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GUIDELINES FOR THE PROCUREMENT, PROCESS AND DISTRIBUTION OF TISSUES FOR TRANSPLANTATION

Approved by the National Transplant Centre, September 2016

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SECTION A OBJECTIVES AND ACTIVITIES……………………………7

A.1 Introduction A.2 Definitions of Facilities Involved in the Process A.2.1 Definition of a Tissue Bank A.2.2 Definition of a Harvesting Facility A.2.3 Definition of a Transplant Facility A.2.4 Use of Tissues for Transplantation A.3 Definitions A.3.1 Procurement A.3.2 Culture A.3.3 Preservation A.3.4 Critical A.3.5 Distribution A.3.6 Cadaveric Donor A.3.7 Living Donor A.3.8 Serious Adverse Event A.3.9 Standard Operating Procedures (SOP) A.3.10 Processing A.3.11 Quarantine A.3.12 Serious Adverse Reaction A.3.13 Traceability A.3.14 Quality System A.3.15 Storage A.3.16 Transplantation A.3.17 Allogenic Use A.3.18 Autologous Use A.3.19 Validation (“confirmation” or for equipment and environments, “qualification”) A.3.20 Audit

SECTION B GENERAL ORGANIZATIONAL REQUIREMENTS FOR TISSUE BANKS…………………………..….10

B.1 Institutional Identity B.2 Organisation of the Bank B.2.1 Supervisor B.2.2 Personnel B.2.2.1 Personnel Training B.3 Quality Management System B.4 Documentation B.4.1 General Information B.4.2 Traceability B.4.3 Data Protection and Confidentiality B.4.4 Archives B.5 Protecting the Health and Safety of Personnel B.6 Facilities and Equipment B.6.1 Facilities B.6.1.1 Access, cleanliness, maintenance and waste disposal B.6.1.2 Processing Facilities B.6.1.3 Storage Facilities B.6.2 Equipment and Reagents B.7 Waste Disposal B.8 External Collaborations B.8.1 Collaborating with Other Facilities B.8.2 Monitoring B.8.3 Collaborating with Other Banks

SECTION C DONOR SELECTION……………….17

C.1 Introduction GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 3 of 55

C.2 Expression of willingness to make Cadaveric Donation C.2.1 Expression of Acceptance of Cadaveric Donation C.2.2 Consent for Retrieval from a Living Donor C.2.3 Documenting Expressed Will C.3 Anonymity C.4 Certification of Death C.5 General Donor Eligibility C.5.1 Assessing Donor’s General Eligibility C.5.2 Exclusion Criteria for an Ineligible Tissue Donor C.5.3 Assessing Paediatric Donors C.5.4 Specific Exclusion Criteria for Various Tissues C.5.4.1 Ocular Tissue C.5.4.2 Musculoskeletal Tissue C.5.4.3 Valves C.5.4.4 Vessels C.5.4.5 Skin C.5.4.6 Amniotic Membrane C.6 Physical Examination of the Donor C.7 Autoptic Examination C.8 Tests for Communicable Diseases C.8.1 Laboratory Analysis C.8.2 Storing Blood Serum C.8.3 Obligatory Serological Tests C.8.4 Follow-up for Living Donors C.8.5 Autologous Donor C.8.6 Serological Reports C.9 Donor Documentation

SECTION D PROCUREMENT……………………………25

D.1 Personnel D.2 Protocol and Procedures D.3 Procurement Facilities and Quality Procedures D.4 Donor D.5 Time Limits for procurement D.6 Procedures for Musculoskeletal Procurement D.6.1 Procurement Procedures D.6.2 Culture Exams D.6.3 Procurement from a Living Donor D.7 Procedures for Ocular Tissues Procurement D.7.1 Corneas Procurement D.7.2 Eyes procurement D.8 Procedures for Valves Procurement D.8.1 Procurement Procedures D.8.2 Living Donors D.9 Procedures for Vascular Procurement D.9.1 Procurement Procedures D.9.2 Cadaveric Donors D.9.3 Living Donors D.10 Procedures for Skin Procurement D.10.1 Procurement Procedures D.10.2 Procurement Sites D.11 Procedures for Placenta Procurement D.11.1 Procurement Procedures D.12 Preservation of Tissues Immediately Following Procurement D.13 Labelling Containers D.14 Reconstruction of the Cadaver D.15 Final Procedures D.15.1 Accompanying Documentation D.16 Transport GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 4 of 55

SECTION E PROCESSING TISSUES………………………..34

E.1 Receiving Tissues at the Bank E.2 Recording Documentation E.3 Distinguishing Among Tissues E.4 Processing Procedures E.5 Culture Media, Reagents and Materials E.6 Ocular Tissue E.6.1 Evaluating Tissue Suitability E.6.1.1 Suitability Criteria for Corneas Used for Perforating Keratoplasty E.6.1.2 Criteria for Suitable Corneas for A nterior Lamellar Keratoplasty E.6.1.3 Criteria for Suitable Corneas for Endothelial Keratoplasty E.6.1.4 Criteria for Suitable Scleral or Corneal Tissue for Tectonic Use E.6.2 Methods of Preserving Cornea E.6.2.1 Short Term Cold Preservation E.6.2.2 Long Term Hot Preservation E.6.2.3 Lyophilisation E.6.2.4 Preserving in Ethanol E.6.2.5 Dehydration E.7 Musculoskeletal Tissue E.7.1 Dimensions of Samples E.7.2 General Information E.7.3 Fresh Small Osteochondral Allograft Segments (Joint Cartilage) E.7.4 Cryopreserved Tissue Retrieved Aseptically and Not Sterilised E.7.4.1 Cryopreserved Osteochondral Allografts E.7.4.2 Cryopreserved Bone and Connective Tissue E.7.4.3 Expiry Dates of Cryopreserved Tissues E.7.5 Lyophilised Tissues: Methods, Controls, and Expiry Dates E.7.6 Simply Dehydrated Tissue: Methods, Controls and Expiry Dates E.7.7 Irradiated Tissues: Methods, Controls and Expiry Dates E.7.8 Tissues Sterilised with Ethylene Oxide: Methods, Controls and Expiry Dates E.7.9 Bone Demineralisation E.8 Vessels and Valves E.8.1 Sample Evaluation E.8.2 Microbiological Controls E.8.3 Disinfection E.8.4 Freezing Procedures E.8.5 Storage E.9 Skin Tissue E.9.1 Microbiological Controls and Maintaining Viability E.9.2 Processing E.9.3 Preservation of Skin Tissue E.9.3.1 Vital Skin Grafts E.9.3.1.1 Preserving Fresh Skin Grafts E.9.3.1.2 Preserving Frozen Skin Grafts E.9.3.2 Non Vital Skin Grafts E.9.3.2.1 Preservation in Glycerol E.10 Amniotic Membrane E.10.1 Isolating the Amniotic Membrane E.10.2 Disinfection E.10.3 Microbiological Quality Controls and Maintaining Viability E.10.4 Anatomical and Morphological Quality Controls E.10.5 Preservation Methods for the Amniotic Membrane E.10.5.1 Cryopreservation of the Vital Amniotic Membrane E.10.5.2 Freezing E.11 Preservation Time Limits E.12 Discarding Unsuitable Tissues E.13 Variations in Procedures

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SECTION F DISTRIBUTION OF TISSUES TO TRANSPLANT CENTRES…………44 F.1 General Information F.2 Preparing Tissue for Distribution to Transplant Centres F.2.1 Checking Documentation F.2.2 Inspection of Containers F.2.3 Packaging and Transport F.2.4 Labelling F.2.5 Accompanying Documentation F.2.5.1 General Information F.2.5.2 Requirements for Accompanying Documentation F.3 Documenting the Receipt of Tissue F.4 Storage After Distribution F.5 Tissue Returned to the Bank

SECTION G RECORDKEEPING AFTER TRANSPLANTATION…………………..47 G.1 Follow-up Documentation G.1.1 Documentation Regarding the Recipient G.2 Keeping Information Confidential

SECTION H MANAGEMENT OF SERIOUS ADVERSE REACTIONS AND EVENTS……… 48 H.1 General Information H.2 Responsibilities of Tissue Banks H.2.1 Procedures H.2.2 Notification H.2.3 Practices H.3 Responsibilities of the Regional Reference Centre H.4 Forms

SECTION I IMPORTING AND EXPORTING TISSUES………………….50 I.1 Importation I.1.1 Requirements for Foreign Banks I.1.2 Initiating the Importation Process I.1.3 Traceability I.1.4 Importation Procedures I.2 Exportation I.2.1 Requirements for Foreign Banks and Transplant Centres I.2.2 Initiating the Exportation Process I.2.3 Exportation Procedures I.3 Records from Importation and Exportation Activities GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 6 of 55

APPLICABLE REGULATIONS ………………………………..……………………………..52

ANNEX I CARDIAC TISSUE CLASSIFICATION………………….…….…..54

ANNEX II ARTERIAL TISSUE CLASSIFICATION……………………….....55 VENOUS TISSUE CLASSIFICATION

ANNEX III CLASSIFYING SEVERITY OF ADVERSE REACTIONS……..56

ANNEX IV CAUSE………………………………………………………………………..57

ANNEX V NOTIFICATION FOR SEVERE ADVERSE REACTIONS…….58

ANNEX VI NOTIFICATION FOR SEVERE ADVERSE EVENTS………….59

ANNEX VII RESULTS OF INVESTIGATIONS INTO SEVERE ADVERSE REACTIONS………..60

ANNEX VIII RESULTS OF INVESTIGATIONS INTO SEVERE ADVERSE EVENTS……………61

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SECTION A

A.0 OBJECTIVES AND ACTIVITIES

A.1 INTRODUCTION

These guidelines shall be applied to the procurement, donation, harvesting, management, processing, preservation and distribution of the following types of human tissue for transplantation: • ocular • musculoskeletal • cutaneous • vascular and valvular • amniotic membrane If patients undergo treatments regulated by other norms, these guidelines shall be applied to the donation, harvesting and monitoring of tissues and for the management of serious adverse events and reactions for the aforementioned types of tissue. Specific guidelines have been issued for pancreatic islets and hepatocytes. Requirements issued per Legislative Decree no. 191/2007 and Legislative Decree no. 16/2010 are valid for all tissue/cells. Tissue and cells utilised for autologous transplantation during the same surgical procedure do not fall under these present guidelines. These guidelines are mindful of national, European, and international provisions and scientific knowledge regarding safety and quality standards for the use of tissue for transplantation. They are revised every two years and occasionally modified for relative legislation, standards and new medical and scientific discoveries.

A.2. Definitions of Facilities Involved in the Process

A.2.1 Definition of a Tissue Bank The term Tissue Bank refers to any public hospitals or non-profit healthcare facilities that handle processing, preservation, storage or distribution of human tissue, as defined in Legislative Decree 191 of 6 November 2007. A Tissue Bank receives authorization through regional jurisdiction and operates in conformity with the present guidelines and applicable regulation.

A.2.2 Definition of a Harvesting Facility This definition refers to any facility where tissue is harvested from living or cadaveric donors. According to regulation, harvested tissue is transferred to a partnering Tissue Bank for processing, preservation, certification and distribution.

A.2.3 Definition of a Transplant Facility This term refers to healthcare facilities or hospital units that carry out the application of tissue on human patients.

A.2.4 Use of Tissue for Transplantation The use of human tissue for transplantation is permitted only after evaluation of suitability and safety by an authorised Tissue Bank where periodic inspections are carried out by the appropriate authorities working in conformity with regulation on quality and safety as provided by European and national directives. Transplantation of tissue without documentation released by a Bank, which certifies the tissue’s safety and quality and guarantees its traceability, is not permitted.

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A.3 Definitions

A.3.1 Procurement: the process that makes tissue available for transplant. It starts with the identification of a potential donor and ends with harvesting the tissue from a living or cadaveric donor.

A.3.2 Culture: within the context of this document it means the phase of processing in which the tissue is preserved under conditions able to ensure normal metabolic activity.

A.3.3 Preservation: the suitable combination of conditions that preserves tissue quality during specified periods of storage. • Cryopreservation: freezing with the use of a cryoprotectant via controlled cooling and preservation at temperatures suitable for maintaining required tissue properties. • Cryoprotectant: a chemical substance that protects the cell during the cooling phase, minimizing the negative effects of increased solute concentrations and the formation of ice crystals (i.e. dimethyl sulfoxide DMSO, glycerol). • Controlled cooling: the tissue and cells are placed in a controlled-rate freezer, which guarantees progressive, steady and reproducible cooling. • Cryogenic containers: vapour (-150˚C) and liquid nitrogen (-196˚C) preservation systems for preserving tissue and cells. • Freezing: the processing and management of tissue and cells at non-cryogenic temperatures to guarantee preservation. • Lyophilisation (freeze-dried): this consists in the removal of water, under vacuum conditions, from previously frozen tissue through a process of sublimation which consists in the direct passage from a solid phase (ice) to a gaseous phase (vapour) surpassing the liquid phase (water). Dehydration of the tissue impedes enzymatic activity and degeneration permitting preservation for long periods of time. • Glycerolisation: preservation procedure using 85%-87% glycerol in a refrigerator at +2˚C/+10˚C. This procedure does not allow for viable sustainability after processing. • Dehydration: preservation of corneal tissue using dehydrating substances. • Ethanol preservation: preservation in 70% ethanol.

A.3.4 Critical: potentially effecting the quality or safety of tissue and cells or is in contact with these.

A.3.5 Distribution: transportation and delivery of tissue for clinical use.

A.3.6 Cadaveric Donor: donor whose death has been documented according to Law no. 578 of 29 December 1993 and the Health Ministry Decree of 11 April 2008 and whose consent to organ donation has been ascertained.

A.3.7 Living Donor: living subject who consents to the donation of his/her own tissue for the purposes of either allogenic or autologous transplantation.

A.3.8 Serious Adverse Event: any negative event connected to the supply, management, processing, storage and distribution of tissue that may trigger the transmission of pathologies, death or conditions that put patients at risk for handicaps or disabilities or can prompt or prolong hospitalization or ill conditions.

A.3.9 Standard Operating Procedures (SOP): this document describes operational methods aimed at the correct execution of a process in an established, logical, detailed and reproducible format.

A.3.10 Processing: all operations tied to the preparation, modification, preservation and packaging of tissue destined for application on human patients. GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 9 of 55

A.3.11 Quarantine: the state of harvested tissues or tissue isolated during wait periods for data necessary to evaluate tissue suitability for transplantation/graft.

A.3.12 Serious Adverse Reaction: an unwanted response from the donor or the recipient, including communicable diseases connected to the supply or application of tissue, which triggers death, puts patients at risk for handicaps or disabilities, or prompts or prolongs hospitalization or ill conditions.

A.3.13 Traceability: the possibility to locate and identify tissue during each phase of supply, processing, management, storage and distribution to the recipient or place of disposal, including the ability to identify the donor and the Bank that receives, processes and preserves the tissue and the ability to identify those responsible for the transplantation of tissue on recipients. Traceability includes the ability to locate and identify all pertinent data regarding products and materials that have come in contact with the tissue.

A.3.14 Quality System: the organizational model aimed at quality management including all of the activities that are directly or indirectly conducive to quality.

A.3.15 Storage: preserving the product in adequate and controlled conditions until it is distributed.

A.3.16 Transplant: within the context of this document this term shall also be used as a synonym for graft, application and use on human patients.

A.3.17 Allogenic Use: tissue or cell harvesting and transplantation/application from one individual to another of the same species.

A.3.18 Autologous Use: tissue or cell harvesting and transplantation/application on the same individual.

A.3.19 Validation (“confirmation” or for equipment and environments, “qualification”): documented proof able that guarantees, with a high level of certainty, that certain procedures, devices or locations provided for a product correspond to predefined specifications and qualitative characteristics. A procedure is validated in order to evaluate if a system functions efficiently in relation to its foreseen purpose.

A.3.20 Audit: systematic and independent examination aimed at verifying if activities performed and results rendered for quality purposes are in accordance as previously established, and to check if they are efficient and suitable for reaching objectives.

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SECTION B

B.0 GENERAL ORGANIZATIONAL REQUIREMENTS FOR TISSUE BANKS

B.1 Institutional Identity

The Bank’s purpose statement must be clearly established and documented. It must have organization, structure and adequate operational procedures to perform activities for which it has been authorised.

The Bank must prepare and maintain a record of its activities in which the type and quantity of tissue received, checked, processed, stored and distributed, or otherwise utilised, must be reported. Tracking of the origin and destination of tissue distributed for transplantation must be kept on file.

The Bank shall present a report on its activities every trimester to the National Transplant Centre (a.k.a. NTC), or to the Regional Centre.

B.2 Organization Within the Bank

B.2.1 Supervisor Every Bank must have a supervisor who satisfies the following conditions and possesses these minimum qualifications: • a university degree in medicine or the biological sciences or equivalent university qualifications • at least three years of work experience in related fields.

The Bank’s procedures and regulations must be carried out under his/her supervision.

The Supervisor: • must assure that harvesting, processing, quality control, storage and distribution of tissue are performed in accordance with applicable legislation and these guidelines • must guarantee the validation of tissue and initiation of procedures necessary for adverse reactions and events.

If the Supervisor is not a medical doctor, one must be appointed for evaluating donor eligibility, the Bank’s clinical activities and clinical assessment of results for tissue that has been utilised.

For Eye Banks, it is suitable to consult with an Ophthalmologist.

In order to guarantee the Bank’s activities, a substitute or representative must also be identified in case of the Supervisor or Medical Supervisor’s absence, even if short-term.

B.2.2 Personnel The Bank’s operating team must include a sufficient number of personnel to perform all necessary work and guarantee continued service. Personnel must be qualified and dedicated to perform all necessary work. Personnel must be evaluated at regular intervals as specified in the quality system manual, in order to uphold the level of competency.

All roles occupied by personnel must be clear, documented and updated. All relative duties, competencies and responsibilities must be well understood and documented.

An organogram must be outlined which clearly identifies working relationships and correlated responsibilities. GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 11 of 55

B.2.2.1 Personnel Training All members of personnel must have adequate initial training and must keep their own competencies up to date with scientific and technical progress by participating in refresher courses, technical meetings and other informative programmes. All personnel must know and periodically review norms and procedures.

The training programme must be described in the Quality System manual and must assure that: • each member is competent to perform their entrusted duties • they understand the organization, Quality System manual and safety norms of the workplace • they are adequately informed about legal and ethical standards.

Completion of these training programmes and subsequent periodical verification of skills must be documented.

B.3 Quality Management System

The Bank must apply and maintain a documented system for quality management. A Supervisor must also be appointed who is not directly involved in the processing of tissue.

The Bank’s Supervisor must assure that all activities comply with legal requirements, the present guidelines and the institution’s Quality Management System manual.

The Quality Management System is comprised of a manual, policies and standard operating procedures (SOP), which describe all critical activities. Furthermore, it includes all other documentation that describes personnel training, management of instruments/devices, and qualifications for the workplace, and must include records of all actions and provide evidence of activities performed.

Procedures must guarantee standardisation of activities performed and traceability in all phases: coding, donor eligibility, supply, processing, preservation, storage, transportation, distribution and disposal, including all aspects of quality control and assurance.

Documentation and procedures must be periodically audited and updated by the Quality Supervisor to comply with any modifications to activities or norms and legal requirements. All modifications must be audited, dated, approved, documented and performed by identified personnel.

Documents must be organised in such a manner that chronology of modifications can be easily provided and assures that only the latest updates are in use.

Copies of manuals and procedures must be made available for all personnel and, upon request, for any persons authorised to inspect the Bank. A reference copy must be kept by the Bank’s Supervisor or by the Quality Management Supervisor.

Backup plans must be organised in case of termination or temporary suspension of the Bank’s activities. Stored tissue should be transferred to another identified Bank including all related data on traceability, quality and safety.

The Bank must organise a system of checking its own activities aimed at certifying the observation of procedures and regulation in order to assure constant, systematic and improved progress. Independent evaluations must be performed at least every two years by qualified and competent persons. Results and corrective interventions must be documented.

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In case of requested changes to quality and safety parameters, documented investigations must be performed and decisions must be made regarding possible corrective and preventative measures.

If unsuitable tissue is detected, relative procedures must be followed under supervision of the Bank’s Supervisor and results must be documented. Investigations, results and corrective measures must be documented. The efficiency of preventative and corrective measures is subject to specific evaluation.

B.4 Documentation

B.4.1 General Information All documentation must be confidential, accurate and complete.

All documents must be legible and indelible. They may be handwritten or typed using a validated system, including electronic support, in which case all security measures must be taken against external access and possible attacks by viruses. Furthermore, backup procedures must be performed to avoid the loss of any data, performed both daily and periodically.

The execution of each procedural phase (harvesting, preparation, lab testing, storage, distribution and transplantation) must be documented so that each step may be clearly traceable, as well as identification of the person who performed the work, including information on all relevant departments. Moreover, documentation must show the results of tests, as well as all related interpretations and all data regarding the products or materials that come into contact with the tissue. Materials, devices and personnel involved in all critical activity must be identified and recorded.

Documentation must be detailed so that each step performed may be clearly understood and must be made available upon request for inspection by authorised persons within limits regarding medical- legal confidentiality.

Access to documentation and data must be limited to subjects authorised by the supervisor, as well as any appropriate authorities who perform inspections.

All documentation regarding donor history and information on tissue processing must be made available upon request by the transplant surgeon, with exception of any information that infringes upon the donor’s confidentiality. Documentation attesting to tissue suitability (in terms of quality and safety) must accompany the tissue when distributed to Transplant Centres.

B.4.2 Traceability Each sample must be assigned an initial and a unique number, which is used to identify the tissue throughout all phases from harvesting to distribution and use. The initial and unique number must match the tissue with its donor. Each tissue distributed must be identified with a Single European Code (SEC), made up of the Donation Identification Sequence (DI SEC), assigned on a national basis by the Italian Transplantation Information System (SIT), and the Product Identification Sequence (PI SEC).

B.4.3 Data Protection and Confidentiality All necessary measures must be adopted to assure that all data collected, including genetic information and any access by third parties, remains anonymous so that neither the donor nor the recipient are identifiable.

Data protection and safeguarding measures must be adopted to prevent addition to, elimination of or unauthorised modification of stored information regarding donors and any other transfer of information. GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 13 of 55

No information shall be divulged that leads to donor identification without authorisation. The identity of recipients must not be revealed to the donor or his/her family and vice versa.

Access to documents must be limited to subjects authorised by the Bank’s Supervisor, as well as appropriate authorities in case of inspection and must comply with confidentiality measures.

B.4.4 Archives All records critical to quality and safety must be archived for at least 30 years after the tissue’s use, disposal or expiry.

All documentation regarding tissue that has not been processed must also be archived for 30 years.

B.5 Protecting the Health and Safety of Personnel

It is the Bank Supervisor’s duty to assure that compliance with Legislative Decree no. 81/2008 and related regulation is enforced in order to protect the health and safety of personnel.

Procedures on how to maintain a safe workplace must be present. These measures must adhere to European, national and local regulation.

Inherent risks due to the use and manipulation of biological materials must be identified and reduced as much as possible while maintaining an adequate level of tissue quality and safety.

B.6 Facilities and Equipment

B.6.1 Facilities Tissue Banks must provide adequate facilities for performing all necessary activities in compliance with parameters presented in these guidelines. The rooms and equipment must therefore be designed, positioned, built, adapted and maintained in such a manner as to ensure compliance with requirements. Layout and design must aim to reduce as much as possible the risk of error and prevent environmental and cross contamination. Access to Tissue Banks must be limited to authorised personnel.

B.6.1.1 Access, Cleanliness, Maintenance and Waste Disposal The Bank must provide written policies and procedures regarding access, cleanliness, maintenance and waste disposal.

B.6.1.2 Processing Facilities When certain activities include the processing of tissues and cells that come into contact with the environment, they must be performed in a location that guarantees a specific type of air quality and filtration in order to minimise the risks of contamination, including contamination among donations. The efficacy of these measures must be validated and controlled.

If tissue comes into contact with the environment during processing without being further subject to microbial inactivation methods, the number of particles and the number of microbial colonies in the air quality must be equivalent to Grade A according to European standards for good manufacturing practices ( Good Manufacturing Practice : GMP), annex 1 to Legislative Decree of 24 April 2006 no. 219 and Directive no. 2003/94/EC, with an adequate background environment for processing the following tissues/cells: • corneas or amniotic membranes that are utilised similarly to the cornea require at least a Grade D background environment GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 14 of 55

• cutaneous tissue and tissue used internally (e.g. vessels, valves, musculoskeletal tissue not subject to sterilisation) the background environment must be at least Grade B (at rest). Tissue that undergoes a final procedure for microbial inactivation may be processed in a background environment that is at least Grade C (at rest).

It is necessary to document and, hence, be able to demonstrate that the selected environment complies with quality and safety requirements. The processing environments and laminar flow hoods must be classified, updated and monitored in compliance with ISO 14644 and EU GMP Annex 1.

Entrance to processing rooms must be rigorously limited to persons directly involved or to visitors authorised by the Supervisor and must be accompanied by internal personnel. Entry must be preceded by dressing in specific garments that minimise contamination from external sources and protect personal safety. Procedures for the admittance of materials and personnel must be accurately reported in the SOP manual and be easily accessible.

Procedures for cleaning and sanitising rooms must be validated and include an adequate rotation of decontaminating cleaning products. The disinfectants and detergents used in Grade A and B rooms must be sterile before use.

Plans for routine and periodic detailed cleaning procedures must be defined and cleaning personnel must be adequately trained. Cleaning activities must be recorded including identification of the personnel responsible.

Sanitising and decontamination procedures must be validated for all of the different types of tissues that may be processed within the same processing room.

Tissues harvested from HIV, HBV or HCV-infected donors intended for autologous transplantation must be processed separately in the facilities conducting such practices. In addition, effective extraordinary cleaning procedures of all surfaces must be carried out using products with certified viral inactivation properties.

If the work environment is shared to conduct services other than those of the Bank, there will need to be written agreements in which responsibilities and duties are specified regarding the use and cleaning of these spaces. Personnel that are not employed by the Bank must be adequately instructed and trained.

If inspection results show that air quality levels do not fall within the intervals indicated for Grade A, a risk assessment must be documented for tissue that has been processed in these conditions. The environment must then be thoroughly sanitised and checked again.

B.6.1.3 Storage Facilities If tissue must be preserved, storage conditions necessary for maintaining required tissue properties must be defined. Critical parameters must be controlled and monitored.

Refrigeration devices and incubators containing tissues/cells must be fit for the use envisaged, and appropriate monitoring procedures of such devices must be adopted to ensure that the tissues/cells are maintained at the desired preservation temperature. Continuous temperature monitoring and recording, together with effective alarm systems must be put in place in all incubators, refrigerators, storage freezers and liquid nitrogen tanks in order to signal any circumstance in which conditions come close to or fall outside the predefined ranges. Storage areas must be organised where tissue is clearly separated and distinguishable from those that are quarantined, those suitable for distribution and those that must be discarded. If quarantined tissue and tissue suitable for distribution are stored in the same location/environment, storage containers must be used that are physically separate or safety dividers must be used inside of these. GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 15 of 55

Tissue preservation that must adhere to special criteria must, nonetheless, guarantee absolute separation among samples.

Cryopreservation with liquid nitrogen must comply with all the requirements laid down to ensure safety of personnel and correct tissue conservation in accordance with the current NTC (National Transplantation Centre) Guidelines for Tissue Bank cryobiological environments.

B.6.2 Equipment and Reagents The arrangement and maintenance of all equipment and the use and preservation of materials must correspond to their intended purpose and minimise every risk for recipients and/or personnel.

All critical technical equipment and devices must be identified, validated and periodically inspected and must preventatively undergo maintenance in compliance with the manufacturer’s instructions. The equipment management system is put in place to ensure that equipment characteristics and reliability remain unaltered over time.

Equipment or materials that affect critical phases of processing or storage (e.g. temperature, pressure, number of particles, level of microbial contamination) must be identified and kept under observation, monitored, have alarms checked and undergo adequate revision and repair to identify any malfunction or defects and guarantee that critical parameters constantly remain within acceptable limits. When possible, all equipment that has a critical function must be calibrated to certain reference parameters.

All equipment and instruments providing critical measurement functions must be calibrated to a specific reference parameter, where this is available. A maintenance plan must therefore be established listing all critical plant/equipment/systems and the specific checks to be carried out (e.g. calibration check on the basis of set accuracy and precision parameters).

New or repaired equipment must be checked during installation and validated before use. The results of all inspections must be documented.

All equipment considered critical needs to periodically receive maintenance, be cleaned, and disinfected.

Each piece of critical equipment needs to be equipped with a reference manual, including its function in compliance with regulation, along with detailed instructions on methods of intervention in case of malfunction or breakdown.

Operating Procedures must indicate in detail all specifics regarding critical materials and reagents. In particular, specifics for additives (e.g. solutions) and packaging materials must be defined. Critical materials and reagents must adhere to documented instructions and, in certain cases, provisions contained in Legislative Decree no. 46 of 24 February 1997 (implementation of directive 93/42/CEE concerning medical devices) and Legislative Decree no. 332 of 8 September 2000 (implementation of directive 98/79/CE relative to in vitro diagnostic medical devices). As a general rule, reagents must be sterile and adapted for human use. In cases where the use of reagents that do not correspond to these requirements is necessary, reasons for choosing them must be documented and the product’s safety must demonstrated.

B.7 Waste Disposal Hazardous waste must be collected with methods that minimise risks for the Bank’s personnel and the environment and must be consistent with European, national and local regulation (Presidential Decree no. 254 of 15/07/2003).

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B.8 External Collaborations

B.8.1 Collaborating with Other Facilities If the Bank is not equipped with all organisational needs and laboratory equipment necessary for optimal processing, packaging and distribution of tissue which allow for the best clinical use possible, it may refer to external facilities that adhere to applicable regulation for certain procedures, but not the entire process.

Referring to these facilities may also be indispensable in times of temporary malfunction in which the Bank itself cannot conduct all necessary operations. These types of collaborations shall be regulated by written agreements or contracts that specify the responsibilities expected of the external facility and list all relative procedures in detail.

Banks shall evaluate and select external facilities based on their abilities to respect these guidelines.

The Bank’s Supervisor must guarantee that all personnel from external facilities involved in activities performed for the Bank are aware of the content and regulations contained within these guidelines and will observe them.

B.8.2 Monitoring The Supervisor must be able to monitor activities of external laboratories and/or facilities, and acquire and archive their certifications for performing activities in perfect compliance with the content of these guidelines and the Bank’s manuals on procedures and quality.

B.8.3 Collaborating With Other Banks The Bank may distribute tissue, for which it has been authorised, from other national Banks and foreign Banks, with which legal agreements have been established.

The label applied by the Bank where the tissue has initially originated from must never be removed, altered or covered.

The original documents that accompany the tissue must be sent to the healthcare facility where the transplant will take place without any omissions or alterations.

If the Bank’s Supervisor deems it necessary to conduct further verification regarding the suitability of either the tissue or the donor, all documentation relative to these findings must be sent to the healthcare facility where the transplant will take place along with the original documentation.

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SECTION C

C.0 DONOR SELECTION

C.1 Introduction

Standard Operating Procedures must be defined for all phases of harvesting, from donor identification to the delivery of tissue to the Bank.

C.2 Expressing Will to Donate

C.2.1 Expression of Willingness to make Cadaveric Donation The expression of the willingness to make a cadaveric donation must be ascertained according to norms established by the applicable legislation. For the retrieval of tissue from cadaveric donors please refer to Law of 1 April 1999, no. 91 - Regulation on retrieval and transplantation of organs and tissue.

C.2.2 Consent for Retrieval from a Living Donor The person responsible for overseeing the donation process must guarantee that the donor has been adequately informed of all the aspects relative to donation and the retrieval process before actually donating. Information must be provided by trained healthcare workers capable of communicating in a clear and adequate manner while responding to all of the donor’s questions. Information shall include: aims and nature of retrieval, risks and consequences, analytical exams, recording and protection of donor’s personal information, medical confidentiality, and treatment goals.

Donors have the right to be informed of the results of tests performed to exclude the risk of any communicable infectious diseases. The mode of communicating any possible positive results must be established. Donors must be guaranteed confidentiality for the use of their information.

C.2.3 Documenting Expressed Will The expression of the intent to make a cadaveric donation must be documented along with the purposes for which tissues and cells may be used (including therapeutic use, clinical experimentation, or both) and any specific instruction relative to disposal if the tissues or cells are not used for the purposes for which they were designated.

C.3 Anonymity Documents and information relative to the donor are confidential and shall be stored and treated with respect to privacy for the donor and his/her family. Anonymity is guaranteed through the use of initials and identification codes to track tissues.

C.4 Certification of death The cadaveric donor’s death certificate is produced according to Law of 29 December 1993, no. 578 and the Ministry of Health Decree of 11 April 2008.

C.5 General Donor Eligibility A person’s eligibility to donate tissue should be based on their medical and behavioural history, clinical state, physical exam, results of serological tests performed on blood drawn and autopsy, if performed, with the goal of reducing the risk of transmitting pathologies from the donor to the recipient.

C.5.1 Assessing Donor’s General Eligibility An extensive collection of information regarding the potential donor’s medical/behavioural/sexual history must be gathered by competent personnel. GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 18 of 55

If the actual person responsible for selecting donors is not part of Bank staff, there must be written agreements on procedures to follow regarding donor assessment and the types of tissue and blood samples to retrieve in compliance with these present guidelines.

Questions regarding medical history posed in the form of a questionnaire or interview shall be directed to the donor if he/she is living. Otherwise, questions will be directed to their relatives, presiding physician, general practitioner or other reliable source, in case of cadaveric donation.

Information from medical charts and results of exams and lab tests must also be taken into consideration.

All documentation must also indicate the source of information.

Living donors must confirm the veracity of all information given.

C.5.2 Exclusion Criteria for an Ineligible Tissue Donor The following conditions are cause for total exclusion for tissue use: • unknown cause of death for cadaveric donors (tissue may be used for transplantation only after an autopsy has pinpointed the cause of death and the following points have been excluded) • disease of unknown aetiology – with the exception of donations in the presence of corneal sarcoidosis, amyloidosis, idiopathic pulmonary fibrosis • personal history or clinical or laboratory evidence of an active infectious disease including HIV, HBV, HCV or unknown aetiology of jaundice • subjects with risk factors for HIV, HBV or HCV: -subjects engaging in at-risk sexual behaviour within the last 12 months -prostitution within the last 12 months - intravenous, intramuscular or subcutaneous narcotic drug use or inhaling cocaine within the last 12 months -haemophilic subjects undergoing infusion of coagulation factors of human origin -percutaneous exposure or exposure to open wounds or mucous membranes potentially infected with HIV, HBV or HCV within the last 12 months -subjects in haemodialysis (longer than one month) for chronic kidney failure -subjects that have spent time in incarceration within the last 12 months -venereal diseases diagnosed or treated within the last 12 months -tattoos, piercing or acupuncture within the last 12 months, if not performed with sterile single-use materials -subjects who have had partners who are at risk of infection with HIV, HBV or HCV, as previously defined, within the last 12 months • systemic infections that have not been checked at the time of donation, including bacterial diseases, systemic viral infections, fungal or parasitic diseases, or serious local infections of tissue and cells destined for donation. Donors suffering from bacterial septicaemia may be evaluated and taken into consideration for donating ocular tissue, only if it is to be preserved via organ culture with the means of identifying any possible contamination.

• subjects with risk factors for prion disease: -subject or family member with Creutzfeldt-Jakob disease or variant GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 19 of 55

-Presence of dementia or chronic degenerative neurological diseases of unknown aetiology (e.g. Alzheimer’s disease, multiple sclerosis, amyotrophic lateral sclerosis, acute sclerosing panencephalitis, Parkinson’s disease, progressive multifocal leukoencephalitis) -subjects that have used derivatives of hypophysary hormones or allotransplantation of the dura mater or have undergone unspecified intracranial procedures -subject has undergone a surgical procedure or transfusion of blood or blood components in Great Britain during the time period from 1980-1996 • Reye’s syndrome • active or previous malignant neoplasm with exceptions for: -basal cell carcinoma -uterine cervical carcinoma in situ -carcinoma in situ of the vocal cords -bladder carcinoma in situ • for only corneal donors, malignant neoplasms are not considered exclusion criteria except in the cases of: -malignant neoplasms of hematopoietic origin -malignant neoplasms of the eye -subjects who have undergone transplants for organs, ocular tissue or xenotransplantation (biological and pharmaceutical products are excluded, as well as medical apparatus deriving from non viable cells and tissues) • ingestion or exposure to toxic substances that may be transmitted in harmful doses (e.g. cyanide, lead, mercury, gold) • pharmacological treatments with immunosuppressants which may render tests unreliable for viral markers • chemotherapy or radiation therapy, excluding corneal donors • autoimmune diseases including collagen diseases that may compromise tissue quality • vaccination with a live virus (measles, rubella, mumps, varicella, yellow fever, smallpox) within the four weeks preceding donation • risk of transmitting infectious diseases related to travelling or exposure to infectious agents, which are not excludable with extensive testing • haemopoietic disorders including: monoclonal gammopathy (MGUS are not included in the exclusion criteria, with the exception of bone tissue, unless this latter undergoes processing (lyophilisation and demineralization) to eliminate the haemopoietic cells), myelodysplasia, polycythemia vera and essential thrombocythemia.

C.5.3 Assessing Paediatric Donors In addition to criteria listed in the previous section, the following must also be considered:

Children born to mothers who are HIV-positive or match one of the other criteria for exclusion in section C.5.2 may not be qualified as donors until all risk of transmitting infectious disease has been definitively excluded. a) Children younger than 18 months who are born to mothers positive for HIV, Hepatitis B, Hepatitis C or HTLV, or at risk of contracting infection, or who have been breastfed by the mother within the last 12 months cannot be qualified as donors, irrespective of analytical tests. b) Children who are born to mothers positive for HIV, Hepatitis B, Hepatitis C or HTLV, or at risk of contracting infection, who have not been breastfed by the mother within the last 12 months and who have tested negative for HIV, Hepatitis B, Hepatitis C and HTLV after analytical and physical testing and reviewing their medical charts, may be qualified as donors. GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 20 of 55

C.5.4 Specific Exclusion Criteria for Various Tissues The following conditions may indicate reduced tissue quality and therefore must be acknowledged during donor selection and tissue evaluation.

C.5.4.1 Ocular Tissue • active eye infections • congenital or acquired eye diseases/disorders that may compromise the results of transplantation • laser or refractive surgeries: tissue may be suitable for endothelial transplantation

C.5.4.2 Musculoskeletal Tissue • significant osteoporosis (in case of donating large skeletal segments for structural support) • diseases, such as acromegaly and hyperparathyroidism, that include structural alterations of the bone matrix • subjects under 15 yrs old and over 65 yrs old for metaphyseal and epiphyseal segments for mechanical support • subjects over 55 yrs old for vital cartilage, osteochondral allotransplant and meniscus • subjects over 65 yrs old for tendon and fascia lata • subjects over 78 years for lyophilised and cancellous tissue.

C.5.4.3 Valvular Transplantation • certified epilepsy and undergoing treatment • anorexia and bulimia • genetic diseases causing valve pathology • frostbite • chronic alcoholism • cardiac valvulopathy of the aortic and pulmonary valves with moderate to severe regurgitation • previous cardio-surgical procedures on cardiac valves or vascular segments that are intended for retrieval • aortic dissection • direct and severe trauma of the retrieval area • pneumonia within the last 30 days without resolution • subject over 65 yrs old

C.5.4.4 Vessels • treatment with anti-epileptic drugs • anorexia and bulimia • Down, Marfan or Noonan syndromes • frostbite • chronic alcoholism • chronic peripheral obliterative arteriopathy (claudicatio intermittens) • arteritis • aneurism • diabetes mellitus type 1 (excluding for retrieval in the femoral-popliteal-tibial area) • long term corticosteroid therapy and/or with somatropin • previous vascular surgeries • advanced atherosclerotic lesions (atherosclerotic plaque calcification, ulceration or haemorrhage) • vessel trauma • arterial dissection • subjects over 70 years for veins, and over 50 for arteries GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 21 of 55

C.5.4.5 Skin • abrasions, acute extensive burns • clinically suspicious nevi, multiple nevi > 100 • presence of systemic diseases compromising the skin • skin infections, dermatitis, extensive local inflammatory pathologies, ectoparasitosis • subjects of < 14 yrs old and > 78 yrs old

C.5.4.6 Amniotic Membrane • pregnancy term < 35 weeks (33 weeks for twin births) • emergency C-section • untimely rupture of membranes • fetal malformations and/or pathologies confirmed via ultrasound • tinted fluids • donor or newborn with genetic disease: Down, Marfan or Noonan syndromes • diabetes type 1 or insulin dependent gestational diabetes • chronic alcoholism

C.6 Physical Examination of the Donor

Before harvesting tissue, the donor must undergo accurate physical examination.

Particular attention must be paid to the presence of: • venereal infections (genital ulcers, anal condylomas, etc.) • signs of infective risk factors (punctured blood vessels, tattoos, piercing) • cutaneous infections, dermatitis, local inflammatory skin infections, ectoparasitosis.

If signs are present upon examination that might indicate exclusion criteria for donation, further in- depth examination of medical history and adequate laboratory or instrumental tests must be performed in order to exclude the presence of contraindications to donation.

Comprehensive evaluations of medical history and physical examination must be signed by the physician responsible for assessing donor eligibility.

C.7 Autoptic Examination The cadaver donor of tissues, with the sole exception of cornea donors, must undergo autopsy or exploration of the thoracic and abdominal cavities during the tissue harvesting procedure to exclude the risk of diseases that could be transmitted with transplantation.

Written communication of the results must take place as soon as they are available and the Bank’s Supervisor or designated person must review them before the tissue is prepared for distribution, except for circumstances listed in clause 9 of the present section.

C.8 Tests for Communicable Diseases Potential donors must be tested in relation to communicable diseases in compliance with this document.

Tests must be carried out on blood samples from the donor. In case of cadaveric donation, all blood samples must be obtained as quickly as possible, preferably within 12 hours of the donor's death and, nevertheless, no more than 24 hours after, in order to reduce the degree of haemolysis within the sample.

Blood samples must be adequately identified. GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 22 of 55

If blood is drawn from a living donor or a cadaveric donor with heartbeat, samples must not be taken in proximity to active infusion sites.

If the donor has experienced significant loss of blood and has undergone blood transfusions, haemocomponents or infusion of plasma-expanders in the 48 hours prior to blood being drawn, tests cannot be performed on samples taken prior to the infusions. Results must be evaluated taking into account dilution. If dilution is greater than 50%, results are not reliable and the donor is therefore to be considered not eligible.

It must be specified in the tissue’s accompanying documentation that the sample does not exceed the levels of dilution indicated, otherwise all necessary parameters must be communicated in order to calculate said datum.

Alternatively, the test findings or the specimen sent for testing must be accompanied by a declaration that haemodilution is not present.

For living donors, blood samples are taken at the same time as the donation, or within an allowable margin of 7 days after the donation.

C.8.1 Laboratory Analysis If possible, tests must be performed in a routine manner, by an authorised laboratory accredited by the province or region, which uses CE marked diagnostic instruments. Tests used must be validated for compliance with current scientific knowledge. The following recommendations are indicated for laboratories to distinguish potential tissue donors: • use of computerised procedures for the transfer of results from measuring instruments to the laboratory’s IT management system, eliminating manual transcription of results • use of computerised procedures and/or graphic tools useful for highlighting results, which determine the absolute ineligibility of the donor (bold, capitals, underlining, etc.) • indication in the report of the numeric value produced by the instrument and the relative limits of positivity, as well as the positive/negative outcome • confirmation by 2 operators of the congruity of instrumental results with reported results, producing a report with two signatures

C.8.2 Storing Blood Serum The blood serum of all donors must be accurately identified and, if tissues have not been discarded, must be adequately sealed and preserved at a temperature less than -40˚C for at least 25 years after utilization of the tissue.

C.8.3 Obligatory Serological Tests Subjects are considered ineligible for donation and their tissue cannot be used for transplantation if they test positive for any of the following obligatory tests: • anti-HIV 1 and 2 antibodies • anti-HCV antibodies • Hepatitis B surface antigens (HBsAg)

Furthermore, it is obligatory to test for antibodies to Hepatitis B core antigen (HBcAb); if results are positive, and HBsAg results are negative, the risk of transmitting HBV must be excluded through adequate testing.

Investigation for TPHA must also be performed or other tests that detect anti treponema antibodies. Results must be evaluated as follows: GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 23 of 55

CLIA/EIA Total antibodies or TPHA/TPPA NEGATIVE = NO ANTIBODIES PRESENT = DONOR IS ELIGIBLE

CLIA/EIA Total antibodies or TPHA/TPPA POSITIVE = ANTIBODIES PRESENT therefore perform VDRL or RPR VDRLR/RPR NEGATIVE = PREVIOUS INFECTION = DONOR ELIGIBLE VDRLR/RPR POSITIVE = RECENT INFECTION = DONOR NOT ELIGIBLE

Anti-HTLV I and II antibodies must be tested for donors who live in high prevalence areas or if they or their parents come from such areas, or if their sexual partners come from such areas. If tests results are positive, their tissue cannot be used for transplantation.

Supplemental testing for specific tissues include: • Toxo IgM for amniotic membranes. If results are positive, the tissue cannot be used for transplant • CMV IgM for skin, cardiac valves and vessels and amniotic membrane. If results are positive, tests for CMV DNA must be performed (on nucleic acid of polymorphonucleates), if negative the donor is considered eligible • CMV IgG for skin. If results are positive, notification must be sent to the centre that made the request for tissue. • CMV Ig testing it is not mandatory in case of derma irradiated with a dose at least of 13 KGray. • CMV Ig testing it is not mandatory in case of decellularized valves and vessels

Further specific tests may be required if the donor comes from geographical areas where other infectious diseases are endemic.

If other tests are performed including NAT exams for HIV, HBV and HCV, in addition to obligatory serological testing, the time frame to consider for risk factors listed under clause 5.2 of this section may be reduced to 6 months.

C.8.4 Follow-up for Living Donors Tests for HIV 1 and 2, HCV and HBsAg antibodies must be repeated on living donors at least 180 days after donation. Alternatively, the blood sample taken at the time of donation or following the donation must be tested with nucleic acid amplification tests (NAT) for HIV, HBV and HCV (nevertheless, it is necessary to perform tests envisaged in clause C.8.3).

The results of these tests must be available before tissue can be released.

C.8.5 Autologous Donor It is necessary to perform at least the same basic lab tests for living allogenic donors. Any positive results do not necessarily prohibit preservation, processing and reimplanting tissues, cells or any other derivative produced, as long as it is possible to preserve them in isolated conditions in order to avoid any risk of cross contamination.

C.8.6 Serological Reports Serological and biomolecular reports must remain attached to the donor’s documentation.

C.9 Donor Documentation There must be a clinical chart for every donor that includes: • personal information (name, surname, date and place of birth) • age, sex, medical history and behaviour history (information gathered must be sufficient enough to compare with exclusion criteria when necessary) GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 24 of 55

• results of physical examination • formula for haemodilution, if applicable • consent form • clinical data, results of lab tests and results of any other tests • if necroscopic examinations have been performed, results must be noted in the clinical chart (if tissue and cells cannot be preserved for long periods of time, a preliminary oral account of examinations must be recorded and it must be noted in documentation that the autopsy is in progress). The virtual autopsy and/or exploration of the thoracic and abdominal cavities during the harvesting procedure shall be considered as diagnostic findings. GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 25 of 55

SECTION D

D.0 PROCUREMENT

D.1 Personnel Procurement is to be performed by personnel who have completed a training programme provided by a specialised medical team or by the Tissue Bank, which has been documented by the Bank or the RRC.

These trained technicians shall undergo regular monitoring and checks of their professional skills as indicated in point B.2.2.1.

A medical doctor shall be appointed head of the procurement team in accordance with the provisions of point D.4. The doctor in question need not take part in the procurement procedure.

D.2 Protocol and Procedures The Bank must define protocol for the procurement of tissues and cells and written agreements must be established including protocol, if the medical team is not employed by the Bank, for all types of tissues, cells or samples to be retrieved.

Procedures must include verification of the following: • donor’s identity • documentation relative to consent/expression of will • evaluation of selection criteria for donors • evaluation of lab exams.

Procedures for procurement, packaging, labelling and transportation of tissues/cells to the Bank must also be specified.

D.3 Procurement Facilities and Quality Procedures Facilities where procurement is performed must be acceptable including air quality that is equivalent to operating theatre standards, except for procurement of corneas and tissues that undergo sterilisation after processing at the Bank.

Procurement shall be conducted with aseptic procedures, in order to safeguard tissue properties and reduce the risk of bacterial contamination, especially when a final sterilisation phase cannot be applied to the product.

For living donors, particular attention needs to be given to the health and safety of the subject.

D.4 Donor Before procurement, the presiding physician must verify the identity of the donor. A record must also be kept of the procedures used for verification including identification of personnel performing such procedures.

He/she must also certify that all donor exclusion criteria have been cleared and the donor has expressed the will to donate.

The removal of invasive devices or instruments (cannulas, catheters, etc.) must be authorised by a pathologist or medical examiner.

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After all tissues have been procured from cadaveric donors, the body should be carefully reconstructed.

D.5 Time Limits for Procurement Procurement must be performed as soon as possible following death (or circulatory arrest for multi- organ donation).

For musculoskeletal tissue, skin, blood vessels and valves, samples must be procured within 12 hours of death. If the body is cooled within the first six hours after death, retrieval may be performed within 24 hours after death, or within 30 hours for musculoskeletal tissue.

The procurement within the first 6-8 hours is preferred for ocular tissues and, nonetheless, no longer than 24 hours after death.

The name of the procurement facility and the length of time passed between death and retrieval, as well as any time in refrigeration, must be included in documentation sent to the Bank.

D.6 Procedures for Musculoskeletal Procurement

D.6.1 Procurement Procedures Procurement must be performed with the same conditions and procedures as a surgical operation and follow procedural standards as indicated here. The medical team must be comprised of 3-4 people, depending on the type of procurement one of which must be the Medical Director. For cadaveric donors, depilation of the procurement site must be performed, as well as covering of the genitals and any other possible surgical incisions.

D.6.2 Culture Exams for Procured Segments Procured segments must undergo culture exams to detect any aerobes, anaerobes and fungi.

In the event of the tissues undergoing post-procurement processing in the Bank’s laboratories, culture exams are not compulsory at the time of procurement but can be carried out during the processing and post-processing phase.

D.6.3 Procurement from a Living Donor Procurement must be performed during surgery and samples must be taken for culture exams, as described in the previous paragraph.

D.7 Procedures for Ocular procurement

D.7.1 Corneas procurement Procurement procedures must be arranged with the Bank.

Generally the process continues as follows: after disinfecting the skin, a blepharostat is applied and 5% povidone iodine is used for disinfection, then proceed with 360˚ limbal conjunctival peritomy, pulling back the conjunctiva as much as possible and avoid having conjunctival tissue remain attached to the limbus.

A full thickness scleral incision is made, about 4mm from the limbus, around 360˚, without damaging the underlying uvea. Then, remove the sclerocorneal cupola avoiding distortions or folding as much as possible. After removing the lens and iris, place the removed cornea with scleral ring in a container with filled with a solution for preservation.

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Proceed to reconstruction, and cover the anterior segment with a plastic former and the conjunctiva. Close the palpebras and apply sutures as necessary. Remove any residues left from disinfecting solutions. Leave the donor’s head raised and check to make sure there is no bleeding.

D.7.2 Eye Procurement procedures must be arranged with the Eye Bank.

Generally the process continues as follows: after disinfecting the skin, a blepharostat is applied and 5% povidone iodine is used for disinfection, then proceed with 360˚ limbal conjunctival peritomy. Cut the rectus muscles avoiding damage to the sclera. After having dislocated the eyeball upwards, cut the optic nerve 5-10mm from the eyeball. Spray the eye with a sterile physiological solution and wrap it firmly with sterile gauze leaving the cornea exposed.

Place the eye in a container inserting some sterile rectangular cotton strips (at least four) between the container wall and the gauze. Once the strips are wet with physiological solution, they will swell and hold the eye firmly.

Proceed to reconstruction: insert a small ball of damp gauze in the orbit and cover with a plastic former and the conjunctiva. Close the palpebras and apply sutures as necessary. Remove any residues left from disinfecting solutions. Leave the donor’s head raised and check to make sure there is no bleeding.

D.8 Procedures for Valves procurement

D.8.1 Harvesting Procedures Harvesting must be performed using the same aseptic conditions and procedures used in cardiovascular surgery. If possible, the medical team must be comprised of 2 persons, one of which must be a surgeon.

D.8.2 Living Donors For patients undergoing a heart transplant, if the heart that is removed does not present valvular lesions, valvular tissues may be recuperated during cardiectomy.

D.9 Procedures for Vascular Segments procurement

D.9.1 Harvesting Procedures Harvesting must be performed using the same aseptic conditions and procedures used in cardiovascular surgery.

D.9.2 Cadaveric Donors Arteries to be harvested include: • arteries and veins of the thoracic and abdominal cavities • arteries and veins of the lower limbs

D.9.3 Living Donors Donations of vascular segments from living donors is possible when a vein retrieved during a saphenectomy is not discarded and may be sent to the Bank.

D.10 Procedures for Skin Procurement

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Procurement must be performed using the same aseptic conditions and procedures used in surgery. Per regulation, the medical team must be comprised of 2 persons, one of which is the procurement surgeon.

Procurement sites must undergo depilation as well as washing with water and detergent. All surfaces are then disinfected.

D.10.2 Procurement Sites Procurement tissue of suitable thickness should be performed in areas that are not visible in order to avoid disfigurement. This particularly includes: anterior and posterior thighs, upper limbs, back. Procurement should be avoided of: the head, neck, hands, perineum and genitals.

De-epidermised dermis (DED) should be procured preferably in the posterior area of the calf (only in men).

Dermis should be procured preferably from the dorsal area (lumbar region).

The label must indicate the donor code, region procurement, batch number, expiry date of the transport medium, type of tissue procured: Skin-DED-Dermis.

D.11 Procedures for Placenta Procurement

D.11.1 Procurement Procedures Placental tissues are procured during an elective caesarean section adhering to aseptic criteria and following the internal protocols of the Bank. After extraction of the fetus and excision of the umbilical cord, the placenta is extracted in a non-traumatic manner, avoiding massive rupture.

D.12 Preservation of Tissues Immediately Following Procurement Immediately after procurement, the operator shall position the tissue or organ, in aseptic conditions, into a sterile container of suitable size containing an acceptable solution for preservation.

Once closed, the container shall not be reopened; the tissue or organ contained inside shall only be removed by personnel of the Bank.

The container is stored at a temperature of +2°C/+10°C until it is to be transported.

Tissues that do not have to undergo further processing must be packed in three layers of bags made of a cryo-resistant material and closed in a suitable way to ensure the integrity of the packaging. In certain cases, the most external bag may be replaced by a rigid container.

D.13 Labelling Containers The container must display a label with: • the identity of the donor (name, surname, date of birth) or donation code • the type of tissue • the following must also be indicated either on the label or in accompanying documentation: - operating facility, if pertinent - date and time of the procedure - for autologous donors the label must indicate the phrase, “exclusively for autologous use” - description of the solution used for preservation during transport - any other necessary precautions

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The external container for transport must display a label that specifies: • the identity of the procurement centre (address, telephone) • identity of the receiving Tissue Bank (address, telephone) • for autologous donations, it must indicate the phrase “only for autologous use”

If third parties are handling transportation, labels must also indicate: • the human origin of the tissue destined for transplantation • description of conditions to be observed for transport and preservation and, if necessary, display the phrase “DO NOT IRRADIATE” • coding as per Ministerial memorandum no.3, 8 May 2003 • date and time of the beginning of transport.

D.14 Reconstruction of the Cadaver After all tissues have been procured from cadaveric donors, the body should be carefully reconstructed.

D.15 Final Procedures The surgeon, who coordinates procurement team, must write up a report noting the date, time, place of the procedure, the names of the operators, donor and the procurement sites. He must sign the document in a legible manner. The autopsy request must be noted for cadaveric donors.

D.15.1 Accompanying Documentation The tissues must be sent to the Bank with at least the following documentation: • procurement notes with the identification of the donor and name of the personnel who identified the donor • description of the tissues sent and any blood samples • for donor’s with no heartbeat, the date and time of death, preservation methods used for the cadaver (refrigerated, yes/no, potential refrigeration), except for cornea donors • batch/preservation solution used for tissues • declaration signed by the physician responsible for evaluating donor eligibility in compliance with these guidelines, including absence of any exclusion criteria for donation based on investigations of medical history and physical examination • results of serological tests, if available • consent for donation of cornea and consent for living donors • name and address of the Bank where tissues and cells are being sent

D.16 Transport Tissue must be sent to the Bank in the shortest amount of time possible and, in any event, in compliance with the timeframes and procedures defined by each Bank to ensure full traceability.

Tissue must be transported in such a manner that the integrity of containers is assured and the temperature is maintained at +2˚C/+10˚C, until further processing at the Bank.

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SECTION E

E.0 PROCESSING TISSUES

E.1 Receiving Tissues at the Bank Once tissue has arrived at the Bank, a documented evaluation must be performed to ascertain that the transport conditions, packaging, labelling and accompanying documentation comply with the present guidelines and with any other specific requests made by the Bank.

The evaluation must be carried out as defined in the SOP. Any variation from the SOP must be reported in proper internal records.

The Bank must provide documented procedures for the management and separation of unsuitable tissues or those with incomplete test results, in order to eliminate contamination risks for other processed and stored tissues and cells.

E.2 Recording Documentation The Bank must keep and record documentation attained and archive it for at least 30 years after confirmation of clinical utilisation of the tissue, or after its disposal.

Among other data, the Bank must record and keep the following: • documentation of expressed will, as described in clause C.2.3 • all necessary documentation regarding harvesting and donor selection, according to clause C.5.

E.3 Distinguishing Among Tissues Tissue from each donor must be processed and stored singularly in order to avoid contact or contamination between each sample and with tissues from other donors.

Each tissue shall be identified by a unique code given by the Bank to ensure full traceability. A Single European Code (SEC) must be attributed to each tissue, at the latest, prior to distribution for clinical use.

E.4 Processing Procedures Sterile instruments, aseptic procedures and acceptable practices must be used throughout all phases of processing and packaging in order to avoid contamination, the growth of microorganisms and to maintain cell viability. All processing phases must be performed in an environment that is microbiologically and climate controlled.

Critical processing procedures must be validated and shall not hinder upon the tissue’s suitability for the clinical use. Validation may be based upon studies performed by the Bank, on data from published studies, or, for fully consolidated processes, on retrospective evaluation of clinical results on tissues provided by the Bank. It must be proven that the validation process can be performed in a coherent and efficient manner at the Bank by its personnel.

Any time a microbial inactivation procedure is applied to tissues, the process must be specified, documented and validated.

All procedures must be documented in the SOP and must be performed in compliance with approved SOP. Any modifications to procedures must be validated and documented before any significant changes can be made. Processing procedures must be periodically evaluated in order to maintain optimal results. GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 31 of 55

E.5 Culture Media, Reagents and Materials Culture media, reagents, materials and containers utilised for harvesting, analysis, preservation and storage of tissues must be sterile and suitable for their intended purposes. These products must be accompanied by certificates of sterility and quality provided by the manufacturer with internationally approved standards. Otherwise, validation or documentation published in national or international papers or in approved guidelines must be provided.

The manufacturer, characteristics, batch number, code, date of production and/or expiry date (pertaining to commercially available products) for media and reagents must be recorded on every tissue’s chart. If reagents are produced at the Bank, the batch number, production and expiry dates, initials and validation must be recorded.

E.6 Ocular Tissue

E.6.1 Evaluating Tissue Suitability Ocular tissue suitability for transplantation is dependent upon its future surgical use. Evaluation criteria and tests used to verify suitability must conform to applicable standards for specific evaluation and preparation of tissues in relation to their final utilisation.

It is the surgeon’s duty to specify the type of tissue requested from the Bank in relation to intended utilisation.

Corneas for transplantation must be evaluated with at least one of the following methods: • slit lamp • specular microscope • optical microscope

Endothelial cell mortality must be evaluated for corneas preserved in culture.

The suitability of examined tissue for transplantation is still subject to donor evaluation and the results of serological investigations in accordance with the provisions presented in these guidelines.

E.6.1.1 Suitability Criteria for Corneas Used for Perforating Keratoplasty • endothelial cell density should be equal to or greater than 2,000 cells/mm² • endothelial cell mosaic is monomorphic and without marked pleomorphism or polymegathism • no signs of, or extremely reduced, endothelial dystrophy or degeneration (pigment deposits, guttae) • The corneal stroma is free from significant opacity, which would compromise transparency.

E.6.1.2 Criteria for Suitable Corneas for A nterior Lamellar Keratoplasty The corneal stroma is considered suitable if it is free from significant opacity, which would compromise transparency.

E.6.1.3 Criteria for Suitable Corneas for Endothelial Keratoplasty • endothelial cell density is equal to or greater than 2,000 cells/mm² • endothelial cell mosaic is monomorphic and without marked pleomorphism or polymegathism • no signs of, or extremely reduced, endothelial dystrophy or degeneration (pigment deposits, guttae).

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Scleral and corneal tissues are considered suitable when there are no signs of alterations and no inflammatory or degenerative pathologies are present (malacic areas or hyperpigmentation).

E.6.2 Methods of Preserving Cornea

E.6.2.1 Short Term Cold Preservation Preserving at hypothermic temperatures (+2˚/+10˚C) allows for short term storage of an isolated cornea. The longest amount of storage time allowed depends upon the characteristics of the culture medium used and is indicated on the information sheet of the product in use. If the Bank itself produces the medium, the maximum amount of storage time is indicated within the procedures for medium validation.

E.6.2.2 Long Term Warm Preservation This type of preservation allows for long term storage of an isolated cornea maintained at +31˚/+37˚C. Storage time may be prolonged more than two weeks. During this time microbiological tests must be performed on the preservation liquid as well as at least one examination of the cornea under an optical microscope to confirm its suitability.

E.6.2.3 Lyophilisation Lyophilisation allows for long-term storage of corneal lamella used for lamellar keratoplasty. Lyophilisation must be conducted via suitable and standardised procedures applicable to corneal tissue. Dehydrated tissues packaged in sterile containers may be stored at room temperature for up to 12 months, unless valid data indicates otherwise, for which sterility controls must be performed.

E.6.2.4 Preserving in Ethanol The sclera may be stored in 70% ethanol at room temperature for a maximum period of 12 months.

E.6.2.5 Dehydration Corneal tissue may be stored in silica gel for a maximum of 6 months at 4˚C.

E.7 Musculoskeletal Tissue

E.7.1 Dimensions of Samples Segments of osseous tissue for massive transplantation may be X-rayed with possibly 2 projections and a measurement system, in order to allow selection of the segment to be used based on its dimensions and characteristics.

E.7.2 General Information The following are current established methods indicated for processing musculoskeletal tissue. Other methods may be used if efficacy has been proven.

E.7.3 Fresh Small Osteochondral Allograft Segments (Joint Cartilage) All fresh small osteochondral allografts must be retrieved aseptically in the operating theatre.

They cannot be used on patients before the donor’s blood tests have been completed in compliance with these guidelines. Furthermore, bacteriological tests must be negative and donor suitability must be approved by the Medical Director or other assigned person.

E.7.4 Cryopreserved Tissue Retrieved Aseptically and Not Sterilised

E.7.4.1 Cryopreserved Osteochondral Allografts Osteochondral allografts are retrieved using aseptic techniques in the operating theatre. A cryopreservation solution may be added to treat the cartilage’s surface before freezing. Bone which has been retrieved and adequately packaged is generally frozen by exposure to temperatures equal GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 33 of 55 to or less than -80˚C, otherwise they may be subject to controlled freezing temperatures using liquid nitrogen and computerised techniques.

E.7.4.2 Cryopreserved Bone and Musculoskeletal Connective Tissue After aseptic retrieval in the operating theatre, the bone or connective tissue (costal cartilage, fascia lata, tendon or meniscus) that is to undergo cryopreservation is situated in an environment with temperatures equal to or less than -80˚C within 12 hours. A cryopreservation solution may be added to treat the cartilage’s surface before freezing. Further tissue processing (e.g. washing, cutting, decontamination) must be performed aseptically.

E.7.4.3 Expiry Dates of Cryopreserved Tissues Tissues that are cryopreserved at temperatures equal to or less than -80˚C may be stored for a period of up to 5 years, after which they will be discarded according to applicable regulation. Cryopreserved tissues ready for use must not be frozen again once thawed.

E.7.5 Lyophilised Tissues: Methods, Controls, and Expiry Dates Lyophilisation is a preservation method but does not encompass sterilisation. Sterilisation must be attained either using aseptic protocol or performing additional sterilisation techniques.

After standardising lyophilisation procedures, a quality control programme must be envisioned and documented to monitor the process. Tissues dehydrated through lyophilisation must be stored at room temperature.

Every cycle must be clearly documented, including the duration, temperature and vacuum pressure of each phase.

Significant samples must be tested for residual water content. Lyophilised tissues that are vacuum packed have an indefinite shelf life, however it is advisable to store tissue no longer than 5 years, unless supporting data exists to extend this time.

E.7.6 Simply Dehydrated Tissue: Methods, Controls and Expiry Dates The use of simple dehydration (evaporation) of tissues as a means of preservation must be controlled in a manner similar to that used for lyophilisation. The temperatures for simple dehydration must be less than 60°C.

Every dehydration cycle must be monitored for temperature levels during the procedure. After dehydration, select samples must be tested for residual humidity.

Dehydrated tissues have the same expiry date as lyophilised tissues.

E.7.7 Irradiated Tissues: Methods, Controls and Expiry Dates Commercial and hospital irradiation facilities are available for gamma ray, ionising irradiation. The minimum recommended dose for bacterial sterilisation is 25 kGray.

Viral inactivation depends upon numerous factors and no specific dose can be recommended. However, it must be validated if possible, and in any case justified by tests, literature or specific evidence. The protocol used must be validated taking into consideration the initial microbial load and must be carried out by facilities following good irradiation practices and in possession of the official authorisation required under law.

Sterilisation by means of ionising radiation must be documented. Processing records must include the name of the facility and the resulting dosimetry for each batch. A unique batch number must be assigned and added to the tissues’ documentation.

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Tissues sterilised through irradiation have the same expiry date as frozen or lyophilised tissues.

E.7.8 Tissues Sterilised with Ethylene Oxide: Methods, Controls and Expiry Date Caution must be practiced when using ethylene oxide since its residues may have toxic effects on musculoskeletal allografts, as has already been demonstrated in literature.

In following appropriate processing practices, tissues are placed in containers that are permeable to ethylene oxide and exposed to mixtures of ethylene oxide gas as suggested by the manufacturer’s guidelines. It may be necessary to formulate an individualised protocol, depending on the nature of the samples that must be sterilised.

A Quality Control programme must demonstrate that equipment complies with specific requirements for temperature, humidity and gas concentration for the period selected. During sterilisation with ethylene oxide, proper aeration procedures must be followed to allow for the removal of ethylene oxide residues and/or its by-products.

Chemical indicator strips must be included in each batch. Validated procedures must be in progress for each tissue batch to certify that sterilisation has been completed. Monitoring of the residual level of chemical products and by-products must be performed on select samples from each batch of finished tissues.

Sterilisation with ethylene oxide has no influence on the expiry date.

E.7.9 Bone Demineralisation Different methods and procedures are available and acceptable for the production of demineralised bone. Quality controlled reagents must be used. Residual calcium levels must be determined for each method. E.8 Vessels and Valves Tissue must always be maintained at a temperature of +2/+10°C until the preparation phase, which should take place if possible within 12 hours (maximum 48 hours for valves and 72 hours for vessels) upon arrival at the bank. Tissues are prepared according to standard surgical procedures for isolating cardiac valves and vascular segments.

E.8.1. Sample Evaluation The characteristic dimensions of each tissue sample are reported including a description of the tissue and any possible lesions, as well as macroscopic anatomical evaluation with reference to classification models in Annexes I and II.

E.8.2 Microbiological Controls One or more samples of all suitable tissues are to be selected to undergo microbiological evaluations, which shall also be performed on the fluids in which they are transported. Each bank must provide a list of pathogens in their operating procedures that call for tissue to be discarded if found prior to disinfection.

E.8.3. Decontamination Each tissue is put into a decontamination solution in a sterile container labelled with a univocal code for each sample.

The composition of the disinfecting fluid, the temperature and time limits for each sample are to be defined by the protocol for disinfection of each individual bank. The efficacy of the disinfecting solution must be validated (based on tests performed by the actual Bank, or on data from published studies, or on consolidated procedures after retrospective evaluation).

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At the end of the time limits for disinfection, samples shall be selected for each suitable tissue for microbiological evaluation, which shall also be carried out on the disinfecting solution.

E.8.4. Freezing Procedures All suitable tissues will be frozen after passing the disinfection stage. Preparation for freezing must be done in the same sterile conditions and with the same precautions used in earlier stages. Each tissue is placed within a cryo-resistant sterile bag along with a quantity of freezing solution predefined by each Bank’s protocol, which may also involve the use of a nutrient medium and a cryoprotectant agent (e.g. DMSO in percentages determined by the tissue type). Each bag should be clearly identified and must state the type of tissue content, the freezing date/ the expiry date and the internal univocal code. Freezing is performed using a programmable cryogenic freezer, which trigger a controlled drop in thermal conditions caused by liquid nitrogen vapours.

E.8.5. Storage Cryogenically frozen tissues are stored at temperatures varying from -140°C and -185°C in storage containers utilising liquid nitrogen. Tissues used for transplantation should be stored no more than 5 years from the date of freezing.

E.9 Skin Grafts

Skin tissue grafts are lifesavers in severe burn cases. Generally, skin and DED are temporary medication measures. Dermis, also called Decellularized Dermis, is a permanent treatment, providing an excellent scaffold that can be applied in a range of surgical procedures.

E.9.1. Microbiological Controls and Maintaining Viability Microbiological evaluations must be carried out on skin tissue at least before the last packaging and, nonetheless, before freezing in order to verify that sterility has not been compromised during the stages of retrieval, temporary storage and transport. No samples shall be cleared for distribution if their microbiological tests show growth (regardless of isolated examples). The microbiological tests on both donor and harvested, processed and distributed skin tissue shall be carried out in full compliance with the Bank’s SOP.

Further tests must be carried out following freezing on surrogate samples in the event of the dispatch for transplantation of cryopreserved skin tissue.

Skin grafts destined for freezing or cryopreservation must undergo viability assessments via microscopic and culture evaluations or tests (i.e. MTT, RN, CFE). These investigations must be performed before freezing and at thawing to validate tissues before use.

E.9.2 Processing

On arrival at the Bank, skin tissue is registered and subsequently processed in compliance with the specific preservation procedures laid down by the Bank. Tissues may be stored in a refrigerator at +2°C/+10°C for a period not exceeding 72 hours.

Skin and DED undergo the same processing-preservation procedures.

Dermis undergoes a separate processing-decellularization-preservation procedure.

Skin tissue must be placed in a washing saline solution to eliminate unwanted residues, and subsequently in a decontamination medium for a pre-determined length of time.

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Microbial inactivation procedures that tissues are subject to must be specified, documented and validated. If antimicrobial solutions are used, the skin must undergo thorough and validated washing to remove as many antimicrobial residues as possible that may invalidate microbiological quality tests later on.

E.9.3 Preservation of Skin Grafts

E.9.3.1 Vital Skin Grafts

E.9.3.1.1 Preserving Fresh Skin Grafts Fresh skin must be stored in a refrigeration system that is electronically monitored, with permanent temperature recordings. It is required to have an alarm system that activates when temperatures exceed the permitted limits. The skin must be stored in suitable sealed containers (hypothermic storage) at a temperature of +2°C/+10°C for no longer than 72 hours.

E.9.3.1.2 Preserving Frozen Skin Grafts A cryoprotectant solution must be used for both freezing and cryogenically freezing skin to minimise any damage to tissues. Skin and DED must remain in a cryoprotectant solution for the time necessary to ensure adequate penetration before cryopreservation while maintaining a suitable temperature for refrigeration, in compliance with the Bank’s SOP.

Skin frozen at -80°C may be stored for a maximum period of two years, and for up to 5 years in liquid nitrogen. Cryopreserved skin that proves to be contaminated after microbiological testing may be thawed in compliance with current techniques and undergo glycerolisation.

E.9.3.2 Non Viable Skin Grafts

E.9.3.2.1 Preservation in Glycerol For long-term preservation of non viable skin, a solution of 85%-87% glycerol is used. Skin is placed in suitable sterile containers or bags using aseptic techniques at a temperature of +2°C/+10°C for a maximum of five years. Glycerolised skin is preserved in a solution of 85%-87% glycerol for a maximum period of five years at +2°C/+10°C.

E.9.3.2.2 Decellularized Dermis

Dermis is decellularized and preserved in various ways: frozen, glycerolised or lyophilised. After a pre-clinical experimental phase in vitro and in vivo, the Banks then proceed to produce, preserve and distribute tissue in compliance with their validated SOP. The preservation timeframes must be documented and validated in compliance with the facility’s SOP.

E.10 Amniotic Membrane

E.10.1 Isolating the Amniotic Membrane The placenta, umbilical cord and the attached amniotic sac are washed with a sterile saline solution (or sterile saline solution mixed with antibiotics and/or antifungals). The amniotic membrane is then prepared according to the Bank's internal procedures, which have been documented and validated.

E.10.2 Decontamination If the amniotic membrane undergoes disinfection, the composition of the disinfecting solution, the temperature and the amount of time samples should be treated must be predefined by each Bank’s protocol. Efficacy of the disinfecting solution must be validated (based on tests performed by the GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 37 of 55 actual Bank, or on data from published studies, or on consolidated procedures after retrospective evaluation).

At the end of the time limits for decontamination, samples shall be selected for each suitable tissue for microbiological evaluation, which shall also be carried out on the disinfecting solution.

E.10.3 Microbiological Quality Controls and Maintaining Viability Microbiological evaluations must be performed throughout all phases of processing for amniotic membranes that are destined for cryopreservation without final disinfection. Each bank must provide a list of pathogens in their operating procedures that call for tissue to be discarded if found prior to disinfection.

Vital amniotic membranes must undergo specific checks for viability, which must be defined within the Bank’s procedures. Furthermore, each bank must also establish cell viability levels, below which tissues cannot be distributed as vital tissue.

E.10.4 Anatomical and Morphological Quality Controls Dimensional and structural characteristics of each strip of amniotic membrane must be identified (epithelial side - stromal side). A description of the vital amniotic membrane tissue and any possible lesions are to be recorded, as well as macroscopic anatomical evaluation as per the Bank’s internal operating procedures.

E.10.5 Preservation Methods for the Amniotic Membrane

E.10.5.1 Cryopreservation of the Vital Amniotic Membrane Every strip of suitable amniotic membrane is to be cryopreserved within 48 hours from retrieval of the placenta in the operating theatre. Preparation for cryopreservation must be done in the same sterile conditions and with the same precautions used in earlier stages.

Every strip of amniotic membrane is placed in an appropriate sterile cryo-resistant bag/container together with a quantity of freezing solution predefined by each Bank’s protocol, which may also involve the use of a nutrient medium and a cryoprotectant agent.

Cryopreservation is obtained using a programmable cryogenic freezer, which triggers a controlled drop in thermal conditions caused by liquid nitrogen vapours. Freezing curves used for amniotic membranes must be previously validated and defined in the Bank’s internal procedures.

Records must be kept for every cryopreservation cycle. Amniotic membranes under cryopreservation may be stored for a maximum period of 5 years.

E.10.5.2 Freezing Each suitable fragment of non vital amniotic membrane is to be packaged in a container or medium and preserved by placing it in a freezer with a maximum temperature of -80°C until the end of the retention period (expiry date). Otherwise, it may be cryopreserved. A programmable freezer may be used but is not essential since it is not necessary to preserve cell viability.

Frozen amniotic membrane may be preserved for a maximum period of 2 years. Membranes may be preserved only for 5 years even if at -80°C and when a cryoprotectant has been added.

E.11 Preservation Time Limits Maximum preservation time limits refer to the time period from the moment of retrieval until utilisation.

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E.12 Discarding Unsuitable Tissues Tissues recognised as unsuitable for any reason must be discarded. The disposal must be recorded, as well as the motivations for doing so.

E.13 Variations in Procedures Risk assessments must be performed and documented with approval of the supervisor to decide the fate of stored tissues before introducing any new selection or screening criteria for donors, or any significant changes to processing.

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SECTION F

F.0 DISTRIBUTION OF TISSUES TO TRANSPLANT CENTRES

F.1 General Information

Transplant Centres may request tissues for transplant/grafting purposes from a Bank that has been identified by their own region or from facilities that have established agreements with the region and are authorised for distributing specific tissues.

The Bank will arrange to supply the tissue complying with distribution criteria and all priorities listed in their own operating procedures. If the Bank does not have the tissue requested, it will search other Banks in the same country. If there is no availability in all of the national facilities, the Bank may request the tissue from a foreign Bank as described in section I.

If there is no Bank within the regional territory of the Transplant Centre that has been identified by the regional authority and no agreements have been stipulated with Bank’s of other regions, the Centre shall send requests to the Regional Reference Centre (RRC), who will send the request to another Italian Bank. If the tissue is not available within national territory, the Transplant Centre shall inform the RRC and ask for international intervention, through an Italian Bank.

Distribution criteria and assignment priorities must be described in the procedure manual and the Bank must be in compliance.

F.2. Preparing Tissue for Distribution to Transplant Centres

F.2.1. Checking Documentation Before any tissue packages can be removed from quarantine and sent for distribution, the Bank’s Medical Director must again control all documentation relative to donor eligibility (medical history, adherence to donor selection criteria, autoptic data when available, blood and microbiological tests and harvesting documentation).

The Bank’s Supervisor must also verify the completeness and conformity of all documentation for processing and tissue quality controls. The tissue destined for distribution must correspond to the predefined standards.

Operations involving the distribution of tissue must be described in the procedures manual.

F.2.2. Inspection of Containers A final inspection of the container and its labelling must be performed to check for integrity, the correct placement of the tissue inside, the data on the label and the identification of the content. Labels for tissues used by the Tissue Bank may not be removed, altered or covered.

F.2.3. Packaging and Transport Transportation of containers is performed with regards to the predefined environmental conditions, based on the type of tissue transported and the storage conditions. Tissues may be sent to transplantation centres at the storage temperature or already in a state fit for use, after thawing and washing, in sealed sterile containers.

Tissues may be sent to transplant centres in sealed sterile containers ready for use, after having been thawed and washed.

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For transportation outside of the facility in which the Bank is located, it is necessary to have a sealed external packaging, suitable to maintain storage or transportation temperatures as indicated in the Bank’s operating procedures. Containers and packaging must be validated for suitability.

If delivery is entrusted to third parties, agreements must be written to guarantee that required conditions are maintained.

F.2.4 Labelling Each single package of tissue must present: • name of the Tissue Bank • type of tissue • tissue identification code (including the batch, if applicable) • tissue dimensions if applicable • expiry date • if tissue is positive for disease markers it must be labelled “biohazard” • for autologous donation it must be labelled “exclusively for autologous use” and include patient identification • if tissue is has a defined consignee, include the identification of the receiver. For the last two scenarios, the name of the receiver may me indicated in the accompanying documentation rather than on the label.

An outside label must be applied on external packaging with the following indications: • name, address and telephone number of the Tissue Bank • Transplant Centre identification where the tissue is being delivered, including the address and telephone number • human origin of the tissue to be transplanted and the phrase, “HANDLE WITH CARE” • for vital tissues, the phrase, “DO NOT IRRADIATE” • any recommendations for transport conditions • the possible presence of CO₂ or other cryogenic gases inside the packaging.

F.2.5 Accompanying Documentation

F.2.5.1 General Information All tissues must be accompanied by documentation that is to be included into the clinical records of the transplant recipient. Specific instructions must be included with the tissue requiring particular treatment.

F.2.5.2 Requirements for Accompanying Documentation The accompanying documentation must contain all the information described on the labelling as well as the following data: • tissue origin (specify if tissue comes from another country) • information and test results from donor screening • storage temperature or time and date of thawing, if tissue is delivered thawed • instructions for opening the package and container and for possible thawing and restoration of the tissue • any transport/storage medium, or possible residues from preserving agents/solutions or from additional processing (e.g. antibiotics, ETOH, ETO, DMSO, etc.) • any sterilising/inactivation procedures used • any negative results from microbiological tests performed • total quantity and number of containers being sent • results from residual cell viability tests • expiry date from the time the package is opened or content is restored. GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 41 of 55

Accompanying documentation must specify that: • each tissue must be used for only one patient • the receiving healthcare facility is responsible for correctly preserving the tissue from the moment of delivery until transplantation • the Transplant Centre may only store tissue received from the Bank temporarily while awaiting transplantation • the receiving healthcare facility must inform the Bank of the tissues destination (date/place of transplant, transplant surgeon, identification and clinical data of the recipient), in order to guarantee traceability • the receiving healthcare facility is responsible for the keeping all records necessary to guarantee traceability of tissues sent • the Bank must be immediately informed of any adverse reactions in the recipient correlated to the transplant.

F.3 Documenting the Receipt of Tissue Confirmation of receipt and use of the tissue must be sent to the Bank by sending documentation with the date of receipt and information about the destination (recipient’s name, date of the procedure, transplant surgeon) along with the signature of the doctor responsible for the transplant.

This declaration implies the suitability of tissue at the moment of delivery. This documentation also guarantees traceability of the tissue.

F.4 Storage After Distribution Tissues must be used within the expiry date indicated by the Bank and must be preserved according to methods and time limits designated by the Bank in written procedures. If frozen materials are distributed, they must be accompanied by instructions for thawing.

The Transplant Centre is responsible for supplying storage conditions adequate for thawed tissue.

F.5 Tissue Returned to the Bank If tissue is returned to the Bank, the Supervisor must evaluate and decide upon its final destination in compliance with written operating procedures.

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SECTION G

G.0 RECORDKEEPING AFTER TRANSPLANTATION

G.1 Follow-up Documentation Each Bank will issue forms to collect follow-up information of the recipient.

G.1.1 Documentation Regarding the Recipient All forms will include: • the patient’s name, date of birth, sex • diagnosis of the illness cured with transplantation • the facility where the procedure was performed • identification code of the tissue used (indicated in the tissue’s accompanying documentation) • surgical procedures performed • date of the surgery • name and surname of the surgeon who performed the transplant • clinical data of the recipient in compliance with instructions from the Bank’s scientific committee or in adherence to any possible indications given by the respective RRC.

G.2 Keeping Information Confidential All information related to reactions to the transplant, or at follow-up and any other information that may be of interest to the Bank for epidemiological purposes and for safety, must be utilised by the Bank in ways that no indications shall appear that may lead to the identification of the recipient according to Article 18 paragraph 2 of Law 91/99.

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SECTION H

H.0 MANAGEMENT OF SERIOUS ADVERSE REACTIONS AND EVENTS

H.1 General Information Serious reactions affect living donors and recipients of human tissues and cells.

Both the Retrieval Centres and the Transplant Centres are affected by such a situation. Annex III shows how to classify the severity of reactions. Adverse reactions that are not considered serious do not need to be reported. Serious adverse events may occur at any stage of the process: from the time a donor is chosen to the actual transplant. The event may take place at any facility that is involved in the process.

Other than those situations that enter under the definition of serious adverse event described in section A.3, the following situations are also considered events to report: • the release of unsuitable tissues destined for transplantation (even if they have not been used) • involvement of other patients or donors in sharing procedures, services, providers or donors • the loss of irreplaceable autologous tissue or allogenic tissue that is highly compatible (for a specific patient) • the loss of a significant amount of allogenic tissue, regardless of its compatibility.

H.2 Responsibilities of Tissue Banks

H.2.1 Procedures All Banks must provide instructions for reporting adverse events and reactions to Transplant Centres and to all facilities that are involved in phases throughout the process. Each facility must have written procedures to comply with this requirement.

Each Bank must establish procedures for communicating with the Regional Reference Centre (RRC) and the National Transplant Centre (NTC) for all serious adverse reactions/events, and to include all of pertinent information available and the results of investigations aimed at ascertaining the causes and outcomes.

Each Bank must have quick, written verifiable procedures that allow for the recall of any deliveries of products that may be associated with serious adverse events/reactions.

H.2.2 Notification Banks must immediately notify the RRC and the NTC’s Tissue and Cell Department of any adverse events/reactions via e-mail or fax. They must also be notified of all provisions adopted to protect other tissues or cells that may be affected or that have been distributed for transplantation/grafting.

Notifications must be immediately sent out even if the involvement of other tissues/cells in the reaction is only suspect, just as if there were only a hypothetical risk tied to such an event.

If a Bank is alerted of a serious adverse event, they must notify the RRC and NTC in order to guarantee safety for any other possible recipients of tissues or organs, regardless if the event itself does not have any influence over the Bank’s own stock of tissues/cells.

H.2.3 Practices Banks must conduct investigations to discover the cause and implications of serious adverse events/reactions for which it has received information. Annex IV shows a method of attributing the cause, which is to be used in investigations. GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 44 of 55

The Bank’s Supervisor must evaluate the possible necessity of recalling tissues and initiating and coordinating any further necessary actions. The Bank must have an effective recall procedure that includes a description of responsibilities and practices to be performed. These practices are to be performed within a defined period of time and call for the identification of tissues and cells affected and retracing of their paths.

Every donor that may have contributed in causing the reaction in the recipient must be identified. All tissues and cells originating from this donor must then be recuperated. Consignees and recipients of tissues and cells retrieved from the same donor must be informed of possible risks that they may be exposed to.

Upon request, the NTC may provide support to the Tissue Bank or the RRC during the investigation.

Outcomes of the investigation must be communicated to the RRC and the NTC as soon as they are available.

Tissue Banks must keep written reports on research conducted with regards to adverse reactions, including outcomes, follow-up and corrective actions.

H.3 Responsibilities of the Regional Reference Centre The RRC must notify all the facilities involved in writing and adopt all necessary preventive and corrective measures. It must also maintain contact with the NTC and provide support in investigations, as necessary.

H.4 Forms Banks must send all pertinent information available and adopted provisions to the RRC and the NTC using the forms in annexes V or VI, and then the results of investigations using annexes VII or VIII.

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SECTION I

I.0 IMPORTING AND EXPORTING TISSUES

All importing and exporting of tissues must be in compliance with Italian Ministerial Decree of 10 th October 2012, amended by Ministerial Decree of 29 th July 2015, and with all eventual subsequent amendments.

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APPLICABLE REGULATIONS

1. DIRECTIVE 2004/23/EC OF THE EUROPEAN PARLIAMENT AND COUNCIL OF 31 March 2004 on the definitions of quality and safety for donations, harvesting, controls, processing, preservation, storage and distribution of human tissues and cells.

2. COMMISSION DIRECTIVE 2006/17/EC of 8 February 2006 putting into effect Directive 2004/23/EC of the European parliament and council regarding certain technical indications for the donation, harvesting and control of human tissues and cells.

3. COMMISSION DIRECTIVE 2000/86/EC of 24 October 2006 putting into effect Directive 2004/23/EC of the European parliament and council regarding indications for traceability, notifications of serious adverse reactions and events and certain technical indications for coding, processing, storage and distribution of human tissues and cells.

4. COMMISSION DIRECTIVE 2012/39/EU of 26 November 2012, which modifies Directive 2006/17/EC regarding certain technical indications for tests performed on human tissues and cells.

5. Legislative Decree 6/11/07 no. 191 putting into effect Directive 2004/23/EC on definitions in regulation for the quality and safety of donating, harvesting, controls, processing, preservation, storage and distribution of human tissues and cells.

6. Legislative Decree 25/01/10 no. 16 putting into effect Directives 2006/17/EC and 2006/86/CE, putting into effect Directive 2004/23/EC regarding technical indications for the donation, harvesting and control of human tissues and cells, as well as for indications relative to traceability, notifications of serious adverse reactions and events and certain technical indications for coding, processing, preservation, storage and distribution of human tissues and cells.

7. Legislative Decree 30/05/12 no. 85 Modifications and additions to Legislative decree 25 January 2010, no. 16, putting into effect Directives 2006/17/EC and 2006/86/EC, putting into effect Directive 2004/23/EC regarding technical indications for the donation, harvesting and control of human tissues and cells, as well as for indications relative to traceability, notifications of serious adverse reactions and events and certain technical indications for coding, processing, preservation, storage and distribution of human tissues and cells.

8. Law 01/04/99 no. 91 Regulation on harvesting and transplanting organs and tissues.

9. Law 12/08/93 no. 301 regulations regarding harvesting and grafting cornea.

10. Legislative Decree 81/08 and other regulations relative to protecting health and safety in the workplace.

11. Legislative Decree 196/03 Code regarding the protection of personal data.

12.Decree 10/10/12 Procedures for the exportation or importation of human tissues, cells and reproductive cells destined for use on humans.

13. Presidential Decree 15/07/03 no. 254 Regulation in reference to provisions for the management of biomedical waste in compliance with paragraph 24 of Law 31 July 2002, no. 179.

14.UNI EN ISO 9000:2005: Quality management systems. Basis and Terminology.

15. UNI EN ISO 9001:2008: Quality management systems. Requirements. GUIDELINES FOR THE Code Tissue Guidelines PROCUREMENT, PROCESS AND Revision 01 DISTRIBUTION OF TISSUES FOR Date 9/2016 TRANSPLANTATION Page p. 47 of 55

16.UNI EN ISO 9004:2009: Managing an organization for long term success – An approach to quality management.

17.UNI EN ISO 14644-1:2001 Clean rooms and associated controlled environments. Classification of air cleanliness.

18.UNI EN ISO 14644-2:2001 Clean rooms and associated controlled environments - Specifications for evaluation and surveillance to demonstrate continued conformity with UNI EN ISO 14644-1.

19.UNI EN ISO 14644-3:2006 Clean rooms and associated controlled environments part 3: evaluation methods.

20.UNI EN ISO 14644-4:2004 Clean rooms and associated controlled environments. Part 4: design, construction and start-up.

21.UNI EN ISO14698-1:2004 Clean rooms and associated controlled environments- Controlling biocontamination part 1: general principles and methods.

22.UNI SPERIMENTALE 10127-1:1992 Guide for determining calibration intervals for measuring devices: general criteria.

23.UNI SPERIMENTALE 10127-2:1992 Guide for determining calibration intervals for measuring devices: intervals advised for linear, angular and geometric measurements.

24.US GUIDANCE FOR INDUSTRY Sterile Drug products produced by Aseptic processing – Current Good Manufacturing Practice (September 2004 Pharmaceutical CGMPs).

25.EC GUIDE TO MANUFACTURING PRACTICE - REVISION TO ANNEX 1. Title: Manufacturing of Sterile Medicinal Products – February 2008.

26.Common Approach for Definition of Reportable Serious Adverse Events and Reactions as put forth in the Tissues and Cells Directive 2004/23/EC and Commission Directive 2006/86/EC Version 1.0 (2009)”.

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ANNEX I

CARDIAC TISSUE CLASSIFICATION

CLASS 1 • valve leaflets are unusable, leaflet coaptation tests are negative • calcification of leaflets and outflow • valvular regurgitation • severe damage due to dissection or harvesting • bicuspid with congenital defects • intimal lesions along entire aortic conduit • valves are to be completely discarded

CLASS 2 • valve leaflets are abnormal, leaflet coaptation tests are negative • presence of atheromas on over 30% of valvular and conduit surfaces • areas of intimal calcification • valves are unusable, except possibly for monocuspid preparation

CLASS 3 • valve leaflets are normal, leaflet coaptation tests are positive • no presence of calcification or atheroma on valve leaflets • mitral valve with atheroma over 15-30% of valvular surfaces • conduit is free of calcification and atheroma covers 15-30% of surfaces • small areas of contusion are present but not in proximity to the valve ring

CLASS 4 • valve leaflets in excellent condition, leaflet coaptation tests are positive • no signs of damage • fenestration over <2% of surfaces • no calcification or atheroma • mitral valve shows small atheromatous areas over <5% of surfaces and no signs of intimal lesions of the conduit with atheroma over <15% of surfaces

CLASS 5 • Tissue and valve leaflets are anatomically perfect

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ANNEX II

ARTERIAL TISSUE CLASSIFICATION

CLASS I: Unsuitable • aneurysm or presence of blisters • diffusion of transmural calcifications (> 30%) • areas of fairly extensive intimal ulceration • negative pressure test

CLASS II: Suitable with reservations • large arteries • Areas of fibrocalcific thickening • segments of calcified atheroma (<15%) protruding into the lumen without ulcerated lesions • positive pressure test • fenestration <5% of the total surfaces

CLASS III: Suitable • anatomically perfect • small collections of fibrolipid material • positive pressure test

VENOUS TISSUE CLASSIFICATION

CLASS I • varicose tissue • areas of collapse > 30% of total surface areas • infiltrating or periadventitial parietal fibrosis (post-phlebitic) > 15% • negative pressure test

CLASS II • segment has thickened, is not dilated, with some limited ectasia

CLASS III • No apparent lesions

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ANNEX III

CLASSIFYING SEVERITY OF ADVERSE REACTIONS

Minor clinical consequences that do not require Not serious hospitalisation and/or do not cause disabilities or consequences for the donor or recipient An adverse reaction that has prompted:

Hospitalisation or prolonged hospital stay and/or Serious • persistent or significant inability or disability • medical or surgical procedures to avoid permanent injury or reduced function • severe communicable infection Following retrieval of tissues/cells or their application: • a major procedure became necessary (vasopressors, At risk of death intubation, transfer to intensive care) to avoid death • an infection was transmitted that put the patient in danger of death Death Death

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ANNEX IV

CAUSE

There is not enough data to determine NA the cause. 0 There is evident data, beyond a Excluded reasonable doubt, that other causes have Unlikely attributed to the adverse reaction. There is not sufficient data to attribute 1 the reaction to the application/retrieval Possible of tissues/cells or other causes. 2 Data clearly attributes the reaction to Likely tissues/cells. Data shows, beyond a reasonable doubt, 3 that the adverse reaction is attributed to Certain tissues/cells.

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ANNEX V

NOTIFICATION OF SERIOUS ADVERSE REACTIONS

Early notification of suspected serious adverse reactions

Tissue Bank Notification number Date of notification (year/month/day) Subject involved (recipient or donor) Date and place of harvesting (if the person suffering a reaction is the donor) or of use on human patients (year/month/day) Unique ID of the donation Date of alleged serious adverse reaction (year/month/day) Type of tissues and cells involved in the alleged serious adverse reaction

Type(s) of alleged serious adverse reaction(s)

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ANNEX VI

NOTIFICATION OF SERIOUS ADVERSE EVENTS

Early notification of suspected serious adverse events

Bank Notification number Date of notification (year/month/day) Date of Serious Adverse Event (year/month/day) Serious adverse events that may Specify affect the quality and safety of tissues Defect of Equipment Human Other and cells due to deviations relative to: tissues failure error (specify) and cells Harvesting

Monitoring

Transport

Processing

Storage

Distribution

Materials

Other (specify)

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ANNEX VII

Results of Investigation Into Serious Adverse Reactions

Tissue Bank Notification number Confirmation date (year/month/day) Date of serious adverse reaction (year/ month/day) Unique ID number of the donation Confirmation of serious adverse reaction (yes / no) Change to the type of serious adverse reaction (yes/no) If yes, please specify Clinical results (if known) - Fully recovery - Mild sequelae - Severe sequelae - Death Results of the investigation and final conclusions

Recommendations for preventive and corrective measures

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ANNEX VIII

Results of Investigation Into Serious Adverse Reactions

Tissue Bank Notification number Confirmation date (year/month/day) Date of serious adverse event (year/ month /day) Assessment of underlying causes (in detail)

Adopted preventive measures (in detail)