Utilizing proficiency testing survey data to create advanced educational content: the virtual crossmatch challenge model.

Proficiency testing (PT) surveys include data from laboratories across the world and are ideal for creating advanced educational content, beyond just consensus grading. Educational challenges provide a unique opportunity to probe common laboratory practices and risk assessment, especially in cases where there is no "analyte" tested. Human leukocyte antigen (HLA) compatibility evaluation between donor and recipient pairs has been traditionally assessed using T-cell and B-cell physical crossmatches. However, advancements in our ability to identify and characterize HLA antibodies using solid phase assays, in combination with changing deceased donor allocation schemes and improved HLA typing, have shifted the paradigm from performing physical crossmatches to the use of the virtual crossmatch (VXM). VXM is a compatibility assessment relying on the interpretation of pre-transplant HLA laboratory data and as such, it is not an "analyte". However, VXM results are used in clinical decision-making. The VXM assessment depends on patient characteristics as well as laboratory and transplant center practices but must ensure safe transplantation outcomes while maintaining equity in access to transplantation. In this manuscript, we describe the American Society for Histocompatibility and Immunogenetics (ASHI) PT Educational VXM Challenge, as a model for creating educational content using PT survey data. We discuss the different components of the VXM Challenge and highlight major findings and learning points acquired from ASHI VXM Challenges performed between 2018-2022, such as the lack of correlation between the VXM and the physical crossmatch in the presence of low level donor-specific antibodies (DSA), or when the DSA were aimed against donor alleles that are not present on the antibody panel, and in the presence of an antibody to a shared eplet. Finally, we show that the VXM Educational Challenge serves as a valuable tool to highlight the strengths and pitfalls of the VXM assessment and reveals differences in testing and result interpretation among participating HLA laboratories.

Posture of Healthy Subjects Modulated by Transcutaneous Spinal Cord Stimulation.

Transcutaneous electrical stimulation of the spinal cord is used to restore locomotion and body weight support in patients with severe motor disorders. We studied the effects of this non-invasive stimulation on postural control in healthy subjects. Stimulation at the L1-L2 vertebrae was performed to activate the extensor muscles of the lower limbs. Because postural regulation depends on the cognitive style, the effects of the stimulation were analyzed separately in field-dependent (FD) and field-independent (FI) participants. During the study, FD and FI participants (N = 16, 25 ± 5 years, all right dominant leg) stood on a force platform in a soundproof chamber with their eyes closed. Stimulation was applied in the midline between the L1 and L2 vertebrae or over the left or right dorsal roots of the spinal cord; under the control condition, there was no stimulation. Stimulation destabilized posture in healthy subjects, whereas patients with movement disorders usually showed an improvement in postural control. In the FD participants, left dorsal root and midline stimulation increased several postural parameters by up to 30%. Dorsal root stimulation on the side of the supporting leg reduced postural control, while stimulation on the side of the dominant leg did not. No significant changes were observed in the FI participants.

COVID-19 infection and vaccination rarely impact HLA antibody profile in waitlisted renal transplant candidates- a multicenter cohort.

Although rare, infection and vaccination can result in antibodies to human leukocyte antigens (HLA). We analyzed the effect of SARS-CoV-2 infection or vaccination on HLA antibodies in waitlisted renal transplant candidates. Specificities were collected and adjudicated if the calculated panel reactive antibodies (cPRA) changed after exposure. Of 409 patients, 285 (69.7 %) had an initial cPRA of 0 %, and 56 (13.7 %) had an initial cPRA > 80 %. The cPRA changed in 26 patients (6.4 %), 16 (3.9 %) increased, and 10 (2.4 %) decreased. Based on cPRA adjudication, cPRA differences generally resulted from a small number of specificities with subtle fluctuations around the borderline of the participating centers' cutoff for unacceptable antigen listing. All five COVID recovered patients with an increased cPRA were female (p = 0.02). In summary, exposure to this virus or vaccine does not increase HLA antibody specificities and their MFI in approximately 99 % of cases and 97 % of sensitized patients. These results have implications for virtual crossmatching at the time of organ offer after SARS-CoV-2 infection or vaccination, and these events of unclear clinical significance should not influence vaccination programs.

Current donor selection strategies for allogeneic hematopoietic cell transplantation.

Since the first allogeneic hematopoietic stem cell transplantation (HCT) was performed by Dr. E. Donnall Thomas in 1957, the field has advanced with new stem cell sources, immune suppressive regimens, and transplant protocols. Stem cells may be collected from bone marrow, peripheral or cord blood from an identical twin, a sibling, or a related or unrelated donor, which can be human leukocyte antigen (HLA) matched, mismatched, or haploidentical. Although HLA matching is one of the most important criteria for successful allogeneic HCT (allo-HCT) to minimize graft vs host disease (GVHD), prevent relapse, and improve overall survival, the novel immunosuppressive protocols for GVHD prophylaxis offered improved outcomes in haploidentical HCT (haplo-HCT), expanding donor availability for the majority of HCT candidates. These immunosuppressive protocols are currently being tested with the HLA-matched and mismatched donors to improve HCT outcomes further. In addition, fine-tuning the DPB1 mismatching and discovering the B leader genotype and mismatching may offer further optimization of donor selection and transplant outcomes. While the decision about a donor type largely depends on the patient's characteristics, disease status, and the transplant protocols utilized by an individual transplant center, there are general approaches to donor selection dictated by donor-recipient histocompatibility and the urgency for HCT. This review highlights recent advances in understanding critical factors in donor selection strategies for allo-HCT. It uses clinical vignettes to demonstrate the importance of making timely decisions for HCT candidates.

Immunological considerations-HLA matching and management of high immunological risk recipients.

Lung transplant outcomes remain inferior largely due to mismatching in human leukocyte antigens (HLAs) that leads to chronic rejection and chronic allograft dysfunction. The mismatched donor HLAs can be recognized by the effector T cells or donor-specific HLA antibodies. This review summarizes mechanisms leading to immune responses as a result of HLA mismatching. It specifically focuses on sensitized lung transplant candidates with preformed anti-HLA antibodies, which represent a significant management challenge for physicians. In this review, we describe the diagnostic histocompatibility testing and therapeutic options for managing the sensitized lung transplant patients and discuss how multidisciplinary approach may help to improve lung transplantation outcomes.

Mismatch epitope load predicts de novo-DSA-free survival in pediatric liver transplantation.

BACKGROUND: Our knowledge of de novo anti-HLA donor-specific antibodies (dnDSA) in liver transplantation continues to be defined. We hypothesized that differences of HLA-DR/DQ mismatches can improve precision in alloimmune risk categorization and be applied to tailor immunosuppression. METHODS: A retrospective chart review of 244 pediatric patients consecutively transplanted at our center between 2003 and 2019 was performed to identify patients tested for dnDSA. Records were queried for: demographics, pre-transplant diagnosis, biopsy-proven T-cell-mediated rejection (TCMR), radiology proven biliary complications, tacrolimus trough levels, dnDSA characteristics, and HLA typing. The eplet mismatch analyses were performed using HLAMatchmaker™ 3.1. All statistical analyses were conducted using R software version 3.40. RESULTS: There were 99 dnDSA-negative patients and 73 dnDSA-positive patients (n = 70 against class II and n = 3 against class I and II). ROC analysis identified optimal cutoff of eplet mismatch load for dnDSA and defined risk groups for an alloimmune outcome. Kaplan-Meier curves and log-rank tests showed high eplet mismatch load was associated with shorter dnDSA-free survival (log-rank p = .001). Multivariable Cox regression models showed that tacrolimus coefficient of variation and tacrolimus mean levels were significantly associated with dnDSA-free survival (p < .001 and p = .036). Fisher's exact test showed that dnDSA was associated with an increased likelihood of TCMR (OR 14.94; 95% CI 3.65 - 61.19; p < .001). Patients without TCMR were more likely to have dnDSA to HLA-DQ7 and less likely to have dnDSA to HLA-DQ2 (p = .03, p = .080). CONCLUSIONS: Mismatched epitope load predicts dnDSA-free survival in pediatric liver transplant, while dnDSA specificity may determine alloimmune outcome.

Development of an industrial cyber-physical platform for small series production using digital twins.

The article describes an industrial cyber-physical platform for small series production using digital twins under development at ITMO University (Saint Petersburg, Russia). The platform is based on the following approaches: group technology, adaptive and selective assembling, and digital twin of production systems and processes. The article presents a mechanism for constructing a unified manufacturing process, and results of an integrated multiscale simulation of an injection moulding process. The issues of ensuring identification and monitoring of objects of the industrial cyber-physical platform are considered. Specific service applications required to implement the smart product concept are discussed. The combination of the considered technologies is used to create digital twins of production system objects. All humans that have different roles in the product value stream can interact with the industrial cyber-physical platform at the three levels, receiving support in performing their tasks. This article is part of the theme issue 'Towards symbiotic autonomous systems'.

Graft Versus Host Disease After Intestinal Transplantation: A Single-center Experience.

BACKGROUND: Graft versus host disease (GVHD) is an uncommon but highly morbid complication of intestinal transplantation (ITx). In this study, we reviewed our 17-y experience with GVHD focusing on factors predicting GVHD occurrence and survival. METHODS: Retrospective review of 271 patients who received 1 or more ITx since program inception in 2003 with survival analysis using Cox proportional hazard modeling. RESULTS: Of 271 patients, 28 developed GHVD 34 (18-66) d after ITx presenting with rash or rash with fever in 26, rectosigmoid disease in 1, and hemolysis in 1; other sites, mainly rectosigmoid colon, were involved in 13. Initial skin biopsy demonstrated classic findings in 6, compatible findings in 14, and no abnormalities in 2. Additional sites of GVHD later emerged in 14. Of the 28 patients, 16 died largely from sepsis, the only independent hazard for death (hazard ratio [HR], 37.4181; P = 0.0008). Significant (P < 0.0500) independent hazards for occurrence of GVHD in adults were pre-ITx functional intestinal failure (IF) (HR, 15.2448) and non-IF diagnosis (HR, 20.9952) and early post-ITx sirolimus therapy (HR, 0.0956); independent hazards in children were non-IF diagnosis (HR, 4.3990), retransplantation (HR, 4.6401), donor:recipient age ratio (HR, 7.3190), and graft colon omission (HR, 0.1886). Variant transplant operation was not an independent GVHD hazard. CONCLUSIONS: Initial diagnosis of GVHD after ITx remains largely clinical, supported but not often confirmed by skin biopsy. Although GVHD risk is mainly recipient-driven, changes in donor selection and immunosuppression practice may reduce incidence and improve survival.

Hypercalcemia in Small Lymphocytic Lymphoma with an Elevated Parathyroid Hormone-Related Peptide Associated with Early Richter Transformation.

Hypercalcemia in malignancy is associated with multiple mechanisms and occurs in up to 20-30% of cancer patients. We report a case of small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) associated with hypercalcemia and an elevation in parathyroid hormone-related peptide (PTHrP) in the setting of a Richter transformation. Real-time reverse transcriptase PCR on lymph node biopsy specimens obtained before and after transformation showed an 8-fold increase in PTHrP mRNA levels and about 2-fold decrease in the levels of its cognate receptor PTHR1. The findings of this case suggest that parathyroid hormone-related peptide might be useful in monitoring a specific group of patients with SLL/CLL who develop hypercalcemia during the course of their disease and could suggest an autocrine-like mechanism involving PTHrP in Richter transformation.

Guiding therapeutic plasma exchange for antibody-mediated rejection treatment in lung transplant recipients - a retrospective study.

Antibody-Mediated Rejection (AMR) due to donor-specific antibodies (DSA) is associated with poor outcomes after lung transplantation. Currently, there are no guidelines regarding the selection of treatment protocols. We studied how DSA characteristics including titers, C1q, and mean fluorescence intensity (MFI) values in undiluted and diluted sera may predict a response to therapeutic plasma exchange (TPE) and inform patient prognosis after treatment. Among 357 patients consecutively transplanted without detectable pre-existing DSAs between 01/01/16 and 12/31/18, 10 patients were treated with a standardized protocol of five TPE sessions with IVIG. Based on DSA characteristics after treatment, all patients were divided into three groups as responders, partial responders, and nonresponders. Kaplan-Meier Survival analyses showed a statistically significant difference in patient survival between those groups (P = 0.0104). Statistical analyses showed that MFI in pre-TPE 1:16 diluted sera was predictive of a response to standardized protocol (R2  = 0.9182) and patient survival (P = 0.0098). Patients predicted to be nonresponders who underwent treatment with a more aggressive protocol of eight TPE sessions with IVIG and bortezomib showed improvements in treatment response (P = 0.0074) and patient survival (P = 0.0253). Dilutions may guide clinicians as to which patients would be expected to respond to a standards protocol or require more aggressive treatment.

Operational tolerance in intestinal transplantation.

By presenting the first case report of true operational tolerance in an intestinal transplant patient, we aim to demonstrate that tolerance is possible in a field that has been hampered by suboptimal outcomes. Although operational tolerance has been achieved in liver and kidney transplantation, and some intestinal transplant patients have been able to decrease immunosuppression, this is the first instance of true operational tolerance after complete cessation of immunosuppression. A patient received a deceased-donor small intestinal and colon allograft with standard immunosuppressive treatment, achieving excellent graft function after overcoming a graft-versus-host-disease episode 5 months posttransplant. Four years later, against medical advice, the patient discontinued all immunosuppression. During follow-up visits 2 and 3 years after cessation of immunosuppression, the patient exhibited normal graft function with full enteral autonomy and without histological or endoscopic signs of rejection. Mechanistic analysis demonstrated immune competence against third party antigen, with in vitro evidence of donor-specific hyporesponsiveness in the absence of donor macrochimerism. This proof of principle case can stimulate future mechanistic studies on diagnostic and therapeutic strategies, for example, cellular therapy trials, that can lead to minimization or elimination of immunosuppression and, it is hoped, help revitalize the field of intestinal transplantation.

Serum dilutions as a predictive biomarker for peri-operative desensitization: An exploratory approach to transplanting sensitized heart candidates.

Antibody-mediated rejection (AMR) of cardiac allografts mediated by anti-HLA Donor Specific Antibodies (DSA) is one of the major barriers to successful transplantation for the treatment of end-stage heart failure. Therapeutic plasma exchange (TPE) is a first-line treatment for pre-transplant desensitization. However, indications for treatment regimens and treatment end-points have not been well established. In this study, we investigated how sera dilutions could guide TPE regimens for effective peri-operative desensitization and early AMR treatment. Our data show that 1:16 dilutions of EDTA-treated sera and 1.5 volume TPE reduce anti-HLA class I and class II antibody levels in the same manner and, therefore, allows to predict which antibodies would respond to peri-operative TPE. We successfully applied this approach to transplanting three highly sensitized cardiac recipients (CPRA 85-93%) with peri-operative desensitization based on a virtual crossmatch performed on 1:16 diluted serum. Furthermore, we have used sera dilutions to guide DSA treatment post-transplant. Although these findings have to be confirmed in a larger prospective study, our data suggest that serum dilutions can serve as a predictive biomarker to guide peri-operative desensitization and post-transplant immunologic management.

Ytterbium increases transmembrane water transport in Zea mays roots via aquaporin modulation.

In our study, the rare earth element ytterbium (Yb3+) was demonstrated to affect water exchange in roots of Zea mays seedlings. Herewith, the overall membrane permeability (Pd) increased. The Pd increase was determined by aquaporin activity but not the membrane lipid component since the closure of aquaporin channels due to low intracellular pH abolished the positive effect of Yb3+ on Pd. Additionally, the expression level of aquaporin genes ZmPIP2;2, ZmPIP2;6 and ZmTIP2;2 was increased when plants were grown in the presence of Yb3+. Our results indicate that previously described positive influence of rare earth metals on plant growth and productivity may be mediated (at least partially) by the modification of the plant hydraulic system.

Discovery of Metabolic Biomarkers Predicting Radiation Therapy Late Effects in Prostate Cancer Patients.

Patients presenting with prostate cancers undergo clinical staging evaluations to determine the extent of disease to guide therapeutic recommendations. Management options may include watchful waiting, surgery, or radiation therapy. Thus, initial risk stratification of prostate cancer patients is important for achieving optimal therapeutic results or cancer cure and preservation of quality of life. Predictive biomarkers for risks of complications or late effects of treatment are needed to inform clinical decisions for treatment selection. Here, we analyzed pre-treatment plasma metabolites in a cohort of prostate cancer patients (N = 99) treated with Stereotactic Body Radiation Therapy (SBRT) at Medstar-Georgetown University Hospital in a longitudinal, quality-of-life study to determine if individuals experiencing radiation toxicities can be identified by a molecular profile in plasma prior to treatment. We used a multiple reaction mass spectrometry-based molecular phenotyping of clinically annotated plasma samples in a retrospective outcome analysis to identify candidate biomarker panels correlating with adverse clinical outcomes following radiation therapy. We describe the discovery of candidate biomarkers, based on small molecule metabolite panels, showing high correlations (AUCs ≥ 95%) with radiation toxicities, suitable for validation studies in an expanded cohort of patients.

Donor-Specific HLA Antibodies as Biomarkers of Transplant Rejection.

This article reviews the current evidence to classify donor-specific antibodies (DSAs) using Food and Drug Administration-National Institutes of Health Biomarkers, EndpointS, and other Tools (BEST) resource terms as diagnostic, prognostic, predictive, monitoring, and risk biomarkers for graft rejection. The emphasis is on DSA characteristics, including the DSA levels determined by mean fluorescence intensity and/or titers, the ability to activate a complement cascade (C1q, C3d, and C4d binding), and specific IgG subclasses to define distinct roles of DSAs as biomarkers in clinical practice. In addition, technical limitation of DSA testing is discussed.

Assessing the utilization of high-resolution 2-field HLA typing in solid organ transplantation.

HLA typing in solid organ transplantation (SOT) is necessary for determining HLA-matching status between donor-recipient pairs and assessing patients' anti-HLA antibody profiles. Histocompatibility has traditionally been evaluated based on serologically defined HLA antigens. The evolution of HLA typing and antibody identification technologies, however, has revealed many limitations with using serologic equivalents for assessing compatibility in SOT. The significant improvements to HLA typing introduced by next-generation sequencing (NGS) require an assessment of the impact of this technology on SOT. We have assessed the role of high-resolution 2-field HLA typing (HR-2F) in SOT by retrospectively evaluating NGS-typed pre- and post-SOT cases. HR-2F typing was highly instructive or necessary in 41% (156/385) of the cases. Several pre- and posttransplant scenarios were identified as being better served by HR-2F typing. Five different categories are presented with specific case examples. The experience of another center (Temple University Hospital) is also included, whereby 21% of the cases required HR-2F typing by Sanger sequencing, as supported by other legacy methods, to properly address posttransplant anti-HLA antibody issues.

Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens.

Historically, it has been difficult to propagate cells in vitro that are derived directly from human tumors or healthy tissue. However, in vitro preclinical models are essential tools for both the study of basic cancer biology and the promotion of translational research, including drug discovery and drug target identification. This protocol describes conditional reprogramming (CR), which involves coculture of irradiated mouse fibroblast feeder cells with normal and tumor human epithelial cells in the presence of a Rho kinase inhibitor (Y-27632). CR cells can be used for various applications, including regenerative medicine, drug sensitivity testing, gene expression profiling and xenograft studies. The method requires a pathologist to differentiate healthy tissue from tumor tissue, and basic tissue culture skills. The protocol can be used with cells derived from both fresh and cryopreserved tissue samples. As approximately 1 million cells can be generated in 7 d, the technique is directly applicable to diagnostic and predictive medicine. Moreover, the epithelial cells can be propagated indefinitely in vitro, yet retain the capacity to become fully differentiated when placed into conditions that mimic their natural environment.

Conditionally reprogrammed normal and primary tumor prostate epithelial cells: a novel patient-derived cell model for studies of human prostate cancer.

Our previous study demonstrated that conditional reprogramming (CR) allows the establishment of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types including breast, lung, colon and prostate. Using CR, we have established matched normal and tumor cultures, GUMC-29 and GUMC-30 respectively, from a patient's prostatectomy specimen. These CR cells proliferate indefinitely in vitro and retain stable karyotypes. Most importantly, only tumor-derived CR cells (GUMC-30) produced tumors in xenografted SCID mice, demonstrating maintenance of the critical tumor phenotype. Characterization of cells with DNA fingerprinting demonstrated identical patterns in normal and tumor CR cells as well as in xenografted tumors. By flow cytometry, both normal and tumor CR cells expressed basal, luminal, and stem cell markers, with the majority of the normal and tumor CR cells expressing prostate basal cell markers, CD44 and Trop2, as well as luminal marker, CD13, suggesting a transit-amplifying phenotype. Consistent with this phenotype, real time RT-PCR analyses demonstrated that CR cells predominantly expressed high levels of basal cell markers (KRT5, KRT14 and p63), and low levels of luminal markers. When the CR tumor cells were injected into SCID mice, the expression of luminal markers (AR, NKX3.1) increased significantly, while basal cell markers dramatically decreased. These data suggest that CR cells maintain high levels of proliferation and low levels of differentiation in the presence of feeder cells and ROCK inhibitor, but undergo differentiation once injected into SCID mice. Genomic analyses, including SNP and INDEL, identified genes mutated in tumor cells, including components of apoptosis, cell attachment, and hypoxia pathways. The use of matched patient-derived cells provides a unique in vitro model for studies of early prostate cancer.

Pre-empting Antibody-Mediated Rejection: A Program of DSA Monitoring and Treatment Can Effectively Prevent Antibody Mediated Rejection.

Antibody-mediated rejection (AMR) remains a problem without a reliable treatment in the care of kidney transplant patients. We proposed and tested a program of screening for donor specific antibodies (DSA) to initiate treatment of patients before AMR was detected and to prevent its occurrence. Starting in April 2012, we stratified patients into high-, medium-, and low-risk groups for the development of DSA and instituted a program of screening for and treatment of these antibodies. We used a historic control group of patients transplanted at our center as a comparator and looked at rates of DSA testing and development as well as rates of development of AMR, cell-mediated rejection, and graft loss. 614 patients were transplanted under the protocol compared with 266 patients in the control group. Length of follow-up was similar in both groups. The group undergoing DSA screening had lower rates of DSA development (17.6% versus 24.8%, p=0.016) and that DSA was found at a significantly earlier time post-transplant (147 versus 248 days, p=0.02). Incidence of AMR was dramatically lower in the screened group (1.3% versus 8.6%, p<0.0001) with no grafts lost due to AMR. AMR was found to occur at an average of 181 days post-transplant. Rates of acute cellular rejection did not decrease in a manner similar to AMR rates. In conclusion, a program of universal risk-stratified DSA testing in kidney transplant patients can dramatically reduce rates of AMR and virtually eliminate graft loss due to AMR.