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An immunohistologic study of bone marrow biopsy frozen sections from 42 cases involved by a variety of reactive and neoplastic disorders is presented. Thirteen cases also were studied using other methods, including cytochemistry, surface marker analysis of cell suspensions, and/or DNA hybridization. Thirty-four of 42 cases (81%) were adequately phenotyped on frozen tissue using a panel of antibodies for hematolymphoid-associated antigens. The immunostains from the remaining eight cases were unsatisfactory, primarily as a result of heavy background staining. Eighteen cases were lymphoproliferative disorders of B-cell phenotype and 12 of these showed surface monotypic immunoglobulin expression by the frozen section technique. Six cases showed B- or pre-B-cell antigens but no surface immunoglobulins. Of the remaining 16 patients, two cases showed myeloid markers and three showed T-cell phenotype. Nine cases showed a mixture of polyclonal B- and T-cell populations. Keratin was demonstrated in a single case of metastatic carcinoma included in the study. These results indicate that the majority of hematopoietic processes can be successfully phenotyped on bone core frozen sections and demonstrate the usefulness of immunohistologic study of the frozen bone marrow biopsy specimens, especially when the specimens for other modalities are not available or are inadequate. The keys to achieving the best results from the frozen bone marrow immunohistochemistry were the gentle handling of the specimens and the preparation of high-quality, cryostat-cut frozen sections.

To provide baseline information on the immunoarchitecture of normal bone marrow, we studied cryostat-cut, frozen, and paraffin-embedded, fixed tissue sections prepared from 21 core biopsies of normal bone marrow obtained during bone marrow harvests for transplantation. A large panel of antibodies was applied that included, for frozen tissue, Leu-6 (CD1), T11 (CD2), Leu-3a (CD4), Leu-1 (CD5), Leu-2a (CD8), J5 (CD10), My7 (CD13), Leu-11 (CD16), B4 (CD19), B1 (CD20), B2 (CD21), Tac (CD25), My9 (CD33), T200 (CD45), NKH-1 (CD56), kappa and lambda chains, beta F1, Ki-67, HLA-DR, TQ1, and keratin, and for fixed tissue, leukocyte common antigen (CD45), L26 (CD20), LN1 (CDw75), LN2 (CD74), LN3, LN4, LN5, MB1 (CD45R), MB2, MT1 (CD43), MT2 (CD45R), UCHL1 (CD45R0), BM1, Ki-1 (CD30), Leu-M1 (CD15), lysozyme, KP1 (CD68), actin, S100, neuron-specific enolase, vimentin, and keratin. On fresh-frozen sections CD19 and CD2 were the most reliable and sensitive markers for B and T cells, staining 5% and 9% of marrow cells, respectively. Immunoglobulins generally showed heavy background staining, which frequently precluded an accurate assessment. The CD4 to CD8 ratio in the bone marrow was reversed from that of peripheral blood. On fixed tissues, leukocyte common antigen was found in 14% of the marrow cells, corresponding roughly to the lymphocyte population. L26, a pan-B-cell marker, stained 3% of the marrow cells. Among the other B-cell markers, LN1 and MB2 stained a large number of cells (40% to 70%), indicating reactivity with cells of the myeloid or erythroid series in addition to lymphocytes. Among the T-cell markers, UCHL1 and MT1 stained 66% and 50% of the cells, respectively, which could be explained by their cross-reactivity with myeloid cells. Nonspecific myelomonocytic markers (Leu-M1, KP1, and lysozyme) also showed reactivity in a high percentage of cells. No particular architectural distribution patterns of B or T lymphocytes were noted in either frozen or fixed bone marrow specimens. The results of this study provide normal baseline data for the immunohistologic application of hematopoietic and lymphoid markers on frozen or fixed bone marrow biopsy specimens.

The pathologic diagnosis of malignant mesothelioma is often difficult, even with the benefit of special studies such as histochemistry, electron microscopy, and immunohistochemistry. Ber-EP4 is a newly characterized monoclonal antibody that reliably labels epithelial tissues but does not react with mesothelial cells. We evaluated Ber-EP4 on formalin-fixed, paraffin-embedded tissue sections from 115 malignant mesotheliomas and 83 adenocarcinomas. Although 72 cases (87%) of adenocarcinoma were positive for Ber-EP4, only one (1%) of the mesotheliomas was reactive. The only adenocarcinomas that did not stain were from the breast (eight of 25 cases nonreactive) and the kidney (all three cases nonreactive). The staining pattern in the positive adenocarcinomas was usually intense and membranous, but additional weak cytoplasmic staining was seen in many cases. The reactivity was diffuse in 59 cases and focal in 13 cases. The results of our study suggest that the Ber-EP4 antibody may have great use in the differential diagnosis of mesothelioma versus adenocarcinoma, particularly when only formalin-fixed tissue is available.

There is a significantly increased incidence of malignant lymphoma in patients with acquired immunodeficiency syndrome (AIDS). The lymphomas are usually of a high grade and of B-cell phenotype. While the frequent presence of reactive monocytoid B lymphocytes in patients with AIDS-related lymphadenopathy has recently been documented in several studies, to our knowledge, there are no reported cases of monocytoid B-cell lymphoma, the neoplastic counterpart of monocytoid B lymphocytes, in patients with AIDS. We now describe a human immunodeficiency virus (HIV)-positive patient with HIV-related lymphadenopathy in whom monocytoid B-cell lymphoma developed during the course of his disease. The morphologic and immunologic features of the lymphoma were characteristic of monocytoid B-cell lymphoma, and the involved lymph node exhibited a reversed CD4/CD8 ratio. Moreover, using the polymerase chain reaction, we were able to demonstrate HIV genome in DNA extracted from the lymph node tissue. To our knowledge, this is the first report of a case of monocytoid B-cell lymphoma occurring in an HIV-positive patient and in which we were able, by using a sensitive molecular biologic technique, to demonstrate HIV sequence in paraffin-embedded, fixed lymph node sections.

Hodgkin's disease (HD) is characterized morphologically by a variable infiltration of tissues by eosinophilic granulocytes. The lesions also contain numerous T cells, predominantly of the CD4+ immunophenotype. To investigate whether the presence or absence of tissue eosinophilia is related to the immunophenotype of the T cells, we studied 43 cases of HD (28 nodular sclerosing, ten mixed cellularity, and five unclassifiable) for the relative numbers of lymphocytes positive for CD2, CD3, CD4, CD5, CD8, CD25, CD38, T9, TQ1, HLA-DR, and beta F1, and for the number of eosinophils in tissue sections. By univariate and multivariate analysis, we determined that there was an inverse relationship between the number of eosinophils and the presence of TQ1+ (P less than .0005) and CD25+ (P less than .0005) lymphocytes. In addition, we observed that TQ1 stained the Reed-Sternberg cells in these lesions. We also determined that the T cells expressed HLA-DR more frequently in the nodular sclerosis subtype than in other subtypes of HD (P less than or equal to .0001). We therefore conclude that the degree of tissue eosinophilia in the lymph nodes of patients with HD may be explained, at least in part, by the immunophenotype of the T cells present in the affected lymph nodes.