Table 1.
Assay parameters | Sites/laboratories |
|||||
---|---|---|---|---|---|---|
Stanford University Medical Center | University of Washington/Harborview Medical Center | Viracor-IBT Laboratories | Primex Clinical Laboratories | Mayo Clinic | Brigham and Women’s Hospital | |
Specimen type | EDTA plasma | Blood, plasma, serum, CSF, DBS, tissue | Plasma, serum, whole blood, bone marrow, CSF, lower respiratory sample, tissue, fecal | Nasopharyngeal swab | Whole blood (for quantitative PCR); whole blood, body fluid (eg, CSF, amniotic, ocular), respiratory fluid (eg, bronchial washing, BAL fluid, NP aspirate, sputum), bone marrow, fresh tissue (for qualitative PCR) | Whole blood |
Specimen volume | 1 mL | 3 mL blood, 1 mL CSF, 1 full circle DBS | Varies | 500 µL | 200 µL required | 50 µL |
EBV DNA extraction method |
|
Automated magnetic bead-based method, with pre-extraction lysis step if DBS or tissue is the source material, depends on specimen type | Easy mag or QiaCube, optimized by specimen type | Automated Roche | Roche MagNA Pure LC 2.0 | Automated total nucleic acid isolation |
Extraction instrument (if automated) | Qiagen QIASymphony SP/AS | Roche MP96 or Magnapure, Promega Maxwell 16 | Easy mag or QiaCube- optimized by specimen type | Roche Magnapure | Roche MagNA Pure LC 2.0 | Roche MagNA Pure 2.0 |
Elution volume | 90 µL | 100 µL | varies by specimen type | 100 µL | 100 µL | 100 µL |
PCR reaction volume | 25 µL | 50 µL; 32.2 µL (for EBV IR1 [large internal repeat]) | _* | 25 µL | 20 µL | 20 µL |
Eluate input volume | 10 µL | 20 µL; 15 µL (for EBV IR1) | _ | 10 µL, samples standardized for DNA content prior to PCR | 5 µL | 5 µL |
PCR enzyme | Qiagen Artus EBV (for routine); Roche FastStart Taqman Probemaster (for BamHI W assay for NPC) | Applied Biosystems AmpliTaq DNA Polymerase; Bio-Rad Sso7d fusion polymerase (for EBV IR1) | _ | Taq polymerase | Roche FastStart | Taq polymerase |
PCR mastermix | Qiagen Artus EBV (for routine); Roche FastStart Taqman Probemaster (for BamHI W) | In-house PCR mix; Bio-Rad SsoAdvanced Probes Supermix with ROX (2x) (for EBV IR1) | _ |
|
Roche analyte-specific reagents | Qiagen artus EBV LC PCR kit (product has been discontinued by manufacturer) |
EBV gene target(s) for PCR amplification | EBNA-1 (for routine); BamHI W (multicopy) | EBER (single copy); EBV IR1 gene (multicopy) | EBNA-1 and EBER - dual target | EBNA-1; EBER1/EBER2 (single copy) | Latent membrane protein (single copy) | 97 bp region of EBNA-1 |
PCR chemistry | TaqMan | TaqMan | TaqMan | _ | FRET | Hydrolysis |
PCR instrument | Qiagen Rotor-Gene Q | ABI 7500; ABI StepOne (for EBV IR1) | ABI | QuantStudio 12K | LightCycler 2.0 | LightCycler 2.0 |
Cycling conditions |
|
50C (2 min), 95C (2 min), 45 cycles (95C [20 sec], 60C [1 min]); (for EBV IR1) 50C (2 min), 95C (20 sec), 45 cycles (95C (3 sec), 60C [30 sec]) | _ | 50C (2 min), 95C (10 min), 40 cycles (95C (10 sec), 60C (1 min) | _ | 45 cycles (3 steps each [95C, 55C, 72C]) |
Calibrator composition | Primary standard WHO, secondary standard = plasmid, Namalwa | Acrometrix EBV Panel – used to calibrate plasmid standard curve, Raji | Primary standard WHO, secondary standard = plasmid |
|
Roche calibrators (200 000 000 copies/mL to 2000 copies/mL), Acrometrix | EBV DNA plasmid |
Calibrator extracted/not extracted | Extracted (WHO standard), not extracted (plasmid) | Extracted | Extracted (WHO standard), but not with every run | Extracted | Not extracted | Not extracted |
Harmonization to IU/mL units (yes/no) | Yes (calibrated to WHO standard but awaiting international sites to adopt IU/mL units before implementing) | Yes | Yes | No | Not currently (data acquired to convert to IU/mL using either WHO or Acrometrix standards) | No |
Unit | Copies/mL and IU/mL | IU/mL | IU/mL | Copies/mL, Ct value | Copies/mL | _ |
Assay contains internal control (yes/no) | Yes (for routine), no (for BamHI W) | Yes | Yes | Yes | Yes | Yes (provided in Qiagen PCR kit) |
Quantitative/qualitative | Quantitative for routine tests; reported as qualitative for NRG-HN001 trial | Quantitative for plasma, CSF, DBS; qualitative for tissue | Quantitative for multiple specimen types; qualitative for fecal and tissue specimens | Quantitative | Quantitative for whole blood; qualitative for multiple specimen types | _ |
No response provided in survey. CSF = cerebrospinal fluid; DBS = dried blood spots; EBNA-1 = Epstein-Barr nuclear antigen 1; EBV = Epstein-Barr virus; IU = international unit; NPC = nasopharyngeal carcinoma; PCR = polymerase chain reaction; WHO = World Health Organization.