This guideline was compiled according to the BSH process at: http://b-s-h.org.uk/guidelines/proposing-and-writing-a-new-bsh-guideline/. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) nomenclature was used to evaluate levels of evidence and to assess the strength of recommendations. The GRADE criteria can be found at http://www.gradeworkinggroup.org. In addition to the authors' comprehensive databases, a specific literature review was conducted on 18th April 2017 on the following databases: MEDLINE (OVID), EMBASE (OVID), CENTRAL (The Cochrane library) and Web of Science (SCI-Expanded, CPCI-S) using the search terms: G6PD activity in heterozygous beta thalassaemia, G6PD activity in reticulocytes, Glock and McLean assay procedure, Leukocyte G6PD activity, Molecular characterization of G6PD variants, "Correction" for the presence of young red cells and reticulocytes. The search covered the period back to 1950. Exclusions included articles not in English, non-human papers and those without abstracts. This yielded 9443 publications which, with exclusions and duplications, resulted in 236 articles which were additionally reviewed. Review of the manuscript was performed by the British Society for Haematology (BSH) Guidelines Committee General Haematology, the BSH Guidelines Committee and the General Haem Task Force sounding board of BSH. It was also on the members section of the BSH website for comment. This guideline is an update of the first G6PD guideline [The Assessment of Glucose-6-Phosphate Dehydrogenase Deficiency; prepared by the General Haematology Task Force, 1991]. Data from recent External Quality Assessment (EQA) exercises show that there is continued variation in both the results obtained and laboratory practice and this may be sufficient to affect clinical outcome. The guideline is for use by staff working in diagnostic laboratories and is intended to promote the harmonisation of analytical methods through sharing best practice in the diagnosis of G6PD deficiency. Glucose-6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme expressed in all tissue cells where it catalyses the first step in the pentose phosphate pathway. In the red blood cell, this is the sole pathway for the production of NADPH, which is required to maintain glutathione in the reduced state (GSH). Failure of this process impairs the ability of the red cell to deal with oxidative stress, which may lead to haemolytic episodes and anaemia that can be severe and in some cases fatal. G6PD deficiency shows marked genetic heterogeneity. Minucci and colleagues described some 186 mutations (Minucci et al., 2012) since when an additional 31 mutations have been reported (Gomez-Manzo et al., 2016). However, of the estimated 400 million people worldwide that have G6PD deficiency, a few polymorphic mutations account for the vast majority (Vulliamy et al., 1988). The most common G6PD variants are found in people who originated from the Mediterranean countries (the Mediterranean type), parts of Africa (the African type; G6PD A-) and parts of India and South East Asia (Beutler, 1971; Grimes, 1980; Wintrobe, 1981; Dacie, 1985). DNA sequence analysis has shown that the vast majority of mutations arise from single amino acid substitutions, mostly leading to a decrease in enzyme stability or to reduced catalytic efficiency. The degrees of enzymatic dysfunction detected in variants have been found to be in accordance with the severity of the clinical manifestations (Vulliamy et al., 1998; Gómez-Manzo et al., 2017). A list of G6PD variants can be found at http://www.bioinf.org.uk/g6pd/db. The gene encoding G6PD is located near the telomeric region of the distal arm of the X chromosome (band Xq28) in a region that includes the genes for haemophilia A, dyskeratosis congenita and colour blindness. The G6PD gene consists of 13 exons and spans 18·5 Kb, with a GC-rich promoter region (Persico et al., 1986). Being X-linked, males can be either hemizygous normal or hemizygous deficient, whereas females may be either homozygous normal, homozygous deficient or heterozygous. A heterozygous female will be a mosaic for cells expressing the wild type enzyme and cells expressing a deficient variant. The variable proportion of normal and deficient red cells, as a consequence of random X-chromosome inactivation (Lyonisation), renders diagnosis in some female heterozygotes difficult. A fraction of red cells in heterozygotes (on average, 50%) is as enzyme-deficient as in hemizygous males and therefore susceptible to haemolysis. Such individuals are usually less severely affected than homozygous females or hemizygous males. The severity of haemolysis and its potential clinical complications are roughly proportional to the fraction of deficient red cells. As a result of the random nature of X-chromosome inactivation during embryogenesis, individual females can manifest skewing in favour of either the normal or deficient G6PD allele. The latter, if extreme, will result in most red cells being G6PD-deficient and therefore susceptible to haemolysis. The great majority of G6PD-deficient individuals have no clinical manifestations in the steady state and the condition remains undetected until they are exposed to an exogenous haemolytic trigger such as bacterial or viral infections, ingestion of fava beans (favism) or drugs. However, some G6PD-deficient individuals suffer from a chronic non-spherocytic haemolytic anaemia (CNSHA), often requiring blood transfusion, in some cases on a long-term basis. These are due to the so-called class 1 variants, in which G6PD activity is less than 10% of normal (Table 1; adapted from Luzzatto et al., 2016). G6PD deficiency is never complete, as this is not compatible with survival, but enzyme activity may be so low as to be undetectable in red blood cells by standard methods. As the red cells age in the circulation there is a gradual reduction in G6PD activity and the mean value in young red cells has been shown to exceed that of old red cells by a factor of 8·5 (Bonsignore et al., 1964). One of the most clinically significant complication of G6PD deficiency is neonatal jaundice (NNJ), which peaks 2–3 days after birth (Luzzatto, 2010). Although highly variable in severity, without effective treatment it can lead to bilirubin encephalopathy (kernicterus) and permanent neurological damage or death. Haemolysis does not seem to contribute as much as impaired bilirubin conjugation and clearance by the liver (Kaplan et al., 1996). G6PD-deficient newborn babies who also inherit a mutation of the uridine-diphosphate-glucuronosyltransferase 1 (UGT1A1) gene promoter, responsible for Gilbert syndrome, are particularly at risk for neonatal jaundice (Kaplan et al., 1997). Neonatal screening for G6PD deficiency is routinely performed in many countries, mainly on dried blood spots, though not in the UK at the time of publication. Both favism and G6PD-related NNJ are often regarded as being primarily disorders of males: there are many more hemizygous deficient males than homozygous deficient females. However, both favism and NNJ are well documented in females, including heterozygotes (Meloni et al., 1983; Meloni et al., 1992; Luzzatto, 1993). The offending chemicals present in fava beans (broad beans), particularly in the fresh green beans, are divicine and isouramil, powerful oxidising agents which result, respectively, from hydrolysis of the alkaloid glucosides vicine and convicine. While intra-uterine haemolysis in G6PD-deficient fetuses exposed passively to fava beans appears rare, neonatal favism has been reported (Mentzer & Collier, 1975; Corchia et al., 1995). Postnatal haemolysis through breast milk intake after maternal ingestion of fava beans is a recognised cause of NNJ (Yeruchimovich et al., 2002; Al-Azzam et al., 2009). Although placental transfer of the culpable agents in fava beans has not been demonstrated, other glucosides are known to be transferred. Accidental ingestion of mothballs containing naphthalene was one of the more frequent causes of acute haemolysis in G6PD deficiency in the UK (Santucci & Shah, 2000), but is less common now as naphthalene-containing products have been banned in the EU since 2008. Bacterial and viral infections are known triggers of acute haemolytic anaemia in G6PD-deficient individuals although the mechanism of haemolysis is not well defined. Reactive oxygen species (ROS) have been implicated in the pathogenesis of many infections and these are known to cause oxidative damage. Severe haemolysis has been attributed to hepatitis viruses A and B, cytomegalovirus, pneumonia and typhoid fever (Cappellini & Fiorelli, 2008). After severe trauma, G6PD-deficient individuals may be at higher risk of sepsis and once infected may have a more severe clinical course (Spolarics et al., 2001). Some antimalarial drugs cause significant oxidative stress to the red cell and therefore it is important to test patients for G6PD deficiency before starting antimalarial therapy with these specific drugs. However, careful consideration for delaying treatment is required in what may be a life-threatening malaria. Although many drugs have been claimed to cause haemolysis in G6PD deficiency, in only a few of these is there a well-documented causal relationship (Table 2; from Luzzatto & Seneca, 2014 and the British National Formulary). G6PD deficiency should also be considered in other clinical situations as described in Table 3. Although it has been claimed that haemolysis is usually not as severe with the African variant, drug-induced acute haemolytic anaemia in G6PD A- subjects can be life-threatening, therefore in clinical terms, the A- type of G6PD deficiency cannot be regarded as benign. The severity and course of a haemolytic episode depend both on the G6PD variant and the type and duration of oxidative stress. In addition, the age of the individual and any coexisting disease conditions are factors. Little is understood about how G6PD deficiency interacts with other genetic traits that affect red-cell structure or function. In populations where G6PD deficiency is prevalent, haemoglobin S and thalassaemia coexist at a significant frequency. Some studies have suggested that co-inheritance of sickle cell anaemia and G6PD deficiency is associated with more severe anaemia and greater risk of cerebrovascular disease, although this has not been found consistently (Rees et al., 2009). G6PD testing in sickle cell disease and thalassaemic disorders is nevertheless recommended to avoid exposure of G6PD-deficient individuals to oxidant drugs or other agents that may exacerbate anaemia in the context of chronic haemolysis. The impact of transfusing blood from G6PD-deficient donors in high-prevalence regions should be considered, especially when transfusing children. For quantitative assays and for the fluorescence screening test, blood anticoagulated with ethylenediamine tetra-acetic acid (EDTA), heparin or acid-citrate-dextrose (ACD) solution can be stored for up to three weeks at 4°C or up to five days at room temperature with less than 10% loss of G6PD activity (Beutler, 1984). Samples containing variants may be less stable than samples containing the normal enzyme. However, a fresh sample (less than 24 h old) is required for dye decolourisation tests and heparin should not be used as an anticoagulant as it may affect the decolourisation time. The cytochemical test must be carried out on the day of blood collection if anticoagulated with EDTA or heparin, or within one week if ACD is used. Since G6PD in haemolysates is unstable at room temperature, 4°C or −20°C, haemolysates should not be stored. In recent years, affordable, qualitative "point of care" (POC) lateral flow tests have become available. These tests generally require capillary blood from finger-prick, and can be performed and interpreted by health workers at the bedside or in the field in <30 min. These cassette-based enzyme chromatographic devices are based on the reduction of colourless nitroblue tetrazolium dye to dark-coloured formazan. However, to address the problem of misclassification of females with intermediate G6PD activities, more sophisticated quantitative POC devices have been developed that accurately measure G6PD activity normalised for haemoglobin concentration by reflectometry/spectroscopy (Bancone et al., 2018; Pal et al., 2019). Cytochemical staining to demonstrate intracellular G6PD activity presents an alternative way to assess G6PD deficiency, and because it assays intact red blood cells, it can identify heterozygous females. Staining allows visualization (by microscopy) or enumeration (by flow cytometry) of the two distinct red-cell populations resulting from the G6PD-normal and G6PD-deficient erythrocytes. If a male patient is suspected of having G6PD deficiency on clinical grounds, either the fluorescent spot or the dye decolourisation screening test are acceptable first-line tests. The complications of a raised white blood cell count, raised retics, low Hb (and the inability to reliably identify female carriers) must be understood. If the screening test is abnormal or equivocal, the quantitative assay should be undertaken to confirm, or exclude, the diagnosis unless the patient is known to the laboratory. It is best to proceed directly to quantitative assay for female patients, who may be heterozygous and thereby possibly misclassified by any screening test. It is up to the individual laboratory to use either the one substrate (WHO) assay or the Glock and McLean (Glock & McLean, 1953) technique. If a woman has an intermediate or equivocal result in the quantitative assay then the cytochemical test should be undertaken. If there is a clinical or genetic reason to suspect that a woman is heterozygous for G6PD deficiency then the cytochemical test should be undertaken even if the quantitative assay is normal because the cytochemical test may be the only way to detect a deficiency in some cases (other than by DNA analysis). After a haemolytic episode all tests may give normal or equivocal results in G6PD deficiency; therefore, it is recommended that a patient is retested following a haemolytic episode of unknown cause to ensure that G6PD deficiency is not missed. In this clinical scenario, the WHO quantitative technique is satisfactory but suffers the disadvantage that the activity of 6-phosphogluconate dehydrogenase is not measured and therefore this test cannot be used to assess the effect of young red cells and reticulocytes. After a haemolytic episode or if the reticulocyte count is raised, the Glock and McLean technique is therefore more informative with respect to interpretation. Alternatively, comparison with the activity of either pyruvate kinase (PK) or hexokinase (HK) assayed in parallel often proves helpful. If G6PD deficiency is confirmed, the implications should be explained to the patient (or parent) who may also be given a "card" or information leaflet containing the relevant information. A suggested format for such a card is given in the Appendix 1. A description of various laboratory tests which may be used for the diagnosis of G6PD deficiency together with useful practical points can be found in the Appendix 2. Laboratories undertaking these screening tests and assays should participate in an External Quality Assessment Scheme. Reagent costs for these procedures are relatively inexpensive and readily obtainable from reliable biochemical supply manufacturers. However, the complexity of the tests requires experienced laboratory staff and some costly equipment (e.g. spectrophotometer). Commercially available kits for screening/assay are more straightforward to perform and generally provide a cost-neutral alternative to the procedures described below. The normal range for G6PD activity should be determined in each laboratory whether using an "in-house" (ICSH) procedure or a commercial kit. It is not acceptable to report the kit manufacturer's reference range, which is too broad and will provide a false-normal result on many heterozygotes. If the ICSH method is used, values should not differ widely from the published values. Results are expressed in international units (iu), which are the µmoles of substrate converted per minute. For adults, these values are 8·7 ± 1·7 iu/g haemoglobin at 30°C; 12·1 ± 2·09 iu/g haemoglobin at 37°C (without correction for 6-PGD activity) and 8·34 ± 1·59 iu/g haemoglobin at 37°C (corrected for 6-PGD activity). Newborns and infants have enzyme activities that deviate appreciably from the adult value (Konrad et al., 1972). In one study, the newborn mean activity was about 150% of the adult mean (Oski, 1969). G6PD activity has been found to be higher in premature infants born between 29 and 32 weeks gestation than in term neonates (Mesner et al., 2004). The report should conform to the requirements of the international standard (currently) "ISO 15189:2012 – Medical laboratories – Requirements for quality and competence". Screening test results should be reported either as "normal", "deficient" or "equivocal – please repeat", and the results of quantitative assays should include the enzyme activity, the unit of measurement (iu/g haemoglobin or 1012 red blood cells) or whether the result is expressed as a ratio. In addition, the assay temperature and the reference interval should be stated, together with the reticulocyte count. An interpretation of the result and recommendation(s) for action that reflect the clinical question being asked should be included. With some G6PD variants, including the African type, young red cells and particularly reticulocytes have much higher G6PD activity than mature red cells. For this reason tests carried out during, or soon after, a haemolytic episode may result in normal or even raised enzyme levels producing a false-normal result. In heterozygous females, the impact of selective haemolysis of G6PD-deficient red cells may compound the effect of reticulocytosis and the enzyme activity of residual non-deficient cells can mask the diagnosis of G6PD deficiency. Where G6PD deficiency is suspected on clinical grounds and no alternative explanation for haemolysis is forthcoming a G6PD assay should be repeated at least 2–3 months after resolution of the haemolytic episode. In this situation, examination of a blood film for typical features of oxidant damage is very important and is usually spectacular — almost diagnostic on its own — only if done very promptly, within two days from the onset of the haemolytic episode. If clinically indicated, to avoid delay it is possible to apply a "correction" for the presence of young red cells and reticulocytes by measuring G6PD activity in the heaviest (oldest) red cells after microhaematocrit centrifugation (Herz et al., 1970). Alternatively, since the activity of several other red-cell enzymes is similarly affected by the red-cell age, comparison of their activity to that of G6PD can be undertaken. This approach also takes into account the effect of older cells lost by haemolysis. HK, PK and 6-phosphogluconate dehydrogenase may be used for this purpose. 6PGD is the most convenient since it is assayed as part of the Glock and McLean procedure (see Appendix 2), which forms the basis of the International Council for Standardization in Haematology (ICSH) recommended method for G6PD assay (ICSH, 1977). If HK, PK or 6PGD activity is raised in the presence of a G6PD level at the lower end of the "reference" range it is likely G6PD activity would be subnormal in the absence of haemolysis and with a "normal" age distribution of red cells. Improved identification of heterozygotes by utilizing the G6PD/6PGD ratio has been reported (Minucci et al., 2009). Samples with a very low mean cell haemoglobin (MCH) (<25 pg), as seen for example in thalassaemia, frequently give G6PD activity levels above the reference range, so values falling within range (especially at the lower end) should be viewed with caution and confirmatory molecular testing considered if clinically indicated. Sanna and colleagues (Sanna, et al., 1980) found this was a particular issue when G6PD levels are expressed as activity per g of haemoglobin and to a lesser extent as activity per number of red cells × 109. Sex chromosome aneuploidy is a rare cause of uncertainty in the interpretation of G6PD activity. Male individuals with Klinefelter syndrome (XXY) may have intermediate levels similar to those of heterozygous females. Conversely in Turner syndrome (XO), G6PD activity may be as low as that seen in hemizygous deficient males or in women with extreme Lyonisation. All screening tests are useful to differentiate between normal and grossly deficient samples, but none of these techniques can reliably detect G6PD deficiency in heterozygous women. Equivocal results are difficult to interpret. It is advisable that the activity of these as well as of all deficient samples be confirmed by quantitative assay wherever possible. Anaemic samples and samples with a high leucocyte count can give misleading results in both screening tests. These problems can be avoided if the buffy coat is removed and packed red cells are used instead of whole blood. G6PD "point of care" (POC) is an area of rapid development with a number of different commercial products on the market and in development. Undoubtedly these will be invaluable both for mandated neonatal screening programs and in the use of 8-aminoquinolines for the elimination of Plasmodium vivax malaria especially in "outreach" areas. However, they need not be limited to out of lab or field testing as they have potential to be used as a laboratory screening test, especially when a rapid result is required such as for anti-malarial treatment or rasburicase administration. It is essential that any kit is evaluated for its fitness for purpose before use, especially with reference to storage temperatures, impact of environmental conditions and whether or not the kit incorporates a reference or control line where appropriate. Molecular analysis should be considered in cases where a precise diagnosis is required for clinical reasons, e.g. to confirm the condition in a recently transfused patient, a heterozygous female or where a deficiency may be masked by a reticulocytosis. It should be remembered that molecular analysis may not detect all variants and that it does not reflect the functional activity of the enzyme in all conditions, which will be affected by the reticulocyte concentration and the erythrocyte lifespan. The majority of G6PD mutations are point mutations and to date, 217 have been identified worldwide in both the coding DNA, introns and the 5′ and 3′ untranslated regions. There are common mutations in certain geographical areas such as the Mediterranean mutation and the G6PD A- in Africa and so one potential approach is to use a targeted direct mutation analysis method such as polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) (Poggi et al., 1990) aimed at these particular mutations. If this method produces a negative result or if there are no clear common mutations or if geographical origin is unclear then direct Sanger sequencing (Sanger & Coulson, 1975; Minucci et al., 2008) can be performed, which will also detect less common and novel mutations. Given the improvements in efficiency and cost of Sanger sequencing and the considerable heterogeneity of the mutations encountered in G6PD deficiency, proceeding directly to Sanger sequencing has become a more widely used approach. A positive result from this sequence analysis can be regarded as a stand-alone diagnostic result. However, if a mutation is not found, then only a quantitative assay can establish G6PD deficiency. The recent advances in next generation sequencing have also made it possible for many genes related to red-cell disorders to be sequenced simultaneously. This means that in cases of unexplained haemolysis, all potential genes related to red-cell enzyme deficiency can be looked at in a single molecular assay rather than several molecular and biochemical tests. It should be noted that in heterozygous females, molecular analysis is likely to be the only method of definitive identification of carrier status. Quality assurance is the process by which the laboratory demonstrates that the diagnostic results it produces are reliable in terms of accuracy and precision. In addition to the inclusion of internal quality control materials with each batch, a key element in quality assurance is participation in an accredited External Quality Assessment (EQA) programme, where available. EQA for G6PD qualitative and quantitative tests is offered by the major national and international EQA organisations and the laboratory should understand the type of programme provided and how it should be used in the laboratory to gain maximum benefit from participation (James et al., 2014). EQA is not available for the cytochemical assay and a laboratory undertaking this test should consider some other means of interlaboratory comparison, e.g. sample exchange. The frequency of EQA distributions, the inclusion of specimens that test the laboratory at clinical decision-making activities and the regular review of EQA results are particularly important. Any out-of-consensus EQA result should be fully investigated. The authors wish to thank Dr Jacky Wilson for help in undertaking the additional literature review. Several recommendations were taken from work previously undertaken by Dr Adrian Stephens & Martin Jarvis. The BSH General Haematology Task Force members at the time of writing this guideline were Dr Wayne Thomas (Chair), Dr Mamta Garg (Secretary), Carol D'Souza, Dr Shivan Pancham, Dr Sarah Lawson, Michael Wright, Dr Barbara De la Salle, Ciaran Mooney, Dr Jules Contesti, Nicola Svenson and Dr Savio Fernandes. The authors would like to thank them, the BSH sounding board, and the BSH guidelines committee for their support in preparing this guideline. None of the authors had conflicts of interest to declare. All authors have made a declaration of interests to the BSH and Task Force Chairs which may be viewed on request. Members of the writing group will inform the writing group Chair if any new evidence becomes available that would alter the strength of the recommendations made in this document or render it obsolete. The document will be reviewed regularly by the relevant Task Force and the literature search will be re-run every three years to search systematically for any new evidence that may have been missed. The document will be archived and removed from the BSH current guidelines website if it becomes obsolete. If new recommendations are made an addendum will be published on the BSH guidelines website (http://www.b-s-h.org.uk/guidelines). While the advice and information in this guidance is believed to be true and accurate at the time of going to press, neither the authors, the BSH nor the publishers accept any legal responsibility for the content of this guidance. Template obtained from BSH guidelines website (http://www.b-s-h.org.uk/guidelines). Reagent kits for both the fluorescent spot test (e.g. Trinity Biotech Qualitative G6PD FST Kit; catalogue number 203-A) and the dye decolourisation test (Trinity Biotech G-6-PDH Deficiency Screen Kit; catalogue number 400k) can be obtained commercially.-8 Such kits have the advantage of being "CE" ("Conformité Européene") marked and meet the requirements of the IVD (In Vitro Diagnostic Device) directive (98/79/EC), ensuring they are "fit for purpose". The new In Vitro Diagnostic Device Regulation (EU) 2017/746, published in the Official Journal of the European Union on 5 May 2017, strengthens the approval system for in vitro diagnostics. A procedure based on the recommended method (ICSH, 1979) is given below. Blood is mixed with an appropriate reaction mixture containing a detergent-like compound to lyse the red cells. After a standard time the mixture is "spotted" onto filter paper, dried and inspected under long-wavelength UV light. The appearance and brightness of the fluorescence due to NADPH gives a measure of the activity of G6PD. Mix the reagents in the volumes stated to make a total volume of 20 ml and then dispense this reaction mixture in 0·2 ml aliquots (e.g. in microfuge tubes) and store frozen at −20°C. This mixture is stable for up to one year at this temperature. Blood may be anticoagulated with EDTA (any sodium or potassium salt), heparin or ACD and dried blood spots collected onto filter paper can also be used. Samples give reliable results even after storage for up to five days at 25°C or for up to 21 days at 4°C. Thaw an aliquot of the reaction mixture and allow it to come to room temperature. Mix 20 μl of whole anticoagulated blood with 0·2 ml of reaction mixture. Spot one drop (20 μl) of this mixture onto non-fluorescent filter paper such as Whatman No. 1, as soon as it has been mixed, and again at intervals of 5 and 10 min from the mixing time. Examine the spots under long-wavelength UV light as soon as they have thoroughly dried. Samples with a very high haemoglobin concentration (Hb) (e.g. cord bloods) should be diluted to match the Hb of the normal control (using plasma from the test sample), because "quenching" of the fluorescence has been noted with such samples. Note. The presence of glutathione disulphide (GSSG) in the reaction mixture increases the sensitivity of the method. This is because the GSSG allows the small amounts of NADPH which may be formed by residual G6PD in mildly deficient samples to be reoxidised by glutathione reductase, another enzyme present in the red-cell haemolysate. At the beginning of the incubation no fluorescence should be visible and the samples from people with normal G6PD activity will fluoresce after 10 min incubation. G6PD deficiency is indicated by delayed or abs

HDAC inhibitors (HDACi) increase transcription of some genes through histone hyperacetylation. To test the hypothesis that HDACi-mediated enhanced transcription might be of therapeutic value for inherited enzyme deficiency disorders, we focused on the glycolytic and pentose phosphate pathways (GPPPs). We show that among the 16 genes of the GPPPs, HDACi selectively enhance transcription of glucose 6-phosphate dehydrogenase (G6PD). This requires enhanced recruitment of the generic transcription factor Sp1, with commensurate recruitment of histone acetyltransferases and deacetylases, increased histone acetylation, and polymerase II recruitment to G6PD. These G6PD-selective transcriptional and epigenetic events result in increased G6PD transcription and ultimately restored enzymatic activity in B cells and erythroid precursor cells from patients with G6PD deficiency, a disorder associated with acute or chronic hemolytic anemia. Therefore, restoration of enzymatic activity in G6PD-deficient nucleated cells is feasible through modulation of G6PD transcription. Our findings also suggest that clinical consequences of pathogenic missense mutations in proteins with enzymatic function can be overcome in some cases by enhancement of the transcriptional output of the affected gene.

Abstract Abstract 977 Transcriptionally active genes, including housekeeping genes, are characterised by co-occupancy and antagonistic actions of histone deacetylases (HDAC) and acetyltransferases (HAT). Transcription is facilitated through the prevailing action of HAT, which maintain histone acetylation. HDAC inhibitors (HDACi) induce widespread histone hyperacetylation and as a consequence are expected to increase expression of transcriptionally active genes. We have previously demonstrated this effect in inherited glycosylphosphatidylinositol (GPI-anchor) deficiency, an autosomal recessive disorder characterised by histone hypoacetylation and transcriptional repression of PIGM due to an in cis mutation which disrupts binding of the transcription factor Sp1 to its core promoter cognate motif. We surmised HDACi-mediated hyperacetylation might lead to increased transcription of other housekeeping genes, such as those of the anaerobic glycolytic and pentose phosphate pathways (GPPP) disruption of which is ameliorated by relatively modest increases in enzymatic activity. HDACi could therefore be of therapeutic value in these disorders. To address these hypotheses, EBV B cell lines were treated with the HDACi sodium butyrate (NaBu; 3mM) and mRNA levels for GPPP genes assessed by RQ-PCR. Of 9 genes tested (glucose-6-phosphate dehydrogenase, G6PD; glucose-6-phosphate isomerase, GPI; triosephosphate isomerase, TPI; pyruvate kinase, PK; 6-phosphogluconolactonase, PGLS; phosphogluconate dehydrogenase, PGD; ribulose-5-phosphate-3-epimerase, RPE; ribose-5-phosphate isomerase A, RPIA; transketolase, TKT), only G6PD mRNA levels increased, in a time-dependent fashion, in response to NaBu (n=3; p<0.01), an effect that was also observed in B cells from a patient with G6PD Brighton a severe, Class I G6PD deficiency (n=3; p<0.01). The increase in G6PD mRNA was observed within 4hrs post NaBu exposure and was not abrogated by the protein synthesis inhibitor cycloheximide suggesting a direct effect of NaBu on G6PD transcription. G6PD protein and enzymatic activity increased commensurately with G6PD mRNA level in both normal (n=4; p<0.01) and G6PD Brighton (n=4; p<0.01) B cells. In G6PD deficient B cells, enzymatic activity was restored to normal levels within 24hrs of treatment with HDACi (n=3; p<0.01). The selective effect of HDACi on transcription of G6PD but not other GPPP genes was also observed in other cell types, including 293T cells, and primary CD36+CD71+ erythroblasts generated from normal cord blood CD34+ cells. Notably, in NaBu-treated (1mM) primary erythroblasts a 2.3-fold increase in G6PD mRNA (n=3; p<0.01) accompanied by a 2.5-fold increase of G6PD protein levels (n=3; p<0.05) and 2.6-fold increase in enzymatic activity (n=3; p<0.05) were observed. The epigenetic correlates of G6PD mRNA induction were assessed by ChIP-Q-PCR. This revealed a dynamic, time-dependent, 3 to 4-fold increase in levels of histone 3 and 4 acetylation and a 4-fold increase in Sp1 and Polymerase II occupancy in the promoter of G6PD but not TPI or GPI. Preliminary pharmacological and shRNA experiments suggest that HDACi-mediated transcriptional upregulation of G6PD is Sp1-dependent. No differences were observed in baseline levels of histone acetylation or Sp1 occupancy of G6PD, TPI and GPI implying a yet to be defined cis acting determinant is required for the selective increase in Sp1 binding and histone hyperacetylation. Finally, in erythroblasts generated from peripheral blood mononuclear cells of two patients with Class I G6PD deficiency (G6PD Brighton and G6PD Harilaou), we confirmed the ability of NaBu to increase mutant G6PD mRNA and protein levels leading to increased G6PD enzymatic activity and its restoration to normal within 24hrs. In conclusion, we show that even within the same metabolic pathway, transcriptional upregulation of active genes in response to HDACi is selective rather than universal and is underpinned by enhanced Sp1 binding and histone hyperacetylation. Our findings raise the prospect of using HDACi to treat severe Class I G6PD deficiency. Disclosures: No relevant conflicts of interest to declare.

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, affecting more than 500 million people worldwide, is one of the most common of inherited disorders. There are 186 G6PD mutations published, with mutational clustering within defined ethnic/racial groups. However comprehensive molecular characterization of ethnically associated G6PD mutants and their clinical implications are lacking.Design and methods: Eighty unrelated Palestinian children hospitalized for hemolysis were studied. G6PD activity was determined by quantitative spectrophotometry and G6PD mutations were analyzed by sequencing of gDNA.Results: 65 of 80 children (81%) had G6PD deficiency, accounting for most of the hemolytic disease in this age group. G6PD Mediterraneari(c.563T), African G6PD A-(c.202A/c.375G), and G6PD Cairo(c.404C) were common with relative allele frequencies of 0.33 [1], 0.26, and 0.18 respectively. Two other variants were discovered, G6PD Beverly Hills(c.1160A) mutation, and a novel G6PD missense mutation c.536G>A (Ser179Asn), designated G6PD "Gaza". Three samples exhibited enzyme deficiency without detectable exonic or exon/intron boundary mutations.Conclusion: G6PD deficiency accounts for the majority of diagnoses for hemolysis in Palestinian children (81%), providing support for newborn G6PD deficiency screening programs. We report unanticipated molecular heterogeneity of G6PD variants among Gaza Strip Palestinians greater than reported in neighboring Arab populations. We report a high proportion of affected children with G6PD Cairo, which was observed previously in only a single Egyptian, and a novel mutation G6PD "Gaza". (C) 2012 Elsevier Inc. All rights reserved.

Antenatal screening/testing of pregnant women should be carried out according to the guidelines of the NHS Sickle Cell and Thalassaemia Screening programme. Newborn screening and, when necessary, follow up testing and referral, should be carried out according to the guidelines of the NHS Sickle Cell and Thalassaemia Screening programme. All babies under 1 year of age arriving in the UK should be offered screening for sickle cell disease. Preoperative screening for sickle cell disease should be carried out in patients from ethnic groups in which there is a significant prevalence of the condition. Emergency screening with sickle solubility tests must always be followed by definitive analysis. Laboratories performing antenatal screening should utilize methods capable of detecting significant variants and be capable of quantitating haemoglobins A 2 and F at the cut-off points required by the national antenatal screening programme. The laboratory must ensure a provisional report is available within three working days from sample receipt. Disorders of globin chain synthesis, both thalassaemias and haemoglobin variants, are common in the UK and constitute a significant public health problem. Diagnosis may be required: (i) to confirm a provisional diagnosis, such as sickle cell disease or β thalassaemia major; (ii) to explain a haematological abnormality, such as anaemia or microcytosis; (iii) to identify an abnormality in the presymptomatic phase, as in neonatal screening; (iv) to identify fetuses at risk of significant haemoglobinopathies and offer the parents informed choice; (v) to permit genetic counselling of prospective parents; (vi) to identify the presence of sickle cell haemoglobin preoperatively. Improved fully automated systems and reagents for techniques such as high-performance liquid chromatography (HPLC) and isoelectric focusing (IEF) have led to their introduction in many laboratories. There is also increasing use of other methods to identify globin gene abnormalities including DNA analysis, mass spectrometry and immunological methods. There is therefore a need for an updated guideline defining the role of new techniques and their place in screening and in specific diagnostic settings. The detection of unstable haemoglobins, methaemoglobins and high and low oxygen affinity haemoglobins is not discussed but laboratories should either have methods for detecting these variant haemoglobins or should refer such samples to a reference laboratory. It should be noted that the identification of haemoglobins is often presumptive, based on electrophoretic mobility or other characteristics in an individual of appropriate family origin. Presumptive identification should be based on a minimum of two techniques based on different principles. Definitive identification usually requires DNA analysis, mass spectrometry or protein sequencing. Family studies are also of considerable importance in elucidating the nature of disorders of haemoglobin synthesis. As testing for haemoglobinopathies has implications for genetic counselling, informed consent should be obtained from individuals prior to testing. Throughout these guidelines the term ‘sickle cell disease’ (SCD) encompasses both homozygous and the compound heterozygous states that lead to symptomatic disease as the result of the presence of haemoglobin S. Sickle cell anaemia refers specifically to those homozygous for βS. These guidelines are intended for UK Haematologists and the approach to screening is that which is considered practical and feasible for the British population. Different strategies may be required for populations with a different prevalence of haemoglobinopathies. These guidelines are an update of previous guidelines [British Committee for Standards in Haematology (BCSH) 1988, BCSH 1994a,b, 1998] and were written by clinical and laboratory experts representing areas of high and low prevalence of haemoglobin disorders. A patient representative was also included. Sections relating to antenatal and newborn screening are based on policies produced by the laboratory subcommittee of the National Health Service (NHS) Sickle Cell and Thalassaemia Screening programme and available in the programme’s Laboratory Handbook (NHS Sickle Cell and Thalassaemia Screening Programme 2009). For this updated guideline PubMed, MEDLINE and EMBASE were searched systematically for publications in English from July 2005 to March 2008 using key words (see Appendix I). Other publications between 1995 and July 2005 were also considered. The writing group produced a draft guideline, which was subsequently reviewed by consensus by members of the General Haematology Task Force of the British Committee for Standards in Haematology. The guideline was then reviewed by a sounding board of approximately 65 UK haematologists, the BCSH and the British Society for Haematology Committee and comments incorporated as appropriate. Criteria used to quote levels and grades of evidence are as outlined in appendix 3 of the Procedure for Guidelines Commissioned for the BCSH (http://www.bcshguidelines.com/process1.asp#appendix7). The normal pattern of haemoglobin synthesis during embryonic, fetal and adult life is summarized in Fig 1. Expression of different haemoglobins during normal development (reproduced from Bain (2006) Haemoglobinopathy Diagnosis, 2nd Edition. With permission of Wiley-Blackwell). Fetal haemoglobin, haemoglobin (Hb) F, (α2γ2) represents 90–95% of all haemoglobin by 34–36 weeks gestation. Adult haemoglobin, Hb A, (α2β2) accounts for 4–13% of total haemoglobin in the fetus. After 34 weeks gestation, Hb A production increases significantly as Hb F production falls. At term, Hb F represents 53–95% of all haemoglobin with Hb A levels reaching 20–30%. In addition to being increased in some haemoglobinopathies, increased levels of Hb F can be seen in infants who are small for gestational age, who have experienced chronic hypoxia or who have trisomy 13. Haemoglobin F percentage remains static for the first 2 weeks of life and then decreases by approximately 3% per week when erythropoiesis recommences and is normally <2–3% of total haemoglobin by 6 months of age. Hb A becomes the predominant haemoglobin by 3 months of age, although this switch may be delayed in sick preterm infants. Haemoglobin A2 (α2δ2) is produced in small amounts from birth and usually reaches adult levels by 6 months of age, although it can rise further for the first 1–2 years of life. Hb A2 and Hb Bart’s (γ4 tetramers) may be detected in normal infants born at term. The pattern of haemoglobin synthesis during development explains why α chain abnormalities cause clinical problems from early fetal life and why β chain abnormalities may be difficult to diagnose in the neonatal period. Pre-conceptual testing for haemoglobinopathies is recommended in at-risk groups (Table I). Pre-conceptual testing is important because it can be difficult to complete antenatal screening and fetal diagnosis within the first 12 weeks of pregnancy if the couple is unaware of the risk. This is most likely to be feasible in general practice but other medical practitioners should be alert to the possibility of a carrier state for a haemoglobinopathy and should offer screening. The individuals concerned must be informed of the result, whether or not an abnormality is found. If an abnormality is detected (Hb variant or possible/probable thalassaemia), partner testing should be offered, according to the antenatal testing algorithm (see below), if appropriate. Pre-conceptual testing should always be performed in women being investigated for infertility and in those having assisted conception. If a woman is found to have or be a carrier for a significant haemoglobinopathy, the partner or other sperm donor should be tested, if appropriate, and the women given counselling. If a donor ovum is to be used, the donor should be screened for relevant haemoglobinopathies. Pre-marriage screening for haemoglobinopathies is not usual in the UK but for some religious/ethnic groups pre-marital screening for β thalassaemia heterozygosity may be more acceptable than pre-conceptual or antenatal screening. National Screening Committee (NSC) policy recommends antenatal screening for haemoglobinopathies (http://sct.screening.nhs.uk/policy). Clinically significant haemoglobinopathies that should be detected are shown in Table II. The recommended procedure differs according to whether the antenatal unit is in a high or low prevalence area for SCD and thalassaemia. For high prevalence areas (fetal prevalence of SCD 1·5 per 10 000 pregnancies or higher), universal laboratory screening and use of the Family Origin Questionnaire (FOQ) (Appendix II) is advised. For low prevalence areas (fetal prevalence of SCD <1·5 per 10 000 pregnancies), screening is based on assessing the individual risk by determining the family origin of the woman and her partner by means of the FOQ (NHS Sickle Cell and Thalassaemia Screening Programme 2009). Whichever screening method is applied, the laboratory must ensure a provisional report is available within three working days from sample receipt. Screening in high prevalence areas. The screening policy for high prevalence areas starts with a full blood count (FBC) and HPLC or suitable alternative technique on a maternal blood sample. The women should also be asked to complete the FOQ or local equivalent. The request form sent to the laboratory should include the period of gestation and the results of the FOQ. Testing of the women should ideally be completed before 11 weeks and the whole process including partner testing, if applicable, should be completed within the first 12 weeks of pregnancy (NHS Sickle Cell and Thalassaemia Screening Programme 2006, 2009, National Collaborating Centre for Women’s and Children’s Health 2008). However, even women presenting for the first time late in pregnancy should be offered testing because the results will be relevant both to this and future pregnancies. Screening in low prevalence areas. For low prevalence areas it is recommended that all women be offered screening and the FOQ used to determine their family origin and that of the baby’s father. All should have an FBC and the red cell indices should be assessed and acted on in a similar manner as for high prevalence areas. Haemoglobin analysis is otherwise confined to those women whose own or the baby’s father’s family origin is not Northern European or is unknown. Detection of a haemoglobin variant. If a significant Hb variant is identified it should be confirmed by a suitable alternative method (e.g. haemoglobin electrophoresis or IEF if the initial method was HPLC) and the baby’s father should be offered screening without waiting for the definitive result if he has not been tested before or his result is not available. Variant haemoglobins of clinical relevance in this context are haemoglobins S, C, D-Punjab, E, H, Lepore and O-Arab. Raised Hb A2 percentage. If no relevant variant haemoglobin is identified, Hb A2 percentage is assessed, when appropriate. This is essential if the mean cell haemoglobin (MCH) is <27 pg. Hb A2 level of ≥3·5% in the presence of a MCH <27 pg indicates heterozygosity for β thalassaemia. A Hb A2 of >4% with a normal MCH should be assessed further as it may indicate a milder β thalassaemia carrier state that would warrant testing of the baby’s father (see below). If the Hb A2 is apparently >10% on HPLC, a diagnosis of Hb Lepore should be considered while a Hb A2 level apparently >15% may indicate Hb E trait. Other variant haemoglobins also have a retention time similar to that of Hb A2 on HPLC. Raised Hb F percentage. In the context of an MCH <27 pg, an isolated raised Hb F of >5% identifies possible heterozygosity for δβ thalassaemia and testing of the baby’s father is required. In the presence of a normal MCH, hereditary persistence of fetal haemoglobin (HPFH) should be considered. Possible α thalassaemia heterozygosity. In the absence of a variant Hb and β or δβ thalassaemia heterozygosity, α thalassaemia carrier states should be considered if the MCH is <27 pg. This should be considered regardless of iron status as there is insufficient time in the antenatal setting to re-assess indices after iron treatment. If the MCH is <25 pg, the individual should be assessed for the possibility of α0 thalassaemia heterozygosity in the light of his or her family origin. Family origins that indicate that α0 thalassaemia is possible are shown in Table I. An alternative explanation for these laboratory findings is homozygosity for α+ thalassaemia or iron deficiency, which may also be seen in these ethnic groups. α+ thalassaemia is found in many ethnic groups, with a high (10–30%) carrier frequency in some parts of Africa and South Asia. Even if both partners are carriers, there is no risk to the fetus. Homozygous α+ thalassaemia is not a clinically significant disorder with respect to genetic or obstetric complications, but can cause diagnostic confusion with α0 thalassaemia trait or iron deficiency. • Heterozygotes (carriers) generally have a MCH of 25–28 pg and a normal Hb A2 level. Approximately one-third of cases are silent. • Homozygotes generally have a MCH <25 pg, as seen in carriers for α0 thalassaemia. If the MCH is <25 pg and α0 thalassaemia is possible, the approach that makes best use of resources is to assess the family origin and red cell indices of the partner and proceed to DNA analysis, simultaneously in the woman and the baby’s father, only if both are at risk of α0 thalassaemia. However, if there is any delay in obtaining a blood sample from the baby’s father or if he is not available, then it is appropriate to test the mother’s DNA. The partners of women with haemoglobin H disease also require assessment for α0 thalassaemia. Consent for DNA testing is a legal requirement; this can be obtained at the initial antenatal clinic consultation to avoid any delay later. Ultrasound to detect fetal anaemia may be offered as an alternative to fetal DNA testing from 12 to 16 weeks in cases where the latter is declined. α0 thalassaemia occurs, rarely, in other ethnic groups, e.g. Pakistanis, Indians, some Middle Eastern populations (from United Arab Emirates, Iran, Yemen, Kuwait, Syria, Jordan) and in individuals originating in North-West England (Wigan and other parts of Lancashire). In the Middle East there is also a significant prevalence of non-deletional α thalassaemia, which can give rise to severe haemoglobin H disease in homozygotes. Individuals from such areas should be assessed individually but in general DNA analysis and partner testing is not recommended. The rare cases of Bart’s hydrops fetalis should be detected on ultrasound screening. It should be noted that a diagnosis of β thalassaemia heterozygosity does not exclude co-existing α0 thalassaemia heterozygosity and, in ethnic groups with a significant prevalence of the latter, DNA analysis is indicated when relevant to reproductive choice. For example, if one partner has β thalassaemia heterozygosity and the other possible α0 thalassaemia heterozygosity, both partners should be offered testing for α0 thalassaemia. National Screening Committee and NHS Policy is that all newborn babies should be screened for SCD. Such screening should also be extended to babies under the age of 1 year newly arrived in the UK (NHS Sickle Cell and Thalassaemia Screening Programme 2009). The main objective of the newborn screening programme is to improve outcomes in SCD through early treatment and care. The screening programme will also detect certain other variant haemoglobins by virtue of the analytical methods currently used. Additionally, the finding of Hb F only or of a very low percentage of haemoglobin A (<1·5%) on the newborn screen will identify the majority of babies with β thalassaemia major. Neonatal screening is based on the mother’s place of residence and is done at the age of 5–8 days as part of the newborn dried blood spot screening programme (http://www.newbornscreening-bloodspot.org). Further testing of samples that show a significant abnormality is required. Informed parental consent is required and parents have the right to opt out of testing although the programme is recommended. Opting out should be documented. Clinically significant conditions that should be detected are shown in Table III. In addition, certain conditions that are asymptomatic or have a mild phenotype will be identified and need to be subsequently distinguished from clinically significant abnormalities. For example, sickle cell/HPFH needs to be distinguished from severe forms of SCD and Hb E homozygosity needs to be distinguished from Hb E/β thalassaemia. Clinical follow-up, counselling and repeat testing is arranged for all babies in whom there is the possibility of a clinically significant abnormality and results are notified to the parents, general practitioner and responsible health care consultant at the place of the baby’s birth. In the case of suspected SCD, confirmatory testing and clinical follow-up should be performed in a timely manner so that penicillin prophylaxis can be started by 3 months of age; conjugate pneumococcal vaccine is now given to all babies from 8 weeks of age but is particularly important if the child has SCD (http://sct.screening.nhs.uk/cms.php?folder=2465). Clinical follow-up is also necessary for all babies with Hb F only. Babies who have been transfused in utero or in the early neonatal period are now tested using DNA techniques for the presence of the sickle gene as part the NHS Sickle Cell and Thalassaemia Screening programme. If this is not available or is declined, repeat testing should be performed 4 months from the date of transfusion. Other variant haemoglobins of potential clinical or genetic significance (e.g. haemoglobins C, D, E, O-Arab) will also be detected and in this case the parents, general practitioner and responsible health care practitioner at the place of the baby’s birth should be informed, and parents offered counselling. Opportunistic testing may be initiated by a general practitioner or other medical practitioner, with the informed consent of the patient, or by a haematology laboratory, where an abnormality that requires explanation is detected, e.g. in individuals found to have red cell indices or morphological appearances suggestive of a haemoglobinopathy. Opportunistic testing for sickle cell heterozygosity is best initiated in general practice. Reflex testing by the laboratory will depend on local policy. It is important to detect SCD prior to anaesthesia because its presence will influence clinical management. Testing should be initiated by clinical staff on the basis of a clinical history and assessment of family origin. All patients from groups with a high prevalence of Hb S (Table I) should be offered testing as some cases of milder disease may be unrecognized and the presence of Hb S heterozygosity may also influence peri-operative techniques. For example, cell salvage techniques carry a theoretical risk of red cell sickling and are probably contraindicated, and the use of limb tourniquets should be considered carefully although there is little evidence on which to base recommendations. Appropriate counselling should be given before testing so that patients are able to give their informed consent as there may be implications for patients who discover they are carriers of the sickle cell gene. The patient/parents or guardian (for children) should be informed of the results of testing, even when negative and the result documented in the patient’s medical record to avoid unnecessary repeat testing. Counselling should be offered if the result of the test is positive [National Institute for Clinical Excellence (NICE) 2003]. For routine operations, FBC and haemoglobin analysis using HPLC or a suitable alternative diagnostic method should be performed at the pre-assessment visit. In an emergency, an FBC and a sickle solubility test should be performed. Results in this situation should be evaluated clinically and must be followed by definitive testing (see below). Patients who present for diagnosis should be distinguished from individuals, often healthy, who are being screened for haemoglobinopathies. Such patients require a clinical history and physical examination and the FBC should be assessed, together with other laboratory tests, in the light of the clinical context and family origin. If there is microcytosis, appropriate tests for iron deficiency and anaemia of chronic disease should be performed and testing for thalassaemia considered in patients of appropriate family origin. Depending on agreed local policies, such tests may be initiated by the laboratory. Some laboratories use various published formulae to decide when to initiate such investigations but it should be noted that such formulae are not likely to be reliable in children or pregnant women or in sick patients who may have multiple medical problems influencing the Hb and red cell indices. Haemoglobinopathy investigations should therefore be considered in any unexplained microcytosis, even if the red cell indices are not typical of thalassaemia or another haemoglobinopathy. Haemoglobinopathy investigations may be indicated in the following circumstances: • In new arrivals in the UK with a history of SCD or significant thalassaemia • In patients with SCD receiving blood transfusion, including exchange transfusion, with the aim of lowering the percentage of Hb S • In patients with SCD or β thalassaemia intermedia being administered hydroxycarbamide or other agents to raise the Hb F percentage. HPLC or Hb electrophoresis is satisfactory but it should be noted that some programmes on some HPLC instruments underestimate Hb F levels (see below). • In the investigation of other unexplained haematological disorders or laboratory findings (see Table IV) General practitioners should consider pre-conceptual screening/testing of their patients of child-bearing age. General practitioners should consider and discuss premarital screening/testing of patients of child-bearing age when prenatal testing is unacceptable to the individuals. Clinics carrying out assisted conception should always carry out relevant screening and, when necessary, testing of both the egg donor and/or the sperm donor. Antenatal screening/testing of pregnant women should be carried out according to the guidelines of the NHS Sickle Cell and Thalassaemia Screening programme. Newborn screening and, when necessary, follow up testing and referral, should be carried out according to the guidelines of the NHS Sickle Cell and Thalassaemia Screening programme. Babies under 1 year of age arriving in the UK should be offered screening for SCD as part of the blood spot screening programme. Preoperative testing should be carried out in patients from ethnic groups in which there is a significant prevalence of sickle cell haemoglobin. Emergency screening with a sickle solubility test and full blood count must always be followed by definitive analysis. The need to investigate for thalassaemia and haemoglobinopathies should be considered in patients with unexplained microcytosis. Patients with SCD who are being treated by blood transfusion or with hydroxycarbamide should be appropriately monitored. The availability of fully automated systems and reagents for techniques, such as cation-exchange HPLC and IEF, have led to their introduction in a large proportion of laboratories, replacing cellulose acetate electrophoresis (CAE) as a first-line screening method. The use of mass spectrometry is becoming more widespread for variant identification and may have potential for screening also. The choice of methodology and equipment will be based on volume of workload, sample material (liquid blood or dried blood spots), ease of handling, reproducibility, local availability and expertise and cost. Although the consumables for CAE are inexpensive, the labour costs are relatively high. Therefore, a switch to HPLC may be cost-neutral when many samples are to be analysed (Phelan et al, 1999). Principles and methodology of the techniques used for haemoglobin analysis are outside the scope of this guideline but are available in standard text books. Haemoglobin electrophoresis at pH 8·4–8·6 using a cellulose acetate membrane is simple, reliable and rapid. It enables the provisional identification of haemoglobins A, F, S/G/D, C/E/O-Arab, H and a number of less common variant haemoglobins. Differentiation between haemoglobins migrating to a similar position can be obtained by using electrophoresis on acid (agarose) gels, HPLC or IEF. The provisional identification of any variant haemoglobin should be supported by at least one further unrelated method. Application of an alternative technique will exclude the possibility that a single band in either the S or C position represents a compound heterozygous state such as SD or SG and CE or CO-Arab respectively. If a patient has microcytosis the possibility that a single band represents compound heterozygosity for a variant haemoglobin and β0 thalassaemia must also be considered. Variant haemoglobins, such as Hb S can be quantified by scanning densitometry after electrophoresis/staining; however quantification of haemoglobin A2 by this method is not recommended as the precision is not good enough for the diagnosis of β thalassaemia trait (BCSH 1998). CAE is time consuming when a large number of samples are to be analysed. High-performance liquid chromatography can be used for the quantification of haemoglobins S, A2 and F and for the detection, provisional identification and quantification of many variant haemoglobins. HPLC usually provides accurate quantification of Hb A2 and is therefore suitable for the diagnosis of β thalassaemia trait. Automated HPLC systems are being used increasingly as the initial diagnostic method in laboratories with a high workload. In comparison with haemoglobin electrophoresis, HPLC has the following advantages: 1 The analysers are automated, therefore require less staff time and permit processing of large batches. 2 Very small sample volumes (5 μl) are sufficient for analysis. 3 Quantification of normal and separated variant haemoglobins is available on every sample. 4 Provisional identification of a larger proportion of variant haemoglobins can be made. 5 δ chain variants (recognition of which is important in the diagnosis of β thalassaemia heterozygosity) are more easily detected. High-performance liquid chromatography usually separates haemoglobins A, A2, F, S, C, D-Punjab and G-Philadelphia from each other. However, both Hb E and Hb Lepore often co-elute with A2 (as other haemoglobins co-elute with A, S and F) but may be recognized by alternative techniques. HPLC has the disadvantage that it also separates glycosylated and other derivative forms of haemoglobin, which can make interpretation more difficult. For example, derivatives of haemoglobin S co-elute with haemoglobin A2, rendering its quantification inaccurate. Careful examination of every chromatogram is essential. As with every method of haemoglobin analysis, controls should be run with every batch. Identification of variants is only provisional, and unrelated second-line methods should be used for confirmation. If HPLC is used as the screening technique, it is essential to check and maintain the positions of the windows, which are used as the first stage identification of any variants found. This is generally done by adjusting the column temperature or the flow rate so that the Hb A2 peak appears at a standard time. This is just as important as the calibration of the Hb A2 and Hb F levels and should be checked daily. Appropriate controls should be included wherever possible. Isoelectric focusing is satisfactory for the analysis of whole blood samples, haemolysates or dried blood spots. IEF gives good separation of Hb F from Hb A and clinically significant variant haemoglobins (S, C, D-Punjab, E and O-Arab). IEF can be semi-automated, rendering the technique suitable for screening large numbers of samples. However, this technique has not been validated for HbA2 quantification. Although IEF has better resolution and the advantage that it separates more variants than CAE, it also has the disadvantage that it separates haemoglobin into its post-translational derivatives e.g. Hb F separates into F1 (acetylated) and F11; Hb A can separate into A0, A1, A(αmet), A(βmet) and A(αβmet) – and similarly for other haemoglobins. This makes interpretation more difficult. Identification of variants is still only provisional, and second-line methods should be used for further analysis. The kits for sickle cell solubility tests that are predominantly used in the UK will detect haemoglobin S down to a concentration of 20% (and sometimes below; in some cases as low as 8%) (BCSH 1998). The method of Lewis et al (2006), although less sensitive than some commercial kits, can detect Hb S down to a concentration of 20%. The methods are therefore capable of detecting all cases of sickle cell trait beyond the period of infancy, even when there is coexisting α thalassaemia trait (but possibly not when there is coexisting Hb H disease). False positives have been described in patients with high plasma protein levels (Canning & Huntsman, 1970) and in anaemic patients when double the volume of blood is used in the test (Arras & Perry, 1972; Lilleyman et al, 1972). The latter problem can be avoided, however, by using a more concentrated sample of blood or washing the red cells. All positive and equivocal sickle solubility tests should be confirmed by HPLC or an alternative technique both for confirmation of the presence of Hb S and to distinguish sickle cell trait from sickle cell anaemia and from compound heterozygous st

Triosephosphate isomerase (TPI) deficiency is an autosomal recessive disorder characterised by congenital haemolytic anaemia and progressive neuromuscular dysfunction. No specific treatment exists to alter the natural history and death usually follows in infancy or early childhood. Biochemically TPI deficiency is distinguished by dramatic accumulation of the triose phosphate dihydroxyacetonephosphate (DHAP). DHAP undergoes spontaneous catabolism to methylglyoxal (MG) a potent mediator of protein and nucleotide glycation. MG levels are elevated in TPI deficiency and correlate with neuromuscular dysfunction. Accumulation of triose phosphates may be inhibited through stimulation of the pentose phosphate pathway by maximizing the activity of transketolase which converts glyceraldehyde-3-phosphate into ribose-5-phosphate. Transketolase activity can be enhanced by supplementation with the co-factor thiamine. Thiamine has been shown to reduce triosephosphate accumulation in human red blood cells in vitro but hitherto this not been studied in vivo. We report the outcome of a trial of thiamine supplementation in a female infant born to consanguineous south Asian parents who presented with a haemolytic anaemia at 3 weeks of age followed by neurological symptoms at 11 months culminating in respiratory failure requiring long-term mechanical ventilation. The diagnosis of TPI deficiency was made on the basis of enzyme assay and subsequent genetic analysis which demonstrated homozygosity for the Glu105Asp mutation of the TPI gene previously described in kindreds of European origin. There was marked elevation of red cell DHAP (550 % normal mean). Oral supplementation was commenced at 12 months of age with a lipophilic thiamine derivative - benzoyloxymethylthiamine - chosen to maximize potential CNS bioavailability at a dose of 5mg/kg/day. Response was assessed clinically and by assay of intermediates and metabolites in urine, blood, and CSF on day 0,1,7 and 14. Following thiamine supplementation there was a transient reduction in ventilatory requirement. A maximal (2–3 fold) increase in red cell thiamine and thiamine diphosphate (TPP) concentration was seen at 7 days. Red cell transketolase activity below that of saturation with TPP cofactor was 10% at day 0 and decreased to 0% thereafter. Despite persistently elevated DHAP levels a marked reduction in the MG metabolite D-Lactate of 90% in urine and 57% in the CSF was seen at day 14. Glyoxal a product of lipid peroxidation was also significantly reduced in CSF (70%). After 6 weeks the patient remained dependent on mechanical ventilation and the trial was discontinued. In conclusion oral thiamine supplementation was well tolerated and led to enhanced transketolase activity associated with indicators of reduced MG flux. Intervention preceding the onset of irreversible neuronal damage should be evaluated. Thiamine supplementation holds promise for the prevention and treatment of other neurodegenerative diseases and diabetic complications in which MG-mediated protein glycation is implicated.

Glucosephosphate isomerase (GPI) deficiency in humans is an autosomal recessive disorder, which results in nonspherocytic hemolytic anemia of variable clinical expression. A 4-year-old female with severe congenital hemolytic anemia had low red cell GPI activity of 15.5 IU/g Hb (50% of normal mean) indicating GPI deficiency. Subsequent DNA sequence analysis revealed a novel homozygous 921C to G mutation in the GPI gene sequence, predicting a Phe307 to Leu replacement. Strikingly, the red cell GPI activity in this patient was higher than that found in a second patient expressing the same GPI variant, with a more severe clinical phenotype. We propose that the hemolysis in the first patient may be modified by an accompanying deficiency of glucose-6-phosphate dehydrogenase (G6PD). The proband’s red cell G6PD activity was reduced at 4.5 IU/g Hb (50% of normal mean) and molecular studies revealed heterozygosity for the G6PD Viangchan mutation and a skewed pattern of X-chromosome inactivation, producing almost exclusive expression of the mutated allele. The G6PD Viangchan variant is characterised by severe enzyme deficiency, but not chronic hemolysis. This study suggests that the metabolic consequences of a combined deficiency of GPI and G6PD might be responsible for a different clinical outcome than predicted for either defect in isolation.

We have determined the causative mutation in 12 cases of glucose-6-phosphate dehydrogenase deficiency associated with chronic non-spherocytic haemolytic anaemia. In 11 of them the mutation we found had been previously reported in unrelated individuals. These mutations comprise seven different missense mutations and a 24 base pair deletion. G6PD Nara, previously found in a Japanese boy. Repeated findings of the same mutations suggests that a limited number of amino acid changes can produce the CNSHA phenotype and be compatible with normal development. The one new mutation we have found, G6PD Serres, is 1082 C-->T causing a 361 Ala-->Val substitution in the dimer interface where most other severe G6PD mutations are found. Now that several patients with the same mutation have been reported we can compare the resulting clinical phenotypes. For each mutation we find a reasonably consistent clinical picture, ranging from mild (G6PD Clinic) through moderate (G6PD Nashville) to severe (G6PD Beverly Hills and G6PD Nara).