Analyzing fibrin clot structure using a microplate reader.
Fibrin clot structure studies are often performed using optical methods. For example, the clot's fiber structure can be assessed by measuring light scattering as a function of wavelength. From these measurements, one can calculate the mass/length ratio (mu), a relative measure of fibrin thickness. Fiber thickness has important functional correlates in terms of clot stability and resistance to fibrinolysis. Typically, measurements to calculate mass/length ratios are carried out on high-end spectrophotometers. However, limitations of this instrument include the large sample volume required and the inability to read multiple samples at one time. To circumvent these limitations, a plate-reading spectrophotometer is more commonly used to monitor clot formation; increases in absorbance indicate clot formation, while decreases indicate clot lysis. However, it is unclear whether plate-reading spectrophotometers can be used to quantitatively evaluate fibrin fiber structure. In the current study, we compared spectrophotometric analysis of fibrin gels on single-sample and plate-reading spectrophotometers. Results show that a plate-reading spectrophotometer does not give accurate measurements of the fiber mass/length ratio. However, the plate-reading spectrophotometer can provide a qualitative measure of fiber structure for both purified fibrinogen and plasma. We suggest that plate-reading spectrophotometers can provide a convenient, rapid, and inexpensive means of analyzing fibrin clot structure.
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27705, USA.
A S
AS
Wolberg
D A
DA
Gabriel
M
M
Hoffman
D002118:Calcium / Q000494:pharmacology; D003911:Dextrans / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005337:Fibrin / Q000032:analysis* / Q000378:metabolism / Q000648:ultrastructure*; D005342:Fibrinolysis; D006801:Humans; D007700:Kinetics; D015203:Reproducibility of Results; D013057:Spectrum Analysis / Q000295:instrumentation / Q000592:standards; D013917:Thrombin / Q000494:pharmacology
D003911:Dextrans; D005337:Fibrin; D013917:Thrombin; D002118:Calcium