofibrosis because this streaming appearance is caused by crush artifact rather than a true change. Bone dust and crush artifact can result from aggressive handling of the core biopsy during sample collection, during removal of the biopsy sample from the needle, during preparation of touch impressions, or from collection from a prior aspiration site (H&E stain, original magnification 100×).

Fig. 27.8  Subgross view of a core biopsy. Excellent-quality core biopsy sample that has a superficial layer of dense cortical bone (thick pink area at left of image) with a subcortical region of the medullary cavity that is naturally hypocellular. The cellular marrow component is evident deeper within the sample (middle to right of image). A short-core biopsy sample may only capture this naturally hypocellular area in the subcortical tissue and be misinterpreted as marrow hypoplasia. Adequate depth of penetration into the marrow cavity and complete severing of the core biopsy sample are necessary to obtain a good-quality sample of sufficient length (H&E stain, original magnification 5×).

necropsy sample, bone marrow can be tested for cellularity by placing a portion of the sample in water to see if it sinks (a cellular sample) or floats (a fatty sample). At the time of slide preparation for cytological assessment, dark-blue aggregates of material or cleared spaces having a chatterlike effect on the slide suggest unit particles are present, whereas smooth pink to blue-gray areas suggest predominantly background blood (see Fig. 27.6). Unit particles are necessary for an estimate of marrow cellularity, so with a poor-quality sample without the presence of unit particles, accurate estimation of marrow cellularity is not possible. Taking an overall averaged estimate from multiple unit particles in multiple areas of the sample is the most accurate approach because cellularity is often not uniform throughout a sample. On microscopic evaluation of a cytological preparation, the unit particles are composed of supportive stromal and vascular

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BOX 27.5  Causes of Hypocellular or

Hypercellular Marrow Hypocellular

Hypercellular

Selective or Multilineage Hypoplasia in Marrow •  Aplastic anemia (replacement by fatty tissue) • Drug-associated • Immune-mediated • Toxin-induced • Chemotherapy/radiation • Infectious • Idiopathic • Selective erythroid hypoplasia (see Box 27.9) • Selective myeloid hypoplasia (see Box 27.10)

Increased Hematopoiesis • Erythroid hyperplasia • Myeloid/granulocytic hyperplasia Nonhematopoietic Cellular Proliferation • Neoplasia • Inflammation Hematopoietic Neoplasia

Stromal Changes Replacing/ Altering Marrow Tissue • Myelofibrosis • Myelonecrosis • Serous atrophy of fat “Hematopoietic hypocellularity” may be used to describe the decrease in hematopoietic cells when there is replacement of bone marrow by something other than fat (fibrosis, necrosis, tumor cells) because the marrow is still cellular, but not composed of hematopoietic cells.

elements with hematopoietic cells and adipocytes. The ratio of hematopoietic cellularity to fatty tissue within these unit particles represents the cytological assessment of cellularity within the marrow itself, whereas the number of unit particles noted on the slide may be reflective of sample adequacy more than of actual marrow cellularity. Marrow cellularity assessment is more accurate with a core biopsy sample than with a cytology sample because tissue architecture is retained with histopathology. Core biopsy is strongly recommended when there is a cytological suspicion for hypocellular marrow to either confirm hypocellular marrow or to identify a hypercellular marrow from a “dry tap” or from myelofibrosis that may have impacted the cytological yield. The marrow cellularity must be interpreted in light of the patient’s age and the concurrent CBC data. Younger animals normally have more cellular marrow compared with older animals. Very young animals have little to no fat within the marrow, whereas juvenile animals have approximately 25% fat and 75% hematopoietic cells, young adult animals have approximately 50% fat and 50% cells, and older adult animals have approximately 75% fat and 25% cells.4,10,12,18 General causes of hypocellular and hypercellular marrow are listed in Box 27.5. Comparison of the cytological and histological appearances of hypocellular, normocellular, and hypercellular marrow is depicted in Fig. 27.9.

Hypocellular Marrow A hypocellular marrow (with <25% hematopoietic cells in the face of peripheral demand and taking the patient age into account; see Fig. 27.9) suggests a defect either in the marrow precursor cells themselves or in the supportive microenvironment that the cells depend on, including cytokines, hormones, and growth factors. The remaining marrow (≥75%) is composed of fatty tissue, fibrosis, necrosis, or

gelatinous transformation (serous atrophy of fat). Marrow hypocellularity can involve all cell lineages or can selectively affect one or more lines. Hypocellularity is best confirmed with core biopsy with histopathology, because there are other potential causes for hypocellular marrow with cytology, such as an inadequate sample, a “dry tap,” or myelofibrosis. With hypocellular marrow, often the resident tissue cells are more prominent and account for a higher proportion of the cells present. This may include macrophages that often contain increased iron, plasma cells and lymphocytes, tissue mast cells, and possibly eosinophils. In some cases, actual hyperplasia of mast cells and plasma cells may be seen in hypocellular marrow, such as with ehrlichiosis.29 Aplastic anemia refers to replacement of the marrow by fat with severely decreased hematopoiesis that leads to subsequent cytopenias. This may begin as neutropenia and thrombocytopenia with later development of anemia as a result of the longer lifespan of red blood cells (RBCs) compared with neutrophils and platelets. General causes of panhypoplasia of the bone marrow lineages (aplastic anemia) include drug-associated, immune-mediated, toxin-induced, chemotherapy/radiation–related, infectious, and idiopathic conditions4,30,31 (see Box 27.5). 

Normocellular Marrow A normocellular marrow (approximately 50% hematopoietic cells; see Fig. 27.9) may be appropriate/normal or inappropriate/abnormal and should be interpreted in the context of the patient’s age, the amount of peripheral demand for hematopoiesis, the M:E ratio, and the presence or absence of neoplasia or inflammation/infection on marrow assessment. Some marrows may appear normocellular with low magnification on cytology, but on closer inspection the cellularity within the unit particles is actually composed of increased numbers of plasma cells and macrophages with decreased numbers of hematopoietic precursors. 

Hypercellular Marrow A hypercellular marrow (>75% hematopoietic cells; see Fig. 27.9) typically indicates that one or more cell lines are increased in response to a peripheral demand for cells. This is commonly secondary to hyperplasia in one specific cell lineage (myeloid hyperplasia in response to inflammation, or erythroid hyperplasia in response to blood loss or hemolysis) but can involve multiple cell lines with a strong or combined stimulus. Hypercellularity may also be caused by the presence of other abnormal cell components, such as with effacement of the marrow by neoplasia, or with inflammation including macrophages/histiocytes, plasma cells, and/or lymphocytes. Even a markedly hypercellular marrow can have a low cytological yield in some cases, a form of “dry tap,” which is why core biopsy with histopathology is often helpful to confirm cellularity, particularly in cases of cytological hypocellularity. 

IRON ASSESSMENT In dogs, marrow storage iron seen as hemosiderin in macrophages is a good indicator of total body iron stores. A few clumps of iron are expected per unit particle in a healthy adult canine patient on cytology of the bone marrow (Fig. 27.10). Depletion of marrow iron can be seen with iron deficiency, and Prussian blue or Perl’s iron staining can be used to highlight any iron pigment present. In dogs, iron may be increased with old age, hemolytic anemia, anemia of chronic disease, multiple blood transfusions, dyserythropoiesis or ineffective erythropoiesis, hemochromatosis or hemosiderosis, or parenteral administration of iron. Iron may be decreased with chronic blood loss (even including as a result of repeated phlebotomy), in newborns or very young animals,

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Bone Marrow

Fig. 27.9  Bone marrow cellularity with cytology and core biopsy. Top left: Hypocellular bone marrow cytology sample from a dog. The unit particle contains only adipose tissue and supportive stroma with little to no hematopoietic tissue (Wright-Giemsa stain, original magnification 100×). Top right: Hypocellular bone marrow core biopsy from a dog. The medullary spaces contain only sheets of adipocytes with rare scattered hematopoietic cells and blood-filled sinuses (H&E stain, original magnification 100×). Middle left: Normocellular bone marrow cytology sample from a dog. The unit particles contain approximately equal proportions of hematopoietic cells and fatty tissue. Few dark brown-black aggregates of iron are present and are expected in an adult dog (Wright-Giemsa stain, original magnification 100×). Middle right: Normocellular bone marrow core biopsy from a dog. The medullary tissue contains 30% to 40% hematopoietic cells and 60% to 70% fatty tissue (H&E stain, original magnification 100×). Bottom left: Hypercellular bone marrow cytology from a dog. The unit particles and interparticle areas have a strong predominance of hematopoietic cells with very little fat (<10%). A small amount of iron is visible, and megakaryocytes can be seen prominently within and adjacent to the unit particles (Wright-Giemsa stain, original magnification 100×). Bottom right: Hypercellular bone marrow core biopsy from a dog. The medullary spaces are almost entirely occupied by hematopoietic tissue with only rare adipocytes (round clear spaces). Note that the trabecular bone is purple rather than pink in this image. This reflects less decalcification of this sample compared with the other core biopsies pictured. The mineral component of bone is deeply basophilic with H&E stain, but as the mineral is removed with decalcification, the osteoid matrix, which stains eosinophilic, is all that remains (H&E stain, original magnification 100×).

477

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Bone Marrow BOX 27.6  Interpretation of Bone Marrow

Iron Stores

Increased Iron Stores • Increasing patient age • Hemolytic anemia • Dyserythropoiesis or ineffective erythropoiesis • Anemia of chronic/inflammatory disease • Repeated blood transfusions • Hemochromatosis or hemosiderosis (rare) • Parenteral administration of iron  Decreased Iron Stores • Chronic blood loss • Newborn or very young animal • Nutritional iron deficiency (rare in small animals) • Repeated chronic phlebotomy

Maturation

Fig. 27.10  Iron stores in bone marrow. Top: Hypercellular bone marrow cytology from an old dog with abundant iron stores. Iron stores with aspiration cytology stain as dark brown-black to gray-brown aggregated material, and are often superimposed on unit particles. In less darkly stained areas of a sample (inset), iron stores have a yellow-brown coloration on cytological preparations (Wright-Giemsa stain, original magnification 200× and inset 500×). Bottom: Hypercellular bone marrow core biopsy from an adult dog with abundant iron stores. Iron stores with core biopsy stain as medium-brown to yellow-brown aggregated material. In addition to old age, given the myeloid hyperplasia and relative erythroid hypoplasia in the background, the increased iron in this patient could reflect a component of anemia of chronic/inflammatory disease (H&E stain, original magnification 400×).

In normal marrow, maturation within each lineage should generally be distributed in the shape of a pyramid with the least number of early/immature precursors; the greatest proportion of cells in the maturation and storage pools, which contain the later stage precursors; and middle-aged precursors in the proliferative pool making up the center of the pyramid. In the erythroid lineage, this distribution is often more of a diamond or pentagon shape with slightly fewer metarubricytes (late stage) compared with rubricytes (middle-aged cells), but early precursors (rubriblasts and prorubricytes) are still in much lower numbers (Box 27.7). If the maturation within a given lineage follows the expected pyramidal (or pentagon-shaped) distribution, then maturation is considered orderly and complete. If there are increased numbers of earlier precursors, this is considered a left shift, which is common with hyperplasia. If there is maturation up to a point in development but with a lack of later stage precursors, this is considered a maturation arrest, which is common with immune-mediated destruction of later stage precursors. Release of large numbers of band and segmented neutrophils into the peripheral circulation with an inflammatory stimulus can have the appearance of a maturation arrest in the marrow, but this can easily be ruled out by examination of peripheral blood for neutrophilia, left shift, and toxic change. 

Myeloid-to-Erythroid Ratio or, less often, with nutritional iron deficiency (Box 27.6). Cats lack stainable iron under normal circumstances, and therefore identification of iron in marrow in cats suggests a pathological state, or it can be a sequela of multiple blood transfusions. In cats, pathological conditions that lead to visible/stainable storage iron include hemolytic anemia or dyserythropoiesis, such as with feline leukemia virus (FeLV) infection or hematopoietic neoplasia. 

GENERAL LINEAGE ASSESSMENT Complete bone marrow assessment requires evaluation of several features within each cell lineage (erythroid, myeloid, and megakaryocytic), including the total number/proportion of cells present in that lineage (hypoplasia versus normal versus hyperplasia), completeness of maturation (complete versus arrested), orderliness of maturation (orderly versus left-shifted), and assessment of cellular morphology. This requires an understanding of the normal expectations for each lineage and knowledge of the morphological differences between the various cell types present in marrow.

Overall assessment of the lineages also includes evaluation of the M:E ratio. In this ratio, “myeloid” refers to all granulocytic and monocytic precursors, including mature segmented neutrophils, and “erythroid” refers to all nucleated erythroid precursors, excluding polychromatophils/reticulocytes and mature RBCs. Lymphocytes, plasma cells, and macrophages are assessed concurrently but not included in the ratio. There are wide reported ranges for the normal M:E ratio in dogs and cats, but as a general rule, in dogs, the M:E ratio is normally 0.75:1 to 2.5:1, and in cats, the normal ratio is 1:1 to 3:1.4,25,32 On cytology, this ratio is often calculated on the basis of a 200- to 500-cell differential count in multiple areas of multiple slides but can also be estimated by more experienced cytologists. An estimate of the M:E ratio is all that is typically performed on a core biopsy sample. The significance of the M:E ratio must be interpreted in light of the overall marrow cellularity and peripheral blood findings, which provide the context for this interpretation (Table 27.4). For example, if there is neutrophilia in the periphery and the marrow cellularity is increased with an increased M:E ratio, then this is interpreted as myeloid hyperplasia as an expected response to peripheral demand for neutrophils, as in inflammation. If there is a

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Bone Marrow

479

BOX 27.7  Pyramidal Distribution of Cells in Marrow Myeloblast Progranulocyte

Megakaryoblast Promegakaryocyte

Myelocyte

Rubriblast Prorubricyte

Basophilic megakaryocyte Rubricyte

Metamyelocyte and band Mature megakaryocyte

Mature granulocyte

TABLE 27.4  Myeloid-to-Erythroid (M:E)

Ratio Assessment M:E Ratio

Overall Marrow Cellularity

Interpretation

Normal M:E ratio

Normocellular

Normal

Hypercellular

Erythroid and myeloid hyperplasia

Hypocellular

Erythroid and myeloid hypoplasia

Normocellular

Myeloid hyperplasia and erythroid hypoplasia

Hypercellular

Myeloid hyperplasia

Hypocellular

Erythroid hypoplasia

Normocellular

Erythroid hyperplasia and myeloid hypoplasia

Hypercellular

Erythroid hyperplasia

Hypocellular

Myeloid hypoplasia

Increased M:E ratio

Decreased M:E ratio

concurrent regenerative anemia caused by blood loss, then the M:E ratio may be normal because myeloid hyperplasia and erythroid hyperplasia both contribute to the increased marrow cellularity. If there is nonregenerative anemia in the periphery with decreased marrow cellularity and an increased M:E ratio, this may be interpreted as erythroid hypoplasia as an insufficient response to the peripheral demand for RBCs. 

Relative Versus Absolute Hypoplasia or hyperplasia within a given cell line can be described as an absolute change (a true increase or decrease in that marrow lineage compared with normal), or as a relative change, whether relative to the other lineages or to what would be expected given the hematopoietic stimulus. For example, if there is marrow hypercellularity with a severely increased M:E ratio but retained erythroid cells are present, then there is myeloid hyperplasia with relative erythroid hypoplasia (hypoplasia relative to the amount of myeloid present), but the absolute erythroid numbers may be normal for the patient. If the patient has a concurrent nonregenerative anemia, such as with anemia of inflammatory/chronic disease, then there may truly be concurrent absolute myeloid hyperplasia and absolute erythroid hypoplasia. Because this distinction can be difficult to confirm, interpretation may be left in relative terms. 

Effective Versus Ineffective Hematopoiesis If a lineage is identified as being hyperplastic, an important component of the assessment is whether hematopoiesis in that lineage is

Metarubricyte

“effective” or “ineffective.” “Effective versus ineffective” refers to the impact that the hyperplasia in the marrow has on peripheral blood in that lineage. Effective hematopoiesis leads to evidence of the regenerative bone marrow response in peripheral blood. Effective erythropoiesis leads to a subsequent regenerative anemia in peripheral blood with reticulocytosis and increasing hematocrit. Effective granulopoiesis typically leads to a developing neutrophilia in the periphery with a left shift in neutrophils with circulating bands. “Ineffective hematopoiesis” refers to hyperplasia within that lineage in bone marrow, but without evidence of that regenerative response in the periphery. Ineffective erythropoiesis indicates an attempt by bone marrow at resolution of peripheral anemia, but the anemia remains nonregenerative without an actual regenerative response seen in the periphery (without increasing hematocrit and without reticulocytosis). Ineffective granulopoiesis indicates an attempt by bone marrow at resolution of a peripheral neutropenia (with myeloid hyperplasia in bone marrow) but without an increasing neutrophil count in the periphery. Distinction between effective and ineffective hematopoiesis can aid in the differential causes to consider in a particular case. Caution is recommended with this interpretation, because a regenerative response can take 3 to 7 days to become apparent in peripheral blood. This determination is therefore best made in cases with chronic cytopenias, rather than in those with more acute anemia or neutropenia that may not have had sufficient time to respond and is still in the pre-regenerative phase. 

Dysplasia Morphological assessment is important for cellular classification, but this assessment should also include evaluation for evidence of dysplasia. Dysplasia refers to abnormal morphological features within a cell line caused by a pathological process. Dysplasia is not exclusively seen with hematopoietic neoplasia but is also commonly seen with severe hyperplasia within a lineage, toxic insult, nutritional deficiency (folate/cobalamin), congenital anomalies, FeLV infection, vector-borne infection, immune-mediated conditions, necrosis/inflammation/fibrosis in the marrow, nonhematopoietic neoplasia, or as an effect of certain drug administration including some chemotherapeutic agents4,5,16,33 (Box 27.8). Some components of the overall lineage assessment are easier and more accurate with cytological samples than with core biopsies given the relative ease of identification of individual cells with cytology compared with histopathology. Individual cells can be more difficult to specifically identify on histopathology, particularly earlier to middle-aged precursors; however, general estimations can still easily be made with histopathology. Identification of some cells may be aided by the “company they keep.” In other words, earlier erythroid precursors may be more apparent when seen in the context of clear

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erythroid hyperplasia with many obvious late-stage erythroid precursors. Evidence of left shift, maturation arrest, or dysplasia can still often be identified with histopathology. Compared with cytology, these histological features may be more difficult to definitively confirm or quantify, particularly in subtle cases. For this reason, complete assessment of bone marrow ideally includes both cytological and histological assessments. 

SPECIFIC LINEAGE ASSESSMENTS The following sections will include descriptions of normal findings and common abnormalities in each of the main hematopoietic lineages in bone marrow (erythroid, myeloid, and megakaryocytes), following the concepts introduced by this preceding discussion.

BOX 27.8  Definition and Causes of

Dysplasia

Definition of Dysplasia Abnormal morphological features within a cell line caused by a pathological process  Causes of Dysplasia • Neoplasia (hematopoietic neoplasia, nonhematopoietic neoplasia) • Severe hyperplasia within a lineage • Toxic insult; drug administration (some chemotherapy) • Nutritional deficiency (folate, cobalamin) • Congenital anomaly (macrocytosis in Poodle, congenital dyserythropoiesis in English Springer Spaniel) • Infectious (FeLV, vector-borne infection) • Necrosis/inflammation/fibrosis in bone marrow FeLV, feline leukemia virus.

Erythroid Normal development and maturation within the erythroid lineage begins with a rubriblast that gives rise to 16 to 32 progenitor cells through 4 to 5 divisions/mitoses over a period of 5 to 7 days.4 Division ceases with the rubricyte stage. With each division and maturation step within the erythroid lineage, the precursor cells undergo decreasing cell size, decreasing nuclear-to-cytoplasmic (N:C) ratio, condensation of the nucleus with eventual extrusion, and hemoglobinization of the cytoplasm with a transition from deep-blue to polychromatophilic (gray-blue) to orthrochromic (pink-orange) (Table 27.5; Fig. 27.11). Cytologically, compared with early myeloid lineage cells, early erythroid lineage cells generally have deeper-blue cytoplasm and a coarser chromatin pattern, whereas later-stage erythroid cells are more easily distinguished from myeloid cells by their polychromatophilic to hemoglobinized cytoplasmic coloration, condensing nucleus, and smaller cell size (Fig. 27.12). Erythroid cells may be seen in close association with a macrophage in erythropoietic islands (Fig. 27.13). On a core biopsy sample, early erythroid cells typically have a coarser chromatin pattern with smaller cytoplasmic volume compared with early myeloid cells, which have a higher volume of pale eosinophilic to clear cytoplasm with a finer chromatin pattern. Early myeloid cells are also most typically located along the paratrabecular regions within the marrow, whereas early erythroid cells are in a more interstitial location among the later stage erythroid cells (see Fig. 27.2). The later-stage erythroid cells have a very dark, round, bulleted nucleus with a small volume of eosinophilic cytoplasm. Assessment within the erythroid lineage, as for any cell line, should include evaluation of cell numbers/proportion (hypoplasia versus normal versus hyperplasia) in absolute and/or relative terms, maturation assessment, and evaluation of morphology, including any evidence of dysplasia. The erythroid lineage development follows a pentagon-shaped distribution with only few rubriblasts and prorubricytes (approximately 2%–4%) with the highest number of rubricytes

TABLE 27.5  Erythroid Lineage Maturation Name of Cell Stage

Cytological Description

Rubriblast

Large-size cell; high nuclear-to-cytoplasmic ratio; small volume of deep-blue cytoplasm; large-size, round nucleus with visible nucleoli and fine to coarse granular chromatin pattern

Prorubricyte

Medium- to large-size cell; small to moderate volume of medium- to deep-blue cytoplasm; medium- to large-size, round nucleus without nucleoli and with a coarse chromatin pattern

Rubricyte

Small- to medium-size cell; small to moderate volume of medium-blue (basophilic) to blue-gray (polychromatophilic) to blue-pink cytoplasm; small- to medium-size, round nucleus without nucleoli and with coarse clumped chromatin pattern

Metarubricyte

Small-size cell; small volume of blue-gray to blue-pink (polychromatophilic) to pink-orange (orthochromic) cytoplasm; small-size, round nucleus without nucleoli and with densely clumped chromatin pattern

Cytological Image

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Bone Marrow

481

Fig. 27.11  Cellular identification in bone marrow cytology. Top: Erythroid lineage cell identification. Rubriblasts (RB), prorubricytes (PR), rubricytes (RC), and metarubricytes (MR) are indicated. In addition, a small lymphocyte (L) is present for comparison. Compared with rubricytes and metarubricytes, lymphocytes have a smaller volume of cytoplasm and a more dense and smooth chromatin pattern. Few ruptured or disrupted cells are not labeled and cannot be accurately classified. See Table 27.5 for additional description of erythroid cell characteristics (Wright-Giemsa stain, original magnification 1000×). Bottom: Myeloid lineage cell identification. Myeloblasts (type I, II, and III; MBI, MBII, and MBIII), progranulocytes (PG), neutrophilic myelocytes (MC), neutrophilic metamyelocytes (MM), band neutrophils (BN), and segmented neutrophils (SN) are indicated. In addition, a small lymphocyte is present (L). Few ruptured or disrupted cells are not labeled and cannot be accurately classified. See Table 27.6 for additional description of myeloid cell characteristics (Wright-Giemsa stain, original magnification 1000×).

(approximately 65%–75%) and with fewer metarubricytes (approximately 20%–35%) (see Box 27.7).25,31 Lack or paucity of metarubricytes may suggest maturation arrest. Maturation assessment with cytology should also include evaluation for polychromasia in the background RBC population, which is not possible with core biopsy. Polychromasia is expected, particularly in dogs and to a lesser degree in cats, with normal maturation in the erythroid lineage, and lack of polychromasia with nonregenerative anemia in the periphery may indicate maturation arrest. Dyserythropoiesis is a general term that refers to abnormal erythroid maturation or morphology and may be used to describe either maturation arrest or other forms of ineffective erythropoiesis or to describe morphological abnormalities, as with erythroid dysplasia.

Erythroid Hyperplasia Erythroid hyperplasia is the expected regenerative response to acute blood loss or hemolysis, and peripheral reticulocytosis would be

expected in 3 to 5 days (peaking at 4 days) if the stimulus is sufficient.4 Effective erythroid hyperplasia (effective erythropoiesis) leads to increasing hematocrit, whereas ineffective erythropoiesis does not result in resolution of the anemia with increasing hematocrit or reticulocytosis (Box 27.9). The causes of effective erythropoiesis mirror the causes of regenerative anemia, including acute blood loss or hemolysis. Effective erythropoiesis can also be seen with primary or secondary polycythemia, although marrow assessment cannot reliably distinguish between primary polycythemia vera and secondary causes of polycythemia. Effective erythroid hyperplasia typically has an accompanying left shift within the erythroid lineage with increased rubriblasts and prorubricytes, although rubricytes and metarubricytes still predominate. Polychromasia is typically seen in the background RBC population (Figs. 27.14 and 27.15). Ineffective erythroid hyperplasia (ineffective erythropoiesis) is an increasingly more recognized pattern of hematological abnormalities and is characterized by erythroid hyperplasia in the marrow without

482

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Fig. 27.12  Cytological characterization of myeloid versus erythroid cells. Both images represent the same cytological field from a dog with a hypercellular bone marrow sample. When performing a differential count to assess for an M:E ratio, each cell is identified as myeloid (M) or erythroid (E), as denoted in the bottom image. Some cells cannot be definitively classified because of disruption or rupture, mitosis, a thicker area of the sample, or overlapping characteristics that prevent identification (denoted with “x”). Lymphocytes, plasma cells, and histiocytes are not included in calculating the M:E ratio (Wright-Giemsa stain, original magnification 1000×).

a regenerative response in the periphery. A common cause is precursor-targeted, immune-mediated anemia (PIMA), also referred to as nonregenerative immune-mediated hemolytic anemia (IMHA) (Figs. 27.16 and 27.17). Destruction of later-stage erythroid precursors in PIMA leads to an inability of the erythroid hyperplasia response in the marrow to effectively increase the hematocrit in the periphery. Other causes of ineffective erythroid hyperplasia include nutritional deficiency (iron deficiency, folate/cobalamin deficiency), congenital dyserythropoiesis (Poodles, English Springer Spaniels; but not usually

associated with anemia), or hematopoietic neoplasia (particularly myelodysplastic syndrome [MDS]).4,34,35 Iron-deficiency anemia can usually be distinguished from the other differentials by the presence or absence of stainable iron in the marrow (see Fig. 27.10). Classic cases of PIMA in dogs may have evidence of phagocytosis of erythroid precursors by macrophages and often have increased marrow iron, as well as possible secondary myelofibrosis (see Figs. 27.16 and 27.17). Although maturation arrest may be identified in cases of PIMA, this finding is not specific for an immune-mediated process and can also

CHAPTER 27 

Fig. 27.13  Bone marrow cytology from a dog; erythropoietic island. Erythroid cells can be seen in erythropoietic islands, in which erythroid precursors in various stages of maturation surround a central macrophage. The macrophage supports erythropoiesis by providing nutrients, playing a role in iron metabolism, and phagocytosing extruded nuclei from metarubricytes. Erythropoietic islands may be more readily apparent with erythroid hyperplasia, as is evident in this patient (Wright-Giemsa stain, original magnification 1000×).

BOX 27.9  Causes of Erythroid Hyperplasia

and Erythroid Hypoplasia Erythroid Hyperplasia

Erythroid Hypoplasia

Effective Erythroid Hyperplasia • Regenerative anemia • Blood loss • Hemolysis • Polycythemia (absolute) • Primary (polycythemia vera) • Secondary • Appropriate (hypoxemia) • Inappropriate (local renal hypoxia, erythropoietin-secreting tumor)

Aplastic Anemia (see Box 27.5)

Ineffective Erythroid Hyperplasia • Precursor-targeting immune-mediated anemia (destruction of latestage precursors) • Nutritional deficiency (iron, cobalamin/folate) •  Congenital dyserythropoiesis (Poodles, English Springer Spaniels) •  Hematopoietic neoplasia (particularly myelodysplastic syndrome)

Selective Erythroid Hypoplasia • Precursor-targeting immunemediated anemia (destruction of early-stage precursors) • Neoplasia • Drug-associated • Hyperestrogenism (dogs) • Feline leukemia virus (FeLV) subgroup C infection (cats) • Recombinant human erythropoietin administration • Mild causes without severe anemia • Chronic renal disease • Endocrinopathy • Anemia of chronic/inflammatory disease

be seen with myeloid neoplasia, congenital dyserythropoiesis, and drug-related causes.4 Increased erythrophagia by macrophages is also not specific for PIMA and can be seen with other causes of ineffective erythropoiesis or secondary to blood transfusion. 

Erythroid Hypoplasia Erythroid hypoplasia can occur as a selective process (affecting only the erythroid lineage) or as a component of panhypoplasia in the

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483

marrow as with causes of aplastic anemia (see Box 27.9). Causes for selective erythroid hypoplasia may include PIMA with destruction of early erythroid precursors. This is in contrast to PIMA with late stage erythroid targeting (described above), which leads to erythroid hyperplasia with persistent nonregenerative anemia.36 Other causes include neoplasia, drug-associated causes, hyperestrogenism in dogs, FeLV infection in cats (subgroup C), or administration of recombinant human erythropoietin.4 Chronic renal disease, endocrinopathy, and anemia of chronic/inflammatory disease can all lead to a minor degree of erythroid hypoplasia in the marrow, but most typically are not associated with as severe anemia as the other differentials. There is variability in the literature with regard to terminology for immune-mediated precursor-targeted conditions in bone marrow in dogs and cats. Pure red cell aplasia (PRCA), which is still thought to be caused by an immune-mediated process, refers to lack or near-absence of erythropoiesis, whereas nonregenerative IMHA (PIMA) still has ongoing erythropoiesis, although erythropoiesis is incomplete and impaired. The definition of PRCA often includes an M:E ratio greater than 75:1 or less than 5% of marrow cells from the erythroid lineage, whereas PIMA often has a less severe decrease in erythropoiesis.37-39 Lymphocytosis or lymphoid aggregates may be seen in bone marrow with these conditions, particularly in cats. Increased plasma cells and myelofibrosis may also be seen in these cases, particularly in dogs (see Fig. 27.17).38,39 In some cases, response to immunosuppressive therapy may be very slow, often longer than 1 to 2 months.38,39 Increased phagocytic activity by macrophages may be seen and is helpful in suggesting an immune-mediated pathogenesis, although it is not present in all cases and is not entirely specific. Myeloid hyperplasia may be seen and may even lead to severe marrow hypercellularity despite marked selective erythroid hypoplasia. 

Erythroid Dysplasia Dysplasia within the erythroid lineage may be recognized as nuclear-tocytoplasmic asynchrony, including megaloblastic change, premature pyknosis in immature cells, nuclear fragmentation or karyolysis, multinucleation, internuclear chromatin bridging, cytoplasmic or nuclear vacuolation, or siderotic inclusions (Fig. 27.18). Possible causes for erythroid dysplasia include severe erythroid hyperplasia, ineffective erythropoiesis, myeloid neoplasia, FeLV infection, folate/cobalamin deficiency, congenital dyserythropoiesis (macrocytosis in English Springer Spaniels, Poodles), drug administration (including chemotherapy or other drugs that interfere with DNA synthesis), marrow necrosis/infection/radiation, or toxin effects.4,31,35 

Myeloid Myeloid lineage refers to granulocytic and monocytic precursors. Inflammatory or phagocytic macrophages will be considered in the section “Other Cell Types” below. The vast majority of the myeloid cells are granulocytic, whereas developing monocytes (discussed below) are a minority (<3%–5% of marrow cells).4,18 Normal development and maturation within the granulocytic lineage occurs over 6 to 7 days and begins with a myeloblast that gives rise to, on average, 16 progenitor cells through 4 divisions/mitoses.4 A division may be skipped or added, and the overall time to production of myeloid cells may be shortened, depending on peripheral demands. Division ceases with the myelocyte stage. With each division and maturation step within the granulocytic lineage, the precursor cells undergo decreasing cell size, decreasing N:C ratio, condensation and lobulation of the nucleus, and increasing pallor and alteration of granularity of the cytoplasm. Primary granules are first seen in middle-aged to late myeloblasts and are most prominent in progranulocytes and then replaced with secondary granules in myelocytes (Table 27.6; see Fig. 27.11). Cytologically, several features

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Fig. 27.14  Bone marrow cytology from a dog with erythroid hyperplasia caused by immune-mediated hemolytic anemia. Severe erythroid hyperplasia is present with a decreased M:E ratio and a left shift in erythroid cells with increased numbers of earlier stage precursors. Complete maturation is evident given the background population of polychromatophilic red blood cells with pale blue-gray cytoplasm (top of image). Spherocytes among these polychromatophils (top of image) have a rounded, smaller, hyperchromic appearance. This is an example of effective erythropoiesis, because this patient has regenerative anemia (reticulocytosis) in the peripheral blood (Wright-Giemsa stain, original magnification 500× [top] and 1000× [bottom] ).

aid in distinction of myeloid cells from erythroid lineage (see Fig. 27.12). Early myeloblasts (type I) generally have paler-blue cytoplasm and a finer chromatin pattern compared with early erythroid lineage cells. Later myeloblasts (type II and type III) and progranulocytes are distinguished from erythroid cells by their increasingly prominent and numerous pink cytoplasmic granules. Later-stage myeloid cells are easily distinguished from later-stage erythroid cells by their lobulated nuclear contour and cytoplasmic coloration and, in eosinophils and basophils, their brightly colored granules. Histologically, early myeloid cells typically have a more finely stippled chromatin pattern with higher cytoplasmic volume compared with early erythroid cells, with clear to granulated eosinophilic cytoplasm. Early myeloid cells are also most typically located along the paratrabecular regions within the marrow, whereas early erythroid cells are in a more interstitial location among the later-stage erythroid cells. Myelocyte stages are difficult to

specifically identify on histopathology because of lack of nuclear lobularity. The later-stage granulocytic cells are clearly distinguished on histopathology by their nuclear lobularity and for eosinophils in particular by their prominent eosinophilic granules (see Fig. 27.2). Basophil precursors are very difficult to distinguish histologically, although are generally in low numbers. Assessment within the granulocytic lineage, as for any cell line, should include evaluation of cell numbers/proportion (hypoplasia versus normal versus hyperplasia) in absolute and/or relative terms, maturation assessment, and evaluation of morphology, including any evidence of dysplasia. The granulocytic lineage development follows a pyramid-shaped distribution with only few myeloblasts (1%), a greater number of the proliferative pool of promyelocytes and myelocytes (approximately 15%) and with the largest number of the maturation/storage pool precursors composed of metamyelocytes, bands,

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proportion of marrow cellularity, myeloid hyperplasia and hypoplasia are generally discussed as granulocytic hyperplasia and hypoplasia, rather than pertaining to monocytic cells. 

Myeloid Hyperplasia

Fig. 27.15 Core biopsy from two dogs with erythroid hyperplasia. In both cases, there is severe hypercellularity with a decreased M:E ratio. On histopathology, erythroid lineage cells are identified by their darker, bulleted nuclei, particularly noticeable in later-stage precursors. Earlier erythroid precursors have a small volume of deeply eosinophilic cytoplasm with coarse chromatin (arrows), but are often most easily identified by the context of surrounding erythroid hyperplasia with many late stage precursor cells. In the top and lower left images from the same dog, there is complete maturation with many late rubricytes and metarubricytes. In contrast, in the lower right image, there are greater numbers of rubricytes with a maturation arrest and only few metarubricytes. This (lower right) is a patient with precursor-targeted immune-mediated anemia (PIMA). Also, note in the lower left image, there is a megakaryocyte (arrowhead). Portions of megakaryocyte cytoplasm may be seen without a nucleus in histological section caused by an artifact of plane of section. These anucleate brightly eosinophilic structures should be included as part of the estimate of megakaryocytic cellularity in core biopsies (H&E stain, original magnification 400× [top], 600× [bottom left], and 1000× [bottom right] ).

and segmented granulocytes (approximately 85%) (see Box 27.7).4,18,25 Relative lack of mature segmented neutrophils may be seen in cases of intense inflammation with immediate release of bands and segmented neutrophils into the periphery upon their production in the marrow, or with maturation arrest (as with an immune-mediated process). On core biopsy with histopathology, left shift may be evident as an increased thickness of the paratrabecular band of immature myeloid cells.

Monocytes Monocytes are generally in very low numbers in bone marrow and monoblasts are not reliably distinguished from myeloblasts, even on cytology, because of overlapping morphology. Promonocytes do not contain the cytoplasmic granules noted in promyelocytes. Promonocytes and mature monocytes may be recognized as myeloid cells but are not easily distinguished from neutrophilic myelocytes or metamyelocytes. Mature monocytes are not retained in the marrow as a storage pool, unlike the abundant storage pool in the granulocytic lineages, and therefore are not generally seen in significant numbers in the marrow (generally <3%–5% of cells).4,18 Because of their low

Myeloid/granulocytic hyperplasia, as with the discussion in the erythroid lineage above, can be effective or ineffective (Box 27.10). Effective myeloid hyperplasia refers to marrow granulocytic hyperplasia that leads to neutrophilia and/or left shift in the peripheral blood neutrophil population (Figs. 27.19 and 27.20). This is most common with inflammatory conditions whether they result from bacterial infection, other infectious etiologies, immune-mediated conditions, tissue necrosis, underlying neoplasia, or other causes. This type of process can also occur secondary to drug administration (granulocyte–colony stimulating factor [G-CSF]), as a paraneoplastic process (neoplasm producing G-CSF or a similar granulopoietic growth factor/stimulant), with the recovery phase of cyclic hematopoiesis in Collie dogs, with leukocyte adhesion deficiency in dogs, or with chronic myeloid leukemia (CML).4 CML can be very difficult or even impossible to differentiate from an inflammatory process on bone marrow examination.4 Myeloid hyperplasia is often accompanied by left shift and possibly with paucity of storage pool cells because they exit the marrow quickly upon their production. With septicemia or severe infection, this robust utilization of the storage pool with a concurrent neutropenia can mimic the marrow appearance of ineffective granulopoiesis. Mild cytoplasmic vacuolation of myeloid precursors can occur with robust myeloid hyperplasia and is considered a minor dysplastic change. Ineffective myeloid hyperplasia (ineffective granulopoiesis) is less common than ineffective erythropoiesis but can occur with immune-mediated neutropenia with destruction of later-stage, mature, segmented neutrophils, with hematopoietic neoplasia, such as MDS or acute myeloid leukemia (AML), or with FeLV infection in cats (Fig. 27.21).4 It should be noted that the appearance of an early myeloid hyperplasia response can overlap that of AML, given the increased proportion of myeloblasts before subsequent completion of the maturation pyramid. In cases where the neutropenia is acute, continued monitoring of CBC for an emerging regenerative response (5–7 days) is recommended before interpreting an expansion of blast cells in the marrow as a neoplastic process. 

Eosinophil Hyperplasia Eosinophil precursors are normally less than 6% of overall marrow cellularity.4 Eosinophil hyperplasia in the marrow is the expected bone marrow finding with peripheral eosinophilia of any cause, including with parasitism, allergy/hypersensitivity, inflammatory conditions, mast cell tumor, hypereosinophilic syndrome, or hematopoietic neoplasia (CML, MDS, or AML) (Fig. 27.22). In dogs that have gray eosinophils (particularly common in greyhounds and other sighthounds), marrow eosinophil precursors also lack the typical eosinophilic coloration of the cytoplasmic secondary granules and instead contain clear cytoplasmic vacuoles (Fig. 27.23). Eosinophils in cytological and histological specimens often appear more prominent compared with other myeloid cells, likely because of their brightly colored granules, and therefore may be misinterpreted as hyperplastic even when in appropriate numbers. 

Basophil Hyperplasia Basophil precursors are normally less than 1% of overall marrow cellularity.4 Basophil hyperplasia in the marrow typically accompanies basophilia in the periphery, and often is concurrent with eosinophilia. Basophil hyperplasia may occur with parasitism, allergy/ hypersensitivity, mast cell tumor, or hematopoietic neoplasia (see Fig. 27.19). 

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Fig. 27.16  Bone marrow cytology from a dog with ineffective erythropoiesis caused by precursor-targeted immune-mediated anemia (PIMA). Macrophages contain phagocytized red blood cells in the cytoplasm, as well as phagocytized erythroid precursors including metarubricytes (left) and rubricytes (right top and bottom). In some cases, phagocytized erythroid cells may be seen within free cytoplasmic fragments that have artifactually broken off from phagocytic macrophages in the marrow (lower right). PIMA is an example of ineffective erythropoiesis because there is severe erythroid hyperplasia in the marrow, but there is a nonregenerative anemia in peripheral blood (Wright-Giemsa stain, original magnification 1000×).

Myeloid Hypoplasia Granulocytic hypoplasia can occur as a component of aplastic anemia with panhypoplasia in the marrow or can be selective (see Box 27.10). Granulocytic hypoplasia may also precede hypoplasia in other cell lines, as in some cases with certain drugs.4 Selective granulocytic hypoplasia can occur with immune-mediated targeting of earlier stage myeloid precursors as a result of certain drugs, parvovirus/panleukopenia infection, FeLV infection in cats,4,8,40 or severe inflammation/infection, such as in the early stages of septicemia (caused by depletion of the storage pool in response to high peripheral demand). Granulocytic hypoplasia can also occur transiently with inherited cyclical hematopoiesis in Collie dogs and is then followed by a restorative granulocytic hyperplasia response. 

Myeloid Dysplasia Dysgranulopoiesis, analogous to dyserythropoiesis in the erythroid lineage, refers to abnormal granulocytic maturation and/or morphology. Dysplasia within the myeloid lineage may be seen as giant neutrophils, abnormal mitotic figures, multinucleation, abnormal granulation, hyposegmentation or hypersegmentation, cytoplasmic vacuolation, or bizarre nuclear contour, including ring-form nuclei (see Fig. 27.18). These changes can be seen with MDS/AML, particularly when associated with FeLV infection, but can also be seen with severe myeloid hyperplasia, including with inflammation/infection, certain drug effects, in Giant Schnauzers with cobalamin malabsorption, and during an early recovery phase following granulocytic hypoplasia or with G-CSF administration.4 

Megakaryocytes Normal development and maturation within the megakaryocytic lineage begins with a megakaryoblast. Unlike in the other lineages, megakaryoblasts undergo endomitosis, rather than mitosis, to give rise to a single large polyploid megakaryocyte. Endomitosis refers to nuclear

division without concurrent cytoplasmic division. With each division and maturation step within the megakaryocyte lineage, the cells become larger, with multiple individualized nuclei that fuse into a single large convoluted and lobular nucleus, with increasing condensation of the chromatin, decreasing N:C ratio, and transition from basophilic cytoplasm to pink granulated cytoplasm in mature megakaryocytes (Table 27.7). Cytologically, megakaryocyte precursors are clearly distinguished by their large size and multinucleation or multilobular nuclei, although megakaryoblasts are often not distinguishable from other immature or blast cells in marrow (Fig. 27.24). Megakaryocytes and their precursors are typically closely associated with the unit particles or may be in peripheral areas of the sample, in more hemodilute regions or near the edges of a smear. Megakaryocytic precursors are often not evenly distributed within a sample; therefore for assessment of cellularity within the megakaryocyte population, an estimate should be made on the basis of evaluation of multiple particles on multiple slides. Core biopsy may provide more accurate samples for assessment of megakaryocyte numbers, particularly if there is a suspicion for megakaryocytic hypoplasia on cytological evaluation. Because of the large size of megakaryocytes, an estimate of cellularity on cytology is often performed at lower magnification (10× objective) than for the other lineages (typically 40×, 50×, and/or 100× objective). In a normal marrow, several megakaryocytes are expected per unit particle on average with a strong predominance of mature megakaryocytes (>80%).4,10,25 The literature varies with regard to specific expected numbers of megakaryocytes, but generally 5 to 10 megakaryocytes are expected per 10× low-power field (lpf) with fewer than 3 to 5 megakaryocytes suggesting megakaryocyte hypoplasia and greater than 10 to 20 megakaryocytes per 10× field suggesting megakaryocyte hyperplasia.4,10,12,18 In more general terms, almost no megakaryocytes is considered “too few”; several per field is considered “adequate”; and

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Fig. 27.17  Bone marrow core biopsy from a dog with precursor-targeted immune-mediated anemia (PIMA) and secondary myelofibrosis. Severe erythroid hyperplasia with phagocytosis of erythroid precursors by macrophages within the marrow supports a diagnosis of PIMA. Histologically, this phagocytic activity by macrophages can be seen from low magnification as areas of vacuolation within the marrow (arrows). On closer inspection, macrophage nuclei (denoted by *) can be seen with surrounding clear cytoplasmic phagocytic vacuoles containing erythroid precursor cells. This may not be evident in all cases of PIMA, but when this feature is identified, it is a useful diagnostic aid. In this case, there is secondary myelofibrosis. The eosinophilic streaming quality within the marrow reflects the increased connective tissue (top and right middle). With myelofibrosis, there is often accompanying bony remodeling with endosteal new bone formation (note the irregular contour to the less mature woven bone extending from the trabecular bone [top]) (H&E stain, original magnification 100× [top], 400× [lower left and right middle], 1000× [bottom right] ).

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Fig. 27.18  Erythroid, myeloid, and megakaryocyte dysplasia on bone marrow cytology. Top: Erythroid dysplasia in two cats. Left: Megaloblast with nuclear to cytoplasmic asynchrony of maturation, binucleate metarubricyte (feline leukemia virus [FeLV]–associated); Right: Binucleate megaloblastic cell with multiple atypical rubricytes with nuclear to cytoplasmic dysmaturation (myelodysplastic syndrome with erythroid predominance [MDS-Er]). In megaloblastic cells, note the altered gray/polychromatophilic coloration to the cytoplasm with decreased N:C ratio and immature chromatin pattern. Middle: Myeloid dysplasia in two dogs. Left: Multiple myeloblasts with vacuolated cytoplasm (zonisamide toxicity). Right: Ring-shaped nucleus in a myeloid cell with few cytoplasmic vacuoles (mild secondary myelodysplasia accompanying severe myeloid hyperplasia). Bottom: Megakaryocyte dysplasia in a cat with FeLV-associated dysplastic changes. Left: Dwarf megakaryocyte with hypolobular nucleus and high N:C ratio. Right: Basophilic megakaryocyte with multiple distinct nuclei (Wright-Giemsa stain, original magnification 1000× [top], 1000× [middle], and 500× [bottom] ).

many megakaryocytes is considered “increased.” Poor-quality or low-cellularity cytological smears often have fewer megakaryocytes because these cells are typically associated with unit particles. On core biopsies, megakaryocytes are distinguishable by their large size (Fig. 27.25). Similar to cytological evaluation, megakaryocytes are often not evenly distributed within the marrow on a core biopsy, and therefore the estimate is based on evaluation of multiple marrow spaces across the sample. Megakaryocytes may appear randomly arranged within a core biopsy but are located adjacent to sinusoids to allow for release of platelets into circulation (see Fig. 27.2). In

general, 1 to 3 megakaryocytes per 2 to 3 high-power fields (hpf; 40×) is considered to be within the reference interval.41 It should be noted that cross-sections of megakaryocytes in histological section can lack a nuclear component, given the high cytoplasmic volume, and therefore megakaryocytes may be missed if these larger eosinophilic cytoplasmic portions are not recognized as megakaryocytes (see Fig. 27.15). In addition, care should be taken to not overinterpret the dysplastic features in megakaryocytes on histopathology, because the convoluted nuclear contour can be quite pronounced with this modality. Both cytologically and histologically, megakaryocytes must be distinguished

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TABLE 27.6  Myeloid Lineage Maturation Name of Cell Stage

Cytological Description

Myeloblast

Large-size cell; high nuclear-to-cytoplasmic ratio; small volume of light to medium blue cytoplasm without granules (type I myeloblast), or with small pink-magenta primary granules in low numbers (<15 granules; type II myeloblast) or high numbers (type III myeloblast); large-size, round nucleus with visible nucleoli and fine lacy chromatin pattern

Progranulocyte (promyelocyte)

Large-size cell; small to moderate volume of light-blue cytoplasm with numerous small pink-magenta primary granules that may also be superimposed on the nucleus; medium- to large-size, round, slightly eccentric nucleus without nucleoli and with a fine to slightly coarse chromatin pattern

Myelocyte

Medium-size cell; small to moderate volume of clear to slightly pale-blue cytoplasm without the primary granules (but with secondary bright-pink granules in eosinophils and pale gray-lavender granules in basophils; secondary granules not visible in neutrophils); small- to medium-size, round to oval, eccentrically located nucleus without nucleoli and with coarse chromatin pattern

Metamyelocyte

Small- to medium-size cell; small volume of clear to slightly pale-blue cytoplasm (with secondary granules visible in eosinophils and basophils but not in neutrophils); small-size, indented/reniform nucleus without nucleoli and with coarse stippled chromatin pattern

Band granulocyte

Small-size cell; small volume of clear to slightly pale-blue cytoplasm (with secondary granules visible in eosinophils and basophils but not in neutrophils); small-size horseshoe- or S-shaped nucleus with coarse clumped chromatin pattern

Mature granulocyte

Small-size cell; small volume of clear cytoplasm (with secondary granules visible in eosinophils and basophils but not in neutrophils); small-size, tightly segmented/lobulated nucleus (often bilobed in eosinophils and less tightly segmented in basophils) with densely clumped chromatin pattern

from other multinucleate cells in marrow, including osteoclasts, multinucleate giant macrophages, or multinucleate tumor cells (Fig. 27.26). Assessment within the megakaryocytic lineage, as for any cell line, should include evaluation of cell numbers/proportion (hypoplasia versus normal versus hyperplasia), maturation assessment, and evaluation of morphology, including any evidence of dysplasia.26,42,43 The megakaryocytic lineage development follows a pyramid-shaped distribution, with only few megakaryoblasts and promegakaryocytes, a greater number of basophilic megakaryocytes, and the largest number of mature megakaryocytes (see Box 27.7). If less than 50% of the megakaryocytic cells are mature, with increased promegakaryocytes or basophilic megakaryocytes, this indicates a left shift and supports a regenerative response.

Megakaryocytic Hyperplasia Megakaryocytic hyperplasia is the expected regenerative response to some causes of thrombocytopenia, including immune-mediated destruction of platelets; to causes of increased platelet consumption, including intravascular coagulation, hypersplenism, and vascular injury; or to an infectious etiology, such as rickettsial disease.

Cytological Image

Megakaryocytic hyperplasia is also expected alongside thrombocytosis, as a reactive response to chronic inflammation; with iron deficiency; in response to some therapeutic drugs (vincristine)44; or occasionally with megakaryocytic neoplasia (megakaryocytic leukemia or essential thrombocythemia).4,18,45 (Box 27.11). A left shift may be seen with megakaryocytic hyperplasia with increased numbers of basophilic megakaryocytes (see Fig. 27.24). This left shift may be more difficult to identify on core biopsy with histopathology compared with cytology, but a pale-gray coloration to the cytoplasm and a more open chromatin pattern can be helpful on a core biopsy (see Fig. 27.25). 

Megakaryocytic Hypoplasia Megakaryocytic hypoplasia can be seen with panhypoplasia in the marrow as with causes of aplastic anemia, but can also rarely be a selective process (see Box 27.11). Selective megakaryocytic hypoplasia may occur with immune-mediated destruction of megakaryocytes, also referred to as amegakaryocytic thrombocytopenia. Selective hypoplasia may also occur with drug effects or infectious etiologies. More commonly, these cause hypoplasia in multiple lineages but can affect the megakaryocytes first in some cases.4,18,44

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BOX 27.10  Causes of Myeloid/Granulocytic

Hyperplasia and Myeloid/Granulocytic Hypoplasia Myeloid/Granulocytic Hyperplasia

Myeloid/Granulocytic Hypoplasia

Effective Granulocytic Hyperplasia • Inflammatory conditions • Bacterial or other infection • Immune-mediated conditions • Tissue necrosis • Neoplasia • G-CSF administration • Paraneoplastic process (neoplasm producing G-CSF or other growth factor) •  Recovery from neutropenia with cyclical hematopoiesis in collie dogs • Leukocyte adhesion deficiency • CML

Aplastic Anemia (see Box 27.5) Selective Granulocytic Hypoplasia • Immune-mediated neutropenia (targeting of early-stage precursors) • Drug administration • Infection (parvovirus/panleukopenia, FeLV in cats, early septicemia with depletion of storage pool) • Cyclical hematopoiesis in collie dogs (transient)

Ineffective Granulocytic Hyperplasia • Immune-mediated neutropenia (targeting of late-stage precursors) • Hematopoietic neoplasia • FeLV infection (in cats) • Trapped neutrophil syndrome in Border Collie Eosinophil Hyperplasia • Parasitism • Allergy/hypersensitivity • Inflammatory conditions • Mast cell tumor; other paraneoplastic • Hypereosinophilic syndrome •  Hematopoietic neoplasia (AML, MDS, CML) AML, acute myeloid leukemia; CML, chronic myelogenous leukemia; FeLV, feline leukemia virus; G-CSF, granulocyte–colony stimulating factor; MDS, myelodysplastic syndrome.

Bone marrow evaluation specifically regarding assessment of megakaryocytic lineage is best utilized to differentiate between various causes of thrombocytopenia and is not usually indicated in thrombocytosis unless there is suspicion for megakaryocytic or other hematopoietic neoplasia. In cases of thrombocytopenia, if there is megakaryocytic hyperplasia in the marrow, this suggests either increased consumption of platelets in the periphery or platelet destruction as with immunemediated thrombocytopenia (IMT). If there is megakaryocytic hypoplasia in the marrow, suppression of production of megakaryocytes is the likely cause. Platelet sequestration in the spleen should not affect megakaryopoiesis. 

Megakaryocyte Dysplasia Dysmegakaryocytopoiesis, analogous to dyserythropoiesis and dysgranulopoiesis in the other lineages, refers to abnormal megakaryocyte maturation and/or morphology. Left shift with maturation arrest can be seen with immune-mediated destruction of megakaryocytes. Dysplastic features in megakaryocytes may include hypolobularity or hyperlobularity of nuclei, multiple separated nuclei in a more mature cell, prominent cytoplasmic vacuolation, or dwarf megakaryocytes (see

Fig. 27.18). Dysplasia can be seen with severe megakaryocytic hyperplasia; immune-mediated conditions; neoplasia; drugs; hereditary conditions, such as macrothrombocytopenia in Cavalier King Charles Spaniel dogs; or MDS/AML.4 One unique feature that can occur in megakaryocytes, and can be seen on cytology or histopathology, is emperipolesis, which is the movement of blood cells (most commonly neutrophils) through the megakaryocyte cytoplasm (see Fig. 27.25). The significance in veterinary species is unknown, but there may be an association with inflammation.18,33 

OTHER CELL TYPES Additional cell types expected in bone marrow include lymphocytes, plasma cells, macrophages, mast cells, and stromal elements. These cell types are typically seen to some degree in normal bone marrow, but under certain disease states each of these cell types may become more prominent or increased in number (Box 27.12). With panhypoplasia of bone marrow, lack of hematopoietic cells often leads to apparent increased proportions of these background cellular elements (Fig. 27.27). With routine cytological bone marrow assessment, lymphocytes, plasma cells, and macrophages are typically counted as separate categories in parallel with the differential cell count of the myeloid and erythroid cells but are not included in the M:E ratio.

Lymphocytes Lymphocytes in bone marrow are most commonly small mature lymphocytes, although a few reactive-appearing or less mature lymphocytes may be seen because a mild degree of lymphopoiesis can occur in bone marrow. Under normal circumstances, lymphocytes are expected to be less than 5% of nucleated cells in marrow in dogs and less than 10% in cats, although some reported reference intervals allow for higher numbers (10% in dogs and 20% in cats).4,18,31 Lymphocytes on cytology appear similar to those in blood or elsewhere in the body, but on histopathology, small lymphocytes and late-stage erythroid precursor cells may be difficult to distinguish46,47 (Fig. 27.28). Lymphoid aggregates are rare under normal circumstances but can be more frequent with chronic antigenic stimulation, such as with immune-mediated or infectious conditions. On cytology, lymphoid aggregates may be seen as a regional increase in lymphocyte numbers, whereas on histopathology their architecture is more clearly apparent (Fig. 27.29). Increased numbers of lymphocytes can vary in significance, depending on the size and maturity of the lymphocytes in addition to their pattern and distribution in the marrow. In some cases, follow-up advanced diagnostic testing, such as immunohistochemistry (IHC; core biopsy sample), flow cytometry (liquid bone marrow sample), or polymerase chain reaction (PCR) for antigen receptor rearrangement (PARR; bone marrow smears or formalin-fixed paraffin-embedded tissue), may be necessary to further investigate for a potential neoplastic lymphoid population (see Box 27.3). Increased small lymphocytes can be seen with antigenic stimulation of any cause, including with infectious etiology (particularly tickborne disease, such as ehrlichiosis18,31), inflammatory conditions, or with immune-mediated disorders (including PIMA or PRCA in cats and IMHA/IMT/ PIMA in dogs38,39), or can occur with small cell lymphoma or bone marrow involvement with chronic lymphocytic leukemia (CLL; see Box 27.12; Fig. 27.30). Classic canine and feline CLL of T-lymphocyte origin typically arises within the spleen, and therefore bone marrow involvement is often not a feature. The majority of dogs with CLL have a CD3 +, CD4− and CD8+ phenotype, whereas cats with CLL are typically CD3+,CD4+, and CD8−.48,49 On histopathology, the pattern and distribution of lymphocytes can aid in the distinction between a reactive and neoplastic population, but if there are only subtly increased lymphocyte numbers, evenly dispersed throughout the marrow, this

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Fig. 27.19  Bone marrow cytology in three cats with myeloid hyperplasia. In each case, there is an increased M:E ratio with complete maturation that is orderly to mildly left-shifted with mildly increased immature forms. Basophil precursors can be identified by their deep purple cytoplasmic granules (arrowheads), and eosinophil precursors can be identified by their bright pink cytoplasmic granules (arrow). The few erythroid cells present have a more basophilic staining pattern to the cytoplasm and denser chromatin compared with the predominant myeloid population (Wright-Giemsa stain, original magnification 1000× [top] and 500× [bottom] ).

distinction may not be possible without additional diagnostic testing.12,46-48 A mixed population of lymphoid cells with IHC or clear B-lymphocyte aggregates would support a reactive process, whereas a sheetlike expansion or well-dispersed proliferation of T lymphocytes would support a neoplastic process. In some cases of lymphoma metastatic to the marrow, there may be a paratrabecular distribution of the lymphoid cells. If significantly increased numbers of a monomorphic population of immature or large-sized lymphocytes are seen, this would support a neoplastic lymphoid population (Fig. 27.31). Distinction between lymphoid leukemia and lymphoma requires correlation to CBC findings and other potential sites of involvement (lymph nodes, internal organs), and potentially flow cytometry. With a robust lymphoid proliferation, particularly with lymphoid neoplasia, there are often background cytoplasmic fragments, referred to as lymphoglandular bodies. These fragments are more typical of lymphoid neoplasia than of other hematopoietic proliferations (see Fig. 27.31). 

Plasma Cells Plasma cells in bone marrow are typically in low numbers (<2%– 3% of nucleated cells) in dogs and cats and have similar morphology to elsewhere in the body4,31 (see Fig. 27.28). Occasional Mott cells may be seen, which are plasma cells that contain cytoplasmic immunoglobulin inclusions (Russell bodies), or occasional flame cells, which have a peripheral pink cytoplasmic fringe of immunoglobulin material. With cytology, plasma cells can be unevenly distributed and are often aggregated within unit particles (see Fig. 27.27). On histopathology, distinction between plasma cells and later-stage erythroid cells can be difficult, but typically plasma cells are identified by their eccentric nuclei, more abundant basophilic cytoplasm, and eosinophilic perinuclear clear zone (Fig. 27.32). Histologically, plasma cells may be in perivascular aggregates. Increased plasma cells in bone marrow can be seen with causes of antigenic stimulation, similar to lymphocytosis. These include immune-mediated conditions; infectious etiologies, such as feline infectious peritonitis (FIP) in cats; chronic ehrlichiosis; leishmaniasis;

Fig. 27.20  Bone marrow core biopsy from two dogs with myeloid hyperplasia. In both cases, there is severe hypercellularity with an increased M:E ratio. On histopathology, myeloid lineage cells are identified by their convoluted lobulated nuclei, particularly in later-stage precursors (bottom right). Earlier myeloid precursors arise adjacent to the bone (paratrabecular) (top and bottom left, note the cells adjacent to bony trabeculae). Compared with early erythroid cells, early myeloid cells (bottom left) are larger and have increased cytoplasmic volume with eosinophilic granularity and a more open chromatin pattern (H&E stain, original magnification 400× [top], 400× [bottom right], and 1000× [bottom left] ).

Fig. 27.21  Bone marrow cytology from a dog with immune-mediated neutropenia. Immune-mediated neutropenia may present as ineffective granulopoiesis characterized by myeloid hyperplasia in the marrow with a peripheral neutropenia. Phagocytosis of neutrophils or other myeloid cells by macrophages can aid in confirmation of precursor destruction. Note the eccentric nucleus (arrowhead) of this highly phagocytic macrophage with cytoplasmic neutrophils and myelocytes (Wright-Giemsa stain, original magnification 1000×).

Fig. 27.22  Bone marrow cytology from a dog with a mast cell tumor. Eosinophil hyperplasia in bone marrow can be seen secondary to mast cell neoplasia. Note the brightly colored pink granules in the eosinophil myelocytes, metamyelocytes, band eosinophils, and mature eosinophils. Metastatic mast cell tumor involving bone marrow can be easily identified if there are sheets or aggregates of mast cells cytologically. However, in some cases, only low numbers of neoplastic mast cells may be seen (arrow). If these cells are atypical in morphology with larger cell size (as pictured here), altered granularity, or atypical nuclear features, this can aid in the interpretation of metastatic neoplasia (Wright-Giemsa stain, original magnification 250×).

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myeloid hyperplasia; multiple myeloma/plasma cell neoplasia; or aplastic anemia, although this may reflect a proportional increase resulting from lack of other hematopoietic cells, rather than a true plasmacytosis4,31 (see Box 27.12; see Figs. 27.32; Fig. 27.33). Distinction between plasma cell neoplasia and reactive plasmacytosis can be challenging in some cases, particularly because multiple myeloma may have well-differentiated and mature plasma cell morphology. These cases may be aided by correlation with additional diagnostic testing results supporting multiple myeloma, including serum protein electrophoresis, radiography to detect lytic bone lesions, assessment for Bence Jones proteinuria, or by ruling out infectious diseases, such as rickettsial infections. Large expansive sheets of plasma cells, even if well differentiated, would suggest multiple myeloma.  Fig. 27.23 Bone marrow cytology from a greyhound dog with gray eosinophils. This bone marrow was sampled during complete staging for a mast cell tumor. Although neoplastic mast cells were not identified, there is eosinophil hyperplasia in the bone marrow. Greyhounds, other sighthounds, and rarely other breeds of dog may have gray eosinophils (arrows). These eosinophils have normal function, but have clear staining of the cytoplasmic secondary granules, rather than the typical bright pink color expected in eosinophils. These cells need to be correctly identified in peripheral blood or bone marrow as eosinophils, rather than interpreted as toxic change within neutrophils or neutrophil precursors (Wright-Giemsa stain, original magnification 1000×).

Mast Cells Mast cells can be seen in low numbers in normal bone marrow on cytology, particularly in unit particles, and are identified by their distinctive purple cytoplasmic granules. With histopathology, mast cells are often too subtle to be seen in normal marrow. Mast cells may be in increased numbers with aplastic anemia, myelofibrosis, inflammatory processes, alongside other neoplasia, such as lymphoma, with various causes of regenerative or nonregenerative anemia, or with metastatic mast cell tumor4,31,50 (see Box 27.12; see Figs. 27.22 and 27.27). Metastatic mast cell tumor can be distinguished from hyperplasia by increased atypia within the mast cell population, dense aggregates of mast cells best seen with histopathology,

TABLE 27.7  Megakaryocyte Lineage Maturation Name of Cell Stage

Cytological Description

Megakaryoblast

Large-size cell; high nuclear-to-cytoplasmic ratio; small volume of deep-blue cytoplasm with peripheral blebbing and possibly few clear vacuoles; large-size, round nucleus with visible nucleoli and fine granular chromatin pattern

Promegakaryocyte

Very-large-size cell; moderate volume of medium- to deep-blue cytoplasm with peripheral blebbing and possibly few clear vacuoles; 2–4 medium-size, round nuclei without nucleoli and with a coarse chromatin pattern

Basophilic megakaryocyte Extremely large-size cell; small to moderate volume of medium-blue cytoplasm with peripheral blebbing and possibly few clear vacuoles; large multilobulated nucleus with coarse to dense chromatin pattern

Mature (granular) megakaryocyte

Extremely large-size cell (50–200 μm diameter); high volume of palepink cytoplasm with numerous fine pink-magenta granules; large multilobulated nucleus with dense clumped chromatin pattern

Cytological Image

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Fig. 27.24  Bone marrow cytology from three dogs with megakaryocyte hyperplasia. Megakaryocytes are identifiable from low magnification because of their large size. Megakaryocytes are often seen in close association with unit particles or can be seen in more hemodiluted areas of the slide. In each of the three cases, there is an increased number of megakaryocytes as well as a left shift with more frequent basophilic megakaryocytes (see Table 27.7 for description of mature versus basophilic megakaryocytes) (Wright-Giemsa stain, original magnification 100× [top] and 500× [bottom] ).

or effacement of marrow elements by sheets of mast cells. With histopathology, if a mast cell population is suspected but is difficult to confirm, special stains, such as Giemsa or toluidine blue, can be used to highlight the metachromatic cytoplasmic granules in the mast cells. 

Macrophages/Histiocytes Macrophages are normally seen in bone marrow in low numbers (<1%–2% of nucleated cells4) and are morphologically typical of macrophages elsewhere in the body. Macrophages may have cytoplasmic vacuolation and phagocytized cytoplasmic debris or hemosiderin pigment, which may aid in distinction from other myeloid cells. Macrophages are normal constituents of erythropoietic islands in which a macrophage is surrounded by developing erythroid precursor cells to provide nutrients and phagocytize extruded nuclei from metarubricytes (see Fig. 27.13). Macrophages may be increased with necrosis; inflammation, including that caused by an infectious etiology; immune-mediated conditions; neoplasia; or histiocytic proliferative disorders (histiocytic sarcoma, neoplastic or non-neoplastic

hemophagocytic syndrome, or reactive histiocytosis)4,31,51 (Figs. 27.34 and 27.35; see Box 27.13). A minor component of phagocytosis by macrophages can be seen in normal bone marrow as a result of phagocytosis of senescent RBCs, extruded metarubricyte nuclei, and removal of apoptotic cellular debris. Increased phagocytic activity by macrophages can be seen secondary to necrosis, inflammation, infection (mycobacteriosis, leishmaniasis, histoplasmosis, cytauxzoonosis), infection by hemoparasites (Babesia spp., Mycoplasma spp.), immune-mediated destruction of precursor cells, dyserythropoiesis, severe erythroid hyperplasia, blood transfusion, neoplasia, or hemophagocytic histiocytic proliferative disorders (hemophagocytic histiocytic sarcoma or neoplastic or non-neoplastic hemophagocytic syndrome)4 (see Box 27.13; see Figs. 27.16, 27.17, and 27.34). 

Stromal Elements Normal stromal elements that comprise bone marrow and associated bone tissue include bone trabeculae with their embedded osteocytes in lacunae and flattened surface bone-lining cells, adipocytes within the

CHAPTER 27 

Fig. 27.25  Core biopsy samples from three dogs with megakaryocyte hyperplasia. In all three cases, there is an increase in megakaryocyte numbers, and left shift is also evident (top and bottom left). On histopathology, megakaryocytes are easily identified by their large size. Mature megakaryocytes have eosinophilic cytoplasm and a tightly lobular nucleus with dense to clumped chromatin. Basophilic megakaryocytes have a blue-gray quality to the cytoplasm and a more open chromatin pattern of the nucleus (several in top, right two megakaryocytes in bottom left). Emperipolesis is a unique feature in megakaryocytes that can be seen with cytology or core biopsy. Emperipolesis is a movement of neutrophils through the megakaryocyte cytoplasm (bottom right), and is a different process than actual phagocytosis of neutrophils. There may be an association with inflammation (H&E stain, original magnification 400× [top], 400× [bottom right], and 1000× [bottom left] ).

medullary spaces, and accompanying supportive interstitial stromal cells, capillaries, and larger blood vessels. 

Osteoblasts and Osteoclasts Osteoblasts can be seen in low numbers in normal marrow, but with mature quiescent bone, osteoblasts are typically not readily apparent. Osteoblasts are large rounded to elongated cells that have a perinuclear clear zone, reminiscent of plasma cells; however, osteoblasts are much larger than plasma cells and have a less condensed chromatin pattern. Osteoblasts may be increased with bone remodeling, which can accompany myelofibrosis or can be seen with inflammation, infection, or neoplasia and in young animals with active bone modeling, chronic renal disease, metabolic bone disease, or microfracture/ trauma. On histopathology, osteoblasts are clearly apparent as plump, stellate-shaped cells lining up along bone trabeculae, often associated with endosteal new bone proliferation. Osteoclasts are multinucleate cells that resorb osteoid matrix with bone remodeling and therefore are often not seen in quiescent bone but can be increased with similar causes of bone reactivity/remodeling or in young animals. Osteoclasts must be differentiated from megakaryocytes, as discussed previously (see Fig. 27.26). On histopathology, osteoclasts can be seen in areas of remodeling, in scalloped areas of the bone referred to as Howship’s lacunae. Osteosclerosis, such as that associated with pyruvate kinase deficiency, and osteopetrosis caused by a hereditary osteoclast defect are syndromes where there is expansion of the bone tissue with

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Fig. 27.26 Bone marrow cytology; megakaryocyte and osteoclast. Megakaryocytes need to be distinguished from osteoclasts cytologically. Mature megakaryocytes have a single, condensed, multilobulated nucleus and eosinophilic, granular cytoplasm (left); osteoclasts have similar cytoplasmic color and granularity but have multiple separate similarly sized nuclei (right); and osteoclasts are uncommonly seen in bone marrow aspirates and may be associated with bone modeling/ remodeling, such as in young animals or animals with chronic renal disease, metabolic bone disease, inflammation, microfracture/trauma, or neoplasia (Wright-Giemsa stain, original magnification 240×). (Reprinted with permission from Grindem CB. Bone marrow biopsy and evaluation. Vet Clin Small Anim. 1989;19[4]:680.)

narrowing of marrow spaces leading to anemia or other cytopenias. These conditions are very rare.4 Stromal spindle cells and capillaries are often more prominent in hypocellular marrow samples on cytology because of relative lack of hematopoietic cells superimposed on these elements. Stromal cells and associated extracellular matrix may also be increased with myelofibrosis, which most typically requires core biopsy with histopathology for confirmation, although in some cases cytological findings may be suggestive (Fig. 27.36). 

Myelofibrosis Myelofibrosis is often accompanied by hematopoietic hypocellularity. Some retained foci of persistent hematopoiesis or compensatory hyperplasia of marrow elements commonly exist, along with increased hemosiderin stores (see Fig. 27.36). Myelofibrosis is rarely a primary form of myeloproliferative neoplasia and is much more commonly secondary (Box 27.14). In dogs, causes include immune-mediated disease, especially PIMA; neoplasia; inflammation, infection, vascular injury or necrosis in the marrow; drug-associated or with irradiation; pyruvate kinase (PK) deficiency; or an idiopathic change.4,18,31,52 Myelofibrosis is less common in cats but can occur with immune-mediated anemia, FIP, chronic renal failure, or neoplasia, such as MDS/AML.52 Myelofibrosis is most commonly accompanied by nonregenerative anemia with normal neutrophil and platelet counts, but neutropenia and/or thrombocytopenia can be seen in more severe cases. Ovalocytes and dacryocytes can be seen in circulation in some cases but are not specific for myelofibrosis.27,53 

Myelonecrosis Myelonecrosis can be seen in some cases and is typically easier to confirm with histopathology than with cytology, although necrosis may be suggested with cytology on the basis of amorphous background debris and smudged cellular details. On histopathology, necrosis can be seen as regional hypereosinophilia with nuclear and cellular debris and is often accompanied by inflammation, edema, fibrin accumulation, and/or hemorrhage, and this may corroborate the suspicion and

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differentiate the changes from an artifact of sample collection or crush artifact (Fig. 27.37). Causes of myelonecrosis are similar to those of necrosis elsewhere in the body and include ischemia or hypoxemia, inflammation or infection, drug-related effects, neoplasia, vascular abnormality, immune-mediated processes, or severe hematopoietic cell injury4,31,54 (Fig. 27.38). 

Serous Atrophy of Fat Gelatinous transformation or serous atrophy of fat in the marrow refers to accumulation of mucoid pink matrix (Alcian blue positive) with withered adipocytes. This change is uncommon but can be seen with chronic anorexia/starvation or cachexia. 

INFLAMMATION/INFECTION IN THE MARROW Detection of inflammation in bone marrow can be difficult in some cases because of the overlap in cell types normally in the marrow and inflammatory cells, including neutrophils, histiocytoid/monocytic

BOX 27.11  Causes of Megakaryocytic

Hyperplasia and Megakaryocytic Hypoplasia Megakaryocytic Hyperplasia

Megakaryocytic Hypoplasia

With Thrombocytopenia • Immune-mediated destruction of platelets (IMT) • Increased platelet consumption (intravascular coagulation, hypersplenism, vascular injury/abnormality) • Infectious etiology (rickettsial disease) • Megakaryocytic leukemia

Aplastic Anemia (see Box 27.5)

With Thrombocytosis • Drug administration (vincristine) • Iron deficiency • Chronic inflammation • Megakaryocytic neoplasia (megakaryocytic leukemia or essential thrombocythemia)

Selective Megakaryocytic Hypoplasia • Amegakaryocytic thrombocytopenia (immune-mediated destruction of megakaryocytes) • Drug administration • Infectious causes

cells, lymphocytes, and plasma cells. Particularly with core biopsy with histopathology, there may be additional evidence for inflammation to corroborate a suspicion for increased inflammatory cells. For acute inflammatory conditions, this corroborative evidence may include edema, fibrin accumulation, necrosis, and/or hemorrhage. Additional evidence to support chronic inflammatory conditions may include fibrosis, granulomatous foci, or bony changes. When there is granulomatous inflammation, in particular, careful investigation for etiological agents is recommended. Common organisms causing granulomatous inflammation in the bone marrow include Mycobacterium spp., fungal infections and in particular systemic mycoses, or Leishmania spp. (see Fig. 27.32; Figs. 27.39 to 27.41). If routine staining does not identify organisms, additional special stains can be considered to further assess for Mycobacterium spp. (acid fast or Fites-Faraco stain) or fungal organisms (PAS or GMS stains) (see Box 27.3). Acute inflammation in the marrow can be caused by infection, including bacterial, protozoal, rickettsial, fungal, or other agents, as well as necrosis, drug-related effects, neoplasia, vascular abnormality, or immune-mediated processes. Other infectious agents that can be discovered in bone marrow include Cytauxzoon felis; other hemoparasites, such as Babesia spp. and Mycoplasma spp.; rickettsial diseases; and other multisystemic protozoal infections, such as Toxoplasma spp.55 In cases of hemoparasites, such as Babesia spp. or Mycoplasma spp., peripheral blood smear evaluation or PCR testing are more likely to confirm the diagnosis compared with bone marrow evaluation. C. felis has a blood phase of infection with merozoites in RBCs but also has a tissue phase of infection, with formation of large schizonts in macrophages, and this component may be captured with bone marrow evaluation (Fig. 27.42). Ehrlichial infection typically has an acute hypercellular phase in the marrow, but with chronic infection, dogs can develop pancytopenia with severe bone marrow hypocellularity, often with accompanying plasmacytosis and mastocytosis in the marrow.29,31 Ehrlichial organisms are rarely seen in bone marrow myeloid cells. Some viral infections, including parvovirus and panleukopenia virus infections, can affect the marrow and are typically associated with bone marrow hypoplasia at an early stage of infection with concurrent neutropenia and subsequent marrow recovery. FeLV retroviral infection in cats commonly has hematopoietic effects, with potential for panhypoplasia/aplastic anemia, PRCA, MDS, or AML. Because of the wide range of hematopoietic disturbances that have been associated with FeLV

BOX 27.12  Causes of Increased Lymphocytes, Plasma Cells, and Mast Cells Increased Lymphocytes

Increased Plasma Cells

Increased Mast Cells

• Small lymphocytes • Antigenic stimulation (lymphoid aggregates can also be seen) • Immune-mediated conditions (particularly nonregenerative IMHA or PRCA in cats and IMHA/IMT, PIMA in dogs) • Infectious etiology (tickborne diseases, such as ehrlichiosis) • Inflammatory conditions • Small cell lymphoma or chronic lymphocytic leukemia • Large immature lymphocytes • Lymphoma • Acute lymphoblastic leukemia

• Antigenic stimulation • Immune-mediated conditions • Infectious etiology (FIP in cats, chronic ehrlichiosis, leishmaniasis) • Alongside myeloid hyperplasia • Multiple myeloma/plasma cell neoplasia • Aplastic anemia

• • • • • •

Aplastic anemia Myelofibrosis Inflammation Metastatic mast cell tumor Other neoplasia (lymphoma) Anemia (regenerative or nonregenerative)

FIP, feline infectious peritonitis; IMHA, immune-mediated hemolytic anemia; IMT, immune-mediated thrombocytopenia; PIMA, precursor-targeted immune-mediated anemia; PRCA, pure red cell aplasia.

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497

infection, FeLV testing is recommended in any cat with unexplained hematological abnormalities. It should be noted that FeLV testing on bone marrow itself may reveal an infection that was not identified via testing of the peripheral blood. PCR testing on bone marrow may identify latent infection not captured with enzyme-linked immunosorbent assay (ELISA) or immunofluorescent antibody assay (IFA) testing on blood.55 

NEOPLASIA

Fig. 27.27  Bone marrow cytology from a dog; prominent plasma cells and mast cells. Marrow is overall hypocellular, with only few groups of myeloid cells adjacent to unit particles. There is increased prominence of well-differentiated mast cells and plasma cells, often in aggregates or closely associated with unit particles. Few small lymphocytes are also noted. With hypocellular marrow of any cause, resident plasma cell, lymphocyte, and mast cell populations may be more prominent. In some cases, such as with chronic ehrlichiosis, there may be actual hyperplasia of the plasma cell and mast cell components (Wright-Giemsa stain, original magnification 500×, inset: 1000×).

Neoplasia in bone marrow can be either primary or metastatic to involve the marrow56 (Table 27.8). Primary neoplasia affecting the bone marrow may be of hematopoietic origin (myeloproliferative, lymphoid, histiocytic, or undifferentiated) or nonhematopoietic (primary bone/stromal tumors that secondarily involve the marrow spaces).6 Bone/stromal tumors, such as osteosarcoma, chondrosarcoma, and fibrosarcoma, are not typically sampled under the guise of bone marrow evaluation because these types of neoplasia are not typically associated with cytopenias or atypical circulating cells, and therefore these are best considered with bone57 and soft tissue tumors elsewhere in this text. Leukemia is defined as neoplasia arising within bone marrow or blood, and most leukemias have a circulating component. Subleukemic or aleukemic leukemias are possible and affect bone marrow but are not seen in circulation. Acute leukemia refers to an increased immature “blast” cell component with a variable amount of maturation/

Fig. 27.28 Bone marrow cytology from a dog with increased small lymphocytes and plasma cells. Well-differentiated plasma cells (PC) and small lymphocytes (L) are indicated. Plasma cells can be distinguished from rubricytes or metarubricytes (MR) by their increased cytoplasmic volume, eccentric nucleus, and perinuclear clear zone. Lymphocytes can be distinguished from rubricytes and metarubricytes by their scant cytoplasm and more finely clumped chromatin pattern containing fewer clear spaces. Lymphocytes can be distinguished from free metarubricyte nuclei (FN) because these free nuclei have a darker, more completely filled-in chromatin pattern and lack a rim of cytoplasm (Wright-Giemsa stain, original magnification 1000×).

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differentiation. Acute leukemias can be further divided into those of myeloid and lymphoid origins. Myeloid leukemias are additionally subdivided into undifferentiated; myeloblastic, with or without maturation; promyelocytic; monoblastic/monocytic; myelomonocytic; erythroblastic; and megakaryocytic variants (Figs. 27.43 and 27.44). Chronic leukemia refers to a neoplasm with a predominance of mature-appearing cells with a retained pyramidal distribution of cells within that lineage, and these neoplasms are now referred to as myeloproliferative neoplasms (MPNs).56 In acute leukemias, typically there is rapid progression of disease, whereas chronic leukemias typically have

Fig. 27.29 Bone marrow core biopsy from a cat with precursor-targeted, immune-mediated anemia (PIMA). Note the large lymphoid aggregate (dark rounded basophilic area at left aspect of image) composed of predominantly small lymphocytes. In the background, there is evidence of erythroid hyperplasia with left shift and megakaryocyte hyperplasia. This cat had concurrent nonregenerative anemia with findings supportive of ineffective erythropoiesis associated with PIMA. Lymphoid aggregates can be seen rarely in normal animals but may be more frequent with chronic antigenic stimulation, and particularly with immune-mediated processes. On cytology, increased numbers of small lymphocytes may be noted in some areas of the sample, whereas on core biopsy the architecture of the lymphoid aggregate is apparent (H&E stain, original magnification 400×).

a more slowly progressive, chronic clinical course. Primary hematopoietic neoplasia in bone marrow can be multifocal in early cases or can be diffusely effacing within the marrow, referred to as myelophthisis. General accepted guidelines for an expanded cell population that would suggest a neoplasm include: greater than 20% blast cells for AML, greater than 30% lymphoid cells for lymphoma/lymphoid leukemia, and greater than 15% plasma cells for plasma cell neoplasia/ multiple myeloma.4,56 Large sheets or aggregates of other cell populations suggest neoplasia of those cell types, such as mast cells for mast cell tumor or histiocytes for histiocytic proliferative disease. When complete marrow effacement by the neoplastic cells is absent, it can be difficult to make a definitive interpretation of neoplasia with

Fig. 27.30  Bone marrow core biopsy sample from a dog with lymphoid neoplasia. The hematopoietic tissue is replaced by sheets of fairly monomorphic small to intermediate-sized lymphocytes. The effacement of the tissue provides clear evidence for neoplasia. Differentials may include lymphoma or involvement with chronic lymphocytic leukemia (which usually arises from the spleen rather than the marrow). This dog had a peripheral lymphocytosis of 22,000/μL. Correlation to physical examination findings, diagnostic imaging results, and potentially flow cytometry or immunohistochemistry may help further characterize this neoplastic process (see Box 27.3) (H&E stain, original magnification 400×).

Fig. 27.31  Bone marrow cytology from two dogs with lymphoma. Both dogs have a predominance of large immature hematopoietic cells with prominent nucleoli (“blasts”) in bone marrow, confirmed to be large cell lymphoma. Note the lymphoglandular bodies (blue cytoplasmic fragments; arrows) in the background. Lymphoma can have a somewhat variable appearance cytologically (note the cytoplasmic vacuoles in the lymphoma on the right, not evident on the left). If lymphoid origin is not clear with morphology alone, additional advanced diagnostic testing can be considered (flow cytometry, PCR for antigen receptor rearrangement (PARR), or biopsy with immunohistochemistry; see Box 27.3) (Wright-Giemsa stain, original magnification 1000× [left] and 500× [right] ).

CHAPTER 27  cytology or with histopathology. Early bone marrow recovery with repopulation of the marrow after a prior insult can have an increased proportion of blasts or other immature precursor cells that can mimic the appearance of neoplasia. This can also occur with a very early regenerative response to a peripheral hematopoietic stimulus, and therefore an increased immature/blast component within the marrow can be misinterpreted as potentially neoplastic. When there is an increased immature cell component, the context of historical CBC data and additional clinical information should be taken into consideration because the marrow may represent just a snapshot in time before a

Fig. 27.32 Bone marrow core biopsy from a dog with leishmaniasis. Hypercellular-appearing marrow with numerous macrophages containing multiple small amastigotes (arrowheads). Note the increased numbers of plasma cells (arrows), which can be seen with antigenic stimulation or chronic inflammation. The presence of Leishmania spp. amastigotes clearly rules out a neoplastic plasma cell population despite the high numbers of plasma cells present. Plasma cells on histopathology may overlap in appearance with middle- to late-stage erythroid cells but are often most identifiable by their perinuclear clear zone (H&E stain, original magnification 1000×).

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499

subsequent repopulation occurs. Reassessment of the CBC and, potentially, resampling of bone marrow in 3 to 5 or even 5 to 7 days may be necessary. With a regenerative or hyperplastic response, this would allow time for maturation of the expanded immature population and restoration of the pyramidal distribution in that lineage. A retained or further expanded immature population after 7 days would lend support for a neoplastic proliferation. The anatomical distribution within the marrow on a core biopsy sample may aid in further characterization of the cells present. When an immature mitotically active population is located along the paratrabecular area and is accompanied by maturing neutrophils with myeloid hyperplasia, this likely reflects an early hyperplastic myeloid population. When an immature cell population is present within the interstitial areas in increased numbers, there may be more concern for an emerging neoplastic component (see Fig. 27.38). Additional considerations for an interstitial expanded immature component on a core biopsy sample include a macrophage/ histiocyte population, a megakaryoblast component, particularly if there is megakaryocytic hyperplasia, or a displaced expanded hyperplastic myeloid population. If there is evidence of dysplasia within multiple cell lineages with unexplained peripheral cytopenias and accompanying marrow hypercellularity with increased immature cells in the marrow (but with <20% blasts), these features suggest MDS. MDS is a type of hematopoietic neoplasia that can eventually transition to AML (Fig. 27.45). In some cases, there can be overlapping features between other causes of ineffective hematopoiesis and MDS. In these difficult cases, response to therapy and correlation with additional clinical information may be the best differentiators. With some types of hematopoietic neoplasia that may have a patchy distribution in the marrow or relatively low numbers of neoplastic cells present, distinction from a hyperplastic process can be very difficult. Examples include mast cell hyperplasia versus metastatic mast cell tumor, small cell lymphoma or chronic lymphocytic leukemia versus reactive lymphocytosis, or plasma cell hyperplasia versus multiple myeloma. A combination of cytology and core biopsy with histopathology, potentially with additional special stains (e.g., Giemsa for mast cell tumor) or ancillary diagnostics (IHC, flow cytometry, PARR for potential lymphoma), may be necessary for definitive diagnosis

Fig. 27.33  Bone marrow cytology from a cat and a dog with plasma cell neoplasia. In both the cat (left) and the dog (right), the vast majority of cells in the marrow are neoplastic plasma cells, with only few hematopoietic precursor cells in the background. Note the plasmacytoid morphology of the neoplastic cells, including eccentric nuclei with a coarsely clumped chromatin pattern, and characteristic blue cytoplasmic coloration with a perinuclear clear zone. Plasma cell neoplasia in bone marrow is typically a component of multiple myeloma, which is a clinical diagnosis based on certain inclusion criteria. Plasma cell neoplasia can be well differentiated, or neoplastic cells can be more pleomorphic and atypical (left) with binucleation, anisocytosis, and anisokaryosis (Wright-Giemsa stain, original magnification 500× left and 1000× right).

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Fig. 27.34  Bone marrow cytology from a dog with histiocytosis in the marrow. Increased numbers of mildly pleomorphic histiocytes are recognized by their eccentric nuclei, high volume of medium-blue cytoplasm, and phagocytic activity with cytoplasmic iron pigment (dark-blue intracellular pigment on left) and occasional phagocytosis of hematopoietic precursors (arrowheads; metarubricytes in top right, neutrophil in bottom right). Further characterization of a histiocytic proliferative population as neoplastic or benign/reactive can be very challenging, particularly because the hemophagocytic variant of histiocytic sarcoma often has bland morphological features. Findings need to be correlated to additional clinical information, bloodwork, and diagnostic imaging results (Wright-Giemsa, original magnification 500× left and 1000× right).

BOX 27.13  Causes of Increased

Macrophages and Increased Phagocytic Activity Increased Macrophages/Histiocytes • Inflammation (including infectious etiology) • Immune-mediated conditions • Necrosis • Neoplasia • Histiocytic proliferative disorders (histiocytic sarcoma, reactive histiocytosis, neoplastic or non-neoplastic hemophagocytic syndrome) 

Fig. 27.35 Bone marrow core biopsy from a Bernese Mountain Dog with histiocytosis in the marrow. This marrow is hypercellular with a combination of myeloid and erythroid hyperplasia and a prominent population of histiocytes (occupying much of the image, delineated from the surrounding marrow elements by arrows). There is abundant iron (yellow-brown pigment) in these histiocytes, but the morphological features in this population are fairly bland. Definitive distinction can be very difficult between a benign/reactive histiocytic proliferation (e.g., with an immune-mediated process, drug reaction, or benign variant of hemophagocytic syndrome) and a neoplastic proliferation (as with hemophagocytic histiocytic sarcoma). Correlation with additional clinical information, bloodwork, diagnostic imaging results, and potentially response to therapy is helpful (H&E stain, original magnification 400×).

Increased Phagocytic Activity by Macrophages • Immune-mediated conditions (particularly precursor-targeting immune-mediated anemia (PIMA) or immune-mediated neutropenia) • Necrosis • Inflammation • Infection (mycobacteriosis, leishmaniasis, histoplasmosis, cytauxzoonosis), hemoparasites (Babesia spp., Mycoplasma spp.) • Dyserythropoiesis • Severe erythroid hyperplasia • Blood transfusion • Neoplasia • Hemophagocytic histiocytic proliferative disorders (hemophagocytic histiocytic sarcoma or hemophagocytic syndrome)

Fig. 27.36  Bone marrow core biopsy and cytology from two dogs with myelofibrosis. Myelofibrosis is often a process that can only be discerned on histopathology because there is preservation of marrow architecture with core biopsy (top). In some cases, the suggestion of myelofibrosis may be evident with aspiration cytology (bottom left; note the fibrillar pink-magenta extracellular matrix material). Myelofibrosis is most commonly secondary and can either be mild and multifocal within the marrow (see Fig. 27.17) or can replace most of the marrow (top: note the streaming eosinophilic fibrous tissue occupying the medullary space). Pockets of active hematopoiesis may be retained, though the overall hematopoietic cellularity is decreased (note groups of cells among the fibrous tissue). Reticulin stain (bottom right: note black-staining fibers) or trichrome stain can be utilized to highlight the fibrous tissue and is particularly useful in suspicious or borderline cases (top: H&E stain, original magnification 400×; bottom left: Wright-Giemsa stain, original magnification 500×; bottom right: reticulin stain, original magnification 200×).

BOX 27.14  Causes of Myelofibrosis Primary Myelofibrosis (Rare Form of Myeloproliferative Neoplasia) Secondary Myelofibrosis (Most Common) • Immune-mediated disease (in particular, precursor-targeted immune-mediated anemia [PIMA]) • Neoplasia • Inflammation, infection, vascular injury, or necrosis in bone marrow • Drug-associated • Irradiation • Pyruvate kinase deficiency (in dogs) • Feline infectious peritonitis (FIP; in cats) • Chronic renal failure (in cats) • Idiopathic

Fig. 27.37 Bone marrow core biopsy from a dog with myelonecrosis. Myelonecrosis, as with other stromal changes, is often most easily identified with core biopsy rather than with aspiration cytology. On histopathology, acute myelonecrosis is identified as areas of eosinophilic smudging of the medullary tissue with cell debris and often has accompanying hemorrhage, as in this case. Causes for myelonecrosis may include ischemia, inflammation/infection, drug effects, neoplasia, vascular abnormality, or immune-mediated processes (H&E stain, original magnification 400×).

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Fig. 27.38  Bone marrow core biopsy from a dog; hematopoietic neoplasia and myelonecrosis. Pancytopenia with atypical cells in circulation was observed in this dog. The marrow is replaced by sheets of monomorphic immature large round cells, consistent with hematopoietic neoplasia. There are areas of necrosis (arrows), which are hypereosinophilic with amorphous/smudged debris. On the basis of the cell morphology, there is a suspicion for acute myeloid leukemia, although definitive characterization would require additional diagnostic testing, such as flow cytometry or immunohistochemistry to rule out lymphoid origin (see Box 27.3). The necrosis may be secondary to the neoplasia in this case (H&E stain, original magnification 400×).

Fig. 27.40  Bone marrow cytology from a dog with disseminated leishmaniasis. Macrophages contain numerous round to oval Leishmania spp. amastigotes; organisms are also free in the background. There are accompanying increased plasma cells (identified by their perinuclear clear zone). Leishmania amastigotes have a “parachute men” appearance with a pale round nucleus and rod-shaped dark-staining kinetoplast (inset) (Wright-Giemsa stain, original magnification 1000×).

Fig. 27.41 Bone marrow from a cat with disseminated histoplasmosis. Several large macrophages are present, each containing numerous Histoplasma capsulatum organisms. Note the half-moon appearance of the organisms (inset). Histoplasmosis is a systemic fungal infection that may affect bone marrow, in addition to the lungs, eyes, and other sites (Wright stain, original magnification 250×).

Fig. 27.39 Bone marrow core biopsy from a dog with disseminated blastomycosis. Among background hypereosinophilic necrotic debris there are groups of pale staining, degenerating round yeast organisms (arrow). With Gomori methenamine silver (GMS) staining (inset), the cell walls are outlined (black) and broad-based budding is noted, which confirms Blastomyces dermatitidis infection. Blastomycosis is a systemic mycotic infection that can affect bone and bone marrow in some cases, along with the skin and lungs being common sites of involvement (H&E stain, original magnification 1000×; inset: GMS stain, original magnification 1000×).

(see Box 27.3). With regard to lymphoma versus reactive lymphocytosis, distribution within the marrow may aid in this distinction as well because interstitial aggregates of lymphocytes are more likely to be reactive, whereas paratrabecular aggregates or diffuse distribution of lymphocytes may favor a neoplastic population.12,46-48,58 With immature blasts in increased numbers or entirely effacing the marrow, an interpretation of hematopoietic neoplasia may be clear, but the subtype of neoplasia may be difficult to further determine (Figs. 27.46 and 27.47). There can be morphological overlap between AML

Fig. 27.42  Postmortem bone marrow cytology from a cat with cytauxzoonosis. A large macrophage containing a schizont of Cytauxzoon felis is surrounded by predominantly myeloid cells with occasional lymphocytes and plasma cells. Cytauxzoon piroplasms were seen in the peripheral blood of this cat before death (inset). Scanning bone marrow smears at low magnification is helpful in identifying the large schizonts, which reflect the tissue phase of Cytauxzoon infection (Wright-Giemsa stain, original magnification 250×). (Case courtesy Dr. Jaime Tarigo.)

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TABLE 27.8  Categories of Neoplasia in

Bone Marrow Type of Neoplasia

Subcategory of Neoplasia

Additional Subtypes

Primary hematopoi- Acute lymphoblas- T versus B cell etic neoplasia tic leukemia (ALL) Acute myeloid leukemia (AML)

Undifferentiated, myeloblastic with or without maturation, promyelocytic, monoblastic/ monocytic, myelomonocytic, erythroblastic, megakaryocytic

Chronic myeloproliferative neoplasia (“chronic leukemias”)

Chronic myelogenous leukemia, polycythemia vera, essential thrombocythemia, primary myelofibrosis

Chronic lymphocytic leukemia (CLL)

T versus B cell (but note that T cell CLL typically arises within the spleen rather than the marrow)

Plasma cell neoplasia

Multiple myeloma

Secondary/ Lymphoid neometastatic/multiplasia centric neoplasia

Lymphoma (various subtypes), chronic lymphocytic leukemia (if arising in the spleen and not primary to the marrow)

Histiocytic neoplasia

Histiocytic sarcoma, hemophagocytic histiocytic sarcoma (can arise in the marrow as a primary site or involve the marrow in multicentric disease)

Mast cell tumor

Metastatic mast cell tumor, mast cell leukemia

Carcinoma

Transitional cell carcinoma, apocrine gland adenocarcinoma of the anal sac, etc.

Fig. 27.43 Bone marrow cytology from a dog with acute monocytic leukemia. The marrow is largely replaced by large immature blast cells with only few background hematopoietic precursor cells noted (note a metarubricyte and a rubricyte). On the basis of morphology alone, considerations may include acute myeloid leukemia and lymphoid neoplasia (lymphoma versus acute lymphoblastic leukemia). Based on the cytoplasmic vacuoles and lobular contour of nuclei, in addition to the presence of markedly increased monocytic cells in circulation (inset of blood smear), the findings support an acute monocytic leukemia. Monocytic origin was confirmed with flow cytometry (Wright-Giemsa stain, original magnification 1000×).

*Table does not include primary bone tumors (e.g., osteosarcoma, chondrosarcoma, and fibrosarcoma) because these only affect the marrow by indirect extension/bone infiltration and are not typically associated with hematological changes.

(erythroid, megakaryocytic, or myeloid), acute lymphoblastic leukemia (ALL), and bone marrow involvement by lymphoma. IHC or PARR could potentially confirm lymphoid origin, but flow cytometry may be the best test for further characterization of these immature neoplasms because this test can assess for makers of lymphoid and myeloid origins in addition to CD34, which can aid in distinction between ALL and stage V lymphoma. Cytochemical stains are available in an academic setting to help distinguish among subtypes of AML, but in diagnostic practice cytochemical stains are not commonly performed, and the distinction often does not affect prognosis or treatment decisions (see Box 27.3). Chronic myeloproliferative neoplasms (“chronic leukemias”) are uncommon and include CML, polycythemia vera, and essential thrombocythemia. Bone marrow evaluation in these chronic neoplasms is often unrewarding. Bone marrow findings in chronic myeloproliferative neoplasia have a similar and indistinguishable pyramidal distribution as with a severe reactive/hyperplastic process until there is a blast crisis, as may occur at the late stage of disease. A mild degree of dysplasia can be seen in either a hyperplastic or a neoplastic process. The potential for chronic leukemia should be correlated with clinical findings, CBC data, and exclusion of inflammatory/infectious conditions (for CML), causes of secondary

Fig. 27.44 Bone marrow cytology from a cat with megakaryocytic leukemia. Most of the cells in this image are atypical megakaryocytic precursors. Note the binucleation or multinucleation and the prominent cytoplasmic blebbing (inset). Megakaryocytic leukemia is rare and may be associated with either thrombocytopenia or thrombocytosis (Wright-Giemsa stain, original magnification 1000×).

polycythemia (for polycythemia vera), or reactive thrombocytosis (for essential thrombocythemia). Some dogs may have a benign-appearing proliferative histiocytic population, which, on cytology, may be dispersed throughout the sample or in aggregates (see Fig. 27.34). On histopathology, these histiocytes may be distributed throughout the interstitium or in a more sheetlike arrangement, and cells may have increased phagocytic activity (see Fig. 27.35). Differentials for this type of benign-appearing histiocytic proliferation may include hemophagocytic syndrome, a reactive proliferation, an immune-mediated response, involvement with systemic reactive histiocytosis, or hemophagocytic histiocytic sarcoma. Typical multicentric or disseminated histiocytic sarcoma

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Fig. 27.45 Bone marrow cytology from a cat with myelodysplastic syndrome (MDS). The marrow contains marked erythroid hyperplasia with atypical features and prominent dysplasia within the erythroid lineage cells. Megaloblastic change, binucleation, and frequent nuclear-to-cytoplasmic asynchrony or dysmaturation are prominent. Iron pigment is noted (dark brown-black globules in bottom left corner), which is abnormal in a cat and is often associated with dyserythropoiesis. Based on a blast percentage of less than 20%, erythroid dysplasia, and the marked predominance of erythroid cells, this was classified as MDS with erythroid predominance (MDS-Er). This type of hematopoietic neoplasia can progress to erythroleukemia and may be associated with feline leukemia virus (FeLV) infection (Wright-Giemsa stain, original magnification 100×).

Fig. 27.46  Bone marrow cytology from a dog with poorly differentiated round cell neoplasia. The marrow contains a high number of immature large blast cells, consistent with poorly differentiated round cell neoplasia. The exceedingly large prominent nucleoli of the blast cells are not typical for myeloid, erythroid, or lymphoid cells, in particular. The basophilic background, numerous cytoplasmic fragments (lymphoglandular bodies), and scattered smaller immature lymphoid cells are suggestive of lymphoma, although additional diagnostic testing would be necessary to confirm (see Box 27.3). Increased eosinophil precursors are noted, which may be associated with lymphoma, in particular T-cell lymphoma (Wright-Giemsa stain, original magnification 1000×).

cells have more prominent atypia with multinucleation and mitotic activity, whereas the hemophagocytic variant of histiocytic sarcoma can be more morphologically bland and typically affects the spleen and potentially bone marrow, liver, or other sites. The more general term hemophagocytic syndrome refers to a secondary process that can occur with immune-mediated, infectious, or neoplastic conditions. With hemophagocytic syndrome, there is pancytopenia or bicytopenia, with increased benign-appearing macrophages (>2% of nucleated cells) in marrow with phagocytized RBCs and/or precursors.4 Distinction between these types of histiocytic proliferative processes is often

Fig. 27.47  Bone marrow core biopsy from a dog with hematopoietic neoplasia. The marrow is largely effaced by sheets of monomorphic large round cells, consistent with hematopoietic neoplasia. Note the prominent nucleoli, immature features, and increased mitotic figures (arrowheads). On the basis of morphology alone, further classification is difficult, but in this case large cell lymphoma was confirmed. Advanced diagnostic testing may be necessary, in some cases, to more fully classify hematopoietic neoplasms, especially with poorly differentiated tumors (see Box 27.3) (H&E stain, original magnification 400×).

Fig. 27.48 Bone marrow cytology from a dog with metastatic carcinoma. Among the background hematopoietic cell population, there is a large cluster of cohesive, vacuolated epithelial cells (arrow). This is metastasis from a pulmonary carcinoma in this dog. More common metastatic carcinomas to bone or bone marrow may include transitional cell carcinoma from the urinary bladder and apocrine gland adenocarcinoma of the anal sac. Scanning a cytological slide at low magnification to identify unusual groups of cells is an important step in assessing for foci of metastatic neoplasia. These groups are often randomly distributed within a marrow sample and therefore may be missed without careful examination (Wright stain, original magnification 250×).

difficult but may be aided by correlation with CBC and chemistry data, potential additional sites of involvement (as with imaging of the spleen and the liver), and additional clinical information, as well as response to therapy. Differentiation between benign and malignant histiocytic proliferations can be very difficult in some cases.51 Metastatic neoplasia to bone marrow is less common than primary neoplasia. Metastatic neoplasms may include carcinomas, such as transitional cell carcinoma, apocrine gland adenocarcinoma of the anal sac, or other subtypes, as well as mast cell tumor, histiocytic sarcoma, or lymphoma (see Figs. 27.22 and 27.31; Figs. 27.48 to 27.50). Core biopsy may be necessary to best capture metastatic neoplasia

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505

Fig. 27.49 Bone marrow core biopsy from a dog with metastatic carcinoma. In both paratrabecular and interstitial distributions, there are large rounded to polygonal cells with very few background normal hematopoietic cells. On the basis of individual cell morphology, these cells overlap with a hematopoietic population, but a cohesive aggregated pattern or acinar-type formation by these cells is noted, consistent with epithelial origin. A cytokeratin immunohistochemical stain (AE1/AE3; inset) confirms carcinoma in this case (note the dark brown cytoplasmic immunoreactivity). Investigation for a primary site of this carcinoma is the next step clinically (H&E stain, original magnification 400×; inset: cytokeratin AE1/AE3 immunohistochemical stain, original magnification 400×).

some cases to monitor a patient’s progress and fine-tune the course of therapy. Consultation and discussion with the pathologists involved in bone marrow interpretation can be helpful and rewarding in many cases and may aid in clinical interpretation as well as inform treatment decisions.

REFERENCES

Fig. 27.50  Bone marrow cytology sample from a dog with multicentric histiocytic sarcoma. The large atypical cells are neoplastic histiocytes. They have an abundant volume of cytoplasm with cytoplasmic vacuolation and a small amount of cytoplasmic iron (deep blue globular material in left cell). Cytological criteria for malignancy include the large cell size and anisokaryosis, but in other areas of the sample (not pictured) there was also multinucleation and karyomegaly. Immunocytochemistry for CD18 (a panleukocytic marker) highlights the cytoplasm of these cells (inset: note the brown cytoplasmic immunoreactivity). This CD18-positivity confirms histiocytic sarcoma versus a primary bone tumor, such as osteosarcoma (Wright-Giemsa stain, original magnification 1000×; inset: CD18 immunocytochemical stain, original magnification 1000×).

because this can be multifocal in the marrow and is often paratrabecular. Cytological sampling may miss these lesions because aspiration usually reflects the interstitial components of the marrow and could miss focal paratrabecular lesions (see Fig. 27.48). 

CONCLUSIONS Bone marrow evaluation and interpretation can be a diagnostic challenge even with complete CBC, blood smear analysis, aspiration cytology, and core biopsy with histopathology. A bone marrow sample provides only a single snapshot in time. Continued monitoring of CBC and potential repeat bone marrow sampling may be necessary in

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