Research Article
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Akım sitometrik ölçüm öncesi pre-analitik yöntem optimizasyonu ve postmenapozal süreçte immünfenotipleme örneği

Year 2020, , 74 - 78, 31.12.2020
https://doi.org/10.35514/mtd.2021.34

Abstract

Giriş: Akım sitometri tıpta geniş bir kullanım alanına sahiptir. Standartlaştırılmış ve tekrarlanabilir veriler üretilmesi amacı ile standart işlem prosedürleri kullanılmaktadır. Sıçan, immünolojik araştırmalar için yaygın olarak kullanılan bir deney hayvanı modelidir, ancak sıçan türlerindeki yayınlar sınırlı sayıdadır. Amaç: Postmenopozal dönemde olan 19 aylık bir Wistar sıçandan aynı anda alınan periferik kan örneklerin iki farklı pre-analitik yöntem ile akım sitometrik ölçümlere hazırlanması ve lenfosit yüzde oranlarının değerlendirilmesi amaçlanmıştır. Materyal ve Metodlar: Periferik kan örneklerinden lizis veya yoğunluk gradyan santrifüj yöntemleriyle eritrositler uzaklaştırılmıştır. Uygun miktarda antikorlar ile T, B, doğal öldürücü (NK), T helper ve T sitotoksik hücreleri tanımlanmış, BD Accuri C6+ akım sitometride analiz edilmiş ve yüzde oranları belirlenmiştir. Bulgular: Eritrositlerin lizis tamponu ve yoğunluk gradyanı santrifüjü ile uzaklaştırıldığı iki yöntem ile de lenfosit ve T hücre alt gruplarının yüzde oranları benzer olarak bulunmuştur. Her iki yöntem ile üretilen sonuçlar sağlıklı genç sıçan ile karşılaştırıldığında B hücrelerinde azalma ve NK hücrelerde artış olduğu gözlenmiştir. Sonuç: Çalışmamızda kullanılan iki pre-analitik yöntemin de sıçan tam kanının immünfenotiplemesi için uygun olduğu düşünülmektedir.

Supporting Institution

Maltepe Üniversitesi

Thanks

Bu çalışma Maltepe Üniversitesi Bilimsel Araştırma Projelerini Destekleme Birimi tarafından desteklenmiştir. Çalışmanın yürütülmesinde teknik ve altyapı desteği sağlayan Maltepe Üniversitesi Kanser ve Kök Hücre Merkezi ve Maltepe Üniversitesi Deney Hayvanları Uygulama ve Araştırma Merkezi’ne ayrıca teşekkür ederiz.

References

  • 1. Cossarizza A, Chang HD, Radbruch A, Akdis M, Andrä I, Annunziato F, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies. European journal of immunology. 2017;47(10):1584-797.
  • 2. Melamed MR. A brief history of flow cytometry and sorting. Methods in cell biology. 2001;63:3-17.
  • 3. Adan A, Alizada G, Kiraz Y, Baran Y, Nalbant A. Flow cytometry: basic principles and applications. Critical reviews in biotechnology. 2017;37(2):163-76.
  • 4. Nováková M, Glier H, Brdičková N, Vlková M, Santos AH, Lima M, et al. How to make usage of the standardized EuroFlow 8-color protocols possible for instruments of different manufacturers. Journal of immunological methods. 2019;475:112388.
  • 5. Alavi N, Khan SH, Saadia A, Naeem T. Challenges in preanalytical phase of laboratory medicine: rate of blood sample nonconformity in a tertiary care hospital. EJIFCC. 2020;31(1):21.
  • 6. Bielohuby M, Popp S, Bidlingmaier M. A guide for measurement of circulating metabolic hormones in rodents: Pitfalls during the pre-analytical phase. Molecular metabolism. 2012;1(1-2):47-60.
  • 7. Festing MF, Altman DG. Guidelines for the design and statistical analysis of experiments using laboratory animals. ILAR journal. 2002;43(4):244-58.
  • 8. Sengupta P. The laboratory rat: relating its age with human's. International journal of preventive medicine. 2013;4(6):624.
  • 9. Jaye DL, Bray RA, Gebel HM, Harris WA, Waller EK. Translational applications of flow cytometry in clinical practice. The Journal of Immunology. 2012;188(10):4715-9.
  • 10. Kanegane H, Hoshino A, Okano T, Yasumi T, Wada T, Takada H, et al. Flow cytometry-based diagnosis of primary immunodeficiency diseases. Allergology International. 2018;67(1):43-54.
  • 11. Abbondanzo SJ, Chang SL. HIV-1 transgenic rats display alterations in immunophenotype and cellular responses associated with aging. PloS one. 2014;9(8):e105256.
  • 12. Flaherty DK, Wagner CA, Gross CJ, Panyik MA. Aging and lymphocyte subsets in the spleen and peripheral blood of the Sprague-Dawley rat. Immunopharmacology and immunotoxicology. 1997;19(2):185-95.
  • 13. Ridge K, Downes N, Finney B. Effects of strain, sex and age on immunophenotyping parameters in the rat and mouse. Comparative Clinical Pathology. 2019;28(1):41-51.

Pre-analytical method optimization for flow cytometric measurement and immunophenotyping example in post-menopausal rat

Year 2020, , 74 - 78, 31.12.2020
https://doi.org/10.35514/mtd.2021.34

Abstract

Introduction: Flow cytometry has a wide range of applications in medicine. Standard operating procedures are used to produce standardized and reproducible data. The rat is a commonly used model for immunological research, yet publications in rat species are limited. Aim: This study aims to prepare samples taken from a 19-month-old Wistar rat in the postmenopausal period with two different pre-analytical methods for flow cytometric measurements and to analyse the lymphocyte subsets. Material and Methods: Erythrocytes were removed from peripheral blood samples by lysis or density gradient centrifugation. With the appropriate amount of antibodies, T, B, NK, T helper and T cytotoxic cells were identified in a BD Accuri C6+ flow cytometer and percentages of the respective cells were determined. Results: Similar percentages of lymphocyte and T subsets were obtained when the erythrocytes were removed by lysis buffer or density gradient centrifugation. A decrease in B cells and an increase in NK cells compared to the healthy young rats were observed. Conclusion: Both pre-analytical methods used in our study are thought to be suitable for immunophenotyping of rat whole blood.

References

  • 1. Cossarizza A, Chang HD, Radbruch A, Akdis M, Andrä I, Annunziato F, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies. European journal of immunology. 2017;47(10):1584-797.
  • 2. Melamed MR. A brief history of flow cytometry and sorting. Methods in cell biology. 2001;63:3-17.
  • 3. Adan A, Alizada G, Kiraz Y, Baran Y, Nalbant A. Flow cytometry: basic principles and applications. Critical reviews in biotechnology. 2017;37(2):163-76.
  • 4. Nováková M, Glier H, Brdičková N, Vlková M, Santos AH, Lima M, et al. How to make usage of the standardized EuroFlow 8-color protocols possible for instruments of different manufacturers. Journal of immunological methods. 2019;475:112388.
  • 5. Alavi N, Khan SH, Saadia A, Naeem T. Challenges in preanalytical phase of laboratory medicine: rate of blood sample nonconformity in a tertiary care hospital. EJIFCC. 2020;31(1):21.
  • 6. Bielohuby M, Popp S, Bidlingmaier M. A guide for measurement of circulating metabolic hormones in rodents: Pitfalls during the pre-analytical phase. Molecular metabolism. 2012;1(1-2):47-60.
  • 7. Festing MF, Altman DG. Guidelines for the design and statistical analysis of experiments using laboratory animals. ILAR journal. 2002;43(4):244-58.
  • 8. Sengupta P. The laboratory rat: relating its age with human's. International journal of preventive medicine. 2013;4(6):624.
  • 9. Jaye DL, Bray RA, Gebel HM, Harris WA, Waller EK. Translational applications of flow cytometry in clinical practice. The Journal of Immunology. 2012;188(10):4715-9.
  • 10. Kanegane H, Hoshino A, Okano T, Yasumi T, Wada T, Takada H, et al. Flow cytometry-based diagnosis of primary immunodeficiency diseases. Allergology International. 2018;67(1):43-54.
  • 11. Abbondanzo SJ, Chang SL. HIV-1 transgenic rats display alterations in immunophenotype and cellular responses associated with aging. PloS one. 2014;9(8):e105256.
  • 12. Flaherty DK, Wagner CA, Gross CJ, Panyik MA. Aging and lymphocyte subsets in the spleen and peripheral blood of the Sprague-Dawley rat. Immunopharmacology and immunotoxicology. 1997;19(2):185-95.
  • 13. Ridge K, Downes N, Finney B. Effects of strain, sex and age on immunophenotyping parameters in the rat and mouse. Comparative Clinical Pathology. 2019;28(1):41-51.
There are 13 citations in total.

Details

Primary Language Turkish
Subjects Clinical Sciences
Journal Section Research Article
Authors

Mustafa Erinç Sitar 0000-0001-5114-8660

Yaprak Dönmez Çakıl 0000-0002-4605-1167

Publication Date December 31, 2020
Submission Date December 16, 2020
Published in Issue Year 2020

Cite

Vancouver Sitar ME, Dönmez Çakıl Y. Akım sitometrik ölçüm öncesi pre-analitik yöntem optimizasyonu ve postmenapozal süreçte immünfenotipleme örneği. Maltepe tıp derg. 2020;12(3):74-8.