Blood XMRV Scientific Research Working Group Report

ixchelkali

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This PowerPoint presentation from the Blood XMRV Scientific Research Working Group has been posted on the Blood Products Advisory Committee meeting website since I last checked the site. You have to scroll clear to the bottom and click on the report by Graham Simmons.
http://www.fda.gov/AdvisoryCommitte.../BloodProductsAdvisoryCommittee/ucm221857.htm
You need to have PowerPoint to read it; I had to download an adapter to read it with my older version.

I think it's good news. This is the kind of systematic research we need to have being done. I was somewhat worried that the "spiked" samples they were testing (so that they'd all be testing the same thing) wouldn't necessarily behave the way the "wild" virus does in real patients, but it looks like they're considering that, too. In phases III and IV they will use samples from real live people.
 

ixchelkali

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Slides 1 thru 6

I'm going to try to copy as much as I can into plain text, but some of the best parts are tables I don't know how to reproduce here.

slide 1
Blood XMRV Scientific Research Working Group

  • Mission - design and coordinate research studies to evaluate whether XMRV poses a threat to blood safety
  • Working Group includes representatives from transfusion medicine, retrovirology, and CFS scientific communities, as well as representatives from key Federal Agencies including HHS, FDA, NCI, CDC and NHLBI
  • Evaluation of blood safety risks includes several steps:
Evaluate XMRV nucleic acid and antibody assays

Establish prevalence of XMRV in blood donors
Determine if XMRV is transfusion-transmitted
Determine if transfusions are associated with CFS or prostate cancer (epidemiology studies)
================
slide 2
Blood XMRV Scientific Research Working Group
NHLBI - Simone Glynn - Chair
HHS - Jerry Holmberg - Co-chair
NIH - Harvey Alter
ABC - Celso Bianco
CDC - William Bower
BSRI - Michael Busch
ARC - Roger Dodd
FDA - Jay Epstein
FDA - Diane Gubernot
NIH - Eleanor Hanna
CDC- Michael Hendry
MVRBC - Louis Katz
AABB - Steven Kleinman
CDC - Stephan Monroe
NCI - Francis Ruscetti
ARC - Susan Stramer
BSRI - Leslie Tobler
CFIDS - Suzanne Vernon

Testing Labs - Investigators
BSRI - Graham Simmons
CDC - Bill Switzer, Walid Heneine
FDA - Indira Hewlett
FDA - Shyh-Ching Lo
NCI - John Coffin
WPI - Judy Mikovits

=================
slice 3
XMRV SRWG REDS-II Panel Study Phases
Phase I - Analytical Panels
Evaluate performance of XMRV NAT assays

Phase II - Pilot Clinical Studies
Whole Blood versus PBMC
Timing of sample preparation

Phase III - Clinical Sensitivity/Specificity Panel
Assay performance on pedigreed clinical samples

Phase IV - Blood Donor Clinical Panel
Initial estimation of XMRV nucleic acids prevalence in blood donors
Initiation of donor seroprevalence studies

================
slide 4
Phase I - Analytical Panels

Analytical performance panels
Comparison of Limit Of Detections and accuracy of Viral Loads of current assays; standardize performance of future XMRV detection assays for blood cells and plasma
Whole blood panel - spiked with XMRV positive cells
Plasma panel - spiked with supernatant containing XMRV

22Rv1 cells
Human prostate cell line chronically infected with XMRV
Contain at least 10 XMRV proviral copies each
Metzger et al (2010) J Virol 84:1874 (PMID:20007266)

Virus supernatant
Supernatant from cultured 22Rv1 cells
Approximate Viral Load of 5 x 109 RNA copies/ml

===============
slide 5
Whole Blood
Whole Blood (WB) unit from pedigreed (NAT and serology) negative donor

22Rv1 cells spiked into WB to yield 9,900 22Rv1 cells per ml of WB

Three-fold dilutions in fresh WB

0.5 ml aliquots to give 15 panels with three replicates at each dilution, plus 6 negative controls

Plasma

Plasma components from two pedigreed (NAT and serology) negative donors

22Rv1 supernatant spiked to give approximately 250,000 RNA copies per ml of plasma

five-fold dilutions in fresh plasma

0.5 ml aliquots to give 15 panels with three replicates at each dilution, plus 6 negative controls

=================
slide 6
Distributed as two blinded panels of 36 samples each

Whole Blood
9,900 cells/ml
3,300
1,100
367
122
41
13.6
4.5
1.5
0.5
0
0
Plasma

250,000 copies/ml
50,000
10,000
2,000
400
80
16
3.2
0.64
0.128
0
0
 

ixchelkali

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Slides 12 thru 15

slide 12
Conclusions and Limitations

XMRV NAT detection assays were sensitive
All WB assays detected at least 136 proviral copies/ml and four out of six [CDC, FDA(Lo), WPI and NCI] assays demonstrated even greater limits of detection

Four out of five plasma RNA assays performed similarly, with limits of detection of at least 80 RNA copies/ml

Limitations
The study is too small to conduct meaningful statistical comparisons or additional analysis (such as probit to derive confidence intervals around limits of detection)

WB panel lacked sufficient dilutions to reach endpoints

XMRV isolate with which 22Rv1 cells are infected may not adequately represent the diversity of XMRV clinical isolates

Further work on analytical panel development will need to be performed

================
slide 13
Phase II Pilot Studies

Whole Blood versus PBMC versus plasma
Original Lombardi et al. study performed on isolated PBMC and plasma

Large scale studies for prevalence facilitated by using WB or plasma

Donor-recipient and other repositories are mainly comprised of frozen WB and/or plasma

Timing of Processing
Processing and freezing of donor samples slotted to be included in the blood donor clinical panel (phase IV) vary from 2-4 days due to requirements for completion of ID testing. Available donor repositories include frozen WB and plasma samples processed 1-3 days post-phlebotomy.

Studies with other cell-associated viruses (HERVs, HTLV, herpesviruses and anelloviruses), demonstrate that levels of viral nucleic acid in plasma and whole blood vary with time from collection to processing and frozen storage.

===================

slide 14
Phase II Pilot Study Set-ups

XMRV-positive samples
WPI has collected blood from four CFS patients previously identified as XMRV positive in the Lombardi et al. study (by PCR, serology and culture)

Samples separated into tubes and were processed immediately, or left at 4oC for 2 or 4 days

Each sample was processed into PBMC, WB and plasma.

Analysis
Panels were distributed by WPI to CDC and NCI for testing; WPI retained one panel for testing. One set of the panel was distributed to BSRI to keep and use for follow-up work as needed.

====================

slide 15
Phase III - Clinical Sensitivity and Specificity

The ability of participant assays to effectively detect clinical XMRV-positive and -negatives will be examined
To attempt to overcome the issues of clinical variation and specificity, larger numbers of pedigreed clinical XMRV-positive and -negative samples are being assembled into coded cell and plasma panels to be distributed to all labs

Positives
25 clinical samples will be collected by WPI. All will be from patients reported in the Lombardi et al. study to be positive for XMRV by PCR, serology and virus culture. Method and timing of processing will be determined based on pilot study data

Negatives
PCR detection assays have the potential to amplify non-specific human derived genomic sequences

Thus, a larger group of pedigreed negative donors is required in order to introduce generic variability (10 donors, with 3 replicates per donor = 30 negative donor samples in each panel)

The donors will be pedigreed negative by PCR at WPI, FDA (Dr. Lo) and CDC labs, and for serology at WPI, CDC and NCI (Dr. Ruscetti) labs
 

ixchelkali

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Slides 16 & 17

slide 16
Phase IV - Clinical Panel for Donor Prevalence

Source and coding of donor specimens:

BSI/CTS processed residual blood in pilot tubes (vacutainer tubes used for routine ID screening) from 396 donations given by apheresis and double-RBC donors in the Reno/Tahoe area in Dec 2009 into replicate frozen WB and plasma aliquots.

Aliquots were anonymized as sequentially coded samples after capturing donor gender, age and zip code of residence

======================

slide 17
Phase IV - Clinical Panel for Donor Prevalence

Blinded panels consisting of approximately 300 blood donor samples, 25 confirmed XMRV-positive clinical samples and about 30 pedigreed-negative samples from 10 independent donors will be created for WB and plasma

Blinded panels will be distributed to at least four of the participating laboratories for testing.

Test results will be analyzed:
Correlation between whole blood and plasma testing will be determined for each lab and between labs.

Preliminary XMRV prevalence in blood donors (proportion positive for XMRV DNA or RNA) will be estimated based on compiled results from each lab.

==========================

[That's all that aren't tables]
 

Otis

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Does seem like that set of slides appeared later, or I missed it too. :confused:

Just to clarify, select "July 27, 2010: Blood Products Advisory Committee Meeting Presentation: Blood XMRV Scientific Research Working Group (PPTX - 237KB)" to download this PPT file.
 

George

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Sounds like phase I and II are complete and they are on Phase III. I would imagine they are happy for the delay in publication of the myrid of papers to be able to get so far in the phases.
 

JT1024

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I saw a different set of slides probably along the same line. Try the link below. I believe the FDA is in the process of standardizing lab testing so large scale testing can begin.

http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/BloodVaccinesandOtherBiologics/BloodProductsAdvisoryCommittee/UCM222106.ppt#524,1,An

They are apparently coming up with the minimal amount of XMRV to be detected in standardized assays as well as positive controls. They also mention licensing.

Lab testing methodologies need to be validated/licensed. I'm not sure of the exact licensing process but many companies provide assays to detect the various infectious agents as well as other substances (human proteins, antibodies, enzymes, etc). This slideshow indicates they are well along in the process.

Below is the link to an overview of how FDA regulates IVD's (in vitro diagnostics):

http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/IVDRegulatoryAssistance/ucm123682.htm#5
 

Hope123

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If you look through the list of presentations (there are a lot!), there are some other ones on XMRV including one by Indira Hewlett at FDA where she talks about a small African study showing no XMRV in HIV subjects there based on PCR. This is the first I've heard of this study. Anyhow, lots to be gleaned from the slides.
 

Stone

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Can someone please remind me again what this group is hoping to accomplish and basically what these slides mean to us? I'm too foggy/fatigued to wade through it. Can I get an overview from some kind person please?
 

JT1024

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There are two separate Powerpoint presentations from the FDA website. The author of both, Indira Hewlett, Ph. D, is the Chief, Laboratory of Molecular Virology, with the FDA's Center for Biologics Evaluation and Research (CBER), Division of Emerging and Transfusion Transmitted Diseases (DETTD).

The first presentation addresses the fact that there are currently no FDA approved assays for XMRV detection and assays used in cohort studies have not been standardized.

For this purpose a Blood XMRV Scientific Working Group led by National Heart, Lung and Blood Institute (NHLBI) was established to standardize and validate assays to evaluate transfusion risk in future studies through the Retrovirus Epidemiology Study Group (REDS).

The second presentation states:

RT-PCR, qPCR assays for XMRV detection have been established. Ongoing assay improvements for whole blood and plasma are underway. I have yet to understand the reasoning for testing areas endemic for HIV that has not been tested for XMRV. Additional blood donor PBMC (Peripheral Blood Mononuclear Cells) and plasma, including US blood donor specimens are being tested for XMRV DNA and RNA respectively.

Of great significance is the statement that "Testing of well pedigreed CFS patient samples is planned for the future". That tells me that the diagnostic criteria for CFS is going to be more clearly defined. Hopefully, they'll relinquish the CDC definition as garbage and accept the Canadian Consensus Criteria as the standard.

The statement that "FDA Lot release panels are used to establish standards for licensure of assays and post market surveillance of licensed assays." indicates they are moving quickly towards standardizing testing so commercial vendors (e.g. Abbott and others) can develop and market test kits for clinical laboratories.
 

Stone

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Thanks! That helps a lot. It's exactly what I needed and I very much appreciate it.

Todah Rabbah (Thanks very much)
 

Bob

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Transcript - Bloods Products Advisory Committee, 26th July 2010

Here's a transcript (from the FDA) of the Bloods Products Advisory Committee, held on 26th July 2010:

http://www.fda.gov/downloads/Adviso.../BloodProductsAdvisoryCommittee/UCM225388.pdf


It includes the following (see Table of Contents):

An Update on CDC XMRV Activities - Michael Hendry

An Update on FDA/OBRR XMRV Activities - Indira Hewlett

An Update on Blood XMRV Working Group Studies - Graham Simmons

An Update on NCI XMRV Assay Development - Stuart Le Grice