Lymphocytic Infiltrates of the Conjunctiva and Orbit:
Immunohistochemical Staining of 16 Cases
RODERICK R. TURNER, M.D., PETER EGBERT, M.D., AND ROGER A. WARNKE, M.D.
The authors have performed frozen section immunologic stains
on 16 cases of ocular lymphocytic infiltrates and correlated the
results with clinical and histologic findings. Their cases included
inflammatory pseudotumor (3), reactive lymphoid hyperplasia
(3), atypical lymphocytic infiltrate (9), and small cleaved cell
lymphoma (1). Seven of the nine cases with an atypical lymphocytic infiltrate expressed one immunoglobulin light chain,
while only one of six cases considered reactive on histologic
evaluation had immunologic results suggestive of a neoplastic
B cell proliferation. The case of follicular small cleaved cell
lymphoma expressed B lineage antigens but did not express
immunoglobulin; this patient died of disseminated lymphoma
two years after conjunctival involvement. Percentages and subset
ratios of T lymphocytes were quantitated and showed similar
results in reactive and neoplastic lesions. There is no apparent
difference in clinical presentation or follow-up information between patients with reactive lesions and those having an atypical
lymphocytic infiltrate with monotypic immunoglobulin. (Key
words: Immunohistochemistry; B and T lymphocytes; Lymphoma; Conjunctiva; Orbit) Am J Clin Pathol 1984; 81:
447-452
SMALL LYMPHOCYTIC INFILTRATES of ocular
structures, similar to those in other extranodal sites, may
cause difficult diagnostic problems.3,4 Clinical features and
routine histopathologic techniques are often not sufficient
for separation of reactive from neoplastic lesions.
Cell suspension immunologic studies6 have shown that
ocular lymphomas are generally monotypic B-cell proliferations (light chain restriction) and that the average
number of T-cells is commonly lower in neoplastic than
in reactive infiltrates. However, the loss of tissue architecture by the cell suspension method may complicate
analysis of cases with an abundant host response.
We have performed frozen section immunologic stains
on 16 cases of ocular small lymphocytic infiltrates, nine
of which were designated atypical lymphocytic infiltrate
on histologic classification, and correlated the results with
clinical findings.
Departments of Pathology and Ophthalmology, Stanford
University Medical Center, Stanford, California
Methods
We reviewed the 16 cases with orbital and conjunctival
small lymphocytic infiltrates for which frozen tissue was
available in the Laboratory of Tissue Immunodiagnosis
at the Stanford University Medical Center. The cases
were classified according to the recommendations of
Knowles and Jacobiec6 as summarized in Table 1. Clinical
information was obtained from hospital charts and local
physicians.
Four-micron frozen sections were fixed in acetone and
sequentially incubated (15 minutes followed by phosphate-buffered saline wash) with monoclonal mouse antihuman antibody, biotinylated goat antimouse IgG
F(ab')2, and avidin-horseradish peroxidase. Diaminobenzidene was the chromogen, followed by copper sulfate
and a methylene blue counterstain. Monoclonal antibodies, with specificities listed in Table 2, were obtained
from Becton Dickinson Monoclonal Center, Mountain
View, California (anti-Leu-1 to 5, kappa, lambda, mu);
Coulter Electronics, Inc., Hialeah, Florida (anti-B|); Ronald Levy, Stanford University Medical Center, Stanford,
California (anti-la, L-203); J. Donald Capra, Dallas, Texas
(63D3); David Mason and Harald Stein, Oxford, England
(TO 15, R4/23). The two cases unreactive for mu, kappa,
and lambda immunoglobulin (Ig) chains were also stained
for gamma, alpha, and delta chains.
Cell counts were performed using an eyepiece grid at
a total magnification of X400. This provided a standard
grid area for which cell counts could be compared. Percentages of T lymphocytes were calculated by dividing
the number of Leu-4 reactive cells by the sum of the Leu4, TO 15, and 63D3 reactive cells.
Received July 5, 1983; received revised manuscript and accepted for
publication September 26, 1983.
Supported in part by Grants 09157 and 34233 from the National
Institutes of Health, Bethesda, Maryland and an unrestricted grant from
Research for the Prevention of Blindness, Inc.
Address reprint requests to Dr. Turner: Department of Pathology,
UCLA Center for Health Sciences, Los Angeles, California 90024.
447
Results
Histologic Findings
The histologic diagnoses, shown in Table 3, included
inflammatory pseudotumor of fibrous (two cases) and
TURNER, EGBERT, AND WARNKE
448
Table 1. Histologic Classification of Ocular Infiltrates
Proposed by Knowles and Jacobiec6
Diagnosis
Description
Inflammatory pseudotumor
Polymorphous type
Hypocellular type
Fibrotic type
Hypercellular type
Variably cellular, polymorphous
(small lymphocytes, immunoblasts, plasma cells, histiocytes,
occasional eosinophils or neutrophils), hyalinization, edema,
prominent endothelial cells.
Germinal centers uncommon.
Reactive lymphoid hyperplasia
Diffuse pattern
Follicular pattern
Cellular lesions of mature small
lymphocytes, with increased
vascularity and prominent endothelial cells. Germinal centers
prominent in follicular type.
Atypical lymphocytic infiltrate*
Admixture of small lymphocytes
and larger atypical lymphoid
cells without germinal centers.
Commonly extends into fat or
muscle.
Lymphocytic lymphoma
Monomorphous population of
atypical small lymphocytes.
May be follicular or diffuse.
* We use the term "atypical lymphocytic infiltrate" rather than "atypical lymphoid hyperplasia."
polymorphous (one case) types, reactive lymphoid hyperplasia of diffuse (two cases) and follicular (one case)
types, and atypical lymphocytic infiltrate (nine cases) and
follicular small cleaved cell lymphoma (one case). A definite reactive or neoplastic designation could not be
reached on histologic examination in cases of atypical
lymphocytic infiltrate (Figures 1, 2).
Polykaryocytes (multinucleated lymphoid cells) were
present infivecases, four of which were classified as atypical lymphocytic infiltrates and had monotypic Ig staining
Table 2. Monoclonal Antibody Specificity
Monclonal Antibody
Anti-Leu-1
Common Reactivity
Anti-B,
Pan T-lymphocyte, some B-CLL
and B cell lymphomas
Cytotoxic/suppressor T-lymphocytes
Helper T-lymphocytes
Pan T-lymphocyte
T-lymphocytes bearing receptors
for sheep erythrocytes
B-lymphocytes and their precur-
T015
B-lymphocytes and their precur-
Anti-mu, gamma, delta, alpha,
kappa, lambda
Anti-la
B-lymphocytes
Anti-Leu-2a
Anti-Leu-3a
Anti-Leu-4
Anti-Leu-5
63D3
R4/23
B-lymphocytes, some monocytes
and macrophages, some activated T-lymphocytes
Monocytes/macrophages
Dendritic reticulum cells
A.J.C.P. • April 1984
patterns. Among the atypical lymphocytic infiltrates, three
cases had focal plasmacytic differentiation with Dutcher
bodies, and two of these were light chain restricted.
Immunologic Findings
The majority of the cells in most cases were B lymphocytes, although substantial numbers of T cells were
present in some cases (Fig. 3A Table 4). Infiltrates with
both kappa and lambda bearing B cells in expected ratios
had a mean of 48% T cells (range 20-67), while monotypic
lesions averaged 34% T cells (range 7-49).
The B cells in all cases expressed B cell antigens, B,
and TO 15. Seven cases expressed mu heavy chains with
a single light chain (three kappa, four lambda; see Figs.
3B and C). Seven other cases showed an admixture of
kappa cells and lambda cells (Fig. 4). In one case, the
monotypic B cell population stained for Leu-1. Two cases
stained for B, and TO 15 but showed no staining for immunoglobulin chains.
In all cases, virtually all cells including T cells stained
for la antigens. There were small numbers of macrophages
(63D3) scattered throughout the infiltrates. The dendritic
reticulum cell antibody (R4/23) highlighted follicular
structures in nine cases, including three cases of atypical
lymphocytic infiltrate with a histologically diffuse pattern
of infiltration.
Clinical Data
The patients ranged in age from 34-83 years (mean
62). There were 11 females and 5 males. Lesions in the
conjunctiva alone accounted for nine cases, while seven
patients had predominantly orbital involvement. The lesions under the conjunctiva had the characteristic clinical
appearance of a smooth, firm, elevated, tan-pink mass.
Two patients had a prior history of lymphoma at another
site: patient 8 had small lymphocytic lymphoma in axillary
and inguinal lymph nodes six years prior to conjunctival
infiltrate but has had no evidence of systemic disease
since then (three years follow-up); and patient 16 had a
two-year history of widely disseminated follicular small
cleaved cell lymphoma prior to ocular involvement and
died of lymphoma two years after biopsy of the conjunctiva.
All patients had a normal complete blood count and
chest radiograph. Other studies on patients with primary
presentation in the eye region were negative when performed, including protein electrophoresis (3), bone marrow biopsy (2), lymphangiogram (2), and abdominal or
head CT scans (6).
Treatment was not standardized and consisted most
often of local radiotherapy (3,000-4,000 rads) or systemic
corticosteroids. Follow-up information was available for
vol. 81-No.4
449
OCULAR LYMPHOCYTIC INFILTRATES
Table 3. Clinical, Histologic and Immunologic Data for 16 Patients with Ocular Infiltrates.
Patient
No.
Age/Sex
Histologic Diagnosis
Immunologic
Results
Inflammatory pseudotumor,
fibrotic type
Inflammatory pseudotumor,
fibrotic type
Inflammatory pseudotumor,
polymorphous type
Reactive lymphoid
hyperplasia, diffuse
Reactive lymphoid
hyperplasia, diffuse
Reactive lymphoid
hyperplasia, follicular
Atypical lymphocytic
infiltrate
Atypical lymphocytic
infiltrate
Atypical lymphocytic
infiltrate
Atypical lymphocytic
infiltrate
Atypical lymphocytic
infiltrate
Atypical lymphocytic
infiltrate
Atypical lymphocytic
infiltrate
Atypical lymphocytic
infiltrate
Atypical lymphocytic
infiltrate
Mixed B & T cell
population
Mixed B & T cell
population
Ig" B-lineage
lymphoma
Mixed B & T cell
population
Mixed B & T cell
population
Mixed B & T cell
population
Monotypic IgM
lambda
Monotypic IgM
lambda
Mixed B & T cell
population
Monotypic IgM
lambda
Monotypic IgM
lambda
Monotypic IgM
kappa
Monotypic IgM
kappa
Mixed B & T cell
population
Monotypic IgM
kappa
Steroids
6 yrs
NED
Steroids
2 yrs
NED
Surgery,
steroids
Steroids
2'/2 yrs
6 mos
Steroids
8 mos
Radiotherapy
6 mos
Died of other
causes, NED
Persistent local
disease
Persistent local
disease
NED
Steroids
2'/2 yrs
N/A
3 yrs
Persistent local
disease
NED
Steroids
4 yrs
NED
Steroids, then
radiotherapy
Steroids, then
radiotherapy
Radiotherapy
4 yrs
Radiotherapy
3 mos
Persistent local
disease
Died of other
causes, NED
Regression of
disease
NED
N/A
N/A
N/A
N/A
N/A
N/A
Follicular lymphoma, small
cleaved cell type
Ig B-lineage
lymphoma
Radiotherapy
2 yrs after eye
involvement
Died of
disseminating
lymphoma
Site of Lesion
1
34F
Orbit
2
45M
Orbit
3
83F
Orbit
4
51M
5
61F
Conjunctiva,
bilateral
Conjunctiva
6
68F
Orbit
7
66F
Orbit
8
65F
Conjunctiva
9
68F
Conjunctiva
10
64M
Conjunctiva
11
65M
12
54F
Conjunctiva
and orbit
Conjunctiva
13
37F
Conjunctiva
14
44F
Conjunctiva
15
52M
16
67F
Eyelids and
orbits,
bilateral
Conjunctiva
Treatment
Follow-up
2 yrs
7 mos
Status
Abbreviations: Ig" means no staining for immunoglobulin heavy or light chains: NED = no
evidence of disease: N/A = not available.
* Histologic review of Case 3. with knowledge of the immunologic staining results, suggested
that the correct diagnosis of plasmacytoid small lymphocytic lymphoma.
14 cases. None of the seven patients with a monoclonal
proliferation and initial presentation in the ocular region
has developed disseminated lymphoma (six cases with
follow-up, longest four years).
demonstrated monotypic staining in six of eight similar
cases.
Seven of the nine cases with atypical lymphocytic infiltrates, in our series, had monotypic immunoglobulin
light chain, supporting a diagnosis of lymphoma. These
patients, however, have done well. Among the patients
with monoclonal infiltrates for whom follow-up information was available, three had persistent local disease
and three others were disease-free. One patient (patient
8) had a prior history of systemic lymphoma, but he is
currently in remission. Although the follow-up periods
are short, these results are consistent with those previously
reported; clinical importance resides in identification of
higher grade lymphomas rather than identification of
monoclonal small lymphocytic proliferations.2
Our findings indicate that an atypical lymphocytic infiltrate, whether monoclonal or polyclonal, represents an
indolent disease that usually remains localized and does
not rapidly disseminate or show an aggressive course.
Discussion
Lymphocytic infiltrates of ocular structures often are
termed atypical lymphocytic infiltrate when histologically
composed of a diffuse admixture of small mature-appearing lymphocytes, slightly atypical small lymphocytes,
and occasional larger transformed lymphocytes. This designation reflects the prognostic uncertainty in these cases.
In the clinical study by Knowles and Jacobiec,4 two of
seven patients with this diagnosis had associated systemic
lymphoma; one probably had unrelated Hodgkin's disease, while another had a 10-year history of generalized
lymphadenopathy, andfivewere disease-free. Preliminary
immunohistochemical findings of Harris and associates'
450
TURNER, EGBERT, AND WARNKE
A.J.C.P. • April 1984
FlG. 1 (upper). A (left). Atypical lymphocytic infiltrate. The conjunctiva is expanded by a diffuse infiltrate of small lymphocytes and slightly
atypical larger lymphocytes Hematoxylin and eosin (X75). B (right). Higher power shows the cytologic appearance. There is an admixture of small
round lymphocytes, atypical small lymphocytes, and large transformed lymphocytes. Hematoxylin and eosin (X480). Immunologic stains of this
case (Figs. 3A and B) showed a monotypic B cell proliferation.
FlG. 2 (lower). A (left). Atypical lymphocytic infiltrate. There is a diffuse lymphocytic infiltrate in the conjunctiva, similar to the case illustrated
in Figure 1. Hematoxylin and eosin (X75). B (right). Admixture of lymphocytes and atypical small lymphocytes. Hematoxylin and eosin (X480).
Immunoperoxidase stains on this case (Figure 4) show a mixed B- and T-cell pattern.
vol. 8i. No. 4
OCULAR LYMPHOCYTIC INFILTRATES
451
Table 4. Percentages and Subsets of T-lymphocytes
FIG. 3/1. T-lymphocyte stain (anti-Leu-1) demonstrates moderate
numbers of T cells adjacent to aggregates of unstained B cells. Same
case as Figure I. Immunoglobulin stains (Fig. 3fl) show monotypic
lambda light chain. Leu-1 immunoperoxidase (X75).
Whether long-term follow-up will show that the monoclonal lesions eventually disseminate and/or become
clinically significant remains to be determined. At our
institution, patients with ocular lymphocytic lymphoma
undergo staging procedures that usually include chest ra-
B-lineage
Phenotype
Per Cent T-cells,
Mean (range)
Helper/Suppressor
Ratio in Tissue,
Mean (Range)
Polyclonal
(7 cases)
Monotypic
(7 cases)
Ig nonexpressing
(2 cases)
48% (20-67)
2.2(1.4-5.1)
34% (7-49)
2.6(1.0-4.2)
34% (24-44)
2.6
diograph, bone marrow biopsy and lymphangiogram,
and/or CT scan of the abdomen; if the results are negative,
then local radiotherapy is recommended. Patients with
a polyclonal infiltrate usually receive steroids.
In addition to the seven cases with monotypic light
chain, two cases were considered lymphomas because of
B antigen staining together with lack of immunoglobulin
staining. One patient had follicular small cleaved cell
lymphoma, while the other was classified histologically
as inflammatory pseudotumor of the polymorphous type.
Review of the second case suggests that it is a plasmacytoid
small lymphocytic lymphoma with abundant amyloid
material.
Cell suspension studies by Knowles and Jacobiec5 suggest that the percentage of T-cells in the infiltrate is helpful
FIGS. 35 and C. Immunoglobulin light chain stains. Same case as Figure I. B (left). Kappa light chain stain. A few scattered kappa-bearing cells
are present, but most cells are unreactive. Kappa immunoperoxidase (X120). C (right). Lambda light chain stain. Almost all cells appear stained,
except for T-cell regions at periphery of B cell aggregates. Lambda immunoperoxidase (X120).
452
TURNER, EGBERT, AND WARNKE
AJ.C.P. • April 1984
found approximately equal THTS ratios for mixed and
monotypic lesions. Of greater diagnostic usefulness is the
Leu-1 expression by some B-cell lymphomas. The neoplastic population in one of our nine neoplastic lesions
and none of our seven reactive lesions stained diffusely
for Leu-1. Knowles and Jacobiec have observed Leu-1
staining in 40% (6/15) of ocular B-cell lymphomas.8
Immunologic staining is a useful adjunct in diagnostic
evaluation of atypical lymphocytic infiltrates of extranodal
sites. However, ourfindingssuggest that there is no clinical
difference between monoclonal and polyclonal groups.
Studies with more patients and longer follow-up are
needed to determine biologic behavior and resolve the
question of appropriate therapy in these patients.
Acknowledgments. The authors thank Phil Home and Rick Coffin
who prepared the photomicrographs and Margaret Beers who typed the
manuscript.
References
FIG. 4. Immunoglobulin light chain stains. Same case as Figure 2. A
(left). Kappa stain shows many positive cells Kappa immunoperoxidase
(X192). B (right). Lambda stain also shows many reactive cells. Lambda
immunoperoxidase (XI92).
in separation of reactive from neoplastic lesions. They
showed that cases with less than 30% T-cells were almost
exclusively lymphoma, while those with greater than 40%
T-cells usually had polyclonal B-cell populations. While
ourfindingsgenerally support thosefindings,the overlap
between our two groups precluded diagnostic value in an
individual case. In tissue section staining, the distribution
of T cells may be more helpful than their percentage.
It has been suggested that the ratio of T helper/T suppressor lymphocytes is increased in reactive compared
with neoplastic ocular infiltrates.7 We, however, have
1. Harris NL, Bhan AK, Pilch BZ, Goodman ML: Immunohistologic
diagnosis of orbital lymphoid infiltrates. Lab Invest (Abstract)
1983; 48(1):33A
2. Jacobiec FA, Iwamoto T, Knowles DM: Ocular adnexal lymphoid
tumors. Correlative ultrastructural and immunologic marker
studies. Arch Ophthalmol 1982; 100:84-98
3. Knowles DM, Halper JP, Jacobiec FA: The immunologic characterization of 40 extranodal lymphoid infiltrates. Usefulness in
distinguishing between benign pseudolymphoma and malignant
lymphoma. Cancer 1982; 49:2321-2335
4. Knowles DM, Jacobiec FA: Orbital lymphoid neoplasms. A clinicopathologic study of 60 patients. Cancer 1980; 46:576-589
5. Knowles DM, Jacobiec FA: Quantitative determination of T cells
in ocular lymphoid infiltrates. An indirect method for distinguishing between pseudolymphomas and malignant lymphomas.
Arch Ophthalmol 1981; 99:309-316
6. Knowles DM, Jacobiec FA: Ocular adnexal lymphoid neoplasms.
Clinical, histopathologic, electron microscopic, and immunologic
characteristics. Hum Pathol 1982; 13:148-162
7. Knowles DM, Jacobiec FA: Identification of T lymphocytes in ocular
adnexal neoplasms by hybridoma monoclonal antibodies. Am
J Ophthalmol 1983; 95:233-242
8. Knowles DM, Jacobiec FA, Wang CY: The expression of surface
antigen Leu-1 by ocular adnexal lymphoid neoplasms. Am J
Ophthalmol 1982; 94:246-254