The Hector-2 cell line (Bioworld Consulting Laboratories, Mt. Airy, MD) was derived from B-lymphocytes of a donor with FISH-confirmed ciHHV-6 (7 (link)) and contains one HHV-6 integration per cell (in chromosome 18). Hector-2 cells and whole blood samples from an IRB-approved ciHHV-6 registry (HHV-6 Foundation, Santa Barbara, CA) were utilized to validate and calibrate the ratiometric ddPCR assay. DNA was extracted from cells previously frozen at −80°C and the duplex ddPCR assay for HHV-6 and RPP30 performed.
Patient buffy coat, plasma, and tissue samples saved frozen at −20 to −80°C from routine clinical HHV-6 and ciHHV-6 real-time PCR testing were evaluated for ciHHV-6 by digital PCR. Plasma and tissue samples from patients with or without real-time PCR-confirmed HHV-6 reactivation, and patients with suspected ciHHV-6 (determined a priori as increasing HHV-6 plasma DNA levels during the first 2 weeks after transplantation and persistent levels ≥ 100 copies per/mL in ≥ 80% of subsequent plasma samples), were selected from a hematopoietic stem cell transplantation study population.(20 (link), 21 (link)) Study subjects with HHV-6 reactivation were selected based on the following criteria: 1) negative sample within the first ten days post-transplant 2) two consecutive positive samples 3) negative sample collected seven to ten days after the last positive. Patients without HHV-6 reactivation had three consecutive negative samples collected within the same range of days post-transplant as those with reactivation as well as a negative sample collected seven to ten days after the last of the three consecutive negative samples. Samples were run blind by a single technician.
DNA extractions of cultured cells and tissue specimens were performed on a Maxwell 16 (Promega, Madison, WI) utilizing the total viral nucleic acid extraction kit with varying volumes of cell or tissue sample extracted to 50–100 µl in water. Plasma samples were extracted on a MagnaPure LC (Roche, Basel, Switzerland) utilizing the DNA Isolation Kit I with a volume of 200µl plasma extracted to 100µl DNA in elution buffer. DNA was stored frozen at −20°C until use. DNA from plasma specimens was run in triplicate ddPCR reactions while DNA from cellular specimens was run as a single ddPCR reaction. Use of all patient specimens was approved by the University of Washington Institutional Review Board.
Patient buffy coat, plasma, and tissue samples saved frozen at −20 to −80°C from routine clinical HHV-6 and ciHHV-6 real-time PCR testing were evaluated for ciHHV-6 by digital PCR. Plasma and tissue samples from patients with or without real-time PCR-confirmed HHV-6 reactivation, and patients with suspected ciHHV-6 (determined a priori as increasing HHV-6 plasma DNA levels during the first 2 weeks after transplantation and persistent levels ≥ 100 copies per/mL in ≥ 80% of subsequent plasma samples), were selected from a hematopoietic stem cell transplantation study population.(20 (link), 21 (link)) Study subjects with HHV-6 reactivation were selected based on the following criteria: 1) negative sample within the first ten days post-transplant 2) two consecutive positive samples 3) negative sample collected seven to ten days after the last positive. Patients without HHV-6 reactivation had three consecutive negative samples collected within the same range of days post-transplant as those with reactivation as well as a negative sample collected seven to ten days after the last of the three consecutive negative samples. Samples were run blind by a single technician.
DNA extractions of cultured cells and tissue specimens were performed on a Maxwell 16 (Promega, Madison, WI) utilizing the total viral nucleic acid extraction kit with varying volumes of cell or tissue sample extracted to 50–100 µl in water. Plasma samples were extracted on a MagnaPure LC (Roche, Basel, Switzerland) utilizing the DNA Isolation Kit I with a volume of 200µl plasma extracted to 100µl DNA in elution buffer. DNA was stored frozen at −20°C until use. DNA from plasma specimens was run in triplicate ddPCR reactions while DNA from cellular specimens was run as a single ddPCR reaction. Use of all patient specimens was approved by the University of Washington Institutional Review Board.
