Long-range structural defects by pathogenic mutations in most severe glucose-6-phosphate dehydrogenase deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common blood disorder, presenting multiple symptoms, including hemolytic anemia. It affects 400 million people worldwide, with more than 160 single mutations reported in G6PD. The most severe mutations (about 70) are classified as class I, leading to more than 90% loss of activity of the wild-type G6PD. The crystal structure of G6PD reveals these mutations are located away from the active site, concentrating around the noncatalytic NADP+-binding site and the dimer interface. However, the molecular mechanisms of class I mutant dysfunction have remained elusive, hindering the development of efficient therapies. To resolve this, we performed integral structural characterization of five G6PD mutants, including four class I mutants, associated with the noncatalytic NADP+ and dimerization, using crystallography, small-angle X-ray scattering (SAXS), cryogenic electron microscopy (cryo-EM), and biophysical analyses. Comparisons with the structure and properties of the wild-type enzyme, together with molecular dynamics simulations, bring forward a universal mechanism for this severe G6PD deficiency due to the class I mutations. We highlight the role of the noncatalytic NADP+-binding site that is crucial for stabilization and ordering two ß-strands in the dimer interface, which together communicate these distant structural aberrations to the active site through a network of additional interactions. This understanding elucidates potential paths for drug development targeting G6PD deficiency.

Cyanosis Due to Methemoglobinemia as the Presenting Sign of Glucose-6-Phosphate Dehydrogenase Deficiency in a Child: Diagnostic and Clinical Implications.

A previously healthy 3-year-old boy presented with pallor, jaundice, cyanosis, and a 24-hour history of vomiting and anorexia following fava bean ingestion. Clinical examination and laboratory findings were consistent with severe nonimmune hemolytic anemia with methemoglobinemia. Given the patient's history, a previously unrecognized glucose-6-phosphate dehydrogenase deficiency was suspected and diagnosed. The aim of this article is to delineate the possible coexistence of methemoglobinemia and glucose-6-phosphate dehydrogenase deficiency in children presented with acute hemolysis and discuss its management while reviewing the existing literature.

Association of alpha-thalassemia and Glucose-6-Phosphate Dehydrogenase deficiency with transcranial Doppler ultrasonography in Nigerian children with sickle cell anemia.

BACKGROUND: Stroke is a devastating complication of sickle cell anemia (SCA) and can be predicted through abnormally high cerebral blood flow velocity using transcranial Doppler Ultrasonography (TCD). The evidence on the role of alpha-thalassemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency in the development of stroke in children with SCA is conflicting. Thus, this study investigated the association of alpha-thalassemia and G6PD(A- ) variant with abnormal TCD velocities among Nigerian children with SCA. METHODS: One hundred and forty-one children with SCA were recruited: 72 children presented with normal TCD (defined as the time-averaged mean of the maximum velocity: < 170 cm/s) and 69 children with abnormal TCD (TAMMV ≥ 200 cm/s). Alpha-thalassemia (the α-3.7 globin gene deletion) was determined by multiplex gap-PCR, while G6PD polymorphisms (202G > A and 376A > G) were genotyped using restriction fragment length polymorphism-polymerase chain reaction. RESULTS: The frequency of α-thalassemia trait in the children with normal TCD was higher than those with abnormal TCD: 38/72 (52.8%) [α-/ α α: 41.7%, α -/ α -: 11.1%] versus 21/69 (30.4%) [α-/ α α: 27.5%, α -/ α -: 2.9%], and the odds of abnormal TCD were reduced in the presence of the α-thalassemia trait [Odds Ratio: 0.39, 95% confidence interval: 0.20-0.78, p = 0.007]. However, the frequencies of G6PDA- variant in children with abnormal and normal TCD were similar (11.6% vs. 15.3%, p = 0.522). CONCLUSION: Our study reveals the protective role of α-thalassemia against the risk of abnormal TCD in Nigerian children with SCA.

A novel G6PD deleterious variant identified in three families with severe glucose-6-phosphate dehydrogenase deficiency.

BACKGROUND: Glucose-6-phosphate dehydrogenase deficiency (D-G6PD) is an X-linked recessive disorder resulted from deleterious variants in the housekeeping gene Glucose-6-phosphate 1-dehydrogenase (G6PD), causing impaired response to oxidizing agents. Screening for new variations of the gene helps with early diagnosis of D-G6PD resulting in a reduction of disease related complications and ultimately increased life expectancy of the patients. METHODS: One thousand five hundred sixty-five infants with pathological jaundice were screened for G6PD variants by Sanger sequencing all of the 13 exons, and the junctions of exons and introns of the G6PD gene. RESULTS: We detected G6PD variants in 439 (28.1%) of the 1565 infants with pathological jaundice. In total, 9 types of G6PD variants were identified in our cohort; and a novel G6PD missense variant c.1118 T > C, p.Phe373Ser in exon 9 of the G6PD gene was detected in three families. Infants with this novel variant showed decreased activity of G6PD, severe anemia, and pathological jaundice, consistent with Class I G6PD deleterious variants. Analysis of the resulting protein's structure revealed this novel variant affects G6PD protein stability, which could be responsible for the pathogenesis of D-G6PD in these patients. CONCLUSIONS: High rates of G6PD variants were detected in infants with pathological jaundice, and a novel Class I G6PD deleterious variants was identified in our cohort. Our data reveal that variant analysis is helpful for the diagnosis of D-G6PD in patients, and also for the expansion of the spectrum of known G6PD variants used for carrier detection and prenatal diagnosis.

Genotyping of Malaysian G6PD-deficient neonates by reverse dot blot flow-through hybridisation.

G6PD deficiency is the commonest enzyme deficiency found in humans. Current diagnostic methods lack sensitivity to detect all cases of G6PD deficiency. We evaluated the reverse dot blot flow-through hybridisation assay designed to detect simultaneously multiple known G6PD mutations in a group of Malaysian neonates. Archival DNA samples from 141 G6PD-deficient neonates were subjected to reverse dot blot flow-through hybridisation assay using the GenoArray Diagnostic Kit (Hybribio Limited, Hong Kong) and DNA sequencing. The method involved PCR amplification of 5 G6PD exons using biotinylated primers, hybridisation of amplicons to a membrane containing oligoprobes designed for G6PD mutations known to occur in the Malaysian population and colour detection by enzyme immunoassay. The assay detected 13 of the 14 G6PD mutations and genotyped 133 (94.3%) out of 141 (102 males, 39 females) cases. Among the 39 female G6PD-deficient neonates, there were 7 homozygous and 6 compound heterozygous cases. The commonest alleles were G6PD Viangchan 871G > A (21%) and G6PD Mahidol 487G > A(20%) followed by G6PD Mediterranean 563C > T, (14%), G6PD Vanua Lava 383T > C (12%), G6PD Canton 1376G > T (10%), G6PD Orissa 131C > G (6.3%) G6PD Coimbra 592C > T (5.6%) plus 6 other mutations. DNA sequencing of remaining cases revealed 6 cases of intron 11 nt 93C > T not previously reported in Malaysia and two novel mutations, one case each of nt 1361G > T and nt 1030G > A. We found the reverse dot blot assay easy to perform, rapid, accurate and reproducible, potentially becoming an improved diagnostic test for G6PD deficiency.

Glucose-6-phosphate dehydrogenase deficiency increases cell adhesion molecules and activates human monocyte-endothelial cell adhesion: Protective role of l-cysteine.

Glucose-6-phosphate dehydrogenase is a major enzyme that supplies the reducing agent nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), which is required to recycle oxidized/glutathione disulfide (GSSH) to reduced glutathione (GSH). G6PD-deficient cells are susceptible to oxidative stress and a deficiency of GSH. Endothelial dysfunction is characterized by the loss of nitric oxide (NO) bioavailability, which regulates leukocyte adhesion to endothelium. G6PD-deficient endothelial cells (EC) demonstrate reduced expression of endothelial nitric oxide synthase (eNOS) and NO levels along with reduced GSH. Whether G6PD deficiency plays any role in EC dysfunction is unknown. The chronic inflammation commonly seen in those with metabolic syndrome, characterized by elevated levels of tumor necrosis factor (TNF) and monocyte chemoattractant protein 1 (MCP-1), provided an incentive for investigation of these cytokines as well. A GSH/G6PD-deficient model was created using human umbilical vein endothelial cells (HUVEC) treated with either buthionine sulfoximine (BSO), a pharmacological inhibitor of the rate-limiting enzyme of GSH biosynthesis (γ-glutamylcysteine synthetase), or with 6-aminonicotinamide (6-AN), an inhibitor of G6PD or G6PD siRNA. Normal and G6PD-deficient cells were also treated with pro-atherosclerotic stimuli such as high glucose, TNF, and MCP-1. After inhibiting or knocking down G6PD/GSH, the capacity of endothelial cells for monocyte recruitment was assessed by determining the expression of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), which was upregulated by G6PD deficiency and accompanied by the presence of the oxidative stress markers NADPH oxidase 4 (NOX4), inducible nitric oxide synthase (iNOS), and reactive oxygen species (ROS). Treatment with the inhibitors BSO and 6-AN caused increased levels of adhesion molecule mRNA and monocyte-EC adhesion. Following treatment with high glucose, G6PD-deficient cells showed an increase in levels of ICAM-1 and VCAM-1 mRNA, as well as monocyte-EC adherence, compared with results seen in control cells. Treatment with l-cysteine (a precursor of GSH) protected endothelial cells by increasing GSH and attenuating ROS, ICAM-1, VCAM-1, and monocyte-EC adhesion. These results suggest that G6PD/GSH deficiency plays a role in endothelial dysfunction and that supplementation with l-cysteine can restore GSH levels and reduce the EC activation markers in G6PD-deficient conditions.

An update on glucose-6-phosphate dehydrogenase deficiency in children from Brazzaville, Republic of Congo.

BACKGROUND: Malaria transmission-blocking anti-malarial drugs, such as primaquine, offers an effective strategy for reducing the incidence of falciparum malaria. However, this drug induces haemolytic anaemia among glucose-6-phosphate dehydrogenase (G6PD) deficient individuals. The distribution of G6PD deficiency in Brazzaville, Republic of Congo and the association of G6PD deficiency with haemoglobin levels and blood cell counts were investigated. METHODS: A total of 212 febrile children were recruited for this study. Plasmodium falciparum diagnosis was conducted by microscopy and nested PCR. Sanger sequencing was used to assess G6PD deficiency by detecting 202G>A (rs1050828) and 376A>G (rs1050829) single nucleotide polymorphisms. RESULTS: Two hundred and twelve children were successfully genotyped for G6PD variants. Overall, 13% (27/212) of the children were G6PD deficient and 25% (25/100) females were heterozygous (11 BA- and 14 A+A-). The remaining 160 children had a normal G6PD genotype. The mean red blood and mean platelet counts were significantly lower in hemizygous male (G6PD A-) participants than in normal male (G6PD A+ or B) participants (p < 0.05). CONCLUSION: This study gives an update on G6PD deficiency among Congolese children. Understanding the distribution of G6PD deficiency in other geographical regions is recommended before primaquine is adopted in the malaria control programme.

A typical presentation of dengue fever in a G6PD deficient patient: A case report.

Dengue is a mosquito-borne viral disease that has rapidly spread in the recent years, particularly in South Asia. While haematologic complications, such as cytopenia and bleeding, may occur in severe dengue infection, reports of haemolytic anaemia in dengue fever are scant. We report a patient who developed haemolytic anaemia following dengue fever. A 45 years old gentleman presented with five-day history of fever and was recently diagnosed with dengue fever. He developed jaundice and started vomiting on the third day of his clinical course. His laboratory investigations revealed deranged liver profile, Coombs negative haemolytic anaemia and G6PD deficiency. He was treated conservatively with fluids and blood transfusions. His liver functions and haemolytic anaemia gradually resolved. This case highlights the importance of recognising dengue fever-induced haemolytic anaemia in a G6PD deficient patient by physicians and pathologists, enabling better diagnosis and improved management of this life-threatening condition.

L-Cysteine in vitro can restore cellular glutathione and inhibits the expression of cell adhesion molecules in G6PD-deficient monocytes.

L-Cysteine is a precursor of glutathione (GSH), a potent physiological antioxidant. Excess glucose-6-phosphate dehydrogenase (G6PD) deficiency in African Americans and low levels of L-cysteine diet in Hispanics can contributes to GSH deficiency and oxidative stress. Oxidative stress and monocyte adhesion was considered to be an initial event in the progression of vascular dysfunction and atherosclerosis. However, no previous study has investigated the contribution of GSH/G6PD deficiency to the expression of monocyte adhesion molecules. Using human U937 monocytes, this study examined the effect of GSH/G6PD deficiency and L-cysteine supplementation on monocyte adhesion molecules. G6PD/GSH deficiency induced by either siRNA or inhibitors (6AN/BSO, respectively) significantly (p < 0.005) increased the levels of cell adhesion molecules (ICAM-1, VCAM-1, SELL, ITGB1 and 2); NADPH oxidase (NOX), reactive oxygen species (ROS) and MCP-1 were upregulated, and decreases in levels of GSH, and nitric oxide were observed. The expression of ICAM-1 and VCAM-1 mRNA levels increased in high glucose, MCP-1 or TNF-α-treated G6PD-deficient compared to G6PD-normal cells. L-Cysteine treatment significantly (p < 0.005) increased G6PD activity and levels of GSH, and decreased NOX, ROS, and adhesion molecules. Thus, GSH/G6PD deficiency increases susceptibility to monocyte adhesion processes, whereas L-cysteine supplementation can restore cellular GSH/G6PD and attenuates NOX activity and expression of cell adhesion molecules.

Cytochemical flow analysis of intracellular G6PD and aggregate analysis of mosaic G6PD expression.

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.

Parvovirus B19-associated Hemophagocytic Lymphohistiocytosis in a Patient With Glucose-6-phosphate Dehydrogenase Deficiency.

We report a case of a 12-year-old male with glucose-6-phosphate dehydrogenase deficiency presenting with clinical signs of sepsis and pancytopenia. Investigations revealed parvovirus B19 (PVB19)-associated hemophagocytic lymphohistiocytosis (HLH). The patient recovered fully and quickly with symptomatic treatment. Current evidence suggests that PVB19-associated HLH has a favorable prognosis. Mild undiagnosed cases of HLH may be the cause of pancytopenia in PVB19 infections.

Glucose-6-phosphate dehydrogenase deficiency induced haemolysis in a woman with newly diagnosed diabetes after normalisation of hyperglycaemia.

BACKGROUND: The association between diabetes and G6PD deficiency is still a matter of debate. Hemolysis due to G6PD deficiency in people with diabetes has been reported, but is uncommon. To date, twenty-three cases have been reported from 12 different countries. CASE REPORT: We reported a 19-year-old Saudi women newly diagnosed with Type 1 diabetes in whom hemolytic crises occurred soon after normalization of hyperglycemia and revealed a G6PD deficiency. We reviewed the pertinent literature of this phenomenon and discussed the relevant theories. CONCLUSION: We conclude that in order to reduce the risk of hemolysis, in an area with high incidence of G6PD deficiency, screening of the enzyme activity should be considered in newly diagnosed people with diabetes. In case of G6PD deficiency, it is advisable to correct plasma glucose level gradually in order to avoid the rapid decline in glucose availability.

Demographics and co-occurring conditions in a clinic-based cohort with Down syndrome in the United Arab Emirates.

The majority of studies describing demographics and co-occurring conditions in cohorts with Down syndrome come from regions outside of the Middle East, mainly from Europe and North America. This paper describes demographics and co-occurring conditions in a hospital-based cohort of individuals with Down syndrome living in the Middle Eastern country of the United Arab Emirates (UAE). The first dedicated Down syndrome clinic in the UAE was established in 2012 at Tawam Hospital in Al Ain. This paper describes a clinic-based cohort of 221 participants over 4 years from the Gulf Down Syndrome Registry, a new Down syndrome database and contact registry created at Tawam Hospital. Key demographic findings include mean maternal age of 37 years, among the highest described in the literature. Sixty-two percent of mothers are >35 years. Over 90% of mothers received post-natal diagnosis of Down syndrome. High sex ratio, parental consanguinity, and large family size also characterize the group. The spectrum of many co-occurring conditions mirrors that of previously described populations, with some notable differences. Cardiovascular malformations are well represented, however, atrioventricular canal is not the most common. Genitourinary conditions are common, as evidenced by 12% of males with hypospadias and 15% with undescended testes. Glucose-6-phosphate dehydrogenase deficiency, alpha thalassemia trait, hypovitaminosis D, and dental caries are common in our cohort. This study describes a large hospital-based group with Down syndrome presenting to a new dedicated Down syndrome clinic in the UAE, highlighting unique demographic and co-occurring conditions found in that population.

Genotypic and phenotypic characterization of G6PD deficiency in Bengali adults with severe and uncomplicated malaria.

BACKGROUND: Control of malaria increasingly involves administration of 8-aminoquinolines, with accompanying risk of haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Few data on the prevalence and genotypic basis of G6PD deficiency are available from Bangladesh, where malaria remains a major problem in the South (Chittagong Division). The aim of this study was to determine the prevalence of G6PD deficiency, and associated G6PD genotypes, in adults with falciparum malaria in southern Bangladesh. METHODS: G6PD status was assessed via a combination of fluorescent spot testing (FST) and genotyping in 141 Bengali patients admitted with falciparum malaria to two centres in Chittagong Division from 2012 to 2014. In addition, an analysis of genomic data from 1000 Genomes Project was carried out among five healthy Indian subcontinent populations. RESULTS: One male patient with uncomplicated malaria was found to have G6PD deficiency on FST and a genotype associated with deficiency (hemizygous Orissa variant). In addition, there were two female patients heterozygous for deficiency variants (Orissa and Kerala-Kalyan). These three patients had a relatively long duration of symptoms prior to admission compared to G6PD normal cases, possibly suggesting an interaction with parasite multiplication rate. In addition, one of 27 healthy local controls was deficient on FST and hemizygous for the Mahidol variant of G6PD deficiency. Examination of 1000 Genomes Project sequencing data across the Indian subcontinent showed that 19/723 chromosomes (2.63%) carried a variant associated with deficiency. In the Bengali from Bangladesh 1000 Genomes population, three of 130 chromosomes (2.31%) carried deficient alleles; this included single chromosomes carrying the Kerala-Kalyan and Orissa variants. CONCLUSIONS: In line with other recent work, G6PD deficiency is uncommon in Bengalis in Bangladesh. Further studies of particular ethnic groups are needed to evaluate the potential risk of wide deployment of primaquine in malaria control efforts in Bangladesh.

Vitamin C Inhibits Aggravated Eryptosis by Hydrogen Peroxide in Glucose-6-Phosphated Dehydrogenase Deficiency.

BACKGROUND/AIMS: The study was aimed to investigate if vitamin C could exert protective effects on development of eryptosis caused by glucose-6-phosphate dehydrogenase (G6PD) deficiency and hydrogen peroxide. METHODS: Isolated erythrocytes with different G6PD activity (normal or deficient) were divided into various groups treated by either Vitamin C or H2O2. Phosphatidylserine (PS) extroversion rate was detected by Annexin V binding. The intracellular Ca2+ concentration was detected by Fluo3-fluorescence, and western blot was used to detect the expression of apoptosis factor caspase 3. RESULTS: Compared with the blank group, the PS extroversion rate (P < 0.001), intracellular Ca2+ concentration (P < 0.001) and active caspase 3 expression level (P < 0.05) of erythrocytes significantly increased after treatment of 0.05% H2O2. Then the index of eryptosis significantly decreased after erythrocytes were treated with Vitamin C (1 mg/ml) for 30 min (all P < 0.05). The decline in erythrocytes with G6PD normal activity was more significant than those with G6PD deficiency. CONCLUSION: Vitamin C could effectively inhibit the eryptosis contributed by H2O2 oxidative stress, and the suppression on eryptosis with G6PD normal activity was more effective than that with G6PD deficiency.

Favism, the commonest form of severe hemolytic anemia in Palestinian children, varies in severity with three different variants of G6PD deficiency within the same community.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common genetic abnormality known to predispose to acute hemolytic anemia (AHA), which can be triggered by certain drugs or infection. However, the commonest trigger is fava beans (Vicia faba) ingestion, causing AHA (favism), which may be life-threatening especially in children. G6PD deficiency is genetically highly heterogeneous, as nearly 200 different mutations have been observed. We have investigated the hematological features of acute favism in the Palestinian Gaza community that is characterized by the polymorphic coexistence of three different G6PD deficiency genes (G6PD A-, G6PD Cairo, G6PD Med). We have found by comparison to the general population (485 adults and 466 newborns) that children with favism, in terms of relative frequency, G6PD A- was under-represented, whereas G6PD Med was over-represented. We also found that the severity of anemia was significantly greater with G6PD Med and G6PD Cairo than with G6PD A-; and with G6PD Cairo, compared to the other two variants, there was greater hyperbilirubinemia, as well as persistence of mild anemia and reticulocytosis for as long as 4months after recovery from favism. This is the first report determining a differential impact of different G6PD mutations on the clinical features of favism in the same population and the same environment.

Rapid detection of G6PD mutations by multicolor melting curve analysis.

The MeltPro G6PD assay is the first commercial genetic test for glucose-6-phosphate dehydrogenase (G6PD) deficiency. This multicolor melting curve analysis-based real-time PCR assay is designed to genotype 16 G6PD mutations prevalent in the Chinese population. We comprehensively evaluated both the analytical and clinical performances of this assay. All 16 mutations were accurately genotyped, and the standard deviation of the measured Tm was <0.3°C. The limit of detection was 1.0ng/µL human genomic DNA. The assay could be run on four mainstream models of real-time PCR machines. The shortest running time (150min) was obtained with LightCycler 480 II. A clinical study using 763 samples collected from three hospitals indicated that, of 433 samples with reduced G6PD activity, the MeltPro assay identified 423 samples as mutant, yielding a clinical sensitivity of 97.7% (423/433). Of the 117 male samples with normal G6PD activity, the MeltPro assay confirmed that 116 samples were wild type, yielding a clinical specificity of 99.1% (116/117). Moreover, the MeltPro assay demonstrated 100% concordance with DNA sequencing for all targeted mutations. We concluded that the MeltPro G6PD assay is useful as a diagnostic or screening tool for G6PD deficiency in clinical settings.

Mean reticulocyte volume: a specific parameter to screen for hereditary spherocytosis.

This study assessed the value of mean reticulocyte volume (MRV) for differential diagnosis of hereditary spherocytosis (HS) so as to develop conventional and new specific screen indexes. Subjects in this study were divided into three groups: 53 cases in HS group, 217 cases in hemolytic anemia control group (109 cases of thalassemia (THAL), 56 cases of glucose-6-phosphate dehydrogenase G6PD deficiency anemia, and 52 cases of autoimmune hemolytic anemia (AIHA)), and 100 cases in healthy control group. We analyzed erythrocyte and reticulocyte parameters including MRV, mean sphered corpuscular volume, mean corpuscular hemoglobin concentration, and immature reticulocyte fraction. Results demonstrated that MRV was significantly lower in the HS group but significantly higher in the AIHA and G6PD deficiency anemia groups than that in the healthy control group (P = 0.000). MRV was not significantly different between the AIHA and G6PD deficiency anemia groups (P = 0.977) and between the healthy control and THAL groups (P = 0.168). The area under the ROC curve of MRV for diagnosis of HS was 0.942, with a standard error of 0.019, 95% confidence interval of 0.905-0.979, and optimal critical diagnosis point of 95.77 fL. When the MRV was ≤95.77 fL, the sensitivity and specificity for diagnosis of HS were 86.80% and 91.20%, respectively. Therefore, MRV is a general and specific new index for screening HS and important for differential diagnosis of different types of hemolytic anemia.