Platelet-Rich Plasma Lysate for Treatment of Eye Surface Diseases

Podsumowanie

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.

Streszczenie

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 −80 °C. Before being dispensed to patients, eye drops are tested for sterility. Patients may store platelet lysates at −15 °C for up to 1 month. The growth factor composition is also assessed from randomly selected aliquots, and the mean values are reported here.

Wprowadzenie

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 integrity^4,5.

DED is characterized by heterogeneity in causes and severity^6,7,8 and may be a consequence of different factors like aging, sex⁹, contact lenses, topical or systemic medications¹⁰, or pre-existing conditions like Sjögren'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 tears^6,11. Platelet-based products are considered a valid alternative^12,13 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 conjunctiva^2,3.

So far, heterogeneity in ophthalmic blood-based products has been documented in the literature; these products can be classified according to the blood donors' 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 costs^16,17. Previous studies, indeed, proved that blood-based products obtained from patients with autoimmune and/or systemic diseases may show altered quality and functionality^6,16,17. 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 efficacy^3,11. 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 −80 °C or even at patients' homes at −15 °C for shorter periods.

PRP-L are highly enriched in GFs, which have been proved to stimulate eye surface regeneration^12,20,21. Nevertheless, there are only few reported clinical studies in this area, and all of them used autologous sources^3,22. 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.

Protokół

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

1. PRP apheresis
   1. 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.
   2. Perform plasma-platelet-apheresis using an automated blood collection system, according to the manufacturers' 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.
2. PRP units' 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's manual of instruction.
   1. 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 > 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.
   2. Store leucodepleted and irradiated PRP for a maximum of 5 days at 22 °C ± 2 °C on a platelet shaker before further manipulation²³.
3. PRP dilution
   1. 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.
   2. Dilute PRP with an adequate amount of sterile 0.9% NaCl to a final concentration of 0.32 x 10⁹ ± 0.03 x 10⁹ PLTs/mL, which simulates the average PLT concentration in peripheral blood.
   3. 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.
   4. 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'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.
   5. Store diluted PRP bags at −80 °C for a maximum of 2 months before thawing.

2. Platelet-rich plasma lysate (PRP-L) preparation

1. Thawing
   1. Before starting the thawing procedure, ensure that a warm bath is set at 37 °C. Put the PRP bags into the warm bath and wait until completely thawed.
2. PRP-L collection
   1. Centrifuge the PRP bags at 3000 x g for 30 min at room temperature.
      ​NOTE: The next steps should be performed in sterility under a class II biohazard hood.
   2. 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.
   3. Seal the connection tube of the PRP-L unit with a bag sealer.
3. 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.
   1. Collect 30-60 mL of PRP-L with a sterile syringe and link the syringe to the Luer/lock connection on the filling line.
   2. According to the manufacturer'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.
   3. 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.
   4. 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.
   5. Repeat the procedure with a new eye drops kit.
   6. 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'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.
4. PRP-L storage
   1. 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.
   2. Store at −80 °C for a maximum of 24 months before patient assignment, according to the Italian law²³ and guidelines²⁴.

3. PRP-L dispensation

1. 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' homes for up to 1 month at −15 °C.

Wyniki

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...

Dyskusje

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...

Materiały

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