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YOUR LAB
HEMATOLOGY SECTION
C. DIFFERENTIAL CELL COUNTS
C.2. WRIGHTS STAIN MANUAL METHOD
C.2.1. PRINCIPLE:
Wrights 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
1.2.
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Properly stained smears will display the following characteristics when examined
microscopically:
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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.
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.
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2.2. Staining:
2.2.1.
2.2.2.
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.
2.3.3.
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
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.
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Date: __________________
Change
QA
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OIC
Med Dir
DATE
PREPARER
QA COORDINATOR
LABORATORY OIC
MEDICAL DIRECTOR
ANNUAL REVIEW
REVIEWER SIGNATURE
DATE
REVIEWER SIGNATURE
DATE: ___________
DATE
# COPIES __________
10
11
4
5
6
7
8
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