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Histological identification of prolonged survival of a skin allograft on an extensively burned patient
164
Burns, 8,
164-l 67
Printedin GreatBritain
Histological identification of prolonged survival of a skin allograft on an extensively burned patient lwao Takiuchi, Dousei Higuchi, Yoshihiro Sei and Teruko Nakajima Department of Dermatology, Showa University, Fujigaoka Hospital, Yokohama, Japan
Summary
We applied skin allogratts obtained from 38 unrelated volunteers to a very extensively burned female patient. Seven months after skin transplantation, one allogratl
from a male still survived clinically. In order to ascertain that the graft site resulted from growth of the male donor skin, the ailografted area and patient’s own unburned normal area were examined by the fluorescent staining of Y-body (Y-chromosomal fluorescence). Y-body was detected in 57 per cent and 65 per cent of cells from the grafted area, whereas no fluorescence was detected in the patient’s own unburned normal area. These observations suggest that one allografi from an unrelated donor survived for 221 days on a very extensively burned subject. INTRODUCTION IN GENERAL, skin allografis obtained from unrelated normal subjects are rejected within 8 to 11 days (Rapaport et al., 1962). However, these allografts were able to survive for a long period of time when the skin was planted on extensively burned patients (Branch et al., 1946; Kay, 1957; Ninnemann et al., 1978). The prolonged survival of skin allografts on extensively burned patients has been noted by many clinicians. But these reports are based merely on gross observations. Recently, we have encountered a case of a burned female patient on whom the skin allograft from an unrelated male donor survived for more than 7 months. The human metaphase chromosome, especially the posterior part of the long arm, has high
binding capacity for fluorescence with acridimine derivatives (Casperson et al., 1969); and a part of the Y-chromosome has strong fluorescence in interphase as well as in metaphase (Casperson et al., 1970; Person and Bobrow, 1970). So, the fluorescence of the interphase Y-chromosome is of help in determining genetic sex (Casperson et al., 1970; Person and Bobrow, 1970). Recently, Korn et al. (1978) have reported that Y-bodies can be detected in cryostat section of skin biopsies. We have used the technique of Kom et al. (1978) to show that the allografied area of the female patient was ofthe skin from a male donor. CASE REPORT On 27 August 1977, a 27-year-old housewife was admitted to our hospital with thermal bums involving 80 per cent of her total body surface. From 2 weeks after her injury, her general condition became quite serious due mainly to sepsis and renal failure. Thus, we were unable to apply escharotomies and skin autografts. On 7 and 8 May, skin specimens (0.38 mm thick) were taken from 38 unrelated volunteers; and on 9 May, her burned area was covered with these allografts. After skin transplantation, her condition and many complications, including sepsis, were greatly improved. Thus, 3 weeks after transplantation, we were able to apply skin autografts successfully. In October, most of her burned area was covered with her normal skin. Almost all the allografis were rejected within about 25 days, however, one allograt? obtained from a male donor still survived 22 I days after transplantation. On 16 December, 221 days after transplantation, two
Takiuchi et al.: ProlongedSurvival of Skin Allograh
165
Fig. I. Two hundred and eleven days after grafting. A is the grafted area and B is the unburned normal area of her left lower abdomen. TaBe 1. Fluorescent bodies (Y-bodies) in epidermal cells Y-body Skin specimen’
Pseudopositive
A verage proportion of Y-body (%I
Positive
Negative
0
97 98
21
0
65 57
21 35
6 8
61
63 70
24 10
13 11
66.5
0 1
99 97
0 2
0.5
Patient’s normal : Patient’s grafted : Normal male A 8 Normal female A 8
*One hundred epidermal cells were calculated in each specimen.
pieces of skin were examined by the fluorescent staining of Y-chromosome.
biopsies were obtained from her skin. These
After skin biopsy the survived allogratl rapidly became encrusted, then on 4 January 1978, that is 239 days after transplantation, that graft was totally rejected. MATERIALS
AND
METHODS
The skin biopsy was taken from an allografted area of the patient’s let? lower abdomen (Fig. 1). Control skin biopsies were taken from an
unburned area of her left lower abdomen (Fig. 1) and from 2 normal males and 2 normal females. Each skin section was frozen immediately and subsequently cut in a cryostat. Cryostat sections of each skin samples, 6 pm thickness, were fixed in 1 : I ether-ethanol mixture for 30 minutes, then air dried. Korn’s method (Korn et al., 1978) was used to stain Y-chromosomal fluorescence. Dried slides were placed in MacIlvaine buffer pH 4.5 (MacIlvaine, I92 I) for 5 minutes at room temperature. Slides were immersed for 5 minutes in O-5 per cent (w/v)
Burns Vol. 8/No. 3
166
Fig. 2. Epidermal
cells of the patient’s grafted area showing fluorescent body
(Y-body, arrows).
Fig. 3. Epidermal cells of her unburned normal area. No Y-body can be detected.
quinacrine dehydrochloride (Sigma), rinsed briefly in deionized water. These samples were dried, mounted in Macllvaine buffer, and then observed with Olympus fluorescence microscope with light transmitted from an HBO-200 mercury lamp and a Ploem vertical illuminator. A BG-12 excitor filter and a 530 nm barrier filter were used. All observations were made using oil immersion objectives. Kodac Tri-X film and an exposure time of 90-120 seconds were used for photography.
The percentage of cells with fluorescent Y-body from a sample of 100 epidermal cells was calculated. RESULTS
The results are presented in Table I. Ychromosomal fluorescence was detected in 57 per cent and 65 per cent of cells from her survived area (Fig. 2), but no Y-chromosomal fluorescence was detected in her normal area (Fig. 3). On the other hand, a fluorescent spot,
167
Takwchi et al.: Prolonged Survival of Skin Allograft
Y-body, was observed in about 66.5 per cent of the epidermal cells with the control skin of 2 males and in only 0.5 per cent of that with 2 females.
From our studies, we have confirmed histochemically that some allografts are able to survive for more than 7 months on extensively burned patients.
DISCUSSION It is standard therapy to apply skin autografts to
REFERENCES
severely burned patients. However, when the burned area is too extensive or the general condition of the patient is too serious, it is often not possible to apply skin autografts. In these cases, skin allografts are of particular value (Zaroff et al., 1966; Chambler and Batchelor, 1969). Allografts are able to survive longer on burned patients than on normal subjects (Ceppellini et al., 1969). This is mainly attributable to the depression of cell-mediated immunity (Ceppellini et al., 1969; Munster et al.. 1973). The prolonged survival of human skin allografts on extensively burned patients has been noted and occasionally published. Kay (1957) reported a severely burned patient on whom a skin allograft obtained from his brother survived for 32 weeks. But previous reports, that some allografts were able to survive for a long time on a patient with severe depression of cell-mediated immunity as in patients with extensive burns, were not convincing enough because they were based on gross observations. In fact, Korn et al. (1978) applied Y-body technique to cutaneous biopsies from the burned area ofa female patient who was grafted with the skin from a male donor 3 months previously. However, they were not able to detect Y-body in that area and they concluded that the skin over the graft was the regenerated tissue ofthe patient and not that of the male skin which was originally grafted. In our case, we have applied skin allografts from 38 unrelated donors. Seven months aAer skin transplantation, one allograft from a male survived almost completely and we were able of that to detect Y-body in a high percentage graft, These results suggest that the skin of her survived area is from grafted male skin.
Branch C. D., Wilkins G. F. and Ross F. P. (1946) The coagulum contact method of skin grafting in the treatment of burns and wounds. Surgery 19,460. Casperson T., Zech L., Modest E. J. et al. (1969) DNA-binding fluorochromes for the study of the organization of metaphase nucleus. E.rp. Ce# Rec. 58, 141.
Casperson T., Zech L., Johansson C. et al. (1970) Fluorescent staining of heteropycnotic chromosome region in human interphase nuclei. ,571. CrN Ro 61,472.
Ceppellini K., Curtoni E. S., Mattiuz P. L. et al. (1969) Survival of test skin grafts in man: effect of genetic relationship and of blood groups incompatibility. Ann. N. Y. Acad. Sci. 129,42 I. Chambler K. and Batchelor J. R. (1969) Influence of defined incompatibilities and area of burn on skinhomograft survival in burned subjects. Lancet 1, 16. Kay G. D. (1957) Prolonged survival of a skin homograft in a patient with very extensive bums. Ann. N. Y. Acad. Sci. 64,767. Korn G. A., Gaulden M. E., Baxter C. R. et al. (1978) Use of the Y-body for identification of skin source on a successfully grafted bum patient. J. Inrersr. Derm. 70,285.
Macllvaine T. C. (I 921) A buffer solution for calorimetric comparison. J. Biol. Chem. 49, 183. Munster A. M.. Eurenius K.. Katz R. M. et al. (1973) Cell-mediated immunity after thermal injury. Ann. Surg. 177,139. Ninnemann J. L., Fisher J. C. and Frank H. A. (1978) Prolonged survival of human skin allografts following thermal injury. T’runsplunf. 2569. Person P. L. and Bobrow M. (1970) Technique for identifying Y chromosomes in human interphase nuclei. Nature 226,78. Rapaport F. T.. Lawrence H. S., Thomas L. et al. (1962) Cross-reactions to skin homografts in man. J Clin. Invest. 41,2166. Zaroff L. I., Mills W. jun., Duckett J. W. jun., et al. (I 966) Multiple uses of viable cutaneous homografts in the burned patient. Surgery59,368. Paper accepted I7 December 1980.
Rrquc~ts /iv wprrnu should be addressed 10: Assistant Professor 1. Takiuchi. Department of Dermatology, Showa University. Fujigaoka Hospital, I-30 Fujigaoka Midoriku, Yokohama, Japan 227.
Burns, 8,
164-l 67
Printedin GreatBritain
Histological identification of prolonged survival of a skin allograft on an extensively burned patient lwao Takiuchi, Dousei Higuchi, Yoshihiro Sei and Teruko Nakajima Department of Dermatology, Showa University, Fujigaoka Hospital, Yokohama, Japan
Summary
We applied skin allogratts obtained from 38 unrelated volunteers to a very extensively burned female patient. Seven months after skin transplantation, one allogratl
from a male still survived clinically. In order to ascertain that the graft site resulted from growth of the male donor skin, the ailografted area and patient’s own unburned normal area were examined by the fluorescent staining of Y-body (Y-chromosomal fluorescence). Y-body was detected in 57 per cent and 65 per cent of cells from the grafted area, whereas no fluorescence was detected in the patient’s own unburned normal area. These observations suggest that one allografi from an unrelated donor survived for 221 days on a very extensively burned subject. INTRODUCTION IN GENERAL, skin allografis obtained from unrelated normal subjects are rejected within 8 to 11 days (Rapaport et al., 1962). However, these allografts were able to survive for a long period of time when the skin was planted on extensively burned patients (Branch et al., 1946; Kay, 1957; Ninnemann et al., 1978). The prolonged survival of skin allografts on extensively burned patients has been noted by many clinicians. But these reports are based merely on gross observations. Recently, we have encountered a case of a burned female patient on whom the skin allograft from an unrelated male donor survived for more than 7 months. The human metaphase chromosome, especially the posterior part of the long arm, has high
binding capacity for fluorescence with acridimine derivatives (Casperson et al., 1969); and a part of the Y-chromosome has strong fluorescence in interphase as well as in metaphase (Casperson et al., 1970; Person and Bobrow, 1970). So, the fluorescence of the interphase Y-chromosome is of help in determining genetic sex (Casperson et al., 1970; Person and Bobrow, 1970). Recently, Korn et al. (1978) have reported that Y-bodies can be detected in cryostat section of skin biopsies. We have used the technique of Kom et al. (1978) to show that the allografied area of the female patient was ofthe skin from a male donor. CASE REPORT On 27 August 1977, a 27-year-old housewife was admitted to our hospital with thermal bums involving 80 per cent of her total body surface. From 2 weeks after her injury, her general condition became quite serious due mainly to sepsis and renal failure. Thus, we were unable to apply escharotomies and skin autografts. On 7 and 8 May, skin specimens (0.38 mm thick) were taken from 38 unrelated volunteers; and on 9 May, her burned area was covered with these allografts. After skin transplantation, her condition and many complications, including sepsis, were greatly improved. Thus, 3 weeks after transplantation, we were able to apply skin autografts successfully. In October, most of her burned area was covered with her normal skin. Almost all the allografis were rejected within about 25 days, however, one allograt? obtained from a male donor still survived 22 I days after transplantation. On 16 December, 221 days after transplantation, two
Takiuchi et al.: ProlongedSurvival of Skin Allograh
165
Fig. I. Two hundred and eleven days after grafting. A is the grafted area and B is the unburned normal area of her left lower abdomen. TaBe 1. Fluorescent bodies (Y-bodies) in epidermal cells Y-body Skin specimen’
Pseudopositive
A verage proportion of Y-body (%I
Positive
Negative
0
97 98
21
0
65 57
21 35
6 8
61
63 70
24 10
13 11
66.5
0 1
99 97
0 2
0.5
Patient’s normal : Patient’s grafted : Normal male A 8 Normal female A 8
*One hundred epidermal cells were calculated in each specimen.
pieces of skin were examined by the fluorescent staining of Y-chromosome.
biopsies were obtained from her skin. These
After skin biopsy the survived allogratl rapidly became encrusted, then on 4 January 1978, that is 239 days after transplantation, that graft was totally rejected. MATERIALS
AND
METHODS
The skin biopsy was taken from an allografted area of the patient’s let? lower abdomen (Fig. 1). Control skin biopsies were taken from an
unburned area of her left lower abdomen (Fig. 1) and from 2 normal males and 2 normal females. Each skin section was frozen immediately and subsequently cut in a cryostat. Cryostat sections of each skin samples, 6 pm thickness, were fixed in 1 : I ether-ethanol mixture for 30 minutes, then air dried. Korn’s method (Korn et al., 1978) was used to stain Y-chromosomal fluorescence. Dried slides were placed in MacIlvaine buffer pH 4.5 (MacIlvaine, I92 I) for 5 minutes at room temperature. Slides were immersed for 5 minutes in O-5 per cent (w/v)
Burns Vol. 8/No. 3
166
Fig. 2. Epidermal
cells of the patient’s grafted area showing fluorescent body
(Y-body, arrows).
Fig. 3. Epidermal cells of her unburned normal area. No Y-body can be detected.
quinacrine dehydrochloride (Sigma), rinsed briefly in deionized water. These samples were dried, mounted in Macllvaine buffer, and then observed with Olympus fluorescence microscope with light transmitted from an HBO-200 mercury lamp and a Ploem vertical illuminator. A BG-12 excitor filter and a 530 nm barrier filter were used. All observations were made using oil immersion objectives. Kodac Tri-X film and an exposure time of 90-120 seconds were used for photography.
The percentage of cells with fluorescent Y-body from a sample of 100 epidermal cells was calculated. RESULTS
The results are presented in Table I. Ychromosomal fluorescence was detected in 57 per cent and 65 per cent of cells from her survived area (Fig. 2), but no Y-chromosomal fluorescence was detected in her normal area (Fig. 3). On the other hand, a fluorescent spot,
167
Takwchi et al.: Prolonged Survival of Skin Allograft
Y-body, was observed in about 66.5 per cent of the epidermal cells with the control skin of 2 males and in only 0.5 per cent of that with 2 females.
From our studies, we have confirmed histochemically that some allografts are able to survive for more than 7 months on extensively burned patients.
DISCUSSION It is standard therapy to apply skin autografts to
REFERENCES
severely burned patients. However, when the burned area is too extensive or the general condition of the patient is too serious, it is often not possible to apply skin autografts. In these cases, skin allografts are of particular value (Zaroff et al., 1966; Chambler and Batchelor, 1969). Allografts are able to survive longer on burned patients than on normal subjects (Ceppellini et al., 1969). This is mainly attributable to the depression of cell-mediated immunity (Ceppellini et al., 1969; Munster et al.. 1973). The prolonged survival of human skin allografts on extensively burned patients has been noted and occasionally published. Kay (1957) reported a severely burned patient on whom a skin allograft obtained from his brother survived for 32 weeks. But previous reports, that some allografts were able to survive for a long time on a patient with severe depression of cell-mediated immunity as in patients with extensive burns, were not convincing enough because they were based on gross observations. In fact, Korn et al. (1978) applied Y-body technique to cutaneous biopsies from the burned area ofa female patient who was grafted with the skin from a male donor 3 months previously. However, they were not able to detect Y-body in that area and they concluded that the skin over the graft was the regenerated tissue ofthe patient and not that of the male skin which was originally grafted. In our case, we have applied skin allografts from 38 unrelated donors. Seven months aAer skin transplantation, one allograft from a male survived almost completely and we were able of that to detect Y-body in a high percentage graft, These results suggest that the skin of her survived area is from grafted male skin.
Branch C. D., Wilkins G. F. and Ross F. P. (1946) The coagulum contact method of skin grafting in the treatment of burns and wounds. Surgery 19,460. Casperson T., Zech L., Modest E. J. et al. (1969) DNA-binding fluorochromes for the study of the organization of metaphase nucleus. E.rp. Ce# Rec. 58, 141.
Casperson T., Zech L., Johansson C. et al. (1970) Fluorescent staining of heteropycnotic chromosome region in human interphase nuclei. ,571. CrN Ro 61,472.
Ceppellini K., Curtoni E. S., Mattiuz P. L. et al. (1969) Survival of test skin grafts in man: effect of genetic relationship and of blood groups incompatibility. Ann. N. Y. Acad. Sci. 129,42 I. Chambler K. and Batchelor J. R. (1969) Influence of defined incompatibilities and area of burn on skinhomograft survival in burned subjects. Lancet 1, 16. Kay G. D. (1957) Prolonged survival of a skin homograft in a patient with very extensive bums. Ann. N. Y. Acad. Sci. 64,767. Korn G. A., Gaulden M. E., Baxter C. R. et al. (1978) Use of the Y-body for identification of skin source on a successfully grafted bum patient. J. Inrersr. Derm. 70,285.
Macllvaine T. C. (I 921) A buffer solution for calorimetric comparison. J. Biol. Chem. 49, 183. Munster A. M.. Eurenius K.. Katz R. M. et al. (1973) Cell-mediated immunity after thermal injury. Ann. Surg. 177,139. Ninnemann J. L., Fisher J. C. and Frank H. A. (1978) Prolonged survival of human skin allografts following thermal injury. T’runsplunf. 2569. Person P. L. and Bobrow M. (1970) Technique for identifying Y chromosomes in human interphase nuclei. Nature 226,78. Rapaport F. T.. Lawrence H. S., Thomas L. et al. (1962) Cross-reactions to skin homografts in man. J Clin. Invest. 41,2166. Zaroff L. I., Mills W. jun., Duckett J. W. jun., et al. (I 966) Multiple uses of viable cutaneous homografts in the burned patient. Surgery59,368. Paper accepted I7 December 1980.
Rrquc~ts /iv wprrnu should be addressed 10: Assistant Professor 1. Takiuchi. Department of Dermatology, Showa University. Fujigaoka Hospital, I-30 Fujigaoka Midoriku, Yokohama, Japan 227.