June 2024 – Daniel Hesse

Disclaimer: This article is intended for informational purposes only and is not written by a medical professional. It has not been reviewed by a medical professional and may include inaccuracies. No analytical values or clinical diagnoses should be based on the information provided in this article. National recommendations and clinical practices may vary, and readers should consult appropriate healthcare professionals for specific medical advice.

    ¤ Summary

Diagnosing acute pancreatitis remains challenging due to high error rates in standard clinical panels. This complexity is heightened by macroamylasemia which is characterized by the presence of elevated levels of macroamylase in the blood and can lead to falsely elevated amylase results unless specifically ruled out. This article discusses the general challenges in diagnosing pancreatitis and specifically highlights how the gel permeation chromatography (GPC) method using Blue Dextran 2000 (BD2000) can be used to rule out pancreatitis by accurately measuring macroamylase levels.

    ¤ Background

Acute pancreatitis is characterized by a sudden onset of epigastric pain radiating to the back, often accompanied by vomiting and elevated serum amylase levels. However, similar amylase elevations can occur in other acute abdominal conditions, which complicates diagnosis. Accurate diagnosis within the first 48 hours is crucial, especially when serum amylase levels exceed three times the normal value. Normal amylase levels may also be observed if there’s a delay in seeking medical care, necessitating serum lipase testing.

Amylase Analysis in Serum and Urine

Amylase is an enzyme that digests carbohydrates and is produced primarily in the pancreas and salivary glands. Elevated levels of amylase in the blood can indicate pancreatic damage or other health issues. Measuring amylase in serum and urine is a common practice in diagnosing pancreatic conditions, including acute pancreatitis.

Reference Intervals for Amylase:

• Serum Amylase (S-Amylas): 0.46-1.48 µkat/L
• Serum Pancreatic Amylase (S-Pankreasamylas): 0.31-0.79 µkat/L
• Urine Amylase (U-Amylas): 0.52-9.6 µkat/L

There are no differences between genders in these reference intervals.

Formation of Macroamylase

Macroamylase, one of the most common types of macroenzymes, is a high molecular weight complex composed of amylase enzymes linked to molecules such as plasma proteins and immunoglobulins. These complexes are too large to be filtered by the kidneys, so they remain in the bloodstream, leading to elevated serum amylase levels without the corresponding clinical symptoms of pancreatitis. Elevated levels of macroamylase are generally benign and seldom indicate serious health issues. The reason behind the formation of macroamylase is not entirely clear, but it can occur in both healthy individuals and in those with a variety of diseases, including liver disease, diabetes, cancer, malabsorption, and autoimmune disorders [1]. Therefore, it is crucial to differentiate between macroamylase and elevated levels of amylase due to acute pancreatitis to avoid misdiagnosis and unnecessary treatments.

Diagnostic Challenges and Current Options

Standard diagnostic tools include clinical assessment, serum lipase determination, ultrasound, and computed tomography (CT). However, conventional methods like abdominal X-rays and ultrasound often fall short during acute attacks due to interfering bowel gas and paralytic ileus. CT is generally reserved for severe cases or when the diagnosis is uncertain. Magnetic resonance imaging (MRI) is emerging but not yet standardized.

In some regions, such as Sweden, a common practice involves running an enzymatic photometric urine amylase analysis when serum amylase levels are elevated. This method offers a longer detection window since amylase remains elevated in urine longer than in serum. However, urine amylase testing is less specific and may still result in false positives, thus requiring confirmation through other means. Additionally, urine amylase analysis is not typically used to rule out macroamylase because macroamylase complexes are too large to be excreted in urine. Specific tests like GPC with Blue dextran 2000 are needed to accurately differentiate macroamylase from other forms of amylase.

The Gel Permeation Chromatography Method Using Blue dextran 2000

The GPC method using BD2000 offers a robust solution for macroamylase analysis. By utilizing gel chromatography, this method effectively differentiates macroamylase from other forms of amylase. This specificity prevents misdiagnosis and ensures accurate clinical decisions. Studies highlight the efficacy of BD2000 in purifying and characterizing large molecules like macroamylase.

    ¤ Gel Filtration Method for Analyzing Macroamylase

Principle Behind the Use of BD2000

BD2000 is used to create a standard curve for identifying and quantifying macroamylase in serum samples. Due to its high molecular weight, BD2000 mimics the large complexes formed by macroamylase, making it an ideal marker for distinguishing these from smaller molecular forms of amylase.

Procedure

1. Preparation of Gel Filtration Column:
   • A column is packed with a suitable gel filtration medium, such as Sephadex G-100.
   • The column is equilibrated with a buffer solution suitable for amylase activity, typically a phosphate buffer at pH 7.0.
2. Sample Preparation:
   • Serum samples to be analyzed for macroamylase are mixed with BD2000 as a standard.
   • The sample and standard solution are applied to the top of the gel filtration column.
3. Separation and Elution:
   • Samples are eluted through the column, and fractions are collected.
   • BD2000 is used as a reference to identify the elution volume for large molecules, including macroamylase.
4. Detection and Quantification:
   • The fractions are analyzed for amylase activity using a standard amylase activity test, such as an enzymatic colorimetric method.
   • The elution profile is compared with the standard curve from BD2000 to identify the fractions containing macroamylase.

Advantages of the GPC Method Using BD2000

• Specificity: Accurately identifies macroamylase, preventing false positives.
• Reliability: Consistent results across various samples and conditions.
• Cost-Effectiveness: Reduces unnecessary treatments and shortens evaluation times, saving healthcare costs.

Comparative Analysis of Urine Amylase and the GPC Method Using BD2000

• Specificity: Urine amylase can be elevated due to various conditions, not just pancreatitis, leading to potential false positives. The GPC method using BD2000 provides high specificity by accurately differentiating macroamylase from other forms of amylase.
• Timing: Urine amylase requires collection over a period, potentially delaying diagnosis. The GPC method using BD2000 offers quicker and more precise results through specific gel chromatography.
• Diagnostic Accuracy: Urine amylase is less reliable as a stand-alone test and best used in conjunction with other tests. The GPC method using BD2000 enhances diagnostic accuracy, improving clinical decision-making.
• Practicality and User-Friendliness: Urine amylase may require multiple collections and can be inconvenient for patients. The GPC method using BD2000 provides a streamlined process, easily integrated into existing lab workflows, reducing patient discomfort.

    ¤ Blue dextran 2000 from TdB Labs

Why the Quality of BD2000 Matters

Quality is critical for accurate macroamylase determination. TdB Labs’ BD2000 is designed for optimal performance in GPC, with key release parameters such as molecular weight and a highly controlled degree of substitution.

• Molecular Weight: Consistency in molecular weight ensures reliable separation of macroamylase from other forms of amylase.
• Degree of Substitution: The degree of substitution affects the binding and elution properties of BD2000, ensuring accurate results.

TdB Labs’ BD2000 meets these stringent criteria, making it fit for purpose in clinical diagnostics. Additionally, our products are produced and quality-controlled according to ISO9001 standards, using raw materials from ISO9001-approved suppliers.

    ¤ Summary

Implementing the GPC method using BD2000 in clinical labs enhances diagnostic precision, minimizes unnecessary treatments, and improves patient outcomes. This method’s adoption can significantly optimize healthcare resources and patient management in acute pancreatitis cases.

Key Points Summary

• Diagnosing acute pancreatitis remains challenging due to high error rates in standard clinical panels.
• Macroamylasemia can cause falsely elevated amylase readings, potentially leading to a misdiagnosis of pancreatitis unless it is specifically identified and accounted for.
• Macroamylase is formed when amylase binds to large molecules such as polysaccharides or immunoglobulins.
• Elevated levels of macroamylase are generally benign and seldom indicate serious health issues.
• The GPC method using BD 2000 can be used to rule out pancreatitis by accurately measuring macroamylase levels.
• Conventional diagnostic tools include clinical assessment, serum lipase determination, ultrasound, and CT.
• Enzymatic photometric urine amylase analysis is less specific and may result in false positives; it is not typically used to rule out macroamylase.
• The GPC method using BD2000 offers high specificity, reliability, and cost-effectiveness.
• Quality of BD2000 from TdB Labs is ensured by strict production under quality-control standards according to ISO9001.
• Reference intervals for amylase and pancreatic amylase levels are provided.

    ¤ References

[1]  M. CUTOLO et al., “MACROAMYLASEMIA: A POSSIBLE CAUSE OF UNEXPLAINED HYPERAMYLASEMIA IN RHEUMATOID ARTHRITIS,” Rheumatology, vol. 34, no. 3, pp. 290–292, 1995, doi: 10.1093/rheumatology/34.3.290.