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.

Identification of and solution for false D-dimer results.

BACKGROUND: Clinically, D-dimer (DD) levels are mainly used to exclude diseases such as deep venous thrombosis (DVT). In clinical testing, DD assays can be subjected to interference that may cause false results, which directly affect the clinical diagnosis. Our hypothesis was that the 95% confidence intervals (CIs) of the fibrin degradation product (FDP)/DD and fibrinogen (Fib)/DD ratios were used to identify these false results and corrected via multiple dilutions. METHODS: In total, 16 776 samples were divided into three groups according to the DD levels detected by Sysmex CS5100 and CA7000: Group A, DD ≥ 2.0 µg/mL fibrinogen equivalent unit (FEU); group B, 0.5 < DD < 2.0 µg/mL FEU; and group C, DD ≤ 0.5 µg/mL FEU. The 95% CIs of the FDP/DD and Fib/DD ratios were calculated. Six abnormal DD results were found according to the 95% CIs. For verification, we performed multiple dilutions, compared the results with those of other instruments, and tested the addition of heterophilic blocking reagent (HBR). RESULTS: The median and 95% CI of the FDP/DD ratio were 3.76 and 2.25-8.15 in group A, 5.63 and 2.86-10.58 in group B, 10.23 and 0.91-47.71 in groups C, respectively. For the Fib/DD ratio, the 95% CIs was 0.02-2.21 in group A, 0.68-8.15 in group B, and 3.82-55.27 in groups C. Six abnormal results were identified after multiple dilutions, by comparison with other detection systems, and after HBR addition. CONCLUSIONS: The FDP/DD ratio is more reliable for identifying false results. If the FDP/DD ratio falls outside the 95% CI, it should be verified by different methods.

The bias between different albumin assays may affect clinical decision-making.

Differences between laboratory assays can have important clinical implications. For creatinine assays this became apparent soon after the introduction of the Modification of Diet in Renal Disease formula and resulted in international efforts towards standardization. Albumin in blood is measured by different assays, either chromogenic using Bromocresol green (BCG) or Bromocresol purple (BCP), or by an immunoassay. Since differences between these assays have received limited attention we evaluated bias and imprecision of BCG and BCP assays in comparison to the immunoassay using blood samples from patients with membranous nephropathy and nephrotic syndrome. For the BCG assay, the mean bias was high (6.2 g/l, with a standard deviation of 2.4 g/l) compared to a bias of 0.3 g/l (standard deviation 1.5 g/l) for the BCP assay. Importantly, we questioned clinical relevance by evaluating the accuracy of the decision toward the use of prophylactic anticoagulant therapy. Notably, nephrologists may reach inappropriate treatment decisions using the BGC assay in up to 59% of patients. Thus, our study should stimulate efforts towards standardization of the albumin assays.

Analytical Performance of a New Immunoturbidimetric Assay for von Willebrand Factor (VWF) Activity Testing.

BACKGROUND: Von Willebrand disease requires laboratory confirmation with quantitative and qualitative measurements of von Willebrand factor (VWF). Qualitative VWF-activity (VWF-Ac) tests have poor inter- and intra-laboratory reproducibility with coefficients of variation (CVs) as high as 64%, often lacking accuracy at low VWF-Ac levels. METHODS: This study evaluated the recently launched immunoturbidometric STAGO® STA-VWF:RCo® reagent for VWF-Ac. Accuracy was evaluated on 32 samples by comparing results using the Siemens® Innovance® reagent. An intra-run reproducibility study was performed on controls. Linearity and lower limit of detection was studied on external-quality-assurance (EQA) material with a known VWF-Ac level. RESULTS: STA-VWF:RCo® reagent results were within clinical interpretation agreement with Siemens® Innovance®. The reproducibility study yielded % CVs of 8.41 for normal and 11.46 for abnormal controls and the assay was linear between 73 and 14.6% and remained linear to 2% with extrapolation. CONCLUSIONS: The STAGO® STA-VWF:RCo® reagent showed clinically meaningful accuracy and acceptable precision.

Validity and application of immunoturbidimetric and enzyme-linked immunosorbent assays for the measurement of adiponectin concentration in ponies.

BACKGROUND: Circulating adiponectin concentrations were lower in ponies with a history of endocrinopathic laminitis and in nonlaminitic ponies that subsequently developed laminitis. The assays used in these studies have been discontinued or are no longer valid. OBJECTIVES: (1) to determine the validity of immunoturbidimetric (IT) and enzyme linkedimmunosorbent (ELISA) assays for equine total and high molecular weight (HMW) [adiponectin] measurement and (2) to investigate the association between [adiponectin] measured using these assays and endocrinopathic laminitis. STUDY DESIGN: Method validation and cohort study. METHODS: Accuracy and precision of IT and ELISA assays for measuring total (TAC) and HMW (HMWAC) [adiponectin] were determined. Using the IT assay, the effects of anti-coagulant and storage temperature were assessed, TAC was measured in previously laminitic (PL) and never laminitic (NL) ponies (n = 6/group). Comparison with a previously validated radioimmunoassay was made in NL ponies (n = 223). Association between TAC and subsequent laminitis development in NL ponies was investigated using univariable logistic regression and ROC curve analysis. RESULTS: The IT assay was precise and demonstrated good agreement with the previously validated radioimmunoassay. TAC was significantly (P<0.01) lower in PL (mean ± s.d. 8.9 ± 2.9 µg/mL) compared to NL (24.2 ± 11.8 µg/mL) ponies and in NL ponies that developed laminitis within 12 months (median 4.8 µg/mL; IQR 2.65-13.4 µg/mL) compared to those that remained nonlaminitic (19.9 µg/mL; 9.95-31.5 µg/mL). TAC was significantly (P = 0.01) associated with laminitis occurrence within 12 months. Use of the area under the ROC curve to distinguish animals that did and did not develop laminitis showed good accuracy (0.76). None of the ELISA methods validated satisfactorily. MAIN LIMITATIONS: Laminitis risk is based on data from ponies in one region. CONCLUSIONS: The IT method is suitable for measurement of equine TAC. TAC is lower in ponies with previous or future laminitis. The ELISA methods are not suitable for measurement of equine HMWAC or TAC.

Quantification of human complement C2 protein using an automated turbidimetric immunoassay.

BACKGROUND: The measurement of complement components is clinically useful where a deficiency is suspected, or where excessive activation and consumption are present in disease. C2 deficiency carries an increased risk of developing systemic lupus erythematosus, recurrent infections and atherosclerosis. In this study, we have evaluated The Binding Site's Human Complement C2 SPAPLUS® assay. METHODS: Linearity was tested using 13 sample dilutions covering the standard measuring range. Within- and between-assay variabilities were calculated using five samples with different C2 concentrations. The correlation between C2 concentrations in EDTA-plasma and serum was assessed, as was the correlation between C2 measurements by the automated assay and radial immunodiffusion. C2 concentrations were compared with CH50 activity, and quantified in individuals with homozygous or heterozygous C2 deficiency, acquired angioedema and patients with chronic inflammatory conditions. RESULTS: The assay was linear across the measuring range (3.8-42.3 mg/L). Intra- and interassay variability were 2.3%-3.8% and 0%-3.3%, respectively. Comparison between C2 measurements in EDTA-plasma and serum provided a strong correlation (p<0.0001, R2=0.82, slope 0.92), as did the correlation between the automated and radial immunodiffusion methods (p<0.0001, R2=0.89, slope 1.07). A positive correlation between C2 concentration and CH50 activity was demonstrated (p<0.0001, R2=0.48). Significant differences were observed between the median C2 concentrations obtained in healthy controls and the patient clinical samples, with homozygous C2-deficient patients giving below detectable results. CONCLUSIONS: This C2 SPAPLUS® assay allows the automated, rapid and precice quantification of complement C2 protein and could therefore be considered as a replacement for older, more time-consuming methods.

Evaluation of a Turbidimetric ß-d-Glucan Test for Detection of Pneumocystis jirovecii Pneumonia.

Currently, diagnosis of Pneumocystis jirovecii pneumonia (PJP) relies on analysis of lower respiratory specimens, either by microscopy or quantitative real-time PCR (qPCR). Thus, bronchoscopy is required, which is associated with increased risk of respiratory failure. We assessed the value of noninvasive serologic ß-d-glucan (BDG) testing for laboratory diagnosis of PJP using a newly available turbidimetric assay. We identified 73 cases of PJP with positive qPCR results from lower respiratory specimens for Pneumocystis and serology samples dating from 1 week before to 4 weeks after qPCR. In addition, 25 sera from controls with suspected PJP but specimens negative for Pneumocystis by qPCR were identified. Sera were tested with a turbidimetric BDG assay (Fujifilm Wako Chemicals Europe GmbH, Neuss, Germany), using an 11-pg/ml cutoff. Sensitivity and specificity were calculated based on qPCR test results as a reference. The turbidimetric BDG assay identified 63/73 patients with positive or slightly positive qPCR tests for an overall sensitivity of 86%; after exclusion of cases with only slightly positive qPCR results, sensitivity was 91%. No correlation between serum BDG levels and respiratory specimen DNA levels was found. Serologic BDG testing was negative in 25/25 controls with negative qPCR for a specificity of 100% using the predefined cutoff. In 22/25 samples (88%), no BDG was detected. Serologic BDG testing using the turbidimetric assay showed high sensitivity and specificity compared to qPCR of lower respiratory specimens for the diagnosis of PJP. Both turnover time and test performance will allow clinicians to delay or in some cases forego bronchoscopy.

Determination of serum albumin, analytical challenges: a French multicenter study.

Among the biological markers of morbidity and mortality, albumin holds a key place in the range of criteria used by the High Authority for Health (HAS) for the assessment of malnutrition and the coding of information system medicalization program (PMSI). If the principle of quantification methods have not changed in recent years, the dispersion of external evaluations of the quality (EEQ) data shows that the standardization using the certified reference material (CRM) 470 is not optimal. The aim of this multicenter study involving 7 sites, conducted by a working group of the French Society of Clinical Biology (SFBC), was to assess whether the albuminemia values depend on the analytical system used. The albumin from plasma (n=30) and serum (n=8) pools was quantified by 5 different methods [bromocresol green (VBC) and bromocresol purple (PBC) colorimetry, immunoturbidimetry (IT), immunonephelometry (IN) and capillary electrophoresis (CE)] using 12 analyzers. Bland and Altman's test evaluated the difference between the results obtained by the different methods. For example, a difference as high as 13 g/L was observed for the same sample between the methods (p <0.001) in the concentration range of 30 to 35 g/L. The VBC overestimates albumin across the range of values tested compared to PBC (p <0.05). PBC method gives similar results to IN for values lower than 40 g/L. For IT methods, one of the technical/analyzer tandem underestimates the albumin values inducing a difference of performance between the immunoprecipitation methods (IT vs IN, p <0.05). Although, the albumin results are related to the technical/analyzer tandem used. This variability is usually not taken into account by the clinician. Thus, clinicians and biologists have to be aware and have to check, depending on the method used, the albumin thresholds identified as risk factors for complications related to malnutrition and PMSI coding.

Validation and determination of a reference interval for canine HbA1c using an immunoturbidimetric assay.

BACKGROUND: Hemoglobin A1c (HbA1c) provides a reliable measure of glycemic control over 2-3 months in human diabetes mellitus. In dogs, presence of HbA1c has been demonstrated, but there are no validated commercial assays. OBJECTIVE: The purpose of the study was to validate a commercially available automated immunoturbidimetric assay for canine HbA1c and determine an RI in a hospital population. METHODS: The specificity of the assay was assessed by inducing glycosylation in vitro using isolated canine hemoglobin, repeatability by measuring canine samples 5 times in succession, long term inter-assay imprecision by measuring supplied control materials, stability using samples stored at 4°C over 5 days and -20°C over 8 weeks, linearity by mixing samples of known HbA1c in differing proportions, and the effect of anticoagulants with paired samples. An RI was determined using EDTA-anticoagulated blood samples from 60 nondiabetic hospitalized animals of various ages and breeds. Hemoglobin A1c was also measured in 10 diabetic dogs. RESULTS: The concentration of HbA1c increased proportionally with glucose concentration in vitro. For repeat measurements, the CV was 4.08% (range 1.16-6.10%). Samples were stable for 5 days at 4°C. The assay was linear within the assessed range. Heparin- and EDTA-anticoagulated blood provided comparable results. The RI for HbA1c was 9-18.5 mmol/mol. There was no apparent effect of age or breed on HbA1c. In diabetic dogs, HbA1c ranged from 14 to 48 mmol/mol. CONCLUSIONS: The assay provides a reliable method for canine HbA1c measurement with good analytic performance.

Evaluación del desempeño analítico de tres métodos de cuantificación de hemoglobina A1c / Analytical performance evaluation of three methods for hemoglobin A1c quantification / Avaliação do desempenho analítico do três métodos de quantificação de hemoglobina A1c

El laboratorio debe garantizar la exactitud de los resultados de HbA1c cumpliendo con los requisitos analíticos internacionales de calidad, cada vez más estrictos y asegurar que una variación de HbA1c de 0,5 puntos porcentuales (%-NGSP) o más entre dos controles consecutivos de un paciente diabético se deba a una variación clínica y no a una variación analítica. En este trabajo se evaluó el desempeño analítico de tres métodos comerciales para HbA1c: inmunoturbidimétrico, enzimático y cromatográfico de intercambio catiónico. Para tal fin, se procesaron por cada método distintos controles comerciales de HbA1c, con trazabilidad al método de referencia IFCC, determinándose en cada caso Coeficiente de Variación Total, Bias, Error Total, Valor de Referencia del Cambio y cambio clínico significativo de HbA1c en el punto crítico 7,0%-NGSP. En las condiciones analíticas de este trabajo, solamente el método inmunoturbidimétrico tuvo un desempeño analítico aceptable, permitiendo atribuir un cambio de 0,5%-NGSP a una variación clínica significativa del paciente. Frente a las recomendaciones internacionales sobre el uso de HbA1c en el control y diagnóstico de diabetes, es indiscutible la importancia de elegir un método que satisfaga los requerimientos analíticos mínimos de calidad para asegurar la utilidad clínica del resultado de HbA1c.

The laboratory must guarantee the accuracy of HbA1c results meeting the increasingly strict international analytical quality standards and assuring that an HbA1c variation of 0.5 percentage points (%-NGSP) or more between two consecutive controls of a diabetic patient is due to a clinical variation and not to an analytical variation. In this paper, the analytical performance of three commercial methods for HbA1c: Immunoturbidimetric, Chromatographic and Enzymatic Cation Exchange, were evaluated. For this purpose, commercial controls with assigned values traceable to the IFCC reference method for HbA1c were processed. For each methodology, total Coefficient of Variation (CV%), Bias%, Total Error (TE%), Change Reference Value and Clinically Significant Change (CSC) at the critical point of HbA1c 7.0%-NGSP were determined. Within the analytical conditions of this study, only the immunoturbidimetric method had an acceptable analytical performance, allowing attribute a change in 0.5%-NGSP to a significant clinical variation. Faced with international recommendations on the use of HbA1c on control and diagnosis of diabetes, the importance of choosing a method that meets the minimum analytical quality requirements to ensure the clinical utility of HbA1c result is undeniable.

O laboratório deve garantir a precisão dos resultados da HbA1c cumprindo com os requisitos analíticos internacionais de qualidade cada vez mais exigentes e garantir que uma variação de HbA1c de 0,5 pontos percentuais (% - NGSP) ou mais entre duas verificações consecutivas de um doente diabético seja devido a uma variação clínica e não a uma variação analítica. Neste trabalho foi avaliado o desempenho analítico de três métodos comerciais para HbA1c: imunoturbidimétrico, enzimático e cromatográfico de intercâmbio catiônico. Para esse fim, foram processados diversos controles comerciais de HbA1c por cada método, com rastreabilidade ao método de referência IFCC, determinando em cada caso Quociente de Variação Total, Bias, Erro Total, Valor de Referência da Alteração e Alteração Clinicamente Significativa de HbA1c no ponto crítico 7,0%-NGSP. Nas condições de análise deste estudo, apenas o método imunoturbidimétrico teve um desempenho analítico aceitável, permitindo atribuir uma alteração de 0,5%-NGSP a uma variação clínica significativa do paciente. Perante as recomendações internacionais sobre o uso da HbA1c no controle e diagnóstico da diabetes, é inegável a importância de escolher um método que atenda os requisitos analíticos mínimos de qualidade de análise para garantir a utilidade clínica do resultado HbA1c.