Intraocular lymphoma: diagnostic approach and immunophenotypic findings in vitrectomy specimens.
Abstract: * Context.--Diagnosis and classification of primary intraocular lymphoma can be challenging because of the sparse cellularity of the vitreous specimens.

Objective.--To classify and clinically correlate intraocular lymphoma according to the World Health Organization (WHO) classification by using vitrectomy specimens.

Design.--Clinical history, cytologic preparations, flow cytometry reports, and outcome of 16 patients diagnosed with intraocular lymphoma were reviewed.

Results.--The study group included 10 women and 6 men. The mean age of the patients was 63 years (range, 19-79 years). Eleven patients had central nervous system involvement and 6 patients had systemic involvement. All cases were adequately diagnosed and classified according to the WHO classification by using combination of cytologic preparations and 4-color flow cytometry with a limited panel of antibodies to CD19, CD20, CD5, CD10, and [kappa] and [lambda] light chains. The cases included 9 primary diffuse large B-cell lymphomas of the CNS type; 2 diffuse large B-cell lymphomas, not otherwise specified; 1 extranodal, low-grade, marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT); 1 precursor B-lymphoblastic lymphoma; and 3 peripheral T-cell lymphomas, not otherwise specified. Of note, all 11 cases of diffuse large B-cell lymphoma were [CD10.sup.-]. All the patients received systemic chemotherapy and radiation therapy. Only 4 patients were free of disease at last follow-up (range, 18 months to 8 years), with severe visual loss.

Conclusions.--Intraocular lymphoma cases can be adequately classified according to the WHO classification. Diffuse large B-cell lymphoma, [CD10.sup.-] and most likely of non-germinal center B-cell-like subgroup, is the most common subtype of non-Hodgkin lymphoma in this site, in contrast to ocular adnexal lymphoma for which MALT lymphoma is the most common subtype.

(Arch Pathol Lab Med. 2009;133:1233-1237)
Article Type: Clinical report
Subject: Lymphomas (Identification and classification)
Lymphomas (Diagnosis)
Immunopathology (Research)
Phenotype (Identification and classification)
Authors: Raparia, Kirtee
Chang, Chung-Che "Jeff"
Chevez-Barrios, Patricia
Pub Date: 08/01/2009
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 College of American Pathologists ISSN: 1543-2165
Issue: Date: August, 2009 Source Volume: 133 Source Issue: 8
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 230247108
Full Text: Primary intraocular lymphoma (PIOL), considered part of primary central nervous system lymphoma (PCNSL), is a rare type of non-Hodgkin lymphoma. Primary intraocular lymphoma is a unique, malignant lympho-proliferation because of its involvement with an immune-privileged site including the retinal pigment epithelium, retina, vitreous, and optic nerve. The incidence of intraocular lymphoma, like that of other lymphomas, has increased in the past 15 years. (1)

Frequently masquerading as a uveitis, PIOL is often misdiagnosed for intraocular inflammation and is treated with corticosteroids or, infrequently, for a viral retinitis and is treated with antiviral medication. (1) Prompt diagnosis of PIOL is imperative because consultation with a neurooncologist and initiation of chemotherapy and/or radiation therapy can extend the patient's life. Furthermore, most cases of PIOL eventually involve the brain (PCNSL) and patients have a poor prognosis. (1)

Diagnosis and classification of intraocular lymphoma can be challenging because of the sparse cellularity and the characteristics of the vitreous specimens. The primary goal of this study is to examine if the integration of clinical, cytologic, and flow cytometric immunophenotyping (FCI) findings may help in classifying the intraocular lymphoma according to the World Health Organization (WHO) classification by using the approach of triaging the specimen upon its arrival to the laboratory.

MATERIALS AND METHODS

Patients

We retrospectively reviewed cytologic preparations from 1999 to 2006 of 161 consecutive diagnostic vitrectomy specimens including vitreous taps and washings. In addition, we reviewed clinical history, cytologic preparations, flow cytometry reports, disease course, and outcome of 16 patients diagnosed with intraocular lymphoma in accordance with Institutional Review Board guidelines and approval.

Cytology Specimens

A standard 3-port pars plana 20-gauge probe approach was used in all cases to obtain a complete core vitrectomy specimen. Undiluted vitreous was first obtained for cytologic analysis. A dilute specimen was then obtained by using the infuser and cutting the remaining vitreous. Vitreous wash fluid, collected in the vitrector cassette, was sometimes used for flow cytometry analysis. To prevent cell death, 1 to 2 ml of vitreous specimen was expediently placed into a tube containing Roswell Park Memorial Institute culture medium for a total volume of 2 to 5 ml of fluid.

Liquid-based cytologic preparations from vitrectomy specimens were prepared for Papanicolaou staining in each case. If liquid-based preparation was suggestive of or cytologically positive for lymphoma, the remaining specimen was sent for FCI. We also submitted specimen for flow cytometry if the clinical suspicion for lymphoma was high and the specimen was composed mainly of lymphocytes.

Sensitivity and specificity of the cytologic diagnosis, which was compared with the immunophenotypic characteristics and final diagnosis, was also obtained. We grouped the cytologic diagnosis of highly suspicious and/or positive for lymphoma in the positive category. The cytologic specimens negative for malignancy were classified in the negative category.

Flow Cytometry and Immunohistochemistry on Cell Block

The specimen was immediately processed and a limited panel of antibodies was used for immunophenotyping including antibodies to CD3, CD30, CD19, CD20, CD5, CD10, and [kappa] and [lambda] light chains. In suspected cases of T-cell lymphoma, markers such as CD2, CD4, CD7, and deoxynucleotidyl transferase (TdT) were used. The specimen was considered of adequate cellularity if it contained 0.1 X [10.sup.6] cells/mL. Cell block was made from the vitreous washings in 2 cases in which additional specimen was left over after it was sent for flow cytometry. Vitreous washing specimen or undiluted aspirate was filtered through a 100-[micro]m filter. Both specimen types were washed in Cellgro (Mediatech, Herndon, Virginia) and resuspended in 1% newborn calf serum (Cambrex, Walkersville, Maryland) with 1X phosphate-buffered saline (PBS; Cambrex). Cell counts were performed on a Sysmex KX-21 (Sysmex America, Mundelein, Illinois). The cells (1 X [10.sup.6]) were incubated with newborn calf serum in PBS at 37[degrees]C for 10 minutes, lysed for 5 minutes at room temperature with 1X ammonium chloride, and then washed in 1X PBS. The specimens were incubated with the antibodies conjugated with fluorescein isothiocyanate, phycoerythrin, peridinin chlorophyll protein, and allophycocyanin for 15 minutes at 4[degrees]C; washed in 1X PBS; and resuspended in 0.5% paraformaldehyde in 1X PBS.

A 4-color FACSCalibur flow cytometer with CellQuest software (Becton Dickinson, San Jose, California) was used to acquire flow cytometric data. Ten thousand cells were acquired for analysis. The instrument setting was established daily by using the normal peripheral blood and Calibrite beads (Becton Dickinson) and the procedures described previously. Paint-A-Gate software (Becton Dickinson) was used for flow cytometric data analysis. Specific cell populations were identified by using cluster analysis, antigen expression patterns, and forward and side angle light-scatter characteristics. Nonviable cells and debris were removed on the basis of forward and orthogonal light-scatter characteristics.

Polymerase Chain Reaction and Gene Rearrangement

T-cell receptor gene rearrangement studies by polymerase chain reaction (PCR) were done in 2 cases on paraffin-embedded tissue to evaluate the clonality of T cells. This test was performed by a reference laboratory (ARUP Laboratories, Salt Lake City, Utah).

The combination of cytologic and clinical findings, FCI, and molecular data served as bases to diagnose and subclassify PIOL according to the latest WHO classification. (2)

RESULTS

Patients

The study group included 10 women and 6 men. The mean age of the patients was 63 years (range, 19-79 years). Eleven patients had central nervous system (CNS) involvement before onset of ocular symptoms and 6 patients had systemic involvement before the time of ocular presentation. Thirteen patients had bilateral involvement of the eyes. The 6 patients with systemic involvement included 1 patient with history of acute lymphoblastic leukemia, 2 with a history of T-cell lymphoma, 1 with a history of mucosa-associated lymphoid tissue (MALT) lymphoma, and 2 with a history of diffuse large B-cell lymphoma. Because all the diffuse large B-cell lymphoma cases were bilateral--7 of which had previous CnS involvement and only 2 of which showed previous systemic involvement--they were classified as 9 cases of primary diffuse large B-cell lymphoma of the CNS and 2 cases were classified as diffuse large B-cell lymphoma, not otherwise specified, according to the latest WHO classification.

The clinical findings are summarized in Table 1. All patients presented with symptoms of vitritis.

Cytology

The liquid-based preparation showed large round or oval nuclei (frequently segmented and often containing prominent nucleoli), occasionally with irregular nuclear membranes, surrounded by scant basophilic cytoplasm (Figure 1). Cytologic examination of the vitreous specimen can be difficult because there may be a relative lack of lymphoma cells compared to the reactive inflammatory cells. In addition, background of necrotic debris can make it difficult to evaluate the atypical lymphoma cells, but if present, it aids in the diagnosis of lymphoma or at least raises a strong suspicion for lymphoma. Of the 16 cases, 7 were highly suggestive of lymphoma, 7 were positive for lymphoma, and 2 were negative by cytologic examination. Immunohistochemical stains with a lymphoma panel showed that CD3 and CD30 (T-cell lymphoma) were positive in 1 case on the cell block, confirming T-cell lymphoma. Cytology was very sensitive and yielded a diagnosis of either suggestive of or positive for lymphoma in 14 of 16 cases (87.5%).

[FIGURE 1 OMITTED]

Flow Cytometry

Although specimen cellularity was adequate in only 7 cases, flow cytometry was essential for classifying these lymphomas with the limited panel of antibodies. Our results included 9 cases of primary diffuse large B-cell lymphoma of the CNS type; 2 cases of diffuse large B-cell lymphoma, not otherwise specified; 1 extranodal, lowgrade, marginal zone B-cell lymphoma of MALT type; 1 precursor B-lymphoblastic lymphoma; and 3 T-cell lymphomas, not otherwise specified. Of note, all 11 cases of diffuse large B-cell lymphoma were [CD10.sup.-] (Figure 2, A and B).

[FIGURE 2 OMITTED]

Polymerase Chain Reaction

T-cell receptor gene rearrangement studies with the PCR performed in 2 cases of T-cell lymphoma showed clonally rearranged T-cell receptor in both cases, thus supporting the diagnosis of T-cell lymphoma (cases 11 and 16; Table 2).

All cases were adequately diagnosed and classified according to the WHO classification by using a combination of clinical findings, cytologic preparations, and 4-color flow cytometry with a limited panel of antibodies to CD19, CD20, CD5, CD10, and [kappa] and [lambda] light chains and/ or molecular techniques (Table 2).

Outcome

The follow-up period for these patients ranged from 18 months to 8 years (Table 1). All patients received either systemic chemotherapy, radiation therapy, or both. One patient underwent surgery for the brain lesions and 2 also had a bone marrow transplant. Eight patients died of the disease and 4 were lost to follow-up. Only 4 patients are free of disease at last follow-up, 3 of whom had uninvolved CNS at time of presentation and all of whom have severe loss of vision.

COMMENT

Primary intraocular lymphoma is a subset of primary central nervous system lymphoma (with predilection for intraocular regions that sit behind the blood-retina barrier) and often masquerades as uveitis.1 It has been estimated that the incidence of PCNSL has risen from 0.04 in 100000 to 0.3 in 100000 in the immunocompetent population. (3) Although PIOL can have a T-cell origin, it is predominately a non-Hodgkin lymphoma of B-cell origin. (3) Although it initially presents intraocularly, 60% to 85% of patients with PIOL will have CNS lymphoma. (4) In our series, 11 patients (69%) had CNS involvement before onset of ocular symptoms.

Cytologic examination of vitreous biopsy specimens has been used to make a diagnosis of PIOL since the mid1970s for cases that were initially diagnosed as uveitis. (5,6) Vitreous specimens may not always contain neoplastic cells and, thus, be negative for the diagnosis of PIOL. This is especially true if there is minimal vitreous involvement by the lymphoid cells, if the cells have degenerated, or if the vitreous component is reactive while the retinal/choroidal lesion is neoplastic; thus, this represents a sampling issue. (7) At times the quality of the cytology is poor, which makes it unfeasible to render a diagnosis. Davis and colleagues (8) submitted vitrectomy specimens from 27 patients, suspected of having an intraocular malignancy, to analysis by cytology and showed that a final diagnosis of lymphoma was achieved in 13 patients, with cytology yielding 4 true-positive lymphoma cases (a sensitivity of 31%) and no false-positive cases. In our series, cytology was very sensitive in yielding a diagnosis of either suggestive of or positive for lymphoma in 14 of 16 cases (87.5%).

Thus, while a positive cytologic evaluation was certain for lymphoma, a negative evaluation does not completely rule out the possibility of intraocular lymphoma. When suspicion for PIOL still runs high, it is essential to perform immunophenotypic analysis by using laboratory tests such as immunohistochemistry, flow cytometry, or molecular analysis by PCR, which can be done on cytologic specimens. (9,10) Recently, Gonzales and Chan (11) recommended external transcleral chorioretinal and transvitreal retinochoroidal biopsies when diagnostic vitrectomy is inconclusive but when a very high suspicion for PIOL remains. Sharara et al (12) used flow cytometric immunophenotyping, in conjunction with histologic analysis and immunohistochemistry, for classifying the tumors of 43 patients suspected of having ocular adnexal lymphoid proliferations. They concluded that FCI refines the histologic diagnosis and classification. Similar to their results, FCI was helpful in diagnosing and accurately classifying the intraocular lymphomas in all 16 cases in our series.

The most common primary lymphoma arising from ocular tissues is a diffuse large B-cell lymphoma (DLBCL). (4,13) In our series, DLBCL was the most frequent intraocular lymphoma (11 of 16 cases). The most common primary lymphoma subtype occurring in the ocular adnexa is the low-grade, malignant, extranodal, marginal zone B-cell lymphoma of MALT type. (13) We did not include any ocular adenexal lymphoma cases in this series and only 1 of 16 cases of PIOL was extranodal, marginal zone B-cell lymphoma.

In our current series, all 11 cases of diffuse large B-cell lymphoma were [CD10.sup.-]. Hans et al (14) showed that immunostains (CD10, BCL6, and MUM1) can be used to determine the germinal center B-cell-like (GCB) and non-GCB subtypes of DLBCL and to predict survival, similar to the results with cDNA microarray. (14) They defined the GCB subtype of diffuse large B-cell lymphoma as expressing CD10 and/or BCL6 without expressing MUM1 and the non-GCB subtype as negative for CD10 and BCL6 or as positive for MUM1 and BCL6, but without expressing CD10. Patients with GCB DLBCL had a significantly better overall survival than those with activated B-cell-like DLBCL. (15,16) Although we did not perform BCL6 and MUM1 assays on any of the 11 cases because of limited sample, our series strongly suggests that PIOL belongs to the activated B-cell-like DLBCL (non-GCB group). This may have contributed to the poor prognosis observed in our patients. In agreement with our results, Coupland et al (17) and Bhagavathi et al (18) showed that PIOL and primary CNS DLBCL has a non-germinal center B-cell phenotype in most cases and is associated with poor prognosis.

In conclusion, intraocular lymphoma cases can be adequately classified according to the WHO classification. Diffuse large B-cell lymphoma is the most common subtype of non-Hodgkin lymphoma in this site, in contrast to ocular adnexal lymphoma in which MALToma (MALT lymphoma) is the most common subtype. Virtually all diffuse large B-cell lymphomas in this site lack CD10 expression, suggesting a non-germinal center B-cell origin and hence a poor prognosis. Early disease detection, accurate staging, and response evaluation by using a combination of different diagnostic modalities including radiology, molecular, and flow cytometry studies are needed. However, improved targeted therapy remains the ultimate goal to provide a better prognosis for these patients. Further studies with a larger sample size are required to investigate the molecular mechanisms of intraocular lymphoma and new novel therapeutic drugs.

References

(1.) Chan CC, Buggage RR, Nussenblatt RB. Intraocular lymphoma. Curr Opin Ophthalmol. 2002;13:411-418.

(2.) Swerdlow SH, Campo E, Harris NL, et al, eds. 4th ed. Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; 2008. World Health Organization Classification of Tumours; vol 2.

(3.) Cote TR, Manns A, Hardy CR, Yellin FJ, Hartge P. Epidemiology of brain lymphoma among people with or without acquired immunodeficiency syndrome. AIDS/Cancer Study Group. J Natl Cancer Inst. 1 996;88:675-679.

(4.) Coupland SE, Heimann H, Bechrakis NE. Primary intraocular lymphoma: a review of the clinical, histopathological and molecular biological features. Graefes Arch Clin Exp Ophthalmol. 2004;242:901-913.

(5.) Akpek EK, Ahmed I, Hochberg FH, et al. Intraocular-central nervous system lymphoma: clinical features, diagnosis, and outcomes. Ophthalmology. 1999; 106:1805-1810.

(6.) Michels RG, Knox DL, Erozan YS, Green WR. Intraocular reticulum cell sarcoma: diagnosis by pars plana vitrectomy. Arch Ophthalmol. 1975;93:1331 1335.

(7.) Blumenkranz MS, Ward T, Murphy S, Mieler W, Williams GA, Long J. Applications and limitations of vitreoretinal biopsy techniques in intraocular large cell lymphoma. Retina. 1992;12(suppl 3):S64-S70.

(8.) Davis JL, Miller DM, Ruiz P. Diagnostic testing of vitrectomy specimens. Am J Ophthalmol. 2005;140:822-829.

(9.) Chan CC, Wallace DJ. Intraocular lymphoma: update on diagnosis and management. Cancer Control. 2004;11:285-295.

(10.) Buggage RR, Chan CC, Nussenblatt RB. Ocular manifestations of central nervous system lymphoma. Curr Opin Oncol. 2001;13:137-142.

(11.) Gonzales JA, Chan CC. Biopsy techniques and yields in diagnosing primary intraocular lymphoma. Int Ophthalmol. 2007;27:241-250.

(12.) Sharara N, Holden JT, Wojno TH, Feinberg AS, Grossniklaus HE. Ocular adnexal lymphoid proliferations: clinical, histologic, flow cytometric, and molecular analysis of forty-three cases. Ophthalmology. 2003;110:1245-1254.

(13.) Coupland SE, Damato B. Lymphomas involving the eye and the ocular adnexa. Curr Opin Ophthalmol. 2006;17:523-531.

(14.) Hans CP, Weisenburger DD, Greiner TC, et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 2004;103:275-282.

(15.) Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000;403:503 511.

(16.) Rosenwald A, Wright G, Chan WC, et al. The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med. 2002;346:1937-1947.

(17.) Coupland SE, Loddenkemper C, Smith JR, et al. Expression of immunoglobulin transcription factors in primary intraocular lymphoma and primary central nervous system lymphoma. Invest Ophthalmol Vis Sci. 2005;46:3957-3964.

(18.) Bhagavathi S, Sharathkumar A, Hunter S, et al. Activated B-cell immunophenotype might be associated with poor prognosis of primary central nervous system lymphomas. Clin Neuropathol. 2008;27:13-20.

Kirtee Raparia, MD; Chung-Che (Jeff) Chang, MD, PhD; Patricia Chevez-Barrios, MD

Accepted for publication March 26, 2009.

From the Department of Pathology, The Methodist Hospital, Houston, Texas.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Patricia Chevez-Barrios, MD, Department of Pathology, The Methodist Hospital, 6565 Fannin Street, Houston, TX 77030 (e-mail: pchevez-barrios@tmhs.org).
Table 1. Demographics of Patients With Primary Intraocular Lymphoma

Case No.   y/Sex            History            CNS Involvement

   1       67/F    Cutaneous T-cell lymphoma       Present
   2       75/F    Negative                        Present
   3       76/F    Negative                        Present
   4       69/F    Negative                        Absent
   5       70/M    Negative                        Present
   6       79/F    Negative                        Present
   7       69/F    Negative                        Present
   8       52/M    Negative                        Absent
   9       64/F    Negative                        Present
   10      73/M    Negative                        Present
   11      57/M    Negative                        Present
   12      49/F    Negative                        Present
   13      71/F    Negative                        Absent
   14      19/F    ALL                             Absent
   15      46/F    Negative                        Present
   16      69/M    Negative                        Absent

Case No.   Diagnosisa                Treatment         Follow-up

   1       T-cell                Chemotherapy, XRT   None
   2       B-cell                Chemotherapy, XRT   Died
   3       B-cell                Chemotherapy        None
   4       B-cell                Chemotherapy, XRT   Living (44 mo)
   5       MALToma               Chemotherapy, XRT   Died
   6       B-cell                Chemotherapy, XRT   Died
   7       B-cell                Chemotherapy        Died
   8       B-cell                Chemotherapy, XRT   Died
   9       B-cell                XRT                 None
   10      B-cell                Chemotherapy, XRT   Died
   11      T-cell                XRT                 Died
   12      B-cell                Chemotherapy, XRT   Living (42 mo)
   13      B-cell                XRT                 Died
   14      Precursor B-cell LL   Chemotherapy, XRT   Living (8 y)
   15      T-cell                Chemotherapy, XRT   None
   16      B-cell                Chemotherapy, XRT   Living (18 mo)

Abbreviations: ALL, acute lymphoblastic leukemia; CNS, central
nervous system; LL, lymphoblastic leukemia; MALToma, mucosa-associated
lymphoid tissue lymphoma; XRT, radiation therapy.

(a) Lymphoma, unless otherwise stated.

Table 2. Different Diagnostic Techniques for the Diagnosis of
Intraocular Lymphoma

Case   Cytologic                    Flow Cytometry
No.    Preparations   Cellularity   Markers

 1     Liquid-        Low           CD4 (+), CD8 (+)
       based, cell
       block

 2     Liquid-        Adequate      CD19 (+), CD20 (+), [lambda]
       based                        chain (+); CD5 (-),
                                    CD10 (-), [kappa]
                                    chain (-)

 3     Liquid-        Scant         CD19 (+), CD20 (+), [lambda]
       based,                       chain (+); CD5 (-),
       cytospin                     CD10 (-), [kappa]
                                    chain (-)

 4     Liquid-        Adequate      CD19 (+), CD20 (+), [lambda]
       based,                       chain (+); CD5 (-),
       cytospin                     CD10 (-), [kappa]
                                    chain (-)

 5     Liquid-        Scant         CD19 (+), CD20 (+),
       based,                       CD5 (+), [kappa]
       cytospin                     chain (+); CD10 (-),
                                    [lambda] chain (-)

 6     Liquid-        Low           CDI9 (+), CD20 (+),
       based                        [kappa] chain (+);
                                    CD5 (-), CDI0 (-),
                                    [lambda] chain (-)

 7     Liquid-        Scant         CD19 (+), CD20 (+),
       based,                       [kappa] chain (+);
       cytospin                     CD5 (-), CD10 (-),
                                    [lambda] chain (-)

 8     Liquid-        Low           CD19 (+), CD20 (+), [lambda]
       based                        chain (+); CD5 (-),
                                    CD10 (-), [kappa]
                                    chain (-)

 9     Liquid-        Adequate      CD19 (+), CD20 (+),
       based,                       [kappa] chain (+);
       cytospin                     CD5 (-), CD10 (-),
                                    [lambda] chain (-)

 10    Liquid-        Scant         CD19 (+), CD20 (+),
       based,                       [kappa] chain (+);
       cytospin                     CD5 (-), CD10 (-),
                                    [lambda] chain (-)

 11    Liquid-        Scant         CD4 (-), CD8 (-)
       based

 12    Liquid-        Adequate      CD19 (+), CD20 (+),
       based                        CD22 (+), [kappa]
                                    chain (+); CD5 (-),
                                    CD10 (-), [lambda]
                                    chain (-)

 13    Cytospin       Low           CD19 (+), CD20 (+),
                                    [kappa] chain (+);
                                    CD5 (-), CD10 (-),
                                    [lambda] chain (-)

 14    Liquid-        Adequate      CD19 (+), CD20 (+),
       based, cell                  CD10 (+), CD34 (+),
       block                        TdT (+); Ig light
                                    chain (-)

 15    Liquid-        Adequate      CD19 (+), CD20 (+),
       based                        [kappa] chain (+);
                                    CD5 (-), CD10 (-),
                                    [lambda] chain (-)

 16    Liquid-based   Adequate      CD2 (+), CD3 (+),
                                    CD7 (+); CD5 (-),
                                    CD4 (-), CD8 (-)

Case   Immunohistochemistry
No.    and PCR

 1     Immunohistochemistry
       CD3 (+), CD30 (+)
       on cell
       block

 2

 3

 4

 5

 6

 7

 8

 9

 10

 11    Gene
       rearrangement

 12

 13

 14

 15

 16    Gene
       rearrangement

Abbreviations: Ig, immunoglobulin; PCR, polymerase chain reaction;
TdT, terminal deoxynucleotidyl transferase; (+), positive;
(-), negative.
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