Standard Operating Procedure

Subject HEMA 3 Differential Stain Procedure

Index Number Lab-1531
Section Laboratory
Subsection Regional/Affiliate
Category Departmental
Contact Munson, Karen
Last Revised 10/12/2018

References
Required document for Laboratory Accreditation by the College of American Pathologists (CAP), Centers
for Medicaid and Medicare Services (CMS) and/or COLA.

Applicable To
Employees of the Gundersen St. Joseph’s Hospital laboratories and Gundersen Palmer Lutheran Hospital
and Clinics laboratories.

Detail
PRINCIPLE:
When a thin film is processed, either by an automatic slide stained or by a manual method and
examined microscopically, the nucleus and the cytoplasm of neutrophils, lymphocytes, monocytes,
eosinophil’s and basophils will show a characteristic blue or red coloration and the relative frequency of
each type of leukocyte can then be established.

CLINICAL SIGNIFICANCE: N/A

SPECIMEN:
Smears are prepared from fresh blood samples and must be air dried, completely. EDTA or sodium
citrate can be used as anticoagulants. Heparin and potassium oxalate will interfere and should not be
used. Smears can also be made directly from the drop of blood from a capillary puncture. Smears
should be made at the time of blood collection, to insure platelets are not clumped.

REAGENTS/MATERIALS:
1. Hema 3 Fixative Solution
2. Hema 3 Solution I
3. Hema 3 Solution II
4. Distilled or DI water in squeeze bottle
5. Coplin jars to hold each solution

Storage and Stability:

1. Store at room temperature (15-30oC)
2. Change stain weekly. Keep original bottles and Coplin jars tightly closed.
3. Do not use after the expiration date.
4. The presence of excessive precipitation in the stain solution or on the stained slides and
inadequate differentiation of cell types may indicate deterioration.

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 Standard Operating Procedure

EQUIPMENT/INSTRUMENTATION: N/A

Implementation
PROCEDURE:
SLIDE PREPARATION
Use whole blood collected in an EDTA tube. Follow acceptable venipuncture procedure. Blood collected
using heparin as an anticoagulant is acceptable if slides are made immediately. A finger stick can also be
performed, with a drop of blood from the puncture site being placed directly onto the slide. In this case,
two (2) slides should be made. The slide should be made before collection of any other blood from this
site as a delay may cause a change in platelets that will appear on the finger stick slide.
1. A drop of fresh capillary or anticoagulant blood should be place on a clean glass slide near the
frosted end of the slide. Using the H-Pette diff maker place the cannula in the center of the
stopper of the collection tube. Gently press until the cannula completely penetrates the septum.
The tube is now ready for use. Invert tube at an angle. Gently press the round edge of the H-
Pette to the glass slide to transfer sample to the slide.
2. Place the slide on a flat surface with the drop of blood on the right.
3. With the thumb and index finger of the left hand, hold the two left edges of the slide. With the
right hand, hold the second slide with the thumb on the edge of the slide. Place the end of the
spreader slide slightly in front of the drop of blood on the other slide. The angle between the
two slides should be approximately 25º.
4. Draw the spreader slide back toward the drop of blood. As the spreader slide comes into
contact with the drop of blood, the blood will begin to spread to the edges of the spreader. If
this doesn’t occur, wiggle the spreader slide slightly until it does so.
5. While maintaining a 25º angle, firmly push the spreader slide against the horizontal slide. This
step should be performed when the drop of blood has spread to within 1/8 inch of the edges of
the horizontal slide.
6. The blood smears should be allowed to dry. If the temperature or humidity is such the slide
drying does not occur rapidly, the slide may be waved rapidly in the air. The small personal fan
in Hematology can also be used. Distortion can occur if slides do not dry quickly enough.

A smear the length of ½ to ¾ of the slide should result. The end should be evenly feathered. This
provides the best type of slide for counting cells. Visual inspection allows the technologist to observe for
poor feathering, air bubble holes, or other discrepancies that might result in difficult reading of the
smear. A drop of blood that is too large or too small may make a poor smear. Jerky movement of the
spreader slide may cause a poor smear, as will slides that are not clean. Failure to keep the spreader
slide at a 25º angle will result in a thicker smear (increased angle) or a thinner smear (decrease angle).
Delay in making the smear once a drop of blood is placed on the slide may cause cell abnormalities.

STAINING PROCEDURE
Reagents/Materials
1. Hema 3 Fixative
2. Hema 3 Solution I
3. Hema 3 Solution II
4. DI water
5. Coplin jars
6. Prepared/dried blood smear

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 Standard Operating Procedure

Procedure
1. Transfer each solution into a Coplin jar.
2. Dip dry blood smear/slide five times for one second each in Hema 3 Fixative Solution. Allow
excess to drain.
3. Dip slide three to five times for one second each in Hema 3 Solution I. Allow excess to drain.
4. Dip slide three to five times for one second each in Hema 3 Solution II. Allow excess to drain.
5. Rinse slide with DI water.
6. Allow to dry.

Staining intensity may be varied by increasing or decreasing the number of dips in Solutions I and II.
Eosinophilic staining may be intensified by increasing dips in Solution I; basophilic staining may be
intensified by increasing dips in Solution II. Two to Six dips in Hema 3 Solutions I and II will provide
desirable results for most staining applications. Any variation of the procedure described may adversely
affect results.

CALCULATIONS: N/A

INTERPRETATION: N/A

LIMITATIONS: N/A

REVIEW AND CHANGES:

This document and all attached forms should be reviewed optimally on an annual basis, with 2 years as
the maximum review date. Review will be done by the Technical Leader, Supervisor, Manager, Medical
Director or designated person. Changes require retyping document or form and review by the Medical
Director.

REFERENCES:
Hema 3 Stain Set Label

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