Introduction

In general, a biopsy should be performed before definitive treatment if the results will change the therapy or influence the owner's thoughts to treat their animals. The purpose of this lecture is to introduce special sampling techniques that are used at the Japan Small Animal Cancer Center. During the presentation, all techniques will be demonstrated by pictures and video clips.

Bone Marrow Aspiration Cytology Using Anticoagulant¹

1.  Indication: Blood cell production abnormality and morphology assessment, staging procedure for hematopoietic and non-hematopoietic malignancies.

2.  Pros: The marrow and blood will not clot during the procedure. Hemodilution of bone marrow sample is minimized.

3.  Cons: A single sample may not be representative of the entire bone marrow. Limitation of cytology.

4.  Precaution: Coagulopathy; significant hemorrhage is rare even for patients with thrombocytopenia.

5.  Instruments and supplies required: Illinois bone marrow needle (15-gauge for middle to large dogs, 18-gauge for small dogs and cats), local anesthesia and systemic analgesia and/or tranquilization, #11 scalpel blade, 12 ml syringe, (watch?) glass, 1–2 ml 4% EDTA 2K isotonic fluid solution (do not use heparin as an anticoagulant)^a, pipets and pipet tips, microscope slides, cytology stains (e.g., Diff-Quick, Wright-Giemsa, other).

6.  Technique and tips to obtain a good sample:

a.  The rostral aspect of the greater tubercle of the humeral head is the ideal location.

b.  Clip the hair and prepare the bone marrow aspiration site aseptically with a surgical scrub.

c.  Using 25-gauge needle, inject a small amount of 2% lidocaine in and around the site where the bone marrow needle will be inserted.

d.  Identify the insertion site and make a small stab incision with a #11 surgical blade.

e.  Advance the bone marrow needle with the stylet in place. When the needle is firmly fixed in the bone, remove the stylet and attach a 12 ml syringe. To prevent extra hemodilution or blood clot in the bone marrow needle, the procedure should be performed as fast as possible once the tip of the needle is in the marrow.

f.  Aspirate the bone marrow sample into the syringe. The author prefers that a small amount (0.5–1ml) of 4% EDTA is inserted in the syringe before the aspiration. The marrow and blood will not clot if the sample is diluted 50:50 with the 4% EDTA solution.

g.  Expel the marrow and blood into a watch glass. The marrow-rich spicules are selected by a pipet and gently smeared on microscope slides. Select the marrow-rich spicules carefully since the spicules often resemble fat drops.

Traumatic Catheter Biopsy for Bladder Mass²

1.  Indication: Urinary bladder mass.

2.  Pros: Tissue samples can be obtained without surgery. No risk of tumor seeding through the abdomen.

3.  Cons: Difficult to obtain samples from a mass located on non-trigone area. Requires general anesthesia or at least sedation and good analgesia.

4.  Precaution: Coagulopathy; biopsy specimens should be taken with caution since the bladder wall is very fragile due to the presence of the underlying disease.

5.  Instruments and supplies required: 5F or 8F polypropylene urinary catheter (open-ended or closed-ended with side holes), 12 ml syringes, 18-gauge needles, pre-cleaned microscope slides, 5–10 centrifuge tubes and centrifuge, sterile saline, pipet, 10 ml of 10% buffered formalin, cytology stains (e.g., Diff-Quick, Wright-Giemsa, other), ultrasound machine (US).

6.  Technique and tips to obtain a good sample:

a.  Identify the bladder mass with US.

b.  Insert the catheter and replace urine with 10–15 ml of sterile saline. Advance the catheter forcefully into the tumor (see Figure 1). This is best accomplished with ultrasonography.

c.  Apply suction while withdrawing the catheter (see Figure 2). Tissue that has been suctioned into the catheter will come out with some saline. The tissue-contained saline is centrifuged (1,500 rpm for 2–3 minutes) and the supernate is discarded.

d.  Repeat the procedures until enough sediment is obtained.

e.  All sediment are collected in one tube and filled up with 10% buffered formalin for histopathological evaluation (cell-pack).

f.  Smears are also made from the sediment for cytological evaluation before the tissue is formalinized.

Figure 1. The catheter is inserted in the mass in the urinary bladder.

Figure 2. The tumor is suctioned from a catheter with a side hole.

Ultrasound-Guided Fine Needle Aspiration for Lytic Bone Lesions³

1.  Indication: Lytic bone lesion.

2.  Pros: Quick, easy, cost-effective, safe and minimal patient discomfort. The risk of pathologic fracture and wound infection are also minimal. FNA is less likely to seed neoplastic cells into the surrounding tissues.

3.  Cons: Limitation of cytology; usually diagnosed as "sarcoma" and often does not allow for further classification of primary bone tumors.

4.  Precaution: Coagulopathy.

5.  Instruments and supplies required: 22 or 23-gauge, 1–1.5 inch needles, 6–12 ml syringes, pre-cleaned microscope slides, ultrasound machine (US).

6.  Technique and tips to obtain a good sample:

a.  After induction of anesthesia or administration of sedation, the aspiration site is clipped and surgically scrubbed.

b.  Identify the region with cortical defects by US. These areas typically appear hypoechoic.

c.  A 22 or 23-gauge needle is inserted through the cortex into the medullary cavity. The author usually does not apply negative pressure to avoid blood dilution.

d.  Remove the needle once serosanguineous fluid or blood is obtained in the hub of the needle.

e.  Make multiple smears in order to increase the accuracy of diagnosis.

Endnotes

a. How to make 4% EDTA solution for bone marrow aspiration:
Ingredients: EDTA 2K Powder 2.0 grams and sterile saline, enough to make a TOTAL volume of 50 ml.
Equipment: 250 ml beaker, weighing scale, 60 ml syringe, sterilizing filter, 18-gauge needles, 21-gauge needle, 50 ml sterile empty vial.
Procedure: Weigh out 2.0 grams of EDTA 2K powder. Place the powder in a beaker. Add 50 ml sterile saline to beaker. Agitate the beaker to confirm that the powder is completely dissolved. Using a 60 ml syringe with 18-gauge needle, draw out EDTA solution from the beaker. Place a sterilizing filter and new 18-gauge needle onto syringe and inject into 50 ml sterile empty vial. May use 21-gauge needle to vent vial to make solution flow into vial easier. Your finished product is a 4% EDTA solution.
Storage: EDTA 4% injection should be stored in the refrigerator. We give it a 30 days expiration date if stored properly.

Japan Small Animal Cancer Center, 2-27-4 Nakatomiminami, Tokorozawa, Saitama, 359-0003, Japan, Phone: 81-4-2943-8699, Fax: 81-4-2943-8698, Web page: www.jsamc.jp

References

1.  Ogilvie GK, Moore AS. Bone marrow aspiration and biopsy. In: Managing the Canine Cancer Patient: A Practical Guide to Compassionate Care. Yardley, PA, Veterinary Learning Systems, 2006:78–81.

2.  Ogilvie GK, Moore AS. Lower urogenital tract biopsy. In: Managing the Canine Cancer Patient: A Practical Guide to Compassionate Care. Yardley, PA, Veterinary Learning Systems, 2006:82–86.

3.  Britt T, Clifford C, Barger A, et al. Diagnosing appendicular osteosarcoma with ultrasound-guided fine-needle aspiration: 36 cases. J Small Anim Pract 2007;(48):145–150.