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. 2022 May 2;11(9):1527.
doi: 10.3390/cells11091527.

Comparison of Whole Blood Cryopreservation Methods for Extensive Flow Cytometry Immunophenotyping

Valentina Serra  1 , Valeria Orrù  1 , Sandra Lai  1 , Monia Lobina  1 , Maristella Steri  1 , Francesco Cucca  1   2 , Edoardo Fiorillo  1
Affiliations

Comparison of Whole Blood Cryopreservation Methods for Extensive Flow Cytometry Immunophenotyping

Valentina Serra et al. Cells. .

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.

Keywords: cryopreservation; flow cytometry; immunophenotyping; method comparison; whole blood.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Flow diagram of the study approach. Five blood samples were characterized for 41 immunophenotypes in fresh and matched thawed samples one week after freezing. Eleven additional immunophenotypes were measured in fresh blood and after their freezing by FM method. Thawed and fresh samples were compared by thawed/fresh ratio. Viability was assessed in three samples per method 18 months after storage. SmT: Smart Tubes; CyD: CytoDelics; FM: Freezing Mix; HeS: HetaSep; TVT: TransFix/EDTA Vacuum Tubes.
Figure 2
Figure 2
Comparison of B-cell subsets using the five cryopreservation methods assessed. The cell frequency variation in thawed (T) with respect to fresh (F) sample (represented as T/F ratio) is shown for each cryopreservation method. The red line indicates the ideal ratio of 1, meaning no cell frequency variation between cryopreserved and fresh whole blood. The variation of (A) B cells, (B) IgD positive B-cells and (C) IgA positive B-cells were represented as mean values of 5 samples ± standard deviation. * p-values from the paired t-test ˂ 0.05.
Figure 3
Figure 3
Representative IgA staining of fresh whole blood and thawed samples. The corresponding fresh whole blood is shown on the same line for each freezing method: fresh sample (A) is the control for SmT (B), and TVT (C) methods; fresh sample (D) is used as the control for CyD (E) method, and fresh sample (F) for HeS (G) and FM (H) methods.
Figure 4
Figure 4
Representative CD27 staining of fresh whole blood and thawed samples. The corresponding fresh whole blood is shown on the same line for each freezing method: fresh blood (A) is the control for SmT (B), and TVT (C) methods; fresh sample (D) is used as the control for CyD (E) method, and fresh sample (F) for HeS (G) and FM (H) methods.
Figure 5
Figure 5
Comparison of CD4 T-cell subsets using the five cryopreservation methods. The cell frequency variation in thawed (T) with respect to fresh (F) sample (represented as T/F ratio) is shown for each cryopreservation method. The red line indicates the ideal ratio of 1 (i.e., with no cell frequency variation between cryopreserved and fresh whole blood). The variation of (A) CD4 T cells, (B) PD1+ CD4 T cells, and (C) Tregs were represented as mean values of 5 samples ± standard deviation. * p-values from the paired t-test ˂ 0.05.
Figure 6
Figure 6
Representative CD45RA vs. CCR7 staining of fresh whole blood and thawed samples. The corresponding fresh whole blood is shown in the same line for each freezing method: fresh blood (A) is the control for SmT (B), and TVT (C) methods; fresh sample (D) is used as the control for CyD (E) method, and fresh sample (F) for HeS (G) and FM (H) methods.
Figure 7
Figure 7
Representative CXCR5 staining of fresh whole blood and thawed samples. The corresponding fresh whole blood is shown on the same line for each freezing method: fresh blood (A) is the control for SmT (B), and TVT (C) methods; fresh sample (D) is used as the control for CyD (E) method, and fresh sample (F) for HeS (G) and FM (H) methods.
Figure 8
Figure 8
Representative leukocyte staining of fresh and thawed samples. Granulocytes CD45 (light blue), monocytes CD45 (yellow), and lymphocytes CD45 (green) are represented. Each freezing method is indicated on one line; the corresponding fresh whole blood is shown at the left.
Figure 9
Figure 9
Cell viability after cryopreservation. Leukocyte viability was determined by 7-aminoactimicyn-D negativity in morphologically gated granulocytes (green), monocytes (gray), and lymphocytes (blue); and in CD45+ granulocytes (pink), CD45+ monocytes (red), and CD45+ lymphocytes (purple). A representative sample staining for each method is shown.
Figure 10
Figure 10
Comparison of cytotoxic T cells, NKs, and monocytes in FM treated and fresh samples. The comparison of cell frequency variation in fresh (F) and thawed samples treated with freezing mix (FM) method is shown. (A) CD8+ T cells, (B) NK subsets and (C) monocyte subsets were represented as mean values of five samples ± standard deviation. * p-values from the paired t-test ˂ 0.05.
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