Saturday, February 18, 2012

Answers to 10 Common Criticisms of the Scientific Research Working Group Study ("Blood Working Group")

Answers to 10 Common Criticisms of the SRWG study
by Graham Simmons, PhD


1. All of the controls were not screened by all of the labs.
Response: Controls were screened by at least five labs: WPI, National Cancer Insitute/NCI-Ruscetti, Food and Drug Administration/FDA-Lo, Centers for Disease Control & Prevention (CDC) and NCI/Drug Resistance Program (DRP).

2. Control peripheral blood mononuclear cells (PBMCs) were not screened prior to blinding, so could not have been ruled as negative.
Response: Three out of the 15 did have their PBMCs extensively screened prior to blinding, yet two of these were still called “positive” in various assays by the WPI and NCI/Ruscetti in the study.

3. No cryopreservative was used for the storage of the PBMCs, which would prevent the WPI’s assay from working. No Trizol was used.
Response: Due to the short-term nature of the study it was not felt that preservatives were required for PBMC cryopreservation. The Lo/Alter study detected sequences in PBMCs stored for 15 years in the absence of preservatives. Trizol is for the extraction of nucleic acid and laboratories were given the option of choosing their own extraction methods

4. The length of time allotted for the serology and culture assays was massively reduced, so that the WPI or NCI/Ruscetti assays were not performed as desired.
Response: All the laboratories were allowed as much time as required to perform their desired assays. The culture and serological assays were performed by WPI and NCI/Ruscetti to their own specifications.

5. The WPI was not given the opportunity to complete virus culture assays.
Response: The WPI encountered mycoplasma contamination of their target cell population, and used the plasma samples without results. This was very unfortunate. There were no further stocks left to perform repeat cultures with. It was deemed by both the WPI and the working group that performing the studies on freeze/thawed material would be invalid.

6. Samples and collection tubes were handled in the same laboratory as 22Rv1 cells used to spike the analytical controls.
Response: As stated in the paper, 22Rv1 cells were handled in a separate facility to where all other activities were performed. The fact that only one laboratory detected PCR and virus culture in clinical samples supports the fact that 22Rv1 contamination did not occur at the central laboratory.

7. Patients were on additional therapies that would produce false negatives.
Response: Lo/Alter patients were not on any additional treatments. It is unclear what additional treatments patients were on at the time of Lombardi et al. There is no published evidence that additional treatments would have positive or negative effects.

8. FDA/Lo used the wrong assay from Lo et al. and instead used the one that could not detect positives.

Response: Lo et al. used their own criteria to decide on which assay(s) to use, but it is clear that both primer sets in their paper are equally capable of amplifying diverse polytropic murine leukemia viruses (MLVs), so it is not obvious that one would be better that the other at detecting “positives.”

9. The NCI-Ruscetti did no PCR and could not use their clinically validated serology and culture assays.
Response: NCI-Ruscetti felt that they were not sufficiently experienced at PCR to participate in the study. They did perform their serology and culture assays – just as performed in Lombardi et al.

10. All the SRWG labs optimized their assays to VP62. VP62 does not exist in nature and Lombardi et al. is now known to have discovered HGRVs. Does your study include HGRVs? Or how do HGRVs relate to XMRV?
Response: As demonstrated in an earlier slide, although this study was initiated after Lombardi et al. as a study of XMRV, as soon as Lo et al. was published the mission of the study was broadened to include all MLV-like viruses. Thus, almost all of the assays were designed to perform against MLVs in general and were optimized and tested as such. As our study has demonstrated there is no such thing as an independently validated clinically positive sample against which to test. Currently there is no such thing as human gammaretroviruses (HGRV). No published virus has been isolated, cloned or sequenced from a human.

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