WHI Biospecimens

Specimens collected from WHI participants are maintained in a biorepository in Rockville, MD. The sample inventory is tracked via a WHI Specimen Tracking database, which includes variables associated with the specimens, such as number of fasting hours before blood draw, extraction and quantitation method, and number of freeze/thaw cycles. All biospecimens collected from WHI participants are available for use by Ancillary Studies (ASs) based on scientific merit. To access WHI biospecimens, investigators must have an approved and funded ancillary study. See our Propose a Study page for details.

Specimen Collection, Processing, and Storage Procedures

Participants were asked to fast for at least 12 hours before blood draws. In some cases participants were not fasting. Fasting status is indicated in the database.

• For WHI sample collection and processing details (Baseline through Year 9 blood collections), please see the Blood and Urine Collection, Processing and Shipment section of the WHI (1993-2005) manual.
  
• For details on the LLS blood draw collection and processing, please see the Long Life Study: Synopsis of Blood Protocol document.

All specimens described below are stored frozen at -80 degrees Celsius at a central repository in Rockville, MD. Specimen processing for ancillary studies (aliquoting, DNA extraction) occurs in a central lab (FHCRC specimen processing lab [SPL]).

Time Points of Collection for Specimens

• 1- Time points: Baseline through Year 9 samples were collected in WHI 1993-2005 at WHI clinical centers; samples for the Long Life Study (LLS) were collected in 2012-2013 at participant homes.
• 2- Buffy coat is not available to researchers. DNA is extracted from buffy coat for samples selected for testing at the WHI central lab and aliquots of DNA are shipped to the testing lab.
• 3- Urine samples were collected at the three Bone Mineral Density (BMD) sites where BMD was also measured (see "BMD" on the Subsample Definitions list for brief description)
• 4- CT 6% subsample (see "CT 6% blood" description on the Subsample Definitions list for brief description). RBCs and buffy coat were not on the collection schedule for the 6% CT subsample at years 3, 6, or 9, but some had RBCs and buffy coat collected at these time points.
• 5- Long Life Study (LLS); N = 7,875 with an LLS visit. See Long Life Study for a description). Serum was collected in separator tubes (SST), plasma was collected in standard EDTA tubes and plasma separator tubes (PST). A participant's LLS study visit occurred sometime between March 2012 and May 2013. Depending on when the participant enrolled in WHI, their LLS visit was Year ~14-20.
  

Serum, Plasma, RBCs, and Urine 

Serum, plasma (EDTA, citrate), RBCs, and urine samples are stored in 1.8 ml parent vials or 0.25 ml subs. When parent vials are accessed, an aliquot is made for the testing lab and the remaining volume is placed into 0.25 ml subs for storage at the WHI repository. All samples will have undergone at least one freeze/thaw before they arrive at the testing lab. Freeze/thaw cycle is captured in our database.  

In 2009/2010, samples from <= 3% WHI RBC draws suffered degradation as the result of storage at -20 degrees Celsius while at the processing lab for aliquoting (as described by Pottala et al., Lipids 2012). The affected vials have been removed from the WHI biorepository inventory. Two RBC processing procedures have since been implemented to prevent RBC sample degradation: (1) RBC samples are consistently stored at -80 degrees Celsius, and (2) the minimum RBC aliquot volume is 250 ul (to prevent oxidation). With these procedural changes, the WHI RBC samples are of good quality for use in ancillary studies.

DNA

Buffy coat is stored frozen in our repository and DNA is extracted from the stored buffy coat when it is requested for an ancillary study. An aliquot is made for the testing lab and the remaining sample is returned to the WHI repository.

DNA has been extracted using different methods over time.

• ~2000-2003: Qiagen Puregene/Bioserve
• ~2004-2005: Salt-precipitation
• ~2005-2008: Phenol Chloroform
• ~2008 - Present: Qiagen/Five Prime
  

The current "Five Prime" extraction method was developed by FHCRC using Qiagen reagents in a method designed to maximize both the quality and yield of extracted DNA. An AS may receive DNA samples extracted by different methods in the past, as well as freshly extracted DNA. Extraction method is tracked in the WHI specimen database. Please note that the DNA is extracted on an "as needed" basis, so the date of extraction does not correlate with the date of the blood draw. All Long Life Study DNA has already been extracted using the Five Prime method.

DNA concentration is assessed at the time of extraction. DNA concentration was first assessed using spectrophotometry. Starting in August 2007, DNA concentration has been assessed using PicoGreen. Concentration method is tracked in the WHI specimen database. 

RNA

A PAXgene tube was collected during the Long Life Study visit. RNA/miRNA was extracted from the PAXgene tube within ~1 month of collection. The concentration of RNA was determined by NanoDrop. The extracted RNA is stored frozen in the WHI repository.

Volume of Specimens Available to Ancillary Studies

Parsimonious use of specimen is an important consideration in review of AS proposals. Without significant scientific justification, ancillary studies are limited to the following samples volumes (including any necessary "dead volume", or padding) from a given specimen collection time point:

• 0.25 ml serum, EDTA plasma, citrated plasma, or red blood cells
• 0.5 ml urine (only available on BMD participants)
  
• 1-2 ug DNA (depending on the application)
• 0.5 ug RNA (only available for Long Life Study participants)
  
• Whole blood: not available
  
• Buffy coat: not available
  

Samples are available for almost all study participants, though for some outcomes particular sample types may be more limited. Investigators working on ancillary study proposals can use the WHI Query Builder, a website that lets users create ad-hoc queries that return the number of WHI participants with various criteria, including specimen availability. For Query Builder access, please contact the WHI Help Desk (helpdesk@whi.org). 

Quality Control

Sample Stability Monitoring

Pooled samples of serum, citrated and EDTA plasma were created close to the start of WHI enrollment to be tested for core analytes and CVD biomarkers alongside participant samples. Results from these samples allow the Lab Working Group to monitor the stability of primary analytes over time.

Blind Duplicates

WHI uses blood samples from women who consented to the screening blood draw, but were not enrolled or randomized into any WHI component. The samples are relabeled to provide blinded duplicate samples that are included in all batches of assays. When preparing budgets, please include the costs for testing the blind duplicates. There is no covariate data associated with these samples.

WHI includes blind duplicates as quality control samples in all sample pulls, as follows:

• DNA: Studies requesting DNA samples are required to include quality control samples: 5% of the total number of participant samples (2.5% blind duplicate pairs). For smaller genotyping studies (e.g. candidate genes, limited SNPs), the WHI-CCC will produce a SNP concordance report that assesses the study's (1) correct identification of the blind duplicates, (2) failure to identify blind duplicates, and (3) unexpected duplicates among all samples provided. For larger genotyping studies (e.g. GWAS, sequencing), the PI is to report to the WHI-CCC all duplicates identified. From this list, the WHI-CCC will confirm the correct/incorrect identification of the blind duplicates and any unexpected duplicates. 
• Plasma, serum, urine, and RBC: WHI includes 10% blind duplicate QA samples (5% pairs) with all blood and urine samples. The correlation coefficient and the average coefficient of variation % is computed based on the test results for these samples and reported to the PI.

Sample Processing Quality Control 

The adequacy of serum and plasma aliquoting by the Specimen Processing Lab is evaluated by testing a subaliquot from each blind quality control sample for total cholesterol. Similarly, urine samples are tested for sodium. The correlation and average CV% between the members of each blind pair is computed and reviewed by the WHI Lab Working Group.

Contact the WHI Help Desk at helpdesk@whi.org if you need assistance or have questions.