CLINICAL PATHOLOGY Bone Marrow Collection Technique Sample Preparation Evaluation

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.  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.  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 needle 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 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 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 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 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  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  Phagocytosis, mediators of inflammation, and microbiocidal actions  Eosinophils  Phagocytosis, parasiticidal, hyersensitivity reactions  Basophils  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  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  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 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.