Detection of renal biomarkers in chronic kidney disease using microfluidics: progress, challenges and opportunities.

Chronic kidney disease (CKD) typically evolves over many years in a latent period without clinical signs, posing key challenges to detection at relatively early stages of the disease. Accurate and timely diagnosis of CKD enable effective management of the disease and may prevent further progression. However, long turn-around times of current testing methods combined with their relatively high cost due to the need for established laboratory infrastructure, specialized instrumentation and trained personnel are drawbacks for efficient assessment and monitoring of CKD, especially in underserved and resource-poor locations. Among the emerging clinical laboratory approaches, microfluidic technology has gained increasing attention over the last two decades due to the possibility of miniaturizing bioanalytical assays and instrumentation, thus potentially improving diagnostic performance. In this article, we review current developments related to the detection of CKD biomarkers using microfluidics. A general trend in this emerging area is the search for novel, sensitive biomarkers for early detection of CKD using technology that is improved by means of microfluidics. It is anticipated that these innovative approaches will be soon adopted and utilized in both clinical and point-of-care settings, leading to improvements in life quality of patients.

Visible-Near Infrared Spectroscopic Assessment of Urogenital Tissue in Premenopausal and Postmenopausal Women.

BACKGROUND: A clinical study was conducted to evaluate the feasibility of using visible and near-infrared (NIR) spectroscopy as a potential noninvasive measure of genital skin health in premenopausal and postmenopausal women. METHODS: A total of 45 female subjects (aged 21-70 years), all of whom gave fully informed consent to participate, were enrolled in the study and assigned to 1 of 3 groups: 15 premenopausal (Pre-M), 15 postmenopausal receiving hormone replacement therapy (Post-M HRT), and 15 postmenopausal receiving no form of hormone replacement therapy (Post-M non-HRT). Spectral measurements were taken at the vaginal mucosa, and spectral data were evaluated for the erythema index (EI), hemoglobin index (HI), bilirubin/ß-carotene, and melanin. The color index (CI; calculated as the ratio of absorbance at 480 nm/540 nm) was also determined. Results were compared with previously published results on biomarkers and physical characteristic of genital tissue measured on the same groups of women. RESULTS: Spectral measurements from the Post-M Non-HRT subjects indicated a significant reduction in HI compared with the Pre-M group (P = .0003) and to the Post-M HRT group (P < .0001). Similarly, EI was reduced in the Post-M Non-HRT (P < .0001 and P = .0041 for the Pre-M and Post-M HRT groups, respectively). In contrast, the Post-M Non-HRT subjects exhibited a significant increase in ß-carotene compared with the Pre-M subjects (P = .0098). Bilirubin and melanin were not significantly affected. The Post-M Non-HRT group exhibited a significant increase in CI, indicating a shift away from the hemoglobin absorption region (510-620 nm wavelength) and toward the bilirubin/ß-carotene absorption region (450-490 nm wavelength). This change was significant when compared with both the Pre-M group (P < .0001) and the Post-M HRT group (P = .0048). The changes in spectral measurements were consistent with previously reported changes in physical parameters (vaginal atrophy, increased pH, decreased skin temperature) and with decreased concentrations of the biomarkers histamine and histidine. CONCLUSIONS: Hemodynamic spectral characteristics differ in postmenopausal vaginal tissue compared with tissue in premenopausal women, with decreased absorbance in the hemoglobin absorption region (510-620 nm wavelength) and an increased absorbance in the bilirubin/ß-carotene absorption region (450-490 nm wavelength). A change in absorbance in the visible and NIR wavelengths is a promising, additional measure of genital skin health related to menopause and vulvovaginal atrophy.

Assessment of tissue oxygenation of periodontal inflammation in patients with coronary artery diseases using optical spectroscopy.

BACKGROUND: We have recently developed a non-invasive periodontal diagnostic tool that was validated in periodontitis patients without systemic disorders like coronary artery disease (CAD). The purpose of present study is to verify whether this optical instrument can also be used in periodontitis patients with CAD. METHODS: A total of 62 periodontitis patients with CAD were recruited along with a control group consisting of 59 age and gender matched periodontitis volunteers without systemic disorders. Using a portable optical near-infrared spectrometer, optical spectra were obtained, processed and evaluated from the two groups. A modified Beer-Lambert unmixing model that incorporates a nonparametric scattering loss function was used to determine the relative contribution of deoxygenated hemoglobin (Hb) and oxygenated hemoglobin (HbO2) to the overall spectrum. The balance between tissue oxygen delivery and utilization in periodontal tissues was then assessed. RESULTS: Tissue oxygen saturation was significantly decreased in the periodontitis sites (p < 0.01), compared to the healthy sites in those individuals with CAD. There was a trend towards increased concentration of Hb and decreased concentration of HbO2 from healthy to diseased sites, without statistical significance (p > 0.05). No statistical differences were found in tissue oxygen saturation between the CAD and control groups either in periodontal healthy or inflammatory sites. CONCLUSION: This study supports the hypothesis that optical spectroscopy can determine the periodontal inflammation in patients with certain systemic disorders like CAD. And the overall periodontal oxygenation profiles in CAD patients resemble those in non-CAD individuals either in healthy or inflammatory sites.

Assessment of tissue oxygenation of periodontal inflammation in smokers using optical spectroscopy.

BACKGROUND: We have recently developed a periodontal diagnostic tool that was validated in non-smokers with periodontitis. Tobacco smoking is a recognized risk factor for periodontal diseases that can mask gingival bleeding and lead to a false negative diagnosis. Therefore, the purpose of current study is to further validate this instrument in smokers with periodontal diseases. METHODS: Using a portable optical near-infrared spectrometer, optical spectra were obtained, processed and evaluated from healthy (n = 108), gingivitis (n = 100), and periodontitis (n = 79) sites of 54 systemically healthy smokers. A modified Beer-Lambert unmixing model that incorporates a non-parametric scattering loss function was used to determine the relative contribution of deoxygenated haemoglobin (Hb) and oxygenated haemoglobin (HbO2 ) to the overall spectrum. The balance between tissue oxygen delivery and utilization in periodontal tissues was then assessed. RESULTS: Tissue oxygen saturation was significantly decreased in the gingivitis (p = 0.016) and periodontitis (p = 0.007) sites, compared to the healthy sites. There was a trend towards increased concentration of Hb and decreased concentration of HbO2 from healthy to diseased sites, without statistical significance (p > 0.05). CONCLUSIONS: Optical spectroscopy can determine tissue oxygenation profiles of healthy and diseased sites in smokers. The spectral profile of periodontal sites in smokers generally resembles those from non-smoking patients.

Diabetes-associated periodontitis molecular features in infrared spectra of gingival crevicular fluid.

BACKGROUND: It has been established previously that infrared spectroscopy (IRS) can be used to identify periodontitis-specific molecular signatures in gingival crevicular fluid (GCF) and to confirm clinical diagnoses. This follow-up study is designed to assess whether this novel technique is also able to differentiate diseased from healthy sites in patients with diabetes mellitus (DM) by analyzing the molecular fingerprints embedded in the GCF. METHODS: A total of 65 patients with DM with moderate-to-severe chronic periodontitis (CP) was recruited, and 15 individuals without DM (65 sites) without periodontal diseases were used as control. Clinical examination and GCF samples were taken from a total of 351 sites, including periodontitis (109), gingivitis (115), and healthy (127) sites. Corresponding absorption spectra of GCF samples were acquired and processed, and the relative contributions of key functional groups in the infrared spectra were identified and analyzed. The qualitative assessment of clinical relevance of these GCF spectra was interpreted with multivariate statistical analysis: linear discriminant analysis (LDA). RESULTS: Spectral analysis revealed several molecular signatures representing vibrations in protein (amide I and II), lipid ester, and sugar moieties in the GCF of patients with DM with CP and non-DM controls. The diagnostic accuracy for distinction between healthy and CP sites in patients with DM determined by LDA of GCF spectra was 95.3% for the training set of samples and 87.5% for the validation set. Additional LDA of GCF spectra from healthy sites of non-DM controls and patients with DM revealed 100% diagnostic accuracy for the training set and 86.7% for the validation set. The regions robotically selected by LDA for the two analyses were slightly different in that first LDA identified major regions clustered with the side chain vibrations originating from protein and DNA contents, whereas the second was predominantly the glycation and protein components. CONCLUSION: IRS is a feasible method to differentiate disease-specific molecular signatures in GCF in the presence of DM and to generate a complex biochemical profile of GCF to identify DM-specific spectral features.

Assessment of local hemodynamics in periodontal inflammation using optical spectroscopy.

BACKGROUND: Among the newly emerging diagnostic approaches for periodontitis, optical spectroscopy is a promising complementary diagnostic tool. The objective of this study is to verify the reproducibility of this method at a geographically distinct location (Suzhou, China) to a broader patient population using similar instrumentation to that in a previous report. METHODS: Using a portable optical near-infrared spectrometer, optical spectra were obtained, processed, and evaluated from healthy (n = 62), gingivitis (n = 98), and periodontitis (n = 47) sites from a total of 51 patients. A modified Beer-Lambert unmixing model that incorporates a non-parametric scattering loss function was used to determine the relative contribution of oxyhemoglobin and deoxyhemoglobin to the overall spectrum. The balance between tissue oxygen delivery and oxygen use in periodontal tissues was then assessed. RESULTS: Tissue oxygenation decreased significantly from healthy sites to sites with gingivitis (P <0.01) and between gingivitis and periodontitis (P = 0.015). This is largely caused by a significant increase in deoxyhemoglobin between normal and gingivitis (P <0.01) and a concomitant decrease in oxyhemoglobin between gingivitis and periodontitis (P = 0.02). CONCLUSION: This study supports previous findings that tissue oxygenation as measured by optical spectroscopy is significantly decreased in periodontitis and that optical spectroscopy can simultaneously determine multiple inflammatory indices related to periodontal disease directly in gingival tissues in vivo.

Diabetes-related molecular signatures in infrared spectra of human saliva.

BACKGROUND: There is an ongoing need for improvements in non-invasive, point-of-care tools for the diagnosis and prognosis of diabetes mellitus. Ideally, such technologies would allow for community screening. METHODS: In this study, we employed infrared spectroscopy as a novel diagnostic tool in the prediction of diabetic status by analyzing the molecular and sub-molecular spectral signatures of saliva collected from subjects with diabetes (n = 39) and healthy controls (n = 22). RESULTS: Spectral analysis revealed differences in several major metabolic components - lipid, proteins, glucose, thiocyanate and carboxylate - that clearly demarcate healthy and diseased saliva. The overall accuracy for the diagnosis of diabetes based on infrared spectroscopy was 100% on the training set and 88.2% on the validation set. Therefore, we have established that infrared spectroscopy can be used to generate complex biochemical profiles in saliva and identify several potential diabetes-associated spectral features. CONCLUSIONS: Infrared spectroscopy may represent an appropriate tool with which to identify novel diseases mechanisms, risk factors for diabetic complications and markers of therapeutic efficacy. Further study into the potential utility of infrared spectroscopy as diagnostic and prognostic tool for diabetes is warranted.

An update on novel non-invasive approaches for periodontal diagnosis.

For decades there has been an ongoing search for clinically acceptable methods for the accurate, non-invasive diagnosis and prognosis of periodontitis. There are several well-known inherent drawbacks with current clinical procedures. The purpose of this review is to summarize some of the newly emerging diagnostic approaches, namely, infrared spectroscopy, optical coherence tomography (OCT), and ultrasound. The history and attractive features of these new approaches are briefly illustrated, and the interesting and significant inventions related to dental applications are discussed. The particularly attractive aspects for the dental community are that some of these methods are totally non-invasive, do not impose any discomforts to the patients during the procedure, and require no tissue to be extracted. For instance, multiple inflammatory indices withdrawn from near infrared spectra have the potential to identify early signs of inflammation leading to tissue breakdown. Morphologically, some other non-invasive imaging modalities, such as OCT and ultrasound, could be employed to accurately measure probing depths and assess the status of periodontal attachment, the front-line of disease progression. Given that these methods reflect a completely different assessment of periodontal inflammation, if clinically validated, these methods could either replace traditional clinical examinations for the diagnosis of periodontitis or at least serve as attractive complementary diagnostic tools. However, the potential of these techniques should be interpreted more cautiously given the multifactorial character of periodontal disease. In addition to these novel tools in the field of periodontal inflammatory diseases, other alternative modalities like microbiologic and genetic approaches are only briefly mentioned in this review because they have been thoroughly discussed in other comprehensive reviews.

The influence of nicotine on granulocytic differentiation - inhibition of the oxidative burst and bacterial killing and increased matrix metalloproteinase-9 release.

BACKGROUND: Neutrophils leave the bone marrow as terminally differentiated cells, yet little is known of the influence of nicotine or other tobacco smoke components on neutrophil differentiation. Therefore, promyelocytic HL-60 cells were differentiated into neutrophils using dimethylsulfoxide in the presence and absence of nicotine (3-(1-methyl-2-pyrrolidinyl) pyridine). Differentiation was evaluated over 5 days by monitoring terminal differentiation markers (CD11b expression and formazan deposition); cell viability, growth phase, kinetics, and apoptosis; assessing cellular morphology and ultrastructure; and conformational changes to major cellular components. Key neutrophil effector functions (oxidative burst, bacterial killing, matrix metalloproteinase release) were also examined. RESULTS: Nicotine increased the percentage of cells in late differentiation phases (metamyelocytes, banded neutrophils and segmented neutrophils) compared to DMSO alone (p < 0.05), but did not affect any other marker of neutrophil differentiation examined. However, nicotine exposure during differentiation suppressed the oxidative burst in HL-60 cells (p < 0.001); inhibited bacterial killing (p < 0.01); and increased the LPS-induced release of MMP-9, but not MMP-2 (p < 0.05). These phenomena may be alpha-7-acetylcholine nicotinic receptor-dependent. Furthermore, smokers exhibited an increased MMP-9 burden compared to non-smokers in vivo (p < 0.05). CONCLUSION: These findings may partially explain the known increase in susceptibility to bacterial infection and neutrophil-associated destructive inflammatory diseases in individuals chronically exposed to nicotine.

Biomolecular characterisation of leucocytes by infrared spectroscopy.

Over the last 15 years, infrared (IR) spectroscopy has developed into a novel and powerful biomedical tool that has multiple applications in the field of haematology. By revealing subtle alterations in both the conformation and concentration of key macromolecules, such as DNA, protein and lipids, IR spectroscopy has been employed to investigate multiple aspects of leucocyte physiology. IR spectroscopy has been used, for example, to diagnose and prognose leukaemia; to characterise differentiation and apoptotic processes; to predict drug sensitivity and resistance in leukaemic patients undergoing chemotherapy; to monitor the response of leucocytes to chemotherapy and to perform human leucocyte antigen matching for bone marrow transplant patients. Such studies have provided insight into pathogenic mechanisms underlying specific leucocyte disorders, especially leukaemia. While it is likely to be some considerable time before IR spectroscopy is sufficiently developed to displace the established technologies, IR spectroscopy has the potential to become a valuable analytic tool in basic and clinical haematology.

Quantification of serum apolipoprotein B by infrared spectroscopy.

While the conventional approach to assessing both the risk of coronary artery disease and the adequacy of therapy is LDL cholesterol testing, there is compelling evidence to suggest that apolipoprotein B (apoB) is superior to LDL cholesterol for both of these purposes. However, the measurement of apoB requires techniques that can be expensive and difficult to standardize. The aim of this study was, therefore, to develop a new method, based on infrared (IR) spectroscopy, for the routine quantification of apoB in human serum. A total of 366 serum samples were obtained from patients with various disorders. Small volumes (2 microl) of serum specimens were dried to films, and duplicate IR absorption spectra measured. The reference apoB concentrations were determined separately using a standard method, and the proposed IR method was then calibrated using partial least squares (PLS) regression analysis to quantitatively correlate the IR spectra with the reference results. The apoB concentrations predicted from the IR spectra of serum were highly correlated and in excellent agreement with those determined by the reference method. The correlation coefficient (r) for apoB was 0.94, with the standard error between IR-predicted and reference values was 0.10 g/L. In combination with earlier work demonstrating the accurate determination of LDL-C, HDL-C, total cholesterol, and triglycerides from a single infrared spectroscopic measurement, the addition of accurate apoB determination from the same spectrum makes the method very attractive for laboratory use in the routine evaluation of coronary artery disease risk.

Molecular determination of liver fibrosis by synchrotron infrared microspectroscopy.

Liver fibrosis is an adaptive response to various injuries and may eventually progress to cirrhosis. Although there are several non-invasive methods available to monitor the progression of liver fibrogenesis, they cannot reliably detect fibrosis in its early stages, when the process can be stopped or reversed by removing or eliminating the underlying etiological agent that cause the hepatic injury. In this study, early fibrosis alterations were characterized biochemically, morphologically, and spectroscopically in a rat bile duct ligation (BDL) model. Progressive elevations in serum alanine transaminase (ALT), aspartate transaminase (AST), and bilirubin levels in the BDL rats were found indicating the dynamic deterioration of hepatocellular function. Immunofluorescence microscopy using monoclonal anti-collagen III antibody further revealed abnormal intertwined networks of collagen fibres surrounding the portal areas and extending into the lobules towards the central veins in all BDL samples starting from week one. Synchrotron infrared microspectroscopy of liver sections was exploited to generate false color spectral maps based upon a unique and strong collagen absorption at 1340 cm(- 1), revealing a collagen distribution that correlated very well with corresponding images provided by immunofluorescence imaging. We therefore suggest that infrared microspectroscopy may provide an additional and sensitive means for the early detection of liver fibrosis.

Molecular and chemical characterization of blood cells by infrared spectroscopy: a new optical tool in hematology.

Infrared (IR) spectroscopy has made important contributions to the arena of hematology in the past decade. The normal physiology and pathologic modifications of the three cellular elements in blood, i.e., leukocytes, erythrocytes and platelets, have been thoroughly investigated by this recently emerged optical tool. By revealing subtle alterations in the structures of macromolecules in these blood cells, IR spectroscopy has become an ideal complementary analytical tool to conventional biochemical assays used to diagnose various common hematological disorders. Such traditional assays include molecular structure measurements that determine erythrocyte membrane fluidity and conformational changes, lipid profiling of platelet membranes, as well as assays of leukocyte proliferation and differentiation. IR spectroscopic-based techniques can be used to analyze DNA alterations, secondary structural changes in proteins, and to profile cellular lipids. From a molecular and biomedical perspective, IR spectroscopy has been explored for the diagnosis and prognosis of leukemia and beta-thalassemia, to predict drug sensitivity and resistance in chemotherapy patients, and more recently to examine apoptotic processes in blood cells. These studies have shown great promise in the early identification of drug-resistant patients and the early diagnosis of hematological disorders, especially malignancies. Furthermore, IR spectroscopic-based investigations will enable specific mechanisms underlying hematological disorders to be elucidated by revealing the molecular changes in the blood cells at a very early pathogenesis stage.

Molecular mapping of periodontal tissues using infrared microspectroscopy.

BACKGROUND: Chronic periodontitis is an inflammatory disease of the supporting structures of the teeth. Infrared microspectroscopy has the potential to simultaneously monitor multiple disease markers, including cellular infiltration and collagen catabolism, and hence differentiate diseased and healthy tissues. Therefore, our aim was to establish an infrared microspectroscopy methodology with which to analyze and interpret molecular maps defining pathogenic processes in periodontal tissues. METHODS: Specific key cellular and connective tissue components were identified by infrared microspectroscopy and using a chemical imaging method. RESULTS: Higher densities of DNA, total protein and lipid were revealed in epithelial tissue, compared to the lower percentage of these components in connective tissue. Collagen-specific tissue mapping by infrared microspectroscopy revealed much higher levels of collagen deposition in the connective tissues compared to that in the epithelium, as would be expected. Thus inflammatory events such as cellular infiltration and collagen deposition and catabolism can be identified by infrared microspectroscopy. CONCLUSION: These results suggest that infrared microspectroscopy may represent a simple, reagent-free, multi-dimensional tool with which to examine periodontal disease etiology using entirely unprocessed tissue sections.

Assessment of skin flaps using optically based methods for measuring blood flow and oxygenation.

The objective of this study was to compare two noninvasive techniques, laser Doppler and optical spectroscopy, for monitoring hemodynamic changes in skin flaps. Animal models for assessing these changes in microvascular free flaps and pedicle flaps were investigated. A 2 x 3-cm free flap model based on the epigastric vein-artery pair and a reversed MacFarlane 3 x 10-cm pedicle flap model were used in this study. Animals were divided into four groups, with groups 1 (n = 6) and 2 (n = 4) undergoing epigastric free flap surgery and groups 3 (n = 3) and 4 (n = 10) undergoing pedicle flap surgery. Groups 1 and 4 served as controls for each of the flap models. Groups 2 and 3 served as ischemia-reperfusion models. Optical spectroscopy provides a measure of hemoglobin oxygen saturation and blood volume, and the laser Doppler method measures blood flow. Optical spectroscopy proved to be consistently more reliable in detecting problems with arterial in flow compared with laser Doppler assessments. When spectroscopy was used in an imaging configuration, oxygen saturation images of the entire flap were generated, thus creating a visual picture of global flap health. In both single-point and imaging modes the technique was sensitive to vessel manipulation, with the immediate post operative images providing an accurate prediction of eventual outcome. This series of skin flap studies suggests a potential role for optical spectroscopy and spectroscopic imaging in the clinical assessment of skin flaps.

Infrared spectroscopic identification of beta-thalassemia.

BACKGROUND: The aim of this study was to investigate the potential of infrared (IR) spectroscopy as a fast and reagent-free adjunct tool in the diagnosis and screening of beta-thalassemia. METHODS: Blood was obtained from 56 patients with beta-thalassemia major, 1 patient with hemoglobin H disease, and 35 age-matched controls. Hemolysates of blood samples were centrifuged to remove stroma. IR absorption spectra were recorded for duplicate films dried from 5 microL of hemolysate. Differentiation between the two groups of hemoglobin spectra was by two statistical methods: an unsupervised cluster analysis and a supervised linear discriminant analysis (LDA). RESULTS: The IR spectra revealed changes in the secondary structure of hemoglobin from beta-thalassemia patients compared with that from controls, in particular, a decreased alpha-helix content, an increased content of parallel and antiparallel beta-sheets, and changes in the tyrosine ring absorption band. The hemoglobin from beta-thalassemia patients also showed an increase in the intensity of the IR bands from the cysteine -SH groups. The unsupervised cluster analysis, statistically separating spectra into different groups according to subtle IR spectral differences, allowed separation of control hemoglobin from beta-thalassemia hemoglobin spectra, based mainly on differences in protein secondary structure. The supervised LDA method provided 100% classification accuracy for the training set and 98% accuracy for the validation set in partitioning control and beta-thalassemia samples. CONCLUSION: IR spectroscopy holds promise in the clinical diagnosis and screening of beta-thalassemia.

Reagent-free, simultaneous determination of serum cholesterol in HDL and LDL by infrared spectroscopy.

BACKGROUND: The purpose of this study was to assess the feasibility of infrared (IR) spectroscopy for the simultaneous quantification of serum LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) concentrations. METHODS: Serum samples (n = 90) were obtained. Duplicate aliquots (5 microL) of the serum specimens were dried onto IR-transparent barium fluoride substrates, and transmission IR spectra were measured for the dry films. In parallel, the HDL-C and LDL-C concentrations were determined separately for each specimen by standard methods (the Friedewald formula for LDL-C and an automated homogeneous HDL-C assay). The proposed IR method was then developed with a partial least-squares (PLS) regression analysis to quantitatively correlate IR spectral features with the clinical analytical results for 60 randomly chosen specimens. The resulting quantification methods were then validated with the remaining 30 specimens. The PLS model for LDL-C used two spectral ranges (1700-1800 and 2800-3000 cm(-1)) and eight PLS factors, whereas the PLS model for HDL-C used three spectral ranges (800-1500, 1700-1800, and 2800-3500 cm(-1)) with six factors. RESULTS: For the 60 specimens used to train the IR-based method, the SE between IR-predicted values and the clinical laboratory assays was 0.22 mmol/L for LDL-C and 0.15 mmol/L for HDL-C (r = 0.98 for LDL-C; r = 0.91 for HDL-C). The corresponding SEs for the test spectra were 0.34 mmol/L (r = 0.96) and 0.26 mmol/L (r = 0.82) for LDL-C and HDL-C, respectively. The precision for the IR-based assays was estimated by the SD of duplicate measurements to be 0.11 mmol/L (LDL-C) and 0.09 mmol/L (HDL-C). CONCLUSIONS: IR spectroscopy has the potential to become the clinical method of choice for quick and simultaneous determinations of LDL-C and HDL-C.