RU2765462C1 - Method for predicting the risk of early cadaveric liver transplant dysfunction - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, namely to transplantology. Risk prediction method early cadaveric liver dysfunction consists in washing the liver transplant with normothermic 0.9 % sodium chloride solution in amount of 1,000 ml with subsequent determination of concentration of potassium (K+) and concentration of glucose (Glu) in the obtained washing liquid before starting blood flow. Then coefficient (C) is calculated by formula: C=Glu/K+and if C<1, in the recipient the risk of early graft dysfunction is predicted.EFFECT: method enables determining the liver transplant condition with subsequent prediction of its functions, as well as provides higher accuracy of predicting the risk of early hepatic transplant dysfunction.1 cl, 3 tbl, 3 ex

Description

SUBSTANCE: invention relates to medicine, namely to organ transplantation, and can be used to predict the risk of early cadaveric liver transplant dysfunction.

Orthotopic liver transplantation (OLT) is the most effective and the only radical method for the treatment of end-stage chronic liver diseases.

Every year, according to the Council of Europe, from 5.6 to 9% of patients who were observed in the year on the OLT waiting list die without waiting for a transplant. From 45.2 to 56.3% of patients undergo surgery. Unfortunately, it is not possible to provide all operations with organs from optimal donors. Therefore, marginal donors or donors with expanded criteria are also considered as a source of a donor organ.

Standard or optimal donors include patients with brain death who died from a traumatic brain injury, aged less than 60 years, with stable hemodynamics or insignificant inotropic support. At the same time, the concentration of serum sodium should not exceed 160 mmol / l, liver tests (AST, ALT, GGT) should not exceed the double norm, body mass index (BMI) <30, and the duration of stay in the intensive care unit should not be more than 5 days.

On the way to expanding the donor pool for liver harvesting, the acceptable donor criteria were revised and the so-called "extended criteria" were adopted, including the age of the donor over 60 years, stay in the intensive care unit for more than seven days, a high level of inotropic support (dopamine more than 15 mcg / kg/min), morbid obesity (body mass index over 30 kg/height/ ^m2 ), serum sodium over 160 mmol/l, cold ischemia time over 15 hours, and histological signs of fatty hepatosis (over 50%). This approach led to an increase in the number of OLTs, but also had an impact on patient and graft survival. In this regard, the problem of the variability of the initial function of the liver graft (LT) has become more urgent. An increased risk of developing early liver graft dysfunction (ELD), which includes concepts such as primary non-functioning graft (PNPT) and poor initial function (PNF), is one of the most negative consequences of expanding the donor pool. RDT of the liver, today, is one of the leading causes of complications and mortality in the early period after cadaveric liver transplantation and determines the duration of PT function. According to the literature, from 15 to 35% of recipients after liver transplantation have RTD (of varying degrees) up to a primary non-functioning graft (PNT). RDT is clinically and prognostically significant for the final result and occurs due to severe ischemic and reperfusion injury of the cadaveric liver. The division of RDTP into two categories - PNF and PNP - characterizes the different severity and reversibility of morphofunctional damage to the liver graft. It is believed that PNF is a reversible condition, the outcome of which depends on the regenerative capacity of the transplanted liver. PNFT is an irreversible condition that inevitably leads to graft loss. PNF and PNF are the most serious complications of the early post-transplant period. As a rule, in recipients with RDTP, the time spent in the intensive care unit (ICU) and in the hospital is prolonged, mortality rates and graft loss increase (Prediction and treatment of early dysfunction of a transplanted liver. Abstract of the thesis for the degree of Candidate of Medical Sciences Moisyuk L. Ya., 2017).

A known method for predicting RTD, described in the patent. BY 21121 “A method for perioperative prediction of the risk of early dysfunction and acute liver transplant rejection in liver transplantation. The authors determined the rs913930 genotype of the TLR4 gene in the donor's blood, during the back-table operation they performed a biopsy from the III segment of the liver and determined the levels of expression of HMGB-1 and CD68 ⁺ in the biopsy specimen, during transplantation 1 hour after portal reperfusion, blood was taken from the left hepatic vein of the recipient with the determination of the level of MIP-1 in it, and 2 hours after reperfusion - blood sampling from the recipient's portal vein with the determination of the concentration of HGF in it, before suturing the skin wound, a post-reperfusion biopsy was performed from segment III of the liver and the levels of HMGB-1 expression were determined in the biopsy and CD68 ⁺ and when a donor of the CC or C/T genotype rs913930 of the TLR4 gene is detected in the blood, and / or when the post-reperfusion biopsy specimen has an increase in the level of HMGB-1 expression by more than 13% compared to the pre-reperfusion biopsy, and / or at the level of CD68 + expression more than 48 cells/mm ² in the post-reperfusion biopsy and/or HGF concentration in the blood of the recipient more than 7900 ng/ml predicted a high risk of early dysfunction and, and at the level of MIP-1 in the blood of the recipient more than 40 ng/ml, a high risk of liver transplant rejection was predicted. Determination of the expression level of HMGB-1 and CD68+ in a biopsy specimen is informative, however, the preparation of a preparation for immunohistochemical studies requires a lot of time and depends on the technologies of a particular medical institution. Therefore, the authors obtain the result of such a study already in the post-transplant period. The duration of graft survival is determined mainly by the degree of immune response of the recipient's body against foreign tissue. Therefore, to maintain PT after surgery, special preparations are used, the so-called immunosuppressants, which are constantly being improved in the direction of increasing the selectivity of their action from “non-specific” to “specific”. In the first case, the ability to suppress the processes of hematopoiesis or lymphopoiesis as a whole is implied, in the second, selective inhibition of executive, more specialized mechanisms (links) of immunity is assumed. On the other hand, immunosuppressants themselves can be the cause of complications. In the method, treatment is prescribed to the recipient even before the result of the immunohistochemical study is obtained. And this can cause the development and complication of the course of RTD.

There are also various methods for predicting the risk of post-transplant complications. Method [Muller T. et al. Transplant Proc. 31 - 1999 - Vol.3B - R.12S-15S] is to determine the concentration in the blood of the recipient of T-lymphocytes. The authors recommend maintaining the concentration of T-lymphocytes in the blood to prevent rejection of the transplanted liver within 50/µl and below. In the method for predicting the risk of complications in recipients after cadaveric liver transplantation (RF patent 2424529), the authors determine the concentration of circulating CD34 hematopoietic stem cells in the peripheral blood at least 2 times a week during the first month after liver transplantation. The risk of post-transplant complications is predicted from the average values for the first 10 and subsequent 20 days with a value below 0.1% in the second or both time intervals. The methods have proven their effectiveness, however, it should be noted that the prediction of the risk of complications was not carried out in the post-transplant period. The authors noted the changes characteristic of the already developed RTD.

In clinical practice, it is generally accepted that changes in the donor liver, which may further lead to RTD, begin from the moment when arterial blood ceases to enter the organ, and continues to develop in the process of subsequent restoration of blood flow (Cheezum MK, Marzilli M., 2014) . And also the occurrence of changes depends on the explantation and conservation of the organ (duration of primary thermal ischemia, duration of cold ischemia of the organ, composition of the preservative solution) and the quality of the implantation stage (duration of secondary thermal ischemia of the organ, prevention and protection against ischemic damage (IP)).

The main problem of hypothermic preservation of organs is the cessation of work (due to ischemia and hypothermia) of the sodium pump, which leads to a gradual change in intracellular and extracellular concentrations of potassium and sodium and cell hydration. The effectiveness of preservative solutions, different in composition, is due to poor diffusion of ions and solutes into the cell (prevention of cell edema) (Compared efficacy of preservation solutions in liver transplantation: A long-term graft outcome study from the european liver transplant registry / R. Adam [ et al.] // American Journal of Transplantation. - 2015).

The world uses 5 main preservative solutions Collins (Collins), Euro-Collins (Euro-Collins), UW, Viaspan, HTK (Custodiol, Custodiol), Celsior (Celsior). Basically, in the Russian Federation, Custodiol (Сustodiol) is a hyperpotassic solution, originally developed for cardioplegia, but successfully used for hypothermic preservation of organs, as a preservative solution for cadaveric liver. It is currently believed that preservation of the liver by Custodiol up to 8 hours is safe, but, nevertheless, does not exclude the occurrence of RTD up to PNF. The organ-preserving effects of Custodiol are primarily due to its osmolarity, which, under conditions of circulatory hypoxia, prevents the development of intracellular edema, and, consequently, the mechanism of osmotic damage to the hepatocyte (Experimental rationale for the use of a combined solution for the conservation of a liver explant. Abstract of a dissertation for the degree of candidate of medical sciences Sokharev A.S.).

Liver ischemia at various stages of organ harvesting and preservation and subsequent reperfusion causes significant damage to hepatocytes, capillary endothelium, and bile duct epithelium (Montalvo-Jave E.E. et al., 2008). Reperfusion injury is considered an etiological factor in graft rejection reactions (in 10% of recipients), as well as liver failure in the postoperative period (Manzini G. Et al., 2013).

One of the most important aspects of the conditioning of liver transplants is the creation of conditions in which hepatocytes are able to maintain homeostasis under conditions of ischemia and generate energy substrates after reperfusion. It is well known that the liver is central to glucose metabolism, synthesizing substrates involved in several metabolic pathways (The role of the liver in metabolic homeostasis: implications for inborn errors of metabolism. JIMD.1991; Human hepatocytes as a tool for studying toxicity and drug metabolism Curr Drug Metab 2003;). Under conditions of ischemia, the aerobic pathway of glucose metabolism is switched to an anaerobic one and the accumulation of metabolites with toxic properties, such as lactic acid, occurs. At the same time, restoration of homeostasis in liver cells is impossible without excessive consumption of ATP. And the conservation of liver transplants, which does not allow the protection of liver cells aimed at maintaining their homeostasis, can lead to a violation of the function of the transplant.

Thus, ischemia triggers a cascade of damage and can be characterized as a pre-apoptotic phase and apoptosis. The preapoptotic phase is characterized by three processes: hypoxia, malnutrition, and inability to remove toxic metabolic waste products. With the cessation of blood circulation, the influx of molecular oxygen ceases. The onset of hypoxia causes the depletion of energy reserves, such as the depletion of ATP reserves in mitochondria. ATP depletion is accompanied by cellular changes, including edema, loss of normal cell integrity, and loss of membrane polarity. Apoptosis of hepatocytes leads to the accumulation of metabolic waste products, activation of proteases and further cell death. In view of the above, before the donor organ is included in the recipient's bloodstream, it is necessary to remove from it the remains of the preservative containing an excess amount of potassium and cellular decay products in order to minimize or prevent hemodynamic disturbances.

Currently, various options for pre-washing the graft using solutions (Ringer's solution with lactate and 100 ml of 25% albumin, 5% albumin solution) are used at the stage of final vascular reconstruction. This method allows for additional laboratory diagnostics of the condition of the liver graft (determination of indicators in the washing solution).

Washing of the vascular bed of the donor organ is performed after the formation of an anastomosis of the suprahepatic section of the inferior vena cava of the donor organ with the inferior vena cava of the recipient and the formation of an anastomosis of the portal vein stump of the recipient with the portal vein stump of the donor organ for safe subsequent inclusion of the liver into the systemic circulation. Undoubtedly, the most physiological is the perfusion of the donor organ with the blood of the recipient. However, this technique is not always applicable due to the condition of the recipient, due to the course of the disease, performed hepatectomy and excessive blood loss.

Thus, the determination of the viability of PT hepatocytes using various biomarkers can be a predictor of the development of RTD.

There are studies in which the viability of donor liver hepatocytes was determined using various biomarkers (urea, albumin, lactate, potassium, glucose, etc.) (Predictor parameters of liver viability during porcine normothermic ex situ liver perfusion in a model of liver transplantation with marginal grafts. Am J Transplant 2019 Transient hyperglycemia during liver transplantation does not affect the early graft function Annals of hepatology 2015. The authors proved that the level of the studied biomarkers can be a predictor of the degree of damage to the liver tissue and the possibility of its recovery. However, all the studied samples were obtained against the background of continuous machine perfusion of the organ or from the systemic blood flow of the recipient's body, as well as from the bile produced by the already transplanted organ. In addition, measurements were performed in a multicomponent solution with the addition of erythrocytes. This reflects the totality of processes occurring in the body as a whole. Therefore, the allocation of processes occurring directly in the organ seems to be problematic and not very informative.

The known method "Peculiarities of interstitial glucose metabolism in early dysfunction of the transplanted liver" (Center for Surgery and Transplantation of the Federal State Budgetary Institution "State Research Center - Federal Medical Biophysical Center named after A.I. Burnazyan" FMBA of Russia, Moscow).

In the proposed method, the authors evaluated the initial function of liver grafts according to generally accepted laboratory and clinical criteria during the first 7 postoperative days. And also determined the dynamics of the concentration of glucose and its metabolites in the graft using the technology of interstitial microdialysis. The study consisted of two parts: retrospective and prospective.

In the retrospective part, we analyzed the features and outcomes of cadaveric liver transplantation and determined the incidence of RDT and PNF, as well as assessed their impact on graft survival.

In the prospective part, patients with underwent liver transplantation and a microdialysis catheter was inserted during the operation (61 Hepatic Microdialysis Catheter, M Dialysis AB, Sweden). The number of observations in the prospective part is 7. The following events and states were established as endpoints: RTD according to the criteria of K. Olthoff; PNF according to UNOS criteria; transplant loss (recipient death or retransplantation); death of the recipient. The duration of the study was 7 days (160–175 h) after the completion of the liver transplantation operation, during this period, continuous collection of microdialysis samples of the transplant interstitial fluid was performed. The composition of peripheral blood (acid-base state, electrolyte level, biochemical analysis, coagulogram) was studied at least once a day. During transplantation, before suturing the wound, the catheter was placed by puncture into the IV segment of the liver, fixed to the falciform ligament, and brought out through the counter-opening to the anterior abdominal wall. A standard isotonic solution (Perfusion Fluid T1, M Dialysis AB, Sweden) was delivered through the inner lumen of the catheter using a micropump (106 MD Pump, M Dialysis AB, Sweden) at a rate of 0.3 μl/min. When the perfusion fluid reached the semipermeable membrane, passive transport of substances from the interstitial fluid to the catheter cavity began along the concentration gradient. The resulting solution was continuously withdrawn at the same rate through the second lumen of the catheter and collected in a microtube. Change of microtubes, i.e. separate samples were obtained at least once every 3 hours. Determination of the concentrations of glucose, lactate, pyruvate and glycerol was carried out using a set of standard reagents on the analyzer "ISCUS Clinical Microdialysis Analyzer" (M Dialysis AB, Sweden) within 24 hours from the moment of receipt samples.

In 7 patients included in the prospective part of the work, all variants of the initial graft function were observed: normal (n=4), RDT (n=2) and PNF (n=1). In addition, in one case, the early postoperative period was complicated by thrombosis of the hepatic artery against the background of RDT.

The results of the study showed that with the normal initial function of the grafts, the following patterns of interstitial concentrations of glucose and its metabolites were observed:

1. glucose ≥5.0 mmol/l with fluctuation range up to 20 mmol/l;

2. rapid (within 24 hours) decrease in lactate concentration to 1.5-3.0 mmol/l and subsequent stable level ranging from 0.5 to 2.0 mmol/l;

3. In the first 12 hours after graft arterial reperfusion, an increase in pyruvate concentration from 0–50 to 250–500 μmol. By the end of the first day and later, a decrease and stabilization of the pyruvate concentration at the level of 50–300 µmol/L was noted.

With the reversible form of RTD, a more pronounced variability of indicators was observed with the upper limit of the range extended to 23–25 mmol/L (for glucose), up to 20–25 mmol/L (for lactate), up to 1200 µmol/L (for pyruvate). Normalization of indicators occurred by the end of the 2nd day of the postoperative period. With thrombosis of the graft hepatic artery, fulminant, developing within 2–4 hours, changes in interstitial glucose concentrations were observed - a decrease to 0.0–0.5 mmol/l; lactate - growth up to 25-30 mmol / l; pyruvate - reduction to 0–10 µmol/l. In PNF, chronologically, the first marker of severe and irreversible dysfunction was a low (3.0 mmol/l) glucose concentration, which decreased to 0 mmol/l over the next 2 hours and remained at this level until the onset of death.

The authors noted that the method showed its high efficiency in determining the concentration of glucose and its metabolites in the intercellular fluid of the donor liver during OLT. However, this technique is associated with additional traumatization of the donor organ in the form of placing a microcatheter and subsequent monitoring after starting blood flow at different stages of liver functioning. Also, with the help of this study, it is not possible to fully evaluate the initial function of liver grafts, since the measurements were carried out after the start of blood flow in the donor liver, i.e. fixed the nature of changes for already implemented events.

Closest to the proposed is a method for studying the level of potassium in the wash fluid to determine its relationship with severe post-reperfusion syndrome in liver transplantation of a deceased donor (PRS) (Association Between Flushed Fluid Potassium Concentration and Severe Postreperfusion Syndrome in Deceased Donor Liver Transplantation. 2017 Oct 29), which we took as a prototype.

The authors in the method determined the level of potassium in the washing liquid (perfusate). To do this, the liver graft was washed through the portal vein (PV) with 5% albumin solution (1 ml per gram of graft) at normothermic temperature at the stage of completion of the formation of the anastomosis of the subhepatic vena cava (SVC) before starting the blood flow. Samples of the liver graft wash solution were collected at the end of the wash and the potassium level was determined using a gas analyzer. According to the results of this study, the authors revealed a direct pattern between the development and severity of post-reperfusion syndrome, which correlates with an increase in the level of potassium in the wash water and the development of RTD.

Taking into account the effectiveness of the proposed method for determining the safety of a cadaveric donor liver, it should be noted that it is not without a number of disadvantages. The authors found that at a potassium concentration of 6.65 mmol/l, the probability of developing the frequency of early graft dysfunction is 63.6%. Therefore, according to their observations, even at higher rates, there are cases without the development of early graft dysfunction. Therefore, the study of the isolated level of potassium is not an absolute predictor of the occurrence of post-transplant complications. Secondly, in addition to determining the level of potassium, in the work, not liver metabolites were used as the studied indicators, but such indicators as the donor risk index, the Child-Turcott-Pugh score, the type of donor, the time of warm ischemia of AT and the assessment of the model of terminal stages of liver disease. These indicators are complex and require a separate study of their impact on the safety of the graft function.

Summing up the above data, it should be emphasized that modern methods for determining the safety of a donor organ do not meet the needs of practical transplantation.

In this regard, one of the main tasks of research in the field of transplantation is to predict the risk of RTD and the search for objective factors on which such a prediction depends.

Therefore, we came to the conclusion that the study of metabolic markers directly in the donor organ (in the washing fluid) under conditions of its isolation from the recipient's systemic circulation seems to be the most reliable and informative for reflecting its condition and subsequent prediction of its functions.

The technical result of the present invention is to increase the accuracy of predicting the risk of early PT dysfunction due to intraoperative determination of the ratio of the concentration of potassium and glucose in the lavage fluid from the liver transplant before starting blood flow.

This result is achieved by the fact that in the known method for predicting the risk of early dysfunction of a cadaveric liver graft, which includes washing the liver graft with a normothermic solution, followed by determining the concentration of potassium in the resulting lavage fluid before starting blood flow, according to the invention, the graft is washed with a 0.9% solution of sodium chloride in an amount of 1000 ml and, additionally, simultaneously with determining the concentration of potassium (K ⁺ ) in the resulting washing liquid, determine the concentration of glucose (Glu), after which the coefficient (K) is calculated according to the formula: K = Glu / K ⁺ and with a value of K < 1 in the recipient predict the risk of early graft dysfunction.

It is known that when blood flow is started in the PT at high potassium concentrations in the perfusate, the recipient's heart rhythm disturbances may develop, up to and including stopping. As mentioned above, modern preservative solutions are hyperpotassic. In this regard, washing the organ from the preservative, followed by measuring the concentration of potassium in the washing fluid from the graft using a gas analyzer, is a standard procedure for OLT. There are observations demonstrating the concentration of potassium in the perfusate after the use of 1000 ml of sodium chloride (NaCl), exceeding this figure in the preservative solution.

It is also known from the literature that the liver occupies a central place in glucose metabolism, synthesizing substrates involved in several metabolic processes. Glucose level correlates with the degree of ischemic damage and post-transplant function (Predictor parameters of liver viability during porcine normothermic ex situ liver perfusion in a model of liver transplantation with marginal grafts. 2019). Along with potassium, glucose, which is determined in all cases of the perfusate study, is not included in both the preservative solution and the NaCl solution. Considering that the samples of the lavage fluid were obtained directly from the vascular bed isolated by the PT, the only possible source of the studied substances can only be different degrees of damage to hepatocytes.

This led us to the conclusion that the determination of the concentration of potassium and glucose in the lavage fluid reflects the metabolism directly in the isolated PT, and their ratio may be more informative for assessing the effect of harvesting and conservation on the organ. To confirm this, we conducted a study and compared the concentration of potassium and glucose in the lavage fluid from PTs recognized as suitable for the operation of PTs with organs that were harvested for liver transplantation, but the suitability of which had to be confirmed by an urgent histological examination, based on the results of which a decision was made to refuse use in the operation. That. these bodies, according to established criteria, should have had RTD up to its primary non-functioning. We used 5 full-sized livers from cadaveric donors, which, after physical and histological examinations, were found unsuitable for OLT. The death of donors was established according to the criteria for brain death. Washing, explantation, preservation of these organs was carried out according to standard methods for use in the transplantation operation. Physically detected changes in organs that raised doubts about their suitability for transplantation were confirmed by urgent histological examination. After the termination of cold conservation, the development of secondary warm ischemia was modeled, the duration corresponding to the average time of formation of the necessary vascular anastomoses in a real surgical intervention. After occlusion of the suprahepatic section of the inferior vena cava (IVC) of the donor organ, the latter was washed through the portal vein with 0.9% normothermic sodium chloride solution in a volume of 1000 ml, similar to the washing of the organ during surgery. Drainage took place through an open sub-portion of the IVC, which fully corresponded to the situation in the conditions of a real operation. After the organ was flushed with the indicated volume, a sample of the flushing fluid was taken from the lumen of the IVC in the retrohepatic section. The standard sample of flushing liquid for the gas analyzer is 1 ml. Then the concentration of potassium and glucose was determined. Washing of the PT with 0.9% normothermic sodium chloride solution in a volume of 1000 ml. effectively eliminates decay products from the vascular bed. In addition, due to its ionic composition and osmotic pressure, sodium chloride has long been recognized as the most suitable blood substitute solution. And also due to its composition, it allows for the most correct determination of the above parameters.

Table 1 shows the data of post-mortem donors considered unsuitable for liver donation, and the results of the study of the washing fluid of liver transplants harvested from them. Where: 1 - PT; 2 - age; 3 - diagnosis (acute cerebrovascular accident (ACV)); 4 - body mass index; 5 - the need for urgent histological examination; 6 -% macrovesicular hepatosis according to urgent histological examination; 7 – preservation time (hour:min); 8 - potassium level in mmol/l after washing the organ with 1000 ml. NaCl; 9 - glucose level in mmol/l after washing the organ with 1000 ml. NaCl; 10 - the ratio of indicators of glucose / potassium, calculation according to the formula according to the formula: K = Glu / K ⁺ .

Table. No. 1. Data of post-mortem donors deemed unsuitable for liver donation and the results of the study of the washing fluid of liver transplants harvested from them.

one 2 3 4 5 6 7 eight 9 10 A 52 ONMK 47.8 Not one hundred 20:10 17.6 11.7 0.66 B 59 ONMK 22.9 cito 80 3:30 13.9 1.1 0.08 C 59 ONMK 33.2 cito one hundred 10:20 15.7 14.4 0.92 D 56 ONMK 24.9 cito 90 17:30 32 19.7 0.62 E 52 ONMK 30.2 cito 90 52:10 42.1 3.1 0.07

As follows from column 10, in all PTs, K was <1. This indicated the coincidence of the data on the morphological and biochemical status of the graft, which suggested the possibility of the occurrence of RTD up to primary non-functioning.

In a similar way, we measured the concentration of potassium and glucose in the PT during OLT and calculated their ratio. Table No. 2 shows the data of post-mortem donors recognized as morphologically suitable for liver donation, and the results of a study of the washing fluid of liver transplants obtained during the transplant operation. Where: 1- Fri; 2 - age; 3 - diagnosis (traumatic brain injury (TBI)); 4 – donor body mass index; 5 -% macrovesicular hepatosis according to histological examination; 6 – preservation time (hour:min); 7 - potassium level in mmol/l after washing the organ with 1000 ml. NaCl; 8 - glucose level in mmol/l after washing the organ with 1000 ml. NaCl; 9 - ratio of indicators glucose/potassium, calculation according to the formula according to the formula: K= Glu/K ⁺ .

Table. No. 2. Data from post-mortem donors found to be morphologically suitable for liver donation and the results of a study of liver graft lavage obtained during the transplant operation.

one 2 3 4 5 6 7 eight 9 A 39 ONMK 27.4 Up to 5 3:55 3.7 5.2 1.41 B 37 ONMK 25.4 Up to 5 4:15 1.6 1.7 1.06 V 53 ONMK 24.2 Up to 5 3:58 8.4 5.8 0.61 G 25 TBI 22.5 Up to 5 5:26 4.7 3.1 0.66 D 47 ONMK 31.8 10 8:30 11.7 16.1 1.38 E 37 ONMK 31.1 Up to 5 11:25 8.1 eleven 1.35 F 52 TBI 32.4 Up to 5 3:55 7.2 7.2 one

As follows from the given data (Table No. 2), when determining the ratio K= Glu/K+ in the washing liquid of PT A, B, E, F, G > 1. The postoperative period in recipients who received PT data proceeded without complications. Signs of RTD were not identified. This made it possible to carry out the postoperative period in recipients against the background of standard immunosuppression.

Recipients who received PT V and G developed signs of RTD, which required a reduction in immunosuppression (reducing the dose of drugs). To confirm the signs of the development of RDT, the level of transaminases and bilirubin was determined in the recipients in the early postoperative period. An increase in these parameters (transaminases more than 2000 U/l, bilirubin more than 100 µmol/l) indicated the beginning of the development of RTD.

Table No. 3 shows the data of recipients of post-mortem organs in the postoperative period, who underwent determination of the content of potassium and glucose in the washing liquid during the surgical intervention.

Tab. No. 3 Data of recipients of post-mortem organs who underwent determination of the content of potassium and glucose in the washing liquid during the surgical intervention. Where: 1 - PT; 2 - gender of the recipient who received PT; 3 - age of the recipient; 4 - increase in transaminases in the early postoperative period more than 2000 IU/l; 5 - increase in the level of bilirubin over 100 µmol/l; 6 – glucose/potassium ratio <1 (+/-); 7 - the presence of signs of dysfunction of the liver graft in the early postoperative period (+/-).

one 2 3 4 5 6 7 A F 39 - - - - B M 37 - - - - V M 53 - + + + G M 25 + + + + D F 47 - - - - E M 37 - - - - F M 52 - - - -

Comparing and analyzing the obtained results, we came to the conclusion that intraoperative determination of the ratio K = Glu / K⁺v lavage fluid from PT is an effective method for predicting the risks of early dysfunction and allows you to adjust immunosuppressive treatment regimens at the earliest stages. All this prompted us to use this determination of the Glu/K+ ratio in the washing liquid from the PT in our clinical practice.

The essence of the method is illustrated by examples. (we need three examples).

Example 1

Patient T., 42 years old. Zh. has been under observation at the Federal State Budgetary Institution “RSRCHT” since 01/14/2020. Considers himself ill since 1995. In September 2019, a clinic of "jaundice" arose, for which the patient was hospitalized in an infectious diseases hospital. Diagnosis of chronic viral hepatitis C since 1995. Sharp deterioration since September 2019.

After the examination, a diagnosis was made: chronic viral hepatitis "C" with an outcome in cirrhosis of the liver. Child-Pugh В-С, MELD - 25; Portal hypertension: Varicose veins of the esophagus IIst; Ascites; esophageal bleeding. He was referred for a consultation and examination at the Federal State Budgetary Institution "RSRCHT".

In January 2020, the patient applied to the Federal State Budgetary Institution "RSRCHT" for a consultation and examination for Child-Pugh class "C" liver cirrhosis with an outcome in liver cirrhosis, as a result of which she was put on the "waiting list" for liver transplantation.

On February 23, 2020, she was hospitalized at the clinic of the Republican Scientific and Practical Center for Chemistry and Surgery.

On February 23, 2020, orthotopic liver transplantation from a post-mortem donor was performed.

Donor characteristics: male, 39 years old. Cause of brain death: Acute cerebrovascular accident of hemorrhagic type. In the intensive care unit: up to 3 days. Body mass index (BMI) - 24.7. The donor met all selection criteria and had a negative epidemiological history. According to the clinical and laboratory examination and visual data, the post-mortem liver was found to be suitable for use in surgery; an urgent histological examination is not required. Also, all time parameters related to cold and warm ischemia were observed. Planned histological examination of the donor liver - a variant of the norm, macrovesicular steatosis up to 5%.

Preservation of the donor liver with Custodiol solution for 4 hours and 25 minutes.

Immediately before the operation, the recipient was determined:

Bilirubin - 83.6 µmol/l; ALT - 19 U / l; AST - 53 IU / l.

Surgery protocol.

Operation: Starzl laparotomy, revision of the abdominal organs, hepatectomy with preservation of the retrohepatic part of the inferior vena cava (IVC), orthotopic liver transplantation of the "piggy back" type, drainage of the abdominal cavity.

The donor liver is placed in an orthotopic position. A caval anastomosis was formed between the IVC of the donor and the recipient in the "piggy back" type. The portal vein (PV) was divided. Thrombectomy was performed using a Fogarty probe. An end-to-end anastomosis was formed between the IV of the donor and the recipient with a Prolen 6/0 thread.

Donor liver was washed with 1000 ml. 0.9% normothermic sodium chloride solution with drainage through the free lumen of the subhepatic segment of the donor inferior vena cava. At the end of the flush, through the free lumen of the subhepatic segment of the donor inferior vena cava, a sample of the flush fluid was taken from its cavity in the amount of 1 ml to determine the parameters on the gas analyzer. The result was obtained: K+ - 3.7 mmol/l; Glucose - 5.2 mmol / l. coefficient (K) was calculated using the formula K=Glu/K ⁺ . K=5.2/3.7. K=1.4.

The resulting coefficient (K) testified to the functional safety of the donor organ.

Postoperative period:

Maximum values: bilirubin - 30.8 µmol/l; ALT - 435 IU / l; AST - 463 IU / l. This indicated the absence of signs of early graft dysfunction.

Immunosuppression was induced with Simulect according to the recommended protocol. Subsequently, three-component therapy - Tacrolimus, Myfortic, steroid hormonal preparations. Appointment of Tacrolimus under the control of concentration.

Normalization of indicators: bilirubin - 2 days; ALT - 15 days; AST - 7 days.

Conclusion: The early postoperative period was uneventful.

The patient was discharged on the 19th day in a satisfactory condition under observation at the outpatient Transplant Center.

Currently, no graft dysfunction has been identified, and the patient is receiving planned immunosuppressive therapy.

Example 2

Patient K., 49 years old. M. has been under observation at the Federal State Budgetary Institution “RSRCHT” since April 2019.

From the anamnesis: Chronic viral hepatitis "C" with an outcome in cirrhosis of the liver. Liver transplantation was performed on 17.03.2010.

On April 2, 2019, the patient applied to the Federal State Budgetary Institution "RSRCHT" for a consultation and examination. The diagnosis was made: loss of function of the transplanted liver. Child-Pugh С, MELD - 30; Portal hypertension: Varicose veins of the esophagus IIIst; Ascites; Esophageal bleeding. As a result, he was put on the "waiting list" for liver transplantation.

Since April 2019, he has been on the waiting list for liver retransplantation.

On March 26, 2020, the patient was hospitalized at the RRCRT clinic.

On March 26, 2020, the patient underwent orthotopic liver retransplantation from a post-mortem donor.

Donor characteristics: M, 53 years old. Cause of brain death: Acute cerebrovascular accident of ischemic type. In the intensive care unit: up to 3 days; BMI - 24.2. The donor met all selection criteria and had a negative epidemiological history. According to the clinical and laboratory examination and visual data, the post-mortem liver was found to be suitable for use in surgery; an urgent histological examination is not required. Also, all time parameters related to cold and warm ischemia were observed. Planned histological examination of the donor liver - a variant of the norm, macrovesicular steatosis up to 5%.

Preservation of the donor liver with Custodiol solution for 3 hours 58 minutes.

Immediately before the operation, the recipient was determined:

Bilirubin - 251.7 µmol / l; ALT - 68 U / l; AST - 148 IU / l.

Surgery protocol.

Operation: Starzl laparotomy, revision of the abdominal organs, hepatectomy with preservation of the retrohepatic part of the inferior vena cava (IVC), orthotopic liver transplantation of the "piggy back" type, drainage of the abdominal cavity.

The donor liver is placed in an orthotopic position. A caval anastomosis was formed between the IVC of the donor and the recipient in the "piggy back" type. The portal vein (PV) was divided. Thrombectomy was performed using a Fogarty probe. An end-to-end anastomosis was formed between the IV of the donor and the recipient with a Prolen 6/0 thread.

Donor liver was washed with 1000 ml. 0.9% normothermic sodium chloride solution with drainage through the free lumen of the subhepatic segment of the donor inferior vena cava. At the end of the flush, through the free lumen of the subhepatic segment of the donor inferior vena cava, a sample of the flush fluid was taken from its cavity in the amount of 1 ml to determine the parameters on the gas analyzer. The result was obtained: K+ - 8.4 mmol/l; Glucose - 5.8 mmol / l.

The coefficient (K) was calculated using the formula K=Glu/K ⁺ . K=5.8/8.4. K=0.69. The resulting coefficient (K) testified to the possibility of the occurrence of RTD up to the primary non-functioning.

Postoperative period:

Maximum values: bilirubin - 320.1 µmol/l; ALT - 2649 IU / l; AST - 2081 IU / l. This indicated the appearance of signs of early graft dysfunction.

Immunosuppression was induced with Simulect according to the recommended protocol. Given the value of the coefficient less than 1, a decision was made to reduce immunosuppression - the onset was postponed until the third day. Mayfortic was not prescribed. Tacrolimus levels were maintained at 50% of the recommended level. Hormonal preparations in full.

Conclusion: the patient's postoperative period required reduction of immunosuppressive therapy. This was realized in the final restoration of the transplanted liver

Normalization of indicators: bilirubin - 31 days; ALT - 34 days; AST - 28 days.

The patient was discharged on the 44th day in a satisfactory condition under observation at the outpatient Transplant Center.

At present, no graft dysfunction has been identified, and the patient is receiving planned immunosuppressive therapy.

Example 3

Patient V., 39 years old. M. has been under observation at the Federal State Budgetary Institution “RSRCHT” since 11/09/2020.

From the anamnesis: he considers himself ill since September 2019, when periodic epigastric pains, a feeling of heaviness and discomfort in the right hypochondrium first appeared. In August 2020, the "jaundice" clinic emerged. The patient was hospitalized in the Botkin hospital. He was diagnosed with chronic viral hepatitis (CVH) "C", cirrhosis of the liver, class "C" according to Child-Pugh. The patient was referred for a consultation at RRCCT.

On January 25, 2021, the patient applied to the Federal State Budgetary Institution "RSRCHT" for a consultation and examination. Based on the results of the examination, a diagnosis was made: chronic viral unverified hepatitis with an outcome in cirrhosis of the liver. Child-Pugh С, MELD - 24; Portal hypertension: Varicose veins of the esophagus Ist; Ascites. As a result, he was put on the "waiting list" for liver transplantation.

On February 11, 2021, the patient was hospitalized at the RRCRT clinic.

On February 11, 2021, the patient underwent orthotopic liver retransplantation from a post-mortem donor.

Donor characteristics: F, 37 years old. Cause of brain death: Acute cerebrovascular accident of hemorrhagic type. In the intensive care unit: up to 7 days; BMI - 31.1 (extended criteria donor). According to the clinical and laboratory examination and visual data, the post-mortem liver was found to be suitable for use in surgery; an urgent histological examination is not required. Also, all time parameters related to cold and warm ischemia were observed. Planned histological examination of the donor liver - a variant of the norm, macrovesicular steatosis up to 5%.

Preservation of the donor liver with Custodiol solution for 11 hours and 25 minutes.

Immediately before the operation, the recipient was determined:

Bilirubin - 97.6 µmol / l; ALT - 74 IU / l; AST - 33 IU / l.

Surgery protocol.

Operation: Starzl laparotomy, revision of the abdominal organs, hepatectomy with preservation of the retrohepatic part of the inferior vena cava (IVC), orthotopic liver transplantation of the "piggy back" type, drainage of the abdominal cavity.

The donor liver is placed in an orthotopic position. A caval anastomosis was formed between the IVC of the donor and the recipient in the "piggy back" type. The portal vein (PV) was divided. Thrombectomy was performed using a Fogarty probe. An end-to-end anastomosis was formed between the IV of the donor and the recipient with a Prolen 6/0 thread.

Donor liver was washed with 1000 ml. 0.9% normothermic sodium chloride solution with drainage through the free lumen of the subhepatic segment of the donor inferior vena cava. At the end of the flush, through the free lumen of the subhepatic segment of the donor inferior vena cava, a sample of the flush fluid was taken from its cavity in the amount of 1 ml to determine the parameters on the gas analyzer. The result was obtained: K+ - 8.1 mmol/l; Glucose - 8.3 mmol / l. The coefficient (K) was calculated using the formula K=Glu/K+. K=8.3/8.1. K=1.

The resulting coefficient (K=1) testified to the functional safety of the donor organ.

Postoperative period:

Maximum values: bilirubin - 98.8 µmol/l; ALT - 1159 IU / l; AST - 1413 IU / l. This indicated the absence of signs of early graft dysfunction.

Immunosuppression was induced with Simulect according to the recommended protocol. Subsequently, three-component therapy - Tacrolimus, Myfortic, steroid hormonal preparations. Appointment of Tacrolimus under the control of concentration.

Normalization of indicators: bilirubin - 26 days; ALT - 21 days; AST - 22 days.

Conclusion: the early postoperative period of this patient, who received a liver transplant from a donor with extended criteria and a long period of conservation (more than 11 hours), was uneventful. A slight slowdown in the decline in liver function tests was found. No therapy adjustments were required.

The patient was discharged and transferred on the 31st day in a satisfactory condition under observation at the outpatient Transplant Center. At present, no graft dysfunction has been identified, and the patient is receiving planned immunosuppressive therapy.

To date, the proposed method has been successfully used in OLT in 10 recipients, while they have not observed the development of early graft dysfunction. All recipients whose coefficient was below 1 had their postoperative therapy adjusted.

The proposed method in comparison with the prototype has a number of advantages, the main of which is that it allows you to determine the state of the PT with subsequent prediction of its functions due to intraoperative determination of the ratio of potassium and glucose concentration in the lavage fluid from the liver transplant before starting blood flow. It should be noted that the use of this method in clinical practice makes it possible to predict the risk of early dysfunction and to correct immunosuppressive treatment regimens at the earliest stages, thereby preventing its further development.

The method was developed in the group of transplantation surgery, the department of interventional radiology and operative surgery "FGBU RNTSRKhT" and has so far been clinically tested in 10 recipients with a positive result.

Claims (1)

A method for predicting the risk of early dysfunction of a cadaveric liver graft, including washing the liver graft with a normothermic solution, followed by determining the concentration of potassium in the resulting lavage fluid before starting blood flow, characterized in that the cadaveric liver graft is washed with 0.9% sodium chloride solution in an amount of 1000 ml and, additionally, simultaneously with the determination of the concentration of potassium (K ⁺ ) in the resulting washing liquid, the concentration of glucose (Glu) is determined, after which the coefficient (K) is calculated according to the formula: K=Glu/K ⁺ and with a value of K<1 in the recipient, the risk is predicted early graft dysfunction.