Fetal Cell Screen
The fetal cell screen is used to assess the degree of Rh (also called Rho [D] or D) sensitization in an Rh-negative mother during gestation of an Rh-positive fetus. Fetal red cells carrying the Rh positive antigen can leak into the maternal circulation and immunize the Rh-negative mother, possibly leading to hemolytic disease of the newborn. The potential degree of isoimmunization and severity of hemolytic disease increases with each Rh-incompatible pregnancy. The fetal cell screen quantifies the extent of the leakage and permits determination of how much Rh immune globulin (RhoGAM) to administer in order to prevent the mother from developing anti-D antibodies against the fetus.
Rho immune globulin is normally administered by intramuscular injection to Rh-negative mothers at 28-30 weeks of gestation and again within 72 hours postpartum. The dosage is higher for mothers who test positive for fetal red blood cells. The treatment is performed earlier when amniocentesis or chorionic villus sampling is performed, or following spontaneous or elective abortion.
Aside from Rh-incompatibility cases, the test may be ordered to investigate the severity of maternal-fetal hemorrhage following a trauma, or during post-mortem examination of a stillbirth.
Although in principle the fetal cell screen is simple to perform, the procedure is subjective, labor-intensive, and time-consuming; and requires skill, experience, and strict attention to detail.
When possible, the procedure should be scheduled with the laboratory in advance. The specimen may be rejected by the laboratory if it is delivered too long after
The possibility that the mother has an inherited blood disorder that might produce fetal hemoglobin should be considered when interpreting a positive result.
The fetal cell screen allows discrimination of fetal from maternal red blood cells in a sample of maternal blood. This procedure is also known as the Kleihauer-Betke test; acid elution for fetal hemoglobin; fetal hemoglobin stain; and fetal-maternal hemorrhage test.
At the hospital or laboratory, 3–7 ml of maternal blood is drawn, usually from a vein on the back of the hand or the inside of the elbow. The venipuncture site is cleaned with antiseptic, a needle is inserted, and the blood sample is collected into a vacutainer containing either EDTA or citrate. The procedure takes about five minutes. The nurse or phlebotomist performing the venipuncture should observe universal precautions for the prevention of transmission of bloodborne pathogens. The sample should be kept at room temperature and transported to the laboratory for analysis within 6 hours.
The fetal screen test may be performed as a qualitative serological (antibody-based) test. In this method, a 3% suspension of the mother's blood in physiological saline is mixed with an antibody to the Rho (D) antigen. The antibodies will bind to any Rh-positive fetal cells present. After the cells are incubated with the antibody, the cells are washed to remove any unbound reagent antibody, and one drop of a 3% suspension of reagent Rh positive red cells is added. These cells serve as indicators. They bind to the remaining antigen-combining sites of the antibodies attached to fetal red cells, causing formation of rosettes. The number of rosettes found in five low-power microscopic fields is determined. When greater than or equal to seven rosettes are seen, the test is considered positive. A fetal-maternal bleed of at least 30 mLs is required to give a positive test result. If this test is positive, a quantitative test is used to more accurately determine the quantity of fetal blood in the maternal circulation.
The quantity of fetal cells in the maternal circulation is most often measured using a special staining technique known as the Kleihauer-Betke method. In urgent cases, such as with trauma, results can be returned in as little as one hour. The blood sample is smeared onto a microscope slide, fixed with ethanol, and allowed to air dry. The blood film is then treated with a weakly acidic phosphate solution and stained with eosin dye. Under acidic conditions, the adult hemoglobin elutes, or leaks, out of the maternal cells, and they appear as pale "ghosts," while the more acid-resistant fetal hemoglobin remains in the fetal red blood cells and becomes stained pink to red. The stained films are viewed under a light microscope, and the number of fetal and maternal cells found in 50 low-power fields is recorded. The number of fetal red blood cells is reported as a percentage of blood cells counted. The greater the amount of fetal blood leakage, the larger the dose of Rho immune globulin to be administered to the mother.
Rho immune globulin is a potent form of anti-D that is given to Rh negative mothers to prevent formation of an antibody to the D antigen. It acts by interfering with the recognition of D antigen by the mother's immune system. One dose can prevent immunization from up to 30 mL of fetal blood. The percentage of fetal cells is multiplied by 50 to give the mLs of fetal blood. (Each percent of fetal red cells represents 1/100th of the blood. Assuming an average maternal blood volume of 5000 mL, each percent is equivalent to 5000/100 = 50 mL.) This number is divided by 30 to give the number of doses needed. If the number to the right of the decimal is lower than 5, one dose is added. Otherwise, two doses are added to the result.
The Kleihauer-Betke is currently the standard method used to detect fetal red blood cells in maternal circulation, although it is labor-intensive, subjective, and not suitable for automation. Since the mid-1990s, automated methods for quantifying fetal cells by flow cytometry have been under development. In these methods, the blood sample is treated with anti-D or anti-fetal hemoglobin antibodies that are labeled with fluoroscein isothiocyanate (FITC). The antibodies react only with the fetal cells. In the flow cytometer, the suspended blood cells in the specimen stream through a sample tube in single file past a beam of ultraviolet light and are counted as the light beam is broken. The light also causes the FITC label to fluoresce, and light detectors in the flow cytometer allow differential counting of the fluorescently tagged fetal cells as they pass. Compared to the Kleihauer-Bekte test, the automated flow cytometry method offers superior objectivity, sensitivity, specificity, and reproducibility.
No special preparation is necessary.
After the blood sample is drawn, pressure should be applied to the puncture site until the bleeding stops to reduce bruising, and a bandage may be applied to the site. The woman may resume normal activities.
Amniocentesis—A prenatal screening procedure in which a needle inserted through the abdominal wall is used to take a sample of amniotic fluid from the amniotic sac in the uterus of a pregnant woman.
Chorionic villus sampling (CVS)—A prenatal screening procedure in which a needle inserted through the vagina and cervix or through the abdominal wall is used to remove chorionic villi.
Flow cytometry—The measurement of cells or cellular properties as they move in a fluid stream past stationary detectors.
Fluorescein isothiocyanate (FITC)—An organic molecule that can be covalently bound to other molecules, and that emits visible light when exposed to ultraviolet radiation.
Hemoglobin—The oxygen-carrying pigmented protein of red blood cells.
Rh-incompatibility—The development of anti-Rh antibodies in an Rh-negative mother in response to antigens from her Rh-positive fetus.
Rhogam—Trade name for Rho [D] immunoglobulin used to immunize Rh-negative mothers.
Vacutainer—A glass tube fitted with a rubber stopper from which air is evacuated to produce a slight vacuum, used for blood specimen collection.
Venipuncture—The puncture of a vein for therapeutic purposes or to collect a specimen.
There is no risk to the mother or fetus. Complications associated with venipuncture are negligible when the procedure is performed correctly. Minor discomfort while blood is drawn or bruising at the puncture site may occur, and there is a slight risk of infection.
The percentage of fetal red blood cells in the circulation of normal non-pregnant adults is generally <0.1%.Alevelof>0.6% fetal cells is reported as positive.
Health care team roles
A physician, usually an obstetrician, orders the test. A nurse, phlebotomist, or laboratory technician collects the blood sample by venipuncture and arranges transport to the laboratory for analysis. The patient should be observed to ensure that bleeding from the puncture site stops.
In the laboratory, a clinical laboratory scientist/medical technologist trained in the Kleihauer-Betke staining procedure prepares, treats, and examines the blood smear, and counts and reports the numbers of maternal and fetal cells. A physician uses the reported ratio of fetal to maternal cells to calculate the amount of Rh immune globulin to be prescribed for the mother.
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Patricia L. Bounds, Ph.D.