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Updated: Jul 21, 2026
A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood
Published on: February 8, 2016
Comparative flow cytometry-based immunophenotyping analysis of peripheral blood leukocytes before and after fixation
E Vistica Sampino1, J Morgan2, A Chorzalska2
1Division of Pediatric Hematology/Oncologyat Rhode Island Hospital and Warren Alpert Medical School at Brown University, Providence, RI, USA; Center for Cancer and Blood Disorders, Connecticut Children's Medical Center, Hartford, CT, USA.
Insights
Paraformaldehyde fixation minimally impacts most immune cell populations in flow cytometry. However, it significantly alters marker expression in CD4 T cells, regulatory T cells, CD8 T cells, monocytes, and basophils, necessitating careful assessment.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Flow cytometry immunophenotyping is crucial for analyzing immune cell populations in research and clinical settings.
- Timely processing of peripheral blood samples can be challenging, often requiring fixation methods.
- Fixation may introduce artifacts, potentially affecting flow cytometry results.
Purpose of the Study:
- To compare flow cytometry immunophenotyping results of whole blood samples processed with and without paraformaldehyde fixation.
- To identify immune cell populations affected by fixation artifacts.
Main Methods:
- Human peripheral blood samples were stained with 33 fluorochrome-conjugated antibodies.
- Samples were analyzed using flow cytometry with and without a paraformaldehyde fixation step.
- Comparative analysis of 13 immune cell populations was performed.
Main Results:
- Paraformaldehyde fixation did not significantly alter the frequency of B cells, Plasmablasts, Dendritic cells, NK cells, Granulocytes, Neutrophils, Eosinophils, or Hematopoietic Stem/Progenitor Cells.
- Significant marker shifts were observed in CD4 T cells, T regulatory cells, CD8 T cells, Monocytes, and Basophils due to fixation.
- Fixation-induced artifacts varied across different immune cell subsets.
Conclusions:
- Paraformaldehyde fixation can introduce significant artifacts in flow cytometry immunophenotyping, particularly affecting T cell subsets, monocytes, and basophils.
- Pre-experimental evaluation of fixation artifacts is essential when using comprehensive immunoprofiling panels.
- The findings highlight the need to consider fixation feasibility and its impact on specific cell populations.
Abstract:
Flow cytometry based immunophenotyping provides prime insight into cellular population composition and characteristics, and is widely used in basic and clinical research. Challenges in processing peripheral blood samples in a timely manner necessitate protocol adaptations and utilization of fixatives. Fixation, however, may introduce artifacts to the flow cytometry readout. We performed a comparative flow cytometry immunophenotyping analysis of 13 immune cell populations in the whole blood using a staining protocol with and without fixation step. Freshly procured human peripheral blood samples were stained with a panel of 33 fluorochrome-conjugated antibodies. Samples were processed using a protocol with or without a paraformaldehyde-based fixation step, and matching sample pairs were analyzed by flow cytometry. Our results show that paraformaldehyde-based fixation, in comparison to matched unfixed samples, did not significantly affect population distribution and frequency for: B cells, Plasmablasts, Dendritic cells, NK cells, Granulocytes, Neutrophils, Eosinophils, or Hematopoietic Stem/Progenitor Cells. However, fixation led to significant marker shifts in the subpopulation distribution in CD4, T regulatory, CD8, Monocytes, and Basophils. These results indicate the importance of pre-experimental assessment of fixation-introduced artifacts in the flow cytometry output when considering the feasibility of fresh processing. This is especially important for samples analyzed using comprehensive exploratory immunoprofiling panels.
