Path. Res. Pract. 187,96-102 (1991)
A Monoclonal Antibody to Human Leukocyte Common Antigen, SHL-l, and its Use for Formalin-Fixed, Paraffin-Embedded Tissues ::. Seong Hoe Park, Geon Kook Lee, Young Mee Bae, Chong Jai Kim, Hyung Geun Song, Chul Woo Kim, Je Geun Chi and Sang Kook Lee Department of Pathology, College of Medicine, Seoul National University, Korea
SUMMARY The authors describe a newly characterized murine monoclonal antibody to the human leukocyte surface antigen, SHL-1. The antigen belongs to the leukocyte common antigen (LCA) family, and its molecular weight is about 180 000 daltons, which is similar to that of some previously characterized LCAs. The SHL-1 antigen is resistant to conventional tissue-fixation and embedding procedures. This antibody can therefore be used in the immunohistochemical staining of paraffin-embedded tissue sections. Wide screening with a sufficient number of both fresh and routinely processed paraffin-embedded tissues was done with indirect immunoperoxidase technique. With this procedure, SHL-1labeled the majority of normal leukocytes and hematopoietic malignancies. Some B-cell malignancies were not stained with this antibody. The non-hematologic malignancies posing diagnostic problems of differentiation from lymphomas or leukemias were completely negative to SHL-l. The immunoreactivity to SHL-1 of samples from 24 leukemic patients and 15 human tumor cell lines was determined by the immunofluorescence method. Of 24 leukemic preparations, 23 were strongly reactive to this antibody. One case of B-cell leukemia did not react with SHL-1. No immunoreactivity was demonstrated in non-hematopoietic tumor cell lines. The overall reaction pattern of SHL-1 proved its usefulness in both diagnostic and research practice in hematological disorders. This antibody detected cell surface antigens ofthe T cell series more effectively than those ofthe B-cell series in terms of the positive number of cells and mean fluorescence intensity.
Introduction There is an increasing number of monoclonal antibodies to various kinds of cellular antigens. Among these, antibodies to leukocyte antigens have been useful in the differential diagnosis of hematologic or non-hematologic neoplasms and identification of subtypes among hematological disorders.
* This study is supported by a genetic engineering grant of the Minister of Education of Korea (1989) and a clinical research grant of Seoul National University Hospital (1989). 0344-0338/91/0187-0096$3.50/0
The leukocyte common antigen (LCA) is known to be widely distributed among T and B lymphocytes, macrophages, granulocytes and thymocytes 3- 5,8, 12, 14, 16, 17. The LCAs on different cell populations differ in mobility on sodium dodecyl sulfate gels, and they are a family of molecules rather than a single molecule. Among LCAs, some have a specific determinant on a particular subset of leukocytes and are not present on all kinds of leukocytes. Monoclonal antibodies to LCA also show some differences in reactivity between fresh and wax-embedded tissue sections. In this study we describe a monoclonal antibody, recognizing LCA, produced in this laboratory. The diag© 1991 by Gustav Fischer Verlag, Stuttgart
Monoclonal Antibody
nostic applications were widely tested on various kinds of formalin-fixed, paraffin-embedded and fresh tissue preparations.
Material and Methods
Material Fresh peripheral blood and bone-marrow biopsy specimens from a total of 24 leukemic patients were obtained for analysis. Various cell suspensions of peripheral blood, thymus and bone marrow and a total of 15 human tumor cell lines were analyzed on FACScan (Becton Oickinson Co.). Paraffin-embedded tissue blocks of various tumors were collected from the files of the Oepartment of Pathology of Seoul National University Hospital. Particular attention was paid to include various types of malignant lymphomas, undifferentiated carcinomas and round-cell neoplasms such as neuroblastoma, rhabdomyosarcoma and Ewing's sarcoma. All these types of cells are quite similar in morphology to that of malignant lymphoma, and differentiation between these types of tumors and malignant lymphoma is of prime importance in pathology. A small number of fresh surgical specimens were also collected for analysis.
to
Human Leukocyte Common Antigen . 97
markers (Smlg, COS, C03, C04, COS, C02, C019, COB and C033) using the FITC (fluorescein isothiocyanate) method.
Immunoperoxidase Method Snap frozen sections or paraffin-embedded tissue sections, after fixation and deparaffinization, were stained with SHL-1, followed by purified biotinylated goat anti-mouse IgG and then avidin-horseradish peroxidase conjugate. The reaction pattern was analyzed based on serial H-E stained sections. Paraffinembedded tissue sections of malignant lymphomas were also stained with other monoclonal antibodies to lymphocyte-specific antigens (MTl, Biotest AG, Frankfurt, FRG; LN1 (COw7S), LN2 (C074), Techniclone International, Santa Ana: Lambda, Kappa, Oako).
Results
Characterization of the SHL-l Target Antigen The molecular weight of the SHL-l antigen was determined by immunoprecipitation and SDS-PAGE. The major precipitation band of the SHL-l antigen corresponded to a molecule of approximately 180000 m.w. (Fig. 1).
Production of the Monoclonal Antibody and Characterization of the Target Antigen Balb/c mice were immunized with human leukemic T-cell line Jurkat, and the hybridoma secreting the SHL-1 was produced by fusion of the spleen cells from Balb/c mice and the mouse myeloma cell line SP 2/0-Ag 14 as previously described?, 10, 15. The antigen was biochemically characterized with immunoprecipitation and analyzed by polyacrylamide gel electrophoresis. The cells were labeled with iodine ( IZ5 1) and lactoperoxidase. Triton X-114 solubilized cellular proteins were subjected to indirect immunoprecipitation with monoclonal antibodies and an excess of goat anti-mouse IgG. Washed immunoprecipitates were solubilized by boiling in a gel sample buffer (10% glycerol/S% 2-mercaptoethanoI/3% NaOodS0 4/62.S mM Tris buffer). After polyacrylamide gel elctrophoresis, the fixed and dried gels were exposed to Kodak RP X-Omat film in the presence of intensifying screens 6.
-r205 -r 180 -r 116 -r 97.4 66
45
Determination of Antibody Isotype The isotype of SHL-1 was determined by enzyme immunoassay using Screen Type™ (Boeringer Mannheim, Biochemicals). Isotyping was performed with goat anti-murine isotype specific antisera (lgG1, IgG2a, IgG2b, IgG3, IgM, kappa, lambda) followed by peroxidase-labeled swine anti-goat IgG as the secondary antibody. With the addition of ABTS (2,2'azinobis[3-ethylbenzothiazoline-6-sulfonic acid] and hydrogen peroxide (HzO z) substrate, positive samples turned an intense bluegreen color.
Immunofluorescence Method Fresh cell suspensions of several tumor cell lines, normal lymphoid tissues, and both fresh bone marrow and peripheral blood of leukemic patients were prepared and analyzed on a fluorescence-activated cell sorter (FACS). After application of a murine unconjugated anti-human leukocyte monoclonal antibody (SHL-1), purified fluorescein-conjugated goat anti-mouse IgG was employed. Fresh bone marrow and peripheral blood specimens from leukemic patients were analyzed for immunologic
KD Fig. 1. Molecular characteristics of leukocyte common antigen recognized by the monoclonal antibody, SHL-l. Human T-cell leukemia cell line, Jurkat is used for lysis, immunopercipitation and SOS-polyacrylamide gel electrophoresis. Control antibody is an isotype-matched monoclonal antibody to COla.
98 . Seong Hoe Park et al. Table 1. Distribution of the SHL-1 antigen in samples from leukemic patients Case No.
Sex/Age (yrs)
Specimen
1 2 3 4 5 6 7 8 9 10
Ml7
PB BM BM PB, BM BM BM PB PB BM BM BM PB BM BM BM BM BM PB PB BM PB BM PB BM
11
12 13
14 15 16 17 18 19 20 21 22
23 24
MIlS FilS M/29 MIS M/24 F/12 F/ll
Ml4 Ml8
F/4 FIlO F123 F/48 F/7 Mo F/4 F/5
Ml4 Ml64 F/30 MIS F/40
Ml24 Ml16
Immunologic Markers Cell Type SmIg CDS CD3 CD4 CD8 CD2 CD19 CDB CD33 SHL-1
+ + +
ND
+ + + +
+ +
+ + +
+ +
+
+
+
+ + + + + +
+
+ +
+
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
+ + +
+
ND ND ND ND ND ND
ND ND
ND
ND
+ +
ND ND
+
ND ND ND
ND ND
+ + + + + + + + + + +
ND ND
+ + + + + + + + + +
+ +
ND ND
ND ND
+
+
+
+
+
+
+
+ + + + + + + + + + + + + + + + + + + + + + +
T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL CALL CALL CALL CALL CALL B-ALL B-ALL B-ALL B-CLL CLL ANLL ANLL ANLL ANLL *
ANLL = Acute non-lymphocytic leukemia; CALL = Common acute lymphocytic leukemia; ND = Not done; * = Inconclusive; PB = peripheral blood; BM = Bone marrow.
Determination of Antibody Isotype The subclass of SHL-1 was determined by enzyme immunoassay, and it was shown to be of the IgG1 subclass.
Distribution of the SHL-l Antigen in Hematopoietic Cells Analyzed by Immunofluorescence Human hematopoietic cells from peripheral blood, thymus and bone marrow showed strong immunoreactivity to SHL-1. Fetal thymocytes at 23 weeks and postnatal thymocytes at different ages (7 months and 11 months) were strongly labeled with SHL-1, but the thymic epithelial cells were completely unlabeled. Strong immunoreactivity to SHL-1 antibody is also verified in adult thymocytes, peripheral blood leukocytes, mixture of B lymphocytes .and macrophages and bone marrow cells. A larger percentage of cells of T-cell lineage were reactive than were other types of hematopoietic cells, including B lymphocytes, macrophages and bone marrow cells.
Distribution of the SHL-l Antigen in Fresh Leukemic Cells Among 24 samples from 24 leukemic patients, 23 were strongly labeled with SHL-1, regardless of cell types. Cell lines of T-cell lineage were well recognized by SHL-1, and the reaction pattern was more prominent than that of the B-ceillineage. It suggested that SHL-1 recognized the LeA
which is relatively T-cell predominant. However, tumors of B-celliineage showed immunoreactivity to SHL-1 even if the reaction pattern was not as prominent as those of the T-cells, except for one leukemic sample of B cell origin, probably in the early stage of B-cell ontogeny. One case of Table 2. Distribution of the SHL-1 antigen in various tumor cell lines Cell Lines
Origin
Results
Jurkat HSB2 Raji KG1 KG1a U937 K562 RDES T98G SNU16 Y79 N417 N146 H69 C14
T lymphocyte T lymphocyte B lymphocyte Myeloid cell Myeloid cell Monocyte Erythroleukemia Ewing's sarcoma Glioblastoma multiforme Gastric adenocarcinoma Retinoblastoma Small cell lung cancer Small cell lung cancer Small cell lung cancer Adenocarcinoma of lung
99 59.5 54.6 76.8 89.2 82.7
(%)1
1.3
0.1 9.4 8.2 9.2 0 0 0 0
1 Percent of positive cells. The results are the mean of 5 experiments.
Monoclonal Antibody to Human Leukocyte Common Antigen . 99
lymphocytic leukemia which had inconclusive cell characteristics was also positive to SHL-1. The results are summarized in Table 1.
Distribution of the SHL-l Antigen in Hematopoietic Cells and Various Tumor Cell Lines Analyzed by Immunofluorescence The 15 tumor cell lines were analyzed using FACS. The tumor cell lines of both myeloid and lymphoid origin expressed SHL-l antigen, showing high immunoreactivity to the antibody. But the cell line K562 (Erythroleukemia) was not reactive to SHL-1. The glioblastoma multiform cell line (T98G), the retinoblastoma cell line (Y79) and the gastric carcinoma cell line (SNU16) showed weak immunoreactivity to SHL-1 antibody around 10% on FACS analysis. These findings were checked with immunoblotting studies, using 35S-methionine metabolic and 125 1 cell
Fig. 2. SHL-1 reactiviry with non-neoplastic lymphoid tissue, palatine tonsil. Virtually all lymphoid cells are SHL-1(+). In two follicles (F), the reactiviry to SHL-1 is relatively weak. Immunoperoxidase-stained cryostat section without counter-stain (x 98).
Fig. 3. High power view of Fig. 2. In the center of the follicles (F), the large cells are stained with SHL-1 rather weakly (x 196).
surface labeling methods, to see whether there are real SHL-l antigens in these cell lines. The weak reactivity was thought to be nonspecific since no precipitation bands were found with these methods. All tumor cell lines of non-hematopoietic origin were not reactive to the antibody. The results of the indirect immunofluorescence study are summarized in Table 2.
Tissue Distribution of the SHL-l Antigen Tested on Fresh and Paraffin-Embedded Sections of Normal and Neoplastic Lymphoid Tissue In cryostat sections of non-neoplastic lymphoid tissues, SHL-1 recognized almost all lymphoid cells. In lymph nodes and tonsils, lymphoid cells in the mantle zones and interfollicular areas showed intense immunoreactivity to SHL-1. The lymphocytes in germinal centers, especially the large immature cells, showed relatively weaker reactivity
100 . Seong Hoe Park et al.
(Fig. 2,3). When paraffin-embedded tissue sections were tested, a similar reaction was observed. Some of the follicular center cells were not stained with SHL-l. Immunoreactivity was mostly confined to the cell surface membrane, although some cells showed faint cytoplasmic staining. Histiocytes, including interdigitating reticulum cells, dendritic reticulum cells and sinus histiocytes, were not stained or stained faintly with this antibody. Plasma cells were also not stained. SHL-l recognized all thymocytes in both the medulla and cortex in the tissue sections of the thymus (Fig. 4). Non-lymphoid cells, including vascular endothelial cells, surface epithelial cells and other mesenchymal cells, were not stained at all (Fig. 5). The non-Hodgkin's lymphomas (NHL) examined were well recognized by this antibody (Table 3A, B). Of 22 cases of NHL, four cases showed no reactivity. Those four cases were of the diffuse, histiocytic type and of B-cell immunophenotype. Of four B-cell NHLs with SHL-l (- ), one case was reactive only with LN-2. This suggested the possibility that the case was of true histiocytic origin. In this series of NHL, a case of B-immunoblastic lymphomawas included.
Fig. 5. High magnification view of Fig. 4. Outlines of blood vessels (arrow heads) are scattered in the section. It appears that vascular endothelial cells are not reactive to SHL-1 (X 213).
The large immature cells were not stained. In contrast, smaller and more differentiated cells showed intense reactivity. In one case of Lennert's lymphoma, all lymphoid neoplastic cells were strongly reactive, but epithelioid cells were not stained with SHL-l. Excluding these four cases not reactive with SHL-l, the immunoreactivity of neoplastic lymphoid cells to SHL-l was not significantly different between the T- and B-cell types. But there were variations in the intensity of the reaction from one area of the section to another. This suggested that fixation and embedding procedures may influence the strength of the reactivity of the antigen recognized by this antibody, as described by others 16 . However, this effect is relative rather than absolute, since immunoreactivity in almost all cases was relatively well-preserved. Non-lymphoid or non-hematopoietic neoplasms were not reactive to SHL-l (Table 4), although infiltrating lymphocytes demonstrated a positive reaction to this antibody. The non-lymphoid, non-neoplastic tissues were not reactive to this antibody, either. Fig. 4. SHL-1 reactivity with non-lymphoid tissue, thymus. All thymocytes are stained with SHL-l. Note that thymic epithelial cells within Hassall's corpuscles are not stained (arrow). Immunoperoxidase-stained cryostat section without counter-stain (x 112).
Discussion The LeA is a cell membrane glycoprotein unique to hematopoietic cells whose functional significanc.~ is still
Monoclonal Antibody to Human Leukocyte Common Antigen, 101 Table 3A. SHL-1 reactivity in Non-Hodgkin's lymphomas
Table 4. SHL-1 reactivity in Non-Hematopoietic tumor tissues
Classification
Tissues
SHL-1
Epithelial tumors Undifferentiated carcinoma of nasopharynx Undifferentiated carcinoma of stomach Adenocarcinoma of rectum Adenocarcinoma of pancreas Squamous cell carcinoma of esophagus Squamous cell carcinoma of uterine cervix Hepatoblastoma
0/5 0/1 0/1 0/1 0/1 0/1 0/1
Non-epithelial tumors Ewing's sarcoma Retinoblastoma Neuroblastoma Wilms' tumor Embryonal rhabdomyosarcoma Chondroid chordoma Astrocytoma
0/2 0/1 0/2 0/1 0/1 0/1 0/1
No. of Positive ReactionslNo. Tested
Diffuse, histiocytic' Diffuse, poorly differentiated lymphocytic Diffuse, mixed lymphocytic-histiocytic Diffuse, Iympholastic Mycosis funfoides
12/16 ,2/2 2/2 1/1 1/1
Total
18/22
, One immunoblastic sarcoma, B-cell type and one Lennert's lymphoma were included in diffuse histiocytic type. They showed diffuse positive reaction to SHL-1 of tumor cells. Table 3B. Results of immunoperoxidase study of Non-Hodgkin's lymphoma SHL-1 Pan-T LN-l LN-2
Case Diagnosis No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic (B immunoblastic sarcoma) ML, diffuse histiocytic (Lennert's lymphoma) ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, poorly differentiated ML, poorly differentiated ML, mixed HL ML, mixed HL ML, mixed HL Mycosis fungoides
ND = not done; ML lymphocytic.
+ +
+
+
+
+
ND
+ + +
+
+ + + +
+ +
ND
+
+
+ +
+
+ +
+
+ + +
+
ND ND ND
+
ND
+
+ + + +
ND ND ND ND
ND ND ND ND
+
+
+
ND ND
+
ND ND
= malignant lymphoma; HL = histiocytic-
unclear. Though antigenically similar, there are differences in molecular weights according to cell populations 11 . The monoclonal antibodies to human leukocyte antigens produced previously have shown different specificities in labeling different subtypes of leukocytes. These antibodies recoglllze certain glycoprotein moieties constituting LCAs l-5,8,9, 12-15, 17. The result of the present study demonstrates that the cell surface molecule identified by monoclonal antibody SHL-l is present on the majority of both neoplastic and non-neoplastic leukocytes in fresh-frozen and wax-embed-
+
ded tissues. This suggests that the SHL-l antigen is one of the determinants of LCAs, which does not become denatured or masked during fixation and embedding. The relatively weaker labeling of normal follicular center cells and the negative reaction of some B-cell, large cell lymphomas suggest decreased antigenic expression or lowered antigen densities in some stages of B cell differentiation 2,8. Positive labeling was attained either by the immunofluorescence method or the immunoperoxidase method for both fresh-frozen sections and routinely processed, formalin-fixed, paraffin-embedded sections, even after decalcification. The strong resistance of the antigen to such processing offers practical use of SHL-l in diagnostic pathology. The SHL-l labeled both bone marrow preparations and peripheral blood samples in over 95% of cases of leukemia. The only non-reacting case was one of B-cell acute lymphocytic leukemia. Most of the malignant lymphomas were also labeled with SHL-l. All of the non-hematologic tumors and normal tissues were completely unlabeled with SHL-l. Non-hematologic round-cell neoplasms often causing diagnostic problems with non-Hodgkin's lymphoma, such as undifferentiated carcinoma, neuroblastoma, Ewing's sarcoma and rhabdomyosarcoma were easily differentiated with SHL-l. As indicated by this study, specific staining of fresh-frozen and formalin-fixed, paraffin-embedded tissue sections and cell suspensions is useful in solving the diagnostic problem of distinguishing lymphomas from undifferentiated carcinomas and in differentiating lymphomas and leukemias from other small round cell tumors of childhood. Immunoreaction with SHL-l antibody indicates, with a high degree of probability, that a tumor is of lymphoid origin. However, negative results cannot be interpreted as indicating that the tumor is of non-leukocyte origin, because the SHL-l did not react with some large B-cell lymphomas; thus it cannot be used as a general marker of leukocytes. For complete evaluation of all kinds of leukocyte subsets, use of other monoclonal antibodies labeling B-cell subsets is helpful for diagnostic purposes. Similarly,
102 . Seong Hoe Park et al. immunohistochemical investigations of undifferentiated neoplasms should include SHL-l as well as B markers to rule out a lymhoid tumor. Other antibodies to LCA have shown similar behavior to SHL-l in that they have failed to react with some large cell or immunoblastic B-cell tumors S, and there have been previously characterized anti-leukocyte antibodies recognizing antigenic determinants whose molecular weights were also around 180 kD. Wood et al.J7 characterized the pan-leukocyte monoclonal antibody L3B12 (lgG2 isotype) and the molecular weight of the L3B12 antigen was 180 kD. The L3B12 antibody labeled most leukocytes and related neoplasms with a similar tissue distribution to that obtained with SHL-l, including lack of immunoreactivity in some large cell immunoblastic lymphomas. Another previously described monoclonal antibody to a LCA, which has an antigenic molecular weight similar to SHL-l, is UCHL-1 9• The UCHL-l antigen has a molecular weight of 180-185 kD, and UCHL-l antibody labeled normal and neoplastic T-cells, macrophages and mature myeloid cells. The UCHL-l antigen was also T-cell predominant in nature, but the antibody gave a diffuse cytoplasmic positive reaction in some large B-cell lymphomas of centroblastic and immunoblastic types, while SHL-l showed a lack of immunoreactivity in centroblastic and immunoblastic lymphomas. Furthermore, most B cell neoplasms were strongly reactive with SHL-l. Thus SHL-l is different from UCHL-l both in terms of the pattern of tissue distribution and in that SHL-l recognizes almost all lymphoid cells without significant difference in reactivity. Two other leukocyte-reactive antibodies, PD7/26 and 2B 11 6,8, which are used as panleukocyte marker, are clearly different from SHL-l in that the molecular weights of the PD7/26 and 2B11 antigens were approximately 200000 daltons. PD7/26 antibody recognizes mainly lymphocytes in germinal centers and mantle zones, and 2B11 antibody detects mainly lymphocytes in T-zones. These patterns of tissue distribution of the antigens are quite different from that of SHL-l. In summary, SHL-l is a monoclonal antibody recognizing a LCA which appears to have a predominantly T-lymphocyte-specific determinant. The relatively potent positive labeling of most leukemias, including myeloid or monocytic leukemias arid most B-cell lymphomas, along with resistance of the antigen to routine surgical processing, makes SHL-l a useful immunologic marker in differentiating malignant lymphomas or leukemic infiltration from non-hematologic round-cell neoplasms.
References 1 Battifora H, Trowbridge IS (1983) A monoclonal antibody useful for the differential diagnosis between malignant lymphoma and non-hematopoietic neoplasms. Cancer 51: 816-821
2 Cartun RW, Coles FB, Pastuszak WT (1987) Utilization of monoclonal antibody L26 in the identification and confirmation of B-celllymphomas. Asensitive and specific marker applicable to formalin- and B5-fixed, paraffin-embedded tissues. Am J Pathol 129:415-421 3 Coffman RL, Weissman IL (1981) B220: a B cell-specific member of the T200 glycoprotein family. Nature 289: 681-683 4 Dalchau R, FabreJW (1981) Identification with a monoclonal antibody of a predominantly Blymphocyte specific determination of the human leukocyte common antigen. Evidence for structural and possible functional diversity of the human leukocyte common molecule. J Exp Med 153: 753-765 5 Dalchau R, Kirkley J, Fabre JW (1980) Monoclonal antibody to a leukocyte-specific membrane glycoprotein probably homologous to the leukocyte-common (L-C) antigen of the rat. Eur J Immunol 10: 737-744 6 Engleman EG, Warnke R, Fox RI, Dilley FJ, Benike q, Levy R (1981) Studies of a human T lymphocyte antigen recognized by a monoclonal antibody. Proc Natl Acad Sci 78: 1791-1795 7 Koehler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-498 8 Kurtin PJ, Pinkus GS (1985) Leukocyte common antigen-A diagnostic discriminant between hematopoietic and non-hematopoietic neoplasms in paraffin sections using monoclonal antibodies: Correlation with immunologic studies and ultrastructural localization. Hum Pathol 16: 353-365 9 Norton AJ, Ramsay AD, Smith SH, Beverly PCL, Isaacson AJ (1986) Monoclonal antibody (UCHL-1) that recognises normal and neoplastic T cells in routinely fixed tissues. J Clin Pathol39: 399-405 10 Nowinski RC, Lostrom ME, Tam MR, Stone MR, Burnette WN (1979) The isolation of hybrid cell lines producing monoclonal antibodies against the p15(E) protein of murine leukemia viruses. Virology 93: 111-126 I I Omary MB, Trowbridge IS, Battifora HA (1980) Human homologue of murine T200 glycoprotein. J Exp Med 152: 842-852 12 Pizzold G, Beverley P, Thomas JA, Bradstock KF, Mattingly S, Janossy G (1980) Differential diagnosis of malignant lymphoma and non-lymphoid tumor using monoclonal anti-leukocyte antibody. Cancer 46: 2640-2647 13 Poppema S, Hollema H, Visser L (1987) Monoclonal antibodies (MTl, MT2, MB1, MB2, MB3) reactive with leukocyte subsets in paraffin-embedded tissue sections. Am J Pathol 127: 418-429 14 Sunderland CA, McMaster WR, Williams AF (1979) Purification with monoclonal natibody of a predominant leukocytecommon antigen and glycoprotein from rat thymocytes. Eur J lmmunol 9: 155-159 15 Trowbridge IS (1983) Interspecies spleen-myeloma hybrid producing monoclonal antibodies against mouse lymphocyte surface glycoprotein, noo. J Exp Med 138: 313-323 16 Warnke RA, Gatter KC, Falini B, Hildreth P, Woolston RE, Pulford K, Cordell JL, Cohen B, De Wolf-Peeters C, Mason DY (1983) Diagnosis of human lymphoma with monoclonal antileukocyte antibodies. N Engl J Med 309: 1275-1281 17 Wood GS, Link M, Warnke RA, Dilley J, Levy R (1984) Pan-leukocyte monoclonal antibody L3B12. Characterization and application to research and diagnostic problems. Am J Clin Pathol81: 176-183
Received January 12, 1990 . Accepted in revised form March 17, 1990
Key words: Leukocyte common antigen - Monoclonal antibody - Formalin-fixed tissue - Immunohistochemistry Seong Hoe Park, M.D., Department of Pathology, College of Medicine, Seoul National University, 28 Yunkun-Dong, Chongno-Ku, Seoul, 110-744, Korea
A Monoclonal Antibody to Human Leukocyte Common Antigen, SHL-l, and its Use for Formalin-Fixed, Paraffin-Embedded Tissues ::. Seong Hoe Park, Geon Kook Lee, Young Mee Bae, Chong Jai Kim, Hyung Geun Song, Chul Woo Kim, Je Geun Chi and Sang Kook Lee Department of Pathology, College of Medicine, Seoul National University, Korea
SUMMARY The authors describe a newly characterized murine monoclonal antibody to the human leukocyte surface antigen, SHL-1. The antigen belongs to the leukocyte common antigen (LCA) family, and its molecular weight is about 180 000 daltons, which is similar to that of some previously characterized LCAs. The SHL-1 antigen is resistant to conventional tissue-fixation and embedding procedures. This antibody can therefore be used in the immunohistochemical staining of paraffin-embedded tissue sections. Wide screening with a sufficient number of both fresh and routinely processed paraffin-embedded tissues was done with indirect immunoperoxidase technique. With this procedure, SHL-1labeled the majority of normal leukocytes and hematopoietic malignancies. Some B-cell malignancies were not stained with this antibody. The non-hematologic malignancies posing diagnostic problems of differentiation from lymphomas or leukemias were completely negative to SHL-l. The immunoreactivity to SHL-1 of samples from 24 leukemic patients and 15 human tumor cell lines was determined by the immunofluorescence method. Of 24 leukemic preparations, 23 were strongly reactive to this antibody. One case of B-cell leukemia did not react with SHL-1. No immunoreactivity was demonstrated in non-hematopoietic tumor cell lines. The overall reaction pattern of SHL-1 proved its usefulness in both diagnostic and research practice in hematological disorders. This antibody detected cell surface antigens ofthe T cell series more effectively than those ofthe B-cell series in terms of the positive number of cells and mean fluorescence intensity.
Introduction There is an increasing number of monoclonal antibodies to various kinds of cellular antigens. Among these, antibodies to leukocyte antigens have been useful in the differential diagnosis of hematologic or non-hematologic neoplasms and identification of subtypes among hematological disorders.
* This study is supported by a genetic engineering grant of the Minister of Education of Korea (1989) and a clinical research grant of Seoul National University Hospital (1989). 0344-0338/91/0187-0096$3.50/0
The leukocyte common antigen (LCA) is known to be widely distributed among T and B lymphocytes, macrophages, granulocytes and thymocytes 3- 5,8, 12, 14, 16, 17. The LCAs on different cell populations differ in mobility on sodium dodecyl sulfate gels, and they are a family of molecules rather than a single molecule. Among LCAs, some have a specific determinant on a particular subset of leukocytes and are not present on all kinds of leukocytes. Monoclonal antibodies to LCA also show some differences in reactivity between fresh and wax-embedded tissue sections. In this study we describe a monoclonal antibody, recognizing LCA, produced in this laboratory. The diag© 1991 by Gustav Fischer Verlag, Stuttgart
Monoclonal Antibody
nostic applications were widely tested on various kinds of formalin-fixed, paraffin-embedded and fresh tissue preparations.
Material and Methods
Material Fresh peripheral blood and bone-marrow biopsy specimens from a total of 24 leukemic patients were obtained for analysis. Various cell suspensions of peripheral blood, thymus and bone marrow and a total of 15 human tumor cell lines were analyzed on FACScan (Becton Oickinson Co.). Paraffin-embedded tissue blocks of various tumors were collected from the files of the Oepartment of Pathology of Seoul National University Hospital. Particular attention was paid to include various types of malignant lymphomas, undifferentiated carcinomas and round-cell neoplasms such as neuroblastoma, rhabdomyosarcoma and Ewing's sarcoma. All these types of cells are quite similar in morphology to that of malignant lymphoma, and differentiation between these types of tumors and malignant lymphoma is of prime importance in pathology. A small number of fresh surgical specimens were also collected for analysis.
to
Human Leukocyte Common Antigen . 97
markers (Smlg, COS, C03, C04, COS, C02, C019, COB and C033) using the FITC (fluorescein isothiocyanate) method.
Immunoperoxidase Method Snap frozen sections or paraffin-embedded tissue sections, after fixation and deparaffinization, were stained with SHL-1, followed by purified biotinylated goat anti-mouse IgG and then avidin-horseradish peroxidase conjugate. The reaction pattern was analyzed based on serial H-E stained sections. Paraffinembedded tissue sections of malignant lymphomas were also stained with other monoclonal antibodies to lymphocyte-specific antigens (MTl, Biotest AG, Frankfurt, FRG; LN1 (COw7S), LN2 (C074), Techniclone International, Santa Ana: Lambda, Kappa, Oako).
Results
Characterization of the SHL-l Target Antigen The molecular weight of the SHL-l antigen was determined by immunoprecipitation and SDS-PAGE. The major precipitation band of the SHL-l antigen corresponded to a molecule of approximately 180000 m.w. (Fig. 1).
Production of the Monoclonal Antibody and Characterization of the Target Antigen Balb/c mice were immunized with human leukemic T-cell line Jurkat, and the hybridoma secreting the SHL-1 was produced by fusion of the spleen cells from Balb/c mice and the mouse myeloma cell line SP 2/0-Ag 14 as previously described?, 10, 15. The antigen was biochemically characterized with immunoprecipitation and analyzed by polyacrylamide gel electrophoresis. The cells were labeled with iodine ( IZ5 1) and lactoperoxidase. Triton X-114 solubilized cellular proteins were subjected to indirect immunoprecipitation with monoclonal antibodies and an excess of goat anti-mouse IgG. Washed immunoprecipitates were solubilized by boiling in a gel sample buffer (10% glycerol/S% 2-mercaptoethanoI/3% NaOodS0 4/62.S mM Tris buffer). After polyacrylamide gel elctrophoresis, the fixed and dried gels were exposed to Kodak RP X-Omat film in the presence of intensifying screens 6.
-r205 -r 180 -r 116 -r 97.4 66
45
Determination of Antibody Isotype The isotype of SHL-1 was determined by enzyme immunoassay using Screen Type™ (Boeringer Mannheim, Biochemicals). Isotyping was performed with goat anti-murine isotype specific antisera (lgG1, IgG2a, IgG2b, IgG3, IgM, kappa, lambda) followed by peroxidase-labeled swine anti-goat IgG as the secondary antibody. With the addition of ABTS (2,2'azinobis[3-ethylbenzothiazoline-6-sulfonic acid] and hydrogen peroxide (HzO z) substrate, positive samples turned an intense bluegreen color.
Immunofluorescence Method Fresh cell suspensions of several tumor cell lines, normal lymphoid tissues, and both fresh bone marrow and peripheral blood of leukemic patients were prepared and analyzed on a fluorescence-activated cell sorter (FACS). After application of a murine unconjugated anti-human leukocyte monoclonal antibody (SHL-1), purified fluorescein-conjugated goat anti-mouse IgG was employed. Fresh bone marrow and peripheral blood specimens from leukemic patients were analyzed for immunologic
KD Fig. 1. Molecular characteristics of leukocyte common antigen recognized by the monoclonal antibody, SHL-l. Human T-cell leukemia cell line, Jurkat is used for lysis, immunopercipitation and SOS-polyacrylamide gel electrophoresis. Control antibody is an isotype-matched monoclonal antibody to COla.
98 . Seong Hoe Park et al. Table 1. Distribution of the SHL-1 antigen in samples from leukemic patients Case No.
Sex/Age (yrs)
Specimen
1 2 3 4 5 6 7 8 9 10
Ml7
PB BM BM PB, BM BM BM PB PB BM BM BM PB BM BM BM BM BM PB PB BM PB BM PB BM
11
12 13
14 15 16 17 18 19 20 21 22
23 24
MIlS FilS M/29 MIS M/24 F/12 F/ll
Ml4 Ml8
F/4 FIlO F123 F/48 F/7 Mo F/4 F/5
Ml4 Ml64 F/30 MIS F/40
Ml24 Ml16
Immunologic Markers Cell Type SmIg CDS CD3 CD4 CD8 CD2 CD19 CDB CD33 SHL-1
+ + +
ND
+ + + +
+ +
+ + +
+ +
+
+
+
+ + + + + +
+
+ +
+
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
+ + +
+
ND ND ND ND ND ND
ND ND
ND
ND
+ +
ND ND
+
ND ND ND
ND ND
+ + + + + + + + + + +
ND ND
+ + + + + + + + + +
+ +
ND ND
ND ND
+
+
+
+
+
+
+
+ + + + + + + + + + + + + + + + + + + + + + +
T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL T-ALL CALL CALL CALL CALL CALL B-ALL B-ALL B-ALL B-CLL CLL ANLL ANLL ANLL ANLL *
ANLL = Acute non-lymphocytic leukemia; CALL = Common acute lymphocytic leukemia; ND = Not done; * = Inconclusive; PB = peripheral blood; BM = Bone marrow.
Determination of Antibody Isotype The subclass of SHL-1 was determined by enzyme immunoassay, and it was shown to be of the IgG1 subclass.
Distribution of the SHL-l Antigen in Hematopoietic Cells Analyzed by Immunofluorescence Human hematopoietic cells from peripheral blood, thymus and bone marrow showed strong immunoreactivity to SHL-1. Fetal thymocytes at 23 weeks and postnatal thymocytes at different ages (7 months and 11 months) were strongly labeled with SHL-1, but the thymic epithelial cells were completely unlabeled. Strong immunoreactivity to SHL-1 antibody is also verified in adult thymocytes, peripheral blood leukocytes, mixture of B lymphocytes .and macrophages and bone marrow cells. A larger percentage of cells of T-cell lineage were reactive than were other types of hematopoietic cells, including B lymphocytes, macrophages and bone marrow cells.
Distribution of the SHL-l Antigen in Fresh Leukemic Cells Among 24 samples from 24 leukemic patients, 23 were strongly labeled with SHL-1, regardless of cell types. Cell lines of T-cell lineage were well recognized by SHL-1, and the reaction pattern was more prominent than that of the B-ceillineage. It suggested that SHL-1 recognized the LeA
which is relatively T-cell predominant. However, tumors of B-celliineage showed immunoreactivity to SHL-1 even if the reaction pattern was not as prominent as those of the T-cells, except for one leukemic sample of B cell origin, probably in the early stage of B-cell ontogeny. One case of Table 2. Distribution of the SHL-1 antigen in various tumor cell lines Cell Lines
Origin
Results
Jurkat HSB2 Raji KG1 KG1a U937 K562 RDES T98G SNU16 Y79 N417 N146 H69 C14
T lymphocyte T lymphocyte B lymphocyte Myeloid cell Myeloid cell Monocyte Erythroleukemia Ewing's sarcoma Glioblastoma multiforme Gastric adenocarcinoma Retinoblastoma Small cell lung cancer Small cell lung cancer Small cell lung cancer Adenocarcinoma of lung
99 59.5 54.6 76.8 89.2 82.7
(%)1
1.3
0.1 9.4 8.2 9.2 0 0 0 0
1 Percent of positive cells. The results are the mean of 5 experiments.
Monoclonal Antibody to Human Leukocyte Common Antigen . 99
lymphocytic leukemia which had inconclusive cell characteristics was also positive to SHL-1. The results are summarized in Table 1.
Distribution of the SHL-l Antigen in Hematopoietic Cells and Various Tumor Cell Lines Analyzed by Immunofluorescence The 15 tumor cell lines were analyzed using FACS. The tumor cell lines of both myeloid and lymphoid origin expressed SHL-l antigen, showing high immunoreactivity to the antibody. But the cell line K562 (Erythroleukemia) was not reactive to SHL-1. The glioblastoma multiform cell line (T98G), the retinoblastoma cell line (Y79) and the gastric carcinoma cell line (SNU16) showed weak immunoreactivity to SHL-1 antibody around 10% on FACS analysis. These findings were checked with immunoblotting studies, using 35S-methionine metabolic and 125 1 cell
Fig. 2. SHL-1 reactiviry with non-neoplastic lymphoid tissue, palatine tonsil. Virtually all lymphoid cells are SHL-1(+). In two follicles (F), the reactiviry to SHL-1 is relatively weak. Immunoperoxidase-stained cryostat section without counter-stain (x 98).
Fig. 3. High power view of Fig. 2. In the center of the follicles (F), the large cells are stained with SHL-1 rather weakly (x 196).
surface labeling methods, to see whether there are real SHL-l antigens in these cell lines. The weak reactivity was thought to be nonspecific since no precipitation bands were found with these methods. All tumor cell lines of non-hematopoietic origin were not reactive to the antibody. The results of the indirect immunofluorescence study are summarized in Table 2.
Tissue Distribution of the SHL-l Antigen Tested on Fresh and Paraffin-Embedded Sections of Normal and Neoplastic Lymphoid Tissue In cryostat sections of non-neoplastic lymphoid tissues, SHL-1 recognized almost all lymphoid cells. In lymph nodes and tonsils, lymphoid cells in the mantle zones and interfollicular areas showed intense immunoreactivity to SHL-1. The lymphocytes in germinal centers, especially the large immature cells, showed relatively weaker reactivity
100 . Seong Hoe Park et al.
(Fig. 2,3). When paraffin-embedded tissue sections were tested, a similar reaction was observed. Some of the follicular center cells were not stained with SHL-l. Immunoreactivity was mostly confined to the cell surface membrane, although some cells showed faint cytoplasmic staining. Histiocytes, including interdigitating reticulum cells, dendritic reticulum cells and sinus histiocytes, were not stained or stained faintly with this antibody. Plasma cells were also not stained. SHL-l recognized all thymocytes in both the medulla and cortex in the tissue sections of the thymus (Fig. 4). Non-lymphoid cells, including vascular endothelial cells, surface epithelial cells and other mesenchymal cells, were not stained at all (Fig. 5). The non-Hodgkin's lymphomas (NHL) examined were well recognized by this antibody (Table 3A, B). Of 22 cases of NHL, four cases showed no reactivity. Those four cases were of the diffuse, histiocytic type and of B-cell immunophenotype. Of four B-cell NHLs with SHL-l (- ), one case was reactive only with LN-2. This suggested the possibility that the case was of true histiocytic origin. In this series of NHL, a case of B-immunoblastic lymphomawas included.
Fig. 5. High magnification view of Fig. 4. Outlines of blood vessels (arrow heads) are scattered in the section. It appears that vascular endothelial cells are not reactive to SHL-1 (X 213).
The large immature cells were not stained. In contrast, smaller and more differentiated cells showed intense reactivity. In one case of Lennert's lymphoma, all lymphoid neoplastic cells were strongly reactive, but epithelioid cells were not stained with SHL-l. Excluding these four cases not reactive with SHL-l, the immunoreactivity of neoplastic lymphoid cells to SHL-l was not significantly different between the T- and B-cell types. But there were variations in the intensity of the reaction from one area of the section to another. This suggested that fixation and embedding procedures may influence the strength of the reactivity of the antigen recognized by this antibody, as described by others 16 . However, this effect is relative rather than absolute, since immunoreactivity in almost all cases was relatively well-preserved. Non-lymphoid or non-hematopoietic neoplasms were not reactive to SHL-l (Table 4), although infiltrating lymphocytes demonstrated a positive reaction to this antibody. The non-lymphoid, non-neoplastic tissues were not reactive to this antibody, either. Fig. 4. SHL-1 reactivity with non-lymphoid tissue, thymus. All thymocytes are stained with SHL-l. Note that thymic epithelial cells within Hassall's corpuscles are not stained (arrow). Immunoperoxidase-stained cryostat section without counter-stain (x 112).
Discussion The LeA is a cell membrane glycoprotein unique to hematopoietic cells whose functional significanc.~ is still
Monoclonal Antibody to Human Leukocyte Common Antigen, 101 Table 3A. SHL-1 reactivity in Non-Hodgkin's lymphomas
Table 4. SHL-1 reactivity in Non-Hematopoietic tumor tissues
Classification
Tissues
SHL-1
Epithelial tumors Undifferentiated carcinoma of nasopharynx Undifferentiated carcinoma of stomach Adenocarcinoma of rectum Adenocarcinoma of pancreas Squamous cell carcinoma of esophagus Squamous cell carcinoma of uterine cervix Hepatoblastoma
0/5 0/1 0/1 0/1 0/1 0/1 0/1
Non-epithelial tumors Ewing's sarcoma Retinoblastoma Neuroblastoma Wilms' tumor Embryonal rhabdomyosarcoma Chondroid chordoma Astrocytoma
0/2 0/1 0/2 0/1 0/1 0/1 0/1
No. of Positive ReactionslNo. Tested
Diffuse, histiocytic' Diffuse, poorly differentiated lymphocytic Diffuse, mixed lymphocytic-histiocytic Diffuse, Iympholastic Mycosis funfoides
12/16 ,2/2 2/2 1/1 1/1
Total
18/22
, One immunoblastic sarcoma, B-cell type and one Lennert's lymphoma were included in diffuse histiocytic type. They showed diffuse positive reaction to SHL-1 of tumor cells. Table 3B. Results of immunoperoxidase study of Non-Hodgkin's lymphoma SHL-1 Pan-T LN-l LN-2
Case Diagnosis No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic (B immunoblastic sarcoma) ML, diffuse histiocytic (Lennert's lymphoma) ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, diffuse histiocytic ML, poorly differentiated ML, poorly differentiated ML, mixed HL ML, mixed HL ML, mixed HL Mycosis fungoides
ND = not done; ML lymphocytic.
+ +
+
+
+
+
ND
+ + +
+
+ + + +
+ +
ND
+
+
+ +
+
+ +
+
+ + +
+
ND ND ND
+
ND
+
+ + + +
ND ND ND ND
ND ND ND ND
+
+
+
ND ND
+
ND ND
= malignant lymphoma; HL = histiocytic-
unclear. Though antigenically similar, there are differences in molecular weights according to cell populations 11 . The monoclonal antibodies to human leukocyte antigens produced previously have shown different specificities in labeling different subtypes of leukocytes. These antibodies recoglllze certain glycoprotein moieties constituting LCAs l-5,8,9, 12-15, 17. The result of the present study demonstrates that the cell surface molecule identified by monoclonal antibody SHL-l is present on the majority of both neoplastic and non-neoplastic leukocytes in fresh-frozen and wax-embed-
+
ded tissues. This suggests that the SHL-l antigen is one of the determinants of LCAs, which does not become denatured or masked during fixation and embedding. The relatively weaker labeling of normal follicular center cells and the negative reaction of some B-cell, large cell lymphomas suggest decreased antigenic expression or lowered antigen densities in some stages of B cell differentiation 2,8. Positive labeling was attained either by the immunofluorescence method or the immunoperoxidase method for both fresh-frozen sections and routinely processed, formalin-fixed, paraffin-embedded sections, even after decalcification. The strong resistance of the antigen to such processing offers practical use of SHL-l in diagnostic pathology. The SHL-l labeled both bone marrow preparations and peripheral blood samples in over 95% of cases of leukemia. The only non-reacting case was one of B-cell acute lymphocytic leukemia. Most of the malignant lymphomas were also labeled with SHL-l. All of the non-hematologic tumors and normal tissues were completely unlabeled with SHL-l. Non-hematologic round-cell neoplasms often causing diagnostic problems with non-Hodgkin's lymphoma, such as undifferentiated carcinoma, neuroblastoma, Ewing's sarcoma and rhabdomyosarcoma were easily differentiated with SHL-l. As indicated by this study, specific staining of fresh-frozen and formalin-fixed, paraffin-embedded tissue sections and cell suspensions is useful in solving the diagnostic problem of distinguishing lymphomas from undifferentiated carcinomas and in differentiating lymphomas and leukemias from other small round cell tumors of childhood. Immunoreaction with SHL-l antibody indicates, with a high degree of probability, that a tumor is of lymphoid origin. However, negative results cannot be interpreted as indicating that the tumor is of non-leukocyte origin, because the SHL-l did not react with some large B-cell lymphomas; thus it cannot be used as a general marker of leukocytes. For complete evaluation of all kinds of leukocyte subsets, use of other monoclonal antibodies labeling B-cell subsets is helpful for diagnostic purposes. Similarly,
102 . Seong Hoe Park et al. immunohistochemical investigations of undifferentiated neoplasms should include SHL-l as well as B markers to rule out a lymhoid tumor. Other antibodies to LCA have shown similar behavior to SHL-l in that they have failed to react with some large cell or immunoblastic B-cell tumors S, and there have been previously characterized anti-leukocyte antibodies recognizing antigenic determinants whose molecular weights were also around 180 kD. Wood et al.J7 characterized the pan-leukocyte monoclonal antibody L3B12 (lgG2 isotype) and the molecular weight of the L3B12 antigen was 180 kD. The L3B12 antibody labeled most leukocytes and related neoplasms with a similar tissue distribution to that obtained with SHL-l, including lack of immunoreactivity in some large cell immunoblastic lymphomas. Another previously described monoclonal antibody to a LCA, which has an antigenic molecular weight similar to SHL-l, is UCHL-1 9• The UCHL-l antigen has a molecular weight of 180-185 kD, and UCHL-l antibody labeled normal and neoplastic T-cells, macrophages and mature myeloid cells. The UCHL-l antigen was also T-cell predominant in nature, but the antibody gave a diffuse cytoplasmic positive reaction in some large B-cell lymphomas of centroblastic and immunoblastic types, while SHL-l showed a lack of immunoreactivity in centroblastic and immunoblastic lymphomas. Furthermore, most B cell neoplasms were strongly reactive with SHL-l. Thus SHL-l is different from UCHL-l both in terms of the pattern of tissue distribution and in that SHL-l recognizes almost all lymphoid cells without significant difference in reactivity. Two other leukocyte-reactive antibodies, PD7/26 and 2B 11 6,8, which are used as panleukocyte marker, are clearly different from SHL-l in that the molecular weights of the PD7/26 and 2B11 antigens were approximately 200000 daltons. PD7/26 antibody recognizes mainly lymphocytes in germinal centers and mantle zones, and 2B11 antibody detects mainly lymphocytes in T-zones. These patterns of tissue distribution of the antigens are quite different from that of SHL-l. In summary, SHL-l is a monoclonal antibody recognizing a LCA which appears to have a predominantly T-lymphocyte-specific determinant. The relatively potent positive labeling of most leukemias, including myeloid or monocytic leukemias arid most B-cell lymphomas, along with resistance of the antigen to routine surgical processing, makes SHL-l a useful immunologic marker in differentiating malignant lymphomas or leukemic infiltration from non-hematologic round-cell neoplasms.
References 1 Battifora H, Trowbridge IS (1983) A monoclonal antibody useful for the differential diagnosis between malignant lymphoma and non-hematopoietic neoplasms. Cancer 51: 816-821
2 Cartun RW, Coles FB, Pastuszak WT (1987) Utilization of monoclonal antibody L26 in the identification and confirmation of B-celllymphomas. Asensitive and specific marker applicable to formalin- and B5-fixed, paraffin-embedded tissues. Am J Pathol 129:415-421 3 Coffman RL, Weissman IL (1981) B220: a B cell-specific member of the T200 glycoprotein family. Nature 289: 681-683 4 Dalchau R, FabreJW (1981) Identification with a monoclonal antibody of a predominantly Blymphocyte specific determination of the human leukocyte common antigen. Evidence for structural and possible functional diversity of the human leukocyte common molecule. J Exp Med 153: 753-765 5 Dalchau R, Kirkley J, Fabre JW (1980) Monoclonal antibody to a leukocyte-specific membrane glycoprotein probably homologous to the leukocyte-common (L-C) antigen of the rat. Eur J Immunol 10: 737-744 6 Engleman EG, Warnke R, Fox RI, Dilley FJ, Benike q, Levy R (1981) Studies of a human T lymphocyte antigen recognized by a monoclonal antibody. Proc Natl Acad Sci 78: 1791-1795 7 Koehler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-498 8 Kurtin PJ, Pinkus GS (1985) Leukocyte common antigen-A diagnostic discriminant between hematopoietic and non-hematopoietic neoplasms in paraffin sections using monoclonal antibodies: Correlation with immunologic studies and ultrastructural localization. Hum Pathol 16: 353-365 9 Norton AJ, Ramsay AD, Smith SH, Beverly PCL, Isaacson AJ (1986) Monoclonal antibody (UCHL-1) that recognises normal and neoplastic T cells in routinely fixed tissues. J Clin Pathol39: 399-405 10 Nowinski RC, Lostrom ME, Tam MR, Stone MR, Burnette WN (1979) The isolation of hybrid cell lines producing monoclonal antibodies against the p15(E) protein of murine leukemia viruses. Virology 93: 111-126 I I Omary MB, Trowbridge IS, Battifora HA (1980) Human homologue of murine T200 glycoprotein. J Exp Med 152: 842-852 12 Pizzold G, Beverley P, Thomas JA, Bradstock KF, Mattingly S, Janossy G (1980) Differential diagnosis of malignant lymphoma and non-lymphoid tumor using monoclonal anti-leukocyte antibody. Cancer 46: 2640-2647 13 Poppema S, Hollema H, Visser L (1987) Monoclonal antibodies (MTl, MT2, MB1, MB2, MB3) reactive with leukocyte subsets in paraffin-embedded tissue sections. Am J Pathol 127: 418-429 14 Sunderland CA, McMaster WR, Williams AF (1979) Purification with monoclonal natibody of a predominant leukocytecommon antigen and glycoprotein from rat thymocytes. Eur J lmmunol 9: 155-159 15 Trowbridge IS (1983) Interspecies spleen-myeloma hybrid producing monoclonal antibodies against mouse lymphocyte surface glycoprotein, noo. J Exp Med 138: 313-323 16 Warnke RA, Gatter KC, Falini B, Hildreth P, Woolston RE, Pulford K, Cordell JL, Cohen B, De Wolf-Peeters C, Mason DY (1983) Diagnosis of human lymphoma with monoclonal antileukocyte antibodies. N Engl J Med 309: 1275-1281 17 Wood GS, Link M, Warnke RA, Dilley J, Levy R (1984) Pan-leukocyte monoclonal antibody L3B12. Characterization and application to research and diagnostic problems. Am J Clin Pathol81: 176-183
Received January 12, 1990 . Accepted in revised form March 17, 1990
Key words: Leukocyte common antigen - Monoclonal antibody - Formalin-fixed tissue - Immunohistochemistry Seong Hoe Park, M.D., Department of Pathology, College of Medicine, Seoul National University, 28 Yunkun-Dong, Chongno-Ku, Seoul, 110-744, Korea
