Yonago Acta medica 1999;42:201–210

Mitotic Langerhans Cells in the Normal Human Epidermis: Light andElectron Microscopic Observations in All Mitotic Phases
Shigeo Oota
First Department of Anatomy, Faculty of Medicine, Tottori University, Yonago 683-0826, Japan
In three experiments of the normal human epidermis under different biopsy and fixative
buffer conditions, Langerhans cells (LCs) in all mitotic phases were found with theThin-Section-Reembedding and Ultrathin-Sectioning (TRUS) technique; two in theprophase, five in the metaphase, two in the anaphase and two in the telophase. The
serial light microscopic examination showed many characteristic features, namely theshapes of mitotic LCs in each section were quite different in one cell as well as between
individual cells, even in the same mitotic phase. No round mitotic LCs were seen. TwoLCs in the prophase were detected in the specimens prefixed in a fixative dissolved indialysate (Kindaly 2) instead of buffer solution. Birbeck granules were witnessed in allphases.
Key words: electron microscopy; epidermis; human; Langerhans cells; mitosis

It has been considered difficult to observe mitotic Langerhans cells (LCs) in the normal humanepidermis, and so far only one electron microscopic observation of the mitotic LC (in theanaphase) has been reported by Konrad andHönigsmann (1973). In the present study, comprising three experiments under different biopsy and fixative buffer conditions, 11 LCs invarious mitotic phases were observed with theThin-Section-Reembedding and UltrathinSectioning (TRUS) technique (Oota, 1999). In
all phases, the mitotic LCs showed markedly
characteristic features. These features of allphases in the human epidermis were obtained
for the first time by light and electron microscopy.

Materials and Methods
Skin biopsies (5 mm × 15 mm) were taken 3times, the 2nd one year after the 1st and the 3rd
two months after the 2nd, from the forearm of a63-year-old healthy male volunteer, who had
given informed consent. The 1st and the 2nd

biopsy specimens were obtained under lidocaine
anesthesia without epinephrine, and the 3rd biopsy specimen was excised under anesthesia
with epinephrine. The biopsy specimens weretreated in the same manner every time: immersedin physiological saline immediately after operating at the dermatological biopsy-operating
room in this Faculty. After 20 min, the specimen was divided into 3 to 5 equal parts and prefixed in the fixative solutions shown in Table 1.
Here Kindaly 2 (Fuso Pharmaceutical Industry,Ltd., Osaka, Japan), acetic dialysate solution
for hemodialysis, was tried as a physiologicalvehicle instead of the buffer. The components
and osmolarity of the chemical fixatives in Experiments 1 and 2 are shown in Table 2. After 1h, each specimen was dissected into small
pieces and the fixation was continued for 24 h at
4˚C. Subsequent procedures were previously
described as the TRUS technique (Oota, 1999).
Although in the technique the so-called “inverted gelatin capsule technique” (Robbins andGonatas, 1964; Miyauchi and Hashimoto, 1987)
was used twice, the polyethylene capsules(TAAB Lab. Equip. Ltd., Berks., United Kingdom)

Abbreviations: CB, cacodylate buffer; LC, Langerhans cell; M, mol/L; PB, phosphate buffer; TRUS, ThinSection-Reembedding and Ultrathin-Sectioning201

S. Oota

Table 1. Primary fixative solutions and the number of sectioned blocks and of mitotic LCs
found in the materials prefixed with the fixatives
Primary
fixative solutions

No. ofsectioned blocks

No. ofmitotic LCs

Observed
mitotic phase

Experiment 1

2.5% GA in 0.05 M CB
302.5% GA in 0.1 M CB
44Met, Met, Tel, Tel
2.5% GA in 0.15 M CB
42Met, Ana
Experiment 2
2.5% GA in 0.05 M PB
1
02.5% GA in 0.1 M PB
302.5% GA in 0.15 M PB
202.5% GA in Kindaly 241ProExperiment 3
2.5% GA in 0.05 M CB
21Ana
2.5% GA in 0.1 M CB
202.5% GA in 0.05 M PB
202.5% GA in 0.1 M PB
21Met
2.5% GA in Kindaly 222
Pro, Met
Ana, anaphase; CB, cacodylate buffer; GA, glutaraldehyde; LC, Langerhans cell; M, mol/L; Met, metaphase;
PB, phosphate buffer; Pro, prophase; Tel, telophase.

were employed instead of gelatin capsules. All
thin-sections in the 3 experiments were cut
parallel to the surface of the epidermis. The oil
immersion pictures were taken from the sections on slides; a drop of water was poured between the section and a cover glass for use of
the section for electron microscopy. The ultrathin sections were placed on a copper grid, stained with uranyl acetate and lead citrate, and observed with a Hitachi electron microscope, H
500, at 80 kV.

Results
In the 1st experiment, 6 mitotic LCs (Fig. 1:Rows 3, 4, 5, 8, 10 and 11) were found in thematerials prefixed with the fixatives in 0.1 M
and 0.15 M cacodylate buffer (CB) (Table 1).
In the 2nd experiment, only 1 LC in the prophase (Fig. 1: Row 1) was observed in the materials prefixed in Kindaly 2, which is commonly used for artificial kidneys, and none in thematerials prefixed with the fixatives in phosphate buffer (PB) (Table 1). Why no mitotic
LCs were observed in these materials is ob-

Table 2. Components and osmolarity of chemical fixatives
Fixative solution

Experiment 1

Osmolarity of Total osmolarity
buffer (vehicle)(mOsm)(mOsm)

2.5% GA in 0.05 M CB
103345
2.5% GA in 0.1 M CB
2024302.5% GA in 0.15 M CB285510
Experiment 2 2.5% GA in 0.05 M PB
94373
2.5% GA in 0.1 M PB
203
470
2.5% GA in 0.15 M PB
296
566
2.5% GA in Kindaly 2255
484
CB, cacodylate buffer; GA, glutaraldehyde; LC, Langerhans cell; M, mol/L;
PB, phosphate buffer.

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Mitotic LCs in the normal human epidermis

RowNo.
1

Light microscopic pictures of serial sections of mitotic LCs
Section No.
23456781234567891011Remarks for Fig. 1Row No. Mitotic phaseFig. 1. Light microscopic pictures of mitotic
Langerhans cells (LCs). Rows 1 and 2, in theprophase; Rows 3 to 7, in the metaphase;Rows 8 and 9, in the anaphase; and Rows 10and 11, in the telophase. All pictures, except
Row 8, were photographed by oil immersion, × 510. Row 8 is the first mitotic LC
encountered by chance, × 255. CB, cacodylate buffer; PB, phosphate buffer.

Buffer*Experiment No.

1Prophase
Kindaly 2
2nd experiment
2Prophase
Kindaly 2
3rd experiment
3Metaphase
0.1 M CB
1st experiment
4Metaphase
0.1 M CB
1st experiment5
Metaphase
0.15 M CB
1st experiment
6
Metaphase
0.1 M PB
3rd experiment
7
Metaphase
Kindaly 2
3rd experiment
8
Anaphase
0.15 M CB
1st experiment
9Anaphase
0.05 M CB
3rd experiment
10Telophase
0.1 M CB
1st experiment
11Telophase
0.1 M CB
1st experiment*Buffer or vehicle used in fixative solution.

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