Analysis of Cross-sectional and Longitudinal HLA and Anti-viral Responses After COVID Infection in Renal Allograft Recipients: Differences and Correlates.

BACKGROUND: Characterization of anti-HLA versus anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) immune globulin isotypes in organ transplant recipients after coronavirus disease 2019 (COVID-19) infection has not been reported. We aimed to determine changes in anti-HLA antibodies in renal transplant patients with COVID-19 and compare the immunoglobulin and epitope-binding pattern versus anti-SARS-CoV-2 antibodies. METHODS: This is a cross-sectional study of 46 kidney transplant recipients including 21 with longitudinal sampling. Using a semi-quantitative multiplex assay, we determined immunoglobulin (Ig) M, IgA, IgG, and IgG1-2-3-4 antibodies against Class I and Class II HLA, and 5 SARS-CoV-2 epitopes including the nucleocapsid protein and multiple regions of the spike protein. RESULTS: Fourteen of 46 (30%) patients had donor-specific anti-HLA antibodies (donor-specific antibody [DSA]), 12 (26%) had non-DSA anti-HLA antibodies and 45 (98%) had anti-SARS-CoV-2 antibodies. Most DSAs targeted HLA-DQ (71%), with a dominant IgG isotype and IgG1 subtype prevalence (93%), and/or IgG3 (64%), followed by IgG2 (36%). Comparatively, there was a higher prevalence of IgA (85% versus 14%, P = 0.0001) and IgM (87%, versus 36%, P = 0.001) in the anti-SARS-CoV-2 antibody profile, when compared to DSAs, respectively. Anti-SARS-CoV-2 antibody profile was characterized by increased prevalence of IgM and IgA, when compared to DSAs. The median calculated panel reactive antibody before COVID-19 diagnosis (24%) tended to decrease after COVID-19 diagnosis (10%) but it was not statistically significant ( P = 0.1). CONCLUSIONS: Anti-HLA antibody strength and calculated panel reactive antibody in kidney transplant recipients after COVID-19 do not significantly increase after infection. Although the IgG isotype was the dominant form in both HLA and SARS-CoV-2 antigens, the alloimmune response had a low IgA pattern, whereas anti-SARS-CoV-2 antibodies were high IgA/IgM.

Aberrant T-cell antigen expression in B lymphoblastic leukaemia.

B lymphoblastic leukaemia (B-ALL) cells are characterized by the expression of various B-cell antigens. Expression of T/Natural Killer-cell antigens, however, has rarely been reported in B-ALL (TAg+ B-ALL), and the significance of this aberrant antigen expression is unclear. We thus analysed the frequency of TAg+ B-ALL at our institution and investigated its significance in the context of immunophenotypes, cytogenetic/molecular findings, and prognosis. We reviewed 134 consecutive cases of B-ALL and found 18 cases (13·4%) of TAg+ B-ALL. The most common aberrant T-cell antigens expressed were CD2, CD5, and CD7 at equivalent rates (each in six cases), CD4 (two cases), and CD56 (three cases). Adverse cytogenetic abnormalities were seen in a significantly larger proportion of the TAg+ cases (72·2%) than the TAg- cases (32·2%; P = 0·003). Multivariate Cox-regression analysis showed that the risk of relapse over time was higher in the TAg+ cases, independent of high risk status (based on age and white blood cell count) and the presence of adverse cytogenetic abnormalities (hazard ratio = 2·256, P = 0·065). These findings suggest that T-cell antigen expression in B-ALL may be an independent predictor of poor prognosis, and a useful marker to identify patients at increased risk for relapse and for harbouring adverse cytogenetic abnormalities.

KHSV(-) EBV(-) post-transplant effusion lymphoma with plasmablastic features: variant of primary effusion lymphoma?

Primary effusion lymphoma (PEL) is a rare type of B-cell non-Hodgkin lymphoma (NHL), which predominantly occurs in HIV-infected individuals, and is pathogenetically linked with Kaposi sarcoma (KS)-associated herpes virus/human herpes virus-8 (KSHV/HHV-8) infection with or without evidence of Epstein-Barr virus (EBV) co-infection. Although uncommon, PELs have been reported in immunocompetent patients and recipients of solid organ allografts. Rare cases of KSHV(-) EBV(+) post-transplant effusion lymphomas resembling PEL have also been described, as have KSHV(-) EBV(-) effusion lymphomas, the latter including those arising in individuals with chronic liver disease. We report a unique KSHV(-) EBV(-) post-transplant effusion lymphoma associated with serum paraproteins, occurring in an HIV(-) individual, which had cytologic features and phenotype similar to PEL, and displayed a complex karyotype including isochromosome 12p and translocation t(8;22), resulting in rearrangement of c-MYC.

G6b-B cell surface inhibitory receptor expression is highly restricted to CD4+ T-cells and induced by interleukin-4-activated STAT6 pathway.

The G6b-B gene encodes a novel cell surface receptor of the immunoglobulin superfamily that activates inhibitory signaling pathways by triggering SHP-1/SHP-2 via immunoreceptor tyrosine-based inhibitory motifs (ITIM) in its cytoplasmic domain. We previously identified decreased G6b-B expression in peripheral blood mononuclear cells (PBMC) during acute cellular cardiac allograft rejection. We studied the expression of G6b-B in different human mononuclear cell populations and its regulation. Real-time polymerase chain reaction (PCR) revealed that G6b-B mRNA is higher in CD4+ T cells or monocytes, but is not different between CD25+ CD4+ T cells and CD25- CD4+ T cells. G6b-B mRNA was increased in CD4+ T cells in presence of interleukin-4 in dose- and time-dependent manners. To understand the regulatory mechanism, we analyzed a 1.9-kb 5'-flanking region of the G6b-B translation start site and found a putative cis-acting element for Signal Transducer and Activator of Transcription (STAT)-6. Luciferase-reporter-gene-assay and electrophoretic mobility shift assays identified the STAT6 site as necessary for the induction of G6b-B by IL-4. Our study demonstrates that G6b-B expression is highly restricted to peripheral CD4+ T cells and up-regulated by the IL-4-induced STAT6 pathway, strongly suggesting that G6b-B is involved in regulation of the immune response by CD4+ T cell-mediated and IL-4 induced regulatory mechanisms.

Expression of inhibitory receptor ILT3 on neoplastic B cells is associated with lymphoid tissue involvement in chronic lymphocytic leukemia.

T cell responses against leukemia-associated antigens have been reported in chronic lymphocytic leukemia (CLL). However, the relentless accumulation of CLL B cells in some patients indicates that anti-tumor immune responses are inefficient. Inhibitory receptors from the Ig-like transcript (ILT) family, such as ILT3 and ILT4, are crucial to the tolerogenic activity of antigen presenting cells. In this study, we examined the expression of ILT3 on CD5+ B cells obtained from 47 patients with CLL. Using flow cytometry and RT-PCR, we found that B CLL cells from 23 of 47 patients expressed ILT3 protein and mature ILT3 mRNA. ILT3 protein and mRNA were not found in normal B cells obtained from donors without CLL. Expression of ILT4 in normal and B CLL cells showed a pattern similar to ILT3. The frequency of ILT3 positive CLL B cells was higher in patients with lymphoid tissue involvement, suggesting that ILT3 may have prognostic value in CLL. Our findings indicate that expression of ILT3 and ILT4 on CLL B cells represents a phenotypic abnormality that may play a role in tolerization of tumor-specific T cells.

Increased access to transplantation of highly sensitized patients under the new kidney allocation system. A single center experience.

We aimed to investigate the impact of the new kidney allocation system (KAS) on the rate of transplantation of sensitized patients at our center. Pre-KAS and post-KAS intervals were Jan 1st to Dec 3rd 2014 and Jan 1st 2015 to Dec 3rd 2015, respectively. The number of deceased-donor crossmatches performed by flow cytometry increased from 715 pre-KAS to 1188 post-KAS. The percent of crossmatches performed for sensitized patients with calculated panel reactive antibody (cPRA)>0% increased from 19% pre-KAS to 26% post-KAS (p<0.0001). The number of deceased-donor kidney transplants performed at our center increased from 115 pre-KAS to 125 post-KAS (9% increase). There was a significant increase in the percentage of deceased-donor kidney transplants received by sensitized candidates (from 14% to 26% pre- and post-KAS, respectively; p<0.0001). The highest increase was seen in the patients with cPRA>98%, from 0% to 9%, followed by the group with cPRA 50-79%, from 5% to 8%. This increase was balanced by a decrease of 12% in the percentage of non-sensitized recipients, and a modest decrease of 1% in the group with cPRA 1-49%. In conclusion, transplant rate has increased in sensitized patients after KAS. The highest increase was observed among highly sensitized patients (cPRA>98%).

Alloantibodies and the outcome of cadaver kidney allografts.

The role of humoral immunity in causing antibody-mediated rejection (AMR) of organ allografts has been extensively documented. For this reason, negative complement-dependent cytotoxicity (CDC) cross-matches between recipient sera and donor T and B lymphocytes have become a mandatory requirement for cadaveric kidney transplantation. However, the significance of donor-specific antibodies (DSAs) detectable only by flow cytometry (FC) or solid phase assays (SPA) but not CDC is still controversial. We have performed a retrospective analysis of FC cross-matching results in 80 consecutive cadaver kidney allograft recipients. Antibodies against HLA class I and class II antigens were measured by CDC and SPA in sequential samples of sera obtained prior to transplantation. The preoperative cross-match was performed by CDC using magnetically sorted T and B cells from donor spleen. Sera obtained from each patient before and at the time of transplantation were included in the final cross-match. The sample of serum obtained at the time of transplantation was cross-matched retrospectively by FC and analyzed for anti-HLA antibody specificity on high resolution SPA. The actuarial kidney allograft survival at one year was 98%. Two of these eighty patients lost the graft, one due to AMR, the other for reasons unrelated to DSAs. Donor-specific antibodies were detected by FC in 17 of 80 patients, yet only 6 of 17 had an early episode of AMR. This episode was successfully reversed by desensitization therapy using intravenous immunoglobin (IVIG) and plasmapheresis. Flow cytomery cross-matching showed 95% specificity but only 35% sensitivity for prediction of AMR (p = 0.002). There was a significant correlation between high panel reactive antibodies (PRA) and positive FC cross-matching (p = 0 .0001), as well as high PRA and AMR (p = 0.0004 by CDC and 0.0011 by Luminex). Reversible AMR occurred 12-30 days post-transplantation in 8 patients. Of these 8 patients, 3 had no detectable DSAs in spite of C4d positivity, 4 had C4d deposition in conjunction with anti-HLA antibodies, and 1 patient had DSAs (anti-MICA) yet no C4d deposition. We conclude that early initiation of desensitization protocols can prevent transplant failure and that retrospective FC cross-matches may facilitate the diagnosis of AMR. Extensive analysis of patients' sera using a comprehensive set of tests may contribute to early treatment and better understanding of the mechanism underlying humoral rejection.

Acute and hyperacute humoral rejection in kidney allograft recipients treated with anti-human thymocyte antibodies.

Equine and rabbit antihuman thymocyte globulins (ATGs) have been used in renal transplantation for prevention and treatment of acute rejection. We now report that hyperacute and acute antibody-mediated rejection of renal allografts occurred in three newly transplanted patients who received ATG for induction therapy. Antibody studies performed using complement-dependent cytotoxicity, flow cytometry, enzyme-linked immunosorbent assay, and Luminex yielded negative results for antilymphocytic and antiendothelial cell antibodies in the pretransplant sera obtained from these patients. ATG treatment was initiated at the time of transplantation. One of the patients experienced hyperacute rejection and required transplant nephrectomy within 24 h of transplantation. The other two patients developed acute antibody-mediated rejection within 14 days after transplantation. None of the patients developed antihuman leukocyte antigen antibodies when humoral rejection occurred. However, xenoantibodies that strongly bound to human lymphocytes and, importantly, to activated endothelial cells, were identified in the sera obtained at the time of humoral rejection. Hence, our results strongly implicate ATG in the induction of antibody-mediated rejection of kidney allografts. Flow cytometry testing of ATG reactivity to endothelial cells may be useful in identifying and discarding the ATG lots containing xenoantibodies that can bind to activated endothelial cells of the transplant.

Regulatory CD8+CD28- T cells in heart transplant recipients.

Human regulatory CD8+CD28- T cells (Ts) generated in vitro were demonstrated to suppress the activation and proliferation of T helper cells (Th) induced by allogeneic cells. This effect requires cell-to-cell contact, is antigen-specific, and results in Th anergy. To study the population of CD8+CD28- T cells present in vivo, flow cytometry was performed on whole blood specimens obtained from 25 heart transplant recipients and 12 normal controls. A significant expansion of CD8+CD28- T cells was found in transplant recipients as compared with normal individuals (p = 0.005). Expression of CD38, human leukocyte antigen-DR, and perforin positive cells within the CD8+CD28- subset was significantly higher in transplant patients than in normal controls, yet there was no correlation between the expression of these markers and acute rejection. Expression of the CD27 marker, however, was significantly higher within CD8+CD28- T cells from patients without rejection as compared with patients in rejection (p = 0.005), indicating that the memory-like CD8+CD28-CD27+ T-cell subset comprises regulatory cells, which play a protective role for the graft. CD8+CD28- T cells isolated from transplant patients did not display cytotoxic activity against donor cells and showed high expression of the killing inhibitory receptor CD94. This study identifies the phenotypic changes that occur in patients with heart transplants and opens new avenues for the induction of specific immunosuppression in transplantation.

Expression of immune inhibitory receptor ILT3 in acute myeloid leukemia with monocytic differentiation.

BACKGROUND: The diagnosis of AML with monocytic differentiation is limited by the lack of highly sensitive and specific monocytic markers. Immunoglobulin-like transcript 3 (ILT3) is an immune inhibitory receptor expressed by myelomonocytic cells and at high levels by tolerogenic dendritic cells. METHODS: Using flow cytometry, we analyzed the expression of ILT3 in 37 patients with AML and 20 patients with no detectable disease. RESULTS: We showed that ILT3 was expressed in all cases of AML displaying monocytic differentiation (FAB M4/M5; N = 18), but not in AML M1/M2 and M3 (N = 19; P < 0.0001). Co-expression of ILT3 and immature cell markers, such as CD34 and CD117, was observed in monoblastic leukemia. ILT3 expression was preserved after treatment in M4/M5 patients with refractory or relapsed disease. ILT3 expression was associated with the presence of cytogenetic abnormalities linked to an intermediate prognosis (P = 0.001). Rare CD45dimCD34+CD117+ILT3+ cells were identified in noninvolved bone marrow, suggesting that ILT3 expression is acquired at an early stage by normal myelomonocytic precursors. CONCLUSIONS: ILT3 is a highly sensitive and specific marker which distinguishes AML with monocytic differentiation from other types of AML. Testing of ILT3 expression should be incorporated into the initial diagnostic work-up and monitoring of patients with AML.

Hematogones are markedly decreased in chronic myeloid leukemia: multiparametric flow cytometric analysis.

Past studies have shown decreased hematogones in the bone marrow of patients with myelodysplastic syndromes and acquired aplastic anemia. In this study, we examined the bone marrow of patients with chronic myeloid leukemia (n = 33, mean age 49 years, age range 2-83 years) for the presence of hematogones and compared their frequency with that of age-matched controls (n = 50). We found that the percentages of total and stage I hematogones were decreased in chronic myeloid leukemia at diagnosis (n = 25) and at follow-up post therapy (n = 8) when compared to age-matched controls (diagnosis, total: 0.29% vs. 0.87%, p = 0.001; diagnosis, stage I: 0.06% vs. 0.20%, p = 0.008; follow-up, total: 0.17% vs. 0.87%, p < 0.001; follow-up, stage I: 0.04 vs. 0.20, p = 0.003). We also found a significant decrease in the number of natural killer cells in patients with chronic myeloid leukemia at diagnosis. Further studies are warranted to elucidate the mechanism of hematogone decrease in chronic myeloid leukemia and whether this finding also applies to other myeloproliferative neoplasms.

Pre- and posttransplantation allosensitization in heart allograft recipients: major impact of de novo alloantibody production on allograft survival.

The involvement of humoral response in allograft rejection has been suggested by both immunologic and histochemistry studies. In the present study, we explored the role of alloantibodies in a large cohort of heart allograft recipients followed for 15 years. Sequential samples of sera were obtained from 950 recipients of heart allografts before and after transplantation at the time when protocol endomyocardial biopsies were performed. The presence of anti-human leukocyte antigen (HLA) antibodies was investigated using complement mediated cytotoxicity and solid phase assay (SPA). Our data reveal an inverse correlation between the development of alloantibodies after transplantation and heart allograft survival. The 15-year graft survival was highest in patients who never developed alloantibodies (70%) or who displayed them only before transplantation (71%); graft survival in recipients who showed antibodies both before and after transplantation (56%), or only after transplantation (47%), was lower. The deleterious effect of antibodies on graft survival started 8 years after transplantation, suggesting that the production of de novo antibodies may have been triggered by some later event. We found that patients with de novo antibodies appearing more than 1 year after transplantation had the poorest survival. Furthermore, the development of de novo antibodies was preceded in 76% of these patients by cellular rejection grade 3 or higher, according to the International Society for Heart Transplantation (ISHT) grading criteria. Development of antibody-mediated rejection (AMR) had a significant negative impact on graft survival (16% in AMR(+) vs 63% in AMR(-) patients, p = 0.0008). Of the 23 patients with AMR, 21 displayed cytotoxic donor-specific antibodies (DSA) at the time of diagnosis, and in 18 of these cases SPA showed that they were directed against the donors' HLA. The data demonstrate that the detection of alloantibodies permits a better definition of AMR in heart allograft recipients. Identification of patients at risk for developing AMR is of great importance for early treatment of rejection episodes.

Hematogones: a review and update.

Our knowledge regarding the nature and function of 'hematogones' has evolved considerably, since the initial description more than 70 years ago. Once considered the 'mystery cells' of the bone marrow, major advances in immunology and flow cytometry have enabled us to better characterize these cells and recognize them as physiologic precursors of B-cells. In this review, we describe the morphologic and phenotypic characteristics and clinical relevance of hematogones, and report recent advances in our understanding and knowledge of these cells as they relate to physiologic and different pathologic conditions.

Membrane and soluble ILT3 are critical to the generation of T suppressor cells and induction of immunological tolerance.

The tolerogenic phenotype of human dendritic cells is characterized by high cell surface expression of the inhibitory receptor ILT3. ILT3 signals both intracellularly inhibiting tyrosine phosphorylation, NF-kappaB and MAPK p38 activity, transcription of certain co-stimulatory molecules, secretion of cytokines and chemokines, and extracellularly into the T cells with which the dendritic cells interact. Both ILT3(high) tolerogenic dendritic cells and soluble ILT3 induce CD4 Th anergy and differentiation of antigen specific CD8 T suppressor cells. Recombinant ILT3-Fc protein has important immunotherapeutic potential acting directly on activated T cells and promoting the induction of immunological tolerance.

Increased adenosine triphosphate production by peripheral blood CD4+ cells in patients with hematologic malignancies treated with stem cell mobilization agents.

Hematopoietic stem cell (HSC) transplantation is an important therapeutic option for patients with hematologic malignancies. To explore the immunomodulatory effects of HSC mobilization agents, we studied the function and phenotype of CD4(+) T cells from 16 adult patients with hematologic malignancies undergoing HSC mobilization treatment for autologous transplantation. Immune cell function was determined using the Immuknow (Cylex) assay by measuring the amount of adenosine triphosphate (ATP) produced by CD4(+) cells from whole blood. ATP activity measured in G-CSF-treated patients was significantly higher than that measured in healthy individuals or "nonmobilized" patients. In patients treated with G-CSF, CD4(+) T cells were predominantly CD25(low)FOXP3(low), consistent with an activated phenotype. However, T-cell depletion did not abrogate ATP production in blood samples from G-CSF-treated patients, indicating that CD4(+) myeloid cells contributed to the increased ATP levels observed in these patients. There was a significant correlation between ATP activity and patient survival, suggesting that efficient activation of CD4(+) cells during mobilization treatment predicts a low risk of disease relapse. Monitoring immune cell reactivity using the Immuknow assay may assist in the clinical management of patients with hematologic malignancies and optimization of HSC mobilization protocols.

Diffuse large B-cell lymphoma with TEL/ETV6 translocation.

Cytogenetic abnormalities of chromosome 12p involving the TEL/ETV6 gene are observed in a variety of hematopoietic neoplasms including acute leukemias, myelodysplastic syndromes, and myeloproliferative disorders. Karyotypic aberrations, including rearrangements, deletions, and amplifications of chromosome 12p, have been documented in B-cell non-Hodgkin lymphoma; however, rearrangements targeting TEL have rarely been reported. Here we describe a diffuse large B-cell lymphoma that had a complex karyotype including t(9;12)(q22;p13), which was confirmed by fluorescence in situ hybridization to represent rearrangement of TEL. Additional cytogenetic abnormalities included t(3;14)(q27;q32) involving the variant, alternative breakpoint region of the BCL6 gene and del(6)(q13q23), resulting in the loss of 1 allele of BLIMP1. This case reiterates the importance of correlating morphologic and phenotypic findings with the results of cytogenetic analysis to avoid errors in diagnosing hematologic neoplasms and highlights the rare association of B-cell non-Hodgkin lymphoma with aberrations of TEL.

Immunoglobulin-like transcript 3: A crucial regulator of dendritic cell function.

Dendritic cells (DC) are key components of the immune system, which actively participate in innate and adaptive immune responses. They are traditionally viewed as the immunologic centerpiece that is able to prime CD4(+) helper and CD8(+) cytotoxic T-cell effector populations. However, accumulated evidence highlights the functional plasticity of DC, which are shown to also be able to display a tolerogenic function eliciting the differentiation of T suppressor (Ts) and regulatory (Treg) cells. This tolerogenic state of DC is characterized by low costimulatory potential and high expression of inhibitory receptors. Conspicuously among the latter is the immunoglobulin-like transcript 3 (ILT3), which independently prevents the activation of both DC and T cells. DC overexpressing ILT3 display lower phosphorylation levels of NF-kappaB and fail to stimulate the full program of Th proliferation and maturation eliciting instead the differentiation of CD8(+) T(S) and CD4(+) Treg. In contrast, ILT3-knockdown DC have robust cytokine and chemokine production, and are able to trigger stronger T-cell responses to viral antigens or alloantigens. Understanding and manipulating the functional immunogenic/tolerogenic dichotomy of DC has important clinical applications for achieving tolerance in organ transplantation, stemming autoimmune diseases or, conversely, generating efficient immunogenic vaccines for immunotherapy in cancer and chronic viral diseases.

Hematogones are markedly reduced in pediatric acquired aplastic anemia: multiparametric flow cytometric analysis.

Acquired aplastic anemia (AA) and myelodysplastic syndromes (MDS) are bone marrow (BM) failure syndromes with overlapping clinical features, and at least a subset appears to share common pathophysiologic mechanisms. Recent studies of MDS have shown down-regulation of genes involved in B-cell development and decreased B-cell precursors (hematogones). We explored the possibility that AA, similar to MDS, might also be associated with defects in development of lymphoid cells, especially B-cells, by using flow cytometry to assess the presence of hematogones and mature lymphocytes in BM samples from 25 children with AA and 41 age-matched controls. We observed that the percentage of total and early (stage I) hematogones were significantly decreased in AA compared to controls, and they returned to normal numbers after hematopoietic stem-cell transplant. This demonstrates early B-cell lineage involvement in AA, similar to recent findings in MDS. Our findings suggest dysfunction of an early multilineage progenitor in the pathogenesis of AA.

Relevance of different antibody detection methods for the prediction of antibody-mediated rejection and deceased-donor kidney allograft survival.

Presensitizing alloantibodies may represent a grave danger in organ transplantation, increasing the risk of antibody-mediated rejection (AMR) and graft loss. However, not all antibodies are harmful to the graft. In our study of a cohort of 325 deceased-donor renal allograft recipients, the patients were determined eligible to receive an allograft based on a negative complement-dependent cytotoxicity (CDC) crossmatch (XM). Yet at the time of transplantation, many candidates displayed donor-specific antibodies (DSA) by more sensitive methods, such as solid-phase assays (SPA, Luminex) or flow cytometry crossmatch (FCXM). The majority of the patients who were DSA positive by either SPA (67%) or FCXM (66%) presented an AMR-free clinical course posttransplantation. Among the patients who developed AMR (N = 29), 76% proved clinically manageable and did not lose the graft. Analysis of the DSA mean fluorescence intensities (MFI) of Luminex indicated no statistically significant difference between patients who experienced AMR episodes and those who did not. Importantly, many of the patients with AMR did not test positive for DSA by SPA (20/29) or FCXM (14/29). Despite false-positive and false-negative results, the detection of DSA by SPA or FCXM was positively associated with AMR, but not with actuarial graft survival. The field of organ transplantation has always struggled to reconcile two opposing goals: improving transplantation outcome while increasing access to transplantation. SPA and FCXM appear to be oversensitive and defining patients as "sensitized" according to these methods would block access to transplantation for many candidates who would otherwise benefit greatly from receiving the allograft. Nevertheless, SPA and FCXM are invaluable tools, assisting clinicians in gauging AMR risk and tailoring immunosuppression of the posttransplantation immunological monitoring accordingly.