SUMMARY. The authors describe the methodology used in their department for obtaining skin and storing it in a skin bank and the use of cryopreserved skin. They indicate the legal requirements for the creation of a live tissue bank specifically for frozen skin, and they present the results of five years’ work.
The health authority is responsible for authorizing the creation of a skin bank. It is essential that the hospital should be authorized for the extraction of organs for transplant and have sufficient back-up in terms of technology and human resources. Usually there is a co-ordinator in charge of the legal aspects, who selects or rejects donors.
Once the director of the team has been appointed, he or she proceeds with the training programme for the other members of the team, which will enable them to gain the approval of the health authority.
Methodology of skin extraction
The skin to be extracted comes from multi-organ donors. The co-ordinator has to check all the requirements for qualification as a legal donation:
<% riquadro "Sepsis
Malignant tumour
Systemic disease (e.g. collagenosis, vasculitis)
Neurological disease
HIV and risk groups" %>
Table I - Absolute controindications
<% riquadro "Age over 75 yr
Arteriosclerosis
High blood pressure
Long-lasting diabetes
Long-lasting pharmacological treatment
Alcoholism
Acupuncture in the past six months" %>
Table II - Relative controindications
A. Evaluation of the potential donor
There are some general contraindications that affect all the organs and others that are more specific for each single organ.
All potential donors are tested as follows:
Haematology Group/Rh
Coagulation
White cell count
Biochemistry UGI, creatinine
Liver profile
Kidney profile
Gasometry
Urine test
Pregnancy test
Bacteriology HIV
B-CHV
CMV
VDRLv
Blood and urine
Gram in sputum
Immunology HLA
Complementary ECG
Abdominal ultrasound
Breast X-ray
Requirements for the selection of skin donor:
<% riquadro "Sepsis
Skin infection or dermatitis
Lung infection or pneumonia
Drug addiction
Jaundice
Toxic or viral hepatitis
Antigen or antibody for Treponema pallidum
Tumours, including sarcoma, carcinoma, lymphoma, and leukaemia" %>
Table III - List of diseas taht exclude a potential donor
B. Harvesting the skin
Skin extraction is carried out in the operating room with maximum asepsis. Any area that is wounded or covered by a plaster is discarded. Visible areas (hands, feet, face, neck) will always be respected.
The fields to extract from are:
Preparation of donor. First of all, every area has to be disinfected independently of the others. The surgeon must discard his gloves and coat after each area has been treated.
The disinfection steps are as follows: shaving, disinfection with clorhexidine, cleansing with saline serum, second disinfection with iodine and re-cleansing, and application of isopropylic alcohol 60%. The skin is then harvested with an electric dermatome and stored in flasks with 200 ml of maintenance liquid (for composition, see Table IV).
Next the bottles are covered with double sterile bags with identification of the field. Then either the cryopreservation process is started or the material is stored at 4 °C for no more than 48 h.
The preservation liquid is prepared in sterile conditions and stored in 500-ml flasks at a temperature of -40 °C. The flasks bear a label with the date of preparation and expiry.
<% riquadro "MEM (minimum essential medium) 85% dilution
FBS (foetal bovine serum) 10% dilutionSepsis
Skin infection or dermatitis
Lung infection or pneumonia
Drug addiction
Jaundice
Toxic or viral hepatitis
Antigen or antibody for Treponema pallidum
Tumours, including sarcoma, carcinoma, lymphoma, and leukaemia
Antibiotics (penicillin, streptomycin, fungizone) 5% dilution Sepsis
Skin infection or dermatitis
Lung infection or pneumonia
Drug addiction
Jaundice
Toxic or viral hepatitis
Antigen or antibody for Treponema pallidum
Tumours, including sarcoma, carcinoma, lymphoma, and leukaemia" %>
Table IV - Composition of maintenance liquid
C. Cryopreservation
Processing the skin. All the work is done under sterile conditions. The liquid used for the process can be glycerol (Table V) or dimethyl sulphoxide (Table VI), which is the one we use.
The skin obtained from the different areas must be kept apart. The strips of skin are put in scoured tulle gras or wrapped in plastic sheets. Freezing bags (model Gambro 1200) are then used, each containing about 500-600 sq. cm. Two samples of skin from each flask are taken for the bacteriological control, after which maintenance liquid (60 ml) is added to the bag and the air is extracted. The bags are double sealed and incubated for one hour.
Bacteriological control. Two samples of the maintenance liquid in each flask are taken for cultivation, both bacteria and fungi. A fragment of skin from each bag is also cultured. Once the incubation time of the bags is over, another sample of the liquid (about 30 ml) is sent for culturing.
Labelling of the bags. For identification purposes in the event of contamination, each bag is labelled with the amount of skin, the area obtained, and the date of processing.
Cryopreservation. The cryopreservation process is carried out in a freezing chamber. It is important that both the probe and the skin should be at room temperature at the beginning of the process. The temperature chart should match that of the freezing monitor, failing which the skin should be discarded. The process lasts 55 minutes, as follows:
– first 20 min, decrease to -20 °C;
– from 20 to 30 min, decrease to -60 °C;
– from 30 to 40 min, maintain at -60 °C;
– from 40 to 50 min, decrease to -80 °C;
– in the last minutes, decrease to below -100 °C.
Next to the bags we freeze a control tube for each bag. The bags are then introduced into a tank with liquid nitrogen where they remain until use or are discharged if contaminated.
* Viability test. The trypan blue stain is used to test viability. Our viability rate is above 70% for more than 24 months.
<% riquadro "32 ml TC-199 medium
43 ml glycerol (33% dilution)
25 ml albumin (20% dilution)" %>
Table V - Composition of preservation liquid per 100 ml
<% riquadro "Dimethyl sulphoxide in 10% or 15% dilution" %>
Table VI - Dimenthyl sulphoxide
Use
Evaluating needs. It should be borne in mind that 1% of body surface area corresponds to about 170 sq. cm in an adult 1.78 m tall weighing 70 kg (for children, use the surface charts).
Uses of the skin.
a. In our burn unit. The bags containing frozen skin are defrosted by immersion before use in water at a temperature of 40 °C. Once the bags are opened, the skin is rinsed with saline serum three times before use;
b. Sent to other centres, using one of two methods: dry ice or liquid nitrogen.
During the last five years we have obtained the results given below (Tables VII-XV).
<% riquadro "Donors 34
Total skin harvested 94950 sq. cm
Suitable for clinic use 89800 sq. cm
Discarded 5150 sq. cm
Percentage discarded 0.53%" %>
Table VII - Use of skin
<% riquadro "Tumour 2700 sq. cm (0.28%)
Living donor (after lipectomy) 350 sq. cm (0.036%)
Contamination 950 sq. cm (0.10%)
Defective freezing 1150 sq. cm (0.12%)" %>
Table VIII - Reson for discarding skin
<% riquadro "Used in our hospital:
Burns 38 patients
Lyell syndrome 1 patient
Other 1 patient
Sent to other hospitals 17450 sq. cm" %>
Table IX - Aetiology
<% riquadro "Used in our hospital:
Burns 38 patients
Lyell syndrome 1 patient
Other 1 patient
Sent to other hospitals 17450 sq. cm" %>
Table X - Number of procedures in each patient
<% riquadro "One procedure 28 patients
Two procedures 8 patients
Three procedures 4 patients" %>
Table XI - Percentage of burns surface of recipient
<% riquadro "Over 70% 0 patients
50-70% 17 patients
30-50% 12 patients
Less than 30% 9 patients
Others 2 patients" %>
Table XII - Recipients with burns in 30-50% TBSA
<% riquadro "Number of patients 17
Alive 14
Total amount of skin used 34350 sq. cm
Average per patient 2453 sq. cm
Died 3
Total amount of skin used 3650 sq. cm
Average per patient 1216 sq. cm" %>
Table XIII - Recipients with burns in 50-70% TBSA
<% riquadro "Number of patients 12
Alive 9
Total amount of skin used 14800 sq. cm
Average per patient 1645 sq. cm
Died 3
Total amount of skin used 3050 sq. cm
Average per patient 1016 sq. cm" %>
Table XIV - Recipients with burns in less than 30 TBSA
<% riquadro "Number of patients 9
Alive 7
Total amount of skin used 4000 sq. cm
Average per patient 571 sq. cm
Died 2
Total amount of skin used 1600 sq. cm
Average per patient 800 sq. cm" %>
Table XV - Other cases
<% riquadro "Number of patients 2
Lyell syndrome 1
Amount of skin used 850 sq. cm
Post-debridement coverage 1
Amount of skin used 400 sq. cm" %>
Table XVI - Absolute controindications
A number of different factors have collectively contributed to the higher survival rates in patients with extensive burns. These include better intensive care treatment, deeper knowledge of burn physiopathology and the requirements of metabolic support, earlier debridement of the eschar, better isolation of the patient, and the use of different temporary coverings until the patient’s skin is available, including cryopreserved skin, which is the source of the temporary skin graft we use in patients with extensive burns above 40% TBSA.1-12
The possibility of using live tissue such as harvested and frozen skin offers the best results in the temporary treatment of burn wounds, although it presents the disadvantage of the cost of the necessary installations and the risk of the transmission of unknown or undetectable diseases.
We think that for the temporary covering of a burn wound any method that involves biosynthetic material can be useful. Also, we should not underrate commercial temporary dressings that supplement the use of biological dressings.
In our environment, and with an experience of more than five years using cryopreserved skin, we are convinced that without this possibility many of our surviving patients would have died, while in the patients we have treated we have not seen a single case of cross-infection.
Conclusions
We are of the opinion that in patients with major burn wounds affecting between 40 and 70% TBSA, the use of allografts with cryopreserved skin is the best therapeutic tool. The cost of setting up a bank for cryopreserved skin is high, but compared with the use of commercial skin substitutes it is cheap.
We probably would all wish to resolve burn wounds definitively with compatible skin, but until we can achieve this we believe that skin banks with frozen skin offer the highest quality for a temporary solution, as well as safety as regards cross-infection.
RESUME. Dans cet article les Auteurs décrivent leur méthodològie pour obtenir la peau et pour la conserver dans une banque de la peau et leur méthode d’emploi de la peau cryopréservée. Ils expliquent les exigences légales pour la création d’une banque de tissus vivants et en manière spécifique pour la peau congélée. En conclusion ils présentent leurs résultats après cinq années de travail.