Comparison of the detection of Helicobacter pylori infection by commercially available serological testing kits and the 13C-urea breath test.

BACKGROUND: Serum Helicobacter pylori (H. pylori) antibody kits (LZ and LIA) using the latex agglutination immunoassay method are commercially available, but few studies have been performed to determine their diagnostic accuracy or to compare their results with those of enzyme-linked immunosorbent assay (ELISA) kits (EP and EIA). METHODS: Sera were obtained from 213 hospital outpatients with dyspeptic symptoms. The serological results were compared with the result of the 13C-urea breath test (UBT) which seems to be reliable. RESULTS: Of the 213 subjects, 154 were diagnosed as positive for H. pylori infection according to the UBT. The sensitivities and specificities of these tests were 97.4% and 76.3%, 98.1% and 78.0%, 99.4% and 74.6%, and 98.1% and 71.2% for the EP, LZ, EIA and LIA tests, respectively. When the 13 subjects whose seropositive results of the four kits were completely opposite to the negative results of the UBT were excluded, the specificities of evaluated kits were all higher than 90%. The concordance rate between the EP and EIA tests was 98.1% (Spearman's rank correlation coefficient = 0.83) and that between the LZ and LIA tests was 97.1% (correlation coefficient = 0.91). The LZ gave higher antibody titer value than EP (p < 0.0001, Z = 9.82; Wilcoxon signed-rank test), and EIA gave higher value than LIA (p < 0.0001, Z = 6.43; Wilcoxon signed-rank test). CONCLUSIONS: The latex immunoassay method provided the same reliability to ELISA in terms of the diagnostic accuracy for current H. pylori infection, although we should take into account the titer value differences by each test method in practical use.

Accuracy of three hemoglobin A1c point-of-care systems for glucose monitoring in patients with diabetes mellitus.

OBJECTIVES: Use of hemoglobin A1c point-of-care devices in physician offices provides immediate results and reduces inconveniences for the patients. We compared the analytical performances of 3 point-of-care HbA1c analyzers to high pressure liquid chromatography (HPLC). MATERIAL AND METHODS: We preselected a pool of 40 EDTA-preserved whole blood samples from our laboratory with HbA1c results obtained by HPLC (mean 6.6% [49 mmol/mol] and range: 4.6-9.9% [27-87 mmol/mol]). Aliquots of theses samples were tested by Afinion AS100, DCA Vantage and In2it point-of-care systems. According the Clinical Laboratory Standards Institute EP-09 protocol we determined linearity (linear regression and correlation coefficient between point-of-care and reference methods), bias (Bland-Altman analysis) and coefficient of variation (%). We used the acceptability criteria endorsed by the National Glycohemoglobin Standardization Program. RESULTS: The calculated correlation coefficients (r) were 0.98, 0.98 and 0.83 for Afinion AS100, DCA Vantage and In2it systems, respectively. The 95% confidence interval of the error between point-of-care systems and the reference method was -0.41% and +0.34% (p =.22) for Afinion AS100, -0.62% and +0.05% (p =.57) for DCA Vantage, and -1.15% and +1.26% (p<.001) for the In2it. The coefficients of variation for Afinion AS100, DCA Vantage and In2it systems were 1.80, 3.74 and 7.14%, respectively. CONCLUSION: Only the Afinion AS100 point-of-care system met all NGSP performance criteria.

Evaluation of an automated assay based on monoclonal anti-human serum amyloid A (SAA) antibodies for measurement of canine, feline, and equine SAA.

Major acute phase proteins (APPs) have proven diagnostically useful in dogs, cats and horses with routine use facilitated by commercially available automated heterologous assays. An automated assay applicable across all three species would highly facilitate further dissemination of routine use, and the aim of this study was to validate an automated latex agglutination turbidimetric immunoassay based on monoclonal anti-human serum amyloid A (SAA) antibodies for measurement of canine, feline and equine SAA. Serum samples from 60 dogs, 40 cats and 40 horses were included. Intra- and inter-assay imprecision, linearity and detection limit (DL) were determined to assess analytical performance. To assess clinical performance, equine and feline SAA measurements were compared with parallel measurements using a previously validated automated SAA assay in a method comparison setting, and by assessing overlap performance of canine SAA in healthy dogs and diseased dogs with and without systemic inflammation. Intra- and inter-assay CVs ranged between 1.9-4.6% and between 3.0-14.5%, respectively. Acceptable linearity within a clinically relevant range of SAA concentrations was observed for all three species. The DL was 1.06 mg/L. Method comparison revealed acceptable agreement of the two assays measuring feline and equine SAA, and the overlap performance of canine SAA was acceptable. The tested assay measured SAA in canine, feline and equine serum with analytical and overlap performance acceptable for clinical purposes so improving practical aspects of clinical APP application. The monoclonal nature of the antibodies suggests strong, long-term inter-batch performance stability.

[The latex agglutination with video digital registration: the enhancement of diagnostic significance of conventional technique].

The rapid semiquantitative latex-tests, because of their analytic characteristics and convenient application, became widespread in the practice of laboratory diagnostics. Though, in spite of high sensitivity and specificity, their diagnostic effectiveness is lower that it could be mainly because of the impossibility to document the results of latex agglutinative re4actions and to manage the objective quality control. The application of systems of video digital registration permits to enhance the clinical significance of these analyses. By means of scanner systems (control and program complex "Expert Lab") the image of analytic objects is received with the results of latex agglutination reaction. The application of program techniques (the programs "Expert Lab - Agglutination" and "Expert Lab - Agglutination - Micros") in data processing permits to get the precise qualitative characteristics of active reactions, to ensure the automatic interpretation of results and gives an opportunity to proceed with the internal laboratory quality control. The saving of analytic object image in computer memory after termination of reaction favors the formation of data base, the implementation of retrospective evaluation of obtained results, additional consultations in dubious cases, including on-line. The application of complex "Expert Lab" permitted to develop the miniaturizes matrix systems permitting to decrease the withdrawal of latex reagents, to increase the productivity of analytical stage of operation preserving all analytical characteristics of method.

Rapid detection of Mycoplasma pneumonia in a microfluidic device using immunoagglutination assay and static light scattering.

We present real-time, rapid detection of Mycoplasma pneumonia in PBS inside a Y-channel PDMS microfluidic device via optical fiber monitoring of latex immunoagglutination. The latex immunoagglutination assay was performed with serially diluted M. pneumonia solutions using highly carboxylated polystyrene particles of 390 and 500 nm diameter conjugated with monoclonal anti-M. pneumonia. Proximity optical fibers were located around the viewing cell of the device, which were used to measure the increase in 45 degrees forward light scattering of the aggregated particles. The detection limit are slightly less than 50 pg mL(-1) both for 390 and 500 nm microspheres and the detection time do not exceed 90 s.

Novel automated pulse immunoassay for human alpha-fetoprotein.

BACKGROUND: To improve current alpha-fetoprotein (AFP) assays, which are expensive and time-consuming, a specific AFP reagent has been developed for practical use in our newly developed high-speed, highly sensitive pulse immunoassay (PIA) system, in which a latex immunoagglutination reaction is carried out under a high-frequency pulse voltage, leading to an enhanced immunological reaction. METHODS: We evaluated the assay performance (reproducibility, sensitivity, dilution linearity, interference) of the newly developed automated AFP PIA compared with the current AFP assay. RESULTS: Using pooled serum samples, the within-run reproducibility resulted in a correlation variation of 3.6-4.7%. The AFP assay detection limit was determined to be 2.5 microg/L. Linear sequential dilution was found up to nearly 700 microg/L. Even up to an AFP concentration of 1.0 g/L, the prozone phenomenon was not observed. Free and conjugated bilirubin, haemolytic haemoglobin, chyle and rheumatoid factor did not show any test interference. Using AFP-positive serum samples from 114 patients, the correlation between our PIA and a chemiluminescence immunoassay resulted in an excellent correlation coefficient of 0.994. CONCLUSIONS: The performance of AFP reagents in the PIA device shows that the system has excellent speed and equal sensitivity and specificity compared with the most highly sensitive conventional method. Our PIA system thus appears ready for use in the clinical diagnosis setting.

[Analytical characteristics micromatrix semiquantitative method used in serial tests with video digital registration].

A multiplex analytical system has been developed to carry out microformat latex agglutination tests with video digital registration. The system makes it possible to apply less than 1 microl drops of latex and samples in the matrix format to a carrier and to make the test in 30 points at once, as well as to record and to interpret the results of the test, by keeping analytical information for each sample. Comparison of the results of determination of the serum levels of C-reactive protein, rheumatoid factor, and antistreptolysine in the system developed by the authors with the results obtained in the macroagglutination test by the immunoturbidimetric technique leads to the conclusion that the methods are comparable. The proposed type of a latex agglutination test based on a matrix approach and miniaturization assures efficient production and the quality of laboratory studies.

The enhanced diffusional mixing for latex immunoagglutination assay in a microfluidic device.

Latex immunoagglutination assay in a microfluidic device is expected to be even easier than its large-sized, commercialized counterpart. However, such demonstration has had a limited success due to the difficulties in mixing in a microfluidic device, especially for the microparticles used in latex immunoagglutination assay. The primary goal of this work is to improve diffusional mixing towards the successful latex immunoagglutination in a microfluidic devices without any non-specific binding. To this end, SDS (sodium dodecyl sulfate, an ionic surfactant) or Tween 80 (polyethylene sorbitol ester, a non-ionic surfactant) was added to the antibody-conjugated polystyrene (PS) microparticle suspension. These surfactant-added particle suspensions were mixed with the target antigen solution at the Y-junction of a microfluidic device. The immunoagglutination and the diffusion behavior were visually identified with an inverted light microscope. Both surfactants showed some problems such as non-specific binding (with SDS) or very poor diffusion (with Tween 80). As an alternative approach, therefore, highly carboxylated PS microparticles, where the surface is saturated with carboxyl-terminated side chains, were evaluated without using any surfactants. These particles showed very low non-specific binding comparable to that with Tween 80 and good diffusional mixing equivalent to that with SDS.

External quality assessment of HbA(1c) for point of care testing.

OBJECTIVES: To evaluate the long term total imprecision of HbA(1c) testing within the county of Uppsala in relation to the Swedish analytical goal of coefficient of variation (CV) < 3% for HbA(1c) and to study the cost of an external quality assurance program for point-of-care HbA(1c) The county uses Bayer DCA 2000 for point-of-care HbA(1c) testing currently having 23 of these instruments. METHODS: Method imprecision was assessed by analysis of patient samples performed as split samples during a 3 year period (2002-2004) as part of the quality assurance program for point-of-care HbA(1c) testing. The samples were first analysed on a Bayer DCA 2000 and the samples were then sent to the centralised laboratory for reanalysis with an HPLC system (Variant II, Biorad). The testing was performed approximately 8 times per year with each instrument. RESULTS: The median CV between the HPLC method and the point-of-care instruments for each unit was slightly higher than 3%. CONCLUSION: The DCA 2000 systems have an acceptable imprecision and agreement with the central laboratory. The test results show acceptable agreements within the county regardless where the patient is tested. The cost of the external quality assurance program is calculated to be approximately SEK 1340 (Euro 150) per instrument.

[A scanner-based system for documentation, objective assessment, and recording of the results of latex-agglutination tests].

The present paper describes the use of the scanner "Expert-Lab Agglutination" system to record the results of latex-agglutination tests in making a reaction on the lids of 96-well immunoassay plates. The described system provides a possibility of having a primary image of all tests carried out on a carrier. Software offers the prospect of contrasting the images of individual and control samples, increasing the size of images, and comparing the latter. The use of special image treating techniques may also yield objective figures characterizing the rate of a reaction. The possibility of archiving primary information and retrospectively appraising the correctness of the result of an analysis at any required moment is of fundamental importance to the latex-agglutination test.

Bacterial antigen detection test in meningitis.

OBJECTIVE: To evaluate the role of bacterial antigen detection test in cerebrospinal fluid (CSF) for a rapid etiological diagnosis of bacterial meningitis. METHODS: The study included 36 cases of bacterial meningitis and 14 controls. Latex particle agglutination test (LPA test) for detection of bacterial antigen was done in the CSF using slidex meningitis kit (Biomeriux, France). RESULTS: Using LPA test, an etiological diagnosis could be made in 83% cases of bacterial meningitis. In contrast, CSF Gram stain and culture showed 36% and 6% positivity, respectively. The sensitivity and specificity of LPA test were 83% and 100%, respectively. The common etiological organisms were S. pneumoniae, H. influenzae type b and N. meningitidis A. S. pneumoniae was encountered in all age groups while H. influenzae type b was found only below one year of age. CONCLUSIONS: LPA test is a rapid and superior diagnostic tool as compared to CSF Gram stain and culture. The study recommends LPA test as an adjunct laboratory test for rapid etiological diagnosis of bacterial meningitis for prompt institution of proper antibiotics.

IgM quantification in the cerebrospinal fluid of sleeping sickness patients by a latex card agglutination test.

An increased IgM concentration in cerebrospinal fluid (CSF), occurring as a consequence of massive intrathecal IgM synthesis, is a marker of interest for diagnosis of the meningo-encephalitic stage in human African trypanosomiasis. However, in current practice, IgM in CSF is not determined because of the lack of a simple and robust test that is applicable in African rural regions where the disease prevails. We describe the development of a sensitive semiquantitative card agglutination test, LATEX/IgM, for IgM quantification in CSF. The test is simple and fast and the lyophilized reagent remains stable even at 45 degrees C. CSF end-titres obtained with LATEX/IgM parallel the IgM concentrations determined by nephelometry and enzyme-linked immunosorbent assay. Detection of intrathecal IgM synthesis is the most sensitive marker for CNS involvement in sleeping sickness. At a cut-off value of >or= 8, the sensitivity and specificity of LATEX/IgM for intrathecal IgM synthesis are 89.4 and 92.7%. As a consequence, patients with LATEX/IgM end-titres >or= 8 are likely to have intrathecal IgM synthesis, thus central nervous system involvement and therefore should be treated accordingly. Further studies should concentrate on the relationship between the LATEX/IgM end-titres, presence of intrathecal IgM synthesis and occurrence of treatment failures in patients treated with pentamidine.

Evaluation of an automated latex D-dimer immunoassay in the clinical assessment of suspected venous thromboembolism.

Because the reliability of clinical signs in venous thromboembolism (VTE) is poor, a highly sensitive, non-invasive test may improve the selection of patients requiring further investigation. We assessed the sensitivity and negative predictive value of an automated D-dimer latex immunoassay (IL-Test ) in 68 patients presenting with suspected VTE. The plasma D-dimer concentration was estimated and an appropriate diagnostic radiological investigation performed. Control values were obtained from healthy young and elderly volunteers. Using a cut-off value of 330 ng/ml, the assay had a sensitivity of 100% and negative predictive value of 100% for VTE. We conclude that the IL-Test. automated D-dimer assay has a suitably high sensitivity and adequate negative predictive value to be included in a pre-test clinical probability protocol for the evaluation of patients with suspected VTE.

New compact-type latex photometric immunoassay system for hemoglobin and three acute inflammation markers: neutrophil count, C-reactive protein, and anti-streptolysin O.

A new compact-type latex photometric immunoassay system, SPOTCHEM IM SI-3510 (ARKRAY, Inc., Kyoto, Japan), which assays three kinds of inflammatory markers-neutrophil count (NPC), C-reactive protein (CRP), and anti-streptolysin O (ASO)-was evaluated. Hemoglobin (Hb), which is a good marker for anemia, can also be measured with it. NPC and CRP are measured using antibodies against neutrophilic elastase and CRP, purified streptolysin O was used for ASO determination, and Hb was measured by an azide-methemoglobin method. Whole blood, serum, and plasma specimens can be used as samples with this system. In this study, whole blood treated with dipotassium ethylenediamine tetraacetic acid was used for evaluation. Linearity and reproducibility were good for all of the items studied. Good correlations were observed between the results obtained by this system and those obtained by routine methods. Since NPC exhibited a high correlation with the routine white blood cell (WBC) counts, it was judged to be useful as a substitute for WBC counting. Since this system is small and easy to operate, and evaluation revealed reliable results, it was judged to be practical for small laboratories, and satellite testing in hospitals and physicians' office laboratories for patients suspected to have acute inflammation.

Performances of a new, automated latex assay for the exclusion of venous thromboembolism.

The performance of a new latex-enhanced turbidimetric assay, D-Dimer PLUS, has been evaluated with two analyzers performing various coagulation assays: the BCS Analyzer and the BCT Analyzer. A precision study showed total coefficients of variation ranging from 2.7 to 11.1% with the BCS Analyzer and from 2.5 to 6.6% with the BCT Analyzer. We investigated the ability of D-Dimer PLUS to exclude venous thromboembolism in 312 outpatients suspected of either pulmonary embolism or deep venous thrombosis. Three months follow-up was available for all patients. With the BCS Analyzer, we determined a cut-off value of 190 ng/ml, which gave a sensitivity of 97.9% [95% confidence interval (CI), 92.6-99.7%], a specificity of 37.9% (95% CI, 30.9-43.8%) and a negative predictive value of 97.6% (95% CI, 91.7-99.7%). With the BCT Analyzer, at a cut-off value of 130 ng/ml, sensitivity was 96.8% (95% CI, 91.0-99.3%), specificity was 45.2% (95% CI, 38.5-51.2%) and the negative predictive value was 97% (95% CI, 91.6-99.4). This new assay is fast and fully automated, and its performance is suitable to exclude venous thromboembolism. Management studies should be performed to assess its utility.

Evaluación del analizador automático Inmuno 1 de Technicon por la determinación de hormonas / The evaluation of the technicom Inmuno 1 automatic analyzer for hormonal component

El analizador automático Inmuno 1 de Bayer realiza procedimientos de medida competitivos y tipo sandwich con separación mediante partículas magnéticas e inmunoanálisis de aglutinación con látex. El objetivo del presente trabajo es la evaluación de los procedimientos de medida de los constituyentes hormonales: tirotropina, tiroxina, triyodotironina, prolactina, folitropina, lutropina y cortisol en el analizador autonático Unmuno 1. Se evaluaron las siguientes características analíticas: repetibilidad intra e interdiaria, la linealidad de la curva de dilución y sensibilidad funcional para la concentración sérica de tirotropina, especificidad analítica de los anticuerpos contra la tirotropina, folitropina y lutropina frente a la Beta coriogonadotropina humana e intercambiabilidad entre los procedimientos de medida utilizados en nuestro laboratorio y los realizados por el analizador Inmuno 1 (AU)

Evaluation of kits for the detection of fibrin(ogen) degradation products in dogs.

The sensitivities and specificities of 3 commercial serum fibrin(ogen) degradation product (FDP) kits and 1 plasma FDP kit for the detection of FDPs in dogs were determined. Blood was collected for measurement of serum and plasma FDP concentrations from 30 healthy dogs and from 20 dogs that fulfilled clinical and laboratory criteria for disseminated intravascular coagulation. To determine the effect of hemolysis on FDP results, blood was collected simultaneously into Bothrops atrox venom-based and thrombin-based serum collection tubes for measurement of FDPs using a single serum FDP kit. The sensitivity (80-95%) and specificity (90-100%) for a positive or negative FDP result, regardless of concentration, was similar for all kits. Kits yielded discordant results in individual dogs and FDP concentrations obtained from 1 serum FDP kit were consistently higher than those from the other kits. Serum prepared from venom-based collection tubes was significantly more hemolyzed than serum prepared from thrombin-based collection tubes or citrated plasma. Hemolysis did not affect the FDP results. On the basis of these results, we conclude that commercial latex agglutination kits for detection of FDPs in serum and plasma samples from human patients are valid for use in dogs. The plasma FDP assay is a viable alternative to currently used serum FDP assays and has the advantage of using a single (citrated plasma) sample for measuring coagulation parameters and FDP concentration.

Development of a latex immunoturbidimetric assay for the automated measurement of alpha2 plasmin inhibitor-plasmin complex in human plasma.

We have developed a rapid, sensitive, and quantitative latex immunoturbidimetric assay for the measurement of alpha2 plasmin inhibitor-plasmin inhibitor complex (PIC) in human plasma. The method is based on the latex agglutination reaction using a suitable pair of monoclonal antibodies. One reacts with plasmin and the other reacts with alpha2 plasmin inhibitor. In this assay, we added 6-aminohexanoic acid to the reaction buffer in order to avoid the nonspecific latex agglutination caused by precursor proteins such as plasminogen. We used this method with an automatic analyzer HITACHI 7150 (Hitachi Ltd., Hitachi, Japan) and measured PIC within the range of 0.2 to about 50 mg/mL in only 15 minutes. The precision indices (CV%) of intra-assays and interassays were <4.4% and <3.4%, respectively. The influence of plasminogen or alpha2 plasmin inhibitor on plasma was negligible. Based on these results, it is considered that this method would be useful for evaluation of a broad spectrum of fibrinolytic disorders, particularly disseminated intravascular coagulation.

The development of a semi-automated latex agglutination test for the detection of antibodies to Anaplasma marginale using a cell culture-derived antigen.

Serologic diagnosis of anaplasmosis is currently done by the complement-fixation, ELISA, and card agglutination tests. These tests have utilized A. marginale harvested from bovine erythrocytes as antigen which is often contaminated with erythrocyte stroma. We are currently testing A. marginale propagated in a Ixodes scapularis cell line as antigen for serologic tests. In this study, we report the use of the cell culture-derived A. marginale as antigen for development of a rapid, semi-automated latex agglutination test. Diluted serum and latex (polystyrene microspheres), sensitized with cell culture-derived A. marginale proteins, were dispensed into 96-well microtiter plates. An initial reading of light transmission was recorded by a computer-interfaced scanning autoreader. After 30 minutes, the plates were mixed and read a second time, recording the delta % light transmittance. The sensitized latex microspheres (latex) agglutinated in the presence of A. marginale antibodies, thus producing an increase in light transmittance. In preliminary tests, 724/977 of the sera were positive for A. marginale antibodies with an apparent agreement of 83.3% when compared with the complement-fixation test. Sensitization and sera dilution buffers were shown to have a marked effect on the sensitivity and specificity of this assay. Results will be presented on the optimization of buffers and the testing of sera from experimentally and field-infected cattle.