scholarly journals CytoBas: Precision component‐resolved diagnostics for allergy using flow cytometric staining of basophils with recombinant allergen tetramers

Allergy ◽  
2021 ◽  
Author(s):  
Craig I. McKenzie ◽  
Nirupama Varese ◽  
Pei Mun Aui ◽  
Bruce D. Wines ◽  
P. Mark Hogarth ◽  
...  
Keyword(s):  
Recombinant Allergen ◽  
Flow Cytometric

scholarly journals CytoBas: Precision component-resolved diagnostics for allergy using flow cytometric staining of basophils with recombinant allergen tetramers

2020 ◽  
Author(s):  
Craig McKenzie ◽  
Nirupama Varese ◽  
Pei Aui ◽  
Bruce Wines ◽  
P Hogarth ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Allergic Sensitization ◽  
Roc Curves ◽  
Specific Ige ◽  
Recombinant Allergen ◽  
Predictive Values ◽  
Allergen Components ◽  
Flow Cytometric ◽  
High Positive Predictive Value ◽  
Direct Fluorescence

Background: Diagnostic tests for allergy rely on detecting allergen-specific IgE. Component-resolved diagnostics incorporate multiple defined allergen components to improve the quality of diagnosis and patient care. Objective: To develop a new approach for determining sensitization to specific allergen components that utilizes fluorescent protein tetramers for direct staining of IgE on blood basophils by flow cytometry. Methods: Recombinant forms of Lol_p_1 and Lol_p_5 proteins from ryegrass pollen (RGP) and Api_m_1 from honeybee venom (BV) were produced, biotinylated and tetramerized with streptavidin-fluorophore conjugates. Blood samples from 50 RGP-allergic, 41 BV-allergic and 26 controls were incubated with fluorescent protein tetramers for flow cytometric evaluation of basophil allergen binding and activation. Results: Allergen tetramers bound to and activated basophils from relevant allergic patients but not controls. Direct fluorescence staining of Api_m_1 and Lol_p_1 tetramers had greater positive predictive values than basophil activation for BV and RGP allergy, respectively, as defined with receiver operator characteristics (ROC) curves. Staining intensities of allergen tetramers correlated with allergen-specific IgE levels in serum. Inclusion of multiple allergens coupled with distinct fluorochromes in a single tube assay enabled rapid detection of sensitization to both Lol_p_1 and Lol_p_5 in RGP-allergic patients and discriminated between controls, BV-allergic and RGP-allergic patients. Conclusion: Our novel flow cytometric assay, termed CytoBas, enables rapid and reliable detection of clinically relevant allergic sensitization. The intensity of fluorescent allergen tetramer staining of basophils has a high positive predictive value for disease and the assay can be multiplexed for a component-resolved and differential diagnostic test for allergy.

Flow cytometric enumeration and immunophenotyping of hematopoietic stem and progenitor cells

2001 ◽  
Vol 38 (2) ◽  
pp. 139-147
Author(s):  
Jan W. Gratama ◽  
D. Robert Sutherland ◽  
Michael Keeney
Keyword(s):  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Flow Cytometric ◽  
Stem And Progenitor Cells

Expression of Platelet Membrane Glycoprotein V in Human Megakaryocytes and Megakaryocytic Cell Lines: A Study Using a Novel Monoclonal Antibody against GPV

1994 ◽  
Vol 72 (05) ◽  
pp. 762-769 ◽  
Author(s):  
Toshiro Takafuta ◽  
Kingo Fujirmura ◽  
Hironori Kawano ◽  
Masaaki Noda ◽  
Tetsuro Fujimoto ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Lines ◽  
Immunohistochemical Analysis ◽  
Specific Protein ◽  
Platelet Membrane ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Flow Cytometric ◽  
Polymerase Chain ◽  
Megakaryocytic Cell

SummaryGlycoprotein V (GPV) is a platelet membrane protein with a molecular weight of 82 kD, and one of the leucine rich glycoproteins (LRG). By reverse transcription-polymerase chain reaction (RT-PCR), GPV cDNA was amplified from mRNA of platelets and megakaryocytic cell lines. However, since there are few reports indicating whether GPV protein is expressed in megakaryocytes as a lineage and maturation specific protein, we studied the GPV expression at the protein level by using a novel monoclonal antibody (1D9) recognizing GPV. Flow cytometric and immunohistochemical analysis indicated that GPV was detected on the surface and in the cytoplasm of only the megakaryocytes in bone marrow aspirates. In a megakaryocytic cell line UT-7, GPV antigen increased after treatment with phorbol-12-myri-state-13-acetate (PMA). These data indicate that only megakaryocytes specifically express the GPV protein among hematopoietic cells and that the expression of GPV increases with differentiation of the megakaryocyte as GPIb-IX complex.

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