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Cytogenetic Diagnosis
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The rapid rate at which human lymphocytes divide allows a high-quality karyotype to be obtained within 48-72 hours. For this reason, PUBS has been used when a rapid karyotype was important for the medical management of a patient. This situation presents itself during the workup of fetuses identified to have a structural anomaly or severe early-onset growth restriction at a gestational age near the limit of viability.
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However, the success rate of chorionic villus sampling (CVS) in the second and third trimesters of pregnancy has been reported to be more than 99%, with a risk of spontaneous loss within 4 to 6 weeks from the procedure lower than 0.3%.65,66 In addition, in a series of 551 late CVSs, fetal karyotype was obtained with direct analysis of uncultured chorionic villus cells within a mean interval of 4.4 days (with a standard deviation of 0.86 days) in 96.3% of cases.67 Therefore, late CVS has become a viable alternative to obtain fetal cells for rapid karyotype analysis. Moreover, the emergence of new molecular cytogenetic technologies, first FISH in the mid-1990s6,7 and then qf-PCR,8,9, and 10 allowed 24 to 48 hours' diagnosis of the most prevalent chromosome abnormalities (trisomy 13, trisomy 18, trisomy 21, and sex chromosome aneuploidy), thus providing rapid reassurance for those women with normal results and early decisions of pregnancy management for abnormal fetuses. These molecular-based tests, followed by full karyotype analysis, are widely used in many centers for those women in which fetal anomalies are detected by ultrasound examination and in cases of late referral, further reducing the need for rapid prenatal diagnosis by PUBS.
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Fetal karyotyping from fetal-blood-derived lymphocytes can be considered as an option when attempts of karyotyping through amniocentesis or CVS fail (ie, laboratory culture failures, maternal cell contamination) or yield inconclusive results (ie, true mosaicism or pseudomosaicism). Mosaicism has been reported in 2% to 3% of amniotic fluid cultures68; however, it has been confirmed by PUBS in only 0.2% to 0.4% of cases.68,69, and 70 Possible explanations for this discrepancy include in vitro changes in cultured cells (pseudomosaicism), a postzygotic error that is confined to extraembryonic tissues (amnion or trophoblast), an unrecognized dizygotic twin pregnancy with early death of a chromosomally abnormal twin, or contamination with maternal cells. If mosaicism is not confirmed in fetal blood, patients should be informed that it is not possible to exclude that the cytogenetically abnormal cell line may be present in fetal tissues other than blood cells (ie, liver, brain, etc). Another ambiguous cytogenetic result that can require PUBS is the finding of a supernumerary chromosome (marker chromosome), a heterogeneous group of disorders characterized by the presence of fragments of chromosomes that can be inherited from one of the parents or that may arise de novo. In cases of de novo occurrence, PUBS can be offered to the couple to rule out mosaicism for the marker chromosome. Regrettably, this extrachromosomal material is easily lost from the leukocytes. Identification of mosaicism for the marker chromosome in fetal blood is diagnostic, but a negative result is not helpful.69
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Congenital Infections
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In the past, fetal blood sampling has been used in the prenatal diagnosis of several congenital infections including toxoplasmosis, rubella, cytomegalovirus, varicella, and parvovirus. The general approach consisted of obtaining fetal blood in which to isolate the infectious agent or document a specific fetal immune response. The magnitude of the fetal immune response, however, may vary according to the maturity of the fetal immune system, thus not consistently detectable prior to 20-22 weeks of gestation. Such response can also be transient, and therefore be affected by the time interval between infection and blood sampling. Moreover, fetal immune response may be weak or undetectable, and the formation of antigen–antibody complexes under conditions of excess antigen may interfere with the immunologic assay used. For these reasons, documentation of a specific fetal immunoglobulin M (IgM) response is considered diagnostic of recent infection; in contrast, its absence is not helpful in the diagnosis. Currently, PCR amplification is used to rapidly detect and quantify the presence of microorganisms within the amniotic fluid. The sensitivity of this technique is high and the overall accuracy is comparable to or better than fetal blood tests by PUBS, with less risk to the fetus.12,13,14, and 15
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Fetal anemia has numerous etiologies, both immune and nonimmune. Overall, the most common is red cell alloimmunization, which still affects 6.3 of every 1000 live births in United States.71 The purpose of perinatal management of suspected fetal anemia is to identify affected fetuses, correct their anemia by transfusion, and deliver them at the appropriate time. Traditionally, identification of the anemic fetus has involved invasive methods such as amniocentesis (to quantify bilirubin, present in the amniotic fluid as the result of fetal hemolysis, and expressed as a change in optical density) or PUBS, which allows direct measurement of fetal hemoglobin. Particularly in situations where fetal anemia was caused by suppression of erythropoiesis (Kell alloimmunization and Parvovirus B19 infection) or by acute fetomaternal hemorrhage, the measurement of amniotic fluid optical density 450 was not effective in the detection of fetal anemia, and accessing the fetal circulation remained the only tool available for the diagnosis. The awareness of risks associated with invasive procedures prompted several investigators to search for noninvasive means of detecting fetal anemia. The measurement of the peak systolic velocity in the middle cerebral artery (MCA) has emerged as an accurate method of identification of fetal anemia.16 Adoption of surveillance with serial MCA Doppler assessments obviates the need of invasive testing, with data suggesting that as many as 70% of invasive procedures can be eliminated when relying on Doppler ultrasonography. The peak MCA velocity has also proved to be useful in the detection of nonhemolytic anemic states, including Kell alloimmunization, fetal Parvovirus infection, and fetomaternal hemorrhage, and following laser therapy for twin-to-twin transfusion.72
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Primary Immunodeficiency Diseases
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Primary immunodeficiency diseases are heritable disorders of immune system function. Many are associated with single gene defects, whereas others may be polygenic or secondary to gene–environment interactions.18,73,74 The 5 main groups of primary immunodeficiencies include humoral immunodeficiencies, cellular deficiencies, combined immunodeficiencies, defects in fagocyte function, and complement deficiencies. In the past 10 years there has been a rapid increase in scientific knowledge about the pathogenesis of a number of inherited primary immunodeficiencies, and in many cases the underlying genetic cause has been recognized and early molecular testing via mutation analysis in the CVS-extracted DNA is available.75 In some cases prenatal testing can be accomplished by measuring the concentration and/or function of the defective gene products in a chorionic villus sample (eg, the enzymes adenosine deaminase and purine nucleoside phosphorylase deficiencies in some forms of severe combined immunodeficiency [SCID]).76 Fetal blood analysis is reserved for cases in which a genetic basis is suspected but the specific defect is unknown. This type of testing, involving phenotypic and functional analysis of immune cells in fetal blood, is suitable in the presence of other affected family members. Because immune cells are only present in the peripheral circulation in significant numbers after 18 to 20 weeks, PUBS should be postponed until this gestational age. This type of testing is less reliable than genetic testing, since a normal result does not allow the exclusion of the possibility that the disease will emerge later in fetal development. Table 26-6 displays the normal values of T cells and B cells at different gestational ages.
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Hemophilias A and B, inherited as X-linked recessive traits, are the most common hereditary hemorrhagic disorders, and are caused by a deficiency or dysfunction of blood coagulation factor VIII and factor IX, respectively. Together with von Willebrand disease, a defect of primary hemostasis, these disorders accounts for 95% to 97% of all inherited bleeding disorders. Fetal cord blood sampling to investigate hemostatic disorders is rarely used and considered only when all the alternative techniques can not be applied or genetic analysis is not informative.77 Most inherited defects of hemostasis are now amenable to first trimester prenatal diagnosis by molecular techniques17 that provide an early diagnosis and have a lower risk of fetal morbidity and mortality; therefore, the use of PUBS for this indication (prenatal diagnosis of coagulopathies) has been largely abandoned.
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Fetal blood sampling can be useful in the prenatal diagnosis and management of congenital quantitative (thrombocytopenias) and qualitative (thrombocytopathies) platelet disorders.
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These include immune-mediated thrombocytopenias and genetic syndromes in which thrombocytopenia is one of the features.
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Immune thrombocytopenia (ITP), also known as idiopathic or autoimmune thrombocytopenic purpura, accounts for about 5% of cases of pregnancy-associated thrombocytopenia78 and is caused by autoantibodies directed against platelet surface glycoproteins (HPA-1a is by far the most common antigen involved). Maternal antiplatelet antibodies may cross the placenta and cause fetal thrombocytopenia. The risk of fetal and neonatal bleeding as a result of thrombocytopenia is low in ITP. No maternal findings are of value in predicting the degree of fetal thrombocytopenia. A PUBS in late pregnancy for measuring the fetal platelet count is not considered justified as a routine practice in ITP, because the associated risks outweigh the risk of serious neonatal hemorrhage.79 Moreover, data from clinical cohort studies do not confirm that altering the route of delivery based on the perinatal platelet count influences the neonatal outcome.80
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Alloimmune thrombocytopenia (AIT) is the most common cause of severe thrombocytopenia in fetuses and term neonates and the most frequent cause of intracranial hemorrhage in neonates born at term. AIT is caused by parental incompatibility of a platelet-specific antigen, with maternal sensitization to the antigen in question resulting in fetal thrombocytopenia. The disease can be considered as the counterpart of Rh immunization in the platelet system, but unlike Rh sensitization the first born is affected in half of the cases. Intracranial hemorrhage occurs in 10% to 22% of severely affected infants, and 75% of the bleeding events occur before delivery. The optimal antenatal management for AIT has not been defined and remains controversial.81 In the past, an invasive approach to therapy involved serial (because of the short half-life, 4 to 5 days of platelets) intrauterine transfusion of platelets directly into the fetal circulation. The estimated cumulative risk of fetal loss with transfusion therapy in AIT is 1.3% per procedure and 5.5% per affected pregnancy.19 This invasive approach is now reserved as a "rescue therapy," when medical treatment has failed to increase the platelet count. Antenatal medical therapy of AIT with high doses of intravenous immunoglobulins (IVIG) and steroids was first introduced in the mid-1980s and it is now clear that this disorder can be successfully treated medically in utero in the vast majority of cases.19 However, in most studies of medical therapy for AIT, serial PUBS procedures are performed to initially assess the severity of thrombocytopenia and subsequently to monitor the effectiveness of its treatment. There is no question, however, that PUBS for diagnostic purposes is associated with an increased fetal morbidity and death in patients with AIT, due to the higher risk of fetal exsanguination. In a series of 79 patients with AIT, serious complications occurred in 6% of the 175 fetal sampling procedures, and this resulted in the emergency delivery or death in utero of 14% of the infants.82 When PUBS is performed in patients with AIT in which the fetal platelet count is less than 50,000/mL, transfusion of maternal-compatible platelets should be administered before withdrawing the needle. Awareness of the potential for morbidity with PUBS has led to efforts to determine optimal therapeutic regimens for the management of women with AIT, using a risk stratification based on the history of previous affected siblings.82 The ultimate objective of therapeutic regimens currently under investigation is to effectively treat the disorder, while avoiding PUBS as much as possible or, if necessary, delaying the procedure until a gestational age when a viable infant can be delivered if a complication arises.
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Thrombocytopenia with absent radius (TAR) syndrome is an autosomal recessive condition characterized by the absence or hypoplasia of the radius and hematologic abnormalities. Fetal blood sampling is indicated in the presence of prenatal sonographic evidence of radial abnormalities.83 Differential diagnosis includes mainly Fanconi pancytopenia syndrome, which is diagnosed by a characteristic high frequency of diepoxybutane-induced chromosomal breakage on karyotype.
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Table 26-7 displays the most common congenital qualitative platelet disorders and cases of prenatal diagnosis reported.
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Hemoglobinopathies refer to a diverse group of inherited disorders characterized by a reduced synthesis of one or more globin chains (thalassemias) or the synthesis of structurally abnormal hemoglobin (hemoglobin variants).
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Originally, the prenatal diagnosis of hemoglobinopathies was accomplished on fetal blood by assessment of the rate of synthesis of different globin chains. Blood cells were incubated with radiolabeled leucine for 2 hours to label the nascent hemoglobin. Red cells were then lysed and the globin precipitated and separated into carboxyl-methylcellulose columns. The presence of only hemoglobin S identifies a fetus with sickle cell anemia. Quantitation of the amount of β chains in relation to the amount of γ chains synthesized was necessary for the diagnosis of β-thalassemia. Now that most of the mutations responsible for hemoglobinopathies have been characterized, the diagnosis of these disorders is possible in many centers by mutation analysis with PCR-based techniques as early as 12 weeks of gestation on specimens collected with CVS.84 Moreover, in recent years, noninvasive prenatal diagnosis of β-thalassemia using individual fetal erythroblasts isolated from maternal blood after enrichment has been reported.85
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Intrauterine Growth Restriction
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In the presence of early onset fetal growth restriction, fetal blood sampling with cordocentesis has been proposed to identify possible causes, such as fetal aneuploidy or infections, as well as to directly assess the fetal acid-base status. Previous studies have attempted to relate blood gas levels determined through PUBS to outcome in chromosomally normal fetuses that are small for gestational age.86 A significant association between the pH of blood obtained by cordocentesis in SGA fetuses and their neurologic development in childhood has been reported.87 However, the information that can be obtained when PUBS is performed in growth-restricted fetuses is of limited value in the prediction of fetal compromise. Prospective studies of growth-restricted fetuses in recent years provided evidence that Doppler interrogation of the venous system offers an accurate prediction of acid-base status at birth.88 Moreover, the combination of Doppler studies and biophysical parameters effectively predicts deterioration prospectively. Therefore, the results of PUBS, which largely contributed to improving the understanding of the pathophysiology of intrauterine growth restriction (IUGR), are not used to guide intervention in current clinical practice, and the delivery of chromosomally normal growth-restricted fetuses is usually timed on the basis of noninvasive tests.
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Direct quantification of fetal thyroid hormones using PUBS enables an accurate determination of thyroid status in fetuses at risk for thyrotoxicosis (ie, transplacental transfer of thyroid-stimulating antibodies) or hypothyroidism (ie, primary congenital hypothyroidism, transplacental transfer of antithyroid drugs or thyroid-blocking antibodies). The rationale of prenatal diagnosis of thyroid dysfunction is to promptly treat this condition in utero. Evidence has been provided that intramniotic injection of thyroxine is useful to reduce the size of the fetal goiter and possibly esophageal compression and polyhydramnios.89 Whether this therapy can optimize fetal growth and neurologic development requires further research. It has been suggested that PUBS should be offered to pregnant women with Grave disease if any of the following are present: (1) a history of previously affected sibling; (2) a history of maternal 131I treatment and high thyroid-stimulating antibodies (>5 IU or >160%); and (3) the presence of fetal tachycardia, growth restriction, fetal goiter, hydrops, or cardiomegaly.90