Donor-specific HLA antibodies (DSA) are related to antibody-mediated rejection (ABMR) and graft failure. The rationale and frequency of screening for anti-HLA antibodies in stable patients are not established. The aim of our study is to evaluate the impact of early DSA appearance in the first month post-transplant on graft outcomes. All kidney transplant recipients between 1/1/2012–12/31/2022 with anti-HLA antibody screening by Luminex during the first month post-transplant were included. Patients with preformed or historical DSA and those with DSA detection after graft loss were excluded. The mean fluorescence intensity cut-off was 1,500. Three hundred fifty-three patients were included and the median time from transplant to first antibody sample was 30.0 days. During 3.8 years of follow-up, graft loss occurred in 9.1% and 19.5% had ABMR. A total of 8.5% developed early-DSA in the first month. Patients with early-DSA detection had more HLA sensitization at the time of transplant (p < 0.001). Multivariable analysis showed that the presence of early-DSA was an independent risk factor for ABMR. In conclusion, sensitized patients at the time of transplant have a higher risk of DSA formation in the first month, probably reflecting alloimmune memory, therefore early HLA antibody screening should be performed in this high-risk population.

Highly sensitized patients awaiting kidney transplantation face substantial challenges due to the presence of potential donor-specific anti-HLA antibodies (DSA). These antibodies increase the risk of antibody-mediated rejection (ABMR), but also complicate their access to HLA compatible transplantation. Although advancements in allocation priority programs, such as the Spanish Program for the Access of Highly Sensitized Patients to Kidney Transplantation (PATHI), have introduced virtual crossmatching (v-XM) to streamline compatibility assessments, patients with >99,5 % virtual panel reactive antibodies (vPRA) often remain on waiting lists for extended periods with minimal chances of receiving a transplant. This article summarizes Spanish guidelines for a harmonized and comprehensive framework for the management of highly sensitized patients. These guidelines focus on strategies to facilitate transplantation in the presence of DSA, including a stepwise approach to delist HLA antigens, prioritizing those recognized as "less deleterious" antibodies, to expand transplant options while minimizing immunological risks. Conventional desensitization techniques are discussed, alongside the innovative use of imlifidase to enable transplants in particularly complex cases. Post-transplant monitoring protocols are also exposed, with a focus on early detection of antibody rebound and effective management of ABMR. Ultimately, this resource offers clinicians a structured framework to navigate the intricate challenges of kidney transplantation in high-risk populations, aiming to enhance access to life-saving procedures and improve patient outcomes.

Background. Antibody-mediated rejection (ABMR) has become one of the leading causes of chronic lung graft dysfunction. However, in lung transplantation, this entity is sometimes difficult and controversial to diagnose. It is mainly caused by the appearance of donor- specific anti-human leukocyte antigen (HLA) antibodies (DSA), although there are situations with Cod deposits in biopsy in the absence of circulating DSA. The aim of this work was to study the potential role of non-HLA antibodies in the development of ABMR without DSA after lung transplantation. Methods. A case-control study was designed with a cohort of lung transplant recipients at our institution. Twenty-seven patients with ABMR and without anti-HLA antibodies were identified after lung transplantation, and a control group of 21 transplant recipients was selected with the same post-transplant follow-up without evidence of rejection. Non-HLA antibodies were studied pretransplant using Luminex (ThermoFisher, One Lambda). Results. The median of the pretransplant non-HLA-positive antibodies in the group with ABMR without DSA is significantly higher than in the control group: 2 (interquartile range, 0-16) vs 0 (interquartile range, 0-1; P .01). Patients with 1.5 pretransplant non-HLA antibodies were more likely to develop ABMR without DSA (sensitivity, 80.95%; specificity, 55.55%; area under the curve, 71.3%). Conclusion. The increase of non-HLA antibodies before lung transplantation has recently been shown to increase the risk of chronic lung allograft dysfunction. These results confirm that patients with a higher number of non-HLA antibodies could be at risk of developing ABMR without DSA. These results point out the possible usefulness of pre-lung transplant non-HLA antibodies to identify patients with end-stage lung disease at risk of developing ABMR without DSA.

The human leukocyte antigen (HLA) system plays a critical role in transplant immunology, influencing outcomes through various immune-mediated rejection mechanisms. Hyperacute rejection is driven by preformed donor-specific antibodies (DSAs) targeting HLAs, leading to complement activation and graft loss within hours to days. Acute rejection typically occurs within six months post-transplantation, involving cellular and humoral responses, including the formation of de novo DSAs. Chronic rejection, a key factor in long-term graft failure, often involves class II DSAs and complex interactions between the innate and adaptive immune systems. Advancements in HLA antibody detection, particularly single antigen bead (SAB) assays, have improved the sensitivity and characterization of DSAs. However, these assays face challenges like false positives from denatured antigens and false negatives due to low antibody titers or complement competition. Furthermore, molecular mismatch (MM) analysis has emerged as a potential tool for refining donor–recipient compatibility but faces some issues such as a lack of standardization. Highly sensitized patients with calculated panel-reactive antibodies (cPRA) of 100% face barriers to transplantation. Strategies like serum dilution, novel therapies (e.g., Imlifidase), and delisting approaches could refine immunological risk assessment and delisting strategies are essential to expand transplant opportunities for these patients.

BACKGROUND/AIM:Despite the donor-exchange program implementation for highly sensitized (HS) patients, no improvement in waiting list in those HS patients with 100% calculated panel reactive of antibodies (cPRA) is observed. Recently, it has been published the treatment with imlifidase in desensitization algorithm. However, there are low-risk strategies to reduce cPRA. A cPRA of <99.95% increase donor offer chances, so delisting (DL) strategies should be addressed in cPRA reduction. We propose an integral approach for DL from low to intermediate risk to assess the 100% HS patients on waiting list. METHODS:The common DL criteria for previously forbidden alleles were that they should neither have been present in previous transplants nor possess complement fixation ability. Low-risk phase of DL is based on historical mean fluorescence intensity (MFI) of <5000 in the last 2 years. The next phase risk is based on single-antigen test in 1/10 diluted serum with MFI value of <3000; without eplet mismatch (low-intermediate risk specificity), or with eplet mismatch from previous transplants (intermediate risk). The molecular mismatch may be assessed with the mismatch calculator tool from registry website (https://www.epregistry.com.br/). CONCLUSIONS:Low-risk DL approaches are now widely used to reduce cPRA in HS patients; however, sometimes it is not enough to get transplanted and new tools are needed. Despite new treatments with imlifidase, some cases had anti-human leukocyte antigen rebound levels with a higher risk of rejection. Here, we propose a scaled DL approach would be a better therapeutic approach for HS patients whenever possible.

Recent concerns regarding the safety of Janus kinase inhibitors (JAKis) have prompted investigation into their impact on immune cell subsets in rheumatoid arthritis (RA) patients. This study aims to analyse alterations in immune cell populations induced by JAKis that may contribute to adverse events, such as infections or malignancies. This study included 78 RA patients meeting ACR/EULAR criteria with an established treatment with JAKis (tofacitinib, baricitinib, upadacitinib, or filgotinib), 20 healthy donors, and 20 RA patients treated with biological disease-modifying antirheumatic drugs (bDMARDs). Peripheral blood mononuclear cells (PBMCs) were immunophenotyped directly after isolation using multiparametric flow cytometry to characterise innate and adaptive immune-cell subsets. JAKi-treated patients showed a significant reduction in cytotoxic NK Dim (CD3-CD56+CD16+) cells and in the percentage of NK Dim cells expressing the activation marker Nkp30. In CD4+ T cells, the percentage of Th17 (CD3+CD4+CD45RA+CCR6+CXCR3-), Th1-17 (CD3+CD4+CD45RA+CCR6+CXCR3+), and central memory (CM, CD3+CD4+CD45RA+CD62L+) cells was lower in the JAKi group, while effector memory (EM, CD3+CD4+CD45RA-CD62L-) and terminally differentiated CD45RA (TEMRA, CD3+CD4+CD45RA+CD62L-) T helper cells were increased compared to healthy and bDMARD-treated controls. The reduction in NK Dim and Th1-17 cells and the increase in exhausted Th subsets suggest a potential compromise in antiviral immunity and balanced immune responses in JAKi-treated RA patients. These alterations may contribute to an increased risk of infections or malignancies.

To improve lung transplant recipient (LungTx) outcome, it would be of great interest to measure the net state of immunosuppression to avoid both infection and rejection. Measurement of Torquetenovirus load (TTV load) has been proposed as a biomarker to monitor solid organ transplantation, but its relationship with immunosuppressive drugs, particularly mycophenolic acid (MPA), is not well understood. We performed a prospective study of 53 LungTx, measuring TTV load before transplantation, at week 3, and at month 3. Tacrolimus and MPA doses and levels were recorded, and an area under the MPA curve (AUC-MPA) was calculated at the third month. LungTx in the fourth quartile of TTV load at the third week and the third month exhibited a low risk of acute rejection (OR 0.113, 95% CI 0.013-0.953, p = 0.045) and a high risk of opportunistic infection from month 3 to 6 (OR 15.200, 95% CI 1.525-151.511, p = 0.020), respectively. TTV load was weakly related to tacrolimus trough level at month 3 (rho = 0.283, p = 0.040). Neither MPA blood levels nor AUC-MPA were related to TTV load, although only patients with a reduction in MPA dose from month 1 to 3 showed a smaller increase in TTV load (0.86, IQR 2.58 log10 copies/mL vs. 2.26, IQR 3.02 log10 copies/mL, p = 0.026). In conclusion, TTV load in LungTx is only partially related to exposure to immunosuppressive drugs. Other variables, such as inflammation, immunosenescence, and frailty, may influence the overall level of immunosuppression and TTV load.

Background/Objectives: Some lung transplant (LungTx) recipients do not achieve the expected lung function within the first year, a condition known as baseline lung allograft dysfunction (BLAD). Our objective was to analyze the risk factors associated with BLAD, focusing on the variables associated with a higher risk of developing a more intense alloimmune response. Methods: We carried out a prospective study including 88 LungTx recipients. BLAD was defined as failure to reach 80% of the predicted value for forced expiratory volume in one second (FEV1) and/or forced vital capacity (FVC) on two tests conducted at least three weeks apart. Tacrolimus time in therapeutic range (TTR) and mycophenolic acid area under the curve (MPA AUC0–12h) were measured at the third month. Donor–recipient compatibility was assessed using HLA eplet mismatch analysis, performed via HLA Matchmaker 3.1. Results: BLAD patients showed greater eplet mismatch burden (67, IQR 20 vs. 55, IQR 22, p = 0.018) and had been exposed to a lower TTR (26.6%, IQR 14.0% vs. 39.6%, IQR 24.3%, p = 0.039) and less frequently to an adequate third-month MPA AUC0–12 > 30 mg × h/L (57.1% vs. 89.2%, p = 0.020). DR/DQ eplet mismatches (β = −0.348, p = 0.002) and third-month MPA AUC0–12 (β = 0.285, p = 0.009) were independently associated with six-month predicted FEV1%. Conclusions: Among other variables, BLAD and initial lung graft function are associated with greater eplet discordance and lower immunosuppressive drug exposure, suggesting a potential role of underlying alloimmune responses in their pathogenesis.

Chronic allograft dysfunction (CLAD) is the main cause of graft loss after lung transplantation (LTR). Within the immunological factors involved in CLAD development, the antibody-mediated rejection (ABMR) has the most impact. However, ABMR diagnosis is difficult due to the limited sensitivity of histopathological, immunhistochemical, and immunological criteria currently used. Growing evidence is demonstrating the impact of molecular mismatch in ABMR; here, we ought to assess the potential role of molecular mismatch in CLAD development. A total of 457 LTR were recruited for the study, with HLA type from donors and recipients to assess molecular mismatch, and with a minimum follow-up of 180 days. The combination of molecular mismatch in class-II (HLA-EMMA and HLA-Matchmaker algorithms) with EMMA DR score >12 and antibody verified eplet mismatch in DRB1345 (AbV DRB1345) > 3 predicts CLAD development independently of ex-smoker, prolonged period of hospitalization (>33 days), acute cellular rejection (ACR), and ABMR. The HR of the prediction model for molecular mismatch in class-II was 1.52 (1.01-2.56, p=0.045). This observation could point to a potential role of poor molecular mismatch in class-II to fill the gap of underdiagnosis of ABMR, previous to CLAD development. Prospective studies should be addressed to confirm the utility of molecular mismatch in the identification of patients at risk of CLAD development.

Background The Omicron variant of SARS-CoV-2 emerged as a new variant of concern, characterized by high transmissibility and lower severity compared with previous variants, and became the majority variant in the sixth wave in Spain. This study aims to assess the impact of SARS-CoV-2 infection on liver transplant recipients (LTRs) during 2023 in the population of Cantabria. Methods The study included 295 LTRs undergoing follow-up at the Liver Transplant Unit of the Marqués de Valdecilla University Hospital. Data on patient characteristics, comorbidities, and vaccination schedules were collected. Humoral response to mRNA vaccines was evaluated using IgG antibodies against the S protein and an adequate response was defined as antibody titers of >260 BAU/mL 1 month after the third vaccine dose. Results In Cantabria, 0.75% of the general population and 7.10% of LTRs were infected with SARS-CoV-2. Most LTRs were men with comorbidities, mainly cardiovascular disease and hypertension. Of the LTRs, 95.2% were fully vaccinated and received 3 doses of the Moderna mRNA vaccine, and all had an adequate response after 4 to 5 doses. Most infections in LTRs were asymptomatic or mild, with lower hospitalization rates. No LTRs required intensive care admission or died owing to SARS-CoV-2 infection. Conclusions LTRs are targets for SARS-CoV-2 vaccination. The Omicron variant has shown greater transmissibility but causes milder disease in vaccinated LTRs. All vaccinated LTRs showed an adequate response after 4 to 5 doses. Therefore, vaccination protects against severe disease and mortality in LTRs, and booster vaccinations with variant-adapted vaccines are recommended.

Donor exchange programs were designed to allocate organs for highly sensitized (HS) patients. The allocation algorithm differs slightly among countries and includes different strategies to improve access to transplants in HS patients. However, many HS patients with a calculated panel reactive of antibodies (cPRA) of 100 % remain on the waiting list for a long time. Some allocation algorithms assume immunological risk, including Imlifidase treatment, to increase the chance of transplantation in very HS patients. Here, we describe our unicenter experience of low-risk delisting strategy in 15 HS patients included in the Spanish donor exchange program without donor offers. After delisting, 7 out of 15 HS patients reduced the cPRA below 99.95 % and impacted the reduction of time on the waiting list (p = 0.01), where 5 out of 7 achieved transplantation. Within those HS that remained above 99.95 %, 1 out of 8 was transplanted. All the HS were transplanted with delisted DSA, and only one with DSA level rebounded early after transplantation. All HS transplanted after delisting maintain graft function. The transplant immunology laboratories are challenged to search intermediate risk assessment methods for delisting high HS patients.

Objectives: Flow cytometry analyses of lymphocyte subpopulations (T, B, NK) are crucial for enhancing clinical algorithms and research workflows. Estimating the total error (TE) values for the percentage and absolute number of lymphocyte subpopulations using the state-of-the-art (SOTA) approach with real data from an external proficiency testing (EPT) scheme was performed. A comparison with previously published Biological Variability (BV)-based specifications was carried out. Methods: A total of 44,998 results from 86 laboratories over 10 years were analysed and divided into two five-year periods (2012-2016) and (2017-2021). Data come from the IC-1 Lymphocytes scheme of the Spanish External Quality Assurance System (EQAS) GECLID Program. This quantitative scheme includes percentages and absolute numbers of CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), CD19(+), and CD3-CD56(+)CD16(+) NK cells. The percentage of TE was calculated as: |reported value - robust mean|*100/robust mean for each laboratory and parameter. The cut-off for TE is set at 80% best results of the laboratories. Results: A significant reduction in the SOTA-based TE for all lymphocyte subpopulations in 2017-2021 was observed compared to 2012-2016. The SOTA-based TE fulfils the minimum BV-based TE for percentages of lymphocyte subpopulations. The parameter with the best analytical performance calculated with SOTA (2017-2021 period)-based TE was the percentage of CD3(+) (TE=3.65 %). Conclusions: The values of SOTA-based specifications from external quality assurance program data are consistent and can be used to develop technical specifications. The technological improvement, quality commitment, standardization, and training, reduce TE. An update of TE every five years is therefore recommended. TE assessment in lymphocyte subsets is a helpful and reliable tool to improve laboratory performance and data-based decision-making trust.

OBJECTIVES:CD34+ hematopoietic stem cell (HSC) enumeration, crucial for HSC transplantation, is performed by flow cytometry to guide clinical decisions. Variability in enumeration arises from biological factors, assay components, and technology. External quality assurance schemes (EQAS) train participants to minimize inter-laboratory variations. The goal is to estimate total error (TE) values for CD34 cell enumeration using state-of-the-art (SOTA) methods with EQA data and to define quality specifications by comparing TE using different cutoffs. METHODS:A total of 3,994 results from 40 laboratories were collected over 11 years (2011-2022) as part of the IC-2 Stem Cells Scheme of the GECLID Program that includes absolute numbers of CD34 cells. The data were analyzed in two periods: 2011-2016 and 2017-2022. The TE value achieved by at least 60 %, 70 %, 80 %, and 90  % of laboratories was calculated across the two different periods and at various levels of CD34 cell counts: above 25, 25 to 15, and under 15 cells/μL. RESULTS:A decrease in the SOTA-based TE for CD34 cell enumeration was observed in the most recent period in 2017-2021 compared with 2012-2016. A significant increase of P75 TE values in the low CD34 range (<15 cells/μL) levels was found (p<0.001). CONCLUSIONS:Technical advancements contribute to the decrease TE over time. The TE of CD34 cell FC counts is measure-dependent, making it responsive to precision enhancement strategies. The TE measured by EQAS in this study may serve as a quality specification for implementing ISO 15189 standards in clinical laboratories for CD34 cell enumeration.

BACKGROUND:CD64 expression on neutrophils surface (CD64N) by flow cytometry has been validated as a rapid biomarker for bacterial infections in both peripheral blood and other biological fluids. Ascites is a common complication in cirrhotic patients that a variety of factors can cause, including bacterial infections. Manual counting of polymorphonuclear (PMN) cells in ascitic fluid and microbiologic culture are essential for its diagnosis. We aimed to validate the determination of CD64N by flow cytometry in ascitic fluid and assess its potential usefulness in the rapid identification of bacterial infections. MATERIALS AND METHODS:A prospective unicentre study was conducted. Flow cytometry was used to analyse the expression of CD64N in 77 ascitic fluid samples from the initial paracentesis of 60 cirrhotic patients in different admission episodes from November 2021 to December 2022. RESULTS:Seventeen samples were diagnosed with bacterial infection based on a positive microbiologic culture or by PMN count (>250 PMN/mm3 in ascitic fluid). The median of CD64N MFI was significantly increased in the bacterial infection group (3690.5 MFI [1635.23-6521.18] vs. 1105.9 MFI [737.3-2048.2], p < 0.001). The CD64 MFI ratio of granulocytes to lymphocytes was elevated in the bacterial infection group (13.06 [6.38-24.58] vs. 5.01 [3.38-7.36], p < 0.001). A CD64N ratio higher than 9.9 identified those patients with bacterial infection with 70.6 and 86.7% sensitivity and specificity, with an area under the curve (AUC) of 79.4%. CONCLUSION:The CD64N determined by flow cytometry on ascitic fluid could help quickly identify bacterial infections in ascites patients, allowing early antibiotic treatment.

Among myeloid regulatory cells (MRCs), some particular subsets termed myeloid-derived suppressor cells (MDSCs) have been described. They are suppressor myeloid cells characterized by their ability to regulate innate and adaptive immune responses and known to accumulate in the context of chronic diseases and cancer. The lack of specific markers makes their classification difficult and requires functional studies to distinguish them from other myeloid cells. In this sense, the in vitro analysis of the proliferation of T lymphocytes cultured with MDSCs provides information about the regulatory function of these cells. Here, we provide a detailed protocol to assess the ability of human Mo-MDSCs to suppress T cell proliferation in vitro after obtaining Mo-MDSCs and CD4+T cell from peripheral blood.

Background. Lung transplantation remains the treatment of choice for end-stage lung diseases, and recipient selection is currently based on clinical urgency, ABO compatibility, and donor size. The risk of allosensitization is classically based on HLA mismatch, but eplet mismatch load is increasingly seen to be important in long-term outcomes in solid organ transplantation. Chronic lung allograft dysfunction (CLAD) is relatively common and relevant, affecting almost 50% of patients 5 y after transplantation and being the first cause of death from the first year after transplantation. The overall class-II eplet mismatch load has been associated with CLAD development. Methods. Based on clinical data, 240 lung transplant recipients were eligible for CLAD, and HLA and eplet mismatch was analyzed using the HLAMatchmaker 3.1 software. Results. A total of 92 (38.3%) lung transplant recipients developed CLAD. The time free-of-CLAD was significantly decreased in patients with presence of DQA1 eplet mismatches (P = 0.015). Furthermore, when other previously described CLAD risk factors were studied in a multivariate analysis, the presence of DQA1 eplet mismatches was found to be independently associated with the early onset of CLAD. Conclusions. The concept of epitope load has arisen as a new tool to better define donor–recipient immunologic compatibility. The presence of DQA1 eplet mismatches potentially would increase the likelihood of developing CLAD.