Lymphocyte Subsets and Activation Antigens in a Reference Population: A Flow Cytometric Study Using Single and Double Antibody Staining

The transferrin receptor (TR), the HLA DR antigen (DR), and the antigen binding OKT10 (T10) are present on activated lymphocyte populations. The authors have studied their expression and that of antigens defined by eight commercial monoclonal antibodies on peripheral blood lymphocytes of 50 healthy hospital workers aged 23–60 years. A whole blood lysis technique was employed and cells were enumerated on a flow cytometer. The percentage of cells bearing the three activation antigens were generally low: mean values for T10 being 7.2%; for TR 1.8%; and for DR 8.8%. There was, however, considerable variability, with occasional subjects having 20% or more cells positive for one of the three antigens. High values of one activation antigen did not correspond with high values of another in the same subject. Nor was there correlation of the presence of activation antigens with the occurrence of cells bearing T11, T4, T8, Leu 1, Leu 2a, Leu 3a, or Leu 7. Double labelling with the following pairs of fluorescein (FITC) and phycoerythrin (PE) labeled antibodies: Leu 2a, DR; Leu 3a, DR; Leu 3a, Leu 2a; TR, DR, indicated that simultaneous expression of the corresponding antigens do not normally occur on lymphocytes of healthy individuals. Double labelling with B1 and DR in five subjects indicated the presence of B1 (+) DR (-) cell population. No pattern of relationship could be detected among common clinical variables or HLA type and an increased expression of activation antigens.     

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.     

Lymphocyte subsets and activation antigens in a reference population: a flow cytometric study using single and double antibody staining

The transferrin receptor (TR), the HLA DR antigen (DR), and the antigen binding OKT10 (T10) are present on activated lymphocyte populations. The authors have studied their expression and that of antigens defined by eight commercial monoclonal antibodies on peripheral blood lymphocytes of 50 healthy hospital workers aged 23-60 years. A whole blood lysis technique was employed and cells were enumerated on a flow cytometer. The percentage of cells bearing the three activation antigens were generally low: mean values for T10 being 7.2%; for TR 1.8%; and for DR 8.8%. There was, however, considerable variability, with occasional subjects having 20% or more cells positive for one of the three antigens. High values of one activation antigen did not correspond with high values of another in the same subject. Nor was there correlation of the presence of activation antigens with the occurrence of cells bearing T11, T4, T8, Leu 1, Leu 2a, Leu 3a, or Leu 7. Double labelling with the following pairs of fluorescein (FITC) and phycoerythrin (PE) labeled antibodies: Leu 2a, DR; Leu 3a, DR; Leu 3a, Leu 2a; TR, DR, indicated that simultaneous expression of the corresponding antigens do not normally occur on lymphocytes of healthy individuals. Double labelling with B1 and DR in five subjects indicated the presence of B1 (+) DR (-) cell population. No pattern of relationship could be detected among common clinical variables or HLA type and an increased expression of activation antigens.     

A histochemical and immunohistochemical study of extra-ocular sebaceous carcinoma

A histochemical and immunohistochemical study of five cases of extra-ocular sebaceous carcinoma was performed using formalin-fixed and paraffin-embedded tissue specimens. Histochemically, the clear cells of sebaceous carcinomas were negative with periodic acid-Schiff and alcian blue staining. Immunohistochemically, the tumour cells of sebaceous carcinomas showed positive reactions for epithelial membrane antigen, human milk fat globules subclass 1, human milk fat globules subclass 2 and Leu M1, but did not express carcinoembryonic antigen, breast carcinoma associated antigen, S-100 protein, gross cystic disease fluid protein-15 or Dako M1. These histochemical and immunohistochemical findings were compared with those of other skin cancers which must be distinguished histopathologically from sebaceous carcinoma. We conclude that sebaceous carcinoma can be distinguished from eccrine porocarcinoma, malignant clear cell hidradenoma, extramammary Paget's disease, malignant trichilemmoma, squamous cell carcinoma and basal cell carcinoma by histochemical and immunohistochemical techniques using formalin-fixed and paraffin-embedded tissue specimens.     

Clonal expression of differentiation and Ia-like antigens on alloreactive human T lymphocytes

A series of human T lymphocyte clones showing specific responses for major histocompatibility complex-encoded alloantigens were isolated from a single culture. They were classified into distinct functional groups based on measurement of alloantigen-specific proliferation and cytotoxicity. Surface antigen expression on these clones was analyzed using a panel of monoclonal antibodies specific for T cell differentiation antigens and for Ia-like antigens. Four distinct groups were identified based on the Leu series of differentiation antigens: (a) Leu1+,2+,3+,4+; (b) Leu1+,3+,4+; (c) Leu1+,2+,4+; and (d) Leu2+,4+. Functionally distinct clones showed not only differential expression of Leu2 and Leu3 , antigens which had previously been shown to distinguish helper/inducer and cytotoxic/suppressor cells, but also of Leu1, an antigen classified as a common T cell marker. All of the clones were found to express Ia antigens as detected by three framework monoclonal antibodies, but none were found to express a determinant associated with the DR3 allospecificity , although it was found on normal B lymphocytes and a B cell line established from the HLA-DR3 individual from whom these clones were derived.     

A human natural killer cell-associated antigen defined by monoclonal antibody anti-Leu (NKP-15): functional and two-color flow cytometry analysis

A monoclonal antibody, anti-Leu 11a (NPK-15), was generated against human large granular lymphocytes (LGL). Anti-Leu 11a reacted with the majority of Percoll gradient-enriched LGL cells, a subpopulation of peripheral blood lymphocytes (PBL) approx (approximately 10-20%), and most granulocytes, but not with a significant number of monocytes, T lymphocytes, or erythrocytes. Cell sorting experiments demonstrated that the Leu 11a+ population encompassed essentially all functional natural killer (NK) cells in the peripheral blood. Two-color flow cytometry analysis of PBL populations stained with anti-Leu 11a and anti-Leu 7 revealed the existence of four distinct populations: Leu 11a-, 7+; Leu 11a+, 7-; Leu 11a+, 7+; and Leu 11a-, 7-. The Leu 11a+ population did not appear to include cells marked with the T cell-associated antigens Leu 1, Leu 2, or Leu 3. The existence of a cell surface antigen common to granulocytes and NK cells, which is capable of distinguishing subpopulations of Leu 7+ cells, provides a useful probe to analyze the nature of the NK lineage.     

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.     

Immunohistological phenotyping of thyroid infiltrating lymphocytes in Graves'' disease and Hashimoto''s thyroiditis.

Subsets of lymphocytes in the thyroid were immunophenotyped by their surface antigens in frozen tissues of Hashimoto's thyroiditis and Graves' disease. Using triple layer immunoperoxidase staining (IP), monoclonal antibodies (T3, Leu 3, T8, anti-Tac and Leu 7) were employed to detect markers of T cell subsets, activated T cells, and a natural killer associated antigen. B cells were identified by 2 step IP with anti-IgD antisera. Excluding those cells forming lymphoid follicles, the density of lymphocytes infiltrating between thyroid epithelial cells was much higher in Hashimoto's thyroiditis than in Graves' disease. However, relative proportions of subsets were similar in both diseases. Most of the infiltrating cells were T3 positive T cells (T3+), with more T8+ (suppressor/cytotoxic T) than Leu 3+ (inducer/helper T). Some Leu 7+ were occasionally seen, but surface IgD positive mature B cells (IgD+) were almost absent. In contrast, IgD+ cells were densely aggregated in primary lymphoid follicles and mantle zones of secondary follicles. In these regions, Leu 3+ cells were about twice as frequent as T8+ cells. Some Leu 7+ and scarce Tac+ cells were also found. The present study indicates a major involvement of immunoregulatory T cells in autoimmune thyroid disease, and also suggested intrathyroidal maturation of B cells.     

Cellular and humoral immune reactions in chronic active liver disease. II. Lymphocyte subsets and viral antigens in liver biopsies of patients with acute and chronic hepatitis B

The characteristics and distribution of the inflammatory infiltrate in liver biopsies of 25 patients with hepatitis B viral (HBV) infection were studied in relation to the distribution and expression of HBV antigens. Mononuclear subsets were characterized with monoclonal (OKT, OKM, Leu) antibodies to surface antigens. For the demonstration of viral antigens directly conjugated antibodies to surface (HBsAg), core (HBcAg) and 'e' (HBeAg) antigen were used. For the study of mutual relations all methods were performed on serial cut tissue sections. In chronic active hepatitis B (CAH-B, n = 12) OKT8+ lymphocytes of T cell origin were the only cell type present in areas with liver cell degeneration and T cell cytotoxicity appears to be the only immune mechanism. In chronic persistent hepatitis B (CPH-B, n = 7) the only conspicuous feature was the presence of many Leu 3+ lymphocytes of the helper/inducer population in the portal tracts. In acute hepatitis B (AHB, n = 6) OKT8+ cells of non-T origin (OKT1-,3-) and Leu 7+ cells of presumed natural killer (NK) potential predominated in the areas with liver cell necrosis, and non-T cell cytotoxicity appears to be the predominant immune mechanism. In none of these disease entities a positive spatial relation could be established between the cytotoxic cells and the demonstrable expression of HBV antigens in hepatocytes. It is concluded that differences in immunological reaction pattern may explain the different course in the three forms of HBV infection studied.     

Cross-reactive antigenicity of nucleoproteins of lyssaviruses recognized by a monospecific antirabies virus nucleoprotein antiserum on paraffin sections of formalin-fixed tissues

Diagnosis of rabies is routinely confirmed by detection of rabies virus antigens in acetone-fixed frozen brain tissues or imprint smears using an immunofluorescence method with commercial antirabies virus antibodies. Since recent molecular analyses disclosed wide heterogeneity in the genome sequences of rabies virus strains and related lyssaviruses, it is necessary to confirm the presence of common epitopes in these lyssaviruses. In this study we confirmed the presence of cross-reactive antigens of various lyssaviruses in paraffin sections of formalin-fixed tissue using a monospecific rabbit antiserum prepared by immunization with a recombinant nucleoprotein of rabies virus. By immunohistochemical application, the antigen was detected predominantly in the cytoplasm of neurons in the brains of mice infected with rabies virus, Duvenhage virus, Mokola virus and European bat lyssavirus-1, while no cross-reaction was observed in uninfected humans and animals including dogs, bats, and raccoons. In addition, we examined one autopsy case that was infected in a rabies-endemic nation and developed the clinical manifestation of rabies after returning to Japan in 1970, and found that the antigen was well preserved in paraffin sections of formalin-fixed tissues. Thus, this suggests that the lyssavirus-specific antigen is recognized by the monospecific antibody against rabies virus nucleoprotein, and that this cross-reactive antigen is detectable on formalin-fixed paraffin-embedded tissues by immunohistochemical analysis.     

Mononuclear-cell subsets in human idiopathic crescentic glomerulonephritis (ICGN): Analysis in tissue sections with monoclonal antibodies

Mononuclear inflammatory cells (MIC) were analyzed in renal biopsies from 16 patients with ICGN (7 with glomerular immune complex deposits, 3 with anti-GBM disease, and 6 without immune deposits) by the avidin-biotin-immunoperoxidase technique utilizing monoclonal antibodies to cell surface antigens: T11 (total T), T4 (inducer/helper T), T8 (suppressor/cytotoxic T), B1 (B cells), M1 (monocytes/granulocytes), and Leu 7 [natural killer (NK) cells]. Total MIC were significantly increased in both glomeruli and interstitial tissues of the patients. Interstitial MIC consisted mainly of lymphocytes (80%) and monocytes (19%), with small numbers of B and NK cells present. In contrast, MIC in renal glomeruli of patients with ICGN were composed of monocytes (65%) rather than T lymphocytes (34%). A majority of T lymphocytes found in renal tissues of patients and controls had the helper/inducer phenotype. Tissue T4/T8 ratios were not significantly different in the glomeruli and interstitium. Monocytes and T lymphocytes accumulating in renal tissues of patients with ICGN may mediate glomerular injury in all forms of human ICGN.Presented in part at the 15th Annual Meeting of the American Society of Nephrology, Chicago, Illinois, 1982.     

Surface markers of primate B and T lymphoid cell lines identified by antibodies to human and simian lymphocyte antigens

Simian B and T lymphoid cell lines were shown to maintain surface markers found on mature lymphocytes in vivo. The T lymphoid cell lines expressed Ia-like antigens on their surfaces, further suggesting that they represent mature, activated T cells. These Ia antigens show a structural similarity to Ia on human cells although some diversity exists. The Ia antigen expressed on T lymphoid cell lines was shown to be very similar to those on B lymphoid cell lines. Owl monkey and marmoset T lymphoid cell lines were also shown to express a VH immunoglobulin-related determinant, a marker which is thought to be associated with T cell antigen receptor. Owl monkey and marmoset T cell lines express a surface antigen which identifies the sheep erythrocyte receptor on human T cells and some of these lines express an antigen found on human helper T cells. It is noteworthy that substantial conservation of surface components has occurred within primate evolution such that monoclonal antibodies to human Ia, OKT-11a and Leu 3a markers can be used to type lymphocytes of lower primates.     

Immunophenotypic and genotypic characterisation of multiple myelomas with adverse prognosis characterised by immunohistological expression of the T cell related antigen CD45RO (UCHL-1).

AIMS: To investigate whether plasma cell expression of early B cell, late B cell/preplasma cell, T cell, and myelomonocytic antigens or myeloma associated lymphocytic infiltrates correlated with prognosis in bone marrow biopsy specimens of patients with multiple myeloma. METHODS: Bone marrow biopsy specimens of 23 patients with multiple myeloma were investigated for plasma cell expression and interstitial lymphocyte expression of T cell related antigen CD45RO (UCHL-1). RESULTS: Eight patients showed plasma cell expression of CD45RO and 16 showed increased tumour infiltrating CD45RO positive lymphocytes, which were correlated with poor survival by multivariate analyses (p = 0.005 and p = 0.04, respectively). B cell antigens (MB2, CD20) but no T cell specific antigens (CD3) or T cell receptor gene rearrangements were expressed by plasma cells in CD45RO positive myelomas. Of 16 patients with myeloma who had increased tumour infiltrating CD45RO positive lymphocytes, four had interstitial lymphocyte expression of B cell antigens and two had interstitial lymphocyte expression of the T cell specific antigen CD3. CONCLUSIONS: The recognition of plasma cell expression of CD45RO and increased interstitial CD45RO lymphocytes in bone marrow biopsy specimens of patients with multiple myeloma is an adverse prognostic finding not indicative of an aberrant T cell phenotype or genotype; it is consistent with B cell/pre-plasma cell antigen expression by myeloma cells and their lymphocytic precursors.     

A monoclonal antibody (OPT1) to T cells which is available for paraffin-embedded materials

A monoclonal antibody (MAb), OPT1, reactive with T cells in formalin-fixed, paraffin-embedded tissue sections, has been identified through immunization with activated T cells from peripheral blood lymphocytes (PBL). The antibody is an IgG1 antibody as demonstrated by the Ouchterlony technique. By cytofluorometric analysis, almost all CD3+ lymphocytes and only a few CD20+ lymphocytes of peripheral blood expressed the OPT1 antigen. Nonhematolymphoid cell lines were negative for OPT1 by the immunoperoxidase staining using acetone-fixed cell lines. On the contrary, peripheral T cells, cells of two T cell lines out of four and a part of the cells of one B cell line out of two were positive for OPT1. The immunoperoxidase staining of paraffin-embedded tissue sections revealed that most of lymphocytes in T cell areas of lymph nodes expressed OPT1 antigen. Some lymphocytes in both cortex and medulla of the thymus and erythroid precursors of the bone marrow were OPT1+. In the malignant lymphoma series, approximately 90% of T cell lymphomas and 6% of B cell lymphomas reacted with OPT1. None of the Reed-Sternberg cells nor Hodgkin cells in Hodgkin's disease were positive. Consequently, OPT1 may be useful for the diagnosis and study of malignant lymphomas and other related lesions.     

Usual interstitial pneumonitis is a T-cell alveolitis

Usual interstitial pneumonitis (UIP) is an idiopathic inflammatory disorder that produces scarring of the lung parenchyma. We studied open-lung biopsies of 13 patients with UIP using immunohistological staining and monoclonal antibodies. T lymphocytes (Leu 4+) accounted for 59% of cells in the alveolar septal infiltrates in UIP and OKT8+ cells accounted for the majority of T lymphocytes in most cases. OKM1+ granulocytes comprised a smaller percentage (14%) of the alveolar infiltrates. Granulocytes were most frequent within cystic airspaces and inflamed small airways. Class II HLA (Ia) antigens were expressed on lymphocytes, macrophages, endothelial cells, and alveolar type II cells in lungs with UIP. This study demonstrates that altered immunoregulatory subsets are present in the lungs of patients with UIP and suggests the possibility that activated T cells may play a role in the pathogenesis of this disorder.     

Enzyme-labeled Antigen Method: Factors Influencing the Deterioration of Antigen-binding Activity of Specific Antibodies during Formalin Fixation and Paraffin Embedding

The enzyme-labeled antigen method is an immunohistochemical technique detecting plasma cells producing specific antibodies in tissue sections. The probe is an antigen labeled with an enzyme or biotin. This immunohistochemical technique is appliable to frozen sections of paraformaldehyde (PFA)-fixed tissues, but it has been difficult to apply it to formalin-fixed, paraffin-embedded (FFPE) sections. In the current study, factors inactivating the antibody reactivity during the process of preparing FFPE sections were investigated. Lymph nodes of rats immunized with horseradish peroxidase (HRP) or a mixture of keyhole limpet hemocyanin/ovalbumin/bovine serum albumin were employed as experimental models. Plasma cells producing specific antibodies, visualized with HRP (as an antigen with enzymatic activity) or biotinylated proteins in 4% PFA-fixed frozen sections, significantly decreased in unbuffered 10% formalin-fixed frozen sections. The positive cells were further decreased by paraffin embedding following formalin fixation. In paraffin-embedded sections fixed in precipitating fixatives such as ethanol and acetone and those prepared with the AMeX method, the antigen-binding reactivity of antibodies was preserved. Fixation in periodate-lysine-paraformaldehyde and Zamboni solution also kept the antigen-binding reactivity in paraffin to some extent. In conclusion, formalin fixation and paraffin embedding were major causes inactivating antibodies. Precipitating fixatives could retain the antigen-binding reactivity of antibodies in paraffin-embedded sections.     

Monoclonal antibodies reactive with normal and neoplastic T cells in paraffin sections

Without fresh or frozen tissue, it previously has been impossible to confirm the T-cell nature of reactive or neoplastic lymphoid cells. The availability of antibodies reactive with T cells in paraffin sections now allows retrospective analysis of a large number of cases. Two commercially available monoclonal antibodies, MT1 and MT2, were tested for their reactivities with T cells in a wide range of formalin-fixed, paraffin-embedded tissues, including 130 cases of immunologically characterized lymphoma. In reactive lymph nodes, MT1 stained the T-cell areas, whereas MT2 stained both the T-cell areas and mantle-zone B lymphocytes. MT1 stained 38 of 55 T-cell lymphomas (69.1%; 94.7% of cases from one hospital that used a shorter fixation time, and 55.6% of cases from another hospital that used a longer fixation time). MT2 stained only 6 (10.9%) of the T-cell lymphomas. Among the 74 cases of B-cell lymphoma, 3 (4.0%) were stained by MT1 and 30 (40.5%) by MT2.MT1 was also reactive with 3 of 4 cases of granulocytic sarcoma, as expected from its reactivity with normal granulocytes. Neither MT1 nor MT2 stained Reed-Sternberg cells or their variants in HodgKin's disease. We conclude that MT1 is a valuable marker for T cells, particularly when used with a panel of antibodies reactive with B cells in paraffin sections. MT2 is of limited value because of its cross-reactivity with many B-cell lymphomas.     

Immunophenotypic diagnosis of leiomyosarcomas and rhabdomyosarcomas with monoclonal antibodies to muscle-specific actin and desmin in formalin-fixed tissue

Two fibrillary proteins, muscle-specific actin (MSA) and desmin, are found only in cells of smooth and skeletal muscle lineages. Among the monoclonal antibodies (MAbs) to these antigens which we have tested, we found several to be reactive in formalin-fixed, paraffin-embedded sections. This finding widened the possibility of using these MAbs in routine diagnostic surgical pathology for the immunodiagnosis of rhabdomyosarcomas (RMS) and leiomyosarcomas (LMS). We therefore conducted a comparative study of three such MAbs which are available commercially and which we applied to paraffin-embedded, formalin-fixed tissues from 15 patients with RMS and 19 patients with LMS. The case selection criteria included typical light-microscopic appearances as well as immunoreactivity with at least one of the MAbs. MSA was detected in all cases of RMS and LMS, whereas desmin was reactive in 12 of 13 RMS and 10 of the 19 LMS. (Desmin antigenicity was judged to be lost in two RMS, since the vascular smooth-muscle tissue present in the specimens failed to react with these antibodies.) In LMS, desmin tended to show focal positivity, whereas the MSA in the same specimens was diffusely positive. These results demonstrate the utility of MAbs for confirmation of the muscle lineage of LMS and RMS in formalin-fixed, paraffin-embedded tissue. The results also indicate that, with the MAbs tested, the antigenicity of MSA is preserved more consistently than that of desmin in formalin-fixed, paraffin-embedded tissue, and that MSA is a more sensitive marker for the detection of muscle differentiation than is desmin, especially in LMS.     

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.     

In situ immune complexes, lymphocyte subpopulations, and HLA-DR-positive epithelial cells in Hashimoto thyroiditis

Surgical specimens from thyroid glands from seven patients with Hashimoto thyroiditis and two patients with non-autoimmune colloid goiter were analyzed by immunohistologic techniques (direct and indirect immunofluorescence and immunoperoxidase tests) using polyclonal antisera against total immunoglobulin, Ig classes (IgM, IgD, IgG, and IgA), and complement component C3 and monoclonal antibodies specific for B cells, T cell subpopulation, macrophages, natural killer cells, granulocytes, and HLA-DR antigen. Complement-fixing immune complexes (IgG+, C3+) were noted predominantly in areas with only slight destruction and only moderate lymphoid infiltration of thyroid follicles. In areas with intense lymphoid infiltration of thyroid follicles, where many well-developed germinal centers and significant perivascular lymphoid infiltration were seen, immune complexes were scarce. In these latter areas T helper cells (OKT4+, Leu3a+), were more abundant than T cytotoxic/suppressor cells (OKT8+), macrophages (OKM1+), and plasma cells (IgG+); only a few B lymphocytes (smIgM+, smIgD+), granulocytes (ViMD5+), and natural killer cells (VEP13+, Leu7+) were noted in the interstitium between thyroid follicles, intruding between thyroid follicular epithelial cells and merging into the thyroid follicular lumen. Many activated T cells (OKT10+, HLA-DR+) were present in these areas of advanced destruction. HLA-DR antigen expression was seen on macrophages, tissue reticulum cells, vascular endothelial cells, lymphoid cells, and, most interestingly, on thyroid epithelial cells. Normal thyroid epithelial cells did not express HLA-DR. Only a few epithelial cells in the vicinity of lymphoid infiltrations were HLA-DR+ in early stages of Hashimoto thyroiditis, and the number of HLA-DR+ epithelial cells was significantly increased in advanced stages of the disease. In our present report the potential role of HLA-DR+ thyroid epithelial cells for the in situ stimulation of the immune system within the thyroid gland of patients with Hashimoto thyroiditis is discussed, and it is hypothesized that HLA-DR+ thyroid epithelial cells may be an important factor for the progression and self-perpetuation of the disease, which is probably initiated by humoral components of the immune system but further propagated by cellular immunopathologic mechanisms.