Deceased organ donor screening for human immunodeficiency virus, hepatitis B virus and hepatitis C virus: Discordant serology and nucleic acid testing results.

BACKGROUND: Before the 2014 policy change pertaining to infectious disease screening, many organ procurement organizations (OPOs) were supplementing serologic screening of deceased organ donors with nucleic acid testing (NAT) for human immunodeficiency virus (HIV-1), hepatitis B virus (HBV), and hepatitis C virus (HCV). The number of seronegative, NAT-positive donors has not been directly measured. METHODS: HIV, HBV, and HCV screening results of 11 229 donor referrals evaluated from 2010 to 2013 were obtained from 3 OPO-affiliated laboratories, capturing 35% of all donors in the United States. Laboratories used either polymerase chain reaction assay or transcription-mediated amplification assay to test 9643 deceased donors by NAT. RESULTS: The NAT results were positive in 21 (0.2%), 1 (0.02%), and 11 (0.1%) donors who were seronegative for HIV, HBV, and HCV, respectively. All discordant HIV-1 results were from one laboratory using a polymrease chain reaction assay. Thirteen of the reactive HIV NAT results in seronegative referrals were repeated and were non-reproducibly positive (NRP). Ten (0.1%), 452 (7.8%), and 197 (2.2%) of HIV-, HBV-, and HCV-seropositive donors, respectively, were negative by NAT. CONCLUSIONS: This study highlights the importance of robust quality assurance to minimize NRP NAT results. NAT may allow for increased utilization of organs from HBV- and HCV-seropositive, NAT-negative donors.

Flow cytometry crossmatch reactivity with pronase-treated T cells induced by non-HLA autoantibodies in human immunodeficiency virus-infected patients.

Pronase treatment is used in the flow cytometry crossmatch (FCXM) to prevent nonspecific antibody binding on B cells. However, we have observed unexpected positive results with pronase-treated T cells in human immunodeficiency virus (HIV)-infected patients. In this study, 25 HIV-infected patients without HLA antibodies were tested with pronase-treated and nontreated cells. HIV-positive sera were pretreated with reducing agents and preabsorbed with pronase-treated and nontreated T or B cells before crossmatching. All patients displayed FCXM reactivity with pronase-treated T cells but not with nontreated T cells. None of the patients exhibited FCXM reactivity with pronase-treated and nontreated B cells. These patients displayed FCXM reactivity with pronase-treated CD4+ and CD8+ T cells but not with their nontreated counterparts. Preabsorption with pronase-treated T cells reduced the T cell FCXM reactivity. Preabsorption with pronase-treated B cells or nontreated T and B cells did not have any effect on the T cell FCXM reactivity. Pretreatment with reducing agents did not affect the T cell FCXM reactivity. 15 of 21 HIV-infected kidney allograft recipients with pronase-treated T cell FCXM reactivity display long-term graft survival (1193±631days). These data indicate that HIV-infected patients have nondeleterious autoantibodies recognizing cryptic epitopes exposed by pronase on T cells.

Improving syphilis screening in deceased organ donors.

BACKGROUND: Current U.S. policy requires screening of all deceased organ donors for syphilis infection. To date, information on syphilis test performance in this population is limited. METHODS: All donors with a positive rapid plasma reagin (RPR) and matched donors with negative RPR who were evaluated by one organ procurement organization from January 1, 2000, to September 30, 2012, were retrospectively tested, using retained, residual serum, with two alternate RPR tests and four treponemal-specific tests: A fluorescent treponemal antibody absorption test, a microhemagglutination test, a chemiluminescence immunoassay (CLIA), and a Treponema pallidum particle agglutination (TP-PA) test. RESULTS: Thirty-two of 3,555 (0.9%) potential deceased organ donors screened during the study period showed a positive RPR; 61 RPR-negative matched donor samples were studied as well. Thirteen (40.6%) of the RPR-positive donors were found to be false-positive based on confirmatory TP-PA. As compared to TP-PA, the sensitivity of the fluorescent treponemal antibody absorption, microhemagglutination, and CLIA was 87.5%, 91.7% and 100%, respectively. The CLIA and TP-PA results were 100% concordant. Only 17 (53.1%) of the RPR-positive donors had a total of 46 organs recovered for transplantation. CONCLUSION: Current screening of deceased organ donors by RPR yields a significant number of false-positive results. Use of alternative tests or the routine use of confirmatory tests may reduce the frequency of false-positive results in deceased organ donors.

Virtual crossmatch by identification of donor-specific anti-human leukocyte antigen antibodies by solid-phase immunoassay: a 30-month analysis in living donor kidney transplantation.

Selection of donors for kidney transplantation depends on accurate prediction of risk factors for immunologic rejection. Historically, cytotoxicity crossmatch (CXM) examining lysis of donor cells by preformed anti-human leukocyte antigen (HLA) antibodies (Abs) has been considered the best predictor of immunologic rejection. However, there is much interest in defining anti-HLA Ab specificity in recipient sera by immunoassay to predict crossmatch results and aid in donor selection. Current immunoassays for anti-HLA Abs are highly sensitive, though correlation between Abs detected by immunoassay and their functional relevance in CXM and subsequent transplantation is not well defined. In this study, we retrospectively examined the predictive value of detection of donor-specific anti-HLA Abs (DSA) by Luminex Single Antigen assay from 149 consecutive living donor kidney transplant recipients. We demonstrate that detection of DSA by immunoassay accurately predicted negative crossmatch and graft survival. However, this approach had limited sensitivity for predicting positive crossmatch, attributable to either limited typing of donor HLA-DQ and -DP alleles or due to non-HLA Abs. False-positive prediction of CXM correlated with detection of "weak" Abs with low mean fluorescence intensity (MFI < 2000). Furthermore, we found that a ratio of the MFI of the DSA bead to the MFI of the positive control bead was a better method for identifying weak DSA that did not result in CXM-positive reactions. Interestingly, patients with weak DSA and negative CXM had equivalent graft survival over an 18 month follow-up period, suggesting that weak DSA may not preclude transplantation.

Living donor renal transplantation in the presence of donor-specific human leukocyte antigen antibody detected by solid-phase assay.

Presensitization of donor human leukocyte antigens (HLA) demonstrated through a positive crossmatch is detrimental to allograft function and best avoided through donor exclusion. The clinical significance of alloantibody detectable by sensitive solid-phase assay is not completely defined and is the focus of this study. Pretransplant sera from 64 consecutive living-donor renal transplant recipients were screened by enzyme-linked immunosorbent assay (ELISA) and Luminex assays. Results were analyzed for correlation with clinical outcome. Luminex proved more sensitive than ELISA for alloantibody detection, with three identifiable patterns. Twenty-eight patients were antibody negative, 24 had non-donor-specific antibody (non-DSA), and 12 had donor-specific antibody (DSA). The highest number of rejections (n = 4) and graft losses (n = 6) occurred in the antibody-negative group. The non-DSA group had two graft losses, as did the DSA group. The two graft losses in the DSA group were caused by recurrent focal segmental glomerulosclerosis (FSGS) at 35 months and death with a functioning graft at 32 months. Overall, there were no cases of antibody-mediated rejection and allograft function to 4 years was comparable among all three groups. Under our standard immunosuppression protocol and crossmatch criteria for histocompatibility, alloantibody detectable by Luminex was not detrimental to successful living-donor transplantation.

HLA class I antibody mediated accommodation of endothelial cells via the activation of PI3K/cAMP dependent PKA pathway.

Allografts transplanted across ABO incompatibility or human leucocyte antigen (HLA)-sensitization undergoes antibody (Ab) mediated hyperacute rejection. Depleting anti-graft Ab from the recipient by plasmapheresis prior to transplantation can prevent this Ab-mediated rejection. Under these conditions, allografts have been shown to function even when the Ab rebound in the recipients. We have developed an in vitro model using human aortic endothelial cells (EC) and elucidated the ability of W6/32 HLA class I monoclonal Ab to provide signals following binding to MHC class I molecules. Using this model, we show that ECs undergo caspase 3-dependent cell death by apoptosis upon exposure to saturating concentrations of W6/32 and complement. In contrast, exposure of ECs to sub-saturating concentrations of W6/32 conferred resistance towards Ab/complement-mediated lysis that has been termed accommodation. Accommodated ECs exhibited a significant increase in the expression of anti-apoptotic genes Bcl-xL, Bcl-2 and Heme Oxygenase-1 and the induction of Phosphatidylinositol 3 kinase (PI3K) and cyclic adenosine monophosphate (cAMP) dependent protein kinase A activities that facilitate the phosphorylation of Bad at positions Ser(136) and Ser(112). In conclusion, exposure of sub-saturating concentrations of HLA class I Ab results in the induction of signals downstream that confers resistance to endothelial cells against Ab-complement mediated cell death. Together, the observations made in this study will provide the basis for delineating the molecular mechanisms involved in mediating accommodation and developing strategies to induce accommodation in grafts prior to transplantation in highly sensitized patients.

Liver transplantation for hepatocellular carcinoma: expanding special priority to include stage III disease.

HYPOTHESIS: After liver transplantation, patients with stage III hepatocellular carcinoma (HCC) experience survivals similar to those of patients with less advanced disease and of matched control subjects. DESIGN: Retrospective review of prospectively collected database. SETTING: University hospital. PATIENTS: Fifty-one adults with HCC and 153 matched adults without HCC who underwent orthotopic liver transplantation. MAIN OUTCOME MEASURES: One-, 3-, and 5-year survivals for all groups. After matching for year of transplantation, age, sex, and underlying liver disease, long-term survival was compared between groups. Rates of recurrence were also measured in the HCC groups. RESULTS: From August 1, 1985, to February 28, 2002, we performed 635 adult liver transplantations, including 51 (8%) in patients with HCC. One hundred fifty-one patients without HCC who underwent transplantation were selected as controls. Patient demographic features were similar between case-control groups. The overall 5-year survival trend was worse for patients with HCC vs their matched controls (48% vs 65%; P = .07); however, this survival disadvantage was eliminated when patients with stages I through III HCC were combined and compared with their matched controls (59% vs 63%; P = .96). Survival of patients with stage III disease was comparable to that of matched controls (65% vs 59%; P = .44). CONCLUSIONS: For patients with stages I through III disease, long-term survival is comparable to that of matched controls, and only patients with stage IV disease experience poorer survival. Consideration should be given to granting exception points to patients with stage III disease.

Hospital-independent organ recovery from deceased donors: a two-year experience.

Early experience with deceased donor (DD) organ recovery outside of the hospital setting was found to be safe, efficient and cost effective. A 2-year experience under current practice protocols implemented to further process improvements is now reviewed. From December 1, 2001 to December 31, 2003, 123 criteria eligible DDs were transferred from local and regional hospitals to the Mid-America Transplant Services (MTS) facility for organ and tissue recovery. In this retrospective analysis, outcome comparisons were made with 79 conventional hospital-based recoveries. Compared to hospital recoveries, MTS facility recoveries were associated with significantly reduced critical care unit time (819 vs. 502 min), time to cross-clamp following brain death (966 vs. 731 min), operating room delay (54 vs. 9 min) and a trend toward reduced organ cold ischemia times which reached significance for heart and lungs when compared to regional hospital recoveries (147 vs. 221 and 192 vs. 327 min). MTS facility recovery afforded substantial cost savings over local and regional hospital recoveries (US 6,690 dollars and US 5,452 dollars per donor, respectively). The current practice of DD recovery at the MTS facility was applicable for most recoveries, improved process efficiency, and afforded substantial cost savings without donor compromise.

Prospective, pilot, open-label, short-term study of conversion to leflunomide reverses chronic renal allograft dysfunction.

Leflunomide (LEF) is a synthetic isoxazole derivative with anti-inflammatory and antiviral properties, which has been reported to prevent acute rejection and delay progression of chronic allograft nephropathy (CAN) in animal models. We performed a pilot, crossover trial in 22 renal transplant recipients who were converted from azathioprine (AZA) or mycophenolate mofetil (MMF) to LEF in an effort to slow progression of renal dysfunction [deteriorating renal function (n = 5), cyclosporine (CyA) nephrotoxicity (n = 4) or biopsy-proven CAN (n = 13)]. Baseline maintenance immunosuppression consisted of CyA, AZA or MMF and prednisone. Six-month postconversion patient and graft survival was 100% and 91%, respectively. Mean serum creatinine 6months preconversion was 2.2 +/- 0.6mg/dL, at initiation was 3.0 +/- 1.1 mg/dL, and 6 months postconversion was 2.8 +/- 1.3 mg/dL. The rate of change in serum creatinine was 35 +/- 39%/6 months preconversion and -5 +/- 21%/6 months postconversion to LEF (p = 0.003). Two patients discontinued LEF for diarrhea and myalgia. No readmissions, increase in liver function tests, infections or acute rejection episodes occurred. Mean CyA levels did not change, 146 +/- 72 ng/ mL pre-LEF vs. 132 +/- 51 ng/mL post-LEF, p = NS. Conversion to LEF reversed progression of chronic renal allograft dysfunction with minimal toxicity.

Cadaveric-donor organ recovery at a hospital-independent facility.

BACKGROUND: Of the many logistic issues addressed throughout the cadaveric organ donation process, timely access to the operating theater for surgical recovery of organs and tissues can be one of the most problematic. Delay in recovery adds to cost, risks organ viability, and compounds donor family anguish with compromise to donation consent. METHODS: From March 1 to November 30, 2001, 25 cadaveric donors were selected and successfully transferred from local donor critical care units to an off-site facility, which was constructed, equipped, and staffed to allow surgical recovery of organs and tissues. Assessment of the recovery process and outcome results was compared to 42 consecutive, hospital-based, organ recoveries within the Mid-American Transplant Services (MTS) organ procurement organization region. RESULTS: Twenty-five MTS-facility and 42 hospital organ recoveries were successfully conducted with no technical losses and satisfactory function in all 206 transplanted organs. From the MTS donor group, 7 hearts, 4 lungs, 21 livers, 28 kidneys, and 5 pancreases were successfully transplanted. Statistically significant in the MTS group was higher donor age (44.1 vs. 30.2 years), shorter total donor management time (539 vs. 718 min), reduced delay in start of surgery (25 vs. 77 min), shorter cold ischemia time for recovered pancreases (355 vs. 630 min), and reduced mean cost per donor ($10,636 vs. $12,918). There was no significant difference in race, gender, cause of death, vasopressor requirements, organs per donor recovered (3.12 vs. 3.62) or transplanted (2.60 vs. 3.36), rate of tissue recoveries (68% vs. 67%), total operating room time (207 vs. 200 min.), or cold ischemia time (excluding pancreas). CONCLUSIONS: Cadaveric-donor multiorgan and tissue recovery at this hospital-independent facility was successfully accomplished in a manner indistinguishable from conventional hospital organ and tissue recovery. The intended objectives of improved access to the operating theater were realized along with the added benefit of significant cost savings and convenience to hospital personnel and surgical recovery teams.

In vitro T cell proliferation from kidney allograft biopsies with unremarkable pathology: new strategies for an old problem.

Acute rejection of renal allografts is mediated by infiltrating alloreactive T cells. The goals of this study were to correlate T cell proliferation with rejection and to determine whether T cell proliferation in the absence of rejection would predict future rejection episodes. Toward this, kidney biopsies (n=100) were cultured in the presence of interleukin-2. Cultures were examined at 4, 24, and 48 hr for T cell proliferation. A strong correlation was observed between T cell proliferation at any time point and rejection. There was not a significant correlation between T cell proliferation in biopsies with no rejection and the occurrence of a rejection episode within 2 months. However, T cell proliferation after 4 hr was a better predictor of the occurrence of rejection within 2 months compared with observations after 24 and 48 hr. Therefore, a subgroup of patients with unremarkable biopsies but T cell proliferation may be at risk for rejection and warrant closer observation and possible tailoring of immunosuppression.