CHAPTER 27 The Bone Marrow

Bone marrow is the major hematopoietic organ of the body. In young animals, active hematopoietic tissue is found throughout both flat and long bones. As growth ceases, hematopoietic activity in the central areas of long bones regresses. In adults, most active hematopoiesis occurs in the flat bones and the extremities of long bones. The central area of long bones contains mostly fat, with very little active hematopoietic tissue.

Active hematopoietic tissue is highly vascular and consists of islands of hematopoietic tissue surrounded by vascular sinuses. The islands of hematopoietic tissue are composed of the following: erythroid, granulocytic, monocytic and thrombocytic series cells; marrow structural cells (adventitial cells); fat cells; and a few macrophages, lymphocytes, plasma cells, and mast cells. The hematopoietic tissue islands are bounded by the endothelium lining the vascular sinuses.

INDICATIONS

The most common indication for bone marrow cytologic examination is recognition of hematologic abnormalities not readily explained by a good history, physical examination, chemistry profile, and/or other clinical procedures. Bone marrow examination is often rewarding in evaluating animals with nonregenerative anemia, persistent neutropenia, or persistent thrombocytopenia.¹^–³

Bone marrow cytologic evaluation can also be used to assess the marrow’s involvement in some neoplastic conditions, such as lymphoid neoplasia and mast cell neoplasia; to identify suspected infectious agents, such as Histoplasma capsulatum, Leishmania donovani, Ehrlichia spp., Cytauxzoon felis, and Toxoplasma gondii; and to evaluate patients with evidence of hyperglobulinemic conditions (e.g., multiple myeloma, lymphoma, ehrlichiosis/anaplasmosis, leishmaniasis, and disseminated histoplasmosis) (Box 27-1).⁴^–⁸

Box 27-1 Bone Marrow Cytologic Evaluation

Abnormal Serum Chemistry Findings

Hypercalcemia

Hyperglobulinemia (monoclonal and polyclonal)

Decreased serum iron

CBC, Complete blood count; WBC, white blood cell; RBC, red blood cell

CONTRAINDICATIONS

Contraindications of bone marrow biopsies are few, and complications are uncommon. Restraint, sedation, and anesthesia usually pose a greater risk to the patient than the biopsy procedure. Bone marrow aspiration biopsies can often be collected without sedation; general anesthesia is seldom necessary. Hemorrhage is a theoretical concern in thrombocytopenic animals, but significant bleeding rarely occurs, even in severely thrombocytopenic animals. Bleeding complications can be controlled with fresh whole blood or appropriate component therapy.^4,^9,¹⁰ Iatrogenic marrow infection is possible, but the risk is miniscule, especially if the area of skin through which the aspirate is collected has been properly prepared. The main contraindication for bone marrow biopsy is performing an unnecessary biopsy. Evaluation of a current blood smear and a thorough clinical workup are mandatory before performing a bone marrow biopsy.

SAMPLE COLLECTION AND PREPARATION

Proper collection and preparation of marrow are necessary for maximal diagnostic usefulness.^4,^5,^7,¹¹^–¹³ Bone marrow degenerates rapidly after collection or the animal’s death; therefore bone marrow samples from dead animals should be collected immediately (within 30 minutes) after the animal’s death. Regardless of whether the animal is alive or dead, bone marrow cytologic preparations should be prepared immediately after collection. Interestingly, granulocytes appear to be the first cells to undergo significant morphologic distortion after marrow sample collection or the animal’s death. Granulocytic nuclei swell and, losing their contorted lobulated pattern, become large and round to ovoid. The nuclear chromatin pattern loses its dense clumped areas and stains less intensely and more uniformly. As a result, neutrophils that have undergone this alteration may be mistaken for blast cells. This error can lead to misdiagnosis of neoplasia. Extreme caution must be used when examining samples from animals dead for 30 minutes or more before slide preparation. These samples can be examined for cellularity, organisms, mast cell infiltration, erythrophagocytosis, plasmacytosis, and accumulation of iron-containing pigments, but examination for evidence of neoplasia can result in misdiagnosis. The same requirements and limitations apply for bone marrow samples submitted for histopathologic examination.

After bone marrow preparations are air-dried, they should be stained as soon as possible. When submitting bone marrow cytologic preparations for consultation, several air-dried unfixed preparations and air–dried stained preparations should be submitted.

Some techniques for collecting, preparing, and staining bone marrow cytologic preparations are discussed subsequently.

Instruments and Supplies

A 15- to 18-gauge, 1- to 2-inch bone marrow aspiration biopsy needle, clear Petri dish or watch glass, 10- to 12-ml syringe, several clean microscope slides and coverslips, the supplies for a surgical prep and, optionally, ethylene-tetra-acetic acid (EDTA) and isotonic fluid solution are required for collecting and preparing bone marrow aspirate biopsies. These instruments and supplies should be located on a clean work counter in close proximity to the work area where the sample is to be collected. If anticoagulant is not used, smears must be prepared from the aspirate immediately (within 30 seconds of collection). Some acceptable bone marrow biopsy needles are the Rosenthal (Popper and Sons, New Hyde Park, NY), Illinois sternal, and Jamshidi needles (Tyco Healthcare-Kendall, Mansfield, MA 02048).

A sterile 2% to 3% EDTA/isotonic saline solution can be prepared using commercially available EDTA blood collection tubes, which contain about 1.5 mg of EDTA (as a freeze-dried powder or liquid solution) per milliliter of blood to be added. Injecting 0.35 ml of sterile isotonic saline into a 7-ml EDTA tube produces about 0.35 ml of 3% EDTA or 0.42 ml of 2.5% EDTA, depending on whether the tube originally contained powdered or liquid EDTA.

Aspiration Biopsy

Site

Bone marrow aspiration biopsies may be collected from the proximal humerus, iliac crest, trochanteric fossa of the femur, and sternebrae of dogs and cats (Figure 27-1).^4,^5,^7,¹¹^–¹³ The proximal humerus and iliac crest in large dogs and the proximal humerus and trochanteric fossa in small dogs and cats are the most accessible. As a result, they are the most commonly used sites. Because of the danger of penetrating the thoracic cavity, the sternebrae should be avoided in cats and small dogs. For the same reason, the ribs should be used only when incisional biopsies are performed. The trochanteric fossa may not be approachable in large, well-muscled, or very obese patients. Also, the cortical bone of the trochanteric fossa may be so dense in older dogs that it prevents easy penetration of the marrow cavity. Because of the small diameter of the bones of cats and small dogs, the trochanteric fossa often supersedes the iliac crest as a site for bone marrow aspiration in these animals.

Figure 27-1 Dog skeleton showing common sites for bone marrow collection. In large dogs, the dorsal approach to the iliac crest (A) is an excellent site for aspiration and core biopsies. In small dogs and cats the lateral approach to the wing of the ilium (A) is a good site for core biopsies, and the trochanter fossa of proximal femur (B) is a good site for aspiration biopsies. For all small animals, the proximal humerus (C) is an excellent site for both aspiration and core biopsies.

(Reprinted with permission from Grindem CB, Neel JA, Juopperi TA: Cytology of bone marrow. Vet Clin Small Anim 32[6]:1316, 2002.)

Collection

To aspirate bone marrow via the humerus or trochanteric fossa of the femur, the animal is placed in lateral recumbency. If the iliac crest is used, the animal can be standing, sitting, or in sternal recumbency. Regardless of the site used, the hair is clipped from the skin several inches around the area where needle puncture is anticipated. The skin is then prepared as for surgery, and a local anesthetic is injected into the skin and under the periosteum at the puncture site.

A small stab incision is made at the biopsy site with a sterile scalpel blade (number 11). The greater trochanter of the proximal femur is located by palpation, and the biopsy needle is passed medial to the trochanter, with its long axis parallel to the long axis of the femur, until the needle reaches the trochanteric fossa. When bone is contacted, moderate pressure is applied and the needle is rotated in an alternating clockwise-counterclockwise motion. The operator usually feels the resistance decrease when the needle enters the bone marrow in the trochanteric fossa or humerus, but this sensation may not be as obvious for the iliac crest. To aspirate the humerus, palpate the greater tubercle and insert the needle into the flat area on the craniolateral surface of the proximal humerus distal to the greater tubercle, avoiding the articular cartilage. For the iliac crest, palpate the greatest prominence of the crest and, once the needle is firmly in bone, it is probably in the marrow cavity. With core biopsies of the iliac crest it is important to keep the needle parallel to the long axis of the wing of the ilium.

When the marrow cavity has been entered, the stylet is removed and a 10- to 20-ml syringe containing 0.3 to 0.5 ml of sterile 2% to 3% EDTA/isotonic saline solution is attached to the needle. Strong negative pressure is applied by rapidly pulling the plunger back as far as possible (usually two thirds to three fourths of the volume of the syringe). Most animals show evidence of pain when bone marrow aspiration begins, and this is good evidence that the needle is in the marrow cavity. After a few drops of marrow are collected, the negative pressure is released. If EDTA is used, the volume of marrow collected should not exceed the volume of EDTA/isotonic saline solution in the syringe. Continued negative pressure contaminates the marrow sample with blood. Whether a marrow sample appears in the syringe or not, negative pressure should not be applied to the syringe twice in the same area. This causes aspiration of excessive amounts of blood.

If marrow is not collected at the first site, the syringe is removed, the stylet is replaced in the biopsy needle, the needle is repositioned by slight advancement and rotation, and negative pressure is reapplied. If marrow is still not collected, the negative pressure is maintained and the needle is slowly withdrawn until marrow is obtained or the needle exits the bone. If this fails to collect an adequate sample, aspiration can be attempted from another site in the same bone or from another bone. If a sample cannot be collected by aspiration, a core biopsy or incisional biopsy is necessary.

Smear Preparation without EDTA

If EDTA/isotonic saline is not used, as soon as a few drops of marrow sample appear in the syringe, the plunger is released, the syringe is detached from the needle, and the stylet is replaced in the needle. The needle remains embedded in the bone.

Then the sample is immediately expelled directly onto a glass microscope slide. Direct smears similar to blood smears can be made and squash preps should also be prepared by tilting a slide 45 to 70 degrees, allowing the blood to drain from the slide into a watch glass or Petri dish (Figure 27-2). Marrow flecks tend to adhere to the glass microscope slide. A second glass microscope slide is placed perpendicularly across the marrow flecks adhered to the first slide, causing the marrow flecks to spread. The two slides are then smoothly pulled apart in a horizontal plane, dispersing the flecks.

Figure 27-2 Smear preparation for bone marrow aspirates not collected in EDTA/isotonic saline solution. A, Some of the aspirate is expelled onto a tilted glass microscope slide. The slide can be propped up with another glass slide braced against the side of a Petri dish. Marrow flecks tend to adhere to the slide as the marrow runs down it. B, Another glass slide is placed over the slide containing the aspirate, which spreads the aspirate. C, The two slides are slid apart. D, This produces two preparations.

In Romanowsky-stained smears, the marrow flecks appear as blue-purple streaks. If the sample does not contain marrow flecks, the needle can be repositioned by slight advancement and rotation. The stylet is removed, another syringe is attached, and the aspiration procedure is repeated. After two or three aspiration attempts or when marrow flecks are recovered, the needle is removed.

Smear Preparation with EDTA

If EDTA/isotonic saline is used, once the marrow sample is collected, the plunger is released to relieve the negative pressure, and the needle is detached from the syringe. The stylet is replaced in the needle and the needle remains embedded in the bone. The contents of the syringe are thoroughly mixed, and the anticoagulated marrow sample is expelled into a watch glass or clear Petri dish. If the sample does not contain marrow flecks, the needle can be repositioned by slight advancement and rotation. The stylet is removed, another syringe containing EDTA/saline solution is attached, and the aspiration procedure is repeated. After two or three aspiration attempts, or when marrow flecks are recovered, the needle is removed.

Marrow samples collected in EDTA/isotonic saline solution and expelled into a Petri dish are prepared as follows. The Petri dish is tilted and/or rotated under a soft light so that the marrow flecks can be seen and distinguished from fat droplets. Marrow flecks are clear to slightly opaque and light gray; fat droplets are clear and glistening. Marrow flecks may be slightly irregular in shape; fat droplets are spherical. Generally, marrow flecks are easily located if an adequate sample has been collected.

Flecks are transferred from the sample in the Petri dish to glass microscope slides by tilting the Petri dish, causing the sample to drain to one side of the dish. Some flecks cling to the bottom of the Petri dish, and the fluid portion of the sample drains away from them. These flecks are harvested with a microhematocrit capillary tube, one end of which is touched to the side of the fleck. Often the fleck is partially aspirated into the capillary tube and can be transferred to the glass microscope slide. If the fleck does not partially aspirate into the capillary tube, the tube is gently advanced, forcing the fleck into it. The marrow fleck is then transferred onto the glass microscope slide by tapping the capillary tube end containing the fleck on the slide. Any excessive fluid transferred can be removed by touching the fluid with a piece of absorbent paper or cloth.

After the fleck is transferred to the glass microscope slide, a 22- × 22-mm coverslip is placed over the fleck at a 45-degree angle to the glass microscope slide, allowing one corner of the coverslip to hang over the edge of the microscope slide (Figure 27-3). When the coverslip is placed over the fleck, the fleck spreads to about twice its previous size. Some smears should be made without any pressure other than that caused by the coverslip, and others should be made with gentle thumb pressure sufficient to cause the smears to spread to about twice the diameter caused by coverslip pressure alone. This ensures that some of the flecks are optimally spread.

Figure 27-3 Smear preparation for bone marrow aspirates collected in EDTA/isotonic saline solution. A, A fleck, collected from the Petri dish containing the sample, is placed on a glass microscope slide. B, A microscope slide coverslip is placed over the fleck at a 45-degree angle to the slide. This spreads the fleck and accompanying fluid. C, The coverslip is slid horizontally and smoothly off the glass slide. D, Both the glass microscope slide preparation and coverslip preparation can be used. However, the coverslip preparation is usually hard to handle during staining and is often discarded. Multiple preparations can be made on a glass slide, and multiple slides can be prepared.

Excessive pressure, which causes cell rupture, is the most common error when preparing cytologic smears of marrow flecks. When making smears from marrow samples in EDTA/isotonic saline solution, using another glass microscope slide instead of a coverslip to spread the fleck may cause excessive pressure and result in rupture of nucleated cells.

Core Biopsy

Core biopsies can be collected with a Jamshidi infant (cats and small dogs) or pediatric (large dogs) marrow biopsy needle. The same procedure is used for collecting core biopsies as described for collecting marrow aspirate biopsies, except after the point of the needle has penetrated the cortex of the bone and entered the marrow cavity, the stylet is removed from the needle and the needle is advanced about 3 mm with a rotating motion.^4,^5,^7,^8,¹¹^–¹³ This cuts and collects a core of bone marrow. The needle is removed from the animal, and the core of marrow is forced onto a glass microscope slide by passing the stylet through the barrel of the needle from the hub end or retrograde from the needle end if the biopsy needle is tapered.

Using the point of the needle, the core of marrow is gently rolled the length of the microscope slide. After making one or two slide preparations in this manner, the core of marrow is placed in a container filled with 10% neutral buffered formalin. If the cytologic preparations are to be sent to an outside laboratory, they should not be mailed with the formalin-filled container because formalin vapors alter the cells’ staining qualities.

COLLECTING MARROW SAMPLES FROM DEAD ANIMALS

Bone marrow samples are occasionally collected from dead animals. If a complete evaluation of the marrow is expected, the samples must be collected and the cytologic preparations made immediately (within 30 minutes) after the animal’s death. When collection and/or preparation is delayed, the preparations can be evaluated for cellularity, organisms, mast cell infiltration, plasmacytosis, erythrophagocytosis, and iron-containing pigments, but should not be evaluated for myeloid or lymphoid neoplasia. Delay in sample preparation can result in rupture of all of the cells.

Although samples can be collected from most flat bones and the extremities of most long bones of dead animals, it is advisable to collect marrow samples from the same location(s) used in live animals (see Figure 27-1). Remember that in adults, the central areas of long bones contain mostly fat. Access to the marrow can be gained by sawing the bone, cutting it with rongeurs, or fracturing it. If the bone is sawed, heat generated at the saw line can damage adjacent cells; therefore samples from sawed bones should be collected well away from the saw line. Marrow is dug from between bony trabeculae, trying to avoid collecting bone spicules. The marrow is placed on one end of a glass microscope slide and gently rolled (by lifting upward with a needle or other instrument at the back of the sample) the length of the slide. Several slides are prepared from samples collected from several different areas of the bone marrow. The slides are air-dried and stained as described later.

STAINING MARROW PREPARATIONS

After fleck smears or core biopsy or necropsy imprints are made, they are air-dried. The general Romanowsky-type staining procedures for staining blood smears are followed, except the stains and buffers are left in contact with the cells for a longer time. The extent to which the times must be lengthened depends on the thickness and cell density of the smears, but staining times must usually be increased two times or more. Macroscopically, the marrow fleck streaks in the smear appear blue-purple to purple when the smear is properly stained.

CELLS IN MARROW

To evaluate cytologic preparations of bone marrow, one must be able to recognize the normal cells that occur in bone marrow, the neoplastic cells that have a propensity for infiltrating bone marrow, the organisms that can infect the marrow, and the processes (e.g., erythrophagia) that occur in some pathologic conditions.⁴^–⁹ ^,13 ^,14

Erythroid Series

During proliferation and maturation, erythroid progenitor cells undergo 4 to 5 mitoses, producing 16 to 32 daughter cells. As they mature, erythroid cells decrease in size, their nuclei condense, and their cytoplasm changes from dark blue to red-orange. The general characteristics of the different cell stages of erythroid production are described later. Table 27-1 lists the different stages of erythroid development and provides an estimate of the relative proportions and a brief description and a generalized schematic of the classic morphology of each stage.

Table 27-1 Nucleated Erythroid Cell Developmental Stages and Their Relative Percentage, Description, and Schematic Morphology

Granulocyte Series

Myeloid progenitor cells usually undergo four mitoses; however, depending on circumstances, a mitosis may be skipped or additional mitoses may occur. The developmental stages, immature to mature, of the granulocyte series are myeloblast, progranulocyte (promyelocyte), myelocyte, metamyelocyte, band cell, and segmented granulocyte (mature neutrophil, eosinophil, and basophil). The myeloblast, myelocyte, and metamyelocyte stages of neutrophilic granulocytes cannot be reliably differentiated from monocytes. Table 27-2 lists the different stages of development of the granulocyte series and gives the relative proportions and a brief description of the classic morphology of each stage.

Table 27-2 Developmental Stages of Neutrophils and Their Relative Percentage, Description, and Schematic Morphology

Myeloblasts

The myeloblast is large and may be round or irregular in shape. It has a large nucleus with a fine to finely stippled chromatin pattern and one or more visible nucleoli (see Figure 27-5). The cytoplasm is blue to blue-gray and does not contain visible granules. The myeloblast has the highest nuclear-to-cytoplasmic ratio of any of the granulocytic developmental stages.

Progranulocytes (Promyelocytes)

The next stage of granulocyte development is the progranulocyte or promyelocyte. Often, the progranulocyte is slightly larger than the myeloblast due to increased cytoplasm. The major differentiating feature of the progranulocyte is the presence of scattered, small, azurophilic (red-purple) primary granules throughout the cytoplasm (Figure 27-8; see Figure 27-5). Primary granules are also called nonspecific granules. Nuclei are the same size to slightly smaller than nuclei of myeloblasts, have lacy to coarse chromatin patterns, and may contain visible nucleoli (which are usually fewer and less prominent than those of myeloblasts) or nucleolar rings. Nucleolar rings are rings of densely staining chromatin that demark obscured nucleoli. The nuclear-to-cytoplasmic ratio of promyelocytes is less than that of myeloblasts. The presence of primary granules allows progranulocytes to be recognized as members of the granulocyte series and differentiates them from the monocyte series. Therefore progranulocytes can be identified more specifically than myeloblasts, neutrophil myelocytes, and neutrophil metamyelocytes, which lack visible primary granules. As a result, the term progranulocyte, instead of promyelocyte, can be used to identify members of this developmental stage.

Figure 27-8 Bone marrow aspirate. Progranulocyte (PG), neutrophilic myelocyte (M), eosinophilic myelocyte (EM), early metamyelocyte (EMM), metamyelocyte (MM), band neutrophil (BN), mature eosinophil (ME), and damaged cell nucleus (D) are indicated. (Wright-Giemsa stain, original magnification 250×.)

Box 27-2 Some Diseases that Can Cause Nonregenerative Anemia, Neutropenia, and/or Thrombocytopenia in Dogs and Cats, Classified by the Overall Marrow Cellularity with which They Can Be Associated

Hypocellular marrow	Normocellular Marrow	Hypercellular Marrow
FeLV suppression (cat)–A	FeLV suppression (cat)–A, d	Histoplasmosis–A
Ehrlichiosis (dog)–A	Ehrlichiosis (dog)–A, d	Ehrlichiosis (dog)–TP, e
Renal insufficiency–NA	Histoplasmosis–A, d	Myeloproliferative disease–A
Marrow necrosis–A, a	Myeloproliferative disease–A, d	Lymphosarcoma–A
Radiation, cytotoxic drugs, etc–A, a	Lymphosarcoma–A, d	Anemia of inflammatory disease (i.e., anemia of chronic disease)–NA
Myelofibrosis (sclerosis)–A, b
Pure red cell aplasia–NA, c
Immune-mediated idiopatic

FeLV, Feline leukemia virus; A, one to all of the marrow cell lines may be affected; NA, nonregenerative anemia; NP, neutropenia; TP, thrombocytopenia; a, anemia may not be present in the acute phase; b, flecks, if any are collected, may appear small but hypercellular because fibrous tissue has replaced most of the marrow elements, but a few pockets of hypercellular marrow may remain; c, rarely, thrombocytopenia and/or neutropenia may also be present; d, usually associated with hypocellular marrow; e, ehrlichiosis can be associated with hypocellular marrow, but then it is usually in the acute to subacute phase of infection and is associated with thrombocytopenia alone—clinical signs are often unapparent.

Myelocytes

The next developmental stage of the granulocyte series is the myelocyte. The disappearance of the primary granules (seen in the progranulocyte stage) aids differentiation of the myelocyte from the progranulocyte. Also, myelocytes are smaller than progranulocytes, and their nuclear chromatin is more dense, giving a coarser pattern (Figure 27-9; see Figures 27-5 to 27-8). Nuclei are round to oval, and nucleoli are not visible. Cytoplasm is light blue to clear and contains secondary (specific) granules, which are poorly visualized in the neutrophil series but give eosinophils and basophils their characteristic appearance.

Figure 27-9 Bone marrow from a cat with eosinophilia and basophilia. Note three basophilic myelocytes, a progranulocyte, and a binucleated promegakaryocyte. (Wright-Giemsa stain, original magnification 250×.)

The granules of feline eosinophils are rod shaped, and the granules of canine eosinophils are pleomorphic (but typically round) and variable in size (see Figures 27-7 and 27-8). Feline basophils contain granules that are oval, tend to stain lavender, and fill the cytoplasm. The oval granules may appear round when viewed end-on. Immature feline basophils contain many red-purple granules (see Figures 27-6 and 27-9). Canine basophils contain a few to many round, variably sized, red-purple (metachromatic) granules. The myelocyte is the last stage capable of mitosis.

Metamyelocytes

The next stage of granulocyte development is the metamyelocyte. Its nucleus is classically described as kidney bean shaped. Generally, cells with nuclei having indentations extending less than 25% of the way into the nucleus are classified as myelocytes, whereas those with nuclei having indentations extending 25% to 75% of the way into the nucleus are classified as metamyelocytes (see Figure 27-6). The cytoplasmic characteristics of metamyelocytes are similar to those of myelocytes, with the neutrophil myelocyte cytoplasm being clear. This stage and subsequent stages do not undergo mitosis.

Band Cells

The next stage of granulocyte development is the band cell. It has a nucleus with a curved band or rod shape and smooth, parallel sides (see Figures 27-7 and 27-8). Some chromatin clumps are present. No area of the nucleus has a diameter less than one-half the diameter of any other area of the nucleus. Membrane irregularity and excessive narrowing of the nucleus warrant classifying of the cell as a mature neutrophil. The cytoplasmic characteristics of band cells are similar to those of metamyelocytes.

Segmented Granulocytes

The final stage of granulocyte development is the segmented granulocyte (mature neutrophil, eosinophil, or basophil). Its nucleus is lobate or has areas of marked constriction (see Figure 27-7). The nuclear border is often irregular with large, dense chromatin clumps. Its cytoplasmic characteristics are similar to those of myelocytes, metamyelocytes, and band cells.

Monocyte Series

Cells of the monocyte series account for only a small percentage of the total marrow cells. As mentioned earlier, monoblasts cannot be differentiated from myeloblasts by light microscopy. Also, promonocytes are morphologically similar to neutrophilic myelocytes and metamyelocytes in bone marrow cytologic preparations stained with Romanowsky-type stains. Mature monocytes in bone marrow smears have the same appearance as monocytes in peripheral blood.

Thrombocyte (Megakaryocyte) Series

Megakaryoblasts

The most immature developmental stage of the megakaryocyte series recognizable by light microscopy is the megakaryoblast. It is not commonly identified in marrow aspirates, however, because it usually occurs in small numbers and is difficult to differentiate from other blast cells.

Promegakaryocytes

The promegakaryocyte is the next recognizable stage. It has deep blue cytoplasm and contains two to four nuclei that may appear separate but are linked by thin strands of nuclear material (see Figure 27-9). Promegakaryocytes are much larger than WBC and RBC precursors.

Megakaryocytes

The megakaryocyte is the next developmental stage. Megakaryocytes are gigantic (50 to 200 microns in diameter) and contain more than four nuclei that are joined and form a lobulated mass (Figures 27-10 and 27-11). The larger the cell, the greater the nuclear ploidy (number). Megakaryocytes with deeply basophilic cytoplasm are less mature than those with light blue cytoplasm containing eosinophilic granules. Immature megakaryocytes are sometimes called basophilic megakaryocytes, and mature megakaryocytes are sometimes called eosinophilic or granular megakaryocytes. Bare nuclei of megakaryocytes may be seen in bone marrow cytologic preparations. They may represent nuclei of megakaryocytes that have shed their cytoplasm as platelets into the peripheral blood or megakaryocytes whose cytoplasm has been torn from their nuclei during smear preparation.