Monoclonal antibodies reactive with normal and neoplastic T cells in paraffin sections

Monoclonal Antibodies Reactive with Normc. and Neoplastic T Cells in Paraffin Sections C. S. NG, MB, BS, MRCPATH,* JOHN K. C. CHAN, MB, BS,w P. K. HUI, MB, BS, MD,* AND STEPHENT. H. LO, AIMLS* Without fresh or frozen tissue, it previously has been impossible to confirm the T-cell nature of reactive or neoplastic lymphoid cells. The availability of antibodies reactive with T cells in para f f i n s e c t i o n s n o w allows r e t r o s p e c t i v e analysis of a large n u m b e r of cases. Two commercially available monoclonal antibodies, MT1 and MT2, were tested for their reactivities with T cells in a wide r a n g e of formalin-fixed, p a r a f f i n - e m b e d d e d tissues, including 130 cases of immunologically characterized lymphoma. I n reactive lymph nodes, MTI stained the T-cell areas, whereas MT2 stained both the T-cell areas and mantlezone B lymphocytes. MT1 stained 38 of 55 T-cell lymphomas (69.1%; 94.7% of cases from one hospital that used a shorter fixation time, and 55.6% of cases from another hospital that used a longer fixation time). MT2 stained only 6 (10.9%) of the T-cell lymphomas. Among the 74 cases of B-cell lymphoma, 3 (4.0%) were stained by MT1 a n d 30 (40.5%) by MT2. MT1 was also reactive with 3 of 4 cases of granulocytic sarcoma, as expected from its reactivity with normal granulocytes. Neither MT1 n o r MT2 stained Reed-Sternberg cells or their variants in Hodgkin's disease. We conclude that MT1 is a valuable marker for T cells, particularly w h e n used with a panel of antibodies reactive with B cells in paraffin sections. MT2 is of limited value because of its cross-reactivity with many B-cell l y m p h o m a s . HuM PATHOL 19:295--303, 1988.

I m m u n o p h e n o t y p i c characterization of lymphoid cells and lymphomas has been greatly facilitated by the availability of a wide panel of monoclonal antibodies. Most of these antibodies work only on fresh or frozen tissue. If only fixed tissue is available, the B cells may be confirmed in some cases by the demonstration of cytoplasmic immunoglobulin or J chain, 1-5 or by using the recently available antibodies such as LN1, LN2, MB1, MB2, and Ki. B35-12 Confirmation of the T-cell nature of lymphoid cells in paraffin sections has not been possible until recently. T h e m o n o c l o n a l antibodies U C H L 1 and MT18-1~ have been r e p o r t e d to be excellent markers for T cells in paraffin sections. In the series reported by Norton and Isaacson, 14 20 (83%) of 24 T-cell lymphomas were stained by MT1. Similar findings have also been reported in smaller series. 9a~ In this study, MT1 and MT2 were applied to a large From the *Department of Morbid Anatomy, Prince of Wales Hospital; ?Institute of Pathology, Queen Elizabeth Hospital; and the :~Department of Pathology, Kwong Wah Hospital, Hong Kong. Revision accepted for publication 1 J u n e 1987. Supported in part by the Croucher Foundation HK. Address correspondence and reprint requests to Dr. Ng: Dep a r t m e n t of Pathology, Caritas Medical Center, Shamshuipoo, Kowloon, Hong Kong. Keywords: B cells; lymphoid cells; lymphoma; monoclonal antibodies; MT1; MT2; T cells. 9 W.B. Saunders Company 0046-8177/88 $0.00 + .25

n u m b e r of cases of neoplastic and nonneoplastic lymphoproliferative lesions and a range of normal tissues to determine their value for application in paraffin sections. MATERIALSAND METHODS

Monoclonal Antibodies MT1 and M T 2 were obtained f r o m Biotest Diagnostics, Dreieich, West Germany. According to the manufacturer, MT1 is an IgG1 murine monoclonal antibody reactive with 110- and 100-kilodahon antigens on T cells and histiocytes. MT2 is an IgG1 antibody reactive with 200- and 190-kilodalton antigens on mature T cells and B cells of mantle and marginal zones.

Tissue Specimens The cases were retrieved from the surgical pathology and autopsy files of the Prince of Wales Hospital and Q u e e n Elizabeth Hospital, H o n g Kong. The tissue was fixed in buffered formalin and embedded in paraffin. Four-micron sections were cut for routine histologic and immunohistochemical studies. Lymphoid Tissue. Lymph nodes showing reactive changes were chosen, including 10 cases of nonspecitric reactive hyperplasia, 5 cases of dermatopathic lymphadenopathy, 5 cases of histiocytic necrotizing lymphadenitis, 5 cases of tuberculous lymphadenitis, and 2 cases of histiocytosis X. Paraffin blocks of thymus, spleen, and marrow were also studied. Non-Hodgkin's Lymphoma. One hundred thirty cases of non-Hodgkin's lymphoma that had been immunologically characterized with a panel of monoclonal antibodies on frozen tissue, as previously described, were chosen, tS-t7 They included 74 B-cell, 55 T-cell, and 1 biphenotypic lymphoma. Hodgkin's Disease. T w e n t y - t h r e e cases o f Hodgkin's disease (3 of lymphocyte-predominant, 10 of nodular-sclerosing, and 10 of mixed-cellularity subtype) were selected. Granulocytic Sarcoma. Four cases of granulocytic sarcoma were chosen for this study. Histiocytic Sarcoma. One case of histiocytic sarcoma (MO1 +, MO2 +, EBM11 +) was included in this study. Organs. Various normal tissues, including the skin, tongue, salivary gland, stomach, small and large intestines, pancreas, liver, gall bladder, nasal mucosa, kidney, thyroid, adrenal, breast, ovary, fallopian 295

HUMANPATHOLOGY Volume19, No, 3 [March 1988] TABLE t.

tube, uterus, testis, prostate, heart, lung, and brain, were studied.

Reactivities of MT~ and MT2 in Normal and Reactive Lymphoid Tissues* MT1

Immunohistochemical Method

T h e a v i d i n - b i o t i n - p e r o x i d a s e c o m p l e x (ABC) t e c h n i q u e was used. is Briefly, the paraffin sections were d e w a x e d in xylol and b r o u g h t to alcohol. End o g e n o u s peroxidase activity was blocked with 0.03% h y d r o g e n p e r o x i d e in m e t h a n o l f o r 25 m i n u t e s . T r y p s i n i z a t i o n was c a r r i e d o u t f o r 30 m i n u t e s at 37~ T h e sections were incubated with the m o n o clonal antibodies (1:5 dilution) overnight at 4~ T h e sections were t h e n incubated with biotinylated antim o u s e i m m u n o g l o b u l i n for 30 minutes and ABC for 45 minutes (Dakopatts, C o p e n h a g e n , Denmark), with t h o r o u g h washing between the steps. T h e color reaction was d e v e l o p e d with 0.03% h y d r o g e n p e r o x i d e and 0.6% diaminobenzidine. H e m a t o x y l i n was used for counterstaining. In cases o f T-cell l y m p h o m a not stained by MT1, i m m u n o s t a i n i n g with MT1 was carried out on 6-~xm f r o z e n sections as well. Frozen sections f r o m five randomly selected cases o f B-cell l y m p h o m a were similarly i m m u n o s t a i n e d with MT1.

RESULTS Lymphoid Tissue

T h e reactivity patterns o f the various lymphoid tissues are shown in table 1. Positive staining by both M T 1 a n d M T 2 was e x p r e s s e d as cell m e m b r a n e staining. MT1 stained the T-cell areas, whereas MT2 stained the T-cell areas as well as the m a n t l e and marginal zones o f l y m p h o i d follicles (figs. 1 and 2). N e u t r o p h i l s also exhibited strong staining with MT1 but not MT2. I n histiocytic n e c r o t i z i n g l y m p h a d e n i t i s , the diagnostic "necrotizing" foci contained an a b u n d a n c e o f activated T cells for study. 19,2~T h e large activated l y m p h o i d cells within these foci stained strongly with M T 1 (fig. 3), b u t only rare cells stained weakly with MT2. T h e epithelioid cells and Langhans' giant cells in tuberculous foci showed variable weak cytoplasmic a n d / o r m e m b r a n e staining with MT1. Interdigitating r e t i c u l u m cells a n d L a n g e r h a n s ' cells in reactive l y m p h nodes and the two cases o f histiocytosis X were not stained by MT1. In the b o n e m a r r o w , megakaryocytes showed variable staining with M T 1. Various Organs

T h e various organs studied showed u n i f o r m l y negative staining with M T 1 and MT2. Non-Hodgkin's Lymphomas

MT2

Reactive lymph node

Lymphocytes in germinal centers Mantle zone lymphocytes Inter follicular lymphocytes P l a s m a cells Tingible body macrophages S i n u s hisfiocytes Interdigitating r e t i c u l u m cells Neutrophils Vascular endothelium Thymus Cortical thymocytes Medullary thymocytes D e n d r i t i c cells (S- 100 p o s i t i v e ) in m e d u l l a Hassall's corpuscles Spleen Lymphocytes in germinal centers Mantle-zone lymphocytes Marginal-zone lymphocytes Periarteriolar

lymphocytes

+ -+ + + (S)

+ + (S)

- -+ + (S)

+ + + + (S)

+ + --+ + + + + (s)

+ + + --+ + + + + (s)

-

-

+ + + + (S)

-

+ + + + (S)

+ + (W)

+ + + (S)

+ + + (M)

-

+ (M)

+ --+ + + (S)

-

+ + + + (S)

-

+ + + + (S)

+++-+ ++++ (S)

+ + + + (S)

Dermatopathic

lymphadenopathy Interdigitating reticulum/ Langerhans cells Phagocytic histiocytes Histiocytic necrotizing lymphadenitis (Kikuchi's disease) Activated lymphoid cells (majority T cells) Phagocytic histiocytes Tuberculous lymphadenifis Epithelioid histiocytes Langhans' giant cells Histiocytosis X Langerhans' cells Bone marrow Granulocytic cells Erythroid cells Megakaryocytes

I

+ + + + (s)

-~+

---+ + + + (M) ---+ + + (M)

-

(W)

+ + + + (S) I

+ + + + (M)

* P e r c e n t a g e o f p o s i t i v e cells q u a n t i t a t e d as follows: - = 0%; + = 1-10%; ++ = 10-25%; +++ = 25-50%; ++++ = >50%. Intensity of staining: W = weak; M = moderate; S = strong.

t h a n 10% o f unequivocally neoplastic cells were positive. All 4 T-lymphoblastic l y m p h o m a s were stained by MT1. T h e staining was diffuse and strong except in 1 case. For p e r i p h e r a l T-cell lymphomas, the posi-

T h e results of staining with MT1 and M T 2 are shown in tables 2 and 3. Most o f the positive cases showed m e m b r a n e staining in over 25% o f the neoplastic cells (figs. 4 and 5). In a minority o f cases, less

296

T-CELL-REACTIVEMONOCLONAL ANTIBODIES[Ng et al9

.

_

_ .

_

.

,

.

.L ~

'

1

.

1

FIGURE 1. Lymph node showing reactive hyperlolasia, with the germinal centers being marked by asterisks. A, Immunostaining with MTI. Only the paracortical cells are stained. B, Immunostaining with MT2. Both the paracoffical cells and mantle-zone lymphocytes are stained, (ABC immunoperoxidase method with hematoxylin counterstain, x 75,]

tivity rate with M T I was 66.7% (fig. 5). However, when the cases of the two contributing hospitals were analyzed separately, the positivity rate for the hospital using a shorter fixation time (8 to 24 hours) was 93.8%, in contrast to 54.3% for the other hospital that used a longer fixation time (8 to 36 hours). No correlation of MT1 staining was seen with the expression of specific T-cell antigens CD1 to CD8, the Hodgkin's disease-associated antigen Ki-1, the activated T-cell markers I2/HLA-DR and interleukin-2 receptor, or natural killer cell markers NKH1, Leu 7, and Leu l lb. Among the 17 negative cases, 13 showed staining of some reactive cells within the tumor, whereas 4 cases showed completely negative staining9 However, all these 17 cases showed strong staining with MT1 on frozen sections (fig. 6). Among the 74 B-cell lymphomas, MT1 stained 2 cases of small lymphocytic lymphoma and 1 case of Burkitt's lymphoma. The cell membrane staining was strong in virtually all neoplastic cells in 1 case of small lymphocytic lymphoma, but weak and focal in the other 2 cases of lymphoma. In the negative cases, small MTl-positive cells were scattered among the neoplastic cells9 The neoplastic cells of 5 cases of Bcell lymphoma were not stained by MT1 on frozen section. MT2 stained only 10.9% o f the T-cell lymphomas, and the staining was often focal and weak

(fig. 5). However, it also stained 30 cases (40.5%) of B-cell lymphoma. MT2 staining occurred in virtually all morphologic types of B-cell lymphoma, but most consistently in small lymphocytic and diffuse small cleaved cell lymphomas (fig. 7). The single case of mixed B- and T-cell lineage lymphoblastic lymphoma 21-24 (CD 1 -,CD2 +,CD3 +, CD4 +,CD5 +,CD7 +,CD8 +,CD 19 +,CD20 +,CD22 +) was stained by MT1 but not MT2.

Hodgkin's Disease R e e d - S t e r n b e r g cells and variants were not stained by MT1 or MT2. Reactive small cells staining with MT1 or MT2 were scattered throughout.

Granulocytic and Histiocytic Sarcomas As expected from the strong staining in granulocytic cells, three of the four cases of granulocytic sarcoma were stained with MT1. The case of histiocytic sarcoma was negative with both MT1 and MT2. DISCUSSION

For reliable immunophenotypic characterization o f l y m p h o i d cells, fresh or frozen tissue is required. 1,25 The major disadvantages, however, are 297

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FIGURE 2 [left]. The paracortical lymphocytes, but not the endothelial celrs, show membrane staining by MTI, [ABC immunoperoxidase method, x 750.] FIGURE 3 [right]. Histiocytic necrotizing lymphadenitis [Kikuchi's disease]. The activated lymphoid cerls, including those in mitosis, within the "necrotizing" foci show strong membrane staining by MT1. The phagocytes [arrows] are not stained. [ABC immunoperoxidase method. x 750.]

that the cytologic details are often suboptimal in cryostat sections and that immunophenotyping becomes impossible when only fixed tissue is available. Attempts have been made to apply the lineage-specific monoclonal antibodies to paraffin sections. Hsu et al. 25 have f o u n d that Leu 1 (CD 5) and BA1 (CD24) may be preserved in Bouin's-fixed tissue, whereas other lineage-specific antigens are uniformly masked or destroyed in paraffin-embedded tissue. Holgate et al.26 have found that with the use of periodate-lysine-paraformaldehyde (PLP) or PLP-dichromate for fixation, most monoclonal antibodies can be successfully applied to paraffin sections, provided TABLE 2.

that the immunogold-silver staining method is used. Stein et al. 27 have shown that monoclonal antibodies can be reliably applied to freeze-dried, paraffin-embedded sections. Tanaka et al. 28 and Sato et al. 29 have also successfully applied a wide range of lineage-specific antibodies on acetone-fixed, paraffin-embedded tissues. However, the techniques mentioned here require special processing, and tissue fixed in formalin can no longer be salvaged. Monoclonal antibodies reactive with T cells in formalin-fixed tissue have not become available until recently. 8-m,13a4 In the present study, two commercially available monoclonal antibodies, MT1 and

Reactivities with MTfl and MT2 in T-Cell Lymphomas* MT1

Lymphoblastic lymphoma Peripheral T-cell lymphoma Total

MT2

Hospital A

Hospital B

Total

Hospital A

Hospital B

Total

3/3 (100) 15/16 (93.8) 18/19 (94.7)

1/1 (100) 19/35 (54.3) 20/36 (55.6)

4/4 (100) 34/51 (66.7) 38/55 (69.1)

0/3 (0) 2/16 (12.5) 2/19 (10.5)

0/1 (0) 4/35 (11.4) 4/36 (11.1)

0/4 (0) 6/51 (11.8) 6/55 (10.9)

* Data given as the number of positive cases/number of cases tested (percentage in parentheses).

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T-CELL-REACTIVEMONOCLONAL ANTIBODIES[Ng et al.]

TABLE $. Reactivities of MT1 and MT2 in Malignant Lymphoproliferative Diseases Other Than TCell Lymphomas

marginal-zone B cells. It stains the activated T cells weakly or not at all.

No. of

Cases tested

MT 1-positive Cases

Application of MTI in Lymphomas

MT2-positive Cases

B-cell lymphomas

Small lymphocytic Chronic lymphocytic leukemia Plasmacytoid Follicular Small cleaved cell Mixed cell Large cell Diffuse Small cleaved cell Mixed cell Large cell Immunoblastic Lymphoblastic Burkitt's Total Biphenotypic lymphoma Histiocytic sarcoma Granulocytic sarcoma Hodgkin's disease

3 3

2 0

4 6 5

0 0 0

6 8 30 4 2 3 74

0 0 0 0 0 1 3 (4.1%)

1 1 4 23

1 0 3 0

3 3*

5 4 6 1

0 0 30 (40.5%)

* Mature plasma cells in the lesions were not stained.

MT2, were studied for their applicability in formalin-fixed, paraffin-embedded tissue. In reactive lymphoid tissues, MT1 appears to stain specifically the T-cell areas. In histiocytic necrotizing lymphadenitis, the activated T lymphocytes2~ are particularly well stained. However, because MT1 cross-reacts with granulocytic cells and some megakaryocytes, it cannot be considered entirely T-lineage specific. MT2, as r e p o r t e d by the m a n u f a c t u r e r , reacts not only with T cells but also with mantle and

FIGURE 4. Peripheral T-cell lymphoma, multilobated cell type. A, The large lymphoma cells have highly lobated nuclei. [Hematoxylin-eosin stain. x 750.] B, The lymphoma cells are stained by MTfl,whereas the histiocyte [arrow] is not stained. [ABC i m m u n o p e r o x i d a s e method, x 750.]

299

In T-cell lymphomas, MT1 stained 69.1% of all cases studied. We initially considered this not an unexpected finding, because loss of T-cell markers is a c o m m o n f i n d i n g in p e r i p h e r a l T - c e l l l y m phomas, a5'3~ particularly those of the nose (which accounted for 27 of 51 peripheral T-cell lymphomas in the present series). 16,34,35 To pursue this possibility further, we immunostained frozen tissue from these negative cases with MT1, and achieved positive results in all cases. This finding indicates that MT 1 recognizes a labile cell membrane antigen that may be masked or destroyed by formalin fixation or tissue processing. The data were therefore reanalyzed according to the source of material. The positivity rate was significantly higher (p < 0.0001) for hospital A, which used the shorter fixation time (table 2). Variations in immunohistochemical techniques could not account for the difference, because all the tests were carried out in one laboratory. One major difference remained that hospital A used on average a shorter fixation time than hospital B. The problem of overfixation causing loss of immunoreactivity of many tissue antigens has been well documented. 2 A m o n g the 17 negative cases, there was not even a single MTl-positive cell in 4 cases. T h o u g h positively stained reactive small cells were seen in the other 13 cases, they were in general fewer t h a n the n u m b e r o f small T cells in the corresponding frozen sections. Therefore, it appears that with prolonged formalin fixation, loss of MT1 reactivity occurs first in the neoplastic T cells (which presumably possess less antigen than their normal counterparts), followed by some reactive T cells, and finally all cells. In this respect, MT1 appears to be an

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FIGURE 5. Monomorphic peripheral T-cell lymphoma of medium-sized cell type. A, Monotonous population of medium-sized cells with moderate amounts of clear cytoplasm. [Hematoxylin-eosin stain, x 300.] B, Positive staining by MTI. [ABC immunoperoxidase method. x 300.] C, Negative staining by MT2. [ABC immunoperoxidase method, x 300.]

FIGURE 6. Pleomorphic T-cell lymphoma. A, The lymphoma cells are predominantly large and pleomorphic, but small and medium-sized neoplastic ceils are also present. [Hemotoxylin-eosin stain, x 600.] B, Completely negative staining by MT1 on paraffin section, [ABC immunoperoxidase method, x 300.] C, The lymphoma cells are, however, stained by MT1 on frozen section, (ABC immunoperoxidase method. x 600.]

300

T-CELL REACTIVEMONOCLONAL ANTIBODIES [Ng et al,]

FIGURE 7. Follicular B-cell lymphoma of mixed cell type, immunostained by MT2. A, The centers of the two neoplastic fo{licles are marked by stars. Staining is mainly in the peripheral portion, B, The small cleaved cells are stained, whereas the large noncleaved cells are weakly or not stained. [ABC immunoperoxidase method. A, x 150; B, x 600,]

excellent marker for T-cell lymphomas, provided that the duration of fixation is kept to the a minimum. When this antibody is applied on consultation materials or for retrospective studies, the lower positivity rate as obtained for hospital B (55.6%) would probably be more realistic. MT1 stained two cases of B-cell small lymphocytic lymphoma and one case of Burkitt's lymphoma in this study, and similar findings have also been reported by Norton and Isaacson recently. 36'a7 These three cases, however, also stained with two to three of the B-cell markers MB1, MB2, LN1, and LN2.12 Therefore, one has to be particularly cautious in accepting a case of small lymphocytic lymphoma as being of T-cell type on the basis of MT1 positivity alone. It is preferable to apply both MT1 and other B-cell-specific antibodies as a panel. On examination of the paraffin-sections of B-cell lymphomas, the background reactive lymphocytes staining with MT1 are not always small round cells with condensed chromatin. The nuclei of some cells are slightly larger than small lymphocytes and are elongated or angulated; the chromatin is finely granular. They might be difficult to differentiate from the small cleaved cells (centrocytes) of small cleaved cell or mixed cell B lymphomas. However, they occur

as isolated cells and should not be mistaken for neoplastic cells, which tend to occur in clusters and sheets. The pattern also corresponds to that of the T11-positive reactive cells as seen in frozen sections, although the suboptimal cytomorphologic preservation in cryostat sections does not permit such detailed morphologic assessment. The cross-reactivity of MT1 with granulocytes and some histiocytes, a problem also of UCHL1, la demands caution in interpretation of a positive result. However, provided that immunostaining with a batch of antibodies such as anti-Leu M1, 2 l y s o z y m e y %-antitrypsin, Cathespin G, a9 and S-100 protein 4~ and demonstration of chloroacetate esterase 41,42 are carried out in the appropriate situation, the problem of mistaking granulocytic sarcoma for T-cell lymphoma can be avoided.

Application of MT2 in Lymphomas MT2 stains only 10.9% of T-cell lymphomas. On the other hand, a significant percentage of B-cell lymphomas is stained, particularly the small cell lymphomas. This result is not unexpected, because MT2 also stains the normal mantle- and marginal-zone B cells. Though MT2 does not stain the normal follic301

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u l a r c e n t e r cells, it stains a b o u t h a l f o f t h e cases o f follicular lymphoma. This preliminary finding might b e u s e d to h e l p solve t h e d i a g n o s t i c p r o b l e m o f f l o r i d reactive follicular hyperplasia versus follicular lymphoma. Conclusion M T 1 is f a i r l y s p e c i f i c f o r T - c e l l l y m p h o m a s (exc e p t f o r t h e s m a l l l y m p h o c y t i c t y p e ) , b u t its sensitivity v a r i e s w i t h t h e d u r a t i o n o f f i x a t i o n o f t h e tissue. O n t h e o t h e r h a n d , M T 2 s h o w s b o t h low s p e c i f i c i t y a n d s e n s i t i v i t y f o r T - c e l l l y m p h o m a s . M T 1 is t h e r e f o r e very useful for staining normal and neoplastic T cells, p a r t i c u l a r l y w h e n u s e d i n c o m b i n a t i o n w i t h o t h e r a n t i b o d i e s a n d e n z y m e r e a c t i o n s t h a t w o r k in f o r m a l i n - f i x e d t i s s u e s . 5-12 H o w e v e r , it c a n n o t r e place the various lineage-specific monoclonal antib o d i e s t h a t w o r k o n l y o n f r e s h tissue, b e c a u s e it d o e s n o t w o r k c o n s i s t e n t l y o n p a r a f f i n sections. T h e m a j o r a p p l i c a t i o n s o f M T 1 a r e m a i n l y in two a r e a s . First, the T-cell nature of a lesion can be confirmed when f r e s h t i s s u e is n o t a v a i l a b l e . S e c o n d , r e t r o s p e c t i v e analysis of a large number of cases of peripheral T-cell lymphoma can be performed, which can help in c l a r i f y i n g t h e b e h a v i o r o f this r a r e t u m o r .

ADDENDUM Since submission o f this manuscript for publication, f o u r f u r t h e r studies on monoclonal antibodies reactive with T lymphocytes in paraffin sections have a p p e a r e d in the literature. 4~-46 T h e conclusions o f P o p p e m a et al. 44 are broadly similar to ours, except for one point. Because MT2 failed to stain their 20 cases o f T-cell l y m p h o m a studied, they s u m m a r i z e d in a flow chart that there was "probably no l y m p h o m a " for a lymphoproliferative lesion having a p h e n o t y p e o f MT1 +, M T 2 +, M B 1 - . W e a r e skeptical about this conclusion because our study shows that u p to 10.9% o f T-cell lymphomas may stain with MT2. T h e antibodies T2/48 and L60 stained significant numbers o f B-cell l y m p h o m a s 43,45 and Reed-Sternberg cells45 and therefore a r e o f limited value in diagnosis o f T-cell l y m p h o m a s . Mason a n d Gatter 46 have r e p o r t e d the use o f polyclonal a n t i s e r a a g a i n s t CD3 which give e x c e l l e n t staining o f n o r m a l a n d n e o p l a s t i c T cells in p a r a f f i n - e m b e d d e d tissues. Acknowledgments. T h e authors t h a n k Coulter Electronics (HK) Ltd. for the supply o f monoclonal antibodies, a n d J. Li, D. S. Y. Lo, and W. Tse for their technical assistance.

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