In vitro evaluation of retinoblastoma seeds using red and green lasers

Abstract

Objective:

To explore the use of red and green lasers in studying retinoblastoma seeds in pathological samples.

Methods:

This study was a retrospective, laboratory-based experimental study, conducted at a tertiary care eye center of northeast India. Medical records were retrospectively analyzed in a 7-year period from 2016 to 2023 to identify enucleated retinoblastoma (RB) eyeballs having tumor seedings. All pathological specimens that were studied using red and green laser pointers (Class III Laser product) were included. Lasers of wavelength 532 nm (green) and 630 nm (red) were used under the objective of compound microscopes to study different RB seeds. The activities of seeds were studied by the use of lasers. The seeds were documented under the objective of the compound microscope. For statistical analysis, IBM SPSS 26 was utilized. The descriptive statistics of the quantitative variables was reported through the metric mean. The categorical variables were described using count and percentages. Independence of categorical variable was carried out using Chi-square test and P values.

Results:

Thirty-eight specimens with RB seedings at different locations were included in the study. RB seeds in anterior segment (n = 2 cases), over ciliary process (n = 2 cases), vitreous seeds (n = 18 cases), retinal seeds (n = 2 cases), retinal pigment epithelium seeds (n = 3 cases), subretinal seeds (n = 5 cases), combined vitreous and subretinal seeds (n = 6 cases), and calcified seeds (n = 2 cases) were noted. With red and green lasers, varied changes were observed in the RB seeds, particularly the presence and absence of electron dense clouding within the seeds. Active seeds could be differentiated from inactive seeds using the laser technique.

Conclusion:

Use of laser in the present study was first of its kind, carried out to visualize RB seeds at different sites in raw or wet specimens of the enucleated eyeballs. By this technique, active seeds could be differentiated from the inactive seeds based on electron dense clouds within the seeds’ configurations.

Retinoblastoma (RB) is the most common primary malignant intraocular tumor in children.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17] RB may present with visible seeds at different intraocular locations.[1,2,3,4,5,7,9] Morphologically, RB seeds are classified as dusts, spheres, and clouds and mixed type.[1] They are thought to influence advancement of cancer.[1,2,3,4,5] RB seeds may involve the anterior segment by depository or infiltrative seedings at different locations.[1,2,3,4,5] These seedings can be picked up by anterior segment optic coherence tomography (OCT), ultrasound biomicroscopy (UBM), and magnetic resonance imaging scans (MRI).[1,2,3,4,5] Ciliary body involvement by the seeds, though rare, is very important with respect to treatment.[1] Understanding RB seedings is very important, and some seeds may be located in areas where they are difficult to visualize by imaging.[6,7,8,9,10,11,12,13,14,15,16,17,18,19,20] Visualizing subretinal seeds by existing imaging systems such as OCTs can be difficult occasionally. RB seeds adjoining the optic nerve head and optic cup sometimes may be invisible to conventional imaging systems. Lasers are low, deviating, high-intensity, narrow-width light, which is delivered in short pulses or continuous pulses.[1,19,20] The spectral region makes laser green or red.[1,19,20] The green-colored laser is about ten times brighter than the red-colored laser.[1,19,20] Tumor seeds or cells are an electrically neutral configuration of two or more molecules in an electron cloud.[1,19,20] Use of lasers utilizing optimum properties of light can detect such seeds. Infrared laser may elicit RB seeds usually not visible by conventional imaging modalities.

Methods

This retrospective, observational, laboratory-based experimental study was carried out in the ophthalmic pathology laboratory at a tertiary eye care center in northeast India from January 2016 to January 2023. Medical records during this 7-year study period were analyzed to screen enucleated specimens of RB cases and identify cases with RB seeds, where lasers were used during microscopic study. Institutional ethics committee (IEC) approval was obtained for the study. Written informed consents were obtained from the guardians of the patients for conducting the study. For statistical analysis, IBM SPSS 26 was utilized. The descriptive statistics of the quantitative variables was reported through the metric mean. The categorical variables were described using count and percentages. Independence of categorical variable was carried out using Chi-square test and P values. Any P value less than 0.05 was considered as significant at 5% level of significance.

The enucleated eyeballs were received in the laboratory and fixed in 10% neutral buffered formalin for 24 to 48 hours. The pathological examination included grossing of the specimens. Morphometric measurements were taken, including the entire eyeball, cornea, pupil, and optic nerve. The cut end of the optic nerve was submitted separately in a block. Transillumination test was done in all RB specimens to get the internal impression of the tumor before the dissection. Cut sections of the eyeball were examined, and tumor dimensions were noted. The bread loaf technique was used for the lateral calottes. A small peripheral calotte was used for the laser experiment. A laser pointer belonging to class-III product was used in the laboratory to visualize the RB seeds. Lasers complying to 21 Code of Federal Regulation (CFR) and wavelengths of 532 nm (green) and 630 nm (red) were used in the laboratory setup under the objective of a compound microscope (ZEISS, Axioskop-40 and Axioscope-5- Axiocam 208 color, Germany) without tagging the different RB seeds [Fig. 1a-f]. Continuous wave lasers emitted electromagnetic waves in spatial and temporal coherence. Both green and red lasers were cost-effective lasers with different wavelengths available commonly as laser pointers. Laser safety spectacles and other precautions were observed during the laser delivery. Laser-induced changes were documented including RB seed activity, sites of location, and calcifications in the tumor. After the digital photography, the tissues in the calottes were washed and transferred to the processing calottes for further fixation of the tissues. Care was taken to avoid tissue destruction during the entire procedure.

Figure 1.

The methodology of LASER application. (a) Protective glasses and laser pointer used in the study; (b) application of laser externally under the microscope Axioscope 5- Axiocam 208 color and Axioskop 40-Axiocam MRc (Germany); (c) magnified view of the laser application. Photographs were taken closing the shutter of the first microscope; (d) green laser emitted from the pointer; (e) RB seed visualized under green laser; (f) digital documentation being performed under different magnifications in the microscope

RB seeds were broadly classified as anterior segment or posterior segment seedings. Meticulous observation in the corresponding hematoxylin and eosin-stained slide was performed to look for Schlemm’s canal and trabecular meshwork invasion and other areas. Intraciliary seeds were distinguished from interciliary RB seeds in the permanent section. Posterior segment seeds were classified as vitreous, retinal pigment epithelium, subretinal, or combined seeds. RB seeds were further categorized as dusts, clouds, spheres, active, inactive, and undetermined (nonspecific). Seed activity was defined based on the morphology of the seeds (presence of sprouting and daughter seeds in active seeds). Types of seed regression were classified as described by Munier[1]: Type 0 = complete disappearance, type I = refringent and/or calcified residues, type II = amorphous nonspherical inactive residues, and type III = a combination of the latter two. Two ocular pathologists validated the findings independently.

Results

In the study period, a total of 63 RB enucleation cases were identified, out of which 38 cases with RB seedings that had undergone laser study were included in the study. Cases without seedings were excluded. Patient profiles of all 38 cases were noted and correlated with the structural change in the eyeballs [Tables 1–5]. The average age of the study population during enucleation was 3.8 years (range: 6 months to 29 years). The study included two adult RB cases. Male preponderance was noted in the study (n = 22, 58%). Involvement of the right eye (n = 21, 55%) was slightly more than the left eye (n = 17). There was no statistically significant correlation of seed activity with age, gender, or laterality of the eye (P > 0.05). The distribution of seeds as per location, activity, characteristics, and regression patterns is tabulated in Table 1. In nine cases (23.6%), active seeds were noted, where laser study could identify activity and distinguish them from inactive seeds (n = 11, 28.9%). Eighteen seeds (47.3%) were indeterminate or partially regressed. Active seeds were noted to have electron-dense clouds, while inactive seeds appeared hollowed and empty [Fig. 2a-d; Video]. Patterns of regression in vitreous seeds were classified as type 0, type 1a and 1b, type 2, and type 3.[1,8] In our study, we were able to observe calcified seeds (type 1a) in three cases, crystalline refractile seeds (type 1b) in two cases, and amorphous nonspherical seeds (type 2) in two cases. We did not observe complete disappearance (type 0) or a combination of regression (type 3) patterns. There was no interpersonal variation observed between two in-house pathologists while interpreting the pathological slides.

Table 1.

Distribution of RB seeds

Sites and types of RB seeds		No of cases (%)
Anterior segment
Anterior chamber		1 (2.6%)
Iris		1 (2.6%)
Posterior segment
Intraciliary		1 (2.6%)
Interciliary		1 (2.6%)
Vitreous		18 (47.3%)
Retinal		2 (5.2%)
Retinal pigment epithelium		3 (7.8%)
Subretinal		5 (13.1%)
Subretinal + vitreous (combined)		6 (15.7%)
Other characteristics
Clouds		14 (36.8%)
Sphere		6 (15.7%)
Dusts		11 (28.9%)
Nonspecific		7 (18.4%)
Activity of seeds
Active seeds		9 (23.6%)
Inactive seeds		11 (28.9%)
Indeterminate/partial regressed seeds		18 (47.3%)

Table 5.

Distributions of eyes (Of 38 cases with RB seeds)

		Count		Percent
Valid
OD		21		55.3
OS		17		44.7
Total		38		100.0

Figure 2.

(a, b) Active RB seeds under green and red lasers. Central electron dense clouds in active seeds were noted (with arrow head); (c, d) Inactive seeds were noted to have central clearing under green and red laser (with arrow head) (×40X)

Table 2.

Distribution of gender (All 63 cases)

		Count		Percent
Valid
Female		28		44.4
Male		35		55.6
Total		63		100.0

Table 3.

Distributions of gender (Of all 38 cases with RB seeds)

		Count		Percent
Valid
Female		16		42.1
Male		22		57.9
Total		38		100.0

Table 4.

Distribution of eyes (All 63 cases)

		Count		Percent
Valid
OD		38		60.3
OS		25		39.7
Total		63		100.0

Discussion

Retinoblastoma seedings offer a therapeutic and management challenge to the ocular oncologist.[1,2,3,4,5] Vitreous and subretinal seedings may be noted in groups C, D, and E RB.[1,2,3,4,5] Vitreous cavity seedings can be primary or secondary.[1,2,3,4,5] Secondary vitreous seeding can appear during the course of the treatment.[1,2,3,4,5] Morphologically, seedings can produce daughter seeds which are protrusion of a seed sprouting [Fig. 3a and b]. Vitreous seeds were the most common observation in our study, while two cases of anterior chamber seedings were also noted. Jakati et al.[8] observed clinicopathological correlation of aqueous seeds in 25 eyes that had undergone primary enucleation. Our study on RB seeds was exclusively on enucleated eyeballs and was not compared with clinical RB seedings. Jakati et al.[8] grouped anterior chamber seedings as type 1, type 2, and type 3. Most of the seeds in anterior chamber were associated with histopathological high-risk factors.[8] Seeding in Schlemm’s canal, Honavar’scanal, and Petit’s canal can pose problems and worsen RB prognosis.[1] Munier et al.[1] found that the involvement of ciliary processes by RB seeds can be often challenging. Interciliary and intraciliary seeds are very difficult to image in vivo. Plaque brachytherapy can be useful in intraciliary involvement,[1] while intracameral injection of chemotherapeutic agents can be useful in interciliary involvement.[1] Thus, tumor seeds play an important role in RB management and prognosis.

Figure 3.

(a) Large vitreous seeds with a daughter seed in the vitreous cavity elicited by green Laser (×100X); (b) Sprouting of the seed was noted in the vitreous cavity by green Laser; (c) Multiple subretinal seeds penetrating the transparent retina were observed by green laser (×10X); (d) Calcified vitreous seeds were noted in red laser (×40X)

The authors have previously documented seedings in raw specimens of an enucleated eyeball by using fluorescein stain.[6] Observing seedings in raw specimens of an enucleated eyeball offers a major advantage over processed specimen study as processing washes out most of the free-floating seeds.[7] The present study was carried out to elicit RB seeding by laser application in a cost-effective manner.

Electron clouds around the RB seeds help to differentiate between active and inactive seeds or calcified and amorphous seeds [Fig. 2]. Sometimes, despite electrical energy of molecules, two additional vibrations can act on the tumor seeds, namely, vibrational and rotational factors. Tumor seeds can be excited to various rotational and vibrational states in the spectral range.[19,20] In active seeds, we noted electron-dense clouds within the seeds, but central clearing was noted in the inactive seeds [Fig. 2]. Subretinal seeds were also documented using this technique [Fig. 3c]. Crystalline seeds which consisted of large atoms and molecules arranged in repetition had irregular surfaces and demonstrated back scattering [Fig. 3d]. The authors have previously performed a study on RB seeds using Raman spectra, which is a well-known type of inelastic scattering of light due to the effect of photons with various matters including cancer seeds.[7,20] During this scattering, the photons gain or lose energy due to the three-dimensional rotational or vibration status of the seeds similar to our present study.[7,20] The authors have found significant use of the Raman spectra technique in analyzing the molecular composition of RB seeds.[7,20] The main usefulness of Raman spectroscopy in analyzing the tumor and seeds is its noninvasiveness. Without damaging the tissue proper, real-time diagnosis is possible during in vivo imaging of the eye.[7,20] Raman spectra provide detailed material information of the chemical bonds within the tumor seeds and specific spectral information can be a biomarker in RB, which may aid in monitoring the effectiveness of treatment on the RB seeds.[7,18,20] Raman study was carried out in a single case of RB in our previous publication.[7] We did not compare the laser detection versus Raman’s usefulness in our study. Both have their separate usefulness in RB seeds. To elaborate, lasers used in the present study detected the active seeds compared to inactive seeds on the basis of electron cloud density in the active seeds in contrast to hollowness within inactive seeds in both green and red laser. But Raman spectroscopy study was effective precisely to find the protein (high spectral change) within the viable RB seeds compared to the adjoining nontumor zone where spectral change was lower in spectroscopy.[7]

While this was an in vitro study, future research must be aimed at expanding and using these concepts for potential use in in vivo environments. Utilizing light waves of spectra safe for living tissue may aid in better detection and management of seeds at difficult sites, particularly the subretinal seeds.

Our study was an experimental lab-based study and had a few limitations. Since it was an experimental retrospective study that included only enucleated eyes, there was a potential selection bias in the current study. Another limitation of the current study is its small sample size, which may have resulted in wider confidence intervals.

Conclusion

Use of laser in the current study was first of its kind, carried out to visualize RB seeds at different locations in raw specimens of the enucleated eyeballs. By this procedure, active seeds could be differentiated from the inactive seeds based on electron dense clouds within the seeds’ structures. Future research must be aimed at applying these concepts to study safety and viability of use in the living tissue and patients with RB.

Authors’ contribution

Dr. Dipankar Das: Concepts, design, definition of intellectual content, literature search, pathological and innovation of Laser in retinoblastoma, pathological studies and descriptions, manuscript preparation, and manuscript review. ‘Guarantor’; Dr. Obaidur Rehman: Manuscript preparation, Review of the manuscript; Dr. Kasturi Bhattacharjee: Clinical studies, Enucleations, Prosthesis; Mr. Apurba Deka: Data Collection and typing of the manuscript; Dr. Bidhan Chandra Das: Pathology reporting; Dr.Sulaikha Zakir Hussain: Manuscript preparation; Prof. Amit Choudhury: Detailed statistical analysis.

Declaration of patient’s guardian(s) consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.