Staining a Peripheral Blood Smear

Principle^[1][2]

A properly stained blood smear is essential for accurate interpretation of cellular morphology. The Wright stain and Wright-Geimsa stain (Romanowsky stains) are the two most common stains used in Hematology for staining peripheral blood and bone marrow smears. The staining gives color to the cells, like the nucleus and granules, making the cells more visible and easier to identify, count and differentiate. We will be learning a quick stain method that allows for sample integrity checks and assessment. We will also be learning an automated method on the Midas III Plus.

This stain is a solution that contains methanol and a combination of eosin and new methylene blue dyes that fixes and stains the blood smears. The methanol fixes the smear to the slide and preserves the cellular morphology. The new methylene blue dye is a basic dye that stains acidic (basophilic) cellular components.  The nucleus, RNA and basophilic granules will all take up the new methylene blue dye giving them a dark blue to purple color. The eosin dye is an acidic dye that stains basic (eosinophilic) components including hemoglobin (salmon) or eosinophilic granules and stains them an orange/reddish color.

The combination of methylene blue and eosin form a complex dye (thiazine eosinate) via oxidation which stains neutral components of the cells. Staining reactions are pH dependent and is usually has a range of about 6.4 to 6.8.

Cytoplasmic granules in the neutrophils have a neutral pH and takes up characteristics of both dyes resulting in a tan/pinkish background.

Materials

• Wright/Giemsa stain
• Camco Quik Stain
• Well-made smear
• Light microscope
• Distilled water
• Coplin Jars with covers
• Kim wipes/towels
• Midas III Plus Stainer
• Buffer
• Rinse

Sample Type

• EDTA anticoagulated blood sample, via venipuncture
• Fresh capillary blood can be used, finger or heel stick

Safety Considerations

• The Methanol in the dye is considered Toxic and flammable!
• Refer to MSD sheets for safety considerations located in the reagent prep room.
• All specimens received in the lab must be regarded as potentially infectious so follow safe practices.
  Refer to regulations

Procedure For Quik Stain

1. Place the dry blood smear into the coplin jar filled with stain. Gently agitate the slide for 10 seconds by dipping the slide in and out of the stain.
2. Allow the excess methylene blue dye to drain onto a Kimwipe towel.
3. Rinse the slide in distilled water for about 30 seconds
4. Using a Kim wipe, wipe the residual dye from the back of the slide, and around the smear without disturbing the smear.
5. Place the slide in a drying rack to air dry thoroughly.
6. Once thoroughly dried, evaluate the slide using a light microscope.

Procedure for Automatic Stain (MIdas III-Plus)

1. Load the staining vessels into the appropriate Station for the program being run.
2. Station 1 will contain the Wright/Giemsa stain and fixative combination. Station 2 will contain Buffer and Station 3 will contain Rinse.
3. Load the slides into the slide carrier by laying it on a flat surface and sliding the slide guard up. Ensure they are placed straight. Slide the carrier onto the tower arm.
4. Turn on the Stainer (Midas III-Plus) and wait for it to initialize. The on/off switch is by the input power cord. The on position is indicated by I and the odd position is indicated by the O on the switch face.
5. Press RUN
6. Press PROG  1 as the desired program and press ENTER. The run will start, the arm will initialize, and the tower will move to the first Station in the sequence. As the slide carrier runs in each Station, the unit will display run information,
7. To pause the run, press STOP. To continue the run, press RUN. To cancel the run press, STOP again.  Wait for the tower to go to the vertical home position and then press STOP 3 times to reset to the home position.
8. An audible beep sounds six times at the completion of the program and the Standby Mode is entered. Scrolling marquee will appear.
9. Gently unload the slide carrier by laying it flat on an absorbent towel. Slide up the slide guard and remove the slide.
10. Evaluate the slide using a light microscope.

Procedural Note

• There are a variety of Wright staining methods available. Refer to Appendix 1.
• To avoid the thick end of the slide from coming off, make sure the smears are completely dried before staining.
• Water or drying artifact can occur if dried incorrectly or not dried properly.
• Humidity in the air may cause the RBC’s to have a moth-eaten or punched out appearance.
• Wright’s stain can gradually become more acidic on standing if not kept tightly closed when not in use.
• Stain must be stored at room temperature.
• It is recommended that a blood smear be made and stained within 2-3 hours of collection to ensure the best staining results.
• Never blow on a smear to dry it. Humidity from your breath can have a similar affect as water/drying artifacts.[

Quality

• Ideally the blood smear should be stained with a few hours of collection to maintain the staining integrity.
• The slides must be completely dry before applying stain to avoid any water artifacts or preventing the thick part of the smear from coming off.

Characteristics of a well stained smear

• Red blood cells – salmon/reddish/pink
• Nuclei – dark blue/purple
• Basophil cytoplasmic granules – dark blue/purple
• Eosinophil cytoplasmic granules – reddish/orange
• Neutrophil cytoplasmic granules – lavender/lilac/light tan
• Background of the smear should be colorless and clear with no stain precipitate.

Sources of Error^[3]

Factors that can affect the quality of a stained blood smear:

1. If the smear is too purple/blue/ dark:
   • Buffer pH is too high (alkaline)
   • Excessive stain solution (new methylene blue) to buffer
   • Evaporation of the alcoholic stain solution before being washed off the smear
   • Inadequate rinsing/washing
   • Overstaining
   • Too thick a smear
   • Heparin samples
   • Protein abnormalities (Multiple Myeloma)
2. Staining smears greater than 1 day old. If the smear is too orange/pink/light:
   • Buffer pH is too low (<6.0)
   • Excess stain solution (eosin) to buffer
   • Too short a staining time
   • Excessive washing
   • Exposure to acid fumes
   • Contaminants in wash water – chlorine
   • Very thin smear
3. The presence of small coarse-granules or seen on the cell or throughout the smear may be due to:
   • Uneven spreading of the stain solution on the smear.
   • Precipitation of the dye may cause artifacts to appear on the smear surface.
   • Unclean slide.
   • Dust particles.
4. When a smear appears too brown or thick it may be due to a very high hemoglobin or a thick smear
5. Retractile spaces in the red blood cells may be due to improper drying of the smear, or very humid environment. May have the appearance of “moth-eaten” RBC’s or punched out hole.

Appendix 1

A basic comparison of the Quick stain, 2 step and automated staining methods

1.  Quick stain:
   As the name implies, it is a fast away to obtain a well stained smear. Quick stains contain a combination of methylene blue dye, eosin Y dye and a fixative (absolute methanol). Can take up to 1 minute to produce a stained blood smear.
2. Two step method:
   A smear is placed on a staining rack over a sink and the Wright stain (containing Methanol) is spread over the smear for about 1-5 minutes. Equal amount of a buffer solution (prevents changes in the stain pH) is flooded on the smear and gently mixed. A green metallic sheen forms on the slide. The smear is rinsed with water. (distilled) and dried.
3. Automated stainers:
   They use belts to carry the slides through the stain or baskets that dip slides in the solutions
   Advantage – Many slides can be stained at the same time.
   Disadvantage – A problem with the stain would affect many slides.