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Comparative study with in situ hybridization and immunocytochemistry in detection of HIV-1 in formalin-fixed
H Daugharty1, E G Long, B L Swisher
1Division of Immunologic, Oncologic, and Hematologic Diseases, Centers for Disease Control, Atlanta, Georgia 30333.
Insights
In situ hybridization (ISH) was most sensitive for detecting human immunodeficiency virus (HIV-1) in cell lines, while immunocytochemistry with polyclonal antibody (ICC-p) showed higher sensitivity and lower variability in cocultured cells.
Area of Science:
- Immunology
- Virology
- Biotechnology
Background:
- Accurate detection of human immunodeficiency virus (HIV-1) is crucial for understanding viral pathogenesis and developing effective treatments.
- Formalin-fixed, paraffin-embedded tissues are commonly used for histological analysis, but their impact on the sensitivity of HIV-1 detection methods needs careful evaluation.
- Standardization of immunohistological techniques is essential for reliable diagnosis and research.
Purpose of the Study:
- To compare the diagnostic sensitivity of three distinct immunohistological techniques for detecting HIV-1 in infected human lymphocytes.
- To evaluate the performance of in situ hybridization (ISH) and two immunocytochemistry (ICC) methods using different antibodies.
- To identify the most reliable method for detecting HIV-1 markers in processed cell samples.
Main Methods:
- Comparison of three techniques: in situ hybridization (ISH) with HIV-1 cDNA, immunocytochemistry with a monoclonal p24 antibody (ICC-m), and immunocytochemistry with a polyclonal AIDS patient antibody (ICC-p).
- Optimization of enzyme digestion and antibody reaction conditions for all tested methods.
- Testing of both HIV-1 infected and non-infected cultured human lymphocytes, formalin-fixed and paraffin-embedded.
Main Results:
- All three methods uniformly yielded negative results for non-infected control cells.
- In situ hybridization (ISH) demonstrated the highest positivity rate in infected continuous cell lines (p ≤ 0.0001).
- Immunocytochemistry with polyclonal antibody (ICC-p) showed higher sensitivity and lower variability compared to ICC-m and ISH in cocultured cells (p ≤ 0.0001).
Conclusions:
- The choice of immunohistological technique significantly impacts the sensitivity of HIV-1 detection, with ISH being superior for cell lines and ICC-p for cocultured cells.
- Variability in test procedures, reagents, and specimen processing are critical technological parameters affecting HIV-1 detection.
- These findings have implications for the detection of other viral markers in infected tissues, emphasizing the need for method standardization.
Abstract:
The sensitivities of three immunohistological techniques were compared in this study for detecting human immunodeficiency virus (HIV-1) in infected cultured human lymphocytes that had been formalin-fixed and paraffin-embedded. The techniques included in situ hybridization (ISH) with HIV-1 cDNA; immunocytochemistry with HIV-1 p24 monoclonal antibody (ICC-m); and immunocytochemistry with HIV-1 polyclonal antibody from a patient with acquired immunodeficiency syndrome (AIDS) (ICC-p). Procedures were optimized for enzyme digestion and for antibody reaction conditions. HIV-1--infected cells and noninfected control cells were tested. Noninfected controls were uniformally negative by all three methods. Infected cells had the highest positivity rate by the ISH method (p less than or equal to 0.0001), and the ICC-p method was more positive than the ICC-m (p less than or equal to 0.0001). Both the ICC-p and the ICC-m techniques were more positive with the cocultivated cell cultures than the ISH, which was more sensitive with the infected continuous cell line (P less than or equal to 0.0001). The ICC-p method had a lower standard deviation on positive results than either the ICC-m or ISH method. The variability observed with these test procedures, reagents, and specimens suggests that these are important technological parameters in detecting p24, with implications for detecting other HIV-1 markers in infected tissues.