G6PD

Overview of G6PD Assay

Definition: A laboratory test used to measure the activity of the enzyme glucose-6-phosphate dehydrogenase (G6PD) in red blood cells (RBCs)
Clinical Significance:
- Diagnosis of G6PD Deficiency: G6PD deficiency is a common inherited enzyme deficiency that can cause hemolytic anemia
- Identifying at-Risk Individuals: Helps identify individuals who are at risk of developing hemolytic anemia after exposure to certain triggers (e.g., drugs, infections, foods)
- Genetic Counseling: Provides information for genetic counseling and family planning
Background:
- G6PD is an enzyme in the pentose phosphate pathway (also known as the hexose monophosphate shunt)
- G6PD catalyzes the first step in the pathway, which produces NADPH
- NADPH is essential for reducing glutathione, which in turn protects RBCs from oxidative damage
- G6PD deficiency impairs NADPH production, making RBCs vulnerable to oxidative stress, which leads to hemoglobin denaturation and hemolysis

Principle of G6PD Assay

G6PD assays are based on measuring the rate of NADPH production. The general principle is:

G6PD catalyzes the reaction of glucose-6-phosphate (G6P) to 6-phosphogluconate, producing NADPH
The rate of NADPH production is measured spectrophotometrically by monitoring the increase in absorbance at 340 nm (NADPH absorbs light at this wavelength)
The rate of NADPH production is directly proportional to the G6PD activity

Methods for Measuring G6PD Activity

Quantitative Spectrophotometric Assay (Gold Standard):
- Principle: Measures the rate of NADPH production by monitoring the change in absorbance at 340 nm over time
- Procedure:
  1. Prepare a Hemolysate: Lyse red blood cells to release G6PD
  2. Add Substrate and Coenzyme: Mix the hemolysate with a buffer containing glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP)
  3. Measure Absorbance: Monitor the absorbance at 340 nm over a fixed period (e.g., 5 minutes)
  4. Calculate G6PD Activity: Calculate the change in absorbance per minute (ΔA/min), which is proportional to the G6PD activity
  5. Normalize to Hemoglobin: Normalize the G6PD activity to the hemoglobin concentration in the sample to correct for variations in RBC number
    - G6PD Activity = (ΔA/min / Hemoglobin Concentration)
- Units:
  - Units per gram of hemoglobin (U/g Hb)
  - International units (IU) per liter
- Advantages:
  - Quantitative: Provides a precise measurement of G6PD activity
  - Most accurate method for diagnosing G6PD deficiency
- Limitations:
  - More complex and time-consuming than screening tests
  - Requires a spectrophotometer and trained personnel
Qualitative Fluorescent Spot Test (Screening Test):
- Principle: Detects NADPH production by observing the fluorescence of NADPH under UV light
- Procedure:
  1. Prepare a Blood Sample: Collect blood in an anticoagulant (e.g., EDTA or heparin)
  2. Incubate: Incubate a drop of blood on filter paper with G6PD substrate (glucose-6-phosphate) and NADP
  3. Observe Fluorescence: Examine the filter paper under UV light after a specific incubation period
- Interpretation:
  - Positive (Normal): The spot fluoresces brightly under UV light, indicating sufficient NADPH production and normal G6PD activity
  - Negative (Deficient): The spot does not fluoresce or fluoresces weakly, indicating impaired NADPH production and G6PD deficiency
- Advantages:
  - Simple and rapid
  - Inexpensive
  - Requires minimal equipment
- Limitations:
  - Qualitative: Only provides a yes/no result, does not quantify G6PD activity
  - Less sensitive than quantitative assays
  - False-negative results can occur in:
    - Females with G6PD deficiency due to X-chromosome inactivation (mosaicism)
    - Patients with recent hemolytic episodes (young red cells have higher G6PD activity)
  - False-positive results can occur due to:
    - Blood Transfusions: recent transfusion will skew the value
    - High Blood Sugar: hyperglycemia
Flow Cytometry:
- Principle: Uses fluorescent dyes to measure G6PD activity in individual RBCs
- Procedure:
  1. Label Blood Cells: Red blood cells are labeled with a fluorescent substrate that is converted to a fluorescent product by G6PD
  2. Flow Cytometry Analysis: The cells are analyzed by flow cytometry, and the fluorescence intensity of each cell is measured
  3. Quantification: G6PD activity is determined based on the fluorescence intensity
- Advantages:
  - More sensitive and quantitative than the fluorescent spot test
  - Can detect G6PD deficiency in heterozygous females due to its ability to measure G6PD activity in individual cells
- Limitations:
  - Requires a flow cytometer and trained personnel

Interpretation of Results

Normal G6PD Activity: Indicates that the individual has sufficient G6PD enzyme activity and is not at risk for hemolytic anemia
Decreased G6PD Activity: Indicates G6PD deficiency
Important Considerations:
- Testing Should Be Performed Remotely From Hemolytic Episode: G6PD activity may be falsely normal during or shortly after a hemolytic episode due to increased numbers of reticulocytes (young RBCs have higher G6PD activity)
- Repeat Testing: It is often recommended to repeat the G6PD assay several weeks or months after the hemolytic episode has resolved to obtain a more accurate result
- Clinical Correlation: G6PD activity should always be interpreted in conjunction with the patient’s clinical history, ethnicity, and family history

Factors Affecting Accuracy

Sample Storage: G6PD activity decreases over time, so testing should be performed as soon as possible after sample collection
Transfusions: Recent blood transfusions can affect the results, as the transfused RBCs may have normal G6PD activity
Hemolysis: Hemolyzed samples should not be used, as they can interfere with the assay
Medications: Certain medications can interfere with the G6PD assay
Reticulocytosis: During hemolytic episodes, the increased reticulocyte count can falsely elevate the G6PD activity
Assay Validation: Ensure that the G6PD assay has been properly validated for the patient population being tested

Quality Control

Run Controls with Known G6PD Activity Levels:
- Include normal and deficient controls with each assay run
- The control results should fall within established reference ranges
Check Reagents and Buffers:
- Use high-quality reagents and buffers
- Prepare reagents according to the manufacturer’s instructions
- Monitor the expiration dates of reagents
Follow Standardized Procedures:
- Adhere to standardized operating procedures for performing and interpreting the G6PD assay
Participate in Proficiency Testing Programs:
- To assess the accuracy and reliability of the laboratory’s G6PD testing

Reporting Results

Report the G6PD activity level in the appropriate units (e.g., U/g Hb)
Include the reference range for the assay
Note the method used (e.g., quantitative spectrophotometric assay, fluorescent spot test)
Provide an interpretation of the results, noting any abnormal findings
Include any comments or qualifications, such as “G6PD activity may be falsely normal during or shortly after a hemolytic episode; repeat testing is recommended”

Key Terms

Glucose-6-Phosphate Dehydrogenase (G6PD): An enzyme that protects red blood cells from oxidative damage
NADPH: A reducing agent that is essential for protecting cells from oxidative stress
Hemolytic Anemia: Anemia caused by increased destruction of red blood cells
Fluorescent Spot Test: A qualitative screening test for G6PD deficiency
Spectrophotometric Assay: A quantitative method for measuring G6PD activity
Heinz Bodies: Inclusions composed of denatured hemoglobin

ESR

Heinz Body