Gerwyn helped me understand that there are at least two important ways that Satterfield et al. can be distinguished from Lombardi et al.
First, the difference between PMBCs and plasma:
Lombardi et al. (remember, this included three different labs- the WPI, the Cleveland Clinic and the National Cancer Institute) used western blot and IFC after 42 days of culturing peripheral blood mononuclear cells (PMBCs). They also used a range of antibodies, including SSFV, which is specific to MLVs.
Thus, Lombardi et al. isolated DNA and RNA from concentrated PMBCs. For the detection of a human gamma retrovirus, the concentration of viral nucleic acid is vital.
Satterfield, on the other hand, attempted to locate HMRV in DNA extracted from 62 microlitres of plasma (not PMBCs). Not only is plasma far more dilute than PMBCs (plasma is 97 percent water) but Satterfield used a mere 62 microliters. The concentration of potential HMRV viral nucleic acid in Satterfield’s assay would have been close to, if not actually, zero.
At least Satterfield is consistent in his methods. Using 62 microliters of plasma for such a research study was reminiscent of his ridiculous commercial Cooperative Diagnostic test, which he claimed was able to detect HMRV in a finger prick's worth of blood before it was pulled off the market.
Second, the Satterfield et al. assay:
An assay that detects HMRV in Macaques monkeys will not necessarily detect HMRV in humans because antibody responses in primates are qualitatively and quantitatively different from those in humans.
Satterfield’s assay was validated only for Macaques monkeys, not humans.
In fact, Satterfield’s serology assay failed to find antibodies to HGRV in clinically validated positive human samples.
So even if he had looked for HMRV in the right place and cultured PMBCs for 42 days, as Lombardi et al did, Satterfield’s assay was not validated to humans.
Just three amino acid changes in the SU region would defeat the Elisa and Western Blot tests completely.
I don't think Gerwyn is correct. There is no indication in the Lombardi paper that they did any culturing for the PCR. I went around and around with him on this. I plucked out the methodology section piece by piece and showed it to him and asked where is the culturing? Finally he said it was inferred or something like that.
If you'll read Satterfields interview a bit more closely you'll see he quantifies the amount of DNA he was looking through and according to him his tests looked
through much more DNA than in the original study. Or you can actually look at the paper a quick look at which indicates the Satterfield et al didn't use plasma at all - they used the same PBMC's in the same manner as Lombardi et al.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3050813/?tool=pubmed
Blood samples were shipped from collection centers overnight. Most were processed immediately upon arrival, but a few samples were incubated in the refrigerator for 1 to 2 days prior to separation of the blood components. For component separation, blood was centrifuged and the buffy coat, including the peripheral blood mononuclear cells (PBMCs), was immediately and carefully removed. The buffy coat was either processed immediately or stored at -20C for later analysis. Nucleic acids were extracted using the Qiagen blood DNA minikit protocol (Qiagen, Valencia, CA). Extracted DNA was quantitated using the Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) and checked for integrity with a minimum 260/280 ratio of 1.8 and by -actin PCR. Plasma was immediately frozen for later analysis.
PCR analysis was performed on PBMC DNA using three previously described tests (Table (Table2),2), two for the polymerase (pol) gene, and one for the gag gene used in Urisman et al., Lombardi et al., and Lo et al. [1,5,9,14].
In the paper Satterfield et al point out how much DNA they examined
PCR was used to test a subset of specimens for which sufficient DNA remained, including 28 samples from "severe CFS" persons, 11 "unclassified CFS" and 9 controls [1,9]. 2.5 μg of DNA (833 ng of PBMC DNA) was used in the pol real-time PCR test, providing for 3.3 to 8.3 times the PBMC DNA used by Lombardi et al. [5,14].
They used the 22RV1 clone to test their samples against....
Dilutions of DNA from XMRV-infected 22Rv1 human prostate carcinoma cells were used as positive controls in this test [15]. 1.0 μg of DNA (333 ng of PBMC DNA) was used in the nested pol and gag PCR tests at the CDC for which 1,000 and 10 copies of the XMRV(VP62) plasmid were used as positive controls [1,9]
The Lombardi paper stated the strains they found were essentially the same as VP62 and, I believe VP34 - which later research showed were essentially the same as 22RVI.
Satterfield’s assay was validated only for Macaques monkeys, not humans.
In fact, Satterfield’s serology assay failed to find antibodies to HGRV in clinically validated positive human samples.
There actually are no clinically validated positive human samples. That's the whole problem isn't it? Except for the labs in the Science paper no other labs have validated Lombardi's finding and that's how you validate tests. If there is a blood sample that everybody could agree was positive then they would all use that - but only the CDC and the WPI could detect XMRV in one of the BWG's tests and only sometimes (and the CDC not in the other one)....That's why Abbot spend $250,000 or whatever it is to infect those monkeys with XMRV. If they had had what they consider to be validated human samples they would have certainly used those to develop their antibody tests.
The labs have, however, except for the culturing replicated all of Lombardi et al's methods. Neither the PCR nor the antibody tests involved culturing. I asked Racaniello about this and he said there's no culturing indicated in the PCR test in the paper.
You know these people aren't stupid. This is what they do for a living and their labs credibility is at stake with every paper they put out. Gerwyn seems to rather consistently find glaring errors big enough to drive a truck through in his analyses of these studies; ie it was apparently impossible for Satterfield to find XMRV given their methods. But consider that given the attention this field is getting Satterfield and his lab are sure to look like idiots rather quickly if they make such silly mistakes as Gerwyn suggests.
What Gerwyn is saying- that the negative studies are all bogus...and that the authors are idiots and they're making simple mistakes......is very enticing - he is saying what everyone wants to hear...But...
If you read Satterfields bio at the top of the paper - his work creating new PCR techniques, his work with the Defense Dept using those techniques, his development of new algorhythms for PCR,... the fact that he founded a company that does nothing but PCR.....I feel like he probably knows what he's doing. I felt the same way with Dusty Miller and his 20 years of work in the field and his 200 plus papers. So if I'm going to have decide whether to trust someone; Gerwyn - or Satterfield or Dusty Miller - I'm going to have to go with them - no matter how enticing what Gerwyn says he believes is going on...
That's just me!
Maybe I respect authority too much! Time will tell.