clinical pathology bone marrow collection technique sample preparation evaluation

CLINICAL PATHOLOGY

Bone MarrowCollection TechniqueSample Preparation

Evaluation

Introduction to the hemopoietic system

Hematopoiesis: formation of blood cellsThe 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 humerusIliac crestRibsSternum

Bone marrow aspiration/biopsy sites continued

Large dogs- iliac crestSmall dogs- trochanteric fossa of the femurCats- trochanteric fossaThe 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.

10-20 ml sterile syringeClean slidesClear petri or watch glassEDTA and salineSurgery prep equipmentSedative/anesthesia

Bone marrow collection technique

Sedate/anesthesizeLocal anesthesia (in some cases)Aseptic prep of areaSkin incision with bladeBiopsy 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 slideAllow the sample to drain from the slide into

a watch glass or petri dishMarrow flecks tend to adhere to the glass

slide.A second slide is placed perpendicularly

across the marrow flecks causing it to spreadThe 2 slides are then pulled apart in a

horizontal planeMarrow clots quickly so work quickly.

Using EDTAAn alternative is to use 3-5 ml of EDTA/isotonic

saline in a syringe and then aspirateUsing 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 dryUse Diff quick stainLet 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 cellsErythroid cellsGranulocytic cells Monocytic cellsMegakaryocytic cellsLymphocytic cellsStromal 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 oxygenThe cells proliferate producing daughter cellsRemember 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 marrowVascular 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

HyperplasiaHypoplasiaNeoplasiaFibrosisInflammationInfarction

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 approach10x 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 leukemiaLymphoblastic leukemia- more blast cells in the

blood and bone marrowPlasma cell myeloma- proliferation of plasma

cells in the bone marrow.