Donor Specificity but Not Broadness of Sensitization Is Associated With Antibody-Mediated Rejection and Graft Loss in Renal Allograft Recipients.

Panel-reactive antibodies are widely regarded as an important immunological risk factor for rejection and graft loss. The broadness of sensitization against HLA is most appropriately measured by the "calculated population-reactive antibodies" (cPRA) value. In this study, we investigated whether cPRA represent an immunological risk in times of sensitive and accurate determination of pretransplantation donor-specific HLA antibodies (DSA). Five hundred twenty-seven consecutive transplantations were divided into four groups: cPRA 0% (n = 250), cPRA 1-50% (n = 129), cPRA 51-100% (n = 43), and DSA (n = 105). Patients without DSA were considered as normal risk and received standard immunosuppression without T cell-depleting induction. Patients with DSA received an enhanced induction therapy and maintenance immunosuppression. Surveillance biopsies were performed at 3 and 6 months. Median follow-up was 5.7 years. Among the three cPRA groups, there were no differences regarding the 1-year incidence of ABMR (p = 0.16) and TCMR (p = 0.75). The 5-year allograft survival rates were similar and around 87% (p = 0.28). The estimated glomerular filtration rate at last follow-up was 50-53 mL/min (p = 0.45). On multivariable Cox proportional hazard analysis, the strongest independent predictor for ABMR and (death-censored) graft survival was pretransplantation DSA. cPRA were not predictive for ABMR, TCMR, or (death-censored) graft survival. We conclude that with current DSA assignment, the broadness of sensitization measured by cPRA does not imply an immunological risk.

Lupus nephritis and non-Hodgkin lymphoma simultaneously diagnosed in a patient on methimazole.

A 78 year old white male on methimazole due to Grave's thyroiditis presented with acute renal failure after a short term history of progressive shortness of breath, malaise, myalgias, arthralgias, and bilateral lower limb swelling. The abdomen was remarkable for splenomegaly and lower extremities for erythema nodosum. No peripheral lymphadenopathy was detected. Serum albumin was 1.7 g/dl and very high erythrocyte sedimentation rate. Urine sediment was very active with dysmorphic red blood cells and casts and significant proteinuria (6.6 grams/day). Serum complements were abnormally low and antinuclear and anti-DNA antibodies were positive. Renal histopathology revealed membranoproliferative glomerulonephritis, along with a full house pattern on IFF consistent with lupus nephritis. Bone marrow aspiration revealed a 40% infiltration by a lymphocyte population of small cells consistent with a B cell non-Hodgkin lymphoma. The patient was treated with methylprednisolone, cyclophosphamide and rituximab and acute dialysis. Over the following weeks the patient became dialysis independent and returned to his baseline GFR.

Direct sequencing of double-stranded PCR products incorporating a chemiluminescent detection procedure.

A simple and reliable method is described for direct sequencing of material generated by the polymerase chain reaction (PCR). Sequencing reactions can be performed directly on PCR products without the need for purification of the template by removal of residual deoxyribonucleoside triphosphates or primers. The coupling of a chemiluminescent detection system with the use of the same primers in the initial and sequencing PCR's allows for sequencing of a number of PCR products on the one gel.

Assessment of PCR of the D17S30 locus for forensic identification.

PCR analysis of the VNTR locus D17S30 was assessed for its potential use in forensic identification analysis. "Allelic drop-out," the inefficient amplification of some alleles, complicates the interpretation of DNA typing at this locus. PCR conditions were varied in an effort to improve amplification of the alleles at this locus. Such changes included the use of denaturants, formamide and DMSO, to overcome any incomplete denaturation of template strands due to GC content or allele size. Lowering the annealing temperature during the PCR cycle enhanced the amplification of a larger fragment, but this was not related to the D17S30 locus. It appears that the structure of the genome of some individuals rendered PCR amplification inefficient at this locus.

Purification of human leucocyte DNA: proteinase K is not necessary.

A rapid nontoxic method for the purification of DNA from human leucocytes is described. Preliminary experiments which tested different methods of DNA purification indicated that digestion of proteins with proteinase K was unnecessary. This led to the development of a simple procedure involving lysis of the cells in SDS followed by extraction with 6 M NaCl. The method described overcomes the requirement for lengthy incubations in the presence of expensive proteinase K and subsequent extraction with toxic chemicals.