Urinary Sediment Cells Research Articles

Atypical cells in urine sediment: a novel biomarker for early detection of bladder cancer

Atypical cells in urine sediment: a novel biomarker for early detection of bladder cancer

Uropathogens and their antibiotic susceptibility patterns among diabetic patients at st. john of god hospital, duayaw nkwanta, Ghana: a cross-sectional study.

Uropathogens are microorganisms that cause urinary tract infections (UTIs). Owing to higher blood glucose levels and compromised immune functions, treatment of uropathogens in diabetic patients is a challenge. This study aims to assess the prevalence of uropathogens and their antibiotic susceptibility among diabetic patients at St. John of God Hospital at Duayaw Nkwanta (SJGHDN) in the Ahafo region of Ghana. The cross-sectional study recruited 175 diabetic patients at SJGHDN between August and September 2023. Questionnaires were used to collect patients' background information. Fasting Blood Sugar (FBS) was assessed by using a glucometer. Urine samples were examined for the presence of uropathogens. A sterile inoculating loop with a calibrated volume of 2 µl was used for plating. Each colony equals 500 CFU/mL. Significant uropathogen was determined by multiplying the counted colonies by 500 to obtain CFU/mL. Positive uropathogen was defined as CFU ≥ 105/mL. Significant uropathogen was defined as ≥200 colonies per sample. The disc diffusion method was used to determine antibiotic susceptibility. Out of the 175 patients, 19.4% expressed various uropathogens with Escherichia coli being the predominant. Suboptimal glucose level was the most significant risk factor (p = 0.038). Glucosuria (p = 0.036), hazy urine (p = 0.028), positive leukocyte esterase (p = 0.001), and pus cells in urine sediment (p = 0.020) were significant indicators of uropathogen occurrence. Klebsiella pneumonia and Proteus mirabilis were resistant to ≥4 antibiotics. Amikacin, nitrofurantoin, levofloxacin, ciprofloxacin, and ceftriaxone demonstrated efficacy against the isolates. This study underscores the notable prevalence of uropathogens in diabetic patients and the alarming levels of antibiotic resistance observed. The results highlight the critical need for vigilant monitoring and customized treatment approaches, particularly for diabetic patients exhibiting risk factors such as elevated urine glucose levels, cloudy urine, and presence of leukocyte esterase and pus cells in urine sediment. The significant resistance to frequently used antibiotics like co-trimoxazole and tetracycline points to the necessity of routine susceptibility testing and the use of alternative antibiotics for effective treatment. These findings can assist healthcare providers in more effectively managing and preventing UTIs in diabetic populations.

Long Non-coding RNA NEAT1 , NOD-Like Receptor Family Protein 3 Inflammasome, and Acute Kidney Injury.

Acute kidney injury (AKI) is common in hospitalized patients and is associated with high mortality. Inflammation plays a key role in the pathophysiology of AKI. Long non-coding RNAs (lncRNAs) are increasingly recognized as regulators of the inflammatory and immune response, but its role in AKI remains unclear. We explored the role of lncRNA Neat1 in (1) a cross-sectional and a longitudinal cohort of AKI in human; (2) three murine models of septic and aseptic AKI and (3) cultured C1.1 mouse kidney tubular cells. In human, hospitalized patients with AKI (n=66) demonstrated significantly increased lncRNA Neat1 levels in urinary sediment cells and buffy coat versus control participants (n=152) from a primary care clinic; and among 6 kidney transplant recipients, Neat1 levels were highest immediately after transplant surgery followed by a prompt decline to normal levels in parallel with recovery of kidney function. In mice with AKI induced by sepsis (via LPS injection or cecal ligation and puncture) and renal ischemia-reperfusion, kidney tubular Neat1 was increased versus sham-operated mice. Knockdown of Neat1 in the kidney using short hairpin RNA preserved kidney function, suppressed overexpression of the AKI biomarker NGAL, leukocyte infiltration and both intrarenal and systemic inflammatory cytokines IL-6, CCL-2 and IL-1β. In LPS-treated C1.1 cells, Neat1 was overexpressed via TLR4/NF-κB signaling, and translocated from the cell nucleus into the cytoplasm where it promoted activation of NLRP3 inflammasomes via binding with the scaffold protein Rack1. Silencing Neat1 ameliorated LPS-induced cell inflammation, whereas its overexpression upregulated IL-6 and CCL-2 expression even without LPS stimulation. Our findings demonstrate a pathogenic role of Neat1 induction in human and mice during AKI with alleviation of kidney injury in 3 experimental models of septic and aseptic AKI after knockdown of Neat1. LPS/TLR4-induced Neat1 overexpression in tubular epithelial cells increases the inflammatory response by binding with the scaffold protein, Rack1, to activate NLRP3 inflammasomes.

Acute Kidney Injury and BK Polyomavirus in Urine Sediment Cells.

Polyomaviruses are widespread, with BK viruses being most common in humans who require immunosuppression due to allotransplantation. Infection with BK polyomavirus (BKV) may manifest as BK virus-associated nephropathy and hemorrhagic cystitis. Established diagnostic methods include the detection of polyomavirus in urine and blood by PCR and in tissue biopsies via immunohistochemistry. In this study, 79 patients with pathological renal retention parameters and acute kidney injury (AKI) were screened for BK polyomavirus replication by RNA extraction, reverse transcription, and virus-specific qPCR in urine sediment cells. A short fragment of the VP2 coding region was the target of qPCR amplification; patients with (n = 31) and without (n = 48) a history of renal transplantation were included. Urine sediment cell immunofluorescence staining for VP1 BK polyomavirus protein was performed using confocal microscopy. In 22 patients with acute renal injury, urinary sediment cells from 11 participants with kidney transplantation (KTX) and from 11 non-kidney transplanted patients (nonKTX) were positive for BK virus replication. BK virus copies were found more frequently in patients with AKI stage III (n = 14). Higher copy numbers were detected in KTX patients having experienced BK polyoma-nephropathy (BKPyVAN) in the past or diagnosed recently by histology (5.6 × 109-3.1 × 1010). One patient developed BK viremia following delayed graft function (DGF) with BK virus-positive urine sediment. In nonKTX patients with BK copies, decoy cells were absent; however, positive staining of cells was found with epithelial morphology. Decoy cells were only found in KTX patients with BKPyVAN. In AKI, damage to the tubular epithelium itself may render the epithelial cells more permissive for polyoma replication. This non-invasive diagnostic approach to assess BK polyomavirus replication in urine sediment cells has the potential to identify KTX patients at risk for viremia and BKPyVAN during AKI. This method might serve as a valuable screening tool for close monitoring and tailored immunosuppression decisions.

Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy Cells.

Decoy cells that can be detected in the urine sediment of immunosuppressed patients are often caused by the uncontrolled replication of polyomaviruses, such as BK-Virus (BKV) and John Cunningham (JC)-Virus (JCV), within the upper urinary tract. Due to the wide availability of highly sensitive BKV and JCV PCR, the diagnostic utility of screening for decoy cells in urine as an indicator of polyomavirus-associated nephropathy (PyVAN) has been questioned by some institutions. We hypothesize that specific staining of different infection time-dependent BKV-specific antigens in urine sediment could allow cell-specific mapping of antigen expression during decoy cell development. Urine sediment cells from six kidney transplant recipients (five males, one female) were stained for the presence of the early BKV gene transcript lTag and the major viral capsid protein VP1 using monospecific antibodies, monoclonal antibodies and confocal microscopy. For this purpose, cyto-preparations were prepared and the BK polyoma genotype was determined by sequencing the PCR-amplified coding region of the VP1 protein. lTag staining began at specific sites in the nucleus and spread across the nucleus in a cobweb-like pattern as the size of the nucleus increased. It spread into the cytosol as soon as the nuclear membrane was fragmented or dissolved, as in apoptosis or in the metaphase of the cell cycle. In comparison, we observed that VP1 staining started in the nuclear region and accumulated at the nuclear edge in 6-32% of VP1+ cells. The staining traveled through the cytosol of the proximal tubule cell and reached high intensities at the cytosol before spreading to the surrounding area in the form of exosome-like particles. The spreading virus-containing particles adhered to surrounding cells, including erythrocytes. VP1-positive proximal tubule cells contain apoptotic bodies, with 68-94% of them losing parts of their DNA and exhibiting membrane damage, appearing as "ghost cells" but still VP1+. Specific polyoma staining of urine sediment cells can help determine and enumerate exfoliation of BKV-positive cells based on VP1 staining, which exceeds single-face decoy staining in terms of accuracy. Furthermore, our staining approaches might serve as an early readout in primary diagnostics and for the evaluation of treatment responses in the setting of reduced immunosuppression.

#6669 ANALYSIS OF KIDNEY BIOPSIES AND URINE SEDIMENT USING SCANNING ELECTRON MICROSCOPY (SEM) AND ATOMIC FORCE MICROSCOPY (AFM)

Abstract Background and Aims The classification of renal diseases is dependent on the evaluation of kidney biopsies using light microscopy techniques. Ultrastructural details (e.g. podocyte foot processes, amyloid fibrils, Fabri's intracellular inclusions) need transmission electron microscopy (TEM) which has high costs, requires specialized personnel and is time consuming. Therefore, alternative techniques are highly desirable. Here we report our experience with scanning electron microscopy (SEM) and atomic force microscopy (AFM). Method Paraffin-embedded kidney sections (3μm thick) were examined after dewaxing and Jones-methenamine methane staining for type IV collagen fibers. Dried urine sediments were prepared using standard methods for urine cytological analysis. Analysis was then performed using atomic force microscopy (nGauge system) on dried specimens and with Scanning Electron Microscopy (Zeiss) after gold-spattering of the surface. Reference images were obtained by standard microscopy. Results The combination of SEM and thin paraffin sections allows the observation of ultrastructural details in kidney biopsies with a very simple and fast preparation. AFM is even faster than SEM, as it does not require any preparation of the surface of the sample. An important advantage of SEM is the possibility to view the sample at low magnification, which makes it easier to identify the location of the glomeruli. It is thus possible to switch from small magnifications (100x) to very extreme magnifications (10000x). Notably, the interpretation of the images requires knowledge of the artifacts induced by paraffin inclusion. Specifically, structures and cells containing lipids (for example foamy cells) appear empty. Chromatin is also condensed in the nuclei around the periphery of the nucleus and in spike structures whit large empty areas. The recognition of nuclei in SEM and AFM is important for the subsequent interpretation of the images. Podocyte pedicles fusion can be identified relatively easily in SEM and in AFM. Both techniques add relevant ultrastructural details of urine sediment cells, from epithelial cells to isolated tubular cells. Also, casts show many ultrastructural features which are not evident in classical optical microscopy. It is also possible to measure the thickness of the basement membrane with great precision both in SEM and with AFM. The major limitation of this approach is the limited experience of pathologists with these methods and the absence of an atlas of normal and pathological kidney. Conclusion Considering the affordable costs of SEM and AFM instruments and the simple preparation of samples for their analysis, these should be considered a valid alternative to TEM. SEM and AFM are alternative methods, simple and relatively cheap and very fast to obtain ultrastructural details of renal biopsies. We thank ANED and AST for the great support given to research.

Analysis of Urine Sediment Images for Detection and Classification of Cells

Urine sediment tests are important in the diagnosis of abnormal diseases related to the urinary tract. The formation of cells such as red blood cells and white blood cells in the urine of patients is important for the diagnosis of the disease. Therefore, cells need to be fully identified in clinical urinalysis. Urinalysis with human eyes; Since it is subjective, time consuming and causing errors, methods have been developed to automate microscopic analysis with the help of computer and software systems. In this study, the YOLO-v7 algorithm, which gives successful results in image processing technology, was used as a method and model. The dataset used in the study was created by using microscopic images of urine sediment taken from the Biochemistry Laboratory of the Faculty of Medicine, Selcuk University. Seven different cell segmentation and classification studies have been carried out, including WBC, RBC, WBCC, Epithelial, Flat Epithelial, Mucs and Bubbles, which have clinical value for the diagnosis of the disease. Experimental studies were carried out with the YOLO-v7 algorithm and the results were presented. The contributions of this study can be summarized as follows. (1) In this study, which is proposed for segmentation of cells on the urine cell images in the Urine Sediment dataset, for the experimental studies carried out with the YOLO model, whose performance was evaluated; Precision, Recall, mAP(0.5) and F1-Score(%) segmentation performance metrics were calculated as 0.384, 0.759, 0.432 and 0.510, respectively. (2) A computer-aided support system to assist physicians in segmenting urine cells is presented as a secondary tool. Classification accuracy for WBC, RBC, WBCC, Epithelial, Flat Epithelial, Mucs and Bubbles cells was calculated as 0.78, 0.94, 0.90, 0.57, 0.92, 0.68 and 0.97, respectively. A mean classification success of 0.822 was achieved for all classes. Thus, it has been seen that the Yolov7 model can be used by experts as a tool for recognizing cells in the urine sediment. As a result, it has been shown that suitable deep learning models can be used to recognize the biometric properties of urinary sediment cells. With the model created using deep learning libraries, urine sediment cells can be easily classified, and it is possible to define many different cells if there is a dataset with sufficient number of images.

Phenotype of Urine Sediment Cells in Patients with Bladder Cancer

Phenotype of urine sediment cells were studied in patients with bladder cancer depending on the cancer stage and recurrence prognosis. In T1N0M0 stage, the number of lymphocytes decreased, in T2N0M0 stage, the most pronounced shift was an increase in the number of erythrocytes. Irrespectively of the disease stage, we observed increased number of innate immunity cells and cells that inhibit antitumor immunity in the composition of the leukocyte fraction of urine sediment cells. At T1N0M0 stage, the epithelial-endothelial fraction was characterized by increased content of cells expressing CD13 marker (responsible for tumor growth and metastasis) and reduced number of cells expressing CD15 marker (responsible for intercellular adhesion). In patients developing relapse of bladder cancer, the number of lymphocytes was decreased in urine sediment cells and the number of epithelial and endothelial cells expressing CD13 marker was increased.

A SEC61A1 variant is associated with autosomal dominant polycystic liver disease.

Autosomal dominant polycystic liver and kidney disease is a spectrum of hereditary diseases, which display disturbed function of primary cilia leading to cyst formation. In autosomal dominant polycystic kidney disease a genetic cause can be determined in almost all cases. However, in isolated polycystic liver disease (PLD) about half of all cases remain genetically unsolved, suggesting more, so far unidentified genes to be implicated in this disease. Customized next-generation sequencing was used to identify the underlying pathogenesis in two related patients with PLD. A variant identified in SEC61A1 was further analysed in immortalized patients' urine sediment cells and in an epithelial cell model. In both patients, a heterozygous missense change (c.706C>T/p.Arg236Cys) was found in SEC61A1, which encodes for a subunit of the translocation machinery of protein biosynthesis at the endoplasmic reticulum (ER). While kidney disease is absent in the proposita, her mother displays an atypical polycystic kidney phenotype with severe renal failure. In immortalized urine sediment cells, mutant SEC61A1 is expressed at reduced levels, resulting in decreased levels of polycystin-2 (PC2). In an epithelial cell culture model, we found the proteasomal degradation of mutant SEC61A1 to be increased, whereas its localization to the ER is not affected. Our data expand the allelic and clinical spectrum for SEC61A1, adding PLD as a new and the major phenotypic trait in the family described. We further demonstrate that mutant SEC61A1 results in enhanced proteasomal degradation and impaired biosynthesis of PC2.

Comparison of six microscopic methods and two operators for estimation of white and red blood cells in canine urine sediment.

Little information is currently available about the analytical variability of urinalysis. We aimed to compare results obtained by two operators using six microscopic methods in the quantification of urinary leukocytes (WBC) and erythrocytes (RBC). Forty urine samples (10mL) were centrifuged (450g, 5minutes) and resuspended in 0.5mL of supernatant. Two operators with different expertise in urinalysis interpreted sediment results using the six methods, obtained by combining the use of microscope slides (Slide) or counting chambers (Chamber) with three different techniques: bright-field (BF) microscopy, phase-contrast (PC) microscopy, and stained sediment (SS) evaluations. The mean WBC and RBC counts from 10 fields (Slide) or squares (Chamber) observed at 400× were used to calculate the difference and agreement between operators and methods. We also estimated the concordance between methods in classifying microhematuric or pyuric samples. Operator 2 counted significantly lower WBC counts using Slide+BF (P= 0.009) and Slide+PC (P= 0.001) than Operator 1, whereas no inter-operator differences were recorded for RBC counts. The concordance between the operators ranged from "good" to "very good." No differences or biases were found for WBC counts among the methods, and concordances were "good" to "very good"; proportional biases were found for RBC counts between Slide+BF vs Slide+SS and Slide+PC vs Slide+SS. Concordance measurements for RBC counts ranged from "good" to "very good." All methods yielded good reproducibility among operators, although stained SS evaluations allowed better identification of WBC by the inexperienced operator. However, we suspected that the SS preparations affected RBC counts. All other methods yielded reproducible WBC and RBC counts.

Urine cytological study in patients with clinicopathologically confirmed neuronal intranuclear inclusion disease.

ObjectiveThe diagnosis of neuronal intranuclear inclusion disease (NIID) is currently based on CGG repeat expansion in the 5′UTR of the NOTCH2NLC gene, or p62-positive intranuclear inclusions in skin biopsy. The purpose of this study is to explore the value of non-invasive pathological findings in urine sediment cells from NIID patients.Materials and methodsTen patients with clinically suspected NIID were enrolled for skin biopsy and gene screening. Morning urine (500 ml) was collected from each patient, and cell sediment was obtained by centrifugation. Urine cytology, including Giemsa staining, p62 immunostaining, and electron microscopic examination, were conducted on cell sediment.ResultsThe main clinical symptoms of 10 patients included episodic disturbance of consciousness, cognitive impairment, tremor, limb weakness, and so on. Cerebral MRI showed that 9 patients had linear DWI high signal in the corticomedullary junction. Genetic testing found that the number of CGG repeat ranged from 96 to 158 in the NOTCH2NLC gene. Skin biopsy revealed that all patients showed p62-positive intranuclear inclusions in 18.5 ± 6.3% of the duct epithelial cells of sweat gland. In contrast, urine sediment smears revealed that only 3 patients had p62 positive intranuclear inclusions in 3.5 ± 1.2% of the sedimentary cells. Ultrastructural examinations showed that intranuclear inclusions were also identified in the cell sediment of the 3 patients.ConclusionUrine cytology may be a new and non-invasive pathological diagnosis technique for some NIID patients, although the positive rate is not as high as that of skin biopsy, which is a sensitive and reliable pathological method for NIID.

A Key Feature of Atypical Cells in Urinary Sediment for Urothelial Carcinoma Screening

A Key Feature of Atypical Cells in Urinary Sediment for Urothelial Carcinoma Screening

Urinary C3 levels associated with sepsis and acute kidney injury-A pilot study.

Acute kidney injury (AKI) is an abrupt deterioration of renal function often caused by severe clinical disease such as sepsis, and patients require intensive care. Acute-phase parameters for systemic inflammation are well established and used in routine clinical diagnosis, but no such parameters are known for AKI and inflammation at the local site of tissue damage, namely the nephron. Therefore, we sought to investigate complement factors C3a/C3 in urine and urinary sediment cells. After the development of a C3a/C3-specific mouse monoclonal antibody (3F7E2), urine excretion from ICU sepsis patients was examined by dot blot and immunoblotting. This C3a/C3 ELISA and a C3a ELISA were used to obtain quantitative data over 24 hours for 6 consecutive days. Urine sediment cells were analyzed for topology of expression. Patients with severe infections (n = 85) showed peak levels of C3a/C3 on the second day of ICU treatment. The majority (n = 59) showed C3a/C3 levels above 20 μg/ml at least once in the first 6 days after admission. C3a was detectable on all 6 days. Peak C3a/C3 levels correlated negatively with peak C-reactive protein (CRP) levels. No relationship was found between peak C3a/C3 with peak leukocyte count, age, or AKI stage. Analysis of urine sediment cells identified C3a/C3-producing epithelial cells with reticular staining patterns and cells with large-granular staining. Opsonized bacteria were detected in patients with urinary tract infections. In critically ill sepsis patients with AKI, urinary C3a/C3 inversely correlated with serum CRP. Whether urinary C3a/C3 has a protective function through autophagy, as previously shown for cisplatin exposure, or is a by-product of sepsis caused by pathogenic stimuli to the kidney must remain open in this study. However, our data suggest that C3a/C3 may function as an inverse acute-phase parameter that originates in the kidney and is detectable in urine.

High SARS-CoV-2 Viral Load in Urine Sediment Correlates with Acute Kidney Injury and Poor COVID-19 Outcome.

AKI is a complication of coronavirus disease 2019 (COVID-19) that is associated with high mortality. Despite documented kidney tropism of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there are no consistent reports of viral detection in urine or correlation with AKI or COVID-19 severity. Here, we hypothesize that quantification of the viral load of SARS-CoV-2 in urine sediment from patients with COVID-19 correlates with occurrence of AKI and mortality. The viral load of SARS-CoV-2 in urine sediments (U-viral load) was quantified by qRT-PCR in 52 patients with PCR-confirmed COVID-19 diagnosis, who were hospitalized between March 15 and June 8, 2020. Immunolabeling of SARS-CoV-2 proteins Spike and Nucleocapsid was performed in two COVID-19 kidney biopsy specimens and urine sediments. Viral infectivity assays were performed from 32 urine sediments. A total of 20 patients with COVID-19 (39%) had detectable SARS-CoV-2 U-viral load, of which 17 (85%) developed AKI with an average U-viral load four-times higher than patients with COVID-19 who did not have AKI. U-viral load was highest (7.7-fold) within 2 weeks after AKI diagnosis. A higher U-viral load correlated with mortality but not with albuminuria or AKI stage. SARS-CoV-2 proteins partially colocalized with the viral receptor ACE2 in kidney biopsy specimens in tubules and parietal cells, and in urine sediment cells. Infective SARS-CoV-2 was not detected in urine sediments. Our results further support SARS-CoV-2 kidney tropism. A higher SARS-CoV-2 viral load in urine sediments from patients with COVID-19 correlated with increased incidence of AKI and mortality. Urinary viral detection could inform the medical care of patients with COVID-19 and kidney injury to improve prognosis.

Survey on reporting of epithelial cells in urine sediment as part of external quality assessment programs in Brazilian laboratories.

IntroductionEpithelial cells (ECs) are structures regularly observed during urine microscopy analysis. The correct identification of EC subtypes can be useful since renal tubular epithelial cells (RTECs) are clinically relevant. We investigate the urinary ECs report and the judgement of its clinical importance by Brazilian laboratories.Materials and methodsA survey with four questions was made available to participants of the Urinalysis External Quality Assessment Program (EQAP) from Controllab. Laboratories composed 3 groups: (1) differentiating ECs subtypes: “squamous”, “transitional” and “RTECs”; (2) differentiating ECs subtypes: “squamous” or “non-squamous” cells; (3) without ECs subtype identification. Participants did not necessarily answer to all questions and the answers were evaluated both within the same laboratory’s category and within different categories of laboratories.ResultsA total of 1336 (94%) laboratories answered the survey; Group 1, 119/140 (85%) reported that ECs differentiation is important to the physician and 62% want to be evaluated by EQAP, while in Group 3, 455/1110 (41%) reported it is useful to them, however only 25% want be evaluated by EQAP. Group 2 laboratories 37/51 (73%) reported that the information is important, but only 13/52 (25%) are interested in an EQAP with differentiation of the 3 ECs subtypes.ConclusionMost of the laboratories do not differentiate ECs in the three subtypes, despite the clinical importance of RTECs. Education of laboratory staff about the clinical significance of urinary particles should be considered a key priority.

Cytomegalovirus in urinary sediment in patients with acute kidney injury

BackgroundImmunosuppression in solid organ transplantation is associated with frequent infections. Renal allograft recipients are susceptible to opportunistic infections and can acquire human cytomegalovirus (HCMV) infections even within the allograft. There, HCMV can be found in both the glomerulus and tubular cells, but is mostly restricted to specific and circumscribed sites. Therefore, not all organ infections are identifiable by immunohistology for HCMV proteins in fine needle core biopsies. Thus, we performed a urinalysis study to search for HCMV-specific RNA transcripts in the urine sediment of patients with acute kidney injury.MethodsUrinary sediment of 90 patients with acute kidney injury (AKI), including 48 renal transplant recipients (RTX) and 42 non-transplant recipients (nRTX), was collected from morning urine for RNA extraction and reverse transcription. The copy number of HCMV transcripts was evaluated using a UL132 HCMV-specific probe set and by real-time quantitative polymerase chain reaction (RT-qPCR).ResultsOf the 48 RTX patients, ten showed HCMV copies in their urine sediment cells. Within this group, three recipients had negative HCMV serology and received an allograft from an HCMV-seropositive donor. In addition, all three RTX patients on a belatacept-based immunosuppressive regimen had HCMV transcripts in their urine. Of the 42 nRTX patients, only two had detectable HCMV transcripts in urine sediment cells and both were under immunosuppression.ConclusionsTen immunosuppressed renal allograft recipients and two immunosuppressed non-transplant patients with AKI showed HCMV copies in urine sediment. Thus, HCMV positivity in urinary sediment appears to be associated with immunosuppression. This study describes a novel noninvasive method for detection of HCMV in urinary sediment. Whether all HCMV infections can be detected or only those with viral replication warrants further investigation.

Using urine to diagnose large-scale mtDNA deletions in adult patients.

ObjectiveThe aim of this study was to evaluate if urinary sediment cells offered a robust alternative to muscle biopsy for the diagnosis of single mtDNA deletions.MethodsEleven adult patients with progressive external ophthalmoplegia and a known single mtDNA deletion were investigated. Urinary sediment cells were used to isolate DNA, which was then subjected to long‐range polymerase chain reaction. Where available, the patient`s muscle DNA was studied in parallel. Breakpoint and thus deletion size were identified using both Sanger sequencing and next generation sequencing. The level of heteroplasmy was determined using quantitative polymerase chain reaction.ResultsWe identified the deletion in urine in 9 of 11 cases giving a sensitivity of 80%. Breakpoints and deletion size were readily detectable in DNA extracted from urine. Mean heteroplasmy level in urine was 38% ± 26 (range 8 ‐ 84%), and 57% ± 28 (range 12 – 94%) in muscle. While the heteroplasmy level in urinary sediment cells differed from that in muscle, we did find a statistically significant correlation between these two levels (R = 0.714, P = 0.031(Pearson correlation)).InterpretationOur findings suggest that urine can be used to screen patients suspected clinically of having a single mtDNA deletion. Based on our data, the use of urine could considerably reduce the need for muscle biopsy in this patient group.

Laboratory changes during adrenocorticotropic hormone therapy associated with renal calcified lesions.

Renal calcified lesions are known as one of the complications during adrenocorticotropic hormone (ACTH) therapy for intractable epilepsy. However, laboratory changes during the therapy or laboratory features of high-risk cases with renal calcified lesions are yet to be clarified. In this study, 43 patients with West syndrome aged ≤2years were included. We retrospectively reviewed age and body mass index at the beginning of ACTH therapy, as well as the amount of fluid intake, daily urinary volume, and laboratory data during therapy. In addition, we studied the urinary sediment of the cases with renal calcified lesions diagnosed by computed tomography. After initiating ACTH treatment, urinary calcium (Ca)/creatinine ratio and urinary pH increased within 2weeks. Urinary crystals and renal tubular epithelial cells (RTECs) in urinary sediment were frequently found in most cases. Urinary Ca levels, proteinuria or frequency of urinary crystals, and number of RTECs in the urinary sediment were significantly higher in patients with epithelial casts (ECs) or hematuria than in patients without these findings. Among the seven patients who underwent abdominal CT, ECs or hematuria were found only in those with renal calcified lesions. These findings suggested that patients with ECs or hematuria were more likely to have calcified lesions. The risk of renal calcified lesions increased after 2weeks of ACTH treatment. Abnormal findings in urinary sediments might be an early sign of renal calcification during ACTH therapy.

Gene mutation detection of urinary sediment cells for NMIBC early diagnose and prediction of NMIBC relapse after surgery.

Early diagnose of bladder cancer could lead to good prognosis and high 5-year-survival rate. Among bladder cancer, about 75% patients with were nonmuscle-invasive bladder cancer (NMIBC). Patients were painful and easily get infected during bladder cancer diagnosis, which mainly depends on invasive cystoscopy and low-sensitivity urine exfoliation cytology. Meanwhile, relapse after surgery was also becoming the major problem for patients. Exploring noninvasive, high-sensitivity, and painless method is very important and meaningful for NMIBC treatment.Firstly, we found potential related gene mutation sites for NMIBC by searching COSMIC database and related study. Urinary sediment cells of patients both in normal group (patients with benign) and NMIMC group were collected before and after operation for potential gene mutation site detecting. Meanwhile, the urinary sediment cells of relapse patients and good prognosis people in NMIBC group after surgery were also collected for further Gene mutation detection and NMIBC relapse after surgery prediction.Fourteen genes (152 mutation sites) were selected between 95 NMIBC patients and 67 control patients, which were FGFR3, TP53, PIK3CA, and others. Compared with control group, mutation ratio of above 14 genes was higher in NMIBC group. NMIBC diagnose model was established by 5 times cross-validation and had a good effects, which included the all mutation site in FGFR3, TP53, PIK3CA, ARID1A, STAG2, and KTM2D. On the contrary, the relapse rate was 30.5% among 95 patients for about 1.5-year follow-up time. Compared with control group, smoking rate and tumor grade were higher in relapse group. Meanwhile, mutation rate of FGFR3, TP53, PIK3CA, ERBB3, and TSC1 in relapse group were higher than that in normal group. According to the mutation sites of FGFR3, TP53, PIK3CA, and ERBB3 and the combination of urinary sediment cells genetic mutation and relapse status, a predicted model for NMIBC relapse was also established, which had 90% accuracy.The diagnosed NMIBC model (based on FGFR3, TP53, PIK3CA, ARID1A, STAG2, and KTM2D gene mutation) and predicted relapse model (based on FGFR3, TP53, PIK3CA, and ERBB3 gene mutation) possess high accuracy and would be applied in early diagnose and early predicting relapse of patients.

Phenol Based RNA Isolation is the Optimum Method for Study of Gene Expression in Human Urinary Sediment

Evaluation of gene expression in urinary sediment has been considered as a promising non-invasive approach for biomarker identification of kidney diseases. Nonetheless, there are several challenges in extraction of RNA from this valuable source of biomarkers, mostly because of the factors that have influence on quality of isolated RNA such as low cellular content. Accordingly, we compared the quality of RNA from urine sediment samples that was isolated by four different methods. TRIzol reagent with basic protocol (method 1), modified procedure of TRIzol (method 2), a column-based protocol (method 3) and combination of method 1 and 3 (method 4) were applied for isolation of RNA from identical aliquots of five healthy urine samples. The quality and yield of isolated RNA were evaluated based on concentration and purity. Expression levels of GAPDH and miR-21 were studied by quantitative RT-PCR. Methods 1 and 2 showed the highest RNA yield while no difference in purity of RNA in different methods was noticed. Quantitative RT-PCR findings indicated that Ct values in samples of method 1 had the lowest level. Although higher concentrations of RNA were isolated by method 2, the declined Ct values in this method might indicate degradation of isolated RNA. Column based protocols (method 3 and 4) were failed to show significant recovery of RNA. It seems that isolation procedure using TRIzol, as a phenol based method, is the most efficient, robust and reliable procedure for RNA isolation from urinary sediment cells.