Smita Vaidya

Frequency, potential risk and therapeutic intervention in end-stage renal disease patients with antiphospholipid antibody syndrome: a multicenter study.

BACKGROUND Antiphospholipid antibody syndrome (APAS) is characterized by the presence of anticardiolipin antibodies (ACA) in association with thrombotic disorders of arterial and/or venus systems, spontaneous abortion(s) or thrombocytopenia. METHODS In this multicenter study, 502 end-stage renal disease (ESRD) patients awaiting renal transplants were screened to determine the frequency of APAS, the potential risk associated with APAS, and strategies for therapeutic intervention. Ninety-three patients (19%) had high titers of ACA. Twenty-three patients had documented evidence of one or more of the thrombotic disorders such as lupus, frequent abortions, frequent thrombosis of arteriovenous shunts, biopsy-proven microrenal angiopathy, or thrombocytopenia and thus were diagnosed with APAS. Of these 23 patients, 11 received kidney transplants either with (4 patients) or without (7 patients), concomitant anticoagulation therapy. RESULTS All seven of the patients with APAS not treated with anticoagulation therapy lost their allografts within 1 week as a result of renal thrombosis. In contrast, three out of four transplant patients with APAS treated with anticoagulation therapy maintained their allografts for over 2 years. The fourth patient lost his graft within a week because of thrombosis. Of the remaining 70 patients with high titers of ACA but no evidence of thrombotic disorders, 37 received kidney transplants. None lost their allografts as a result of thrombosis. Our data suggest that, although 19% of our ESRD patients exhibit high titer of ACA, only 5% of the patients have APAS. CONCLUSION In conclusion, our data suggest that the patients with APAS are at high risk of posttransplant renal thrombosis. Anticoagulation therapy could prevent patients from posttransplant thrombosis in patients with APAS.

Prediction of crossmatch outcome of highly sensitized patients by single and/or multiple antigen bead luminex assay

Background. We used a solid-phase assay to identify human leukocyte antigen (HLA) Class I and II specificities in highly reactive sera, and applied this information to predict crossmatch outcome with greater than 90% accuracy. Methods. Sera from 20 highly sensitized end-stage renal disease patients reactive to 70–100% of HLA Class I and II antigen panel were analyzed by single and/or multiple antigen flow microparticle bead assay using Luminex platform (Luminex assay). These sera contained antibodies directed against high frequency public HLA class I and/or II epitopes accounting for 70–100% of serum’s total reactivity. More than 2,000 complement dependent cytotoxicity (CDC) and 200 flow crossmatches (FLXM) were performed using sera from these patients and deceased donor T and B lymphocytes. Results. Luminex serum analysis was able to correctly predict outcomes of 95% of T and B cell FLXM. In contrast, predictive values for the CDC T and B cell crossmatches by Luminex serum analysis were only 77% and 75%, respectively. The use of serum analysis in donor selection would have reduced the total number of required FLXM by more than 50%. The frequency of negative T cell FLXM was 56% when donors were selected randomly; however, if serum antibody analysis had been used to preselect the donors by excluding donors that were likely to be positive, the frequency of corresponding negative crossmatches would have increased up to 93%. Conclusion. This approach to donor selection may allow wider geographic sharing of cadaver donor organs without actually performing the crossmatch.

Transient poration and cell surface receptor removal from human lymphocytes in vitro by 1 MHz ultrasound

The study objective was to gain insight into ultrasound-induced, sub-lytic cell surface modifications. Two primary hypotheses were tested by flow cytometric methods; viz., sonication will: 1. remove all or part of a specific cell surface marker in lymphocytes surviving insonation, and 2. induce transient pores in the cell membranes of some surviving cells. RPMI 1788 human lymphocytes were exposed in vitro to 1-MHz, continuous-wave ultrasound (approximately 8 W/cm2 ISP) for 30 s, which lysed approximately 50% of the cells. Insonation: 1. altered cell morphology, increasing the population of cells of reduced size but high structure (designated as population R2), many of which were nonviable, and diminishing the population of cells of large size and high structure (designated as population R1), most of which were viable, 2. diminished the fluorescence signal from the pan B lymphocyte marker CD19 in populations R1 and R2 to equivalent extents, and 3. increased by approximately 7-fold the number of transiently permeabilized cells in R1, as evidenced by simultaneous uptake of propidium iodide and fluorescein diacetate. The results indicate that ultrasound-induced CD19 removal from R1 cells can occur without accompanying gross membrane loss. The cell morphology/mortality shifts indicate that the ultrasound-induced morphological change is associated with lethal membrane poration, suggesting that the diminished CD19 fluorescence signal from insonated R2 cells arises partly by simultaneous loss of membrane fragments, CD19 and cytoplasm.

Improved flow cytometric detection of HLA alloantibodies using pronase: Potential implications in renal transplantation

Background. Flow cytomeric crossmatch (FCXM) has grown in popularity and has become the “standard of practice” in many programs. Although FCXM is the most sensitive method for detecting alloantibody, the B cell FCXM has been problematic. Difficulties with the B cell FCXMs have been centered around high nonspecific fluorescence background owing to Fc-receptors present on the B cells and autoantibodies. To improve the specificity and sensitivity of the B cell FCXM, we utilized the proteolytic enzyme pronase to remove Fc receptors from lymphocytes before their use in FCXM. Methods. Lymphocytes isolated from peripheral blood, spleen, or lymph nodes were treated with pronase and then used in a three-color FCXM. A total of 167 T- and B cell FCXMs using pronase-treated and untreated cells were performed. Testing used serial dilutions of HLA allosera (22 class I and 6 class II), with the titer of each antibody at one dilution past the titer at which the complement-mediated cytotoxicity anti-human globulin crossmatch became negative. Results. After pronase treatment, the actual channel values of the negative control in both T cell and B cell FCXMs declined from 78±10 to 57±4 (P <0.05) and 107±11 to 49±3 (P <0.00001), respectively. Pronase treatment resulted in improved sensitivity of the T and B cell FCXM in detecting class I antibody by 20% and 80%, respectively. In no instance was a false-positive reaction observed. In this study, pronase treatment improved the specificity of B cell FCXM for detecting class II antibodies from 75% to 100% (P =0.03). In no instance was a false-negative reaction recorded. Lastly, on the basis of these observations we re-evaluated three primary transplant recipients who lost their allografts because of accelerated rejection. One of the patients was transplanted across negative T and B cell FCXM, whereas the other two patients were transplanted across a positive T cell, but negative B cell, FCXM. After pronase treatment, T and B cell FCXMs of each patient became strongly positive, and donor-specific anti-HLA class I antibody was identified in each case. Conclusion. Utilization of pronase-treated lymphocytes improves both the sensitivity and specificity of the FCXM.

Clinical Importance of Anti-Human Leukocyte Antigen-Specific Antibody Concentration in Performing Calculated Panel Reactive Antibody and Virtual Crossmatches

Background. Highly sensitized patients develop multi-human leukocyte antigen (HLA) specific antibodies. This study measures concentrations of anti-HLA antibodies in multispecific sera by converting fluorescence intensity into molecules of equivalent soluble fluorochrome (MESF) units. This was used to determine MESF units required for a positive T and B flow crossmatches (FLXM). Methods. MESF values of negative controls and sera from patients devoid of HLA antibodies were measured by FLXM and flow panel reactive antibody (PRA) screening beads. Fluorescence intensity values of anti-HLA specific antibodies determined by FlowPRA single antigen beads of highly sensitized patients were converted into MESF units. In addition, endpoint titers, MESF units, and % PRA of 26 sera were established. Results. MESF analysis accurately predicted the outcomes of 100% of T and B FLXM of sera with strong (MESF units>18,000) donor-specific antibody (DSA). The predictive values of T and B FLXM declined to 95% and 88% with weak DSA (6,000 MESF<10,000). Endpoint titers of sera from highly sensitized patients ranged from 1:512 to 1:8 with corresponding MESF values of 452,596 to 20,000 units. However, there was no statistical difference in PRA values among these sera (95%–100%). We successfully transplanted five patients who had weak donor-specific HLA antibodies (MESF units>2,000). The graft survival at 1 year was 100% and there was no evidence of DSA posttransplant. Conclusion. MESF analysis is both a time and cost efficient way of measuring antibody strength. The strength of the antibody present in the sera of transplant candidates is critical for crossmatch prediction.

Efficacy of anticoagulation therapy in end-stage renal disease patients with antiphospholipid antibody syndrome.

Background. End-stage renal disease (ESRD) patients with antiphospholipid antibody syndrome (APAS) remain at high risk for the development of renal thrombosis without the benefit of anticoagulation therapy. This study examines the efficacy of anticoagulation therapy in this high-risk patient population. Method. Of nine APAS renal-transplant patients, seven were treated with coumadin, whereas two were treated with heparin. Results. Of the two patients treated with heparin, one had early allograft loss, whereas the other patient is doing fine at 5 years posttransplant. Of the seven 7 patients treated with coumadin, two patients are doing well at 2 and 3 years posttransplant, two had early allograft loss, the remaining three patients returned to dialysis after they were taken off of the coumadin at 6, 12, and 20 months posttransplant because of bleeding complications. Conclusions. Anticoagulation therapy is beneficial to some but not all APAS patients. In addition, bleeding complications are a serious side effect of this therapy.

Pretransplant soluble CD30 is a better predictor of posttransplant development of donor-specific antibodies and acute vascular rejection than panel reactive antibodies.

Background. This study tests a hypothesis that pretransplant concentration of soluble CD30 (sCD30) is a better predictor of posttransplant development of donor-specific HLA antibodies (DSA) and acute vascular rejection (AVR) than panel reactive HLA antibodies (PRA). Methods. Pretransplant sera from 115 patients were evaluated for their PRA and sCD30 concentrations. All patients received calcineurin-inhibitor based immunosuppressive therapy. Objective measurements for rejection were biopsy-proven AVR episodes within first 6 months of the transplant. Posttransplant sera of patients with or without AVR were tested for the presence of DSA. Results. AVR rate was 16% (18/115). Patients positive for PRA and sCD30 tests were at significantly higher risk for AVR compared to those patients negative for both tests (36% versus 5%, p=0.01). Among negative PRA patients risk for AR was significantly elevated if they were also tested positive for sCD30 concentrations (21% versus 5%, p=0.04). Of the 18 patients with AVR, 14 were positive for sCD30, and 13 of them (93%) developed DSA posttransplant (p=0.001) Nineteen patients without AVR were tested for DSA and sCD30 concentrations. Only two of these 19 patients were positive for sCD30 and DSA. AVR was strongly associated with the patients tested positive for both the tests: DSA and sCD30 (p=0.00007). Furthermore, patients with AVR are more likely to produce DSA than those without AVR (p=0.02). Conclusion. These data support our hypothesis that patients positive for sCD30 contents are at high risk the development of DSA and AVR posttransplant regardless of their pretransplant PRA.

Campath and renal transplant rejection

Abstract:  No clear guidelines exist for the treatment of acute vascular rejection following renal transplantation. This report documents one patient who was treated with plasmapheresis, immunoglobulin and Campath with good initial response. However, rejection recurred resulting in graft loss and, in addition, the patient developed post‐transplant lymphoma.

Soluble CD30 concentrations in ESRD patients with and without panel reactive HLA antibodies

Abstract:  Background:  In this retrospective study we compared accuracy of panel reactive antibodies (PRA) with serum soluble CD30 (sCD30) contents in predicting acute rejection crisis post‐renal transplant.

Contributions and clinical significance of IgM and autoantibodies in highly sensitized renal allograft recipients.

The contributions of auto and IgM antibodies in the levels of serologic reactivities of 30 highly sensitized patients were assessed by autologous T cell crossmatches at 4 degrees C and 22 degrees C and dithiothreitol (DTT) reduction of IgM antibodies. The range of panel reactivities of sera from these patients was 30-100%, median 55%. A monthly screen of these sera against a 30-member T cell panel was performed with and without addition of DTT (final concentration = 0.005 M). The results were divided into 3 groups. Group 1 consisted of 17 sera whose PRA values did not change following the DTT treatment. Also none of these sera had autoantibodies, suggesting that these sera contained DTT-resistant (IgG) antibodies, most likely directed against allogeneic targets. Group 2 consisted of 10 sera whose PRA values declined substantially (20-42%) following the DTT treatment, but only 1 serum derived from a patient with systemic lupus erythematosus had autoantibodies. These results suggested that although these sera contained IgM and IgG antibodies, these antibodies were most likely directed at allogeneic target structures with only one exception. Group 3 consisted of 3 sera that became completely unreactive to panel lymphocytes following the DTT treatment. All 3 sera had autoantibodies that were also removed with DTT, suggesting that these sera contained predominantly IgM antibodies directed at autologous target cells. All 3 patients from whom these sera were derived received successful kidney transplants across donor-specific positive T cell crossmatches that became negative following the DTT treatment. We conclude that although 13 out of 30 patients have IgM antibodies, only a small subset of these patients have autoantibodies. Renal transplantation in the presence of auto/IgM antibodies may be safe.