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Schematic diagram of the Flow Cytometry crossmatch (FACS- CM). In contrast to the CDC-CM with a complement mediated vital-or lethal staining of lymphocytes, the FACS-CM is based upon an indirect immunostaining using fluorescence dye-labelled secondary antibodies. In contrast to the FACS-CM of only HLA-class I bearing T-cells (A) the outcome of the B-cell crossmatch (B) may be falsified by irrelevant immune complexes (C) which through their Fc-fragments directly bind to Fc-receptors (black horseshoes) of B-cells.
Source publication
The detection of donor-specific anti-HLA antibodies by standard procedures such as complement-dependent cytotoxicity assay (CDC) or flow cytometric (FACS) analysis is limited by its low sensitivity and the quality of the donor cells. Therefore, an ELISA-based technique was employed using solid phase-immobilized monoclonal antibodies to capture HLA...
Context in source publication
Context 1
... comparison to the conventional CDC-CM the 1117 FACS-CM procedure has a higher sensitivity which is in the range of that of the AHG-enhanced CM ( Scornik et al., 1997). This procedure is not based on vital or lethal staining as demonstrated for the CDC-CM but on an indirect immune staining procedure using secondary fluorescence-labelled antibodies (Fig. 3). Therefore, it allows the detection of both low complement-activating, but also of complement-independent anti-donor antibodies. The outcome of this assay may be influenced by irrelevant antigen-antibody complexes in the recipient's serum through binding of Fc-fragments to the Fc-receptors. These are expressed at high quantities on the ...
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Citations
... doi: bioRxiv preprint 8100E microscope. Crossmatch scores were determined by using values 1 (≤ 10%), 2 (10-20%), 3 (20-40%), 6 (40-80%) and 8 (80-100%) for increasing frequencies of dead cells according to standard protocols of the National Institute of Health (USA) (11). ...
In the present study, we developed a novel cell therapy approach to selectively combat antibody-mediated rejection (AMR), a major and unresolved complication after solid organ transplantation (SOT) caused by donor-HLA-specific, alloreactive B cells. Current treatment options including B-cell depletion protocols are inefficient and result in complete loss of humoral immunity. To selectively eliminate alloreactive B cells characterized by corresponding anti-donor-HLA B-cell receptors (BCRs), we engineered T cells with a novel chimeric receptor comprising a truncated HLA molecule fused to intracellular 4-1BB/CD3ksi signaling domains to generate T cells overcoming rejection by antibodies (CORA-Ts). As proof-of-concept, CORA receptors based on HLA-A*02 were shown to bind anti-HLA-A*02 antibodies from the serum of kidney transplant recipients, indicating their suitability to also target the respective membrane-bound anti-HLA-A*02 BCRs on alloreactive B cells. In co-cultures with B-cell lines expressing and releasing anti-HLA-A*02 antibodies, CORA-Ts were specifically activated, released pro-inflammatory cytokines (e.g. IFN-gamma, granzyme B), and exhibited strong cytotoxicity resulting in an effective reduction of anti-HLA-A*02 antibody release. A modification of the HLA-A*02 alpha3-domain within the CORA receptor effectively abrogated T-cell sensitization. Additionally, using CRISPR/Cas9-mediated knockout of a selected binding protein, CORA-Ts were able to resist immunosuppressive treatment to ensure high efficiency in transplant patients. Our results demonstrate that CORA-Ts are able to specifically recognize and eliminate alloreactive B cells, and thus selectively prevent formation of anti-HLA antibodies even under immunosuppressive conditions. This suggests CORA-Ts as potent novel approach to specifically combat AMR and improve long-term graft survival in SOT patients while preserving their overall B-cell immunity.
... Donor-recipient monkey selection. Anti-donor autoantibody for selection of transplantation pairs was analyzed via flow cytometry [35][36][37][38][39][40] . Briefly, isolated donor PBMCs were incubated with recipient sera for 30 min at 4 °C than washed with FACS buffer (BD, San Jose, CA, USA). ...
Many groups are working to improve the results of clinical allogeneic islet transplantation in a primate model. However, few studies have focused on the optimal islet dose for achieving normal glycemia without exogenous insulin after transplantation in primate models or on the relationship between rejection and islet amyloid polypeptide (IAPP) expression. We evaluated the dose (10,000, 20,000, and > 25,000 islet equivalents (IEQ)/kg) needed to achieve normal glycemia without exogenous insulin after transplantation using eleven cynomolgus monkeys, and we analyzed the characteristics exhibited in the islets after transplantation. 10,000 IEQ/kg (N = 2) failed to control blood glucose level, despite injection with the highest dose of exogenous insulin, and 20,000 IEQ/kg group (N = 5) achieved unstable control, with a high insulin requirement. However, 25,000 IEQ/kg (N = 4) achieved normal glycemia without exogenous insulin and maintained it for more than 60 days. Immunohistochemistry results from staining islets found in liver biopsies indicated that as the number of transplanted islets decreased, the amount of IAPP accumulation within the islets increased, which accelerated CD3+ T cell infiltration. In conclusion, the optimal transplantation dose for achieving a normal glycemia without exogenous insulin in our cynomolgus monkey model was > 25,000 IEQ/kg, and the accumulation of IAPP early after transplantation, which depends on the transplanted islet dose, can be considered one factor in rejection.
... As donor lymphocytes are directly incubated with the serum of the selected recipients, the test is termed de facto (physical) crossmatching. Crossmatching is performed using CDC-based assays as well as other assays (flow cytometry or solid phase-based) in which whole donor cells or HLA-molecules extracted from the donors are incubated with the recipients' sera to detect a traceable signal or reaction [4][5][6]. Besides de facto crossmatching, there is a theoretical approach called virtual crossmatching. ...
... The first assays performed using this technique consisted of cell trays combining panels of living lymphocytes to determine the specificities of merely cytotoxic anti-HLA antibodies. Today, native or recombinant HLA antigens, after their isolation and purification, are adhered to small plastic carrier particles used as solid matrices, and then assessed by the Luminex platform to detect the carrier bead and fluorescence dye-labeled secondary antibodies bound to the primary anti-HLA antibodies [5]. In 1998, it was first suggested to abstain from de facto CDC-based crossmatching for non-immunized patients [10]. ...
The specification of anti-human leukocyte antigen (HLA) antibodies is an important task for patients awaiting kidney allografts. Especially the patients immunized in previous transplantations, transfusions, or pregnancies must be carefully observed, since grafting patients with HLA antigens/phenotypes recognized by their pre-formed antibodies are the main cause of harmful hyperacute and acute rejection. The complement-dependent lymphocytotoxicity-based de facto (physical) crossmatching (CDC-CM) has thus been implemented as the last diagnostic obstacle before kidney allografting. Here, an assay is performed by incubating the donors’ lymphocytes with the sera of the prospective recipients, and a negative outcome was desired for eligibility of the underlying organ allocation. Furthermore, valid antibody specification has to be performed at least quarterly for each patient on the kidney waiting list, as defined by certain guidelines, for example, the Eurotransplant guidelines. Based on the exclusion of these specificities, also referred to as virtual crossmatching, certain donors are a priori listed as unacceptable for these recipients. In this case report, we showed that defining unacceptable antigens may be difficult if the recipients’ antibodies are allele-specific after being generated in the patient who is expressing the HLA-class II antigen DQ6 and also developing antibodies against this antigen. Low resolution (two-digit) typing is used before kidney allografting. Thus, these antibodies are generally not definable, as donors and recipients share the same antigen (allelic group). Here, we demonstrate the diagnostic approaches required to exclude inadequate kidney donors for a patient exhibiting antibodies only against the HLA-DQB1*06:04 allelic variant and not against the common phenotype HLA-DQ6. In practice, the patient’s HLA-class II high resolution (four-digit) typing, as well as his antibody specification at the highest (single antigen) resolution, are included. Furthermore, we critically discuss, according to the Eurotransplant guidelines, the missing possibility to declare own HLA-antigens unacceptable, which may be very helpful for recipients who exhibit allele-specific antibodies.
... All cases were crossmatched using the complement-dependent cytotoxicity (CDC) method, and results were interpreted based on the standard complement-dependent cytotoxicity-crossmatch (CDC-CM) assay scoring system. 7 This system utilizes semi-quantitative analysis of the reaction, with a result of 40% and above interpreted as a firm positive. In our centers, however, the decision to proceed to transplantation has been based on whether the crossmatch testing result does not exceed 30%. ...
... b Chronic rejection: progressive dysfunction of kidney, characterized by gradual increase of creatinine serum level in the absent of other causes such as dehydration, infection, or drug intoxication (only for the purpose of our study). c DGF was defined as the presence of needs for dialysis within the first seven days after kidney transplantation.6,7 d Graft survival (non-censored for death) was calculated from the date of transplantation up to the time when graft failure developed, which was characterized by a return to long-term dialysis, re-transplantation or the last follow-up when the transplant was still functioning or up to the date of death. ...
Background
Pediatric kidney transplantation was only introduced in Indonesia in 2013. We therefore aimed to assess the characteristics and outcomes of transplants performed from its inception to January 2019.
Method
The study had a dual‐center retrospective design. We examined the records of kidney transplant recipients and then calculated patient and graft survival rates by Kaplan‐Meier survival analysis with 95% confidence intervals (95% CI).
Results
In total, 12 kidney transplantations were performed in eleven children during the study period; among these, ten were boys, and nine had renal failure caused by congenital anomaly of the kidney or urinary tract. All donors were living, and all recipients were on dialysis at the time of transplantation, when their median age was 14.5 years (range, 8‐19 years). Three patients died of infection in the first year of follow‐up and two lost their allograft by the time of their last follow‐up (median, 13 months; range, 4‐69 months). The 1‐year patient survival rate was therefore 68.18% (95% CI, 29.72%‐88.61%), which remained unchanged at 3 and 5 years. However, the non‐death‐censored graft survival rates at 1, 3, and 5 years were 68.18% (95% CI, 29.72%‐88.61%), 51.14% (95% CI, 14.5%‐79.46%), and 25.57% (95% CI, 1.38%‐64.78%), respectively.
Conclusion
Patient and graft survival rates after pediatric kidney transplantation in Indonesia are lower than those reported in other countries. Closer patient follow‐up and stricter adherence to guidelines could improve transplant outcomes, but we must seek to improve the balance between infection and rejection.
... Even today, a negative crossmatch outcome in such a test is regarded as the best predictor for shortterm survival of renal allografts. The standard technique established originally was the complement-dependent lymphocytotoxicity (CDC) assay and the work-flow of this assay has been described in detail previously (Altermann et al., 2006). Using this functional vitality assay, only those DSA were detectable that exert their detrimental function by their complement-activating features, ultimately leading to the lysis of donor lymphocytes. ...
... The second drawback of this assay is its low sensitivity leading to its general inability to detect low concentrations of DSA. Consequently, the CDC was modified by introducing secondary anti-human immunoglobulin (Ig) antibodies, which recognize the primary DSA, thereby supplementing this assay's technical design in order to enhance its complementactivating potency (Gebel and Bray, 2000;Karpinski et al., 2001;Altermann et al., 2006). The procedure was termed anti-human globulin-(AHG-) enhanced CDC-XM. ...
... In order to circumvent some of the CDC-XMspecific problems the flow-cytometric crossmatch (FACS-XM) was established, which allowed the detection of complement-activating as well as complement-independent DSA (Lobo et al., 1981;Garovoy et al., 1983;Scornik et al., 1994Scornik et al., , 1997Bittencourt et al., 1998;Christiaans et al., 1998;Karpinski et al., 2001;Altermann et al., 2006). Its sensitivity is in the range of the AHG-enhanced CDCcrossmatch. ...
Transplant recipients who have undergone sensitizing events, such as pregnancy, blood transfusion or previous transplants, frequently develop antibodies directed against the highly polymorphous human leukocyte antigen (HLA)-molecules. These pre-formed, donor-specific antibodies (DSA) present a high risk of causing organ failure or even complete loss of the grafted organ as a consequence of antibody-mediated, hyper-acute or acute allograft rejection. In order to detect DSA, the so-called functional complement-dependent lymphocytotoxicity assay (CDC-XM) was established about 50 years ago. Although effective in improving the outcome of solid organ allo-grafting, for the last ten years this assay has been controversially discussed due to its low sensitivity and especially because of its high susceptibility to various artificial factors, which generally do not yield reliable results. As a consequence, novel immunochemical test systems have been developed using ELISA- or bead-based solid phase assays as replacements for the traditional CDC-based assays. Because these assays are independent of single or vital cells, which are frequently not available, they have provided an additional and alternative diagnostic approach compared with the traditional CDC-based and flow-cytometric analyses. Unfortunately, however, the AMS-ELISA (Antibody Monitoring System), which was the first system to become commercially available, was recently discontinued by the manufacturer after seven years of successful use. Alternative procedures, such as the AbCross-ELISA, had to be either considerably modified, or did not yield reliable results, as in the case of the Luminex-based assay termed DSA. We draw the conclusion that due to the unique features and fields of application reviewed here, the implementation of solid phase cross-matching still represents an urgent requirement for any HLA-laboratory's routine tasks.
... For patients with circulating HLA antibodies, a pretransplant cross-match is usually performed by mixing serum from the patient with lymphocytes derived from the potential donor (Altermann, Seliger, Sel, Wendt, & Schlaf, 2006). Progression to transplant, or the determination of transplant risk and choice of antirejection therapy, is therefore based on the measurement of the circulating HLA antibody using bead-based assays on a Luminex platform, and the binding of antibody with HLA antigens on the surface of donor T and B cells. ...
The detection and semiquantitative measurement of circulating human leucocyte antigen (HLA)‐specific antibodies is essential for the management of patients before and after transplantation. In addition, the pretransplant cross‐match to assess the reactivity of recipient HLA antibody against donor lymphocytes has long been the gold standard to prevent hyperacute rejection. Whilst both of these tests assume that recipient HLA‐specific antibody is the only variable in the assessment of transplant risk, this is not the case. Transplant immunologists recognize that some HLA antigens are expressed at levels a magnitude lower than others (e.g., HLA‐C, HLA‐DQ), but within loci, and between different cell types there are many factors that influence HLA expression in both resting and activated cells. HLA is not usually expressed without the specific promoter proteins NLRC5, for HLA class I, and CIITA, for class II. The quantity of HLA protein production is then affected by factors including promoter region polymorphisms, alternative exon splice sites, methylation and microRNA‐directed degradation. Different loci are influenced by multiple combinations of these control mechanisms making prediction of HLA regulation difficult, but an ability to measure the cellular expression of each HLA antigen, in conjunction with knowledge of circulating HLA‐specific antibody, would lead to a more informed algorithm to assess transplant risk.
... This assay allowed both the detection of complement-independent and of complement-activating i.e. cytotoxic DSA [6][7][8]. A general drawback of this assay, the sensitivity of which is in the range of the AHGenhanced CDC-based crossmatch, however, is its artefact-influenced outcome through the "irrelevant/unspecific" binding of antibodies through their Fc-parts to the Fc-receptors expressed on B-lymphocytes which all over the years represented rather a common than a rare event [9,10]. The proposal to perform FACS-based B-cell cross-matching only after pre-incubation of these donor cells with heat-denatured rabbit serum has been the first reliable approach to overcome this methodical drawback [11]. ...
... Due to this methodical aspect i.e. in order to act independently of the availability of separated vital cells or their insufficient quality, additional crossmatch assays were established using the design of solid phase-based assays (SPA). Two diagnostic systems, both representing enzyme-linked immunosorbent assays (ELISA) were implemented in our tissue typing laboratory for various groups of patients suffering from artificially influenced CDCcrossmatch outcomes [9,[14][15][16][17][18] or characterized by the general lack of single donors' lymphocytes [19]. Both assays, however, the pros and cons of which will afterwards be discussed, were discontinued by their manufacturers for mere commercial reasons in the years 2013 and 2016, respectively. ...
... Furthermore, there are several studies which point onto the detrimental effects of HLA-specific alloantibodies of the IgM isotype and thus clearly advise to detect and not to destroy these antibodies [26,27]. Unfortunately these IgM alloantibodies as well as so-called weak (low titer) IgG alloantibodies are eliminated using DTE/DTT although they may easily be detectable using solid phase crossmatch techniques modified with secondary anti-IgG/M antibodies [9,16]. These arguments have for years challenged the general diagnostic approach to use reducing agents in order to specify anti-HLA alloantibodies, and it has to be concluded that reducing agents are not at all applicable in order to selectively eliminate autoantibodies. ...
Antibodies directed against HLA antigens of a given donor represent the most prominent cause for hyper-acute and acute rejections. In order to select recipients without donor-specific antibodies the complement-dependent cytotoxicity (CDC-) crossmatch was first established representing the standard procedure up to the present. Its negative pre-transplant outcome is currently regarded as the most important requirement for a successful short term kidney graft survival. As a functional assay, however, it strongly depends on the availability of isolated donor lymphocytes and in particular on their vitality. Moreover, during the last ten years several disadvantages of the CDCbased
procedure have increasingly been discussed with respect to this assay’s high susceptibility to disruptive factors which frequently lead to false positive outcomes. In this context several autoimmune diseases especially of the immune complex type (type III) or pharmacological treatment of a given recipient have been shown to lead to
unexpected “false-positive” outcomes of the CDC-crossmatch. As methodical alternatives for anti-HLA antibody specific cross-matching two ELISA-based procedures i) the Antibody Monitoring System (AMS-) ELISA and ii) the AbCross-ELISA were established in our tissue typing laboratory and those of some other groups. Both systems, however, were discontinued for mere commercial reasons in the years 2013 and 2016, respectively. Using the same set of diagnostic antibodies, the AMS-ELISA, now named Donor-Specific Antibodies/DSA, was afterwards again manufactured as a microbead-based array using the Luminex platform. With a view to establish the DSA-assay as the only remaining solid phase-based crossmatch system commercially available, this procedure was systematically evaluated in our laboratory. Primarily but not exclusively based on drawbacks of the evaluation software, however, 69 (32.5%) of the virtually defined crossmatch results (n=212 independent anti-HLA class I and II specifications and
their corresponding DSA-assays, respectively) were classified as divergent using the DSA-assay whereas only 143 results (67.5%) were classified as accordant by this assay’s software. Referring to the chosen cohort of recipients (n=106) not less than 62 (58.4%) of them are characterized by findings which are not supported by virtual crossmatching.
We here provide evidence that for various reasons the outcomes provided by the DSA-assay, in contrast to those of the AMS-ELISA as its precursor system, have critically to be challenged. We therefore conclude that modifications are urgently required to be introduced by the manufacturer in order to lead again to a system of sufficient validity usable for any laboratory's Routine diagnostice
... Later, anti-human immunoglobulin (AHG) was added to the assay (AHG-CDC) to increase the sensitivity and to detect non-complement fixing antibodies [12]. Although this assay has been widely adopted over years, it has some limitations including low sensitivity, detecting autoreactive antibodies and non-HLA antibodies as false positive, and failure to distinguish complement-binding (CB) and non-complement-binding (NCB) antibodies [13][14][15]. Flow cytometry-based cross matching (FCXM) introduced 30 years ago has increased sensitivity over CDC method and identifies specific cell types as DSA targets [16][17][18][19]. However, this approach requires access to viable donor lymphocytes, may detect autoreactive or non-HLA antibodies, and fails to detect extremely low DSA levels [20]. ...
Purpose of Review
Despite advanced immunosuppression, donor-specific antibodies (DSA) remain the leading cause of acute and chronic transplant tissue injury. Comprehensive evaluation of anti-donor humoral immune responses is critical for successful prevention, diagnosis, and treatment of antibody-mediated rejection. This review summarizes the evolution of techniques used for this purpose in experimental and clinical transplantation.
Recent Findings
For decades, measuring DSA serum levels was the only way to assess recipient humoral immunity. Recently, the interest shifted from quantifying circulating DSA to the analyses of various B cell subsets and most importantly, of donor antigen-specific B cells. State-of-the-art approaches have been developed by studies of model antigens, infectious agents, and autoimmunity. These methods are now being adopted by the transplantation field.
Summary
The complexity of humoral immunity caused by organ transplantation necessitates complementary approaches assessing both DSA and various B cell subsets to successfully target antibody-mediated rejection.
... CDC detects DSA directed against HLA molecules. [4] It is performed by incubating T-and B-lymphocytes of the donor with serum from the recipient in a multiwell plate with subsequent addition of complement. Complement is activated when recipient antibodies bind to the donor cells. ...
The significance of pretransplant anti-human leukocyte antigen antibody levels that are detectable by more sensitive platforms (including the Luminex platform) yet undetected by complement-dependent cytotoxicity (CDC) assay remains unclear. The aim of this study was to determine the clinical significance of the donor-specific antibody (DSA) assay Luminex crossmatch and its impact on short-term renal graft outcome such as acute rejections, graft survival, and graft function. The results of pretransplant DSA-lymphocyte crossmatching (LCXM) assay in 126 renal allograft recipients whose CDCs crossmatches were negative were retrospectively analyzed for correlation with posttransplant outcomes. Of the 126 recipients, 32 (25.4%) had pretransplant DSA positive. Statistically significant association was found between DSA-LCXM positivity with 14th day estimated glomerular filtration rate (eGFR) (P = 0.05), DSA Class I with 3rd (P = 0.014) and 6th month (P = 0.02) eGFR, DSA Class II with 14th day (P = 0.06) and 1st month (P = 0.10) eGFR, mean fluorescent intensity (MFI) DSA with 7th day (P = 0.08) and 14th day (P = 0.09) eGFR, and maximum MFI DSA with 7th day eGFR (P = 0.09). The posttransplant eGFR was higher at various time intervals in DSA-LCXM-negative patients as compared to DSA-positive patients. However, pretransplant DSA-LCXM results did not predict the rejection episodes, graft loss, and 1-year posttransplant 24 h urine protein. Pretransplant DSA detected by LCXM in patients with a negative CDC does not predict adverse short-term outcomes. However, the difference in posttransplant eGFR supports further investigation in long-term effects.
... NIH-CDC is being widely used in developing countries and has the capacity of detecting the complement fixing antibodies; however, it has relatively low sensitivity and it is subjective. [3] On the other hand, FCXM is far more sensitive for donor B and T cells, but its specificity is limited by its inability to differentiate HLA from non-HLA specific; and complement from noncomplement fixing antibodies. [4] This may lead to a patient being unduly denied/delayed a transplant. ...
... NIH-CDC is a standard method, however; there is always an associated risk of primary and memory alloantibody response in nonsensitized and presensitized recipient of transplant, respectively. [3] FCXM assay is extremely sensitive however there may be cases where the results for B-cells are affected by high background due to nonspecific binding of IgG to Fc receptors. It can be overcome by the use of proteolytic enzyme (pronase) making the assay more reliable, sensitive, and specific; [24] however, in this study, the overall results were unaffected with both pronase treated and untreated cells. ...
Background and Objectives: Various methods have been reported for the detection of antibodies in recipient sera, which can be human leukocyte antigens (HLAs) or non-HLA specific, complement- or noncomplement fixing, as well as donor T (HLA-Class-I) and/or B cell (HLA-Class-I and II) specific. These alloantibodies play a pivotal role in antibody-mediated renal transplantation rejection. Deposition of C4d in peritubular capillaries of a kidney biopsy is a marker of antibody-mediated rejection. The C4d flow-panel reactive antibodies (PRAs) are a screening method for HLA-specific and complement fixing antibodies. However, the method is limited by the lack of donor specificity.
Design and Settings: Here, we present a new and simple flow cytometric method referred to as C4d-flow cytometry crossmatch (C4d-FCXM) for the detection of donor-specific (T and/or B cell) and C4d-fixing alloantibodies.
Results: The method was applied in a series of clinical cases and judged to be useful. The method may limit unwanted deferral of the donor due to positivity in C4d Flow-PRA and/or FCXM and may be helpful in prediction of antibody mediated rejections. Furthermore, this method can provide information pretransplant in contrast to kidney biopsy and C4d evaluation done posttransplant.
Conclusions: We postulate that this method incorporates most of the features of all the available modalities (i.e., National Institute of Health-complement dependent lymphocytotoxicity, FCXM, cytotoxic FCXM and C4d-flowPRA) yet cost-effective and best suited for resource-limited laboratory/ies which is a common scenario in developing countries.
