IntroductionThe humoral response after SARS-CoV-2 vaccination and boosters in kidney transplant recipients (KTRs) is heterogeneous and depends on immunosuppression status. There is no validated immune measurement associated with serological response in clinical practice. Multicolor flow cytometric immunophenotyping could be useful for measuring immune response. This study aimed to study B- and T-cell compartments through Standardized EuroFlow PID Orientation after SARS-CoV-2 vaccination and their association with IgG SARS-CoV-2 seropositivity status after two doses or boosters.MethodsWe conducted a multicenter prospective study to evaluate humoral response after SARS-CoV-2 vaccination in KTRs. Heterologous regimen: two doses of inactivated SARS-CoV-2 and two boosters of BNT162b2 mRNA (n=75). Homologous vaccination: two doses of BNT162b2 mRNA and one BNT162b2 mRNA booster (n=13). Booster doses were administrated to KTRs without taking into account their IgG SARS-CoV-2 seropositivity status. Peripheral blood samples were collected 30 days after the second dose and after the last heterologous or homologous booster. A standardized EuroFlow PID Orientation Tube (PIDOT) and a supervised automated analysis were used for immune monitoring cellular subsets after boosters.ResultsA total of 88 KTRs were included and divided into three groups according to the time of the first detected IgG SARS-CoV-2 seropositivity: non-responders (NRs, n=23), booster responders (BRs, n=41), and two-dose responders (2DRs, n=24). The NR group was more frequent on mycophenolate than the responder groups (NRs, 96%; BRs, 80%; 2DRs, 42%; p=0.000). Switched memory B cells in the 2DR group were higher than those in the BR and NR groups (medians of 30, 17, and 10 cells/ul, respectively; p=0.017). Additionally, the absolute count of central memory/terminal memory CD8 T cells was higher in the 2DR group than in the BR and NR groups. (166, 98, and 93 cells/ul, respectively; p=0.041). The rest of the T-cell populations studied did not show a statistical difference.Conclusionswitched memory B cells and memory CD8 T-cell populations in peripheral blood were associated with the magnitude of the humoral response after SARS-CoV-2 vaccination. Boosters increased IgG anti-SARS-CoV-2 levels, CM/TM CD8 T cells, and switched MBCs in patients with seropositivity after two doses. Interestingly, no seropositivity after boosters was associated with the use of mycophenolate and a lower number of switched MBCs and CM/TM CD8 T cells in peripheral blood.

Introduction: In Uruguay with 3,286,314 inhabitants (2011 census), renal transplantation is performed in 3 transplant centers, is regulated by the Instituto Nacional de Donación y Trasplante (INDT) and financed by the Fondo Nacional de Recursos. The allocation is made on a national algorithm and it is an INDT´s responsibity. In the last 5 years, including the Health Emergency, an average of 139 kidney transplants were performed per year. The average rate for these 5 years of renal transplantation is 42.33 pmp. Objective: Describe the renal waiting list (WL) admission process and the recipient population. Methods: The study algorithm for admission to the WL include: □ABO and Rh blood group typing. □HLA typing by PCR-SSO (Luminex) for A, B, C, DRB1, DQA1 and DQB1 locus. □HLA antibody screening (Luminex), class I and/or II. If positive, a specificity search is performed. □Search for autoantibodies by microlymphocytotoxicity technique (MLCT). □Patient’s serum is preserved (serum collection). □PRA (wall reactive antibodies) by MLCT, defining hyperimmunized PRA>80%. During the period in WL the following procedures are performed: □serum collection update every 3 months or after sensitizing events. Failure to comply with this requirement excludes recipients from the allocation. □HLA antibody screening and specificity analysis are performed annually and PRA every six months. Results: There are currently 434 patients in the WL, including one vascular emergency. Distribution - by age: 1.8% <18 years old, 85% 18-64 years old and 13.2%>65 years old; by sex: 43.8% female, 56.2% male. Blood group distribution: 54% are 0.35% A, 8% B and 2% AB. The highest immunological risk group: 18%, who aspire to be retransplanted, 1.4% hyperimmunized. There are 26.5% with positive HLA antibody screening, see table and distribution in graphs. Conclusions: A single national protocol is applied at the inclusion on the WL. In the renal WL, the adult population (18 to 65 years old), blood group 0, with a male/female ratio of 1.3 prevails. The immune risk group does not present significant changes with respect to previous evaluations. The prevalence of sensitized to both classes and hyperimmunized is maintained.

Introduction: The Instituto Nacional de Donación y Trasplante (INDT) implemented two years ago the new kidney allocation model (new model -NM), previously evaluated with mathematical model that allowed to simulate scenarios and evaluate the impact on allocations. The NM maintains a mixed flow and scoring system as the Previous model (PM), but establishes changes in ABO compatibility and the weighing of: time wait list (TWL), HLA compatibility and donor (D) and recipient (R) age (Table 1). The aim of this work is to characterize and compare the groups of patients assigned under both models (NM and PM), in relation to: recipient age, TWL, ABO compatibility and HLA compatibility score. Methods: The population studied included the recipients assigned during 1/2018-02/2020 (PM) and during 03/2020-03/2022 (NM) which were obtained from the database of the INDT´s Assignment Unit (Table). We reviewed the following variables of 1st and 2nd recipients assigned: D and R ABO, R age, TWL, and HLA compatibility recorded as numerical value. Variables categorized were: age (0-18/19-60/over 61 years), ABO compatibility with donor (isogroup/compatible). Statistic analysis was applied. Results: Total allocation records analysed were N=405, 205 corresponding to PM and 200 to NM. With the new model, 25(12%) compatible ABO recipients were assigned, while in PM all allocations were ABO isogroup. NM allocated patients with higher WLT than PM (U13887, p<0.05- Mann Whitney U test). NM allocated patients with lower HLA compatibility score than PM (D=0.3145, p<0.05, Kolmogorov-Graph). Age range comparison did not show differencies (p > 0.05 Chi-square 11.8176). Conclusions: The comparative analysis of both models shows differences in HLA compatibility and TWL in recipients assigned. The decrease on HLA compatibility score is explained in the first two years of NM implementation by the allocation on patients with long TWL.

Introduction Predictive value of donor specific antibody (DSA) for the ocurrence of antibody mediated rejection (AMR) and graft loss in the individual patient is variable. In Uruguay, these techniques for detecting antibodies began to be implemented since 2016.The objective of this study was to assess the impact of presenting pre-formed antibodies in the first year of kidney transplantation outcome. Materials and Methods Retrospective multi-centric study, which included adult patients who received a kidney transplant with a cadaveric donor between 1/1/16 and 4/30/17, in the 3 transplant centers of Uruguay. All patients underwent study of HLA class I and II antibodies and specificity study for determination of DSA, performed by solid-phase technique, Luminex. At the transplant, they had a negative cross-test due to microliphocytotoxicity. Comparison off the incidence of acute rejection (AR) and survival of grafts and patients were calculated between the groups of patients with and without preformed antibodies. Results Of 125 patients analyzed, 73% did not present preformed antibodies, while 27% were sensitized with the presence of anti-HLA antibodies both class I and/or class II. Of this 27% of patients, 11% had anti-HLA antibodies pre-transplant positive, both class I and/or class II without DSA, and 16% had DSA. Patients were divided into 3 groups, according to absence of antibodies (G1), presence of antibodies but without DSA-positive (G 2) and presence of antibodies with DSA-positive (G3). G2 and G3 had a significantly higher percentage of females (p = 0.002), retransplants (p = 0.001), time on dialysis (p = 0.01), higher PRA (p = 0.001) as well as a higher number of transfusions (p = 0.02), versus G1 patients. The 3 groups did not have induction differences nor maintenance immunosuppression. There was a significant difference in perioperative desensitization, with infusion of gamma globulin (G3 75% versus G1 and G2 4.6% and 8%, p = 0.001). Month follow-up of the groups was of G1, G2 and G3, 11 ± 5.12 ± 5 and 9 ± 5.5 months of mean respectively. The incidence of AR in G2 and G3 was 46% and 45% versus 15% G1, (p = 0.001). Diagnosis of AR was performed at 19 ± 17.16 ± 26 and 18 ± 32 days post transplant for groups G1, G2 and G3 respectively, without differences. Patients survival was similar between groups, with 1 death in each group, all due to sepsis. Censored by death graft survival at one year was 95% G1, 100% G2 and 93% G3 (p = ns). Conclusion Patients transplanted with preformed antibodies, were most frequently females, retransplant and had more time on dialysis. Although this group mostly received desensitization treatment, at 3 months it presents a higher acute rejection rate, but with similar renal graft survival at 1 year.

Introduction: allogenic hematopoietic stem cell transplantation (HSCT) is the only currently available curative treatment for a number of high-risk hematologic malignancies and for a range of inherited and acquired non-malignant diseases. Haploidentical (HI) HSCT is a valid option for patients who lack an HLA-identical brother. Objective: to describe the results obtained with HI HSCT in pediatrics. Method: in 2005 an HI HSCT program was started at the Pediatric Hemato-Oncological Department of the Centro Hospitalario Pereira Rossell, for patients who lack an HLA-matched related donor. Results: Thirty two patients were transplanted, 24 of them with hematological malignancies and 8 with non-malignant diseases. Two strategies were used to prevent the Graft-Versus-Host-Disease (GVHD), in-vitro T-cell depletion (28 patients) and in-vivo depletion of aloreactives T-cells with high-dose post-transplantation cyclophosphamide (4 patients). Successful engraftment occurred in 27 patients (84%), with full-donor chimerism. Incidence of acute and chronic GVHD was 26.9% and 11.8% respectively. One year non-relapsed mortality was 21.9%. With a median follow-up of 32 months, the overall survival was 52.4%. Conclusions: HI HSCT has proved to be a feasible option in our country for those patients without an HLA-identical donor. The results obtained are comparable to those obtained with other alternative donors and costs are more reasonable. Today, Uruguay is in a better position to offer a HSCT to patients who need it.

Introducción: el trasplante alogénico de progenitores hematopoyéticos (TPH) es actualmente la única opción de tratamiento curativo disponible para un número de neoplasias hematológicas de alto riesgo, así como para algunas enfermedades no malignas hereditarias o adquiridas. El TPH haploidéntico (HI) es una opción válida para pacientes que no tienen un hermano HLA-idéntico. Objetivo: describir los resultados obtenidos con TPH HI en pediatría. Material y método: en el año 2005 se inició en el Centro Hemato-Oncológico Pediátrico del Centro Hospitalario Pereira Rossell un programa de TPH HI para aquellos pacientes sin donante relacionado HLA-idéntico. Resultados: se trasplantaron 32 pacientes, 24 con neoplasias hematológicas y 8 con enfermedades no malignas. Se utilizaron dos estrategias de prevención de la enfermedad injerto contra huésped (EICH), depleción de linfocitos T (DLT) in vitro (28 pacientes) y DLT alorreactivos in vivo con altas dosis de ciclofosfamida postrasplante (4 pacientes). Veintisiete pacientes (84%) tuvieron un implante con quimerismo total del donante. La incidencia de EICH agudo y crónico fue de 26,9% y 11,8%, respectivamente. La muerte no relacionada a recaída al año del trasplante fue de 21,9%. Con una mediana de seguimiento de 32 meses, la sobrevida global a dos años fue de 52,4%. Conclusiones: el TPH HI ha demostrado ser una opción factible en nuestro medio para aquellos pacientes sin donante HLA-idéntico. Los resultados son comparables a los obtenidos con otros donantes alternativos y con costos más accesibles. Uruguay está hoy día mejor posicionado para ofrecer un TPH a los pacientes que así lo requieran.

Introduction: tests of Human Leucocyte Antigen (HLA), antibodies for patients on the waiting list for renal transplant, are necessary to allocate organs and to define the appropriate immunomodulator treatment that result in the best tranplant survival rates.Methods: we looked for HLA antobodies in 488 patients on the national waiting list for renal transplant. We defined patients with reactivity > 20% (41 pacients) as immunized, and those with reactivity > 80% (6 patients) as highly immunized, by using the microlymphocitotoxicity technique against a lymphicyte test panel, detecting specificity by ELISA and flow cytometryResults and conclusions: we found HLA antibodies class I and class II by ELISA, and identified HLA specificities by flow cytometry: 41 (8.4%) of immunized patients showed HLA antibodies class I and 22 (4.5%) evidenced class II. The most frequently found specificities for these antibodies were : A24, A23, BW6, B44, CW6, CW2, DR8, DR7, DQ2, DQ7. We compared the distribution by age, sex, retransplantation, blood type, previous blood transfusions, time on the waiting list and diagnosis of the immunized population with those non-immunized. We found that in the immunized group most of them are candidates for a second transplant (X-2= 130,47), have received previous transfusions (X-2= 119,2) and have been on the waiting list longer(p<0,0001). NO differences were found in the distribution by age, sex or etiological diagnosis of chronic renal failure (CRF), being the non-determined nephropaty and glomerulopathies, the most frequent types. Distribution by blood type showed significant differences: in the immunized group there were more patients belonging to group 0 (X-2 = 7,9) and less of them belonging to group A (X-2 = 3,94).

Introducción: en el trasplante renal, la investigación en los pacientes en lista de espera de anticuerpos (Ac) contra Human Leucocyte Antigen (HLA) resulta necesaria para adjudicar el órgano y para adecuar el tratamiento inmunomodulador que promueva mayor sobrevida al trasplante. Material y método: realizamos búsqueda de anticuerpos HLA en 488 pacientes en lista de espera nacional de trasplante renal (2005). Definimos como inmunizados a aquellos con reactividad > 20% (41 pacientes), y altamente inmunizados con > 80% (6 pacientes), por técnica de microlinfocitotoxicidad enfrentados a un panel linfocitario, detectando especificidad por ELISA y citometría de flujo. Resultados y conclusiones: detectamos anticuerpos HLA clase I y II por ELISA, e identificamos especificidades HLA por citometría de flujo: 41 (8,4%) pacientes inmunizados presentaron anticuerpos HLA clase I y 22 (4,5%) asocian clase II. Las especificidades de estos anticuerpos más frecuentemente encontradas fueron: A24, A23, BW6, B44, CW6, CW2, DR8, DR7, DQ2, DQ7. Comparamos distribución por edad, sexo, retrasplante, grupo sanguíneo, transfusiones previas, tiempo en lista de espera y diagnóstico en población inmunizada respecto a no inmunizada. Constatamos que en el grupo de inmunizados predominan los candidatos a un segundo trasplante (X2=130,47), quienes han recibido transfusiones previas (X2=119,2) y aquellos con mayor tiempo en lista de espera (p<0,0001). No hay diferencias en la distribución por edad, sexo ni diagnóstico etiológico de insuficiencia renal crónica, predominando la nefropatía indeterminada y glomerulopatías. La distribución por grupo sanguíneo mostró diferencias significativas: en el grupo de inmunizados hay más pacientes del grupo 0 (X2 =7,9) y menos del grupo A (X2 =3,94).

Nitric oxide ((*)NO) has been implicated in multiple physiological and pathological immune processes. Different methods have been developed to detect and quantify (*)NO, where one of the principal difficulties are the accurately detection in cellular system with low levels of (*)NO production. The choice of the (*)NO detection method to be used depends on the characteristics of the experimental system and the levels of (*)NO production which depend on either the organism source of samples or the experimental conditions. Recently, high sensitive methods to detect and image (*)NO have been reported using 4,5-diaminofluorescein-based fluorescent probes (DAF) and its derivate 4,5-diaminofluorescein diacetate (DAF-2 DA). This work was aimed to adapt and optimize the use of DAF probes to detect and quantify the (*)NO production in systems of high, moderate and low out-put production, especially in human PBMC and their subpopulations. Here, we report an original experimental design which is useful to detect and estimate (*)NO fluxes in human PBMC and their subpopulations with high specificity and sensitivity.

Introduction: The IgVH mutational profile ia a strong prognostic indicator in CLL patients (pts.), particularly in early stages, where clinical staging fails to accurately predict outcomes. Our center designed a prospective trial in newly diagnosed CLL pts; our aim was to evaluate peripheral blood inmunophenotype including CD38 and the IgVH and correlate these parameters with clinical evolution and treatment requirements.