Approach to BM aspiration

Indications
• Investigation of peripheral blood abnormality

• Leukemia, MPN, MDS

• PUO

• Staging of lymphoma

• Unexplained splenomegaly; storage disorders

• Evaluation of a patient who does not follow the

predicted course of initial diagnosis
 Decision making variables in
Peripheral film examination

• Unexplained cytopenias
• Abnormal cells/ blasts
• Single or multilineage dyspoiesis
Cytopenias when BM is NOT indicated

• MAHA
• Infectious causes
• Hypersplenism
• Is a BM Bx necessary?

• Unilateral or bilateral?
 BM Biopsy - Advantages

• Focal lesions – lymphoma, granuloma,

metastasis
• Assessment of marrow fibrosis
• Assessment of cellularity, topography
of cells, interrelationships, cell
distribution
• Assessment of bone, blood vessels,
stromal elements, lymphoid
aggregates, necrosis
 Bilateral BM Bx
• Marrow infiltration; staging of
lymphoma

• PUO
 Contraindications

• None, other than physical limitations e.g. pain or

restricted mobility.

• Avoid sites of previous radiotherapy (inevitably

grossly hypocellular and
not representative).
 Procedure

• Performed under sterile conditions.

• The skin at the site of the biopsy is shaved, if

necessary, and cleaned with a disinfectant
solution.
 Procedure
(Anaesthesia)

1. Bone marrow aspiration may be performed under local

anaesthesia such as 1% lidocaine, using a 25-gauge
needle.

2. Short acting intravenous benzodiazepines (e.g.

midazolam)
may be administered—with appropriate monitoring (pulse
oximetry), oxygen administration and available
resuscitation equipment—when trephine biopsy is
performed.
General anaesthesia rarely used (except in children).
Numbing Skin
3. Skin and periosteum over the posterior iliac spine are
infiltrated with local anaesthetic.
Care must be taken to fully anesthetize the periosteum,
where most of the bone pain fibers are located.

4. A small cutaneous incision is made, the aspirating needle

is introduced through this and should penetrate the
marrow cortex 3–10mm before removal of the trocar.

5. No more than 0.5–1mL marrow should be aspirated

initially, and smears made promptly.
 Procedure

Aspiration may cause a very brief, sharp pain. If

no pain is noted
and no marrow is obtained, the needle may be
rotated and suction applied again. If no
marrow is obtained, another sampling site
may be required.
6. Further material can be aspirated and placed in EDTA
or other anticoagulant media for other studies.
7.
An Islam or Jamshidi needle is preferred for
trephine biopsy.

1.The needle is advanced through the same puncture

site to penetrate the cortex.
2.The trocar is removed and using firm hand
pressure the needle is rotated clockwise and should
be advanced as far as possible.
3.The needle is removed by gentle anti-clockwise
rotation. In this manner an experienced operator
should regularly obtain biopsy
samples of 25–35mm in length.
4.Simple pressure dressings are sufficient after care
and minor discomfort at the location
Bone marrow aspirate
• A bone marrow film should first be
examined macroscopically to
make sure that particles or
fragments are present.

• Bone marrow aspirates which

lack particles may be diluted with
peripheral blood and may
therefore be unrepresentative.
An ideal bone marrow film with
particles is shown.
Normal ranges of bone marrow cells
• M:E ratio 2.4 • Eosinophils
(1.3-4.6) 2.2 (0.3-4.2)
• myeloblasts 1.4 • Basophils
(0-3) 0.1 (0-0.4)
• Promyelocytes • Monocytes
7.8 (3.2-12.4) 1.3 (0-2.6)
• Myelocytes • Erythroblasts
7.6
25.9 (13.6-
(1.9-13.3)
38.2)
• Metamyelocytes • Lymphocytes
4.1 (2.3-5.9) 13.1 (6-20)
• Neutrophils plus band cells • Plasma cells
34.2 (23.4-45) 0.6 (0-1.2)
 Complications

Complication Comment
 Bleeding Hematoma Most common complication
 Wound infection
 Neuropathy Very rare, usually lateral cutaneous nerve,
mostly obese patients
 Fracture Older individuals
 Osteomyelitis Very rare, requires systemic therapy
 Needle breakage Osteopetrosis
 Death Very rare, retroperitoneal hemorrhage
• There are a variety of "Romanowsky-type" stains with
mixtures of methylene blue, azure, and eosin
compounds
 Wright’s stain

• It is named for James Homer Wright,

who devised the stain, a modification of
the Romanowsky stain, in 1902.
 Wright’s stain

Principle:
• Wright’s stain is a Romanowsky stain that contains Eosin and
Methylene blue.

• Eosin (acid dye) stains basic cell structures such as hemoglobin,

eosinophil granules, and primary granules a red-orange color.

• Methylene blue (basic dye) stains acid cell structures such as

RNA in nucleolus and cytoplasm, nuclear chromatin, and basophil
granules a blue color.

• A combination of both dyes stains neutral cell structures such

as neutrophil granules a pinkish-tan color.
 Wright Stain Procedure

Specimen-
• Should be prepared from a freshly drawn
blood specimen (EDTA purple top tubes).
Allow smears to air dry completely before
staining.
• Please use an X or check mark to indicate
that all stains and reagents are present on
the bench before manual staining of rack.
Reagents needed for manual testing are listed
below.
 Reagents checklist

• Methanol for Wright Stain

• Wright Stain
• Wright Stain / Buffer mixture
• Please mix the Wright Stain and buffer mixture
before blood smears are made or before the smears
are air drying.

• Combine 15ml of Wright Stain with 75ml of Wright

Stain Buffer, mix well and allow to stand for at least
0 minutes. Make sure that a metallic sheen is
observed on the surface before staining.

• Allow to stand 10 minutes prior to use.

 WRight stain PROceDURe

• Place freshly prepared and air dried blood smears in a manual staining rack.______
• Place slide rack with air dried blood smears in Methanol for 30 seconds. ______
(Adjust timer for 30 seconds.) . 2. . 2.
• Take a disposable pipette and flood the Wright Stain on the appropriately labeled
slides. ______
• Set timer for 3 minutes. ______
• Place oxidizing Wright Stain and Wright Stain Buffer Mixture on Wright stained slides
laying on slide rack (Displacing the Wright Stain off the slides with the pipette filled
with Wright Stain/buffer mixture and viewing a metallic sheen on the top of
slides.)____
• Set timer for 6 minutes. ______
• Place slides in Wright Stain Buffer for .5 minutes.
• Set timer for .5 minutes. _________
• Retrieve slide rack with stained slides from buffer rinse and allow to air dry before
examination with immersion oil and 00X objective.
 staineD sliDe tiPs

• Wipe excess stain from back of slides with methanol

soaked gauze, being careful not to wipe off the
stained side of the slides.

• Place drop of immersion oil onto slide to be viewed

making it is completely air dried after staining.
• A properly stained smear looks pinkish-purple macroscopically; the
red cells appear red-orange microscopically.

• 1. The RBCs may appear blue and the WBC nuclei deeply stained if:
a. The staining time is too long.
b. Washing is inadequate.
c. The stain or buffer is excessively alkaline.
d. The smear is too thick.

• ●Correct by decreasing stain time, decreasing pH of buffer, or

making a thinner slide.
• 2. The RBCs may appear excessively red and
the WBC nuclei poorly stained if:
• a. The staining time is inadequate.
• b. Washing of slide is excessive.
• c. The stain or buffer is too acidic.
• ●Correct by increasing stain time or increasing
pH of the buffer.
• 3. Excess precipitate is caused by insufficient
washing or inadequate filtering of the stain.