A new high-sensitive nephelometric method for assaying serum C-reactive protein based on phosphocholine interaction

Background: The measurement of C-reactive protein (CRP) concentrations has been of interest as a classical marker of acute phase response; in addition, it has been of particular interest in cardiovascular risk stratification where high-sensitive measurements are necessary. Since CRP is able to bind phospholipids (mainly phosphocholine) in the presence of calcium ions, we explored the possibilities of developing a high-sensitive affordable nephelometric CRP assay based on diluted soy oil emulsions. Methods: Serum (or heparinized plasma) was mixed with Intralipid 20% in Tris-calcium buffer (pH 7.5). After 12 min of incubation at 37 degrees C, the CRP-phospholipid complexes were measured by nephelometry (840 nm) using a BN II nephelometer (Siemens). Results (n=97) were compared with those obtained using a typical immunoturbidimetric method (Roche). Results: Imprecision of the functional nephelometric assay was evaluated using three human serum pools. Within-run coefficients of variation (CVs) for level 1, 2 and 3 were 6.1%, 4.7% and 4.5%, respectively, and between-run CVs were 17.6%, 18.8% and 11.3%, respectively. Good agreement was obtained between the functional nephelometric and the immunoturbidimetric CRP assay in a concentration range from 0.1 mg/L to 50 mg/L (r=0.884). A logit-log calibration curve was made between 0.056 mg/L and 1.785 mg/L. The limit of detection was 0.5 mg/L. Conclusions: The functional nephelometric CRP assay allowed high-sensitive CRP determinations in serum and plasma. Since the assay is species independent, the described functional CRP assay could be used for veterinary purposes as well

Development of an affordable dye-stained microalbuminuria screening test

Background. A simple spot test was developed, which allows quantification of microalbuminuria. Evaluation was carried out according to the ISO 15189 guidelines. Methods. Urine was spotted on cellulose acetate strips and stained using different sensitive protein binding dyes (nigrosin, Coomassie Blue R-250, amido black). The colour intensity of the stained spots was quantified using a Kodak Image 450 station. Results. Analytical sensitivity of the Coomassie Blue based method (18 mg/L) was better than that for nigrosin (50 mg/ L) or amido black (100 mg/L) based methods. Within-run coefficient of variation (CV) and between-run CV of the Coomassie blue assay were, respectively, 8.4% and 9.7% (50 mg/L), and 3% and 4.5% (400 mg/L). For nigrosin, these data were, respectively, 8.4 and 9.4 (50 mg/L), and 3.4 and 6.4% (400 mg/L). Coomassie Blue showed a preferential binding selectivity towards albumin. The method was found to be linear between 20 and 600 mg/L. A good correlation (r2 = 0.89) was obtained between Coomassie Blue based and immunonephelometric measurements. Immuno-unreactive albumin (prepared by protease treatment) could be detected by the spot test, which offers an advantage of the method versus immunochemical tests. Ammonium sulphate precipitation could further increase the specificity of the assay by eliminating effects of free light chains. Conclusion. The described method is very simple and extremely cheap, which makes it potentially suited for screening programmes, particularly in third world countries

A new turbidimetric method for assaying serum C-reactive protein based on phosphocholine interaction

Background: C-reactive protein (CRP) is able to bind phospholipids (mainly phosphocholine) in the presence of calcium ions. We investigated the use of this property for developing an affordable turbidimetrical CRP assay based on diluted soy oil. Methods: Serum (or heparinized plasma) was mixed with Intralipid® 20% in Tris-calcium buffer (pH 7.5). After 30 min of incubation at 37°C, the CRP-phospholipids complexes were measured by turbidimetry (660 nm/700 nm) with a Cobas 6000 analyzer (Roche). Results were compared with those obtained using a typical immunoturbidimetric method (Roche). Results: Good correlation (r2=0.931) was obtained between the functional and the immunoturbidimetric CRP assay. Within-run and between-run %CV values for the functional assay were 2.4% (100 mg/L); 6.0% (50 mg/L); 10% (20 mg/L), and 3.6% (100 mg/L); 8.0% (50 mg/L); 11% (20 mg/L), respectively. The limit of detection was 7 mg/L. Results were not affected by serum calcium, triglyceride, or phospholipid concentrations. Conclusions: The functional CRP assay allowed measurement of CRP in serum and plasma in the range of 7 mg/L–400 mg/L. The assay is particularly suited in conditions where resources are restricted. Since the assay is species independent, the described functional CRP assay could be used for veterinary purposes as well. Clin Chem Lab Med 2009;47:1417–22.Peer Reviewe

Agglutination of intravenously administered phosphatidylcholine-containing lipid emulsions with serum C-reactive protein

Background: C-reactive protein (CRP) is able to bind phospholipids in the presence of calcium. We wanted to investigate the reaction of CRP with various commercial fat emulsions and to explore the impact of CRP agglutination on serum CRP levels. Materials and Methods: Serum specimens were mixed with Intralipid 20% (soybean oil-based fat emulsion), Structolipid (structured oil-based fat emulsion), Omegaven (fish oil-based fat emulsion), or SMOFlipid (mixed soybean oil-, olive oil-, and fish oil-based emulsion) in Tris-calcium buffer (pH 7.5). After 30 minutes of incubation at 37 degrees C, CRP-phospholipid complexes were turbidimetrically quantified and flow cytometric analysis was performed. Similarly, CRP complexes were monitored in vivo, following administration of fat emulsion. Results: CRP was able to agglutinate phospholipid-containing lipid droplets present in the soybean oil-based fat emulsion and the structured oil-based fat emulsion. To a lesser extent, agglutination was observed for fish oil-containing fat emulsions, whereas no agglutination was noticed for the mixed soybean oil-, olive oil-, and fish oil-based emulsion. Results for propofol-containing emulsions were comparable. Agglutination correlated with phospholipid content of the emulsions. When in vivo agglutination occurred, plasma CRP values dropped due to consumption of CRP by phospholipid-induced agglutination. Conclusions: In this in vitro experiment, we demonstrated agglutination of CRP with phospholipids in various fat emulsions. Research studies are required in patients to determine which effects occur with various intravenous fat emulsions

A functional turbidimetric method to determine C-reactive protein in horses

A turbidimetric method to determine serum C-reactive protein (CRP) concentration, based on soybean oil phosphocholine interaction, was performed on horse serum samples to evaluate its potential diagnostic value in veterinary medicine. Intralipid 20% in 0.1 M Tris-calcium buffer (pH 7.5) was added to horse serum. After 30 min of incubation at 37 degrees C, the CRP phosphocholine complexes were turbidimetrically, bichromatically (660 nm/700 nm) quantified on a commercial analyzer. Furthermore, comparison between CRP and other inflammatory markers, including white blood cell and neutrophil counts, was performed to evaluate the diagnostic value of both tests. Standardization of the assay was done using a commercial human CRP calibrator. The CRP measurements were performed on serum samples (296 patients and 34 controls). Reference values were found to be lower than 10 mg/l. The method was found to be linear between 1 and 400 mg/l. A moderate correlation was observed between CRP values and the relative neutrophil counts. Receiver-operating characteristics analysis demonstrated the area under the curve for CRP was 0.928, which was superior (P < 0.001) to the neutrophil count (0.804) and the leukocyte count (0.664) in detecting the presence of inflammation. This CRP assay showed reliable results as an acute phase test in horses, confirming its species-independent capability to detect CRP in various mammals, including horses

Malaria is more prevalent than iron deficiency among anemic pregnant women at the first antenatal visit in rural South Kivu

Anemia is common during pregnancy and is associated with poor outcomes. Objectives were not only 1) to determine the prevalence of anemia and iron deficiency (ID) but also 2) to identify other factors associated with anemia in pregnant women from South Kivu province, in the eastern Democratic Republic of Congo. Between December 2013 and March 2014, 531 women attending the first antenatal visit in their second trimester of pregnancy were recruited. Sociodemographic, clinical, and biological data were collected. Hemoglobin (Hb) was determined by a portable photometer (Hemocue® Hb201+), and anemia was defined as altitude-adjusted Hb 5 mg/L and/or α-1-acid glycoprotein > 1 g/L) whereas hypoalbuminemia was defined as serum albumin < 35 g/L. A Giemsa-stained blood smear was used to diagnose malaria. The median age (interquartile range) was 25.5 (21.1–31.3) years, with anemia in 17.6% and ID in 8%. Malaria was present in 7.5% and hypoalbuminemia among 44%. Soluble transferrin receptor concentration was higher in the presence of inflammation and/or malaria. In the final logistic regression model, factors independently associated with anemia were malaria (adjusted odds ratio [aOR]: 11.24 (4.98–25.37) P < 0.001), hypoalbuminemia [aOR: 2.14 (1.27–3.59); P = 0.004] and elevated CRP [aOR: 1.94 (1.10–3.45); P = 0.022]. ID was not highly prevalent and not associated with anemia in our population. Effective control of anemia during pregnancy in this region should consider fighting malaria and other infectious diseases in combination with measures to improve women’s nutrition, both before and during pregnancy.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population

Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type. Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non-species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations within reference ranges for healthy domestic cats. In contrast, unhealthy cheetahs had higher (P < 0.001) serum amyloid A, alpha(2)-globulin, and haptoglobin concentrations compared with the healthy subgroup. Moreover, serum amyloid A (P = 0.020), alpha(2)-globulin (P < 0.001) and haptoglobin (P = 0.001) concentrations in cheetahs suffering from chronic kidney disease were significantly greater compared to the reportedly healthy cheetahs. Our study indicates that serum proteins in the cheetah can be analyzed by routine capillary electrophoresis, whereas acute-phase proteins can be measured using available immunoassays or non-species-specific techniques, which are also likely to be applicable in other exotic felids. Moreover, results suggest that serum amyloid A and haptoglobin are important acute-phase proteins in the diseased cheetah and highlight the need to evaluate their role as early-onset markers for disease

High prevalence of anemia but low level of iron deficiency in preschool children during a low transmission period of Malaria in rural Kivu, Democratic Republic of the Congo

Anemia is a worldwide public health concern especially in preschool children in developing countries and iron deficiency (ID) is generally assumed to cause at least 50% of the cases. However, data on this contribution are scarce. To close this gap, we determined in 2013 the contribution of ID in the etiology of anemia and measured others factors associated to noniron deficiency anemia (NIDA) in 900 preschool children randomly selected during a two-stage cluster nutritional survey in the Miti-Murhesa health zone, in eastern Democratic Republic of the Congo. In these children, we collected sociodemographic, clinical, and biological parameters and determined the nutritional status according to the World Health Organization 2006 standards. Anemia was defined as altitudeadjusted hemoglobin < 110 g/L and ID was defined as serum ferritin < 12 μg/L or < 30 μg/L in the absence or presence of inflammation, respectively. Median (interquartile range) age was 29.4 (12-45) months. The prevalence of anemiawas 46.6% (391/838) among whom only 16.5% (62/377) had ID. Among children without signs of inflammation, only 4.4%(11/251) met the ferritin-based (unadjusted) definition of ID. Logistic regression analysis identified ID, history of fever during the last 2weeks andmid-upper armcircumference < 125mmas the only independent factors associated to anemia. In conclusion, anemia is a severe public health problem in the Miti-Murhesa health zone, but NIDA is mostly predominant and needs to be further studied. Control of infections and prevention of acute undernutrition (wasting) are some of appropriate interventions to reduce the burden anemia in this region.SCOPUS: ar.jinfo:eu-repo/semantics/publishe