ACTA O P H T H A L M O L O G I C A VOL. 4 5 1967

From the Department o / Ophthalmology, Kommunehos@italet, Copenhagen, Denmark.

(Head: Professor P. Brendstrup, M . D.).

TRYPAN BLUE::.)

Vital Staining of Cornea and Conjunctiva.

BY

M. S. Norn

Trypan blue is a blue dye, a diazo compound. The molecule is composed of three elements connected by two azo-links (-N= N-). Each element consists of two hexagonal rings (fig. 1, after Evans).

The dye contains acid groups. It therefore becomes linked to alkaline groups in the tissue, which latter it stains. The stained tissue areas may accordingly be characterized as acidophilic.

Vital staining with trypan blue may proceed in two ways differing in prin- ciple (Baker).

The dye may be taken up in living cells by a kind of phagocytosis. However, only cells of the reticulo-endothelial system and similar cells are able to absorb the dye actively. Trypan blue is then visible as coarse grains in the cytoplasm.

Other dyes may likewise be phagocytized, but trypan blue seems to be the most suitable dye for this purpose.

Trypan blue can also stain dead or degenerate cells (Pappenheimer), even any kind of such cells. The staining is diffuse.

Fig. I .

") Received Sept. 28th 1966. *'-) This work was supported by Kommunehospitalets 100 Ars Jubilaeumsfond and by

Det videnskabelige Sekretariat, Ksbenhavns Hospitalsvmen.

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Trypan blue stains all dead cells as well as the cells having reached a certain degree of degeneration. Slightly degenerate cells remain unstained.

The early stage of degeneration can be disclosed by other dyes (neutral red, acridine orange) or by a reduced growth rate in tissue culture.

The above results have been achieved from experiments with leucocytes in blood, pus, and urine (neutral red, Fleishmaizn et al.) and bone marrow cells (acridine orange, Hataway). They may not be of universal validity, however.

Examples of the former type of staining have been seen in the conjunctiva after subconjunctival injection of the dye (Sousa, Lohlein): Phagocytosis of dye grains by living cells, manifesting itself by presence of such grains in histiocytes.

The latter type of staining: "general vital staining", is seen after instillation of the dye into the conjunctival sac. This is employed for vital staining of the cornea and conjunctiva, e. g. of wearers of contact lenses.

The object of the present study has been to compare the vital-staining capa- city of trypan blue with those of rose bengal (directly) and methylene blue (indirectly), the latter two dyes having been subjected to similar investigations by the author (Norn 1964 a, 1964 b, 1966).

PRESENT INVESTIGATIONS

These comprised examinations of 3 1 microscopical preparations, 15 experimen- tal studies, and examination of a clinical series of 100 eyes from 100 patients.

The dye used was a 1 'O/O aqueous solution of trypan blue from the British firm Gurr. The colour intensity may vary somewhat. The highest colour intensity was used in most of the cases, a lower intensity having been used in no more than 14 per cent.

The hydrogen ion concentration in the solution emplyed was 6.44 (the Central Laboratory, Kommunehospitalet).

MICROSCOPY Biopsy of the Conjunctiva.

Biopsy 25 minutes after subconjunctival injection of trypan blue showed dye gxains to have been phagocytized in histiocyte-like cells situated round the vessels. Otherwise no staining was seen.

In a case of pronounced corneal ulcer with loss of substance trypan blue was instilled in the conjunctival sac two hours before the enucleation (photo 3 b).

This specimen showed not only dye grains in phagocytizing cells of the connective tissue, but also nuclear staining of connective tissue cells. The latter must be supposed to be dead cells. A small number of superficial squamous cells were likewise slightly stained.

38 1

Subconjunctival injection of the mucus-specific alcian blue (NorTt 1963) caused staining of connective tissue fibrils. No such fibrils were stained by try- pan blue, which only gave phagocytized dye grains or staining of degenerate cells.

Aspirated Con junctiual Fluid. The conjunctival fluid was aspirated with a special pipette (Norn 1960)

shortly after vital staining with rose bengal and trypan blue. Most of the cells had blue nuclei and red or blue cytoplasm (photos 1 and 2).

Some of the cells were only slightly stained. Neutrophilic leucocytes were often only red or completely unstained.

Mucous Thread. The mucous thread in the inferior conjunctival fornix (Norn 1963) was

removed by means of two wooden pegs after vital staining with rose bengal and trypan blue, and transferred to a glass slide.

The preparation was stained most intensely b y the last dye employed (try- pan blue or rose bengal).

Practically all the cells, even neutrophilic leucocytes, were stained by trypan blue. The nuclei obtained the strongest colour. The cytoplasm was oftcn only stained by rwe bengal. Definitely blue or definitely red cells were rare. A few instances were seen of red nuclei in blue cytoplasm.

The mucous fibrils were stained a paler colour by trypan blue or rose Lengal. Precipitated dye grains were practically never found (photo 4).

The above two dyes contrast in this respect with methylene blue, which is most often seen as coarse, precipitated grains, only occasionally with staining of odd cells round the dye grains (Norn 1966 and photo 5) .

Epithelial Scrapings. Intensely vital-stained areas were scraped with a knife, after superfluous

vital stain had been carried off with the tear fluid. Microscopy showed the stained areas to consist of nests of cells having blue

nuclei and red or blue cytoplasm. These were surrounded by non-stained cells.

Conclusion

and, to a smaller extent, mucus. Trypan blue is a reliable vital stain, which stains dead and degenerate cells

EXPERIMENTAL STUDIES

Trypan blue does not tend to precipitate. I t can be mixed with rose bengal and fluorescein. This stable mixture of three vital stains serves as a specific

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vital stain giving some areas a blue colour (trypan blue), others a red (rose bengal), while some show yellow fluorescence (fluorescein).

Trypan blue does not precipitate in acid (pH 2) or alkaline buffer (pH 13). Previous instillation of a buffer, p H 4.5 - pH 10, into the conjunctival sac hardly alters the stainability.

Subsequent staining of the conjuntiva and cornea with the mucus-specific green alcian blue gives no possibility of distinguising definitely between green mucus and blue cells, the colour contrast being too slight.

T i m e Factor. The trypan-blue-stained areas of the conjunctiva or cornea will fade slowly,

approximately like rose-bengal-stained areas, the colour having disappeared after 20 to 60 minutes.

Permanent discoloration has not been observed.

CLINICAL STUDZES

The clinical material comprised 100 eyes, the great majority with external ocular diseases, where vital staining was supposed to occur. The diagnoses are shown in table 1 .

Table I. The clinical material, vital-stained by trypan bltie, following staining by a mixture of

rose bengal and fluorescein. The group of other diseases comprises solitary or few cases of ectropion, exophthalmos,

facial palsy, episcleritis, phlyctena, chemosis, glaucoma, etc.

normal erosion and foreign body in cornea dentritic keratitis keratitis zoster other keratitis keratoconjunctivitis sicca wearers of contact lenses infectious conjunctivitis papillomatous conjunctivitis chronic simple conjunctivitis blepharoconjunctivitis subconjunctival haemorrhage other diseases

4 16 2 4

10 3 6

14 6 7 4 5

19

Total 100

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Procedure. A small drop (0.01 ml) of a mixture of rose bengal and fluorescein was first

instilled into the conjunctival sac (rose bengal 50 mg, fluorescein 50 mg, sodium chloride 45 mg, distilled water to 5 g).

The vital staining was observed in a slit lamp and was recorded in tabular form. The degree of staining was estimated in arbitrary grades 1 to 5 . Grade 3 indicates moderate staining, grades 2 and 4 weak and intense staining respectively, while grades I and 5 represent the extremes.

Next 0.01 ml of trypan blue was instilled. The blue colour was observed and recorded similarly.

+Try Total

Side-Elfects. Unlike methylene blue, which frequently (in 30 per cent) causes the eyes

to smart, often excessively so, instilled trypan blue made only few patients (4 per cent) complain of smarting eyes.

Local anaesthesia was employed in no more than 3 per cent of the presmt series.

-Try +Try -Try -Try -Try -R.B. +R,B, +Flu +R.B.or -R.B. -Flu +Flu --Flu

Cornea. Fig. 2 A illustrates the mean grades of staining by trypan blue (black column)

and rose bengal (white column). The cornea was on the average a little more intensely stained by rose bengal than by trypan blue.

Table 2 shows that 40 per cent were stained by trypan blue and - together with a.n additional 10 per cent - by rose bengal or fluorescein, whereas there was no instance of staining by trypan blue alone.

Table 2. Staining of different sites by trypan blue (Try) following staining by rose bengsl (R.B.)

and fluorescein (Flu). 100 patients. The figures indicate the percentages of patients whose eyes were stained by the dye

concerned (+) or remained unstained by the dye concerned (-).

cornea 40 0 7 6 10 50 bulbar conj. 60 10 7 1 7 33 plica semilun. 70 23 7 0 7 23 caruncle 95 8 2 0 2 3 inf. fornix 9 4 0 0 0 91 inf. tarsus 48 24 1 0 1 51 Marx' line 74 0 26 0 26 0

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Photo 1. Conjunctival fluid, vital-stained by rose bengal and trypan blue

prior to suction with pipette. X 250.

Photo 2 . Froin the same preparation as fig. 1. X 400.

Photo 3 n . Pronounced corneal

ulcer, vital-stained by rose bengal.

Photo 3 b. Same eye as photo 3 a , vital-stained by trypan blue. Marx’ line is just

visible.

Photo 4. Mucous thread from the inferior conjunctival fornix, vital-stained

by rose bengal and trypan blue. X 100.

PhCJto 5 . Mucous thread from the inferior conjunctival fornix, vital-stained

by rose bengal and inethylene blue. X 100.

The ten cases with failing trypan-blue staining comprised one of mild corneal lesion caused by a contact lens, one of foreign body in the cornea, and two cases of each of the following lesions: marginal keratitis, corneal erosion, and corneal oedema with burst bullae.

In these eight cases important diagnostic data would be missed by vital stain- ing with trypan blue alone. The rose bengal and fluorescein mixture, on the other hand, disclosed the stated morbid conditions.

In the last two cases such a slight punctate rose bengal staining was seen that it could not be said for certain to be pathological.

Bulbar Conjunctiva. The meail grades of staining seemed to be alike for trypan blue and rose

bengal (fig. 0 A). In 10 per cent staining was only obtained with trypan blue, and in 7 per cent

only with rose bengal (table 2). In no more than one case was there a clinically significant difference between

the two dyes: a slight corrosion due to hydrochloric acid was stained by rose bengal and fluorescein, but not by trypan blue. In one of the cases of keratocon- junctivitis sicca the grades of staining were identical, while in two rose bengal stained more intensely. The same was true for the cornea and the plica semi- lunaris.

Plica Semilunaris and Caruncle. The mean grades of staining were approximately equal for the two dyes. Trypan blue stained more often than rose bengal. No clinical importance was

recognizable of punctate staining in these areas, which may also be intensely stained in normals.

A vertical line of trypan blue along the lateral border of the plica was found in a small number of cases (6 per cent).

A blue ring round the ciliary root was observed in 12 per cent.

Inferior Fornix and Con junctiva of Inferior Tarsus. Trypan blue was seen to stain more intensely and more frequently than rose

bengal in these areas of the conjunctiva. The staining was represented by scat- tered dots and seemed to be of no clinical importance.

Marx' Line. Marx' line is a line presenting punctate, often intense staining seen along

the palpebral border over the distribution of the lacrimal streak. It was found to be stained in all cases by rose bengal, and most intensely by this dye. 74 per cent were also stained by trypan blue (photo 3 b). The blue dots were found scattered among the red.

385 Acta Ophthalmcl 45. 111 27

In five instances a suggestion of a double line was seen: red anteriorly and blue posteriorly. The phenomenon was more frequent in the cases of combined staining with methylene blue and rose bengal.

The failing trypan blue staining seemed to be of no clinical importance.

DISCUSSION

In 100 cases a direct comparison was drawn between the vital-staining capa- cities of trypan blue and rose bengal, that is a blue versus a red dye. Both dyes possess acid groups.

On microscopy trypan blue was found to be a reliable vital stain, which, however, perhaps stains less frequently than rose bengal. This suggests that cells at an early stage of degeneration are only stained by rose bengal, whereas not by trypan blue.

Neutrophilic leucocytes in the conjunctival fluid are often only stained by rose bengal, while the corresponding cells in the mucous thread are stained by both dyes.

This suggests that the neutrophilic leucocytes in the conjunctival fluid are alive, emigrated from the conjunctival vessels on their way to combating bac- teria and other injurious elements. The dead and dying leucocytes. on the other hand, are caught by the mucous thread and carried off with this.

This difference in staining capacity may also explain why Marx’ line and cases of keratoconjunctivitis sicca are more intensely stained by rose bengal than t y trypan blue.

The blue colour gives the best contrast to the conjunctiva, while the red colour of rose bengal gives the best contrast to the cornea (Sjogren) . The latter fact contributes towards explaining the poor staining of the cornea by trypan blue.

In a previous paper I have reported a similar comparative investigation using methylene blue and rose bengal (Norn 1966). The series investigated likewise comprised 100 cases, and the results regarding the mean grade of staining are shown in fig. 2 B.

The rose bengal staining differing little in the two series, these are com- parable.

Trypan blue can therefore be compared indirectly with methylene blue. This means that two blue dyes can be compared, one of which possesses acid groups (trypan blue) and the other alkaline groups (methylene blue).

Marx’ line is stained more intensely and more frequently by trypan blue than by methylene blue. This probably means that Marx’ line is acidophilic, perhaps due to a certain prekeratinisation.

Methylene blue stains the conjunctiva better than trypan blue. This suggests that the conjunctiva is basophilic.

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n

CORNEA CONJ. PLlCA CARUN- FORNIX TARSUS MARX’

Fig. 2 A Mean grades o f staining o f the cornea and different areas o f the conjunctiva

Trypan blue (black columns) and rose bengal (white columns). Arbitrary grading 1-5 (ordinate).

BULBl SEMILUN. CEL INF. INF. LINE

’1

CORNEA CONJ. PLICA CARUN- FORNIX TARSUS M BULBl SEMILUN. CEL INE INF LINE

Fig. 2 B Mean grade o f staining of the cornea and different areas of the conjunctiva.

Methylene blue (black columns) and rose bengal (white columns).

Microscopy has shown, however, that methylene blue precipitates as grains in

The “better” staining is therefore possibly represented solely by the pre-

Rose bengal stains pathological areas of the cornea more intensely than both

the conjunctiva (photo 5 ) , whereas proper staining is rarely seen.

cipitated grains, which are caught by the conjunctival mucus.

387 21 *

methylene blue and trypan blue, partly because the red colour forms a better contrast in this area.

Trypan blue offers no clinical advantages over a mixture of rose bengal and fluorescein. Though trypan blue can be added, I judge that in the routine work one is best served with the mixture of rose bengal and fluorescein alone.

This mixture of two dyes has the advantage that it offers a possibility of after-staining with the mucus-specific alcian blue and thus of distinguishing in doubtful cases between degenerate cells (red) and mucus (blue) (Norn 1964 B).

SUMMARY

The vital-staining capacity of trypan blue in the conjunctiva and on the cornea has heen subjected to microscopical, experimental, and clinical studies.

Trypan blue has been campared directly with rose bengal and fluorescein, and indirectly with methylene blue.

Trypan blue was found to stain degenerate cells and mucus. The conjunctiva was stained more intensely by trypan blue than by rose

Pathological processes of the cornea were stained more poorly by trypan blue

Attempts have been made to explain these differences in staining capacity

For clinical vital staining a mixture of rose bengal and fluorescein is pre-

bengal, but no clinical significance of this staining was demonstrable.

than by rose bengal.

on the basis of colour contrast and acidophilia versus basophilia.

ferable to trypan blue.

REFERENCES

Baker, John R.: Principles of biological microtechnique. A study of fixation and dye- ing. London: Methuen Ltd. 1958. pp. 357.

Evans, Herbert M & Werner Schulemann: The action of vital stains belonging to the benzidine group. Science. 39: 443-453 (1914).

Fleishmann, Wal ter & Karl F. Pollaczek: Zur priifung der vitalitat in Leukocyten mittels Farbstoffen. Klin. Wchnschr. 10: 1808-1810 (1931).

Hathaway, Wi l l iam E, Newby, Louis A & John H . Githens: the acridin orange vita- bility test applied to bone marrow cells. I Correlation with trypan blue and eosin dye exclusion and tissue transformation. Blood 23: 517-525 (1964)

Lohlein, Walther: in Henke, F. & 0. Lubarsch: Handbuch der speziellen pathologischen Anatomie und Histologie. XI11 Springer: Berlin. (1928)

Norn, M. S.: Cytology of the conjunctival fluid. Experimental and clinical studies based on a quentitative pipette method. pp 152 Acta ophth. suppl . 59 (1960).

Norn, M. S.: mucus on conjunctiva and cornea. Acta ophth. 41: 13-23 (1963). Norn, M. S.: specific double vital staining of the cornea and conjunctiva with rose

bengal and alcian blue. Acta ophth. 42: 84-96 (1964A).

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Norn, M. S.: vital staining in practice, using a mixed stain and alcian blue. Acta

Norn, M. S.: Methylene blue (methylthionine) vital staining of the cornea and con-

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