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


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American National Red Cross

Subject: Medical Laboratory Technology


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



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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1. Hanson CA. Reticulocyte analysis by flow cytometry. In: Keren DF, Hanson CA, Hurtubise PE, eds. Flow cytometry and clinical diagnosis. Chicago: American Society of Clinical Pathologists, 1994:368–83.

2. Fayek MH, Saad AA, Eissa DG, Tawfik LM, Kamal G. Role of gel test and flow cytometry in diagnosis of Coombs' negative autoimmune haemolytic anaemia. Int J Lab Hematol 2012;34:311–9.

3. Lin JS, Hao TC, Lyou JY, et al. Clinical application of a flow cytometric direct antiglobulin test. Transfusion 2009;49: 1335–46.

4. Chaudhary R, Das S, Gupta R, Khetan D. Application of flow cytometry in detection of red cell bound IgG in Coombs negative AIHA. Hematology 2006;11:295–300.

5. Nance SJ, Garratty G. Application of flow cytometry to immunohematology. J Immunol Methods 1987;101:127–31.

6. Garratty G. Immune hemolytic anemia associated with negative routine serology. Semin Hematol 2005;42:156–64.

7. Garratty G. Immunobiology of transfusion medicine. New York: Marcel Dekker, 1994.

8. McGann PT, McDade J, Mortier NA, Combs MR, Ware RE. IgA-mediated autoimmune hemolytic anemia in an infant. Pediatr Blood Cancer 2011;56:837–9.

9. Duarte-Rey C, Bogdanos DP, Leung PS, Anaya JM, Gershwin ME. IgM predominance in autoimmune disease: genetics and gender. Autoimmune Rev 2012;11:A404–12.

10. Stroncek DF, Njoroge JM, Procter JL, Childs RW, Miller J. A preliminary comparison of flow cytometry and tube agglutination assays in detecting red blood cell-associated C3d. Transfus Med 2003;13:35–41.

11. Fueger JT, Gottschall JL, Curtis BR, Johnson ST. DAT negative immune hemolytic anemia: the role of enhanced techniques [abstract]. Transfusion 2003;43:103A.

12. Green GA. Autologous IgM, IgA and complement binding to sickle erythrocytes in vivo: evidence or the existence of dense sickle cell subsets. Blood 1993;82:985–92.

13. Beres W, Nance S. Stability of in vitro red blood cell (RBC)bound IgG, IgA and IgM by flow cytometric (FC) analysis [abstract]. Transfusion 2013;53:182A.

14. Beres W, Nance SJ. Evaluation of normal donor pools for RBCbound immunoglobulin (Ig) levels measure by flow cytometry [abstract]. Transfusion 2014;54:179A.

15. Hunt AF, Reed MI. Tannic acid and chromic chlorideinduced binding of protein to red cells: a preliminary study of possible binding sites and reaction mechanisms. Med Lab Sci 1990;47:189–94.

16. Fung ML, Grossman BJ, Hillyer CD, Westhoff CM, eds. Technical manual. 18th ed. Arlington, VA: American Association of Blood Banks, 2014:425–7.

17. The instrument controls in CellQuest. In: Operator training manual FACSCalibur. San Jose, CA: Becton Dickinson Immunocytometry Systems, 1999;5:6, 10.

18. Beres W, McGuire L, Nance S, Meny G. Detection of in vivo red blood cell (RBC) bound IgG measured by flow cytometry (FC) [abstract]. Transfusion 2010;50:166A.

19. Arndt PA, Garratty G. A critical review of published methods for analysis of red cell antigen-antibody reactions by flow cytometry, and approaches for resolving problems with red cell agglutination. Transfus Med Rev 2010;24:172–94.