Luminex, Tissue typing and HLA matching.

luminex, HLA typing

1. Tissue Typing and HLA Luminex Ken Chau

2. Contents HLA What is Luminex, Multiplex What is MFI How is MFI interpreted, correlate clinically

3. LUMINEX

4. What is LUMINEX immunoassay? Luminex assay is a magnetic microparticle-based immunoassay which utilizes the same sandwich principles as traditional ELISAs ( Enzyme-linked immunosorbent assay ).

5. The sample is mixed with the bead mixture, allowing the analytes to bind to their respective capture antibodies on the beads. The beads are incubated to allow for the binding of analytes from the sample. After washing away unbound materials, samples are incubated with a mixture of biotinylated detection antibodies and a streptavidin-phycoerythrin (PE) reporter , a fluorescent dye.

6. The beads are passed through a dual-laser flow-based detection system in the Luminex analyzer. One laser excites the internal dyes to identify the bead set and hence the analyte. The second laser excites the PE , generating a fluorescence signal that correlates with the amount of analyte bound. Multiple readings are taken at each bead region, ensuring robust detection.

7. What is Multiplex? Multiplex allows multiple biological target analytes to be simultaneously examined and quantified in a single sample. Benefits of Multiplex include : Maximizes limited sample – allows data collection from just 25mcL or less of undiluted sample Minimizes experimental variability – samples are processed only once, so multiple data points are derived from a single manipulation Economical – examining multiple analytes in a single sample saves time and resources.

8. Median Fluorescence Intensity (MFI) MFI is how Luminex results are read out. Fluorescence Measurement : The fluorescence intensity of PE on each bead is measured. The intensity reflects the quantity of the analyte captured on that bead. Median Calculation : For each bead set (specific analyte), the fluorescence intensity is measured for numerous beads. The median value of these fluorescence intensities is calculated. The median, rather than the mean, is used to reduce the impact of outliers and provide a more robust measure of central tendency. Interpreting MFI : MFI is directly proportional to the concentration of the analyte in the sample. Higher MFI values indicate higher concentrations of the target analyte. Standard Curves and Quantification : Standard curves are generated using known concentrations of the analyte. The MFI values from the samples are compared to the standard curve to quantify the analyte concentration.

9. Typical Bead Count per Analyte Minimum Bead Count : Most Luminex instruments and assays are designed to use at least 50 to 100 beads per analyte to ensure robust statistical analysis. This count provides a sufficient sample size to calculate a reliable median value and reduces the impact of any potential outliers. Optimal Bead Count : For higher precision and reliability, many assays use around 100 to 200 beads per analyte. This range ensures a good representation of the population of beads, allowing for more accurate median calculations. High Throughput and Variability : In high-throughput settings or assays with more variability, using even more beads (up to several hundred) per analyte might be preferred to enhance data robustness.

10. How do we correlate fluorescence intensity to actual level of antigen/antibody? The correlation of fluorescence intensity to the actual level of antigen or antibody in a Luminex immunoassay involves generating a standard curve using known concentrations of the target analyte 1) after preparing a series of standard solutions with known concentrations (using serial dilution methods) of target antigen/antibody. 2) run Luminex assay on each solutions and measure fluorescence intensity (FI) 3) plot FI against different concentration to generate standard curve

11. Mean Fluorescence Intensity (MFI) cut offs Baranwal et al. Comparative analysis of Luminex-based donor-specific antibody mean fluorescence intensity values with complement-dependent cytotoxicity & flow crossmatch results in live donor renal transplantation. Indian J Med Res. 2017 Feb

12. Conclusion 60 years have passed since the introduction of complement-dependent cytotoxicity (CDC) as the first technique for the detection of HLA antibodies in recipients before undergoing renal transplantation. M ethodologies to detect HLA antibodies have progressed from purely target donor cell-based assays , such as CDC or flow cytometry to the more sensitive and specific HLA protein-based solid-phase assay systems in the form of an enzyme-linked immunosorbent assay ( ELISA ) or HLA antigen-coated fluorescence bead assay based on a Luminex platform. In Luminex , the mean fluorescence intensity (MFI) is a measure of the degree of saturation of total antigens present on the beads by antibodies and is used as a surrogate marker for the level of antibody titres . Currently, there is a lack of consensus with regard to the optimum MFI cut-offs for classifying antibodies as positive or those that are significant.

13. Thank you. Any question?