Wright–Giemsa stain is used on prepared peripheral blood smears in hematology laboratories. The blood smear is prepared as a thin film on a clean glass slide and allowed to air dry before staining.
The thickness of the blood film, proper drying, and the condition of the blood sample affect the quality of microscopic examination. A properly prepared blood film is required for the clear observation of blood cells under the microscope.
What is Wright–Giemsa Stain?
Wright–Giemsa stain is a polychromatic Romanowsky-type differential stain used to distinguish the cellular structures present in peripheral blood and bone marrow smears. It contains a combination of eosin and methylene-blue-derived dyes, including azure compounds. These dyes produce different colours in the nucleus, cytoplasm, and granules of blood cells. The staining of blood and bone marrow smears using this stain is known as Wright–Giemsa staining.
Wright, Giemsa, and Wright–Giemsa stains belong to the same group of Romanowsky stains. They have similar staining properties, but their dye formulations and staining procedures may differ. In some references, the terms Wright stain and Wright–Giemsa stain are used for related or overlapping staining methods.
Objectives of Wright–Giemsa Staining
The following are the objectives of Wright–Giemsa staining:
- To differentiate red blood cells, white blood cells, and platelets in peripheral blood smears.
- To identify different types of white blood cells and perform a differential leukocyte count.
- To examine the size, shape, and staining characteristics of red blood cells.
- To observe the nucleus, cytoplasm, and granules of white blood cells.
- To detect abnormal and immature blood cells based on their morphological characteristics.
- To examine the size and shape of platelets and estimate their number in blood smears.
- To study the morphology of developing blood cells present in bone marrow smears.
- To detect certain blood parasites, such as Plasmodium, in stained blood smears. Giemsa staining is preferred for detailed examination of malaria parasites.
Principle of Wright–Giemsa Staining
The principle of Wright–Giemsa staining is based on the binding of acidic and basic dyes to different cellular components. The acidic dye eosin Y binds to the basic components of the cells, including haemoglobin and certain cytoplasmic proteins. It stains the red blood cells pink to orange and the eosinophil granules orange-red.
The basic dyes, mainly azure B and other methylene blue derivatives, bind to the negatively charged components of the cells, such as DNA and RNA. Azure B stains the RNA-rich cytoplasm blue and also binds to the nuclear chromatin.
The characteristic purple colour of the nuclear chromatin and neutrophil granules is produced by the interaction of azure B with eosin Y. The two dyes form azure–eosin complexes in these cellular structures. This is known as the Romanowsky effect.
The staining reaction is also affected by the pH of the phosphate buffer. The pH influences the binding of acidic and basic dyes to the cellular components. A suitable buffer pH is required to obtain the characteristic staining colours of the blood cells.
Wright-Giemsa Stain Composition
Wright-Giemsa stain is a mixture of acid and basic dyes that are dissolved in methanol. The following are the composition of Wright-Giemsa stain:
- Eosin Y- It is an acidic stain that stains haemoglobin and some cytoplasmic components on blood films pink to red in colour.
- Methylene blue- It is a basic dye that is present in the solution of Wright-Giemsa stain. It stains the acidic components of cells blue in colour
- Azure Dyes- azure dyes are obtained from methylene blue. Azure B is the main azure dye used in the production of Wright-Giemsa stain; some formulations contain additional Azure A as well. Azure B combines with eosin Y in Wright-Giemsa stain to give the characteristic purple colour to nuclear chromatin and some granules.
- Methanol- It is a solvent for dyes and fixative for blood films.
- Glycerol- In some preparations of Wright-Giemsa stain, this substance is used to dissolve the dyes and prevent them from precipitating.
- Phosphate Buffer- This is used to maintain the pH of the stain wash solution, generally 6.8 for blood smears. It is present in some formulations of Wright-Giemsa stain separately as a wash solution.
Reagents and Materials Required for Wright–Giemsa Staining
The following are the reagents and materials required for Wright–Giemsa staining:
Reagents Required
- Wright–Giemsa Stain– Wright–Giemsa staining solution containing eosin and methylene blue derivatives. It is available as a ready-to-use solution or as a stain that requires dilution before use.
- Phosphate Buffer– Phosphate buffer, commonly of pH 6.8, is used to maintain the required pH during staining. The buffer pH may vary depending upon the staining method and the kit used.
- Methanol– Absolute methanol is used for fixing the blood smear. It is required separately in methods that include a separate fixation step.
- Rinsing Solution– Distilled water, deionized water, or the rinsing solution supplied with the staining kit is used to remove excess stain from the slide.
Some ready-to-use Wright–Giemsa staining solutions contain the stain, buffer, and fixative in a single solution. Other staining kits contain separate reagents that are prepared or mixed before use.
Materials and Equipment
- Clean glass microscope slides
- Spreader slides for preparing thin blood smears
- Staining rack or Coplin jars
- Pipettes or droppers for adding staining solutions
- Light microscope with an oil immersion objective (100×)
- Immersion oil for microscopic examination
- Fresh peripheral blood sample or EDTA-anticoagulated blood sample
- Blood collection materials, such as sterile lancets, needles, syringes, and EDTA collection tubes, as required
- Prepared and air-dried peripheral blood or bone marrow smears
- Timer or stopwatch
- Forceps or slide holders
- Laboratory coat, protective gloves, and safety goggles
Preparation of Wright–Giemsa Staining Reagents
Wright–Giemsa stain is available as a ready-to-use solution or as a stock staining solution that requires dilution before use. The preparation of the staining reagents depends upon the formulation used.
Preparation of Ready-to-Use Wright–Giemsa Stain
Some Wright–Giemsa staining solutions are supplied ready to use. For example, Sigma-Aldrich Wright–Giemsa Stain, Modified (Procedure No. WG), is a methanol-based solution that does not require dilution before staining.
The staining solution is used directly from the bottle. Phosphate buffer or deionized water is used separately according to the staining method.
Preparation of Working Wright–Giemsa Stain
Some Wright–Giemsa staining kits require the stain to be mixed with phosphate buffer before use.
The following is the preparation of working Wright–Giemsa stain using the Abcam Wright–Giemsa Stain Kit (ab245888):
- Take 25 mL of Wright–Giemsa staining solution in a clean container.
- Add 25 mL of phosphate buffer solution of pH 6.8.
- Mix the two solutions properly to prepare the working Wright–Giemsa stain.
- The prepared working stain is used for staining the blood smears.
Other stock or concentrated Wright–Giemsa stains may require different dilutions. The dilution should be carried out according to the instructions supplied with the particular stain.
Preparation of Phosphate Buffer
Phosphate buffer is used to maintain the required pH during staining. It may be supplied as a ready-to-use solution or as a concentrated reagent that requires dilution.
For example, the working phosphate buffer supplied as Sigma-Aldrich Phosphate Buffer (P3288) is prepared as follows:
- Take one vial of phosphate buffer reagent.
- Dissolve the contents in deionized water to make a final volume of 3.8 litres.
- Mix properly until the reagent is completely dissolved.
- The prepared buffer has a pH of 7.2 and is stored at 2–8°C.
The Abcam Wright–Giemsa Stain Kit (ab245888) contains a phosphate buffer solution of pH 6.8 that is supplied ready to use.
Preparation of Methanol and Rinsing Solution
Absolute methanol is used directly without dilution when a separate fixation step is required. The methanol present in some ready-to-use staining solutions also acts as a fixative.
Distilled or deionized water is used for rinsing the stained slides. Some staining methods require phosphate buffer in place of water.
Procedure of Wright–Giemsa Staining
The procedure of Wright–Giemsa staining using Sigma-Aldrich Wright–Giemsa Stain, Modified (Procedure No. WG), is given below:
Preparation and Fixation of Blood Smear
- Collect a fresh peripheral blood sample by finger prick or venipuncture. Fresh blood or blood collected in an EDTA tube can be used.
- Take a clean glass slide and place a small drop of blood near one end.
- Hold another clean slide at an angle of 30–45° and bring it in contact with the blood drop.
- Allow the blood to spread along the edge of the spreader slide and push it forward to prepare a thin blood smear with a feathered edge.
- Allow the blood smear to air dry completely at room temperature.
- The modified Wright–Giemsa staining solution contains methanol, which acts as a fixative during staining. Separate methanol fixation is not required for freshly prepared smears in this method.
- If the smear cannot be stained immediately, fix it in methanol for 1 minute and allow it to dry before staining.
Wright–Giemsa Staining Steps
The following are the steps of the horizontal staining method using Sigma-Aldrich modified Wright–Giemsa stain:
- Place the completely dried blood smear horizontally on a staining rack.
- Cover the blood smear with 1–2 mL of Wright–Giemsa staining solution.
- Allow the stain to remain on the slide for 1 minute.
- Add an equal volume of deionized water or phosphate buffer of pH 6.8–7.2 to the staining solution.
- Mix the stain and buffer gently over the slide.
- Allow the diluted stain to remain on the smear for another 1–3 minutes.
- Wash the slide thoroughly with deionized water to remove the excess stain.
- Keep the slide in an upright position and allow it to air dry completely.
The staining times and reagent volumes given above are for Sigma-Aldrich Wright–Giemsa Stain, Modified. Other Wright–Giemsa staining solutions may require different staining times and buffer volumes.
Rapid Dip Staining Method
The rapid dip staining method is another procedure used for staining peripheral blood smears. The following are the steps using Sigma-Aldrich Wright–Giemsa Stain, Modified:
- Take approximately 50 mL of Wright–Giemsa staining solution in a Coplin jar.
- Fill another Coplin jar with deionized water or phosphate buffer of pH 6.8–7.2.
- Place the completely dried blood smear in the staining solution with the feathered edge facing downward.
- Keep the slide in the staining solution for approximately 30 seconds.
- Remove the slide and transfer it to the deionized water or phosphate buffer for 1–10 minutes. Do not agitate the slide while it is immersed in the solution.
- Remove the slide and rinse it briefly with running deionized water.
- Allow the stained blood smear to air dry completely before microscopic examination.
The rapid dip method is not recommended for bone marrow smears because these preparations generally require longer staining times. In the Sigma-Aldrich method, bone marrow smears should be stained for at least 3 minutes and kept in the buffer for 3–10 minutes.
Other rapid staining kits may use different reagents, dipping times, and staining procedures.
Microscopic Examination
The microscopic examination of Wright–Giemsa-stained blood smears is carried out as follows:
- Place the completely dried and stained blood smear on the microscope stage.
- Examine the slide under the low-power objective (10×) to observe the distribution of blood cells and staining quality.
- Select a suitable area near the feathered edge where the red blood cells are present in a single layer without overlapping.
- Examine the selected area under the high-power objective (40×) to observe the distribution and morphology of blood cells.
- Place a drop of immersion oil on the selected area and examine it under the oil immersion objective (100×).
- Observe the nucleus, cytoplasm, and granules of white blood cells. The red blood cells and platelets are also examined for their morphological characteristics.
- Examine a known positive-control blood smear stained in the same run. The red blood cells should appear pink to orange, the nuclei of white blood cells purple, and the eosinophil granules orange.
- If the control slide shows improper staining, the staining results should be checked before reporting the findings.
Observation and Results of Wright–Giemsa Staining
Observation
When a properly stained peripheral blood smear is examined under the microscope, the different types of blood cells show characteristic staining colours.
The observations of Wright–Giemsa staining are as follows:
- The mature red blood cells are stained pink to orange. They appear as round, biconcave cells with a pale central region.
- A few immature red blood cells may appear blue-grey due to the presence of residual RNA. These cells are known as polychromatophilic erythrocytes.
- The nuclei of white blood cells are stained in different shades of purple. The cytoplasm may appear light pink, blue, or grey-blue depending upon the type of cell.
- Neutrophils have a dark purple, multilobed nucleus. Their cytoplasm is pale pink with fine lilac or light purple granules.
- Eosinophils have a bilobed nucleus and large orange-red granules in the cytoplasm.
- Basophils contain dark blue to purple-black granules. These granules may cover the nucleus.
- Lymphocytes have a large, round, dark purple nucleus with a small amount of light blue cytoplasm. Some lymphocytes may contain a few reddish-purple granules.
- Monocytes have a kidney-shaped or folded purple nucleus. Their cytoplasm appears grey-blue and may contain fine granules or vacuoles.
- Platelets are seen as small, irregular cell fragments with light blue cytoplasm and violet-purple granules.
The staining colours of different blood cells are given below:
| Blood Cell | Staining Colour |
|---|---|
| Mature erythrocytes | Pink to orange with central pallor |
| Polychromatophilic erythrocytes | Blue-grey or grey-purple |
| Leukocyte nuclei | Purple to dark purple |
| Neutrophils | Pale pink cytoplasm with fine lilac granules |
| Eosinophils | Orange-red cytoplasmic granules |
| Basophils | Dark blue to purple-black granules |
| Lymphocytes | Dark purple nucleus with light blue cytoplasm |
| Monocytes | Purple nucleus with grey-blue cytoplasm |
| Platelets | Light blue cytoplasm with violet-purple granules |
Result
In a properly stained blood smear, red blood cells appear pink to orange, while the nuclei of white blood cells are stained purple. The different types of white blood cells can be identified by their nuclear shape, cytoplasmic colour, and granules. Platelets are seen as small purple granular structures.
The stained smear is examined for changes in the size, shape, colour, and arrangement of red blood cells. Abnormal nuclear structures, immature white blood cells, and changes in platelet morphology may also be observed.
A small number of blue-grey polychromatophilic erythrocytes may be present in a normal blood smear. Their number may increase when more immature red blood cells are released into the blood.
The background of a properly stained smear is clear and free from stain deposits. Excessive blue or red staining, stain precipitates, and water artifacts may interfere with the examination of blood cells. Abnormal staining colours alone are not sufficient for the diagnosis of a disease.
Differences Between Wright–Giemsa, Wright, Giemsa and Diff-Quik Stains
Wright, Giemsa and Wright–Giemsa stains belong to the Romanowsky group of stains. Diff-Quik is a rapid modified Romanowsky stain. These stains contain acidic and basic dyes, but their composition, staining procedures and applications differ.
The differences between Wright–Giemsa, Wright, Giemsa and Diff-Quik stains are given below:
| Features | Wright–Giemsa Stain | Wright Stain | Giemsa Stain | Diff-Quik Stain |
|---|---|---|---|---|
| Type of Stain | Polychromatic Romanowsky-type differential stain | Romanowsky-type differential stain | Romanowsky-type differential stain | Rapid modified Romanowsky stain |
| Composition | Contains eosin Y and methylene blue derivatives, including azure dyes | Contains eosin and oxidized methylene blue dyes | Contains eosin Y, methylene blue and azure dyes, usually dissolved in methanol and glycerol | Contains separate eosinophilic and basophilic staining solutions |
| Fixation | Methanol may be present in the stain or used separately | Methanol in the staining solution usually acts as a fixative | Thin smears are usually fixed separately with methanol | A separate methanol fixative is used |
| Staining Procedure | The smear is stained with Wright–Giemsa solution and buffer. Horizontal and dip methods are available | The smear is covered with Wright stain, followed by buffer | The smear is stained with a diluted Giemsa solution | The smear is passed through fixative, eosinophilic stain and basophilic stain |
| Staining Time | Usually a few minutes for routine blood films, but depends upon the formulation | Generally a few minutes | Often requires longer staining than Wright stain, depending upon the method | Usually completed within a short time by repeated dipping |
| Main Applications | Examination of peripheral blood and bone marrow smears | Routine blood cell examination and differential leukocyte count | Blood parasite examination, blood cell morphology and cytogenetic staining | Rapid examination of cytological smears, fine-needle aspirates and other cellular preparations |
| Cellular Details | Shows nuclear chromatin, cytoplasm and cytoplasmic granules | Shows the morphology of red blood cells, white blood cells and platelets | Shows nuclear chromatin and certain fine intracellular structures | Shows nuclear and cytoplasmic details, including cytoplasmic granules |
| Parasite Examination | Can be used to detect blood parasites, but some parasite details may not stain clearly | Some blood parasites may be observed, but it is less reliable for detailed examination | Preferred for the detection and identification of malaria parasites | Not generally used as the standard method for malaria parasite identification |
Wright Stain vs Wright–Giemsa Stain
Wright stain is mainly used for the routine examination of peripheral blood smears. Wright–Giemsa stain contains a combination of Romanowsky dyes that is used for staining both blood and bone marrow smears.
Both stains differentiate the nucleus, cytoplasm and granules of blood cells. Wright–Giemsa stain may provide different staining intensity and nuclear details depending upon the proportion of dyes and the buffer used.
Wright Stain vs Giemsa Stain
Wright stain is commonly used for differential white blood cell counting and the examination of red blood cell morphology.
Giemsa stain is used for the examination of blood cells and the identification of blood parasites, particularly Plasmodium species. It can show certain parasite structures, such as Schüffner’s dots, more clearly than Wright stain.
In malaria examination, thick blood smears are stained without methanol fixation, while thin blood smears are fixed before Giemsa staining.
Wright–Giemsa Stain vs Diff-Quik Stain
Diff-Quik staining is a rapid staining method that uses separate fixative, acidic dye and basic dye solutions. It is commonly used for the examination of air-dried cytological smears.
Wright–Giemsa staining is mainly used in hematology for the examination of blood and bone marrow cells. Diff-Quik is often used for the rapid examination of fine-needle aspiration smears and other cytological specimens.
The staining colours and cellular details obtained with both methods depend upon the formulation, staining time and preparation of the specimen.
The names Wright and Wright–Giemsa are sometimes used for related staining methods. Commercial staining solutions may contain different proportions of eosin and azure dyes, and their staining procedures are not always the same.
Applications of Wright–Giemsa Staining
The following are the applications of Wright–Giemsa staining:
- It is used to stain peripheral blood smears for the microscopic examination of red blood cells, white blood cells, and platelets.
- To identify different types of white blood cells and perform a differential leukocyte count.
- It is used to examine changes in the size, shape, and colour of red blood cells in the investigation of anaemia.
- Detection of abnormal and immature white blood cells, which may help in the investigation of leukaemia and other blood disorders.
- It is used to examine the size, shape, and distribution of platelets and estimate their number in blood smears.
- To study the developing blood cells in bone marrow aspirate smears and identify abnormalities in their morphology.
- It can be used to detect certain blood parasites, such as Plasmodium species. However, Giemsa staining is preferred for the identification of malaria parasites.
- It is used to detect certain blood-borne microorganisms and intracellular inclusions, such as those associated with Anaplasma and Ehrlichia infections. The staining method has limited sensitivity for these organisms.
- Wright–Giemsa staining is also used in hematology laboratories for studying normal and abnormal blood cell morphology.
Common Wright–Giemsa Staining Errors and Troubleshooting
Common problems associated with Wright-Giemsa Staining
- Excess Blue Stain – The red blood cells will have been coloured blue or purple. The white blood cell nuclei will also be too dark. This can occur due to an alkaline buffer, long staining time, thick blood films and inadequate washing. Ensure that the pH of the buffer is correct. Reduce the staining time. Ensure adequate washing of the slide.
- Excess Pink Stain – The blood film will appear too pink/red. This can occur due to an overly acidic buffer. It can also occur due to a short staining time and over-washing. Ensure the buffer has the correct pH. Ensure correct staining time and washing time.
- Red Blood Cells Grey/Green – The red blood cells are appearing grey/green. Eosinophil granules will also appear off colours (not the normal orange-red). This is common due to an overly alkaline stain/buffer, long staining time and inadequate washing. Ensure the buffer pH is correct. Blood which has been heparinised can cause abnormal colours.
- Light Staining – The blood cells can appear very light. This can occur due to a short staining time, over washing. Or due to using old, diluted or incorrectly prepared stain. Ensure the correct stain is used and staining time is correct.
- Precipitation – Black/blue/purple particles can appear on the slide due to precipitation of the dye. This can occur due to using old, unfiltered stain. Or leaving stain on the slide. Using dirty glass slides and inadequate washing can cause this. Clean slides should be used. If necessary filtrate the stain. Do not leave stain on the smear.
- Water Artefacts – Irregular refractive areas can appear in the red blood cells. This can occur due to water in the methanol. Or improper drying of the blood film. Use fresh anhydrous methanol and ensure the blood film is fully dry.
- Poor Cell Differentiation – The nuclei, cytoplasm and granules of the white blood cells cannot be seen. This can occur due to incorrect buffer pH. Poor fixation. Incorrect staining time. Or thick/uneven blood films. Ensure the blood film is thin. Ensure correct buffer pH, fixation and staining times.
- Inconsistent Staining – Different colours can appear on different slides. This can occur due to changes in buffer pH, concentrations of stain and staining times. Evaporation of the stain can affect results. Use fresh reagents. Ensure consistent conditions during staining. Ensure a well-stained control smear is viewed.
A few red blood cells can appear bluish-grey. These are known as Polychromatophilic erythrocytes. These contain leftover RNA. They are usually larger than normal red blood cells. This is seen in single cells, not most of the blood film like excess blue.
Limitations of Wright–Giemsa Staining
The following are the limitations of Wright–Giemsa staining:
- Wright–Giemsa staining is used to study the morphology of blood cells. The exact cause of a blood disorder cannot be determined by this staining method alone.
- The staining colour depends upon the pH of the buffer, concentration of the stain, and staining time. Changes in these conditions may produce abnormal staining colours.
- Thick or uneven blood smears make it difficult to observe the nucleus, cytoplasm, and granules of blood cells.
- Improper drying or fixation of blood smears may produce artifacts. These can change the normal appearance of red blood cells and other cellular structures.
- Wright–Giemsa stain is not preferred for the detailed examination of blood parasites. Certain structures of malaria parasites, such as Schüffner’s dots, may not be clearly seen. Giemsa stain is preferred for the identification of Plasmodium species.
- Blood parasites present in very small numbers may not be detected during microscopic examination. A negative blood smear does not exclude a parasitic infection.
- Some microorganisms and cellular inclusions are difficult to identify using Wright–Giemsa stain. Additional laboratory tests may be required for their identification.
- Some abnormal and immature blood cells have similar staining characteristics. Their identification requires microscopic examination by trained personnel.
- It cannot be used to measure the total blood cell count or haemoglobin concentration accurately. These measurements require other laboratory methods.
- Different formulations of Wright–Giemsa stain may give different staining colours and intensity. The buffer requirements and staining procedures may also differ.
Precautions of Wright–Giemsa Staining
- All blood samples should be treated as potentially infectious. Standard precautions should be taken when obtaining and preparing blood films.
- A lab coat, single-use gloves and eye protection should be worn.
- Protect any cuts or wounds on your hands.
- Single-use needles and lancets should be used to collect blood samples. Used needles and sharps should be placed into a sharps container.
- Avoid splashing blood. Do not use mouth pipetting techniques.
- Hands should be washed properly once gloves have been removed. The working surface should be cleaned and disinfected.
- Methanol is a toxic and highly flammable substance. Methanol and methanol based staining solutions should be used in a well ventilated area away from naked flames, heat or sparks.
- Avoid direct contact with methanol and staining solutions. Keep containers of reagents sealed.
- Clean glass slides should be used. They should be dry and not greasy. Blood films should be thin and even.
- Air dry the blood film. Do not use heat to fix.
- Fresh anhydrous methanol should be used to fix the samples if required. Some Wright-Giemsa solutions contain methanol and fixing is not required.
- Suitable phosphate buffer or water at the correct pH should be used. The pH of the buffer will depend on the method and reagent used.
- Correct staining and washing times should be used for the chosen Wright-Giemsa stain. Ensure that the staining solution does not dry out.
- Correctly stored staining solutions should be used. Do not use solutions with visible precipitates.
- A known control blood film should be stained. This will ensure that the red blood cells, white blood cells and platelets have been stained correctly.
- Blood stained items should be placed in the correct bio-hazard bins. Staining solutions containing methanol and other chemical waste should be collected and disposed of according to laboratory safety protocols.
References
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