Immunocomplex Capture Fluorescence Analysis Research Articles

Intra-Liver Allograft C3d-Binding Donor-Specific Anti-HLA Antibodies Predict Rejection After Liver Transplantation

There is no doubt that antibody-mediated rejection (AMR) due to donor-specific anti-HLA antibodies (DSA) brings a poor outcome for liver transplant recipients. However, the relationship between intragraft DSA (g-DSA), complement-binding abilities, and AMR remains unknown. We enrolled a total of 20 liver transplant recipients who underwent protocol or episode graft biopsies in the mid to long term after liver transplant (median 48.5, range 6-198 months), and their status of g-DSA and complement 3d (C3d)-binding abilities was assessed with the graft immunocomplex capture fluorescence analysis (ICFA) technique. The prevalence of g-DSA was 15.0 % in liver transplant recipients (3/20), and serum DSA (s-DSA) also existed in 15.0% of recipients. The number of g-DSA+/s-DSA+, g-DSA+/s-DSA-, g-DSA-/s-DSA+, and g-DSA-/s-DSA- cases are 1, 2, 2, and 15, respectively. The g-DSA+ group demonstrated a significant high rejection activity index: 3.67 ± 1.53, compared with the g-DSA- group: 1.24 ± 1.15 (P=.0045). Moreover, C3d-binding reaction was notably higher in the g-DSA+ group (C3d index: 1.87 ± 0.38 vs 0.76 ± 0.35) (P < .0001). Overall, the g-DSA+ group was more associated with liver allograft rejection-not only AMR, but also T cell-mediated rejection (P=.031). These results suggest that the existence of g-DSA and intragraft C3d-binding reaction had a negative impact on the liver allografts, but in contrast s-DSA did not have any significant impact.

Improved detection of donor-specific HLA-class II antibody in kidney transplant recipients by modified immunocomplex capture fluorescence analysis

Immunocomplex capture fluorescence analysis (ICFA) which basic principle is same as Luminex crossmatch (LXM), could detect donor-specific HLA antibody (DSA). The advantages of ICFA are (i) detection of DSA and (ii) no requirement of viable cells over the flow cytometry crossmatch (FCXM). However, FCXM has been widely used because of its higher sensitivity than ICFA, in particular HLA-class II antibody detection. In this study the accuracy of DSA detection against HLA-class II was investigated by modifying the original method of ICFA. Increment of the sensitivity was found when purified peripheral blood mononuclear cells (PBMCs) were used instead of whole blood. An ICFA-PBMC in addition to FCXM-T/B was conducted for 118 patients before kidney transplantation and 13 patients with de novo DSA against HLA-class II after transplantation. Significantly positive correlation was observed between the values of ICFA-PBMC and DSA mean fluorescence intensity (MFI) targeting class II (p < 0.0001). When the cutoff level of 1.4 was determined by receiver operating characteristic curve analysis, the average DSA MFI was found to be significantly higher in the ICFA-PBMC (class II) positive group comparing to that in the negative group (12,217 vs 3885, p = 0.0027). ICFA-PBMC and optimized cutoff level could provide valid information in cases of suspected DSA.

Establishment of a novel cell-based assay using HLA-transfected cells to detect HLA antibodies

The detection of HLA antibodies is important in clinical practice, such as platelet transfusion refractoriness and transfusion-related lung injury. However, difficulties are associated with the preparation of panel cells for conventional HLA detection systems using intact cells, such as the immunocomplex capture fluorescence analysis (ICFA).Based on an ICFA analysis, HEK293 cells stably transfected with the HLA-A locus were used instead of peripheral blood mononuclear cells (PBMC). The reactivity, sensitivity, and stability of transfectants were examined.All 20 antisera to HLA-A identified by LABScreen® Single Antigen class I (LS-SA1) were reactive to our modified-ICFA (m-ICFA) and showed the same specificities as those in LS-SA1, indicating the cell surface expression and correct antigenicity of the HLA-A locus in transfectants. The expression of HLA class I antigens was similar between transfectants frozen for 6 years and those prior to freezing. In the reaction of the anti-A24 or anti-A33 antibody vs each transfectant, the index of m-ICFA was higher than that of WAKFlow® ICFA. Our m-ICFA also showed that false negative reactions sometimes observed in capture assays may be avoided.By using HLA-A transfectants as ICFA targets, we herein developed m-ICFA. Our m-ICFA may avoid false negative reactions of capture assay like enzyme-linked immunosorbent assay and can also be carried out in almost any laboratory without cell culture facilities.

Detection of Intragraft Anti-Blood Group A and B Antibodies Following Renal Transplantation

BackgroundGraft immunocomplex capture fluorescence analysis is an attractive method to detect intragraft donor-specific anti-HLA antibodies. In ABO-incompatible transplantation, anti-A and B antibodies are also considered to be important donor specific antibodies (ABO-DSA). Therefore, it is useful to monitor intragraft ABO-DSAs to assess antibody-mediated rejection. MethodsTo capture A and B antigens, anti-Band III, von Willebrand factor (VW), and plasmalemma vesicle-associated protein (PLVAP) beads were produced. The allograft specimen was homogenized in a lysis buffer. Subsequently, A and B antigens were captured by anti-Band III, VW, or PLVAP beads. The immune complexes were then detected by phycoerythrin-conjugated anti-human IgG antibodies and analyzed using a Luminex system. ResultsAlthough Band III and VW beads yielded false positives and false negatives, PLVAP beads captured A and B antigens with high sensitivity (91.7%) and specificity (100%) when an index > 1.5 was considered positive. The proximity in A and B antigens and PLVAP expression was confirmed using immunohistochemical evaluation. Furthermore, sodium dodecyl sulfate polyacrylamide gel electrophoresis supported that PLVAP is an A and B antigen carrier protein. Case ReportBiopsies were conducted following an ABO-incompatible renal transplant (type A to O) and evaluated for ABO-DSA. Graft immunocomplex capture fluorescence analysis was demonstrated as follows: 3.19 (1 h, serum creatinine [s-Cr] 3.95 mg/dL, titer IgG 1:512, glomerulitis [g] 0, peritubular capillaritis [ptc] 0, complement 4d [C4d] 1); 1.8 (4 d, s-Cr 2.29 mg/dL, titer 1:256, g 0, ptc 0, C4d 3); 1.2 (22 d, s-Cr 1.58 mg/dL, titer 1:128, g 0, ptc 2, C4d 3). This result indicated that the remnant ABO-DSA were adsorbed and subsequently removed from the allograft successfully. ConclusionsThis novel application could be used to detect intragraft ABO-DSAs, which could lead to a correct diagnosis and shed light on the ABO-DSA kinetics following ABO-incompatible transplantation.

Clearance of Intra-graft Donor Specific Anti-HLA Antibodies in the Early Stage of Antibody-Mediated Rejection Following Rituximab and Apheresis Therapy in Renal Transplantation

BackgroundThe management of acute or, in particular, chronic antibody-mediated rejection (AMR) resulting from donor-specific HLA antibodies (DSA) is a critical barrier to obtaining better long-term graft survival. To ascertain the efficacy of anti-AMR therapies, the transition of intra-graft DSA (g-DSA) was assessed. MethodsAllograft biopsy specimens were analyzed by graft immunocomplex capture fluorescence analysis, as previously described. One hundred recipients who underwent graft biopsies between April 2016 and December 2017 were enrolled for this study. Fifteen recipients diagnosed with g-DSA positive (+) received anti-humoral treatments and underwent follow-up biopsies. g-DSA levels were assessed again by a follow-up biopsy at 6–12 months following the treatments. ResultsWith anti-humoral treatments, 9 out of 15 recipients comprised a g-DSA negative (-) (3.59 ± 2.82-.58 ± .25): g-DSA6-12- group, while the remaining 6 recipients comprised a g-DSA +(20.6 ± 17.0-14.9 ± 14.1): g-DSA6-12+ group. The initial g-DSA scores were significantly higher in the g-DSA6-12+ group (P = .01). All samples were diagnosed as chronic AMR in the g-DSA+ groups, whereas there were 3 chronic AMR, 4 acute AMR, and 2 incomplete AMR samples in the g-DSA- group. Interestingly, the frequency of responsible DSA belonging to class II tended to be higher in the g-DSA6-12+ group (4/6) compared to the g-DSA6-12- group (2/9) (P = .14). ConclusionThese results imply that chronic exposure to DSA causes significant and irreversible damage to the allograft. Timely and adequate anti-humoral intervention might reverse the early phase of AMR with complete clearance of g-DSA.

Graft Immunocomplex Capture Fluorescence Analysis Can Detect Intragraft Anti–Major Histocompatibility Complex Antibodies in Mice Cardiac Transplant

BackgroundImmunocomplex capture fluorescence analysis has recently been applied as a method for detection of intragraft donor-specific anti–major histocompatibility complex (MHC) antibodies (DSA) in humans. Although intragraft DSA in humans is an intense topic of investigation, there is no report to assess intragraft DSA in murine organ transplantation. MethodsA model of presensitized mouse cardiac transplantation by donor splenocytes was used. To capture mouse MHC, anti-MHC class I/II antibodies were immobilized on Luminex beads. The MHC/DSA complexes were captured by the Luminex beads followed by detection of phycoerythrin-conjugated antimouse IgG antibodies where DSA had already reacted with the allograft in vivo. ResultsLuminex beads were capable of detecting class I DSA in the cardiac allograft, though results for class II DSA were negative. Immunohistochemical investigation revealed that cardiac allografts had abundant MHC class I expression but only minor expression of MHC class II. Furthermore, MHC/class II DSA complexes were successfully detected in splenocytes and serum from a presensitized recipient. ConclusionsThese data suggested that graft immunocomplex capture fluorescence analysis can be also applied in murine cardiac transplantation. This novel application in mice would accelerate our comprehension of DSA through mechanistic studies.

Human leukocyte antigen antibody detection technologies in platelet transfusion refractoriness, with special emphasis on functional test

In the clinical settings of transfusion medicine, human leukocyte antigen (HLA) antibody detection and platelet cross-matching are important tests applied in the management of patients with platelet transfusion refractoriness (PTR). Presently, fluorescence beads technologies using extracted and purified HLA antigens are applied for the HLA antibody screening, and highly sensitive methods such as flow cytometry crossmatch (FCXM) and immunocomplex capture fluorescence analysis (ICFA), based on the use of live intact cells, are used for the cross-matching. Such highly sensitive methods have importantly contributed for the prevention of refractoriness caused by low titer antibodies but, on the other hand, they also detect those low titer antibodies or naturally occurring antibodies without or with unknown clinical relevance. Moreover, the use of recombinant antigens has been shown to be associated with specific issues. In addition, although the relationship between the antibody features and the clinical implications, such as the relevance of antibody subclass in platelet clearance, has been investigated in the field of transplantation, presently, few evidence exists in the literature related to blood transfusion. Here, we reviewed on the available HLA antibody detection methods, the issues of the present methods, and present a new method for the evaluation of clinically significant HLA antibodies using in vitro platelet phagocytosis assay.

Comparison of Sensitivity of Immunocomplex Capture Fluorescence Analysis for Detecting Donor-specific Anti-HLA Class II Antibodies in Kidney Transplant Patients

BackgroundImmunocomplex capture fluorescence analysis (ICFA) detects donor-specific antihuman leukocyte antigen (HLA) antibodies (DSA), but the detection sensitivity of HLA class II antibodies using conventional ICFA is as low as 57%. The aim of the study was to improve the detection sensitivity of HLA class II antibodies by ICFA, and compare the ICFA results with the Luminex single-antigen bead test. MethodsSix DSA-negative kidney transplant donors and recipient pairs and 10 HLA class II DSA-positive pairs were included in the study. The detection sensitivity of modified ICFA was compared with conventional ICFA, and the ICFA results were compared with the Luminex single-antigen bead test. ResultsThe index value of modified ICFA was higher than that of conventional ICFA. The cutoff value of conventional ICFA was 30,686 (MFI), which was improved to 19,405 using modified ICFA. Regarding the HLA-DQ antibody, 5 samples found to be positive by Luminex single-antigen bead testing were all negative using modified ICFA. The reason for this discrepancy could be related to: (1) the difference in detection sensitivity; (2) the difference in HLA antigen surface expression between naive lymphocytes and synthetic beads; or (3) the structure of synthetic HLA DQ antigen on the Luminex single-antigen beads. ConclusionThe index value of the modified ICFA was higher than that of conventional ICFA, and the detection sensitivity of HLA class II antibodies was improved by modified ICFA. Further assessment is necessary to clarify the reasons for divergence between ICFA and Luminex single-antigen bead test results.

P241 A study of immunocomplex capture fluorescence analysis using lymphocyte and monocyte fraction by freezing isolation

Aim Immunocomplex capture fluorescence analysis (ICFA) is a novel method to predict rejection/acceptance responses in organ transplantation. The method is less susceptible to the effect of drug compared with traditional methods. The method uses fluorescent beads to sensitively detect antibodies directed against HLA I/II antigens on blood cells.In this study, the changes in mean fluorescence intensity (MFI) control values of lymphocyte and monocyte fraction from donor cells before cryopreservation and donor cells after one month of cryopreservation were monitored using ICFA for a lymphocyte crossmatch. Methods The subjects were patients who were going to undergo living-donor kidney transplantation. ICFA method for a lymphocyte crossmatch was performed. The stability of MFI control values was monitored for donor cells before cryopreservation and those after one month of cryopreservation. Results The monitoring results showed that the MFI control value of lymphocyte and monocyte fraction from donor cells after one month of cryopreservation was 9.4% higher than that of lymphocyte and monocyte fraction from donor cells before cryopreservation. On the other hand, the MIF control value of leukocytes after one month of cryopreservation using the conventional method was 100%. Conclusions More reliable donor-specific HLA antibody test results can be obtained by lymphocyte crossmatch test using lymphocyte and monocyte fraction by freezing isolation from cryopreserved donor cells.

Influences of Pre-formed Donor-Specific Anti–Human Leukocyte Antigen Antibodies in Living-Donor Renal Transplantation: Results With Graft Immunocomplex Capture Fluorescence Analysis

BackgroundAdvances in immunosuppressants enable organ transplantation for sensitized patients. However, influences of pre-formed donor-specific anti–human leukocyte antigen (HLA) antibodies (DSA) have not been fully understood in renal transplantation (RT). On the other hand, immunocomplex capture fluorescence analysis (ICFA) is a reliable method to detect donor-specific anti-HLA antibodies and HLA antigen complexes. Graft ICFA can detect DSA in an allograft (g-DSA). MethodsTo elucidate the consequences of pre-formed DSA, 198 patients who underwent living-donor RT were enrolled for this study (observation period: 57.8 ± 34.9 months); 187 patients in the DSA− group (excluding ABO-incompatible cases) and 11 patients in the DSA+ group. Before RT, all DSA+ patients had undergone rituximab administration and plasmapheresis. For a graft ICFA, the biopsy specimen (1 × 105 cells) was dissolved, and HLA antigens were captured by anti-HLA beads. Finally, DSA-HLA complexes were detected by means of PE-conjugated anti-human IgG antibodies and analyzed by use of a Luminex system. A ratio (sample/blank beads, mean of fluorescence intensity) was calculated: ≥1.0 was determined as positive g-DSA. ResultsThere were no significant differences in 5-year graft survival (87.9%/100% in the DSA−/DSA+ groups, respectively). In terms of antibody-mediated rejection (AMR), within 1 month after RT, pathologically determined AMR occurred 3.2% and 63.4% in the DSA− and DSA+ groups, respectively (P < .0001). However, interestingly, more than half of them (57.1%) indicated only subclinical AMR, that is, no fluctuation of S-Cr. As representative of 2 cases of subclinical AMR, g-DSA deposition could be confirmed (1.15 ± 0.04) at 1 hour after reperfusion by graft ICFA. Furthermore, g-DSA shifted to 2.20 ± 0.98 at 3 weeks after transplantation, along with a decline in s-DSA mean of fluorescence intensity (1718-506.5). ConclusionsAlthough pathologically determined AMR occurred more frequently in pre-formed DSA+ recipients, it can be argued that a successful de-sensitization protocol inhibits further production of DSA and graft destruction.

Males without apparent alloimmunization could have HLA antibodies that recognize target HLA specificities expressed on cells.

Human leukocyte antigen (HLA) antibodies, which are involved in the development of transfusion-related side effects such as transfusion-related lung injury, are sometimes found in males without a history of alloimmunization (eg, transplantation and transfusion). Whether HLA antibodies in male donors can interact with their target HLA specificities expressed on cells have not been completely investigated. The HLA antibodies detected in 7 male donors were characterized. Flow cytometry and immunocomplex capture fluorescence analysis were performed to evaluate the ability of these antibodies to bind with target HLA specificities expressed on cells. The association of these antibodies with complement was examined using anti-C1q antibody. Sustainability of HLA antibodies over time was compared in 26 male vs 57 female donors. The antibodies from all 7 donors recognized intact HLA molecules coated onto microbeads. The antibodies in 2 of 7 donors also recognized their target HLA specificities expressed on cells. Furthermore, the antibodies in one of these 2 donors showed HLA specificities that involved complement binding. Twenty-one of 26 initially positive male donors had turned negative for HLA antibody at least 1 year after their initial positive screening, whereas HLA antibody positivity was maintained for a long time in most female donors. Males without apparent alloimmunization could have HLA antibodies that recognize their target HLA specificities on cells and that could potentially modify molecular events in affected cells.

Graft Immunocomplex Capture Fluorescence Analysis to Detect Donor-Specific Antibodies and HLA Antigen Complexes in the Allograft

ABSTRACTBackground: Immunocomplex capture fluorescence analysis (ICFA) is an attractive method to detect donor-specific anti-HLA antibodies (DSA) and HLA antigen complexes. Currently, antibody-mediated rejection (AMR) due to DSA is usually diagnosed by C4d deposition and serological DSA detection. Conversely, there is a discrepancy between these findings frequently. Thereupon, our graft ICFA technique may contribute to establish the diagnosis of AMR.Methods: Graft samples were obtained by a percutaneous needle biopsy. Then, the specimen was dissolved in PBS by the lysis buffer. Subsequently, HLA antigens were captured by anti-HLA beads. Then, DSA–HLA complexes were detected by PE-conjugated anti-human IgG antibodies, where DSA had already reacted with the allograft in vivo, analyzed by a Luminex system.Results: A ratio (sample MFI/blank beads MFI) was calculated: ≥ 1.0 was determined as positive. We found that DSA–HLA complexes in the graft were successfully detected from only slight positive 1.03 to 79.27 in a chronic active AMR patient by graft ICFA. Next, positive graft ICFA had predicted the early phase of AMR (MFI ratio: 1.38) even in patients with no serum DSA. Finally, appropriate therapies for AMR deleted DSA deposition (MFI ratio from 0.3 to 0.7) from allografts.Conclusions: This novel application would detect early phase or incomplete pathological cases of AMR, which could lead to a correct diagnosis and initiation of appropriate therapies. Moreover, graft ICFA might address a variety of long-standing questions in terms of DSA.Abbreviations: AMR: Antibody-mediated rejection; DSA: Donor-specific antibodies; ICFA: Immunocomplex capture fluorescence analysis.

Excellent Results of Immunocomplex Capture Fluorescence Analysis-I for Cross-Match Test in Renal Transplantation

A flow cytometry cross-match (FCXM) test is the gold standard for detection of human leukocyte antigen (HLA) antibodies in renal transplantation because of its high sensitivity. However, this technique can produce false-positive results when non-HLA antibodies or low-titer donor-specific antibodies (DSA) are detected. To determine the clinical relevance of the recently introduced novel cross-match test termed immunocomplex capture fluorescence analysis (ICFA), we retrospectively compared the results of ICFA and FCXM, including a single-antigen bead test for detection of DSA in renal transplant recipients. We found a correlation of 71.4% (235/329) between the results of ICFA-I and FCXM-T, whereas that between ICFA-II and FCXM-B was 41.1% (134/326). Ninety-four patients were ICFA-I negative and FCXM-T positive, and 188 were ICFA-II negative and FCXM-B positive, whereas 46.8% (44/94) and 61.7% (116/188) were found to be DSA-I and DSA-II negative, respectively, which classified them into the non-HLA antibody and low-titer DSA groups, respectively. The mean value of molecules of equivalent soluble fluorochrome for DSA-I was 22,994 in the ICFA-I–positive group, which was significantly higher than 2117 in the negative group (P < .0001), whereas there was no significant difference for DSA-II between the ICFA-II–positive and ICFA-II–negative groups. Graft survival in the ICFA-I–negative group was significantly higher than that in the ICFA-I–positive group (P = .0058). Our results indicate that ICFA-I does not respond to non-HLA antibodies or low-titer DSA, which have influence on graft survival. Therefore, this novel hybrid test, which combines cross-match testing and HLA antibody detection functions, may be useful for clinical pretransplantation evaluation of renal transplantation patients.

リンパ球クロスマッチにおける immunocomplex capture fluorescence analysis（ICFA） の有用性についての検討

リンパ球クロスマッチにおける immunocomplex capture fluorescence analysis（ICFA） の有用性についての検討

非血縁者間臍帯血移植におけるレシピエントHLA抗体と生着の関連性について ; HLAミスマッチ移植におけるICFA法を用いた交差適合試験の有用性

2006年12月までに東京都赤十字血液センター臍帯血バンクを介して移植が行われた患者391例についてHLAクラスI, クラスII抗体の検査を行い, 臍帯血移植における患者(レシピエント)HLA抗体と生着の関連性について解析した. 患者HLA抗体の特異性が臍帯血HLA抗原に反応しない場合, 抗体の有無による生着率の差は認められなかったが, 患者HLA抗体の特異性が臍帯血のHLA不一致抗原に反応する場合, 生着を認めたのは8例中4例で, 生着不全が多いことが確認された. この8例についてimmunocomplex capture fluorescence analysis(ICFA)法にて確認検査を行ったところ, 4例で陽性となり生着不全の結果と一致した. HLA不一致抗原を有する臍帯血移植前には患者のHLA抗体を高感度抗体検査法で検査し, 臍帯血のHLA不一致抗原が抗体と反応しないように選択することにより生着不全の危険性を回避することが期待できる. また, ICFA法はHLA不一致抗原を有する臍帯血移植における確認検査法として有用と考えられた.

Application of bead array technology to simultaneous detection of human leucocyte antigen and human platelet antigen antibodies

Detection of antibodies against human leucocyte antigens (HLA) and human platelet antigens (HPA) is crucial for patients refractory to platelet transfusion therapy. However, a reliable and high-throughput method for HLA cross-matching and detecting HPA antibodies has not yet been described. Immunocomplex capture fluorescence analysis (ICFA) was developed for high-throughput, simultaneous detection of HLA and HPA antibodies. Microarray beads were separately coupled with monoclonal antibodies specific for CD36, CD41, CD42b, CD49b, CD61 and HLA class I antigens. Platelets reacting with patient serum were lysed and the lysates were incubated with the bead mixture to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins or HLA antigens. The beads capturing immunocomplexes were then subjected to flow cytometric analysis. Immunocomplex capture fluorescence analysis was validated using 50 serum samples containing HLA antibodies and 20 serum samples containing HPA antibodies. The method enabled the detection of all the HLA antibodies with a sensitivity comparable to that of the purified HLA antigen-coated pooled-bead assay (FlowPRA, One Lambda, Canoga Park, CA, USA). The method also enabled the detection of all the HPA antibodies with a sensitivity higher than that of the mixed passive haemagglutination. In this study, we developed a rapid, simple and reliable method for the simultaneous analysis of HLA and HPA antibodies. ICFA can also be used as an alternative to the lymphocyte cytotoxicity test for HLA cross-matching.