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CD3-positive B cells: a storage-dependent phenomenon.

Nagel A, Möbs C, Raifer H, Wiendl H, Hertl M, Eming R - PLoS ONE (2014)

Bottom Line: Since cellular subsets are often classified by phenotypic analyses, our results indicate that ex vivo cellular classification in peripheral blood might result in misleading interpretations.Therefore, in order to obtain results reflecting the in vivo situation, it is suggested to minimize times of ex vivo blood storage after isolation of PBMC.Moreover, to enable reproducibility of results between different research groups and multicenter studies, we would emphasize the necessity to specify and standardize the storage conditions, which might be the basis of particular findings.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology and Allergology, Philipps University Marburg, Marburg, Germany; Institute of Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

ABSTRACT
The majority of clinical studies requires extensive management of human specimen including e.g. overnight shipping of blood samples in order to convey the samples in a central laboratory or to simultaneously analyze large numbers of patients. Storage of blood samples for periods of time before in vitro/ex vivo testing is known to influence the antigen expression on the surface of lymphocytes. In this context, the present results show for the first time that the T cell antigen CD3 can be substantially detected on the surface of human B cells after ex vivo storage and that the degree of this phenomenon critically depends on temperature and duration after blood withdrawal. The appearance of CD3 on the B cell surface seems to be a result of contact-dependent antigen exchange between T and B lymphocytes and is not attributed to endogenous production by B cells. Since cellular subsets are often classified by phenotypic analyses, our results indicate that ex vivo cellular classification in peripheral blood might result in misleading interpretations. Therefore, in order to obtain results reflecting the in vivo situation, it is suggested to minimize times of ex vivo blood storage after isolation of PBMC. Moreover, to enable reproducibility of results between different research groups and multicenter studies, we would emphasize the necessity to specify and standardize the storage conditions, which might be the basis of particular findings.

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Numbers of CD3lowCD20+ B cells are time- and temperature-dependent.The increase in the number of CD3lowCD20+ B cells was time-dependent, but independent of storage conditions (4°C versus room temperature (RT) versus humidified atmosphere at 37°C, 5% CO2). CD3lowCD20+ B cells were detectable at earlier time points and more pronounced at 4°C storage compared to RT and 37°C incubation, respectively. Shown are the results of two independent experiments performed in the same patient.
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pone-0110138-g006: Numbers of CD3lowCD20+ B cells are time- and temperature-dependent.The increase in the number of CD3lowCD20+ B cells was time-dependent, but independent of storage conditions (4°C versus room temperature (RT) versus humidified atmosphere at 37°C, 5% CO2). CD3lowCD20+ B cells were detectable at earlier time points and more pronounced at 4°C storage compared to RT and 37°C incubation, respectively. Shown are the results of two independent experiments performed in the same patient.

Mentions: Following B cells over time of storage revealed increasing numbers of CD3lowCD20+ cells, independent of the storing condition (humidified atmosphere at 37°C and 5% CO2versus RT versus 4°C) compared to freshly isolated cells (Figure 6). However, the largest increase in CD3lowCD20+ cells was observed after storage of isolated PBMC at 4°C. Moreover, each of the tested storing conditions resulted in a time-dependent increase in CD3lowCD20+ B cells compared to freshly isolated cells (Figure 6). Thus, it seems that non-physiological storing conditions (4°C>RT >37°C) of blood samples might favor the generation of CD3lowCD20+ B cells.


CD3-positive B cells: a storage-dependent phenomenon.

Nagel A, Möbs C, Raifer H, Wiendl H, Hertl M, Eming R - PLoS ONE (2014)

Numbers of CD3lowCD20+ B cells are time- and temperature-dependent.The increase in the number of CD3lowCD20+ B cells was time-dependent, but independent of storage conditions (4°C versus room temperature (RT) versus humidified atmosphere at 37°C, 5% CO2). CD3lowCD20+ B cells were detectable at earlier time points and more pronounced at 4°C storage compared to RT and 37°C incubation, respectively. Shown are the results of two independent experiments performed in the same patient.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199681&req=5

pone-0110138-g006: Numbers of CD3lowCD20+ B cells are time- and temperature-dependent.The increase in the number of CD3lowCD20+ B cells was time-dependent, but independent of storage conditions (4°C versus room temperature (RT) versus humidified atmosphere at 37°C, 5% CO2). CD3lowCD20+ B cells were detectable at earlier time points and more pronounced at 4°C storage compared to RT and 37°C incubation, respectively. Shown are the results of two independent experiments performed in the same patient.
Mentions: Following B cells over time of storage revealed increasing numbers of CD3lowCD20+ cells, independent of the storing condition (humidified atmosphere at 37°C and 5% CO2versus RT versus 4°C) compared to freshly isolated cells (Figure 6). However, the largest increase in CD3lowCD20+ cells was observed after storage of isolated PBMC at 4°C. Moreover, each of the tested storing conditions resulted in a time-dependent increase in CD3lowCD20+ B cells compared to freshly isolated cells (Figure 6). Thus, it seems that non-physiological storing conditions (4°C>RT >37°C) of blood samples might favor the generation of CD3lowCD20+ B cells.

Bottom Line: Since cellular subsets are often classified by phenotypic analyses, our results indicate that ex vivo cellular classification in peripheral blood might result in misleading interpretations.Therefore, in order to obtain results reflecting the in vivo situation, it is suggested to minimize times of ex vivo blood storage after isolation of PBMC.Moreover, to enable reproducibility of results between different research groups and multicenter studies, we would emphasize the necessity to specify and standardize the storage conditions, which might be the basis of particular findings.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology and Allergology, Philipps University Marburg, Marburg, Germany; Institute of Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

ABSTRACT
The majority of clinical studies requires extensive management of human specimen including e.g. overnight shipping of blood samples in order to convey the samples in a central laboratory or to simultaneously analyze large numbers of patients. Storage of blood samples for periods of time before in vitro/ex vivo testing is known to influence the antigen expression on the surface of lymphocytes. In this context, the present results show for the first time that the T cell antigen CD3 can be substantially detected on the surface of human B cells after ex vivo storage and that the degree of this phenomenon critically depends on temperature and duration after blood withdrawal. The appearance of CD3 on the B cell surface seems to be a result of contact-dependent antigen exchange between T and B lymphocytes and is not attributed to endogenous production by B cells. Since cellular subsets are often classified by phenotypic analyses, our results indicate that ex vivo cellular classification in peripheral blood might result in misleading interpretations. Therefore, in order to obtain results reflecting the in vivo situation, it is suggested to minimize times of ex vivo blood storage after isolation of PBMC. Moreover, to enable reproducibility of results between different research groups and multicenter studies, we would emphasize the necessity to specify and standardize the storage conditions, which might be the basis of particular findings.

Show MeSH