Flow Cytometric Crossmatch Tests Research Articles

OR52 Ides desensitization therapy: Breaking down antibody barriers to transplantation

Despite increased transplant rates for highly sensitized candidates, inequities for those with CPRA >99.7% remain. This case highlights the efficacy of an endopeptidase (IdeS) to cleave IgG HLA-specific antibodies and complications for donor-specific HLA antibody (DSA) monitoring in the presence of therapeutic agents. DSA was evaluated using flow cytometric crossmatch tests (Flow-XM) and Luminex immunoassays (Lifecodes classes I and II single antigen; Immucor-Lifecodes, Stamford, CT and Single Antigen Beads; One Lambda, Canoga Park, CA). Sera were pretreated using Melon Columns™. A 43 year old male with a CPRA of 99.99% was entered into a clinical trial investigating the efficacy of IdeS to cleave IgG molecules and render them ineffective in eliciting complement activation or antibody-dependent cellular cytotoxicity. A deceased donor was identified to whom the patient had HLA-DP DSA yielding a positive prospective Flow-XM. Within 2 h of IdeS administration, immunoassays and a Flow-XM revealed no detectable DSA. Due to concern for DSA rebound, alternate day DSA monitoring and intense post-transplant immunosuppression were employed. Two single antigen panels confirmed DSA reappearance on POD7 prior to high dose immunoglobulin (IVIG) dosing. Administration of IVIG produced an increase in the negative and positive control beads and reactivity with self HLA-antigens (3400 mean fluorescent intensity, MFI). Utilization of multiple bead assays, Flow-XM tests, and HLA pattern analysis rather than MFI was needed to monitor further increases in DSA that would prompt therapeutic plasmapheresis (TPE). Cumulative DSA test analysis revealed a stabilization of DSA accompanied by declining serum creatinine values with no initiation of TPE. New therapeutics hold promise for removing DSA barriers; however, post transplant monitoring can be challenging due to test interference that accompany these biologics. This case illustrates the use of multiple assays and HLA pattern analysis to guide post transplant care.Download : Download high-res image (270KB)Download : Download full-size image A.M. Jackson: 3. Speaker’s Bureau; Company/Organization; Thermo Fisher Scientific.

HLA donor-specific antibodies in allogeneic hematopoietic stem cell transplantation: challenges and opportunities.

Allogenic hematopoietic stem cell recipients may have preformed antibodies directed against foreign HLA antigens. The use of partially HLA-mismatched allogeneic hematopoietic stem cell donors allows for the possibility of the presence of circulating HLA donor-specific antibodies (DSAs) in the recipient. The presence of DSAs at the time of stem cell infusion increases the risk of primary graft failure. More recently developed technology using solid phase immunoassays (SPIs) with fluorochrome-conjugated beads has greatly improved the ability to detect and classify DSAs. When used in combination with the classic lymphocytotoxic complement-dependent and flow cytometric crossmatch tests, SPIs help provide DSA strength assessment. Parous females frequently harbor DSAs. DSAs tend to be of higher intensity when directed against haploidentical first-degree relatives. DSA assessment requires frequent monitoring as their relative strength can change over time. Although the criteria that constitutes a prohibitive DSA is unknown, desensitization techniques can result in engraftment rates as experienced in fully HLA-matched allogeneic blood or marrow transplantation recipients.

P156 An unexpected discovery complicates an otherwise uneventful HLA identical hemapoetic stem cell transplant

Oncology patients frequently require platelet transfusions during chemotherapy regimens. This case involves a 46 year old woman diagnosed with myelodysplastic syndrome, who developed HLA antibodies following platelet transfusions. The patient was then evaluated for a Hematopoietic Stem Cell Transplant (HSCT) with her sister. HLA typing was performed using rSSO, (One Lambda, Inc., Canoga Park, CA) and SBT with BigDye V1.1 Terminator reagents on a 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA). HLA-specific antibody was assessed using phenotypes, LMID, (Lifecodes®, Immucor, Norcross, GA) and single antigen panels (One Lambda Inc., Canoga Park, CA). Flow cytometric crossmatch tests were performed on a BD FACSCanto II. Intermediate typing failed to identify the patient’s A-Locus antigen using peripheral blood and buccal samples. SBT typing revealed a novel HLA-A∗02 allele that differed from the HLA-A∗02:01 present in her otherwise HLA identical sibling. Sequence alignments showed an apparent HLA-A∗02:01/A∗03:XX gene conversion event involving exon 3. HLA antibody assessments of the patient’s serum showed low level sensitization toward HLA-A2, A9, A28, A25, A26 and A66. A negative flow cytometric crossmatch confirmed the low strength of the HLA-A2 antibody. The urgency for transplant and low likelihood of finding a donor for which the patient was not sensitized (CPRA = 77) precipitated her placement into our desensitization HSCT protocol. A non-myeloablative, HLA-A mismatched HSCT with her sibling occurred following desensitization using plasmapheresis and low dose IvIg. Identification of HLA antibodies is crucial for HSCT programs that support HLA mismatched transplantation. New or rare alleles can pose additional challenges in HSCT with regards to matching with potential donors and avoiding sensitization to common HLA antigens found in blood products.

Variable HLA expression on deceased donor lymphocytes: Not all crossmatches are created equal

Flow cytometric crossmatch tests are used to detect donor-specific antibody and determine eligibility for transplantation. Crossmatch sensitivity is dependent on lymphocyte quality, to include HLA expression on the cell surface. The impact of HLA expression variability on crossmatch reactivity was examined using lymphocytes isolated from different donor types: deceased donor (DD) versus living donor (LD) and tissue sources (blood, spleen, or lymph nodes). HLA class I expression was similar on B cells isolated from LD blood, DD spleen, and DD lymph nodes, but significantly lower on B cells isolated from DD blood (p=0.0004). In contrast, class II expression on B cells and class I on T cells were significantly higher in LD blood than all DD tissues. Within DD tissues, spleen provided the highest expression of class II on B cells and class I on T cells. HLA expression on B cells, but not T cells, was impacted by memory (CD27+) versus non-memory status. Importantly, HLA expression differences on lymphocytes isolated from the same donor but different tissues impacted crossmatch outcomes. HLA expression is impacted by multiple factors and should be routinely monitored to ensure crossmatch sensitivity and to reconcile crossmatch strength with solid phase HLA antibody analyses.

Immunogenicity of decidual stromal cells in an epidermolysis bullosa patient and in allogeneic hematopoietic stem cell transplantation patients.

Allogeneic mesenchymal stromal cells (MSCs) are widely used in regenerative medicine, but little is known about their immunogenicity. In this study, we monitored the therapeutic and immunogenic effects of decidual stromal cells (DSCs) from term placentas when used as a therapy for generalized severe junctional epidermolysis bullosa (JEB) (previously termed Herlitz JEB), a lethal condition caused by the lack of functional laminin-332. An 11-month-old JEB patient was treated with five infusions of allogeneic DSCs within a 3-month period. Amniotic membranes (AMs) were applied to severe wounds. After the treatment, wounds started to heal in the middle of the blisters, but the improvements were transient. After two infusions of DSCs, the JEB patient had developed multispecific anti-HLA class-I antibodies. No antibodies to laminin-332 were detected, but the patient had high levels of anti-bovine serum albumin antibodies, which could bind to DSCs. Peripheral blood mononuclear cells (PBMCs) from the patient had a higher proliferative response to DSCs than to third-party PBMCs, which contrasts with the pattern observed in healthy donors. Human DSCs and MSCs induced similar xenoreactivity in mice. Two of 16 allogeneic stem cell-transplanted patients, treated with DSCs for graft-versus-host disease or hemorrhagic cystitis, showed a positive flow cytometric crossmatch test. One patient had anti-HLA antibodies before DSC infusion, whereas the other had no anti-HLA antibodies at any time. AM and DSC infusions may have improved the healing process in the JEB patient, but DSCs appeared to induce anti-HLA antibodies. The risk of alloimmunization by DSCs seems to be low in immunocompromised patients.

19-OR

Aim The development and refinement of new assays for HLA antibody detection are leading the way for the virtual crossmatch. Assays such as Luminex™, allow for the simultaneous detection of HLA Class I and class II antibodies and the ability to distinguish them through the use of multiplex platforms. The sensitivity of these solid phase assays provides an accurate assessment of a patient’s sensitization status and the identification of HLA antigens that specifically target them. Methods In August 2012, a pilot study was initiated within the framework of the International Cell Exchange to provide a means to correlate antibody testing and virtual crossmatch predication through the comparison of MFI data with crossmatch outcome. A group of 11 leading laboratories tested 2 cells against 4 unknown sera. T and B Flow crossmatch results along with class I and class II single antigen IgG results were reported to UCLA for analysis. For each cell/serum combination, coefficient of variations (%CV) were calculated using the 11 center-specific MFI and MESF values. MFI and MESF values were compared using standard linear regression. Results For T cell outcomes, %CV was greater for MESF values (median %CV = 59%) than MFI values (median %CV = 23%), P Conclusions Our results suggest that results from MFI values can be used to accurately predict the results of both T and B flow cytometric crossmatches. Incorporation of solid phase assays into proficiency testing surveys based on flow cytometric crossmatch tests should be considered.

Clinical Relevance and IgG Subclass Determination of Non-HLA Antibodies Identified Using Endothelial Cell Precursors Isolated From Donor Blood

ABO and human leukocyte antigen (HLA) alloantibodies provide major immunologic barriers to successful transplantation; however, there is increasing recognition for the role of anti-endothelial cell antibodies (AECAs) in allograft rejection. We investigated the relationship between AECAs identified using donor-derived endothelial cell precursors (ECPs) and kidney allograft rejection and function. Sixty live donor kidney recipients were tested pretransplant for AECAs and HLA-antibodies using flow cytometric crossmatch tests and solid-phase bead immunoassays. Renal allograft function was assessed by serum creatinine (SCr) values collected at early (mean, 50 days) and late (mean, 815 days) time points posttransplant and by incidence and type of rejection. Immunoglobulin G (IgG) subtype determination of both AECAs and HLA antibodies bound to ECPs was performed using flow cytometry. Fourteen patients (23%) tested positive for donor-reactive IgG AECAs and had statistically higher SCr values and incidences of cellular rejection early posttransplant compared with 46 patients who tested negative (P=0.014 and P<0.05). SCr values were not statistically different late posttransplant. IgG subclass determination showed AECAs to be enriched for IgG2 and IgG4, subclasses that do not activate complement effectively. Detection of donor-reactive immunoglobulin M (IgM) AECAs did not correlate with increased SCr or incidence of rejection. Crossmatch tests performed using donor-derived ECPs allow for the identification of alloantibodies that are associated with cellular rejection and are distinct from alloantibodies detected using lymphocytes.

Characterization of an antibody specific for HLA-A36 and not reactive with HLA-A1

Humoral sensitization to HLA often results in antibodies to public determinants shared among two or more antigens. Although monoclonal antibodies to A36 have been produced, there are no reports of polyclonal antibodies that react with A36 but not A1. We report here sera from a heart transplant recipient that reacted with A36 but not A1 in tests with both phenotype and single antigen panels on the Luminex platform. Flow cytometric crossmatch tests yielded positive results with an A36 bearing phenotype but not with a phenotype containing A1. A36 reactivity in solid phase assays was abrogated by absorption with cells bearing A36, but not with A1-positive cells. The frequency of B cells in this patient specific for A1 was comparable to that for individuals not sensitized to A1. These data indicate that reactivity was to an epitope present on A36 but absent from A1.

A flow cytometric crossmatch test for simultaneous detection of antibodies against donor lymphocytes and endothelial precursor cells

Complement-dependent cytotoxicity or flow cytometric lymphocyte crossmatch (LXM) tests may fail to detect clinically significant antibodies (Abs) against non-human leukocyte antigen (HLA). A flow cytometric endothelial precursor cell crossmatch (EPCXM) test (XM-ONE) is available for detection of Abs against donor endothelial precursor cells (EPCs). We showed that lymphocytes co-purified with EPCs can be used in LXM tests allowing simultaneous detection of Abs reactive with donor EPCs and lymphocytes. The lymphocyte population co-purified with EPCs on anti-Tie-2 Ab-coupled magnetic beads contained CD 8(+) and CD 4(+) T-cells, B-cells, and natural killer (NK)- and natural killer T (NKT)-cells. HLA class I antigen expression was slightly higher on CD 3(+) lymphocytes co-purified on Tie-2 Ab beads than on unseparated lymphocytes, whereas HLA class I and II antigen levels on CD 19(+) lymphocytes were not significantly different. Sera from 10 patients with panel-reactive Abs were tested on cells from nine donors using flow cytometric LXM and EPCXM tests. There was a very good correlation (R(2) = 0.94) between the channel shift values obtained on unseparated and Tie-2 Ab bead-isolated T-lymphocytes, whereas the correlation between the channel shift values obtained on the two B-lymphocyte populations was lower (R(2) = 0.71). T- and B-lymphocytes co-purified with EPCs can be used in LXM tests enabling simultaneous detection of donor lymphocyte- and EPC-reactive Abs in a single-tube XM-ONE assay.

28-P: A flow cytometric crossmatch test for simultaneous detection of antibodies against donor lymphocytes and endothelial cells

28-P: A flow cytometric crossmatch test for simultaneous detection of antibodies against donor lymphocytes and endothelial cells

Kidney transplantation in a recipient with anti-HLA antibody IgM positive

In general, anti-HLA antibody belongs to the IgG subclass, but there are very few reports of detection of anti-HLA IgM antibodies. In the present study, we report a renal transplant recipient with a positive NIH-complement dependent cytotoxicity (NIH-CDC) test. The patient was a 24-year-old male with focal segmental glomerulosclerosis (FSGS) as the underlying kidney disease. He had been on maintenance hemodialysis since December 2003 and finally received a living-donor allograft from his mother in October 2008. Pre-transplantation, the NIH-CDC test was positive for both B and T cells, but the flow-cytometric crossmatch test (FCXM) was negative for both cells. The result of the panel-reactive antibody assay (PRA)-single beads test using anti-IgM antibody as the second antibody demonstrated that the positive NIH-CDC test was due to the presence of anti-HLA IgM antibody against the donor-specific antigen A24. Biopsy specimens showed thrombus formation in a small number of glomeruli immediately after the transplantation, but this finding was no longer seen at three months postoperatively. We report successful renal transplantation in a case with anti-HLA IgM antibody.

Comparison of Antibody Monitoring System with Flow Cytometric Crossmatch Test in Renal Transplant Recipients with High Panel-Reactive Antibody

Background/Aims: The antibody monitoring system (AMS) is a recently developed enzyme-linked immunosorbent assay (ELISA) crossmatch assay to detect donor-specific anti-HLA immunoglobulin G antibodies (DS-HLA Abs). This study was conducted to compare the AMS with the flow cytometric crossmatch (FCXM) test in renal transplant recipients with high panel-reactive antibody (PRA). Methods: Thirty-two sera were obtained from 10 patients with panel reactivity above 50%. When anti-HLA Ab was detected by ELISA PRA and the matched donor had the corresponding HLA antigen, it was considered to indicate DS-HLA Ab. The results of the AMS assay and FCXM were compared with the DS-HLA Abs. Results: Twenty-three (71.9%) sera were positive for DS-HLA Abs by ELISA PRA. The AMS assay showed that the number of compatible sera with DS-HLA Abs was 27 (84.4%), and it was significantly concordant (κ = 0.649, p < 0.0001). For FCXM, the number of compatible sera with DS-HLA Abs was 26 (81.3%), and it was also significantly concordant (κ = 0.614, p < 0.0001). There was a significant degree of concordance between the AMS assay and FCXM in detection of DS-HLA Abs (κ = 0.452, p = 0.010). Conclusion: The AMS assay is comparable to FCXM in detecting DS-HLA Abs in high PRA recipients.

Detection of Donor-Specific Anti-HLA Class I and II Antibodies Using Antibody Monitoring System

The antibody monitoring system (AMS, GTI Inc) is a solid enzyme-linked immunosorbent assay (ELISA) crossmatch test for the detection of immunoglobulin G (IgG) antibody to donor-specific solubilized HLA class I and class II antigens. The objective of this study was to compare the results of the AMS assay with donor-specific anti-HLA IgG antibodies (DS-HLA Abs), as determined by ELISA panel reactive antibody (PRA) and the flow cytometric crossmatch test (FCXM). A total of 107 sera were screened for the presence of HLA Abs by ELISA PRA (LAT-M, One-Lambda Inc), the DS-HLA Abs were determined in 34 serum samples (31.8%) by an ELISA panel (LAT class I and class II, One-Lambda Inc) and FCXM. The FCXM and AMS assays were performed with matched lymphocytes from 56 donors. There was a significant degree of concordance (89.7%) between the two tests ( P < .001). The sensitivity, specificity, positive predictive value, and negative predictive value of AMS assay to detect DS-HLA Abs was 88.2%, 94.5%, 88.2%, and 94.5%, respectively. The AMS is a simple, objective test, which has several advantages over the cell-based crossmatch test, such as elimination of non-HLA antibody reactivity, elimination of non-donor-specific antibody reactivity, no need for viable cells, and preparation of the donor’s HLA antigens in advance. In summary, this study suggested that AMS may be useful as a supportive crossmatch test or as a monitoring test after transplantation to detect class I or class II DS-HLA Abs.

Liver transplantation and the lymphocyte crossmatch

Hyperacute rejection related to donor-specific antibodies rarely occurs in liver transplantation, probably because of the role of Kupffer cells, sinusoidal cells, and the dual blood supply. However, the long-term outcome of liver grafts, transplanted in the context of a positive lymphocytotoxic crossmatch, is approximately 20% lower than that of grafts with a negative crossmatch. Without recipient selection based on the crossmatch, which is currently the generally accepted policy, 11% to 24% of the liver transplants will be performed with a positive crossmatch. Using the level of panel reactive antibodies, (eg > 30%) as an indicator for sensitization, a lymphocytotoxic crossmatch can be performed before transplantation on a limited number of patients, to avoid a positive crossmatch. Sometimes the medical urgency does not allow the patient to wait for a negative crossmatch. Therefore, plasmapheresis should be performed before transplantation to diminish the antibody level and the posttransplant use of prostaglandin-E and high-dose steroids should be considered to avoid the short-term immunologic complications. These measures require more study before they can become standard practices. Possibly, Mycophenolate Mofetil might be effective in preventing chronic rejection. The introduction of the flowcytometry crossmatch procedure will not only provide a more sensitive, but also a faster procedure (~4 hours), allowing a wider use of the pretransplant crossmatch test in liver transplantation. However, the increased sensitivity and speed are balanced by the much lower specificity of the positive flowcytometric crossmatch test. The most appropriate way to use flow techniques for lymphocyte crossmatching has not yet been clearly determined because of the specificity problem. In the setting of other solid organ transplantation, the presence of preformed donor-specific antibodies is linked with occurrence of hyperacute or accelerated rejection, and is likely to result in graft failure in a short period of time. Therefore, a positive crossmatch directed against donor HLA antigens is a concern in organ transplantation, especially in kidney and heart transplantation. A positive IgG crossmatch test precludes transplantation in most cases. In liver transplantation, the picture is not so clear, but probably represents a relative contraindication to transplantation.

The occurrence of cytotoxic and non-complement-fixing antibodies in the crossmatch serum of patients with early acute rejection episodes.

In an earlier study we found a strong correlation between the presence of donor-reactive HLA-specific antibodies in the crossmatch serum and early acute rejection episodes. Our experience was also that some of these antibodies were not cytotoxic and could therefore not be detected using the microcytotoxicity assays. In the present study, 11 patients from the earlier study who had weakly positive B cell reactive cytotoxic antibodies of the IgG class were further characterized. In addition, 14 new patients were selected who experienced early acute rejections but had a completely negative donor-B cell cytotoxicity crossmatch. A group of 12 controls without immunological complications was added, as well as 5 patients with early graft losses due to nonimmunological causes. Using the flow cytometric crossmatch test we confirmed the presence of HLA-specific antibodies in all 11 patients from the earlier study. In addition, positive flow cytometric crossmatches shown to be caused by HLA antibodies were observed in 11 of the 14 patients with acute rejections and negative cytotoxic crossmatch. One of 17 control patients had antibodies that were not HLA-reactive. IgA antibodies as well as IgG subclass determinations were performed in all positive sera. A substantial proportion of patients had HLA-specific antibodies of non-complement-binding classes (IgG2, IgG4, IgA) often of higher titers than IgG1 and IgG3. The subclass distribution pattern was heterogeneous and often included several subclasses. We conclude that non-complement-fixing antibodies can also contribute to the risk for development of early acute rejections after necrokidney transplantation. Immunological mechanisms for these findings are discussed.