Staining of bone marrow smear

The staining of bone marrow smears and the preparation of histological sections in a haematology lab are critical procedures for diagnosing various haematological disorders, such as leukaemia, anaemias, and myelodysplastic syndromes. Below is a detailed guide to these processes.

Staining of Bone Marrow Smear

Bone marrow smears allow for the detailed study of cellular morphology. Staining is essential to differentiate between various types of blood cells and detect abnormalities.

Steps for Preparing and Staining a Bone Marrow Smear:

1. Bone Marrow Aspiration:
   • A fine needle collects a bone marrow aspirate, usually from the iliac crest.
   • The sample contains a mixture of marrow cells suspended in a small amount of marrow fluid.
2. Making the Smear:
   • Place a small drop of bone marrow aspirate on a clean glass slide.
   • Use a second slide to spread the drop by gently pulling it across the surface, creating a thin smear.
   • Allow the smear to air dry for a few minutes.
3. Fixation (optional, depending on the stain):
   • Some protocols may include fixing the smear in Methanol to preserve cellular structures.

4. Staining:

   • Wright-Giemsa Stain is the most commonly used stain for bone marrow smears. It provides detailed cellular differentiation of the nucleus, cytoplasm, and granules.
   • Procedure:
     • Fix the smear in methanol if necessary (typically 1-3 minutes).
     • Apply Wright-Giemsa stain for 3-10 minutes.
     • Add a buffer solution to allow the stain to develop for 5-10 minutes.
     • Rinse the slide with a gentle stream of water or buffer.
     • Let the slide air dry.
   • Result:
     • Nuclei stain purple or blue.
     • Cytoplasm stains shades of pink to blue, depending on the cell type.
     • Granules stain with distinct colours for different types of white blood cells.

5. Examination:
   • The stained smear is examined under a microscope by a pathologist or haematologist.
   • Cellular morphology, myeloid-erythroid ratio, presence of blasts, dysplasia, and other features are assessed.

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Preparation of Bone Marrow Histological Section

A bone marrow biopsy provides a core of marrow tissue, essential for assessing the bone marrow’s architecture, cellularity, and infiltrative or fibrotic processes.

Steps for Preparing Bone Marrow Histological Sections:

1. Bone Marrow Biopsy:
   • A core biopsy is typically taken from the posterior iliac crest.
   • This provides a solid piece of bone marrow tissue.
2. Fixation:
   • The bone marrow biopsy is immediately placed in 10% neutral buffered formalin to fix the tissue.
   • The biopsy is usually left in fixative for a few hours to preserve the tissue structure overnight.
3. Decalcification:
   • Since the biopsy contains bone, it must be decalcified before sectioning.
   • Decalcification is typically performed using EDTA or a mild acid solution, which softens the bone without damaging the marrow tissue.
   • The time required for decalcification depends on the size and density of the bone tissue (usually a few hours).
4. Dehydration and Embedding:
   • After decalcification, the tissue is dehydrated by passing it through increasing ethanol concentrations (from 70% to 100%).
   • The biopsy is then cleared in xylene and embedded in paraffin wax.
   • Once embedded, the tissue block is cooled and hardened.
5. Sectioning:
   • Thin sections (approximately 3-5 micrometres) are cut from the paraffin block using a microtome.
   • These thin sections are floated on a warm water bath to remove any wrinkles and then placed onto glass slides.
6. Deparaffinization and Rehydration:
   • The paraffin sections are deparaffinized in xylene or another clearing agent.
   • The slides are then rehydrated by passing them through decreasing ethanol concentrations, followed by water.

7. Staining:

   • Hematoxylin and Eosin (H&E) Stain is the standard stain used for histological sections.
   • Procedure:
     • Stain the sections with hematoxylin for a few minutes (stains nuclei dark blue).
     • Rinse with water and differentiate with an acid-alcohol solution to remove excess stain.
     • Stain with eosin to counterstain the cytoplasm and extracellular matrix (cytoplasm stains pink).
     • Dehydrate the slide in ethanol, clear it with xylene, and mount it with a coverslip.
   • Result:
     • Nuclei appear blue to purple.
     • Cytoplasm, extracellular matrix, and collagen appear in shades of pink.
8. Special Stains:
   • • Reticulin Stain (e.g., silver stain) for assessing fibrosis in the bone marrow.
     • Prussian Blue Stain for evaluating iron storage within the marrow.
     • Immunohistochemistry (IHC) for identifying specific cell surface markers (e.g., CD markers in leukaemia).

8. Examination:
   • The stained section is examined under a microscope to assess the overall architecture of the marrow, the presence of normal and abnormal cell populations, fibrosis, and any signs of malignancy.