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Multicolor flow cytometric analysis of the CD45 antigen provides improved lymphoid cell discrimination in bone marrow
R Festin1, A Björkland, T H Tötterman
1Section for Clinical Immunology and Transfusion Medicine, University Hospital, Uppsala, Sweden.
Insights
This study introduces a new multiparametric method using CD45 antigen expression to accurately identify lymphoid cells. This technique improves lymphocyte gating in bone marrow and tissue biopsies, especially when light scatter alone is insufficient.
Area of Science:
- Immunology
- Cell Biology
- Flow Cytometry
Background:
- Non-leukocytes can mimic lymphocyte light scatter, complicating accurate cell identification in bone marrow and tissue samples.
- Standard lymphocyte gating methods based solely on light scatter are often insufficient for complex biological samples.
Purpose of the Study:
- To develop and validate a multiparametric flow cytometry method for precise lymphoid cell identification.
- To improve the gating strategy for lymphocyte subsets in challenging samples like bone marrow and solid organ biopsies.
Main Methods:
- A triple-staining technique incorporating an anti-CD45 antibody with standard FITC/PE double staining was employed.
- Analysis involved gating on CD45-positive cells combined with light scatter parameters (orthogonal and forward scatter).
- The method was applied to human bone marrow and liver biopsy samples.
Main Results:
- The CD45 gating strategy effectively resolved lymphocyte subsets from debris in liver biopsies.
- Accurate identification of T cells, NK cells, and B cells in bone marrow was achieved.
- Distinguishing cell types in tissue biopsies was enhanced by gating on CD45 expression and light scatter.
Conclusions:
- The described multiparametric CD45 gating method enhances lymphocyte identification in flow cytometry.
- This technique is valuable for immune monitoring of non-malignant tissues and aids lymphocyte gating when light scatter is ambiguous.
- The method is broadly applicable to single-cell samples requiring precise immune cell enumeration.
Abstract:
Samples from bone marrow or non-hematopoietic tissue such as solid organ biopsies often contain an excess of non-leukocytes exhibiting lymphocyte-like light scatter characteristics, making it sometimes difficult to define satisfactory light scatter lymphocyte gates. To circumvent this, we describe here a multiparametric method of identifying lymphoid cells by expression of the CD45 antigen, in conjunction with light scatter parameters. A 'third color'-conjugated anti-CD45 antibody was included with every FITC/PE double staining, thereby permitting live or list mode analysis gating on CD45 positive cells. The triple-staining technique was applied to (a) human bone marrow, showing that special attention has to be given to the enumeration of B cells, and (b) to liver biopsies, where gating on CD45 fluorescence and orthogonal light scatter was shown to clearly resolve all lymphocyte subsets from debris. All cell types examined in tissue biopsies as well as T and NK cells in bone marrow were best distinguished by gating on bright CD45 expression in conjunction with low orthogonal light scatter, while accurate identification of marrow B cells relied upon including all levels of CD45 intensity. The multicolor gating procedure, aimed mainly at immune-monitoring of non-malignant tissues, is applicable to most kinds of single cell samples, and may prove to be an aid for lymphocyte gating in cases where leukocyte populations are not clearly resolved on a light scatter basis alone.
