RU2424529C1 - Method for prediction of risk of complications of cadaveric liver transplantation in recipients - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: not less than twice a week for the first month following the liver transplantation, recipient's peripheral blood is examined for the concentration of circulating stem haemopoetic cells CD 34+ to find its average values for the first 10 and the following 20 days, and if their value is less than 0.1% in the second or both time intervals, a risk of post-transplantation complications is predicted in the recipient.

EFFECT: invention provides high accuracy of prediction of the risk of early, most dangerous post-transplantation liver rejection.

2 ex, 1 dwg

Description

The invention relates to medicine, more specifically to surgery, and may find application in transplantation of donor organs.

The steady growth of liver diseases, including cirrhosis, is often associated with the need for transplantation of a donor organ to a patient. This, in turn, is associated with the possibility of post-transplant complications, the most formidable of which is rejection of the donor liver as a foreign one. The greatest danger is its rejection in the early postoperative period - usually 4-10 days and up to 1-1.5 months [Ekka-Zohar A. et al. IMAJ (Israel). 2006 Vol. 8 (6). P.400-405].

Currently, it is generally accepted that transplant survival is determined solely by the degree of the immune response of the recipient organism against foreign tissue. The result of such a reaction against foreign information entering the body with the graft is the inclusion of specific cellular and humoral mechanisms (reactions) of active rejection of the transplanted tissue. Therefore, to preserve the transplant, special drugs are used, the so-called immunosuppressants, which historically 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 in general is implied, in the second, selective inhibition of executive, more specialized immunity mechanisms (links) is assumed.

One of the main tasks of research in the field of transplantology is to predict the risk of rejection of the donor liver and the search for objective factors on which such a forecast depends.

A known method for predicting the risk of post-transplant complications by the concentration in the blood of the recipient of T-lymphocytes. The authors recommend maintaining the concentration of blood T-lymphocytes to prevent rejection of the transplanted liver within 50 / μl and below [Muller T. et al. Transplantation Proc. 31 - 1999 - Vol.3B - P.12S-15S]. This method is taken as a prototype. Its authors, like many others, associate transplant survival with its immunological compatibility with the recipient's circulating lymphocytes. However, the maintenance of a blood T-lymphocyte concentration of 50 / μl recommended by the authors after liver transplantation raises some doubts. So, in our practice, successful cases of transplantation were accompanied by significantly higher concentrations of T-lymphocytes (more than 50 / μl) and, conversely, below 50 / μl, it was the least successful. Moreover, the liver transplantation performed in one of the failed cases, which turned out to be successful, was accompanied by a gradual increase in the concentration of T-lymphocytes and exceeded 50 / μl. This fact, in our opinion, indicates that it is necessary to look for other, probably no less substantial and specific mechanisms of the recipient’s organism influence on the functional viability of the liver transplant, not limited to the principle of immunological compatibility. And the prototype method, which is based on this concept, cannot provide an accurate prediction of post-transplant complications. The disadvantages of the prototype method include the fact that the authors estimate the content of T-lymphocytes only in the first 10 days (in the prototype Fig. 1-2 P.13S), while the most dangerous period from the point of view of the possibility of transplant rejection is, as noted above, the first 30 days.

The technical result of the present invention is to improve the accuracy of prediction of post-transplant complications due to the use of CD34 + for circulating hematopoietic stem cells.

This result is achieved by the fact that in the recipient’s peripheral blood at least 2 times a week during the first month after liver transplantation, the concentration of circulating CD34 + hematopoietic stem cells is determined, its average values are found for the first 10 and subsequent 20 days and when their value is below 0.1% during the second or both time intervals in the recipient predict the risk of post-transplant complications.

To solve this problem, we conducted a thorough analysis of the literature data and the results of our own studies on the possible impact on the rejection of the liver of various blood parameters of the recipients.

So, in the literature there is evidence that the success of cadaveric liver transplantation is not determined by the degree of compatibility with the leukocyte complex of human antigens (HLA) [Liver Transplantation. 2007. Vol.13. P.80-90, P.1405-1413].

Recommendations for the level of blood T-lymphocytes range from 50 / μl (in the prototype) to 1 / μl or less [Transplantation Proceedings 13-1999. Vol.1 No. 3B. P.16S-18S].

This led us to the conclusion that it is necessary to search for other factors affecting the functional viability of the liver transplant.

To this end, we monitored the subpopulation composition of the mononuclear fraction of blood in patients after cadaveric liver transplantation for 7 months after surgery [Medical immunology. 2009. No. 4-5 (vol. 11). p.464]. At the same time, we noted fluctuations in various subpopulations of mononuclear cells and at different times after surgery, but we failed to come to a single conclusion about the advantages of this or that indicator.

It is known that in case of malignant processes against the background of antitumor therapy (immunosuppressive therapy), an excessive decrease in the level of mononuclear cell recipients circulating in the blood is accompanied by an increased risk of early mortality.

Earlier, we noted the beneficial effect of autologous bone marrow transplantation on the functional activity of the kidney in glomerulonephritis [Ryabov SI, Rakityanskaya IA, Shutko AN In the book "Kidneys and the immune system." L-d., "Science." 1989, p. 131].

Positive results of the same procedure have been reported with cirrhosis [Medical immunology. 2009. No. 4-5 (vol. 11). p. 463].

Based on these data, we hypothesized the possible participation of bone marrow progenitor cells in maintaining the viability of a liver transplant [Granov A.M., Shutko A.N. Paradoxes of malignant growth and tissue incompatibility, Ed. "Hippocrates", 2002]. Later, against the background of fluctuations in the content of different fractions of lymphocytes, we found significant fluctuations in both stem hematopoietic cells (HSC) and progenitor cells of lymphocytopoiesis in the fraction of peripheral blood mononuclear cells of liver recipients at different time periods after transplantation, i.e. state of "turbulent" hematopoiesis. According to a number of studies, SGCs are able to migrate to various tissues and organs, to the liver, lungs, gastrointestinal tract, blood vessels, and heart, supporting proliferative processes in them [Nat. Med. 2000. Vol. 6. P.1229-1234, Stem Cells. 2001. Vol.19. P.304-312]. Since the capillary network is also the target of such influences during ischemic damage and rejection, it is important to note that a certain part of the circulating SGC carries markers of endothelial cell precursors.

Since SGK are the ancestors for all types of circulating cells, fluctuations in the content of stem cells in the blood determine the fluctuations of other populations. We have shown [European Radiation Research (University of Leicester UK). 2005. P.166], that the concentrations of the main subpopulations of lymphocytes are in very different phases with respect to the value of SGK. It follows that SGCs are a fundamental reference marker of the entire lymphoproduction system, which makes them a priority when interpreting the results.

In connection with the above analysis of known, sometimes conflicting data, and our own observations, we decided to conduct a study to elucidate the possible pathogenetic role of circulating SGC in the early post-transplant period in cadaveric liver recipients (during the first month after surgery).

The study included 36 patients after liver transplantation performed at the Russian Center for Science and Technology. 23 conditionally healthy people without liver diseases, including after extensive surgical interventions examined at the Military Medical Academy, made up the control group. All cadaveric liver transplant recipients received conventional 3-component immunosuppressive therapy, including calcineurin inhibitors (cyclosporine or tacrolimus), prednisone, and azathioprine.

For each of 36 patients of the main group, the following cell markers of blood mononuclear cells were determined for an average frequency of 2 times a week within 30-40 days after surgery and 23 patients from the control group: CD3 +, CD4 +, CD8 +, CD11b +, CD19 +, CD25 +, CD31 +, CD34 + , CD45 +, CD56 +, CD62 +, CD133 + (direct immunofluorescence method with antibodies produced by "Dako," BD ") and CD154 +, HLA-DR +, TdT + (terminal deoxynucleotidyl transferase), VEGF + (vascular-endothelial growth factor) - indirect method with antibodies Dako "," BD "," Sorbent ". The measurements were carried out on a flow cytofluorimeter FACScan , Becton Dickinson: The number of cells having one marker or another was estimated as a percentage of the total number of mononuclear cells.

Statistical data processing was performed using Student's t-test to assess the probability of differences (p) between the mean values of M ± m.

The obtained results were analyzed by us to identify specific features of the dynamics of each of the indicators during the first month after transplantation. The monthly period was divided into 2 intervals: up to 10 and after 10 days. For these time intervals, the mean concentrations of the analyzed subpopulations were calculated taking into account fluctuations of their individual values. Based on a review of the average concentration values of these subpopulations, we did not find a single kinetics type for all patients, with the exception of SGK (CD34 +). Only on the basis of this indicator the group of patients was statistically divided.

A preliminary assessment in the control group showed that the concentration of SGK in their blood is 0.15 + 0.05% in the mononuclear fraction. The results of measurements of SGC in the study group of patients were analyzed within two 99% confidence intervals: 1-10 days, 11-30 days. Individual changes in the levels of SGC were determined by selecting approximating curves in the Microsoft Excel program. This classification of the shape of individual kinetic curves made it possible to group them into four statistically different variants of changes occurring within the indicated (1 and 2) time intervals (see drawing).

Option I (see drawing, white columns) was characterized by an abnormally high level of SGC in both time intervals (first and second) (p = 0.001).

In option II (see the drawing, gray columns), which is more common among liver transplant recipients, the initial increase in the indicator is noted, but it is relatively small, and in the second time period there are no significant differences from the control.

In option III (see the drawing, dash), a larger, even compared to option I, initial increase in SGC was replaced by a deep drop to a level 4 times lower than that for control in the corresponding time interval of 10-30 days (p <0.001 )

In option IV (see the drawing, black columns) the first rise of the SGC was absent, and in the second period of time there was a deep, 2.6 times less than the level of control, drop (p <0.001).

From the data shown in the drawing, it followed that by 10-30 days after the operation (second time period) the levels of SGK naturally decreased from option I to option II and further to options III-IV, while in option I the level of SGK was higher at 3, 5 times of the control indicated by an arrow on the ordinate axis (see drawing), and in variants III and IV - lower by 3.5 (p <0.001) and 2.3 times (p <0.001), respectively.

Among the distinguished variants of the dynamics of SGC, the first two (I and II) were characterized by a favorable course of the early postoperative period (average CD34 + values were higher than 0.1% in the first time interval or equal to 0.1% in the second interval), and in variants III and IV we noted different complications (the average CD34 + values in this case in variant III were much higher than 0.1% in the first time interval, and in the second interval - much lower than 0.1%, in variant IV these values were lower than 0.1% in both time intervals). Based on this, a conclusion was drawn about the positive pathogenetic effect of circulating SGC on the state of the liver transplant in the early period after transplantation, as well as on the dependence of the result of the operation on the ability of the hematopoietic system to produce the necessary amount of SGC and maintain their concentration in the blood circulation when using immunosuppressive therapy at the physiological level norms (0.15 + 0.05%). This formed the basis of the present invention.

The essence of the method is as follows.

The recipient 2 times a week for the first month after cadaveric liver transplantation measures the concentration of circulating SGK (CD34 +) in the blood, and finds its average values for the first 10 and the next 20 days. If the value of these values is 0.1% or more in both time intervals, the recipient is assigned to the group of patients with a favorable transplant outcome. If these values are below 0.1% in the second or both time intervals, the risk of complications in the postoperative period is predicted for the recipient.

The essence of the method is illustrated by examples.

EXAMPLE 1

Patient A., born in 1962, came to the clinic of the Russian Scientific Center for Radiology and Surgical Technologies (RRCRC) in April 2009 with cirrhosis of the liver of unspecified etiology and signs of liver cell failure.

From the anamnesis it is known that in 2003 she was treated in an infectious diseases hospital in St. Petersburg with a diagnosis of Acute viral hepatitis B. Subsequently, markers of viral hepatitis B were not determined. It was observed at the place of residence of the infectious disease specialist. Since 2008, noted constant jaundice, skin itching, weight loss.

According to the survey, the selection committee decided to include the patient on the waiting list for orthotopic liver transplantation. The diagnosis was clarified: Autoimmune hepatitis type I. Encephalopathy The patient was on the waiting list from April to September 2009 (5.5 months).

09/18/09, in the presence of a cadaveric organ suitable for the patient, orthotopic liver transplantation was performed at the Russian Center for Science and Technology. From the first day after the operation, the traditional three-component immunosuppressive therapy was started: prograf, azathioprine, prednisone.

In the patient’s peripheral blood 2 times a week during the first month after liver transplantation, the concentration of circulating CD34 + hematopoietic stem cells was determined, which amounted to the following values: September 22, 2009 - 0.028%, September 28 - 0.093%, i.e. the average value for the first 10 days was 0.061%. Further, until September 19, 2009 (the next 20 days), 6 tests of peripheral blood were performed on CD34 +: 01.2010 - 0.076%, 05.10 - 0.0124%, 08.10 - 0.0058%, 12.10 - 0.084%, 15.10 - 0.064%, 19.10 - 0.23%, and the average value was 0.079%. Since the CD34 + values were less than 0.1% in both time modes, post-transplant complications could be expected in the patient.

Observation of it showed: the transplant function did not recover immediately. On the 6th day after the operation, a puncture biopsy of the liver was performed, acute rejection was detected, estimated by the Banff classification, Ib, pulse therapy with solu-medrol 500 mg was carried out, immunosuppression was strengthened, azathioprine was canceled, mycophenolic acid was prescribed 720 mg per day. The further course was accompanied by a slow restoration of liver function (transplant). Bilirubin, as the main indicator of liver function, was 630 μmol / L on day 6, 290 μmol / L on day 15, and 40 μmol / L at discharge, with a normal value of 20 μmol / L. The hospitalization period was 58 days, with an average of 28 days.

Currently, the patient is undergoing outpatient monitoring by the Russian Center for Science and Technology, the liver transplant is functioning, its function is supported by constant use of the prograph 3 mg / day, myfortic 720 mg / day.

EXAMPLE 2

Patient K., born in 1987, was observed at the RSCRHT clinic since January 2007. Diagnosed with chronic viral hepatitis C with an autoimmune component. Cirrhosis of the liver. Portal hypertension.

In August 2008, the selection committee decided to add the patient to the waiting list. The length of stay on the waiting list was 7 months, 02.02.2009, the patient underwent transplantation of cadaveric liver.

In the recipient’s peripheral blood, as in Example 1, at least 2 times a week during the first month after liver transplantation, the concentration of circulating CD34 + hematopoietic stem cells was determined, which was: 02.09.2009 - 0.68%, 08.09 - 0.38%, the average value was 0.53% . In the next 20 days, these values were as follows: September 12 - 0.34%, September 15 - 0.53%, September 18 - 0.21%, September 22 - 0.68%, September 25 - 0.41%, October 1 - 0.37%, and the average value was 0.42%. Due to the fact that both average CD34 + concentrations were higher than 0.1%, no post-transplant complications were predicted.

Observations of the patient showed: the course of the early postoperative period was smooth. Immunosuppressive therapy was traditional three-component: prograph, azathioprine, prednisone. The levels of the main indicators of transplant functioning had a regular dynamics (gradual decrease in bilirubin content, to discharge 24 μmol / L, ALT, ACT normal), no clinical signs of rejection were detected. The hospital stay was 30 days.

Currently, the patient is observed in the RSCRHT on an outpatient basis, liver function indices do not differ from the norm. Constantly the patient takes prograf 5 mg / day, Mayfortic 360 mg / day for the prevention of rejection.

EXAMPLE 3

Patient G., born in 1965, was observed at the Russian Center for Science and Technology for 6 months in a waiting list for liver transplantation with a diagnosis of Cirrhosis of the liver of unspecified etiology. Portal hypertension. Ascites. Encephalopathy

10/17/09, she underwent orthotopic liver transplantation. Three-component immunosuppressive therapy: cyclosporine, prednisone, mayfortic. The values of peripheral blood parameters: in the first time period, they amounted to 10/19/2010 - 0.078%, 22.10. - 0.085%, 26.10 - 0.24%, while the average value was 0.135%. In the second time period, these values were: 29.10 - 0.062%, 02.11 - 0.118%, 12.11 - 0.41%, 16.11 - 0.28%, 18.11 - 0.22% and the average value - 0.218%. Both values were higher than 0.1%, and therefore postoperative complications were not expected.

The postoperative period was smooth, therapy was planned, the patient was discharged 30 days after surgery. By statement, the level of bilirubin is 32 μmol / L, ALT, ACT within the acceptable range.

To date, the patient is observed on an outpatient basis at the Russian Center for Science and Technology, after surgery for 6 months. He is constantly taking immunosuppressive drugs: sandimmune-neoral, mayfortic. The liver transplant function is satisfactory, biochemical parameters (bilirubin, ALT, AST, alkaline phosphatase, GGTP within normal values).

To date, the proposed method for predicting post-transplant complications has been clinically tested in 45 recipients - all of them have been able to timely determine the course of the postoperative period and prescribe one or another immunosuppressive therapy. In 11 recipients, the prognosis was unfavorable, but the early appointment of adequate therapy avoided serious complications.

The proposed method compared with the known has significant advantages in that it:

1. provides a higher accuracy in predicting post-transplant complications, since it uses the parent cells (SGC CD34 +), and not their derivatives (T-cells), the level of which does not allow to unambiguously judge the risk of complications;

2. Improves the reliability of the forecast by using the average values of the concentration of SGK in 2 time intervals of the critical period and taking into account their ratio.

The method was developed in the group of transplantation and vascular surgery in conjunction with the laboratory of immunological methods of the Russian Science Center for Chemotherapy, and underwent clinical testing in 45 recipients after transplantation of a cadaveric liver with a positive result.

Claims (1)

A method for predicting the risk of complications in recipients after cadaveric liver transplantation, including a study of peripheral blood in them, characterized in that the peripheral blood of the recipient at least 2 times a week during the first month after liver transplantation determines the concentration of circulating CD34 + hematopoietic stem cells, find it the average values for the first 10 and subsequent 20 days and when their value is below 0.1% in the second or both time intervals at the recipient, the risk of post-transplant donations is predicted neny.

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