Paraffin Embedding Technique for Specimens Obtained by Vitrectomy

Histologic and immunohistochemical preparation of pathologic fluidscontaining cellular material is difficult, because the techniques are limitedby the amount of cellular material. Similar problems limit the preparationof vitrectomized material. Cytopreparatory techniques, including milliporefilter and celloidin bag cell block, have been introduced since 1980 for diagnosticvitrectomies.^1,2 These techniquesare limited by the fact that the specimens cannot be stained using differenthistologic and immunohistochemical techniques. A second disadvantage of thesetechniques is that long-term storage of obtained material for later histologicevaluation is not possible. Direct paraffin embedding of vitrectomized specimensis possible, but specimens with low cell numbers are difficult to prepare.^3,4

Kawan et al⁵ introduced a brush cellblock technique for diagnosis of bronchial neoplasms, in which histologicevaluation of the tissue can be performed with a small amount of aspiratedcells. Herein, we introduce a modification of the technique by Kawan et al⁵ to embed vitrectomized material in paraffin.

Eyes from 101 patients underwent vitrectomy for different pathologicindications (Table 1). The operatingtechnique was similar in all patients.⁶ Dilutedvitreous material was obtained from the receptacle attached to the aspirationline at the end of a pars plana vitrectomy and then centrifuged at 3000 revolutionsper minute for 10 minutes. The collected pellet was mixed with sodium citrateplasma (1:1, Ci-Trol; Dade Behring, Vienna, Austria). Afterward, the sameamount of thromboplastin (Thromborel S; Dade Behring) was added to the pellet.The material was then coagulated to a fibrin block and the block centrifugedat 3000 revolutions per minute for 5 minutes and fixed in 10% buffered formaldehydefor 1 hour (Figure 1). The blockwas embedded in paraffin wax with a melting point of 58°C to 60°C(Histosec; Merck, Vienna, Austria). Five-micrometer sectioning was performed.Routine hematoxylin-eosin, periodic acid–Schiff, and Masson trichromestaining was performed in all cases. Depending on the clinical diagnosis,immunohistochemical staining for CD3, CD20, CD45, S100, and HMB45 (Table 2) was performed. This latter techniquehas been previously described.⁷

Examples of routine histologic and immunohistochemical staining areshown in Figure 2, Figure 3, Figure 4, and Figure 5. Paraffin embedding of vitrectomymaterial was performed easily in all cases. The tissue may be prepared andsectioned within hours.

Routine histologic examination following hematoxylin-eosin, periodicacid–Schiff, and Masson trichrome staining demonstrated excellent morphologicdifferentiation of the vitrectomized cells. Immunohistochemical staining showedgood staining quality and morphologic delineation. Using the appropriate antibodyconcentration, background staining was not observed.

The paraffin embedding technique of vitrectomized material introducedherein is an easy and fast method for paraffin embedding of intraocular fluids,even with a small number of cells. The technique enables ophthalmic pathologistswho are not experienced with vitreous samples to handle this material easily.

In contrast, direct paraffin embedding of vitrectomized specimens requiresa large number of cells, as in cases of vitreous hemorrhage, endophthalmitis,or diabetic retinopathy. In these instances, a considerable amount of cellswill be lost.^3,4

Paraffin embedding of vitrectomized intraocular fluids using the fibrinand paraffin method has the following important advantages. Specimens withsmall numbers of cells from patients with macular pucker or retinal detachmentwithout hemorrhage can be analyzed using all histologic techniques, includingimmunohistochemical analysis, which is not possible with cytopreparation techniques.The second major advantage is the possibility of archiving specimens for futurestudies.

The authors have no relevant financial interest in this article.

We thank Arthur Fu, MD, Moorfields Eye Hospital, London, England, forcritical reading of the manuscript.

Correspondence: Dr Ardjomand, Department of Ophthalmology, MedicalUniversity Graz, Auenbruggerplatz 4, 8036 Graz, Austria (navid.ardjomand@meduni-graz.at).