Glucose-6-Phosphate Dehydrogenase (G6PD) Fluorescent Spot Test 

Introduction

The G6PD fluorescent test is a rapid and reliable method for diagnosing G6PD deficiency. The test involves measuring the activity of G6PD in a sample of red blood cells. A low level of G6PD activity indicates a deficiency. 

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited red blood cell disorder that affects approximately 400 million people worldwide. G6PD is an enzyme that plays a crucial role in protecting red blood cells from damage caused by oxidative stress. When G6PD is deficient, red blood cells become more susceptible to hemolysis, a condition in which red blood cells rupture and release hemoglobin, the protein that carries oxygen. This will subsequently lead to anemia if the exposure to oxidative stress is prolonged.

G6PD deficiency is an X-linked recessive genetic disorder. This means that the gene responsible for G6PD production is located on the X chromosome, one of the two sex chromosomes (X and Y). Females have two X chromosomes, while males have one X chromosome and one Y chromosome. In order for a female to have G6PD deficiency, she must inherit a defective G6PD gene from both of her parents (carrier mother and affected father). Males, on the other hand, only need to inherit one defective G6PD gene from their mother to be affected by the disorder.

In most cases, G6PD deficiency does not cause any symptoms. However, when red blood cells are exposed to certain triggers, such as certain medications, infections, or fava beans, it can lead to a sudden episode of hemolysis called a hemolytic crisis. Symptoms of a hemolytic crisis can include:

• Pale skin
• Jaundice (yellowing of the skin and eyes)
• Fatigue
• Weakness
• Dark urine
• Shortness of breath
• Abdominal pain
• Fever

There is no cure for G6PD deficiency. However, there are treatments that can help to prevent and manage hemolytic crises. The most important treatment is to avoid triggers that can cause hemolysis. This includes avoiding certain medications, infections, and fava beans. In some cases, individuals with G6PD deficiency may need to receive blood transfusions to treat severe anemia.

Principle of G6PD fluorescent spot test

The G6PD fluorescent spot test is a rapid and reliable method for diagnosing glucose-6-phosphate dehydrogenase (G6PD) deficiency. The test is based on the principle that G6PD catalyzes the reduction of nicotinamide adenine dinucleotide phosphate (NADP+) to nicotinamide adenine dinucleotide phosphate hydrogen (NADPH). NADPH is a fluorescent molecule that can be visualized under ultraviolet (UV) light.

The chemical reaction that underlies the G6PD fluorescent test is as follows:

G6P + NADP+ + H2O → 6-phosphogluconate + NADPH + H+

The test procedure involves incubating a sample of blood with glucose-6-phosphate (G6P) and NADP+ in a substrate reagent. If G6PD is present, it will catalyze the reduction of NADP+ to NADPH. The amount of NADPH produced is directly proportional to the G6PD activity in the sample.

After incubation, the sample is spotted onto a filter paper and allowed to dry. The filter paper is then viewed under long-wave UV light. If G6PD activity is normal, the spot will fluoresce bright green, indicating the presence of NADPH. If G6PD activity is deficient, the spot will either have reduced fluorescence or no fluorescence at all.

Method differs slightly according to the manufacturer’s protocol. 

Materials

• 5 uL cord blood sample with sodium citrate anticoagulant (BLUE TOP). 
• Dilution buffer
• G6PD reagent 
• Distilled water
• QC vials: normal control, intermediate control, deficiency control
• Filter paper (“Guthrie Test Paper”, Schleicher & Schuell No. 2992) 
• Microcentrifuge tubes
• Long Wave UV box

Protocol

1. Prepare G6PD reagent stock substrate solution: Add 5 mL dilution into G6PD reagent freeze-dried powder vial. Leave for 30 minutes until completely dissolved and mixed properly. This reagent is stable for four weeks at +4°C and two months at -20°C. 
2. Prepare quality control (QC) solution: Add 50 uL distilled water into each QC freeze-dried powder vial. Leave for 30 minutes until completely dissolved and mixed properly.  
   • Normal Control
   • Intermediate Control
   • Deficiency Control

3. Label 4 microcentrifuge tube with the following, respectively:
   • Normal Control
   • Intermediate Control
   • Deficiency Control
   • Patient

4. Add 100 uL G6PD into each of the microcentrifuge tubes, respectively. 
5. Add 5 uL of QC material and patient sample into their respective labeled tube. 
6. Mix the solution properly. 
7. Allow to incubate for 10 minutes at room temperature. 
8. After 10 minutes, take 10 uL of the Normal Control mixture and place it on a labeled filter paper. 
9. Repeat with the rest of the tubes on their own labeled filter paper or space. 
10. Let the filter papers dry completely at room temperature. 
11. After the filter papers are completely dry (approximately 1 hour), view the results under a long-wave UV lamp in a darkened room.  
12. Compare the results of the patient sample to the QCs for reporting purposes.

Interpretation

The G6PD fluorescent spot test is a qualitative test, meaning that it provides a visual indication of G6PD activity rather than a quantitative measurement. The interpretation of the test results is based on the intensity of fluorescence observed under long-wave UV light.

• Normal G6PD activity: A sample with normal G6PD activity will exhibit bright green fluorescence, indicating the presence of a sufficient amount of NADPH.
• Intermediate G6PD activity: A sample with intermediate G6PD activity may show reduced fluorescence compared to a normal sample. The fluorescence may be dim or have a yellowish hue.
• Deficient G6PD activity: A sample with deficient G6PD activity will show no or very weak fluorescence, indicating a lack of NADPH production.

Troubleshooting

The accuracy of the G6PD fluorescent test can be affected by several factors, including:

• Sample preparation: Proper sample collection, storage, and handling are crucial to ensure accurate results.
• Reagent quality: The quality of the reagents used in the test can impact the sensitivity and specificity of the results.
• Incubation time: The incubation time of the blood sample with the substrate reagent should be controlled to ensure optimal NADPH production.
• UV light source: The intensity and wavelength of the UV light source can affect the visibility of fluorescence.
• In some G6PD deficiencies, newly formed red blood cells exhibit normal enzyme activity, thus patients who have recently experiences a hemolytic crisis must have their older RBCs to be isolated from the young ones for proper interpretation.
• If the patient has received a blood transfusion, this test should only be considered clinically relevant after 30 days have passed. This is because donor red blood cells typically exhibit normal G6PD activity, which can influence the test result before this time frame.

Frequently Asked Questions (FAQs)

What is the G6PD blood spot test and how does it work?

The G6PD fluorescent spot test, also known as FST, is a simple and quick way to screen for Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a common genetic condition affecting red blood cells. 

Principle

• G6PD is an enzyme in red blood cells that helps protect them from damage.
• The test relies on the natural fluorescence of a molecule called NADPH, which is produced by the G6PD enzyme.
• In individuals with normal G6PD activity, enough NADPH is produced, resulting in a bright green fluorescence under ultraviolet (UV) light.
• In individuals with G6PD deficiency, less or no NADPH is produced, leading to little or no fluorescence.

The methodology and interpretation of the test has been described in the protocol above. 

What is the normal fluorescence of G6PD?

In the G6PD fluorescent spot test, “normal fluorescence” refers to a bright green color observed under UV light when the test is performed on a sample with normal G6PD activity. This green fluorescence arises because the G6PD enzyme converts a specific molecule called glucose-6-phosphate (G6P) into another molecule called NADPH. NADPH has the natural property of fluorescing green when exposed to UV light.

Can the G6PD test be wrong?

Yes, the G6PD test can be wrong in a few different ways.

False-positives:

• Borderline cases: The test is qualitative, meaning it only indicates presence or absence of G6PD activity, not the exact level. Sometimes, individuals with mild G6PD deficiency might have an intermediate level of enzyme activity that falls near the threshold between normal and deficient. This can lead to a false-positive result.
• Interfering substances: In rare cases, substances in the blood, such as certain medications or vitamins, might interfere with the test and cause a false-positive result.
• Technical errors: Mistakes during sample collection, handling, or processing can also lead to inaccurate results.

False-negatives:

• Timing of the test: If the test is done during or shortly after an episode of hemolytic anemia (where red blood cells are destroyed), most of the affected cells might already be gone. This can lead to a false-negative result, as the remaining cells might have normal G6PD activity.
• Severity of deficiency: Individuals with very mild G6PD deficiency might have enough enzyme activity to pass the test, even though they have the condition.

Here are some additional factors to consider:

• Different test methods: There are several types of G6PD tests available, each with its own limitations. Some are more specific and accurate than others.
• Experience of the healthcare professional: Interpreting the test results requires expertise and experience. A healthcare professional with a good understanding of G6PD deficiency is better equipped to interpret borderline results and consider other factors that might influence the accuracy.

What is a methemoglobin reduction test for G6PD?

The methemoglobin reduction test, also known as the methemoglobin dye test or glutathione stability test, is an older method used to screen for glucose-6-phosphate dehydrogenase (G6PD) deficiency. It is a qualitative test, meaning it only indicates the presence or absence of G6PD activity, not the exact level.

How the test works:

1. Red blood cells are treated with sodium nitrite: This chemical converts hemoglobin in the red blood cells to methemoglobin, which is a form of hemoglobin that cannot carry oxygen.
2. A dye and a substrate are added: The dye, such as methylene blue, can only be reduced (activated) by NADPH, a molecule produced by the G6PD enzyme. The substrate, such as glucose, provides the necessary energy for the reaction.
3. The mixture is incubated: This allows time for the G6PD enzyme to convert G6P to NADPH, which can then reduce the methemoglobin back to hemoglobin.
4. The color of the mixture is observed:
   • Normal G6PD activity: The dye is reduced, and the mixture turns blue.
   • G6PD deficiency: The dye is not reduced, and the mixture remains brown or greenish-brown.

Limitations of the methemoglobin reduction test:

• Not very sensitive or specific: It can miss mild cases of G6PD deficiency and give false-positive results in some individuals.
• Labor-intensive and time-consuming: Requires specialized equipment and trained personnel.
• Not considered a reliable diagnostic tool: Other, more accurate tests are now available for diagnosing G6PD deficiency.

Current recommendations:

The methemoglobin reduction test is no longer widely used for G6PD deficiency screening due to its limitations. The quantitative fluorescent spot test (FST) is now the preferred screening method. It is more accurate, faster, and easier to perform.

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. Beutler E. Drug-induced hemolytic anemia and non-spherocytic hemolytic anemia. In Glucose-6-Phosphate Dehydrogenase (Yoshida A. and Beutler E., Eds) pp. 3-12, 1986. Academic, Orlando.
2. Beutler E. A series of new screening procedures for pyruvate kinase deficiency, glucose – 6 – phosphate dehydrogenase deficiency and glutathione reductase deficiency. Blood 1966;28:553-562.
3. Dow PA, Petteway MB, Alperin JB. Simplified method for G6PD screening using blood collected on filter paper. Am J Pathol 1974;61:333-336.
4. Luzzatto, L., Mehta, A. and Vulliamy, T. (2001) Glucose 6-Phosphate Dehydrogenase Deficiency. In: Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D., Eds., The Metabolic and Molecular Bases of Inherited Disease, 8th Edition, McGraw-Hill, New York, 4517-4533.
5.  Herz F, Kaplan E, Scheye ES. Diagnosis of erythrocyte glucose-6-phosphate dehydrogenase deficiency in the negro male despite hemolytic crisis. Blood 1970;35:953-954.