Table 1.

Tissue preservation/stabilization variables associated with biomarker analysis

Variables	Possible reasons	Potential solutions
Pre-analytical
Tissue evaluation time  (grossing)	Delay in transport	Transport and process tissues as soon as possible post  excision
Time delay to  preservation	Heavy work load or back-log of cases	Communicate tissue processing requirements with clinical  and laboratory staff
Phosphatase, kinase and  proteinase activity in  tissue	Type of preservative/fixation	Use rapid preservative method compatible with downstream  analysis
	Lack of inhibitors/stabilization in  transport medium	Use preservative/fixative containing phosphatase, kinase,  proteinase inhibitors or with inhibitor activity
	Delay in transport/preservation/  stabilization	Transport, preserve and process tissues as soon as possible  post excision
	Storage temperature	Store tissue at appropriate temperature for preservation  solution
	Introduction of phosphatases/  proteinases from the environment	Maintain clean tissue processing/grossing areas
Degree of fixative cross-  linking	Preservative/fixative containing  formalin, gluteraldehyde or other  cross-linker	Use minimal amount of cross-linking agent to achieve  adequate fixation
	Excessive tissue size in relation to  fixative volume	Reduce tissue size. Increase volume of fixative
	Penetration time of preservative  solution	Reduce tissue size. Use preservative with permeabilization  reagents
	Elevated storage temperature	Store tissue at appropriate temperature for preservation  solution. Reduce storage temperature
Tissue hydration state	Low ambient humidity. Open  specimen container	Transport tissue in a closed container
	Delay in transport/preservation
Post-analytical
Time delay to analysis	Lack of proper equipment, reagents,  storage conditions	Prepare reagents in advance. Plan procedure prior to  retrieving samples
	Malfunctioning equipment	Develop a plan to use an alternate source of equipment
Poor/variable quality  protein and/or nucleic  acids results	Delay in stabilizing tissue sample	Transport/preserve/fix tissue as soon as possible post  excision
	Elevated ambient temperature during  tissue transport/processing	Place samples on wet ice or in chilled container
	Inadequate fixation/preservation	Follow directions for type of fixation/tissue
	Use of post-mortem tissue samples	Limit ischemic conditions. Procure samples as soon as  possible post-mortem
	Differences in sample handling and  processing	Stabilize/preserve tissue as soon as possible post excision  (time <20 min)
		Obtain highly pure cell populations (>75%)
		Freeze tissue in such a method as to ensure rapid, uniform  cooling (<5 min).
		Use RNAse-free precautions: wear gloves, use nucleic acid  free plastic ware, clean work area with RNAse  decontamination solutions
		Add protease inhibitors to appropriate reagents
	Extensive cross-linking due to fixation	Determine type of fixation, volume or size of tissue sample,  depth of tissue within the tissue block
	Inadequate removal of paraffin from  formalin-fixed paraffin embedded  tissue	De-paraffinize in two changes of xylene for 5–15 min each