scholarly journals Evaluation of an Alternative Staining Method Using SYTO 13 to Determine Reticulated Platelets

2019 ◽  
Vol 119 (05) ◽  
pp. 779-785 ◽  
Author(s):  
Laura Hille ◽  
Marco Cederqvist ◽  
Julia Hromek ◽  
Christian Stratz ◽  
Dietmar Trenk ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Reactivity ◽  
Dependent Manner ◽  
Reticulated Platelets ◽  
Automated Assay ◽  
Rna Quantification ◽  
Time Period ◽  
Immature Platelet Fraction ◽  
Syto 13

AbstractReticulated platelets reflect the rate of platelet turnover and represent the youngest circulating platelets in peripheral blood. Reticulated platelets contain residual ribonucleic acid (RNA) from megakaryocytes which is lost in a time-dependent manner and can be transcribed into proteins even in the absence of a nucleus. An increased proportion of reticulated platelets is associated with higher platelet reactivity, cardiovascular events and mortality. At present, a fully automated assay system (SYSMEX haematology analyser) is available for analysis. This method, however, is not suitable for extended laboratory investigations like subsequent cell sorting. Flow cytometry analysis after staining with thiazole orange (TO) is frequently used in such settings despite several limitations. Here, we describe a new assay for determination of reticulated platelets by flow cytometry using the nucleic acid staining dye SYTO 13 and compare it with SYSMEX and TO staining as current standards. A significant correlation between immature platelet fraction (IPF) determined by SYSMEX XE-2100 analyser and results obtained with the SYTO 13-based assay was observed (r = 0.668, p < 0.001) which was stable during a reasonable time period. In contrast, the correlation between TO staining and IPF was weaker (r = 0.478, p = 0.029) and lost after 90 minutes of staining. SYTO 13 staining of platelets enabled sorting of RNAlow and RNArich platelets which was confirmed by RNA quantification of sorted platelets. Except for fixation of platelets, sorting of these platelet sub-populations was stable under various experimental settings. In summary, determination of reticulated platelets with the new SYTO 13 assay offers distinct technical advantages enabling further laboratory processing.

Decreased platelet reactivity identified by whole blood flow cytometry in Fanconi anaemia patients

2007 ◽  
Vol 98 (12) ◽  
pp. 1291-1297 ◽  
Author(s):  
Susanne Holzhauer ◽  
Ana-Gabriela Sitaru ◽  
Wolfram Ebell ◽  
Detlev Schindler ◽  
Helmut Hanenberg ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Blood Flow ◽  
Platelet Function ◽  
Whole Blood ◽  
Platelet Reactivity ◽  
Bone Marrow Failure ◽  
Receptor Expression ◽  
Platelet Glycoprotein ◽  
Bleeding Tendency ◽  
Reticulated Platelets

Summarydisorder characterized by congenital anomalies and a high risk for bone marrow failure and cancer. Bleeding is a frequent complication in FA, leading to substantial morbidity and mortality. Thrombocytopenia is a major factor leading to this complication, but the bleeding tendency of FA patients often exceeds what one might expect based on their platelet counts. We therefore investigated if alterations of platelet function contribute to the bleeding tendency of FA patients. We assessed platelet function in 11 FA patients and 23 controls with whole blood flow cytometry. We analyzed the expression of platelet membrane glycoprotein receptors, reactivity of platelets to physiologic agonists and the proportion of young platelets. In FA patients platelet PAC-1 after stimulation with thrombin receptor activating peptide (TRAP) and adenosine diphosphate (ADP) were 15–70% lower than in controls. We found no or only minor differences of platelet glycoprotein receptor expression between groups. While the proportion of reticulated platelets was not different, the absolute number of reticulated platelets was markedly lower in FA patients. Our data show that FA is associated with reduced platelet reactivity, which may contribute to the high bleeding tendency in FA patients. Whole blood flow cytometry is a suitable method for analysis of platelet function in FA patients.

scholarly journals Detection ofVibrio choleraeO1 and O139 in environmental waters of rural Bangladesh: a flow-cytometry-based field trial

2014 ◽  
Vol 143 (11) ◽  
pp. 2330-2342 ◽  
Author(s):  
L. RIGHETTO ◽  
R. U. ZAMAN ◽  
Z. H. MAHMUD ◽  
E. BERTUZZO ◽  
L. MARI ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Flow Cytometer ◽  
Environmental Waters ◽  
Fast Determination ◽  
River Waters ◽  
Quantitative Measurements ◽  
Time Period ◽  
Measurement Protocol ◽  
Hydrological System

SUMMARYPresence ofVibrio choleraeserogroups O1 and O139 in the waters of the rural area of Matlab, Bangladesh, was investigated with quantitative measurements performed with a portable flow cytometer. The relevance of this work relates to the testing of a field-adapted measurement protocol that might prove useful for cholera epidemic surveillance and for validation of mathematical models. Water samples were collected from different water bodies that constitute the hydrological system of the region, a well-known endemic area for cholera. Water was retrieved from ponds, river waters, and irrigation canals during an inter-epidemic time period. Each sample was filtered and analysed with a flow cytometer for a fast determination ofV. choleraecells contained in those environments. More specifically, samples were treated with O1- and O139-specific antibodies, which allowed precise flow-cytometry-based concentration measurements. Both serogroups were present in the environmental waters with a consistent dominance ofV. choleraeO1. These results extend earlier studies whereV. choleraeO1 and O139 were mostly detected during times of cholera epidemics using standard culturing techniques. Furthermore, our results confirm that an important fraction of the ponds’ host populations ofV. choleraeare able to self-sustain even when cholera cases are scarce. Those contaminated ponds may constitute a natural reservoir for cholera endemicity in the Matlab region. Correlations ofV. choleraeconcentrations with environmental factors and the spatial distribution ofV. choleraepopulations are also discussed.

The Immature Platelet Fraction Is Sensitive to the Platelet Size and a Useful Parameter for Screening for Macrothrombocytopenia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1097-1097
Author(s):  
Koji Miyazaki ◽  
Yukako Koike ◽  
Mikio Danbara ◽  
Ryouichi Horie ◽  
Masaaki Higashihara
Keyword(s):  
Cold Storage ◽  
Conflicts Of Interest ◽  
Large Cell ◽  
Dependent Manner ◽  
Invasive Treatment ◽  
Distribution Width ◽  
Reticulated Platelets ◽  
Platelet Size ◽  
Immature Platelet Fraction ◽  
Platelet Aggregates

Abstract Abstract 1097 The immature platelet fraction (IPF) is a useful parameter indicating thrombopoietic activity to differentiate the causes of thrombocytopenia. We previously reported that the percentage of IPF (%IPF) is negatively correlated to the platelet count among ITP patients, and not among myelodysplastic syndrome (MDS) patients. We also noticed that some MDS patients exhibited extremely high %IPF values, which were dissociated from the percentages of reticulated platelets (%RP) measured by flow cytometry. Such discrepancies were also observed in hereditary macrothrombocytopenias, which are sometimes difficult to be distinguished from ITP, because ITP also exhibits increased number of reticulated platelets in a slightly larger size. Once misdiagnosed, a hereditary macrothrombocytopenia patient might be subjected to an invasive treatment such as splenectomy. In order to avoid such mistreatments, a clear marker to differentiate macrothrombocytopenia is desperately needed. In this study, we investigated the IPF values of 16 individuals from 12 families with various hereditary macrothrombocytopenia in order to clarify whether the IPF could be a useful marker to distinguish macrothrombocytopenia from ITP, and examined the IPF during EDTA aggregation and cold-storage to elucidate how platelet size may affect the IPF value. The IPF values were about 5 times higher in MYH9 disorders (%IPF 48.0 ± 1.8) and about 1.5 times higher in other macrothrombocytopenias (%IPF 17.0 ± 2.2) than immune thrombocytopenic patients with similar platelet counts (%IPF 9.3 ± 0.4). These results suggested that the platelet size can affect the IPF value. However it still remains the possibility that some factors specific to these macrothrombocytopenias other than the platelet size might make an influence on the IPF, because the characteristic of large platelets in hereditary macrothrombocytopenia has not been fully understood, and no one knows whether large platelets are functionally identical to normal platelets except for the size. In order to exclude the possibility, we next examined the changes of IPF values during EDTA aggregation and cold-storage. The IPF was significantly increased during storage in a time dependent manner along with forming platelet clumps. The IPF was strongly influenced by a few tiny platelet aggregates rather than other platelet indices, such as mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet distribution width (PDW). In conclusion, the IPF is susceptible to the platelet size, and could be a useful parameter for screening of macrothrombocytopenia from ITP. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Modeling Population Spatial-Temporal Distribution Using Taxis Origin and Destination Data

2021 ◽  
Vol 13 (7) ◽  
pp. 3727
Author(s):  
Fatema Rahimi ◽  
Abolghasem Sadeghi-Niaraki ◽  
Mostafa Ghodousi ◽  
Soo-Mi Choi
Keyword(s):  
Regression Analysis ◽  
Mean Squared Error ◽  
Population Distribution ◽  
Temporal Distribution ◽  
Coefficient Of Determination ◽  
Temporal Modeling ◽  
Location Data ◽  
Time Period ◽  
Proposed Model

During dangerous circumstances, knowledge about population distribution is essential for urban infrastructure architecture, policy-making, and urban planning with the best Spatial-temporal resolution. The spatial-temporal modeling of the population distribution of the case study was investigated in the present study. In this regard, the number of generated trips and absorbed trips using the taxis pick-up and drop-off location data was calculated first, and the census population was then allocated to each neighborhood. Finally, the Spatial-temporal distribution of the population was calculated using the developed model. In order to evaluate the model, a regression analysis between the census population and the predicted population for the time period between 21:00 to 23:00 was used. Based on the calculation of the number of generated and the absorbed trips, it showed a different spatial distribution for different hours in one day. The spatial pattern of the population distribution during the day was different from the population distribution during the night. The coefficient of determination of the regression analysis for the model (R2) was 0.9998, and the mean squared error was 10.78. The regression analysis showed that the model works well for the nighttime population at the neighborhood level, so the proposed model will be suitable for the day time population.

A Cuticle Collagen Encoded by the lon-3 Gene May Be a Target of TGF-β Signaling in Determining Caenorhabditis elegans Body Shape

Genetics ◽  
2002 ◽  
Vol 162 (4) ◽  
pp. 1631-1639
Author(s):  
Yo Suzuki ◽  
Gail A Morris ◽  
Min Han ◽  
William B Wood
Keyword(s):  
Caenorhabditis Elegans ◽  
Body Shape ◽  
Small Body ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Cellular Mechanisms ◽  
C Elegans ◽  
Gene Expression Analyses ◽  
Dose Dependent

Abstract The signaling pathway initiated by the TGF-β family member DBL-1 in Caenorhabditis elegans controls body shape in a dose-dependent manner. Loss-of-function (lf) mutations in the dbl-1 gene cause a short, small body (Sma phenotype), whereas overexpression of dbl-1 causes a long body (Lon phenotype). To understand the cellular mechanisms underlying these phenotypes, we have isolated suppressors of the Sma phenotype resulting from a dbl-1(lf) mutation. Two of these suppressors are mutations in the lon-3 gene, of which four additional alleles are known. We show that lon-3 encodes a collagen that is a component of the C. elegans cuticle. Genetic and reporter-gene expression analyses suggest that lon-3 is involved in determination of body shape and is post-transcriptionally regulated by the dbl-1 pathway. These results support the possibility that TGF-β signaling controls C. elegans body shape by regulating cuticle composition.

scholarly journals The Past, Present and Future of Flow Cytometry in Central Nervous System Malignancies

2021 ◽  
Vol 4 (1) ◽  
pp. 11
Author(s):  
Evrysthenis Vartholomatos ◽  
George Vartholomatos ◽  
George A. Alexiou ◽  
Georgios S. Markopoulos
Keyword(s):  
Central Nervous System ◽  
Flow Cytometry ◽  
Nervous System ◽  
Therapeutic Agents ◽  
Phenotypic Characterization ◽  
Cell Phenotype ◽  
Analytical Technique ◽  
The Past ◽  
Current Review

Central nervous system malignancies (CNSMs) are categorized among the most aggressive and deadly types of cancer. The low median survival in patients with CNSMs is partly explained by the objective difficulties of brain surgeries as well as by the acquired chemoresistance of CNSM cells. Flow Cytometry is an analytical technique with the ability to quantify cell phenotype and to categorize cell populations on the basis of their characteristics. In the current review, we summarize the Flow Cytometry methodologies that have been used to study different phenotypic aspects of CNSMs. These include DNA content analysis for the determination of malignancy status and phenotypic characterization, as well as the methodologies used during the development of novel therapeutic agents. We conclude with the historical and current utility of Flow Cytometry in the field, and we propose how we can exploit current and possible future methodologies in the battle against this dreadful type of malignancy.

scholarly journals KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

2021 ◽  
Author(s):  
Diana Spiegelberg ◽  
Jonas Stenberg ◽  
Pascale Richalet ◽  
Marc Vanhove
Keyword(s):  
Flow Cytometry ◽  
Live Cells ◽  
Time Resolved ◽  
Surface Receptors ◽  
Full Characterization ◽  
End Point ◽  
Titration Curves ◽  
Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

Association of Lipoprotein(a) levels with intrinsic and on-clopidogrel platelet reactivity

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
A Kille ◽  
T Nuehrenberg ◽  
C.M Valina ◽  
F.J Neumann ◽  
W Hochholzer
Keyword(s):  
Flow Cytometry ◽  
Platelet Function ◽  
Platelet Reactivity ◽  
Laboratory Data ◽  
Secondary Analysis ◽  
Structural Similarity ◽  
Lipoprotein A ◽  
Causal Risk Factor ◽  
Premature Cardiovascular Disease ◽  
Potential Association

Abstract Background Lipoprotein(a) [Lp(a)] is an independent, genetic, and causal risk factor for premature cardiovascular disease (CVD). Laboratory data have suggested an interaction of Lp(a) with platelet function, potentially caused by its structural similarity to plasminogen. So far, the potential association of Lp(a) with platelet activation and reactivity has not been well established in larger clinical cohorts. Methods This secondary analysis of the EXCELSIOR study analyzed intrinsic platelet reactivity before loading with clopidogrel 600mg and on-treatment platelet reactivity tested 24 hours following loading in patients undergoing elective coronary angiography. Platelet reactivity was tested by optical aggregometry as final aggregation after 5 min following stimulation with 5μM ADP. Platelet reactivity was also assessed by flow cytometry (expression of CD62P and PAC1) following stimulation with ADP and TRAP. Levels of Lp(a) on admission of each patient were immediately measured from fresh samples in a central laboratory. Results The present analysis included 2046 patients. Levels of Lp(a) ranged between 0 and 332 mg/dl. Results for intrinsic (p=0.80) and on-clopidogrel platelet reactivity (p=0.81) did not differ between quartiles of Lp(a) levels (Figure). Flow cytometry analyses confirmed these findings. Conclusion The present data do not support the hypothesis of an interaction of Lp(a) with platelet function. This finding might be important to define the safety of evolving therapeutic options for lowering Lp(a). Funding Acknowledgement Type of funding source: None

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