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

Yerpasheva D. Kemaykin V. Zhunis G. Aisyn Z. Vorobjev I.
2023 National Scientific Medical Center

Journal of Clinical Medicine of Kazakhstan
2023 #20 Issue 5 47 - 54 pp.

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.

B cells , cell suspension , flow cytometry , immunophenotyping , lymph node , lymphoma , sample fixation , T cells

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Department of Biology, School of Sciences and Humanities, Nazarbayev University, Astana, Kazakhstan
Center for Oncohematology and BMT, National Research Oncology and Transplantology Center, Astana, Kazakhstan
Laboratory of Cell Motility, National Laboratory Astna, Astana, Kazakhstan

Department of Biology
Center for Oncohematology and BMT
Laboratory of Cell Motility

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