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Chapter 24 : In Situ Gene Amplification and Hybridization Assays: Disease Monitoring Applications Using Flow Cytometry
Author:
Bruce K. Patterson
Content Type: Reference
Publication Year:
2006
Category: Immunology
Book DOI:
10.1128/9781555815905
Chapter DOI:
10.1128/9781555815905.ch24
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In Situ Gene Amplification and Hybridization Assays: Disease Monitoring Applications Using Flow Cytometry, Page 1 of 2
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Abstract:
The ideal situation in diagnostics would be if one could in a sensitive and specific manner detect certain disease-causing genes, including infectious agents, without destroying the cells. From a diagnostic perspective, the detection of genes and proteins within a cell would need to be high throughput on a user-friendly platform capable of an expanded menu. This chapter describes multiple approaches of performing molecular biology in a cell. It also describes how to use the assays to gain a better understanding of disease processes and ultimately how the assays can be adopted in clinical laboratories to provide the most comprehensive information on response to a variety of therapies. The powerful combination of single-copy detection in cells combined with the high-throughput cellular analysis platform of flow cytometry led to the elucidation that human immunodeficiency virus type 1 (HIV-1) infected enough cells to account for the severe immune destruction leading to AIDS. In situ self-sustained sequence replication (IS-3SR) is based on the use of primers with attached RNA polymerase initiation sites and the combination of three different enzymes in the same reaction mixture (DNA polymerase, RNase H, and RNA polymerase), resulting in accumulation of target mRNA through the combination of reverse transcription, DNA synthesis, and in vitro transcription. The development of HIV-1 entry inhibitors, most notably the CCR5 inhibitors, will present an exciting opportunity for cell-based diagnostics.
Citation:
Patterson B.
2006.
In Situ Gene Amplification and Hybridization Assays: Disease Monitoring Applications Using Flow Cytometry,
p 222-230.
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.ch24
Figures
In Situ Gene Amplification and Hybridization Assays: Disease Monitoring Applications Using Flow Cytometry
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Citation:
Patterson B.
2006.
In Situ Gene Amplification and Hybridization Assays: Disease Monitoring Applications Using Flow Cytometry,
p 222-230.
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.ch24
/content/10.1128/9781555815905.ch24.ch24fig01
FIGURE 1
Schematic of the HIV-1 life cycle in T lymphocytes and the ideal tests to determine the response to therapy.
10.1128/9781555815905/f0223-01_thmb.gif
10.1128/9781555815905/f0223-01.gif
FIGURE 1
Schematic of the HIV-1 life cycle in T lymphocytes and the ideal tests to determine the response to therapy.
Citation:
Patterson B.
2006.
In Situ Gene Amplification and Hybridization Assays: Disease Monitoring Applications Using Flow Cytometry,
p 222-230.
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.ch24
/content/10.1128/9781555815905.ch24.ch24fig02
FIGURE 2
Representative dot plot of simultaneous immunophenotyping for CD4 and real-time PCR in situ (FISNA) detecting cells containing HIV-1 DNA. Biotinylated or 2,4-dinitrophenol (DNP)-conjugated antibodies survive the thermocycling process (
30
).
10.1128/9781555815905/f0224-01_thmb.gif
10.1128/9781555815905/f0224-01.gif
FIGURE 2
Representative dot plot of simultaneous immunophenotyping for CD4 and real-time PCR in situ (FISNA) detecting cells containing HIV-1 DNA. Biotinylated or 2,4-dinitrophenol (DNP)-conjugated antibodies survive the thermocycling process ( 30 ).
Citation:
Patterson B.
2006.
In Situ Gene Amplification and Hybridization Assays: Disease Monitoring Applications Using Flow Cytometry,
p 222-230.
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.ch24
/content/10.1128/9781555815905.ch24.ch24fig03
FIGURE 3
Representative laser confocal image analysis of real-time PCR in situ (FISNA) detecting cells in the prostate containing HIV-1 DNA following long-term therapy with antiretroviral drugs. These data suggest that reservoirs of HIV-1 DNA-containing cells are not necessarily cleared by long-term therapy (
26
).
10.1128/9781555815905/f0225-01_thmb.gif
10.1128/9781555815905/f0225-01.gif
FIGURE 3
Representative laser confocal image analysis of real-time PCR in situ (FISNA) detecting cells in the prostate containing HIV-1 DNA following long-term therapy with antiretroviral drugs. These data suggest that reservoirs of HIV-1 DNA-containing cells are not necessarily cleared by long-term therapy ( 26 ).
Citation:
Patterson B.
2006.
In Situ Gene Amplification and Hybridization Assays: Disease Monitoring Applications Using Flow Cytometry,
p 222-230.
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.ch24
/content/10.1128/9781555815905.ch24.ch24fig04
FIGURE 4
Monitoring HIV-1 replication in patients on highly active antiretroviral therapy by simultaneous immunophenotyping and ultrasensitive fluorescence in situ hybridization in T lymphocytes (CD4+ CD45RO+) and in monocytes/macrophages (CD14/CD16).
10.1128/9781555815905/f0226-01_thmb.gif
10.1128/9781555815905/f0226-01.gif
FIGURE 4
Monitoring HIV-1 replication in patients on highly active antiretroviral therapy by simultaneous immunophenotyping and ultrasensitive fluorescence in situ hybridization in T lymphocytes (CD4+ CD45RO+) and in monocytes/macrophages (CD14/CD16).
Citation:
Patterson B.
2006.
In Situ Gene Amplification and Hybridization Assays: Disease Monitoring Applications Using Flow Cytometry,
p 222-230.
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.ch24
/content/10.1128/9781555815905.ch24.ch24fig05
FIGURE 5
Combining plasma viral load (pVL) and simultaneous immunophenotyping and ultrasensitive fluorescence in situ hybridization in T lymphocytes (CD4+ CD45RO+) and in monocytes/macrophages (CD14/CD16) to determine response of multiple tissue compartments to highly active antiretroviral therapy. This extremely powerful test bundle identifies individuals with a maximal response to therapy in all compartments (left) and those individuals with persistent viral replication (in cells) despite undetectable plasma viral load (right). As one might predict for an individual with persistent viral replication, plasma viral load increased during the course of therapy.
10.1128/9781555815905/f0226-02_thmb.gif
10.1128/9781555815905/f0226-02.gif
FIGURE 5
Combining plasma viral load (pVL) and simultaneous immunophenotyping and ultrasensitive fluorescence in situ hybridization in T lymphocytes (CD4+ CD45RO+) and in monocytes/macrophages (CD14/CD16) to determine response of multiple tissue compartments to highly active antiretroviral therapy. This extremely powerful test bundle identifies individuals with a maximal response to therapy in all compartments (left) and those individuals with persistent viral replication (in cells) despite undetectable plasma viral load (right). As one might predict for an individual with persistent viral replication, plasma viral load increased during the course of therapy.
Citation:
Patterson B.
2006.
In Situ Gene Amplification and Hybridization Assays: Disease Monitoring Applications Using Flow Cytometry,
p 222-230.
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.ch24
/content/10.1128/9781555815905.ch24.ch24fig06
FIGURE 6
Simultaneous detection of the HIV-1 coreceptor CCR5 and HIV-1 replication. This combination can be used to monitor response to HIV-1 entry and CCR5 inhibitors. Combining this with CXCR4 in a third color will allow the monitoring of potential tropism shifts.
10.1128/9781555815905/f0227-01_thmb.gif
10.1128/9781555815905/f0227-01.gif
FIGURE 6
Simultaneous detection of the HIV-1 coreceptor CCR5 and HIV-1 replication. This combination can be used to monitor response to HIV-1 entry and CCR5 inhibitors. Combining this with CXCR4 in a third color will allow the monitoring of potential tropism shifts.
Citation:
Patterson B.
2006.
In Situ Gene Amplification and Hybridization Assays: Disease Monitoring Applications Using Flow Cytometry,
p 222-230.
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.ch24
/content/10.1128/9781555815905.ch24.ch24fig07
FIGURE 7
In situ detection of HPV E6 and E7 mRNA in cervical cytology samples from women with highgrade squamous intraepithelial lesions (HSIL) and women without cytologic abnormalities (WNL). The difference in the percentage of E6 and E7 mRNA-expressing cells is highly statistically significant (
24
).
10.1128/9781555815905/f0228-01_thmb.gif
10.1128/9781555815905/f0228-01.gif
FIGURE 7
In situ detection of HPV E6 and E7 mRNA in cervical cytology samples from women with highgrade squamous intraepithelial lesions (HSIL) and women without cytologic abnormalities (WNL). The difference in the percentage of E6 and E7 mRNA-expressing cells is highly statistically significant ( 24 ).
Citation:
Patterson B.
2006.
In Situ Gene Amplification and Hybridization Assays: Disease Monitoring Applications Using Flow Cytometry,
p 222-230.
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.ch24
/content/10.1128/9781555815905.ch24.ch24fig08
FIGURE 8
Ultrasensitive fluorescence in situ hybridization detection of HPV E6 and E7 mRNA expression in liquid-based cervical cytology samples. Hybrid Capture II high-risk HPV-positive samples (top row) and Hybrid Capture II high-risk HPV-negative samples were hybridized and run on a Guava Technologies PCA-96 capillary cytometer.
10.1128/9781555815905/f0228-02_thmb.gif
10.1128/9781555815905/f0228-02.gif
FIGURE 8
Ultrasensitive fluorescence in situ hybridization detection of HPV E6 and E7 mRNA expression in liquid-based cervical cytology samples. Hybrid Capture II high-risk HPV-positive samples (top row) and Hybrid Capture II high-risk HPV-negative samples were hybridized and run on a Guava Technologies PCA-96 capillary cytometer.
Citation:
Patterson B.
2006.
In Situ Gene Amplification and Hybridization Assays: Disease Monitoring Applications Using Flow Cytometry,
p 222-230.
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.ch24
References
/content/book/10.1128/9781555815905.ch24
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4.
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5.
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9.
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16.
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17.
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18.
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19.
Korber, B. T.,, K. J. Kunstman,, B. K. Patterson,, M. Furtado,, M. M. McEvilly,, R. Levy, and , S. M. Wolinsky. 1994. Genetic differences between blood- and brain-derived viral sequences from human immunodeficiency virus type 1-infected patients: evidence of conserved elements in the V3 region of the envelope protein of brainderived sequences. J. Virol 68: 7467– 7481.
20.
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21.
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23.
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24.
Narimatsu, R., and , B. K. Patterson. 2005. High-throughput cervical cancer screening using intracellular human papillomavirus E6 and E7 mRNA quantification by flow cytometry. Am J. Clin. Pathol. 123: 716– 723.
25.
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26.
Paranjpe, S.,, J. Craigo,, B. Patterson,, M. Ding,, P. Barroso,, L. Harrison,, R. Montelaro, and , P. Gupta. 2002. Subcompartmentalization of HIV-1 quasispecies between seminal cells and seminal plasma indicates their origin in distinct genital tissues. AIDS Res. Hum. Retrovir. 18: 1271– 1280.
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29.
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30.
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31.
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32.
Patterson, B. K.,, S. McCallister,, M. Schutz,, J. N. Siegel,, K. Shults,, Z. Flener, and , A. Landay. 2001. Persistence of intracellular HIV-1 mRNA correlates with HIV-1-specific immune responses in infected subjects on stable HAART. AIDS 15: 1635– 1641.
33.
Patterson, B. K.,, V. L. Mosiman,, L. Cantarero,, M. Furtado,, M. Bhattacharya, and , C. Goolsby. 1998. Detection of HIV-RNA-positive monocytes in peripheral blood of HIV-positive patients by simultaneous flow cytometric analysis of intracellular HIV RNA and cellular immunophenotype. Cytometry 31: 265– 274.
34.
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35.
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37.
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38.
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40.
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41.
Spira, A. I.,, P. A. Marx,, B. K. Patterson,, J. Mahoney,, R. A. Koup,, S. M. Wolinsky, and , D. D. Ho. 1996. Cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques. J. Exp. Med. 183: 215– 225.
42.
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44.
Zhang, L.,, B. Ramratnam,, K. Tenner-Racz,, Y. He,, M. Vesanen,, S. Lewin,, A. Talal,, P. Racz,, A. S. Perelson,, B. T. Korber,, M. Markowitz, and , D. D. Ho. 1999. Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy. N. Engl. J. Med. 340: 1605– 1613.