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Chapter 11.16 : Whole-Blood Lymphocyte Immunophenotyping Using Cell Surface Markers by Flow Cytometry

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Whole-Blood Lymphocyte Immunophenotyping Using Cell Surface Markers by Flow Cytometry, Page 1 of 2

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Abstract:

Whole blood can be stained with fluorochrome-labeled monoclonal antibodies against antigen markers found on the surfaces of lymphocytes. The stained samples are treated with a lysing solution to destroy erythrocytes. The flow cytometer is an instrument capable of rapid, quantitative, multiparameter analysis of heterogeneous cell populations on a cell-by-cell basis (single-cell analysis). During acquisition, the cells travel past the laser beam and scatter the laser light. The stained cells fluoresce. These scatter, and fluorescence signals provide information about the cells' size, internal complexity, and relative fluorescence intensity. The percentage of fluorescent cells is determined for each antibody. In addition, the use of the light scatter measurements from the individual cells along with fluorescence allows identification of the lymphocyte population.

Citation: Garcia L. 2010. Whole-Blood Lymphocyte Immunophenotyping Using Cell Surface Markers by Flow Cytometry, p 242-249. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch11.16
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Figures

Image of Figure 11.16-1
Figure 11.16-1

(A) Forward versus side light scatter histogram showing the smaller light scatter gate (lymphocytes) which is used for analyzing the remaining tubes from that specimen. (B) CD45/CD14 histogram from gated R1. The lymphocyte purity is the percentage of positive cells within the gate that express bright CD45-positive cells that are negative for CD14 (quadrant LR). The purity in this sample is 97%. The number of cells in quadrant LR which are bright CD45 positive and negative for CD14 is the number of gated lymphocytes (3,812 cells). (C) The same light scatter gate as in panel A except that it has a rather large light scatter region drawn around the lymphocytes (R2). (D) The twoparameter histogram of CD45/CD14 fluorescence is gated on R2. The number of cells in quadrant LR which are bright CD45 positive and negative for CD14 is the total number of lymphocytes (3,847 cells). The lymphocyte recovery is 99%, obtained as follows: (3,812/3,847) × 100. FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Citation: Garcia L. 2010. Whole-Blood Lymphocyte Immunophenotyping Using Cell Surface Markers by Flow Cytometry, p 242-249. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch11.16
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Image of Figure 11.16-2
Figure 11.16-2

Four-color immunophenotyping using CD45 and side scatter gating. (A) Gate (R1) on the bright CD45 nongranular population (lymphocytes). This is the region used for analysis of both tubes 1 and 2. (B) CD3 CD4 (quadrant 2) from tube 1. (C) CD3 CD8 (quadrant 2) from tube 1. (D) CD CD4 (quadrant 1) from tube 2. (E) CD3 CD(1656) (quadrant 1) from tube 2. FITC, fluorescein isothiocyanate; APC, allophycocyanin; PE, phycoerythrin.

Citation: Garcia L. 2010. Whole-Blood Lymphocyte Immunophenotyping Using Cell Surface Markers by Flow Cytometry, p 242-249. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch11.16
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Download as Powerpoint

References

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1. Centers for Disease Centrol and Prevention. 1997. Revised guidelines for performing CD4+ T-cell determinations in persons infected with human immunodeficiency virus (HIV). MMWR Morb. Mortal. Wkly. Rep. 46(RR-2):129.
2. NCCLS. 1998. Clinical Applications of Flow Cytometry: Quality Assurance and Immunophenotyping of Lymphocytes. Document H42-A, vol. 18, no. 21. NCCLS, Wayne, PA.
3. Nicholson, J.,, P. Kidd,, F. Mandy,, D. Livnat,, and J. Kagan. 1996. Three-color supplement to the NIAID DAIDS guideline for flow cytometric immunophenotyping. Cytometry 26:227230.

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