Diagnostic immunology最新文献

The application of immunostaining techniques to the study of sections of formalin-fixed, paraffin-embedded tissues has deeply influenced the practice of surgical pathology of tumors. We have favored in our laboratory the avidin-biotin complex immunoperoxidase method and have gradually substituted monoclonal antibodies for polyclonal sera. Better results are sometimes obtained with trypsinization prior to the application of the primary antibody. The more common primary antibodies used are directed against the following categories of cellular antigens: intermediate filaments, oncofetal products, hormones or hormone-related peptides, enzymes or enzyme-related peptides, cell- or tissue-"specific" products, lymphocyte-leukocyte antigens, and immunoglobulin chains. Controls are essential to every immunohistochemical reaction. Because of the pitfalls of immunohistochemical techniques, immunohistochemistry as applied to the study of tumors should be used with utmost care and as an extension of routine surgical pathology.

One hundred and eighty-four serum specimens were assayed for antibodies to the human immunodeficiency virus. All specimens were screened with a commercial enzyme immunoassay and confirmed by two indirect immunofluorescence assays. Sera were also assayed by Western blot. Results from sera of 48 healthy heterosexual volunteers were all negative by EIA, IFA, and Western blot. Sera from 50 healthy homosexual men negative by EIA were also negative by IFA and Western blot. Sixty-two patients with persistent generalized lymphadenopathy or newly diagnosed AIDS all were positive by EIA, IFA, and Western blot. Of 24 sera from patients with autoantibodies, with no evidence of AIDS-related diseases, five appeared to be false-positive by EIA, since they were nonreactive by IFA and Western blot. In addition, three other samples contained both autoantibodies and human immunodeficiency virus antibodies. False-positive results were observed in both the EIA and IFA with monoclonal antibodies directed toward the MHC class II antigens DQ and DR. The reactivity of these antibodies could not be distinguished from positive patients' sera, in either EIA or IFA. We conclude that in general indirect immunofluorescence performed well as a confirmatory test after screening by enzyme immunoassay for human immunodeficiency virus antibodies.

Twelve patients with a histologic diagnosis of lymphoblastic lymphoma (LBL) were studied immunologically using the methodologic refinement of comparative serial section immunochemistry. By this means, we demonstrate complex LBL phenotypic profiles, revealing 3 major immunologic subtypes: immature T cell, 7 cases; intermediate or mature T cell, 3 cases; immature B cell (pre-pre-B), 2 cases. This phenotypic diversity challenges the basic belief that all LBL are the same. Our immature T-cell cases with frequent simultaneous Leu 2/3/6/9/CALLA/Tdt expression correspond to cortical thymic phenotypes; our mature T-cell phenotypes with Leu 9/la expression and absent L6/Tdt correspond either to medullary thymocytes or post-thymic T cells; our pre-pre-B phenotypes with simultaneous Tdt/CALLA/B4 expression correspond to common acute lymphocytic leukemia (ALL) phenotypes. Mature T-LBL phenotypes are similar to "novel" peripheral T-cell lymphoma phenotypes. Scant or absent Tdt expression in mature LBL is not an isolated antigenic change but a complete phenotypic profile difference from immature T-LBL. The major T- and B-cell phenotypes of LBL might have therapeutic significance. Treatment among LBL phenotypes may need to vary as with acute lymphocytic leukemia phenotypes. Further study is needed; in the meantime, comparative serial section immunotyping promises substantial utility in revealing the immunologic complexity of the lymphomas.

The functional response of peripheral blood T lymphocytes was studied in patients with end-stage renal disease treated by chronic hemodialysis for over 1 year. Proliferation after phytohemagglutinin stimulation of patients' peripheral blood mononuclear cells and of T lymphocyte fractions isolated by either sheep erythrocyte rosetting or by use of a nylon wool column was significantly reduced as compared with that of corresponding fractions from healthy control subjects (P less than 0.001). The induction of suppressor cell activity by concanavalin A in rosetted T cell fractions was higher with cells of hemodialyzed patients than with control cells (P less than 0.025). The expression of class II MHC antigen (HLA-DR) by the T8 lymphocyte subset after concanavalin A induction, as determined by staining with monoclonal antibodies and two-color fluorescence analysis by flow cytometry, was also higher in hemodialyzed subjects (P less than 0.025). Since contamination by non-T cells in such cell fractions and increases in proliferation after indomethacin treatment of peripheral blood mononuclear cells were similar in hemodialyzed and control subjects, it is unlikely that the depressed T lymphocyte responses and the increased suppressor cell activity can be attributed to increased peripheral blood monocyte counts observed in patients undergoing hemodialysis. Studies of the biological events associated with the activation of lymphocytes of hemodialyzed patients revealed a reduction in expression of interleukin 2 receptor in the plasma membrane of phytohemagglutinin-stimulated lymphocytes as determined by staining with monoclonal antibody (P less than 0.01). In addition, a very low secretion of interleukin 2 by stimulated peripheral blood mononuclear cell populations was observed in about one-half of patients receiving hemodialysis.

A non-enzymatic immuno assay was optimized for detection of immunoglobulin A in serum and cerebrospinal fluid specific in acquired and congenital toxoplasmosis. An antihuman IgA monoclonal antibody was coated onto a polystyrene to capture total IgA. Suspensions of Toxoplasma gondii were used as a visible antigen. Eight hundred specimens (sera, cord blood serum, and cerebrospinal fluid) were tested. These were collected from 300 patients with acquired toxoplasmosis and from 28 children with congenital toxoplasmosis. In acquired toxoplasmosis, the assay allowed early detection of specific IgA, with kinetics similar to those of specific IgM expression. In congenital toxoplasmosis, anti-T. gondii IgA could be detected in the neonatal period. This assay was useful for diagnosis, follow-up, and posttherapeutic evaluation of toxoplasmosis. Specific IgA was also detected in the cerebrospinal fluid of infected newborn children. This simple IgA capture assay improves serological diagnosis of acquired and congenital toxoplasmosis when used in combination with analysis of T. gondii specific IgG and IgM.

The subclass distribution of human anti-thyroglobulin IgG antibodies (TgAb) was studied in 24 sera using an indirect enzyme-linked immunosorbent assay (ELISA) with monoclonal anti-subclass antibodies. An IgG4/IgG1 predominance was found. TgAb were purified from seven sera by affinity chromatography. The antigen-specific indirect ELISA was repeated in the purified TgAb, in which IgG subclasses were measured by a competitive assay with monoclonal antibodies. The latter method showed an IgG2 predominance. Comparison of the results obtained by the three approaches showed discrepancies that probably reflect bias introduced by both the indirect ELISA and the affinity chromatography.

Activated B cells that spontaneously secrete immunoglobulin are found in homosexual men with AIDS or lymphadenopathy. These cells constitute a very small percentage of peripheral blood lymphocytes (usually less than 1%), making identification of their surface antigens difficult. To identify surface antigens on immunoglobulin-secreting cells, peripheral blood mononuclear cells were first reacted with monoclonal antibody, followed by a fluorescein-conjugated goat antimouse globulin reagent. Secretion of immunoglobulin was then assessed in a reverse hemolytic plaque assay, with a modified Cunningham chamber in which an individual plaque-forming cell could be examined with a fluorescence microscope. All plaque-forming cells were found to be reactive with OKT 10 and 4F2 monoclonal antibodies; there was moderate reactivity with anti-la and B4. The same results were found when normal pokeweed mitogen-stimulated lymphocytes were tested. The surface phenotype of these cells is consistent with that of a preplasma cell.

We tested the premise that measurement of interleukin 2 receptor (IL2R) and transferrin receptor (TR) can be used to assess proliferative responses to pokeweed mitogen (PWM) and tetanus toxoid (TT). Our study group consisted of patients with Human Immunodeficiency Virus (HIV) infection, including patients with acquired immunodeficiency syndrome (AIDS, n = 10), AIDS-related complex (n = 14), lymphadenopathy syndrome (n = 7), or homosexual men seropositive for HIV (n = 6). Controls were 40 healthy seronegative blood donors. IL2R and TR expression by stimulated mononuclear cells were assessed using specific monoclonal antibodies and flow cytometry, and results were analyzed using the 3H-thymidine assay for DNA synthesis as a standard for comparisons. For identifying low PWM responses, both the IL2R+ cell percent and the IL2R+ cell number (no.) per lymphocyte trigger region (a relative measure of IL2R+ cell no. per culture) on day 3 (72 h) were sensitive (greater than 90%) and specific (80%); day 3 TR+ cell no. was also sensitive (92%) and specific (100%). For detecting low TT-induced responses, day 7 IL2R+ cell no. proved the most sensitive (100%) and specific (78%) parameter. These findings indicate that cytofluorometric analysis of IL2R and/or TR expression is a reliable method for detecting impaired proliferative responses to PWM and TT in these patients. Such a method offers an attractive alternative to the regulatory and disposal problems associated with radioactivity in the conventional DNA synthesis assay, as well as providing insight to the mechanism(s) responsible for impaired proliferation.

Sera from rabbits immunized with culture filtrates and homogenates of Nocardia asteroides B1042 gave at least eight precipitin bands by immunoelectrophoresis. At least 20 proteins with isoelectric points (pls) in the pH 4 to 5.4 range were observed in isoelectric focusing patterns. The enzyme-linked immunoelectro-transfer blot (EITB) assay showed that several of the isofocused proteins reacted with rabbit antisera and with sera from nocardiosis and tuberculosis patients. Antibodies against three proteins with pls of 4, 4.43, and 4.68 (antigenic factors 1,6,8) were present in nocardiosis patients' sera. The proteins were excised from isofocused gels, and IgG monoclonal antibodies (MAbs) were produced by the hybridoma method. MAbs against factors 1 and 6 did not crossreact with cytoplasmic antigens of Mycobacterium chelonae, M. intracellulare serotypes 4B and 8A, M. fortuitum, M. gordonae, or M. kansasii in the EITB method. Factor 8 (MAb) crossreacted with antigens of M. intracellulare and M. fortuitum.