Fetal fibronectin in patients at increased risk for premature birth.

OBJECTIVE: The objective of this study was to evaluate fetal fibronectin as a screening test for subsequent preterm birth in asymptomatic pregnant women. STUDY DESIGN: Eighty-seven pregnant women at increased risk for preterm birth underwent weekly sampling of cervicovaginal secretions beginning in the middle of the second trimester and continuing until delivery or until 34 weeks of gestation, with quantitative measurement for fetal fibronectin. In addition, assessment of cervical dilatation, uterine activity, and tocolytic therapy was performed with each sampling. Preterm birth was the specific outcome measured, and the correlation of fetal fibronectin with this outcome was determined. RESULTS: Overall, 31% of the patients experienced a spontaneous preterm birth. As a predictor for delivery before 37 completed weeks of gestation, the presence of fetal fibronectin had a sensitivity of 92.6%, a specificity of 51.7%, a positive predictive value of 46.3%, and a negative predictive value of 93.9%. For delivery before 34 weeks, fetal fibronectin had a sensitivity of 92.3% and a negative predictive value of 97.8%. By means of logistic regression analysis a positive fetal fibronectin result was highly significantly correlated with preterm birth (odds ratio 3.8, p < 0.001) and more so than the presence of four or more uterine contractions per hour, tocolytic therapy, or cervical dilatation of > or = 2 cm. The addition of contractions, tocolytic therapy, or cervical dilatation to a positive fetal fibronectin result did not increase the predictive capacity of a positive fetal fibronectin alone. CONCLUSION: Fetal fibronectin in the cervicovaginal secretions of asymptomatic patients has potential value as a screening test in the identification of patients at risk for preterm birth. This test had equally high sensitivity and negative predictive value for birth before 37 weeks.

Fetal cells in the maternal circulation. Technical considerations for practical application to prenatal diagnosis.

Recent advances in cell separation technology and DNA analytic techniques leave little doubt as to the presence of fetal cells in the maternal circulation. The potential of using these cells for genetic analysis is compelling. The practical aspects of establishing a universal method utilizing the new capabilities in clinical practice have not been addressed to date. The major hurdles that still need to be traversed before this technology is universally adopted include the identification of appropriate sampling and separation methods yielding fetal cells amenable to genetic analysis by rapid DNA technologies, clinical studies of appropriate statistical power to validate and compare this approach to current genetic testing, and comparison of this approach to other noninvasive paradigms such as triple screening. Despite the tremendous value of noninvasive genetic screening, the rigorous course required to progress from description of scientific capability to validation of a clinical test must not be ignored or rushed for financial considerations.

Fetal fibronectin in cervical and vaginal secretions as a predictor of preterm delivery.

BACKGROUND: Preterm delivery is the leading cause of neonatal mortality in the United States, but efforts to address the problem are hampered by the inability to predict accurately which pregnancies are at risk. We postulated that damage to the fetal membranes may release fetal fibronectin into the cervix and vagina, giving rise to a biochemical marker for preterm delivery. METHODS: We measured fetal-fibronectin concentrations in cervical and vaginal secretions, amniotic fluid, and maternal plasma with a sensitive immunoassay using the monoclonal antibody FDC-6. Immunohistochemical studies were used to determine the distribution of fetal fibronectin in the placenta and amniochorionic membranes and to ascertain its cell of origin. RESULTS: Women with uncomplicated pregnancies (n = 163) who delivered at term rarely had cervicovaginal fetal-fibronectin concentrations above 0.05 micrograms per milliliter between 21 and 37 weeks of gestation (11 of 267 cervical samples [4 percent] and 9 of 267 vaginal samples [3 percent]. High levels of fetal fibronectin were detected in amniotic fluid and in the cervical or vaginal secretions of 93.8 percent of the women with preterm rupture of membranes (n = 65). Cervical or vaginal fetal fibronectin was also present in 50.4 percent of the women with preterm uterine contractions and intact membranes (n = 117), and its presence identified the women who delivered before term (n = 60) with a sensitivity of 81.7 percent and a specificity of 82.5 percent. In the placenta and membranes, fetal fibronectin was found at points of contact with the uterine wall. CONCLUSIONS: The presence of cervicovaginal fetal fibronectin in the second and third trimesters of pregnancy identifies a subgroup of women who are at high risk for preterm delivery. This phenomenon may reflect the separation of the chorion from the decidual layer of the uterus, with the release of intact or degraded chorionic components of the extracellular matrix into the cervical and vaginal secretions.

In vivo alteration of RES phagocytosis by magnetic albumin microspheres.

The effect of non-localized magnetically responsive albumin microspheres (MR-AMS) on RES phagocytic function was investigated. MR-AMS and MR-AMS containing Adriamycin (MR-AMS-ADR) were injected intravenously in rats at a dose which saturated all RES phagocytes. The ability of the RES to clear the bloodstream of a subsequent suspension of MR-AMS or streptococci was then analyzed over a three day period. MR-AMS clearance was enhanced after initial RES saturation with MR-AMS but depressed when the saturating agent was MR-AMS-ADR. Bloodstream clearance of streptococci was depressed by MR-AMS and MR-AMS-ADR. Localization of MR-AMS in the RES might depress RES phagocytosis by physical saturation of phagocytes or by a direct toxic effect of encapsulated chemotherapeutic agents.

Magnetic targeting of microspheres in blood flow.

Magnetically responsive albumin microspheres can be targeted to the vasculature of specific organs, using extracorporeal magnetic sources. Experiments have been performed on targeting these microspheres to specific regions of normal and tumorous rat tails. This paper quantitatively analyzes the relationship between magnetic forces and the observed microsphere holding. The magnetic forces are determined by the magnetic responsiveness of the microspheres, and by the spatial field of the magnet; both of these are measured. The microsphere holding is defined as that fraction of the microspheres perfusing the tail which are held at a particular site; this is measured at various positions in the tail. The holding as a function of magnetic force is thereby established. To interpret the data, the dynamics of microspheres in blood flow is considered, including motion to a vessel wall, shear forces at the wall, and intersphere attraction. Overall, the method appears favorable for targeting therapeutic drugs to tumor sites in humans.

Selective targeting of magnetic albumin microspheres containing low-dose doxorubicin: total remission in Yoshida sarcoma-bearing rats.

Magnetically responsive albumin microspheres containing doxorubicin hydrochloride were selectively localized in Yoshida sarcoma tumors. Tumors were implanted subcutaneously in the tail of Holtzman rats and allowed to grow to at least 200 mm2 size before initiation of experimental treatment. Drug-bearing microspheres at a dose level of either 0.5 or 2.5 mg/kg were infused proximal to the tumor via the ventral caudal artery. A bipolar permanent magnet was placed adjacent to the tumor during the infusion to effect localization. Control animals were treated with free doxorubicin infused intra-arterially at 5.0 mg/kg or 0.5 mg/kg. In other test groups animals received placebo microspheres localized in the tumor via influence of the external magnetic field, or drug-containing microspheres were infused without utilization of the magnet to effect localization. Of the 22 animals receiving magnetically localized doxorubicin microspheres 17 had total histological remission of the tumor. The remaining animals demonstrated marked tumor regression representing as much as 500-600 mm2 decrease in tumor size. While no deaths or metastases occurred in the groups receiving localized drug, animals treated with free doxorubicin, placebo microspheres or non-localized doxorubicin microspheres exhibited a significant increase in tumor size with metastases and subsequent death in 90-100% of the animals. No significant differences were noted in tumor regression/remission data between the 0.5 and 2.5 mg/kg dose levels of magnetically localized doxorubicin spheres. These results represent a significant advance in targeted chemotherapy in that 77% of the animals in the magnetically localized doxorubicin microsphere treatment groups exhibited total remission after only one regimen of drug therapy.

Selective targeting of magnetic albumin microspheres to the Yoshida sarcoma: ultrastructural evaluation of microsphere disposition.

Magnetic albumin microspheres (1 micron average diameter) were selectively targeted to subcutaneous solid Yoshida sarcoma tumors (average size 450 mm2) in Holtzman rats. This was accomplished by placing an external magnet adjacent to the tumor while the microspheres were infused. Microspheres contained ultra-fine particles of Fe3O4 and no drug (placebo). Placebo microspheres were used due to the previously demonstrated rapid tumoricidal effect of targeted low-dose doxorubicin microspheres. Animals were killed 10 min, 60 min, 30 min, 24 hr and 72 hr after microsphere administration and tumors were examined by transmission electron microscopy to determine the in vivo disposition of the magnetically targeted microspheres. Using placebo microspheres, we have demonstrated microspheres endocytosed in endothelial cells as early as 10 min after infusion. By 30 min microspheres can be seen in the extravascular compartment, sitting adjacent to tumor cells and occasionally in tumor cells. By 24 hr the majority of microspheres have been endocytosed by tumor cells. Microspheres were still observed within tumor cells as late as 72 hr after administration. The rapid extravasation and cellular uptake of magnetically focused microspheres explains the extremely rapid tumoricidal effect previously observed when doxorubicin-containing microspheres were targeted to the tumor.

A rapid method for immunofluorescent staining of paraffin sections using iron-containing protein A microspheres.

A method to rapidly perform immunofluorescence or light microscopic staining on formalin-fixed paraffin sections has been devised utilizing magnetic albumin microspheres containing Staphylococcal protein A. Because the protein A constituent of the microspheres has the property of binding the Fc portion of immunoglobulin G (IgG) class antibodies, the microspheres can be used to rapidly bind antigen-antibody complexes by the Fc portion of the antibody. Deparaffinized sections were stained with fluorescein isothiocyanate-conjugated antibody (IgG fractions) by standard techniques, after which the protein A microspheres were layered over the sections. Distinct fluorescence of sections was noted with the addition of the microspheres, whereas only autofluorescence was present with direct staining alone. The microspheres were also visualized by light microscopy by a subsequent Prussian blue reaction, staining the Fe3O4 within the microsphere matrix. This method represents a more rapid method for identifying antigens in tissues embedded in paraffin than has previously been reported.

Specific cell binding using staphylococcal protein A magnetic microspheres.

Protein A, a protein derived from Staphylococcus aureus, was incorporated into the matrix of magnetic albumin microspheres. Because staphylococcal protein A binds most subclasses of immunoglobulin G through their Fc portions, immunoglobulin may be rapidly bound to microspheres without chemical coupling agents or a diamino-heptane spacer group. Microspheres so prepared bind specifically to a given cell type when incubated in vitro with a heterogeneous cell population. The use of these microspheres as a drug carrier capable of cellular specificity as well as their ability to isolate homogeneous cell populations rapidly is discussed.

In vivo kinetics of magnetically targeted low-dose doxorubicin.

The in vivo kinetics of low-dose doxorubicin (0.05 mg/kg), entrapped in a carrier and magnetically targeted, were characterized in a rat tail model. Tissue concentrations of doxorubicin at a preselected target site and in various organs were followed over time. As late as 60 min postinjection, 3.7 microgram/g of drug was found at the target site with no detectable drug levels found in any organ. In comparison, a 100-fold higher dose (5.0 mg/kg iv) of free doxorubicin yielded drug concentrations of 1.8 microgram/g at the target site and 15.0 microgram/kg in the pooled organs. Therefore, 1% of the free intravenous dose targeted magnetically yielded approximately twice the local doxorubicin concentration at a preselected target site with no detectable systemic distribution. Magnetic targeting of particulate drug carriers to localized disease sites is suggested as an efficient method of obtaining high local drug concentrations and may reduce many unwanted side effects from unrestricted systemic circulation.

Tumor remission in Yoshida sarcoma-bearing rts by selective targeting of magnetic albumin microspheres containing doxorubicin.

Magnetically responsive albumin microspheres containing doxorubicin and magnetite (Fe3O4) were selectively targeted to Yoshida sarcoma tumors in rats by utilizing an extracorporeal magnet. Tumor cells were inoculated subcutaneously in the tail of rats, and the tumors were allowed to grow to an average size of 9 X 45 mm prior to initiating treatment. Drug-bearing microspheres (0.5 mg of doxorubicin per kg of body weight) were infused proximal to the tumor through the ventral caudal artery while the tumor was exposed to an external magnetic field of 5500 Oe for 30 min. Control animals received free doxorubicin administered either intravenously (5 mg/kg) or infused intraarterially (5 and 0.5 mg/kg), drug-bearing microspheres infused intraarterially (0.5mg/kg), without the external magnet, or placebo microspheres with magnetic localization. Of the 12 animals treated with a single dose in the experimental group, 9 exhibited total remission of the tumor, representing a disappearance of tumors as large as 60 mm in length. Marked tumor regression was observed in the remaining three rats, and no deaths or metastases occurred in the experimental group. In contrast, significant increases in tumor size with widespread metastases occurred in all control groups and most rats died. These experiments indicate that targeting of oncolytic agents to solid neoplasms by magnetic microspheres may be a means of increasing the efficacy and decreasing the toxicity of antitumor agents.

In vitro release of biologically active adriamycin by magnetically responsive albumin microspheres.

The kinetic release of therapeutically active Adriamycin from two different heat-stabilized preparations of magnetically responsive albumin microspheres (1 micron) has been evaluated using a rapid in vitro bioassay-harvesting system. Release products are added to freshly plated monolayers of a malignant Fisher 344 rat fibrosarcoma cell line, and the inhibition of [3H]uridine incorporation into trichloroacetic acid-precipitable material (RNA) and whole cells is determined in a 6-hr microtiter assay. The latter harvesting technique utilizes semiautomated cell collection using a multiple sample harvester. Standard fluorometric drug analyses are used to quantitate the chemical release rates of Adriamycin and related degradation products (aglycones). By altering the temperature of albumin matrix stabilization from 22 to 135 degrees, the half-time for the release of therapeutically active drug has been varied from 15 min to 9 hr. The biological activity of drug products released by the highest temperature (135 degrees) preparation is 78% of that for the native free drug. These in vitro antitumor assays are used to predict the maximal rates of release that could occur at the tissue level under optimal conditions of in vivo targeting.

Interaction of living cells with polyionenes and polyionene-coated surfaces.

Polyionenes have been shown recently (A. Rembaum, Appl. Polym. Symp. No. 22, 299, 1973) to produce the following biological effects: 1) bactericidal action, 2) formation of insoluble complexes with DNA and heparin, 3) neuromuscular blocking action, 4) cell aggregation and lysis, and (5) cell adhesion. In present study, polyionenes of various structures (mainly I3, 3, I6, 10) were used as molecular probes to gain an understanding of the cell surface phenomena of adhesion on glass- and polyionenes-treated surfaces. Since tumor cells show different durface cell properties, including an increase in the anodic mobility, they bind preferentially to polyionene-treated surfaces. Normal human diploid WI-38 cells were found to adhere at a lower rate than SV-transformed WI-38 cells. However, cell spreading was accelerated in both cases. A study of the interaction of polyionenes in solution in vitro and in vivo and polyionenes covalently bound to polymeric microspheres with leukemic murine EL4 cells and normal thymocytes showed specific cytotoxity towards the leukemic cells.