Pediatric Hematology/Oncology and ImmunopathologyPediatric Hematology/Oncology and ImmunopathologyВопросы гематологии/онкологии и иммунопатологии в педиатрии1726-17082414-9314Fund Doctors, Innovations, Science for Children34810.24287/1726-1708-2020-19-2-141-151ORIGINAL ARTICLESОРИГИНАЛЬНЫЕ СТАТЬИDevelopment of flow cytometry assay for Wiskott–Aldrich syndrome diagnosis by WASP protein evaluationРазработка метода диагностики синдрома Вискотта–Олдрича путем оценки экспрессии белка WASP с использованием проточной цитофлуориметрииhttps://orcid.org/0000-0002-6148-7209PershinD. E.ПершинД. Е.
Russian Federation

Junior Researcher, Doctor of Medical Laboratory, Laboratory of Hematopoietic stem cell transplantation and Immunotherapy,

Russia, 117997, Moscow, Samory Mashela st., 1

младший научный сотрудник, врач клинической лабораторной диагностики лаборатории трансплантационной иммунологии и иммунотерапии гемобластозов,

117997, Москва, ГСП-7, ул. Саморы Машела, 1

dimprsh@icloud.comhttps://orcid.org/0000-0002-2874-0014LodoevaO. B.ЛодоеваО. Б.
Russian Federation
https://orcid.org/0000-0002-6553-2505FadeevaM. S.ФадееваМ. С.
Russian Federation
https://orcid.org/0000-0003-0471-2956MersiyanovaI. V.МерсияноваИ. В.
Russian Federation
https://orcid.org/0000-0002-8697-4206KhorevaA. L.ХореваА. Л.
Russian Federation
https://orcid.org/0000-0003-1435-665XVladimirovI. S.ВладимировИ. С.
Russian Federation
https://orcid.org/0000-0001-7578-9657VoroninK. A.ВоронинК. А.
Russian Federation
https://orcid.org/0000-0003-0079-7761BrilliantovaV. V.БриллиантоваВ. В.
Russian Federation
https://orcid.org/0000-0002-0501-8686VarlamovaT. V.ВарламоваТ. В.
Russian Federation
https://orcid.org/0000-0001-7247-4844VedmedskayaV. A.ВедмедскаяВ. А.
Russian Federation
https://orcid.org/0000-0001-9857-4456RodinaYu. A.РодинаЮ. А.
Russian Federation
https://orcid.org/0000-0002-7634-2053RaykinaE. V.РайкинаЕ. В.
Russian Federation
https://orcid.org/0000-0003-1735-0093MaschanM. A.МасчанМ. А.
Russian Federation
https://orcid.org/0000-0002-3113-4939ShcherbinaA. Yu.ЩербинаА. Ю.
Russian Federation
Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, Immunology Ministry of Healthcare of Russian FederationФГБУ «Национальный медицинский исследовательский центр детской гематологии, онкологии и иммунологии им. Дмитрия Рогачева» Минздрава России020720201921411510107202001072020Copyright ©; 2020, «D. Rogachev NMRCPHOI»Copyright ©; 2020, ФГБУ «НМИЦ ДГОИ им. Дмитрия Рогачева» Минздрава России2020«D. Rogachev NMRCPHOI»ФГБУ «НМИЦ ДГОИ им. Дмитрия Рогачева» Минздрава Россииhttps://creativecommons.org/licenses/by/4.0https://hemoncim.com/jour/article/view/348

Wiskott–Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by microplatelet thrombocytopenia, eczema, frequent infections and an increased risk of autoimmune disorders and malignant neoplasms. Mutation detection in WAS gene is the gold standard for diagnosis of this disorder. This gene encodes a WASP protein, which works as regulator of cell cytoskeleton and is involved in the transmission of many intracellular signals. Nowadays there is no rapid and reliable method that allows to confirm WAS in a short period of time. Early detection of WAS in patients enables initiation of a donor search and preparation for the HSCT procedure. It also helps to avoid the development of severe and life-threatening conditions during waiting for genetic confirmation of the diagnosis by using pathogenetic therapy. Currently flow cytometry is one of the leading laboratory methods that permits to get the information about the expression of a protein in several hours. The study below describes rapid and reliable based on flow cytometry assay for WAS diagnosis. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. The study included 46 patients with suspected WAS from 2 months to 17 years old. Patients were examined from January 2018 to January 2020. WAS gene defect was confirmed in 35 patients. It was calculated that normal threshold value for WASP expression is 7.07 with sensitivity and specificity 100% and 93.1% respectively. Besides negative correlation between WASP expression index and WAS clinical severity was shown (r = –0.63). This flow cytometry assay can be used for chimerism detection in WAS patients after HSCT. The flow cytometry assay for WASP protein evaluation is rapid, highly sensitive and highly specific. It allows to speed up diagnosis of this disorder.

Синдром Вискотта–Олдрича (СВО) является редким X-сцепленным первичным иммунодефицитом, характеризующимся микротромбоцитопенией, экземой, частыми инфекционными эпизодами, повышенным риском аутоиммунных проявлений и злокачественных новообразований. «Золотым стандартом» диагностики этого заболевания является выявление мутации в гене WAS. Данный ген кодирует белок WASP, являющийся регулятором цитоскелета и участвующий в передаче множества внутриклеточных сигналов. В настоящий момент отсутствует быстрый и надежный метод, позволяющий в кратчайшие сроки установить диагноз СВО. Ранняя диагностика у пациентов с подозрением на СВО позволяет не только своевременно инициировать поиск донора и начать подготовку к трансплантации гемопоэтических стволовых клеток (ТГСК), но также избежать развития тяжелых, жизнеугрожающих состояний в ожидании генетического подтверждения диагноза путем использования патогенетической терапии. На сегодняшний день проточная цитофлуориметрия является одним из ведущих лабораторных методов, позволяющих в течение нескольких часов получить информацию об экспрессии в клетках того или иного белка. В данной работе описана разработка надежной и быстрой лабораторной методики для диагностики СВО, основанной на проточной цитофлуориметрии. Данное исследование одобрено независимым этическим комитетом и утверждено решением ученого совета ФГБУ «НМИЦ ДГОИ им. Дмитрия Рогачева» Минздрава России. В исследование были включены 46 пациентов с подозрением на СВО в возрасте от 2 месяцев до 17 лет, обследованных в период с января 2018 г. по январь 2020 г. Из них у 35 человек диагноз был подтвержден выявлением дефекта гена WAS. В данной работе было рассчитано нормальное пороговое значение индекса экспрессии WASP, составляющее 7,07, показано, что чувствительность и специфичность данного метода в диагностике СВО составляют 100% и 93,1% соответственно, отмечена отрицательная корреляция тяжести СВО и индекса экспрессии белка WASP (r = –0,63). Также показана практическая значимость метода для определения линейного химеризма у пациентов с СВО после ТГСК. Метод оценки внутриклеточной экспрессии белка WASP с помощью проточной цитофлуориметрии у пациентов с подозрением на СВО является быстрым, высокочувствительным и высокоспецифичным, что позволяет максимально ускорить диагностику этого заболевания.

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