The objective : immunohistochemical and electron microscopic imaging of the SARS-CoV-2 coronavirus in lung, lymph nodes and kidney tissues of patients who died of COVID-19. Subjects and Methods . For immunohistochemical tests, specimens of sections of formalin-fixed and paraffin-embedded tissues of the lungs, lymph nodes and kidneys of patients who died from COVID-19 were used. Quantitative assessment of the SARS-CoV-2 viral load level was carried by the original RT qPCR and calculated by the formula: SARS-CoV-2 copy number/ABL1 х 100 copies, expressed as the ratio of the true number of SARS-CoV-2 cDNA copies per 100 copies cDNA of the ABL1 gene. For morphological tests, samples of native lung, lymph node and kidney tissues were taken. Results . Immunohistochemical and electron microscopic tests revealed particles of the SARS-CoV-2 coronavirus in the cytoplasm of endothelial cells of air-blood barrier of the lungs, the vascular glomerulus of the kidneys, in the cytoplasm of macrophages of the lymph node, and also in cytoplasm of lymphocytes in the lumen of lung capillaries.

Multi-organ failure is one of the common causes of fatal outcome in COVID-19 patients. However, the pathogenetic association of the SARS-CoV-2 viral load (VL) level with fatal dysfunctions of the lungs, liver, kidneys, heart, spleen and brain, as well as with the risk of death in COVID-19 patients remains poorly understood. SARS-CoV-2 VL in the lungs, heart, liver, kidneys, brain, spleen and lymph nodes have been measured by RT qPCR using the following formula: NSARS-CoV−2/NABL1 × 100. Dissemination of SARS-CoV-2 in 30.5% of cases was mono-organ, and in 63.9% of cases, it was multi-organ. The average SARS-CoV-2 VL in the exudative phase of diffuse alveolar damage (DAD) was 60 times higher than in the proliferative phase. The SARS-CoV-2 VL in the lungs ranged from 0 to 250,281 copies. The “pulmonary factors” of SARS-CoV-2 multi-organ dissemination are the high level of SARS-CoV-2 VL (≥4909) and the exudative phase of DAD. The frequency of SARS-CoV-2 dissemination to lymph nodes was 86.9%, heart–56.5%, spleen–52.2%, liver–47.8%, kidney–26%, and brain–13%. We found no link between the SARS-CoV-2 VL level in the liver, kidneys, and heart and the serum level of CPK, LDH, ALP, ALT, AST and Cr of COVID-19 patients. Isolated detection of SARS-CoV-2 RNA in the myocardium of COVID-19 patients who died from heart failure is possible. The pathogenesis of COVID-19-associated multi-organ failure requires further research in a larger cohort of patients.

Lymphopenia is a frequent hematological manifestation, associated with a severe course of COVID-19, with an insufficiently understood pathogenesis. We present molecular genetic immunohistochemical, and electron microscopic data on SARS-CoV-2 dissemination and viral load (VL) in lungs, mediastinum lymph nodes, and the spleen of 36 patients who died from COVID-19. Lymphopenia <1 × 109/L was observed in 23 of 36 (63.8%) patients. In 12 of 36 cases (33%) SARS-CoV-2 was found in lung tissues only with a median VL of 239 copies (range 18–1952) SARS-CoV-2 cDNA per 100 copies of ABL1. Histomorphological changes corresponding to bronchopneumonia and the proliferative phase of DAD were observed in these cases. SARS-CoV-2 dissemination into the lungs, lymph nodes, and spleen was detected in 23 of 36 patients (58.4%) and was associated with the exudative phase of DAD in most of these cases. The median VL in the lungs was 12,116 copies (range 810–250281), lymph nodes—832 copies (range 96–11586), and spleen—71.5 copies (range 0–2899). SARS-CoV-2 in all cases belonged to the 19A strain. A immunohistochemical study revealed SARS-CoV-2 proteins in pneumocytes, alveolar macrophages, and bronchiolar epithelial cells in lung tissue, sinus histiocytes of lymph nodes, as well as cells of the Billroth pulp cords and spleen capsule. SARS-CoV-2 particles were detected by transmission electron microscopy in the cytoplasm of the endothelial cell, macrophages, and lymphocytes. The infection of lymphocytes with SARS-CoV-2 that we discovered for the first time may indicate a possible link between lymphopenia and SARS-CoV-2-mediated cytotoxic effect.

The objective: to perform quantitative analysis of SARS-CoV-2 viral load (VL) levels in lung tissues in deceased patients with COVID-19 and to evaluate its association with the nature of histological changes in the lungs and the duration of stay in ICU till the lethal outcome.Subjects and Methods. Sections of formalin-fixed and paraffin-embedded lung tissues of 36 deceased patients with COVID-19 were used. The SARS-CoV-2 viral load was quantitatively assessed using the original qPCR. VL was calculated using the following formula: copies SARS-CoV-2 / copies ABL1 × 100, expressed as the ratio of the true number of SARS-CoV-2 cDNA copies per 100 copies of ABL1 gene cDNA.Results. In cases with no histological changes typical of diffuse alveolar lung injury (DAI), the detection rate of SARS-CoV-2 RNA and the average level of the SARS-CoV-2 viral load were 62.5% (5 out of 8 observations) and 104.75 (range 0-313) copies of SARS-CoV-2 cDNA per 100 copies of human ABL1 gene cDNA. The average level of the SARS-CoV-2 viral load in the lungs with prevailing histological changes characteristic of the proliferative and exudative phases of DAI differed by 60 times and amounted to 909 (18-2,657) and 54,924 (834-250,281) copies of SARS-CoV-2 cDNA per 100 copies of human ABL1 cDNA, respectively. The average duration of stay in the intensive care unit in the group of patients with exudative and proliferative phases of DAI was 10.64 (1-22) and 8.14 (1-21) bed-days, respectively. The detection rate of the SARS-CoV-2 RNA in patients with diffuse alveolar lung injury was 100%.

JAK2 (Janus kinase 2) V617F, CALR (Calreticulin) exon 9, and MPL (receptor for thrombopoietin) exon 10 mutations are associated with the vast majority of Ph-negative chronic myeloproliferative neoplasms (MPNs). These mutations affect sequential stages of proliferative signal transduction and therefore, after the emergence of one type of mutation, other types should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Our study includes DNA samples from 1958 patients with clinical evidence of MPN, admitted to the National Research Center for Hematology for genetic analysis between 2016 and 2019. In 315 of 1402 cases (22.6%), CALR mutations were detected. In 23 of these 315 cases (7.3%), the JAK2 V617F mutation was found in addition to the CALR mutation. In 16 from 24 (69.6%) cases, with combined CALR and JAK2 mutations, V617F allele burden was lower than 1%. A combination of JAK2 V617F with MPL W515L/K was also observed in 1 out of 1348 cases, only. JAK2 allele burden in this case was also lower than 1%. Additional mutations may coexist over the low background of JAK2 V617F allele. Therefore, in cases of detecting MPNs with a low allelic load JAK2 V617F, it may be advisable to search for other molecular markers, primarily mutations in exon 9 of CALR. The load of the combined mutations measured at different time points may indicate that, at least in some cases, these mutations could be represented by different clones of malignant cells.

Introduction. The pathogenesis of myeloproliferative neoplasms is associated with the chimeric gene BCR-ABL1 or with one of the driver mutations in the genes JAK2, MPL and CALR (Calreticulin). However, the classifi cation of the World Health Organization lists no myeloid neoplasms with more than one driver genetic abnormality. Aim. To search for mutations in the genes JAK2, MPL and CALR in patients with BCR-ABL1-positive chronic myeloid leukemia (CML), as well as to evaluate the kinetics of the discovered mutations during tyrosine kinase inhibitor (TKI) therapy. Materials and methods. mRNA and DNA samples isolated from blood and bone marrow cells of 567 CML patients, who underwent periodic monitoring of the BCR-ABL1 transcript level over the 2012–2019 period were included in the study The BCR-ABL1 transcript level was determined using a highly sensitive quantitative real-time polymerase chain reaction. The mutations JAK2V617F and MPLW515L/K were detected using real-time quantitative allele-specifi c polymerase chain reaction. Mutations in the CALR gene were investigated using fragment analysis followed by Sanger sequencing. Results. The combination of the BCR-ABL1, JAK2 and CALR gene mutations among CML patients receiving TKIs was 1.23 % (7/567). Out of these, the combination of BCR-ABL1 with JAK2V617F and the combination of BCR-ABL1 with CALR gene mutations were detected in 0.88 % (5/567) and 0.35 % (2/567) of cases, respectively. During TKI therapy, in 5 out of 7 patients, the level of BCR-ABL1 reached major molecular response (MR). In 4 of these patients, the therapy was discontinued. These patients are currently in molecular remission. In the remaining 2 patients, major MR was not achieved, despite the use of second-generation TKI preparations. Conclusions. The combination of the BCR-ABL1 chimeric gene with gene mutations Jak2 or CALR was a rare event and amounted to 0.88 and 0.35 % of cases, respectively. The combination of BCR-ABL1 with Jak2V617F and CALR mutations does not always impede the achievement of major MR.

Primary myelofibrosis is a myeloproliferative neoplasm that occurs de novo, characterized by clonal proliferation of stem cells, abnormal expression of cytokines, bone marrow fibrosis, hepatosplenomegaly as a result of extramedullary hematopoiesis, symptoms of tumor intoxication, cachexemia, peripheral blood leukoerythroblastosis, leukemic progression and low survival. Primary myelofibrosis is a chronic incurable disease. The aims of therapy: preventing progression, increasing overall survival, improving quality of life. The choice of therapeutic tactics is limited. Allogenic hematopoietic stem cell transplantation is the only method that gives a chance for a cure. The role of mutations in a number of genes in the early identification of candidates for allogeneic hematopoietic stem cell transplantation is being actively studied. The article describes the clinical case of the detection ofASXL1gene mutations in a patient with prefibrous primary myelofibrosis. The diagnosis was established on the basis of WHO criteria 2016. The examination revealed a mutation ofASXL1. Interferon alfa therapy is carried out, against the background of which clinico-hematological remission has been achieved. Despite the identified mutation, the patient is not a candidate for allogeneic hematopoietic stem cell transplantation. Given the unfavorable prognostic value of theASXL1mutation, the patient is subject to active dynamic observation and aggressive therapeutic tactics when signs of disease progression appear.

Background Frequency of BCR-ABL1 translocation and JAK2V617F or CALRtype-1,2 mutation co-occurrence in chronic myeloid leukemia (CML) patients varies in different populations. According to Soderquist et al., it amounts to 0.4% of cases in USA; Martin-Cabrera et al. – 0.2% in Germany; Lewandowski et al. – 0.7% in Poland. Combination of BCR-ABL1 translocation with CALRtype-1,2 mutations in CML patients during tyrosine kinase inhibitor (TKI) therapy has not been reported yet. Prognostic value of the BCR-ABL1 transcript level kinetics and of JAK2V617F or CALRtype-1,2 mutations allele burden (AB) in Russian CML patients undergoing TKI therapy has not been assessed so far. Aims To evaluate frequency of coexistence and kinetics of the BCR-ABL1 transcript level and AB of JAK2V617Fand CALRtype-1,2 mutations in CML patients undergoing TKI therapy. Materials and Methods mRNA and DNA samples isolated from blood and bone marrow cells of 683 CML patients, who underwent periodic monitoring of the BCR-ABL1 transcript level over the 2012–2020 period were included in the study. The BCR-ABL1 transcript level and AB of JAK2V617F mutation were determined using a highly sensitive (Log-5) quantitative real-time polymerase chain reaction. CALRtype-1,2 mutations were investigated using fragment analysis followed by Sanger sequencing. Results The combination of the BCR-ABL1, JAK2V617F, and CALRtype-1,2 mutations among CML patients receiving TKIs occurred in 8 of 683 cases (1.17%). Out of these, the combination of BCR-ABL1 with JAK2V617F, and the combination of BCR-ABL1 with CALRtype-1,2 mutations were detected in 0.88% (6/683) and 0.29% (2/683) of cases, respectively. During TKI therapy, in 6 out of 8 cases, the level of BCR-ABL1 reached major molecular response (MR). In 4 of these cases, the therapy was discontinued. These patients are currently in molecular remission. In the remaining 2 patients, major MR was not achieved, despite the use of second generation TKIs. Conclusions The combination of the BCR-ABL1 translocation with JAK2V617F or CALRtype-1,2 mutations is a rare event in our cohort of patients and occurred in 0.88 and 0.29% of cases, respectively. The combination of the BCR-ABL1, JAK2V617F, and CALRtype-1,2 mutations may not interfere with the achievement of major MR under TKI treatment. Frequency of BCR-ABL1 translocation and JAK2V617F or CALRtype-1,2 mutation co-occurrence in chronic myeloid leukemia (CML) patients varies in different populations. According to Soderquist et al., it amounts to 0.4% of cases in USA; Martin-Cabrera et al. – 0.2% in Germany; Lewandowski et al. – 0.7% in Poland. Combination of BCR-ABL1 translocation with CALRtype-1,2 mutations in CML patients during tyrosine kinase inhibitor (TKI) therapy has not been reported yet. Prognostic value of the BCR-ABL1 transcript level kinetics and of JAK2V617F or CALRtype-1,2 mutations allele burden (AB) in Russian CML patients undergoing TKI therapy has not been assessed so far. To evaluate frequency of coexistence and kinetics of the BCR-ABL1 transcript level and AB of JAK2V617Fand CALRtype-1,2 mutations in CML patients undergoing TKI therapy. mRNA and DNA samples isolated from blood and bone marrow cells of 683 CML patients, who underwent periodic monitoring of the BCR-ABL1 transcript level over the 2012–2020 period were included in the study. The BCR-ABL1 transcript level and AB of JAK2V617F mutation were determined using a highly sensitive (Log-5) quantitative real-time polymerase chain reaction. CALRtype-1,2 mutations were investigated using fragment analysis followed by Sanger sequencing. The combination of the BCR-ABL1, JAK2V617F, and CALRtype-1,2 mutations among CML patients receiving TKIs occurred in 8 of 683 cases (1.17%). Out of these, the combination of BCR-ABL1 with JAK2V617F, and the combination of BCR-ABL1 with CALRtype-1,2 mutations were detected in 0.88% (6/683) and 0.29% (2/683) of cases, respectively. During TKI therapy, in 6 out of 8 cases, the level of BCR-ABL1 reached major molecular response (MR). In 4 of these cases, the therapy was discontinued. These patients are currently in molecular remission. In the remaining 2 patients, major MR was not achieved, despite the use of second generation TKIs. The combination of the BCR-ABL1 translocation with JAK2V617F or CALRtype-1,2 mutations is a rare event in our cohort of patients and occurred in 0.88 and 0.29% of cases, respectively. The combination of the BCR-ABL1, JAK2V617F, and CALRtype-1,2 mutations may not interfere with the achievement of major MR under TKI treatment.

Introduction . The advent of tyrosine kinase inhibitors (TKIs) in clinical practice drastically improved prognosis in patients with chronic myeloid leukaemia (CML). Adverse events of the TKI therapy and its high financial burden warrant the trend to gradually abandon this treatment. Aim . To assess the results of CML patient monitoring after the withdrawal of TKI therapy. Patients and methods . This prospective study included 98 chronic phase CML patients satisfying the criteria: any receiving of TKI therapy for ≥3 years; deep molecular response (DMR, BCR-ABL ≤ 0.01 % IS) during ≥ 2 years. The withdrawal was followed by quantitative BCR-ABL estimation performed monthly for the first 6 months of the survey, bimonthly for 1 year and every 3 months from the second year onwards. Therapy was resumed at a loss of major molecular response (MMR, BCR-ABL ≥ 0.1 % IS). Results . The MMR loss upon the TKI withdrawal was observed in 48 (49 %) patients. Survival without MMR loss was 52 % past 24 months since withdrawal, with a median of 35 months (23–52). The duration of therapy, MR and the MR depth at the time of withdrawal significantly correlated with a conserved post-therapy MMR. Gender, age, a Sokal risk group, type and line of TKI therapy at withdrawal, and imatinib resistance in history were not observed to significantly impact molecular relapse-free remission. MMR was recovered in all 48 patients with TKI therapy resumed in molecular relapse. In 65 % of the patients, adverse therapy events observed during treatment completely resolved by 6 months of post-therapy monitoring. Musculoskeletal pain (withdrawal syndrome, WS) was reported in 42 % patients in the post-therapeutic period, which did not lead to TKI resumption. The WS development correlated with an elder age and longer therapy prior to withdrawal. Conclusion . Molecular relapse-free survival in CML patients with treatment-free remission (TFR) is comparable to other published evidence. Monitoring safety during TFR is attested by the lack of disease progression and MMR recovery upon TKI resumption in all patients.

Introduction Duration of treatment with tyrosine kinase inhibitors (TKI) is an important favorable prognostic factor for treatment-free remission (TFR) and deep molecular response (DMR) in patients (pts) with chronic myeloid leukemia (CML). Aim To analyze the impact of first-line treatment duration with imatinib and 2G TKI on molecular relapse-free survival (MRFS) in CML pts with sustained DMR. Patients and methods In total, 174 CML pts in chronic phase with ≥3 years treatment duration and sustained DMR (MR4-MR5, BCR-ABL IS <0,01%) discontinued TKIs in the prospective multicenter trial RU-SKI and in the “historical” cohort with TKI cessation. A molecular relapse was defined as a major molecular response (MMR) loss (BCR-ABL IS >0.1%). Median (Me) age of pts was 43 (range 21-86) years; 39% were males. Me TKI treatment duration was 86 months (IQR 57-125 mo), Me DMR duration was 45 (IQR 29-66 mo). Imatinib, 2G TKI in first-line and 2G TKI in second-line were discontinued in 116 (66.7%), 15(8.6%) (11 nilotinib, 3 dasatinib, 1 bosutinib) and 43 (24.7%) (34 nilotinib, 9 dasatinib) pts respectively. Results Me follow-up time after treatment cessation was 33 months (range 1-133). MRFS at 6, 12, 24 and 36 months was 62% (CI: 55-70%), 53% (CI: 45-60%), 50% (CI: 42-57%) and 48% (CI: 40-56%) respectively. Treatment duration and MR4.5 vs MR4 were significant for MRFS both in univariate (p=0.009 and p=0.015) and multivariate analysis (p=0.017 and p=0.037). No difference in 36 months MRFS was observed for imatinib (47% (CI: 42-60%)), first-line 2G TKI (59% (CI: 33-84%) p=0.58) and second-line 2G TKI (47% (CI: 32-63%) p=0.97) groups. However, significantly shorter treatment duration was observed in first-line 2G TKI group (Me=44 mo (IQR 40-52 mo)) vs imatinib (Me=93 mo (IQR 69-127 mo)), p< 0.0001). The 36 months MRFS was 25% (CI: 1-50%) in shortly treated pts from imatinib group (n=21, Me treatment time 48 mo (IQR 42-56 mo)) and 59% (CI: 33-84%) in first-line 2G TKI group (p=0.08). Conclusion The 2G TKI as a first-line seem to be an effective option to reduce the treatment duration before TFR in CML pts although MRFS rate was not significantly improved in our study.

Introduction. The pathogenesis of myeloproliferative neoplasms is associated with the chimeric gene BCR-ABL1 or with one of the driver mutations in the genes JAK2, MPL and CALR (Calreticulin). However, the classifi cation of the World Health Organization lists no myeloid neoplasms with more than one driver genetic abnormality. Aim. To search for mutations in the genes JAK2, MPL and CALR in patients with BCR-ABL1-positive chronic myeloid leukemia (CML), as well as to evaluate the kinetics of the discovered mutations during tyrosine kinase inhibitor (TKI) therapy. Materials and methods. mRNA and DNA samples isolated from blood and bone marrow cells of 567 CML patients, who underwent periodic monitoring of the BCR-ABL1 transcript level over the 2012–2019 period were included in the study The BCR-ABL1 transcript level was determined using a highly sensitive quantitative real-time polymerase chain reaction. The mutations JAK2V617F and MPLW515L/K were detected using real-time quantitative allele-specifi c polymerase chain reaction. Mutations in the CALR gene were investigated using fragment analysis followed by Sanger sequencing. Results. The combination of the BCR-ABL1, JAK2 and CALR gene mutations among CML patients receiving TKIs was 1.23 % (7/567). Out of these, the combination of BCR-ABL1 with JAK2V617F and the combination of BCR-ABL1 with CALR gene mutations were detected in 0.88 % (5/567) and 0.35 % (2/567) of cases, respectively. During TKI therapy, in 5 out of 7 patients, the level of BCR-ABL1 reached major molecular response (MR). In 4 of these patients, the therapy was discontinued. These patients are currently in molecular remission. In the remaining 2 patients, major MR was not achieved, despite the use of second-generation TKI preparations. Conclusions. The combination of the BCR-ABL1 chimeric gene with gene mutations Jak2 or CALR was a rare event and amounted to 0.88 and 0.35 % of cases, respectively. The combination of BCR-ABL1 with Jak2V617F and CALR mutations does not always impede the achievement of major MR.

Background. Ph-negative chronic myeloproliferative neoplasms (MPNs) are characterized by proliferation of one or more myeloid cell lineages and include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Somatic Jak2, MPL and CALR gene mutations are responsible for more than 90% of NPM cases. These mutations affect sequential stages of prolipherative signal transduction and therefore after the emergence of one type of mutation another types basically should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Aim. To evaluate frequencies of MPL and CALR mutations in Jak2 positive MPN cases for Russian cohort of patients. Methods. Archival DNA samples from MPN patients followed up at the National Research Center for Hematology between 2014 and 2019 included into retrospective study. DNAs and RNAs were extracted from blood using reagent kit from Interlabservice (Russia). Jak2 V617F mutation was quantified by real-time PCR kit from Syntol (Russia) according to manufacturers instructions. CALR exon 9 deletions/insertions were analyzed by fragment analysis (sensitivity >= 3%). MPL W515L/K mutations were assessed by in-house allele specific PCR. All cases were tested for phi-negativity using BCR-ABl p210 PCR kit from Interlabservice (Russia). Results. At least one of the mutations was found in 3863 cases. Jak2 V617F mutation - 3385 cases (87.6%); CALR insertion or deletion - 471 case (12.2%); MPLW515L/K mutation - 31 case (0.8%). We have found 28 cases (0.7%) with Jak2 and CALR mutations combined and 3 cases (0.1%) with Jak2 and MPL mutations in the cohort studied. Matched measures were obtained at least twice at different time points during the course of disease for these cases. No cases with simultaneous CALR and MPL mutations were detected. In 23 from 31 (74%) cases with combined mutations Jak2 V617F allele burden was lower than 3%. Among cases with combined mutations 5 were diagnosed with PV, 8 - with ET, 8 - with PMF and 10 with unclassified MPN. No correlations between diagnosis, mutation combination or allele burden were found. Conclusions. Based on the data, obtained on retrospective DNA samples we cannot state whether combined mutations are present in different clones of myeloid cells or in one. Indirectly, the fact that more often mutations in CALR and MPL genes were found in the cases with a low Jak2 V617F allele burden may indicate that additional mutations occur in the "competing" cell clone. Further prospective studies with mutation monitoring over the therapy are required to assess the value of combined mutations for MPN pathogenesis. Disclosures No relevant conflicts of interest to declare.

Thalassemia and qualitative hemoglobinopathy are hereditary disorders of Hb synthesis that lead to change in the Hb conformation or a decrease in the synthesis of structurally normal Hb, and consequently, to erythron pathology. Many variants of Hb are unstable or have altered affinity for oxygen, and, in heterozygous form can be associated with clinical and hematological manifestations (hemolytic anemia, hypochromic microcytic anemia, erythrocytosis). HbD-Punjab [β121 (GH4) Glu → Gln; HBB: C.364G> C] is variant of Hb carrying the amino acid substitution in the 121 position of β-globin chain. In all cases reported so far, patients with HbD-Punjab/β+-thalassemia (IVSI+5 G-C) combination experienced typical thalassemia with hypochromic microcytosis. HbD-Punjab was detected by electrophoresis from 37 to 94% of total Hb. The article describes rare clinical case of the cohabitation of HbD-Punjab/β+-thalassemia (IVSI+5 G-C) in a patient with homozygous variant of Gilbert's syndrome observed in AS Loginov Moscow Clinical Scientific Center.

The detection of somatic mutations in the 9 exon of the calreticulin gene (CALR) is regulated by the clinical recommendations as a diagnostic criterion for chronic Ph-negative myeloproliferative neoplasms (MPN). Some methods of nucleic acids testing are used to identify CALR gene mutations with different requirements for special skills of personnel and expensive equipment. The purpose of this work is to compare the results of the detection of CALR gene mutations in venous blood samples by allele-specific RT-PCR with subsequent electrophoresis, fragment analysis and Sanger- or pyro- sequencing. We used 1284 blood samples of patients with suspected MPN and 20 blood donor samples. Mutations in the CALR gene of the I and II type were identified using PCR-RT with the original primers and TaqMan probes. Also, all samples were tested for mutations in the CALR gene by electrophoretic detection of PCR results in an agarose gel. The use of allele-specific RT-PCR followed by electrophoretic detection made it possible to determine clinically significant mutations in the CALR gene in 81 venous blood samples of JAK2- and MPL-negative patients, including 42 cases of type I mutation, 33 cases of type II mutation and 8 rare CALR mutations. Mutations in the 9 exon of the CALR gene were not detected in any of the 20 blood donor samples or in 121 blood samples of patients with polycythemia vera. In randomly selected 20 negative samples, CALR gene mutations were also not detected using Sanger sequencing. All positive samples were confirmed by fragment analysis, as well as with Sanger- sequencing and pyro- sequencing. The described combined approach to detect mutations of the CALR gene in peripheral blood samples can be used in clinical diagnostic laboratories that have a standard set of equipment for electrophoresis of nucleic acids and a PCR-RT. We also propose a confirmatory test based on the pyrosequencing of DNA using the system of genetic analysis "PyroMark Q24".