Validation of rapid fecal calprotectin assay using particle enhanced turbidimetric immunoassay for inflammatory bowel disease.

Fecal calprotectin (FC) is a promising biomarker for diagnosis and treatment of inflammatory bowel disease, ulcerative colitis (UC), and Crohn's disease. An enzyme immunoassay (EIA) is widely used for FC detection, though the considerable lag time, up to several days, causes clinical management delay. This study was performed to examine the new rapid kit fCAL-turbo, which is based on a particle-enhanced turbidimetric immunoassay (15 min), by comparing FC values with other EIAs (EliA, PhiCal, Bühlmann) and endoscopic scores. Using 94 samples, fCAL-turbo showed strong significant positive correlations with the other kits (Spearman's r = 0.9178-0.9886). Of 74 UC patients, 69 underwent an endoscopy and fCAL-turbo reflected endoscopic activity with a moderate correlation with Mayo endoscopic subscore (MES) (r = 0.6945, others r = 0.6682-0.7013). Receiver operating characteristic analyses based on MES 0 versus 1-3 showed a similar efficacy as compared to the other kits (cut-off and area under the curve: 89.70 µg/g and 0.8592, respectively, others 62.35-138.4 µg/g and 0.8280-0.8611, respectively). Furthermore, multiple regression analysis confirmed that fCAL-turbo results significantly contributed to prediction of MES 0 with a higher t-value as compared to the other biomarkers. fCAL-turbo showed strong correlations with the other kits and also demonstrated excellent performance for predicting endoscopic remission of UC.

Validation of the fCAL turbo immunoturbidimetric assay for measurement of calprotectin in porcine and bovine fecal samples.

The concentration of calprotectin in feces is a well-studied marker of gastrointestinal inflammation in humans. However, little is known about fecal calprotectin in farm animals. In this work, we have validated an immunoturbidimetric method for fecal calprotectin (Bühlmann fCAL® turbo assay, Schönenbuch, Switzerland) in porcine and bovine fecal samples. Linearity was evaluated by serial dilution (R2 > 0.97 was obtained for both species). Accuracy was assessed by a recovery study, with results between 80 and 120% for low, medium, and high samples in both species. Intra- and inter-assay variability was <20%. Limit of detection was 6.4 µg/g in pig and 5.3 µg/g in cow. Limit of quantification was 13.4 µg/g (pig) and 11.1 µg/g (cow). Additionally, clinical validation has been included to evaluate the ability of the assay to detect inflammatory status in the intestine under different management conditions. In experiments with porcine, it was found that piglets treated with ZnO had lower concentrations of fecal calprotectin. In a second experiment in bovine, calves with diarrhea had higher concentration of fecal calprotectin. The Bühlmann fCAL® turbo assay is suitable for measurement of calprotectin in porcine and bovine fecal samples. Moreover, fecal calprotectin could be a good biomarker of intestinal inflammation in both species.

Development and characterization of a latex turbidimetric immunoassay using rabbit anti-CRP single-chain Fv antibodies.

In this study, we developed and demonstrated a latex turbidimetric immunoassay (LTIA) using latex beads immobilized with rabbit monoclonal single-chain variable fragments (scFvs) selected from an scFv-displayed phage library. Sixty-five different anti-c-reactive protein (anti-CRP) scFv clones were identified after biopanning selection using antigen-coupled multi-lamellar vesicles. By ranking antigen-binding clones using the apparent dissociation rate constant (appkoff) as a sorting index, scFv clones with a dissociation constant (KD free) ranging from 4.07 × 10-9 M to 1.21 × 10-11 M were isolated. Among them, three candidates (R2-6, R2-45, and R3-2) were produced in the culture supernatant at concentrations of 50 mg/L or higher in flask culture and maintained at considerably high antigen-binding activity in immobilized state on the CM5 sensor chip surface. All the scFv-immobilized latexes (scFv-Ltxs) prepared were well-dispersed in 50 mM MOPS at pH 7.0, without additives for dispersion, and their antigen-dependent aggregation was sufficiently detectable. The reactivity of scFv-Ltx to antigen differed among the scFv clones, in particular, R2-45 scFv-Ltx detected the CRP with the highest signal. Furthermore, the reactivity of scFv-Ltx varied significantly with salt concentration, scFv immobilization density, and the type of blocking protein. Particularly, antigen-dependent latex aggregation improved significantly in all rabbit scFv clones when scFv-Ltx was blocked with horse muscle myoglobin compared with conventional bovine serum albumin; while their baseline signals in the absence of antigen were fully stable. Under optimal conditions, R2-45 scFv-Ltx exhibited greater aggregation signals with antigen concentrations higher than those produced by conventional polyclonal antibody-immobilized latex for CRP detection in LTIA. The methodology for rabbit scFv isolation, immobilization, and antigen-dependent latex aggregation demonstrated in the present study can be applicable to scFv-based LTIA for various target antigens.

Falsely decreased vancomycin caused by rheumatoid factor: A case report.

BACKGROUND: Vancomycin is associated with potential nephrotoxicity and trough concentrations need to be monitored in certain patients. Falsely decreased vancomycin measurement may result in overtreatment and need to be identified promptly by clinicians and pharmacists to avoid toxicities. METHODS AND RESULTS: We report a case of rheumatoid factor-mediated falsely low vancomycin measurement with Abbott particle-enhanced turbidimetric inhibition immunoassay (PETINIA) method. Reanalyzing the sample using an alternative method, removing the interferences using heterophile blocking reagent as well as rheumatoid factor clean-up solution all helped to solve the false results. Results from alternative method and interference studies showed vancomycin concentrations reached toxic concentrations in the patient and administration of the drug was immediately terminated. The patient experienced a transient increase in serum creatinine. CONCLUSIONS: Even though most modern immunoassays use blocking agents to neutralize interfering antibodies such as rheumatoid factor, it is important for health care professionals to understand that occasional interference still occurs due to the heterogeneous nature of rheumatoid factor.

Evaluation of Latex Immunoturbidimetric Assay Thresholds and HIT in Cardiothoracic Surgery.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a common differential diagnosis in cardiothoracic surgery. The latex immunoturbidimetric assay (LIA) is an enhanced immunoassay that has recently been introduced for the detection of total HIT immunoglobulin and retains a higher specificity of 95% compared to the enzyme-linked immunosorbent assay. OBJECTIVES: To investigate if a semiquantitative relationship exists between increasing LIA levels beyond the current positivity threshold and its correlation to positive serotonin release assay results in cardiothoracic surgery. METHODS: This was a multicenter, observational cohort of cardiothoracic surgery patients initiated on anticoagulation with heparin-based products. To conduct sensitivity and specificity analysis of LIA values, HIT positive was defined as a LIA value ≥1 unit/mL and HIT negative was defined as a LIA level <1 unit/mL. A receiver operating characteristic (ROC) analysis was utilized to evaluate the predictive performance of the LIA. RESULTS: At manufactures' cutoffs of ≥1.0 unit/mL, LIA sensitivity and specificity was 93.8% and 22%, respectively, yielding a false positive rate of 78%. At a higher cutoff of 4.5 units/mL, LIA sensitivity and specificity was 75% and 71%, respectively, yielding a false positive rate of 29% and an area under the ROC curve of 0.75 (P = .01; 95% confidence interval: 0.621-0.889). Bivalirudin was initiated in 84.6% of false positive LIA results. CONCLUSION: This study suggests that the diagnostic accuracy of the LIA can be optimized by increasing the LIA positivity threshold. Proposing a higher LIA cutoff, may mitigate unwarranted anticoagulation and bleeding outcomes.

Validation of new automated turbidimetric immunoassays for the measurement of haptoglobin and inter-α-trypsin inhibitor heavy chain H4 specific for the bovine species.

BACKGROUND: Good strategical programs are required for the early detection of disease even in the absence of evident clinical signs, which is crucial in satisfying animal welfare. Haptoglobin (Hp) and inter-α-trypsin inhibitor heavy chain H4 (ITIH4) are acute phase proteins and good biomarkers of early inflammation in cattle, with plasma levels that significantly increase after injury or infection. OBJECTIVES: We aimed to develop and validate two new immunoturbidimetric methods for Hp and ITIH4. METHODS: Species-specific antibodies were obtained and used to develop the immunoassays. For the Hp assay, antibodies were fixed to latex microparticles to enhance detection. The immunoassays were set up in an automated analyzer to carry out validation studies. Reference intervals were calculated using Reference Value Advisor. RESULTS: The Hp immunoturbidimetric method had a linear analytical range up to 0.40 mg/mL. The limit of detection (LoD) was 0.005 mg/mL, and the limit of quantification (LoQ) was 0.007 mg/mL. Total imprecision was less than 7%. Comparison with ELISA and single radial immunodiffusion (SRID) showed good correlation, whereas the comparison with the colorimetric method showed constant and proportional differences. The ITIH4 immunoassay showed linearity up to 5 mg/mL, and the LoD was 0.002 mg/mL. Total imprecision was less than 6%. Method comparison showed a good correlation with single radial immunodiffusion, both methods being equivalent. Bilirubin, triglycerides, and hemoglobin presented no interference in any of the assays. Reference intervals were 0.007-0.017 mg/mL for Hp and 0.2-0.7 mg/mL for ITIH4 in dairy cows 10 days before parturition. CONCLUSIONS: Immunoturbidimetric methods developed for Hp and ITIH4 can measure basal and increased levels of these proteins, showing adequate precision, accuracy, and robustness.

Study on the Mechanism of False Low Measurement of IgG-Binding (Affinity) IgM Type M Protein by Turbidimetric Immunoassay.

BACKGROUND: The purpose of this study was to investigate the immunological and physical characteristics of IgM-λ type M-protein from patients who were measured low in the turbidimetric immunoassay (TIA) IgM assay without error codes for high concentration to determine the cause of the false low levels and to clarify the mechanism of their occurrence. METHODS: Materials were IgM patient samples and 8 serum samples from other IgM M-protein patients as controls. Patient samples were assayed by the TIA method, in which five manufacturers and six models (two reagent manufacturers) share the principle, and the BN ProSpec method (nephelometric method), which has a different principle. Dilution linearity tests, IgG addition experiments, isoelectric point electrophoresis, and hydrophobic chromatography were performed on patients and subjects. In addition, the binding capacity of γ-globulin by BIACORE was also examined. RESULTS: The reaction curve of the patient IgM curved downward when the concentration of IgM exceeded 20 g/L, and no error code was obtained. In the measurement by the TIA method of five manufacturers and six models, patient IgM was measured at a false low level with no error code obtained in undiluted dilution by any of the instruments and reagents, but could be measured without any problem by the nephelometric method. In addition, in the patient IgG addition experiment, only patient IgM showed a false low level under high IgG concentration. Furthermore, the binding capacity of patient IgM to γ-globulin (IgG) by BIACORE was significantly higher than that of the control IgM-type M protein. CONCLUSIONS: Patient IgM has an affinity (binding capacity) for IgG and forms an IgM-IgG complex under conditions of high IgG concentration. It was speculated that this complex inhibited the reaction with the anti-IgM antibody and the absorbance of the second reaction did not increase, suggesting a false low.

Ultrasensitive Detection of C-Reactive Protein by a Novel Nanoplasmonic Immunoturbidimetry Assay.

Nanotechnology has attracted much attention, and may become the key to a whole new world in the fields of food, agriculture, building materials, machinery, medicine, and electrical engineering, because of its unique physical and chemical properties, including high surface area and outstanding electrical and optical properties. The bottom-up approach in nanofabrication involves the growth of particles, and we were inspired to propose a novel nanoplasmonic method to detect the formation of nanoparticles in real time. This innovative idea may contribute to the promotion of nanotechnology development. An increase in nanometer particle size leads to optical extinction or density (OD)-value changes in our nanosensor chip at a specific wavelength measured in a generic microplate reader. Moreover, in applying this method, an ultrasensitive nanoplasmonic immunoturbidimetry assay (NanoPITA) was carried out for the high-throughput quantification of hypersensitive C-reactive protein (CRP), a well-known biomarker of cardiovascular, inflammatory, and tumor diseases. The one-step detection of the CRP concentration was completed in 10 min with high fidelity, using the endpoint analysis method. The new NanoPITA method not only produced a linear range from 1 ng/mL to 500 ng/mL CRP with the detection limit reduced to 0.54 ng/mL, which was an improvement of over 1000 times, with respect to regular immunoturbidity measurement, but was also effective in blood detection. This attractive method, combined with surface plasmon resonance and immunoturbidimetry, may become a new technology platform in the application of biological detection.

Validation of an automated immunoturbidimetric assay for feline serum amyloid A.

BACKGROUND: Serum Amyloid A (SAA) is a major acute phase protein in cats, increasing rapidly in response to various inflammatory diseases. An automated latex-enhanced immunoturbidimetric assay for human SAA (LZ-SAA, Eiken), previously validated for use in cats, has had further major modification (VET-SAA, Eiken) for specific use in veterinary diagnostic laboratories but has yet to be validated in cats. RESULTS: Intra-assay and inter-assay CVs for the VET-SAA assay ranged from 1.88-3.57% and 3.98-6.74%, respectively. Linearity under dilution was acceptable with no prozone effect observed. Limit of detection was 1.65 mg/L and limit of quantification was 6 mg/L. Haemoglobin and triglyceride showed no adverse interference, but bilirubin produced positive bias in samples with low SAA. Comparison with the LZ-SAA assay showed significant correlation with proportional bias increasing as SAA concentration increased, likely related to differing calibration standards. SAA was significantly higher in patients with inflammatory disease compared with non-inflammatory disease, and in patients with moderate to highly elevated α1-AGP compared with patients with normal α1-AGP. Improvement of the assay range may be required to fully evaluate differences between disease groups at low SAA levels. Based on ROC curve analysis, at a cut-off point of 20.1 mg/L the VET-SAA assay discriminated between inflammatory and non-inflammatory disease with sensitivity of 0.93 and specificity of 0.99. CONCLUSIONS: The automated VET-SAA assay is a robust, precise, and accurate method for measurement of feline SAA which can clearly identify patients with inflammatory disease. It should be a valuable biomarker for use in feline medicine.

Verification of the fCAL turbo immunoturbidimetric assay for measurement of the fecal calprotectin concentration in dogs and cats.

The concentration of calprotectin in feces (fCal) is a clinically useful marker of chronic gastrointestinal inflammation in humans and dogs. No commercial assay is widely available to measure fCal in small animal medicine, to date. Thus, we verified the immunoturbidimetric fCAL turbo assay (Bühlmann) of fCal for canine and feline fecal extracts by determining linearity, spiking and recovery, and intra-assay and inter-assay variability. We determined RIs, temporal variation over 3 mo, and effect of vaccination and NSAID treatment. Observed:expected (O:E) ratios (x̄ ± SD) for serial dilutions of feces were 89-131% (106 ± 9%) in dogs and 77-122% (100 ± 12%) in cats. For spiking and recovery, the O:E ratios were 90-118% (102 ± 11%) in dogs and 83-235% (129 ± 42%) in cats. Intra- and inter-assay CVs for canine samples were ≤19% and ≤7%, and for feline samples ≤22% and ≤21%. Single-sample RIs were <41 µg/g for dogs and <64 µg/g for cats. With low reciprocal individuality indices, using population-based fCal RIs is appropriate, and moderate fCal changes between measurements (dogs 44.0%; cats: 43.2%) are considered relevant. Cats had significant (but unlikely relevant) fCal increases post-vaccination. Despite individual fCal spikes, no differences were seen during NSAID treatment. The fCAL turbidimetric assay is linear, precise, reproducible, and sufficiently accurate for measuring fCal in dogs and cats. Careful interpretation of fCal concentrations is warranted in both species during the peri-vaccination period and for some patients receiving NSAID treatment.

Comparison of three different HbA1c measurement methods - the Atellica®CH930, Capillary 3 Tera, and BioRad Variant Turbo II.

AIM: HbA1c measurement is very useful for the follow-up and detection of glycemic disorder, since it is easier and faster test and is independent of the patient's fasting status. In this study, we aimed to perform the comparative evaluation of 3 different methods for HbA1c measurement including capillary electrophoresis, immunoturbidimetric assay and high-performance liquid chromatography-HPLC. MATERIALS AND METHODS: This study comprised 134 leftover whole blood samples obtained from the subjects submitted for routine HbA1c testing. All blood samples were collected in EDTA-containing vacutainer tubes. The HbA1c levels were measured simultaneously using three different methods. Bias estimation, method agreement and concordance between the pairwise methods comparisons were evaluated by Bland-Altman plot and Passing-Bablok regression test. RESULTS: HbA1c levels ranged from 3.8% to 13.4% and measured by three different methods to make the comparison. The median values of samples based on immunoturbidimetric method (6.05%, IQR = 1.80) were higher than capillary electrophoresis method (5.90%, IQR = 1.80) and HPLC (5.85%, IQR = 1.80) method. The study group was classified into three subgroups based on the HbA1c levels measured with the HPLC method: Group 1 (n = 57) was composed of subjects with HbA1c levels less than 5.7%, Group 2 (n = 35) had HbA1c levels between 5.7% and 6.4%, Group 3 (n = 42) had HbA1c levels equal and more than 6.5%. CONCLUSION: To our knowledge, there is no study evaluating the HbA1c measurement on the Atellica® CH 930 Analyzer. We compared the Atellica®CH930 Analyzer with both HPLC and capillary electrophoresis. The Atellica®CH930 Analyzer showed acceptable performance and a strong correlation with both mentioned methods.

Evaluation of D-dimer levels measured by different analytical methods in COVID-19 patients.

Clinicians experience some challenges due to the lack of standardization of test, although D-dimer is a prognostic marker for COVID-19. We compared the clinical and analytical performances of D-dimer results obtained from different devices, kits and methods in patients with a diagnosis of COVID-19. Thirty-nine patients with a diagnosis of COVID-19 and 24 healthy individuals were included in the study. D-dimer levels were measured with Innovance D-DIMER kit (immunoturbidimetric method) on Sysmex CS-2500 and BCS XP and VIDAS D-Dimer Exclusion II kit (enzyme-linked fluorescence method) on mini VIDAS. The studies of precision, method comparison and clinic performance were performed. The variation coefficients in all systems were within the acceptable imprecision (7.8%). Bias%(12.5%) between BCS XP and Sysmex CS-2500 was lower than the acceptable Bias%(15.5%). Bias% values (19.2% and 33.3%, respectively) between Mini VIDAS with BCS XP and Sysmex CS-2500 were higher than the acceptable Bias%. The correlation coefficients among all systems were 0.89-0.98. For 500 ng/ml FEU, there was almost perfect agreement between BCS XP and Sysmex CS-2500, a moderate agreement between Mini VIDAS and BCS XP and Sysmex CS-2500. The cut-off values for distinguishing between individuals with and withoutCOVID-19 were Mini VIDAS, Sysmex CS-2500 and BCS XP 529, 380 and 390 ng/ml FEU, respectively. The immunoturbidimetric method can be used as an alternative to the enzyme-linked fluorescent method because of satisfactory agreement at the different thresholds proposed for venous thromboembolism. However, it is recommended to follow up COVID-19 with the D-dimer results obtained by the same assay system.

Investigation of the quantitative detection of serum Helicobacter pylori antibody in clinical laboratories in China.

BACKGROUND: This study aimed to investigate the implementation and quality control of the quantitative detection of serum Helicobacter pylori (H. pylori) antibody in clinical laboratories in China. METHODS: Online external quality assessment (EQA) questionnaires were distributed to the clinical laboratories by National Center for Clinical Laboratories (NCCL) of China. We collected information on the quantitative detection procedures of serum H. pylori antibody in clinical laboratories, including detection reagents, methods, instruments, calibrators, and internal quality control (IQC). We distributed quality control products to some select laboratories that conducted quantitative detection and analyzed the obtained test data. We evaluated the quantitative detection procedure based on the standard evaluation criteria set at a target value of ±30%. RESULTS: 70.9% (146/206) of the laboratories conducted quantitative detection of H. pylori antibody; 29.1% (60/206) of the laboratories performed qualitative detection. Domestic reagents and matching calibrators accounted for more than 97.1% (200/206) of all reagents. Latex-enhanced immunoturbidimetry was used in 89.7% (131/146) of the laboratories for quantitative determination, while the colloidal gold method was used in 66.7% (40/60) of the laboratories for qualitative determination. A total of 130 laboratories participated in the EQA; 123 completed the assessment, and the pass rate was 75.6% (93/123). CONCLUSION: Clinical quantitative detection of serum H. pylori antibody is performed at a high rate in China. Thus, further studies on the specificity of commercial detection reagents are needed. EQAs are useful to monitor and improve the detection quality of H. pylori antibodies.

Comparison of a point-of-care serum amyloid A analyzer frequently used in equine practice with 2 turbidimetric immunoassays used in human and veterinary medicine.

Rapid, accurate detection of serum amyloid A (SAA) is needed in equine practice. We validated a patient-side point-of-care (POC) assay (Stablelab; Zoetis) compared to the turbidimetric immunoassays LZ-SAA (TIA-Hum) and VET-SAA (TIA-Vet; both Eiken Chemical). Analytical performance was assessed at 3 different concentration ranges and with interferences. Inter-method comparison using 49 equine serum samples revealed a significant difference between median SAA results (p < 0.0001), with the strongest bias between the POC and TIA-Vet (median 1,093 vs. 578 mg/L). The median SAA value obtained with the TIA-Hum method was 752 mg/L. Correlation between POC/TIA-Hum and between POC/TIA-Vet was fair (rs = 0.77 and 0.69) and excellent between both TIAs (rs = 0.93). Bias between POC/TIA-Hum, POC/TIA-Vet, and TIA-Hum/TIA-Vet was -56.7%, -80.9%, and -28.2%, respectively. POC intra- and inter-assay CVs (16.1-30% and 19.8-35.5%) were higher than TIA CVs (generally <12%). Bilirubin and hemoglobin had a negative bias on POC and TIA-Vet results (-16.6 to -45.6%); addition of intralipid yielded a positive bias (35.9-77.4%). The POC had good linearity of SAA concentrations up to 10,312 mg/L (R2 = 0.92). A hook effect was present at SAA >3,000 mg/L for the POC assay. Equine serum SAA was stable over a median period of 2.5 y when stored at -80°C. Overall, there was excellent-to-moderate correlation between tests, but imprecision and hook effect of the POC, as well as bias between the methods, must be considered.

Analytical validation of a point-of-care test and an automated immunoturbidimetric assay for the measurement of canine C-reactive protein in serum.

C-reactive protein (CRP) is an acute phase protein, which is used to evaluate and monitor the response of the innate immune system to a variety of inflammatory processes in the dog. The purpose of this study was to analytically validate a point-of-care assay (IDEXX Catalyst CRP Test) and an immunoturbidimetric assay (Gentian Canine CRP Immunoassay) for the measurement of serum CRP concentrations in dogs. These 2 assays (Catalyst, Gentian) were compared to a previously validated enzyme-linked immunosorbent assay (Tridelta Development EIA Canine CRP Assay). Linearity, precision, reproducibility, and accuracy were assessed using leftover serum samples. Agreement between assays was assessed using leftover serum samples and serum from clinically healthy dogs. Observed to expected ratios (O/E) for dilutional parallelism were 83.9 to 163.1% and 108.3 to 160.6% for the Catalyst and the Gentian assays, respectively. Coefficients of variation for intra-assay variability ranged from 6.4 to 9.5% for the Catalyst assay and 1.5 to 2.6% for the Gentian assay. Coefficients of variation for inter-assay variability ranged from 3.8 to 18.2% for the Catalyst assay and 4.5 to 5.8% for the Gentian assay. The mean O/E for recovery were 97.9% and 98.5% for the Catalyst and Gentian assays, respectively. Correlations between assays were as follows: Catalyst and Tridelta (R 2 = 0.76), Gentian and Tridelta (R 2 = 0.79), and Catalyst and Gentian (R 2 = 0.98). The Catalyst and Gentian assays are both acceptable for measuring CRP in dog serum, but their results are not directly comparable with the Tridelta assay.

La protéine C réactive (CRP) est une protéine de phase aiguë, qui est utilisée pour évaluer et surveiller la réponse du système immunitaire inné à une variété de processus inflammatoires chez le chien. Le but de cette étude était de valider analytiquement un test au point de service (test IDEXX Catalyst CRP) et un test immunoturbidimétrique (Gentian Canine CRP Immunoassay) pour la mesure des concentrations sériques de CRP chez le chien. Ces deux tests (Catalyst, Gentian) ont été comparés à un test immuno-enzymatique précédemment validé (Tridelta Development EIA Canine CRP Assay). La linéarité, la précision, la reproductibilité et l'exactitude ont été évaluées à l'aide d'échantillons de sérum restants. La concordance entre les tests a été évaluée à l'aide d'échantillons de sérum restants et de sérum provenant de chiens cliniquement sains. Les rapports observés/attendus (O/E) pour le parallélisme de dilution étaient de 83,9 à 163,1 % et de 108,3 à 160,6 % pour les tests Catalyst et Gentian, respectivement. Les coefficients de variation pour la variabilité intra-test variaient de 6,4 à 9,5 % pour le test Catalyst et de 1,5 à 2,6 % pour le test Gentian. Les coefficients de variation pour la variabilité inter-test variaient de 3,8 à 18,2 % pour le test Catalyst et de 4,5 à 5,8 % pour le test Gentian. L'O/E moyen pour la récupération était de 97,9 % et de 98,5 % pour les tests Catalyst et Gentian, respectivement. Les corrélations entre les tests étaient les suivantes : Catalyst et Tridelta (R 2 = 0,76), Gentian et Tridelta (R 2 = 0,79) et Catalyst et Gentian (R 2 = 0,98). Les tests Catalyst et Gentian sont tous deux acceptables pour mesurer la CRP dans le sérum de chien, mais leurs résultats ne sont pas directement comparables avec le test Tridelta.(Traduit par Docteur Serge Messier).

Soluble urokinase plasminogen activator receptor (suPAR) predicts critical illness and kidney failure in patients admitted to the intensive care unit.

Soluble urokinase plasminogen activator receptor (suPAR) is an inflammatory biomarker and risk factor for kidney diseases, with a potential prognostic value in critically ill patients. In this monocentric prospective study, we measured plasma suPAR levels immediately after ICU admission in unselected 237 consecutive patients using a turbidimetric assay. Primary objective was the prognostic value for ICU- and 28-day mortality. Secondary objectives were association with sequential organ failure assessment (SOFA) score, coagulation and inflammation markers, AKI-3 and differences in prespecified subgroups. Median suPAR levels were 8.0 ng/mL [25th-75th percentile 4.3-14.4], with lower levels in ICU survivors than non-survivors (6.7 vs. 11.6 ng/mL, p < 0.001). SuPAR levels were higher in COVID-19, kidney disease, moderate-to-severe liver disease, and sepsis. ICU mortality increased by an odds ratio (OR) of 4.7 in patients with the highest compared to lowest quartile suPAR. Kaplan-Meier overall survival estimates at 3 months were 63% and 49%, in patients with suPAR below/above 12 ng/mL (log-rank p = 0.027). Due to an observed interaction between SOFA score and suPAR, we performed a random forest method identifying cutoffs. ICU mortality was 53%, 17% and 2% in patients with a SOFA score > 7, SOFA ≤ 7 & suPAR > 8 ng/mL, and SOFA score ≤ 7 & suPAR ≤ 8 ng/mL, respectively. suPAR was a significant predictor for AKI-3 occurrence (OR per doubling 1.89, 95% CI: 1.20-2.98; p = 0.006). suPAR levels at ICU admission may offer additional value for risk stratification especially in ICU patients with moderate organ dysfunction as reflected by a SOFA score ≤ 7.

Development of an agglutination-enhancement immunoturbidimetric tacrolimus assay with improved sensitivity.

Accurate quantification of low level of blood drugs by Latex enhanced immunoturbidimetric assay (LEITA) remains a challenge due to its inherent limited sensitivity. To deal with this problem, we designed a new agglutination-enhancement strategy, and applied it for development of a highly sensitive and accurate tacrolimus LEITA. By this principle, a very small amount of biotin labeled anti-tacrolimus monoclonal antibodies (BLATMA) can arise agglutination strong enough for accurate reading of the increased absorbance since the BLATMA bears multiple biotin molecules, and the agglutination mediated by BLATMA can be inhibited by a similarly small amount of tacrolimus when the drug binds to BLATMA, giving rise to an improved sensitivity. The limit of detection (LOD) and functional sensitivity obtained by the proposed tacrolimus LEITA was 0.22 ng/mL and 0.59 ng/mL, respectively, 8-20 times more sensitive than the conventional drug-latex or antibody-latex based direct inhibition LEITA formats. Good precision was observed in the whole range of clinically significant tacrolimus concentration. The reliability of the tacrolimus LEITA was demonstrated by its strong correlation with both liquid chromatography-tandem mass spectrometry (LC-MS/MS) (R2 = 0.977, slope = 0.998) and the ABBOTT tacrolimus chemiluminescent magnetic immunoassay (CMIA) (R2 = 0.982, slope = 1.01) in the analysis of 119 clinical samples. It's concluded that the agglutination-enhancement strategy can be applied to construct highly sensitive LEITA for accurate tacrolimus analysis; owing to the improved sensitivity, this technique can be expected not only to improve the reliability of LEITA for low-level drug monitoring, but also to broaden the scope of analytes detectable by LEITA.

Prospective comparison of (1,3)-beta-D-glucan detection using colorimetric and turbidimetric assays for diagnosing invasive fungal disease.

Serum (1→3)-ß-D-glucan (BDG), an pan fungal antigen, is detected in some invasive fungal diseases (IFDs). We compared two commercial kits, the Fungitell assay (FA) (colorimetric) and the Wako assay (WA) (turbidimetric) over a 4-month period to prospectively test 171 patients who mainly had hematological conditions (62%) and experienced episodes (n = 175) of suspected invasive fungal infection. Twenty-three episodes due to BDG-producing fungi were diagnosed (pneumocystosis, n = 12; invasive aspergillosis, n = 5; candidemia, n = 3; invasive fusariosis, n = 2; hepato-splenic candidiasis, n = 1).Both assays provided similar areas under the curves (AUC = 0.9). Using the optimized positivity thresholds (≥120 pg/ml for FA and ≥ 4 pg/ml for WA), the sensitivity and specificity were 81.8% (CI95: 61.5-92.7), 94.8% (90.1-97.3) for FA and 81.8% (61.5-92.7), 95.4% (90.9-97.8) for WA. Negative predictive value was 97.3% (93.3-99.0) for both tests. If the manufacturer's positivity threshold (≥11 pg/ml) was applied, the WA sensitivity decreased to 50%. Among 71 patients with bacterial infections, 21.1% were FA-positive and 5.6% were WA-positive (p < 10-2).The WA performed similarly as compared to the FA with an optimized cutoff value. The WA is a single sample test that is clinically relevant when a prompt therapeutic decision is required. LAY SUMMARY: Serum (1→3)-ß-D-glucan testing is dominated by two kits including Fungitell colorimetric assay (FA) and the Wako turbidimetric assay (WA). We compared them prospectively and observed that they both perform similarly when selecting their optimal threshold (≥120 pg/ml for FA and ≥ 4 pg/ml for WA).

Preparation and characterization of cyclic citrullinated peptide-immobilized latex beads for measurement of anti-citrillinated protein antibody through latex particle-enhanced turbidimetric immunoassay.

The anti-citrillinated protein antibody (ACPA) plays an important role in early diagnosis of rheumatoid arthritis (RA), and is usually detected by using cyclic citrullinated peptide (CCP) as antigen. The ACPA against CCP test is usually performed utilizing enzyme-linked immunosorbent assay (ELISA), but the ELISA is expensive and time-consuming. Here, latex particle-enhanced turbidimetric immunoassay (LTIA) based on CCP-immobilized latex bead was proposed for fast measurements of ACPA of RA patients. CCP-immobilized latex bead was fabricated through three methods, including direct coupling, overall coupling and layer by layer coupling. According to the optimized experiments, layer-by-layer coupling was the best method with advantages of time-saving, simple operation and good repeatability. In addition, a spacer arm of appropriate length between latex beads and CCP could avoid stereoscopic obstacles and make ACPA closer to CCP. The CCP-immobilized latex bead based on layer by layer coupling (CCP-LB-LLC) was used for assembling the homemade kit, which was applied in fast measurements of ACPA through LTIA. The homemade kit possessed a low limit of detection (0.2 U/mL) and an acceptable the batch-to-batch reproducibility. In addition, the homemade kit can be stored at 4 °C for at least one month. When used to detect 20 clinical samples, the results of homemade kit were consistent with commercial ELISA. Furthermore, LTIA based on the homemade kit was simpler and cheaper than ELISA. These results demonstrated that the homemade kit could be useful for diagnosis of RA patients.