Comparative Analysis of the Clarus Aspergillus Galactomannan Enzyme Immunoassay Prototype for the Diagnosis of Invasive Pulmonary Aspergillosis in Bronchoalveolar Lavage Fluid.

BACKGROUND: Galactomannan (GM) testing using Platelia Aspergillus enzyme immunoassay (Platelia AGM) from bronchoalveolar lavage fluid (BALF) aids in early diagnosis of invasive pulmonary aspergillosis (IPA). Globally, only a minority of laboratories have the capability to perform on-site GM testing, necessitating accessible and affordable alternatives. Hence, we conducted a comparative evaluation of the new clarus Aspergillus GM enzyme immunoassay prototype (clarus AGM prototype) with Platelia AGM using BALF samples. METHODS: This is a single-center, prospective, cross-sectional study, where Platelia AGM testing was routinely performed followed by clarus AGM prototype testing in those with true positive or true negative AGM test results according to the 2020 EORTC/MSG and the 2024 FUNDICU consensus definitions. Descriptive statistics, ROC curve analysis, and Spearman's correlation analysis were used to evaluate analytical performance of the clarus AGM prototype assay. RESULTS: This study enrolled 259 adult patients, of which 53 (20%) were classified as probable IPA, while 206 did not fulfill IPA-criteria. Spearman's correlation analysis revealed a strong correlation between the two assays (rho = 0.727, p < 0.001). The clarus AGM prototype had a sensitivity of 96% (51/53) and a specificity of 74% (153/206) for differentiating probable versus no IPA when using the manufacturer recommended cut-off. ROC curve analysis showed an AUC of 0.936 (95% CI 0.901-0.971) for the clarus AGM prototype, while the Platelia AGM yielded an AUC of 0.918 (95% CI 0.876-0.959). CONCLUSIONS: Clarus AGM prototype demonstrated a strong correlation and promising test performance, comparable to Platelia AGM, rendering it a viable alternative in patients at risk of IPA.

Performance of the clarus Aspergillus galactomannan enzyme immunoassay prototype for the diagnosis of invasive pulmonary aspergillosis in serum.

BACKGROUND: Serum galactomannan (GM) testing is essential for diagnosing invasive aspergillosis (IA), particularly in immunocompromised individuals. The global lack of on-site GM testing capacities necessitates cost-effective alternatives, such as .the clarus Aspergillus GM enzyme immunoassay prototype (clarus AGM prototype). METHODS: This single-centre, cross-sectional study compared the diagnostic performance of the clarus AGM prototype (IMMY, Norman, Oklahoma) with the serological gold standard (=Platelia AGM assay; Bio-Rad, Marnes-la-Cocquette, France). IA was classified according to modified 2020 EORTC/MSG consensus and 2024 FUNDICU criteria. In total, 300 prospectively (May-Dec 2023) and retrospectively (2012-2015) collected samples were included. RESULTS: Among 300 samples from 232 patients, 49 (16%) were classified as proven (n = 1) or probable IA (n = 48). In non-IA cases (n = 250), one patient was classified as possible IA. With the manufacturer recommended cut-off of ≥0.2, sensitivity and specificity of the clarus AGM prototype were 27% (13/49; 95% confidence interval [CI]: 15%-41%) and 99% (248/250; 95% CI: 97%-100%), respectively, while sensitivity and specificity were 78% and 79% when using the optimised Youden's cut-off of 0.0045 ODI. ROC curve analysis demonstrated an area under the curve (AUC) of 0.829 (95% CI: 0.760-0.898) for the clarus AGM prototype in distinguishing between proven/probable IA and non-IA. The AUC for the Platelia AGM was 0.951 (95% CI: 0.909-994). Spearman's correlation analysis showed a weak correlation between the two assays (0.382; p < .001). CONCLUSIONS: The weak correlation between the clarus AGM prototype and Platelia AGM highlights the need for further investigation into the clinical performance of the clarus AGM prototype, giving the different antigen epitopes addressed.

Diagnosis of Coccidioidomycosis with the Second-Generation Miravista IgG and IgM Enzyme Immunoassay and the Role of Adding Miravista Coccidioides Antigen Detection to Immunodiagnostic Assays.

In the present study, we validate and compare the second-generation Miravista Coccidioides IgG and IgM enzyme immunoassays (EIA) (MiraVista Diagnostics [MVD] Ab EIA) to Meridian Diagnostics Coccidioides IgG and IgM EIA (Meridian Ab EIA), immunodiffusion (ID) and complement fixation (CF). We also evaluated whether the addition of Coccidioides antigen testing to anti-Coccidioides antibody testing increased the sensitivity for the diagnosis of currently active coccidioidomycosis. We retrospectively studied 555 patients evaluated at Valleywise Health Medical Center between January 2013 and May 2017 for whom coccidioidomycosis was suspected and samples were submitted to MVD for testing. Specimens were tested for antigen in the MVD antigen enzyme immunoassay (MVD Ag EIA) and for IgG and IgM antibodies with MVD and Meridian Diagnostics EIAs. ID and CF were obtained from medical records. Sensitivity and specificity were 83.0% and 91.1% or MVD Ab EIA, 69.3% and 99.7% for Meridian Ab EIA, 85.4% and 100% for ID and 65.5% and 100% for CF. Combined MVD antigen and antibody detection by EIA and ID resulted in increased sensitivity in disseminated and pulmonary disease (MVD Ag/MVD Ab: 100%, 88.3%; MVD Ag/Meridian Ab: 98.2%, 78.6%; and MVD Ag/ID: 100%, 91.7%). The detection of antibodies by MVD EIA was more sensitive than Meridian EIA or CF but similar to ID. This study supports the use of antigen testing in immunocompromised patients and those with suspected disseminated disease. Furthermore, the addition of antigen detection by EIA to antibody detection resulted in higher sensitivity of all serological tests.

The most common methods for the diagnosis of moderate or severe coccidioidomycosis rely on the detection of antibodies or antigens. Here we present the validation of a new Miravista Coccidioides antibody detection test combined with antigen detection and compare it to other immunodiagnostics.

Plasmonic enzyme immunoassay via nanobody-driven controllable aggregation of gold nanoparticles for detection of ochratoxin A in pepper.

Ochratoxin A (OTA) in food poses a serious challenge to public health. Herein, using the nanobody-driven controllable aggregation of gold nanoparticles (AuNPs) in a glucose oxidase-tyramine-horseradish peroxidase (GOx-TYR-HRP) system, we propose a direct competitive plasmonic enzyme immunoassay (dc-PEIA) for OTA detection. The OTA-GOx conjugate catalyzes glucose to produce hydrogen peroxide (H2O2), and then HRP catalyzes H2O2 to generate hydroxyl radical which induces the crosslink of TYR. Crosslinked TYR leads to aggregation of AuNPs through strong electrostatic interactions, which is tunable based on the competition of OTA-GOx and free OTA for binding the immobilized nanobody. The optimized dc-PEIA achieves an instrumental limit of detection (LOD) of 0.275 ng/mL and a visual LOD of 1.56 ng/mL. It exhibits good selectivity for OTA and accuracy in the analysis of pepper samples, with the confirmation of high-performance liquid chromatography. Overall, the dc-PEIA is demonstrated as a useful tool for detecting OTA in food.

Protocol to detect senescence-associated ß-galactosidase and immunoperoxidase activity in fresh-frozen murine tissues.

Double labeling to identify different markers in the same tissue section represents a useful tool either for in situ diagnosis or characterization of molecular associations. Here, we present a protocol to detect senescence-associated ß-galactosidase (SA-ßGal) and immunoperoxidase (IPO) activity in fresh-frozen murine tissues. We describe steps for tissue collection, solution preparation, SA-ßGal staining, IPO staining, hematoxylin counterstaining, microscopic observation, and signal quantification. This protocol can be used to detect in situ proteins alongside SA-ßGal activity. For complete details on the use and execution of this protocol, please refer to Pacheco-Rivera et al.1.

Investigating the cut-off values of captopril challenge test for primary aldosteronism using the novel chemiluminescent enzyme immunoassay method: a retrospective cohort study.

The measurement evolution enabled more accurate evaluation of aldosterone production in hypertensive patients. However, the cut-off values for novel assays have been not sufficiently validated. The present study was undertaken to validate the novel chemiluminescent enzyme immunoassay for aldosterone in conjunction with other methods. Moreover, we also aimed to establish a new cut-off value for primary aldosteronism in the captopril challenge test using the novel assay. First, we collected 390 plasma samples, in which aldosterone levels measured using liquid chromatography-mass spectrometry ranged between 0.18 and 1346 ng/dL. The novel chemiluminescent enzyme immunoassay showed identical correlation of plasma aldosterone with liquid chromatography-mass spectrometry, in contrast to conventional radioimmunoassay. Further, we enrolled 299 and 39 patients with primary aldosteronism and essential hypertension, respectively. Plasma aldosterone concentrations measured using the novel assay were lower than those measured by radioimmunoassay, which resulted in decreased aldosterone-to-renin ratios. Subsequently, positive results of the captopril challenge test based on radioimmunoassay turned into "negative" based on the novel assay in 45% patients with primary aldosteronism, using the conventional cut-off value (aldosterone-to-renin activity ratio > 20 ng/dL per ng/mL/h). Receiver operating characteristic curve analysis demonstrated that aldosterone-to-renin activity ratios > 8.2 ng/dL per ng/mL/h in the novel assay was compatible with the conventional diagnosis (sensitivity, 0.874; specificity, 0.980). Our study indicates the great measurement accuracy of the novel chemiluminescent enzyme immunoassay for aldosterone, and the importance of measurement-adjusted cut-offs in the diagnosis of primary aldosteronism.

Adrenal venous sampling criteria for chemiluminescent enzyme immunoassay as a preferable alternative to radioimmunoassay in primary aldosteronism.

Plasma aldosterone concentration (PAC) was routinely measured using radioimmunoassay (RIA); however, the RIA kit was discontinued in March 2021 in Japan. This study examined PAC conversion in adrenal venous sampling (AVS) and AVS criteria when measured using chemiluminescent enzyme immunoassay (CLEIA). PAC of 415 adrenal venous blood samples from AVS (including segmental AVS) of 63 patients with primary aldosteronism was measured using RIA (Spac-S aldosterone kit; Fujirebio Inc.) and CLEIA (Lumipulse Presto Aldosterone; Fujirebio Inc.). PAC of 70 AVS samples was also measured using liquid chromatography-mass spectrometry (LC-MS/MS, ASKA Pharma Medical Co., Ltd.). PAC conversion formulas were determined for each AVS sample assay. PAC measured using CLEIA was significantly correlated with that measured using RIA (correlation coefficient = 0.971). The PAC conversion formula was PAC (CLEIA) = PAC (RIA) × 0.772 - 1,199 pg/mL. The PAC of 14,000 pg/mL in RIA was equivalent to 9,613 pg/mL in CLEIA. PAC measured using CLEIA was also correlated with that measured using LC-MS/MS, and the PAC conversion formula was PAC (CLEIA, pg/mL) = 0.97 × PAC (LC-MS/MS, pg/mL) + 211. The inter-assay coefficient of variability (CV) was 1.1-1.3% and intra-assay CV was 1.0-1.7%, measured using CLEIA. The PAC conversion formula for AVS samples was obtained using CLEIA and RIA, and the conversion formula was different from that for peripheral blood. PAC values measured by CLEIA showed preferable accuracy and high concordance with those measured by LC-MS/MS, even in AVS samples. The study outcomes are useful for interpreting AVS results using non-RIA measurement methods.

SOBA: Development and testing of a soluble oligomer binding assay for detection of amyloidogenic toxic oligomers.

The formation of toxic Amyloid ß-peptide (Aß) oligomers is one of the earliest events in the molecular pathology of Alzheimer's Disease (AD). These oligomers lead to a variety of downstream effects, including impaired neuronal signaling, neuroinflammation, tau phosphorylation, and neurodegeneration, and it is estimated that these events begin 10 to 20 y before the presentation of symptoms. Toxic Aß oligomers contain a nonstandard protein structure, termed α-sheet, and designed α-sheet peptides target this main-chain structure in toxic oligomers independent of sequence. Here we show that a designed α-sheet peptide inhibits the deleterious effects on neuronal signaling and also serves as a capture agent in our soluble oligomer binding assay (SOBA). Pre-incubated synthetic α-sheet-containing Aß oligomers produce strong SOBA signals, while monomeric and ß-sheet protofibrillar Aß do not. α-sheet containing oligomers were also present in cerebrospinal fluid (CSF) from an AD patient versus a noncognitively impaired control. For the detection of toxic oligomers in plasma, we developed a plate coating to increase the density of the capture peptide. The proof of concept was achieved by testing 379 banked human plasma samples. SOBA detected Aß oligomers in patients on the AD continuum, including controls who later progressed to mild cognitive impairment. In addition, SOBA discriminated AD from other forms of dementia, yielding sensitivity and specificity of 99% relative to clinical and neuropathological diagnoses. To explore the broader potential of SOBA, we adapted the assay for a-synuclein oligomers and confirmed their presence in CSF from patients with Parkinson's disease and Lewy body dementia.

The Detection of SARS-CoV2 Antigen in Wastewater Using an Automated Chemiluminescence Enzyme Immunoassay.

The SARS-CoV-2 virus, which is driving the current COVID-19 epidemic, has been detected in wastewater and is being utilized as a surveillance tool to establish an early warning system to aid in the management and prevention of future pandemics. qPCR is the method usually used to detect SARS-CoV-2 in wastewater. There has been no study using an immunoassay that is less laboratory-intensive than qPCR with a shorter turnaround time. Therefore, we aimed to evaluate the performance of an automated chemiluminescence enzyme immunoassay (CLEIA) for SARS-CoV-2 antigen in wastewater. The CLEIA assay achieved 100% sensitivity and 66.7% specificity in a field-captured wastewater sample compared to the gold standard RT-qPCR. Our early findings suggest that the SARS-CoV-2 antigen can be identified in wastewater samples using an automated CLEIA, reducing the turnaround time and improving the performance of SARS-CoV-2 wastewater monitoring during the pandemic.

Is the Medium Still the Message? Culture-Independent Diagnosis of Gastrointestinal Infections.

Infectious diarrhea is caused by a variety of pathogens, including viruses, bacteria, and parasitic organisms. Though the causative agent of diarrhea has historically been evaluated via stool cultures, recently, culture-independent diagnostic tests (CIDT) have been developed and utilized with increasing frequency. Current practice guidelines recommend their use as adjuncts to stool cultures for diagnosing acute and chronic diarrhea. The three principal CIDT are microscopy, enzyme-based immunoassays (EIAs), and molecular based polymerase chain reaction (PCR). This review explores the common causes of infectious diarrhea, the basics of stool culture, the diagnostic utility of these three culture-independent modalities, and the strengths and weaknesses of all currently available clinical techniques. It also outlines considerations for specific populations including returning travelers and those with inflammatory bowel disease.

Clinical Significance of Toxigenic Clostridioides difficile Growth in Stool Cultures during the Era of Nonculture Methods for the Diagnosis of C. difficile Infection.

The importance of the detection of relevant toxins or toxin genes to diagnose Clostridioides difficile infection (CDI) or the prediction of clinical outcomes of CDI has been emphasized in recent years. Although stool culture of C. difficile is not routinely recommended in the era of nonculture methods as the preferred tools for CDI diagnosis, the clinical significance of toxigenic C. difficile growth (tCdG) in stool cultures was analyzed. A clinical study was conducted in medical wards of Tainan Hospital, Ministry of Health and Welfare, in southern Taiwan. Diarrheal adults with fecal glutamate dehydrogenase and C. difficile toxin between January 2013 and April 2020 were included. Of the 209 patients with CDI, 158 (75.6%) had tCdG found in stool cultures, and the rest (51, 24.4%) had no tCdG in stool. Only prior ceftazidime or ceftriaxone therapy was independently associated with no tCdG in stool (odds ratio [OR] 2.17, P = 0.02). Compared to the patients with tCDG in stool, those without tCdG in stool experienced treatment success more often (97.1% versus 67.0%, P < 0.001) if treated with metronidazole or vancomycin but had a similar in-hospital mortality or recurrence rate. In the multivariate analysis among 114 patients with CDI treated with metronidazole or vancomycin, treatment success was independently associated with no tCdG in stool (OR 12.7, P = 0.02). Despite the limited utility of stool cultures in CDI diagnoses, no tCdG in stool culture heralds a favorable therapeutic outcome among adults with CDI treated with metronidazole or vancomycin. IMPORTANCE The importance of detecting toxins or toxin genes when diagnosing Clostridioides difficile infections (CDIs) or predicting the severity and outcomes of CDI has been emphasized in recent years. Although the yielding of C. difficile from stool cultures might implicate higher bacterial loads in fecal samples, in an era of nonculture methods for the standard diagnosis of CDIs, clinical significance of positive stool cultures of toxigenic C. difficile was analyzed in this study. Despite the limited ability of stool cultures in CDI diagnoses, no yielding of C. difficile growth might predict the successful CDI therapy.

An Immunoenzymatic Method for the Determination of Ochratoxin A in Biological Liquids (Colostrum and Cow's Milk).

Ochratoxins are mycotoxins that have been extensively studied lately due to the multiple toxic effects such as nephrotoxicity, hepatotoxicity, and carcinogenicity. These toxins contaminate plant and animal foods and after ingestion they reach into body fluids. The method of competitive direct enzyme immunoassay, in the solid phase, was validated through the determination of specific parameters (performance, linearity, recovery percentage, limit of detection, limit of quantification). The validated method was used to determine ochratoxin A in colostrum and cow's milk. The method applied for the determination of ochratoxin A was linear for the concentration range of 0.0-0.5 ng/mL, the value for the regression coefficient (r) was 0.9838. Ochratoxin A was present in 91.67% of the colostrum and in 93.33% of cow's milk samples. The linearity of the method, demonstrated for very low concentrations of analyte, the detection limit as well as the limit of quantification recommend the method for the determinations of micro-pollutants from foods, including biological fluids.

Development of a highly sensitive chemiluminescent enzyme immunoassay for fragmented cytokeratin 18 using new antibodies.

Fragmented cytokeratin 18 (fCK18) released from epithelial cells undergoing apoptosis is widely studied in various diseases. However, fCK18 measurement is not utilized in clinical practice due to imprecise disease-state cutoff values. Therefore, we set out to generate new monoclonal antibodies (mAbs) and a recombinant fCK18 (rfCK18) calibrator in an effort to develop a highly sensitive chemiluminescent enzyme immunoassay (CLEIA). New capture mAb (K18-624) had a high binding ability compared to the current commercial antibody. New detection mAb (K18-328) recognized 323S-340G of CK18. A rfCK18 was expressed in the soluble fraction of E. coli when the N-terminal region (260 amino acid residues) of CK18 was truncated. Analysis of performance and measurement of human fCK18 were evaluated using K18-624 and K18-328 in a highly sensitive CLEIA. The coefficients of variation (CV) for within-run and between-day repeatability were below 10% and the recoveries were in the range of 15%. The detection sensitivity was 0.056 ng/mL. Serum fCK18 levels were significantly increased in non-alcoholic steatohepatitis (NASH) patients when compared to healthy individuals. Our new fCK18 mAbs showed high affinity and sensitivity. CLEIA using our new antibodies will be useful in measuring fCK18 in human blood thereby generating accurate clinical diagnoses of human liver diseases.

Fully automated chemiluminescence enzyme immunoassays showing high correlation with immunoprecipitation mass spectrometry assays for ß-amyloid (1-40) and (1-42) in plasma samples.

Blood based ß-amyloid (Aß) assays that can predict amyloid positivity in the brain are in high demand. Current studies that utilize immunoprecipitation mass spectrometry assay (IP-MS), which has high specificity for measuring analytes, have revealed that precise plasma Aß assays have the potential to detect amyloid positivity in the brain. In this study, we developed plasma Aß40 and Aß42 immunoassays using a fully automated immunoassay platform that is used in routine clinical practice. Our assays showed high sensitivity (limit of quantification: 2.46 pg/mL [Aß40] and 0.16 pg/mL [Aß42]) and high reproducibility within-run (coefficients of variation [CVs]: <3.7% [Aß40] and <2.0% [Aß42]) and within-laboratory (CVs: <4.6% [Aß40] and <5.3% [Aß42]). The interference from plasma components was less than 10%, and the cross-reactivity with various lengths of Aß peptides was less than 0.5%. In addition, we found a significant correlation between the IP-MS method and our immunoassay (correlation coefficients of Pearson's r: 0.91 [Aß40] and 0.82 [Aß42]). Our new method to quantify plasma Aß40 and Aß42 provides clinicians and patients with a way to continuously monitor disease progression.

Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping.

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell-cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.

Comparison of 4 commercial enzyme immunoassays for serology testing of human parvovirus B19 infection.

BACKGROUND: Parvovirus B19 is a pathogenic virus often diagnosed by serology, yet little is known about analytical performance of commercial enzyme immunoassays (EIAs). OBJECTIVE: To investigate performance of 4 EIAs for parvovirus B19 IgM and IgG: Liaison, Euroimmun, Mikrogen and Virion/Serion. STUDY DESIGN: To compare 4 EIAs to Biotrin's ELISA on 168 samples and determine consensus score for discordant samples using Mikrogen's confirmatory line assay. RESULTS: Two thirds of results for IgM/IgG were identical for all 4 EIAs and Biotrin. Liaison shows the highest IgM sensitivity, but has low specificity. Euroimmun lacks IgM sensitivity. Mikrogen had a good overall performance, but had the lowest IgG specificity. Virion/Serion had variable performance with a low IgM specificity and the most borderline and cross-reactive results. CONCLUSIONS: Liaison and Mikrogen have similar performance to Biotrin's ELISA. Euroimmun lacks sensitivity and Virion/Serion produced many borderline and cross-reactive results.

Comparison of the analytical and clinical performances of two different routine testing protocols for antinuclear antibody screening.

BACKGROUND: The diagnosis of systemic autoimmune rheumatic diseases (SARD) is based on the detection of serum antinuclear antibodies (ANA) for which indirect immunofluorescence (IIF) is the golden standard. New solid-phase immunoassays have been developed to be used alone or in combination with the detection of extractable antinuclear antibodies (ENA) to improve SARD diagnosis. The purpose of this study was to compare the clinical performances of different ANA screening methods alone or in combination with ENA screening methods for SARD diagnosis. METHODS: A total of 323 patients were screened for ANA by IIF, EliA™ CTD Screen, and ELISA methods. Agreements were calculated between the methods. Then, EliA™ CTD Screen positive samples were screened for ENA by line immunoassay (LIA) and fluorescence enzyme immunoassay (FEIA). RESULTS: The diagnostic accuracy of EliA™ CTD Screen (79% sensitivity and 91% specificity) was better than that of ELISA or IIF. The combination of EliA™ CTD plus IIF had the highest sensitivity (93%). ENA determination revealed that Ro52 and Ro60 were the most prevalent specificities. The use of IIF alone was not able of detecting up to 36% of samples positive for Ro52, and 41% for Ro60. CONCLUSIONS: EliA™ CTD Screen has a better diagnostic performance when compared to IIF and ELISA. The combined use of EliA™ CTD Screen and IIF clearly improves the rate and accuracy of SARD diagnosis. The use of EliA™ CTD Screen as first-line screening technique allows the detection of antibodies, which could not be detected by IIF alone.

A performance evaluation of chemiluminescence enzyme immunoassays on the Sysmex CN-6500 haemostasis analyser.

BACKGROUND: The Sysmex CN-6500 is a new haemostasis analyser with an integrated immunoassay module that performs chemiluminescence enzyme assay (CLEIA) in addition to coagulation, turbidimetric, chromogenic and platelet aggregation tests. AIMS: To evaluate the analytical performance of the CN-6500 against the predicate device (Sysmex HISCL-800) for soluble thrombomodulin (TM), thrombin-antithrombin (TAT), tissue plasminogen activator/plasminogen activator inhibitor 1 complex (tPAI-C) and plasmin α2 plasmin inhibitor complex (PIC) assays. METHODS: Imprecision was assessed by testing two levels of quality control plasmas 10 times on 5 separate days. Comparability was studied in 230 plasmas from normal donors (n = 30), patients with suspected disseminated intravascular coagulation (DIC, n = 100), sepsis (n = 20) or liver disease (n = 20), lipaemic (n = 20), haemolysed (n = 20) and icteric samples (n = 20). Limit of detection, limit of quantitation and linearity were determined by testing serial dilutions of normal plasma. Sample carryover was assessed by testing samples with high and low normal levels of the analytes concerned. RESULTS: The CN-6500 performed 21 CLEIA tests per hour, while simultaneously performing coagulation tests. Acceptable between-run imprecision was obtained using commercial controls with normal and high activity for each analyte (%CV <4%), for all four assays. Excellent linearity was observed (slope 0.89-1.03; r2 >0.99) across the measurement range. The lower limits of detection and quantitation were as follows: TM <0.3/0.6 TU/ml, TAT >0.1/<0.2 ng/ml, PIC <0.004/<0.008 µg/ml and tPAI-C < 0.01/<0.1 ng/ml, respectively. All four assays showed excellent correlation between analysers and were unaffected by haemolysis, icterus or lipaemia. No carryover was observed. CONCLUSIONS: Our data demonstrate that the performance of the CLEIA assays on the CN-6500 is comparable to that of a stand-alone immunoassay analyser.