HLA Laboratory in the Current Era: Flow cytometry, Luminex Cytometry and Transplant Registries to the Rescue!

Question 1

What can a flow cytometer measure ?

Answer 1

• size (forward scatter)
• granularity/ internal complexity (side scatter)
• fluorescence intensity

Question 2

Advantages of Flow Cytometry over CDC

Answer 2

• More sensitive than CDC
• Cell viability is less of an issue- especially for B Cells
• Less subjective readings
• Detects only IgG antibodies
  But more expensive

CDC = complement-dependent cytotoxicity

Question 3

3-color Flow Cytometry Crossmatch

Answer 3

• isolate T cells and B cells
• Incubation: if patient has antibodies = attach
  — CD19-APC = B cell
  — CD 3-PerCP = T cells
  — FTIC anti-IgG = how many patient antibodies attached to T and B cells

Question 4

Properties of Secondary Antibody

Answer 4

• Affinity Purified
• F(ab)’ 2 Fragment (partially cleaved Fc region)
• binds Fc Specifically; not Fab
• No Crossreactivity with mouse, rabbit Immunoglobulins
• Must be titred for maximum sensitivity (Optimized for low Ab
  Concentrations)

Question 5

Negative Flow Crossmatch

Answer 5

peaks for FITC-A are on the left

Question 6

Positive Flow Crossmatch

Answer 6

FITC-A peaks are shifted more right

Question 7

Inferences in the Fluorescense XM

Answer 7

• 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

Question 8

Luminex

Question 9

Luminex Cytometry

Answer 9

• 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

Question 10

Luminex Cytometry: Antibody Analysis

Answer 10

- 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

Question 11

Advantages (and disadvantage) of Luminex

Answer 11

• 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
• BUT…. Cannot run cells, thus can’t be used for crossmatch**

Question 12

PRA

Answer 12

Calculated Panel Reactive Antibody:
- Refers to the positivity of a patient sample against a panel of cells representative of the donor population

Question 13

Calculated PRA (cPRA)

Answer 13

• When antibody testing moved to single antigen beads, it lost the ability to be representative of the population frequency of an antigen
• A ‘true’ PRA is not captured by single antigen beads
• %PRA based on the known frequency of antigens in the population
• Online cPRA tools allow the calculation of %PRA
• Provide clinicians with a sense of donor availability