Abstract
Background
Cytological examination of rapidly transported, unfixed vitreous specimens is considered the gold standard in exclusion of primary intraocular lymphoma (PIOL) in patients with idiopathic steroid resistant chronic uveitis. These specimens are difficult to interpret, and reports of “false negatives” or “false positives” are common. Fixation using HOPE solution (Herpes-glutamic acid buffer mediated Organic solvent Protection Effect) has been successfully applied in the investigation of cytospin preparations using immunocytology, in situ hybridisation and polymerase chain reaction (PCR). The purpose of this study was to compare the cytologic features of vitrectomy specimens from patients with clinical reactive vitritis and PIOL prepared following HOPE fixation with those in conventional cytospins and to identify any diagnostic pitfalls.
Methods
Pars plana vitrectomy was performed in 15 cases of patients with chronic uveitis. The vitreous samples were halved: one half was fixed in HOPE solution; the other half remained unfixed. All samples were subject to conventional staining, immunocytology and clonality analysis using polymerase chain reaction (IgH-PCR) and GeneScan. The specimens were assessed following by two pathologists who were masked to the cytological preparation method. The specimens were evaluated for cellularity, cellular appearance, cytoplasmic and nuclear features as well as quality of the immunostains.
Results
Twelve of the fifteen vitreous samples were diagnosed as reactive vitritis; in three specimens, a primary intraocular lymphoma of B-cell type was diagnosed. Compared to the unfixed vitreal specimens, the quality of the cytomorphology and immunocytology improved in the HOPE-fixed specimens. IgH-PCR and GeneScan analysis demonstrated polyclonal amplification products in the reactive cases, and monoclonal B-cell populations in the B-PIOL.
Conclusion
Our results demonstrate that cytomorphology and immunoreactivity of vitreous specimens are well preserved following HOPE fixation. DNA of sufficient quality could be extracted from HOPE-fixed vitreous biopsies, in order to perform clonality analyses. HOPE fixation appears to be promising in simplifying the transportation of these specimens, and may improve the diagnostic reliability of vitreous specimens in patients with masquerade syndrome.
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References
Barkan G, Rubin M, Michael C (2002) Diagnosis of melanoma aspirates on thinPrep: the University of Michigan experience. Diagn Cytopathol 26:334–339
Blumenkranz MS, Ward T, Murphy S et al (1992) Applications and limitations of vitreoretinal biopsy techniques in intraocular large cell lymphoma. Retina 12:64–70
Cassoux N, Merle-Beral H, Leblond V et al (2000) Ocular and central nervous system lymphoma: clinical features and diagnosis. Ocul Immunol Inflamm 8:243–250
Char DH, Ljung BM, Deschenes J et al (1988) Intraocular lymphoma: immunological and cytological analysis. Br J Ophthalmol 72:905–911
Cordell JL, Falini B, Erber WN et al (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 32:219–229
Coupland SE, Anastassiou G, Hummel M et al (2005) Primary intraocular lymphoma of T-cell type: report of a case and review of the literature. Graefe Arch Clin Exp Ophthalmol 243:189–197 (Dec. 10; EPubmed)
Coupland SE, Bechrakis NE, Anastassiou G et al (2003) Evaluation of vitrectomy specimens and chorioretinal biopsies in the diagnosis of primary intraocular lymphoma in patients with masquerade syndrome. Graefe Arch Clin Exp Ophthalmol 10:860–870
Coupland SE, Heimann H, Bechrakis NE (2004) Primary intraocular lymphoma: a review of the clinical, histopathological and molecular biological features. Graefe Arch Clin Exp Ophthalmol 242:901–913
Coupland SE, Willerding G, Jahnke K et al (2005) Demonstration of identical clonal derivation in a case of oculocerebral lymphoma. Br J Ophthalmol 89:238–239
Davis JL (2004) Diagnosis of intraocular lymphoma. Ocul Immunol Inflamm 12:7–16
Davis JL, Solomon D, Nussenblatt RB et al (1992) Immunocytochemical staining of vitreous cells. Indications, techniques, and results. Ophthalmology 99:250–256
Davis JL, Viciana AL, Ruiz P (1997) Diagnosis of intraocular lymphoma by flow cytometry. Am J Ophthalmol 124:362–372
Engel HM, Green WR, Michels RG et al (1981) Diagnostic vitrectomy. Retina 1:121–149
Farkas T, Harbour JW, Davila RM (2004) Cytologic diagnosis of intraocular lymphoma in vitreous aspirates. Acta Cytol 48:487–491
Freeman LN, Schachat AP, Knox DL et al (1987) Clinical features, laboratory investigations, and survival in ocular reticulum cell sarcoma. Ophthalmology 94:1631–1639
Goldey SH, Stern GA, Oblon DJ et al (1989) Immunophenotypic characterization of an unusual T-cell lymphoma presenting as anterior uveitis. A clinicopathologic case report. Arch Ophthalmol 107:1349–1353
Hsu SM, Raine L, Fanger H (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577–580
Jensen OA, Johansen S, Kiss K (1994) Intraocular T-cell lymphoma mimicking a ring melanoma. First manifestation of systemic disease. Report of a case and survey of the literature. Graefe Arch Clin Exp Ophthalmol 232:148–152
Liu K, Klintworth GK, Dodd LG (1999) Cytologic findings in vitreous fluids. Analysis of 74 specimens. Acta Cytol 43:201–206
Ljung BM, Char D, Miller TR et al (1988) Intraocular lymphoma. Cytologic diagnosis and the role of immunologic markers. Acta Cytol 32:840–847
Lobo A, Larkin G, Clark BJ et al (2003) Pseudo-hypopyon as the presenting feature in B-cell and T-cell intraocular lymphoma. Clin Exp Ophthalmol 31:155–158
Merle-Beral H, Davi F, Cassoux N et al (2004) Biological diagnosis of primary intraocular lymphoma. Br J Haematol 124:469–473
Michels RG, Knox DL, Erozan YS et al (1975) Intraocular reticulum cell sarcoma. Diagnosis by pars plana vitrectomy. Arch Ophthalmol 93:1331–1335
Olert J, Wiedorn KH, Goldmann T et al (2001) HOPE fixation: a novel fixing method and paraffin-embedding technique for human soft tissues. Pathol Res Pract 197:823–826
Palexas GN, Green WR, Goldberg MF et al (1995) Diagnostic pars plana vitrectomy report of a 21-year retrospective study. Trans Am Ophthalmol Soc 93:281–308
Qualman SJ, Mendelsohn G, Mann RB et al (1983) Intraocular lymphomas. Natural history based on a clinicopathologic study of eight cases and review of the literature. Cancer 52:878–886
Read RW, Zamir E, Rao NA (2002) Neoplastic masquerade syndromes. Surv Ophthalmol 47:81–124
Rothova A, Ooijman F, Kerkhoff F et al (2001) Uveitis masquerade syndromes. Ophthalmology 108:386–399
Scroggs MW, Johnston WW, Klintworth GK (1990) Intraocular tumors. A cytopathologic study. Acta Cytol 34:401–408
Sen Gupta R, Hillemann D, Kubica T et al (2003) HOPE-fixation enables improved PCR-based detection and differentiation of Mycobacterium tuberculosis complex in paraffin-embedded tissues. Pathol Res Pract 199:619–623
Umland O, Ulmer AJ, Vollmer E et al (2003) HOPE fixation of cytospin preparations of human cells for in situ hybridization and immunocytochemistry. J Histochem Cytochem 51:977–980
van Dongen JJ, Langerak AW, Bruggemann M et al (2003) Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 17:2257–2317
Whitcup SM, Chan CC, Buggage RR et al (2000) Improving the diagnostic yield of vitrectomy for intraocular lymphoma. Arch Ophthalmol 118:446
Whitcup SM, de Smet MD, Rubin BI et al (1993) Intraocular lymphoma. Clinical and histopathologic diagnosis. Ophthalmology 100:1399–1406
White VA, Gascoyne RD, Paton KE (1999) Use of the polymerase chain reaction to detect B- and T-cell gene rearrangements in vitreous specimens from patients with intraocular lymphoma. Arch Ophthalmol 117:761–765
Wiedorn KH, Olert J, Stacy RA et al (2002) HOPE—a new fixing technique enables preservation and extraction of high molecular weight DNA and RNA of >20 kb from paraffin-embedded tissues. Hepes-glutamic acid buffer mediated organic solvent protection effect. Pathol Res Pract 198:735–740
Wilson DJ, Braziel R, Rosensbaum JT (1992) Intraocular lymphoma. Immunopathologic analysis of vitreous biopsy specimens. Arch Ophthalmol 110:1448–1455
Zaldivar RA, Martin DF, Holden JT et al (2004) Primary intraocular lymphoma. Clinical, cytologic and flow cytometric analysis. Ophthalmology 111:1762–1767
Acknowledgements
The authors would like to thank Dr. Torsten Goldmann of the Research Centre Borstel, Clinical and Experimental Pathology, Borstel for the provision of the HOPE solution for initial testing in vitrectomy specimens. We would also like to thank him for his critical analysis of the manuscript. Furthermore, the authors are grateful for the excellent technical assistance of Mrs. E. Seibt, Mrs. P. Noack and Mr. Henning Müller.
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Coupland, S.E., Perez-Canto, A., Hummel, M. et al. Assessment of HOPE fixation in vitrectomy specimens in patients with chronic bilateral uveitis (masquerade syndrome). Graefe's Arch Clin Exp Ophthalmol 243, 847–852 (2005). https://doi.org/10.1007/s00417-005-1166-1
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DOI: https://doi.org/10.1007/s00417-005-1166-1