Monoclonal antibodies reactive in routinely processed tissue sections of malignant lymphoma, with emphasis on T-cell lymphomas

The immunoreactivity of eight monoclonal antibodies was evaluated on 45 routinely processed lymphomas (22 T-cell lymphomas, 11 B-cell lymphomas, and 12 cases of Hodgkin's disease). Two antibodies reactive with leukocyte common (T200) antigens (PD7/26 and 2B11) stained most of the B- and T-cell lymphomas but did not stain the Reed-Sternberg cells and variants in Hodgkin's disease. Two antibodies known to stain B cells (LN-1 and LN-2) reacted with some of the B-cell lymphomas, but LN-2 also reacted with the neoplastic cells in six of 22 T-cell lymphomas and with the Reed-Sternberg variants in eight of 12 cases of Hodgkin's disease. The granulocyte antibody anti-Leu M1 reacted with most cases of Hodgkin's disease but also reacted with two of 11 B-cell non-Hodgkin's lymphomas. An antibody to epithelial membrane antigen (anti-EMA) stained some cases of T-cell lymphoma, B-cell lymphoma, and Hodgkin's disease. Leu 7 was expressed in one T-cell lymphoma and in one case of Hodgkin's disease. A novel antibody reactive with T cells (L60) stained all cases of T-cell lymphoma but also stained some cases of B-cell lymphoma and one case of Hodgkin's disease. We conclude that none of these antibodies, when used alone on routinely fixed paraffin-embedded material, is completely sensitive and specific for T-cell lymphoma, B-cell lymphoma, or Hodgkin's disease. However, a panel of antibodies is useful in distinguishing Hodgkin's disease from non-Hodgkin's lymphoma and in suggesting the B- or T-cell phenotype of non-Hodgkin's lymphomas.     

Comparison of monoclonal and polyclonal antibodies directed against immunoglobulin light and heavy chains in non-Hodgkin's lymphoma

A study comparing the usage of monoclonal and polyclonal antibodies specific for immunoglobulin light and heavy chains was performed on frozen-tissue sections of 30 B-cell non-Hodgkin's lymphomas. In 16 cases, monotypic staining for an immunoglobulin light chain was demonstrated with monoclonal antibodies using a three-step avidin-biotin peroxidase complex (ABC) method; 13 cases were positive for kappa. In 14 cases, no immunoglobulin light-chain production was demonstrated. Repeat staining of 11 of these 14 cases with polyclonal anti-sera by a direct immunoperoxidase method demonstrated monotypic staining for light chain in 10 cases, 9 of which were positive for lambda. In 22 of 30 non-Hodgkin's lymphomas, an immunoglobulin heavy chain was identified using monoclonal anti-sera. In eight cases, however, no heavy chain was found. Repeated staining with polyclonal sera of additional sections in three of eight cases demonstrated heavy-chain production in each case. Decreased sensitivity, especially for the detection of the lambda light chain, rendered this particular lot of monoclonal antibodies unsuitable for immunophenotyping non-Hodgkin's lymphomas. Variability of antigenic sites on the immunoglobulin molecule seems a likely explanation for these observations.     

Monoclonal antibody Leu-22 (L60) permits the demonstration of some neoplastic T cells in routinely fixed and paraffin-embedded tissue sections

Monoclonal antibody Leu-22 (L60) detects a T cell-associated antigen which is stably expressed in routinely fixed and paraffin-embedded tissue sections. We investigated the utility of monoclonal antibody Leu-22 to immunophenotype routinely processed lymphoid neoplasms by determining its reactivity in 105 archival pathologic specimens of lymphoid neoplasia that had been previously immunophenotyped by standard cell suspension and frozen tissue section techniques. Monoclonal antibody Leu-22 reacted with 69% of T cell non-Hodgkin's lymphomas (NHLs), including cases belonging to each of the major clinicopathologic categories, and with 22% of B cell NHLs, but did not react with the Reed-Sternberg (RS) cells of Hodgkin's disease (HD). We concluded that monoclonal antibody Leu-22 reacts preferentially but not exclusively with T cell NHLs. Therefore, we performed parallel analyses of the same 105 cases with monoclonal antibodies leukocyte common antigen (LCA), Leu-M1, LN1, and LN2, which detect various paraffin-resistant antigens, and of 80 of these cases with monoclonal antibody UCHL1, which detects a paraffin-resistant T cell-associated antigen. UCHL1 reacted with 61% of the T cell NHLs studied. Sixty-nine percent of T cell NHLs expressed the LCA+, Leu-22+ or Leu-M1+, LN1- phenotype and 47% of B cell NHLs expressed the LCA+, Leu-22-, Leu-M1-, LN1+ phenotype. These phenotypes had a false-positive rate of only 7%. The substitution of UCHL1 for Leu-22 or the combined use of UCHL1 and Leu-22 in this panel did not improve our ability to correctly predict the T cell phenotype of these lymphoid neoplasms. LN1 and LN2 reacted with 13% and 56% of T cell NHLs, respectively, and LN2 reacted with RS cells in 85% of cases of HD. In summary, our results demonstrate that the judicious use of monoclonal antibody Leu-22 in combination with other selected commercially available monoclonal antibodies permits the determination of the B cell or T cell origin of a high proportion of NHLs, and is helpful in the differential diagnosis between HD and NHL among cases that have been routinely fixed and paraffin-embedded.     

Immunophenotyping of non-Hodgkin's lymphomas in paraffin-embedded tissue sections. A comparison with genotypic analysis.

Several monoclonal antibodies (MoAbs) are now available for immunophenotyping non-Hodgkin's lymphomas (NHLs) in paraffin-embedded tissue sections. To determine the reliability of these reagents in predicting the genotype, 44 cases of NHL were studied with the alkaline phosphatase-anti-alkaline phosphatase technique with the use of the following MoAbs: leukocyte common antigen (CD45), Mac 387, L26, 4KB5, MB1, MB2, LN2, UCHL1, MT1, and MT2. The lineage of the neoplastic cells was determined in all cases by gene rearrangement studies for immunoglobulin heavy chain and for the T-cell receptor beta-chain. Genotypic results showed B-cell lineage in 33 cases (75%), T-cell lineage in 6 cases (14%), and mixed or undetermined lineage in 5 cases (11%). A concordance of lineage assignment by paraffin section immunophenotyping with gene rearrangement studies was observed in 37 of 39 (95%) lymphomas with an unequivocally defined genotype. MoAb L26 was the most sensitive in detecting B-cell genotype; MoAbs MT1 and UCHL1 were the most sensitive and specific, respectively, in detecting T-cell genotype. The authors conclude that lineage assignment of NHLs in paraffin sections is reflective of the corresponding genotype when an appropriate panel of MoAbs is used.     

Nature of non-B, non-T lymphomas: an immunohistological study on frozen tissues using monoclonal antibodies

In a previous study employing conventional immunological marker analysis we found that 17% of high grade malignant lymphomas were devoid of cytoplasmic and membrane immunoglobulin and also sheep erythrocyte receptors. Cryostat sections from 24 of these cases (four of low grade and 20 of high grade malignancy) were stained with a panel of 30 monoclonal antibodies and six polyclonal antisera using a sensitive immunoperoxidase method. All tumours expressed the leucocyte common antigen (detected by monoclonal antibody 2D1) and all lacked epithelial cytokeratin (monoclonal antibody LE61), confirming their haematopoietic origin. All but one of the lymphomas expressed antigens characteristic of either B cells (17 cases) or T cells (six cases), while one case (morphologically a centroblastic lymphoma) had an unusual dual phenotype in which strong staining for T6 (marker of immature T cells) was associated with expression of the pan B lymphocyte antigens detectable with To15, anti-B1, anti-Leu12. This case was therefore classified as a B cell lymphoma showing aberrant expression of the T6 antigen. The pan B cell antibodies (To15, anti-B1, anti-Leu12) all appeared highly specific and sensitive, but the simultaneous use of all three monoclonal antibodies was necessary to detect the B cell nature in each of the 18 lymphomas. A wider panel of monoclonal antibodies was required to detect T lymphomas since these often disclosed atypical and restricted phenotypes. To15 and UCHT1 were the most reliable antibodies for the detection of B and T cell neoplasms, respectively. We conclude that most, if not all, "non-B, non-T" lymphomas are of either B or T lymphocyte origin and that monoclonal antibodies provide indispensable tools in their classification and diagnosis.     

The role of immunocytochemistry in diagnostic pathology.

This review suggests that immunocytochemistry in diagnostic pathology can be performed using relatively small panels of antibodies and that it should be reserved for situations in which, for one reason or another, the pathologist cannot exert his or her conventional diagnostic skills. Examples include the diagnosis of tumours the true nature of which is uncertain because of anaplasia or poor morphological preservation; the demonstration of small numbers of cells which are otherwise too rare to be recognised in conventionally stained preparations; and the immunophenotyping of non-Hodgkin's lymphomas. Recently progress has been made in the context of non-Hodgkin's lymphomas by the development of monoclonal antibodies that detect T and B cell associated markers in paraffin wax sections. Most of these reagents, however, recognise either lineage associated (but not lineage specific) variants of the leucocyte common antigen CD45, or antigens that are poorly characterised. A recent promising development has therefore been the demonstration that polyclonal antisera raised against the CD3/T3 T cell specific marker (purified by affinity chromatography) are suitable for staining T cells in paraffin sections. This approach will hopefully enable antibodies to be produced which react with other well defined white cell associated markers in routine biopsy material.     

Methods in pathology. Identification of T-cell lymphomas in paraffin-embedded tissues using polyclonal anti-CD3 antibody: comparison with frozen section immunophenotyping and genotypic analysis.

While L26 (CD20) is now well established as a B-cell marker of high specificity for use in paraffin-embedded tissues, paraffin-reactive T-cell antibodies (UCHL1, MT1, Leu-22, DF-T1, and MT2) have not shown comparable lineage specificity. A new commercially available polyclonal antibody directed against a synthetic peptide sequence of the CD3 (T-cell) antigen has recently become available for use on paraffin sections. In order to evaluate the utility of this antibody, we studied CD3 expression in conjunction with L26 and leukocyte common antigen (LCA) in 15 T-cell and 20 B-cell non-Hodgkin's lymphomas (NHL), all genotypically confirmed by DNA hybridization and immunophenotyped by immunoperoxidase studies in frozen tissue. Ten of 15 T-cell NHLs (67%) showed unequivocal immunolabeling of neoplastic cells with anti-CD3 in paraffin-embedded tissue. Of the five negative cases, three were lymphoblastic lymphomas, and two were peripheral (postthymic) lymphomas (one anaplastic large cell, Ki-1 positive and one large cell, immunoblastic). CD3 expression was identical in paraffin and cryostat sections (100% concordance). Twenty of 20 B-cell NHLs were positive with L26 and LCA but were negative with anti-CD3. Other neoplasms examined, including three granulocytic sarcomas and 45 nonhematopoietic tumors, were similarly negative with anti-CD3. We conclude that polyclonal anti-CD3 is a sensitive and highly specific T-cell marker in paraffin-embedded tissue and, when used in conjunction with LCA and L26, that it can determine cell lineage in the majority of non-Hodgkin's lymphomas.     

Immunophenotypic analysis of acute lymphoblastic leukemia using routinely processed bone marrow specimens

Monoclonal antibodies have been recently developed that react with antigens expressed on T and B lymphocytes in routinely processed, paraffin-embedded lymphoid tissues. In this study, we assessed bone marrow clot and/or core biopsy sections of 19 cases of acute lymphoblastic leukemia (ALL) using routinely decalcified, B5- or formalin-fixed, paraffin-embedded sections and a panel of monoclonal antibodies, including LN1, LN2, L26, Leu-22, UCHL-1, and LCA. Each case had been previously phenotyped using freshly obtained aspirate material and a standard immunophenotypic protocol. Our results demonstrate the utility of the LN2 antibody in differentiating between precursor B-cell (pre-B) and precursor T-cell ALL. The LN2 antibody stained 11 of 12 cases of pre-B ALL and did not react with any of the seven T-cell ALLs. The other antibodies tested were less helpful. The Leu-22 antibody stained both pre-B and T-cell ALLs, while the results with UCHL-1 revealed peculiar nuclear staining of pre-B and T-cell ALLs; this we attributed to processing artifact. The L26 antibody reacted with only one case of pre-B ALL (also CD20 antigen positive), while the LN1 antibody did not react with any pre-B ALLs. Neither L26 nor LN1 stained any cases of T-cell ALL. The LCA antibody stained in only four (21%) of 19 cases, two pre-B and two T-cell ALLs. The results also suggest that this panel of antibodies may be useful in differentiating ALL from mature B-cell and T-cell lymphomas involving the bone marrow.     

New marker of B lymphocytes, MB2: comparison with other lymphocyte subset markers active in conventionally processed tissue sections.

The use of the murine monoclonal antibody MB2 for identifying B lymphocytes in routinely processed tissue was evaluated and contrasted with the use of the monoclonal antibody UCHL1 for identifying T cells. One hundred and sixty eight surgical biopsy specimens were immunostained with these antibodies, including a wide range of normal and neoplastic non-lymphoid tissues, as well as normal lymphoreticular tissues and lymphomas. Sixty four non-Hodgkin's lymphomas were also examined, of which 51 had been previously phenotypically defined. In selected cases the results were compared with those obtained using two other monoclonal antibodies MB1 and MT1, used for identifying B and T cells, respectively, in paraffin sections. MB1 stained a smaller proportion of B cell tumours than MB2 and staining was, in general, weaker, except in one case of centroblastic lymphoma. MT1 immunoreactivity was comparable with that of UCHL1, except in one case of T lymphoblastic lymphoma (MT1 positive, UCHL1 negative). None of the antibodies is ideal, but, if used as a panel, they permit the separation of B cells and T cells in paraffin sections.     

Morphologic and immunophenotypic characterization of primary brain lymphomas using paraffin-embedded tissue

Primary lymphomas of the brain constitute about 1% of all primary intracranial neoplasms, but recent studies suggest an increasing incidence. Most cases are associated with an immunosuppressed state. We reviewed 29 cases of primary brain lymphoma from the Yorkshire Health Authority Region between 1970 and 1988 and found a striking increase in incidence over this period. No overt evidence of immunosuppression was found in any case. All were non-Hodgkin's in type and were classified morphologically using Kiel criteria and immunophenotypically using a panel of antibodies. Cryo-preserved tissue was available in five cases for parallel immunophenotyping. The majority of tumours were high-grade lymphomas together with three of lymphoplasmacytoid type. Thirteen tumours showed a striking pleomorphic morphology with plasmacytoid features. A reactive, predominantly perivascular monomorphic T-cell population was seen in all tumours. Most tumours were of B-cell lineage. No cases of Hodgkin's disease, T-cell or histiocytic lymphoma were present. Light chain restriction was present in only 46% of cases. The results of tumour immunophenotyping on cryostat sections were comparable with those from paraffin blocks. Our study emphasizes the value of a panel of antibodies reactive in paraffin-embedded tissue, allowing simultaneous evaluation of morphology and immunophenotype, and suitable for small biopsies received from stereotactic procedures.     

Expression of vimentin and epithelial membrane antigen in human malignant lymphomas

Immunoreactivity with monoclonal antibodies against the intermediate filament protein, vimentin, and epithelial membrane antigen (EMA) was examined in 330 cases of lymphoma (317 non-Hodgkin's and 13 Hodgkin's lymphomas), 12 reactive lymph nodes and mononuclear cells of the peripheral blood using either indirect immunoperoxidase staining or the avidin-biotin immunoperoxidase complex technique. The cell origin of each tumor was established using a panel of monoclonal antibodies against lymphocyte differentiation antigens. There were 41 T-cell, 247 B-cell and 29 undetermined lymphomas, and 13 cases of Hodgkin's disease in the series. Vimentin was expressed in 24 T-cell lymphomas (58.5%) and 60 B-cell lymphomas (24.2%). This difference in frequency was statistically significant. Vimentin expression in follicular lymphomas was less frequent than in diffuse B-cell lymphomas. In diffuse lymphomas, small and medium cell types were more reactive with anti-vimentin than large cell types. Reed-Sternberg cells (R-S cells) in Hodgkin's disease were positive for vimentin in 11 cases (84.6%). The frequency of EMA reactivity in lymphomas was low, particularly in T-cell lymphomas. No positive cases were found among follicular lymphomas. In diffuse non-Hodgkin's lymphomas, EMA was expressed only in mixed and large cell types, but never in smaller ones. In conclusion, monoclonal antibodies against vimentin and EMA appear to be of limited usefulness for the diagnosis of non-Hodgkin's lymphomas, but anti-vimentin antibody may be used as an adjunct to the diagnosis of R-S cells in Hodgkin's disease.     

Expression of Vimentin and Epithelial Membrane Antigen in Human Malignant Lymphomas

Immunoreactivity with monoclonal antibodies against the intermediate filament protein, vimentin, and epithelial membrane antigen (EMA) was examined in 330 cases of lymphoma (317 non-Hodgkin's and 13 Hodgkin's lymphomas), 12 reactive lymph nodes and mononuclear cells of the peripheral blood using either indirect immunoperox-idase staining or the avidin-biotin immunoperoxidase complex technique. The cell origin of each tumor was established using a panel of monoclonal antibodies against lymphocyte differentiation antigens. There were 41 T cell, 247 B cell and 29 undetermined lymphomas, and 13 cases of Hodgkin's disease in the series. Vimentin was expressed in 24 T-cell lymphomas (58.5%) and 60 B cell lymphomas (24.2%). This difference in frequency was statistically significant. Vimentin expression in follicular lymphomas was less frequent than in diffuse B-cell lymphomas. In diffuse lymphomas, small and medium cell types were more reactive with anti-vimentin than large cell types. Reed-Sternberg cells (R-S cells) in Hodgkin's disease were positive for vimentin in 11 cases (84.6%). The frequency of EMA reactivity in lymphomas was low, particularly in T cell lymphomas. No positive cases were found among follicular lymphomas. In diffuse non Hodgkin's lymphomas, EMA was expressed only in mixed and large cell types, but never in smaller ones. In conclusion, monoclonal antibodies against vimentin and EMA appear to be of limited usefulness for the diagnosis of non Hodgkin's lymphomas, but anti vimentin antibody may be used as an adjunct to the diagnosis of R-S cells in Hodgkin's disease.     

Paraffin section immunophenotyping of non-Hodgkin's lymphoma, using a panel of monoclonal antibodies

The monoclonal antibodies F8-11-13, 4KB5, MB1, and MB2 recognize largely B-cell-restricted antigenic determinants that resist routine processing. Similarly, MT1, MT2, and UCHL1 react with fixation-resistant T-cell-restricted antigens. In order to evaluate the diagnostic potential of these antibodies, the authors have assessed their immunoreactivity with a series of 81 formalin-fixed and paraffin-embedded non-Hodgkin's lymphomas (48 B-cell, 33 T-cell) encompassing a wide variety of histologic subtypes, which had been fully characterized by frozen-section immunophenotyping. Ninety-six percent of B-cell lymphomas reacted with one or more of the B-cell-associated antibodies, whereas 100% of T-cell lymphomas reacted either with MT1, UCHL1, or both antibodies. MT2 was of no value in distinguishing between B- and T-cell lymphomas. None of the antibodies was entirely lineage specific; furthermore, a proportion of cases failed to react with one or more of the B- or T-cell-associated antibodies. Although these antibodies provide useful information in distinguishing between T- and B-cell lymphomas, the authors suggest that a panel of these antibodies is necessary for accurate determination of the histogenesis of these tumors. As with any immunohistochemical marker, interpretation of the immunostaining must be in the context of the morphologic features.     

Paraffin-immunohistochemical analysis of 226 non-Hodgkin's malignant lymphomas in the endemic area of human T-cell leukemia virus type 1

A study was conducted to evaluate the usefulness of paraffin-immunohistochemistry for histopathological classification of non-Hodgkin's malignant lymphomas (NHML). the phenotypes of lymphoma cells and other cells were examined using 11 monoclonal and 3 polyclonal antibodies by the ABC method on paraffin-embedded tissue sections of 226 cases of NHML, comprising 94 B-cell lymphomas (B-ML) and 132 T-cell lymphomas (T-ML). In 219 NHML cases (96.8%), lymphoma cells reacted with more than one of these antibodies. A set of MB-1, Mx-pan B, L26, LN-1, LN-2 and anti-immunoglobulin light chain antibodies characterized each subtype of B-MLs, categorized according to the Kiel classification. Mantle-zone lymphoma (MzML) was added as one subtype. L26 stained the largest number of B-MLs (82.8%). B-cell chronic lymphocytic leukemia (B-CLL) was labeled most frequently by MB-1. MzML was characterized by reactivity of lymphoma cells with LN-2 and by the appearance of monoclonal immunoglobulin light chain along the cell membrane. Follicle center cell lymphomas were stained by LN-1 and LN-2, although a small number of proliferating cells were labeled by LN-1 in B-CLL, MzML and the immunocytoma lymphoplasmacytic/cytoid variant. MT-1 and/or UCHL-1 showed various degrees of reactivity with the cell membranes of lymphoma cells in 94.8% of T-MLs. Among the T-cell pleomorphic lymphomas of Suchi and Lennert, the adult T-cell leukemia/lymphoma type, defined by stippled heterochromatin distribution and peculiar huge cells, reacted selectively (p less than 0.05) with anti-phosphokinase C antibody. Anaplastic large cell T-ML reacted with a set of Ber H2, LN-2 and Leu M1. In T-zone lymphomas without hyperplastic follicles, angioimmunoblastic lymphadenopathy with dysproteinemia-type T-ML, lymphoepithelioid cell lymphomas and some pleomorphic lymphomas comprising clear large lymphoma cells, there were many intermingling B cells, and their constitution varied. In some lymphoblastic lymphomas of both the T cell and B-cell type, phenotypes of T cells and B cells were expressed. Consequently, it was shown that paraffin immunohistochemistry was useful for the practical histopathological diagnosis of NHML even in the area where human T-cell leukemia virus type 1 is endemic.     

An immunocytochemical study of T-cell lymphomas using monoclonal and polyclonal antibodies effective in routinely fixed wax embedded tissues

Formalin fixed and paraffin wax embedded tissue from 24 cases of T-cell lymphoma diagnosed using immunocytochemistry on cryostat sections was examined using a panel of eight monoclonal and three polyclonal antisera. The monoclonal antibodies UCHL1 and MT1 proved to be comparable and reliable markers of neoplastic cells in T-cell lymphomas. The B-cell specific marker, MB1, strongly stained all cells in two cases of pleomorphic large cell T-cell lymphoma, large cells in two cases of pleomorphic mixed medium and large cell lymphoma, and isolated clusters of blast cells in four cases of T-zone and angioimmunoblastic lymphadenopathy-like T-cell lymphoma. The cells stained by MB1 expressed T suppressor/cytotoxic surface markers on frozen section. Epithelial membrane antigen, as detected by a polyclonal anti-EMA and the monoclonal antibody HMFG2, was expressed in 36% of tumours especially those of monomorphic large cell and pleomorphic large cell phenotype. Single granules or finely dispersed cytoplasmic granularity was seen in four tumours using the anti-granulocyte reagent Leu M1. Tumour cells in one case stained in a pattern identical to Reed-Sternberg cells in Hodgkin's disease. Granular alpha-1-antitrypsin staining was found in 10 cases of pleomorphic large cell and monomorphic large cell lymphoma. No staining was observed using anti-lysozyme or the monoclonal macrophage specific marker Mac411. Monomorphic and pleomorphic large cell lymphomas tended to show a common immunophenotype with the majority of cells co-expressing alpha-1-antitrypsin HLA-DR and epithelial membrane antigen. Scattered large transformed blast cells in cases of angioimmunoblastic lymphadenopathy-like T-cell lymphomas and T-zone lymphomas shared a similar immunophenotype with the large cell lymphomas. Using a panel of monoclonal antibodies effective in paraffin embedded tissue, diagnostically useful staining profiles which correlate with the morphological phenotype can be established in T-cell lymphomas.     

The demonstration of B-cell, T-cell and myeloid antigens in paraffin sections

The monoclonal antibodies MB1 and MT1, which detect B cells and T cells respectively, have been applied to human lymphoid tissues. The distribution of staining within paraffin sections was compared with that observed using frozen sections and was found to be identical. The antibodies were then applied to paraffin sections of 19 B-cell lymphomas and 10 T-cell lesions in which full immunophenotyping had been performed. The B lymphomas all consisted of a large majority of MB1 positive cells with a variable infiltrate of small MT1 positive lymphocytes. The T cell lesions consisted of MT1 positive cells with few MB1 positive cells except in residual B cell areas of lymph nodes. In paraffin sections from cases of Hodgkin's disease anti-Leu M1 identified Reed-Sternberg cells and their variants and MB1 and MT1 showed a similar distribution of B cells and T cells to that demonstrated in previous studies using frozen sections. The results show that MB1 and MT1 are useful markers for B and T cells in routinely fixed paraffin embedded tissue. In conjunction with anti-Leu M1 they provide a valuable panel of antisera for the examination of lymph nodes and other biopsies when frozen tissue is not available.     

Utilization of monoclonal antibody L26 in the identification and confirmation of B-cell lymphomas. A sensitive and specific marker applicable to formalin-and B5-fixed, paraffin-embedded tissues.

Immunophenotypic analysis of paraffin-embedded tissues of lymphoproliferative disorders has been facilitated by recent developments of monoclonal antibodies that react with epitopes that survive histologic processing. Leukocyte common antigen (LCA) antibody has made a significant contribution to the immunocytochemical separation of non-Hodgkin's lymphomas from nonlymphoid neoplasms. However, a small percentage of lymphomas, particularly some large cell or immunoblastic B-cell tumors, will not label with LCA antibody. Other antibodies, directed against B lymphocytes, experience problems of specificity and a lack of sensitivity when applied to formalin-fixed specimens. The authors recently investigated a monoclonal antibody (L26) that demonstrates excellent specificity and sensitivity for B lymphocytes, and tumors derived from them, in formalin- and B5-fixed, paraffin-embedded tissue. The avidin-biotin peroxidase complex (ABC) technique was utilized for immunostaining 95 cases of malignant lymphoproliferative disorders and a variety of normal and neoplastic nonlymphoid tissues. When applied to sections of benign lymphoid tissue, the L26 antibody labeled germinal center cells, mantle zone and scattered interfollicular lymphocytes, but not histiocytes or plasma cells. L26 marked 100% (44/44) of the large cell and immunoblastic B-cell lymphomas, along with 1 case of pre-B cell lymphoblastic lymphoma. This included 8 cases that were LCA-negative. None of the T-cell lymphomas or plasma cell tumors studied demonstrated L26 immunostaining. No normal, benign, or neoplastic nonlymphoid tissues examined stained with this antibody. L26 successfully labels B lymphocytes and B-cell lymphomas in routinely processed tissues, often with greater sensitivity and intensity than LCA. This antibody should prove invaluable in the investigation of atypical lymphoid proliferations and the identification of B-cell derived lymphomas, when fresh or frozen tissue is unavailable for analysis.     

Immunohistochemical staining for terminal deoxynucleotidyl transferase (TDT). An enhanced method in routinely processed formalin-fixed tissue sections

This report describes an improved technique for sensitive and specific localization of terminal deoxynucleotidyl transferase (TdT) in routinely processed paraffin-embedded, formalin-fixed tissue sections using DNAse pretreatment and the avidin-biotin complex (ABC) technique. This method is useful in identifying lymphoblastic lymphomas (14/15 cases positive), with all other B- and T-cell lymphomas tested negative for the reaction. Used in conjunction with monoclonal antibodies immunoreactive for T- and B-cells in paraffin sections this technique should prove helpful in immunophenotyping malignant lymphomas where fresh tissue is unavailable for study.     

Essential Markers in Malignant Lymphoma: A Diagnostic Approach

Abstract

There has been a continuous production of antibodies to lymphocytes that are immunoreactive in routinely fixed, paraffin-embedded sections so that the reliance for fresh tissue for the immunophenotyping of malignant lymphoid proliferations and their mimics is greatly reduced. Although antigen-retrieval procedures are necessary for the demonstration of many antigens, reliable immunophenotyping can be performed in fixed tissues for most lymphomas without resorting to frozen sections, cell imprints, and homogenized tissue preparations for flow cytometry. This article uses the panel approach for the immunotyping of the lymphoid proliferations using antibodies that are directed to diagnostic categories of tumors with common morphologic characteristics. Tumors are grouped according to cell size as large or small cell lymphomas and according to pattern as diffuse or follicular lymphomas. The immunophenotypic profile of Hodgkin's disease also is discussed. Finally, the properties and characteristics of useful antibodies for each of these categories of lymphomas are discussed in some detail. (The J Histotechnol 25:215, 2002)     

Immunophenotypic diagnosis of non-Hodgkin's lymphoma in paraffin sections. Co-expression of L60 (Leu-22) and L26 antigens correlates with malignant histologic findings

The recent availability of monoclonal antibodies immunoreactive to T- or B-lineage antigens in formalin-fixed, paraffin-embedded tissue has permitted the adaptation of frozen-section immunodiagnostic criteria to paraffin-embedded tissue. Among a variety of reactive lymphoid processes, monoclonal antibody L26 showed a pattern of staining consistent with pan-B reactivity. Antibodies L60 (Leu-22) and UCHL-1 showed a pan-T and T-subset pattern of reactivity, respectively. In benign processes, L26 and L60 (or UCHL-1) were not coexpressed. In contrast, among 77 B-lineage non-Hodgkin's lymphomas, 42% showed aberrant co-expression of L26 and L60. The L26+/L60+ phenotype was most common in small lymphocytic lymphomas (80%) but was also noted in one third of diffuse large cell lymphomas. Expression of UCHL-1 was not identified in B-lineage neoplasms but was found, along with L60, on four of five T-lineage lymphomas studied as controls. The authors conclude that the anomalous coexpression of L60 and L26 antigens is a unique feature of B-lineage lymphomas and can be used for the immunodiagnosis of these malignancies in routinely processed tissue.