Positive Lymphocytes Flow Cytometry Crossmatch Due to AT1r Antibodies in Absence of HLA-DSA.
1Department of Pathology, University of Michigan School of Medicine, Ann Arbor, MI
2Department of Internal Medicine, UMICH, Ann Arbor
3Department of Surgery, UMICH, Ann Arbor.
Meeting: 2016 American Transplant Congress
Abstract number: 120
Keywords: Antibodies, Flowcytometry crossmatching
Session Information
Session Name: Concurrent Session: Kidney AMR: Making the Diagnosis
Session Type: Concurrent Session
Date: Sunday, June 12, 2016
Session Time: 4:30pm-6:00pm
Presentation Time: 4:54pm-5:06pm
Location: Veterans Auditorium
The angiotensin II type 1 receptor (AT1r) plays a key role in the renin-angiotensin system. Anti-AT1R antibodies (Abs) have been associated with both acute and chronic injuries of kidney allografts and detected in 17-59% of the pre-transplant samples. AT1R is widely distributed among different organs and tissues as well as in circulating leukocytes. The aim of this work is to investigate if positives flow cytometric crossmatch (FCXM) in the absence of HLA-DSA can be produced by AT1r Ab.
We query from our laboratory database all the positive FCXM in the absence of HLA-DSA Ab. We tested for anti AT1r with an ELISA assay (One Lambda, CA). Two serum samples (SS) with high titers (>40u/ml) of AT1r Ab were incubated with a surrogate donor's cells known of produce positive FCXM. After 30 min incubation the cells were washed and then incubated with an elution buffer to remove the Abs absorbed by the cells. After centrifugation the supernatant was collected and tested with the AT1r ELISA assay.
Eleven of seventeen (64.7%) SS with a positive T or B cells FCXM were also AT1r positive using a 12 U/ml cut off. Two of seventeen positive T or B FCXM were AT1r negative. For comparison we tested 22 SS with negative FCXM selected from our database as a control, and 4/22 samples (18.2%) were positive for AT1r Ab, two with result of 12 U/ml and the other two with 16 and 20 U/ml.
To demonstrate that positive FCXM was produced by AT1r IgG Abs we tested the eluted supernatant after the incubation of two SS with a surrogate donor cells with the same AT1r ELISA assay. The OD from the eluted samples was only 6-16% lower than the original SS. These results demonstrate that IgG AT1r Ab produce the positive FCXM result.
Our data shows that AT1r Abs are capable of producing a positive T and B cell FCXM. A positive FCXM in absence of HLA-DSA should lead to screening for AT1r Ab to evaluate immunologic risk and guide post-transplant monitoring. 
CITATION INFORMATION: Ramirez M, Kershaw D, Huang Y, Magee J, Samaniego M, Williams T, Schauss D, Franks T, Kleyman Y, Ramon D. Positive Lymphocytes Flow Cytometry Crossmatch Due to AT1r Antibodies in Absence of HLA-DSA. Am J Transplant. 2016;16 (suppl 3).
To cite this abstract in AMA style:
Ramirez M, Kershaw D, Huang Y, Magee J, Samaniego M, Williams T, Schauss D, Franks T, Kleyman Y, Ramon D. Positive Lymphocytes Flow Cytometry Crossmatch Due to AT1r Antibodies in Absence of HLA-DSA. [abstract]. Am J Transplant. 2016; 16 (suppl 3). https://atcmeetingabstracts.com/abstract/positive-lymphocytes-flow-cytometry-crossmatch-due-to-at1r-antibodies-in-absence-of-hla-dsa/. Accessed May 21, 2025.« Back to 2016 American Transplant Congress