Dr. P.

Thiruselvame,
M. V. Sc.

VCM – 512 Exercise No.: 6 26.09.2011

 Definition:

Bone marrow aspiration and

biopsy are diagnostic procedures involving
the introduction of a rigid, hollow needle into
the marrow-containing, cancellous part of
either a long or flat bone.
Schematic depiction of the hematopoietic compartment of the medullary cavity.
• Any single cytopenia unresponsive to therapy
• Markedly high blood cell counts
• Suspect leukemia
• Looking for organisms that cause systemic infection
– (Histoplasma spp., Leishmania spp., Cytauxzoon spp.)
• Looking for occult neoplasia
– Hypercalcemia
– Bony lysis on radiographs
• Evaluating iron stores
 Performing Bone Marrow Sampling

Two procedures
1. Bone marrow aspirates
2. Bone marrow core biopsy
Steps:
I. Preparing equipment
II. Patient preparation and sedation
III. Placement of the sampling needle
IV. Procuring the sample
V. Preparing slides
VI. Confirming adequate sampling
VII. Submitting samples to the lab or interpreting results
in house
 Comments about the Bone Marrow Aspiration

• Different sites depending upon the patient (Large or

small animal)

• Smaller volume of Lidocaine (< 3 ml) may be adequate.

– Careful of Lidocaine in cats

• Drape the area, to keep hands sterile

• Coat the aspiration syringe with an anticoagulant

 Common sites of collection:

• Proximal end of the femur (Trocantric fossa)

• Proximal end of the humerus

• Iliac crest

• Ribs

• Sternum
 Choosing a Site

Proximal humerus
– As an IM pin would be placed.
– Preferred for severe thrombocytopenia
• Less soft tissue to go through
• Direct pressure for primary hemostasis easily applied

Proximal femur
– Easier in the cat

Wing of the ilium

– Easier in the cat
– 2 approaches – dorsal and lateral
 Choosing a Needle

Rosenthal needle
– 100 % metal (steel) Rosenthal can be re-autoclaved
– May need to be sharpened occasionally
– 16 - 18 Gauge - not for biopsy

Illinois sternal-iliac needle

– Guide piece prevents deep penetration when penetrating wing of ileum
– Take guide piece off for long bones
– 16 - 18 Gauge – not for biopsy
– Can be re-autoclaved a few times or gas sterilized
– Aluminum rather than steel – doesn’t sharpen well
Jamshidi needle
– 13 or 8 Gauge
– For core biopsy or aspiration
– Fine wire use to remove the core biopsy
– End of the needle is tapered to retain the core
 Preparing equipment

Check the needle (sterile technique)

– needle is sharp and without chips

– stylet completely occludes the hollow needle

– Stylet is properly seated in the needle

 Preparing equipment

• Sterile supplies:
– Supplies for animal sedation
– Surgical gloves
• keep paper wrap for sterile field
– Drape
– #11 blade
– Bone marrow needle
– 10 ml syringes
– 18 Gauge needles
 Preparing equipment
• Non-sterile supplies:
– Surgical prep equipment

– Lidocaine and syringes

– EDTA or heparin

– Petri dish

– Pipettes

– Microscope slides

– Quick Stain (e.g., Diffquick)

– Microscope

– Formalin and container if doing biopsy

 Patient Preparation

• Sedate

• Clip and surgically prep site

– 4 inches square

• Lidocaine block down to bone, including periosteum

• Re-scrub

• Drape aseptically
 Proximal Humerus –
“IM pin” method
• Lateral recumbency

• Rotate elbow medially & push humerus cranially to expose the shoulder

• Stab incision # 11 blade on “flat spot” between the greater tubercle and the
humeral head

• Thumb of other hand holding elbow along long axis of humerus for reference

• Line needle up parallel with the long axis of the humerus (other thumb)

• Slowly twist clockwise and counter-clockwise until needle is seated in cortical

bone

• Then screwdriver-like motion until needle well seated in the marrow cavity

• Check needle by “wiggling” to make sure firmly seated in bone

• Coat a 10 ml syringe with anticoagulant

– Have assistant attach sterile 18 Gauge needle to syringe &

remove cap

– Assistant holds vial of anticoagulant

– Draw up 1 - 1.5 ml anticoagulant

– Coat syringe by drawing plunger to highest ml mark on

the syringe

– Squirt anticoagulant into Petri dish

– Remove needle

• Remove the cap and stylet from the bone marrow needle and
place on the sterile field
• Firmly attach the coated syringe
• Rapidly pull plunger back to 8 - 10 ml
– This hurts
• As soon as you see blood, release pressure, and get 1 ml
or less of bone marrow
• Very quickly squirt the marrow into the Petri dish, and
swirl
• Look for spicules (“flecks”)
• If no spicules, remove needle and try again
• If spicules, replace the marrow needle cap and prepare
slides to confirm good sample
Proximal Humerus – lateral technique
 Iliac Crest – dorsal technique:
• Sternal recumbency, hind limbs tucked under

• Palpate “flat spot” on the iliac crest (Impossible in obese dogs; Easy in cats)

• Insert needle at widest point of the iliac crest

• Axis of the needle is roughly perpendicular to the table - Direct slightly

caudally rather than cranially

• The needle has a tendency to slide off the iliac crest medially or laterally, so

do this step slowly and firmly Using the guide-piece of an Illinois needle

can help control how far the needle penetrates if it slips off the crest
 Iliac Wing – lateral technique:
• Not recommended for cats or small dogs (less than 10 Kg)
• Lateral recumbency
• Palpate “flat spot” on the iliac crest
• Insert the needle 1 - 2 cm ventral to the center of the iliac crest
• Axis of the needle is perpendicular to the long axis of the ilium and
perpendicular to the table
• Careful not to advance the needle through the opposite cortex
• Jamshidi needle can be used to take a full thickness marrow biopsy
 Proximal femur
• Easier in the cat
• Lateral recumbency
• Rotate stifle slightly medially
• Place thumb along long axis of femur,
pointing proximally, and ending on greater
trochanter
• Insert the needle under thumb, into the
inter-trocantric fossa
• Axis of the needle is parallel to the long
axis of the femur
• Remember the sciatic nerve runs caudal to
the femur
Slide Preparation
 Aspiration or Core Biopsy?
Advantages of aspiration
• Cellular morphology is more clear
– Better identification of cell lineages
– Characteristics of malignancy

• Can calculate E:M ratios

– Estimate regenerative responses
– Interpret with respect to CBC and reticulocyte count
– Normal 3:1 to 5:1

• Maturation sequence counts are easier

– More mature cell stages should be present in successively greater numbers
– More younger cells means leukemia, maturation arrest
 Aspiration or Core Biopsy?
Advantages of core biopsy

• If repeated attempts to aspirate produce no fluid (“packed marrow”)

– myelophthisic disease

– Myelofibrosis

• If repeated attempts to aspirate produce blood only with flecks of fat

– Aplastic anemia

• Can evaluate marrow cellularity

• Can evaluate tissue architecture

– Invasion by normal looking lymphocytes indicates lymphoma
 Bone Marrow Core Biopsy
• Use Jamshidi needle in any approach
– As soon as the needle is well seated through the
cortex, remove the stylet, and replace the cap
and handle
– Advance the needle 1-2 cm further, rotating in a
single direction
– “stir” the needle to break loose the core
– Remove the needle rotating in a single direction
– Pass the wire or stylet backward to pop the core
out the top of the needle
– Core 0.75 - 1 cm long is sufficient
• Cytology can be made by rolling the core on
slides, or scraping it
• Place cores in formalin for histopathology
Evaluation of Adequate Sampling
Gross examination
• Unstained slide – blank spot
• Stained slide – very dark purple spot
– Bone marrow may require longer staining
Microscopic examination
• Large nucleated cells confirm presence
of bone marrow
– Blue round cells are Erythroid
– “pink squigglies” are myeloid
• Peripheral blood only indicates poor
sample
• May see dark brown iron stores
• May see multi-nucleated
megakaryocytes
Evaluation of Adequate Sampling
Iron stores (Hemosiderin)

• Can see them reasonably well on

Diffquick

• Increased with chronic

inflammatory disease
– Anemia of chronic disease (non-
regenerative)

• Decreased with chronic blood loss

– Need iron supplementation
 Bone Marrow Particles:

Fat (adipose)

Both of the illustrated particles have > 75% cellularity (< 25 % fat)
 Bone Marrow Cells Types:

• Stem cells
• Erythroid cells
• Granulocytic cells
• Monocytic cells
• Megakaryocytic cells
• Lymphocytic cells
• Stromal cells (supporting cells)
Erythrocyte and Granulocyte Maturation
 Megakaryocytes
Scan at low power for large purple multi-nucleated (30 - 50) cells
Increased if regenerative thrombocytopenia or iron deficiency
Decreased if non-regenerative or acute severe thrombocytenia
 Megakaryocytes

Megakaryocytes

Megakaryocytes numbers can be assessed at low power (4 - 10x)

Mature Megakaryocytes
Immature
Megakaryocytes

• Compared to mature cells, have:

– Smaller size.
– Higher nucleus: cytoplasm (N:C) ratio
– Bluer (more basophilic) cytoplasm.
 Mitotic Figures:

• Cells may be “caught” in division on smear.

• May appear as “fragmented” nucleus / as two nuclei.

• May occur in any of the marrow cell lines.

Basic pathologic lesions of the Bone
Marrow:

• Hyperplasia
• Hypoplasia
• Neoplasia
• Fibrosis
• Inflammation
• Infarction
 Other cells found in bone marrow:
• Vascular system cells
– Supply nutrients to the marrow

• Reticular cells
– Give structure to the marrow

• Osteoclasts and osteoblasts

– Occasionally found in an aspirate

• Sometimes infectious organisms

– Ehrlicia spp., Fungal, Leishmania spp.
 Finishing Up
Once adequate samples are confirmed:

• Remove the aspiration needle

• Take core biopsies if needed

• Recover the patient

Temporary lameness on the sampled leg is not

unusual

• Seldom lasts for more than a few days

• Seldom happens when iliac crest is sampled

Ow! Dude, That’s my bone marrow !!

Always cover
with Pain management!
This hurts!
 Submitting Samples

Always confirm adequate samples prior to shipping (2 - 4

unstained slides along with stained slides)
• Make and dry slides quickly
• Unstained slide should probably be fixed in methanol
Always submit same day CBC and unstained blood smear
with bone marrow samples
• Impossible to interpret M:E ratio without it
• Reticulocyte count also if anemic
– PCV < 25 in the dog
– PCV< 20 in the cat
 Submitting Samples
Don’t send cytology smears in the same box as
formalin fixed core samples

• Formalin will damage cellular uptake of stain

Request special stains if indicated

• Prussian blue for iron

• Immuno-histochemistry if neoplasia of unknown lineage

Can submit for culture if PUO

• Red top tube and sterile swab culture tube

• Never EDTA – it kills bacteria

 Interpreting reports
Abnormal Bone Marrow Cells
• Atypical cells – characteristics of malignancy
– leukemia or myelodysplasia
• Malignant cells in clusters – metastasis
• Mast cell tumors
– Can be present in normal marrow
– Clusters suggests neoplasia
• Plasma and Mott cells – chronic antigenic
stimulation
– Ehrlichiosis or immune mediated disease
– Large numbers may indicate myeloma
• Osteoblasts and Osteoclasts - rare
 References:

• Grindem, C. B., Neel, J. A. and T. A. Juopperi (2002) Cytology of

bone marrow. Vet Clin North Am Small Anim Pract; 32:
1313 - 1374.

• Grindem, C. B., Tyler, R. D. and R. L. Cowell. The bone marrow.

In Diagnostic Cytology of the Dog and Cat, 3rd Ed. Eds,
Cowell, R. L., Tyler, R. D., Meinkoth, J. H. and D. B.
DeNicola (2008) Mosby Elsiever, pp. 422 - 450.