S. Iannelli

Validity of flow cytometry for cross-match evaluation in clinical renal transplantation.

This paper reports a 2-year experience of more than 5,000 cross-match tests for renal transplantation. Tests were performed by means of both standard light microscopy and an innovatory method based on flow cytometry, an up-to-date investigative technique for computerized analysis of individual cell characteristics. Flow cytometry allowed a better detection of weak positive reactions (false-negative cross-matches) than light microscopy, thus reducing the risk of selecting candidates with donor presensitization. Transplant clinical outcome supported the value of this original and advanced technological method.

ELISA ANTI-HLA ANTIBODY SCREENING IDENTIFIES NON-COMPLEMENT-FIXING ANTIBODIES RESPONSIBLE FOR ACUTE GRAFT REJECTION. A CASE REPORT

We report on a kidney transplant recipient experiencing an unexpected early acute vascular graft rejection. Retrospective analysis of patient serum samples, utilizing a new ELISA HLA screening technique, revealed that the rejection crisis and the subsequent graft loss were due to a pretransplant donor‐specific pre‐sensitization caused by a non‐complement‐fixing antibody of IgG2 class. The case illustrates the clinical significance of non‐complement‐fixing anti‐HLA antibodies. In addition it is shown that ELISA methods are suitable for detecting potentially harmful donor pre‐sensitization in waiting‐list patients not detectable by standard lymphocytotoxicity techniques. Hence ELISA could be an alternative to flow cytometry for this purpose. It is concluded that screening and cross‐matching techniques which detect non‐complement‐fixing anti‐HLA antibodies could improve graft outcome, and should form part of the immunological monitoring of kidney transplant waiting‐list patients.

Influence of the immunogenetic KIR and HLA systems on long-term renal transplant outcome

BACKGROUND Numerous studies have established the importance of innate immunity, particularly natural killer (NK) cells, in transplantation tolerance. NK cells express killer cell immunoglobulin-like receptors (KIRs) on their surface. By recognizing and binding major histocompatibility complex class I antigens, KIRs prevent autologous cell killing and promote lysis of non-self antigen-presenting cells. This study investigated the role of 16 KIR genes and donor-recipient KIR/HLA combinations on 5-year outcomes in a population of deceased donor kidney transplant recipients. MATERIAL/METHODS We genotyped 126 renal transplant patients and their donors for HLA A, B, C, DR, and KIR genes. Patients underwent standardized transplantation and immunosuppressive protocols and were followed-up for 5 years. Graft function was evaluated by serum creatinine level and glomerular filtration rate calculated using the 4-variable modification of diet in renal disease (MDRD) equation. RESULTS The presence of KIR2DS3 in the recipients was associated with better graft function indexes over time (p<0.05), but this effect was not confirmed by multivariate analysis. Conversely, the presence KIR2DS3 in the recipients combined with the presence of its HLA ligand in the donor had a detrimental effect on the trends of serum creatinine levels and eGFR trends, also confirmed by multivariate analysis. Kidney transplant recipients negative for the KIR2DL1 gene displayed higher creatinine levels after 5 years. Lastly, transplantation of HLA-A3/A11-negative donor kidneys into KIR3DL2-positive patients exerted a protective effect in terms of 5-years outcome (p<0.05). CONCLUSIONS The present study demonstrates an important role of the KIR immunogenetic system in the long-term immune response to kidney transplantation.

Flow cytometry evaluation of urinary sediment in renal transplantation

The value of exfoliative urinary cytology for the diagnosis of different pathological conditions in renal transplantation is widely recognized. The method, however, has not yet gained full acceptance, mainly because identification of the different cells is not always possible by means of standard staining techniques. In view of its characteristics, flow cytometry (FC) seems to represent a consistently reliable, rapid and innovative approach for differentialing the various cells present in the urinary sediment and assessing their number. This study gives the examination result of 223 urinary specimens from 127 transplanted patients selected according to pathology. Sediment cells, collected from fresh urine samples, were washed, treated with a lysing solution, resuspended in saline solution and directly analysed in a FACSCAN cytometer. Morphological evaluation showed: a small number of cells in patients with stable renal function; a larger number of cells, with predominance of lymphocytes, during acute rejection episodes; an absolute predominance of neutrophils during bacterial infection; large-sized cellular debris in cases of post-transplant tubular necrosis; and small cell debris in cases of cyclosporine cytotoxicity. Lymphocyte surface-marker evaluation made it possible to differentiate lymphocyte populations observed during acute rejection episodes (cytotoxic T-cell, CD8 and HLA class II and NK cells) from those detected during bacterial infection (T-cell CD4 positive). These results suggest that urinary FC may be a reliable diagnostic tool in clinical renal transplantation.

Value of panel reactive antibodies (PRA) as a guide to the treatment of hyperimmunized patients in renal transplantation

Patient presensitization represents a considerable problem in candidacy for renal transplantation. While it is well known that hyperimmunized patients--panel reactive antibody (PRA) higher than 60%--create difficulties in donor matching and have a worse outcome than non-hyperimmunized patients, less information is available on patients with an intermediate degree of sensitization (30-60%). In order to evaluate how graft outcome relates to such degrees of sensitization, 241 consecutive transplanted patients were divided into two groups on the basis of their previous years PRA peak: group A, PRA 0-29%; group B, PRA 30-60%. Group A showed a significantly better survival both in the first year (90% vs 79%, P < 0.05) and in the third year (82% vs 64%, P < 0.01). However, detailed analysis of group B demonstrated that some parameters may significantly influence graft outcome: (1) better compatibility on locus DR; (2) a primary kidney transplant; (3) a dialysis duration of less than 6 months; and (4) the prophylactic use of antilymphocyte globulin (ALG).

Mononuclear Cell Antigen Expression after Contact with One-Day Disposable Contact Lens Materials

Purpose: To analyse the effects of 1-day disposable contact lens (CL) material on mononuclear cell activation antigens and adhesion molecules, in an in vitro model set up in order to simulate the first 2 h of wear. Methods: Etafilcon A and Hilafilcon A materials were analysed. Mononuclear cells, collected from peripheral blood samples of normal human donors, were placed in contact with the materials, both new and coated with artificial tear solution. As a control, Thermanox coverslips and a group with no material were included. Immunofluorescence reactions were carried out on cells, to study the distribution of activation (HLA-DR, CD25, CD69) and adhesion (CD11a, CD18, CD54) molecules; the flow cytometry technique was used to analyse cells. Results: Statistically significant results were demonstrated for the CD69 and all the 3 adhesion molecules. Artificial tear coating enhanced the mononuclear cell response. Conclusions: The daily mononuclear cell activation can lead, with time, to production of inflammatory mediators that may be responsible for discomfort or, at worst, intolerance and definitely to CL wear remittance.

Application of flow cytometry in clinical renal transplantation

Flow cytometry (FC) may be considered as a fundamental technique in studying cell biology and pathology. It combines the quantitative character of biochemical methods with the multiparametric capacities of microscope analysis in a high-precision process for rapid analysis of individual cell characteristics. Three original FC techniques routinely applied in the field of renal transplantation are reported in the present study. They concern the donor-recipient cross-match test, the morphological analysis of urinary sediment and the modulation of the density of various membrane antigens on the lymphocyte surface. A common factor underlies all these methods: they aim to provide the physician with a reliable diagnostic tool in clinical renal transplantation.