Validation of a novel and accurate ApoE4 assay for automated chemistry analyzers.

The allele ε4 of the apolipoprotein E gene (APOE ε4) is the major genetic risk factor for non-dominantly inherited Alzheimer's Disease (AD). Current techniques for APOE ε4 carriers identification show good accuracy but have several disadvantages that limit its implementation in a clinical laboratory. These include the need for sample preprocessing, poor automation, low throughput, requirement of additional equipment, and high cost. We followed ISO 13485 guidelines to validate the e4Risk test, a new latex-enhanced immunoturbidimetric blood assay for apolipoprotein E4 (ApoE4) determination in human plasma samples. The test showed high performance in terms of lot to lot variability, precision, interferences, reagents stability, prozone, and detectability. Furthermore, diagnostic accuracy is almost equal (99%) to the gold standard, APOE ε4 genotyping by polymerase chain reaction (PCR). Furthermore, we demonstrated that the e4Risk test can be adapted to any clinical chemistry analyzer, including the high throughput analyzers present in most hospitals and clinical laboratories. The e4Risk test versatility, low cost, and easiness provides an excellent solution for APOE ε4 carriers identification using the same blood sample drawn for biochemical diagnostic work-up of AD patients, which can have important advantages for patient stratification in clinical trials, preventative strategies for AD, and clinical assessment of risk for brain amyloidosis.

Comparison of Enterprise Point-of-Care and Nova Biomedical Critical Care Xpress analyzers for determination of arterial pH, blood gas, and electrolyte values in canine and equine blood.

BACKGROUND: Point-of-care analyzers can provide a rapid turnaround time for critical blood test results. Agreement between the Enterprise Point-of-Care (EPOC) and bench-top laboratory analyzers is important to determine the clinical reliability of the EPOC. OBJECTIVES: The aim of the study was (1) to evaluate the precision (repeatability) of blood gas values measured by the EPOC and (2) to determine the level of agreement between the EPOC and Nova Critical Care Express (Nova CCX) for the assessment of arterial pH, blood gases, and electrolyte variables in canine and equine blood. METHODS: Arterial blood samples from dogs were analyzed on the EPOC and Nova CCX analyzers to determine precision and agreement of pH, PaCO2 , PaO2 , and HCT. The same analytes plus Na+ , K- , and Cl- were analyzed for agreement using equine blood. Statistical analyses included assessment of precision using the coefficient of variation (CV%), and agreement using the Deming regression, Pearson correlation, and Bland-Altman plots. RESULTS: Both analyzers provided precise results of pH, PaCO2 , PaO2, and HCT, meeting CV% quality requirement values. In both species, Deming regression results were acceptable and correlation values were above 0.93 for arterial pH and blood gases, but lower for sodium and chloride. Bland-Altman plots demonstrated varying degrees of bias, but good agreement between the 2 analyzers was seen when arterial blood gases and electrolytes were measured, except for PaCO2 and Cl-. CONCLUSION: The EPOC analyzer provides consistent, reliable results for canine arterial blood gas values and for equine arterial blood gas and electrolyte values. Cl- results could be acceptable with the application of a correction factor, but the PaCO2 results were more variable.

Clinical application evaluation of two fourth-generation human immunodeficiency virus (HIV) screening assays in West China Hospital.

BACKGROUND: Fourth-generation human immunodeficiency virus (HIV) screening assays have been used in many laboratories. The Elecsys® HIV combi PT assay is a new kind of fourth-generation HIV screening assay developed to allow earlier detection of seroconversion. METHODS: A total of 271,845 routine specimens were detected using the Elecsys® HIV combi assay and Elecsys® HIV combi PT assay from September 2010 to December 2012 in a large university hospital. Repeatedly, reactive screening samples were confirmed according to recommended confirmatory algorithms. RESULTS: The false-positive rate and positive predictive value (PPV) of two assays are 0.08 and 78.35%, respectively, for the Elecsys® HIV combi assay and 0.07 and 82.21% for the Elecsys® HIV combi PT assay. Ninety-four percent cases with cutoff index ratio <15.0 were false-positive. When we set the specificity as 95.0 and 99.0%, PPV could increase to 98.7, 99.6, 98.8, and 99.7%, and sensitivity reduced to 99.2, 98.4, 98.5, and 96.8% for the Elecsys® HIV combi assay and the Elecsys® HIV combi PT assay, respectively. CONCLUSIONS: The Elecsys® HIV combi PT assay shows a better performance in specificity than the Elecsys® HIV combi assay. Most weakly reactive results were false-positive, this means it still need to be improved and it will need laboratory personnel to communicate with the clinical doctor and patients more properly about the result of the assay.

[Assessment of the capability of the fluid model of automatic blood cell analyzer in white blood cell count and classification].

OBJECTIVE: To evaluate the application of XT-4000i automatic blood cell analyzer in white cell count and classification of cerebrospinal fluid (CSF). METHODS: The fluid model of XT-4000i automatic blood cell analyzer was directly used to detect the white cell count and classification in 200 samples of CSF, and compared with the results obtained by manually microscopic counting method. White blood cell classification was performed by manual method under microscope with Wright's staining,and instrumental method with fluorescence staining and flow cytometry. RESULTS: There is no statistical difference between instrumental and manual method in detecting the absolute counting of WBC, RBC, mononuclear cell and multinucleate cells (P>0.05), and there is a good correlation between the two methods (r=0.987, 0.999, 0.981 and 0.983 for WBC, RBC, mononuclear cell and multinucleate cell counts). Tumor cells in the samples with high fluorescent staining by instrumental method can be identified with Wright's staining by microscope method, which were consistent with the clinical diagnosis. CONCLUSION: The fluid model of XT-4000i automatic blood cell analyzer was rapid and accurate in CSF white cell count and classification,and it also can provide preliminary information for tumor cells screening. The fluid model of XT-4000i automatic blood cell analyzer in white cell count and classification of CSF has the value of popularization in clinical application.

Long-term automated sampling of PCDD/PCDF flue gas: current status and critical issues.

After entry into force of the Stockholm Convention and Aarhus Protocol and in order to implement the upcoming European legal background, the European countries are asked to apply control measures to reduce the release of persistent organic pollutants (POPs) such as dioxins and furans (PCDD/PCDF) and polychlorinated biphenyls as well as to establish POPs release inventories. In this perspective, development of measuring techniques of emissions is a focal issue in acquiring useful information. In this paper, results of various measurement campaigns at different municipal waste incineration (MWI) plants using long-term automated sampling of PCDD/PCDF are presented. The samples collected from both manual and automated campaigns were analyzed following the European Standard EN-1948:2006 by high-resolution gas chromatograph/high-resolution mass spectrometer. Performances of two different commercial systems have been investigated. Anomalous values occurred during one long-term campaign (22.16 pg I-toxic equivalent (TEQ)/Nm(3)), compared to average values (4-5 pg I-TEQ/Nm(3)) of the MWI. At this maximum value, a main occurrence of abnormal and instable operating conditions has been found. Sampling based on long-term basis was found to be more reliable to monitor PCDD/PCDF emissions than occasional short-term sampling. Nevertheless, the results of long-term campaigns demonstrate that emission levels detected in 15-30 days campaigns, when unsteady operating conditions can occur, as start-up and shut down, are not immediately comparable to the typical levels in a 6-8 h, when operating conditions are generally stable. Moreover, there are often differences observed in the congener profiles between short- and long-term campaigns.

New generation IQ-200 automated urine microscopy analyzer compared with KOVA cell chamber.

OBJECTIVE: The examination of the urine remains to be one of the most commonly performed tests in laboratory practice. Currently, laboratories also need to accredit their urine diagnostics by comparing their measurement methods to acceptable references. In this study we compared particle counts obtained by new generation automated technique, image capture analysis (IQ-200) with those of a standardized chamber counts. DESIGN AND METHODS: The same 258 urine samples from different departments of a hospital assayed by IQ-200 were analyzed in parallel with the KOVA cell chamber system. Clinically significant discrepancy results (positive vs. negative) for red blood cell (RBC) and white blood cell (WBC) were also compared with those obtained by dipstick testing. RESULTS: There was a good agreement between the automated system and sediment microscopy for RBCs, WBCs, and squamous epithelial cells (SCs) (r=0.90; r=0.80; r=0.72, respectively: P<0.001). The IQ-200 was more sensitive for determining RBCs, WBCs, and SCs than other formed elements. CONCLUSIONS: IQ-200 can perform accurate quantification of microscopic element in urine. However, automated techniques are not completely free of error. Therefore, by adopting an appropriate algorithm and combining the results with stript analysis and other laboratory tests allows further reduction of clinically important errors.

Benefits of the iQ200 automated urine microscopy analyser in routine urinalysis.

BACKGROUND: Urine microscopic analysis is hampered by its lack in standardisation and semi-quantitative reports, resulting in limited reliability. Automation of urinalysis could overcome these problems. METHODS: We compared the performance of the iQ200 with traditional microscopy and strip analysis in routine urinalysis. A total of 1482 routine samples, positive in dipstick testing, were evaluated for erythrocytes, leukocytes, casts, dysmorphic erythrocytes and bacteria using the iQ200 and traditional microscopy. The results of 320 of these samples were linked to underlying urological pathology as well as results from bacterial culturing. RESULTS: Analytically, the iQ200 surpasses traditional microscopy. The identification of casts and dysmorphic erythrocytes in routine samples improves when using the iQ200, although the sub-classification of casts required well-trained technicians. The auto-classification of particles was least reliable for yeast and bacterial cocci. The quantitative reports, and therefore the use of precise cut-off points allowed earlier and improved detection of urinary tract pathology. CONCLUSIONS: The performance of the iQ200 is equal to traditional microscopy, but it strongly improves the reliability of urinalysis by standardisation, quantitative reports and improved workflow. From a clinical point of view, renewed attention and improvement of routine urinalysis aids in the efficient detection of renal and urinary tract pathology.

Intermethod differences in results for total PSA, free PSA, and percentage of free PSA.

Serum prostate-specific antigen (PSA) assays differ in calibration and response to different PSA forms. We examined intermethod differences in total PSA (tPSA) and free PSA (fPSA) measurements. We tested 157 samples with tPSA concentrations of 2 to 10 ng/mL (2-10 microg/L) using 6 PSA/fPSA method pairs and 1 tPSA method: ADVIA Centaur (complexed and total; Siemens Diagnostics, Tarrytown, NY), ARCHITECT i 2000(SR) (Abbott Diagnostics, Abbott Park, IL), AxSYM (Abbott Diagnostics), IMMULITE 2000 (Siemens Diagnostics), Modular E170 (Roche Diagnostics, Indianapolis, IN), UniCel DxI 800 (Beckman Coulter, Brea, CA), and VITROS ECi (tPSA only; Ortho-Clinical Diagnostics, Raritan, NJ). Regression analysis was performed for PSA, fPSA, and percentage of fPSA with the ARCHITECT i 2000(SR) comparison method. Differences between test and comparison methods were estimated at 2.5, 4.0, and 10.0 ng/mL (2.5, 4.0, and 10.0 microg/L) for tPSA and 15%, 20%, and 25% for percentage of fPSA. Relative differences were more than 10% at 4.0 ng/mL (4.0 microg/L) tPSA for the Centaur, IMMULITE, ECi, and DxI methods. At 20% fPSA, the relative difference was more than 10% for all methods except the AxSYM. Additional harmonization is needed for tPSA and fPSA methods.

On-line monitoring of gas-phase bioreactors for biogas treatment: hydrogen sulfide and sulfide analysis by automated flow systems.

Biogas is produced by biological processes under anaerobic conditions and may contain up to 20,000 ppm(v) hydrogen sulfide (H(2)S), a corrosive substance that attacks power engines and can affect the health of the industrial staff. H(2)S must be removed from the biogas, especially in co-generation facilities where the biogas is burnt for energy production. Nowadays, biofiltration is being studied and considered as an interesting alternative for removing H(2)S from the biogas besides classical chemical processes. The novelty of this work is the design and construction of an automated H(2)S on-line analyser to assess the composition of the liquid and gas phases of gas-phase bioreactors. The analyser is made of two parallel flow configurations which share the same detection device. The first configuration is a single-channel flow injection analyser (FIA) to detect S(2-) in the liquid phase. The second configuration is a continuous flow analyser (CFA) with a gaseous diffusion step (GD-CFA) for detecting H(2)S in the gas phase. The diffusion step enables separation of the H(2)S((g)) from the sample and its conversion into a detectable chemical species (S(2-)). S(2-) detection was performed with an Ag(2)S ion-selective electrode (ISE) selective to S(2-)(aq). The main response parameters of the FIA system are a linear range between 3 x 10(-5) and 1 x 10(-1) mol L(-1) S(2-) (0.61-3,200 mg L(-1)), with a sensitivity of 27.9 mV decade(-1) and a detection limit of 1.93 x 10(-5) mol L(-1) S(2-). The GD-CFA configuration presents a linear range between 400 and 10,000 ppm(v) H(2)S((g)) with a sensitivity of 26.1 mV decade(-1) and a detection limit of 245 ppm(v) H(2)S. The proposed analyser was used by analysing real gas and liquid samples with optimal results at a full-scale biotrickling filter for biogas treatment at a municipal wastewater treatment plant.

Rheumatoid factor interference in the determination of carbohydrate antigen 19-9 (CA 19-9).

BACKGROUND: Investigation of a 61-year-old Caucasian male suffering from fatigue and weight loss led to the finding of a carbohydrate antigen 19-9 (CA 19-9) concentration of 80 kU/L using an ADVIA Centaur analyser. Determination of CA 19-9 on Vidas, AxSYM and Architect i2000 systems gave normal results. His rheumatoid factor concentration was very high (900 kIU/L) and assay interference was suspected. METHODS: Besides using several laboratory procedures to show the cause of the interference, we tried to estimate the frequency of the suspected interference. Therefore, two studies were performed. The first was carried out in a multicentre setting using four different CA 19-9 methods on 51 randomly selected samples with high rheumatoid factor concentrations and ten samples containing no or very low rheumatoid factor. In the second study we used heterophilic blocking tubes for 68 routinely analysed samples with CA 19-9 concentrations ranging between 37 and 250 kU/L using an ADVIA Centaur analyser. RESULTS: In the multicentre study we found eight discrepant CA 19-9 results, but only one was clearly due to interference. We showed that the interference detected, just as in the index case, was caused by rheumatoid factor. The other discrepancies could not be explained, but are probably related to method-dependent differences. In the 68 routinely analysed samples, no interference could be shown using the heterophilic blocking tubes. CONCLUSIONS: Although interferences in the CA 19-9 assay are not frequent, the ADVIA Centaur system appears to be more sensitive to rheumatoid factor interference. The lack of standardisation remains an important issue for this assay. The determination of CA 19-9 during the follow-up of patients should be performed using a single method. If, however, there is any clinical doubt about a result, CA 19-9 should be determined using another method to exclude possible interferences.

Sequence determination of lychnin, a type 1 ribosome-inactivating protein from Lychnis chalcedonica seeds.

The complete amino acid sequence of lychnin, a type 1 ribosome-inactivating protein (RIP) isolated from Lychnis chalcedonica seeds, has been determined by automated Edman degradation and ESI-QTOF mass spectrometry. Lychnin consists of 234 amino acid residues with a molecular mass of 26 131.14 Da. All amino acid residues involved in the formation of the RIP active site (Tyr69, Tyr119, Glu170, Arg173 and Trp203) are fully conserved. Furthermore, a fast MALDI-TOF experiment showed that two out of three cysteinyl residues (Cys32 and Cys115) form a disulfide bridge, while Cys214 is in the thiol form, which makes it suitable for linking carrier molecules to generate immunotoxins and other conjugates.

Performance characteristics of seven automated CA 125 assays.

Cancer antigen 125 (CA 125) is a high-molecular-mass glycoprotein that is used as a tumor marker to monitor disease progression and response to therapy and in early detection of recurrence after treatment for ovarian cancer. The Access 2 (Beckman Coulter, Brea, CA), ADVIA Centaur (Bayer Diagnostics, Tarrytown, NY), ARCHITECT i2000 (Abbott Diagnostics, Abbott Park, IL), AxSYM (Abbott Diagnostics), Elecsys 2010 (Roche Diagnostics, Indianapolis, IN), IMMULITE 2000 (Diagnostic Products, Los Angeles, CA), and VITROS ECi (Ortho Clinical Diagnostics, Raritan, NJ) assays for CA 125 were evaluated for detection limit, dilution linearity, imprecision, correlation, and reference intervals. The maximum average deviation from target recoveries for dilution linearity studies ranged from 3.7% for the ADVIA Centaur to 18.2% for the IMMULITE 2000. Imprecision studies yielded total coefficients of variation of 2.0% to 8.3% at CA 125 concentrations of 35 and 114 U/mL (35 and 114 kU/L). Method comparison studies revealed good agreement with the VITROS ECi comparison method, with slopes ranging between 0.88 to 1.19 and correlation coefficients of more than 0.95. All methods show acceptable performance characteristics and generally compare well. However, for some samples, substantial differences exist between methods, necessitating parallel testing when introducing a new method.

Cholesterol crystals causing falsely elevated automated cell count.

This is a report of 3 cases of body fluid containing numerous cholesterol crystals that caused falsely elevated cell counts on an automated cell counter. Two of the cases were pleural effusion fluid from patients with long-standing rheumatoid arthritis. Fluid in the third case was from upper extremity cystic lesions of a patient with squamous cell carcinoma of the head and neck. Microscopic examination revealed abundant cholesterol crystals in all fluid samples. In all 3 cases, initially, the automated cell counter reported very elevated WBC and RBC counts that were much higher than those from the manual count. This interference by cholesterol in the automated cell counter is discussed. In addition, possible pathophysiology of cholesterol formation in the body fluid is discussed and chylous and pseudochylous (chyliform) effusions are reviewed. Finally, the use of automated instruments in the evaluation of body fluid is reviewed.

Semiautomated determination of pesticides in water using solid phase extraction disks and gas chromatography-mass spectrometry.

A method based on semiautomated solid phase extraction using octadecyl-bonded silica disks and gas chromatography-mass spectrometry, operated in selected ion monitoring mode, allows detection and quantification of approximately 100 pesticides and transformation products in drinking water. Samples (500 mL) were passed through the disk, and the retained pesticides were eluted with acetone and ethyl acetate. Typical recoveries for pesticides at 0.1 microg L(-1) in water were in the range of 72-120% with relative standard deviations less than 20%. Calibration curves were linear over the range of 0.025-0.5 microg mL(-1) (equivalent to a concentration range in drinking water of 0.05-1.0 microg L(-1)).

[Adaptation of Biomerieux enzymatic UV ammonia on Konelab analysers]. / Adaptation de l'ammonium enzymatique UV de BioMérieux sur automates Konelab.

INTRODUCTION: Circulating ammonia in normal patients is relatively low, despite the fact that ammonia is continually produced from endogenous amino acid metabolism. The physiopathological interest of plasmatic ammonia determination lies primarily in its relationships to hepatic insufficiency (cirrhotic or neoplasic), or the diagnosis and the forecast of the Reye's syndrome. OBJECTS: This study describes an evaluation of plasmatic ammonia determination by the UV end point enzymatic method using GLDH on KONELAB(TM) analyzers. METHODS: The glutamate dehydrogenase (GLDH : EC.1.4.1.3) catalyses the reducing amination of alpha-cetoglutarate in the presence of NH(4)(+) and of NADPH, H(+) to form glutamate and NADP(+). The reduction of NADPH,H(+)'s concentration, directly proportional to ammonia rates, is evaluated at 340 nm. All the conditions were met to optimize the method, while covering a satisfying field of measurement. RESULTS AND COMMENTS: The evaluation of the modified method showed a good precision (repeatability: CV < 4 %; interserial reproducibility: CV from 2.01 to 2.93 %; Intraserial reproducibility: CV equal to 0.67%) and a very good accuracy. The field of measurement extends from 27 to 250 micromol/L, with a limit of detection (L(D)) lowered to 0.325 micromol/L. CONCLUSION: The adapted technique is simple, fast, inexpensive and especially automatizable. It is in addition reliable and chiefly more sensitive, adapting particularly to the determination of plasmatic ammonia in urgency as in routine within our laboratory.

Rapid, fully automated flow injection antioxidant capacity assay.

A flow injection method for antioxidant capacity assessment based on a low-cost laboratory-made analyzer is reported. A sample of 30 microL is injected in acetate buffer stream, pH 4.6, that converges with ABTS*(+) reagent stream. Detection is achieved by monitoring absorbance at 414 nm. The proposed method achieves a sample throughput of up to 120 samples h(-1), the detection limit being 1.3 microM trolox. Precision was better than 5% relative standard deviation (n = 4) and the linear range was 4-100 microM, expanded to 250 microM trolox utilizing concentration gradients formed along the injected sample bolus. Information on reaction kinetics is obtained through a single injection. The method was applied to pure compounds and wine and honey samples. Good correlation was found between antioxidant capacity assessed through the proposed method and phenolic content: r = 0.94 for red wines, r = 0.96 for white and rose wines, and r = 0.89 for honeys.

Evaluation of an automated instrument for viability and concentration measurements of cryopreserved hematopoietic cells.

Two important parameters for determination of deleterious effects of cellular processing on hematopoietic progenitor cells are cell viability and concentration. The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Hospital evaluated the Beckman Coulter Vi-Cell automated instrument for the measurement of these two parameters. Using 33 thawed hematopoietic progenitor cell samples, automated Vi-Cell viability results were compared to those obtained using the standard trypan blue manual method. In addition, cell concentrations from these samples were compared with results from the Model Z2 Coulter Counter. Chinese Hamster Ovary cells were used for the evaluation of Vi-Cell linearity at the Beckman Coulter Cellular Analysis Development Center. Significant correlation was obtained when the two methods were compared for both cell concentration and percentage viability (P < .0001). The results of the linearity study indicated that the Vi-Cell is linear from approximately 5 x 10(4) to greater than 1 x 10(7) cells/mL. The Vi-Cell uses sample volumes as low as 0.5 mL; cell diameters may be 2 to 70 microns. The Vi-Cell automated instrument offers many significant advantages for cell analyses in today's busy laboratory environment.

Direct analysis of pesticide residues in olive oil by on-line reversed phase liquid chromatography-gas chromatography using an automated through oven transfer adsorption desorption (TOTAD) interface.

A fully automated on-line reversed phase liquid chromatography-gas chromatography system is described. The system uses a prototype of the automated through oven transfer adsorption desorption interface. The system is demonstrated by presenting a new rapid method for the determination of pesticide residue in olive oil, which is injected directly with no sample pretreatment step other than filtration. Methanol:water is used as the eluent in the LC preseparation step, while the LC fraction containing the pesticide is automatically transferred to the gas chromatograph. Detection limits of pesticides varied from 0.18 to 0.44 mg/L when a flame ionization detector was used. As an example, relative standard deviation and linear calibration are presented for terbutryne.

A microcapillary trap cartridge-microcapillary high-performance liquid chromatography electrospray ionization emitter device capable of peptide tandem mass spectrometry at the attomole level on an ion trap mass spectrometer with automated routine operation.

Reversed-phase microcapillary chromatography (RP-microLC) combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) is one of two prevailing techniques in proteomic analysis, the other being matrix-assisted laser desorption/ionization (MALDI). Despite the arguably better dynamic range obtainable with ESI, MALDI is increasingly popular due to ease of use, ruggedness and the ability to decouple separation from ionization. By contrast, in order to take advantage of the sensitivity and dynamic range afforded by the concentration-dependent nature of ESI, it is directly coupled to separations that take place in small i.d. RP-microLC columns. This gain in sensitivity often comes at a loss of ruggedness due to clogging of the small i.d. RP-microLC columns, one result of which is limited sample throughput. Here we describe a combined micropre-column-microLC-ESI device that is sensitive, rugged and modular in design allowing facile construction and troubleshooting. Due to low signal-to-noise as little as 1 attomole of a peptide can be selected by data-dependent methods for collision-induced dissociation. Importantly, the resulting tandem mass spectrum is of high enough quality to identify the peptide sequence by a database search against a complex database using SEQUEST. Finally, the device is demonstrated to be rugged as judged by >60 consecutive reversed-phase microLC separations on complex peptide mixtures before chromatographic resolution is degraded.