Cell biology

1. CHAPTER 7
PREPARATION, STAINING AND
EXAMINATION OF BONE MARROW
SMEARS
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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
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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
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4. Outline
 Bone marrow
 Sites of bone marrow aspiration
 Preparation and examination and of bone marrow
smears
 Myeloid : Erythroid ratio
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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)
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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.
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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
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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

10. BONE MARROW BIOPSY INDICATION
• Mast cell disease
• Idiopathic thrombocytopenic purpura
• Primary amyloidosis
• Metabolic bone disease
• Therapeutic follow-up
– Chemotherapy/bone marrow transplantation
– Treatment of isolated cytopenia

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.
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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
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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
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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.
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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

16. 16

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
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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
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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.
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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.
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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.
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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
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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.
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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
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27. Bone marrow Films cont’d
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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
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29. Particle/Crush Smears cont’d
Squash preparation and meandering smear for
the cytological analysis of bone marrow
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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
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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%
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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
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33. Cellularity of Bone marrow cont’d
NORMAL/NORMOCELLULAR MARROW BIOPSY 33

34. Cellularity of Bone marrow cont’d
Hypocellular marrow
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Aplastic marrow

35. Cellularity of Bone marrow cont’d
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Hypercellular marrow

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)
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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
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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
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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
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40. Marrow Differential Counts in Adults
• Myeloblasts: 0.0-3.5%
• Promyelocytes: 0.0- 6.0%
• Myelocytes: 8.0-15.0%
• Metamyelocytes: 9.0-25.0%
• Band and Segmented: 15.0-27%
• Neutrophils: 7.0-25.0%
• Eosinophils: 0.0-4.0%
• Basophils: 0.0-1.0%
• Pronormoblasts: 0.0-3.0%
• Basophilic normoblasts: 1.0-5.0%
• Polychromatophilc normoblasts: 5.0-20.0%
• Orthochromatic normoblasts: 1.0-15.0%
• Lymphocytes + Precurssors: 3.0-20.0%
• Plasmacytes + Precurssors: 0.0-3.5%
• Monocytes + Precurssors: 0.0-2.0%
• M:E Ratio: 1.5-3.5%
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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?
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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.
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