clinical pathology bone marrow collection technique sample preparation evaluation
TRANSCRIPT
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.