Introduction
Similar to the Leishman stain, MGG is part of the Romanowsky stains and adopts the same principle. MGG stain consists of May-Grünwald stain and Giemsa stain. MGG stain is used for morphology assessment of bone marrow aspirates especially in diseases like chronic lymphocytic leukemia, chronic myeloid leukemia, aplastic anemia.
Principle of MGG stain
MGG stain works by differentially staining different cellular components based on their acidic or basic properties. Acidic components, such as DNA and chromatin, stain blue or purple, while basic components, such as cytoplasm and proteins, stain pink or red. The stain also highlights nuclear structures, such as nucleoli and nuclear membranes.
MGG stain is used to visualize cells in a variety of tissues, including blood, bone marrow, and lymph nodes. It is particularly useful for identifying and classifying different types of white blood cells, such as lymphocytes, granulocytes, and monocytes. It can also be used to detect abnormalities in cells, such as the presence of cancer cells or infection.
Method differs slightly according to the manufacturer’s protocol.
Materials
- May-Grünwald dye
- Absolute methanol
- Giemsa dye
- Glycerol
- Phosphate buffer pH 6.8
Protocol
Preparation of May-Grünwald stain
- Dissolve 0.3 g of May-Grünwald dye in 100 mL absolute methanol in a 250 mL conical flask.
- Warm the mixture to 50°C in a water bath for a few hours and allow it to cool to room temperature.
- Stir the mixture on a magnetic stirrer and leave it stirring for 24 hours.
- Filter the mixture and stain is ready for use.
Preparation of Giemsa stain
- Add 1.0 g of Giemsa dye into 66 mL of glycerol and warm the mixture in a conical flask for 1-2 hours at 50°C.
- Cool the mixture to room temperature and add 66 mL of absolute methanol.
- Leave the mixture to dissolve for 2-3 days, mixing it at intervals.
- The stain is then ready for use after filtering.
Staining
- Prepare a solution of May-Grünwald stain and phosphate buffer at 1:1 ratio and mix well.
- Prepare a 1:10 dilution of Giemsa stain with phosphate buffer pH 6.8 and mix well.
- Fix bone marrow aspirate smear in absolute methanol for 10 -15 minutes.
- Fully cover the slide with May-Grünwald-phosphate mixture and incubate for 10 minutes.
- Decant the May Grunwald-phosphate mixture from the slide and fully cover the slide with the Giemsa-phosphate mixture.
- Incubate for 15 minutes.
- Decant the mixture and rinse the slide with slow running tap water.
- Wipe the back of the slide and edges with Kim wipes. Be careful not to touch the smear.
- Dry the slide using a hair dryer on the lowest speed (not more than 10 seconds at a time) or air dry in a tilted position.
- Mount the slide with Depex and cover the zone of morphology with a long cover slip.
- This slide is now ready for viewing.
Interpretation
To assess the bone marrow aspirate morphology accurately, prior knowledge of the patient’s history and also clinical indication for the procedure is necessary.
Microscopy examination usually covers 10X magnification, 40X magnification as well as 100X (oil immersion) magnification assessment.
Under 10X objective magnification, things to note include:
- Hypercellular or hypocellular bone marrow fragment
- Normal or diluted or hypercellular cell trails
- Number and morphology of megakaryocytes available
- Presence of abnormal cell clumps are suggestive of marrow infiltration
Under 40X objective magnification, things to note include:
- Whether the erythropoiesis are normoblastic, megaloblastic or dysplastic in nature
- Presences of all stages of normal maturation for the myeloid and lymphoid lineage cells including myeloblasts and lymphoblasts
- Presence of normal or abnormal plasma cells and macrophages and whether their number is increased.
- Presence of any metastatic cells.
Troubleshooting
- Expired or contaminated reagents: Using expired or contaminated stains can lead to inconsistent or weak staining.
- Incorrect stain concentration: Using solutions at incorrect concentrations can cause under- or overstaining.
- Improper staining times: Deviating from the recommended staining times can lead to under- or overstaining.
- Incorrect pH: Improper buffer solution pH can affect stain interaction with cellular components.
- Insufficient rinsing: Inadequate rinsing can leave behind residual buffer salts, affecting stain color and clarity.
Frequently Asked Questions (FAQs)
What is the MGG stain used for?
MGG stain, or May-Grünwald Giemsa stain, has several important uses in various fields, primarily focusing on visualizing and analyzing cells.
Hematology
- Differential counting of blood cells (CBC with differential): This is the most common use of MGG stain. It effectively distinguishes and colors different types of white blood cells (neutrophils, eosinophils, basophils, lymphocytes, monocytes) and red blood cells, allowing for accurate assessment of their numbers and morphology. MGG stain highlights various cellular features such as nuclear size, shape, chromatin pattern, and cytoplasmic granules. This detailed visualization aids in identifying abnormal cell characteristics associated with different diseases like leukemia, anemia, and infections.
Cytology
- Examination of body fluids: MGG stain is used to analyze cells present in body fluids like cerebrospinal fluid, pleural fluid, and synovial fluid for the presence of abnormal cells, potentially indicating infections, inflammation, or malignancies.
- Fine-needle aspiration biopsies: MGG stain helps in examining cell samples obtained through minimally invasive procedures like FNAs, providing valuable information for diagnosing various tumors and other tissue abnormalities.
Histology
- Visualization of hematopoietic tissues: MGG stain can be used on tissue sections, particularly bone marrow biopsies, to assess the composition and morphology of blood-forming cells, aiding in diagnosing blood disorders.
- Detection of parasites and microorganisms: In some cases, MGG stain can help identify certain parasites and microorganisms within tissue samples.
What cell components are better seen in MGG staining?
MGG staining excels at visualizing various cell components, offering detailed information about cell morphology and function.
- Nuclear size and shape: MGG clearly distinguishes variations in nuclear size and shape, crucial for identifying abnormal cells like blasts in leukemia.
- Chromatin pattern: The staining allows visualization of chromatin condensation, revealing irregularities like clumping or dispersed chromatin, indicative of potential issues like DNA damage.
- Visualizing immature cells: Due to their larger nuclei and prominent nucleoli, immature cells like blasts and stem cells are readily identifiable with MGG.
- Good color differentiation: The combination of acidic and basic dyes in MGG creates a spectrum of colors, allowing for better distinction between different cell components compared to other stains.
What are the advantages and disadvantages of using MGG stain compared to other methods?
Advantages of MGG stain
- Versatility: MGG stain effectively visualizes both nuclei and cytoplasm, making it suitable for analyzing various cell types and components.
- Fast and simple: The staining process is relatively quick and straightforward, requiring minimal steps and readily available components.
- Cost-effective: The reagents for MGG staining are generally inexpensive compared to some specialized staining techniques.
- Good standardization: Established protocols and readily available commercial kits ensure consistent staining results across laboratories.
- High-quality details: MGG provides excellent visualization of nuclear and cytoplasmic morphology, including size, shape, chromatin pattern, and cytoplasmic granules.
- Durable results: Properly prepared slides stained with MGG can be stored for long periods without significant fading or deterioration.
Disadvantages of MGG stain
- Limited specificity: Although it stains various components, MGG doesn’t offer the same level of specificity as some specialized stains designed for specific structures or cell types.
- Not ideal for all cell types: Some cell types, like mast cells or reticulocytes, might require different staining techniques for optimal visualization.
- Potential for artifacts: Improper technique or suboptimal conditions can lead to staining artifacts that might interfere with accurate interpretation.
- Not ideal for automation: Automating MGG staining can be challenging due to the multiple steps involved and potential variability in results.
- Health hazards: The components of MGG stain are considered hazardous materials and require proper handling and disposal procedures.
- Unable to differentiate between different types of leukemias: While MGG can identify leukemia, it might not provide enough details to pinpoint the specific subtype, requiring further tests like immunophenotyping.
- Inadequate detection of rare parasitic infections: MGG might not be sensitive enough to identify specific parasites present in low numbers, necessitating more specific diagnostic tests.
- Miss subtle changes in cell morphology: Early stages of some malignancies might exhibit subtle changes that MGG might miss, requiring more sensitive techniques like flow cytometry.
Compared to other methods
Here’s a brief comparison of MGG to other commonly used staining methods:
| Method | Advantages | Disadvantages |
|---|---|---|
| Wright stain: Similar to MGG in versatility and simplicity, but offers less cytoplasmic detail | Faster than MGG, less expensive | Lower cytoplasmic detail, more prone to artifacts |
| Giemsa stain: Can be used alone for some applications, but offers less nuclear detail than MGG | Simpler than MGG, good for specific cell types | Less nuclear detail, not as versatile as MGG |
| Immunohistochemistry: Highly specific for visualizing specific proteins | More complex and expensive, requires additional antibody preparation | |
| Flow cytometry: Can analyze large cell populations quickly | Limited morphological information, requires specialized equipment |
What are the components of May-Grünwald Giemsa stain?
May-Grünwald Giemsa (MGG) stain is actually a combination of two different stains: May-Grünwald stain and Giemsa stain. Both stains contain different components and work together to achieve the characteristic staining of various cell components.
May-Grünwald stain
- Eosin Y: This is an acidic dye that stains acidic components in cells, such as cytoplasm and some granules, in pink or orange tones.
- Methylene Blue: This is a basic dye that stains basic components in cells, such as nuclei and other granules, in blue or purple tones.
Giemsa stain
- Azures (Azure B and Azure II): These are oxidized methylene blue dyes that contribute to the characteristic purple-blue staining of nuclei.
- Eosin Y: This is the same component as in May-Grünwald stain, further enhancing cytoplasmic staining.
- Methylene Blue Chloride: This component interacts with the Azures to achieve the characteristic Romanowsky-Giemsa effect, resulting in the final purple coloration.
Disclaimer: This protocol is intended for informational purposes only and may need to be modified depending on the specific laboratory procedures and patient circumstances. Always consult with a qualified healthcare professional for guidance. See additional information.
