Need for procedural details in the protocol for specimen processing by the MagNA Pure LC Instrument.

Abstract

We read with interest the favorable findings of Holzl et al. (1) showing good agreement of plasma HIV-1 RNA measurements (by the ultrasensitive COBAS AMPLICOR HIV-1 Monitor test) by using the conventional (manual) RNA purification method and the fully automated method performed with the MagNA Pure LC instrument. Their findings would enable diagnostic laboratories to use a commercially available automated nucleic acid purification system for a complex molecular assay and to save considerable resources. The authors stated that they subjected the low-positive, high-positive, and negative controls provided with the COBAS AMPLICOR HIV-1 Monitor test to RNA purification and reverse transcription (RT)-PCR analysis in both the conventional and automated methods, but no further procedural details on this part of their evaluation were provided. In the manufacturer-specified manual RNA purification procedures for the ultrasensitive assay, 12.5 μl of each control reagent is added to the respective tube containing 600 μl of lysis buffer and the centrifuged pellet of the normal human plasma provided in the assay kit. For processing clinical specimens, 500 μl of each patient plasma specimen is concentrated in a tube by centrifugation and then subjected to lysis (600 μl of lysis buffer). However, when using the MagNA Pure LC large-volume total nucleic acid protocol for automated nucleic acid purification, the instrument does not allow differential pipetting and processing of assay controls versus patient specimens during the same run. How did Holzl et al. use the MagNA Pure LC instrument to process the assay controls and patient specimens within the same run? Were modified procedures used for handling the controls so that they could be processed along with clinical specimens within the same run? If the controls were not processed on the same run as the clinical specimens, the validity of the automated nucleic acid purification process and the assay results obtained from the clinical specimens would be uncertain. Procedural details from the authors regarding proper processing of assay controls by the MagNA Pure LC instrument are critically important to diagnostic laboratories planning to validate and adopt this fully automated nucleic acid purification method for use with the ultrasensitive COBAS AMPLICOR HIV-1 Monitor test.