**2. Gross pathology**

The first step of grossing an enucleated eye in preparation for histopathological microscopic examination is establishing the laterality. Several anatomical landmarks provide useful cues to orient the globe properly, and these include the cornea, oblique muscles, and ciliary arteries. The corneal horizontal diameter is larger than its vertical diameter by around 1 mm, and this produces an oval shape (**Figure 1A**). The insertion of the superior oblique muscle tendon after originating from the trochlea is in the superior outer (temporal) quadrant just behind the insertion of the superior rectus muscle tendon. The inferior outer (temporal) quadrant receives the insertion of inferior oblique muscle, just lateral to the optic nerve (**Figure 1B**). Locating the horizontal planes can be done affirmatively by identifying the long posterior ciliary arteries that run horizontally at 3 and 9 o'clock [9].

In the past, the enucleated eye was grossed and processed to produce one pupiloptic nerve (PO) section, which is then studied histopathologically. This practice was abandoned following the consensus of the International Retinoblastoma Staging Working Group (IRSWG) in 2009 where the efforts of 85 members from

#### **Figure 1.**

*Grossing an enucleated eye with retinoblastoma: (A) vertical corneal measurements; (B) inferior oblique muscle insertion located temporal (lateral) to the optic nerve (asterisk); (C, D) pupil-optic nerve (PO) section; (E) cassettes containing both calottes cut in bread-loaf pattern (red arrows), while the PO section is submitted in a separate cassette.*

**91**

**Figure 2.**

*calcification.*

*Histopathological Characteristics and Classification for Prognostic Indicators*

24 different countries were joined to establish guidelines for tissue processing and handlining as well as staging. They concluded that the entire enucleated globe should be examined microscopically. This can be achieved by dividing the eye into four blocks. The optic nerve, tumor, and anterior chamber structures are included in the central pupil-optic nerve (PO) section composing one block as demonstrated in (**Figure 1C, D**). Two mirrored blocks composed of the calottes, representing the remaining eye tissue after harvesting the PO section, are usually cut consecutively in a bread-loaf fashion and embedded on edge to increase the examined surface area of the choroid, and this will subsequently improve the chances of detection of choroidal involvement (**Figure 1E**). The last block comprises the optic nerve margin cross section, and this is usually taken initially before cutting the eye. The optic nerve head, lamina cribrosa, and optic nerve posterior to the lamina cribrosa in a single section plane are usually displayed in the PO section as shown

Enucleation of the eye not only enables histopathological diagnosis, but it also yields fresh ocular tissue on which molecular and genetic testing can be carried out. Such tests are of paramount importance as their results are needed for family counseling and prognosis prediction. To facilitate this, guidelines and protocols were proposed to ensure preservation of the harvested ocular tissue for examination to obtain the best histopathological and molecular testing results. This guideline states that the enucleated globe should be processed and opened soon after surgery in order to prevent proteins and nucleic acid denaturation. Then, the optic nerve length is measured and documented in mm, and this is followed by preparing the block of the optic nerve margin cut section before proceeding with opening the eye. The next step involves opening the globe with the aid of transillumination, which is helpful in localizing the margins of the intraocular mass in addition to planning the collection of the PO section. Opening the eye can be accomplished using one of two techniques. The first proposed technique involves creating a window opening in the sclera adjacent to the edge of the bulk of the tumor. This scleral window is ideally created using a trephine. The second method is done utilizing a large bore 22-gauge needle that is used under sterile conditions to aspirate fresh tumor cells/tissue. The needle is inserted obliquely under direct visualization via the sclera into the posterior chamber behind the lens, and aspiration takes place once it is inside the tumor mass. Finally, the globe is fixed in an adequate amount of formalin for a minimum

*(A) Gross photo showing the encephaloid appearance of the tumor. (B) White flecks representing the* 

*DOI: http://dx.doi.org/10.5772/intechopen.89410*

in (**Figure 1D**) [9].

of 48 hours [10].

#### *Histopathological Characteristics and Classification for Prognostic Indicators DOI: http://dx.doi.org/10.5772/intechopen.89410*

*Retinoblastoma - Past, Present and Future*

The first step of grossing an enucleated eye in preparation for histopathological microscopic examination is establishing the laterality. Several anatomical landmarks provide useful cues to orient the globe properly, and these include the cornea, oblique muscles, and ciliary arteries. The corneal horizontal diameter is larger than its vertical diameter by around 1 mm, and this produces an oval shape (**Figure 1A**). The insertion of the superior oblique muscle tendon after originating from the trochlea is in the superior outer (temporal) quadrant just behind the insertion of the superior rectus muscle tendon. The inferior outer (temporal) quadrant receives the insertion of inferior oblique muscle, just lateral to the optic nerve (**Figure 1B**). Locating the horizontal planes can be done affirmatively by identifying the long

In the past, the enucleated eye was grossed and processed to produce one pupiloptic nerve (PO) section, which is then studied histopathologically. This practice was abandoned following the consensus of the International Retinoblastoma Staging Working Group (IRSWG) in 2009 where the efforts of 85 members from

*Grossing an enucleated eye with retinoblastoma: (A) vertical corneal measurements; (B) inferior oblique muscle insertion located temporal (lateral) to the optic nerve (asterisk); (C, D) pupil-optic nerve (PO) section; (E) cassettes containing both calottes cut in bread-loaf pattern (red arrows), while the PO section is* 

posterior ciliary arteries that run horizontally at 3 and 9 o'clock [9].

**2. Gross pathology**

**90**

**Figure 1.**

*submitted in a separate cassette.*

24 different countries were joined to establish guidelines for tissue processing and handlining as well as staging. They concluded that the entire enucleated globe should be examined microscopically. This can be achieved by dividing the eye into four blocks. The optic nerve, tumor, and anterior chamber structures are included in the central pupil-optic nerve (PO) section composing one block as demonstrated in (**Figure 1C, D**). Two mirrored blocks composed of the calottes, representing the remaining eye tissue after harvesting the PO section, are usually cut consecutively in a bread-loaf fashion and embedded on edge to increase the examined surface area of the choroid, and this will subsequently improve the chances of detection of choroidal involvement (**Figure 1E**). The last block comprises the optic nerve margin cross section, and this is usually taken initially before cutting the eye. The optic nerve head, lamina cribrosa, and optic nerve posterior to the lamina cribrosa in a single section plane are usually displayed in the PO section as shown in (**Figure 1D**) [9].

Enucleation of the eye not only enables histopathological diagnosis, but it also yields fresh ocular tissue on which molecular and genetic testing can be carried out. Such tests are of paramount importance as their results are needed for family counseling and prognosis prediction. To facilitate this, guidelines and protocols were proposed to ensure preservation of the harvested ocular tissue for examination to obtain the best histopathological and molecular testing results. This guideline states that the enucleated globe should be processed and opened soon after surgery in order to prevent proteins and nucleic acid denaturation. Then, the optic nerve length is measured and documented in mm, and this is followed by preparing the block of the optic nerve margin cut section before proceeding with opening the eye. The next step involves opening the globe with the aid of transillumination, which is helpful in localizing the margins of the intraocular mass in addition to planning the collection of the PO section. Opening the eye can be accomplished using one of two techniques. The first proposed technique involves creating a window opening in the sclera adjacent to the edge of the bulk of the tumor. This scleral window is ideally created using a trephine. The second method is done utilizing a large bore 22-gauge needle that is used under sterile conditions to aspirate fresh tumor cells/tissue. The needle is inserted obliquely under direct visualization via the sclera into the posterior chamber behind the lens, and aspiration takes place once it is inside the tumor mass. Finally, the globe is fixed in an adequate amount of formalin for a minimum of 48 hours [10].

**Figure 2.** *(A) Gross photo showing the encephaloid appearance of the tumor. (B) White flecks representing the calcification.*

Macroscopically, the tumor has an encephaloid appearance, and this is not surprising given that it arises from the retina which resembles the neurological tissue (**Figure 2A**). The tumor is typically white in color, and it encompasses lightly colored flecks. In fact, these flecks are analogous to the dystrophic calcification within the necrotic tissue microscopically (**Figure 2A**) [10].
