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Background: The introduction of novel short course treatment regimens for the radical cure of Plasmodium vivax requires reliable point-of-care diagnosis that can identify glucose-6-phosphate dehydrogenase (G6PD) deficient individuals. While deficient males can be identified using a qualitative diagnostic test, the genetic make-up of females requires a quantitative measurement. SD Biosensor (Republic of Korea) has developed a handheld quantitative G6PD diagnostic (STANDARD G6PD test), that has approximately 90% accuracy in field studies for identifying individuals with intermediate or severe deficiency. The device can only be considered for routine care if precision of the assay is high., Methods and Findings: Commercial lyophilised controls (ACS Analytics, USA) with high, intermediate, and low G6PD activities were assessed 20 times on 10 Biosensor devices and compared to spectrophotometry (Pointe Scientific, USA). Each device was then dispatched to one of 10 different laboratories with a standard set of the controls. Each control was tested 40 times at each laboratory by a single user and compared to spectrophotometry results. When tested at one site, the mean coefficient of variation (CV) was 0.111, 0.172 and 0.260 for high, intermediate, and low controls across all devices respectively; combined G6PD Biosensor readings correlated well with spectrophotometry (rs = 0.859, p<0.001). When tested in different laboratories, correlation was lower (rs = 0.604, p<0.001) and G6PD activity determined by Biosensor for the low and intermediate controls overlapped. The use of lyophilised human blood samples rather than fresh blood may have affected these findings. Biosensor G6PD readings between sites did not differ significantly (p = 0.436), whereas spectrophotometry readings differed markedly between sites (p<0.001)., Conclusions: Repeatability and inter-laboratory reproducibility of the Biosensor were good; though the device did not reliably discriminate between intermediate and low G6PD activities of the lyophilized specimens. Clinical studies are now required to assess the devices performance in practice., Competing Interests: The authors have declared that no competing interests exist.

Certain clinical indications and treatments such as the use of rasburicase in cancer therapy and 8-aminoquinolines for Plasmodium vivax malaria treatment would benefit from a point-of-care test for glucose-6-phosphate dehydrogenase (G6PD) deficiency. Three studies were conducted to evaluate the performance of one such test: the STANDARD™ G6PD Test (SD BIOSENSOR, South Korea). First, biological interference on the test performance was evaluated in specimens with common blood disorders, including high white blood cell (WBC) counts. Second, the test precision on fingerstick specimens was evaluated against five individuals of each, deficient, intermediate, and normal G6PD activity status. Third, clinical performance of the test was evaluated at three point-of-care settings in the United States. The test performed equivalently to the reference assay in specimens with common blood disorders. High WBC count blood samples resulted in overestimation of G6PD activity in both the reference assay and the STANDARD G6PD Test. The STANDARD G6PD Test showed good precision on multiple fingerstick specimens from the same individual. The same G6PD threshold values (U/g Hb) were applied for a semiquantitative interpretation for fingerstick- and venous-derived results. The sensitivity/specificity values (95% confidence intervals) for the test for G6PD deficiency were 100 (92.3-100.0)/97 (95.2-98.2) and 100 (95.7-100.0)/97.4 (95.7-98.5) for venous and capillary specimens, respectively. The same values for females with intermediate (> 30% to ≤ 70%) G6PD activity were 94.1 (71.3-99.9)/88.2 (83.9-91.7) and 82.4 (56.6-96.2)/87.6(83.3-91.2) for venous and capillary specimens, respectively. The STANDARD G6PD Test enables point-of-care testing for G6PD deficiency., Competing Interests: The authors have declared that no competing interests exist.

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common enzyme deficiency, prevalent in many malaria-endemic countries. G6PD-deficient individuals are susceptible to hemolysis during oxidative stress, which can occur from exposure to certain medications, including 8-aminoquinolines used to treat Plasmodium vivax malaria. Accordingly, access to point-of-care (POC) G6PD testing in Brazil is critical for safe treatment of P. vivax malaria., Methodology/principal Findings: This study evaluated the performance of the semi-quantitative, POC STANDARD G6PD Test (SD Biosensor, Republic of Korea). Participants were recruited at clinics and through an enriched sample in Manaus and Porto Velho, Brazil. G6PD and hemoglobin measurements were obtained from capillary samples at the POC using the STANDARD and HemoCue 201+ (HemoCue AB, Sweden) tests. A thick blood slide was prepared for malaria microscopy. At the laboratories, the STANDARD and HemoCue tests were repeated on venous samples and a quantitative spectrophotometric G6PD reference assay was performed (Pointe Scientific, Canton, MI). G6PD was also assessed by fluorescent spot test. In Manaus, a complete blood count was performed. Samples were analyzed from 1,736 participants. In comparison to spectrophotometry, the STANDARD G6PD Test performed equivalently in determining G6PD status in venous and capillary specimens under varied operating temperatures. Using the manufacturer-recommended reference value thresholds, the test's sensitivity at the <30% threshold on both specimen types was 100% (95% confidence interval [CI] venous 93.6%-100.0%; capillary 93.8%-100.0%). Specificity was 98.6% on venous specimens (95% CI 97.9%-99.1%) and 97.8% on capillary (95% CI 97.0%-98.5%). At the 70% threshold, the test's sensitivity was 96.9% on venous specimens (95% CI 83.8%-99.9%) and 94.3% on capillary (95% CI 80.8%-99.3%). Specificity was 96.5% (95% CI 95.0%-97.6%) and 92.3% (95% CI 90.3%-94.0%) on venous and capillary specimens, respectively., Conclusion/significance: The STANDARD G6PD Test is a promising tool to aid in POC detection of G6PD deficiency in Brazil., Trial Registration: This study was registered with ClinicalTrials.gov (identifier: NCT04033640)., Competing Interests: The authors have declared that no competing interests exist.

Background: Point-of-care glucose-6-phosphate dehydrogenase (G6PD) testing has the potential to make the use of radical treatment for vivax malaria safer and more effective. Widespread use of G6PD tests as part of malaria case management has been limited, in part due to due concerns regarding product usability, user training, and supervision. This study seeks to assess how well end users can understand the Standard™ G6PD Test (SD Biosensor, Suwon, South Korea) workflow, result output, and label after training. This will ultimately help inform test registration and introduction., Methods: Potential G6PD test users who provide malaria case management at three sites in Brazil, Ethiopia, and India were trained on the use of the SD Biosensor Standard G6PD Test and assessed based on their ability to understand the test workflow and interpret results. The assessment was done through a questionnaire, designed to assess product usability against key technical product specifications and fulfill regulatory evidence requirements. Any participant who obtained 85% or above correct responses to the questionnaire was considered to adequately comprehend how to use and interpret the test., Results: Forty-five participants, including malaria microscopists, laboratory staff, nurses, and community health workers took part in the study. Seventy-eight percent of all participants in the study (35/45) obtained passing scores on the assessment with minimal training. Responses to the multiple-choice questions indicate that most participants understood well the test intended use, safety claims, and warnings. The greatest source of error regarding the test was around the correct operating temperature. Most test results were also read and interpreted correctly, with the haemoglobin measurement being a more problematic output to interpret than the G6PD measurement., Conclusions: These data results show how a standardized tool can be used to assess a user's ability to run a point-of-care diagnostic and interpret results. When applied to the SD Biosensor Standard G6PD Test, this tool demonstrates that a range of users across multiple contexts can use the test and suggests improvements to the test instructions and training that can improve product usability, increase user comprehension, and ultimately contribute to more widespread effective use of point-of-care G6PD tests., Trial Registration: NCT04033640.

Background: The radical cure of Plasmodium vivax and P. ovale requires treatment with primaquine or tafenoquine to clear dormant liver stages. Either drug can induce haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, necessitating screening. The reference diagnostic method for G6PD activity is ultraviolet (UV) spectrophotometry; however, a universal G6PD activity threshold above which these drugs can be safely administered is not yet defined. Our study aimed to quantify assay-based variation in G6PD spectrophotometry and to explore the diagnostic implications of applying a universal threshold., Methods and Findings: Individual-level data were pooled from studies that used G6PD spectrophotometry. Studies were identified via PubMed search (25 April 2018) and unpublished contributions from contacted authors (PROSPERO: CRD42019121414). Studies were excluded if they assessed only individuals with known haematological conditions, were family studies, or had insufficient details. Studies of malaria patients were included but analysed separately. Included studies were assessed for risk of bias using an adapted form of the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool. Repeatability and intra- and interlaboratory variability in G6PD activity measurements were compared between studies and pooled across the dataset. A universal threshold for G6PD deficiency was derived, and its diagnostic performance was compared to site-specific thresholds. Study participants (n = 15,811) were aged between 0 and 86 years, and 44.4% (7,083) were women. Median (range) activity of G6PD normal (G6PDn) control samples was 10.0 U/g Hb (6.3-14.0) for the Trinity assay and 8.3 U/g Hb (6.8-15.6) for the Randox assay. G6PD activity distributions varied significantly between studies. For the 13 studies that used the Trinity assay, the adjusted male median (AMM; a standardised metric of 100% G6PD activity) varied from 5.7 to 12.6 U/g Hb (p < 0.001). Assay precision varied between laboratories, as assessed by variance in control measurements (from 0.1 to 1.5 U/g Hb; p < 0.001) and study-wise mean coefficient of variation (CV) of replicate measures (from 1.6% to 14.9%; p < 0.001). A universal threshold of 100% G6PD activity was defined as 9.4 U/g Hb, yielding diagnostic thresholds of 6.6 U/g Hb (70% activity) and 2.8 U/g Hb (30% activity). These thresholds diagnosed individuals with less than 30% G6PD activity with study-wise sensitivity from 89% (95% CI: 81%-94%) to 100% (95% CI: 96%-100%) and specificity from 96% (95% CI: 89%-99%) to 100% (100%-100%). However, when considering intermediate deficiency (<70% G6PD activity), sensitivity fell to a minimum of 64% (95% CI: 52%-75%) and specificity to 35% (95% CI: 24%-46%). Our ability to identify underlying factors associated with study-level heterogeneity was limited by the lack of availability of covariate data and diverse study contexts and methodologies., Conclusions: Our findings indicate that there is substantial variation in G6PD measurements by spectrophotometry between sites. This is likely due to variability in laboratory methods, with possible contribution of unmeasured population factors. While an assay-specific, universal quantitative threshold offers robust diagnosis at the 30% level, inter-study variability impedes performance of universal thresholds at the 70% level. Caution is advised in comparing findings based on absolute G6PD activity measurements across studies. Novel handheld quantitative G6PD diagnostics may allow greater standardisation in the future., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: LvS receives a stipend as a Specialty Consulting Editor for PLOS Medicine and serves on the journal's editorial board.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a common genetic blood condition, can result in kernicterus at birth, and later in life as severe hemolysis on exposure to certain infections, foods, and drugs. The unavailability of point-of-care tests for G6PD deficiency is a barrier to routine curative treatment of Plasmodium vivax malaria with 8-aminoquinolines, such as primaquine. Two quantitative reference tests (Trinity Biotech, Bray, Ireland and Pointe Scientific, Canton, MI; Cat No. G7583) and the point-of-care STANDARD ™ G6PD test (SD Biosensor, Suwon, South Korea) were evaluated. The STANDARD G6PD test was evaluated at multiple temperatures, in anticoagulated venous and capillary samples, including 79 G6PD-deficient and 66 intermediate samples and across two laboratories, one in the United States and one in Thailand. The STANDARD test performed equivalently to a reference assay for its ability to diagnose G6PD deficiency (< 30% normal) with a sensitivity of 100% (0.95 confidence interval [CI]: 95.7-100) and specificity of 97% (0.95 CI: 94.5-98.5), and could reliably identify females with less than 70% normal G6PD activity with a sensitivity of 95.5% (0.95 CI: 89.7-98.5) and specificity of 97% (0.95 CI: 94.5-98.6). The STANDARD G6PD product represents an opportunity to diagnose G6PD deficiency equally for males and females in basic clinical laboratories in high- and low-resource settings. This quantitative point-of-care diagnostic test for G6PD deficiency can provide equal access to safe radical cure of P. vivax cases in high- and low-resource settings, for males and females and may support malaria elimination, in countries where P. vivax is endemic.

Introduction: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in the human population affecting an estimated 8% of the world population, especially those living in areas of past and present malaria endemicity. Decreased G6PD enzymatic activity is associated with drug-induced hemolysis and increased risk of severe neonatal hyperbilirubinemia leading to brain damage. The G6PD gene is on the X chromosome therefore mutations cause enzymatic deficiency in hemizygote males and homozygote females while the majority of heterozygous females have an intermediate activity (between 30-80% of normal) with a large distribution into the range of deficiency and normality. Current G6PD qualitative tests are unable to diagnose G6PD intermediate activities which could hinder wide use of 8-aminoquinolines for Plasmodium vivax elimination. The aim of the study was to assess the diagnostic performances of the new Carestart G6PD quantitative biosensor., Methods: A total of 150 samples of venous blood with G6PD deficient, intermediate and normal phenotypes were collected among healthy volunteers living along the north-western Thailand-Myanmar border. Samples were analyzed by complete blood count, by gold standard spectrophotometric assay using Trinity kits and by the latest model of Carestart G6PD biosensor which analyzes both G6PD and hemoglobin., Results: Bland-Altman comparison of the CareStart normalized G6PD values to that of the gold standard assay showed a strong bias in values resulting in poor area under-the-curve values for both 30% and 80% thresholds. Performing a receiver operator curve identified threshold values for the CareStart product equivalent to the 30% and 80% gold standard values with good sensitivity and specificity values, 100% and 92% (for 30% G6PD activity) and 92% and 94% (for 80% activity) respectively., Conclusion: The Carestart G6PD biosensor represents a significant improvement for quantitative diagnosis of G6PD deficiency over previous versions. Further improvements and validation studies are required to assess its utility for informing radical cure decisions in malaria endemic settings.

Background: Medicines that exert oxidative pressure on red blood cells (RBC) can cause severe hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Due to X-chromosome inactivation, females heterozygous for G6PD with 1 allele encoding a G6PD-deficient protein and the other a normal protein produce 2 RBC populations each expressing exclusively 1 allele. The G6PD mosaic is not captured with routine G6PD tests., Methods: An open-source software tool for G6PD cytofluorometric data interpretation is described. The tool interprets data in terms of % bright RBC, or cells with normal G6PD activity in specimens collected from 2 geographically and ethnically distinct populations, an African American cohort (USA) and a Karen and Burman ethnic cohort (Thailand) comprising 242 specimens including 89 heterozygous females., Results: The tool allowed comparison of data across 2 laboratories and both populations. Hemizygous normal or deficient males and homozygous normal or deficient females cluster at narrow % bright cells with mean values of 96%, or 6% (males) and 97%, or 2% (females), respectively. Heterozygous females show a distribution of 10-85% bright cells and a mean of 50%. The distributions are associated with the severity of the G6PD mutation., Conclusions: Consistent cytofluorometric G6PD analysis facilitates interlaboratory comparison of cellular G6PD profiles and contributes to understanding primaquine-associated hemolytic risk., (© 2017 The Authors. European Journal of Haematology Published by John Wiley & Sons Ltd.)

Individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency are at risk of severe haemolysis following the administration of 8-aminoquinoline compounds. Primaquine is the only widely available 8-aminoquinoline for the radical cure of Plasmodium vivax. Tafenoquine is under development with the potential to simplify treatment regimens, but point-of-care (PoC) tests will be needed to provide quantitative measurement of G6PD activity prior to its administration. There is currently a lack of appropriate G6PD PoC tests, but a number of new tests are in development and are likely to enter the market in the coming years. As these are implemented, they will need to be validated in field studies. This article outlines the technical details for the field evaluation of novel quantitative G6PD diagnostics such as sample handling, reference testing and statistical analysis. Field evaluation is based on the comparison of paired samples, including one sample tested by the new assay at point of care and one sample tested by the gold-standard reference method, UV spectrophotometry in an established laboratory. Samples can be collected as capillary or venous blood; the existing literature suggests that potential differences in capillary or venous blood are unlikely to affect results substantially. The collection and storage of samples is critical to ensure preservation of enzyme activity, it is recommended that samples are stored at 4 °C and testing occurs within 4 days of collection. Test results can be visually presented as scatter plot, Bland-Altman plot, and a histogram of the G6PD activity distribution of the study population. Calculating the adjusted male median allows categorizing results according to G6PD activity to calculate standard performance indicators and to perform receiver operating characteristic (ROC) analysis.

Glucose-6-phosphate dehydrogenase (G6PD) activity is essential for redox equilibrium of red blood cells (RBCs) and, when compromised, the RBCs are more susceptible to haemolysis. 8-aminoquinolines (primaquine and tafenoquine) are used for the radical curative treatment of Plasmodium vivax malaria and can cause haemolysis in G6PD deficient subjects. Haemolytic risk is dependent on treatment dose and patient G6PD status but ultimately it correlates with the number of G6PD deficient RBCs. The G6PD spectrophotometric assay reliably identifies deficient subjects but is less reliable in heterozygous females, especially when other blood conditions are present. In this work we analysed samples with a range of G6PD phenotypes and haematologic conditions from 243 healthy volunteers of Asian or African-American heritage using both the spectrophotomeric assay and the G6PD flow-cytometric assay. Overall 18.5% of subjects (29.3% of Asian females) presented with anaemia, associated with decreased RBCs volume (MCV) and reticulocytosis; the flow-cytometric assay showed good correlation with the spectrophotometric assay (Pearson's r 0.918-0.957) and was less influenced by haemoglobin concentration, number of RBCs and number of reticulocytes. This resulted in more precise quantification of the number of G6PD deficient RBCs and presumably higher predictive power of drug induced haemolytic risk.

Background: A large gap for the support of point-of-care testing is the availability of reagents to support quality control (QC) of diagnostic assays along the supply chain from the manufacturer to the end user. While reagents and systems exist to support QC of laboratory screening tests for glucose-6-phosphate dehydrogenase (G6PD) deficiency, they are not configured appropriately to support point-of-care testing. The feasibility of using lyophilized recombinant human G6PD as a QC reagent in novel point-of-care tests for G6PD deficiency is demonstrated., Methods: Human recombinant G6PD (r-G6PD) was expressed in Escherichia coli and purified. Aliquots were stored at -80°C. Prior to lyophilization, aliquots were thawed, and three concentrations of r-G6PD (representing normal, intermediate, and deficient clinical G6PD levels) were prepared and mixed with a protective formulation, which protects the enzyme activity against degradation from denaturation during the lyophilization process. Following lyophilization, individual single-use tubes of lyophilized r-G6PD were placed in individual packs with desiccants and stored at five temperatures for one year. An enzyme assay for G6PD activity was used to ascertain the stability of r-G6PD activity while stored at different temperatures., Results: Lyophilized r-G6PD is stable and can be used as a control indicator. Results presented here show that G6PD activity is stable for at least 365 days when stored at -80°C, 4°C, 30°C, and 45°C. When stored at 55°C, enzyme activity was found to be stable only through day 28., Conclusions: Lyophilized r-G6PD enzyme is stable and can be used as a control for point-of-care tests for G6PD deficiency.

Primaquine and other 8-amnoquinoline based anti-malarials can cause haemolysis in subjects with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Correct diagnosis of G6PD status in patients is crucial for safe treatment of both relapsing stages of Plasmodium vivax and transmitting forms of Plasmodium falciparum. Lack of suitable point-of-care tests has hampered a much needed wide use of primaquine for malaria elimination. In this study we have assessed the performances of two qualitative tests, the fluorescent spot test (FST) and the G6PD CareStart test (CST), against the gold standard quantitative spectrophotometric assay in a population of 1000 random adult healthy volunteers living in Yangon, Myanmar. The prevalence of G6PD deficiency in the Bamar, Karen and in the whole sample set was 6.6% (10.1% in males), 9.2% (21.0% in males) and 6.8% (11.1% in males) respectively. The FST and CST showed comparable performances with sensitivity over 95% and specificity over 90%, however for cases with severe G6PD activity the FTS had improved performance. If used with a conservative interpretation of the signal, the CareStart test has the potential to be used in the field and, by allowing a wider use of primaquine, to help malaria elimination.

The only currently available drug that effectively removes malaria hypnozoites from the human host is primaquine. The use of 8-aminoquinolines is hampered by haemolytic side effects in glucose-6-phosphate dehydrogenase (G6PD) deficient individuals. Recently a number of qualitative and a quantitative rapid diagnostic test (RDT) format have been developed that provide an alternative to the current standard G6PD activity assays. The WHO has recently recommended routine testing of G6PD status prior to primaquine radical cure whenever possible. A workshop was held in the Philippines in early 2015 to discuss key challenges and knowledge gaps that hinder the introduction of routine G6PD testing. Two point-of-care (PoC) test formats for the measurement of G6PD activity are currently available: qualitative tests comparable to malaria RDT as well as biosensors that provide a quantitative reading. Qualitative G6PD PoC tests provide a binomial test result, are easy to use and some products are comparable in price to the widely used fluorescent spot test. Qualitative test results can accurately classify hemizygous males, heterozygous females, but may misclassify females with intermediate G6PD activity. Biosensors provide a more complex quantitative readout and are better suited to identify heterozygous females. While associated with higher costs per sample tested biosensors have the potential for broader use in other scenarios where knowledge of G6PD activity is relevant as well. The introduction of routine G6PD testing is associated with additional costs on top of routine treatment that will vary by setting and will need to be assessed prior to test introduction. Reliable G6PD PoC tests have the potential to play an essential role in future malaria elimination programmes, however require an improved understanding on how to best integrate routine G6PD testing into different health settings.

The use of primaquine and other 8-aminoquinolines for malaria elimination is hampered by, among other factors, the limited availability of point-of-care tests for the diagnosis of glucose-6-phosphate dehydrogenase (G6PD) deficiency. Historically, the most used source of blood for G6PD analyses is venous blood, whereas diagnostic devices used in the field require the use of capillary blood; data have shown that the two sources of blood often differ with respect to hemoglobin concentration and number of red blood cells. Therefore, we have analyzed, in both capillary and venous blood drawn from the same healthy donors, the correlation of G6PD activity assessed by two qualitative tests (the Fluorescent Spot test and the CareStart test) with the gold standard quantitative spectrophotometric assay. Results obtained on 150 subjects with normal, intermediate, and deficient G6PD phenotypes show that, although differences exist between the aforementioned characteristics in capillary and venous blood, these do not impact on the quantitative assessment of G6PD activity after corrected for hemoglobin concentration or red blood cell count. Furthermore, we have assessed the sensitivity and specificity of the two qualitative tests against the gold standard spectrophotometric assay at different activity thresholds of residual enzymatic activity in both blood sources., (© The American Society of Tropical Medicine and Hygiene.)

A barrier to eliminating Plasmodium vivax malaria is inadequate treatment of infected patients. 8-Aminoquinoline-based drugs clear the parasite; however, people with glucose-6-phosphate dehydrogenase (G6PD) deficiency are at risk for hemolysis from these drugs. Understanding the performance of G6PD deficiency tests is critical for patient safety. Two quantitative assays and two qualitative tests were evaluated. The comparison of quantitative assays gave a Pearson correlation coefficient of 0.7585 with significant difference in mean G6PD activity, highlighting the need to adhere to a single reference assay. Both qualitative tests had high sensitivity and negative predictive value at a cutoff G6PD value of 40% of normal activity if interpreted conservatively and performed under laboratory conditions. The performance of both tests dropped at a cutoff level of 45%. Cytochemical staining of specimens confirmed that heterozygous females with > 50% G6PD-deficient cells can seem normal by phenotypic tests., (© The American Society of Tropical Medicine and Hygiene.)

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common human enzyme deficiency. It is characterized by abnormally low levels of G6PD activity. Individuals with G6PD deficiency are at risk of undergoing acute haemolysis when exposed to 8‒aminoquinoline-based drugs, such as primaquine. For this reason it is imperative to identify individuals with G6PD deficiency prior to administering these anti-malarial drugs. There is a need for the development and evaluation of point-of-care G6PD deficiency screening tests suitable for areas of the developing world where malarial treatments are frequently administered. The development and evaluation of new G6PD tests will be greatly assisted with the availability of specimen repositories., Methods: Cryopreservation of erythrocytes was evaluated as a means to preserve G6PD activity. Blood specimens from 31 patients including ten specimens with normal G6PD activity, three with intermediate activity, and 18 with deficient activity were cryopreserved for up to six months., Results: Good correlation in G6PD activity between fresh and cryopreserved specimens (R2 = 0.95). The cryopreserved specimens show an overall small drop in mean G6PD activity of 0.23 U/g Hb (P=0.23). Cytochemical staining showed that intracellular G6PD activity distribution within the red blood cell populations is preserved during cryopreservation. Furthermore, the mosaic composition of red blood cells in heterozygous women is also preserved for six months or more. The fluorescent spot and the BinaxNOW qualitative tests for G6PD deficiency also showed high concordance in G6PD status determination between cryopreserved specimens and fresh specimens., Conclusions: A methodology for establishing a specimen panel for evaluation of G6PD tests is described. The approach is similar to that used in several malaria research facilities for the cryopreservation of parasites in clinical specimens and axenic cultures. Specimens stored in this manner will aid both the development and evaluation of current and emerging G6PD tests. The availability of G6PD tests is a critical bottleneck to broader access to drugs that confer radical cure of Plasmodium vivax, a requirement for elimination of malaria.