The immuassay handbook parte79

757© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/B978-0-08-097037-0.00062-2

PregnancyKevin Spencer1 ([email protected])

Tim Chard2

From the earliest stages of gestation the human fetus and placenta synthesize a number of compounds that are con-sidered to be qualitatively and quantitatively specific to pregnancy. Measurement of these compounds is widely used in the diagnosis of various pregnancy disorders. In addition, maternal tissues such as the uterine epithelium (endometrium) and ovary (corpus luteum) secrete materials that may be of diagnostic value.

Many of these materials circulate at remarkably high levels. For example, the serum concentration of estrogens near term is 100 times greater than the highest level found in a woman who is not pregnant. Still more surprisingly, the functions of many of these materials are still unknown. Rare pregnancies occur in which estriol is almost totally absent from maternal blood, for example in Smith–Lemli–Opitz syndrome and Steroid Sulfatase Deficiency, and this absence causes varied manifestations (see below).

As with functions, there is a similar dearth of informa-tion on control mechanisms. The types of feedback rela-tionships that exist in the physiology of the normal adult are not found with the placental hormones or similar com-pounds. The circulating levels of most of the compounds show no diurnal rhythm or change with physiological events such as sleep, exercise, or meals. The rate of synthe-sis of placental products seems to relate solely to the mass of the tissue of origin (the placental trophoblast) and to the rate of uteroplacental blood flow. For most clinical pur-poses this is, of course, advantageous; a single measure-ment of a fetoplacental product can be taken without concern for the physiological status of the mother.

One special feature of the measurement of most fetopla-cental and related products is the noticeable change in lev-els in relation to the stage of pregnancy; some decreasing and others increasing. For this reason it is usually not pos-sible to quote a single reference interval for a given analyte; instead, a separate reference interval has to be used for each week and sometimes each day of pregnancy. This has two important implications. First, large number of samples are required to establish a normal reference interval. Second, it is almost impossible for the clinician or analyst to recall the reference interval for each week without some kind of prompt. Furthermore many of the compounds that are measured during pregnancy have no defined primary stan-dard by which to standardize the assay method and, in addi-tion, the very nature of immunoassay (the usual method of choice for the measurement of most placental and preg-nancy analytes) can introduce methodological biases. To remove gestational age variation effects and to allow some degree of standardization from center to center and between different methods, biochemical parameters, and some

biophysical (Ultrasound and Blood Pressure) parameters have been expressed as Multiples of the normal Median (MoM). Essentially the analyte concentration is expressed as a ratio of the measured value to the median values found in normal pregnancies of the same gestational age. Thus a value of 1.00 MoM is normal, a value of 2.00 MoM is ele-vated and a value of 0.50 MoM is reduced. Thus, when the action limit for alphafetoprotein (AFP) in screening for Neural Tube Defects (NTDs) is stated as being ‘2.5 times the median’ or ‘2.5 MoM’ this will be the same regardless of the week of pregnancy and it will be universal from center to center and assay to assay. The median will, of course, change but the action limit expressed as a MoM will not.

The relationship between analyte levels and gestational age places a heavy demand on the accuracy of gestational dating. A given marker value could be normal for 16 weeks but above the reference interval 1 week earlier. In clinical practice, dating errors of up to 4 weeks or more are relatively common. Dating is usually based on the first day of the last menstrual period (LMP). This can be confirmed by ultra-sound measurements of the fetus either by crown-rump length (CRL) before 14 weeks of pregnancy; biparietal diameter (BPD) or head circumference (HC) thereafter, although it is recognized that the latter is more accurate. If the LMP is in doubt, or there is a gross discrepancy with ultrasound then the ultrasound date is accepted as the ‘gold standard’. When biochemical markers are being used in the context of aneuploidy screening it is usually common to base median calculations on the dating by gestational age or indeed on the CRL measurement itself. The latter is usually most preferred because there are a number of different ultra-sound charts in use that would result in different gestational days being calculated for the same fixed CRL.

Clinical Conditions and DisordersDETECTION OF EARLY PREGNANCYDetection of early gestation by measurement of human chorionic gonadotropin (hCG) is probably the commonest immunoassay test in pregnancy. The indication is almost always maternal concern (which may reflect a positive or a negative frame of mind). In a woman without symptoms there are very few medical indications for a urine preg-nancy test. However, it is by far the commonest and most popular of all ‘home-use’ tests, a marketplace supplied by highly convenient dipstick procedures.

THREATENED ABORTIONThreatened abortion can occur at any time during the first 24 weeks of pregnancy. The woman presents with

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clinical features of an abortion process (pain and bleeding) but the outcome is uncertain. Many cases of this type will proceed normally to term. There is no specific treatment for threatened abortion but if the fetus is dead the contents of the uterus are often surgically evacuated (dilatation and curettage).

ECTOPIC PREGNANCYFertilization takes place at the outer end of the Fallopian tube. The fertilized ovum then passes along the tube and implants in the uterus 6–7 days later. If the process is inter-rupted then implantation takes place in the tube itself. This is known as ectopic pregnancy. It is often followed by rupture of the tube and hemorrhage into the abdomen, a life-threatening emergency that may become apparent at any time in the first 8 weeks of pregnancy. The treatment is laparotomy or laparoscopy and removal of the ectopic pregnancy. Over recent years the medical management of ectopic pregnancy with the use of a single dose of intra-muscular methotrexate has been shown to be a valuable alternative. Successful outcome is dependent on the initial serum hCG levels (the likelihood of treatment failure is greater at higher serum hCG concentrations), the size of the ectopic pregnancy on transvaginal ultrasound (TVS) and the presence or absence of fetal heart activity with resolution occurring in 85% of cases. Single-dose metho-trexate provides significant cost savings when compared to laparoscopy.

Whilst most pregnancies are obviously within the uterus, Pregnancy of Unknown Location (PUL) is used to describe cases where, whilst there is a positive preg-nancy test, there is no sign of a pregnancy inside or outside the uterus, on transvaginal ultrasound or even at laparos-copy. Up to 31% of women attending early pregnancy assessment centers have a PUL though the experience of the sonographer can reduce this to 10%.

The majority of PULs are at low risk of being ectopic (outside the uterus) in location. However, correct detection is essential as, even though at least 15% of ectopic pregnan-cies resolve spontaneously, there is no method of differen-tiating these from those that will develop, eventually rupturing and putting the woman’s life in danger.

There are four possible outcomes of PUL: disappear-ance of the pregnancy (44–69% of cases); progression to confirmation of a normal intrauterine pregnancy (up to 75% develop into viable intrauterine pregnancies); ectopic pregnancy (8.1–42.8%); persisting PULs which account ultimately for 2% of cases and are defined as those where the hCG levels do not decrease, there are no signs of tro-phoblastic disease and the location of the pregnancy can-not be identified. Expectant management of PUL has been shown to be safe and effective in reducing the need for surgical intervention but does require several visits to an early pregnancy assessment unit.

CHROMOSOME DEFECTS OF THE FETUSAneuploidy is a condition in which a cell has an incorrect number of chromosomes. Human cells are supposed to have 46 chromosomes. Females have 23 pairs of chromosomes,

whereas males have 22 pairs but then a final pair containing an X and a Y (females have two X chromosomes). In aneu-ploidy, a cell might have three copies of a particular chro-mosome—making 47 chromosomes—or only one copy of a particular chromosome, making 45 chromosomes in the cell. Any change in the number of chromosomes can affect health.

The best-known defect of chromosome number is Down syndrome or trisomy 21 (three copies of chromo-some 21 in place of the usual two copies). Langdon Down first described in his 1866 essay on “Observation of an eth-nic classification of idiots” the phenotypic expression of the syndrome which was described as “mongoloid”. How-ever since a major campaign by scientists and clinicians in the 1960s the syndrome has been known as Down syn-drome. Although Shuttleworth in 1909 described the asso-ciation between the syndrome and increased maternal age, it was not until 1959 that Lejune and Jacobs demonstrated that the condition resulted from an extra copy of chromo-some 21. This extra copy results either from nondisjunc-tion (failure of paired chromosomes to disjoin (separate) during cell division so that both chromosomes go to one daughter cell and none to the other) or from a transloca-tion (a chromosome abnormality caused by the rearrange-ment of parts between nonhomologous chromosomes). The syndrome requires either the whole or a segment of the long arm of chromosome 21, the distal portion of which is now known to determine the facial features, heart defects, mental IQ and other clinical features. Most cases result from nondisjunction and the additional genetic material is invariably of maternal origin. The major clini-cal consequences of trisomy 21, apart from the learning disability with IQ scores ranging from 20 to 70, are the congenital heart defects occurring in 50% of individuals. Gastrointestinal problems occur in around 30% with visual, ear, nose, and throat problems in 50% of children. The frequency of hypothyroidism, epilepsy, and leukemia is increased. Advances in medical treatment have increased the median life expectancy of Down syndrome individuals to about 50 years, although with a significant risk of Alzheimer’s disease in later life.

Prenatal detection and the offer of termination of the pregnancy is the only way to reduce the incidence of this condition. The condition occurs in around 1 in 600 preg-nancies and is common among older mothers (one in 1000 at age 30, one in 428 at age 35, one in 130 at age 40 (term risks)). Screening for Down syndrome is almost universal in obstetric practice. Originally this was based solely on maternal age (35 or over). Since 1990 years various bio-chemical tests have been introduced (see below) initially used in the second trimester (14–20 weeks) of pregnancy and in the last 10 years ultrasound tests have been described, including measurement of nuchal translucency (NT). The latter, together with some biochemical tests, enables detection in the late first trimester (10–13 weeks of pregnancy). This combination of ultrasound and biochem-ical tests has become known as the Combined Test and this now forms part of National Screening Strategies in the UK and many European countries.

Those identified as being at risk by one or more of the screening tests are offered amniocentesis (collection of

759CHAPTER 9.8 Pregnancy

amniotic fluid through a needle), usually done at 16–18 weeks or chorionic villus sampling (CVS) performed from 11 weeks onwards. This latter technique essentially takes a biopsy of the placental tissue. The fetal skin cells from the amniotic fluid or the placental tissue can be examined by standard cytogenetic techniques to make a firm diagnosis usually involving karyotyping. In many centers standard karyotyping has been replace by the molecular technique of Quantitative fluorescent polymerase chain reaction (QF-PCR) as the primary test, with standard karyotyping reserved for QF-PCR positive cases or those that present with very high NT. However, amniocentesis or CVS should not be carried out in all women because the proce-dures themselves carry an additional risk (now approxi-mately one in 100–200) over and above the natural background fetal loss of causing an abortion.

Trisomy 18: In 1960 Edwards first described this con-dition in which the median survival time was less than 1 week, with 90% dying within 6 months of birth and 5% surviving to 1 year. Growth deficiency begins in utero and continues postnatally. Congenital heart disease is the most common cause of death. Survivors have profound physical and mental retardation.

Trisomy 13: Also in 1960, Pateau described this condi-tion. The median survival time is less than 1 week, with more that 80% dying in the first month and only 3% sur-viving 6 months. Severe congenital heart disease and renal defects are the most common causes of death.

Sex chromosomal aneuploidy: These are the most problematic to classify as a serious birth defect. Turner’s syndrome or monosomy X (45,X0) is characterized by short stature, gonadal dysgenesis, lymphedema, and con-genital heart defects and typically present clinically at puberty. Intellectual function is normal in most cases. Sim-ilar to most major autosomal trisomies the in utero inci-dence of Turner’s syndrome is much higher than at birth. The same genetic condition appears to be expressed as three different phenotypes; one which results in early preg-nancy loss (before 14 weeks), a group which survives until around 22 weeks and then is spontaneously lost and the third group reach term with no clinical consequence until puberty. Such phenotypic variability makes screening and counseling very difficult. Other sex aneuploidies include Klinefelter syndrome (47,XXY), which occurs with a preva-lence of around one in 1000 male births, whilst 47,XXX occurs in around one in 1000 female births. These later two chromosomal disorders have minor clinical consequences.

NEURAL TUBE DEFECTS OF THE FETUS (NTD)The term neural tube defect is used to describe various degrees of failure of formation of the central nervous sys-tem (brain and spinal cord) and its coverings. The neural crest is the basic embryological structure which folds upon itself to form the brain and spinal cord. This folding pro-cess is normally complete around day 25 and defects in this folding process can lead to various neural tube defects (NTDs). These are usually classified based on embryologi-cal considerations and the presence (open) or absence (closed) of exposed neural tissue. There are two main types

based on whether the brain or the spine is affected: in brief these are anencephaly and spina bifida. In anencephaly most of the brain is absent, together with the cranial bones. This is incompatible with extrauterine life and the fetus dies either before or immediately after delivery. In spina bifida there are defects of the lower spinal nerve roots and their coverings; spinal membranes bulge through the defect as a myelomeningocele. Around one-third of these children survive, often helped by surgery. However, the quality of life is variable: they are usually paralyzed from the waist down (paraplegia) and incontinent.

Screening for NTDs has been available since the mid 1970s, based on the measurement of maternal serum AFP; diagnosis in the years prior to the late 1990s was usually by finding elevated levels of AFP in amniotic fluid and con-firmed by the presence of increased levels of acetyl cholin-esterase. More recently, detection and diagnosis by ultrasound has become the norm with the presence of the so-called fruit signs (lemon and banana) in the cranium. With an increasing focus on screening for Aneuploidies in the first trimester and the use of ultrasound at 11–13 weeks many cases are now being identified much earlier in preg-nancy and in the UK many centers no longer offer routine maternal serum NTD screening in the 14–20 week period.

Women who have had a previous child with a neural tube defect have a very high recurrence risk (about 1 in 30). This risk is greatly reduced by folic acid supplements of 0.4 mg per day. In some countries staple foodstuffs are fortified with folic acid and in others folic acid is recom-mended for all women who plan to conceive. The role of folic acid is thought to promote the folding of the neural crest.

Since the introduction of screening and subsequent improvements in diet and the use of folic acid supplemen-tation the birth incidence of NTDs in the UK has fallen from 3.2 per 1000 in the early 1970s to 0.1 per 1000 in the late 1990s. Approximately 40% of this decline is suggested to be due to antenatal screening and termination and 60% due to a decline in incidence of which improved diet and folic acid supplementation are contributors.

PREMATURE LABORLabor occurring after 24 and before 37weeks is referred to as premature labor or preterm delivery. The child is small (typically less than 2500 g) and at risk of potentially lethal complications such as the respiratory distress syn-drome. In the longer term they may suffer permanent brain damage (mental retardation and cerebral palsy). Pre-mature labor is treated (often unsuccessfully) with drugs that inhibit uterine contractions. If the child is delivered it may need to be transferred to a neonatal intensive care unit. The risk of spontaneous preterm birth is increased in women with a previous late miscarriage or preterm deliv-ery and is inversely related to cervical length measured by transvaginal ultrasound at 20–24 weeks of gestation. In women with a short cervix the use of progesterone sup-positories reduces the risk of early delivery by some 40%. An alternative treatment in those with a very short cervix is cervical cerclage—the insertion of a stitch around the cer-vix. Despite some promising leads, there is no predictive


biochemical test for premature labor. Biochemical meth-ods that have been investigated include the measurement of fetal fibronectin in cervicovaginal secretions after 24 weeks—the presence of which suggests increased risk for preterm delivery. However a recent Cochrane review has concluded that, although fetal fibronectin is marketed and commonly used in labor and delivery units to help in the management of women with symptoms of preterm labor, currently there is not sufficient evidence to recom-mend its use.

PLACENTAL INSUFFICIENCYPlacental insufficiency is a condition of usually ill-defined cause and pathology in which there is partial failure of pla-cental transfer (nutrients to the fetus and waste-product removal). This can lead to fetal growth retardation, fetal distress, or fetal death. The failure is also reflected by reduced secretion of the specific products of the placenta.

Fetal growth retardation, more commonly referred to as small for gestational age fetuses (SGA), is the situation in which, as a result of placental insufficiency, the fetus is born at less than its expected weight. Typically, a neonate is defined as ‘growth retarded’ or ‘small for dates’ if the birth weight is less than the 10th centile (some units use the 5th centile) for the relevant week of gestation. The child may display features of poor nutrition (increased ratio of head to abdominal circumference) and there is an association with hypoxia during delivery and also long-term brain damage. These risks are substantially reduced in cases of SGA iden-tified prenatally, compared to those detected after birth. Screening for SGA (in the absence of preeclampsia) using a combination of maternal characteristics, obstetric history, and a combination of biophysical and biochemical markers at 11–13 weeks could potentially identify 75% of pregnan-cies delivering SGA neonates before 37 weeks (at a false-positive rate of 10%). Such combinations would include increased NT, increased uterine artery pulsatility index and mean arterial pressure, and biochemical markers such as Pregnancy-Associated Plasma Protein-A (PAPP-A), free β-hCG and Placental Growth Factor (PlGF), which are reduced. Although such screening programs are yet to become mainstream, there is evidence that early identifica-tion resulting in increased surveillance and the prophylactic use of low dose aspirin can halve the incidence.

Fetal death in utero in the last 12 weeks of pregnancy is referred to as stillbirth whilst that prior to 24 weeks is referred to as miscarriage. The cause is sometimes pla-cental insufficiency but in many cases no specific reason is found. Miscarriage and stillbirth are associated with abnor-mal results of first trimester screening for aneuploidies, characterized by increased NT and reduced levels of PAPP-A. Although screening algorithms using combina-tions of maternal characteristics, biophysical and biochem-ical markers at best can identify about 35% of cases of either condition, management of the risk is the same as that of growth restriction.

Fetal distress: As a result of placental insufficiency, or sometimes because of mechanical problems in the delivery process, the fetus may become hypoxic during labor. Clin-ical signs include slowing of the fetal heart and passage of

feces by the fetus (meconium). Death or brain damage may result if the child is not delivered rapidly.

PREECLAMPSIAPreeclampsia is a disorder of widespread vascular endo-thelial malfunction that occurs after 20 weeks’ gestation. It is clinically defined by hypertension and proteinuria, with or without pathologic edema. The incidence of preeclamp-sia is estimated to range from 2 to 6% in healthy, nullipa-rous women. Among all cases of preeclampsia, 10% occur in pregnancies of less than 34 weeks’ gestation. The global incidence of preeclampsia has been estimated at 5–14% of all pregnancies.

In developing nations, the incidence of the disease is reported to be 4–18%, with hypertensive disorders being the second most common obstetric cause of stillbirths and early neonatal deaths in these countries.

Mild and Severe PreeclampsiaPreeclampsia is mild in 75% of cases and severe in 25% of them. In its extreme, the disease may lead to liver and renal failure, disseminated intravascular coagulopathy (DIC), and central nervous system (CNS) abnormalities. If preeclampsia-associated seizures develop, the disorder has developed into the condition called eclampsia.

The classification of mild and severe preclampsia is arbitrarily based on the severity of the blood pressure changes and the level of proteinuria. Others have chosen to classify the disease based on the gestational age at onset of the disease. Such classifications are referred to as early (prior to 34 weeks), late (after 37 weeks), and intermediate (34–37 weeks).

The etiology of the disease lies with the impaired placen-tation that takes place prior to 14 weeks in which the tro-phoblastic invasion of the spiral arteries of the placenta are inhibited. This poor vascular development, whilst being a primary cause, is then influenced by a variety of other maternal, genetic and environmental factors during the development of the full-blown disease. Many authorities believe that there may be multiple etiologies.

In recent years there has been considerable interest in the search for biochemical markers of preeclampsia to be used in the context of diagnosis and screening. Many of these bio-chemical markers are those associated with the placenta such as PAPP-A, PlGF, soluble endoglin, soluble fms-like tyro-sine kinase-1 (sFlt-1 or sVEGFR-1 (vascular endothelial growth factor)), activin A, and inhibin A and other angio-genic and antiangiogenic factors. These along with some inflammatory molecules such as cytokines are altered only at the time of disease onset. In the first trimester, algorithms combining maternal characteristics and biophysical and bio-chemical tests have been shown to potentially identify 90% of cases developing early preeclampsia and 60% of those developing late preeclampsia for a false-positive rate of 5%. These biophysical markers include mean arterial pressure and uterine artery pulsatility index, whilst the biochemical markers include PAPP-A and PlGF. Because the general eti-ology of the disease lies in the early pregnancy remodeling of the spiral arteries of the placenta it is thought early screening


is the only option for allowing therapeutic intervention. Recent studies with calcium and vitamins (C and E), and antioxidant therapy have been largely dismissed as ineffec-tive. Low dose aspirin started prior to 14 weeks can poten-tially halve the incidence of preeclampsia.

Treatment, in severe cases, includes reduction of blood pressure and early delivery.

MISCELLANEOUS DISORDERS

Placental AbruptionIn placental abruption, part of the placenta separates with the formation of a large clot between the placenta and maternal tissues. This is sometimes associated with pre-eclampsia. It can lead to severe external bleeding, to a hypercoagulable state, to premature labor, and to placental insufficiency. If severe, the treatment is urgent delivery.

Uncontrolled maternal diabetes leads to overgrowth of both fetus and placenta, with elevated levels of fetoplacen-tal products. If untreated, fetal death is likely in the last 4–6 weeks of pregnancy. Some cases of diabetes appear only during pregnancy (gestational diabetes). Today, with careful control of the mother’s metabolic state, the diabetic pregnancy is virtually normal from every biological and clinical standpoint. Screening for gestational diabetes usually involves an oral glucose tolerance test and is usually offered in women with associated risk factors such as a Body Mass Index (BMI) greater than 30, previous history of gestational diabetes or family history of diabetes or a mac-rosomic baby (greater than 4.5 kg) in a previous pregnancy. Macrosomia is associated with increased levels of PAPP-A in the first trimester. Since PAPP-A is a protease that cleaves insulin-like growth factor binding protein it is likely that increased levels result in increased concentrations of insulin-like growth factor, which stimulates fetal growth.

Rhesus (Rh) DiseaseHemolytic disease of the fetus and newborn (HDFN) is a prevalent pathology of pregnancy and was a major obstetric problem. The condition is due to a maternal alloimmune reaction to a paternally inherited antigen. The Rh-positive fetus can sensitize the Rh-negative mother and in subsequent pregnancies, maternal antibodies cross the placenta causing a hemolytic anemia in the fetus, hydrops, and ultimately still-birth. If less severe the fetus survives but the newborn may develop severe jaundice and brain damage. The condition is associated with placental hypertrophy and elevated levels of fetoplacental products. Treatment is by exchange transfu-sion (either in utero or after birth) and early delivery. The most common causes are reactions to the Rhesus antigen D (RhD). Intrauterine blood transfusion for anemic fetuses was one of the first therapeutic approaches. Latterly the use of prophylactic anti-D administration to all RhD negative mothers has reduced the incidence markedly. More recently approaches to identifying whether the fetus has a D geno-type have involved the isolation of cell free fetal DNA (cffDNA) from maternal plasma in early pregnancy and this has led to new avenues for diagnosis, monitoring, and admin-istration of prophylactic anti-D in pregnant women.

Sickle and Thalassemia ScreeningThalassemia is an inherited autosomal recessive blood dis-ease that originated in the Mediterranean region. In thalas-semia the genetic defect, which can be either mutation or deletion, results in reduced rate of synthesis or no synthesis of one of the globin chains that make up hemoglobin. This can cause the formation of abnormal hemoglobin mole-cules, thus causing anemia, the characteristic-presenting symptom of the thalassemias. Thalassemias usually result in underproduction of normal globin proteins, often through mutations in regulatory genes. Thalassemia is a quantita-tive problem of too few globins synthesized, whereas sickle-cell disease (a hemoglobinopathy) is a qualitative problem of synthesis of an incorrectly functioning globin. Sickle hemo-globin (HbS) is a hemoglobin variant in which the sixth amino acid on the β globin chain, glutamic acid, is replaced by valine. Other much rarer hemoglobins have been reported that have this same glutamic acid to valine substi-tution but also an additional substitution elsewhere in the beta chain. All of these variants have a positive sickle solu-bility test, though their electrophoretic characteristics may be different, and all will cause sickle-cell disease. Hemoglo-binopathies imply structural abnormalities in the globin proteins themselves. The sickling disorders are associated with severe life-threatening vaso-occlusive crises, over-whelming sepsis, splenic sequestration, aplastic crises, stroke, priapism, pulmonary hypertension, proliferative retinopathy, and chronic organ damage, such as avascular necrosis of the hips and shoulders. The two conditions may overlap, however, since some conditions that cause abnor-malities in globin proteins (hemoglobinopathy) also affect their production (thalassemia). Thus, some thalassemias are hemoglobinopathies, but most are not. Either or both of these conditions may cause anemia.

Identification of these two disease groups are now tar-geted in the UK through blood spot screening programs as part of newborn screening programs. However in addition there are now antenatal screening programs. The overall aim of the antenatal screening program is to offer sickle cell and thalassemia screening to all women and couples in a timely manner in pregnancy. The screening program facilitates informed choices regarding participation in the program and provides help for those couples identified by screening as being at higher risk. For all pregnant women presenting to maternity services in England, sickle cell and thalassemia screening is an integral part of the early antenatal care offered at first presentation to primary care or first booking. The screening program is based on the use of hematological indi-ces and measurement of hemoglobin (Hb) variants by HPLC in conjunction with the use of a family origin questionnaire. If a maternal carrier is found then partner testing is carried out to determine if the partner is also a carrier and hence the fetus is at high risk of inheriting the disease.

Smith–Lemli–Opitz Syndrome (SLO)Smith–Lemli–Opitz Syndrome (SLO) was first described by Smith, Lemli, and Opitz in 1964. It is an inherited auto-somal recessive disorder caused by mutations in the sterol delta-7-reductase gene, which maps to chromosome 11q12-q13. It is the final enzyme in the sterol synthetic pathway


that converts 7-dehydrocholesterol (7DHC) to cholesterol. The enzyme defect produces low plasma cholesterol and reduced myelination in the cerebral hemispheres, cranial nerves, and peripheral nerves. It occurs in relatively high frequency with approximately one in 20,000–30,000 births in populations of northern and central European back-ground. Mental retardation is almost always present, together with growth restriction, behavioral difficulties and a spectrum of skeletal, genital, cardiac, pulmonary, and renal malformations. The differential diagnosis is often between Ambiguous Genitalia and Intersexuality, Congeni-tal Adrenal Hyperplasia, or Edwards syndrome (trisomy 18). As a result of the deficiency in the sterol pathway con-centrations of unconjugated estriol in the second trimester of pregnancy with fetuses affected by this condition are usu-ally very low. In 29 published cases the median AFP was 0.72 MoM with Unconjugated Estriol (uE3) at 0.21 and total hCG 0.76. It has been suggested that screening using an SLO specific algorithm may be beneficial. Prenatal test-ing with a view to termination of pregnancy is possible. Amniocentesis is performed around 15 weeks’ gestation and analysis by gas chromatography–mass spectrometry dem-onstrates an amniotic fluid cholesterol concentration that is low and a 7DHC concentration that is markedly elevated.

Steroid Sulfatase DeficiencySteroid sulfatase (STS) deficiency is one of the most common human inborn errors of metabolism. This X-linked disorder has an estimated frequency of 1 in 2000 to 1 in 6000 males, and 1 in 2000 women are estimated to be carriers. The clinical phenotype varies greatly in the pre-natal period as compared to postnatal life. STS deficiency results in decreased production of maternal-fetal estrogen due to lack of STS activity in the placenta. This deficiency of estrogen is associated with delayed progression of partu-rition. Newborns with STS deficiency are usually of appro-priate weight and size for their gestational age and have no perinatal complications resulting from the enzyme defi-ciency. Patients with STS deficiency usually present in the first 3 months of life with prominent skin peeling of ante-rior and posterior surfaces of the upper and lower extremi-ties resulting in a condition called ichthyosis.

Cornelia de Lange SyndromeCornelia de Lange (CDL) syndrome is a developmental malformation syndrome characterized by mental handicap, growth retardation, limb reduction abnormalities, and dis-tinctive facial features. Congenital heart defects, gastro-esophageal reflux, and hearing impairment may also be present. There is no known specific biochemical or chromo-somal abnormality associated with the syndrome, and diag-nosis is dependent on the recognition of the characteristic phenotype and in particular the distinctive facial features with both classical and mild cases being recognized. The incidence is estimated at around 1 in 40,000 births with a recurrence risk of less than 1%. Despite this low recurrence risk parental anxiety in subsequent pregnancies is usually high. Prenatal diagnosis, based on recognition of manifesta-tions of the syndrome by ultrasonography, is rarely achieved. In 1983, a deficiency of PAPP-A was reported in maternal

serum samples obtained between 20 and 35 weeks’ gestation from a patient who gave birth to a male infant with Cornelia de Lange syndrome. No PAPP-A could be demonstrated in trophoblast tissue by subsequent immunocytochemical stud-ies of the placenta or in a further patient whose pregnancy also resulted in the birth of a male infant with Cornelia de Lange syndrome. In a study of 18 cases with CDL the median MoM of PAPP-A in the second trimester was 0.21 (range 0.03–0.71). It was concluded that second-trimester maternal serum PAPP-A measurements may be of value as an adjunct to ultrasonography in the prenatal diagnosis of Cornelia de Lange syndrome.

FETAL ORIGINS OF ADULT DISEASE (THE BARKER HYPOTHESIS)Dr. David Barker discovered the relationship between birth weight and the lifetime risk for coronary heart disease in 1989. He showed that the lower the weight of a baby at birth and during infancy, the higher the risk for coronary heart disease in later life. The risk of heart disease falls across the entire range of birth weight. This implies that normal varia-tions in the transfer of food from mothers to babies have profound long-term implications for the health of the next generation. Later studies showed that low birth weight is associated with an increased risk of hypertension, stroke, and type 2 diabetes. This led to the ‘Fetal Origins Hypothesis,’ which proposes that coronary heart disease, and the diseases related to it, originates through responses to undernutrition during fetal life and infancy. These responses permanently change the body’s structure, physiology, and metabolism.

Recent findings have shown that a woman’s body com-position and diet at the time of conception and during pregnancy have important effects on the subsequent health of her offspring. The risk of later chronic disease is further increased if a baby has low weight gain after birth so that at 2 years it is thin or stunted. After the age of two, rapid gain in fatness further increases the risk of later coronary heart disease, hypertension, and type 2 diabetes. These findings point to the importance of:

� protecting the nutrition and health of young women before and during pregnancy,

� protecting the growth of infants, � avoiding rapid increase in fatness after the age of

2 years, especially in children who were thin at around 2 years of age, as part of the strategy to prevent chronic disease in later life.

The intrauterine programming by prenatal determinants and the impact on life course events in adult life (Fig. 1) has become an important area for further research in adverse pregnancy outcomes.

AnalytesAFPAFP is composed of a single polypeptide chain with a relative molecular mass of 69,000. It consists of 590 amino acids and 39% of the amino acid sequence corresponds to that of


albumin. Unlike albumin it is a glycoprotein with 4% carbo-hydrate residues. In amniotic fluid there are two types of AFP, differing in the structure of their carbohydrate chains and therefore in their ability to bind to concanavalin A.

In the fetus, AFP is synthesized by the liver, the gastro-intestinal tract, and the yolk sac (Fig. 2). The concentration of AFP in fetal serum rises rapidly to reach a peak at 12–14 weeks’ gestation. Thereafter it falls until term; the fall con-tinues in the newborn with a half-life of 3–4 days during the first weeks of life, eventually reaching adult levels at 8 months. In amniotic fluid there is a steady fall in AFP levels from a peak in the first trimester; the source is complex and includes maternal blood, fetal urine, and the yolk sac. In the mother, circulating AFP levels rise progressively to reach a peak at 32 weeks, then decrease toward term (Fig. 3).

There is no information on mechanisms that may con-trol the synthesis of AFP by the fetus and thus determine the concentrations in maternal and fetal fluids. The only physiological factor that influences AFP levels is fetal sex, the levels in umbilical blood at term being substantially higher if the child is a boy. The functions of AFP are also unknown. Probably the most convincing suggestion is that it serves a function similar to that of albumin. Other pos-sibilities include estrogen binding, though the activity of human AFP in this respect is far less than that of the more specific sex hormone-binding globulin. It has also been suggested that AFP has immunosuppressive activity and thus plays a role in the maternal–fetal graft relationship.

Reference IntervalMethods for measuring AFP are usually calibrated to the National Institute for Biological Standards and Con-trol (NIBSC)/World Health Organization (WHO)

FIGURE 1 Fetal development and the origins of adult disease. (Reproduced from Heart, Jarvelin M. 84, 219–226 (2000) with permission from BMJ Publishing Group Ltd.). (The color version of this figure may be viewed at www.immunoassayhandbook.com).

FIGURE 2 Schematic drawing showing the production and distribution of alpha-fetoprotein (AFP) into its three compartments: fetal tissues, amniotic fluid (AF), and maternal serum (MS). FS fetal serum. Reproduced with permission from Diagnostic Imaging of Fetal Anomalies. Nyberg et al. Eds. 2003; p998. Lippincott Williams & Wilkins. (The color version of this figure may be viewed at www.immunoassayhandbook.com).


International Standard for AFP (72/225), either directly or by some secondary calibrator. Measurements are therefore expressed either in kU/L or more rarely in mass units (ng/mL) when a typical conversion factor between kU/L and ng/mL is 1.0. There are low levels (<10 kU/L) of AFP in normal nonpregnant subjects.

Indicative reference intervals for AFP in maternal serum at 14–20 weeks’ gestation are shown (Table 1) as determined in the author’s laboratory using the ThermoFisher Scientific Kryptor platform (Brahms Biomarkers, Berlin). Laborato-ries are advised to establish their own reference intervals in their own laboratory using the commercial assay platform in use as defined by best laboratory practice. Constant moni-toring of reference values when screening for NTDs and Down Syndrome is advised to ensure systematic drifts and reagent change shifts do not introduce errors into screening program performance. It is usual practice that in risk calcula-tion software the median values are fitted to a polynomial regression to allow calculation of medians by gestational day.

Assay TechnologyA wide range of immunoassay techniques are available for AFP, including competitive and immunometric assays

with a variety of signal generation systems, although as is common with many routine analytes, radioimmunoassays (RIAs) are now very much a minority and most laborato-ries utilize automated systems with either chemilumines-cent or fluorescent end points (such as Siemens XP or Immulite and Delfia Xpress, AutoDelfia, or Kryptor). Apart from factors common to all immunoassays there are no very special demands on an assay for AFP. Specificity is not an issue in practice because there are no known inter-fering compounds. In amniotic fluid there are variant forms of AFP differing in the extent of glycosylation. These forms can be separated by lectin-affinity chroma-tography. Although some of these forms are considered to be characteristic of NTD, this detailed analysis is rarely done. Extreme sensitivity is not required; all current assays are capable of measuring the levels present in pregnancy. A wide operating range is advantageous: this should embrace both the low normal levels found in Down syndrome and the high normal levels associated with NTD. Typically this would cover the range 1–700 kU/L. The need for pre-cision over a wide range gives a theoretical advantage to labeled antibody (immunometric) assays. Typically between-day precision in the author’s laboratory is around 2% at levels between 10 and 100 kU/L. AFP is used as a screening test and samples are usually assayed in large batches. Some degree of automation is usually essential.

Types of SampleSerum, plasma, or amniotic fluid. As with most immunoas-says, serum is generally preferred to plasma because it is less liable to interference from anticoagulants, precipitates formed on storage etc.

Frequency of UseCommon.

Clinical ApplicationsNeural tube defects. As a result of the defect in the fetal body surface, fetal AFP leaks across exposed capillaries into amniotic fluid, and from there across the membranes into the maternal circulation (Fig. 2). Measurement of AFP in maternal serum therefore provides a screening test for neural tube defects (NTD). The test is most effective when applied at 16, 17, and 18 weeks; the predictive value is less before 15 weeks and after 20 weeks. A positive result of the screening test (defined as more than 2 MoMs in some units and 2.5 MoMs in others) leads to a series of follow-up investiga-tions. The end result of the whole process is detection and termination of 80–90% of spina bifida and 90–100% of anencephaly.

There is considerable overlap in maternal serum AFP (MSAFP) levels between normal and NTD pregnancies (see Fig. 4).

Other causes of elevated AFP are listed in Table 2A woman with an elevated AFP level is subjected to a

careful ultrasound examination, which can identify most of the alternative diagnoses shown, including many cases of NTD. On the whole ultrasound is now capable of diagnos-ing more than 95% of NTD, and amniocentesis and mea-surement of AFP is not considered necessary. If amniotic

FIGURE 3 Concentrations of alpha-fetoprotein (AFP) values in fetal serum, amniotic fluid, and maternal serum. Note the logarithmic scale, indicating much higher levels of AFP in the fetal serum compared to maternal serum. (Reproduced with permission from Haddow J.E. Prenatal screening for open neural tube defects, Down’s syndrome, and other major fetal disorders. Semin. Perinatol. 14, 488 (1990)).

TABLE 1 Typical Weekly Median Levels of AFP in the Second Trimester

Completed Gestational Weeks Maternal Serum AFP kU/L

14 24.4815 28.5916 33.2817 38.6218 44.6719 51.4920 59.16


fluid AFP is measured then with a level less than 2 MoMs, no further action is taken. If it is greater than 2 MoMs, acetylcholinesterase is usually measured. Positive findings on ultrasound or raised levels of both amniotic fluid AFP and acetylcholinesterase, are an indication for termination of the pregnancy (subject, of course, to full counseling of the woman).

Down syndrome. The use of low levels of MSAFP is dis-cussed later in the chapter (see SCREENING FOR ANEUPLOIDY).

Placental insufficiency. In some cases with elevated MSAFP levels but no NTD there is evidence of increased fetal risk, especially low birth weight, later in the preg-nancy. A woman with raised MSAFP levels should always be regarded as ‘at risk’ for other adverse pregnancy outcomes.

HUMAN CHORIONIC GONADOTROPINhCG is one of a family of glycoprotein hormones, the other members being luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyrotropin (TSH). Each of these consists of two subunits: an α subunit (92 amino acids) which is virtually identical in all four; and a β sub-unit which is characteristic of the individual hormone. The two subunits are noncovalently linked to form the dimer, which is known as intact hCG (hCGαβ), having a molecular weight of 39.5 kDa. The β subunit of hCG is a

single chain of 145 amino acids. The first 121 N-terminal amino acids share 80% sequence homology with β-LH; the C-terminus of β-hCG has a 24 amino acid extension not present in β-LH. Both subunits of the molecule are needed for biological activity but the β subunit deter-mines the specificity of the action. In maternal serum during pregnancy, hCG can be found in at least two forms, free β−hCG (hCGβ) which is the β subunit not linked to the α subunit (hCGα) and the intact dimer (hCGαβ). It is important to differentiate between these two forms because their use in screening for aneuploidy shows them to have different specificity. It is also impor-tant from an analytical point of view that assays are cor-rectly identified, with which form is being measured and specified (see below).

Chorionic gonadotropin is produced by the syncytiotro-phoblast (and possibly also by the cytotrophoblast) of the placenta. The synthesis involves independent translation of the respective messenger RNAs for the α and β sub-units. At least six genes from chromosome 19 are known to code for the β subunit. One gene on chromosome 6 codes for the α subunit. Posttranslational glycosylation of the subunits occurs before they are released as free α hCG (hCGα), free β hCG (hCGβ), and intact hCG (hCGαβ). Regulation of synthesis of intact hCG appears to be lim-ited by production of the β subunit.

In the placenta, serum, and urine, hCG is present in multiple related forms (Fig. 5). Urine is the major route for clearance of hCG from the circulation and β-core is the major degradation product of the β subunit produced by the kidney. The half-life of the intact hormone shows mul-tiple components, the initial faster phase being 6 h and the later phase 35.6 h. For the free β subunit the clearance is much quicker with an initial faster phase of 0.68 h and a later phase of 3.93 h.

Intact hCG appears in maternal blood shortly after implantation and then rises rapidly until 8 weeks’ gesta-tion. Levels show little change at 8–12 weeks, then decline until 18 weeks and remain fairly constant until term. Free β-hCG levels tend to peak around 9–10 weeks and then fall gradually through the rest of the first and second trimes-ters. Relatively speaking the concentration of free β hCG is around 0.05–0.10% of that of intact hCG. The pattern of hCG in fetal blood is similar to that in the mother but at 2–3% of the concentration. At term the levels in the female fetus are substantially higher than those in the male. This pattern of different expression of hCG is also evident in the maternal serum during the first and the second trimester.

The levels and pattern of hCG in amniotic fluid are similar to those in blood. In urine some hCG is in the form of intact hormone (20–25%) whereas the remainder con-sists of free β subunit and, in particular, a fragment known as ‘β core’.

The mechanisms that determine the levels of hCG in maternal blood are unknown. Unlike the steroid hormones, there is no relationship to the mass of tissue of origin.

In the first trimester hCG may be the major luteotro-phic factor from the implanting embryo ensuring mainte-nance of the corpus luteum. In the second trimester it has been postulated that hCG is the stimulus for testosterone synthesis by the fetal testis.

FIGURE 4 Distribution of maternal serum AFP MoM in normal pregnancies and those with spina bifida or anencephaly.

TABLE 2 Associations with Elevated MSAFP

1. Normal pregnancy (upper end of distribution)2. NTDs3. Other congenital anomalies such as exomphalos and ventral

wall defects4. Multiple pregnancy5. Inaccurate gestational dating (underestimate)6. Fetal death7. Maternal tumors (hepatoma, gonadal teratomas)8. Maternal liver disease9. Renal anomalies e.g., congenital nephrosis

10. Infant sacrococcygeal tumor11. Other potential adverse pregnancy outcome


Reference IntervalMost commercially available diagnostic immunoassays for hCG are calibrated against World Health Organization (WHO) 3rd International Standard (IS) 75/537 or 4th IS 75/789 (which are essentially identical), for hCGβ against 1st International Reference Preparation (IRP) 75/551, and for hCGα against 1st IRP 75/569.

These preparations were originally intended for bioas-say rather than immunoassay. The hCG was assigned units based on bioactivity, with 70 µg corresponding to 650 IU in the 3rd and 4th IS. The nonbioactive α and ß subunits were assigned units based on mass, with 1 µg of each corresponding to 1 IU of the relevant IRP.

Recent advances in purification technology have led a working group of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) to develop the first WHO reference reagents that selectively target hCG and its specific subunits (see Table 3). Six prepara-tions have now been established as the first WHO Interna-tional Reference Reagents (IRRs) for immunoassays with the following codes.

These are the first immunoassay standardization reagents for human glycoprotein analytes with values assigned in molar units rather than based on bioreactivity or arbitrary units. Their availability will facilitate charac-terization of current immunoassays for hCG-related mol-ecules and should ultimately lead to improvements in standardization and between-method comparability.

FIGURE 5 Various human chorionic gonadotropin (hCG) related molecules in placenta, blood, and urine and the proposed degradation pathway. (Reproduced with permission from Cole, L.A. Immunoassay of human chorionic gonadotropin, its free subunits, and metabolites. Clin. Chem. 43, 2233–2243 (1997)).

TABLE 3 IFCC Nomenclature and WHO Codes for First IRRs for Six Important Isomers of hCG

hCG IsomersIFCC Nomenclature WHO Code

Intact hCG hCG IRR 99/688Nicked hCG hCGn IRR 99/642HCG beta subunit hCGβ IRR 99/650Nicked hCG beta subunit hCGβn IRR 99/692HCG beta core fragment hCGβcf IRR 99/708HCG alpha subunit hCGα IRR 99/720


The range of hCG levels in the second trimester using methods that measure Intact hCG (hCGαβ), total hCG (hCGαβ+β), or free β-hCG are shown in Table 4. For the first trimester only free β-hCG is used to screen for Down syndrome and the levels as used in the author’s laboratory using the Kryptor platform are shown in Table 5.

Assay TechnologyBecause of the very similar nature of the β subunits of LH and hCG, early immunoassays showed considerable cross-reaction between the two. Unambiguous detection of early pregnancy was therefore not possible. The introduction of assays with an antibody directed to the β subunit of hCG yielded assays that were considerably more specific, and a further enhancement resulted from the use of immuno-metric assays and monoclonal antibodies. For practical purposes, most current hCG assays can be regarded as entirely specific to this analyte; interference by LH is so unlikely that it can safely be ignored.

An important and on-going problem of hCG assays arises from the fact that blood (and especially urine) con-tains a mixture of intact hCG, free β subunit, and free α subunit. Individual assays may respond quite differently to each of these: for example, some competitive assays are much more sensitive to free subunit than to intact hor-mone, whereas some immunometric assays react with intact hormone but not at all with free subunit. Because the intact/subunit composition of calibrators is usually unlike that of samples, different assays can give very differ-ent results on the same sample. The major practical effect of this is the chaotic picture that emerges from external quality assessment schemes.

Assays can be classified based on the molecular form that is targeted in the assay. If an assay uses a capture antibody that is to an epitope on the β-subunit and then uses a

signaling antibody that is also to an epitope on the β-subunit then this assay will measure both intact hCG and free β-hCG—these assays are usually referred to as total hCG assays. If the capture antibody is to an epitope on the β-subunit and the signaling antibody is to an epitope on the α-subunit (or vice versa) then this assay will measure only intact hCG. Assays that are specific for free β-hCG use a capture antibody with an epitope deep in the binding cleft on the β subunit—this epitope is hidden when the β subunit is bound to the α subunit (as in intact hCG) and thus only captures free β-hCG. In such assays the signaling antibody is to a different epitope on the β subunit. Provided that a labo-ratory uses one assay, and adheres to the reference intervals derived from that assay, no problems should arise. However, there is now very good evidence that an assay specific to the free β subunit (i.e., not reacting with intact hCG) has greater efficiency than intact or total hCG assays in screening for aneuploidy, particularly so in the first trimester.

Because of the wide biological range and the elevated levels seen in maternal serum in pregnancies with Down Syndrome a wide analytical range is required. This would be typically 0.1–150 IU/L for free β-hCG and 150 kU/L for total or intact hCG.

Typical between-day precision for free β-hCG in the author’s laboratory using the Kryptor platform is around 2–2.5% at levels between 7 and 80 IU/L.

As already noted, qualitative (yes/no) immunoassays for urine hCG are widely used in clinical practice. Some of the older forms are based on agglutination, others on micro-fluidic devices using immunochromatography and enzyme labels with a color endpoint. Ease of use is essential because the tests will often be used by untrained clinicians in emer-gency circumstances. The type of pregnancy test sold over the counter to the general public is perfectly adequate for this purpose.

Types of SampleSerum, plasma, or urine. Urine is widely used for qualitative pregnancy tests and gives results virtually identical to blood. Some analytical platforms cannot use ethylenediaminetet-raacetic acid (EDTA) plasma because of interference in various parts of the immunoassay or detection process.

Frequency of UseCommon.

Clinical ApplicationsDetection of early pregnancy. This is the commonest use of hCG immunoassays. The result is qualitative (i.e., yes or no). Most commercial tests have a detection limit of 25 IU/L. This permits detection of pregnancy in the major-ity of subjects at approximately 7 days postimplantation (i.e., around the time of the missed period). More sensitive research assays can detect pregnancy as early as 1–2 days after implantation. At one time there was much concern about false-negative and false-positive results. With current technology the chance of either of these errors by 7 days after the missed period must be virtually zero.

Threatened abortion. The hCG levels are generally low in those cases in which the pregnancy will be lost and normal in those that proceed satisfactorily. However, there

TABLE 4 Median Values Different hCG Assays in the Second Trimester of Pregnancy

Completed Gestational Weeks

Intact hCG (kU/L)

Total hCG (kU/L)

Free β-hCG (IU/L)

14 48.5 40.7 21.715 34.9 31.5 16.216 26.5 25.4 12.417 21.2 18.8 9.818 18 17.5 7.9

TABLE 5 Median Value of Free β-hCG in the First Trimester of Pregnancy

Completed Gestational Weeks Free β-hCG (IU/L)

8 51.99 71.210 68.311 57.112 45.713 36.7


is considerable overlap between the two groups and a use-ful distinction is not possible before 8 weeks gestation. By this stage the fetal heart can be seen on ultrasound, and the presence or absence of the fetal heart provides a more definitive test. Some workers have advocated serial mea-surements of hCG; because the doubling-time in early pregnancy is 2 days, failure to increase over a period of 4 days or more is an unfavorable sign.

Ectopic pregnancy. The hCG levels are positive in all cases of ectopic pregnancy. Many women present with lower abdominal pain of unknown cause. If hCG is nega-tive, then a pregnancy-related condition can be safely excluded. If the test is positive, and even if there is no sign of pregnancy on ultrasound, then further investigation must focus on the possibility of an ectopic pregnancy (ultrasound and laparoscopy) or a PUL.

Serum levels of hCG (pregnancy hormone) are used to help determine location: approximately 70% of women with ectopic pregnancy will have a rise in hCG that is slower than the minimum for normal pregnancy or a fall that is slower than the minimum for spontaneous miscar-riage. However, 15% of normal pregnancies will have an abnormal doubling-time. This can make it very difficult to differentiate between a failing intrauterine pregnancy, a healthy intrauterine pregnancy and an ectopic pregnancy.

Diagnosis of ectopic pregnancy on transvaginal ultra-sound should be based on the positive detection of an extrauterine sac and indirect signs such as a complex

adnexal mass or echogenic fluid. In combination, these methods have a 93.5–100% positive detection rate.

Down syndrome. The use of elevated levels of hCG is discussed later in the chapter (see SCREENING FOR ANEUPLOIDY).

Late pregnancy. Low levels are found in placental insufficiency and high levels in preeclampsia. The test is used rarely if ever clinically.

ESTRIOLEstriol is one of the four classical ovarian estrogens (estriol, estradiol, estrone, and estetrol); it has the characteristic aro-matic A-ring of all estrogens, together with hydroxyl groups at the 3, 15, and 16 positions (Fig. 6). The relative levels of these steroids are approximately in the ratio 200:20:10:1.

The unique feature of estriol is that it is produced by the syncytiotrophoblast from fetal precursors (Fig. 7). The

FIGURE 6 Estriol.

FIGURE 7 Metabolic pathway involved in the synthesis of estrogens, showing that once in the maternal compartment they undergo conjugation with glucuronic acid or sulfate by the liver. DHEAS, dehydroepiandrosterone sulfate. Reproduced with permission from Diagnostic Imaging of Fetal Anomalies. Nyberg et al. Eds. 2003; p998. Lippincott Williams & Wilkins.


fetal adrenal produces dehydroepiandrosterone (DHEA) and the fetal liver converts this to 16-hydroxy-DHEA. Both of these compounds circulate in the fetus as the sul-fate conjugate. In the placenta the 16-hydroxy-DHEA-S is deconjugated by a sulfatase: the A-ring of the molecule is acted on by an aromatase to yield the form characteristic of estrogens. Estriol is then secreted into the maternal circu-lation. A small portion (less than 10%) of total estriol is not conjugated and remains as unconjugated estriol (uE3). The pattern in maternal blood is a progressive rise throughout gestation. Approximately 70% of all estrogens are bound to sex hormone-binding globulin and are thought to be biologically inactive. Diurnal variation has been reported in both total estriol and uE3 with levels 15% lower in the morning.

The factors determining the levels of estriol in the maternal circulation are the supply of fetal precursors, and the conversion of these to estriol in the placenta. The lat-ter is the rate-limiting step and the time-to-time control of estriol secretion is the same as that of products such as human placental lactogen (hPL), dependent on the mass of the trophoblast and the rate of uteroplacental blood flow. Because of the unique synthetic pathway of estriol, there are a number of unusual observations in experimental and pathological situations. These include:

� Estriol levels fall after administration of corticosteroids to the mother. These compounds cross the placenta, suppress the fetal pituitary–adrenal axis, and hence the production of DHEA.

� Anencephaly (with an inactive fetal pituitary) and absence or hypoplasia of the fetal adrenal gland are associated with very low levels of estriol.

� Low levels of estriol are associated with the rare condi-tion of placental sulfatase deficiency in which the pre-cursor (16-hydroxy-DHEA-S) cannot be deconjugated and hence is not available for conversion in the placenta.

� Low levels of estriol are also associated with the rare condition of Smith–Lemli–Opitz syndrome in which there is a deficiency in 3β hydroxysterol reductase and an inability to synthesize cholesterol, and hence estriol cannot be produced from its precursors.

Reference IntervalBecause production of uE3 requires well-functioning fetal adrenal gland, fetal liver, and placenta, the original use of uE3 was as a test for fetal well-being in later pregnancy. Early assays for uE3 were optimized for the third-trimester concentrations that are approximately 5–10 times higher than the second-trimester concentrations. The uE3 assays currently used for maternal serum screening have been recalibrated to attain the necessary performance at the lower concentrations required for Down syndrome screen-ing in the second trimester.

There is no standard reference material for estriol assays. Such a reference preparation is not essential because the molecule is small, can be synthesized, and can be mea-sured by physical methods. Isotope-dilution gas chroma-tography/mass spectrometry is considered the reference method. To convert results from nanograms per milliliter to nanomoles per liter, multiply by 3.467.

Most uE3 assays are designed to measure naturally occurring uE3 in maternal serum. Artificial samples, including calibrators from other kits, certain proficiency samples, and certain controls, may produce results as much as 2–3 times lower than expected. Proficiency surveys must be graded by peer group, rather than across all methods. The median values in use in the author’s lab using the Beckman Access II platform are shown in Table 6.

Assay TechnologyAll immunoassays for estriol are competitive in design. Sen-sitivity is not a requirement because the levels in pregnancy are very high. Specificity is not an issue because estriol is the predominant estrogen in pregnancy blood; interference by other estrogens would have only minimal effects. Some assays include enzymes that cleave conjugates so that all estriol is measured (total estriol). There are also assays with very specific antisera that only measure unconjugated estriol. In the latter, sensitivity may become an issue. Most methods in clinical use today tend to be based on chemilu-minescence or fluorescence detection systems.

Typical between-day precision for uE3 in the author’s laboratory using the Beckman Access II platform is around 6–9% at levels between 4 and 20 nmol/L.

Type of SampleSerum is preferable for the only common current application: Down syndrome screening.

Frequency of UseFairly common.

Clinical ApplicationsDown syndrome. The use of low levels of unconjugated estriol is discussed later in the chapter (see SCREENING FOR ANEUPLOIDY).

Placental insufficiency. In the 1960s and 1970s measure-ment of urine estriol was the most widely used special test of fetal well-being. In the late 1970s and 1980s the urine sample was replaced by blood. In the later 1980s and 1990s estriol measurement was replaced by biophysical techniques such as ultrasound. However, there is no doubt that placental insufficiency is reflected by reduced levels of estriol.

INHIBIN AInhibins and activins are members of the transforming growth factor-beta superfamily, which are a group of

TABLE 6 Median Values for uE3 in the Second Trimester of Pregnancy

Completed Gestational Weeks

Unconjugated Estriol nmol/L

14 1.6815 2.3316 3.1717 4.1718 5.33


structurally but functionally diverse growth factors. Mature inhibin is a 32 kDa heterodimeric glycoprotein composed of an α subunit linked by disulfide bridges to one of two possible β subunits. If the beta subunit is type A (βA), it is called inhibin A (also referred to as dimeric inhibin A); if the beta subunit is type B (βB), it is called inhibin B. When the two β subunits combine they form the homodimer Activin for which three forms can exist, Activin A (bA–bA), Activin B (bB–bB), and Activin AB (bA–bB). Many differ-ent molecular forms of these subunits can be found in biological fluids (Fig. 8).

During pregnancy, inhibin is produced by the syncytio- and cytotrophoblast cells of the placenta, although smaller amounts are produced by the decidua, fetal membranes, and the developing fetus. The control of secretion from the placenta is not fully understood and the function dur-ing pregnancy is not known. Inhibin A is a placental prod-uct, and placental products tend to be increased in pregnancies associated with T21. It is an effective screen-ing marker in combination with existing serum markers and maternal age. Both Activin A and inhibin B have been shown to be increased in women with established pre-eclampsia and also before the onset of the disease in both the second and first trimester of pregnancy.

Reference IntervalThe WHO 1st International Reference Standard for human inhibin A (91/624) was developed in 1994. IRP 91/624 is a lyophilized recombinant preparation that has

assigned IU (1 pg/mL is approximately equal to 0.037 IU/mL). It is recommended that inhibin A assays use this material for calibration. The expected median values of inhibin A in the second trimester of pregnancy used in the author’s laboratory using the Beckman Access II platform are shown in Table 7.

Assay TechnologyImmunoassays are used to measure inhibin A due to the requirement for picomolar sensitivity. Because of the structural similarity between inhibin A and other members of the transforming growth factor-β superfamily proteins (in particular inhibin B and activin A), it has been difficult to design assays specific for inhibin A. There are a limited number of assays available commercially for the measure-ment of inhibin A. The majority of inhibin A assays employ

FIGURE 8 Many different molecular forms of unprocessed and partially processed inhibin subunits have been described in follicular fluid, amniotic fluid, and serum. (Reproduced with permission from Knight P.G. Roles of inhibins, activins, and follistatin in the female reproductive system. Front Neuroendocri-nol. 17, 476–509 (1996)).

TABLE 7 Median Values for Inhibin A in the Second Trimester of Pregnancy

Completed Gestational Weeks Inihibin A ng/L

14 20115 18216 17017 16618 170


a microtiter plate enzyme-linked immunosorbent assay (ELISA) format; however, an automated assay using che-miluminescent detection has recently become available making it ideal for use in population screening. One of the difficulties for companies in developing such assays is the Intellectual Property Rights, which are assigned to only one diagnostics company.

Typical between-day precision for inhibin A in the author’s laboratory using the Beckman Access II platform is around 4% at levels between 100 and 400 ng/L

Type of SampleSerum is preferable for the only common current application: Down syndrome screening.


Clinical ApplicationsDown syndrome. The use of increased levels of inhibin A is discussed later in the chapter (see SCREENING FOR ANEUPLOIDY).

Preeclampsia. At the time of onset of the disease, levels of inhibin have been reported to be elevated by up to 8 fold. In the second trimester of those presenting later with preeclampsia, levels are still elevated but at around 2 MoM. Even in the first trimester levels of inhibin A are around 1.6 MoM in cases that develop early preeclampsia. Studies combining biophysical measurements such as Mean Arte-rial Pressure and Uterine Artery Pulsatility Index, with maternal history along with the measurement of PAPP-A, PlGF, and inhibin A in the first trimester, suggest that 90% of early preeclampsia could be identified at a 10% screen-positive rate. Such screening programs are in their infancy but could identify a cohort of women who may benefit from the prophylactic use of low dose aspirin, which could reduce the incidence of the disease by up to 50%.

PREGNANCY-ASSOCIATED PLASMA PROTEIN-APAPP-A is a zinc-containing metalloproteinase glycopro-tein ubiquitously expressed at low levels in human tissues, and at much higher concentrations in the placenta. During pregnancy, PAPP-A is produced by the trophoblast and exists in a 2:2 complex with the preform of eosinophil major basic protein in the maternal circulation. The molecular weight of the complex is approximately 500 kDa and is comprised of 1547 amino acids and derived from a larger precursor of placental origin. The gene for PAPP-A has been localized on the long arm of chromosome 9. At term, approximately 1% of PAPP-A is in its free, proteo-lytically active form. Proteolytic release of insulin-like growth factor binding proteins by PAPP-A allows insulin growth factors (IGFs) to stimulate IGF receptors, which function in fetal development and postnatal growth.

After delivery PAPP-A is cleared from the body with a two component clearance model with an initial clearance half-life of 52.9 h and a second phase clearance half-life of 142.9 h. However in the first trimester clearance rates after

first trimester termination are considerably longer with an initial rate of 93.9 h and a secondary rate of 362.9 h. This has implications for interpreting data in pregnancies when there has been fetal demise of one twin (a vanished twin).

Studies suggest that reduced concentrations of PAPP-A in pregnancy are associated with chromosome abnormali-ties in the fetus in the first trimester of pregnancy, espe-cially T21 but also in other major aneuploidies such as T13 and T18. Thus, PAPP-A can be used for first trimester screening, together with hCGβ and NT to form the com-bined test. In the second trimester, levels in T21 are rela-tively normal and thus PAPP-A is of no value in screening for this disorder in the second trimester. Although levels are still low in cases of T13 and T18, this observation is not in clinical use. Although elevated levels of PAPP-A have not been associated with adverse pregnancy outcomes, low levels of PAPP-A in the first trimester (≤5th percentile or 0.4 MoM) have been statistically significantly associated with several adverse obstetrical outcomes including spon-taneous fetal loss at ≤24 weeks’ gestation, intrauterine growth restriction (IUGR) (birth weight <5th percentile), preeclampsia, gestational hypertension, preterm delivery, and fetal death >24 weeks’ gestation.

Reference IntervalThe WHO Standard 78/610 for pregnancy-specific beta 1-glycoprotein (SP1) was developed in 1983 and composed of pooled sera from women at the time of delivery. This reference preparation was found to contain PAPP-A and other placental and pregnancy-associated proteins, and a PAPP-A concentration was later assigned to IRP 78/610. Most available assays are either standardized to IRP 78/610 or traceable to a method standardized to IRP 78/610; how-ever, IRP 78/610 is no longer available. A recently formed IFCC working group has been assigned to develop a new IRP for PAPP-A. As with uE3 there are significant method differences in unitary value thus proficiency surveys must be graded by peer group, rather than across all methods. The median values in use in the author’s laboratory using the Brahms Kryptor platform are shown in Table 8.

Assay TechnologyAlthough PAPP-A is present in men and nonpregnant women, concentrations are typically <1 mIU/L. Most of the assays available for PAPP-A are nonisotopic noncompetitive immunoassays. Automated sandwich chemiluminescence formats are available as are a time-resolved amplified cryptate

TABLE 8 Median Value of PAPP-A in the First Trimester of Pregnancy

Completed Gestational Weeks PAPP-A U/L

8 0.129 0.3210 0.6711 1.1912 1.8913 2.86


emission method (Brahms Kryptor) and a time-resolved flu-orometric immunoassay (PerkinElmer Delfia). In addition to screening for T21, PAPP-A has also been investigated as a marker of plaque rupture in acute coronary syndrome, and some manufacturers have developed PAPP-A assays specifi-cally designed to detect cardiac abnormalities.

Typical between-day precision for PAPP-A in the author’s laboratory using the Kryptor platform is around 2–2.5% at levels between 0.3 and 4.2 IU/L.

Clinical ApplicationsDown syndrome. The use of reduced levels of PAPP-A in the presence of a fetus with T21, T13, or T18 is discussed later in the chapter (see SCREENING FOR ANEUPLOIDY).

Adverse Pregnancy Outcome. Although elevated lev-els of PAPP-A have not been associated with adverse preg-nancy outcomes, low levels of PAPP-A in the first trimester (≤5th percentile or 0.4 MoM) have been statistically signifi-cantly associated with several adverse obstetrical outcomes including spontaneous fetal loss at ≤24 weeks’ gestation, IUGR (birth weight <5th percentile), preeclampsia, gesta-tional hypertension, preterm delivery, and fetal death >24 weeks’ gestation. It has been suggested that a screening program using maternal factors, biophysical parameters and biochemical markers such as PAPP-A and PlGF may provide a screening program that could identify approxi-mately 90% of women (at a 10% screen positive rate) who would develop early preeclampsia enabling these women to be offered prophylactic low dose aspirin to prevent the dis-ease progression in approximately 50% of cases.

Type of SampleSince PAPP-A contains zinc atoms as part of its structure EDTA plasma cannot be used since the EDTA will seques-ter the zinc, causing the PAPP-A molecule to change in conformation, and can prevent binding of the antibodies, resulting in ultralow apparent concentrations. Serum is the preferred sample type. When samples are collected at the same time as for hematological indices, or for tests requir-ing plasma or whole blood, the clotted blood tube should always be the first tube collected and never after an EDTA tube has been collected. Ultra low PAPP-A results should have serum calcium and/or potassium analyzed to check for potential potassium EDTA contamination.


PLACENTAL GROWTH FACTOR (PLGF)PlGF is a dimeric glycoprotein belonging to the VEGF factor family, and shares 53% amino acid homology with VEGF. It is expressed predominantly by trophoblasts and is able to cause endothelial proliferation, migration, and activation. In pregnancy, it is believed that PlGF and other angiogenic factors regulate trophoblast invasion of the spi-ral arteries. Impaired trophoblast invasion leads to insuffi-cient vascular remodeling of the spiral arteries compared to normal pregnancy. This leads to reduced perfusion of the placenta which then can lead to a variety of adverse

pregnancy outcomes such as small for gestational age or preeclampsia. In these conditions, levels of PlGF are reduced and its inclusion in screening programs for pre-eclampsia has been proposed. The biological activity of PlGF is attributed to binding VEGF-R1, also known as fms-related tyrosine kinase 1 (Flt-1) thereby activating angiogenic pathways for normal placentation. The soluble form of this receptor (sFlt-1) acts as a competitive inhibitor by binding PlGF in the circulation making it unable to bind to receptors therefore losing its proangiogenic activity. In pregnancies with T21 it has been suggested also that levels of PlGF may be disturbed to a small degree—although there is confusion as to whether this is decreased in the first trimester and increased in the second trimester. A role for PlGF in aneuploidy screening is not established.

Reference IntervalThere is no developed International Reference Prepara-tion for PlGF. Most commercial assays use a recombinant protein as standard and this is then calibrated either by mass alone or by comparison with competitor assays. Lev-els of PlGF increase throughout pregnancy. In the first trimester when PlGF may appear to have clinical value the expected median levels at this time, using the PerkinElmer Delfia Xpress assay, are shown in Table 9.

Assay TechnologyCommercial assays for PlGF have only recently started to become available. Initially assays were based on standard sandwich immunoassays using ELISA technology. Recently high-throughput assays suitable for screening purposes have become available from companies such as Roche and Perki-nElmer using sandwich immunoassays with either chemilu-minescent or fluorescence end points. Alere also produce a point of care type application on their Triage platform. The majority of recent assays are targeted at measuring free PlGF (i.e., not including the PlGF bound to sFLT-1).

Typical between-day precision for PlGF in the author’s laboratory using the Delfia Xpress platform is around 6% at levels between 18 and 40 pg/mL.

Clinical ApplicationPlGF has potential to be used as part of a screening pro-gram for preeclampsia based on a combination of mater-nal risk factors, biophysical measurements such as Mean Arterial Pressure, Uterine Artery Pulsatility index, and the biochemical markers PlGF and PAPP-A.

TABLE 9 Median Value of PlGF in the First Trimester of Pregnancy

Completed Gestational Weeks PlGF pg/mL

9 16.910 19.811 23.712 27.213 33.6


Type of SampleSerum is the preferred sample type.

Frequency of UseNot commonly used as yet.

OTHER ANALYTES

ProgesteroneProgesterone is produced by the corpus luteum in the first 6 weeks of pregnancy and by the placenta thereafter. It is believed to be the hormone principally responsible for the maintenance of early pregnancy. Measurements of serum pro-gesterone and serum hCG have been used to predict the preg-nancy outcome. A serum progesterone of <20 nmol/L has been shown to be predictive of a failing pregnancy. Levels above 25 nmol/L are “likely to indicate” and levels above 60 nmol/L are “strongly associated with” pregnancies subse-quently demonstrated to be viable. Serum hCG and proges-terone levels at defined times can be used to predict the immediate viability of a PUL, but cannot be used reliably to predict its location. Clinical experience does not significantly improve the ability to assess PUL outcome and invariably serial hCG measurements are those used clinically. A meta-analysis of 26 studies has demonstrated that, whilst a single serum progesterone measurement has a good discriminative capacity to distinguish between pregnancy failure and a viable intrauterine pregnancy, a single measurement cannot discrim-inate between ectopic pregnancy and non ectopic pregnancy.

Screening for AneuploidyScreening for Down syndrome (T21) over the past two decades has become an established part of obstetric prac-tice largely through the use of maternal serum biochemi-cal screening in the second trimester of pregnancy. In the late 1980s and early 1990s levels of the biochemical mark-ers AFP, Total hCG, uE3, and free β hCG were shown to be altered in pregnancies with T21. In the mid to late 1990s inhibin A was shown to be another potential marker.

It is well documented that increasing maternal age is related to an increased risk for T21 (and also T18 and T13) and maternal age risk forms the basis or a priori risk for calcu-lating a new risk based on the measurement of the biochemi-cal markers. In some instances a previous pregnancy with one of the above aneuploidies requires further adjustment of the background age risk and this adjustment is commonly applied to increase the background risk by around 0.75%.

The methodology for calculating risk is based on a well known statistical procedure called Bayes Theorem in which a revised risk is calculated by adjusting the prior risk by a likelihood ratio (LR) derived from the biochemical measure-ment. An LR is an expression of how likely it is that the mea-sured result comes from a population of pregnancies with T21 compared with how likely it has come from a popula-tion of normal pregnancies. LRs can be calculated using standard gaussian statistical techniques. Most biochemical and some biophysical markers in pregnancy do not fit a

gaussian distribution unless the measure is log transformed. Figure 9 shows the distribution of hCG in normal pregnan-cies and those with T21 after log transformation of the hCG MoM. If the measured value of hCG in mothers blood is 2.0 MoM then the LR can be determined from the ratio of the heights of the two gaussian curves at that point and in this example the ratio is approximately 2. Then the mother’s age at the date of delivery (e.g., 30 years old) is used to calcu-late the risk of T21 (in this example, 1 in 685). In the exam-ple above of hCG, when the LR for a marker level of 2 MoM is 2 then the revised risk is 1/(685/LR) = 1/(685/2) = 1 in 342.

If more than one marker is being used, then in simple terms the first LR could be multiplied by a second or third or fourth LR to obtain a combined LR that adjusts the maternal age in the same way. In reality there is some level of correlation between the markers and this has to be taken into account but the mathematics are complex and beyond the scope of this review.

Once a revised risk has been obtained then the vast majority of screening programs will use a specific risk cut-off that enables classification of the women into a high-risk (Screen-positive) or a low-risk (Screen-negative) group. Of course after screening there is always still a risk because the very nature of screening is to identify women at sufficiently high enough risk to warrant invasive testing such as CVS or amniocentesis, which allows karyotyping of fetal cells and is diagnostic. The risk cut-off ultimately defines the Detec-tion Rate and False-Positive Rate because all three are interlinked: if you set out to increase the Detection Rate then the False-Positive Rate increases and the cut-off has to move to lower risk (e.g., changing from 1:150 to 1:200). In the UK both second and first trimester programs are now standardized on a risk cut-off of 1:150 at term.

In the second trimester, screening for aneuploidies is primarily for T21 and T18—the pattern of biochemical markers is such that T13 and the other aneuploidies are similar to normal with perhaps the exception of Triploidy (3 sets of each chromosome). The patterns expressed are shown in Table 10.

FIGURE 9 Gaussian distributions for hCG and the estimation of the Likelihood Ratio (LR). (The color version of this figure may be viewed at www.immunoassayhandbook.com).


The expected median levels in pregnancies with the two common anomalies of T21 and T18 for the main markers used in the second trimester are shown in Table 11.

Screening performance using a combination of the dif-ferent markers shows that for a two-marker protocol using AFP and free β-hCG a detection rate of around 64% can be achieved for a 5% screen-positive rate. If a three marker protocol was used, including uE3 then this would increase to around 67% and if a four marker protocol was used, including inhibin A, then this would increase to around 72%. In more than 20 published prospective intervention studies this modeled screening performance has been con-firmed in routine practice. The four-marker protocol commonly referred to as the “Quad Test” is generally the standard in use when screening is performed in the second trimester.

The past decade and a half has seen a considerable focus on moving screening earlier into the first trimester. Ear-lier screening is seen as providing women with an earlier reassurance and if termination of pregnancy is desired, this can be completed by a safer procedure and before fetal movements are usually evident. The fact that some aneu-ploid pregnancies detected in the first trimester will be spontaneously lost before term is not a valid argument against early screening. For these women it is important information to know, as it can shape future reproductive decision making.

Screening markers in the first trimester consist of the ultrasound marker NT and the maternal serum biochemi-cal markers free β-hCG and PAPP-A and these form what has become known as the combined test. This test has now become the National Screening Standard in many countries.

NT is the term used for an ultrasound measurement of the thickness of an echogenic area of fluid that exists in all fetuses between the fetal skin and the soft tissue overlying the cervical spine seen in a mid-sagittal view of the face of

the fetus. NT (see Fig. 10) is the single most discrimina-tory marker of T21 in the first trimester. The measure-ment of NT, unlike the measurement of serum markers, depends on operator skill, especially when one considers the small size of the measurement of 1–3 mm being nor-mal. The NT measurement may be made at the same time as the early pregnancy dating scan, between 11 weeks 0 days and 13 weeks 6 days. Most experienced sonographers can obtain the measurement within a few minutes. The CRL is measured during the same examination. NT increases with advancing pregnancy, and the CRL (a pre-cise measure of gestational age) is used to standardize NT measurement for gestational age, either converting NT to MoM values (observed NT measurements divided by the median NT measurement for the corresponding CRL) or to delta values (observed NT measurements minus the median NT measurement for the corresponding CRL). The Fetal Medicine Foundation (www.fetalmedicine.com/fmf/ ) and Nuchal Translucency Quality Review Program (www.ntqr.org) are two organizations that provide ultraso-nographer training, certification, and ongoing audit. Con-tinuing audit of individual sonographers is required by many screening programs and in the UK this is carried out along with the monitoring of biochemical markers by the Downs Syndrome Screening Quality Assurance Support Service (DQASS) at a national level.

Not only is increased NT a good marker for T21, it is also a very good marker for T13 and T18, and can be supra

TABLE 10 Second Trimester Marker Patterns in Common Aneuploidies

Aneuploidy AFP HCG Inhibin A UE3

T21 Low High High LowT18 Low Low Normal LowT13 Small increase Normal Normal NormalTurners (45×0) Small decrease High and low High and low Small decreaseOther sex Normal or high Normal or high Unknown NormalTriploidy Type I High HighTriploidy Type II Normal Low

TABLE 11 Marker Levels in Second Trimester Cases of T21 and T18

Serum MarkerT18 Median MoM

T21 Median MoM

AFP 0.65 0.75Total hCG 0.32 2.06Unconjugate Estriol 0.42 0.72Free β-hCG 0.33 2.2Inhibin A 0.87 1.92

FIGURE 10 Midsagittal view of the fetal head profile showing the area of Nuchal Translucency (NT).


elevated in hydropic Turner’s syndrome. Increased NT is also associated with a number of adverse pregnancy out-comes and an increased risk for fetal cardiac defects. Other ultrasound markers have been identified that may aid as second-line investigations and these include looking for the presence or absence of the nasal bone (absent is T21), measurement of the fronto-maxillary facial angle (which is increased in T21), the presence of tricuspid regurgitation and abnormal blood flow through the ductus venosus. All of these latter markers require skill in measurement and in the case of the latter two, high energy color Doppler. They are not advised as routine screening markers and are best left to skilled tertiary centers.

Combining first trimester maternal serum biochemistry and NT measurement is an effective screening procedure because the two modalities do not appear to be correlated. Retrospective and now many prospective studies have shown that 90% of cases of T21 can be detected for a false-positive rate of 5% or less, and studies have also shown that approximately 90% of all other major chromosomal anomalies can be detected for an additional 1% false-positive rate. Table 12 summarizes the basic pattern of changes in the various markers associated with aneuploidy.

Attempts to improve the accuracy of individualized risks in both the second and first trimester have been carried out by correcting many of the biochemical markers for preg-nancy variables that influence the levels of these markers. Some of the variables are outlined in Table 13.

One other individual factor that needs consideration when screening, is: how does the overall quoted detection rate and false-positive rate equate to the individual being screened? Quoted screening performance data relates to the overall population but what an individual is concerned about is: what is the detection rate for a person of that age? As we know, increased maternal age brings about increased risk for T21 and although the risk is modified by pregnancy variables, as described, there is still an age-related detection rate and false-positive rate effect, such that in a 20 year old with first trimester screening the detection rate is not 90% at a 5% false-positive rate but is 73% for a 1.1%; and in second trimester it is not 75% at a 5% but is 55% at a 1.8%. Likewise in women of 45 the first trimester detection and false-positive rate is 97% at 29.6 and 98% at 45.9%.

Alternative screening strategies have been described that are more complex than the simple Quad Test or the Combined Test. One such test is the Integrate Test, which attempts to combine the tests used in the first trimester with those used in the second trimester. Thus PAPP-A and NT are measured at 11–13 weeks, the patient is not given

a risk assessment but then returns at 16 weeks for further biochemical tests that include AFP, Free β-hCG, uE3, and inhibin A. Only when all the data are available is a risk produced. The theoretical benefits of this approach are a perceived increase in detection rate or lowering of the false-positive rate based on data from two trials (SURRUS and FASTER) however the practicalities of this approach make screening more complex. Also many have voiced serious ethical and moral issues with respect to withholding infor-mation at the end of the first trimester part of the test. In the UK this is not a nationally recommended screening test, but it is popular in the USA.

TABLE 12 Changes in Marker Patterns in the First Trimester of Aneuploid Pregnancies

Aneuploidy NT CRL Fetal Heart Rate Free β-hCG PAPP-A

Trisomy 21 ↑ 2.5 ↔ ↑ ↑2.2 ↓0.5Trisomy 18 ↑ 3.5 ↓ ↓ ↓0.3 ↓0.5Trisomy 13 ↑ 2.5 ↔ ↑ ↓0.5 ↓0.3Turner’s ↑ 7.0 ↔ ↑ ↔ ↓0.5Triploidy I ↑ 2.5 ↔ ↓ ↑8.0 ↓0.8Triploidy II ↔ ↓ ↓ ↓0.2 ↓0.2

TABLE 13 Maternal and Pregnancy Factors Influencing Biochemical Marker Levels

Factor First Trimester Second Trimester

Gestational age PAPP-A increased, free β-hCG decreased after 9 weeks

AFP, uE3 increased, hCG decreased, inhibin A little change

Maternal weight All decreased with increasing weight

All decreased with increasing weight, uE3 least affected

Multiple pregnancy

Twice as high in twins—chorionicity effect

Twice as high in twins

Insulin-dependent diabetes mellitus

PAPP-A decreased AFP decreased, uE3 and hCG increased

Fetal Sex Free β-hCG and PAPP-A increased with a female fetus

HCG increased, AFP decreased with female fetus

Assisted Conception

Free β-hCG increased, PAPP-A decreased

HCG increased, uE3 decreased

Ethnicity Afro-Caribbean PAPP-A 50% higher free β-hCG higher by 10%

AFP, hCG higher in Asian and Afro-Caribbean, inhibin lower in Afro- Caribbean

Smoking PAPP-A increased HCG, uE3 decreased, AFP increased and inhibin increased by 60%

Gravidity/Parity Both increased as pregnancy number increases

HCG decreased

Previous Pregnancy

2–3 times more likely to be high risk if previous was high risk

3–5 times more likely to be high risk if previous was high risk


Other more complex screening strategies that have been proposed are based around setting risk cut-offs such that those with very high risks (e.g., 1:50) are offered CVS, those with a very low risk (e.g., 1:1000) are reassured and those in the middle are offered further testing. This further testing can be to refer those women to a specialist fetal medicine/ultrasound unit for the measurement of nasal bone, facial angle, tricuspid regurgitation and ductus veno-sus in the first trimester—this information is then com-bined with the earlier information from combined testing and a revised single risk calculated. A second alternative would be to offer the intermediate group second trimester biochemical screening with the Quad test and to combine all of the test elements together in a single risk. The modi-fications add little to the overall detection rate but can mean the false-positive rate falls well below 2%.

Another proposal to improve performance overall has been as a result of the observation that in T21 the specifici-ties of the individual markers change across the first and second trimester gestational windows. Thus PAPP-A has better clinical discrimination between 9 and 11 weeks than between 11 and 13 whilst with free β-hCG this is reversed. Since NT is measured optimally at 11–13 weeks and the observation of other ultrasound features is best performed around 12 weeks it has been suggested that further improvements could be made by taking a blood sample at 9–10 weeks and using this to measure PAPP-A and then at 12 weeks collect a second sample at the time of NT for measuring free β-hCG. Such protocols allow detection rates of 90% to be achieved with a false-positive rate of less than 2%, which is the aspirational detection rate for the UK National program for 2011.

Whilst the past 20 years has seen some significant devel-opments in screening initially for T21 and more recently for other aneuploidies in the first trimester, it may not be long before we see a new range of tests that may eventually compete with screening or compete with conventional QF-PCR in the diagnosis of T21 and possibly some other aneuplodies. In 1997, Dennis Lo (then in Oxford) described the presence in the maternal circulation of small amounts of cell-free fetal DNA (cffDNA) during pregnancy. Ten years later, a number of studies had shown that this cffDNA could be quantified by a variety of techniques and that the additional amount of chromosome 21 could be distin-guished from normal in those women carrying a fetus with T21. In the last year, four studies have shown, using new DNA sequencing technologies, that this technique could be another tool in the array of methods available. Whether such techniques become replacements for conventional karyotyping, or become a replacement for screening, or become techniques applied to intermediate risk group women, is yet to be established. Clearly one issue is that of

cost and accessibility to such technologies and whether such techniques can really apply to all types of chromo-somal disorders.

If the last 20 years has seen rapid change in methods of pregnancy screening and evaluation, clearly the next 10 years could be equally as exciting and challenging.

Further ReadingAitken, D.A., Ireland, M., Berry, E. et al. Second-trimester pregnancy associated

plasma protein-A levels are reduced in Cornelia de Lange Syndrome pregnan-cies. Prenat. Diagn. 19, 706–710 (1999).

Barker, D.J.B. (ed), Fetal and Infant Origins of Adult Disease. (BMJ Publishing Group, London, 1992).

Barker, D.J.P. Mothers, Babies, and Disease in Later Life. (BMJ Publishing Group, London, 1994).

Berghella, V., Hayes, E., Visintine, J. and Baxter, J.K. Fetal fibronectin testing for reducing the risk of preterm birth. Cochrane Database of Systematic Reviews (Issue 4) (2008)10.1002/14651858.CD006843.pub2. Art. No.: CD006843.

Chitty, L.S. and Lau, T.K. 1st trimester screening & diagnosis, Prenat. Diagn. 31, 1–130 (2011). (Whole Issue).

Chiu, R.W., Akolekar, R., Zheng, Y.E. et al. Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale valid-ity study. BMJ 342, c7401 (2011).

Clejan S., Ashwood E.R., Bashirians G., Rasmussen S.A., Snyder J.A., Spencer K., Wald N., Whitley R. Clinical and Laboratory Standards Institute (CLSI). Maternal Serum Screening; Approved Standard—2nd edn CLSI document I/LA25–A2 (ISBN 1-56238-000-0).

Conde-Agudelo, A., Papageorghiou, A.T., Kennedy, S.H. and Villar, J. Novel biomarkers for the prediction of the spontaneous preterm birth phenotype: a systematic review. BJOG 118, 1042–1054 (2011).

Condous, G., Van Calster, B., Kirk, E., Haider, Z., Timmerman, D., Van Huffel, S. and Bourne, T. Prediction of ectopic pregnancy in women with a pregnancy of unknown location. Ultrasound Obstet. Gynecol. 29, 680–687 (2007).

Craig, W.Y., Haddow, J.E., Palomaki, G.E. et al. Identifying Smith-Lemli-Opitz syndrome in conjunction with prenatal screening for Down syndrome. Prenat. Diagn. 26, 842–849 (2006).

Fetal Medicine Foundation website http://www.fetalmedicine.com/fmf/Gagnon, A., Wilson, R.D., Audibert, F. et al. Obstetrical complications associated

with abnormal maternal serum markers analytes. Obstet. Gynaecol. Can. 30, 918–949 (2008).

Kashork, C.D., Sutton, V.R., Fonda, J.S. et al. Low or absent unconjugated estriol in pregnancy: an indicator for steroid sulfatase deficiency detectable by fluores-cence in situ hybridization and biochemical analysis. Prenat. Diagn. 22, 1028–1032 (2002).

Maron, J. and Bianchi, D.W. Prenatal diagnosis using cell free nucleic acids in maternal body fluids: a decade of progress. Am. J. Med. Genet. 145C, 5–17 (2007).

NHS Sickle Cell and Thalassaemia Screening Programme: http://sct.screening.nhs.uk

Nice Guidelines for Antenatal Care: Routine Care for the Healthy Pregnant Woman. (2008).Nice Guidelines for Multiple Pregnancy: The Management of Twin and Triplet Pregnancy

in the Antenatal Period. (2011).Nicolaides, K.H. Turning the pyramid of prenatal care. Fet. Diagn. Ther. 29,

183–196 (2011).Palomaki, G.E., Kloza, E.M., Lambert-Messerlian, G.M. et al. DNA sequencing of

maternal plasma to detect Down Syndrome: an international clinical validation study. Genet. Med. 13, 913–920 (2011).

Pre-eclapmsia web casts:- http://www.goodmood.fi/perkinelmer/?videoId=31787638

30th Anniversary Issue. Prenat. Diagn. 30, 599–711 (2010).Sonek, J. First trimester ultrasonography in screening and detection of fetal

anomalies. Am. J. Med. Genet. 145C, 45–61 (2007).Spencer, K. Aneuploidy screening in the first trimester. Am. J. Med. Genet. 145C,

18–32 (2007).Whole issue supplement. Placenta 32, S1–S76 (2011).

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