Introduction
Leukocyte alkaline phosphatase (LAP) / Neutrophil alkaline phosphatase (NAP) stain stands as a valuable diagnostic tool, offering insights into the functionality of neutrophils, a critical component of the immune system. This enzymatic stain specifically targets alkaline phosphatase, an enzyme abundantly present in the cytoplasm of mature neutrophils. The intensity of the stain reflects the level of LAP/NAP activity, providing a measure of neutrophil maturity and overall health.
LAP / NAP staining serves as a diagnostic aid in various hematological conditions, particularly those involving neutrophil dysfunction. It plays a crucial role in differentiating between reactive neutrophilia, a normal response to infection or inflammation, or chronic myeloid leukemia which has a higher number of immature neutrophils or blast cells.
Principle of NAP / LAP stain
Leukocyte alkaline phosphatase (LAP) / Neutrophil alkaline phosphatase (NAP) stain relies on the principle of diazotization and coupling reactions to visualize the activity of alkaline phosphatase (ALP), an enzyme abundant in mature neutrophils. The intensity of the stain reflects the level of ALP activity, providing a measure of neutrophil maturity and overall health.
The staining procedure begins with the application of a diazonium salt solution to blood smears. Diazonium salts are highly reactive compounds that readily form azo dyes when coupled with aromatic compounds. In this case, the diazonium salt reacts with naphthol AS-BI phosphoric acid, a substrate for ALP.
ALP, when present in the cytoplasm of LAP/NAP-positive neutrophils, catalyzes the hydrolysis of naphthol AS-BI phosphoric acid, releasing naphthol. This naphthol then reacts with the diazonium salt, forming an insoluble blue-violet or reddish brown precipitate (depending on the dye) used within the neutrophil cytoplasm.
The intensity of the stain depends on the amount of ALP present in the neutrophil. Mature neutrophils, with their high ALP content, exhibit strong staining, while immature neutrophils, with lower ALP activity, show weaker staining. This differential staining pattern allows for the assessment of neutrophil maturity and the identification of potential abnormalities in neutrophil function.
Method differs slightly according to the manufacturer’s protocol.
Materials
- Fresh unstained peripheral blood smear slide, a positive control slide from a hamster and a negative control slide from mice
- Freshly prepared fixative: 1:9 ration of 40% formaldehyde: absolute methanol
- Naphthol AS phosphate
- 0.2 mol/L Tris buffer pH 9.0
- Fast Blue BB salt (coupling azo dye)
- Neutral red, 0.02% aqueous solution (counterstain)
- Filter paper
- Funnel
Protocol
- Prepare stock substrate solution: Dissolve 30 mg naphthol AS phosphate in 0.5 mL N,N-dimethylformamide and add 100 mL 0.2 mol/L Tris buffer.
- Prepare working buffered substrate solution: Warm 40 mL of stock substrate solution to room temperature and add 24 mg of Fast Blue BB salt. Mix well until the salt is fully dissolved. Filter the mixture using a filter paper and funnel before use.
- Fix all the blood smear slides in the freshly prepared fixative.
- Rinse the slide with slow running tap water for 2 – 3 minutes.
- Incubate the slides in working buffered substrate solution for 15 minutes.
- Rinse the slide with slow running tap water for 1 – 2 minutes.
- Air dry the slides.
- Incubate the slides in counterstain for 3 minutes.
- Rinse the slides briefly and allow to air dry.
- Mount the slide with Depex and cover the zone of morphology with a cover slip.
- This slide is now ready for viewing.
Interpretation
LAP / NAP scores are used to grade the intensity of the LAP / NAP stain on a scale of 0 to 4+. A LAP / NAP score of 0 indicates no staining, while a LAP / NAP score of 4+ indicates the strongest staining. LAP / NAP scores are interpreted in the context of the patient’s clinical presentation and other laboratory findings. The LAP / NAP score has a possible scoring of 0 – 400.
In general, a LAP / NAP score of 3+ or 4+ is considered normal, indicating that the neutrophils are mature and functioning normally. A LAP / NAP score of 2+ or less may suggest impaired neutrophil maturation or function. This could be due to a variety of factors, including chronic myeloid leukemia (CML).
However, it is important to note that LAP / NAP scores should not be interpreted in isolation. The presence of immature neutrophils, as indicated by a low LAP / NAP score, must be considered in conjunction with other laboratory findings, such as a complete blood count (CBC) and a bone marrow biopsy, to make an accurate diagnosis.
Presence of alkaline phosphatase is based on the precipitates of blue or red granules in the neutrophils. A positive control (hamster) and a negative control (mice) is used as comparison for the measurements of the LAP / NAP score. Assessment of LAP / NAP scoring is as follows:
- Count 100 neutrophils (including band forms)
- Rate the LAP / NAP score for each neutrophil as below and add the score for 100 neutrophils.
| Score | Neutrophil staining | Interpretation |
| 0 | Absence of stained granules | Negative |
| 1 | Occasional stained granules in the cytoplasm | Positive |
| 2 | Moderate number of stained granules | Positive |
| 3 | Numerous stained granules in the cytoplasm | Strong positive |
| 4 | Numerous stained granules crowding and overlying the nucleus in the cytoplasm | Heavy positivity |
3. Normal range: 40 – 100.
4. A high score may indicate a leukemoid reaction for e.g. severe infections such as sepsis and pneumonia and tissue necrosis such as from burns or pancreatitis.
5. A low score may indicate certain disorders for e.g. chronic myeloid leukemia, myeloproliferative neoplasm or aplastic anemia.
Frequently Asked Questions (FAQs)
A LAP / NAP score indicates the maturity and activity level of neutrophils, which are a type of white blood cell crucial for fighting infections. It is measured through a neutrophil alkaline phosphatase (NAP) stain, where the intensity of the stain corresponds to the enzyme activity within the neutrophils.
Here’s a breakdown of the interpretation:
- Higher LAP / NAP score (darker staining): Indicates more mature neutrophils with higher enzyme activity, usually signifying normal function.
- Lower LAP / NAP score (lighter staining): May suggest less mature neutrophils with lower enzyme activity, potentially linked to impaired function.
However, interpreting LAP / NAP scores is always done in the context of a patient’s overall clinical picture and other laboratory findings. It’s not a standalone diagnostic tool but adds valuable information when used in conjunction with other tests.
What does it mean if alkaline phosphatase is high?
Having a high alkaline phosphatase (ALP) level can be concerning, but it’s crucial to remember that it doesn’t automatically diagnose any specific condition. High ALP levels can point to various possibilities, some more serious than others. Here’s what you need to know:
Possible Causes
- Liver problems: This is the most common cause, including conditions like:
- Blocked bile ducts: Gallstones, tumors, or inflammation can hinder bile flow, leading to high ALP.
- Hepatitis: Viral infections like hepatitis B or C can damage liver cells, increasing ALP release.
- Cirrhosis: Scarring of liver tissue can also elevate ALP levels.
- Bone disorders: ALP is also produced in bones, so conditions like:
- Paget’s disease: This causes abnormal bone growth and remodeling, leading to high ALP.
- Fractures or bone tumors: These can temporarily increase ALP as bones heal or rebuild.
- Rapid bone growth in children and adolescents: This is natural and doesn’t usually require concern.
- Other possible causes: Less common contributors include:
- Certain medications: Some drugs can interfere with ALP levels.
- Pregnancy: Cholestasis of pregnancy can cause a temporary rise in ALP.
- Heart failure: In rare cases, heart problems can affect ALP levels.
Why is LAP / NAP score low in Paroxysmal Nocturnal Hemoglobinuria (PNH)?
In Paroxysmal Nocturnal Hemoglobinuria (PNH), a low Leukocyte Alkaline Phosphatase (LAP) score reflects a fundamental abnormality in the affected cells.
PNH and GPI-anchored proteins: PNH is caused by a genetic mutation that affects a specific type of molecule called glycosylphosphatidylinositol (GPI). GPI anchors various proteins to the cell membrane, including:
- Decay-accelerating factor (DAF): Crucial for protecting red blood cells from complement-mediated destruction.
- Neutrophil alkaline phosphatase (NAP): The enzyme measured in the LAP score.
Loss of GPI-anchored proteins: In PNH, the mutation disrupts the GPI anchor, causing these vital proteins to be deficient on affected blood cells, including red blood cells and neutrophils.
Consequences on LAP / NAP: Since LAP / NAP is a GPI-anchored protein, PNH patients have defective anchoring and reduced activity of the enzyme. This translates to a low LAP score, indicating less mature and potentially less functional neutrophils.
Limitations of LAP score: While a low LAP score is often seen in PNH, it is not specific to the disease. Other conditions like Chronic Myelogenous Leukemia (CML) can also have low scores. Additionally, LAP score is not routinely used for PNH diagnosis due to the availability of more definitive tests like flow cytometry.
Can infections affect the LAP / NAP score?
Yes, infections can definitely affect the LAP / NAP score, and it’s important to consider this during interpretation.
Impact of Infections on LAP / NAP Score
- Increased LAP / NAP Score
- Infections can trigger an increased production of white blood cells, including neutrophils. This increased production, often called a leukemoid reaction, can lead to higher NAP activity and consequently, a higher LAP / NAP score.
- Some specific infections, like viral hepatitis or Epstein-Barr virus, can also directly stimulate NAP activity in neutrophils, leading to an elevated LAP / NAP score.
- Decreased LAP / NAP Score
- In some cases, particularly severe infections, the body’s immune system may be overwhelmed, leading to immature neutrophils with lower NAP activity. This can result in a decreased LAP / NAP score.
- Additionally, certain medications used to treat infections, like some antibiotics, can affect NAP activity and potentially lower the LAP / NAP score.
Importance of Considering Infections
- When interpreting a LAP / NAP score, it’s crucial to factor in the presence of any recent or ongoing infections. Knowing the type of infection and its severity can help explain potential changes in the score.
- If you have an abnormal LAP / NAP score and your doctor suspects an infection, they may order additional tests to confirm the diagnosis and identify the specific cause.
Limitations of LAP Score in Infections
- It’s important to remember that LAP / NAP score is not a specific test for infections. Many other factors, besides infections, can influence the score.
- The type and severity of the infection can also impact how it affects the LAP / NAP score, making interpretation complex.
What factors can affect the LAP / NAP score?
The LAP / NAP score can be influenced by several factors. It’s crucial to consider these factors when interpreting the results and determining potential causes for abnormal scores.
1. Underlying Medical Conditions
- Myeloproliferative disorders: Conditions like chronic myeloid leukemia (CML) historically showed lower LAP / NAP scores due to abnormal neutrophil production. Although more specific tests like cytogenetic analysis are preferred for CML diagnosis nowadays, LAP / NAP score may still be considered in specific contexts.
- Bone marrow disorders: Conditions affecting bone marrow function, like Paget’s disease or bone fractures, can affect LAP / NAP score due to their impact on neutrophil production and maturation.
- Vitamin deficiencies: Deficiencies in vitamin B12 or folate can lead to lower LAP / NAP scores due to their importance in neutrophil development.
- Infections: Both acute and chronic infections can influence LAP / NAP score depending on the severity and type of infection. Some infections trigger higher production of immature neutrophils with lower NAP activity, while others stimulate NAP activity, leading to higher scores.
- Immune system disorders: Certain autoimmune diseases and immune deficiencies can affect neutrophil function and, consequently, LAP / NAP score.
2. Medications
- Corticosteroids: These drugs can stimulate neutrophil production and maturation, leading to higher LAP / NAP scores.
- Chemotherapy drugs: These drugs can damage bone marrow and suppress neutrophil production, resulting in lower LAP / NAP scores.
- Antibiotics: Some antibiotics can stimulate NAP activity, potentially raising the score, while others might affect neutrophil maturation and lower it.
- Methotrexate: This immunosuppressant drug can decrease NAP activity and lower the LAP / NAP score.
- Antithyroid medications: These medications can suppress neutrophil production and lower the LAP / NAP score.
3. Other Factors:
- Age: LAP / NAP score tends to be higher in children and adolescents due to increased bone growth and neutrophil turnover.
- Pregnancy: LAP / NAP score naturally increases during pregnancy due to hormonal changes and placental influences.
- Diet: Certain vitamin and mineral deficiencies can affect LAP / NAP score indirectly through their impact on neutrophil function.
What is the difference between serum alkaline phosphatase and LAP score?
Serum alkaline phosphatase (ALP) and LAP / NAP score are both related to the enzyme alkaline phosphatase (ALP), but they differ in several key aspects:
What they measure
- Serum ALP: Measures the total activity of ALP in the blood, regardless of the cell type it originates from. This includes ALP from the liver, bones, intestines, kidneys, and placenta.
- LAP / NAP score: Specifically measures the activity of neutrophil alkaline phosphatase (NAP), an isoenzyme of ALP found mainly in neutrophils, which are a type of white blood cell.
Purpose of the test
- Serum ALP: Used to assess various potential issues, including liver problems (most common), bone disorders, bile duct blockages, and some cancers.
- LAP / NAP score: Primarily used to:
- Investigate abnormal white blood cell counts (neutropenia or leukocytosis).
- Aid in differentiating certain blood disorders, especially historically for chronic myeloid leukemia (CML) – however, more specific tests like cytogenetic analysis are now preferred for CML diagnosis.
- Monitor treatment response in some cases involving neutrophil function.
Interpretation
- Serum ALP: Interpretation depends on the specific situation and other contributing factors. Increased levels can indicate various issues, while low levels are less common.
- LAP / NAP score: Higher scores generally suggest more mature neutrophils, while lower scores may indicate less mature or impaired neutrophils. However, interpretation needs to consider other factors like age, medications, and infections.
Limitations
- Serum ALP: Not specific to a single organ or condition, requiring further investigations for definitive diagnosis.
- LAP / NAP score: Cannot diagnose specific conditions definitively and should be interpreted alongside other clinical information and tests.
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.
