Comparison of polymerase chain reaction and flow cytometry for measuring telomere length of human leukocytes.

Telomere length can be measured by polymerase chain reaction (PCR), allowing to obtain the absolute length of telomeres (ALT) in base pair, and by flow cytometry, which can only estimate the relative telomere length. The aim of the study was to compare the results of the two methods and to develop an accurate and reliable way of converting the relative telomere length to absolute. The peripheral blood from 21 donors was analyzed. Measurement of leukocyte telomere length by flow cytometry was carried out using a commercial Telomere PNA Kit / FITC (Dako, Denmark) with two CytoFLEX flow cytometers (Beckman Coulter, China) and BD FACSCanto II (Becton Dickinson, USA), obtaining the molecular equivalent of fluorescence (MEF). To measure telomere length by real-time PCR, calibrators with a known number of telomeric repeats were prepared. Two quantitative PCRs were carried out: one for telomeric repeats, the other for determining the number of genome-equivalents of DNA, three times for each sample, which made it possible to calculate ALT. A strong direct relationship was found between the MEF obtained with BD FACSCanto II and CytoFLEX (r = 0.97). Analysis of PCR and flow cytometry results showed a significant correlation between ALT and MEF. We calculated the regression equations of ALT and MEF for CytoFLEX - y = 0.0043x (r = 0.84) and for BD FACSCanto II - y = 0.0051x (r = 0.82). Correlation analysis showed a high comparability of telomere lengths measured by two methods. The obtained regression equations allow converting the results of flow cytometry into absolute values, allowing the comparison of the results of different research groups and the use of this method in clinical trials.

Bone Marrow Multipotent Mesenchymal Stromal Cells in Patients with Diffuse Large B-Cell Lymphoma.

In diffuse large B-cell lymphoma, bone marrow involvement is rarely diagnosed. We compared the properties of bone marrow stromal progenitor cells and the concentration of fibroblast CFU in patients with diffuse large B-cell lymphoma without bone marrow involvement and in healthy donors. It was found that the properties of multipotent mesenchymal stromal cells in patients in the debut of the disease differed considerably from those in healthy donors. In particular, the total cell production in patients was significantly higher than in donors. In multipotent mesenchymal stromal cells of patients, some cell parameters were changes; the mean fluorescence intensity of the adhesion molecule ICAM1 on the cell surface was increased. The mean fluorescence intensity of mesenchymal stromal cell markers (HLA-ABC, CD73 and CD90) was significantly elevated. The relative expression of BMP4, MMP2, FGFR1, and ICAM1 genes in mesenchymal stromal cell was reduced, while the expression of FGFR2 gene was enhanced. Despite the absence of proven involvement of the bone marrow, the properties of mesenchymal stromal cells, the components in the stromal microenvironment niche regulating hemopoiesis are altered in patients with diffuse large B-cell lymphoma.

Individual Differences of Multipotent Mesenchymal Stromal Cells Manifesting in during Interaction with Lymphocytes.

Analysis of changes in lymphocyte subpopulations during co-culturing with multipotent mesenchymal stromal cells (MSC) revealed two distinct MSC groups: one group (A) increased HLA-DR expression on lymphocytes during co-culturing and the other (B) did not change it in comparison with lymphocyte monoculture. In stromal cells interacting with lymphocytes, expression of HLA-DR molecules was initiated, but only in samples that induced enhanced expression on lymphocytes and irrespectively of whether allogeneic or autologous lymphocytes were used for co-culturing with MSC. In group A, the relative expression of IDO1 significantly increased in comparison with group B. The revealed individual differences in MSC can explain why not all MSC samples are effective in the treatment of autoimmune diseases, acute "graft-versus-host" disease, and other pathologies.

Co-Culturing of Multipotent Mesenchymal Stromal Cells with Autological and Allogenic Lymphocytes.

We studied the effect of autologous and allogeneic lymphocytes on multipotent mesenchymal stromal cells in co-culture. It is shown that changes in multipotent mesenchymal stromal cells and in lymphocytes did not depend on the source of lymphocytes. Contact with lymphocytes triggers expression of HLA-DR molecules on multipotent mesenchymal stromal cells and these cells lose their immune privilege. In multipotent mesenchymal stromal cells, the relative level of expression of factors involved in immunomodulation (IDO1, PTGES, and IL-6) and expression of adhesion molecule ICAM1 increased, while expression of genes involved in the differentiation of multipotent mesenchymal stromal cells remained unchanged. Priming of multipotent mesenchymal stromal cells with IFN did not affect these changes. In turn, lymphocytes underwent activation, expression of HLA-DR increased, subpopulation composition of lymphocytes changed towards the increase in the content of naïve T cells. These findings are important for cell therapy.

[Absolute numbers of peripheral blood CD34+ hematopoietic stem cells prior to a leukapheresis procedure as a parameter predicting the efficiency of stem cell collection]. / Absoliutnoe kolichestvo gemopoéticheskikh stvolovykh kletok CD34+ v perifericheskoi krovi pered protseduroi leikafereza kak parametr, prognoziruiushchii éffektivnost' sbora stvolovykh kletok.

AIM: To identify a parameter predicting a collection of at least 2·106 CD34+ hematopoietic stem cells (HSC)/kg body weight per leukapheresis (LA) procedure. SUBJECTS AND METHODS: The investigation included 189 patients with hematological malignancies and 3 HSC donors, who underwent mobilization of stem cells with their subsequent collection by LA. Absolute numbers of peripheral blood leukocytes and CD34+ cells before a LA procedure, as well as a number of CD34+ cells/kg body weight (BW) in the LA product stored on the same day were determined in each patient (donor). RESULTS: There was no correlation between the number of leukocytes and that of stored CD34+ cells/kg BW. There was a close correlation between the count of peripheral blood CD34+ cells prior to LA and that of collected CD34+ cells calculated with reference to kg BW. CONCLUSION: The optimal absolute blood CD34+ cell count was estimated to 20 per µl, at which a LA procedure makes it possible to collect 2·106 or more CD34+ cells/kg BW.

[Multiple myeloma: Maintenance therapy after autologous hematopoietic stem cell transplantation, depending on minimal residual disease]. / Mnozhestvennaia mieloma: podderzhivaiushchaia terapiia posle transplantatsii autologichnykh gemopoéticheskikh stvolovykh kletok v zavisimosti ot minimal'noi ostatochnoi bolezni.

AIM: To determine the efficiency of maintenance therapy with bortezomib in patients with multiple myeloma (MM) who have achieved complete remission (CR) after autologous hematopoietic stem cell (auto-HSCT), depending on the presence of minimal residual disease (MRD). SUBJECTS AND METHODS: In January 2014 to February 2016, fifty-two MM patients (19 men and 33 women) aged 24 to 66 years (median 54 years), who had achieved CR after auto-HSCT, were randomized to perform maintenance therapy with bortezomib during a year. On day 100 after auto-HSCT, all the patients underwent immunophenotyping of bone marrow plasma cells by 6-color flow cytometry to detect MRD. Relapse-free survival (RFS) was chosen as a criterion for evaluating the efficiency of maintenance therapy. RESULTS: After auto-HSCT, MRD-negative patients had a statistically significantly higher 2-year RFS rate than MRD-positive patients: 52.9% (95% confidence interval (CI), 35.5 to 70.5%) versus 37.2% (95% CI, 25.4 to 49.3%) (p=0.05). The presence of MRD statistically significantly increased the risk of relapse (odds ratio 1.7; 95% CI, 1.2 to 3.4; p=0.05). Two-year cumulative risk of relapse (using the Kaplan-Meier) after auto-HSCT did not statistically significantly differ in MRD-negative patients receiving (n=15) and not receiving (n=10) maintenance therapy with bortezomib (p=0.58). After completion of maintenance treatment, 42% of the MRD-positive patients achieved a negative status. In the MRD-positive patients who had received maintenance therapy, the average time to recurrence was 5 months longer than that in the naïve patients: 17.3 versus 12.3 months. CONCLUSION: The MRD status determined in MM patients who have achieved CR after auto-HSCT is an important factor for deciding on the use of maintenance therapy.

Changing the Properties of Multipotent Mesenchymal Stromal Cells by IFNγ Administration.

We studied changes in the population of human multipotent mesenchymal stromal cells activated by IFNγ. The cells were cultured under standard conditions; IFNγ was added in various concentrations for 4 h or over 2 passages. It was shown that the total cell production significantly decreased after long-term culturing with IFNγ, but 4-h exposure did not affect this parameter. After 4-h culturing, the expression levels of IDO1, CSF1, and IL-6 increased by 300, 7, and 2.4 times, respectively, and this increase persisted 1 and 2 days after removal of IFNγ from the culture medium. The expression of class I and II MHC (HLA) on cell surface practically did not change immediately after exposure to IFNγ, but during further culturing, HLA-ABC (MHC I) and HLA-DR (MHC II) expression significantly increased, which abolished the immune privilege in these cells, the property allowing clinical use of allogenic multipotent mesenchymal stromal cells. Multipotent mesenchymal stromal cells can suppress proliferation of lymphocytes. The degree of this suppression depends on individual properties of multipotent mesenchymal stromal cell donor. Treatment with IFNγ did not significantly affect the intensity of inhibition of lymphocyte proliferation by these cells.

[The dynamics of paroxysmal nocturnal hemoglobinuria clone in patients with aplastic anemia in process of immune suppressive therapy].

The implementation of principles of highly sensitive flow cytometry into diagnostic of paroxysmal nocturnal hemoglobinuria increased rate of detection of paroxysmal nocturnal hemoglobinuria clone in patients with aplastic anemia already at early stages of diagnosis establishment (up to 79%). However, detection of paroxysmal nocturnal hemoglobinuria clone attracts interest not only from point of view of progression in % of patients with aplastic anemia). The occurrence of paroxysmal nocturnal hemoglobinuria clone in patients with aplastic anemia can be accompanied by hidden disorders of haemopoesis with increasing risk in conditions of proliferative stress. Hence, it is necessary to monitor the given clone during all period of observation. The study is a prospective investigation analyzing dynamics of paroxysmal nocturnal hemoglobinuria clone in process of immune suppressive therapy applied to 44 patients with aplastic anemia. The mentioned clone was initially detected in 59.6% of patients. The median of observation amounted to 27 (9-48) months. Depending on size of granulocytic paroxysmal nocturnal hemoglobinuria clone patients were allocated in four conditional groups: group I - from 0.01% to 0.99% (n=11); group II - from 1% to 9.99% (n=8); group III - from10% to 49.9% (n=4); group IV - from 50% and more (n=5). In the course of study the differently directed dynamics of paroxysmal nocturnal hemoglobinuria clone was revealed. In 3 out of 11 patients from group I median of paroxysmal nocturnal hemoglobinuria clone increased from minor values (less than 1%) to 3.55%; at that in one patient occurred total elimination of paroxysmal nocturnal hemoglobinuria clone to 12th month of observation. The noticeable unidirectional dynamics was established in patients of group III: already to 3d month of observation, simultaneously with becoming of remission, median of size of paroxysmal nocturnal hemoglobinuria clone in group diminished from 22.9% (18.39%-24.77%) to 5.6% (1.5%-6.7%). Among patients of groups II and IV paroxysmal nocturnal hemoglobinuria clone remained stable. The development of hemolytic form of paroxysmal nocturnal hemoglobinuria was observed in all patients of group IV i.e. in 18% of patients with aplastic anemia with primarily detected paroxysmal nocturnal hemoglobinuria clone. In the process of observation, in 37% of patients with aplastic anemia without primarily detected paroxysmal nocturnal hemoglobinuria clone its occurrence and persistence (median - 0.34% (0.1%-6.2%)) was noticed. According to the results of study, alteration of sizes of paroxysmal nocturnal hemoglobinuria clone or its occurrence develop in case of response to ISP and, most probably, depend on advantage of growth in the process of repair of normal (GPI positive) or clonal (GPI negative) hemopoiesis. To acquire more reliable conclusions will be possible through development of techniques of molecular diagnostic simultaneously with dynamic observation of course of disease in the given patients.