Protocol for Tear Sample Collection

Protocol for tear sample collection

4 μl of tears^[1] are collected from eye of the patient, (form external canthus, avoiding tear reflex as much as possible) using a 4 μl^[2] glass capillary micropippetes (Drummond Scientific Company, Broomall, PA; Cat No. 1-000-0040, 4 μl, 32mm).^[3] Tears can be attempted to be collected from the tear meniscus, or from the cul de sac by smoothly pulling the inferior eyelid. For this maneuver is important to hold the capillary tube from the distal superior end, avoiding heating it with the hand and maintaining the superior orifice open. Tears will flow into the tube gradually.

Once collected, and with the aid of the provided squeezing bulb or an electronic pipette samples are immediately and carefully placed into an sterile collection tube with a tight seal (10mm O.D. tubes with gasket 0.5ml tubes from Sarstedt (catalog72.730.106) ^[4] already containing 16 μl of ice-cold Assay Buffer (either from the Cytokine Assay kit, or you may use also this buffer: PBS pH 7.4, 1% BSA, 0.05 Tween 20, 0.05% sodium azide), and wrap the top of the tube with parafilm.  (Keep vials open only for the required amount of time avoiding evaporation of sample). The buffers herein mentioned are intended for analysis of tear cytokines with the multiplex bead-based immunoassay (Luminex).

Place tubes with samples in the freezer, ice, or keep at 4ºC until all samples have been collected, then transfer to -80ºC until assayed.