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Updated: Sep 23, 2025
Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry
Published on: June 26, 2018
Comparison of Whole Blood Cryopreservation Methods for Extensive Flow Cytometry Immunophenotyping
Valentina Serra1, Valeria Orrù1, Sandra Lai1
1Institute for Genetic and Biomedical Research, National Research Council (CNR), Cittadella Universitaria di Monserrato, 09042 Cagliari, Italy.
Insights
Cryopreservation of blood samples is crucial for large studies. Dimethyl sulfoxide-based methods best preserve immune cell populations for flow cytometry analysis, closely matching fresh blood results.
Area of Science:
- Immunology
- Biotechnology
Background:
- Flow cytometry is essential for studying the human immune system.
- Maintaining sample quality during large-scale, multicenter studies is challenging.
- Effective cryopreservation of whole blood is vital for reliable immunophenotyping.
Purpose of the Study:
- To compare immunophenotypic data quality from fresh blood versus five cryopreservation methods.
- To evaluate the impact of cryopreservation on 41 distinct immune cell populations.
Main Methods:
- Blood samples were cryopreserved using three fixative-based and two dimethyl sulfoxide (DMSO)-based methods.
- Immunophenotyping was performed using flow cytometry to quantify 41 immune cell populations.
- Data from cryopreserved samples were compared to fresh blood controls.
Main Results:
- Fixative-based methods compromised the detection of critical B and T cell markers (e.g., CD27, CXCR3, CCR6).
- DMSO-based methods allowed reliable discrimination of most immune cell populations post-thaw.
- One DMSO-based method, using media with DMSO, yielded results most similar to fresh blood.
Conclusions:
- Cryopreservation significantly impacts immunophenotyping data quality.
- DMSO-based cryopreservation is superior to fixative-based methods for preserving immune cell populations.
- Specific DMSO-containing media offer the most reliable approach for long-term storage of blood for flow cytometry analysis.
Abstract:
Fresh blood immunophenotyping by flow cytometry, based on the reliable simultaneous detection of several markers in a cell, is the method of choice to study the circulating human immune system. Especially in large and multicenter studies, high sample quality is difficult to achieve, and adequate collection and storage of samples with fine-tuned whole blood cryopreservation is mandatory. Here, we compared the quality of immunophenotypic data obtained from fresh blood with those obtained after five cryopreservation methods by quantifying the levels of 41 immune cell populations. They comprised B and T lymphocyte subsets and their maturation stages, as well as monocytes and granulocytes. Three methods used fixative solutions and two other methods used dimethyl sulfoxide solutions to preserve cell viability. The fixative methods prevented detection of markers critical for identification of B and T cell subsets, including CD27, CXCR3, and CCR6. The other two methods permitted reliable discrimination of most immune-cell populations in thawed samples, though some cell frequencies varied compared to the corresponding fresh sample. Of those two methods, the one preserving blood in media containing dimethyl sulfoxide produced results that were most similar to those with fresh samples.
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