The role of sampling in the detection of microbial contamination on cadaveric allograft skin used as a biological wound dressing

36

Burns (1985) 12,36-48

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The role of sampling in the detection of microbial contamination on cadaveric allograft skin used as a biological wound dressing S. Randolph May and James F. Wainwright Southeastern Burn Research Institute, Humana Hospital Augusta Burn Center, and the Department of Surgery, Medical College of Georgia, Augusta, Georgia, USA

Frederick A. DeClement Saint Agnes Medical Center and Burn Center, and the Department of Surgery, Hahnemann University School of Medicine, Philadelphia, Pennsylvania, USA Summary The availability of cryopreservation and low temperature storage techniques for cadaveric allograft skin allows it to be preserved while microbial assessments arc made before its use as a temporary biological dressing on burn wounds. In a 300-donor, 5-year prospective study, we tested ten skin samples from defined areas on each donor for microbiological contamination. Although the skin from 52.3 per cent of the donors possessed some detectable residual microbial contamination after surgical body preparation and skin removal, such contamination was limited to an average of 1.4 areas per body, leaving 86 per cent of all skin obtained free from detectable contamination and suitable for use as biological wound dressings. The number of skin samples tested per donor body determined the accuracy of detection of the presence of contamination. Testing one skin sample per donor body yielded a correct skin assessment 92 per cent of the time, while testing five skin samples increased the accuracy to 96 per cent, and testing ten skin samples yielded a 99.9 per cent accuracy in detection of skin contamination. Thus, it is within the ability of a skin bank to set the limits of microbiological risk to patients receiving processed cadaveric allograft skin.

INTRODUCTION THE usefulness of cadaveric allograft skin for temporary burn wound closure has been confirmed many times (Brown and McDowell, 1942; Artz et al., 1955; MacMillan, 1962; Bondoc and Burke, 1971; Rudolph et al., 1979; Shuck, 1979;

Wachtel et al., 1979), but the potential for the transmission of infection from such skin onto debilitated, immunologically incompetent burn patients remains an issue of some concern (Monafo et al., 1976; May and DeClement, 1981b; Heck et al., 1981; DeClement and May, 1983; May, 1983,1985; May et al., 1985). During the past decade, the increased number of skin banks associated with burn treatment centres in the United States has facilitated the low temperature storage of allograft skin between procurement and utilization, and this storage period has provided the time necessary for the adequate microbiological assessment of the skin prior to grafting onto patients. Indeed, current standards promulgated by the American Association of Tissue Banks state that an important responsibility of the modern skin bank is to minimize the potential for infection of patients’ burn wounds with contaminated allograft dressings (Standards Committee, Skin Council, American Association of Tissue Banks, 1979; Standard C1.320, Standards Committee, American Association of Tissue Banks, 1984). Transmission of microbial contamination may be stopped by first testing the allograft skin for the presence of contaminant organisms, and then by excluding contaminated skin from transplantation onto patients’ wounds. The skin excluded from grafting may be any allograft with

May et al.: Contamination

on cadaver skin biological dressings

(May and DeClement, 1981b; DeClement and May, 1983; May, 1983, 1985; May et al., 1985), or may be only that skin which is contaminated with pathogenic bacteria, fungi or yeast (Heck et al., 1981). The usefulness of a contaminant exclusion technique for allograft skin depends upon defining the minimum body surface area over which the presence or absence and the type of microbiological contamination remain constant. A single microbiological quality assessment sample obtained and tested from each of these minimal body surface areas can accurately reveal what skin is uncontaminated and useful for grafting, and what skin is contaminated and thus might be excluded from grafting. We have undertaken a prospective investigation of 300 cadaveric allograft skin donors obtained over a S-year period. Microbiological assessments were performed on skin samples from ten defined body areas on these donors. The data were used to determine to what degree analyses of skin samples from fewer than ten body areas would decrease the accuracy of the microbiological assessment of the skin. The object of the study was to determine the number of skin samples per donor body which needed to be tested in order to maintain a defined probability that contaminated skin would not be grafted onto the wounds of burn patients, and to determine the relative costs of such assessment. contamination

MATERIALS AND METHODS Procurement of skin donors Between 5 April 1978 and 24 June 1983, skin was obtained post-mortem from 300 donors by the Saint Agnes Medical Center Skin Bank. The sources of these donors were the Office of the Philadelphia Medical Examiner and hospitals in the Philadelphia regional area. The parameters of the donor population used in this study have been previously reported in this journal (May et al., 1985). The total amount of skin obtained from the 300 donors was 147.23 m* (1583.16 ft*). To assure that the dermal side of the skin was free from microbiological contamination present locally or systemically in the donor (DeClement and May, 1983; May et al.. 1984, 1985), skin was removed and processed only from donors free from the following conditions: sepsis or bacteraemia. dermatitis or other infection of the skin, pneumonia or other respiratory infection, addiction to controlled substances or overdose from a controlled substance (due to risk of hepatitis), toxic or viral hepatitis, jaundice (potentially due to viral hepatitis), treponemal antigen or anti-

37

body and leprosy. To prevent the possible transmission of oncogenic viruses, other donor exclusions included malignancy, cancer chemotherapy or radiation therapy. Poisoning was excluded because of potentially adverse effects to the wound bed or system of the graft recipient. Finally, logistical considerations mandated the exclusion of skin donation from bodies which were undernourished at death (due to difficult skin removal), which possessed autoimmune or collagen diseases affecting the integrity of the skin, or which had sustained burns greater than 50 per cent of the body surface area. Independent body area skin removal protocol To assure that the skin to be removed from the donors was surgically clean on its epidermal side, an independent body area (IBA) surgical preparation was carried out on ten defined body areas (May and DeClement, 1981b; DeClement and May, 1983; May et al., 1985). The areas and their abbreviations in this study were as follows: left chest (LC), left back (LB), left anterior thigh (LA), left posterior thigh (LP), left lower leg (LL), right chest (RC), right back (RB), right anterior thigh (RA), right posterior thigh (RP) and right lower leg (RL). The areas were surgically prepared by the sequential application of povidone-iodine and detergent, sterile water rinse, povidone-iodine solution, and 70 per cent isopropanol according to a previously published protocol (May and DeClement, 1980; May and DeClement, 1981b; DeClement and May, 1983; May et al.. 1984, 1985). After surgical draping of the donor body, a Brown dermatome (Zimmer USA, Warsaw, Indiana, USA) was used to remove skin 7.6cm (3 in.) wide, at least 2O.Ocm (gin.) in length, and 0.38mm (0.015 in.) thick sequentially from single IBA sites, and the dermatome blades were changed and the dermatome cleaned with 70 per cent isopropanol between the removal of skin from each body area. A blood sample of at least 7 ml volume was obtained from each donor, usually by intraventricular cardiac puncture with an l&gauge, 8.9 cm (3.5 in.) needle mounted on a disposable plastic syringe. The cumulative amount of skin obtained from body areas on 300 donors was as follows: LC, 9.037 m2 (97.17 ft*); LB, 18.445 m’ (198.33 ft*); LA, 20.383 m* (219.17 ft2); LP, 17.252 m* (185.50 ft*); LL. 8.448 m* (90.83 ft’); RC, 9.347 m’ (100.50 ft*); RB, 18.631 m* (200.33 ft*); RA, 20.491 m* (220.33 ft2); RP. 16.926 m* (182.00 ft*); RL, 8.277 m* (89.00 ft2). The ten mean amounts of skin obtained per

38

donor from each of the ten body areas ranged from 0.02760.0683 m2 (0.287-0.734 ft2).

Skin processing Skin from each body area was placed into a separate air-tight container of 4” C transport medium (TM). After harvest, the skin was placed into 4°C cryopreservation medium (CM) for at least 1 h prior to the taking of microbiological samples. In this study, TM was composed of either Tis-u-Sol (Travenol Laboratories, Deerfield, Michigan, USA) or Eagle’s Minimal Essential Medium-Earle’s Balanced Salt Solution with 10 per cent (v/v) pooled human sera and 2mM fresh L-glutamine (Eagle, 1959). Cryopreservation medium was composed of Eagle’s Minimal Essential Medium-Earle’s Balanced Salt Solution with 10 per cent (v/v) pooled human sera, 2 mM fresh L-glutamine and 15 per cent glycerol (reagent grade, J. T. Baker Chem. Co., Phillipsburg, New Jersey, USA), in keeping with previously published work (May and DeClement, 1980; May and DeClement, 1981b, 1981~; May and DeClement, 1982; DeClement and May, 1983; May, 1983; May et al., 1984; May and Wainwright, 1985a, 1985b; May, 1985). Constituents of TM and CM were purchased from Difco Laboratories, Detroit, Michigan, USA; Microbiological Associates, Walkersville, Maryland, USA; or Flow Laboratories, Inc., McLean, Virginia, USA.

Microbiological evaluation The skin from whole bodies was tested for contamination with syphilis and hepatitis B virus based upon assays of each donor’s serum for (a) Treponema pallidurn based on a microhaemagglutination assay (MHA-TP, Seratek Kit, Ames Co., Div. Miles Labs, Elkart, Indiana, USA) performed after a positive syphilis screening test (RPR, Hyson, Westcott and Dunning, Inc., Baltimore, Maryland, USA), and (b) tests for the presence of hepatitis B surface antigen and antisurface antigen antibody (Ausria II-125 and Ausab Diagnostic Kits, Abbott Laboratories, North Chicago, Illinois, USA) (May and DeClement, 1981b). Skin from individual body areas was evaluated for the presence of bacteria by a previously published method (May and DeClement, 1981b; DeClement and May, 1983; May, 1983; May included et al., 1984, 1985). That method homogenizing l-cm’ full-thickness skin biopsies

Burns (1985)Vol. 12/No. 1

from each body area in 2 ml of sterile 0.9 per cent NaCl using a 4-ml Teflon pestle tissue grinder driven by a slow-speed motor (Arthur Thomas, Philadelphia, Pennsylvania, USA), and then placing a O-1 ml aliquot of each homogenate onto two plates of tripticase-soy agar with 5 per cent sheep red blood cells (one each for aerobic and anaerobic incubation), one chocolate agar plate (CO2 incubation), and one eosin-methylene blue agar. Bacterial media were incubated at 37” C for 48 h. Fungal determinations were performed by placing 0.25 ml of the homogenate onto a Sabouraud dextrose agar slant and O-25 ml onto a Mycosel agar slant. Fungal media were incubated at 30” C for 4-6 weeks. The presence of acid-fast bacilli, including Mycobacterium tuberculosis, was tested by placing 0.25 ml of the homogenate onto a Lowenstein-Jensen agar slant and O-25 ml onto a Middlebrook and Cohn agar slant. Cultures for acid-fast bacilli were incubated at 37” C for 8 weeks. Media were obtained from BBL (Cockeysville, Maryland, USA). Microbial isolates obtained from the agar plates were identified according to routine microbiological procedures.

Validity of the microbiological evaluation After the procurement of the initial skin samples for microbiological testing, skin was cryopreserved according to a previously published protocol consisting of - 1”C mm’ cooling, - 196” C storage, and warming at rates of approximately by immersion +260” C min-’ to +458” C mini in 20” C-37” C water (May and DeClement, 1980; May and DeClement, 1981~; May, 1983; DeClement and May, 1983; May and Wainwright, 1985a, 1985b; May, 1985). When packets of skin which were contamination-free according to tests performed on the initial samples were opened after warming just prior to grafting, a l-cm2 full-thickness biopsy was obtained for a second microbiological evaluation. This procedure was performed on 1477 packets of sequentially processed skin. The skin from only two of these packets was found to possess microbiological contamination; the contamination in both cases Staphylococcus epidermidis (May and was DeClement, 1981b; DeClement and May, 1983). This finding revealed that when a IO-area IBA testing protocol was used for donor body preparation, skin removal and skin processing, there was a 0.14 per cent misclassification of skin as uncontaminated when in fact it was contaminated. This extremely small error rate validated the testing procedure.

May et al.: Contamination on cadaver skin biological dressings

Experimental design for comparison of microbial sampling rates We undertook an investigation of the

relationship between the number of microbiological assessment samples per donor and the accuracy

of that assessment using a protocol we developed previously (May and DeClement, 1981b; DeClement and May, 1983; May, 1983, 1985). We conducted analyses which determined the specific body areas which were contaminated on the skin obtained from each of the 300 donors. We also determined the amount of skin obtained from each body area (see Independent body area skin removal protocol). Next, we defined microbiological assessment groups made up of the ten original body areas (Table I). Each assessment group consisted of contiguous body areas which were assigned to a single skin sample for microbiological evaluation. Body areas were arbitrarily assigned to the role of test sites for contiguous body areas, since in a previous analysis of this group of 300 donors there was a uniform distribution of contamination across all ten body areas (May et al., 1985). For example, if only one skin sample were to be used to classify skin from the whole body, then a skin sample tested from the right chest (RC) was arbitrarily chosen to apply to all ten body areas (7’abfe I). In a similar fashion, if the body were to be tested using only three skin samples, then the evaluation of the sample obtained from the right chest (RC) would apply to the anterior and posterior trunk (RC, LC. RB, LB), the evaluation of the sample obtained from the right anterior thigh (RA) would apply to the right leg (RA, RP, RL), and the evaluation of the sample obtained from the left posterior thigh (LP) would apply to the left leg (LA, LP, LL) (Table I). Using this method, we determined which body areas would be assigned to contaminated and uncontaminated skin groupings if l-9 skin samples were evaluated per donor body. Of course, the actual classification of skin from each body area into contaminated and uncontaminated groupings was known from the IO-area IBA tests, and the assignment of skin based upon l-9 samples per donor body could be compared directly with the lo-area classification. From this experimental design, the amount of skin mistakenly assigned to contaminated and uncontaminated groups could be computed for each donor body, and for the 300 body sample as a whole (Table V). There were two types of skin misclassification: contaminated skin misclassified as uncontaminated, and uncontaminated skin misclassified as contaminated (Table V). The former represented a risk of

39

wound contamination to the allograft recipient, while the latter represented the loss of usable Table 1. Assignment microbial

assessment

of independent

Number of microbial Body area assessment groups tested per donor body

RC

4

5

6

7

8

9

10

body

areas

to

group9

RC LB RC RA LP RC LB RA LP RC LP LB RA LP RC LB LA RA LP RP RC LB LA RA LP RP RL LC LB LA LP RA RP LL RL LC RC LB RB LA RA LP RP LL

Body area(s) covered by tested body area All ten RC, LC, RB, LB, RC, LC, RA. RP. LA; LP; RC, LC RB, LB RA, RP, LA, LP, RC, LC LA, LP RB; LB RA, RP RL. LL Rt, LC RB, LB LA RA LP, LL RP, RL RC. LC RB, LB LA RA LP RP RL, LL LC, RC LB, RB LA LP RA RP LL RL LC RC LB RB LA RA LP RP LL, RL

!?A, LA RP, LP, RL, LL RB, LB RL LL

RL LL

Each area tested independently

a Abbreviations: RC, right chest; FIB, right back; RA, right anterior thigh; RP, right posterior thigh; RL, right lower leg; LC, left chest; LB, left back; LA, left anterior thigh; LP. left posterior thigh; LL, left lower leg.

Burns (1985) Vol. 12/No. 1

40

skin and lost cost reimbursement for the procurement and processing of the skin. While in an earlier study (May and DeClement, 1981b), we assumed relatively equal body areas in determining the percentage of skin misclassification as a function of number of body areas sampled, in the present work we have used the amounts of skin actually obtained from each individual body area tested from each donor in the computation of the percentages of skin misclassified in terms of the presence or absence of microbiological contamination. Skin bank survey Written surveys of skin banks were undertaken by one of the authors (S.R.M) in 1979 (May and DeClement, 1981a, 1981~) and in 1983 (unpublished data) on behalf of the Standards Committee of the Skin Council of the American Association of Tissue Banks. The survey documents were sent to all skin banks in the United States which were known to the Skin Council. One of the questions asked in both surveys was the routine number of skin samples obtained per donor body by individual skin banks. Information on this question was obtained from 9 skin banks in 1979 and from 12 skin banks in 1983. Statistical methods The differences between the amount of contamination present on whole donor bodies and that present on independent body areas was tested by the method of Chi-square. The frequency of isolation of residual contaminant species on whole bodies and independent body areas was also tested by the Chi-square method. After best curve fitting by the method of least squares, Pearson’s linear regression analyses were used to determine the degree of correlation (r) between (a) the percentage of uncontaminated skin misclassified as contaminated and the number of body areas tested per donor, and (b) the percentage of contaminated skin misclassified as uncontaminated and the number of body areas tested per donor. Student’s t-test for independent samples (twotailed) was utilized to test whether the mean number of skin samples obtained per donor body for microbiological assessment differed between surveys of US skin banks conducted in 1979 and 1983. Statistical significance in each test was defined as PSO.05. The effect of lost cost reimbursement due to the discarding of uncontaminated skin misclassified as contaminated was computed based upon

a cost of $329.52 (US Dollars) per O-093 m* (l.Of?) of skin. This skin cost was the mean amount charged by 26 US skin banks according to a 19831984 survey (Hornbokel et al., 1985). The cost estimate for microbiological evaluation of a single body area was based upon the cost of bacterial growth media, diagnostic reagents and personnel time needed to (a) homogenize and plate the sample on three types of media for bacteria, two types of media for fungi and yeast, and two types of media for acid-fast bacilli, (b) observe the cultures for the defined periods and (c) identify any contaminants isolated. RESULTS Residual contamination on cadaveric allograft skin Table II lists the number of donors in our sample which yielded contaminated skin from O-10 body areas. It can be seen from the table that the skin from 46.7 per cent of donors was contaminationfree, while 52.3 per cent of donors yielded contaminated skin from at least one body area. In 300 donors, the mean number of areas per donor from which contaminated skin was obtained was 1.39, while the mean number of body areas from which uncontaminated skin was obtained was 8.61. Thus, 86.1 per cent of all skin obtained by the IO-area IBA protocol on 300 donors was determined to be contamination-free by the tests employed. Because jkin from only a few areas per donor body was contaminated, there was a significant difference between the number of donor bodies yielding contaminated skin and the number of Table II. Number of microbiologically contaminated areas per donor body in=300 donors) Number of contaminated body areas per donor 0 1 2 3 4 5 6 7 8 9 IO Mean Standard deviation Standard error of the mean Median

Frequency

(%)

140 (46.7%) 61 (20.3%) 34 (11.3%) 28 (9.3%) 14 (4.7%) 10 (3.3%) 5 (1.7%) 3 (1.0%) 2 (0.7%) 0 (0.0%) 3 (1.0%) 1.39 1.92 0.11 1.00

May et al.: Contamination

41

on cadaver skin biological dressings

Table 111.Comparison of the residual microbiological contamination rate of cadaveric allograft skin based upon whole body assessment and independent

body

area

assessmenta

Percentages Whole

Contaminating

49.0% 7.0% 0.3%

Mycobacterium

tuberculosis

acid-fast

(147) (21) (I)

0.0%

(Frequencies)

Independent

body

areas

(n=3000)

(n=300)

agent

i3acteriab Fungi Yeast and other

bodies

(0)

12.8% 1.0% 0.03%

0.0%

(384) (31) (1)

(0)

bacilli

a The presence of Treponema pallidurn in 8 or 2.7 per cent of the donors, and the potential of hepatitis B virus in 7 or 2.3 per cent of the donors (antiHBs+/HBsAg+ occurred in 3 or 1 per occurred in 4 or 1.3 per cent of the donors, and anti-HBsilHBsAgcent of the donors) are detected on the basis of serum assays which apply to both whole bodies and to all independent body areas on those donor bodies, so the comparative contamination rates are the same. b Excluding Treponema pallidurn.

body areas yielding contaminated skin (Table III). For example, some skin from 49 per cent of the 300 donors was contaminated with bacteria, but only 13 per cent of all 3000 donor body areas tested were contaminated by these organisms. Similarly, fungi contaminated some skin from 7 per cent of the donors, but only 1 per cent of all body areas tested were contaminated with fungi. Finally, yeast contaminated some skin from 0.3 per cent of the donors, but only 0.03 per cent of all body areas tested were contaminated with yeast. The differences between the amount of contamination present on each donor body taken as a whole and that present on independent body areas was significant statistically in the case of bacterial (x2=264.7, 1 df, P
Species of residual microbiological contamination The number of donor bodies and the number of donor body areas which yielded skin contaminated with specific microbial species in this 300donor study are listed in Table IV. We have found that the frequencies of recovery of individual species of microorganisms from any piece of skin from a donor body and from the independent donor body areas are significantly different statistically (x2 values for the differences within individual species ranged from 4.05 to 303.13, 1 df, P values ranged from
which the difference was not statistically significant (x2=1.88, 1 df,P=O.17). Residual contamination by Staphylococcus epidermidis was by far the most prevalent, and was recovered from nearly 10 per cent of all the donor body areas. S. epidermidis accounted for 63 per cent (299/478) of all microbial isolates recovered (Table IV). Many donor bodies possessed multiple independent areas with residual so that a total of S. epidermidis contamination, 299 areas were contaminated with this organism on 137 donor bodies. Other frequently isolated contaminants included Staphylococcus uureus, Micrococcus spp., Escherichia coli, and Asper@ lus spp. (Table IV). Ail other microbial contaminants were isolated at levels below 0.8 per cent of skin obtained. Generally, then, the tested skin samples exhibited normal skin flora, primarily of the staphylococcus genus. Very few anaerobic bacteria, pathogenic bacteria, tungl or yeast were isolated from the skin samples. Multiple species were isolated from 61 body areas, or 2 per cent of the total. The multiple isolates were usually S. epidermidis and E. coli (25 body areas), S. epidermidis and S. aureus (16 body areas), or S. epidermidis and Micrococcus spp. (8 body areas). Only one body area out of 3000 possessed more than two species of residual microbiological contamination. Bacterial and fungal contamination were never found together on a single body area. Skin misclassification The data presented thus far have shown that more provably uncontaminated skin might be obtained if skin were removed from the donor by

42

Burns 11985) Vol. 12/No. 1

Table IV. Comparison of the frequency of isolation of residual microbiological contaminants from cadaveric allograft skin collected from 300 donors

Contaminating

agent

Bacteria Staph ylococcos epidermidis Staphylococcus aureus Micrococcus spp. Escherichia coli Bacillus subtilis Enterobacter cloacae Enterococcus ~pp.~ Propionibacterium acnes Streptococcus viridans Clostridium perfringens Corynebacterium spp. Klebsiella pneumoniae Pseudomonas aeruginosa Neisseria meningitidis Streptococcus agalactiaeb Fungi Aspergillus spp. Scopulariopsis spp. Penicillium spp. Petriellidium bodyii Rhizopus spp. Yeast Torulopsis glabrata

Frequencies Whole bodies (n=300)

137 27 19 13 7 5 5 4 3 2 2 2 2 1 1

(45.7%) (9.0%) (6.3%) (4.3%) (2.3%) (1.7%) (1.7%) (1.3%) (1.0%) (0.7%) (0.7%) (0.7%) (0.7%) (0.3%) (0.3%)

13 4 3 1 1

(4.3%) (1.3%) il.O%i (0.3%) (0.3%)

1 (0.3%)

(percentages) Donor body areas (n=3000)

299 (9.96%) 42 (1.40%) 24 (0.80%) 37 (1.23%) 11 (0.37%) 5 (0.17%) 6 (0.20%) 4 (0.13%) 3 (0.10%) 2 (0.07%) 2 (0.07%) 2 (0.07%) 7 (0.23%) 1 (0.03%) 1 (0.03%) 21 5 3 1 1

(0.70%) (0.15%) (0.09%) (0.03%) (0.03%)

1 (0.03%)

a All Group D Streptococci. b A Group B Streptococcus.

a method which separated the body into ten independent areas, rather than treating the donor as a single entity. The next step was to determine the number of areas into which each donor might best be divided. The decision on such partitioning was based upon the amount of skin misclassification and the testing costs associated with partitioning of the donor into l-10 body areas. The amount of skin misclassification depended upon the amount of skin obtained from each defined body area as well as the presence or absence of detectable bacteria. Two types of skin misclassification were possible: that in which contaminated skin was mistakenly classified as uncontaminated, and that in which uncontaminated skin was misclassified as contaminated. The actual amounts and percentages of such misclassification are shown for 300 donors in Table V. Also shown in the table is the amount of cost reimbursement lost by the misclassification of uncontaminated skin as contaminated, and its subsequent loss as transplantable material. From

Table V, it can be seen that 95.7 per cent, 98.2 per cent, 996 per cent and 99.9 per cent correctness in the classification of skin contamination could be obtained by testing five, seven, nine and ten skin samples per donor body, respectively.* Graphs of the decrease in the two types of skin misclassification are shown in Fig. 1. The best-fit linear regression lines correspond to equations (1) and (2) below: C=8.30-0.89N

(1)

U=9,43-l.OON

(2)

where C was the percentage of all skin obtained which was mistakenly classified as uncontaminated when in fact it was contaminated, U was the * These misclassification percentages were determined by averaging the percentages for the two types of misclassification, except in the case of ten skin samples per donor body, where only the contaminated skin misclassified as uncontaminated was used (see Table V, Footnote c).

May et al.: Contamination

V. Mistaken

Table samples

tested

Number of body areas tested per donor 1

2 3 4 5 6 7 8 9 10

classification

per donor

of skin

and

associated

costs

(US

Dollars)

resulting

from

one

to ten

body=

Amount of contaminated skin misclassified and retainedb mz

43

on cadaver skin biological dressings

ft2

IO-96 117.8 10.62 114.2 7.95 85.5 74.3 6.91 5.95 64.0 3.16 34.0 2.33 25.0 1.79 19.2 6.7 0.62 0.09 0.009

%d

7.44 7.21 5.40 4.69 4.04 2.15 1.58 1.21 0.42 0.009

Gross loss per donor if discardede $129 $125 $94 $82 $70 $37 $27

$21 ;i

Amount of uncontaminated skin misclassified and discardedb m2

ft2

13.04 140.2 10.72 115.3 9.69 104.2 7.84 84.3 6.90 74.2 4.07 43.8 3.10 33.3 2.46 26.5 0.51 5.5 0.00 0.0

%d

8.86 7.28 6.58 5.33 4.69 2.77 2.10 1.67 0.35 0.00

Net gain per donor Gross gain per (gross gain donor if minus gross retained loss) $154

$25

$127 $114 $93 $82 $48 $37 $29

$E $11 $11 $11 $9

::

-:': $0

cost of microbial assessment per body” $6.74 $13.48 $20.22 $26.96 $33.70 $40.44 $47.18 $53.92 $60.66 $67.40

a From 147.23 mz (1583.16 ft*) of skin obtained from a total of 3000 independent body areas on a total of 300 donor bodies. b Based upon the assumption that 10 IBA tests per donor accurately detect all contamination, and allow correct classification of skin from each body area. c Excluding serum tests for syphilis and hepatitis B virus. d Per cent of all allograft skin obtained from 300 donors in this study. e Revenue lost per donor due to skin being discarded as a result of correct classification as contaminated using the listed number of body area tests per donor. Cost estimate is based upon the mean amount charged by 26 US skin banks of $329.52 per 0.093 m* fl.Oft*) according to a 19831984 survey (Hornbokel et al., 1985). ‘Revenue gained per donor due to skin being retained and used as a result of correct classification as uncontaminated using the listed number of body area tests per donor. Cost estimate is based upon the data cited in the footnote above. g These zeros indicate that the ten body area comparison is identical to the original data, which was based upon 10 independent body areas. The actual values in the case of contaminated skin misclassified as uncontaminated are 0.031 m*, 0.33 ft2, and 0.14 per cent of all skin (see Materials and Methods), and thus the resultant correct classification is 99.86 per cent.

percentage of all skin obtained which was mistakenly classified as contaminated when in fact it was uncontaminated, and N was the number of body areas sampled per donor. The Pearson linear correlation coefficients (r) for both equations were 0.99. In both cases, the percentage of misclassification of skin was decreased about 1 per cent for every added skin sample tested per body.

COST A single body area microbiological test included sample preparation and the specific culture tests for bacteria, fungi and yeast ($6.50), plus Grampositive ($1.00) and Gram-negative ($5.62) bacterial identification. However, only 2.1 per cent of cultures grew Gram-negative bacteria requiring identification, and only 11.9 per cent of cultures grew Gram-positive bacteria requiring identification. Thus, the total mean cost for a microbiological testing of a single body area was $6.74 (US dollars in 1985).

SKIN BANK SURVEYS Figure 2 shows the distribution of the number of skin banks with regard to the number of skin samples that they tested per donor body in 1979 and 1983. Both surveys showed that very few banks relied on fewer than three or more than seven samples per donor body. The major difference between the results obtained by the two surveys was a narrowing of the distribution with a clustering centred around 56 samples tested per donor body in 1983. The difference between the mean number of body samples (+s.d.) obtained in 1979,6.OOf2.78, was not significantly different from the mean number obtained in lY83, 5.33+ 1.92 (t=0.65, 19 df,P=O.26). DISCUSSION Before the recent proliferation of skin banking facilities in the United States, cadaveric allograft skin employed as a temporary biological wound dressing was used directly from the donor cadaver without microbiological evaluation (Brown

Burns (1985) Vol. 12/No. 1

Contaminated Skin Retained Y = 8.30 - 0.89X r=0.99 Data from 300 donors

0

1

2

3

4

5

6

7

8

9

10

Uncontaminated Skin Discarded Y=9.43-1.00x r=0.99 Data from 300 donors 65432l0

12

3

4

5

6

Number of Areas Sampled

7

8

91

Per Body

Fig. 1. Relationships between the number of areas sampled per donor for microbiological testing and (A) percentage of all skin obtained which was mistakenly classified as contaminated when in fact it was uncontaminated, and (B) percentage of all skin obtained which was mistakenly classified as uncontaminated when in fact it was contaminated.

and McDowell, 1942; Brown et al., 1953; Artz et al., 1955; MacMillan, 1962; Haynes, 1963; Zaroff et al., 1966; Shuck et al., 1969; Shuck, 1979). However, numerous reports concur that both human allograft and canine xenograft skin possess residual pathogenic and non-pathogenic bacteria (Switzer et al., 1966; Monafo et al., 1976; Heck et al., 1981; May and DeClement, 1981b; May et al., 1985; and the present work). The bacterial cleanliness of cadaveric allograft skin used as a biological wound dressing is impor-

tant for three reasons. First, the skin is grafted onto wounds of immunologically compromised and physiologically debilitated burn patients (Ninnemann, 1981, 1983), whose status makes them easily susceptible to infection from contamination remaining on the skin (Monafo et al., 1976). Second, allograft skin requires structural integrity and durability for mechanical wound coverage in order to provide an adequate barrier to the loss of water, protein and heat, and to prevent contamination of the wound by environ-

May et al.: Contamination

on cadaver

skin biological dressings 1979

3 2 2

2

z

1

3

n

k

1963 4

9

z

3

2

2 1 0 0

i

2

NUMBER

j

4

5

6

i

OF MICROBIAL

s

91’0

SAMPLES

Fig. 2. Results of surveys conducted in 1979 and 1983 which determined the number of microbial samples obtained and tested for each donor body by responding skin banks.

mental microorganisms. Many bacteria isolated from processed allograft skin are members of species which have been shown to produce and secrete potent degradative enzymes capable of digesting the structural elements of skin (Davies et al., 1973), potentially reducing the skin’s integrity and durability.* Third, it has been stated that cadaveric skin performs better as a biological dressing if it is viable (see below); however, bacterial enzymes and toxins capable of destroying skin cells may reduce skin viability. The risk of microbial contamination on cadaveric allograft skin obtains because of the requirements of skin viability (Brown et al., 1953; Artz et al., 1955; MacMillan, 1962; Zaroff et al., 1966; Bondoc and Burke, 1971; Rudolph et al., 1979; Wachtel et al., 1979). This requirement precludes treatment of the skin with bacteriocidal agents such as harsh chemicals or irradiation as has been reported (Korlof et al., 1972), since such procedures destroy viability. Instead, * For example, streptococci produce streptolysin, streptokinase, DNAase, hyaluronidase, protease, amylase. and esterase; staphylococci produce haemolysins, lipasc. DNAase, hyaluronidase, and staphylokinase; and the genus Clostridium produces lecithinase, collagenasc and hyaluronida~e.

45

allograft cleanliness can be provided by a combination of prevention, based upon the selection of non-infected donors, and effective aseptic surgical preparation of the skin before its removal from the donor. In a previous paper in this journal (May et al., I985), we have shown that the most important factor in the cleanliness of cadaveric allograft skin is the performance of the surgical operator or team which prepared the donor body and removed and processed the skin. In the present work, we have shown that contamination on cadaveric allograft skin donor bodies is rare and sporadic. Thus, to maximize the probability that all available clean skin will be recognized as such and retained for transplantation, a number of areas must be tested from each body, and the cleanliness of the skin determined in each individual local area. The independent body area (IBA) procurement method was developed by us in 1978 to meet the requirement to maximize the procurement of contamination-free skin. After a pilot investigation on 100 cadaveric donors, we reported that the surgical body cleansing protocol we adopted was sufficient to allow contamination-free skin to be obtained from 92 per cent of all body areas (May and DeClement, 1981b). We would like to state categorically that no cadaveric allograft skin procured and processed by our method should be considered to be sterile. However, such cadaver skin is contaminationfree by defined microbiological testing methods, that is, the level of microbial contamination is lower than the detection limits of the solid agar tests used. A discussion of the relative merits of solid and liquid bacterial testing methods for allograft skin has been published (DeClement and May, 1983). The major difference between liquid and solid agar growth media is that the liquid media are more sensitive in their ability to detect the presence of very small numbers of bacteria; however, there are liabilities inherent in the choice of highly sensitive media when applied to the testing of cadaveric allograft skin. Basically, the liabilities of liquid bacterial growth media can be summarized as follows: (a) they allow fastergrowing organisms to be detected with greater certainty than slower-growing ones due to overgrowth, (b) they preclude quantitative bacterial assessment by serial sample dilution prior to plating onto the growth agar. and (c) they are susceptible to false-positive results generated by lapses in technique and media contamination. For these reasons, we have recommended that solid agar bacterial growth media be used in the

48

microbiological assessment of skin even though such media are slightly less sensitive in the detection of microbial contamination (DeClement and May, 1983). The number of independent areas which are sampled and tested per donor body determines the accuracy of detection of contamination, and determines the risk to recipient patients (May and DeClement, 1981b; DeClement and May, 1983; May, 1983, 1985). In the 300-donor investigation reported here, the ten mean amounts of skin obtained per donor from each of ten body areas ‘ranged from 0.0276-0.0683 m2 (0.2870.734ft2), and that range defined the minimum body surface area requiring testing for a 99.9 per cent accuracy in skin classification with respect to microbial contamination. Put another way, the results of our study indicate that if one out of every three packets of skin, or every 450cm* (72in.*) of allograft, are tested for contamination, then a 99.9 per cent accuracy in detection of skin contamination can be obtained. In a previous study, we found that burn patients in our treatment centre whose wounds were covered with cadaveric allograft received an average of 0.383 m2 (4.12 ft2) of skin (May and DeClement, 1981d). Since that amount of skin represented about 25 packets using our system, or 3800 cm2 (600 in.*) in area, then the present study indicates that if five samples were tested per donor body, the risk of mistakenly placing a contaminated piece of skin onto a recipient patient’s wounds would be 4.04 per centx2.5 packets, or 100 per cent, on average, assuming random independent packet selection. If ten samples are tested per donor, the probability of unknowingly transplanting a piece of contaminated skin onto a patient would be 0.14 per centx25 packets, or 3.5 per cent on average. For this reason, we have chosen to follow a conservative approach in that (a) we have used ten tests per donor body to maximize the probability of detecting microbial contamination, and (b) we have excluded all skin which possesses detectable microbiological contamination from transplantation onto burn wounds. This decision is tenable only because a relatively small percentage (14 per cent) of the skin we procure and process is contaminated; thus, we can hold to a conservative standard yet obtain sufficient allograft skin for our burn patients’ needs. Different surgical operators may procure skin with a different contamination rate than that presented in this report, however, an analysis similar to the one presented here can be performed, curves such as those presented in Figs. la and b can be con-

Burns (1985)Vol. 12/No. 1 strutted, and a considered decision made as to the level of contamination which is acceptable to the individual skin bank or to the surgeons utilizing the allograft skin. We believe that calculations (similar to the ones presented in this paragraph) bearing on the risk to the recipient patient must also be undertaken so that a relatively informed decision can be made as to the number of microbiological samples which should be tested per donor body. While the 1979 survey of US skin banks revealed a wide dispersion in the number of skin samples tested per donor body by the individual banks, the 1983 survey revealed a more central tendency, with a mean of about five samples per body. This coalescence on five samples per body may have been influenced by the Guidelines for the Banking of Skin Tissues published in 1979 by the Standards Committee of the Skin Council of the American Association of Tissue Banks (Standards Committee, Skin Council, American Association of Tissue Banks, 1979). These guidelines state that ‘skin samples one cm2 in size should be cultured for each 20 per cent of the total body area collected’, that is, five samples should be obtained per donor body. The results of the investigation presented here indicate that the choice of five skin samples per donor can result in a skin classification accuracy of about 95-96 per cent (Table V). The usual constraint on testing as many as ten body areas for the presence of microbial contamination is the assumption that the required costs and personnel time will not be worth the gains obtained (Heck et al., 1981). In the present study, we have shown that the gains obtained are improved accuracy of skin classification, while the increased costs involved are relatively modest. Technically, only small amounts of money were saved by the improved accuracy of skin assignment (see net gain in Table v), since there was an approximately equal reassignment of skin to contaminated and uncontaminated classifications with increasing numbers of areas tested per body. In summary, our protocol cost $6.74 for a complete microbiological evaluation of every 450 cm2 (72 in.2) of skin obtained. Thus, for ten samples per donor body, it cost a total of $67.40 for these tests. In our opinion, it was reasonable to pay that price for a 99.9 per cent accuracy in skin classification with respect to microbiological contamination. Similar cost analyses can allow each skin bank to determine the relationship between cost and accuracy in their individual situation, and to choose a donor body sampling rate based upon both parameters.

May et al.: Contamination

on cadaver

skin biological

dressings

Acknowledgements The Saint Agnes Medical Center Skin Bank was founded and is operated in part by a grant from the Research Fund Board of Trustees of Philadelphia General Hospital, and further sustained in part by grants from the Burn Foundation of Greater Delaware Valley (now The Burn Foundation, Philadelphia, Pennsylvania). The data presented here were collected during the tenure of Dr May and Mr Wainwright on the staff of the Saint Agnes Medical Center Skin Bank. We wish to thank Carole M. Ehleben for suggestions regarding the analysis of the financial data. The analysis of portions of the data was supported by a grant from the Burn Foundation, Augusta, Georgia, to the Southeastern Burn Research Institute. Portions of this work were presented at the 16th Annual Meeting, American Burn Association, San Francisco, California, 11-14 April 1984.

REFERENCES Artz C. P., Becker J. M., Sako Y. et al. (1955) Postmortem skin homografts in the treatment of extensive burns. Arch. Surg. 71, 682. Bondoc C. C. and Burke J. F. (1971) Clinical experience with viable frozen human skin and a frozen skin bank. Ann. Surg. 174, 371. Brown .I. B., Fryer M. P.. Randall P. et al. (1953) Postmortem homografts as ‘biological dressings’ for extensive burns and denuded areas. Ann. Surg. 138, 618. Brown J. B. and McDowell F. (1942) Massive repairs of burns with thick split skin grafts: Emergency ‘dressing’ with homografts. Ann. Surg. 115, 658. Davis B. I)., Delbecco R.. Eisen H. N. et al. (1973) Microbiology, 2nd Ed. New York: Wiley, p. 636. DeClement F. A. and May S. R. (1983) Procurement, cryopreservation and clinical application of skin. In: Classman A. B. and Umlas J. (eds.) Cryopreservation of Tissue and Solid Organs for Transplantation. Arlington, VA.. American Association of Blood Banks, p. 29. Eagle H. (1959) Amino acid metabolism in mammalian cell cultures. Science 130, 432. Haynes B. W. Jr. (1963) Skin homografts: A life-saving measure in severely burned children. J. Trauma 3, 217. Heck E., Blood S. and Baxter C. R. (1981) The importance of the bacterial flora in a cadaver homograft donor skin: Bacterial flora in cadaver homograft. J. Burn Care Rehabil. 2. 212. Hornbokel H. E., Simon M. R. and Jordan M. H. (1985) Cadaver allograft skin-supply and demand. Proceedings of the American Burn Association, Volume 17 (Seventeenth Annual Meeting, 27-30 March, 1985, Orlando, Florida, USA), Abstract number 87.

47

Korlof B., Simoni E., Baryd I. et al. (1972) Radiation sterilized split skin: A new type of biological dressing. Stand. J. Plast. Reconstr. Suw. 6. 126. Macfiillan B. G. (1962) Homograft ikin: A valuable adjunct to the treatment of thermal burns. J. Trauma 2, 130. May S. R. (1983) Cryopreservation and clinical use of human allograft skin. In: Aso K. and Sumida S. (eds.) Low Temperature Medicine. Tokyo: Asakura Publishing Co., p. 156. May S. R. (1985) Recent developments in skin banking. In: May S. R. and Dogo G. (eds.) Care of the Burn Wound. Basel: S. Karger, p. 175. May S. R. and DeClement F. A. (1980) Skin banking methodology: An evaluation of package format, cooling and warming rates, and storage efficiency. Cryobiology 17, 34. Mav S. R. and DeClement F. A. (1981a1 Skin bankine. P&t I. Procurement of tra&plan&ble cadaver:c allograft skin for burn wound coverage. J. Burn Care Rehabil. 2, 7. May S. R. and DeClement F. A. (1981b) Skin banking. Part II. Procurement of low contamination cadaveric dermal allograft for temporary burn wound coverage. .I. Burn Care Rehabil. 2. 64. Ma;S. R. and DeClement F. A. (1981~) Skin banking. Part III. Cadaveric alloeraft skin viability. J. Burn Care Rehabil. 2, 128. May S. R. and DeClement F. A. (1981d) Skin banking. Part IV. Cadaveric dermal allograft donor and recipient selection. J. Burn Care Rehabil. 2, 184. May S. R. and DeClement F. A. (1982) Development of a radiometric metabolic viability testing method for human and porcine skin. Cryobiology 19. 362. May S. R., Still J. M. Jr. and Atkinson W. B. (1984) Recent developments in skin banking and the clinical uses of cryopreserved skin. .I. Med. Assoc. Georgia 73, 233. May S. R. and Wainwright J. F. (1985a) Integrated study of the structural and metabolic degeneration of skin during 4°C storage in nutrient medium. Cryobiology 22, 18. May S. R. and Wainwright J. F. (1985b) Optimum warming rates to maintain glucose metabolism in porcine skin cryopreserved by slow cooling. Cryobiology 22, 196. May S. R., Wainwright J. F. and DeClement F. A. (1985) The effect of variables determining the amount of microbial contamination on cadaveric allograft skin used as a biological wound dressing. Burns 11, 242. Monafo W. W., Tandon S. N., Bradley R. E. et al. (1976) Bacterial contamination of skin used as a biological dressing. A potential hazard. JAMA 235, 1248. Ninnemann J. L. (ed.) (1981) The Immune Consequences of Thermal Injury. Baltimore: Williams and Wilkins. Ninnemann J. L. (ed.) (1983) Traumatic Injury. Infection and Other Immunological Sequelae. Baltimore: University Park Press.

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Rudolph R., Fisher J. C. and Ninnemann J. L. (1979) Skin Grafting. Boston: Little, Brown and Co., p. 155. Shuck J. M. (1979) Biologic dressings. In: Artz C. P., Moncrief J. A. and Pruitt B. A. Jr. (eds.) Burns: A Team Approach. Philadelphia: W. B. Saunders, p. 211. Shuck J. M., Pruitt, B. A. Jr. and Moncrief J. A. (1969) Homograft skin for wound coverage. Arch. Surg. 98, 472. Standards Committee, American Association of Tissue Banks (1984) Standards for Tissue Banking. Arlington, Virginia, American Association of Tissue Banks, p. 12.

Standards Committee, Skin Council, American Association of Tissue Banks (1979) Guidelines for the banking of skin tissues. Am. Assoc. Tin. Banks Newslett. 3(l), 5. Switzer W. E., Moncrief J. A., Mills W. Jr. et al. (1966) The use of canine heterografts in the therapy of thermal injury. J. Trauma 6, 391. Wachtel T. L., Ninnemann J., Fisher J. C. et al. (1979) Viability of frozen allografts. Am. J. Surg. 138, 783. Zaroff I., Mills W. Jr., Duckett J. Jr. et al. (1966) Multiple uses of viable cutaneous homografts in the burned patient. Surgery 59, 368. Paper

accepted

22 May 1985.

Correspondence should be addressed 10: S. Randolph May, PhD, Director, Southeastern Burn Research Institute, Suite 303, 3623 J. Dewey Gray Circle, Augusta, Georgia 30909, USA.

G. WHITAKER INTERNATIONAL BURNS PRIZE - PALERMO (ITALY) UNDER THE PATRONAGE OF THE AUTHORITIES OF THE SICILIAN REGION FOR 1986

By law No. 57 of 14 June 1983 the Sicilian Regional Assembly authorized the President of the Region to grant the ‘Giuseppe Whitaker Foundation’, a non-profit making organization under the patronage of the Accademia dei Lincei, with headquarters in Palermo, an annual contribution for the establishment of a ‘G. Whitaker International Burns Prize’ aimed at recognizing the activity of the most qualified experts from all countries in the field of burns pathology and treatment. The amount of the prize is fixed at twenty million Italian lire. The prize will be awarded every year by the month of June at the Palermo headquarters of the G. Whitaker Foundation. The adjudicating committee is composed of the President of the Foundation, the President of the Sicilian Region, the representative of the Accademia dei Lincei within the G. Whitaker Foundation, the Dean of the Faculty of Medicine and Surgery of Palermo University, the President of the Italian Society of Plastic Surgery, a legal expert, the previous year’s prize-winner, and not fewer than three experts in the burns field nominated in agreement with the President of the Region as a guarantee of the respect for the scientific purposes which the legislators intended to achieve when establishing the prize. Anyone who considers himself to be qualified to compete for the award may send by 31 January 1986 his detailed curriculum vitae to Dr Michele Masellis M.D., Secretary-Member of the Scientific Committee, G. Whitaker Foundation, via Dante 167, 90141Palermo, Italy.