A Study of the Argentaffin (Kultschitzky) Cells in frozen-dried Tissue by Phase-Contrast Microscopy and Ultra-Violet Light By A. C.

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1 289 A Study of the Argentaffin (Kultschitzky) Cells in frozen-dried Tissue by Phase-Contrast Microscopy and Ultra-Violet Light By A. C. CHRISTIE (From the Royal Cancer Hospital, London. Present address, Royal Hospital for Women, Paddington, Sydney, Australia) With one plate (fig. 1) SUMMARY 1. The argentaffin cells in guinea-pig intestine have been studied by phase-contrast microscopy and in ultra-violet light. Frozen-dried tissue has been used. 2. One such cell in a section 5-7^ thick was selected and studied throughout. In an unfixed section mounted in nonane, the cytoplasm is packed with fine granules which emit a greenish-yellow fluorescence in ultra-violet light of wavelength 2,750 A. There is also photographic evidence of absorption of light of this wavelength. 3. After formaldehyde fixation the fluorescence changes to orange-yellow and becomes much weaker. The absorption of light by the granular cytoplasmic contents is also greatly reduced. 4. There is no fluorescence and no photographic evidence of absorption of light of wavelength 2570 A by the granular cytoplasmic contents, either before or after formaldehyde fixation. 5. The nuclear chromatin pattern in these cells is unaltered by formaldehyde fixation, and is well demonstrated in photographs taken in ultra-violet light of both the wavelengths mentioned above. INTRODUCTION I T has recently been reported that the granular material in the argentaffin (Kultschitzky) cells is a formalin artifact (anon., 1954). Also, Eros (1932) reported that the granules in formaldehyde-fixed tissue fluoresce in ultraviolet light. This phenomenon was re-examined by Jacobson (1939) in the case of formaldehyde-fixed human argentaffin cell (so-called 'carcinoid') tumour, the maximum amount of absorption being in the region of 2,700 A. Lison (1953) stated that the yellow fluorescence is only obtained after formaldehyde fixation. Erspamer and Asero (1952) claimed that the substance 'enteramine' within the argentaffin cells is 5-hydroxytryptamine. They, employing the picrate derivative, and Rapport, Green, and Page (1948), and Rapport (1949), working with the tryptamine-creatinine-sulphate complex, studied the ultra-violet absorption spectrum and found that each compound shows maximal absorption at 2,750 A. They did not employ formaldehyde at any stage. It is therefore important to ascertain whether, in fact, the granular argentaffin substance does or does not fluoresce in ultra-violet light and absorb such a wavelength, without previous treatment with formalin. [Quarterly Journal of Microscopical Science, Vol. 96, part 3, pp , 1955.]

2 290 Christie A Study of the Argentaffin (Kultschitzky) cells Although the granular material within these cells in human and guineapig material is fixed by osmium tetroxide (Christie, 1955) and by potassium dichromate (unpublished observation), it was considered advantageous to avoid chemical fixatives if possible. Frozen-dried tissue was the obvious choice, provided the granular material is retained in the unfixed cell in sections thus prepared. This was found to be the case, and a study of the argentaffin cells both by phase-contrast microscopy and in ultra-violet light will be the subject of the present communication. First, cells were studied in unfixed tissue; then the same cells were re-examined after formaldehyde fixation, and finally stained by Gomori's (1948) hexamine silver nitrate technique to ensure correct identification of the cells under examination. MATERIAL AND METHODS After a guinea-pig had been killed instantly, a piece of duodenum (or upper jejenum) was transferred to liquid propane at C. within 45 seconds. Frozen-dried material was obtained by drying the tissue at 40 0 C. for 3 days and then embedding in paraffin wax at 58 0 C. over a period of 2 minutes. Sections 5-7/x thick were cut and mounted on quartz slides. After removal of the paraffin by flooding the section with nonane, a quartz coverslip was applied and sealed round the edges with molten paraffin wax in order to prevent the nonane from evaporating. The following procedures were then carried out: (1) Examination under ordinary light, first by ordinary and then by phasecontrast microscopy, and finally by ultra-violet light of wavelengths 2,750 A and 2,570 A. (2) After removing the coverslip carefully and allowing the nonane to evaporate, the section was exposed to formaldehyde vapour (from a solution of commercial formalin of 40% strength in a Coplin jar) for 4 hours and then immersed in 10% formaldehyde with 1% calcium chloride for 16 hours. After washing in distilled water for half an hour the section was remounted in nonane and examined as in (1) above. (3) The coverslip was again removed and the section stained by Gomori's (1948) hexamine silver nitrate technique for approximately 18 hours, when the granules in the argentaffin cells appeared a light brown colour against a practically unstained background. RESULTS In frozen-dried unfixed paraffin sections of guinea-pig small intestine, the argentaffin cells are clearly visible under ordinary light microscopy, but are even more clearly discernible when phase-contrast is used. In fig. 1, A a plump, roughly spherical cell can be seen filled with fine granules. On formaldehyde fixation and subsequent staining it is seen to contain the silver-reducing granules typical of argentaffin (Kultschitzky) cells, a confirmation of its identity.

3 Christie A Study of the Argentaffin (Kultschitzky) cells 291 Under ultra-violet light of wavelength 2,750 A this cell emits a clearly visible greenish-yellow fluorescence, and contains cytoplasm which strongly absorbs light of this wavelength (fig. 1, B). After formaldehyde fixation this cell shows some shrinkage (fig. 1, D), but no more than is apparent in nearby cells. However, when viewed under ultraviolet light of wavelength 2,750 A there is a considerable difference in the cell's appearance. The bright greenish-yellow fluorescence has now changed to orange-yellow and its intensity is considerably diminished. Fig. 1, E shows that the intensity of absorption of light of this wavelength by the granular cytoplasmic contents is now not detectable photographically. The reduction of the intensity of both the fluorescence and the absorption after formaldehyde fixation could possibly be explained on the basis of alterations in the intensity of the ultra-violet light source overnight (for a lapse of this time is required for formaldehyde fixation to be accomplished). To overcome this possible source of error, sections from the same paraffin block of frozen-dried unfixed material were cut and examined in conjunction with the formaldehyde fixed ones at the same time. Over a period of about 15 minutes the sections, fixed and unfixed, were repeatedly interchanged and examined by myself and two colleagues, without knowledge as to which one was fixed and which not so treated, in order to eliminate the possibility of personal factors prejudicing decisions as to colour and intensity changes, as well as eliminating errors due to changes in the intensity of the light source. A change in both the colour and the intensity of the flourescence after formaldehyde fixation was clearly apparent. The unfixed cell was also examined in light of wavelength 2,570 A, both before and after formaldehyde fixation. Fig. 1, F depicts the same cell after, and fig. 1, c the cell before formaldehyde fixation, when photographed in light of this wavelength. In both figures strong absorption by the nuclear chromatin is shown, but not by the granular cytoplasmic contents; nor do they fluoresce. There is also absorption of light of wavelength 2,750 A by the nuclear chromatin, and comparison of fig. 1, B and 1, c with 1, E and 1, F show that formaldehyde fixation has not materially altered the typical nuclear pattern; or rather, since the pattern was first depicted in formaldehyde-fixed tissue as, for example, in Ciaccio's (1906) illustration, it is more correct to say that the characteristic pattern is not produced by such fixation. The granules have the same appearance and are of about the same size in unfixed and fixed tissue, when studied by phase-contrast microscopy, as can be seen from fig. 1, A and 1, D. DISCUSSION Gomori (1948) maintained that formalin either alone or in mixtures is essential for the fixation of the argentaffin cell with its content of cytoplasmic granular material. On the other hand, Cordier (1926) reported that he could see and stain the granules with neutral red, in teased fresh tissue suspended in 'serum artificief.

4 292 Christie A Study of the Argentaffin (Kultschitzky) cells It has now been shown that, in frozen-dried guinea-pig tissue, the granules are present in a morphologically similar form to those seen in formaldehydefixed tissue. In such unfixed tissue sections prepared by the Altmann-Gersh technique they also emit a greenish-yellow flourescence when viewed in ultra-violet light of wavelength 2,750 A. After formaldehyde fixation the colour changes to orange-yellow and the intensity is diminished. The intensity of light absorption as assessed by photographic means is also greatly diminished by treatment with formalin. In the cell depicted in fig. 1, E it was not sufficient to affect the photographic plate, though in other argentaffin cells examined in the same and in other sections there was often slight darkening of the cytoplasm. Thicker sections, such as can be employed when one is examining carcinoid tumours, would almost certainly show considerable absorption amongst groups of cells rich in granular material; but throughout this work only single cells in sections 5-7 /x. thick have been examined. These observations are opposed to the hypothesis that the granular material is a formalin artifact, if by this is meant that in tissue fixed in fixatives not containing formalin the granules are not demonstrable. However, they confirm that formalin almost certainly has some chemical action on the granules a deduction previously made from the observation that previous fixation by formaldehyde prevents the subsequent darkening of them by osmium tetroxide (Christie, 1955). It appears that the above findings favour the hypothesis of Cordier (1926), who also worked with guinea-pig material, that the argentaffin substance is present in granular form in fresh tissues examined immediately after removal from the animal. I should like to thank Mr. R. King for taking the photographs, and Miss Shirley Charter for preparing the frozen-dried sections. FIG. 1 (plate), A, a frozen-dried, unfixed, and unstained paraffin section of guinea-pig duodenum, showing a crypt of Lieberkiihn with a relatively large, spheroidal argentaffin cell containing cytoplasm packed with fine granules. (Phase-contrast.) B, the same crypt of Lieberkiihn as shown in fig. 1, A, viewed by ultra-violet light of wavelength 2,750 A. The cytoplasmic contents of the argentaffin cell show strong absorption of this light. Although it is better seen in the next figure, the typical chromatin pattern within the nucleus can be discerned. C, the same crypt as in fig. 1, A, B, photographed in ultra-violet light of wavelength 2,570 A. Although there is still strong absorption by the nuclear chromatin, the cytoplasmic contents of the argentaffin cell do not absorb light of this wavelength. D, the same crypt viewed by phase-contrast microscopy after formaldehyde fixation. Apart from considerable shrinkage, the cytoplasmic contents of the argentaffin cell have been otherwise unaltered by fixation. E, the same crypt viewed by ultra-violet light of wavelength 2,750 A, after formaldehyde fixation. Although the nuclear chromatin still shows strong absorption and reveals a pattern unchanged by fixation, the cytoplasmic contents now show no photographically detectable absorption. F, the same crypt, now photographed in ultra-violet light of wavelength 2,570 A, after formaldehyde fixation. Nuclear chromatin still shows strong absorption, but, as in fig. 1, c, there is no cytoplasmic absorption by the argentaffin cell.

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6 Christie A Study of the Argentaffin (Kultschitzky) cells 293 REFERENCES ANON., Lancet, Annotation, 2, 372. CHRISTIE, A. C, (In the press.) CIACCIO, M. C, C.R. Soc. biol. Paris, 60, 76. CORDIER, R., Arch. Biol. Paris, 36, 427. EROS, G., Allg. Path. Anat., 54, 385. ERSPAMER, V., and ASERO, B., Nature Lond., 169, 800. GOMORI, G., Arch. Path., 45, 48. JACOBSON, W., J. Path. Bact., 49, 1. LlSON, L-, Histochimie et cytochimie animales. Paris (Gauthier-Villars). RAPPORT, M. M., GREEN, A. A., and PAGE, I. H., J. biol. Chem., 176, RAPPORT, M. M., Ibid., 180, 961.