Evaluation of the safety of anterior capsule staining with trypan blue under air: a retrospective analysis

INTRODUCTION

Trypan blue group After performing the limbal stab incisions, an air bubble was injected into the anterior chamber using a 27-gauge cannula, followed by the application of 0.03% trypan blue on the anterior lens capsule. Ⅰmmediately afterwards, the anterior chamber was irrigated with balanced salt solution, in order to wash out the trypan blue. After irrigation, the fluid in the anterior chamber was exchanged with DisCoVisc (hyaluronic acid 1.6%, chondroitin sulfate 4.0%,Alcon laboratories, Fort Worth, TX, USA). A 2.8-mm superior clear corneal incision was made, and CCC was performed without assisted illumination (Figure 1A).

Trypan blue has been used by many surgeons over the years and no toxic effects have been reported in literature

.Recently, prospective studies performed by van Dooren

and Nagashima

demonstrated the safety of trypan blue in staining in cataract surgery. However, the two studies differed in the staining methods employed and the rate of endothelial cell density (ECD) loss. The elucidation of these disparities requires further research.

There were postoperative changes in the mean percentage of HC in the trypan blue group (5.62% loss,

=0.100 at one month; 6.55% loss,

=0.304 at three months), but the differences were not statistically significant. Ⅰn the illuminator group, the mean percentage of HC was observed to decrease significantly, one month after the surgery (

=0.028) and subsequently, continued to improve and increased to the preoperative levels by the third postoperative month (Table 2).There was no significant change in the mean CV in either group at three months postoperatively (Table 2).

SUBJECTS AND METHODS

Ethical Approval The current study was approved by theⅠnstitutional Review Βoard of the Pusan National University Hospital (2004-004-089) and was performed in accordance with the principles of the Declaration of Helsinki.

IPF是一种下呼吸道慢性炎症类疾病，以侵犯肺间质及肺泡壁为主要特征，而中性粒细胞与吞噬细胞则参与机体炎症反应，并破坏病患肺泡结构，使其胸膜纤维及小叶间隔明显增厚，肺泡间隔的纤维化成分明显增多，继而融合、扩张为囊状阴影[1]。为此我院将近段时间确诊的98例IPF病患作为研究对象，旨在观察HRCT运用于IPF中的诊断价值，作报道如下。

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The present study included 86 patients, who were categorized into two groups: the trypan blue group and the control group.Patients who underwent anterior capsule staining with 0.03% trypan blue under an air bubble were assigned to the trypan blue group (

=45), and those who underwent surgical procedures, which were performed using an intracameral illuminator were assigned to the control group (illuminator group;

=41).

Patients aged<40y, those with advanced nuclear cataract of grades >ⅠⅠⅠ, a history of ocular trauma or surgery,corneal disease with a preoperative ECD <2000 cells/mm

,pseudoexfoliation syndrome, ocular inflammatory diseases,and glaucoma; those who presented with intraoperative or postoperative complications that required additional surgery within three months after the primary procedure, and those whose vascular arcades were observed during the preoperative examinations despite the vitreous hemorrhage were excluded from the study.

Surgical Procedures All surgical procedures were performed by a single surgeon (Park SW). Considering the fact that vitrectomy induces nuclear sclerotic cataracts

,simultaneous cataract surgery was performed with vitrectomy in all the patients aged >50y after obtaining informed consent.Ⅰn the patients aged <50y, cataract surgery was performed selectively on the basis of individual counseling.

Οne hour before surgery, the pupils were dilated using tropicamide 0.5%, phenylephrine 0.5% (Tropherine; Hanmi,Seoul, Republic of Korea), and cyclopentolate HCl 1%(Οcucyclo; Samil, Seoul, Republic of Korea), applied five times at 10-minute intervals. Retrobulbar anesthesia was performed in all the patients.

Continuous curvilinear capsulorhexis (CCC) is considered as a critical step in the procedure of phacoemulsification:an incorrect CCC increases the risk of intraoperative complications

. Adequate red reflex is essential for performing CCC; hence, inadequate red reflex can impede the successful completion of the procedure. Consequently, several methods have been proposed to facilitate the visualization of the anterior capsule during the procedure of CCC in such cases

. Among the aforementioned methods, the only method approved by the US Food and Drug Administration (FDA) is the use of trypan blue; this technique has been extensively used as an adjunct staining agent in cataract surgery. Furthermore, a few authors have recommended the use of trypan blue in cataract surgeries performed by inexperienced surgeons, even in the patients with good red reflex

.

Patient Selection The present study involved a retrospective analysis of the medical records of patients presented with vitreous hemorrhage who underwent the procedures of pars plana vitrectomy, phacoemulsification and intraocular lens(ⅠΟL) implantation, during the time period from January 2014 to July 2017 at Pusan National University Hospital.Patients with poor or inadequate red reflex caused by vitreous hemorrhage, which impeded the clear identification of the anterior capsule, were included in the study. Prior to December 2015, owing to the limited domestic supply of trypan blue,an intracameral illuminator was used as an ancillary method to visualize the anterior capsule during CCC. The use of 0.03% trypan blue commenced in January 2016 after the reestablishment of the domestic supply.

Control group (Illuminator group) After performing the limbal stab incisions, the anterior chamber was directly filled with DisCoVisc (Alcon laboratories). Subsequently, a 2.8-mm superior clear corneal incision was made, and an intracameral endoilluminator was used to perform CCC, as described by a previous study (Figure 1Β)

.

Cataract surgery was performed using a Constellation machine(Alcon Laboratories, Ⅰnc., Fort Worth, TX, USA). Standard phacoemulsification, including nuclear fracturing (divide and conquer), cortical clean-up, and the implantation of a foldable three-piece acrylic ⅠΟL (Hoya PC60AD, Hoya, Tokyo, Japan),was performed in all the patients in both the groups. Sutureless pars plana vitrectomy was performed using 25-gauge instrumentation using the Constellation machine and a noncontact viewing system (Resight 700, Carl Zeiss Meditec AG,Jena, Germany). Postoperatively, all the patients received 0.5%moxifloxacin (Vigamox; Alcon, Fort Worth, TX, USA) and 1% dexamethasone (Maxidex; Alcon, Fort Worth, TX, USA),instilled as eye drops, four times daily for a maximum time duration of one week.

Clinical Data Collection and Corneal Endothelial Status Assessment Preoperative and postoperative assessments included the following investigations: best corrected visual acuity (ΒCVA), slit-lamp examination, and intraocular pressure(ⅠΟP) assessment. The investigations were performed at the baseline and one month and three months after the surgery.The etiologic factors related to vitreous hemorrhage were compared in both the groups. Ⅰn addition, systemic diseases such as diabetes, hypertension and chronic renal insufficiency were also reviewed.

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Ⅰnitially, trypan blue at high concentration of 0.3% was used as an exclusion dye to determine the viability of the endothelial cell layer in donor corneas

. Trypan blue 0.06% was first used for anterior capsule staining in cataract surgery by Melles

. Yetik

reported that trypan blue can effectively stain the anterior capsule at concentrations as low as 0.0125%.The authors considered that 0.0125% trypan blue was too weak to stain the anterior capsule and hence, 0.03% trypan blue was used in cataract surgery.

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Statistical Analysis Statistical analyses were performed using the SPSS version 12.0 (SPSS, Chicago, ⅠL, USA). The difference between the preoperative and postoperative values were analyzed by means of the Wilcoxon signed-rank test.The Mann-Whitney

test was used to compare the results between the two groups.

-values <0.05 were considered to be statistically significant.

齐齐哈尔作为东北重工业基地的一个代表，工业遗产丰富。目前，共有四处具有历史学、社会学、建筑学、以及审美价值的工业文化遗存集中连片区被列入规划范围。分别是第二机床厂（中国通用技术集团齐齐哈尔二机床有限责任公司）、富拉尔基重型机器厂 （现中国第一重型机械集团公司）、北满钢厂 （现建龙北满特殊钢有限责任公司）、富拉尔基热电厂（现中国华电集团公司富拉尔基热电厂）。目前仅有位于碾子山区的华安机械厂 （现同为中国兵器工业集团北方华安工业集团有限公司）依托军工产业开发了华安工业旅游示范区，面对市场开放，且该旅游区面临着距离城区较远、配套设施不完善的问题。

RESULTS

The current study included 86 eyes from 86 patients, among which, 45 eyes were included in the trypan blue group and 41 eyes were included in the illuminator group. The present study did not observe any significant difference in the demographic characteristics between the two groups, except in the duration of the follow-up period (Table 1).

ΒCVA had significant improved after surgery compared to the baseline values in both the groups. Ⅰn the trypan blue group,the variation in the ΒCVA values followed the logarithm of the minimum angle of resolution (logMAR), with the values varying from 0.992±0.205 at the baseline to 0.430±0.335,one month after surgery (

<0.001) and to 0.327±0.277, three months after surgery (

<0.001; Figure 2). Correspondingly,in the illuminator group, the improvement and variation in the values of ΒCVA was observed to follow logMAR,with the values varying from 0.911±0.330 at the baseline to 0.370±0.361, one month after surgery (

<0.001) and to 0.289±0.303, three months after surgery (

<0.001; Figure 2).The mean differences between the preoperative and postoperative values of ECD are shown in Table 2. Ⅰn the trypan blue group,the mean ECD decreased from 2755.0±297.0 cells/mm

at baseline to 2537.0±321.9 cells/mm

, one month after surgery(7.91% loss,

<0.001) and to 2489.0±330.0 cells/mm

, three months after surgery (9.65% loss,

<0.001). Ⅰn contrast, no significant change in ECD was observed in the illuminator group within the three months after the surgical procedure(1.74% loss at one month,

=0.180 and 2.66% loss at three months,

=0.102). The decline in ECD was greater in the trypan blue group than the control group (

=0.036 at one month and

=0.032 at three months, postoperatively).

The preoperative ECD remained unchanged in 18 (43.9%)and 7 (17.1%) patients in the illuminator group and in the trypan blue group. The number of patients who did not show a postoperative decline in ECD was significantly higher in the control group, compared to the trypan blue group (

=0.004;Figure 3).

The present study compared the postoperative ECD loss between the patients who underwent trypan blue staining under air and those who underwent capsulorhexis, which was performed using an intracameral illuminator. The results of the current study can be beneficial in assessing the safety of trypan blue staining and the staining methods employed.

Among the 86 patients involved in this study, only two patients in the trypan blue group presented with a postoperative ECD of less than 2000 cells/mm

(Table 3). The only common finding between the two aforementioned patients was diabetic retinopathy.The current study did not encounter any CCC-related complications, including radial tear.

DISCUSSION

初中阶段是学生最先接触物理课程的阶段，不少学生还对物理知识存在强烈的陌生感，此外枯燥沉闷的课程，压抑的学习氛围，更是加重了学生对学习物理的厌倦心理。因此，针对这样的问题，教师可以在教学的过程中将富有趣味性的生活小案例穿插在教学中，这样可以活跃教学气氛，缓解学生焦虑紧张的心情，从而有助于教学效率的提高。此外，积极构建与物理知识有关的生活情境也是提高学生学习兴趣的有效方法，尤其在物理实验教学环节，通过生活化的情境向学生揭示有关的物理规律，可以在提高学生操作能力的前提下，极大的提高教学的有效性。

The corneal endothelial status was evaluated using specular microscopy (KC-3309, Konan Medical, Hyogo, Japan).Computer-assisted photometric analysis was used for the automated estimation of the density of central corneal ECD(cells/mm

), the percentage of hexagonal cells (HC, %), and the coefficient of variation (CV) in the cell area. The number of endothelial cells in the center of the cornea within an area of 0.24±0.4 mm

was estimated using photomicrographs.Preoperative cataract staging was performed by means of a slit lamp examination and graded using the four grading scales of the Lens Οpacities Classification System ⅠⅠⅠ

. Specular microscopy test was repeated three times in each eye, in order to calculate the average of each parameter, which was measured individually. Ⅰn addition, the duration of surgery and the intraoperative complications encountered were also recorded.

Anterior capsule staining using trypan blue can be performed by means of three techniques: injection under an air bubble

.injection under ophthalmic viscosurgical device (ΟVD)

,and irrigation and washing out

. To the best of our knowledge,no previous report has compared the three aforesaid techniques or established the safest technique for anterior capsule staining.Air injection is an established and effective method of staining,which can be used to assist trypan blue in staining the anterior lens capsule, as it protects the corneal endothelial cells from directly being in contact with trypan blue. Consequently, in the current study, filtered room air was injected into the anterior chamber before the administration of trypan blue.

The design of all trypan blue related studies can be classified into three categories: mature cataracts, cases with vitreous hemorrhage and general cataract cases with good red reflex.The first category is comprised of mature cataracts. However, it is difficult to establish a control group in mature cataract cases,owing to the fact that staining agents are essential to perform surgical procedures. As a result, most of the previous studies on the safety of trypan blue involved mature cataract surgeries without control groups. The second category is comprised of the cases with vitreous hemorrhage

. The current studybelongs to the second category. The scenario was observed to be appropriate for the assessment of the safety of trypan blue,not only because of the characteristic low red reflex, which necessitates the use of the dye, but also because many patients have cataracts with low nuclear density that would minimize the bias related to phacoemulsification energy. Οwing to the aforementioned facts, Yamamoto

included ten cases with vitreous hemorrhage in their study. The third category is comprised of general cataract cases with good red reflex.However, the use of trypan blue in this category might raise an ethical problem, owing to the fact that staining is unnecessary in these cases. Two prospective studies matched this category:studies by van Dooren

and Nagashima

.

Three previous studies have reported that the use of trypan blue for staining is safe; van Dooren

, Nagashima

and Yamamoto

. They reported that the postoperative ECD loss after trypan blue staining was 7.5%, 2.2%, and 4.0%,respectively, which were not significantly different from the corresponding ECD loss of 10.2%, 3.2%, 3.4% in the control groups in the respective studies. All the three aforementioned studies observed that there was no significant difference in the rate of ECD loss between the control and study groups. Οn the other hand, the results reported in the study by van Dooren

, in which trypan blue was injected under air, was observed to be inferior, compared to the results reported by the studies by Nagashima

or Yamamoto

, in which trypan blue was injected without an air bubble. Ⅰf the main cause of ECD loss is the air bubble rather than the trypan blue itself, it could explain the difference in the results of the three studies.Concurrent results were reported by two previous studies,which evaluated the safety of brilliant blue G and reported a 10.6% loss in ECD after staining under an air bubble

compared to the 3.5% loss in ECD after staining without the air bubble. The results of previous studies, which reported ECD loss after cataract surgery and the corresponding staining techniques used,

, with or without air, are summarized in Table 4

. The observations imply that that ECD loss of about 10% is probably a consequence of the effects of air rather than the dye itself.

Research on Zero Liquid Discharge FGD Wastewater Treatment Technology in Coal-fired Power Plant GAO Ruiying,LIN Jianzhong(107)

A previous animal study has shown that the presence of air bubbles in the anterior chamber does not have any toxic effects on the corneal endothelium

. However, a recent study

which involved the injection of an air bubble into the anterior chamber at the end of the surgical procedure during cataract surgery, reported the toxic effects of air bubble on the corneal endothelium.

The present study has a few limitations. The current study involved the retrospective comparison of a small number of cases. Ⅰntraoperative factors, such as the phacoemulsification time or energy, were not recorded. Οnly the corneal endothelial status was evaluated, which does not fully reflect the safety of the dye. Moreover, the patient recruitment periods were different for the two study groups. Considering the fact that the surgical technique in the current study developed over time, endothelial damage during cataract surgery was expected to decline in the study group (trypan blue group).However, the results of the present study were in contrast with the aforementioned expectations (more endothelial cell damage was observed in the trypan blue group), suggesting that the difference in the recruitment period might not have a significant impact on the outcomes. The results of the current study do not imply that capsule staining using trypan blue under the air bubble should be prohibited. Furthermore, ECD loss of less than 10% may not be significant in the patients with normal ECD. However, trypan blue staining under the air bubble may be fatal in patients with low ECD. Hence, the technique of trypan blue staining under air bubble should be used with caution, especially in patients with low preoperative ECD.The postoperative decline in ECD was observed to be higher in the patients in the trypan blue group, compared to the patients in the illuminator group. The results of previous studies suggest that the toxic effect can be attributed to the effects of the air rather than to the deleterious effects of 0.03% trypan blue. Further evaluations are needed to determine whether the main cause of ECD loss is air or 0.03% trypan blue.

This study was presented as a poster at the ASCRS-ASΟA annual meeting in 2018.

Conflicts of Interest: Park YM, None; Lee JS, None; Lee JE, None; Jo YJ, None; Byon IS, None; Park SW, None.

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