Improved Biocompatibility by Modified Cellulosic Membranes: The Case of Hemophan

The rising problem of biocompatibility is encouraging the development of new dialysis membranes, but the high cost of synthetic ones precludes their wide use. The authors compared the biocompatibility of cuprophan (CU), cellulose acetate (CA), and hemophan (HE), evaluating both in vitro and in vivo polymorphonuclear leukocyte (PMN) oxidative metabolism activation by resting chemiluminescence and complement activation by C3a; in vivo PMN counts during dialysis were also performed. The lowest increase in in vitro PMN resting chemiluminescence using HE was + 71.3% with CA, +49.3% with CU, and + 21.4% with HE (p less than 0.001 versus CA and CU); furthermore, HE did not significantly stimulate PMN resting chemiluminescence during in vivo hemodialysis: + 56.6% with CA, + 38.8% with CU, and + 3.7% with HE (p less than 0.01 versus CU and p less than 0.001 versus CA). C3a concentration increased with all membranes both in vitro and in vivo, but HE (in both experimental conditions) showed the lowest increase at any time (p less than 0.001 versus CA and CU). After 15 min of dialysis, PMN count dropped to 20.3% of basal values with CU, to 49.8% with CA, and to 76.5% with HE (p less than 0.001 versus CU and CA). Among cellulosic membranes, HE is the most biocompatible and appears to be an important step in preventing blood-membrane interactions and related complications.     

The Development of a Multiorgan Ex Vivo Perfused Model: Results With the Porcine Liver‐Kidney Circuit Over 24 Hours

We already developed an ex vivo liver‐kidney model perfused for 6 h in which the kidney acted as a homeostatic organ to improve the circuit milieu compared to liver alone. In the current study, we extended the multiorgan perfusions to 24 h to evaluate the results and eventual pitfalls manifesting with longer durations. Five livers and kidneys were harvested from female pigs and perfused over 24 h. The extracorporeal circuit included a centrifugal pump, heat exchanger, and oxygenator. The primary end point of the study was the evaluation of the organ functions as gathered from biochemical and acid‐base parameters. In the combined liver‐kidney circuit, the organs survived and maintained an acceptable homeostasis for different lengths of time, longer for the liver (up to 19–23 h of perfusions) than the kidney (9–13 h of perfusions). Furthermore, glucose and creatinine values decreased significantly over time (from the 5th and 9th hour of perfusion onward). The addition of a kidney to the perfusion circuit improved the biochemical environment by removing excess products from ongoing metabolic processes. The consequence is a more physiological milieu that could improve results from future experimental studies. However, it is likely that long perfusions require some nutritional support over the hours to maintain the organ's vitality and functionality throughout the experiments.     

Laparoscopic Nephrectomy,Ex Vivo Repair,and Autotransplantation for a Renal Artery Aneurysm: Report of a Case

A 57-year-old woman was hospitalized with a left renal artery aneurysm (RAA). The aneurysm measured 35 mm in diameter and was located at the renal artery bifurcation. We performed a laparoscopic nephrectomy using a retroperitoneal approach and performed an ex vivo repair of the renal artery. The reconstructed kidney was then autotransplanted at the left iliac fossa. The patient's postoperative course was uneventful. A laparoscopic nephrectomy and ex vivo repair are both considered to be effective for treating complex RAA.     

Lipid Apheresis by Hemoperfusion: In Vitro Efficacy and Ex Vivo Biocompatibility of a New Low-Density Lipoprotein Adsorber Compatible with Human Whole Blood

Abstract: To date, selective extracorporeal low-density lipoprotein (LDL) removal can only be performed from plasma; that is, a plasma-cell separation step using a centrifuge or a plasma membrane separator is necessary initially. This article characterizes a new polyacrylate-based LDL adsorber directly applicable to whole blood . In vitro single-pass hemoperfusion tests using pooled donor blood showed quantitative adsorption of atherogenic LDL-cholesterol (LDL-C) and complete recovery of protective high-density lipoprotein C. Fibrinogen, another independent risk factor of atherosclerosis, was also adsorbed to a lesser extent. Single-pass ex vivo biocompatibility using fresh donor blood on-line was excellent and resulted in minimal cell loss. Neither signs of hemolysis nor activation of monocytes (interleukin-l production) were detected. Only slight activation of leukocytes (elastase release) and thrombocytes (platelet factor 4 secretion) as well as of coagulation (thrombin-antithrombin complex formation) and complement (C3a, C5a generation) was observed. Under the experimental conditions used, the optimal anticoagulation regimen was 0.5 IU heparin plus 0.375 mg citratelml blood. Priming the column with a buffer of pH 7.4 containing heparin, citrate, and Ca²⁺ is recommended. In conclusion, this new adsorber exhibited selective LDLC adsorption in vitro combined with excellent ex vivo biocompatibility and thus holds great promise for a successful clinical application in a closed-loop system in patients.     

Ex Vivo Preclinical Evaluation of Membrane Plasma Separators

Four different types of hollow-fiber membrane plasma separators, constructed of cellulose acetate, polyvinyl alcohol, polyethylene, and polymethylmethacrylate membranes, were evaluated in ex vivo dog perfusions under conditions simulating their clinical use. An arteriovenous (A-V) fistula constructed in the dogs for blood access enabled repeated access to be achieved without surgical intervention. All modules produced transient leukopenia and a reduction of platelet counts. The polymethylmethacrylate module showed minimum reductions of white blood cell counts and CH50. The early leukocyte count reduction in membrane plasmapheresis is most likely related to the magnitude of complement activation by the membrane, as is seen with hemodialysis.     

Validation of Lymphatic Mapping in Colorectal Cancer: In Vivo, Ex Vivo, and Laparoscopic Techniques

Background:The use of lymphatic mapping (LM) is being investigated to improve the staging of colorectal cancer (CRC) and thereby identify patients who might benefit from adjuvant chemotherapy. This study evaluated in vivo, laparoscopic, and ex vivo approaches for the ultrastaging of CRC.Methods:Seventy-five CRC patients were enrolled in a study of LM with peritumoral injection of isosulfan blue dye. LM was undertaken during open colon resection (OCR) in 64 patients, during laparoscopic colon resection (LCR) in 9 patients, and after specimen removal (ex vivo) in 2 patients. Ex vivo LM was also undertaken in 6 patients after unsuccessful in vivo LM. All nodes were examined by hematoxylin and eosin (H&E) staining; in addition, sentinel lymph nodes (SNs) were multisectioned and examined by immunohistochemical staining with cytokeratin (CK-IHC).Results:At least one SN was identified in 72 patients (96%). In vivo LM identified SNs in 56 of 64 (88%) patients undergoing OCR and in 9 of 9 (100%) patients undergoing LCR. Ex vivo LM was undertaken as the initial mapping procedure in 2 cases of intraperitoneal colon cancer and after in vivo LM had failed in 6 cases of extraperitoneal rectal carcinoma; an SN was identified in 7 of the 8 cases. Focused examination of the SN correctly predicted nodal status in 53 of 56 OCR cases, 9 of 9 LCR cases, and 6 of 7 ex vivo cases. Multiple sections and CK-IHC identified occult micrometastases in 13 patients (17%), representing 10 OCR, 1 LCR, and 2 ex vivo cases.Conclusions:LM of drainage from a primary CRC can be accurately performed in vivo during OCR or LCR. Ex vivo LM can be applied when in vivo techniques are unsuccessful and may be useful for rectal tumors. During LCR, colonoscopic injection can be used to mark the primary tumor and define the lymphatic drainage so that adequate resection margins are obtained. These LM techniques improve staging accuracy in CRC.Presented at the 53rd Annual Meeting of the Society of Surgical Oncology, New Orleans, Louisiana, March 16-19, 2000     

Successful Emergent Lung Transplantation After Remote Ex Vivo Perfusion Optimization and Transportation of Donor Lungs

A recent clinical trial provided evidence that ex vivo lung perfusion (EVLP) results in optimized human donor lungs for transplantation. Excellent recipient outcomes were documented after 4 h of normothermic perfusion. We report a clinical case utilizing remote EVLP to assess and improve function of initially otherwise unacceptable injured donor lungs followed by transportation and subsequent bilateral lung transplantation in a patient with virally induced refractory respiratory failure supported with extracorporeal membrane oxygenation. This is the first lung transplantation with the application of remote EVLP, wherein the donor lungs were transported from the donor hospital to a center for EVLP and then transported to another hospital for transplantation. It is also the first case of lung transplantation in the United States utilizing EVLP for functional optimization leading to successful transplantation. Organ procurement data, EVLP assessment, and the pre‐ and postoperative course of the recipient are presented. The available evidence supporting EVLP, the humanitarian and cooperative utilization of lungs otherwise discarded, are discussed.     

Comparison of Rapid In Situ, Regular In Situ, and Ex Vivo Flushing on Hepatic Function

The efficacy of three flushing techniques on subsequent liver function was assessed using the in vivo isolated liver perfusion model (ILPM). Livers from brain-dead mongrel dogs were flushed with cold Euro-Collins as follows: Group I, rapid in situ flushing (10 min); Group II, regular in situ flushing (45 min); Group III, ex vivo flushing (10 min). All livers were then heterotopically transplanted into recipients, using the ILPM, by anastomosis of the portal vein, vena cava, and hepatic artery to the recipient's portal vein, iliac vein, and iliac artery. Reperfusion followed for 30 min. Laboratory samples collected at 0, 5, 15, and 30 min showed that hepatic function was not altered by ex vivo flushing and was only slightly altered by rapid in situ flushing. Regular in situ flushing proved to be damaging to livers. Histological analysis confirmed these findings. Therefore, either rapid in situ or ex vivo flushing can be safely used by the transplant specialist.     

Effect of FTY720 and Ex Vivo Graft Irradiation in Rat Small Bowel Transplantation: Expression of Mucosal Addressin Cell Adhesion Molecule-1

Purpose We performed a semiquantitative analysis of the expression of Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and gut-associated tissues (GALT) during small bowel graft rejection in the rat to confirm the effect of FTY720 and ex vivo graft irradiation. Methods Small bowel transplantations (SBT) were performed from BN rats to LEW rats. Four groups of SBT animals were studied on days 3, 5, and 7 after operations (untreated, FTY720, ex vivo graft irradiation, FTY720+ex vivo graft irradiation). Indirect immunoperoxidase staining was performed against CD4 and MAdCAM-1. The number of CD4-positive cells in the allografts was also analyzed by flow cytometry. Results The graft survival was prolonged only in the FTY720-treated groups. The SBT allografts treated by FTY720 demonstrated less infiltration, but the ex vivo graft irradiation group did not show any effect on its expression. In the FTY720-treated groups, MAdCAM-1 expression on high endothelial venules (HEVs) in Peyer's patches (PPs) was upregulated and its expression on endothelial cells of vessels in the lamina propria was downregulated in comparison with the allograft group without FTY720. Conclusions It is important to prevent the infiltration of CD4-positive cells, the downregulation of MAdCAM-1 expression on HEVs in PPs and the upregulation of MAdCAM-1 expression on endothelial cells of vessels in the lamina propria for the prolongation of graft survival.     

A Hemoperfusion Column Based on Activated Carbon Granules Coated with an Ultrathin Membrane of Cellulose Acetate

A hemoperfusion system has been developed which makes use of activated carbon encapsulated with cellulose acetate. Studies have revealed that there are no stagnant flow regions in the column, there is minimal particle release and the coating is 30 A thick. The relationships between pore size, pore volume and surface area have been examined. Twenty-five patients in grade IV coma have been treated with the column for treatment of drug overdose or agricultural chemical poisoning; the clinical course of one meprobamate-poisoned patient is described in detail.     

Failure of Platelet Markers to Evaluate Membrane Biocompatibility During a Combined Hemodialysis-Hemoperfusion Treatment

Plasma levels of the platelet markers beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) are among the most sophisticated indexes of biocompatibility available to evaluate new members for hemodialysis. This investigation was designed to determine the extent of platelet activation by measuring the alpha-granule release products, beta-TG and PF4; anticoagulation and thrombogenesis by monitoring plasma heparin; and fibrinopeptide A (FPA) and thromboxane B2 (TX B2) levels during treatment with a combined hemodialysis-hemoperfusion system. Both in vivo and in vitro results showed that the platelet markers had a pattern different from that generally observed during treatment with hemodialysis alone. This is due to the avidity of charcoal for the markers studied, which therefore cannot be used to evaluate the biocompatibility of the system.     

In Vitro and In Vivo Evaluation of Protamine–Heparin Membrane for Microencapsulation of Rat Langerhans Islets

Abstract: Rat pancreatic islets were microencapsulated with multilayer protamine–heparin (PH) membrane. Basal and stimulatory insulin secretion of microencapsulated islets was similar to the controlled free islets in vitro. During the long–term culture (up to 2 weeks) mean insulin release of encapsulated islets did not significantly differ from the mean of free ones (the ratio of mentioned means was 54–167%). Empty PH microcapsules transplanted into Wistar rats intraperitoneally and under the kidney capsule were generally harmless up to 4 months. In only a few cases traces of fibrotic tissue around capsules entrapped in the omentum were found. No damage of microcapsules structure was observed. The worst results were obtained in the instance of retroperitoneal transplantation. We conclude, therefore, that PH membrane was proved to be highly biocompatible, nontoxic for islets, and did not impair viability and glucose–dependent insulin secretion of Langerhans islets in in vitro culture.     

Ex vivo extended left hepatectomy with caval preservation, temporary portacaval shunt, and reconstruction of the right hepatic vein outflow using a reversed portal vein bifurcation graft

Liver resections that require ex vivo techniques occur rarely, but when done are generally performed on veno-veno bypass to maintain venous return and decompress the portal circulation during the anhepatic phase of the procedure. We describe an ex vivo extended left hepatectomy that was performed with preservation of the inferior vena cava and the use of a temporary portacaval shunt to eliminate the need for veno-venous bypass. Ex vivo resection allowed reconstruction of right hepatic vein branches, using the patient's reversed portal vein bifurcation as a graft to provide venous outflow.     

Clinical Evaluation of a New Dialyzer, FLX-12 GW, with a Polyester-Polymer Alloy Membrane

Abstract: The performance of a membrane in renal failure therapy is determined by its structure, its overall mass transfer properties, and its blood compatibility. In this regard. removal of β₂-microglobulin (β2M) has become a major objective of dialysis therapy. In the present study, a newly developed high-flux membrane composed of a polyester-polymer alloy (PEPA) with the components of polyarylate and polyethersulfone (dialyzer FLX-12 GW; Nikkiso Co., Japan) has been evaluated with regard to both hiocompatibility and elimination capacity for β2M during hemodialysis of 8 stable chronic uremic patients. The clearance values of low molecular weight solutes were in the same range as those reported for high-flux dialyzers of comparable surface area. There was no drop in leukocyte counts and only a minimal fall in platelet counts nearly in the same range as has been observed by other investigators using polyamide membrane. C3a Des Arg generation was low, and C5a Des Arg formation was not significantly influenced. There was a sharp drop in the serum β2M level (-35%) during dialysis with a clearance between 59.7 ± 5.6 ml/min ( Q_B 200 ml/min) and 70.1 ± 9.7 ml/min ( Q_B 300 ml/min), respectively. Accordingly, the sieving coefficient was calculated to be 0.2 at 30 min after start of dialysis and 0.6 1 h later. The membrane was able to remove 184.0 ± 22.3 mg/4 h due to an apparent rate of adsorption during the first hour of treatment in combination with high transmembrane transfer in the following time.     

Stable Control of Physiological Parameters,But Not Infection,in Preterm Lambs Maintained on Ex Vivo Uterine Environment Therapy

Ex vivo uterine environment (EVE) therapy is an experimental neonatal intensive care strategy wherein gas exchange is performed by membranous oxygenators attached to the umbilical vessels. Our aim was to assess the ability of a newly refined EVE system to maintain key physiological parameters in preterm lambs within optimal ranges for 48 h. EVE group; n = 6: Preterm lambs were delivered under general anesthesia at 115 ± 2 days of gestational age. Animals were submerged in a bath of artificial amniotic fluid on EVE therapy for 48 h. Physiological parameters were monitored in real‐time over the length of the experiment. Control group; n = 11: Ewes carrying a single fetus (115 ± 2 days of gestational age) underwent recovery surgery to allow placement of a fetal carotid artery catheter. Fetuses received an infusion of sterile saline only. After euthanasia, EVE and Control group fetuses underwent necroscopy to perform static pressure–volume curves and for sampling of lung and cord blood plasma for molecular analyses. Five out of six fetuses in the EVE group completed the study period with key physiological variables remaining within their respective reference ranges for the duration of the 48 h study. Bacteremia was identified in four out of five EVE fetuses, and was associated with a systemic inflammatory response. Using our refined EVE therapy platform, preterm lambs were maintained in a stable physiological condition for 48 h. These findings represent a significant advance over earlier work with this system; however, the identification of bacteremia and a fetal inflammatory response suggests that further refinement to the EVE therapy platform is required.     

Activation of Basophils Is a New and Sensitive Marker of Biocompatibility in Hemodialysis

The hemodialysis procedure involves contact between peripheral blood and the surface of dialyzer membranes, which may lead to alterations in the pathways of innate and adaptive immunity. We aimed to study the effect of blood–membrane interaction on human peripheral basophils and neutrophils in hemodialysis with high‐ and low‐permeability polysulfone dialyzers. The surface expression of CD203c (basophil selection marker) and CD63 (activation marker) after activation by the bacterial peptide formyl‐methionyl‐leucyl‐phenylalanine (fMLP) or anti‐Fcε receptor I (FcεRI) antibody and the absolute number of basophils was investigated before and after hemodialysis with each of the dialyzers. Moreover, the expression on neutrophils of CD11b, the CD11b active epitope, and CD88 was analyzed in the same groups of individuals. The expression of CD63 in basophils following activation by fMLP was significantly higher in the patient group compared with that in healthy controls, but no differences were observed after activation by anti‐FcεRI. During the hemodialysis procedure, the low‐flux membrane induced up‐regulation of CD63 expression on basophils, while passage through the high‐flux membrane did not significantly alter the responsiveness. In addition, the absolute number of basophils was unchanged after hemodialysis with either of the dialyzers and compared with healthy controls. We found no significant differences in the expression of the neutrophil activation markers (CD11b, the active epitope of CD11b, and CD88) comparing the two different dialyzers before and after dialysis and healthy controls. Together, these findings suggest that alterations in basophil activity may be a useful marker of membrane bioincompatibility in hemodialysis.     

The Effects of a Polyacrylonitrile Membrane and a Membrane Made of Regenerated Cellulose on the Plasma Concentrations of Erythropoietin During Hemodialysis

In vitro studies have shown that some dialysis membranes significantly adsorb erythropoietin (EPO), a fact that might have an effect on anemia in long-term hemodialysis (HD) patients and on anemia treatment with recombinant human EPO. The purpose of the study was to determine whether the ability of adsorption demonstrated in vitro also has an effect on EPO concentrations in vivo. In a crossover study, the plasma concentrations of EPO were examined in 11 patients on chronic HD during HD using a polyacrylonitrile (AN69) membrane (high in vitro adsorption) plus EPO administered subcutaneously after the HD session, HD using a Cuprophan membrane (low in vitro adsorption) plus EPO administered subcutaneously after the HD session, HD using an AN69 membrane plus EPO administered subcutaneously after the HD session plus EPO administered intravenously immediately before HD, or HD using a Cuprophan membrane plus EPO administered subcutaneously after the HD session plus EPO intravenously immediately before HD. The intradialysis plasma concentrations of EPO (not detectable in the dialysate) determined at the dialyzer inlet and outlet at Minutes 5 and 240 of the procedure did not differ significantly after its subcutaneous administration from its predialysis concentrations with either the Cuprophan or AN69 membrane. A comparison of EPO concentrations between AN69 and Cuprophan did not reveal marked differences either. The course of concentrations after additional EPO intravenous administration was similar with no statistically demonstrable difference between the 2 membranes. In conclusion, under clinical conditions, AN69 and Cuprophan membranes do not differ in their effects on plasma EPO concentrations. The differences in EPO adsorption between AN69 and Cuprophan, demonstrated in vitro, do not seem to be of clinical importance.