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Blood Film Staining Effects Educational Document
Blood Film Staining Effects Educational Document
Introduction
The stained peripheral blood film is one of the world’s most widely and frequently used tests. Since its introduction in
the late nineteenth century, basic elements of the blood film preparation and analysis have changed little. Modern
technology improvements and refinements have enhanced the availability of good quality commercial Romanowsky
stains, automated stainers and semi–automated slide makers.
A fresh, well-made, peripheral blood film is crucial for accurate cell morphology assessment. (Refer to The Peripheral
Blood Film: Staining, Cell Estimation and Review, CPSA ALQEP: May 2003.) 1
Blood films that are too thin or too thick present a problem. Extremely thin films (caused by too small a drop, too slow
spreading or too low a spreader angle), may result in RBCs that appear as spherocytes and increased WBCs, such as
monocytes and neutrophils, in the tails. An incorrect differential will result. Always scan the film under low power to
detect this aberration.
In extremely thick films, the counting area is too small. At least ten low-powered fields where fifty percent of the RBCs
do not overlap are required for an accurate WBC differential.
Blood films with excessive tails or gritty feathered ends indicate a spreader edge that is rough or dirty, or an
accumulation of leukocytes due to either slow spreading or a very high leukocyte count.
Prior to staining, cells must be fixed to the glass slide with acetone-free methanol, either alone or in solution with dye.
Addition of a buffer solution to the dye changes the pH of the solution and ionizes the reactants to initiate the pH-
dependent staining process. Acidic cellular elements such as nucleoproteins, nucleic acids and primitive cytoplasmic
proteins, react with the basic dyes, methylene blue and its oxidative products. These elements are basophilic and stain
variations of blue. Basic cellular elements such as hemoglobin molecules and some cytoplasmic constituents in
leukocytes, have an affinity for the acidic dye, eosin. These elements are acidophilic and stain orange-red. A neutrophil
has neutral staining characteristics and stains blended shades of purple or pink, representing combinations of acidic and
basic molecular groups. Azure dyes stain the primary or non-specific granules in most myeloid cells red-purple, hence,
the term azurophilic granules.
Romanowsky Stains
Romanowsky stains are universally used in hematology. They are composed of methylene blue, oxidative products of
methylene blue (Azure A, Azure B, Azure C and Thionin) and eosin dyes. Giemsa, a commonly used stain does not
adequately stain red blood cells, platelets or white blood cell cytoplasms when used alone. A second Romanowsky stain
is therefore often used in combination with Giemsa and contains azure dyes to intensify the staining of nuclear features
and of azurophilic and toxic granulation. Wright, Wright-Giemsa and May-Grünwald Giemsa are commonly used
combinations of Romanowsky stains. Rapid or “quick” stains, developed for ‘stat’ situations or for small laboratories,
are adequate for assessing normal cell morphology.
Historically Romanowsky stains presented large variations in staining properties between stain batches and
manufacturers. This variation in stain quality is a result of the continued oxidation of methylene blue. A simple
combination of pure azure B and eosin Y, as used in the new polychrome stains, is preferable as oxidation is complete in
these solutions producing a standardized stain. Most laboratories purchase commercial ready-to-use staining solutions
for convenience and for reproducibility, however raw materials are available for “homemade” stains. 2
Staining Methods
Thoroughly dried blood films may be stained manually or with an automated staining instrument.
Note:
Slides cannot be added once the staining process is started.
The staining process takes approximately twenty minutes.
Produces good quality, reproducible staining.
Macroscopically, a properly prepared and well-stained blood film should appear pink in the thin area and have a
purplish-blue tint in the thicker area.
Cell/Component; Color;
1. Stain too acidic (red) Buffer or stain too acid Correct pH, remake buffer
-RBCs are bright red-orange Excess buffer for stain Shorten buffer time/amount
-WBC nuclei are pale blue Insufficient staining time Prolong staining time/amount
-Eosin granules are brilliant orange-red Very thin films Correct film thickness
Old stain (oxidized alcohol) Check expiration date/stain
2. Stain too alkaline (blue) Buffer or stain too alkaline Correct pH, remake buffer
-RBCs are blue-green Insufficient buffer for stain Increase buffer time/amount
-Eosin granules are gray/blue Excessive staining time Decrease staining time/amount
-WBC nuclei are blue-purple Very thick films Correct film thickness
-Neutrophil granules are too dark
-Lymph cytoplasm is gray
4. WBC nuclei too dark Stain too concentrated Check Giemsa dilution (dip-type stainers)
Adjust metered stain volumes (platen-type
stainers)
Staining time incorrect Check timing on stain stations (dip- type
stainers)
Reduce staining interval (manual dip-method)
References
1. Clarke, Dr. Gwendolyn, The Peripheral Blood Film: Staining, Cell Estimation and Review, CPSA ALQEP: May, 2003.
2. Dacie, Sir J.V., Lewis, S.M., Practical Haematology, 7th edition, pages 77 to 81, Churchill Livingstone, 1991.
3. Stiene-Martin, E.A., Lotspeich-Steininger, C.A., Koepke, J.A., Clinical Hematology - Principles, Procedures and
Correlations, 2nd edition, pages 22 to 34, Lippincott, 1998.
4. O’Connor, Barbara, H., A Color Atlas and Instruction Manual of Peripheral Cell Morphology, pages 15 to 18,
Williams & Wilkins, 1984.
5. Bain, Barbara, J., Blood Cells - A Practical Guide, 3rd edition, pages 11 to 13, Blackwell Science, 2002.
6. Dynacare Kasper Medical Laboratories, Staining Procedures, and Operating Instruction Manuals.