Skills Laboratory: How to collect diagnostic bone marrow samples

Bone marrow aspirate evaluation may not be in your primary diagnostic toolbox, but it is essential for diagnosing many disorders. Challenges in obtaining high-quality, diagnostic samples may deter many practitioners from confidently using this technique when the need arises.

In this article, we provide direction in determining when a bone marrow aspirate is indispensable to your workup and outline the techniques for obtaining diagnostic samples. Guidelines for interpretation of bone marrow aspirates are available from other sources.1-10 However, we describe the general cytologic criteria of good-quality aspirates. We also discuss pathologic conditions that render aspirates difficult to collect and provide diagnostic alternatives, such as bone marrow core biopsies.

INDICATIONS FOR BONE MARROW SAMPLING

The primary indication for bone marrow aspiration is abnormal complete blood count results. Once extra-marrow causes have been excluded, abnormalities, such as nonregenerative anemia, neutropenia, thrombocytopenia, or the presence of immature blast cells or mature cells with atypical morphology, should prompt bone marrow evaluation. Findings from the physical examination (fever of unknown origin), radiography (bony lysis), and the serum chemistry profile (hyperproteinemia, hypercalcemia) may also be indications for examining bone marrow. Pathogenic organisms found in the marrow cavity that may lead to fever or hyperglobulinemia include Leishmania, Cytauxzoon, and Histoplasma species. The definitive diagnosis of some neoplasms, such as plasma cell myeloma, and the staging of certain malignancies, such as lymphoma and mast cell tumors, require marrow examination. Finally, bone marrow aspiration is indicated to evaluate marrow iron stores.

There are few contraindications to bone marrow aspiration. Thrombocytopenia is not a contraindication, and, in fact, bone marrow aspiration is often performed to rule out decreased production as the cause of low platelet numbers. However, care should be taken to minimize trauma. Severe bleeding disorders characterized by prolonged coagulation times are more of a concern, and marrow aspiration should be delayed until the coagulopathy is controlled. Risk of bone fracture is ordinarily low, but a fracture may occur if the biopsy needle is inappropriate for the patient's size or if cortical integrity is compromised by disease.

BONE MARROW ASPIRATE VS. BONE MARROW CORE BIOPSY

The advantage of bone marrow aspiration over bone marrow core biopsy is the ability to distinguish individual cellular morphology, which is important in identifying cell lineages; characterizing morphologic abnormalities, especially subtle ones, within a lineage; and calculating ratios, such as myeloid-to-erythroid and maturation ratios. The extensive processing required to prepare bone core biopsy samples alters cellular morphology, rendering individual cellular identification more difficult. On the other hand, bone marrow core biopsies are required to evaluate overall marrow cellularity, architectural relationships, and the presence of myelofibrosis or necrosis. Appreciation of marrow architecture, which maintains spatial relationships, facilitates the diagnosis of osteomyelitis and metastatic disease within the marrow space. Accordingly, bone marrow aspirates and core biopsies are complementary techniques, and both may be required for full and accurate interpretation of the pathophysiologic process.

EQUIPMENT

Selecting appropriate equipment is necessary for acquiring high-quality samples (see boxed text titled "Supply checklist for bone marrow collection"). Bone marrow aspirates are usually obtained with a Rosenthal needle or an Illinois sternal-iliac aspiration needle, whereas bone marrow core biopsies are performed with a Jamshidi biopsy needle. Newer biopsy needles, such as the Goldenberg Snarecoil needle (Ranfac), offer several advantages, but we have no experience with them. Several companies carry both reusable (autoclavable) and disposable instruments, including Becton, Dickinson; Cardinal Health; Dyna Medical; and Jorgensen Laboratories. Disposable needles with plastic handles can be reused after gas sterilization but should be disposed of when they become dull or bent.

Supply checklist for bone marrow collection

Bone marrow aspiration and biopsy needles come in several sizes determined by needle gauge. At least two gauges should be available. A 15- or 16-ga aspiration needle is appropriate for most patients; an 18-ga needle should be available for cats and small dogs. A 1-in needle may be sufficient for many animals, but a 1 5/16-in needle is required for large-breed dogs. Thirteen-gauge Jamshidi biopsy needles are appropriate for most patients; 8-ga needles can be used in larger dogs.

A basic marrow aspiration or biopsy needle consists of two pieces: a hollow needle portion with a proximal collar and a solid stylet that fits the needle bore (Figure 1). The stylet usually has a notch that locks it into the collar of the needle portion. Most needles also have a cap that fits over the proximal end to maintain sterility and facilitate handling.

Figure 1. A bone marrow aspiration needle (left) and a Jamshidi biopsy needle (right) are shown with the stylets removed. The Jamshidi biopsy needle narrows at the tip to retain the sample within the needle bore. To remove the biopsy sample, the crooked wire is inserted retrograde into the needle to push the core through the wider, proximal end.

Additional supplies are common to every clinic. A watch glass, Petri dish, or similar container can be used to separate and collect bone marrow particles from the harvested sample before making smears.

COLLECTION SITES AND PATIENT PREPARATION

Hematopoiesis is most active in the flat bones, such as the skull and ribs; however, bone marrow is collected from sites that are more easily and safely accessed. These include the greater tubercle of the proximal humerus, the iliac crest of the pelvis, and the trochanteric fossa of the proximal femur (Figure 2). Site preference depends on patient conformation and body condition. Precise landmarks and animal positioning are described in the procedure section below.

General anesthesia may be required for fractious animals, but heavy sedation is adequate in most patients. Shave and surgically prepare the site, and perform a local anesthetic block of the skin, subcutaneous tissues, and periosteum with 1% to 2% lidocaine. Endosteal innervation remains intact and is the site of pain on aspiration. Bleeding is typically minimal and requires only local compression when the procedure is completed. If necessary, provide postprocedural analgesia with nonsteroidal anti-inflammatory agents given short-term.

Figure 2. Bone marrow collection sites. (A) The greater tubercle of the proximal humerus. (B) The iliac crest of the pelvis. (C) The trochanteric fossa of the proximal femur.

BONE MARROW ASPIRATION AND SLIDE PREPARATION

There are three keys to collecting high-quality bone marrow aspirates:

1. Place the needle securely within the marrow cavity.

2. Avoid hemodilution.

3. Prevent clot formation once the sample is obtained.

Procedure

Wear sterile surgical gloves when handling the aspiration needle and collecting samples. Small surgical drapes around the site help maintain a sterile field. To facilitate passage of the aspiration needle, make a small stab incision in the skin over the aspiration site with a No. 11 scalpel blade.

A secure grip on the biopsy needle is fundamental to placing it correctly within the marrow cavity. With the stylet locked in place, hold the needle in a modified pencil grip for precise control (Figure 3). Securely lodge the proximal end of the biopsy instrument against the palm of your hand or against the first metacarpophalangeal joint. This contact keeps the stylet in place and permits generation of the force necessary to penetrate the cortex, while the fingers provide stability and control. Compressing the skin and soft tissue around the site with the opposite hand also improves stability.

Figure 3. Hold the collar of the aspiration needle securely between your thumb, index, and ring finger to provide fine control and stability. With the cap in place, lodge the proximal end of the instrument tightly against your first metacarpophalangeal joint.

Proximal humerus. For sampling the proximal humerus, place the animal in lateral recumbency. With your nondominant hand, grasp the elbow to stabilize the limb and flex the shoulder, aligning the humerus parallel to the body wall. The site of aspiration is the flattened area between the greater tubercle and the humeral head.1-4,7,9 Align the needle along the long axis of the bone, and apply penetrating force (forward pressure) perpendicular to the cortical surface (Figure 4).

Figure 4. With the stylet in place, align the needle along the long axis of the humerus and perpendicular to the cortical surface of the greater tubercle. To penetrate the cortex, apply forward pressure while rotating the needle clockwise and counterclockwise.

Femur. For the femur, place the animal in lateral recumbency. Stabilize the femur by grasping the stifle with your nondominant hand. Slight internal rotation of the stifle will enhance exposure of the proximal femur. Position the needle medial to the greater trochanter within the fossa, and direct it toward the stifle aligned with the femoral shaft.1-4,7,9 As with the humerus, apply penetrating force perpendicular to the cortical surface (Figure 5 [figure no longer available]).

Iliac crest. For the iliac crest, place the animal in sternal recumbency with the hindlimbs under the animal so the crest protrudes. This site may be difficult to approach in an obese animal. Identify the widest portion of the dorsal aspect of the wing of the ilium, placing a finger on either side. Position the aspiration needle perpendicular to the crest, and direct it ventrally (Figure 6 [figure no longer available]).1-4,7,9

Failure to maintain a perpendicular relationship to the cortex causes the needle to travel along (or within) the cortex instead of through it. Use steady forward pressure combined with a twisting rotation of the needle to penetrate the cortex and advance the needle into the marrow space 1 to 2 cm depending on the animal's size. Decreased resistance when the needle enters the marrow cavity may or may not be appreciated. If the needle is correctly seated, it will have absolutely no give or wiggle room and will move in unison with the humerus or femur upon flexion of the shoulder or hip, respectively.7

After removing the stylet (Figure 7), attach a 10- or 12-ml syringe to the end of the biopsy needle. Rinsing the syringe barrel with 4% disodium- or dipotassium-EDTA reduces sample clotting during collection. Rapidly apply full suction (pull the syringe plunger to 8 to 10 ml) until the briefest flash of blood is seen (Figure 8), and then immediately release the vacuum. Immediate cessation of suction is crucial to preventing hemodilution.

Figure 7. Once the needle is firmly seated in the medullary cavity, remove the cap, and withdraw the stylet.Gently remove the syringe, and expel the contents onto glass slides or into a watch glass containing EDTA. Alternatively, the entire needle with the syringe attached can be removed from the bone, and the contents of the syringe and needle can be expelled onto glass slides or into a watch glass.

Figure 8. Rapidly apply suction (8 to 10 ml). As soon as a flash of blood appears in the hub of the syringe, immediately release the suction to prevent hemodilution, a common cause of poor-quality samples.

If you place the marrow sample onto glass slides, work quickly to prepare the slides to avoid sample clotting; it is helpful to have a second individual assist with slide preparation. Tilt each slide to allow blood to run off the slide (Figure 9A), place a second clean slide perpendicularly to the first slide, and use it to gently squash the marrow particles or spicules (Figure 9B). Gently pull apart the two slides, spreading the marrow spicules across the slides.

Figure 9A. Express a small amount of marrow onto a slide, and tilt the slide to allow excessive blood to flow away from the spicules.

If you place the sample in a watch glass, tilt the container to allow the blood to drain away, revealing marrow spicules. Marrow spicules look like pale yellow, glistening grains of sand. Gently retrieve the spicules with the end of a pipette, microhematocrit tube, or hypodermic needle. Place the spicules on a glass slide, and gently crush them with a second clean glass slide. The marrow and blood remaining in the watch glass can be transferred to an EDTA vial and submitted with the smears in case additional slides need to be prepared. Clotted marrow samples can be placed in formalin and processed like a biopsy sample.5

Figure 9B. Place a second slide on top of the spicules perpendicular to the first slide, and gently squash the spicules as you pull the second slide across the first.

Allow the smears to dry before staining. Marrow spicules on an unstained slide appear as light grainy areas (Figure 10) and are usually surrounded by blood.

Figure 10. The unstained slide (left) has several light grainy areas (arrow). The light areas are the squashed marrow particles surrounded by blood. The stained smear (right) contains deeply basophilic cellular spicules surrounded by blood.

Sample review and submission

Before moving the patient to recovery, stain and examine one slide to ensure sample adequacy. Diff-Quik stain (Dade Behring) or another rapid stain is acceptable for this review. On gross examination of the stained slide, a high-quality cellular sample should contain several small, deep-blue streaks within a pink background of blood (Figure 10). On microscopic examination, there should be several dense blue spicules surrounded by monolayers of hematopoietic cells (Figure 11A). If the smear is poorly cellular (Figure 11B), a second aspiration should be attempted slightly away from the original collection site or from a different site. If a cellular aspirate cannot be obtained, collect a core biopsy sample at this time as the marrow pathology may preclude collection of a cellular aspirate.

Figure 11A. Note the highly cellular spicule with abundant iron (white arrows), several large megakaryocytes (black arrows), a heterogeneous population of hematopoietic cells, and large, lipid vacuoles.

Label and submit two to four unstained slides containing the bone marrow aspirate to a veterinary clinical pathologist along with the results of a recent complete blood count, preferably performed at the same time as the aspirate, and a blood smear. Accurate interpretation of a bone marrow sample depends on knowledge of the peripheral hematologic findings. Include the patient's signalment, a brief history that includes information about previous and concurrent illnesses and recent drug therapy and the reason for taking the aspirate on the submission form. If bone marrow core biopsy samples in buffered formalin accompany the aspirate, the formalin vial and cytology slides must be completely isolated from one another because formalin vapors will fix the cytology slides, rendering them unstainable. Additional unstained smears should be submitted if special stains are requested (Prussian blue for iron) or anticipated (cytochemistry or immunochemistry for determination of lineage in leukemias).

Figure 11B. The microscopic appearance of a poorly cellular spicule, which may be secondary to poor sampling or reflect true hypocellularity.

Nondiagnostic samples

Low-cellularity or poor-quality samples may be a result of inadequate technique, including improper seating of the needle in the marrow cavity, excessive aspiration resulting in hemodilution, or sample clotting before slide preparation. However, several pathologic conditions prevent acquisition of cellular marrow aspirates. Myelofibrosis often results in poorly cellular samples because of abundant collagen and reticulin fibers. Aspirates from patients with aplastic anemia typically yield only adipose tissue with few poorly cellular to no hematopoietic spicules. In cases of myelofibrosis or aplastic anemia, core biopsies are required to identify the presence of fibrosis or the replacement of hematopoietic tissue by adipose tissue, respectively. As mentioned in the introduction, bone marrow aspiration and core biopsy are complementary techniques, so providing both samples will maximize the diagnostic information.

BONE MARROW CORE BIOPSY

A bone marrow core biopsy can be performed from the same bone by selecting a site a few millimeters away from the aspiration site, permitting preparation of a single site. Alternatively, the biopsy sample can be obtained from a different site. Biopsy sampling from the ilium can be performed by using the same approach as for aspiration or by using a lateral-to-medial approach through the dorsal aspect of the wing.7,9 The latter may be preferable in cats and small dogs with thin ilial wings.

Figure 12A. Once the needle has gained purchase in the cortex, remove the stylet. Then drive the biopsy needle into the medullary cavity in a manner identical to collecting an aspirate.

Remaining perpendicular to the cortex, insert the needle in the same manner as for the aspiration, but remove the stylet once the needle has purchase in the cortex (Figure 12A). After removing the stylet, continue to advance the needle into the marrow for about 1 cm, depending on patient size. After the needle is firmly seated, rock the needle vigorously and rapidly back and forth in two or three directions (Figure 12B). There will be little or no obvious lateral motion in the needle if it is securely seated. Then slightly retract, redirect, and slightly advance the needle to cut the core, which will be retained within the needle bore. Remove the biopsy needle from the bone with a continued twisting motion. Expel the core from the needle by placing the wire in the cutting end of the Jamshidi biopsy needle and gently pushing the small piece of cortical bone and marrow retrograde from the needle.

Figure 12B. After the needle is well-seated, vigorously rock it back and forth in two or three directions (red arrows) to cut the core and retain it within the biopsy instrument. Then withdraw the needle with a rapid twisting motion.

If the biopsy is performed correctly, the sample will have a white end (cortex) and a red strip (marrow) (Figure 13). A core that is 0.75 to 1 cm long is sufficient. If the first attempt is unsuccessful, another attempt can be made adjacent to the first. Before placing the sample in 10% buffered formalin for submission to a surgical pathology service, you can gently roll it on a glass slide for cytologic samples if an adequate aspirate has not been obtained. Be careful not to crush the sample, resulting in a nondiagnostic biopsy sample.

Figure 13. A gross specimen of a bone marrow core biopsy (unfixed). Note the white cortex (right) and red marrow (left). Shorter core samples (0.75 cm or more) are acceptable as long as sufficient red marrow is present. Marrow with decreased erythroid activity may be pale. The marrow tissue often appears gelatinous before formalin fixation.

CONCLUSION

This review is intended to provide the tools you need to become proficient in acquiring diagnostic bone marrow samples. Once confident in your skills, you may be surprised how often you will include this modality in your diagnostic work-up to provide high-quality care for your patients.

Kristen R. Friedrichs, DVM, DACVP

Karen M. Young, VMD, PhD

Department of Pathobiological Sciences

School of Veterinary Medicine

University of Wisconsin

Madison, WI 53706

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