Re shipped at ambient temperature to a central lab by overnight

Re shipped at ambient temperature to a central lab by overnight courier, where they were promptly frozen at -70 until being shipped frozen to the London Regional Cancer Program laboratory for ELISA assessment. Prior to initiation of plasma collection for the MA.14 trial, in a pilot study to assess the potential effect of this collection/storage process, we assessed OPN plasma levels from eight blood samples maintained at room temperature for 4 vs. 24, 48 or 72 hours, and found that measured values did not differ with time at room temperature. The 1-(Cyclopropylsulfonyl)-1,4-diazepane total number of plasma samples available for OPN assays was 2,540, with 624 (94 ) of 667 patients having plasma collected at some time. The time-points for collection and numbers of available patients and samples are summarized in Table 1. Primary statistical analyses utilized all samples at each time point, but some exploratory analyses examined data from paired samples for recurrent and non-recurrent patients. Prognostic analyses examined baseline IHC and plasma OPN effects on multiple survival outcomes. To maximize inclusion of patients in these investigations and given the low PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/10714611 likelihood of tumor recurrence (2 in 388 patients) PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/10572343 in the first four months after randomization, we Benzothiophene-3- boronic acid combined pre-randomization and four-month results, giving a "baseline" assessment of OPN in 388 (58 ) of 667 patients. When there were multiple assessment results for a patient during the pre-randomization and four-month time-points, they were averaged to yield a baseline OPN plasma value.Bramwell et al. Breast Cancer Research 2014, 16:R8 http://breast-cancer-research.com/content/16/1/RPage 3 ofTable 1 Available samplesTotal accrual Total patients with samples for OPN analyses Plasma - baseline/4 m - any time Tumor tissue for IHC Total number plasma samples Timing - plasma samples/number of patients Baseline (pre-randomization) 4m 8m 12 m 16 m* 24 m 36 m 48 m* 60 m Recurrence 314/301 374/361 396/391 406/401 33/29 540/487 206/159 29/29 220/211 22/20 388 (58 ) 624 (94 ) 462 (69 ) 2,540 667 patients*later protocol amendment eliminated these sample collections. IHC, immunohistochemistry; OPN, osteopontin.For measurement of tumor tissue OPN, it was possible to obtain primary tumor blocks/slides for OPN IHC in 462 (69 ) of 667 patients accrued to MA.14. In total, 647 (97 ) of patients had either measurement of OPN in primary tumor (by IHC), or one or more measurements of plasma OPN (by ELISA). For comparison with MA.14 study samples, 90 plasma samples from healthy women were collected under a protocol approved by the University of Western Ontario Research Ethics Board. Volunteer donors were hospital staff who contributed blood samples for the purpose of establishing normal levels of various blood components, including OPN. The only data collected on these individuals were sex and age.OPN analytic methodsThe primary antibody used was the monoclonal antibody mAb53 (Enzo Life Sciences, catalogue #ADI-905-629). Biotinylated secondary antibody was from the LSAB2 kit (Dako, Burlington, ON, Canada). Stained slides were evaluated by light microscopy in a blinded fashion by two pathologists (ABT, WAK). Tumor cell staining for OPN was determined as a continuous variable of the percentage of cells staining (regardless of intensity), as well as using two different semi-quantitative systems. The first was the non-linear system of Allred et al. [17], which assigns proportion and intensity scores as follows: proportion as the estimated fra.