Bone marrow aspirates allow for detailed assessment of cell morphology and so can identify relatively subtle changes such as dysplastic features, disordered or arrested development of an individual cell line and allow for detailed evaluation of a leukaemic infiltrate. However it is difficult to assess marrow cellularity or for the presence of fibrosis with aspiration and these are better assessed with a core biopsy. Since the aspirate and core give different information the two samples are often taken at the same time.
Depending on the site of aspiration, the bone marrow aspiration and core biopsy can be performed under general anaesthesia or with sedation and local anaesthesia. The proximal humerus is generally the preferred site although general anaesthesia will be required for appropriate positioning of the patient and because the procedure is painful. Where there is a contraindication for general anaesthesia, the iliac crest can be used with a combination of sedation analgesia and local anaesthesia. The trachanteric fossa and tibial crest are other possible sites.

A high quality bone marrow needle with stylet is required. The main choice is between locked stylet (Jamshidi type) where the stylet cannot be pushed back during insertion and the non locking stylet (Klima and Rosenthal needles) where care has to be taken to keep the stylet in position during insertion. The Jamshidi can be used to obtain both aspirates and core samples, whilst the Klima is only used for aspirates. Do not attempt to perform this procedure a spinal needle. The size of needle should be appropriate to the patient and the site being aspirated.

Other required equipment includes microscope slides, a scalpel blade, lignocaine, sterile gloves and drape, a 20 ml syringe and sterile anticoagulant. CPDA collected from a blood transfusion bag is preferable, although a solution of EDTA prepared from a blood collection tube is adequate. EDTA solution is prepared by filling an EDTA tube with the appropriate amount of sterile saline. Ten or so microscope slides are placed leaning on a near vertical slope, eg against a sandbag. Further slides are required for making smears and are positioned nearby.

Procedure for sampling from the greater tubercle of the humerus.

• The patient is anaesthetised and be placed in lateral recumbency. This should be right lateral recumbency for a right handed operator and left lateral recumbency for a left handed operator
• The area over the shoulder joint is clipped and surgically prepared
• An assistant flexes both elbow and shoulder joints and rotates the elbow inward towards the body wall, such that the shoulder joint is turned outward. The spine of the scapular and the acromium process at its distal end are palpated. The next bony prominence distal to this is the greater tubercle of the humerus which is the site of the aspirate
• Local anaesthetic e.g. 2% lidocaine is instilled (0.25ml in cats and up to 1-2ml in dogs) into the area, firstly into the subcutaneous tissues using a 23 gauage needle. The needle is advanced down to the surface of the greater tubercle and lignocaine is injected into the periosteum by applying pressure on the needle/syringe
• A small skin incision is made using a scalpel blade over the distal end of the greater tubercle
• The Jamshidi needle is primed with the anticoagulant solution. The stylet is withdrawn from the needle and with the 20ml syringe anticoagulant is flushed through the needle and emptied. The stylet is replaced and locked in place
• The Jamshidi needle is held in the palm of the hand using a pistol grip, with the heel of the needle in the palm of the hand and the needle being supported by the index finger
• The needle is inserted into the greater tubercle parallel to the long axis of the humerus, aiming for the point of the elbow. It may be difficult to get a purchase in the bone, but with repeated twisting of the needle to and fro with firm pressure the needle tip enters the bone cortex. Now further firm pressure is applied whilst rotating the needle to and fro to advance the needle. Similarly firm counter pressure needs to be applied by an assistant to stop the animal being pushed off the table
• Once in the medullary cavity the needle should feel firmly lodged and it should not be possible to wobble the needle from side to side. Indeed the animal could be lifted off the table by raising the needle
• The stylet is removed and the 20ml syringe attached. The syringe is then firmly aspirated by pulling sharply on the plunger 2 or 3 times, applying around 15 mls of suction each time and holding the barrel of the syringe firmly. Bone marrow (thick bloody material) should appear in the syringe. Once marrow appears further aspiration should not be attempted since this could result in haemodilution
• The syringe is removed, leaving the Jamshidi needle in place and marrow is quickly transferred to glass slides before it clots
• A drop of marrow is placed at the top of each of the tilted glass slides. Blood runs down to the base of the slides, leaving marrow spiccules on the slides. Squash smears are made by placing a second slide flat over the first slide at right angles to it, (flattening spiccules) and the drawing the second slide quickly and smoothly over the first slide
• One or two slides should be stained with a rapid dunking stain kit to ensure marrow spiccules have been harvested. Spiccules appears as densly stained blue areas, but it is preferable to check under the microscope that the samples are of diagnostic quality and not excessively haemodiluted
• A core biopsy sample can then be obtained by advancing the needle a further 2-3cm down the humeral shaft without the stylet in place. Once advanced, the needle is moved sideways quickly in different directions or rotated repeatedly in the same direction, to ensure the core is sectioned at its base
• The needle is then withdrawn. The core is pushed out of the needle by placing a blunt probe in the needle tip end and pushing the core sample out of the handle end
• Impression smears can be made by rolling the core down a glass slide, before placing it in formalin for histopathological analysis
• A sterile dressing is placed and opioid analgesia given for 4-6 hours post procedure.

If attempts at marrow aspiration are unsuccessful this may be the result of poor technique or may be as a result of myelofibrosis or myelopthesis. Aspiration can be attempted at another site such as the other greater tubercle or iliac crest. If repeated aspirates are unsuccessful a core biopsy should be taken. An impression smear of the core can be made by rolling the core onto a glass slide. Lysine coated slides may harvest more cells than standard cells.

Making the Smear (see images above)

An air-filled syringe is attached to the needle (if the syringe was already attached to the needle it should be disconnected from needle, filled with air and then re-attached to the needle). Holding the tip of the needle over a clean glass slide, the plunger is briskly depressed, thus expelling the contents of the needle onto the slide (image 1 above) (now referred to as slide 1), or, if there is a high harvest,  distributing between 2 or 3 slides laid side by side.The sample is then smeared using the squash preparation technique. NB it is very important to smear  the sample rather than just squirting it on to a slide, since the cells must be in a monolayer to be evaluated.
A. Squash preparation

• Slide 1 is held in the left hand (for right handed people)
• A second side (now referred to as slide 2) held in the right hand and placed flat onto slide 1 at right angles to it
• This spreads the sample (image 2 and 3 above)
• If necessary gentle pressure may be applied using the right thumb to encourage the cells to spread out. Excessive pressure will result in cell rupture.

The smear is on the lower surface of slide 2
With experience this technique produces excellent smears. Practice is required!

Smearing fluid samples

For turbid samples the blood smear technique is used (see preparing a blood film), or can be modified with the line concentration technique.

Line concentration technique

The sample is smeared as for the blood smear technique, but when the spreader slide has been advanced about two-thirds of the way across the slide the spreader slide is abruptly lifted upwards. This produces a smear with a concentrated line of cells at its end, instead of a feathered edge.

Impression smears

• • Impression smears are made before and after cleaning the surface of the ulcerated mass with a saline-moistened swab and blotting dry
  • A clean glass slide is gently touched to the lesion and immediately lifted off without smearing the slide across the lesion

Impression smears may also be made from the cut surface of tissues obtained at surgery or post mortem. Blood and tissue fluid should be removed by blotting the sample dry before touching the sample onto a slide.

Scrapings

• The lesion is first cleaned and blotted dry
• A scalpel blade is scraped across the lesion’s surface several times
• Material collected on the blade is transferred to a slide by gently teasing it off the blade using a needle or the blade can be used like a paintbrush to gently smear the sample onto a slide
• If the material is thickly spread a squash preparation is made as described for fine needle aspirates.

Swab smears

A swab or cotton bud is used to collect samples from fistulous tracts and from the vagina. Unless the lesion is very moist the swab must first be moistened in isotonic saline. The swab is rubbed against the surface of the lesion and then gently rolled along a slide, taking care not to rub the swab across the slide since this would damage the cells.

• A spreader slide is used. This is narrower than the smear slide to avoid spreading the cells over the edge of the slide. To make a spreader break the corner off a normal slide, having first scored it with a diamond writer or a glass cutter
• A drop of blood from a microhaematocrit tube is placed onto one end a slide
• The spreader slide is placed at an angle of about 30° in front of the sample and slid backwards until it comes into contact with the sample drop
• The sample then spreads out along the width of the spreader slide
• The spreader slide is advanced forwards, creating a smear with a feathered edge
• If too much blood is applied to the slide or taken up by the spreader slide the smear will be too long and the cells will be pushed over then end of the slide
• The angle of the spreader slide can be varied to change the size of the smear – a lower angle produces a longer smear. Care should be taken not to make the smear too long. The smear should extend to the middle or distal 2/3rd of the slide so that the examination area is fairly central
• Slides with a frosted end are preferred since these are very smooth and produce good smears which can be easily labelled.