Penny Stevens
Procedure Number
Date
Author(s), Name & Title
International QA/QC Coordinator
Pro64-B-04 Version 1
Jan 06
SMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your lab’s
specific processes and/or specific protocol requirements. Users are directed to countercheck facts when considering
their use in other applications. If you have any questions contact SMILE.
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YOUR LAB
HEMATOLOGY SECTION
C. DIFFERENTIAL CELL COUNTS
C.2. WRIGHT’S STAIN MANUAL METHOD
C.2.1. PRINCIPLE:
Wright’s stain is a polychromatic stain consisting of a mixture of eosin and methylene
Blue. When applied to blood cells, the dyes produce multiple colors based on the ionic
charge of the stain and the various components of the cell. The eosin ions are negatively
charged and stain basic cell components an orange to pink color. The methylene blue
ions are positively charged and stain the acid cell components in varying shades of blue.
The neutral components of the cell are stained by both components of the dye producing
variable colors.
C.2.2. SPECIMENS
1. Thin wedge blood smears are prepared on pre-cleaned glass slides from freshly drawn
capillary or venous blood anticoagulated with EDTA.
2. The blood smear slide must be properly prepared, labeled and completely dry before
commencing with the staining procedure.
3. For best results, prepare and stain smears within two (2) to three (3) hours of
specimen collection.
C.2.3. EQUIPMENT AND REAGENTS:
1. EQUIPMENT:
1.1. Four (4) coplin jars
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C.2. WRIGHTS STAIN MANUAL METHOD
1.2. Pre-clean glass slides
1.3. Whatman Grade 41 filter paper – 110mm diameter
1.4. Funnel – short neck
2. REAGENTS:
2.1. Diff Quick Fixative Solution
2.2. Diff Quick Solution I (Red)
2.3. Diff Quick Solution II (Blue)
2.4. Distilled Water
NOTE: The reagents are flammable and must be stored in the flammable cabinet.
Keep only the minimum working supply, appropriately labeled with hazard warnings,
in the section.
C.2.4. PROCEDURE:
1. Dip the blood smear into the Diff Quick fixative solution five times for one (1)
second each time. Wipe the back of the slide with gauze to prevent carry over into
the next stain. Do not allow the fixative to dry on the slide as this will result in an
over-fixed wet appearance. Immediately proceed to the next step while the slide is
still wet.
2. Dip the fixed blood smear into the Diff Quick solution I five times for one (1) second
each time. Wipe the back of the slide with gauze to prevent carry over into the next
stain. Do not allow the stain to dry on the slide. Immediately proceed to the next step
while the slide is still wet.
3. Dip the fixed blood smear into the Diff Quick solution II five times for one (1) second
each time. Wipe the back of the slide with gauze to prevent carry over into the next
stain. Do not allow the stain to dry on the slide. Immediately proceed to the next step
while the slide is still wet.
4. Rinse the fixed blood smear with distilled water five times for one (1) second each
time. Wipe the back of the slide to decrease drying time. Set the slide upright to air
dry. Allow the slide to dry thoroughly before reading.
5. To decrease drying time, slides may be blown dry. Set the blow dryer on cool and
hold the slide at least 12 inches from the air flow until completely dry.
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C.2. WRIGHTS STAIN MANUAL METHOD
C.2.5. PROCEDURAL NOTES:
1. Properly stained smears will display the following characteristics when examined
microscopically:
1.1. Red to buff pink colored red blood cells.
1.2. White blood cell nucleus coloration:
1.2.1.
1.2.2.
Lymphocyte and Neutrophil nucleus are dark purple.
Monocyte nucleus is light blue to purple.
1.3. White blood cell and platelet cytoplasm appearance:
1.3.1.
1.3.2.
1.3.3.
1.3.4.
1.3.5.
1.3.6.
Neutrophil cytoplasm is light pink with lilac granules.
Lymphocyte cytoplasm varies from light to dark blue.
Monocyte cytoplasm is gray-blue with few fine reddish granules.
Basophil cytoplasm is clear with few blue-black granules.
Eosinophil cytoplasm is pink to clear with many large red to orange
granules.
Platelets appear as light blue to colorless with red violet granules.
NOTE:
The Diff Quick Fixative is insufficient to adequately fix the
basophil granules. As a result, most of the granules are lost in the staining
process and the basophils appear ‘agranular’, which can result in
misidentification. This basophil appearance is inconsistent with images
presented in current literature.
1.4. Precipitation: Rare to slight precipitation is acceptable. As stain solution II
evaporates, excess precipitation will form. Filter the stain solution II as needed
to remove excess precipitation.
1.5. Contamination: Bacterial or semen contamination can occur as a result of shared
use between microbiology and hematology. If either is present in the stain
solution, replace all reagents and perform maintenance as indicated in section
C.2.7.
2. Sources of error:
2.1. Fixation:
2.1.1.
Inadequate fixation may result in a detached specimen, indistinct
nuclear detail or poor granule staining. This is especially evident in
basophils and primary myeloid granules.
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C.2. WRIGHTS STAIN MANUAL METHOD
2.1.2.
Dry freshly prepared smears for at least fifteen (15) minutes to prevent
drying artifacts. Use only pre-cleaned glass slides. Slides other than
pre-cleaned may result in improper fixation and the presence of haze
over the slide due to acid-base disturbance. Pre-cleaned slides are
necessary for quality stain appearance.
2.1.3.
Use only anhydrous methanol for fixative. Keep it tightly closed and
away from moisture or chemical fumes.
2.1.4.
If fixation problems occur or increased granularity visualization is
necessary, increase the fixation period to one (1) minute.
2.2. Staining:
2.2.1.
Excessively blue stain appearance may be due to improper smear
preparation, prolonged staining time or increased stain alkalinity.
2.2.2.
Excessively pink stain appearance may be due to insufficient or
decreased staining time, prolonged washing time or increased stain
acidity.
2.3. Rinse:
2.3.1.
Use only distilled water or phosphate buffered saline for the final rinse.
Tap water is never acceptable as a substitute for the rinse since it can
disturb the acid base balance and may contain chlorine.
2.3.2.
Vigorous or prolonged washing may dislodge cell from under-fixed
smears, cause nuclear clumping or under-staining.
2.3.3.
Insufficient rinsing may result in a blue smear or excessive precipitate.
2.4. Drying: Air drying is the preferred method. Forced rapid drying may alter the
color intensities by shortening the exposure time to the wash water because the
red spectrum of colors continues to develop as long as the cellular elements are
wet.
C.2.6 QUALITY CONTROL
1. Prepare one differential slide daily using a patient sample with a normal MCV, MCH,
MCHC and total white count. Stain the slide as indicated in procedure section C.2.4.
Review the slide for Color, Precipitation and Contamination.
2. If the color does not meet the specifications identified in the SOP as indicated above
or precipitation and/or contamination are present, the quality is determined to be
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DIFFERENTIAL CELL COUNTS
C.2. WRIGHTS STAIN MANUAL METHOD
unsatisfactory. Indicate unsatisfactory on the Quality Control (QC) form and replace
the stain as indicated in the following section. Document the problem and corrective
action in the appropriate section of the QC form. If the stain is determined to be
satisfactory, indicate satisfactory on the QC sheet.
C.2.7. MAINTENANCE
1. Perform maintenance weekly or as needed for problems indicated in previous
sections.
2. Discard reagents into the gram stain hazardous waste containers located in the
microbiology department.
3. Clean the coplin jars using mild soap and tap water. Perform a final rinse using
distilled water and allow the jars to air dry.
4. Use two sets of coplin jars and rotate the sets weekly allowing the jars to dry
thoroughly before use.
5. Replace the fixative, stain and water into the appropriately labeled jars with accurate
hazard warning labels. Filter the stain solution II (blue stain) using a short neck
funnel and 110 mm diameter filter paper folded into a cone.
NOTE: The gravimetric filtration process is slow – do not attempt to speed it up or it will
force particulates into the filtered stain.
B.2.8. APPENDICES:
1. Diff Quick Maintenance / Quality Control Form.
2. SOP Validation Form and SOP Change Control.
3. SOP Approval.
B.2.9. REFERENCES:
1. Brown, Barbara A.; Hematology: Principles and Procedures, Sixth Edition, Lea and
Febriger Book Publisher, 1993, Pages 97-100.
2. Hoffman, Donald; Hematology: Principles and Practice, Second Edition, American
Book Publisher, 1995, Pages 2202-2203.
3. Carr, Jacqueline H. and Rodak, Bernadette F.; Clinical Hematology Atlas, W.B.
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DIFFERENTIAL CELL COUNTS
C.2. WRIGHTS STAIN MANUAL METHOD
Saunders Company, 1999, Pages 33-86.
4. Laboratory CQI SOP, 8 April 2004.
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DIFFERENTIAL CELL COUNTS
C.2. WRIGHTS STAIN MANUAL METHOD
SOP VALIDATION
SOP NAME:
C.2. WRIGHT’S STAIN MANUAL METHOD
Clear and specific title and principle: yes / no
Comments:
All necessary supplies, equipment, and materials are listed: yes / no
Comments:
SOP is sufficiently detailed to be understood but not overly complex: yes / no
Comments:
SOP text adequately describes process/procedure: yes / no
Comments:
SOP accomplishes purpose: yes / no
Comments:
Reviewed by: (Name & Title)
Signature: __________________________
Date: __________________
SOP CHANGE CONTROL
Date
Change
QA
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OIC
Med Dir
DIFFERENTIAL CELL COUNTS
C.2. WRIGHTS STAIN MANUAL METHOD
SOP APPROVAL
SIGNATURE
DATE
PREPARER
QA COORDINATOR
LABORATORY OIC
MEDICAL DIRECTOR
ANNUAL REVIEW
REVIEWER SIGNATURE
DATE
DOCUMENT COPY CONTROL
LOCATIONS
REVIEWER SIGNATURE
DATE: ___________
DATE
# COPIES __________
1
9
2
10
3
11
4
5
6
7
8
SUPERSEDES: _21 Jan 1998_____
DATE SOP RETIRED: __________
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