Morphometric and visual evaluation of fibrosis in renal biopsies.

Interstitial fibrosis is an outcome measure of increasing importance in clinical trials of both renal transplantation and native disease, but data on the comparative advantages of fibrosis measurement methods are limited. We compared four morphometric techniques and contrasted these with two visual fibrosis-scoring methods on trichrome-stained slides. Two morphometric methods included whole-slide digital images: collagen III immunohistochemistry and a new technique using trichrome and periodic acid-Schiff subtraction morphometry; the other two methods included Sirius Red with and without polarization on multiple digital fields. We evaluated 10 serial sections from 15 renal biopsies with a range of fibrosis extent and diagnoses on duplicate sections with each method on separate days. Three pathologists performed visual scoring on whole-slide images. Visual and morphometric techniques had good to excellent interassay reproducibility (R(2) = 0.62 to 0.96) and interobserver reproducibility (R(2) = 0.75 to 0.99, all P < 0.001). Morphometry showed less variation between observers than visual assessment (mean of 1% to 5% versus 11% to 13%). Collagen III, Sirius Red unpolarized, and visual scores had the strongest correlations (R(2) = 0.78 to 0.89), the greatest dynamic range, and the best correlation with estimated GFR (R(2) = 0.38 to 0.50, P < 0.01 to 0.001). Considering efficiency, reproducibility, and functional correlation, two current techniques stand out as potentially the best for clinical trials: collagen III morphometry and visual assessment of trichrome-stained slides.

Posttransplant lymphoproliferative disorder associated with an Epstein-Barr-related virus in cynomolgus monkeys.

BACKGROUND: Human posttransplant lymphoproliferative disorder (PTLD) has been shown to be associated with Epstein-Barr virus (EBV) infection. Primate animal models of PTLD and the use of molecular markers in its diagnosis have not been reported. This study was designed to evaluate the frequency, pathology, and molecular characteristics of PTLD in cynomolgus kidney allograft recipients. METHODS: Over a 5-year period (January 1995 to November 2000), 160 primate renal transplants were performed at the Massachusetts General Hospital (MGH). Of these, all cases (n=9) that developed PTLD were included. H&E stained paraffin sections of all available tissue samples from the cases were evaluated for the presence of PTLD. Immunoperoxidase staining for T cells (CD3), B cells (CD20), kappa and lambda light chains as well as EBV nuclear antigens (EBNA2) and latent membrane proteins (EBV LMP-1) was done on paraffin sections using standard immunohistochemical (IHC) methods. In situ hybridization for EBV encoded RNA (EBER) was performed in all tissue samples with atypical lymphoid proliferations, using a novel EBER nucleotide probe based on consensus gene sequences from EBV and the related herpes lymphocryptoviruses (LCV) infecting baboons and rhesus macaques. RESULTS: Of 160 consecutive primate renal transplants performed at MGH, 5.6% developed PTLD 28-103 days after transplantation. In all cases, the lymph nodes were involved and effaced by an atypical polymorphous lymphoid proliferation of EBER+ B cells, diagnostic for PTLD. Focal staining for EBNA-2 was noted in tumor cells. In 67% (six of nine) the PTLD infiltrates were present in extra nodal sites, notably liver (56%), lung (44%), heart (44%), renal allograft (44%), and native kidney (22%). The spleen was involved by PTLD in all four animals that had not undergone a pretransplant splenectomy. The PTLD morphology was similar in all cases and predominantly of the polymorphous type, however, some of these showed areas that appeared minimally polymorphous. No cases of monomorphic PTLD were seen. CONCLUSIONS: By in situ hybridization, expression of the RNA product, homologous for EBV-encoded RNA (EBER) was identified in the PTLD tumor cells of all cases, indicating latent primate EBV- related infection. This report identifies a novel animal model of EBV associated PTLD in the setting of kidney transplantation, with valuable implications for managing and understanding human PTLD and oncogenesis.

C4d deposition in cardiac allografts correlates with alloantibody.

BACKGROUND: The presence of C4d along the peritubular capillaries in kidney allografts correlates with the presence of anti-donor serum alloantibodies. We applied C4d staining to cardiac allograft and non-allograft biopsies to determine if C4d staining in heart allografts correlates with anti-donor serum alloantibodies. METHODS: We stained for C4d all available frozen tissue biopsies from cardiac transplant recipients between 1997 and 2002, including autopsies. Two hundred twenty-one tissue samples from 124 patients were analyzed. Included in both groups were a variety of International Society for Heart and Lung Transplantation (ISHLT) grades of rejection plus post-implant cardiac ischemic injury (PIMI), and biopsies from patients who had received OKT3. Patients were matched by age, gender and interval after transplantation. Forty-four additional controls were included from patients biopsied for non-transplant-related cardiac disease. RESULTS: C4d staining of the myocardial capillaries correlated well with the presence of anti-donor alloantibodies. Twenty-one of 25 biopsies from patients with anti-donor alloantibodies showed C4d staining (84%), whereas only 7 of 60 without anti-donor alloantibodies stained for C4d. C4d staining did not correlate with ischemia or OKT3 therapy. Only 4 of 44 non-transplant biopsies stained for C4d (9%). An example of the clinical utility of C4d staining in patient care is presented. CONCLUSIONS: C4d staining of the capillaries in cardiac allografts correlates well with anti-donor serum alloantibodies, is a useful assay to verify alloantibody deposition, and can be used to establish one of the criteria for antibody-mediated cardiac rejections.