Flow Cytometric Detection of Procoagulant Properties of Plasma from Patients with Clinically Confirmed Vaccine-Induced Immune Thrombotic Thrombocytopenia

Abstract Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines including ChAdOx1 nCoV-19 (AstraZeneca) vaccine. The putative mechanism involves formation of pathological anti-PF4 antibodies that activate platelets via the FcγRIIa receptor to drive thrombosis and the associated thrombocytopenia. Functional assays are important in the VITT diagnostic pathway as not all detectable PF4 antibodies are pathogenic. Detection of procoagulant platelets (platelets supporting thrombin generation) in presence of PF4 has been proposed as a diagnostic assay for VITT (Althaus et al). Procoagulant platelets are not typically generated in response to low level agonist stimulation; however, combination of ligand binding of G-protein coupled receptors (GPCR) (eg. PAR1) and ITAM linked receptors (eg. GPVI, CLEC2 and FcγRIIa) synergistically induce procoagulant platelet formation. Here, we describe an alternative flow cytometric assay to diagnose VITT. We hypothesized that priming of platelets with a PAR1 agonist at a level sufficient to release PF4, but insufficient to generate a significant procoagulant response in donor platelets, would provide a platform in which procoagulant response would be dependent on presence of FcγRIIa dependent procoagulant antibodies in patient plasma, without requirement for additional PF4. Methods: Our previously established flow cytometry-based procoagulant platelet assay (using cell death marker GSAO and P-selectin) was modified to incorporate exogenous patient plasma and performed on whole blood from healthy donors screened for FcγRIIa responsiveness (aggregation response to anti-CD9 antibody, ALB6), primed with 5 μM SFLLRN. The assay was performed on Australian patients referred for confirmatory VITT testing with probable VITT (confirmed thrombosis within 4-42 days of ChAdOx1 nCov-19 vaccination, D-Dimer > 5x ULN, platelets < 150 x 10 9/L or falling platelet count) after screening on PF4/heparin ELISA (Asserachrom HPIA IgG Assay, Stago Diagnostics). Procoagulant response was also measured in presence of 0.5 U/mL and 100 U/mL heparin, monoclonal FcγRIIa blocking antibody, IV.3, and intravenous immunoglobulin, IVIg. Some plasmas were incubated with ChAdOx1 nCoV-19 or SARS-CoV-2 spike protein. Flow cytometry positive patients were also tested by serotonin release assay (SRA) and multiplate aggregometry. Clinical correlation was obtained. Results: Citrated plasma from 49 unique patients with suspected VITT are reported. Plasma from ELISA+ve patients with clinical picture consistent with VITT (n=31), significantly increased the procoagulant platelet proportions in healthy donors in presence of 5 μM SFLLRN (p<0.0001, Figure 1A). This increase was not seen with plasma from healthy donors (n=14); or individuals exposed to ChAdOx1 nCov-19 vaccine without VITT: thrombocytopenic thrombosis patients who were ELISA-ve and SRA-ve (n=14); or low-level ELISA+ve patients without thrombocytopenia who were negative by either multiplate or SRA (n=4). The procoagulant platelet response induced by VITT positive plasma was reduced with low dose heparin (0.5 U/mL, p<0.01) except for 3 patients who showed a heparin-enhancing effect (Figure 1B). High dose heparin (100 U/mL, p<0.0001), IV.3 (10 µg/mL, p<0.0001) or IVIg (10 mg/mL, p<0.0001) abolished the procoagulant response (Figures 1C-D). The in vitro effect of IVIg was predictive of the in vivo response to IVIg therapy (Figure 1E). Addition of SARS-CoV-2 spike protein had no effect on the procoagulant platelet response. ChAdOx1 nCov-19 had an inconsistent effect on procoagulant platelet formation in presence of VITT plasma. Use of donors without a robust aggregation response to ALB6 resulted in false negative results. Conclusion: Induction of FcγRIIa dependent procoagulant response by patient plasma, suppressible by high dose heparin and IVIg, is highly indicative of VITT in the correct clinical circumstance. This assay modification of priming donor platelets from known FcγRIIa responsive donors with a GPCR agonist to potentiate the ITAM signaling from platelet activating immune complexes, results in a sensitive and specific assay. This may represent a functional platform that can be adopted into diagnostic laboratories to identify patients with platelet-activating antibodies and potentially predict treatment responses. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Is Zap-70 Protein Detection by Flow Cytometry a Reliable Tool in CLL Prognostic Evaluation?.

Blood ◽

10.1182/blood.v104.11.4771.4771 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4771-4771

Author(s):

Magali Le Garff-Tavernier ◽

Michel Ticchioni ◽

Rémi Letestu ◽

Martine Brissard ◽

Frédéric Davi ◽

...

Keyword(s):

Flow Cytometry ◽

T Cell ◽

B Cells ◽

Protein Expression ◽

B Cell ◽

Flow Cytometric Analysis ◽

Protein Detection ◽

Flow Cytometric ◽

Mutational Status ◽

Healthy Donors

Abstract Background : Expression of ZAP-70 protein has been shown to be correlated with mutational status of immunoglobulin heavy-chain variable region (IgVH) genes, a major prognostic factor in CLL. We investigated whether the detection of ZAP-70 protein by flow cytometric analysis using unconjugated and conjugated monoclonal antibodies (mAbs) could be applied securely in the workup of patients with CLL. Methods: Flow cytometric analysis of ZAP-70 protein was performed using the method described by Crespo et al (N Engl J Med2003;348:1764) with minor modifications. Both fresh and cryopreserved mononuclear cells from CLL patients and healthy donors were fixed and permeabilized using Fix and Perm kit (Caltag Laboratories), incubated with anti-ZAP-70 mAb (clone 2F3.2, Upstate Biotechnology) and then revealed with goat antimouse FITC mAb (Immunotech). Finally cells were incubated with CD3-APC, CD56-APC and CD19-PC5. We also tested 3 mAbs conjugated to various fluorochromes: 2F3.2-FITC (Upstate), 1E7.2-PE (eBioscience), 1E7.2-PE or -Alexa 488 (Caltag). ZAP-70 protein detection in B-cells was expressed either as a percentage of its expression in the T and NK-cells or as a ratio (R) of T-cell mean cell fluorescence (MCF) to B-cell MCF. Western blotting of protein lysates from purified B-cells was carried out to control results obtained by cytometry in 55 samples. Mutational status was defined using a cutoff of 98%. Results: In 13 healthy donors, the mean percentage of ZAP-70 protein expression obtained by flow cytometry with unconjugated mAb (clone 2F3.2) was 4.69% ± 1.93 [range 2–9%] and the R ratio was 6.64 ± 1.54 and > 4.8. In 83 B-CLL samples, ZAP-70 expression was determined using the same method and compared to IgVH mutational status. Results in table below show a 75% concordance between gene mutations and ZAP-70 expression when considering a percentage of positive B-cells > 20%. A better concordance (81%) is obtained with a threshold T-cell MCF/ B-cell MCF at 4 determined by Youden’s index. To note the high concordance (90%) between unmutated status and ZAP-70 + expression (19/21). Comparison with at least 1 of the 3 conjugated mAbs has been performed for 63 samples, with discordant results in our laboratories. 62 mutated IgVH samples 21 unmutated IgVH samples ZAP-70 B-Cells + ≤ 20 % : 43 > 20% : 19 T-cell MCF/B-cell MCF ≥ 4 : 48 < 4 : 19 Conclusions: Our data document the concordance between IgVH gene mutational status and ZAP-70 protein expression measured by flow cytometry, particularly in ZAP-70 negative samples. We found that the indirect method of labelling with unconjugated anti-ZAP-70 mAb remains until now, in our hands, the gold standard method compared to the available dyes conjugate mAbs.

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Diagnostic utility of flow cytometric immunophenotyping in myelodysplastic syndrome

Blood ◽

10.1182/blood.v98.4.979 ◽

2001 ◽

Vol 98 (4) ◽

pp. 979-987 ◽

Cited By ~ 187

Author(s):

Maryalice Stetler-Stevenson ◽

Diane C. Arthur ◽

Nicholas Jabbour ◽

Xiu Y. Xie ◽

Jeff Molldrem ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Diagnostic Utility ◽

Flow Cytometric Immunophenotyping ◽

Flow Cytometric ◽

Healthy Donors ◽

Number Of Patients ◽

Highly Sensitive ◽

History Of ◽

Diagnosis And Classification

The myelodysplastic syndromes (MDSs) are characterized by bilineage or trilineage dysplasia. Although diagnostic criteria are well established for MDS, a significant number of patients have blood and bone marrow findings that make diagnosis and classification difficult. Flow cytometric immunophenotyping is an accurate and highly sensitive method for detection of quantitative and qualitative abnormalities in hematopoietic cells. Flow cytometry was used to study hematopoietic cell populations in the bone marrow of 45 patients with straightforward MDS. The results were compared with those obtained in a series of patients with aplastic anemia, healthy donors, and patients with a history of nonmyeloid neoplasia in complete remission. The immunophenotypic abnormalities associated with MDS were defined, and the diagnostic utility of flow cytometry was compared, with morphologic and cytogenetic evaluations in 20 difficult cases. Although morphology and cytogenetics were adequate for diagnosis in most cases, flow cytometry could detect immunophenotypic abnormalities in cases when combined morphology and cytogenetics were nondiagnostic. It is concluded that flow cytometric immunophenotyping may help establish the diagnosis of MDS, especially when morphology and cytogenetics are indeterminate.

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Flow cytometry of bone marrow aspirates from neuroblastoma patients is a highly sensitive technique for quantification of low-level neuroblastoma

F1000Research ◽

10.12688/f1000research.53133.1 ◽

2021 ◽

Vol 10 ◽

pp. 947

Author(s):

Neha Jain ◽

Shaista Sattar ◽

Sarah Inglott ◽

Susan Burchill ◽

Jonathan Fisher ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Bone Marrow Involvement ◽

Flow Cytometric Analysis ◽

Standard Of Care ◽

Tumour Cells ◽

Street Hospital ◽

Low Level ◽

Bone Marrow Trephine ◽

Flow Cytometric

Background: Bone marrow involvement is an important aspect of determining staging of disease and treatment for childhood neuroblastoma. Current standard of care relies on microscopic examination of bone marrow trephine biopsies and aspirates respectively, to define involvement. Flow cytometric analysis of disaggregated tumour cells, when using a panel of neuroblastoma specific markers, allows for potentially less subjective determination of the presence of tumour cells. Methods: A retrospective review of sequential bone marrow trephine biopsies and aspirates, performed at Great Ormond Street Hospital, London, between the years 2015 and 2018, was performed to assess whether the addition of flow cytometric analysis to these standard of care methods provided concordant or additional information. Results: There was good concurrence between all three methods for negative results 216/302 (72%). Positive results had a concordance of 52/86 (61%), comparing samples positive by flow cytometry and positive by either or both cytology and histology. Of the remaining samples, 20/86 (23%) were positive by either or both cytology and histology, but negative by flow cytometry. Whereas 14/86 (16%) of samples were positive only by flow cytometry. Conclusions: Our review highlights the ongoing importance of expert cytological and histological assessment of bone marrow results. Flow cytometry is an objective, quantitative method to assess the level of bone marrow disease in aspirates. In this study, flow cytometry identified low-level residual disease that was not detected by cytology or histology. The clinical significance of this low-level disease warrants further investigation.

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Flow cytometry of bone marrow aspirates from neuroblastoma patients is a highly sensitive technique for quantification of low-level neuroblastoma

F1000Research ◽

10.12688/f1000research.53133.2 ◽

2021 ◽

Vol 10 ◽

pp. 947

Author(s):

Neha Jain ◽

Shaista Sattar ◽

Sarah Inglott ◽

Susan Burchill ◽

Jonathan Fisher ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Bone Marrow Involvement ◽

Flow Cytometric Analysis ◽

Standard Of Care ◽

Tumour Cells ◽

Street Hospital ◽

Low Level ◽

Bone Marrow Trephine ◽

Flow Cytometric

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Residual Clonal Plasma Cells Detected By Flow Cytometry at 10-4level within Autologous Stem Cell Grafts Is Associated with Significantly Less Overall Survival

Blood ◽

10.1182/blood-2018-99-111794 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3438-3438

Author(s):

Guldane Cengiz Seval ◽

Klara Dalva ◽

Sule Mine Bakanay ◽

Mustafa Merter ◽

Ugur Sahin ◽

...

Keyword(s):

Flow Cytometry ◽

Overall Survival ◽

Hematopoietic Cell Transplantation ◽

Plasma Cells ◽

High Dose ◽

Statistical Tool ◽

Aberrant Expression ◽

Post Transplant ◽

Flow Cytometric ◽

The Impact

Abstract Introduction:High-dose Melphalan followed by autologous hematopoietic cell transplantation (ASCT) compared to conventional chemotherapies has been shown to improve survival and progression free survival (PFS) in eligible patients with multiple myeloma (MM). However cells that are resistant to the high-dose therapy and remaining in the patient and/or PBSC grafts may eventually lead to relapse. Over the past decade, data have been published on the role of circulating plasma cells (CPCs) as a poor prognostic feature at the time of diagnosis and prior to auto HSCT. But these studies have not sought this issue exclusively in PBSC grafts. The aim of this study was to analyze the impact of flow cytometric measurement of residual clonal cells within the apheresis products on outcome following ASCT. Materials and Methods:Patients with a diagnosis of MM who underwent auto HSCT at our center between January 2008- Mart 2018 were prospectively analyzed. PBSC grafts were tested for the presence of abnormal PCs (APC) and the number of normal PCs (NPC) by multi-parameter flow cytometry (FCM). Standard panel was set up with CD138FITC/CD38PE/CD45ECD/CD56PC5, CD45-CD56+PCs were identified as APC if any aberrant expression (including; CD20(loss)CD27(loss)CD28(gain)CD33(loss)CD34(gain)CD81(loss) CD117(gain)) is detected at diagnosis, the corresponding antibody was also added to the panel. Since maintenance was not an approved treatment in myeloma most patients did not receive any. Outcome was determined as response to transplant, PFS and overall survival (OS) in months by Kaplan-Meier analysis using SPSS (IBM SPSS Statistics 21; IBM Corp., Chicago, IL) statistical tool kit. Results: A total of 209 patients with MM with routine assessment for APC in the apheresis product were included in the analysis. Of these patients, 195 who underwent ASCT, met the predetermined criteria for inclusion. There were 81 (41.5%) female and 114 (58.5%) male patients. The median age at diagnosis of MM was 56 years (range, 31-71 years). The interval from the time of diagnosis to ASCT was median 7 months and median follow-up from ASCT was 49 months (range, 3-198 months). Among 195 patients, 36 (18.5%) had evidence of APC contamination in the PBSC grafts ranging between 1x10-4-12.8 x10-4of total cells. Subtypes of MM were similar among those w/wo APC. Seventy-four patients had pre and post ASCT PET-CT imaging done with 28.2% still active lesions post-ASCT. There was no correlation between PET-CR and absence of APC in grafts. Neither was there a statistically significant association between disease response <VGPR at mobilization and the number of APC in the apheresis product. Post-transplant responses were closely associated with pre-transplant responses. A total of 25 and 57 patients relapsed/progressed in respectively 6 and 12 months after ASCT. Post-transplant response was significantly associated with relapse at 6 months (≥VGPR vs. <VGPR; 37.5% vs. 62.5%, p=0.000). Estimated median OS was significantly different between patients w/o APC contamination; 20.4±8.1 and 63.3±3.8, respectively (p=0.000) (Figure 1). There were no differences in OS among the patients achieving VGPR/CR at the time of mobilization and also two months after the ASCT. We performed a subgroup analysis of patients in grouping together VGPR/CR or only CR and among those patients presence of APC was correlated with worse OS (Figure 1). PFS at 2 years were 60.3±4.3% and 75.6±9.7% in patients receiving APC free PBSC grafts compared with grafts with evidence of APC contamination, respectively (p=0.595). Discussion:PostASCT immune response was predictive for OS only when combined with APC results. Our study provides new insight suggesting a survival advantage value of residual APC detected by FCM, even at a low sensitivity level, on OS but not PFS following ASCT. The impact of such analysis may be more relevant on PFS with better assessment of residual APC with next generation flow cytometric/sequencing approaches while taking into account maintenance. Disclosures Ilhan: Roche: Speakers Bureau; BMS: Speakers Bureau; Alexion: Speakers Bureau; Celgene: Speakers Bureau.

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A Multiplex Flow Cytometry Bead Assay For Simultaneous Detection Of IgG, IgA, IgM Heparin-Dependent Antibodies

Blood ◽

10.1182/blood.v122.21.3551.3551 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3551-3551

Author(s):

Mia Sullivan ◽

Scott Grady ◽

Janice G McFarland ◽

Brian R Curtis

Keyword(s):

Flow Cytometry ◽

Sensitivity And Specificity ◽

Simultaneous Detection ◽

High Sensitivity ◽

Thrombin Inhibitors ◽

High Dose ◽

Good Sensitivity ◽

Antibody Isotype ◽

Igm Antibodies ◽

Dose Heparin

Abstract Heparin-Induced Thrombocytopenia (HIT) is an adverse reaction to heparin, in which affected patients produce platelet activating antibodies specific for complexes of platelet-factor 4 (PF4) and heparin (HPF4) resulting in a prothrombotic condition that in severe cases can be life-threatening. HIT is not rare, since as many as 600K people/year are affected. It is therefore important that a quick and accurate diagnosis of HIT be made, which requires both compatible clinical findings and serologic evidence of a heparin dependent antibody. First line testing consists of immunologic assays that detect IgG, IgA and IgM antibodies targeting PF4 in a complex with heparin or another polyanion. The most widely used assay is the PF4 ELISA, in which HPF4 bound to the wells of a microtiter plate is the target antigen. The ELISA has high sensitivity (near 100%) for antibody detection but a low positive predictive value for HIT diagnosis. Therefore, samples giving positive ELISA results are often retested using a functional assay like the serotonin release assay (SRA) that is more specific for HIT diagnosis. However, the SRA is difficult to perform and interpret and is not widely available. Further, the ELISA is time-consuming, costly, and most versions cannot distinguish IgG, IgA, IgM (IgG/A/M) antibodies. Knowledge of the antibody isotype can be useful when ELISA and SRA results are discrepant. For instance, strong ELISA results due to clinically insignificant IgM antibodies would not be detected by SRA, and additional testing to confirm IgM antibody isotype can lead to a delay in HIT diagnosis, and in some cases continuation of high cost treatments with direct thrombin inhibitors (DTI). Moreover, there is evidence that some IgA and IgM antibodies may cause HIT. Despite the high sensitivity of the ELISA and high specificity of the SRA, the shortcomings mentioned for these tests highlight the need for improved methods to support a diagnosis of HIT. We report development of a multiplex flow cytometry bead assay (MFBA) for simultaneous detection of IgG/A/M heparin-dependent antibodies that allows higher throughput and has sensitivity and specificity comparable to the PF4 ELISA. For the MFBA, polystyrene beads, to which chemically depolymerized heparin is covalently linked are incubated with purified platelet PF4 to produce HPF4-bead targets. Lyophilized HPF4 beads are stable for at least 3 months. HPF4-beads are incubated with serum, washed, and incubated with anti-human -IgG, -IgA, and –IgM, each with different fluorescent labels for detection of HIT antibodies by flow cytometry. A total of 164 patient samples could be tested by a single technologist in 8 hours. Serum samples from 300 patients with suspected HIT and previously tested by SRA were tested by both MFBA and an in-house PF4 ELISA including high dose heparin (100 U/L) confirmation step. Studies were approved by institutional IRB. MFBA results showed good sensitivity (87% IgG, 63% IgA, 36% IgM) for IgG, good specificity (96% IgG, 91% IgA, 93% IgM) for all isotypes when compared to the ELISA, and good sensitivity/specificity (93%/91%) compared to SRA. The area under the curve (AUC) calculated for the receiver operating characteristic graphs compared to the SRA were excellent (0.9-1.0) for both MFBA (AUC = 0.95) and PF4 ELISA (AUC = 0.98). All samples positive by MFBA inhibited with high dose heparin suggesting that this extra step is not required, and 3 samples that tested IgG positive in both MFBA and SRA gave indeterminate results (not inhibited with high dose heparin) in ELISA. Overall, these data show that the MFBA has accuracy, sensitivity, and specificity for detection of IgG antibodies comparable to the PF4 ELISA and the “gold standard” SRA with the additional advantage of simultaneously distinguishing IgG/A/M antibody isotypes, which will lead to reduced confirmation testing. Sensitivity of MFBA for detection of IgA and IgM antibodies was significantly lower compared to ELISA, but this reflects the assay design intended to increase specificity of the MFBA for these two isotypes. In addition to its multiplex capabilities, the MFBA allows for higher sample throughput and shortened turn-around times that will contribute to overall improved patient care. Disclosures: No relevant conflicts of interest to declare.

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Precision therapeutic targets for COVID-19

Virology Journal ◽

10.1186/s12985-021-01526-y ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Zachary A. Krumm ◽

Grace M. Lloyd ◽

Connor P. Francis ◽

Lith H. Nasif ◽

Duane A. Mitchell ◽

...

Keyword(s):

Distress Syndrome ◽

Replication Cycle ◽

Spike Protein ◽

Small Subset ◽

High Dose ◽

Drug Cocktail ◽

Proteolytic Activation ◽

Main Protease ◽

Clinical Drugs ◽

Length Of Hospitalization

AbstractBeginning in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a novel pathogen that causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 has infected more than 111 million people worldwide and caused over 2.47 million deaths. Individuals infected with SARS-CoV-2 show symptoms of fever, cough, dyspnea, and fatigue with severe cases that can develop into pneumonia, myocarditis, acute respiratory distress syndrome, hypercoagulability, and even multi-organ failure. Current clinical management consists largely of supportive care as commonly administered treatments, including convalescent plasma, remdesivir, and high-dose glucocorticoids. These have demonstrated modest benefits in a small subset of hospitalized patients, with only dexamethasone showing demonstrable efficacy in reducing mortality and length of hospitalization. At this time, no SARS-CoV-2-specific antiviral drugs are available, although several vaccines have been approved for use in recent months. In this review, we will evaluate the efficacy of preclinical and clinical drugs that precisely target three different, essential steps of the SARS-CoV-2 replication cycle: the spike protein during entry, main protease (MPro) during proteolytic activation, and RNA-dependent RNA polymerase (RdRp) during transcription. We will assess the advantages and limitations of drugs that precisely target evolutionarily well-conserved domains, which are less likely to mutate, and therefore less likely to escape the effects of these drugs. We propose that a multi-drug cocktail targeting precise proteins, critical to the viral replication cycle, such as spike protein, MPro, and RdRp, will be the most effective strategy of inhibiting SARS-CoV-2 replication and limiting its spread in the general population.

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

Cancer Biomarkers ◽

10.3233/cbm-203049 ◽

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.

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

Diagnostics ◽

10.3390/diagnostics11081320 ◽

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.

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

Botanica Marina ◽

10.1515/bot-2020-0050 ◽

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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