Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human genetic enzyme disorder globally, affecting the survival of red blood cells (RBCs). This condition involves a shortfall of the G6PD enzyme, which protects RBCs from oxidative damage. Without adequate G6PD, exposure to certain infections, medications, or foods can trigger the premature destruction of red blood cells, leading to hemolytic anemia. Testing identifies at-risk individuals and prevents these potentially harmful hemolytic episodes. Understanding the methods used for diagnosis is the first step in managing this inherited condition.
Screening and Diagnostic Testing
Testing for G6PD deficiency occurs in two main clinical contexts: large-scale screening and targeted diagnostic evaluation. Newborn screening is an important public health measure, often mandatory in high-prevalence populations, and is typically performed shortly after birth using a small blood sample. This screening identifies infants at risk before exposure to hemolytic triggers, allowing for early preventive measures. Targeted diagnostic testing is performed later in life when a patient presents with symptoms of unexplained acute hemolytic anemia, such as jaundice, dark urine, and fatigue. It is also necessary before prescribing certain medications, like primaquine, which are known to trigger hemolysis in deficient individuals.
Assessing G6PD Enzyme Activity
The most common initial method for identifying G6PD deficiency is the Fluorescent Spot Test (FST), a qualitative screening method. This rapid, inexpensive test relies on the enzyme’s function to produce Nicotinamide Adenine Dinucleotide Phosphate (NADPH). When the blood sample is incubated with specific reagents, a normal sample will fluoresce brightly under ultraviolet light due to NADPH production, while a deficient sample will show little to no fluorescence.
The definitive diagnosis and classification of severity require the quantitative Spectrophotometric Assay, considered the gold standard. This laboratory test measures the precise amount of G6PD enzyme activity in red blood cells. It monitors the rate of NADPH formation over time, which is directly proportional to the enzyme’s activity, using a spectrophotometer to track the change in light absorption. This quantitative measurement allows clinicians to classify the deficiency as mild, moderate, or severe. Genetic testing, which identifies the specific mutation, is generally reserved for cases where enzyme assays are inconclusive.
Factors Influencing Test Accuracy
Accurate interpretation of G6PD enzyme activity tests is complicated by the age of the red blood cells (RBCs) in the sample. RBCs naturally lose G6PD activity as they age, meaning that older cells are more deficient than younger cells. This is particularly relevant during an active hemolytic crisis, which causes the rapid destruction of the oldest, most enzyme-deficient RBCs. In response to the rapid loss of cells, the body releases a surge of young RBCs, called reticulocytes, which contain normal or near-normal levels of G6PD activity.
Testing a patient during this period can lead to a falsely normal or “false negative” result because the remaining circulating cells are the younger, less-deficient ones. This temporary masking effect can dangerously delay a correct diagnosis and management plan. Similarly, a recent blood transfusion using healthy donor blood will temporarily elevate the total G6PD activity in the patient’s circulation. The transfused healthy cells dilute the patient’s deficient cells, also causing a false negative result that does not reflect the patient’s true baseline status. To avoid these misleading results, enzyme activity testing should be deferred for two to three months after a hemolytic episode or a blood transfusion.