Mononuclear cells in normal colon and colonic carcinoma express the same T cell activation markers.

The proportion of mononuclear cells (MC) expressing some of the T cell activation markers in normal colon and colonic carcinomas was determined by immunoperoxidase staining and computer-assisted video image analysis (VIA). Tissue sections were stained with monoclonal antibodies against the T cell activation markers which included the MHC class II antigens DR, DP and DQ, interleukin 2 receptor (CD25) and the p180 (CD45RO) form of the leucocyte common antigen. The mean values for the proportion of leucocytes expressing these activation markers were 92% for DR, 61% for DP, 68% for DQ and 60% for CD45RO in tumours and 90, 62, 70 and 55% in normal tissue respectively. Few cells were stained for the IL2 receptor (mean values 7% for tumours and 5% for normal tissue) or the p220 form (CD45RA) of the leucocyte common antigen (mean values 6% for tumours and 4% for normal tissue). The proportion of MC bearing any of these markers in the normal colon was not significantly different from the matched colonic carcinomas.     

Density and phenotype of tumour-associated mononuclear cells in colonic carcinomas determined by computer-assisted video image analysis.

The density and phenotypes of tumour-associated mononuclear cells (TAMC) in tissue sections of colonic carcinomas was determined by the technique of video image analysis (VIA). This technique allowed an accurate and objective enumeration of both total mononuclear cells (MC) in H&E stained sections and individual types of cells as revealed by immunoperoxidase staining with monoclonal antibodies in frozen sections. This enumeration allowed reliable statistical analysis of the differences between sample groups. Using this technique it was found that the density of MC in histiologically normal tissue was significantly higher than in tumour tissue. Tumours from patients with the best prognosis (stage A) had significantly higher numbers of TAMC than stage B (P less than 0.02), C (P less than 0.002) and D (P less than 0.002) tumours. The differences in the density of TAMC between tumours obtained from stage B and C and that between C and D were not significant, whereas stage B had a significantly higher TAMC density than stage D tumours (P less than 0.05). Comparing tumour differentiation, well differentiated adenocarcinomas had a significantly higher (P less than 0.05) TAMC density than poorly differentiated tumours but not moderately differentiated tumours. Moderately and poorly differentiated adenocarcinomas did not differ significantly in the density of TAMC. In examining the phenotype of these cells, it was found that T lymphocytes formed the majority of the TAMC with the CD4+ subset predominating in 28 of 29 cases. Similarly, all sections of normal colon (taken at least 4 cm away from the tumour) had more CD4+ than CD8+ cells. The proportion of the total leucocyte population that was CD3+ was comparable in normal and tumour tissue. Generally, few macrophages were present in either tumour or normal tissues. B cells (CD21%) and subset of NK cells (CD57+) were not detected in the tumours. There were no significant differences in the proportion of leucocytes which were CD4+, CD8+ and CD14+ (macrophages) between the normal colon and the tumour tissues. The types of cells in the TAMC population did not differ with tumour stage or differentiation or with the density of the TAMC itself.     

Immunophenotyping of non-Hodgkin''s lymphomas using a panel of antibodies on paraffin-embedded tissues.

The use of monoclonal and polyclonal antibodies for the immunophenotyping of non-Hodgkin's lymphomas in paraffin-embedded tissue has been limited by the fact that most antigens on lymphoid cells are denatured by histologic fixation, dehydration, and embedment. In this article the authors have analyzed a small panel of antibodies which represent exceptions to this rule, in that they identify denaturation-resistant determinants on leukocyte antigens in paraffin-embedded tissue. Monoclonal antibodies L26 [corrected] and 4KB5 label preferentially B cells, monoclonal antibody UCHL1 stains predominantly T cells, and monoclonal antibody MAC 387 reacts with granulocytes and some macrophages. A polyclonal antiserum raised against purified CD3 (T3) antigen, a T-cell-specific molecule, was also employed. This antibody panel was used to immunophenotype routinely processed tissue biopsy specimens from 61 non-Hodgkin's lymphomas (all of which had been previously phenotyped in cryostat sections). The lineage of the neoplastic cells was correctly identified in 32 of 34 (94%) cases of B-cell lymphoma, in 19 of 19 (100%) cases of T-cell neoplasm, and in 2 of 4 (50%) cases of histiocytic malignancy. It is concluded that this combination of antibodies is helpful in immunophenotyping non-Hodgkin's lymphomas when only paraffin-embedded tissue sections are available, although additional reagents of higher specificity are required to improve the identification of lymphomas.     

Analysis of leukocyte surface antigens on ethanol-fixed paraffin-embedded tissue material

Ethanol-fixed paraffin-embedded specimens of human tissues were studied whether the surface antigens of leukocytes in these tissues can be stained and analyzed. Three-layer indirect immunoperoxidase staining was performed on the ethanol-fixed paraffin-embedded sections by the use of several monoclonal antibodies for whole human leukocytes (Dako LC), B cells (Dako CD-22, 4KB5, and L26; Leu 14), T cells and their subsets (Dako UCHL-1, T1, T3, T4 and T8; Leu 4, 3a and 2a) and monocyte/macrophage lineage (Dako macrophage, Leu M1, M3 and M5). The results were compared with those on fresh-frozen sections. No essential differences were obtained between the paraffin-embedded and the fresh frozen sections stained by the following antibodies; Dako LC for whole human leukocytes; Dako UCHL-1, T3 and Leu 4 for T cells; Dako CD22, 4KB5, L26 and Leu 14 for B cells; Dako macrophage, Leu M1 and M5 for monocyte/macrophage lineage. On the other hand, the subsets of T cells could only be detected on the fresh-frozen sections. The results of the leukocyte analysis on the paraffin-embedded specimens of several renal diseases were very similar to those reported by other investigators on fresh-frozen sections or PLP-fixed materials. Thus, by the use of appropriate monoclonal antibodies, the ethanol-fixed paraffin-embedded material can be used for leukocyte analysis except for the definition of T cell subsets.     

Monoclonal antibody (UCHL1) that recognises normal and neoplastic T cells in routinely fixed tissues.

UCHL1 is a murine monoclonal antibody that recognises a 180-185 kD determinant on CD4 (72%) and CD8 (36%) positive T cells. This antibody is effective in formalin fixed and paraffin embedded tissues, using the immunoperoxidase method. One hundred and forty three cases of malignant lymphoma were examined. Neoplastic cells in 100% of cases of Mycosis fungoides (n = 10), 83% of cases of peripheral T cell lymphoma (n = 25), and 78% of cases of (T-ALL) T acute lymphoblastic lymphoma (n = 9) were stained by this antibody. In addition, staining was seen in 100% of cases of malignant histiocytosis of the intestine (n = 13), a condition now thought to be a T cell lymphoma. Two cases of true histiocytic lymphoma were also positive. This antibody stained neither the neoplastic cells in a wide range of B cell lymphomas (n = 62) nor Reed-Sternberg cells in 16 cases of Hodgkin's disease. UCHL1 also stained neoplastic cells in four cases of granulocytic sarcoma. A panel of normal tissues was similarly studied. Staining was seen in normal T cells and mucosal intraepithelial lymphocytes, macrophages, mature myeloid cells, and endometrial stromal granulocytes. UCHL1 is a monoclonal antibody that identifies T cells in formalin fixed paraffin embedded tissues, and should prove useful for diagnosing T cell lymphomas, especially when only formalin fixed tissue is available for diagnosis.     

Use of the monoclonal antibody WR17, identifying the CD37 gp40-45 Kd antigen complex, in the diagnosis of B-lymphoid malignancy

The distribution of the gp40-45 Kd antigen bound by the WR17 monoclonal antibody of IgG2 subclass in normal lymphoid tissue was characterized by immunohistochemistry and immunofluorescence staining with flow cytometric analysis. The predominant staining pattern observed was characteristic of an anti-pan-B-lymphocyte reagent. Weak reactions were observed by immunofluorescence staining of viable cell suspensions with all neutrophils and T-lymphocytes in some normal donors. In tissue sections, B-lymphocytes were stained and no cross reactions were observed with T-lymphocytes, although macrophages stained in some sections. A range of T- and B-cell malignancies were stained with WR17 and the reactivity compared to that observed with other monoclonal antibodies in the CD19, CD21 and CD22 clusters. All B-non-Hodgkin's lymphomas, B-chronic lymphocytic, prolymphocytic and hairy cell leukaemia cells examined were stained by WR17 in indirect immunofluorescence assays, whilst the T-cell tumours were negative. The same pattern was observed in cryostat sections of malignant tissue and in addition some tissue macrophages expressed the CD37 antigen cytoplasmically. Intra-tumour heterogeneity of staining was observed with all the monoclonal antibodies tested, although overall WR17 consistently stained B-cell tumours even when expression of the CD19 pan-B-lymphocyte antigen could not be detected with some monoclonals. Monoclonal antibodies, such as WR17, within the CD37 cluster and binding to the gp 40-45 Kd molecule, bind to mature B-lymphocytes and identify the majority of B-cell malignancies.     

A novel monoclonal antibody (OPD4) recognizing a helper/inducer T cell subset. Its application to paraffin-embedded tissues.

A novel monoclonal antibody (MAb), OPD4, reactive with a helper/inducer (H/I) subset of T cells in formalin-fixed, paraffin-embedded tissue sections, has been identified through immunization with an activated H/I T cell line, namely DL40. The antibody is an IgG1 antibody and it recognizes an antigen with a molecular weight of 200 kd, corresponding to that of leukocyte common antigen. OPD4+/CD4+ T cells provided better help for pokeweed mitogen-stimulated polyclonal IgG production than OPD4-/CD4+ T cells. OPD4 recognized the H/I T cell subset even in paraffin-embedded tissue sections, but did not recognize nonhematopoietic cells, suppressor/cytotoxic T cells, B cells, monocytes in the peripheral blood, or other normal hematopoietic cells as examined by the flow cytometric and immunoperoxidase methods. Besides the lymphoid cells, OPD4 reacted with a number of histiocytes (epithelioid cells) in tissues from sarcoidosis and tuberculosis. For the neoplastic lesions, OPD4 reacted with approximately half of the cases of T cell lymphomas. Consequently, OPD4 may be useful for the diagnosis and study of malignant lymphomas and other related lesions.     

Monoclonal antibody OPD4 is reactive with CD45RO, but differs from UCHL1 by the absence of monocyte reactivity.

Monoclonal antibody OPD4 was described as a novel monoclonal antibody recognizing a helper/inducer T-cell subset in paraffin-tissue sections. The authors have analyzed this reagent with respect to its molecular weight, reactivity with leukocyte common antigen (LCA) transfectants and cell and tissue distribution. OPD4 was found to be reactive with the 180 kd molecular weight isoform of the leukocyte common antigen family, similar to antibody UCHL1 and therefore can be considered as a CD45RO reagent. The only difference in distribution was found to be the absence of OPD4 reactivity with peripheral blood monocytes. Since OPD4 recognizes the same antigen on T cells as UCHL1, both reagents do not need applied in the immunophenotyping of lymphomas.     

Immunohistological detection of T lymphocytes with monoclonal antibody BL-T2

Using the monoclonal Pan-T cell antibody BL-T2 T lymphocytes are identified on tissue sections with an indirect immunofluorescence technique. The T cell areas between the lymphoid follicles (B cells) are stained on cryostat sections of human palatine tonsils. A conventional heterologous anti-Ig antiserum was used for the detection of B lymphocytes. The identification of T and B lymphocytes by immunofluorescence allows statements about the distribution of both populations in tissue sections.     

The differential diagnosis of hairy cell leukemia with a panel of monoclonal antibodies

A panel of monoclonal antibodies reactive with normal human lymphoid cells and with hairy cells has been applied to the immunocytochemical analysis of hairy cell leukemia. Staining was performed by immunoenzymatic methods on frozen sections of bone marrow trephines and extramedullary tissues and on cell smears. Hairy cells reacted with antibodies against HLA-DR, leukocyte common antigen, B-cell antigens (antibodies To15 and B1) and with three anti-hairy cell monoclonal antibodies (S-HCL3, HC1, and HC2). Neoplastic cells in other B-cell lymphoproliferative disorders also expressed HLA-DR, leukocyte common, and B-cell antigens but were consistently negative for the antigen detected by monoclonal antibody S-HCL3. Furthermore, hairy cells differed from other neoplastic B-cells in that they were unreactive with monoclonal antibodies against C3b receptors, anti-Leu-1, Tü1, Tü33, and lacked a meshwork of dendritic reticulum cells. These findings establish a distinctive antigenic phenotype for hairy cell leukemia and indicate that it may be diagnosed reliably by immunoenzymatic labeling of tissue sections or cell smears.     

Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue.     

Stimulation of mucosal T cells in situ with anti-CD3 antibody: location of the activated T cells and their distribution within the mucosal micro-environment.

Mucosal T cells in explants of human fetal small intestine (17-20 weeks gestation) in organ culture were activated in situ using monoclonal anti-CD3 antibody. Changes in the distribution of T cells within the mucosa, and their phenotype, were monitored by immunohistochemistry on frozen sections. Anti-CD3 stimulated T cells (as determined by expression of CD25) were predominantly in the lamina propria and were rarely seen in the epithelium. In control cultures, after 72 h, CD3+ IEL decreased to low numbers compared to day zero. However, in cultures treated with anti-CD3, IEL numbers were maintained and in some experiments significantly increased compared to day zero levels. At onset of culture 50-60% of CD3+ IEL were CD4-, 8-, and virtually all were HML-1+. The T cell infiltrate into the epithelium induced by activation of lamina propria T cells with anti-CD3 was also mostly CD3+, 4-, 8-, HML-1+. These experiments provide strong evidence that increases in IEL numbers can be a consequence of lamina propria T cell activation.     

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.     

Alpha beta T cell receptor expression in rat intestinal epithelium

Intra-epithelial lymphocytes (IEL) in normal rat small intestine have been analysed for alpha beta T cell receptor (TcR) expression by immunoperoxidase histochemistry on frozen sections of gut, and by immunofluorescence on isolated cells. In frozen sections, a mean value of 61% of IEL were stained by the monoclonal antibody R73, which is specific for an invariant determinant of the alpha beta TcR. Analysis of isolated IEL by flow cytometry gave similar results and showed that 40-62% of IEL were stained by R73. Of the IEL population as a whole, 98% of cells were LCA+, 90% CD8+ and 10% CD4+. These results for alpha beta TcR expression in rat intestinal IEL closely parallel our recent data for mice, and are at variance with the view that this lymphoid compartment is dominated by gamma delta T cells.     

Immunohistochemical identification of mast cells in formaldehyde-fixed tissue using monoclonal antibodies specific for tryptase

An avidin-biotin enhanced immunoperoxidase procedure using monoclonal antibodies (AA1, AA3, and AA5) prepared against human mast cell tryptase resulted in intense staining of mast cells in paraffin-embedded tissue. The distribution of mast cells observed was similar to that seen when adjacent serial sections were stained using a standard procedure with toluidine blue, though the immunoperoxidase technique permitted the identification of significantly more mast cells. With monoclonal antibody AA1, immunostaining was entirely specific for mast cell granules, and there was negligible background staining in a range of tissues including lung, tonsil, colon, gastric mucosa, skin, and pituitary. There was no staining of antibody on basophils or on any other normal blood leukocyte. The technique was effective with tissue fixed in either Carnoy's or neutral buffered formalin, though the internal mast cell structure was better preserved with formaldehyde fixation. The immunoperoxidase staining procedure with monoclonal antibody AA1 is a highly specific and sensitive means for the detection of mast cells in routinely processed tissues.     

Interstitial mononuclear cell infiltrates in chronic rejection of the kidney and correlation with peripheral blood.

To investigate the characteristics of interstitial inflammatory cells and possible involvement of nudelta T cells, 16 renal allograft biopsies showing chronic rejection were stained by immunohistochemical method and correlated with the data of peripheral blood evaluated by flow cytometry. For immunophenotyping, fresh frozen sections were stained with monoclonal antibodies against CD3, CD4, CD8, CD68, CD56, TCRdelta1 and HLA DR. Paraffin embedded tissue was stained with CD45RO, CD20-Cy and CD68. Nine cases of nonspecific tubulointerstitial change and 4 cases of nonallograft tubulointerstitial nephritis were used as a control. Inflammatory infiltration was present in all cases studied. T cells predominated in the interstitium of chronic rejection and were followed by macrophages and B cells. The degree of interstitial infiltration of frozen section was not accordant with that of paraffin sections. Allografts with nonspecific tubulointerstitial changes or tubulointerstitial nephritis of native kidneys showed similar distribution pattern in terms of type and degree. However, the degree of infiltrate did not give any statistical significance among groups. The CD4/CD8 ratios in interstitial infiltrates were less than 1.0 in 6 cases and was not accordant with those of peripheral blood. Proportion of nudelta T cells increased over 10% in 2 cases in tissue and in 3 cases in peripheral blood. In 3 cases of chronic rejection in which both tissue and blood results were available, there was no concordance of CD4/CD8 or nudeltaT/CD3 between them. Tubular expression of HLA DR was, however, present only in 4 cases of chronic rejection. In conclusion, T lymphocytes were predominant regardless of diagnosis or disease activity. T lymphocyte subset did not give any suggestion as to the diagnosis or disease activity in chronic rejection. Furthermore nudelta T cells had only limited value. Lymphocytic subsets in peripheral blood would not be predictors of tissue destruction in chronic rejection.     

Localization of T-Lymphocyte Areas in Human Lymphoid Tissues by Fluorochrome-Labelled Helix pomatia A Haemagglutinin

Fluorochrome-labelled Helix pomatia A haemagglutinin (HP) stained T-dependent areas in cryostat tissue sections of human lymph node, tonsil, and spleen and thymocytes within thymic medulla but not within thymic cortex. Neuraminidase treatment of the tissue section was a prerequisite for the positive cell staining. The staining was cell-membrane-associated, and the histological pattern was essentially the same as that observed in parallel sections treated with monoclonal antibodies to T cells (OKT3, Leu-I). In addition, blood vessel walls and connective tissue were also stained by HP conjugates. The latter staining was independent of neuraminidase treatment, resistant to α-galactosidase treatment and was not blocked by pretreatment with anti-fibronectin antibody. Both types of staining reaction were specific inasmuch as unlabelled HP and the competitive sugar hapten N-acetyl-D-galactosamine blocked the reaction. Frozen, formalin-fixed tissue sections showed the same stamability and reaction pattern as unfixed sections.     

Desmin as an immunochemical marker of human decidual cells and its expression in menstrual fluid

The expression of intermediate filament proteins in human endometrial tissue was examined. Desmin was selectively expressed in decidualized stroma, as demonstrated by SDS-PAGE analysis and positive response with a monoclonal antibody specific for desmin in ELISA and in western blot analysis. The same monoclonal antibody specifically stained human decidual cells in decidualized endometrium (secretory endometrium) in formalin-fixed paraffin-embedded sections prepared from diagnostic curettage samples. Desmin was also detected in menstrual fluid. Therefore, desmin might serve as a biochemical and histochemical marker of human decidualized endometrium.     

Immunohistochemical application of an antibody specific for human CD1a to the diagnosis of canine mast cell tumour

Canine mast cell tumours (MCTs) may be graded microscopically for prognostic purposes. Grade I (well-differentiated) and grade II (intermediate differentiation) tumours have an abundance of metachromatic granules within the cytoplasm; however, grade III (poorly differentiated) MCTs may be difficult to diagnose as they frequently have fewer discernable granules. Herein we report that a cross-reactive anti-human CD1a monoclonal antibody (clone O10) may be used in immunohistochemistry to identify canine MCTs of all grades. The antibody was applied to tissue sections from 48 canine MCTs of different histological grades. Serial sections from each tumour were stained with toluidine blue and safranin O to compare diagnostic sensitivity. All MCTs were labelled positively by the CD1a antibody, but histochemical staining was often equivocal and identification of mast cells was extremely difficult in some cases. This antibody did not label neoplastic cells in cases of canine histiocytoma, plasmacytoma or amelanotic melanoma; therefore, the reagent may be a valuable marker for the diagnosis of canine MCTs, especially those tumours of histological grade III.     

Immunoarchitecture of normal human bone marrow: a study of frozen and fixed tissue 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.