Comparison of solutions for preservation of the rabbit liver as tested by isolated perfusion

The University of Wisconsin (UW) solution consists of a relatively complex mixture of agents. In this study we compared simpler preservation solutions, namely, histidine-tryptophan-ketoglutarate glutarate (HTK) and phosphatebuffered sucrose (PBS) with different compositions of UW solution in the isolated perfused rabbit liver model. Livers were stored cold for 24 and 48 h. After 24 h of preservation, the amount of bile produced in UW-preserved livers was significantly greater (P<0.05) than that in HTK-preserved livers. Also, there was less LDH released into the perfusate in UW-preserved livers. There was more edema and lower K+/Na+ rations in HTK-preserved livers than in UW-preserved livers (all data P<0.05). After 48 h of preservation, the differences between livers preserved in UW or HTK solution were less noticeable than at 24 h and bile production was similar. LDH and AST release were greater in HTK-preserved livers than in UW livers, but these differences were not statistically significant. Preservation in PBS for 48 h was worse than in either UW or HTK solution. Substitution of polyethylene glycol (PEG) for hydroxyethyl starch (HES) in 48-h UW-preserved livers was not effective. We conclude that solutions simpler in composition than UW solution may be effective in kidney transplantation but do not appear suitable for successuful liver preservation.     

The importance of a colloid in canine pancreas preservation.

The role of hydroxyethyl starch (HES), the colloid component of the UW solution, was tested in canine pancreas preservation. Segmental pancreatic autografts were preserved for 48 hr cold storage with UW solution with HES (group 1) or UW solution without HES (group 2). After preservation, the pancreas was transplanted, and survival, serum glucose, serum amylase, intravenous glucose tolerance tests, tissue water content, and histology were compared between groups. In group 1 (with HES), 9/10 dogs were long-term survivors with one dog dying due to causes unrelated to preservation failure. In group 2 (without HES), 3/6 dogs died due to graft loss within one week posttransplant (P = 0.01). No graft failure occurred in group 1 (0/9) versus graft loss in 4/6 dogs in group 2 (P = 0.04). All animals in group 1 (with HES) showed normal serum glucose and amylase concentrations postoperatively, normal tissue water values after preservation, k values comparable to those observed after segmental autotransplantation without preservation, and relatively good histology. In group 2 (without HES), in 4/6 dogs graft failure occurred that led to the death (3 dogs) of the animals or to a diabetic state (1 dog). After 48-hr cold storage without HES, a significant increase in tissue water content, glucose and amylase levels was seen. After transplantation, hyperglycemia, hyperamylasemia, and clinical diabetes were observed in 4/6 dogs. Autopsy and histological evaluation showed evidence of thrombosis and ischemic insult. Two of 6 dogs in group 2 remained normoglycemic during follow-up with borderline k values. The results suggested that for consistently successful 48-hr preservation of the pancreas, HES is an important component of the UW solution. Although a colloid may not be essential for short-term preservation of kidney and liver, it appears to be an important factor in successful pancreas preservation.     

A novel dextran 40-based preservation solution

Although the University of Wisconsin (UW) solution has become the standard solution for the preservation of kidneys for transplantation, the importance of the colloid hydroxyethylstarch (HES), one of the key compounds of the UW solution, has been questioned repeatedly. It is now established that HES is not necessary for routine kidney preservation. However, colloids may still be advantageous in UW like solutions for the purpose of multiorgan procurements and the preservation of organs from marginal donors. It has been shown in various experimental models that dextran 40 may successfully substitute for HES. Dextran 40 is not only cheaper but also has a variety of biological effects that may be beneficial during the graft reperfusion phase. The aim of this clinical study was to examine the efficacy of a dextran 40-based preservation solution (Dex-PS) for its use in human kidney graft preservation and to compare the transplantation results with kidneys preserved with UW solution. A total of 87 kidneys were preserved with Dex-PS and matched with 87 kidneys preserved with UW solution. Both groups were comparable in terms of donor and recipient characteristics and both had a high proportion of kidneys from nonheart-beating donors. Patient survival and graft survival after 1 year were 95% and 86% for the Dex-PS group and 94% and 90% for the UW group, respectively (P=NS). Primary nonfunction, delayed graft function, postoperative need for dialysis, and follow-up of serum creatinine were statistically comparable between these two groups. We conclude that dextran 40 can safely replace HES in UW solution for the purpose of clinical kidney preservation. There were no statistically detectable differences in graft performance between the kidneys preserved with UW and those preserved with Dex-PS.     

Evaluation of UW solution in a rat kidney preservation model. I. Effect of hydroxyethyl starch and electrolyte composition.

Preservation of rat kidneys by simple ice-storage has been demonstrated after 48 hr using the University of Wisconsin (UW) solution; hypothermic preservation of the rat kidney was dramatically improved. A modified UW solution without hydroxyethyl starch (HES) also gave uniform survival after 48 hr of storage, and even better function and morphology. Substitution of HES from another source also improved renal function when compared with UW (Dupont) and it is suggested that prolonged storage of UW solution prior to use may reduce its effectiveness. Reversing the Na:K ratio of the solution still allowed successful preservation, but significantly worsened morphology of the surviving kidney.     

Efficacy of polyethylene glycols in University of Wisconsin preservation solutions: a study of isolated perfused rat liver

Recent reports argue that the performance of University of Wisconsin (UW) solution is limited by the presence of hydroxyethyl starch (HES) as an additive, since HES could be responsible for human red blood cell aggregation. We investigated the effect on rat liver preservation of replacing HES in UW solution by polyethylene glycols (PEG20 and PEG35) at two concentrations. An isolated perfused rat liver model was used. Six groups of preserved livers (n = 7 for each group) were compared to controls (nonpreserved livers, n = 7). The following preservation solutions were assayed: UW without oncotic supply, UW-HES (0.25 mmol/L), UW-PEG20 (0.03 and 0.25 mmol/L), and UW-PEG35 (0.03 and 0.25 mmol/L). After 24-hour cold storage, the livers were perfused for 120 minutes at 37 degrees C with oxygenated Krebs-Henseleit solution. During perfusion, transaminase release, portal and bile flows, and bromosulfophthalein (BSP) clearance were assessed. Results showed that the omission of oncotic supply in UW statistically increased ALT and AST release in perfusate and decreased bile and portal flows. PEG addition in UW solution, especially PEG35 at 0.25 mmol/L, effectively protected the rat liver graft from the onset of hypothermic ischemia/reperfusion damage. In conclusion, data reported here reveal that oncotic supply is essential for liver preservation and that HES can be effectively replaced by PEG in UW solution.     

Effects of polyethylene glycol and hydroxyethyl starch in University of Wisconsin preservation solution on human red blood cell aggregation and viscosity

University of Wisconsin (UW) preservation solution is considered an effective flush and cold storage liquid. However, recent studies have provided evidence of the hyperaggregating effect on human red blood cells (RBC) of hydroxyethyl starch (HES), one of the components of the UW solution. In contrast, preservation solutions containing polyethylene glycol (PEG) have been found to be effective for organ preservation. The aim of this study was to compare the effects of HES (50 g/L); PEG 20 kDa (50 and 30 g/L), and PEG35 kDa (1.05 g/L) added to UW on the rheologic parameters of human RBC at 4 degrees C. Sedimentation rate was measured by the Westergren procedure and blood viscosity evaluated at high shear rates using a cone/plate viscometer. Alterations in RBC morphology and aggregation were evaluated by light microscopy. RBC sedimentation and viscosity were not affected by the inversion of Na+ and K+ concentrations in UW, but were increased by HES. PEGs appeared to reduce RBC deformability with concomitant inhibition of RBC aggregation. These results were consistent with reduced viscosity for PEG-containing solutions. In conclusion, the use of PEG did not change the physiologic function of human RBCs and thus may be an alternative to HES in UW liquids.     

Effect of University of Wisconsin organ-preservation solution on haemorheology

In conventional cold-storage organ preservation, the donor organ is flushed with University of Wisconsin (UW) solution at 0–4°C. The initial flush is used to wash out blood from the microcirculation to allow optimal preservation with the UW solution. The component hydroxyethyl starch (HES) of UW is known to cause relatively high viscosity and a possible interaction with blood, i.e. increased red blood cell (RBC) aggregation. The aim of this study was to investigate the influence of the HES component on the viscosity of UW and the aggregation behaviour of blood during washout. Viscosity aspects were measured with a cone-plate rheometer. HES-induced RBC aggregation was studied by means of an optical aggregation measuring device. The experiments were carried out with rat whole blood and mixtures of rat whole blood with UW-solution and UW without HES (UWmod), at 4°C. The viscosity of blood at 4°C is two-times higher than at 37°C; the UW/blood mixture at 4°C is 1.3-times more viscous than blood at 37°C; the 4°C UWmod/blood mixture equals the viscosity of blood at 37°C. The UW/blood mixture shows a ninefold increased aggregation compared with whole blood. These aggregates are larger than the diameter of the sinusoids in the rat liver. A mixture of whole blood and UWmod shows a lower aggregation than blood. Apart from an increased viscosity, HES in UW causes increased RBC aggregation. The aggregates are larger than the diameter of the sinusoids. Initial washout could be optimised by pre-flushing to improve the viability of the liver and to decrease delayed graft function.     

An examination of the effects of solutions containing histidine and lactobionate for heart, pancreas, and liver preservation in the rat.

Fifty-five rat pancreas transplants, 18 rat heart transplants, and 41 rat liver transplants were performed using standard UW solution, the new HL solution (HL-I), or a modified HL solution (HL-II). Storage times of 18 hr were used in the heart preservation experiments, 24 hr in the liver preservation experiments, and 48 or 72 hr in the pancreas preservation experiments. HL-I solution was superior to both HL-II and UW solution for heart preservation (1-week graft survival rates of 100% [7/7], 0% [0/5], and 50% [3/6], respectively). HL-I and HL-II were superior to UW for 24 hr liver preservation (1-week graft survival rates of 78% [11/14], 80% [8/10], and 29% [5/17], respectively). In contrast, HL-II was superior to both HL-I and UW solutions for pancreas preservation following both 48-hr preservation and 72-hr preservation. Satisfactory graft function was achieved in 100% (7/7), 40% (6/15), and 44.4% (4/9) of pancreases transplanted after 48 hr using HL-II, HL-I, and UW solutions, respectively, and in 50% (4/8), 0% (0/8), and 0% (0/8) following 72-hr preservation. Histidine- and lactobionate-containing solutions thus represent a further improvement in organ preservation by simple cold storage.     

Prolonged rat pancreas preservation using a solution with the combination of histidine and lactobionate

A newly formulated solution consisting of lactobionate with or without histidine was tested in the preservation of the rat pancreas. Adult male Lewis rats weighing 120–250 g were used as donors and recipients. Fifty-four rat pancreas transplants were performed to investigate the effectiveness of this test solution and to compare it with the standard University of Wisconsin (UW) solution. The final osmolarity of the new test solution was 290–320 mosmol/l. This solution had a higher sodium content and lower potassium content (Na: 110 mEq/l, K: 50 mEq/l). Adenosine, insulin, hydroxyethyl starch and dexamethasone, which are components of the UW solution, were not present in this test solution. Histidine was used as a buffer. Rat pancreases were stored at 4 °C in either standard UW solution, or high-Na ⁺-histidine solution, or high-Na ⁺ -lactobionate solution for 48 h and 72 h prior to heterotopic transplantation into rats with streptozotocin-induced diabetes mellitus. Functional success rates for rats receiving pancreases that had been preserved in high-Na*-histidine and in high-Na ⁺-lactobionate solutions at 4°C were 100% (5/5) and 100% (7/7) after 48 h preservation, and 50% (4/8) and 14% (1/7) after 72 h preservation, respectively. By contrast, standard UW solution gave only a 44% (4/9) success rate after 48 h preservation and a 0% (0/8) success rate after 72 h preservation. These results demonstrated that the high-Na ⁺-histidine solution was superior to standard UW solution for rat pancreas preservation. This was probably due to the buffer, histidine, which prevented the acidosis of ischemic tissue during the period of preservation.     

Role of Hydroxyethyl Starch in Ischemia-Reperfusion Injury in Rat Intestinal Transplantation

Objective

This study was designed to evaluate the role of 0%, 3%, 6% hydroxyethyl starch (HES) and University of Wisconsin (UW) perfusion and preservation solutions on ischemia-reperfusion injury (IRI) of rat intestinal transplantations, solutions, respectively.

Materials and methods

Rats underwent orthotopic intestinal transplantation (Lewis to Lewis) after using perfusion and preservation saline (group l), 3% HES (group 2), 6% HES (group 3), or UW (group 4) solutions. The change in weight was recorded from preoperative to postoperative day (POD) 30. At 30 minutes after reperfusion, we harvested intestinal juice preoperatively as well as at 30 minutes after reperfusion and on POD 1 and 3 when recipients underwent open surgery for maltose absorption tests and sampling. The Park' scores of IRI were evaluated by light microscopy after hematoxylin and eosin (H&E) staining.

Results

An increased weight was more evident in group 2 than the other groups, particularly the on POD 1 and POD 3 (P < .05). It was significantly greater than groups 1 and 3 on POD 7 (P < .05). Compared with the other groups, the 30-minute post-reperfusion. Park score and intestinal juice content in group 2 was decreased significantly (P < .01), while in group 3 the Park score was increased, and the maltose absorption level decreased significantly (P < .05).

Conclusion

Three percent HES solution attenuated IRI in rat intestinal transplantation. High-concentration HES solutions were unfit for intestinal preservation. Thus the adverse effects of UW solution may be attribute at least in part to its high HES, concentration.     

Pancreas preservation with University of Wisconsin and Celsior solutions: a single-center, prospective, randomized pilot study

BACKGROUND: Celsior is an extracellular-type, low-viscosity, preservation solution already used for heart, lung, liver, and kidney transplantation. We report the results of a single-center, prospective, randomized pilot study specifically designed to compare the safety profile of Celsior solution with University of Wisconsin (UW) solution in clinical pancreas transplantation. METHODS: A total of 105 consecutive procurements were randomized to graft preservation with UW (n=53) solution or Celsior (n=52) solution. The groups were comparable with regard to all donor and recipient characteristics. RESULTS: Five grafts were discarded and 100 grafts (50 UW vs. 50 Celsior) were transplanted. Mean cold and warm ischemia times were 11.0 +/- 2.1 hr and 37.2 +/- 6.0 min for UW compared with 10.8 +/- 1.8 hr and 38.1 +/- 5.9 min for Celsior (P =not significant). Delayed endocrine pancreas function was recorded in one graft preserved with UW solution. Eleven recipients (UW 12% vs. Celsior 10%, P =not significant) required a relaparotomy. The mean serum levels of glucose, amylase, and lipase remained comparable between the study arms at equivalent intervals after transplantation. One recipient died with functioning grafts in each study arm; two further grafts were lost to arterial thrombosis (Celsior) and chronic rejection (UW), respectively. Actuarial 1-year patient and graft survival rates overlapped in the two study arms (98% and 96%, respectively). CONCLUSIONS: Within the range of cold ischemia time reported in this study, UW and Celsior solutions have similar safety profiles for pancreas preservation.     

Comparative study of the University of Wisconsin solution versus Eurocollins in kidney transplant

UW (University of Wisconsin) solution, formulated by Belzer's team in Madison, has already been proved to increase cold ischemia time in liver and pancreas preservation. A multicentre clinical trial is being conducted to compare renal preservation in human transplantation using two different solutions: UW and Eurocollins (EC). This paper, whose results will be included in the multicentre trial, reports local comparative results between UW and EC perfused Kidneys. The two donor populations UW (28 cases) and EC (47 cases) were not randomized. They were however comparable in renal function prior to harvesting but not in age (35 +/- 13.4 years EC versus 27.7 +/- 12.4 years UW). The two recipient populations (48 EC versus 48 UW) were more homogeneous. Comparative results were significant with better graft function in the UW group: creatinine at one week: 499.2 +/- 296.3 EC versus 277.6 +/- 226.2 mumol/l, p less than 0.0001; creatinine at one month: 228.7 +/- 135 EC versus 159.7 +/- 135.6 mumol/l, p less than 0.02 and a decrease in acute tubular necrosis (39.5% EC versus 14.5% UW) and hospital stay. These results justify the use of UW solution by intraaortic flush especially during multi-organ procurement.     

Rat liver preservation: II. Combining UW solution with Eurocollins solution or Ringer's lactate abrogates its protective effect

Abstract. The mechanisms by which cold preservation solutions exert their protective effects are only partially understood. The consequences of mixing different solutions, with presumably different modes of action, may be additive and beneficial or may be deleterious. It is commonplace in clinical liver preservation to use Ringer's lactate (RL), Eurocollins (EC), and University of Wisconsin (UW) solution in sequence for washout of blood, precooling, and cold storage of the organ. In this study, 114 Sprague Dawley rats received orthotopic liver transplants that were flushed in various sequences with RL, EC, and UW solutions. One-week animal survival served as the criterion of preservation success. The results demonstrated that liver preservation with UW solution alone is significantly superior ( P < 0. 01) to any combination of RL, EC, and UW solutions and may explain some of the instances of primary nonfunction in clinical liver transplantation.     

Rat liver preservation

The mechanisms by which cold preservation solutions exert their protective effects are only partially understood. The consequences of mixing different solutions, with presumably different modes of action, may be additive and beneficial or may be deleterious. It is commonplace in clinical liver preservation to use Ringer's lactate (RL), Eurocollins (EC), and University of Wisconsin (UW) solution in sequence for washout of blood, precooling, and cold storage of the organ. In this study, 114 Sprague Dawley rats received orthotopic liver transplants that were flushed in various sequences with RL, EC, and UW solutions. One-week animal survival served as the criterion of preservation success. The results demonstrated that liver preservation with UW solution alone is significantly superior (P<0.01) to any combination of RL, EC, and UW solutions and may explain some of the instances of primary nonfunction in clinical liver transplantation.     

Efficacy and safety of Celsior preservation fluid in liver transplantation: one-year follow up of a prospective, multicenter, non-randomized study

The purpose of this prospective, nine-center, non-randomized study was to assess the efficacy and safety of Celsior preservation fluid in liver transplantation using unselected donors. As data comparing allograft outcomes following liver transplantation using Celsior and University of Wisconsin (UW) preservation fluids are limited, we also compared our cohort with matched controls selected from the European Liver Transplant Registry (ELTR) who received total liver grafts preserved with UW solution during the same period. One hundred and forty patients who received livers preserved with Celsior were included. The primary endpoint, graft loss at one-yr post-transplantation, was observed in 24 patients (17.1%) which was not significantly different from the 20.0% pre-defined threshold rate (95% confidence interval [CI] 10.9, 23.4; p=0.398). Predictive factors for graft loss on univariate analysis were moderate-to-severe steatosis on the donor graft (5/22 patients with graft loss vs. 8/107 patients without, p=0.046) and duration of warm ischemia (1.4±1.1 h in patients with graft loss vs. 0.9±0.5 h in patients without, p=0.034). Hepatic artery thrombosis and stenosis occurred in seven (5.0%) and six (4.3%) patients, respectively. The comparison of our patients to 420 ELTR controls showed that one-yr graft survival rates (Celsior: 82.9%, 95% CI 75.8, 88.2; UW: 78.6%, 95% CI 74.4, 82.2) and Kaplan-Meier one-yr graft survival distributions (p=0.285) were similar. Within the cold ischemia time achieved in our study, liver preservation with Celsior appeared efficient and safe. Comparison with ELTR patients suggested that liver allograft survival was similar using Celsior or UW solution for preservation of unselected donor grafts.     

Comparison of the efficacy of University of Wisconsin solution and Newcastle organ perfusion fluid in the preservation of livers for transplantation

Abstract University of Wisconsin solution (UW) is now used widely for the preservation of livers for transplantation. However, the use of commercial solutions has added considerably to the cost. We were able to produce a local version of organ perfusion fluid (NOPF) incorporating all the constituents of UW except for the hydroxyethyl starch and adenosine. We compared graft outcome using NOPF with imported grafts perfused with commercial UW solution. The two recipient groups (15 patients each) were similar with respect to age and sex distribution, urgency of transplantation, regraft status and patient and graft survival. Postoperative duration of ventilation, dialysis requirements, peak bilirubin, peak ALT and lowest unsupported prothrombin time were also similar in both groups. In conclusion, local perfusion fluids based on UW can be produced without detriment to graft outcome with considerable financial savings. At our institution, this represents a reduction of 33 % in the cost of perfusion fluids.     

Liver preservation with UW solution. I. Evidence that hydroxyethyl starch is not essential.

Preservation of rat livers by simple ice-storage has been demonstrated after 24 hr using the University of Wisconsin (UW) solution. Equally good preservation could be obtained by substituting hydroxyethyl starch (HES) from another source or by omission of HES from the UW formulation. Survival, early and late function, and morphology at the end of a 3-month follow-up period were essentially similar for all three test groups. It is concluded that HES is not required for optimum preservation of the rat liver in this treatment model.     

Luminescence-Based Assay to Screen Preservation Solutions for Optimal Ability to Maintain Viability of Rat Intestinal Grafts

Background

Segmental intestinal transplantations from living, genetically related donors provide advantages compared with those from cadaveric subjects. However, successful preservation during ischemic cold storage is critical for living donor grafts. Thus, the development of preservation solutions that maintain graft viability is essential for success. Herein we have reported application of a cell-based viability assay in multiwell plates to assess the effectiveness of various solutions to preserve intestinal grafts.

Methods

Freshly isolated intestinal chips from luciferase transgenic rats were placed in 96-well tissue culture plates for incubation at 4°C for 24 hours in various preservation solutions: ET-Kyoto (ET-K), University of Wisconsin (UW) solution, Euro-Collins (EC) solution, histidine-tryptophan-ketoglutarate (HTK) solution, lactated Ringer’s (LR) solution, or saline.

Results

As indicated by a higher level of luminescence, intestinal chips preserved in UW, HTK, or ET-K solution contained more viable cells, than those preserved in EC, LR, or saline solution. After exposure to the preservation solutions for 1 hour, the mucosal layer chips showed lower cell viability than the muscle layer chips.

Conclusion

Our data demonstrated that ET-K and UW solutions used together with intestinal chips of Luciferase transgenic rat and in vivo imaging provided optimal viability during ischemic cold storage prior to transplantation. Further development of preservation conditions to minimize the loss of viability of intestinal grafts before clinical transplantation is essential to improve outcomes.     

Trimetazidine reduces renal dysfunction by limiting the cold ischemia/reperfusion injury in autotransplanted pig kidneys

Ischemia/reperfusion injury leads to delayed graft function, which is a major problem in kidney transplantation. This study investigated the effects of adding trimetazidine (TMZ) to the perfusate of cold-stored kidneys on the function of reperfused autotransplanted pig kidney. The left kidney was removed and cold-flushed with Euro-Collins (EC), or University of Wisconsin (UW) solutions with or without 10(-6)M TMZ and stored for 48 h at 4 degrees C. The kidneys were then autotransplanted and the contralateral kidneys were removed. Several parameters were analyzed over the 14 d after transplantation. The survival rate was 57% in pigs transplanted with kidneys cold-flushed with UW and 43% for those flushed with EC solution; it was 100% for pigs having kidneys cold-flushed with TMZ-supplemented UW and EC solutions. The functions of the transplanted kidneys were also better preserved after cold flush with TMZ-supplemented solutions than with TMZ-free solutions. Creatinine clearance was higher and the urinary excretion of trimethylamine-N-oxide and dimethylamine, used as markers of renal medulla injury, were lower in animals transplanted with kidneys cold-flushed with TMZ-supplemented solutions than with TMZ-free solutions. The cytoprotective action of TMZ also reduced interstitial and peritubular inflammation and the numbers of infiltrating mononuclear CD45+and CD3+ T cells. These results indicate that the tissue damage due to ischemia/reperfusion injury may be prevented, at least in part, by adding TMZ to preservation solutions.     

24-hour rabbit heart storage with UW solution. Effects of low-flow perfusion, colloid, and shelf storage

A new technique for 24-hr cardiac preservation is described utilizing very low flow perfusion (microperfusion) with a cold flush solution. Rabbit hearts were arrested with UW solution and then perfused with the same solution through the aortic root at 0 degrees C at a rate of 3-6 ml/gm heart weight/24 hr. When tested on an ex vivo working heart model, the cardiac output (CO) was 28.72 +/- 7.69 ml/g/min compared with fresh UW flushed controls of 26.48 +/- 2.25 ml/g/min. Both oxygenated highflow perfusion with a more conventional perfusate and 24-hr ice storage with UW led to inferior results. Omission of the colloid, hydroxyethyl starch (HES), from the UW solution or prolonged shelf storage were also significantly detrimental. When a previously untested colloid, polyethylene glycol 20,000, was substituted for HES for microperfusion, excellent cardiac function was obtained. In fact, the mean CO of this group, 31.91 +/- 5.70, was significantly above that of fresh HES-UW unstored controls. The suggestion that the UW solution might be improved by this substitution warrants further study.