Nature and Clonality of the Fluoresceinated Secondary Antibody in Luminex Multiplex Bead Assays Are Critical Factors for Reliable Monitoring of Serum HLA Antibody Levels in Patients for Donor Organ Selection, Desensitization Therapy, and Assessment of the Risk for Graft Loss.

Luminex multiplex immunoassays enable simultaneous monitoring of Abs against multiple Ags in autoimmune, inflammatory, and infectious diseases. The assays are used extensively to monitor anti-HLA Abs in transplant patients for donor organ selection, desensitization, and assessing the risk for graft rejection. To monitor IgG Abs, fluoresceinated IgG constant H chain-binding polyclonal F(ab') () is used as the fluoresceinated secondary Ab (2nd-Ab), whereas IgG subclasses are monitored with Fc-specific monoclonal whole IgG ().

Conformational Variants of the Individual HLA-I Antigens on Luminex Single Antigen Beads Used in Monitoring HLA Antibodies: Problems and Solutions.

Background: Single antigen beads (SAB) are used for monitoring HLA antibodies in pretransplant and posttransplant patients despite the discrepancy between virtual and actual crossmatch results and transplant outcomes. This discrepancy can be attributed to the presence of conformational variants of HLA-I on SAB, assessment of which would increase the concordance between SAB and flow cytometry crossmatch (FCXM) results, thus enabling improved organ accessibility for the waiting list patients and a better prediction of antibody-mediated rejection.

Methods: The conformational variants were examined on HLA-I beads, iBeads, acid-/alkali-treated beads, and T cells using HLA-I monoclonal antibodies (W6/32, TFL-006, and heavy chain (HC)-10).

Prevalence and Clinical Impact of Donor-Specific Alloantibody Among Intestinal Transplant Recipients.

Background: Rejection remains the leading cause of allograft loss, and a major barrier to improving long-term outcomes after intestinal transplantation. Our aim is to define the prevalence and investigate the role of donor-specific antibody (DSA) on intestinal graft outcomes.

Methods: The study includes 109 transplants performed in 95 recipients at a single center.

Dynamics and epitope specificity of anti-human leukocyte antibodies following renal allograft nephrectomy.

Background: A considerable proportion of patients awaiting kidney transplantation is immunized by previous transplantation(s). We investigated how allograft nephrectomy (Nx) and withdrawal of maintenance immunosuppression (WD-MIS) in patients with a failed renal allograft contribute to allosensitization.

Methods: HLA antibodies (HLAabs) were analyzed before and after Nx and/or WD-MIS using a single antigen bead assay.

Significance of the differences in the prevalence of anti-HLA antibodies in matched pairs of mother's and cord blood.

The presence of IgG against pathogens in the cord blood (CB) of vaccinated mothers is attributed to transplacental transfer. However, previous studies using lymphocytotoxicity assay showed anti-HLA IgG in mother's blood (MB) but not in CB, perhaps due to non-transfer of anti-HLA IgG or assay limitations in detecting anti-HLA IgG. Anti-HLA IgG of native and purified sera of 16 MB and CB pairs were measured using an array of microbeads coated with HLA-I/-II molecules on a Luminex platform.

Complement-fixing antibodies against denatured HLA and MICA antigens are associated with antibody mediated rejection.

Background: We have found antibodies against denatured HLA class I antigens in the serum of allograft recipients which were not significantly associated with graft failure. It is unknown whether transplant recipients also have denatured HLA class II and MICA antibodies. The effects of denatured HLA class I, class II, and MICA antibodies on long-term graft outcome were further investigated based on their ability to fix complement c1q.

The Monospecificity of Novel Anti-HLA-E Monoclonal Antibodies Enables Reliable Immunodiagnosis, Immunomodulation of HLA-E, and Upregulation of CD8+ T Lymphocytes.

In human cancers, over-expression of HLA-E is marked by gene expression. However, immunolocalization of HLA-E on tumor cells is impeded by the HLA-Ia reactivity of commercial anti-HLA-E monoclonal antibodies (MAbs). So there was a clear need to develop monospecific anti-HLA-E MAbs for reliable immunodiagnosis of HLA-E, particularly considering the prognostic relevance of HLA-E in human cancer.

Risk Stratification of Human Leukocyte Antigen Class II Donor Specific Antibody Positive Patients by Immunoglobulin G Subclasses.

Background: Human leukocyte antigen (HLA) antibodies are a major cause of graft loss in mismatched transplant recipients. However, the time to graft loss resulting from antibody induced injury is unpredictable. The unpredictable nature of antibodies may be related to the subclass of antibodies.

Pre-existing anti-HLA antibodies negatively impact survival of pediatric aplastic anemia patients undergoing HSCT.

Graft failure and survival are the major problems for patients with aplastic anemia undergoing hematopoietic stem cell transplantation (HSCT). Previous studies showed that anti-HLA antibodies negatively impact engraftment in HSCT. This retrospective study of 51 pediatric patients with acquired aplastic anemia who underwent allogeneic HSCT at a single institution between 2006 and 2012 investigated the influence of anti-HLA antibodies on the outcome of HSCT.

Acute liver allograft antibody-mediated rejection: an inter-institutional study of significant histopathological features.

Acute antibody-mediated rejection (AMR) occurs in a small minority of sensitized liver transplant recipients. Although histopathological characteristics have been described, specific features that could be used (1) to make a generalizable scoring system and (2) to trigger a more in-depth analysis are needed to screen for this rare but important finding. Toward this goal, we created training and validation cohorts of putative acute AMR and control cases from 3 high-volume liver transplant programs; these cases were evaluated blindly by 4 independent transplant pathologists.

Association of anti-human leukocyte antigen and anti-angiotensin II type 1 receptor antibodies with liver allograft fibrosis after immunosuppression withdrawal.

Background: Many pediatric patients who receive a living-donor liver transplant undergo withdrawal of immunosuppression (IS). For them, the high incidence of long-term progressive graft fibrosis is of particular concern.

Methods: We conducted a cross-sectional study including 81 pediatric patients who underwent IS withdrawal after living-donor liver transplant at Kyoto University Hospital and whose serum samples and pathological data could be obtained during the analysis period.

Changes in successive measures of de novo donor-specific anti-human leukocyte antigen antibodies intensity and the development of allograft dysfunction.

Background: Many patients develop de novo donor-specific anti-human leukocyte antigen antibodies (dnDSA) after transplantation. Despite development of dnDSA, not all patients will immediately fail. This study analyzes dnDSA intensity and longitudinal trends as prospective clinical parameters to assess subsequent allograft function.

Donor-specific alloantibodies are associated with fibrosis progression after liver transplantation in hepatitis C virus-infected patients.

Hepatitis C virus (HCV) fibrosis progression after liver transplantation (LT) is accelerated in comparison with fibrosis progression before transplantation. The vast majority of the risk factors for fibrosis progression after LT are not modifiable. With the goal of identifying modifiable risk factors for fibrosis progression, we evaluated the impact of preformed and de novo donor-specific human leukocyte antigen alloantibodies (DSAs) on fibrosis progression after LT in HCV-viremic patients.

Impact of IgM and IgG3 anti-HLA alloantibodies in primary renal allograft recipients.

Background: With standard IgG donor-specific anti-HLA antibody (DSA) testing, it is unclear which immunoglobulin-G (IgG) DSA positive patients will fail. We looked further into the immune response by studying immunoglobulin-M (IgM) and IgG subclass 3 (IgG3) DSA to determine if these identify the IgG DSA patients at highest risk for allograft loss.

Methods: In 189 consecutively transplanted primary renal allograft recipients, sera were collected sequentially pre- and posttransplant.

Antibody-mediated rejection as a contributor to previously unexplained early liver allograft loss.

We analyzed 60 patients with idiopathic early allograft loss (defined as death or retransplantation at <90 days) to determine the relative contribution of preformed donor-specific human leukocyte antigen alloantibodies (DSAs) to this endpoint, and we defined strict criteria for the diagnosis of antibody-mediated rejection (AMR) in liver allografts. The inclusion criteria encompassed the availability of a pretransplant serum sample and both postreperfusion and follow-up tissue specimens for a blinded, retrospective re-review of histology and complement component 4d (C4d) staining. AMR was diagnosed on the basis of the presence of all 4 of the following strict criteria: (1) DSAs in serum, (2) histopathological evidence of diffuse microvascular injury/microvasculitis consistent with antibody-mediated injury, (3) diffuse C4d staining in the portal microvasculature with or without staining in the sinusoids or central veins in at least 1 sample, and (4) the exclusion of other causes of a similar type of injury.

Improved Long-Term Survival in Kidney Transplant Recipients with Donor-Specific HLA Antibodies After Mycophenolic Acid Escalation.

The development of donor specific antibodies (DSA) post transplant has been associated with chronic rejection and graft failure. In a longitudinal study, we have shown that increases in DSA precede rejection by months, thus allowing time for intervention. We hypothesized that mycophenolic acid (MPA) dose increases may reduce and/or stabilize DSA strength and also preserve renal function.

Gastric cancer progression may involve a shift in HLA-E profile from an intact heterodimer to β2-microglobulin-free monomer.

Phenotypic expression of human leukocyte antigen (HLA)-E on the surface of tumor lesions includes intact heterodimer [HLA-E heavy chain and β2-microglobulin (β2m)] and β2m-free monomer. Anti-HLA-E monoclonal antibodies (mAbs), MEM-E/02 or 3D12 bind to the peptide sequences in β2m-free HLA-E, which is common and shared with HLA-Ia monomers. A newly developed monospecific anti-HLA-E mAb (TFL-033) recognizes HLA-E-restricted peptide sequences on α1 and α2 helices away from β2-m-site.

Trends and characteristics in early glomerular filtration rate decline after posttransplantation alloantibody appearance.

Background: Approximately 7% to 9% of patients with donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) fail within 1 year post-DSA onset. However, little is known as to how this DSA-associated failure temporally progresses. This longitudinal study investigates DSA's temporal relationship to allograft dysfunction and identifies predictors of allograft function's progressive deterioration post-DSA.

Preformed class II donor-specific antibodies are associated with an increased risk of early rejection after liver transplantation.

Preformed donor-specific human leukocyte antigen antibodies (DSAs) are considered a contraindication to the transplantation of most solid organs other than the liver. Conflicting data currently exist on the importance of preformed DSAs in rejection and patient survival after liver transplantation (LT). To evaluate preformed DSAs in LT, we retrospectively analyzed prospectively collected samples from all adult recipients of primary LT without another organ from January 1, 2000 to May 31, 2009 with a pre-LT sample available (95.

HLA class II DQA and DQB epitopes: recognition of the likely binding sites of HLA-DQ alloantibodies eluted from recombinant HLA-DQ single antigen cell lines.

Donor-specific antibodies (DSA) in sera of sensitized transplant patients are often produced against the specific epitopes on mismatched HLA antigens. In this study, we selected sera from 30 kidney transplant patients with DSA and AMR to define DQ epitopes. Using adsorption and elution assays, we identified 18 antibody reaction patterns to define 6 new epitopes and to confirm 12 previously defined epitopes.

Impact of alloantibody strength in crossmatch negative DSA positive kidney transplantation.

Objectives: The clinical relevance of pre-transplant "low-level" donor specific anti-HLA antibodies (DSAs) in crossmatch negative kidney transplant recipients remains unclear. To determine what level of DSA associates with antibody mediated rejection (AMR) could be the way to measure the clinical relevance of pre-transplant "low-level" donor specific anti-HLA antibodies (DSAs) in crossmatch negative kidney transplant recipients.

Design And Methods: A retrospective analysis of 221 patients from October 2008 to December 2009 was included in this study.

The role of immunoglobulin-G subclasses and C1q in de novo HLA-DQ donor-specific antibody kidney transplantation outcomes.

Background: Anti-HLA-DQ antibodies are the predominant HLA class II donor-specific antibodies (DSAs) after transplantation. Recently, de novo DQ DSA has been associated with worse allograft outcomes. The aim of this study was to determine the further complement-binding characteristics of the most harmful DQ DSA.

The clinical impact of humoral immunity in pediatric renal transplantation.

The development of anti-donor humoral responses after transplantation associates with higher risks for acute rejection and 1-year graft survival in adults, but the influence of humoral immunity on transplant outcomes in children is not well understood. Here, we studied the evolution of humoral immunity in low-risk pediatric patients during the first 2 years after renal transplantation. Using data from 130 pediatric renal transplant patients randomized to steroid-free (SF) or steroid-based (SB) immunosuppression in the NIH-SNSO1 trial, we correlated the presence of serum anti-HLA antibodies to donor HLA antigens (donor-specific antibodies) and serum MHC class 1-related chain A (MICA) antibody with both clinical outcomes and histology identified on protocol biopsies at 0, 6, 12, and 24 months.

Humoral theory of transplantation: some hot topics.

Introduction: Antibody is a major cause of allograft injury. However, it has not been routinely tested post-transplant.

Sources Of Data: A literature search was performed using PubMed on the topics of 'antibody monitoring', 'autoantibody and allograft dysfunction' and 'prevention and treatment of antibody-mediated rejection (AMR)'.

Incidence and impact of de novo donor-specific alloantibody in primary renal allografts.

Background: To date, limited information is available describing the incidence and impact of de novo donor-specific anti-human leukocyte antigen (HLA) antibodies (dnDSA) in the primary renal transplant patient. This report details the dnDSA incidence and actual 3-year post-dnDSA graft outcomes.

Methods: The study includes 189 consecutive nonsensitized, non-HLA-identical patients who received a primary kidney transplant between March 1999 and March 2006.