Novel Flow Cytometry-Based Method for Detection of Anti-HLA Complement Activating Donor-Specific Antibodies in Renal Transplant Recipients and Its Comparison With Other Conventional Detection Methods

• Published in: Transplantation proceedings 2023-01, Vol.55 (1), p.134-139
• Main Authors: Rani, Lekha, Singh, Heera, Saikia, Biman, Aggarwal, Ritu, Kumar, Yashwant, Kumar, Mahendra, Chhabra, Seema, Kumar, Manoj, Kumar, Bhuvnesh, Kumar, Vinkesh, Das, Prabir, Sharma, Ashish, Ramchandran, Raja, Kohli, Harbir Singh, Minz, Ranjana Walker
• Format: Article
• Language: English
• Subjects: Antibodies; Complement System Proteins; Flow Cytometry - methods; Graft Rejection; Histocompatibility Testing - methods; HLA Antigens; Humans; Isoantibodies; Kidney Transplantation
• ISSN: 0041-1345; 1873-2623
• DOI: 10.1016/j.transproceed.2022.11.007

Presence of preformed donor specific antibodies (DSAs) detected by complement-dependent cytotoxicity (CDC-XM) is a strong contraindication for transplant. However, it has limitations including its sensitivity and its inability to distinguish between HLA-specific and other non-HLA-specific antibodies. In this study, we standardized CDC-XM by flow cytometry and determined its relevance by comparing its results with other methods of DSA detection, such as routine CDC-XM, antibody binding assay by flow cytometry (FC-XM), and Luminex-based crossmatch assays, such as Luminex crossmatch (LXM) and virtual crossmatch (VXM). A total of 79 serum samples were tested for DSAs by the flow cytometric complement-dependent cytotoxicity crossmatch assay (FC-CDC-XM) and then the results of FC-CDC-XM were compared with other detection methods such as CDC-XM, FC-XM, LXM, and VXM. We found that the FC-CDC-XM assay is more sensitive than routine CDC-XM. Out of total 79 sera, 24 sera were detected positive (T cells positive: 1 case and B cells positive: 23) by FC-CDC-XM as compared with 3 sera using CDC-XM; these 3 sera also showed positivity by FC-CDC-XM. After FC-XM assay, 23 samples were positive by FC-XM and out of these 23 samples, 13 were also positive by FC-CDC-XM. On comparing the FC-CDC-XM results with VXM and LXM, 10 sera of 24 FC-CDC-XM positive had HLA class II antibodies detected on a Luminex platform. The FC-CDC-XM is a more sensitive and specific method for detection of HLA-specific complement-fixing antibodies than CDC-XM and FC-XM. FC-CDC-XM should be used in tissue-typing laboratories after intra- and inter- laboratory validation.