NMD2024
DAVAO MEDICAL SCHOOL FOUNDATION
BONE MARROW PREPARATIONS (HISTOLOGY)
AIJE HOPE BRUZON-HORTEL, MD
BONE MARROW PREPARATIONS
1. Bone Marrow Aspirate (BMA Smear)
- assess cellular morphology
- conduct specialized test in the bone marrow
- in general aspiration is used for evaluation of
hematologic condition, cancer, metastatic disorder,
storage disorders, chronic systemic conditions
2. Bone Marrow Biopsy
- performed as part of the Bone Marrow aspiration
procedure (get a tissue)
- provide more specific info about cellularity of
marrow and the extent of disease as compared to
BMA
STERNAL PUNCTURE
Source: Elsevier Website
1. With the patient in the supine position, locate the
sternal angle using gentle digital palpation. Use the
needle cover to locate the intersection between the
sternal angle and the midsternal line (Fig. 3A and
B). To facilitate the procedure, ask the patient to
extend their neck (Fig. 3C). Disinfect the area and
cover it with a surgical aperture drape.
Sternal Puncture
A. Location of the sternal puncture site. The sternal
angle, formed by the junction of the manubrium
and the body of the sternum, lies 2cm below the
sternal notch.
B. Gently mark the puncture site.
C. Inject 10ml lidocaine, initially at the level of the
skin, and then in the periosteum.
D. Identify the parts of the Osgood needle before
performing aspiration.
2. Administer local anesthetic with 1% lidocaine, using
3ml to anaesthetize the skin and subcutaneous
tissue. Then, gently inject lidocaine at various points
on the cortical bone, administering 1ml anesthetic
with each injection.
3. After a waiting time of between 3 and 5min, start the
procedure. First, identify the parts of the bone
marrow needle (Fig. 3D). Hematologists usually use
size 16 and 18 needles.
4. Insert the needle at the previously marked site,
through the skin and subcutaneous tissue until it
abuts the bone.
5. Push the needle through the bony cortex, rotating it
on its axis until it is fixed in place at the desired
depth (Fig. 4A and B).
Sternal Puncture (continued).
A. Introduce the needle by rotating it on its axis.
B. Continue until the needle is fixed in place.
C. Remove the stylet.
D. Draw the aspirate using a 20ml syringe. Only
1ml will be needed for the smear.
6. Withdraw the stylet (Fig. 4C), attach a 20ml syringe,
and aspirate gently (Fig. 4D).
7. For a smear sample, aspirate only 1ml of fluid.
8. The smear technique used will usually vary from
one hospital to another. Fig. 5 shows the steps
involved to prepare the smear.
Preparing the Bone Marrow Smear.
A. When the bone marrow aspiration needle is in
place, use a 20ml syringe to draw 1ml of
aspirate.
B. Place a few drops of aspirate on a slide held
vertically to facilitate visualization of the bone
marrow spicules.
C. Using the edge of a second slide, separate and
isolate a few bone marrow spicules from the rest
of the sample.
D. Transfer these to a third slide.
E. Without applying pressure, distribute the
spicules in the center of the slide.
F. Place the second slide across the third slide to
form a cross with the spicules in the center.
G. Gently but quickly slide the second slide over the
third slide to extend the spicules along its length.
H. Make sure the aspirate is spread evenly over the
entire slide.
I. Allow to dry for 5min, and then send it to the lab
for tincture.
9. Five smears are prepared, with each slide being
appropriately identified.
JBS 3/8/2021
 NMD2024
DAVAO MEDICAL SCHOOL FOUNDATION
BONE MARROW PREPARATIONS (HISTOLOGY)
AIJE HOPE BRUZON-HORTEL, MD

BONE MARROW EXAMINATION

1. Bone Marrow Aspiration (BMA Smear)
- cell morphology and enumeration of
marrow cellular elements
2. Bone Marrow Biopsy
- cellularity, fibrosis, marrow architecture or
vasculature, infections or infiltrative
disorders

Bone Marrow Aspirate vs Peripheral Smear

- presence of SPICULES in BMA
- presence of FEATHERY EDGE in PBS

Bone Marrow Cellularity

Bone Marrow Biopsy Section - volume ratio of hematopoiesis and fat
- get tissue from cancellous bone - cellularity is age dependent (normal)
- adequate size: o newborn: 100% Cellularity
o contain 3 or 4 intertrabecular spaces § all marrow is hematopoietic, as we
o 20-30 mm (2-3 cm) in length age, amount of fat increases
- Which one is adequate? o adult: 30 – 70% Cellularity

Which one is Hypercellular, Hypocellular, and Normal?

How do you know that this is a tissue and not a BMA?

- TRABECULAE !!! – not present in smear
Example of a Normal Cellularity: LEFT PICTURE

- Why it should be adequate?

o So that interpretation quality and detection
of focal lesions will be reliable
- Microscopically, this tissue will appear as this:
o observe the TRABECULAE
o from cancellous bone, we can see marrow
cavity filled with hematopoietic cells

- This picture will change under pathologic

condition:
o Hypocellular - anemia
o Hypercellular - leukemia

JBS 3/8/2021
 NMD2024
DAVAO MEDICAL SCHOOL FOUNDATION
BONE MARROW PREPARATIONS (HISTOLOGY)
AIJE HOPE BRUZON-HORTEL, MD

Hypercellular Marrow (> 70% Cellularity) Erythroid Cells

o Bone Marrow Morphology
§ Erythroid colonies can be readily identified
on biopsy sections by their uniformly
round nuclear contours, prominent cell
membranes, and the dense homogenous
chromatin of the numerous orthochromic
normoblasts
§ These erythroid colonies are generally
small and non-paratrabecular

A. Bone Marrow Biopsy B. Bone Marrow Aspirate

Bone Marrow Tissue

o Lesser Fat Cells, More Hematopoietic Cells
Bone Marrow Smear
o Lesser Fat Cells, More Hematopoietic Cells
o Red Circle – megakaryocyte

Hypocellular Marrow (< 30% Cellularity)

Total Proportion of Cells in the Bone Marrow

ERYTHROPOIESIS

Differential Cell Count

o cell lineage and M:E ratio can be determined
here
o performed on aspirate smear
§ count minimum of 500 cells
§ enumerate types
o performed on several different preparations to
avoid focal cellular distribution anomalies
o there should be correlation with overall cellularity
o good to have both BMA and BMB

JBS 3/8/2021
 NMD2024
DAVAO MEDICAL SCHOOL FOUNDATION
BONE MARROW PREPARATIONS (HISTOLOGY)
AIJE HOPE BRUZON-HORTEL, MD

Erythroid Cells BMB Showing Myeloblast and Promyelocytes in their

- Found in clusters known as Erythroid normal paratrabecular position
Islands
- Located in intertrabecular space (arrow) NUCLEOLUS

NUCLEAR MEMBRANE

CYTOPLASM

- Immature granulocytes are found

preferentially adjacent to bony spicules and
around arterioles
- Maturing granulocytes are seen extending
into the intertrabecular space
- Red Arrow: myeloblast
o High N:C Ratio
Erythroid Cell on HPO o Earliest immature cell of myeloid
- erythroblasts at various stages of maturation series
- Red Arrow: Intermediate Erythroblast o Cytoplasm is agranular
o Larger in SIZE - Green Arrow: promyelocyte
- Blue Arrow: Late Erythroblast o Different from myeloblast aside from
size is the presence of granules in
cytoplasm
o Stage where Primary Granules
appear
- Visible Nucleoli – because these cells are
immature cells

Maturing Cells
ENDOTHELIAL CELLS

MATURE CELLS
– transported to
circulation via the sinusoid

Granulocytes
- predominant cells in the normal bone marrow in all
ages
- 50-70% of nucleated cell
- maturation sequence: mature forms predominate
- Bone Marrow Morphology
o on biopsy sections, the earliest identifiable - Can already identify which granulocyte it is
myeloid precursor generally reside adjacent based on the secondary granules
to bony trabeculae - Blue Arrow: Neutrophil Myelocyte
- can be used to confirm presence of leukemia o Size, Location of Nucleus,
o because mature myeloid form predominate Cytoplasm (granules)
o in leukemia: increase in immature! - Red Arrow: Eosinophil Myelocyte
- Granulopoiesis can increase 10x in infection aside o Red-orange granules in cytoplasm
from leukemia or other inflammatory conditions - Green Arrow: Endothelial Cells
o In marrow cavities, there are
sinusoids, and within the sinusoids
we can see mature cells.
- Once cells mature, they will be transported
to the circulation via the sinusoids in the
marrow cavities
- Mature neutrophils adjacent to sinusoid

JBS 3/8/2021
 NMD2024
DAVAO MEDICAL SCHOOL FOUNDATION
BONE MARROW PREPARATIONS (HISTOLOGY)
AIJE HOPE BRUZON-HORTEL, MD

o In cases of infection, they will - Bone Marrow Sections

immediately go to the area of o Best for evaluating quantity and
infection distribution
- Bone Marrow Morphology
Myeloid Maturation Series o Large cells with multilobulated nuclei
o Voluminous pale pink cytoplasm
o Reside adjacent to bone marrow sinuses
and randomly dispersed
§ If there is a need for platelet, they
just have to extend their
cytoplasm to the sinusoid to
release platelet
o 3-4 megakaryocytes/hpf on bone marrow
biopsy sections
o Immature megakaryocytes are difficult to
recognize
Red Marrow

- the more immature the cells is, the bigger it

is; specifically, the nucleus and cytoplasm or
N:C ratio
- bigger nucleus, small amount of cytoplasm
- as it matures, nucleus decreases and
cytoplasm increases
Morphology of Immature Granulocytes
For bone marrow preparations
- Red Marrow - mostly hematopoietic cells
o Fat Cells can still be seen but
hematopoietic cells are abundant
- Yellow Marrow - mostly fat cells

Megakaryocyte

- Debatable because of the obstruction

Reticulin Fibers

Maturation Sequence:
1. Myeloblast - round nucleus, prominent
nucleolus
2. Promyelocyte - smaller nucleus, more
cytoplasm, appearance of
primary granules
3. Myelocyte - size of nucleus decreases and
is located on one side,
appearance of secondary
- Normal component of bone marrow stroma
granules, can identify which
- Reticular fiber forming mesh work
granulocyte it is
- If there is an increase in reticulin fiber, this
4. Metamyelocyte - characteristic indentation of
nucleus but is 50% of the can correlate to bone marrow fibrosis
o Associated with benign and
diameter
malignant condition
5. Band Cell - size of nucleus decreases,
- Special stain: Reticulin Stain
more prominent invagination or
indentation of nucleus until such
time that the nucleus is divided
into lobe/segment
6. Neutrophil - nucleated

Megakaryocytes

- Least numerous hematopoietic element:

o < 1% of nucleated cells

JBS 3/8/2021
 NMD2024
DAVAO MEDICAL SCHOOL FOUNDATION
BONE MARROW PREPARATIONS (HISTOLOGY)
AIJE HOPE BRUZON-HORTEL, MD

Bone Marrow Biopsy Try to identify:

E

D
A

B C

G
Try to identify:
A - OSTEOCLAST

Try to identify:

MEGAKARYOCYTE

JBS 3/8/2021