Вопросы гематологии/онкологии и иммунопатологии в педиатрии. 2021; 20: 58-65
Особенности пула хранения и морфологии тромбоцитов у детей с неуточненным геморрагическим синдромом
https://doi.org/10.24287/1726-1708-2021-20-1-58-65Аннотация
Несмотря на современные возможности лабораторной диагностики геморрагического синдрома, у некоторых пациентов причины кровоточивости остаются неуточненными. Среди данных причин потенциально могут скрываться легкие дефекты тромбоцитарного звена гемостаза. Цель работы: выявить особенности функции тромбоцитарного звена гемостаза у детей с неуточненным геморрагическим синдромом. Данное исследование одобрено независимым этическим комитетом и утверждено решением ученого совета ФГБУ «НМИЦ ДГОИ им. Дмитрия Рогачева» Минздрава России. Обследованы 50 пациентов в возрасте от 2 до 17 лет с различными проявлениями кровоточивости и отсутствием лабораторных данных за коагулопатию и/или тромбоцитопению, проведена цитофлуориметрия тромбоцитов с активацией. Оценены морфологические характеристики тромбоцитов по размеру/гранулярности (FSC/SSC), плотность рецептора CD62р как маркера секреции a-гранул, d-гранулы тромбоцитов по флуоресценции загруженного мепакрина. Активация тромбоцитов производилась смесью CRP + TRAP. Сравнение проводили с результатами обследования 50 условно здоровых детей (контрольная группа, КГ) в возрасте от 2 до 17 лет. Выраженность геморрагического синдрома оценивалась при помощи стандартизованной шкалы ISTH BAT. Тяжесть геморрагических проявлений по шкале ISTH BAT составила от 2 до 6 баллов. В результате проведенного исследования были выделены 2 группы пациентов, различающиеся по расчетному параметру отношения FSC/SSC, для неактивированных тромбоцитов. В КГ медиана FSC/SSC составила 1,235 (от 1,1 до 1,4), в группе 1 (n = 19) – 0,97 (от 0,9 до 1,05), и в группе 2 (n = 31) – 1,24 (от 1,11 до 1,43). Количество тромбоцитов достоверно не различалось между КГ и группами пациентов 1 и 2. В группах пациентов наблюдалась достоверная корреляционная связь между уменьшением количества тромбоцитов и увеличением их размера и гранулярности с сохранением высокой корреляционной связи между размером и гранулярностью. В группе 1 общая гранулярность была повышена независимо от размера и количества тромбоцитов, были увеличены объем плотных гранул и мембранный CD62p, но снижен гранулярный CD62p. Механизм дегрануляции не был нарушен в обеих группах пациентов. Полученные нами результаты убедительно указывают на вклад нарушений пула хранения и морфологии тромбоцитов в развитие геморрагических проявлений у детей с неуточненным геморрагическим синдромом.
Список литературы
1. Gresele P., Harrison P., Bury L., Falcinelli E., Gachet C., Hayward C.P., et al. Diagnosis of suspected inherited platelet function disorders: results of a worldwide survey. J Thromb Haemost 2014; 12: 1562–9.
2. Gresele P., Falcinelli E., Bury L. Laboratory diagnosis of clinically relevant platelet function disorders. Int J Lab Hem 2018; 40 (Suppl 1): 34–45.
3. Quiroga T., Goycoolea M., Panes O., Aranda E., Martínez C., Belmont S., et al. High prevalence of bleeders of unknown cause among patients with inherited mucocutaneous bleeding. A prospective study of 280 patients and 299 controls. Haematologica 2007; 92 (3): 357–65.
4. Andres O., Henning K., Straub G., Pfl ug A., Manukjan G., Schulze H. Diagnosis of platelet function disorders: A standardized, rational, and modular fl ow cytometric approach. Platelets 2018; 29 (4): 347–56.
5. Rand M.L., Reddy E.C., Israels S.J. Laboratory diagnosis of inherited platelet function disorders. Transfus Apher Sci 2018; 57: 485–93.
6. Ramström S., Södergren A.L., Tynngård N., Lindahl T.L. Platelet Function Determined by Flow Cytometry: New Perspectives? Semin Thromb and Hemost 2016; 42 (3): 268–81.
7. Ignatova A.A., Ponomarenko E.A., Polokhov D.M., Suntsova E.V., Zharkov P.A., Fedorova D.V., et al. Flow cytometry for pediatric platelets. Platelets 2019; 30 (4): 428–37.
8. Boknäs N., Ramström S., Faxälv L., Lindahl T.L. Flow cytometry-based platelet function testing is predictive of symptom burden in a cohort of bleeders. Platelets 2018; 29: 512–9.
9. Gresele P., Harrison P., Gachet C., Hayward C., Kenny D., Mezzano D., et al. Diagnosis of inherited platelet function disorders: Guidance from the SSC of the ISTH. J Thromb Haemost 2015; 13: 314– 22.
10. Rodeghiero F., Tosetto A., Abshire T., Arnold D.M., Coller B., James P., et al. ISTH/SSC bleeding assessment tool: a standardized questionnaire and a proposal for a new bleeding score for inherited bleeding disorders. JTH 2010; 8 (9): 2063–5.
11. Suntsova E.V., Demina I.M., Ignatova A.A., Ershov N.M., Trubina N.M., Dobrynina J., et al. Bleeding tendency and platelet function during treatment with romiplostim in children with severe immune thrombocytopenic purpura. Int J Hematol 2017; 105: 841–8.
12. Ignatova A.A., Karpova O.V., Trakhtman P.E., Rumiantsev S.A., Panteleev M.A. Functional characteristics and clinical effectiveness of platelet concentrates treated with ribofl avin and ultraviolet light in plasma and in platelet additive solution. Vox Sang 2016; 110: 244–52.
13. Balduini C.L., Melazzini F., Pecci A. Inherited thrombocytopenias – recent advances in clinical and molecular aspects. Platelets 2017; 28: 3–13.
14. Michelson A.D., Barnard M.R., Krueger L.A., Valeri C.R., Furman M.I. Circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin. Circulation 2007; 104: 1533–7.
15. Michelson A.D., Barnard M.R., Krueger L.A., Frelinger A.L. 3rd, Furman M.I. Evaluation of platelet function by flow cytometry. Methods 2000; 21 (3): 259– 70.
16. Chaudhary P.K., Kim S., Jee Y., Lee S.H., Park K.M., Kim S. Role of GRK6 in the regulation of platelet activation through selective g protein-coupled receptor (GPCR) desensitization. Int J Mol Sci 2020; 21: 1–14
17. Mahmoodian R., Salimian M., Hamidpour M., Khadem-Maboudi A.A., Gharehbaghian A. The eff ect of mild agonist stimulation on the platelet reactivity in patients with type 2 diabetes mellitus. BMC Endocr Disord 2019; 19: 1–8
18. Koltsova E.M., Balashova E.N., Ignatova A.A., Poletaev A.V., Polokhov D.M., Kuprash A.D., et al. Impaired platelet activity and hypercoagulation in healthy term and moderately preterm newborns during the early neonatal period. Pediatr Res 2019; 85 (1): 63–71.
19. Hézard N., Potron G., Schlegel N., Amory C., Leroux B., Nguyen P. Unexpected persistence of platelet hyporeactivity beyond the neonatal period: a flow cytometric study in neonates, infants and older children. Thromb Haemost 2003; 90 (1): 116–23.
20. Polokhov D.M., Ershov N.M., Ignatova A.A., Ponomarenko E.A., Gaskova M.A., Zharkov P.A., et al. Platelet function and blood coagulation system status in childhood essential thrombocythemia. Platelets 2019; 31 (8): 1001–11
21. Yip C., Linden M.D., Attard C., Monagle P., Ignjatovic V. Platelets from children are hyper‐responsive to activation by thrombin receptor activator peptide and adenosine diphosphate compared to platelets from adults. Br J Haematol 2015;168: 526–32.
Pediatric Hematology/Oncology and Immunopathology. 2021; 20: 58-65
Specifities of the storage pool and morphology of platelets in children with unspecified hemorrhagic syndrome
https://doi.org/10.24287/1726-1708-2021-20-1-58-65Abstract
Despite modern possibilities of laboratory diagnosis of hemorrhagic syndrome, in some patients, the causes of bleeding remain unspecified. Among these reasons, mild defects in the platelet link of hemostasis can potentially be hidden. The aim of the work is to identify the features of the function of the platelet hemostasis in children with unspecified hemorrhagic syndrome. This study is supported by the Independent Ethics Committee and approved by the Academic Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. We examined 50 patients aged 2 to 17 years with various manifestations of bleeding and lack of laboratory data proving coagulopathy and/or thrombocytopenia; platelet cytofluorometry with activation was performed. The morphological characteristics of platelets in terms of size/granularity (FSC/SSC), the density of the CD62p receptor as a marker of a-granule secretion, and d-granules of platelets were assessed by the fluorescence of loaded mepacrine. Platelet activation was performed with a CRP + TRAP mixture. Comparison was carried out with the results of examination of 50 healthy children (control group - CG) aged 2 to 17 years. The severity of hemorrhagic syndrome was assessed using the standardized ISTH BAT score. The severity of hemorrhagic manifestations according to BAT ISTH score ranged from 2 to 6 points. As a result of the study, two groups of patients differing in the calculated parameter of the FSC/SSC ratio for non-activated platelets were identified. In the CG, the median FSC/SSC was 1.235 (from 1.1 to 1.4), in group 1 (n = 19), the median was 0.97 (from 0.9 to 1.05), and in group 2 (n = 31), the median was 1.24 (from 1.11 to 1.43). The number of platelets of the CG and the groups of patients did not differ significantly. A significant correlation between a decrease in the number of platelets and an increase in their size and granularity, while maintaining a high correlation between size and granularity was observed in groups of patients. In group 1, the overall granularity was increased regardless of the size and number of platelets, the volume of dense granules and membrane CD62p was increased, but the granular CD62p was decreased. The degranulation mechanism was not impaired in both groups of patients. Our results indicate convincingly the contribution of the storage pool and platelet morphology disorders to the development of hemorrhagic manifestations in children with unspecified hemorrhagic syndrome.
References
1. Gresele P., Harrison P., Bury L., Falcinelli E., Gachet C., Hayward C.P., et al. Diagnosis of suspected inherited platelet function disorders: results of a worldwide survey. J Thromb Haemost 2014; 12: 1562–9.
2. Gresele P., Falcinelli E., Bury L. Laboratory diagnosis of clinically relevant platelet function disorders. Int J Lab Hem 2018; 40 (Suppl 1): 34–45.
3. Quiroga T., Goycoolea M., Panes O., Aranda E., Martínez C., Belmont S., et al. High prevalence of bleeders of unknown cause among patients with inherited mucocutaneous bleeding. A prospective study of 280 patients and 299 controls. Haematologica 2007; 92 (3): 357–65.
4. Andres O., Henning K., Straub G., Pfl ug A., Manukjan G., Schulze H. Diagnosis of platelet function disorders: A standardized, rational, and modular fl ow cytometric approach. Platelets 2018; 29 (4): 347–56.
5. Rand M.L., Reddy E.C., Israels S.J. Laboratory diagnosis of inherited platelet function disorders. Transfus Apher Sci 2018; 57: 485–93.
6. Ramström S., Södergren A.L., Tynngård N., Lindahl T.L. Platelet Function Determined by Flow Cytometry: New Perspectives? Semin Thromb and Hemost 2016; 42 (3): 268–81.
7. Ignatova A.A., Ponomarenko E.A., Polokhov D.M., Suntsova E.V., Zharkov P.A., Fedorova D.V., et al. Flow cytometry for pediatric platelets. Platelets 2019; 30 (4): 428–37.
8. Boknäs N., Ramström S., Faxälv L., Lindahl T.L. Flow cytometry-based platelet function testing is predictive of symptom burden in a cohort of bleeders. Platelets 2018; 29: 512–9.
9. Gresele P., Harrison P., Gachet C., Hayward C., Kenny D., Mezzano D., et al. Diagnosis of inherited platelet function disorders: Guidance from the SSC of the ISTH. J Thromb Haemost 2015; 13: 314– 22.
10. Rodeghiero F., Tosetto A., Abshire T., Arnold D.M., Coller B., James P., et al. ISTH/SSC bleeding assessment tool: a standardized questionnaire and a proposal for a new bleeding score for inherited bleeding disorders. JTH 2010; 8 (9): 2063–5.
11. Suntsova E.V., Demina I.M., Ignatova A.A., Ershov N.M., Trubina N.M., Dobrynina J., et al. Bleeding tendency and platelet function during treatment with romiplostim in children with severe immune thrombocytopenic purpura. Int J Hematol 2017; 105: 841–8.
12. Ignatova A.A., Karpova O.V., Trakhtman P.E., Rumiantsev S.A., Panteleev M.A. Functional characteristics and clinical effectiveness of platelet concentrates treated with ribofl avin and ultraviolet light in plasma and in platelet additive solution. Vox Sang 2016; 110: 244–52.
13. Balduini C.L., Melazzini F., Pecci A. Inherited thrombocytopenias – recent advances in clinical and molecular aspects. Platelets 2017; 28: 3–13.
14. Michelson A.D., Barnard M.R., Krueger L.A., Valeri C.R., Furman M.I. Circulating monocyte-platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin. Circulation 2007; 104: 1533–7.
15. Michelson A.D., Barnard M.R., Krueger L.A., Frelinger A.L. 3rd, Furman M.I. Evaluation of platelet function by flow cytometry. Methods 2000; 21 (3): 259– 70.
16. Chaudhary P.K., Kim S., Jee Y., Lee S.H., Park K.M., Kim S. Role of GRK6 in the regulation of platelet activation through selective g protein-coupled receptor (GPCR) desensitization. Int J Mol Sci 2020; 21: 1–14
17. Mahmoodian R., Salimian M., Hamidpour M., Khadem-Maboudi A.A., Gharehbaghian A. The eff ect of mild agonist stimulation on the platelet reactivity in patients with type 2 diabetes mellitus. BMC Endocr Disord 2019; 19: 1–8
18. Koltsova E.M., Balashova E.N., Ignatova A.A., Poletaev A.V., Polokhov D.M., Kuprash A.D., et al. Impaired platelet activity and hypercoagulation in healthy term and moderately preterm newborns during the early neonatal period. Pediatr Res 2019; 85 (1): 63–71.
19. Hézard N., Potron G., Schlegel N., Amory C., Leroux B., Nguyen P. Unexpected persistence of platelet hyporeactivity beyond the neonatal period: a flow cytometric study in neonates, infants and older children. Thromb Haemost 2003; 90 (1): 116–23.
20. Polokhov D.M., Ershov N.M., Ignatova A.A., Ponomarenko E.A., Gaskova M.A., Zharkov P.A., et al. Platelet function and blood coagulation system status in childhood essential thrombocythemia. Platelets 2019; 31 (8): 1001–11
21. Yip C., Linden M.D., Attard C., Monagle P., Ignjatovic V. Platelets from children are hyper‐responsive to activation by thrombin receptor activator peptide and adenosine diphosphate compared to platelets from adults. Br J Haematol 2015;168: 526–32.
