Closed System Protocol for Autologous Serum Eyedrop ASE Production

1
Closed System Protocol for Autologous Serum
Eyedrop (ASE) Production Jay C. Bradley, MD1, Jan
Simoni, DVM, PhD2, David L. McCartney, MD1 Dept
of Ophthalmology and Visual Sciences1 and Dept of
Surgery2 Texas Tech University Health Sciences
Center, Lubbock, Texas
Correspondence Jay C. Bradley, MD Texas Tech
University Health Sciences Center Department of
Ophthalmology Visual Sciences 3601 4th St, STOP
7217, Lubbock, TX 79430-7217 E-mail
jay.bradley_at_ttuhsc.edu Tel [phone]
Fax [phone]
Introduction Recent clinical reports have
suggested beneficial effects of ASE in treatment
of numerous ocular surface disorders, including
dry eye syndrome and post-LASIK neurotrophic
keratopathy. The published ASE production
protocols lack validation. To fulfill this
biotechnological gap, our current study focused
on the development of an effective method for ASE
production.

• Protocol
• A 19 gauge winged infusion set , a double male
  Luer lock, and an 18 gauge needle are used for
  blood draw using standard aseptic technique.
• A 150 mL evacuated container is used for 100 mL
  of whole blood collection (Note top of all
  containers cleaned with 100 ethanol prior to
  access). After collection, an 18 gauge needle
  with attached Pall 0.2 µm 32 mm Supor filter is
  used to eliminate vacuum and then removed. A
  sterile glove is placed over top of evacuated
  container for transport.
• A clotting time of 90 minutes at room temperature
  is performed.
• Centrifugation at 3000 x g for 15 minutes is
  performed.
• Serum is extracted using a 3.5 inch 18 gauge
  spinal needle, Statlock IV with clear link
  system, Pall 1.2 µm 32 mm Supor filter, double
  male Luer lock, and an 18 gauge needle and
  transferred to another 150 mL evacuated
  container.
• Serum is filtered using an 18 gauge needle,
  Statlock IV with clear link system, Pall 0.45 µm
  32 mm Supor filter, double male Luer lock, and an
  18 gauge needle and transferred to a 500 mL
  evacuated container.
• Serum is diluted with BSS Plus to 20
  concentration using an 18 gauge needle, Statlock
  IV with clear link system, double male Luer lock,
  and 18 gauge needle and transferred to another
  500 mL evacuated container.
• The bottle is inverted and then filtered using an
  18 gauge needle, Statlock IV with clearlink
  system, Pall 0.2 µm 32 mm Supor filter, double
  male Luer lock, and 18 gauge needle and
  transferred to another 500 mL evacuate container.
• The final bottle is then inverted and an 18 gauge
  needle, Statlock IV with clearlink system,
  stopcock, and 18 gauge needle is used to transfer
  the 20 serum into individual disposable
  ethylene gas-sterilized daily-use 2 mL plastic
  bottles. Amount instilled into each bottle is
  determined by placing exactly 1 gram into each
  bottle using a balance.

Conclusions This method, based on FDA-approved
disposable medical devices and powered by vacuum,
showed to be highly reproducible and easily
validated. The 1.2 µm and 0.45 µm filters were
effective in the removal of the blood clotting
debris and corpuscular elements, respectively,
and the 0.2 µm filter served as an ASE
sterilization device. The protocol did not induce
significant lipid peroxidation or alter its serum
protein spectrum. The resulting ASE was sterile
and pyrogen free, meeting requirements for
ophthalmic drugs. Contrary to prior reports,
clotting did not significantly alter growth
factor levels, based on TGFb1 levels. The closed
system produces therapeutically active ASE
effectively and economically and can be used in
any ambulatory setup, providing patients with a
high quality ophthalmic product.