Blood and Bone Marrow Preparation Techniques

Blood and Bone Marrow

• Blood smears are prepared using a drop of blood, air-dried, and fixed with methanol.
• The smears are then stained using May-Grunwald Giemsa stain to visualize cell features.

Blood Film Preparation

• Different quality blood films are shown, from well-made films to films that are too thick, too thin, or have inconsistent pressure.
• Proper technique is crucial for clear cell visualization.
• Inconsistent pressure and uneven spreading during blood film preparation can lead to problems like elongated tails in the film.

Blood Film Staining

• Fixation in methanol, crucial for cells, for 5 minutes is standard procedure.
• Bone marrow fixation requires 15-20 minutes.
• May-Grunwald is diluted 50:50 in buffered water (pH 6.8)
• Giemsa stain is diluted 1:10 in buffered water (pH 6.8)
• A final step is rinsing in buffered water (pH 6.8).

Blood Smear Examination

• Oil immersion microscopy is used to examine the blood smear.
• The appropriate area shows red blood cells just touching, enabling detailed visualization.
• The smear is examined systematically from side-to-side, or from the thick to thin area.

Blood Smear Quality Considerations

• Too thick films obstruct cell detail.
• Too thin films make RBCs appear spherocytic.

Red Cell Morphology

• Important parameters for red cells include hypochromia and polychromasia, and anisocytosis.

White Cell Morphology

• Different types of white blood cells (WBCs), like lymphocytes, neutrophils, monocytes, eosinophils, basophils, metamyelocytes, myelocytes, and promyelocytes, are examined for their morphology.
• Slides of each WBC type are presented.

Bone Marrow

• Normal bone marrow has a myeloid to erythroid ratio of 3:1 (with variations from 2:1 to 4:1).
• Cell types present are both mature and immature.
• The cellular and fatty components are distributed evenly.

Bone Marrow - Trephine Biopsies

• Fixation using 10% formal saline is performed up to 48 hours.
• Decalcification methods depend on the laboratory process.
• EDTA is used as one type of preservation method.
• Staining techniques are dependent on the laboratory procedure
• H&E, iron (Perls' reaction), immunohistochemistry, and reticulin (silver impregnation) stains are examples.

Required Samples

• A full blood count (FBC) is collected at the same time.
• Trephine biopsies are not always necessary.
• Bone marrow aspirates are sometimes required.
• Multiple slides are typically made of the samples depending on the need for various stains and tests. (e.g., MGG (May-Grunwald-Giemsa) and cytochemistry)
• An EDTA blood sample is crucial for cytogenetic analysis and usually is obtained at 2 x 5 ml.

Normal Bone Marrow - Quantitative Data

• Cell counts for different bone marrow types are shown in a table.

This quiz covers techniques for preparing and staining blood smears and examining them under oil immersion microscopy. It includes essential steps for fixation and the importance of smear quality. Test your knowledge on the methods and procedures related to blood and bone marrow preparation.