BLOOD FILM STAINING EFFECTS

An Educational Supplement prepared by ALQEP – May 2004

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.

Blood Film Preparation

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.

Staining the Blood Film

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.

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 BLOOD FILM STAINING EFFECTS

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.

Manual Staining Methods

1. Dip Method (Rapid):

The dip method is a quick staining method that uses a modified Wright-Giemsa stain buffered in methanol at pH
6.8. Slides are immersed in the stain in a coplin jar for a user-determined length of time. It is important that the
Wright-Giemsa stain be kept tightly sealed in coplin jar when not in use and be replaced when water artifact
appears in red cells (see: Causes and Corrections of Stain Deviations).
Note: Staining time may be increased for greater cellular detail as required.

Automated Staining Methods

1. Hema-Tek Slide Stainer - Miles Scientific (platen-type stainer):

The Hema-Tek slide stainer is a self-contained bench top slide stainer that uses a Hema-Tek Stain Pak. Three
sensing switches are triggered sequentially to activate three solution pumps which deliver metered volumes of
stain, buffer and rinse solution from the stain pack. A Wright-Giemsa Pak is most commonly used. Slides are
advanced by two parallel conveyer spirals with the stain, buffer and rinse solutions pumped up between the blood
film and platen.
Note:
 Slides can be randomly added if required
 The staining process takes approximately ten minutes
 Time phases are constant
 Pump volumes are user adjusted by control knobs
 A stain/buffer ratio of 1:2 is desirable
 Cleaning procedures and daily maintenance are vitally important to prevent stain deposit artefact.

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 BLOOD FILM STAINING EFFECTS

2. Hemastainer Automatic Slide Stainer - Miles Scientific (dip-type stainer):

This is an automated staining instrument that stains up to fifty slides at one time. The slides are loaded into a slide
basket and dipped into each of six stations in the staining process. Five individual, adjustable timers control the
first five stations. The six stations consist of: methanol, stain, a second stain, buffered-water rinse, a phosphate
buffer rinse and lastly, forced-air drying. The two stains commonly used in the stainer are May-Grunwald and
diluted Giemsa.

3. Midas II Automatic Slide Stainer - EM Diagnostic (dip-type stainer):

This instrument is a completely automatic slide stainer that stains up to twenty slides at one time. The slides are
loaded into a bucket and are cycled through six stations: methanol, stain, a second stain, water rinse, phosphate
buffer rinse, followed by the drying station. The first five stations are on individual, adjustable timers.

The Midas II stainer is similar in operation to the Hemastainer.

Note:
 Slides cannot be added once the staining process is started.
 The staining process takes approximately twenty minutes.
 Produces good quality, reproducible staining.

Evaluating the Stain Quality

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.

Optimal microscopic staining characteristics with a Romanowsky stain are as follows:

Cell/Component; Color;

Red blood cells Salmon pink

Nuclei of neutrophils Deep blue-purple
Specific granules of neutrophils, granules of Light purple or violet
lymphocytes, granules of platelets
Specific granules of basophils Deep purple
Specific granules of eosinophils Orange
Chromatin (including Howell-Jolly bodies) Purple
Dohle bodies Blue-grey
Promyelocyte granules and Auer rods Purplish-red
Cytoplasm of lymphocytes Blue
Cytoplasm of monocytes Blue-grey (ground glass appearance)
Cytoplasm of neutrophils Light pink
Cytoplasm of platelets Purple-blue to lilac

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 BLOOD FILM STAINING EFFECTS
Causes and Corrections of Stain Deviations
Stain Deviation
1. Stain too acidic (red)
-RBCs are bright red-orange
-WBC nuclei are pale blue
-Eosin granules are brilliant orange-red
2. Stain too alkaline (blue)
-RBCs are blue-green
-Eosin granules are gray/blue
-WBC nuclei are blue-purple
-Neutrophil granules are too dark
-Lymph cytoplasm is gray
3. Stain too pale
-Little contrast in WBCs
4. WBC nuclei too dark
5. Water/drying artifact
6. Precipitation on slides
Summary
A properly made, properly stained and correctly assessed blood film is a critically important diagnostic tool. A poorly
made or poorly stained blood film is of little value and may result in erroneous patient results and missed or incorrect
diagnosis.
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Causes
Buffer or stain too acid
Excess buffer for stain
Insufficient staining time
Very thin films
Old stain (oxidized alcohol)
Buffer or stain too alkaline
Insufficient buffer for stain
Excessive staining time
Very thick films
Weak stain solution
Stain too concentrated
Staining time incorrect
Water contamination
Film drying too slowly
Unclean platen or lines
Precipitate in stains
Insufficient rinsing
Correction
Correct pH, remake buffer
Shorten buffer time/amount
Prolong staining time/amount
Correct film thickness
Check expiration date/stain
Correct pH, remake buffer
Increase buffer time/amount
Decrease staining time/amount
Correct film thickness
Change stain in station 2 and 3
(Hemastainer or Midas stainer)
Lengthen staining interval (manual dip-method)
Check Giemsa dilution (dip-type stainers)
Adjust metered stain volumes (platen-type
stainers)
Check timing on stain stations (dip- type
stainers)
Reduce staining interval (manual dip-method)
Replace methanol and/or stain
Keep dishes covered (prevent water
absorption)
Check humidity in air/increase drying speed,
if possible
Check for severe anemia
Clean platen/lines, do maintenance
Filter or replace stains
Check rinsing time
Check rinse filter (dip-type stainers)
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 BLOOD FILM STAINING EFFECTS

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.

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