Biosensor for Point-of-Care Analysis of Immunoglobulins in Urine by Metal Enhanced Fluorescence from Gold Nanoparticles.

Biosensors are easy-to-use and cost-effective devices that are emerging as an attractive tool, not only in settling diagnosis or in disease monitoring, but also in mass screening tests, a timely topic that impacts on daily life of the whole society. Nanotechnologies lend themselves to the development of highly sensitive devices whose realization has become a very interdisciplinary topic. Relying on the enhancement of the fluorescence signal detected at the surface of patterned gold nanoparticles, we report the behavior of an analytical device in detecting immunoglobulins in real urine samples that shows a limit of detection of approximately 8 µg/L and a linear range of 10-100 µg/L well below the detection limit of nephelometric method, which is the reference method for this analysis. These performances have been reached thanks to an effective surface functionalization technique and can be improved even more if superydrophobic features of the substrate we produce will be exploited. Since the analyte recognition is realized by antibodies the specificity is very high and, in fact, no interference has been detected by other compounds also present in the real urine samples. The device has been assessed on serum samples by comparing IgG concentrations values obtained by the biosensor with those provided by a nephelometer. In this step we found that our approach allows the analysis of the whole blood without any pretreatment; moreover, it is inherently extendable to the analysis of most biochemical markers in biological fluids.

Immune Checkpoint Human Endogenous Retrovirus-H Long Terminal Repeat-Associating Protein 2 is Upregulated and Independently Predicts Unfavorable Prognosis in Bladder Urothelial Carcinoma.

BACKGROUND/AIMS: human endogenous retrovirus-H long terminal repeat-associating protein 2 (HHLA2) is highly expressed in multiple solid malignant tumors, making it a potential biomarker for tumorigenesis and invasion. However, the expression and clinical significance of HHLA2 in bladder urothelial carcinoma (BUC) have not been extensively studied. This study aimed to investigate the relationship between HHLA2 expression and clinicopathological characteristics of BUC. METHODS: A total of 212 patients pathologically diagnosed with BUC were included in this study. HHLA2 expression was analyzed by immunohistochemical staining and qRT-polymerase chain reaction. Correlations of HHLA2 expression and pathological characteristics, including 5-year recurrence-free survival (RFS) and overall survival (OS) were examined, and the diagnostic value of HHLA2 was estimated by using the receiver operating characteristic (ROC) curve. RESULTS: Immunohistochemical staining showed that the expression of HHLA2 was significantly upregulated in BUC tissues compared with normal bladder tissues. In BUC tissues, HHLA2 expression was significantly associated with tumor size, tumor stage, tumor grade, and lymph node metastasis (all p < 0.05). HHLA2 expression was an independent prognostic factor of tumor metastasis (p < 0.05). The Kaplan-Meier survival curve revealed that high HHLA2 expression was significantly correlated with the poor RFS and OS of BUC patients (both p < 0.05), and the ROC curve showed HHLA2 could be a good diagnostic marker. CONCLUSIONS: HHLA2 can independently predict unfavorable prognosis in BUC.

Identification of urine protein biomarkers with the potential for early detection of lung cancer.

Lung cancer is the leading cause of cancer-related deaths and has an overall 5-year survival rate lower than 15%. Large-scale clinical trials have demonstrated a significant relative reduction in mortality in high-risk individuals with low-dose computed tomography screening. However, biomarkers capable of identifying the most at-risk population and detecting lung cancer before it becomes clinically apparent are urgently needed in the clinic. Here, we report the identification of urine biomarkers capable of detecting lung cancer. Using the well-characterized inducible Kras (G12D) mouse model of lung cancer, we identified alterations in the urine proteome in tumor-bearing mice compared with sibling controls. Marked differences at the proteomic level were also detected between the urine of patients and that of healthy population controls. Importantly, we identified 7 proteins commonly found to be significantly up-regulated in both tumor-bearing mice and patients. In an independent cohort, we showed that 2 of the 7 proteins were up-regulated in urine samples from lung cancer patients but not in those from controls. The kinetics of these proteins correlated with the disease state in the mouse model. These tumor biomarkers could potentially aid in the early detection of lung cancer.

High incidence of intact or fragmented immunoglobulin in urine of patients with multiple myeloma.

In this prospective study we determined the incidence of intact/fragmented immunoglobulin and Bence Jones protein in urine immunofixation using Sebia reagents and HydrasysTM 2 apparatus and compared the results to concentrations of serum free light chains (FLC) assessed using Siemens BNTM II nephelometer and the immunoassay Freelite (Binding Site) in 289 patients with multiple myeloma at diagnosis. It was found that in one third of IgG, IgA and IgD myeloma patients, intact/fragmented immunoglobulin can be detected in urine and is connected with impaired renal function and reduced survival. Urine immunofixation detects monoclonal protein (FLC and intact/fragmented immunoglobulin) in 66-79% of IgG and IgA myeloma patients while serum FLC immunoassay detect it in 82-94% of IgG and IgA myeloma patients. However, the latter method is inadequate for detection of intact/fragmented immunoglobulin in urine. Serum FLC immunoassay and urine immunofixation are complementary methods in diagnosing and monitoring monoclonal protein in patients with myeloma.

Clinicopathological features of renal amyloidosis: a single-center study on 47 cases.

The correlations between the clinicopathological features and the long-term outcomes of renal amyloidosis (RA) were analyzed with a view to develop strategies for improving diagnosis and prognosis of RA. We retrospectively reviewed the clinicopathological characteristics of 47 patients diagnosed with RA between 2004 and 2014 at the Wuhan Union Hospital. The data on the renal histology, clinical manifestations, and prognosis of RA patients were retrieved from the hospital records and characteristic patterns were identified. The histological changes in the kidneys were correlated with the clinical manifestations of RA. Additionally, most RA patients in this study had decreased serum levels of κ light chain and increased urine levels of κ and λ light chains as well as presence of M-protein in the urine and serum. Patients with early RA showed no specific pathognomonic symptoms. Bleeding associated with diagnostic renal biopsy was rare. We recommend that the routine work-up of patients aged over 40 years and presenting with non-diabetic nephropathy includes the non-invasive tests for the measurement of serum and urine levels of κ and λ light chains as well as protein electrophoresis tests for the presence of urinary and serum M-protein. Additionally, such patients should undergo renal biopsy screening with Cong-red staining to ensure early diagnosis of RA and improve their survival, since the risk of hemorrhage related to renal biopsy screening is low at early stages of RA.

Diagnosis of monoclonal gammopathy of renal significance.

Monoclonal gammopathy of renal significance (MGRS) regroups all renal disorders caused by a monoclonal immunoglobulin (MIg) secreted by a nonmalignant B-cell clone. By definition, patients with MGRS do not meet the criteria for overt multiple myeloma/B-cell proliferation, and the hematologic disorder is generally consistent with monoclonal gammopathy of undetermined significance (MGUS). However, MGRS is associated with high morbidity due to the severity of renal and sometimes systemic lesions induced by the MIg. Early recognition is crucial, as suppression of MIg secretion by chemotherapy often improves outcomes. The spectrum of renal diseases in MGRS is wide, including old entities such as AL amyloidosis and newly described lesions, particularly proliferative glomerulonephritis with monoclonal Ig deposits and C3 glomerulopathy with monoclonal gammopathy. Kidney biopsy is indicated in most cases to determine the exact lesion associated with MGRS and evaluate its severity. Diagnosis requires integration of morphologic alterations by light microscopy, immunofluorescence (IF), electron microscopy, and in some cases by IF staining for Ig isotypes, immunoelectron microscopy, and proteomic analysis. Complete hematologic workup with serum and urine protein electrophoresis, immunofixation, and serum-free light-chain assay is required. This review addresses the pathologic and clinical features of MGRS lesions, indications of renal biopsy, and a proposed algorithm for the hematologic workup.

Current treatment landscape for relapsed and/or refractory multiple myeloma.

Recent developments in the treatment of multiple myeloma have led to improvements in response rates and to increased survival; however, relapse is inevitable in almost all patients. Recurrence of myeloma is typically more aggressive with each relapse, leading to the development of treatment-refractory disease, which is associated with a shorter survival. Several phase II and III trials have demonstrated the efficacy of recently approved agents in the setting of relapsed and/or refractory multiple myeloma, including immunomodulatory agents, such as lenalidomide and pomalidomide, and proteasome inhibitors, such as bortezomib and carfilzomib. Currently, however, there is no standard treatment for patients with relapsed and/or refractory disease. This Review discusses the current treatment landscape for patients with relapsed and/or refractory multiple myeloma and highlights disease-related and patient-related factors--such as pre-existing comorbidities or toxicities--that are important considerations for clinicians when selecting an appropriate treatment regimen.

Response evaluation and monitoring of multiple myeloma.

Monitoring multiple myeloma (MM) is essential during the evaluation of response to each therapy line, after transplantation and at the time of relapse or progression in all patients. An initial complete workup, including appropriate protein studies in serum and urine is mandatory. The use of uniform criteria is particularly important in the context of clinical trials. Complete remission (CR) definition, the goal for the majority of patients, is now in constant evolution, with immunophenotypic and molecular minimal residual disease measurement in bone marrow as well as imaging techniques. Identification of relapse/progression with traditional and novel techniques for eventual prompt intervention with rescue treatment is a current issue of debate.

Immunoglobulin light chain amyloidosis.

Primary light chain amyloidosis is the most common form of systemic amyloidosis and is caused by misfolded light chains that cause proteotoxicity and rapid decline of vital organ function. Early diagnosis is essential in order to deliver effective therapy and prevent irreversible organ damage. Accurate diagnosis requires clinical skills and advanced technologies. The disease can be halted and the function of target organs preserved by the prompt reduction and elimination of the plasma cell clone producing the toxic light chains in the bone marrow. Heart damage is the major determinant of survival, and staging with cardiac biomarkers guides treatment. Two-thirds of patients can benefit from treatment with improved quality of life and extended survival. Future efforts should be directed at early diagnosis, improving the tolerability and efficacy of anti-plasma cell therapy, accelerating recovery of organ function via promoting resorption of amyloid deposits, and developing novel approaches to counter light chain proteotoxicity.

The utility of isotope-coded protein labeling for prioritization of proteins found in ovarian cancer patient urine.

Urine offers a number of attractive features as a sample type for biomarker discovery, including noninvasive sampling, quantity and availability, stability, and a narrow dynamic range. In this study we report the first application of isotope coded protein labeling (ICPL), coupled with in-solution isoelectric fractionation and LC-MALDI-TOF/TOF, to examine and prioritize urinary proteins from ovarian cancer patients. Following the definition of stringent exclusion criteria a total of 579 proteins were identified with 43% providing quantitation data. Protein abundance changes were validated for selected proteins by ESI-Qq-TOF MS, following which Western blot and immunohistochemical analysis by tissue microarray was used to explore the biological relevance of the proteins identified. Several established markers (e.g., HE4, osteopontin) were identified at increased levels in ovarian cancer patient urine, validating the approach used; we also identified a number of potential marker candidates (e.g., phosphatidylethanolamine binding protein 1, cell-adhesion molecule 1) previously unreported in the context of ovarian cancer. We conclude that the ICPL strategy for identification and relative quantitation of urine proteins is an appropriate tool for biomarker discovery studies, and can be applied for the selection of potential biomarker candidates for further characterization.

Diagnosis and inflammatory response of patients with candiduria.

Candiduria is common in hospitalised patients, but the clinical relevance is still unclear. This study was done to further our knowledge on detection of and host responses to candiduria. Urines and clinical data from 136 patients in whom presence of yeast was diagnosed by microscopic urinalysis were collected. Diagnosis by standard urine culture methods on blood and MacConkey agar as well as on fungal culture medium (Sabouraud dextrose agar) was compared. Inflammatory parameters (IL-6 and IL-17, Ig) were quantified in the urine and compared with levels in control patients without candiduria. Standard urine culture methods detected only 37% of Candida spp. in urine. Sensitivity was especially low (23%) for C. glabrata and was independent of fungal burden. Candida specific IgG but not IgA was significantly elevated when compared with control patients (P < 0.0001 and 0.07 respectively). In addition, urine levels of IL-6 and IL-17 were significantly higher in candiduric patients when compared with control patients (P < 0.001). Multivariate analysis documented an independent association between an increased IgG (odds ratio (OR) 136.0, 95% confidence interval (CI) 25.7-719.2; P < 0.0001), an increased IL-17 (OR 17.4, 95% CI 5.3-57.0; P < 0.0001) and an increased IL-6 level (OR 4.9, 95% CI 1.9-12.4; P = 0.001) and candiduria. In summary, our data indicate that clinical studies on candiduria should include fungal urine culture and that inflammatory parameters may be helpful to identify patients with clinically relevant candiduria.

Elevated urinary excretion of immunoglobulins in nonproteinuric patients with type 1 diabetes.

Increased albuminuria is a hallmark of early diabetic nephropathy, whereas the role of immunoglobulins (Igs), such as IgG (its 1-4 subtypes), IgA, and IgM, different in charge and size, has not been examined in early nephropathy in the past due to lack of a sensitive and reliable method. Our study group consisted of subjects with type 1 diabetes (T1D) and normoalbuminuria (n = 78), microalbuminuria (n = 78), and of 75 healthy subjects (HS). A Luminex-based immunoassay (1,000 time more sensitive than nephelometry-based method) was validated for the urine matrix and used for the measurements of IgG1-4, IgA, and IgM in our study groups. The Luminex-based assay detected Igs in 87% of HS subjects and in 100% of T1D subjects. Recovery of known amounts of Igs added to urine was 92-118%. In the normoalbuminuria group, urinary concentrations of albumin, IgG2, IgA, and IgM were significantly higher than in HS, whereas in the microalbuminuria, further elevation of IgG2, IgG4, and IgA was the most pronounced. In all three groups, fractional excretion of Igs was at least 100 times lower than that of albumin. Fractional excretion of IgG2 was the highest among all Igs. We validated a sensitive method for measuring Igs in urine using Luminex. We found that elevated concentrations of Igs, particularly in IgG2 and IgA, is present in subjects with T1D and no proteinuria. Elevation of those particular Ig subtypes suggests a contribution of novel mechanisms in early diabetic nephropathy, different from charge and size barrier impairment.

Diagnostic values of parasite-specific antibody detections in saliva and urine in comparison with serum in opisthorchiasis.

Infection by the liver fluke (Opisthorchis viverrini) causes hepatobiliary disease and bile duct cancer (cholangiocarcinoma, CCA) in endemic areas in Southeast Asia. Measurements of humoral immune response particularly parasite-specific antibodies are useful not only for serodiagnosis but they have been implicated as risk factors of CCA. In this study, we used indirect Enzyme Immunosorbent Assay (ELISA) to measure O. viverrini-specific immunoglobulins in serum, urine and saliva and assessed efficacies in diagnosis of opisthorchiasis and evaluated the relationship of antibodies among clinical specimens in a sample population in endemic areas in Khon Kaen, Thailand. By employing the Receiver Operation Characteristics (ROC) analysis, diagnostic efficacy based upon the area under the curve (AUC) revealed that serum, salivary IgG and IgA performed better than urine for diagnosis of opisthorchiasis. Seropositive cases were found in both parasite egg-negative as well as O. viverrini egg-positive groups. The levels of serum IgG correlated with intensity of O. viverrini infection (P<0.05). Diagnostic sensitivities based on serum and salivary IgG, IgA also positively associated with the intensity of infection. Correlations between serum antibodies and those in saliva were found to be greater in egg-negative than egg-positive individuals for O. viverrini. Our findings indicated a complex interrelation between antibody responses in different clinical specimens triggered by liver fluke infection. More comprehensive examinations are needed to determine the potential utility of salivary antibody detection which, in combination with the conventional fecal examination method, may better assist in the identification of individuals with opisthorchiasis. Furthermore, it may provide a better indicator of the risk of disease, particularly CCA.

Long-term biological variation of serum protein electrophoresis M-spike, urine M-spike, and monoclonal serum free light chain quantification: implications for monitoring monoclonal gammopathies.

BACKGROUND: We analyzed serial data in patients with clinically stable monoclonal gammopathy to determine the total variation of serum M-spikes [measured with serum protein electrophoresis (SPEP)], urine M-spikes [measured with urine protein electrophoresis (UPEP)], and monoclonal serum free light chain (FLC) concentrations measured with immunoassay. METHODS: Patients to be studied were identified by (a) no treatment during the study interval, (b) no change in diagnosis and <5 g/L change in serum M-spike over the course of observation; (c) performance of all 3 tests (SPEP, UPEP, FLC immunoassay) in at least 3 serial samples that were obtained 9 months to 5 years apart; (d) serum M-spike ≥10 g/L, urine M-spike ≥200 mg/24 h, or clonal FLC ≥100 mg/L. The total CV was calculated for each method. RESULTS: Among the cohort of 158 patients, 90 had measurable serum M-spikes, 25 had urine M-spikes, and 52 had measurable serum FLC abnormalities. The CVs were calculated for serial SPEP M-spikes (8.1%), UPEP M-spikes (35.8%), and serum FLC concentrations (28.4%). Combining these CVs and the interassay analytical CVs, we calculated the biological CV for the serum M-spike (7.8%), urine M-spike (35.5%), and serum FLC concentration (27.8%). CONCLUSIONS: The variations in urine M-spike and serum FLC measurements during patient monitoring are similar and are larger than those for serum M-spikes. In addition, in this group of stable patients, a measurable serum FLC concentration was available twice as often as a measurable urine M-spike.

[Dynamics of the protein content of human urine during 5-day immersion].

Variations in the protein spectrum (12 groups) of native daily urine were studied during 5-day dry immersion (DI) of 14 subjects at the age of 19 to 26 years using gradient electrophoresis in polyacrylamide gel. Protein excretion with urine did not alter in the course of the experiment. However, the urine proteins spectrum trended to some shifts. Excretion of low-molecular proteins decreased and of albumin increased pointing to remodulation of tubular reabsorption initiated by the experimental conditions. Steady growth of the Tamm-Horsfall protein concentration seems to be a defense reaction. There was an incremental decrease in immunoglobulins that could be associated with a reduction of glomerular filter permeability for high-molecular proteins.

The value of IgG-uria in predicting renal failure in idiopathic glomerular diseases. A long-term follow-up study.

BACKGROUND: Proteinuria is the hallmark of glomerular disease and non-selective proteinuria is often associated with progression to renal failure. The predictive value of urine IgG excretion was studied comprehensively in patients with nephrotic syndrome. In the present follow-up study, we examine the predictive value of IgG-uria in patients with idiopathic glomerular diseases with a wide range of proteinuia. METHODS: A total of 189 (113 males and 76 females) patients with idiopathic glomerulonephritis and serum creatinine of less than 150 µmol/L diagnosed between 1993 and 2004 were followed up to their last visit in 2009. Measurement of urine excretion of albumin, IgG, and protein HC were performed in the early morning of spot urine samples collected at the time of the diagnostic renal biopsy. Patients were stratified according to urine protein concentrations and the progression rate to end-stage renal disease (ESRD) calculated using Kaplan-Meier survival analysis. ESRD was defined as the start of renal replacement therapy. RESULTS: During the study follow-up time of 1429 person-years; 26 (13.8%) patients reached ESRD. The overall mean kidney survival time of studied patients with serum creatinine less than 150 were 13.4 years. The incidence rate of ESRD was ∼18 per 1000 person-years. Stratified analysis identified urinary excretion of IgG, but not albuminuria, as predictor of ESRD. The progression rate to ESRD was 36 per 1000 person-years in patients with urine IgG concentration exceeding 5 mg/mmol urine creatinine, compared to a progression rate of 6/1000 person-years for patients with lower levels of urine IgG. CONCLUSION: The findings of the study suggest that at early stages, the level of IgG-uria is useful to be used in risk stratification of patients with proteinuric glomerular diseases.

Falsos negativos en la detección de componentes monoclonales por electroforesis capilar / False negatives in the analysis of monoclonal components by capillary electrophoresis

La electroforesis capilar es una técnica rápida y automatizada que permite la detección de componentes monoclonales en suero con alta sensibilidad, estando ampliamente instaurada en los laboratorios clínicos. Se han descrito sin embargo, falsos positivos y negativos que plantean problemas a la hora de interpretar el trazado electroforético. Los falsos positivos son debidos fundamentalmente a sustancias exógenas no proteicas presentes en la muestra que absorben radiación en la región ultravioleta dando lugar a picos anómalos en el proteinograma. Los falsos negativos se deben en su mayoría a la baja concentración del componente monoclonal, aunque se han descrito también cuando se encuentran en alta concentración, debidos principalmente a las propiedades físico-químicas de la paraproteína que provocan una separación incorrecta de la misma. Presentamos una serie de casos con componentes monoclonales de tipo IgA e IgM, detectados inicialmente por electroforesis de acetato de celulosa y presentes en la muestra en alta concentración, en los que la electroforesis capilar tuvo problemas para su detección (AU)

Capillary electrophoresis is a fast, automated and highly sensitive technique for the detection of monoclonal components in serum that is being increasingly introduced in clinical laboratories. Nevertheless, false negative and positive results have been reported, and thus the electrophoretic pattern may be difficult to interpret. False positive results are chiefly due to exogenous substances other than proteins present in serum, which absorb at UV wavelengths and can produce an abnormal spike. False negative results are mainly due to very low concentrations of monoclonal components. However, high concentration monoclonal components may not be correctly detected due to the physicochemical properties of the paraproteins, which may cause anomalous separation. In this work, we study three cases with high concentration monoclonal components of the IgA and IgM classes. They were initially detected on cellulose acetate electrophoresis, but the capillary electrophoresis was not able to correctly detect them (AU)