Myeloperoxidase Reaction (MPO) Stain

Introduction

Myeloperoxidase is a lysosomal enzyme, located in the azurophilic granules of the neutrophils and its precursors, eosinophils and monocytes. This MPO stain is mainly used to differentiate acute myeloid leukaemia from acute lymphoblastic leukaemia. MPO stain can be a valuable tool for:

• Diagnosing and classifying leukemia: By differentiating between MPO-positive myeloid blasts (characteristic of AML) and MPO-negative lymphoid blasts (seen in ALL).
• Monitoring treatment response: Tracking changes in MPO expression in bone marrow samples can indicate the effectiveness of leukemia treatment.
• Identifying inflammatory processes: Increased MPO levels in tissues can suggest the presence of inflammation.
• Studying myeloid differentiation: MPO expression patterns can help visualize the differentiation process of myeloid stem cells.

Principle of MPO

Peroxidase in leukocyte granules oxidizes benzidine to form insoluble, stable and non-diffusible reaction product in the presence of hydrogen peroxide (H₂O₂).  This product is colourless to blue or brown derivatives which is localized at the site of the enzyme. Copper sulphate or nitrate can be used to enhance the staining.

But how does the stain differentiate between different cell types? The key lies in the selective expression of MPO. Neutrophils and certain other myeloid cells like monocytes and some basophils are rich in MPO, while lymphocytes, another white blood cell type, lack it. Therefore, when applied to a tissue sample or blood smear, the stain paints only MPO-containing cells with vibrant hues, leaving MPO-negative cells unstained.

Materials

• Air dried peripheral blood or bone marrow smear (within 24 hours of collection)
• Fixative – 10% formal ethanol (10 mL of 40% formalin and 90 mL of absolute ethanol)
• The staining solution:
  • 30% Ethanol: 100 mL
  • Benzidine dihydrochloride: 0.3 g
  • 0.132M (3.8%) ZnSO₄·7H₂O: 1 mL
  • Sodium acetate: 1 g
  • 3% hydrogen peroxide: 0.7 mL
  • 1 N sodium hydroxide: 1.5 mL
• Giemsa stain

Protocol

1. Prepare air dried film – preferable within 24 hours of collection (peroxidase is unstable in the light).
2. Fix slide in fixative at room temperature for 60 seconds.
3. Rinse in slow running tap water for 15 – 30 seconds and air dry.
4. Place slide on staining rack and fully cover the air-dried slide with the staining solution and incubate for 1 minute. 
5. Rinse in slow running tap water for 1 minute.
6. Counterstain with Giemsa stain at 1:10 dilution for 10 minutes. 
7. Wash off counter stain. 
8. Wipe the back of the slide and edges with Kim wipes. Be careful not to touch the smear. 
9. 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.
10. Mount the slide with Depex and cover the zone of morphology with a cover slip. 
11. This slide is now ready for viewing. 

Enzyme activity may be preserved for as long as 3 weeks if preparations are stored in the dark.

Interpretation

Positive MPO Reaction: A positive MPO reaction is characterized by a distinct brown to green cytoplasmic staining in granulocytes, reflecting the presence and activity of the enzyme. This indicates the presence of mature neutrophils (positive control), eosinophils, and some basophils in the sample. It is also positive for myeloblasts (except those at a very primitive stage), promyelocytes and metamylocytes. A strong, consistent positive reaction suggests normal granulocyte maturation and function.

Negative MPO Reaction: Conversely, a negative MPO reaction reveals the absence of brown to green cytoplasmic staining for example in lymphoblasts and lymphocytes. Monoblasts and monocytes may be positive or negative.

Pathological Variations: Deviations from the expected staining pattern can unveil underlying pathologies for example in congenital deficiency of neutrophil, where results will be negative or in dysplastic neutrophils.

For Optimal Result

• Adherence to standardized protocols for sample collection, handling, and processing is crucial.
• Using high-quality validated reagents is essential.
• Maintaining proper laboratory practices and quality control measures helps ensure consistent and reliable results.
• Experienced pathologists who are familiar with MPO staining patterns should interpret the results.
• Correlation with other diagnostic tests and clinical findings is necessary for accurate diagnosis and management decisions.

Frequently Asked Questions (FAQs)

What cells are MPO positive?

The primary cells that are MPO positive are neutrophils, a crucial type of white blood cell responsible for battling infections. MPO is the most abundant enzyme within these cells, playing a key role in their antimicrobial activity.

However, MPO isn’t exclusive to neutrophils. Here’s a broader picture of MPO-positive cells:

Myeloid Lineage

• Monocytes: These white blood cells, involved in inflammation and antigen presentation, can also express MPO, although typically in lower levels than neutrophils.
• Eosinophils: While their primary function isn’t directly linked to MPO, a small subset of eosinophils might exhibit MPO positivity.
• Myeloid blasts: In acute myeloid leukemia (AML), the abnormal blasts arising from myeloid stem cells are typically MPO-positive, making the MPO stain crucial for diagnosis and subclassification.

Other Cells

• Myeloid precursors: During the differentiation process of myeloid stem cells into mature cells, MPO expression gradually increases, making some precursor cells also MPO-positive.
• Rare cases: In exceptional circumstances, MPO expression has been observed in cells outside the myeloid lineage, such as some subsets of lymphocytes or even certain epithelial cells. However, these instances are not typical and require further investigation.

What is the function of the myeloperoxidase (MPO)?

The function of MPO depends on the context.

Innate Immune Defense

• Primary function: MPO plays a crucial role in our innate immune defense. It’s most abundant in neutrophils, white blood cells that fight infections. Within neutrophils, MPO produces powerful reactive oxygen species (ROS) like hypochlorous acid (similar to bleach) when hydrogen peroxide and chloride ions interact. These ROS directly kill bacteria, fungi, and other invading pathogens engulfed by the neutrophil.

Wound Healing and Inflammation

• Tissue remodeling: After an injury, MPO contributes to wound healing by breaking down damaged proteins and other molecules, aiding tissue remodeling.
• Inflammatory response: MPO is involved in the body’s inflammatory response. While it helps eliminate pathogens during inflammation, excessive MPO activity can also damage healthy tissues. Some research suggests it might contribute to chronic inflammation associated with various diseases.

What color does MPO stain positive?

MPO itself is colorless, but when detected using an MPO stain, it produces a colored product that allows visualization under a microscope. The specific color of the stained cells depends on the chromogen used in the staining procedure.

Here are some common chromogens and the colors they produce with MPO:

• 3,3′-diaminobenzidine (DAB): This is the most widely used chromogen, and it reacts with MPO to produce a brown precipitate.
• Benzidine: This chromogen is no longer commonly used due to its carcinogenic properties, but it also produces a brown precipitate with MPO.
• 3,3′-diaminobenzidine tetrahydrochloride (DAB-TH): This is a variant of DAB that produces a black precipitate, offering better contrast for microscopic observation.
• Alpha-naphthyl acetate esterase (ANAE): This is a different type of stain that targets specific enzymes within neutrophils, including MPO. It produces a red precipitate when MPO is present.

Therefore, depending on the chosen chromogen, MPO-positive cells can appear brown, black, or red when stained. It’s crucial to know the specific chromogen used in the context of your inquiry to interpret the color correctly.

What are the components of MPO stain?

The content of an MPO stain typically consists of the following components:

Antibody

• This is the most crucial component of the stain and is specifically designed to recognize and bind to the MPO enzyme. Antibodies are highly specific proteins produced by the immune system in response to foreign substances. In the case of MPO stains, the antibody is engineered to target and bind only to the MPO enzyme, leaving other cellular components unlabeled.

Chromogen

• Once the antibody binds to MPO, a chromogen is introduced. This is a colorless compound that gets activated by the enzyme, undergoing a chemical reaction that produces a visible colored product. The specific color of the product depends on the chosen chromogen. Some common chromogens used in MPO stains include:
  • 3,3′-diaminobenzidine (DAB): This produces a brown precipitate.
  • 3,3′-diaminobenzidine tetrahydrochloride (DAB-TH): This is a variant of DAB that produces a black precipitate, offering better contrast for microscopic observation.
  • Alpha-naphthyl acetate esterase (ANAE): This targets specific enzymes within neutrophils, including MPO, and produces a red precipitate.

Buffer

• The staining solution usually contains a buffer to maintain a specific pH and ionic strength, which are crucial for optimal antibody and enzyme activity.

Other components

• Depending on the specific protocol, the staining solution might also include additional components like preservatives, stabilizers, or detergents to optimize the staining process and prevent degradation of the reagents.

It’s important to note that the exact composition of an MPO stain might vary depending on the manufacturer, the intended application, and the specific needs of the laboratory. However, the core components mentioned above are essential for the stain to function effectively and specifically detect MPO expression in cells.

Do monoblasts stain with MPO?

In general, monoblasts typically do not stain positively for MPO. Here’s a breakdown of MPO expression in monoblasts and related cells:

MPO and Myeloid Differentiation

• MPO is primarily expressed in mature myeloid cells like neutrophils, monocytes, and dendritic cells.
• During myeloid differentiation, the process by which stem cells mature into specific myeloid cells, MPO expression gradually increases.
• Early precursors, including monoblasts, generally express very little or no MPO.

Monoblast MPO Staining

• While monoblasts are considered part of the myeloid lineage, they are the earliest stage and haven’t fully differentiated yet.
• Therefore, most monoblasts do not exhibit positive MPO staining.
• In rare cases, particularly with certain rare types of leukemia, some monoblasts might show weak or partial MPO positivity. However, this is not typical and requires further investigation in such instances.

Importance of Context

• MPO staining is used in various contexts, primarily for differentiating leukemias.
• When interpreting MPO staining results, it’s crucial to consider the specific cell type, disease context, and patient history.
• Absence of MPO staining in monoblasts is consistent with their early stage in differentiation.

What are the limitations of the MPO stain?

The MPO stain, while valuable in various clinical settings, has several limitations that need to be considered for accurate diagnosis and interpretation.

Specificity

• Not entirely specific: While MPO is primarily found in myeloid cells, trace amounts can be present in other cell types like some lymphocytes or even epithelial cells in rare cases. This can lead to misinterpretation, especially in challenging diagnostic scenarios.
• Variable expression: MPO expression levels can vary within different subtypes of the same cell type, adding a layer of complexity to interpreting stain intensity.

Sensitivity

• Weak expression: Some AML subtypes have inherently low MPO expression, potentially leading to false negatives if the stain sensitivity is insufficient.
• Technical factors: Improper tissue processing, suboptimal staining protocols, or inadequate observer experience can further affect sensitivity, potentially missing positive cases.

Diagnostic Limitations

• Not definitive alone: MPO staining should always be interpreted alongside other diagnostic tests like flow cytometry, cytogenetics, and molecular analysis for a comprehensive picture.

What are the alternative tests to the MPO stain?

While the MPO stain remains a valuable tool in various clinical settings, particularly for leukemia diagnosis and monitoring, it has limitations like potential lack of specificity and sensitivity. In some cases, alternative tests might offer additional insights or overcome these limitations.

Flow cytometry

• This technique utilizes lasers and fluorescent antibodies to analyze individual cells, offering detailed information about their size, granularity, and surface marker expression.
• In leukemia diagnosis, flow cytometry can precisely identify and characterize blasts based on their specific markers, including myeloperoxidase (MPO).
• Compared to MPO stain, flow cytometry provides quantitative data on MPO expression levels and can detect even small populations of MPO-positive blasts.

Immunohistochemistry (IHC) with different markers

• While MPO is a helpful myeloid marker, other IHC markers can provide complementary information depending on the specific diagnostic question.
• Examples include myeloperoxidase receptor (MPOR), which can be expressed in some MPO-negative leukemias, or CD34, a marker for progenitor cells helpful in specific diagnostic scenarios.
• Combining MPO staining with IHC for other markers can improve diagnostic accuracy and aid in subclassifying certain leukemias.

Cytogenetics and molecular analysis

• These techniques analyze the genetic makeup of cells, identifying chromosomal abnormalities or specific gene mutations associated with different types of leukemia.
• While not directly detecting MPO, these tests can provide crucial information about the underlying cause of leukemia and guide treatment decisions.
• In some cases, cytogenetic or molecular abnormalities might help differentiate between MPO-positive and negative leukemias with overlapping features.

Emerging techniques

• Newer technologies like mass cytometry and next-generation sequencing are constantly evolving and hold promise for even more detailed and precise characterization of cells, potentially offering alternatives to traditional MPO staining in the future.

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.

References

1. Bain BJ. A Practical Guide. 6th Edition (Wiley). 2022.
2. Bain BJ, Bates I, Laffan MA. Dacie and Lewis Practical Haematology: Expert Consult: Online and Print 12th Edition (Elsevier). 2016.
3. Carr JH. Clinical Hematology Atlas 6th Edition (Elsevier). 2021.