Bone Marrow
Collection Technique
Sample Preparation
Evaluation
CLINICAL PATHOLOGY
Introduction to the hemopoietic system
 Hematopoiesis: formation of blood cells
 The bone marrow is the major hematopoietic organ of
the body.
 In the adult (under normal circumstances), much of the
bone marrow is hematopoietically inactive and filled with
fat.
 Active bone marrow remains in the flat bones and the
ends of long bones. The central area has mainly fat.
 Active bone marrow can expand into fat filled areas in
response to increases peripheral use, loss, or destruction.
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Remember red vs. yellow bone marrow?
 In young animals, active hematopoietic tissue is found
throughout both flat and long bones.
Indications for Bone Marrow Cytology
 Hematologic abnormalities not readily explained by
a good history, physical exam, chemistry panel,
and/or other tests.
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Nonregenerative anema
Persistent neutropenia
Persistent thrombocytopenia
Pancytopenia
Neoplasia
Proliferative disorders- myleofibrosis
Types of Bone Marrow Collection
 Aspiration
 Core biopsy
Problems with Bone Marrow Aspiration/Biopsy
 Need to sedate or anesthetize patient
 May be a risk
 Hemorrhage
 Concern with bleeding disorders or thrombocytopenic animals
 Iatrogenic marrow infection
 We cause an infection/issue
Common sites of collection of Bone Marrow
Aspiration/Biopsy
 Proximal end of the femur (trochanteric fossa)
 Proximal end of the humerus
 Iliac crest
 Ribs
 Sternum
Bone marrow aspiration/biopsy sites continued
 Large dogs- iliac crest
 Small dogs- trochanteric fossa of the femur
 Cats- trochanteric fossa
 The ribs and the sternum should be avoided in small
dogs and cats- risk of puncturing the thoracic cavity
 Biopsy of the trochanteric fossa may be difficult in
obese or well-muscled animals
Instruments and Supplies
 16 to 18 gauge , 1 to 1 ¾ inch bone marrow biopsy
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needle.
10-20 ml sterile syringe
Clean slides
Clear petri or watch glass
EDTA and saline
Surgery prep equipment
Sedative/anesthesia
Bone marrow collection technique
 Sedate/anesthesize
 Local anesthesia (in some cases)
 Aseptic prep of area
 Skin incision with blade
 Biopsy needle is introduced and advanced into the
cortical bone
 Need is rotated in alternating clockwise and
counterclockwise motions
Bone Marrow Collection Continued
 Once in marrow cavity, the stylet is removed.
 10-20 ml syringe is attached and negative pressure is
used to collect the marrow.
 Apply suction until blood is seen within the hub of
the syringe.
 Stop at this point to avoid contamination with
peripheral blood.
 Place a drop of the sample onto clean slides.
Sample Preparation
 Immediately place sample on a tilted slide
 Allow the sample to drain from the slide into a watch
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glass or petri dish
Marrow flecks tend to adhere to the glass slide.
A second slide is placed perpendicularly across the
marrow flecks causing it to spread
The 2 slides are then pulled apart in a horizontal
plane
Marrow clots quickly so work quickly.
Using EDTA
 An alternative is to use 3-5 ml of EDTA/isotonic
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saline in a syringe and then aspirate
Using this method will allow collection of a greater
amount of sample and more slides may be made
The syringe contents are expelled into a watch glass
or petri dish
The petri dish is tilted and/or rotated to examine the
sample for marrow flecks
Marrow flecks are opaque/tan and irregular in
shape.
Flecks cling to the bottom of the dish, the fluid
drains to the bottom
Using EDTA continued
 Harvest the flecks with microhematocrit capillary
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tube or pipette.
Place on slide, may need to blow gently over the top
of the tube to dislodge the fleck
Using a coverslip use the horizontal pull apart
technique.
Let air dry
Use Diff quick stain
Let stain and buffers allow a longer contact time
Core Biopsy technique
 Same procedure except:
 Jamshidi biopsy needle is used when the biopsy needle enters
the marrow cavity, the stylet is removed and the needle is
advanced about 3 mm with a rotating mtion. This cuts the
core.
 This fills the bore of the needle
 The stylet or probe pushes the core out the top of the biopsy
instrument
 The core is rolled on the slide with the needle.
 The remainder of the core is placed in formalin.
Bone Marrow Cells Types
 Stem cells
 Erythroid cells
 Granulocytic cells
 Monocytic cells
 Megakaryocytic cells
 Lymphocytic cells
 Stromal and sustencacular cells (suppoting cells)
Stem Cells
 Give rise to all blood cells depending on the body’s
need
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Erythroid series
Granulocytic series
Lymphocytic series
Monocytic series
Megakaryocytic series
Erythroid cells
 Functions: to carry oxygen
 The cells proliferate producing daughter cells
 Remember Rubriblast to Reticulocyte
Granulocytic Cells
 Functions depend on cell type
 Neutrophils
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Eosinophils
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Phagocytosis, mediators of inflammation, and microbiocidal
actions
Phagocytosis, parasiticidal, hyersensitivity reactions
Basophils
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Inflammation and parasiticidal
Monocytic Cells
 Function:
 Tissue phagocytes (clean up functions), secrete mediators or
inflammation, stimulate lymphocytes, and process antigens for
presentation to lymphocytes
Megakaryocytic cells
 Function:
 Production of thrombocytes (important in hemostasis)
Lymphocytic cells
 Function:
 Mediation of the immune response (T-cells), antibody
production (B-cells and plasma cells).
Other cells found in bone marrow
 Vascular system cells
 Supply nutrients to the marrow
 Reticular cells
 Give structure to the marrow
 Osteoclasts and osteoblasts
 Ocassionally found in an aspirate
 Sometimes infectious organisms
 Ehrlicia
 Fungal
 Leishmania
Basic pathologic lesions of the Bone Marrow
 Hyperplasia
 Hypoplasia
 Neoplasia
 Fibrosis
 Inflammation
 Infarction
Evaluation of the cellularity of the Marrow
 Depends on the age of the animal
 Young animals contain very little fat 25%, 75% cells
 Adults contain 50% fat, 50% cells
 Old animals contain 75% fat, 25% cells
 Need to use other labwork to help differentiate the
different causes of cellularity changes in the marrow
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CBC
Chem panel
Clinical signs
History
Felv/FIV test
Ehrlichia, etc
Evaluating Bone Marrow Slides
 Systematic approach
 10x scan slide- note degree of cellularity and amount
of fat
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Note the number of megakaryocytes
>50/large fleck suggests megakaryocyte hyperplasia
80% of granulocytes should be more mature form
90% of erythroid should be rubricytes and metarubricytes
Example of Bone Marrow Responses
 Regenerative anemias tend to have hyperplastic
erythroid compartment
 Neutrophilia due to inflammation-hyperplastic with
increased numbers of neutrophils
 With Neutrophilia may see myeloid hypoplasia
Neoplastic Disorders
 Leukemia is a neoplastic proliferation of
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hematopoietic cells within the bone marrow
Diagnosis is based on CBC and bone marrow exam
The bone marrow is replaced by proliferating
immature cells
Lymphocytic leukemia
Lymphoblastic leukemia- more blast cells in the
blood and bone marrow
Plasma cell myeloma- proliferation of plasma cells in
the bone marrow.