Trends in HLA Antibody Screening and Identification and Their Role in Transplantation

Screening Methods

HLA screening assays are used to detect the presence or absence of HLA antibodies and do not typically provide any information on HLA specificity of the detected antibodies. The function of the screening assay is to detect the presence or absence of anti-HLA IgG antibodies in a rapid and specific manner. Newly developed techniques include assays that use a multiplex platform, such as the Luminex™, as well as bead assays that use standard flow cytometry. These solid-phase tests can simultaneously detect and distinguish MHC class I and class II antibodies, thus providing information that was previously problematic to obtain. HLA class I antibodies were identifiable by the microcytotoxicity assay, but HLA class II antibodies often remained unidentified due to the overlapping distribution of MHC class I and II antigens. The FlowPRA™ test consists of pools of microparticle beads, approximately 2-4 µm in diameter, that are coated with a full HLA class I or class II phenotype derived from purified HLA-bearing cell lines.^[24] Concomitant measurement of HLA class I and class II beads is possible because HLA class II beads are labeled with fluorochrome similar to phycoerythrin (PE), whereas HLA class I beads are nonfluorescent (Figure 1). The beads at the top of Figure 1 represent MHC class II-coated beads that are labeled with fluorescence, whereas the beads at the bottom of the flow cytometry dot plot show nonfluorescent MHC class I-coated beads. The percentage of panel-reactive antibodies (PRAs) can be determined by calculating the percentage of beads that react positively with patient sera. Figure 2 shows schematic histograms of positive and negative sera. In Figure 2A & B, the patient serum is negative for anti-MHC class I and class II antibodies, respectively. By contrast, Figure 2C & D illustrate patient sera that are positive for both anti-MHC class I and class II antibodies. Sera that contain no antibody have a single homogenous peak, whereas sera that contain HLA antibodies show multiple peaks, reflecting a heterogeneous population. The pool of antigens represented in the FlowPRA assay contains all common HLA antigens as well as the majority of rare antigens. The assay has a rapid turnaround time of approximately 2.5 h which allows flexibility for testing, such as expedited listing of cardiac recipients. Previously, AHG-CDC testing could take as long as 4-5 h to complete and was more susceptible to technical errors.

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Flow cytometry dot plot showing flow screen beads. The R2 group represents HLA class II screen beads that are coated with fluorochrome (PE) versus group R3 HLA class I beads that have no fluorescent dye attached to the beads, thus allowing simultaneous detection of anti-HLA class I and class II antibodies. PE: Phycoerythrin.

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FlowPRA™ screen beads. (A) A patient serum negative for HLA class I antibodies: 0% PRAs; (B) A serum negative for HLA class II antibodies: 0% PRAs; (C) A serum that is positive for anti-HLA Class I antibody: 89% PRAs; (D) A serum positive for anti-HLA class II antibody: 75% PRAs. FITC: Fluoroisothyocyanate; PRA: Panel-reactive antibody.

It is well known that prolonged cold ischemia time causes delayed graft function in deceased donor transplants, resulting in worse long-term graft survival. Therefore, preventing any unnecessary cold ischemia time is crucial for deceased donor transplantation.^[25,26,27] Typically, a prospective crossmatch between donor and recipient is required before transplantation to detect any circulating donor-specific antibodies. Interestingly, under certain circumstances (e.g., import kidneys with long ischemia times), the flow screening procedure has been used to determine the presence or absence of HLA antibody in recipients in advance of transplantation. This has decreased the ischemia time by allowing transplantation to proceed without a prospective crossmatch if no anti-HLA antibody was detected in the recipient.^[9]

The development of Luminex multiplex platforms has advanced the field of HLA antibody testing by providing a technique that allows the simultaneous detection of multiple analytes. Up to 100 color-coded microspheres can be detected in a single well, thus providing a powerful tool for antibody screening and identification. In the screening assay, purified HLA antigens are coated onto color-coated microspheres, incubated with patient sera and analyzed for shifts in fluorescent intensity. The utility of the method lies in the ability to assay high volumes of samples in a rapid manner as the sample preparation and acquisition of samples is performed in a 96-well microtiter plate. Once the presence of HLA class I or class II antibodies has been determined by a screening assay, the specificity of HLA antibody can be determined using specificity assays.

Expert Rev Clin Immunol. 2008;4(3):391-399. © 2008  Expert Reviews Ltd.

Cite this: Trends in HLA Antibody Screening and Identification and Their Role in Transplantation - Medscape - May 01, 2008.