Isolation-free measurement of single urinary extracellular vesicles by imaging flow cytometry

Wu, Liang; Woud, Wouter W.; Baan, Carla C.; Hesselink, Dennis A.; Pol, Edwin van der; Jenster, Guido; Boer, Karin

doi:10.1016/j.nano.2022.102638

Cited by 4 publications

(9 citation statements)

References 37 publications

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“…For uEV isolation measurement using IFCM, 10 µL of the total 150 µL uEV solution was diluted in 990 µL of PBS to prevent swarm detection resulting from an excess concentration of EVs (approximately 1×10 9 objects/mL). 23 , 24 , 37 To account for the 40 mL of urine used for EV isolation, the final concentration of uEV isolates used for IFCM staining is 2.67-fold more concentrated than the initial urine. Similarly, as a positive control, platelet-poor plasma was diluted 12.7-fold to avoid the swarm effect.…”

Section: Methodsmentioning

confidence: 99%

“…For showing non-biological background signals, such as antibody aggregates, samples were treated with detergent to lyse phospholipid and lipid particles. 23 , 38 After each IFCM measurement, all labeled samples were incubated with TritonX-100 (final concentration of 0.05%) at room temperature for 30 min and measured again.…”

Section: Methodsmentioning

confidence: 99%

“…IFCM acquisition was performed on an ImageStreamX Mark II imaging flow cytometer (ISx; Cytek Biosciences, Fremont, CA, USA) using INSPIRE ® software (version 200.1.0.765; Cytek Biosciences) as reported previously for measuring unprocessed urine and plasma. 21 , 23 Purified uEVs were measured with the same acquisition protocol as urine and cell supernatant. In brief, the settings of INSPIRE ® were: flow speed velocity of 40 mm/s, 6 μm diameter of the flow core, 60× magnification, 405-nm laser at channel 01 (Ch01) with power: 120 mW, 488-nm laser at channel 02 (Ch02) with power: 200 mW, 642-nm laser at channel 05 (Ch05) with power: 150 mW, 785-nm laser for exciting side scatter (SSC) at channel 06 (Ch06) with power: 1.25 mW, and channel 04 (Ch04) used for presenting bright field.…”

Section: Methodsmentioning

confidence: 99%

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Polarized HLA Class I Expression on Renal Tubules Hinders the Detection of Donor-Specific Urinary Extracellular Vesicles

Wu,

van Heugten,

van den Bosch

et al. 2024

IJN

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Purpose Kidney transplantation is the optimal treatment for patients with end-stage kidney disease. Donor-specific urinary extracellular vesicles (uEVs) hold potential as biomarkers for assessing allograft status. We aimed to develop a method for identifying donor-specific uEVs based on human leukocyte antigen (HLA) mismatching with the kidney transplant recipients (KTRs). Patients and Methods Urine and plasma were obtained from HLA-A2+ donors and HLA-A2- KTRs pre-transplant. CD9 (tetraspanin, EV marker) and HLA-A2 double-positive (CD9+ HLA-A2+) EVs were quantified using isolation-free imaging flow cytometry (IFCM). Healthy individuals’ urine was used to investigate CD9+ HLA-class-I+ uEV quantification using IFCM, time-resolved fluoroimmunoassay (TR-FIA), and immunogold staining cryo-electron microscopy (cryo-EM). Culture-derived CD9+ HLA-class-I+ EVs were spiked into the urine to investigate urine matrix effects on uEV HLA detection. Deceased donor kidneys and peritumoral kidney tissue were used for HLA class I detection with histochemistry. Results The concentrations of CD9+ HLA-A2+ EVs in both donor and recipient urine approached the negative (detergent-treated) control levels for IFCM and were significantly lower than those observed in donor plasma. In parallel, universal HLA class I+ uEVs were similarly undetectable in the urine and uEV isolates compared with plasma, as verified by IFCM, TR-FIA, and cryogenic electron microscopy. Culture supernatant containing HLA class I+ vesicles from B, T, and human proximal tubule cells were spiked into the urine, and these EVs remained stable at 37°C for 8 hours. Immunohistochemistry revealed that HLA class I was predominantly expressed on the basolateral side of renal tubules, with limited expression on their urine/apical side. Conclusion The detection of donor-specific uEVs is hindered by the limited release of HLA class I+ EVs from the kidney into the urine, primarily due to the polarized HLA class I expression on renal tubules. Identifying donor-specific uEVs requires further advancements in recognizing transplant-specific uEVs and urine-associated markers.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Polarized HLA Class I Expression on Renal Tubules Hinders the Detection of Donor-Specific Urinary Extracellular Vesicles

Wu,

van Heugten,

van den Bosch

et al. 2024

IJN

Self Cite

View full text Add to dashboard Cite

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“…In particular, the simultaneous detection of both EV surface proteins and internal miRNAs from captured EVs demonstrated that disease-related biomarker signals could be normalized by EV-related biomarker signals. , However, most methods involve EV capture, which is laborious and requires many rinsing steps to remove unbound probes, making them difficult to use in clinics. To facilitate a more practical and efficient technique, flow cytometric analysis can be adopted for EV biomarker detection because this method does not involve rinsing steps to remove unbound probes. , …”

Section: Introductionmentioning

confidence: 99%

“…To facilitate a more practical and efficient technique, flow cytometric analysis can be adopted for EV biomarker detection because this method does not involve rinsing steps to remove unbound probes. 21,22 In this study, we identified breast cancer cell-derived EVs by flow-cytometry-based simultaneous detection using programmed cell death-ligand 1 (PD-L1) aptamer and miR-21 molecular beacon (MB-21) (Figure 1). To overcome the limitations of conventional flow cytometry in recognizing nanosized EVs, 21,23,24 the biomarker detection signal was enhanced by forming EV clusters using 1,2-distearoyl-snglycero-3-phosphoethanolamine (DSPE)-polyethylene glycol (PEG)-DSPE.…”

Section: Introductionmentioning

confidence: 99%

In Situ Simultaneous Detection of Surface Protein and microRNA in Clustered Extracellular Vesicles from Cancer Cell Lines Using Flow Cytometry

Lee,

Lee

et al. 2023

ACS Biomater. Sci. Eng.

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Extracellular vesicles (EVs) are becoming increasingly important in liquid biopsy for cancer because they contain multiple biomarkers, including proteins and RNAs, and circulate throughout the body. Cancer cellderived EVs are highly heterogeneous, and multiplexed biomarker detection techniques are required to improve the accuracy of diagnosis. In addition, in situ EV biomarker detection increases the efficiency of the detection process because EVs are difficult to handle. In this study, in situ simultaneous detection of EV surface proteins, programmed cell death-ligand 1 (PD-L1), and internal miRNA-21 (miR-21) analyzed by conventional flow cytometry was developed for a breast cancer liquid biopsy. However, the majority of EVs were not recognized by flow cytometry for biomarker detection because the size of EVs was below the detectable size range of the flow cytometer. To solve this problem, the formation of EV clusters was induced by 1,2-distearoyl-snglycero-3-phosphoethanolamine (DSPE)-polyethylene glycol-DSPE during biomarker detection. Consequently, both PD-L1 and miR-21 detection signals from cancer cell-derived EVs were drastically increased, making them distinguishable from normal cell-derived EVs. The in situ simultaneous cancer biomarker detection from EV clusters analyzed by flow cytometry contributes to an increase in the sensitivity and accuracy of the EV-based liquid biopsy for cancer.

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DNA four-way junction-driven dual-rolling circle amplification sandwich-type aptasensor for ultra-sensitive and specific detection of tumor-derived exosomes

Zhao,

Yang,

Tang

et al. 2024

Biosensors and Bioelectronics

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Isolation-free measurement of single urinary extracellular vesicles by imaging flow cytometry

Cited by 4 publications

References 37 publications

Polarized HLA Class I Expression on Renal Tubules Hinders the Detection of Donor-Specific Urinary Extracellular Vesicles

Polarized HLA Class I Expression on Renal Tubules Hinders the Detection of Donor-Specific Urinary Extracellular Vesicles

In Situ Simultaneous Detection of Surface Protein and microRNA in Clustered Extracellular Vesicles from Cancer Cell Lines Using Flow Cytometry

DNA four-way junction-driven dual-rolling circle amplification sandwich-type aptasensor for ultra-sensitive and specific detection of tumor-derived exosomes

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