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Sayed D.,Flow Cytometry Laboratory | El-Badawy O.,Assiut UniversityAssiutEgypt | Afifi N.,Assiut UniversityAssiutEgypt | Hamza W.S.,Assiut UniversityAssiutEgypt | El-Sayed Y.,Sohag University
Journal of Medical Virology | Year: 2016

Anti-HBs levels wanes with time. Many studies discussed the B cell response to HBV vaccine. However, the data about memory T cell response are limited. To evaluate the efficacy of hepatitis B vaccine via evaluating anti-HBs levels and HBsAg specific memory T-lymphocytes through descriptive study. The study was conducted in a tertiary care setting. This study included 440 vaccinated persons during infancy. Group I: 6 to less than 10 years old; Group II: 10 to less than 14 years old; Group III: 14 to less than 17 years old; Group IV: 17 years old. The serum samples were screened for HBV markers. Cytokines secretion by HBsAg-specific memory CD45RO+ CD4+ T cells was measured after in vitro culture using flow cytometry. The mean titer of anti-HBs was higher in group I in comparison to others (P-value=0.000 for each). IFN-γ and IL-4 secreted by memory CD4+ T cells were positive in all with anti-HBs >100mIU/ml, while positive in 87% and 75% of participants with anti-HBs <10mIU/ml and positive in 73% and 32% of participants with absent anti-HBs. The percentage of cells secreting IFN-γ and those secreting IL-4 were higher among participants with serum anti-HBs >100mIU/ml than those having <10mIU/ml or absent (P<0.001 for each). Anti-HBs positivity decreased with time since childhood vaccination. Breakthrough infections are rare in vaccinated persons. Hepatitis-B vaccine is efficient in controlling HBV infection. Flow cytometry is a useful tool to assess the long term persistence of T cell memory after childhood vaccination. © 2016 Wiley Periodicals, Inc.


Uherkova L.,Institute of Hematology and Blood Transfusion IHBT | Vancurova I.,Institute of Hematology and Blood Transfusion IHBT | Vancurova I.,Charles University | Vyhlidalova I.,Institute of Hematology and Blood Transfusion IHBT | And 13 more authors.
Leukemia Research | Year: 2013

We established and characterized a new IL-6 dependent multiple myeloma (MM) cell line UHKT-893 from the bone marrow of a relapsed 57-year-old woman. Results: Using nephelometry, cells with plasma cell phenotype and morphology were found to secrete IgG and free kappa (κ)-light chain of immunoglobulin. κ-Light chain was also recognized intracellularly by flow cytometry and by mass spectrometry. VH4-39 region of IgVH genes was rearranged and somatically hypermutated. Cytogenetic analysis of cells revealed new chromosome abnormalities in all breakpoints unique in both MM patients and cell lines - t(1;6), t(1;11), t(5;15), t(5;21), +der(11;15) and der(16). IL-6 independent subline UHKT-893a was established by adaptation to descending IL-6 concentration, while the original cell line keeps on maintaining its IL-6 dependency. Conclusion: The cell line provides a suitable material for cellular and molecular studies of tumor abnormalities, with potentially unique mutagenic features of myeloma disease. It may be utilized for human hybridoma construction and vaccine development. Both IL-6 dependent and independent cell clones represent an important model for studies of myeloma cell growth and resistance emerging during targeted therapy. © 2012 Elsevier Ltd.


Borowitz M.J.,Johns Hopkins Medical Institutions | Craig F.E.,University of Pittsburgh | DiGiuseppe J.A.,Hartford Hospital | Illingworth A.J.,Flow Cytometry Laboratory | And 4 more authors.
Cytometry Part B - Clinical Cytometry | Year: 2010

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder characterized by a somatic mutation in the PIGA gene, leading to a deficiency of proteins linked to the cell membrane via glycophosphatidylinositol (GPI) anchors. While flow cytometry is the method of choice for identifying cells deficient in GPI-linked proteins and is, therefore, necessary for the diagnosis of PNH, to date there has not been an attempt to standardize the methodology used to identify these cells. Methods: In this document, we present a consensus effort that describes flow cytometric procedures for detecting PNH cells. Results: We discuss clinical indications and offer recommendations on data interpretation and reporting but mostly focus on analytical procedures important for analysis. We distinguish between routine analysis (defined as identifying an abnormal population of 1% or more) and high-sensitivity analysis (in which as few as 0.01% PNH cells are detected). Antibody panels and gating strategies necessary for both procedures are presented in detail. We discuss methods for assessing PNH populations in both white blood cells and red blood cells and the relative advantages of measuring each. We present steps needed to validate the more elaborate high-sensitivity techniques, including the need for careful titration of reagents and determination of background rates in normal populations, and discuss technical pitfalls that might affect interpretation. Conclusions: This document should both enable laboratories interested in beginning PNH testing to establish a valid procedure and allow experienced laboratories to improve their techniques. © 2010 Clinical Cytometry Society.


Mutzke E.,Healthy Environmental | Chomyshyn E.,Flow Cytometry Laboratory | Nguyen K.C.,Healthy Environmental | Blahoianu M.,Healthy Environmental | Tayabali A.F.,Healthy Environmental
Analytical Biochemistry | Year: 2015

Flow cytometry was evaluated for its capacity to detect and distinguish a wide size range (20-2000 nm) of fluorescent polystyrene particles (PSPs). Side scatter and fluorescence parameters could predict dispersed PSP sizes down to 200 nm, but the forward scatter parameter was not discriminatory. Confocal microscopy of flow-sorted fractions confirmed that dispersed PSPs appeared as a single sharp peak on fluorescence histograms, whereas agglomerated PSPs were detected as smaller adjacent peaks. Particles as small as 200 nm could also be detected by flow cytometry after they were first phagocytized by J774A.1 murine macrophages. Confocal microscopy demonstrated that these PSPs were internalized within the cytoplasm. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and calcein-AM (acetoxymethyl ester) assays showed that they were not cytotoxic. Internalized PSP size correlated to both cellular side scatter (R2 = 0.9821) and fluorescence intensity (R2 = 0.9993). Furthermore, PSPs of various sizes could be distinguished when J774A.1 cells were loaded with a single size of PSP and mixed with cells containing other sizes. However, spectra of cells loaded with a mixture of PSP sizes resembled those containing only the largest PSP. These data demonstrate the capacity and limitations of phagocytosis-coupled flow cytometry to distinguish between dispersed and agglomerated states and detect a wide size range of particles. Crown Copyright © 2015 Published by Elsevier Inc.


PubMed | Flow Cytometry Laboratory
Type: Case Reports | Journal: Cytometry. Part B, Clinical cytometry | Year: 2012

The loss of CD45, the leukocyte-common antigen, has been described in rare cases of large B-cell lymphoma (LBCL) subtypes with extranodal involvement by immunohistochemical methods. Here we report a case of a patient with LBCL, with no extranodal lesions, which is CD45 negative by flow cytometry (FC) immunophenotyping.Immunophenotyping and DNA content analysis was performed by multiparametric FC on lymph node and bone marrow aspirate obtained from a 65 year old male patient.Malignant B-lymphocytes were CD5-, CD10+/++, CD11c-, CD19+, CD20+/++, CD23-, CD34-, CD38-/+, CD45-, CD79b++/+++, BCL2 overexpressed, FMC7++, IgM++/+++, TdT- with Lambda light chain restriction. This pathological cellular population showed near-diploid DNA content, with a high proliferate rate.To our knowledge, we describe the first case of a CD19+ B-cell non-Hodgkin lymphoma without expression of CD45 detected by FC, and the first case without extranodal involvement presentation. This case is reported not only because it is a rare one but also to raise awareness of FC users of its correct diagnosis.

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