Letters to the Editor Trypan Blue: Is It Suitable for Staining of the Endometrium? I was pleased to read the interesting article by Tam et al [1] published in the Journal of Minimally Invasive Gynecology. In this prospective pilot case study analysis, the authors examined endometrial dye instillation as a novel approach that may improve the histopathological assessment of morcellated specimens. Even though their method is useful, I would like to comment on the authors’ preference of trypan dye. First, trypan is toxic to living cells [2]. Trypan toxicity–related morphologic changes in surface and glandular epithelial cells were not stressed in the article by Tam et al. Especially in an atrophic endometrium, such toxicity-associated morphologic changes may either shadow or imitate nuclear atypia associated with endometrial intraepithelial carcinoma because these atypia are usually limited to the surface. Second, because trypan specifically stains dead cells [3,4], the living endometrial component might not be stained with dye instillation. Hence, the unstained endometrial component may be missed from sampling for histopathological assessment. Third, the longer exposure of endometrial tissue to this dye would increase trypan toxicity–associated changes. I believe that limiting the use of trypan to in premenopausal patients whose endometrial intraepithelial carcinoma risk is relatively lower and avoiding it use in patients in whom operation time is expected to be longer would contribute to the quality of this method and serve as a future reference for similar studies. Mustafa Ozturk, MD Ibrahim Alanbay, MD Haldun Umudum, MD Ankara, Turkey
References 1. Tam T, Harkins G, Caldwell T, Zaino R, Hazard D. Endometrial dye instillation: a novel approach to histopathologic evaluation of morcellated hysterectomy specimens. J Minim Invasive Gynecol. 2013;20: 667–671. 2. Awad D, Schrader I, Bartok M, Sudumbrekar N, Mohr A, Gabel D. Brilliant Blue G as protective agent against trypan blue toxicity in human retinal pigment epithelial cells in vitro. Graefes Arch Clin Exp Ophthalmol. 2013;251:1735–1740. 1553-4650/$ - see front matter Ó 2014 AAGL. All rights reserved.
3. Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol. 2001. May:Appendix 3:Appendix 3B. 4. Fauque P, Amor AB, Joanne C, Agnani G, Bresson JL, Roux C. Use of trypan blue staining to assess the quality of ovarian cryopreservation. Fertil Steril. 2007;87:1200–1207. http://dx.doi.org/10.1016/j.jmig.2013.11.003
Reply to Dr. Ozturk We would like to express our appreciation for Dr. Ozturk’s comments on our original article [1]. A proper discussion of this novel approach evaluating trypan blue instillation for endometrial staining of morcellated hysterectomy specimens is important for patient safety and diagnostic efficiency and accuracy. The first comment regarding trypan being toxic to living cells is a valid concern. Fortunately, we were able to use VisionBlue (Dutch Ophthalmic USA, Kingston, NH), a sterile solution of trypan blue. Each milliliter of VisionBlue contains 0.6 mg trypan blue, 1.9 mg sodium monohydrogen orthophosphate (Na2HPO4.2H2O), 0.3 mg sodium dihydrogen orthophosphate (NaH2PO4*2H2O); 8.2 mg sodium chloride (NaCl), and water [2]. Trypan blue dye has been used in numerous surgical settings and in diagnostic evaluations to aid in tissue identification or anatomic evaluation. VisionBlue (trypan blue ophthalmic solution) is a selective tissue staining agent used as a medical aid in cataract surgery. The use of trypan blue dates back to the 1970s when it was used preoperatively to stain the corneal endothelium [3], and is currently used frequently in cataract surgery [4,5]. The in vivo use of trypan blue did not show deleterious effects in patients who underwent vitreoretinal surgery [4]. The American Academy of Ophthalmology has recently reported a technology assessment on the use of trypan blue in cataract surgery further suggesting the dye’s extensive use and safety in ophthalmologic surgeries [6]. This further elucidates the safety of trypan blue use in surgeries. Concern regarding trypan-related morphologic changes either shadowing or imitating nuclear atypia associated with endometrial intraepithelial carcinoma is not a problem our pathologists encountered. Trypan blue provided pale but distinct staining of the endometrium on the fresh specimens, which persisted even after overnight fixation. The dye was
Letters to the Editor
not associated with any artifactual histologic deterioration and consequently did not interfere with any diagnostic interpretation. The absence of any blue color in sections stained only with eosin provides evidence that the trypan blue stain is lost in processing or dehydration through graded alcohols or xylene. As for trypan blue specifically staining dead cells and not staining the living endometrial component, this concern was not encountered. Figures 2 and 3 in the article showed gross tissue staining and the persistence of light blue staining after overnight fixation. The concern over longer exposure of endometrial tissue to trypan blue increasing trypan toxicity, although a valid concern, is not an issue as was discussed earlier. Prolonged exposure did not cause any adverse patient outcome from dye instillation. Given the dye’s safety and the dilute solution being used in endometrial instillation, we were reassured of the patient’s well-being during surgery. The largest specimen from this study was obtained from a supracervical hysterectomy performed for a 635-g uterus. The total operating time, from incision to closure of port sites, was 57 minutes. This is the longest operating time from the cohort of patients in the study because of the additional morcellating time. The patient had no adverse events from the use of trypan blue during the surgery. Thank you for your comments suggesting that we should be limiting the use of trypan blue in premenopausal patients whose endometrial carcinoma risk is relatively lower along with avoiding dye use in cases in which the operating time is expected to be longer; your reason was that these precautions would contribute to the quality of this method, particularly because our methodology will later serve as a future reference for similar studies. In our opinion, we believe that this dye is safe for endometrial staining, and the small amount used (0.5 mL) is not
153
sufficient enough to adequately stain the endometrium of a large myomatous uterus. Also, in our opinion, because of the volume relationship between infused dye and cavity size, a longer instillation time along with a larger volume of dye (possibly 1 mL) is a better choice because it provides more effective endometrial staining. Teresa Tam, MD, FACOG Chicago, Illinois Gerald Harkins, MD, FACOG Danielle Hazard, MD, FACOG Trevor Caldwell, MD Richard Zaino, MD Hershey, Pennsylvania References 1. Tam T, Harkins G, Caldwell T, Zaino R, Hazard D. Endometrial dye instillation: A novel approach to histopathologic evaluation of morcellated hysterectomy specimens. J Minim Invasive Gynecol. 2013;20:667–671. 2. VisionBlue (trypan blue ophthalmic solution) [package insert]. The Netherlands: Dutch Ophthalmic Research Center; 2004. 3. Norn MS. Per operative trypan blue vital staining of corneal endothelium. Eight years’ follow up. Acta Ophthalmol (Copenh). 1980;58: 550–555. 4. Abdelkader EA, McBain VA, Anand M, et al. In vivo safety of trypan blue use in vitreoretinal surgery. Retina. 2011;31:1122–1127. 5. Jhanji V, Chan E, Das S, et al. Trypan blue dye for anterior segment surgeries. Eye (Lond). 2011;25:1113–1120. 6. Jacobs DS, Cox TA, Wagoner MD, Ariyasu RG, Karp CL. American Academy of Ophthalmology; Ophthalmic Technology Assessment Committee Anterior Segment Panel. Capsule staining as an adjunct to cataract surgery: a report from the American Academy of Ophthalmology. Ophthalmology. 2006;113:707–713. http://dx.doi.org/10.1016/j.jmig.2013.11.006
References 1. Tam T, Harkins G, Caldwell T, Zaino R, Hazard D. Endometrial dye instillation: a novel approach to histopathologic evaluation of morcellated hysterectomy specimens. J Minim Invasive Gynecol. 2013;20: 667–671. 2. Awad D, Schrader I, Bartok M, Sudumbrekar N, Mohr A, Gabel D. Brilliant Blue G as protective agent against trypan blue toxicity in human retinal pigment epithelial cells in vitro. Graefes Arch Clin Exp Ophthalmol. 2013;251:1735–1740. 1553-4650/$ - see front matter Ó 2014 AAGL. All rights reserved.
3. Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol. 2001. May:Appendix 3:Appendix 3B. 4. Fauque P, Amor AB, Joanne C, Agnani G, Bresson JL, Roux C. Use of trypan blue staining to assess the quality of ovarian cryopreservation. Fertil Steril. 2007;87:1200–1207. http://dx.doi.org/10.1016/j.jmig.2013.11.003
Reply to Dr. Ozturk We would like to express our appreciation for Dr. Ozturk’s comments on our original article [1]. A proper discussion of this novel approach evaluating trypan blue instillation for endometrial staining of morcellated hysterectomy specimens is important for patient safety and diagnostic efficiency and accuracy. The first comment regarding trypan being toxic to living cells is a valid concern. Fortunately, we were able to use VisionBlue (Dutch Ophthalmic USA, Kingston, NH), a sterile solution of trypan blue. Each milliliter of VisionBlue contains 0.6 mg trypan blue, 1.9 mg sodium monohydrogen orthophosphate (Na2HPO4.2H2O), 0.3 mg sodium dihydrogen orthophosphate (NaH2PO4*2H2O); 8.2 mg sodium chloride (NaCl), and water [2]. Trypan blue dye has been used in numerous surgical settings and in diagnostic evaluations to aid in tissue identification or anatomic evaluation. VisionBlue (trypan blue ophthalmic solution) is a selective tissue staining agent used as a medical aid in cataract surgery. The use of trypan blue dates back to the 1970s when it was used preoperatively to stain the corneal endothelium [3], and is currently used frequently in cataract surgery [4,5]. The in vivo use of trypan blue did not show deleterious effects in patients who underwent vitreoretinal surgery [4]. The American Academy of Ophthalmology has recently reported a technology assessment on the use of trypan blue in cataract surgery further suggesting the dye’s extensive use and safety in ophthalmologic surgeries [6]. This further elucidates the safety of trypan blue use in surgeries. Concern regarding trypan-related morphologic changes either shadowing or imitating nuclear atypia associated with endometrial intraepithelial carcinoma is not a problem our pathologists encountered. Trypan blue provided pale but distinct staining of the endometrium on the fresh specimens, which persisted even after overnight fixation. The dye was
Letters to the Editor
not associated with any artifactual histologic deterioration and consequently did not interfere with any diagnostic interpretation. The absence of any blue color in sections stained only with eosin provides evidence that the trypan blue stain is lost in processing or dehydration through graded alcohols or xylene. As for trypan blue specifically staining dead cells and not staining the living endometrial component, this concern was not encountered. Figures 2 and 3 in the article showed gross tissue staining and the persistence of light blue staining after overnight fixation. The concern over longer exposure of endometrial tissue to trypan blue increasing trypan toxicity, although a valid concern, is not an issue as was discussed earlier. Prolonged exposure did not cause any adverse patient outcome from dye instillation. Given the dye’s safety and the dilute solution being used in endometrial instillation, we were reassured of the patient’s well-being during surgery. The largest specimen from this study was obtained from a supracervical hysterectomy performed for a 635-g uterus. The total operating time, from incision to closure of port sites, was 57 minutes. This is the longest operating time from the cohort of patients in the study because of the additional morcellating time. The patient had no adverse events from the use of trypan blue during the surgery. Thank you for your comments suggesting that we should be limiting the use of trypan blue in premenopausal patients whose endometrial carcinoma risk is relatively lower along with avoiding dye use in cases in which the operating time is expected to be longer; your reason was that these precautions would contribute to the quality of this method, particularly because our methodology will later serve as a future reference for similar studies. In our opinion, we believe that this dye is safe for endometrial staining, and the small amount used (0.5 mL) is not
153
sufficient enough to adequately stain the endometrium of a large myomatous uterus. Also, in our opinion, because of the volume relationship between infused dye and cavity size, a longer instillation time along with a larger volume of dye (possibly 1 mL) is a better choice because it provides more effective endometrial staining. Teresa Tam, MD, FACOG Chicago, Illinois Gerald Harkins, MD, FACOG Danielle Hazard, MD, FACOG Trevor Caldwell, MD Richard Zaino, MD Hershey, Pennsylvania References 1. Tam T, Harkins G, Caldwell T, Zaino R, Hazard D. Endometrial dye instillation: A novel approach to histopathologic evaluation of morcellated hysterectomy specimens. J Minim Invasive Gynecol. 2013;20:667–671. 2. VisionBlue (trypan blue ophthalmic solution) [package insert]. The Netherlands: Dutch Ophthalmic Research Center; 2004. 3. Norn MS. Per operative trypan blue vital staining of corneal endothelium. Eight years’ follow up. Acta Ophthalmol (Copenh). 1980;58: 550–555. 4. Abdelkader EA, McBain VA, Anand M, et al. In vivo safety of trypan blue use in vitreoretinal surgery. Retina. 2011;31:1122–1127. 5. Jhanji V, Chan E, Das S, et al. Trypan blue dye for anterior segment surgeries. Eye (Lond). 2011;25:1113–1120. 6. Jacobs DS, Cox TA, Wagoner MD, Ariyasu RG, Karp CL. American Academy of Ophthalmology; Ophthalmic Technology Assessment Committee Anterior Segment Panel. Capsule staining as an adjunct to cataract surgery: a report from the American Academy of Ophthalmology. Ophthalmology. 2006;113:707–713. http://dx.doi.org/10.1016/j.jmig.2013.11.006
