Bone Marrow Examination Techniques

Bone Marrow Smear Preparation, Staining, and Examination

• Bone marrow examination is a crucial diagnostic tool in hematology
• It involves the analysis of bone marrow samples obtained through bone marrow aspiration and biopsy
• The aspirate yields semi-liquid bone marrow, which can be examined under a light microscope or analyzed by flow cytometry, chromosome analysis, or polymerase chain reaction (PCR)
• Trephine biopsy provides a solid piece of bone marrow, examined microscopically for cellularity and possible infiltrative processes

Bone Marrow Aspiration

• Common sites for aspiration in adults are the posterior iliac crest
• Posterior iliac crest is preferred because there is less risk of injury to blood vessels and easier observation of the procedure
• In cases of children, the sternum is useful and considered a good site when aspiration is needed.
• For infants, the proximal portion of the tibia is often the preferred site due to thinner bone
• A minimum of approximately 0.3 mL of marrow is needed for analysis

Bone Marrow Biopsy

• This involves collecting a solid piece of bone marrow often obtained with a trephine biopsy or needle biopsy
• This is critical for analyzing cellularity and architectural structure, with a view to examine for potential focal lesions which can be associated with Hodgkin's, non-Hodgkin's lymphoma, granuloma, and multiple myeloma

Bone Marrow Film Preparation

• Films are obtained with a smooth-edge spreader and 2 cm in width to create films of the aspirated bone marrow 3 to 5 cm in length.
• Films are fixed and stained with Romanowsky dyes
• Additional staining options, like Perl's method, help detect the presence of iron
• Films need adequate staining procedure to yield high-quality results
• Over night staining procedure to achieve optimal results

Bone Marrow Examination Objectives

• Identify conditions warranting bone marrow examination
• Indicate bone marrow aspiration sites based on age groups
• Describe techniques for bone marrow smear preparation and examination
• Evaluate bone marrow cellularity (normal/hypercellular/hypocellular)
• Assess myeloid-to-erythroid ratio
• Interpret differential counts

Indications for Bone Marrow Examination

• Unexplained anemia, unexpected cytopenia, or cytosis
• Multilineage abnormalities of circulating blasts in adults
• Abnormal peripheral blood smear morphology
• Diagnosis, staging, and follow-up of hematological malignancy
• Investigation of suspected bone marrow metastases
• Unexplained focal bony lesions or organomegaly
• Investigations for pyrexia or specific infections (e.g., granulomatous disease, tuberculosis, leishmaniasis, fungal infections)
• Evaluation of iron stores
• Monitoring after chemotherapy, radiation therapy, or stem cell transplantation

Contraindications

• Severe bleeding disorders

Complications

• Mild soreness lasting 12-24 hours is common, but severe complications are rare

Particle/Crush Smears

• Useful to assess cellularity and detect abnormal cells, megakaryocytes numbers, and certain focal diseases

Systemic Scheme for Examining Aspirated BM Films

• Low power (x10): assess cellularity, identify megakaryocytes, and abnormal cell clumps. Identify macrophages
• High power (x40, x100): assess maturation stages of myeloid and erythroid cells, determine myeloid:erythroid ratio(M:E), perform differential counts, assess iron content, check for necrosis.

Assessment of Bone Marrow Cellularity

• Cellularity is assessed by comparing the areas occupied by fat spaces and nucleated cells.
• Normal marrow is considered normocellular or normoplastic
• Cellularity varies based on patient age and site of aspiration

Myeloid to Erythroid Ratio

• The myeloid to erythroid ratio (M:E ratio) expresses the proportion of myeloid (granulocytic) versus erythroid cells in bone marrow
• In normal adult bone marrow, myeloid cells usually outnumber erythroid cells

Differential Count on Aspirated Bone Marrow

• Expression of cell types as percentages is not routinely performed because of long preparation times and limited clinical utility.
• The count may be unreliable due to irregular distribution of cells and possible inclusion of cells from peripheral blood
• A minimum of 200 cells should be assessed

Differential Counts in Adults

• Includes specific percentages of different cell types (myeloblasts, promyelocytes, myelocytes, metamyelocytes, band/segmented neutrophils, eosinophils, basophils, normoblasts, lymphocytes, plasma cells, and monocytes)

Review Questions

• Include questions about the conditions for examination, the sites in different age groups, preparation techniques, cellularity, the myeloid-to-erythroid ratio, and differential counts.

Hematological Cell Markers

• Identifying proteins on cell surfaces that are associated with cell lineage and maturation stage.
• Immunophenotyping is necessary for diagnosing and characterizing Acute Leukaemia, chronic myeloid leukaemia, and chronic lymphoproliferative disorders

Cell Marker Studies by Flow Cytometry

• Using monoclonal or polyclonal antisera and immunofluorescent methods to analyze cell markers and CD (cluster differentiation) markers which are specific to particular cells
• Flow cytometry is suitable for small samples

Terminal Deoxynucleotidyl Transferase (TdT) Test

• TdT is a nuclear enzyme marker. High levels are in lymphoblastic leukemias but not AML. TdT is helpful in differentiating between ALL and AML.

Molecular Methods (e.g., FISH)

• DNA probes and PCR used clinically are used to diagnose leukemia.
• Confirm chromosomal abnormalities and monitor residual disease after therapy
• Helpful in diagnosing certain leukemias and determining presence of mutations/markers associated with certain leukemias.

Systemic Lupus Erythematosus (SLE)

• Chronic rheumatic disease that affects many parts of the body.
• Immunologic disorder characterized by inflammation
• Associated with skin rashes, arthralgia, fever, and renal, cardiac, and vascular lesions
• Common in women of childbearing age.

LE Cell Phenomenon

• Presence of an immunoglobulin in serum that causes depolymerization of nuclear chromatin in polymorphs; phagocytosis occurs.
• The phagocyte (usually a neutrophil) incorporates a denatured nucleus from another cell into the LE cell.

Examination of Films for LE Cells

• Examine films for LE cells, especially the edges and tails.
• At least 500 polymorphs must be examined/counted.
• Differentiate from “tart cells” which are usually monocytes that have ingested nuclei of lymphocytes.