Intraocular

Lymphoma

Immunopathologic Analysis David J.

Wilson, MD; Rita Braziel, MD; James

T.

of Vitreous

Biopsy Specimens

Rosenbaum, MD

Immunologic analysis of cell surface (immunophenotyping) has become a standard procedure in the evaluation of systemic lymphomas. However, attempts to apply these techniques to intraocular lymphoma have not been uniformly successful. We successfully immunophenotyped five consecutive cases of intraocular lymphoma using immunoperoxidase surface marker analysis of cytocentrifuged specimens in two cases and flow cytometry in three. In all five cases, a monoclonal B-cell population was unequivocally present. Contrary to previous reports, we found surface marker analysis of vitreous biopsy specimens to be helpful in the diagnosis and treatment of intraocular lymphoma. Not only did it support the cytologic diagnosis but it allowed comparison of the immunophenotype of vitreous infiltrates with that of previous or subsequent lymphomatous lesions from nonocular sites. (Arch Ophthalmol. 1992;110:1455-1458)

rather than uveal, and (2) systemic lymphoma with secondary ocular in¬ volvement, usually of the uvea.1 Pri¬ mary lymphomas of the CNS have received more attention in the ophthalmologic literature because of their pro¬ pensity to masquerade as chronic pos¬ terior uveitis. Immunophenotyping of primary CNS lymphomas has been performed on post¬ mortem tissue, brain biopsy specimens, or cells obtained by lumbar puncture.2"4 These studies indicate that most CNS lymphomas are of B-cell origin and have either cytoplasmic or cell-surface immunoglobulin. Immunophenotyping is helpful in the diagnosis and classifica¬ tion of these tumors and may potentially be of some prognostic benefit. Unfortu¬ nately, comparable information on pri¬ mary ocular lymphomas is lacking. We present our experience indicating that primary ocular lymphomas are consis¬ tently of B-cell origin.

lymphomas rarely in"^ -Hodgkin's can volve the but

PATIENTS AND METHODS Patients

\s=b\

markers

-^

eye,

they

present with either of two general clinicopathologic patterns: (1) primary lym¬ phomas of the central nervous system (CNS) (microgliomatosis, reticulum cell sarcoma) in which the intraocular in¬ volvement is

principally

retinovitreal

for publication March 17, 1992. From the Departments of Ophthalmology (Drs Wilson and Rosenbaum), Medicine (Dr Rosenbaum), Cell Biology (Dr Rosenbaum), and Pathology (Dr Braziel), Oregon Health Sciences University, Portland. Reprint requests to Casey Eye Institute, 3375 SW Terwilliger Blvd, Portland, OR 97201-4197 (Dr

Accepted

Wilson).

Between November 1984 and January cases of intraocular lymphoma were diagnosed at the Uveitis Clinic of the Casey Eye Institute, Oregon Health Sci¬ ences University, Portland. Four of these cases were presented at the Association for Research in Vision and Ophthalmology meeting in 1990, but none have been included in prior publications.5 The case histories of these patients are presented in Table 1. Patient 1 developed a progressive leukoencephalopathy without any radiologie or cytologie evidence of re¬ current lymphoma. She died on March 17, 1988, without any clinical evidence of re¬ currence. The other four patients were alive at the time of this writing. Patient 2

1991, five

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developed

a recurrent lymphoma of the left ethmoidal and sphenoidal sinus 1 year after diagnosis, which was treated with ra¬ diation. Patient 3 was found to have a re¬ currence of her CNS lymphoma when atypical lymphocytes were found in her cerebrospinal fluid in June 1990.

Methods Vitreous specimens were collected with automated vitrectomy instruments with si¬ multaneous infusion of balanced salt solution to maintain intraocular pressure. In most cases all the readily accessible vitreous was removed to maximize the number of cells available for study. Specimens were trans¬ ported to the Cytology and Immunology Laboratory in balanced salt solution. A cytologie diagnosis was made based on examination of Wright-stained cytocentrifuged specimens or modified Papanicolaoustained filtered specimens.6 The immunophenotype was established in patients 1 and 2 using a standard avidin-biotin complex technique on cytocentrifuge preparations of vitreous cells. The antibody panel used for immunologie analysis included some or all of the anti¬ bodies shown in Table 2. As a negative control, mouse ascites fluid was substi¬ tuted for the primary antibody. Vitreous cells from cases 3 through 5, and cerebrospinal fluid cells from case 3, were immunophenotyped with flow cytometry on a fluorescence-activated cell sorter. Because most of the vitreous cells were trapped within the vitreous, the vitreous specimens were first digested with an enzyme, hyaluronidase (1 g/L), to put the cells in suspen¬ sion. A cell count and viability assay were performed prior to analysis. This informa¬ tion was used to determine the markers that were used (Table 3). Dual staining tech¬ niques were used to increase the number of markers on the small numbers of cells ob¬ tained from most of these vitreous speci¬ mens. This procedure entailed using a com-

Table 1.—Clinical Histories* Date and Patient No.

Age at Diagnosis,

y

70

Findings

MRI and/or

Date and Method

Ocular Involvement Bilateral vitritis and subretinal infiltrates

Diagnosis 9/84, vitrectomy of

CT Scan of the Brain 9/84, CT scan, normal

Lumbar Puncture

Other 9/84, abdominal CT scan, normal

9/84, positive

Date of Onset of Symptoms 11/84, blurred vision

Treatment Whole brain and ocular

radiation, intravenous and intrathecal

chemotherapy 11/85, vitrectomy Bilateral vitritis

57

11/85, negative

11/85, MRI,

without subretinal infiltrates

bilateral

11/86,

2/85, blurred

abdominal CT

parietal

scan,

10/86,

lesions

Whole brain and ocular

vision

radiation, chemotherapy

normal;

lymphoma left ethmoidal and

with blood brain barrier

disruption2

sphenoidal

sinuses

2/88, brain

66

1/88, CT scan,

Bilateral vitritis and subretinal infiltrates

biopsy

left frontal lesion

4/88,

5/89, vitrectomy,

rare

atypical lymph

positive for lymphoma

1987, decreased Radiation fine motor eyes, coordination

to

chemotherapy with blood brain barrier

disruption2 5/89, vitrectomy

72

69

*MRI indicates

1/91, vitrectomy

magnetic

Table

resonance

CD2 CD4 CD5 CD8 CD19 CD20 None None None None None None

CT scan, normal 1/91, MRI and CT scan, diffuse CNS involvement

5/89, negative

5/89, abdominal CT scan, normal

1/91, positive

2.—Antibody Panel

Used for

-rosette receptor, Helper cells cells, rare cell Suppressor cells cells cells cells cells cells cells cells cells

igM igD IgG IgA

by Coulter, Hialeah, Fla;

Table

Radiation to eyes and whole brain

vision

Table

Source* Coulter Coulter BD Coulter Coulter Coulter

cells

Dilution 1:10

3.—Diagnostic Panel Based Cell Count and Viability*

Antibody Panel Used CD2, 4, 8, 19, 20, IgM,

30 000-80000

CD2, 20,

IgG,

Cells,

10s

,

Insufficient cells for

immunophenotyping cytopreparation

Caltag Caltag Caltag

*ln all cases, a Wright-stained was also used for cytologie study.

Caltag BD BD

Becton Dickenson

1:5

(BD), Mountain View,

4.—Immunophenotype and

Analysis*

Method of

%

Method of

CD2

Not done Not done 3.0

CD4

CD8

CD19

CD20

65

1.0

93 80

1.6

immunoperoxidase; FC,

,

,

Total No.

of

on

Total Cell Count >80000 20000-30000

Intraocular lymphoma. Immunopathologic analysis of vitreous biopsy specimens.

Immunologic analysis of cell surface markers (immunophenotyping) has become a standard procedure in the evaluation of systemic lymphomas. However, att...
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