Tuesday, September 19, 2017
Project co-funded by the European Commission in the framework of the 2nd Health Programme
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E.3.1. ACTIVITIES

E.3.1.1. Reception

See generic requirements in section A.3.3.1.

Information required concerning the donor and the acquisition of human material:

a)     Donor identification. The unique identification code of tissues and cells is attributed by the bank of tissues and cells or a national/international system;

b)    Donor sex and age;

c)     Donor type: living (domino)donor, deceased heart-beating organ donors, deceased non heart-beating (organ)donor, cadaveric (tissue) donor;

d)    Informed consent (living donors):  a copy of the original or signed consent result of the consultation of the national register;

e)     Identification of the recovery centre and the responsible person of donation and recovery;

f)     Personal medical history of the donor (absence of the exclusion criteria);

g)     Cause of death;

h)    Results of the clinical examination and autopsy in relevant cases ;

i)      Date and time of circulatory arrest (for deceased donors) ;

j)      Date and time of cooling of the body (for the non-heart beating donors);

k)    Place, date and hour of the recovery ;

l)      Description and identification of the recovered material (arteries, veins).

 

E.3.1.2. Access to the processing facilities

See generic requirements in section A.3.3.2.

E.3.1.3. Processing

E.3.1.3.1.General

See generic requirements in section A.3.3.3.1.

E.3.1.3.2. Processing methods
E.3.1.3.2.1. Processing of tissues and cells

In order to avoid the complementary warm ischemia time during the processing, the tissues must remain humid and have a low temperature (if possible below 10°C).

In, order to avoid further warm ischemia during the dissection of recovered blood vessels, the tissues must remain humid and the temperatures need to be low (as low as possible, below +10°C).

E.3.1.3.2.1.1. Processing of valves

During the heart dissection, the aortic and the pulmonary roots are separated and in some cases the mitral valve as well.  During this phase the morphological description, and measuring of the dissected allografts is performed, as well as functional testing of the valves.

The allograft needs to be described in detail; this description must mention all the tissue malformation and the iatrogenic lesions.

The functional tests, leaflet coaptation and level of leak, must be checked.  The results of this test must be noted in the allograft record.

The heart valves are sized on the basis of the internal diameter of the valva anulus, expressed in millimetres (mm).  The valve annulus may be neither distended nor distorted.

The length and the internal diameter of the arterial conduits (aortic, pulmonary trunk as well as the left and right pulmonary branches) must be mentioned as well.

GRADING

GRADE 1 (valves to be discarded):

Unusable valve leaflet;

Negative leaflet coaptation test;

Conduit and valvular leaflet calcification;

Valvular insufficiency;

Severe damage due to dissection or recovery;

Bicuspid congenital defects;

Intimal injuries along the aortic conduit.

 

GRADE 2 (unusable valves except for monocuspid preparation):

Abnormal valve leaflets;

Negative leaflet coaptation test;

Atheromas on over 30% of the valvular and conduit surface;

Calcification areas at intimal level.

 

GRADE 3

Normal valve leaflet;

Positive leaflet coaptation test;

Absence of calcification and atheromas on the valve leaflets;

Mitral valve with atheromas on 15- 30% of the valve surface;

Absence of calcification on the conduit and presence of atheromas on 15-30% of the surface;

Presence of small bruising areas but not in proximity of the valvular ring.

 

GRADE 4

Excellent valvular leaflet;

Positive leaflet coaptation test;

No damages;

Fenestrations on <2% of the area;

Absence of calcifications and atheromas; Small atheromatous areas on <5% of the mitral valve surface;

Absence of intimal injuries of the conduit of the conduit with atheromas on <15% of the surface.

 

GRADE 5

Tissue and valve leaflet anatomically perfect.

E.3.3.3.2.1.2. Processing of vessels

The allograft needs to be described in detail (aspect and consistence); this description must mention all the tissue malformation and the iatrogenic lesions.

The vessels are sized on the basis of the diameter, expressed in millimetres (mm) and the length in centimetres (cm).

 

GRADING ARTERIAL VASCULAR TISSUE

GRADE I; (Not eligible)

Aneurysmatic tissue or presence of blisters;

Transmural diffuse calcifications (>30%);

Extensive intimal ulceration areas;

Negative pressure test.

 

GRADE II: (Subject to evaluation)

Megaarteries;

Fibro-calcific thickening areas;

Segmental calcific atheromas (<15%)protruding into the lumen without ulcerative lesions;

Positive pressure test;

Fenestration on >5% of the total surface.

 

GRADE III: (eligible) Anatomically perfect;

Little collection of fibrolipidic material;

Positive pressure test.

 

GRADING VENOUS VASCULAR TISSUES

GRADE I:

Varicose tissue;

Failure areas on >30% of the total surface;

Parietal fibrosis infiltrating or periavventitial areas on >15%;

Negative pressure test.

 

GRADE II:

Thickened segment;

Not dilated;

Some limited ectasias

 

GRADE III: No apparent lesions

 

QUALITY CODES DEFINITION LIST, VALVES:

 

Acceptable for further processing:

Code 01: No visible morphological abnormalities

Code 02: Minimal atheroma in basal attachment of leaflet;

Minimal fibrosis in (basal attachment of) leaflet / vascular wall;

Fenestration(s) in otherwise perfect graft;

Petechiae in otherwise perfect graft.

Code 03: Minor atheroma in vascular wall (conduit);

Atheroma in < 1/3 of the basal attachment of the leaflet;

Fibrosis in < 1/3 of (the basal attachment of) the leaflet;

Fenestrations.

Code 04: Atheroma in the vascular wall;

Atheroma in < 2/3 of the basal attachment of the leaflet;

Fibrosis in < 2/3 (of the basal attachment) of the leaflet;

Fenestrations.

Code 05: Discrete atheroma in the vascular wall or the basal attachment of the leaflet;

Discrete fibrosis in (basal attachment of) leaflets;

Pinpoint calcification in vascular wall;

Minor adhesions of leaflets at the commisures;

Fenestrations.

* Codes 04 and 05 are not acceptable for aortic valves from donors ≥ 56 years of age.

 

Not acceptable for further processing:

Code 06: Extensive fibrosis or atheroma and/or calcification in vascular wall, calcification in leaflets, Major leaflet adhesion, fenestrations > 1/3 the length of free edge Rigid vascular wall (conduit) Large areas of detached intima / tears in vascular wall (conduit).

Code 07: Damaged during removal of the heart.

Code 08: Damaged during dissection of the heart.

Code 09: Incompetent valve.

Code 10: Other abnormalities (anatomical or procedural).

Code 11: Failure to meet one or more of the time limits adherence requirements detailed in SOP P-01.

 

QUALITY CODES DEFINITION LIST, VESSELS:

 

Acceptable*:

Code 01: No visible morphological abnormalities.

Code 02: Superficial atheroma, minor spots of fibrosis.

Code 03: Minor fatty streaks, minor fibrosis.

 

Not acceptable:

Code 06: Discrete atheroma: patches in > 1/3 of the surface area of the vessel wall;

Calcification.

Code 07: Damaged during removal of the aorta segment from the donor.

Code 08: Damaged during dissection in the Bank.

Code 10: Other abnormalities (anatomical or procedural).

Code 11: Failure to meet any of the time limits adherence requirements detailed in SOP P-01.

 

EVALUATION OF VALVES

Not acceptable valves:

Unusable valve leaflet

Calcification and extensive fibrosis in leaflets 

Negative leaflet coaptation test (Incompetent valve)

Valvular insufficiency

Major leaflet adhesion

Fenestrations > 1/3 the length of free edge

Conduit calcification

Intimal injuries along the aortic conduit (Large areas of detached intima / tears in vascular wall)

Rigid vascular wall (conduit)

Severe damage due to dissection or recovery

Other abnormalities (anatomical or procedural) e.g. Bicuspid congenital defects;

 

Not acceptable vessels

Discrete atheroma: patches in > 1/3 of the surface area of the vessel wall

Aneurysmatic tissue or presence of blisters

Transmural diffuse calcifications

Extensive intimal ulceration areas;

Aneurysmatic tissue or presence of blisters;

Transmural diffuse calcifications

Extensive intimal ulceration areas;

Negative pressure test.

Damaged during removal from the donor

Damaged during dissection in the Bank

Other abnormalities (anatomical or procedural)

 

E.3.1.3.2.2. Decontamination of valves and vessels

E.3.1.3.2.2.1. Decontamination of valves

Allografts are microbiologically sampled and cultured for aerobic and anaerobic germs as well as for fungi and yeasts before antibiotic incubation.

Samples on which analyses must be performed are:

a)     The transport medium at the beginning of processing

b)    Subvalvular (aortic and pulmonary) myocardial tissue

c)     End sample after decontamination and rinsing, before final packaging

Each bank should establish in its operating procedures a list of pathogens whose presence leads to the elimination of the tissue even though disinfection was successful.

Microorganisms that never be found in a cardiovascular tissue sample (pre-antibiotic or post-antibiotic sample)

Clostridium sp.

Clostridium perfringens

Clostridium tetani

Enterococcus sp

Flavobacterium meningiosepticum

Klebsiella rhinoscleromatis

Listeria monocytogenes

Neisseria gonorohoeae

Nocardia sp.

Pseudomonas aeruginosa or pseudomallei

Staphylococcus aureus (MRSA)

Salmonella sp.

Shigella sp.

Streptococcus pyogenes (group A)

Aspergillus spp.

Candida spp.

Mucor spp.

Penicillium spp.

Other yeasts and fungi

Mycobacteria (in an “at risk donor”)

 

Each tissue is placed in a disinfecting solution in a sterile container labelled with a specific internal code. The composition of the solution, the incubation temperature and the duration are defined by the bank’s standard operating procedures.

The tissues shall be incubated with antibiotics during 5 to 6 hours at 37 °C. An ampoule with 2 ml of the antibiotic cocktail is transferred to a solution of Medium 199 obtaining an endvolume of 100 ml (50 times dilution).

The antibiotic cocktail consists of:

Ciprofloxacin, 0.15 mg/ml

Amikacin, 0.6   mg/ml

Metronidazole, 0.6   mg/ml

Vancomycin, 0.6   mg/ml

Flucytosine, 1.5 mg/ml

Since there are different protocols of decontamination and different temperature conditions and duration, each tissue establishment would have to be free to decide the sterilization conditions of the allografts.

E.3.1.4. Quality control

The quality control tests on cardiovascular grafts should consider at least the following minimum quality criteria:

a)     Absence of transmissible disease agents and malignant cells;

b)    Functional competence;

c)     Good morphology (no fissures, congenital defects,  no/minimal calcification etc.);

d)    Anatomical suitability; accurate length of conduit and diameter of annulus;

e)     Tissue matrix structure intact;

f)     Biomechanical strength.

 

See generic requirements in section A.3.3.4.

E.3.1.4.1. Microbiological control
E.3.1.4.1.1. General principle

See generic requirements in section A.3.3.4.1.

E.3.1.4.1.2. Microbiological controls
E.3.1.4.1.3. Methods
E.3.1.4.2.Other controls

E.3.1.5. Packaging and labelling

See generic requirements in section A.3.3.5.

At the end of the time stipulated by the antibiotic schedule, the tissues are packaged for cryopreservation.

The cryobags used have to assure the nitrogen liquid resistance as well the isolation of the samples.

When the storage of the tissues are in liquid nitrogen two bags are mandatory in order to prevent a possible liquid nitrogen contamination if the bag breaks.

E.3.1.6. Storage/ preservation

E.3.1.6.1. General

See generic requirements in section A.3.3.6.1.

In order to assure the tissue quality, it is essential that the processing and preservation take as less time as possible, with a total ischemia time of 72 hours!

The maximum defined delays are 24 hrs for the start of recovery, 40 hrs for the start of processing and 48 hrs for the start of cryopreservation (starting from the moment of circulatory arrest in case of deceased donors). The moment of the death (or the circulatory arrest) that of recovery (in case of living donors), the beginning of processing and of the cryopreservation are indicated on the record of concerned tissue.

The processing should be performed within 24 hours of cardiac arrest/recovery and finish with cryopreservation at maximum 72 hours of cardiac arrest.

Any extension of these delays may eventually be accepted only after specific validation of the particular case.

 

E.3.1.6.2. Methods of preservation and storage

The standard operating procedures (SOP) describe the solutions, the duration and the temperature of incubation as well as the freezing parameters.

This information along with other information is communicated to the implanting surgeon at the moment of the allograft delivery or by means of a periodically distributed protocol.

E.3.1.6.2.1. Storage at +4°C

If the antibiotics incubation can not be directly followed by cryopreservation, the allografts may be stored at a temperature of +4°C in an appropriate solution (Medium 199) until cryopreservation (maximum is 40 hours after circulation stop). Any longer storage periods need to be subjected to thorough research to determine if the quality of the tissue is high enough to be used safely.

E.3.1.6.2.2. Cryopreservation and storage at -80°C

During the cryopreservation, the speed of the freezing with the intermediary temperature of the cryopreservation procedure (for the chamber as well as for the control tissue) must be recorded.  Storage at -80°C is acceptable for a short period (maximum 1 months).  Longer periods of storage at -80 °C are not acceptable.

This issue is for discussion. In the literature is mentioned that you can store up to 6 months maximally.  However, the majority of TE-s are accepting actually to 3 months.  For me this modification can be acceptable!

Allografts to be shipped need to be kept in dry ice, at the temperatures between -70°C and -80°C (-76°C).

The shipment and temporary storage of the vascular allograft in the dry ice at -70°C to -80°C is acceptable with the condition that the allograft remains in these (controlled) temperature conditions until use.  It may not be re-stored in the initial storage temperature (-170°C or lower) and its storage duration is limited to maximum 1 month from the moment of removal from the initial storage conditions

E.3.1.6.2.3. Cryopreservation and storage in Liquid Nitrogen

Tissues are cryopreserved by using a controlled rate freezer.  With a specially programmed protocol (implementing various freezing parameters) the tissues reach a temperature of -100 °C.  During the cryopreservation protocol, the speed of the freezing must be recorded, as well as any consistencies that might have occurred during the freezing run.  After cryopreservation the frozen tissues are transferred to a temperature monitored liquid nitrogen tank.

Freezing must happen in controlled rate freezer, decreasing the temperature very slowly (-1°C) down to -40°C (to avoid recrystallization at the level of -23°C) and then rapidly (-5°C) down to -1000°C.

The storage in the vapour phase of liquid nitrogen (between -170°C and -190°C) (-150°C and -187°C) is acceptable for a period of 5 years.

After longer storage periods the tissues expire and need to be discarded. Another option is using these expired tissues in validated research experiments.

Thawing, removal of cryoprotection medium (dilution) and re-establishment of the isotonic state of the vascular allograft are of critical importance in order to guaranty the integrity of the cryopreserved tissue.  Also, among the other information on tissues and cells, the accompanying record of the cryopreserved tissue must contain the detailed protocol of thawing and dilution, tissue reconstitution and a list of the necessary material.

 

E.3.1.6.3. Storage area

See generic requirements in section A.3.3.6.3.

E.3.1.7. Documentation and release

E.3.1.7.1. General

See generic requirements in section A.3.3.7.1.

E.3.1.7.2. Release

See generic requirements in section A.5.1.6.2.

E.3.1.7.3. Processing file contents

See generic requirements in section A.3.3.7.3.

E.3.1.7.4. Availability for inspection

See generic requirements in section A.3.3.7.4.

E.3.1.7.5. Traceability

See generic requirements in section A.3.3.7.5.

E.3.1.7.6. Archive

See generic requirements in section A.3.3.7.6.

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