Transplantation Immunology and Laboratory Support

The histocompatibility barrier

HLA proteins are the dominant immunologic barrier to transplantation. Class I molecules, the products of HLA-A, HLA-B, and HLA-C, pair with β2-microglobulin, appear on essentially all nucleated cells, and are surveyed by CD8-positive cytotoxic T cells. Class II molecules, the products of HLA-DR, HLA-DQ, and HLA-DP, are restricted mainly to antigen-presenting cells such as dendritic cells, monocytes and macrophages, and B lymphocytes, and are surveyed by CD4-positive helper T cells. A molecule of either class must bind a short peptide to be stably expressed on the cell surface, so every HLA molecule presents a peptide derived from proteins inside or around its cell.^{11 Bloch EM, et al. Technical Manual. 2026.}

The genes of both classes are closely linked on the short arm of chromosome 6 and are inherited together as a haplotype, one from each parent. Mendelian segregation gives any two full siblings a 25% chance of sharing both haplotypes (HLA-identical), a 50% chance of sharing one (haploidentical), and a 25% chance of sharing neither. These inheritance probabilities are the basis of related-donor searches in hematopoietic transplantation.^{22 Jimenez Jimenez AM, et al. Allogeneic hematopoietic cell donor selection. 2025.}

The HLA region is the most polymorphic in the human genome. This variation helps populations respond to diverse pathogens and means that virtually any two unrelated individuals are HLA-mismatched. Molecular typing resolves tens of thousands of named alleles.^{3,43 Marsh SGE, et al. Nomenclature for factors of the HLA system, 2026. 2026.4 IPD-IMGT/HLA Database. Database statistics, release 3.65.}

Allele counts increase with each database release. In HLA-DRB1*13:01:01:02, the fields identify the allele group, protein sequence, synonymous coding variation, and noncoding variation. Expression suffixes indicate expression status. HLA-A*24:09N is a null allele whose product is not expressed. This level of resolution can affect a matching decision.^{3,43 Marsh SGE, et al. Nomenclature for factors of the HLA system, 2026. 2026.4 IPD-IMGT/HLA Database. Database statistics, release 3.65.}

ABO is the conventional blood group system with the greatest direct impact on solid-organ transplantation, because A and B antigens are expressed on vascular endothelium. Preformed anti-A or anti-B in the recipient can bind a newly reperfused incompatible graft, activate complement, and produce hyperacute rejection within minutes to hours. For ordinary biological ABO compatibility, a group A recipient may receive an A or O organ, a group B recipient may receive B or O, a group O recipient receives O, and a group AB recipient may receive any ABO group. Organ-specific allocation rules and non-A₁ subtype provisions can narrow or broaden those pairings, and any intended ABO-incompatible pathway follows the transplant program's protocol.^{55 United Network for Organ Sharing. Organ Transplant.}

Graft types and allorecognition

Most clinical transplants are allografts, and HLA disparity accounts for much of their rejection risk. Recipient T cells encounter foreign HLA through two routes.^{66 Sureda A, et al. The EBMT Handbook. 2024.}

Direct allorecognition. Recipient T cells bind an intact allogeneic HLA-peptide complex displayed on donor antigen-presenting cells carried within the graft. An unusually large fraction of recipient T cells can respond, in part because cross-reactive memory T cells join the alloreactive pool. The mixed lymphocyte reaction is the historical in-vitro correlate of this pathway. Routine donor selection is now based on molecular HLA typing and antibody-based compatibility testing.^{1,61 Bloch EM, et al. Technical Manual. 2026.6 Sureda A, et al. The EBMT Handbook. 2024.}

Indirect allorecognition. Recipient antigen-presenting cells take up shed donor HLA protein, process it, and present donor-derived fragments on the recipient's own HLA. This pathway supports alloantibody production and chronic rejection.^{66 Sureda A, et al. The EBMT Handbook. 2024.}

Either route can lead to direct CD8-mediated cytotoxicity, delayed-type hypersensitivity, or antibody-mediated injury through complement fixation and antibody-dependent cellular cytotoxicity. The dominant mechanism helps determine the timing and pathology of rejection.^{66 Sureda A, et al. The EBMT Handbook. 2024.}

Rejection patterns and their prevention

Histocompatibility testing

Transplantation requires HLA typing, HLA antibody screening and identification, crossmatching, and post-transplant monitoring, alongside ABO compatibility and infectious disease evaluation of the donor. Allogeneic hematopoietic transplantation uses high-resolution molecular HLA typing for donor selection. Detailed typing, antibody-detection, and crossmatch methods are covered in serologic and molecular testing practice.^{22 Jimenez Jimenez AM, et al. Allogeneic hematopoietic cell donor selection. 2025.}

Calculated panel-reactive antibody (cPRA) expresses a candidate's sensitization for organ allocation. The transplant program designates clinically relevant antibody specificities as unacceptable antigens after laboratory and clinical interpretation. The cPRA estimates the proportion of the donor population expected to express at least one unacceptable antigen from donor-population genotype and haplotype frequencies. A higher cPRA means a smaller expected compatible donor pool. The laboratory reports donor-specific antibody, crossmatch results, assay limits, and the expected compatible-donor pool. The transplant program decides whether to proceed or use a different clinical pathway.^{77 Organ Procurement and Transplantation… OPTN policies.}

Post-transplant monitoring differs by transplant type. Solid-organ recipients are followed for donor-specific antibodies (DSAs), commonly with multiplex single-antigen bead arrays. A DSA can be clinically silent or accompany antibody-mediated graft injury, so the result is interpreted with graft function, histology, and other clinical evidence.^{11 Bloch EM, et al. Technical Manual. 2026.} Hematopoietic recipients are followed for chimerism by short tandem repeat (STR) analysis. PCR amplification of informative pretransplant donor and recipient STR loci and the post-transplant specimen, followed by capillary electrophoresis, estimates donor and recipient fractions. The desired donor fraction depends on disease, cell lineage, conditioning, and transplant strategy. A serial decline in donor fraction can indicate graft instability or relapse, so report each result with the previous results.^{66 Sureda A, et al. The EBMT Handbook. 2024.}

Hematopoietic progenitor cell transplantation

Hematopoietic progenitor cell (HPC) transplantation provides hematopoietic recovery after cytoreductive therapy, replaces defective or destroyed hematopoiesis, and can supply a donor immune graft-versus-leukemia (GVL) effect against residual malignant cells.^{88 Granot N, et al. History of hematopoietic cell transplantation. 2020.}

Graft sources and donor selection

• Autologous transplantation returns the patient's collected HPCs after high-dose therapy, an established approach in multiple myeloma and selected lymphomas. Conventional donor-versus-recipient GVHD and donor-derived GVL are absent, and occult malignant cells can return with a contaminated graft.^{88 Granot N, et al. History of hematopoietic cell transplantation. 2020.}
• Allogeneic transplantation supplies donor hematopoiesis and may provide GVL activity, as needed in selected marrow-failure syndromes, inherited hematopoietic diseases, and leukemias. It introduces graft-failure and GVHD risks. When a suitable family donor is unavailable, unrelated-donor and cord-blood registries broaden the search. Matched unrelated donors undergo high-resolution DNA-based HLA typing.^{2,82 Jimenez Jimenez AM, et al. Allogeneic hematopoietic cell donor selection. 2025.8 Granot N, et al. History of hematopoietic cell transplantation. 2020.}
• Syngeneic transplantation from an identical twin is rare. Expected HLA and minor-histocompatibility disparities are minimal, so classic alloreactive GVHD and GVL are markedly reduced. An HLA-identical sibling who is not a twin can still differ at minor histocompatibility antigens and produce GVHD or GVL activity.^{88 Granot N, et al. History of hematopoietic cell transplantation. 2020.}
• Umbilical cord blood can supply a pediatric recipient whose smaller body requires a lower total cell dose. Its low T-cell dose and predominantly naive lymphocytes permit more HLA mismatch and reduce chronic GVHD compared with many adult-derived grafts, although acute GVHD remains possible.^{2,62 Jimenez Jimenez AM, et al. Allogeneic hematopoietic cell donor selection. 2025.6 Sureda A, et al. The EBMT Handbook. 2024.}

Donor eligibility under FDA HCT/P rules

Federal rules for human cells, tissues, and cellular and tissue-based products (HCT/Ps) establish communicable-disease eligibility for allogeneic HPC donors. Appropriate FDA-licensed, approved, or cleared donor-screening assays cover HIV-1 and HIV-2 antibodies plus HIV-1 nucleic acid testing (NAT); HBsAg, total anti-HBc, and HBV NAT; anti-HCV and HCV NAT; and syphilis. Viable leukocyte-rich HCT/Ps add anti-HTLV-I/II and CMV antibody testing, and living donors receive West Nile virus NAT. Autologous HCT/Ps are exempt from the federal donor-eligibility determination. Applicable labels identify autologous use and reactive or unevaluated infectious-disease results. Any additional testing and storage controls follow the program's own procedures.^{9,109 Donor-eligibility testing and exceptions for…, et al. 21 CFR §§1271.80, 1271.85, 1271.90 (2025). 2025.10 US Food and Drug Administration. Testing HCT/P donors for relevant communicable disease agents… 2025.}

Collection methods

• Bone marrow (HPC, Marrow) is collected by multiple aspirations from the posterior iliac crests into a sterile anticoagulated container using aseptic technique.^{1111 Foundation for the Accreditation of Cellular… FACT-JACIE International Standards for Hematopoietic Cellular… 2026.}
• Apheresis (HPC, Apheresis) collects circulating progenitors after the transplant service selects a mobilization regimen. The cellular therapy laboratory monitors the peripheral-blood CD34-positive count, times collection under the program protocol, and measures each product's viable CD34-positive yield. The laboratory uses the selected regimen to interpret specimen timing and expected yield.^{6,116 Sureda A, et al. The EBMT Handbook. 2024.11 Foundation for the Accreditation of Cellular… FACT-JACIE International Standards for Hematopoietic Cellular… 2026.}
• Cord blood (HPC, Cord Blood) is collected aseptically by gravity through an umbilical vein into a validated sterile anticoagulant system after cord clamping. Collection may occur before or after placental delivery, with maternal and infant care taking priority.^{1111 Foundation for the Accreditation of Cellular… FACT-JACIE International Standards for Hematopoietic Cellular… 2026.}

CD34 dose and the collection target

CD34 is a transmembrane protein expressed by hematopoietic progenitors and several nonhematopoietic progenitor or endothelial populations. A validated viable CD34-positive count is the operational surrogate for progenitor dose. Targets vary by indication, graft source, recipient, and manipulation. For adult peripheral-blood collections, the 2024 EBMT handbook gives generally accepted minimums of 2 × 10⁶ CD34-positive cells/kg for one autologous transplant and 4 × 10⁶/kg for one allogeneic transplant, with optimal ranges of 2.5 to 5 × 10⁶/kg and 5 to 8 × 10⁶/kg, respectively. A program may require a higher collection target when processing loss is expected. CD34-positive cells express HLA. A and B antigen expression on these cells is limited or heterogeneous and changes with erythroid differentiation. Donor matching is therefore based on HLA, and product processing and transfusion support manage ABO incompatibility.^{6,116 Sureda A, et al. The EBMT Handbook. 2024.11 Foundation for the Accreditation of Cellular… FACT-JACIE International Standards for Hematopoietic Cellular… 2026.}

For an 82-kg allogeneic recipient with a protocol goal of 4 × 10⁶ CD34-positive cells/kg, the required total is 82 × 4 × 10⁶ = 3.28 × 10⁸ CD34-positive cells. A first apheresis containing 1.64 × 10⁸ viable CD34-positive cells supplies half of that planned total. The remaining collection plan accounts for measured yield, viability, and expected processing loss.

Laboratory processing

On receipt, the cellular therapy laboratory verifies labeling and container integrity and inspects the product for abnormal appearance or evidence of mishandling or contamination. The laboratory uses aseptic technique, qualified supplies, and a validated environment whenever the system is opened. Product testing includes total nucleated-cell count and viability. HPC products intended to restore hematopoiesis also require a viable CD34-positive count. Additional CBC, differential, platelet, and product assays follow the product specification. After processing, the laboratory performs validated microbial testing, notifies the physician of any positive result, assesses the risk, and documents how the result is managed. Bone marrow is filtered to remove fat, bone particles, and debris. Red-cell reduction, plasma reduction, mononuclear-cell enrichment, and resuspension use validated product-specific procedures that preserve recovery, viability, and sterility.^{6,116 Sureda A, et al. The EBMT Handbook. 2024.11 Foundation for the Accreditation of Cellular… FACT-JACIE International Standards for Hematopoietic Cellular… 2026.}

Cryopreservation and thawing

Cryopreservation procedures may reduce red cells, plasma, and total volume to limit incompatible material, hemolysis, infusion volume, and cryoprotectant exposure. Validated HPC protocols commonly add dimethyl sulfoxide (DMSO) gradually to a final concentration of 5% to 10%, then use controlled-rate freezing and cryogenic nitrogen storage. The product is thawed rapidly by its validated method, commonly near 37 °C, and infused promptly. Washing or dilution can reduce DMSO and hemolysate exposure when clinically indicated, with potential cell loss considered in the decision.^{6,116 Sureda A, et al. The EBMT Handbook. 2024.11 Foundation for the Accreditation of Cellular… FACT-JACIE International Standards for Hematopoietic Cellular… 2026.}

Graft manipulation

CD34-positive selection may use validated immunomagnetic enrichment or flow-sorting platforms. Tumor-cell contamination of an autologous product is possible, and ex-vivo selection or purging remains disease- and protocol-specific. Donor T-cell depletion or CD34-positive selection can reduce GVHD. It can also delay immune recovery and increase infection, graft-failure, or relapse risk in some settings. The outcome depends on the depleted cell subsets, graft source, conditioning, and post-transplant prophylaxis.^{6,116 Sureda A, et al. The EBMT Handbook. 2024.11 Foundation for the Accreditation of Cellular… FACT-JACIE International Standards for Hematopoietic Cellular… 2026.}

Cord blood testing

The bank uses validated procedures to characterize a cord blood unit for identity and HLA type, ABO/Rh, total nucleated-cell and viable CD34-positive content, viability, hemoglobinopathy status, maternal donor eligibility, and product sterility. Infectious-disease testing follows the FDA requirements for viable leukocyte-rich HCT/Ps. Release and confirmatory testing also verify that the selected unit and recipient records agree.^{10,1110 US Food and Drug Administration. Testing HCT/P donors for relevant communicable disease agents… 2025.11 Foundation for the Accreditation of Cellular… FACT-JACIE International Standards for Hematopoietic Cellular… 2026.}

ABO incompatibility in HPC transplantation

In allogeneic HPC donor selection, HLA compatibility takes priority over ABO compatibility. ABO-incompatible grafts are therefore common, and laboratory support distinguishes major, minor, and bidirectional mismatch.^{1212 Matteocci A, et al. Immuno-hematologic complexity of ABO-incompatible allogeneic… 2024.}

Engraftment, graft rejection, and GVHD

HPC infusion reactions can resemble transfusion reactions. Conditioning-related liver injury, sinusoidal obstruction syndrome, chemotherapy or radiation lung injury, infection, and drug toxicity can mimic GVHD. Autologous grafts lack conventional donor-versus-recipient alloreactivity, although rare autologous GVHD-like syndromes have been reported.^{66 Sureda A, et al. The EBMT Handbook. 2024.}

Engraftment is reported using standard thresholds. Neutrophil recovery is the first of 3 successive days with an absolute neutrophil count of at least 500/µL after the post-transplant nadir. Platelet recovery is the first of 3 consecutive days with a platelet count of at least 20,000/µL after 7 consecutive days without platelet transfusion. Red cell recovery, immune reconstitution, and donor chimerism are separate outcomes. Graft source, viable CD34-positive dose, processing, conditioning, donor and recipient factors, and post-transplant care all influence timing.^{1313 Sureda A, et al. Harmonizing definitions for hematopoietic recovery, graft… 2024.}

Graft rejection is immune-mediated failure of donor-cell establishment. Graft failure is the broader clinical category and includes immune and nonimmune causes. Reduced-intensity or nonmyeloablative conditioning can leave more host T- and NK-cell activity and increase rejection risk in some settings. HLA disparity, donor-specific anti-HLA antibody, low cell dose, graft manipulation, and a female-donor-to-male-recipient pairing can also contribute, with their effect depending on the transplant platform.^{6,136 Sureda A, et al. The EBMT Handbook. 2024.13 Sureda A, et al. Harmonizing definitions for hematopoietic recovery, graft… 2024.}

Graft-versus-host disease. Donor alloreactive T lymphocytes mediate GVHD. Conditioning injury, antigen-presenting cells, and cytokines also contribute to tissue damage. Cord blood produces less chronic GVHD than many adult-derived grafts despite greater HLA disparity, although acute GVHD remains a substantial risk. NIH criteria classify GVHD by clinical phenotype. Classic acute GVHD occurs within 100 days after hematopoietic cell transplantation (HCT) or donor lymphocyte infusion (DLI). Persistent, recurrent, or late acute GVHD has acute features beyond day 100 after HCT or DLI without chronic features. Classic chronic GVHD has chronic diagnostic features, and overlap chronic GVHD has both acute and chronic features.^{14,1514 Jagasia MH, et al. National Institutes of Health Consensus Development Project on… 2014.15 Lee SJ, et al. NIH Chronic Graft-Versus-Host Disease Consensus Conference 2025… 2025.}

The clinical transplant service interprets GVHD findings and selects treatment. Graft T-cell depletion can lower GVHD risk. It can also delay immune recovery and raise graft-failure or relapse risk in some settings, so the cellular therapy laboratory documents the manipulation and final cell content. A donor lymphocyte infusion is a lymphocyte-rich product, usually from the original donor, that may be collected fresh or supplied from an eligible stored or engineered source under protocol. The clinical service determines its use. The collection and processing laboratory verifies identity, eligibility, cell content, and release requirements.^{15,1615 Lee SJ, et al. NIH Chronic Graft-Versus-Host Disease Consensus Conference 2025… 2025.16 Center for International Blood and Marrow… Cellular Therapy Forms Instruction Manual. 2026.}

Transfusion support for transplant recipients

Transfusion support during HPC transplantation commonly uses prestorage-leukocyte-reduced and irradiated cellular components. CMV-safe selection depends on recipient serostatus, graft type, component, and program policy.^{66 Sureda A, et al. The EBMT Handbook. 2024.}

HLA alloimmunization. Leukoreduction reduces HLA alloimmunization and platelet refractoriness but does not eliminate either. In the Trial to Reduce Alloimmunization to Platelets, conducted during induction therapy for acute myeloid leukemia, lymphocytotoxic antibodies developed in 45% of controls, 18% of recipients of filtered pooled platelets, 21% of recipients of UV-B-treated pooled platelets, and 17% of recipients of filtered apheresis platelets.^{1717 Trial to Reduce Alloimmunization to… Leukocyte reduction and ultraviolet B irradiation of platelets… 1997.} Anti-HLA class I antibody is a major immune cause of poor posttransfusion platelet increments. Donor-specific anti-HLA antibody, especially high-level or complement-binding antibody, is associated with poor engraftment or graft failure in several HCT settings and is interpreted by antibody level, locus, assay, and transplant platform.^{18,1918 Kongtim P, et al. ASTCT consensus recommendations on testing and treatment of… 2024.19 Cohn CS. Platelet transfusion refractoriness. 2020.}

CMV transmission. A CMV-seronegative HCT recipient is at risk of primary transfusion-transmitted CMV. For a seropositive recipient, reactivation during immunosuppression is the main concern. CMV-seronegative or leukocyte-reduced cellular components are accepted CMV-safe strategies for seronegative recipients, with pathogen-reduced platelets accepted in relevant settings. Duration varies with recipient serostatus, graft type, immune reconstitution, GVHD, and program policy.^{2020 Hakki M, et al. American Society for Transplantation and Cellular Therapy series. 2021.}

Transfusion-associated GVHD (TA-GVHD). Viable donor T lymphocytes in a transfused cellular component can proliferate in a susceptible recipient and attack marrow, skin, liver, and gastrointestinal tissue. TA-GVHD is rare, and the current Circular of Information describes its fatality rate as nearly 100%. Fever, generalized maculopapular rash, diarrhea, liver injury, and marrow aplasia or pancytopenia usually begin 2 to 30 days after transfusion. Irradiation prevents donor T-cell proliferation. The Circular of Information specifies 2,500 cGy targeted to the center of the container and a minimum of 1,500 cGy delivered to every part of the component. The transplant program defines which components require irradiation and the duration of support. Check each component against the recipient's irradiation and CMV requirements before issue, because TA-GVHD is nearly always fatal once it develops.^{2121 AABB. Circular of Information for the Use of Human Blood and Blood… 2024.}

Practice