Abstract G6PD deficiency affects approximately 500 million people globally, necessitating enzymatic screening before antimalarial treatment to prevent drug-induced hemolysis. Despite the expansion of quantitative point-of-care testing (POCT), standardized lyophilized controls are still limited. This study aims to develop these materials to ensure diagnostic accuracy and support EQA program. Control materials were prepared from G6PD-normal and deficient packed red blood cells (PRBCs). PRBCs were washed and mixed with 5% (w/v) sucrose at the optimized 1:5, 1:10, 1:15 and 1:20 ratios. G6PD activity and hemoglobin (Hb) levels were quantified using the STANDARD™ G6PD test. Stability was evaluated at 4 and -20°C through 6 months.  The 1:20 formulation demonstrated optimal reconstitution and clear phenotype differentiation. At 4°C, G6PD activity of G6PD normal control materials remained stable for 1 month followed by a fluctuation below the normal threshold at month 2 (3.9 U/g Hb) while at -20°C, it maintained stability for up to 6 months, despite a gradual decline to 4.0 U/g Hb. Whereas the G6PD deficient control materials exhibited remarkable stability at both temperatures. In addition, Hb levels appeared relatively stable across all tested conditions throughout the 6-month period. In conclusion, lyophilized G6PD control materials with 5% sucrose solution at a 1:20 ratio are suitable for quantitative POCT. For long-term stability, -20°C storage is recommended. This material provides a robust foundation for developing EQA programs compliant with ISO/IEC 17043:2023, ensuring reliable G6PD quantitative Point-of-Care testing.

Keywords: Lyophilization, Control materials, G6PD activity, Point-of-Care testing, Stability

Funding: This work was supported by a part of the research grant from the Faculty of Associated Medical Sciences, Chiang Mai University, Thailand.

Citation:  Mongkham, W., Chaisonying, T., Cheammanee, N., Pongpunyayuen, P., Ruengdit, C., Punyamung, M., and Pornprasert, S. 2027. Development of lyophilized control material for G6PD quantitative Point-of-Care testing. Natural and Life Sciences Communications. 26(1): e2027010.

Graphical Abstract:

INTRODUCTION

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway (PPP), responsible for producing nicotinamide adenine dinucleotide phosphate (NADPH). This co-enzyme plays a pivotal role in maintaining the cellular redox balance and protecting cells against oxidative stress (Mohiuddin, 2023). In mature erythrocytes, G6PD activity is of paramount importance because these cells lack a nucleus and protein-synthesizing machinery to replenish enzyme levels during their lifespan. Consequently, the PPP serves as the sole source of NADPH in red blood cells. Given their primary function in oxygen transport, erythrocytes are continuously exposed to reactive oxygen species (ROS). A deficiency in NADPH leads to oxidative damage to the cellular membrane, ultimately resulting in hemolysis (Luzzatto et al., 2016).

G6PD deficiency is an X-linked inherited disorder, leading to distinct phenotypic expressions between sexes. While hemizygous males and homozygous females typically manifest clear enzymatic deficiency, heterozygous females exhibit a broad spectrum of G6PD activity. This clinical variability is attributed to lyonization (X-inactivation), which creates a mosaic population of both G6PD-normal and G6PD-deficient erythrocytes within the same individual (Luzzatto et al., 2020). Globally, G6PD deficiency affects approximately 500 million people. In Thailand, the prevalence ranges from 3% to 18%, varying by ethnicity and geographic region (Luzzatto et al., 2020; Nuinoon et al., 2022). In contrast, the African continent reports a higher average prevalence of approximately 24%. Notably, the global distribution of G6PD deficiency strongly correlates with regions where malaria is endemic, as certain genotypes have been shown to offer a selective survival advantage by reducing the severity of malarial infection (Amoah et al., 2021). In 1985, the World Health Organization (WHO) established a classification system for G6PD variants based on clinical manifestations, residual enzymatic activity, and molecular characteristics (World Health Organization, 2022). Under this framework, Class I represents the most severe phenotype, characterized by less than 10% residual activity and the presence of chronic non-spherocytic hemolytic anemia (CNSHA). Class II variants also exhibit severe deficiency (<10% activity). Class III is defined by moderate-to-mild deficiency (10-60% activity), while Class IV and Class V represent very mild-to-no deficiency (60-150% activity) and increased enzymatic activity (>200% activity), respectively. Conversely, Classes II and III exhibit overlapping enzymatic activities and clinical features, including susceptibility to drug-induced hemolysis, favism, and neonatal hyperbilirubinemia. Notably, variants such as G6PD Mediterranean and G6PD A- often show overlapping activity levels. To better align genetic expression with clinical severity and patient management, the classification was recently revised to consolidate Classes II and III into Group B (Watchko and Bhutani, 2025). While most adults with G6PD deficiency remain asymptomatic unless triggered by exposure to oxidative agents, which lead to acute hemolytic anemia, jaundice, and hemoglobinuria. In neonates, G6PD deficiency significantly increases the risk of neonatal hyperbilirubinemia, which may lead to severe neurological complications if left untreated (Cappellini and Fiorelli, 2008). Consequently, the WHO recommends G6PD screening before the administration of antimalarial drugs, particularly for Plasmodium vivax infections (the most common malaria parasite species in Thailand) (Shimizu et al., 2020), to eliminate dormant liver-stage parasites (hypnozoites) and prevent malaria relapse (WHO guidelines for malaria, 2025; World Health Organization, 2024). Reliable G6PD testing is therefore indispensable to mitigate the risk of drug-induced hemolysis in these patients.

Various methodologies are available for G6PD deficiency test, with spectrophotometric method as the gold standard by measuring NADPH absorbance at 340 nm. However, due to resources and budgetary constraints, the fluorescent spot test (FST) remains a widely used qualitative screening method. While cost-effective, FST is limited by a detection threshold of approximately 30-40% enzymatic activity, often failing to accurately identify heterozygous females whose activity levels typically fall within the 30-80% range (Alam et al., 2018a; Domingo et al., 2019; Neamyanon et al., 2026). To address these limitations, Point-of-Care testing (POCT) has gained significant traction, especially in remote and malaria-endemic regions, by providing rapid, decentralized diagnostics. The STANDARD™ G6PD test (SD Biosensor, South Korea) is currently a WHO-prequalified portable device that offers rapid quantitative results, making it ideal for primary healthcare settings (World Health Organization, 2025). The clinical demand for such quantitative accuracy has increased following WHO recommendations for G6PD quantification prior to tafenoquine administration, requiring an activity threshold of ≥70% for safe usage (WHO guidelines for malaria, 2025).

Despite the rising global and local implementation of quantitative POCT, robust quality control (QC) infrastructure remains insufficient. Data from the External Quality Assessment (EQA) unit at Chiang Mai University (CMU) indicates a growing demand for proficiency testing programs specifically tailored for G6PD POCT users. While previous successes have been achieved in developing lyophilized QC materials for qualitative FST (Pienthai et al., 2019; Pornprasert et al., 2022), these legacy formulations are incompatible with the advanced optical and biosensor-based technologies of POCT devices. Currently, there is a critical lack of standardized, lyophilized quantitative QC materials for G6PD POCT. Consequently, this study aims to develop lyophilized control material specifically optimized for G6PD quantitative POCT, ensuring diagnostic accuracy and supporting the expansion of EQA programs in accordance with international standards. Our preliminary findings indicate that while the lyophilization process led to a predictable reduction in enzymatic activity, the optimized 5% sucrose formulation successfully preserved sufficient G6PD activity to distinguish between G6PD normal and deficiency phenotypes for up to six months at -20°C, although minor fluctuations in activity were observed during the storage period. Importantly, the developed materials maintained clear clinical differentiation between normal and deficient groups, demonstrating their potential utility as a stable substrate for point-of-care quality assurance despite the anticipated activity loss.

MATERIALS AND METHODS

Ethical approval

Ethic procedures of the study were reviewed and approved by Ethics Committee of the Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand (approval number AMSEC-68EX-088). All participants received written and verbal explanations regarding study objectives and procedures before enrollment. Written informed consent was obtained from each participant. To eliminate phenotypic variability related to X-linked mosaicism, only adult male subjects were included. This approach ensured definitive classification of G6PD status based on enzymatic activity. Participants were screened for infectious markers, including anti-HIV antibody, hepatitis B surface antigen (HBsAg), and anti-hepatitis C virus (anti-HCV) antibody,
to ensure biosafety during preparation of control materials.

Blood sample collection and G6PD activity analysis by standard method

Peripheral venous blood samples (50 mL per participant) of G6PD deficiency and non-deficiency donors were collected into EDTA tubes under aseptic conditions. Samples were processed within 4 hours of collection to minimize enzymatic degradation. The G6PD activity was measured using a gold standard method, a quantitative spectrophotometric assay (GPD0204, Mindray, Shenzhen, China) performed on an automated UV enzymatic analyzer (BS-360E, Mindray). The assay quantifies G6PD activity by measuring the rate of increase in absorbance at 340 nm, corresponding to the reduction of NADP⁺ to NADPH during the enzymatic conversion of glucose-6-phosphate to 6-phosphogluconate. The G6PD activity was normalized to hemoglobin (Hb) and expressed as units per gram of Hb (U/g Hb). Therefore, samples with activity <3.8 U/g Hb were classified as G6PD deficient, while values ≥3.8 U/g Hb were considered normal according to manufacturer specifications.

Preparation of lyophilized G6PD control materials

Control materials were prepared from blood samples of donors with G6PD activity normal (19.97 U/g Hb) and deficient (1.31 U/g Hb) samples measured by the gold standard method. The process of preparation was previously described (Pienthai et al., 2019). Briefly, EDTA whole blood was centrifuged at 1,000 × g for 10 minutes at 4°C to separate plasma and buffy coat. Plasma and leukocytes were removed, and packed red cells were washed three times with 0.85% (w/v) isotonic saline to eliminate residual plasma proteins and cellular debris. Washed packed RBCs were mixed with 5% (w/v) sucrose solution at an optimized ratio (1:20, 1:15, 1:10, and 1:5) to serve as a cryoprotectant. The mixture was frozen at -20°C for 24 hours to induce hemolysis. Following thawing, 5 mL aliquots were dispensed into sterile 10 mL amber glass vials. Samples were lyophilized under controlled vacuum conditions until complete dehydration was achieved. The lyophilized materials were sealed and stored under defined temperature conditions pending stability evaluation.

Quantification of G6PD activity by Point-of-Care testing (POCT)

The POCT (STANDARD™ G6PD test; SD Biosensor, Suwon, Republic of Korea) was used for quantitative measurement of G6PD activity before lyophilization and at each stability time point. This device provides semi-quantitative enzymatic activity results normalized to total hemoglobin (U/g Hb). The assay is based on a colorimetric enzymatic reaction in which G6PD catalyzes the oxidation of glucose-6-phosphate with concomitant reduction of NADP⁺ to NADPH. The generated NADPH reduces nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) via a diaphorase-mediated reaction, producing a violet chromogen proportional to enzymatic activity. Moreover, a total Hb is measured in a separate reaction zone on the test strip. Testing was performed according to the manufacturer’s instructions. Briefly, a calibration chip specific to the test lot was inserted into the STANDARD™ G6PD analyzer. Ten microliters of control materials before lyophilization or reconstituted control materials were mixed with extraction buffer, and 10 µL of the mixture were dispensed onto the sample pad. After closing the chamber flap, quantitative results were automatically displayed within 2 minutes. In accordance with the manufacturer’s classification criteria, G6PD enzymatic activity was interpreted based on the following thresholds: deficient (≤4.0 U/g Hb) and normal (>4.0 U/g Hb). The analytical performance of the device encompassed a measuring range of 4-25 g/dL for total Hb and 0-20 U/g Hb for G6PD activity.

Characteristics and short-term stability of lyophilized control materials

To evaluate the characteristics and G6PD activity of lyophilized G6PD control materials, the lyophilized control materials were reconstituted with 300 and 500 µL of distilled water, followed by gentle swirling and incubation at room temperature for 10 minutes. The solubility and homogeneity were visually inspected before determining the Hb concentration and G6PD activity by STANDARD™ G6PD analyzer. In addition, a stability study was conducted by storing the vials at room temperature, 4°C, and -20°C for a 4-week period. Each week, one vial from each storage condition was randomly selected to monitor the total Hb and G6PD activity by this POCT analyzer. Acceptable stability involved normal G6PD control materials to show G6PD activity >4.0 U/g Hb throughout the study period.

Long-term stability of lyophilized control materials

Based on the preliminary short-term stability results, the formulation demonstrated optimal solubility, minimal sample volume requirement, and superior enzymatic stability was selected for long-term evaluation using a six-month stability model. At each predefined monthly interval, two vials were randomly selected from each storage condition and tested in duplicate to ensure analytical precision. Each vial was reconstituted with 300 µL of distilled water and incubated at room temperature for 10 minutes to achieve complete dissolution. Analytical stability was defined as the maintenance of total Hb and G6PD enzymatic activity for 6 months. Acceptable stability involved normal G6PD control materials to remain >4.0 U/g Hb G6PD activity throughout the study period.

RESULTS

Characteristics and G6PD activity of lyophilized control materials after reconstitution

The physical characteristics of the lyophilized control materials were evaluated at various ratios (1:20, 1:15, 1:10, and 1:5). All formulations demonstrated well-reconstitution within 10 minutes when using 500 µL of distilled water. However, the 1:5 ratio showed incomplete dissolution with 300 µL of diluent, thus the ratio was not tested. The Hb concentrations of the control materials before lyophilization were 14.5 and 14.0 g/dL for samples with G6PD normal and deficiency, respectively (Table 1). Following lyophilization and reconstitution with 300 µL of distilled water, Hb recovery varied across different ratios. As shown in Table 1, the 1:20 ratio yielded measured Hb levels of 12.3 and 10.0 g/dL for samples with G6PD normal and deficiency, respectively. In contrast, the 1:15 ratio showed improved recovery with values of 16.0 and 13.4 g/dL, for samples with G6PD normal and deficiency, respectively. However, samples prepared at 1:10 ratio resulted in elevated Hb levels (>20 g/dL) for both G6PD normal and deficiency. The G6DP activity was also analyzed by the STANDARD™ G6PD analyzer. The results showed that enzymatic activities before lyophilization were 9.8 and 1.1 U/g Hb in control materials of G6PD normal and deficient samples, respectively. The decreased activities in both groups of samples were observed after reconstitution. In addition, the proportional decrease in both Hb concentration and G6PD activity across all tested reconstitution volumes (300 and 500 µL) and ratios (1:20, 1:15, and 1:10), as summarized in Table 1. Based on these findings, the 1:20 and 1:15 ratios were selected for a preliminary 4-week stability study.

Table 1. G6PD activity determined by Point-of-Care testing of each ratio between packed red cells and 5% sucrose solution.

Short-term (4 weeks) stability of lyophilized control materials

The results revealed that both 1:20 and 1:15 ratios effectively maintained total Hb and G6PD activity throughout the 4-week period across all tested storage temperatures: room temperature, 4°C, and -20°C (Table 2). Since no differences were observed between the two ratios, the 1:20 ratio was further prioritized for the 6-month long-term stability evaluation.

Table 2. Stability of G6PD activity and hemoglobin concentration of lyophilized control materials at different formulation ratios and storage temperatures over 4 weeks.

Long-term (6 months) stability of lyophilized control material

The new lot of lyophilized control materials were prepared for analyzing long-term stability which monitored over a 6-month period (Supplementary Table 1). At -20°C, the G6PD normal control materials exhibited a baseline (Day 0) G6PD activity of 4.8 ± 0.26 U/g Hb and declined to 4.0 ± 0.41 U/g Hb, reaching the manufacturer's deficiency threshold (≤ 4.0 U/g Hb) at month 6 (Figure 1). In contrast, the G6PD deficient control materials stored at -20°C remained stable throughout the study, maintaining a G6PD activity of 0.9 ± 0.05 U/g Hb at baseline (Day 0) to 0.9 ± 0.05 U/g Hb at month 6 (Figure 1). Similar trends were observed at 4°C; the G6PD normal control materials (baseline 4.4 ± 0.34 U/g Hb) remained stable for one month before fluctuation below the normal threshold at month 2 (3.9 ± 0.33 U/g Hb), followed by a return to 4.3 ± 0.50 U/g Hb at month 6 (Figure 1). Whereas the G6PD deficient control materials stored at 4°C remained stable G6PD activity at values of 0.8 U/g Hb through 6 months (Figure 1). Notably, hemoglobin concentrations remained remarkably consistent across all conditions throughout the study. At -20°C, Hb levels ranged from 11.1 to 13.9 g/dL for G6PD normal control materials and 11.5 to 13.4 g/dL for G6PD deficient control materials (Figure 2). Similarly, at 4°C, hemoglobin values varied between 11.5 and 13.9 g/dL in the G6PD normal control materials, and 11.8 and 14.3 g/dL in the G6PD deficient control materials (Figure 2). Moreover, regression analysis was performed and results showed that storage time had no significant effect on G6PD activity during the period of the study in both 4°C and -20°C (Supplementary Figures 1 and 2, respectively).

Figure 1. Stability of G6PD activity stored at -20 and 4°C for 6 months. The values are expressed as mean and error bars of the standard deviation (SD) calculated from two independent vials of control materials, each vial was measured in duplicate (n=4). The horizontal reference line at 4.0 U/g Hb is an acceptable stability involved normal G6PD control materials.

Figure 2. Stability of hemoglobin concentration stored at -20 and 4 °C over 6 months. The values are expressed as mean and error bars of the standard deviation (SD) calculated from two independent vials of control materials, each vial was measured in duplicate (n=4).

DISCUSSION

The optimization of lyophilized control materials focused on rapid reconstitution, complete solubility, distinct differentiation between G6PD-normal and G6PD-deficient phenotypes, and stability throughout the study period. Initial assessments of various ratios revealed that the 1:5 ratio, when reconstituted with 300 µL of distilled water, exhibited incomplete dissolution and excessive viscosity. Although reconstitution with 500 µL improved solubility, a 300 µL volume was ultimately selected as the optimal condition to maximize Hb concentration, ensuring the control material is closely required for analyzer performance and using a minimized sample to be prepared. The observed reduction in Hb concentration following lyophilization, beyond the expected dilution from the 5% sucrose solution, may be attributed to the formation of nanobubbles during the reconstitution phase. As described in previous literature (Snell et al., 2020), nanobubbles possess a high specific surface area that facilitates the adsorption and accumulation of Hb proteins at the gas-liquid interface. This phenomenon likely induces structural alterations in the Hb molecules, reducing the fraction of proteins in a soluble monomeric state and potentially interfering with the spectrophotometric transmittance (%T) used for Hb measurement. Besides, nanobubbles may cause volumetric inaccuracy during sample aspiration by displacing the effective liquid volume, collectively resulting in lower measured Hb concentrations.

Furthermore, the freeze-drying process and prolonged exposure to -20°C are well-documented factors that can accelerate protein degradation (Jiang and Nail, 1998; Lu et al., 2025). To mitigate these effects, the incorporation of a preservative or cryoprotectant is essential. Previous research has demonstrated that sugar-based cryoprotectants are effective in maintaining cellular and protein integrity (Jung et al., 2025). In this study, the 5% sucrose solution provided protection for protein. Although minor protein loss is inherent to the lyophilization, the preserved levels were potentially suitable for clinical diagnostic purposes. These findings suggest that while sucrose functions as a stabilizer, further optimization may be required to fully offset the degradative effects of the freeze-drying process.

While preliminary short-term (4-week) stability results indicated that the lyophilized control materials maintained highly comparable activities across all storage temperatures, including room temperature, long-term preservation at this condition remains limited. According to previous studies, G6PD activity is relatively stable at room temperature, but it is not always consistent and can decrease by approximately 5% in 24 hours (Alam et al., 2018b). Consequently, the 1:20 ratio was prioritized for the 6-month evaluation of long-term stability at 4°C and -20°C. This selection was based on its superior reconstitution properties, cost-effectiveness due to the lower sample volume required for preparation, and its ability to provide clear differentiation between G6PD-normal and G6PD-deficient phenotypes. Notably, the enzymatic activity of the 1:20 ratio remained distinct from the manufacturer's cut-off values throughout the study period, ensuring reliable diagnostic interpretation.

Regarding long-term stability at 4°C, the observed decline into the deficient range during Month 2, followed by a return to normal levels in subsequent months, suggests analytical variability rather than true enzymatic degradation. On the other hand, at -20°C, although the control material showed stability, the mean activity gradually declined to the borderline of the manufacturer's deficiency threshold (4.0 U/g Hb) by Month 6. These fluctuations may be attributed to inconsistencies in the manual reconstitution process or potential homogeneity issues within the lyophilized material. In addition, the lack of internal quality control (IQC) makes the difference between systematic and random analysis errors.

The value of 19.97 U/g Hb represents the G6PD activity of the original donor blood measured by the spectrophotometric reference method, whereas the value of 9.8 U/g Hb was obtained from the prepared control material using the POCT device. Thus, these values were obtained using different analytical methods and from different sample preparations, and they should not be considered directly interchangeable. The observed difference may be attributable to differences in assay principles, calibration, sample matrix, and other analytical characteristics of the two methods. Because a direct method-comparison study was not performed, the relative contribution of these factors cannot be determined from the present data. Moreover, the further decrease in measured activity from 9.8 U/g Hb before lyophilization to approximately 5 U/g Hb after lyophilization may reflect changes in measurable enzyme activity associated with the lyophilization and reconstitution processes.

This study had some limitations. In this study, we used POCT test kits as they are widely available in several facilities from the on-field services to the bedside in the hospital. However, according to the classification criteria of the STANDARD™ G6PD POCT, the measured G6PD activity of the lyophilized material designated as the “normal control” did not fall within the device-defined normal range (>6.0 U/g Hb) of female individuals. Although the material was initially prepared from samples with normal G6PD activity, a reduction in enzyme activity was observed following
the lyophilization process, resulting in activity levels predominantly within the intermediate range of female (4.1-6.0 U/g Hb) of female individuals. Thus, we selected male donors to avoid the variability of phenotypes, male donors were chosen to ensure the consistency of the G6PD activity. Heterozygous females could show normal G6PD activity which depend on variable factors such as genotypes, X-inactivation (Au et al., 2006), number of reticulocytes, etc. Furthermore, G6PD genotypes were not performed as male only explicit 2 possible phenotypes, such variations of G6PD were not observed.

Since the control material prepared from only one donor, the number of prepared vials is limited. Additionally, the high cost of the POCT test kits barred a large-scale statistical analysis following ISO 13528:2022 (statistical methods for proficiency testing providers) guidelines. For future studies, evaluating the impact of transportation effects is essential to simulate logistics. To further improve these control materials, future research should consider recruiting donors with higher baseline G6PD activity. This would provide a wider margin from the manufacturers cut-off, potentially extending the shelf-life and clinical utility of G6PD control materials. Optimizing preservative and cryoprotectant concentrations is essential to enhance long-term stability and to suppress microbial growth, particularly since sucrose may act as a nutrient source in the lyophilized matrix. Most importantly, ensuring stringent homogeneity across all vials during the aliquoting and lyophilization processes remains critical to minimize variability. In addition, future studies implement a pilot EQA program to evaluate impact of transportation on the stability of control materials across network laboratories.

CONCLUSION

This study is the first to develop control materials specifically for quantitative G6PD Point-of-Care testing (POCT) and it demonstrates that a lyophilized G6PD control material, formulated from packed red cells with 5% sucrose solution at a 1:20 ratio provides a viable foundation for G6PD quantitative POCT. The prepared control material effectively differentiates between G6PD normal and deficient phenotypes using the STANDARD™ G6PD test. Stability profiles indicate that storage at 4°C maintains enzymatic integrity for up to one month, though subsequent fluctuations suggest analytical variables. On the other hand, storage at -20°C provides a more stable environment, despite G6PD normal control materials, with a gradual decline in G6PD activity reaching the borderline of the manufacturer's deficiency threshold by month 6. These observations underscore the critical influence of manual reconstitution and the necessity for rigorous calibration to ensure measurement reliability. Collectively, these findings provide a robust scientific foundation for the development of quality control materials in alignment with ISO/IEC 17043:2023 standards. Furthermore, this work supports the strategic design of External Quality Assessment (EQA) programs for G6PD quantitative POCT, thereby ensuring diagnostic reliability in clinical settings.

ACKNOWLEDGEMENTS

The authors thank the technicians at the Associated Medical Sciences Clinical Service Center, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand for their assistance.

AUTHOR CONTRIBUTIONS

Wachiraporn Mongkham: Conceptualization (Lead), Methodology (Lead), Formal Analysis (Lead), Writing – Original Draft (Lead), Project Administration (Lead); Thanatip Chaisonying: Methodology (Equal), Formal Analysis (Equal), Writing – Review & Editing (Equal); Nathaphon Cheammanee: Methodology (Equal), Formal Analysis (Equal), Writing – Review & Editing (Equal); Panida Pongpunyayuen: Data Curation (Equal), Formal Analysis (Equal), Writing – Review & Editing (Equal), Project Administration (Supporting); Chedtapak Ruengdit: Data Curation (Equal), Formal Analysis (Equal), Writing – Review & Editing (Equal), Project Administration (Supporting); Manoo Punyamung: Data Curation (Equal), Formal Analysis (Equal), Writing – Review & Editing (Equal), Project Administration (Supporting); Sakorn Pornprasert: Conceptualization (Lead), Methodology (Lead), Formal Analysis (Lead), Writing – Original Draft (Lead), Project Administration (Lead), Supervision (Lead).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

REFERENCES

Alam, M.S., Kibria, M.G., Jahan, N., Price, R.N., and Ley, B. 2018a. Spectrophotometry assays to determine G6PD activity from Trinity Biotech and Pointe Scientific G6PD show good correlation. BMC Research Notes. 11(1): 855. https://doi.org/10.1186/s13104-018-3964-7

Alam, M.S., Kibria, M.G., Jahan, N., Thriemer, K., Hossain, M.S., Douglas, N.M., Phru, C.S., Khan, W.A., Price, R.N., and Ley, B. 2018b. Field evaluation of quantitative point of care diagnostics to measure glucose-6-phosphate dehydrogenase activity. PLoS One. 13(11): e0206331. https://doi.org/10.1371/journal.pone.0206331

Amoah, L.E., Asare, K.K., Dickson, D., Abankwa, J., Busayo, A., Bredu, D., Annan, S., Asumah, G.A., Peprah, N.Y., Asamoah, A., et al. 2021. Genotypic glucose-6-phosphate dehydrogenase (G6PD) deficiency protects against Plasmodium falciparum infection in individuals living in Ghana. PLoS One. 16(9): e0257562. https://doi.org/10.1371/journal.pone.0257562

Au, W.Y., Lam, V., Pang, A., Lee, W.M., Chan, J.L., Song, Y.Q., Ma, E.S., and Kwong, Y.L. 2006. Glucose-6-phosphate dehydrogenase deficiency in female octogenarians, nanogenarians, and centenarians. The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences. 61(10): 1086–1089. https://doi.org/10.1093/gerona/61.10.1086

Cappellini, M.D. and Fiorelli, G. 2008. Glucose-6-phosphate dehydrogenase deficiency. Lancet. 371(9606): 64–74. https://doi.org/10.1016/s0140-6736(08)60073-2

Domingo, G.J., Advani, N., Satyagraha, A.W., Sibley, C.H., Rowley, E., Kalnoky, M., Cohen, J., Parker, M., and Kelley, M. 2019. Addressing the gender-knowledge gap in glucose-6-phosphate dehydrogenase deficiency: Challenges and opportunities. International Health. 11(1): 7–14. https://doi.org/10.1093/inthealth/ihy060

Jiang, S. and Nail, S.L. 1998. Effect of process conditions on recovery of protein activity after freezing and freeze-drying. European Journal of Pharmaceutics and Biopharmaceutics. 45(3): 249–257. https://doi.org/10.1016/s0939-6411(98)00007-1

Jung, S.E., Moon, Y., Lim, M., Jeong, H., and Seo, H. 2025. Glucose-enhanced cryopreservation of hCAR-T cells: Improved recovery and reduced apoptosis. Biomolecules & Therapeutics (Seoul). 33(6): 963–974. https://doi.org/10.4062/biomolther.2025.144

Lu, X., Domingo-Yenes, B., Cohen, N., and Grzincic, E. 2025. Freezing-induced protein aggregation in a bispecific antibody: Characterization and mechanistic insights. Journal of Pharmaceutical Sciences. 114(5): 103711. https://doi.org/10.1016/j.xphs.2025.103711

Luzzatto, L., Ally, M., and Notaro, R. 2020. Glucose-6-phosphate dehydrogenase deficiency. Blood. 136(11): 1225–1240. https://doi.org/10.1182/blood.2019000944

Luzzatto, L., Nannelli, C., and Notaro, R. 2016. Glucose-6-phosphate dehydrogenase deficiency. Hematology/Oncology Clinics of North America. 30(2): 373–393. https://doi.org/10.1016/j.hoc.2015.11.006

Mohiuddin, H.A.S.S. 2023. Biochemistry, Hexose monophosphate pathway. StatPearls.

Neamyanon, J., Saejeng, A., Wongwian, P., Kiti, S., Nanjai, S., Jaiyasen, T., and Pornprasert, S. 2026. Performance comparisons of three rapid screening methods for the G6PD deficiency test in newborns. Journal of Associated Medical Sciences. 59(1): 47–53. https://doi.org/10.12982/jams.2026.007

Nuinoon, M., Krithong, R., Pramtong, S., Sasuk, P., Ngeaiad, C., Chaimusik, S., Kanboonma, J., and Sarakul, O. 2022. Prevalence of G6PD deficiency and G6PD variants amongst the southern Thai population. PeerJ. 10: e14208. https://doi.org/10.7717/peerj.14208

Pienthai, N., Pongpunyayuen, P., Ruengdit, C., and Pornprasert, S. 2019. Development of suitable external quality control material for G6PD deficiency screening with the fluorescent spot test. Clinical Chemistry and Laboratory Medicine. 58(1): e18–e20. https://doi.org/10.1515/cclm-2019-0421

Pornprasert, S., Pongpunyayuen, P., Chairatanapiwong, S., Lawapakull, P., Kulawong, P., Jaiyasen, T., Khamphikham, P., and Intasai, N. 2022. Efficiency of lyophilized hemolysate control material in proficiency testing program for G6PD deficiency screening in Thailand. International Journal of Laboratory Hematology. 44(4): e160–e163. https://doi.org/10.1111/ijlh.13816

Shimizu, S., Chotirat, S., Dokkulab, N., Hongchad, I., Khowsroy, K., Kiattibutr, K., Maneechai, N., Manopwisedjaroen, K., Petchvijit, P., Phumchuea, K., et al. 2020. Malaria cross-sectional surveys identified asymptomatic infections of Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi in Surat Thani, a southern province of Thailand. International Journal of Infectious Diseases. 96: 445–451. https://doi.org/10.1016/j.ijid.2020.05.022

Snell, J.R., Kumar, N.S.K., Suryanarayanan, R., and Randolph, T.W. 2020. Nanobubbles in reconstituted lyophilized formulations: Interaction with proteins and mechanism of formation. Journal of Pharmaceutical Sciences. 109(1): 284–292. https://doi.org/10.1016/j.xphs.2019.05.005

Watchko, J.F. and Bhutani, V.K. 2025. Revised World Health Organization (WHO) classification of G6PD gene variants: Relevance to neonatal hyperbilirubinemia. Seminars in Fetal and Neonatal Medicine. 30(1): 101619. https://doi.org/10.1016/j.siny.2025.101619

World Health Organization. 2025. WHO Guidelines for Malaria. Geneva: World Health Organization; 2025. https://www.who.int/teams/global-malaria-programme/report/world-malaria-report-2025. Accessed March 20, 2026.

World Health Organization. 2022. Meeting report of the technical consultation to review the classification of glucose-6-phosphate dehydrogenase (G6PD) (Vol. 2026). https://cdn.who.int/media/docs/default-source/malaria/mpac-documentation/mpag-mar2022-session2-technical-consultation-g6pd-classification.pdf?sfvrsn=1f36be5e_12&download=true. Accessed March 20, 2026.

World Health Organization. 2024. World malaria report 2024: Addressing inequity in the global malaria response. https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2024. Accessed March 20, 2026.

World Health Organization. 2025. Public report for STANDARD G6PD test (PQDx 0581 117-00). https://extranet.who.int/prequal/WHOPR/public-report-standard-g6pd-test pqdx-0581-117-00. Accessed March 20, 2026.

OPEN access freely available online

Natural and Life Sciences Communications

Chiang Mai University, Thailand. https://cmuj.cmu.ac.th

Supplementary

Figure S1. Regression analysis of normal G6PD activity stored at 4°C at each time point.

Figure S2. Regression analysis of normal G6PD activity stored at -20°C at each time point.

Table S1. G6PD activity and Hb concentration measured by STANDARDTM G6PD POCT kit at each time point stored at 4 and -20°C.