Chris Cornell

How to improve the quality of kidneys from non-heart-beating donors: a randomised controlled trial of thrombolysis in non-heart-beating donors.

Background. The growth in the prevalence of end-stage renal failure has been accompanied with a rise in the waiting list for renal transplantation, which has not been matched by an increase in the kidney donor pool. Non–heart-beating donors (NHBD) offer a potential source of kidneys that are not currently being significantly used. Cardiac arrest for a protracted period of time leads to in situ thrombosis, and, as a consequence, the discard rates for harvested kidneys is higher than brain–stem-dead donors. Methods. A double-blinded, randomised, controlled trial of streptokinase preflush or placebo for NHBD was performed. An initial 30 donors were entered into the study. After routine nephrectomy, NHBD kidneys were machine perfused as part of viability screening before transplantation. Kidneys were then transplanted within 24 hours of cardiac arrest. The primary objectives were the improvements of viability parameters (perfusion, enzyme levels, and histopathology) of the kidneys. The secondary objective was to increase the number of kidneys passing the viability tests and thus transplanted. Results. The two groups of NHBD donors and their kidneys were similar in their descriptive epidemiologic characteristics. The NHBD kidneys from the streptokinase-treated donors had a better appearance at procurement (P <0.001) and performed better during machine preservation (P <0.001). Enzyme biomarkers present in the kidney perfusate were all significantly reduced by the use of streptokinase. These included glutathione S-transferase (P <0.001), fatty acid binding protein (P <0.001), and alanine aminopeptidase (P <0.001). However, although there was a higher proportion of kidneys transplanted through the use of streptokinase (63.6% with streptokinase vs. 42.6% with placebo), this did not achieve significance. There was no difference with respect to postoperative bleeding and transfusion requirements in the recipient whether streptokinase preflush or placebo was used. Conclusion. This study using streptokinase preflush in the NHBD was found to improve the condition of the kidneys retrieved. The improvement in the quality of the donor kidneys was not associated with an increased morbidity in the recipient.

Use of Two Biomarkers of Renal Ischemia to Assess Machine-perfused Non-Heart-beating Donor Kidneys

Renal transplantation is a recognized treatment for end-stage renal failure, offering a better quality of life, independence from dialysis, better survival rates, and decreased cardiovascular complications compared with renal replacement therapy (1). Recently, continued increases in the prevalence of end-stage renal failure and the length of transplantation waiting lists have not been matched by an increased supply of donors from the traditional brainstem dead donors and living donors. Non-heart-beating donor (NHBD) kidneys are regaining importance to substantially increase the kidney donor pool, with estimates indicating a potential increase of 20–40% (2)(3). The use of NHBD kidneys is associated with the development of two adverse conditions, primary nonfunction (PNF) and delayed graft function (DGF), which are caused by the ischemic injury that occurs after cardio-respiratory arrest. Therefore, in centers with NHBD programs, it has become necessary to screen-out damaged kidneys that will never work. Machine preservation using continuous hypothermic pulsatile perfusion has been adopted in NHBD kidney screening initiatives, and perfusion characteristics (flow, pressure, resistance, temperature, weight gain) with enzyme analysis of kidney effluents are used to assess viability. Since its isolation and identification as ligandin, glutathione S -transferase (GST) has evolved as a suitable biomarker in the pretransplantation assessment of machine-perfused NHBD kidneys. Different isoforms have been identified from the distal and proximal renal tubules, and the commonest isoform, α-GST, has previously been used as a biomarker of renal ischemia as assessed by ELISA (4). However, we have previously demonstrated that a spectrophotometric assay for total GST activity (tGST) could reliably be substituted for α-GST (5). Original work by the Maastricht NHBD group has established threshold limits for tGST activity in kidney perfusates for the selection of “viable” kidneys for transplantation (6). This criterion has been introduced in Newcastle, England, where 69 NHBD renal transplants have …

Assessment of non-heart-beating donor (NHBD) kidneys for viability on machine perfusion.

Abstract The shortage of organs has resulted in renewed interest in organs from non-heart-beating donors (NHBD). Viability assessment of such organs may reduce the incidence of delayed graft function and primary nonfunction. In Phase III of the NHBD programme, introduction of machine perfusion enabled the assessment of these marginal donors. Since then the graft survival has been 88.4% compared with the previous phase where machine perfusion or viability assessment was not done (45.5%). The parameters used were total glutathione S-transferase (GST) in the perfusate, the intrarenal vascular resistance (IRVR) and flow characteristics over time. Methods: All NHBD kidneys were machine perfused through a locally developed perfusion system. The viability was assessed by serial measurements of the above-mentioned parameters. Results: Forty-two local NHBD kidneys were retrieved and one kidney was imported, of which 19 donors (i.e. 38 kidneys) were of the uncontrolled (category II) donors. After viability assessment on machine perfusion; two kidneys were discarded due to positive tests for syphilis, four kidneys had high total GST levels, five kidneys due to high IRVR and poor flow characteristics and one did not flush on retrieval. Three kidneys were exported after viability tests. In 28 NHBD kidney recipients, immediate graft function was seen in two kidneys, 22 (84.6%) developed delayed graft function. One kidney had primary non-function, and two recipients lost their grafts, due to chronic rejection and renal vein thrombosis. There were two deaths, unrelated to transplantation. Graft survival was achieved in 88.4% (23/26 graft survival in phase III) of cases. Conclusion: Machine perfusion and assessment of NHBD kidneys has been successfully introduced to the Newcastle NHBD programme. This approach, using renal transplants from largely category II donors produced a success rate of 88.4% which was significantly better than the phase II period (45.5%) of the program p=0.023, Fisher 2 tail test).

Comparison of perfusate activities of glutathione S-transferase, alanine aminopeptidase and fatty acid binding protein in the assessment of non-heart-beating donor kidneys

Background: With a universal shortage of donor organs, screening and selection of marginal kidneys from non-heart-beating donors (NHBDs) provides a valuable source for transplantation. Pre-transplant viability assessment is based on a combination of flow characteristics and assessment of ischaemic tissue injury during NHBD kidney machine perfusion by measurement of total glutathione S-transferase (GST) activity. Successful viability screening has facilitated 69 renal transplants from 60 NHBDs within our transplant centre since 1998, with a first-year graft survival of 90·5%. Methods: We have investigated alanine aminopeptidase (Ala-AP) and fatty acid binding protein (FABP) as alternative biochemical markers to GST for pre-transplantation viability assessment. They were measured, together with GST, in tissue perfusate samples from machine-perfused kidneys from controlled and uncontrolled NHBDs. Results: During machine perfusion, a parallel response was seen for each of the three markers in the perfusates of controlled and uncontrolled NHBD kidneys over the 4-h perfusion. A highly significant correlation was obtained between GST and Ala-AP activities (P<0·0001) and between GST activity and FABP concentration (P<0·0001) in corresponding samples. Conclusion: In this study, GST, Ala-AP and FABP represent equivalent biochemical markers in terms of their ability to quantitate renal tissue injury in human NHBD kidneys. However, there may be some advantage in using all three analytes to provide complementary information on kidney allograft viability.

Improving the quality of kidneys from non-heart-beating donors, using streptokinase: An animal model

Background. Non-heart-beating donors (NHBD) offer a promising potential to increase the cadaveric organ donor pool, especially for kidneys. However, almost half of NHBD kidneys are discarded after viability assessment. This wastage is costly in both human and monetary terms. Intravascular thrombosis at the time of donor death is an event leading to failure in the viability assessment. We have developed an animal model to investigate the effects of the addition of streptokinase to the in situ flush medium before transplant in an attempt to redress the situation. Methods. Two groups of eight healthy young Landrace Yorkshire white pigs were entered into the study. Both groups were subjected to approximately 70 min warm ischemia. Both groups received an intravascular flush with 4 L of Marshall’s solution with heparin (1000 IU/L); one group of pigs also had streptokinase (1.5 MIU/L) added. After donor nephrectomy, all kidneys were machine perfused for 4 hr. Data on perfusion characteristics were taken and samples of kidney effluent were assayed for tissue damage biomarkers, glutathione S-transferase (GST) and alanine aminopeptidase (Ala-AP). Wedge sections of porcine kidneys were taken at the end of perfusion, for histological analysis. Results. Data on machine perfusion parameters (temperature, mean pressure index, resistance) indicate better cooling, lower resistance, and lower mean pressure index in the streptokinase-treated group of pigs. GST and Ala-AP levels were increased in the nonstreptokinase group perfusates. Histopathological analysis of porcine kidneys indicated more ischemic injury and tissue damage in the nonstreptokinase group. Conclusion. The use of streptokinase in this porcine NHBD model conferred benefits to donor kidneys when assessed by machine perfusion. Markers of biochemical injury indicated that less renal tissue damage occurred with the incorporation of streptokinase in the in situ flush medium.

Ischemia-Reperfusion Injury in Cadaveric Nonheart Beating, Cadaveric Heart Beating and Live Donor Renal Transplants

PURPOSE Ischemia-reperfusion injury is gaining importance in transplantation as being responsible for allograft dysfunction. Ischemia occurs during kidney procurement, which is shortest in LDs, and prolonged in cadaveric HBDs and NHBDs. MATERIALS AND METHODS Renal transplants from 17 LDs, 15 HBDs and 19 NHBDs were assessed during reperfusion for biochemical markers of ischemia-reperfusion injury and assessed clinically. Central venous blood sampling was assayed for free radicals using electron spin resonance and tissue injury biomarkers, namely lactate dehydrogenase, fatty acid binding protein, alanine aminopeptidase, lactate and total antioxidants. RESULTS The return to stable renal function was more rapid in LD renal transplants, while recovery continued from 3 months after hospital discharge in NHBD renal transplants. Injury markers, such as lactate dehydrogenase, fatty acid binding protein, alanine aminopeptidase and lactate, were raised at the time of reperfusion, especially in NHBD renal transplants. Free radical release measured by electron spin resonance showed 2 phase release, that is early (0 to 10-minute) and late (20 to 40-minute) release. In NHBD, HBD and LD renal transplants the index of free radical release in the early phase was 1.43, 1.36 and 1.20, and in the late phase it was 1.43, 1.38 and 0.97, respectively (each ANOVA p <0.05). CONCLUSIONS NHBD renal transplants were accompanied by a greater release of free radicals at reperfusion (NHBD > HBD > LD), which was associated with an increase in tissue injury markers at reperfusion. This was reflected in a slower return to stable renal function in NHBD compared to HBD and LD renal transplants.

Comparison of Glutathione S-Transferase and Alanyl Aminopeptidase as Viability Markers in a Porcine NHBD Model

Abstract: A critical shortage in the kidney donor pool has led to a renewed interest in non-heart beating donors (NHBDs). As warm ischaemic tissue injury is a characteristic of NHBD, it is therefore crucial to screen such kidneys for viability to disqualify kidneys that will never work, i.e. develop primary non-function. Viability screening is somewhat time-consuming, usually involving a combination of donor history, visual assessment of the donor kidney at procurement and monitoring during hypothermic machine preservation. Machine perfusion of NHBD kidneys enables physical characteristics to be evaluated by perfusion flow rate and pressure. Glutathione S-transferase (GST) is an established kidney injury biomarker, but its prolonged assay time may result in delays in any potential transplantation. Alanyl aminopeptidase (Ala-AP) has been developed as an alternative novel biomarker of ischaemic renal injury. A porcine NHBD model with differential ischaemic renal injury, using heparin ± streptokinase in the initial in situ flush solution, was developed to evaluate the two biomarker enzymes, GST and Ala-AP. Two groups of 16 porcine NHBD kidneys (heparin alone or heparin + streptokinase) were machine perfused for 4 h, with documentation of pressure, flow, resistance and temperature and samples of kidney perfusate were taken at hourly intrevals. Wedge sections of the kidneys were analysed at termination of perfusion. Histopathology of renal wedge sections, machine perfusion physical parameters and biomarkers demonstrated the differential renal ischaemic insult. The two biomarkers, GST and Ala-AP appear to be correlate with the degree of ischaemic injury and with each other. Mild ischaemic injury occurs in the streptokinase-treated group, with lower efflux of GST and Ala-AP. Assays for Ala-AP activity in humans are significantly shorter than the comparative assays for GST, enabling early decision making on viability. This can, therefore, facilitate transplantation of the potential NHBD kidney within the recommended 24-hour period of cardiac arrest of donor.

Pseudohyperphosphataemia in multiple myeloma.

We read the article by Hawkins! with great interest and would like to report our findings on the subject. Serum phosphate concentrations were measured in 41 patients who have multiple myeloma with identified serum paraproteins by four methods, the first three methods being performed on a Cobas Mira analyser. (1) Direct single step method using ammonium heptamolybdate in sulphuric acid (absorbance: 340 nm); (2) a modification of the molybdate method with the addition of albumin and formate(absorbance: 340 nm); and (3) an enzymatic method in which phosphate ions react with inosine in the presence of nucleoside phosphorylase to give ribose-l-phosphate and hypoxanthine. Hypoxanthine is oxidized to xanthine, then to uric acid and the hydrogen peroxide formed measured as the quinoneimine product when it reacts with 4-aminophenazone (absorbance: 550 nm). (4) Technicon SMAC 3 (absorbance: 340 nm) by courtesy of Mr Ian Gibb, Department of Clinical Biochemistry, Royal Victoria Infirmary, Newcastle upon Tyne, UK. Paraprotein concentration was determined by integration of the electrophoretic peak area using a Corning 720 Scanning Densitometer on 33 samples. Spuriously high phosphate concentrations were obtained in the direct acid/molybdate method in 11 out of 41 samples due to the occurrence of turbidity in the reaction cuvettes. Turbidity, and hence spuriously high phosphate concentrations, was noted in two out of 41 samples by the albumin/formate method which had previously been reported by Bakker et al? to be free of this problem. The enzymatic method and the Technicon SMAC which incorporates a dialysis step were not susceptible to turbidity problems. We did not find any association between paraprotein type and the occurrence of turbidity by chi-squared analysis. The mean of 10gIO paraprotein concentrations in samples where turbidity was noted by methods 1 and/or 2, n = 11, was significantly higher than the mean of Ann Clin Biochem 1992; 29: 237-238