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Published on: March 8, 2012
Detection of human T-cell lymphotrophic virus type I in archival tissue specimens
G S Wood1, A Ruffo, A Salvekar
1Department of Dermatology, Case Western Reserve University, Cleveland, Ohio, USA.
Insights
A new polymerase chain reaction (PCR) method detects human T-cell lymphotropic virus type I (HTLV-I) in archival biopsy specimens. This technique enables the study of HTLV-I in various tissues, aiding research into associated diseases.
Area of Science:
- Virology
- Molecular Biology
- Pathology
Background:
- Human T-cell lymphotropic virus type I (HTLV-I) is linked to adult T-cell leukemia/lymphoma (ATL).
- Analyzing archival biopsy specimens is crucial for understanding HTLV-I's role in disease pathogenesis.
- Detecting HTLV-I in preserved tissues presents technical challenges due to DNA degradation.
Purpose of the Study:
- To develop a sensitive and specific polymerase chain reaction (PCR)-based method for detecting HTLV-I proviral DNA in archival paraffin-embedded biopsy specimens.
- To validate the method's specificity using Southern blot analysis and nucleotide sequencing.
- To establish controls for DNA integrity and T-cell adequacy in archival samples.
Main Methods:
- Development of a PCR assay targeting the HTLV-I pX region.
- Use of Southern blot analysis with a nested oligonucleotide probe for specificity confirmation.
- Nucleotide sequencing for precise identification of PCR products.
- Inclusion of nucleophosmin and T-cell receptor-gamma genes as controls for DNA quality and quantity.
- Testing on archival skin and lymph node biopsy specimens from Japanese patients with HTLV-I-seropositive ATL.
Main Results:
- Formalin fixation and paraffin embedding reduced assay sensitivity by 100-fold compared to fresh samples.
- HTLV-I pX sequences were reliably detected in paraffin-embedded MT4 T cells and ATL biopsy specimens.
- Both formalin and B5 fixation methods proved suitable for the assay.
- The developed PCR assay demonstrated 100% specificity for HTLV-I-infected tissues.
Conclusions:
- The established PCR method facilitates the investigation of HTLV-I's role in human diseases by enabling analysis of diverse archival tissue specimens.
- The integrated controls enhance the reliability of PCR-based studies on T cells, mitigating false-negative results from DNA degradation or insufficient cell density.
- This technique offers a valuable tool for retrospective studies on HTLV-I-associated conditions.
Objective And Design:
To develop a method for the detection of human T-cell lymphotropic virus type I (HTLV-I) in archival biopsy specimens. A polymerase chain reaction-based gene amplification method was developed to detect HTLV-I proviral DNA in paraffin-embedded specimens. The specificity of the polymerase chain reaction products was controlled by Southern blot analysis using a nested oligonucleotide probe and by nucleotide sequencing. The nucleophosmin gene and the T-cell receptor-gamma gene were used as controls for the integrity and adequacy of total DNA and T-cell DNA, respectively. This study was conducted with patients referred to an academic medical center. Biopsy specimens were obtained from lesional skin or lymph node from Japanese patients with HTLV-I seropositive adult T-cell leukemia/lymphoma. The main outcome measure was the ability to detect HTLV-I pX region proviral DNA.
Results:
Comparative analysis of DNA extracted from fresh samples of the HTLV-I infected MT4T-cell line demonstrated that formalin fixation and paraffin embedding resulted in a 100-fold reduction in sensitivity of the assay. Nevertheless, HTLV-I pX sequences were still readily detectable in paraffin-embedded samples of MT4 T cells and adult T-cell leukemia/lymphoma specimens. Both formalin and B5 fixation were suitable for the assay that was 100% specific for HTLV-I-infected tissues.
Conclusions:
The use of this method should greatly facilitate investigation of the role of HTLV-I in human diseases by allowing analysis of a wide variety of archival tissue specimens. In addition, the controls designed for the current study can be used in a variety of other polymerase chain reaction-based studies of T cells to ensure against false-negative results caused by DNA degradation or inadequate T-cell density.
