  Heparins are are sulfated polysaccharides that have tremendous clinical
importance as anticoagulant drugs. Monitoring of heparin blood levels can
improve patient safety. In clinical practice, heparins are monitored indirectly
by their inhibtory effect on coagulation proteases. Drawbacks of these
established methods have stimulated the development of simple direct detection
methods with cationic dyes that change absorbance or fluorescence upon binding
of polyanionic heparin. Very few such dyes or assay kits, however, are
commercially and widely available to a broad community of researchers and
clinicians. This study compares the performance of three commercial dyes for
the direct quantification of unfractionated heparin and the widely used
low-molecular-weight heparin enoxaparin. Two traditional metachromatic dyes,
Azure A and Toluidine Blue, and the more recently developed fluorescent dye
Heparin Red were applied in a mix-and-read microplate assay to the same
heparin-spiked human plasma samples. In the clinically most relevant
concentration range below 1 IU (international units) per mL, only Heparin Red
is a useful tool for the determination of both heparins. Heparin Red is at
least 9 times more sensitive than the metachromatic dyes which can not reliably
quantify the heparins in this concentration range. Unfractionated heparin
levels between 2 and 10 IU per mL can be determined by all dyes, Heparin Red
being the most sensitive.
