The alkaline haematin detergent (AHD575) method for the determination of haemoglobin in blood--a candidate reference measurement procedure

The article describes the development and evaluation of the alkaline haematin detergent (AHD575) method for the determination of haemoglobin in blood without the need for toxic materials and suitable for use in laboratories in countries with limited resources and restricted import of toxic materials. The validation of the method has been performed in accordance with the requirements set out in the international standard ISO 15193, which describes the procedures necessary for development of a candidate reference measure-ment procedure. The main results were: The trueness of the haemiglobin cyanide (HiCN) method depends upon the diluent used. The international haemiglobin cyanide reference material BCR CRM 522 does not have the same chemical properties as that derived from fresh lysed erythrocytes and cannot be used for calibrating the AHD575 method. The transformation of oxyhaemoglobin to haematin at pH 13 proceeds 5-8 times more rapidly than its conversion to haemiglobin cyanide. The AHD575 method yields results comparable with the HiCN method and uses a readily available crystalline standard of high purity. The introduction of the AHD575 method does not require new reference intervals, the values being directly transferable from (commutable with) the established HiCN procedure.     

The determination of total haemoglobin as haemiglobin

This study describes a new method for routine total haemoglobin determination by conversion to haemiglobin (Hi), and compares it with the haemiglobin cyanide (HiCN) method. Experiments were performed in two phases. Firstly, the absorbance coefficient (epsilon Hi,500mm) at pH = 6.8, determined from 24 blood samples, was found to be 998 +/- 31 m2.mol-1. Secondly, using this value of epsilon, the range of haemoglobin concentration measured was 3.7 to 19.2 mmol.l-1 (6 to 31 g.dl-1). The correlation data shows an excellent correlation (r = 0.982, p less than 0.001) with the reference procedure. Reproducibility and accuracy were shown to be good.     

Stability constants of haemoglobin cyanide and azide measured by two-wavelength spectrophotometric method

A two-wavelength spectrophotometric procedure for the simultaneous determination of haemoglobin and haemoglobin cyanide (HiCN) (or of haemoglobin and haemoglobin azide (HiN3)) concentrations in mixtures has been developed and applied to the determination of the stability constants of HiCN and HiN3. The analytically reliable procedure allowed stability constants to be estimated with about 10% (relative standard deviation, coefficient of variation) uncertainty. Values of 1.9 X 10(6) and 2.0 X 10(5) 1 X mol-1 were obtained for HiCN and HiN3, respectively. These results are discussed in relation to the optimal composition of the reagents for blood haemoglobin assay by the two methods.     

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).     

Plasma haemoglobin determination using chlorpromazine as a non-carcinogenic reagent

Plasma haemoglobin was assayed with the non-carcinogenic reagent phenothiazine. This method is sensitive and allows the measurement of plasma haemoglobin concentrations in the range 4-500 mg/l with a within-run CV of 2.1%, and a between-run CV of 4.3%. A spectrophotometric scanning method (x) based on the determination of haemoglobin as haemiglobin cyanide using the Soret band at 419 nm correlated well with the phenothiazine method (y): y = 1.07x + 15.8, r = 0.995, n = 31. It was found that the absorbances in the phenothiazine method were markedly dependent on the concentration of phosphoric acid.     

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.     

氰化高铁血红蛋白国家一级标准物质的研制

目的 研制氰化高铁血红蛋白(HiCN)国家一级标准物质,用作血红蛋白测定结果溯源的标准.方法 参照国际血液学标准化委员会(ICSH)的要求,制备HiCN标准物质;按照ISOGuide 35的要求,评价标准物质的均匀性和稳定性,在二者合乎要求的基础上,由多个实验室使用溯源至美国国家标准技术研究所(NIST)标准滤光片的分光光度计为HiCN标准物质定值;为验证定值结果的可靠性,使用定值仪器测定国际标准物质,将测定结果与WHO参考实验室的定值进行比较,此外,对所研制标准物质和国际标准物质的扫描图形进行了比较.结果 HiCN标准物质均匀性的不确定度为0.000 4 g/L,变异系数(CU)为0.09%;长期稳定性的不确定度为0.000 6 g/L;HiCN标准物质的定值为0.6159 g/L,不确定度为0.000 4 g/L;当扩展因子取2时,标准物质的扩展不确定度为0.001 8 g/L;定值仪器对国际标准物质的测定结果与国际标准物质定值的相对偏差为0.08%.结论 HiCN标准物质均匀性和稳定性良好,定值方法准确、可靠.     

Sulfhaemoglobin. Absorption spectrum,millimolar extinction coefficient at λ = 620 nm,and interference with the determination of haemiglobin and of haemiglobincyanide

The spectrophotometric properties of sulfhaemoglobin (SHb) and some derivatives were investigated using an improved technique for measuring the SHb fraction induced in human blood samples. The millimolar extinction co-efficient of SHb at λ = 620 nm was found to be 20.8 (S.D. 1.48; S.E. = 0.44; n = 11). In addition it was demonstrated that the spectral changes occurring in SHb containing haemoglobin solutions upon the addition of KCN, K₃Fe(CN)₆ and K₃Fe(CN)₆ + KCN invalidate the KCN addition method for the determination of haemiglobin. The influence of clinically occurring SHb fractions on the internationally standardized total haemoglobin determination were shown to be insignificant.     

Evaluation of two commercially available photometers for blood donor haemoglobin measurement

Two commercially available portable haemoglobin measuring devices (Delphi Haemoglobin Meter and HemoCue) were evaluated both within the laboratory and under routine operating conditions in blood donation collection centres. Both meters demonstrated satisfactory accuracy when compared to the reference cyanmethaemoglobin (HiCN) method. Within-run and between-run precision of both meters was satisfactory. There was no significant difference evident between capillary and venous haemoglobin results using either meter. In the main blood collection centre, using Delphi meters, 63.1% of results returned from an external proficiency testing program were acceptable (± 2SD of Quality Control Laboratory result). In satellite centres using Delphi meters 36.5% were acceptable while 60.0% of HemoCue results from satellite centres were acceptable. Assay time was almost twice as long using the HemoCue. The study confirmed the necessity to evaluate equipment and operators under both controlled and routine operating conditions. External proficiency testing programs are recommended to monitor operator performance on a regular basis.     

Ethylene glycol-stabilized haemolysates as control material in haemoglobinometry

Human blood haemolysates containing ethylene glycol (final volume fraction 0.35) were prepared and stored at -20 degrees C (in the liquid state) up to 372 days. During the whole period, the total haemoglobin concentration (assayed in the material by means of the reference HiCN method) was found to be stable; spectral analysis also failed in detecting any deterioration, Hi formation being low. Good stability was also recorded on storage at 2-4 degrees C for 15 days, but only for 1-2 days at room temperature. The stabilised haemolysate is suggested as a material for long-term control of accuracy in hemoglobinometry.     

不同方法测定血红蛋白溶液中总蛋白量的比较

目的采用三种方法对含有大量血红蛋白溶液的样本测定总蛋白量,并进行比较,确定一种可行且简单的测定血红蛋白溶液中总蛋白量的方法.方法分别采用BCA法、Bradford法和Lowry法测定溶液总蛋白量,并采用HiCN法测定溶液中血红蛋白的含量.比较不同方法测得数据的差异.结果 5份样本用BCA法测得总蛋白量为76.33 g/L,Bradford法测得值为179.59 g/L,Lowry法测得值为22.81 g/L.HiCN法测定血红蛋白量为133.65 g/L.结论 Bradford法是一种可行且重复性好的测定血红蛋白溶液中总蛋白量的方法,BCA法和Lowry法的测定结果明显低于实际值.     

血红蛋白测试仪在献血者初筛中的应用及效果评价

目的 探讨采供血机构所使用的血红蛋白微片法测定血红蛋白含量(Hbg/L)的实际意义和效果评价.方法 分别采用氰化高铁血红蛋白(HiCN)法、硫酸铜比重法、血红蛋白微片法、血球计数仪检测法,测定血红蛋白含量为高、中、低三种浓度的样本,各50～60次;并对500份随机献血者血样用硫酸铜比重法、血球计数仪及血红蛋白微片法同时进行检测,使用SPSS统计软件对测定结果 进行统计分析.结果 除硫酸铜比重法外,其他三种方法 的检测结果 均存在统计学差异,但对仪器进行校验后的再次检测结果 表明,微片法和血球计数仪的检测结果 不存在统计学差异.结论 当计量局无法对采供血机构所使用的计量仪器进行强检时,我们可以通过其他方法 对仪器进行对比,以达到对仪器进行校验的目的 .血红蛋白微片法测试血红蛋白含量(Hb)可代替硫酸铜法用于流动采血车上献血者的血红蛋白初筛实验.     

Standardization of a simple method for the estimation of haemoglobin A2

An improved method for the estimation of haemoglobin A2, at the same time precise, simple and cheap, is proposed. Haemoglobin A is separated from haemoglobin A2 by electrophoresis on Cellogel in discontinuous buffer at alkaline pH. The strips of cellulose acetate containing the haemoglobin fractions are completely dissolved in 80% acetic acid. The percentage of haemoglobin A2 present in each sample is calculated from the values for the spectrophotometric absorbance at 396 nm. The average percentage of haemoglobin A2 (+/- standard deviation) determined by this method was 2.31 +/- 0.37 in 51 normal subjects, and 4.64 +/- 0.53 in 29 subjects with heterozygous beta-thalassaemia.     

十六烷基三甲基溴化铵溶血剂的探讨

本文用扫描图形证明十六烷基三甲基溴化铵与血红蛋白的反应液有恒定图形，其５４０ｎｍ的吸光度与血红蛋白的浓度成正比，与氰化高铁血红蛋白在５４０ｎｍ的吸光度高度相关。用本法测定血红蛋白精密度、准确度、稳定性、线性均可满足临床需要。高胆红素、高血脂、高球蛋白对本试验无干扰，可用定值溶血液校准仪器，试剂无毒性、稳定易于保存且可同时测定白细胞，白细胞测定的精密度及稳定性都较为理想。     

Analytical goal setting prior to selection of a method for glycated haemoglobin

Without a consensus on either a reference method or a single glycated haemoglobin standard, the individual laboratory has to establish and secure its own assay method. Prior to selection of a method for glycated haemoglobin we used an assessment model and defined our clinical goals. Based on clinical goals it was possible to set up goals of analytical quality and evaluate available literature concerning the performance characteristics of different assay methods. We wanted a method that measured HbAlc, without measuring the labile intermediate pre-HbAlc, and that provided separate detection of haemoglobin variants. Low imprecision was required to secure the measured, non-diabetic reference interval, and to minimize random analytical error. We found the isoelectric focusing method the only one able to meet our goals, and a method evaluation was carried out. Clinical goals of analytical quality should always be defined before method selection and implementation. Using this model, we were able to establish a precise isoelectric focusing method that fulfilled our clinical goals by measuring HbAlc with a total coefficient of variation of 2.1% and a non-diabetic reference interval from 5.2% to 6.8% HbAlc.     

The determination of free and protein-bound haemoglobin in plasma using a combination of HPLC and absorption spectrometry

The aim of the study was to develop a method for the determination of haemoglobin in plasma suitable for use to set target values for external quality assessment schemes for this analyte using commercially available test kits and equipment. In the early phase of the method development it became clear that the use of a single method, namely HPLC, would not be possible. However, by combining HPLC and absorption spectrophotometry, both qualitative and quantitative rapid determinations of protein-bound and free haemoglobin were able to be performed on equipment present in most routine clinical chemistry laboratories. The separation of protein-bound and free haemoglobin could be carried out using commercial HPLC equipment for the determination of haemoglobin A1c (HbA1c) without modification of the conditions used. Instead of haemolysed blood, the same volume of plasma (10 microl) was injected. The eluate was not discarded, but collected in 1-minute fractions so that the void volume (protein-bound Hb) and the haemoglobin peaks (free Hb) were available for the colorimetric determination of haemoglobin using the pseudoperoxidase activity of the haem moiety on hydrogen peroxide and a chromogen (3,3',5,5'-tetramethylbenzidine) in concentrated acetic acid and optimal determination at 600 nm. (In this publication at 578 nm due to the use of a spectrophotometer with Hg-discharge lamp and filter). The appearance of a blue colour in the reaction tube or cuvette indicated the presence of haemoglobin. The use of the above chromogen, with its absorption maximum around 600 nm excluded interference from serum components such as bilirubin, which may interfere in the conventional method often used to determine plasma haemoglobin. The method can be used quantitatively by including an aqueous human haemoglobin standard in the run. This elutes from the HPLC column only as free haemoglobin in the concentration range from 0.1 to 10 g/l. Addition of human haemoglobin to haemoglobin-free plasma resulted in the binding of all Hb to plasma proteins up to a concentration between 2 and 3 g/l (void-volume fraction). At higher concentrations free Hb appeared in the 3-5 minute fractions. These observations agree with published data on the scavenging capacity of plasma for Hb released from erythrocytes. The method is rapid, (HPLC-run maximally 6 min, quantitative colorimetric results 5-10 min) precise (inter-assay coefficients of variation < 8%) and suitable for answering the question as to whether the protein-binding (scavenging) system which prevents the nephro- and cerebrotoxic effects of haemoglobin has been saturated or not, an important question in patients with acute haemolysis problems. A qualitative result is obtainable within 10 minutes of injecting the sample into the HPLC-system. The use of this assay in controlling blood transfusion and haemolytic events arising from surgery, intravascular haemolytic bacteria or artificial heart valves can help in rapid corrective action, if needed.     

A simple and rapid method for the estimation of serum haemoglobin binding capacity on ultrogel® AcA 44

A method is described for the estimation of serum haemoglobin binding capacity. The method is based on the separation of the high molecular weight haemoglobin haptoglobin complex formed after addition of haemoglobin in excess to serum from uncomplexed haemoglobin by gel filtration through Ultrogel AcA 44. Agar gel electrophoresis and benzidine staining proved that the first fraction was exclusively the haemoglobin haptoglobin complex. The second peak consisted of free haemoglobin. In this method the amount of haemoglobin bound to haptoglobin is calculated from the absorbance of the chromatographed complex at 412 nm. The results obtained are reproducible and comparable with those of an electrophoretic reference method, but less precise. The method is simple, and accurate, and is faster than gel filtration methods described earlier.     

A comparative analysis of factors influencing haemoglobin content in RBC units

Background and objectivesThe increment in a patient’s haemoglobin level is based upon the haemoglobin content of the transfused RBC units. The total haemoglobin present in the blood bags can vary because of the blood donor, processing method, volume and type of bag used. The study is done to analyse the factors causing variation of haemoglobin content in RBC units.Materials and MethodsA total of 260 RBC units were tested for the haemoglobin content and analysed with the donor variables (age, gender, weight & capillary haemoglobin). The blood bags were then separated into two groups based on the donor capillary haemoglobin (normal 12.5–15.0 g/dL vs high 15.1–18.0 g/dL), volume (350 vs 450 mL), processing method (Platelet rich plasma vs buffy coat) and further analysis was carried out.ResultsThe mean haemoglobin content was 54.7 g ranging from 34.2–80 g per unit. The factors which significantly influenced (p < 0.0001) were capillary haemoglobin, gender and weight of donor, volume of blood collected and the processing method. There was a significant difference (p < 0.0001) in haemoglobin content between the two groups in all the three categories (capillary haemoglobin, volume and processing method). Regression analysis showed all three of them contributed to 80 % variability of haemoglobin content in the RBC unit.ConclusionThe marked variation of haemoglobin content in our study revealed that there is a need for standardizing RBC unit. Labelling of units with haemoglobin content and transfusion based on it will result in better patient care.     

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.     

Haemoglobin A2 measurement using high performance liquid chromatography.

Haemoglobin A2 levels were assessed using a modular high performance liquid chromatography (HPLC) system with a protocol designed for the measurement of haemoglobin A1C. There was good correlation (r = 0.96; P < 0.001) between this technique and the International Committee for Standardisation in Haematology (ICSH) recommended method of microchromatography. The range of haemoglobin A2 was found to be 2.3-3.2% in apparently normal individuals, and 4.0-6.7% in those with beta-thalassaemia trait. The HPLC system produced reliable results quickly for haemoglobin A2 with no alteration to the protocol used for measuring haemoglobin A1C.