The document explains Luminex immunoassay technology and its application in tissue typing, highlighting its benefits such as multiplexing capabilities for analyzing multiple targets simultaneously and the measurement of mean fluorescence intensity (MFI) for quantifying analyte concentrations. It discusses standard curve generation for correlating fluorescence intensity with actual levels of antigens or antibodies and emphasizes the evolution of HLA antibody detection methodologies over the past 60 years. There is a noted lack of consensus on optimal MFI cut-offs for classifying antibodies as significant in clinical settings.

1. Tissue Typing and
HLA Luminex
Ken Chau

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

6. LUMINEX

7. 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).

8. Color-coded
microspheres, or
beads, are internally
dyed with different
proportions of red
and infrared
fluorophores that
correspond to a
distinct spectral
signature, or bead
region.
Beads are coated
with specific capture
antibodies or
oligonucleotides.

9. • 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.

10. • 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.

11. 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.

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

13. Typical Bead Count per Analyte
1.Minimum Bead Count:
1. Most Luminex instruments and assays are designed to use at least 50 to 100 beads
per analyte to ensure robust statistical analysis.
2. This count provides a sufficient sample size to calculate a reliable median value and
reduces the impact of any potential outliers.
2.Optimal Bead Count:
1. For higher precision and reliability, many assays use around 100 to 200 beads per
analyte.
2. This range ensures a good representation of the population of beads, allowing for
more accurate median calculations.
3.High Throughput and Variability:
1. 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.

14. 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

16. 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

26. 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.
• Methodologies 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.

27. Thank you.
Any question?