**3.1 Sample collection**

280 Biomarker

al., 2011; Kang et al., 2011). Microbial DNA is also extracted in urine. Through the expanding discipline of microbial metagenomics, we now understand that the relative distribution of microbial DNA has important clinical utility (Nelson et al., 2010; Virgin & Todd, 2011). New improvements in next generation sequencing and microarray technology are showing how the interactions between microbial communities and their host are measurable and are correlated with the health of the host. Urine, like feces, has the potential to provide an easily accessed fluid type, whose flora may provide an exquisitely sensitive measure of pathological state. For example, the microbiome of urine can be used to monitor asymptomatic sexually transmitted disease and is highly correlated to data generated from the urethra swabs (Dong et al., 2011; Nelson et al., 2010). As more work is done in this field,

The breast is a complex organ whose architecture is intertwined with its biology. Even the structure of the nipple is multifaceted and not completely well understood (Love & Barsky, 2004). However, it does provide unique access to fluid that can be leveraged for biomarker development. Nipple aspirate fluid (NAF) and ductal lavage contain cells that have been used for the diagnosis and monitoring of breast cancer (Lang & Kuerer, 2007; Li et al., 2005; Mendrinos et al., 2005; Sauter et al., 1997). NAF is generally obtained either through spontaneous emission or suction, while ductal lavage requires the use of a microcatheter to enter the duct orifice to rinse and collect fluid. Although more invasive, ductal lavage yields more cells (Dooley et al., 2001; Li et al., 2005). These cells originate from the ductal epithelium and by studying them in the NAF, we can glean important information about the active biology within the ducts without the risks associated with biopsy (Dooley et al., 2001; King & Love, 2006; Miller et al., 2006). Much of this work has focused on the early identification of neoplasia using proteomic or cytological analysis of the cells isolated from this fluid (Dooley et al., 2001; Harigopal & Chhieng, 2010; King & Love, 2006; Mendrinos et al., 2005; Wrensch et al., 1992; Wrensch et al., 2001). Recent work has focused on the genomic profiling of NAF cells in order to identify early biomarkers that may predict progression, before morphological changes are evident. For example, the methylation of key tumor suppressor genes can be a highly effective means of predicting tumorgenesis. Preliminary work using NAF samples has demonstrated this as a feasible biomarker of early cancer detection (Krassenstein, 2004). However, measuring the methylation status of key genes in NAF-derived cells is generally not a sensitive enough technique on its own to diagnose disease or predict progression (Euhus et al., 2007; Fackler et al., 2006; Locke et al., 2007).

Mitochondrial sequencing has been shown to be a sensitive way of identifying neoplastic tissues (Czarnecka et al., 2006; Jakupciak et al., 2008; Jakupciak et al., 2008). Mutations in the mitochondrial genome are often found at higher rates than in normal tissues. It is likely that in many cases, these mutations are directly linked to disease pathogenesis, while in others this linkage may only be an effect of other processes. Various groups have applied different techniques to sequence mtDNA from NAF. Zhu and colleagues showed that mutations in mtDNA can be detected non-invasively from NAF using sequencing (Zhu et al., 2005). Jakupciak and colleagues used a mitochondrial resequencing microarray and were able to demonstrate the detection of mutations and a high correlation to traditional sequencing methods (Jakupciak et al., 2008). These methods show great promise for clinical use, although further work is required to validate the approaches. Interestingly, traditional

it is likely that more examples will be uncovered.

**2.8 Nipple aspirate fluid** 

The utility of a given sample to yield a clinically meaningful result is dependent on many factors. These include when and how samples were collected, the preservation method used to stabilize the analytes, shipping and storage effects, and the correct association of patient data with the sample. Variation in any of these areas can have a substantial impact on the usefulness of a sample.

There is conflicting data as far as the effect of time delay between sample collection and the time of extraction of RNA. Some studies report that any delay in getting the sample from the living state to a preserved state (frozen, in formalin (FFPE) or RNAlater) will decrease the quality of the sample (Hong et al., 2010). There are other studies that indicate that there is at least a 16 hour window in which the sample collection and the QC metrics of BioAnalyzer assessment do not show any degradation (Micke et al., 2006). In our experience, we have found that any interruption of sample collection state en route to preservation could lead to degradation of the RNA (unpublished observation). Lisowski and colleagues found that as FFPE sample slices aged, signal intensity by in situ hybridization (ISH) was impacted. If they sliced from the block right before extracting RNA, the signal was clearer and stronger (Lisowski et al., 2001). While some tissues are considered homogenous, studies by Irwin and Dyroff show that there are different physiological responses to different sections of liver in response to drugs (Dyroff et al., 1986; Irwin et al., 2005).

### **3.2 Shipping and storage**

With the advent of electronic tracking by the shipping industry, as well as a societal expectation of overnight shipments, samples can safely and quickly travel from a clinical

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Generally, projects that use this kind of labeling do not have any computer connection from the collection sites to the database storing the sample information. Before the advent of ubiquitous computers and hand held devices, associating the sample label information to a

The major drawback with the naïve barcode system (barcoded tubes that are associated at the point of collection with the sample) is that if the association of sample to barcode is not made by the collection site, then the container is just a tube of tissue, useless for further study. To effectively use the naïve barcode, sites benefit from having access to the database while collecting samples. This can be as simple as barcode scanners that allow some amount of data entry. In some instances, double barcode labels can be supplied to the sites, one is affixed to the form and one is placed on the tube, with the association in to the database to

One method of association, which is a compromise between the intelligent barcode method and the naïve barcode method, is done by associating barcoded containers into a kit at a central laboratory assembly site. Then the kits are shipped to various collection sites. As the kit leaves the facility, the internal containers are still a naïve barcoded container, however at this point, they are associated with a tube type and a destination, all of this information is tracked at a the central laboratory, not on the containers. At the collection site, the kit is associated to a patient. This reduces the amount of data entry needed. The practice of associating the kit barcode at the site of collection to the patient ID allows some flexibility, while still allowing tracking of the tubes within the kits to be organized. This method

In addition, given the current increase of hand held scanners with WiFi access, immediate computer access is no longer a large barrier. Car rental agencies and store inventory systems have been using portable scanners to track inventory for decades; similarly, it isn't too difficult to adopt similar technology for use in clinical trial data collection. The New York subway system integrates data from barcoded tickets, generated from identified machines, all with customer anonymity, to track where passenger flow is most active. There are some groups who have started to study the benefits of this type of live data association in studies involving human donors or patients (Avilés et al., 2008). While it is not essential for the sites to have computer access, as the paper trail of requisition forms is still common,, instant computer contact by the collection site does make the tracking easier. Handwriting barcodes and manual association outside of the database defeats the efficiency of the naïve barcode system, although downstream sample processing can make use of the barcoding system if

In addition, there is an added benefit of naïve barcodes for double blind studies. Double blind studies mask the sample identity, including patient and treatment information. This is to prevent bias in the study and to protect the identity of the study patients. In the past a double tier system of identification numbers would cryptically hide the patient information from those involved in the collection or the analysis of the study. Only a select few would have access to source information about both the patient and drug information. Unique barcodes on the container, without any study information on the label, can provide a double

blind labeling system, as long as the sample is always tracked in the LIMS system.

ensures the highest quality association between a given sample and the donor.

there is a barcode and the association is made to the patient identifier.

matching piece of paper seemed an effective way to track samples.

be made later.

site to a separate processing facility. FedEx pioneered the idea of hub shipments and overnight travel, but others have adopted and emulated their practices. Some couriers will replenish dry ice on shipments traveling more than 24 hours (World Courier). Coupled with this is the need for the initial shipper to pack the samples in such a fashion that they will be held at the correct temperature for at least 24 hours. Written or web based guidance should be given to all collection sites with explicit details as to size of shipping containers and amount of dry ice to use to ensure safe passage of the samples.
