<abstract xmlns="http://www.w3.org/1999/xhtml"><p>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. <italic><bold>Immunohematology</bold></italic><bold>2016;32:161–169.</bold></p></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. Immunohematology2016;32:161–169.

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

Immunohematology

Flow cytometric methods are commonly used to analyze white blood cell surface antigen expression. We developed a flow cytometric method to detect red blood...

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

Article Category: Original Report

Published Online: Oct 09, 2019

Page range: 161 - 169

DOI: https://doi.org/10.21307/immunohematology-2019-061

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

© 2016 S.A. Koepsell et al., published by Sciendo

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

Article Category: Original Report

Published Online: Oct 09, 2019

Page range: 161 - 169

DOI: https://doi.org/10.21307/immunohematology-2019-061

Keywordsred blood cell, direct antiglobulin test, autoimmune hemolytic anemia, tannic acid, flow cytometry, immunoglobulins

© 2016 S.A. Koepsell et al., published by Sciendo

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