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Cut-off values for diagnosis of G6PD deficiency by flow cytometry in Thai population
Anchalee Thedsawad1, Wanchai Wanachiwanawin1, Orathai Taka1
1Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Wanglang Road, Bangkoknoi, Bangkok, 10700, Thailand.
Insights
Flow cytometry offers a reliable method for screening glucose-6-phosphate dehydrogenase (G6PD) deficiency. This technique accurately identifies G6PD deficiency in males and distinguishes between heterozygous and homozygous females.
Area of Science:
- Hematology
- Clinical Diagnostics
- Genetics
Background:
- Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked genetic disorder.
- X-inactivation in heterozygous females complicates G6PD deficiency screening.
- Existing G6PD screening tests are less reliable for diagnosing heterozygous females.
Purpose of the Study:
- To establish flow cytometric cut-off values for G6PD deficiency screening.
- To accurately diagnose G6PD deficiency in hemizygous males and heterozygous/homozygous females.
- To compare flow cytometry with genetic analysis for G6PD deficiency diagnosis.
Main Methods:
- Utilized 205 leftover blood samples for quantitative methemoglobin reduction (MR) screening.
- Employed multiplex amplification refractory mutation system-polymerase chain reaction and direct DNA sequencing as the gold standard.
- Analyzed test accuracy, including sensitivity, specificity, and predictive values, using MedCalc software.
Main Results:
- Established optimal flow cytometry cut-off values for classifying females (homozygous normal, heterozygous, homozygous deficient) and males (hemizygous normal, hemizygous deficient).
- Achieved high sensitivity and specificity in both female and male G6PD deficiency screening.
- Flow cytometry successfully differentiated intermediate phenotype heterozygous females but not extreme phenotype ones from homozygous females.
Conclusions:
- Flow cytometry provides a reliable and efficient method for screening G6PD deficiency.
- The established cut-off values enable accurate identification of G6PD deficiency in hemizygous males and heterozygous/homozygous females.
- This method enhances diagnostic capabilities for G6PD deficiency, particularly in females.
Abstract:
In heterozygous females, X-inactivation causes a change in glucose-6-phosphate dehydrogenase (G6PD) activity from normal to deficient. Most G6PD screening tests are used to accurately diagnose hemizygous males, but they are less reliable for diagnosing heterozygous females. This study established flow cytometric cut-off values for screening of G6PD deficiency in hemizygous males and heterozygous or homozygous females. We studied 205 (125 females, 80 males) leftover blood samples from quantitative methemoglobin reduction (MR) screening. G6PD gene mutations determined by multiplex amplification refractory mutation system-polymerase chain reaction and direct DNA sequencing were used as the gold standard reference. Accuracy of the test, including the sensitivity, specificity, and positive and negative predictive values, was analyzed using MedCalc software. The optimal cut-off values for classification of %red blood cells with normal G6PD activity or %bright cells into homozygous normal, heterozygous, and homozygous deficiency in females were 85.4-100%, 6.3-85.3%, and 0-6.2%, respectively (sensitivity 93.2%, specificity 100%). The cut-offs for classification into hemizygous normal and hemizygous deficiency in males were 76.5-100% and 0-76.4%, respectively (sensitivity 100%, specificity 96.5%). Flow cytometry can be used to differentiate heterozygous females with intermediate phenotype from homozygous females, but cannot distinguish between heterozygous females with extreme phenotype and homozygous females. By flow cytometry, heterozygous and homozygous deficiency was detected in 29.6% and 3.2% of females, respectively. Among males, hemizygous deficiency was found in 31.3%. Flow cytometry can be used to screen patients with G6PD deficiency, and reliably and efficiently identify heterozygous and homozygous females, and hemizygous males based on cellular G6PD activity.
