RU2430370C2 - Method of diagnosing kidney allotransplant rejection - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method of diagnosing rejection of kidney allotransplant includes determination of phase height PH of peripheral blood live lymphocytes by method of phase-interference microscopy, determination of quantity of lymphocytes with phase height PH≤1.5 mcm, 1.5 mcm<PH≤2 mcm, 2 mcm<PH≤2.5 mcm, PH>2.5 mcm, selection of lymphocyte activity coefficients for each limit, said phase heights of lymphocytes equal k₃=3, k₂=2, k₁=1, k₀=0 respectively. Obtained data are used to determine functional activity of lymphocytes in sample by formula: FA=(k₃n₃+k₂n₂+k₁n₁+k₀n₀)/n, where n is number of lymphocytes in sample, n₃ is number of lymphocytes with PH≤1.5 mcm, n₂ is number of lymphocytes with 1.5 mcm<PH≤2 mcm, n₁ is number of lymphocytes with 2 mcm<PH≤2.5 mcm, n₀ is number of lymphocytes with PH>2.5 mcm, k₃, k₂, k₁, k₀ are coefficients of lymphocyte activity, and if value of lymphocyte functional activity is within FA=1.8-2.0, rejection of kidney transplant is diagnosed.

EFFECT: application of claimed method makes it possible to set diagnosis in due time, which considerably increases efficiency of anti-crisis therapy in post-transplantation period.

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Description

The invention relates to medicine, in particular to laboratory research in transplantology.

Allotransplantation of the kidney is the method of choice in the treatment of patients with the "terminal stage of chronic renal failure (CRF), due to which the patient's life is supported, his social and medical rehabilitation is achieved.

An important factor in the deterioration of renal allograft function is acute rejection in the early postoperative period; therefore, its timely diagnosis and adequate treatment are important predictors of a favorable clinical outcome.

Rejection is a specific immune response of an organism to transplantation donor antigens, causing inflammatory damage to the graft. The main morphological components of this process are tubulo-interstitial nephritis in the cell type of rejection and arteritis in the vascular type of rejection. Moreover, almost all mechanisms of the cellular and humoral response are involved in the rejection reaction. Despite the use of modern immunosuppressive drugs, acute rejection is a common cause of transplant loss.

Currently, the diagnosis of acute rejection reaction is based on a comprehensive assessment of clinical symptoms and signs, changes in laboratory parameters and special instrumental methods of research. Monitoring the development of the acute rejection reaction is carried out on the basis of clinical, instrumental and laboratory immunological data.

A known method for the diagnosis of transplant rejection (Transplantology. Edited by Academician V. I. Shumakov. M .: MIA, 2006. - S.500-506) is a percutaneous biopsy of a renal allograft, allowing morphological analysis of pathological processes in ATP with using histological, immunological and electron microscopic studies. A percutaneous renal transplant biopsy is performed by the surgeon under aseptic conditions using a special biopsy needle under the control of an ultrasound transducer, followed by fixation, staining and preparation of a micropreparation.

The disadvantages of this method are invasiveness with the possibility of complications, the involvement of several specialists, the use of expensive reagents for the preparation of micropreparations.

A known method for the diagnosis of transplant rejection ("Transplantology". Edited by Academician V. I. Shumakov. M .: "MIA", 2006. - S.350), based on the determination of a set of laboratory parameters of peripheral blood and urine of the recipient. Laboratory signs of rejection are as follows: in the blood - a sharp increase in creatinine (by 25% from the original) and urea, leukocytosis, lymphocytosis, a sharp increase in ESR, hyperkalemia, hypercoagulation; in urine, an increase in proteinuria and a decrease in the specific gravity of urine. Research is carried out according to standard generally accepted methods, daily.

In addition to the acute rejection reaction, the above symptoms can occur in various combinations for infectious complications, surgical complications, toxicity of calcineurin inhibitors and other drugs.

The disadvantages of this method: the lack of strict specificity for the acute rejection reaction, it is impossible to diagnose rejection reactions in the subclinical stage, rejection reactions without clinical transplant dysfunction.

The closest is a method for diagnosing renal transplant rejection, including determining the phase height of the pH of living peripheral blood lymphocytes by phase interference microscopy, the number of lymphocytes in the sample (RF patent for the invention No. 2348932, IPC G01N 33/48, publ. 2009). The method is based on the assessment of morphological and functional parameters of live unfixed and unpainted peripheral blood lymphocytes of kidney transplant recipients. To achieve a technical result, a comprehensive rapid assessment of linear (geometric - diameter, perimeter, area) and volume-structural (refractive - height and volume) indicators of each lymphocyte is carried out using a laser interference microscope, which allows to study living cells in transmitted light. Diagnosis is based on the evaluation of phase portraits of lymphocytes. The value of the indicator of heterogeneity of the nuclear structures of living lymphocytes is determined and when the value of this indicator is lower than 0.2 in not less than 20% of the cells, a conclusion is made about the beginning of renal transplant rejection.

The disadvantages of the method include its high complexity and lack of accuracy, since the use of both geometric and phase parameters to assess the state of lymphocytes does not allow us to take into account the constancy of the amount of nuclear material of cells, because activation of small blood lymphocytes may not be estimated correctly due to the use of diameter in the calculations. The introduction of an indicator of lymphocyte activity into clinical practice is difficult, since it requires two stages of mathematical analysis of data.

The objective of the invention is to remedy these disadvantages due to the fact that only the phase parameters of the nuclear structures of peripheral blood lymphocytes are taken into account, which most accurately characterize the processes of lymphocyte activation in the reaction of rejection of a renal allograft.

For this, in a method for diagnosing renal transplant rejection, which includes determining the phase height of pH of living peripheral blood lymphocytes by phase interference microscopy, the number of lymphocytes in the sample is proposed to determine the number of lymphocytes with a phase height of pH ≤ 1.5 μm, 1.5 μm ≤ pH ≤ 2 μm, 2 μm ≤ pH ≤ 2.5 μm, pH ≥ 2.5. Also new is the fact that lymphocyte activity coefficients are chosen for each limit of the indicated phase heights of lymphocytes equal to k ₃ = 3, k ₂ = 2, k ₁ = 1, k ₀ = 0, respectively; according to the data obtained, the functional activity of lymphocytes in the FA sample is determined from the formula:

FA = (k ₃ n ₃ + k ₂ n ₂ + k ₁ n ₁ + k ₀ n ₀ ) / n,

where n is the number of lymphocytes in the sample,

n ₃ - the number of lymphocytes with pH ≤ 1.5 μm,

n ₂ is the number of lymphocytes with 1.5 μm ≤ pH ≤ 2 μm,

n ₁ - the number of lymphocytes with 2 μm ≤ pH ≤ 2.5 μm,

n ₀ - the number of lymphocytes with pH ≥ 2.5,

k ₃ , k ₂ , k ₁ , k ₀ - activity coefficients of lymphocytes,

and with the value of the functional activity of lymphocytes within FA = 1.8-2.0, renal transplant rejection is diagnosed.

The selected diagnostic indicator FA, which determines renal transplant rejection, characterizes the refractive index for all components of living blood lymphocytes and depends on the nature of the intracellular substance, its concentration and conformational state.

The core of the lymphocyte is the nucleus, and nuclear proteins make a decisive contribution to the optical parameters of the cell, such as phase height. The amount of chromatin in a somatic cell with a standard (diploid) set of chromosomes is constant and does not depend on most factors, the content of histone and nonhistone proteins remains practically unchanged, which suggests a constant concentration of nuclear material. The refractive index for all components of cell structures is on average 1.088 and can vary within small limits when the conformational state of the molecules changes - the smaller the size of the complex of proteins, DNA and RNA, the lower the refractive index. Thus, the phase height of lymphocytes depends primarily on the degree of chromatin packing in the nucleus and the activity of protein synthesis processes. That is, this value allows you to indirectly assess the process of renal allograft rejection. For the phase height of lymphocytes, there is an inverse dependence on the degree of activation — the lower the pH value, the higher the cell activity. In practice, the limits of phase heights were established for different levels of activation. The most active are cells with pH less than 1.5 μm, which corresponds to the maximum degree of chromatin decondensation and activation of lymphocytes, this group was assigned an activity coefficient equal to 3. Similarly, activity coefficients were determined for the remaining phase height limits.

According to the results of the study, it was found that the number of lymphocytes, taking into account their activity coefficient in relation to the total number of lymphocytes in the sample, determines the amount of activity of the entire population.

The method is as follows.

Lymphocytes were isolated from peripheral blood (PC) stabilized with heparin at a concentration of 25 PIECES per 1 ml by the standard Woushp density gradient method for Ficoll-Paque lymphocytes (p = 1.077 g / cm ³ ) manufactured by Amersham Biosciences (Sweden). Blood was diluted with saline 1: 1, then PC was layered in centrifuge tubes on a gradient layer. Centrifuged at 1500 rpm for 45 minutes. A ring of mononuclear cells at the boundary of the gradient was collected and washed with saline twice for 5-7 minutes at 1000 rpm.

Cells were placed in a mirror-coated Goryaev chamber and examined using a CPM Cytoscan. The sample for one study was 50-100 cells. The obtained phase portraits of lymphocytes were processed using the mathematical package MatLab.

As a criterion for diagnosing the rejection reaction, we determined the functional activity of a sample of FA lymphocytes, as a value inversely proportional to the phase height of the pH of each cell in the sample, which characterizes the conformational state of nucleolar proteins and the degree of chromatin decondensation: FA = (k ₃ n ₃ + k ₂ n ₂ + k ₁ n ₁ + k ₀ n ₀ ) / n, where FA is the functional activity of lymphocytes; n is the number of cells in the sample, n ₃ is the number of lymphocytes with pH ≤ 1.5 μm, n ₂ is the number of cells with 1.5 μm ≤ pH ≤ 2 μm, n ₁ is the number of cells with 2 μm ≤ pH ≤ 2.5 μm, n ₀ is the number of cells with pH ≥ 2.5, k ₃ , k ₂ , k ₁ , k ₀ is the coefficient of activity of lymphocytes.

Studies of the nuclear structures of lymphocytes were carried out by computer phase morphometry in healthy individuals. This group included 30 people, more than 2,000 phase-interference portraits were studied with the determination of the functional activity of the nuclei of vital unpainted peripheral blood lymphocytes:

FA = (3 · 100 + 2 · 196 + 89 + 0 · 41) / 426 = 1.83 for T-lymphocytes;

FA = (3 · 109 + 2 · 19.8 + 90 + 0 · 42) / 439 = 1.85 for B-lymphocytes.

In practically healthy individuals, the FA value was 1.83 ± 0.1 for T- and 1.85 ± 0.1 for B-lymphocytes. The obtained high rates are associated with the predominance of lymphocytes with medium and low values of phase height, i.e. functionally active cells.

We examined 18 patients with end-stage chronic renal, receiving renal replacement therapy, who subsequently underwent kidney transplantation. This group is represented by 50% of women (n = 9) and 50% of men (n = 9), the average age was 42 ± 9.5 years.

The causes leading to the terminal stage of chronic renal failure were: chronic glomerulonephritis - 61.1% (n = 11), chronic pyelonephritis - 22.2% (n = 4), chronic tubulo-interstitial nephritis - 5.6% (n = 1), Alport syndrome - 5.6% (n = 1) and gout 5.6% (n = 1).

The method of renal replacement therapy in kidney transplant recipients in this group was peritoneal dialysis in 39% (n = 7), hemodialysis in 61% (n = 11). The average duration of dialysis was 29.7 ± 18.4 months.

For this group, the functional activity of the nuclei of lymphocytes was also determined:

FA = (3 · 15 + 2 · 210 + 1 · 102 + 0 · 36) / 363 = 1.56 for T-lymphocytes;

FA = (3 · 12 + 2 · 192 + 1 · 99 + 0 · 33) / 336 = 1.54 for B-lymphocytes.

In table 3.1.1. the values of the functional activity of the nuclei of T- and B-lymphocytes of patients with ESRD undergoing treatment with dialysis and healthy individuals (donors) are presented.

Table 3.1.1 FA T- and B-lymphocytes of patients with ESRD (M ± σ) Survey groups The value of FA T-lymphocytes The value of FA b-lymphocytes Donors N = 30 1.83 ± 0.1 1.85 ± 0.1 Patients with ESRD N = 18 1.56 ± 0.15 * 1.54 ± 0.2 * Note: significance of differences p <0.05; * - different from control

Both T- and B-lymphocytes are characterized by a significant decrease in the functional activity of cell nuclei. Based on the results obtained, it can be assumed that there are signs of functional insufficiency in the T- and B-link of immunity, which is obviously associated with the suppression of cellular activation. At the same time, the data obtained do not allow us to evaluate the proliferative potential of lymphocytes, but only characterize a change in the state of immunoreactivity in patients with end-stage CRF.

After kidney transplantation, nuclear lymphocyte structures were studied by computer phase morphometry in the early postoperative period.

Eleven patients had an uncomplicated course with primary graft function. The indicator of the functional activity of the nucleus was 1.4 ± 0.27 for both T- and B-lymphocytes.

In 6 patients with delayed nitrogen and water-releasing graft function without immunological conflict (absence of infectious complications), FA was 1.43 ± 0.3 for all cell types.

Thus, there were no significant differences in the functional activity of nuclei in patients with primary and delayed graft function.

In 3 patients with ORO, before the manifestation of clinical manifestations, the value of the functional activity of the nucleus increased to 1.9 ± 0.1 for T and / or B cells, depending on the type of reaction.

After anti-crisis therapy with “pulses” of methylprednisilone to a total dose of 1.5-3.0 g and antithymocytic globulin - ATG (Frezenius), FA was comparable to that in the group with uncomplicated course.

Example 1

Patient B., born in 1973, with a diagnosis of chronic glomerulonephritis, chronic renal failure, terminal stage, arterial hypertension, myocardial dystrophy has been observed in MONIKI since 2005.

Considers himself ill for more than 10 years, when the examination revealed high blood pressure values, changes in urinalysis. Deterioration since 2003, when there was an increase in creatinine, urea, proteinuria. Conducted conservative therapy with prednisone. Since 2005, chronic renal failure, terminal stage. Beginning of treatment with peritoneal dialysis in March 2005 using low- and medium-osmolar solutions. In 2006, he suffered 2 episodes of dialysis peritonitis.

October 14, 2007, an allotransplantation of a cadaveric kidney was performed with a match for two antigens from a donor of 40 years old who died from a head injury. The time of primary thermal ischemia is 25 minutes, the conservation period was 30 hours. Immunosuppressive therapy included prograph, prednisone, metipred, sellcept, zenapax. The graft function is delayed, treatment with peritoneal dialysis is continued. Diuresis on days 1, 3, 5, 7, 9, 11 was 400, 270, 250, 230, 330, 350 ml, respectively. Plasma creatinine levels on days 1, 3, 5, 7, 9, 11 1.24; 1.3; 1.0; 1.03; 0.89; 0.8 mmol / L, respectively.

According to a set of laboratory parameters of peripheral blood and urine of the recipient, a computer was prescribed. On the 5th and 11th days after allotransplantation, phase morphometry of peripheral blood T and B lymphocytes was performed. The sample for populations of each type of lymphocyte ranged from 70 to 100 cells. The phase height of each lymphocyte was determined and the corresponding activity coefficient from 0 to 3 was assigned to the cell by the value of this indicator. The total number of lymphocytes and the number of cells in each group were calculated and the functional activity of T-lymphocytes was calculated:

for 5 days FA = (k ₃ · 8 + k ₂ · 50 + k ₁ · 20 + k ₀ · 2) / 80 = (3 · 8 + 2 · 50 + 1 · 20 + 0 · 2) / 80 = 1 , 86;

for 11 days, FA = (3 · 6 + 2 · 20 + 1 · 36 + 0 · 38) / 100 = 0.94;

and B lymphocytes:

for 5 days FA = (3 · 10 + 2 · 44 + 1 · 18 + 0 · 4) / 76 = 1.79;

for 11 days, FA = (3 · 16 + 2 · 50 + 1 · 14 + 0 · 12) / 92 = 1.76.

FA of B-lymphocytes, despite standard post-transplant drug therapy, remained high, which allowed us to suggest the presence of a rejection reaction of a vascular type.

10.24.07, (on the 10th day) a biopsy of the renal transplant was performed.

Histological examination of a kidney biopsy.

Light microscopy was carried out with stains - hematoxylin-eosin, SIC reaction, Mason trichrome. The preparation has 13 glomeruli, there is a slight delay of lymphocytes in the lumen of the capillary loops, the rest of the glomerulus is without features. Interstitial edema. Diffuse rare infiltration with mononuclear cells without a pronounced tendency to invasion into the tubules (tubulitis up to 4-5 lymphocytes per tubule section). The tubules look enlarged by lowering the height of the tubular epithelium. In 1 artery of medium caliber, there are individual lymphocytes in the subendothelial region. In small arteries and arterioles pronounced vacuolization of myocytes.

Immunofluorescence results: IgG - no, IgA - no, IgM - no, C3 - no, fibrin - no, C4D - is determined on the vascular endothelium.

Conclusion: acute vascular rejection 2a (according to the Banff classification), residual effects of acute tubular necrosis.

On the 8th day, anti-crisis therapy of ATH was started in a total dose of 500 mg.

After anti-crisis therapy, the functional activity of lymphocytes decreased:

T-lymphocytes on day 14 FA = (3 · 0 + 2 · 16 + 1 · 38 + 0 · 26) / 80 = 0.87;

B-lymphocytes on day 14 FA = (3 · 0 + 2 · 16 + 1 · 34 + 0 · 30) / 80 = 0.83.

The graft function stabilized, the growth of diuresis was noted: on the 14th, 16th, 18th day - 600, 1100 and 2100 ml. The plasma creatinine level on days 14, 16, 18 was 0.65; 0.49; 0.28 mmol / l and reached normal by 25 days.

November 15, 2007 the operation was performed: removal of the peritoneal catheter. 11/22/07 removed the stent from the ureter of the nephrotransplant.

November 29, 07 the patient was discharged with satisfactory renal transplant function.

Example 2

Patient M., 49 years old, was admitted to MONIKI with a diagnosis of chronic glomerulonephritis, chronic renal failure, arterial hypertension, myocardial dystrophy, and peritoneal dialysis. Sick for 25 years on treatment with peritoneal dialysis since February 2004

Cadaveric kidney transplantation was performed on July 13, 2007. Donor male, 22 years old, cause of death - traumatic brain injury. The time of primary thermal ischemia is 20 minutes. The conservation period is 30 hours.

Immunosuppressive therapy included SuA, prednisone, metipred, sellcept, zenapax. The graft function is delayed, treatment with peritoneal dialysis is continued. Diuresis on days 1, 3, 5, 7, 9, 11 was 400, 500, 350, 300, 400, 400 ml, respectively. Plasma creatinine level on days 1, 3, 5, 7, 9, 11; 0.97; 0.92; 0.88; 0.95; 0.88 mmol / L, respectively.

On the 7th day after allotransplantation, phase morphometry of peripheral blood T and B lymphocytes was performed. The sample for populations of each type of lymphocyte ranged from 65 to 100 cells. The phase height of each lymphocyte was determined and the corresponding activity coefficient from 0 to 3 was assigned to the cell by the value of this indicator. The total number of lymphocytes and the number of cells in each group were calculated and the functional activity was calculated:

T-lymphocytes on the 7th day FA = (3 · 14 + 2 · 27 + 1 · 16 + 0 · 8) / 65 = 1.72;

B-lymphocytes on day 7 FA = (3 · 16 + 2 · 40 + 1 · 8 + 0 · 8) / 72 = 1.89.

07/20/07, a kidney transplant biopsy was performed.

Histological examination of a kidney biopsy specimen: two renal biopsy specimens contain 6-8 glomeruli, no sclerosis. Part of proximal glomeruli with slightly widened lumen, their epithelium in a state of protein dystrophy. Interstitium was noted with diffuse focal lymphoplasm cell infiltration (i2) with a tendency to penetrate into the wall and lumen of the tubules, there are foci of partial destruction of the tubule wall (t3). Arterioles are not changed. In one partially cut artery, marginal standing of mononuclear cells is noted. Conclusion: Acute rejection 1B according to Banff classification.

On the 10th day, anti-crisis therapy with methylprednisolone in a total dose of 2500 mg was started.

A study of the functional activity of lymphocytes on days 11 and 14 showed the effectiveness of treatment.

T-lymphocytes:

on day 11 FA = (3 · 0 + 2 · 8 + 1 · 28 + 0 · 44) / 80 = 0.55;

on day 14 FA = (3 · 0 + 2 · 10 + 1 · 30 + 0 · 32) / 72 = 0.69;

B lymphocytes:

on day 11 FA = (3 · 12 + 2 · 52 + 1 · 18 + 0 · 18) / 100 = 1.58;

on day 14 FA = (3 · 0 + 2 · 16 + 1 · 34 + 0 · 30) / 80 = 0.83.

The graft function stabilized, an increase in diuresis was noted: on the 14th, 16th, 18th day - 1000, 1700 and 2000 ml. The plasma creatinine level on days 14, 16, 18 was 0.68; 0.29; 0.21 mmol / L and reached normal by 20 days.

Thus, the effectiveness of anti-crisis therapy can be quantified by the dynamics of the values of the functional activity of lymphocytes. A decrease in FA to 1.3-1.4, which corresponds to values in the uncomplicated course of the post-transplant period, indicates the effectiveness of the treatment.

Claims (1)

A method for the diagnosis of renal transplant rejection, including determining the phase height of the pH of living peripheral blood lymphocytes by phase interference microscopy, the total number of lymphocytes in the sample, characterized in that the number of lymphocytes with a phase height of pH ≤ 1.5 μm, 1.5 μm ≤ pH is determined ≤ 2 μm, 2 μm ≤ pH ≤ 2.5 μm, pH ≥ 2.5 μm, and the coefficients of activity of lymphocytes for each limit of the indicated phase heights of lymphocytes are chosen equal to k ₃ = 3, k ₂ = 2, k ₁ = 1, k ₀ = 0, respectively, is determined from the data f nktsionalnuyu FA activity of lymphocytes in the sample by the formula:
FA = (k ₃ n ₃ + k ₂ n ₂ + k ₁ n ₁ + k ₀ n ₀ ) / n, where
n is the number of lymphocytes in the sample,
n ₃ - the number of lymphocytes with pH ≤ 1.5 μm,
n ₂ is the number of lymphocytes with 1.5 μm ≤ pH ≤ 2 μm,
n ₁ - the number of lymphocytes with 2 μm ≤ pH ≤ 2.5 μm,
n ₀ - the number of lymphocytes with pH ≥ 2.5 μm,
k ₃ , k ₂ , k ₁ , k ₀ are the coefficients of activity of the lymphocytes, and with a value of the functional activity of the lymphocytes within FA = 1.8-2.0, renal transplant rejection is diagnosed.