Abstract 4521: Flow cytometric measurement of increased erythrocyte bound IgG: A potential screening method for colorectal cancer
Authors: Kitao Akihito , Kawamoto Shinichiro , Kurata Keiji , Hayakawa Ikuyo , Matsuoka Hiroshi , Sumi Yasuo , Kakeji Yoshihiro , Kamesaki Toyomi , Minami Hironobu 
Published: August 2018

Abstract
Introduction: Colorectal cancer (CRC) incidence is increasing worldwide. While screening for CRC has led to early detection, thus reducing mortality, checking for fecal occult blood, as the accepted screening method for CRC worldwide, has some shortcomings including false-negative results in early disease stages and/or advanced CRC of the right ascending colon. We have previously reported on the ectopic expression of Band3 (encoded by SLC4A1), an antigen abundant in the cytoplasmic membrane of erythrocytes, in a case of CRC with autoimmune hemolytic anemia (AIHA), and suggested that Band3 expression in CRC might stimulate production of the autoantibody that binds to the erythrocyte membrane. Besides, the amount of erythrocyte-bound IgG may be higher in CRC patients compared with healthy people, even though no symptoms or signs of AIHA are seen. We conducted a prospective observational study to compare the amount of erythrocyte-bound IgG, including anti-Band 3 antibody, between CRC patients and healthy controls.
Patients and Methods: From October 2016 to November 2017, 30 CRC patients and six healthy controls were enrolled. Direct Coombs test was carried out, using test tube and column methods, immediately after blood collection. Cell suspensions were incubated with either anti-human IgG antibody or a control isotype conjugated with FITC. Fluorescence was measured with a flow cytometer. Mean fluorescence intensity (MFI) was calculated as the difference in fluorescence intensity between anti-human IgG and control isotypes. Total serum IgG was collected using protein G sepharose. The amount of serum erythrocyte-bound IgG was quantified by ELISA using erythrocyte membrane protein coated 96-well plates and anti-human IgG antibody. Erythrocyte membrane protein was immunoprecipitated and subjected to immunoblotting using anti-Band3 IgG to confirm its proportion to total serum IgG.
Results: Clinical AIHA was not observed in any patient, but a direct Coombs test was positive in eight patients (one on the test tube method, five on the column method, and two patients on both methods). The median±SD MFI was 40±17.4 in patients with CRC and 23±11.6 in controls (p=0.005). Moreover, with the MFI cutoff value set at 31, ROC curve analysis showed the sensitivity, specificity, and area under the curve to be 100%, 70% and 0.867, respectively. In CRC patients, the median MFI was not affected by positive or negative results for fecal occult blood, involvement of the left or right colon, and/or disease stage. The serum erythrocyte-bound IgG and the proportion of anti-Band3 to total serum IgG was higher in CRC patients than in controls.
Conclusion: Whether flow cytometric measurement of total erythrocyte-bound IgG, including anti-Band 3 antibody, in CRC patients can be a useful screening tool warrants further evaluation in a larger cohort.
Citation Format: Akihito Kitao, Shinichiro Kawamoto, Keiji Kurata, Ikuyo Hayakawa, Hiroshi Matsuoka, Yasuo Sumi, Yoshihiro Kakeji, Toyomi Kamesaki, Hironobu Minami. Flow cytometric measurement of increased erythrocyte bound IgG: A potential screening method for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4521.