Diagnostic bone marrow sampling in cats. Currently accepted best practices

Abstract

Practical relevance:

Hematologic disorders are relatively common in cats, as inflammatory, immune-mediated and infectious diseases have the potential to impact erythroid, myeloid and thrombopoietic lines within the bone marrow.

Clinical challenges:

Clinicians often benefit from information obtained from bone marrow cytology and histopathology in diagnosing hematologic disorders in feline patients. However, these sampling procedures are ones that many veterinarians are intimidated by and thus not comfortably performing in clinical practice.

Audience:

This review, aimed at all veterinarians who treat cats, highlights the reasons a clinician may elect to sample bone marrow, and details the collection of bone marrow for both cytology and histopathology. Potential complications and limitations of each procedure are also reviewed.

Evidence base:

The collection of bone marrow for cytology and histopathology is not new to feline medicine and several research studies have investigated various sampling modalities in cats. This article offers a comprehensive review of the currently accepted best practices.

Bone marrow aspiration and core biopsy are safe, reliable and effective techniques used to diagnose many important clinical conditions. Intriguingly, despite these minimally invasive procedures being accessible tools for diagnosing various hematological malignant and non-malignant conditions, many practitioners are deterred from performing bone marrow sampling due to a misperception that the procedures are technically demanding. While proper technique is key to obtaining diagnostic samples, adequate training will allow any veterinarian to confidently perform bone marrow sampling when the patient history warrants this level of diagnostic investigation.

Reasons to sample bone marrow

The primary indication for a clinician to perform bone marrow sampling is persistent complete blood count abnormalities. Common irregularities include neutropenia, poorly and non-regenerative anemias, thrombocytopenia, thrombocytosis, circulating blast cells, and mature cells with atypical morphology (see box below).^1–8 It should be noted that leukemia may be occult, with marked marrow infiltration despite few to no circulating blast cells.^9,10 Thus, bone marrow should be evaluated in patients with suspected marrow malignancy. Concurrent assessment of a complete blood count and peripheral blood smear is strongly recommended whenever bone marrow is evaluated. A thorough diagnostic investigation is of paramount importance for eliminating extra-marrow causes of the documented derangements.

Equipment

Needles

Several types of reusable and disposable bone marrow aspiration and core biopsy needles are available (see box below). Reusable needles are typically made of surgical steel and may be re-sharpened. Disposable needles have a steel cannula and stylet with a plastic handle and syringe hub.

Bone marrow aspiration needles commonly used in feline medicine are the Illinois sternal/iliac type (Figure 1), Osgood needles and Rosenthal needles (Figure 2). The use of 13–19 G needles is most appropriate in cats, dependent on the sampling site and size of the patient. An Illinois sternal/iliac aspiration needle has an adjustable depth guard to allow precise control over the depth of penetration. A sharp stylet easily penetrates the bone, and a twist-off cap securely holds it in place. These needles are compatible with both Luer slip and Luer lock syringes. Osgood needles are extremely fine and of small gauge, which makes them particularly useful in cats. Rosenthal needles are reusable and have large spool-type female Luer hubs and sharp-pointed cannulas. The fitted stylets also have matched beveled points.

Figure 1.

(a) Illinois sternal/iliac aspiration needle. (b) Individual components (anticlockwise from left): needle, cap, stylet and adjustable depth guard

Figure 2.

(a) Rosenthal aspiration needle. (b) Individual components: stylet (left) and needle (right)

Similar to bone marrow aspiration needles, many types of reusable and disposable core biopsy instruments have historically been used. Arguably the most common type of bone marrow collection needle currently used in veterinary medicine is the Jamshidi biopsy needle (Figure 3). As with any biopsy needle, this instrument may be used to obtain both cytology and histopathology samples. The needles are available in many sizes and have lightweight handles, as well as adapters to accommodate Luer lock syringes. Importantly, each has a tapered bevel-ended cannula and a bevel-ended locking stylet that extends beyond the tip of the cannula. The tapered cannula helps to retain the marrow core.

Figure 3.

(a) Jamshidi biopsy needle. (b) Individual components (from left): needle, cap and tip shield, stylet and bone marrow expulsion stylus

Rotary battery-powered sampling devices have recently gained popularity. Two systems are most commonly used in veterinary medicine: the EZ-IO Intraosseous Infusion System and OnControl biopsy system. Such instruments are commonly used in human medicine, particularly in infant and pediatric patients. Studies have suggested these sampling devices yield better diagnostic samples, are easier to use compared with manual systems, and take less time to obtain quality samples (see later).^11,12

Priming solution

To prepare a 5–10% EDTA priming solution, thoroughly mix 0.5 ml of sterile saline in a 7 ml EDTA Vacutainer tube (purple/lavender top tube). Aspirate this mixture into a 12 ml sterile collection syringe.

Patient preparation

The intended sampling location should be shaved and aseptically prepared. The author favors shaving the preferred site prior to administering sedation whenever possible. Patients experience discomfort upon aspiration of bone marrow due to intact endosteal innervation. This discomfort is not adequately eliminated by systemic or local anesthesia alone. Rather, pre-emptive multimodal analgesia is recommended. Local anesthesia with 1–2% lidocaine provides temporary analgesia to the skin, subcutaneous tissues and periosteum. While some patients require general anesthesia, most only need heavy sedation. The sedative/anesthetic protocol should be chosen based on patient-centered considerations. Intubation is recommended to protect and ensure airway patency. Post-procedural analgesia for 48–72 h is appropriate.

The sampling site, typically 6–15 cm², should be surgically prepared. Veterinarians should wear sterile gloves, and the use of a sterile drape is beneficial for maintaining a sterile field. A #11 or #15 scalpel blade is used to make a stab incision in the skin at the intended sampling site. The biopsy needle should be primed by removing the stylet and flushing it with a small volume of the 5–10% EDTA priming solution. The stylet should then be replaced in the core biopsy needle. Approximately 0.05–0.2 ml of the priming solution should be left in the collection syringe.

Aspiration

Flat bones, including the ribs and bones of the skull, have very active hematopoiesis. Unfortunately, sampling bone marrow from these locations is not practical. Common alternative anatomic locations in cats are the greater tubercle of the proximal humerus (Figure 4), the iliac crest (Figures 5 and 6), and the trochanteric fossa of the femur (Figure 7). Occasionally, bone marrow may be sampled from the sternum or costochondral junction. The sampling location ultimately depends on several factors, including patient body conformation, clinician preference and evidence-based data.

Figure 4.

(a,b) Proper placement of an Illinois iliac/sternal aspiration needle at the flattened region between the greater tubercle and the head of the left humerus

Figure 5.

(a,b) Proper placement of an Illinois sternal/iliac aspiration needle in the right iliac crest using a dorsal approach

Figure 6.

Proper placement of an Illinois sternal/iliac aspiration needle in the left iliac crest using a transilial approach

Figure 7.

Proper placement of a sterile hypodermic needle in the trochanteric fossa of the left femur

The general procedure for obtaining a bone marrow aspirate is the same regardless of anatomic location. The aspiration needle should be advanced through the stab incision to the level of the periosteum by firmly and slowly rotating the needle clockwise and counterclockwise. The author recommends stabilizing the aspiration needle by placing the proximal end firmly against the palm of the hand or first metacarpophalangeal joint. Furthermore, holding the needle with the tips of the thumb, index and middle fingers as distally as possible allows maximal stability and control. When the aspiration needle is properly seated in the bone marrow cavity, it should not be necessary to hold it because of its secure positioning.

Once the aspiration needle is properly seated in the desired anatomic location, the veterinarian should remove the stylet and attach the collection syringe containing 0.05–0.2 ml of the priming solution. Bone marrow may then be aspirated by applying one to three short bursts of negative pressure to the plunger of the collection syringe to dislodge bone marrow particles, and then holding the plunger at maximum aspiration volume. Excessive negative pressure bursts on the syringe plunger are not recommended, as this action heats the syringe barrel and promotes coagulation. A variation of this technique involves not precoating the collection syringe with priming solution. Rather bone marrow particles are dislodged as described and subsequently expelled immediately into a Petri dish containing priming solution.

The volume of marrow collected should not exceed 0.4–0.5 ml. If there is no yield initially, the stylet should be replaced and the needle advanced to repeat the aspiration. Aspiration needles should not be advanced without their stylets in place. If there is no yield after three attempts, another sampling site should be used. Alternatively, if a needle is too deeply embedded, it should be gradually withdrawn while gently rotating it and maintaining negative pressure on the plunger of the collection syringe.

The aspirated contents should be gently expelled into a Petri dish to look for marrow spicules/particles (Figure 8); these typically appear granular or as tiny glistening specks. The presence of fat globules confirms the sample is of marrow origin. Smears should be made immediately following collection (see box below).

Figure 8.

Marrow spicules/particles. Courtesy of Dr Ty McSherry

Figure 9.

(a,b) Appropriately prepared marrow slides. Courtesy of Dr Ty McSherry

Core biopsy

The core biopsy needle should be advanced through the stab incision at the site of sampling and seated in the proximal cortex, similar to performing a bone marrow aspiration. As already mentioned, a properly embedded needle should not need to be held in position. Once securely in place, the stylet is removed and the biopsy needle is advanced with clockwise and counterclockwise rotation as far as possible. The cannula should then be withdrawn with rotation 1–2 mm and subsequently re-advanced at a slightly different angle. This process is repeated two or three times in an effort to completely isolate the core specimen securely within the cannula. Once the biopsy needle has been completely withdrawn, the specimen should be extruded in a retrograde fashion using the biopsy probe.

Unfragmented core samples at least 0.5 cm long are considered to be of appropriate quality for histopathologic evaluation. ¹⁶ When bone marrow aspiration is not fruitful, a marrow core can be rolled onto a microscope slide to make a cytology preparation. Bone marrow core samples should be placed in 10% buffered formalin for fixation prior to sending to a reference veterinary laboratory for evaluation by a board-certified veterinary clinical pathologist.

Sample type and site selection

The author recommends routinely obtaining bone marrow samples for both cytology and histopathology, as each yields unique information. Cytology provides superb morphological detail of marrow cells (Figure 10). Examination of histopathologic samples affords excellent evaluation of stromal changes, including myelofibrosis, inflammation and neoplasia. Cytology cannot provide architectural details about marrow adipose tissue and/or fibrous connective tissue. Similarly, it can be challenging to differentiate proliferating bone marrow cells from abnormal ones based on morphology alone. Histopathology cannot elucidate the mechanisms for hypoplasia and aplasia.

Figure 10.

Normal cat bone aspirate cytology at × 10 (a) and × 100 (b) magnification

Ideally one site should be used to obtain the core biopsy and another site to obtain a sample for cytology. However, if only one site is used, the marrow core biopsy should be collected after aspiration, to prevent hemodilution of the aspirated sample. Note that marrow core biopsies cannot be collected from the sternum and costochondral junction (see page 763).

Defarges et al evaluated sternal bone marrow aspiration in healthy cats, ¹⁴ and showed that while the technique was safe and easy to perform, samples were of lower quality compared with those collected from the ilium. In another study by the same group, bone marrow core biopsy was performed in 10 healthy cats, using 13 G Jamshidi type needles to collect samples from the left iliac crest and 15 G needles from the EZ-IO Intraosseous Infusion System to sample the left humeral head. There were no significant differences between the 15 G and 13 G samples. However, the 15 G humeral biopsies were assessed to be easier to obtain than the 13 G iliac biopsies. ¹⁷ These authors also investigated 15 G bone marrow biopsy samples collected from humeral heads in cats with hematologic disorders. They found the procedure was well tolerated and that the results helped facilitate the diagnosis of hematopoietic diseases. ¹⁸

Dhumeaux et al compared manual and power driver-assisted bone marrow sampling techniques in 12 healthy cats. ¹² On day 1, half of the cats were randomized to have marrow aspiration performed at both the proximal humerus and the iliac crest, as well as a marrow biopsy obtained at the iliac wing using an automated power driver device (OnControl). The other half of the cats had the same procedures performed using a manual technique. On day 3, each cat had marrow aspirated from the contralateral humerus and iliac crest, as well as a marrow core biopsy collected from the proximal humerus using the opposite technique from day 1. For all procedures and at all sites, the ‘ease of use’ was determined to be better for the automated technique than for the manual technique (P <0.05). Using the proximal humerus as the site for bone marrow sampling was associated with a higher rate of success (P <0.05), a lower number of attempts (P <0.05), a shorter duration procedure (P<0.05), a higher-rated ‘ease of use’ of the technique (P <0.05) and a better quality sample (P <0.05) when compared with the iliac crest.

Conclusions

Bone marrow examination is an essential procedure for the examination of various hematologic disorders in cats. Obtaining quality specimens for pathologic review is unquestionably important. Use of proper technique is critical to yield an accurate diagnosis and prognostic information. Information gained from bone marrow aspirates and biopsies also allows veterinarians to logically and appropriately prescribe medical interventions for patients.

Key Points

• The clinical skills and confidence to reliably and safely perform bone marrow sampling procedures are within the reach of every veterinary practitioner.

• Patients should be provided with pre-emptive multimodal analgesia.

• The most common sites for sampling bone marrow in cats are the proximal humerus, proximal femur and iliac crest.

• Bone marrow core biopsies cannot be collected from the sternum and costochondral junction.

• The proximal humerus is the preferred location for obtaining a bone marrow core biopsy. Research suggests that use of a rotary battery-powered device (EZ-IO Intraosseous Infusion System) may be the easiest method for sampling at this location.

• Bone marrow core biopsy needles may also be used to aspirate marrow. Ideally the two sample types should be collected from different sites. However, if using only one site, the core sample should be collected prior to aspiration.

• An EDTA blood sample should be submitted for a concurrent complete blood count whenever bone marrow is sampled.

• One bone marrow cytology slide should be stained and evaluated in hospital to ensure adequate sampling. All unstained slides should be submitted for evaluation by a board-certified veterinary clinical pathologist.

Case notes

Sebastian, a 4-year-old male neutered Devon Rex from Sioux Falls, South Dakota, USA, was presented for evaluation of pancytopenia.

Recent history Two weeks prior to presentation, Sebastian had been evaluated by his referring veterinarian for lethargy, hyporexia and exercise intolerance.

A reference voided urinalysis and biochemical profile had been unremarkable. A reference complete blood count identified pancytopenia.

Long-term history Sebastian had been adopted from a local breeder at 8 weeks of age. He had no travel history, and received year-round monthly flea and tick preventative. He was the sole animal in his residence, and was housed indoors only. His vaccinations were current, but he had not received any immunizations or antimicrobials in the past 8 weeks.

Physical examination findings Quiet, alert. Temperature 38ºC; heart rate 210 beats/min; respiratory rate 40 rpm; body condition score 4/9. Thoracic auscultation and abdominal palpation unremarkable. Rectal examination normal. Oral mucous membranes moist and pale pink; capillary refill time 3 s.

Laboratory findings

Complete blood count
Parameter	Result	Reference interval
Hematocrit	20%	26–42%
Red blood cells	4.23 × 106/l	5.0–10.5 × 106/l
Mean cell volume	47 fl	39–55 fl
Mean cell hemoglobin concentration	33 g/dl	30–36 g/dl
Reticulocytes	5.9 × 109/l	<60 × 109/l
White blood cells	2.92 × 106/l	3.8–19.5 × 106/l
Neutrophils	1.7 × 106/l	2.5–12.0 × 106/l
Platelets	187 × 106/l	300–800 × 106/l

Serum biochemistry and urinalysis No abnormalities detected

Infectious disease screening Feline leukemia virus negative; feline immunodeficiency virus negative; PCR panel for vector-borne pathogens (including Bartonella species) negative

Diagnostic imaging Thoracic and abdominal radiographs unremarkable. No overt architectural abnormalities on abdominal ultrasonography

Immunologic testing Saline slide agglutination test negative; red blood cell surface antibodies (IgM, IgA, C3) negative

Bone marrow sampling Marrow cytology and histopathology identified erythroid hyperplasia with maturation arrest evidenced by relatively low numbers of rubricytes, metarubricytes and reticulocytes. Hematopoietic cell necrosis, inflammation and lymphoid aggregates also present

Treatment and follow-up Sebastian was diagnosed with primary non-regenerative immune-mediated hemolytic anemia based on clinical signs, clinicopathologic testing and exclusion of other causes of anemia. Given the signs compatible with anemia (tachycardia, tachypnea), he was blood typed and crossmatched to receive a transfusion of packed red blood cells (pRBCs; 10 ml/kg IV over 4 h). Therapy with prednisolone (40 mg/m2 PO q24h), ciclosporin (5 mg/kg PO q12h), melatonin (1 mg PO q24h), doxycycline (5 mg/kg PO slurry q12h) and folate (500 µg PO q24h) was initiated. Based on clinical signs, two additional pRBC transfusions were required over 4 weeks. Doxycycline was discontinued after 14 days. The hematocrit began to improve and reticulocytosis was noted after 41 days of therapy. Sebastian was successfully weaned from all medications and supplements after 7.5 months.

• What this case demonstrates: Myelonecrosis is relatively uncommon in cats, and should be suspected in patients with pancytopenia. Confirmation of diagnosis requires evaluation of bone marrow. With aggressive therapeutic intervention, patients can make a complete recovery, although response time is expected to be prolonged dependent on the extent of marrow involvement.