Typing for all known HLA-DR alleles by group-specific PCR and flow cytometry-based multiplexed single nucleotide extension

2003 ◽  
Vol 64 (10) ◽  
pp. S38
Author(s):  
Yanzheng Zhang ◽  
Charlie Costin ◽  
Christopher Giang ◽  
Robert Vorhaben ◽  
Peter Stastny
Keyword(s):  
Flow Cytometry ◽  
Single Nucleotide ◽  
Specific Pcr ◽  
Hla Dr

scholarly journals Alveolar compartmentalization of inflammatory and immune cell biomarkers in pneumonia-related ARDS

Critical Care ◽  
2021 ◽  
Vol 25 (1) ◽  
Author(s):  
Inès Bendib ◽  
Asma Beldi-Ferchiou ◽  
Frédéric Schlemmer ◽  
Mathieu Surenaud ◽  
Bernard Maitre ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Hospital Mortality ◽  
Distress Syndrome ◽  
Immune Cell ◽  
Routine Care ◽  
Clinical Endpoint ◽  
Blood Samples ◽  
Hla Dr ◽  
Serum Ratio ◽  
Immunocompetent Patients

Abstract Background Biomarkers of disease severity might help individualizing the management of patients with the acute respiratory distress syndrome (ARDS). Whether the alveolar compartmentalization of biomarkers has a clinical significance in patients with pneumonia-related ARDS is unknown. This study aimed at assessing the interrelation of ARDS/sepsis biomarkers in the alveolar and blood compartments and explored their association with clinical outcomes. Methods Immunocompetent patients with pneumonia-related ARDS admitted between 2014 and 2018 were included in a prospective monocentric study. Bronchoalveolar lavage (BAL) fluid and blood samples were obtained within 48 h of admission. Twenty-two biomarkers were quantified in BAL fluid and serum. HLA-DR+ monocytes and CD8+ PD-1+ lymphocytes were quantified using flow cytometry. The primary clinical endpoint of the study was hospital mortality. Patients undergoing a bronchoscopy as part of routine care were included as controls. Results Seventy ARDS patients were included. Hospital mortality was 21.4%. The BAL fluid-to-serum ratio of IL-8 was 20 times higher in ARDS patients than in controls (p < 0.0001). ARDS patients with shock had lower BAL fluid-to-serum ratio of IL-1Ra (p = 0.026), IL-6 (p = 0.002), IP-10/CXCL10 (p = 0.024) and IL-10 (p = 0.023) than others. The BAL fluid-to-serum ratio of IL-1Ra was more elevated in hospital survivors than decedents (p = 0.006), even after adjusting for SOFA and driving pressure (p = 0.036). There was no significant association between alveolar or alveolar/blood monocytic HLA-DR or CD8+ lymphocytes PD-1 expression and hospital mortality. Conclusions IL-8 was the most compartmentalized cytokine and lower BAL fluid-to-serum concentration ratios of IL-1Ra were associated with hospital mortality in patients with pneumonia-associated ARDS.

Vitamin D modulates the expression of HLA-DR and CD38 after in vitro activation of T-cells

2017 ◽  
Vol 29 (3) ◽  
Author(s):  
Simon Villegas-Ospina ◽  
Wbeimar Aguilar-Jimenez ◽  
Sandra M. Gonzalez ◽  
María T. Rugeles
Keyword(s):  
Vitamin D ◽  
Flow Cytometry ◽  
Healthy Subjects ◽  
Mononuclear Cells ◽  
Activation Markers ◽  
Hla Dr ◽  
In Vitro Activation ◽  
Cells Cultured

AbstractObjective:Vitamin D (VitD) is an anti-inflammatory hormone; however, some evidence shows that VitD may induce the expression of activation markers, such as CD38 and HLA-DR. We explored its effect on the expression of these markers on CD4Materials and methods:CD38 and HLA-DR expression was measured by flow cytometry in PHA/IL-2-activated mononuclear cells cultured under VitD precursors: three cholecalciferol (10Results:Cholecalciferol at 10Conclusion:Although no significant correlations were observed in vivo in healthy subjects, VitD treatment in vitro modulated immune activation by increasing the expression of CD38 and decreasing the proliferation of HLA-DR

Immunophenotypic Study of Basophils by Multiparameter Flow Cytometry

2008 ◽  
Vol 132 (5) ◽  
pp. 813-819
Author(s):  
Xiaohong Han ◽  
Jeffrey L. Jorgensen ◽  
Archana Brahmandam ◽  
Ellen Schlette ◽  
Yang O. Huh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Chronic Myelogenous Leukemia ◽  
Peripheral Blood ◽  
Myelogenous Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Color Flow ◽  
Aberrant Expression ◽  
Flow Cytometric ◽  
Hla Dr

Abstract Context.—The immunophenotypic profile of basophils is not yet fully established, and the immunophenotypic changes in chronic myelogenous leukemia are not fully characterized. Objective.—To establish a comprehensive immunophenotypic spectrum of normal basophils and to assess the range of immunophenotypic aberrations of basophils in chronic myelogenous leukemia. Design.—Using 4-color flow cytometry, we compared the immunophenotypic profile of basophils in peripheral blood or bone marrow samples from 20 patients with no evidence of neoplasia to basophils from 15 patients with chronic myelogenous leukemia. Results.—Basophils in control cases were all positive for CD9, CD13, CD22, CD25 (dim), CD33, CD36, CD38 (bright), CD45 (dimmer than lymphocytes and brighter than myeloblasts), and CD123 (bright), and were negative for CD19, CD34, CD64, CD117, and HLA-DR. Basophils in all chronic myelogenous leukemia patients possessed 1 to 5 immunophenotypic aberrancies. The most common aberrancies were underexpression of CD38, followed by aberrant expression of CD64 and underexpression of CD123. CD34 and CD117 were present in cases with basophilic precursors. Myeloblasts showed a distinct immunophenotypic profile, as they typically expressed CD34 and CD117, showed dimmer expression (compared with basophils) of CD38, CD45, and CD123, and lacked expression of CD22. Conclusions.—Flow cytometric immunophenotyping can identify immunophenotypic aberrations of basophils in chronic myelogenous leukemia, and discriminate basophils from myeloblasts.

scholarly journals Genome-Wide Linkage Mapping for Preharvest Sprouting Resistance in Wheat Using 15K Single-Nucleotide Polymorphism Arrays

2021 ◽  
Vol 12 ◽  
Author(s):  
Lingli Li ◽  
Yingjun Zhang ◽  
Yong Zhang ◽  
Ming Li ◽  
Dengan Xu ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Linkage Mapping ◽  
Cost Effective ◽  
Wheat Breeding ◽  
Preharvest Sprouting ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Specific Pcr ◽  
Grain Color ◽  
Sprouting Resistance

Preharvest sprouting (PHS) significantly reduces grain yield and quality. Identification of genetic loci for PHS resistance will facilitate breeding sprouting-resistant wheat cultivars. In this study, we constructed a genetic map comprising 1,702 non-redundant markers in a recombinant inbred line (RIL) population derived from cross Yangxiaomai/Zhongyou9507 using the wheat 15K single-nucleotide polymorphism (SNP) assay. Four quantitative trait loci (QTL) for germination index (GI), a major indicator of PHS, were identified, explaining 4.6–18.5% of the phenotypic variances. Resistance alleles of Qphs.caas-3AL, Qphs.caas-3DL, and Qphs.caas-7BL were from Yangxiaomai, and Zhongyou9507 contributed a resistance allele in Qphs.caas-4AL. No epistatic effects were detected among the QTL, and combined resistance alleles significantly increased PHS resistance. Sequencing and linkage mapping showed that Qphs.caas-3AL and Qphs.caas-3DL corresponded to grain color genes Tamyb10-A and Tamyb10-D, respectively, whereas Qphs.caas-4AL and Qphs.caas-7BL were probably new QTL for PHS. We further developed cost-effective, high-throughput kompetitive allele-specific PCR (KASP) markers tightly linked to Qphs.caas-4AL and Qphs.caas-7BL and validated their association with GI in a test panel of cultivars. The resistance alleles at the Qphs.caas-4AL and Qphs.caas-7BL loci were present in 72.2 and 16.5% cultivars, respectively, suggesting that the former might be subjected to positive selection in wheat breeding. The findings provide not only genetic resources for PHS resistance but also breeding tools for marker-assisted selection.

scholarly journals Developing a Simple Algorithm Based on Multiparameter Flow Cytometry for Fast Screening of Acute Promyelocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 25-26
Author(s):  
Vitória Ceni Ceni ◽  
Katia B Pagnano ◽  
Gislaine B O Duarte ◽  
Marina DB Pellegrini ◽  
Bruno Kosa Duarte ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Discriminant Analysis ◽  
Acute Promyelocytic Leukemia ◽  
Simple Algorithm ◽  
Diagnostic Algorithm ◽  
Promyelocytic Leukemia ◽  
Advisory Board ◽  
Multiparameter Flow Cytometry ◽  
Fast Screening ◽  
Hla Dr

Introduction: Acute promyelocytic leukemia (APL) is a genetically and molecularly well-defined type of acute leukemia that is curable but has a frequent early mortality due to bleeding. So, there is a need for a fast diagnostic screening in order to start appropriate therapy. Multiparameter flow cytometry (MFC) is usually performed in all types of acute myeloid leukemias (AMLs) but only few features have been described as characteristic of APL. Aim: to develop a diagnostic algorithm based on the intensity of expression of several antigens examined by MFC in AML that could reliably discriminate between APL and the other types of AML. Material and Methods: Consecutive newly diagnosed AMLs treated in our Institution during the last 2 years entered the study. Immunophenotyping was included in the diagnostic workup. An 8-color platform based on the Euroflow recommendations was used. The mean fluorescence intensity (MFI) of each antigen tested was assessed and those best discriminating between APL and all other types of AML were obtained by a discriminant analysis. Phenotypic characteristics of normal myeloblasts taken from examinations of bone marrow (BM) MFC performed for the diagnosis of cytopenias were used as controls. Results: 24 cases of APL and 56 cases of other primary AML entered the study. Median age: 39 (23-56) and 62(26-81) years respectively. Concerning ELN risk groups of non-APL cases, 13 were favorable risk, 26 were intermediate and 09 were adverse risk. In 8 cases risk assessment was not possible due to the absence of cytogenetics. Moreover, among APL patients, 7 cases had a FLT3-ITD mutation. Among non-APL AMLs, 4 had FLT3-ITD mutation, 4 had NPM1 and 10 had FLT3-ITD and NPM1mutation. Concerning antigen expression, CD34 was expressed in only 1/24 APL samples, and in 18/56 samples from non-APL AMLs. The following flow features were differentially expressed in both groups: SSC (p &lt;0.0001), CD45 (p=0.02), CD13 (p=0.001), CD64 (p=0.004), HLA-DR (p&lt;0.0001) and CD33 (p&lt;0.0001) (Table 1). In the discriminant analysis, MFI CD34 and MFI HLA-DR were able to accurately classify APL and non-APL AML in only 62.5%. However, after the addition of the ratio of SSC between blasts and lymphocytes, these 3 parameters were able to differentiate APL from non-APL AML in 91.2% of the cases. Conclusion: MFC was adequate for a fast screening of APL in most cases. Expression of CD34 was not very useful, as many AMLs do not express this antigen, similar to APL, but SSC, together with HLA-DR could discriminate both types of leukemia in most cases. Disclosures Pagnano: Astellas: Other: Advisory Board and lecture; Novartis: Other: Advisory Board; Pintpharma: Other: Lecture; EMS: Other: Lecture. Duarte:Janssen: Other: Lecture; Astellas: Other: Lecture.

Rapid mapping of a chlorina mutant gene cn-A1 in hexaploid wheat by bulked segregant analysis and single nucleotide polymorphism genotyping arrays

2019 ◽  
Vol 70 (10) ◽  
pp. 827 ◽  
Author(s):  
H. B. Jiang ◽  
N. Wang ◽  
J. T. Jian ◽  
C. S. Wang ◽  
Y. Z. Xie
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Single Nucleotide Polymorphism Array ◽  
Bulked Segregant Analysis ◽  
Recessive Gene ◽  
Mutant Gene ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Chlorophyll Metabolism ◽  
Specific Pcr ◽  
Leaf Mutant

The yellow–green leaf mutant can be exploited in photosynthesis and plant development research. A Triticum aestivum mutant with the chlorina phenotype, here called B23, was produced by treatment with the chemical mutagen sodium azide. This B23 mutant showed significantly lower chlorophyll content than wild-type Saannong33, and its chloroplast structure was abnormal. All its yield-related traits, except for the number of spikes per plant, were also significantly decreased. Genetic analysis confirmed that the mutant phenotype was controlled by a recessive gene, here designated cn-A1. Using bulked segregant analysis and the wheat 660K single nucleotide polymorphism array, the cn-A1 gene was mapped to chromosome 7AL, and 11 polymorphic markers were developed. Further analysis showed that cn-A1 was located in a 1.1-cM genetic region flanked by Kompetitive allele specific PCR (KASP) markers 660K-7A12 and 660K-7A20, which corresponded to a physical interval of 3.48 Mb in T. aestivum cv. Chinese Spring chromosome 7AL containing 47 predicted genes with high confidence. These results are expected to accelerate the process of cloning the cn-A1 gene and facilitate understanding of the mechanisms underlying chlorophyll metabolism and chloroplast development in wheat.

scholarly journals Phenotype analysis of hematopoietic CD34+ cell populations derived from human umbilical cord blood using flow cytometry and cDNA-polymerase chain reaction

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2103-2114 ◽  
Author(s):  
SJ Thoma ◽  
CP Lamping ◽  
BL Ziegler
Keyword(s):  
Flow Cytometry ◽  
Polymerase Chain Reaction ◽  
Cell Surface ◽  
Chain Reaction ◽  
Hematopoietic Stem ◽  
Hla Dr ◽  
Cd34 Cells ◽  
Rare Populations ◽  
Pcr Method ◽  
Polymerase Chain

Abstract A strategy to phenotype rare populations of hematopoietic cells expressing the cell-surface marker CD34 was studied. The antigenic phenotype of umbilical core blood (CB) CD34+ cells was investigated using flow cytometry and compared with the mRNA-phenotype determined by cDNA-polymerase chain reaction (cDNA-PCR) analysis. The cDNA-PCR method allowed an mRNA evaluation of small numbers of cells. Monoclonal antibodies and oligonucleotide primers that recognize myeloid, lymphoid, erythroid and platelet/megakaryocytic cell membrane antigens or corresponding mRNA transcripts were used. Evaluation by flow cytometry showed that the vast majority of CD34+ CB cells coexpressed CD38, CD18, HLA-DR, and CD33. Rare subpopulations of CD34+CD38-, CD34+CD18-, CD34+HLA-DR-, and CD34+CD33- were also identified. A large proportion of CD34+ CB cells expressed CD13, CD45R, and to a lesser extent CD71. The CD36, CD51, and CD61 antigens were identified on a small number of CD34+ cells. The three-color flow cytometry analysis showed that CD34+ cells stained with antibodies to CD61 and CD36 or CD51 can be divided into subsets that may represent progenitor cells committed to the erythroid and/or megakaryocytic lineage. A variety of other lineage-specific cell-surface antigens including pre-T-cell marker CD7 and markers of early B cells, ie, CD10 and CD19, were not coexpressed with CD34+. Using the cDNA-PCR it was seen that the mRNA phenotype of a small number of sorted CD34+ cells (purity > 98%) was negative for the markers CD2, CD14, CD16, CD20, CD21, CD22, CD41b, and glycophorin A that are expressed on differentiated cells but positive for CD34, CD7, CD19, CD36, and CD61. The results suggest that circulating CD34+CD7+ and CD34+CD19+ CB cells cannot be distinguished by flow cytometry but can be detected by cDNA-PCR. This indicates that CB either contains very low numbers of these progenitors or that the antigen density of CD7 and CD19 on CD34+ cells is below the detection limit of the flow cytometer. In contrast to flow cytometry, cDNA-PCR allows the phenotypic analysis of cells even if their number is small. Thus, the cDNA-PCR method can be useful in linking phenotype analyses, ie, markers of differentiation, to studies on gene expression within rare populations of hematopoietic stem cells.

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