Immunoradiometric assay for the N-terminal fragment of proatrial natriuretic peptide in human plasma

Recently, the N-terminal fragment of proatrial natriuretic peptide (N-terminal proANP) has been proposed as a marker of chronic congestive heart failure. In this study, we established a two-step immunoradiometric assay using monoclonal antibodies and synthetic N-terminal proANP (1-67) as a standard. It allows us to measure plasma N-terminal proANP in only 4 h without prior extraction. The detection limit of this assay was 15 pmol/L for a 100 microL sample of plasma. Within-run CVs ranged from 1.7% to 2.9% and between-run CVs ranged from 4.2% to 5.1%. The dilution curves of plasma samples showed good linearity and analytical recovery was 89-104%. The mean (+/-SD) N-terminal proANP in plasma of 33 healthy subjects was 188 (+/-71) pmol/L and 1030 (+/-411) pmol/L in 25 patients with heart failure. Our immunoradiometric assay is rapid and precise enough for routine determination of N-terminal proANP in human plasma.     

Immunoradiometric assay of thyroglobulin in patients with differentiated thyroid carcinomas: need for thyroglobulin recovery tests

As interference from thyroglobulin autoantibodies appears to have been overcome in new commercial thyroglobulin assays by the use of monoclonal antibodies, the need for thyroglobulin recovery tests became uncertain. Sera (n=45) from patients with differentiated thyroid carcinomas were selected on the basis of a thyroglobulin recovery value below 70% in the Dynotest Tg immunoradiometric assay (Brahms) routinely used in our laboratory. Serum thyroglobulin levels were then measured using three other commercial immunoradiometric assays: thyroglobulin ERIA (Pasteur), HTGK (Sorin) and ELSA HTG (Cis Bio International). Thyroglobulin autoantibodies were measured using the Thyrak assay (Brahms). Although many patients were thyroglobulin antibodies-negative (< 200 U/ml, n=26), most immunoradiometric assays failed to detect thyroglobulin in patients with evidence of recurrence. Low thyroglobulin values associated with low thyroglobulin recovery in thyroglobulin antibody-negative patients appear to be more biologically relevant than a single low thyroglobulin value, which can lead to lack of medical intervention. We conclude that the thyroglobulin recovery test is a prerequisite for the correct interpretation of serum thyroglobulin levels determined with immunoradiometric assays in the follow-up of thyroglobulin autoantibody-negative patients treated for differentiated thyroid carcinomas.     

Automated nephelometric assay for seminal fluid transferrin

Wilson's disease responds to a variety of treatments including D-penicillamine and trientene. Nephrotic syndrome is a late complication of D-penicillamine treatment. We report a pediatric patient with Wilson's disease who developed nephrotic syndrome 2 wk after beginning D-penicillamine. His nephrosis resolved and his disease is quiescent with trientene treatment.     

Levels of Cathepsin D in breast cyst fluid]

Cathepsin-D was assayed in serum and in breast cyst fluid of 60 non neoplastic patients with GCD. The results are independent from cytological type or possible cyst relapse. Although the study confirms the high levels of Cat-D in breast cyst fluid no predictive value has been demonstrated. Its expression may be related to systemic endocrine factors.     

Calcium oxalate crystals in benign breast cyst fluid

We have recently observed a strongly birefringent material of varying shapes and sizes in a benign breast cyst fluid specimen from a 52-yr-old woman with suspicious mammographic microcalcifications. The finding of calcium oxalate crystals in the breast cyst fluid should be regarded as a recognition of a particular type of calcification, easily overlooked with the conventional light microscopy. To date, we are unaware of any cytologic reports of this type of calcification in the breast.     

Wuchereria bancrofti microfilariae in the dentigerous cystic fluid: an unusual presentation

The Glidewire (Microvasive, Natick, MA) or Terumo wire (Terumo, Japan) is constructed with a hydrophilic polymer surface that enables easier passage through narrowed lumens in the urinary tract. This study examined the effects of gas sterilization on Glidewire surface structure, slipperiness, and ability to support bacterial growth. Light microscopy at 100x and 400x and scanning electron microscopy at 100 to 1300x were used to compare the surface tips of five new 0.038-inch Glidewires with those resterilized one or three times. The tips were immersed in water prior to standard gas sterilization for operating room equipment. Subjective evaluation of slipperiness involved asking 10 blinded urologists to assess the nature of new and resterilized wires by feel. Support of bacterial growth was assessed by comparing cultures performed on new wires (control) with those of wires incubated with Bacillus stearothermophilus. Microscopy, reviewed by a pathologist, revealed no perceivable surface differences after one and three gas sterilizations. Eight of the urologists noted similar or improved slipperiness of resterilized wires compared with new wires. Bacterial cultures of intentionally infected wire segments showed no growth after standard gas sterilization in all cases. In this study, gas sterilization did not adversely affect the lubricious nature or the surface coating of the hydrophilic coating of Glidewires. Also, gas resterilization was bactericidal to new and used wires that had been infected with a heat-tolerant organism.     

Degradation of annexin I in bronchoalveolar lavage fluid from patients with cystic fibrosis

BACKGROUND/AIMS: The number of perisinusoidal myofibroblasts has been shown to be increased in hepatocellular carcinoma, as compared to cirrhosis. This increase might suggest a cooperative relationship between tumour cells and myofibroblasts. To assess this relationship, we undertook: (a) an immunohistochemical study to confirm the existence of an increased number of perisinusoidal myofibroblasts in human hepatocellular carcinoma, as compared to cirrhosis with or without liver cell dysplasia, (b) an in vitro study testing the role of normal or tumoral human hepatocytes in myofibroblast proliferation. METHODS: Forty explanted cirrhotic livers, including 14 with hepatocellular carcinoma and 24 with liver cell dysplasia, were studied. Myofibroblasts were detected by immunohistochemistry using an antibody directed against alpha-smooth muscle actin. Hepatic myofibroblasts in culture were obtained by outgrowth from human liver explants. RESULTS: There was a progressive increase in the number of perisinusoidal myofibroblasts, from cirrhotic nodules without dysplasia to liver cell dysplasia and hepatocellular carcinoma. Conditioned medium from isolated normal human hepatocytes had only minor mitogenic effects on myofibroblasts, as assessed by measuring DNA synthesis and cell growth. In contrast, conditioned medium from a human hepatoma cell line (HepG2 cells) markedly stimulated the proliferation of human myofibroblasts. This mitogenic activity was stored in HepG2 cells and secreted in the extracellular medium rather than being simply released following cell lysis. CONCLUSIONS: These results suggest that the increased number of myofibroblasts in hepatocellular carcinoma might be due to a paracrine mechanism involving soluble mitogenic factor(s) secreted by tumour cells.     

Ubiquitin in cerebrospinal fluid: a rapid competitive enzyme-linked immunoflow assay

The aims of this study were to develop a rapid immunoassay to determine the levels of ubiquitin in cerebrospinal fluid and to establish the ubiquitin levels in the spinal fluid of normal aged individuals. A competitive enzyme-linked immunoflow assay was developed. In this assay, ubiquitin is bound to nitrocellulose membrane, after which the primary antibody-test sample mixture and the enzyme-labeled secondary antibody under vacuum are applied sequentially. The final reaction product is collected in a microtiter plate by suction. This competitive assay requires only approximately 4 h and is potentially useful for determining in biological fluids the levels of any antigen or its antibodies that might be present. Employing this immunoassay, cerebrospinal fluid ubiquitin levels were found to be 147.5 +/- 5.2 ng ml-1 in non-neurological aged cases.     

A terminal-labelling microcytotoxicity assay with 125I-iododeoxyuridine as a label for target cells

The development of a terminal-labelling microcytotoxicity assay is described in which target cells (fetal fibroblasts) were labelled with 125I-iododeoxyuridine after effector (lymphoid) cells had been incubated with them for 24 h. The time-course for the development of cell-mediated cytotoxicity was assessed following allogeneic skin grafting. 'Non-specific' cytotoxicity detracts from the sensitivity of all microcytotoxicity assays and the terminal-labelling assay using 125I is no exception. The non-specific effects can be reduced but not eliminated by the removal of adherent cells. The optimum target cell/effector cell ratio would seem to be between 1:100 and 1:250. Residual lymph node cells did not appear to incorporate enough label to affect the test results. In vivo correlates of in vitro findings are still not easy to determine.     

Expression of gross cystic disease fluid protein-15/Prolactin-inducible protein in rat salivary glands

Gross cystic disease fluid protein-15 (GCDFP-15)/prolactin-inducible protein (PIP) is present at moderate levels in human submandibular and sublingual glands and is barely detectable in human parotid gland. The rodent homologue, PIP, has previously been identified in adult submandibular and lacrimal glands. Here we present the molecular characterization of rat PIP and show that this protein is a product of neonatal and adult rat submandibular, sublingual, and parotid glands. cDNA clones encoding rat PIP were isolated and sequenced. The deduced amino acid sequence of rat PIP shows 56% overall identity and 80% similarity with mouse PIP. By SDS-PAGE, secreted rat PIP has an apparent Mr of 17,000, with a minor proportion present as Mr 20-22,000 N-glycosylated forms. PIP was localized in rat salivary glands by immunogold silver staining. PIP was identified in acinar cells of developing and mature submandibular and parotid glands and at very low levels in sublingual gland serous demilunes. Typically, rat submandibular gland secretory proteins are produced by either acinar cell progenitors (Type III cells) or mature acinar cells. The expression pattern observed for PIP is similar to that previously reported for salivary peroxidase, an important component of nonimmune mucosal defense.     

A new biochemical assay in the diagnostic management of nasal cerebrospinal fluid leakage

A new method for the detection of cerebrospinal fluid (CSF) leakage is described, and is a refinement of the method originally reported by Oberascher and Arrer in 1986. Immuno-electrophoretic measurements are performed in a two-buffer system, making the test easier to do and providing qualitatively better images. Genetic variants of transferrin (which has a general population incidence of 2-4%) can be discriminated from false-positive test results in affected families. The test described is recommended as the method of choice for initial screening of suspected CSF leakage.     

IS-RT-PCR assay detection of MT-MMP in a human breast cancer cell line

The effect of the host system on the pathogenicity, immunogenicity, and antigenicity of infectious bursal disease virus (IBDV) was investigated. One classic (SAL) and one variant strain (IN) of IBDV were passaged separately six times in three host systems, namely BGM-70 continuous cell line, primary chicken embryo fibroblast (CEF) cells, or embryonating chicken eggs (embryos) or one time in the bursa of Fabricius (BF) of specific-pathogen-free (SPF) chickens. Passage in BGM-70 cells or CEF cells resulted in loss of pathogenicity, but viruses passaged in embryos or BF maintained their pathogenicity. For the immunogenicity study, the viruses described above were used to prepare live and inactivated vaccines, containing 10(3) mean embryo infectious doses (EID50s) and 10(5) EID50s respectively. These vaccines induced different levels of protection. It was concluded that the antigen titration methodology employing embryonating chicken eggs was not suitable for titration of viruses propagated in other host systems because of varying degrees of adaptation and/or pathogenicity of the viruses resulting in variability in antigen mass of the tested vaccines. To test this assumption, an antigen-capture enzyme-linked immunosorbent assay was used as a titration system to compare the antigenicity of viruses propagated in BGM-70 cells or BF. Preparations containing similar antigen masses were inactivated then inoculated into two age groups of SPF chickens and antibody titers were monitored. During the experimental period, the geometric mean virus-neutralizing (VN) antibody titers of the vaccinated groups did not differ significantly (P > 0.05).     

CA-125 and carcinoembryonic antigen assay vs. cytodiagnostic experience in the classification of benign ovarian cysts

OBJECTIVE: To compare the relative strengths of two factors involved in making an accurate differentiation between functional and epithelial ovarian cysts, along with their combination: (1) the cytologist's level of experience in interpreting ovarian cytology, (2) the use of the tumor markers carcinoembryonic antigen (CEA) and CA-125 in cyst fluid, and (3) a combination of (1) and (2). STUDY DESIGN: Papanicolaou-stained sediments from fluid aspirated from 31 resected ovarian cysts (6 functional and 25 epithelial) were blindly and independently evaluated by five pathologists with varying experience in ovarian cytology. Cyst fluid supernatant was used for CEA, enzyme-linked immunosorbent assay, and CA-125 radioimmunoassay; CEA levels > 5 ng/mL or CA-125 > 5,000 U/mL were considered elevated. Cysts were categorized cytologically and histologically as functional or epithelial and by tumor markers as "neither elevated" or "either or both elevated" (EBE). RESULTS: The agreement of histologic diagnosis with each pathologist's cytologic diagnosis ranged from 53% to 84% (53%, 71%, 83%, 82%, 84%), corresponding to increasing level of experience. The percentage of agreement with EBE was 77%, whereas combined experienced pathologist's diagnosis and EBE was 87%. Kappa equaled .45 for experienced cytopathologist's diagnosis or EBE alone. Kappa equaled .53 when the pathologist or EBE diagnosed an epithelial cyst, indicating results unlikely to occur by chance. CONCLUSION: The distinction of functional from epithelial ovarian cysts is best achieved by combining measurement of the tumor markers CEA and CA-125 with a high level of cytopathology experience.     

Adenoid cystic carcinoma of the breast: mammographic appearance and pathologic correlation

OBJECTIVE: The objective of our study was to describe the mammographic features of adenoid cystic carcinoma of the breast and to correlate mammography findings with histopathologic findings. CONCLUSION: Adenoid cystic carcinoma is a rare type of breast neoplasm that usually appears as a slowly enlarging nodule. In spite of its low incidence, recognition is important because early detection ensures good prognosis. On mammography, these tumors often appear as moderately circumscribed, lobulated nodules that are similar to other types of benign and malignant tumors. Therefore, cytologic and histologic evaluations are needed for accurate diagnosis.     

Rapid diagnosis of tuberculous meningitis by polymerase chain reaction assay of cerebrospinal fluid

A polymerase chain reaction (PCR) method for the rapid diagnosis of tuberculous meningitis (TBM) was used to study prospectively 47 cerebrospinal fluid (CSF) samples from 45 patients. Twenty CSF samples were from patients with clinically suspected TBM and another 27 samples came from patients without clinically suspected TBM. Mycobacterial DNA was detected in 15 CSF samples (14 from patients with clinically suspected TBM and 1 from a patient not suspected of having TBM). Of the PCR-positive samples, 4 were also positive for mycobacterial culture. However, 32 PCR-negative samples were all culture-negative. All samples were negative for the acid-fast bacillus by direct smear. The single PCR-positive patient in the clinically unsuspected TBM group was initially diagnosed as suffering from aseptic meningitis on the basis of his clinical features. The mycobacterial culture of his CSF specimen was also positive and a revised diagnosis of an aseptic type of TBM was made. The estimations of specificity and sensitivity in this study were 100% and 70% respectively. The results showed that using a PCR to detect mycobacterial DNA in CSF for the early diagnosis of TBM is not only a rapid but also an accurate method.     

Adenoid cystic carcinoma of the breast: value of histologic grading and proliferative activity

Using vitreous fluorophotometry and quantitative fluorescence microscopy the authors studied the permeability of the blood-retinal barrier (BRB) to fluorescein in control and in 8 days streptozotocin-induced diabetic rats. Vitreous fluorophotometry showed that fluorescein permeates BRB in control and in diabetic rats. However, in diabetic rats the permeability to fluorescein was significantly increased as compared to control rats. The vitreous penetration ratio (VPR) values for total and free fluorescein at 60 min, were higher for diabetic rats (231.2+/-12.9 min-1 for total fluorescein and 1299.24+/-58.0 min-1 for free fluorescein) than for control rats (95.5+/-3.5 min-1 for total fluorescein and 646.6+/-55. 0 min-1 for free fluorescein) (P<0.05). Quantitative confocal fluorescence microscopy confirmed these findings and identified the site of leakage across the BRB by comparing the relative importance of the fluorescein leakage across the outer and inner BRB. In control rats the fluorescence levels remained relatively low in the photoreceptor layer, next to the outer BRB but in the inner nuclear layer, next to the inner BRB reached values that were almost ten times higher. These results suggest that in retinas of control rats fluorescein penetrates predominantly through the inner BRB. In diabetic rats the fluorescence levels in the photoreceptor and in the inner nuclear layer were significantly increased as compared to the fluorescence levels in controls rats. Nevertheless, in the inner nuclear layer the fluorescence levels were also generally higher than the fluorescence levels at the photoreceptor layer. The rates of fluorescence levels between the inner nuclear layer and the photoreceptor layer were apparently 3:1, 60 min after the single intravenous injection of fluorescein. Also, the fluorescein penetration in the inner nuclear layer of the diabetic rats is higher than that observed in the inner nuclear layer of the control rats (P<0.001). These findings suggest that the permeability to fluorescein of both components of the BRB is increased 8 days after the induction of diabetes by streptozotocin and that the permeability of the retinal vasculature is preferentially affected.     

Adenoid cystic cancer of the breast. Case report and meta-analysis of the literature

Adenoid-cystic carcinoma of the breast is considered a rare entity with a comparatively favourable prognosis. We report the case of a 58 year old woman and review another 150 cases published to date in the pertinent literature. The clinical course and outcome of 99 patients was evaluated by meta-analysis. Recurrence-free ten-year survival was calculated at 85.1 percent following mastectomy and at 45.7 percent after breast-conserving therapy (p < 0.05). Six of ten local recurrences and 7 of 8 distant metastases occurred 5 years or later after initial treatment. It is concluded that patients with adenoid-cystic carcinoma of the breast should be treated by primary mastectomy and extended follow-up is recommended.     

A comparison between radioligand and immunohistochemical assay of hormone receptors in primary breast cancer

The detection of oestrogen and progesterone receptor (ER and PgR) levels in human breast carcinoma has traditionally been performed using a biochemical radioligand binding method. This method has several disadvantages including the requirement for generous tissue samples, the production of radioactive waste products and the inability to exclude non-malignant cellular material from the assay process. An alternative method for detecting hormone receptors is available with the use of a monoclonal antibody specific for the ER or PgR receptor using immunocytochemical assay (ER-ICA or PgR-ICA). Although designed for use on frozen section material, with modifications this method can be used on paraffin sections of routinely fixed and processed tissue, on archival material and on very small specimens. Further, an objective assessment or scoring of staining intensity is possible using computerized video-image analysis. Forty-three cases of primary breast carcinoma, treated from 1989 to 1991 at Goulburn Valley Base Hospital, Shepparton were assessed for ER and PgR content using both the radioligand method and immunohistochemistry with video-image analysis, and the results were compared. Of the 43 cases, ER-ICA and ER had a concordance of 81% (P < 0.001, r = 0.58) and in 39 cases, PgR and PgR-ICA had a concordance of 87% (P < 0.001, r = 0.54). Because the sample for radioligand assay is of uncertain composition and the immunohistochemical stain can be scored specifically for malignant epithelium, a degree of discordance is thought to be mostly attributable to the limitations of the radioligand assay.