Quantitative nephelometric assay for determining myoglobin evaluated

A recently introduced automated nephelometric immunoassay involving shell/core particles for determination of myoglobin (Behringwerke) was evaluated with the BNA Nephelometer. Method precision was good: the intra-assay CV varied between 1.5% and 6.1%; with daily calibration, the interassay CV ranged between 1.5% and 7.5%. For usual sample dilutions, the assay response varied linearly with myoglobin concentrations up to 23.1 nmol/L. After automatic dilution by the instrument, concentrations up to 2310 nmol/L could be measured without high-dose "hook" effect. Further manual dilution allowed measurement of myoglobin concentrations up to 26,000 nmol/L. Calibration was stable for at least seven days. We detected no significant interferences from hemoglobin, haptoglobin, bilirubin, iodine-containing contrast media, and rheumatoid factors. Treating lipemic samples with Lipoclean (Behringwerke) decreased test results. Simultaneously drawn serum and plasma samples from the same subject showed no consistent differences in myoglobin concentrations. The mean reference myoglobin concentration was 1.380 (SD 0.82) nmol/L for men and 0.878 (SD 0.45) nmol/L for women. In patients with renal insufficiency, serum creatinine values were moderately related to serum myoglobin values (r = 0.465). Although a commercial radioimmunoassay (Byk-Sangtec) and the nephelometric assay intercorrelated well (r = 0.929), values obtained by nephelometry were significantly lower (P less than 0.05). By both assays, results for heart and skeletal muscle tissue extracts showed no correlation, a finding that suggests the existence of multiple forms of myoglobin in human tissues. We conclude that immunonephelometry is a rapid, practical, and reliable method for measuring myoglobin in serum.     

Examination of a competitive enzyme-linked immunoassay (CELIA) technique and a laser nephelometric immunoassay technique for the measurement of apolipoprotein B

A competitive enzyme-linked immunoassay (CELIA) technique for quantitative measurement of apolipoprotein B (Apo B) was developed. The method is a non-isotopic immunoassay that utilizes a soluble enzyme/antibody complex as a universal labeling reagent. The method was characterized according to precision, sensitivity, recovery and parallelism. The CELIA Apo B method was compared to a commercially available laser nephelometric immunoassay. We found that the nephelometric results were highly correlated with triglyceride levels and the nephelometric assay was susceptible to interference from lipemia or turbidity. The range of values obtained on 56 apparently healthy, fasting young adults was 0.35-1.25 g/l by the CELIA method and 0.40-1.00 g/l by the nephelometric immunoassay. The nephelometric method was more precise (coefficient of variation 5%) than the CELIA technique (CV 10%); however, the CELIA method seems to be less sensitive to interferences.     

Kinetic determination of serum haptoblobin with a centrifugal analyzer.

We describe a method for determining haptoglobin with a centrifugal analyzer that is based on haptoglobin combining stoichiometrically with hemoglobin to form a complex that has peroxidase-like activity proportional to the quantity of haptoglobin present. Under assay conditions, unbound hemoglobin exhibits only a small fraction of the total peroxidase activity. Activity is measured colorimetrically at 405 nm after reaction with o-dianisdine and ethyl hydrogen peroxide. The procedure is standardized by saturating aknown amount of hemoglobin with a serum whose hemoglobin binding capacity exceeds the amount of hemoglobin in the assay system. The mean and mean within-run precision of our method, determined by performing 17 replicate assays of both a pooled normal serum and a 10-fold dilution of the serum, was 1.13 g/liter (CV, 2.9%), and 106 mg/liter (cv, 5.8%), respectively. The 95 percentile estimate of the normal range by our method is 0.45-1.85 g/liter hemoglobin binding capacity. When results by our automated method were compared to those by a manual method [Scand. J. Clin. Lab. 2nvest. 18, 80 (1965)], the slope of the unweighted linear least-squares regression line was .970 the y-intercept 26 mg/liter, and the correlation coefficient .995.     

Development of a microparticle-enhanced nephelometric immunoassay for quantitation of human lysozyme in pleural effusion and plasma

A microparticle-enhanced nephelometric immunoassay, based on polystyrene beads coated with antihuman lysozyme antibody, has been developed for lysozyme quantification in sera and pleural effusions. The standard curve extends from 0.58 mg/l to 18.75 mg/l and no antigen effect was observed. The results showed a good serial precision. The intra-assay precision (n = 20) expressed as CV was between 2.2 and 4.2 in three different concentrations. The inter-assay precision, with different calibration curves (n = 12) was between 6.4 and 7.1. The analytical assay showed a sufficient linearity (r > 0.999). There were no interferences either with haemoglobin (up to 4 g/l), lipids (up to 0.5%, expressed as 1% Lipofundina content), or bilirubin (up to 5 mg/dl). The analytical sensitivity was lower than 0.6 mg/l. The correlation with a Micrococcus lysodeikticus turbidimetric assay showed a correlation coefficient of 0.915. We have studied 92 patients with pleural effusion. In each case, pleural fluid adenosine deaminase activity and pleural fluid to plasma lysozyme ratio were determined. The lysozyme ratio showed similar clinical sensitivity and specificity as to adenosine deaminase.     

全自动血液分析仪 Sysmex XT-2000i 与 Mindray BC-5800的比较分析

目的：比较分析 Sysmex XT-2000i 和 Mindray BC-58002类全自动血液分析仪各项检验参数结果的精密度和相关性。方法选取60例 EDTA-K2抗凝静脉全血,对2台仪器各参数进行相关性分析;选取低、中、高值标本分别用2台仪器检测10次,对其进行精密度分析。结果测定 Sysmex XT-2000i 血细胞计数各参数变异系数（CV）为0．02％～5．70％,白细胞分类各参数 CV 为0．06～1．66％,Mindray BC-5800血细胞计数各参数 CV 为0．02％～5．30％,白细胞分类各参数 CV 为0．09％～1．17％。2台仪器间血细胞计数各参数的决定系数（r2）为0．185～0．995。结论2台仪器各参数精密度与设计范围相符合,二者之间的相关性较好。     

Measurement of eleven serum proteins by microparticle-enhanced nephelometric immunoassay.

Eleven proteins (immunoglobulins IgG, IgA, IgM, orosomucoid, alpha 1-antiproteinase, haptoglobin, ceruloplasmin, C-reactive protein, transferrin, prealbumin and alpha 2-macroglobulin) in human serum were quantitated by a new microparticle-enhanced nephelometric immunoassay. This is a one step competitive assay, based on the nephelometric measurement of light scattered by clusters of protein-coated microparticles specially synthesized for that use. Statistical evaluation (precision, recovery and method comparison) shows that the determination of serum proteins is reliable and accurate for wide ranges of concentration and that the method is quite adequate for strongly increased concentrations. This microparticle-enhanced nephelometric immunoassay appears to offer an alternative method for routine measurement of a great variety of serum proteins at high and intermediate concentrations, which are usually quantified by radial immunodiffusion or conventional immunonephelometry. On account of its sensitivity, it can also be used for the determination of relatively low concentrations of analytes.     

Analysis of inflammatory response in human plasma samples by an automated multicapillary electrophoresis system.

A new automated multicapillary zone electrophoresis instrument with a new high-resolution (HR) buffer (Capillarys with HR buffer) for analysis of human plasma proteins was evaluated. Albumin, alpha(1)-antitrypsin, alpha(1)-acid glycoprotein, haptoglobin, fibrinogen, immunoglobulin (Ig)A, IgG and IgM were determined nephelometrically in 200 patient plasma samples. The same samples were then analyzed on the Capillarys system (Sebia, Paris, France). The albumin concentration from the nephelometric determination was used for quantification of the individual peaks in the capillary electrophoresis (CE) electropherogram. There was strong linear correlation between the nephelometric and electrophoretic determination of alpha(1)-antitrypsin (R(2) = 0.906), alpha(1)-acid glycoprotein (R(2) =0.894) and haptoglobin (R(2) = 0.913). There was also good correlation between the two determinations of gamma-globulins (R(2) = 0.883), while the correlation was weaker for fibrinogen (R(2) = 0.377). The Capillarys instrument is a reliable system for plasma protein analysis, combining the advantages of full automation, good analytical performance and high throughput. The HR buffer in combination with albumin quantification allows the simultaneous quantification of inflammatory markers in plasma samples without the need for nephelometric determination of these proteins.     

Comparison of two methods for rapid determination of C-reactive protein with the Tina-quant

C-reactive protein (CRP) as an acute phase protein is an important diagnostic marker for the presence and course of human processes. Out of the acute phase proteins it is one of those the concentrations increase most rapidly with its sensitivity being superior to other markers of inflammation, such as leukocytosis, erythrocytic sedimentation rate, and fever. This study compared two-point-of-care assays with the standard laboratory method Tina-quant CRP processed on a Hitachi 917: the immunofiltration assay NycoCard CRP Whole Blood and the turbidimetric immunoassay Micros CRP. Both methods are carried in the presence of a patient, by using capillary or venous blood. Seventy-eight blood samples were analyzed first in the standard laboratory routine and then by both rapid test assays. The precision of both assays was determined from the confidence interval. The results were statistically analyzed by arithmetic standard deviation mean method, variation coefficient, Spearman correlation index, Wilcoxon and Bland-Altman tests, and Passing-Bablock regression. NycoCard CRP Whole Blood showed a correlation coefficient of R = 0.9838; the precision had a coefficient of variation of CV = 1.8759% while As compared with Tina-quant CRP had R = 0.9934 and CV = 0.9160%. Both assays indicated the same results as Tina-quant CRP. Both Tina-quant CRP and NycoCard CRP Whole Blood give the best fit for the rapid determination of CRP.     

Diana-5全自动血液分析仪白细胞分类与镜检的相关性研究

目的对Diana-5全自动血液分析仪白细胞五分类与镜检的相关性进行研究与评价。方法系统研究Diana-5全自动血液分析仪白细胞分类测定的精密度、总变异、镜检率、敏感性、特异性和过筛符合率,并分析比较与镜检结果的相关性。结果仪器分类测定中性粒细胞(Neu)、淋巴细胞(Lym)、单核细胞(Mon)、嗜酸性粒细胞(Eos)和嗜碱性粒细胞(Bas)的精密度分别为1.25%、1.53%、5.96%、7.58%和15.85%,总变异分别为1.82%、2.86%、7.84%、9.32%和17.32%。与镜检结果的相关系数分别为0.982、0.956、0.842、0.536和0.328。分类镜检率为20.2%,敏感性98.0%,特异性87.2%,过筛符合率88.1%。该仪器可提示异常白细胞的检测信息,其中非成熟细胞占异常提示信息的51.5%。结论Diana-5血液分析仪白细胞分类测定的自动化程度高,结果重复性好,具有良好的相关性,能有效发挥仪器的过筛作用,非常适合血常规的快速检测。     

气质联用技术检测尿琥珀酰丙酮及其在酪氨酸血症诊断中的应用

目的建立气相色谱质谱用技术（GC-MS）测定尿琥珀酰丙酮（SA）的方法,用于临床上鉴别诊断酪氨酸血症Ⅰ型患者。方法尿SA先用盐酸羟胺进行污化反应,然后用乙酰乙酸2次萃取,双三甲基硅烷-三氟乙酰胺（BSTFA-TMCS）衍生化,衍生化后的SA用气相色谱质谱仪进行测定分析。质谱分析模式采用选择离子监测（SIM）模式进行检测,SA选择212/182 m/z,内标丙二酸选择233/248 m/z。分析该法的精确性、稳定性、样本回收率、残留分析和实测浓度与加入浓度之间的相关性。用该法检测肝肿大伴酪氨酸升高的13例患者。结果SA批内变异系数（CV）为6.8%;批间CV为7.8%;回收率为94.0%-102.0%;前处理CV为12.4%;残留分析〈1%。在5-120 mg/mL之间,实测浓度与加入浓度之间的相关系数（r）为0.997。内标丙二酸回收率为95.0%-103.0%。确诊酪氨酸血症Ⅰ型患儿2例,尿SA浓度分别为26、54 mmol/mol肌酐。结论GC-MS测定尿SA具有较高的回收率、精确性和准确性,为临床上鉴别诊断酪氨酸血症Ⅰ型提供了新的方法。     

Automated latex nephelometric immunoassay for the measurement of serum lipoprotein (a)

A sensitive immunoassay based on latex particle agglutination has been developed for measuring lipoprotein Lp(a) concentrations in serum or plasma. Carboxylated latex particles (diameter 240 nm) covalently coated with F(ab')₂ fragments of anti-lipoprotein Lp(a) antibodies are incubated with diluted sample (400-fold) for 12 min at room temperature, with the resulting agglutination quantified by measuring the change of light-scatter produced. The assay has been automated on the Behring nephelometer analyzer with a sampling rate of 150 samples/hour. This assay generates a standard curve in the range of 27 to 1750 mg/L, showing inter-assay precision of less than 8%. There were no interferences from plasminogen, bilirubin, Intralipid?, haemoglobin, rheumatoid factor, and apolipoprotein B. No significant differences were observed when fresh and frozen samples were compared. Sample pretreatment with “Lipoclean” clearing agent and sample lyophillization decreased the agglutinating reaction. In two separate studies using 77 and 112 patient sera the Lp(a) values, determined by the latex nephelometric method, the Terumo Macra? Lp(a) ELISA test, and the Pharmacia Apo(a) radioimmunoassay method, gave correlation coefficients of 0.948 and 0.974, respectively. Physiological lipoprotein (a) values were determined in a blood donor group, with the distribution of serum Lp(a) highly skewed, with a mean (SD) and median values of 213(236) mg/L and 116 mg/L, respectively. Concentrations of Lp(a) were found to be age-and sex-independent. This latex nephelometric procedure is a convenient method and an interesting alternative to other immunoassays for routine measurement of human lipoprotein (a). © 1993 Wiley-Liss, Inc.     

Three immunoassay methods evaluated for quantifying prealbumin (transthyretin) in serum

We developed an immunoturbidimetric assay for prealbumin on the Cobas Bio centrifugal analyzer and compared results from this assay with those from a rate nephelometric assay (Beckman Instruments Inc.) and a radial immunodiffusion kit (Behring Diagnostics). All three assays were evaluated for precision, linearity, and correlation to each other for analysis of sera from pediatric patients. All assays gave similar results for patients' samples. Values were higher by the radial immunodiffusion assay than by the other two methods, which gave similar results for the same specimens. We conclude that the immunoturbidimetric and rate nephelometric assay for prealbumin are acceptable alternatives for quantifying prealbumin in serum and also have a faster turnaround time than radial immunodiffusion.     

Analytical variability of the Fibrotest proteins

OBJECTIVES: The analytical variability of the Fibrotest (FT) parameters raises the issue of the test's reliability for routine use. Whereas standardization has been proposed by the International Federation of Clinical Chemistry (IFCC) for specific proteins, few data are available concerning the actual transferability of the FT proteins, i.e. haptoglobin, apolipoprotein A1 and alpha2 macroglobulin. The aim of this study was to evaluate the analytical variability of the FT proteins. DESIGN AND METHODS: During the FIBROPACA study, we evaluated 112 sera from patients with hepatitis C infection who underwent liver biopsy. We compared measurements of haptoglobin, apolipoprotein A1 and alpha2 macroglobulin by the autoanalyzers Immage (Beckman-Coulter) and the FT reference BNProspec (Dade-Behring). RESULTS: Optimal concordance was found for haptoglobin (correlation: y = 1.05x -0.09; correlation coefficient = 0.98). However, apolipoprotein A1 as determined with Immage was globally 12% lower than with BNProspec (correlation: y = 0.88x -0.05; correlation coefficient = 0.91) and alpha2 macroglobulin values were 40% greater with Immage than with BNProspec (correlation: y = 1.40x -0.46; correlation coefficient = 0.96). CONCLUSIONS: Inter-technique analytical variability of the Fibrotest parameters remains a major issue. After IFCC standardization of specific proteins, some discrepancies remain for alpha2 macroglobulin and, to a lesser extent, for apolipoprotein A1. National and international quality control programs would be useful to monitor analytical performance of protein assays.     

EP10-T2用于TPO-Ab检测的方法学评价

目的以EP 10-T 2作为临床实验方法评定标准,对微粒子酶免疫发光法(M E IA)检测血清甲状腺过氧化物酶抗体(TPO-A b)并进行方法学评价。方法根据国际临床实验室标准化委员会(NCCLS)颁布的《定量临床实验室方法初步评价试剂准则(EP 10-T 2)》的要求,用美国雅培公司AXSYM全自动化学发光仪进行TPO-A b的测定。结果低值、高值TPO-A bD的批内CV<5%,批间CV<5%。TPO-A b的总不精密度<2%,TPO-A b的线性相关系数为0.997。TPO-A b实验值均等于或接近于靶值,TPO-A b的携带污染率为0.4%,与临床诊断相关系数为1。结论用M E IA检测TPO-A b具有灵敏度高、特异性强、重复性好、精密度高及携带污染率低等优点。     

磺基水杨酸检测尿液和脑脊液蛋白自动比浊法的建立

目的对磺基水杨酸比浊法进行改良,并对其测定尿液和脑脊液蛋白的自动化分析进行评价。方法应用改良磺基水杨酸仪器比浊法(简称仪器法),在全自动生化分析仪上建立测定参数,评价其方法的精密度、线性范围、回收率,并与手工磺基水杨酸比浊法(简称手工法)进行比较。结果批内和批间变异系数(CV)分别为1.2%～1.5%和2.1%～3.5%。线性范围为0.037～2.38 g/L。回收率为85.6%～109.2%。与手工法比较,Y仪器=0.980 4X手工+0.010 8,相关系数(r)=0.982。结论改良的磺基水杨酸比浊法测定微量总蛋白具有较高的精密度和准确度,适合于全自动生化分析仪。     

Radioimmunoassay of total urinary estrogens.

We describe a radioimmunoassay for total estrogens in urine. Estrogens are extracted by adsorption on XAD-2 resin, eluted with methanol, and acid-hydrolyzed. Estrogens are then determined in a diluted aliquot of the hydrolysate. Within-run coefficients of variation for estriol values between 6.0 and 100 micrograms/L were less than 5%; between-run CV was 19.6% at an estriol concentration of 16.8 microgram/L. A correlation coefficient of 0.96 was found on comparison of the radioimmunoassay (y) with a standard fluorometric assay (x); the regression equation is y = 2.18 x - 6.70 micrograms/L. Determination on pregnancy and non-pregnancy urine can be done within 4 h.     

A novel use of endpoint nephelometry to standardize the rate nephelometric assay of human and rat plasma apoprotein A

A sensitive, accurate, and reliable method is described for calibrating the rate immunonephelometric assay of rat and human plasma apolipoprotein A (Apo A). Pure Apo A and high-density lipoprotein (HDL) of known Apo A concentration were used in endpoint nephelometry to determine Apo A concentrations of rat and human plasma pools. The endpoint method had coefficients of variation of 7.96% and 4.35% for rat and human plasma pools, respectively. These plasma pools were then used as secondary standards for the rate nephelometric assay. Excellent agreement (+/- 6%) existed between the plasma Apo A values determined by endpoint nephelometry and rate nephelometry. The Apo A concentration of a frozen human plasma pool determined by endpoint nephelometry was 125.2 +/- 9.6 mg/dl. The value of the same pool determined by rate nephelometry over a 1-year period with the Centers for Disease Control WHO lyophilized plasma standard was 125.4 +/- 21.2 mg/dl. Furthermore, it was found that the rat HDL was also a suitable standard in the rate nephelometric assay of Apo A. In contrast, Apo A, purified to homogeneity, showed different reaction kinetics from that of Apo A in the whole plasma and therefore was not a suitable standard in the rate nephelometric assay. We therefore conclude that primary standard Apo A, purified to homogeneity, can be used by endpoint nephelometry to calibrate plasma pools that can then be used as secondary standards in the rate nephelometric determination of rat and human plasma Apo A. The ready applicability of this method in the accurate determination of plasma Apo A under well-defined experimental conditions such as in chronic ethanol-fed rats and in human subjects with normal lipid levels and those with hyperlipidemia is demonstrated.     

A simple method for determination of serum catalase activity and revision of reference range

A rapid, cost-efficient, spectrophotometric assay for serum catalase activity was developed. It was a combination of optimized enzymatic conditions and the spectrophotometric assay of hydrogen peroxide based on formation of its stable complex with ammonium molybdate. Lipemic and icteric sera increased the absorbance without influencing the catalase assay. Due to the high catalase activity in erythrocytes artificial hemolysis increased serum catalase activity. The imprecision of the method was CV less than 5.8% within run as well and day-to-day. The catalase assay performed using polarographic and spectrophotometric determination of hydrogen peroxide yielded a good correlation (r = 0.9602, b = 1.011, a = -0.648, n = 440). In 742 healthy individuals the mean and SD values of serum catalase were 50.5 +/- 18.1 kU/l with 17.7% higher activity in males than in females. Between 14-60 yr the serum catalase increased with age.     

GLP体系下全自动血凝分析仪的3Q验证

目的以Sysmex CA7000全自动血凝分析仪为例,探究良好实验室规范(GLP)体系下全自动血凝分析仪3Q验证过程。方法选择凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、凝血酶时间(TT)、纤维蛋白原(FIB)4项检验指标,对仪器的批内精密度、日间精密度、准确性、线性、携带污染率进行性能验证。结果 PT、APTT、TT、FIB的批内精密度CV值分别为1.33%、1.57%、1.47%、1.90%;日间精密度CV值分别为1.73%、1.52%、1.55%、2.14%;在准确度方面,PT、APTT、TT、FIB的正常质控CV值分别为7.45%、3.88%、-4.98%、4.36%,PT、APTT的异常质控CV值分别为8.11%、8.77%;FIB的线性相关系数(r)值为0.999 3,a值为1.02;标本携带污染率最高CV值为2.15%;所测结果均符合行业通用标准和仪器厂家要求。结论通过GLP体系下的3Q验证,Sysmex CA7000血凝分析仪各方面性能优良,可以用于临床检验部的检验工作。     

老年患者应用D-二聚体床旁测定的性能评价

目的 探讨AQT90 FLEX免疫分析仪床旁快速检测全血D-二聚体(D-dimer)的临床性能并评估其在老年患者中的应用价值.方法 参考我国卫生行业标准《D-二聚体定量检测》(WS/T 477-2015)中提供的设备性能评价方法,对AQT90 FLEX免疫分析仪检测全血D-二聚体(D-dimer)进行性能评价.选择2015年北京大学第一医院老年科60岁及以上住院患者,同时采取静脉血193份(男158份,女35份),比较AQT90 FLEX免疫分析仪和ACL TOP全自动凝血仪测定D-二聚体结果的相关性.结果 高值和低值样品的批内不精密度分别为2.619％和2.767％;携带污染率为0.12％;AQT90 FLEX免疫分析仪与ACL TOP全自动凝血仪检测结果的相关系数为0.9491 (P＜0.01),AQT90 FLEX免疫分析仪检测结果为2.52,ACL TOP全自动凝血仪检测结果+0.15,在女性、高龄患者,倍数关系略增加.结论 AQT90 FLEX免疫分析仪床旁测定全血D-二聚体浓度精密度高,与ACL TOP全自动凝血仪检测结果相关性好,AQT90 FLEX免疫分析仪的检验结果约为ACL TOP全自动凝血仪检验结果的2.52倍.将其应用于门急诊及重症监护室老年人群的血栓性疾病筛查结果可靠.