Enzyme-linked immunosorbent assay (ELISA) detects qualitatively and quantitatively the presence of antibodies or antigens in a sample. Due to its simplicity, high sensitivity, and user-friendliness, the test is widely used in laboratory research, clinical diagnoses, and food testing. This chapter describes the indirect semiquantitative ELISA protocol used to monitor antibody levels in animals and analyze the titer levels of specific antibodies against a target antigen in serum and saliva.

OBJECTIVE: To develop a classification tree via semiquantitative analysis for ultrasonographic breast composition assessment using routine breast ultrasonography examination images.
METHODS: This study retrospectively enrolled 100 consecutive normal women who underwent screening mammography and supplemental ultrasonography. Based on sonographic breast composition, the patients\' breasts were classified as nondense or dense, which were correlated with mammographic breast composition. Ultrasonographic breast composition was classified based on the fibroglandular tissue (FGT) thickness-to-subcutaneous fat and retromammary fat (FAT) thickness ratio. In addition, the presence of a high glandular tissue component (GTC) in FGT or the presence of evident fat lobules in FGT was investigated. The cutoff point between the nondense and dense breasts was calculated from the area under the curve (AUC).
RESULTS: All cases with a high GTC were dense breasts, and all cases with evident fat lobules in the FGT were nondense breasts. The AUC of the FGT thickness-to-FAT ratio of all cases, the group without a high GTC, the group without evident fat lobules in the FGT, and the group without a high GTC or evident fat lobules in the FGT were 0.93, 0.94, 0.99, and 1, respectively.
CONCLUSIONS: The presence of a high GTC indicated dense breasts, and the presence of evident fat lobules in the FGT represented nondense breasts. For the remaining cases, the cutoff point of the FGT thickness-to-FAT thickness ratio was 0.93 for ultrasonographic two-grade scale breast composition assessment with 100% accuracy.

Ziziphi Spinosae Semen refers to the dried seed of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou. The seed is composed of a reddish brown coat and a yellow kernel. A comparative study was conducted to investigate differences in the chemical composition and their relative contents between the seed coat and kernel of Ziziphi Spinosae Semen. First, the chemical compounds found in the seed coat and kernel were characterized and identified using ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). The analytical results tentatively identified 57 chemical compounds based on reference-compound comparison, literature retrieval, and chemical-database (e. g., MassBank) searches; these compounds included 14 triterpenes, 23 flavonoids, 7 alkaloids, 6 carboxylic acids, and 7 other types of compounds. The mass error of the identified compounds was within the mass deviation range of 5×10-6 (5 ppm). Next, two methods of multivariate statistical analysis, namely, principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA), were used to compare the differential compounds between the two seed parts. A total of 17 differential compounds were screened out via OPLS-DA based on a variable importance in projection (VIP) value of >5. The results revealed that betulinic acid, betulonic acid, alphitolic acid, and jujuboside Ⅰ mainly existed in the seed coat whereas the 13 other compounds, such as spinosin, jujuboside A, and 6‴-feruloylspinosin, mainly existed in the seed kernel. Therefore, these 17 differential compounds can be used to distinguish between the two seed parts. Finally, a semiquantitative method was established using UPLC and a charged aerosol detector (CAD) with inverse gradient compensation in the mobile phase. Six representative compounds with different types were selected to examine the CAD response consistency: magnoflorine (alkaloid), spinosin (flavone), 6‴-feruloylspinosin (flavone), jujuboside A (triterpenoid saponin), jujuboside B (triterpenoid saponin), and betulinic acid (triterpenoid acid). The results showed that the relative standard deviation (RSD) of the average response factors at different levels of these six compounds was 7.04% and that their response intensities were similar. Moreover, each compound in the fingerprint demonstrated good response consistency, and the peak areas obtained directly reflected the contents of each compound. Based on the semiquantitative fingerprints obtained, betulinic acid and oleic acid were considered the main components of the seed coat. The betulinic acid content in the seed coat was approximately 7 times higher than that in the seed kernel. Spinosin, jujuboside A, linoleic acid, betulinic acid, and oleic acid were the main components of the seed kernel. The spinosin content in the seed kernel was 18 times higher than that in the seed coat. In addition, the jujuboside A content in the seed kernel was 24 times higher than that in the seed coat. The proposed method can accurately determine the main components and compare the relative contents of these components in different seed parts. In summary, this study identified the differences in chemical components between the seed coat and kernel of Ziziphi Spinosae Semen and clarified the main components and their relative contents in these parts. The findings can not only provide a basis for the identification of chemical compounds and quality research on different parts of Ziziphi Spinosae Semen but also promote the development and utilization of this traditional Chinese medicine.

In classic semiquantitative metabolomics, metabolite intensities are affected by biological factors and other unwanted variations. A systematic evaluation of the data processing methods is crucial to identify adequate processing procedures for a given experimental setup. Current comparative studies are mostly focused on peak area data but not on absolute concentrations. In this study, we evaluated data processing methods to produce outputs that were most similar to the corresponding absolute quantified data. We examined the data distribution characteristics, fold difference patterns between 2 metabolites, and sample variance. We used 2 metabolomic datasets from a retail milk study and a lupus nephritis cohort as test cases. When studying the impact of data normalization, transformation, scaling, and combinations of these methods, we found that the cross-contribution compensating multiple standard normalization (ccmn) method, followed by square root data transformation, was most appropriate for a well-controlled study such as the milk study dataset. Regarding the lupus nephritis cohort study, only ccmn normalization could slightly improve the data quality of the noisy cohort. Since the assessment accounted for the resemblance between processed data and the corresponding absolute quantified data, our results denote a helpful guideline for processing metabolomic datasets within a similar context (food and clinical metabolomics). Finally, we introduce Metabox 2.0, which enables thorough analysis of metabolomic data, including data processing, biomarker analysis, integrative analysis, and data interpretation. It was successfully used to process and analyze the data in this study. An online web version is available at http://metsysbio.com/metabox.

UNASSIGNED: Persistent dyspnea is frequent in post-COVID patients, even in the absence of pulmonary embolism (PE). In this scenario, the role of lung perfusion scintigraphy is unclear. The present study correlated scintigraphy-based semiquantitative perfusion parameters with chest high-resolution computed tomography (hrCT) volumetric indexes and clinical data in post-COVID patients with persistent dyspnea.
UNASSIGNED: Sixty patients (30 post-COVID and 30 not previously affected by COVID-19) with persistent dyspnea submitted to lung perfusion scintigraphy and hrCT were retrospectively recruited. Perfusion rates of the pulmonary fields and hrCT-based normalized inflated, emphysematous, infiltrated, collapsed, and vascular lung volumes were calculated. Inflammatory and coagulation biomarkers were collected. PE at imaging was an exclusion criterion.
UNASSIGNED: Compared to controls, reduced perfusion rates of the lower pulmonary fields and higher perfusion rates of the middle ones were observed in post-COVID patients, while hrCT findings were superimposable between the two groups. Perfusion rates of lower pulmonary fields were significantly associated only with abnormal lung volumes at hrCT.
UNASSIGNED: In post-COVID dyspnea without PE, lung perfusion scintigraphy may reveal a pulmonary involvement not detectable by hrCT. Post-COVID patients may show decreased perfusion rates of lower pulmonary fields in the presence of normal vascular density and markers of inflammation/coagulation.

The most common gene responsible for autosomal recessive retinitis pigmentosa (RP) is EYS. The manner of decay of genetically defective EYS gene transcripts varies depending on the type of mutation using our cellular model, which consists of induced photoreceptor-directed fibroblasts from EYS-RP patients (EYS-RP cells). However, disease-specific profiles have not been clarified in EYS-RP cells. Herein we investigated comprehensive gene expression patterns and restoration of altered expression by low molecular weight molecules in EYS-RP cells.
Using induced photoreceptor-like cells by CRX, RAX, NeuroD, and OTX2, we employed qRT-PCR and DNA microarray analysis to compare expression levels of disease-related genes in EYS-RP cells. We investigated the effect of antiapoptotic or anti-endoplasmic reticulum (ER) stress/antioxidant reagents on the restoration of altered gene expression.
Expression levels of phototransduction-related genes (blue opsin, rhodopsin, S-antigen, GNAT1, GNAT2) were lower in EYS-RP cells. CRYGD was extracted by global gene expression analysis, as a downregulated, retina-related and apoptosis-, endoplasmic reticulum (ER) stress- or aging-related gene. Pathway enrichment analysis suggested that \"complement and coagulation cascades,\" \"ECM-receptor interaction\" and \"PI3K-Akt signaling pathway\" could be involved in EYS-RP-associated pathogenesis. Among the matching/overlapping genes involved in those pathways, F2R was suggested as an EYS-RP-associated gene. The downregulation of CRYGD and F2R was completely restored by additional 4-PBA, an inhibitor of ER stress, and partially restored by metformin or NAC. In addition, 4-PBA normalized the expression level of cleaved caspase-3.
Our cellular model may reflect the ER stress-mediated degenerative retina and serve as a pathogenesis-oriented cost-effective rescue strategy for RP patients.

OBJECTIVE: White blood cell (WBC) scintigraphy is considered the gold-standard nuclear imaging technique for diagnosing fracture-related infection (FRI). Correct interpretation of WBC scans in FRI is important since a false positive or false negative diagnosis has major consequences for the patient in terms of clinical decision-making. The European Association of Nuclear Medicine (EANM) guideline for correct analysis and interpretation of WBC scans recommends semiquantitative analysis of visually equivocal scans. Therefore, this study aims to assess the diagnostic accuracy of semiquantitative analysis of visually equivocal WBC scans for diagnosing FRI.
METHODS: A retrospective single-center study was performed in consecutive patients who received WBC scintigraphy in the diagnostic work-up for FRI between February 2012 and January 2017. All the visually equivocal scans were analysed using semiquantitative analysis by comparing leukocyte uptake in the manually selected suspected infection focus with the contralateral bone marrow (L/R ratio). Cut-off points for a \'positive\' scan result of >0%, >10% and >20% leukocyte increase between the early and late scans were used in separate analyses. The discriminative ability was quantified by calculating the sensitivity, specificity and diagnostic accuracy.
RESULTS: In total, 153 WBC scans were eligible for inclusion. After visual assessment of all the scans, 28 visually equivocal scans were included. Dichotomization of the ratios using the cut-off of >0% resulted in a sensitivity of 30%, a specificity of 45% and a diagnostic accuracy of 40%. The >10% cut-off point resulted in a sensitivity of 18%, a specificity of 82% and a diagnostic accuracy of 66%. The >20% cut-off point resulted in a sensitivity of 0%, a specificity of 89% and a diagnostic accuracy of 67%.
CONCLUSIONS: Semiquantitative analysis of visually equivocal WBC scans is insufficient for correctly diagnosing FRI.

BACKGROUND: Programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) blockade immunotherapies have changed the landscape of cancer therapy. However, the main limitation of these therapies is the lack of definitively predictive biomarkers to predict treatment response. Whether PD-L1 expression on circulating tumor cells (CTCs) is associated with the clinical outcomes of immunotherapy remains to be extensively investigated.
METHODS: One hundred fifty-five patients with different advanced cancers were enrolled in this study and treated with anti-PD-1/PD-L1 monoclonal antibodies. Using the Pep@MNPs method, CTCs were isolated and enumerated. The PD-L1 expression levels were analyzed by an immunofluorescence assay for semiquantitative assessment with four categories (negative, low, medium, and high).
RESULTS: Prior to immunotherapy, 81.93% (127/155) of patients had PD-L1-positive CTCs, and 71.61% (111/155) had at least one PD-L1-high CTC. The group with PD-L1-positive CTCs had a higher disease control rate (DCR) (71.56%, 91/127), with a DCR of only 39.29% (11/28) for the remaining individuals (p = .001). The objective response rate and DCR in PD-L1-high patients were higher than those in the other patients (32.44% vs. 13.64%, p = .018 and 75.68% vs. 40.91%, p < .0001, respectively). The reduction in the counts and ratios of PD-L1-positive CTCs and PD-L1-high CTCs reflected a beneficial response to PD-1/PD-L1 inhibitors. Furthermore, patients with PD-L1-high CTCs had significantly longer progression-free survival (4.9 vs. 2.2 months, p < .0001) and overall survival (16.1 vs. 9.0 months, p = .0235) than those without PD-L1-high CTCs.
CONCLUSIONS: The PD-L1 level on CTCs may serve as a clinically actionable biomarker for immunotherapy, and its dynamic changes could predict the therapeutic response.
CONCLUSIONS: This study was designed to investigate the role of programmed death-ligand 1 (PD-L1) expression on circulating tumor cells in predicting and monitoring response to programmed death-1 (PD-1)/PD-L1 blockade immunotherapies in patients with advanced cancer. The results of the study showed that PD-L1-high-expression circulating tumor cells (CTCs) were both a predictive biomarker and a prognostic factor in patients with advanced cancer treated with anti-PD-1/PD-L1 monoclonal antibodies. These observations suggest that PD-L1 level on CTCs is a potential clinical biomarker for immunotherapy.

Various studies have reported to the superiority of semiquantitative (SQ) analysis over visual analysis in detecting metabolic changes in the brain. In this study, we aimed to determine the limitations of SQ analysis programs and the current status of 18F- fluorodeoxyglucose (FDG)-positron emission tomography (PET) scan in dementia. 18F- FDG-PET/computed tomography (CT) brain images of 39 patients with a history of dementia were analyzed both visually and semiquantitatively. Using the visually markedly abnormal 18F- FDG-PET images as standard, we wanted to test the accuracy of two commercially available SQ analysis programs. SQ analysis results were classified as matching, partially matching and nonmatching with visually markedly abnormal studies. On visual analysis, 18F- FDG-PET showed marked regional hypometabolism in 19 patients, mild abnormalities in 8 and was normal in 12 patients. SQ analysis-1 results matched with visual analysis in 8 patients (42.1%) and partially matched in 11. SQ analysis-2 findings matched with visual analysis in 11 patients (57.8%) and partially matched in 7 and did not match in 1. Marked regional hypometabolism was either on the left side of the brain or was more significant on the left than the right in 63% of patients. Preservation of metabolism in sensorimotor cortex was seen in various dementia subtypes. Reviewing images in color scale and maximum intensity projection (MIP) image was helpful in demonstrating and displaying regional abnormalities, respectively. SQ analysis provides less accurate results as compared to visual analysis by experts. Due to suboptimal image registration and selection of brain areas, SQ analysis provides inaccurate results, particularly in small areas and areas in close proximity. Image registration and selection of areas with SQ programs should be checked carefully before reporting the results.

The emerging role of molecular imaging has made possible to evaluate and quantify biochemical changes of molecular targets in specific neurochemical systems involved in movement disorders, providing neurochemical information of clinical changes before the pathological features occurred. In detail, radionuclides imaging techniques are frequently used for the in vivo study of neurotransmitter and receptor function, alterations in cerebral blood flow and metabolic activity, abnormal protein deposition, and inflammation, with a central role in molecular imaging for preclinical and clinical studies. The present chapter represents an overview of main use of single-photon emission computed tomography (SPECT) in movement disorders, with a focal attention on specific radiotracers used, recent advances in SPECT technology and reconstruction algorithm and added specific value of semiquantitative methods for images analysis. Finally, a brief paragraph is dedicated to description of SPECT/CT devices and advantages of using hybrid technology.