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There are three main objectives of prenatal and perinatal testing:
Prenatal Testing Current recommendations for prenatal testing are summarized in the following table. Prenatal Testing Guidelines All women should be tested for ABO and D as early as possible in pregnancy, preferably during their first trimester visit. ABO typing is done primarily for patient identification. The results should not conflict with historical records. Any discrepant results must be fully investigated. A record of the maternal ABO type is also helpful should the newborn infant develop signs and symptoms consistent with ABO HDN. D typing should be done on at least two separate occasions and the results should be identical. This recommendation is especially important as a safeguard to prevent an Rh negative woman from being falsely typed as Rh positive and denied RhIG. Serologic confirmation of the D type is also recommended at the beginning of each subsequent pregnancy. Historically, if a patient typed as Rh negative, additional testing was then performed to determine if they had Rh Du or weak D expression. In the past several years, weak D testing has been eliminated for all patients except obstetric patients. More recently, the American Association of Blood Banks has determined that weak D testing is no longer necessary for obstetric patients. The main reason is that today's blood typing reagents are much more potent and most of the patients who were previously typed as weak D are now typed as Rh positive. All women are now typed as either Rh negative or positive. The clinical implication of this change is that a few women who actually have weak expression of the D antigen will be classified as Rh negative and will be candidates for Rh immune globulin. Giving Rh immune globulin to these women is not harmful. All women, regardless of their D type, should be tested during each pregancy for clinically significant antibodies, ideally at their first obstetrician visit. Antiglobulin testing should be done with anti-IgG reagent to detect clinically significant antibodies that are capable of crossing the placenta and causing hemolytic disease of the newborn (HDN). The same ehnancement methods (LISS, PEG) used to detect unexpected antibodies during pretransfusion testing can be used for prenatal antibody detection. An additional antibody screen may be ordered for Rh negative women at 26 to 28 weeks gestation to determine if active immunity to D has developed, before administration of RhIG prophylaxis. The risk of a woman developing anti-D between the first trimester and 28 weeks gestation is very low, occuring in only 2 of every 1000 Rh negative pregnancies. This antibody screen is not required by regulatory agencies and is probably not cost effective. In most cases, Rh positive patients need be screened for antibodies only once during their initial visit. Only 1 in every 1000 women develops new antibodies capable of causing HDN between the first and third trimesters. Routine testing for unexpected antibodies in the third trimester or at delivery rarely provides useful clinical information. Regardless of D type, additional antibody screening in the third trimester is indicated when there is a history of significant antibodies, blood transfusions or traumatic deliveries. Antibody screening is also necessary prior to antepartum transfusion. If the antibody screen is positive at any time during pregancy, the blood group specificity of the antibody should be identified. It should not be assumed that an antibody present in a D negative woman is anti-D, even after RhIG therapy. A limited panel or Rh negative RBCs, consisting of r'r (dCe/dce), r''r (dcE/dce), and rr (dce/dce) cells, can be used to exclude significant antibodies other than D. If a pregnant woman's plasma is reactive with one or more of the limited panel cells, then a comprehensive antibody panel needs to be run to identify antibody specificity. Antibody identification is necessary to determine if the antibody is likely to cause HDN (see table in Chapter 1). IgG antibodies to Rh, Kell, Duffy, Kidd and S antigens are likely to cause HDN. Some IgG antibodies, such as antibodies toChido/Rodgers, Knops and Cromer system antigens, usually do not cause HDN. Antibodies to Lewis, P1 and M antigens are usually IgM and do not cross the placenta. Also, these antigens are poorly expressed on fetal RBCs. Even if antibody did cross the placenta, it would not bind to fetal RBCs. Therefore, HDN is unlikely. Once these IgM antibodies are identified, no further testing is required. Once a clinically significant antibody is identified, the titer needs to be measured to assist the obstetrician in determining the severity of HDN and the need for fetal monitoring, ultrasonography, amniocentesis, and cordocentesis. Invasive procedures such as amniocentesis and cordocentesis may introduce additional fetal cells into the maternal circulation and cause the antibody titer to increase dramatically. Therefore, obstetricians usually wait until the antibody titer reaches a critical level before performing an invasive procedure. Amniocentesis is usually performed if there is a twofold or greater increase in titer during pregnancy or if the titer equals or exceeds a critical level. In the first pregnancy affected with anti-D, either a rising titer or a critical titer of 16 indicates the need for amniocentesis and OD450 analysis. The critical titer varies from insitution to institution, depending on the titer method, but is usually between 8 and 32. For anti-D, the use of R2R2 RBCs are recommended for titrations, because they have a uniform expression of D antigen from one donor to another. Transfusion services usually use the same critical titer as anti-D for other Rh antibodies. Very little data exists concerning critical titers for non-Rh antibodies encountered in pregancy. Transfusion services may use a higher titer of 32 or 64 for these antibodies. Repeat titers at 2 to 4 week intervals after 18 weeks gestation is helpful in determining the need for invasive monitoring. If a previous sample from the current pregnancy is available, it should be tested in parallel with the current sample. Once the decision has been made to monitor HDN with an invasive procedure, subsequent antibody titrations are not necessary. Not all clinically significant antibodies detected during pregnancy will cause HDN. The mother's antibody may have been induced during a previous pregnancy by a different consort, in which case the current fetus may be antigen negative and not at risk for HDN. It is worthwhile to phenotype RBCs from the putative father whenever the potential for HDN exists. The probability that a fetus carries the antigen to which the maternal serum contains antibody can be estimated from maternal and paternal phenotypes. If there is a high likelihood that the father is heterozygous for the gene encoding the offending antigen, molecular genotyping of amniotic fluid cells or chorionic villi can performed. PCR assays are available for for Rh, Kell, Duffy or Kidd genotypes. Testing at Delivery At delivery, the following tests should be performed on the mother.
The Kleihauer Betke test relies on the principle that red cells containing fetal hemoglobin (HbF) are less susceptible to acid elution than cells containing HbA. A thin smear of maternal blood is exposed to citric acid, which elutes hemoglobin from maternal red cells, resulting in pale ghost cells. Fetal red cells are resistant to acid and retain their hemoglobin. Consequently, they stain pink with erythrosin B dye. The smear is examined microscopically to determine the percentage of fetal red blood cells. Results are reported as the percent of fetal RBCs seen. The sensitivity of the method is approximately 0.1 mL of fetal blood in the maternal circulation. This corresponds to about 1 fetal cell per 50,000 maternal cells. The Kleihauer-Betke stain may occasionally underestimate the number of fetal RBCs present due to the fact that the fetus begins to synthesize hemoglobin A in the last trimester of pregnancy. Fetal cells, which had completed the switch to adult hemoglobin, would be counted as adult cells. False positive reactions may occur when maternal RBCs have increased levels of hemoglobin F such as occurs in various hemoglobinopathies including hereditary persistence of fetal hemoglobin, thalassemias, and sickle cell anemia. This test involves a considerable amount of subjective interpretation. The quality of the stain must be very good so that red cells can be clearly distinguished from leukocytes. Several published studies and proficiency surveys have demonstrated that the precision and accuracy of this method are poor. Variation from laboratory to laboratory is 50% and the rate of fetal cell detection is only 90%. The flow cytometric method utilizes a fluorescently labeled monoclonal antibody to the gamma chain of the HbF molecule (anti-HbF). A sample of whole blood is fixed with glutaraldehyde to crosslink hemoglobin inside the cells and then cell membranes are permeabilized with a detergent to ensure access and binding of anti-HbF. A flow cytometer determines the percentage of fetal cells by analyzing approximately 65,000 cells. Fetal red cells are clearly distinguished from adult cells by their significantly higher fluorescent signal. Proficiency surveys have shown this method to be more accurate and precise. The coefficient of variation is < 7.5%. After delivery the following tests should be performed on a cord or newborn infant capillary or venous blood sample.
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