Urinary Sediment Cells Research Articles

Maternal germline mosaicism in Fabry disease.

Fabry disease (FD) is an X-linked monogenic disorder caused by mutations in the GLA gene which leads to a deficiency of the functionally active lysosomal α-galactosidase A enzyme. Here, we report on a family of five members: unaffected parents, one unaffected son, and another son and daughter both carrying the same mutation (p.G138E) in the GLA gene. Genotype analysis using intragenic GLA markers confirmed the maternal origin of the mutation. The affected son and daughter carried the same mutation; however, it was not detected in the peripheral blood, buccal cells, and urinary sediment cells of their mother. Moreover, the unaffected son without the alteration in the GLA gene carried the same maternal chromosome X (disease-associated) haplotype. To the best of our knowledge, this study represents the first case of maternal germline mosaicism in FD.

Von Hansemann Cells from Fresh Urine Sediment Samples in the Diagnosis of Malakoplakia.

Background:Malakoplakia is characterized by the presence of plaques with macrophages containing inclusion bodies. The diagnosis of this disease is carried out by biopsy of the lesion. The objective of this paper was to assess the value of fresh urine sediment in the diagnosis of malakoplakia.Materials and Methods:Five suspected cases of malakoplakia that showed macrophages with inclusions called bodies of Michaelis-Gutmann (von Hansemann cells) in unstained urine sediment were processed with Papanicolaou, Giemsa, and periodic acid-Schiff (PAS) stains. Four of the five patients had a history of cystitis and had developed antibiotic resistance. The other patient had the characteristics cells in a routine urinalysis.Results:Papanicolaou stain revealed intracytoplasmic eosinophilic or basophilic bodies, single or multiple in macrophages. Such bodies were stained deep red with PAS technique. Giemsa stain showed these bodies with a faint basophilic coloration, sometimes with a central core. Bladder biopsies established the definitive diagnosis, showing bodies within and outside macrophages, with a concentric “birds-eye” or “owl-eye” (targetoid) appearance.Conclusions:Finding of von Hansemann cells in fresh urine sediment of patients with cystitis and a history of resistance to antibiotic scan leads to the diagnosis of malakoplakia. Giemsa stain can show in some cases the characteristic central core of Michaelis-Gutmann bodies. Malakoplakia is probably the result of an acquired defect in macrophage function causing impairment of bactericidal activity. A correct diagnosis is important because the spread to ureters with bilateral stenosis and obstruction can lead to kidney failure.

Henoch-schonlein Purpura Nephritis with Renal Interstitial Lesions.

ObjectiveTo investigate the clinical pathology and prognosis & outcome of Henoch-Schonlein purpura nephritis (HSPN) with renal interstitial lesions.MethodsAll 148 patients were analyzed for clinical, renal pathological, and prognostic features. Patients with no, mild, and moderate- severe renal tubulo-interstitial lesions were included in group A, B and C, respectively.ResultsThe estimated glomerular filtration rate (eGFR) of group B was significantly lower than that of group A. The levels of serum creatinine and blood urea nitrogen in group C were significantly higher than those in groups A and B. Clinical type II was correlated with pathological types II and IIIa; pathological type IV and IIIb were correlated with clinical type VI and IV. There were significant differences in the level of red blood cells in urinary sediment, levels of urine occult blood and in the prognosis among the 3 groups.ConclusionClinically, Type II is the most common cause; pathologically, Type IIIa is more common. The severity of renal tubulo-interstitial lesions is positively correlated with a decline in renal function and GFR. There is a correlation between the severity of renal tubulo-interstitial lesions and the severity of hematuria. Most patients with HSPN have a good prognosis.

Using Genetic and Epigenetic Markers to Improve Differential Diagnosis of Prostate Cancer and Benign Prostatic Hyperplasia by Noninvasive Methods in Mexican Patients.

Prostate cancer (PCa) is the most common malignancy in Mexican men. Serum prostate-specific antigen (PSA) is the usual noninvasive biomarker used for its detection. Its low specificity can increase the number of unnecessary prostate biopsies and the incidence of unpleasant complications for patients. The androgen-receptor gene (AR-CAG) repeat length and the percentage of promoter methylation (PPM) of genes glutathione-S-transferase P1 (GSTP1) and Ras association domain family 1 isoform A (RASSF1A) improve PCa detection. As an option for noninvasive assessment, we evaluated a combined analysis of all these biomarkers. A total of 186 patients scheduled for biopsy were included in the present study. PSA and AR-CAG repeats were analyzed in blood samples. The PPM of GSTP1 and RASSF1A genes was estimated in prostate tissue and urinary sediment cells (USCs) and plasma DNA using quantitative methylation-specific polymerase chain reaction. The predictive values for PCa and benign prostatic hyperplasia (BPH), logistic regression analysis, receiver operating characteristic curve, and decision curve analysis were used to assess the differential diagnosis. Statistically significant differences between PCa and BPH patients were observed for all biomarkers, with higher positive and negative predictive values when all biomarkers were included in the analysis, attaining USC values of 89.2% and 78.0%, respectively. The differential diagnosis accuracy of PSA (area under the curve, 0.59) increased to 0.70 and 0.68, respectively, when the combined analysis of PPM of RASSF1Aplasma or GSTP1AUSC and AR-CAG repeats was performed. Decision curve analysis showed the utility of the combined analysis to decrease the number of unnecessary biopsies. The results showed that combined analysis of the proposed biomarkers in plasma and USCs significantly increased the confidence for the differential diagnosis for PCa and BPH. This noninvasive practice might help in the early detection of PCa and patient follow-up, avoiding to some extent unnecessary prostate biopsies.

Prediction of Early BK Virus Infection in Kidney Transplant Recipients by the Number of Cells With Intranuclear Inclusion Bodies (Decoy Cells).

BackgroundBK virus (BKV) is the cause of nephropathy. Because BKV nephropathy can progress to graft loss, early diagnosis of BKV infection is very important. In this study, we aimed to investigate the utility of quantifying cells with intranuclear inclusion bodies (decoy cells) in urinary sediment for the screening and monitoring of BKV infection in renal transplant recipients at our hospital.MethodsThis was a retrospective single-center study. Urine sediment examination was performed at each outpatient visit, and the number of decoy cells was measured in the whole microscopic field. Patients (n = 41) were divided into the BK viremia group (blood positive for BKV DNA by polymerase chain reaction [PCR]) and non-BK viremia group (blood negative for BKV DNA by PCR), and the decoy cell count in urinary sediments was examined.ResultsThe maximum decoy cell count was significantly higher (P = 0.04) in the BK viremia group than in the non-BK viremia group. In the receiver operating characteristic curve for the maximum decoy cells, the cutoff value was 507 cells. The area under the receiver operating characteristic curve was 0.8774 (95% confidence interval, 0.7739-0.9810). The number of decoy cells at the time of appearance in the BK viremia group was not significantly different from that in the non-BK viremia group. However, the BK viremia group showed an increasing trend, whereas the non-BK viremia group showed a decreasing trend, in the number of decoy cells. There was a positive correlation between the number of decoy cells and the data from the urine BKV-DNA PCR quantification (correlation coefficient [r] = 0.74).ConclusionsMeasurement of decoy cells in urinary sediments may predict early BKV infection, and if performed quickly, it may be useful for screening and continuous monitoring of BKV infection in renal transplant recipients.

Determination of red blood cells in urinary sediment: Do pH and specific gravity of urine matter?

It has been postulated that erythrocyte cell lysis in urine is common in the case of low urine specific gravity and high urine pH. We aimed to verify the influence of urine dipstick pH and specific gravity on erythrocyturia in the urine sediment in the case of positive dipstick hemoglobinuria. We retrospectively collected data on dipstick specific gravity, pH, and urine sediment analysis done by nephrologists in the clinical and research urine laboratory at the Department of Nephrology, University Medical Center Ljubljana. During the 6-year observation period, we analyzed 843 second morning midstream urine samples with positive dipstick hemoglobinuria. Erythrocyturia in urinary sediment was detected significantly less often in urine samples with concomitant hemoglobinuria 1+ and pH < 6.0, (odds ratio (OR) 0.40; 95% confidence interval (CI) 0.21-0.76, p = 0.005). The difference was maintained in multivariate analysis including patient age, gender, and specific gravity (OR 0.32; 95% CI 0.15-0.65, p = 0.002). In samples with higher grade of hemoglobinuria (≥2+), the impact of cell lysis in the case of low pH was negligible. Specific gravity did not have any influence on erythrocyte detection in urinary sediment. Urinary pH <6.0 impaired the detection of erythrocytes in urinary sediment, while the dipstick measured urine specific gravity did not have any impact on it. Urine samples with low-grade hemoglobinuria and low pH without erythrocyte detection in urinary sediment should be evaluated again to avoid false-negative results. .

Enhancing the Detection of Dysmorphic Red Blood Cells and Renal Tubular Epithelial Cells with a Modified Urinalysis Protocol

Urinary sediment is used to evaluate patients with possible urinary tract diseases. Currently, numerous protocols are applied to detect dysmorphic red blood cells (RBCs) and renal tubular epithelial cells (RTECs) in urinary sediment. However, distinct protocols are used by nephrologists and medical technologists for specimen concentration and observation, which leads to major discrepancies in the differential counts of formed elements such as dysmorphic RBCs and RTECs and might interfere with an accurate clinical diagnosis. To resolve these problems, we first tested a modified urinalysis protocol with an increased relative centrifuge force and concentration factor in 20 biopsy-confirmed glomerulonephritis patients with haematuria. We successfully improved the recovery ratio of dysmorphic RBCs in clinical specimens from 34.7% to 42.0% (P < 0.001). Furthermore, we confirmed the correlation between counts by the modified urinary protocol and Sysmex UF-1000i urinary flow cytometer (r ≥ 0.898, P < 0.001). A total of 28 types of isomorphic and dysmorphic RBCs were detected using a bright field microscope, with results comparable to those using a standard phase contrast microscope. Finally, we applied Sternheimer stain to enhance the contrast of RTECs in the urinary sediments. We concluded that this modified urinalysis protocol significantly enhanced the quality of urinalysis.

PHENOTYPIC FEATURES OF CELLS IN URINARY SEDIMENT OF PATIENTS WITH NON-INVASIVE BLADDER CANCER

More than 430,000 new cases of bladder cancer are diagnosed every year in the world, and approximately 70–80 % of patients present with non-invasive disease. To improve the effectiveness of early detection of bladder cancer, high sensitivity diagnostic tools should be used. In our study we used flow cytometry to determine surface markers in urinary sediment cells of patients with bladder cancer. The analysis of the phenotypic features of urinary sediment cells showed an increase in the relative content of CD13+ cells in patients with non-invasive bladder cancer in comparison with the control group. In addition, there was a 4.8-fold increase in cells expressing CD15 surface antigen (p<0.001) and 4.9-fold increase in cells expressing CD45 surface antigen (p<0.001). Considering the fact, that most patients with bladder cancer develop recurrence after treatment, we studied the relationship between the expression level of surface markers of urinary sediment cells and the development of disease recurrence. The absence of differences in the level of CD15+ cells in urinary sediment of patients with recurrent bladder cancer can be explained by the absence of differences in proliferative activity of recurrent and non-recurrent tumors. The higher level of CD13+ cells in patients with recurrent bladder cancer indicates greater depth of tumor invasion. It is obvious that the deeper the tumor invasion into the submucosal layer of the bladder, the greater the likelihood of non-radical transurethral resection and the development of disease recurrence. Thus, the analysis of the level of CD13+ and CD45+ cells in the urinary sediment can provide information on tumor invasion into the bladder submucosal layer and the risk of bladder cancer recurrence.

Reduced expression of E-cadherin and increased sialylation level in clear cell renal cell carcinoma.

Cancer cells are characterized by an aberrant increase in protein N-glycosylation and by disruption of E-cadherin-mediated adherens junctions. However, the relationship between alterations in N-glycosylation process and loss of E-cadherin adhesion in cancer remains unclear. The mechanisms of altered expression of adhesive glycoproteins in cancer cells have not been fully elucidated. Thus, the aim of this study was to examine the expression of E-cadherin and sialyl Lewisa/x, NeuAcα2-3Gal, NeuAcα2-6Gal/GalNAc structures in the normal renal tissue and intermediate and cancerous tissues from patients with clear cell RCC. Moreover, we attempted to correlate the E-cadherin expression with some specific sugar residues of renal cancer tissue glycoproteins. The expression of E-cadherin was analysed using ELISA test and immunoblotting. Oligosaccharide structures and sialylation level were detected with ELISA test using specific biotinylated lectins or antibodies. A significant decrease of E-cadherin expression as well as a significant increase in sialylated oligosaccharides level in intermediate zone and renal cancer tissue in comparison to normal renal tissue are reported. Significant decrease in expression of cadherins and increase in sialylation of oligosaccharide structures in renal cancer tissue in comparison to normal renal tissue, and in renal cancer tissue in comparison to intermediate zone of renal tissue, are important for the future research concerning detection and quantification of cadherins and sialylated oligosaccharide structures in urine and cells of urinary sediment as possible non-invasive marker of early RCC.

Diagnostic pitfall of carryover: in automatic urine analyzers

Abstract Objective We aimed to find out whether there is significant carryover effect which causes false-positive hematuria on red blood cells (RBCs) in automatic urine chemistry (DIRUI H-800) and sediment (DIRUI FUS-200) analyzers. Methods Twenty-four samples with gross hematuria selected as containing high RBC concentration and forty-eight samples which had both negative result in dipstick and 0/hpf in microscopic examination selected as containing low RBC concentration. Carryover% was calculated via the formula [carryover%=100×(b1−b2)/(a2−b2)]. Carryover effect within results was analyzed with Wilcoxon test. Results Carryover% was very high (67%) in urine chemistry analyzer. Carryover% of urine sediment analyzer was found 0.4% whilst false-positive hematuria percentage was 87.5% for the first samples came after gross hematuria and 6.6% for the second samples. The first samples analyzed after gross hematuria had significantly higher (p&lt;0.001) results than the second samples in both analyzers. Conclusion In urine sediment analyzer, carryover% calculated by formula was found analytically sufficient, but it causes highly false-positive results due to diagnostic limit of hematuria (RBC&gt;3/hpf) is low. To prevent carryover in both urine analyzers; washing procedures should be revised and the diagnostic effect of carryover should also be taken into account by biochemists.

Novel round cells in urine sediment and their clinical implications

BackgroundVoided urine contains a variety of cells from the kidney and urinary tract and urinalysis provides us various information by investigating cellular components. We investigated urine sediment from renal impaired patients. ResultsWe found ‘round cell’ to be distinct from known cells in sediment and is close to proximal convoluted tubule-derived cells based on morphology and molecular marker expression (GGT1 but not podocalyxin). Also it was positive for undifferentiated cell markers, including PAX2, WT1, OSR1, and SIX2. They were observed in end-stage renal failure patients and the number of cells was correlated with the severity of chronic kidney disease. A prospective analysis revealed that patients who had more round cells were more likely to require hemodialysis within a year. ConclusionRound cells are a novel marker that can be used to predict the need for hemodialysis.

Establishment of Simple and Routine Methods in Early Diagnosis of Gentamicin-Induced Kidney Injury Based on a Rat Model.

The changes in biomarkers of gentamycin- (GM-) induced kidney injury have been studied by using simple and routine methods and also assessed the efficacy and utility of these routine biomarkers in early diagnosis. Eighty Sprague-Dawley (SD) rats were randomly divided into 4 groups: three experimental groups treated with different GM dosages (4, 20, and 100 mg·kg−1) and a control group. The experimental groups were given intramuscular GM injections once daily for 14 days, and the control group was given intramuscular sterile water. Blood and urine samples were collected on treatment days 1, 3, 7, and 14 to test for total protein (TP), albumin (ALB), blood urea nitrogen (BUN), creatinine (CRE), uric acid (UA), pH, specific gravity (SG), proteins (PRO), and cells in urinary sediment. Histopathology and kidney coefficient were performed on excised kidney specimens. The result indicated that serum CRE, BUN, and TP, urine PRO, and urinary hyaline casts and low-transitional epithelium showed an immediate and highly sensitive response to kidney injury, and the combined diagnosis with the above methods could be used in early diagnosis. Particularly, the process of the test was simple and quick, no special equipment, so it is more suit for primary medical institution.

Application of molecular imaging combined with genetic screening in diagnosing MELAS, diabetes and recurrent pancreatitis.

We report molecular imaging combined with gene diagnosis in a family with 7 members who carried an A3243G mutation in mitochondrial tRNA and p.Thr 137 Met in cationic trypsinogen (PRSS1) gene presented with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), diabetes, and recurrent pancreatitis. DNA sequencing was used to detect and validate mitochondrial DNA and PRSS1. We also verified that mitochondrial heterozygous mutations and c.410 C>T mutation causing p.Thr 137 Met could be detected in oral epithelial cells or in urine sediment cells. In addition, molecular imaging was carried out in the affected family members. In this pedigree, MELAS syndrome accompanied by pancreatitis was an important clinical feature, followed by diabetes. Heteroplasmy of the mtDNA A3243G and c.410 C>T mutation of PRSS1 was found in all tissue samples of these patients, but no mutations were found in 520 normal control and normal individuals of the family. However, based on molecular imaging observations, patients with relatively higher lactate/pyruvate levels had more typical and more severe symptoms, particularly those of pancreatic disease (diabetes or pancreatitis). MELAS syndrome may be associated with pancreatitis. For the diagnosis, it is more reasonable to perform molecular imaging combined with gene diagnosis.

Abstract 4614: Mutation detection in urine from bladder cancer patients as non-invasive prognostic tool

Abstract In Europe, bladder cancer (BC) is the second most common cause of death among patients with genitourinary tract malignancies. Patients with non-muscle invasive bladder cancer (NMIBC) have excellent survival; however two-thirds develop recurrences. For the diagnosis of BC, we mainly rely on urine cytology and cystoscopy. Although cystoscopy is the mainstay of the diagnosis, it is an invasive and costly procedure. Cytology has high specificity but has low sensitivity especially in low-grade, low-stage tumors. Urine sediment cells are a source of tumor DNA, they are easily accessible, and their sampling is not invasive. The identification of a urine-based biomarker could partly replace cystoscopies, thereby reducing the frequency and associated costs, improving patient's quality of life and even leading to earlier detection of recurrences. Tumor-specific mutations can be used to detect recurrences in urine, presenting a non-invasive diagnostic procedure respect to cystoscopy. The present study involves 252 male subjects diagnosed with BC recruited between 1994 and 2012 from the Turin Bladder Cancer Study. Urine samples have been collected for the first time at the time of diagnosis, and exfoliated cells have been extracted. A subgroup of 83 patients has been followed-up for three years, collecting a urine sample every six months. We have investigated the diagnostic and prognostic potentiality of a panel of 40 mutations in hTERT, FGFR3, PIK3CA, and Ras genes detected in DNA from exfoliated cells by SNApShot assay. For each gene we calculated crude and adjusted (by age, smoking status, chemotherapy, low/intermediate/high risk, grade) Hazard Ratios (HR) and the corresponding 95% Confidence Intervals (95% CI) using the Cox proportional-hazard regression model. The results were correlated with stage and tumor grade, with response to therapy and the occurrence of recurrences, in order to assess the validity of exfoliated cells as a source of DNA for the development of a non-invasive test for the follow-up of patients. Mutations in hTERT gene promoter and FGFR3 gene are the most frequent somatic mutations in BC. In particular, hTERT mutations were significantly correlated with high risk, high grade and muscle invasive tumors (p&amp;lt;0.0001). In contrast, for FGFR3 the prevalence of mutations was significantly higher in non-high grade (p&amp;lt;0.0001) and in NMIBC (p = 0.01). Cox regression analysis shows an increased risk of recurrence and recurrence/progression in association with hTERT mutations (adjp&amp;lt;0.007 and p&amp;lt;0.008, respectively). At first follow-up, the significant association between high grade and hTERT mutations was confirmed (p = 0.004). Cox regression analysis confirms an increased risk of recurrence and recurrence/progression in association with hTERT mutations (adjp&amp;lt;0.007 and adjp&amp;lt;0.008, respectively). These findings could suggest novel approach to identify biomarkers of BC at diagnosis and for prognostic reasons. Citation Format: Rossana Critelli, Francesca Fasanelli, Manuela B. Assumma, Ellen C. Zwarthoff, Marco Oderda, Silvia Polidoro, Carlotta Sacerdote, Paolo Vineis, Giuseppe Matullo, Alessio Naccarati. Mutation detection in urine from bladder cancer patients as non-invasive prognostic tool. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4614. doi:10.1158/1538-7445.AM2015-4614

Somatic mosaicism and variant frequency detected by next-generation sequencing in X-linked Alport syndrome.

X-linked Alport syndrome (XLAS) is a progressive, hereditary nephropathy. Although men with XLAS usually develop end-stage renal disease before 30 years of age, some men show a milder phenotype and develop end-stage renal disease later in life. However, the molecular mechanisms associated with this milder phenotype have not been fully identified. We genetically diagnosed 186 patients with suspected XLAS between January 2006 and August 2014. Genetic examination involved: (1) extraction and analysis of genomic DNA using PCR and direct sequencing using Sanger's method and (2) next-generation sequencing to detect variant allele frequencies. We identified somatic mosaic variants in the type VI collagen, α5 gene (COL4A5) in four patients. Interestingly, two of these four patients with variant frequencies in kidney biopsies or urinary sediment cells of ≥50% showed hematuria and moderate proteinuria, whereas the other two with variant frequencies of <50% were asymptomatic or only had hematuria. De novo variants can occur even in asymptomatic male cases of XLAS resulting in mosaicism, with important implications for genetic counseling. This is the first study to show a tendency between the variant allele frequency and disease severity in male XLAS patients with somatic mosaic variants in COL4A5. Although this is a very rare status of somatic mosaicism, further analysis is needed to show this correlation in a larger population.

ALA-induced fluorescence detection with photoresist-based microfluidic cell sorter for bladder cancer diagnosis

We present a photoresist-based microfluidic fluorescence-activated cell sorter (μFACS) for bladder cancer diagnosis and assess its potential for clinical application. In the developed μFACS system, bladder cancer cells in urine sediment are detected using aminolevulinic acid (ALA)-based fluorescence detection, and then sorted at the Y-junction of the microchannel using an off-chip pinch valve. Once cancer cells are collected, they can be analyzed using polymerase chain reaction (PCR) for detailed gene alteration analysis. Generally, time and cost-effective advantages of negative photoresist material and fabrication technologies have been widely pronounced; however, autofluorescence is a significant drawback when negative photoresist is used as a structural material for μFACS applications. To achieve sensitive fluorescence detection, autofluorescence in the detection area is minimized by thinning the photoresist cover layer of the microchannel and inclining the optical angle of the detection system. Through system calibrations and clinical tests using urine sediments, our experimental results demonstrate a combination of ALA-based photodynamic diagnostic tests using μFACS systems and subsequent PCR analysis is a promising diagnostic approach for bladder cancer.

Atypical/malignant urothelial cells in routine urinary sediment: Worth knowing and reporting

BackgroundUrinary cytology (Ucytol), which is performed in pathology laboratories on fixed and stained samples, represents the gold standard for the identification of atypical/malignant urothelial cells (A/MUC) due to urothelial carcinoma. In this paper we describe three patients in whom A/MUC, due to a bladder carcinoma, were identified with conventional urine sediment (Used) examination on unfixed and unstained samples. MethodsIncluded are urine samples prepared with conventional and standardized techniques as currently used in general clinical laboratories. Samples were examined with phase contrast microscopy. A/MUC were identified according to the criteria currently used for Ucytol. ResultsA/MUC (i.e., cells with unusual and pleomorphic size and shape, increased nuclear/cytoplasmic ratio, increased number of nuclei, irregular nuclear borders and irregular chromatin patterns, either isolated or in clusters) were identified in the urine of three patients, all of whom were found to have bladder carcinoma by cystoscopy. ConclusionsAt variance with the common and widespread view, A/MUC can also be identified with conventional Used examination, even though Ucytol still represents the gold standard method.

Micro RNA142-3p Is Differentially Expressed in the Peripheral Blood of Kidney Transplant Recipients With Graft Dysfunction Without Acute Rejection.

Background and aims. The uncovering of non invasive molecular biomarkers of graft dysfunction would be an important accomplishment in the clinical management of kidney transplant recipients (KTR). We evaluated the transcriptional expression of micro RNA 142-3p (miRNA 142-3p) in urinary sediment cells and peripheral blood cells of KTR in order to evaluate it as a non-invasive biomarker for graft dysfunction. Patients and methods. Seventy-six KTR who underwent 98 graft biopsies for the evaluation of renal dysfunction were included. There were forty-nine indication biopsies obtained for evaluation of acute graft deterioration, 42 surveillance biopsies obtained during the period of delayed graft function and 7 were protocol biopsies from stable patients at the third post-transplant month. Peripheral blood and urine were obtained immediately before the biopsy for the evaluation of the miRNA 142-3p by real-time polymerase chain reaction (RT-PCR). Biopsies were classified according to the Banff squema into three groups: acute rejection (AR, n = 23), other causes of dysfunction (OD, n = 68) and normal kidney transplant biopsy (NKT, n = 7). Results. The molecular expression of miRNA 142-3p in the urinary sediment cells of NKT was [(0.7 (95%CI 0.6 - 2.0)] and no difference was found in the comparison with the AR group [(9.0 (95%CI 0.3 - 38.2)] and OD group [(8.0 (95%CI 0.5 - 80.3)], (P = 0.29). In the peripheral blood, miRNA 142-3p expression values were [(1.1 (95%CI 0.4 - 2.4)] for the NKT group and presented lower expression in the group of patients with AR [(1.3 (95%CI 0.5 - 1.8)] as compared with the OD group [(2.39 (95%IC 1.06 - 5.82)], (P = 0,01). Conclusions. Micro RNA 142-3p presented increased expression in the peripheral blood of patients with kidney graft function not due to acute graft reejction. The uncovering of the clinical situation in which this miRNA is increased may lead to the development of an accurate biomarker for such situation.

Non-Invasive Tests to Help Stratify Risk of Rejection in Renal Transplant Recipients.

Molecular biomarkers in blood and urine samples could help risk stratify kidney transplant recipients (KTR) and reduce the need for invasive testing with biopsies to diagnose acute rejection (AR). Changes at molecular level are likely to precede both histological findings and graft dysfunction, allowing earlier diagnosis and treatment to limit graft damage. The aim of our study is to identify and characterize biomarkers that can diagnose and predict AR in KTR. Blood and urine samples were collected from KTR before the transplant and over the first year post-transplant at 26 time-points and during episodes of graft dysfunction. RNA was extracted from blood collected into Tempus Blood RNA tubes and from urine sediment cells. cDNA was synthesised and pre-amplified for urine. Quantitative real time PCR was done for 20(blood) or 23(urine) target and 4 control genes. A pilot study consisting of 18 biopsy proven acute rejection (BPAR) and 26 stable KTR revealed a significant over-expression of 5 genes in blood (SEMA7A, PF4, TGFB, ITGAM, C6orf25) at week 2 post transplantation in the BPAR group, thereby predicting the subsequent development of rejection episodes occurring up to one year post-transplantation (Wilcoxon test, p<0.05, FDR <5%). Using the expression of these 5 genes in a multivariate prediction model returned a probability score that predicted BPAR with a sensitivity of 0.7, specificity of 0.95, and an AUC of 0.80. Rise in SEMA7A, TGFB and IP-10 gene expression in blood of BPAR occurred 3-4 weeks prior to biopsy compared to creatinine rise that was 1 week pre-biopsy. For urine, the difference of gene expression 1-2 weeks and 3-4 weeks pre-biopsy was analysed in 14 BPAR and 14 stable samples at matched time points. Difference in Perforin, PDCD1, FasL and IP-10 expression were detected between these groups. Thus it might be possible to use a urine test to predict a rejection episode with high specificity and sensitivity up to 4 weeks before it occurs. Our pilot study suggests that mRNA levels in blood and urine could help risk-stratify patients for AR in KTR before clinical evidence of AR allowing better management and potential individualisation of anti-rejection therapy. Validation of these findings in a larger cohort is clearly needed.

Editorial Comment from Dr Borkowska to Recurrence and progression in patients with non‐muscle invasive bladder cancer: Prognostic models including multicolor fluorescence in situ hybridization molecular grading

In this issue of the journal, Lodde et al. assess the usefulness of “molecular grading fluorescence in situ hybridization (mG-FISH)” in a prognostic model for recurrence and progression using patients with non-muscle invasive bladder cancer.1 These were selected to be tumors of intermediate risk (according to the European Association of Urology definition), and FISH was carried out using multicolor probes to the centromeres of chromosomes 3, 7, 17 and the 9p21 locus. The authors found subclassification by mg-FISH into low risk (a diploid chromosomal pattern or only loss of 9p16 locus or chromosome 3 aneuploidy) and high risk (aneuploidy of chromosomes 7or 17) enabled a slight increase in the accuracy of the prognostic models, when compared with those derived using clinicopathological variables, such as tumor stage, grade, multiplicity and size, the patient's sex and age, and whether the patient received intravesical chemotherapy after resection. The study is notable for several reasons. Although it is retrospective in design, the authors used the UroVysion test as a prognostic tool (rather than diagnostic) to supplement the histopathological details of the tumor. As the authors suggested, patients with low mG-FISH could have less intensive and shorter treatment follow ups, which might reduce treatment costs. The downside to a less intensive surveillance strategy is potential delayed recognition of disease progression (which occurred in 16% of this cohort). This test causes no extra problems to a patient, as it is carried out in the urine sediment cells. Unfortunately, the relatively frequent problem (approximately 20%) is the insufficient number of cells (“no cells”) in the urine sediment specimen, which makes both the UroVysion test and the mG-FISH assessment impossible.2 As the proposed mG-FISH is treated as a supplement to the histopathological assessment of the tumor, perhaps it could be better to use this technique in the same tissue; that is, in the tumor, if, of course, after the initial recognition, this test is not carried out separately in order to monitor the disease. The authors underline that the use of the European Organization for the Research and Treatment of Cancer calculator, recommended by the European Association of Urology, did not allow selecting from the tested group those patients who will have disease recurrence or progression. It is a frequently mentioned imperfection of this calculator.3 Additionally, the introduction of the histopathological subclassifications might not be completely useful, and requires a great deal of experience in assessing specimens under the microscope. Bladder cancer is a heterogeneous disease – both interpatient and intratumor heterogeneity is important here.4 Hence, there are difficulties in finding markers that would be useful in routine practice. It is possible that heterogeneity itself and the variety of genetic changes are the reason for false negative results of the UroVysion test, whereas the histopathological recognition is positive. Therefore, undoubtedly, the proposed study shows an interesting approach, yet it requires analysis on a bigger and, what I find even more important, on a histopathologically homogeneous group of patients, so that clear conclusions can be drawn (patients with grade G3 should not be tested in the intermediate risk group). A similar approach with slightly different criteria for low- and high-risk positive FISH is presented in the work of Matsuyam et al.5 They found that the percentage of 9p21 loss (>12%) was an independent prognostic factor for recurrence, and the sum of the non-modal copy number fraction of chromosomes 3, 7 and 17 (>16%) was a prognostic factor for disease progression. None declared.