The role of quality control in a skin bank: tissue viability determination.
New surgical procedures requiring viable skin have increased rapidly over the last few years. The cell viability assessment in allograft skin is a major step forward in burn treatment, since it is well-known that taking is correlated with grafted tissue viability. Various methods, both qualitative and quantitative, are currently used. Although qualitative assays (histomorphology, immunocytochemistry) are routinely performed in our laboratory, there arose a need to set up a standardised quantitative assay in an attempt to obtain a cut-off value so that the skin sample could be determined valid or not for grafting. Therefore, two different tetrazolium salt compounds MTT and WST-1, were compared in order to determine their efficacy in the evaluation of tissue viability. Several experimental conditions were analysed: 1- cellular cultures of keratinocytes and fibroblasts, 2- fresh skin tissue samples, 3- the same specimen tested daily for at least 2 weeks, 4- after cryopreservation and thawing. Viable cells were analysed by the cleavage of tetrazolium salts to formazan by cellular enzymes. The formazan dye produced by metabolically active cells was then quantified by measuring the absorbance of the dye solution at the appropriate wavelength. It was seen that WST-1 is easier to handle, more stable, has a wider linear range, accelerated colour development and is more sensitive than MTT on fresh specimens and cell suspension. However, after 72 hours of storage at 4 degrees C, most of the WST-1 tested specimens no longer gave any absorbance signal, whilst MTT specimens were seen to give a signal for more than two weeks. Moreover, after thawing WST-1 tested samples were almost negative, whilst MTT samples continued to give strong signals. In conclusion, WST-1 assay offers rapid and precise results as to the cell viability of fresh allografts and cell cultures, whilst the MTT method is much more useful in establishing viability after long conservation and cryopreservation. In our clinical experience, allografts transplanted at 72 hr post-harvesting or after cryopreservation showed a mean of take more than of 80%, demonstrating that the MTT system is more reliable for the determination of allograft viability. Studies are ongoing with larger clinical cohorts to establish the precise cut-off value for skin graft validation.
Alotto
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Ospedale CTO, Dept. of Plastic Surgery and Burn Unit "Skin Bank", Via Zuretti 29, Turin, 10126, Italy.
Ariotti
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Graziano
S
S
Verrua
R
R
Stella
M
M
Magliacani
G
G
Castagnoli
C
C