Modified quantitative approach for assessing peripheral blood TREC and KREC levels in immunodeficient patients
- 作者: Saitgalina M.A.1, Ostankova Y.V.1, Liubimova N.E.1, Semenov A.V.2, Kuznetsova R.N.1,3, Totolian A.A.1,3
-
隶属关系:
- St. Petersburg Pasteur Institute
- Ekaterinburg Research Institute of Viral Infections of SRC VB Vector of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing
- I.P. Pavlov First St. Petersburg State Medical University of the Ministry of Healthcare of Russian Federation
- 期: 卷 12, 编号 5 (2022)
- 页面: 981-996
- 栏目: METHODS
- URL: https://journals.rcsi.science/2220-7619/article/view/119186
- DOI: https://doi.org/10.15789/2220-7619-MMF-2039
- ID: 119186
如何引用文章
全文:
详细
Introduction. The immune status is a multifaceted parameter quantitatively and qualitatively analyzing functional activity immune system state in immune organs as well as some non-specific mechanisms of antimicrobial protection. Peripheral blood level of T-receptor excision rings (TREC) and B-cell excision rings (KREC), respectively, can serve as surrogate markers of T- and B-cell maturation. Currently, the diagnostic kits available on the market have two significant disadvantages: i) the kits are aimed at diagnosing immunodeficiency conditions only in newborns and children, while keeping adult patients uncovered; ii) essentially, use solely single reference normalization gene for data normalization resulting in increased variability and decreased sensitivity of the assay data. The aim: to develop a highly sensitive method for laboratory assessment of the state of immunity in immunodeficient patients by using real-time PCR for assessing TREC and KREC level in children and adults. Materials and methods. There were used whole blood and dry blood spot samples obtained from newborns and adults, apparently healthy individuals as well as patients with verified PID and HIV-infection. A total of 2577 samples were examined. Commercial kits were used as comparison methods. Results. Multiplex PCR was carried out, analyzing the number of target molecules TREC and KREC, as well as fragments of the HPRT and RPP30 normalization genes analyzed with the developed series of plasmid calibrators. The established analytical range of TREC/KREC DNA measurements comprised 103 to 109 copies/mL. The accuracy of measurements on a tablet-type instrument (CFX) was 95.84%, on a rotary-type instrument (Rotor-Gene 3000) — 95.11%, which corresponds to the standard indicator. The equivalence between the data obtained after assessing whole blood samples and dry blood drops was shown. The data analysis allowed to find out 100%-diagnostic specificity and sensitivity of the method proposed. Conclusion. The method developed by us allows to diagnose decline in T- and/or B-cell immunity in children and adults and can be used to detect TREC and KREC molecules both in peripheral whole blood samples and dry blood spots using Guthrie cards. Moreover, the uniform values of reference norms can be used regardless of the type of analyzed clinical material. The study data evidence about potential for effective use of multiplex PCR diagnostics both for complex primary testing/screening of newborns and assessing state of immunity to identify adult patients with PID and as a part of the diagnostic monitoring of patients with secondary immunodeficiencies, e.g., HIV infection.
作者简介
M. Saitgalina
St. Petersburg Pasteur Institute
Email: Sajgalinam@mail.ru
Junior Researcher, Laboratory of Molecular Immunology
俄罗斯联邦, St.PetersburgYu. Ostankova
St. Petersburg Pasteur Institute
编辑信件的主要联系方式.
Email: shenna1@yandex.ru
PhD (Biology), Head of the Laboratory of HIV Immunology and Virology; Senior Researcher, Laboratory of Molecular Immunology
俄罗斯联邦, St. PetersburgN. Liubimova
St. Petersburg Pasteur Institute
Email: natelu@mail.ru
PhD (Biology), Researcher, Laboratory of Molecular Immunology
俄罗斯联邦, St. PetersburgA. Semenov
Ekaterinburg Research Institute of Viral Infections of SRC VB Vector of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing
Email: alexvsemenov@yahoo.com
PhD, MD (Biology), Director
俄罗斯联邦, EkaterinburgR. Kuznetsova
St. Petersburg Pasteur Institute; I.P. Pavlov First St. Petersburg State Medical University of the Ministry of Healthcare of Russian Federation
Email: kuznetzova.rais@yandex.ru
ORCID iD: 0000-0003-1668-6716
PhD (Medicine), Allergologist-Immunologist, Associate Professor, Department of Immunology
俄罗斯联邦, St. Petersburg; St. PetersburgA. Totolian
St. Petersburg Pasteur Institute; I.P. Pavlov First St. Petersburg State Medical University of the Ministry of Healthcare of Russian Federation
Email: totolian@spbraaci.ru
RAS Full Member, PhD, MD (Medicine), Professor, Director, Head of the Department of Immunology
俄罗斯联邦, St. Petersburg; St. Petersburg参考
- Гордукова М.А., Корсунский И.А., Чурсинова Ю.В., Бяхова М.М., Оскорбин И.П., Продеус А.П., Филипенко М.Л. Определение референсных интервалов TREC и KREC для скрининга новорожденных с иммунодефицитными состояниями в РФ // Медицинская иммунология. 2019. Т. 21, № 3. C. 527–538. [Gordukova M.A., Korsunsky I.A., Chursinova Yu.V., Byakhova M.M., Oscorbin I.P., Prodeus A.P., Filipenko M.L. Determining reference ranges for TREC and KREC assays in immune deficiency screening of newborns in Russian Federation. Meditsinskaya immunologiya = Medical Immunology (Russia), 2019, vol. 21, no. 3, рр. 527–538. (In Russ.)] doi: 10.15789/1563-0625-2019-3-527-538
- Гордукова М.А., Оскорбин И.П., Мишукова О.В., Зимин С.Б., Зиновьева Н.В., Давыдова Н.В., Смирнова А.С., Никитина И.А., Корсунский И.А., Филипенко М.Л., Продеус А.П. Разработка набора реагентов для количественного определения молекул ДНК TREC и KREC в цельной крови и сухих пятнах крови методом мультиплексной ПЦР в режиме реального времени // Медицинская иммунология. 2015. Т. 17, № 5. С. 467–478. [Gordukova M.A., Oskorbin I.P., Mishukova O.V., Zimin S.B., Zinovieva N.V., Davydova N.V., Smirnova A.S., Nikitina I.A., Korsunsky I.A., Filipenko M.L., Prodeus A.P. Development of real-time multiplex PCR for the quantitative determination of TREC’s and KREC’s in whole blood and in dried blood spots. Meditsinskaya immunologiya = Medical Immunology (Russia), 2015, vol. 17, no. 5, pp. 467–478. (In Russ.)] doi: 10.15789/1563-0625-2015-5-467-478
- Евгина С.А., Савельев Л.И. Современные теория и практика референтных интервалов // Лабораторная служба. 2019. T. 8, № 2. С. 36–44. [Evgina S.A., Saveliev L.I. Сurrent theory and practice of reference interval. Laboratornaya sluzhba = Laboratory Service, 2019, vol. 8, no. 2, pp. 36–44. (In Russ.)] doi: 10.17116/labs2019802136
- Корсунский И.А., Гордукова М.А., Мунблит Д.Б., Козлов И.Г., Продеус А.П., Корсунский А.А. Клинические и эпидемиологические аспекты первичных иммунодефицитных состояний и их раннего обнаружения // Медицинская иммунология. 2017. Т. 19, № 5. С. 505–512. [Korsunskiy I.A., Gordukova M.A., Munblit D.B., Kozlov I.G., Prodeus A.P., Korsunskiy A.A. Clinical and epidemiological aspects of primary immunodeficiency diseases (PID) and early diagnosis options. Meditsinskaya immunologiya = Medical Immunology (Russia), 2017, vol. 19, no. 5, pp. 505–512. (In Russ.)] doi: 10.15789/1563-0625-2017-5-505-512
- Латышева Т.В., Латышева Е.А., Мартынова И.А., Аминова Г.Э. Пульмонологические проявления у взрослых пациентов с дефектом гуморального звена иммунитета // Терапевтический архив. 2016. Т. 88, № 8. С. 127–134. [Latysheva T.V., Latysheva E.A., Martynova I.A., Aminova G.E. Pulmonary manifestations in adult patients with a defect in the humoral link of immunity. Terapevticheskii arkhiv = Therapeutic Archive, 2016, vol. 88, no. 8, pp. 127–134. (In Russ.)] doi: 10.17116/terarkh2016888127-134
- Патент № 2756979 Российская Федерация, МПК C12Q 1/686 (2018.01), C12Q 1/6876 (2018.01). Способ лабораторной персонифицированной диагностики состояния иммунитета новорожденных и набор олигодезоксирибонуклеотидных праймеров и флуоресцентно меченных зондов: № 2019142752; заявлено 17.12.2019: опубликовано 07.10.2021 / Семенов А.В., Останкова Ю.В., Любимова Н.Е., Тотолян Арег А. Патентообладатель: ФБУН НИИ эпидемиологии и микробиологии имени Пастера. 20 с. [Patent No. 2756979 Russian Federation, Int. Cl. C12Q 1/686 (2018.01), C12Q 1/6876 (2018.01). A method for laboratory personalized diagnosis of the state of immunity in newborns and a set of oligodeoxyribonucleotide primers and fluorescently labeled probes: No. 2019142752; application: 17.12.2019: date of publication 07.10.2021 / Semenov A.V., Ostankova Yu.V., Liubimova N.E., Totolian Areg A. Proprietor: FBUN NII epidemiologii i mikrobiologii imeni Pastera. 20 p. (In Russ.)]
- Тузанкина И.А., Каракина М.Л., Власова Е.В. Анализ клинических проявлений дебюта первичных иммунодефицитов у взрослых // Медицинская иммунология. 2014. Т. 4, № 16. C. 367–374. [Tuzankina I.A., Karakina M.L., Vlasova E.V. Analysis of the clinical manifestations of the onset of primary immunodeficiencies in adults. Meditsinskaya immunologiya = Medical Immunology (Russia), 2014, vol. 4, no. 16, pp. 367–374. (In Russ.)] doi: 10.15789/1563-0625-2014-4-367-374
- Arismendi M.I., Kallas E.G., Santos B.A., Carneiro-Sampaio M.M., Kayser C. Thymopoiesis and regulatory T cells in healthy children and adolescents. Clinics, 2012, vol. 67, no. 5, pp. 425–429. doi: 10.6061/clinics/2012(05)04
- Baker M.W., Grossman W.J., Laessig R.H., Hoffman G.L., Brokopp C.D., Kurtycz D.F., Cogley M.F., Litsheim T.J., Katcher M.L., Routes J.M. Development of a routine newborn screening protocol for severe combined immunodeficiency. J. Allergy Clin. Immunol., 2009, vol. 124, no. 3, pp. 522–527. doi: 10.1016/j.jaci.2009.04.007
- Dar N., Gothelf D., Korn D., Frisch A., Weizman A., Michaelovsky E., Carmel M., Yeshayahu Y., Dubnov-Raz G., Pessach I.M., Simon A.J., Lev A., Somech R. Thymic and bone marrow output in individuals with 22q11.2 deletion syndrome. Pediatr. Res., 2015, vol. 4, no. 77, pp. 579–585. doi: 10.1038/pr.2015.14
- Dyavar S.R., Ye Z., Byrareddy S.N., Scarsi K.K., Winchester L.C., Weinhold J.A., Fletcher C.V., Podany A.T. Normalization of cell associated antiretroviral drug concentrations with a novel RPP30 droplet digital PCR assay. Sci. Rep., 2018, vol. 8, no. 1: 3626. doi: 10.1038/s41598-018-21882-0
- Esber R., Theresa S., Hjort M., Wiley V. Newborn screening for primary immunodeficiencies — a method evaluation. Twin Res. Hum. Genet., vol. 21, no. 5: Special Section: Abstracts for the 42nd Human Genetics Society of Australasia Annual Scientific Meeting, Sydney, New South Wales, 2018, pp. 429–476. doi: 10.1017/thg.2018.52
- Hazenberg M.D., Borghans J.A., de Boer R.J., Miedema F. Thymic output: a bad TREC record. Nat. Immunol., 2003, vol. 4, no. 2, pp. 97–99. doi: 10.1038/ni0203-97
- Kamae C., Nakagawa N., Sato H., Honma K., Mitsuiki N., Ohara O., Kanegane H., Pasic S., Pan-Hammarström Q., van Zelm M.C., Morio T., Imai K., Nonoyama S. Common variable immunodeficiency classification by quantifying T-cell receptor and immunoglobulin -deleting recombination excision circles. J. Allergy Clin. Immunol., 2013, vol. 5, no. 131, pp. 1437–1440. doi: 10.1016/j.jaci.2012.10.059
- Klein S.L., Flanagan K.L. Sex differences in immune responses. Nat. Rev. Immunol., 2016, vol. 16, pp. 626–638. 10.1038/nri.2016.90
- Kwok J.S.Y., Cheung S.K.F., Ho J.C.Y., Tang I.W.H., Chu P.W.K., Leung E.Y.S., Lee P.P.W., Cheuk D.K.L., Lee V., Ip P., Lau Y.L. Establishing simultaneous T cell receptor excision circles (TREC) and K-deleting recombination excision circles (KREC) quantification assays and laboratory reference intervals in healthy individuals of different age groups in Hong Kong. Front. Immunol., 2020, vol. 11: 1411. doi: 10.3389/fimmu.2020.01411
- Laitala V., Ylikoski A., Raussi H.M., Ollikka P., Hemmilä I. Time-resolved detection probe for homogeneous nucleic acid analyses in one-step format. Anal. Biochem., 2007, vol. 361, no. 1, pp. 126–131. doi: 10.1016/j.ab.2006.11.015
- Pido-Lopez J., Imami N., Aspinall R. Both age and gender affect thymic output: more recent thymic migrants in females than males as they age. Clin. Exp. Immunol., 2001, vol. 125, pp. 409–413. doi: 10.1046/j.1365-2249.2001.01640.x
- Profaizer T., Slev P. A multiplex, droplet digital PCR assay for the detection of T-cell receptor excision circles and kappa-deleting recombination excision circles. Clin. Chem., 2019, vol. 66, pp. 229–238. doi: 10.1373/clinchem.2019.308171
- Quiros-Roldan E., Serana F., Chiarini M., Zanotti C., Sottini A., Gotti D., Torti C., Caimi L., Imberti L. Effects of combined antiretroviral therapy on B- and T-cell release from production sites in long-term treated HIV-1+ patients. J. Transl. Med., 2012, vol. 10, no. 94. doi: 10.1186/1479-5876-10-94
- Rechavi E., Lev A., Simon A.J., Stauber T., Daas S., Saraf-Levy T., Broides A., Nahum A., Marcus N., Hanna S., Stepensky P., Toker O., Dalal I., Etzioni A., Almashanu S., Somech R. First year of israeli newborn screening for severe combined immunodeficiency-clinical achievements and insights. Front. Immunol., 2017, vol. 8: 1448. doi: 10.3389/fimmu.2017.01448
- Resino S., Seoane E., Pérez A., Ruiz-Mateos E., Leal M., Muñoz-Fernández M.A. Different profiles of immune reconstitution in children and adults with HIV-infection after highly active antiretroviral therapy. BMC Infect. Dis., 2006, vol. 6: 112. doi: 10.1186/1471-2334-6-112
- Serana F., Chiarini M., Zanotti C., Sottini A., Bertoli D., Bosio A., Caimi L., Imberti L. Use of V(D)J recombination excision circles to identify T- and B-cell defects and to monitor the treatment in primary and acquired immunodeficiencies. J. Transl. Med., 2013, vol. 11: 119. doi: 10.1186/1479-5876-11-119
- Sottini A., Serana F., Bertoli D., Chiarini M., Valotti M., Vaglio Tessitore M., Lmberti L. Simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) by real-time PCR. J. Vis. Exp., 2014, vol. 94: 52184. doi: 10.3791/52184
- Tessitore M.V., Sottini A., Roccaro A.M., Ghidini C., Bernardi S., Martellosio G., Serana F., Lmberti L. Detection of newly produced T and B lymphocytes by digital PCR in blood stored dry on nylon flocked swabs. J. Transl. Med., 2017, vol. 15, no. 1: 70. doi: 10.1186/s12967-017-1169-9
- Ye P., Kirschner D.E., Kourtis A.P. The thymus during HIV disease: role in pathogenesis and in immune recovery. Curr. HIV Res., 2004, vol. 2, no. 2, pp. 177–183. doi: 10.2174/1570162043484898
- Van Zelm M., Van der Burg M., Langerakand A., Van Dongen J. PID comes full circle: applications of V(D)J recombination excision circles in research, diagnostics and newborn screening of primary immunodeficiency disorders. Front. Immunol., 2011, vol. 2, no. 12, pp. 1–9. doi: 10.3389/fimmu.2011.00012
补充文件
