Behring Nephelometer Analyzer Research Articles

Microalbuminuria is a negative correlate for cognitive function in older adults with peripheral arterial disease: results from the U.S. National Health and Nutrition Examination Survey 1999–2002

Microalbuminuria (MA) has been increasingly identified as a marker of cardiovascular risk. Although poor cognitive function has been implicated as a sequelae of increased cardiovascular burden, little is known about the association between MA and cognitive function. Population-based cross-sectional study. National Health and Nutrition Examination Survey 1999-2002 in the USA. 2049 noninstitutionalized adults (>/=60 years) with nonmissing values in cognitive test, urinary albumin-to-creatinine ratio (UACR) and ankle-brachial blood pressure index (ABPI) was analysed. Participants with UACR >300 microg mg(-1) were excluded. The UACR, in the unit of microg mg(-1), was calculated by dividing the urinary albumin value by the urinary creatinine concentration. MA was defined as UACR between 30 and 300 microg mg(-1). Cognitive function was measured by a 2-min Digit Symbol Substitution Test (DSST). Peripheral artery disease (PAD) was defined as an ABPI <0.9 in either leg. Overall speaking, MA was inversely associated with DSST score after controlling for age, sex, race, body mass index and educational level (regression coefficient = -2.8, P = 0.002). There was an effect modification of PAD on the association between MA and the DSST score. Amongst participants with PAD, the DSST score for those with MA was lower than those without MA (beta = -6.3, P = 0.003) after multivariate adjustment. Moreover, participants with PAD in the highest quartile of UACR had significantly lower DSST score compared to those in the lowest quartile (beta = -8.7, P = 0.001). There was no association between MA and cognitive function amongst participants without PAD. We observed an additive effect of MA and PAD on DSST score. Participants with both MA and PAD had a lower mean DSST score compared to those without both conditions (beta = -6.2, P = 0.003). The presence of MA or a higher level of urinary albumin excretion was inversely associated with cognitive function in participants with PAD.

Apolipopretein A-I and B Distribution among the Employees and their Partners in Korea

This study was performed to investigate the distribution of apolipoproteins A-I and B among Korean employees and their partners. The study population consisted of 7,633 men and women (4,578 men and 3,054 women) residing in Seoul and Kyung-gee Do, with an average age of 43.5 +/- 8.3 years. Blood samples were collected following at least 12 hours of fasting. Apolipoproteins A-I and B were measured using a Behring Nephelometer analyzer. The body mass index (BMI) for each participant was calculated as weight (kg) divided by height squared (m2). Information on health-related behaviors such as exercise, alcohol intake, and smoking habits was collected through self-administrated questionnaires. The mean concentrations of Apo A-I were 132.6 +/- 22.3 mg/dL and 142.9 +/- 24.8 mg/dL in the men and women, respectively. The concentration of Apo A-I increased significantly across all age categories of men. The mean concentrations of Apo B were 101.7 +/- 23.2 mg/dL and 87.8 +/- 23.5 mg/dL in the men and women, respectively, and Apo B increased significantly across all age categories for both the men and women. Exercise and BMI were major determinants for Apo A-I and B levels. The 10th percentile of Apo A-I concentration was 109 mg/dL in the men and 113 mg/dL in the women, and the 90th percentile of Apo B concentration was 131 mg/dL in the men and 118 mg/dL women. For the prevention of coronary artery disease, we recommend that for individuals in the 10th percentile of concentration for Apo A-I and the 90th percentile of concentration for Apo B, active preventive interventions such as weight loss and exercise should be taken. This study, within its limitations, may be useful for evaluating apolipoprotein A-I and B concentrations in Korean adults.

High Prevalence of C-Reactive Protein Elevation with Normal Triglycerides (100–149 mg/dL): Are Triglyceride Levels Below 100 mg/dL More Optimal in Coronary Heart Disease Risk Assessment?

The National Cholesterol Education Program defines a fasting triglyceride level below 150 mg/dL as normal. However, observational data suggest that triglyceride levels above 100 mg/dL may predict coronary heart disease (CHD) events. To determine the prevalence of systemic inflammation with a normal triglyceride level (100-149 mg/dL), data obtained from the population-based cross-sectional study of 4412 men and women in the third National Health and Nutrition Examination Survey were reviewed. Measurements included fasting lipids and lipoproteins and serum C-reactive protein (CRP) level measured using a Behring Nephelometer Analyzer System. High CRP level was equally prevalent whether the designated triglyceride cutpoint was greater than or equal to 100 (unadjusted OR, 2.0; 95% CI, 1.7-2.3) or greater than or equal to 150 (unadjusted OR, 1.9; 95% CI, 1.6-2.2). After adjustment for other covariates, the triglyceride range of 100 to 149 mg/dL remained independently associated with elevated CRP level (OR, 1.3; 95% CI, 1.02-1.67). In addition, an approximately fivefold higher likelihood of elevated CRP level was observed with triglyceride levels between 100 and 149 mg/dL and normal body mass index (BMI; 24-24.9) compared with lower triglyceride level (<65 mg/dL) and BMI (<22) (P < 0.0001). These data indicate that "normal" triglyceride levels (100-150 mg/dL) are associated with systemic inflammation and that lower fasting triglyceride levels (eg, <100 rather than <150 mg/dL) may be a more optimal cutpoint in CHD risk assessment.

Development of ELISA on microplate for serum C-reactive protein and establishment of age-dependent normal reference range

Background: C-reactive protein (CRP), a marker of systemic inflammation, has been proposed to predict outcome in patients with unstable angina; and elevated levels of CRP were found to be associated with an increased risk of coronary events. Methods: Two enzyme-linked immunosorbent assays (ELISA) of different sensitivities were developed on microplate for CRP. Both ELISA established used Dako polyclonal anti-CRP antibody for coating and Dako horse radish peroxidase (HRP)-conjugated polyclonal anti-CRP antibody for detection. Results: The sensitivity of the high and regular sensitivity ELISA was 0.16 and 0.6 mg/l, respectively. Our assays demonstrated an excellent correlation with commercial CRP assays performed on a Behring Nephelometer Analyzer II (BNII) at both regular and ultrasensitive levels, with both correlation coefficients above 0.98 and slopes of approximately 1. Using our microplate assays, we established normal reference value for serum CRP. Based on ANOVA statistical test, we found that the mean±S.D. was 1.3±1.27 mg/l ( n=202) for normal individuals of 50–80 years and 0.43±0.42 mg/l ( n=148) for the group of 20–50 years. Conclusions: The normal serum CRP mean concentrations for two age groups were distinctively different ( p value<0.001). Our study suggests two different normal cutoffs of serum CRP to be employed for individuals in different age groups.

The effect of reaction temperature for nephelometric assays for rheumatoid factor

Even using the same assay parameter, reagent and calibrator (N-latex RF kit II), the results of the assay for serum rheumatoid factors (RFs) with the Behring Nephelometer Analyzer (BNA) were higher than those with the Behring Nephelometer II Analyzer (BNII) ([BNII]=0.76 [BNA]−5.7 kIU/l, r=0.997, Sy/ x=60.73, n=99). The mean bias (BNA minus BNII)±S.D. was 52.7±85.5 using the Bland and Altman plot method, and the bias was not constant. The only difference in the assay condition with the two methods was the reaction temperature with the BNA being performed at room temperature (25±1°C) and the BNII being performed at 37°C. The ratio of the results with the BNII to the BNA (BNII/BNA) ranged from 0.23 to 1.18. A significant difference was observed in the BNII/BNA ratio in patients with high levels of C-reactive protein (CRP) over 2.0 mg/l (mean BNII/BNA ratio; 0.78) in comparison to patients with normal CRP levels under 2.0 mg/l (mean BNII/BNA ratio; 0.65) ( P<0.01). The RF concentrations with the BNA were reduced by addition of urea, which has been used as a mild protein-denaturing agent, and there was a significant correlation between the values calculated as (1-value treated with urea/original value without urea)×100 and the BNII/BNA ratio ( r=0.652, P<0.01). These data suggested that the bias between the RF values obtained by the BNA and BNII might be caused by the variation in the reactivity of autoantibodies, which might be decreased in some inflammatory diseases.

Evaluation of the analytical performances of a new fully automated commercial immunonephelometric assay for lipoprotein(a).

Several commercial methods have been proposed for lipoprotein(a) (Lp(a)) measurements over the past decades. However, only a few of them appear completely suitable in terms of analytical performance, costs and practicability. We evaluated the analytical performance of a new commercial fully automated immunonephelometric assay for Lp(a) measurements on the IMMAGE Immunochemistry System. Mean within- and between-run coefficients of variation were 2.7% (range 1.2-4.7%) and 3.8% (range 1.8-7.9%), respectively. The linearity of the assay was confirmed up to 102 mg/dl and the deviation from the expected values did not exceed 4% (mean deviation: 1.9%). Moreover, the relative non-linearity was acceptable, ranging from 1.4% to 1.6% and hence constantly lower than the 2.5% upper limit. Since there is no reference method for Lp(a) measurements, 100 routine random serum samples measured by the IMMAGE Immunochemistry System immunonephelometric assay were further compared with two other commercial immunonephelometric assays (Array LPA immunonephelometric assay and BNA Latex-Enhanced Lp(a) nephelometric assay). Non-parametric regression and relative Spearman's correlation coefficients were satisfactory, (y=1.009x - 1.38; r=0.998 IMMAGE Immunochemistry System vs. Array LPA and y=0.922x - 0.40; r=0.989 IMMAGE Immunochemistry System vs. Behring Nephelometer Analyzer (BNA) Latex Lp(a) assay). On the basis of the results of the present evaluation we conclude that the analytical performance and the main technical features of the IMMAGE Immunochemistry System immunonephelometric assay make it a suitable method for Lp(a) measurement in clinical laboratories.

Development and validation of an automated particle-enhanced nephelometric immunoassay method for the measurement of human plasma C1q.

We have developed a sensitive immunoassay based on latex particle agglutination for measuring C1q concentrations in human plasma. In this simple and fast particle-enhanced immunoassay, we used carboxylated latex particles (diameter 210 nm) covalently coated with F(ab')2 fragments of anti-C1q antibodies. These particles are incubated with diluted sample (400-fold) for 6 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/hr. This assay generates a standard curve in the range of 24-775 mg/L, showing intraassay and interassay precision of < 8% and < 10%, respectively. Dilution linearity was validated throughout the dynamic range of the assay. There were no interferences from bilirubin, Intralipid, haemoglobin, and rheumatoid factor. Results obtained in 45 clinical samples correlated well with those obtained by a commercial radial immunodiffusion method (r = 0.936), and with those obtained by the Behring immunoprecipitation nephelometric test (r = 0.950). The mean concentration in plasma from healthy subjects was 180 mg/L and the reference interval was from 128 to 237 mg/L. This latex nephelometric procedure is a convenient method and an interesting alternative to other immunoassays for routine measurement of human C1q.

Discrepancies between lipoprotein(a) concentrations in icteric sera measured by immunonephelometry and electroimmunodiffusion

We compared the lipoprotein(a) [Lp(a)] levels in 32 icteric sera determined both ba an electroimmunodiffusion assay (EIA), using the Hydragel Lp(a) kit (Sebia, France) and by two immunonephelometric assays, one on a Behring Nephelometer Analyzer (BNA), using antiserum from Immunofrance, and the other on a Beckman analyzer (Array), using antiserum from, Dako (Denmark). With the EIA assay, the Lp(a) level was 0.09 ± 0.09 (mean ± SD in g/L), with the BNA assay, 1.01 ± 1.51 and with the Array assay, 0.05 ± 0.05. Sample blanks values (0.76 ± 1.28 g/L) demonstrated that the high Lp(a) levels obtained in the BNa assay are caused by nonspecific precipitation. Analysis of the precipitate indicated the presence of Lipoprotein X, and abnormal lipoprotein that appears in the serum of patients with obstructive jaundice or with lecithin-cholesterol acyltransferase deficiency. The precipitant seems to be polyethyleneglycol (PEG) that was added to the reaction medium in both the BNA and the Array assays to stabilize the Lp(a)-anti Lp(a) immune complex. In the Array assay, interference by this nonspecific precipitation is eliminated by preliminary centrifugation of the diluted sample. However, coprecipitation of Lp(a) could occur during this step. Consequently, the results of Lp(a) measurement in serum from patients with hepatobiliary diseases should be interpreted with caution when immunonephelometric assays are used with a medium containing PEG.

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 lyophilization 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).

Automated quantitative nephelometric latex immunoassay for determining ferritin in human serum

We describe a rapid and sensitive latex nephelometric immunoassay for quantifying ferritin in human serum. This latex immunoassay procedure uses commercially available ready-for-use reagents [Tina-Quant (a) Ferritin, Boehringer Mannheim] that have a long shelf life. The assay consists of incubating the diluted serum sample (5-fold) for 12 min at room temperature with latex particles covalently coated with anti-ferritin antibodies, and then quantifying the change of light-scatter produced. The assay is fully automated on the Behring nephelometer analyzer with a sampling rate of 150 samples/hour. The method has an analytical range of 3 to 260 micrograms/l. Maximal intra- and inter-assay CVs were 4.0 and 6.2%, respectively. Analytical recoveries ranged from 91.3 to 103.6%. Assay detection limit was less than 3 micrograms/l. Linearity of the test is given throughout the measuring range. There was no interference from bilirubin (up to 340 mumol/l), haemoglobin (up to 7 g/l), or rheumatoid factor (up to 1,100 IU/ml). Turbid and lipemic samples interfere. This interference may be avoided by pretreating these samples prior to assay. Results correlated well with those obtained by an automated ELISA test (r = 0.995) and with those of two commercial RIA methods (r greater than 0.97). This latex nephelometric procedure is a convenient method and represents an interesting alternative to other immunoassays for measuring ferritin in human serum.

Serum C-reactive protein (CRP) values of patients infected with Taenia saginata.

Elevated serum C-reactive protein (CRP) values are identified in patients with infections, some tumors or various types of tissue destruction, and clinical measurement of serum CRP is valuable as a screening test for organic diseases and as a sensitive index of disease activity and response to therapy in the disease mentioned above (Pepys, 1981). But, to our knowledge, CRP has been given but scant attention in cases of tapeworm infection. Human infection with Taenia saginata is uncommon in Japan and clinical manifestations of this tapeworm infection are mild or symptomless, but infection with T saginata is an important parasitic diseases because of its world wide distribution.From April 1987 to March 1991, we treated 5 patients with T. saginata infection. All of the patients were Japanese males aged 23 to 66 years old. None of them had a previous history of the tapeworm infection. All of the infections were asymptomatic, except for the discomfort and embarrassment occasioned by the crawling of proglottids from the anus. The place of contraction of 3 patients was Ethiopia, 1 patient Japan and 1 patient unknown. All of the patients expelled a single T. saginata with intraduodenal gastrographin injection or oral administration of paromomycin sulphate. Before the therapy, serum CRP levels were measured with a Behring Nephelometer Analyzer with CRP-Latex reagent kit.The serum CRP value of all patients was under 0.3 mg/dl and this value was in normal range. The results are shown in Table 1.The results reported here show that CRP values of patients infected with a single T. saginata are in the normal range. It has been reported that CRP was found in a human liver culture but not in bone marrow, spleen, thyroid, mammary gland or thoracic duct lymphocytes in culture (Hurlimann et al., 1966) and that interleukin-1 (IL-1) and interleukin-6 (IL-6) both stimulated liver synthesis of CRP and that probably IL-6 played a key role in stimulation of CRP (Hazenberg et al., 1988). The results of these reports indicate that the liver is an important organ for producing CRP and that the production is stimulated by interleukins stimulated by tissue damage. T. saginata, whose head has four suckers lives by attaching to the small intestinal wall. Our results suggest that single infections with T. saginata may not induce interleukins sufficient to increase the CRP value. It is thought that injury to the intestinal wall by a single T. saginata is so mild that intestinal infection by the tapeworm does not stimulate production of CRP in the liver to clinically detectable levels. We think that if an elevated serum CRP value is observed in an asymptomatic person infected with a T. saginata, other abnormal accompanying conditions such as neoplasma or collagen disease should be considered.

Quantitative automated latex nephelometric immunoassay for determination of myoglobin in human serum

We have evaluated a new latex nephelometric test for the quantitation of myoglobin in human serum. The assay consists of incubating the diluted serum sample (20-fold) for 12 min at room temperature with latex particles covalently coated with anti-myoglobin antibodies and then quantifying the change of light-scatter produced. The assay is fully automated on the Behring nephelometer analyzer with a sampling rate of 150 samples/hour. There is no interference from bilirubin (up to 340 mumol/l), haemoglobin (up to 7,000 mg/l), or rheumatoid factor (up to 1,100 int. units/ml). Myoglobin standard curve extends from 20 to 380 micrograms/l. Assay detection limit lies around 6 micrograms/l. Coefficient of variation ranged from 2.7 to 7.6%. Correlation coefficient between latex immunoassay and an RIA method was 0.987, calculated from the assay of 37 samples. A statistically significant difference was found between the distribution for females and males. The serum level of myoglobin showed an age-dependent variation. Concentrations up to 60 micrograms/l are considered to be normal.

Immunonephelometric Determination of the Apolipoprotein A-II

A fully mechanized immunonephelometric method is described for the rapid and specific determination of apolipoprotein A-II in serum. The method utilizes commercially available sheep antiserum against human apolipoprotein A-II. Nephelometry was performed with the Behring Nephelometer Analyzer (BNA). A single determination can be performed in 12 minutes, requiring 10 microliters sample volume. The measuring range is about 0.08 to 1.25 g/l apolipoprotein A-II. Precision is characterized by intra-assay coefficients of variation of 3.37%, 3.93% and 4.49% for apolipoprotein A-II concentrations of 1.22 g/l, 0.376 g/l and 0.185 g/l, and inter-assay coefficients of variation of 4.27% for an apolipoprotein A-II concentration of 0.404 g/l, respectively. Accuracy of the method is shown by the close correlation of results with those from radial immunodiffusion (r = 0.913, y = 1.091 x -0.033, n = 75).

Automated nephelometric immunoassays with novel shell/core particles

AbstractNephelometry has become a very reliable and convenient method for immunoassays. Recent innovations in polymer chemistry have led to the design of highly sensitive and reproducible immunoassays for the Behring Nephelometer Analyzer. The principle of particle‐enhanced immunoassays has led to the development of new polymer particles. Key advances include the preparation and use of particles with polystyrene cores covered by thin shells with chemically reactive groups. The cores are prepared by emulsion polymerization, giving particles with a well‐defined size. The particle size has been optimized to fulfill the requirements for nephelometric analysis. The shells consist of a polystyrene/polymethacrylate/polymethacrylamidoacet‐aldehyde dipentylacetal copolymer, which allows a covalent binding of the immunochemicals of interest. Using these particles, a method for the determination of C‐reactive protein has been worked out.