Contraindications

Bone marrow aspiration biopsy is a relatively innocuous procedure in horses. Excessive hemorrhage is theoretically possible after bone marrow aspiration, but this is extremely rare.⁴ If severe thrombocytopenia (<20,000 platelets/μl), clotting factor deficiencies, or disseminated intravascular coagulation is present, any hemorrhage usually can be corrected by applying direct pressure to the aspiration site for 4 to 5 minutes.¹ When aspirating marrow from sternebrae, the needle should be placed carefully to prevent cardiac puncture. One horse died after sternal bone marrow aspiration because of left ventricular laceration and subsequent cardiac tamponade. Monoclonal gammopathy and defective hemostasis in this horse probably contributed to development of cardiac tamponade.⁵ Iatrogenic infection is theoretically possible but is unlikely when the aspiration site is properly prepared and aseptic technique is followed.⁴

Specimen Collection

Marrow aspiration is simply tissue aspiration through cortical bone. Bone marrow can be collected from the sternum, ribs, or ilium using an 18-gauge spinal needle. The aspiration site should be clipped, cleaned with surgical disinfectant, and locally anesthetized with 2% lidocaine. Both the subcutis and the periosteum should be infiltrated with the local anesthesia solution.

A small stab incision is made in the skin with a #11 scalpel blade. The needle is inserted until the periosteum is penetrated and marked resistance encountered. The cortical bone is penetrated by applying steady pressure while rotating the spinal needle. The extent of bone penetration and effort required to obtain bone marrow depend on the site of aspiration and the animal’s age. Generally, 1 to 2 cm of penetration of the tuber coxae are sufficient in the adult, and similar sites in the foal require less penetration. Cortical bone is more resistant to needle penetration in adults, and considerably less effort is required to penetrate cortical bone in foals.

Once the spinal needle is properly seated in the marrow cavity, the stylet is removed from the needle, and bone marrow is aspirated into a 12-ml syringe containing 1 to 2 drops of 10% disodium ethylenediaminetetraacetate (EDTA) or 1 to 2 ml of a 2% EDTA solution. Strong suction usually is necessary to aspirate bone marrow; the syringe plunger can be withdrawn to the 10-ml mark with little damage to hematopoietic cells. About 0.2 ml (2 to 4 drops) of marrow are aspirated into the syringe. If marrow cannot be aspirated, the stylet is replaced, and the needle is repositioned slightly (proximally or distally) depending on clinical judgment. Another attempt to aspirate bone marrow is then made. When marrow aspiration is complete, the syringe may be detached, and the specimen and anticoagulant are mixed with a swirling motion. If small bone marrow particles can be observed along the barrel of the syringe, the spinal needle is removed.

Occasionally, bone marrow may be collected at necropsy. Specimens must be obtained within 30 minutes of death because subsequent cellular degeneration may preclude adequate examination. Bone marrow is most easily and quickly obtained by cracking a rib, carefully removing cortical bone fragments, and gently recovering the gelatinous marrow. The marrow specimen can subsequently be used to prepare cytologic touch imprints or smears, after which the remaining marrow can be preserved in 10% neutral-buffered formalin solution for histologic evaluation.

Specimen Preparation and Staining

A few drops of marrow suspension are expelled onto a slide that is held vertically (Fig. 15-1). Marrow particles adhere, whereas blood flows down the slide. Squash preparations are made by holding a second slide at a right angle to the first slide and pressing gently while pulling the second slide laterally (Fig. 15-1). Alternatively, the marrow may be expelled into a Petri dish, where particles may be selected with a Pasteur pipette. Particles are placed on a slide, and squash preparations are made as described earlier. For marrow core biopsies, the core can be rolled gently between two slides to produce cytologic specimens before tissue fixation. For necropsy specimens, a small portion of marrow is used to prepare cytologic touch imprints or squash preparations.

Fig. 15-1 Bone marrow squash preparation. Left, Marrow is expelled onto a slide held vertically. Marrow particles adhere as blood flows downward. A second slide (smear slide) is held at a right angle to the first slide. Right, Smears are made by pressing gently and pulling the second slide laterally. The completed smear is labeled, air-dried, and stained.

Once air-dried, bone marrow specimens may be stained by the classic Wright’s stain technique or rapid modification of the Wright’s stain procedure using Diff-Quik stain. With the classic Wright’s stain, the staining and buffering times should be extended to 5 minutes each because of increased cellularity of the preparations. With the Diff-Quik procedure, bone marrow smears should be dipped in each solution a total of 10 to 20 times to account for increased cellularity. Under low microscopic magnification (10× objective), red, blue, and purple colors should be apparent. Lack of purple color, especially in cell nuclei, indicates insufficient staining. The preparation can be salvaged by repeating the staining procedure on the original smear until the desired tinctorial properties are achieved. (See Chapter 14 for more information on staining procedures and problems.)

Particle Cellularity

Precise evaluation of bone marrow cellularity requires histologic examination of bone marrow core biopsies taken from a site of active hematopoiesis or disease. Bone marrow core biopsy is not performed routinely in many private practices, so marrow cellularity is assessed cytologically using stained marrow smears and the 10× microscope objective. Bone marrow particles in cytologic preparations consist of fat (appearing as clear spaces or balloon-like adipocytes) and clusters of hematopoietic cells, in which dense aggregates of cells stain dark bluish purple. Normocellular particles observed in healthy adult horses consist of 50% fat and 50% cells (Fig. 15-2). Hypocellular particles consist mainly of fat (Fig. 15-3), and hypercellular particles are composed of dense cellular aggregates (Fig. 15-4). Hypocellular bone marrow particles suggest decreased hematopoiesis. In contrast, hypercellular particles suggest increased hematopoiesis (normal or abnormal response), infiltrative disease, or primary or metastatic neoplasia. Examination of the cellular composition of the marrow at higher magnification determines which of these conditions exists.

Fig. 15-2 Normocellular bone marrow particle consisting of 50% fat (clear spaces) and 50% hematopoietic cells.

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