Limits...
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.

Show MeSH

Related in: MedlinePlus

CD3lowCD20+ cells belong to the B lymphocyte compartment.Phenotyping of CD3lowCD20+ cells revealed CD19 expression in all of the cells, confirming the assignment of CD3lowCD20+ cells to the population of B lymphocytes. B cells showing characteristic features of an activated (CD80, CD86, CD5) and memory (IgG) phenotype are elevated in CD3lowCD20+ B cells compared to the CD3-CD20+ B cell subset. Additionally, expression of the specific T cell markers αβ- and γδ-TCR were only marginally found on the cell surface of CD3lowCD20+ and CD3-CD20+ B cells, respectively, excluding that the CD3lowCD20+ cells are T-B-cell doublets. Extreme values are illustrated as asterisks, outliers as circles. Shown are the results from at least 5 individual patients. Comparative statistical evaluation of different B cell subset regarding cell surface antigen expression was not performed due to the small sample size.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4199681&req=5

pone-0110138-g003: CD3lowCD20+ cells belong to the B lymphocyte compartment.Phenotyping of CD3lowCD20+ cells revealed CD19 expression in all of the cells, confirming the assignment of CD3lowCD20+ cells to the population of B lymphocytes. B cells showing characteristic features of an activated (CD80, CD86, CD5) and memory (IgG) phenotype are elevated in CD3lowCD20+ B cells compared to the CD3-CD20+ B cell subset. Additionally, expression of the specific T cell markers αβ- and γδ-TCR were only marginally found on the cell surface of CD3lowCD20+ and CD3-CD20+ B cells, respectively, excluding that the CD3lowCD20+ cells are T-B-cell doublets. Extreme values are illustrated as asterisks, outliers as circles. Shown are the results from at least 5 individual patients. Comparative statistical evaluation of different B cell subset regarding cell surface antigen expression was not performed due to the small sample size.

Mentions: Phenotypic characterization by means of flow cytometry revealed that both CD3lowCD20+ and CD3-CD20+ cells belong to the population of B lymphocytes (Figure 3). The cells of both subsets were found to express CD19 (Figure 3) and BAFF-R (>98%; data not shown) on their cell surface. Furthermore, both barely expression of specific T cell markers such as αβ-TCR and γδ-TCR (Figure 3) and doublet discrimination by flow cytometry (Figure S1) confirmed that the CD3lowCD20+ lymphocyte population did not consist of T-B-cell clusters. Compared to CD3-CD20+ B cells the population of CD3lowCD20+ B cells contained a substantially higher amount of CD5+, CD80+, CD86+ and surface IgG+ cells (Figure 3). Thus, the CD3lowCD20+ subset predominantly encompasses B cells with an activated or memory phenotype.


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

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

CD3lowCD20+ cells belong to the B lymphocyte compartment.Phenotyping of CD3lowCD20+ cells revealed CD19 expression in all of the cells, confirming the assignment of CD3lowCD20+ cells to the population of B lymphocytes. B cells showing characteristic features of an activated (CD80, CD86, CD5) and memory (IgG) phenotype are elevated in CD3lowCD20+ B cells compared to the CD3-CD20+ B cell subset. Additionally, expression of the specific T cell markers αβ- and γδ-TCR were only marginally found on the cell surface of CD3lowCD20+ and CD3-CD20+ B cells, respectively, excluding that the CD3lowCD20+ cells are T-B-cell doublets. Extreme values are illustrated as asterisks, outliers as circles. Shown are the results from at least 5 individual patients. Comparative statistical evaluation of different B cell subset regarding cell surface antigen expression was not performed due to the small sample size.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110138-g003: CD3lowCD20+ cells belong to the B lymphocyte compartment.Phenotyping of CD3lowCD20+ cells revealed CD19 expression in all of the cells, confirming the assignment of CD3lowCD20+ cells to the population of B lymphocytes. B cells showing characteristic features of an activated (CD80, CD86, CD5) and memory (IgG) phenotype are elevated in CD3lowCD20+ B cells compared to the CD3-CD20+ B cell subset. Additionally, expression of the specific T cell markers αβ- and γδ-TCR were only marginally found on the cell surface of CD3lowCD20+ and CD3-CD20+ B cells, respectively, excluding that the CD3lowCD20+ cells are T-B-cell doublets. Extreme values are illustrated as asterisks, outliers as circles. Shown are the results from at least 5 individual patients. Comparative statistical evaluation of different B cell subset regarding cell surface antigen expression was not performed due to the small sample size.
Mentions: Phenotypic characterization by means of flow cytometry revealed that both CD3lowCD20+ and CD3-CD20+ cells belong to the population of B lymphocytes (Figure 3). The cells of both subsets were found to express CD19 (Figure 3) and BAFF-R (>98%; data not shown) on their cell surface. Furthermore, both barely expression of specific T cell markers such as αβ-TCR and γδ-TCR (Figure 3) and doublet discrimination by flow cytometry (Figure S1) confirmed that the CD3lowCD20+ lymphocyte population did not consist of T-B-cell clusters. Compared to CD3-CD20+ B cells the population of CD3lowCD20+ B cells contained a substantially higher amount of CD5+, CD80+, CD86+ and surface IgG+ cells (Figure 3). Thus, the CD3lowCD20+ subset predominantly encompasses B cells with an activated or memory phenotype.

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
Related in: MedlinePlus