Panel of primers, kit and method for determining donor genetic chimerism in patients after allogeneic hematopoietic stem cell transplantation

Abstract

FIELD: molecular biology; transplantology.SUBSTANCE: it can be used to assess the engraftment of donor cells after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of hemoblastoses. The claimed invention is based on a study of changes in the levels of donor-specific genetic markers – short tandem repeats (STR) – in the recipient relative to the donor. To do this, an original primer panel is used to simultaneously detect at least 20 loci using quantitative fluorescence PCR (QF-PCR), where one of the primers in the pair contains a fluorescent dye. The chimerism determination kit also contains reagents for PCR, DNA fragments of a given length as an internal size standard, a control DNA sample, and a set of reference fragments of alleles of the listed loci. The method includes DNA extraction from samples of biological material from the donor and recipient, amplification using PCR of the CTP loci, fragment analysis by capillary electrophoresis, identification of unique alleles and calculation of the proportion of donor chimerism for each locus with subsequent averaging.EFFECT: creation of a rapid and reproducible method for determining the genotypes of the CTP using DNA isolated from a sample of peripheral blood of a donor, recipient before and after transplantation, as well as increase in the accuracy in determining chimerism in related donors and recipients, in particular, in determining the proportion of donor hematopoietic cells to the proportion of those in the recipient after allo-HSCT.6 cl, 2 dwg, 6 tbl

Description

[0001] Field of technology to which the invention relates

[0002] The invention relates to molecular biology, medicine, molecular hematology, genetics, and transplantology, specifically to the determination of chimerism in a DNA sample isolated from a patient's biological fluid. It can be used for the quantitative and qualitative assessment of donor cell engraftment after allogeneic hematopoietic stem cell transplantation (allo-HSCT), which is performed in the treatment of hemoblastoses. The claimed invention is based on the study of changes in the levels of donor-specific genetic markers—short tandem repeats (STRs)—in the recipient relative to the donor.

[0003] Technology Level

[0004] Hemoblastoses are a large group of hematopoietic disorders. These diseases disrupt the normal balance of blood cells and affect immune function. Chemotherapy aimed at eliminating pathogenic cells is used to treat severe forms of hemoblastoses. To restore hematopoietic function, the patient may undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT). Donor cellular material allows for the restoration of hematopoiesis after HSCT by repopulating the recipient's cell niches. Assessing the completeness of graft engraftment by comparing the proportion of unique donor and recipient genetic markers in the recipient's blood after transplantation is crucial for monitoring treatment effectiveness. Donor chimerism is also used to assess graft engraftment.Complete donor chimerism (CDC) is defined as the replacement of the recipient's hematopoietic cells by the donor's hematopoietic cells after allo-HSCT, with only a few remaining patient cells. In mixed chimerism (MC), both donor and recipient hematopoietic cells are present in the body, and over time, the recipient's cells may be lost, leading to the development of CDC, or the transplant may be rejected.

[0005] In addition to short tandem repeats (STRs), tandem repeat number variation (TRNV), single nucleotide repeats (SNRs), or variations associated with nucleotide insertions and deletions (indels) are used as genetic regions to determine chimerism. STRs are genomic regions containing a variable number of repeats. Typically, such repeats share a common sequence of 2-8 nucleotides. The number of such repeats can range from two to several dozen. The number of repeats within STR loci varies across the population, allowing a combination of the most polymorphic loci to determine an individual's "genetic fingerprint" and compare individuals using these genetic markers. TNRs are also based on repeats, but each repeat is significantly longer, ranging from 8 to 100 nucleotides. SNPs are single nucleotide substitutions, and therefore population variation is typically limited to two variants, which reduces the discriminatory power of SNPs.Indels, like SNPs, have two states—whether the variation is present or not. This limitation of SNPs and indels requires the simultaneous analysis of a larger number of events to create a unique "genetic fingerprint" for an individual.

[0006] Currently, there is no established method for determining donor chimerism in practical medicine. The methods differ in sensitivity, time, cost and convenience. Chimerism can be determined based on the study of blood group, antibody isotypes, cytogenetic analysis of metaphase plates or fluorescence in situ hybridization for sex chromosomes. Among the main molecular biological methods for determining post-transplant donor chimerism, the following are distinguished: STR-PCR - PCR for short tandem repeats; RT-PCR - PCR with real-time fluorescence fixation; dPCR - digital PCR; RFLP - restriction fragment length polymorphism; MPS - massively parallel sequencing [DOI:10.3390 / diagnostics11040621]. For RFLP analysis, DNA is treated with restriction enzymes specific to the regions where the SNP is located [https: / / doi.org / 10.1097 / 00007890-198703000-00016; PMID:1356057].This method was one of the first to appear; however, it is labor-intensive, has low sensitivity, and requires a large amount of good-quality DNA for analysis. With the advent of PCR, it became possible to work with smaller amounts of DNA, including when determining chimerism [https: / / doi.org / 10.1111 / j.1365-2141.1989.tb00253.x]. RT-PCR is a simple to perform method; however, it requires the use of qualified personnel to perform the test and is limited in assessing chimerism when the proportion of patient (or donor) DNA is above 30%. RT-PCR is performed using calibration curves based on donor and recipient DNA, obtained before allo-HSCT [https: / / doi.org / 10.1182 / blood.v99.12.4618]. dPCR is a third-generation PCR method that allows for absolute assessment of chimerism without calibration curves [https: / / doi.org / 10.4161 / chim.25400]. dPCR is significantly more applicable for assessing chimerism at a single DNA fraction from 0.05% to 100% [https: / / doi.org / 10.1371 / journal.pone.0212708]. Methods based on the determination of SNPs or indels have low discriminatory power, since they are unable to distinguish between samples in multiple transplantations, and can give false positive results due to high sensitivity [https: / / doi.org / 10.1016 / j.bbmt.2014.07.030]. MPS allows for the simultaneous assessment of SNPs, indels, CTPs, and HFTPs. The sensitivity of MPS depends on the sequencing depth [https: / / doi.org / 10.3892 / mmr.2016.5593]. Performing MPS is the most labor-intensive and time-consuming method for assessing chimerism, is expensive, and is a poorly standardized method. Moreover, MPS allows for the simultaneous evaluation of hundreds of SNPs and indels, reducing the specific cost of analysis [https: / / doi.org / 10.3390 / jcm6090087]. With deep sequencing, the method can be used for small amounts of DNA with a small proportion of cells being analyzed.

[0007] Currently, the preferred method for assessing chimerism is the QTP-PCR method, with a reported sensitivity in the range of 1-5% [https: / / doi.org / 10.1159 / 000077555]. The method consists of two stages: first, the QTP or HFTP is amplified during conventional PCR, then subjected to fragment analysis - electrophoresis, with determination of the amplicon lengths. HFTP was initially used to determine chimerism, but the small number of loci available for analysis made their use impossible for a number of donor-recipient pairs [https: / / doi.org / 10.1093 / ajcp / 107.3.292]. Thus, CTPs have become the main genetic determinant for studying chimerism; they are shorter and have greater variability [https: / / doi.org / 10.1038 / sj.leu.2402017]. CTP-PCR is a rapid, reliable, accurate, and reproducible method that does not require additional equipment for existing molecular hematology laboratories. Moreover, both commercial and in-house CTP kits can be used for chimerism analysis.Furthermore, the method requires a small amount of DNA for analysis, which is important when assessing linear chimerism in small cell subpopulations. However, post-transplant monitoring of chimerism after allo-HSCT faces the challenge of detecting chimerism after transplantation from a sibling whose genotype may be very close to that of the recipient. Therefore, searching for a combination of loci with an increased number for analysis will facilitate the identification of markers for genotype comparison and chimerism calculation. Furthermore, it is important to improve the reliability and accuracy of chimerism determination, including by searching for a combination of loci and their number, which will allow for the averaging of the measurement error introduced by the variability of the PCR procedure and the refinement of the results.At the same time, it is also important to find solutions that achieve all the above-mentioned advantages without the need for additional reactions for a single sample, so as not to complicate, increase the time and cost of the tests. From a clinical perspective, the optimal method should be informative, sensitive, accurate, reproducible, simple, and easy to implement.

[0008] A method for determining chimerism is known from the prior art (WO 2008 / 056937 A1, 07.11.2006), based on genotyping of the hypervariable region of mitochondrial DNA. The method for genomic typing of mitochondrial DNA polymorphism alleles in a mixture of donor and recipient DNA comprises: a) amplifying DNA regions containing at least one polymorphism using at least one pair of primers that specifically hybridize with a conserved target region, wherein at least one primer is covalently linked to a fluorescent dye; b) fragment analysis of at least one PCR reaction product; c) determining from the result of step "b" which alleles of the hypervariable region genes, nucleotide positions 303polyC and 16184polyC from mitochondrial DNA are present in the sample; d) calculation of at least one allele of the CTP that is different for the donor and recipient before transplantation; d) calculation of the proportion of donor chimerism.Furthermore, the present invention describes primers for use in the above methods. Diagnostic kits for implementing these methods are also provided. In the invention, identifying alleles of just one hypervariable region requires two sequential steps: polymerase chain reaction (PCR) to generate a gene fragment containing the polymorphism, and acrylamide gel electrophoresis. However, such a kit contains a small number of discriminatory targets and may not be suitable for some donor-recipient pairs.

[0009] The closest to the claimed group of inventions are a kit and method for determining chimerism based on genomic typing of CTP alleles in a DNA sample isolated from biological material (US 2010 / 0099082 A1, 01.06.2007).In order to determine chimerism in a DNA sample, the following is carried out: a) bringing genomic polynucleic acids in the sample into contact with at least one pair of primers, as a result of which the 5' and / or 3' primer of at least one pair of primers specifically hybridizes with target regions including polymorphic nucleotides of the alleles, and carrying out an amplification reaction of fragments of the alleles containing at least one allele of the CTP; b) identifying for each of the at least one pair of primers whether an amplification product is formed at step "a"; c) determining from the result of step b which allele of the CTP is present in the sample D2S1360, D7S1517, D8S1132, D9S1118, D10S2325, D11S554, D12S1064, D12S391, D17S1290, D19S253, MYCL1, P450CYP19 and SE-33; d) performing steps a-c for the donor; d) performing steps a-c for the recipient before transplantation; e) performing steps a-c for the recipient after transplantation and calculating chimerism.The present invention describes primers used to detect chimerism. Diagnostic kits for implementing these methods are also provided. However, the known solution suffers from insufficient coverage of the CTP for detecting chimerism in related donors and recipients, as well as difficulty combining more than five markers in a single reaction. These factors make the known method unsuitable for routine genotyping of many CTP loci required for chimerism detection.

[0010] The claimed invention aims to optimize a method for determining chimerism in a DNA sample isolated from a patient's biological fluid after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The invention expands the arsenal of technical means used to determine chimerism, an important objective indicator used to evaluate the effectiveness of treatment for patients with blood cancers.

[0011] Disclosure of the essence of the invention

[0012] The technical result consists of the creation of a rapid and reproducible method for determining hematopoietic stem cell genotypes using DNA isolated from a peripheral blood sample of a donor and recipient before and after transplantation. Another technical result is increased accuracy in determining chimerism in related donors and recipients, particularly in determining the ratio of donor hematopoietic cells to recipient hematopoietic cells after allo-HSCT.

[0013] The technical result is achieved by using a primer panel for determining chimerism after hematopoietic stem cell transplantation using quantitative fluorescence polymerase chain reaction (QF-PCR), including pairs of forward and reverse primers for the simultaneous determination of the following short tandem repeat (STR) loci: D5S818, D21S11, D7S820, CSF1PO, D2S1338, D3S1358, vWA, D8S1179, D16S539, D1S1677, PentaD, TPOX, TH01, D22S1045, D18S51, FGA, D6S1043, D13S317, D12S391 and PentaE, wherein one of the primers in the pair contains a covalently linked fluorescent dye.

[0014] The panel may additionally contain pairs of forward and reverse primers for detecting the following CTP loci: D2S441, D1S1656, D19S433, D10S1248, D11S4463, D1S1627, D3S4529, D1GATA113, D17S974, D6S1017, D4S2408, D9S2157, D6S474, D2S1776, D18S853, D20S482 and D14S1434.

[0015] The technical result is achieved by using a kit for determining chimerism after hematopoietic stem cell transplantation using quantitative fluorescence polymerase chain reaction (QF-PCR), including:

[0016] a. primer pairs - forward and reverse, for the simultaneous detection of the above loci;

[0017] b. reagents for PCR;

[0018] c. DNA fragments with a given length as an internal size standard;

[0019] d. DNA control sample;

[0020] e. A set of reference fragments of alleles of the listed loci.

[0021] The technical result is achieved by a method for determining chimerism after hematopoietic stem cell transplantation, including:

[0022] collection of samples of biological material containing the nuclear cell component of blood from the donor and the recipient before transplantation and from the recipient after transplantation;

[0023] DNA extraction from each selected sample of biological material;

[0024] carrying out amplification using quantitative fluorescence PCR (QF-PCR) of the listed short tandem repeat (STR) loci by contacting the isolated DNA from each selected biological sample with primers and reagents for carrying out PCR;

[0025] conducting fragment analysis of all obtained amplicons using capillary electrophoresis in acrylamide gel with the determination of the CTP loci containing unique alleles for both the donor and recipient;

[0026] determination of donor chimerism for a DNA sample from a recipient after transplantation at selected loci, calculated for each locus of the KTP;

[0027] averaging the obtained chimerism values ​​for all KTP loci.

[0028] In a specific embodiment of the invention, the proportion of the donor's unique allele in the recipient after transplantation for each locus is determined by the peak magnitude in the chromatogram, measured in relative fluorescent units, divided by the sum of the fluorescence intensities of all peaks of the locus, expressed in relative fluorescent units. The length of the loci is determined using the components of the above-mentioned kit.

[0029] The genomic DNA of the recipient and donor can be isolated from biological material, which is whole blood, bone marrow biopsy or leukocyte mass.

[0030] Brief description of drawings

[0031] The invention is illustrated by drawings, where Figure 1 shows an algorithm for determining chimerism, which can be implemented using programming tools, which takes into account the results of FT-PCR for 37 loci; Figure 2 shows an example of an electropherogram for one donor DNA sample obtained based on the results of FT-PCR with subsequent fragment analysis.

[0032] Implementation of the invention

[0033] Determination of donor genetic chimerism in a patient after allogeneic hematopoietic stem cell transplantation is based on the identification of genetic discrepancies in donor-recipient pairs using KTP markers.

[0034] During the implementation of the method, quantitative fluorescence PCR is carried out for at least 20 CTP markers presented in Table 1 Nos. 1-20. In one embodiment of the invention, an additional 17 CTP markers presented in Table 1 Nos. 21-37 can be used. Thus, the total number of CTP markers located on somatic chromosomes that can be used in the claimed method is 37 (see Table 1).

[0035] Table 1. KTP markers used to determine chimerism

[0036] № Locus Chromosome region Repeat Fluorescent label Main loci 1 D2S1338 2q35-37.1 TGCC / TTCC FAM 2 D3S1358 3p21.31 AGATE / AGAC TET 3 D5S818 5q21-31 AGATE FAM 4 D7S820 7q11.21-22 GATA FAM 5 D8S1179 8q24.13 TCTG / TCTA TET 6 D12S391 12p13.2 AGATE / AGAC NED 7 D13S317 13q22-31 TATC NED 8 D16S539 16q24-qter AGATE TET 9 D18S51 18q21.33 AGAA HEX 10 D19S433 19q12-13.1 AAGG / AAAG / AAGG / TAGG ROX 11 D21S11 21q21.1 TCTA / TCTG FAM 12 CSF1PO 5q33.5-34 AGATE FAM 13 FGA 4q28 TCTT / CTCC / TTCC HEX 14 TH01 11p15.5 AATG HEX 15 TPOX 2p25.3 AATG HEX 16 vWA 12p13.31 TCTA / TCTG TET 17 D1S1656 1q42 TAGA / TG / TAGG ROX 18 D2S441 2p14 TCTA ROX 19 D10S1248 10q26.3 GGAA ROX 20 D22S1045 22q12.3 ATT HEX Additional loci 21 D6S1043 6q16.1 GATA NED 22 PentaD 21q22.3 AAAGA TET 23 PentaE 15q26.2 AAAGA NED 24 D11S4463 11q25 TATC Alexa 633 25 D1S1677 1q23.3 TTCC TET 26 D1S1627 1p21.1 ATT Alexa 594 27 D3S4529 3q12.1 ATYT Alexa 594 28 D1GATA113 1p36.23 GATA Alexa 594 29 D17S974 11q25 CTAT Alexa 594 30 D6S1017 6q21 AGATE / GATA Alexa 594 31 D4S2408 4p15.1 ATCT Alexa 594 32 D9S2157 9q34.2 ATA Alexa 594 33 D6S474 6q21 AGATE / GATA Alexa 633 34 D2S1776 2q24.3 AGATE Alexa 633 35 D18S853 18p11.31 ATA Alexa 633 36 D20S482 20p13 AGATE Alexa 633 37 D14S1434 14q32.13 CTRT Alexa 633

[0037] The method for determining chimerism based on the use of markers from Table 1 contains the following steps:

[0038] 1. Conducting a polymorphism analysis of the lengths of the KTP markers presented in Table 1 for the donor and recipient before allo-HSCT;

[0039] 2. Conducting a length polymorphism analysis for the given markers for the recipient after allo-HSCT to calculate the proportion of donor markers.

[0040] The result analysis includes the following steps:

[0041] 1. Conducting length polymorphism analysis for the KTP markers presented in Table 1 for the donor and recipient before allo-HSCT; and for the recipient after allo-HSCT;

[0042] 2. Determination of discrepancies in KTP markers between donor and recipient after allo-HSCT;

[0043] 3. Calculation of the proportion of donor markers, as the ratio of the height, expressed in relative fluorescence units, of unique donor alleles to the unique alleles of the patient's markers before transplantation; followed by averaging the obtained values ​​for all markers used for the calculation.

[0044] The chimerism detection kit includes forward primers labeled with a detectable label and reverse primers for the loci shown in Table 1 selected as markers.

[0045] For length polymorphism analysis, quantitative fluorescence PCR (QF-PCR) is used with a forward primer labeled with a detectable label and a reverse primer to the loci listed in Table 1. Forward primers may have sequences according to SeqID No. 1-37 (see Table 2), respectively. Reverse primers may have sequences according to SeqID No. 38-74 (see Table 2), respectively.

[0046] Table 2. Primers for FT-PCR

[0047] Seq ID Locus Subsequence Fluorescent label 1 D2S1338 AATAAAGACTTCATGGTCCTGACTA FAM 2 D3S1358 GTTTCAGAGCAAGACCCTGTCTC TET 3 D5S818 AGTGCTTTTTAGCCAAGTGATT FAM 4 D7S820 ATAGAACACTTGTCATAGTTTAGAAC FAM 5 D8S1179 CCTATCCTGTAGATTATTTTCACTG TET 6 D12S391 CAACAGGATCAATGGATGC NED 7 D13S317 GCCCAAAAAGACAGACAGAAAG NED 8 D16S539 CAGGAGCTGGGGGGTCTA TET 9 D18S51 CCTGTAGTCTCAGCTACTTGC HEX 10 D19S433 ACCCGAATAAAATTCTTCTCTCT THIRD 11 D21S11 ATGTTCTCCAGAGACAGACTAATA FAM 12 CSF1PO ACCCTGTGTCTCAGTTTTCC FAM 13 FGA TTAGGACATCTTAACTGGCATTC HEX 14 TH01 TATCTGGGCTCTGGGGT HEX 15 TPOX GAACCCKCACAGGTTAATTAAGA HEX 16 vWA GCAGATGATAAATACATAGGATGG TET 17 D1S1656 AGGATTCTTCAGAGAAATAGAATCAC THIRD 18 D2S441 GGCTACAGGAATCATGAGC THIRD 19 D10S1248 TAATAGCTATTATTACCAAAGCATG THIRD 20 D22S1045 TCTGATCCTGGCTCCACC HEX 21 D6S1043 CCAGAGATAGAAGCAATAGTG NED 22 PentaD AAGGTATCTTTTGAAGAGTAGAACTT TET 23 PentaE TCTTATTTGGGTTATTGAGAA NED 24 D11S4463 GCAATGCTGCTATTGGCACT Alexa 633 25 D1S1677 TTCTGTTGGTATAGAGCAGTGTTT TET 26 D1S1627 CATGAGGTTTGCAAATACTATCTTAAC Alexa 594 27 D3S4529 CCCAAAATTACTTGAGCCAAT Alexa 594 28 D1GATA113 TCTTAGCCTAGATAGATACTTGCTTCC Alexa 594 29 D17S974 GCACCCAAAACTGAATGTCATA Alexa 594 30 D6S1017 CCACCCGTCCATTTAGGC Alexa 594 31 D4S2408 AAGGTACATAACAGTTCAATAGAAAGC Alexa 594 32 D9S2157 CAAAGCGAGACTCTGTCTCAA Alexa 594 33 D6S474 GCAGGACTGTGAGTTCTAACT Alexa 633 34 D2S1776 TGAACACAGATGTTAAGTGTGTATATG Alexa 633 35 D18S853 GCACATGTACCTAAAACTTAAAAT Alexa 633 36 D20S482 CAGAGACACCGAACCAATAAGA Alexa 633 37 D14S1434 TTCCCAGCCTCCATAATCAG Alexa 633 38 D2S1338 GAGTTATTCAGTAAGTTAAAGGATTG NO 39 D3S1358 CAACAGAGGCTTGCATGTATC NO 40 D5S818 GGTGATTTTCCTCTTTGGTAT NO 41 D7S820 AAATACTGAAACCAGTGTGAAC NO 42 D8S1179 GTATTTTTGTATTTCATGTGTACATTCG NO 43 D12S391 CTGTCATGAGATTTTTCAGCCT NO 44 D13S317 ACCCATCTAACGCCTATCTGTATT NO 45 D16S539 GTTTGTGTGTGCATCTGTAAGCATG NO 46 D18S51 ACATGTTGGCTTCTCTCTGT NO 47 D19S433 GTTTAGGCATGTTGGCACATT NO 48 D21S11 ATGGTCTGTTATGGGACTTTTC NO 49 CSF1PO GTTTCTGTGTCAGACCCTGTTC NO 50 FGA AATCGAAAATATGGTTATTGAAGTAGC NO 51 TH01 AGGTCACAGGGAACACAGACTC NO 52 TPOX ACAGAACAGGCACTTAGGGA NO 53 vWA TGTGAAAGCCCTAGTGGATGATA NO 54 D1S1656 ATCCCCATATAAGTTCAAGCCTG NO 55 D2S441 GCAAGTATAATCATTCTTATTTAACATC NO 56 D10S1248 TTATTCTGACAGTTCTCTTTTTCC NO 57 D22S1045 CCCACTATGGGCAAACCTTAA NO 58 D6S1043 GCCTACCATGTTTTGAAGGC NO 59 PentaD AGCCAGGCATGGTGAGG NO 60 PentaE GGCCGATGCAGGTGTATT NO 61 D11S4463 TGATGATCTGCAAAGGAGGA NO 62 D1S1677 GTGACAGGAAGGACGGAATG NO 63 D1S1627 GTTTTAATTTTCTCCAAATCTCCA NO 64 D3S4529 GAGACAAAATGAAGAAACAGACAG NO 65 D1GATA113 GTCAACCTTTGAGGCTATAGGAA NO 66 D17S974 GGTGAGAGTGAGACCCTGTC NO 67 D6S1017 GTGAAAAAGTAGATATAATGGTTGGTG NO 68 D4S2408 GTGAAATGACTGAAAAATAGTAACCA NO 69 D9S2157 GAAAATGCTATCCTCTTTGGTATAAAT NO 70 D6S474 AGATTTAAACAGTGTACAGCAA NO 71 D2S1776 GTCTGAGGTGGACAGTTATGAAA NO 72 D18S853 GTCAACCAAAACTCAACAAGTAGTAA NO 73 D20S482 GCCACATGAATCAATTCCTATAATAAA NO 74 D14S1434 TGCAAATGCACACAGATTTC NO

[0048] To visualize PCR results, a fragment analysis procedure is used, which involves electrophoresis in acrylamide gel with the addition of a fluorescently labeled internal size standard (ISS) to each sample. Fragment sizes in nucleotides are determined relative to the ISS. The allele of each marker (CTP locus) is then determined relative to predefined parameters containing information on the size range and fluorescent label for each allele of each marker.

[0049] QF-PCR with genotyping of KTP alleles for the analysis of length polymorphisms of the given markers is performed on donor and recipient samples before allo-HSCT, and on recipient samples after allo-HSCT. To determine chimerism, unique alleles are first selected for the donor and recipient before transplantation. These informative alleles are used to calculate chimerism. For this purpose, donor and recipient samples after allo-HSCT are compared, and the proportion of chimerism is calculated for each marker using the informative alleles using the formula: Donor chimerism (%) = 100% * (donor allele fluorescence level) / (sum of fluorescence levels at the locus).

[0050] Chimerism determination can be implemented using software that takes into account PCR results for 37 loci. The software algorithm is shown in the image (Fig. 1). The system used for chimerism determination includes a computer with pre-installed software capable of performing the following steps:

[0051] a. Import data files obtained using a genetic analyzer.

[0052] b. Determining the lengths of fragments in accordance with the internal dimensional standard.

[0053] c. Determination of genotypes of selected markers for the donor and recipient before and after transplantation.

[0054] d. Identification of unique alleles that differ between the donor and recipient before transplantation, with storage of this information in the database.

[0055] e. Analysis of the proportion of donor and recipient alleles after transplantation for all selected markers and averaging the results.

[0056] f. Generating an analytical report.

[0057] The use of software and hardware for implementing the invention makes it possible to simplify the fragment analysis of the results of the PCR reaction products and to reduce the variability of the value of the measured chimerism introduced by the analysis method.

[0058] Examples of the invention implementation

[0059] The claimed invention was used to determine the chimerism of human DNA samples obtained from donors and recipients before and after allogeneic hematopoietic stem cell transplantation.

[0060] Step 1. Set of primers for QTP and conditions for performing PCR.

[0061] Table 1 lists the CTPs used, their genetic locations, and repeat structures. It should be noted that primer concentrations are selected individually for each round of primer synthesis. Table 2 lists the primer sequences for amplifying the selected CTPs, as well as the fluorescent label used. Table 3 lists the primer concentrations for compiling the final primer mix. All primers are mixed in the required proportions and added to the reaction as a primer mix. Table 4 lists the composition of the CTP-PCR.

[0062] Table 3. Concentration of each primer pair in the final reaction mixture

[0063] № Locus Primer concentration (nM) 1 D2S1338 80 2 D3S1358 160 3 D5S818 100 4 D7S820 200 5 D8S1179 360 6 D12S391 80 7 D13S317 80 8 D16S539 80 9 D18S51 80 10 D19S433 160 11 D21S11 160 12 CSF1PO 160 13 FGA 300 14 TH01 120 15 TPOX 80 16 vWA 100 17 D1S1656 100 18 D2S441 100 19 D10S1248 240 20 D22S1045 240 21 D6S1043 140 22 PentaD 120 23 PentaE 300 24 D11S4463 160 25 D1S1677 240 26 D1S1627 240 27 D3S4529 240 28 D1GATA113 300 29 D17S974 160 30 D6S1017 300 31 D4S2408 80 32 D9S2157 80 33 D6S474 80 34 D2S1776 120 35 D18S853 120 36 D20S482 240 37 D14S1434 80

[0064] Table 4. Composition of one QTP-PCR

[0065] Component Volume (µl) Water for molecular work 13,4 5x PCR buffer 5 dNTP (25 mM) 0,4 MgCl2 (25 mM) 2 Primer mix 2 Taq-HS polymerase (Biolabmix, 5 units / µl) 0,2 DNA (0.5 ng / µl) 2 Total volume 25

[0066] Any suitable nucleic acid amplifier, such as ThermoFisher ProFlex, can be used for QTP-PCR with cycling protocol parameters such as those shown in Table 5 (elongation times may vary between amplifiers).

[0067] Table 5. Cycling protocol parameters

[0068] Step (°C) Time (min:sec) Cycles 95 2:00 1 94 0:10 2 63 1:10 2 94 0:10 28 60 1:10 28 60 5:00 1 12 Storage 1

[0069] Stage 2. Capillary electrophoresis with detection of fluorescently labeled fragments.

[0070] 1 μl of the PCR product was mixed with 10 μl of high-purity formamide (GTZ, Russia) and 0.25 μl of the internal size standard G500 (GTZ, Russia). After denaturation at 95°C for 3 minutes, the mixture was transferred to capillary electrophoresis plates and electrophoresed using suitable equipment (e.g., GTZ G16, Russia). Capillary electrophoresis was performed under standard conditions using the appropriate compensation matrix for eight detection channels. Figure 2 shows an example of an electropherogram for one donor DNA sample obtained by FT-PCR followed by fragment analysis. The parameters for injection of applicons during capillary electrophoresis were selected to obtain relative fluorescence values ​​in the range of 5,000–10,000 units.

[0071] Step 3. Detection limit and quantification of donor chimerism.

[0072] To detect the presence of a small fraction of cells against a large fraction of cells with a different genotype, rigorous sensitivity testing of each individual marker was conducted. The feasibility of estimating the proportion of confounded DNA for chimerism calculations for individual CTP markers was also tested.

[0073] To test the ability of the present invention to quantify chimerism, serial dilutions of DNA containing different proportions of donor DNA were analyzed.

[0074] Table 6. KTP markers for chimerism and the detection limit (for the first ten loci) The number of dropped loci depending on the proportion of admixed DNA

[0075] DNA share Marker number 0,8 1,6 3,2 6,4 Total 1 10 4 3 1 18 2 15 5 4 1 25 3 10 10 5 2 27 4 34 3 2 1 40 5 25 5 7 0 37 6 20 6 1 1 28 7 31 3 5 0 39 8 23 11 1 1 36 9 15 8 4 0 27 10 11 3 0 1 15 11 35 9 6 2 52 12 16 9 6 2 33 13 14 8 6 1 29 14 14 6 1 0 21 15 34 6 3 2 45 16 12 6 6 0 24 17 34 4 2 2 42 18 27 4 1 0 32 19 30 5 3 0 38 20 16 3 7 1 27 21 16 7 5 0 28 22 14 6 2 2 24 23 10 10 7 1 28 24 14 10 7 1 32 25 20 10 2 0 32 26 14 8 2 1 25 27 31 5 1 2 39 28 30 4 6 1 41 29 17 6 4 2 29 30 12 9 5 1 27 31 32 3 7 0 42 32 27 9 0 2 38 33 22 5 1 2 30 34 20 5 0 2 27 35 23 3 6 0 32 36 34 5 7 0 46 37 20 10 2 1 33 Total 194 58 32 8 292

[0076] The maximum sensitivity (limit of detection) was equal to or lower than 1.6% (Table 5). Samples containing 1 and 5 ng of DNA showed similar sensitivities. The observed detection limits are suitable for quantitative determination of chimerism in clinical settings.

[0077] The present invention utilizes a large panel of CTP markers, enabling the identification of loci suitable for assessing donor chimerism, even in cases of consanguineous transplantation. Furthermore, the larger number of loci used for assessing chimerism allows for a more precise definition of chimerism and mitigates interlocus variation in measurement associated with PCR characteristics. Furthermore, the present invention can be used even with small amounts of DNA and enables precise determination of the size, area, and peak height of each locus, enabling quantitative determination of the proportion of chimerism. Thus, the invention is useful not only for determining overall blood chimerism but also for determining linear chimerism in small cell subpopulations.

Claims

1. A primer panel for the detection of chimerism after hematopoietic stem cell transplantation using quantitative fluorescence polymerase chain reaction (QF-PCR), comprising pairs of forward and reverse primers for the simultaneous detection of the following short tandem repeat (STR) loci: D5S818, D21S11, D7S820, CSF1PO, D2S1338, D3S1358, vWA, D8S1179, D16S539, D1S1677, PentaD, TPOX, TH01, D22S1045, D18S51, FGA, D6S1043, D13S317, D12S391 and PentaE, wherein one of the primers in the pair contains a covalently linked fluorescent dye.

2. The panel according to claim 1, characterized in that it additionally contains pairs of forward and reverse primers for determining the following KTP loci: D2S441, D1S1656, D19S433, D10S1248, D11S4463, D1S1627, D3S4529, D1GATA113, D17S974, D6S1017, D4S2408, D9S2157, D6S474, D2S1776, D18S853, D20S482 and D14S1434.

3. A kit for the determination of chimerism after hematopoietic stem cell transplantation using quantitative fluorescence polymerase chain reaction (QF-PCR), including: a) a panel according to paragraph 1 or according to paragraphs 1 and 2; b) reagents for PCR; c) DNA fragments of a given length as an internal size standard; d) control DNA sample; e) a set of reference fragments of alleles of the listed loci.

4. A method for determining chimerism after hematopoietic stem cell transplantation, including: selection from the donor and the recipient before transplantation and from the recipient after transplantation of samples of biological material containing the nuclear cell component of the blood; DNA extraction from each selected sample of biological material; carrying out amplification using quantitative fluorescence PCR (QF-PCR) of short tandem repeat (STR) loci by bringing the isolated DNA from each selected sample of biological material into contact with the components of the kit according to paragraph 3; conducting a fragment analysis of all obtained amplicons using capillary electrophoresis in acrylamide gel, with the determination of the CTP loci containing unique alleles for both the donor and recipient; determination of donor chimerism for a DNA sample from a recipient after transplantation at selected loci, calculated for each locus of the CTP; averaging the obtained chimerism values ​​for all KTP loci.

5. The method according to paragraph 4, characterized in that the proportion of the donor unique allele in the recipient after transplantation for each locus is determined by the magnitude of the peak on the chromatogram, measured in relative fluorescent units, divided by the sum of the fluorescence intensities of all peaks of the locus, expressed in relative fluorescent units.

6. The method according to paragraph 4, characterized in that the genomic DNA of the recipient and the donor is isolated from biological material, which is whole blood, a bone marrow biopsy, or leukocyte mass.

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