Collagen Screening: Type I and Type III Collagen Synthesis
The collagen proteins made in skin and bone are synthesized by cultured dermal fibroblasts. These cells can be used to examine the structure and synthesis of type I and type III procollagen/collagen.
For collagen screening, cultured fibroblasts are shipped from the referring laboratory. When facilities are not available for growing fibroblasts, the skin biopsy can be shipped directly. Once the cells are adapted to our culture conditions, they are plated at high density (250,000 per 35mm culture dish) and allowed to attach and spread overnight. They are then incubated for 16 hours with radioactively labeled proline in the presence of ascorbic acid to enhance collagen production. The cell layer and the secreted medium proteins are harvested separately and examined by gel electrophoresis. In addition, we cleave the collagenous proteins in both samples with protease to remove the non-triple helical domains from both ends of the central triple helix, and examine the protease resistant molecules by gel electrophoresis. Turnaround time 4 weeks from cultured cells, 6-8 weeks from biopsy.
Sensitivity: 85-90% of individuals with OI and 99% of individuals with EDS type VII are identified by alterations in chains mobility and amount of type I collagen. Greater than 95% of individuals with EDS type IV are identified by alterations in the chains of type III collagen.
Reflex Testing: If collagen screening studies are requested and the biochemical results are consistent with vascular EDS, cDNA sequencing will be initiated automatically to confirm the diagnosis. In these instances cDNA sequencing is completed on the segments of the COL3A1 cDNA suspected to house the mutation based on collagen testing.
Sample type: Skin biopsy or Cultured Cell Fibroblasts
Turnaround time (TAT): 6-8 weeks