R Gunson on behalf of SCVCG p1 of 3

Comparison of Scottish labs Adenovirus, CMV and EBV tests

Background

The aim was to compare both the sensitivity and quantitative values of the PCR tests for adenovirus, CMV and EBV DNA in all 4 Scottish labs, Glasgow, Edinburgh, Aberdeen and Dundee.

A panel of 5 serial dilutions of adenovirus, CMV and EBV samples was prepared, tested in Glasgow and distributed to the other 3 Scottish labs (Edinburgh, Aberdeen and Dundee). These were designed to test the whole process of extraction and amplification. In addition, one already extracted DNA sample of the three viruses was also sent. On receiving the returned results, it was apparent that the results of the samples were lower than expected. Based on this finding we were concerned that the virus titre might have declined during the transport at room temperature. To determine this we removed an aliquot from the -80oC freezer and retested this after 36 hrs at room temperature. On retesting a significant decline in titre of the samples especially at the bottom end of the range was observed. The titre of the extracted DNA remained constant. Due to uncertainty about the exact amount of sample decline, we felt unable to fully interpret the results

Comparison of QCMD results

As an additional QC measure and because we were concerned the results might not be a true reflection of the loss of titre during transport and storage, we also compared the most recent QCMD results of all 4 labs.

1. For adenovirus, none of the labs issued a quantitative result but all 4 performed satisfactorily, detecting all samples greater than 1000 copies/ml (Table 1). Lab 3 failed to detect 1 adenovirus sample of 1,000 copies/ml.

2. For CMV, all labs detected samples greater than 500 copies/ml. For CMV Glasgow has a quantitative value cut-off of 2,500 copies/ml. The quantitative results of all 4 labs were comparable to each other and the QCMD value (within 0.5 log) for values greater than 2,500 copies/ml, the required level of accurate quantitation (Table 2).

3. For EBV, Glasgow and lab 3 detected all samples. Lab 2 failed to detect one sample at 3,500, whilst Lab 4 failed to detect 1 sample at 700. For EBV Glasgow has a quantitative value cut-off of 5,000 copies/ml. The quantitative results of Glasgow, lab 2 and lab 3 were comparable to each other and the QCMD value (within 0.3 log). Lab 4 quantitated 0.5 - 1.0 log lower than the other labs and QCMD (Table 3).

Conclusions All 4 Scottish labs performed equivalently and satisfactorily for adenovirus and CMV detection and for CMV quantitation. All 4 Scottish labs performed equivalently and satisfactorily for EBV detection. Glasgow, Lab 2 and Lab 3 performed equivalently and satisfactorily for EBV quantitation; Lab 4 EBV 0.5 – 1 log lower than the other labs and QCMD. R Gunson on behalf of SCVCG p2 of 3

Table 1: Comparison of Scottish labs adenovirus QCMD results

QCMD Glasgow number QCMD log log Lab 2 log Lab 3 log Lab 4 log ADV11-01 2.99 pos pos neg pos ADV11-02 3.13 pos pos pos pos ADV11-03 2.43 pos pos pos pos ADV11-04 neg neg neg neg neg ADV11-05 4.03 pos pos pos pos ADV11-06 4.14 pos pos pos pos ADV11-07 3.87 pos pos pos pos ADV11-08 3.1 pos pos pos pos

Table 2: Comparison of Scottish labs CMV QCMD results

QCMD QCMD Glasgow number log/ml log/ml Lab 2 log Lab 3 log Lab 4 log

CMV12-01 4.3 3.97 4.12 4.33 4 CMV12-02 3.74 3.38 3.67 4.11 4.04 CMV12-03 2.24 1.12 neg 2.33 neg CMV12-04 2.08 neg pos 2.56 neg CMV12-05 2.91 2.86 2.96 3.41 3 CMV12-06 3.3 2.9 3.12 3.22 pos CMV12-07 3.33 2.8 3 3.33 3.34 CMV12-08 neg neg neg neg neg CMV12-09 3.67 3.29 3.49 3.75 3.76 CMV12-10 2.73 2.72 2.82 2.95 pos R Gunson on behalf of SCVCG p3 of 3

Table 3: Comparison of Scottish labs EBV QCMD results

QCMD Glasgow number QCMD log log Lab 2 log Lab 3 log Lab 4 log EBV12-01 2.76 2.92 pos 2.99 neg EBV12-02 3.8 4.15 4.18 4.49 3.18 EBV12-03 5.21 5.54 5.55 5.64 4.76 EBV12-04 4.82 4.93 5.12 5.21 4.32 EBV12-05 4.33 4.63 4.66 4.96 3.79 EBV12-06 4.49 4.88 4.76 5.04 3.87 EBV12-07 4.79 4.96 4.73 5.27 4.38 EBV12-08 3.5 3.89 neg 3.92 pos EBV12-09 4.3 3.43 3.57 3.77 pos EBV12-10 neg neg neg neg neg