HUPO reference sample collection protocol

HUPO reference samples were collected at, and using materials from BD Diagnostics. For each ethnic group (Caucasian, Asian American, African American), blood was acquired by venipuncture from one male donor and one female donor. Donors were tested and determined negative for HIV-1 and HIV-2 antibody, HIV-1 antigen (HIV-1), Hepatitis B surface antigen (HBsAg), Hepatitis B core antigen (anti-HBc), Hepatitis C virus (anti-HCV), HTLV-I/II antibody (anti-HTLV-I/II), and syphilis. Blood intended for plasma preparation was collected into the following tubes: BD Plus Plastic K2EDTA, 10 mL, reorder# 367525; BD Glass Sodium Citrate, 0.105 m, 10 mL, reorder# 366007; BD Plus Plastic Lithium Heparin, 10 mL, 16 x 100 mm, reorder# 367880; BD Glass Serum with silica clot activator, 10 mL, 16 x 100 mm, reorder# 367820. The collection procedure was as follows. Under the direction of a qualified and licensed physician, trained phlebotomists collected blood from each donor into evacuated blood collection tubes. From each individual consenting donor (n = 6 donors), approximately 400 mL of blood was collected into 40 tubes by two venipunctures, 20 tubes per venipuncture. Discard tubes (serum) were drawn first, with each venipuncture, to prime the tubing. With the first venipuncture, the order of draw was to fill 10 citrate tubes followed by 10 serum tubes. With the second venipuncture, the order of draw was 10 heparin tubes first followed by 10 K2EDTA tubes. Tube handling conditions are detailed in Tab. 1.

The specimens were centrifuged appropriately (see Tab. 1) under refrigerated conditions (2-6°C). The resultant serum and plasma from the 10 spun tubes of each type from each donor were pooled into one secondary 50 mL conical bottom BD (TM) Falcon tube for each tube type. The secondary tube was centrifuged at 2400 RCF for 15 min to remove potentially remaining cellular material from the serum and to prepare platelet poor plasma from the EDTA, heparin and citrate secondary tubes. Equal volumes of serum or plasma were pooled (across gender,

Tab. 1 Tube handling conditions

Tube type

No. of inversions

Clotting time

Centrifugation speed

Centrifugation time

K2EDTA

10

None

<1300 x g

10 min

Lithium heparin

10

None

<1300 x g

10 min

Serum

None

30 min

<1300 x g

10 min

Sodium citrate

4

None

1500 x g

15 min

but within ethnicities) from each secondary tube into media bottles, leaving approximately 10% at the bottom of the secondary tube to ensure no cellular material is collected. Serum/plasma was mixed gently and kept on ice while being distributed into 250 mL aliquots in cryovials. All aliquoting was completed within 75 to 90 min of specimen collection.

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