Mitigating the Prozone-Like Effect in HLA Antibody Testing: A Case Report of Therapeutic Plasma Exchange for Antibody-Mediated Rejection Post-Heart Transplantation.

Therapeutic plasma exchange (TPE) is a cornerstone treatment for antibody-mediated rejection (AMR) post-organ transplantation, aiming to eliminate pathogenic donor-specific HLA antibodies (DSA). However, limitations in HLA antibody interpretation due to the prozone-like effect (PLE) can lead to inaccurate assessment of treatment efficacy. We present a case of a heart transplant recipient with suspected AMR, where an unexpected increase in DSA levels post-TPE prompted investigation into PLE. Solid-phase Luminex assays were employed to detect HLA antibodies. Serum was run neat as well as after treatment with ethylenediaminetetraacetic acid (EDTA). Nephelometry was used to detect complement levels. Laboratory analysis of pre-TPE serum revealed higher DSA levels with EDTA treatment, characteristic of PLE. Complement measurements supported complement-mediated interference in the pre-TPE sample. This case underscores the importance of being aware that PLE can occur in HLA testing and can impact the interpretation of TPE efficacy for AMR.

HLA Genotype Imputation Results in Largely Accurate Epitope Mismatch Risk Categorization Across Racial Groups.

Background: Biomarkers that predict posttransplant alloimmunity could lead to improved long-term graft survival. Evaluation of the number of mismatched epitopes between donor and recipient HLA proteins, termed molecular mismatch analysis, has emerged as an approach to classify transplant recipients as having high, intermediate, or low risk of graft rejection. When high-resolution genotypes are unavailable, molecular mismatch analysis requires algorithmic assignment, or imputation, of a high-resolution genotyping. Although imputation introduces inaccuracies in molecular mismatch analyses, it is unclear whether these inaccuracies would impact the clinical risk assessment for graft rejection. Methods: Using renal transplant patients and donors from our center, we constructed cohorts of surrogate donor-recipient pairs with high-resolution and low-resolution HLA genotyping that were racially concordant or discordant. We systemically assessed the impact of imputation on molecular mismatch analysis for cohorts of 180-200 donor-recipient pairs for each of 4 major racial groups. We also evaluated the effect of imputation for a racially diverse validation cohort of 35 real-world renal transplant pairs. Results: In the surrogate donor-recipient cohorts, imputation preserved the molecular mismatch risk category for 90.5%-99.6% of racially concordant donor-recipient pairs and 92.5%-100% of racially discordant pairs. In the validation cohort, which comprised 72% racially discordant pairs, we found that imputation preserved the molecular mismatch risk category for 97.1% of pairs. Conclusions: Overall, these data demonstrate that imputation preserves the molecular mismatch risk assessment in the vast majority of cases and provides evidence supporting imputation in the performance of molecular mismatch analysis for clinical assessment.

Advances in Human Leukocyte Antigen Testing Technologies and Management Strategies for Platelet Transfusion Refractoriness.

CONTEXT.­: The blood bank is often consulted for transfusion support of patients with suspected platelet transfusion refractoriness (PTR). The workup is complex because testing includes specialized assays that are uncommonly ordered with limited availability. Add to this the variety of possible products-crossmatched platelets, human leukocyte antigen (HLA)-matched platelets, HLA antigen-negative platelets-and the approach to PTR can be overwhelming. Moreover, most literature on the subject is published in transfusion medicine journals aimed at transfusion medicine physicians and blood bank specialists in academic settings. Resources tailored to community hospital blood banks are lacking. OBJECTIVE.­: To provide pathologists who may not have subspecialized training in transfusion medicine and who direct blood banks algorithmic workflows based on clinical scenario and test availability to provide appropriate transfusion support for patients with PTR. DATA SOURCES.­: This review is a comprehensive overview of terminology, HLA testing procedures, interpretations, and practical recommendations for managing PTR in various scenarios based on expert opinion as well as relevant medical literature published from 2007 to 2022. CONCLUSIONS.­: Consultation on PTR is complicated and encompasses many clinical and laboratory aspects. The lack of guidelines derived from high-quality prospective studies poses challenges in the workup and management of PTR. Hindering the process further are limited test availability, unfamiliarity with the technical assays, and the various specialized platelet products. The clinical evaluation algorithm presented herein along with the workflow pathways offer pathologists user-friendly and best-practice guidelines with different options based on the clinical scenario and the tests available.

Optimizing autologous stem cell collections for patients with multiple myeloma receiving G-CSF and Plerixafor: A single center project.

BACKGROUND: Increasing indications for cellular therapy collections have stressed our healthcare system, with autologous collections having a longer than desired wait time until apheresis collection. This quality improvement initiative was undertaken to accommodate more patients within existing resources. STUDY DESIGN AND METHODS: Patients with multiple myeloma who underwent autologous peripheral blood stem cell collection from October 2022 to April 2023 were included. Demographic, mobilization, laboratory, and apheresis data were retrospectively collected from the medical record. RESULTS: This cohort included 120 patients (49.2% male), with a median age of 60 years. All received G-CSF and 95% received pre-emptive Plerixafor approximately 18 hours pre-collection. Most (79%) had collection goals of at least 8 × 106/kg CD34 cells, with 63% over 70 years old having this high collection goal (despite 20 years of institutional data showing <1% over 70 years old have a second transplant). With collection efficiencies of 55.9%, 44% of patients achieved their collection goal in a single day apheresis collection. A platelet count <150 × 103/µL on the day of collection was a predictor for poor mobilization; among 27 patients with a low baseline platelet count, 17 did not achieve the collection goal and 2 failed to collect a transplantable dose. CONCLUSIONS: With minor collection goal adjustments, 15% of all collection appointments could have been avoided over this 6-month period. Other strategies to accommodate more patients include mobilization modifications (Plerixafor timing or substituting a longer acting drug), utilizing platelet counts to predict mobilization, and modifying apheresis collection volumes or schedule templates.

Three patients highlighting potential pitfalls in platelet refractory testing.

BACKGROUND: Platelet-transfusion refractory (PR) patients do not achieve expected post-transfusion platelet counts. We investigate suspected PR patients with post-transfusion platelet counts, indirect platelet antibody screens (ind-PAS), Class I HLA antibody tests (HLA-Scr), and physical platelet crossmatch (PXM) studies. STUDY DESIGN AND METHODS: The three following cases describe possible pitfalls of laboratory tests used in PR workup and management. RESULTS: Case #1: Antibody testing detected antibodies to only HLA-B13, corresponding to a 4% calculated panel reactive antibodies (CPRA; 96% predicted donor compatibility). However, PXM showed the patient compatible with 11/14 (79%) donors; two of the PXM-incompatible units were ABO-incompatible. Case #2: PXM revealed compatibility with 1/14 screened donors; however, the patient did not respond to the product from the compatible donor. The patient did respond to HLA-matched product. Dilution studies provided evidence of the prozone effect, which caused negative PXM despite clinically relevant antibodies. Case #3: There was a discrepancy between the ind-PAS and HLA-Scr. Ind-PAS was negative for HLA antibodies, while HLA-Scr was positive and specificity testing corresponded to 38% CPRA. Per the package insert, the sensitivity of ind-PAS is ~85% compared to HLA-Scr. DISCUSSION: These cases highlight the importance of investigating incongruent results. Cases #1 and #2 demonstrate PXM pitfalls: ABO incompatibility can result in positive PXM and false-negative PXM can occur in the setting of the prozone effect. Case #3 reveals the importance of knowing a test's sensitivity. Centers that only perform ind-PAS may fail to detect HLA antibodies.

Seventy-five years of service: an overview of the College of American Pathologists' proficiency testing program in histocompatibility and identity testing.

The Histocompatibility and Identity Testing Committee offers an overview of the College of American Pathologists' (CAP) Proficiency Testing (PT) program, commemorating its significant 75th anniversary in 2024. The CAP PT program has undergone significant growth and evolution over the years, ultimately achieving Centers for Medicare and Medicaid Services approval. In 1979, CAP's partnership with the American Association for Clinical Histocompatibility Testing marked a pivotal moment, leading to the creation of the first proficiency testing survey in 1980. This laid the foundation for various PT programs managed by the CAP Histocompatibility and Identity Testing Committee, including HLA antibody testing, HLA molecular typing, engraftment monitoring, parentage/relationship testing, HLA disease associations and drug risk, and HLA-B27 typing. Each program's distinctive considerations, grading methodologies, and future prospects are detailed here, highlighting the continual evolution of histocompatibility and identity testing PT to support emerging technologies and evolving laboratory practices in the field.

A case of Tn polyagglutination discovered by an ABO blood group discrepancy.

BACKGROUND: Tn syndrome is an acquired form of polyagglutination arising from somatic mutations of hematopoietic stem cells. Tn red blood cells (RBCs) are agglutinable by naturally occurring anti-Tn antibodies in most adult sera. Current ABO typing reagents are monoclonal and do not detect polyagglutination on forward typing. However, herein we describe a case of Tn activation that was suspected due to cross-reactivity with a monoclonal anti-A reagent. STUDY DESIGN AND METHODS: A 63-year-old man with myeloproliferative neoplasm, who historically typed as group O, demonstrated unexpected mixed field reactivity with anti-A reagent using a gel-based method. However, manual tube testing was consistent with the patient's historical group O type. RESULTS: Lectin testing demonstrated reactivity with Salvia sclarea and Glycine soja, but not Arachis hypogea. The patient's RBCs produced positive crossmatches with healthy donor sera, but reactivity was eliminated by ficin pretreatment of the RBCs. Ficin treatment also resolved typing discrepancies on gel-based typing. No reactivity was noted using cord blood sera, and N antigen expression was diminished upon phenotyping. Tn activation was confirmed by detection of a novel 4-nucleotide deletion (c.395-398del) in exon 3 of C1GALT1C1 resulting in a truncated glycosyltransferase. CONCLUSION: This case of acquired Tn polyagglutination due to a novel mutation was first suspected from an ABO phenotyping discrepancy. It highlights the cross-reactivity of anti-A reagent with Tn antigen when tested on a common gel-based method. Furthermore, the case demonstrates that review of patient history and test information can clarify discrepancies and guide resolution.

Implementing virtual crossmatch based diagnostic management teams in human leukocyte antigen laboratories and transplant programs.

Histocompatibility testing has continuously evolved since its inception. One such advancement is the implementation of the virtual crossmatch (VXM). Recent changes to allograft allocation schemes have resulted in increased organ sharing over greater distances, resulting in expanded utilization of the VXM to assess donor: recipient compatibility. In fact, the VXM has become a major arbitrator of pre-transplant compatibility assessment prior to both deceased and living donor organ allocation. This shift in pre-transplant practice is concurrent with the US healthcare systems' move towards more inclusive and coordinated team-based management approach to disease diagnosis. Diagnostic Management Teams (DMTs) exemplify this shift in patient care. Our institution seized the opportunity to build and implement a VXM DMT to improve and streamline pre-transplant assessment. This VXM DMT is compliant with US regulatory standards and provides a consultative report containing relevant pre-transplant information, test interpretation as well as recommendations for HLA additional (if any) testing. Herein we describe the development of and experience with the VXM DMT a year after its launch.

Convalescent Plasma: Therapeutic Hope or Hopeless Strategy in the SARS-CoV-2 Pandemic.

As the world faces the current SARS-CoV-2 pandemic, extensive efforts have been applied to identify effective therapeutic agents. Convalescent plasma collected from recovered patients has been a therapeutic modality employed for over a hundred years for various infectious pathogens. Specifically, it has been used in the treatment of many viral infections with varying degrees of clinical efficacy. As we consider the use of convalescent plasma in the battle against this new strain of coronavirus, it is prudent to review what is known from past experiences. Accordingly, the aim of this review is to examine in detail studies of convalescent plasma used during previous viral outbreaks and pandemics with particular focus on hemorrhagic fevers, influenza, and other coronaviruses. The concluding sections of this review address the potential use of convalescent plasma during the present-day SARS-CoV-2 pandemic, not only insofar as its clinical benefit but also the steps required to make convalescent plasma treatments readily available for an exponentially growing patient population. By the end, the authors hope to address the extent to which convalescent plasma represents a realistic therapeutic approach, or a distraction from other potentially useful treatments.

CD8+ T cells mediate antibody-independent platelet clearance in mice.

Platelet transfusion provides an important therapeutic intervention in the treatment and prevention of bleeding. However, some patients rapidly clear transfused platelets, preventing the desired therapeutic outcome. Although platelet clearance can occur through a variety of mechanisms, immune-mediated platelet removal often plays a significant role. Numerous studies demonstrate that anti-platelet alloantibodies can induce significant platelet clearance following transfusion. In fact, for nearly 50 years, anti-platelet alloantibodies were considered to be the sole mediator of immune-mediated platelet clearance in platelet-refractory individuals. Although nonimmune mechanisms of platelet clearance can often explain platelet removal in the absence of anti-platelet alloantibodies, many patients experience platelet clearance following transfusion in the absence of a clear mechanism. These results suggest that other processes of antibody-independent platelet clearance may occur. Our studies demonstrate that CD8(+)T cells possess the unique ability to induce platelet clearance in the complete absence of anti-platelet alloantibodies. These results suggest a previously unrecognized form of immune-mediated platelet clearance with significant implications in the appropriate management of platelet-refractory individuals.