Romanowsky Stain – Principle, Types, Composition, Procedure and Uses

Romanowsky stain is commonly used for the microscopic examination of peripheral blood smears in the laboratory. A thin blood film is prepared on a clean glass slide and allowed to air-dry before Romanowsky staining. The stained film is then examined under a microscope to observe the size and shape of erythrocytes, the nuclei of leukocytes, and the granules present in their cytoplasm.

The blood smear must be prepared properly to preserve the cellular morphology. Improper preparation or drying may change the appearance of blood cells and affect microscopic examination.

Different Romanowsky staining methods require suitable staining reagents and buffer solutions. The preparation of the blood film and the staining reagents are carried out separately. The appearance of stained blood cells also depends on the staining conditions used for the selected method.

What is Romanowsky Stain?

Romanowsky stain refers to a group of differential, polychromatic stains used for the microscopic examination of blood and bone marrow smears. These stains contain a combination of acidic eosin dyes and basic thiazine dyes, particularly azure dyes derived from methylene blue.

The term Romanowsky stain does not refer to a single stain with a fixed composition. Different formulations contain varying proportions of these dyes. The stain is named after Dmitri Leonidovich Romanowsky, a Russian physician who developed an early method of polychromatic staining in the late 19th century.

Purpose of Romanowsky Staining

The following are the objectives of Romanowsky staining:

• To increase the contrast between blood cells and their cellular components during microscopic examination.
• To distinguish the nuclear chromatin and observe the shape of leukocyte nuclei.
• To observe differences in the cytoplasmic staining of different leukocytes.
• The cytoplasmic granules of granulocytes can be identified and examined.
• To examine the size, shape, and staining characteristics of erythrocytes.
• Platelets can be identified and their size and granules can be observed.

Composition of Romanowsky Stain

The main staining dyes present in Romanowsky stains are azure B and eosin Y. Azure B is a basic (cationic) thiazine dye, while eosin Y is an acidic (anionic) xanthene dye. These two dyes are sufficient for the preparation of a standardized Romanowsky stain.

Traditional Romanowsky stains also contain methylene blue and other thiazine dyes formed during the polychroming of methylene blue. These include azure A, azure B, and azure C. The proportions of these dyes vary depending on the staining formulation.

Methanol is commonly used as a solvent for the preparation of Romanowsky stain solutions. Some formulations also contain glycerol, while certain standardized preparations use other solvents such as dimethyl sulfoxide.

The stock staining solution is diluted with buffered water before use. Phosphate buffer is commonly used to maintain the required pH of the working staining solution. The solvent and buffer composition may differ according to the staining method.

Principle of Romanowsky Staining

Romanowsky staining is based on the different affinities of acidic and basic dyes for the cellular components of blood and bone marrow smears.

Differential Affinity of Romanowsky Dyes

Azure B is a basic, positively charged dye that binds to negatively charged cellular structures, particularly the phosphate groups of DNA and RNA. It initially stains nuclear chromatin and RNA-rich cytoplasm blue.

Eosin Y is an acidic, negatively charged dye that binds to positively charged sites in cellular proteins. It stains haemoglobin in erythrocytes and the granules of eosinophils pink to red. The different affinities of these dyes produce contrasting colours in the cellular components.

Romanowsky Effect

The Romanowsky effect or Romanowsky–Giemsa effect is the development of a purple colour due to the interaction between azure B and eosin Y on certain cellular structures.

Azure B first binds to negatively charged sites in the cellular material. Eosin Y then combines with the bound azure B to form azure B–eosin complexes. These complexes produce a purple colour that cannot be obtained with either dye alone.

The Romanowsky effect is mainly observed in nuclear chromatin, neutrophil granules, and platelets. The formation of the purple complexes depends on the cellular structure and the rate at which eosin enters the azure B-stained material.

Factors Influencing Romanowsky Dye–Cell Interactions

The following are the factors that influence the interaction of Romanowsky dyes with cellular components:

• pH of Staining Solution– The pH of the staining solution affects the ionization of cellular groups. At a lower pH, eosin staining is generally increased, while a higher pH favours the uptake of basic dyes.
• Dye Concentration– The amount of azure B and eosin Y taken up by the cells and the formation of the purple Romanowsky colour depend on the concentration and relative proportions of these dyes.
• Ionic Strength– The concentration of buffer salts determines the ionic strength of the staining solution, which affects the electrostatic attraction between charged dyes and cellular components.
• Solvent– Methanol and other solvents affect the solubility of the dyes and their entry into cellular structures. The staining pattern may also change with the amount of methanol present in the working solution.
• Nature of Cellular Components– Nucleic acids contain negatively charged phosphate groups. Proteins have both acidic and basic groups that provide binding sites for the different dyes.
• Fixation– The chemical and structural properties of cellular material may change during fixation. Methanol generally preserves the Romanowsky effect, whereas formaldehyde fixation may reduce or inhibit the characteristic purple staining.
• Staining Time– A short staining period may produce weak colours, while prolonged staining can change the relative amounts of azure B and eosin Y taken up by the cells.
• Temperature– The temperature of the staining solution affects the rate of dye uptake. The staining intensity and formation of purple dye complexes may also vary with temperature.
• Purity of Dyes– Impurities present in the staining dyes may affect their binding to cellular structures. Different amounts of azure B, eosin Y and other dye components can produce variations in the staining results.

Types of Romanowsky Stains

Romanowsky stains are divided into different types based on their dye composition and staining methods. The commonly used Romanowsky stains include Giemsa, Wright, Leishman, May–Grünwald, Jenner and Field stains. Some modified and combined staining methods are also used in hematology and cytology.

1. Giemsa Stain

Giemsa stain contains a mixture of azure dyes, methylene blue and eosin. The staining powder is usually dissolved in methanol and glycerol to prepare the stock solution.

It is commonly used for staining thick and thin blood smears, particularly for the examination of malaria parasites. Giemsa stain is also used for bone marrow smears and other cytological preparations.

2. Wright Stain

Wright stain is a Romanowsky-type stain containing polychromed methylene blue and eosin in methanol. The methanol present in the solution also acts as a fixative for air-dried blood films.

It is mainly used for the examination of peripheral blood smears and differentiation of leukocytes. Wright staining is commonly used in hematology laboratories.

3. Leishman Stain

Leishman stain consists of polychromed methylene blue and eosin dissolved in methanol. It is used for staining thin blood films, where the methanol in the staining solution also fixes the cells.

Leishman stain is commonly used for examining the morphology of erythrocytes, leukocytes and platelets. It may also be used for the examination of blood parasites.

4. May–Grünwald Stain

May–Grünwald stain contains methylene blue, eosin and related thiazine dyes in a methanolic solution.

It is used for staining peripheral blood and bone marrow smears. This stain is commonly used with Giemsa stain for obtaining better nuclear and cytoplasmic staining.

5. Jenner Stain

Jenner stain is one of the simplest Romanowsky-type stains. It consists mainly of methylene blue eosinate dissolved in methanol.

It is used for staining blood films and may also be combined with Giemsa stain. The combined method is known as Jenner–Giemsa staining.

6. Field Stain

Field stain is a rapid Romanowsky staining method that uses two separate aqueous staining solutions. One solution contains basic dyes, including methylene blue and azure dyes, while the other contains eosin.

It is mainly used for rapid staining of thick blood smears during the microscopic examination of malaria parasites. Modified Field staining methods can also be used for thin blood films.

7. Jaswant Singh–Bhattacharji (JSB) Stain

JSB stain is a water-based Romanowsky staining method developed for the rapid detection of malaria parasites.

It consists of two staining solutions. The first contains polychromed methylene blue, while the second contains eosin. The stain is used for the examination of blood films in malaria microscopy.

8. Wright–Giemsa Stain

Wright–Giemsa staining combines the staining properties of Wright and Giemsa dyes. It may be used as a combined staining solution for blood and bone marrow smears.

This method is used for examining blood-cell morphology, nuclear structures and cytoplasmic granules. It is also used in routine hematological examination.

9. May–Grünwald–Giemsa (MGG) Stain

May–Grünwald–Giemsa staining is a combined Romanowsky staining method in which May–Grünwald stain is followed by Giemsa stain.

It is commonly used for the examination of peripheral blood smears, bone marrow cells and cytological specimens. The method provides distinct staining of nuclear chromatin and cytoplasmic components.

10. Diff-Quik Stain

Diff-Quik is a rapid modified Romanowsky staining method. It generally consists of three solutions, including a methanol-based fixative, an eosin-containing solution and a basic thiazine dye solution.

It is mainly used for rapid staining of air-dried cytological smears, including fine-needle aspiration specimens and body-fluid preparations.

Comparison of Different Types of Romanowsky Stains

Difference Between Romanowsky Stain and Wright Stain

Romanowsky stain refers to a group of differential stains containing acidic and basic dyes. Wright stain is one of the individual staining methods belonging to this group.

The term Romanowsky stain includes Giemsa, Leishman, Wright and other related staining methods, whereas Wright stain refers to a particular formulation of polychromed methylene blue and eosin dissolved in methanol.

Requirements for Romanowsky Staining

The following materials are required for Romanowsky staining of blood and bone marrow smears:

• Blood Sample– Fresh peripheral blood or blood collected in an EDTA tube is used for preparing blood smears. Bone marrow aspirate smears can also be stained.
• Glass Slides– Clean, dry and grease-free glass slides are required for preparing the blood films.
• Spreader Slide– A glass slide with a smooth edge is used for spreading the blood sample to prepare a thin film.
• Romanowsky Stain– A suitable staining solution such as Giemsa, Wright, Leishman or Wright–Giemsa stain is required. The stain is selected according to the specimen and staining method.
• Methanol– Absolute methanol is used for fixing air-dried thin blood smears when separate fixation is required. Some Romanowsky stains contain methanol and do not require a separate fixation step. Thick blood films for malaria examination are stained without methanol fixation.
• Phosphate Buffer– A phosphate buffer of suitable pH is required for staining. A pH of about 6.8 is commonly used for routine blood-smear staining, while pH 7.2 is used for Giemsa staining of malaria blood films.
• Distilled Water– Distilled or deionized water is used for preparing the buffer and rinsing the stained slides.
• Staining Rack or Jar– A staining rack, staining tray or Coplin jar is used to hold the slides during staining.
• Pipettes and Measuring Cylinders– These are required for measuring and transferring the staining reagents. Droppers may also be used for adding stain and buffer to the slides.
• Timer– It is used to maintain the required staining time according to the selected method.
• Microscope and Immersion Oil– A light microscope with a 100× oil-immersion objective is used for examining the stained smears. Immersion oil is required for examination under this objective.
• Protective Equipment– Laboratory gloves, a protective coat and suitable waste containers are required for handling blood specimens and staining reagents.

Procedure of Romanowsky Staining

Romanowsky staining is carried out on air-dried blood smears using a suitable staining solution. The blood film is prepared, fixed and stained before microscopic examination.

The procedure of Romanowsky staining using the modified Wright–Giemsa horizontal staining method is given below.

1. Take a clean, dry and grease-free glass slide.
2. Place a small drop of fresh peripheral blood or EDTA-anticoagulated blood about 1 cm from one end of the slide.
3. Hold a spreader slide at an angle of 30–45° and move it backwards to touch the blood drop. Allow the blood to spread along the edge of the spreader and push it forward in a smooth movement to prepare a thin film.
4. Allow the blood film to air-dry completely.
5. Place the completely dried blood smear horizontally on a staining rack.
6. Add 1–2 mL of Wright–Giemsa staining solution to cover the blood film.
7. Allow the stain to remain on the slide for 1 minute.
8. Add an equal volume of phosphate buffer of pH 7.2 to the stain. Mix the solution gently on the slide.
9. Leave the mixture on the blood film for another 1–3 minutes.
10. Wash the stained slide thoroughly with deionized water to remove the excess stain.
11. Allow the slide to air-dry completely.
12. Place the dried slide on the microscope stage and examine the stained blood film under low magnification.
13. Add a drop of immersion oil to the suitable examination area and observe the blood cells using the 100× oil-immersion objective.

Thin blood films are fixed with methanol before or during staining. For the modified Wright–Giemsa method, the staining solution itself contains methanol. If staining cannot be carried out immediately, the dried smear may be fixed in methanol for 1 minute and allowed to dry.

Thick blood films prepared for malaria examination are not fixed with methanol. They are dried completely and stained without fixation to allow the erythrocytes to undergo lysis.

The staining time, dye concentration, buffer pH and fixation method depend on the Romanowsky stain used. The modified Wright–Giemsa method described here uses phosphate buffer at pH 7.2, within the recommended pH range of 6.8–7.2. Other formulations, including Leishman and Giemsa stains, require their respective staining conditions.

Results and Interpretation of Romanowsky Staining

A properly stained blood film shows pink erythrocytes, purple leukocyte nuclei and different colours in the cytoplasm and granules of white blood cells.

The staining characteristics of the different blood cells are given below.

Staining Characteristics of Red Blood Cells

Mature erythrocytes appear pink to salmon pink with a lighter central area. The pink colour is due to the staining of haemoglobin by eosin.

Normal erythrocytes are round, non-nucleated cells with a central pale area. Their size, shape and staining intensity can be examined under the microscope.

Hypochromic erythrocytes have a larger central pale area and reduced staining intensity. Polychromatophilic erythrocytes appear grey-blue due to the presence of residual RNA. Changes in cell size and shape can also be observed in abnormal blood films.

Staining Characteristics of White Blood Cells

The nuclei of leukocytes are stained purple to reddish-purple. The nuclear chromatin, cytoplasm and granules show different staining characteristics in different leukocytes.

• Neutrophils have dark purple nuclei with two to five lobes. Their cytoplasm is pale pink and contains fine lilac or light-purple granules.
• Eosinophils contain large orange-red to brick-red granules in the cytoplasm. The nucleus is usually bilobed and appears purple.
• Basophils have coarse dark-purple to purple-black granules. These granules may cover the nucleus, making its shape difficult to observe.
• Lymphocytes have a round or slightly indented dark-purple nucleus with a narrow rim of blue cytoplasm. The cytoplasm of reactive lymphocytes may show a deeper blue colour and increased volume.
• Monocytes have a folded or kidney-shaped purple nucleus. Their cytoplasm appears grey-blue and may contain fine azurophilic granules and vacuoles.

Abnormal leukocytes may show changes in nuclear shape, chromatin structure, cytoplasmic granules and cell size. Hypersegmented neutrophils, immature leukocytes and abnormal granulation can be identified during microscopic examination.

Staining Characteristics of Platelets and Cellular Inclusions

Platelets appear as small purple-stained cell fragments with fine purple granules and a pale-blue peripheral area. They are normally smaller than erythrocytes and may be seen singly or in small groups.

Different cellular inclusions can also be observed in Romanowsky-stained blood films.

• Howell–Jolly bodies appear as small, round, dark-purple inclusions within erythrocytes. These contain remnants of nuclear DNA.
• Basophilic stippling is observed as multiple fine or coarse blue granules distributed throughout the erythrocyte cytoplasm.
• Pappenheimer bodies appear as small blue-purple granules, usually near the periphery of erythrocytes. These contain iron and can be confirmed by iron staining.
• Döhle bodies are pale-blue inclusions present near the periphery of neutrophil cytoplasm.
• Toxic granulation appears as coarse, dark-purple granules in the cytoplasm of neutrophils.
• Malaria parasites may be observed within erythrocytes. In Giemsa-stained blood films, the parasite cytoplasm is blue and its chromatin is red to purple. Schüffner’s dots are more reliably demonstrated with Giemsa staining than with Wright–Giemsa staining.

The presence of abnormal cells or inclusions is interpreted along with their size, shape, distribution and other blood findings. Staining colour alone is not sufficient for the diagnosis of a particular disease.

Typical Staining Colours of Cellular Components in Romanowsky Staining

The exact shades may vary with the selected Romanowsky formulation. Wright, Giemsa, Leishman and May–Grünwald–Giemsa stains may produce differences in cytoplasmic and granule colours, although the main staining patterns are similar.

Factors Affecting Romanowsky Staining Quality

The following are the factors affecting Romanowsky staining quality:

• Blood smears should be prepared soon after collection. When EDTA blood is stored for a long time, erythrocytes may change their shape, leukocytes may develop cytoplasmic vacuoles and platelets may form clumps.
• A thick blood film contains overlapping cells, making it difficult to examine their morphology. The thin blood film should have a feathered edge and an area where the erythrocytes are present in a single layer.
• The blood smear must be allowed to air-dry completely before fixation, as slow drying or exposure to moisture may produce crenated erythrocytes and irregular pale areas.
• Grease, dirt and other materials present on the glass slide may produce gaps and streaks in the blood film. The cells may also be stained unevenly.
• Methanol is used to fix the dried blood film and preserve the cellular morphology. If the methanol contains moisture, clear refractile areas may appear in the erythrocytes. Inadequate fixation can also cause damage to the cells during washing.
• An alkaline staining buffer may produce excessive blue staining, whereas an acidic buffer gives a more reddish appearance with weak nuclear staining. The buffer pH should be maintained according to the selected Romanowsky staining method.
• The staining solution is diluted with buffer in the proportion required for the selected method. A weak solution gives pale staining, while an excessively concentrated stain may produce darkly stained cells.
• The blood film should be stained for the required time. When the staining time is too short, the nuclear and cytoplasmic structures appear faint. Prolonged staining may produce dark cells and stain deposits.
• Old or contaminated staining solutions may contain precipitated dye particles. These appear as dark-blue or purple granules on the blood film and may be mistaken for platelets, bacteria or other cellular inclusions.
• Insufficient washing leaves excess stain and deposits on the slide, while prolonged washing may remove the stain from the cellular components.
• Distilled or deionized water is used for preparing the staining buffer and washing the slides. Impurities in water or an unsuitable pH may change the staining colours.
• The stained slide should be dried without excessive heating. Heat and moisture may change the cellular morphology. Thick blood films prepared for malaria examination must be dried completely before staining, as insufficient drying may cause the film to detach during washing.
• A properly prepared control blood smear is used to check the quality of the staining solution. The staining colours of erythrocytes, leukocyte nuclei and cytoplasmic granules are examined for any changes.

Applications of Romanowsky Staining

The following are the applications of Romanowsky staining:

• Romanowsky staining is used for the microscopic examination of peripheral blood smears.
• It is used to identify different types of leukocytes and perform differential white blood cell counts.
• The size, shape and staining characteristics of erythrocytes can be examined to help in the morphological assessment of anaemia.
• Platelets can be examined for their size, shape, granules and distribution in the blood film.
• To identify immature leukocytes and abnormal blood cells during the examination of peripheral blood smears.
• Bone marrow smears are stained to observe the different stages of blood cell development and identify abnormal haematopoietic cells.
• Cellular inclusions such as Howell–Jolly bodies, basophilic stippling and Pappenheimer bodies can be observed in erythrocytes.
• It is used to examine morphological changes in leukocytes, including toxic granulation, Döhle bodies and cytoplasmic vacuoles.
• Giemsa staining is used for the detection and identification of Plasmodium parasites in thick and thin blood films.
• Other blood parasites, including Babesia and Trypanosoma, can also be examined using suitable Romanowsky staining methods.
• Giemsa stain is used to detect Leishmania amastigotes in tissue smears and aspirates.
• May–Grünwald–Giemsa and Diff-Quik stains are used for the examination of fine-needle aspiration smears, body fluids and other cytological specimens.
• Giemsa stain is also used for G-banding of metaphase chromosomes during cytogenetic examination.

Advantages of Romanowsky Staining

The following are the advantages of Romanowsky staining:

• Different cellular components are stained in different colours using the same staining solution.
• The nuclear chromatin, cytoplasm and granules of blood cells can be distinguished clearly under a light microscope.
• It helps to differentiate the five types of leukocytes based on their nuclear shape and cytoplasmic staining characteristics.
• Erythrocytes, leukocytes and platelets can be examined together in a single blood film.
• The characteristic purple staining of nuclear chromatin and certain cytoplasmic granules is obtained by the Romanowsky effect, which cannot be produced by azure B or eosin Y alone.
• Romanowsky staining is simple to perform and does not require expensive laboratory equipment.
• Rapid Romanowsky staining methods, such as Diff-Quik, can be completed within a few minutes.
• Different Romanowsky stains can be used for peripheral blood smears, bone marrow smears and air-dried cytological specimens.
• Giemsa staining allows the detection of malaria parasites in thick blood films. Thin films can be used for the identification of parasite species and estimation of parasite density.

Limitations of Romanowsky Staining

The following are the limitations of Romanowsky staining:

• Romanowsky staining shows the morphology of blood cells but cannot determine the exact cause of an abnormality. Other laboratory tests may be required for diagnosis.
• The examination of stained blood films requires an experienced microscopist. Immature and abnormal blood cells may be difficult to identify based on their morphology alone.
• A properly prepared blood smear is required for microscopic examination. Thick or uneven blood films contain overlapping cells, making it difficult to observe their nuclear and cytoplasmic structures.
• The staining colours depend on the pH of the buffer, dye concentration, staining time and formulation used. Changes in these conditions may produce differences in staining results.
• Stain precipitates and other materials present on the blood film may be mistaken for platelets, parasites or cellular inclusions.
• Romanowsky staining is not suitable for the accurate counting of reticulocytes. Supravital stains such as new methylene blue or brilliant cresyl blue are used for this purpose.
• Heinz bodies are not reliably visible with routine Romanowsky staining. These inclusions require supravital staining for proper examination.
• Pappenheimer bodies can be observed in Romanowsky-stained erythrocytes, but their iron content is confirmed using Perls’ Prussian blue stain.
• Thin blood films are less sensitive than thick films for detecting malaria parasites, particularly when the number of parasites is low.
• Thick blood films do not preserve the normal morphology of erythrocytes. The identification of malaria parasite species may be difficult without examining a thin blood film.
• Some parasite structures are not equally visible with all Romanowsky stains. For example, Schüffner’s dots are demonstrated more reliably with Giemsa stain than with Wright–Giemsa stain.
• Romanowsky staining does not measure haemoglobin concentration or absolute blood cell counts. These require separate haematological tests.

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