Laser Immunonephelometry Research Articles

Interleukin-10 and interleukin-1 receptor antagonist distinguish between patients with sepsis and the systemic inflammatory response syndrome (SIRS)

The current study evaluated the potential of clinical parameters and circulating biomarkers to distinguish sepsis from SIRS in patients admitted with systemic inflammation. Clinical parameters, leukocyte counts and platelets were measured on admission. Circulating C-reactive protein (CRP), procalcitonin (PCT) and cytokine concentrations were quantified using laser immunonephelometry, immunoluminescence and a Bio-Plex suspension bead array system respectively. Blood, sputum, urine, peritoneal and cerebrospinal fluid were sent for microscopy and culture. Based on clinical information and the results of microbiological testing, 62 patients were classified retrospectively into 2 groups, those with sepsis (n = 37) or SIRS (n = 25). Mean body temperature was higher and blood pressure lower in the sepsis patients. Circulating concentrations of CRP, PCT, interleukin (IL)-10 and IL-1 receptor antagonist (IL-1Ra) were significantly higher in patients with sepsis, with IL-10 identified as the best biomarker in differentiating sepsis from SIRS. The biomarkers that best predicted overall mortality were platelet counts >PCT ≥ CRP > IL-6 > IL-1Ra. These findings demonstrate that patients with sepsis have significantly increased levels of the immunosuppressive/anti-inflammatory cytokines, IL-1Ra and IL-10, compared to those with SIRS, consistent with a more intense counteracting anti-inflammatory response, while a biomarker profile including platelets, PCT, CRP, IL-6 and IL-1Ra may be useful to predict mortality.

Laser Immunonephelometry Reference Intervals for Eight Serum Proteins in Healthy Children

Eight serum proteins were analyzed with the Behring nephelometer in samples from 479 healthy French children, ages three to 16 years. Girls had higher concentrations of IgM and albumin than boys had. Age appeared to be a main factor of variation for the proteins tested. Reference intervals are presented for IgG, IgA, IgM, albumin, transthyretin (prealbumin), retinol-binding protein, alpha 1-acid glycoprotein, and C-reactive protein. The significance of increased concentrations of C-reactive protein within a community is discussed.

Screening for microalbuminuria in patients with diabetes mellitus: frozen storage of urine samples decreases their albumin content.

The influence of storage on urinary albumin concentration was prospectively studied with use of overnight urine specimens (Albustix negative) from 73 diabetic patients. From each urine sample four aliquots were taken. One was stored at 4 degrees C and assayed within two weeks, the other three were stored at -20 degrees C and assayed within two weeks and after two and six months. Albumin concentration was measured with laser immunonephelometry. The detection limit, 1 mg/L, suffices for the screening of diabetic patients for microalbuminuria. After storage for two and six months at -20 degrees C, significantly lower albumin concentrations were found. The difference was mainly caused by lower concentrations found in urine samples in which a precipitate had formed, which was the case in 22 and 25 samples, respectively. Thus, freezing of urine samples for determination of low concentrations of albumin may yield falsely low results. Urine samples are best stored at 4 degrees C and assayed within two weeks.

Host serum protein levels in cysts of human hydatidosis

8 proteins (albumin, IgG, IgM, IgA, C 3c, C 4, orosomucoid and α 1antitrypsin) were determined by laser immunonephelometry in hydatid cyst fluid from cysts and sera from 16 patients. The cystic level of albumin was 34.5 ± SD 59.1 μ/ml (range 3.6–85); of IgG 12.9 ± 18.7 μg/ml (1.9–75); of IgM 10.5 ± 10.4 μg/ml (3.37); and of IgA 7.2 ± 3.5 μg/ml (4–19.7). The 4 other proteins represented a smaller fraction: C 3c, 0.9 ± 0.60 μg/ml (0.5–2.5); C 4, 1.3 ± 0.9 μg/ml (0.5–3.5); orosomucoid, 2.8 ± 2.3 μg/ml (1.4–9.2); and α 1-antitrypsin, 5 ± 4.5 μg/ml (2–19). These 8 host proteins constituted 24.6 ± 24.5% (2.4–76) of the total hydatid cyst fluid proteins (343.7 ± 172.1 ug/ml, range 180–900). The albumin/IgG ratio of 3 ± 2.8 (0.4–10.8) in hydatid cyst fluid was more variable than that in sera, 2 ± 0.5 (1.2–2.7).

Effects of two antihypertensive agents on lipids, lipoproteins, and apoproteins A and B: Comparison of prazosin and atenolol

To compare the effects of prazosin and atenolol on plasma lipid concentrations, 60 patients with arterial hypertension (diastolic 90 to 120 mm Hg, systolic 160 to 200 mm Hg) were allocated at random to one of two treatment groups. The lipid profile included the determination of total cholesterol and triglyceride levels by ultracentrifugation, and assays of apoproteins A 1 and B by laser Immunonephelometry. Determinations were performed 30 days before and on the first, 90th, and 180th days of treatment. Both drugs had similar reducing effects on blood pressure. A significant decrease in total cholesterol, low-density lipoprotein cholesterol, and apoprotein B, and an increase in high-density lipoprotein cholesterol and apolipoprotein A 1 were observed in patients receiving prazosin, whereas patients receiving atenolol exhibited opposite changes in plasma lipids. Although the clinical significance of these findings is uncertain, such metabolic effects should be taken Into account when evaluating the risk/benefit ratio of antihypertensive treatments.

Rapid assessment of albumin concentration by immunoturbidimetry.

The dye-binding methods involving bromcresol green and bromcresol purple may seriously overestimate albumin in serum that has a below-normal albumin/globulin ratio, particularly in nephrotic syndrome hypoalbuminemia. To overcome this problem, we developed a new immunoturbidimetric method for albumin with use of a centrifugal analyzer. Results by this method (y) and by radial immunodiffusion (x) agreed well and yielded the regression equation: y = 0.91x + 1.33 (n = 40, r = 0.96). Compared with laser immunonephelometry, our method is easier and faster (15 min for the overall determination). Thus, the proposed immunoturbidimetric method is particularly suitable for the rapid indirect assessment of oncotic pressure and indirectly for monitoring intravascular body fluids.

Variations du taux de fibronectine plasmatique après chirurgie pour cancer digestif

Fibronectin is a large, adhesive glycoprotein, also called opsonic α 2 surface-binding glycoprotein. This glycoprotein which participates in all adhesions may modulate wound healing; plasma fibronectin, because it is opsonically active, may mediate reticulo-endothelial activity. Citrated plasma samples were analysed from twenty surgical patients with digestive cancer. The samples were obtained preoperatively and on 1st, 3rd, 7th, 14th and 28th postoperative days. Fibronectin assay was made by laser immunonephelometry. Other samples were obtained from a population of 340 healthy male and female subjects whose age ranged from 10 to 70 years, so that reference could be made to the standard mean of fibronectin related to the age of each patient. In the surgical patients, there was a significant decrease in fibronectin from the first to the third postoperative day. On the 7th day, there was a secondary increase of fibronectin levels which exceeded the preoperative values at the 14th and 28th days. There was no plasma dilution when the samples were collected, this being shown by protidaemia, osmolarity and haematocrit which were measured at the same time. « Septicand « non septicpatients were compared. For septic patients, fibronectin levels were lower and the secondary increase occurred later than in the non septic patients. Patients with no secondary increase did not recover and died. Fibronectin levels were compared with other classical parameters of nutritional assessment, i.e. albumin, prealbumin, transferrin and urinary creatinine indexes. These parameters were not well correlated. However, this study may indicate the usefulness of fibronectin as an indicator of nutritional status and of non specific host defence.

Étude comparative des effets de la prazosine et de l'aténolol sur les lipides plasmatiques de patients hypertendus

In order to investigate comparatively the effects of prazosin and atenolol on plasma lipid concentrations, 60 patients from two centres, presenting with arterial hypertension (diastolic 90-120 mmHg, systolic 160-200 mmHg), were allocated at random to one or the other of these drugs. The lipid profile, stable during the period of observation, included total cholesterol and triglycerides, study by ultracentrifugation, and assays of apoproteins A1 and B by laser immunonephelometry. Determinations were performed 30 days before, and on the 1st, 90 th and 180 th days of treatment. Both drugs had similar lowering effects on blood pressure. A significant decrease in total cholesterol, LDL cholesterol and apoprotein B and an increase in HDL cholesterol and apolipoprotein A1 were observed in patients under prazosin, whereas patients under atenolol exhibited opposite variations in plasma lipids. Although the clinical significance of these findings is uncertain, such metabolic effects should be taken into account when evaluating the risk/benefit ratio of antihypertensive treatments.

Influence of polyunsaturated fats and fat restriction on serum lipoproteins in humans

This study was designed to determine the effects of polyunsaturated fats and of reducing intake of total fat on serum lipids, lipoproteins, and apolipoproteins. Twenty-two normolipidemic women living in a nunnery were given a reference diet (fat/carbohydrate 42 46% of energy, P S ratio 0.16), a polyunsaturated diet ( 42 46% , P S 1.0 ), and a low-fat, polyunsaturated diet ( 32 56% , P S 1.0 ) for 6 weeks each. Serum and lipoprotein lipids were determined by standard procedures, apolipoproteins either by laser immunonephelometry or by rocket immunoelectrophoresis. Consumption of the polyunsaturated diet decreased cholesterol and apolipoprotein B levels in VLDL (−33.1% and −23.8%) and in LDL (−13.5% and −8.8%) without affecting HDL. Consumption of the low-fat, polyunsaturated diet resulted in a reincrease of VLDL triglycerides, but not of VLDL cholesterol. Concentration of VLDL apolipoprotein B further fell (−41.6%) and that of apolipoprotein E decreased (−25.9%), resulting in an increased VLDL lipid/apolipoprotein mass ratio. This study indicates that responses to therapeutic polyunsaturated diet are lowered levels of VLDL and LDL, but unchanged levels of HDL. Additional restriction of dietary fat intake alters the VLDL composition with a decrement in apolipoprotein E enriched VLDL particles.

Xanthomes plans normolipémiques et myélome IgGĸ à activité anti-lipoprotéine

Clinicopathologic findings are reported of a woman with plane xanthomatosis, multiple myeloma (IgGĸ) and normolipemia. Plasma lipoproteins were bound to the monoclonal immunoglobulin. The complex was separated by ultracentrifugation, then the proteins were measured by radial immunodiffusion and laser immunonephelometry. Monoclonal IgGĸ interact with the low-density lipoproteins. The literature about the association between normolipemic or hyperlipemic xanthomatosis and myeloma was reviewed and the physiopathology of this association discussed. Several hypotheses are suggested but, at present, it is shown that lipoprotein-paraprotein complexing in some patients may be due to autoantibody activity of the myeloma protein against serum lipoprotein; immune complexes interfere with normal lipoprotein catabolism resulting in xanthomas and normolipemia or hyperlipemia.

Relationship between microtubules and Golgi apparatus in hepatocytes: a quantitative study during experimental nephrosis.

In many cell types, microtubules are preferentially associated with the Golgi apparatus. However, the existence of a functional link between these two organelles is still hypothetical. To gain insight into this question, the relationships between microtubules and the Golgi apparatus were studied in rat hepatocytes during experimental nephrosis induced by the aminonucleoside of puromycin. This condition is known to cause prolonged stimulation of plasma protein production by the hepatocytes. Rats were studied 2, 4, 5, 10 and 20 days after aminonucleoside injection. The amount of albumin was measured in serum and hepatic microsomes by laser immunonephelometry. The volume densities of microtubules around the Golgi apparatus and in the remaining cytoplasm were measured by ultrastructural morphometry. Changes of the Golgi apparatus were analysed by measuring the volume density of the whole organelle and the respective proportion of saccules and vesicles. Proteinuria began 5 days after aminonucleoside injection and was accompanied by a decrease in serum albumin and a rise in microsomal albumin. These changes were still more striking after 10 days, but protein and albumin levels were almost back to normal after 20 days. Concomitantly, the volume density of the microtubules increased significantly around the Golgi apparatus (32% after 10 days), and not in the remaining cytoplasm. The Golgi apparatus was enlarged (80% after 10 days) with a higher ratio of secretory vesicle to saccule volume densities. These results show that additional microtubules are present around the Golgi apparatus during the enhanced production of plasma proteins which occurs in nephrosis. They suggest that in hepatocytes, microtubules play a part in the Golgi apparatus function of plasma protein processing.

Immunonephelometric quantitation of apolipoprotein E in human serum.

A laser immunonephelometric procedure for the determination of apolipoprotein E in human serum and lipoprotein fractions was developed. Coefficients of variation were 2.3% within-run, 4.7% between-run. The distribution of apolipoprotein E in various lipoprotein fractions revealed a higher proportion of apolipoprotein E in VLDL in hypertriglyceridaemic subjects (especially marked in Type III patients) than in normal subjects. Laser immunonephelometry appears suitable for the routine measurement of apolipoprotein E.

Rapid quantification of factor VIII R:AG by laser immunonephelometry : Influence of ethylene diamine tetra acetic acid (EDTA)

Rapid quantification of factor VIII R:AG by laser immunonephelometry : Influence of ethylene diamine tetra acetic acid (EDTA)

Quantitative determination of human plasma apolipoprotein A-I by laser immunonephelometry.

A technical procedure is described for quantitation of human apolipoprotein A-I (apo A-I) in normal plasma or serum by immunonephelometry. Dilution of the plasma samples with 6 mol/L guanidine chloride ensures maximum exposure of the antigenic sites of the apoprotein and enables optimum quantitation of the apo A-I without requiring extraction with organic solvents. Similar data are obtained by this assay and with radioimmunoassay for normal subjects (1.2--1.5 g/L), and the results obtained on 31 patients are correlated with a coefficient of 0.92. The apo A-I values are correlated with values for plasma high-density lipoprotein cholesterol (r = 0.64). The interassay CV for immunonephelometry is about 7% and the standard curve is linear between 0.1 and 1.0 microgram of apo A-I per sample, corresponding to a 150-fold dilution of serum or plasma. The assay is applicable to plasma samples containing as much as 4 g of triglycerides per liter. At higher concentrations plasma delipidation is required.

Plasma levels of VLDL- + LDL-cholesterol, HDL-cholesterol, triglycerides and apoproteins B and A-I in a healthy population Influence of Several Risk Factors

Plasma apo B and apo A-I were determined in 477 subjects (206 males and 271 females) by laser immuno-nephelometry. Measurements of VLDL- + LDL-cholesterol, HDL-cholesterol and triglycerides were done simultaneously. VLDL- + LDL-cholesterol and apo B values were similar in males and females and increased with age. HDL-cholesterol and apo A-I values were higher in females but stable with age. Different regression curves (HDL-cholesterol vs apo A-I) were obtained in males and females and a negative correlation was found between HDL-cholesterol or apo A-I and triglycerides. Increased body weight was associated with higher values of VLDL- + LDL-cholesterol, apo B and triglycerides in both sexes but lower values of HDL-cholesterol and apo A-I essentially in males. Finally, the study provides evidence of a relationship between smoking and alcohol consumption on the one hand, and HDL-cholesterol and apo A-I on the other.

Amniotic fluid alpha-fetoprotein measurements by laser immunonephelometry

A method is described for measuring human alpha-fetoprotein concentration (AFP) in amniotic fluid by laser immunonephelometry showing results comparable with those obtained by radioimmunoassay. Advantages gained with the laser technique lie in its simplicity as a method which avoids use of radioactive materials, a more rapid 2 h turnaround time and a relatively small capital equipment outlay with minimal operating costs. Laser immunonephelometry appears to be an excellent alternative to radioimmunoassay for measuring AFP in amniotic fluid.

Cost containment in laser immunonephelometry

Cost of measuring serum specific proteins on the Hyland Laser Nephelometer (PDQ) is reduced from $1.60 (U.S.) per test to an average of 35 cents (range: 18–76) per patient result by preparing dilutions in polyethylene glycol of antibodies that are available commercially (Dakopatts a/s, Denmark). An expensive, cumbersome dilutor-dispenser used in the prescribed method is replaced by simple pipetting procedure. Antibodies employed were verified to be monospecific immunoglobulins raised each against one 11 human specific proteins studied, namely albumin, orosomucoid, alpha-1-antitrypsin, alpha-2-macroglobulin, haptoglobin, transferrin, beta-1-C-globulin (C3), beta-1-A-globulin (C4), and immunoglobulins IgG, IgA and IgM. Development of relative light scatter, measured nephelometrically, was shown to be stabilized within 40 minutes of adding sample to antibody. Linearity of graphs for six calibrating references in each method, relating concentration of standard to nephelometric relative light scatter, was excellent. Furthermore, very reproducible results were obtained from day-to-day (CV of 2 – 10%) for standard concentrations within ranges usually encountered clinically. Precision for Hyland Immunochemistry Control, measured against Hyland calibrating references, was acceptable (average CV: 7.6%; range: 5 – 11%). Due to lack of uniformity found previously in expressing concentration in different batches of Hyland Calibrators and Accuracy Controls, correlation of results obtained with Hyland antibodies versus those obtained with Dako antibodies was not carried out. Intermanufacturer comparison of results of the 11 proteins in commercial immunochemical quality control standards from Hyland and two other sources (Atlantic Antibodies, Behring Diagnostics) was disappointing. However, precision in measuring the same 11 specific proteins in controls of three commercial sources (Hyland, Atlantic, Behring) studied with Dako antibodies and Hyland calibration (average CV of 7.6, 10.5 and 11.1% respectively) was similar to that previously reported using Hyland antibodies and Hyland calibration (corresponding average CV of 7.6, 10.7 and 12.5% respectively). Dako antibodies were found to be well suited for use on the Hyland Laser Nephelometer. Need for proper standardization of calibration between various manufacturers is again emphasized.

Quality of commercially available controls in laser immunonephelometry.

Lack of accuracy in expressing concentrations of specific proteins in different lots of Hyland calibrating reference and quality control materials, supplied for the Hyland PDQ laser nephelometer, prompted an attempt to find alternative and more reliable calibrating materials available commercially. Laser grade (Atlantic Antibodies) and radial immunodiffusion grade materials (Behring Diagnostics) were evaluated in this study. Using Atlantic Antibodies' calibrator, only five of 11 specific proteins assayed in Atlantic control serum were within acceptable ranges; precision was less than that using Hyland materials of identical lots. Mean values for each of 10 of the same 11 specific proteins of Behring control plasma assayed against Behring stabilised human serum, were within manufacturers' acceptable ranges; however, precision was less than that with Hyland materials from identical lots. Nine of the 11 specific proteins in a frozen serum pool sample, measured on the laser nephelometer, agreed well with results of the specific proteins measured by radial immunodiffusion, using the same Behring calibration and quality control. Only the Hyland materials were prediluted for use, which explains the greater precision that was obtained. Intercomparison of concentrations, as expressed by respective manufacturers, was largely unacceptable. Based on these results there is need, until satisfactory reagents become available, to standardise on one parent Hyland reference sample giving best precision and to calibrate all subsequent working standards against the parent reference.

Recent progress and some problems in quantification of serum C-reactive protein

Advantages and disadvantages on the conventional semiquantitative CRP tests are discussed. Quantitative methods of determining serum CRP levels include radioimmuno assay, radio-electroimmuno-precipitation and laser immuno-nephelometry. Laser immunonephelometry has been most frequently applied in recent years. Some important problems related to laser immuno-nephelometry are discussed.The authors recommend that polyethylene glycol is not to be added, that purified CRP standards are to be dissolved in absorbed human serum, and that inactivation of serum samples is preferable.

Comparison of specific protein assays in biological fluids by radial immunodiffusion and laser nephelometer

Laser immuno-nephelometry is presently being used for measuring concentrations of specific proteins in biological fluids. Our findings have not substantiated the highly correlative results with radial immunodiffusion that have been reported. The reason for our poor correlation was found in the lack of uniformity in expressing concentrations of the different specific proteins in the calibrating standards. One value of the laser nephelometer was the greater precision obtained when comparing results to those measured by radial immuno-diffusion. Also, the laser was found to be more sensitive for measuring low level concentrations.