CHT Crossmatch Procedures & Interpretation 2 — Questions and Answers
Question 1: Flow cytometric crossmatch (FCXM) is considered more sensitive than standard CDC crossmatch because:
- It uses a larger volume of recipient serum in each test run
- It detects antibody binding directly, independent of complement fixation (Correct answer)
- It only requires T cells, simplifying cell preparation
- It uses fresh rather than frozen archived serum samples
Correct answer: It detects antibody binding directly, independent of complement fixation
FCXM uses a fluorescent-labeled secondary anti-human antibody to detect IgG binding to cell-surface HLA, allowing detection of low-level antibodies insufficient to fix complement.
Question 2: In a flow cytometric T-cell crossmatch, a positive result is typically defined by:
- Greater than 10% of T cells showing cell death by 7-AAD exclusion
- A fluorescence channel shift exceeding the laboratory-defined threshold vs. negative control (Correct answer)
- Greater than 50% of T cells showing surface antibody binding
- An MFI lower than the negative control sample
Correct answer: A fluorescence channel shift exceeding the laboratory-defined threshold vs. negative control
A positive FCXM is defined by a fluorescence shift (in channels or MFI) beyond a laboratory-established threshold relative to the negative control, indicating specific antibody binding.
Question 3: A positive B-cell crossmatch with a concurrent negative T-cell crossmatch most likely indicates:
- Anti-HLA class I antibodies directed at HLA-A, B, or C antigens
- Anti-HLA class II antibodies directed at DR, DQ, or DP antigens (Correct answer)
- ABO blood group incompatibility
- Nonspecific complement activation from aggregated immunoglobulin
Correct answer: Anti-HLA class II antibodies directed at DR, DQ, or DP antigens
B cells express both HLA class I and class II; a positive B-cell but negative T-cell crossmatch suggests anti-class II specificity, since T cells lack constitutive HLA class II expression.
Question 4: A 'virtual crossmatch' in transplantation is performed by:
- Using computer modeling to simulate complement-mediated lysis in silico
- Comparing recipient antibody specificities (from solid-phase assay) against the donor's HLA type (Correct answer)
- Generating in vitro donor cell lines from stored DNA for physical testing
- Testing recipient serum against a virtual panel of 100 random donor cells
Correct answer: Comparing recipient antibody specificities (from solid-phase assay) against the donor's HLA type
Virtual crossmatch matches the recipient's HLA antibody profile (from Luminex single antigen bead testing) against the donor's HLA typing to predict physical crossmatch outcome without requiring donor cells.
Question 5: EDTA is sometimes added to crossmatch reaction tubes in order to:
- Enhance HLA antigen expression on lymphocyte surfaces
- Chelate divalent cations to inhibit complement activation (Correct answer)
- Preserve lymphocyte viability during extended incubation
- Reduce nonspecific binding of the secondary fluorescent antibody
Correct answer: Chelate divalent cations to inhibit complement activation
EDTA chelates Ca²⁺ and Mg²⁺ ions that are required for complement cascade activation, thereby preventing complement-mediated cytotoxicity and allowing detection of complement-independent antibodies.
Question 6: The antihuman globulin (AHG)-enhanced CDC crossmatch increases sensitivity by:
- Replacing the requirement for rabbit complement entirely
- Crosslinking IgG bound to cell-surface HLA, increasing complement-fixing complexes (Correct answer)
- Specifically targeting IgM antibodies in the recipient serum
- Reducing false-positive results caused by autoimmune antibodies
Correct answer: Crosslinking IgG bound to cell-surface HLA, increasing complement-fixing complexes
AHG (anti-human IgG) binds to IgG already attached to cell-surface HLA antigens, crosslinking them and creating larger immune complexes that more efficiently fix complement.
Question 7: Which cell preparation is most appropriate as a target for a T-cell crossmatch?
- Washed red blood cells isolated from donor whole blood
- Peripheral blood T lymphocytes isolated by rosetting or immunomagnetic separation (Correct answer)
- Bone marrow-derived dendritic cells from the donor
- Donor-derived platelet-rich plasma
Correct answer: Peripheral blood T lymphocytes isolated by rosetting or immunomagnetic separation
Peripheral blood T lymphocytes are used because they constitutively express HLA class I antigens and are readily isolated from blood by rosetting with neuraminidase-treated sheep RBCs or CD3 magnetic beads.
CHT Crossmatch Procedures & Interpretation 2
Flow cytometric crossmatch (FCXM) is considered more sensitive than standard CDC crossmatch because: