Biopsy assessment and HLA antibody detection have been detailed elsewhere ( 5 ). Biopsies were processed for histology (Periodic acid-Schiff, hematoxylin and eosin, and trichrome) and tested for DSA, as per local standard of care. The anti-HLA antibody testing method varied depending on the center (enzyme-linked immunosorbent assay, CDC-AHG [complement-dependent cytotoxicity plus anti-human globulin] panels, FlowPRA (One Lambda Inc., Canoga Park, CA) screen or Luminex (One Lambda, Canoga Park, CA; single-antigen bead testing), as did the criteria for assigning positivity. The local C4d staining methods (immunofluorescence [n = 530] or immunoperoxidase [n = 131]) and grading were described previously (33 33. Sellares, J • Reeve, J • Loupy, A ... ). The methods for assessing HLA antibody data were based on standard of care for the center at the time of biopsy and thus were variable among centers.
Flowpra
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FlowPRA is a flow cytometry-based assay that measures the presence and reactivity of anti-HLA antibodies in patient samples. It provides quantitative data on the strength and specificity of these antibodies, which is important for monitoring transplant patients and assessing the risk of rejection.
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Lab products found in correlation
Labscreen single antigen beads, by Thermo Fisher Scientific (3 mentions)
Labscreen single antigen bead assay, by Thermo Fisher Scientific (1 mentions)
Labscreen pra, by Thermo Fisher Scientific (1 mentions)
Labscreen mixed, by Thermo Fisher Scientific (1 mentions)
C1qscreen, by Thermo Fisher Scientific (1 mentions)
Labtype xr, by Thermo Fisher Scientific (1 mentions)
Single antigen beads, by Thermo Fisher Scientific (1 mentions)
9 protocols using flowpra
1
Biopsy-Based Assessment of HLA Antibodies
2
HLA Antibody Assessment and Donor Mismatch
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HLA antibody assessments were performed on all patients at baseline and regular intervals per protocol thereafter. Antibody screening was performed using solid-phase flow cytometry screening (FlowPRA, One Lambda, Inc., Canoga Park, CA). Sera from patients with anti-HLA antibodies were subsequently analyzed using LABScreen single-antigen bead assay (One Lambda, Inc.) to determine antibody specificity and the presence/absence of donor-specific antibodies (DSAs) (mean fluorescence intensity). Sera are not pretreated or diluted before single-antigen bead testing. Mismatch was determined by comparing donor–recipient phenotype at the antigen/allele level for the A, B, and C class I loci and DQ and DR class II loci.
3
Monitoring Anti-HLA Antibodies in Transplant Recipients
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For all recipients, anti-HLA antibodies were analyzed before transplantation and monitored annually after transplantation. Serum samples collected from 2009 to 2019 were examined for IgG antibodies against HLA class I or II using methodologies including Flow PRA, LABScreen Mixed and LABScreen PRA (One Lambda). Any positive evaluations were re-screened and the DSA was identified using LABScreen Single Antigen and Supplement (One Lambda). Mean fluorescence intensity (MFI) values above 1,000 for DSAs against HLA-A, -B, -DR, and -DQ at the 4-digit level were scored as positive.
4
Kidney Transplant Outcomes and Antibody Analysis
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Data on transplantations and hospital stays, as well as follow-up data, were collected from hospital records. Baseline characteristics, such as recipient age and gender, donor type (deceased or living), number of previous transplants, cold ischemia time, number of HLA mismatches, pretransplant panel reactive antibody (PRA) percentages divided into groups (0–10%, >10%–50%, and ≥50%), and preformed DSA, were collected and analyzed. In addition, early clinical outcomes, including the generation of de novo DSA, rate of delayed graft function (DGF), the frequency and type of acute allograft rejection (cellular or antibody-mediated rejection), and one-year graft and patient survival, were analyzed. DGF was defined as the need for more than 1 dialysis during the first week after transplant. HLA class I and II antibody screenings were performed using FlowPRA® (One Lambda, Inc., Canoga Park, CA), and the specificity determination was measured by Luminex using LABScreen® single antigen beads (One Lambda, Inc., Canoga Park, CA).
5
Evaluation of Donor-Specific Antibodies in Transplant Recipients
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The serum from the recipients was screened for anti-HLA antibody with Flow PRA (One Lambda, Inc., Canoga Park, CA). The LTR was considered sensitized when the panel-reactive assay (PRA) was positive (greater than 0%) and regarded unsensitized when PRA was negative. The sensitized recipients were further tested for donor-specific antibody (DSA) with LABScreen single-antigen beads (One Lambda, Inc.). The positive DSA was defined by mean fluorescence intensity (MFI) units greater than 1000 or more. The complement-binding function in DSA was measured with C1q Screen (One Lambda Inc.). The test for anti-HLA antibody was done in ReproCELL Japan Inc. Yokohama.
6
HLA Antibody Monitoring for Kidney Transplants
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Serum collected within 6 months before transplantation was analyzed by SABA (FlowPRA; One Lambda, Canoga Park, CA, USA) as previously reported [10 (link)]. Luminescence was read on a LABScan 100 Luminex system (One Lambda) and a mean fluorescence intensity >1000 was considered a positive result.
Polymerase chain reaction sequence-specific oligonucleotide technology (LABType XR; One Lambda) was used for HLA typing of recipients and donors (HLA-A, -B, -C, -DQ and -DR). Until 2016, routine HLA typing was performed only for HLA-A, HLA-B and HLA-DR; HLA-C and HLA-DQ were typed only if antibodies against them were detected by SABA, whereupon typing was performed to determine whether the antibodies were DSAs or non-DSAs. Patients with anti-HLA antibodies that recognized and cross-reacted with epitope groups specific to donor-mismatched HLAs were considered to have DSAs.
Serum analysis by SABA was again performed 6 months after KTx and then followed up annually. SABA was also performed when a patient underwent for-cause biopsy or when rejection was suspected on clinical evaluation.
During the follow-up period, dnDSAs sometimes disappeared and reappeared. Patients who tested positive for dnDSAs even once during this period were included in the dnDSA group.
Polymerase chain reaction sequence-specific oligonucleotide technology (LABType XR; One Lambda) was used for HLA typing of recipients and donors (HLA-A, -B, -C, -DQ and -DR). Until 2016, routine HLA typing was performed only for HLA-A, HLA-B and HLA-DR; HLA-C and HLA-DQ were typed only if antibodies against them were detected by SABA, whereupon typing was performed to determine whether the antibodies were DSAs or non-DSAs. Patients with anti-HLA antibodies that recognized and cross-reacted with epitope groups specific to donor-mismatched HLAs were considered to have DSAs.
Serum analysis by SABA was again performed 6 months after KTx and then followed up annually. SABA was also performed when a patient underwent for-cause biopsy or when rejection was suspected on clinical evaluation.
During the follow-up period, dnDSAs sometimes disappeared and reappeared. Patients who tested positive for dnDSAs even once during this period were included in the dnDSA group.
7
Long-term Kidney Transplant Outcomes
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Efficacy and safety outcomes from randomization to month 84 (year 7), including time to death and/or graft loss, acute rejection, renal function, safety and de novo DSA incidence, are summarized. As in prior analyses 28 , 29 , 30 , acute rejection was defined as central biopsy–proven rejection that was either clinically suspected for protocol‐defined reasons or clinically suspected for other reasons and treated. A combined end point comprising time to first occurrence of death, graft loss or estimated GFR (eGFR) <20 mL/min per 1.73 m2 was examined post hoc. GFR was estimated using the six‐variable MDRD equation 31 . Adverse events (AEs) were mapped to terms from the Medical Dictionary for Regulatory Activities version 17.0 (MedDRA MSSO, McLean, VA) and expressed as incidence rates adjusted per 100 person‐years of exposure to assigned treatment. Serious AEs are defined in the supplementary material. De novo DSA development was assessed centrally by solid‐phase flow cytometry (FLowPRA; One Lambda, Inc., Canoga Park, CA), with HLA class specificity (class I or II) determined by LABScreen single antigen beads (One Lambda, Inc.).
8
HLA Antibody Screening and Quantification
9
Alloantibody Screening for Transplant Eligibility
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