Bone Marrow Analysis and Results Interpretation

Presenters: Muwanguzi Enoch

&

Elizabeth John

Moderator: Okongo Benson

UMLTA CPD SERIES

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Presentation Outline

• Objectives
• Introduction
• Specimen collection
• BM analysis
• Lab Tests on BM Biopsies
• Typical BM report
• Results interpretation
• Indications and contraindications of BM examination.
• Conclusion

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Objectives of the Presentation

• To State the composition of the bone Marrow.
• To mention indications for bone marrow examination.
• To List the various tests that can be performed on the bone marrow sample.
• To prepare various bone marrow film.
• To identify and characterize features of hemopoietic and metastatic tumor cells
• To perform bone marrow differential count and compute M:E ratio
• To be able to prepare a systematic bone marrow film comment/report.

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THE BONE MARROW�Introduction …1/3

• Bone marrow or medulla ossea
• is the soft tissue found in the hollow interior of bones.
• Bone marrow is an organ
• 3.4% - 5.9% of body weight
• Major Haemopoietic site in normal adults
• Produces ≈ 6 billion cells per kg per day

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THE BONE MARROW�Introduction …2/3

• Types of marrow
• Red Marrow
• Myeloid tissue
• More at birth
• More in flat bones like
• Hip bone, breast bone, skull, ribs, vertebrae and shoulder blades, and in the cancellous ("spongy") material at the proximal ends of the long bones femur and humerus
• Yellow Marrow
• Fat cells
• Hollow interior of the middle portion of long bones
• Bone marrow stroma

NB: In Health or Disease the body can convert yellow marrow back to red marrow in order to increase blood cell production

Cellularity of BM Core biopsy

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A

B

C

Hypercellularity

Normocelluarity

Hypocellularity

THE BONE MARROW�Introduction …3/3

Sites of BM sampling

• Sternum
• Iliac crest
• Spines of vertebrae
• Tibia.

NB:

The type of marrow specimen is described by how it is extracted.

• BM Biopsy
• Aspirate
• Trephine Biopsy
• Open surgical biopsy
• BM Postmortem

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Cellularity of BM Core biopsy

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Trephine

Aspiration

Value of BM Examination or BM Biopsies

• Without exception the PB should be examined carefully first – it is relatively by uncommon circumstance to find haematological disease in BM without evidence in the PB.
• Haematological and Non-haematological malignancies
• Cytology and Histological examinations are now major aspects of BM investigation.
• Recent techniques include:
• BM culture for cytogenetic and cytokinetic studies
• Isotopic labelling
• Processing for electron microscopy
• Clonal culture

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• Cytogenetic: cell structure and function: the study of the relationship between inheritance and the structure and function of cell components
• Cytokinetic (Adj):cytokinesis(n) cell division: division of the cytoplasm of a cell during mitosis or meiosis.

BM specimen collection - 1

• Invasive procedure
• Clinical judgement and exclusion criteria
• Before BM sample collection collect blood sample for CBC and PBF within 24hrs.
• Many factors dictate the choice of one site over the other – Sternum - best

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Do not collect blood for CBC or PBF after BM collected; there will be stress related WBC elevated count

BM specimen collection - 2

• Collected by who?
• Skilled physician/(hematologist/pathologist)
• Minimum amount of marrow (0.5 – 1.0mL) should be aspirated – dilution with blood
• What is the key role of laboratory personnel?
• Specimen management Does not collect

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Lab analysis of BM Sample -1

1. Gross examination (Naked eye or low power): Visual inspection of the specimen for color, consistency, and any visible abnormalities. NORMAL BM appears Uniform reddish-pink in color, soft, friable texture; Homogenenous in consistency, No visible cycts or cavities; no visible scarring or fibrosis; No haemorrage or bleeding; Normal fat distribution (30 – 50%); No visible infiltration or replacement
2. Histopathological examination: Microscopic examination of stained bone marrow biopsy sections to evaluate:
• Cellularity
• Architecture
• Presence of abnormal cells or infiltrates
• Iron stores
• Fibrosis
3. Cytogenetic analysis: Examination of chromosomes for abnormalities, such as translocations or deletions, which can indicate cancer or other disorders.
4. Flow cytometry: Analysis of cell surface markers to identify and characterize immune cells, detect minimal residual disease, or diagnose hematological malignancies.
5. Molecular testing: DNA or RNA analysis to detect specific genetic mutations or aberrations associated with various diseases.�

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Lab Procedures applicable to BM biopsies

Imprint: pressed-in shape: a pattern, design, or mark that is made by pressing something down on or into something else

Conventional tests

and Cytochemistry

Preparations of BM to be studied

1. Trephine Biopsy
• Includes both Marrow and Bone
• Fixed in Bouin’s fluid or Zenkers fixative
• Decalcified
• Embedded in paraffin wax or plastic
• Stained with H&E/ Giemsa
1. Applications: Assessing cellularity, Quantitative cellular relationships, detection of fibrosis, Granulomas, focal infiltration with lymphoma cells, Hodgkins disease, or metastatic carcinoma.
2. Poor Preparation for:
• Appreciating morphology of individual cells
• Evaluation of iron stores – decalcification
• Enzyme cytochemistry
• Immunophenotyping

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1. X
2. x

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Preparations of BM to be studied

2. Touch Preparation

• An imprint of trephine on a glass slide
• Usually stained with May-Grunwald Giemsa stain
• Key when aspiration can not be obtained – heavy infiltration by abnormal cells
• Provides information on individual cell morphology and enzyme cytochemistry can be done

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Handling of Aspirated Bone Marrow

Routine Handling of Aspirated BM

1. Films made of material aspirated.
2. Marrow concentrated and films made.
3. Particle smears (direct and crush preparations)
4. Histological sections.

May be stained with Romanowsky

Special handling of aspirated BM

• Electron microscopy
• For cytogenetic studies
• Isotopic labelling and metabolic studies
• Colony culture techniques
• Immunofluorescent studies

Histological sections

• These commonest form BM is processed.
• They may be from:
• Needle biopsy (trephine) – cannot be used for Fe demonstration due to decalcification process.
• Clotted marrow ( No decalcification; fatty marrow may be removed because it floats)
• Aspirated fragments (No decalcification)
• Fixation in Zenker’s acetic acid solution (overnight)
• Embedding – Paraffin was or plastic material

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Tests on histological sections

• H&E
• Romanowsky staining (Giemsa)
• PAS
• Immunocytochemistry/histochemistry
• Iron store demonstration
• Demonstration of reticulin fibres
• The following can be demonstrated on sections:
• AFB and fungi
• Metastatic cancer or lymphoma cells (immunochem)
• Molecular methods like PCR can be done to detect molecular gene rearrangements and minimal residual disease or defection of infectious diseases.

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Advantages Histological sections

• Provide a clear picture of the architecture and cellularity
• Superior to smears for demonstration of non-haemopoietic elements such as maligmant cells (infiltration).
• Assessment of Fe stores is probably more reliable from sectioned biopsy.

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Disadvantages Histological sections

• The above clear picture of the architecture and cellularity is somewhat inferior for study of cytological details.
• These details are lost during processing; hence sections may not be valuable for diagnosis of leukaemia and refractory anaemias.
• Particles adequate for histoloical sections are always not obtained especially in conditions in which diagnosis depends on marrow evidence.
• Myelofibrosis and metastatic cancer

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Bone marrow examination reports

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Bone marrow examination reports

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BM Biopsy Results interpretation

The following are the key elements that inform the interpretation:

1. PBF & CBC
2. Cellularity
3. Distribution of cells
4. Myeloid-to-Erythroid (M:E) ratio
5. Iron stores
6. Estimation of megakaryocytes
7. Maturation pattern and morphology of @ cell series
8. Presence of rare cell types or abnormal cells

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1. Peripheral blood examination

• More info if:
• Film is well prepared
• Clinical notes available
• Absolute values used
• Macroscopy done
• FBC done on same day of BM withdraw
• Reticulocyte count done
• Results of FBC, Film and Reticulocyte count are incorporated in the report.

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2. Cellularity of the marrow

• NCC of aspirated marrow is unreliable due to variable dilution with blood even with the list possible samples
• BM cellularity: the relationship of the developing haemopoietic cells to the fat spaces within the BM is present as a relatively fixed proportion.
• The ratio of marrow cells to marrow fat=1:1 – 2:1
• Ratio > 2:1 constitutes hypercellularity (hyperplastic marrow).
• The nature of cell line that is more abundant can give important clues to mechanisms of anaemia or leucocyte abnormalities.

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3. Distribution of cells

• Distribution of various cells can be ascertained in two ways:
• Several slides are scanned under low & high magnification and on basis of previous experience one estimates the number and distribution of cells.
• One actually makes a differential of 300 – 1000 cells and calculates the % of each type of cell (Myelogram).
• A combination of the above methods is preferable.

NOTE: The differential is the most essential because it affords an objective record from which future changes may be measured.

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3. Distribution of cells..

• Handing the differential:
• From the diff. Myeloid to Erythroid ratio (MER) is calculated or estimated.
• MER is the ratio of total granulocytes to total normoblasts. In adults the range is broad varying from approximately 1.2 : 1 and 5:1. Generally quoted as 4:1.

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Interpretation of MER

INCREASED (e.g. 6 – 1)

• Infections
• Chronic myelogenous leukaemia (CML)
• Erythroid hypoplasia.

REDUCED (e.g. Less than1.2 – 1)

• Depression of leucopoiesis

or

• Normoblastic hyperplasia

[Depending on BM cellularity]

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NOTE:

MER does not indicate if the elements are hypoplasia or hyperplasia.

MER is interpreted alongside BM cellularity.

Estimation of megakaryocytes

• The number is reliably estimated in BM sections.
• Scanning films with good cellularity under low power (x100 total Magnification) –
• on average 1 – 3 megakaryocytes @ field should be found normally.

NOTE:

• Megakaryocytes are found at the edges of films and in the vicinity of fragments and their number and maturation should be recorded

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Reference ranges of marrow aspirate in adults

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4. Maturation pattern and morphology of @ cell series

• During differential count one should evaluate whether maturation is normal i.e.
• If nuclear & cytoplasmic development is in balance
• Impaired cytoplasmic maturation in normoblasts e.g.in impaired Hb synthesis,
• Impaired nuclear maturation may occur in
• Certain drugs
• Some leukaemia (acute)
• Note: Hyperplasia of a cell series also alters its maturation pattern

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5. Presence of rare cell types or abnormal cells

BM should be scanned to look for rare or unexpected cells like:

1. Tissue mast cells (Tissue Basophils)
2. Osteoblasts
3. Clusters of metastatic neoplastic cells
4. Possible presence of parasites or microorganisms
5. Lipid loaded (laden) macrophages

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These are some of the non-haemopoietic elements assessed in the bone marrow cytology

Presence of rare cell types or abnormal cells

1. Tissue mast cells (Tissue Basophils)
• Normally very frequently increased in
• Aplastic or refractory anaemias
• Lymphoproliferative disorders

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Presence of rare cell types or abnormal cells

b) Osteoblasts

• These are cells that synthesize the collagen matrix of the bone.
• Increased in:
• Aspirates of infants and children – Normally
• Adults when Bone marrow destruction or repair is occurring as in:
• Hyperparathyroidism,
• Metastatic tumour
• Recent biopsy at the same site

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Presence of rare cell types or abnormal cells

c) Clusters of metastatic neoplastic cells

• In patients with metastatic cancer.
• Note:
• Some neoplastic cells resemble myeloblasts and other primitive blasts.
• Clue: metastatic cells appear in clusters or clumps

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Presence of rare cell types or abnormal cells

d) Possible presence of parasites or microorganisms.

• Fungi
• Cryptococcus neoformans
• Protozoa:
• Trypanasomes [metacylic trypomastigotes]
• T.b.rhodesience (African trypanosomiasis)
• T.b.gambiense
• Leishmania donovani (Visceral leishmaniasis) [Promastigotes]

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Presence of rare cell types or abnormal cells

e) Lipid loaded (laden) macrophages

• As in:
• Gaucher’s disease and
• Due to lack of β-glucosidase
• Niemann-Pick disease
• Lack of sphingomyelinase

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Inherited disturbances in lipid metabolism associated with specific enzymes

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Gaucher’s cells in a bone marrow aspirate

Gaucher cells are macrophages - monocytic cells engorged by the presence in the lysosome of the incompletely degraded lipid glucocerebroside, which stains Positive with the Periodic Acid Schiff (PAS) reagent.

At high magnification, these cells present with a fibrillary type of cytoplasm (“crumpled tissue paper appearance”) and an eccentrically displaced nucleus

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A pair of pseudo-Gaucher cells seen in a BM aspirate from a patient with CML

Niemann-Pick cell in BM

Other diagnostic features during BM examination

• Erythrophagocytosis – autoimmunity
• Abnormal number of phagocytic reticulum cells (macrophages)
• Excess plasma cells
• Non-haemopoietic cells
• Degenerative or necrotic cells
• Characterized by:
• Stain irregularly with blurred outlines
• Cytoplasmic shrinkage and nuclear pyknosis
• Found in:
• SCD
• Lymphomas (occasionally)
• ALL &CLL (occasionally)
• Metastatic cancer

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Indications of BM examination

1. Primary diagnosis of haematolyphoid malignancies

• Acute leukaemia
• Chronic myeloproliferative disorders
• Chronic lymphoproliferative disorders
• Myelodysplastic syndromes
• Hodgkin’s and non-hodgkin lymphomas
• Multiple myeloma

2. Staging of lymphoid malignancies and solid tumours.

3. Post treatment follow-up

• Post chemotherapy and radiation therapy
• Post bone marrow transplant

4. Detection of infection and/or source of fever of unknown origin (PUO)

• Mycobacterium and fungal infections
• Granulomas
• Unknown infectious agents using cultures and special stains
• Haemophagocytic syndrome

5. Primary diagnosis of systemic diseases (Non-haematological malignancies)

• Metabolic disorders (Gaucher’s disease. Etc)
• Systemic mastocytosis

6. Miscellaneous

• Evaluation of storage iron
• Evaluation of unexplained cytopenias.

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Contraindications of BM examination

1. As work-up for most anaemia (ancillary tests are done) especially:
• IDA - ferritin levels than BM iron stores
• Megaloblastic anaemia. –Folate, Vit B12 levels & Schilling test
• Haemolytic anaemia- Biochemical tests
• BM – May be indicated in unexplained macrocytic anaemia- myelodysplastic syndrome.
2. Patients with Thrombocytopenia
3. Patients with Coagulopathies

NB: Most cases of unexplained anaemia with no abnormality in WBCs or Platelets are secondary to systemic disease (ACD) rather than haematological disorder.

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Causes of Dry Tap during BM extraction

1. Sparse haemopoietic activity – virtually no marrow exists to be withdrawn (severe hypocellularity & marrow aplasia)
2. Marrow contains tightly packed, sticky highly immature cells which cannot be aspirated (some acute leukaemias)
3. Marrow fibrosis (scar tissue) or metastic cancer is present.

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Conclusion

• BM Examination is a very important aspect in diagnosis, monitoring and classification of haematological malignancies.
• BM analysis is a focal point for Clinical and Laboratory haematologists.
• Laboratory networking can enable us achieve complete laboratory analysis of the BM specimen.

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References

• Bain, B. J., Bates, I., Laffan, M. A., & Lewis, S. M. (2016). Dacie and Lewis practical haematology: expert consult: online and print. Elsevier Health Sciences.
• Joan H. Howanitz & Peter J. Howanitz (1991). Laboratory Medicine – Test Selection and Interpretation. Churchill Livingstone.
• Roger Hall and Robert G. Malia, Medical Laboratory Haematology: Butterworths London 1984.
• Monica Cheesbrough, District Laboratory Practice in Tropical Countries, Part 2, CambridgeUniversity Press, 2000.
• Sir John V. Dacie and S.M. Lewis, Practical haematology, 8th Ed., ELBS Churchill Livingstone, 1994.
• Jacqueline H. Carr and Bernadette F. Rodack, Clinical Hematology Atlas, W.B. Saunders Company, 1999.
• Hoffbrand and Lewis, Postgraduate Haematology 2nd Ed., William Heinemann Medical Books Ltd, London, 1981.
• F.J. Baker and R.E. Silverton, Introduction to Medical Laboratory Technology, Butterworths London Boston
• John Bernard Henry, Clinical Diagnosis and Management by Laboratory Methods, 18th Ed., W.B.Saunders Company, 1991.

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THE END

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THANK YOU

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