Detection of human T-cell lymphotrophic virus type I in archival tissue specimens.
OBJECTIVE
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.
Wood
G S
GS
Department of Dermatology, Case Western Reserve University, Cleveland, Ohio, USA.
Ruffo
A
A
Salvekar
A
A
Henghold
W
W
Takeshita
M
M
Kikuchi
M
M
D000328:Adult; D000368:Aged; D001706:Biopsy; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis; D015368:Human T-lymphotropic virus 1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004279:DNA, Viral