2. Acknowledgements
• Addisa Ababa University
• Jimma University
• Haramaya University
• Hawassa University
• University of Gondar
• American Society for Clinical Pathology
• Center for Disease Control and Prevention-Ethiopia
2
3. Objectives
At the end of this chapter the student would be able to:
Indicate the conditions for which bone marrow
examination is indicated
Indicate the sites of bone marrow aspiration in the
different age groups
Discuss the techniques for preparation and examination
of bone marrow smears
Discuss bone marrow cellularity
Discuss myeloid to erythroid ratio
3
4. Outline
Bone marrow
Sites of bone marrow aspiration
Preparation and examination and of bone marrow
smears
Myeloid : Erythroid ratio
4
5. Bone marrow examination
• Refers to the pathologic analysis of samples of bone marrow
obtained by bone marrow aspiration and bone marrow
biopsy(often called a trephine biopsy)
• The aspirate yields semi-liquid bone marrow, which can be
examined
– Under a light microscope as well as
– Analyzed by flow cytometry (immunophenotyping)
– Chromosome analysis, or
– Polymerase chain reaction(PCR)
5
6. Introduction
Bone marrow examination – an indispensable aspect of
the hematological investigation and diagnosis
Marrow examination is necessary for:
Discovering or confirming a variety of diagnoses
The monitoring of Hematologic and non hematologic
diseases (solid tumors) and leukemic patients
undergoing intensive chemotherapy
Cytological and histological examination are the major
aspects of bone marrow investigation although in recent
years:
Bone marrow culture for cytogenetic and kinetic
studies
Isotopic labeling
Processing for electron microscopy
Clonal studies
Culture and other methods have become established
7. Bone marrow examination…
•Bone marrow examinations may be used to:
•Diagnose and stage hematologic and nonhematologic neoplasia,
•Determine the cause of cytopenias
•Confirm or exclude metabolic and
•Infectious conditions.
•Each bone marrow procedure is ordered after consideration of
clinical symptoms and peripheral blood findings
•Bone marrow puncture is prohibited in patients with
coagulopathies such as hemophilia or vitamin K deficiency,
although thrombocytopenia is not an absolute contraindication.
7
8. Indications for bone marrow examination
• Unexplained anemia, Cytopenia or cytosis
• Abnormal peripheral blood smear morphology
• Diagnosis, staging and follow-up of haematological malignancy
• Acute and chronic leukaemias,
• Myelodysplastic syndromes,
• Chronic myeloproliferative disorders,
• Lymphomas,
• Plasma cell myeloma,
• Amyloidosis,
• Mastocytosis
• Investigation of suspected bone marrow metastases
8
9. BONE MARROW BIOPSY INDICATION
Hypercalcemia, lytic lesions, hypergammaglobulinemia
Lymphoma Staging (classification)
Infections (Granuloma)
Fever of Unknown origin
Metastatic (transfer of disease from one organ to another)
workup
Small cell tumors of childhood
11. Types of Bone Marrow Collection
Aspiration Vs Trephine biopsy
Bone marrow aspiration
– Is a procedure to collect a sample of bone marrow fluid.
– This fluid is then looked at under the microscope.
– It is checked for abnormal cells. It may also be tested in other
ways.
– usually simple, safe and relatively painless
Bone marrow biopsy
– A trephine biopsy yields a narrow, cylindrically shaped solid
piece of bone marrow which is examined microscopically
(sometimes with the aid of immunohistochemistry) for
cellularity and infiltrative processes.
11
12. Bone Marrow Aspiration Vs Biopsy
Bone Marrow Aspiration
Fine cytological details
Cytochemical stains
Microbiological culture
Flow cytometry
Cytogenetic and molecular studies
Bone marrow Biopsy
Assessment of cellularity and architecture
Detect lesions
Assessment of aplastic anemia, metastasis
Archival material
12
13. Bone Marrow Specimens
Samples of bone marrow can be obtained by:
Aspiration using a special needle and syringe, e.g.,
Salah, Klima, and Islam’s aspiration needles
Percutaneous trephine in which a section of bone is
taken for examination
Open surgical biopsy or open trephine that requires
full operating theatre practice
Most bone marrow samples for hematological purposes
are obtained by aspiration often combined with needle or
trephine biopsy
The aspiration procedure is usually simple, safe and
relatively painless
13
14. Sites of Bone marrow Aspiration and Biopsy
The site selected for the aspiration
depends on:
The age of the patient
Whether or not a needle or
trephine biopsy is required
The appropriate sites in an adult
include the posterior iliac crest
(preferred site), anterior iliac crest,
and sternum.
Under 12 years – iliac crest
The tibia may be used in infants
younger than 18 months of age.
14
15. The sternum
The best site when aspiration only is needed
The easiest to puncture
Considered to yield the most cellular samples
A disadvantage is that the patient has a clear view of the procedure
which may cause distress
Anterior or posterior iliac spines
Have the advantage that if no material is aspirated, a micro trephine
biopsy can be performed immediately
In disorders associated with replacement of hemopoietic marrow
by other tissues or cells(e.g., malignancies in the bone marrow)
Marrow aspiration may be difficult or impossible, the so-called dry
tap
In such cases, a needle or trephine biopsy is essential 15
17. Aspiration Biopsy cont’d
Bone marrow biopsy from the
superior part of the posterior iliac
spine (back of the hipbone) 17
18. Aspiration Biopsy cont’d
Infants and children: the sternum is naturally thin and an
alternative site is preferred
Under 12 years – iliac crest
Under 2 years – the presence of active marrow in the long
bones makes the proximal anterior portion of the tibia a
possible site
In disorders associated with replacement of hemopoietic
marrow by other tissues or cells (e.g., malignancies in the
bone marrow)
Marrow aspiration may be difficult or impossible, the so-
called dry tap
In such cases, a needle or trephine biopsy is essential
18
19. Aspiration and Biopsy cont’d
A minimum amount of marrow should be aspirated
Volumes over 0.5ml will almost certainly be diluted with
blood making processing and interpretation more difficult
Careful preparation is essential
It is desirable, if possible, to concentrate the marrow cells at the
expense of the blood in which they are diluted
19
20. Preparations prior to the procedure
The procedure should be explained in detail to the patient.
The past clinical history of the patient should be obtained
Informed consent should be obtained from the patient.
A blood count and smear should be obtained
Adequate sedation and analgesia determined
Assessment of thrombocytopenia or coagulopathic risks
Consider site for BM examination carefully 20
21. Procedure for Bone marrow aspiration
Patient preparations should be ensured.
A needle is inserted in to the iliac crest or spine
The needle and stylet are pushed into the bone with a slight rotary
motion.
When it is felt that the needle is firmly in place, the stylet is
removed and a syringe with out anticoagulant is attached and
<0.5ml of bone marrow and blood aspirated.
Bone marrow smears should be prepared immediately following
aspiration.
21
22. Procedure for Bone marrow aspiration…
A second syringe should be attached to the aspiration needle to
draw additional samples for supplementary tests, such as
Flow cytometry,
Cytogenetic analysis and Molecular genetic studies,
microbiology, Electron microscopy or BM culture.
In the event of a ‘dry tap’, or if no particles (‘fragments’) have
been obtained, the BM aspirate can be repeated at a slightly
different angle or at another site.
22
23. Procedure for Trephine Biopsy of bone marrow
It is obtained by inserting the biopsy needle into the bone and
using a to-and-fro rotation to obtain a core of tissue.
Needles are both re-usable and disposable.
The bone marrow biopsy is then placed on a slide, where imprints
are made before processing for cytologic investigations.
Specimen should fixed in 10% formal saline
After decalcification, the biopsy specimen is embedded in paraffin
wax and sections cut on a microtome.
23
24. Bone marrow Films
Method
1. Deliver single drops of aspirate on two slides about 1cm from
one end and then quickly remove most of the blood with a
pipette applied to the edge of each drop.
Alternatively, place the slides on a slop to allow the blood
to drain away
The irregularly shaped marrow fragments tend to adhere
to the slide and most of them will be left behind
24
25. Bone marrow Films cont’d
2. Prepare films using ¾ of a glass slide of the marrow
fragments and the remaining blood using a smooth-edged
glass spreader of not more than 2cm in width
The marrow fragments are dragged behind the spreader
and leave a trail of cells behind them
It is in these cellular trails that the differential counts be
made commencing from the marrow fragments and
working back towards the head of the film; in this way,
smaller numbers of cells from the peripheral blood
become incorporated in the differential count
The preparation can be considered satisfactory only when
marrow particles as well as free marrow cells can be
seen in stained films.
25
26. 4.4 Bone marrow Films cont’d
3. Fix the films of bone marrow and stain them with
Romanowsky dyes as used for peripheral films
However, a longer fixation time (at least 20 minutes in
methanol) is essential for high quality staining
The staining time should also be increased if the marrow
is hypercellular
26
27. Bone marrow Films cont’d
27
Film of aspirated bone marrow.
The marrow particles are easily
visible, mostly at the tail of the
film
28. Particle/Crush Smears
Aspirated marrow particles are isolated crush preparations
made by gentle pressure of a second slide combined with the
sliding apart of the two slides either in one movement or by a
series of interrupted movements
Technique gives preparations of authentic marrow cells
Squashing and smearing out the particles causes
disruption and distortion of cells
The resultant thick preparations are difficult to stain
well
28
30. Examination and Assessment of Stained Bone
marrow Preparations
The first thing to do is to look with the naked eye at a
selection of slides and to choose from them the best spread
films containing easily visible marrow particles
The particles should then be examined with a low power
objective with particular reference to their:
cellularity and
an estimate of whether the marrow is:
Hypoplastic/hypocellular
Normoplastic/normocellular
Hyperplastic/hypercellular
30
31. Cellularity of Bone marrow
It is expressed as the ratio of the volume of hemopoietic cells
to the total volume of marrow
It is judged by comparing the areas occupied by fat spaces and
by nucleated cells in the particles
Normal marrow is normocellular or normoplastic
Cellularity varies with the age of the subject and the site from
which the bone marrow is taken
For example, in an individual 50 years of age:
vertebrae = 75%
sternum = 60%
iliac crest = 50%
31
32. Cellularity of Bone marrow cont’d
If the percentage is increased for the age of the patient
The marrow is said to be hypercellular or hyperplasic
Such hyper cellular marrow is seen in:
Myeloproliferative disorders (e.g. CML,Polycythemia)
Lymphoproliferative disorders (e.g. ALL, CLL)
Infections
Acute leukemia
32
36. Cellularity of Bone marrow cont’d
If the percentage is decreased for the age of the patient
The marrow is said to be hypo cellular or hypo plastic
It is a finding in conditions associated with marrow failure
Aplastic anemia
Toxicity (e.g. drugs, chemicals)
36
37. Myeloid to Erythroid Ratio (M:E)
It is an expression of the myeloid and erythroid compartments
relative to each other
It is calculated after classifying at least 200 cells (leukocytes of
all types and stages of maturation are counted together)
In normal adult bone marrow, the myeloid cells always
outnumber the erythroid cells with a mean value of 4:1
An increased M:E ratio shows:
An increase in the number of leukocytes, and
Depression of the erythroid series
A decrease in the ratio indicates:
The presence of erythroid hyperplasia and suppression of
granulocytes
37
38. Differential Count on Aspirated Bone marrow:
The Myelogram
Expression of the incidence of the various cell types as
percentages is not a mandatory part of bone marrow
examination because of:
The relatively long time required to perform the count
Little clinical usefulness of such an effort
The count is also unreliable due to:
Irregular distribution of the marrow cells, and
Inclusion of cells from the peripheral blood for which there
is no compensation
If at all to be attempted, a minimum of 200 cells should be
studied
38
39. Differential Count on… cont’d
Because of the naturally variegated pattern of the bone
marrow and the irregular distribution of the marrow cells:
Differential counts on marrow from normal subjects vary
widely
Minor degrees of deviation from the normal occurring in
disease are difficult to establish
39
41. Review Questions
1. What elements of the stained bone marrow architecture are
mainly assessed in bone marrow examination?
2. What is the normal myeloid to erythroid ratio?
3. What is the significance of a bone marrow examination?
41
42. Bibliography
• MA Lichtman, E Beutler, U Seligsohn, K Kaushansky,
TO Kipps (Editors). William’s Hematology. 7th Ed.
McGraw-Hill Co. Inc. 2008.
• Dacie, John V and Lewis, S.M. Practical Hematology
10th Edition Churchill-Livingstone 2006.
• Wintrobe, Maxwell M. Clinical Hematology 11th
Edition Lea and Febiger, Philadelphia 2003.
42