What is the workup for a patient with consistently incompatible crossmatch results?

Workup for Consistently Incompatible Crossmatch Results

A patient with persistently incompatible crossmatch results requires systematic investigation to distinguish between clinically significant HLA alloantibodies, non-HLA antibodies (including autoantibodies and antibodies to low-frequency antigens), and technical artifacts—this distinction is critical because only true HLA incompatibility contraindicates transplantation.

Initial Serologic Evaluation

Confirm True Incompatibility vs. Technical Issues

• Repeat ABO blood typing twice to exclude ABO incompatibility as the cause, as this is mandatory before proceeding with further workup 1
• Perform both sensitive crossmatch methods: Use anti-human globulin-enhanced complement-dependent cytotoxicity (AHG-CDC) and flow cytometry crossmatching, as these membrane-dependent assays protect against hyperacute rejection but don't necessarily identify the target antigen 1
• Test with multiple sera samples: Use historically reactive (highest PRA), current, and immediately pre-procedure sera, as antibody levels fluctuate and immune memory responses can occur 1

Distinguish IgG from IgM Antibodies

• Eliminate IgM reactivity through heat treatment or chemical methods (such as dithiothreitol), as IgM-positive crossmatches are NOT contraindications to transplantation 1, 2
• Only IgG antibodies are clinically relevant for transplant compatibility 1

HLA Antibody Characterization

Determine if Antibodies are HLA-Specific

• Perform solid-phase assays using ELISA or Flow Bead technology with specific soluble HLA antigens as targets to definitively identify whether antibodies are directed against HLA antigens 1
• These newer assays are superior to membrane-dependent methods because traditional crossmatches cannot distinguish HLA from non-HLA reactivity 1
• Calculate panel reactive antibody (PRA) percentage using sensitive methods to quantify the degree of sensitization 1

Identify Specific HLA Antibody Class and Specificity

• Perform comprehensive HLA typing for both Class I (HLA-A, B, C) and Class II (DP, DQ, DR) antigens using molecular genotyping rather than serologic methods, as serotyping has high discrepancy rates (up to 36% in African Americans) 1
• Use donor-specific antibody (DSA) testing with single-antigen bead assays to identify the exact HLA specificities of antibodies present 3
• Consider DSA-specific flow crossmatch methods that can distinguish Class I from Class II HLA antibodies and are not impacted by autoantibodies or therapeutic antibodies 3

Non-HLA Antibody Investigation

Screen for Autoantibodies

• Perform autoantibody testing as autoantibodies can cause positive crossmatches but are not contraindications to transplantation when HLA-specific antibodies are absent 1, 3
• Up to 30% of multiparous women have anti-HLA antibodies, but autoantibodies can confound interpretation 1

Evaluate for Antibodies to Low-Frequency Antigens

• Test for antibodies to private (low-frequency) antigens that may not be represented on standard screening panels 1, 4, 5
• These antibodies (such as anti-Kpa in red cell transfusion) can cause incompatible crossmatches despite negative antibody screens 4, 5
• Perform crossmatches with fresh donor cells when possible, as certain antigens may be disrupted by freezing 1

Consider ABO-Related Antibodies

• Perform A subtype testing when relevant, as anti-A or anti-B antibodies can bind to blood group substances on lymphocytes and cause false-positive crossmatches 1, 2
• Absorption studies using type A or B red blood cells can eliminate ABO antibodies to clarify if they are causing the incompatibility 2

Specialized Testing for Persistent Incompatibility

When Standard Workup is Negative

• Use at least two different antibody detection methods (glycoprotein-specific assays like MAIPA and whole-cell assays) to reduce false-negative results, as low-affinity antibodies may be missed by single methods 1
• Consider delayed antibody appearance: Repeat testing 2-8 weeks later if initial results are inconclusive, as certain alloantibodies appear weeks after initial exposure 1, 6
• Test for antibodies to unstable or weakly expressed antigens that may require fresh cells from high expressers 1

Crossmatch with Specific Donor Cells

• Perform crossmatch with fresh paternal/donor platelets or lymphocytes to detect antibodies to private antigens not identified by panel testing 1
• This is particularly important when solid-phase assays are negative but physical crossmatches remain positive 1

Critical Interpretation Pitfalls

• A positive flow cytometry crossmatch with negative AHG-CDC requires careful interpretation—approximately 60% of positive traditional crossmatches are not directed to donor HLA, and 36% of negative traditional crossmatches miss true HLA DSA 3
• Positive crossmatches without identified HLA antibodies may represent non-HLA reactivity and are not absolute contraindications to transplantation 1, 2
• False-positive results occur in 79% of incompatible crossmatches when antibody screens are negative, often due to technical factors or clinically irrelevant antibodies 7, 5
• Incompatibility detected only at immediate spin or room temperature (rather than 37°C/antiglobulin phase) is less likely to be clinically significant 5

Management Based on Findings

• If HLA-specific IgG antibodies are confirmed: Transplantation requires desensitization protocols or identification of HLA-matched donors 1
• If only non-HLA antibodies or autoantibodies are present: Proceed with transplantation using standard protocols, as these are not contraindications 1, 2, 3
• For highly sensitized patients (PRA ≥50%): Avoid blood transfusions or use leukocyte-poor products to prevent further sensitization 1