Influence of Chemical and Genetic Manipulations on Cellular Organelles Quantified by Label-Free Optical Diffraction Tomography.

Label-free optical diffraction tomography provides three-dimensional imaging of cells and organelles, along with their refractive index (RI) and volume. These physical parameters are valuable for quantitative and accurate analysis of the subcellular microenvironment and its connections to intracellular biological properties. In biological and biochemical cell analysis, various invasive cell manipulations are used, such as temperature change, chemical fixation, live cell staining with fluorescent dye, and gene overexpression of exogenous proteins. However, it is not fully understood how these various manipulations affect the physicochemical properties of different organelles. In this study, we investigated the impact of these manipulations on the cellular properties of single HeLa cells. We found that after cell fixation and an increase in temperature, the RI value of organelles, such as the nucleus and cytoplasm, significantly decreased overall. Interestingly, unlike the cell nuclei, cytoplasmic RI values were hardly detected after membrane permeation, indicating that only intracytoplasmic components were largely lost. Additionally, our findings revealed that the expression of GFP and GFP-tagged proteins significantly increased the RI values of organelles in living cells compared to the less effective RI changes observed with chemical fluorescence staining for cell organelles. The result demonstrates that distinct types of invasive manipulations can alter the microenvironment of organelles in different ways. Our study sheds new light on how chemical and genetic manipulations affect organelles.

Diagnosis of Ischemic Renal Failure Using Surface-Enhanced Raman Spectroscopy and a Machine Learning Algorithm.

To diagnose renal function using a biochip capable of detecting SERS and to assess Raman measurements taken from a bilateral renal ischemia model and the feasibility of early diagnosis was done. After generating a bilateral renal ischemia rat model, blood and urine were collected. After confirming the presence of renal injury and function, liquid drops were placed onto a Raman chip whose surface had been enhanced with Au-ZnO nanorods. SERS biomarkers that diffused into the nanogaps were selectively amplified. Raman signals varied based on the severity of the renal function, and these differences were confirmed statistically. These results confirm that renal ischemia leads to renal dysfunction and that surface-enhanced Raman spectroscopy and a machine learning algorithm can be used to track signals in the urine from the release of SERS biomarkers.

Minimizing Motion Artifacts in Intravital Microscopy Using the Sedative Effect of Dexmedetomidine.

Among intravital imaging instruments, the intravital two-photon fluorescence excitation microscope has the advantage of enabling real-time 3D fluorescence imaging deep into cells and tissues, with reduced photobleaching and photodamage compared with conventional intravital confocal microscopes. However, excessive motion of organs due to involuntary movement such as breathing may result in out-of-focus images and severe fluorescence intensity fluctuations, which hinder meaningful imaging and analysis. The clinically approved alpha-2 adrenergic receptor agonist dexmedetomidine was administered to mice during two-photon fluorescence intravital imaging to alleviate this problem. As dexmedetomidine blocks the release of the neurotransmitter norepinephrine, pain is suppressed, blood pressure is reduced, and a sedation effect is observed. By tracking the quality of focus and stability of detected fluorescence in two-photon fluorescence images of fluorescein isothiocyanate-sensitized liver vasculature in vivo, we demonstrated that intravascular dexmedetomidine can reduce fluorescence fluctuations caused by respiration on a timescale of minutes in mice, improving image quality and resolution. The results indicate that short-term dexmedetomidine treatment is suitable for reducing involuntary motion in preclinical intravital imaging studies. This method may be applicable to other animal models.

Micro-endoscopy for Live Small Animal Fluorescent Imaging.

Intravital microscopy has emerged as a powerful technique for the fluorescent visualization of cellular- and subcellular-level biological processes in vivo. However, the size of objective lenses used in standard microscopes currently makes it difficult to access internal organs with minimal invasiveness in small animal models, such as mice. Here we describe front- and side-view designs for small-diameter endoscopes based on gradient-index lenses, their construction, their integration into laser scanning confocal microscopy platforms, and their applications for in vivo imaging of fluorescent cells and microvasculature in various organs, including the kidney, bladder, heart, brain, and gastrointestinal tracts, with a focus on the new techniques developed for each imaging application. The combination of novel fluorescence techniques with these powerful imaging methods promises to continue providing novel insights into a variety of diseases.

Label-Free Raman Spectroscopic Techniques with Morphological and Optical Characterization for Cancer Cell Analysis.

Using the Raman spectroscopic analysis system that gives the chemical information of the biomaterials, classification is performed through the acquisition of fingerprint signals for each cell line, and the basis of the diagnosis is provided. The origin of diagnosis can be clarified by precise analysis through comparison of local signals and morphology in cells, including measurement at tissue level. In this result, normal breast cell line (MCF-10A) and breast cancer cell lines (MDA-MB-231, MDA-MB-453) were characterized using Raman spectroscopy, atomic force microscopy (AFM), and optical microscopy. These three modalities were combined in order to not only separate cancerous and noncancerous cell lines but to analyze their morphological and optical properties. From the results, the inherent optical properties of cancer cells separated from normal cells in terms of local variation were observed. Bright-field (BF) transmission imaging is also compared to the morphological height difference obtained from AFM and is correlated with surface Raman spectra.

A Wi-Fi-Based Mask-Type Laryngoscope for Telediagnosis During the COVID-19 Pandemic: Instrument Validation Study.

BACKGROUND: Owing to the COVID-19 pandemic, social distancing has become mandatory. Wireless endoscopy in contactless examinations promises to protect health care workers and reduce viral spread. OBJECTIVE: This study aimed to introduce a contactless endoscopic diagnosis system using a wireless endoscope resembling a mask. METHODS: The Wi-Fi-based contactless mask endoscopy system comprises a disposable endoscope and a controller. First, the effective force applied by the tip during insertion was evaluated in a simple transoral model consisting of a force sensor on a simulated oropharynx wall. Second, the delay in video streaming was evaluated by comparing the frame rate and delays between a movement and its image over direct and Wi-Fi connections. Third, the system was applied to a detailed laryngopharyngeal tract phantom. RESULTS: The smartphone-controlled wireless endoscopy system was successfully evaluated. The mean, maximum, and minimum collision forces against the wall of the transoral model were 296 mN (30 gf), 363 mN (37 gf), and 235 mN (24 gf), respectively. The delay resulting from the wireless connection was 0.72 seconds. Using the phantom, an inexperienced user took around 1 minute to orient the endoscope to a desired area via the app. CONCLUSIONS: Device articulation does not pose a significant risk of laryngopharyngeal wall penetration, and latency does not significantly impede its use. Contactless wireless video streaming was successful within the access point range regardless of the presence of walls. The mask endoscope can be controlled and articulated wirelessly, minimizing contact between patients and device operators. By minimizing contact, the device can protect health care workers from infectious viruses like the coronavirus.

Mobility of Nucleostemin in Live Cells Is Specifically Related to Transcription Inhibition by Actinomycin D and GTP-Binding Motif.

In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility.

Poly(A)+ Sensing of Hybridization-Sensitive Fluorescent Oligonucleotide Probe Characterized by Fluorescence Correlation Methods.

Ribonucleic acid (RNA) plays an important role in many cellular processes. Thus, visualizing and quantifying the molecular dynamics of RNA directly in living cells is essential to uncovering their role in RNA metabolism. Among the wide variety of fluorescent probes available for RNA visualization, exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes are useful because of their low fluorescence background. In this study, we apply fluorescence correlation methods to ECHO probes targeting the poly(A) tail of mRNA. In this way, we demonstrate not only the visualization but also the quantification of the interaction between the probe and the target, as well as of the change in the fluorescence brightness and the diffusion coefficient caused by the binding. In particular, the uptake of ECHO probes to detect mRNA is demonstrated in HeLa cells. These results are expected to provide new insights that help us better understand the metabolism of intracellular mRNA.

Targeting CXCR4-dependent immunosuppressive Ly6Clow monocytes improves antiangiogenic therapy in colorectal cancer.

Antiangiogenic therapy with antibodies against VEGF (bevacizumab) or VEGFR2 (ramucirumab) has been proven efficacious in colorectal cancer (CRC) patients. However, the improvement in overall survival is modest and only in combination with chemotherapy. Thus, there is an urgent need to identify potential underlying mechanisms of resistance specific to antiangiogenic therapy and develop strategies to overcome them. Here we found that anti-VEGFR2 therapy up-regulates both C-X-C chemokine ligand 12 (CXCL12) and C-X-C chemokine receptor 4 (CXCR4) in orthotopic murine CRC models, including SL4 and CT26. Blockade of CXCR4 signaling significantly enhanced treatment efficacy of anti-VEGFR2 treatment in both CRC models. CXCR4 was predominantly expressed in immunosuppressive innate immune cells, which are recruited to CRCs upon anti-VEGFR2 treatment. Blockade of CXCR4 abrogated the recruitment of these innate immune cells. Importantly, these myeloid cells were mostly Ly6Clow monocytes and not Ly6Chigh monocytes. To selectively deplete individual innate immune cell populations, we targeted key pathways in Ly6Clow monocytes (Cx3cr1-/- mice), Ly6Chigh monocytes (CCR2-/- mice), and neutrophils (anti-Ly6G antibody) in combination with CXCR4 blockade in SL4 CRCs. Depletion of Ly6Clow monocytes or neutrophils improved anti-VEGFR2-induced SL4 tumor growth delay similar to the CXCR4 blockade. In CT26 CRCs, highly resistant to anti-VEGFR2 therapy, CXCR4 blockade enhanced anti-VEGFR2-induced tumor growth delay but specific depletion of Ly6G+ neutrophils did not. The discovery of CXCR4-dependent recruitment of Ly6Clow monocytes in tumors unveiled a heretofore unknown mechanism of resistance to anti-VEGF therapies. Our findings also provide a rapidly translatable strategy to enhance the outcome of anti-VEGF cancer therapies.

Longitudinal micro-endoscopic monitoring of high-success intramucosal xenografts for mouse models of colorectal cancer.

Colorectal cancer (CRC) is one of the most frequently lethal forms of cancer. Intramucosal injection allows development of better mouse models of CRC, as orthotopic xenografts allow development of adenocarcinoma in the submucosa of the mouse colon wall. In this paper, a method of orthotopic injection is monitored longitudinally using cellular-resolution real-time in vivo fluorescence microendoscopy, following the injection of three different cell lines: 3T3-GFP to confirm immunosuppression and HCT116-RFP cells to model CRC. Adenoma formation is first observable after 7 to 10 days, and by use of 33 G needles a tumor induction rate of greater than 85% is documented. An additional experiment on the injection of rapamycin reveals drug efficacy and localization between 24 and 48 hours, and suggests the promise of real-time cellular-resolution fluorescence micro-endoscopy for developing longitudinal therapy regimes in mural models of CRC.

Micro-endoscopic In Vivo Monitoring in the Blood and Lymphatic Vessels of the Oral Cavity after Radiation Therapy.

Radiotherapy, although used worldwide for the treatment of head, neck, and oral cancers, causes acute complications, including effects on vasculature and immune response due to cellular stress. Thus, the ability to diagnose side-effects and monitor vascular response in real-time during radiotherapy would be highly beneficial for clinical and research applications. In this study, recently-developed fluorescence micro-endoscopic technology provides non-invasive, high-resolution, real-time imaging at the cellular level. Moreover, with the application of high-resolution imaging technologies and micro-endoscopy, which enable improved monitoring of adverse effects in GFP-expressing mouse models, changes in the oral vasculature and lymphatic vessels are quantified in real time for 10 days following a mild localized single fractionation, 10 Gy radiotherapy treatments. Fluorescence micro-endoscopy enables quantification of the cardiovascular recovery and immune response, which shows short-term reduction in mean blood flow velocity, in lymph flow, and in transient immune infiltration even after this mild radiation dose, in addition to long-term reduction in blood vessel capacity. The data provided may serve as a reference for the expected cellular-level physiological, cardiovascular, and immune changes in animal disease models after radiotherapy.

High sensitivity temperature measurement via mask-free hybrid polymer long period fiber grating.

Long-period fiber gratings (LPFGs) are useful for environmental sensing under conditions of high corrosiveness and electromagnetic interference. Most LPFGs are fabricated by coherent or high-power UV illumination of an optical fiber under an amplitude mask, resulting in narrow and environmentally-dependent band rejection. We present a hybrid LPFG waveguide fabricated without an amplitude mask through polymer self-assembly under low-power incoherent UV illumination, which demonstrates high-temperature sensitivity in its transmission spectrum compared to LPFG sensors based purely on silica waveguides. A sensitivity of 1.5 nm °C -1 is obtained experimentally for attenuation near 1180 nm, and a sensitivity of 4.5 nm °C -1 with a low random error was obtained with a composite of attenuation bands. Finite element method simulations and coupling mode theory reveal this to be due to a thermo-optic coefficient one order of magnitude greater than that of fused silica. The device has potential for a simple and inexpensive transmission intensity based temperature sensor consisting of an infrared light source, the LPFG, a bandpass filter, and a photodiode.

In Vivo Fluorescence Microendoscopic Monitoring of Stent-Induced Fibroblast Cell Proliferation in an Esophageal Mouse Model.

PURPOSE: To evaluate the feasibility of self-expanding metal stent (SEMS) placement and fluorescence microendoscopic monitoring for determination of fibroblast cell proliferation after stent placement in an esophageal mouse model. MATERIALS AND METHODS: Twenty fibroblast-specific protein (FSP)-1 green fluorescent protein (GFP) transgenic mice were analyzed. Ten mice (Group A) underwent SEMS placement, and fluoroscopic and fluorescence microendoscopic images were obtained biweekly until 8 weeks thereafter. Ten healthy mice (Group B) were used for control esophageal values. RESULTS: SEMS placement was technically successful in all mice. The relative average number of fibroblast GFP cells and the intensities of GFP signals in Group A were significantly higher than in Group B after stent placement. The proliferative cellular response, including granulation tissue, epithelial layer, submucosal fibrosis, and connective tissue, was increased in Group A. FSP-1-positive cells were more prominent in Group A than in Group B. CONCLUSIONS: SEMS placement was feasible and safe in an esophageal mouse model, and proliferative cellular response caused by fibroblast cell proliferation after stent placement was longitudinally monitored using a noninvasive fluorescence microendoscopic technique. The results have implications for the understanding of proliferative cellular response after stent placement in real-life patients and provide initial insights into new clinical therapeutic strategies for restenosis.

One-pot sequential synthesis of tetrasubstituted thiophenes via sulfur ylide-like intermediates.

Herein, we describe a novel approach for the practical synthesis of tetrasubstituted thiophenes 8. The developed method was particularly used for the facile preparation of thienyl heterocycles 8. The mechanism for this reaction is based on the formation of a sulfur ylide-like intermediate. It was clearly suggested by (i) the intramolecular cyclization of ketene N,S-acetals 7 to the corresponding thiophenes 8, (ii) 1H NMR studies of Meldrum's acid-substituted aminothioacetals 9, and (iii) substitution studies of the methoxy group on Meldrum's acid containing N,S-acetals 9b. Notably, in terms of structural effects on the reactivity and stability of sulfur ylide-like intermediates, 2-pyridyl substituted compound 7a exhibited superior properties over those of others.

Fluoroscopic removal of retrievable self-expandable metal stents in patients with malignant oesophageal strictures: Experience with a non-endoscopic removal system.

OBJECTIVES: To evaluate clinical outcomes of fluoroscopic removal of retrievable self-expandable metal stents (SEMSs) for malignant oesophageal strictures, to compare clinical outcomes of three different removal techniques, and to identify predictive factors of successful removal by the standard technique (primary technical success). METHODS: A total of 137 stents were removed from 128 patients with malignant oesophageal strictures. Primary overall technical success and removal-related complications were evaluated. Logistic regression models were constructed to identify predictive factors of primary technical success. RESULTS: Primary technical success rate was 78.8 % (108/137). Complications occurred in six (4.4 %) cases. Stent location in the upper oesophagus (P=0.004), stricture length over 8 cm (P=0.030), and proximal granulation tissue (P<0.001) were negative predictive factors of primary technical success. If granulation tissue was present at the proximal end, eversion technique was more frequently required (P=0.002). CONCLUSIONS: Fluoroscopic removal of retrievable SEMSs for malignant oesophageal strictures using three different removal techniques appeared to be safe and easy. The standard technique is safe and effective in the majority of patients. The presence of proximal granulation tissue, stent location in the upper oesophagus, and stricture length over 8 cm were negative predictive factors for primary technical success by standard extraction and may require a modified removal technique. KEY POINTS: • Fluoroscopic retrievable SEMS removal is safe and effective. • Standard removal technique by traction is effective in the majority of patients. • Three negative predictive factors of primary technical success were identified. • Caution should be exercised during the removal in those situations. • Eversion technique is effective in cases of proximal granulation tissue.

Endoscopic time-lapse imaging of immune cells in infarcted mouse hearts.

RATIONALE: High-resolution imaging of the heart in vivo is challenging owing to the difficulty in accessing the heart and the tissue motion caused by the heartbeat. OBJECTIVE: Here, we describe a suction-assisted endoscope for visualizing fluorescently labeled cells and vessels in the beating heart tissue through a small incision made in the intercostal space. METHODS AND RESULTS: A suction tube with a diameter of 2 to 3 mm stabilizes the local tissue motion safely and effectively at a suction pressure of 50 mm Hg. Using a minimally invasive endoscope integrated into a confocal microscope, we performed fluorescence cellular imaging in both normal and diseased hearts in live mice for an hour per session repeatedly over a few weeks. Real-time imaging revealed the surprisingly rapid infiltration of CX3CR1(+) monocytes into the injured site within several minutes after acute myocardial infarction. CONCLUSIONS: The time-lapse analysis of flowing and rolling (patrolling) monocytes in the heart and the peripheral circulation provides evidence that the massively recruited monocytes come first from the vascular reservoir and later from the spleen. The imaging method requires minimal surgical preparation and can be implemented into standard intravital microscopes. Our results demonstrate the applicability of our imaging method for a wide range of cardiovascular research.

Identification of cromolyn sodium as an anti-fibrotic agent targeting both hepatocytes and hepatic stellate cells.

Liver fibrosis and cirrhosis, the late stage of fibrosis, are threatening diseases that lead to liver failure and patient death. Although aberrantly activated hepatic stellate cells (HSCs) are the main cause of disease initiation, the symptoms are primarily related to damaged hepatocytes. Thus, damaged hepatocytes, as well as HSCs, need to be simultaneously considered as therapeutic targets to develop more efficient treatments. Here, we suggest cromolyn sodium as an anti-fibrotic agent to commonly modulate hepatocytes and hepatic stellate cells. The differentially expressed genes from 6 normal and 40 cirrhotic liver tissues which were collected from GEO data were assessed by pharmacokinetic analysis using a connectivity map to identify agents that commonly revert abnormal hepatocytes and HSCs to normal conditions. Based on a series of analyses, a few candidates were selected. Candidates were tested in vitro to determine their anti-fibrotic efficacy on HSCs and hepatocytes. Cromolyn, which was originally developed as a mast cell stabilizer, showed the potential to ameliorate activated HSCs in vitro. The activation and collagen accumulation for HSC cell lines LX2 and HSC-T6 were reduced by 50% after cromolyn treatment at a low concentration without apoptosis. Furthermore, cromolyn treatment compromised the TGF-ß-induced epithelial mesenchyme transition and replicative senescence rate of hepatocytes, which are generally associated with fibrogenesis. Taken together, cromolyn may be the basis for an effective cure for fibrosis and cirrhosis because it targets both HSCs and hepatocytes.

In vivo imaging of Lgr5-positive cell populations using confocal laser endomicroscopy during early colon tumorigenesis.

BACKGROUND AND STUDY AIMS: A diagnostic molecular marker for pre-neoplastic lesions, particularly before polyposis, is still lacking. Lgr5 has been broadly accepted as a marker for intestinal cancer stem cells. The aim of this study was to investigate the monitoring of Lgr5( + ) cells as a useful tool for the early diagnosis of premalignant lesions before polyp formation. METHODS: In vivo molecular imaging was performed to examine colon tumorigenesis in Lgr5-EGFP mice treated with azoxymethane and dextran sodium sulfate. eGFP( +) Lgr5( +) regions in the descending colon were longitudinally monitored using side-view confocal endomicroscopy. Based on the eGFP signal intensity on the luminal surface, polyps were classified into two groups - Lgr5-high and Lgr5-low. White light colonoscopy was used to monitor polyp formation. RESULTS: Approximately 75 % of the polyps originated from foci containing Lgr5-eGFP( +) cells, whereas 25 % of the polyps emerged from Lgr5( -) foci. Among eGFP( +) foci, Lgr5-high foci grew faster than Lgr5-low foci. CONCLUSIONS: Polyps developed at Lgr5( +) regions. Luminal Lgr5 expression was correlated with the growth rate of early-stage adenomas. Lgr5 is a promising molecular marker for the early diagnosis of colon tumors.

Early-stage diagnosis of bladder cancer using surface-enhanced Raman spectroscopy combined with machine learning algorithms in a rat model.

Early diagnosis and accurate assessment of tumor development facilitate early bladder cancer resection and initiation of drug therapy. This study enabled an early, accurate, label-free, noninvasive diagnosis of bladder tumors by analyzing nano-biomarkers in a single drop of urine through surface-enhanced Raman spectroscopy (SERS). In a standard N-butyl-N-4-hydroxybutyl nitrosamine-induced rat model of bladder cancer, cancer stage and polyp tumor development were monitored using a small endoscope with a diameter of 1.2 mm in a minimally invasive manner without the need to kill the rats. Samples were divided into cancer-free, early-stage, and polyp-form cancer. Training data were classified according to micro-cystoscopic 5-aminolevulinic acid fluorescence diagnosis, and specimens were postmortem verified through histopathological analysis. A drop of urine from each sample group was placed on an Au-coated zinc oxide nanoporous chip to filter nano-biomaterials and selectively enhance the Raman signals of nanoscale analytes via SERS. Principal component analysis was used to reduce the dimensionality of the collected Raman spectra, and partial least squares discriminant analysis was used to find diagnostic clusters based on the labeled samples. The combination of SERS and machine learning achieved an accuracy ≥99.6% in diagnosing both early- and polyp-stage bladder tumors. With an area under the receiver operating characteristic curve greater than 0.996, the accuracy of the diagnosis in the rat model suggests that SERS-based diagnostic methods are promising when coupled with machine learning. Low-cost, label-free, and noninvasive surface-enhanced Raman spectra are ideal for developing clinically relevant point-of-care diagnostic techniques.

Diagnosis and classification of kidney transplant rejection using machine learning-assisted surface-enhanced Raman spectroscopy using a single drop of serum.

The quest to reduce kidney transplant rejection has emphasized the urgent requirement for the development of non-invasive, precise diagnostic technologies. These technologies aim to detect antibody-mediated rejection (ABMR) and T-cell-mediated rejection (TCMR), which are asymptomatic and pose a risk of potential kidney damage. The protocols for managing rejection caused by ABMR and TCMR differ, and diagnosis has traditionally relied on invasive biopsy procedures. Therefore, a convergence system using a nano-sensing chip, Raman spectroscopy, and AI technology was introduced to facilitate diagnosis using serum samples obtained from patients with no major abnormality, ABMR, and TCMR after kidney transplantation. Tissue biopsy and Banff score analysis were performed across the groups for validation, and 5 µL of serum obtained at the same time was added onto the Au-ZnO nanorod-based Surface-Enhanced Raman Scattering sensing chip to obtain Raman spectroscopy signals. The accuracy of machine learning algorithms for principal component-linear discriminant analysis and principal component-partial least squares discriminant analysis was 93.53% and 98.82%, respectively. The collagen (an indicative of kidney injury), creatinine, and amino acid-derived signals (markers of kidney function) contributed to this accuracy; however, the high accuracy was primarily due to the ability of the system to analyze a broad spectrum of various biomarkers.