13: Computational Analysis of the Single Antigen Beads Used in Solid Phase Assays for the Detection of Anti-HLA Antibodies

Abstract Background and Aims The introduction of more sensitive and specific tools as solid phase immunoasssays has improved the ability to detect HLA antibodies. The techniques based on solid phase immunoassays in Luminex system can be performed either as screening assays (with pooled antigen panels that use bead populations coated in affinity-purified class I or class II HLA molecules) or to assess the specificity with single-antigen bead (SAB) assays (single cloned allelic HLA antigen).The sensitivity of each assay Is different, and their results may be discordant. Our objective was to evaluate the potential discrepancies between tests in a cohort of patients on our waiting list with no previous sensitizing events, for their potential influence in the access to transplantation. Method Observational study of 184 patients included in the kidney-transplant waiting list on March 31st, 2019. All possible sensitizing events (pregnancy, transfusion and previous transplants) were registered. HLA antibodies where analyzed both by screening and SAB tests. Results We limited the observations to males (64.7% of the cohort), as most women (95.4%) had a known sensitizing event, not being possible to discard possible pregnancies in the remaining. Of men, 46.2% had a sensitizing event and 63.8% did not. All 63 unsensitized patients showed a negative screening test, and 49 (77,78%) had at least one SAB done being positive in 73.4% (36/49). The positive SAB yielded a cPRA >10% in 30 cases (between 21-85%), mostly (27/30) due to HLA class II antibodies (88.8% with at least one DQ antibody) with a median MFI of 1571 (range:752-17650). A thorough review of specificities showed that 81% DQB1* and 89.6% DQA1* antibodies did not show consistency in all beads included for the same allele. Conclusion Screening and SAB assays show a significant discordancy in unsensitized men included in the waiting list for kidney transplantation. Automated detection of antibodies by SAB without a proper specialized interpretation may wrongly limit access to transplantation.

Download Full-text

The impact of anti-HLA antibodies on unrelated cord blood transplantations

Blood ◽

10.1182/blood-2009-10-249219 ◽

2010 ◽

Vol 116 (15) ◽

pp. 2839-2846 ◽

Cited By ~ 162

Author(s):

Minoko Takanashi ◽

Yoshiko Atsuta ◽

Koki Fujiwara ◽

Hideki Kodo ◽

Shunro Kai ◽

...

Keyword(s):

Cord Blood ◽

Solid Phase ◽

Human Leukocyte ◽

Antibody Detection ◽

Red Cross ◽

Hla Antibodies ◽

Platelet Recovery ◽

Neutrophil Recovery ◽

Unrelated Cord Blood ◽

The Impact

Abstract The majority of cord blood transplantations (CBTs) have human leukocyte antigen (HLA) disparities. We investigated the impact that patients' pretransplantation anti-HLA antibodies have on the outcome of CBTs. Testing for anti-HLA antibody and its specificity was performed retrospectively at the Japanese Red Cross Tokyo Blood Center with sensitive solid-phase antibody detection assays. Among 386 CBTs, which were first myeloablative stem cell transplantations for malignancies and used a single unit of cord blood, 89 tested positive. Among the antibody-positive group, the cord blood did not have the corresponding HLA type for the antibody in 69 cases (ab-positive), while 20 cases had specificity against the cord blood HLA (positive-vs-CB). Cumulative incidence of neutrophil recovery 60 days after transplantation was 83% (95% confidence interval [CI], 79%-87%) for the antibody-negative group (ab-negative), 73% (95% CI, 61%-82%) for ab-positive, but only 32% (95% CI, 13%-53%) for the positive-vs-CB (P < .0001, Gray test). With multivariate analysis, the ab-positive showed significantly lower neutrophil recovery than the ab-negative (relative risk [RR] = 0.69, 95% CI, 0.49-0.96, p = .027). The positive-vs-CB had significantly lower neutrophil recovery (RR = 0.23, 95% CI, 0.09-0.56, P = .001) and platelet recovery (RR = 0.31, 95% CI, 0.12-0.81, P = .017) than the ab-negative. Patients' pretransplantation anti-HLA antibodies should be tested and considered in the selection of cord blood.

Download Full-text

DONOR-SPECIFIC HLA ANTIBODIES DETECTED BY SINGLE ANTIGEN BEADS IN RENAL TRANSPLANT PATIENTS: IMPACT ON CLINICAL OUTCOME

Transplantation Journal ◽

10.1097/00007890-201007272-00158 ◽

2010 ◽

Vol 90 ◽

pp. 83

Author(s):

J. P. Peräsaari ◽

L. Kyllönen ◽

K. Salmela ◽

J. Merenmies

Keyword(s):

Renal Transplant ◽

Clinical Outcome ◽

Hla Antibodies ◽

Transplant Patients ◽

Single Antigen ◽

Renal Transplant Patients

Download Full-text

Conformational Variants of the Individual HLA-I Antigens on Luminex Single Antigen Beads Used in Monitoring HLA Antibodies

Transplantation Journal ◽

10.1097/tp.0000000000001420 ◽

2017 ◽

Vol 101 (4) ◽

pp. 764-777 ◽

Cited By ~ 14

Author(s):

Vadim Jucaud ◽

Mepur H. Ravindranath ◽

Paul I. Terasaki

Keyword(s):

Hla Antibodies ◽

Single Antigen ◽

The Individual

Download Full-text

The Kinetics of Anti-HLA Antibodies in the First Year after Kidney Transplantation: In Whom and When Should They Be Monitored?

Journal of Transplantation ◽

10.1155/2018/8316860 ◽

2018 ◽

Vol 2018 ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

Maria Cristina Ribeiro de Castro ◽

Erick A. Barbosa ◽

Renata P. Souza ◽

Fabiana Agena ◽

Patrícia S. de Souza ◽

...

Keyword(s):

Acute Rejection ◽

De Novo ◽

First Year ◽

Sharp Drop ◽

Hla Antibodies ◽

Single Antigen ◽

The Impact ◽

Kinetics Of ◽

Time Point ◽

Rejection Rates

The impact of the kinetics of the anti-HLA antibodies after KTx on the occurrence of acute rejection as well as the better time-point to monitor anti-HLA Abs after transplantation is not completely defined. This prospective study followed 150 patients over 12 months after transplantation. Serum IgG anti-HLA Abs were detected by single antigen beads after typing donors and recipients for loci A, B, C, DR, and DQ. Before KTx, 89 patients did not present anti-HLA Abs and 2% developed “de novo” Abs during the 1st year, 39 patients were sensitized without DSAs, and 13% developed DSA after surgery; all of them presented ABMR. Sensitized patients presented higher acute rejection rates (36.4% versus 13.5%, p<0.001), although 60% of the patients did not present ABMR. Patients, in whom DSA-MFI decreased during the first two weeks after surgery, did not develop ABMR. Those who sustained their levels presented a rate of 22% of ABMR. 85% of patients developed ABMR when MFIs increased early after transplantation (which occurred in 30% of the DSA positive patients). In the ABMR group, we observed an iDSA-MFI sharp drop on the fourth day and then an increase between the 7th and 14th POD, which suggests DSA should be monitored at this moment in sensitized patients for better ABMR prediction.

Download Full-text

62-P: Significance of anti-HLA antibodies detected by solid-phase assay in heart allograft recipients

Human Immunology ◽

10.1016/j.humimm.2009.09.095 ◽

2009 ◽

Vol 70 ◽

pp. S41

Author(s):

Hugo P. Sondermeijer ◽

Eric Ho ◽

Geo Serban ◽

George Vlad ◽

Donna Mancini ◽

...

Keyword(s):

Solid Phase ◽

Hla Antibodies ◽

Heart Allograft ◽

Solid Phase Assay

Download Full-text

C1q-Binding HLA Antibodies Do Not Predict Platelet Transfusion Failure in TRAP Study Participant

Blood ◽

10.1182/blood.v124.21.1562.1562 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1562-1562

Author(s):

Rachael P. Jackman ◽

Douglas Bolgiano ◽

Mila Lebedeva ◽

Sherrill J. Slichter ◽

Philip J. Norris

Keyword(s):

Conflicts Of Interest ◽

Fluorescent Antibody ◽

Study Participant ◽

Class I ◽

Detection Methods ◽

Antibody Detection ◽

Hla Antibodies ◽

Luminex Assay ◽

Single Antigen ◽

Antibody Levels

Abstract Introduction: In the Trial to Reduce Alloimmunization to Platelets (TRAP) study, 101 of the 530 subjects became clinically refractory (CR) to platelet transfusions in the absence of detectable antibodies against HLA as measured by the lymphocytotoxicity assay (LCA). Using more sensitive bead-based detection methods we have previously demonstrated that while many of these LCA- patients do have anti-HLA antibodies, that these low to moderate level antibodies do not predict refractoriness. In addition to being less sensitive then bead based methods, the LCA screen only detects complement-binding antibodies. As these antibodies could be important for platelet rejection, we assessed if previously undetected complement-binding antibodies could account for some of the refractoriness seen in LCA- patients. Methods: 169 LCA- (69 CR, 100 non-CR) and 20 LCA+ (10 CR, 10 non-CR) subjects were selected from the TRAP study. Anti-class I HLA IgG and C1q binding antibodies were measured in serum or plasma using two different multi-analyte, semi-quantitative, bead-based fluorescent antibody detection assays: the LabScreen mixed Luminex assay, and the LabScreen single antigen class I assay with or without added EDTA (One Lambda). Groups were compared using an unpaired t-test, a=0.05, and correlation between variables was also assessed. Results: New measurements of anti-class I HLA IgG antibodies reliably reproduced earlier data with a strong correlation between the old and new measurements (R2=0.9736, p<0.0001). While some of the LCA- subjects did have detectable C1q-binding anti-class I HLA antibodies, and some LCA+ subjects did not, levels of these antibodies were significantly higher among LCA+ subjects (p<0.0001). Levels of C1q-binding anti-class I HLA antibodies were not significantly different between CR and non-CR among either the LCA- or LCA+ subjects. Conclusions: While complement-binding anti-class I HLA antibody levels were higher in the LCA+ subjects, higher levels of these antibodies were not seen in CR LCA+ patients as compared with non-CR LCA+ patients. Complement-binding anti-class I HLA antibodies do not account for refractoriness seen among the LCA- TRAP subjects. This work confirms that low to mid level anti-class I antibodies do not drive refractoriness to platelets, and suggests that antibody-independent mechanisms cause refractoriness in patients lacking higher levels of anti-HLA antibodies. Disclosures No relevant conflicts of interest to declare.

Download Full-text