Read SVF_CMVDNA10_sgw_010710.xlsx text version

QUALITY CONTROL for MOLECULAR DIAGNOSTICS

The Altum Building, Todd Campus,

West of Scotland Science Park, Glasgow, G20 0XA Scotland Tel: +44 (0) 141 945 6474 Fax: +44 (0) 141 945 5795 www.qcmd.org [email protected]

Final Report QCMD 2010 Human Cytomegalovirus DNA (CMVDNA10) EQA Programme

Rob Schuurman, Sophie Alain & Anton M van Loon

on behalf of QCMD and its Scientific Council August 2010

Not to be reproduced or quoted without permission of QCMD. Any queries about this report should be addressed to the QCMD Neutral Office.

The QCMD programme is organised in collaboration with the European Society for Clinical Virology and the European Society for Clinical Microbiology & Infectious Diseases.

Registered in Scotland Reg No: SC219746 Registered Office: 7 Castle Street, Edinburgh EH2 3AH

1. Programme aims

The primary aim of this External Quality Assessment Programme was to assess the proficiency of laboratories in the detection and quantitation of CMV.

2. Programme details

Table 1: Programme details CMVDNA10 Date of panel distribution 10/05/2010 Number of respondents Number of participants 233 Number of datasets submitted Number of countries 35 Number of qualitative datasets submitted Number of qualitative and quantitative datasets submitted Five participants did not return results; none officially withdrew.

228 (98%) 262 44 (17%) 218 (83%)

3. Panel composition

This EQA panel for the detection of Human Cytomegalovirus (CMV) consisted of nine samples containing various concentrations of CMV strain AD169 in either plasma or VTM and one plasma sample negative for CMV. The panel materials were derived from cultured CMV (AD169 reference strain). Negative plasma was obtained from the Groningen blood bank (The Netherlands). The QCMD EQA panels contain a range of samples, designed to look at different aspects of assay performance. Panel members are designated `core proficiency samples' on the basis of scientific information, clinical relevance and clinical experience (published literature and professional clinical guidelines) and, where available and appropriate, established target performance limits taken from previous QCMD EQA distributions. Laboratories are expected to correctly analyse and report the core proficiency samples in order to show acceptable proficiency. Table 2: Panel composition

Sample CMV10-06 CMV10-03 CMV10-08 CMV10-10 CMV10-04 CMV10-01 CMV10-07 CMV10-09 CMV10-02 CMV10-05 Sample content CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV Negative Sample * matrix VTM VTM VTM Plasma Plasma Plasma Plasma Plasma Plasma Plasma Sample conc. Copies/ml 2,552,701 275,423 23,988 16,904 5,534 1,879 1,799 684 230 Sample status Frequently detected Frequently detected Frequently detected Frequently detected Frequently detected Frequently detected Frequently detected Detected Detected Negative Core Sample type Core Core Core Core Core Core Core Core

Key to Table 2 Sample: QCMD panel sample codes for the samples distributed to participants. Sample content: viral content of the panel samples. Sample matrix: material used as a matrix in preparation of the panel samples. Sample conc.: consensus values calculated from all of the data returned by participants, once outliers had been removed. The values are not technology specific and should not be used by participants for method comparison or as a target for individual laboratory assessment. Sample type: panel samples classified as core proficiency samples. * Plasma: Human plasma negative for CMV and interfering viruses. VTM: Virus Transport Medium.

Additional information on panel samples

In VTM Extended dilution series: panel samples CMV10-06, -03 and -08. These samples were identical to those used in previous distributions and consisted of 10-fold dilutions to allow for linearity analysis of the applied assays and technologies. In plasma Extended dilution series: panel samples CMV10-10, -04, -01, -07, -09 and -02. These samples consisted of 3-fold dilutions in the lower but still clinically relevant ranges of viral load. This allowed qualitative and quantitative assessment of assay performance, assay sensitivity and assay linearity both within and between technologies and assay types. In addition the dilutions in the panel allowed for analysis of a laboratory's capability to determine viral load differences between paired samples. Duplicate samples: panel samples CMV10-01 and -07 were duplicate samples, included to assess assay reproducibility. Negative: panel sample CMV10-05 was included to evaluate the frequency of false-positive results.

QCMD 2010 Human Cytomegalovirus DNA (CMVDNA10) EQA Programme

PAGE 2 of 11

4. Programme results

4.1. Qualitative performance on the core proficiency samples

Figure 1 shows the performance of participants on the core proficiency samples. In this round of the EQA 89.7% of datasets returned to QCMD reported all core proficiency samples correctly. Figure 1: Performance on the core proficiency samples for all participants 100.0% 90.0% Percentage of datasets 80.0% 70.0% 60.0% 50.0% 40.0% 30.0% 20.0% 10.0% 0.0% 9/9 8/9 7/9 6/9 5/9 4/9 3/9 Number of core samples correct 2/9 1/9 0/9 7.6% 1.5% 0.4% 0.4% 0.0% 0.0% 0.0% 0.4% 0.0% 89.7%

4.2. Qualitative analysis of the EQA data for all panel samples

The number (percentage) of correct qualitative results are presented in Table 3. Qualitative data were returned by participants as 'positive', 'negative' or 'not determined'. Not determined results were counted as incorrect for all panel samples (positive or negative). QCMD organises datasets according to commercial and in-house technology groups, which are Conventional PCR, Real time PCR, NASBA, SDA, TMA and bDNA. Where datasets were reported as `other' for a technology or kit method this was reviewed by the QCMD Neutral Office and assigned to an appropriate group where possible. Table 3: Number of correct qualitative results per panel member and technology type

PCR Sample Sample content Sample conc. Copies/ml Total datasets n=262 n CMV10-06 CMV10-03 CMV10-08 CMV10-10 CMV10-04 CMV10-01 CMV10-07 CMV10-09 CMV10-02 CMV10-05 CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV Negative 2,552,701 275,423 23,988 16,904 5,534 1,879 1,799 684 230 259 260 261 259 260 258 257 240 199 261 % 98.9 99.2 99.6 98.9 99.2 98.5 98.1 91.6 76.0 99.6 Conventional Commercial n=19 n 19 19 19 19 19 19 19 18 12 19 % 100.0 100.0 100.0 100.0 100.0 100.0 100.0 94.7 63.2 100.0

a

NASBA e Real time

c

In-house b n=6 n 5 6 6 6 6 6 6 5 5 6 % 83.3 100.0 100.0 100.0 100.0 100.0 100.0 83.3 83.3 100.0

Commercial n=111 n 110 110 111 110 110 110 108 101 87 110 %

In-house d n=125 n 124 124 124 124 124 122 123 115 94 125 % 99.2 99.2 99.2 99.2 99.2 97.6 98.4 92.0 75.2 100.0 n 1 1 1 0 1 1 1 1 1 1

n=1 % 100 100 100 0 100 100 100 100 100 100

99.1 99.1 100.0 99.1 99.1 99.1 97.3 91.0 78.4 99.1

Key to Table 3 Sample: QCMD panel sample codes for the samples distributed to participants. Sample content: viral content of the panel samples. Sample conc.: consensus values calculated from all of the data returned by participants, once outliers had been removed. The values are not technology specific and should not be used by participants for method comparison or as a target for individual laboratory assessment. Total datasets: number and percentage of datasets reporting the correct qualitative result for each panel sample. A breakdown of the results for all datasets is also provided based on technology type. a: Genomica CLART Entherpex (n=1), Mobidiag Prove-it Herpes (n=1), Roche Amplicor CMV Monitor (n=4), Roche COBAS Amplicor CMV Monitor (n=12), Seegene Seeplex Meningitis ACE Detection (n=1). b: Details not presented. c: Abbott CMV PCR Kit (n=3), Argene CMV HHV6,7,8 R-gene (n=6), Argene CMV R-gene (n=18), astra GmbH Real Star CMV PCR Kit (n=2), Cepheid affigene CMV trender (n=7), Cepheid Smart CMV (n=2), Diagenode CMV Quantitative Detection (n=1), Diagenode CMV quantitative real-time pcr (n=1), Epoch Biosciences MGB Eclipse CMV Assay (n=1), GeneProof CMV PCR Kit (n=1), Nanogen CMV ELITe MGB (n=1), Nanogen Q-CMV Real time Complete Kit (n=21), QIAGEN artus CMV PCR Kit (LC) (n=6), QIAGEN artus CMV PCR Kit (RG) (n=17), QIAGEN artus CMV PCR Kit (TM) (n=10), Roche LightCycler CMV Quant Kit (n=12), Sacace Biotechnologies CMV Real-TM Quant (n=1), Sacace CMV PCR Kit (n=1). d: Details not presented. e: bioMerieux NucliSENS CMV pp67 (n=1).

QCMD 2010 Human Cytomegalovirus DNA (CMVDNA10) EQA Programme

PAGE 3 of 11

4.3. Qualitative performance scores for all panel samples

Table 4: Qualitative performance scores per technology type

Total Sample Sample Status n=262 0 CMV10-06 CMV10-03 CMV10-08 CMV10-10 CMV10-04 CMV10-01 CMV10-07 CMV10-09 CMV10-02 CMV10-05 Frequently detected Frequently detected Frequently detected Frequently detected Frequently detected Frequently detected Frequently detected Detected Detected Negative 259 260 261 259 260 258 257 240 199 261 1 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 22 63 0 3 3 2 1 3 2 4 5 0 0 1 0 19 19 19 19 19 19 19 18 12 19 All technologies n=19 1 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 1 7 0 3 0 0 0 0 0 0 0 0 0 0 0 5 6 6 6 6 6 6 5 5 6 1 0 0 0 0 0 0 0 0 0 0 Conventional Commercial

a

PCR Real time

b

NASBA Commercial n=111

c

e

In-house n=6 2 0 0 0 0 0 0 0 1 1 0

In-house n=125 3 1 1 0 1 1 1 3 0 0 1 0 124 124 124 124 124 122 123 115 94 125 1 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 10 31 0

d

n=1 3 1 1 1 1 1 3 2 0 0 0 0 1 1 1 0 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 3 0 0 0 1 0 0 0 0 0 0

3 1 0 0 0 0 0 0 0 0 0

0 110 110 111 110 110 110 108 101 87 110

1 0 0 0 0 0 0 0 0 0 0

2 0 0 0 0 0 0 0 10 24 0

Key to Table 4 Sample: QCMD panel sample codes for the samples distributed to participants. Sample status: the sample status assigned to each panel sample. Please see Appendix A for more information. Total. All technologies: number of datasets awarded each score (0 to 3). A breakdown of the results for all datasets is also provided based on technology type. These data are presented graphically in Figure 2. a: Genomica CLART Entherpex (n=1), Mobidiag Prove-it Herpes (n=1), Roche Amplicor CMV Monitor (n=4), Roche COBAS Amplicor CMV Monitor (n=12), Seegene Seeplex Meningitis ACE Detection (n=1). b: Details not presented. c: Abbott CMV PCR Kit (n=3), Argene CMV HHV6,7,8 R-gene (n=6), Argene CMV R-gene (n=18), astra GmbH Real Star CMV PCR Kit (n=2), Cepheid affigene CMV trender (n=7), Cepheid Smart CMV (n=2), Diagenode CMV Quantitative Detection (n=1), Diagenode CMV quantitative real-time pcr (n=1), Epoch Biosciences MGB Eclipse CMV Assay (n=1), GeneProof CMV PCR Kit (n=1), Nanogen CMV ELITe MGB (n=1), Nanogen Q-CMV Real time Complete Kit (n=21), QIAGEN artus CMV PCR Kit (LC) (n=6), QIAGEN artus CMV PCR Kit (RG) (n=17), QIAGEN artus CMV PCR Kit (TM) (n=10), Roche LightCycler CMV Quant Kit (n=12), Sacace Biotechnologies CMV Real-TM Quant (n=1), Sacace CMV PCR Kit (n=1). d: Details not presented. e: bioMerieux NucliSENS CMV pp67 (n=1).

Figure 2: Percentage of qualitative performance scores per technology type

100 90 80 70 60 % 50 40 30 20 10 0

a b c d a b c d a b c d a b c d a b c d a b c d a b c d a b c d a b c d a b c d CMV10-06 CMV10-03 CMV10-08 CMV10-10 CMV10-04 CMV10-01 CMV10-07 CMV10-09 CMV10-02 CMV10-05

3 2 1 0

Technology group per panel sample

a: Conventional commercial PCR, b: Conventional in-house PCR, c: Real time commercial PCR, d: Real time in-house PCR. e: the single dataset submitted using NASBA technology is not presented in this graph.

QCMD 2010 Human Cytomegalovirus DNA (CMVDNA10) EQA Programme

PAGE 4 of 11

4.4. Qualitative analysis of the EQA data by assay target gene

Participants were asked to specify the target gene of their assay when submitting their results. Out of the 262 datasets received by QCMD 216 (82%) contained information on the target gene. These data are summarised in Table 5. Table 5: Analysis of the qualitative data by assay target gene

Sample Sample content Sample conc. Copies/ml Total datasets n=262 n CMV10-06 CMV10-03 CMV10-08 CMV10-10 CMV10-04 CMV10-01 CMV10-07 CMV10-09 CMV10-02 CMV10-05 CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV (AD169) CMV Negative 2,552,701 275,423 23,988 16,904 5,534 1,879 1,799 684 230 259 260 261 259 260 258 257 240 199 261 % 98.9 99.2 99.6 98.9 99.2 98.5 98.1 91.6 76.0 99.6 n 55 55 56 55 55 56 55 49 42 56 UL122 n=56 % 98.2 98.2 100.0 98.2 98.2 100.0 98.2 87.5 75.0 100.0 n 25 25 25 25 25 24 25 24 20 24 UL123 n=25 % 100.0 100.0 100.0 100.0 100.0 96.0 100.0 96.0 80.0 96.0 n 45 46 46 46 46 44 46 43 33 46 UL54 n=46 % 97.8 100.0 100.0 100.0 100.0 95.7 100.0 93.5 71.7 100.0 n 22 22 22 22 22 22 22 21 18 23 UL55 n=23 % 95.7 95.7 95.7 95.7 95.7 95.7 95.7 91.3 78.3 100.0 n 36 36 36 36 36 36 34 33 27 36 UL83 n=36 % 100.0 100.0 100.0 100.0 100.0 100.0 94.4 91.7 75.0 100 n 8 8 8 8 8 8 8 8 6 8 US8 n=8 % 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 75.0 100.0 n 22 22 22 22 22 22 22 21 21 22 Other* n=22 % 100.0 100.0 100.0 100.0 100.0 100.0 100.0 95.5 95.5 100.0 n 46 46 46 45 46 46 45 41 32 46 Not reported n=46 % 100.0 100.0 100.0 97.8 100.0 100.0 97.8 89.1 69.6 100.0

Key to Table 5 Sample: QCMD panel sample codes for the samples distributed to participants. Sample content: viral content of the panel samples. Sample conc.: consensus values calculated from all of the data returned by participants, once outliers had been removed. The values are not technology specific and should not be used by participants for method comparison or as a target for individual laboratory assessment. Total datasets: number and percentage of datasets reporting the correct qualitative result for each panel sample. A breakdown of the results for all datasets is also provided based on target gene. * 5' untranslated region (n=1), Glycoprotein H (n=1), mtrII (n=3), UL55 and UL123 (n=1), UL93 (n=2), US17 (n=4), UL86 (n=5), UL32 (n=2), UL111A (n=1), UL89 (n=1), US18 (n=1).

4.5. Quantitative performance on the paired samples

Assays may differ relatively in the quantitative values they report. Therefore QCMD assesses quantitative proficiency based on the difference reported between paired samples, which provides participants with a measure of proficiency that is independent of assay bias. The median difference between the paired samples is calculated from the log concentration of participants' results. In this round of the EQA, quantitative EQA performance was assessed using paired samples containing CMV in plasma. For each quantitative dataset, the reported difference in concentration between sample CMV10-10 and -07 was compared with the median difference in concentration reported by all laboratories reporting quantitative data. Participants were expected to report to within 0.5 log10 copies/ml of the median in order to show acceptable proficiency (median difference: 1.000 log10 copies/ml). 91.7% (n=200/218) of quantitative datasets reported to within 0.5 log10 copies/ml of the median difference. 3.2% (n=7/218) of quantitative datasets did not return a quantitative result for one or both samples. Figure 3: Performance on the paired samples for all quantitative datasets 100.0% 90.0% Percentage of datasets 80.0% 70.0% 60.0% 50.0% 40.0% 30.0% 20.0% 10.0% 0.0% 0 - <0.5 0.5 - <1 1 - <1.5 1.5 Log10 units from the median difference NR 3.7% 0.5% 0.9% 3.2% 91.7%

QCMD 2010 Human Cytomegalovirus DNA (CMVDNA10) EQA Programme

PAGE 5 of 11

4.6. Quantitative analysis of the EQA data and performance scores for all panel samples Consensus concentration score

The consensus concentration score relates to scoring on the basis of a consensus mean for each positive panel sample. Table 6: Quantitative scores by technology type in comparison to the consensus concentration

Total Sample Consensus Log10 virus concentration Mean CMV10-06 CMV10-03 CMV10-08 CMV10-10 CMV10-04 CMV10-01 CMV10-07 CMV10-09 CMV10-02 6.407 5.440 4.380 4.228 3.743 3.274 3.255 2.835 2.362 SD 0.446 0.450 0.421 0.433 0.443 0.408 0.444 0.460 0.455 0 142 155 152 154 161 146 146 129 91 1 38 32 52 48 35 45 51 46 42 n=218 2 17 18 11 10 14 14 9 12 5 3 4 5 3 5 8 4 6 3 2 17 8 0 1 0 9 6 28 78 All technologies n=16 2 2 1 0 6 2 1 0 15 1 0 0 15 1 0 0 15 1 0 0 13 3 0 0 15 1 0 0 13 1 0 0 4 2 0 0 11 7 0 0 0 0 0 2 10 Conventional Commercial

a

PCR Real time

b

NASBA

c

e

In-house n=1

Commercial n=105 0 1

In-house n=95 0 1 2

d

n=1 4 0 0 0 0 3 0 8 28 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 1 0 0 1 0 0 1 0 0 0 0 0 1 0 0 0 0 0

LOD/NR 0 1 2 3 LOD/NR 0 1 2 3 LOD/NR 0 0 0 1 0 0 0 1 0 0 1 0 0 0 0 1 0 0 0 1 0 0 0 1 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1

2 3 LOD/NR 2 1 0 0 0 6 6 18 39

3 LOD/NR 0 1 2 3 LOD/NR 1 2 1 2 3 1 1 1 1

80 18 4 1 84 13 6 1 80 20 4 1 78 22 3 2 87 12 3 3 73 20 5 1 74 19 2 4 63 19 3 2 37 27 1 1

60 18 12 65 17 11 57 31 61 25 60 22 57 31 53 26 50 12 6 7 9 6 7 4

59 22 11

Key to Table 6 Sample: QCMD panel sample codes for the samples distributed to participants. Consensus Log10 virus concentration: the mean quantitative value and standard deviation value for each panel sample expressed in log10 units and calculated once outlying values had been removed. Total. All technologies: number of datasets awarded each score (0 to 3). LOD/NR refers to datasets where an upper or lower limit of detection was reported or no value was reported (these data were excluded from the scoring). SD refers to the Standard Deviation. A breakdown of the results for all datasets is also provided based on technology type. These data are presented graphically in Figure 4. a: Roche Amplicor CMV Monitor (n=4), Roche COBAS Amplicor CMV Monitor (n=12). b: Details not presented. c: Abbott CMV PCR Kit (n=3), Argene CMV HHV6,7,8 R-gene (n=6), Argene CMV R-gene (n=16), astra GmbH Real Star CMV PCR Kit (n=1), Cepheid affigene CMV trender (n=7), Cepheid Smart CMV (n=2), Diagenode CMV Quantitative Detection (n=1), Diagenode CMV quantitative real-time pcr (n=1), Epoch Biosciences MGB Eclipse CMV Assay (n=1), GeneProof CMV PCR Kit (n=1), Nanogen CMV ELITe MGB (n=1), Nanogen Q-CMV Real time Complete Kit (n=21), QIAGEN artus CMV PCR Kit (LC) (n=4), QIAGEN artus CMV PCR Kit (RG) (n=17), QIAGEN artus CMV PCR Kit (TM) (n=10), Roche LightCycler CMV Quant Kit (n=12), Sacace Biotechnologies CMV Real-TM Quant (n=1). d: Details not presented. e: bioMerieux NucliSENS CMV pp67 (n=1).

Figure 4: Percentage of quantitative performance scores in comparison to the consensus concentration

100 90 80 70

LOD/NR

60

%

3 2 1 0

50 40 30 20 10 0

a c d a c d a c d a c d a c d a c d a c d a c d a c d CMV10-06 CMV10-03 CMV10-08 CMV10-10 CMV10-04 CMV10-01 CMV10-07 CMV10-09 CMV10-02

Technology group per panel sample

a: Conventional commercial PCR, c: Real time commercial PCR, d: Real time in-house PCR. e: the single datasets submitted using NASBA and conventional in-house PCR technology are not presented in this graph.

QCMD 2010 Human Cytomegalovirus DNA (CMVDNA10) EQA Programme

PAGE 6 of 11

Technology consensus score

The technology consensus score relates to scoring on the basis of a technology group consensus mean per positive panel sample. Table 7: Quantitative scores by technology type in comparison to the technology consensus concentration

Sample Tech. Consensus Log10 virus concentration Mean CMV10-06 CMV10-03 CMV10-08 CMV10-10 CMV10-04 CMV10-01 CMV10-07 CMV10-09 CMV10-02

Sample

Conventional Commercial n=16 0 3 6 13 13 12 13 9 9 5 1 2 3 2 2 3 2 7 5 1 2 0 0 1 1 1 0 0 0 0 3 0 0 0 0 0 1 0 0 0

a

Sample

Tech. Consensus Log10 virus concentration

Real time Commercial n=105 0 73 74 74 74 79 65 70 62 40 1 23 22 25 25 20 27 22 20 24 2 6 6 5 4 3 6 3 3 1 3 1 2 1 2 3 1 4 2 1

c

SD 0.445 0.410 0.202 0.149 0.152 0.237 0.130 0.185 0.195

LOD/NR 11 7 0 0 0 0 0 2 10

Mean CMV10-06 CMV10-03 CMV10-08 CMV10-10 CMV10-04 CMV10-01 CMV10-07 CMV10-09 CMV10-02 6.404 5.422 4.341 4.197 3.708 3.195 3.201 2.764 2.231

SD 0.372 0.392 0.384 0.399 0.392 0.373 0.394 0.444 0.448

LOD/NR 2 1 0 0 0 6 6 18 39

5.972 5.181 4.441 4.362 3.965 3.445 3.553 2.990 2.687

Tech. Consensus Log10 virus concentration Mean SD 0.516 0.508 0.481 0.496 0.522 0.462 0.517 0.503 0.475 0 65 67 65 66 64 63 64 56 49

Real time In-house d n=95 1 23 20 25 23 26 23 26 24 13 2 3 8 5 5 3 6 5 6 4 3 0 0 0 1 2 0 0 1 1

CMV10-06 CMV10-03 CMV10-08 CMV10-10 CMV10-04 CMV10-01 CMV10-07 CMV10-09 CMV10-02

6.435 5.485 4.413 4.240 3.743 3.328 3.260 2.881 2.460

Key to Table 7 Sample: QCMD panel sample codes for the samples distributed to participants. LOD/NR Tech. Consensus Log10 virus concentration: the mean quantitative value and standard deviation value for each panel sample expressed in log10 units and 4 calculated once outlying values had been removed. The number of datasets awarded 0 each score (0 to 3) is then presented. LOD/NR refers to datasets where an upper or 0 lower limit of detection was reported or no value was reported (these data were 0 excluded from the scoring). SD refers to the Standard Deviation. These data are 0 presented graphically in Figure 5.

3 0 8 28

a: Roche Amplicor CMV Monitor (n=4), Roche COBAS Amplicor CMV Monitor (n=12).

c: Abbott CMV PCR Kit (n=3), Argene CMV HHV6,7,8 R-gene (n=6), Argene CMV R-gene (n=16), astra GmbH Real Star CMV PCR Kit (n=1), Cepheid affigene CMV trender (n=7), Cepheid Smart CMV (n=2), Diagenode CMV Quantitative Detection (n=1), Diagenode CMV quantitative real-time pcr (n=1), Epoch Biosciences MGB Eclipse CMV Assay (n=1), GeneProof CMV PCR Kit (n=1), Nanogen CMV ELITe MGB (n=1), Nanogen Q-CMV Real time Complete Kit (n=21), QIAGEN artus CMV PCR Kit (LC) (n=4), QIAGEN artus CMV PCR Kit (RG) (n=17), QIAGEN artus CMV PCR Kit (TM) (n=10), Roche LightCycler CMV Quant Kit (n=12), Sacace Biotechnologies CMV Real-TM Quant (n=1). d: Details not presented.

Figure 5: Percentage of overall quantitative performance scores in comparison to the technology consensus concentration

100 90 80 70 60

%

LOD/NR 3

50 40 30 20 10 0

a c d a c d a c d a c d a c d a c d a c d a c d a c d CMV10-06 CMV10-03 CMV10-08 CMV10-10 CMV10-04 CMV10-01 CMV10-07 CMV10-09 CMV10-02

2 1 0

Technology group per panel sample

a: Conventional commercial PCR, c: Real time commercial PCR, d: Real time in-house PCR.

QCMD 2010 Human Cytomegalovirus DNA (CMVDNA10) EQA Programme

PAGE 7 of 11

Figure 6: Assay linearity by technology type in relation to the overall consensus results - VTM 7.500 Technology consensus (log10 Copies/ml) 7.000 6.500 6.000 5.500 5.000 4.500 4.000 3.500 4.000 4.500 5.000 5.500 6.000 Consensus (log10 Copies/ml) 6.500 Conventional commercial (n=16) y = 0.75x + 1.12 R² = 0.9979 Real time commercial (n=105) y = 1.018x - 0.12 R² = 1 Real time in-house (n=95) y = 1.00x + 0.05 R² = 0.9999

Key to Figure 6 The technology consensus values are presented for panel samples CMV10-06, -03 and -08, which contained CMV in VTM, in relation to the overall consensus data. Error bars represent one standard deviation either side of the consensus values.

Figure 7: Assay linearity by technology type in relation to the overall consensus results - plasma 5.000 Technology consensus (log10 Copies/ml) 4.500 4.000 3.500 3.000 2.500 2.000 1.500 2.000 2.500 3.000 3.500 Consensus (log10 Copies/ml) 4.000 4.500 Conventional commercial (n=16) y = 0.93x + 0.45 R² = 0.989 Real time commercial (n=105) y = 1.05x - 0.23 R² = 0.9995 Real time in-house (n=95) y = 0.95x + 0.19 R² = 0.9988

Key to Figure 7 The technology consensus values are presented for panel samples CMV10-10, -04, -01, -07, -09 and -02, which contained CMV in plasma, in relation to the overall consensus data. Error bars represent one standard deviation either side of the consensus values.

Figure 8: Assay reproducibility by technology type 4 Technology consensus (log10 Copies/ml) 3.5 3 2.5 Conventional commercial (n=16) 2 1.5 1 0.5 0 CMV10-01 CMV10-07

Key to Figure 8 The technology consensus values are presented for the duplicate panel samples (CMV10-01 and -07) which contained CMV in plasma. Error bars represent one standard deviation either side of the consensus values.

Real time commercial (n=105) Real time in-house (n=95)

QCMD 2010 Human Cytomegalovirus DNA (CMVDNA10) EQA Programme

PAGE 8 of 11

5. Comments

General comments Two hundred and thirty-three participants registered for the QCMD 2010 Human Cytomegalovirus DNA EQA programme, an increase of 14% on the 204 who registered in 2009. The number of datasets submitted increased by 17% to 262 (2009: 223) (Table 1). Qualitative analysis of the EQA data Over 90% of participants were able to detect CMV DNA down to a concentration of 684 copies/ml. Panel sample CMV10-02, which was present at a concentration of 230 copies/ml, was correctly reported by 76% of participants. This was comparable to the qualitative performance in the 2009 programme, in which 78.9% of participants correctly detected the sample with a concentration of 238 copies/ml. One 'not determined' result was submitted for the negative panel sample (CMV10-05). The false positivity rate for this round of the EQA was 0%. This compared favourably to the 1.8% false positivity rate recorded in 2009. The percentage of false positive results ranged from 0.6% to 3.1% between 2003 and 2008. Quantitative analysis of the EQA data Quantitative data were submitted in 83% (n=218/262) of datasets, compared to 82% in 2009. Standard deviation values for the 2010 programme ranged from 0.408 to 0.460 log10 units for viral loads from 2.362 to 6.407 log10 units (Table 6). In comparison, the standard deviation values in the 2009 programme ranged from 0.418 to 0.614 log10 units for viral loads from 2.325 to 6.348 log10 units. Consensus concentration values were highly comparable between real time commercial and real time in-house technologies (Table 7) although regression coefficients show some variation (Figures 6 and 7). Individual participants should investigate these coefficients and linearities using the results from their own laboratory. The consensus concentration values for the conventional commercial PCR technologies were higher than for the real time technologies for all panel samples in human plasma (Table 7). All technology groups showed a high degree of reproducibility on the duplicate panel samples.

Acknowledgements

Data analysis and report generation were performed by Stuart West and William MacKay of the QCMD Neutral Office.

QCMD © 2010. The QCMD EQA programme samples, associated reports and data generated during this programme are intended for External Quality Assessment (EQA) and Proficiency Testing (PT) purposes only. QCMD operates according to a strict Code of Practice which is in line with ISO/IEC 17043 and associated standards. Data reported in QCMD programmes is representative of a laboratory's standard diagnostic testing protocols irrespective of the technology they use. The data provided in the reports are based on technical information provided by the individual laboratories as part of the assessment process, as such it does not constitute a formal technology method comparison. All text and images produced by QCMD are the property of QCMD unless otherwise stated. The reproduction and use of these materials is not permitted without the express written consent of QCMD. The use of the information provided in QCMD reports for commercial purposes is strictly prohibited.

QCMD 2010 Human Cytomegalovirus DNA (CMVDNA10) EQA Programme

PAGE 9 of 11

Appendix A

Assigning the sample status

Sample status is assigned by peer-group consensus, based on the qualitative results returned by all participants. It is not a measure of the 'strength' of a positive sample nor is it technology-dependent, and is used solely for the scoring of the EQA data. The rationale for the sample status is: Frequently detected: More than 95% of datasets recorded the correct positive result. Detected: Between 65% and 95% of datasets recorded the correct positive result. Infrequently detected: Less than 65% of datasets recorded the correct positive result. Negative: A panel sample that does not contain the target and produces an unequivocal negative result.

Scoring system for qualitative EQA data

The scores awarded for qualitative EQA data were based on the sample status. The scoring system is represented in the following table, where 0 is 'highly satisfactory' and 3 is 'highly unsatisfactory'. Colour has been included as an extra visual aid.

Scoring system based on the assigned sample status

Sam ple status Negative Frequently detected Detected Infrequently detected Negative 3 2 1 0 Participant's result Not determ ined 3 2 1 3 Positive 0 0 0 3

QCMD 2010 Human Cytomegalovirus DNA (CMVDNA10) EQA Programme

PAGE 10 of 11

Scoring system for quantitative EQA data

In order to compare participants' results within specific technologies or kit methods, where sufficient datasets were reported (5 or more) methods or kits were assigned to a technology group e.g. Real time PCR, bDNA or NASBA etc. Where datasets were reported as 'other' for a technology or kit method this was reviewed by the QCMD Neutral Office and assigned to an appropriate group where possible. The negative panel samples were not included in these analyses. The parameters of the normal distribution was estimated from the log of the positive datasets submitted by participants for each panel sample. Scores were assigned based on the distance from the mean value for each panel sample. The scoring system used for quantitative EQA data ranged from 0 (highly satisfactory) to 3 (highly unsatisfactory).

An assigned value was calculated for each panel sample using two methods. These were: 1. Consensus concentration - the mean of the participants' results once outliers had been removed. 2. Technology consensus concentration - the mean of the participants' results per technology group once outliers had been removed. The standard deviation was calculated as the square root of the mean square for error from the ANOVA table where the response was the log concentration of participants' results with outliers removed. The factor was technology group. Outliers were defined as values with a standardised residual with modulus greater than three. Although outliers were removed for the calculation of the assigned values they were included in the data analysis and scored accordingly. Scores were awarded based on the distance from the calculated mean value for each panel sample. Zero points was awarded if the quantitative value returned was within one standard deviation from the mean. One point was awarded if the quantitative value was between one and two standard deviations, two points if the value was within two and three standard deviations and three points for quantitative values more than three standard deviations from the mean.

±1SD -2SD -3SD

0 1 2 3 1 2 3

+2SD +3SD

Each coloured division represents one standard deviation (SD) from the mean, so that zero points were awarded for quantitative values that were within one standard deviation and three points for quantitative values that were more than three standard deviations from the mean.

Further information about the QCMD method of analysing EQA data can be found in the following peer-reviewed publication: Staines HJ, Garcia-Fernandez L, Pogothata R, Wallace PS, MacKay WG, Van Loon AM. Monitoring performance of nucleic acidbased diagnostic measurement system users by EQA. Accred Qual Assur. 2009; 14: 243­252.

QCMD 2010 Human Cytomegalovirus DNA (CMVDNA10) EQA Programme

PAGE 11 of 11

Information

SVF_CMVDNA10_sgw_010710.xlsx

11 pages

Report File (DMCA)

Our content is added by our users. We aim to remove reported files within 1 working day. Please use this link to notify us:

Report this file as copyright or inappropriate

1323683


Notice: fwrite(): send of 193 bytes failed with errno=104 Connection reset by peer in /home/readbag.com/web/sphinxapi.php on line 531