Simple 3D spheroid cell culture model for studies of prion infection.

Mouse neuronal CAD 5 cell line effectively propagates various strains of prions. Previously, we have shown that it can also be differentiated into the cells morphologically resembling neurons. Here, we demonstrate that CAD 5 cells chronically infected with prions undergo differentiation under the same conditions. To make our model more realistic, we triggered the differentiation in the 3D culture created by gentle rocking of CAD 5 cell suspension. Spheroids formed within 1 week and were fully developed in less than 3 weeks of culture. The mature spheroids had a median size of ~300 µm and could be cultured for up to 12 weeks. Increased expression of differentiation markers GAP 43, tyrosine hydroxylase, ß-III-tubulin and SNAP 25 supported the differentiated status of the spheroid cells. The majority of them were found in the G0/G1 phase of the cell cycle, which is typical for differentiated cells. Moreover, half of the PrPC on the cell membrane was N-terminally truncated, similarly as in differentiated CAD 5 adherent cells. Finally, we demonstrated that spheroids could be created from prion-infected CAD 5 cells. The presence of prions was verified by immunohistochemistry, western blot and seed amplification assay. We also confirmed that the spheroids can be infected with the prions de novo. Our 3D culture model of differentiated CAD 5 cells is low cost, easy to produce and cultivable for weeks. We foresee its possible use in the testing of anti-prion compounds and future studies of prion formation dynamics.

Large extracellular vesicles transfer more prions and infect cell culture better than small extracellular vesicles.

Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans and animals. Extracellular vesicles, especially small exosomes, have been extensively studied in connection with various diseases. In contrast, larger microvesicles are often overlooked. In this work, we compared the ability of large extracellular vesicles (lEVs) and small extracellular vesicles (sEVs) to spread prions in cell culture. We utilized CAD5 cell culture model of prion infection and isolated lEVs by 20,000×g force and sEVs by 110,000×g force. The lEV fraction was enriched in ß-1 integrin with a vesicle size starting at 100 nm. The fraction of sEVs was partially depleted of ß-1 integrin with a mean size of 79 nm. Both fractions were enriched in prion protein, but the lEVs contained a higher prion-converting activity. In addition, lEV infection led to stronger prion signals in both cell cultures, as detected by cell and western blotting. These results were verified on N2a-PK1 cell culture. Our data suggest the importance of lEVs in the trafficking and spread of prions over extensively studied small EVs.

Quantitative proteomic analysis of cerebrospinal fluid of women newly diagnosed with multiple sclerosis.

PURPOSE: The lack of reliable diagnostic and/or prognostic biomarkers for multiple sclerosis (MS) is the major obstacle to timely and accurate patient diagnosis in MS patients. To identify new proteins associated with MS we performed a detailed proteomic analysis of cerebrospinal fluid (CSF) of patients newly diagnosed with relapsing-remitting MS (RRMS) and healthy controls. MATERIAL: Reflecting significantly higher prevalence of MS in women we included only women patients and controls in the study. To eliminate a potential effect of therapy on the CSF composition, only the therapy-naïve patients were included. METHODS: Pooled CSF samples were processed in a technical duplicate, and labeled with stable-isotope coded TMT tags. To maximize the proteome coverage, peptide fractionation using 2D-LC preceded mass analysis using Orbitrap Fusion Tribrid Mass Spectrometer. Differential concentration of selected identified proteins between patients and controls was verified using specific antibodies. RESULTS: Of the identified 900 CSF proteins, we found 69 proteins to be differentially abundant between patients and controls. In addition to several proteins identified as differentially abundant in MS patients previously, we observed several linked to MS for the first time, namely eosinophil-derived neurotoxin and Nogo receptor. CONCLUSIONS: Our data confirm differential abundance of several previously proposed protein markers, and provide indirect support for involvement of copper-iron disbalance in MS. Most importantly, we identified two new differentially abundant CSF proteins that seem to be directly connected with myelin loss and axonal damage via TLR2 signaling and Nogo-receptor pathway in women newly diagnosed with RRMS.

Prion Strains Differ in Susceptibility to Photodynamic Oxidation.

Prion disorders, or transmissible spongiform encephalophaties (TSE), are fatal neurodegenerative diseases affecting mammals. Prion-infectious particles comprise of misfolded pathological prion proteins (PrPTSE). Different TSEs are associated with distinct PrPTSE folds called prion strains. The high resistance of prions to conventional sterilization increases the risk of prion transmission in medical, veterinary and food industry practices. Recently, we have demonstrated the ability of disulfonated hydroxyaluminum phthalocyanine to photodynamically inactivate mouse RML prions by generated singlet oxygen. Herein, we studied the efficiency of three phthalocyanine derivatives in photodynamic treatment of seven mouse adapted prion strains originating from sheep, human, and cow species. We report the different susceptibilities of the strains to photodynamic oxidative elimination of PrPTSE epitopes: RML, A139, Fu-1 > mBSE, mvCJD > ME7, 22L. The efficiency of the phthalocyanine derivatives in the epitope elimination also differed (AlPcOH(SO3)2 > ZnPc(SO3)1-3 > SiPc(OH)2(SO3)1-3) and was not correlated to the yields of generated singlet oxygen. Our data suggest that the structural properties of both the phthalocyanine and the PrPTSE strain may affect the effectiveness of the photodynamic prion inactivation. Our finding provides a new option for the discrimination of prion strains and highlights the necessity of utilizing range of prion strains when validating the photodynamic prion decontamination procedures.

Changes in cellular prion protein expression, processing and localisation during differentiation of the neuronal cell line CAD 5.

BACKGROUND INFORMATION: Cellular prion protein (PrPC ) is infamous for its role in prion diseases. The physiological function of PrPC remains enigmatic, but several studies point to its involvement in cell differentiation processes. To test this possibility, we monitored PrPC changes during the differentiation of prion-susceptible CAD 5 cells, and then we analysed the effect of PrPC ablation on the differentiation process. RESULTS: Neuronal CAD 5 cells differentiate within 5 days of serum withdrawal, with the majority of the cells developing long neurites. This process is accompanied by an up to sixfold increase in PrPC expression and enhanced N-terminal ß-cleavage of the protein, which suggests a role for the PrPC in the differentiation process. Moreover, the majority of PrPC in differentiated cells is inside the cell, and a large proportion of the protein does not associate with membrane lipid rafts. In contrast, PrPC in proliferating cells is found mostly on the cytoplasmic membrane and is predominantly associated with lipid rafts. To determine the importance of PrPC in cell differentiation, a CAD 5 PrP-/- cell line with ablated PrPC expression was created using the CRISPR/Cas9 system. We observed no considerable difference in morphology, proliferation rate or expression of molecular markers between CAD 5 and CAD 5 PrP-/- cells during the differentiation initiated by serum withdrawal. CONCLUSIONS: PrPC characteristics, such as cell localisation, level of expression and posttranslational modifications, change during CAD 5 cell differentiation, but PrPC ablation does not change the course of the differentiation process. SIGNIFICANCE: Ablation of PrPC expression does not affect CAD 5 cell differentiation, although we observed many intriguing changes in PrPC features during the process. Our study does not support the concept that PrPC is important for neuronal cell differentiation, at least in simple in vitro conditions.

Affinity depletion versus relative protein enrichment: a side-by-side comparison of two major strategies for increasing human cerebrospinal fluid proteome coverage.

Cerebrospinal fluid (CSF) is in direct contact with the central nervous system. This makes human CSF an attractive source of potential biomarkers for neurologic diseases. Similarly to blood plasma, proteomic analysis of CSF is complicated by a high dynamic range of individual protein concentrations and by the presence of several highly abundant proteins. To deal with the abundant human CSF proteins, methods developed for blood plasma/serum are routinely used. Multiple affinity removal systems and protein enrichment of less abundant proteins using a combinatorial peptide ligand library are among the most frequent approaches. However, their relative impact on CSF proteome coverage has never been evaluated side-by-side in a single study. Therefore, we explored the effect of CSF depletion using MARS 14 cartridge and ProteoMiner ligand library on the number of CSF proteins identified in subsequent LC-MS/MS analysis. LC-MS/MS analysis of crude (non-treated) CSF provided roughly 500 identified proteins. Depletion of CSF by MARS 14 cartridge increased the number of identifications to nearly 800, while treatment of CSF using ProteoMiner enabled identification of 600 proteins. To explore the potential losses of CSF proteins during the depletion process, we also analyzed the "waste" fractions generated by both methods, i.e., proteins retained by the MARS 14 cartridge, and the molecules present in the flow-through fraction from ProteoMiner. More than 250 proteins were bound to MARS 14 cartridge, 100 of those were not identified in the corresponding depleted CSF. Similarly, analysis of the waste fraction in ProteoMiner workflow provided almost 70 unique proteins not found in the CSF depleted by the ligand library. Both depletion strategies significantly increased the number of identified CSF proteins compared to crude CSF. However, MARS 14 depletion provided a markedly higher number of identified proteins (773) compared to ProteoMiner (611). Further, we showed that CSF proteins are lost due to co-depletion (MARS 14) or exclusion (ProteoMiner) during the depletion process. This suggests that the routinely discarded "waste" fractions contain proteins of potential interest and should be included in CSF biomarker studies.

Precision in the design of an experimental study deflects the significance of proteinase-activated receptor 2 expression in scrapie-inoculated mice.

Proteinase-activated receptor 2 (PAR2) is suspected to modulate the pathogenesis of various neurodegenerative conditions. We previously described delayed onset of clinical symptoms and prolonged survival of PAR2-deficient mice after intracerebral inoculation with prions. Here we report the results from a refined blinded study that aimed to investigate the effects of PAR2 deletion on scrapie pathogenesis after peripheral infection. This study failed to confirm that PAR2 deficiency impacts on the length of the incubation period, with PAR2-/- and PAR2+/+ littermates developing scrapie at the same time. To clarify the discrepancy between the two observations, we repeated the intracerebral inoculation study while utilizing our refined protocol, which aimed to limit possible sources of experimental bias. The study again failed to confirm the significant effect of PAR2 expression on the course of prion infection. Our report emphasizes and discusses the importance of unbiased experimental design and the selection of proper genetic controls when using genetically altered animal models for prion pathogenesis studies.

Expression of the cellular prion protein affects posttransfusion recovery and survival of red blood cells in mice.

BACKGROUND: Cellular prion protein (PrP(C) ) is expressed on various cell types including red blood cells (RBCs). The PrP(C) plays a key role in the pathogenesis of prion diseases, but its physiologic function remains unclear. PrP(C) is expressed on CD34+ hematopoietic stem cells and its expression is regulated during blood cell differentiation including the erythroid line. STUDY DESIGN AND METHODS: We investigated the role of PrP(C) in RBC survival in circulation by transfusing a mix of biotin-labeled RBCs from wild-type (WT) and PrP knockout (KO) mice to groups of recipient mice (WT and KO). The proportion of biotinylated RBCs in peripheral blood was estimated by flow cytometry. RESULTS: KO RBCs displayed a markedly higher first-day posttransfusion recovery but had a decreased survival in circulation when compared to WT RBCs. Similar results were obtained in all groups of transfused mice, irrespective of RBCs biotinylation level. In addition, we confirmed this finding in an analogous study using Tga20 mice overexpressing PrP(C) and KO mice of a different genetic background. CONCLUSION: Our results demonstrate that PrP(C) expression affects RBC recovery and survival in circulation.

Toxicity of carboxylated carbon nanotubes in endothelial cells is attenuated by stimulation of the autophagic flux with the release of nanomaterial in autophagic vesicles.

Carbon nanotubes (CNTs) exhibit a number of unique properties that make them attractive for various nanomedicine applications including their intravascular use. Therefore, the vascular toxicity of CNTs is a critical safety concern and methods of CNTs toxicity modulation are of great interest. Here, we report that carboxylated multiwalled carbon nanotubes (MWCNTs) induce a decrease in viability of cultured human umbilical vein endothelial cells (HUVECs) associated with the profound accumulation of autophagosomes. This autophagosome accumulation was mTOR kinase independent and was caused by blockade of the autophagic flux rather than by activation of autophagy. Stimulation of the autophagic flux with 1nmol/L bafilomycin A1 attenuated the cytotoxicity of carboxylated MWCNTs in HUVECs and was associated with the extracellular release of the nanomaterial in autophagic microvesicles. Thus, pharmacological stimulation of the autophagic flux may represent a new method of cytoprotection against toxic effects of nanomaterials. FROM THE CLINICAL EDITOR: This study investigates the mechanisms of toxicity of multiwalled carbon nanutubes on human endothelial cells, concluding that pharmacological stimulation of autophagic flux may represent a new method of cytoprotection against the toxic effects of these nanomaterials.

Detection of prions in matching post-mortem skin and cerebrospinal fluid samples using second-generation real-time quaking-induced conversion assay.

Real-time quaking-induced conversion assay (RT-QuIC) exploits templating activity of pathogenic prion protein for ultrasensitive detection of prions. We have utilized second generation RT-QuIC assay to analyze matching post-mortem cerebrospinal fluid and skin samples of 38 prion disease patients and of 30 deceased neurological controls. The analysis of cerebrospinal fluid samples led to 100% sensitivity and 100% specificity, but some samples had to be diluted before the analysis to alleviate the effect of present RT-QuIC inhibitors. The analysis of the corresponding skin samples provided 89.5% sensitivity and 100% specificity. The median seeding dose present in the skin was one order of magnitude higher than in the cerebrospinal fluid, despite the overall fluorescent signal of the skin samples was comparatively lower. Our data support the use of post-mortem cerebrospinal fluid for confirmation of prion disease diagnosis and encourage further studies of the potential of skin biopsy samples for intra-vitam prion diseases´ diagnostics.

Quinacrine reactivity with prion proteins and prion-derived peptides.

Quinacrine is a drug that is known to heal neuronal cell culture infected with prions, which are the causative agents of neurodegenerative diseases called transmissible spongiform encephalopathies. However, the drug fails when it is applied in vivo. In this work, we analyzed the reason for this failure. The drug was suggested to "covalently" modify the prion protein via an acridinyl exchange reaction. To investigate this hypothesis more closely, the acridine moiety of quinacrine was covalently attached to the thiol groups of cysteines belonging to prion-derived peptides and to the full-length prion protein. The labeled compounds were conveniently monitored by fluorescence and absorption spectroscopy in the ultraviolet and visible spectral regions. The acridine moiety demonstrated characteristic UV-vis spectrum, depending on the substituent at the C-9 position of the acridine ring. These results confirm that quinacrine almost exclusively reacts with the thiol groups present in proteins and peptides. The chemical reaction alters the prion properties and increases the concentration of the acridine moiety in the prion protein.

Detection of the GPI-anchorless prion protein fragment PrP226* in human brain.

BACKGROUND: The accumulation of the misfolded forms of cellular prion protein, i.e. prions (PrPSc), in the brain is one of the crucial characteristics of fatal neurodegenerative disorders, called transmissible spongiform encephalopathies (TSEs). Cellular prion protein is normally linked to the cell surface by the glycosylphosphatidylinositol (GPI) anchor. There is accumulating evidence that the GPI-anchorless prion protein may act as an accelerator of formation and propagation of prions. In the TSE affected human brain we have previously discovered a novel GPI-anchorless prion protein fragment, named PrP226*, which ends with the tyrosine 226. This fragment can be labeled specifically by the monoclonal antibody V5B2. METHODS: We developed a DELFIA based assay for quick and sensitive detection of the PrP226* fragment in human brain tissue homogenates. By calculating the ratio between the signals of native (N) and denatured (D) samples applied to the assay we were able to observe significant difference between 24 TSE affected brains and 10 control brains. The presence of PrP226* in brain tissue was confirmed by western blot. RESULTS: Our results demonstrate that PrP226* is present in small quantities in healthy human brain, whereas in degenerated brain it accumulates in prion aggregates, proportionally to PrPSc. Samples with high D/N ratio generally comprised more proteinase K resistant PrP, while no correlation was found between the quantity of PrP226* and standard classification of Creutzfeldt-Jakob disease (CJD). CONCLUSIONS: In the present study we show that the PrP226* fragment accumulates in prion aggregates and after being released from them by a denaturation procedure, could serve as a proteinase K digestion independent biomarker for human TSEs. The PrP226* assay described in this paper offers a tool to follow and study this unique anchorless PrP fragment in various parts of human brain and possibly also in other tissues and body fluids.

Gerstmann-Sträussler-Scheinker syndrome with the P102L pathogenic mutation presenting as familial Creutzfeldt-Jakob disease: a case report and review of the literature.

Gerstmann-Sträussler-Scheinker syndrome is a rare autosomal dominant disease caused by a mutation in the prion gene, usually manifesting as progressive ataxia with late cognitive decline. A 44-year-old woman with a positive family history developed early personality and behavior changes, followed by paresthesias and ataxia, later associated with memory problems, pyramidal signs, anosognosia and very late myoclonus, spasticity, and severe dysexecutive impairment. Magnetic resonance showed caudate, mesio-frontal, and insular hyper-intensities, electroencephalography revealed generalized triphasic periodic complexes. A pathogenic P102L mutation in the prion gene was detected. Our case differed from classical Gerstmann-Sträussler-Scheinker syndrome by rapid progression, severe dementia, abnormal electroencephalography and magnetic resonance findings, which were highly suggestive of familial Creutzfeldt-Jakob disease.

Photodynamic inactivation of prions by disulfonated hydroxyaluminium phthalocyanine.

Sulfonated phthalocyanines (Pcs) are cyclic tetrapyrroles that constitute a group of photosensitizers. In the presence of visible light and diatomic oxygen, Pcs produce singlet oxygen and other reactive oxygen species that have known degradation effects on lipids, proteins and/or nucleic acids. Pcs have been used successfully in the treatment of bacterial, yeast and fungal infections, but their use in the photodynamic inactivation of prions has never been reported. Here, we evaluated the photodynamic activity of the disodium salt of disulfonated hydroxyaluminium phthalocyanine (PcDS) against mouse-adapted scrapie RML prions in vitro. PcDS treatment of RML brain homogenate resulted in a time- and dose-dependent inactivation of prions. The photodynamic potential of Pcs offers a new way to inactivate prions using biodegradable compounds at room temperature and normal pressure, which could be useful for treating thermolabile materials and liquids.

Deletion of protease-activated receptor 2 prolongs survival of scrapie-inoculated mice.

Proteinase-activated receptor 2 (PAR2) has recently been identified to be a possible modulator of neurodegeneration. To investigate whether PAR2 plays a role in prion infection, we inoculated PAR2-deficient (PAR2(-/-)) and wild-type (WT) mice intracerebrally with the Rocky Mountain Laboratory strain of scrapie. PAR2(-/-) mice demonstrated a delayed onset of clinical symptoms, including weight loss, and demonstrated moderate but highly significant prolongation of survival over WT controls. Concomitantly, no apparent differences in brain pathology, infectivity or features of brain prion protein between deceased WT and PAR2(-/-) mice were found. Our study suggests that PAR2 deletion modulates dynamics of the disease without gross perturbation of its pathogenesis.

Flow Cytometry Analysis of Blood Large Extracellular Vesicles in Patients with Multiple Sclerosis Experiencing Relapse of the Disease.

The number of people living with multiple sclerosis (MS) in developed countries is increasing. The management of patients is hindered by the absence of reliable laboratory tests accurately reflecting the disease activity. Extracellular vesicles (EVs) of different cell origin were reportedly elevated in MS patients. We assessed the diagnostic potential, with flow cytometry analysis, of fresh large EVs (lEVs), which scattered more light than the 590 nm silica beads and were isolated from the blood plasma of relapsing remitting MS patients. Venous blood was collected from 15 patients and 16 healthy controls (HC). The lEVs were isolated from fresh platelet-free plasma by centrifugation, labelled with antibodies and the presence of platelet (CD41+, CD36+), endothelial (CD105+), erythrocyte (CD235a+), leukocyte (CD45+, CD19+, CD3+) and phosphatidylserine (Annexin V+) positive lEVs was analyzed using standard flow cytometry. Cryo-electron microscopy was used to verify the presence of EVs in the analyzed plasma fractions. MS patients experiencing acute relapse had slightly reduced relative levels (% of positive lEVs) of CD105+, CD45+, CD3+, CD45+CD3+ or CD19+ labelled lEVs in comparison to healthy controls. An analysis of other markers or a comparison of absolute lEV counts (count of lEVs/µL) did not yield any significant differences. Our data do not support the hypothesis that the exacerbation of the disease in RRMS patients leads to an increased numbers of circulating plasma lEVs which can be monitored by standard flow cytometry.

Underestimation of the expression of cellular prion protein on human red blood cells.

BACKGROUND: Recent transmissions of variant Creutzfeldt-Jakob disease by blood transfusion emphasize the need for the development of prion screening tests. The detection of prions in blood is complicated by the presence of poorly characterized cellular prion protein (PrP(C) ) in both plasma and blood cells. According to published studies, most of PrP(C) in blood cells resides in platelets (PLTs) and white blood cells. STUDY DESIGN AND METHODS: To clarify conflicting reports about the quantity of PrP(C) associated with human red blood cells (RBCs), quantitative flow cytometry, Western blot (WB), and enzyme-linked immunosorbent assay (ELISA) were used to measure protein levels in healthy donors. RESULTS: RBCs expressed 290 ± 140 molecules of PrP(C) per cell, assuming equimolar binding of monoclonal antibody (MoAb) 6H4 to PrP(C). Binding of alternate PrP(C) MoAbs, FH11 and 3F4, was substantially lower. WB estimated the level of PrP(C) per cell on RBCs to be just four times lower than in PLTs. A similar level of PrP(C) was detected using ELISA. The weak binding of commonly used MoAb 3F4 was not caused by PrP(C) conformation, truncation, or glycosylation, suggesting a covalent modification, likely glycation, of the 3F4 epitope. CONCLUSIONS: Taken together, human RBCs express low but significant amounts of PrP(C) /cell, which makes them, due to high RBC numbers, major contributors to the pool of cell-associated PrP(C) in blood. Previous reports utilizing MoAb 3F4 may have underestimated the amount of PrP(C) in RBCs. Likewise, screening tests for the presence of the abnormal prion protein in blood may be difficult if the abnormal protein is modified similar to RBC PrP(C).

Development of monoclonal antibodies specific for glycated prion protein.

Transmissive spongiform encephalopathies (TSE) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrP(TSE)) in brain. PrP(TSE) is at present the only specific biochemical marker of human and animal TSE. As deposits of PrP(TSE) remain in the body for long periods, there is substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrP(TSE) may have potential to serve as a diagnostic marker. Monoclonal antibodies specific for carboxymethyl lysine/arginine-modified prion protein were prepared. Recombinant human prion protein (rhPrP) was bacterially expressed and purified by affinity chromatography. rhPrP was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of 6 mice, and 960 hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of four promising clones. One of them (EM-31) strongly reacts with human and mouse recombinant PrP-CML, and three other clones react also with CML in vitro modified human and mouse brain PrP. Besides possible implication in TSE diagnostics, the antibodies may serve as tolls to advance our knowledge regarding the role of glycation in the prion pathophysiology.

Detection of Prions in Brain Homogenates and CSF Samples Using a Second-Generation RT-QuIC Assay: A Useful Tool for Retrospective Analysis of Archived Samples.

The possibilities for diagnosing prion diseases have shifted significantly over the last 10 years. The RT-QuIC assay option has been added for neuropsychiatric symptoms, supporting biomarkers and final post-mortem confirmation. Samples of brain homogenates used for final diagnosis, archived for many years, provide the possibility for retrospective studies. We used a second-generation RT-QuIC assay to detect seeding activity in different types of sporadic and genetic prion diseases in archival brain homogenates and post-mortem CSF samples that were 2 to 15 years old. Together, we tested 92 archival brain homogenates: 39 with definite prion disease, 28 with definite other neurological disease, and 25 with no signs of neurological disorders. The sensitivity and specificity of the assay were 97.4% and 100%, respectively. Differences were observed in gCJD E200K, compared to the sporadic CJD group. In 52 post-mortem CSF samples-24 with definite prion disease and 28 controls-we detected the inhibition of seeding reaction due to high protein content. Diluting the samples eliminated such inhibition and led to 95.8% sensitivity and 100% specificity of the assay. In conclusion, we proved the reliability of archived brain homogenates and post-mortem CSF samples for retrospective analysis by RT-QuIC after long-term storage, without changed reactivity.

Cord Blood Extracellular Vesicles Analyzed by Flow Cytometry with Thresholding Using 405 nm or 488 nm Laser Leads to Concurrent Results.

Extracellular vesicles (EVs) from liquid biopsies are extensively analyzed by flow cytometry, a technology that is continuously evolving. Thresholding utilizing a violet 405 nm laser side scatter (VSSC) has recently been implemented. Here, we collected set of large EV (lEV) samples from cord blood, which we analyzed using a standard flow cytometer improved via a 405 nm laser side scatter. Samples were analyzed using two distinct thresholding methods-one based on VSSC, and one based on VSSC combined with fluorescence thresholding on stained phosphatidylserine. Through these thresholding methods, we compared lEVs from pre-term births and control cord blood. Double-labeled lEVs with platelet CD36+/CD41+, activated platelet CD41+/CD62P+ and endothelial CD31+/CD105+ antibodies were used. Apart from comparing the two groups together, we also correlated measured lEVs with the thresholding methods. We also correlated the results of this study with data analyzed in our previous study in which we used a conventional 488 nm laser SSC. We did not find any difference between the two cord blood groups. However, we found highly concurrent data via our correlation of the thresholding methods, with correlation coefficients ranging from 0.80 to 0.96 even though the numbers of detected lEVs differed between thresholding methods. In conclusion, our approaches to thresholding provided concurrent data and it seems that improving the cytometer with the use of a VSSC increases its sensitivity, despite not being particularly critical to the validity of flow cytometric studies that compare pathological and physiological conditions in liquid biopsies.