Reticulocytes are transitional cells between late normoblasts and fully mature erythrocytes. Since basophilic RNA substances still exist in their cytoplasm, vital staining with brilliant cresyl blue staining solution will reveal a light blue or dark blue reticular structure in the cytoplasm under microscopic examination.
New Methylene Blue Staining Solution is prepared from New Methylene Blue, phosphate, deionized water and other components, and is used for the vital staining and counting of reticulocytes. The results stained with this solution feature a bright and clear background, and are not affected by prolonged over-staining. After staining, reticulocytes exhibit a light blue or dark blue reticular structure. This reagent is for research use only and not intended for clinical diagnosis or any other applications.
Materials to Be Prepared by the User:
1. Fresh whole blood or EDTA-anticoagulated whole blood
2. Small test tubes or centrifuge tubes, glass slides, microscope (oil immersion lens)
Operating Procedures (For Reference Only):
1. Take a small test tube or centrifuge tube and add 2–3 drops of New Methylene Blue Staining Solution.
2. Add another 2–3 drops of fresh whole blood or EDTA-anticoagulated whole blood to it and mix well.
3. Incubate at room temperature for 15–20 minutes or longer.
4. Take one drop of the mixture to prepare a blood smear.
5. Count the number of reticulocytes among at least 1000 erythrocytes under an oil immersion lens.
Staining Results:
After staining, the cytoplasm of reticulocytes contains a light blue or dark blue reticular structure.
Calculation:
Reticulocyte percentage = Number of reticulocytes counted among 1000 erythrocytes / 1000
Absolute reticulocyte count (cells/L) = Reticulocyte percentage × Erythrocyte count (cells/L)
Precautions:
1. This staining method is only applicable for the test tube procedure.
2. Ensure sufficient staining time; do not prepare blood smears immediately after mixing. If the room temperature is low, appropriately extend the staining time or incubate the mixture in a 37℃ constant temperature incubator.
3. Prepare blood smears with a uniform thickness and avoid erythrocyte overlapping to prevent interference with staining effects.
4. Fresh whole blood or EDTA-anticoagulated blood is required for blood cell smear staining.
5. Use the reagent as soon as possible after opening to avoid compromising subsequent experimental outcomes.
6. For your safety and health, wear a lab coat and disposable gloves during operation.
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