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Journal of Visualized Experiments : JoVE Jul 2012The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue,...
The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate (5,7). Often, changes in response to hypoxia begin before the tissue can be preserved (12). The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures (3). It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.
Topics: Animals; Fixatives; Formaldehyde; Perfusion; Polymers; Rats; Tissue Fixation
PubMed: 22871843
DOI: 10.3791/3564 -
Transplant International : Official... 2022Currently, static cold storage (SCS) of hearts from donations after brainstem death remains the standard clinically. However, machine perfusion (MP) is considered an... (Review)
Review
Currently, static cold storage (SCS) of hearts from donations after brainstem death remains the standard clinically. However, machine perfusion (MP) is considered an approach for donor organ management to extend the donor pool and/or increase the utilization rate. This review summarizes and critically assesses the available clinical data on MP in heart transplantation. We searched Medline (PubMed), Cochrane, Embase, and clinicaltrials.gov, along with reference lists of the included publications and identified 40 publications, including 18 articles, 17 conference abstracts, and five ongoing clinical trials. Two types of MP were used: hypothermic MP (HMP) and normothermic MP (NMP). Three studies evaluated HMP, and 32 evaluated NMP. Independent of the system, MP resulted in clinical outcomes comparable to traditional SCS. However, NMP seemed especially beneficial for high-risk cases and donation after circulatory death (DCD) hearts. Based on currently available data, MP is non-inferior to standard SCS. Additionally, single-centre studies suggest that NMP could preserve the hearts from donors outside standard acceptability criteria and DCD hearts with comparable results to SCS. Finally, HMP is theoretically safer and simpler to use than NMP. If a machine malfunction or user error occurs, NMP, which perfuses a beating heart, would have a narrower margin of safety. However, further well-designed studies need to be conducted to draw clear conclusions.
Topics: Heart; Heart Transplantation; Humans; Organ Preservation; Perfusion; Tissue Donors
PubMed: 35401041
DOI: 10.3389/ti.2022.10258 -
Machine perfusion preservation versus static cold storage for deceased donor kidney transplantation.The Cochrane Database of Systematic... Mar 2019Kidney transplantation is the optimal treatment for end-stage kidney disease. Retrieval, transport and transplant of kidney grafts causes ischaemia reperfusion injury.... (Meta-Analysis)
Meta-Analysis
BACKGROUND
Kidney transplantation is the optimal treatment for end-stage kidney disease. Retrieval, transport and transplant of kidney grafts causes ischaemia reperfusion injury. The current accepted standard is static cold storage (SCS) whereby the kidney is stored on ice after removal from the donor and then removed from the ice box at the time of implantation. However, technology is now available to perfuse or "pump" the kidney during the transport phase or at the recipient centre. This can be done at a variety of temperatures and using different perfusates. The effectiveness of treatment is manifest clinically as delayed graft function (DGF), whereby the kidney fails to produce urine immediately after transplant.
OBJECTIVES
To compare hypothermic machine perfusion (HMP) and (sub)normothermic machine perfusion (NMP) with standard SCS.
SEARCH METHODS
We searched the Cochrane Kidney and Transplant Register of Studies to 18 October 2018 through contact with the Information Specialist using search terms relevant to this review. Studies in the Register are identified through searches of CENTRAL, MEDLINE, and EMBASE, conference proceedings, the International Clinical Trials Register (ICTRP) Search Portal and ClinicalTrials.gov.
SELECTION CRITERIA
All randomised controlled trials (RCTs) and quasi-RCTs comparing HMP/NMP versus SCS for deceased donor kidney transplantation were eligible for inclusion. All donor types were included (donor after circulatory (DCD) and brainstem death (DBD), standard and extended/expanded criteria donors). Both paired and unpaired studies were eligible for inclusion.
DATA COLLECTION AND ANALYSIS
The results of the literature search were screened and a standard data extraction form was used to collect data. Both of these steps were performed by two independent authors. Dichotomous outcome results were expressed as risk ratio (RR) with 95% confidence intervals (CI). Continuous scales of measurement were expressed as a mean difference (MD). Random effects models were used for data analysis. The primary outcome was incidence of DGF. Secondary outcomes included: one-year graft survival, incidence of primary non-function (PNF), DGF duration, long term graft survival, economic implications, graft function, patient survival and incidence of acute rejection.
MAIN RESULTS
No studies reported on NMP, however one ongoing study was identified.Sixteen studies (2266 participants) comparing HMP with SCS were included; 15 studies could be meta-analysed. Fourteen studies reported on requirement for dialysis in the first week post-transplant (DGF incidence); there is high-certainty evidence that HMP reduces the risk of DGF when compared to SCS (RR 0.77; 95% CI 0.67 to 0.90; P = 0.0006). HMP reduces the risk of DGF in kidneys from DCD donors (7 studies, 772 participants: RR 0.75; 95% CI 0.64 to 0.87; P = 0.0002; high certainty evidence), as well as kidneys from DBD donors (4 studies, 971 participants: RR 0.78, 95% CI 0.65 to 0.93; P = 0.006; high certainty evidence). The number of perfusions required to prevent one episode of DGF (number needed to treat, NNT) was 7.26 and 13.60 in DCD and DBD kidneys respectively. Studies performed in the last decade all used the LifePort machine and confirmed that HMP reduces the incidence of DGF in the modern era (5 studies, 1355 participants: RR 0.77, 95% CI 0.66 to 0.91; P = 0.002; high certainty evidence). Reports of economic analysis suggest that HMP can lead to cost savings in both the North American and European settings.Two studies reported HMP also improves graft survival however we were not able to meta-analyse these results. A reduction in incidence of PNF could not be demonstrated. The effect of HMP on our other outcomes (incidence of acute rejection, patient survival, hospital stay, long-term graft function, duration of DGF) remains uncertain.
AUTHORS' CONCLUSIONS
HMP is superior to SCS in deceased donor kidney transplantation. This is true for both DBD and DCD kidneys, and remains true in the modern era (studies performed in the last decade). As kidneys from DCD donors have a higher overall DGF rate, fewer perfusions are needed to prevent one episode of DGF (7.26 versus 13.60 in DBD kidneys).Further studies looking solely at the impact of HMP on DGF incidence are not required. Follow-up reports detailing long-term graft survival from participants of the studies already included in this review would be an efficient way to generate further long-term graft survival data.Economic analysis, based on the results of this review, would help cement HMP as the standard preservation method in deceased donor kidney transplantation.RCTs investigating (sub)NMP are required.
Topics: Delayed Graft Function; Graft Rejection; Graft Survival; Humans; Incidence; Kidney; Kidney Transplantation; Organ Preservation; Perfusion; Randomized Controlled Trials as Topic; Refrigeration; Time Factors; Tissue Donors
PubMed: 30875082
DOI: 10.1002/14651858.CD011671.pub2 -
Annals of Transplantation Aug 2021A shortage of available organs for liver transplantation has led transplant surgeons and researchers to seek for innovative approaches in hepatoprotection and... (Review)
Review
A shortage of available organs for liver transplantation has led transplant surgeons and researchers to seek for innovative approaches in hepatoprotection and improvement of marginal allografts. The most exciting development in the past decade has been continuous mechanical perfusion of livers with blood or preservation solution to mitigate ischemia-reperfusion injury in contrast to the current standard of static cold storage. Two variations of machine perfusion have emerged in clinical practice. During hypothermic oxygenated perfusion the liver is perfused using a red blood cell-free perfusate at 2-10°C. In contrast, normothermic machine perfusion mimics physiologic liver perfusion using a red blood cell-based solution at 35.5-037.5°C, offering a multitude of potential advantages. Putative effects of normothermic perfusion include abrogation of hyperfibrinolysis after reperfusion and inflammation, glycogen repletion, and regeneration of adenosine triphosphate. Research in normothermic machine perfusion focuses on development of biomarkers predicting allograft quality and susceptibility to ischemia-reperfusion injury. Moreover, normothermic perfusion of marginal allografts allows for application of a variety of therapeutic interventions potentially enhancing organ quality. Both methods need to be subjected to translational investigation and evaluation in clinical trials. A clear advantage is transformation of an emergency procedure at night into a planned daytime surgery. Current clinical trials suggest that normothermic perfusion not only increases the use of hepatic allografts but is also associated with milder ischemia-reperfusion injury, resulting in a reduced risk of early allograft dysfunction and less biliary complications, including ischemic cholangiopathy, compared to static cold storage. The aim of this review is to give a concise overview of normothermic machine perfusion and its current applications, benefits, and possible advances in the future.
Topics: Aged; Biomarkers; Humans; Liver; Liver Transplantation; Organ Preservation; Perfusion; Reperfusion Injury
PubMed: 34426566
DOI: 10.12659/AOT.931664 -
Transplant International : Official... 2022normothermic perfusion (EVNP) is an emerging strategy in kidney preservation that enables resuscitation and viability assessment under pseudo-physiological conditions... (Review)
Review
normothermic perfusion (EVNP) is an emerging strategy in kidney preservation that enables resuscitation and viability assessment under pseudo-physiological conditions prior to transplantation. The optimal perfusate composition and duration, however, remain undefined. A systematic literature search (Embase; Medline; Scopus; and BIOSIS Previews) was conducted. We identified 1,811 unique articles dating from January 1956 to July 2021, from which 24 studies were deemed eligible for qualitative analysis. The perfusate commonly used in clinical practice consisted of leukocyte-depleted, packed red blood cells suspended in Ringer's lactate solution with Mannitol, dexamethasone, heparin, sodium bicarbonate and a specific nutrient solution supplemented with insulin, glucose, multivitamins and vasodilators. There is increasing support in preclinical studies for non-blood cell-based perfusates, including Steen solution, synthetic haem-based oxygen carriers and acellular perfusates with supraphysiological carbogen mixtures that support adequate oxygenation whilst also enabling gradual rewarming. Extended durations of perfusion (up to 24 h) were also feasible in animal models. Direct comparison between studies was not possible due to study heterogeneity. Current evidence demonstrates safety with the aforementioned widely used protocol, however, extracellular base solutions with adequate oxygenation, supplemented with nutrient and metabolic substrates, show promise by providing a suitable environment for prolonged preservation and resuscitation. https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021231381, identifier PROSPERO 2021 CRD42021231381.
Topics: Animals; Extracorporeal Circulation; Humans; Kidney; Kidney Transplantation; Organ Preservation; Perfusion
PubMed: 35634582
DOI: 10.3389/ti.2022.10236 -
Clinical Hemorheology and... 2021Extracorporeal perfusion (EP) is moving into focus of research in reconstructive and transplantation medicine for the preservation of amputates and free tissue...
BACKGROUND
Extracorporeal perfusion (EP) is moving into focus of research in reconstructive and transplantation medicine for the preservation of amputates and free tissue transplants. The idea behind EP is the reduction of ischemia-related cell damage between separation from blood circulation and reanastomosis of the transplant. Most experimental approaches are based on a complex system that moves the perfusate in a circular course.
OBJECTIVE AND METHODS
In this study, we aimed to evaluate if a simple perfusion by an infusion bag filled with an electrolyte solution can provide acceptable results in terms of flow stability, oxygen supply and viability conservation for EP of a muscle transplant. The results are compared to muscles perfused with a pump system as well as muscles stored under ischemic conditions after a one-time intravasal flushing with Jonosteril.
RESULTS
With this simple method a sufficient oxygen supply could be achieved and functionality could be maintained between 3.35 times and 4.60 times longer compared to the control group. Annexin V positive nuclei, indicating apoptosis, increased by 9.7% in the perfused group compared to 24.4% in the control group.
CONCLUSIONS
Overall, by decreasing the complexity of the system, EP by one-way infusion can become more feasible in clinical situations.
Topics: Humans; Ischemia; Organ Preservation; Perfusion; Plastic Surgery Procedures
PubMed: 28759964
DOI: 10.3233/CH-170298 -
Transplantation Aug 2022Ischemia-reperfusion injury remains a primary concern in upper extremity transplantation. Ex vivo normothermic perfusion (EVNP) enables near-physiological organ...
BACKGROUND
Ischemia-reperfusion injury remains a primary concern in upper extremity transplantation. Ex vivo normothermic perfusion (EVNP) enables near-physiological organ preservation, avoiding the deleterious effects of hypoxia and cooling. We investigated the effectiveness of human limb EVNP compared with static cold storage (SCS).
METHODS
Twenty human upper extremities were procured. Ten were perfused at 38 °C with an oxygenated red blood cell-based solution, and contralateral limbs served as SCS control (4 °C). EVNP was terminated with systolic arterial pressure ≥115 mm Hg, compartment fullness, or a 20% decline in oxygen saturation. Weight, contractility, compartment pressure, tissue oxygen saturation, and uptake rates were assessed. Perfusate fluid dynamics, gases, electrolytes, and metabolites were measured. Myocyte injury scores and liquid chromatography-mass spectrometry analysis were performed.
RESULTS
EVNP duration was 41.6 ± 9.4 h. Vascular resistance averaged 173.0 ± 29.4 mm Hg × min/L. Weight change and compartment pressures were 0.4 ± 12.2% ( P = 0.21) and 21.7 ± 15.58 mm Hg ( P = 0.003), respectively. Arterial and venous carbon dioxide partial pressure, oxygen saturation, and pH were 509.5 ± 91.4 mm Hg, 15.7 ± 30.2 mm Hg, 87.4 ± 11.4%, and 7.3 ± 0.2, respectively. Oxygen uptake rates averaged 5.7 ± 2.8 mL/min/g. Lactate reached 20 mmol/L after 15 (interquartile range = 6) h. Limb contractility was preserved for 30.5 (interquartile range = 15.8) h ( P < 0.001) and negatively correlated with perfusate potassium (ρ = -0.7, P < 0.001). Endpoint myocyte injury scores were 28.9 ± 11.5% (EVNP) and 90.2 ± 11.8% (SCS) ( P < 0.001). A significant increase in taurine ( P = 0.002) and decrease in tryptophan ( P = 0.002) were detected. Infrared thermography and indocyanine green angiography confirmed the presence of peripheral perfusion.
CONCLUSIONS
EVNP can overcome the limitations of cold preservation by extending preservation times, enabling limb quality assessment, and allowing limb reconditioning before transplantation.
Topics: Extracorporeal Circulation; Humans; Organ Preservation; Organ Preservation Solutions; Perfusion; Upper Extremity
PubMed: 35152257
DOI: 10.1097/TP.0000000000004045 -
Nature Communications Aug 2023Current machine perfusion technology permits livers to be preserved ex situ for short periods to assess viability prior to transplant. Long-term normothermic perfusion...
Current machine perfusion technology permits livers to be preserved ex situ for short periods to assess viability prior to transplant. Long-term normothermic perfusion of livers is an emerging field with tremendous potential for the assessment, recovery, and modification of organs. In this study, we aimed to develop a long-term model of ex situ perfusion including a surgical split and simultaneous perfusion of both partial organs. Human livers declined for transplantation were perfused using a red blood cell-based perfusate under normothermic conditions (36 °C) and then split and simultaneously perfused on separate machines. Ten human livers were split, resulting in 20 partial livers. The median ex situ viability was 125 h, and the median ex situ survival was 165 h. Long-term survival was demonstrated by lactate clearance, bile production, Factor-V production, and storage of adenosine triphosphate. Here, we report the long-term ex situ perfusion of human livers and demonstrate the ability to split and perfuse these organs using a standardised protocol.
Topics: Humans; Liver Transplantation; Liver; Perfusion; Bile; Preservation, Biological
PubMed: 37553343
DOI: 10.1038/s41467-023-40154-8 -
Transplantation Dec 2022Normothermic ex situ liver perfusion is increasingly used to assess donor livers, but there remains a paucity of evidence regarding criteria upon which to base a...
BACKGROUND
Normothermic ex situ liver perfusion is increasingly used to assess donor livers, but there remains a paucity of evidence regarding criteria upon which to base a viability assessment or criteria predicting early allograft function.
METHODS
Perfusate variables from livers undergoing normothermic ex situ liver perfusion were analyzed to see which best predicted the Model for Early Allograft Function score.
RESULTS
One hundred fifty-four of 203 perfused livers were transplanted following our previously defined criteria. These comprised 84/123 donation after circulatory death livers and 70/80 donation after brain death livers. Multivariable analysis suggested that 2-h alanine transaminase, 2-h lactate, 11 to 29 mmol supplementary bicarbonate in the first 4 h, and peak bile pH were associated with early allograft function as defined by the Model for Early Allograft Function score. Nonanastomotic biliary strictures occurred in 11% of transplants, predominantly affected first- and second-order ducts, despite selection based on bile glucose and pH.
CONCLUSIONS
This work confirms the importance of perfusate alanine transaminase and lactate at 2-h, as well as the amount of supplementary bicarbonate required to keep the perfusate pH > 7.2, in the assessment of livers undergoing perfusion. It cautions against the use of lactate as a sole indicator of viability and also suggests a role for cholangiocyte function markers in predicting early allograft function.
Topics: Bicarbonates; Alanine Transaminase; Liver Transplantation; Perfusion; Liver; Lactates; Allografts; Organ Preservation
PubMed: 36044364
DOI: 10.1097/TP.0000000000004263 -
Current Topics in Medicinal Chemistry Jan 2002Organ perfusion techniques bridge the methodological gap between in vivo studies on the one hand and in vitro studies on the other. In drug candidate selection and... (Review)
Review
Organ perfusion techniques bridge the methodological gap between in vivo studies on the one hand and in vitro studies on the other. In drug candidate selection and subsequent development the differences between these systems should be considered carefully in study design, as one approach may be more suitable than the other depending on the question(s) being asked and, in particular, how the data will be used. This article is not concerned with the mechanics, the surgery or composition of perfusates as there are numerous reviews/books covering these aspects. Instead, using perfused gut, liver, lung, kidney and brain as examples, the emphasis is on the usefulness (or otherwise) of the data generated with respect to drug absorption, metabolism, pharmacokinetics (PK) and the factors which affect these parameters. Perfusion systems are not difficult to set up but do require 'high maintenance' for routine use. For this reason they have been used sparingly by the pharmaceutical industry mainly for problem solving or mechanistic studies. The latter part of this article shows how simultaneous dosing of numerous compounds followed by multiple--component analysis using LC/MS/MS has proved to be an effective way to improve the throughput of absorption, pharmacokinetics and metabolism screening ex vivo.
Topics: Animals; Drug Evaluation, Preclinical; Humans; In Vitro Techniques; Mass Spectrometry; Models, Biological; Organ Specificity; Perfusion; Pharmacokinetics
PubMed: 11899066
DOI: 10.2174/1568026023394623