scholarly journals O-GlcNAcylation in early stages of chronic lymphocytic leukemia; protocol development for flow cytometry

2021 ◽  
pp. 1-10
Author(s):  
Viktória Temesfői ◽  
Kinga Molnár ◽  
Péter Kaltenecker ◽  
Barbara Réger ◽  
Árpád Szomor ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Lymphocyte Count ◽  
Blood Parameters ◽  
Cell Number ◽  
Metabolic Changes ◽  
Protein Glycosylation ◽  
Lymphocytic Leukemia ◽  
Tween 20 ◽  
Protocol Development ◽  
Flow Cytometric

BACKGROUND: Recent studies proved that metabolic changes in malignant disorders have an impact on protein glycosylation, however, only a few attempts have been made so far to use O-GlcNAc analysis as a prognostic tool. Glucose metabolism is reported to be altered in hematological malignancies thus, we hypothesized that monitoring intracellular O-GlcNAc levels in Rai stage 0-I (Binet A) CLL patients could give deeper insights regarding subtle metabolic changes of progression which are not completely detected by the routine follow-up procedures. OBJECTIVE: In this proof of concept study we established a flow cytometric detection method for the assessment of O-GlcNAcylation as a possible prognostic marker in CLL malignancy which was supported by fluorescence microscopy. METHODS: Healthy volunteers and CLL patients were recruited for this study. Lymphocytes were isolated, fixed and permeabilised by various methods to find the optimal experimental condition for O-GlcNAc detection by flow cytometry. O-GlcNAc levels were measured and compared to lymphocyte count and various blood parameters including plasma glucose level. RESULTS: The protocol we developed includes red blood cell lysis, formalin fixation, 0.1% Tween 20 permeabilisation and employs standardized cell number per sample and unstained controls. We have found significant correlation between O-GlcNAc levels and WBC (R2= 0.8535, p< 0.0029) and lymphocyte count (R2= 0.9225, p< 0.0006) in CLL patients. Interestingly, there was no such correlation in healthy individuals (R2= 0.05664 for O-GlcNAc vs WBC and R2= 0.04379 for O-GlcNAc vs lymphocytes). CONCLUSION: Analyzing O-GlcNAc changes in malignant disorders, specifically in malignant hematologic diseases such as CLL, could be a useful tool to monitor the progression of the disease.

Clinical Predictors of Abnormal Peripheral Blood Lymphocytoses Diagnosed by Flow Cytometry: An Algorithmic Approach That Can Be Applied in Routine Clinical Practice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3932-3932
Author(s):  
Jared M. Andrews ◽  
Mitchel T. Holm ◽  
Jerome B. Myers
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Lymphocyte Count ◽  
Flow Cytometric Analysis ◽  
Clinical History ◽  
Lymphocytic Leukemia ◽  
Western World ◽  
Abnormal Phenotype ◽  
Flow Cytometric ◽  
The Mean

Abstract Background Elevated peripheral blood lymphocyte counts in adults can occur in benign reactive conditions as well as malignant disease processes. Chronic lymphocytic leukemia (CLL) is the most common adult hematologic malignancy of the western world affecting the middle aged and elderly. Less commonly B, T, and Natural Killer (NK) cell leukemia / lymphomas may also present with lymphocytosis. Flow cytometry has greatly improved the ability to detect low levels of abnormal lymphocyte populations in peripheral blood. It is, however, a relatively expensive test and clinical guidelines for its appropriate usage are not well defined. Methods We conducted a retrospective review of peripheral blood lymphocytoses that were submitted for flow cytometric analysis at Madigan Army Medical Center, Tacoma, WA from 2002 – 2004. Under laboratory protocol, all patients ≥ 50 years of age with an absolute lymphocyte count (ALC) of &gt; 4 X 109 Cells/L had a peripheral smear evaluated by both a hematology technician and pathologist. Specimens determined to warrant flow cytometric analysis based on review of clinical history, prior lab values, degree of lymphocytosis, and morphology were either recommended for flow cytometry in a comment; or sent directly for analysis with the clinician’s approval. We reviewed complete blood counts (CBCs), previous flow cytometry results, as well as bone marrow and electronic clinical history. All patients with previous diagnoses of lymphoproliferative disorders (LPDs) or ALC &lt; 4 X 109 Cells/L were excluded. Results Approximately 7,300 CBC specimens/month (3,400 from patients ≥ 50 years of age) were performed. Of these, an average of 44 specimens/month had a lymphocytosis of ≥ 4 X 109 Cells/L, from approximately 28 different patients. From this group 71 flow cytometric cases (an average of 2/month) were performed over the 2 year period. 42 cases (59%) had an abnormal phenotype. 27 had a phenotype consistent with CLL, and the other 15 were a mixture of LPDs involving B and T-lymphocytes as well as NK cells. Comparing normal phenotype to abnormal phenotype showed statistically significant differences between the mean age (n-60.4 ±7.5, abn-69.8±8.7), ALC (n-4.9±0.8, abn-9.2±8.1), and relative lymphocyte count (RLC) (n-43.9±7.5%, abn-59.3±8.8%). Conclusion Absolute lymphocyte counts ≥ 4 X 109 Cells/L in adults ≥ 50 years of age represent approximately 1% of the CBCs performed in our laboratory. Review of these cases by a pathologist is logistically feasible due to the low incidence. Our method of reviewing for morphology, clinical history, and past lymphocyte counts with comments to the ordering clinician yielded a high incidence of abnormal phenotype diagnoses when evaluated by flow cytometric analysis (59%). Age, ALC, and relative lymphocyte counts are variables that can be used to develop guidelines for determining the appropriateness of flow cytometric analysis. Patients &lt; 52.4 years of age fall below two standards of deviation from the mean age of the abnormal phenotype group. The standard of deviation for mean ALC is very small (4.9±0.8), which indicates that counts &gt; two standards of deviation above the mean, or 6.5 X 109 Cells/L, would correlate strongly with an abnormal phenotype. The same conclusion could be made with a RLC &gt; 58.9%. In conclusion, patients ≥ 50 years of age with an ALC &gt; 6.5 X 109 Cells/L or a RLC &gt; 58.9% are likely to have a lymphoproliferative disorder and flow cytometric analysis is indicated.

Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1) - a Putative Diagnostic Marker for Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1587-1587
Author(s):  
Sabrina Uhrmacher ◽  
Magdalena Hertweck ◽  
Julian Paesler ◽  
Felix Erdfelder ◽  
Alexandra Filipovich ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Tyrosine Kinase ◽  
Healthy Volunteers ◽  
Receptor Tyrosine Kinase ◽  
Peripheral Blood ◽  
Lymphocytic Leukemia ◽  
Orphan Receptor ◽  
Flow Cytometric

Abstract Abstract 1587 Poster Board I-613 In chronic lymphocytic leukemia (CLL) WNT signaling is constitutively active and several members of this signaling pathway are uniformely upregulated in these cells. Apart from classical WNT receptors like FZD and LRP6, receptor tyrosine kinase-like orphan receptor 1 (ROR1) has been shown to function as a receptor for WNT proteins, too. Furthermore, it could recently be demonstrated that ROR1 is frequently expressed on the surface of CLL cells and might therefore serve as a therapeutic target in this disease. However, so far only little is known about the expression status of this protein in different patients. Moreover, a diagnostic antibody for flow cytometric investigations is lacking. Thus, the aim of our study was to i) establish a directly labelled anti-ROR1 antibody for flow cytometry, ii) to confirm previous results on ROR1 expression in CLL, iii) to investigate ROR1 expression in different cell compartments and iv) correlate our findings to known markers of risk and disease progression. Peripheral blood of CLL patients as well as healthy volunteers was subjected to flow cytometric analysis. Besides standard determination of leukocyte subpopulations ZAP70 and CD38 status was assessed according to current diagnostic recommendations. In addition, ROR1 surface expression was first detected by flow cytometry using a specific primary antibody directed against ROR1 and a fluorescent labelled secondary antibody. Using this experimental setting we found that ROR1 is expressed on 63.4% of all neoplastic CLL cells and also on 30.5% of T cells in the peripheral CLL blood. In contrast, no ROR1 expression could be detected on NK cells, B cells, CD8+- or CD4+-T cells of healthy individuals. To improve the analytical technique the ROR1 antibody was directly conjugated with Phycoerythrin (PE) and the experiments were repeated. With the conjugated antibody we detected ROR1 expression on 97.1% of neoplastic CLL cells and virtually on no T lymphocytes. ROR1 expression levels correlated neither with the expression of ZAP70 nor with CD38. Again, we could not detect ROR1 expression on peripheral blood cells of our healthy volunteers. Taken together, ROR1 expression appears to be highly restricted to CLL cells. If in addition to CD5 and CD19 ROR1 detection is included into diagnostic flow cytometric panels the specificity and sensitivity of immunophenotypic CLL diagnostics may be greatly enhanced. Disclosures Hallek: Roche: Consultancy, Honoraria, Research Funding.

ROR-1 Is a Highly Discriminative Marker in Flow Cytometric Minimal Residual Disease (MRD) Detection in Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3197-3197 ◽  
Author(s):  
Michaela Patz ◽  
Barbara Pentok ◽  
Kathrin Cremer ◽  
Stefanie Linnartz ◽  
Esther Lilienweiss ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Research Funding ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
Cell Populations ◽  
Discrimination Analysis ◽  
Flow Cytometric ◽  
Correct Discrimination ◽  
Minimal Residual

Abstract Introduction:With the advent of new potent therapies for chronic lymphocytic leukemia (CLL) minimal residual disease (MRD) detection becomes increasingly important to assess remission depth. While molecular MRD detection for CLL remains laborious and time consuming flow cytometry is a fast, economic and sensitive method in detecting low frequencies of CLL cells. The usefulness of the antigens CD81, CD5, CD20, CD43 and CD79b has been previously described for this purpose. ROR-1 has recently been identified as a signature gene in CLL and mantle cell lymphoma. The potential utility of ROR-1 in flow cytometric minimal residual cell analysis has not been evaluated yet. Methods: 10 normal samples and 77 remnants of randomly selected samples from diagnosed patients undergoing CLL therapy were analyzed by flow cytometry. A customized dry formulation of an antibody panel was used, comprising antibodies directed against CD5, CD19, CD20, CD43, CD45, CD79b, CD81 and ROR-1 (DuraClone RE CLB). Linearity, repeatability and inter-operator variability of data analysis of the method were examined. B cell populations comprising at least 50 positive events (46 normal B cell populations, 25 CLL populations, paired and unpaired) were analyzed for their expression profile as assessed by respective mean fluorescence intensities of the antibody labels within classified populations. The expression profiles were subject to supervised discrimination analysis (DA). Results: Between124,000 and 2,122,000 (683,000 ± 450,000) CD45+ events were acquired from the 87 samples. The background of cells with a CLL-like phenotype in the normal samples was determined as <0.001% of CD45+ events. Linearity was confirmed in the range from 1% to 0.0025%. The Repeatability analysis and the inter-operator variability showed concordance with typical Poisson distribution characteristics. The 46 populations with a typical normal B cell phenotype ranged from 0.014% to 9.592% with an average of 2.45% ± 2.75 of CD45+ events. The 25 populations with a classical or non-classical CLL phenotype ranged from 0.007% to 5.459% with an average of 1.41% ± 1.65 of CD45+ events. Posterior discrimination analysis revealed 100% correct discrimination for CLL populations and 96% correct discrimination for normal populations when relying on ROR-1 expression alone in CD19+CD45+ B cells. This result was only surpassed by the complete antibody combination (100% / 100%) but not by any other of the markers, neither in single use nor in combination Conclusion: The 8-color dry flow cytometry panel comprising CD5, CD19, CD20, CD43, CD45, CD79b, CD81 and ROR-1 demonstrated sensitive, linear and specific detection of residual CLL cells in a relevant low range of frequency. ROR-1 revealed to be a highly discriminative marker in the analysis of residual CLL cells by flow cytometry. Utilizing this flow cytometry approach, MRD detection showing sensitivity comparable to molecular techniques can be achieved in CLL. Disclosures Hallek: AbbVIe: Consultancy, Honoraria; Mundipharma: Consultancy, Honoraria; Glaxo-SmithKline: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau; Celgene: Consultancy, Honoraria; Roche: Consultancy, Research Funding, Speakers Bureau. Kreuzer:Gilead Sciences: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche Pharma GmbH and Mundipharma GmbH: Consultancy, Honoraria, Research Funding, Speakers Bureau.

scholarly journals Linking Microenvironmental Signals to Metabolic Switches and Drug Responses in Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 479-479
Author(s):  
Zhenghao Chen ◽  
Gaspard Cretenet ◽  
Hanneke ter Burg ◽  
Gabriela Andrejeva ◽  
Jeffrey C Rathmell ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Glucose Uptake ◽  
Metabolic Pathways ◽  
Research Funding ◽  
Tca Cycle ◽  
Metabolic Changes ◽  
Lymphocytic Leukemia ◽  
Biopsy Material ◽  
Mitochondrial Mass

Chronic lymphocytic leukemia (CLL) has become a paradigm for a cancer that depends on signals from the microenvironment. In lymph nodes (LN), CLL cells receive from surrounding cells proliferative and pro-survival signals, which can protect against chemotherapy and also the Bcl-2 inhibitor venetoclax (VEN). To avoid drug resistance, combination of VEN with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (IBR) is currently explored. IBR has the potency to drive CLL cells out of the LN to eventually die by VEN-induced apoptosis. The activation status in the LN likely affects tumor metabolism, though it is yet unclear how, and whether such putative metabolic changes can be linked to VEN resistance. In this study,we aimed to investigate the altered metabolism of CLL cells in the tumor microenvironment (TME), what signals determine these changes, and how to counteract VEN resistance at the metabolic level. We compared the metabolic status of CLL cells in LN biopsy material and paired peripheral blood (PB) CLL cells. Both higher mitochondrial mass and glucose uptake (flow cytometry) were found in CLL cells derived from LN as compared to PB. To determine which TME signals affect metabolism, we mimicked 3 LN signals: CD40, B cell receptor (BCR) and toll-like receptor (TLR) signaling.Invitrostimulation of PB CLL was followed by mitochondrial mass and glucose uptake (flow cytometry), oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measured on Seahorse XF Analyzer. The datademonstrated that CD40 but surprisingly not BCR or TLR signaling recapitulated the metabolic changes observed in LN cells. Next, two sets of metabolomics were performed for samples both in vitro and in vivo. Firstly, PB samples of 10 patients were stimulated with/without CD40L for 48 hours and analyzed by mass-spec for metabolic intermediates. In parallel, metabolomics was performed on PB samples from 13 patients sampled before and after 3 months of ibrutinib treatment. The data show various metabolic pathways are activated in the TME, particularly citric acid (TCA) cycle, pyruvate metabolism, glycolysis, and fatty acid metabolism. Apart from these general changes, the highest-ranking shifts in metabolites point to involvement of amino acids to fuel the TCA cycle, which in turn drives oxidative phosphorylation (OXPHOS). Overall, CD40 signaling results in increased glycolysis, but more dominantly, increased OXPHOS, while IBR in fact has opposing effects (Figure 1), indicating that TME-driven metabolic alterations are mitigated by IBR treatment. In order to link the altered metabolic state to VEN resistance, PB-derived cells were treated with OXPHOS inhibitors during CD40 stimulation. Remarkably, OXPHOS inhibition by oligomycin (ATP synthase inhibitor) did not affect CLL activation, yet counteracted strongly for VEN resistance. Of several BCL-2 family members induced by CD40 ligation, both anti-apoptotic MCL-1 and BCL-XL were downregulated by oligomycin. These data suggest that OXPHOS inhibition affects CD40 signals to counteract VEN resistance. In conclusion, metabolic changes of CLL in LN are recapitulated by CD40 signal, while IBR treatment shows opposite effects, together providing indirect insight into the LN metabolism. In LN, most of the key metabolic pathways are enhanced, particularly OXPHOS. Finally, we found OXPHOS inhibition reverses CLL VEN resistance. Our findings link CLL metabolism in LN microenvironment to VEN resistance, and may provide novel clues for therapeutic targeting in the treatment of VEN resistance patients. Disclosures Forconi: Roche: Honoraria; Gilead Sciences: Research Funding; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Menarini: Consultancy; Novartis: Honoraria; Janssen-Cilag: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau. van der Windt:Genmab: Employment. Eldering:Celgene: Research Funding; Janssen Pharmaceutical Companies: Research Funding; Roche: Research Funding.

scholarly journals Time to Diagnosis of Chronic Lymphocytic Leukemia in a Health System Setting

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5835-5835
Author(s):  
Carl Shultz ◽  
Lauren Sigler ◽  
Jesse Manikowski ◽  
Amanda Young ◽  
Joseph Vadakara
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Health System ◽  
Median Time ◽  
Lymphocyte Count ◽  
Pearson Correlation ◽  
Lymphocytic Leukemia ◽  
Low Grade ◽  
Significant Difference ◽  
Time To Diagnosis

Abstract Introduction Chronic lymphocytic leukemia (CLL) is a low-grade malignancy that is generally indolent and often asymptomatic at presentation. While it can present with anemia, thrombocytopenia, progressive splenomegaly and adenopathy, it is usually diagnosed on routine laboratory studies with confirmatory flow cytometry. Complications of CLL include lack of appropriate immune response and surveillance, which may lead to more frequent infections and the development of second primary neoplasms, most commonly cutaneous malignancies. Prior studies have investigated the time to diagnose CLL from the onset of clinical signs and symptoms. One study looking at the Surveillance, Epidemiology, and End-Results (SEER) database found that the median time to diagnosis from the onset of signs or symptoms was approximately 63 days (1). While early diagnosis may not change the natural history of the disease, it may lead to earlier initiation of preventive strategies such as vaccinations and skin cancer screening. The purpose of this study is to investigate the time to diagnosis of patients with CLL from the onset of lymphocytosis meeting criteria for CLL to confirmatory flow cytometry and its correlation with patient characteristics within a rural health system. Methods A retrospective cohort analysis of patients with CLL who had an absolute lymphocyte count (ALC) greater than 5,000/microL and a flow cytometry was performed within the Geisinger Health System from 1997 to 2018. Patient age, sex, date of first lymphocytosis meeting diagnostic criteria, and date of flow cytometry was electronically extracted from the EMR. This data was then cross-referenced with the cancer registry database for accuracy of the diagnosis. To determine any difference between groups and the time from first ALC meeting criteria to confirmatory flow cytometry performed, the Wilcoxon Two-Sample Test was utilized. Associations between two continuous variables (age and time from first ALC meeting criteria to confirmatory flow cytometry performed, initial lymphocyte count and time from first ALC meeting criteria to confirmatory flow cytometry performed) were measured using the Pearson Correlation Coefficients. Results A total of 268 patients were identified. Univariate statistics are reported in Table 1. The median time from lymphocytosis to flow for our study was 7.5 days (IQR: 0 - 335) with a mean of 347 days (range: 0 - 5335 days). Just over half (53.8%) had a diagnosis within the first two weeks; however, greater than 20% were diagnosed after one year and just over 5% after five years (table 2). There was a positive correlation between age and time from lymphocytosis to flow (r=0.178, p=0.0034). Therefore, an increase in age at diagnosis was associated with an increase in time from initial lymphocytosis to flow. A weak negative correlation was seen between the degree of initial lymphocytosis and time from lymphocytosis to flow (r=-0.105, p=0.0867). Therefore, a lower lymphocyte count was associated with a slightly longer time to diagnosis. There was no significant difference between median time from lymphocytosis to flow in females (7 days IQR: 0 - 510) and males (8 days IQR: 0 - 271) with a p-value 0.4570. Conclusion This study demonstrates that there can be a broad range of time to diagnosis of CLL (figure 1). While gender did not play a significant role, increased age and lower initial lymphocyte value were associated with a delay in diagnosis. Larger studies are needed to include a more diverse population to confirm these predictive associations. Further data analyses are required to assess for co-morbidities and other characteristics in the identified at risk patient groups to help facilitate earlier diagnosis and appropriate preventative measures. References 1. Friese CR, Earle CC, Magazu LS, et al. Timeliness and quality of diagnostic care for medicare recipients with chronic lymphocytic leukemia. Cancer. 2011;117(7):1470-1477. doi:10.1002/cncr.25655 Disclosures No relevant conflicts of interest to declare.

scholarly journals Detection of Emerging Ibrutinib Resistance by Flow Cytometry in Patients with Chronic Lymphocytic Leukemia

2021 ◽  
Author(s):  
Ferenc Takács ◽  
Lili Kotmayer ◽  
Ágnes Czeti ◽  
Gábor Szalóki ◽  
László Tamás ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Kinase Inhibitor ◽  
Resistance Mutation ◽  
Lymphocytic Leukemia ◽  
Flow Cytometric ◽  
Clinical Resistance ◽  
Treatment Naïve ◽  
Droplet Pcr ◽  
Ibrutinib Resistance

Abstract Purpose: Bruton’s tyrosine kinase inhibitor ibrutinib has revolutionized the treatment of chronic lymphocytic leukemia (CLL). Although ibrutinib is a highly effective drug, during the treatment acquired ibrutinib resistance may occur and its early detection is an important issue. Our aim was to investigate several phenotypic markers on CLL cells to reveal changes in their expression during ibrutinib treatment.Methods: In our study 28 (treatment naive, ibrutinib sensitive, clinically ibrutinib resistant) peripheral blood (PB), and 6 paired PB and bone marrow (BM) samples were examined. The expression of several surface markers (CD69, CD184, CD86, CD185, CD27) was assessed by flow cytometry in each sample. Furthermore, the presence of the BTKC481S resistance mutation was tested using digital droplet PCR. In addition, we investigated the changes of the phenotype of CLL cells during ibrutinib treatment in one patient with acquired ibrutinib resistance.Results: The expression of CD27 decreased during ibrutinib therapy but increased again at the onset of clinical resistance. Expressions of CD69 and CD86 were also elevated at the onset of clinical ibrutinib resistance. The expression of CD86 showed correlation between PB and BM samples. Relapsed cases with high CD86 expression were positive for BTKC481S mutation. Our prospective study showed that the increases in the expression of CD27, CD69 and CD86 were detectable up to several months before the onset of clinical resistance.Conclusion: Our research suggests that the flow cytometric measurements of certain markers, especially CD86, may predict development of ibrutinib resistance, however, confirmatory experiments are still required.

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

Diagnostics ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1320
Author(s):  
Kristýna Pekárková ◽  
Jakub Soukup ◽  
Marie Kostelanská ◽  
Jan Širc ◽  
Zbyněk Straňák ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cord Blood ◽  
Extracellular Vesicles ◽  
Correlation Coefficients ◽  
Flow Cytometer ◽  
Liquid Biopsies ◽  
Physiological Conditions ◽  
Flow Cytometric ◽  
Term Births ◽  
And Control

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.

scholarly journals Flow cytometric measurements as a proxy for sporulation intensity in the cultured macroalga Ulva (Chlorophyta)

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Omri Nahor ◽  
Cristina F. Morales-Reyes ◽  
Gianmaria Califano ◽  
Thomas Wichard ◽  
Alexander Golberg ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Life Cycle ◽  
Abiotic Factors ◽  
Physiological Status ◽  
Seaweed Cultivation ◽  
Reproductive Growth ◽  
Biotic And Abiotic Factors ◽  
Flow Cytometric ◽  
Abiotic Stimuli ◽  
Fundamental Shift

Abstract Controlling the life cycle of the green macroalga Ulva (Chlorophyta) is essential to maintain its efficient aquaculture. A fundamental shift in cultivation occurs by transforming the thallus cells into gametangia and sporangia (sporulation), with the subsequent release of gametes and zoids. Sporulation occurrence depends on algal age and abiotic stimuli and is controlled by sporulation inhibitors. Thus, quantification of sporulation intensity is critical for identifying the biotic and abiotic factors that influence the transition to reproductive growth. Here, we propose to determine the sporulation index by measuring the number of released gametes using flow cytometry, in proportion to the total number of thallus cells present before the occurrence of the sporulation event. The flow cytometric measurements were validated by manually counting the number of released gametes. We observed a variation in the autofluorescence levels of the gametes which were released from the gametangia. High autofluorescence level correlated to phototactically active behaviour of the gametes. As autofluorescence levels varied between different groups of gametes related to their mobility, flow cytometry can also determine the physiological status of the gametes used as feedstock in seaweed cultivation.

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