EMS26077-20
Introduction
New Methylene Blue N Stain, Aqueous, provides a staining technique for reticulum of immature erythrocytes. Immature erythrocytes (reticulocytes) contain RNA which is lost as cells age. When New Methylene Blue N Stain, Aqueous, is used to supravitally stain viable erythrocytes, the RNA in young cells is precipitated and stained deep blue. The stained RNA appear as blue granules which may be connected into a reticulo-filamentous pattern.
Staining Procedure
- Filter New Methylene Blue N Stain, Aqueous, prior to use.
- Collect appropriate blood sample for reticulocyte count, per laboratory protocol. Proceed with staining procedure as soon as possible after blood draw.
- Pre-label microscopic slide(s) with appropriate patient identifiers.
- Mix five drops of New Methylene Blue N Stain, Aqueous with five drops of whole blood; mix gently with a pipette.
- See Procedure Notes #1, #2, and #3.
- Incubate mixture at room temperature for 10-15 minutes.
- See Procedure Note #4.
- Thoroughly remix stain/blood suspension after incubation.
- See Procedure Note #5.
- Prepare wedge smear(s) on pre-labeled microscopic slide(s) with the remixed stain/blood suspension.
- Allow slide(s) to thoroughly air-dry.
- Evaluate reticulocyte count under oil immersion.
Results
Reticulocytes | Pale blue with dark blue granular/reticular material |
Red cells | Pale blue or blue-green |
Procedure Notes
- A small test tube, vial, or centrifuge tube can be used for mixing purposes.
- Smaller or larger amounts of stain and blood can be mixed as long as volumes are of equal proportions.
- Separate pipettes should be used for each solution and step to avoid any possibility of sample contamination.
- Incubating longer than 15 minutes may increase the possibility that mature erythrocytes will also be darkly stained.
- Reticulocytes have lower density than mature erythrocytes and will be near the top during incubation. Remixing prior to preparing smears allows for equal cell distribution.
References
- Bauer, John D. Clinical Laboratory Methods. 9th ed. St. Louis: Mosby, 1982. 195-198.
- Lillie, R.D., and Harold Fullmer. Histopathologic Technic and Practical Histochemistry. 4th ed. New York: McGraw-Hill, 1976. 752-753.
- McPherson, Richard and Matthew Pincus. Henry's Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Philadelphia: Elsevier Saunders, 2011. 514. 544.
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