How fixation affects the results of lymph node immunophenotyping by flow cytometry

Dana Yerpasheva 1, Vadim Kemaykin 2, Gulzhanat Zhunis 2, Zhasulan Aisyn 2, Ivan Vorobjev 3 *
More Detail
1 Department of Biology, School of Sciences and Humanities, Nazarbayev University, Astana, Kazakhstan
2 Center for Oncohematology and BMT, National Research Oncology and Transplantology Center, Astana, Kazakhstan
3 Laboratory of Cell Motility, National Laboratory Astna, Astana, Kazakhstan
* Corresponding Author
J CLIN MED KAZ, Volume 20, Issue 5, pp. 47-54. https://doi.org/10.23950/jcmk/13762
OPEN ACCESS 784 Views 1006 Downloads
Download Full Text (PDF)

ABSTRACT

Aim: Flow cytometric diagnosis of lymphoma and leukemia is of high clinical and research importance. However, performing flow cytometry analysis on the day of biopsy might be of challenge due to several reasons, including late sample delivery, problems of preparing the reliable panel for immunophenotyping based on other diagnostic studies, etc. This problem could be partially solved if cell suspension could be fixed and stained on another day or after several days after standard FFPE (formalin-fixed and paraffin-embedded) procedure.
Material and methods: Addressing this issue, we compared staining of live lymphocytes in suspension obtained from lymph node biopsies and same specimens fixed using 2-4%-paraformaldehyde, 1-3%-glyoxal, and 0.1-1% glutaraldehyde with subsequent immunostaining on the next day or later.
Results: Staining after fixation could be partially representative only after paraformaldehyde fixation for 20 min and subsequent storage of cell suspension in phosphate-buffer saline within not more than 3 days. Probes stained after fixation always shows lower stain index compared to staining of live cells.
Conclusion: Staining after fixation cannot be used for determining of the percentage of CD45-positive cells and for testing B-cell lymphomas since antigens against light chains of IgG cannot be properly detected in fixed specimens.
Keywords: flow cytometry, immunophenotyping, cell suspension, lymph node, sample fixation, B cells, T cells, lymphoma

CITATION

Yerpasheva D, Kemaykin V, Zhunis G, Aisyn Z, Vorobjev I. How fixation affects the results of lymph node immunophenotyping by flow cytometry. J CLIN MED KAZ. 2023;20(5):47-54. https://doi.org/10.23950/jcmk/13762

REFERENCES

  • Reichard A, Asosingh K. Best practices for preparing a single cell suspension from solid tissues for flow cytometry. Cytometry Part A. 2019;95(2):219-226. https://doi.org/10.1002/cyto.a.23690
  • Jamur MC, Oliver C. Cell fixatives for immunostaining. Immunocytochemical methods and protocols. 2010:55-61. https://doi.org/10.1007/978-1-59745-324-08
  • Suthipintawong, C, Leong ASY, Vinyuvat S. Immunostaining of cell preparations: a comparative evaluation of common fixatives and protocols. Diagnostic cytopathology. 1996;15(2):167-174. https://doi.org/10.1002/(SICI)1097-0339(199608)15:2<167::AID-DC17>3.0.CO;2-F
  • Babcock GF, Dawes SM. Immunophenotyping using fixed cells. Methods in Cell Biology. 1994;41:81-93. https://doi.org/10.1016/s0091-679x(08)61710-0
  • Sampino EV, Morgan J, Chorzalska A, Nguyen L, Rodriguez CYuA, Pardo M, et al. Comparative flow cytometry-based immunophenotyping analysis of peripheral blood leukocytes before and after fixation with paraformaldehyde. Journal of Immunological Methods. 2022;511:113379. https://doi.org/10.1016/j.jim.2022.113379
  • Sędek Ł, Kulis J, Słota Ł, Twardoch M, Pierzyna-Świtała M, Perkowski B, et al. The influence of fixation of biological samples on cell count and marker expression stability in flow cytometric analyses. Central European Journal of Immunology. 2020;45(2):206-213. https://doi.org/10.5114/ceji.2020.95858
  • Paredes RM, Tadaki DK, Sooter A, Gamboni F, Sheppard F. Cryopreservation of human whole blood allows immunophenotyping by flow cytometry up to 30 days after cell isolation. Journal of Immunological Methods. 2018;452:32-38. https://doi.org/10.1016/j.jim.2017.08.013
  • Eltoum I, Fredenburgh J, Myers RB, Grizzle WE. Introduction to the theory and practice of fixation of tissues. Journal of Histotechnology. 2001;24(3):173-190. https://doi.org/10.1179/his.2001.24.3.173
  • Reichard A, Wanner N, Stuehr E, Alemagno M., Weiss K, Queisser K, et al. Quantification of airway fibrosis in asthma by flow cytometry. Cytometry Part A. 2018;93(9):952-958. https://doi.org/10.1002/cyto.a.23373
  • Levitt D, King M. Methanol fixation permits flow cytometric analysis of immunofluorescent stained intracellular antigens. Journal of immunological methods.1987; 96(2):233-237. https://doi.org/10.1016/0022-1759(87)90319-X
  • Lal RB, Edison LJ, Chused TM. Fixation and long‐term storage of human lymphocytes for surface marker analysis by flow cytometry. Cytometry: The Journal of the International Society for Analytical Cytology. 1988;9(3):213-219. https://doi.org/10.1002/cyto.990090305
  • Richter KN, Revelo NH, Seitz KJ, Helm MS, Sarkar D, Saleeb RS, et al. Glyoxal as an alternative fixative to formaldehyde in immunostaining and super‐resolution microscopy. The EMBO journal. 2018;37(1):139-159. https://doi.org/10.15252/embj.201695709
  • Thomas S, Sadanandan J, Blackburn SL, McBride DW, Dienel A, Hong S, et al. Glyoxal Fixation Is Optimal for Immunostaining of Brain Vessels, Pericytes and Blood-Brain Barrier Proteins. International Journal of Molecular Sciences. 2022;23(14):7776. https://doi.org/10.3390/ijms23147776
  • Rizzoli S. Techniques for sample processing and super-resolution imaging for investigating neuronal and muscular samples. Nervenheilkunde. 2019;38(05):264. https://doi.org/10.1055/s-0039-1684945
  • Vallangeon BD, Tyer C, Williams B, Lagoo AS. Improved detection of diffuse large B‐cell lymphoma by flow cytometric immunophenotyping—Effect of tissue disaggregation method. Cytometry Part B: Clinical Cytometry. 2016;90(5):455-461. https://doi.org/10.1002/cyto.b.21322
  • Bellesi S, Fiorita A, Corina L, D'Agostino G, Maiolo E, Scarano E., et al. The role of flow cytometry for the timely diagnosis of lymphoma in the head and neck district. Oral Oncology Reports. 2023;6, 100045. https://doi.org/10.1016/j.oor.2023.100045
  • Maecker HT, Frey T, Nomura LE, Trotter J. Selecting fluorochrome conjugates for maximum sensitivity. Cytometry Part A: the journal of the International Society for Analytical Cytology. 2004;62(2):169-173. https://doi.org/10.1002/cyto.a.20092
  • Bigos, M., Stovel, R., & Parks, D. Evaluating multi-color fluorescence data quality among different instruments and different laser powers-methods and results. In CYTOMETRY PART A. 2004;59(1): 42-42.
  • Maecker H, Trotter J. Selecting reagents for multicolor flow cytometry with BD™ LSR II and BD FACSCanto™ systems. Nat Methods. 2008;5(an6–an7). https://doi.org/10.1038/nmeth.f.229
  • Soh KT, Wallace PK. Monitoring of measurable residual disease in multiple myeloma by multiparametric flow cytometry. Current protocols in cytometry. 2019;90(1):e63. https://doi.org/10.1002/cpcy.63
  • O'Brien MC, Bolton WE. Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry. Cytometry: The Journal of the International Society for Analytical Cytology. 1995;19(3):243-255. https://doi.org/10.1002/cyto.990190308
  • Almohammed H, Alhafiz GA, Alghatam FH, Hussen J. The Impact of Camel Leukocytes Fixation on Cell Count and Monoclonal Antibodies Reactivity in Flow Cytometry. World's Veterinary Journal. 2022;12(1):74-80. https://doi.org/10.54203/scil.2022.wvj10
  • Pinto LA, Trivett MT, Wallace D, Higgins J, Baseler M, Terabe M, et al. Fixation and cryopreservation of whole blood and isolated mononuclear cells: Influence of different procedures on lymphocyte subset analysis by flow cytometry. Cytometry Part B: Clinical Cytometry: The Journal of the International Society for Analytical Cytology. 2005;63(1):47-55. https://doi.org/10.1002/cyto.b.20038
  • Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV. Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry Part A: the journal of the International Society for Analytical Cytology. 2005;67(1):4-17. https://doi.org/10.1002/cyto.a.20167
  • Kim SO, Kim J, Okajima T, Cho NJ. Mechanical properties of paraformaldehyde-treated individual cells investigated by atomic force microscopy and scanning ion conductance microscopy. Nano convergence. 2017;4(1):1-8. https://doi.org/10.1186/s40580-017-0099-9
  • Stewart JC, Villasmil ML, Frampton MW. Changes in fluorescence intensity of selected leukocyte surface markers following fixation. Cytometry Part A: the journal of the International Society for Analytical Cytology. 2007;71(6):379-385. https://doi.org/10.1002/cyto.a.20392
  • Van Ewijk W, Van Soest PL, Verkerk A, Jongkind JF. Loss of antibody binding to prefixed cells: fixation parameters for immunocytochemistry. The Histochemical Journal. 1984;16:179-193. https://doi.org/10.1007/bf01003548
  • Murrell-Bussell S, Nguyen D, Schober WD, Scott J, Simpson JL, Elias S, et al. Optimized fixation and storage conditions for FISH analysis of single-cell suspensions. Journal of Histochemistry & Cytochemistry. 1998;46(8):971-973. https://doi.org/10.1177/002215549804600811
  • Hobro AJ, Smith NI. An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging. Vibrational Spectroscopy. 2017;91:31-45. https://doi.org/10.1016/j.vibspec.2016.10.012
  • Ng AA, Lee BT, Teo TS, Poidinger M, Connolly JE. Optimal cellular preservation for high dimensional flow cytometric analysis of multicentre trials. Journal of immunological methods. 2012;385(1-2):79-89. https://doi.org/10.1016/j.jim.2012.08.010
  • Caldwell CW. Preservation of B‐cell‐associated surface antigens by chemical fixation. Cytometry: The Journal of the International Society for Analytical Cytology. 1994;16(3):243-249. https://doi.org/10.1002/cyto.990160308
  • Abay A, Simionato G, Chachanidze R, Bogdanova A, Hertz L, Bianchi P, et al. Glutaraldehyde–a subtle tool in the investigation of healthy and pathologic red blood cells. Frontiers in physiology. 2019;10:514. https://doi.org/10.3389/fphys.2019.00514
  • Lanier LL, Warner NL. Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis. Journal of immunological methods.1981;47(1):25-30. https://doi.org/10.1016/0022-1759(81)90253-2
  • Troussellier M, Courties C, Zettelmaier S. Flow cytometric analysis of coastal lagoon bacterioplankton and picophytoplankton: fixation and storage effects. Estuarine, Coastal and Shelf Science. 1995;40(6):621-633. https://doi.org/10.1006/ecss.1995.0042
  • Diks AM, Bonroy C, Teodosio C, Groenland RJ, De Mooij B, De Maertelaere E, et al. Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research. Journal of immunological methods. 2019; 475:112616. https://doi.org/10.1016/j.jim.2019.06.007
  • Brincat P, Degaetano J, Donaldson C. Evaluation of a method allowing preservation of fresh lymph nodes for flow cytometric immunophenotyping. Cytometry Part B: Clinical Cytometry. 2012;82(4):245-251. https://doi.org/10.1002/cyto.b.21021
  • McCarthy DA, Macey MG, Cahill MR, Newland AC. Effect of fixation on quantification of the expression of leucocyte function‐associated surface antigens. Cytometry: The Journal of the International Society for Analytical Cytology. 1994;17(1):39-49. https://doi.org/10.1002/cyto.990170106