Publications by authors named "Galina A Novichkova"

14 Publications

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Healthy pediatric platelets are moderately hyporeactive in comparison with adults' platelets.

Platelets 2021 Nov 9:1-8. Epub 2021 Nov 9.

National Medical Research Center of Pediatric Hematology, Oncology and Immunology Named after Dmitry Rogachev, Russian Ministry of Healthcare, Cellular Hemostasis and Thrombosis Lab, Moscow, Russian Federation.

Studies on platelet function in children older than neonatal period are few and their results are controversial. The pediatric platelets were alternatively reported to be more active or less active than adults' ones. We compared platelet function in the several age groups of children to adults and evaluated the age when platelet function reaches the adults' status. The study included 76 healthy children and 49 healthy adult volunteers. Types of platelet activation used included: collagen-related peptide (CRP) and PAR-1 activating peptide SFLLRN; SFLLRN, PAR-4 activating peptide AYPGKF and adenosine diphosphate (ADP); ADP. The parameters determined included forward (FSC) and side scatter (SSC), CD42b, CD61, CD62P, PAC-1, annexin V binding and mepacrine release levels. Resting pediatric platelets were similar to adults' platelets except for 1.2-fold decreased FSC and dense granules volume in youngest children, and 2.5-fold increased annexin V level in children aged 1-10 years. After CRP+SFLLRN stimulation, pediatric platelets had a 1.2-fold lower alpha- and 1.1-fold lower dense granule release than adults. For SFLLRN+AYPGKF+ADP stimulation, this was observed only for youngest children. The response to ADP stimulation was identical for pediatric platelets and adults. Pediatric platelets have lower granular release than adults' platelets, which persists until the age of 18.
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http://dx.doi.org/10.1080/09537104.2021.1981848DOI Listing
November 2021

Unique Combination of Diamond-Blackfan Anemia and Lynch Syndrome in Adult Female: A Case Report.

Front Oncol 2021 16;11:652696. Epub 2021 Apr 16.

Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia.

We present an extremely rare clinical case of a 38-year-old Russian patient with multiple malignant neoplasms of the uterus and colon caused by genetically confirmed two hereditary diseases: Diamond-Blackfan anemia and Lynch syndrome. Molecular genetic research carried out by various methods (NGS, Sanger sequencing, aCGH, and MLPA) revealed a pathogenic nonsense variant in the gene: NM_000179.2: c.742C>T, p.(Arg248Ter), as well as a new deletion of the chromosome 15's locus with the capture of 82,662,932-84,816,747 bp interval, including the complete sequence of the gene. The lack of expediency of studying microsatellite instability in endometrial tumors using standard mononucleotide markers NR21, NR24, NR27, BAT25, BAT26 was demonstrated. The estimated prevalence of patients with combination of Diamond-Blackfan anemia and Lynch syndrome in the world is one per 480 million people.
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http://dx.doi.org/10.3389/fonc.2021.652696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085342PMC
April 2021

Platelet function and bleeding at different phases of childhood immune thrombocytopenia.

Sci Rep 2021 04 30;11(1):9401. Epub 2021 Apr 30.

National Medical Research Center of Pediatric Hematology, Oncology and Immunology Named After Dmitry Rogachev, Russian Ministry of Healthcare, 1 Samory Mashela Str, Moscow, Russia, 117997.

Immune thrombocytopenia (ITP) is believed to be associated with platelet function defects. However, their mechanisms are poorly understood, in particular with regard to differences between ITP phases, patient age, and therapy. We investigated platelet function and bleeding in children with either persistent or chronic ITP, with or without romiplostim therapy. The study included 151 children with ITP, of whom 56 had disease duration less than 12 months (grouped together as acute/persistent) and 95 were chronic. Samples of 57 healthy children were used as controls, while 5 patients with leukemia, 5 with aplastic anemia, 4 with MYH9-associated thrombocytopenia, and 7 with Wiskott-Aldrich syndrome were used as non-ITP thrombocytopenia controls. Whole blood flow cytometry revealed that platelets in both acute/persistent and chronic ITP were increased in size compared with healthy donors. They were also pre-activated as assessed by PAC1, CD62p, cytosolic calcium, and procoagulant platelet levels. This pattern was not observed in other childhood thrombocytopenias. Pre-activation by CD62p was higher in the bleeding group in the chronic ITP cohort only. Romiplostim treatment decreased size and pre-activation of the patient platelets, but not calcium. Our data suggest that increased size, pre-activation, and cytosolic calcium are common for all ITP platelets, but their association with bleeding could depend on the disease phase.
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http://dx.doi.org/10.1038/s41598-021-88900-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087794PMC
April 2021

Primary Immunodeficiencies in Russia: Data From the National Registry.

Front Immunol 2020 6;11:1491. Epub 2020 Aug 6.

Tatarstan Pediatric Republican Clinical Hospital, Kazan, Russia.

Primary immunodeficiencies (PID) are a group of rare genetic disorders with a multitude of clinical symptoms. Characterization of epidemiological and clinical data via national registries has proven to be a valuable tool of studying these diseases. The Russian PID registry was set up in 2017, by the National Association of Experts in PID (NAEPID). It is a secure, internet-based database that includes detailed clinical, laboratory, and therapeutic data on PID patients of all ages. The registry contained information on 2,728 patients (60% males, 40% females), from all Federal Districts of the Russian Federation. 1,851/2,728 (68%) were alive, 1,426/1,851 (77%) were children and 425/1,851 (23%) were adults. PID was diagnosed before the age of 18 in 2,192 patients (88%). Antibody defects (699; 26%) and syndromic PID (591; 22%) were the most common groups of PID. The minimum overall PID prevalence in the Russian population was 1.3:100,000 people; the estimated PID birth rate is 5.7 per 100,000 live births. The number of newly diagnosed patients per year increased dramatically, reaching the maximum of 331 patients in 2018. The overall mortality rate was 9.8%. Genetic testing has been performed in 1,740 patients and genetic defects were identified in 1,344 of them (77.2%). The median diagnostic delay was 2 years; this varied from 4 months to 11 years, depending on the PID category. The shortest time to diagnosis was noted in the combined PIDs-in WAS, DGS, and CGD. The longest delay was observed in AT, NBS, and in the most prevalent adult PID: HAE and CVID. Of the patients, 1,622 had symptomatic treatment information: 843 (52%) received IG treatment, mainly IVIG (96%), and 414 (25%) patients were treated with biological drugs. HSCT has been performed in 342/2,728 (16%) patients, of whom 67% are currently alive, 17% deceased, and 16% lost to follow-up. Three patients underwent gene therapy for WAS; all are currently alive. Here, we describe our first analysis of the epidemiological features of PID in Russia, allowing us to highlight the main challenges around PID diagnosis and treatment.
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http://dx.doi.org/10.3389/fimmu.2020.01491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424007PMC
May 2021

Platelet function and bleeding in chronic lymphocytic leukemia and mantle cell lymphoma patients on ibrutinib.

J Thromb Haemost 2020 10;18(10):2672-2684

City Clinical Hospital named after S.P. Botkin, Moscow, Russia.

Background: Therapy with irreversible Bruton's tyrosine kinase inhibitor ibrutinib in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) is associated with bleeding.

Objectives: To propose the predictive markers of such bleeding, as well as mechanisms responsible for decreased bleeding at later therapy stages.

Patients/methods: We investigate platelet functional activity in 50 CLL and 16 MCL patients on ibrutinib using flow cytometry and light transmission aggregometry.

Results: Prior to treatment, both patient groups had decreased platelet counts; impaired aggregation with adenosine diphosphate (ADP); and decreased binding of CD62P, PAC1, and annexin V upon stimulation. Bleeding in patients treated with ibrutinib was observed in 28 (56%) CLL patients, who had decreased aggregation with ADP and platelet count before therapy. Their platelet count on therapy did not change, platelet aggregation with ADP steadily improved, and aggregation with collagen first decreased and then increased in anticorrellation with bleeding. Bleeding in MCL was observed in 10 (62%) patients, who had decreased dense granule release before therapy. ADP and ristocetin induced platelet aggregation in ibrutinib-treated MCL patients increased on therapy, while collagen-induced aggregation evolved similarly to CLL patients.

Conclusions: Our results suggest that ibrutinib-dependent bleeding in CLL patients involves three mechanisms: decreased platelet count (the most important discriminator between bleeding and non-bleeding patients), impaired platelet response to ADP caused by CLL, and inhibition by ibrutinib. Initially, ibrutinib shifts the balance to bleeding, but then it is restored because of the improved response to ADP.
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http://dx.doi.org/10.1111/jth.14943DOI Listing
October 2020

Heterogeneity of Integrin αβ Function in Pediatric Immune Thrombocytopenia Revealed by Continuous Flow Cytometry Analysis.

Int J Mol Sci 2020 Apr 25;21(9). Epub 2020 Apr 25.

National Medical Research Centеr of Pediatric Hematology, Oncology and Immunology named after Dmitry Rogachev, 1 Samory Mashela St, Moscow 117198, Russia.

Immune thrombocytopenia (ITP) is an autoimmune condition primarily induced by the loss of immune tolerance to the platelet glycoproteins. Here we develop a novel flow cytometry approach to analyze integrin αβ functioning in ITP in comparison with Glanzmann thrombasthenia (GT) (negative control) and healthy pediatric donors (positive control). Continuous flow cytometry of Fura-Red-loaded platelets from whole hirudinated blood was used for the characterization of platelet responses to conventional activators. Calcium levels and fibrinogen binding were normalized to ionomycin-induced responses. Ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Platelets from all ITP patients had significantly higher cytosolic calcium concentration in the quiescent state compared to healthy donors (15 ± 5 nM vs. 8 ± 5 nM), but calcium increases in response to all activators were normal. Clustering analysis revealed two subpopulations of ITP patients: the subgroup with high fibrinogen binding (HFB), and the subgroup with low fibrinogen binding (LFB) (8% ± 5% for LFB vs. 16% ± 3% for healthy donors in response to ADP). GT platelets had calcium mobilization (81 ± 23 nM), fibrinogen binding (5.1% ± 0.3%) and thrombus growth comparable to the LFB subgroup. Computational modeling suggested phospholipase C-dependent platelet pre-activation for the HFB subgroup and lower levels of functional integrin molecules for the LFB group.
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http://dx.doi.org/10.3390/ijms21093035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7246588PMC
April 2020

Platelet function and blood coagulation system status in childhood essential thrombocythemia.

Platelets 2020 Nov 19;31(8):1001-1011. Epub 2019 Dec 19.

Oncology and Immunology, Federal Research and Clinical Centre of Pediatric Hematology , Moscow, Russian Federation, Russia.

Childhood essential thrombocythemia (ET) is a rare chronic myeloproliferative disorder. The quality of life of ET patients may decrease as a result of ischemic and hemorrhagic complications of unclear origin. Our goal was to characterize the hemostatic system in children with ET. We genotyped and investigated blood samples from 20 children with ET in a prospective case series study using platelet aggregation, functional flow cytometry (FC) assay and standard clotting assays. Three children had a mutation, 4 had mutations in and 13 were triple-negative. Myelofibrosis in stage 1-2 was detected in 3 children. Three patients had bleeding episodes and seven had ischemic events. Aggregation in response to collagen, adenosine diphosphate, and ristomycin was decreased in all patients. In FC, significant changes in the whole patient group compared to the healthy children control group were decrease in the resting forward scatter and PAC1 binding (activated GPIIb/IIIa) level. For the activated platelets, dense granules release (by mepacrine), PAC1, and GPIIb/IIIa levels were significantly decreased. GPIb/V/IX, -selectin, and phosphatidylserine levels manifested only moderate differences. Forward and side scatter changes in response to stimulation (representing shape change) and dense granules release were significantly lower in the 3 patients with bleeding than in the 17 patients without hemorrhage. Activated partial thromboplastin time was slightly prolonged, prothrombin index was slightly shortened and thrombin time was normal, while fibrinogen was mildly decreased in the ET patients. It could be concluded that the observed platelet function defects could be related to bleeding in ET, and be potentially used as a marker.
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http://dx.doi.org/10.1080/09537104.2019.1704710DOI Listing
November 2020

Mechanisms of increased mitochondria-dependent necrosis in Wiskott-Aldrich syndrome platelets.

Haematologica 2020 04 5;105(4):1095-1106. Epub 2019 Jul 5.

National Scientific and Practical Center of Pediatric Hematology, Oncology and Immunology named after Dmitry Rogachev, Moscow

Wiskott-Aldrich syndrome (WAS) is associated with thrombocytopenia of unclear origin. We investigated real-time cytosolic calcium dynamics, mitochondrial membrane potential and phoszphatidylserine (PS) exposure in single fibrinogen-bound platelets using confocal microscopy. The WAS platelets had higher resting calcium levels, more frequent spikes, and their mitochondria more frequently lost membrane potential followed by PS exposure (in 22.9% of platelets 3.9% in controls; <0.001) after the collapse of the last mitochondria. This phenomenon was inhibited by the mitochondrial permeability transition pore inhibitor cyclosporine A, as well by xestospongin C and lack of extracellular calcium. Thapsigargin by itself caused accelerated cell death in the WAS platelets. The number of mitochondria was predictive of PS exposure: 33% of platelets from WAS patients with fewer than five mitochondria exposed PS, while only 12% did among those that had five or more mitochondria. Interestingly, healthy donor platelets with fewer mitochondria also more readily became procoagulant upon PAR1/PAR4 stimulation. Collapse of single mitochondria led to greater cytosolic calcium increase in WAS platelets if they had one to three mitochondria compared with platelets containing higher numbers. A computer systems biology model of platelet calcium homeostasis showed that smaller platelets with fewer mitochondria could have impaired calcium homeostasis because of higher surface-to-volume ratio and greater metabolic load, respectively. There was a correlation (C=0.81, <0.02) between the mean platelet size and platelet count in the WAS patients. We conclude that WAS platelets readily expose PS via a mitochondria-dependent necrotic mechanism caused by their smaller size, which could contribute to the development of thrombocytopenia.
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http://dx.doi.org/10.3324/haematol.2018.214460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7109739PMC
April 2020

Thrombopoietin receptor agonist switch in children with persistent and chronic severe immune thrombocytopenia: A retrospective analysis in a large tertiary center.

Pediatr Blood Cancer 2019 06 11;66(6):e27704. Epub 2019 Mar 11.

Dmitri Rogachev National Research Center for Pediatric Hematology, Oncology and Immunology, Moskva, Russia.

We retrospectively analyzed sequential therapy with romiplostim and eltrombopag in 23 children with immune thrombocytopenia: switching from romiplostim to eltrombopag (10 patients) or vice versa (13 patients). The median age of patients at enrollment in the study was 5.6 years (2-15 years). Switching from romiplostim to eltrombopag was effective in eight (80%) patients, whereas switching from eltrombopag to romiplostim was effective in eight (62%) patients. The response rate was similar in patients failing the first thrombopoietin receptor agonist and those who had previous response. To date, all responders continue to maintain platelets over 50 × 10 /L at 13-39 months after switching.
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http://dx.doi.org/10.1002/pbc.27704DOI Listing
June 2019

Flow cytometry for pediatric platelets.

Platelets 2019 4;30(4):428-437. Epub 2018 Oct 4.

a Cellular Hemostasis and Thrombosis Lab , National Medical Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitry Rogachev, Russian Ministry of Healthcare , Moscow , Russian Federation.

The ability of platelets to carry out their hemostatic function can be impaired in a wide range of inherited and acquired conditions: trauma, surgery, inflammation, pre-term birth, sepsis, hematological malignancies, solid tumors, chemotherapy, autoimmune disorders, and many others. Evaluation of this impairment is vitally important for research and clinical purposes. This problem is particularly pronounced in pediatric patients, where these conditions occur frequently, while blood volume and the choice of blood collection methods could be limited. Here we describe a simple flow cytometry-based screening method of comprehensive whole blood platelet function testing that was validated for a range of pediatric and adult samples (n = 31) in the hematology hospital setting including but not limited to: classic inherited platelet function disorders (Glanzmann's thrombasthenia; Bernard-Soulier, Wiscott-Aldrich, and Hermasky-Pudlak syndromes, MYH9-dependent thrombocytopenia), healthy and pre-term newborns, acute and chronic immune thrombocytopenia, chronic lympholeukemia, effects of therapy on platelet function, etc. The method output includes levels of forward and side scatter, levels of major adhesion and aggregation glycoproteins Ib and IIb-IIIa, active integrins' level based on PAC-1 binding, major alpha-granule component P-selectin, dense granule function based on mepacrine uptake and release, and procoagulant activity quantified as a percentage of annexin V-positive platelets. This analysis is performed for both resting and dual-agonist-stimulated platelets. Preanalytical and analytical variables are provided and discussed. Parameter distribution within the healthy donor population for adults (n = 72) and children (n = 17) is analyzed.
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http://dx.doi.org/10.1080/09537104.2018.1513473DOI Listing
May 2019

Bleeding tendency and platelet function during treatment with romiplostim in children with severe immune thrombocytopenic purpura.

Int J Hematol 2017 Jun 7;105(6):841-848. Epub 2017 Mar 7.

National Scientific and Practical Center of Pediatric Hematology, Oncology and Immunology, 117198, Moscow, Russia.

It has been suggested that platelet function in chronic immune thrombocytopenic purpura (ITP) may be abnormal. Thrombopoietin mimetics used for treatment can affect it, but the data remain limited. We investigated platelet function of 20 children diagnosed with severe ITP (aged 1-16 years, 12 females and eight males). Platelet functional activity in whole blood was characterized by flow cytometry before and after stimulation with SFLLRN plus collagen-related peptide. Levels of CD42b, PAC1, and CD62P, but not CD61 or annexin V, were significantly increased (P < 0.05) in resting platelets of patients before treatment compared with healthy donors. On average, PAC1 and CD62P in patients after activation were also significantly elevated, although some patients failed to activate integrins. Romiplostim (1-15 μg/kg/week s.c.) was prescribed to seven patients, with clinical improvement in six. Interestingly, one patient had clinical improvement without platelet count increase. Eltrombopag (25-75 mg/day p.o.) was given to four patients, with positive response in one. Others switched to romiplostim, with one stable positive response, one unstable positive response, and one non-responding. Platelet quality improved with romiplostim treatment, and their parameters approached the normal values. Our results suggest that platelets in children with severe ITP are pre-activated and abnormal, but improve with treatment.
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http://dx.doi.org/10.1007/s12185-017-2207-3DOI Listing
June 2017

Clinical and genomic heterogeneity of Diamond Blackfan anemia in the Russian Federation.

Pediatr Blood Cancer 2015 Sep 6;62(9):1597-600. Epub 2015 May 6.

Dmitry Rogachev Federal Clinical Research Center for Pediatric Hematology, Oncology and Immunology, Moscow, Russia.

Background: Diamond Blackfan anemia (DBA) is a genetically and clinically heterogeneous ribosomopathy and inherited bone marrow failure syndrome characterized by anemia, reticulocytopenia, and decreased erythroid precursors in the bone marrow with an increased risk of malignancy and, in approximately 50%, physical abnormalities.

Methods: We retrospectively analyzed clinical data from 77 patients with DBA born in the Russian Federation from 1993 to 2014. In 74 families there was one clinically affected individual; in only three instances a multiplex family was identified. Genomic DNA from 57 DBA patients and their first-degree relatives was sequenced for mutations in RPS19, RPS10, RPS24, RPS26, RPS7, RPS17, RPL5, RPL11, RPL35a, and GATA1.

Results: Severe anemia presented before 8 months of age in all 77 patients; before 2 months in 61 (78.2%); before 4 months in 71 (92.2%). Corticosteroid therapy was initiated after 1 year of age in the majority of patients. Most responded initially to steroids, while 5 responses were transient. Mutations in RP genes were detected in 35 of 57 patients studied: 15 in RPS19, 6 in RPL5, 3 in RPS7, 3 each in RPS10, RPS26, and RPL11 and 1 each in RPS24 and RPL35a; 24 of these mutations have not been previously reported. One patient had a balanced chromosomal translocation involving RPS19. No mutations in GATA1 were found.

Conclusion: In our cohort from an ethnically diverse population the distribution of mutations among RP genes was approximately the same as was reported by others, although within genotypes most of the mutations had not been previously reported.
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http://dx.doi.org/10.1002/pbc.25534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4515145PMC
September 2015

Spread of vancomycin-resistant Enterococcus faecium in two haematological centres in Russia.

Int J Antimicrob Agents 2010 Feb 14;35(2):177-81. Epub 2009 Dec 14.

National Research Center for Hematology, Moscow, Russia.

This paper describes the clonal diversity of vancomycin-resistant Enterococcus faecium isolated from patients with haematological malignancies in Russia. Pulsed-field gel electrophoresis (PFGE) typing of 129 vanA-positive E. faecium strains revealed 23 independent restriction profiles with two predominant clonal types. Multilocus sequence typing (MLST) of 16 strains selected from two predominant PFGE types showed that they belong to the epidemic clonal complex (CC) 17. Tn1546-like elements of isolates were compared with the prototype element from E. faecium BM4147 by polymerase chain reaction (PCR). Four different Tn1546 types were distinguished according to structural alternations. Polymorphism in the orf1 and vanSH genes was detected. However, a significant prevalence of the prototype Tn1546 was revealed. Tn1546-like elements with the same structures were observed in strains of different PFGE types. The virulence genes esp, gelE and hyl were detected by PCR in 118 isolates (91%), 87 isolates (67%) and 35 isolates (27%), respectively. In contrast, agg and cylA genes were not found. The detection frequency of esp was higher in epidemic strains than in sporadic ones (100% vs. 56%; P<0.05). This study describes a genetically variable population of vancomycin-resistant E. faecium in two Russian haematological centres. The spread of vancomycin resistance was mostly due to the distribution of the two subclones of E. faecium CC17, enriched with the virulence marker esp. At the same time, dissemination of an altered Tn1546 also occurred.
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http://dx.doi.org/10.1016/j.ijantimicag.2009.10.006DOI Listing
February 2010
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