CHT Antibody Detection & Characterization 1 — Questions and Answers
Question 1: Which assay platform is the current gold standard for HLA antibody detection and specificity determination in US transplant labs?
- Luminex-based Single Antigen Bead (SAB) assay (Correct answer)
- Complement-dependent cytotoxicity (CDC) PRA
- ELISA-based PRA panel
- Western blot
Correct answer: Luminex-based Single Antigen Bead (SAB) assay
Luminex SAB assays provide high sensitivity and specificity for detecting and identifying HLA antibodies against individual antigens, making them the standard in most US transplant centers.
Question 2: What does a prozone effect (hook effect) in Luminex SAB testing refer to?
- High antibody concentrations cause falsely low MFI due to steric inhibition of detection (Correct answer)
- Very low antibody concentrations produce falsely elevated MFI
- Complement in serum activates beads producing false positives
- The assay reaches maximum fluorescence and cannot distinguish concentrations
Correct answer: High antibody concentrations cause falsely low MFI due to steric inhibition of detection
In the prozone effect, very high antibody concentrations saturate bead antigen sites and sterically hinder detection antibody binding, leading to falsely reduced MFI values.
Question 3: How does EDTA treatment of serum improve HLA antibody detection in Luminex SAB assays?
- It chelates calcium to inactivate complement, preventing complement-mediated bead interference (Correct answer)
- It denatures IgM antibodies to isolate IgG only
- It removes lipids that interfere with bead binding
- It reduces background fluorescence from non-specific binding
Correct answer: It chelates calcium to inactivate complement, preventing complement-mediated bead interference
EDTA chelates calcium ions required for complement activation, preventing complement already present in serum from binding C1q to beads and generating false-positive signals.
Question 4: What is the significance of a 'mean fluorescence intensity' (MFI) threshold in SAB assay interpretation?
- MFI values above the threshold are considered positive for antibody presence and used to define DSA (Correct answer)
- MFI directly measures antibody concentration in mg/mL
- MFI values below the threshold indicate tolerance
- MFI is only used for CDC crossmatch interpretation
Correct answer: MFI values above the threshold are considered positive for antibody presence and used to define DSA
Each lab establishes an MFI cutoff (e.g., 1,000 or 2,000) above which a result is called positive; thresholds are validated internally and affect clinical decision-making.
Question 5: Which HLA antibody class is most commonly detected in sensitized transplant patients?
- IgG (Correct answer)
- IgM
- IgA
- IgE
Correct answer: IgG
IgG HLA antibodies are the most clinically relevant and most commonly detected class in transplant patients, as they cross the placenta, fix complement, and mediate AMR.
Question 6: What is a 'Panel Reactive Antibody' (PRA) test historically designed to detect?
- Antibodies in a patient's serum reactive against a panel of randomly selected donor lymphocytes (Correct answer)
- The percentage of viable lymphocytes in a donor panel
- Autoantibodies against patient's own HLA
- Complement levels in post-transplant serum
Correct answer: Antibodies in a patient's serum reactive against a panel of randomly selected donor lymphocytes
The PRA test measures the breadth of HLA sensitization by determining what percentage of a random donor panel is lysed by the patient's serum, indicating how broadly sensitized the patient is.
CHT Antibody Detection & Characterization 1
Which assay platform is the current gold standard for HLA antibody detection and specificity determination in US transplant labs?