Abstract
Determine if the COBAS® AmpliPrep/COBAS® TaqMan® System (COBAS® System) could be used to quantify viral loads of HIV and HBV in FFPE biopsies using as input sample purified RNA and DNA, respectively.
Cell lines containing HIV (CRL-8993 producing multiple non-infective copies of the HIV genome), HBV (CRL-2235 containing the genome of the HBV) and negative (Jurkat Clone E6-1) were used. Formalin treated (fixed) and non-treated cells suspended in SPEX buffer were analyzed in COBAS® System. RNA from non-treated cells was purified using QIAamp RNA Blood Mini Kit (Qiagen). RNA or DNA of fixed cells and FFPE cells were purify in a Maxwell® 16 LEV (Promega) with the corresponding Maxwell® 16 LEV FFPE RNA or DNA extraction kit. RNA and DNA were diluted in SPEX buffer for analysis.
HIV cp/ml obtained in the COBAS® System with fixed CRL-8993 cells were 100 times lower than with non-treated cells. HIV cp/ml obtained with non-treated cells was similar to counts obtained with RNA from the same number of cells. Fixed cells RNA gave about half the cp/ml obtained by non-treated cells RNA. The drop was due to lower fixed cells RNA recovery, as adjusted values to cp/ng of RNA added showed no difference between fixed and non-treated cells. FFPE cells were prepared from dilution series of CRL-8993 into jurkat cells, maintaining a constant cellular density. The cp/ml and cp/μg of RNA obtained with RNA from FFPE curls of each cellular dilution showed high degree of correlation with the number of FFPE CRL-8993 cells. Quantitation of HBV was possible in CRL-2235 cells suspended in SPEX buffer. However, the same was not achieved in fixed cells. Quantitation of HBV in DNA from fixed and non-treated cells was very similar, with superimpose best fit equations.
The COBAS® System can be used in the clinical laboratory to quantitate HIV and HBV in purified RNA and DNA from FFPE samples, respectively. There was no detectable loss of input RNA. This was not directly tested for DNA. It was revealed that the origin of nucleic acid was not a factor in the test as long as the appropriate purification method was applied. The methodology of this study could be extended to quantify HIV and HBV in RNA and DNA from fresh tissue biopsies. The procedure can be extended to other RNA and DNA viruses like HCV and CMV, respectively, for which kits are available.
