Extraction Time of Kidneys From Deceased Donors and Impact on Outcomes.

Cold ischemia time (from flush to out-of-ice) and warm ischemia time (from out-of-ice to reperfusion) are known to impact delayed graft function (DGF) rates and long-term allograft survival following deceased donor kidney transplantation. We propose an additional ischemia time, extraction time, beginning with aortic cross-clamp and perfusion/cooling of the kidneys, and ending with removal of the kidneys and placement on ice on the backtable. During this time the kidneys rewarm, suffering an additional ischemic insult, which may impair transplant function. We measured extraction times of 576 kidneys recovered and transplanted locally between January 2006 and December 2008, then linked to Scientific Registry of Transplant Recipients (SRTR) data for outcomes. Extraction time ranged from 14 to 123 min, with a mean of 44.7 min. In SRTR-adjusted analyses, longer extraction time and DGF were statistically associated (odds ratio [OR] = 1.19 per 5 min beyond 60 min, 95% confidence interval [CI] 1.02-1.39, p = 0.03). Up to 60 min of extraction time, DGF incidence was 27.8%; by 120 min it doubled to nearly 60%. Although not statistically significant (OR = 1.19, 95% CI 0.96-1.49, p = 0.11), primary nonfunction rate also rose dramatically to nearly 20% by 120 min extraction time. Extraction time is a novel and important factor to consider when evaluating a deceased donor kidney offer and when strategizing personnel for kidney recovery.

Loss of pediatric kidney grafts during the "high-risk age window": insights from pediatric liver and simultaneous liver-kidney recipients.

Pediatric kidney transplant recipients experience a high-risk age window of increased graft loss during late adolescence and early adulthood that has been attributed primarily to sociobehavioral mechanisms such as nonadherence. An examination of how this age window affects recipients of other organs may inform the extent to which sociobehavioral mechanisms are to blame or whether kidney-specific biologic mechanisms may also exist. Graft loss risk across current recipient age was compared between pediatric kidney (n = 17,446), liver (n = 12,161) and simultaneous liver-kidney (n = 224) transplants using piecewise-constant hazard rate models. Kidney graft loss during late adolescence and early adulthood (ages 17-24 years) was significantly greater than during ages <17 (aHR = 1.79, 95%CI = 1.69-1.90, p < 0.001) and ages >24 (aHR = 1.11, 95%CI = 1.03-1.20, p = 0.005). In contrast, liver graft loss during ages 17-24 was no different than during ages <17 (aHR = 1.03, 95%CI = 0.92-1.16, p = 0.6) or ages >24 (aHR = 1.18, 95%CI = 0.98-1.42, p = 0.1). In simultaneous liver-kidney recipients, a trend towards increased kidney compared to liver graft loss was observed during ages 17-24 years. Late adolescence and early adulthood are less detrimental to pediatric liver grafts compared to kidney grafts, suggesting that sociobehavioral mechanisms alone may be insufficient to create the high-risk age window and that additional biologic mechanisms may also be required.

The aggressive phenotype revisited: utilization of higher-risk liver allografts.

Organ shortage has led to increased utilization of higher risk liver allografts. In kidneys, aggressive center-level use of one type of higher risk graft clustered with aggressive use of other types. In this study, we explored center-level behavior in liver utilization. We aggregated national liver transplant recipient data between 2005 and 2009 to the center-level, assigning each center an aggressiveness score based on relative utilization of higher risk livers. Aggressive centers had significantly more patients reaching high MELDs (RR 2.19, 2.33 and 2.28 for number of patients reaching MELD>20, MELD>25 and MELD>30, p<0.001), a higher organ shortage ratio (RR 1.51, 1.60 and 1.51 for number of patients reaching MELD>20, MELD>25 and MELD>30 divided by number of organs recovered at the OPO, p<0.04), and were clustered within various geographic regions, particularly regions 2, 3 and 9. Median MELD at transplant was similar between aggressive and nonaggressive centers, but average annual transplant volume was significantly higher at aggressive centers (RR 2.27, 95% CI 1.47-3.51, p<0.001). In cluster analysis, there were no obvious phenotypic patterns among centers with intermediate levels of aggressiveness. In conclusion, highwaitlist disease severity, geographic differences in organ availability, and transplant volume are the main factors associated with the aggressive utilization of higher risk livers.

The aggressive phenotype: center-level patterns in the utilization of suboptimal kidneys.

Despite the fact that suboptimal kidneys have worse outcomes, differences in waiting times and wait-list mortality have led to variations in the use of these kidneys. It is unknown whether aggressive center-level use of one type of suboptimal graft clusters with aggressive use of other types of suboptimal grafts, and what center characteristics are associated with an overall aggressive phenotype. United Network for Organ Sharing (UNOS) data from 2005 to 2009 for adult kidney transplant recipients was aggregated to the center level. An aggressiveness score was assigned to each center based on usage of suboptimal grafts. Deceased-donor transplant volume correlated with aggressiveness in lower volume, but not higher volume centers. Aggressive centers were mostly found in regions 2 and 9. Aggressiveness was associated with wait-list size (RR 1.69, 95% CI 1.20-2.34, p = 0.002), organ shortage (RR 2.30, 95% CI 1.57-3.37, p < 0.001) and waiting times (RR 1.75, 95% CI 1.20-2.57, p = 0.004). No centers in single-center OPOs were classified as aggressive. In cluster analysis, the most aggressive centers were aggressive in all metrics and vice versa; however, centers with intermediate aggressiveness had phenotypic patterns in their usage of suboptimal kidneys. In conclusion, wait-list size, waiting times, geographic region and OPO competition seem to be driving factors in center-level aggressiveness.

Pregnancy outcomes in kidney transplant recipients: a systematic review and meta-analysis.

Approximately 50,000 women of reproductive age in the United States are currently living after kidney transplantation (KT), and another 2800 undergo KT each year. Although KT improves reproductive function in women with ESRD, studies of post-KT pregnancies are limited to a few voluntary registry analyses and numerous single-center reports. To obtain more generalizable inferences, we performed a systematic review and meta-analysis of articles published between 2000 and 2010 that reported pregnancy-related outcomes among KT recipients. Of 1343 unique studies, 50 met inclusion criteria, representing 4706 pregnancies in 3570 KT recipients. The overall post-KT live birth rate of 73.5% (95%CI 72.1-74.9) was higher than the general US population (66.7%); similarly, the overall post-KT miscarriage rate of 14.0% (95%CI 12.9-15.1) was lower (17.1%). However, complications of preeclampsia (27.0%, 95%CI 25.2-28.9), gestational diabetes (8.0%, 95%CI 6.7-9.4), Cesarean section (56.9%, 95%CI 54.9-58.9) and preterm delivery (45.6%, 95%CI 43.7-47.5) were higher than the general US population (3.8%, 3.9%, 31.9% and 12.5%, respectively). Pregnancy outcomes were more favorable in studies with lower mean maternal ages; obstetrical complications were higher in studies with shorter mean interval between KT and pregnancy. Although post-KT pregnancy is feasible, complications are relatively high and should be considered in patient counseling and clinical decision making.

Abnormal macrophage development in Hodgkin's disease: a morphometric study.

The development of macrophages from blood monocytes from 22 patients with untreated Hodgkin's disease and 20 normal subjects has been studied at intervals over a 6-day period of suspension culture in the presence of autologous serum and lymphocytes. Morphometric measurements were made on electron micrographs and the results subjected to multivariate and univariate analyses of variance. The cells from the Hodgkin's group showed highly significant differences from normal. The whole-cell volume, surface area, and cell membrane excess, as well as the mitochondrial volume and surface area, showed smaller increases over the period of culture, and the normal increase in mitochondrial profile numbers was not seen. It would appear that although the patients' monocytes transformed into macrophages in culture, their development was seriously deranged.

Morphometry of blood monocytes in malignant lymphoma.

Morphometric methods were used to study the ultrastructure of blood monocytes in 23 patients with Hodgkin's disease, 12 patients with non-Hodgkin's lymphoma, and 20 normal subjects. The results were analysed using both univariate and multivariate methods. Both analyses supported the interpretation that the total mitochondrial contribution to the cellular ultrastructure was less in Hodgkin's disease than in the other two groups, with smaller mitochondrial volume fractions, volumes, and surface areas being found. In the patients with non-Hodgkin's lymphoma univariate analysis suggested that monocyte nuclei were larger, with more euchromatin and a relatively smaller heterochromatin-euchromatin interface; the cell profile area was larger and the surface to volume ratio was smaller compared with normal subjects. Morphological changes have not previously been described in blood monocytes in malignant lymphoma, but they may be the counterpart of the known mononuclear phagocyte dysfunction.

Morphometry of peptide-containing nerves in gut muscle layers: a quantitative approach to the study of autonomic neuro-muscular junctions.

A method for the quantitative assessment of peptide-containing nerves in the gastrointestinal muscle layers is described. Tissue samples are sectioned obliquely, at 45 degrees, in the plane bisecting the long axes of longitudinal and circular muscle fibres, and peptide-containing nerve fascicles are revealed by immunofluorescence. Cross-sectioned fascicles are counted (number/area of muscle) and their length density per volume of muscle layer is calculated. The composition of immunostained nerve fascicles is also assessed, by counting peptide-containing fibres in each, while peptide transmitter co-distributions are studied in parallel using adjacent tissue sections. Thus, the approach described provides a means for the delineation of both quantitative tissue distribution and composition of autonomic neuro-muscular junctions in the gastro-intestinal tract.

Lipopigment in rat hippocampal and Purkinje neurones after chronic phenytoin administration.

The accumulation of lipopigment may indicate ageing, certain diseases and cellular damage, while phenytoin, which has been claimed to cause selective clinical cerebellar dysfunction and degeneration, has been reported to produce increased lipopigment accumulation in rat Purkinje neurones. In the present study, 8 rats received phenytoin, 300 mg/kg/day for 20 weeks, and were compared with a control group of 9 rats in respect of lipopigment in Purkinje and hippocampal neurones. Neuronal lipopigment was identified by fluorescence microscopy. The results did not indicate that phenytoin administration was associated with an increase in the area corresponding to (i.e. within the outlines of) neuronal lipopigment in Purkinje neurones, although the relatively small number of animals limits the power of the study. However, in hippocampal neurones, a two-way analysis of variance for numbers of discrete regions of lipopigment demonstrated a significant interaction (P = 0.003) between, firstly, size categories of discrete regions of lipopigment and, secondly, phenytoin administration or a control procedure. In hippocampal neurones, phenytoin administration was accompanied by a decrease in the numbers of discrete lipopigment regions in the smaller size categories and an increase in the numbers in the larger size categories. This finding indicates the need for further investigation into the effects of phenytoin on brain regions other than the cerebellum, as intellectual deterioration may be related to chronic use of this drug.

A stereological analysis of capillaries in normal and hypertrophic muscle.

A realistic model of the distribution of the partially orientated capillaries in skeletal muscles has been introduced for stereological analyses. Distributional parameters not previously estimated for capillary networks in muscles have been quantified. These include the lengths of capillary per unit volume of tissue ((L(v)) and a dimensionless index of orientation (omega). The present study demonstrates that surgical techniques for inducing skeletal muscle hypertrophy can be an effective stimulus for the proliferation of additional capillaries. In the hypertrophic muscles studied the capillaries become more highly orientated. This suggests that the growth of new capillaries occurs preferentially along the long axis of the muscle.

Common statistical errors in morphometry.

Morphometry is the quantitative measurement of morphological features. Data is usually obtained by probabilistic sampling techniques and is often markedly variable due to intrinsic variations in sampled specimens. Statistical analyses are required both to allow for and to control such variability. Care is needed in the analysis of morphometric data if false conclusions are to be avoided. Examination of any reasonably sized sample of publications in morphometry usually results in the detection of at least several common errors of statistical practice. Commonest errors involve statistics being carried out on untransformed percentage data, statistics on ratios, repeated multiple applications of tests designed for single comparisons, misuse of correlation and violations of statistical assumptions.

Measurement of red blood cell-bound C3b and C3d using an enzyme-linked direct antiglobulin test.

Complement has a complex role in immune mediated red blood cell (RBC) destruction and usually induces extravascular hemolysis of C3b-coated RBCs by erythrophagocytosis and by acting synergistically with cell-bound immunoglobulins. A sensitive two-stage enzyme-linked direct antiglobulin test (ELDAT) was developed and used to measure RBC-bound C3b and C3d in 120 healthy adult individuals and in 60 patients suffering from a variety of conditions, including warm- and cold-type autoimmune hemolytic anemia, neoplasia, and collagen diseases. The results were compared with those of standard agglutination tests employing polyclonal and monoclonal antiglobulin reagents. Small amounts of C3b and C3d were detected on RBCs of the healthy individuals only by the ELDAT and probably reflected the continuing low-grade activation of complement necessary for the maintenance of homeostasis of a variety of physiological systems. The quantity did not vary with age or gender. In the patients, increased amounts of RBC-bound C3b and C3d were relatively common and probably resulted from autoantibody activity, immune-complexes, and nonspecific adsorption. There was no association between positive ELDAT results and the presence of active hemolysis. The ELDAT was far more sensitive than the agglutination tests for detecting RBC-bound C3b and also for C3d if the monoclonal reagent was employed.

The effects of ageing and a vitamin E-deficient diet on the lipopigment content of rat hippocampal and Purkinje neurones.

This study examined associations between a vitamin E-deficient diet, ageing and aspects of the morphology of neuronal lipopigment in rat hippocampal and Purkinje neurones. Groups of rats given a standard diet were killed at 6, 12, 18 and 25 months of age, while a group which had received a vitamin E-deficient diet from 1-18 months were killed at 18 months of age. Lipopigment within a neuronal cell body consists of a number of discrete regions of varying size. These were identified by fluorescence microscopy and a photograph for each individual neurone was projected onto paper, so that the outlines of the discrete regions of lipopigment could be drawn and subjected to morphometric measurements. Both ageing and vitamin E deficiency in relation to hippocampal neurones and vitamin E deficiency in relation to Purkinje neurones (in which ageing effects were not examined), were associated with a significant (< 0.05) increase in the mean total area (per rat) enclosed by the lipopigment outlines. For both vitamin E deficiency and ageing this increase was associated with both an increase in the number of relatively large discrete lipopigment regions and a decrease in the number of relatively small discrete lipopigment regions. The findings in relation to vitamin E deficiency could be explained by an increased rate of lipopigment formation, involving processes which also occur in ageing.

Comparison of three DNA extraction methods on bone and blood stains up to 43 years old and amplification of three different gene sequences.

Extraction of amplifiable DNA-from degraded human material in the forensic context remains a problem, and maximization of yield and elimination of inhibitors of the Polymerase Chain Reaction (PCR) are important issues which rarely feature in comparative studies. The present work used PCR amplification of three DNA sequences (HLA DPB1, amelogenin and mitochondrial) to assess the efficiency of three methods for extracting DNA (sodium acetate, magnetic beads and glass-milk) from 32 skeletal samples and 25 blood stains up to 43 years old. The results, analyzed using multivariate statistics, confirmed that the extraction method was crucial to the subsequent detection of amplification products; the glass-milk protocol performed better than sodium acetate, which was better than magnetic beads. Successful amplification also depended on gene sequence, multiple copy mitochondrial sequences performing best; however, with the singly copy sequences, the longer HLA DPB1 (327 bp) being detected just as often as the shorter amelogenin (106/112 bp). Amplification products were obtained more frequently from blood stains than bone, perhaps reflecting differences inherent in the material, and from younger compared with older specimens, though plateauing seemed to occur after 10 years. PCR inhibitors were more frequent in sodium acetate extracts.