Introduction

Anterior capsule staining in mature cataract can be done by using various vital dyes like trypan blue, ^{indocyanine green, and sodium fluorescein[1] to facilitate capsulorhexis. Sodium fluorescein is not used now. Because of its small molecular size, it is not able to remain confined in the anterior chamber but diffuses to lens and vitreous.[2] While trypan blue and indocyanine green provides excellent visualization of the capsule, staining with trypan blue was definitely more intense and persistent.[3] So trypan blue is preferred dye for staining of anterior capsule. Trypan blue was initially available as 0.1% but now it is available in 0.06% concentration. There is a report that even with lower concentration; it can effectively stain the anterior capsule.[4]}

The purpose of this study was to determine the minimum concentration of trypan blue that stains the anterior capsule. Blueness of dye which decides the staining of capsule was quantified with the help of RGB scale (Red, Green, Blue scale) on “Adobe Photoshop Image Reader” software (Version 7.0).

Methods

This prospective double blind randomized study was carried out in the department of Ophthalmology, Medical College and Sir T. Hospital, Bhavnagar after approval from institutional ethics committee. The study was conducted in 50 patients with senile mature cataract undergoing small incision cataract surgery under peribulbar block. Patients with systemic diseases, any acute ocular inflammation or ocular complication in other eye, all congenital cataracts, glaucoma, traumatic cataract and pregnant patients were excluded from the study. After taking informed written consent, patients were randomized in five different groups according to different concentrations of trypan blue dye used; 0.06% in group A(n=10), 0.03% in group B(n=10), 0.015% in group C(n=10), 0.0075% in group D(n=10) and 0.00375% in group E(n=10).

Method of preparation of different concentration of trypan blue

Dosage and administration

Trypan blue is available in 1ml /vial.

The volume of the dye used for capsular staining was 0.1 ml in all groups. The dye was applied for staining capsule by tuberculin syringe with hydro cannula under classic air bubble technique^[5] and kept in contact with capsule for 40 seconds.

Patients were operated by same surgeon using same microscope and illumination. Video recording was done with same video camera. Surgical procedure was transported through SV video port of microscope to computer with facility of TV tuner card. The required still image was taken and was opened with “Adobe Photoshop Image Reader” software version 7. A 6 mm center circle in stained capsule was cut in opened file and pasted in to a new file. Window menu bar was opened and RGB scale was opened in work space. RGB scale was calculated by the average of 5 readings which were taken from the centre, and from 12, 3, 6 & 9 o’clock position. Comparative study of satisfactory staining of capsule was done by using RGB scale values in “Adobe Photoshop Image Reader” Software (Version-7). In RGB scale, white color means R=255, B=255 and G=255.

Image 1 and 2 shows RGB scale values before and after stain in “Adobe Photoshop Image Reader” Software (Version-7).

Figure 1

Image 1: RGB scale on Adobe Photoshop (Before stain)

Figure 2

Image 2: RGB scale on Adobe Photoshop (After stain)

Data collected were subjected to statistical analysis. The difference between the pre and post stain R, G & B value were calculated for each concentration of trypan blue. Then the mean difference (MD) of R and G was compared with MD of B separately for each concentration of trypan blue. The paired t test was used to compare the MDs. P value of <0.05 was considered as statistically significant.

Results

The R and G values were significantly decreased, while B value remained nearly same after staining the capsule in group A.Figure 1

Figure 3

Figure 1: RGB values before and after trypan blue staining in group A

The R and G values were significantly decreased, while B value remained nearly same after staining the capsule in group B.Figure 2

Figure 4

Figure 2: RGB values before and after trypan blue staining in group B

The R and G values were significantly decreased, while B value remained nearly same after staining the capsule in group C.Figure 3

Figure 5

Figure 3: RGB values before and after trypan blue staining in group C

The R, G and B values remained nearly same before and after staining the capsule in group D Figure 4

Figure 6

Figure 4: RGB values before and after trypan blue staining in group D

The R, G and B values remained nearly same before and after staining the capsule in group E. Figure 5

Figure 7

Figure 5: RGB values before and after Trypan blue staining in group E

The difference between before and after stain values of R, G and B were calculated and then mean of the differences (MD) in R, G and B were calculated for each concentration. Then the MD of R and G was compared with MD of B separately for each concentration of trypan blue. Table 1

Figure 8

Table 1: Comparison of R and G with B in all the groups

There was statistically significant difference (p<0.05%) when MD of R and MD of G was compared with MD of B in groups A, B and C which indicate that capsular staining was satisfactory.

There was no statistical significant difference (p>0.05%) when MD of R and MD of G was compared with MD of B in groups D and E which indicate that capsular staining was not satisfactory.

Discussion

Trypan blue is used to stain anterior capsule for successful capsulorhexis in mature cataract,^[6] cataract surgery in corneal scars or edema, dense anterior cortical spokes, a mature brown nucleus, vitreous hemorrhage and asteroid hyaloids and dye-aided anterior and posterior capsulorhexis in pediatric cataracts.^[7-8] It is also useful for teaching phaco technique because staining helps residents to find and grasp the anterior capsular flap; it is particularly valuable for any surgeon learning new phaco techniques such as phaco flip or phaco chop. In the later method, a stained capsulorhexis edge helps the transitioning surgeon to avoid overly distending it with the chopper’s shaft or misplacing the horizontal chopper’s tip peripherally, outside the capsular bag.^[9-10]

Though trypan blue is widely used in ophthalmology, some study showed that it has carcinogenic properties ^[11] and induces apoptosis in human retinal pigment epithelial cell at a higher concentration.^[12] Staining of hydrophilic acrylic intraocular lens with trypan blue has been reported in literature.^[13] Trypan blue was toxic in vitro to corneal endothelium and corneal fibroblasts at higher concentrations and notably longer exposure time.^[14]

Initially trypan blue was available in 0.1% concentration, but now-a-days it is available and used as 0.06% concentration. A study showed that even lower concentration can stain the capsule.^[3] Hence, we wished to determine minimum concentration of trypan blue which stains the capsule in mature cataract.

This study showed that in group A, B and C; R and G value decreased more compared to B after staining and MD of R and G before and after stain was statistically significant compared to MD of B value (p<0.05%). As blueness was not much altered in comparison of red and green, staining of capsule was satisfactory in all the three groups. In group D and E; R, G and B value remains almost same before and after staining so MD of R and G before and after stain was not statistically significant compared to MD of B value (p>0.05%). So capsule remains nearly white and visualization of capsule was not possible which indicates concentration of 0.0075% or lower could not stain the capsule satisfactorily.

We could not find all the patients with white mature cataract (R, G and B value 255) due to awareness of people and timely detection of cataract.

Conclusion

We conclude that trypan blue in concentration of 0.03% and 0.015% is as effective as 0.06% for staining the capsule. In view of the animal studies showing carcinogenicity of trypan blue with higher concentration and long term results in human being are awaited, we recommend 0.015% of trypan blue as the optimal concentration for staining the capsule in mature cataract.