Tuesday, August 22, 2017
Project co-funded by the European Commission in the framework of the 2nd Health Programme
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D.3.1. ACTIVITIES

D.3.1.1. Reception

See generic requirements in section A.5.1.5.

The recovered skin should be transferred to the tissue establishment as soon as possible after recovery or must be put in the first processing fluid direct after recovery.  After the skin has been received at the tissue establishment the processing of the skin should commence within 72 hours of recovery having taken place.  In expectation of the processing, the skin should be kept at 2-8°C, in a physiological medium with sufficient buffering capacity.

D.3.1.2. Access to the processing facilities

See generic requirements in section A.5.1.2.

D.3.1.3. Processing

D.3.1.3.1.General

See generic requirements in section A.5.1.3.

The recovered skin is processed to allow longer storage periods until transplantation in suitable patients.  The methods used must be validated and must kept up with the scientific state-of-the-art in due time.

Critical processing procedures must be validated and may not inactivate human tissues and cells, or make them harmful to the recipient.  This validation can be on the basis of studies undertaken by institutions themselves, on details of published studies or, where accepted processing procedures are concerned, retrospective evaluation of clinical results achieved with human tissues and cells provided by the establishment.

D.3.1.3.2. Processing methods
D.3.1.3.2.1. Processing of tissues

Unsuitable pieces of skin are cut away and irregular edges trimmed after which the remaining section can be measured in order to calculate the surface area of the skin allograft in question. The dimensions of the skin allograft are expressed in square centimetres (cm²).

 
 
D.3.1.3.2.2. Decontamination of tissues

Unsuitable pieces of skin are cut away and irregular edges trimmed after which the remaining section can be measured in order to calculate the surface area of the skin allograft in question. The dimensions of the skin allograft are expressed in square centimetres (cm²).

D.3.1.3.2.3. Sterilisation of tissues

Whether or not the skin may be sterilised is dependent on the clinical application and the quality requirement set. Conventional sterilisation methods (for example radiation, heat, gas and immersion in chemical agents) have a detrimental effect on the structural integrity and viability of the skin.

D.3.1.3.2.4. Inactivation with regard to prions

Specific inactivation with regard to prions is possible if the viability of the skin allograft is not a prerequisite for the intended clinical use.  In all other cases all the products used that constitute a risk should be accompanied by the necessary certificates in order to reduce the risk to a minimum.

D.3.1.4. Quality control

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

a)   Absence of transmissible disease agents and malignant cells;

b)   Correct thickness;

c)   Integrity (to provide barrier function);

d)   Accurately sized pieces and clean edges;

e)   Cell viability (optional, depending on the intended application);

f)    Sterile if labelled as such.

See generic requirements in section A.5.1.4.

D.3.1.4.1. Microbiological control
D.3.1.4.1.1. General principle

See generic requirements in section A.5.1.4.1.

D.3.1.4.1.2. Microbiological controls

1. Skin is an inherently non-sterile tissue and is also not sterile at the time of recovery.  This makes it a complex matter to draw up standards that are functionally achievable and ethically responsible within the framework of the tissue transplant standards.

2. With the bacteriological and mycological evaluation of the skin allografts every effort must be made to ensure the absence of relevant pathogens (Table 1 and others) and to determine an appropriate threshold of asepticism (bio burden).

3. Pathogens may at no time be allowed to occur on skin allografts, irrespective of their density.  The bacteriological and mycological investigations may however reveal the presence of low density micro-organisms, which form a part of the inherent resident skin flora.

4. Sampling making use of a swab is not permitted.  A correlation exists between the positive culture and the quantity of skin used.  The taking of representative samples (± of a minimum 2 cm ²) is thus indispensable.

5. All processes are carried out on representative samples in accordance with standard clinical-microbiological techniques over a period of 14 days (this is still in discussion in Belgium, 7 days may be also appropriate).  The samples are cultured in an appropriate fluid enrichment media for aerobic and anaerobic bacteria, yeasts and fungi and any growth in the medium is monitored on a daily basis.  Should growth be observed (turns opaque/murky, turbid) within 7 days then the tissue is rejected.  Should the growth be observed after day 7, then the micro-organisms should be identified according to standard clinical-microbiological techniques.  The tissue is rejected when pathogenic germs are identified.

6. The choice for this culture media and the protocol for the microbiological tests fall under the auspices and responsibility of the recognised laboratory to which the tissue establishment sends the samples to be tested.

7. Should antibiotics, or other products with an antimicrobial action, be made use of in the transportation and/or conditioning process, then these should be removed (for example by copious rinsing) or inactivated prior to the bacteriological and mycological inspections.  Validated protocols concerning the rinsing or inactivation processes must be in place at the tissue establishment.

8. Hemocultures have value as a source of information but have more than likely no added value with respect to the quality and safety of the skin allografts.

 

 

 

 

Table 1. Germs that definitely must not occur on skin allografts

 

Acinetobacter baumannii

Beta-hemolytic streptococci

Burkholderia cepacia

Clostridium perfringens

Clostridium tetani

Corynebacterium diphteriae

Enterobacteriaceae (coliforms)

Pseudomonas aeruginosa

Staphylococcus aureus (MRSA/MSSA)

Aspergillus spp.

Candida spp.

Mucor spp.

Penicillium spp.

Other yeasts and fungi

Mycobacteria (in an ‘at risk donor’)

 

Table 2. Summary

Incubation period for skin samples

1 to 7 days inclusive

8 to 14 days inclusive

Growth

â tissue rejected

Presence of commensal flora only

â tissue approved

Presence of a pathogen

â tissue rejected

 

9. In order to detect fungal growth on the skin allografts it is sufficient for the sample to be cultured for 14 days.  If the tissue does not meet the above stated conditions, validated bacterial inactivation is acceptable.

D.3.1.5. Packaging and labelling

See generic requirements in section A.5.1.5.

 

 

D.3.1.5.1. Primary packaging and labelling

The primary packaging (the packaging that comes into contact with the skin allografts) should be suitable for this purpose (if available, medical device class IIa packaging).

In order to offer extra protection to the skin allograft it is preferable that an additional secondary packaging is utilised.

D.3.1.6. Storage/ preservation

D.3.1.6.1. General

See generic requirements in section A.6.

D.3.1.6.2. Methods of preservation and storage

Depending on the clinical application and the quality requirement stipulated, skin can be preserved in a number of different ways.  Cryopreservation in liquid nitrogen and/or glycerolisation are the methods normally used.  Cyropreserved or glycerolised skin is used as ‘biological dressing’ for temporary coverage of (burn) wounds.  Compared to the other tissues such as heart valves or cornea, the donor skin is present much shorter in the patient’s body when used as biological dressing.

Skin can also made acellular in order to use it as dermal implant; for instance in the Cuono method or under an autologous very thin split skin to reduce donor site morbidity on the patient and achieve a better scar quality.  In this type of treatment, the donor skin is gradually remodelled and replaced by the patient’s own tissue within a couple of months.

Every preservation and storage process should be specified (in the standard operating procedures of the tissue establishment), documented and validated.

Each type of storage condition should specify the maximum shelf life.  This would include, among other matters, taking into account any deterioration in the required properties of the skin.  This maximum shelf life should be documented and validated and reported to the surgeon carrying out the transplantation.  Regular critical evaluation of the preservation method used by the TE must be carried out to ensure that the intended results are still achieved.  Prior to the implementation of a significant process change the altered method has to be validated and documented.

D.3.1.6.2.1. Preservation and storage at 37ºC

This method used fluids with antibiotics and nutrients to keep the cells in the skin viable. This method can be used for a short period, 2-3 days.

D.3.1.6.2.2. Preservation and storage at +4°C

This is a method for storage viable skin allografts, maintaining their structural integrity, for short periods of time (days to weeks).  A physiological preservation medium with nutrients and possessing sufficient buffering capacity is recommended.

D.3.1.6.2.3. Cryopreservation at between -60°C and -80°C

This is a method for preserving viable skin allografts, maintaining their structural integrity, for medium term periods (months).  A controlled (refrigeration curve) and validated refrigeration procedure is recommended.

D.3.1.6.2.4. Cryopreservation in liquid nitrogen

This is a method for preserving viable skin allografts, maintaining their structural integrity, for longer periods (years).  A controlled (refrigeration curve) and validated refrigeration procedure is recommended.  The skin allografts are stored in liquid nitrogen (gases) at a temperature below -130°C.

D.3.1.6.2.5. Glycerolisation with a high glycerol concentration

This is a method for preserving non-viable skin allografts.  Glycerol concentrations of between 85 and 98% are the norm.  The glycerol concentration is increased in a gradual manner.  The glycerol solutions used are preferably of Ph. EU quality but at least from vegetable origin.

Glycerol has an anti-microbial action and will result in the skin being less immunogenic. Glycerolised skin allografts should be kept at 2 to 8°C.

D.3.1.6.2.6. Lyophilisation and dehydration

Rarely used and quite roundabout methods for storage non-viable skin allografts. Lyophilised skin can be kept at room temperature.

D.3.1.6.2.7. Decelluralisation

These are methods to lower the antigenicity of the skin further compared to glycerolisation. Removing the donor cells can be done by incubation of the skin in high concentration on NaCl (<1 M) or low concentrations of NaOH

D.3.1.6.2.8. Other preservation and storage procedures

A number of other procedures for the preserving and storage of skin allografts have been described in academic journals. These include:

a)     Vitrification;

b)    Preservation in high concentrations of propylene glycol;

c)     Preservation at 22°C in anhydrous NaCl (sodium chloride).

D.3.1.6.3. Storage area

See generic requirements in section A.6.

D.3.1.7. Documentation and release

D.3.1.7.1. General

See generic requirements in section A.3.3.7.1.

D.3.1.7.2. Processing file contents

See generic requirements in section A.5.1.6.3.

D.3.1.7.3. Availability for inspection

See generic requirements in section A.5.1.6.4.

D.3.1.7.4. Traceability

See generic requirements in section A.5.1.6.5.

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