Crossmatch Testing

1. Introduction

2. Cell Types in HLA Crossmatch

• T-cell Crossmatch: Detects complement-fixing anti-Class I antibodies which can cause immediate, hyperacute rejection. A positive T-cell crossmatch is generally a contraindication to transplant because it strongly implies the recipient has cytotoxic anti-donor antibodies ().
• B-cell Crossmatch: Helps detect anti-Class II antibodies (or weak Class I) that T-cell crossmatch might miss. Isolated B-cell positivity (with T-cell negative) usually indicates Class II DSAs against donor HLA-DR/DQ (). These antibodies can mediate acute or chronic rejection, though they are less associated with instantaneous hyperacute rejection than Class I DSAs. Historically, B-cell crossmatches have a higher false-positive rate (due to non-HLA IgM binding or Fc-receptor uptake of IgG on B cells) and some centers previously deemed them less reliable (). However, in modern practice both T and B cell crossmatches are performed to fully assess donor-specific antibody, as Class II antibodies are clinically significant for graft outcomes.

• Peripheral Blood Lymphocytes (PBL): Often used for living donors or deceased donors when a blood sample is available before organ procurement. Advantages: Readily accessible via blood draw, minimal processing (lymphocytes isolated from blood), and usually high cell viability. Enables performing a crossmatch before organ retrieval in deceased donors, expediting decision-making (). Disadvantages: In deceased donors, pre-retrieval blood may be limited or unavailable (e.g. trauma cases), and blood from critically ill donors can have lower HLA expression on lymphocytes (). Additionally, if the donor received recent blood transfusions, there is a slight chance of transient passenger lymphocytes or immune complexes that could complicate results (though this is rare).
• Spleen: Spleen tissue (removed at organ procurement) is rich in lymphocytes. Advantages: Yields a large quantity of T and B cells, which is especially useful if multiple or repeated crossmatches are needed. Often provides enough B cells for a robust B-cell crossmatch. Disadvantages: Available only post-organ retrieval (for deceased donors) (), which means the crossmatch is done on the day of surgery, potentially extending cold ischemia time. Handling is critical: spleen cells can suffer viability loss if not processed and kept in cold media immediately () (). Cell viability can drop if the spleen tissue dries out or is delayed in transport, risking an inadequate or artifactual result. Moreover, if the donor was given therapies like rituximab (an anti-B cell drug), residual drug on splenic B cells can cause false positive reactions (since complement or detection reagents might target the drug on those cells).
• Lymph Nodes: One or more lymph nodes taken at organ procurement. Advantages: Like spleen, provides donor lymphocytes post-retrieval; easier to manage than a whole spleen (small tissue, simpler cell isolation). Often used in deceased donors as a source of viable lymphocytes for crossmatch and HLA typing. Disadvantages: Also only available after organ retrieval (), so it cannot be used for a preemptive crossmatch prior to organ removal. Yields a more limited number of cells compared to spleen (a single lymph node has fewer total lymphocytes), which can be a constraint if multiple assays are needed. Proper preservation in cold transport media is required to maintain cell viability () ().

3. Crossmatch Methods

• Standard CDC (NIH Technique): This is the basic test performed at room temperature (around 20–22°C) for a short incubation. It primarily detects high-titer IgG antibodies that readily fix complement, as well as IgM antibodies if present () (). A positive result (cell lysis) in the T-cell CDC indicates dangerous Class I DSAs, whereas a B-cell CDC positive (with T-cell negative) suggests Class II DSAs. Standard CDC is simple but relatively insensitive – it may miss low-level or non-complement-binding antibodies (). It also cannot distinguish antibody class or specificity without further testing (one must test T vs B cells separately to infer Class I vs II).
• Amos Wash Modification: To improve CDC accuracy, the Amos technique introduces wash steps after the serum incubation and before adding complement. By washing the donor cells, unbound or low-affinity antibodies (especially IgM) and other serum proteins are removed, reducing background complement activation (). This “three-wash” method (and one-wash variants, often called modified Amos) eliminates anti-complementary factors and weak IgM that could cause nonspecific cell death (). The result is a cleaner assay that detects only clinically meaningful IgG antibodies with less noise. In practice, the Amos modification increases test specificity and sensitivity by filtering out irrelevant binding (). (For example, IgM that binds at cold temperatures but is clinically unimportant at body temperature is washed off.) Many labs incorporate at least one wash step in CDC crossmatches to reduce artifactual positives due to serum impurities.
• AHG-Augmented CDC (Antihuman Globulin Potentiated CDC): This is a sensitized CDC method developed to catch antibodies that standard CDC might miss (). After incubating donor cells with recipient serum, a secondary anti-human globulin reagent is added before complement. This antihuman Ig (often targeting IgG heavy chains or light chains) binds to any Ig already attached on the cell, effectively clustering a greater number of antibodies on the cell surface (). By aggregating IgG molecules, it amplifies complement activation when complement is later added, leading to cell lysis even if each antibody alone would not suffice to trigger lysis (). AHG-CDC can reveal IgG antibodies that are non-complement-fixing (e.g. IgG2 or IgG4 subclasses) or present at low titer (). In doing so, it significantly increases sensitivity, detecting weak Class I or Class II DSAs that would be false-negative in a direct CDC. The trade-off is some loss of specificity: AHG-augmented crossmatch may produce positives of uncertain clinical significance (since it forces cell death even with antibodies that naturally might not cause harm) (). Nonetheless, AHG-CDC (sometimes called “enhanced CDC”) became a common second-tier crossmatch for sensitized patients.
• Cold vs Warm CDC: Performing the CDC crossmatch at different temperatures can differentiate IgM from IgG antibodies. IgM antibodies (like ABO isoagglutinins or certain naturally occurring anti-HLA IgM) often react at cold temperatures (4°C) and may not bind as well at 37°C, whereas IgG antibodies are detected at 37°C. Thus, a crossmatch done at 4°C (sometimes called cold CDC) can reveal cold-reactive IgM, and one at 37°C (warm CDC) targets clinically relevant IgG () (). For example, a serum that causes donor cell lysis at 4°C but not at 37°C suggests IgM antibodies that likely won’t cause rejection in vivo (since the body is warm). Conversely, activity at body temperature indicates IgG DSAs with potential to harm the graft. Many protocols include incubating separate aliquots at both 37°C and 4°C for comprehensive assessment. Extended incubation time (e.g. 60–120 minutes instead of 30) is another modification to increase sensitivity, allowing weaker antibodies more time to bind and activate complement ().

• Principle: If recipient DSAs have bound to the donor cells, the secondary fluorescein-tagged anti-IgG will bind to those antibodies. Each cell that has attached antibody will show increased fluorescence. The flow cytometer quantitates this by measuring the fluorescence intensity of thousands of individual T and B cells (). The readout is often given as a shift in median fluorescence channel or mean fluorescence intensity (MFI) of the donor cells with patient serum compared to a negative control serum (). A significant rightward shift indicates the presence of recipient antibody bound to those cells.
• Sensitivity: Flow cytometry can detect very low levels of antibody – far lower than what is required to cause complement-mediated lysis. It will identify IgG antibodies that do not activate complement (e.g. IgG4 DSAs) or that are present in too low a concentration for CDC () (). In fact, flow crossmatch is often 10–100 fold more sensitive than standard CDC. This high sensitivity means that a CDC-negative/Flow-positive result is common when the recipient has weak DSAs. Flow XM thus serves as an early warning for donor-specific antibodies that could still injure the graft (even if they didn’t cause immediate lysis in vitro). Studies found that patients with flow-only positive crossmatches (CDC–/Flow+) have a higher risk of rejection than those who are completely crossmatch-negative ().
• Ability to Distinguish Class I vs II: By simultaneously analyzing T cells and B cells, the Flow XM indicates the class of antibodies present. For example, fluorescence on B cells only (with T cells at baseline) signifies class II–specific antibodies (since B cells carry class II, T cells do not) (). Fluorescence on both T and B cells implies class I antibodies (because both cell types express class I) – possibly along with class II if B-cell signal is higher. This dual-target analysis in one test is a major advantage; earlier CDC methods required separate setups for T and B. Flow cytometry thus provides a clearer picture of the antibody specificity (class I vs class II) in the patient’s serum.
• Quantitative/Objective Readout: Unlike CDC, which is read by microscope and yields a subjective cell death percentage, Flow XM produces a numeric fluorescence shift that can be objectively measured and standardized. This allows for semi-quantitative assessment of antibody strength. While it’s not an exact antibody titer, the magnitude of fluorescence shift often correlates with antibody levels. This objectivity reduces inter-technician variability and can be automated for high-throughput testing (). Many labs set threshold values of channel shifts to call a crossmatch positive or negative, and use control sera for calibration.
• Enhanced Specificity Measures: Flow crossmatch, being so sensitive, can also detect irrelevant or nonspecific binding. To improve specificity, labs implement techniques such as pronase treatment of donor cells – an enzyme that digests Fc receptors on B cells and monocytes. This prevents nonspecific adherence of immune complexes or Ig to the cell surface via Fc receptors. Adding pronase prior to the crossmatch markedly reduces background fluorescence in B-cell flow assays and improves discrimination between true and false positives (). It is especially helpful because B cells naturally have FcγR that can bind IgG in the patient’s serum, causing false positives; pronase cleaves those receptors (). Other steps include using dithiothreitol (DTT) treatment of serum to break up IgM (if IgM interference is suspected) and careful gating strategies to exclude dead cells or autofluorescent cell populations (e.g. monocytes) that could skew results.
• Advantages: Flow XM’s main advantage is its high sensitivity and ability to detect any bound IgG, complement-fixing or not (). It has revealed DSAs that were missed by CDC but nonetheless correlated with graft rejection, thereby improving risk assessment (). It also consolidates class I and II detection in one assay and provides an objective result. Moreover, it can be completed fairly quickly (a few hours) and is amenable to automation and standardization. In the modern era, flow crossmatch is often considered the gold standard crossmatch for kidney transplantation, used in combination with solid-phase assays.
• Limitations: Despite its strengths, Flow XM has some downsides. It requires a flow cytometer and technical expertise, making it costlier and more complex than CDC. It is so sensitive that it may detect antibodies of questionable clinical relevance (such as very low-level DSAs or antibodies against denatured antigens), leading to potential over-caution (). Indeed, flow crossmatches have a higher false-positive rate – one analysis showed up to ~20% of crossmatches may be false positives, often due to nonspecific Ig binding (). Interpretation can be tricky in the gray zone of a borderline fluorescence shift. Additionally, Flow XM typically focuses on IgG; IgM DSAs (if present) might not be detected unless specific steps are taken. Finally, like CDC, it still requires viable donor cells, which can be a logistical limitation in urgent situations (though much less cell quantity is needed for flow than for CDC).

4. Comparison of CDC vs. Flow Crossmatch

• CDC Crossmatch – Pros: Well-established with decades of correlation to outcomes; detects strongly complement-fixing antibodies (usually the most dangerous ones); a positive CDC is highly predictive of hyperacute rejection (). Equipment needs are minimal (no flow cytometer required). Cons: Lower sensitivity – misses many DSAs that can cause less immediate forms of rejection (); cannot detect non-complement-binding IgG; requires subjective interpretation; live cells and fresh complement are required (quality of both can affect results); some false negatives (due to prozone, complement inhibitors, etc.) and occasional false positives (due to autoantibodies, IgM, etc.) occur. Limited ability to characterize the antibody beyond T vs B cell.
• Flow Crossmatch – Pros: Very sensitive – can detect virtually any donor-specific IgG antibody (); objective, quantitative readout; differentiates class I vs II by simultaneous T/B analysis; does not require complement; can be automated and standardized. A negative Flow crossmatch provides high confidence of the absence of donor-specific antibodies. Cons: Can yield false positives or clinically irrelevant positives (e.g. due to nonspecific Ig binding) (); more technical expertise and equipment needed; an isolated Flow-XM positive result requires careful interpretation with other tests (e.g. antibody identification by Luminex) to decide if it’s truly prohibitive. In some cases, Flow XM might be too sensitive, detecting low-level DSAs that one might choose to ignore if the risk is low – thus, centers must define what level of Flow positivity is considered significant.

5. Interpretation of Results

• CDC T–/B– & Flow T–/B– (All Negative): Interpretation: No detectable donor-specific HLA antibodies. The recipient’s serum did not react against donor T or B cells in either assay. This is the ideal scenario – a completely negative crossmatch indicates immunological compatibility (). Clinically, a transplant can proceed with minimal risk of hyperacute antibody-mediated rejection. (Standard monitoring for de novo antibody development post-transplant is still necessary, but no pre-existing DSAs are evident.)
• CDC T+ / B+ & Flow T+ / B+ (All Positive): Interpretation: Strong positive crossmatch; indicates the presence of robust donor-specific antibodies, likely against HLA Class I (and possibly Class II as well) (). T-cell positivity means the serum has complement-fixing Class I antibodies; B-cell positivity could be due to Class I and/or Class II antibodies. In essence, the recipient has high-level DSAs that both kill cells with complement and are readily detected by flow. Clinical implications: A positive CDC (particularly on T cells) is associated with hyperacute or immediate rejection if transplantation is attempted (). Such a donor is generally contraindicated unless extraordinary desensitization is feasible. The presence of broad reactivity suggests the patient is strongly sensitized; transplant would typically be aborted to avoid catastrophic graft loss.
• CDC T– / B+ & Flow T– / B+ (Isolated B-cell Positive in both): Interpretation: Pattern consistent with HLA Class II–specific antibodies. The recipient’s serum reacts with donor B cells but not T cells. Donor B cells express HLA class II antigens (DR, DQ, DP), whereas T cells do not; thus a positive B-cell crossmatch with negative T-cell crossmatch indicates antibodies directed at Class II HLA (). These could be anti-HLA-DR or -DQ DSAs. In CDC, it means those Class II antibodies were strong enough to fix complement on B cells. Clinical implications: Class II DSAs can cause antibody-mediated rejection (AMR), often manifested as early humoral rejection or chronic rejection of the graft. A CDC-positive Class II DSA is significant; many centers will avoid the transplant or require desensitization/treatment (e.g. plasmapheresis, IVIG) before proceeding. However, some historical protocols considered isolated B-cell CDC positives (with T-cell negative) as less acute than T-cell positives – since class II antibodies typically do not cause hyperacute rejection of a kidney, for instance. Nevertheless, they portend a high risk of acute rejection episodes and poorer long-term outcome if ignored. A Flow-positive B-cell result reinforces that these antibodies are present. In summary, isolated class II DSA scenario: transplant may proceed only with caution (aggressive immunosuppression or desensitization), or be deferred if antibody strength is high.
• CDC T+ / B– & Flow T+ / B– (Isolated T-cell Positive in both): Interpretation: Suggests the presence of antibodies that target an antigen on T cells but not on B cells. Since B cells share all HLA Class I with T cells (and also have more antigens), a true HLA Class I DSA would bind to B cells as well. Therefore, a pattern of T-cell crossmatch positive while B-cell is negative is usually due to non-HLA antibodies or an unusual situation (). For example, the recipient might have an antibody against a T-cell specific antigen (like CD3, CD4, or CD8) or another T-cell surface protein not found (or not expressed) on B cells. Another possibility is an antibody to a private HLA-A,B,C epitope that happens to be expressed strongly on T cells of the donor but perhaps the donor’s B cells (if tested separately) were not available or got lost – but generally, T vs B expression of class I should be similar. So the most likely interpretation is a false positive due to something like an autoantibody or a cross-reactive epitope on T cells. Clinical implications: Non-HLA T-cell reactive antibodies (for instance, anti-CD3) are not known to cause hyperacute rejection of an organ, but they could indicate autoimmune tendencies. Labs will investigate an isolated T-cell positive by doing an autocrossmatch (patient serum vs patient T cells) to see if it’s an autoantibody, and by testing for known non-HLA antibodies. If truly only T-cell reactive and not donor-HLA-specific, the transplant may still be feasible (since the antibody isn’t targeting donor HLA). However, caution is warranted. In practice, this pattern might also prompt re-testing because it’s uncommon to see T+ B– unless there was a technical issue (e.g., B cells were not present in sufficient number or were coated with something like rituximab that masked the flow result). In summary, isolated T-cell positivity is atypical for HLA and usually considered a spurious or non-HLA finding ().
• CDC T–/B– & Flow T+ / B+ (CDC Negative, Flow Positive for T and B): Interpretation: The recipient serum has donor-specific antibodies that bind to both T and B cells (so likely Class I DSAs, possibly alongside Class II), but these antibodies did not cause complement lysis in the CDC assay. This is a classic CDC–/Flow+ scenario indicating low-titer or non-complement-fixing DSAs. For example, the patient may have IgG antibodies against donor HLA-A or -B that are below the threshold of CDC detection or are of a subclass like IgG4 that doesn’t trigger complement well (). Flow, being more sensitive, picks them up on both T and B cells. Clinical implications: This pattern means the patient does have donor-specific HLA antibody, even though the CDC was reassuringly negative. Clinically, such patients are at increased risk of antibody-mediated rejection post-transplant compared to patients who are crossmatch-negative by all methods (). Many centers would treat a CDC–/Flow+ as a positive crossmatch for decision purposes, especially if the antibody strength is moderate to high on flow or if confirmed by solid-phase assay. Some might proceed with transplantation in a CDC–/Flow+ case if the antibody MFI is low and it’s an urgent situation, but they would intensify perioperative immunosuppression and closely monitor for rejection. The key is recognizing that Flow-positive DSAs are real – studies show they can still fix complement in vivo and cause C4d deposition in the graft, even though they failed to fix complement in vitro in the CDC test (). Therefore, a Flow T+/B+ result, even with CDC negative, is taken seriously as an incompatibility sign.
• CDC T–/B– & Flow T– / B+ (CDC Negative, Flow Positive B-cell only): Interpretation: This indicates a low-level Class II–specific DSA. The antibody targets an HLA class II antigen on donor B cells (e.g. HLA-DR or DQ), and is detectable by flow (binding to B cells) but did not cause any CDC cytotoxicity. Perhaps the antibody titer is low or it’s an IgG subclass that doesn’t activate complement. T cells are negative in flow because they lack class II. Essentially, this is a case of a pure class II DSA that is only detected by the sensitive flow assay. Clinical implications: Pure Class II DSAs (especially DQ or DP antibodies) are often observed in sensitized patients and can lead to rejection episodes post-transplant, even if CDC is negative. This flow-positive B-cell result should be correlated with antibody identification assays; if a known DSA corresponds, it confirms the finding. Clinically, many centers would either avoid such a donor or implement therapies (IVIG, plasmapheresis) to lower the antibody before transplant. However, compared to a class I DSA, a small amount of class II antibody might be seen as slightly less acute in terms of hyperacute risk. It mainly raises risk for early antibody-mediated rejection and graft dysfunction. The decision may depend on the MFI/strength of the antibody. In any case, a Flow B-cell only positive is a red flag that the recipient has class II antibodies against the donor, and transplant without intervention could be perilous.
• CDC T–/B– & Flow T+ / B– (CDC Negative, Flow Positive T-cell only): Interpretation: This rare pattern implies an antibody that bound donor T cells but not B cells in the flow assay. Since T cells and B cells share class I HLA, a class I DSA should have been detected on both. Class II doesn’t apply because T lack class II. Thus, an isolated flow T-cell positivity likely means a non-HLA antibody or some artifact affecting T cells. One possibility: the patient has an IgG autoantibody against a T-cell specific antigen (e.g. an anti-CD3 or anti–HLA class I heavy chain epitope that is only accessible on resting T cells). Alternatively, if the donor’s B cells were inadvertently not present or were all bound by something like rituximab (if donor was treated), the flow might show only T cell binding. Generally, though, this pattern calls for repeat testing or additional investigation. Clinical implications: If confirmed as a non-HLA antibody, it might not contraindicate transplant. For example, antibodies against MHC Class I-related chain A (MICA) or other minor antigens can sometimes cause crossmatch positivity in flow; their clinical impact is less clear, but they are not the classic HLA antibodies. The transplant team would proceed cautiously, if at all, and monitor. Often an isolated Flow T-cell+ leads to reviewing the test for technical errors (such as improper gating of B cells). It is usually treated similarly to the CDC/flow T+ B– scenario discussed above – likely an artifact or non-HLA reactivity.
• CDC T+ / B– & Flow T– / B– (CDC Positive, Flow Negative): Interpretation: This discordant result is unusual, but when it occurs, the culprit is often IgM antibodies. CDC can pick up IgM (because IgM fixes complement strongly, especially at colder temperatures), but the standard flow crossmatch typically detects IgG (most labs use an anti-IgG reagent). For instance, if the recipient has an IgM anti-donor antibody – such as an anti-ABO blood group antibody or a cold-reactive IgM anti-HLA – it could cause complement lysis of donor cells in CDC, yet the flow (looking for IgG binding) would be negative (). Another scenario: the patient’s serum might have non-HLA IgM (or even IgG) that triggers complement non-specifically (so CDC shows cell death) but the flow reagent might not recognize it if it’s not typical IgG binding to the cell. Clinical implications: A classic example is ABO-incompatible crossmatch – ABO isoagglutinins (often IgM class) can lyse donor lymphocytes in CDC, yielding a positive result, but if the flow assay is only detecting IgG, it might be negative. Obviously, an ABO-incompatible transplant is a major issue; such a result would indicate the transplant cannot proceed without ABO antibody removal. Another example is a patient with strong IgM autoantibodies (say, cold agglutinins): the CDC might be positive with any cells (including auto), but flow IgG is negative. The laboratory should perform an autocrossmatch and possibly treat the serum with DTT to confirm this (DTT will abolish IgM and make the CDC negative if IgM was the cause) () (). In general, whenever CDC is positive but flow is negative, technical verification is needed. The lab would check complement source, repeat with AHG-augmented CDC (if not already done), and consider serum treatment. From a clinical standpoint, one would not ignore a CDC positive just because the flow is negative – especially a T-cell CDC positive. It may be prudent to assume an incompatible transplant unless the cause is clearly identified as an innocuous IgM or autoantibody.

6. Autocrossmatch

• Purpose: A positive autocrossmatch indicates that the recipient has antibodies that react with self-antigens on their lymphocytes. Often these are autoimmune antibodies (for example, certain autoimmune diseases or infections can induce lymphocytotoxic antibodies) or broadly reactive antibodies that bind to common antigens on any lymphocytes. IgM autoantibodies are a frequent cause – for instance, cold-reactive IgM (like cold agglutinins) can bind to lymphocyte surfaces and activate complement, causing cell lysis in vitro (). By performing an autocrossmatch, the lab identifies if a positive donor crossmatch might be due to such autoantibodies rather than true alloantibodies.
• Interpretation:
  • Negative Autocrossmatch: If the patient’s serum does not react against their own cells, then any positivity seen in a donor crossmatch is more likely to be donor-specific alloantibody. A negative autocrossmatch is the usual finding in allosensitized patients (they have antibody to someone else’s HLA, not their own). In crossmatch interpretation, a negative auto gives confidence that a positive result with donor cells is meaningful and not just an artifact of a generally “reactive” serum.
  • Positive Autocrossmatch: If the patient’s serum kills their own lymphocytes or shows significant binding, it signals the presence of autoantibodies or other nonspecific reactivity (). In this case, a positive crossmatch with a donor might be a false positive – the serum might react with everyone’s cells. For example, a patient with lupus might have antilymphocyte antibodies that make every crossmatch positive. When autocrossmatch is positive, labs employ further steps: one common approach is treating the serum with DTT (dithiothreitol), which destroys IgM. Since many autoantibodies are IgM, a DTT-treated serum may lose its reactivity if IgM autoantibody was the cause (). If the autocrossmatch becomes negative after DTT, it confirms that IgM autoantibodies were behind it. Any residual positivity after removing IgM would suggest an IgG autoantibody. Another approach is absorption techniques – absorbing the serum with autologous cells or B cells to remove autoantibodies.
• Impact on Donor Crossmatch Decisions: A positive autocrossmatch complicates interpretation. For instance, if a donor crossmatch is weakly positive and the auto is also positive, the transplant center might interpret the donor crossmatch as “clinically negative” (i.e., no donor-specific antibody, only autoantibody). Some centers in such cases proceed with transplant if they conclude the antibody is purely auto-immune and not specifically directed at donor antigens. However, it requires caution and often additional testing (such as testing the serum against panel cells or performing a DAT/ELISA for autoantibodies). In contrast, if the autocrossmatch is negative but donor crossmatch is positive, one can be more confident that the donor crossmatch signifies true donor-specific HLA antibodies. Autocrossmatch results can also explain high background in flow crossmatches. Laboratories routinely document autocrossmatch results to decide if crossmatch positivity is due to allo- or autoantibodies ().

7. False Positives and False Negatives

• Autoantibodies: As noted, patient autoantibodies can cause cytotoxicity in CDC or fluorescence in flow even against any cells. Autoimmune conditions or infections can induce such antibodies. A classic sign is a positive autocrossmatch (). In CDC, autoantibodies (often IgM) will kill autologous cells; in flow, they will bind autologous cells. Solution: Perform an autocrossmatch for every positive result. If positive, treat serum with DTT to eliminate IgM; a reduction of reactivity after DTT suggests the false positive was due to IgM autoantibodies () (). Persistent reactivity after DTT might indicate IgG autoantibodies (less common). Labs may also test patient serum against a panel of random donor cells – if reactive against all, it’s likely nonspecific (so-called “polyspecific” antibodies).
• Nonspecific IgM (Cold reactive antibodies): IgM that reacts at cold temperature or nonspecifically activates complement can produce CDC positivity. For example, ABO isoagglutinins: if the donor is ABO-incompatible, the recipient’s anti-A or anti-B IgM can lyse donor lymphocytes in CDC (lymphocytes do express ABO antigens at low levels). In flow IgG crossmatch, these IgM wouldn’t be detected, highlighting the discrepancy of CDC+/Flow– in such cases (). Another sign is if reactivity is seen only in the cold incubation (4°C) CDC and not at 37°C () (). Solution: Pre-warm the serum to inactivate cold IgM or use 37°C incubation only; neutralize ABO antibodies if doing an ABO-incompatible transplant (through plasmapheresis or enzyme treatment of cells).
• Anti-complementary substances: Some patient sera contain immune complexes or antiphospholipid antibodies that spontaneously activate complement or make cells fragile. This can cause high background cell death in CDC (). The technologist might notice that even the negative control well (with no patient serum, or with an inert serum) has cell death when mixed with the patient’s serum – indicating the serum itself is toxic. Solution: Serum cleansing steps (e.g. heat-inactivation of complement in patient serum, though that also removes IgM), or using serum adsorption to remove immune complexes can help. The Amos wash (multiple washes) also removes nonspecific immune complexes prior to adding complement, thus reducing this type of false positive ().
• Non-HLA Alloantibodies: These are antibodies against donor antigens that are not HLA, for example, antibodies to endothelial antigens or minor histocompatibility antigens on lymphocytes. One example is anti-MICA antibodies (MICA is a stress-induced antigen) or antibodies to CD15 (a granulocyte antigen that might be present if some granulocytes contaminate the lymphocyte prep). These can bind donor cells and cause a positive flow crossmatch or even CDC if they fix complement. They are “false” positives in the sense of HLA compatibility, but they are real antibodies. Often, their presence is suspected if the patient has no HLA antibodies on solid-phase assays, yet crossmatch is positive. Solution: Identify the antibody via additional testing (there are assays for MICA antibodies, etc.). If a non-HLA antibody is suspected and considered not clinically relevant, the transplant could potentially proceed, but this is done cautiously as knowledge on non-HLA antibody impact is evolving.
• Fc-receptor binding (Flow Crossmatch specific): B cells and monocytes have Fc receptors (FcγR) that can capture IgG from serum non-specifically. In a flow crossmatch, patient IgG can “stick” to these cells via Fc receptors, even if the IgG isn’t specific for those cells. This yields increased fluorescence, mimicking a positive crossmatch. B-cell flow crossmatches are notorious for this, contributing to up to 50% false positive rates historically (). Solution: Use pronase digestion of donor cells to cleave Fc receptors, greatly reducing nonspecific IgG binding (). Also, include an appropriate negative control serum – often AB serum with no HLA antibodies – to set a baseline. If the patient’s serum fluorescence is only slightly above the control, it might be just Fc-binding. Additionally, one can pre-block Fc receptors by incubating cells with IVIG or irrelevant immunoglobulin before adding patient serum (though pronase is more effective).
• Rituximab and other Therapeutic Monoclonal Antibodies: If the donor has been treated with monoclonal antibody drugs (such as rituximab, alemtuzumab, daratumumab, etc.), these drugs can be bound to donor lymphocytes at the time of crossmatch and cause false positives. For example, rituximab (anti-CD20) bound on donor B cells will be detected by the anti-IgG reagent in a flow crossmatch – appearing as though the patient has anti-donor B cell antibody (). In CDC, rituximab on donor B cells can directly activate complement (since rituximab is an IgG that will fix complement on B cells), causing B-cell CDC to be positive even if patient serum has no antibody. Similarly, alemtuzumab (anti-CD52) would coat all lymphocytes. Recognition: If a donor was known to have received such a drug (e.g., a donor with lymphoma treated with rituximab), the lab should be alerted. A flow crossmatch false positive due to drug often shows B-cell positive while T-cell is negative (for rituximab specifically, since it targets B cells), and the pattern might not align with the patient’s antibody profile (e.g., the patient’s historical antibody screen is negative). Solution: Treating donor cells to remove the drug is challenging (washing might not remove firmly bound IgG). Some labs use an anti-idiotype antibody to block the therapeutic antibody, or perform additional washes. In the case of daratumumab (anti-CD38), which can interfere with flow XM, treating serum with DTT or specific blocking peptides has been shown to negate the effect (). Recognizing these interferences is critical to avoid falsely labeling a crossmatch as positive – literature reviews have catalogued known monoclonal therapies that cause false positives and recommend mitigation strategies () ().
• Laboratory errors: Simple issues like improper cell isolation (e.g., RBC contamination causing clotting or hemolysis), using the wrong serum, or reading errors can produce false positive readings. Strict controls (using known negative serum as a control on donor cells) help flag if something is amiss. If a false positive is suspected, repeating the crossmatch is often warranted.

• Low Titer or Low Affinity DSA: The patient may have a donor-specific antibody, but in such a low concentration that it falls below the detection threshold of the assay. In CDC, a small amount of antibody might not induce enough cell death to be scored as positive. In Flow, if the fluorescence shift is very small, it might be considered within noise and called negative. This can happen early after a sensitizing event or as antibodies gradually fade. Mitigation: Use the most sensitive assays (e.g., single-antigen bead tests) to detect any antibody the crossmatch might miss. If the patient has a known history of a certain DSA but the current crossmatch is negative, be cautious – the immune memory is still there (). Such a patient could have an anamnestic response post-transplant. Therefore, historical data and SAB assays complement the crossmatch to avoid this pitfall.
• Non-complement-fixing IgG subclasses (CDC specific): A DSA that is IgG4 or IgG2 might bind to donor cells but not activate complement effectively, yielding a negative CDC (). The CDC would falsely reassure in this case. Likewise, “blocking antibodies” (theoretical antibodies that bind but prevent complement binding) could mask a reaction. Mitigation: The AHG-augmented CDC is specifically designed to catch these – by clustering IgG, even IgG4 can cause complement activation (). So using AHG-CDC can reduce false-negative CDC results (). Also, performing a Flow XM will catch IgG4 even if CDC misses it.
• Prozone/Hook effect in Flow: Extremely high levels of antibody can paradoxically cause underestimation in certain assays. In flow crossmatch, this is less common than in bead assays, but if a patient has a super high-titer DSA, sometimes the saturating amounts of IgG can lead to cell agglutination or very high fluorescence that the software might miscalculate (though generally the flow cytometer will still show a large shift). In solid-phase assays like Luminex, this “high-dose hook effect” can give false low readings (), and labs are aware to dilute patient serum if a prozone effect is suspected. In a cell-based crossmatch, a related effect is if too much antibody causes complement consumption before full cytotoxicity (in CDC) – theoretically, if complement is exhausted by a massive amount of antibody binding, some cells might survive leading to an underestimation of positivity. Mitigation: Perform the test with serum dilutions if suspicion arises (for example, a highly sensitized patient with PRA 100% suddenly showing a negative crossmatch might warrant a diluted crossmatch to ensure it’s not prozone).
• Poor cell quality or antigen expression: If donor cells are not viable or express low levels of HLA, an existing antibody might not bind or cause a detectable reaction – yielding a false negative. For instance, a brain-dead donor in shock might have lymphocytes with downregulated HLA expression (). If those cells are used, a weak antibody could be missed. Similarly, if the cells have been inappropriately stored and many are dead, the antibody may have nothing to bind (dead cells might have shed their HLA or the antibody binds but dead cells can’t be lysed further). Mitigation: Always check cell viability and count. If viability is poor (<50% live), the CDC assay in particular may be unreliable (background death obscures additional death). In flow, gating can exclude dead cells (using a viability dye), but if too many are dead, the live subset might be biased. The lab may request a new donor specimen (another lymph node or spleen piece) if cell quality is in doubt.
• Inhibitory substances (Flow specific): Occasionally, patient serum may contain substances that inhibit the binding of detection reagents. For example, human anti-mouse antibodies (HAMA) in a patient could, in theory, bind to the mouse monoclonal anti-IgG reagent and block it, reducing fluorescence. Or if the patient is on certain drugs that quench fluorescence or interfere with immunoassays, it might reduce the signal. These are rare, but to troubleshoot, labs might test the serum reactivity on a standard panel to see if it uniformly blocks things.
• Improper gating or analysis (Flow): A technical oversight could cause missing a positive. For example, if the flow cytometer gate meant to capture B cells is set too strictly, one might gate out the B cells with bound antibody (if they have slightly altered scatter properties). Or if an incorrect negative control was used, the analysis might subtract too much, masking a true shift. Mitigation: Use standardized analysis protocols and always review dot plots manually especially when things don’t match expectation.

• For CDC, inclusion of positive and negative control wells is crucial. A positive control (e.g., serum with a known HLA antibody to the donor or a standard anti-lymphocyte serum) ensures the complement is working and cells can be lysed; a negative control (e.g., AB serum with no antibodies) ensures the assay is clean. If the patient’s test is negative but the positive control fails to kill cells, the CDC result might be a false negative due to complement failure – one would repeat with a fresh complement source (). If the patient’s test is positive but the negative control shows similar lysis, it’s likely a false positive due to serum issues.
• For Flow, use of a negative control serum on donor cells is standard. Many labs use serum from a male AB donor with no antibodies as a baseline. If the patient’s fluorescence is not significantly above this baseline, the crossmatch is considered negative. If both control and patient show high fluorescence, it suggests a problem like autofluorescent cells or general Ig uptake – possibly a false positive. Also, running a autologous flow crossmatch (patient serum on patient cells in flow) can reveal if the serum tends to bind indiscriminately.
• Cross-check with solid-phase assays (Luminex single-antigen beads): If crossmatch is positive, one should look at the patient’s HLA antibody specificities. Do they have antibody to a specific donor antigen? If yes, that confirms the positive is “true.” If the crossmatch is positive but SAB is negative for donor’s antigens, it raises suspicion of a false positive (or a non-HLA antibody). Conversely, if SAB shows strong donor-specific antibody but crossmatch is negative, that might be a false negative crossmatch – perhaps due to prozone or low cell expression. The lab could dilute the serum and repeat the crossmatch in such a case.
• DTT treatment: As mentioned, treating serum with DTT (which breaks IgM pentamers and some disulfide bonds in IgG) is a useful tool. For instance, a patient with a suspected IgM causing positivity can have the crossmatch repeated with DTT-treated serum. If the result goes from positive to negative, it confirms an IgM false positive (). If it remains positive, an IgG antibody is likely present. DTT is also used to mitigate interference from drugs like daratumumab (which is an IgG-kappa monoclonal – DTT can dissociate it) ().

8. Common Assay Issues and Troubleshooting

• Cell Viability Problems: Donor cell viability is paramount for reliable results. If cells are dying or of poor quality, CDC assays may show high background killing and flow cytometry gating becomes difficult. Issue: Lymphocytes from spleen/lymph node that were not kept cold or in media may degrade (); prolonged ischemia or shipping delays can reduce viability. Troubleshoot: Always check viability with trypan blue before the assay. If viability is low (e.g., < 80%), request a new specimen if possible (another lymph node sample) or proceed with caution. For CDC, one can increase complement concentration slightly to ensure even weakened cells can be lysed by strong antibody (though this also can increase background). Document background death from control wells – if high, consider the result equivocal. In flow, gate on a live/dead stain to exclude dead cells. If viability is a known issue (e.g., an ailing donor), relying more on virtual crossmatch or solid-phase assays might be prudent rather than a questionable cell-based result.
• Contamination of Cell Prep: Sometimes donor lymphocyte preparations carry contaminating cells or substances. Red blood cells contamination can cause issues in CDC (hemoglobin release can be toxic to cells or consume complement). Platelets or cell debris can bind antibodies non-specifically and contribute to fluorescence in flow. Troubleshoot: Use proper gradient separation to get a clean lymphocyte population. If RBCs are present, lyse them (with ammonium chloride buffer) prior to crossmatch. Filter out clumps/debris. Ensure cells are washed and resuspended in appropriate buffer (e.g., PBS or media without human serum that could cause nonspecific uptake). Monocyte contamination is a known problem for flow: monocytes express CD19 at low levels and have many Fc receptors, so they can masquerade as B cells and give false positive fluorescence. To mitigate this, use gating that excludes large, auto-fluorescent monocytes (gating on lymphocyte forward/side scatter and a tighter CD19 vs side scatter gate). Pronase treatment also strips Fc receptors on monocytes.
• Inadequate Complement (CDC specific): The quality of complement (usually rabbit serum as complement source) is crucial. If complement is weak or inactive (e.g., from improper storage or an old lot), a true positive CDC might appear negative (false negative). Conversely, some complement lots have high spontaneous toxicity, causing false positives. Troubleshoot: Titrate new complement lots with known positive and negative sera. Always include a positive control when doing CDC – if that control fails to kill, suspect complement issues (). Store complement in small aliquots at –70°C and avoid repeated freeze-thaws. If a positive crossmatch is unexpectedly negative, repeat CDC with a different batch of complement or perform AHG-CDC which can amplify weak reactions.
• Serum Interference: Patient serum can have factors that interfere, such as clotting factors (if sample not fully coagulated), residual medications, or high Ig levels. Troubleshoot: If a serum is lipemic or hemolyzed, try to obtain a better sample. If the patient is on plasma exchange or IVIG, be aware that IVIG can cause transient positivity (IVIG contains IgG that might deposit on cells non-specifically). In such cases, wash the cells more or delay crossmatch until IVIG is cleared if possible. Heparinized samples should be avoided for CDC (heparin can inhibit complement). Use serum separated from a clot (red-top tube or serum separator tube) for CDC.
• Prozone/High-Dose Hook Effect: In bead assays this is common; in crossmatches it’s less so, but if suspected (e.g., known high-titer antibodies but unexpectedly negative or weak result), perform the crossmatch with serial dilutions of the patient’s serum. If a dilution produces a stronger reaction than neat, that indicates prozone. Also check solid-phase results: extremely high SAB MFI (> 15,000) sometimes correlate with prozone in cell assays – dilution then is advisable.
• Borderline Flow Crossmatch Results: Sometimes the flow cytometry result is just around the cut-off. This could be due to slight nonspecific binding or a very low-level DSA. Troubleshoot: Analyze the data carefully – look at histograms or dot plots rather than just the computed channel shift. Compare with the negative control serum result. Some labs use a statistical cutoff (e.g., crossmatch considered positive if channel shift > 2 standard deviations above negative control). If still uncertain, repeat the flow crossmatch or run a different assay: for example, a C1q binding assay on the serum (to see if any of the patient’s antibodies fix complement) or a cell-based ELISA. Consulting the patient’s antibody profile can help: if they have a known DSA with MFI ~1000–2000, the flow crossmatch could be borderline. In such cases, increasing the serum concentration (using neat serum if normally diluted, or repeating with a more sensitive secondary antibody) might clarify. Ultimately, if a result is truly equivocal, the lab should report it as such, and the clinician might treat it as cautiously positive if the clinical risk is high.
• Crossmatch vs. Virtual crossmatch discrepancies: Occasionally the lab might get a donor’s HLA type and predict (virtual crossmatch) that the patient is incompatible due to known antibody, yet the physical crossmatch comes out negative. This could be a false negative of the crossmatch or a false positive antibody test. Troubleshoot: Consider the timing – if the patient had treatments that lowered antibody levels (IVIG, plasmapheresis), the antibody might now be below detectable levels even though historically present. Or, the antibody detected by SAB could have been an artifact (e.g., binding to denatured antigen on beads, not actually binding to native HLA on cells ()). To resolve, perform an AHG-CDC or a more sensitive flow, and run a C4d or C3d binding assay on the patient’s serum with donor cells or donor HLA coated beads. If those show no complement binding, the antibody might not be clinically relevant. Communication between the lab and clinicians is key here – they may decide to proceed if they trust the crossmatch, or err on side of caution if the antibody is known.
• Documentation and Communication: Every unusual finding (like autoantibodies or drug interferences) should be well-documented. If a patient has known interfering substances (say, a positive autocrossmatch each time due to autoimmune disease), flag the record so future crossmatches are interpreted with that in mind. Also, close communication with the organ procurement team helps – e.g., knowing the donor had rituximab allows the lab to adjust the crossmatch protocol or interpretation.

9. Recent Research and Best Practices

• Virtual Crossmatch (VXM): With the advent of sensitive solid-phase assays (Luminex single-antigen bead tests) for detecting anti-HLA antibodies, the concept of a “virtual crossmatch” has gained traction (). A virtual crossmatch means that if a candidate’s known HLA antibody specificities are compared against a donor’s HLA typing and found to have no incompatible matches, the transplant can proceed without a physical crossmatch on the day of transplant. This approach is used to save time, especially in deceased donor kidney allocation. For example, UNOS allocation now utilizes unacceptable antigen listings to perform a virtual crossmatch in the computer match run, aiming to avoid offering organs to patients who would be positive crossmatch (). Best practice is to maintain up-to-date antibody profiles on sensitized patients (through regular panel reactive antibody screenings and SAB tests). Centers like Cambridge (UK) reported that in carefully selected patients (e.g., never sensitized, or consistently antibody-negative), relying on virtual crossmatch allowed them to skip the physical crossmatch and save ~3 hours of cold ischemia time, which improved transplant outcomes () (). Current practice: Many programs perform an immediate “virtual crossmatch” upon donor offer; if it’s negative (no known DSA), they may fast-track the transplant while a confirmatory physical crossmatch is done in parallel. If the virtual crossmatch is positive (known DSA exists), they might decline the organ or initiate desensitization. The physical crossmatch is thus becoming a confirmatory step in some cases, rather than a first-line screen, thanks to virtual crossmatching accuracy.
• Integration of Solid-Phase Assays: Modern histocompatibility labs use a combination of CDC, flow crossmatch, and Luminex SAB assays. Solid-phase assays identify the exact antibody specificities and give an semiquantitative strength (MFI value). Best practice is to interpret crossmatch results in the context of these antibody assays. For instance, if a flow crossmatch is positive, one looks at the SAB results to see which donor antigen is the target and how strong the antibody is. Conversely, if SAB shows a donor-specific antibody with high MFI, a negative flow crossmatch might be re-evaluated (was there a technical miss?). In fact, the sensitivity hierarchy now is: SAB > Flow XM > AHG-CDC > CDC. Research has shown that some antibodies detected by SAB might not cause a positive flow crossmatch (especially weak DQ antibodies), yet they could still be clinically relevant in causing rejection (). Therefore, many centers will avoid transplant if there is a strong DSA on SAB, even if the flow XM is negative – an example of virtual crossmatch trumping physical crossmatch in decision-making. However, SAB assays can sometimes detect antibodies that are not harmful (like those against denatured HLA epitopes or very low-level IgM), so the challenge is distinguishing clinically significant DSAs (). Current best practice guidelines (e.g., AST and ASHI guidelines) recommend using SAB results to characterize risk but confirm critical decisions with cell-based crossmatch whenever feasible.
• C1q/C3d Binding Assays: A development in research is assessing the functional capacity of DSAs to bind complement. For example, a patient might have a DSA with MFI 5000, but if it’s mostly IgG4, it won’t fix C1q. Specialized assays (C1q binding or C3d assays) can identify whether antibodies can initiate complement. Studies correlate C1q-positive DSAs with higher risk of acute rejection. Some labs incorporate this: if a DSA is present but C1q assay is negative, they might be more inclined to proceed (especially if Flow XM is negative), whereas C1q-positive DSAs raise a red flag. This isn’t yet universal practice, but it’s an emerging best practice in immunological risk assessment of crossmatch-positive cases.
• Flow Cytometry Enhancements: There is ongoing research to standardize flow crossmatch interpretation. One initiative is creating uniform cutoff values or scoring systems that can be shared across labs (since currently one lab’s “weak positive” might be another’s “negative”). New fluorescent reagents (e.g., using Fcγ-specific reagents to detect only IgG that are bound via Fab, not ones passively stuck via Fc) are being tried to reduce background. The Halifax protocol (and a faster variant “Halifaster”) are new methodologies that streamline flow XM into a 96-well format, shortening test time and enabling semi-automation () (). These protocols could become best practice for deceased donor crossmatching to minimize delays.
• Desensitization and Positive Crossmatch Transplants: In cases of positive crossmatch that cannot be avoided (e.g., highly sensitized patients with no other donor options), recent research focuses on desensitization therapies. Protocols using plasmapheresis, IVIG, Rituximab, Bortezomib (proteasome inhibitor), and newer agents (eculizumab, IdeS enzyme to cleave IgG) have been developed to overcome positive crossmatches () (). Best practice in such scenarios is to convert a strong positive crossmatch into a negative or weak-positive one before proceeding with transplant. For instance, a patient might undergo plasmapheresis until the flow crossmatch becomes negative, then go to transplant with immunosuppressive support. The success of these interventions is monitored by crossmatch tests – e.g., repeating flow XM after treatments to guide when it’s safe to transplant. The field of transplantation has recognized that not all crossmatch positives are absolute barriers if therapies can mitigate them, which is a shift from earlier eras. However, these are high-risk transplants and done in specialized centers with close antibody monitoring.
• Organ-Specific Practices: As a best practice, the stringency of crossmatch requirements can differ by organ. For kidney and heart transplants, a negative T-cell crossmatch is considered mandatory due to risk of hyperacute rejection. In heart transplantation, time is critical, so sometimes a heart transplant is done with only a virtual crossmatch or an immediate retrospective crossmatch – but any unexpectedly positive result prompts aggressive post-op therapy. For liver transplants, interestingly, some centers will proceed even with a positive crossmatch, because the liver is more tolerant and can sometimes “absorb” antibodies (the liver’s vast endothelium and Kupffer cells can clear circulating immune complexes). Studies have noted that liver transplants can succeed despite positive crossmatch, and a liver transplant can even reduce antibody levels (possibly protecting a subsequent kidney transplant) (). Best practice here is organ-specific: e.g., in liver-alone transplants, a positive crossmatch is not an absolute contraindication if the benefits outweigh risks, whereas in kidney, it mostly is – unless desensitization is done. These nuances are reflected in current guidelines which acknowledge that, for example, heart and kidney require prospective crossmatch, while liver can be transplanted in sensitized patients with careful monitoring.
• Unacceptable Antigen Policies: Another best practice driven by research is the use of unacceptable antigen listing for highly sensitized patients. Instead of doing crossmatches with multiple donors, the patient’s unacceptable HLA antigens (to which they have antibodies) are listed in the allocation system, so those donors are automatically bypassed. This is an application of virtual crossmatching on a national scale that significantly improves transplant rates for sensitized patients by avoiding offers that would crossmatch positive. Research (like the collaborative transplant study and UNOS data) has shown better outcomes and shorter wait times when virtual crossmatch and unacceptable antigen screening are employed proactively () ().
• Continuous Improvements in Assays: Recent studies are also focusing on the quality of HLA antigen presentation in various assays. Some DSAs bind to cells but not to beads, or vice versa, due to antigen conformation differences (). Research is ongoing to create better cell-based surrogate assays (e.g., using donor-derived cell lines or pseudovirus displaying donor HLA) to test binding in a more natural context. Also, the use of endothelial cell crossmatches (crossmatching with donor endothelial cells rather than lymphocytes) is being explored, since endothelial cells are the actual target in the graft and they express HLA class I and II plus other antigens. This may identify certain non-HLA antibodies. However, this is still research-phase and not routine.
• Data-Driven Risk Models: Combining all these test results, researchers are developing scoring systems or algorithms that incorporate antibody strength, complement binding ability, IgG subclass, etc., to predict the risk of rejection if transplanted. The goal is personalized risk assessment – not just “positive or negative” crossmatch, but “how positive” and what that means for outcome. Best practice is moving towards this nuanced interpretation. For instance, a weak Flow positive with a DQ antibody might be considered low risk with proper treatment, whereas a strong Flow positive with C1q-binding Class I DSA is high risk.