in this study , we retrospectively selected 75 cases of formalin - fixed , paraffin - embedded hair - bearing adult skin samples between 2002 and 2009 at asan medical center . samples were taken from the margins of routine surgical procedures such as excisional biopsy or excision for benign / malignant skin lesions that were diagnosed as following : various inflammatory lesions with parakeratosis ( n=16 ) , keratinocytic tumors ( n=11 ) , benign tumors with apocrine and eccrine differentiation ( n=14 ) , malignant tumors with follicular differentiation ( n=1 ) , benign tumors with follicular differentiation ( n=17 ) , and tumors with sebaceous differentiation ( n=12 ) . the rest were retrieved from mature cystic teratoma ( n=2 ) and normal scalp tissues biopsied for therapeutic reasons ( n=2 ) . to analyze cd99 expression patterns in fetal skin , gestational ages of the collected cases were 16 , 17 , 19 ( 2 cases ) , 20 , 25 , 26 , and 28 weeks . all tissues were immediately fixed in 10% buffered formalin , and then processed in paraffin wax using standard procedures . sections were serially cut into 4-m sections and stained with hematoxylin and eosin for examination . the study protocol was approved by the institutional review board ( project number 2009 - 440 ) of asan medical center . when multiple blocks were available in a single case , one block containing representative tissue was selected . ihc staining for cd99 was performed using a benchmark xt autoimmunostianer ( ventana medical systems , tucson , az , usa ) according to the manufacturer s instructions and using the reagents supplied with the kit . in brief , 4-m sections were mounted on silanized charged slides , dried for 10 minutes at room temperature and then further dried for 20 minutes at 65c . after deparaffinization , heat - induced epitope retrieval using standard cell conditioning solution 1 was performed for 24 minutes . subsequently , primary anti - cd99 ( 1:100 , clone dn16 , dinona , seoul , korea ) was applied by an automated immunostaining system with the ultraview dab detection kit ( ventana medical systems ) . analysis of whole tissue section slides stained for cd99 by ihc was performed under a light microscope as previously described [ 14 - 16 ] . the results of cd99 immunostaining were classified into the following groups : consistently complete and strong membrane staining was assigned a value + + , weak to moderate membrane staining was assigned a value of + , variable membrane staining was assigned a value of + / , and no staining was assigned a value of . occasional cytoplasmic staining was minimal and weak . for immunofluorescence ( if ) staining , 4-m sections were incubated with mouse anti - cd99 ( 1:200 , clone dn16 , dinona ) and rabbit anti - cd34 ( 1:10 , # ab64480 , rabbit polyclonal , abcam , cambridge , uk ) for 60 minutes at room temperature . sections were then incubated with tetramethylrhodamine - conjugated host anti - mouse igg antibody and fluorescein isothiocyanate - conjugated host anti - rabbit igg antibody for 60 minutes at room temperature . sections incubated with the appropriate isotype control primary antibodies and fluorescently labeled secondary antibodies were utilized as necessary . results were analyzed using confocal microscopy ( leica tcs_np / sp , leica microsystems , mannheim , germany ) . in this study , we retrospectively selected 75 cases of formalin - fixed , paraffin - embedded hair - bearing adult skin samples between 2002 and 2009 at asan medical center . samples were taken from the margins of routine surgical procedures such as excisional biopsy or excision for benign / malignant skin lesions that were diagnosed as following : various inflammatory lesions with parakeratosis ( n=16 ) , keratinocytic tumors ( n=11 ) , benign tumors with apocrine and eccrine differentiation ( n=14 ) , malignant tumors with follicular differentiation ( n=1 ) , benign tumors with follicular differentiation ( n=17 ) , and tumors with sebaceous differentiation ( n=12 ) . the rest were retrieved from mature cystic teratoma ( n=2 ) and normal scalp tissues biopsied for therapeutic reasons ( n=2 ) . to analyze cd99 expression patterns in fetal skin , gestational ages of the collected cases were 16 , 17 , 19 ( 2 cases ) , 20 , 25 , 26 , and 28 weeks . all tissues were immediately fixed in 10% buffered formalin , and then processed in paraffin wax using standard procedures . sections were serially cut into 4-m sections and stained with hematoxylin and eosin for examination . the study protocol was approved by the institutional review board ( project number 2009 - 440 ) of asan medical center . all hematoxylin and eosin  stained slides were reviewed to ensure quality . when multiple blocks were available in a single case , one block containing representative tissue was selected . ihc staining for cd99 was performed using a benchmark xt autoimmunostianer ( ventana medical systems , tucson , az , usa ) according to the manufacturer s instructions and using the reagents supplied with the kit . in brief , 4-m sections were mounted on silanized charged slides , dried for 10 minutes at room temperature and then further dried for 20 minutes at 65c . after deparaffinization , heat - induced epitope retrieval using standard cell conditioning solution 1 was performed for 24 minutes . subsequently , primary anti - cd99 ( 1:100 , clone dn16 , dinona , seoul , korea ) was applied by an automated immunostaining system with the ultraview dab detection kit ( ventana medical systems ) . analysis of whole tissue section slides stained for cd99 by ihc was performed under a light microscope as previously described [ 14 - 16 ] . the results of cd99 immunostaining were classified into the following groups : consistently complete and strong membrane staining was assigned a value + + , weak to moderate membrane staining was assigned a value of + , variable membrane staining was assigned a value of + / , and no staining was assigned a value of . occasional cytoplasmic staining was minimal and weak . for immunofluorescence ( if ) staining , 4-m sections were incubated with mouse anti - cd99 ( 1:200 , clone dn16 , dinona ) and rabbit anti - cd34 ( 1:10 , # ab64480 , rabbit polyclonal , abcam , cambridge , uk ) for 60 minutes at room temperature . sections were then incubated with tetramethylrhodamine - conjugated host anti - mouse igg antibody and fluorescein isothiocyanate - conjugated host anti - rabbit igg antibody for 60 minutes at room temperature . sections incubated with the appropriate isotype control primary antibodies and fluorescently labeled secondary antibodies were utilized as necessary . results were analyzed using confocal microscopy ( leica tcs_np / sp , leica microsystems , mannheim , germany ) . to examine the expression pattern of cd99 in epidermal keratinocytes , immunostaining for cd99 was performed on nonpathologic adult skin samples ( fig . 1a ) . in the epidermis , cell membranes in the basal cell layer were strongly stained ( fig . 1b ) , whereas the keratinocytes in the prickle cell layer just above the basal cell layer showed markedly reduced staining and cells in the granular and hornified layers did not express cd99 . we also identified intense cd99 expression in langerhans cells in the epidermal layer ( fig . infundibula of hair follicles possessed a cd99 expression pattern similar to that of the epidermis . 1c ) at the bulges , and basal cells of the follicular ors were also strongly stained ( fig . other subpopulations of hair follicles showed varying immunoreactivity . when observing cd99 staining in the central shaft of hair follicles , the cuticle was negative for cd99 , the lower irs was weakly positive , and the ors was strongly positive ( fig . to flat cell - lined apocrine glands were negative for cd99 , but relatively tall cell - lined apocrine glands were positive for cd99 . the apical luminal surfaces of both flat and tall cell - lined glands were cd99-negative ( fig . secretory coils of eccrine sweat glands are composed of outer clear cells and inner cuboidal cells ; the outer clear cells were immunonegative and scattered inner cuboidal cells were immunopositive for cd99 ( fig . the excretory duct is composed of two - layered cuboidal cells , and both layers were immunopositive for cd99 ( fig . the apical luminal surfaces of inner eccrine cells and inner excretory ducts also lacked cd99 expression ( fig . 2b , c ) . in the sebaceous glands , basal cells surrounding the sebaceous alveolus were cd99 immunopositive , but mature sebocytes containing central lipid droplets were immunonegative for cd99 ( fig . cells that were positive for cd99 immunostaining in normal adult epidermis and skin appendages are summarized in table 1 . eight developing fetal human skin samples were obtained from aborted fetuses of 1628 gestational weeks . in early stages , fetal skin is called periderm and is composed of 23 epidermal cell layers . at 16 weeks of gestation , the periderm , including the epidermal basal layer and hair follicle germ , was negative for cd99 , whereas mesenchymal cells around the hair germ were strongly positive for cd99 ( fig . , the fetal epidermis and skin appendages start to resemble normal adult skin ; basal cell layer , intermediate cell layer , hair follicles , sweat glands , and sebaceous glands appear at this stage . cd99 was expressed in the epidermal basal cell layer , hair bulge , ors , and dermal papilla ( fig . fetal skin is histologically similar to adult skin , showing cd99 immunoreactivity patterns identical to those of adult skins ( fig . 3c , d ) . in our study , cd99 staining was particularly strongly positive in immature epidermal basal cells and cells at the bulge . we therefore compared cd99-positive subpopulations of normal epidermis and skin appendages to cd34-positive subpopulations that have been reported as cutaneous precursor cells using double - if . we focused on expression patterns of these markers in the basal cells of epidermis and hair follicles . in the adult epidermis and skin appendages , cd99 and cd34 expression partially overlapped at the lower ors ( fig . c ) . in the 20-week - old fetal skin , cd99 and cd34 were not co - expressed in the basal cells of epidermis ( fig . 5c ) , or ors ; however , mesencymal cells of dermal papilla showed co - expression ( fig . to examine the expression pattern of cd99 in epidermal keratinocytes , immunostaining for cd99 was performed on nonpathologic adult skin samples ( fig . 1a ) . in the epidermis , cell membranes in the basal cell layer were strongly stained ( fig . 1b ) , whereas the keratinocytes in the prickle cell layer just above the basal cell layer showed markedly reduced staining and cells in the granular and hornified layers did not express cd99 . we also identified intense cd99 expression in langerhans cells in the epidermal layer ( fig . infundibula of hair follicles possessed a cd99 expression pattern similar to that of the epidermis . 1c ) at the bulges , and basal cells of the follicular ors were also strongly stained ( fig . other subpopulations of hair follicles showed varying immunoreactivity . when observing cd99 staining in the central shaft of hair follicles , the cuticle was negative for cd99 , the lower irs was weakly positive , and the ors was strongly positive ( fig . secretory apocrine glands showed variable cd99 expression . relatively thin to flat cell - lined apocrine glands were negative for cd99 , but relatively tall cell - lined apocrine glands were positive for cd99 . the apical luminal surfaces of both flat and tall cell - lined glands were cd99-negative ( fig . secretory coils of eccrine sweat glands are composed of outer clear cells and inner cuboidal cells ; the outer clear cells were immunonegative and scattered inner cuboidal cells were immunopositive for cd99 ( fig . the excretory duct is composed of two - layered cuboidal cells , and both layers were immunopositive for cd99 ( fig . the apical luminal surfaces of inner eccrine cells and inner excretory ducts also lacked cd99 expression ( fig . basal cells surrounding the sebaceous alveolus were cd99 immunopositive , but mature sebocytes containing central lipid droplets were immunonegative for cd99 ( fig . cells that were positive for cd99 immunostaining in normal adult epidermis and skin appendages are summarized in table 1 . eight developing fetal human skin samples were obtained from aborted fetuses of 1628 gestational weeks . in early stages , fetal skin is called periderm and is composed of 23 epidermal cell layers . at 16 weeks of gestation , the periderm , including the epidermal basal layer and hair follicle germ , was negative for cd99 , whereas mesenchymal cells around the hair germ were strongly positive for cd99 ( fig . , the fetal epidermis and skin appendages start to resemble normal adult skin ; basal cell layer , intermediate cell layer , hair follicles , sweat glands , and sebaceous glands appear at this stage . cd99 was expressed in the epidermal basal cell layer , hair bulge , ors , and dermal papilla ( fig . fetal skin is histologically similar to adult skin , showing cd99 immunoreactivity patterns identical to those of adult skins ( fig . in our study , cd99 staining was particularly strongly positive in immature epidermal basal cells and cells at the bulge . we therefore compared cd99-positive subpopulations of normal epidermis and skin appendages to cd34-positive subpopulations that have been reported as cutaneous precursor cells using double - if . we focused on expression patterns of these markers in the basal cells of epidermis and hair follicles . in the adult epidermis and skin appendages , cd99 and cd34 expression partially overlapped at the lower ors ( fig . c ) . in the 20-week - old fetal skin , cd99 and cd34 were not co - expressed in the basal cells of epidermis ( fig . 5c ) , or ors ; however , mesencymal cells of dermal papilla showed co - expression ( fig . the aim of the current study was to examine cd99 expression in normal human skin and skin appendages ; no such study has been performed to date . we demonstrated that cd99 was strongly expressed in the immature epidermal basal cells , but the fully differentiated granular layer did not show cd99 positivity in normal adult skin . this result is consistent with the findings of a previous study , in which cd99 expression was evaluated in a large series of cutaneous melanoma samples and was described briefly as a crisp membranous staining on normal epidermal basal cells . our study showed that in skin appendages , cd99 showed differential immunoreactivity in each subpopulation . bulge cells , basal cells of the ors , and eccrine inner cuboidal cells were consistently and strongly positive . however , cd99 expression was variably positive in apocrine secretory glands . relatively tall cells with luminal decapitation tended to be cd99-positive , but thin to flat cells without decapitation tended to be negative . analysis of cd99 expression was also performed during different developmental stages of fetal skin . in fetal skin , peridermal cells persist until about week 21 and gradually disappear as stratum corneum . in the early stages of epidermal development , mitotic activity occurs in all the epidermal layers ; however , when differentiation begins , mitotic activity becomes restricted to the cells of the basal layer . angiotensin converting enzyme ( ace ) , keratin 19 , 1-integrin , and p63 were used as stem cell markers in examining the various developmental stages of fetal skin by double if . the expression of ace , 1-integrin , keratin 19 , and p63 was seen in all epidermal layers of the developing fetal skin at 1120 weeks of gestation . from 21 weeks of gestation onward , their expression was mainly confined to the basal layer of epidermal cells . in our study , cd99 was not seen in the periderm at 16 weeks of gestation but was consistently expressed at 19 weeks of gestation . based on this result , we suggest that cd99 expression precedes the expression of ace , 1-integrin , keratin 19 , and p63 in the fetal basal layer . based on the particularly strong cd99 expression in immature thymic t - lineage cells , tonsillar lymphoid progenitor cells , and subventricular zone of fetal brain ( c .- s . park , unpublished data ) , we attempted to correlate cd99 expression with immature subpopulations of epidermis and appendages . there is evidence for populations of stem cells located at the basal layer of the epidermis and the hair follicle bulge . cd99 is also strongly expressed in the bulge . according to previous reports that analyzed expression patterns of various precursor markers , cd34 is more specific for the peripheral layer of the ors , cytokeratin ( ck ) 15 for bulge and epidermal basal cells , nestin for inner portions of the ors , ck19 for basal keratinocytes in the upper and lower third of the ors , p63 for the basal / suprabasal layer , hair matrix , and ors , and cd200 for the bulge [ 20 - 24 ] . we showed that cd34 , which was thought to have similar expressions patterns to cd99 , shows different expression by double if . in the present study , cd34 and cd99 were only co - expressed in the lower ors . therefore , we assumed that precursor cell subpopulations in the skin are more varied than previously thought . in conclusion , this study examined cd99 expression in normal adult and fetal skin . cd99 is strongly expressed in the immature epidermal basal cells in the bulge and basal cells of the hair follicle ors . these findings suggest that cd99 may carry out a specific function in the structures where it is expressed . therefore , we present that cd99 is a unique and previously unreported marker of epidermis and its appendages . future studies should focus on functions of cd99 in the epidermis and its appendages and investigate its feasibility as a novel target for the treatment of dermatologic lesions .