ABO COMPATIBILITY AND TISSUE TYPING IN ORGAN TRANSPLANTATION

Organ transplantation is a life-saving medical procedure that replaces a diseased or damaged organ with a healthy one from a donor. However, the success of transplantation hinges on a complex immunological process where the recipient’s immune system must accept the foreign organ, known as an allograft. Two crucial aspects of this immunological compatibility are ABO blood group matching and Human Leukocyte Antigen (HLA) compatibility, often referred to as tissue typing.

ABO Compatibility in Organ Transplantation

The ABO blood group system is a fundamental determinant of compatibility between a donor and recipient. It is based on the presence or absence of specific antigens (A and B) on the surface of red blood cells. Individuals possess antibodies against the ABO antigens they lack. For instance, a person with blood group A has A antigens on their red blood cells and anti-B antibodies in their plasma. Conversely, a person with blood group O lacks both A and B antigens but possesses both anti-A and anti-B antibodies.

The principle of ABO compatibility in transplantation is to avoid transfusion reactions, particularly those involving red blood cells. In the context of solid organ transplantation, the critical consideration is the presence of pre-formed antibodies in the recipient’s plasma that can target ABO antigens on the donor organ’s vascular endothelium.

Here’s a breakdown of ABO compatibility for different recipient blood groups:

• Recipient Blood Group O:
  • Compatible Donor Blood Groups: O
  • Explanation: Individuals with blood group O are often referred to as “universal donors” for red blood cells because their red blood cells lack both A and B antigens. However, in solid organ transplantation, recipients with blood group O have both anti-A and anti-B antibodies. Therefore, they can only safely receive organs from donors who also have blood group O, as these organs will not express A or B antigens that could be targeted by the recipient’s antibodies.
• Recipient Blood Group A:
  • Compatible Donor Blood Groups: A, O
  • Explanation: Recipients with blood group A have A antigens on their red blood cells and anti-B antibodies. They can receive organs from donors with blood group A (matching antigens) and blood group O (no A or B antigens to react with).
• Recipient Blood Group B:
  • Compatible Donor Blood Groups: B, O
  • Explanation: Recipients with blood group B have B antigens on their red blood cells and anti-A antibodies. They can receive organs from donors with blood group B (matching antigens) and blood group O (no A or B antigens to react with).
• Recipient Blood Group AB:
  • Compatible Donor Blood Groups: AB, A, B, O
  • Explanation: Individuals with blood group AB have both A and B antigens on their red blood cells and lack both anti-A and anti-B antibodies. Consequently, they are considered “universal recipients” for red blood cells and can receive organs from donors of any ABO blood group. This is because their immune system will not mount an antibody response against the A or B antigens present on the donor organ.

It is important to note that while ABO compatibility is a fundamental prerequisite for transplantation, it is not the sole determinant of success. HLA compatibility plays an equally critical role.

Hyperacute Rejection

Hyperacute rejection is a rare but severe and immediate form of allograft rejection that can occur within minutes to hours after the reperfusion (restoration of blood flow) of the transplanted organ. It is a pre-formed antibody-mediated rejection process.

Possible Causes of Hyperacute Rejection:

The primary cause of hyperacute rejection is the presence of pre-formed antibodies in the recipient’s circulation that are directed against antigens expressed on the donor organ’s vascular endothelium. These antibodies rapidly bind to the endothelial cells of the transplanted organ’s blood vessels, triggering a cascade of immune responses. The most significant of these pre-formed antibodies are:

1. Anti-ABO Antibodies: As discussed earlier, these are naturally occurring antibodies in individuals who have a different ABO blood group than the donor. For example, a recipient with blood group A who receives a kidney from a blood group B donor will have anti-B antibodies that can bind to the B antigens on the donor kidney’s blood vessels, leading to hyperacute rejection.
2. Anti-HLA Antibodies: These antibodies are directed against specific HLA antigens present on the donor organ and are typically acquired through previous sensitization. Sensitization can occur through:
   • Previous Transfusions: Blood transfusions from donors with different HLA types can induce the formation of anti-HLA antibodies.
   • Previous Transplantations: Exposure to donor antigens from a prior transplant can also lead to sensitization.
   • Pregnancy: Exposure to paternal antigens during pregnancy can sensitize a woman to develop anti-HLA antibodies.

Usual Measures Taken to Avoid Hyperacute Rejection:

The prevention of hyperacute rejection relies heavily on careful pre-transplant immunological assessment and matching:

1. ABO Blood Group Matching: Strict adherence to ABO compatibility rules is the primary strategy to prevent hyperacute rejection mediated by anti-ABO antibodies. Patients are only transplanted with organs that are ABO compatible with their own blood group.
2. Crossmatching: Before transplantation, a retrospective crossmatch and a prospective crossmatch are performed.
   • Retrospective Crossmatch: This involves testing the recipient’s serum against donor lymphocytes that were cryopreserved from the time of donation. This helps identify any existing anti-HLA antibodies against the donor.
   • Prospective Crossmatch: This is performed immediately before transplantation, where the recipient’s current serum is mixed with donor lymphocytes. A positive prospective crossmatch, indicating the presence of recipient antibodies that bind to donor cells, typically leads to the cancellation of the transplant to avoid hyperacute rejection.
3. HLA Typing and Screening: Comprehensive HLA typing of both donor and recipient is performed. Furthermore, recipients are routinely screened for the presence of pre-formed anti-HLA antibodies against a panel of common HLA antigens. If a recipient is found to have antibodies against a significant proportion of the HLA antigens present on a potential donor organ, that organ would be deemed incompatible.
4. Desensitization Protocols (in specific cases): For recipients who are highly sensitized and have a positive crossmatch due to clinically significant antibodies, desensitization protocols may be employed. These protocols aim to reduce the levels of these antibodies before transplantation, often involving plasmapheresis (removal of plasma) and intravenous immunoglobulin (IVIG) or specific immunosuppressive agents. However, these are generally for preventing antibody-mediated rejection (AMR) rather than hyperacute rejection, which is usually avoided by strict pre-transplant screening.

High- and Low-Resolution Molecular Testing and Serologic Techniques for MHC Typing

Human Leukocyte Antigen (HLA) typing, also known as tissue typing, is a critical process used to assess the genetic compatibility between a donor and recipient based on the Human Leukocyte Antigen (HLA) system. HLA molecules are a group of proteins found on the surface of most cells in the body, playing a crucial role in the immune system’s ability to distinguish self from non-self. In transplantation, HLA mismatches can trigger an immune response against the allograft.

There are two main categories of HLA typing: high-resolution and low-resolution, employing both molecular and serologic techniques.

1. Serologic Techniques:

Historically, serologic techniques were the primary method for HLA typing. These methods rely on the use of antisera (laboratory reagents containing antibodies) that are specific for particular HLA antigens.

• Mechanism: Donor or recipient lymphocytes are incubated with a panel of different antisera. If the antibodies in an antiserum bind to the HLA antigens on the lymphocytes, a reaction is observed, indicating the presence of that specific HLA antigen. Various detection methods are used, such as complement-dependent cytotoxicity (CDC), where antibody binding leads to cell lysis.
• Resolution: Serologic typing generally provides low-resolution typing. It can identify HLA antigens at the “phenotypic” level, meaning it can determine the presence or absence of an antigen (e.g., HLA-A2). However, it often cannot distinguish between closely related alleles that have very similar antigenic determinants but differ in their DNA sequences. For example, serology might identify an individual as HLA-A2 positive but cannot differentiate between the different subtypes of A2 (e.g., A*02:01, A*02:03).
• Clinical Uses:
  • Initial Screening and ABO Compatibility: Serologic methods are still used for initial HLA screening and crossmatching, particularly in the context of antibody-mediated hyperacute rejection prevention.
  • Historical Data: Much of the existing knowledge and historical data in transplantation are based on serologic typing.
  • Some Specific Antibody Detection: In some cases, specific antibodies against non-HLA antigens or certain polymorphic HLA epitopes can be detected using serologic methods.

2. Molecular Techniques:

Molecular techniques have largely replaced serologic methods for definitive HLA typing due to their superior accuracy and ability to provide high-resolution information. These methods directly analyze the DNA sequences that encode HLA molecules.

• Mechanism: These techniques involve amplifying specific HLA gene regions using the Polymerase Chain Reaction (PCR) and then determining the DNA sequence. Various molecular methods are employed:
  • Sequence-Specific Oligonucleotide Probes (SSOP): Labeled DNA probes that are complementary to specific HLA sequences are used to hybridize with amplified DNA fragments.
  • Sequence-Specific Primers (SSP): Primers designed to amplify only specific HLA alleles are used in PCR.
  • Sequencing-Based Typing (SBT): The actual DNA sequence of the amplified HLA genes is determined.
  • Next-Generation Sequencing (NGS): This advanced technology allows for the simultaneous sequencing of multiple HLA genes with high throughput and accuracy, providing the most comprehensive high-resolution typing.
• Resolution: Molecular techniques provide high-resolution typing. They can identify HLA alleles at the “genotypic” level, meaning they can determine the specific DNA sequence of an individual’s HLA genes. This allows for the precise identification of each allele (e.g., HLA-A*02:01:01:01). This level of detail is crucial because even minor differences in HLA alleles can lead to immune responses.
• Clinical Uses:
  • Precise HLA Matching: High-resolution molecular typing is essential for identifying the most compatible donor for a recipient, minimizing the risk of T-cell mediated rejection.
  • Identification of Sensitization: It helps precisely identify the specific HLA antigens against which a recipient has developed antibodies.
  • Donor Selection for Stem Cell Transplantation: In hematopoietic stem cell transplantation, HLA matching is even more critical, and high-resolution typing is mandatory to ensure the best possible match.
  • Research and Registry Matching: Facilitates effective matching of potential donors in national and international bone marrow registries.
  • Diagnosis of HLA-Associated Diseases: Certain HLA alleles are associated with specific autoimmune diseases, and molecular typing is used for diagnosis and genetic counseling.

Distinguishing Between High- and Low-Resolution Typing:

The key distinction lies in the level of genetic detail provided. Low-resolution (often serologic) gives a general idea of antigen presence, while high-resolution (molecular) reveals the precise HLA allele. This precision is critical because T-cells recognize specific peptide epitopes presented by HLA molecules, and even single amino acid differences can influence this recognition. Therefore, for optimal long-term allograft survival, especially in kidney, heart, and lung transplantation, high-resolution HLA matching is increasingly the standard.

Donor-Specific Antibodies (DSAs)

Definition:

Donor-Specific Antibodies (DSAs) are antibodies found in the recipient’s serum that are specifically directed against antigens present on the donor organ’s cells. These antigens can be HLA molecules or, less commonly, non-HLA antigens (e.g., ABO antigens, molecules involved in the renin-angiotensin system). DSAs can be pre-formed (present before transplantation, often due to prior sensitization) or de novo (developed after transplantation).

Implication for Long-Term Allograft Survival:

The presence of DSAs, particularly pre-formed DSAs, has a profound negative implication for long-term allograft survival.

1. Immediate Graft Damage (Hyperacute Rejection): If pre-formed DSAs are present and bind to graft antigens upon reperfusion, they can trigger rapid complement activation and endothelial cell damage, leading to hyperacute rejection, which is immediately graft-threatening.
2. Accelerated Acute Antibody-Mediated Rejection (AMR): Even if hyperacute rejection is avoided through careful pre-transplant screening, the presence of DSAs can lead to accelerated and more severe forms of acute AMR after transplantation. These antibodies activate complement, recruit immune cells, and cause microvascular thrombosis and inflammation within the allograft.
3. Chronic Antibody-Mediated Rejection (Chronic AMR): DSAs are a major driver of chronic AMR, a slow and progressive form of rejection. Chronic AMR is characterized by diffuse intimal thickening, fibrosis, and transplant glomerulopathy, leading to the gradual loss of graft function over months or years.
4. Reduced Graft Longevity: The continuous presence or emergence of DSAs leads to ongoing inflammatory insults to the allograft vasculature and parenchyma. This sustained damage compromises the long-term viability and function of the transplanted organ, significantly reducing its lifespan.
5. Therapeutic Challenges: Patients with DSAs are often more difficult to manage. Standard immunosuppressive regimens may not be sufficient to control DSA-mediated damage. Treatment often requires more aggressive immunotherapy, including antibody-depleting therapies (like rituximab) or plasmapheresis, which carry their own risks and may not always be effective in preventing irreversible damage.

Organ Allocation: A Balancing Act of Need and Compatibility

The allocation of donated organs is a highly regulated and meticulously managed process, governed by national and international organ sharing networks. The primary objective is to match available organs with suitable recipients, prioritizing those with the greatest medical need and the highest probability of a successful transplant outcome. Several factors influence this delicate balancing act:

• Medical Urgency: Patients are typically categorized based on their severity of illness and their expected survival time without a transplant. Those in critical condition with limited alternative treatment options are generally placed higher on the waiting list.
• Time on Waiting List: While not the sole determinant, the duration a patient has been waiting for a suitable organ is also considered.
• Blood Type and Tissue Typing (HLA Matching): This is where the concept of compatibility becomes paramount. The Human Leukocyte Antigen (HLA) system is a group of genes that play a crucial role in the immune system’s ability to distinguish self from non-self. Differences in HLA antigens between the donor and recipient can trigger an immune response, leading to organ rejection. Therefore, matching HLA types as closely as possible is a key objective in organ allocation.
• Geographic Proximity: To minimize cold ischemic time (the time the organ is without blood supply), organs are typically offered to recipients within a specific geographic radius first.
• Patient-Specific Factors: Other factors, such as body size, presence of certain antibodies, and specific medical conditions, can also influence allocation decisions.

The Crossmatch: A Final Compatibility Check

While HLA typing provides a predictive measure of compatibility, it is not the definitive answer. The crossmatch is a laboratory test performed just before transplantation to detect the presence of pre-formed antibodies in the recipient’s serum that are directed against the donor’s antigens. These antibodies, if present, can bind to the donor organ and initiate a rapid and destructive immune attack, known as hyperacute rejection.

The crossmatch serves as a critical “go/no-go” decision point. A positive crossmatch indicates the presence of significant donor-specific antibodies and usually contraindicates the transplant, as the risk of immediate rejection is too high. A negative crossmatch, on the other hand, suggests that the recipient does not have detectable antibodies that would target the donor organ, making the transplant a more viable option.

Methodologies for Crossmatching: Detecting the Immune Threat

Over the years, various laboratory techniques have been refined to perform crossmatches. These methods primarily aim to detect the binding of recipient antibodies to donor lymphocytes (cells from the donor’s immune system). The main differences lie in how this binding is detected and amplified.

1. Cytotoxic Crossmatch (Terasaki Crossmatch)

The cytotoxic crossmatch is the traditional and historically most widely used method. It relies on the principle of complement-dependent cytotoxicity (CDC).

• Principle: Donor lymphocytes (typically T and B cells) are incubated with the recipient’s serum. If the recipient has antibodies against antigens present on these donor lymphocytes, these antibodies will bind to the cell surface. In the presence of complement (a component of blood serum that can be activated by antibody-antigen complexes), the antibody-antigen binding will trigger a cascade that leads to the lysis (rupture) of the lymphocyte membrane.
• Procedure:
  1. Lymphocyte Isolation: Donor lymphocytes are isolated from the donor’s blood.
  2. Incubation: Donor lymphocytes are incubated with dilutions of the recipient’s serum for a set period (e.g., 30-60 minutes).
  3. Complement Addition: Fresh complement (usually from a rabbit or other animal source) is added to the mixture.
  4. Incubation with Complement: The mixture is incubated again, allowing for antibody-complement interaction.
  5. Dye Exclusion: A vital dye, such as trypan blue or eosin, is added. This dye can only enter cells with damaged or disrupted cell membranes. If the lymphocytes have been lysed by the complement reaction, the dye will enter and stain the cell.
  6. Microscopic Examination: Under a microscope, the technician observes the lymphocytes. Lymphocytes that have taken up the dye are considered “killed,” indicating a positive reaction. The percentage of killed cells is reported.
• Advantages: Relatively simple, cost-effective, and has a long history of use, providing extensive clinical experience.
• Disadvantages: Can be less sensitive than newer methods, may produce false positives or negatives, and is time-consuming. It also primarily detects IgM antibodies, which are less implicated in chronic rejection.

2. Anti-Kappa and Anti-Lambda Light Chain Crossmatch

While the cytotoxic crossmatch detects overall antibody binding leading to cell death, specific antibody detection methods focus on the type of antibody bound. This often involves using reagents that bind to specific components of the antibody molecule.

• Principle: This method focuses on detecting antibodies bound to donor cells by using anti-human immunoglobulin reagents. Specifically, it targets the kappa (κ) and lambda (λ) light chains that are part of all human antibodies. This approach is particularly relevant when assessing the significance of antibodies detected byflow cytometry.
• Procedure (often performed as part of flow cytometry):
  1. Donor lymphocytes are incubated with recipient serum.
  2. After washing to remove unbound serum components, the lymphocytes are incubated with fluorescently labeled secondary antibodies that specifically bind to either kappa or lambda light chains on the bound donor-specific antibodies.
  3. The presence of fluorescence indicates that recipient antibodies have bound to the donor cells.
• Relevance: This technique is crucial in distinguishing between the presence of donor-specific antibodies and non-specific binding of other serum proteins. It helps to confirm the humoral (antibody-mediated) nature of any detected reactivity and is often incorporated into more sensitive flow cytometry crossmatches.

3. Flow Cytometry Crossmatch (FCXM)

Flow cytometry crossmatch is a more sensitive and sophisticated technique that has largely replaced the cytotoxic crossmatch in many transplant centers. It offers a more precise and quantifiable measure of antibody binding.

• Principle: This method uses fluorescently labeled antibodies to detect the binding of recipient antibodies to donor lymphocytes. Instead of relying on cell lysis, it quantifies the amount of antibody that binds to the cell surface by measuring the intensity of fluorescence.
• Procedure:
  1. Lymphocyte Isolation: Donor lymphocytes are isolated, often separated into T-cell and B-cell populations, as antibodies can target different cell types.
  2. Incubation: Donor lymphocytes are incubated with the recipient’s serum.
  3. Washing: Unbound antibodies are washed away.
  4. Incubation with Fluorescently Labeled Secondary Antibodies: The cells are then incubated with fluorescently labeled anti-human immunoglobulin antibodies. These secondary antibodies are designed to bind to the Fc portion of the bound recipient antibodies, or specifically to the kappa or lambda light chains of the bound antibodies (as described above).
  5. Flow Cytometry Analysis: The cells are passed through a flow cytometer, a machine that analyzes individual cells in a fluid stream. The laser beam excites the fluorescent labels attached to the bound antibodies, causing them to emit light at specific wavelengths. The intensity of this fluorescence, measured by detectors, indicates the degree of antibody binding.
• Advantages: Significantly more sensitive than the cytotoxic crossmatch, can distinguish between T-cell and B-cell reactivity, provides quantitative data (mean fluorescence intensity – MFI), can detect clinically significant antibodies that the cytotoxic method might miss, and can be adapted to detect specific antibody specificities.
• Disadvantages: Requires specialized equipment and trained personnel, can be more expensive, and the increased sensitivity can sometimes lead to a higher rate of “weakly positive” crossmatches that may require careful interpretation in the context of the patient’s antibody profile.

Panel Reactive Antibody (PRA): Quantifying the Risk of Sensitization

Beyond the immediate pre-transplant crossmatch, a broader assessment of a recipient’s immune system’s reactivity to a wide range of potential donor antigens is crucial. This is where the Panel Reactive Antibody (PRA) test comes into play.

• Definition: Panel Reactive Antibody (PRA) is a measure of the percentage of the population of potential donor lymphocytes to which the recipient has pre-formed antibodies. In essence, it quantifies how “sensitized” a recipient is.
• How it’s Measured: The PRA test involves incubating the recipient’s serum with a panel of lymphocytes from multiple different donors, each with a distinct set of HLA antigens. If the recipient’s antibodies react with the lymphocytes from a particular donor in the panel, it indicates that the recipient has antibodies against the HLA antigens present on that donor’s cells. The PRA score is calculated as the percentage of donors in the panel to which the recipient’s serum reacts.
• Relevance to Transplantation:
  • Waiting List Priority: A high PRA score significantly increases the difficulty of finding a compatible organ. Because the recipient has antibodies against a broad range of HLA antigens, the chance of encountering a donor whose organs will not be rejected by these antibodies is lower. This often translates to longer waiting times.
  • Allocation Adjustments: Transplant centers and organ allocation systems often provide priority adjustments for highly sensitized patients (those with a high PRA) to help them access compatible organs more readily.
  • Immunosuppression Strategy: A high PRA influences the intensity and type of immunosuppressive medications prescribed after transplant to prevent rejection.
  • Increased Risk of Rejection: Patients with a high PRA are at a greater risk of developing antibody-mediated rejection, even if they have a negative crossmatch.

High-Throughput Assays for Anti-HLA Antibodies: The Era of Luminex

The increasing understanding of the role of specific HLA antibodies, particularly non-HLA antibodies and low-level HLA antibodies, has led to the development of more sophisticated and efficient antibody detection methods.

• Definition: High-throughput assays for anti-HLA antibodies refer to laboratory techniques that can simultaneously screen a recipient’s serum for antibodies against a large number of different HLA antigens performed in a rapid and automated manner. These assays are designed to be highly efficient and capable of processing multiple samples and antibody specificities concurrently.
• The Luminex Platform: The most prominent example of a high-throughput assay system utilizes bead-based technology, such as the Luminex platform.
  • Principle: In this system, sets of microspheres (beads) are dyed with distinct fluorescent intensities. Each unique bead set is coated with specific purified HLA antigens. The recipient’s serum is incubated with this mixture of antigen-coated beads. If the recipient has antibodies against a particular HLA antigen, these antibodies will bind to the corresponding beads. After washing, fluorescently labeled secondary antibodies that bind to human antibodies are added. As the beads pass through a flow cytometer, the system detects both the bead’s fluorescent signature (identifying the HLA antigen) and the fluorescence from the bound antibodies (indicating the presence and strength of the antibody).
  • Capabilities: Luminex assays can simultaneously test for antibodies against hundreds of different HLA alleles (both Class I and Class II) in a single test. This provides a detailed “antibody profile” of the recipient.
• Significance:
  • Detailed Sensitization Assessment: Provides a much more granular understanding of a patient’s immune sensitization compared to traditional PRA methods.
  • Identification of Specific Antibody Targets: Allows for the identification of antibodies against specific HLA mismatches, which can be crucial for risk stratification and management.
  • Improved Donor Selection: Enables more precise matching between donor and recipient by identifying donors with HLA antigens that the recipient is unlikely to have antibodies against.
  • Monitoring and Desensitization: Facilitates the monitoring of antibody levels over time and guides desensitization protocols for highly sensitized patients.
  • Facilitating Research: Supports research into the role of specific HLA antibodies in transplant outcomes.

Conclusion: A Multi-faceted Approach to Transplant Success

The crossmatch, in its various forms, along with comprehensive antibody screening like PRA and advanced high-throughput assays, represents a cornerstone of modern organ transplantation. These tests are not merely diagnostic tools but are integral components of the organ allocation process, directly influencing the likelihood of transplant success and the long-term well-being of recipients. By meticulously assessing immune compatibility, transplant teams strive to minimize the risk of rejection, optimize graft survival, and ultimately, restore quality of life to those in need. The continuous evolution of these laboratory methodologies underscores the commitment to advancing the science and practice of organ transplantation.