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Appendix O Methods of blood analysis and quality control

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  • 1. Final Draft : 25/10/20021 Appendix O Methods of blood analysis and quality control The assays described in sections 1 and 2 below were conducted at the Department of Haematology, Great Ormond Street Hospital, London. Throughout fieldwork, samples of coagulated and ethylenediaminetetraacetate (EDTA) anticoagulated blood were sent directly to the laboratory by post after their collection. Serum samples were obtained by centrifugation of the coagulated blood sample. The assays described in sections 3 to 14 below were conducted at Medical Research Council Human Nutrition Research (HNR) in Cambridge. Samples of lithium heparin anticoagulated blood were collected and stored in a coolbox, at about 4°C, and delivered to a local processing laboratory in the region of the fieldwork typically within 5 hours of collection. These local laboratories undertook the processing and initial stabilisation of this blood sample into whole blood, red cells, plasma and metaphosphoric acid stabilised plasma portions. The metaphosphoric acid had been previously prepared, aliquotted at HNR and delivered to each local laboratory. The blood sample subfractions were stored frozen, typically at -40°C at these laboratories until their removal on dry ice to HNR, where they were stored frozen, at -80°C, until further subdivided and analysed. The assays described in sections 15 and 16 below were conducted at the SAS Trace Element Unit at the University of Southampton. Throughout fieldwork, samples of ethylenediaminetetraacetate (EDTA) anticoagulated blood were sent directly to the laboratory by post after their collection. Plasma samples were obtained by centrifugation of the anticoagulated blood sample. For some of the analytes measured in the samples that were sent through the post at room temperature, there was a significant linear correlation between the assay values and the magnitude of the delay time. In some there was also a small but significant difference in values obtained from the small number of slightly haemolysed samples and the majority which exhibited no visible haemolysis. In order to correct for these two potential errors, the following mathematical corrections were employed. For each analyte where there was a significant correlation with delay time, each result was corrected (up or down) by the product of the number of hours delay and the slope of the overall rate of change per hour. For each analyte where there was a significant effect of haemolysis, the results from the haemolysed
  • 2. Final Draft : 25/10/20022 samples were multiplied by a correction factor (between 0.969 and 1.023) which represented the ratio of the overall mean values of the haemolysed and non-haemolysed samples. Tables showing the results of the quality control procedures are given at the end of this appendix. 1 Full blood count Full blood counts were performed using a Bayer H3 Haematology Analyser. The analyser uses a colorimeter for measuring haemoglobin at wavelength 546nm. From samples of EDTA anticoagulated blood, red cells, white cells, differential counts and platelets were diluted and hydrodynamically focused through a flow cell and were counted using light and laser detection systems. The samples were analysed on the day of receipt. Quality control procedures comprised both internal and external procedures. Daily internal quality control checks were used to establish the running means of the stable red cell indices, mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC) and daily commercial controls (Bayer Testpoint Haematology control) were used to monitor drift in all measured and calculated parameters. External quality assessment schemes included the National External Quality Assessment Scheme (NEQAS) for haematology and External Quality Assessment Scheme (EQAS) for haematology run by Addenbrookes Hospital, Cambridge. (Table O1) 2 Serum ferritin, vitamin B12, folate and red cell folate These assays were performed on the Abbott IMx semi automated analyser which uses Microparticle Enzyme Immunoassay (MEIA) technology. Individual assay kits were used for each of the analytes, but all are based on the same principle. Microparticles coated with analyte-specific ‘capture’ molecules bind to the analyte in the sample. The resulting immune complex binds to a glass fibre matrix. An alkaline phosphatase-labelled conjugate bound to the matrix then reacts with a fluorogenic substrate and the rate of increase of fluorescence is measured. This is proportional to the concentration of analyte originally present in the sample. Concentrations were determined by comparison with a curve constructed from standards from known concentrations. Samples were analysed as soon as possible after receipt. Quality control procedures comprised both internal and external procedures. For the serum assays, an internal pooled serum sample was used as a drift control with each run. Drift
  • 3. Final Draft : 25/10/20023 control for the red cell folate assay was by use of a commercial red blood cell folate control. External quality assessment was by NEQAS for the haematinic assays. (Table O2) 3 Plasma homocysteine This was measured by the Abbott IMx assay on the IMx analyser. Homocysteine in samples and standards is converted to S-adenosyl-L-homocysteine, followed by coupling with an antibody which is quantified by a fluorescence polarisation immunoassay. To ensure that all of the homocysteine is in the reduced, free form, dithiothreitol is added to the samples. Quality control is achieved by participation in an international external quality assessment scheme based in Denmark1 , as well as by the manufacturer’s QC samples. A quality assurance running check uses a subdivided pool of heparinised plasma. (Table O3) 4 Retinol, tocopherols, lutein/zeaxanthin, lycopene, cryptoxanthin, α-carotene and ß-carotene in plasma These determinations were achieved by high performance liquid chromatography using a method derived from that of Thurnham et al2 . Rapidly-thawed subsamples of plasma, typically 250µl, were extracted with n-heptane in the presence of absolute ethanol, butylated hydroxytoluene (BHT) and α-tocopherol acetate (internal standard). The upper organic phase was evaporated nearly to dryness under vacuum, and was then redissolved in 250µl of the mobile phase, with sonication to achieve dissolution. If necessary a small volume of dichloromethane was added to achieve complete dissolution. 50µl aliquots were then injected onto a 4µ Waters C18 column which was preceded by a 0.5µ reduced stainless steel filter frit, to remove any particles. The mobile phase was acetonitrile 44%, methanol 44%, dichloromethane 12%, by volume, with added BHT at 10mg/l. The flow rate was 1.5ml/min and the column temperature control jacket was maintained at 25˚C. A Waters Millennium- controlled HPLC system, with a photodiode array detector, was used. A triple internal standard of retinyl acetate, tocopherol acetate and ethyl β-apo-8’-carotenoate was used. Retinol and retinyl palmitate are estimated at 325nm, and tocopherols at 292nm, at which wavelength the tocopherol acetate is also measured. All the carotenoids are measured at 450nm. Peak area response factors were obtained from semi-pure, commercially available carotenoids, and from retinol, retinyl palmitate, α- and γ-tocopherols. These were then corrected to 100% purity, by means of their HPLC patterns, and from their absolute optical densities and known extinction coefficients. This procedure was able to separate and quantify all of the following plasma components: retinol and retinyl palmitate at 325nm; α-
  • 4. Final Draft : 25/10/20024 and γ-tocopherols at 292nm; α- and ß-carotenes, lycopene and ß-cryptoxanthin at 450nm. Lutein and zeaxanthin eluted as a single peak and were estimated together at 450nm. Run time was 13 minutes, thus permitting a throughput of about four samples per hour. A mixed standard was run with every batch of extracted samples to check the performance characteristics of the column and detectors, the former being replaced when necessary, to ensure adequate peak separation. Extraction performance is based on the recovery of each of the three internal standards, and column performance is based on retention time shift and peak area recovery of standards. Quality control procedures comprised both internal and external procedures including: • two internal subdivided pools of heparinised human plasma from the Cambridge Blood Transfusion Service, used for long-term drift control and to provide an early warning of any changes in sensitivity of the assay. These are run every 20 samples. • External freeze-dried plasma samples, including ‘SRM 968c Fat-Soluble Vitamins, Carotenoids and Cholesterol in Human Serum’, provided by the National Institute of Standards and Technology (NIST), USA which have assigned values for all of the analytes of interest, and participation in the regular NIST round robin exchange scheme for these analytes. The criterion of adequacy is a 5% coefficient of variation (CV) or better for each internal standard, for both unknowns and for quality assurance (QA) samples. The method used to determine these analytes in plasma samples in this survey was very similar to that used in the NDNS of children aged 11 /2 to 41 /2 years3 , of people aged 65 years and over4 , and of young people aged 4 to18 years5 . (Table O4) 5 25-hydroxyvitamin D in plasma The DiaSorin (previously Incstar, Minnesota, USA) 25(OH)-vitamin D radioimmunoassay (RIA) kit assay was used, which was based on the developmental work of Hollis et al 6 . The antibody to 25(OH)-vitamins D (D2 + D3) had been generated in goats by the vitamin D analogue, 23,24,25,26,27-pentanor-C(22)-carboxylic acid of vitamin D coupled to bovine serum albumin. Firstly, duplicate extraction of the fat-soluble analyte from the plasma samples and from standards was achieved into pure acetonitrile, precipitating plasma proteins. The extracted 25-hydroxyvitamin D was then diluted with tracer 25-hydroxyvitamin D labelled with 125 I. Exposure of this impure mixture to a specific goat antibody against 25- hydroxyvitamin D resulted in specific binding of a proportion of the labelled vitamer,
  • 5. Final Draft : 25/10/20025 dependent on its concentration. Addition of a second antibody then achieved precipitation. Separation of this precipitated protein-bound fraction was achieved by centrifugation and was followed by gamma-counting of the sedimented fraction. Quality control procedures comprised both internal and external procedures including: • an internal subdivided pool of heparinised human plasma from the Cambridge Blood Transfusion Service, used for long-term drift control and to provide an early warning of any changes in sensitivity of the assay • a spiked serum with an assigned 25(OH)-vitamin D value, provided with the kit • serum with an assigned (DiaSorin) 25(OH)-vitamin D level, obtained from BioRad Inc • participation in the ‘DEQAS’ external quality assurance scheme for vitamin D metabolites, run from Charing Cross Hospital, London. We chose not to use the second high spiked serum that was provided with the kit because its concentration lay outside the region of good precision of the assay, and it was preferable to dilute any survey samples which fell into this range. This method was also used to measure 25-hydroxyvitamin D in plasma in the NDNS of children aged 11 /2 to 41 /2 years7 , of people aged 65 years and over8 , and of young people aged 4 to18 years9 . (Table O5) 6 Erythrocyte transketolase for thiamin status, in washed red blood cells This assay was based on that of Vuilleumier et al10 . It depends on the coupling of pyridine nucleotide (NADH) oxidation to glycerol phosphate dehydrogenase, which produces glycerol- 3-phosphate after the transketolase-catalysed conversion of ribose-5-phosphate. The rate of oxidation of NADH was monitored at 340nm, on the Cobas Bio analyser. The reaction rate was measured in both the absence and presence of the transketolase enzyme cofactor, thiamine pyrophosphate (cocarboxylase). Thiamin status was measured by both the basal enzyme activity, expressed per unit of haemoglobin in the sample, and by the activation coefficient, which was the ratio of cofactor-stimulated activity to the basal activity without any added cofactor. Haemoglobin was measured separately by the cyanomethaemoglobin procedure. Quality assurance was achieved with stored red cell preparations from heparinised blood obtained from the Cambridge Blood Transfusion Service and a pooled sample from
  • 6. Final Draft : 25/10/20026 Tanzanian blood. No commercial materials with assigned values or EQAS were available for this analyte. (Table O6) 7 Erythrocyte glutathione reductase activation coefficient for riboflavin status in washed red cells This assay has been adapted ‘in-house’ for use with a Cobas Fara centrifugal analyser from the manual technique developed by Glatzle et al11 . Washed red cell samples were thawed, diluted in water and buffer, centrifuged and the extract was incubated with and without flavin adenine dinucleotide (FAD). Addition of assay reagents, oxidised glutathione and reduced pyridine nucleotide coenzyme, took place in the centrifugal analyser, and was followed by a 5 minute measurement of the reaction rate at 340nm and 37˚C. The ratio of FAD-stimulated to unstimulated activity is the erythrocyte glutathione reductase activation coefficient (EGRAC) and is a reliable and robust measure of riboflavin status. The initial reactivation of the unsaturated apoenzyme in the sample was carried out for a relatively long period, 30 minutes at 37˚C, in order to ensure full reactivation of apoenzyme. The assay is conducted at a low final concentration of (FAD) (1.5µM). We have found this to be necessary, in order to eliminate activation coefficients (ratios) less than 1.0, which can result from enzyme inhibition by FAD, or its breakdown products, if the final concentration of FAD is too high. Quality control samples comprised pools of United Kingdom, Gambian and Tanzanian red cell haemolysates, stored in aliquots at -80˚C and thawed on the day of analysis. No commercial materials with assigned values or EQAS were available for this analyte. (Table O6) 8 Erythrocyte aspartate aminotransferase activation coeficient (EAATAC) for vitamin B6 status in washed red cells This method is based on the procedure described by Vuilleumier et al7 and uses the Cobas Bio centrifugal autoanalyser to monitor the stimulation of erythrocyte aspartate aminotransferase (EAAT) by pyridoxal-5-phosphate (PLP) at 340nm and 37˚C. Washed red cell samples were thawed, diluted in water and buffer, centrifuged, and the extract was incubated with and without PLP. The ratio of PLP-stimulated activity to the basal unstimulated activity is known as EAATAC. Quality control samples comprised separate pools of United Kingdom, Gambian and Tanzanian red cell haemolysates, stored frozen and thawed on the day of analysis. No commercial QC materials or EQAS were available for this analyte.
  • 7. Final Draft : 25/10/20027 (Table O6) 9 Vitamin C in plasma The assay was based on the procedure described by Vuilleumier and Keck12 . The assay is performed on a Roche Cobas Bio centrifugal analyser with fluorescence attachment. It begins with conversion of ascorbic acid in the metaphosphoric acid stabilised plasma sample to dehydroascorbic acid by a specific enzyme, ascorbate oxidase purified from cucumbers, obtained from Sigma, London. This is followed by coupling of the resulting dehydroascorbate with o-phenylene diamine to give a fluorescent quinoxaline. The formation of this quinoxaline is linearly related to the amount of vitamin C in the sample, at least over the range 0-10µg/ml (0-5µM), which is a typical range for vitamin C in plasma, after its pre- storage dilution 1:2 with 10% metaphosphoric acid. The assay was calibrated daily with freshly prepared vitamin C standards. The validity of the fluorimetric assay procedure used was by cross-correlation with HPLC-based assays, and by vitamin C spiking experiments. Preliminary trial runs and literature-assessment verified the stability of the vitamin C under the collection, stabilisation and storage conditions used. A selection of internal quality controls were included in each run, which comprised aliquots of heparinised plasma spiked with each of three levels of vitamin C and stored at -80˚C in metaphosphoric acid. No commercial quality control materials or EQAS were available for this analyte. (Table O7) 10 Plasma iron, Total Iron Binding Capacity (TIBC) and iron % saturation (Roche Unimate Iron; Iron binding capacity test) Measurement of plasma iron by this method depends on the reaction of free ferrous iron with ferrozine, after iron liberation from protein-binding with guanidine, followed by reduction of ferric to ferrous iron with ascorbic acid. The colour was measured on a Hitachi 912 analyser at 546nm. The calibrator was a Roche human serum with assigned value. For samples in which there was obvious lipaemia, centrifugation ensured that the sample used for analysis was free from potentially interfering lipids. Quality control procedures comprised both internal and external procedures including heparinised human plasma samples from the Cambridge Blood Transfusion Service, Roche human sera ‘N’ and ‘P’ (normal and pathological) at stated, half and quarter dilution and NEQAS for plasma iron.
  • 8. Final Draft : 25/10/20028 For assay of TIBC (total iron binding capacity), plasma samples were first mixed with a fixed amount of ferrous chloride in excess of the unsaturated iron-binding capacity. The excess unbound iron was then physically removed by addition of basic magnesium carbonate powder, followed by mixing and centrifugation, leaving only the transferrin-bound iron in solution. This transferrin-bound iron was then measured by the ferrozine reaction in the presence of guanidine giving a direct measure of the total amount of transferrin (iron-binding protein) present in the sample. Percent saturation of transferrin was calculated as 100x [plasma iron/TIBC], both being expressed on a molar basis. Again for this assay the calibrator was a Roche human serum with assigned values. Quality control procedures comprised internal procedures including heparinised human plasma samples from the Cambridge Blood Transfusion Service and Roche human sera ‘N’ and ‘P’ (normal and pathological) at stated and half dilution. (Table 08) 11 Glutathione peroxidase in whole blood (selenium status) This assay was based on that of Paglia and Valentine13 . It was further developed into a standardised procedure during a European Community FLAIR Concerted Action: “Measurement of Micronutrient Absorption and Status’’14 . The assay involves the coupling of glutathione peroxidase-catalysed oxidation of reduced glutathione, in the presence of diothiothreitol, cyanide, ferricyanide and tertiary butyl hydroperoxide, with the glutathione reductase-catalysed reduction of the resulting oxidised glutathione. This results in oxidation of NADPH, measured as a rate reaction at 340nm and 37˚C on the Cobas Bio analyser. The samples were diluted with dithioreitol and Drabkins reagent in a two-stage dilution procedure before the assay. Necessary precautions included precise pH control (pH 7.0) conservation of reagent stability on ice, and rapid processing of the samples. The enzyme was measured in diluted whole blood and its activity was expressed in nmol/mg haemoglobin/min, the latter having been measured on separate, but equivalent subsamples of whole blood at Great Ormond Street Haematology Laboratory. Running quality assurance was achieved with aliquots of heparinised whole blood from the Cambridge Blood Transfusion Service. The enzyme is stable during storage in the frozen state, and the samples were assayed after only a single freeze-thaw cycle after storage. No commercial QC materials or EQAS were available for this analyte. (Table O9)
  • 9. Final Draft : 25/10/20029 12 α1-antichymotrypsin in plasma Different plasma acute phase proteins respond at different rates following the onset of an inflammatory stimulus15 . α1-antichymotrypsin was selected as the most suitable choice of acute phase reactant, with respect to this time course of response as it remains elevated for longer than other acute phase proteins. This Hitachi 912-based nephelometric assay relied on a specific antibody to α1-antichymotrypsin, raised in rabbits, purchased from Dako and diluted in buffer containing polyethylene glycol. The six-point calibration curve used calibration sera with assigned values. The assay has proved robust and reliable over several years of use in the NDNS laboratory and samples were analysed after not more than two freeze-thaw cycles. The analyte is stable in frozen plasma, and the assay is highly sensitive, requiring only a few microlitres of sample. Internal quality control procedures included heparinised human plasma from the Cambridge Blood Transfusion Service and serum samples with assigned values from Dako. (Table O10) 13 Cholesterol and HDL-cholesterol in plasma These colourimetric assays were performed on the Cobas Fara analyser. Cholesterol was measured by the oxidation of cholesterol (liberated by cholesterol esterase), by cholesterol oxidase to 7-hydroxy-cholesterol. Hydrogen peroxide thus liberated then reacts with phenol and 4-amino-antipyrine in the presence of peroxidase, to yield a quinoneimine chromophore measurable at 520nm. The cholesterol assay was calibrated by use of the Roche human calibrator. HDL-cholesterol has been defined as that fraction of total cholesterol which remains in solution after precipitation of low density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterol with magnesium chloride plus phosphotungstic acid. For this assay magnesium/phosphotungstic acid reagent was added to the plasma sample. The sample was then centrifuged, and the clear supernate was assayed by the cholesterol assay described above. The HDL assay was calibrated by the use of Roche P human calibrator. Studies have shown that this precipitation methodology yields results very similar to those of ultracentrifugal separation, which is the reference method for this assay. Quality control procedures for the cholesterol assay comprised an internal procedure using heparinised human plasma from the Cambridge Blood Transfusion Service and a double-
  • 10. Final Draft : 25/10/200210 strength Roche N sample. External quality control comprised NEQAS for cholesterol. For HDL-cholesterol, an ABX control serum N was used at x0.5, x1.0 and x2.0 concentrations. (Table O11) 14 Creatinine in plasma (Roche Unimate 7 CREA) This Hitachi 912 assay is based on the Jaffé reaction (alkaline picrate). It is a rate assay and was calibrated with Roche human serum calibrator of known creatinine concentration. Samples were analysed after not more than two freeze-thaw cycles. Quality control was achieved with Roche human serum samples with assigned values, and for the running quality assurance human heparinised plasma from the Cambridge Blood Transfusion Service was used. External quality control procedures were NEQAS for creatinine. (Table O12) 15 Selenium in plasma and red cells Plasma and whole blood selenium concentrations were measured by using inductively coupled plasma mass spectrometry (ICP-MS)16 following 1 + 15 dilutions of 200µl sample volumes with a diluent which contained 1.0% v/v butan-1-ol, 0.66% m/v Triton X-100, 0.01M ammonia, 0.0002M ammoniumdihydrogen ethylenediaminetetraacetic acid and 0.002M ammoniumdihydrogen phosphate. This diluent destabilised argon-adduct ion species which otherwise would interfere with ICP-MS measurements of selenium and allows accurate analyses at 78 Se. Matrix-matched standards prepared from bovine serum were used for calibration. Red cell selenium was calculated from the whole blood and plasma concentrations, together with the haematocrit. Internal quality control sera were prepared by adding selenium to pools of bovine sera to give increases of 0, 0.40 and 1.60 µmol/l. An additional internal quality control was provided by using a Seronorm preparation. The internal quality controls were analysed at a frequency of not less than one set of four internal quality controls per 10 duplicate test samples. Participation in quality assessment schemes from Centre du Toxicologie de Quebec and TEQAS provided external quality control. (Table O13) 16 Mercury in blood
  • 11. Final Draft : 25/10/200211 Measurements of mercury in whole blood were made by inductively coupled plasma mass spectrometry17 . The diluents used were: (a) 2% w/v Virkon; (b) 25% w/v tetramethyl ammonium hydroxide; (c) 0.14M ammonia, 0.003M diammonium-dihydrogen EDTA, 0.03M ammonium dihydrogen phosphate, and (d) 0.7M ammonia in 1% v/v Triton X-100. To 300µl blood was added 300µl (a), then 100µl (b), then after 30 min, 5.0ml (c), 5.0ml (d), 0.3ml water and 40µl 1.0mg/l thallium solution. For calibration, mercuric nitrate standards were prepared in the presence of control bovine blood containing <1µg mercury/l. These contained the equivalent of 0, 5, 10, 20, 40 and 80 µg mercury/l blood. The validity of the blood mercury data was established by use of 3 Seronorm samples with assigned values and participation in two external quality assessment schemes, one in the UK and one in Canada. (Table O14) Acknowledgements We wish to acknowledge the following who were involved in the blood and urine analyses at the micronutrient Status Laboratory of MRC HNR: Mr S Austin, Mr R Carter, Mr N Matthews, Mr G Harvey, Mr J Swain, Dr S Nigdikar, Miss F Liuni, Miss K Giddens and Miss H Martindale ; the team from Great Ormond Street’s Clinical Haematology Laboratory and the team from University of Southampton’s Clinical Biochemistry Department. We would also like to thank Dr L Jackson and Dr M Birch for acting as survey doctor; Mrs S Levitt, Miss G Bramwell and Mr M Garratt for computing and data handling, advice and data entry, Dr J Perks, Miss J van der Pols, Mrs E Proud, Mrs L Winter, Mr R Quigley, Miss C Treacy and Dr R Re for co-ordination and management of the Survey Office and fieldwork and Miss K Edwards for office-work assistance. We are also indebted to personnel at the following hospitals for their assistance in local sample processing and storage: Ashford St Peter's Hospital NHS Trust, Chertsey Axiom Vet Laboratories, Teignmouth Barnsley District General Hospital Basildon & Thurrock General Hospital Bassetlaw Hospital, Worksop Blackpool Victoria Hospital Bradford Royal Infirmary Brighton Health Care NHS Trust Bronglaif Hospital, Aberystwyth
  • 12. Final Draft : 25/10/200212 Broomfield Hospital, Chelmsford BUPA Norwich Hospital, Colney Bury General Hospital Carlisle Hospitals NHS Trust, Cumberland Central Middlesex Hospital, London Chelsea & Westminster Healthcare, London Chesterfield and North Derbyshire Royal Hospital, Chesterfield Crawley Hospital, Crawley Darent Valley Hospital, Dartford Darlington Memorial Hospital Derby City General Hospital Diana Princess of Wales Hospital, Grimsby Dorset County Hospital, Dorchester East Kent Hospitals NHS Trust, Margate Eastbourne District Hospital Edinburgh Royal Infirmary Essex Rivers Healthcare NHS Trust, Colchester Farnborough Hospital, Opington Gartnavel General Hospital, Glasgow Glenfield General Hospital, Leicester Grampian University Hospitals NHS Trust, Aberdeen Hammersmith Hospital, London Heartlands Hospital, Birmingham Hereford County Hospital Hexham General Hospital Hillingdon Hospital, Uxbridge Kettering General Hospital NHS Trust Liverpool Royal Hospital Luton & Dunstable Hospital NHS Trust, Luton Maidstone Hospital Medway Maritime Hospital, Gillingham Middlesborough General Hospital Milton Keynes General Hospital New Hall Hospital, Salisbury North Bristol NHS Trust, Westbury on Trym Northampton General Hospital Trust Northwick Park Hospital, Harrow Nottingham City Hospital Pembury Hospital, Tunbridge Wells Peterborough District Hospital Pinderfield General Hospital, Wakefield Plymouth Hospitals NHS Trust Princess Margaret Hospital, Swindon Queen Elizabeth Hospital, Birmingham Queen Elizabeth Hospital, Kings Lynn Royal Berkshire Hospital, Reading
  • 13. Final Draft : 25/10/200213 Royal Bournemouth Hospital Royal Cornwall Hospital, Truro Royal Glamorgan Hospital, Llantrisant Royal Gwent Hospital, Newport Royal Lancaster Hospital Royal Oldham Hospital Royal Shrewsbury Hospital Royal Sussex County Hospital, Brighton Russell Hall Hospital, Dudley Salisbury District Hospital Scarborough Hospital Southampton General Hospital Southmead Hospital, Bristol Southport General Infirmary St Georges Hospital, London St James University Hospital, Leeds St Peter's Hospital, Chertsey Steppinghill Hospital, Stockport Stirling Royal Infirmary Stoke Mandeville Hospital, Aylesbury Sunderland Royal Hospital Swansea NHS Trust Tayside University Hospitals NHS Trust, Dundee University Hospital of North Durham Victoria Hospital, Kirkcaldy Victoria Infirmary, Glasgow Warrington Hospital Watford General Hospital West Wales General Hospital, Carmathen Western General Hospital, Edinburgh Whipps Cross Hospital, London Wirral Hospital NHS Trust Withington Hospital, Manchester York District Hospital References and endnotes 1 Moller J, Christensen L, Rasmussen K. An external quality assessment study of the analysis of methylmalonic acid and total homocysteine in plasma. Scand J Clin Lab Invest 1997; 57: 613- 619. 2 Thurnham DI, Smith E, Flora PS. Concurrent liquid-chromatographic assay of retinol, α- tocopherol, ß-carotene, α-carotene, lycopene and ß-cryptoxanthin in plasma, with tocopherol acetate as internal standard. Clin Chem 1988; 34: 377-381.
  • 14. Final Draft : 25/10/200214 3 Gregory JR, Collins DL, Davies PSW, Hughes JM, Clarke PC. National Diet and Nutrition Survey: children aged 11 /2 to 41 /2 years. Volume 1: Report of the diet and nutrition survey. HMSO (London, 1995). 4 Finch S, Doyle W, Lowe C, Bates CJ, Prentice A, Smithers G, Clarke PC. National Diet and Nutrition Survey: people aged 65 years and over. Volume 1: Report of the diet and nutrition survey. TSO (London, 1998). 5 Gregory J, Lowe S, Bates CJ, Prentice A, Jackson LV, Smithers G, Wenlock, R, Farron M. National Diet and Nutrition Survey: young people aged 4 to 18 years. Volume 1: Report of the diet and nutrition survey. TSO (London, 2000). 6 Hollis BW, Kamerud JQ, Selvaag SR, Lorenz JD, Napoli JL. Determination of vitamin D status by radioimmunoassay with an 125 I-labelled tracer. Clin Chem. 1993; 39: 529-533. 7 Gregory JR, Collins DL, Davies PSW, Hughes JM, Clarke PC. National Diet and Nutrition Survey: children aged 1½ to 4½ years. Volume 1: Report of the diet and nutrition survey. HMSO (London, 1995). 8 Finch S, Doyle W, Lowe C, Bates CJ, Prentice A, Smithers G, Clarke PC. National Diet and Nutrition Survey: people aged 65 years and over. Volume 1: Report of the diet and nutrition survey. TSO (London, 1998). 9 Gregory J, Lowe S, Bates CJ, Prentice A, Jackson LV, Smithers G, Wenlock, R, Farron M. National Diet and Nutrition Survey: young people aged 4 to 18 years. Volume 1: Report of the diet and nutrition survey. TSO (London, 2000). 10 Vuilleumier JP, Keller HE, Keck E: Clinical chemical methods for routine assessment of the vitamin status of populations. Part III. The apoenzyme stimulation tests for vitamins B1, B2 and B6, adapted to the Cobas Bio analyser. Internat J Vit Nutr Res 1990; 60: 126-135. 11 Glatzle D, Korner WF, Christeller S, Wiss O. Method for the detection of a biochemical riboflavin deficiency. Stimulation of NADPH2-dependent glutathione reductase from human erythrocytes by FAD in vitro. Investigations on the vitamin B2 status in healthy people and geriatric patients. Internat J Vit Nutr Res 1970; 40: 166-183. 9 Vuilleumier JP, Keck E. Fluorimetric assay of vitamin C in biological materials using a centrifugal analyser with fluorescence attachment. J Micronutrient Anal 1989; 5: 25-34. 10 Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterisation of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70: 158-169. 11 EC-Food Linked Agro-Industrial Research (EC-FLAIR) Concerted Action No. 10 (Rapporteurs: JL Belsten and AJA Wright). European Community - FLAIR common assay for whole-blood glutathione reductase (GSH-Px); results of an inter-laboratory trial. Eur J Clin Nutr 1995; 49: 921-927. 12 Calvin J, Neale G, Fotherby KJ, Price CP. The relative merits of acute phase proteins in the recognition of inflammatory conditions. Ann Clin Biochem. 1988; 25: 60-66. 13 Delves HT, Sieniawska CE. Simple method for the accurate determination of selenium in serum by using inductively coupled plasma mass spectrometry. J. Analyt Atom Spectrom 1997; 12: 387-389. 14 Moreton JA, Delves HT. Simple direct method for the determination of total mercury levels in blood and urine and nitric acid digests of fish by inductively coupled plasma mass spectrometry. J Analyt At Spectrosc 1998; 13: 659-665.

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