Blood XMRV Scientific Research Working Group

From ME/CFS Wiki

Jump to: navigation, search

Encyclopedia | Forum | Main Page | XMRV & MLV's


Contents



Health & Human Services/National Heart, Lung, and Blood Institute (HHS/NHLBI) Blood XMRV Scientific Research Working Group first convened in December 2009. [1]


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. Six labs are participating—the CDC, FDA (2), National Cancer Institute (NCI), WPI, and Blood Systems Research Institute (major contractor for the REDS 2 study).


Evaluation of blood safety risks includes several steps:

  1. Evaluate XMRV nucleic acid and antibody assays
  2. Establish prevalence of XMRV in blood donors
  3. Determine if XMRV is transfusion-transmitted
  4. Determine if transfusions are associated with CFS or prostate cancer (epidemiology studies)
  5. If XMRV is transfusion transmitted, what is the clinical impact on recipients of blood transfusions?


The Blood XMRV Scientific Research Working Group will report to the Department’s Blood, Organ and Tissue Senior Executive Council through established mechanisms.


Funding for currently launched studies via NHLBI REDS-II Program


Members

HHS

  • Jerry Holmberg - Co-chair of XMRV SRWG.
HHS, Senior Advisor for Blood Policy, Blood Safety and Availability, Office of Public Health and Science (OPHS)

NIH

  • NHLBI - Simone Glynn – Chair of XMRV SRWG.
Director of Blood Resources Program at the National Heart, Lung, and Blood Institute (NHLBI) and oversees the Retrovirus Epidemiology Donor Study (REDS) established in early 1990s.
  • CC - Harvey Alter
  • NCI - Francis Ruscetti
  • NCI – John Coffin
  • OD - Dennis Mangan

CDC

  • OID/NCEZID - Stephan Monroe
  • OID/NCEZID - William Bower
  • OID/NCHHSTP -Michael Hendry

FDA

  • CBER - Jay Epstein
  • CBER - Diane Gubernot
  • CBER – Hira Nakhasi

AABB

  • Steven Kleinman

ABC

  • Celso Bianco

ARC

  • Roger Dodd
  • Susan Stramer

BSRI

  • Michael Busch
  • Leslie Tobler

CFIDS

  • Suzanne Vernon

Central Lab

  • BSRI - Graham Simmons

Harvard Medical School

  • Anthony Komaroff

MVRBC

  • Louis Katz


NAT Lab Investigators

  • Graham Simmons - Blood Systems Research Institute (BSRI)
  • Bill Switzer, Walid Heneine - CDC
  • Indira Hewlett - FDA
  • Shyh-Ching Lo - FDA
  • John Coffin - NCI
  • Judy Mikovits - WPI


NOTE: Members that were not on this list when presented at the 1st Intl. Workshop on XMRV (7-8 September 2010):

  • OD - Dennis Mangan
  • CBER – Hira Nakhasi
  • Harvard Medical School - Anthony Komaroff



Summary of study design

Phase I - Analytical Panels

  • Evaluate performance of XMRV NAT assays


Phase II - Pilot Clinical Studies

  • Whole Blood versus PBMC
  • Timing of sample preparation
  • REDS 2 is scheduled to conclude in December 2010, with REDS 3 scheduled to begin soon after.


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


Phase 1 - Analytical Panels

The first stage will be to standardize and validate laboratory methods and reagents for XMRV testing. (Variations in sample collection and laboratory procedures can produce discrepant results.) Each of the six labs analyse blood samples spiked with different amounts of the retrovirus XMRV, in order to determine whether or not the labs' tests are able to detect XMRV in the blood. Samples distributed in March 2010.


  • Blinded
  • Begun after quantitation of the number of viral copies in the cell or the supernate.
  • 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
  • Developed 15 panels based on the number of laboratories that expressed interest both in and outside the United States.
  • Cell line used was human prostate 22Rv1. (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))
  • Lo (FDA) & WPI lab both had a false positive with the Whole blood assay. After decoding the labs went back and sequenced these products, and found them to be non-sepcific bands of human genomic origin. So it was likely a false positive introduced by non-speific priming in the PCR. As these labs perform sequencing for any confirmation of clinical samples, it wasn't believed to be a major issue in terms of assay reproducibility.


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
  • The overall similarity of results suggests sensitivity of assays cannot explain differences in XMRV detection in clinical samples reported by the participating laboratories [2]


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
  • Use of two units of plasma as diluent for preparation of the spiked plasma panel may have compromised the accurate dilution of virus [3]
  • Further work on analytical panel development will need to be performed


Phase II - Pilot Clinical Studies

Scheduled to begin in the June/July timeframe. In this second phase the WPI collected blood from four CFS patients they had found to be XMRV-positive and sent the samples to CDC and NCI for testing. [4] WPI retained one panel for testing, and one set of the panel was distributed to BSRI to keep and use for follow-up work as needed.

  • Must identify the processing time between the collection and preparation of the samples. Culturing is gold standard testing, but takes too long for large scale testing/screening.
  • Must identify the differences between the whole blood preps, Plasma and the peripheral blood mononuclear cells. Ideally large scale studies for prevalence need to use whole blood or plasma, as donor-recipient and other repositories are mainly comprised of frozen whole blood and/or plasma.
  • Two neutral individuals will be collecting results for the analytical and the clinical panel, then report results to the working group.


Results are expected at the beginning of November 2010.


Results

On the 14 December 2010, Phase II results of the Blood Working Group were announced at the Blood Products Advisory Committee Meeting in Washington. [5]



Phase IIa - Setup

4 samples collected by WPI, previously identified as XMRV positive in Lombardi et al.

  • Four Females
  • 26-53 Years of age
  • Three were diagnosed with CFS over 20 years ago, one is a family member of a long-time CFS patient
  • Specimens separated into tubes and were processed immediately, or left at 4oC for 2 or 4 days
  • Each specimen was processed into replicate peripheral blood mononuclear cells, whole blood, and plasma samples that were frozen for panel preparation
  • Unblinded panels were distributed to WPI and CDC and a blinded panel to NCI for testing
  • Patient number 4 had also begun to take Anti- retroviral drugs since May 2010.
  • Not blinded (except NCI/DRP who requested it, and extra negatives sent)


Phase IIa - Findings

LABS:


CDC

  • All 4 patients positive at some point (Day 2, 4, not day 0 though) using plasma. One Nested PCR (N XGAG) assay, one qRT-PCR (pro - protease) assay, and one qRT-PCR (int - integrase) assay used.
  • Western Blot all negative for plasma
  • negative for whole blood and PBLs (peripheral blood lymphocyte - AKA PBMC's) DNA/RNA test. 2 Nested PCR assays used (N Xpol PCR & N XGAG PCR), and one qRT-PCR(pro) assay.
  • Using the Nested PCR results they sequenced the products and found them to be XMRV related. (the products for qRT-PCR were too small to use, and too highly conserved to differentiate between XMRV & other MLV-related viruses)
  • All PCR positive samples tested negative for mouse mitochondrial DNA.


WPI

  • Used same Nested PCR for gag as used in Lombardi et al.
  • All positive bands of the correct size are sequenced to ensure it is true XMRV or another MLV-related virus.
  • All 4 patients positive using plasma nested RT-PCR for XMRV gag. (Using Qiagen Viral RNA kit) Only one of the four positive on Day 0, others on Day 2 & 4.
  • Whole blood samples were all negative for DNA.
  • PBMC were not tested. (samples were lost)


NCI/DRP

  • Used single copy qRT PCR for gag
  • This assay and the CDC ultracentrifuged the plasma, whereas WPI directly extract nucleic acid from plasma. Not clear if this is an issue or not.
  • All samples (Plasma, Whole blood and Peripheral Blood Mononuclear Cells) and all time-points (Day 0, 2, 4) were negative.


Phase IIa - Conclusions and Limitations

  • Two out of three labs detected XMRV in clinical samples
  • Plasma out-performed whole blood in both labs, and Peripheral Blood Mononuclear Cells in the one lab (CDC) that tested Peripheral Blood Mononuclear Cells.
  • Day 2 and day 4 samples out-performed day 0 plasma samples in both labs. Could be that infected cells are dying and releasing bits into the plasma, and is therefore easier to detect at later time points. Not sure yet.
  • Panel was distributed mostly unblinded (except NCI/DRP)
  • Small sample size.
  • Third laboratory failed to detect virus despite sensitive assay.
  • Decided to run a phase IIb and be more structured.


Phase IIb - Setup
  • Blood collected by BSRI, and study blinded.
  • Blood collected into EDTA tubes by an independent phlebotomist at the 4 patients home or work. (one patient had independently initiated anti-retroviral therapy)
  • The pedigreed negative subject used for the analytical panel and phase IIa panel was bled by the same phlebotomist as a control. Tested multiple times by all of the labs involved, with all methods.
  • Phlebotomist directly supplied the samples to BSRI for processing
  • Samples separated into tubes and were processed the same day, or left at 4oC for 2 days
  • Each sample was processed into Peripheral Blood Mononuclear Cells, whole blood, and plasma
  • Blinded panels were distributed by BSRI to WPI, CDC, NCI and Gen-Probe for testing. Two sets of the panel were retained by BSRI to distribute for follow-up work as needed
  • Panels of PBMCs, whole blood & plasma.
  • Results were reported back to BSRI and decoded


Phase IIb - Findings

LABS:


NCI/DRP

  • All Plasma and Peripheral Blood Mononuclear Cell (PBMC) samples at both time points (Day 0, 2) were negative.
  • Ultracentrifuged the plasma or directly extract nucleic acid from plasma.
  • Used single copy qRT PCR for gag.


CDC

  • All Plasma and Peripheral Blood Mononuclear Cell samples at both time points (Day 0, 2) were negative.
  • Same assays as before & new assays. (Nested RT-PCR for XMRV gag & env, qRT-PCR for MLV-related gag & int)


Gen-Probe

  • All Plasma and Peripheral Blood Mononuclear Cell samples at both time points (Day 0, 2) were negative
  • Used target Capture/Transcription-Mediated Amplification (TMA)/chemiluminescent detection (high throughput test) Based on a duplex assay (targets conserved sequences in two separate regions of XMRV genome)
  • Their assay is only validated for XMRV not other MLV-related retroviruses.


WPI

  • 3 patients were positive, patient on Anti-retroviruals was negative, and the negative control was positive using PBMCs. Investigation following decoding of results determined that there was a procedural error during PBMC sample extraction involving reuse of needles (employed to lyse cells and shear DNA) on sequential PBMC cell pellets. So no conclusions can be drawn from this.
  • Plasma all negative.
  • Same Nested RT-PCR for MLV-related gag.
  • Whole blood samples not completed yet - results to be announced at a later date.


Serology only done by CDC & NCI/Ruscetti.


NCI (Ruscetti)

  • 3/4 patients positive for serology & the negative control
  • Used flow cytometry (similar to that used in Lombardi et al.) expressing spleen focus-forming virus env. It detects anti XMRV antibodies.


CDC

  • Serology all negative (Switzer et al. WB for multiple MLVs)
  • Used WB (Western Blot) for multiple MLV-related antigens.


WPI

  • During the Blood Products Advisory Committee Meeting, Mikovits announced that there is complete concordance between Ruscetti's (NCI) and WPIs results by serology. [6]
"There was complete concordance between Frank Ruscetti's serology results and our work with those patient samples. We don't do direct PCR. We were asked to do that for the purpose of this study."
Dr Judy Mikovits, 'December 14, 2010: Blood Products Advisory Committee Meeting Transcript' (FDA, 14 December 2010) [7]



Whole blood not completed yet - results to be announced at a later date.


Phase IIb - Conclusions and Limitations

  • All four labs negative with Plasma. Not sure why, may be level of virus in plasma, or processing issues
  • Only one out of four labs detected XMRV in clinical samples and only in Peripheral Blood Mononuclear Cells.
  • Ultracentrifugation or direct extraction of plasma did not seem to make a difference in detection by NCI lab
  • Sensitive NAT assay from a diagnostic company was unable to detect XMRV/MRV
  • Not designed or performed as a clinical evaluation or longitudinal study
  • Impossible to draw any conclusions from this extremely small study. (Loss of plasma positivity for all samples, false positivity in PCR due to processing issues, false positivity in serology - or was it?)
  • Based on Phase II findings, no clear advantage to delayed processing
  • Include serology in parallel with NAT in future studies
  • Continue with collection of Phase III panel. Include more positive and negative samples and differently sourced samples, peripheral Blood Mononuclear Cells, whole blood, and plasma will be used, with a standard next day processing protocol.


Vote on CFS Blood Donation Referral

At the Blood Products Advisory Committee Meeting on the 14, the FDA asked the Blood Products Advisory Committee (BPAC) to vote on whether the scientific data support asking donors about a medical history and/or diagnosis of CFS as a basis for indefinite deferral. The panel voted 9 to 4 in favour of indefinite deferral of CFS patients based on all evidence that it will promote donor and recipient safety. This recommendation will now be reviewed by the FDA, which typically follows the advice of such panels but is not required to do so. There is no timetable yet on a final decision.


Phase III - Clinical Sensitivity/Specificity Panel

The ability of participant assays to effectively detect XMRV positive and negative clinical samples will be examined.


According to the BWG slides from the 1st XMRV International Workshop (7-8 September 2010) [8], in the third phase, WPI will collect 25 samples from XMRV-positive CFS patients and send them — as well as 30 XMRV-negative samples from 10 healthy donors — to all the other labs for testing. [9] The donors will be pedigreed negative by PCR at WPI and CDC, and for serology at WPI, CDC and NCI (Ruscetti/Bagni) labs.

  • Again, had to wait for IRB approval for taking samples from 25 CFS patients.
  • Plasma and whole blood panels will be matched. (Method and timing of processing will be determined based on pilot study data.)


The design for Phase III had altered slightly when the BWG slides were published by the CAA on the 17 December 2010. [10] Instead of 25 positive clincal samples being collected by the WPI, 30 were now to be collected by the WPI and Harvard. All will be from patients reported by multiple assays as XMRV/MRV+ in the Lombardi et al. or Lo et al. studies. Method and timing of processing will be determined based on pilot study data.


On the 27 December 2010, the XMRV SRWG published a statement to say that they had discussed the findings from the four studies published in Retrovirology on December 20, 2010. That those studies confirmed the importance of carefully checking XMRV/MLV related-positive results for any evidence of contamination with mouse genetic materials. And that the labs involved in the XMRV SRWG will continue to apply best practices and check to the best of their ability that no contamination with mouse DNA is present before reporting any positive results. They also stated that the reports also substantiate the importance of employing tests that not only detect viral DNA and/or RNA but can also detect the virus itself (culture) and/or an immunological reaction to the virus. And that these tests are reflected in the Working Group planned phase III study. [11]


On the 4 August 2011, CFS Central reported that the BWG had begun to break the codes for Phase III of the study and that they were expected the following month. [12]

Phase IV - Blood Donor Clinical Panel

Phase four will analyze blood from 300 blood donors, 25 confirmed XMRV-positive patients (According to the BWG slides from the 1st International XMRV Workshop) [13], but now stated as being 30 (according to the BWG slides from the CAA 17 December 2010 webinar) [14]), and 30 XMRV-negative samples from 10 independent blood donors will be created for WB and plasma. [15] 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.


Links

Phase II

Phase III

Articles

FDA meetings

Notes

  • NAT - Nucleic Acid Amplification Testing. Direct detection of viral genetic material (RNA or DNA) in plasma, cells or blood using amplification methods, such as Polymerase chain reaction (PCR) or Transcription-mediated amplification (TMA)
  • NCI/DRP - National Cancer Institute/Drug Resistance Program.
  • Nested PCR – A second round of PCR performed on product of the first round. Very sensitive.
  • PCR - Polymerase chain reaction. Amplification of viral DNA sequence using highly specific primers yielding product of a certain size.
  • Quantitative PCR (qPCR) or real-time PCR – PCR amplification is followed in real-time by the detection of product using a fluorescent probe
  • REDS-II - Retrovirus Epidemiology Donor Study II. NHLBI’s REDS was created in 1989 to address risks associated with HIV-1, HIV-2, HTLV-1 and HTLV-2 in the general blood supply. [16]
  • RT-PCR - Reverse transcription PCR. The conversion of viral RNA to DNA followed by PCR
  • Serology – The presence of specific antibodies in plasma or serum directed against a virus is evidence of a past or present infection. Such as EIA (Enzyme ImmunoAssay) or Western blot.
  • Supernatant - The clear fluid that results after centrifuging, and contains no cells, though it may contain free virus particles.
  • TMA - Transcription-mediated amplification. Amplification of viral RNA or DNA by rounds of in vitro transcription.
  • Viral culture – Direct isolation and amplification of infectious virus from plasma or cells in a laboratory cell line
  • WB - Western Blot.
  • XMRV SRWG - XMRV Scientific Research Working Group.
Retrieved from "http://www.mecfsforums.com/wiki/Blood_XMRV_Scientific_Research_Working_Group"
Personal tools