Infections and Birth Defects (NIH, 1966)

August 30, 2019

[A woman walks up to a medical clinic.] [Narrator:] Today in the United States, one pregnancy in five fails to produce a living, healthy child 126,000 mentally retarded children are born each year. Millions more suffer from various defects. Infections are among the preventable causes of abortions, stillbirths, and damaged infants. Research on the relationship between infections and birth defects is the major assignment of the Section on Infectious Diseases, Perinatal Research Branch, National Institute of Neurological Diseases and Blindness. The section works directly with other institutes, including Allergy and Infectious Diseases, and Child Health and Human Development, all within the federal government’s Department of Health, Education, and Welfare. A number of infections can cause abnormal pregnancy outcomes. These include rubella, cytomegalic inclusion disease, rubeola, mumps, herpes simplex disease, western equine encephalitis, chicken pox, smallpox, gloxinia, polio, hepatitis, Coxsackie B virus, influenza, syphilis, tuberculosis, toxoplasmosis. The Perinatal Research Branch serves as research headquarters for 14 medical centers throughout the nation which participate with the Neurology Institute in the Collaborative Perinatal Research Project, a study of birth defect causes. This young woman, Mrs. Owens, represents one of 50,000 mothers who, with their infants, are taking part in this collaborative project. In addition to infections, genetic, obstetrical, and other perinatal factors will be considered in the study. Mrs. Owens will give blood at intervals to aid in the study of infections of pregnancy. Her child will contribute smaller amounts of sera at specified intervals. Each time Mrs. Owens’ blood is taken, 40 ml is collected in two 20 ml vacutainer tubes. The specimens provided by Mrs. Owens are allowed to clot at room temperature. Then, placed in a refrigerator overnight to provide maximal retraction of the clot. This procedure allows the technicians to obtain a maximal yield of serum, that fraction of the blood containing the reactive particles to be studied. Tubes are ringed to loosen the clot prior to separating clot from serum in the centrifuge. Specimens are spun for 15 minutes at 2,000 revolutions per minute in a refrigerated centrifuge. [Samples are removed from the centrifuge.] Next, a technician employs a 20 or 25 ml pipette to pull the serum from the two tubes into a single container. [The technician examines the solution inside the long glass tube.] The serum is then transferred to four 1 gram vials. The contents of each vial can be used several times for different tests. The blue line indicates a critical 3 ml mark. If filled beyond this mark, the vial might break during freezing or thawing. Packets of sera are stored at minus 15 to minus 20 degrees centigrade. Cartons are filled with the packets. Fifty pounds of dry ice will keep the specimens frozen for four days, even at summer temperatures. Shipments are made to the Serum Center, Section on Infectious Disease, Bethesda, Maryland. The specimens are logged in by coding clerks at the Serum Center. Each specimen is checked for quantity and quality. Good quality frozen serum is straw-colored. Records are prepared. The packets are placed in aluminum trays, then they are stored at minus 20 degrees Centigrade in one of the four Serum Center freezers. More than one million specimens are available for testing. Priorities for study are determined at staff conferences. For example, during the German measles epidemics, rubella virus was investigated extensively. As a result of the staff conference, statistical personnel using the records in the Serum Center, select the sera of patients to be tested. A tally sheet prepared after the selection enabled personnel to locate the sera. The vials to be tested are taken from their trays, placed in polystyrene blocks and transported to the laboratory. These lightweight polystyrene blocks, tailored to the needs of the section, each hold up to 200 vials. A micro-technique has been perfected to simplify, extend, and speed up the testing. It employs smaller tools, smaller test amounts, yet gives reliable results. With countless blood tests to perform, ordinary procedures would not be feasible. When the vials of sera have thawed, an automatic diluting machine mixes one part of thawed serum with three parts of saline. The dropper takes up the serum, then flushes it out with the saline. In preparation for the test, each cup in this disposable plastic plate receives a drop of saline solution. Long-handled loops calibrated to pick up .025 ml of solution are dipped into the various sera specimens. Several loops can be handled simultaneously. Each contains a different specimen. One of the loops holds the solution made from the sera contributed by Mrs. Owens. The stylus of each loop is rotated in the fingertips to ensure good mixing. The loops carry solution from one row to another for serial dilutions. Specific antigen is added by pipette. A drop measures .025 ml. Mumps antigen, for example, is added. By using different antigens derived from various microorganisms, the section is testing for more than 100 infections, which are of importance to man, to determine to what extent they may contribute to birth defects. The red line marks a serum control which receives no antigen. This will pick up false positive reactions. Few false-positive reactions occur. Next, complement is added to the antibody/antigen mixture. Complement is a substance that takes part in the reaction of antibody with antigen and provides an indirect measurement of the reaction. The plates are refrigerated at 4 degrees Centigrade for 16 to 18 hours. This gives time for the fixation of complement, if any, to take place. If Mrs. Owens’ sera has mumps antibodies, evidence that she has been exposed to this virus, the antibodies would react with the antigen. At the same time, complement is being fixed, or used up as part of the reaction. Next day, sensitized sheep red blood cells are added with a .05 ml dropper. They will provide a way to detect whether complement remains in the test solution or has been fixed. The plates are sealed with a press, taken to a 37 degrees centigrade walk-in incubator, and placed in the shaking machine for 15 minutes. After shaking, the plates remain in the incubator for an additional 30 minutes. A sedimentation period of four hours at room temperature is routine to allow any red cells that were not broken up, or lysed, by unfixed complement to settle. A centrifuge speeds up the process of concentrating intact cells. The test plates are ready to read. Infection is evident when the reaction between antibody and antigen fixes complement, then complement is not free to lyse the red cells. As a result, the positive test appears as a button of red cells. In a negative test, there is no such accumulation of intact red cells. They are broken up, and the liquid appears pink. The more diluted samples had less antibody and a less-apparent button of red cells. In a positive test, the button is decidedly visible. The data on infections is a vital part of the mother’s history. Other variables, such as age, number of previous pregnancies, manner of delivery, also are recorded. The IBM cards become masses of information to be fed into modern electronic computers. These can give answers in days that formerly would have taken months to acquire. The computers enable scientists to correlate millions of bits of information…to view related events from new perspectives. However, the judgment of highly trained investigators is essential in analyzing this data on birth abnormalities correctly. The section of infectious diseases conducts a number of related studies employing a variety of test methods. For example, the fluorescent antibody technique permits the viewer to see fluorescent-labeled antibody attached to specific antigen. Ultraviolet light excites the fluorescence. Literally, spotlighting the reaction. Tissue cultures of placenta and cord are studied to detect pathogenic effects of viruses. A characteristic pattern of cell destruction identifies certain viruses. Animals are studied, including ferrets, and monkeys. Pregnant Rhesus monkeys are injected with rubella virus or other agents to determine at what stage in development of the fetus effects might be observed. An infant monkey is delivered by cesarean to see whether virus can be recovered from organs or if defects are occurring. Mrs. Owens and other mothers participating in the study, the laboratory workers, hospital and clerical personnel, special testing procedures, all are a part of the research story on infections and birth defects. The accomplishments of this research and of the overall perinatal studies will become part of medical communications useful to family doctors, obstetricians, pediatricians, and other specialists throughout the world.

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