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Plasma tissue inhibitor of metalloproteinases-1 as a biological marker? Pre-analytical considerations
Anne F Lomholt1, Camilla B Frederiksen, Ib J Christensen
1Department of Surgical Gastroenterology, 435, Hvidovre University Hospital, Kettegaards Allé 30, DK-2650 Hvidovre, Denmark. annelomholt@hotmail.com
Unlabelled:
Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) may be a valuable biological marker in Colorectal Cancer (CRC). However, prospective validation of TIMP-1 as a biological marker should include a series of pre-analytical considerations. TIMP-1 is stored in platelets, which may degranulate during collection and storage. The aim of this study was to evaluate the influence of platelet TIMP-1 contamination on plasma TIMP-1 levels in healthy volunteers.
Materials And Methods:
All four parts of this study were done on EDTA-plasma. 1: The effect of stasis was evaluated in plasma collected with and without tourniquet. The collected whole blood was centrifuged at three different g-values. The effect of cellular contamination was evaluated 2: by adding plasma from just above the buffy-coat to one of four tubes containing plasma from the same sample and 3: by separating the plasma into three layers: upper, middle and lower. 4: The effect of temperature was studied by collection and handling of corresponding samples on ice and at room temperature. Prior to analysis samples were stored at -80 degrees C. TIMP-1 was determined using a validated in-house ELISA.
Results:
1: TIMP-1 levels in plasma collected with or without stasis were not significantly different. Similarly TIMP-1 levels were not affected by the studied differences in centrifugation force. 2: TIMP-1 levels were significantly increased in plasma potentially contaminated with platelets (p<0.0001). 3: Separation of plasma into an upper, middle and lower layer did not affect the levels of plasma TIMP-1. 4: Samples kept at room temperature following collection showed significantly higher plasma TIMP-1 levels than samples kept on ice (p<0.0001).
Conclusion:
Contamination with platelets during handling and storage of plasma may have significant effect on TIMP-1 levels. The results can define a standard operating procedure for sample collection and handling, which is important in obtaining uniform, comparable and reproducible plasma TIMP-1 levels.
