Topic 2: Preparation and Evaluation of Bone Marrow

Learning Objectives

At the end of this topic, students will be able to:

1. Determine the collection, processing and staining of marrow specimen

2. Recognize the normal marrow cells.
3. Identify the marrow differential count

Activating Prior Learning

Have you ever thought of the importance of the bones in the body? Have you ever
asked of yourself if how many bones are there in a human body, which among these
bones that have a significant role in the formation of our blood cells and why are Medical
Technologists as a laboratory staff needed in the preparation and evaluation of bone
marrow assessment?

Presentation of Contents

BONE MARROW
- Soft spongy tissue that lies in the interior of bones.
- Bone marrow forms around 4% of total body weight (around 2.6 kg in a
healthy adult)
- Serves an active function in the body by producing all three types of
blood cells, as well as lymphocytes, which support the immune system.
- Major source of stem cells

Types of Bone Marrow:

1. RED MARROW
> directly involved in cell production.
- “medulla ossium rubra”

2. YELLOW MARROW
> storage for fat tissues
> tends to be located in the center-most cavities of long bones, and
is generally surrounded by a layer of red marrow
- inactive in the formation of blood cells

29
 - “medulla ossium flava”

* A child under 2 years has 100% red marrow

Functions of Bone Marrow:

- to produce new blood cells and play a key role in the body’s lymphatic
system.
a. Red blood cells (erythrocytes)
b. Platelets or thrombocytes (derived from metamegakaryocytes)
c. Granulocytes (neutrophils, basophils and eosinophils)
d. Macrophages (collectively known as myeloid cells)
e. B-lymphocytes
f. T-lymphocytes

Sites of Bone Marrow Examination:

- POSTERIOR ILIAC CREST (Posterior Superior Iliac Spine)
= the preferred site for marrow aspiration and biopsy

For Adults:
Sternum – aspiration only
Anterior Iliac Crest – less preferred because of the crest’s thick
cortical bone

For Newborns:
Proximal Tibia

29
PROCESSING:
A bone marrow examination usually consists of two parts : 

a. BONE MARROW ASPIRATION

- a syringe is used to aspirate a sample
of the liquid portion of the bone marrow

b. BONE MARROW BIOPSY/TREPHINE BIOPSY

- a core of marrow is taken out
- a needle puncture to obtain
a sample of bone marrow

Processing of Specimens:
a. ASPIRATE
For Particle Preparation
- After the excess fluid is removed from the remaining particles, they
are aggregated and clotted together by adding drops of 0.015 M
CaCl2 around the outside of the aggregate to prevent dispersing the
particles.
- A drop of the marrow is expressed onto the slides and then
distributed with a spreader device (marrow spicules)
- When they can be collected as clot, the particles are transferred to
Zenker’s fixative.
For Fluid Specimen
- Transferred in a Wintrobe tube: centrifuged for 8 – 10 minutes at
apprx 2800 rpm
- Normally four layers are present after centrifugation.

b. TREPHINE Specimens
- First decalcified before processing

29
 - the biopsies are put into vials with Zenker’s working solution, which is
prepared fresh daily.

Equipment for BM collection:

1. Bone marrow aspiration needle
- (UNIVERSITY OF ILLINOIS STERNAL
NEEDLE)
2. Bone marrow biopsy needle
- (JAMSHIDI NEEDLE)

3. Frosted Glass slide

4. Vials with EDTA or EDTA tubes
5. Freshly prepared Zenker’s fixative
6. 1 -2% Lidocaine buffered with Sodium bicarbonate
7. 5ml syringe and 25 gauge needle

Staining of Specimens:

29
 1. Routine staining:
- Romanowsky
2. Special stains
a. Cytochemical Stains
1. Periodic Acid Schiff (PAS)
2. Myeloperoxidase
3. Sudan Black
4. Chloroacetate esterase
5. Non-specific esterase
6. Tartrate-Resistant Acid Phosphatase (TRAP
b. Immunocytochemical stains
- Terminal deoxynucleotidyl transferase (TdT)
3. Stain for sectioned material
- Hematoxylin and Eosin (H & E)

NORMAL MARROW CELLS

- in addition to the developing hematopietic cells, several other cell types are
present in a normal marrow

1. Macrophage (Histiocytes)
- Most mature cell in the mononuclear phagocyte system
- Functions: Perform essential phagocytic
Immunologic functions
In bone marrow:
Normal marrow: macrophages comprises <1% of the nucleated
cells
Increased:
- Hemolytic anemia
- Idiopathic thrombocytopenic purpura
- Solid tumors

Abnormal morphology:
- Gaucher’s disease

2. Mast cells (Tissue Basophil)

- Functions: Inflammatory and hypersensitivity reactions
(which they release their granule contents outside the cell
causing localized edema and inactivation of toxic agents)
- Granules contain Heparin and Histamine
- May be confused with BASOPHIL

In bone marrow:
Rarely seen in bone marrow

Increased:

29
 - Refractory Anemia
- Chronic Renal Failure
- Lymphoproliferative disorders
- Systematic mastocytosis

3. Osteoblasts
- Function: Specialized cells that synthesize new bone matrix
(bone forming cells)
- Often confused with PLASMA CELLS
- often appears in clusters or aggregates and may be mistaken for TUMOR
CELLS

In bone marrow films:

Children and infants : occasionally seen
Adult: Rare

Increased:
-bone formation or repair is occuring
- Paget’s Disease
- Metastatic tumor
-at the site of recent marrow biopsy

OSTEOBLASTS

4. Osteoclasts

PLASMA CELLS
29
 - Functions: involved in Bone demineralization and Resorption
(bone destroying cells)
- Formed from the fusion of circulating monocytes and macrophages
- Often misidentified as MEGAKARYOCYTE

In bone marrow films:

Children and infants : Commonly seen
Adult: Rare

Increased:
- bone remodeling or destruction is occuring
- Osteolytic bone disease
- Metastatic tumor
- at the site of recent marrow biopsy

EVALUATION OF MARROW PREPARATIONS

Prior to the microscopic examination of any bone marrow, Peripheral Blood Smear
obtained at the time of collection should be evaluated for:
- Erythrocyte
- Platelet
- Leukocyte morphology
In extreme Leukopenia:
300 cell differential (100 cells on each three slides) is performed

Bone Marrow Cellularity

- Percentage of marrow space occupied by hematopoietic cells compared with fat
- uses core biopsy

Normocellular Marrow
Adult: Fat – YELLOW MARROW – 10 TO 15%
Hematopoietic elements – RED MARROW – 40 TO 60%
Child under 2 years: RED MARROW -100%

Hypercellular / Hyperplasia
- increase in one or more cell lines

Hypocellular / Hypoplasia
- loss of cellularity or incomplete development in one or more cell lines.

1 : 2 – Normal ratio of marrow fat to hematopoietic elements in adults

29
- Aspirate smears are first evaluated at LOW MAGNIFICATION
= the sample contains SPICULES and is representative of the marrow cavity
- Examination should be directed to areas near spicules containing intact marrow
cells devoid of cytoplasmic stripping and excessive air-drying artifact.
- Abnormal cellular aggregates (e.g., clumps of metastatic cancer cells), and
numbers of megakaryocytes are also best appreciated at low power

- Using LOW-POWER MAGNIFICATION, the following are evaluated

1. Cellularity (Normocellular, Hypercellular, Hypocellular)
2. Cellular Distribution (compare to film preparations)
3. Megakaryocytes
4. Abnormal aggregates or infiltrates (eg.tumor nodules or granulomata)
5. Fibrosis (confirm with reticulin stain)
6. Abnormal intracellular and extracellular material

MARROW DIFFERENTIAL:

29
- Concentrated (or buffy coat) specimen is preferred
- Recommended to count and classify:
= ALL NUCLEATED HEMATOPOETIC CELLS at least 500 and preferably
1000 cells

Myeloid: Erythroid (M:E) Ratio

- Numeric expression comparing the relative number of granulocytic
precursors (neutrophils, eosinophils and basophils) with the relative
erythroid precursors in the bone marrow

NORMAL: 2:1 to 4:1

Infection : 6:1 Leukemia : 25:1

29