Wright’s stain procedure, principle, and results

James Homer Wright devised Wright’s stain procedure in 1902 by modifying the Romanowsky stain. The Wright Stain stains cellular elements in peripheral blood and bone marrow smears. It is a polychromatic stain composed of methylene blue and eosin dyes. 
A Romanowsky stain is a group of staining techniques used in hematology, pathology, and histology to visualize and differentiate cells, tissues, and microorganisms. These techniques use dyes that interact with specific cellular structures, selectively staining different structures based on their staining properties. 
Other Romanowsky stains include Giemsa, Wright-Giemsa, Diss-Quik, and May-Grunwald-Giemsa. 

Wrights Stain Vs. Other Romanowsky stains

The protocol for Wright’s, Wright-Giemsa, and Giemsa stains is the same, except for the second step, where you use the preferred stain. Wright-Giemsa is the most widely used Classical stain and produces more intense basophilic/nuclear staining in blood cell morphology. The May–Grünwald stain has a more intense coloration and takes longer to perform.

Differences between Wrights and Wrights Giemsa stain

The key difference between Wrights and Wrights Giemsa stain is that Wright-Giemsa stain produces more intense basophilic/nuclear staining, while Wright stain achieves a more eosinophilic appearance. The protocol for both stains is identical, with the second step being replaced by the preferred stain.
Wright-Giemsa stain is commonly used for general blood cell analysis, counting immature red and white blood cells, platelet counts, and neutrophil counts. It is also frequently used to evaluate bone marrow specimens, and if the Wright-Giemsa stain is not clear enough, you can use Giemsa stain to enhance stain intensity. On the other hand, Wright stain is often used to test for Immunoglobulin E (IgE) antibodies because these antibodies bind to eosinophils in the cytoplasm of white blood cells, resulting in a more intense red color.

Wright’s stain principle

Wright’s polychromatic stain comprises a combination of Eosin and Methylene blue. Since Wright’s stain is methanol-based, it does not require a fixation step before staining. However, fixation by air drying can help reduce water artifacts that may occur on humid days or with aged stains.
Methanol fixes the cells to the slide, while Eosin Y, an acidic anionic dye, and Methylene blue, a basic cationic dye, ionize when diluted in buffered water. Eosin stains basic components such as hemoglobin and eosinophilic granules an orange to pink color, while Methylene blue stains acidic cellular components such as nucleic acid and basophilic granules in varying shades of blue. Neutral components of the cells are stained by both components of the dye, resulting in variable colors.

Phosphate buffer at pH 6.5 dilutes the stain enabling ionization.

Wright’s Stain Procedure

1. Make a thin smear of your specimen. The smear should be a cell thick and made on a sterile slide. Leave the specimen to air dry.
2. Place the slide on a staining rack.
3. Cover the smear with undiluted Wright’s stain for 2 to 3 minutes. The stain fixes and stains the smear.
4. Cover the stained smear with equal phosphate buffer (pH 6.5). DO NOT allow it to overflow. 
5. Rock the preparation gently. A metallic sheen should appear on top of the preparation. Allow it to stand for 5 minutes.
6. Flood and rinse the stains with distilled water until the edges of the smear appear pinkish-red.
7. Air-dry the film at room temperature and examine it under a microscope under oil immersion.

Related: Differences between a thick and a thin blood smear

Wright’s Staining Results

Different components of the cells pick up different shades of the dyes depending on their pH. Basophil cells appear as dark purple granules with a sky-blue cytoplasm when stained with it. Lymphocytes have a sky-blue cytoplasm and a dark purple nucleus, while erythrocytes appear yellow-red and platelets as purple-violet granules.

Cell Component	Color
Platelets	purple – violet granules
Monocytes	mosaic pink and blue cytoplasm, dark purple nucleus
Lymphocytes	sky blue cytoplasm, dark purple nucleus
Basophils	dark purple granules, sky blue cytoplasm
Eosinophils	blue cytoplasm, orange-red granules, blue nucleus
Neutrophils	pale pink cytoplasm, reddish ileac granules, dark purple nucleus
Erythrocytes	yellow- red

Applications of Wright Stain Procedure

• Staining cellular elements in peripheral blood and bone marrow smears.
• Detect parasites such as malaria, babesia, trypanosoma, leishmania, and filaria in blood.
• Differentiation of white blood cells when conducting a white blood cell count.
• Wright stain is also suitable for fine needle aspirate (FNA) cell preparations. 
• In cytogenetics, scientists use it to stain chromosomes to facilitate the diagnosis of syndromes and diseases.
• Suitable for urine cytology. Urine samples stained with Wright’s stain will identify eosinophils, which can indicate Urinary Tract Infections (UTI) or interstitial nephritis.
• It is combined with fluorescent antibodies or dyes for immunofluorescence studies.
• Test for Immunoglobulin E (IgE) antibodies because they bind to eosinophils.

Limitations of Wright Stain

• To preserve cell morphology, films must be fixed promptly and not left unfixed for more than a few hours.
• The methanol used as a fixative must be completely free of water, as even as little as 1% water can affect the appearance of the films, and higher water content can cause significant changes.
• Red cells may be affected by traces of detergent on inadequately washed slides.
• When thick films are stained, they may become covered by a residue of stain or spoiled by the envelopes of lysed red cells.
• Overstaining can result in poor differentiation of red blood cells.
• Blood samples tested during the relapse cycle of the Plasmodium parasite in the blood may result in false negative results.

Sources of error or artifacts in Wright’s stain procedure

• Inadequate fixation, which can cause poor staining, cell distortion, or water artifacts.
• Overstaining or understaining, which can affect the differentiation and identification of cells and structures.
• Contamination of reagents, slides, or equipment, which can cause unwanted colors, precipitates, or deposits on the film.
• Improper washing or drying, which can cause streaks, spots, or uneven staining on the film.

Quality Control of Wright Stain

• Appearance: Blue-coloured solution
• Clarity: Clear without particles.

Storage and Shelf Life of Wright Stain

• Store between 10 – 30⁰C in a tightly closed container.
• Store away from direct sunlight. 
• Use before the expiry date on the label.
• Stored in a dry, ventilated area protected from extremes of temperature.
• Store away sources of ignition/ fire.
• Seal the container tightly after use.