Flow cytometric analysis of DNA in diagnostic cytology

Flow cytometric analysis of nuclear DNA content and cell surface immunologic phenotyping were used in the evaluation of cytologic samples obtained from four patients. In each sample, a lymphoid cell population was present, which was difficult to evaluate by traditional cytopathologic methods. In two of the cases, the flow cytometric demonstration of monoclonal populations of lymphoid cells characterized by abnormal amounts of nuclear DNA gave support to the cytologic interpretation of malignancy. In a third sample, a lymphoid cell population that could not be cytologically distinguished from malignant lymphoma cells of small lymphoid cell type was shown to be composed of euploid, polyclonal cells. In the fourth case, the demonstration of euploidy in morphologically distinctive cell populations was helpful in interpreting the fine-needle aspirate of the thyroid gland. The authors conclude that flow cytometry can be used to great advantage in the evaluation of cytologic samples as well as in predicting biologic behavior.     

Light and electron microscope analysis of lectin binding to adult rat liver in situ

A comprehensive mapping of lectin receptors on adult rat liver in situ was performed at light and ultrastructural levels by using 12 biotin-labeled lectins and an avidin-biotin-peroxidase complex. In addition, concanavalin A conjugated directly to peroxidase was utilized to study intracellular membrane glycoconjugates. To achieve optimal preservation of these membrane sugar moieties, several fixatives and fixation procedures were evaluated. A periodate-lysin-paraformaldehyde combination provided the best compromise between preservation of ultrastructural details and lectin-binding reactivity. Hepatocyte cell surfaces reacted intensely with concanavalin A, Lens culinaris agglutinin, and Pisum sativum agglutinin (all specific for alpha-D-mannosyl and alpha-D-glucosyl groups) as well as Ricinus communis agglutinin type I (specific for alpha or beta-D-galactose) and wheat germ agglutinin (specific for neuraminic acid and beta-NAc-glucosaminyl groups). In addition, R. communis agglutinin and wheat germ agglutinin exhibited an extremely strong reactivity for bile canaliculi which surpassed the binding of concanavalin A, L. culinaris agglutinin, and P. sativum to these structures. Phaseolus vulgaris agglutinin (specific for beta-D-galactose-glucosyl-NAc and D-mannosyl groups), which exhibited a moderate binding to hepatocyte plasma membranes, reacted more strongly with the endothelium of sinusoids and portal vessels. Although all six of these lectins plus Bandeiraea simplicifolia stained Kupffer cells, B. simplicifolia lectin (an alpha-D-galactosyl marker) was unique in showing a strong reactivity for only this cell type. The avidin-biotin-peroxidase procedure is a sensitive method for detection of sugar moieties on cell surfaces of rat liver at both light and electron microscopic levels. In this study, the procedure was used to localize differential binding of lectins to several anatomical structures of the organ, and furthermore, we were able to map preferential localizations of carbohydrate residues in the glycocalyx of the rat hepatocyte in situ.     

Comparison of the genomes of two sympatric iridescent viruses (types 9 and 16)

Summary A map of the sites in the genome ofCostelytra zealandica iridescent virus (CzIV), using the restriction enzymesBamHI,KpnI, andPstI, showed the genome size to be 170.2 kbp in length. It was found that the genome was cyclically permuted and that 39% of the genome of CzIV contained repetitive sequence elements. The genome was found to hybridize with the genome of another iridescent virus, type 9 (WIV), in DNA-DNA hybridization experiments. A region of the WIV DNA genome (23.4 kbp) did not hybridize with CzIV DNA and this region is similar in size to the total genomic size difference between CzIV and WIV (22.4 kbp). A unique repeat sequence from iridescent virus type 6 (CIV) was shown to be present in the genome of WIV but not that of CzIV. Finally, the positions of the major capsid protein genes, VP53 and VP52, in the restriction enzyme maps for type 16 and type 9 respectively were determined.     

Chronic graft-versus-host disease (cGVHD) following unrelated donor hematopoietic stem cell transplantation (HSCT): higher response rate in recipients of unrelated donor (URD) umbilical cord blood (UCB).

We present a comparative analysis of clinical presentation and response to treatment in 170 patients with chronic graft versus host disease (cGVHD) (123 following transplant from an unrelated donor [URD] and 47 from umbilical cord blood [UCB]). URD transplant recipients were significantly younger (median age 25 versus 39 years, P = .002; and the donor grafts were mostly HLA matched (67% versus 10%, P < .0001). UCB recipients had more frequent responses (complete remission [CR] + partial remission [PR]) to treatment (URD 48% versus UCB 74% at 2 months [P = .005]; 49% versus 78% at 6 months [P = .001] and 51% versus 72% at 1 year [P = .03] in the URD and UCB groups, respectively). Nonrelapse mortality (NRM) after diagnosis of cGVHD was worse after URD grafts. (1 year NRM 27% [19%-35%] URD versus 11% [2%-20%] UCB, P = .055). Separate multivariate analyses were performed in each cohort. In both, thrombocytopenia and no CR or PR at 2 months were independently associated with increased mortality. In addition, progressive onset of cGVHD was a significant predictor of increased mortality in URD cohort. These data suggest that cGVHD following UCB transplant may be more responsive to therapy and also lead to a lower NRM.     

Development of a rapid diagnostic test for pertussis: direct detection of pertussis toxin in respiratory secretions.

Monoclonal antibodies (MAb) were produced against the specific Bordetella pertussis antigen pertussis toxin (PT). In preliminary studies, one MAb (IB12) was selected and used in an enzyme-linked dot blot immunoassay to evaluate the ability of the method to detect known amounts of PT in control experiments and to test its potential for direct detection of PT in nasopharyngeal secretion (NP) specimens from patients with confirmed cases of whooping cough. The dot blot assay was able to detect PT at levels as low as 10 ng per dot in either buffer or control NP specimens. The assay demonstrated specificity, reacting only with dot blots of whole B. pertussis and not Bordetella bronchiseptica, Bordetella parapertussis, or other bacterial strains. In preliminary studies, NP aspirate, swab, and wash specimens were compared. The specimen of choice was found to be the NP aspirate, for which 100% positive results were found in the assay. These initial studies suggest that the dot blot immunoassay in which a MAb is used for direct detection of PT in NP specimens may be useful as a rapid diagnostic test for pertussis.     

Transport across endodermal cells of the chick yolk sac during early stages of development

The endoderm of the chick yolk sac mediates the transfer of materials from the yolk mass to the embryonic circulation. There is little evidence of endocytotic activity in the area pellucida, but the endodermal cells of the area vasculosa possess many microvilli and bristle-coated pits and vesicles, as well as a canalicular system and vacuoles in the apical cytoplasm. Three tracers, horseradish peroxidase, ferritin, and latex spheres, were injected beneath the endoderm of both cultured embryos and embryos in ovo to study the pathway of uptake of extracellular materials. All tracers were sequestered in bristle-coated pits (200–500 nm in diameter) of the endodermal cells of the area vasculosa, but not those of the area pellucida. Both horseradish peroxidase and latex spheres (and probably ferritin) were incorporated into pleomorphic intracellular yolk drops through bristle-coated pits and vesicles, and then into apical vacuoles, which fuse with the intracellular yolk drops. Horseradish peroxidase and ferritin were also found within apical canaliculi. The apical junctions between endodermal cells prevented the intercellular passage of the tracers. A ‘topping-up’ hypothesis is proposed whereby endodermal cells of the area vasculosa continually sequester extracellular yolk material, which is incorporated into a digesting intracellular yolk drop while, at the same time, digested yolk products are being transported across the vascular pole of the endoderm to the extraembryonic circulation and thence to the embryo.     

Isolation of the MHC genes encoding the tumour-specific class I antigens expressed on a murine fibrosarcoma

The C3H UV-induced fibrosarcoma, 1591, is highly immunogenic and, therefore, is readily rejected when transplanted into immunocompetent syngeneic recipients. Previous analysis of 1591 with tumour-specific or H-2-reactive monoclonal antibodies revealed that this antigenicity might be due to the expression of two novel class I major histocompatibility complex (MHC) antigens. In this report we describe the molecular cloning and initial characterization of three genes which account for all of the unique serological class I reactivities observed on this tumour. These include two distinct, but highly conserved, H-2L-like genes, and a third gene the product of which bears determinants which are characteristic of both the tumour and of class I products of the H-2k haplotype. Moreover, each of these genes contains a polymorphic restriction enzyme fragment which is detected in the class I sequences of 1591 relative to normal C3H tissue. Since the expression of these polymorphic class I sequences is relevant to the immunogenicity of 1591, the mutational events by which these genes were generated may be significant to the immunobiology of this tumour.     

Novel Recombinant-Antigen Enzyme Immunoassay for Serological Diagnosis of Syphilis

Enzyme immunoassay (EIA) is an ideal method for screening large numbers of patients for syphilis. We evaluated a novel immune-capture EIA (ICE Syphilis; Murex Diagnostics) that uses three recombinant Treponema pallidum antigens (TpN15, TpN17, and TpN47) and compared the results with those obtained by the native T. pallidum antigen EIA (Captia SelectSyph-G; Centocor) that we currently use for the serodiagnosis of syphilis. Specificity was evaluated by screening 1,184 unselected serum specimens in parallel by the ICE Syphilis and SelectSyph-G assays, while sensitivity was tested with a panel of 101 serum specimens containing antitreponemal antibodies (treated and untreated) from patients with various stages of infection. The specificity of the ICE Syphilis EIA (99.8%) on screening was significantly higher (P < 0.02) than that of the SelectSyph-G EIA (99.2%). The sensitivity of the ICE Syphilis EIA was significantly higher (P < 0.01) than that of the SelectSyph-G EIA on both initial (99 versus 91.4%) and repeat (100 versus 92.4%) testing. The ICE Syphilis EIA was also significantly more sensitive (P < 0.01) than the fluorescent treponemal antibody-abs (92.4%) but not the T. pallidum hemagglutination assay (97.1%). Sera containing antitreponemal antibodies gave a much higher antibody index (absorbance of test serum/kit cutoff) by the ICE Syphilis EIA than by the SelectSyph-G EIA. This combined with the overall high sensitivity makes the ICE Syphilis EIA an ideal test for excluding or detecting treponemal infection in human immunodeficiency virus (HIV)-infected patients. The ICE Syphilis EIA was positive with sera from all 15 HIV-infected patients in the study, whereas sera from 3 HIV-infected patients were negative by the SelectSyph-G EIA. We conclude that the high sensitivity and specificity of the ICE Syphilis EIA and its suitability for automation make it an ideal screening test.     

The expression of the urinary forms of human luteinizing hormone beta fragment in various populations as assessed by a specific immunoradiometric assay

Human gonadotrophins undergo metabolic transformations which result in the presence of several smaller, structurally and immunologically related forms of gonadotrophins in the urine. For luteinizing hormone (LH), a beta core fragment (LHbeta cf) has been isolated from the pituitary and characterized. The corresponding urinary fragment is inferred from mass spectral and immunochemical analysis of chromatographically separated urinary forms. Physicochemical characteristics, primarily mass spectral and chromatographic, indicate that the pituitary and urinary forms of LHbeta cf have a different structure, probably in the carbohydrate moieties. This communication characterizes the expression of LHbeta cf in the urine of both reproductive and post-reproductive age women and in men, employing assays highly specific for the pituitary form of the fragment. It was found that LHbeta cf is the predominant LH associated molecular form in the urine during peri-ovulatory period, peaking 1-3 days later than intact LH and reaching a concentration of approximately 600 fmol/mg creatinine, 7-fold higher than either LH or LH free beta subunit. Corresponding concentrations of human chorionic gonadotrophin (HCG) beta cf were <1% that of LHbeta cf. LHbeta cf cross-reaction with some LH or LHbeta monoclonal antibodies may well interfere with the accurate estimation of the day of the LH surge when urinary tests are utilized.