Viscoelastic Capillary Flow Cytometry Serhatlioglu, Murat; Jensen, Emil Alstrup; Niora, Maria ...
Advanced NanoBiomed Research (Online),
February 2023, 2023-02-00, 2023-02-01, Volume:
3, Issue:
2
Journal Article
Peer reviewed
Open access
A compact microfluidic flow cytometer is demonstrated, comprising viscoelastic flow focusing in fused silica capillaries and a fiber optical interface. Viscoelastic flow focusing enables simple ...device design and operation with a single‐inlet/outlet fluidic configuration. Fused silica capillaries with different inner diameters are effortlessly interchanged to eliminate blockage ratio limitations and enable single‐train particle focusing for a wide range of particle sizes and geometries. The compact system is mounted on an inverted microscope for easy integration with optical imaging and other optofluidic modalities, such as optical trapping and particle sorting. A real‐time cytometric analysis of three channels, forward scattering, side scattering, and fluorescence detection, is performed on LABVIEW. A throughput of 3500 events s−1 is performed on particles of sizes ranging from 2 to 20 μm, using capillaries of different inner diameters ranging from 30 to 75 μm. The outer diameter of all capillaries is identical to the cladding diameter of the applied optical fibers. This enables easy exchange and precise optical alignment of fibers and capillaries on a microfabricated jig. The performance of the microfluidic flow cytometer is benchmarked using polystyrene calibration beads, poly(lactic‐co‐glycolic acid) particles, erythrocytes, THP‐1 leukemic monocytes, and human metaphase chromosomes.
A compact microfluidic flow cytometer (MFC) comprising viscoelastic flow focusing fused silica capillaries and a fiber optical interface is presented. The MFC is mounted on an inverted microscope for easy integration with other optofluidic modalities. Viscoelastic MFC with a throughput of 3500 events s−1 is performed on various type of particles, with sizes ranging from 2 to 20 μm.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
23.
How to evaluate PCR assays for the detection of low-level DNA Clausen, Frederik Banch; Urhammer, Emil; Rieneck, Klaus ...
APMIS : acta pathologica, microbiologica et immunologica Scandinavica,
September 2015, Volume:
123, Issue:
9
Journal Article
Peer reviewed
Open access
High sensitivity of PCR‐based detection of very low copy number DNA targets is crucial. Much focus has been on design of PCR primers and optimization of the amplification conditions. Very important ...are also the criteria used for determining the outcome of a PCR assay, e.g. how many replicates are needed and how many of these should be positive or what amount of template should be used? We developed a mathematical model to obtain a simple tool for quick PCR assay evaluation before laboratory optimization and validation procedures. The model was based on the Poisson distribution and the Binomial distribution describing parameters for singleplex real‐time PCR‐based detection of low‐level DNA. The model was tested against experimental data of diluted cell‐free foetal DNA. Also, the model was compared with a simplified formula to enable easy predictions. The model predicted outcomes that were not significantly different from experimental data generated by testing of cell‐free foetal DNA. Also, the simplified formula was applicable for fast and accurate assay evaluation. In conclusion, the model can be applied for evaluation of sensitivity of real‐time PCR‐based detection of low‐level DNA, and may also assist in design of new assays before standard laboratory optimization and validation is initiated.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Background and ObjectivesFor 5 years, routine genotyping has been performed for selected blood groups of blood donors in the Copenhagen Capital Region, Denmark. The result is summarized in the ...following.Materials and MethodsGenotyping was carried out by an external service provider using the competitive allele specific PCR (KASP) technology. The genotypes were returned to the blood bank and translated into phenotypes by a proprietary IT application.ResultsIn total, 65 alleles from 16 blood group systems (ABO, MNS, Rh, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Dombrock, Colton, Landsteiner‐Wiener, Cromer, Knops, Vel, secretor status) and the HPA1, HPA5 and HPA15 antigens were interrogated. After translation, phenotypes were imported into the laboratory information management system of the blood bank. The results from 31,538 genotyped blood donors were used to calculate the allele frequencies for a Danish blood donor population. ABO genotyping was done for sample ID purposes. Determination of the 1061delC single nucleotide polymorphism (SNP) (NM_020469.2), most frequently characteristic of ABO*A2, was validated against a series of 1287 samples with Dolichos biflorus lectin determination of the A1 phenotype.ConclusionWe report allele frequencies and phenotype frequencies for 16 blood groups from a total of 31,538 genotyped blood donors. Blood products were supplied from a total of 64,312 active blood donors, and of these active blood donors 25,396 (39.5%) were genotyped. These donors represent a valuable resource for patient treatment. This genotyping has enabled the provision of rare genotyped donor blood for patients with alloantibodies and rare reagent cells for serology.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The defective generation or function of regulatory T (Treg) cells in autoimmune disease contributes to chronic inflammation and tissue injury. We report the identification of FoxA1 as a transcription ...factor in T cells that, after ectopic expression, confers suppressive properties in a newly identified Treg cell population, herein called FoxA1(+) Treg cells. FoxA1 bound to the Pdl1 promoter, inducing programmed cell death ligand 1 (Pd-l1) expression, which was essential for the FoxA1(+) Treg cells to kill activated T cells. FoxA1(+) Treg cells develop primarily in the central nervous system in response to autoimmune inflammation, have a distinct transcriptional profile and are CD4(+)FoxA1(+)CD47(+)CD69(+)PD-L1(hi)FoxP3(-). Adoptive transfer of stable FoxA1(+) Treg cells inhibited experimental autoimmune encephalomyelitis in a FoxA1-and Pd-l1-dependent manner. The development of FoxA1(+) Treg cells is induced by interferon-β (IFN-β) and requires T cell-intrinsic IFN-α/β receptor (Ifnar) signaling, as the frequency of FoxA1(+) Treg cells was reduced in Ifnb(-/-) and Ifnar(-/-) mice. In individuals with relapsing-remitting multiple sclerosis, clinical response to treatment with IFN-β was associated with an increased frequency of suppressive FoxA1(+) Treg cells in the blood. These findings suggest that FoxA1 is a lineage-specification factor that is induced by IFN-β and supports the differentiation and suppressive function of FoxA1(+) Treg cells.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Abstract
Background and Objectives
For 5 years, routine genotyping has been performed for selected blood groups of blood donors in the Copenhagen Capital Region, Denmark. The result is summarized in ...the following.
Materials and Methods
Genotyping was carried out by an external service provider using the competitive allele specific PCR (KASP) technology. The genotypes were returned to the blood bank and translated into phenotypes by a proprietary IT application.
Results
In total, 65 alleles from 16 blood group systems (ABO, MNS, Rh, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Dombrock, Colton, Landsteiner‐Wiener, Cromer, Knops, Vel, secretor status) and the HPA1, HPA5 and HPA15 antigens were interrogated. After translation, phenotypes were imported into the laboratory information management system of the blood bank. The results from 31,538 genotyped blood donors were used to calculate the allele frequencies for a Danish blood donor population.
ABO
genotyping was done for sample ID purposes. Determination of the 1061delC single nucleotide polymorphism (SNP) (NM_020469.2), most frequently characteristic of
ABO*A2
, was validated against a series of 1287 samples with
Dolichos biflorus
lectin determination of the A1 phenotype.
Conclusion
We report allele frequencies and phenotype frequencies for 16 blood groups from a total of 31,538 genotyped blood donors. Blood products were supplied from a total of 64,312 active blood donors, and of these active blood donors 25,396 (39.5%) were genotyped. These donors represent a valuable resource for patient treatment. This genotyping has enabled the provision of rare genotyped donor blood for patients with alloantibodies and rare reagent cells for serology.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Immunoglobulinsfrom individuals with immunity to malaria have a strong antiparasiticeffect when transferred to Plasmodium falciparum malariainfected patients. One prominent target of antiparasitic ...antibodies isthe merozoite surface antigen 3 (MSP-3). We have investigated theantibody response against MSP-3 residues 194 to 257(MSP-3₁₉₄₋₂₅₇) on the molecular level. mRNA fromperipheral blood leukocytes from clinically immune individuals was usedas a source of Fab (fragment antibody) genes. A Fab-phage displaylibrary was made, and three distinct antibodies designated RAM1, RAM2,and RAM3 were isolated by panning. Immunoglobulin G1 (IgG1) and IgG3full-length antibodies have been produced in CHO cells. Reactivity withthe native parasite protein was demonstrated by immunofluorescencemicroscopy, flow cytometry, and immunoblotting. Furthermore, theantiparasitic effect of RAM1 has been tested in vitro in anantibody-dependent cellular inhibition (ADCI) assay. Both the IgG1 andthe IgG3 versions of the antibody show an inhibitory effect on parasitegrowth.
RhD negative pregnant women who carry an RhD positive fetus are at risk of immunization against the D antigen, which may result in hemolytic disease of the fetus and the newborn. Predicting the fetal ...RhD status by noninvasive antenatal screening for the fetal RhD gene (RHD) can guide targeted use of antenatal anti-D prophylaxis.Cell-free fetal DNA is extracted from maternal plasma from RhD negative pregnant women at a gestational age of 25 weeks. A real-time PCR-based detection of two RHD exons enables reliable prediction of the fetal RhD status to determine the administration of antenatal prophylaxis, as well as postnatal prophylaxis.
PURPOSE OF REVIEWThe aim of this review is to summarize the most recent developments in the area of detection of fetomaternal hemorrhage by flow cytometry.
RECENT FINDINGSMaternal red blood cell ...chimerism is readily detectable by flow cytometry. Fetal and maternal red blood cells differ in their content of fetal hemoglobin (α2γ2). Fetal red blood cells contain fetal hemoglobin, and normal maternal red blood cells contain some percentage of fetal hemoglobin in a background of normal adult hemoglobin. All blood group systems with allelic differences between mother and fetus are readily applicable for detection of fetomaternal hemorrhage by fetal hemoglobin.
SUMMARYFetal hemoglobin for detection of fetomaternal hemorrhage is an accurate clinical diagnostic procedure for investigation of anemia in fetus and newborn.
Susceptibility to severe acute respiratory syndrome coronavirus 2 shows individual variability in un-vaccinated and previously un-exposed individuals. We investigated the impact of ABO blood group, ...titers of anti-A and anti-B, other blood group antigens, and the extracellular deposition of ABH antigens as controlled by secretor fucosyltransferase 2 (FUT2) status.
We studied incidents in three different hospitals between April to September 2020, where un-diagnosed coronavirus disease 2019 (COVID-19) patients were cared for by health care workers without use of personal protection and with close contact while delivering therapy. We recruited 108 exposed staff, of whom 34 were diagnosed with COVID-19. ABO blood type, titer of anti-A and -B, blood group specific alleles, and secretor status were determined.
Blood group O was associated with lower risk of COVID-19 (OR 0.39, 95 %CI (0.16–0.92), p = 0.03) compared to non-O, i.e., blood groups A, B and AB. High titer anti-A immunoglobulin G (IgG) compared to low titer was associated with lower risk of COVID-19 (OR 0.24 95 %CI (0.07–0.78), p = 0.017). High titer of anti-B immunoglobulin M (IgM) compared to no anti-B (IgM) was associated with lower risk of COVID-19 (OR 0.16, 95 %CI (0.039–0.608), p = 0.006) and the same applies to low titer anti-B (IgM) compared to no titer (OR 0.23, 95 %CI (0.07–0.72), p = 0.012).
The 33Pro variant in Integrin beta-3, that is part of human platelet antigen 1b (HPA-1b), was associated with lower risk of COVID-19 (OR 0.23, 95 %CI (0.034–0.86), p = 0.028).
Our data showed that blood group O, anti-A (IgG) titer, anti-B (IgM) titer as well as HPA-1b are associated with lower risk for COVID-19.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK, ZRSKP
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