Second Specimen collection

Second Specimen Collection (Sisters Changing Lives)

The Sisters Changing Lives (SCL) follow-up was a second specimen collection from a subset of Sister Study participants conducted from Oct. 15, 2013 – Mar. 2, 2015.

Participants in the second specimen collection were from a random sample of the cohort (n=2036) or had developed breast cancer (LCIS, DCIS or invasive breast cancer) since baseline (n=2223). There was some overlap in the two groups since participants selected in the random sample could also be breast cancer cases (n=100). After exclusions for conditions that precluded specimen collection (e.g. prior study refusal, deceased), 3,845 women were invited to participate (random cohort n=1949; additional breast cancer cases n=1896). Specimens were either collected in the home of the participant or at an alternate agreed-upon site, such as a doctor’s office.

	Fielded for Contact (N)	Deceased/ Incapacitated1 (N)	Completed Sample Collection (N)	Response Rate2 (%)
Random cohort No BrCa3 BrCa4 Additional BrCa5	1,949 1,856 93 1,896	14 12 2 13	1,268 1,215 53 1,188	65.5 65.9 58.2 63.1
Total	3,845	27	2,456	64.3

¹ Found to be deceased or incapacitated after contact
² % = Completed/(Fielded minus Deceased or Incapacitated after fielding)*100
³ No diagnosis of LCIS, DCIS or invasive breast cancer at time of SCL sampling
⁴ Diagnosis of LCIS, DCIS or invasive breast cancer as of SCL sampling
⁵ Excludes self-reported BrCa that was later disavowed [as of DR5] (n=8)

Participants were sent a kit in advance for collecting first morning void urine, toenails, and household dust. Blood collections in the home were performed by examiners from a national in-home phlebotomy service (EMSI), and participants were instructed to fast for at least eight hours prior to their blood draw. The examiner collected approximately 45 ml of blood in seven BD Vacutainer® (Becton, Dickinson and Company) tubes, two EDTA tubes, two serum tubes, two ACD-B tubes, and a Tempus RNA tube. They also retrieved the urine and environmental samples, self-administered questionnaires, and took physical measurements according to standardized instructions. Specimens were shipped the same day as collection by FedEx Priority Overnight to the Sister Study lab, where samples were further processed and aliquots were created. 

 

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Second Specimen Processing (Sisters Changing Lives)

Serum and blood clot: Two red-top tubes were centrifuged in the field immediately after collection in order to isolate the serum from the red cells as soon as possible. The time between collection and centrifugation was documented. The serum was transferred to a standard transport tube for return to the lab. Upon arrival, the serum was stored in approximately eight 0.5 ml CBS™ straws and eight 0.5 mL cryovials in liquid nitrogen vapor phase for future analysis of hormones, metabolism analytes, and vitamin levels. Blood clots were stored in a cryovial (VWR 10.0 ml CryoPro).

Plasma and EDTA whole blood: A 10.0 ml EDTA BD Vacutainer® tube (purple top) provided whole blood, plasma, and dried blood. The Vacutainer was shipped to the lab at ambient temperature. Upon arrival, a 1.0 ml aliquot of whole blood was stored in a cryovial and two types of dry blood storage cards were spotted and stored (60 µl per spot). One was a treated card chemically impregnated to lyse cells and stabilize DNA (Whatman FTA Classic Card) and the other was an untreated card (Whatman 903 Protein Saver Card). The remaining whole blood was centrifuged, and the plasma was isolated and stored in 0.5 ml CBS™ straws and cryovials in liquid nitrogen vapor phase for future analysis of factors such as HgbA1C, IGF-1, and ferritin.

A 3.0 ml EDTA BD Vacutainer® tube (tan top) was stored in the original Vacutainer® tube at -20°C for future analysis of metals, trace elements, and environmental contaminants.

ACD-B whole blood: One of the ACD-B Vacutainer® tubes (yellow top) was either aliquoted into six 1.0 ml cryovials and cryopreserved with 10% DMSO or was selected for lymphocyte isolation. 

The second ACD-B tube was transferred to an 8 mL vial for storage in liquid nitrogen vapor phase.

Tempus RNA: Blood was collected in a 3 mL Tempus RNA tube and frozen at -20°C.

Saliva collection: In the event that a blood sample could not be collected due to an unsuccessful phlebotomy, participants were asked to do a saliva collection using an Oragene™ DNA self-collection saliva kit. Although this has its limitations compared to blood, it does provide a DNA source for genetic analyses.

Urine: On the morning of the examiner visit, participants collected approximately 60 ml of their first urine of the day in a collection cup that was pre-tested for phthalates. The cup was placed in a plastic bag and kept cool in the refrigerator until the examiner arrived for collection. The participant wrote the date and time of collection on a pre-printed label. Urine was transferred from the urine collection cup into two leak-proof and pressure-proof transport tubes prior to shipping. Approximately 20 ml were obtained. Urine was aliquoted into twenty 0.5 ml CryoBioSystem™ (CBS™) straws, five 1.0 ml vials, and one 3.6 ml vial. Urine aliquots were stored in liquid nitrogen vapor phase.

Toenail: After removing any nail polish, the participant took a clipping from each toenail on both feet. Big toenails were stored separately from other toenails in provided envelopes. The participant wrote the collection date on a pre-printed label. Toenails were stored at ambient temperature.

Dust: Participants were given eight alcohol swabs (two for testing). Three door frames from three different rooms in the house were swiped for dust resulting in six dirty swabs from three different locations. Swabs were stored in a plastic zipper bag. The participant wrote the collection date on a pre-printed label. The dust was stored at -20°C. A pilot was conducted successfully at Southwest Research Laboratories for the measurement of polycyclic aromatic hydrocarbons (PAHs), phthalates, and pesticides in the dust.

Material type	Total Number of Aliquots Stored
Serum	24,730
Plasma	16,510
Blot clot	4,712
Whole blood*	21,592
Filter cards with whole blood	4,732
Lymphocytes	500
Packed cells	2,351
Urine	59,171
Dust	2,449
Toenails	4,662

*Whole blood includes special metal analysis Vacutainer tube, Tempus RNA tube, whole blood with EDTA, and cryopreserved whole blood.

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