SoGAT Clinical Diagnostics III- A recent UK NEQAS pilot distribution highlights the need for standardisation of EBV DNA quantification. Brigitte Senechal, Habib Seyedzadeh, Anneline Rossouw, Vivienne James UK NEQAS EBV DNA quantification pilot distribution 1
INTRODUCTION-1 EBV EBV infects more than 95% of the population and persists life-long mostly in a latent state in B-lymphocytes. The infection is usually subclinical and occasionally results in infectious mononucleosis syndrome in adolescents. In immunosuppressed patients, including AIDS patients and graft recipients, the viral reactivation (and primary infection children) can occur and may induce lymphoproliferative disorders. EBV is also strongly associated with neoplasms such as Burkitt lymphomas and nasopharyngeal carcinoma. In all these conditions, EBV DNA can be detected in the blood but not in the blood of healthy donors with a past infection. UK NEQAS EBV DNA quantification pilot distribution 2
INTRODUCTION-2 Prevention of the post-transplant lymphoproliferative disorder (PTLD) EBV DNA-emia is common in immunosuppressed patients (>95% seropositive) Rarely causes significant disease PTLD is an important complication / poor outcome (50% mortality) Hematopoietic Stem Cell Transplantation (allogeneic): 1 to 25% in high risk HSCT (unrelated/mismatch/ Tdepletion / cord blood T/ serology mismatch) Prospective monitoring of EBV DNA-emia (Qt) is recommended for High-risk allo HSCT to allow pre-emptive treatment: rituximab, reduction of immunosuppression (treatment efficacy) Solid Organ Transplantation: European Conference on Infection in Leukemia dec. 2009 Adult: 1.0% (kidney) to 20% (small bowel) insufficient data to recommend surveillance Children: 1.2% (kidney) to 30% (small bowel) children children at risk of primary infection routine surveillance is likely to aid British Committee for Standards in Haematology october 2009 UK NEQAS EBV DNA quantification pilot distribution 3
INTRODUCTION-3 When to start prevention of PTLD? European recommendations for HSCT: European Conference on Infection in Leukemia dec. 2009 Indication of pre-emptive therapy: when EBV DNA-emia is high or rising whole blood/plasma/serum Weekly for - / > weekly for + / 3 months or more (earlier+) UK guidelines for SOT, BJH 2010, 149: 675-692- (BCSH & SBTS joint working group) UK NEQAS EBV DNA quantification pilot distribution 4
QUESTIONNAIRE / april 2009 Sent to CMV DNA quantification scheme participants 78.8% use molecular methods to quantify EBV DNA (all real time PCR) Matrix: 62.5% use whole blood 54.2% use plasma 16.6% use both Median detection limit: 250c/mL [10-2000] (all using c/ml) Median number of samples examined per week: 22.5 [0.5-200] Patient types: 68.8% for HSCT / 36.8% for SOT / 7.9% for tumor Clinical cut-off: viral load rise over a definite period of time & confirm high viral load UK NEQAS EBV DNA quantification pilot distribution 5
Pilot /Specimen design A Namalwa/mL 78,000 Expected VL c/ml Log 156,000 5.19 Pre-distribution Artus Homogeneity c/ml Log SD 102300 5.01 0.118 B 7,800 15,600 4.19 31600 4.50 0.149 Log diff. 1.00 Log diff. 0.49 (Qiagen column) (20 vials) A total of 88 sets of specimens were distributed with 74 participants returning results, 13 participants did not examine the specimens 61 sets of results UK NEQAS EBV DNA quantification pilot distribution 6
Log (copies/ml) Pilot / Results by specimen SPECIMEN A expected/ml 5.19 log (156,000) median+/-sd 5.01 +/- 0.57 range 3.00-6.84 SPECIMEN B expected/ml 4.19 log (15,600) median+/-sd 4.02 +/- 0.47 range 3.05-5.49 8.00 8.00 7.00 7.00 6.00 6.00 5.00 median 5.00 4.00 4.00 median 3.00 3.00 2.00 2.00 61 results sorted in increasing order UK NEQAS EBV DNA quantification pilot distribution 7
Log (copies/ml) Pilot / Results by laboratory Specimen A Specimen B 8.00 8.00 7.00 7.00 6.00 6.00 5.00 5.00 4.00 4.00 3.00 3.00 2.00 2.00 Specimen-A results sorted in increasing order Specimen-B results sorted in increasing order UK NEQAS EBV DNA quantification pilot distribution 8
Argene Biosoft Argene Biosoft Other PCR: Single target Real-Time Multiplex Roche: LightCycler Roche: LightCycler Sacace Real-TM Unspecified Log (copies/ml) unknown LMP BXF-1 EBNA-1 unknown EBNA-1 variable LMP-2 Pilot / Results by amplification methods 7.00 6.50 6.00 5.50 5.00 4.50 4.00 3.50 3.00 median Median 5.10+/-0.582 [4.38-6.84] Median 4.84+/-0.351 [3.67-5.40] Inter-laboratory variations: same amplification assay Median 5.27+/-0.509 [4.15-6.15] different amplification platforms different extraction assays UK NEQAS EBV DNA quantification pilot distribution 9
Pilot / Extraction and amplification methods Amplification method n % 21 34.4% 17 27.9% 14 23.0% Roche: LightCycler 2 3.3% Argene Biosoft 2 3.3% PCR: Single target 1 1.6% Real Time Multiplex 1 1.6% Sacace Real-TM 1 1.6% Unspecified 1 1.6% Other 1 1.6% >23 combinations of extraction and amplification assays for 61 sets of results 5 most frequent combinations: 26.2% in house assays Extraction method Amplification method n % NucliSENS easymag 11 18.0 Qiagen* 11 18.0 Qiagen* 6 9.8 NucliSENS easymag 5 8.2 MagnaPure 4 6.6 73.8% commercial assays: no automation / all use IC All open systems: free choice of extraction methods: Extraction method n % Qiagen* 24 36.9% NucliSENS easymag 19 29.2% MagnaPure 9 13.8% Magnetic beads 2 3.1% Nucleo Spin 1 1.5% Unspecified 3 4.6% Other 3 4.6% * BioRobot MDx QIAsymphony UK NEQAS EBV DNA quantification pilot distribution 10
Log (copies/ml) Pilot / Results for the 5 most frequent E&A assay combinations-1 7.50 Specimen-A Median Max/Min Specimen-B Median Max/Min 6.50 5.50 4.50 3.50 2.50 In-house Extraction Amplification NucliSENS easymag NanogenAD: Real-Time Alert Qiagen* Qiagen* NanogenAD: Real-Time Alert NucliSENS easymag MagnaPure n (%) Median A +/- SD Median B +/- SD 11 (18%) 4.83 +/- 0.194 3.86 +/- 0.168 11 (18%) 5.19 +/- 0.397 4.15 +/- 0.383 6 (9.8%) 5.06 +/- 0.624 4.15 +/- 0.660 5 (8.2%) 5.28 +/- 0.111 4.25 +/- 0.202 4 (6.6%) 5.09 +/- 0.991 4.12 +/- 0.827 *BioRobot MDx, QIAsymphony UK NEQAS EBV DNA quantification pilot distribution 11
Log (copies/ml) Pilot / Results for the 5 most frequent E&A assay combinations-2 7.50 Specimen-A Median Max/Min Specimen-B Median Max/Min 6.50 5.50 4.50 3.50 2.50 Extraction Amplification NucliSENS easymag NanogenAD: Real-Time Alert Qiagen* Qiagen* NanogenAD: Real-Time Alert NucliSENS easymag MagnaPure n (%) Median A +/- SD Median B +/- SD 11 (18%) 4.83 +/- 0.194 3.86 +/- 0.168 11 (18%) 5.19 +/- 0.397 4.15 +/- 0.383 5 (8.2%) 5.08 +/- 0.250 4.21 +/- 0.455 5 (8.2%) 5.28 +/- 0.111 4.25 +/- 0.202 3 (4.9%) 4.94 +/- 0.318 3.94 +/- 0.359 *BioRobot MDx, QIAsymphony UK NEQAS EBV DNA quantification pilot distribution 12
Conclusion This pilot study illustrates the lack of standardisation, with participants results ranging 3.84 log for specimen A (4 log) and 2.44 log for specimen B (5 log) using 5 different commercial assays and in house assays: all assays are manual/semi-manual offering free choices of extraction and amplification platforms: variations of 2 log and more within same amplification assay Standardisation: Matrix Extraction assay Amplification platform Amplification/detection assay Needed to establish guidelines for the surveillance of HSCT and SOT at risk of developing PTLD: clinical cut-off for pre-emptive therapy Frequency of assessment (+) Treatment efficacy UK NEQAS EBV DNA quantification pilot distribution 13
IU/mL Copies/mL 2001 First WHO international HBV DNA 2010 First WHO international CMV DNA 100000 90000 80000 70000 60000 50000 40000 30000 20000 10000 0 median 17,000 IU/mL Laboratories (n=109) 100000 12/2008- serum 05/2010-plasma 80000 70000 60000 50000 40000 median 26,000 c/ml 30000 20000 10000 0 Laboratories (n=81) median+/-0.2log: 81% median+/-0.2log: 41% 90000 UK NEQAS EBV DNA quantification pilot distribution 14
Log A - Log B Pilot / Results currently analysed as a log difference 98.4% (60/61) detected 5 log of EBV DNA (specimen A) 93.4% (57/61) detected 4 log of EBV DNA (specimen B) -> 57 log differences calculated: expected 1.00 log median+/-sd 0.96+/-0.21 range 0.05-1.49 96.5% (55/57) reported a 0.46 to 1.46 log difference 1.96 1.46 0.96 median 0.46-0.04 UK NEQAS EBV DNA quantification pilot distribution 15