scholarly journals Sampling, Logistics, and Analytics of Urine for RT-qPCR-based Diagnostics

Cancers ◽  
2021 ◽  
Vol 13 (17) ◽  
pp. 4381
Author(s):  
Rosel Kretschmer-Kazemi Kretschmer-Kazemi Far ◽  
Kirsten Frank ◽  
Georg Sczakiel
Keyword(s):  
Bladder Cancer ◽  
Cancer Patients ◽  
Primary Source ◽  
Urine Samples ◽  
Current Status ◽  
Quantitative Detection ◽  
Human Genomics ◽  
Pcr Products ◽  
Transcriptomics Data ◽  
Data Collections

Body fluids in the context of cancer diagnosis are the primary source of liquid biopsy, i.e., biomarker detection that includes blood and serum, urine, and saliva. RNA represents a particular class of biomarkers because it is thought to monitor the current status of gene expression in humans, in organs, and if present, also in tumors. In case of bladder cancer, we developed a scheme that describes, in detail, all steps from the collection of urine samples from patients, stabilization of samples, their transportation, storage, and marker analysis by qPCR-based technology. We find that urine samples prepared according to this protocol show stability of RNA over more than 10 days at unchilled temperatures during shipping. A specific procedure of primer design and amplicon evaluation allows a specific assignment of PCR products to human genomics and transcriptomics data collections. In summary, we describe a technical option for the robust acquisition of urine samples and the quantitative detection of RNA-based tumor markers in case of bladder cancer patients. This protocol is for general use, and we describe that it works for any RNA-based tumor marker in urine of cancer patients.

scholarly journals Molecular Detection of Bladder Cancer by Fluorescence Microsatellite Analysis and an Automated Genetic Analyzing System

2007 ◽  
Vol 7 ◽  
pp. 1553-1557 ◽  
Author(s):  
Sarel Halachmi ◽  
Michal Cohen ◽  
Raymond Szargel ◽  
Nadin Cohen
Keyword(s):  
Bladder Cancer ◽  
Transitional Cell ◽  
Experimental System ◽  
Microsatellite Analysis ◽  
Urine Samples ◽  
Genetic Changes ◽  
Resected Tumor ◽  
Pcr Products ◽  
Ion Laser ◽  
Tumor Biopsies

To investigate the ability of an automated fluorescent analyzing system to detect microsatellite alterations, in patients with bladder cancer. We investigated 11 with pathology proven bladder Transitional Cell Carcinoma (TCC) for microsatellite alterations in blood, urine, and tumor biopsies. DNA was prepared by standard methods from blood, urine and resected tumor specimens, and was used for microsatellite analysis. After the primers were fluorescent labeled, amplification of the DNA was performed with PCR. The PCR products were placed into the automated genetic analyser (ABI Prism 310, Perkin Elmer, USA) and were subjected to fluorescent scanning with argon ion laser beams. The fluorescent signal intensity measured by the genetic analyzer measured the product size in terms of base pairs. We found loss of heterozygocity (LOH) or microsatellite alterations (a loss or gain of nucleotides, which alter the original normal locus size) in all the patients by using fluorescent microsatellite analysis and an automated analyzing system. In each case the genetic changes found in urine samples were identical to those found in the resected tumor sample. The studies demonstrated the ability to detect bladder tumor non-invasively by fluorescent microsatellite analysis of urine samples. Our study supports the worldwide trend for the search of non-invasive methods to detect bladder cancer. We have overcome major obstacles that prevented the clinical use of an experimental system. With our new tested system microsatellite analysis can be done cheaper, faster, easier and with higher scientific accuracy.

Developing a new urine cell flow cytometry analysis of PD-L1 expression and DNA content for diagnosis of bladder cancer.

2019 ◽  
Vol 37 (8_suppl) ◽  
pp. 60-60
Author(s):  
Shaheen Riadh Alanee ◽  
Mustafa Deebajah ◽  
Pin-I Chen ◽  
Alice (Xiaoyang) Wang ◽  
Bruce Kendrick Patterson
Keyword(s):  
Flow Cytometry ◽  
Bladder Cancer ◽  
Epithelial Cells ◽  
Cancer Patients ◽  
Dna Content ◽  
The United States ◽  
Urine Samples ◽  
Normal Donor ◽  
Urine Testing ◽  
In Cells

60 Background: Bladder cancer is the fifth most common cancer in the United States. PD-1/PD-L1, a pathway used by cancer cells to evade immune response, correlates with bladder cancer severity and has emerged as a target in bladder cancer treatment. Chromosomal instability is also a prominent feature associated with the development of bladder cancer. A method for unbiased analysis of PD-L1 expression and DNA content in cells from urine samples promises to be a new test for diagnosis of bladder cancer. Methods: To evaluate the PD-L1 expression and DNA content, we developed a 4-color flow assay. Cells in voided urine samples were pelleted, fixed in incellMAX (IncellDx Inc.) and stained with antibodies against pan-cytokeratin (CK), CD45, PD-L1 and a cell cycle dye. The stained samples were analyzed by a flow cytometer alongside stained control cells. Results: Fifty bladder cancer patient and 15 normal donor urine samples were collected and tested with this assay. We could distinguish epithelial cells (pan-CK+) and white blood cells (WBCs, CD45+) in urine samples and obtain PD-L1 expression and DNA content information simultaneously from these cell populations. The patient samples showed a significantly higher percentage of WBCs with substantial PD-L1 expression (P < 0.001). The percentage of PD-L1 positive epithelial cells was not distinguishable between normal donor and patient samples. However increased post G1 epithelial cells ( > 5%) were observed in a majority of bladder cancer patients, with around 25% of samples showing a DNA index above 1.05. Conclusions: We developed a flow cytometry-based method to investigate PD-L1 and DNA content simultaneously in cells from urine samples that could provide us with a new method to accurately identify bladder cancer patients through urine testing.

Urine cell flow cytometry analysis of PD-L1 expression and DNA content for bladder cancer.

2019 ◽  
Vol 37 (7_suppl) ◽  
pp. 466-466
Author(s):  
Pin-I Chen ◽  
Alice (Xiaoyang) Wang ◽  
Mustafa Deebajah ◽  
Shaheen Alanee ◽  
Bruce Kendrick Patterson
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Bladder Cancer ◽  
Epithelial Cells ◽  
Cancer Patient ◽  
Cancer Patients ◽  
Dna Content ◽  
Urine Samples ◽  
Normal Donor ◽  
Bladder Cancer Patient

466 Background: Bladder cancer is the fifth most common cancer in the United States. PD-1/PD-L1, a pathway used by cancer cells to evade immune response, correlates with bladder cancer severity and has emerged as a target in bladder cancer treatment. Chromosomal instability is also a prominent feature associated with the development of bladder cancer. A method to unbiasedly analyze PD-L1 expression and DNA content in cells from urine samples will help us better understand bladder cancer. Methods: To evaluate the PD-L1 expression and DNA content, we developed a 4-color flow assay. Cells in urine samples were pelleted, fixed/permeabilized (in incellMAX, IncellDx Inc.) and stained with antibodies against pan-cytokeratin (CK), CD45, PD-L1 and a cell cycle dye. The stained samples were analyzed by a flow cytometer alongside stained control cells. Results: Fifty bladder cancer patient and 15 normal donor urine samples were collected and tested with this assay. We could distinguish epithelial cells (pan-CK+) and white blood cells (WBCs, CD45+) in urine samples and obtain PD-L1 expression and DNA content information simultaneously from these cell populations. The patient samples showed a significantly higher percentage of WBCs with substantial PD-L1 expression. The percentage of PD-L1 positive epithelial cells was not distinguishable between normal donor and patient samples. However, increased post G1 epithelial cells ( > 5%) were observed in a majority of bladder cancer patients, with around 25% of samples showing a DNA index above 1.05. In addition, a comparison of urine collection fixatives showed that incellMAX-fixed samples had the best single cell recovery and DNA content measurement, as shown by lower cell cycle dye staining variability (lower rCV). Statistically significant differences were found between cancer patients and normal samples. Conclusions: We developed a flow cytometry-based method to investigate PD-L1 and DNA content simultaneously in cells from urine samples. Comparing urine samples from bladder cancer patients and normal yielded statistically significant differences that could provide valuable information for bladder cancer patient management.

C31 SECOND VOIDED URINE SAMPLES GIVE BETTER YIELDS OF CELL-FREE DNA THAN FIRST MORNING URINE IN BLADDER CANCER PATIENTS AND CONTROLS

2012 ◽  
Vol 11 (4) ◽  
pp. 89
Author(s):  
J. Hrbácek ◽  
E. Pazourková ◽  
A. Brisuda ◽  
V. Soukup ◽  
J. Mareš ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Cancer Patients ◽  
Urine Samples ◽  
Cell Free Dna ◽  
Free Dna

Evaluation of miR-146a Urinary Level as a Marker for Recurrence of Superficial Bladder Cancer after Transurethral Resection of Bladder Tumor (TUR-BT)

2020 ◽  
Vol 29 (3) ◽  
pp. 145-151
Author(s):  
Amany K. Shahat ◽  
Rabea G. Omar
Keyword(s):  
Bladder Cancer ◽  
Cancer Patients ◽  
Normal Level ◽  
Transurethral Resection ◽  
Cancer Biology ◽  
Bladder Tumor ◽  
Superficial Bladder Cancer ◽  
Urine Samples ◽  
Superficial Cancer

Background: The most prevalent malignancy affect the urinary system is bladder cancer which exhibits a markedly high recurrence rate. The level of urinary microRNA-146a (miR-146a) was significantly higher in superficial bladder cancer patients, and were decreased to normal range after transurethral resection of the bladder tumor (TUR-BT). Its level was increased again if there is recurrence of the superficial bladder tumor diagnosed by follow up cystoscopy. miR-146 may be a clinically important marker for diagnosis and recurrence of superficial bladder cancer and is used instead of follow up operations as follow up cystoscopy for follow up of recurrence . Objective: our work aims to detect a simple, reliable and noninvasive diagnostic and surveillance methods for follow up of superficial bladder cancer. Methodology: The level of miR-146a in urine of 30 superficial bladder cancer patients was evaluated by quantitative reverse transcription polymerase chain reaction assay using voided urine samples before, after TUR-BT and after 3, 6,9,12 months of resection and compared with the result of follow up cystoscopy. Results: miR-146a was significantly increased in urine samples from patients with superficial cancer bladder than in those from the normal individuals (P <.000). Elevated urinary miR-146a levels in patients with bladder cancer were lowered to the normal level after TUR-BT and increased again in those who have tumor recurrence and remain in a normal level in those who have no recurrence after follow up for 3 ,6, 9,12 months after surgery(P=. 007, P=.000 respectively).Conclusion: Our study concluded that urinary miR-146a may be useful as a novel noninvasive diagnostic and follow up marker, anticancer agent or therapeutic target for superficial cancer bladder, also for increasing our knowledge of cancer biology.

Lab in a Tube: Sensitive Detection of MicroRNAs in Urine Samples from Bladder Cancer Patients Using a Single-Label DNA Probe with AIEgens

2015 ◽  
Vol 7 (30) ◽  
pp. 16813-16818 ◽  
Author(s):  
Xuehong Min ◽  
Yuan Zhuang ◽  
Zhenyu Zhang ◽  
Yongmei Jia ◽  
Abdul Hakeem ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Cancer Patients ◽  
Dna Probe ◽  
Urine Samples ◽  
Sensitive Detection

Expression profile of miRNAs in urine samples of bladder cancer patients

2018 ◽  
Vol 12 (12) ◽  
pp. 1311-1321 ◽  
Author(s):  
Leila Nekoohesh ◽  
Mohammad Hossein Modarressi ◽  
Seyed Javad Mowla ◽  
Esmaeil Sadroddiny ◽  
Masoud Etemadian ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Cancer Patients ◽  
Expression Profile ◽  
Urine Samples
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