HLA Laboratory in the Current Era: Flow cytometry, Luminex Cytometry and Transplant Registries to the Rescue! Flashcards
What can a flow cytometer measure ?
- size (forward scatter)
- granularity/ internal complexity (side scatter)
- fluorescence intensity
Advantages of Flow Cytometry over CDC
- More sensitive than CDC
- Cell viability is less of an issue (especially B Cells)
- Less subjective readings
CDC = complement-dependent cytotoxicity
Describe 3-color Flow Cytometry Crossmatch
- Incubate T cells and B cells:
— CD19-APC = B cell
— CD 3-PerCP = T cells - if patient has antibodies:
— FTIC anti-IgG = patient antibodies attached to T and B cells
Properties of Secondary Antibody
- F(ab)’ 2 Fragment (partially cleaved Fc region)
- binds Fc Specifically; not Fab
- No cross-reactivity with mouse/ rabbit Immunoglobulins
- Must be titred for maximum sensitivity (Optimized for low Ab Concentrations)
Negative Flow Crossmatch
peaks for FITC-A are on the left
Positive Flow Crossmatch
FITC-A peaks are shifted more right
Interferences in the Fluorescense XM
- IVIg = false pos
- Thymoglobulin (rabbit polyclonal antibody to thymocytes) = false pos
- Ritiximab (humanized anti-CD20)
- Campath (humanized anti-CD52); removes B cells = false neg
- Auto-reactivity in patient serum = false pos
- Any other antibody that targets T or B cells
Luminex Cytometry
- more simple than Flow Cytometry; only measures 3 colours and allows determination of 500 individual parameters in one tube
- 500 polystyrene beads labeled with varying degrees of 2 colours
- The third colour is used to detect antibody or probe attachment to the bead
Describe Luminex Cytometry: Antibody Analysis
- Each polystyrene bead can be labeled with an individual HLA antigen
- The MFI (mean fluorescence intensity) of each bead is measured
- Because the panel is large, almost all HLA antigens are included in one well
Advantages (and disadvantage) of Luminex
Pros:
- Can measure many more parameters in fewer tubes (ie less processing/ washing etc of samples)
- This cytometer is a lot less expensive than standard flow cytometer
- Methods available for antibody analysis as well as HLA typing
Con:
- Cannot run cells = can’t be used for crossmatch
PRA
Calculated Panel Reactive Antibody:
- Refers to the positivity of a patient sample against a panel of cells representative of the donor population
Calculated PRA (cPRA)
- When antibody testing moved to single antigen beads, it lost the ability to be representative of the population frequency of an antigen
- Online cPRA tools allow the calculation of %PRA (based on the known frequency of antigens in the population)
- Provide clinicians with a sense of donor availability
Disadvantages of Flow Cytometry
- Detects only IgG antibodies
- More expensive
