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International Journal of Blood Transfusion and Immunohematology - IJBTI - Review Articles, Original Articles, Short Communication, Case Series, Case Reports, Letter to Editors
 
     
Original Article
 
Evaluation of erythrocyte autoantibodies with flow cytometric phagocytosis assay
Shoichi Ito1, Tomoko Hishinuma1, Yoshiko Ogiyama1, Tomomi Asano1, Haruka Kagaya1, Michiyo Irino1, Hideya Hasegawa1, Hiroshi Shimizu1, Kenneth E. Nollet2, Masayoshi Minegishi3, Hitoshi Ohto2
1Japanese Red Cross Tohoku Block Blood Center, Sendai, Miyagi, Japan
2Department of Blood Transfusion and Transplantation Immunology, Fukushima Medical University, Fukushima, Japan
3Japanese Red Cross Fukushima Blood Center, Fukushima, Japan

Article ID: 100039Z02SI2018
doi: 10.5348/100039Z02SI2018OA

Corresponding Author:
Hitoshi Ohto, MD, PhD,
Department of Blood Transfusion and Transplantation Immunology and Department of Advanced Cancer Immunotherapy,
Fukushima Medical University, Hikariga-oka,
Fukushima City, Fukushima, 960-1295, Japan

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How to cite this article
Ito S, Hishinuma T, Ogiyama Y, Asano T, Kagaya H, Irino M, Hasegawa H, Shimizu H, Nollet KE, Minegishi M, Ohto H. Evaluation of erythrocyte autoantibodies with flow cytometric phagocytosis assay. Int J Blood Transfus Immunohematol 2018;8:100039Z02SI2018.


ABSTRACT

Aims: Cellular assays such as the monocyte monolayer assay have been used to evaluate the clinical significance of antibodies to red blood cells (RBCs). Flow cytometry (FCM) has also been applied to antibody screening and diagnosing autoimmune hemolytic anemia (AIHA).

Methods: RBC eluate from 131 patients with positive direct antiglobulin tests, along with three human monoclonal antibodies (two anti-D and one anti-Dib) were used to evaluate a new FCM two-color phagocytosis assay using markers that bind RBCs and monocytes. Dual-positive event percentages were compared with antibody titers and trimmed mean fluorescence intensity (MFI) from the 131 eluates and three monoclonal antibodies.

Results: Percent positive by the FCM phagocytosis assay correlated well (R2=0.755) with trimmed-MFI in serial dilutions of two human IgG1 monoclonal antibodies, anti-D and anti-Dib, and 131 clinical samples with titers ranging from 2 to 2048. IgG2 antibodies showed lower phagocytosis relative to titers and MFI associated with these antibodies.

Conclusion: The FCM phagocytosis assay can be used to evaluate AIHA-associated antibodies, with great potential to standardize immunohematology testing, especially in regard to establishing the clinical significance of RBC alloantibodies.

Keywords: Direct anti-globulin test, Flow cytometry, IgG subclass, Phagocytosis


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Author Contributions
Shoichi Ito – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published
Tomoko Hishinuma – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Yoshiko Ogiyama – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Tomomi Asano – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Haruka Kagaya – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Michiyo Irino – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Hideya Hasegawa – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Hiroshi Shimizu – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Final approval of the version to be published
Kenneth E. Nollet – Analysis and interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published
Masayoshi Minegishi – Substantial contributions to conception and design, Acquisition of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published
Hitoshi Ohto – Substantial contributions to conception and design, Analysis and interpretation of data, Drafting the article, Revising it critically for important intellectual content, Final approval of the version to be published
Guarantor of Submission
The corresponding author is the guarantor of submission.
Source of Support
None
Consent Statement
Written informed consent was obtained from the patient for publication of this study.
Conflict of Interest
Author declares no conflict of interest.
Copyright
© 2018 Shoichi Ito et al. This article is distributed under the terms of Creative Commons Attribution License which permits unrestricted use, distribution and reproduction in any medium provided the original author(s) and original publisher are properly credited. Please see the copyright policy on the journal website for more information.



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