Technical standards and guidelines: Prenatal screening for open neural tube defects: This new section on “Prenatal Screening for Open Neural Tube Defects,” together with the new section on “Prenatal Screening for Down Syndrome,” replaces the previous Section H of the American College of Medical Genetics Standards and Guidelines for Clinical Genetics Laboratories

Abstract

This specific technical standards and guidelines statement is intended to augment the current general American College of Medical Genetics (ACMG) Standards and Guidelines for Clinical Genetics Laboratories and to address validation guidelines specific to second trimester maternal serum screening. Individual laboratories are responsible for meeting the CLIA/CAP quality assurance standards with respect to appropriate sample documentation, assay validation, general proficiency, and quality control measures. 182940, Spina bifida; 206500 Anencephaly Neural tube defects (NTDs) result when the embryonic neural tube fails to close properly by approximately 4 (four) weeks' gestation. The clinical consequences of NTDs are dependent on the site and severity of the defect. Failed closure of the anterior neural tube (anencephaly) is lethal and usually results in miscarriage, stillbirth, or early death. Defects lower along the neural tube may be open or have a thin covering membrane (about 80% of cases) or may be closed. Clinical effects of open spina bifida (OSB) cover a wide spectrum that can include paralysis, hydrocephalus, and incontinence. A second trimester maternal serum screening program can aid in the identification of open neural tube defects (ONTD): OSB and anencephaly.1.Wald N.J. Cuckle H.S. Brock J.H. Peto R. Polani P.E. Woodford F.P. Maternal serum alpha-fetoprotein measurement in antenatal screening for anencephaly and spina bifida in early pregnancy: Report of the U.K. Collaborative Study on Alpha-fetoprotein in relation to neural tube defects.1:STN:280:DyaE2s3htFCiug%3D%3D69055Lancet. 1977; 1: 1323-1332Google Scholar Most cases of NTDs are sporadic and inheritance most likely is multifactorial. Kindreds have been described that suggest autosomal or X-linked recessive inheritance. NTDs also can be associated with specific single gene disorders and chromosome disorders. Environmental factors that increase the risk that a fetus will be affected with an NTD include exposure to anticonvulsant medications (e.g., valproate) and inadequate maternal folate intake. Preconceptional folic acid supplementation/fortification can reduce the incidence of NTDs up to 80%, dependent on dose.2.Wald N.J. Law M.R. Morris J.K. Wald D.S. Quantifying the effect of folic acid. Lancet 2001;358:2069–2073. MRC Vitamin Study Research Group Prevention of neural tube defects: results of the Medical Research Council vitamin study.10.1016/0140-6736(91)92605-2Lancet. 1991; 338: 131-137Google Scholar Although the prenatal screening laboratory utilizes clinical chemistry methods such as enzyme immunoassays, the role of the laboratory extends beyond the performance of the tests because the results require a unique kind of interpretation. This interpretation puts the results of the test into the appropriate context of a priori risks as determined by race, gestational age, and family history. The laboratory director is often called upon to provide consultation regarding these risks and options for further action. To address these unique requirements, the laboratory director should meet the standards set out in Section B3 of the ACMG Guidelines. When prenatal screening for ONTDs is performed in a clinical chemistry laboratory in which the director does not meet these standards, the laboratory should have a demonstrated relationship with an individual who does meet the standards set out in Section B3 and who is available in a timely fashion to aid in interpretation and provide consultation when requested. Prenatal testing for ONTDs by measurement of alpha-fetoprotein (AFP) is considered a screening test when performed in maternal serum and a diagnostic test when performed in amniotic fluid. The distinction between a screening and a diagnostic test is important because the goals and expectations for sensitivity, specificity, costs, and acceptable level of invasiveness differ. Maternal serum AFP screening results are not diagnostic of any condition. Rather, the screening process identifies pregnancies that are at sufficient risk for ONTDs to warrant genetic counseling and the offer of additional testing such as ultrasound and amniocentesis. The detection and false-positive rates of this screening test will be a function of several factors, including assay precision, the gestational age when the sample is obtained, the method of gestational age assignment, and the AFP cut-off level used to determine “screen positive” results. The measurements of AFP and acetylcholinesterase (AChE) in amniotic fluid, in combination with ultrasound, are diagnostic for fetal ONTDs.3.Wald N. Cuckle H. Nanchahal K. Amniotic fluid acetylcholinesterase measurement in the prenatal diagnosis of open neural tube defects: Second report of the collaborative acetylcholinesterase study.1:STN:280:DyaK3c7nsFartg%3D%3D10.1002/pd.1970091202Prenat Diagn. 1989; 9: 813-829Google Scholar Prenatal screening for ONTDs is best implemented in the context of a comprehensive program that coordinates preanalytic, analytic, and postanalytic components of the process. Laboratories should either provide educational materials (e.g., brochures, videotape) for use by patients in consultation with their providers or, at a minimum, provide information about where such materials can be obtained. Many laboratories and professional organizations (e.g., ACOG, National Society of Genetic Counselors, regional genetics groups) have produced, and in some cases formally evaluated, materials that are in effective formats, at appropriate reading levels, and available in multiple languages. These materials provide general information about the disorder, test performance, patient rights, eligibility, test interpretation, treatment options, costs, risks and benefits of testing, and what to expect if the screening test is positive. Laboratories should supply providers with informational materials that include the following: ONTD3.1.2 (a) Detailed information about the sampling process and how samples should be labeled and transported to the laboratory; ONTD3.1.2 (b) Samples of test requisitions that must accompany samples to provide information needed for identification and accurate test interpretation; ONTD3.1.2 (c) General information on testing, such as laboratory turn-around time and whether results will be phoned/faxed or mailed; ONTD3.1.2 (d) Information about expectations for test performance (sensitivity, specificity, and failure rate) and reporting formats. Patients should be informed about the benefits and limitations of prenatal screening before testing. It is the duty of the health care professional, not the laboratory, to inform and obtain consent for testing, but the laboratory may be required to document such consent (e.g., in New York state). It is the laboratory's responsibility to provide sufficient information about prenatal screening to the health care provider to ensure that an appropriate specimen is obtained and to facilitate educating the patient and obtaining informed consent. For the most reliable interpretation, laboratories should have a mechanism to collect pretest clinical information that includes: Basic required demographic information (see Sections C2.4 and C3); Gestational age (see also Section ONTD3.5.3.3); Maternal weight; Maternal race (at least Caucasian and African American); Presence of maternal insulin dependent diabetes mellitus prior to pregnancy; Number of fetuses; Previous screening in the current pregnancy (e.g., initial or repeat serum sample); Family history of NTDs. The laboratory may choose to contact health care providers if critical patient information does not accompany the specimen. If the laboratory does not obtain this information, the written report should indicate that the information is missing and what information, if any, was used in the interpretation. In some cases, including information on the report about the potential impact of the missing information may be warranted (e.g., maternal weight, race). In other cases, full interpretation may not be possible (e.g., no gestational age). Blood samples should be collected using standard phlebotomy techniques. The laboratory should specify what samples are acceptable (e.g., whole blood, serum separator tube, spun serum separator tube). Specimen containers should be labeled with the patient's name and draw date. Acceptable specimen handling from collection site to the laboratory should be specified, including packaging, mode of transportation (e.g., courier, United States mail, overnight transport), and temperature range (AFP is very stable and samples can be shipped at ambient temperature). Variables that can affect the acceptability of a sample for ONTD screening or a specific AFP assay protocol should be established by the laboratory and may include both clinical (e.g., gestational age out of range) and sample-related characteristics (e.g., inappropriate sample type, insufficient quantity, gross hemolysis). See also Sections C2.4 through C2.6. Protocols should be designed to avoid contamination, tampering, or substitution. Handling samples must be in accordance with OSHA guidelines, with the express understanding that any human fluids may harbor infectious agents. AFP can be reliably determined in sera stored at 4° to 8°C for several days and at −20°C for years. See also Sections C2.5 through C2.6. See Sections C2.7 through C2.8 for more information. Guidance on developing assay protocols is available. See C5, C6, and C8.3 (Validation). In the United States, the FDA licenses AFP kits as an aid in the diagnosis of ONTDs. As Class III devices, these kits are approved to reliably measure AFP in second trimester maternal serum samples and amniotic fluid. Available kits include immunometric and radioimmunoassay methods, all capable of measuring AFP reliably in the range of values important for ONTD screening (25 to 150 IU/mL). AFP standards can be calibrated in either mass units (ng/mL) or International Units (IU/mL). Each AFP kit manufacturer provides a factor for converting mass units into International Units. Conversion factors should be considered manufacturer-specific. Commercially available AFP kits provide calibrators and specific calibration protocols. Laboratories utilizing assays developed in their own laboratory (“home brew”) or modifying AFP kit assay protocols, are responsible for determining calibration protocols and validating performance. In-house pooled controls, commercially available controls, or controls received in kits serve as checks on reagents and technical performance. Advantages of in-house pooled controls include a sample matrix that more closely resembles patient samples, AFP levels set for ONTD clinical action points, and control lots prepared with long expiration dating to aid in assessment of kit master reagent lot changes and long-term assay drift. An alternative for long-term monitoring is commercial controls bought in sufficient quantity to last a year or more. Repeat assay controls (RACs) are also helpful for monitoring performance variability. To assess short-term performance, unfrozen patient samples are chosen at random from recent assays and reassayed to monitor intra- and interassay precision. Because serum AFP levels are stable when frozen and thawed, reassaying stored patient samples from the time period when the current median values were established can help to identify long-term drift and determine if reference data need to be updated. Each batch assay should contain at least two quality control samples that fall at clinical action points (three controls may be required to comply with some licensure requirements). For example, low controls could be targeted at serum AFP values falling at 0.5 multiples of the median (MoM) for 16 weeks of gestation. Normal or midrange controls could be targeted near the 16-week median (1.0 MoM), and high controls at a value near commonly used ONTD cut-off levels (2.0 to 2.5 MoM). After preparation and aliquoting, performance ranges for in-house pooled controls can be set using standard clinical laboratory quality control approaches. Controls received with AFP kits have an acceptable target range specified by the manufacturers but laboratories may wish to establish an in-house range. This information is used to accept or reject individual control results or a whole assay so care should be taken to set appropriate ranges and avoid unnecessary result rejection. Standard approaches used in the clinical laboratory are appropriate for internal quality control (QC) of AFP assays, including the type and frequency of assessments. As part of the initial method validation, the laboratory should demonstrate that intra- and interassay variation reported by the manufacturer can be reproduced. Standard approaches to routine equipment calibration and preventive maintenance used in the clinical laboratory are appropriate. In many cases, calibration and maintenance protocols are set by the product/equipment manufacturer (see Section ONTD3.4.3). In order for AFP measurements to be interpreted, each result in mass or International Units must first be converted to a MoM for a given gestational age. The resulting MoM levels can then be adjusted for other factors, such as maternal weight. It has been established that values obtained from different lots from the same manufacturer or from different manufacturers may demonstrate systematic bias. Therefore, it is essential that each laboratory establish its own normative data or, at a minimum, demonstrate that data obtained from another source are appropriate for its screened population. Maternal serum AFP levels increase by a constant percentage per week (approximately 10% to 15%) in the second trimester. Package insert (commercial) medians should not be used, even for a short time. Several methods exist that can be utilized to establish reliable medians. Ideally, 100 samples for each gestational week from 15 through 20 would be used to calculate median values. Because AFP is stable, it is possible to use stored frozen specimens collected over several years. It is not necessary that all samples used be from unaffected singleton pregnancies. Using regression analysis (see ONTD3.5.2.4) allows use of fewer samples (e.g., 300 over the 15- to 20-week period) to establish reasonable medians.4.Knight Palomaki G.E. Epidemiologic monitoring of prenatal screening for neural tube defects and Down syndrome.10.1016/S0272-2712(03)00023-4Clin Lab Med. 2003; 23: 531-551Google Scholar “Smoothing” the observed median values by weighted log-linear regression analysis (logarithms of medians regressed vs. gestational age in days or completed weeks, weighted by the square root of the number of observations in each category) provides reliable and accurate medians. This method also allows median values to be extrapolated for weeks in which little data are available. Using median values that are specific to each day of gestation will further improve screening performance. The optimal time for ONTD screening is 16 to 18 weeks but screening is acceptable between 15.0 and 20.9 weeks.1.Wald N.J. Cuckle H.S. Brock J.H. Peto R. Polani P.E. Woodford F.P. Maternal serum alpha-fetoprotein measurement in antenatal screening for anencephaly and spina bifida in early pregnancy: Report of the U.K. Collaborative Study on Alpha-fetoprotein in relation to neural tube defects.1:STN:280:DyaE2s3htFCiug%3D%3D69055Lancet. 1977; 1: 1323-1332Google Scholar Screening performance is significantly decreased in the 14th week of gestation. Under special circumstances, laboratories may accept samples later than 20 weeks gestation with the understanding that clinical options may be limited. Gestational age may be expressed in completed weeks (15 weeks and 5 days is 15 completed weeks). Expressing results in rounded weeks (15 weeks and 5 days is 16 weeks) is not recommended. Screening performance is improved by expressing gestational age as weeks and days or decimal weeks (15 weeks and 5 days is 15.7 weeks). The most common method for determining gestational age is dating by the first day of the last menstrual period (LMP). Although LMP dating is sufficiently accurate for ONTD screening, gestational age estimation based on ultrasound measurements is more accurate and its use improves both the sensitivity and specificity of screening. Ultrasound measurement of crown-rump length (CRL) in early pregnancy provides an accurate estimate of gestational age. In the second trimester, ultrasound dating based on multiple measurements (composite) is accurate to within 10 days. Composite ultrasound dating is preferable to LMP dating. For ONTD screening, ultrasound dating based on biparietal diameter (BPD) measurement alone is recommended for pregnancies at 14 weeks gestation or later, or when the LMP is uncertain or discrepant with physical examination. BPD dating rules out anencephaly and because OSB cases have, on average, BPD measurements equal to a 2-week younger fetus, dates based on BPD can significantly improve detection of open spina bifida.5.Wald N. Cuckle H. Boreham J. Small biparietal diameter of fetuses with spina bifida: implications for antenatal screening.1:STN:280:DyaL3c3itV2ksg%3D%3D10.1111/j.1471-0528.1980.tb04522.xBr J Obstet Gynaecol. 1980; 87: 219-221Google Scholar,6.Wald N.J. Cuckle H.S. Boreham J. Alpha-fetoprotein screening for open spina bifida: effect of routine biparietal diameter measurement to estimate gestational age.1:STN:280:DyaL2c7psFeluw%3D%3DRev Epidem et Sante Publ. 1984; 32: 62-69Google Scholar The method of determining gestational age can be taken into account when providing interpretations in two ways. First, separate medians can be calculated for those pregnancies dated by LMP and those dated by ultrasound measurements. Secondly, separate Gaussian population parameters can be utilized in determining risk (see Sections ONTD3.5.5.2 through ONTD3.5.5.3). The following are interpretive refinements based on patient demographics and other pregnancy-related information that are less critical than taking gestational age into account, but will improve screening performance by optimizing the interpretation. Currently, most laboratories take the following factors into account. AFP levels are, on average, higher in lighter weight women and lower in heavier weight women. Adjusting AFP values for maternal weight improves ONTD screening performance and is recommended.7.Crandall B.F. Lehbhez R.B. Schroth P.C. Matsumoto M. Alpha-fetoprotein concentrations in maternal serum: relation to race and body weight.1:CAS:528:DyaL3sXhsVWhtL8%3D6186415Clin Chem. 1983; 29: 531-533Google Scholar, 8.Neveux L.M. Palomaki G.E. Larrivee D.A. Knight G.J. Haddow J.E. Refinements in managing maternal weight adjustment for interpreting prenatal screening results.1:STN:280:DyaK2s7kslensA%3D%3D10.1002/(SICI)1097-0223(199612)16:12<1115::AID-PD3>3.0.CO;2-6Prenat Diagn. 1996; 16: 1115-1119Google Scholar, 9.Johnson A.M. Palomaki G.E. Haddow J.E. The effect of adjusting maternal serum alpha-fetoprotien levels for maternal weight in pregnancies with fetal open spina bifida: A United States collaborative study.1:STN:280:DyaK3czivVamtw%3D%3D10.1016/S0002-9378(11)90655-3Am J Obstet Gynecol. 1990; 163: 9-11Google Scholar Laboratories should only utilize published weight adjustment formulas for a short time until in-house data are collected and new laboratory-specific formulas derived. Correction for maternal race is recommended, because AFP levels in Black/African American women are about 10% to 15% higher than in Caucasian women.7.Crandall B.F. Lehbhez R.B. Schroth P.C. Matsumoto M. Alpha-fetoprotein concentrations in maternal serum: relation to race and body weight.1:CAS:528:DyaL3sXhsVWhtL8%3D6186415Clin Chem. 1983; 29: 531-533Google Scholar,10.Wald N.J. Kennard A. Hackshaw A. McGuire A. Antenatal screening for Down's syndrome.1:STN:280:DyaK1c7ls1Siuw%3D%3D10.1177/096914139700400402J Med Screen. 1997; 4: 181-246Google Scholar If sufficient data are available, the preferred adjustment method is to calculate a separate set of medians for each of the groups. If too few observations are available in one of the groups, a correction factor can be applied to the MoM when screening those pregnancies. Correction for maternal insulin-dependent diabetes mellitus (IDDM) is recommended. AFP levels have been reported to be 10% to 20% lower in women with IDDM before and during pregnancy.10.Wald N.J. Kennard A. Hackshaw A. McGuire A. Antenatal screening for Down's syndrome.1:STN:280:DyaK1c7ls1Siuw%3D%3D10.1177/096914139700400402J Med Screen. 1997; 4: 181-246Google Scholar Most programs take this into account by using an adjustment factor for AFP MoM levels in IDDM women. There is no consensus on whether this correction should be applied to gestational diabetic women. Calculating MoM results for a 200-pound, Black/African American woman with IDDM would require at least three adjustments to the AFP MoM.7.Crandall B.F. Lehbhez R.B. Schroth P.C. Matsumoto M. Alpha-fetoprotein concentrations in maternal serum: relation to race and body weight.1:CAS:528:DyaL3sXhsVWhtL8%3D6186415Clin Chem. 1983; 29: 531-533Google Scholar,10.Wald N.J. Kennard A. Hackshaw A. McGuire A. Antenatal screening for Down's syndrome.1:STN:280:DyaK1c7ls1Siuw%3D%3D10.1177/096914139700400402J Med Screen. 1997; 4: 181-246Google Scholar Although data are sparse, programs can make the assumption that the effects are independent. Although most data are derived from studies of mainly Caucasian women, the assumption is usually made that a similar effect will be seen in Black/African American women. Laboratories should be able to compute risks for ONTD and OSB even though they may not be routinely reported.11.Estimating an individual's risk of having a fetus with open spina bifida and the value of repeat AFP testing. Report of the Fourth UK Collaborative Study on AFP in relation to neural tube defects. J Epidem Comm Health 1982; 36: 87–95.Google Scholar Some licensing agencies require routine reporting of the risk, and it is important to be able to provide this information to health care professionals for clinical counseling and pregnancy management. The use of specialized software applications is generally considered a necessity for ONTD screening, due to the complex nature of calculating and interpreting the results, the need for patient-specific interpretive reports, and because of the large number of samples processed. Patient-specific risks are generated by complex mathematical algorithms that are integral to prenatal screening. Software to perform these calculations can be obtained commercially or developed in-house. Software must be verified before routine clinical use. The commonly used algorithm to assign a patient-specific risk utilizes the MoM results (adjusted for variables such as weight and race as discussed above) to calculate a likelihood ratio based on the overlapping Gaussian distributions defined by the affected and unaffected distribution parameters. The population or prior risk for OSB or anencephaly is then multiplied by the corresponding likelihood ratio to generate the patient-specific risks.11.Estimating an individual's risk of having a fetus with open spina bifida and the value of repeat AFP testing. Report of the Fourth UK Collaborative Study on AFP in relation to neural tube defects. J Epidem Comm Health 1982; 36: 87–95.Google Scholar Risk algorithms utilize published or in-house population parameters for AFP, expressed as log means and log standard deviations of AFP distributions in unaffected pregnancies and in pregnancies affected with OSB or anencephaly. Population parameters for each of these disorders can vary based on the gestational age at the time of testing and gestational dating method (see ONTD3.5.7.1 and ONTD3.5.7.7). There is no formal consensus on which adjustments to the result or prior risk to include, specifically how to include them or how inclusion influences screening performance. These decisions are left to the laboratory director. Definition of screen Positive and negative results: for ONTD screening, determination of “screen positive” results most commonly relies on AFP MoM cut-off levels. Few laboratories choose the ONTD risk estimate as the screening variable. Reasonable AFP cut-off levels for ONTD screening range between 2.0 and 2.5 MoM. Screen positive results are defined as those with an AFP MoM greater than or equal to the cut-off level. The prevalence, or background risks may be higher or lower in certain populations. In such cases, the screening cut-off level may be modified in order to keep the risk of an ONTD at the cut-off roughly equal (isorisk screening). Either approach is acceptable, but the laboratory should understand the trade-offs associated with the different approaches. For example, Black/African American pregnancies are at about half the risk of ONTDs. Screening programs that use a 2.0 MoM cut-off level in Caucasian pregnancies may use 2.5 MoM for Black/African American pregnancies because the risk is similar for the two groups at these specified levels. Another approach is to keep the cut-off level the same in order to maintain the detection rate for ONTDs at the cut-off equivalent (isodetection screening). In the previous example, modifying the cut-off level in Black/African American pregnancies would reduce both detection and false-positive rates. Keeping the cut-off level at 2.0 MoM would maintain both rates. These may be genetic or environmental. Several of these have been discussed already. The optimal time for ONTD screening is 16 to 18 weeks.1.Wald N.J. Cuckle H.S. Brock J.H. Peto R. Polani P.E. Woodford F.P. Maternal serum alpha-fetoprotein measurement in antenatal screening for anencephaly and spina bifida in early pregnancy: Report of the U.K. Collaborative Study on Alpha-fetoprotein in relation to neural tube defects.1:STN:280:DyaE2s3htFCiug%3D%3D69055Lancet. 1977; 1: 1323-1332Google Scholar Before that time, the unaffected and OSB populations have more overlap. This has the effect of decreasing screening performance, particularly in the 14th week of gestation. After 18 weeks of gestation, performance is relatively unchanged but fewer clinical options may be available. Recent large studies have shown that obese women have a 2- to 3-fold increase risk of ONTDs.12.Waller D.K. Mills J.L. Simpson J.L. Cunningham G.C. Conley M.R. Lassman M.R. Are obese women at higher risk for producing malformed offspring.1:STN:280:DyaK2c7mtFSqtw%3D%3D10.1016/S0002-9378(94)70224-1Am J Obstet Gynecol. 1994; 170: 541-548Google Scholar,13.Werler M.M. Louik C. Shapiro S. Mitchell A.A. Prepregnant weight in relation to risk of neural tube defects.1:STN:280:DyaK287osVegtg%3D%3D10.1001/jama.1996.03530380031027JAMA. 1996; 275: 1089-1092Google Scholar The birth prevalence of ONTDs in the Black/African American population is approximately half of the prevalence in Caucasians.14.Greenberg F. James L.M. Oakley Jr, G.P. Estimates of birth prevalence rates of spina bifida in the United States from computer-generated maps.1:STN:280:DyaL3s7ltVSjsQ%3D%3D10.1016/0002-9378(83)91198-5Am J Obstet Gynecol. 1983; 145: 570-573Google Scholar The birth prevalence of ONTDs is increased several-fold in women with IDDM.15.Kucera J. Rate and type of congenital anomalies among offspring of diabetic women.1:STN:280:DyaE38%2Fgs1emsg%3D%3D5095696J Reprod Med. 1971; 7: 73-82Google Scholar,16.Mills J. Baker L. Goldman A. Malformations in infants of diabetic mothers occur before the seventh gestational week: Implications for treatment.1:STN:280:DyaE1M7ntVCnsg%3D%3D10.2337/diab.28.4.292Diabetes. 1979; 28: 292-293Google Scholar Twin pregnancies are known to have AFP levels approximately two times the levels in singleton pregnancies. Distribution parameters for serum AFP measurements have been defined for unaffected twin pregnancies and for twin pregnancies in which one or both of the fetuses are affected with OSB or anencephaly.17.Cuckle H. Wald N. Stevenson J.D. May H.M. Ferguson-Smith M.A. Ward A.M. Maternal serum alpha-fetoprotein screening for open neural tube defects in twin pregnancies.1:STN:280:DyaK3c3lvF2gsw%3D%3D10.1002/pd.1970100202Prenat Diagn. 1990; 10: 71-77Google Scholar The birth prevalence of ONTDs is also higher in twin pregnancies, with one report estimating that an ONTD is 2.28 times more likely than in a singleton pregnancy. Maternal serum AFP cut-off levels for recommendation of amniocentesis should be determined separately for twin pregnancies (generally, cut-off levels fall between 4.0–5.0 MoM). Other factors, such as the acceptability of performing amniocentesis on twin gestations and the difficult options should one affected fetus be identified, should also be considered. Obtaining a second specimen for repeat testing may be beneficial when the initial specimen has a slightly elevated AFP (relative to the screening cut-off level) and the gestational age is early enough to allow time for appropriate follow-up. Most laboratories do not combine results for the two tests but rather use a simple set of rules for interpreting results of repeat testing. Methods for combining the results of the two tests have been published,11.Estimating an individual's risk of having a fetus with open spina bifida and the value of repeat AFP testing. Report of the Fourth UK Collaborative Study on AFP in relation to neural tube defects. J Epidem Comm Health 1982; 36: 87–95.Google Scholar and the laboratory should develop a policy that indicates the method to be used. If the pregnancy was originally misdated and the new gestational age is too early for interpretation (e.g., 14 weeks or earlier), the subsequent sample can be considered to be the first usable sample. Family history of ONTDs may increase the prior risk, depending on the number of affected relatives and the degree of relatedness. Family history can be incorporated i