How to Obtain a Diagnostic Bone Marrow Sample From the Sternum of an Adult Horse

1. Introduction

Equine bone marrow analysis is an often underused technique that can provide invaluable diagnostic and prognostic information for horses with quantitative or qualitative abnormalities of blood cells [1-3]. The primary function of bone marrow is the production of erythrocytes, leukocytes, and platelets. Consequently, abnormalities in the bone marrow are often reflected in the numbers or morphology of circulating blood cells. Conversely, systemic disease that alters the number or function of blood cells often results in compensatory changes in the bone marrow production of those cells. Abnormalities in the number or morphology of circulating erythrocytes, platelets, or leukocytes that cannot be adequately explained by consideration of history, physical examination, or routine laboratory testing are indications for bone marrow analysis. These indications include unexplained prolonged anemia, polycythemia, pancytopenia, leucocytosis, thrombocytopenia or thrombocytosis, or observation of atypical or neoplastic cells in the peripheral circulation (Table 1). Bone marrow analysis may also be included in the diagnostic plan for horses with chronic unexplained hypercalcemia or hyperproteinemia, which are laboratory abnormalities that are occasionally seen in horses with lymphosarcoma or multiple myeloma, respectively. Bone marrow aspirates and core biopsies are relatively easy to perform and are associated with minimal complications. Although bone marrow sampling is not often indicated in equine medicine, properly obtained samples can provide invaluable diagnostic and prognostic information that cannot be easily obtained in any other manner.

2. Materials and Methods

Diagnostic bone marrow aspirates and core biopsies may be obtained from the sternebrae in most adult horses [1]. Before beginning the procedure, all necessary equipment should be assembled (Table 2). A variety of bone marrow aspirate and biopsy needles are available for use in horses. The author prefers to use a Jamshidi disposable Illinois sternal/ iliac-aspiration needle (1.875 in, 15-g) for aspiration of marrow from the sternebrae of adult horses. These needles have a plastic guard over the distal aspect of the needle that can be removed if more depth of penetration is needed (as is frequently the case in adult horses). Jamshidi disposable bone marrow biopsy/aspiration-needles (11-g, 4-in) are preferred for obtaining core biopsies from the sternebrae of horses (Fig. 1).

MCV = Mean corpuscular volume; M:E = Myeloid to erythroid ratio; TIBC = Total iron binding capacity.

Bone Marrow Aspirates

The horse should be appropriately restrained using sedation and/or stocks as indicated by the temperament of the patient. IV administration of xylazine at 0.3 mg/kg and butorphanol at 0.01 mg/kg provides sufficient restraint for most adult horses. The sternebrae are palpated on ventral midline in the cranial thorax at the level of the elbows. Hair is clipped, and a wide area is surgically prepped; ≈6 ml of 2% lidocaine is infiltrated through the subcutis and muscle layers and into the periosteum to provide local anesthesia (Fig. 2). The area is briefly prepped again, and the clinician wears sterile gloves to perform the procedure.

Table 2. Equipement ans supplies needed to obtain a bone marrow aspirate and core biops from an adult horse.

Figure 1. Jamshidi bone marrow biopsy needle (11-g, 4-in). The unit consists of an external needle (left), stylet (middle), screw cap to cover the stylet (top), and a separate hooked probe (right) that is inserted into the needle to forcibly eject the biopsy specimen from the needle after sample collection.

Figure 2. Infiltration of the subcutis, muscle, and periosteum with 2% lidocaine.

A #10-surgical blade is used to make a stab incision through the anesthetized skin (Fig. 3). The bone marrow aspirate needle is introduced through the stab incision and passed through the muscle until it makes contact with the surface of the sternebrae, just lateral to midline. Some horses have a marked angulation to the ventral surface of the more cranial sternebrae. In these horses, the needle should be angled slightly toward midline, perpendicular to the surface of the bone, so that it is less likely to slip when pressure is applied. A slow, back-and-forth, rotational movement is used to advance the needle until it is firmly seated in bone to a depth of ≈1 - 2 cm. The stylet is removed, and a 12-ml syringe (containing 0.5 - 1.0 ml of 2 - 3% ethylenediamine tetra-acetic acid [EDTA] in saline or sodium citrate anticoagulant) is attached to the hub. Quick, repeated, forceful aspirations are applied to dislodge spicules and marrow particles with minimal peripheral blood contamination. When blood is evident in the hub of the syringe and within the anticoagulant fluid, the syringe is detached from the needle.

Figure 3. Stab incision through the skin to facilitate needle placement.

Figure 4. An equine bone marrow aspirate has been obtained using anticoagulant in the aspiration syringe. Bone marrow spicules are observed clinging to the plastic Petri dish.

The contents of the syringe are ejected into a clear Petri dish and grossly examined for evidence of small, grayish marrow spicules and fat particles that adhere loosely to the plastic (Fig. 4). If spicules and fat are observed, indicating that a sample of adequate quality has likely been obtained, the marrow aspiration needle is removed, and sterile gauze with antibiotic or povidone iodine ointment is held over the area with light pressure to facilitate hemostasis.

If a marrow sample of sufficient quality is not obtained on the first attempt, needle placement is altered, and the procedure is repeated. If multiple attempts to obtain a diagnostic aspirate are unsuccessful, a core biopsy should be considered.

A Pasteur pipette is used to transfer bone marrow particles from the Petri dish to a glass slide. A second slide is placed on top of the first slide, light pressure is applied, and the two slides are slowly pulled apart. As many as 5 - 10 slides should be prepared in this way in case special stains are deemed necessary. Slides are allowed to air dry, and at least one is stained with a Wright’s-Giemsa- type stain (e.g., Dif Quik). The stained slide is scanned microscopically on low power to ensure that the sample is of sufficient quality and contains hematopoietic precursor cells. If marrow particles are not observed or if the number of nucleated cells is low, reaspiration or core biopsy should be considered. If samples are considered to be of sufficient diagnostic quality, any remaining marrow in the Petri dish may be placed in an appropriate sample tube (e.g., Vacutainer tube with EDTA anticoagulant) and submitted to the lab with the stained and unstained slides. This will enable laboratory personnel to prepare additional slides if needed or to perform ancillary testing, such as microcentrifugation, to obtain buffy-coat cells. An EDTA-anticoagulated peripheral blood sample should be simultaneously submitted for a complete blood count (CBC) to facilitate accurate interpretation of the bone marrow results.

Figure 5. Placement of the Jamshidi bone marrow biopsy instrument so that it is lodged in the cortex of the bone before removal of the stylet and advancement into the marrow cavity.

Bone Marrow Core Biopsy

The technique for obtaining a bone marrow core biopsy is similar to that for marrow aspiration. The site is prepped and blocked, and a stab incision is made. The needle is inserted into the bony cortex (Fig. 5). The screw cap is removed, and the stylet is withdrawn before the needle is advanced an additional 1 - 2 cm into the marrow cavity. The needle is forcibly rotated at that site to facilitate detachment of the biopsy specimen. The needle is withdrawn from the bone, and the hooked probe is inserted to force the biopsy specimen from the needle. A good-quality marrow biopsy should include 1 - 2 cm of obvious red marrow, often with some white cortex as well (Fig. 6). The biopsy specimen may be rolled gently across a glass slide to obtain touch impressions for immediate cytologic evaluation. It is then placed in 10% neutral buffered formalin for submission to an appropriate diagnostic laboratory. A CBC should be obtained at the same time as the biopsy specimen.

Figure 6. Cylindrical bone marrow biopsy specimen with deep red marrow and white cortex.

3. Results and Discussion

Diagnostic bone marrow samples are most consistently obtained from the sternebrae of adult horses [1]. The tuber coxae is an excellent alternative site for marrow collection in foals and young adult horses (<3 yr), but, the marrow cavity is comparatively deep at this site in older horses, making it more difficult to obtain quality samples. The marrow cavity of the ribs remain hematopoietically active throughout the life of the horse; however, inadvertent penetration of the intercostal muscles with associated complications including possible pneumothorax or hemothorax is possible if the needle slips from its intended placement on the rib.

The choice of bone marrow sampling technique (aspirate or core biopsy) is made after consideration of the information desired from the sample, the practitioner’s level of expertise with each technique, the temperament of the horse, and the availability and cost of appropriate laboratory support. Aspirates are easier to obtain than core biopsies and require minimal laboratory processing before evaluation so that results are often available more quickly. However, these samples may be significantly hemodiluted because of hemorrhage at the time of sampling, occasionally making it difficult to determine if low cellularity is indicative of a primary disease process or poor sample quality. Bone marrow core biopsies are technically more difficult to obtain than aspirates, but they are essential for confirmation of suspected generalized bone marrow suppression, hypocellularity, myelofibrosis, or aplastic anemia. In many horses, simultaneous collection of both marrow aspirate and core biopsy is recommended.

The addition of anticoagulant to the syringe used for bone marrow aspiration is not essential; however, it does prevent clot formation, increasing the amount of time available for completing the procedure and enhancing the diagnostic quality of the sample. In general, EDTA is the preferred anticoagulant, because it better preserves cellular morphology and permits more accurate cytologic evaluation than sodium citrate. If anticoagulant is not used, samples should be transferred to glass slides immediately after aspiration. Direct smears and squash preparations are made.

These techniques have been used successfully to obtain diagnostic bone marrow aspirate and core biopsy samples from numerous horses with a variety of clinical problems. They have also been used by the author without complication to serially evaluate the bone marrow in horses experimentally infected with equine infectious-anemia virus [4]. Reported complications of sternal bone marrow sampling in horses include rare cases of cardiac puncture [5]. Risk can be minimized by careful placement of the biopsy instrument at right angles to the surface of the sternebrae and closely monitoring and controlling depth of penetration of the needle during the procedures. Infection in the bone marrow or SC tissues is a theoretical concern after bone marrow collection; however, the risk seems to be minimal if appropriate sterile technique is maintained. Hemorrhage is a potential complication, especially in horses with coagulation abnormalities such as severe thrombocytopenia. Direct pressure at the biopsy site is sufficient to control hemorrhage in most horses. The author has successfully obtained diagnostic bone marrow samples from patients with platelet counts of <10,000/µl at the time of sampling. Owners of horses with possible coagulation problems should be warned of the potential for hemorrhage. If the patient is severely compromised, a fresh, whole-blood transfusion before biopsy should be considered.