Distinguishing B and T lymphocytes by scanning electron microscopy.

This work demonstrates differences between B lymphocytes and T lymphocytes as seen in the scanning electron microscope (SEM). Slides of routinely prepared benign and malignant lymphoid tissues were viewed in the SEM. The location of prominent populations of T lymphocytes and B lymphocytes was verified by immunocytochemical staining respectively with CD3 and CD20 antisera. Benign tissues, including infant thymus and adult hyperplastic lymph node, were compared with malignant lymphomas of T-cell and B-cell types. The SEM appearance of benign B and T lymphocytes is compared utilizing the backscattered electron (BSE) mode and secondary electron (SE) mode in adult hyperplastic lymph node and infant thymus, respectively. The BSE and SE modes reveal that the sectioned T-lymphocyte nucleus has a more complex configuration than that of the B lymphocyte. T lymphocytes appear more discrete and separated one from another, while B lymphocytes exhibit close cellular association to form a syncytial array. These features are noted also in malignant lymphomas of B-lymphocyte and T-lymphocyte types, respectively. The SEM can distinguish between B and T lymphocytes by studying the differences in nuclear and chiefly the cell to cell appearances. The syncytial configuration of B lymphocytes may reflect prominent expression of cell adhesion molecules, e.g., ICAM-1, as noted in the literature.

Plural immunoglobulin synthesis in a single cell: an ultrastructural study of two cases with three M-proteins.

Electrophoresis revealed two cases of malignant lymphoma that each contained three M-proteins (IgM lambda.lgG kappa.lgG lambda and IgM lambda.IgM kappa.lgG kappa) in the sera. To determine cellular origin of each M-protein, atypical lymphoid and plasmacytoid cells of both cases were examined by electron microscopy. Atypical lymphoid and plasmacytoid cells possessed rough endoplasmic reticula (RERs) in varying degrees, as seen by conventional electron microscopy, and showed double-stainability for plural antibodies against immunoglobulins following double stainings of immunoelectron microscopy using immunogold staining. Rabbit antibodies against human IgM, lgG, free kappa-light chain and free lambda-light chain were used for the immunoelectron microscopic staining. By the double staining method, plural immunoglobulins, IgM/IgG, IgM/free kappa, IgM/free lambda, IgG/free kappa, IgG/free lambda and free kappa/free lambda, were simultaneously detected in varying degrees in the Golgi area, RERs, and dense bodies of lymphoid and plasmacytoid cells. In conclusion, this study directly exhibited, through electron microscopy, that plural immunoglobulins were synthesized at the same time in a single cell, and that the process of immunoglobulin synthesis in the lymphoid and plasmacytoid cells was different from that in a normal B-cell.

Use of CD23 (BU38) on paraffin sections in the diagnosis of small lymphocytic lymphoma and mantle cell lymphoma.

The CD23 antigen is a low-affinity immunoglubulin E receptor that is expressed during B-cell activation. Recently, it has been shown to be of diagnostic utility in distinguishing between small lymphocytic lymphoma (SLL) and mantle cell lymphoma (MCL), two entities that can have similar morphologic and immunophenotypic features. Such studies, however, generally required viable cells in cell suspension or cryostat sections for detection of CD23. We evaluated staining for the CD23 antigen in paraffin sections, using BU38, an antibody that detects a fixation-resistant epitope of the antigen. We analyzed 44 SLLs, 3 lymphoplasmacytoid lymphomas, and 39 MCLs. Staining was performed on formalin- or B5-fixed paraffin-embedded tissue sections using L26 (CD20), CD3, Leu22 (CD43), and BU38 (CD23) antibodies. All of the cases were of B-cell phenotype (CD20+), and 42/44 SLLs, 3/3 lymphoplasmacytoid lymphomas, and 33/39 MCLs coexpressed the CD43 antigen. CD23 was positive in 41 (93%) of 44 SLLs. The majority of neoplastic cells (75% or more) stained positively, with a membranous pattern of staining. The staining was moderate in intensity and easily interpreted. Only 1/39 MCLs and 1/3 lymphoplasmacytoid lymphomas were CD23 positive. CD23-positive follicular dendritic cells were, however, present in all of the MCLs, either in residual follicles or in large, disordered meshworks. These results demonstrate that the BU38 antibody can detect CD23 on the cells of SLLs in paraffin sections and that this antibody can have diagnostic utility in routine diagnosis.

Richter's syndrome showing pronounced lymphadenopathy in response to administration of granulocyte colony-stimulating factor.

A patient with Richter's syndrome developed rapid generalized lymph node enlargement with a decrease of peripheral blood lymphocytes after recombinant human granulocyte colony-stimulating factor (rhG-CSF) therapy for neutropenia induced by chemotherapy. The lymphadenopathy subsided spontaneously following discontinuation of rhG-CSF medication. Reinstitution of rhG-CSF therapy was followed by the same response as during initial therapy. Histopathologically, the lesions were characteristic of diffuse large cell lymphoma (DLL) with no evidence of myeloid cell involvement. No spontaneous contraction of enlarged lymph nodes followed withdrawal of the second course, but the enlargement subsided with chemotherapy. The patient died of myocardial infarction. All residual tumors examined post mortem presented microscopic features of small lymphocytic lymphoma (SLL), and G-CSF receptor was demonstrated on these neoplastic cells by Northern blot hybridization analysis. This observation indicates that some B cell malignancies may retain G-CSF receptor and respond to G-CSF.

Distinction between diffuse cutaneous malignant follicular center cell lymphoma and lymphoid hyperplasia by computerized nuclear image analysis.

The difficult differential diagnosis between the diffuse variants of cutaneous lymphoid hyperplasia (CLH; synonym; pseudolymphoma) and malignant follicular center cell lymphomas (FCCL) often requires a multidisciplinary approach. Eighteen CLH and 11 FCCL, diagnosed by conventional histology and immunophenotyping and subsequently examined with a polymerase chain reaction to show clonal immunoglobulin heavy-chain gene rearrangements, were subjected to a novel type of automated nuclear image analysis. Of all nuclear parameters tested in azure A-stained semithin sections, the mean nuclear profile area (TN) of lymphoid cells was the best criterion to distinguish between CLH and FCCL (p = 9 x 10(-6)). Additional distinctive features, in the order of decreasing significance, were the SD of TN; all chromatin textural parameters combined; and the light and the dark fractions of the central nuclear profile areas. Parameters related to the chromatin pattern were independent of nuclear profile size in FCCL, but not in CLH. Two lesions registered as CLH displayed the nuclear characteristics favoring this diagnosis, but showed B-cell monoclonality at the DNA level. In conclusion, computerized nuclear image analysis is a helpful additional diagnostic tool in the evaluation of diffuse CLH and cutaneous FCCL.

Lymphoplasmacytic lymphoma in a stallion.

Lymphoplasmacytic lymphoma found in a 6-year-old Anglo-Arabian stallion was investigated histologically, immunohistochemically and ultrastructurally. The animal showed a large mediastinal mass and generalized lymph node involvement. The neoplastic cells were in various differentiation stages of small lymphocyte, centrocyte, centroblast, immunoblast and plasma cell. Some neoplastic cells showed positive cytoplasmic reactivity for mu and lambda chains. There were well developed rough endoplasmic reticulum (RER) and Golgi complexes in plasmacytoid cells, and slightly developed RER or a few long strands of RER in medium-sized to large lymphoid cells. These findings suggest that this neoplasm is of B-cell origin.

Morphometrical investigation of medulloblastoma nuclei by S.A.M. (Shape Analytical Morphometry) software system.

In order to characterize medulloblastomas and to get over the difficulties sometimes encountered in differential diagnosis, a double morphometric procedure has been applied to its nuclei. The first consisted of size measurements (maximum diameter, area and perimeter), the latter is represented by S.A.M. (Shape Analytical Morphometry) software-system specifically implemented to describe shape of biological structure by analytical parameters. Analytical and dimensional parameters submitted to Hotelling's multivariate discriminant analysis gave the best results when used together in convenient discriminant subsets, thereby allowing a good distinction between medulloblastoma in comparison with neuroblastoma, Ewing's tumor, lymphoblastic and lymphocytic lymphoma. These results underline the usefulness of morphometric characterization also for practical diagnostic purposes.

Report of an unusual small lymphocytic B-cell lymphoma selectively involving the B-zone of lymph node.

Selective involvement of the B-cell compartment of lymph node by B-cell malignant lymphomas is an occasional finding related to early phases of lymph node infiltration. The authors have observed a unique case of diffuse small lymphocytic lymphoma that consisted of immunohistologically and genotypically proven B-clonal population exhibiting a repetitive pattern of infiltration in three lymph node samples obtained from the patient during a 9-year period. This pattern consisted of a selective and complete replacement of the B-areas with disappearance of follicles and widening of the medullary cords, an expanded T-zone showing features consistent with dermatopathic lymphadenitis and well-preserved sinuses. Clinically, multiple involved sites at presentation (lymph nodes, spleen, skin, bone marrow, and peripheral blood) and during the 9-year follow-up (testis) were detected, and the disease was associated with a relative indolent course like other low-grade lymphomas. The phenotypic profile of lymphoma cells studied by immunoperoxidase method, and by single-labeling and double-labeling flow cytometric analyses (SIg+, K+, LN2+, MB1+, MB2+, HLA-DR+, CD 9+, CD19+, CD 20+, CD 21+, CD 22+, CD 24+, Leu 8+, CD 5-, CD 10-, CD 11b-, CD 11c-, CD 25-, CD 38-, PCA-1-, FMC-7-, CD 23-) was consistent with a B-cell proliferation at an intermediate stage of differentiation but distinct from other well-defined B-cell neoplasms. Whether such unique B-zone pattern was due to an intrinsic property of this lymphoma or it is to be related to the coexisting reactive T-zone expansion remains controversial.

Spontaneous nonthymic T cell lymphomas in young Wistar rats.

Spontaneous acute lymphomas and related leukemias, occurring in three of 2,974 male Wistar rats used as controls in toxicity studies during the last 14 years (1974-1987), were examined by light and electron microscopy and by using immunohistochemistry. At autopsy, conspicuous hepatosplenomegaly was noted. Morphologically, tumor cells of all three rats were medium-sized lymphocytes with many mitotic figures proliferating mainly in the spleen, liver, and bone marrow. Virus-like particles were not detected. Immunohistochemically, almost all tumor cells were positive for thy-1 antigen but negative for hematopoietic and differentiation markers such as W3/13, W3/25, OX4, OX8, and OX12. The results suggest that the lymphomas in these three rats were derived from T cells.

Intravascular lymphomatosis (malignant angioendotheliomatosis). A B-cell neoplasm expressing surface homing receptors.

We report the clinical and immunohistochemical features of six cases of intravascular lymphomatosis. All patients presented with symptoms referable to central nervous system (CNS) involvement. In two patients, the diagnosis was made on brain biopsy specimens. In the remaining four cases, diagnosis was made at autopsy. Three of the four autopsied patients had focal extravascular lymphoma, diffuse large cell type. In all five cases tested, immunohistochemical stains on paraffin-embedded sections confirmed the lymphoid nature of the malignant cells. Stains on frozen sections in one case, and on paraffin-embedded tissue in the other four cases, demonstrated B-lymphocyte lineage. In the remaining case, electron microscopy confirmed the lymphoid nature of the tumor cells. All three cases tested expressed the Hermes 3-defined homing receptor antigen and lacked peanut agglutinin receptors. Our results are consistent with other reports confirming the lymphoid nature of so-called malignant angioendotheliomatosis. Our limited analysis of surface receptor molecules suggests that the expression of the homing receptor for high endothelial venules is not in itself responsible for the unusual intravascular location of these cells.

Correlation between NOR sizes and numbers in non-Hodgkin's lymphomas.

A series of 36 cases of non-Hodgkin's lymphomas (NHL) has been studied by means of the argyrophil (AgNOR) method for nuclear organizer regions (NORs). Morphometric analysis of highly magnified photographic images of light microscope preparations of the AgNORs was performed by means of an interactive image analysis system. It was observed that in the low-grade specimens (where NORs are less frequent than in those of high-grade histology), the AgNOR sites were highly significantly (P less than 0.001) larger than in high-grade NHL. In the low-grade lymphomas, the AgNOR maximum diameter (Dmax) ranged from 0.7 to 1.7 micron 2 (mean 1.11 micron 2) and area ranged from 0.48 to 1.99 micron 2 (mean 1.11 micron 2). In contrast, in the high-grade specimens, Dmax was from 0.33 to 0.51 micron (mean 0.41 micron) and the area ranged from 0.082 to 0.19 micron 2 (mean 0.13 micron 2). Thus, a well-defined inverse relationship was observed between AgNOR numbers and their sizes. There was total separation between low- and high-grade values in this series. This light microscope technique offers some advantages over ultrastructural morphometry of interphase NORs (fibrillar centres).

Establishment of two Epstein-Barr virus negative Burkitt cell lines from a patient with AIDS and B-cell lymphoma.

To analyze the pathogenesis of B-cell lymphomas in patients with acquired immunodeficiency syndrome (AIDS), we studied two cell lines, Es I and Es III, established from one such lymphoma for the presence of sequences of the Epstein-Barr virus (EBV) and the human immunodeficiency virus [HIV; lymphadenopathy-associated virus (LAV/HTLV-III)] as well as for the presence of cytogenetic abnormalities and monoclonal rearrangements of immunoglobulin and T-cell receptor genes. Both cell lines expressed the same IgM, kappa phenotype as the original lymphoma. The karyotype of Es I was 46, XY, t(8;14), 2 p+, inv (6p), 17p-, and the cells of Es III had an additional i(7q). Immunoglobulin gene studies demonstrated the identical monoclonal rearrangements in both cell lines. Neither EBV nor HIV sequences were detectable in the malignant B cells at the genomic level, leading to the conclusion that mechanisms other than transformation by EBV or HIV may have contributed to the B-cell lymphoma in this patient and possibly also to the generally increased frequency in patients with AIDS.

Morphologic studies of lymphocyte nuclei in follicular and diffuse mixed small- and large-cell (lymphocytic-histiocytic) lymphoma.

Twelve examples of mixed small- and large-cell lymphoma (eight follicular, one follicular and diffuse, and three diffuse) were investigated morphometrically using plastic-embedded tissue in order to study nuclear characteristics of lymphocyte populations in this form of non-Hodgkin's lymphoma (NHL) and to test morphologic bases for current NHL classification systems. This study illustrates that there are many inaccuracies, illusions, and misconceptions in the morphologic criteria currently used to classify mixed small- and large-cell lymphoma. A principal finding was that lymphocyte nuclear profiles in mixed-cell lymphomas tend to be smaller in size (P less than .005) and more irregular in shape (P = .0001) than the morphologically similar counterparts in germinal centers of lymph nodes with reactive hyperplasia. Intercase comparison of mixed small- and large-cell lymphomas revealed a considerable range of mean nuclear area values, some of which were within the size range of normal, small lymphocytes. At the magnifications used for morphometric assessment, a high proportion of lymphocyte nuclear profiles had shallow invaginations, but only a limited number of profiles (4% to 14%) had deep (cleaved) indentations. Contrary to current definitions for this subtype of NHL, lymphocytes with "small" nuclei had the same proportion of the nuclear diameter occupied by nuclear invaginations as lymphocytes with "large" nuclei and, in fact, mean nuclear invagination depth was shallower in "small" nuclei than in "large" nuclei. Furthermore, regardless of whether it is nuclear area or shape that is evaluated, lymphocytes in mixed-cell lymphoma do not separate into two populations of small-cleaved and large noncleaved cells. Morphometry reveals that only four of the 12 examples of mixed small- and large-cell lymphoma had a proportion of the lymphocytes in the size range of fully transformed germinal center lymphocytes that exceeded 25%, and none of the cases approached 50% even though the population of lymphocyte nuclei appearing "transformed," and therefore "large," ranged from 28% to 57%. Such results indicate that the large, noncleaved and cleaved component, as seen in histologic sections of mixed small- and large-cell lymphoma, do not have nuclei of uniform size and many, in fact, are not actually large. The morphometric findings indicate reasons for the poor observer reproducibility in classifying this subtype of NHL.

B lymphocytic lymphoma (large cell) of possible splenic marginal zone origin presenting with prominent splenomegaly and unusual cordal red pulp distribution.

Two cases of large cell lymphoma, B-cell type, primarily involving the red pulp of the spleen rather than the white pulp are described. A number of unusual features suggest that this may be a lymphoma originating from a distinct splenic B-cell lymphocyte whose origin may be the marginal zone of the spleen or the splenic cords. The patients presented with splenomegaly, cytopenias, and no peripheral lymphadenopathy. The gross appearance of the spleens was beefy red without tumor nodules. The tumor cells were primarily in the splenic cords and surrounding residual normal white pulp. There was a minimal hemic phase. The tumor cells had abundant cytoplasm, surface IgM, IgD, kappa, and FC receptors, tartrate-resistant acid phosphatase, but no alkaline phosphatase or interleukin-2 receptors. They had a similar DNA aneuploidy. The most unusual feature was that tumor cells in both cases had phagocytic properties. These lymphomas may be clinically more indolent than their follicular center counterparts.

Primary T-cell lymphoma of the thyroid.

A rare case of non-Hodgkin's malignant T-cell lymphoma of the thyroid in a 79-year-old woman is reported. Light microscopically, the tumor showed diffuse proliferation of lymphoid cells, revealing prominent nuclear irregularity and occasional multilobulated nuclei, and was diagnosed as a diffuse lymphoma of medium-sized cell type. Electron microscopically, the tumor cells were seen to possess irregularly indented nuclei. Immunologic surface marker studies showed that the lymphoma cells were positive for T-lymphocyte-lineage antigens, OKT4 and OKT11, and did not react with B-lymphocyte-associated antigens. These findings indicated that the lymphoma had a T-cell phenotype.

Observer variability in microcomputer-assisted morphometric study of nuclear parameters.

To evaluate observer variability during microcomputer-assisted planimetry, nuclear features (area, perimeter, and form factor) were studied in a case of malignant lymphoma. Tissue fixed in glutaraldehyde, embedded in plastic and sectioned at 1 micron was studied using a Zeiss Kontron IBAS 2000 image analysis system. Manual tracing of nuclear outline was performed to obtain the parameters. Both interindividual and intraindividual reproducibilities were evaluated, the former among 8 observers and the latter by the observers repeating the measurements. With the present technique, intraindividual reproducibility was high and correlation was excellent for area, perimeter and form factor measurements. A high degree of interindividual inconsistency was demonstrated for all parameters. Interindividual correlation was poor for form factor. It is concluded that poor interindividual reproducibility even when the objective method of morphometry is used may be a significant problem in tissue investigations, especially when results from different laboratories are being compared.

Immunoelectron microscopic localization of immunoglobulin in B-cell lymphomas.

Subcellular localization of immunoglobulin (Ig) by immunoelectron microscopy was performed on 20 B-cell lymphomas of low- and high-grade malignancy. The efficiency in demonstrating Ig by pre-embedding technique depends on the antibodies used. F(ab')2 fragments of antibodies were more sensitive than both intact polyclonal and monoclonal antibodies in detecting cytoplasmic Ig. With immunoelectron microscopy Ig could be demonstrated in all cell types of B-CLL and LP-immunocytoma, even in some of the small lymphocytes in B-CLL. Thus, the presence of intracytoplasmic Ig has no diagnostic relevance in differentiating B-CLL from LP-immunocytoma. However, the amount of Ig in the tumor cells of LP-immunocytoma seemed to be greater than in B-CLL. Centrocytic lymphoma and centroblastic/centrocytic lymphoma could be differentiated by their different localization of Ig. In centrocytic lymphoma Ig was localized mainly on the surface membrane, whereas in centroblastic/centrocytic lymphoma moderate amounts of Ig could be detected in the rough endoplasmic reticulum and perinuclear space of the centroblasts and in roughly one third of the centrocytes. In malignant lymphomas of high-grade malignancy (ML centroblastic, ML immunoblastic, and ML lymphoblastic) Ig was localized mainly in the rough endoplasmic reticulum and sometimes in the perinuclear space.