Tim James

Advantages of normothermic perfusion over cold storage in liver preservation.

Background. To minimize the ischemia-reperfusion injury that occurs to the liver with the current method of preservation and transplantation, we have used an extracorporeal circuit to preserve the liver with normothermic, oxygenated, sanguineous perfusion. In this study, we directly compared preservation by the standard method of simple cold storage in University of Wisconsin (UW) solution with preservation by perfusion. Methods. Porcine livers were harvested from large white sows weighing between 30 and 50 kg by the standard procedure for human retrieval. The livers were preserved for 24 hr by either cold storage in UW solution (n=5) or by perfusion with oxygenated autologous blood at body temperature (n=5). The extracorporeal circuit used included a centrifugal pump, heat exchanger, and oxygenator. Both groups were then tested on the circuit for a 24 hr reperfusion phase, analyzing synthetic function, metabolic capacity, hemodynamics, markers of hepatocyte and reperfusion injury, and histology. Results. Livers preserved with normothermic perfusion were significantly superior (P =0.05) to cold-stored livers in terms of bile production, factor V production, glucose metabolism, and galactose clearance. Cold-stored livers showed significantly higher levels of hepatocellular enzymes in the perfusate and were found to have significantly more damage by a blinded histological scoring system. Conclusions. Normothermic sanguineous oxygenated perfusion is a superior method of preservation compared with simple cold storage in UW solution. In addition, perfusion allows the possibility to assess viability of the graft before transplantation.

Evidence of oxidative stress in chronic venous ulcers

Reactive oxygen species have been implicated in the impaired healing of chronic leg ulcers but little direct evidence is available. We have observed a significant (p < 0.01) elevation of the allantoin : uric acid percentage ratio, a marker of oxidative stress, in wound fluid from chronic leg ulcers (median 17, range 8–860) compared to both paired plasma (median 2, range 1–8) and acute surgical wound fluid (median 4, range 3–7). However, the allantoin : uric acid percentage ratio did not differ significantly between chronic wounds that healed and those that failed to heal. Neutrophil elastase was elevated 30‐ to 1300‐fold in chronic wound fluid compared to plasma and there was a correlation (r 2 = 0.742) between wound fluid elastase and the allantoin : uric acid percentage ratio. Total antioxidant capacity of wound fluid, as measured with a chemiluminescence assay, did not show a correlation (r 2 = 0.03) with the observed oxidative stress. These observations suggest that conditions of localized oxidative stress, possibly related to neutrophil‐associated production of reactive oxygen species, are present in chronic leg ulcers. It is possible that future therapeutic strategies aimed at reducing oxidative stress, in addition to good standard care, could improve healing rates of chronic wounds. (WOUND REP REG 2003;11:172–176)

Non-heart-beating donor porcine livers: the adverse effect of cooling.

Normothermic preservation has been shown to be advantageous in an experimental model of preservation of non‐heart‐beating donor (NHBD) livers, which have undergone significant warm ischemic injury. The logistics of clinical organ retrieval might dictate a period of cold preservation prior to warm perfusion. We have investigated the effects of a brief period of cold preservation on NHBD livers prior to normothermic preservation. Porcine livers were subjected to 60 minutes of warm ischaemia and then assigned to following groups: Group W (n = 5), normothermic preservation for 24 hours; and Group C (n = 6), cold preservation in University of Wisconsin solution for 1 hour followed by normothermic preservation for 23 hours (total preservation time, 24 hours). Synthetic function (bile production and factor V production) and cellular damage were compared on the ex vivo circuit during preservation. There was no significant difference in the synthetic function of the livers (bile production and factor V production). Markers of hepatocellular damage (alanine aminotransferase and aspartate aminotransferase release), sinusoidal endothelial cell dysfunction (hyaluronic acid), and Kupffer cell injury (β‐galactosidase) were significantly higher in Group C. The histology of the livers at the end of perfusion was similar. In conclusion, a brief‐period cold preservation prior to normothermic perfusion maintains the synthetic function and metabolic activity but results in significant hepatocellular damage, sinusoidal endothelial cell dysfunction, and Kupffer cell injury. Transplant studies are required to establish whether livers treated in this way are viable for transplantation. (Liver Transpl 2005;11:35–38.)

Assessment of High-Sensitivity C-Reactive Protein Levels as Diagnostic Discriminator of Maturity-Onset Diabetes of the Young Due to HNF1A Mutations

OBJECTIVE Despite the clinical importance of an accurate diagnosis in individuals with monogenic forms of diabetes, restricted access to genetic testing leaves many patients with undiagnosed diabetes. Recently, common variation near the HNF1 homeobox A (HNF1A) gene was shown to influence C-reactive protein levels in healthy adults. We hypothesized that serum levels of high-sensitivity C-reactive protein (hs-CRP) could represent a clinically useful biomarker for the identification of HNF1A mutations causing maturity-onset diabetes of the young (MODY). RESEARCH DESIGN AND METHODS Serum hs-CRP was measured in subjects with HNF1A-MODY (n = 31), autoimmune diabetes (n = 316), type 2 diabetes (n = 240), and glucokinase (GCK) MODY (n = 24) and in nondiabetic individuals (n = 198). The discriminative accuracy of hs-CRP was evaluated through receiver operating characteristic (ROC) curve analysis, and performance was compared with standard diagnostic criteria. Our primary analyses excluded ∼11% of subjects in whom the single available hs-CRP measurement was >10 mg/l. RESULTS Geometric mean (SD range) hs-CRP levels were significantly lower (P ≤ 0.009) for HNF1A-MODY individuals, 0.20 (0.03–1.14) mg/l, than for any other group: autoimmune diabetes 0.58 (0.10–2.75) mg/l, type 2 diabetes 1.33 (0.28–6.14) mg/l, GCK-MODY 1.01 (0.19–5.33) mg/l, and nondiabetic 0.48 (0.10–2.42) mg/l. The ROC-derived C-statistic for discriminating HNF1A-MODY and type 2 diabetes was 0.8. Measurement of hs-CRP, either alone or in combination with current diagnostic criteria, was superior to current diagnostic criteria alone. Sensitivity and specificity for the combined criteria approached 80%. CONCLUSIONS Serum hs-CRP levels are markedly lower in HNF1A-MODY than in other forms of diabetes. hs-CRP has potential as a widely available, cost-effective screening test to support more precise targeting of MODY diagnostic testing.

Simple biochemical markers to assess chronic wounds.

We investigated the potential for the biochemical analysis of chronic wound fluid to predict healing using simple and widely available analytes in an out‐patient clinic setting. Wound fluid was collected from 12 patients attending a leg ulcer clinic and analyzed for a variety of analytes, including lactate, total protein, and albumin. Twelve weeks after collection the wound was assessed for healing (defined as complete healing or greater than 50% reduction in wound size). The median total protein (44.3 ± 8.8 g/l) and albumin (25.0 ± 2.3 g/l) concentrations in exudate collected from four healing wounds were significantly higher (p < 0.05) than in exudate from eight nonhealing wounds (median total protein 29.7 ± 7.6 g/l, median albumin 17.0 ± 4.3 g/l). No significant difference was observed for lactate. A second specimen of wound fluid was collected from four of the patients (three nonhealing and one healing). The protein analysis confirmed the pattern observed for the first collection: nonhealing wounds had total protein and albumin which remained low compared to healing wounds. No wound with an exudate albumin of less than 20 g/l healed. Both total protein and albumin are stable analytes which can be easily measured in any laboratory and may offer a simple biomarker of healing in chronic wounds.

Ultrasensitive Label Free Electrical Detection of Insulin in Neat Blood Serum

Electrical assays potentially offer a highly sensitive, cheap, portable, automated, and multiplexed means of protein biomarker detection, characteristics with an ability to underpin both disease stratification and the development of point of care diagnostics. Most conveniently applied in a reagent free manner, all sensitive assays such as these suffer, however, from profound problems when applied in complex fluids such as blood serum. We report herein, the development, and clinical application, of a highly sensitive and selective electrical insulin biosensor based on a chemisorbed zwittorionic polymer support and a novel reagentless sensing technique based on phase monitoring electrochemical impedance spectroscopy. The polymer adlayer is exceptionally effective in both reducing background response and maintaining receptive antibody binding efficacy, while the non-Faradaic analysis avoids potential interference from background electro-active molecules. Applied to the detection of even a low molecular weight protein (here, insulin), a linear range from 0.1 to 200 pM and an unprecedented femtomolar detection limit are possible in undiluted blood serum.

Utility of cardiac biomarkers for the diagnosis of type V myocardial infarction after coronary artery bypass grafting: insights from serial cardiac MRI

Objectives To examine, using cardiac magnetic resonance (CMR), the utility of cardiac biomarkers for the determination of myocyte necrosis and function after coronary artery bypass grafting (CABG), and to test the recently updated guidelines for the diagnosis of postoperative myocardial infarction (type V MI). Methods and results Forty patients included in a single-centre randomised trial of two surgical techniques for performing CABG underwent serial assessment with CMR biochemical markers. Cine and delayed enhancement CMR (DE-CMR) for assessment of left ventricular (LV) function and irreversible myocyte necrosis was performed and levels of troponin I (TnI) and creatine kinase-MB isoform (CK-MB) were determined. The area under the curve for TnI strongly correlated with the mass of new myocyte necrosis as assessed by DE-CMR (r=0.83, p<0.001), compared with CK-MB (r=0.39, p=0.06). Furthermore, routine assessment of TnI alone at 24 h (>6.6 μg/l) predicted type V MI on DE-CMR with a sensitivity of 88% and specificity of 97%, whereas CK-MB predicted type V MI with a sensitivity of 75% and specificity of 87%. Conclusions Biomarkers alone (TnI), at an appropriate threshold appear robust for the detection of type V MI, independently of supplementary evidence, as suggested by the ESC/ACCF/AHA/WHF criteria. Clinical trial registration information The study is listed on the Current Controlled Trials Registry: ISRCTN41388968. URL: http://www.controlled-trials.com.

Redox and label-free array detection of protein markers in human serum.

A substantial outstanding challenge in diagnostics and disease monitoring is an ability to rapidly and conveniently assay for protein biomarkers within complex biological media. Label-free electroanalytical methods present, arguably, the most promising and scalable means of achieving this but, as with all label-free assays, can struggle with response selectivity issues that arise from nonspecific surface interactions. Impedimetric methods are ultrasensitive and have been applied to the quantification of a wide range of proteins but have not previously been utilized in a multiplexed format capable of operation in complex analytical fluid. Herein, we present the use of thermally cross-linked poly(ethylene glycol) (PEG) polymer sensory array interfaces in the ultrasensitive quantification of two protein markers, insulin and C-reactive protein (CRP). This was achieved with detection limits of 171 ± 19 fM and 150 ± 10 pM, respectively. Significantly, the arrays not only enable the simultaneous, fast, nonamplified, and label-free detection of both markers without reagent addition but do so with little cross talk, even in human serum. A blind analysis of 17 real patient samples generated results in excellent agreement with those obtained through a clinically approved chemiluminescence assay.

Optimisation of an enzymatic method for β-galactosidase

Abstract Background : The enzyme β-galactosidase present in the Kupffer cells of the liver has potential as a marker of liver dysfunction prior to transplantation. Spectrophotometric methods have insufficient sensitivity. Methods : Fluorimetric methods have the required sensitivity and we have optimised such a method in a microtitre plate format to improve its utility. β-galactosidase acts on the substrate 4-methylumbelliferyl-galactoside (MUG) to produce 4-methylumbelliferone (4-MU), detected fluorimetrically with excitation wavelength 355 nm and emission wavelength 460 nm. Results : Reaction conditions in a citrate–phosphate buffer were optimised to give maximal enzyme activity: pH was optimal at 4.4 (range investigated 3.6–5.0) and substrate concentration at 3.33 mmol/l. A small specimen volume (10 μl) in 80 μl of substrate solution produced adequate fluorescent yield after an incubation period of 30 to 60 min at 37 °C. Reaction was terminated by addition of 200 μl of glycine–NaOH, pH 12.8. The assay is linear to 3000 U/ml. The intra-assay coefficient of variation (CV%) at 50, 502, and 2012 U/ml was 4.7, 3.1, and 3.4, respectively ( n =10). Inter-assay CV% at 51, 496, and 1986 U/ml was 7.0, 4.0, and 3.9, respectively ( n =10). Conclusions : The assay has greater practical utility and demonstrated significant differences in the perfusate β-galactosidase between cold-stored and warm-perfused livers in a porcine model of transplantation.

The effects of precision, haematocrit, pH and oxygen tension on point-of-care glucose measurement in critically ill patients: a prospective study.

Background Critical care glycaemic control protocols commonly have treatment adjustment (target) ranges spanning ≤2 mmol/L. These require precise point-of-care glucose measurement, unaffected by other variables, to avoid measurement errors increasing glycaemic variability and hypoglycaemic episodes (both strongly associated with mortality in critically ill patients). Methods A prospective 206 intensive care patient study was carried out. Arterial glucose concentrations were measured in duplicate using three point-of-care instruments (MediSense Precision PCχ, HemoCue DM and Radiometer 700), a central laboratory instrument (Siemens ADVIA), and in whole blood and plasma using the Yellow Springs Instruments 2300 instrument. Results Coefficients of variation for the MediSense, HemoCue, Radiometer and Siemens instruments were 5.1%, 2.5%, 2.1% and 2.3%, respectively. Compared with the Siemens instrument, the bias (95% limits of agreement) for the MediSense, HemoCue and Radiometer instruments were 0.0 (−1.4 to 1.4), 0.0 (−1.2 to 1.1) and −0.2 (−0.9 to 0.6) mmol/L, respectively. The whole blood–plasma glucose concentration difference was significantly affected by the haematocrit. MediSense and HemoCue instrument performances were substantially affected by haematocrit. MediSense instrument performance was also affected by pH and PaO2. Radiometer instrument performance was not affected by haematocrit, pH or PaO2. Conclusions The MediSense instrument was too imprecise for use in critically ill patients. The haematocrit range seen is too great to allow fixed-factor conversion between whole blood and plasma values, substantially affecting the accuracy of both glucose meters. However, the Radiometer instrument was unaffected by the haematocrit, pH or pO2, resulting in a performance equivalent to the laboratory method. Instrument performance differences may therefore partially explain the differing results of tight glycaemic control therapy trials.