Anti-native DNA antibodies were studied using an immunoglobulin class-specific enzyme-linked immunosorbent assay (ELISA) in 450 sera, virtually all of which were antinuclear antibody positive. ELISA was positive in about 85% of systemic lupus erythematosus (SLE) sera, usually at high titer and for two or three isotypes. Virtually all sera with antibodies of the three main classes were collected from SLE patients. Very high titers were unique to SLE. In contrast, low-titer antibodies of a single, mostly IgM, class were found in certain patients without evidence of autoimmune disease or with non-SLE autoimmune diseases. The isotypy and titer of the antibodies hence appear to be critical parameters for a correct interpretation of results. Under these conditions, ELISA seems to be usable as single screening test for the demonstration of anti-DNA antibodies.
Human peripheral blood mononuclear cells when cultured for the short term (11 days) produce an optimal IgM plaque-forming cell (PFC) response to sheep erythrocytes, which is dependent on macrophages and T suppressor and helper lymphocytes. With this system we have investigated the PFC response of individuals with rheumatoid arthritis and compared this to the response of normal sex- and age-matched controls and of patients with osteoarthritis. The response of rheumatoid arthritis patients was significantly lower than that of subjects in the other two categories.
The diagnostic evaluation of infants with suspected DiGeorge syndrome includes peripheral blood lymphocyte phenotype analysis by flow cytometry. Mononuclear cells are typically concentrated from infants' blood samples by Ficoll-Hypaque (FH) density gradient centrifugation prior to monoclonal antibody (Mab) staining. Having observed that lymphocyte percentages in samples prepared by this technique were low more often than anticipated based on the prevalence of the suspected diagnosis, we undertook a prospective study of 55 infants with congenital heart disease comparing FH with a whole-blood (WB) technique. Sixty healthy adult controls were similarly studied. We report the observation that FH-prepared mononuclear cells from blood samples of infants less than 4 months of age yield substantially different phenotypes than WB-prepared samples. The differences are related to red blood cell (RBC) contamination. No such difference was seen with adult samples. Unlike FH-prepared adult samples, contaminating RBCs are indistinguishable from lymphocytes through at least 4 months of age when assessed by the standard cytometric gating parameters of forward and 90 degrees light scatter. The use of a WB ammonium chloride lysis technique is recommended as most appropriate for the preparation of infants' blood samples.
A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to tetanus in dried blood samples spotted on filter paper discs has been developed. A good correlation was found between the two methods, filter paper disc assay and direct blood assay (r = 0.91). The specificity of the test was determined by a competitive inhibition assay using goat anti-tetanus-toxoid antibodies coupled to horseradish peroxidase. The interassay coefficient of variation is 10-12%. The antibodies were measurable until 4 months of storage of filter paper discs at room temperature (25 degrees C). This procedure is useful for mass screening of anti-tetanus antibodies using dried blood samples on filter paper discs.
Weekly blood samples from five healthy volunteers were obtained for 13 weeks to assess the variations in the immunological phenotypes of peripheral blood lymphocytes and monocytes. Monoclonal antibodies to eleven lymphocyte and two monocyte antigens were evaluated using two-color flow cytometry. Average week-to-week differences were less than 5% for all monoclonal antibodies and combinations except for I2+, NKH-1+, MO1+, and MO2+ cells. The observed variations presumably reflected both technical variables and responses of the immune system to daily environmental insults.
The marriage of monoclonal antibody technology and flow cytometry has provided clinical researchers with a powerful tool for the characterization of leukocytes from various sources. In this regard, flow cytometry has been primarily used for the immunophenotyping of peripheral blood lymphocytes in leukemias and various immunodeficiency disorders. Flow cytometry is also useful for the evaluation of leukocyte function in vitro and in vivo. This review discusses the various applications of flow cytometry for the assessment of leukocyte function. Since several cell surface antigens are important constituents involved in cell function, immunofluorescence identification of these markers can provide significant information regarding cell function. Analysis of activation antigen expression by monoclonal antibodies and flow cytometry can provide significant insights about the presence of functional leukocyte populations in patients. Flow cytometry can also be used to directly analyze leukocyte function. Procedures for the quantitative flow cytometric analysis of proliferation of activated lymphocyte subsets are reviewed. Early cell activation is also amenable to flow cytometric measurement. Early activation events such as alterations in membrane potential, intracellular free calcium redistribution, intracellular pH, and changes in membrane fluidity, as well as the direct measurement of enzyme activity, are also described.
Circulating immune complexes isolated from clinical filarial patients' sera by 3% polyethylene glycol precipitation were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on vertical slab gel. Silver staining of the gel after electrophoresis revealed 20 and 30 protein bands without and with reduction of immune complexes, respectively, in the molecular weight range of 148-18K. Filarial antigen in protein eluates from gel slices was detected by sandwich enzyme-linked immunosorbent assay. The eluates from four gel slices, viz., IC-2, IC-4, IC-7, and IC-9, showed filarial antigenic activity. These antigen fractions were characterized and explored for their diagnostic use. IC-7 and IC-9 fractions and microfilariae excretory-secretory (mf ES) antigen share common antigenic determinants as revealed by the fact that saturation of immobilized antibodies with IC-7 or IC-9 inhibited the binding of mf ES antigen coupled to penicillinase. IC-9 fraction appears to be useful in serological differentiation of Wuchereria bancrofti infected persons from those with disease manifestations. Biochemical characterization of the IC-9 fraction revealed the protein nature of the antigen. Comparison of the electrophoretic profiles of immune complexes and W. bancrofti mf ES antigen revealed several common protein bands. The 55 K protein band with antigenic activity was observed in both preparations.
Human IgG responses to carbohydrate antigens, such as those found on many infectious bacteria, are primarily restricted to the IgG2 subclass. This phenomenon, known as isotype restriction, has led us and others to examine the prevalence of IgG2 deficiency among infection-prone individuals. IgG2 deficiency (below 3 SD of the age group mean) is fairly common among patients suffering from chronic bacterial infections and suggests the utility of IgG subclass measurements in evaluating such patients. However, we also describe exceptions to isotype restriction in human responses to carbohydrate antigens. Furthermore, we describe individuals who, while responding very poorly to carbohydrate antigens, have normal IgG2 levels for their age group. These findings indicate the complexity of IgG subclass regulation and suggest that evaluation of infection-prone individuals should include measurements of both IgG subclasses and antibodies to carbohydrate antigens such as phosphocholine.
Multimarker studies were conducted on 195 lymph node, 59 bone marrow, 44 peripheral blood, eight body fluid, and eight internal organ specimens. The markers were identified by fluorochrome-labeled antibodies quantified with flow cytometry. T-cell receptor gene rearrangements were used for the determination of T-cell clonality. These studies confirmed that CD 19 (B4, Leu 12) is highly sensitive for B-lymphoblastic leukemia, CD 7 (Leu 9) is highly sensitive for T-lymphoblastic leukemia, and CD 5 (Leu 1) is highly sensitive for chronic lymphocytic leukemia. When these markers were compared to antigens of the same cell lineage (e.g., CD 19 to CD 20 [Leu 16] or to surface immunoglobulin, CD 7 to CD 3 [Leu 4], and CD 5 to CD 3), a marked discrepancy between them was diagnostic of the corresponding tumor. T-cell marker discrepancy (CD3 vs. CD 7) was demonstrated in T-cell lymphomas, but it was also shown occasionally in polyclonal T-cell populations. On the other hand, a marked discrepancy between the percentages of a B-lineage (CD 19 or CD 20)-positive and a surface-immunoglobulin-positive population is a reliable phenotype for the diagnosis of a surface-immunoglobulin-negative B-cell lymphoma.
Proliferation and differentiation of B cells can be promoted by a number of inducing factors, including interleukin-2 (IL-2). In vitro-stimulated normal B cells usually express IL-2 receptors (TAC). Furthermore, it has been reported that some minor sub-populations of B cells in normal lymphoid tissue also express TAC antigen, as do some leukemic B cells. In the present study, the reactivity of 20 specimens of non-Hodgkin's lymphoma or B-cell origin was examined for a wide panel of monoclonal antibodies (MAb), including an anti-TAC MAb. The ability of the isolated and purified B cells to proliferate in the presence of B-cell mitogens and culture supernatants (containing various growth factors, including IL-2) was also investigated. These experiments provided some evidence that the TAC-negative cells remained unreactive, whereas the TAC-positive cells entered the cell cycle and proliferated. IL-2 responsiveness of neoplastic B cells is therefore discussed.
The standardized fluorescence intensity (FI) of immunostained normal and malignant B-cells was measured as a method of evaluating cellular heterogeneity between lymphoid malignancies. Mature B-cells from peripheral blood lymphocytes (PBL) of different individuals demonstrated characteristic peaks of FI when specific monoclonal antibodies (MoAbs) were employed in standard flow cytometric procedures. Malignant cells from patients with lymphoid leukemias demonstrated FI that differed from that of normal B-cells with various MoAbs and that differed among categories of leukemia. By using a panel of MoAbs reactive with B-cells, as well as a T200 (CD45) antigen, a scheme of malignant cell differentiation may be produced that approximates stages of normal B-cell differentiation. When the FI of malignant cells differs from that of normal cells, or when atypical peaks (or additional peaks) of FI are present in flow cytometric histograms, the investigator should be alerted to the probability of abnormal cell populations. In addition, it is frequently possible to use this information to help classify malignancies, thereby contributing to identification of small numbers of malignant B-cells in otherwise equivocal situations.
An H9-HTLV-III-infected cell line was used as a target in a short-term (3-hr) Cr release assay to assess its sensitivity to lysis by peripheral blood lymphocytes (PBL) from normal donors. The single cell cytotoxicity assay (SCCA) on poly-L-lysine-coated coverslips was used to investigate further the mechanism of binding and killing. Uninfected H9 and K562 cell lines were studied as controls. Our results argue in favour of a natural killer (NK) mechanism being operative on an H9-HTLV-III-infected cell line owing to the following findings: (1) the cell line is sensitive to lysis in a short-term assay; (2) its sensitivity is significantly higher than K562; and (3) the kinetics of lysis, as assessed by SCCA, is similar to that of K562, with a more efficient killing being detectable against H9-HTLV-III. Furthermore, a phenotypic analysis of effector cells suggests that CD4+ lymphocytes are also involved in the lysis of this target. Our data provide evidence for an immune mechanism that may be operative in HTLV-III infection. We then studied, by this method, five groups of patients: one (n = 20) affected by acquired immunodeficiency syndrome (AIDS), one (n = 20) by AIDS-related complex (ARC), one (n = 20) by lymphadenopathy syndrome (LAS), one group (n = 40) of HTLV-III seropositive, apparently healthy people, and one (n = 40) of healthy HTLV-III seronegatives.(ABSTRACT TRUNCATED AT 250 WORDS)
Using a newly developed enzyme-linked immunosorbent assay, antibodies to histones (H1, H2A, H2B, H3 and H4) were found in 32 out of 40 rheumatoid arthritis patients with antinuclear antibodies at a titer greater than or equal to 100 as measured by indirect immunofluorescence. The anti-H2A reactivity was higher in patients with secondary Sjögren's syndrome than in those without, but the highest antihistone reactivity and the most heterogeneous patterns were observed in patients who were receiving D-penicillamine.
A mouse IgG1 monoclonal antibody (MAbC27) with high affinity for human carcinoembryonic antigen (CEA) has been used in vitro and in vivo for study of human colorectal carcinoma (CRC). After introduction to tumor-bearing mice, the distribution of radiolabeled MAbC27 and its F(ab')2 fragments was analyzed by direct measurement of radioactivity in the tumor and in normal organs as well as by whole-body scanning. Radiolabeled MAbC27 and its F(ab')2 were used successfully in localizing tumor xenografts in mice 40 and 20 hr after inoculation, respectively. A tumor as small as 60 mg (4.2 mm in diameter) was visualized by labeled F(ab')2. Circulating CEA, up to 200 micrograms per mouse did not prevent successful tumor imaging. This study indicates that the MAbC27 is highly useful in detection and localization of CRC in mice.
The development of a flow-cytometry (FCM) technique for the direct detection of antiplatelet globulins bound to the surface of platelets (platelet-associated immunoglobulins, or PAIg) is described and utilized to evaluate patients with idiopathic thrombocytopenic purpura (ITP). Elevated levels of platelet-bound globulin were detected by FCM in the ITP patients studied. When plasma was analyzed by FCM for the presence of antiplatelet antibodies, only one patient demonstrated elevated levels. The use of FCM in the direct analysis of PAIg may result in earlier diagnosis and better management of immune thrombocytopenias.
The immunoreactivity of a PLA1 antibody-containing serum to frozen/thawed platelets prepared by four different procedures was measured to assess the practicality of preparing a platelet reagent that could be easily stored and readily used in an immunofluorescent platelet antibody detection system. These frozen aliquots were analyzed at intervals up to 23 weeks after initial freezing and storage. For analysis, the frozen platelets were thawed, washed, resuspended to 2.5 x 10(5) platelets/mL and incubated with a dilution of fresh autologous serum that lacked platelet antibody or with a similar dilution of frozen human serum containing IgG anti-PLA1 antibody. At each interval, a freshly drawn platelet sample from the same donor was incubated in the same manner and used for comparison. All platelet mixtures were then washed and incubated with fluorescein conjugated goat F (ab')2 antihuman IgG. After repeat washing, each mixture was then analyzed with a flow cytometer for the extent of fluorescent antibody bound to each platelet mixture. Anti-PLA1 antibody reactivity with either the fresh or the frozen/thawed platelets remained stable over the period of analysis, with no significant difference in immunofluorescence with either fresh or frozen platelets as target cells. Platelet recovery following the thawing step ranged from 12% to 36% and was independent of the storage time. These studies suggest that frozen platelets can be readily used as reagents for platelet antibody assays.
The immunoreactivity of five sera containing either iso-, auto- or allo-antiplatelet antibodies to frozen-thawed human lymphocyte antigen (HLA)-typed pooled platelets was assessed to determine the practicality of using such a platelet preparation in an immunofluorescent platelet antibody system. The platelet reagent was analyzed over an 8-week period after initial freezing and storage. Mixtures of platelets with each serum or dilutions of it were initially incubated for 1 hour, then extensively washed and incubated with fluorescein-conjugated goat F(ab')2 antihuman IgG, A, M. After repeat washing, each mixture was then analyzed using a flow cytometer to determine the relative amount of fluorescent antibody bound. Not only was each of the five sera consistently positive for the presence of an anti-platelet antibody, but the extent of immunoreactivity for each remained relatively stable over the 8-week period of analysis. The results suggest that frozen pooled HLA-typed platelets should be an invaluable reagent for screening serum samples for the presence of antiplatelet antibodies in patients who are thrombocytopenic or who are refractory to platelet transfusion therapy.
Several deficits of immune response have been reported in hemophiliacs. In order to evaluate the overall immune function in this disease, we have investigated peripheral blood mononuclear cells (PBMC) surface phenotype, lymphokine production, B cell responsiveness, monocyte- and polymorphonuclear (PMN) cell-mediated responses in a group of 25 A or B hemophiliac patients. They were treated with concentrates (greater than 30,000 U/year) for at least 8 years, and ten of them were positive for human immunodeficiency virus (HIV) infection. With particular reference to lymphocyte surface markers, a low CD4+/CD8+ ratio and an increased frequency of CD25+ and surface immunoglobulin (sIg) positive lymphocytes were noted in HIV+ subjects. By contrast, CD3+ and CD4+ cell frequency was decreased in HIV- patients, whereas in the same individuals CD11+ and sIg+ cell percentage was augmented. Regardless of HIV infection, a quite variable degree of B-cell response was seen in hemophiliacs with either a T-independent or a T-dependent polyclonal B cell activator. PMN chemotaxis, phagocytosis, and killing were significantly diminished, whereas similar monocyte functions were basically unaffected. Finally, hemophiliacs were characterized by a profound impairment of leukocyte inhibiting factor (LIF) and lymphocyte-derived chemotactic factor (LDCF) production. Our findings clearly indicate an impairment of immune function in hemophiliacs regardless of HIV infection.
Some nonspecific components (IgG, C3c, C4,) of circulating immune complexes (IC) precipitable by 3.5% PEG were assayed by laser nephelometry in the sera of 71 patients with solid tumours, and of 39 patients with autoimmune diseases. PEG-IgG, PEG-C3c, and PEG-C4 were higher in cancer and in autoimmune diseases than in controls. In cancer patients, PEG-C4 levels and PEG-C4 positivity rate correlated well with tumour burden. PEG-C3c and PEG-IgG were present at higher levels and PEG-C4 was present at lower concentration in cancer than in autoimmune diseases. The results of the present study, performed on a large population of patients with cancer and autoimmune diseases, indicate a different composition of circulating IC in these diseases. Particularly, these data suggest a preferential involvement of the alternative pathway of complement activation in cancer, as already described by other authors. The aggregation of C3c to IC, mediated by the alternative pathway, seems to be the "key" event in the process of IC solubilization. In cancer patients' sera, the presence of "solubilized" IC could partly explain their peculiar biological behaviour as well as the disagreement observed among the different assay methods based upon the binding with complement factors.
