Accessing Biological Samples

Ideally, biomarkers should be measurable in a versatile and easily accessible body fluid, such as serum or urine, to maximize clinical use in terms of patient compliance and readiness to interface with the diagnostic 

After acquiring a minimal amount of specimen, ranging from a few hundred to a few million cells, current proteomic analyses are able to generate MS patterns in a relatively short time span. In one study, less than 2 hours was needed to process human breast tissue that had been removed from the patient in the operating room until the MS data had been acquired. Mammary tissue containing normal breast epithelium and invasive carcinoma was compared, and over 40 peaks were identified that significantly differed in intensity [54]. This study employed LCM to acquire the tissue specimens. 

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Direct injection of pretreated biological samples (also called online sample cleanup) greatly simplified sample preparation for LC/MS/MS analysis. The normal process involves sample aliquot steps, internal standard addition, and centrifugation. Compared to traditional off-line LLE and SPE sample preparation procedures, online methods are easier and faster. Two types of online SPE columns are commercially available. One is the restricted access media (RAM) column. The other is the turbulent flow chromatography (TFC) column. 

Many other types of solid phase adsorbents, including those based on conventional and specialty materials like restricted access media (RAM), can increase analysis speed and improve assay performance. These types of materials, also known as internal reversed-phase packings, are especially useful for assaying target compounds in biological samples such as serum and plasma. They are chemically modified porous silicas that have hydrophilic external surfaces and restricted-access hydrophobic internal surfaces. The ratio of interior to external surface areas is large. Macromolecules such as proteins cannot enter the pores of the RAM (they are excluded from the hydrophobic internal surface) and they elute quickly through the column. However, the smaller analyte molecules that can enter the pores are retained via interactions with the hydrophobic bonded phase within... 

The CBD requires that permission must be obtained before biological samples can be taken. To comply with the CBD, an Access and Beneht-Sharing Agreement (ABA) has to be agreed between the researcher and the source country providing the natural products. The ABA sets out the clauses with respect to the observation, development, and beneht sharing accruing from the use of natural products for medical applications. 

The packing material first described for direct injection of biological samples was prepared by simply saturating the accessible adsorption sites of a Cis reversed-phase silica with human plasma proteins (105). After saturation, the human plasma proteins were denatured at the external surface, and their native conformation was destroyed. With this treatment, the proteins formed a hydrophilic layer with weak ion-exchange properties, which provided protection from contact with the sample proteins, whereas the alkyl ligands inside the pores remained unchanged and thus served for analyte retention. The retention behavior of the saturated phase did not alter with this treatment, but the efficiency was reduced dramatically. Such protein-coated columns have shown a lifetime of several months (106). 

Investigations with more limited goals designed to measure intermediate or short-term changes in biochemical and molecular markers may lessen some of these difficulties. Biological markers that can detect early and subtle differences in individual response would be necessary for this purpose. The most suitable are markers that can be detected in small samples of tissue or body fluids that are accessible to sampling they should be expressed differentially in accordance with differences observed in normal and abnormal responders and they should have a low probability of spontaneous change. 

Having confirmed that a certain depository is recognized by the patent office of the country where the patent application is due to be filed and that the depository is able to handle the specimens in question (for safety reasons certain samples may not be acceptable in some institutions, and some countries may not accept certain biological samples for quarantine reasons), the deposit must be made by the date of filing of the application. The institution where the deposit was made, the accession number which identifies it and the date of the deposit must be mentioned in the patent description. 

In recent years, special SPE supports possessing restricted access properties have been developed [61-65] to allow the direct injection of untreated biological samples into online SPE-LC systems. 

Other possible downsides are the relative paucity of central laboratory facilities especially for assays, and that are accredited to International Standards and also both GLP and GCP compliant. Transport of clinical trial supplies, access of sites to monitoring visits may require days of rail transport rather than air with complications of humidity and temperature extremes on supplies and biological samples. Finally, sectarian violence can break out at any time causing disruption to monitoring of studies and possible danger to foreign monitors. 

Two recently introduced direct injection techniques have been developed to deal with the special problems posed by biological samples. These techniques involve the use of restricted access media (RAM) [11,12] and turbulent flow chromatography [13] and are described later. 

There are a host of new solid phases being brought to the market. Some of the most interesting currently include graphitized carbon, functionalized styrene-divinylbenzene copolymers, restricted access solid-phase reversed phase, affinity chromatography with antibodies, and molecular imprinted polymers. These five types of sorbents have different applications from environmental to biological sample preparation. 

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