Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Pre-analytical sample handling effects on tear fluid protein levels.

Author(s): Gijs M, Arumugam S, van de Sande N, Webers CAB, Sethu S, Ghosh A, Shetty R, Vehof J, Nuijts RMMA

Publication: Sci Rep, 2023, Vol. 13, Page 1317

PubMed ID: 36693949 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the migration of tears on two different types of Schirmer’s Strips and to compare protein content in consecutively collected tear specimens and tear strips subjected to different storage temperatures (fresh, room temperature, 4°C and -20°C versus -80°C) and conditions (frozen dry versus in extraction buffer). The authors also investigated the potential relationship between migration length and protein content and explored potential effects of extraction buffer volume, use of sonication, and albumin concentration on recovery of albumin from TrueBlue Strips. Tears were collected from each eye of 115 healthy subjects using TrueBlue Optics Schirmer’s Strips and migration length was recorded after 5 min.  Unless otherwise specified, strips were immediately frozen at -80°C. Proteins were extracted from strips by placing the strip in phosphate buffered saline (PBS) with protease inhibitors at 4°C for 90 min followed by centrifugation. Total protein content was quantified by the bicinchoninic acid (BCA) assay. To test effects of collection method, different volumes (2, 5, 10 and 20 µL) of tears collected by a microcapillary pipet and stored at -20°C were applied to both TrueBlue than Dina Schirmer’s Strips and wetting lengths were compared. To test the effects of repeated collection, tears were collected from both eyes of four healthy volunteers on TrueBlue Schirmer’s Strips over the course of 90 min. To test the effects of storage, tears from three healthy volunteers were cut in half and one piece was stored at -80°C (control) while the remaining half was analyzed fresh or stored in a tube with extraction buffer at -80°C and stored for 3 or 24 h at room temperature, 4°C, or -20°C (durations not specified). The effect of elution volume was investigated by adding albumin stock solutions to TrueBlue Strips and, after -80°C storage, eluting in 50-400 µL of extraction buffer; specimens were exposed to ambient conditions or submerged in a sonicating water bath for 5 min.

   Summary of Findings:

   At each tested volume, tears migrated further on TrueBlue Strips than Dina Strips. Tears migrated a shorter distance than an equal volume of PBS but further than an equal volume of plasma; and for all fluids migration was not at a constant of 1mm per µL.  In tear specimens from 115 patients, migration length was positively and modestly correlated with protein content (r=0.6671 P<0.0001).  Tear flow rate was highest in the first 10 sec (0.375 mm/s) and then declined. When tear strips were sequentially sampled, protein content decreased with each successive strip until the fourth strip, but increased in the fifth and sixth strip collected.

   Protein content was 21.7% higher in fresh tears than tears stored at -80°C. While protein content was comparable among tears stored at -80°C and -20°C, protein content was lower when tears were stored at room temperature (-11.7% after 3 h and -8.4% at 24 h) or 4°C (-8.4%) than -80°C.  When the tear strip was submerged in extraction buffer prior to frozen storage, protein content was slightly higher than when the strip was frozen directly at -80°C but the magnitude of the difference varied among the three patients (1.6%, 10.3% and 11.1%).

   An average of 83% of tear volume was recovered from collection strips. Despite different sizes, recovery was comparable for glutathione (0.307 kDa, 68.34 ± 3.36%), lysozyme (14.4 kDa, 61.95 ± 5.09%), and albumin (66 kDa, 61.13 ± 3.82%). Protein recovery from strips wetted with stock solutions of albumin increased as the volume of extraction buffer increased, and the percent recovery increased with albumin concentration but was not affected by sonication. 

   Biospecimens

   • Bodily Fluid - Tears

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Colorimetric assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot sequential collection	1st collection 2nd collection 3rd collection 4th collection 5th collection 6th collection
   Biospecimen Acquisition	Type of collection container/solution	TrueBlue Strip Dina Strip
   Storage	Storage temperature	Room temperature 4°C -20°C -80°C
   Analyte Extraction and Purification	Analyte isolation method	400 µL extraction buffer 300 µL extraction buffer 200 µL extraction buffer 100 µL extraction buffer 50 µL extraction buffer Sonicated No Sonication
   Storage	Time at room temperature	3 h 24 h
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh)
   Storage	Storage conditions	In extraction buffer As dry strip

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