Blood transfusions in organ transplant patients: mechanisms of sensitization and implications for prevention.

Sensitization by previous pregnancies or transplants is considered unavoidable, but it is transfusions given to these patients that leads most often to broad sensitization. Both leukocytes and red cells carry a significant HLA antigen load, and residual leukocytes and/or red cell HLA may explain why leukocyte-reduced units are unable to prevent sensitization to any significant degree. Prevention of sensitization will require a more active effort to avoid blood transfusions, whenever possible. When transfusions are required, there is evidence that the use of HLA-matched blood or immunosuppression in selected situations may reduce sensitization, even in patients previously exposed to alloantigens. These additional measures are not logistically straightforward or devoid of risks and need to be confirmed by rigorous studies. However, remaining as passive observers when patients become broadly sensitized should no longer be considered an acceptable alternative for potential transplant recipients.

Reduced-intensity conditioning using fludarabine with either antithymocyte globulin and BU, or low-dose TBI allowing allogeneic hematopoietic SCT.

In a single-center study, we analyzed the outcomes of 66 patients with advanced hematological malignancies receiving two reduced-intensity conditioning regimens for allogeneic transplantation: fludarabine and low-dose TBI (flu/TBI, n=25), or fludarabine, antithymocyte globulin and BU (flu/ATG/BU, n=41). The selection criteria were based on the hypothesis that flu/TBI patients were expected to achieve autologous recovery in the event of non-engraftment. Sixty-three patients (95%) engrafted. Regimen-related mortality at day 100 and 1 year was 6 and 15%, respectively. With median follow-up of 50.4 months, survival did not differ by regimen. Multivariate analysis confirmed that the type of regimen did not affect relapse. In patients achieving full donor chimerism by day 30, those conditioned with flu/TBI showed greater overall survival (P=0.02). Engraftment failure occurred in two patients (3%), both of whom received flu/TBI. We conclude that conditioning with flu/TBI or flu/ATG/BU yields comparable survival and remission outcomes. By contrast to our hypothesis, patients receiving flu/TBI who subsequently failed engraftment did not achieve autologous recovery. Yet, rapid attainment of full donor chimerism after flu/TBI is associated with greater survival than after flu/ATG/BU. Further, larger prospective randomized studies are required to define the advantage of one regimen over the other.

Value of posttransplant antibody tests in the evaluation of patients with renal graft dysfunction.

Posttransplant HLA antibodies correlate with C4d positive rejection and decreased graft survival. However, the diagnostic value of various antibody tests in the management of patients presenting with graft dysfunction is uncertain. Whether all or some patients should be tested, how often, what antibodies to test for and how to interpret results in presensitized or transfused patients, are issues still unresolved. We tested for HLA and non-HLA antibodies by flow cytometry assays in 103 consecutive patients with graft dysfunction. The results show that: (1) C4d positive rejection was diagnosed in 75% of patients who developed posttransplant HLA antibodies, but only in 2% in antibody negative patients. (2) The correlation existed for donor specific IgG antibodies but not for IgM or nondonor specific IgG antibodies. (3) Weak antibody reactivity required confirmation by alternative testing as there were false positive results. (4) Posttransplant transfusions did not induce de novo HLA antibodies. (5) Negative antibody results were unlikely to turn positive after several months of follow-up. (6) Antibodies to the angiotensin II type 1 receptor, HLA-DP and MICA did not correlate with C4d+ rejection. We conclude that testing for posttransplant HLA antibodies is critical in narrowing the diagnostic alternatives in patients with graft dysfunction.

Analysis of early deaths after isolated lung transplantation.

STUDY OBJECTIVES: To determine the causes of death in patients dying within 30 days after lung transplantation at the University of Florida, to assess the importance of several diagnostic modalities for determining the causes of their decline, and to construct an algorithm for the evaluation of patients with severe respiratory compromise occurring early after lung transplantation. DESIGN: Retrospective review of medical records and pathology slides from all patients dying within 30 days after lung transplantation, and biopsy specimen diagnoses from all lung allograft recipients at the University of Florida. PATIENTS: Nine deaths occurred during the first 30 days after transplantation among 117 patients undergoing 123 isolated lung transplantation operations. RESULTS: Infections accounted for the greatest number of deaths (bacterial pneumonia, four patients; catheter-related bacteremia, one patient). Persistent pneumonia confirmed by biopsy specimen was usually accompanied by histologic manifestations of acute cellular rejection and was associated with poor patient outcome (ie, death or subsequent development of bronchiolitis obliterans syndrome). In two patients, antibody-mediated rejection either was the immediate cause of death (hyperacute rejection, one patient) or preceded a fatal case of pneumonia (accelerated antibody-mediated rejection, one patient). Other causes of death included hypoxic-ischemic encephalopathy secondary to an intraoperative cardiac arrest (one patient), pulmonary venous thrombosis with bacterial colonization of the thrombotic material (one patient), and ischemic reperfusion injury (one patient). In most patients, more than one type of diagnostic technique was needed to ascertain the cause of the catastrophic decline. CONCLUSIONS: The causes of early posttransplant death in our patient group included infections, antibody-mediated rejection, hypoxic-ischemic encephalopathy secondary to cardiac arrest, pulmonary venous thrombosis, and ischemic reperfusion injury. Because these processes often demonstrate overlapping clinical and morphologic features requiring multiple diagnostic techniques for resolution, a systematic multimodality approach to diagnosis is advantageous for determining the causes of decline in individual patients and for estimating the incidences of the different causes of early graft and patient loss in the lung transplant population.

What happens to renal transplant recipients who lose their grafts?

Little attention has been given to the fate of patients who lose their grafts. We reviewed outcomes of 438 recipients of first renal allografts who underwent transplantation at our institution between January 1, 1988, and December 31, 1997, and lost their grafts or died with a functioning transplant. Of the 438 patients, 168 patients died with a functioning transplant. The most common causes of death were cardiac disease, infection, and cancer. Patients who died with a functioning graft were older (>49 years, 64.3%) than patients who died after returning to dialysis therapy or who are still alive (>49 years, 25.9%). Eighty-six patients (39%) who returned to dialysis therapy were again placed on a cadaveric waiting list. Only 44 patients received a second transplant, of which 30 transplants (68.2%) are still functioning. Our study shows that relatively few patients who lose kidney transplants are returned to the cadaveric waiting list and even fewer undergo retransplantation.

Outcome of kidney transplants in patients known to be flow cytometry crossmatch positive.

BACKGROUND: The clinical significance of the flow cytometry crossmatch has been addressed in several retrospective studies, but the results have been controversial. There are no prospective studies in which patients known to be antibody positive underwent transplantation. METHODS: The flow cytometry crossmatch was performed prospectively in 1130 renal transplant recipients. A decision to perform transplantation was based on whether the positive results were on T or B cells, in the current or peak specimen, and taking into account the presence or absence of other immunological risk factors. One hundred antibody-positive patients received a transplant. Graft survival and rejection episodes were analyzed in this group and compared with 100 crossmatch-negative patients matched for age, sex, race, and time of transplantation. RESULTS: The incidence of rejection at 1 month was higher in antibody-positive patients (26%) than in antibody-negative patients (12%, P<0.01). Early rejection seemed to be more frequent in antibody-positive patients regardless of whether the antibodies were current or historic, or against T or B cells. There were more steroid-resistant rejections in antibody-positive than in antibody-negative patients. However, biopsy specimens showed that vascular lesions that can be associated with humoral rejection were not more frequent in the antibody-positive patients than in the controls. There were no differences in graft survival between the two groups. CONCLUSIONS: Low-level preformed alloantibodies detected by flow cytometry represent a risk of rejection even for patients purposely selected for having no additional immunological risk factors. The risk seems to be due to donor-specific memory rather than to a direct effect of the antibodies. The results indicate that flow cytometry provides useful information to assess donor-recipient compatibility.

Susceptibility of liver allografts to high or low concentrations of preformed antibodies as measured by flow cytometry.

Liver grafts are more resistant to damage by HLA antibodies than other organ allografts, but it is not clear if the antibodies are associated with graft rejection or graft loss, or if different antibody concentrations have different effects. To explore potential associations between antibody concentrations and outcome, preformed IgG antibodies against donor cells were quantified by flow cytometry in 465 consecutive liver transplant recipients. Antibody-positive patients were classified according to whether they had high or low antibody concentrations and analyzed for possible correlation with graft rejection or graft loss. The results showed that the incidence of rejection was not significantly different between antibody-positive and negative patients. However, patients with high antibody concentrations had a higher incidence of steroid-resistant rejections (31% at 1 year) than patients with low antibody (4%) or no antibody (8%, p < 0.0004). These effects were mainly due to T-cell (HLA class 1) antibodies. The overall incidence of rejection at 1 year was 69% for high antibody patients, 51% for patients with low antibodies and 53% for patients with no antibodies (p not significant). In an apparent paradox, antibody-positive patients underwent fewer early graft losses. Thus, the associations of preformed antibodies and outcome depend, on the one hand, on antibody concentrations, and on the other hand on whether the outcome measured is steroid-sensitive rejection, steroid-resistant rejection or graft survival. These complex interactions may explain the controversial results observed in previous studies.

Infusion of donor spleen cells and rejection in liver transplant recipients.

Intact or inactivated donor lymphoid cells have been found to downregulate the alloimmune response in a number of experimental models. We conducted a randomized, prospective, double blind, and placebo-controlled trial to determine whether heat-treated donor spleen cells would affect early rejection after liver transplantation. Donor spleen was obtained during organ procurement for 40 patients undergoing liver transplantation. All patients were treated with cyclosporine, azathioprine and steroids. The patients were randomized after surgery to receive either heat-treated (45 degrees C for 1 h) spleen cells or placebo. Patients underwent protocol biopsies at 1 wk, 4 and 12 months, or as needed. Biopsies were reviewed in a blind fashion and scored according to the Banff consensus criteria. Randomization resulted in 19 patients in the spleen cell group and 21 in the placebo group. One-yr graft survival was 94 and 100%, respectively. Early rejection was more frequent in the spleen cell group (61 vs. 35%, p, not significant). The histopathological rejection activity index at 7 d was also higher for the patients in the spleen cell group: 39% of spleen cell treated patients had a score of 4 or higher as opposed to 5% in the placebo group (p < 0.01). The mean score was 2.9 +/- 2.8 for the spleen cell group versus 1.3 + 1.7 for the placebo group (p = 0.034). It is concluded that heat-treated donor spleen cells given within 24 h after liver transplantation were not clinically beneficial and increased the intensity of rejection in 7-d protocol liver biopsies.

Susceptibility of lung transplants to preformed donor-specific HLA antibodies as detected by flow cytometry.

BACKGROUND: Preformed anti-HLA antibodies are known to have the potential to induce early graft damage in organ transplant recipients. However, in lung transplant recipients, little information exists about the significance of preformed antibodies directed to either class I or class II HLA antigens. METHODS: A two-color flow cytometry cross-match was performed in 92 consecutive lung transplant recipients using serum obtained immediately before transplantation. The presence of preformed antibodies was correlated with the incidence of severe graft dysfunction manifested as pulmonary infiltrates and severe hypoxemia with onset in the first few hours after transplantation. RESULTS: Six patients (6.5%) had low-level anti-donor IgG antibodies detected by flow cytometry, four against T and two against B lymphocytes. Three patients (50%) developed severe graft dysfunction with pulmonary infiltrates and hypoxemia. Two patients responded to treatment, but the third, who had an antibody highly specific for HLA-DR11, died at 48 hr after transplant. Results of histopathologic studies in this patient are consistent with hyperacute rejection and support a pathogenic role of these antibodies. In contrast, of 86 (93.5%) cases with a negative flow cytometry cross-match, only 4 (5%) had severe but reversible early graft dysfunction with pulmonary infiltrates and hypoxemia, attributed to ischemia-reperfusion injury (P<0.005). CONCLUSIONS: Class II, and perhaps class I HLA antibodies at relatively low concentrations represent a risk factor for severe early pulmonary graft dysfunction, with the potential to progress to hyperacute rejection and death.

Poststreptococcal reactive arthritis: clinical characteristics and association with HLA-DR alleles.

OBJECTIVE: To assess the relationship of poststreptococcal reactive arthritis (ReA) to other forms of ReA and rheumatic fever by comparing the frequency of HLA-B27 and DRB1 alleles in these diseases. METHODS: The diagnosis of poststreptococcal ReA was established in 25 children seen in a pediatric rheumatology clinic. HLA-B27 and DRB1 genotyping was performed in 18 of the white American patients. The DRB1 genotyping results were compared with those in 33 patients with rheumatic fever and 190 normal individuals. RESULTS: HLA-B27 was positive in 3 of the 18 poststreptococcal ReA patients, a frequency not different from that found in normal individuals. Compared with normal controls, the frequency of the DRB1*01 allele was higher in poststreptococcal ReA patients (odds ratio [OR] 2.7, P=0.044), while DRB1*16 was increased in patients with rheumatic fever (OR 4.3, P=0.028). CONCLUSION: The association of poststreptococcal ReA with HLA-DRB1*01, but not with HLA-B27, suggests that its pathogenesis may be more similar to that of rheumatic fever than to that of ReA associated with enteric pathogens.

Multicenter evaluation of the flow cytometry T-cell crossmatch: results from the American Society of Histocompatibility and Immunogenetics-College of American Pathologists proficiency testing program.

BACKGROUND: The performance characteristics and interlaboratory comparisons of the T-cell flow cytometry crossmatch remain largely unknown. METHODS: This study was performed using data from the ASHI-CAP proficiency testing program. Four unknown sera and two unknown cells were sent to participating laboratories twice a year for 4 years. RESULTS: In one survey in which different crossmatch techniques were compared, flow cytometry was slightly more sensitive than the antiglobulin method and considerably more sensitive than direct cytotoxicity. However, the proportion of participants in any given survey detecting antibodies in all sera expected to be positive was 50-60% and has not changed over the years. Failure to detect antibodies correlated with low antibody concentration, diluting the unknown serum by the testing laboratory, and with the instrument used. False positive results with normal sera were infrequent. Fluorescence intensity values were not standardized and were highly variable, but when fluorescence units reported by individual laboratories were divided by their own positive-negative cutoff values, results from different centers were more comparable. In general, fluorescence-to-cutoff ratios >5 correlated with complement binding activity, whereas values <5 denoted concentrations below those required to fix complement. CONCLUSIONS: Flow cytometry, as used by most centers, is highly sensitive and allows relative antibody quantitation. Furthermore, the data define objective parameters that may help to standardize the test and improve its predictive value in clinical transplantation.

Antibody screening by enzyme-linked immunosorbent assay using pooled soluble HLA in renal transplant candidates.

The enzyme-linked immunosorbent assay (ELISA) using HLA class I molecules purified from pooled platelets has the potential to detect HLA antibodies with increased efficiency without sacrificing sensitivity or specificity. This test, which was originally developed in our institution, has been independently validated by recent studies and is now commercially available. We now present evidence of its usefulness as a routine HLA antibody screening test for renal transplant patients. A total of 515 patients were tested monthly by ELISA (13.9 tests/patient) and by antiglobulin-enhanced panel reactivity (6.3 tests/patient). In patients found to be unsensitized, the incidence of false-positive results was less for ELISA than for the panel studies. In patients who were highly sensitized, both tests performed equally well, whereas discordant results were registered mainly in cases of mild sensitization. Because 66% of our patients were not sensitized, the ELISA was effective in reducing the number of more involved tests aimed at characterizing the antibodies. These results provide a foundation to use the pooled platelet HLA ELISA on a routine basis for HLA antibody screening.

Significance of preformed anti-donor antibodies in liver transplantation.

The significance of a positive cross-match in liver transplantation remains controversial, as documented by a number of recent conflicting reports. In this study, we evaluated 195 consecutive orthotopic liver transplant recipients in whom the cross-match was either negative or positive for T or B cells. Special emphasis was placed on the outcome of patients with high levels of preformed IgG antibodies directed against donor T cells. IgG anti-donor antibodies were confirmed by flow cytometry in all cases. Of 10 patients with strong T-cell antibodies, there was one early death due to nonimmunological causes. Transplantation was successful in 9/10 patients followed for 3 months to 3 years. Graft survival, incidence of acute rejection, and number of liver biopsies in patients with a positive cross-match (strong T, weak T, or B cell) were not significantly different from those of patients with a negative cross-match. In the strong T cell antibody group, one patient had early graft dysfunction, with extensive hepatic necrosis and histological signs of antibody-induced damage. Two other patients also showed some evidence of possible antibody-mediated events, such as neutrophil infiltration and hepatocyte swelling. These lesions were reversible, and the patients had uneventful recoveries. Thus, in our experience, preformed antibodies did not preclude good graft survival.