A detailed flow cytometric method for detection of low-level in vivo red blood  cell–bound IgG, IgA, and IgM

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Immunohematology

American National Red Cross

Subject: Medical Laboratory Technology

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ISSN: 0894-203X
eISSN: 1930-3955

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VOLUME 32 , ISSUE 4 (December 2016) > List of articles

A detailed flow cytometric method for detection of low-level in vivo red blood  cell–bound IgG, IgA, and IgM

Wendy Beres * / Geralyn M. Meny / Sandra Nance

Keywords : red blood cell, direct antiglobulin test, autoimmune hemolytic anemia, tannic acid, flow cytometry, immunoglobulins

Citation Information : Immunohematology. Volume 32, Issue 4, Pages 161-169, DOI: https://doi.org/10.21307/immunohematology-2019-061

License : (Transfer of Copyright)

Published Online: 09-October-2019

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ABSTRACT

Flow cytometric methods are commonly used to analyze white blood cell surface antigen expression. We developed a flow cytometric method to detect red blood cell (RBC)-bound immunoglobulin (Ig)G, IgA, and IgM. RBCs were washed; incubated with fluorescein isothiocyanate (FITC)-conjugated anti-IgG, -IgA, or -IgM; washed; and analyzed on the flow cytometer. The method was optimized by determining the dilution of FITC-conjugated anti-IgG, -IgA, and -IgM providing the greatest amount of fluorescence when tested with Ig-coated RBCs and the least amount of fluorescence when tested with naive RBCs. Tannic acid was used to prepare Ig-coated RBCs. Cross-reactivity of FITC-conjugated anti-IgG, -IgA, and -IgM with Ig-coated RBCs was evaluated, and a reference range was established. Use of this method may assist in clinical evaluation of patients who present with hemolysis and a negative direct antiglobulin test.

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