Instructor: Elaine Anthony CVT

 

Bone Marrow Aspiration

 

Surgical prep materials needed

  • 2 - 3 (12 ml syringes
  • B.M needle with stylet
  • local or general anesthesia
  • numerous clean slides (about 20)
  • EDTA
  • #10 blade (to nick skin)

 

  • Bone Marrow Needle with stylet inserted

 

Where to Obtain:

  • 1. trochanteric fossa

 

Where to Obtain:

  • iliac crest (larger animals)

also use:

  • sterum
  • humerus

 

General anesthesia or local into periosteom

  • Insert needle into bone (flushed with EDTA)
    • (Bone will move with needle when properly inserted)
  • * use forward pressure white twisting
    • (stylet is still in needle)

 

  • Remove Stylet

 

  • attach syringe
  • apply negative pressure - quickly
  • release
  • repeat till you see material in hub
  • Stop - ( or you will get blood contamination)

 

  • Beware: do not remove syringe until negative pressure is released!
  • Take syringe off
  • transfer to slides

 

  • Slides should be lined up on slant, so blood contaminant runs downward.
  • Put small drops on many slides

 

  • Make squash prep smear from top portion (bottom is contaminant)
  • (Beware: Place top slide on sample so that smear begins at frosted edge. This gives more room for smear on slides)

 

  • Stain 2 times as long with Dif Quick
  • Evaluate smear for : (low power magnification)
    • 1. Cellularity
      • older animals have more fat, younger animals have less fat
      • hypo cellular, normocellular or hypercellular
  • This sample above is hypercellular

 

  • This sample is hypocellular (low magnification)

 

Maturation Sequence for RBC

Immature to mature (left to right)

  • A. cytoplasm shape, color, size
  • B. Nuclear shape, color, size
  • C. Chromatin pattern
  • D. All 3 together

 

 

Rubricyte Series

A. Rubriblast

  • Red series
    • Nucleus remains round
    • Dark color is cytoplasm

Rubrilast ( note nucleus with in nucleus)

 

Rubricytic Series

  • Rubriblast
    • dark color cytoplasm
    • round nucleus
    • nucleolus with in nucleus

 

Rubricytic Series

  • A. prorubricyte (nucleolus gone)
  • B. Rubricyte
  • C. Metarubricyte

Note - As the RBC matures it gets smaller, but the cytoplasmremains dark and nucleus remains round.

 

Rubricytic Series

  • A. rubricyte
  • B. metarubricyte
  • C. mitotic figure (metarubricyte in division)

 

Rubricytic Series

  • A. Metarubricyte

 

  • A. metarubricyte
  • B. metarubricyte extruding it's nucleus
  • C. segmented neutrophil

 

Reticulocytes - NMB Stain

  • Do not count non - nucleated cells in the M:E RATIO, only nucleated cells of myeloid (white) or Erytroid (red) series.

 

Signs of Regeneration

  • anisocytosis (variation in cell size)
  • howell jolly body (retained nuclear remnant)
  • polychromasia (variation in cell color)
  • NRBC ( not seen here)

 

  • Top - rubriblast divides into 2 prorubricytes
  • Division of each cell continues
  • Thus one rubriblast results in 32 metarubricytes

 

Myelocytic Series

A - Myeloblast

  • lighter cytoplasmic color than rubricytic
  • round nucleus only in blast and progranulocytic stage, then the nucleus indents in the more mature stages.

* Look for :

  • lighter color
  • indented nucleus

 

Maturation Sequence of Myeloblast

  • Myeloblast and Promyelobast have not differentiated into which granulocyte it will be.
  • Look after progran. stage for granulation to distinquish eosinophil, neutrophil, and basophil.

 

A - Progranulocyte

  • azurephillic granules in an abundant cytoplasm are classic for this stage beyond the myeloblast.
  • * The progranulocyte is larger than the blast due to the abundant cytoplasm.

 

M:E Ratio (white or red?)

  • A-E
  • B-E
  • C-M (blast stage, prominent nucleolus within nucleus, light cytplasm)
  • D-E
  • E-E (darker cytoplasm, though is subtle, nucleous within nucleus)
  • F-M
  • G-mitotic figure (don't count either way)
  • H-E
  • I-E
  • J-M

 

Myelocytic

  • abundant light colored cytoplasm
  • indented nucleus
  • early mylocytic neutrophil or late progranulocyte

 

M:E

  • A-M
  • B-M
  • C-M
  • D-E

 

M:E

  • A-M (early band)
  • B-M (band)
  • C-M (late band)

 

M:E

  • A-E
  • B-M
  • C-M
  • D-M
  • E-E
  • F-M
  • G-M
  • H-M
  • I-E
  • J-M

 

A - Plasmic Cell

  • They are B-lymphocytes that are extremely reactive
  • Characteristics:
    • eccetrically located nucleus
    • perinuclear clear zone (golgi apparatus near nucleus doesn't take up stain)
    • trailing dark blue cytoplasm

 

Mott Cell

  • "constipated" plasma cell, is a B-lymphocyte plasma cell with cytoplasm full of Russell bodies (immunoglobulin packets)
  • Can find occassional plasma cells or Mott cells in bone marrow. If more seen, may be indicaton of disease. (myeloproliferative)

 

Megakaryocyte

  • Dil immersion magnification
  • Find first on scan or low power to count - per slide or # per spicule.
  • Then go to high power and oil

 

Immature Megakaryocyte

  • * the more cytoplasm, the more mature the cell

 

M:E Ratio

Whats Wrong?

  • All same type and size
  • Need to see and evalluate for:
    • megakarycyte 8-12 per slide
    • cellularity (hypo, normo or hyper?)
    • maturation sequence (all stages present
    • proper ratio (equal # M to E)

 

Questions?

Elaine AnthonyCVT 

 

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