- Email: [email protected]
Stored skin—stored trouble?
Brrrtsh Jwnzol 0
,,, P/u\/,<
Suryery
1994 The Erifwh Asxiation
(19941, 47. 24 29 of Plastic Surpms
Stored skin-stored
trouble?
0. G. Titley, M. Cooper, A. Thomas and K. Hancock West Midlands
Regional
Plastic and Jaw Surgery
Unit, Wordsley Hospital,
Wordsley,
West Midlands
SUMMARY. Quantitative bacteriology is presented on 102 consecutive split-skin grafts. A sample of graft was cultured whenever skin was taken and whenever stored skin was used. Stored skin unused at 21 days was also cultured. The percentage take of grafts was inversely proportional to number of organisms/g of skin (r = - 0.24, p < 0.05). Commonly used storage conditions facilitated bacterial multiplication. There was no significant difference in organisms/g contaminating grafts for different surgeons, skin preparations, types of grafts, cutting tools or mode of anaesthesia. Male patients had a significantly greater count for initial grafts (p = 0.02), but after 3 weeks storage there was no sex difference.
to prevent bacterial proliferation. Whilst low temperatures do not kill microorganisms, bacterial multiplication to levels detrimental to graft take should be prevented. In 1967, Krizek and co-workers” used quantitative bacteriological methods to demonstrate that the number of organisms present in a wound was relevant to skin graft take. Studying graft take on 50 healthy, granulating wounds they demonstrated success was largely predictable based on the number, rather than the type, of bacteria which were present. When > IO5 organisms/g of tissue were present skin graft take was poor, averaging less than 20 % ; if < 1O5 organisms/g were present takes of over 90% were achieved. This finding paralleled those of other workers who noted that about lo5 or lo6 organisms/g or ml seemed to be a critical level producing clinically significant sepsis, irrespective of the contaminating organism.“-‘” There are exceptions to this, notably the group A Beta haemolytic streptococcus. The objective of this study was the quantitative and qualitative identification of organisms colonising splitthickness skin grafts at the time of harvesting and after 3 weeks of refrigerated storage; to relate the number of organisms/g on fresh skin graft to percentage take, and finally to compare the number of organisms/g contaminating grafts taken under different conditions.
Between 1896 and 1903 Wentscher studied autotransplantation of refrigerated skin grafts. He described successful autotransplantation of human skin which had been stored in an ice box for up to 14 days.‘,’ Carrel demonstrated that refrigerated skin did not undergo autolysis, retained its normal histological appearance over several months and could be successfully autotransplanted after 2 weeks storage.3 Despite these experimental observations, little clinical use of refrigerated skin grafts was made until World War II. Writing in The Lancet, Squadron Leader D. N. Matthews of the Royal Airforce suggested the use of refrigerated skin autografts to reduce the “large number of operations, under general anaesthesia”, required to resurface the skin losses of burnt airmen.” He stored skin with its raw surfaces opposed and wrapped in tulle gras, which was in turn wrapped in saline dampened gauze. This was put into an airtight, screw-capped bottle and then placed in a domestic refrigerator at a temperature of between 3 and 6 “C. In 50 grafts applied he noted good take after storage periods of between 3 and 8 weeks. Around the same time Webster in the USA5 and Flatt in the UK” reported similar methods and results. Many investigators have since studied refrigerated skin storage and found the methods described by Matthews and his contemporaries to be far from ideal. A vast array of storage media and conditions have been shown to be superior to those early descriptions;’ nevertheless refrigerated skin storage as routinely practised today differs little from that described nearly 50 years ago. Today, with safer general anaesthesia, most skin autograft is stored in case of graft failure or for “delayed” grafting. Refrigerated skin is usually stored for up to 3 weeks, after which. cellular respiration ceases.x,s Therefore. after this time stored skin is considered non-viable in terms of take; but its application to wounds is suggested as a biological dressing. In addition to retarding autolysis, refrigeration aims
Methods
Consecutive patients undergoing split-thickness skin grafting as part of their surgery were entered into the study. There were no exclusion criteria. Skin grafts were taken according to the operating surgeons’ preferences. All defects were grafted at the time of primary surgery and skin was not applied to a previously ungrafted defect on the ward. The following data were recorded: age of patient; sex of patient; indication for surgery ; grade of surgeon (SHO, registrar or consultant); skin preparation used 24
25
Stored Skin-Stored Trouble? on donor site (CH(alc), CH(aq), Bet)*; cutting tool (hand knife or dermatome); type of skin graft (sheet, hand perforated or meshed); mode of anaesthesia. There was no attempt to randomise these variables. A sample of every graft taken was sent for qualitative and quantitative microbiology (SSGl) and a piece stored in the “skin-fridge” (BOC-Linde, model FKS 36 10) ; an upright refrigerator of similar design to most domestic appliances, but without an icebox. All skin for storage was placed on tulle gras and wrapped in saline moistened gauze before being placed into a sterile universal container. At 3 weeks any unused skin was sent for microbiology (SSG2). In the interim, if stored skin was applied to a patient a further sample was sent for analysis. At the first dressing change percentage graft take was recorded. The percentage take of any stored skin which had been applied was assessed independently of the rest of the graft. Each sample of skin was sent to the laboratory in its sterile universal container. The skin was aseptically separated from the dressings and transferred into a further sterile universal container and weighed. 2 ml of sterile nutrient broth (Gibco, Paisley, Scotland) were added and this was vortexed for 3 min. Immediately after this, 100 fold dilutions of the broth were made with fresh nutrient broth. 100 microlitres of each dilution were plated out onto an entire blood agar plate. The plates were incubated aerobically for 48 h at 37°C. If any growth was present the dilution that yielded between 20 and 2000 colonies was chosen for counting and the number of each type of colony on this plate was recorded. From this the number of organisms per gram of skin was calculated. Standard laboratory methods were used to identify organism to genus and, for selected isolates, species level. The number of organisms/g of skin was compared for SSGl and SSG2; the degree of association between the number of organisms/g on SSGl and percentage take of graft was calculated and the number of organisms/g was compared for sex of patient, grade of surgeons, skin preparations, cutting tools, types of skin graft and mode of anaesthesia for SSGl and SSG2.
malignant neoplasia (53.9 %), chronic lower limb ulceration (16.7 %) and upper limb trauma (13.7 %>. The staphylococci (S. aUYeUS and coagulase negative staphylococci) were the organisms most commonly isolated from the first skin sample (SSGl). 58 grafts showed no growth. More than one organism was grown from 11 grafts. 83 skin samples were sent for analysis after 3 weeks storage. The stored skin was applied to patients in 10 cases. SSG2 was not sent for analysis in 9 cases. At 3 weeks staphylococci still predominated, but the number of grafts colonised by coliforms, acinetobacter species and pseudomonas species had increased markedly. Moraxella species, which were not found on SSG 1, appeared and alpha-haemolytic streptococci were no longer found. The complete data for both time periods are shown in Table 1. The number of organisms contaminating grafts ranged from CUl.1 x 10’ (median 0) for SSGl and 0-5.0x 10’ (median 9.6 x 103) for SSG2. These data were skewed and transformations did not improve the shape. Therefore non-parametric statistical analyses were used. Percentage take was plotted against organisms/g of skin for SSGl (Fig. 1). A negative correlation was found (r = -0.24; p < 0.05-Spearmans rank correlation). That is, as organisms/g of skin increased, percentage take of the skin graft decreased. In 83 patients SSG2 was analysed after 3 weeks storage. For these patients the number of organisms/g of skin colonising SSGl and SSG2 were compared (Fig. 2). A highly significant increase was demonstrated in the median number of organisms/g contaminating SSG2 (9.6 x 103) compared to SSGl (0) (p < O.OOOl-Wilcoxon rank sum test). Secondly, 48 grafts (57.8%) in the first group showed no growth; after 3 weeks storage this had fallen to 18 (21.7 %) (chisquared = 22.64, p < 0.001). Similarly, the number of grafts growing greater than lo5 organisms/g of skin was significantly greater for the second samples, 32 (38.6 %), compared to the first, 3, (3.6 %) (chi-squared = 30.44, p < 0.001). There was no significant difference between the median number of organisms/g contaminating grafts
Table 1 Results
Over a 4-month period 103 consecutive patients requiring split-thickness skin grafts were entered into the study. One patient was subsequently excluded from all analyses as no samples were sent for microbiology. There were 56 males, mean age 59.2 years (range 5-90) and 46 females, mean age 66.4 years (range 7-88) (t = 1.74; p = 0.08, n.s., two sample t-test). The indications for surgery were predominantly *
CH(alc) = chlorhexidine gluconate 0.5% w/v in 70% IMSDePuy Healthcare. Pearce Laboratories, Leeds. UK. CH(aq) = aqueous chlorhexidine gluconate 0.05% w/v. Sterets Unisept--Seton Healthcare Group, Oldham. UK. Bet = Betadine, Povidone-iodine USP 7.5 % w/v-Napp Laboratories Ltd. Cambridge Science Park, Cambridge, UK.
Organisms grown from SSGl and SSG? skin
samples
Orgarzism
SSGI
Coagulase negative staphylococci s. auW_eus Alpha haemolytic streptococcus
28 II 4
SSG2
I8 10 0
Bacillus sp
3
4
Coliforms
4 I
I4 I3
Acinetobmter sp Micrococcus sp Pseudot?lorlos sp
**Coryneforms Fungi Moroselln sp Agrobncterium
No growth Total number
”
sp
of grafts
I
3
I
6
I
3
I
4
0 0
5
58 I02
I IX 83
26
British Journal 100
AA .nAauYI M A
MA&l
A
AA A
90
of Plastic Surgery
A A A
A
80
AA
A
A
A
A
A
70
A A 10 -A 00 0
A
I
I
I
1
1
/
I
1
1
2
3
4
5
6
7
8
Organisms/g (log) for SSGl Fig. 1 Figure l-Organisms/g
(log 10) colonising SSGl v percentage take of SSGl.
A
A
0
’ SSGl GRAFT Fig. 2
Figure 2-Organisms/g log for SSGl & SSGZ. The broken line on the graph represents the log,,,, of 10’ organisms/g of skin. It should be noted that the open triangles for SSGl and SSG2 at 0 represent 48 grafts and 18 grafts respectively.
for the different grades of surgeon, skin preparations used, types of skin graft, mode of anaesthesia (Kruskal Wallis one way analysis of variance); or cutting tools (Wilcoxon rank sum test) for both sets of samples. Male patients had a significantly higher median number of organisms/g of graft than females for SSGl
(p = 0.02). However, there was no significant sex difference after three weeks storage (Wilcoxon rank sum test). It was assumed that the temperature of the refrigerator at Wordstey was appropriate for skin storage. To test this hypothesis its temperature was
Stored Skin-Stored
27
Trouble?
-TOP
16
-----
MIDDLE
--.------
BOTTOM
14 c
12
f 5
10
E c”
6 6
Figure STemperature
Table 2 Bacteriology
changes in Wordsley skin fridge over 24 h.
and percentage
take of stored
skin
applied to 8 patients Take
Bacteriolog?
Count
S. aureus Ps. aeruginosa
2.2 x 10’ 1.6 x lo” 6.7 x lo4
60 % 80% 80%
9.7 x 104
80 %
“Coryneforms” Coagulase negative staphylococci “ Coryneforms ” Proteus sp Esch. cofi
2.4 x 10”
0
Coagulase negative staphylococci
3.3 x 106
100%
2.3 x 10’
0
5.3 x 10’
0
S. aureus Acinetobacter sp Acinetobucter sp
monitored over a 24-h period. Probes were placed on the top, middle and bottom shelves and the temperature recorded at 30 min intervals. The results are presented graphically in Figure 3. The temperature on the top shelf oscillated between 8.6 and 13.9”C with a mean of 9.8; the middle shelf (where our skin was kept) ranged from 6-l 1°C with a mean of 7.3 and the bottom ranged from 0.3-6.7”C with a mean of 4. The refrigerator door was opened once during the period monitored (at about 1500h) and the temperature on the top shelf peaked at 13.9”C (arrowed). These storage conditions may have been peculiar to our unit and are perhaps not applicable elsewhere. However, on contacting 44 major plastic surgery units by phone, the majority (20) were found to store skin grafts in a domestic refrigerator. Most others used the smaller ward drug fridges (16). Only 2 units used a fridge designed specifically for tissue storage and 6 units were unable to provide the information re-
quested. About 53 % (23) stored only skin in the fridge and 45% (20) stored skin with a variety of other things, including drugs and blood. Stored skin was applied to 10 patients, between 6 and 15 days after harvesting. Data were available for 8 patients and are shown in Table 2. All grafts were contaminated by organisms with counts ranging from 2.2 x lo’-5.3x lo7 organisms/g of skin. Seven different organisms were identified. The number of grafts involved was too small to draw firm conclusions; however, there seemed to be a correlation between a high number of organisms/g and significant failure of these grafts. Discussion
Refrigerated storage of split-thickness skin grafts is a widespread, if not universal, practice in plastic surgery. Despite advances in storage techniques described in the literature, most units use methodology unchanged for nearly 50 years. Built into this method is the use of non-standardised refrigerators. This study has clearly demonstrated that these storage conditions allow significant bacterial proliferation over a 21-day storage period. Earlier work seems to have largely neglected the bacteriology of refrigerated, stored skin grafts. Matthews4 stated some grafts were sterile and others were “infected with normal skin contaminants”. He stated that this had no harmful effects, but did not give any details. Skoog,g using full thickness grafts from white laboratory rats, found “considerable numbers of bacteria present after a week at 3°C” and that the addition of antibiotics provided “satisfactory pro-
28
tection” against this for about three weeks. Krizek’s worklo demonstrated the relevance of bacterial count/g of tissue in the graft bed to skin graft take. It was shown that when counts exceeded lo5 organisms/g then poor graft take resulted. We believe our study is the first to demonstrate that number of organisms/g of skin graft applied is also relevant to graft take; take was shown to decrease as organisms/g increased. As with earlier quantitative bacteriological studies, relevant clinical effects occurred irrespective of the actual contaminating organism. However, in contrast to these studies, an effect was shown for levels of less than lo5 organisms/g. In our study the bacteriology of the graft bed was not assayed. This may be a relevant factor, particularly for delayed application of stored skin. However, the vast majority of defects grafted were primarily created by surgery and delayed grafting was not used. Therefore, one would expect the graft bed to have had an insignificant bacterial count. This is in contrast to the studies of Krizek, who grafted onto granulations, which were colonised by a significant number of organisms. The association demonstrated between skin graft take and organisms/g for SSGl was relatively weak (r = - 0.24) however, 96.4 % of these grafts were contaminated by less than lo5 organisms/g. In contrast, after 3 weeks storage nearly 40% of grafts were contaminated by greater than lo5 organisms/g and the number of grafts showing no growth fell significantly. After 3 weeks, with such bacterial levels, it seems reasonable to assume that a greater association between take and count would be demonstrable. This hypothesis is supported by the Krizek data previously discussed. This is clearly of concern if skin stored for long periods is regularly used in treating patients. Stored skin was only applied to 10 patients (9.8 %) and data were available for only 8 of these. However, we feel it is worth noting that all grafts were contaminated and 3 grafts failed completely. There appeared to be a correlation between a high number of organisms/g and graft failure. However, it would be unwise to draw firm conclusions from such a small database and this area of the study warrants further investigation. A further problem when assessing take of stored skin graft is the less than ideal graft bed compared to the original defect. The degree of contamination of initial and 2 1-dayold stored skin grafts was shown to be independent of grade of surgeon, skin preparation used, type of skin graft, cutting tool used and mode of anaesthesia. We accept that these variables were not compared in a true controlled, randomised fashion, as we felt the numbers necessary for this would have made the study impracticable. Male patients were found to have more organisms/g than females for the initial grafts. The reason for this is not clear but may have been related to shaving donor sites in males. Preoperative shaving causes thousands of microscopic wounds which may potentiate bacterial multiplication, particularly if the patient is shaved on the ward several hours before theatre. Prospective studies have confirmed that shaving increases postoperative wound infection rates.14.‘”
British
Journal
of Plastic
Surgery
The refrigerator used to store skin grafts in our unit was basically a domestic unit adopted for the storage of human tissue. Such practice is not exceptional; almost half of major UK plastic surgery units used such appliances. These refrigerators were shown to have a variable and cyclical temperature range with different peaks and troughs on different shelves of the appliance. This investigation has shown the necessity of strict temperature monitoring of such refrigerators, in the same way as is routine in blood banks. It is of concern to note that some units stored drugs and blood products together with skin grafts in appliances which have the potential for facilitating bacterial (and fungal) growth on skin grafts. With the exception of Acinetobacter species the organisms found in this study do not multiply at temperatures below 4’-‘C.16Therefore we conclude that the type of refrigerator in which skin was stored in our unit facilitated significant bacterial proliferation. Clearly the domestic type of refrigerator is not appropriate for skin storage. The lowest temperature range was at the bottom of our refrigerator and until a better alternative is found grafts should probably be stored at that level. Further studies are planned to investigate the quantitative bacteriology of grafts stored in the smaller ward drug refrigerators and of grafts stored in designated tissue refrigerators, such as found in a blood bank.
Acknowledgements We gratefully Russell’s Hall not have been allowing their
acknowledge the help of the staff at Wordsley and Hospitals, without whose assistance this study would possible and thank the consultant plastic surgeons for patients to be included in this study.
1. Wentscher J. Die verwendung konservieter hautlappen bei der transplantation nack Thiersch. Klin Wschr 1896; 31: 979. Cited by Perry VP. A review of skin preservation. Cryobiology 1966; 3: 109930. 2. Wentscher J. Ein weiter beitrag zur uberlebenfahigkeit der menschlichen epidermiszellen. Deutsch Z Chir 1903: 70: 2144. Cited by Perry VP. A review of skin preservation. Cryobiology 1966: 3: 109-30. 3. Carrel A. The preservation of tissues and its application in surgery. JAMA 1912; 59: 523-7. 4. Matthews DN. Storage of skin for autogenous grafts. Lancet 1945; 1: 775-8. 5. Webster JP. Refrigerated skin grafts. Ann Surg 1944; 120: 43 149. 6. Flatt AE. Refrigerated autogenous skin grafting. Lancet 1948; 2: 249-51. 7. Perry VP. A review of skin preservation. Cryobiology 1966; 3: 109-30. 8. Georgiade N, Peschel E. Georgiade R. Brown 1. A clinical and experimental investigation of the preservation of skin. Plast Reconstr Surg 1956; 17: 267-75. 9. Skoog T. An experimental and clinical investigation of the effect of low temperature on the viability of excised skin. Plast Reconstr Surg 1954; 14: 403-16. 10. Krizek TJ, Robson MC, Kho E. Bacterial growth and skin graft survival. Surgical Forum 1967; 18: 518-9. I 1. Elek SD. Experimental staphylococcal infections in the skin of man. Ann-NY Acad Sci-1956; 65: 85590. 12. Kass EH. Bacteriuria and the diagnosis of infections of the urinary tract. Arch Inter Med 1977; 100: 709-14. 13. Lindberg RB. Moncrief JA. Switzer WE. Order SE, Mills W.
Stored Skin-Stored
29
Trouble?
The successful control of burn wound sepsis. J Trauma 1965; 5: 601-16. 14. Cruse PJE. Foord R. A five year prospective study of 23,649 surgical wounds. Arch Surg 1973; 107: 20610. 15. Alexander JW, Fischer JE, Boyajian M. Palmquist J. Morris MJ. The influence of hair removal methods on wound infections. Arch Surg 1983; 118: 347-52. 16. Parker MT. Collier LH, editors. Topley and Wilson’s principles of bacteriology. virology and immunity, 8th ed. Edward & Arnold. 1990.
Unit, Wordsley Hospital, Stream Road. Wordsley. West Midlands. DY8 5QX. Michael Cooper, MB ChB, MRCPath, Senior Registrar Microbiology. Russell’s Hall Hospital. Dudley, West Midlands, DYl 2HQ. Andrea Thomas, BSc, Statistician. West Midlands Regional Health Authority. 142. Hagley Road, Edgbaston, Birmingham, B16 9PA. Kevin Hancock, MB BS, FRCS, Senior Registrar Plastic Surgery, West Midlands Regional Plastic and Jaw Surgery Unit, Wordsley Hospital. Stream Road. Wordsley. West Midlands, DY8 5QX.
The Authors
Requests for reprints to Mr 0. G. Titley, Department of Plastic Surgery, Royal Victoria Infirmary. Newcastle Upon Tyne.
Oliver Garth Titley, MB ChB, MSc, FRCS, Senior House Officer Plastic Surgery, West Midlands Regional Plastic and Jaw Surgery
Paper received 4 May 1993. Accepted 20 July 1993. after revision.
,,, P/u\/,<
Suryery
1994 The Erifwh Asxiation
(19941, 47. 24 29 of Plastic Surpms
Stored skin-stored
trouble?
0. G. Titley, M. Cooper, A. Thomas and K. Hancock West Midlands
Regional
Plastic and Jaw Surgery
Unit, Wordsley Hospital,
Wordsley,
West Midlands
SUMMARY. Quantitative bacteriology is presented on 102 consecutive split-skin grafts. A sample of graft was cultured whenever skin was taken and whenever stored skin was used. Stored skin unused at 21 days was also cultured. The percentage take of grafts was inversely proportional to number of organisms/g of skin (r = - 0.24, p < 0.05). Commonly used storage conditions facilitated bacterial multiplication. There was no significant difference in organisms/g contaminating grafts for different surgeons, skin preparations, types of grafts, cutting tools or mode of anaesthesia. Male patients had a significantly greater count for initial grafts (p = 0.02), but after 3 weeks storage there was no sex difference.
to prevent bacterial proliferation. Whilst low temperatures do not kill microorganisms, bacterial multiplication to levels detrimental to graft take should be prevented. In 1967, Krizek and co-workers” used quantitative bacteriological methods to demonstrate that the number of organisms present in a wound was relevant to skin graft take. Studying graft take on 50 healthy, granulating wounds they demonstrated success was largely predictable based on the number, rather than the type, of bacteria which were present. When > IO5 organisms/g of tissue were present skin graft take was poor, averaging less than 20 % ; if < 1O5 organisms/g were present takes of over 90% were achieved. This finding paralleled those of other workers who noted that about lo5 or lo6 organisms/g or ml seemed to be a critical level producing clinically significant sepsis, irrespective of the contaminating organism.“-‘” There are exceptions to this, notably the group A Beta haemolytic streptococcus. The objective of this study was the quantitative and qualitative identification of organisms colonising splitthickness skin grafts at the time of harvesting and after 3 weeks of refrigerated storage; to relate the number of organisms/g on fresh skin graft to percentage take, and finally to compare the number of organisms/g contaminating grafts taken under different conditions.
Between 1896 and 1903 Wentscher studied autotransplantation of refrigerated skin grafts. He described successful autotransplantation of human skin which had been stored in an ice box for up to 14 days.‘,’ Carrel demonstrated that refrigerated skin did not undergo autolysis, retained its normal histological appearance over several months and could be successfully autotransplanted after 2 weeks storage.3 Despite these experimental observations, little clinical use of refrigerated skin grafts was made until World War II. Writing in The Lancet, Squadron Leader D. N. Matthews of the Royal Airforce suggested the use of refrigerated skin autografts to reduce the “large number of operations, under general anaesthesia”, required to resurface the skin losses of burnt airmen.” He stored skin with its raw surfaces opposed and wrapped in tulle gras, which was in turn wrapped in saline dampened gauze. This was put into an airtight, screw-capped bottle and then placed in a domestic refrigerator at a temperature of between 3 and 6 “C. In 50 grafts applied he noted good take after storage periods of between 3 and 8 weeks. Around the same time Webster in the USA5 and Flatt in the UK” reported similar methods and results. Many investigators have since studied refrigerated skin storage and found the methods described by Matthews and his contemporaries to be far from ideal. A vast array of storage media and conditions have been shown to be superior to those early descriptions;’ nevertheless refrigerated skin storage as routinely practised today differs little from that described nearly 50 years ago. Today, with safer general anaesthesia, most skin autograft is stored in case of graft failure or for “delayed” grafting. Refrigerated skin is usually stored for up to 3 weeks, after which. cellular respiration ceases.x,s Therefore. after this time stored skin is considered non-viable in terms of take; but its application to wounds is suggested as a biological dressing. In addition to retarding autolysis, refrigeration aims
Methods
Consecutive patients undergoing split-thickness skin grafting as part of their surgery were entered into the study. There were no exclusion criteria. Skin grafts were taken according to the operating surgeons’ preferences. All defects were grafted at the time of primary surgery and skin was not applied to a previously ungrafted defect on the ward. The following data were recorded: age of patient; sex of patient; indication for surgery ; grade of surgeon (SHO, registrar or consultant); skin preparation used 24
25
Stored Skin-Stored Trouble? on donor site (CH(alc), CH(aq), Bet)*; cutting tool (hand knife or dermatome); type of skin graft (sheet, hand perforated or meshed); mode of anaesthesia. There was no attempt to randomise these variables. A sample of every graft taken was sent for qualitative and quantitative microbiology (SSGl) and a piece stored in the “skin-fridge” (BOC-Linde, model FKS 36 10) ; an upright refrigerator of similar design to most domestic appliances, but without an icebox. All skin for storage was placed on tulle gras and wrapped in saline moistened gauze before being placed into a sterile universal container. At 3 weeks any unused skin was sent for microbiology (SSG2). In the interim, if stored skin was applied to a patient a further sample was sent for analysis. At the first dressing change percentage graft take was recorded. The percentage take of any stored skin which had been applied was assessed independently of the rest of the graft. Each sample of skin was sent to the laboratory in its sterile universal container. The skin was aseptically separated from the dressings and transferred into a further sterile universal container and weighed. 2 ml of sterile nutrient broth (Gibco, Paisley, Scotland) were added and this was vortexed for 3 min. Immediately after this, 100 fold dilutions of the broth were made with fresh nutrient broth. 100 microlitres of each dilution were plated out onto an entire blood agar plate. The plates were incubated aerobically for 48 h at 37°C. If any growth was present the dilution that yielded between 20 and 2000 colonies was chosen for counting and the number of each type of colony on this plate was recorded. From this the number of organisms per gram of skin was calculated. Standard laboratory methods were used to identify organism to genus and, for selected isolates, species level. The number of organisms/g of skin was compared for SSGl and SSG2; the degree of association between the number of organisms/g on SSGl and percentage take of graft was calculated and the number of organisms/g was compared for sex of patient, grade of surgeons, skin preparations, cutting tools, types of skin graft and mode of anaesthesia for SSGl and SSG2.
malignant neoplasia (53.9 %), chronic lower limb ulceration (16.7 %) and upper limb trauma (13.7 %>. The staphylococci (S. aUYeUS and coagulase negative staphylococci) were the organisms most commonly isolated from the first skin sample (SSGl). 58 grafts showed no growth. More than one organism was grown from 11 grafts. 83 skin samples were sent for analysis after 3 weeks storage. The stored skin was applied to patients in 10 cases. SSG2 was not sent for analysis in 9 cases. At 3 weeks staphylococci still predominated, but the number of grafts colonised by coliforms, acinetobacter species and pseudomonas species had increased markedly. Moraxella species, which were not found on SSG 1, appeared and alpha-haemolytic streptococci were no longer found. The complete data for both time periods are shown in Table 1. The number of organisms contaminating grafts ranged from CUl.1 x 10’ (median 0) for SSGl and 0-5.0x 10’ (median 9.6 x 103) for SSG2. These data were skewed and transformations did not improve the shape. Therefore non-parametric statistical analyses were used. Percentage take was plotted against organisms/g of skin for SSGl (Fig. 1). A negative correlation was found (r = -0.24; p < 0.05-Spearmans rank correlation). That is, as organisms/g of skin increased, percentage take of the skin graft decreased. In 83 patients SSG2 was analysed after 3 weeks storage. For these patients the number of organisms/g of skin colonising SSGl and SSG2 were compared (Fig. 2). A highly significant increase was demonstrated in the median number of organisms/g contaminating SSG2 (9.6 x 103) compared to SSGl (0) (p < O.OOOl-Wilcoxon rank sum test). Secondly, 48 grafts (57.8%) in the first group showed no growth; after 3 weeks storage this had fallen to 18 (21.7 %) (chisquared = 22.64, p < 0.001). Similarly, the number of grafts growing greater than lo5 organisms/g of skin was significantly greater for the second samples, 32 (38.6 %), compared to the first, 3, (3.6 %) (chi-squared = 30.44, p < 0.001). There was no significant difference between the median number of organisms/g contaminating grafts
Table 1 Results
Over a 4-month period 103 consecutive patients requiring split-thickness skin grafts were entered into the study. One patient was subsequently excluded from all analyses as no samples were sent for microbiology. There were 56 males, mean age 59.2 years (range 5-90) and 46 females, mean age 66.4 years (range 7-88) (t = 1.74; p = 0.08, n.s., two sample t-test). The indications for surgery were predominantly *
CH(alc) = chlorhexidine gluconate 0.5% w/v in 70% IMSDePuy Healthcare. Pearce Laboratories, Leeds. UK. CH(aq) = aqueous chlorhexidine gluconate 0.05% w/v. Sterets Unisept--Seton Healthcare Group, Oldham. UK. Bet = Betadine, Povidone-iodine USP 7.5 % w/v-Napp Laboratories Ltd. Cambridge Science Park, Cambridge, UK.
Organisms grown from SSGl and SSG? skin
samples
Orgarzism
SSGI
Coagulase negative staphylococci s. auW_eus Alpha haemolytic streptococcus
28 II 4
SSG2
I8 10 0
Bacillus sp
3
4
Coliforms
4 I
I4 I3
Acinetobmter sp Micrococcus sp Pseudot?lorlos sp
**Coryneforms Fungi Moroselln sp Agrobncterium
No growth Total number
”
sp
of grafts
I
3
I
6
I
3
I
4
0 0
5
58 I02
I IX 83
26
British Journal 100
AA .nAauYI M A
MA&l
A
AA A
90
of Plastic Surgery
A A A
A
80
AA
A
A
A
A
A
70
A A 10 -A 00 0
A
I
I
I
1
1
/
I
1
1
2
3
4
5
6
7
8
Organisms/g (log) for SSGl Fig. 1 Figure l-Organisms/g
(log 10) colonising SSGl v percentage take of SSGl.
A
A
0
’ SSGl GRAFT Fig. 2
Figure 2-Organisms/g log for SSGl & SSGZ. The broken line on the graph represents the log,,,, of 10’ organisms/g of skin. It should be noted that the open triangles for SSGl and SSG2 at 0 represent 48 grafts and 18 grafts respectively.
for the different grades of surgeon, skin preparations used, types of skin graft, mode of anaesthesia (Kruskal Wallis one way analysis of variance); or cutting tools (Wilcoxon rank sum test) for both sets of samples. Male patients had a significantly higher median number of organisms/g of graft than females for SSGl
(p = 0.02). However, there was no significant sex difference after three weeks storage (Wilcoxon rank sum test). It was assumed that the temperature of the refrigerator at Wordstey was appropriate for skin storage. To test this hypothesis its temperature was
Stored Skin-Stored
27
Trouble?
-TOP
16
-----
MIDDLE
--.------
BOTTOM
14 c
12
f 5
10
E c”
6 6
Figure STemperature
Table 2 Bacteriology
changes in Wordsley skin fridge over 24 h.
and percentage
take of stored
skin
applied to 8 patients Take
Bacteriolog?
Count
S. aureus Ps. aeruginosa
2.2 x 10’ 1.6 x lo” 6.7 x lo4
60 % 80% 80%
9.7 x 104
80 %
“Coryneforms” Coagulase negative staphylococci “ Coryneforms ” Proteus sp Esch. cofi
2.4 x 10”
0
Coagulase negative staphylococci
3.3 x 106
100%
2.3 x 10’
0
5.3 x 10’
0
S. aureus Acinetobacter sp Acinetobucter sp
monitored over a 24-h period. Probes were placed on the top, middle and bottom shelves and the temperature recorded at 30 min intervals. The results are presented graphically in Figure 3. The temperature on the top shelf oscillated between 8.6 and 13.9”C with a mean of 9.8; the middle shelf (where our skin was kept) ranged from 6-l 1°C with a mean of 7.3 and the bottom ranged from 0.3-6.7”C with a mean of 4. The refrigerator door was opened once during the period monitored (at about 1500h) and the temperature on the top shelf peaked at 13.9”C (arrowed). These storage conditions may have been peculiar to our unit and are perhaps not applicable elsewhere. However, on contacting 44 major plastic surgery units by phone, the majority (20) were found to store skin grafts in a domestic refrigerator. Most others used the smaller ward drug fridges (16). Only 2 units used a fridge designed specifically for tissue storage and 6 units were unable to provide the information re-
quested. About 53 % (23) stored only skin in the fridge and 45% (20) stored skin with a variety of other things, including drugs and blood. Stored skin was applied to 10 patients, between 6 and 15 days after harvesting. Data were available for 8 patients and are shown in Table 2. All grafts were contaminated by organisms with counts ranging from 2.2 x lo’-5.3x lo7 organisms/g of skin. Seven different organisms were identified. The number of grafts involved was too small to draw firm conclusions; however, there seemed to be a correlation between a high number of organisms/g and significant failure of these grafts. Discussion
Refrigerated storage of split-thickness skin grafts is a widespread, if not universal, practice in plastic surgery. Despite advances in storage techniques described in the literature, most units use methodology unchanged for nearly 50 years. Built into this method is the use of non-standardised refrigerators. This study has clearly demonstrated that these storage conditions allow significant bacterial proliferation over a 21-day storage period. Earlier work seems to have largely neglected the bacteriology of refrigerated, stored skin grafts. Matthews4 stated some grafts were sterile and others were “infected with normal skin contaminants”. He stated that this had no harmful effects, but did not give any details. Skoog,g using full thickness grafts from white laboratory rats, found “considerable numbers of bacteria present after a week at 3°C” and that the addition of antibiotics provided “satisfactory pro-
28
tection” against this for about three weeks. Krizek’s worklo demonstrated the relevance of bacterial count/g of tissue in the graft bed to skin graft take. It was shown that when counts exceeded lo5 organisms/g then poor graft take resulted. We believe our study is the first to demonstrate that number of organisms/g of skin graft applied is also relevant to graft take; take was shown to decrease as organisms/g increased. As with earlier quantitative bacteriological studies, relevant clinical effects occurred irrespective of the actual contaminating organism. However, in contrast to these studies, an effect was shown for levels of less than lo5 organisms/g. In our study the bacteriology of the graft bed was not assayed. This may be a relevant factor, particularly for delayed application of stored skin. However, the vast majority of defects grafted were primarily created by surgery and delayed grafting was not used. Therefore, one would expect the graft bed to have had an insignificant bacterial count. This is in contrast to the studies of Krizek, who grafted onto granulations, which were colonised by a significant number of organisms. The association demonstrated between skin graft take and organisms/g for SSGl was relatively weak (r = - 0.24) however, 96.4 % of these grafts were contaminated by less than lo5 organisms/g. In contrast, after 3 weeks storage nearly 40% of grafts were contaminated by greater than lo5 organisms/g and the number of grafts showing no growth fell significantly. After 3 weeks, with such bacterial levels, it seems reasonable to assume that a greater association between take and count would be demonstrable. This hypothesis is supported by the Krizek data previously discussed. This is clearly of concern if skin stored for long periods is regularly used in treating patients. Stored skin was only applied to 10 patients (9.8 %) and data were available for only 8 of these. However, we feel it is worth noting that all grafts were contaminated and 3 grafts failed completely. There appeared to be a correlation between a high number of organisms/g and graft failure. However, it would be unwise to draw firm conclusions from such a small database and this area of the study warrants further investigation. A further problem when assessing take of stored skin graft is the less than ideal graft bed compared to the original defect. The degree of contamination of initial and 2 1-dayold stored skin grafts was shown to be independent of grade of surgeon, skin preparation used, type of skin graft, cutting tool used and mode of anaesthesia. We accept that these variables were not compared in a true controlled, randomised fashion, as we felt the numbers necessary for this would have made the study impracticable. Male patients were found to have more organisms/g than females for the initial grafts. The reason for this is not clear but may have been related to shaving donor sites in males. Preoperative shaving causes thousands of microscopic wounds which may potentiate bacterial multiplication, particularly if the patient is shaved on the ward several hours before theatre. Prospective studies have confirmed that shaving increases postoperative wound infection rates.14.‘”
British
Journal
of Plastic
Surgery
The refrigerator used to store skin grafts in our unit was basically a domestic unit adopted for the storage of human tissue. Such practice is not exceptional; almost half of major UK plastic surgery units used such appliances. These refrigerators were shown to have a variable and cyclical temperature range with different peaks and troughs on different shelves of the appliance. This investigation has shown the necessity of strict temperature monitoring of such refrigerators, in the same way as is routine in blood banks. It is of concern to note that some units stored drugs and blood products together with skin grafts in appliances which have the potential for facilitating bacterial (and fungal) growth on skin grafts. With the exception of Acinetobacter species the organisms found in this study do not multiply at temperatures below 4’-‘C.16Therefore we conclude that the type of refrigerator in which skin was stored in our unit facilitated significant bacterial proliferation. Clearly the domestic type of refrigerator is not appropriate for skin storage. The lowest temperature range was at the bottom of our refrigerator and until a better alternative is found grafts should probably be stored at that level. Further studies are planned to investigate the quantitative bacteriology of grafts stored in the smaller ward drug refrigerators and of grafts stored in designated tissue refrigerators, such as found in a blood bank.
Acknowledgements We gratefully Russell’s Hall not have been allowing their
acknowledge the help of the staff at Wordsley and Hospitals, without whose assistance this study would possible and thank the consultant plastic surgeons for patients to be included in this study.
1. Wentscher J. Die verwendung konservieter hautlappen bei der transplantation nack Thiersch. Klin Wschr 1896; 31: 979. Cited by Perry VP. A review of skin preservation. Cryobiology 1966; 3: 109930. 2. Wentscher J. Ein weiter beitrag zur uberlebenfahigkeit der menschlichen epidermiszellen. Deutsch Z Chir 1903: 70: 2144. Cited by Perry VP. A review of skin preservation. Cryobiology 1966: 3: 109-30. 3. Carrel A. The preservation of tissues and its application in surgery. JAMA 1912; 59: 523-7. 4. Matthews DN. Storage of skin for autogenous grafts. Lancet 1945; 1: 775-8. 5. Webster JP. Refrigerated skin grafts. Ann Surg 1944; 120: 43 149. 6. Flatt AE. Refrigerated autogenous skin grafting. Lancet 1948; 2: 249-51. 7. Perry VP. A review of skin preservation. Cryobiology 1966; 3: 109-30. 8. Georgiade N, Peschel E. Georgiade R. Brown 1. A clinical and experimental investigation of the preservation of skin. Plast Reconstr Surg 1956; 17: 267-75. 9. Skoog T. An experimental and clinical investigation of the effect of low temperature on the viability of excised skin. Plast Reconstr Surg 1954; 14: 403-16. 10. Krizek TJ, Robson MC, Kho E. Bacterial growth and skin graft survival. Surgical Forum 1967; 18: 518-9. I 1. Elek SD. Experimental staphylococcal infections in the skin of man. Ann-NY Acad Sci-1956; 65: 85590. 12. Kass EH. Bacteriuria and the diagnosis of infections of the urinary tract. Arch Inter Med 1977; 100: 709-14. 13. Lindberg RB. Moncrief JA. Switzer WE. Order SE, Mills W.
Stored Skin-Stored
29
Trouble?
The successful control of burn wound sepsis. J Trauma 1965; 5: 601-16. 14. Cruse PJE. Foord R. A five year prospective study of 23,649 surgical wounds. Arch Surg 1973; 107: 20610. 15. Alexander JW, Fischer JE, Boyajian M. Palmquist J. Morris MJ. The influence of hair removal methods on wound infections. Arch Surg 1983; 118: 347-52. 16. Parker MT. Collier LH, editors. Topley and Wilson’s principles of bacteriology. virology and immunity, 8th ed. Edward & Arnold. 1990.
Unit, Wordsley Hospital, Stream Road. Wordsley. West Midlands. DY8 5QX. Michael Cooper, MB ChB, MRCPath, Senior Registrar Microbiology. Russell’s Hall Hospital. Dudley, West Midlands, DYl 2HQ. Andrea Thomas, BSc, Statistician. West Midlands Regional Health Authority. 142. Hagley Road, Edgbaston, Birmingham, B16 9PA. Kevin Hancock, MB BS, FRCS, Senior Registrar Plastic Surgery, West Midlands Regional Plastic and Jaw Surgery Unit, Wordsley Hospital. Stream Road. Wordsley. West Midlands, DY8 5QX.
The Authors
Requests for reprints to Mr 0. G. Titley, Department of Plastic Surgery, Royal Victoria Infirmary. Newcastle Upon Tyne.
Oliver Garth Titley, MB ChB, MSc, FRCS, Senior House Officer Plastic Surgery, West Midlands Regional Plastic and Jaw Surgery
Paper received 4 May 1993. Accepted 20 July 1993. after revision.