PPP reference specimens

The primary specimens were sets of four reference specimens prepared under the direction of the HUPO PPP Specimens Committee by BD Diagnostics for each of three ethnic groups: Caucasian-American (B1), African-American (B2), and Asian-American (B3). Each pool consisted of 400 mL of blood each from one male and one post-menopausal female healthy, fasting donor, collected into 10 mL tubes in a prescribed sequence (see Supplementary Protocol) after informed consent. Very large pools were rejected as requiring too prolonged specimen handling and processing unlike the collection ofindividual specimens; even a protocol for two males and two females proved to require more than the 2 h limit we set. Equal numbers of tubes and aliquots were generated with appropriate concentrations of K2-EDTA, lithium heparin, or sodium citrate for plasma or permitted to clot at room temperature for 30 min to yield serum (with micronized silica as clot activator). The additives were dry-sprayed on the inner walls of the tubes, except for 1.0 mL of 0.105 m buffered sodium citrate, which gave a final ratio of 9:1 for blood to citrate in a 10 mL final volume, causing an 11% dilution of the blood. No protease inhibitor cocktails were used. This procedure required 2 h, mostly at 2 to 6°C. After cen-trifugation, volumes from the male and female donors in each donor pair for each specimen type were pooled and then aliquoted into numerous 250 mL portions in vials which were frozen and stored at —70°C. The centrifugation conditions with citrate consistently produced platelet-poor plasma (platelet count <103/mL). Aliquots tested negative for HIV, HBV, HCV, HTLV-1, and syphilis. We supplied four x 250 mL aliquots for each of the four plasma/serum specimens in each set. These vials were shipped on dry ice via courier in early May 2003 (and later to additional laboratories which petitioned to join the project, some of which could no longer be supplied the B1 set). No reshipping was permitted.

The Chinese Academy of Medical Sciences (CAMS) used a variant of the BD protocol to generate similar reference serum and plasma specimens, as described by Li et al. [5] and He et al. [6]. Pools were prepared after review by the CAMS Ethics Committee and informed consent by ten male and ten female donors in Beijing. Donors were fasting and avoided taking medicines or drinking alcohol for the 12 h before sampling. A subsequent pooling of 20 mL from each of the male and female serum or plasma specimens created the C1-CAMS PPP reference specimens which were sent to the 15 laboratories requesting these specimens after storage at —80°C. They were shipped on dry ice using the same courier in September 2003. C1-CAMS specimens were centrifuged originally, and then again upon thawing, at 4°C [6].

Finally, the UK National Institute of Biological Standards and Control (NIBSC) made available to the PPP their lyophilized citrated plasma standard prepared for hemostasis and thrombosis studies from a pool of 25 donors [1].

A standard questionnaire was sent to all laboratories expressing interest. Of 55 laboratories that originally committed to participate, 41 received the BD B1 specimens, 27 the B2 and B3 specimens, 15 the CAMS specimens, and 45 the NIBSC specimens. Laboratories varied on how many of the specimens they actually analyzed.

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