Search
Search
Crossmatching (XM) is a critical procedure in the field of organ transplantation, serving as a cornerstone for ensuring compatibility between donor and recipient. This process involves a series of laboratory tests designed to detect any pre-existing antibodies in the recipient's blood that may react adversely with the donor's tissue antigens. By analyzing these interactions, healthcare professionals can significantly reduce the risk of transplant rejection, thereby improving the overall success rates of transplantation1. Crossmatching helps ensure safety of organ transplants and plays a vital role in the success of the transplant procedure.
A crossmatch is an important procedure in organ transplantation for several critical reasons:
Compatibility assessment: Crossmatching helps determine the immunological compatibility between the donor and recipient. By identifying potential antibody-mediated reactions, healthcare providers can help ensure that the recipient's immune system will not aggressively attack the transplanted organ, thereby improving the chances of a successful transplant. Crossmatching can predict, and therefore help prevent two key types of transplant rejection, hyperacute rejection, and (accelerated) acute antibody mediated rejection.
Hyperacute rejection: One of the primary reasons for conducting a crossmatch is to help prevent hyperacute rejection, which can occur within minutes to hours after transplantation. This type of rejection is caused by pre-existing antibodies in the recipient's blood that react against the donor's antigens, leading to immediate and severe damage to the transplanted organ.
(Accelerated) acute antibody mediated rejection: Whilst circulating donor specific antibodies may not be enough to cause hyperacute rejection, the presence of memory B cells can cause a rapid increase in circulating donor specific antibody in the first 7-10 days post-transplant. This can lead to accelerated acute antibody mediated rejection. Sensitive physical and virtual crossmatch techniques can help identify this risk.
Personalized treatment plans: The results of a crossmatch test can guide the medical team in tailoring immunosuppressive therapy for the recipient. For example, in urgent transplant cases, the medical team may choose to proceed to transplant in the presence of a positive crossmatch, but tailor immunosuppression appropriately.
Resource optimization: Organ transplantation is a resource-intensive process, often involving scarce donor organs. Crossmatching helps ensure that these valuable organs are allocated to recipients with the highest likelihood of success, thereby optimizing the use of available resources.
Patient safety: Ultimately, the primary goal of crossmatching is to help ensure patient safety as transplant rejection can increase the risk of morbidity and mortality for the recipient. By identifying and mitigating risks associated with antibody-mediated rejection, crossmatching helps play a crucial role in protecting the health and well-being of transplant recipients.
Human Leukocyte Antigens (HLA) are the main transplant antigens as these are present on transplanted tissues and often differ between donor and recipient.
Recipients can form HLA-specific antibodies which can cause antibody-mediated rejection of donor tissues. HLA-specific antibodies are formed by prior sensitization where the recipient is exposed to ‘foreign’ HLA. This can occur through pregnancy, blood transfusion, or previous transplant. Therefore, it is important to detect and define such preexisting antibodies in each patient to assess compatibility of potential donors.
Crossmatch procedures encompass both virtual crossmatching and physical crossmatch.
The basis of the physical crossmatch assay is to combine recipient’s serum with donor lymphocytes to detect donor-specific antibodies to mismatched HLA antigens.
The first step of the crossmatch procedure is isolation of lymphocytes from peripheral blood or donor tissue such as spleen or lymph nodes for cadaveric donors. Since T cells express HLA Class I antigens while B cells express both HLA Class I and Class II antigens, separation of cell types helps detection of antibodies to each HLA Class. However in cases where both T and B cells are crossmatch positive it is not possible to tell which class of antibodies are causing reactivity since both cell types express Class I antigens, thereby masking reactivity to Class II antigens that are also expressed on B cells.
Two main types of physical crossmatch assays are Complement-Dependent Cytotoxicity (CDC) assay and the Flow Cytometry Crossmatch (FCXM) assay.
A more recent assay is the bead-based crossmatch that combines conventional cellular crossmatch with microbead technology to selectively capture donor HLA antigens onto beads for testing against recipients’ serum. Its key innovation lies in its ability to distinguish HLA-specific antibodies from other antibodies, thereby helping to potentially reduce false positives caused by non-HLA reactivity.
In their landmark 1969 study, Patel and Terasaki1 showed that a high percentage of kidney transplants performed across a positive complement‑dependent cytotoxicity crossmatch (CDC‑XM) failed because of accelerated rejection. Their demonstration that a simple crossmatch test could predict such rapid graft loss marked a major advance, establishing the CDC‑XM assay as a widely trusted pre‑transplant immunological risk assessment in solid organ transplantation.
The traditional method involves mixing patient serum with cells expressing HLA antigens and addition of complement to detect cell lysis indicating the presence of HLA-specific antibodies.
One of the main advantages of the CDC assay is it detects clinically significant complement-fixing antibodies which are associated hyperacute rejection, the most dangerous and immediate type of rejection.
A main disadvantage of the CDC assay is the low detection sensitivity compared to more modern assays (such as FCXM). This is partly due to the assay not being able to detect non-complement-fixing antibodies or low titer antibodies. It can also be prone to false positive results due to autoantibodies which are often IgM that reduce the test specificity. A summary of advantages and disadvantages of the CDC assay are shown in Table 1.
Over the years, several modifications have been made to the standard CDC-XM assay to enhance its sensitivity, including extended incubation times, the addition of washing steps, and the use of anti-human globulin (AHG) reagent2. Additionally, serum treatment with dithiothreitol (DTT) was introduced to differentiate true positive crossmatch reactions from those caused by clinically insignificant IgM antibodies3.
The CDC crossmatch is still useful in certain transplant settings, especially where access to more advanced technology is limited. It may also be useful in assessment of transplant risk in highly sensitized urgent cases, where surgeons may transplant across a FCXM positive, CDC negative crossmatch with significantly elevated immunosuppression. However, it is often considered outdated as a standalone test due to lower sensitivity and specificity, and it's frequently used in combination with newer methods for a comprehensive immunological risk assessment.
Table 1. A summary of advantages and disadvantages of the CDC assay.
| Aspect | Pros | Cons |
| Sensitivity | Detects complement-activating antibodies | May miss low-level or non-complement-binding antibodies |
| Specificity | Identifies high risk antibodies | Can't determine exact antibody specificity |
| Practicality | Simple and inexpensive and requires low sample volume (usually 1ul of serum and 1ul of cells) | Requires fresh donor cells |
| Clinical Usefulness | Predicts hyperacute rejection | Less predictive of acute or chronic rejection |
| Modern Relevance | Still used in some protocols | Often replaced by FCXM or Luminex in advanced centers |
| Interpretation | Binary output of positive or negative based on cell death under microscope | Interpretation can be subjective, particularly in cases of weak or borderline reactivity |
The CDC AHG (Anti-Human Globulin) assay is an enhanced version of the standard Complement-Dependent Cytotoxicity (CDC) crossmatch used in organ transplantation. It’s designed to be more sensitive than the standard CDC crossmatch to detect low level, but clinically relevant donor specific antibodies.
The CDC AHG assay adds anti-human globulin (AHG) after the addition of serum in the standard assay. This increases the total number of IgG molecules bound to the target cell and therefore enhances the sensitivity of the assay.
The CDC-AHG assay is a more sensitive version of the CDC crossmatch, capable of detecting lower concentration or non-complement-binding IgG antibodies. It’s useful for refining immunologic risk assessment before transplantation, but is often supplemented or replaced by newer, more precise methods (like flow crossmatch or Luminex testing) in many transplant centers. Table 2 shows a summary of advantages and disadvantages of AHG.
Table 2: summary of advantages and disadvantages of AHG-CDC.
| Category | Details |
| Advantages | |
| Sensitivity | Detects low-titer or non-complement-fixing IgG antibodies |
| Clinical Utility | Helps identify high-risk patients missed by standard CDC |
| Use Case | Especially useful in sensitized recipients (e.g., prior transplants, pregnancies, transfusions) |
| Disadvantages | |
| Specificity | Lower specificity; may detect clinically irrelevant antibodies → false positives |
| Sensitivity Limit | Still less sensitive than flow cytometric crossmatch (FCXM) |
| Interpretation | Results may be subjective and harder to interpret compared to newer methods |
The flow cytometric crossmatch (FCXM) was first introduced in 1983 by Garovoy et al.4, as a more sensitive alternative to the traditional CDC crossmatch.
The goal was to detect low-level donor-specific antibodies (DSAs) that the CDC crossmatch might miss, helping to reduce antibody-mediated rejection and improve graft survival.
The traditional method involves mixing patient serum with donor lymphocytes that express HLA antigens. Fluorescent markers are used to identify T and B cell populations and a fluorescent marker (usually anti-human IgG) to detect any bound donor-specific antibodies.
FCXM is a semi-quantitative assay that measures parameters such as median channel shift (MCS) and relative mean fluorescence (RMF) to assess the strength and specificity of donor-specific antibodies (DSAs). These values play a key role in clinical risk assessment during transplantation.
When combined with Luminex-based single antigen bead (SAB) assays, FCXM could potentially enable more precise antibody identification and immunologic risk stratification. Importantly, the detection of low-level DSAs is not necessarily a contraindication to transplantation.
Compared to the traditional complement-dependent cytotoxicity (CDC) crossmatch, FCXM offers greater sensitivity. Used alongside Luminex technology, it can support a risk-based approach to transplantation, helping clinicians make informed decisions based on the presence or absence of DSAs. See Table 3 fontages and disadvantages of the FCXM assay.
Table 3. Advantages and disadvantages of the FCXM assay.
| Advantages | Description |
| High Sensitivity | Detects low levels of donor-specific antibodies (DSAs) that CDC crossmatch may miss. |
| Semi-Quantitative | Provides data like median channel shift (MCS) and relative mean fluorescence (RMF) to estimate antibody strength. |
| Differentiates Cell Types | Can distinguish between T-cell (class I HLA) and B-cell (class I and II HLA) reactivity. |
| Specific for IgG | The use of anti-human IgG to detect recipient antibody helps discriminate from the non-clinically significant IgM antibody which affects the CDC test.4 |
| Better Prediction of AMR | Strong correlation with antibody-mediated rejection (AMR), especially when paired with Luminex assays. |
| Useful for Sensitized Patients | More informative for patients with prior sensitization (e.g., due to transfusion, pregnancy, or previous transplants). |
| Can Be Standardized | With proper protocols (e.g. Halifax6), it can be optimized for speed and scalability. |
| Disadvantages | Description |
| False Positives | May detect non-HLA antibodies or clinically irrelevant antibodies, leading to unnecessary concern. |
| Requires Viable Donor Cells | Needs fresh, viable lymphocytes for accurate results—may be challenging in deceased donor settings. |
| Interpretation Can Be Complex | Requires trained personnel and well-defined cutoffs; borderline results can be ambiguous. |
| Time and Resource Intensive | Compared to virtual crossmatch, FCXM is more labor- and time-intensive (unless rapid protocols like Halifax6 are used). |
| Less Quantitative Than SAB | Doesn’t provide exact antibody specificity or MFI values—must be interpreted alongside Luminex SAB testing. |
A more recent assay is the bead-based crossmatch that combines conventional cellular crossmatch with microbead technology. These solid-phase assays capture HLA antigens of donor cells onto beads away from other cell-based antigens which may cause false reactivity. The goal is to increase specificity by only detecting donor-specific antibodies (DSAs) that are HLA-specific in a recipient's serum.
The virtual crossmatch (VXM) is the process of comparing a patient HLA-specific antibody profile to a donor’s mismatched HLA antigens without performing a traditional physical crossmatch, such as a complement-dependent cytotoxicity (CDC) or flow cytometric crossmatch. It has become an important tool in organ allocation and pre-transplant decision-making as it enables an assessment of transplant compatibility in the absence of donor blood samples.
A negative VXM is when a patient has no detectable pre-existing HLA-specific antibodies directed towards the donor HLA antigens. Such donors are considered antibody-compatible and transplantation can occur.
A positive VXM is when a patient has pre-existing HLA-specific antibodies directed towards the donor HLA antigens. Such donors are deemed antibody incompatible and the transplantation may be possible but would require additional testing such as but not limited to a physical crossmatch.
VXM plays a critical role in deceased donor organ allocation systems by rapidly identifying recipients who are immunologically compatible with a specific donor5. Some centers proceed to cadaveric transplantation on the sole basis of a negative VXM without performing a pre-transplant physical crossmatch which typically takes around 4-5 hours. This reduces the cold ischemia time of the organ before transplant which is associated with better outcomes.
In these cases, a retrospective post-transplant physical crossmatch is often performed to confirm the absence of any preformed donor-specific antibodies.
In living donor cases where time allows for confirmatory testing, a VXM is used alongside a physical crossmatch to confirm compatibility. The VXM can often be used to exclude incompatible donors where a number of living donors have come forward, helping save time and cost.
False negatives may occur if:
False positives may result from:
Does not replace physical crossmatch in all cases, especially for:
The information presented on this website is intended solely for educational and general informational purposes. It is not intended to provide medical advice, constitute clinical guidance, or serve as a substitute for professional judgment in patient care.