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) questionnaire, which is routinely used by clinicians to monitor dry eye syndrome symptoms. Figure 1A shows an outline of the protocol followed, while the pictures in Figure 1B depict the main steps of the procedure.
1. Platelet-rich plasma (PRP) collection
<ol>
	<li>PRP apheresis
	<ol>
		<li>For this protocol, select platelet donors according to Italian laws: platelet donors must be 18-65 years old, with normal pressure and blood count parameters and a platelet count not less than 180 x 109 platelets/L23. Eligible donors cannot take antiplatelet or anticoagulant drugs within 1 week before donation.</li>
		<li>Perform plasma-platelet-apheresis using an automated blood collection system, according to the manufacturers&#39; instructions and national laws23, to obtain 1 unit of single-donor platelet-rich plasma (PRP). Collect PRP in Adenine Citrate Dextrose Solution A (ACD-A) anticoagulant solution. 
		NOTE: Platelet-apheresis is conducted with a continuous procedure; the time of collection is in a range between 40 min and 90 min. The amount of ACD delivered to the donors and the time of the procedures depend on the donor characteristics, e.g., hematocrit and needle gauge.</li>
	</ol>
	</li>
	<li>PRP units&#39; characteristics 
	NOTE: The following step is usually performed automatically by the automated blood collection system during the plasma-platelet-apheresis procedure. Please check the manufacturer&#39;s manual of instruction.
	<ol>
		<li>Resuspend the PRP units collected by apheresis in an adequate amount of preservative solution with the minimum amount of residual plasma, necessary to maintain pH &#62; 6.4 during the whole storage time, to a mean final volume of 180 mL net of the anticoagulant solution (around 40 mL). 
		NOTE: According to Italian law, quality controls have to assess that the platelet (PLTs) count is at least 2.0 x 1011 PLTs/unit, while residual leucocytes have to be less than 1 x 106 cells/unit.</li>
		<li>Store leucodepleted and irradiated PRP for a maximum of 5 days at 22 &#176;C &#177; 2 &#176;C on a platelet shaker before further manipulation23.</li>
	</ol>
	</li>
	<li>PRP dilution
	<ol>
		<li>Immediately before starting PRP dilution, perform a PLT count with a hemocytometer using the sample collected from the main bag through a piercing spike. 
		NOTE: Perform the next steps in sterility under a class II biohazard hood. Wear personal protection equipment (lab coat, gloves, and goggles) during the procedure.</li>
		<li>Dilute PRP with an adequate amount of sterile 0.9% NaCl to a final concentration of 0.32 x 109 &#177; 0.03 x 109 PLTs/mL, which simulates the average PLT concentration in peripheral blood.</li>
		<li>Taking advantage of a piercing spike for blood bags, split the diluted PRP into 300 mL empty collection bags to reach a net volume of 190 mL/bag.</li>
		<li>Use an aliquot of residual diluted PRP (usually 1 mL) to perform quality controls assessing possible microbial contaminations. Perform a sterility assay following the manufacturer&#39;s instructions in a microbiology laboratory (see Table of Materials). 
		NOTE: Use culture vials specific for aerobic blood cultures, which are able to perform the qualitative culture and recovery of aerobic microorganisms (mainly bacteria and yeast) from small volume blood specimens.</li>
		<li>Store diluted PRP bags at &#8722;80 &#176;C for a maximum of 2 months before thawing.</li>
	</ol>
	</li>
</ol>
2. Platelet-rich plasma lysate (PRP-L) preparation
<ol>
	<li>Thawing
	<ol>
		<li>Before starting the thawing procedure, ensure that a warm bath is set at 37 &#176;C. Put the PRP bags into the warm bath and wait until completely thawed.</li>
	</ol>
	</li>
	<li>PRP-L collection
	<ol>
		<li>Centrifuge the PRP bags at 3000 x g for 30 min at room temperature. 
		&#8203;NOTE: The next steps should be performed in sterility under a class II biohazard hood.</li>
		<li>Exploiting the piercing spike of the transfer bag, connect the centrifuged bag with an empty sterile 300 mL transfer bag. Carefully, transfer the PRP-L supernatant, while avoiding debris, into the new bag. When possible, use a bag press.</li>
		<li>Seal the connection tube of the PRP-L unit with a bag sealer.</li>
	</ol>
	</li>
	<li>PRP-L aliquotation 
	NOTE: A starting unit containing 190 mL PRP (see step 1.3.3.) is sufficient to fill two eye drops kits (for details on the specific medical devices used for the application and preservation of eye drops from blood components, see the Table of Materials). Eye drops kits should be opened under a class II hood with the whole string vials positioned above the pre-connected syringe and the central arrow of the stopcock pointing leftward to exclude the anti-bacterial filter.
	<ol>
		<li>Collect 30-60 mL of PRP-L with a sterile syringe and link the syringe to the Luer/lock connection on the filling line.</li>
		<li>According to the manufacturer&#39;s instructions, turn the stopcock by half of a turn to open the line between the PRP-L-containing syringe and the pre-connected syringe. Fill the pre-connected syringe with PRP-L.</li>
		<li>Disconnect the PRP-L syringe, close the tube cap of the luer/lock connection and rotate the stopcock to the original position. Use the eye drops kit syringe to fill the vials with PRP-L.</li>
		<li>Repeat the procedure from steps 2.3.1.-2.3.3. until all the applicator vials are filled. Ensure that each applicator is properly filled, then individually seal them with a bag sealer.</li>
		<li>Repeat the procedure with a new eye drops kit.</li>
		<li>Use a small aliquot of residual diluted PRP-L to assess possible microbial contamination (see step 1.3.4.). 
		NOTE: If the liquid accidentally reaches the anti-bacterial filter at the end of the string, the suction syringe may oppose resistance, hindering the filling. To continue the filling cycle, raise the end section of the string for about 5/6 aliquots from the anti-bacterial hydrophobic filter at the end of the string. In this position, use a new sterile syringe (of 30 mL volume) that has already been filled with air. Connect the female luer/lock of the anti-bacterial filter and press hard and repeatedly on the syringe&#39;s plunger to remove all residues of blood component and render the membrane of the anti-bacterial filter free of the liquid. Remove the syringe and fill the remaining vials.</li>
	</ol>
	</li>
	<li>PRP-L storage
	<ol>
		<li>Properly label each applicator and put them into a plastic bag. Label the plastic bag too, taking care to highlight the donor&#39;s blood group.</li>
		<li>Store at &#8722;80 &#176;C for a maximum of 24 months before patient assignment, according to the Italian law23 and guidelines24.</li>
	</ol>
	</li>
</ol>
3. PRP-L dispensation
<ol>
	<li>Perform patient assignment preferably by matching the PRP-L blood group. Deliver PRP-L applicator vials using a cool box and ensure that each applicator vial contains 1.45 mL of PRP-L, which corresponds to approximately 45 drops. Instruct the patient that applicator vials can be stored at the patients&#39; homes for up to 1 month at &#8722;15 &#176;C.</li>
</ol>

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<li>Bernabei, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blood-based+treatments+for+severe+dry+eye+disease%3A+The+need+of+a+consensus%5BTitle%5D)+AND+%22Journal+of+Clinical+Medicine%22%5BJournal%5D)">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/pubmed?term=((Dry+eye+disease%3A+When+to+treat+and+when+to+refer%5BTitle%5D)+AND+%22Australian+Prescriber%22%5BJournal%5D)">Dry eye disease: When to treat and when to refer.</a> Australian Prescriber. 41 (5), 160-163 (2018).</li>
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</ol>

The authors declare no conflicts of interest.

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 describe the step-by-step processing of platelet-rich plasma lysates (PRP-L) obtained by apheresis for the treatment of DED.
PRP-L is an optimal source for the production of eye drops since it contains more GFs than the other blood-based products22,&#160;and, when compared to serum or PRP, its production or storage are inexpensive and simple. To obtain PRP-L, platelets are subjected to lysis (usually through one or more freeze and thaw cycles) to release their content. This process guarantees a solution enriched in active molecules that stimulate tissue regeneration22,26. An increasing number of diseases have been treated with PRP-L22, but the indication for use in ophthalmology is still weak due to the low standardization criteria in platelet collection and PRP-L production3,22.
Allogenic platelet-based products should be preferred since they are more standardizable than autologous ones, both in terms of donor characteristics and method of preparation. The patient health status may affect the quality of the product6,16,17, while the in-house kits to collect autologous platelets from whole blood when transfusion services are not directly available do not meet the standard quality required in transfusion medicine31.
To our knowledge, there are no clinical studies characterizing the usage of allogenic PRP-L in ophthalmology3, while there are few reports regarding autologous PRP-L eye drops3 and only one study using allogenic PRP-L obtained from cord blood to treat patients with eye surface diseases32. Although allogenic PRP-L is listed in the clinical guidelines24 and its usage has been proposed3,30,&#160;there is still a lack of evidence on its efficacy compared to other treatments and to other blood-based products (e.g., serum). Here, the protocol presented aims to help the scientific community to develop common methods of production and to shed light on the methodological differences.
Here, we described PRP-L production starting from allogenic PRP collected by apheresis. Platelet-based allogenic products to obtain platelet lysates may also be collected from buffy-coats (BCs), and both sources have been equally reported31. BCs are obtained from pooled donors (usually four or five), thus minimizing the inter-individual differences. Conversely, pooling increases the risk of transmitting infectious agents or prions or stimulating an allogenic response31,33. Apheresis is a complex and invasive procedure, and only a minority of donors are eligible for or compliant with it34. Nevertheless, platelet-based products obtained by apheresis are free from other residual circulating blood cells and contain a higher amount of PLTs35. For these reasons, the current work is focused on developing clinical studies to compare PRP-L from these two different sources.
In this protocol, the starting concentration of PLTs in PRP units collected by apheresis was on average 1 x 109/mL, which is consistent with the reported concentrations for other platelet-based products22. In this method, PLTs are diluted afterward with 0.9% NaCl solution to 0.3 x 109/mL. Other protocols report the use of plasma for the dilution22.
Few studies have reported the use of PRP-L in ophthalmology; in these cases, autologous eye drops were prepared at concentrations of PLTs ranging from 0.5 x 109/mL-1 x 109/mL36,37,38. As discussed earlier, standardization markers are desirable and would also help in determining the proper dilution. Here, for instance, we report the concentration of some pivotal GFs in the PRP-L. EGF and PDGF content influence the efficacy of PRP-L22, while TGF&#946; isoforms are involved in regulating immune signaling21,39, and their concentration is finely regulated. Therefore, TGF&#946; concentration in platelet-based eye drops may influence not only the efficacy but also elicit potential detrimental effects39; thus, it should be carefully investigated before defining the proper dilution. Nonetheless, the dilution selected-0.3 x 109 PLTs/mL-was based on the GFs content in tears21,25. Zhang et al. previously compared serum-based eye drops, both autologous and allogenic, and platelet-based lysates for their content in GFs and for their capability to promote the regeneration of corneal cells in vitro30. The study showed how these products have comparable features, with PRP-L having higher EGF concentration but lower fibronectin. In their protocol, the freeze/thaw process was repeated twice, the centrifuge performed at 3500 x g for 30 min, and the platelet lysate stored at &#8722;80 &#176;C30.
Freezing and thawing is indeed a critical step; most of the protocols (including this one) were developed with &#8722;80 &#176;C freezing and 37 &#176;C thawing, but freezing has also been reported at &#8722;24 &#176;C, &#8722;196 &#176;C, and &#8722;150 &#176;C22,33 Even the number of freeze/thaw cycles performed are variable, ranging from 1 to 522,33. A limited number of studies also reported sonication or solvent/detergent treatment to obtain platelet lysates22,33. Other methodological variables previously reported in the preparation of PRP-L concern the centrifugation step-between 300 x g and 10000 x g, from 2 min to 60 min-and the long-term storage, which, in most cases, is at &#8722;80 &#176;C, albeit that similar products have been also been directly stored at &#8722;20 &#176;C22. Storage conditions, in particular, should be carefully monitored since they might affect the availability and activity of GFs contained in lysates. In this highly heterogeneous context, quality controls and clinical studies taking into consideration the release of biogenic factors and the differences in the therapeutic effect should be urgently evaluated.
Finally, here we show how this method has been significantly evaluated with a positive result from an aggregate analysis of patients with dry eye disease receiving PRP-L for 6 months (OSDI questionnaire3). Although promising, OSDI alone is not sufficient to determine the efficacy of PRP-L in the treatment of DED and other ocular surface diseases, and clinical studies on the usage of allogenic PRP-L are warranted. Moreover, possible product composition differences due to alternative methodological steps (i.e., freeze and thaw, centrifugation, storage) should be compared in order to optimize the methodological procedure.
In conclusion, the high heterogeneity of blood sources and protocols still hampers the definitive translation of blood-based products to the clinical treatment of ocular surface diseases. Although PRP-L is an emerging product with some advantageous features, further studies are necessary to validate its use and to develop common guidelines. Sharing and detailing the protocol of preparation may expand the usability and shed light on the critical steps.

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><tbody><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</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>&lt;strong&gt;Materials&lt;/strong&gt;</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 least 0.9 x 109-1 x 109 PLTs/mL. Therefore, to obtain a product that simulates the efficacy of serum eye drops, PRP should be diluted to the physiological platelet content before lysate preparation.
Nevertheless, since tissue repair is mainly driven by platelet-derived GFs, the PLT scount alone might be misleading for an effective therapy of eye surface diseases. In DED, which is the eye disease most commonly treated with blood-derived eye drops, tear film production and homeostasis are impaired. Platelet-based products for the treatment of DED, therefore, should also mimic the physiological content of tears.
To identify the most suitable PRP-L for treating eye surface diseases, described in step 1.3.2. of the present protocol, we preliminarily assessed different PRP dilutions, according to their PLTs content (between 0.7 x 109/mL and 0.3 x 109/mL), and some representative GFs from those that are known to be involved in eye tissue repair12,20,21.
Platelet count was performed with a hemocytometer, while GFs were assessed by means of a multiplex protein quantification assay. The assay was performed as previously described25 according to the manufacturer&#39;s instructions. GFs shown in this manuscript were selected for quantification after a preliminary screening of 36 GFs and GFRs performed on PRP lysate with a semi-quantitative protein array. Luminex quantification was performed on 3 out of the 36 screened GFs: EGF and PDGF (which turned out to be the most abundant ones in our PRP lysates) and TGF&#946;-1,2,3 isoforms (for which the content is important for the eye surface treatment21). EGF and PDGF content were measured as they may influence the efficacy of PRP-L22, while TGF&#946; isoforms were selected for their known role in immune signaling regualtion21.
Since protein arrays are part of another in vitro study on the characterization of different PRP26, those data are not presented in this manuscript.
We quantitatively assessed EGF, PDGF, and TGF&#946; in PRP lysates from two different donors (D1 and D2), previously diluted between 0.7 x 109-0.3 x 109 PLTs/mL in 0.9% NaCl. Figure 2 shows the results of the 0.3 x 109 PLTs/mL dilution, which turned out to be most similar to tear composition.
The 0.3 x 109 PLTs/mL dilution was selected based on literature data on tear composition. The EGF values were found to be quite low compared to the mean tear value but still in the range of normality27. Even PDGF, despite being highly variable between the two donors considered, was always comparable to the concentration found in normal tears20. Finally, TGF&#946;-1 was found to be the most abundant isoform in PRP-L, similar to tears21.
Once the most suitable PLT dilution to prepare apheresis PRP-L had been identified, the Transfusion Medicine Unit started distributing these products to patients affected by eye surface disorders in 2015. The ophthalmologists routinely collected the OSDI questionnaires to monitor DED symptoms; the OSDI test assesses quality of life measures, such as the perception of ocular irritation and how it affects the functioning related to vision. The questionnaire, created by the Outcomes Research Group at Allergan Inc. in 1995 and now accepted as a valid instrument to monitor DED, is submitted to patients and analyzed as previously described28,29.
Here, we show the aggregate results of OSDI tests of DED patients treated between January 2020 and January 2021 (n = 27). After a 6 month therapy with PRP-L, the OSDI scores decreased from 56 &#177; 21 to 45 &#177; 21, indicating an improvement in patients&#39; quality of life (Figure 3).
Despite these data still being in the severe range and not relating to clinical outcomes of efficacy, they suggest that DED patients consider PRP-L a useful product that ameliorates ocular discomfort; this aspect should be further investigated in prospective clinical trials aimed at assessing its efficacy in treating ocular surface diseases.
In Table 1, we report a comparison of the present method of production with another method for preparing allogenic PRP-L for eye drops30 and for other purposes22. To our knowledge, Zhang&#39;s30 protocol and the current protocol are the only published methods to produce PRP-L for the eye surface. In both, PRP-L is obtained from apheresis; differences between the two protocols, mainly relating to the number of freeze and thaw cycles and centrifugation steps, should be compared in order to improve PRP-L production. Nevertheless, these methodological differences have not been proved to be detrimental to the regenerative capacity of PRP-L tested on other tissues22.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63772/63772fig01v2.jpg" /> 
Figure 1: Main steps of the protocol for the preparation of PRP-L. (A) Scheme of the protocol, from PRP collection to PRP-L preparation and dispensation.&#160;(B)&#160;Representative pictures of the main steps of the protocols. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63772/63772fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63772/63772fig02.jpg" /> 
Figure 2: Luminex quantification of platelet-derived growth factors for the 0.3 x 109/mL dilution of PRP-L. (A) Epidermal growth factor (EGF); (B) platelet-derived growth factor (PDGF); (C) transforming growth factor-beta isoform 1 (TGF&#946;1); (D) transforming growth factor-beta isoform 2 (TGF&#946;2); (E) transforming growth factor-beta isoform 3 (TGF&#946;3). Values are expressed as pg/mL, mean &#177; standard deviation of three independent measurements. D1 and D2 are two different platelet donors. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63772/63772fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63772/63772fig03v3.jpg" /> 
Figure 3: Aggregate OSDI scores of DED patients treated with PRP-L between January 2020 and January 2021 at the Ophthalmology Unit of the AUSL-IRCCS di Reggio Emilia. N = 27 patients. OSDI score aggregate results are represented as mean &#177; standard error, p-value was calculated with a paired t-test with data analysis software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63772/63772fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>This article</td>
			<td>PRP-L for the eye (in vitro study)29</td>
			<td>PRP-L for other purposes21</td>
		</tr>
		<tr>
			<td>Source</td>
			<td>PLTs apheresis</td>
			<td>PLTs apheresis</td>
			<td>Apheresis and whole blood</td>
		</tr>
		<tr>
			<td>Freeze and thaw cycles</td>
			<td>1 (at -80 &#176;C)</td>
			<td>2 (at -80 &#176;C)</td>
			<td>1-3 (at -20 &#176;C and -80 &#176;C)</td>
		</tr>
		<tr>
			<td>Storage temperature</td>
			<td>at -80 &#176;C</td>
			<td>at -80 &#176;C</td>
			<td>at -20 &#176;C and -80 &#176;C</td>
		</tr>
		<tr>
			<td>Centrifugation speed before storage</td>
			<td>3000 x g/30 min</td>
			<td>3500 x g/30 min</td>
			<td>400-3000 x g/6 min -30 min</td>
		</tr>
		<tr>
			<td>Filtration before storage</td>
			<td>No</td>
			<td>Yes</td>
			<td>No/Yes</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of protocols to prepare allogenic PRP-L from platelet-based products collected by apheresis.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Medicine

4.0K Views.  AUSL-IRCCS di Reggio Emilia. PRP Lysate is een opkomend product op basis van bloedplaatjes voor de behandeling van oculaire oppervlakteziekten. Hier willen we ons productieprotocol delen en detailleren om de ontwikkeling van gemeenschappelijke methodologische richtlijnen te dekken. Halo-genogene producten zijn verspreid als een alternatief voor autologe behandelingen.Omdat ze hogere werkzaamheidsnormen garanderen. PRP Lysaat bevat een hoge concentratie groeifactoren. De productie is goedkoop en eenvoudig en kan vo...

Video: Platelet-Rich Plasma Lysate for Treatment of Eye Surface Diseases

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Immunology and Infection

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

Bioengineering

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Biology

Preparing Activated Platelet-Rich Plasma for Culturing Human Adipose-Derived Stem Cells

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of cultured cells, implying a possible use in regenerative medicine. Here, a double-spin method was used to prepare PRP from whole blood. PRP was further activated by autologous thrombin. The platelet count was measured in the activated PRP and the proliferation-stimulating effect in human adipose-derived stem cells (hASCs) was examined. The resulting platelet count was 11.5-times higher in PRP than in whole blood plasma. The proliferation of hASCs was markedly enhanced by incubation with 1% PRP. The described method can be used to reproducibly prepare PRP with a high concentration of platelets. PRP prepared by this method markedly promotes proliferation of hASCs.

In this report, we describe a double-spin method for preparing activated platelet-rich plasma (PRP). Using autologous thrombin, human adipose-derived stem cells (hASCs) were cultured. Activated PRP was shown to promote proliferation of hASCs.

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of ...

Developmental Biology

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient&#8217;s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is ...

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a lesion in the Achilles tendon; prepare PRP and inject it (or the saline solution); remove the tendon; and perform biomechanical, molecular, and histological evaluations. At each step, all the procedures and methods are described in detail, so they can be reproduced easily.
Achilles tendons have been surgically sectioned (removal of a 5-mm long section). Afterwards, PRP or saline solution was injected to study whether PRP has a positive effect on the healing of the tendon. Three groups of 40 animals (a total of 120 rats were used in this study) were subdivided into 2 subgroups: PRP injection group and a saline injection control group. Rats were sacrificed at increasing time points (Group A: 5 days; Group B: 15 days; Group C: 30 days) and tendons were removed. 90 tendons underwent biomechanical testing before performing transcriptomic analysis and the 30 remaining tendons were submitted to histological analysis.

Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This protocol describes the evaluation process of healing tendons in rats that have been injected with allogeneic platelet rich plasma (PRP) or saline solution after removing part of the Achilles tendon. The progress of tendon healing is evaluated at several time points using different types of analyses.

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a ...

Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited effect on the repair of cartilage and on bone regeneration. Platelet-rich plasma (PRP) is an autologous bioactive substance that can repair cartilage injury and accelerate bone regeneration effectively. However, reporting of PRP preparation protocols in clinical studies is highly inconsistent, with the majority of studies providing insufficient information to allow the protocol to be reproduced. We describe a repeatable method of preparing PRP visually, the treatment of KOA using PRP intra-articular injection, and methods of evaluating the outcome. PRP was prepared using manual double centrifugation. The PRP layer was extracted from peripheral blood and used for knee joint cavity injection. Evaluations included assessments of blood platelet concentrations and clinical outcomes. Preparation of PRP by manual centrifugation requires less apparatus and is less costly than plasma filtration or centrifugation using equipment. The centrifugation time of our double centrifugation method was 6 and 5 minutes for the respective centrifugations at forces of 800 and 1400 x g, respectively, to allow for the consistent preparation of standardized PRP. However, a manual method is susceptible to operator error, and PRP batch preparation is not available. Intra-articular injection of PRP proved to be an effective treatment for knee osteoarthritis. The entire treatment procedure took less than 30 minutes, the blood platelet concentration of PRP could be standardized, and treatment was proven to be effective when evaluated by follow-up.

Knee osteoarthritis is frequently seen in the orthopedic department. We introduce in detail the entire knee osteoarthritis treatment process with platelet-rich plasma injection, including preparation, procedures, and evaluation.

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched in epitheliotropic growth factors that are essential in corneal wound healing. Unlike AS, PRP is based on a differential centrifugation system, yielding more platelet-derived growth factors. Autologous conditioned serum (ACS) not only preserves the preparation of AS and PRP, but also focuses on immune-modulating properties, which are important in inflammatory diseases.
The lack of standardized protocols and high preparation costs are limitations for the clinical application of ACS. This video experiment demonstrates a standard operating procedure for preparing modified autologous conditioned serum (mACS) eye drops. First, glycerol was added into heparin syringes as the blood cell stabilizer during hypoxic incubation. To activate the blood cells, a 4 h incubation at 37 &#176;C was initiated. Then, the blood samples were centrifuged at 3,500 &#215; g for 10 min at room temperature. After filtration of the supernatant through a 0.22 &#181;m filter, the mACS eye drops were fully prepared.
A tentative try-out of the therapeutic effect of mACS showed that it may have competitive advantages over conventional AS in the corneal wound healing in ex vivo mouse eyes. The AS used in this study was prepared according to published studies and the clinical practice in our hospital. Therefore, the efficacy of mACS on ocular surface diseases could be evaluated in future research through in vivo animal studies and clinical trials.

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

This article describes a protocol to simplify the process and render the preparation of autologous conditioned serum (ACS) less expensive. No special syringes or surface-coated glass beads are needed. Moreover, the modified ACS (mACS) has competitive advantages over conventional autologous serum in the corneal wound healing of murine eyes ex vivo.

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched ...

Preparation of Platelet-Rich Plasma from Human Peripheral Blood

This video demonstrates the preparation of high-quality Platelet-Rich Plasma (PRP) by screening for platelet count, collecting and centrifuging blood, and isolating platelets. This PRP is used in therapeutic applications for tissue regeneration and healing.

This video demonstrates the preparation of high-quality Platelet-Rich Plasma (PRP) by screening for platelet count, collecting and centrifuging blood, and ...

Platelet-Rich Plasma Lysate for Treatment of Eye Surface Diseases

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