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Published on: September 16, 2012
Single human leukocyte antigen flow cytometry beads for accurate identification of human leukocyte antigen antibody
Rui Pei1, Jar-How Lee, Neng-Jen Shih
1Research Department, One Lambda. Inc., Canoga Park, CA, USA. rpei@onelambda.com
Insights
A new single human leukocyte antigen (HLA) bead panel improves antibody identification for highly sensitized patients. This flow cytometry method offers greater accuracy and sensitivity in detecting HLA antibodies, aiding transplant readiness.
Area of Science:
- Immunology
- Transplantation Science
- Biotechnology
Background:
- Assigning antibody specificity in highly sensitized patients is challenging with conventional multi-antigen cell panels.
- Accurate human leukocyte antigen (HLA) antibody identification is crucial for successful organ transplantation and transfusion management.
Purpose of the Study:
- To develop and validate a single antigen bead panel for precise identification of HLA antibody specificities.
- To enhance the accuracy and sensitivity of HLA antibody detection in flow cytometry.
Main Methods:
- Produced 110 single recombinant human leukocyte antigens (HLAs) across A, B, and C loci using a mammalian expression system.
- Coated single HLAs onto distinct colored microbeads for simultaneous detection of antibodies against eight antigens per test.
- Utilized flow cytometry for simultaneous detection of HLA antibodies.
Main Results:
- Single HLA beads demonstrated specific reactions with monoclonal antibodies, offering higher resolution than traditional cell panels.
- The single antigen approach uncovered masked specificities and showed increased sensitivity in serum dilution studies.
- In kidney transplant rejection patients, single antigen beads identified antibodies to mismatched donor antigens with high accuracy.
Conclusions:
- A novel, accurate, and sensitive method for HLA antibody detection using recombinant single HLA-coated flow cytometry beads has been established.
- This single antigen bead technology is valuable for predicting negative crossmatches in highly sensitized organ transplant recipients and patients needing platelets.
Background:
It is difficult to assign antibody specificity for highly sensitized patients using a cell panel with multiple antigens per reaction. We describe here a single antigen bead panel for accurate identification of human leukocyte antigen (HLA) antibody specificities by flow cytometry.
Methods:
A total of 110 single recombinant HLAs, including 34 A locus alleles, 57 B locus alleles, and 19 C locus alleles, were produced by a mammalian expression system. These single antigens were coated onto eight different colored microbeads, which were mixed together in one tube for simultaneous detection of HLA antibodies against eight different antigens per flow cytometry test.
Results:
Single HLA reacted specifically with the serologically defined monoclonal antibodies. The single antigen panel provided higher resolution than the regular cell panel for antibody detection by uncovering the masked specificities. Single antigens also provided higher sensitivity than the multiple antigens coated onto beads for HLA antibody detection as demonstrated by serum dilution studies. In 10 sera from patients who had rejected a kidney transplant, single antigen beads identified antibodies to 31 of 35 antigens that were mismatched in the donor. Most important, none of the reactions were against antigens present in the recipient.
Conclusion:
An accurate and sensitive HLA antibody detection method is described using flow cytometry beads coated with single HLAs produced by recombinant technology. The single antigen beads should be useful in predicting negative crossmatch in highly sensitized organ recipients and highly sensitized patients requiring platelets.
