Crossmatch Assay Research Articles

Flow cytometric crossmatching in human organ transplantation

When donors class I directed ‘warm’ antibodies are detected by the conventional cytotoxic crossmatch preoperatively, there is a negative correlation with outcome in renal, liver, cardiac and pancreatic transplantation. Whn IgG antibodies to T donor crossmatch assay is negative and IgG antibodies to T donor lymphocytes are shown to be present in the recipient sera by flow cytometry then there is an increase in postoperative complications in renal transplants. This includes primary nonfunction and treatable rejection which in some cases results in graft failure. Since its introduction into clinical practice for recipient selection in Newcastle, the complication rate of rejection episodes, primary nonfunction and graft failure has been reduced. When applied to other organs there seems to be a definite detrimental association between a positive flow cytometric crossmatch preoperatively and rejection in cardiac transplantation, in some cases leading to death. The role in liver and pancreas transplants to date has not been determined. In the postoperative period there appears to be a limited role for the flow cytometric crossmatch in the detection of antibody prior to the first clinical episode of rejection. This has been applied to renal transplants but to no other organ.

Clinical and Blood Bank Factors in the Management of Platelet Refractoriness and Alloimmunization

AbstractNumerous independent and interdependent factors are involved in the posttransfusion platelet response. Factors such as ABO match and platelet age are related to circumstances potentially under the control of the blood bank physician and therefore may permit circumvention by an active transfusion service. On the other hand, factors such as fever or sepsis may be unavoidable, being related more to the individual patient or clinical condition. To evaluate which factors could be circumvented, we prospectively followed the 1-hour corrected count increments (CCIs) for 962 single-donor apheresis platelet transfusions to 71 refractory hematologic oncology inpatients, with concomitant recording of implicated factors. Stepwise regression analysis allowed for determination of which concurrent and confounding clinical-, patient-, and blood bank-related factors significantly affected the CCIs. Although many implicated factors proved to be independently associated with an increased or decreased CCI, we found that no single variable consistently explained the CCI variation across the patient population. Each patient appeared sensitive to one or a few particular factors, but because of marked intraindividual variation, it was not possible to identify a priori which factors were important for a given patient. The single exception was a solid-phase red blood cell adherence assay used to cross-match platelets, but only for alloimmunized patients. We also evaluated the utility of requesting FILA-matched platelets from the local suppliers and maintained a clear distinction between platelets simply ordered as HLA matched and actually H LA-identical platelets. Accounting for the confounding clinical-, patient-, and blood bank—related factors, the cross-match assay was a better predictor of an adequate CCI than ordering platelets as FILA matched.

Clinical and blood bank factors in the management of platelet refractoriness and alloimmunization

Numerous independent and interdependent factors are involved in the posttransfusion platelet response. Factors such as ABO match and platelet age are related to circumstances potentially under the control of the blood bank physician and therefore may permit circumvention by an active transfusion service. On the other hand, factors such as fever or sepsis may be unavoidable, being related more to the individual patient or clinical condition. To evaluate which factors could be circumvented, we prospectively followed the 1-hour corrected count increments (CCIs) for 962 single-donor apheresis platelet transfusions to 71 refractory hematologic oncology inpatients, with concomitant recording of implicated factors. Stepwise regression analysis allowed for determination of which concurrent and confounding clinical-, patient-, and blood bank-related factors significantly affected the CCIs. Although many implicated factors proved to be independently associated with an increased or decreased CCI, we found that no single variable consistently explained the CCI variation across the patient population. Each patient appeared sensitive to one or a few particular factors, but because of marked intraindividual variation, it was not possible to identify a priori which factors were important for a given patient. The single exception was a solid-phase red blood cell adherence assay used to cross-match platelets, but only for alloimmunized patients. We also evaluated the utility of requesting HLA- matched platelets from the local suppliers and maintained a clear distinction between platelets simply ordered as HLA matched and actually HLA-identical platelets. Accounting for the confounding clinical-, patient-, and blood bank-related factors, the cross-match assay was a better predictor of an adequate CCI than ordering platelets as HLA matched.

Prolonged neutropenia resulting from antibodies to neutrophil‐specific antigen NB1 following marrow transplantation

Marrow graft failure is a significant cause of morbidity following bone marrow transplantation. A case is reported of marrow graft failure due to neutrophil antibodies. A 13-year-old girl with a large granular lymphocytosis and chronic neutropenia was treated with granulocyte transfusions prior to undergoing a transplant with bone marrow from a partially matched, unrelated donor. Following the transplant, a bone marrow biopsy showed engraftment of donor myeloid cells, but the recipient remained neutropenic. Testing of the serum for neutrophil antibodies found that the recipient's serum had a high-titer neutrophil antibody. Immunoprecipitation studies using the marrow recipient's serum and 125I surface-labeled neutrophils showed that the antibody reacted to the neutrophil-specific antigen NB1. Phenotyping of neutrophils from the marrow donor found that they expressed NB1 antigen, and, in a crossmatch assay, the recipient's serum reacted with donor neutrophils. Despite treatment with granulocyte-macrophage--colony-stimulating factor, the marrow transplant recipient remained neutropenic and died of polymicrobial sepsis and aspergillosis 38 days after the transplant. The presence of high-titer antibodies to neutrophil-specific antigen NB1 in this patient following transplant likely prevented the recovery of her peripheral blood neutrophils and contributed to her death.

Flow cytometric crossmatching and outcome one year after renal transplantation.

Previous studies have shown that flow cytometric crossmatch assays can identify an at risk population in renal transplantation. We used the assay for recipient selection for 1 year. Recipients with donor T cell directed IgG were excluded from transplantation and those with B cell directed IgG were treated with increased immunosuppression. The transplants performed over this period (n = 126) were compared with an earlier series (n = 118) in which flow cytometric crossmatch results did not influence patient management. The results were evaluated for mortality and graft outcome at 3 months and 1 year. In addition, postoperative complications and duration of hospital stay were also assessed.

Crossmatch Procedures Used in Organ Transplantation

Several lymphocyte crossmatch procedures used in clinical histocompatibility laboratories, including complement dependent (CDC, AHG-CDC) and complement independent (flow cytometric, chromium release) techniques, are used to assess the likelihood of allograft rejection due to preformed donor specific antibody. Crossmatch assays can be extremely sensitive and detect very low levels of donor reactive antibody present in the potential recipient. Since positive crossmatches are usually associated with allograft rejection, a positive crossmatch is generally a contraindication to organ transplantation. Recent data, however, suggests that not all positive crossmatches lead to graft rejection, particularly those due to autoantibodies. This underscores the need to critically evaluate any positive crossmatch to determine if the antibodies involved in the reaction are relevant to allograft rejection.

Platelet antibody: review of detection methods.

The driving force behind development of in vitro methods for platelet antibodies is identification of plasma factors causing platelet destruction. Early methods relied on measurement of platelet activation. Current methods are more specific and use a purified antibody against immunoglobulin or complement, which is usually labeled with 125I or tagged with an enzyme or fluorescein. Comparisons of quantitation of platelet-associated IgG show wide variability between different methods. The disparate results can be related both to differences in binding of secondary antibodies to immunoglobulin in solution compared to immunoglobulins attached to platelets and to the improper assumption that the binding ratio between the secondary detecting and primary antiplatelet antibody is one. Most assays can 1) identify neonatal isoimmune thrombocytopenia and posttransfusion purpura, 2) help to differentiate between immune and nonimmune thrombocytopenias, 3) help to sort out the offending drug when drug-induced thrombocytopenia is suspected, and 4) identify platelet alloantibodies and potential platelet donors via a cross match assay for refractory patients. However, the advantages of quantitative assays over qualitative methods with respect to predictions of patients clinical course and response to different treatments remain to be investigated.

Relationship of HLA and platelet-reactive antibodies in alloimmunized patients refractory to platelet therapy.

Platelet crossmatching assays have been used to predict the outcome of platelet transfusions in alloimmunized patients by detecting antibodies against platelets. The transfusion failure of HLA-matched platelets predicted by platelet crossmatching may be related to HLA antibodies undetected by lymphocytotoxicity but detected by platelet immunoglobulin-binding assays or platelet-specific antibodies (both antibodies defined here as platelet-reactive antibodies). To differentiate platelet-reactive antibodies from lymphocytotoxic HLA antibodies, we used HLA characterized lymphocytes in parallel with platelets from individuals to form separate frozen panels. Sera from 10 allosensitized patients were studied in the lymphocyte panel by lymphocytotoxicity and in the platelet panel by enzyme-linked immunoassay (ELISA). By comparing pattern and percent wells reacting in each panel, lymphocytotoxic HLA antibodies and antibodies reactive with platelets in ELISA were detected separately. In all 10 allosensitized patients, platelet-associated antibodies were present and 7 had additional lymphocytotoxic HLA antibodies. Using this double parallel panel technique, we found platelet-reactive antibodies important in platelet alloimmunization, unrecognized by lymphocytotoxicity. These data indicate platelet-crossmatching be solely used in the selection of platelets for allosensitized patients.

3 Prognostic Significance of Chromosomal Abnormalities in Acute Leukaemias and Myelodysplastic Syndromes

The impact of COVID-19 vaccination on the alloimmunity of transplant candidates is unknown. We report a case of positive B cell flow cytometry crossmatch in a patient waiting for second kidney transplantation, 37 days after receiving the COVID-19 vaccine. The preliminary crossmatch, using sample collected before COVID-19 vaccination, was negative. The antibodies to mismatched donor HLA-DR7 were detected only with multi-antigen beads but not with single-antigen beads, excluding possible prozone effects in solid-phase antibody assays. The crossmatches were positive with HLA-DR7–positive surrogates (n = 2) while negative with HLA-DR7–negative surrogates (n = 3), which confirms the HLA-DR7 alloreactivity. The antigen configurations on B lymphocytes are similar to that on the multi-antigen beads while distinct from the single-antigen beads. HLA-DR7 was the repeating mismatched antigen with the failing first kidney allograft. The newly emerged antibody to HLA-DR7 probably is the consequence of bystander activation of memory response by the COVID-19 vaccination. This case highlights the importance of verifying allo-sensitization history and utilizing multiple assays, including cell-based crossmatch and solid-phase assays with multi-antigens. COVID-19 immunization may deserve special attention when assessing the immunological risk before and after organ transplantation.

Four crossmatch methods to select platelet donors

We employed four crossmatch techniques to select platelet donors for refractory patients. Forty-four donor-recipient pairs were studied in 32 patients. Analysis of effectiveness of platelet transfusions revealed that only 18 percent of transfusions gave a borderline response; the remainder were either effective or not effective at all. The corrected predictive values of three crossmatch tests were as follows: enzyme-linked immuno-specific assay, 81 percent; platelet immunofluorescence test, 73 percent; and lymphocytotoxicity, 70 percent (p greater than 0.05). The predictive value of these tests did not differ in HLA-matched versus unmatched platelet transfusions. Donor selection by lymphocytotoxicity compatibility did not appear to be useful if donors were selected by either of the other two methods. The fourth test, antiglobulin-modified lymphocytotoxicity, offered no advantage over lymphocytotoxicity. Our data suggest that platelet crossmatching assays are a useful adjunct to the selection process for the platelet donor in addition to ABO, Rh, and HLA matching.

The Relationship between Donor‐Recipient Lymphocytotoxicity and the Transfusion Response using HLA‐Matched Platelet Concentrates

Matching donor-recipient pairs for HLA antigens provides a logical starting point for selecting platelet donors for thrombocytopenic recipients who have demonstrated refractoriness to pooled random donor platelet transfusions. However, not all recipients achieve a compatible transfusion response with platelets selected by HLA type, indicating that additional selection methods are required. Because of studies indicating that a positive lymphocyte cross-match assay between recipient serum (antibody) and donor lymphocytes predict acute renal allograft rejection, it was hoped that this assay might be useful in predicting the platelet transfusion response for alloimmunized patients. The data herein indicate that the results of this assay in no way correlate with the platelet transfusion response. The lymphocyte cross-match would seem to be helpful in the selection of donors where known HLA antigen mismatches occur between donor and recipient. However, even in this situation, no correlation was observed between the transfusion response and donor-recipient lymphocytotoxicity reaction. Clearly additional assays are needed for the selection of compatible donors from those matched by HLA antigens.

KIDNEY TRANSPLANTABILITY ACROSS A POSITIVE CROSS-MATCH: Cross-match Assays and Distribution of B Lymphocytes in Donor Tissues

Lymphocyte subpopulations were determined in peripheral blood, abdominal lymph-nodes, and spleens from renal allograft donors. Percentages of B lymphocytes were low in peripheral blood (mean±S.E.,10.0±1.0%), variable in lymph-nodes (28.8±3.8), and high in spleens (41.4±2.1). Microlymphocytotoxicity cross-match assays in which sera with B-cell-specific antibody were used were invariably negative with peripheral-blood lymphocytes but positive with lymph-node or spleen preparations. Four renal allografts were transplanted when the standard cross-match was positive with spleen or lymph-node but negative with blood. No hyperacute or accelerated rejection was observed. Potential recipients are often denied allografts because of a positive cross-match with either lymph-node or splenic preparations, and this could be avoided in some cases if cross-matches were performed on peripheral blood or B-lymphocyte-depleted splenic or lymph-node preparations. These results accord with those of other workers who found that B-cell pre-sensitisation is not a contraindication to transplantation.