COMPARISON OF THREE DIFFERENT TECHNIQUES FOR ISOLATION OF NEUTROPHILS FROM BLOOD AND THEIR UTILITY IN PERFORMING NITROBLUE TETRAZOLIUM TEST
DOI:
https://doi.org/10.29121/granthaalayah.v7.i12.2019.305Keywords:
Dextran, Ficoll-Hypaque, Formazon, Gelatin, NBT, NeutrophilsAbstract [English]
Background & Objectives: Assays for neutrophils constitute an important component of screening tests in clinical immunology. There are no standard protocols for performing many of these tests and procedures vary from one laboratory to another. In addition, normal ranges for these assays in healthy Indian population have not been defined. Hence, an attempt is made to evaluate and present a simple technique for WBC isolation and NBT test.
Methods: The study involved participation of 30 healthy adult volunteers. Ten of blood sample collected from each subject was subjected to three different procedures for isolation of WBCs - Ficoll-Hypaque gradient, dextran sedimentation and gelatin sedimentation methods. Cells isolated from these procedures were then used to perform NBT test. Smears were prepared, stained with Giemsa and results were expressed as % of stimulated and unstimulated cells.
Results: The mean cell yield from both dextran and gelatin methods was comparable (2921.67cells vs 2806.67cells/cu mm). The cell yield from Ficoll-Hypaque method was much lower (1408.33 cells/cu mm). In NBT test, the mean readings of stimulated (61%) and unstimulated cells (18%) were almost similar in all three procedures of cell isolation.
Conclusions: Comparison of procedures show that gelatin and dextran sedimentation methods yield high amount of relatively purified WBCs. The efficacy of cells isolated from all three procedures in NBT test was almost similar. The range of stimulated and unstimualted cells in the subjects were within expected levels. Gelatin sedimentation is economical, easy to perform and can be adopted to any clinical laboratory for WBC isolation.
Downloads
References
Kuhns DB. Assessment of Neutrophil function. In Rich RR, Eds Clinical Immunology Principles and Practice, Elsevier, 3rd Ed 2008 Mosby. DOI: https://doi.org/10.1016/B978-0-323-04404-2.10099-5
Nauseef WM. Isolation of human Neutrophils from venous blood. In Quinn MT, DeLeo FR, Bokoch GM Eds. Neutrophil methods and protocols, Methods in Molecular Biology No 412, 2007, Humana press, 15-20. DOI: https://doi.org/10.1007/978-1-59745-467-4_2
Luciano YM, Mantovani B Lysosomal enzyme release from polymorphonuclear leukocytes induced by immune complexes of IgM and IgG. Journal of Imunology 1984, 132, 2015-2020.
Gentle TA, Thompson RA. Neutrophil tests in clinical immunology in Eds Gooi and Chapel H. Clinical immunology: A practical approach. New York, Oxford University press, 1990.
Perutelli P, Catellani S, Scarso L, Cornaglia-Ferraris P, Dini G. Processing of human cord blood by three different procedures for red blood cell depletion and mononuclear cell recovery. Vox Sang 1999, 76, 237-240. DOI: https://doi.org/10.1046/j.1423-0410.1999.7640237.x
Metcalf JA, Gallin JI, Nauseef WM, Root RK. Laboratory Manual of Neutrophil Function. New York, Ravwn press, 1986.
Kielland LG, Vage RA, Eide GE, Sornes S, Naess A. Antituberculosis drugs and human polymorphonuclear leukocyte functions. Chemother 2011, 57, 339-344. DOI: https://doi.org/10.1159/000330442
M. S. Ganachari, Shiv Kumar, K. G. Bhat. Effect of ziziphus jujuba leaves extract on phagocytosis by human neutrophils. Journal of Natural Remedies, 2004, 4, 47-51.
Rahul S. Bhansali, R. K. Yeltiwar, K. G. Bhat. Assessment of peripheral neutrophil functions in patients with localized aggressive periodontitis in Indian population. Journal of Indian Society of Periodontology, 2013, 17, 731-736. DOI: https://doi.org/10.4103/0972-124X.124485
Quach A, Ferrante A. The Application of Dextran Sedimentation as an Initial Step in Neutrophil Purification Promotes Their Stimulation, due to the Presence of Monocytes. Journal of Immunology Research, 2017, 1254792. DOI: https://doi.org/10.1155/2017/1254792
Henson PM The immunologic release of constitients from neutrophil leukocytes I. The role of antibody and complement on nonphagocytosable surfaces or phagocytosable particles. J Immunol 1971; 107: 1535-1546.
Nagler A,Peacock M, Tantoco M, Lamons D, Okarma TB, Okrongly DA. Separation of hematogenic progenitor cells from human umbilical cord. Journal of Hematology & Oncology. 1993, 2. https://doi.org/10.1089/scd.1.1993.2.243. DOI: https://doi.org/10.1089/scd.1.1993.2.243
Antoniewicz-Papis J, Dzieciatkowska A, Lachert E, Letowska KM, Podstawka U, Apel D et al. Sedimentation as effective method of preliminary isolation of stem cells from cord blood. Reports of Practical Oncology and Radiotherapy, 2001, 6, sup1: s19-s25. DOI: https://doi.org/10.1016/S1507-1367(01)70476-2
Marchi LF, Sesti-Costa R, Chedraoul-Silva S, Mantovani B. Compariosn of four methods for the isolation of murine blood neutrophils with respect to the release of reactive oxygen and nitrogen species and the expression of immunological receptors. Comparative Clinical Pathology, 2014; 23: 1469-1476. DOI: https://doi.org/10.1007/s00580-013-1808-3
Paoliello-Paschoalato AE, Azzolini AECS, Cruz MFC, Marchi LF, Kabeya LM, Donadi EA et al. Isolation of healthy individuals' and rheumatoid arthritis patients' peripheral blood neutrophils by the gelatin and Ficoll-Hypaque methods: Comparative efficiency and impact on the neutrophil oxidative metabolism and Fcγ receptor expression. Journal of Immunological Methods, 2014, 412, 70-77. DOI: https://doi.org/10.1016/j.jim.2014.07.001
Wilkinson PC. Neutrophil leucocyte function tests in Thompson RA ed Techniques in clinical immunology, Blackwell Scientific Publications. 2nd ed., 273-293.
Downloads
Published
How to Cite
Issue
Section
License
With the licence CC-BY, authors retain the copyright, allowing anyone to download, reuse, re-print, modify, distribute, and/or copy their contribution. The work must be properly attributed to its author.
It is not necessary to ask for further permission from the author or journal board.
This journal provides immediate open access to its content on the principle that making research freely available to the public supports a greater global exchange of knowledge.