The authors wish to thank &#34;Casa del Dono di Reggio Emilia&#34; for providing donor-derived platelet concentrates.

PRP-L used for the quantitative assessment of growth factors were collected within a wider study on the characterization of PRP products for regenerative purposes, carried out at the AUSL-IRCCS di Reggio Emilia and approved by the Area Vasta Emilia Nord Ethical Committee on 10 January, 2019 (protocol number 2019/0003319). Donors gave their informed consent as per the Declaration of Helsinki. No ethical approval was necessary for collecting the aggregated, anonymous data of the Ocular Surface Disease Index (OSDI) question...

<ol>
	<li>Everts, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+plasma+and+platelet+gel:+A+review.">Platelet-rich plasma and platelet gel: A review.</a> The Journal of Extra-Corporeal Technology. 38 (2), 174 (2006).</li><li>Giannaccare, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+derived+eye+drops+for+the+treatment+of+cornea+and+ocular+surface+diseases.">Blood derived eye drops for the treatment of cornea and ocular surface diseases.</a> Transfusion and Apheresis Science. 56 (4), 595-604 (2017).</li><li>Bernabei, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-based+treatments+for+severe+dry+eye+disease:+The+need+of+a+consensus.">Blood-based treatments for severe dry eye disease: The need of a consensus.</a> Journal of Clinical Medicine. 8 (9), 1478 (2019).</li><li>Findlay, Q., Reid, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+eye+disease:+When+to+treat+and+when+to+refer.">Dry eye disease: When to treat and when to refer.</a> Australian Prescriber. 41 (5), 160-163 (2018).</li><li>Clayton, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+eye.">Dry eye.</a> New England Journal of Medicine. 378 (23), 2212-2223 (2018).</li><li>Jones, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TFOS+DEWS+II+management+and+therapy+report.">TFOS DEWS II management and therapy report.</a> The Ocular Surface. 15 (3), 575-628 (2017).</li><li>Holland, E. J., Darvish, M., Nichols, K. K., Jones, L., Karpecki, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+topical+ophthalmic+drugs+in+the+treatment+of+dry+eye+disease:+A+systematic+literature+review.">Efficacy of topical ophthalmic drugs in the treatment of dry eye disease: A systematic literature review.</a> The Ocular Surface. 17 (3), 412-423 (2019).</li><li>Shih, K. C., Lun, C. N., Jhanji, V., Thong, B. Y. H., Tong, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+review+of+randomized+controlled+trials+in+the+treatment+of+dry+eye+disease+in+Sjogren+syndrome.">Systematic review of randomized controlled trials in the treatment of dry eye disease in Sjogren syndrome.</a> Journal of Inflammation. 14, 26 (2017).</li><li>Rusciano, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+dry+eye+lactoferrin+and+lactobionic+acid.">Age-related dry eye lactoferrin and lactobionic acid.</a> Ophthalmic Research. 60 (2), 94-99 (2018).</li><li>Craig, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TFOS+DEWS+II+definition+and+classification+report.">TFOS DEWS II definition and classification report.</a> The Ocular Surface. 15 (3), 276-283 (2017).</li><li>Drew, V. J., Tseng, C. L., Seghatchian, J., Burnouf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflections+on+dry+eye+syndrome+treatment:+Therapeutic+role+of+blood+products.">Reflections on dry eye syndrome treatment: Therapeutic role of blood products.</a> Frontiers in Medicine. 5, 33 (2018).</li><li>Giannaccare, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+derived+eye+drops+for+the+treatment+of+cornea+and+ocular+surface+diseases.">Blood derived eye drops for the treatment of cornea and ocular surface diseases.</a> Transfusion and Apheresis Science. 56 (4), 595-604 (2017).</li><li>Acebes-Huerta, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-derived+bio-products:+Classification+update,+applications,+concerns+and+new+perspectives.">Platelet-derived bio-products: Classification update, applications, concerns and new perspectives.</a> Transfusion and Apheresis Science. 59 (1), 102716 (2020).</li><li>Quartieri, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomics+comparison+of+cord+and+peripheral+blood-derived+serum+eye+drops+for+the+treatment+of+dry+eye+disease.">Metabolomics comparison of cord and peripheral blood-derived serum eye drops for the treatment of dry eye disease.</a> Transfusion and Apheresis Science. 60 (4), 103155 (2021).</li><li>Badami, K. G., McKellar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allogeneic+serum+eye+drops:+Time+these+became+the+norm.">Allogeneic serum eye drops: Time these became the norm.</a> British Journal of Ophthalmology. 96 (8), 1151-1152 (2012).</li><li>Hwang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+clinical+efficacies+of+autologous+serum+eye+drops+in+patients+with+primary+and+secondary+Sj&#246;gren+syndrome.">Comparison of clinical efficacies of autologous serum eye drops in patients with primary and secondary Sj&#246;gren syndrome.</a> Cornea. 33 (7), 663-667 (2014).</li><li>Chiang, C. C., Lin, J. M., Chen, W. L., Tsai, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allogeneic+serum+eye+drops+for+the+treatment+of+severe+dry+eye+in+patients+with+chronic+graft-versus-host+disease.">Allogeneic serum eye drops for the treatment of severe dry eye in patients with chronic graft-versus-host disease.</a> Cornea. 26 (7), 861-863 (2007).</li><li>Jonsdottir-Buch, S. M., Lieder, R., Sigurjonsson, O. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+lysates+produced+from+expired+platelet+concentrates+support+growth+and+osteogenic+differentiation+of+mesenchymal+stem+cells.">Platelet lysates produced from expired platelet concentrates support growth and osteogenic differentiation of mesenchymal stem cells.</a> PLoS One. 8 (7), 68984 (2013).</li><li>Altaie, A., Owston, H., Jones, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+platelet+lysate+for+bone+regeneration+-+Are+we+ready+for+clinical+translation.">Use of platelet lysate for bone regeneration - Are we ready for clinical translation.</a> World Journal of Stem Cells. 8 (2), 47-55 (2016).</li><li>Vesaluoma, M., Teppo, A. M., Gr&#246;nhagen-Riska, C., Tervo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-derived+growth+factor-BB+(PDGF-BB)+in+tear+fluid:+A+potential+modulator+of+corneal+wound+healing+following+photorefractive+keratectomy.">Platelet-derived growth factor-BB (PDGF-BB) in tear fluid: A potential modulator of corneal wound healing following photorefractive keratectomy.</a> Current Eye Research. 16 (8), 825-831 (1997).</li><li>Zheng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+transforming+growth+factor+&#946;+activity+in+normal+and+dry+eye+human+tears+by+CCL-185+cell+bioassay.">Evaluation of the transforming growth factor &#946; activity in normal and dry eye human tears by CCL-185 cell bioassay.</a> Cornea. 29 (9), 1048 (2010).</li><li>Zamani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+therapeutic+approaches+in+utilizing+platelet+lysate+in+regenerative+medicine:+Are+we+ready+for+clinical+use.">Novel therapeutic approaches in utilizing platelet lysate in regenerative medicine: Are we ready for clinical use.</a> Journal of Cellular Physiology. 234 (10), 17172-17186 (2019).</li><li>Ministro della Salute. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disposizioni+relative+ai+requisiti+di+qualit&#224;+e+sicurezza+del+sangue+e+degli+emocomponenti.">Disposizioni relative ai requisiti di qualit&#224; e sicurezza del sangue e degli emocomponenti.</a> Italian Ministry of Health. , (2015).</li><li>Aprili, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raccomandazioni+SIMTI+sugli+emocomponenti+per+uso+non+trasfusionale.">Raccomandazioni SIMTI sugli emocomponenti per uso non trasfusionale.</a> Societ&#224; Italiana di Medicina Trasfusionale e Immunoematologia. , (2012).</li><li>Schiroli, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+alternative+procedures+to+obtain+packed+red+blood+cells+for+&#946;-thalassemia+major+transfusion+therapy.">Comparison of two alternative procedures to obtain packed red blood cells for &#946;-thalassemia major transfusion therapy.</a> Biomolecules. 11 (11), 1638 (2021).</li><li>Pulcini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apheresis+platelet+rich-plasma+for+regenerative+medicine:+An+in+vitro+study+on+osteogenic+potential.">Apheresis platelet rich-plasma for regenerative medicine: An in vitro study on osteogenic potential.</a> International Journal of Molecular Science. 22 (16), 8764 (2021).</li><li>Ohashi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+epidermal+growth+factor+in+human+tears.">Presence of epidermal growth factor in human tears.</a> Investigative Ophthalmology &amp; Visual Science. 30 (8), 1879-1882 (1989).</li><li>Vitale, S., Goodman, L. A., Reed, G. F., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+NEI-VFQ+and+OSDI+questionnaires+in+patients+with+Sj&#246;gren's+syndrome-related+dry+eye.">Comparison of the NEI-VFQ and OSDI questionnaires in patients with Sj&#246;gren's syndrome-related dry eye.</a> Health Quality of Life Outcomes. 2, 44 (2004).</li><li>Schiffman, R. M., Christianson, M. D., Jacobsen, G., Hirsch, J. D., Reis, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+and+validity+of+the+Ocular+Surface+Disease+Index.">Reliability and validity of the Ocular Surface Disease Index.</a> Archives of Ophthalmology. 118 (5), 615-621 (2000).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+platelet+lysate+compared+to+autologous+and+allogeneic+serum+eye+drops.">Characteristics of platelet lysate compared to autologous and allogeneic serum eye drops.</a> Translational Vision Science and Technology. 9 (4), 24 (2020).</li><li>Henschler, R., Gabriel, C., Schallmoser, K., Burnouf, T., Koh, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+lysate+current+standards+and+future+developments.">Human platelet lysate current standards and future developments.</a> Transfusion. 59 (4), 1407-1413 (2019).</li><li>Samarkanova, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+evaluation+of+allogeneic+eye+drops+from+cord+blood+platelet+lysate.">Clinical evaluation of allogeneic eye drops from cord blood platelet lysate.</a> Blood Transfusion. 19 (4), 347-356 (2021).</li><li>Strunk, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+Forum+on+GMP-grade+human+platelet+lysate+for+cell+propagation:+Summary.">International Forum on GMP-grade human platelet lysate for cell propagation: Summary.</a> Vox Sanguinis. 113 (1), 80-87 (2018).</li><li>Schiroli, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+COVID-19+outbreak+on+the+Transfusion+Medicine+Unit+of+a+Northern+Italy+Hospital+and+Cancer+Centre.">The impact of COVID-19 outbreak on the Transfusion Medicine Unit of a Northern Italy Hospital and Cancer Centre.</a> Vox Sanguinis. 117 (2), 235-242 (2021).</li><li>Klatte-Schulz, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+different+platelet+lysates+and+platelet+rich+preparations+to+stimulate+tendon+cell+biology:+An+in+vitro+study.">Comparative analysis of different platelet lysates and platelet rich preparations to stimulate tendon cell biology: An in vitro study.</a> International Journal of Molecular Science. 19 (1), 212 (2018).</li><li>Fea, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+autologous+platelet+lysate+eye+drops:+An+in+vivo+confocal+microscopy+study.">The effect of autologous platelet lysate eye drops: An in vivo confocal microscopy study.</a> BioMed Research International. 2016, 8406832 (2016).</li><li>Abu-Ameerh, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+lysate+promotes+re-epithelialization+of+persistent+epithelial+defects:+A+pilot+study.">Platelet lysate promotes re-epithelialization of persistent epithelial defects: A pilot study.</a> International Ophthalmology. 39 (7), 1483-1490 (2019).</li><li>Geremicca, W., Fonte, C., Vecchio, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+components+for+topical+use+in+tissue+regeneration:+evaluation+of+corneal+lesions+treated+with+platelet+lysate+and+considerations+on+repair+mechanisms.">Blood components for topical use in tissue regeneration: evaluation of corneal lesions treated with platelet lysate and considerations on repair mechanisms.</a> Blood Transfusion. 8 (2), 107-112 (2010).</li><li>De Paiva, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+TGF-&#946;+signaling+improves+ocular+surface+epithelial+disease+in+experimental+autoimmune+keratoconjunctivitis+sicca.">Disruption of TGF-&#946; signaling improves ocular surface epithelial disease in experimental autoimmune keratoconjunctivitis sicca.</a> PLoS One. 6 (12), 29017 (2011).</li></ol>

In recent years, the clinical use of platelet-based products for ocular surface pathologies has increased, but their diffusion is hampered by the lack of scientific robustness. This is mainly caused by wide heterogeneity in donor sources and preparation protocols, which are often not fully disclosed or not specifically designed for the purpose for which they are dispensed. Particularly, information about platelet-based products collected by apheresis is still lacking. Therefore, the aim of the present work was to describ...

Blood-derived products are widely used in wound care1, maxillofacial and orthopedic surgery, and for the treatment of different ocular surface diseases2 such as dry eye disease (DED)3. In DED, the tear film homeostasis is impaired as a consequence of the abnormal functioning of different factors involved in tear production and ocular surface integrity4,5.
DED is characterized by heterogeneity in causes and severity6,7,8 and may be a consequence of different factors like aging, sex9, contact lenses, topical or systemic medications10, or pre-existing conditions like Sj&#246;gren&#39;s syndrome10. Despite having mild symptoms, DED affects millions of people worldwide, impacting their quality of life and the health system as well6.
Many treatments have been reported for this pathology, but there is still no consensus on the most effective solution12. To date, artificial tears are the first line of therapy aimed at restoring the aqueous composition of the tear film, albeit these substitutes do not contain the main biologically active solutes of natural tears6,11. Platelet-based products are considered a valid alternative12,13 to artificial tears, although their clinical efficacy, recommendations for use, and methods of preparations are still a matter of debate3.
Blood-based products share with tears a similar composition in terms of metabolites14, proteins, lipids, vitamins, ions, growth factors (GFs), antioxidant compounds11 and osmolarity (300 mOsm/L)11. Through the synergistic activity of their components, they promote the regeneration of the corneal epithelium, inhibit the release of inflammatory cytokines, and increase the number of goblet cells and the expression of mucins in the conjunctiva2,3.
So far, heterogeneity in ophthalmic blood-based products has been documented in the literature; these products can be classified according to the blood donors&#39; origin, i.e., autologous, or allogenic, as well as the blood source, i.e., peripheral blood, cord blood, serum, or platelets.
Although autologous products were the most widespread3, allogenic ones are now becoming the preferred choice, since they ensure higher efficacy and safety standards15, together with a significant reduction in costs16,17. Previous studies, indeed, proved that blood-based products obtained from patients with autoimmune and/or systemic diseases may show altered quality and functionality6,16,17. Despite the fact that serum-based eye drops are the most widespread, platelets-based products are recently becoming affirmed as a valid alternative, as they can be easily prepared while maintaining significant levels of efficacy3,11. Currently available platelet-based products can be divided in platelet-rich plasma (PRP), platelet-rich plasma lysate (PRP-L), and plasma rich in growth factors (PRGF)3.
Among them, PRP-L has the advantage of being a long-life frozen product. PRP-L can be prepared from apheresis, buffy-coats, or even from expiring platelets (PLTs)18,19, preciously reducing their wastage. The aliquots can be stored for months in the blood transfusions centers at &#8722;80 &#176;C or even at patients&#39; homes at &#8722;15 &#176;C for shorter periods.
PRP-L are highly enriched in GFs, which have been proved to stimulate eye surface regeneration12,20,21. Nevertheless, there are only few reported clinical studies in this area, and all of them used autologous sources3,22. PRP-L still needs further validation and characterization before it can be routinely used for the treatment of eye surface diseases, since there are no standardized guidelines for its preparation, dispensation, and storage3.
Herein, a detailed protocol is shared for the production of PRP-L used at the Transfusion Medicine Unit in AUSL-IRCCS di Reggio Emilia, Italy, and dispensation to patients with DED. We aim to help the scientific community to develop standard methods of preparation, which may increase homogeneity and consistency in worldwide studies and clinical approaches.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CompoSeal Mobilea II</td>
			<td>Fresenius Kabi, Germany</td>
			<td></td>
			<td>bag sealer</td>
		</tr>
		<tr>
			<td>HeraSafe hood</td>
			<td>Heraeus Instruments, Germany</td>
			<td></td>
			<td>Class II biohazard hood</td>
		</tr>
		<tr>
			<td>MCS+ 9000 Mobile Platelet Collection System</td>
			<td>Haemonetics, Italy</td>
			<td></td>
			<td>automated plasma and multicomponent collection equipment for donating platelet, red cell, plasma, or combination blood components</td>
		</tr>
		<tr>
			<td>Platelet shaker, PF396i</td>
			<td>Helmer, USA</td>
			<td></td>
			<td>Platelet shaker</td>
		</tr>
		<tr>
			<td>Raycell X-ray Blood Irradiator</td>
			<td>MDS Nordion, Canada</td>
			<td></td>
			<td>X-ray Blood Irradiator</td>
		</tr>
		<tr>
			<td>ROTIXA 50RS</td>
			<td>Hettich Zentrifugen, Germany</td>
			<td></td>
			<td>High speed entrifuge</td>
		</tr>
		<tr>
			<td>Sysmex XS-1000i</td>
			<td>Sysmex Europe GMBH, Germany</td>
			<td></td>
			<td>haemocytometer for platelet count</td>
		</tr>
		<tr>
			<td>Warm bath, WB-M15</td>
			<td>Falc Instruments, Italy</td>
			<td></td>
			<td>Warm bath</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACD-A anticoagulant solution A</td>
			<td>Fenwal Inc., USA</td>
			<td>DIN 00788139</td>
			<td>anticoagulant solution for platelet apheresis (1000 ml)</td>
		</tr>
		<tr>
			<td>BD BACTEC Peds Plus/F Culture vials</td>
			<td>BD Biosciences, USA</td>
			<td>BD 442020</td>
			<td>Sterility assay</td>
		</tr>
		<tr>
			<td>BD BACTEC Peds Plus/F Culture vials</td>
			<td>BD Biosciences, USA</td>
			<td>442020</td>
			<td>At least 2 vials for sterility assay</td>
		</tr>
		<tr>
			<td>BD Luer Lok Syringe</td>
			<td>BD Plastipack, USA</td>
			<td>300865</td>
			<td>At least 4 sterile syringes (50 ml)</td>
		</tr>
		<tr>
			<td>Bio-Plex Human Cancer Panel 1</td>
			<td>BioRad Laboratories, USA</td>
			<td>171AC500M</td>
			<td>Standard panel for PDGF isoforms assessment</td>
		</tr>
		<tr>
			<td>Bio-Plex Human Cancer Panel 2</td>
			<td>BioRad Laboratories, USA</td>
			<td>171AC600M</td>
			<td>Standard panel for EGF assessment</td>
		</tr>
		<tr>
			<td>Bio-Plex MAGPIX Multiplex Reader</td>
			<td>BioRad Laboratories, USA</td>
			<td>Magpix</td>
			<td>This instrument allows multiple immunoassays using functionalized magnetic beads.</td>
		</tr>
		<tr>
			<td>Bio-Plex Pro TGF-b Assay</td>
			<td>BioRad Laboratories, USA</td>
			<td>10024984</td>
			<td>Set and standards for TGFb isoforms assessment</td>
		</tr>
		<tr>
			<td>BioRet</td>
			<td>ARIES s.r.l., Italy</td>
			<td>A2DH0020</td>
			<td>At least 4 piercing spike for blood bags</td>
		</tr>
		<tr>
			<td>Blood collection tube</td>
			<td>BD Vacutainer, USA</td>
			<td>367835</td>
			<td>1 tube, necessary to perform platelet counts</td>
		</tr>
		<tr>
			<td>Eye drops kit. COL Medical Device for the application and preservation of eye drops from haemocomponents</td>
			<td>Biomed Device s.r.l., Italy</td>
			<td>COLC50</td>
			<td>Eye drops kit. At least 2 kits for each PRP unit collected</td>
		</tr>
		<tr>
			<td>Human Cancer PDGF-AB/BB Set 1x96well</td>
			<td>BioRad Laboratories, USA</td>
			<td>171BC511</td>
			<td>Set for PDGF isoforms assessment</td>
		</tr>
		<tr>
			<td>Human Cancer2 EGF Set 1x96well</td>
			<td>BioRad Laboratories, USA</td>
			<td>171BC603M</td>
			<td>Set for EGF assessment</td>
		</tr>
		<tr>
			<td>NaCl 0.9% sterile solution</td>
			<td>Baxter S.p.A., Italy</td>
			<td>B05BB01</td>
			<td>1000 ml</td>
		</tr>
		<tr>
			<td>OSDI Questionnaire</td>
			<td>Allergan Inc., USA</td>
			<td>OSDI</td>
			<td>Ocular Surface Disease Index Questionnaire</td>
		</tr>
		<tr>
			<td>Piercing spike</td>
			<td>BioRet ARIES s.r.l., Italy</td>
			<td>BS051004</td>
			<td>Spike</td>
		</tr>
		<tr>
			<td>Platelet Additive Solution A+ T-PAS+</td>
			<td>TERUMO BCT Inc., Italy</td>
			<td>40842</td>
			<td>preservative solution for platelet concentrates (1000 ml)</td>
		</tr>
		<tr>
			<td>Software Excel</td>
			<td>Microsoft, USA</td>
			<td>Excel</td>
			<td>Data analysis software</td>
		</tr>
		<tr>
			<td>Teruflex Transfer bag 1000 ml</td>
			<td>TERUMO BCT Inc., Italy</td>
			<td>BB*T100BM</td>
			<td>1 for PRP dilution</td>
		</tr>
		<tr>
			<td>Teruflex Transfer bag 300 ml</td>
			<td>TERUMO BCT Inc., Italy</td>
			<td>BB*030CM</td>
			<td>At least 6 for each PRP unit collected</td>
		</tr>
	</tbody>
</table>

platelet-rich plasma lysate for treatment of eye surface diseases

Various ocular surface diseases are treated with blood-derived eye drops. Their use has been introduced in clinical practice because of their metabolite and growth factor content, which promotes eye surface regeneration. Blood-based eye drops can be prepared from different sources (i.e., whole blood or platelet apheresis donation), as well as with different protocols (e.g., different dilutions and freeze/thaw cycles). This variability hampers the standardization of clinical protocols and, consequently, the evaluation of their clinical efficacy. Detailing and sharing the methodological procedures may contribute to defining common guidelines. Over the last years, allogenic products have been diffusing as an alternative to the autologous treatments since they guarantee higher efficacy standards; among them, the platelet-rich plasma lysate (PRP-L) eye drops are prepared with simple manufacturing procedures. In the transfusion medicine unit at AUSL-IRCCS di Reggio Emilia, Italy, PRP-L is obtained from platelet-apheresis donation. This product is initially diluted to 0.3 x 109 platelets/mL (starting from an average concentration of 1 x 109 platelets/mL) in 0.9% NaCl. Diluted platelets are frozen/thawed and, subsequently, centrifuged to eliminate debris. The final volume is split into 1.45 mL aliquots and stored at &#8722;80 &#176;C. Before being dispensed to patients, eye drops are tested for sterility. Patients may store platelet lysates at &#8722;15 &#176;C for up to 1 month. The growth factor composition is also assessed from randomly selected aliquots, and the mean values are reported here.

The rationale for the use of serum-derived eye drops (which is the blood-based product most frequently used for the treatment of eye surface diseases) lies in their content of GFs, which are almost completely derived from circulating platelets. PRP contains a significantly higher number of platelets (and, consequently, of platelet-derived GFs) compared to peripheral blood serum, ranging between 0.15 x 109-0.45 x 109 PLTs/mL. According to Italian laws, the platelet count in PRP units should be at lea...

Watch this Scientific Journal Video about Platelet-Rich Plasma Lysate for Treatment of Eye Surface Diseases at JoVE.com

Platelet-Rich Plasma Lysate for Treatment of Eye Surface Diseases

Platelet lysates represent an emerging tool for the treatment of ocular surface diseases. Here, we propose a method for the preparation, dispensation, storage, and characterization of platelet lysate collected from platelet donors.

Various ocular surface diseases are treated with blood-derived eye drops. Their use has been introduced in clinical practice because of their metabolite ...

PRP Lysate is an emerging platelet based product for the treatment of ocular surface diseases. Here we want to share and detail our protocol of production to cover the development of common methodological guidelines. Halo genic products have been diffusing as an alternative to autologous treatments.Since they guarantee higher efficacy standards. PRP Lysate contains a high concentration of growth factors. Its production is inexpensive and simple and it can be stored for a long period of time.The usage of blood products for the treatment of eyes or face diseases is largely documented in the literature. Nevertheless, their applications still has not found a consensus. One of the reasons is the variability of the methods used for their production.Our aim is to share our protocol in order to develop common methodologies. To begin, perform a platelet count with a hemo cystometer using the sample collected from the main bag through a piercing spike. Dilute platelet-rich plasma with an adequate amount of sterile 0.9%sodium chloride.Split the diluted platelet-rich plasma into 300 milliliter empty collection bags to reach a net volume of 190 milliliters per bag. Use an aliquot of residual diluted platelet-rich plasma to perform quality controls, assessing possible microbial contamination. Prepare a sterility assay following the manufacturer's instructions to be tested in a microbiology laboratory.Store diluted platelet-rich plasma bags at minus 80 degrees Celsius for a maximum of two months before thawing. Ensure that a warm bath is set at 37 degrees Celsius. Put the platelet-rich plasma bags into the warm bath and wait until completely thawed.Centrifuge the platelet-rich plasma bags at 3000 times G for 30 minutes at room temperature. Exploiting the piercing spike of the transfer bag, connect the centrifuged bag with an empty sterile 300 milliliter transfer bag. Carefully transfer the PRP Lysate supernatant while avoiding debris into the new bag.Seal the connection tube of the PRP Lysate unit with a bag sealer. Collect 30 to 60 milliliter of PRP Lysate with a sterile syringe, and link the syringe to the Luer Lock connection on the filling line. According to the manufacturer's instructions, turn the stopcock by half of a turn to open the line between the PRP Lysate containing syringe and the pre-connected syringe.Fill the pre-connected syringe with PRP Lysate. Disconnect the PRP Lysate syringe. Close the tube cap of the Luer Lock connection and rotate the stopcock to the original position.Use the eyedrops kit syringe to fill the vials with PRP Lysate. Ensure that each applicator is properly filled. Then individually seal them with a bag sealer.Repeat the procedure with a new eyedrops kit. Use a small aliquot of residual diluted PRP Lysate to assess possible microbial contamination. Properly label each applicator and put them into a plastic bag.Label the plastic bag too. Taking care to highlight the donor's blood group. Store at minus 80 degrees Celsius for a maximum of 24 months before patient assignment, according to the Italian Law and Guidelines.A quantitative assessment of the epidermal growth factor, platelet derived growth factor, and transforming growth factor Beta isoform in PRP Lysate from two different donors shows that the results of 0.3 times 10 to the ninth platelets per milliliter dilution turned out to be most similar to tear composition. After a six month therapy with PRP Lysate ocular surface disease index scores decreased from 56 plus minus 21 to 45 plus minus 21. Indicating improvement in patient's quality of life.The methodological differences between different methods of preparing allogeneic PRP Lysate did not prove to be detrimental to the regenerative capacity of the PRP Lysate tested on the other tissues. Dilution of plasma with sodium chloride and storing of platelet rich plasma needs to be performed with care. As this might alter the composition of the PRP Lysate.Comparative analysis are necessary to investigate variations in these steps.

platelet-rich-plasma-lysate-for-treatment-of-eye-surface-diseases

Research

JoVE Journal

Medicine

3.5K Aufrufe.  AUSL-IRCCS di Reggio Emilia. PRP Lysat ist ein neues plättchenbasiertes Produkt zur Behandlung von Augenoberflächenerkrankungen. Hier möchten wir unser Produktionsprotokoll teilen und detailliert beschreiben, um die Entwicklung gemeinsamer methodischer Richtlinien abzudecken. Halo-Genprodukte haben sich als Alternative zu autologen Behandlungen verbreitet.Da sie höhere Wirksamkeitsstandards garantieren. PRP-Lysat enthält eine hohe Konzentration an Wachstumsfaktoren. Seine Herstellung ist kostengünstig und ei...