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Chapter 24 : In Situ Gene Amplification and Hybridization Assays: Disease Monitoring Applications Using Flow Cytometry

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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

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Human immunodeficiency virus 1
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Highly Active Antiretroviral Therapy
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Figures

Image of FIGURE 1
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
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Image of FIGURE 2
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 ( ).

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
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Image of FIGURE 3
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 ( ).

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
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Image of FIGURE 4
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
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Image of FIGURE 5
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
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Image of FIGURE 6
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
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Image of FIGURE 7
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 ( ).

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
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Image of FIGURE 8
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
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References

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