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Chapter 18 : Flow Cytometry-Based Immunophenotyping Method and Applications

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Abstract:

This chapter provides a broad description of the principles of flow cytometric immunophenotyping, provides specific methods for lymphocyte enumeration with particular emphasis on CD4 T-cell counting, and reviews quality assurance issues that improve the accuracy and reproducibility of overall immunophenotyping results. Samples for immunophenotyping are prepared by incubation with fluorochrome-labeled monoclonal antibodies (MAbs), and red blood cells are removed by lysis to prepare the sample for analysis on the flow cytometer. MAbs are created by the fusion of B-cell tumors and primary B cells previously selected to make antibodies to only one epitope of a specific antigen. The lyse/no-wash (LNW) method permits single-platform (SPT) absolute counting. A second protocol is provided for laboratories that perform more advanced immunophenotyping, using MAbs that must be used in lyse/wash (LW) sample procedures prior to running on the cytometer. The use of a sample handling device which can be loaded with sample tubes inside the biosafety cabinet and attached to the flow cytometer for automated processing reduces sample handling and samples can be safely vortexed throughout sample acquisition. CD4 T-cell levels are important measures for staging human immunodeficiency virus (HIV)-1 disease and predicting disease progression. In HIV-1-infected adults the CD4 T-cell absolute count is the most useful measure of disease progression, while in children CD4 T-cell percentage is preferred because of high variability in lymphocyte counts.

Citation: Hultin L, Hultin P. 2006. Flow Cytometry-Based Immunophenotyping Method and Applications, p 147-157. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch18

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Figures

Image of FIGURE 1
FIGURE 1

LNW sample preparation. Plot A demonstrates optimal CD45 lymphocyte gating with SSC adjusted to reveal distinct leukocyte populations. Plots B, C, D, and E show lymphocyte subsets with the R1 gate applied and cursor settings guided by the negatively stained populations. Results would be reported as follows: CD4 = 45%, CD8 = 41%, NK cells = 4%, and B cells = 10%. The total T-cell percentage (CD3) is 85% in both tubes sampled, providing confidence in the overall result. Plot F is gated on CD45 CD3 lymphocytes, illustrating the additional information available when the four-color immunophenotyping panel is used. Plot G is an ungated display of FL1 versus FL2 used for two different applications. The R3 gating region enumerates counting beads for SPT absolute-count determinations. The R2 gating region includes all CD8 stained events, which are then backgated through the original lymphocyte gate (R1) in plot H to determine recovery for quality assurance. Finally, plot I shows a sample that bound antibody nonspecifically. This staining artifact was successfully corrected for reporting subset percentages by restaining an aliquot of washed blood.

Citation: Hultin L, Hultin P. 2006. Flow Cytometry-Based Immunophenotyping Method and Applications, p 147-157. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch18
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Image of FIGURE 2
FIGURE 2

LW sample preparation. Plots A, B, C, and D are analyzed together to establish an FSC-versus-SSC lymphocyte gate with the highest purity and recovery of lymphocytes. Plots E and F show poor backgating of CD8 stained events due to cell clumping (escapees). Plot G measures elevated coexpression of HLA-DR versus CD38 gated on CD3 CD8 lymphocytes from an HIV-1-infected donor. Plots H and I use CD45RA in combination with either CD27 (H) or CD31 (I) to define CD4 naïve cells.

Citation: Hultin L, Hultin P. 2006. Flow Cytometry-Based Immunophenotyping Method and Applications, p 147-157. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch18
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References

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Tables

Generic image for table
TABLE 1

MAb panels for HIV immunophenotyping

Citation: Hultin L, Hultin P. 2006. Flow Cytometry-Based Immunophenotyping Method and Applications, p 147-157. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch18
Generic image for table
TABLE 2

Expanded T-cell panels for HIV immunophenotyping

Citation: Hultin L, Hultin P. 2006. Flow Cytometry-Based Immunophenotyping Method and Applications, p 147-157. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch18

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