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Melanocyte Counts on Human Forearm Skin1
MELANOCYTE COUNTS ON HUMAN FOREARM SKIN* AODAN S. BREATHNACH, M.Sc., M.O.
Accurate estimates of the number of melanocytes in corresponding areas of different individuals, and in different areas of the same individual, are necessary if many problems relating to variations and disorders of skin pigmentation are to be solved. The only systematic estimates available to date are those published by Szabó (1), who carried out counts on the skin of head and neck, upper arm, forearm, and thigh of a number of individuals. Szabó's forearm specimens were limited to three, in two of which, No. 45, and No. 62, the total area counted was comparatively small (0.1150 mm.2 and 0.0804 mm.2 respectively). In the course of a present investigation into a possible correlation between melanocyte concentra-
tion and differences in skin color in white (Caucasian) subjects, counts were carried out on corresponding areas of forearm skin of ten individuals. Since the results comprise a larger series than has been hitherto available for this region, and since they differ in some respects from those arrived at by Szabó, it is considered that their publication may be of value. MATERIALS AND METHODS
The cell-counts were carried out on dopa-treated epidermal sheets obtained by the "skin-splitting" technic of Billingham and Medawar(2), as modified by Szabó (3). All subjects were male students of this Medical School, to whom thanks are due for their co-operation. Specimens were taken from the upper half of the inner portion of the flexor aspect of the forearm. From each individual, a piece of skin approximately 30 mm.2 in area, and of slightly more than Thierschgraft thickness, was removed under local anesthesia with Procaine 2 %. After digestion for 8 to 10 hours in a buffered 0.5 % solution of commercial trypsin at 2° to 5°C., the dermis was removed, and the epidermal sheet fixed for 1 to 2 hours in formal calcium chloride. It was then incubated at 37°C. in a 1 in 1000 solution of L(-)-dihydroxyphenylalanine at a pH of 7.4 until the melanocytes were stained. Fixation for a further period of 12 hours in formal calcium chloride was then carried out, followed by dehydration, clearing, and mounting "dermal side up" in Canada balsam. The actual counting was done as follows. In each case, the number of melanocytes in ten randomly chosen areas of 0.064 mm.2 (giving a total area counted for each specimen of 0.64 mm.2) was counted, and from the figures obtained, the mean number of cells per square millimetre, and the standard error of this mean was calculated. A magnification of 300 X was found to be the most suitable for accurate counting. Some melanocytes which are weakly dopa-positive, and of *
From the Department of Anatomy, St. Mary's Hospital Medical School, London, W. 2, England. Received for publication February 19, 1957. 181
182
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
"fibroblastic" form, are liable to be missed at appreciably lower magnifications. The higher magnification is also of value in excluding epithelial cells which may be passively stained by the dopa reagent, and in distinguishing between closely superimposed melanocytes along the epidermal ridges. The skin complexion of the subjects chosen varied from fair to dark, and three of the fair-skinned (Nos. 1, 2, and 5) were freckled. In the case of these latter, the piece of skin removed was from a non-freckled area. RESULT5
The results are set out in Table 1. The overall mean was found to be 2024 52 per mm.,2 and the range extended from 1743 to 2327 per mm.2 Seven of the specimens lie within the range 1900 to 2200 per mm.2 These results differ markedly from those arrived at by Szabo (1) for forearm skin. His figures for the three specimens numbered 24, 45, and 62, are 820 50, 1070 60, and 1210 60, respectively. The overall mean for the three is 1033 per mm.2, which is approximately half that of the mean (2024 per mm.2) for the present series. In a personal communication, Dr. Szabo states that only one of his specimens (No. 62) was obtained from the flexor aspect of the forearm, as was the case with all specimens making up the present group. Strictly speaking therefore, this is the only one of his specimens which can be used for comparison with the preseut series, as it would appear (Szabó (1)) that there may be considerable intrinsic differences in the number of melanocytes in different areas of a limb. Since, in addition, the total area counted in this particular specimen was comparatively small (0.0804 mm.2), there does not appear to be much point in discussing further the apparent discrepancy in results between the two series. It may be worthwhile emphasizing however, that in this matter of cell-counts
carried out on different material by different observers, apparently minor differences in technic, and such factors as the personal element involved in deTABLE 1 Mean number of melaaocytes per square millimeter in skin front the flexor aspect of the forearm Specimen
Age
Area counted
1 2 3 4 5 6 7 8 9 10
22 34 19 20 26 25 20 19 19 21
0.64 mm.' 0.64 mm.' 0.64 mm.' 0.64 mm.' 0.64 mm.'
2/Jelanneytes per mm.'
1743
52
1860 1905
19
1963
0.64 mm.' 0.64 mm.' 0.64 mm.'
2005 2060 2105 2122 2155
0.64 mm.2
2327
0.64 mm.2
Overall mean: 2024
70 51 61
Complexion
Fair (freckled) Fair (freckled)
Dark Dark Fair (freckled)
74 73
Fair
79 25 76
Fair Dark Dark
52
Fair
MELANOCYTE COUNTS ON HUMAN FOREARM SKIN
183
ciding whether a particular cell is, or is not, a melanocyte, are bound to lead to some discrepancy in the final results. This can be reduced to a minimum if a standardized technic is adopted in all instances. As a result of experience gained with the present material, it is suggested that when counting melanocytes, a magnification of the order indicated above should be used, if accurate and comparable results are to be obtained. In the last column of Table 1, an indication is given of the general skin complexion of the individuals comprising the group. It is of some interest to note that the two lowest counts were obtained from fair-skinned subjects, and the two highest from dark-skinned ones. This suggests a possibility that there may be some correlation between melanocyte concentration and complexion, although the fact that counts intermediate between these extremes were obtained from fair and dark-skinned individuals alike, would appear to suggest the contrary. However, the present group is not sufficiently large to allow one to make any final pronouncement on this question. In order to be able to do so, further counts on a larger series of specimens must be undertaken. An answer, one way or another, to this problem is desirable in view of the suggestion that the frequency incidence of malignant melanoma at a given site may be related to the local population density of melanocytes, and of the oft-repeated one that melanoma is commoner in fair, than in dark-skinned individuals. SUMMARY
Melanocyte counts were carried out on dopa-treated "split-skin" preparations
of human epidermis, obtained from the upper part of the inner aspect of the flexor surface of the forearm of ten male white subjects of varying complexions. 52 The mean number of melanocytes per mm.2 was found to range from 1743 76, giving an overall mean for the ten of 2024 52. to 2327 REFERENCES 1. SZAB6, G.: The number of melanocytes in human epidermis. Brit. M. J., 1: 1016, 1954. 2. BILLINGHAM, R. E. AND MzDAWAR, P. B.: A study of the branched cells of the mammalian
epidermis with special reference to the fate of their division products. Philos. Tr. Roy. Soc., London, B., 237: 151, 1953. 3. SZAB6, G.: A modification of the technique of "skin splitting" with trypsin. J. Path. & Bact., 70: 545, 1955. AnDENDUM
Since this paper went to press, results of extensive melanocyte counts have been published by Staricco and Pinkus (J. invest. Berm., 8: 33—45, 1957). These authors found the number of melanocytes in human forearm skin to range from 1150 to 2500 per mm.2 The higher of these two figures was obtained from a specimen specifically labelled "Upper forearm", and is not very different from the figures obtained from the present specimens, all of which came from a similar area. Staricco and Pinkus's figures for their other forearm specimens (exact area unspecified) are considerably lower, and range from 1150 to 1500 per mm.2.
184
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Consideration of these figures underlines the fact that considerable variations in melanocyte concentration occur within the forearm, and that mean figures quoted for this entire region may be very different from the actual figures obtained from particular portions of it. Accordingly, it would seem desirable when the results of melanocyte counts are being published, that authors should indicate as explicitly as possible the exact area within a region from which their specimens were obtained. Otherwise, direct comparison of results obtained by different workers may be difficult.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
Accurate estimates of the number of melanocytes in corresponding areas of different individuals, and in different areas of the same individual, are necessary if many problems relating to variations and disorders of skin pigmentation are to be solved. The only systematic estimates available to date are those published by Szabó (1), who carried out counts on the skin of head and neck, upper arm, forearm, and thigh of a number of individuals. Szabó's forearm specimens were limited to three, in two of which, No. 45, and No. 62, the total area counted was comparatively small (0.1150 mm.2 and 0.0804 mm.2 respectively). In the course of a present investigation into a possible correlation between melanocyte concentra-
tion and differences in skin color in white (Caucasian) subjects, counts were carried out on corresponding areas of forearm skin of ten individuals. Since the results comprise a larger series than has been hitherto available for this region, and since they differ in some respects from those arrived at by Szabó, it is considered that their publication may be of value. MATERIALS AND METHODS
The cell-counts were carried out on dopa-treated epidermal sheets obtained by the "skin-splitting" technic of Billingham and Medawar(2), as modified by Szabó (3). All subjects were male students of this Medical School, to whom thanks are due for their co-operation. Specimens were taken from the upper half of the inner portion of the flexor aspect of the forearm. From each individual, a piece of skin approximately 30 mm.2 in area, and of slightly more than Thierschgraft thickness, was removed under local anesthesia with Procaine 2 %. After digestion for 8 to 10 hours in a buffered 0.5 % solution of commercial trypsin at 2° to 5°C., the dermis was removed, and the epidermal sheet fixed for 1 to 2 hours in formal calcium chloride. It was then incubated at 37°C. in a 1 in 1000 solution of L(-)-dihydroxyphenylalanine at a pH of 7.4 until the melanocytes were stained. Fixation for a further period of 12 hours in formal calcium chloride was then carried out, followed by dehydration, clearing, and mounting "dermal side up" in Canada balsam. The actual counting was done as follows. In each case, the number of melanocytes in ten randomly chosen areas of 0.064 mm.2 (giving a total area counted for each specimen of 0.64 mm.2) was counted, and from the figures obtained, the mean number of cells per square millimetre, and the standard error of this mean was calculated. A magnification of 300 X was found to be the most suitable for accurate counting. Some melanocytes which are weakly dopa-positive, and of *
From the Department of Anatomy, St. Mary's Hospital Medical School, London, W. 2, England. Received for publication February 19, 1957. 181
182
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
"fibroblastic" form, are liable to be missed at appreciably lower magnifications. The higher magnification is also of value in excluding epithelial cells which may be passively stained by the dopa reagent, and in distinguishing between closely superimposed melanocytes along the epidermal ridges. The skin complexion of the subjects chosen varied from fair to dark, and three of the fair-skinned (Nos. 1, 2, and 5) were freckled. In the case of these latter, the piece of skin removed was from a non-freckled area. RESULT5
The results are set out in Table 1. The overall mean was found to be 2024 52 per mm.,2 and the range extended from 1743 to 2327 per mm.2 Seven of the specimens lie within the range 1900 to 2200 per mm.2 These results differ markedly from those arrived at by Szabo (1) for forearm skin. His figures for the three specimens numbered 24, 45, and 62, are 820 50, 1070 60, and 1210 60, respectively. The overall mean for the three is 1033 per mm.2, which is approximately half that of the mean (2024 per mm.2) for the present series. In a personal communication, Dr. Szabo states that only one of his specimens (No. 62) was obtained from the flexor aspect of the forearm, as was the case with all specimens making up the present group. Strictly speaking therefore, this is the only one of his specimens which can be used for comparison with the preseut series, as it would appear (Szabó (1)) that there may be considerable intrinsic differences in the number of melanocytes in different areas of a limb. Since, in addition, the total area counted in this particular specimen was comparatively small (0.0804 mm.2), there does not appear to be much point in discussing further the apparent discrepancy in results between the two series. It may be worthwhile emphasizing however, that in this matter of cell-counts
carried out on different material by different observers, apparently minor differences in technic, and such factors as the personal element involved in deTABLE 1 Mean number of melaaocytes per square millimeter in skin front the flexor aspect of the forearm Specimen
Age
Area counted
1 2 3 4 5 6 7 8 9 10
22 34 19 20 26 25 20 19 19 21
0.64 mm.' 0.64 mm.' 0.64 mm.' 0.64 mm.' 0.64 mm.'
2/Jelanneytes per mm.'
1743
52
1860 1905
19
1963
0.64 mm.' 0.64 mm.' 0.64 mm.'
2005 2060 2105 2122 2155
0.64 mm.2
2327
0.64 mm.2
Overall mean: 2024
70 51 61
Complexion
Fair (freckled) Fair (freckled)
Dark Dark Fair (freckled)
74 73
Fair
79 25 76
Fair Dark Dark
52
Fair
MELANOCYTE COUNTS ON HUMAN FOREARM SKIN
183
ciding whether a particular cell is, or is not, a melanocyte, are bound to lead to some discrepancy in the final results. This can be reduced to a minimum if a standardized technic is adopted in all instances. As a result of experience gained with the present material, it is suggested that when counting melanocytes, a magnification of the order indicated above should be used, if accurate and comparable results are to be obtained. In the last column of Table 1, an indication is given of the general skin complexion of the individuals comprising the group. It is of some interest to note that the two lowest counts were obtained from fair-skinned subjects, and the two highest from dark-skinned ones. This suggests a possibility that there may be some correlation between melanocyte concentration and complexion, although the fact that counts intermediate between these extremes were obtained from fair and dark-skinned individuals alike, would appear to suggest the contrary. However, the present group is not sufficiently large to allow one to make any final pronouncement on this question. In order to be able to do so, further counts on a larger series of specimens must be undertaken. An answer, one way or another, to this problem is desirable in view of the suggestion that the frequency incidence of malignant melanoma at a given site may be related to the local population density of melanocytes, and of the oft-repeated one that melanoma is commoner in fair, than in dark-skinned individuals. SUMMARY
Melanocyte counts were carried out on dopa-treated "split-skin" preparations
of human epidermis, obtained from the upper part of the inner aspect of the flexor surface of the forearm of ten male white subjects of varying complexions. 52 The mean number of melanocytes per mm.2 was found to range from 1743 76, giving an overall mean for the ten of 2024 52. to 2327 REFERENCES 1. SZAB6, G.: The number of melanocytes in human epidermis. Brit. M. J., 1: 1016, 1954. 2. BILLINGHAM, R. E. AND MzDAWAR, P. B.: A study of the branched cells of the mammalian
epidermis with special reference to the fate of their division products. Philos. Tr. Roy. Soc., London, B., 237: 151, 1953. 3. SZAB6, G.: A modification of the technique of "skin splitting" with trypsin. J. Path. & Bact., 70: 545, 1955. AnDENDUM
Since this paper went to press, results of extensive melanocyte counts have been published by Staricco and Pinkus (J. invest. Berm., 8: 33—45, 1957). These authors found the number of melanocytes in human forearm skin to range from 1150 to 2500 per mm.2 The higher of these two figures was obtained from a specimen specifically labelled "Upper forearm", and is not very different from the figures obtained from the present specimens, all of which came from a similar area. Staricco and Pinkus's figures for their other forearm specimens (exact area unspecified) are considerably lower, and range from 1150 to 1500 per mm.2.
184
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Consideration of these figures underlines the fact that considerable variations in melanocyte concentration occur within the forearm, and that mean figures quoted for this entire region may be very different from the actual figures obtained from particular portions of it. Accordingly, it would seem desirable when the results of melanocyte counts are being published, that authors should indicate as explicitly as possible the exact area within a region from which their specimens were obtained. Otherwise, direct comparison of results obtained by different workers may be difficult.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.