The determination of haemoglobin as cyanhaemiglobin or as alkaline haematin D-575. Comparison of method-related errors

In order to compare the accuracy of haemoglobin (Hb) determination methods, the commonly used cyanhaemiglobin (HiCN) method and the recently developed alkaline haematin D-575 (AHD) method (R. Zander, W. Lang & H. U. Wolf (1984) Clin. Chim. Acta 136, 83-93; H. U. Wolf, W. Lang & R. Zander (1984) Clin. Chim. Acta 136, 95-104) were tested with respect to method-related errors such as plasma, cell, and Hb errors. Both methods yield a series of more or less significant errors which generally lead to an overestimation of the Hb concentration in the order of 1%. However, in all three cases of plasma errors, i.e. normal plasma error, plasma error in lipaemic blood, and plasma error in bilirubinaemic blood, the AHD method shows significantly lower values of errors than the HiCN method. In the case of cell errors such as ghost and leukocyte errors, the overestimation of the Hb concentration by the HiCN method is 60% higher than that by the AHD method. In the case of Hb errors such as fetal Hb and carboxy Hb errors, there is a significant overestimation of the Hb concentration by the HiCN method, which amounts 3 min after mixing of blood and HiCN solution to 0.7% in the case of fetal Hb and to 13.2% in the case of carboxy Hb. The latter value yields an overestimation of 1.3%, when 10% carboxy Hb in a blood sample is present. In contrast, there is no detectable overestimation after 3 min in the case of the AHD method. Thus, the AHD method provides a higher accuracy in Hb determination than the commonly used HiCN method.     

Observations on the alkaline haematin/detergent complex proposed for measuring haemoglobin concentration

The "alkaline haematin D-575" method for determining haemoglobin, described by Zander et al. [1984) Clin. Chim. Acta 136, 83-93) was tested. Claims that different non-ionic detergents in the reagent result in identical values for the haemoglobin concentration being measured could not be verified. It could also not be verified that a stable end-product with unique spectral characteristics is always reached within approximately 2 min and that conversion of fetal haemoglobin is faster than that with the haemiglobin cyanide method. Because of the many questions regarding the nature and characteristics of the alkaline haematin/detergent complex or complexes, it is not yet possible to recommend this method for routine haemoglobinometry.     

Alkaline haematin D-575, a new tool for the determination of haemoglobin as an alternative to the cyanhaemiglobin method. I. Description of the method

A new method for the rapid and accurate measurement of haemoglobin has been developed as an alternative to the conventional cyanhaemiglobin method. This method is based on the conversion of all haeme, haemoglobin, and haemiglobin species into a stable end product by an alkaline solution of a non-ionic detergent ('AHD reagent'). The reaction product, designated as alkaline haematin D-575, is extremely stable and shows a characteristic absorption peak at 575 nm. As compared to the cyanhaemiglobin method, the determination of haemoglobin by alkaline haematin D-575 offers several advantages such as (1) extreme stability of the AHD reagent and the conversion product, (2) decreased conversion time of all haemoglobin species into the end product, (3) decreased amounts of plasma and cell errors, and errors caused by delayed conversion of carboxy- and fetal haemoglobins, and (4) standardisation by a primary standard (purified crystalline chlorohaemin).     

Alkaline haematin D-575, a new tool for the determination of haemoglobin as an alternative to the cyanhaemiglobin method. II. Standardisation of the method using pure chlorohaemin

Chlorohaemin with high purity (greater than 99%), a stable and well-defined compound, can be used as a primary standard for the standardisation of a haemoglobin assay based on alkaline haematin D-575 [6]. Dissolved in a solution of 25 g Triton X-100 per litre of 0.1 mol/l NaOH ('AHD solution'), the millimolar absorbance coefficient of the end product (alkaline haematin D-575) is 6.960 +/- 0.046 [l X mmol-1 X cm-1] at 575 nm. Within the range of haemoglobin concentrations of 5 to 25 g/100 ml there is a strong linear relation between chlorohaemin concentration and absorbance with a deviation of less than or equal to 2% from the theoretical values. As compared to the conventional cyanhaemiglobin standard solutions, standardisation with pure chlorohaemin is the method of choice because of the simplicity of the preparation of standard solutions, which can be done in every laboratory, and the stability of both the solid compound chlorohaemin and its solutions in alkaline Triton X-100. For the first time a real standard for quality control in haemoglobinometry is recommended: a concentrated solution which behaves like blood, i.e. the simulation of all steps in haemoglobin determination (dilution and photometry) is possible.     

A candidate reference method for the determination of magnesium in serum

A candidate reference method for the determination of magnesium in serum (analytical range 0.5 to 2.0 mmol/l) by flame atomic absorption spectrometry was commissioned. The relative standard deviation of the 4 replicates of each value ranged from 0.18 to 1.07%, and the standard error of the mean ranged from 0.63 to 8.34 mumol/l. In the analysis of 3 different standard solutions (prepared by weighing the analyte), which was performed in each of three different experiments the values recorded by the candidate reference method deviated by -0.15, 0.44 and 0.24%, respectively. The reference method value did not differ significantly from the definitive value. As the method seems to be as reliable as comparable reference methods for the determination of sodium, potassium or calcium, tests of transferability should now be undertaken.     

A candidate reference method for coupled sodium-water determination in human serum

A candidate reference method is described for coupled sodium-water determination based on ion-exchange sodium separation from the serum matrix followed by gravimetry as Na2SO4, and serum water determination by means of microwave evaporation. For sera with normal sodium and water contents, the mean relative standard deviation is 0.6% (0.8 mmol/l). Mean inaccuracy for the coupled sodium-water determination is -0.3% (0.4 mmol/l). The candidate reference method can be considered a reference method because the reference method value did not differ significantly from the definitive value, there is no known source for interferences or bias, and misinterpretation due to abnormal protein or lipid levels is excluded because serum sodium is determined on a plasma water basis. Sodium concentrations determined by the candidate reference method are used for comparing field methods with the candidate reference method. If the resulting regression equation is used in the calibration procedure, good correlation between all (in)direct field methods and the candidate reference method is ensured, and accurate results are produced. Results of proficiency testing show a good correlation between (in)direct field methods and the candidate reference method, because sera with approximately normal water contents are used.     

Proposal of a candidate international conventional reference measurement procedure for free thyroxine in serum.

In the present paper the IFCC WG-STFT recommends and provides the rationale to establish metrological traceability of serum free thyroxine (FT4) measurements to a candidate international conventional reference measurement procedure. It is proposed that this procedure be based on equilibrium dialysis combined with determination of thyroxine in the dialysate with a trueness-based reference measurement procedure. The measurand is thus operationally defined as "thyroxine in the dialysate from equilibrium dialysis of serum prepared under defined conditions". With regard to the trueness-based reference measurement procedure, the WG-STFT recommends use of an isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC/tandem MS) procedure for total thyroxine that has been optimized towards measurement at picomolar concentration levels and that is listed in the database of the Joint Committee for Traceability in Laboratory Medicine (JCTLM). For calibration, the purified thyroxine material IRMM-468 (resulting from a project funded by the European Commission and recently submitted to the JCTLM) is proposed. The WG-STFT stresses that according to this recommendation it is a prerequisite to strictly adhere to the defined equilibrium dialysis procedure, whereas it is permissible to introduce variants in the ID-LC/tandem MS procedure.     

Gas-chromatographic determination of cholesterol in serum: candidate reference method

We present a candidate Reference Method for determination of total cholesterol in serum. The method is based on high-resolution capillary gas chromatography, and we took special precautions with respect to weighing, calibration, and chromatographic peak integration. Analytical recovery of added cholesterol was essentially complete (99.99%, SD 0.48%) and reproducibility was excellent (total CV 0.35-0.50%). When cholesterol was determined in a reference serum certified by means of an established candidate Definitive Method, no significant bias could be detected.     

A reference method for measurement of alkaline phosphatase activity in human serum

We present an official AACC reference method for the measurement of alkaline phosphatase, the culmination of optimization experiments conducted by a group of independent laboratories. The details of this method and evaluation of factors affecting the measurement are described. A metal ion buffer has been incorporated that maintains optimal and constant concentrations of zinc(II) and magnesium(II) ions. Final reaction conditions are: pH (30 degrees C), 10.40 +/- 0.05; 2-amino-2-methyl-1-propanol buffer, 0.35 mol/L; 4-nitrophenyl phosphate, 16.0 mmol/L; magnesium acetate, 2.0 mmol/L; zinc sulfate, 1.0 mmol/L; and N-(2-hydroxyethyl)ethylenediaminetriacetic acid, 2.0 mmol/L.     

Isotachophoretic determination of urea-ammonium in plasma: a candidate reference method

Separation and determination of sample constituents by capillary isotachophoresis are entirely based on physical phenomena. The method has therefore been proposed as a universal reference method for ionic constituents. The present paper shows that even neutral species can be adequately determined after suitable preceding reactions. Urea was completely hydrolysed by urease (EC 3.5.1.5) to ammonia and bicarbonate, followed by direct measurement of the ammonium ion concentration by capillary isotachophoresis. Standard Reference Material No. 912a urea (National Bureau of Standards) was used as a primary standard. The analytical linear range of the method extends to 64 mmol urea per litre. The precision of the method was in the range of 1.05-2.64% (CV) and the analytical recovery of added urea was excellent (99.4%, SD 1.13%). Further proof of accuracy was obtained by analysing the NBS human reference serum (standard reference material 909). The mean result by the capillary isotachophoretic method, 9.52 +/- 0.085 mmol/l, agrees well with the reference value, 9.64 mmol/l. The results obtained by capillary isotachophoresis showed good agreement with those obtained by the coupled-enzyme method (r = 0.995).     

糖化血红蛋白液相色谱-质谱参考测量程序测量结果的不确定度评定

目的探讨液相色谱-质谱(LC-MS)参考测量程序(又称:参考方法)测量人全血中糖化血红蛋白(HbA_(1c))结果的不确定度评定方法。方法建立国际检验医学溯源联合委员会(JCTLM)推荐的HbA_(1c)参考测量程序——LC-MS法,按照国际临床化学与检验医学联合会(IFCC)HbA_(1c)标准化工作组实验方案对样品进行测量,分析测量不确定度分量来源,评定各分量,合成标准不确定度,计算扩展不确定度,确定校准和测量能力(CMC)。结果人全血HbA_(1c)浓度为32.8 mmol/mol、71.8 mmol/mol、52.4 mmol/mol、100.9 mmol/mol、59.6 mmol/mol时,其相对扩展不确定度分别为2.9%、1.5%、1.9%、1.3%和1.7%(95%置信区间,包含因子k=2)。参考测量能力范围为32.8 mmol/mol至100.9 mmol/mol时,其CMC为1.3%。结论该参考实验室评定了LC-MS参考测量程序HbA_(1c)检测结果的测量不确定度,满足了对HbA_(1c)参考测量能力的要求。     

Comparison of five routine methods with the candidate reference method for the determination of bilirubin in neonatal serum

Using five routine methods and the candidate reference method of Doumas (Clin. Chem. 31, 1779-1789 (1985)), total bilirubin was determined in 77 neonatal serum samples (concentration range 63-444 mumol/l, average value 227 mumol/l). Four of the routine methods (Jendrassik & Grof's (Biochem. Z. 297, 81-89 (1938)) method, Hertz's (Scand. J. Clin. Lab. Invest. 33, 215-230 (1974)) method, the bilirubinometer procedure, and the method employing 2,5-dichlorophenyldiazonium (Scand. J. Clin. Lab. Invest. 29, Suppl. 126, Abstr. 11. 12. (1972]) gave values that were generally higher than those of the reference method. In contrast, the results from Vink's (Clin. Chem. 34, 67-70 (1988] direct spectrophotometric method differed only negligibly from those of the reference method. The accuracy of Jendrassik & Grof's method, and to a limited extent that of the 2,5-dichlorophenyldiazonium method, can be improved by redetermination of the molar absorption coefficient, or by using a standard containing a matrix of human albumin, with an assigned value determined by the reference method. It was found that Hertz's direct spectrophotometric method can be replaced by that of Vink. The accuracy of the bilirubinometer results could be improved only by using calibrators with assigned values specific for the bilimeter, or by calibration with a serum pool.     

Liquid chromatography as candidate reference method for the determination of vitamins A and E in human serum

BackgroundOwing to the increasing interest in public health research of antioxidant micronutrients and the inaccuracy of routine serum concentrations of the fat‐soluble vitamins A (retinol) and E (DL‐α‐tocopherol) measurements, we developed a reliable, highly sensitive, robust and rapid method for the quantification of two clinically important lipophilic antioxidants in serum using a reverse‐phase HPLC/DAD method.MethodSample preparation and analytical conditions that would affect extraction efficiency and quantitative results of vitamins A and E were investigated and optimized. Vitamins A and E were extracted from serum via liquid‐liquid extraction (LLE). After adequate sample preparation, the samples were injected directly into the HPLC system with diode‐array detector (DAD). Chromatographic separation was completed in 7 minutes for vitamins A and E. With vitamin A acetate and vitamin E acetate as internal standards, the method was applied to the measurement of vitamins A and E in human serum.ResultsWe evaluated method linearity, accuracy (recovery rate and trueness), precision, carryover, limit of quantitation and limit of detection, and measurement uncertainty. The method was evaluated for trueness using NIST Standard Reference Material SRM 968f. The serum concentration of the studied compounds had a good linear relationship in the range of 0.05 ~ 3.0 μg/mL concentration (r ＝ 0.9998), with 0.0077 μg/mL detection limit and 0.025 μg/mL quantitative limit for vitamin A, respectively, and 1.0 ~ 60.0 μg/mL concentration (r ＝ 0.9999), with 0.40 μg/mL detection limit and 0.50 μg/mL quantitative limit for vitamin E, respectively. The intra‐ and inter‐assay coefficients of variation were calculated by using three concentrations (1, 2, and 3) of the studied compounds in human serum samples. Intra‐assay and inter‐assay precision were 1.23%‐4.97% and 0.97%‐3.79% for vitamin A, respectively, and 0.64%‐4.07% and 0.81%‐5.96% for vitamin E, respectively. The average recovery rates were 100.98% for vitamin A, and 99.21% for vitamin E, respectively. The carryover rate of vitamins A and E was below 1%. As for the evaluation of accuracy, the biases were <± 5% by comparing with NIST standard reference material SRM 968f.ConclusionThe method is a simple sample treatment procedure for the determination of fat‐soluble vitamins A and E in human serum with high sensitivity and specificity. The proposed method could be recommended as a candidate reference method for the determination of serum concentrations of the fat‐soluble vitamins A and E in human serum.     

Determination of the cardiac glycosides digoxin and digitoxin by liquid chromatography combined with isotope-dilution mass spectrometry (LC-IDMS)--a candidate reference measurement procedure

This article describes a method of high analytical sensitivity, reproducibility and trueness for the determination of digoxin and digitoxin in serum or plasma at therapeutic levels using a combination of high-pressure liquid chromatography (HPLC), isotope-dilution mass spectrometry (IDMS) and caesium-adduct formation. A method for threefold deuterium substitution in the glycosides was developed, which could be performed within 24 hours without distillation giving yields > 98% of the theoretical value. Extraction from a serum or plasma matrix was performed using a liquid-phase extraction with ammonium acetate buffer/tertiary butylmethyl ether/ethyl acetate at pH 9.5. The HPLC-separation used a 10 x 2 mm LiChrospher RP-18 5 microm guard column in combination with a 125 x 2 mm main column of the same material and a gradient containing methanol, caesium ions and formic acid. Quantification of digoxin and digitoxin was made with IDMS using deuterated internal standards and the system run in single ion monitoring (SIM) mode. The methods had a lower limit of determination of 0.25 microg/l for digoxin and digitoxin, a trueness between 97.5 and 104% for digoxin and between 98 and 101% for digitoxin, respectively and had a coefficient of variation of less than 3% in the therapeutic range for both glycosides. Maximally 1 ml serum or plasma was needed for the procedure. The method is used to set target values for materials used in external quality assessment surveys (EQAS) run by INSTAND as part of a national EQAS-programme.)     

离子色谱法测定血清锂离子候选参考方法的建立及性能评估

目的：以离子色谱法为基础,建立血清总锂离子测定的候选参考方法并评价其性能。方法采用一种简单的样本前处理方法,用新鲜患者血清样本、IFCC-RELA 样本等对方法的精密度进行评估;用加标回收实验评价方法的准确性。结果锂离子标准曲线线性方程为：Y ＝0．8171X -0．0013,r^2＝0．99995,方法的检出限为6μg/L;测量血清样本的不精密度（CV）小于1．0％;样本的加标回收率在99％～101％之间。结论成功建立了血清锂候选参考方法,可用于血清锂离子项目的量值溯源和标准化,为血清锂常规检测系统向参考方法/参考物质溯源提供了有效途径。     

A semi-automated method for the determination of glycosylated haemoglobin.

A new routine, automated method for the determination of glycosylated haemoglobin is presented. The method offers improvements in analytical capacity, and precision, together with reduced running costs over existing methods, which should assist in making the determination of glycosylated haemoglobin more readily available to the clinician treating diabetes.