Practical determination of LODP (limit of detection for microarray platform) for the evaluation of microarray platforms.

The performance indicator called limit of detection for microarray platform (LODP) was defined in ISO 16578:2013. The methods to determine practical LODP were explored. In general, + 3 SD of the background is used as the signal strength of limit of detection and criteria for dividing positive and negative results. Since the negative signal had been defined differently for each microarray platform, signals obtained from Non-Probe Spots (NPS) installed on the microarrays were defined as the "background" of microarrays. LODP was determined as the lowest concentration of which the average signal exceeded Avg. + 3 SD of the background (NPS) and the signal was significantly different from those of the lower and higher adjacent concentration points measured with a diluted series of reference materials. For reliable qualitative analysis, the positive results can be defined as signals higher than those corresponding to LODP and negative results as lower signals, without determining limit of detection for all target probes. The use of LODP also enables comparisons of platform performances without checking sequence dependencies, and assists to select reliable and fitting platforms for experimental purposes.

Clinical significance of the LacdiNAc-glycosylated prostate-specific antigen assay for prostate cancer detection.

To reduce unnecessary prostate biopsies (Pbx), better discrimination is needed. To identify clinically significant prostate cancer (CSPC) we determined the performance of LacdiNAc-glycosylated prostate-specific antigen (LDN-PSA) and LDN-PSA normalized by prostate volume (LDN-PSAD). We retrospectively measured LDN-PSA, total PSA (tPSA), and free PSA/tPSA (F/T PSA) values in 718 men who underwent a Pbx in 3 academic urology clinics in Japan and Canada (Pbx cohort) and in 174 PC patients who subsequently underwent radical prostatectomy in Australia (preop-PSA cohort). The assays were evaluated using the area under the receiver operating characteristics curve (AUC) and decision curve analyses to discriminate CSPC. In the Pbx cohort, LDN-PSAD (AUC 0.860) provided significantly better clinical performance for discriminating CSPC compared with LDN-PSA (AUC 0.827, P = 0.0024), PSAD (AUC 0.809, P < 0.0001), tPSA (AUC 0.712, P < 0.0001), and F/T PSA (AUC 0.661, P < 0.0001). The decision curve analysis showed that using a risk threshold of 20% and adding LDN-PSA and LDN-PSAD to the base model (age, digital rectal examination status, tPSA, and F/T PSA) permitted avoidance of even more biopsies without missing CSPC (9.89% and 18.11%, respectively vs 2.23% [base model]). In the preop-PSA cohort, LDN-PSA values positively correlated with tumor volume and tPSA and were significantly higher in pT3, pathological Gleason score ≥ 7. Limitations include limited sample size, retrospective nature, and no family history information prior to biopsy. LacdiNAc-glycosylated PSA is significantly better than the conventional PSA test in identifying patients with CSPC. This study was approved by the ethics committee of each institution ("The Study about Carbohydrate Structure Change in Urological Disease"; approval no. 2014-195).

Wisteria floribunda Agglutinin and Its Reactive-Glycan-Carrying Prostate-Specific Antigen as a Novel Diagnostic and Prognostic Marker of Prostate Cancer.

Wisteria floribunda agglutinin (WFA) preferably binds to LacdiNAc glycans, and its reactivity is associated with tumor progression. The aim of this study to examine whether the serum LacdiNAc carrying prostate-specific antigen-glycosylation isomer (PSA-Gi) and WFA-reactivity of tumor tissue can be applied as a diagnostic and prognostic marker of prostate cancer (PCa). Between 2007 and 2016, serum PSA-Gi levels before prostate biopsy (Pbx) were measured in 184 biopsy-proven benign prostatic hyperplasia patients and 244 PCa patients using an automated lectin-antibody immunoassay. WFA-reactivity on tumor was analyzed in 260 radical prostatectomy (RP) patients. Diagnostic and prognostic performance of serum PSA-Gi was evaluated using area under the receiver-operator characteristic curve (AUC). Prognostic performance of WFA-reactivity on tumor was evaluated via Cox proportional hazards regression analysis and nomogram. The AUC of serum PSA-Gi detecting PCa and predicting Pbx Grade Group (GG) 3 and GG ≥ 3 after RP was much higher than those of conventional PSA. Multivariate analysis showed that WFA-reactivity on prostate tumor was an independent risk factor of PSA recurrence. The nomogram was a strong model for predicting PSA-free survival provability with a c-index ≥0.7. Serum PSA-Gi levels and WFA-reactivity on prostate tumor may be a novel diagnostic and pre- and post-operative prognostic biomarkers of PCa, respectively.

Prognostic Impact of Circulating Tumor Cell Detected Using a Novel Fluidic Cell Microarray Chip System in Patients with Breast Cancer.

Various types of circulating tumor cell (CTC) detection systems have recently been developed that show a high CTC detection rate. However, it is a big challenge to find a system that can provide better prognostic value than CellSearch in head-to-head comparison. We have developed a novel semi-automated CTC enumeration system (fluidic cell microarray chip system, FCMC) that captures CTC independently of tumor-specific markers or physical properties. Here, we compared the CTC detection sensitivity and the prognostic value of FCMC with CellSearch in breast cancer patients. FCMC was validated in preclinical studies using spike-in samples and in blood samples from 20 healthy donors and 22 breast cancer patients in this study. Using spike-in samples, a statistically higher detection rate (p=0.010) of MDA-MB-231 cells and an equivalent detection rate (p=0.497) of MCF-7 cells were obtained with FCMC in comparison with CellSearch. The number of CTC detected in samples from patients that was above a threshold value as determined from healthy donors was evaluated. The CTC number detected using FCMC was significantly higher than that using CellSearch (p=0.00037). CTC numbers obtained using either FCMC or CellSearch had prognostic value, as assessed by progression free survival. The hazard ratio between CTC+ and CTC- was 4.229 in CellSearch (95% CI, 1.31 to 13.66; p=0.01591); in contrast, it was 11.31 in FCMC (95% CI, 2.245 to 57.0; p=0.000244). CTC detected using FCMC, like the CTC detected using CellSearch, have the potential to be a strong prognostic factor for cancer patients.

Fluorescent immunochromatography for rapid and sensitive typing of seasonal influenza viruses.

Lateral flow tests also known as Immunochromatography (IC) is an antigen-detection method conducted on a nitrocellulose membrane that can be completed in less than 20 min. IC has been used as an important rapid test for clinical diagnosis and surveillance of influenza viruses, but the IC sensitivity is relatively low (approximately 60%) and the limit of detection (LOD) is as low as 10³ pfu per reaction. Recently, we reported an improved IC assay using antibodies conjugated with fluorescent beads (fluorescent immunochromatography; FLIC) for subtyping H5 influenza viruses (FLIC-H5). Although the FLIC strip must be scanned using a fluorescent reader, the sensitivity (LOD) is significantly improved over that of conventional IC methods. In addition, the antibodies which are specific against the subtypes of influenza viruses cannot be available for the detection of other subtypes when the major antigenicity will be changed. In this study, we established the use of FLIC to type seasonal influenza A and B viruses (FLIC-AB). This method has improved sensitivity to 100-fold higher than that of conventional IC methods when we used several strains of influenza viruses. In addition, FLIC-AB demonstrated the ability to detect influenza type A and influenza type B viruses from clinical samples with high sensitivity and specificity (Type A: sensitivity 98.7% (74/75), specificity 100% (54/54), Type B: sensitivity 100% (90/90), specificity 98.2% (54/55) in nasal swab samples) in comparison to the results of qRT-PCR. And furthermore, FLIC-AB performs better in the detection of early stage infection (under 13 h) than other conventional IC methods. Our results provide new strategies to prevent the early-stage transmission of influenza viruses in humans during both seasonal outbreaks and pandemics.

High-sensitivity immunoassay with surface plasmon field-enhanced fluorescence spectroscopy using a plastic sensor chip: application to quantitative analysis of total prostate-specific antigen and GalNAcß1-4GlcNAc-linked prostate-specific antigen for prostate cancer diagnosis.

A high-sensitivity immunoassay system with surface plasmon field-enhanced fluorescence spectrometry (SPFS) was constructed using a plastic sensor chip and then applied to the detection of total prostate-specific antigen (total PSA) and GalNAcß1-4GlcNAc-linked prostate-specific antigen (LacdiNAc-PSA) in serum, to discriminate between prostate cancer (PC) and benign prostate hyperplasia (BPH). By using this automated SPFS immunoassay, the detection limit for total PSA in serum was as low as 0.04 pg/mL, and the dynamic range was estimated to be at least five digits. A two-step sandwich SPFS immunoassay for LacdiNAc-PSA was constructed using both the anti-PSA IgG antibody to capture PSA and Wisteria floribunda agglutinin (WFA) for the detection of LacdiNAc. The results of the LacdiNAc-PSA immunoassay with SPFS showed that the assay had a sensitivity of 20.0 pg/mL and permitted the specific distinction between PC and BPH within the PSA gray zone. These results suggested that high-sensitivity automated SPFS immunoassay systems might become a powerful tool for the diagnosis of PC and other diseases.

Broad-spectrum detection of H5 subtype influenza A viruses with a new fluorescent immunochromatography system.

Immunochromatography (IC) is an antigen-detection assay that plays an important role in the rapid diagnosis of influenza virus because the protocol is short time and easy to use. Despite the usability of IC, the sensitivity is approximately 10(3) pfu per reaction. In addition, antigen-antibody interaction-based method cannot be used for the detection of influenza viruses with major antigenic change. In this study, we established the use of fluorescent immunochromatography (FLIC) to detect a broad spectrum of H5 subtype influenza A viruses. This method has improved sensitivity 10-100 fold higher than traditional IC because of the use of fluorescent conjugated beads. Our Type-E FLIC kit detected all of the H5 subtype influenza viruses that were examined, as well as recombinant hemagglutinin (HA) proteins (rHAs) belonging to the Eurasian H5 subtype viruses and the Type-N diagnosed North American H5 subtype influenza A viruses. Thus, this kit has the improved potential to detect H5 subtype influenza viruses of different clades with both Type-E and Type-N FLIC kits. Compared with PCR-based diagnosis, FLIC has a strong advantage in usability, because the sample preparation required for FLIC is only mix-and-drop without any additional steps such as RNA extraction. Our results can provide new strategies against the spread and transmission of HPAI H5N1 viruses in birds and mammals including humans.