A new method for direct detection of heparin on surface-modified intraocular lenses. A modification of Jaques' toluidine blue staining method.
BACKGROUND
Examination of surface-bound heparin on synthetic polymers can only be performed by few staining methods. These methods are limited by an only approximate detection of heparin visible at high magnification. Other methods only measure heparin quantitatively (per square dimension) and are rather sensitive to artifacts. Due to its homogeneous staining pattern, the modified toluidine blue staining technique, using a non-protein-based substance, allows examination and analysis of the homogeneity of the monomolecular heparin layer even under critical conditions like scanning electron microscopy.


METHODS
For critical examination of the heparin layer, this method was used for 5 sterile heparin surface-modified (HSM) monofocal (Pharmacia, 809C, 5 mm optical zone), 2 multifocal PMMA (Pharmacia, 811E, 6 mm optical zone) and 1 standard PMMA intraocular lenses (IOLs; Pharmacia, 809P, 5 mm optical zone). A special mixture containing a sodium borate buffer was used to avoid cross-reactions of toluidine with phenylimines, which permit a covalent surface linkage of heparin to synthetic polymeric materials via reductive amination. This resulted in less coarse-grained complex agglutination. After light and spectral microscopy of the surface of stained IOLs, scanning electron microscopy was performed to investigated the reliability and validity of this modified staining method.


RESULTS
All HSM IOLs showed a homogeneous heparin structure and coating, which could be demonstrated even under critical photographic circumstances.


CONCLUSIONS
The modification of the original toluidine blue staining method introduced by Jaques in 1943 is a reliable and reproducible technique for the detailed in vitro analysis of surface-bound heparin with a low artifact rate. Because of the detailed detection of heparin on polymeric surfaces, this staining method is recommended for special problems, e.g. in cataract surgery.
Dick
B
B
Department of Ophthalmology, University of Mainz, Germany.
Schmidt
K G
KG
Eisenmann
D
D
Pfeiffer
N
N
D004396:Coloring Agents; D006493:Heparin / Q000032:analysis* / Q000737:chemistry; D007910:Lenses, Intraocular / Q000592:standards; D020366:Methylmethacrylate; D008768:Methylmethacrylates / Q000737:chemistry*; D008855:Microscopy, Electron, Scanning; D015394:Molecular Structure; D011786:Quality Control; D015203:Reproducibility of Results; D014048:Tolonium Chloride
D004396:Coloring Agents; D008768:Methylmethacrylates; D014048:Tolonium Chloride; D020366:Methylmethacrylate; D006493:Heparin