Chapter 26: FETAL BLOOD SAMPLING

Fetal blood sampling was performed for the first time more than 30 years ago under fetoscopic visualization.¹ In the early 1980s, cordocentesis under ultrasound guidance,² also referred to as "percutaneous umbilical blood sampling" (PUBS), was introduced, bringing forth a new era in fetal assessment and therapy. Fetal blood obtained by PUBS was the optimal specimen to directly measure fetal hematologic, metabolic, and endocrine parameters; to conduct genetic testing (including chromosomal and single-gene studies); to determine whether or not there was fetal hypoxemia and/or acidemia; as well as in the diagnosis and follow-up of suspected congenital infections.^{3,4, and 5}

In recent years, progress in molecular genetics as well as the advances in noninvasive techniques of antenatal surveillance, have largely obviated the need for PUBS. For instance, rapid analytical techniques, such as fluorescent in situ hybridization (FISH), and quantitative fluorescent polymerase chain reaction (qf-PCR), enable the examination of chromosomes within a few hours, without the need to access the fetal circulation.^{6,7,8,9, and 10} Finally, studies of free fetal DNA in maternal blood to define paternally inherited fetal genes, and noninvasive prenatal diagnosis of aneuploidy may further reduce the need for analysis that required PUBS in the past.¹¹ In pregnancies at risk for fetal infection, such as in the case of toxoplasmosis, cytomegalovirus (CMV), and varicella-zoster virus, amniotic fluid analysis with subsequent PCR amplification of bacterial/viral RNA/DNA has proven to be a valuable alternative diagnostic tool, carrying lower risks and with high positive and negative predictive values.^{12,13,14, and 15} One of the most common indications to perform PUBS, which is screening, identification, and follow-up of fetuses at risk for anemia, can be delayed or avoided by serial assessment with Doppler velocimetry of the peak systolic velocity in the fetal middle cerebral artery (MCA).¹⁶ Moreover, phenotypic diagnosis of inherited defects of hemostasis and primary immunodeficiency diseases have been superseded by first trimester molecular diagnosis in chorionic villi.^17–18 Over the last decade, attempts have been made to derive a risk-stratified approach to patient-specific therapy of alloimmune thrombocytopenia (AIT), while keeping invasive procedures for fetal platelet count to a minimum.¹⁹

In conclusion, even though less invasive or noninvasive techniques can now overcome the need for performing PUBS, direct access to the fetal circulation still remains crucial in selected cases, both for diagnostic and therapeutic purposes, including intrauterine platelet or red cell transfusion. The purpose of this chapter is to briefly review the techniques as well as the current indications of cordocentesis and to discuss its role in clinical practice.

Cordocentesis

Before fetal viability, PUBS can be performed in an outpatient facility. However, after fetal viability is reached, the procedure should be performed in proximity to an operating room because emergency cesarean delivery may be required if complications arise during or after the procedure.²⁰

An ultrasound examination should be performed before the procedure to determine fetal viability, position, and biometry, to conduct a careful anatomical examination, as well as to identify the insertion of the umbilical cord in the placenta. Clear identification of the umbilical cord insertion within the placenta is critical; therefore, when technically feasible, many prefer to access the umbilical vein within 1 to 2 cm of its placental insertion site (Figure 26-1). When the placenta is in the posterior wall of the uterus, direct access to this target portion of the umbilical cord may be hampered by the fetus. Under these circumstances, puncture of a free loop can be attempted if access to the sampling site is not achieved upon external manipulation on the maternal abdomen to move the fetus (Figure 26-2).

The abdomen is cleaned with an antiseptic solution and draped. The use of local anesthesia for diagnostic procedures is a matter of choice, reducing the patient's discomfort in cases of prolonged procedures. In addition, maternal sedation (diazepam) can be used for prolonged therapeutic procedures. Diazepam is preferred to meperidine because the latter can induce nausea and vomiting. Prophylactic administration of antibiotics is routinely given in many centers because the risk of chorioamnionitis far outweighs the risks of adverse reactions to the antibiotics; in some reports chorioamnionitis was responsible for up to 40% of the fetal losses.²¹ Collection of a maternal blood sample prior the procedure is indicated to ensure quality control of the fetal samples.

Several different approaches can be used for cordocentesis. All require the needle to be inserted under sonographic monitoring; however, the choice of the transducer, whether handled by the operator or by an assistant, as well as the use of a needle-guiding device connected to the probe or a "free-hand technique" are options. Most experienced operators prefer the free-hand technique because of its flexibility in the adjustment of the needle's path.²² In fact, needle guidance has the advantage of decreasing the risk of cord laceration or needle displacement when the tip is properly placed,²³ but it restricts the lateral motion of the needle, which may be required in cases where the needle should be repositioned. If this is the case, removal of the guiding device during the procedure may become necessary.

Spinal needles of 20- to 25-gauge are used for cordocentesis. Most centers use a 20- to 22-gauge needle. Larger-bore needles are not necessary for diagnostic or therapeutic procedures. Smaller-bore needles have the disadvantage of prolonging the period of time required to obtain fetal blood; in addition, they are more difficult to guide in cases where intraoperative manipulations are required. Alternatively, a biopsy guide 20-gauge needle can be used to gain access to the amniotic cavity; once in proximity of the umbilical cord, a 25-gauge needle, which is threaded into the 20-gauge needle, is advanced into the umbilical vein.²⁴ The incidence of procedure-related complications using this needle-within-needle technique is claimed to be lower than that reported using a single-needle technique (bleeding from puncture site, 23.1%; fetal bradycardia, 0%; and pregnancy loss rate, 0.9%).

The standard length of spinal needle is 8.89 cm, excluding the hub. The length of the needle should take into account the thickness of the maternal adipose tissue and the location of the target segment of cord within the uterus. A few additional centimeters should be considered to avoid underestimation due to intervening events, such as uterine contractions. Priming of the needle with sodium citrate solution immediately before the procedure has been proposed to prevent clot formation.²

If concomitant amniocentesis is planned, the amniotic fluid should be collected before the cordocentesis to avoid contamination of the specimen. In these cases, the path of needle insertion should be such to allow accomplishment of amniocentesis and cordocentesis with only 1 insertion. Once the needle is inserted into the amniotic cavity, the amniotic fluid is aspirated. The needle is subsequently advanced toward and into the cord or, in cases of anterior placenta, it can be withdrawn within the placental mass, reoriented, and advanced into the cord. Within the umbilical cord, it is easier and safer to sample the vein rather than an artery. Puncture of the artery has been associated with a greater incidence of bradycardia and longer postprocedural bleeding.²³

Upon entering the umbilical cord, the stylet is removed, and fetal blood is withdrawn into a syringe attached to the hub of the needle. The syringe may be primed with a small amount of anticoagulant, such as heparin or citrate. After flow is documented, an initial sample should be submitted for blood cell size determination with an electronic cell analyzer (Coulter Electronics, Hialeah, FL, USA) to distinguish fetal from maternal cells (Figure 26-3).^25,26 The proper positioning of the needle can be confirmed by injection of saline solution into the cord and observation of turbulence along the vessel.

The use of paralytic agents to decrease fetal movement is left to the discretion of the operator. When indicated, atracuronium (0.4 mg/kg fetal weight intravenously) or pancuronium (0.1 mg/kg fetal weight) may be used. Once the needle is in place, the samples required for diagnostic purposes are collected in different syringes. Transfer to the appropriate tubes should occur immediately after collection. Tubes with ethylene-diaminetetraacetic acid are indicated for complete blood cell count and platelets; blood group typing requires serum, whereas karyotype and molecular biologic analysis requires anticoagulation with heparin.²⁷ Blood gas analysis can be performed directly on a sample in a heparin syringe. The volume of blood collected varies according to the number of tests required and should take into account the gestational age at sampling. In general, an amount corresponding to a maximum of 6% to 7% of the fetoplacental blood volume for that gestational age may be removed. The fetoplacental blood volume, as expressed in milliliters per kilogram of fetal weight, decreases with advancing gestational age.²⁸ Several studies have evaluated fetoplacental blood volume using different techniques. The average estimate of fetoplacental blood volume is 125 mL/kg of ultrasonographically estimated fetal weight.

Monitoring of the fetal heart rate during fetal transfusions can be performed by placing a Doppler gate on an umbilical artery near the sampling area. This approach avoids the necessity to move the transducer from the sampling site to the fetal chest. After the required volume of blood is withdrawn, 2 to 3 mL of saline can be injected as volume replacement and to confirm the correct position of the needle tip. If turbulence is not observed in the umbilical cord vessel, it may be prudent to draw a final sample to confirm the fetal origin of the blood.

The needle is withdrawn, and the puncture site is monitored for bleeding. If the fetus is viable, fetal heart rate is monitored for 1 to 2 hours after the procedure.

Intrahepatic Vein Blood Sampling

This technique consists of the introduction of a 20-gauge needle into the fetal abdomen aiming for the intrahepatic portion of the umbilical vein or the left portal vein. No significant changes in the concentrations of liver enzymes have been noted after the procedure in fetuses undergoing sampling at the intrahepatic vein (IHV) as opposed to the umbilical cord, suggesting absent or minimal fetal liver injury.²⁹ Fetal liver necrosis after blood sampling from the IHV has been described in an isolated case report.³⁰

Although IHV blood sampling has some advantages over sampling of the umbilical cord (such as avoidance of the umbilical arteries, certainty of the venous origin of the sample, and lack of the need for immediate laboratory support to confirm the fetal origin of the sample), it has been traditionally considered as a second choice in cases of failed cordocentesis because higher rates of fetal losses were reported with this route of sampling in early series (6.2% in low-risk patients).^29,31 Aina-Mumuney et al have recently reported a 20-year experience of a single center with fetal blood sampling, comparing the results of 130 IHV samplings and 110 cordocenteses.³² Success rates were significantly higher with IHV sampling than with cordocentesis (84.6% vs 69.1%, P = .004). There was no difference between IHV and cordocentesis in the incidence of nonreassuring fetal heart rate and the need for immediate delivery. IHV sampling had a significantly lower incidence of streaming from the fetal vasculature puncture site (0.79% vs 30.8%, P < .0001), and this difference was maintained when a subanalysis of cases with fetal thrombocytopenia was performed. The authors, though they acknowledge the inner limitations of the retrospective study design, suggest that IHV sampling may convey a particular advantage in cases of suspected fetal thrombocytopenia.

Cardiocentesis

This method for fetal blood sampling has very limited indications because of the high rate of fetal loss. In a large series of 158 cardiocenteses during the second trimester for the prenatal diagnosis of hemoglobinopathies, the procedure-related fetal loss rate was 5.6%.³³ No cases of fetal cardiac trauma or hemopericardium were encountered among living infants. Cardiocentesis should be reserved for cases in which PUBS or IHV sampling are technically impossible. Ideally, the needle should enter the fetal heart, preferably the right ventricle, through the anterior thoracic wall.³⁴ To prevent fetal movements from interfering with the procedure, the needle should be introduced swiftly into the fetal thorax.

Quality Control of the Specimen

A crucial step is the assessment of the quality of the blood sample. Contamination with maternal blood and amniotic fluid can alter the diagnostic value of the specimen. For example, contamination with amniotic fluid of as little as 1% leads to a significant increase in factor V and VIII activity; therefore, it interferes with the diagnostic accuracy of some congenital hemostatic disorders.³⁵ To assess the purity of the fetal blood sample, the profile of the mean corpuscular volume of red blood cells (RBCs) has been the method commonly used, because fetal RBCs are larger than maternal RBCs. This difference decreases, however, with advancing gestational age. Furthermore, the profile can be misinterpreted in the presence of some maternal hematologic disorders, such as macrocytic anemia. Other hematologic indices can be used to differentiate maternal from fetal blood. For instance, lymphocytes are the predominant white blood cells (WBCs) in the fetus, whereas neutrophils are in the mother. Tables 26-1, and 26-2 present normal hematologic and biochemical values in fetal blood. The Kleihauer-Betke test may be used for second trimester samples, at which time it can accurately detect a maternal contamination.²⁷ The utility of the test is limited during the late third trimester of pregnancy because fetal erythrocytes contain increasing amounts of hemoglobin A. Blood typing can confirm the fetal origin of a sample because antigen I is present only in adult RBCs. Monoclonal antibodies for antigen I can detect as little as a 5% maternal blood contamination.²⁶ Human chorionic gonadotropin (hCG) determination is the best marker for detection of maternal blood contamination in the specimen.²⁷ Maternal blood contains high concentrations of this hormone, whereas fetal blood is practically devoid of it. The ratio of fetal to amniotic fluid to maternal blood of hCG is 1:100:400. Determination of this hormone by an enzyme-linked assay using monoclonal antibodies against the β-subunit of hCG can detect as little as 0.2% contamination with maternal blood.²⁷

The hCG concentration can also be used to rule out contamination of the blood sample with as little as 1% of amniotic fluid.²⁷ Alternatively, dilution with amniotic fluid can be inferred by a proportional decrease in the number of RBCs, WBCs, and platelets in the specimen, or by the presence of ferning or multiple desquamated epithelial cells on a smear.^35,36 The most accurate test for amniotic fluid contamination is the increased activity of factors V and VIII. Factors II and IX are not activated by amniotic fluid and can be used as internal controls.²⁷

Duration of the Procedure

The duration of the procedure is related to placental location, fetal interference, quality of ultrasound image, and operator expertise. In a series of 606 cordocenteses, 70% of the first 100 procedures were performed in less than 10 minutes, whereas the percentage rose to 90% in the last 100 cases.² A second needle insertion was required in 2.9% of the cases. In cases of polyhydramnios with posterior placenta, it may be necessary to perform therapeutic amniocentesis to gain access to the cord insertion.

Training

Fetal blood sampling is not part of general obstetric training, and in the United States it is performed only by specialists in maternal–fetal medicine. Training is difficult to obtain because of the limited number of cases in which this procedure is indicated at any given institution, and because of the risks associated with the procedure. Ideally, the operator should have a strong background in obstetric ultrasonography and experience with other invasive procedures of prenatal diagnosis. Training can first be provided with in vitro models.³⁷ Once proficiency is acquired with in vitro models, the procedure can be performed in patients having elective mid-trimester termination of pregnancy.³⁸

Normal Values for Fetal Blood

Tables 26-3, and 26-4 display the distribution of the most common hematologic and acid–base parameters in fetal blood in relation to the gestational age.³⁹

Maternal Complications

The main maternal risk of PUBS is red cell alloimmunization, which can be minimized with the administration of anti-D immunoglobulins when indicated. Chorioamnionitis is a rare (1%) complication, but it is responsible for up to 40% of pregnancy losses after PUBS.^21,40,41 An isolated case of maternal respiratory distress syndrome in association with Chorinebacterium amnionitis after cordocentesis has also been reported.⁴² For these reasons, antibiotic prophylaxis has been advocated and implemented in many centers, despite the lack of evidence to support this policy.

Fetal Losses

The risk of procedure-related pregnancy loss is difficult to assess because many centers do not have long-term follow-up on all cases. Losses are usually considered to be procedure related if they occur within a short time interval from PUBS (eg, 2-week cutoff for procedure-related loss). However, several investigators include postprocedural loss rates of up to 28 weeks of gestation or up to term. A review of the published series of low-risk cases reported an overall risk of fetal loss before 28 weeks of 1.4% and an additional 1.4% risk of perinatal death after 28 weeks.⁴³ The most important risk factors implicated in the occurrence of complications are as follows:

1. Indication for the procedure. The loss rate after PUBS is a combination of the procedure-related loss rate and the background loss rate. In the presence of chromosomal abnormalities or among critically compromised fetuses (ie, those with hydrops), the risks are substantially higher due to a higher background loss rate. Moreover, some medical disorders that indicate PUBS, such as alloimmune thrombocytopenia, have the potential for increasing the procedure-related fetal mortality, due to a higher risk of exsanguination.⁴⁴ A review of the risks of PUBS in relation to the indication showed that the total loss rate of desired ongoing pregnancies within 2 weeks of the procedure was 1.3% when the indication was prenatal diagnosis of genetic disorders or karyotyping, 6.6% in the presence of structural fetal anomalies, 13.8% among small-for-gestational-age (SGA) fetuses, and 25% in fetuses with nonimmune hydrops.⁴⁰

2. Operator experience. Studies show that the smaller the series size, the higher the incidence of fetal losses.²¹ Given the limited indications for PUBS, dispersion of the case load should be avoided in order to allow referral centers to have enough volume to maintain skills.

3. Gestational age at procedure. Gestational age at sampling is an important determinant of fetal loss rate because a viable fetus can be delivered if a complication occurs. Even though PUBS before 20 weeks is a more challenging procedure, it is not associated with a significantly higher risk of fetal loss.⁴⁵ Smaller-bore needles (25 gauge), however, ought to be used at 12 to 15 weeks rather than at later gestational ages (20 to 22 gauge after 16 weeks).

Hemorrhage

Bleeding from the puncture site is the most common, although usually benign, complication of cordocentesis. It occurs in up to 53% of cases, with a mean duration of 35 seconds.^2,21,46 At a gestational age of less than 21 weeks, postprocedural bleeding has been associated with nearly a 3-fold increase of fetal loss rate.⁴⁵ Bleeding seems to be dependent on the technique used. Use of a 25-gauge rather than a 21-gauge needle is associated with a lower bleeding rate (23%).²⁴ Similarly, the use of a fixed needle guide has a lower bleeding rate from the puncture site (29%).²³ This has been attributed to decreased lateral motion of the needle when compared to the free-hand technique, thus decreasing the risk of laceration of the cord. Puncture of the umbilical artery is associated with a significantly longer duration of bleeding than venipuncture.²³ Fetuses affected by defects in platelet number or function (eg, Glanzmann thrombasthenia or alloimmune thrombocytopenia) are at significant risk for potentially fatal bleeding from the puncture site.^47,48 When PUBS is performed to rule out these conditions, it is prudent to slowly transfuse concentrated irradiated maternal platelets while awaiting the fetal platelet count, because dislodgment of the needle can have serious consequences for the fetus.

Cord Hematoma

In a pathologic study of 50 umbilical cords collected between 1 and 20 hours after PUBS, hematomas were noted in as many as 17% of cases.⁴⁹ No relationship could be documented between transient fetal bradycardia or bleeding from the cord puncture site and the size of the hematoma. A hematoma is generally asymptomatic, but it can be associated with prolonged bradycardia and rapid fetal deterioration.^46,49,50 On occasion, cord hematomas can be visualized by ultrasound as an echogenic area near the puncture site.^51,52 In the absence of signs of fetal distress, expectant management is recommended.

Bradycardia

Transient fetal bradycardia has been reported in 3% to 12% of cases.^2,4,21,23 It is usually a self-limited phenomenon. In most cases, it is thought to be the manifestation of a vasovagal response caused by local vasospasm; this hypothesis is supported by a higher incidence of bradycardia in cases of puncture of an umbilical artery.²³ The occurrence of bradycardia does not seem to be related to the sampling site (placental insertion or free-floating loop of the cord) or to the number of puncture attempts.⁵³ Ulm et al⁵³ reported a series of 339 cordocenteses in which perinatal mortality was significantly higher among cases with bradycardia (61.5%) than those without bradycardia (3.1%). A significantly higher incidence of bradycardia has also been noted among SGA fetuses than appropriately grown fetuses (17.2% vs 4.1%, respectively; P = .001) and among fetuses with absent diastolic flow by Doppler analysis of the umbilical artery when compared to those with present end-diastolic velocities in this vessel (21.4% vs 5.3%; P < .001).²³

Fetomaternal Hemorrhage

Cordocentesis performed for therapeutic purposes is often associated with fetomaternal transfusion (FMT). In a study of consecutive diagnostic cordocenteses, FMT (defined as a ≥50% postprocedural increase in maternal serum α-fetoprotein concentration) occurred in about 40% of cases, and it was more common when the procedure was performed in the presence of an anterior rather than a posterior placenta (65.6% vs 16.6%, P < .001).⁵⁴ These findings have been confirmed by independent investigators.^55,56 The mean estimated volume of hemorrhage was 2.4 mL (3.1% of the total fetoplacental blood volume). FMT is more frequent when the procedure takes longer than 3 minutes and when 2 or more needle insertions are required.⁵⁷ In contrast, no relationship has been reported between the degree of fetomaternal hemorrhage, gestational age, or type of procedure (diagnostic vs therapeutic cordocentesis).⁵⁷

Abruptio Placentae

Despite the use of a transplacental approach, the incidence of this complication is negligible, and only 1 documented case has been reported thus far.⁵⁸

Preterm Delivery

In 7% of cases, cordocentesis is associated with an irregular pattern of uterine contractions.⁴¹ The rate of preterm delivery in a low-risk population was 5.0% to 5.7%.^2,45 Therefore, cordocentesis does not seem to increase the frequency of preterm birth due to spontaneous preterm labor, but rather indicated deliveries if the procedure is performed after viability and there is a complication such as a prolonged bradycardia. In our experience, however, spontaneous rupture of membranes can occur if the procedure is complicated and requires multiple needle insertions.

Fetal Resuscitation

The risk of life-threatening fetal complications during or immediately after cordocentesis is small. After viability, the option of immediate abdominal delivery and postnatal resuscitation is possible. If fetal lung maturity has not been documented or immaturity is suspected, administration of a course of steroids to the patient before the procedure to enhance fetal lung maturity is prudent; however, the risk-to-benefit ratio of this recommendation has not been evaluated. Before viability or when the procedure is performed at an extremely preterm gestational age, fetal resuscitation can be attempted in utero.⁵⁹ In the presence of persistent fetal bradycardia, the common maneuvers to improve uterine perfusion and enhance fetal oxygenation should be implemented immediately. These include placing the patient on their left side, intravenous hydration, and administering oxygen.⁶⁰ If the bradycardia is associated with uterine contractions, subcutaneous or intravenous tocolytics can be administered to the mother.⁶¹ If the bradycardia persists or worsens, a more aggressive approach may be necessary because little or no flow would be present in the fetal circulation to provide peripheral oxygenation and fetomaternal gas and drug exchange. Only anecdotal case reports of in utero resuscitation have been published, and this may reflect a publication bias in favor of successful procedures.^{21,61,62, and 63} If a bradycardia occurs with the needle still in the umbilical vessel, some have recommended immediate administration of epinephrine or volume expanders may be attempted. Table 26-5 displays a protocol for potentially useful approaches that have been derived from recommendations of the American Academy of Pediatrics and the American Heart Association for neonatal resuscitation.⁶⁴ The doses have been adjusted for the estimated fetal weight and the fetoplacental blood volume. If the bradycardia is secondary to documented or suspected prolonged bleeding from the puncture site, hypovolemic shock is likely to occur and volume replacement would be the treatment of choice. Under these circumstances, cardiocentesis has been proposed as a heroic measure for resuscitation. This has been accomplished successfully in a 20-week fetus affected with von Willebrand disease. Cordocentesis was followed by a severe 5-minute bradycardia secondary to a large FMT. An intracardiac puncture and transfusion with 30 mL of maternal blood was used to resuscitate the fetus.⁶³ This discussion suggests that individuals performing PUBS should be prepared for emergency resuscitation and the equipment and medications necessary should be available when the procedure is undertaken.

Cytogenetic Diagnosis

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.

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-1990s^6,7 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.

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

Congenital Infections

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}

Fetal Anemia

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

Primary Immunodeficiency Diseases

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.

Coagulopathies

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.

Platelet Disorders

Fetal blood sampling can be useful in the prenatal diagnosis and management of congenital quantitative (thrombocytopenias) and qualitative (thrombocytopathies) platelet disorders.

Thrombocytopenias

These include immune-mediated thrombocytopenias and genetic syndromes in which thrombocytopenia is one of the features.

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

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.

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.

Thrombocytopathies

Table 26-7 displays the most common congenital qualitative platelet disorders and cases of prenatal diagnosis reported.

Hemoglobinopathies

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).

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

Intrauterine Growth Restriction

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.

Thyroid Disease

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

There are limited data regarding the procedure-related loss rates for cordocentesis in multiple pregnancies. Antasaklis et al, in a cohort of 84 twin gestations, reported a procedure-related fetal loss rate of 8.2% after fetal blood sampling, which is about 4-fold higher than that reported from the same institution when the procedure was performed in singletons.⁹¹ Similarly, a total fetal loss rate of 9.5% (including 1 case of lethal fetal anomaly, 1 case of intrauterine fetal death at 34 weeks of gestation, and 2 cases of extremely preterm birth) has been reported in a series of 59 procedures performed in 30 multifetal pregnancies (29 sets of twins and 1 set of triplets). However, there were no fetal losses within 2 weeks after the procedure.⁹² The possibility that the higher risk of fetal loss in twin pregnancies may be due to the higher background loss rate in this population should be considered. Thus far, there are no studies appropriately comparing the outcome of twin gestations undergoing PUBS with that of twin pregnancies in which no invasive procedures are performed.

The indications for PUBS in twin pregnancies do not differ from those in singletons. In the past, a role for cordocentesis in the diagnosis of twin-to-twin transfusion syndrome (TTTS) has been proposed.⁹³ Specifically, a difference in fetal hemoglobin concentration greater than 2.4 g/dL was reported to have a sensitivity of 50% (4 of 8) and a specificity of 100% for the diagnosis of TTTS with a stuck twin in a series of 38 cases.⁹⁴ In addition, recovery of adult cells in the circulation of the suspected recipient twin, after their injection into the suspected donor during cordocentesis, had a sensitivity of 83% (10 of 12) in the diagnosis of TTTS.^95,96 The diagnosis and staging of TTTS currently rely on sonographic criteria.⁹⁷ Similarly, other proposed indications for diagnostic PUBS in cases of TTTS, such as the surveillance of the occurrence of fetal anemia and anemia–polycythemia sequence in the survivor, after intrauterine death of one twin following laser therapy have been overcome by the introduction of noninvasive techniques, such as the Doppler velocimetry of the MCA.

Access to the fetal circulation may be required to infuse biologic (eg, transfusions) and pharmacologic agents for therapeutic purposes. Fetal anemia is the most common indication for intravascular transfusion. Platelet transfusion in cases of alloimmune thrombocytopenia has been previously discussed. Fetal intravascular infusion of pharmacologic agents may be advantageous when the transplacental passage is poor (eg, digoxin administration to hydropic fetuses with supraventricular tachycardia) or when the drug must be administered as a bolus (eg, adenosine or amiodarone in the presence of persistent supraventricular tachycardia resistant to digoxin).⁹⁸ Direct access to the fetal circulation may be indicated in cases of inherited disorders amenable to treatment with stem cell transplantation.

A new approach to genetic disorders involves the in utero transplantation of stem cells. Stem cells can be administered either into the fetal circulation by PUBS or into the fetal peritoneum with a single injection. Clinical experience suggests that some lymphohematopoietic disorders prenatally diagnosed can be successfully treated in utero using stem cells derived from parental bone marrow or fetal tissues, before life-threatening sequelae become manifested.⁹⁹ The immunological naiveté, together with the sterile intrauterine environment, offer ideal conditions for transplantation, inasmuch that even mismatched cells have the potential to engraft. Moreover, given the plasticity of stem cells, transplant procedures have also been attempted for the treatment of genetic disorders other than hematopoietic (ie, inborn errors of metabolism). However, even though engraftment of allogeneic or xenogeneic cells has been achieved, the levels of donor-cell chimerism in most circumstances have been below what would be considered therapeutic for most target diseases. Mechanisms preventing successful engraftment, as well as strategies capable of selectively increasing donor stem cell homing and engraftment in the fetal recipient, are currently under investigation. The ability to achieve a sustained engraftment after in utero transplantation is also crucial for those seeking to bring fetal gene therapy closer to clinical practice.

Recently, Chan et al¹⁰⁰ reported successful fetal blood sampling in the first trimester (mean gestational age 10 weeks) in 66% (4/6) of fetoscopic-directed and 66% (8/12) of ultrasound guided procedures. This study demonstrates the feasibility of the ex-vivo fetal gene therapy approach.