RU2581925C2 - Method for assessing allogenic immune response in short-term mixed mononuclear culture of unrelated donors - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and can be used for evaluation of Allogenic immune response in short-term mixed mononuclear culture of unrelated donors. For this purpose, with help of flow cytometry quick analysis is carried out with phenotype CD mononuclear b-lymphocyte subpopulation CD3⁺HLA-DR⁺, CD3^-HLA-DR⁺, CD45⁺HLA-DR⁺ each donor, separated and gated by different fluorescent dye in mixed culture. Followed by calculating gain coefficients for each subpopulation. At positive values of CP_CD3-HLA-DR+, and CP_CD45+HLA-DR+ presence of immune according alloHLA factor CP_CD3+HLA-DR+ indicates features of interacting genotypes HLA-DRB1*.

EFFECT: invention enables assessing immune reaction and detecting allogeneic cell sensitisation at allogenic antigens main complex tissue compatibility.

1 cl, 1 tbl, 2 dwg, 3 ex

Description

Application area

The invention relates to medicine, namely to laboratory diagnostics. It is based on modern cultural and immunological methods for determining the expression of allogeneic compatibility molecules and intercellular contacts on mononuclear cells of unrelated donors interacting in their mixed culture.

The mixed lymphocyte culture method is a classic example of studying the effectiveness of immune recognition by allogeneic antigens of the main tissue compatibility complex (for humans - HLA) [1, 2]. It has been proven that the main molecules that stimulate immune responses when mixing allogeneic lymphocytes are class II HLA (HLA-DR, HLA-DQ, HLA-DP). That is why this method has found wide application in typing the HLA-D locus [3]. This study must be carried out for the selection of donor bone marrow during its transplantation [4, 5]. The significance of a mixed culture of lymphocytes in the study of the immune causes of reproductive loss has been shown [6]. It is known that the formation and gestation of pregnancy is an HLA-restricted immune process [7]. By the letter of the Ministry of Health of the Russian Federation “Prevention, diagnosis, treatment of miscarriage” dated 04/11/2003 No. 2510/3796-03-32, in case of miscarriage it is recommended to use methods for determining factors that block the alloimmune response to the fetal HLA. This method can only be a mixed culture of lymphocytes. In the classical version, a mixed culture of lymphocytes is performed strictly under sterile conditions, with an incubation time of 120 hours and with the use of radioisotope markers. The method reflects the ability of lymphocytes to transform into blast cells under the influence of alloHLA [8]. These conditions are not acceptable for a modern diagnostic laboratory, if it concerns the diagnosis of immune causes of reproductive loss. In this case, it is necessary to determine the nature of the immune response to alloHLA for various subpopulations of mononuclear cells as soon as possible. And in the development of this approach as a method for determining the blocking rejection of factors in women’s blood, knowledge of their target cells is especially necessary. Thus, the relevance of developing modern methods for assessing alloimmune interactions by HLA between unrelated donors using flow cytofluorimetry is an urgent task. This kind of research has not been done before.

Analogs of the invention

Currently, a large number of evaluation methods have been proposed using flow cytofluorimetry of antigen-induced activation of lymphocytes. Korolkova O.Yu., Senyukova VV, Kozhevnikova B.C. showed that stimulation of mononuclear cells with anti-CD3 antibodies leads to increased expression of CD25 and HLA-DR molecules on them in the range of 1-7 days [9].

Another analogue is a method for assessing the proliferative activity of lymphocytes using cytofluorimetry. The method is based on the identification of a fraction of mononuclear cells expressing the c-terminal fragment of the b23 / nucleofozmin protein [10].

A similar method is to determine the proliferative activity of lymphocytes by expression of another marker - nucleolar protein a3 [11].

The main disadvantages of the above methods are the complexity, complexity and cost of their adaptation to evaluate alloimmune interactions by HLA.

A prototype of the proposed invention is a method for assessing the interaction of allogeneic lymphocytes by expression of a CD2 activation marker on their surface [12]. This work indicates the possibility of short-term incubation of a mixed culture of lymphocytes and an assessment of their activation by changing the expression of intercellular contact molecules.

The main disadvantage of this method is the lack of new technologies for evaluating the expression of activation molecules. At the same time, according to the technical nature and the achieved result, this method of assessing the interaction of lymphocytes in vitro is the closest to the claimed method.

SUMMARY OF THE INVENTION

The purpose of the invention is to improve the effectiveness of the diagnosis of allogeneic immune recognition for two unrelated donors by comparing the subpopulation composition of the mononuclear cells of each of the donors in their mixed culture with that in the individual cultures of each of the donors. It is known that HLA-DR expression is a dynamic process that is associated with the activation of immunocompetent cells, including in alloimmune interactions [13]. It was proved that the level of HLA-DR expression on the surface of immunocompetent cells is not associated with its synthesis and its density can change over 24 hours [14]. These two messages formed the basis of a new method for evaluating alloimmune interactions in a mixed mononuclear culture, where the activation of various subpopulations of lymphocytes was assessed by the level of change in HLA-DR expression detected by flow cytometry.

The main stages of the presented method for assessing allogeneic interactions of mononuclear cells of unrelated donors are:

1) the allocation of mononuclear cells of two unrelated donors using ficoll-verographin with a density gradient of 1.077 kg / m ³ [8];

2) washing the mononuclear cells twice with a single sodium phosphate buffer (Phosphate buffered saline, PBS) for 7 minutes at a temperature of 10 ° C and a centrifugation speed of 1500g;

3) primary staining of all mononuclear cells with a conjugate of monoclonal antibodies to CD45 (leukocyte marker) with peridinin-chlorophyll (PC-5) for the first donor and with alofikocyanin (APC) for the second donor;

4) incubation of the mixed culture of mononuclear cells for 24 hours at a temperature of 37 ° C and 5% CO ₂ in complete medium (RPMI-1640 with 15% fetal calf serum) in a concentration for each donor of 200,000 cells per 1 μl (in full 400 μl), simultaneous incubation under the same conditions and concentrations of individual cultures of each donor;

5) additional staining of all mononuclear cells (mixed culture and individual cultures) using conjugates of fluorescent dyes (fluorescein isothiocyanate - FITC and phycoerythrin - PE) with monoclonal antibodies to CD3 and HLA-DR, respectively;

6) taking into account the expression of HLA-DR on various subpopulations of mononuclear cells using flow cytofluorimetry;

7) a simultaneous assessment of the degree of increase in HLA-DR expression of each donor on a subpopulation of mixed culture mononuclear cells with respect to those in the corresponding control cultures.

The method is as follows:

1. Venous blood sampling for each unrelated donor is carried out in a boxed treatment room in vacutainers with ethylenediamine acetic acid (EDTA) in a volume of 3 ml, followed by mixing to stabilize the blood coagulation system. To clarify the number of leukocytes and lymphocytes from a test tube, 40 μl of blood is taken and a general blood test is performed on an automatic analyzer. With their values in the normocentric limits, further counting of mononuclear cells at the stages of the method is not required.

Further, all actions are carried out in a boxed room for cultural work, in a laminar cabinet, designed for work with microorganisms 3-4 pathogenicity group.

2. The venous blood of each donor is diluted 2 times with a single sodium phosphate buffer, adding 3 ml of 1 × PBS to 3 ml of blood.

3. In a plastic centrifuge tube with a total volume of 10 ml, a sterile solution of ficoll-verographin with a density gradient of 1.077 kg / m ³ in a volume of 3 ml is added and the previously diluted venous blood of each donor (paragraph 2) in a volume of 6 ml is layered on this solution.

4. Next, centrifugation of both tubes containing ficoll-verographin and diluted venous blood is carried out for 500 g, 30 minutes at a temperature of 10 ° C.

5. After centrifugation is completed, an interphase ring containing mononuclear cells is taken from each tube and transferred to a new centrifuge plastic tube. To wash ficoll-verographin solution from impurities, add 7 ml of 1 × PBS to tubes with mononuclear cells. Then, by careful pipetting, the mononuclear cells are mixed with PBS.

6. Centrifuge both tubes containing donor mononuclear cells and 1 × PBS at 500 g for 7 minutes at 10 ° C.

7. After centrifugation, the supernatant is removed, and procedures 5 and 6 are repeated.

8. After centrifugation, the supernatant is removed. The residual volume of fluid in the tubes should not exceed 50 μl.

9. In the first test tube (donor No. 1) add 5 μl of monoclonal antibodies to CD45 conjugated with fluorescent dye Peridinin-chlorophyll (PC-5), in the second test tube (donor No. 2) - 5 μl of monoclonal antibodies to CD45 conjugated with alofikocyanin . It is recommended to use monoclonal antibodies from Beckman coulter (USA), Biolegend (USA).

10. Tubes with donor mononuclear cells and corresponding conjugates of monoclonal antibodies with fluorescent dyes are incubated for 15 minutes at room temperature in complete darkness (in a closed cabinet).

11. Wash off unbound monoclonal antibodies and centrifuge as described in paragraphs 5 and 6 once.

12. After washing, 1000 μl of complete medium is added to each tube and the mononuclear cells are mixed by careful pipetting. The basis of the complete incubation medium is RPMI-1640 culture medium containing 15% fetal calf serum (ETS), 2 mmol of L-glutamine, 10 mmol of Hepes buffer, 5 × 10 ^-5 mol of 2-mercaptoethanol and gentamicin sulfate at a concentration of 50 μg / ml.

13. In six plastic tubes for flow cytometry (Beckman coulter, USA or Becton dickinson, USA), 200 μl of mononuclear cells prepared in complete medium are added. All sets are duplicated, so for each stage two tubes are used. The first two tubes (No. 1 and No. 2) are designed for a mixed culture of lymphocytes, therefore, mononuclear cells of the first and second donors in a total volume of 200 μl are introduced into each tube (No. 1 and No. 2) and then mixed by careful pipetting for 15 seconds. In tubes No. 3 and No. 4, 200 μl of mononuclear cells of the first donor (monoculture of mononuclear cells of the first donor) are added. In tubes No. 5 and No. 6, 200 μl of mononuclear cells of the second donor (monoculture of mononuclear cells of the second donor) are added.

14. Next, all tubes are placed in a CO ₂ incubator for 24 hours. Incubation conditions: temperature 37 ° C, full humidity, 5% CO ₂ . The top of the tubes is covered with a lid from a 96-well plate.

15. After incubation, a single washing of the mononuclear cells of each tube is carried out. To do this, add 2 ml of 1 × PBS to each tube, mononuclear cells are mixed by pipetting and centrifuged for 5 minutes at 500 g and a temperature of 10 ° C. The supernatant is removed. The residual volume of liquid should not exceed 50 μl.

16. Staining of all mononuclear cells (mixed culture and individual monocultures) is carried out using conjugates of fluorescent dyes (FITC and PE) with monoclonal antibodies to CD3 (add a separate tip to each tube of 5 μl) and HLA-DR (add a separate tip to each tube 5 μl), respectively. It is recommended that commercial systems containing one sample of anti-CD3-FITC and anti-HLA-DR-PE from Beckman coulter, USA be used.

17. Tubes with donor mononuclear cells and the corresponding conjugates of monoclonal antibodies with fluorescent dyes are incubated for 15 minutes at room temperature in complete darkness (in a closed cabinet).

18. After incubation is completed, double washing is carried out in accordance with paragraph 15.

19. 200 μl of 1x fixative is added to each tube to bind the monoclonal antibodies to the corresponding receptors on the mononuclear cells. It is recommended to use OptiLyse (Beckman coulter, USA).

20. Preparation for flow cytometry.

The protocol for flow cytofluorimetry is shown in Figures 1 and 2.

Six windows are prepared (correspond to 1aR2, 1b, 1c, 2aR3, 2b, 2c). The gate corresponding to figures 1b and 1c is tied to the R2 gate (Figure 1a); and to gate R3 (Figure 2a) - windows corresponding to figures 2b and 2c.

21. Taking results. Alternately, flow cytofluorimetry of mononuclear cells of mixed culture of test tube No. 1 and No. 2 is carried out to duplicate the result. In this analysis, all six windows are simultaneously involved. In gates R2 (Figure 1a) and R3 (Figure 2a), mononuclear cells with the CD45 marker accumulate and then for each donor the distribution of three subpopulations is visible: CD3 ^- HLA-DR ⁺ ; CD3 ⁺ HLA-DR ⁺ ; CD45 ⁺ HLA-DR ⁺ . At the next stage, flow cytofluorimetry of monocultures of the first donor (tubes No. 3 and No. 4 for duplicating the result) and the second donor (tubes No. 5 and No. 6 for duplicating the result) is performed. Here cells accumulate only in three corresponding windows: for the first donor - 1a, 1b, 1c (tubes No. 3 and No. 4); for the second - 2a, 2b, 2c (tubes No. 5 and No. 6).

22. The calculation of the results. For each subpopulation, the growth coefficient (KP) of the specific gravity of the subpopulation in a mixed culture with respect to its specific gravity in the corresponding monoculture is calculated:

KP _{CD3 + HLA-DR + = (} (CD3 + HLA-DR + _skl -CD3 ⁺ HLA-DR ⁺ _con) × 100) / CD3 ⁺ HLA-DR ⁺ _con. where:

CD ^{3 +} HLA-DR + _skl - the relative number of cell subpopulation CD3 ⁺ HLA-DR ⁺ in a mixed lymphocyte culture (MLC),%;

CD ³ + HLA-DR ⁺ _con - the relative number of cells of the subpopulation of CD3 ⁺ HLA-DR ⁺ in the monoculture of the analyzed donor,%, con. - the control;

KP _{CD3-HLA-DR + = (} (CD3 - HLA-DR + _skl -CD3 ^- HLA-DR ⁺ _con) × 100) / CD3 ^- HLA-DR ⁺ _con. where:

CD3 ^- HLA-DR ⁺ _SCL - the relative number of cells of the subpopulation of CD3 ^- HLA-DR ⁺ in a mixed culture of lymphocytes,%;

CD3 ^- HLA-DR ⁺ _con - the relative number of cells of a subpopulation of CD3 ^- HLA-DR ⁺ in the monoculture of the analyzed donor,%;

KP _{CD45 + HLA-DR + = (} (CD45 + HLA-DR + _skl -CD45 ⁺ HLA-DR ⁺ _con) × 100) / CD45 ⁺ HLA-DR ⁺ _con. where:

CD45 ⁺ HLA-DR ⁺ _sc - the relative number of cells of a subpopulation of CD45 ⁺ HLA-DR ⁺ in a mixed culture of lymphocytes,%;

CD45 ⁺ HLA-DR ⁺ _con - the relative number of cells of the subpopulation of CD45 ⁺ HLA-DR ⁺ in the monoculture of the analyzed donor,%.

The most significant for evaluating the effectiveness of alloimmune recognition by HLA are the subpopulations of CD3 ^- HLA-DR ⁺ and CD45 ⁺ HLA-DR ⁺ . The presence of an alloimmune response is documented with a positive CP for these subpopulations.

According to the above protocol, a study of a mixed culture of mononuclear cells from 23 pairs of various unrelated donors was performed. To study the reproducibility of the method, four duplicate statements from six pairs of unrelated donors were performed. It was shown that after incubation in a mixed culture of mononuclear cells, the ratio of their subpopulations to those in the corresponding separate culture changes. The data are presented in table 1.

After incubation the number of cells subpopulation of CD3 ⁺ HLA-DR ⁺ mononuclear cells in the mixed culture was 2.33 ± 0.13% versus 1.09 ± 0.11% in individual cultures, p> 0.05. There were no statistically significant differences (when using the Mann-Whitney test) for these indicators. For each case of this subpopulation calculated gain coefficient: KP _{^{= (. (CD3 + HLA-}} DR + _skl -CD3 ⁺ HLA-DR ⁺ _con) × 100) / CD3 ⁺ HLA-DR ⁺ _con.

The spread of growth factors for this subpopulation of mononuclear cells ranged from -34.7% to + 115.3%, which indicates a change in marker expression characterizing this subpopulation during allogeneic immune interactions by HLA from inhibition to pronounced activation. The percentage of pairs of unrelated donors with negative growth rates was 21.73% (5 pairs). In general, the average growth rate for this population was 43.81 ± 6.31%.

The relative number of CD3 ^- HLA-DR ⁺ subpopulation cells after incubation in a mixed culture of mononuclear cells was 21.87 ± 2.16%, and in monocultures it was 15.63 ± 1.08%, p <0.05. A similar growth rate was calculated for this subpopulation. Not a single negative coefficient was found, and KP values ranged from 26.71% to 64.21%, averaging 44.13 ± 2.23%. These data indicate that the above subpopulation of mononuclear cells reflects the severity of the immune response to alloHLA for unrelated donors.

The relative content of cell subpopulation of CD45 ⁺ HLA-DR ⁺ after incubation of mononuclear cells in a mixed culture composes 24,11 ± 3,05%, as in monocultures - 16,72 ± 1,79%, p <0.05. The calculation of the growth coefficient for this subpopulation showed the absence of negative results, the limit of its changes was from 19.33% to 57.17%, averaging 38.19 ± 3.56%. It should be noted that this subpopulation is formed due to CD3 ⁺ / HLA-DR ⁺ and CD3 ^- / HLA-DR ⁺ , and since the latter dominate, the assessment of activity on alloHLA by CD45 ⁺ / HLA-DR ⁺ actually coincides with CD3 ^- / HLA-DR ⁺ .

Considering the fact that in five married couples the growth rate for the CD3 ⁺ / HLADR ⁺ subpopulation was negative, we calculated the percentage of negative immune response for all subpopulations and obtained a value of 7.25%. Thus, the effectiveness of this method for assessing alloimmune interactions by HLA was taken as 92.75%.

Studies of the reproducibility of the method showed that for a subpopulation of CD3 ⁺ / HLA-DR ^{+, the} average deviation from the minimum to maximum growth coefficient in duplicate settings is 10.57 ± 1.22%; for the subpopulation of CD3 ⁺ / HLA-DR ⁺ - 9.33 ± 0.96%, and for the subpopulation of CD45 ⁺ / HLA-DR ⁺ - 9.25 ± 0.93%. The average deviation for all indicators is 9.71 ± 0.59%. Based on this, we can conclude that the reproducibility of the method is above 90.25%.

In general, the data presented indicate that the proposed method for evaluating alloimmune interactions of unrelated donor mononuclear cells is an effective (92.75%) and reproducible (90.25%) diagnostic method.

To confirm the effectiveness of the method, studies were conducted using the developed method of mixed culture of mononuclear cells to recognize allogeneity by HLA-DR of two unrelated donors and a pair of twins.

Example 1. Donor N. (No. 1), a woman, 34 years old, underwent pregravid training at ZAO Modern Medical Technologies at the stage of pregnancy planning (outpatient card of ZAO Modern Medical Technologies No. 5-14-983 / f). Clinical examination showed that the woman has no chronic somatic and genital pathology. When examined for sexually transmitted diseases, the presence of urogenital infections was not detected.

Donor E. (No. 2), male, age 35, is the husband of donor No. 1. Together with his wife, he underwent examination at ZAO Modern Medical Technologies (outpatient card at ZAO Modern Medical Technologies No. 5-14-983 / m). Like his wife, studies were conducted on urogenital infections, examined by a therapist and urologist. The general diagnosis is healthy.

When conducting a mixed culture of mononuclear cells, it was found that the immune response of female mononuclear cells to male ones (donor No. 1 against donor No. 2) was characterized by the following growth factors: KP _CD3 ⁺ _HLA-DR ⁺ = (+) 23%; KP ^{_{CD3 - HLA-DR + = (}} +) 54% and _SD45 KP ^{_{+ HLA-DR + = (+}} ) 51%.

When assessing the immune response of male mononuclear cells to female mononuclear cells (donor No. 2 versus donor No. 1), the following growth factors were obtained: KP _CD3 ⁺ _HLA - _DR + = (+) 38%; CP _CD3 ^- _HLA-DR ⁺ = (+) 61% and _{CP CD45} ⁺ _HLA-DR ⁺ = (+) 58%.

Thus, in the mixed culture of mononuclear cells “donor No. 1 against donor No. 2” and “donor No. 2 against donor No. 1”, an increase in subpopulations is noted, which indicates the presence of an allogeneic immune response induced by the difference in donors for HLA-DRB1 *. The latter fact was documented by molecular genetic typing on commercial DNA-technology NPF kits. In the woman, the HLA-DRB1 * 01.07 genotype was found, in the spouse - the HLA-DRB1 * 04.10 genotype.

Thus, according to the results of immunological and molecular genetic studies in a married couple (donor No. 1 and donor No. 2) no common alleles were found.

Example 2. Donor L. (No. 1), a woman, 25 years old, underwent pregravid preparation at ZAO Modern Medical Technologies at the stage of pregnancy planning (outpatient card at ZAO Modern Medical Technologies No. 5-14-631 / f). Clinical diagnostic examination showed that the woman has no chronic genital and extragenital pathology. All studies on urogenital infections were negative.

Donor M. (No. 2), a man, 32 years old, is the husband of donor No. 1. Together with his wife, he underwent examination at ZAO Modern Medical Technologies (outpatient card at ZAO Modern Medical Technologies No. 5-14-631 / m). Donor No. 2 conducted a study on urogenital infections, it was examined by narrow specialists and therapist. The diagnosis after the examination is healthy.

The mixed culture of mononuclear cells showed that the immune response of female mononuclear cells against male ones (donor No. 1 against donor No. 2) was characterized by the following growth factors: KP _CD3 ⁺ _HLA-DR ⁺ = (+) 19%; KP _CD3 ^- _HLA-DR ⁺ = (+) 42% and KP _CD45 ⁺ _HLA-DR ⁺ = (+) 39%.

The immune response of male mononuclear cells to female (donor No. 2 versus donor No. 1) was manifested by the following growth factors: KP _CD3 ⁺ _HLA-DR ⁺ = (-) 11%; KP ^{_{CD3 - HLA-DR + = (}} +) 34% and _SD45 KP ^{_{+ HLA-DR + = (+}} ) 31%.

It follows from the KP _CD3 data - _HLA - _DR + and KP _SD45 + _HLA - _DR + were always positive for immune "donor №1 against donor №2» response, as well as for the reverse situation: "donor №2 against donor number one". These data prove that the difference in donors for HLA-DRB1 * is always reflected in the increase in the number of subpopulations of CD3 ^- HLA-DR ⁺ and CD45 ⁺ HLA-DR ⁺ . In accordance with the presented data, there was no increase in the CD3 ⁺ HLA-DR ⁺ subpopulation in the mixed mononuclear culture with the orientation “donor No. 2 against donor No. 1”. It is likely that this is due to the functional genotype of donor No. 1 (HLA-DRB1 * 11.13). It is known that the HLA-DRB1 * 13 allele (HLA-DR6 antigen) has low antigenic properties comparable to the antigenic properties of erythrocyte antigen 0 (I) of a blood group [15]. It is this functional property that determined the absence of immune stimulation through a subpopulation of CD3 ⁺ HLA-DR ⁺ . This _suggests that the coefficients KP _{CD3-HLA-DR +} and KP _{CD45 + HLA-DR +} are the main ones for differentiating donor allogeneity with respect to HLA-DRB1 *, and the KP _{CD3 + HLA-DR +} coefficient indicates the functional features of the interacting HLA-DRB1 * genotypes .

When conducting molecular genetic typing of the HLA-DRB1 gene with commercial DNA-technology NPF kits (Moscow), the woman revealed the HLA-DRB1 * 11.13 genotype. The molecular genetic typing of the HLA-DRB1 locus revealed the HLA-DRB1 * 01.17 genotype in a man.

Thus, according to the results of immunological and molecular genetic studies, no common alleles were found in this family pair.

Example 3. Donor X. (No. 1), a man, 21 years old, underwent a laboratory examination at ZAO Modern Medical Technologies (outpatient card ZAO Modern Medical Technologies No. 3-14-711). Donor S. (No. 2), 21 years old (twin brother of donor No. 1), also underwent a laboratory examination at ZAO Modern Medical Technologies (outpatient card ZAO Modern Medical Technologies No. 3-14-712). Both donors underwent molecular genetic typing of the HLA-DRB1 gene using commercial DNA technology kits (Moscow). It was revealed that donor No. 1 and donor No. 2 had a common genotype HLA-DRB1 * 14.15.

When conducting a mixed culture of mononuclear cells, using well-known formulas, the following coefficients were revealed for a pair of donor No. 1 against donor No. 2:

KP _{CD3 + HLA-DR + = (} (CD3 + HLA-DR + _skl -CD3 ⁺ HLA-DR ⁺ _con.) × 100) / CD3 ⁺ HLA-DR ⁺ _con

KP _{CD3 + HLA-DR +} = ((6.21% -6.23%) * 100%) / 6.23% = - 0.32%;

KP _{CD3-HLA-DR + = (} (CD3 - HLA-DR + _skl -CD3 ^- HLA-DR ⁺ _con) × ^{_{100.) / CD3 - HLA-DR}} + _con

KP _{CD3-HLA-DR +} = ((15.33% -16.02%) * 100%) / 16.02% = - 4.31%;

KP _{CD45 + HLA-DR + = (} (CD45 + HLA-DR + _skl -CD45 ⁺ HLA-DR ⁺ _con.) × 100) / CD45 ⁺ HLA-DR ⁺ _con

KP _{CD45 + HLA-DR +} = ((21.54% -22.25%) * 100%) / 22.25% = - 3.19%.

When assessing the immune response in the direction of donor No. 2 against donor No. 1, according to the formulas below, the following growth factors were obtained:

KP _{CD3 + HLA-DR + = (} (CD3 + HLA-DR + _skl -CD3 ⁺ HLA-DR ⁺ _con.) × 100) / CD3 ⁺ HLA-DR ⁺ _con

KP _{CD3 + HLA-DR +} = ((6.11% -6.13%) * 100%) / 6.13% = - 0.33%;

KP _{CD3-HLA-DR + = (} (CD3 - HLA-DR + _skl -CD3 ^- HLA-DR ⁺ _con) × ^{_{100.) / CD3 - HLA-DR}} + _con

KP _{CD3-HLA-DR +} = ((14.28% -15.01%) * 100%) / 15.01% = - 4.87%;

KP _{CD45 + HLA-DR + = (} (CD45 + HLA-DR + _skl -CD45 ⁺ HLA-DR ⁺ _con.) × 100) / CD45 ⁺ HLA-DR ⁺ _con

KP _{CD45 + HLA-DR +} = ((20.39% -21.14%) * 100%) / 21.14% = - 3.55%.

Analyzing the foregoing, we can conclude that for all three coefficients for the immune responses “donor No. 1 against donor No. 2” and “donor No. 2 against donor No. 1”, there was no increase in the number of subpopulations in a mixed culture of mononuclear cells with respect to the control. Moreover, the proportion of estimated populations in a mixed culture of mononuclear cells was lower than in monocultures, therefore, all calculated coefficients were negative. This indicates the absence of an allogeneic immune response according to HLA, which is true because the donors were twins and had a common genotype HLA-DRB1 *. The latter fact was documented by molecular genetic typing on commercial DNA-technology NPF kits.

Thus, the advantages of the proposed invention is as follows:

1. A method of evaluating an allogeneic immune response in a mixed culture of mononuclear short unrelated donors comprises a one-time analysis of subpopulations of mononuclear cells with the phenotype of ^{CD3 + / HLA-DR +,} CD3 - / HLA-DR +, CD45 + / HLA-DR + each donor (separated and gated different fluorescent dye) in their mixed culture using flow cytometry, followed by comparison of the degree of expression of HLA-DR on the respective mononuclear cells of the mixed culture and control individual cultures of mononuclear cells of each donor.

2. According to the level of HLA-DR expression in a mixed culture of mononuclear cells, the immune response of one donor to allogeneic antigens of another is simultaneously evaluated and, conversely, with a reproducibility error of less than 10%.

3. The method does not require additional control series of studies.

4. A pathogenetically substantiated assessment of expression by HLA-DR mononuclear cells is carried out which, on the one hand, are the main molecules of intercellular contacts, and on the other hand, the main allogeneic molecules of tissue compatibility.

5. The research method is quite specific for laboratory studies of immune interactions for allogeneic antigens of two unrelated donors, which can be used in transplantation or in the examination of spouses to diagnose the immune causes of reproductive losses.

The proposed method is not laborious and can be used in clinical diagnostic laboratories.

Information sources

1. Bain B, Vas Mr., Lowenstein L. The development of large immature mononuclear cells in mixed leucocyte cultures // Blood. - 1964. - Jan. - V. 23. - P. 108-116.

2. Park B.H. and Good R.A. Third Party Mixed-Leukocyte Culture Test: A Potential New Method of Histocompatibility Testing // Proc Natl Acad Sci USA. - Jun. - 1972.- V. 69 (6). - P. 1490-1493.

3. Albrechtsen D., Bratlie Α., Nousiainen H., Solheim B.G., Winther N., Thorsb E. Serological typing of HLA-D: Predictive value in mixed lymphocyte cultures (MLC) // Immunogenetics. - 1978. - V. 6, Issue 1. - P. 91-100.

4. Hajek-Rosenmayr A. Mixed lymphocyte culture as a compatibility test for bone marrow transplantation // Wien Klin Wochenschr. - 1986. - Jan 24. - V. 98 (2). - P. 44-49.

5. Order of the Ministry of Health of the RSFSR “On the implementation of bone marrow transplantation in healthcare practice”, No. 31 of 02.25.1991.

6. Pandey M.K., Saxena V. and Agrawal S. Characterization of mixed lymphocyte reaction blocking antibodies (MLR-Bf) in human pregnancy // BMC Pregnancy and Childbirth. - 2003. - V. 3 (2). - P. 1471-2393.

7. Govallo, V.I. Immunology of reproduction / M .: Medicine, 1987. - 304 p.

8. Fremel G. Immunological methods / M .: Medicine, 1987. - 476 p.

9. Korolkova O.Yu., Senyukov VV, Kozhevnikov B.C. Expression of ergotot-associated markers with activated T-lymphocytes // Medical Immunology. - 2009. - T. 11. - No. 2-3. - S. 234-235.

10. Bulycheva T.I., Deineko N.L. The method of immunocytochemical assessment of the proliferative state of lymphocytes by the nature of the expression of the c-terminal fragment of the protein b23 / nucleofosmin in the reaction of indirect immunofluorescence, patent for the invention RU 2438135.

11. Grigoriev A.A., Deineko N.L., Bulycheva T.I. Method for immunochemical evaluation of proliferation of human lymphocytes using a new marker - nucleolar protein a3, patent for invention RU 2431670.

12. Sambur M.B. A method for assessing the interaction of lymphocytes in vitro, based on the determination of their rosette-forming ability // Immunology. - 1991. - No. 2. - S. 30-33.

13. Yarilin A.A. Immunology / M .: GEOTAR-Media, 2010 .-- 752 p.

14. Leplina O.Yu. Characterization of interferon-alpha-induced dendritic cells and their therapeutic potential in the treatment of cancer and infectious diseases: abstract. diss. ... cand. honey. Sciences, Novosibirsk. - 2011 .-- 28 s.

15. Zaretskaya Yu.M., Clinical immunogenetics / M .: Medicine, 1989. - 234 p.

Claims (1)

A method for assessing an allogeneic immune response in a short-term mixed culture of unrelated donor mononuclear cells based on an immunological study of venous blood, characterized in that it includes a one-stage analysis of subpopulations of mononuclear cells with the phenotype CD3 ⁺ HLA-DR ⁺ , CD3 ^- HLA-DR ⁺ , CD45 ⁺ HLA-DR ⁺ of each donor, separated and gated by a different fluorescent dye in their mixed culture, using flow cytofluorimetry, followed by calculation of growth factors for each subpopulation according to the formulas:

Where:

- the relative number of cells of a subpopulation of CD3 ⁺ HLA-DR ⁺ in a mixed culture of lymphocytes,%;

- the relative number of cell subpopulation CD3 ⁺ HLA-DR ⁺ - monoculture analyte donor%;

Where:

- the relative number of cells of the CD3 ^- HLA-DR ⁺ subpopulation in a mixed culture of lymphocytes,%;

- the relative number of cells of the CD3 ^- HLA-DR ⁺ subpopulation in the monoculture of the analyzed donor,%;

Where:

- the relative number of cells of the subpopulation of CD45 ⁺ HLA-DR ⁺ in a mixed culture of lymphocytes,%;

- the relative number of cells of the subpopulation of CD45 ⁺ HLA-DR ⁺ in the monoculture of the analyzed donor,%;
and with positive values of KP _{CD3-HLA-DR +} and KP _{CD45 + HLA-DR +} make a conclusion about the presence of immune recognition by allo HLA, and the coefficient of KP _{CD3 + HLA-DR +} indicates the functional features of the interacting genotypes HLA-DRB1 *.

Images