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Position paper: screening for high-risk pregnancies with maternal serum alpha-fetoprotein MSAFP
Clin Biochem, Vol. 23, pp. 469---476, 1990 Printed in Canada. All rights reserved.
0009-9120/90 $3.00 * .00 Copyright e 1990 The.C,,a,~dian Society of Clinical Chemists.
Position Paper: Screening for High-Risk Pregnancies with Maternal Serum Alpha-fetoprotein MSAFP ANDREW R. MACRAE AND THE MEMBERS OF THE CANADIAN SOCIETY OF CLINICAL CHEMISTS' TASK FORCE ON MSAFP SCREENING Introduction
A
positive association between the risk of open neural tube defects (ONTD) and increased concentrations ofammotic fluid alpha-fetoprotein (AFAFP) was first reported in 1972 (1). An elevated concentration of AFAFP, when combined with an abnormal result of a second amniotic fluid marker, the acetylcholinesterase (ACHE) activity, is highly diagnostic of ONTD (2,3). Since these diagnostic tests require amniocentesis which is itself associated with a risk of fetal loss, the tests on amniotic fluid are only performed in pregnancies where the risk of ONTD is sufficiently high to warrant amniocentesis. Increased maternal serum alpha-fetoprotein (MSAFP) concentrations are also predictive ofONTD, but with a lesser diagnostic accuracy than A F A F P (4). However, MSAFP concentrations, when applied in a carefully structured screening program, will identify those patients who are at sufficient risk of ONTD to support counselling for amniocentesis (5). Depending on the screening cut-off selected, most MSAFP screening programs will identify between 70 and 85% of all ONTD by selecting between 2.5 and 5.0% of the pregnant population for follow-up testing and possible amniocentesis. In the past decade, evidence has accumulated that increased concentrations of MSAFP are also predictive of other adverse pregnancy outcomes and complications during the course of the pregnancy, including ventral wall defects, recent fetal demise, imminent miscarriage, premature delivery and low birth weight (6). It has long been known that advanced maternal age is associated with an increased risk of Down's syndrome. On the basis of this association, it is common practice to offer amniocentesis to all patients with a maternal age greater than 34 years at expected date of confinement. This screening by maternal age detects approximately 15-25% of all
Correspondence: Dr. A.R. MacRae, Department of Laboratory Medicine, Oshawa General Hospital, 24 Alma Street, Oshawa, Ontario, Canada L1G 2B9. Manuscript received July 26, 1990; revised October 10, 1990; accepted October 11, 1990. CLINICAL BIOCHEMISTRY,VOLUME 23, DECEMBER 1990
Down's syndrome pregnancies, depending on the proportion of the total pregnant population aged 35 or greater. Recently, low concentrations of MSAFP have been found to be associated with an increased risk of Down's syndrome (7). The risk of Down's syndrome associated with low MSAFP concentrations can be combined with the risk associated with the maternal age (8). If the combined age/MSAFP risk approaches that of the amniocentesis procedure, amniocentesis may be counselled. The combined age/ MSAFP screen typically identifies 5% of the pregnant population under the age of 35 as candidates for amniocentesis. This high-risk sub-population includes approximately 30% of all Down's syndrome pregnancies (9). The end-product of MSAFP screening is information concerning the risk of an adverse pregnancy outcome. A screening procedure, by design, selects patients at increased risk; however, it must be clearly understood by physicians and the public that the majority of patients with a positive MSAFP screen will have a normal pregnancy outcome. The power of the MSAFP screen in predicting the risk of having an affected pregnancy is dependent on rigorous attention to a wide variety of factors which alter the concentration of MSAFP, and hence the assessment of risk and the appropriate counselling of patients on follow-up procedures. The success of an MSAFP screening program is dependent on the education of, and communication between the various medical specialties that provide care to pregnant patients. The Canadian Society of Clinical Chemists has prepared this Position Paper to support the establishment of MSAFP screening programs of the highest quality. P r e r e q u i s i t e s f o r t h e e s t a b l i s h m e n t of a n MSAFP screening program A screening program can be defined as follows: "The process of identification, among apparently healthy individuals, of those who are sufficiently at risk of a specific disorder to justify a subsequent diagnostic test or procedure."
Screening procedures provide patients with information concerning their risk of a specific disorder, 469
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and a positive screening result is almost invariably a source of anxiety for patients. It is therefore mandatory that patients make an informed choice to enter an MSAFP screening program, and be free to alter their choice at any stage in the process. Some programs record, by means of a signature, the process of informing the patient of the intent and possible outcome of the screen; however, many do not, since the signature does not bind the patient to any consent or refusal to be screened. It is the role of the ordering physician to inform the patient, and it is the responsibility of the local director of the screening program to supply physicians with appropriate educational material concerning the screening protocol, the detection rate and the false-positive rate of the screen. The educational material for both the physician and the public must be furnished in advance of establishing a comprehensive screening program. The false-positive rate of the screen will be determined by the screening cut-off value. The selection of the cut-off value represents a balance between the desire to detect a high proportion of affected individuals and to limit the false-positive rate. In establishing this cut-off value, the availability of resources for follow-up procedures must be considered. Representatives of all participating health care professions should be involved in establishing the cut-off value (clinical biochemists, geneticists, ultrasonographers, obstetricians and family practitioners). The need for frequent interaction between the providers and consumers of screening information requires close communication with a laboratory specifically qualified to provide the screening procedures. The screening laboratory must be prepared to commit substantial resources in support of the screening procedure; in addition to analytical materials and registered technologists specifically trained in the procedures, the laboratory requires a director to oversee the entire laboratory program, a coordinator to provide communication with ordering physicians and referral centres, and clerical support staff skilled in the use of a computer keyboard.
Location of service
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tumour marker does not provide a sufficient basis of knowledge for an MSAFP screening service. The structures for health care delivery in Canada vary from province to province. The standards outlined in this Position Paper should be implemented in the context of the provincial and regional health care systems in existence. The requirements for effective communication with ordering physicians, rapid turnaround time (not to exceed three working days), liaison with genetic counselling services and follow-up testing should define the appropriate locations of service in any provincial setting.
Scope of responsibility of the clinical biochemistry laboratory The scope of laboratory practice for which the laboratory director is responsible includes all activities associated with the laboratory analysis and the provision of the laboratory report. The laboratory report should contain, in addition to other information detailed below, an assessment of the risk of the patient for both ONTD and Down's syndrome, together with interpretive text designed to educate and assist the physician in counselling the patient. The report should represent the current expertise based on the population being screened. It is the responsibility of the physician to interpret this information in light of the patient's individual presentation, history and predisposition. In accepting the responsibility for maintaining expertise in the interpretation of MSAFP results, the laboratory director must also accept a role in the education, where necessary, of physicians, allied medical professionals and the public. The laboratory director should emphasize the factors that affect the laboratory service and the interpretive report, such as the optimum and acceptable gestational ages, the necessity for complete clinical information, the limitation of this test as a screening tool, and the method by which the risk assessment in the laboratory report is calculated.
Required information for the a s s e s s m e n t of risk
The provision of an MSAFP testing service requires a foundation of analytical expertise, implicit in the profession of the clinical biochemist. The advent of new markers for antenatal assessment will further enhance the requirement for a clinical biochemist to direct the analytical component of such a program. Of no less importance is the need for interpretive expertise, efficient transportation systems and good communication with the ordering physicians and local geneticists. A deficiency in any of these components would significantly diminish the overall quality of service. Expertise in the use of AFP as a
An optimal assessment of risk requires that all of the information listed in Table 1 be supplied with the sample. A requisition specifically designed to gather this information should be distributed throughout the catchment area. It is preferable that the requisition should also contain information on the appropriate sample requirements for the assay, together with transportation instructions and the address and telephone number of the laboratory performing the assay. Where more than one screening centre exists within a province, a common provincial requisition should be designed for patient-specific information, with the laboratory-specific information added by each assaying laboratory.
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• • • • • • • • • • • • • • •
TABLE 1 Required Patient Information patient demographic data, sufficient to uniquely identify the patient; date of birth; racial origin (10,11); diabetic status (12); multiple gestation status of the current pregnancy (13); last menstrual period (LMP) date; gestational age as determined by the ordering physician; date for which the gestational age applies; method by which the gestational age has been determined; specimen date and specimen type (serum or amniotic fluid); patient's weight in specified units 110,14); any specimen numbers assigned; existence and nature (high or low) of any previous abnormality in AFP concentration in the current pregnancy; family history of any affected pregnancies (15); results of any chromosome studies in the current pregnancy.
A n a l y t i c a l considerations Monoclonal antibodies, employed in non-radioisotopic assays, provide the analyst with methods ensuring stable results, and enable comparable results to be obtained in separate laboratories across wide geographical areas. These assay characteristics allow the individual laboratory directors to adopt a method t h a t will reduce inconsistencies between laboratories. Polyclonal assays may also be considered if they are accompanied by rigorous attention to the consistency of reagent lots over an extended period of time. Similarly, the major components of the assay procedure, such as the number of replicate assays on each specimen, should be consistent between laboratories. It is strongly preferred t h a t any centre performing amniocentesis should perform both the MSAFP and AFAFP assays, and t h a t a single assay method should be employed for both these analyses. The selected assay must be sufficiently precise to support the decisions made on the basis of MSAFP results. In practice, this means t h a t a value of 10 ~g/L should be significantly distinct from both 9 ~g/L and 11 ~g/L. Laboratories must attain a between-assay coefficient of variation (precision) of 5% or less at all levels greater than 10 ~g/L, or they must attain the same precision through replication of critical samples. AFP concentrations are expressed in the SI units of ~g/L (serum) or mg/L (amniotic fluid), and also as the multiple of the median (MoM) value of the reference population for a given gestational age, If the correct gestational age (and hence the correct
CLINICALBIOCHEMISTRY,VOLUME23, DECEMBER 1990
median) is assigned to a sample, the MoM result will be independent of the gestational age. The risk assessments in the literature for both open spina bifida (OSB) and Down's syndrome are based on MoM values for reference populations comprising the initial samples from the Caucasian, non-diabetic, singleton-pregnancy population. The median value is used rather than the mean, in order to obviate the skew to elevated levels t h a t would otherwise be present. Medians should be recalculated with reasonable frequency in each laboratory. A common set oi median values (e.g., one for each week of gestational age) may be adopted by laboratories within a region that use the same method, provided t h a t this practice enhances the consistency of the reported MoM results. Since the confidence of the median calculations increases with the number of samples in the population, a central data base will be required for most regions or provinces. In order to monitor the consistency of service, a quality control program is required t h a t entails frequent assessment of proficiency, not only of analytical procedures, but also of the assessment of risk in the final reports issued. A m i n i m u m bimonthly challenge of a number of specimens, including both maternal serum and amniotic fluid, is required. The enrollment of all laboratories in a national or international proficiency survey is strongly recommended.
Factors to be employed in the interpretation MSAFP concentrations are affected by a number of factors that are population specific. It is essential t h a t these factors be applied uniformly in the assessment of risk (16). There must also be agreement across medical disciplines on the factors to be used in assigning risk. A consensus process is suggested as the method of attaining this degree of consistency, and the expertise of all appropriate medical disciplines should be sought in the process. The factors that must be common include: • method of expressing gestational age, such as completed weeks, derived from the gestational age assigned by the ordering physician; • r a n g e of gestational ages for which an interpretation is made; • median correction factors for different patient racial origins, if separate log-Gaussian population distributions are not determined; • median correction factor for the insulin-dependent diabetic patient population, if a separate log-Gaussian population distribution is not determined; • maternal weight correction formula for the MoM value; • log-Gaussian population curve parameters for the unaffected, OSB, anencephalic, twin gestation and Down's syndrome populations;
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• b a c k g r o u n d p r e v a l e n c e of ONTD and Down's syndrome in the population; • ONTD r a c i a l prevalence factors for adjusting the background prevalence to that of the different patient racial origins; • ONTD insulin-dependent diabetes mellitus prevalence factor for adjusting the background prevalence to that of the insulin-dependent diabetic population; • any other a priori r i s k f a c t o r , such as previous family history; • OSB r i s k s t h a t discriminate between normal, moderately elevated and high-risk patients, for both initial samples and repeat samples, when appropriate; • method of e x p r e s s i n g D o w n ' s s y n d r o m e risk, as either the risk at term or the risk at midtrimester; • range of maternal ages and MSAFP values for which a Down's syndrome risk will be calculated; • m e t h o d of a s s e s s i n g r i s k in m u l t i p l e gestation p r e g n a n c i e s , and reporting the risk of OSB and Down's syndrome in these patients; • p r o t o c o l for recommending non-directive consideration of further assessment of risk through subsequent procedures.
Screening cut-off values A risk of Down's syndrome at term of 1:385 (that of a 35.5-year-old woman) or greater is a widely accepted standard of practice for recommending amniocentesis. In contrast, the selection of the screening cut-off values for OSB must be appropriate for the local setting. (It is appropriate to consider only the risk of OSB in setting the cut-off value, since anencephaly causes substantial elevations in MSAFP that are easily detected.) The screening cut-off for OSB establishes a balance between the desired detection rate for OSB and the burden of the false-positive rate t h a t is associ-
ET AL.
ated with t h a t detection rate. Therefore, the falsepositive rate should be selected bearing in mind its effect on available resources, namely, repeat assays, genetic consultations, level II ultrasound examinations and amniocentesis. The detection/'ate should be sufficiently high to support the public expectation of a screening program, and the a t t e n d a n t reduction of health care expenditures t h a t are associated with screening protocols. Examples of wellestablished perinatal screening programs are those for phenylketonuria (PKU) and hypothyroidism. For MSAFP screening, the MoM values of 2.00 and 2.50 are common cut-off values. Any value between these limits can be justified based on the considerations indicated above. The local prevalence of OSB will not affect the false-positive rate, or the detection rate (the fraction of the affected pregnancies detected). However, the prevalence will determine the odds of being affected, given a positive screening result. Before a cut-off is implemented, these odds at the cut-off (as distinct from the odds at all levels above the cut-off) should be calculated, and they should be appropriate for the nature of the recommendations for follow-up procedures. As an example, i f a repeat testing protocol is used, a significant fraction of those initially determined as screen positive will revert to negative. Similarly, the local reliance on the noninvasive procedure of level II ultrasound will influence the acceptability of the odds at the cut-off. For these reasons, the risk at the cut-off for OSB need not be the same as t h a t used for Down's syndrome. As a guide for the selection of a cut-off, Table 2 provides the approximate projected relationships between MoM cut-offs, the predicted false-positive rate and detection rate, and the odds of being affected given a positive screening result (OAPR) for three levels of OSB prevalence.
Contents of the patient report The patient report should contain all of the information used in the assessments of risk, such t h a t
TABLE 2 Projected MSAFP Screening Parameters at Various OSB Cut-offs MoM Cut-off Value 2.00 2.10 2.20 2.30 2.40 2.50
Prevalence of OSB 1:2000 1:1000 1:500
FalsePositive Rate (c2)
Detection Rate (%}
4.5 3.9 3.4 2.9 2.6 2.5
85 83 80 77 74 70
OPAR* at the Cut-off 1:1500 1:1180 1:940 1:750 1:600 1:480
1:750 1:590 1:470 1:375 1:300 1:240
1:375 1:295 1:235 1:185 1:150 1:120
*Odds of being affected, given a positive result.
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SCREENING FOR HIGH-RISKPREGNANCIESWITH MSAFP the ordering physician will be aware of the data elements used in the interpretation. This requires t h a t the patient report must contain all of the information listed in Table 1. In addition, the report must contain all calculated values and limits: • patient's calculated age at expected date of confinement; • gestational age used in the calculations, and the method by which it was derived; • AFP result, in SI units (~g/L for serum; mg/L for amniotic fluid) expressed to one decimal place, and in MoM units expressed to two decimal places; • an indication if the MoM unit result has been adjusted for patient weight, racial origin and diabetic status; • OSB screen cut-off value in MoM units; • calculated patient-specific risk of OSB; • absolute risk of Down's syndrome, and the ageequivalent risk expressed in years; • date of the report, the identity of the individual responsible for the report, and the identity and telephone number of the assaying laboratory. If assumptions are made in instances where date elements have not been obtained, then both the data used, and the fact t h a t these data were not specified should be noted in the report; (e.g., a singleton pregnancy should be assumed if not otherwise specified, and this assumption must be indicated). There should be agreement on the factor values to be employed in the absence of patientspecific data, for example, the general population prevalence should be assumed in the absence of information on the racial origin and/or the patient's diabetic status. If the gestational age as of the sample date, and the patient's date of birth are not provided, then the report should withhold the MSAFP screening interpretation until these essential data are supplied.
Central data base Where more t h a n one laboratory is offering an MSAFP service within a province, each laboratory should make their data available to a central data base in the province (16). By inspection of these data, consistency of service between laboratories will be enhanced, and the collective data will assist in updating the factors t h a t are used in interpretation. The data base should include the results of follow-up procedures and pregnancy outcome data which can be supplied by the genetic centres and physicians involved. To enable tracking of patients within the province, an identical method should be used for assigning unique patient identifications. The use of a common interpretive computer program is required for maintaining a consistent service and creating the central data base, which could encompass the data from several or all provinces.
CLINICALBIOCHEMISTRY,VOLUME23, DECEMBER 1990
Protocol for r e c o m m e n d a t i o n of confirmatory procedures As indicated in the preceding sections, a common protocol for the recommendation of confirmatory tests should be established. This protocol should be based on a consensus of the laboratory and medical professionals involved in the care of pregnant patients within the region. As a guide to such a consensus, several MSAFP screening programs for ONTD recommend a repeat MSAFP sample in situations where: (i)the risk of OSB on the basis of MSAFP is moderate (for example, above the normal risk cut-off but less than 1:120); and (ii) there is sufficient time for a second sample, preferably one week later (i.e., the gestational age at the time of the initial sample is 18 completed weeks or less}. The benefits of a repeat MSAFP sample one week following a moderately elevated result include: a stimulus and opportunity to reassess the gestational age of the patient; an enhanced separation of the affected and unaffected populations through regression towards their respective medians; an opportunity to counsel the patient prior to offering more invasive follow-up procedures; a reduction in health care expenses through enhanced exclusion of the unaffected population (up to one third of patients with first sample elevations will have a normal second sample as a result of the above factors). A repeat MSAFP sample is not recommended for: (i) low MSAFP values t h a t indicate an increased risk of Down's syndrome (17}; (ii) very elevated samples t h a t indicate a risk of OSB that exceeds 1:120; and (iii)elevated MSAFP levels at a gestational age greater t h a n 18 weeks. In these instances, level II ultrasound and amniocentesis are recommended. A level I ultrasound examination, to confirm gestational age, is frequently recommended for any patient with an abnormality in MSAFP levels, where an ultrasound has not been performed at a gestational age greater than five weeks. A level II ultrasound, to identify fetal anomalies, is often recommended for any patient with a persistent moderately elevated MSAFP, and any patient considering amniocentesis on the basis of an elevated MSAFP result.
Confirmatory procedures for ONTD following a high M S A F P The confirmatory tests available for detecting ONTD are the combination of level II ultrasound with the analyses of AFAFP and amniotic fluid AChE activity. Collaborative studies recommend t h a t the combination of level II ultrasonography with amniocentesis optimizes the diagnosis of ONTD (18). A negative level II ultrasound in the presence of only a moderate (< 1:400) risk of OSB on the basis
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of MSAFP may be considered by the physician as sufficient evidence against the existence of an ONTD to forego further assessment (191. Ultrasonographers should be aware t h a t physicians and patients might substitute this procedure for the more definitive amniotic fluid assays; therefore, ultrasonographers might be advised to indicate the relative certainty of their findings. If amniocentesis is selected, a normal AFAFP concentration (<2.00 MoM) essentially rules out the presence of ONTD (18). The calculation of an assessment of risk for OSB on the basis of AFAFP concentrations requires the same attention to analytical procedures, clinical information and interpretation as for MSAFP measurement. Because sufficient data are not available at present, an adjustment of the median value and prevalence of ONTD is not performed on the basis of racial origin or diabetic status. Maternal weight and multiple gestation status have no established effect on AFAFP concentrations. With these exceptions, the interpretation of AFAFP concentrations requires the same consistency of factors between assaying laboratories as outlined for MSAFP screening. Both of these procedures should be performed in each screening laboratory. In the presence of a moderately elevated AFAFP result (->2.00 MoM), AChE measurement significantly adds to the diagnostic certainty of the AFAFP test (18). As with any specialized diagnostic laboratory technique, AChE analysis requires constant attention. Since it is reserved for the rare instances of an elevated AFAFP level, it is strongly recommended t h a t the AChE assay be performed at one centre for a province. It follows that efficient means of transportation and communication be established between the AFAFP laboratories and the single AChE laboratory. The laboratories that refer specimens to a common AChE testing centre should adopt a common protocol for selection of samples for AChE testing.
Confirmatory procedures for D o w n ' s syndrome following a l o w M S A F P The confirmatory test for Down's syndrome is amniocentesis followed by chromosome analysis. There is no association between ONTD and Down's syndrome. Furthermore, since these two disorders have opposite effects on MSAFP concentrations, a patient determined to be at risk of Down's syndrome on the basis of a low MSAFP will have a risk of ONTD t h a t is far below the background population risk. The question arises, should AFAFP analyses be performed on samples from patients at risk of Down's syndrome either on the basis of age alone, or age in combination with MSAFP? The prevalence of ONTD in these patients will be quite low, and the diagnostic potential of any test is affected by the prevalence of the disorder in the population tested. The lower the prevalence, the less the likelihood of the screenpositive patient having ONTD; hence the diagnostic potential of the test is diminished (20). For this 474
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reason it is recommended that, if AFAFP measurements are performed in these patients, a positive AFAFP result must be combined with an AChE analysis before counselling on the risk of ONTD. Laboratories may initially choose to accept amniotic fluid samples from patients at risk for Down's syndrome on the basis of advanced maternal age only, for the purpose of establishing median values. Samples from patients at risk of Down's syndrome on the basis of a low MSAFP must not be included in any amniotic fluid median study, since the median values will be slightly negatively biased.
N e w markers for Down's s y n d r o m e In recent years, two additional maternal serum markers for the risk of Down's syndrome have been reported: unconjugated estriol (uE3) (21), and human chorionic gonadotrophin (hCG) (22}. Preliminary indications are t h a t these mid-trimester markers, when combined with maternal age and MSAFP, will enable the detection of approximately 60% of Down's syndrome pregnancies with a falsepositive rate of less t h a n 5~ (23). This detection rate is approximately twice t h a t seen with maternal age and MSAFP in combination, and approximately triple the rate with age as the only screening variable. Of these four risk indices, an increased concentration of mid-trimester maternal serum hCG is the most powerful single indicator of Down's syndrome risk. Conscientious application of current methods for serum hCG would enable adequate measurement of this marker. In contrast, uE3 levels in mid-trimester are low, and assays currently available for third-trimester estriol concentrations are not sufficiently precise at the mid-trimester levels. The addition of uE3 to a screening protocol has only a small beneficial effect on the detection rate; however, it has a significant effect on reducing the false-positive rate of the combined "triple marker" screen (e.g., from 6.7c~ to 4.7~1 (23). Whereas the additional markers for Down's syndrome risk have great promise, it is not recommended t h a t they be incorporated into a newly established MSAFP screening program. The major reasons for this are:
The lack of sufficiently precise methods for uE3; Because of its beneficial effect on the false-positive rate, uE3 should be incorporated into a screening program at the same time as hCG. The currently available uE3 methods must be optimized for the mid-trimester levels. As with AFP, the methods for uE3 (and hCG) must be comparable between laboratories in order to achieve a consistent performance.
The concept of risk volatility; Risk is a difficult concept for physicians to convey to their patients. For each of the serum markers, CLINICAL BIOCHEMISTRY, VOLUME 23, DECEMBER 1990
SCREENING FOR HIGH-RISK PREGNANCIES WITH MSAFP the c a l c u l a t i o n of r i s k is u l t i m a t e l y d e p e n d e n t on the g e s t a t i o n a l age of t h e p a t i e n t . Since g e s t a t i o n a l age is a c o m m o n factor in all t h r e e r i s k d e t e r m i n a n t s , a l t e r a t i o n s in t h e assessed g e s t a t i o n a l age will s u b s t a n t i a l l y c h a n g e t h e c a l c u l a t e d c o m b i n e d risk. W i t h t h r e e m a r k e r s , a difference of two w e e k s in g e s t a t i o n a l a g e could c h a n g e the r i s k e s t i m a t e tenfold. W i t h the single m a r k e r of M S A F P , t h e effect of g e s t a t i o n a l age on r i s k is less t h a n twofold. T h e c o m p o u n d e d effect of g e s t a t i o n a l age w i t h t h r e e m a r k e r s is r e f e r r e d to as " r i s k v o l a t i l i t y . " In fact, the p o t e n t i a l for s u b s t a n t i a l c h a n g e in the r i s k e s t i m a t e b a s e d on the i n f o r m a t i o n supplied is an indication of the p o w e r of t h e screen, if the i n f o r m a tion supplied is correct. P h y s i c i a n s m u s t be a w a r e of t h e s e concepts before the triple m a r k e r s are employed. T h e s e issues which c u r r e n t l y i m p e d e the use of the new m a r k e r s can be a d d r e s s e d t h r o u g h r e s e a r c h a n d education. In t h e i n t e r i m , new studies will be p u b l i s h e d w h i c h will aid in p r e d i c t i n g t h e economic b e n e f i t s of triple m a r k e r s c r e e n i n g for D o w n ' s syndrome.
3. 4.
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8. 9.
Quality assurance It is m a n d a t o r y t h a t a s c r e e n i n g p r o g r a m , which by design is offered to a l a r g e p o p u l a t i o n , should be subjected to a n ongoing q u a l i t y a s s u r a n c e e v a l u a tion. T h e r e are s e v e r a l p a r a m e t e r s of the s c r e e n i n g p r o g r a m t h a t should be assessed: the false-positive rate; p r e g n a n c y o u t c o m e s in t h e s c r e e n e d a n d uns c r e e n e d population; t h e u t i l i z a t i o n of follow-up resources; t h e level of c o m p r e h e n s i o n of the s c r e e n i n g p r o g r a m a m o n g its c o n s u m e r s ; the d e g r e e to which new p a r a m e t e r s are successfully i n t e g r a t e d into the s c r e e n i n g protocol. An M S A F P s c r e e n i n g p r o g r a m r e q u i r e s a subs t a n t i a l a n d c o n t i n u o u s c o m m i t m e n t by t h e laboratory director. T h e q u a l i t y a s s u r a n c e of the p r o g r a m r e q u i r e s t h a t the l a b o r a t o r y director b e c o m e a fully p a r t i c i p a t i n g m e m b e r of the h e a l t h care d e l i v e r y team.
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Acknowledgements T h e C a n a d i a n Society of Clinical C h e m i s t s g r a t e fully a c k n o w l e d g e s the a s s i s t a n c e of Dr. J. E. H a d dow, Mr. G. E. P a l o m a k i a n d Dr. G.J. K n i g h t f r o m the F o u n d a t i o n for Blood R e s e a r c h , S c a r b o r o u g h , Maine, in r e v i e w i n g this paper.
References 1. Brock DJH, Sutcliffe RG. Alpha-fetoprotein in the antenatal diagnosis of anencephaly and spina bifida. Lancet 1972; 2: 197-99. 2. Milunsky A. Prenatal detection of neural tube defects CLINICAL BIOCHEMISTRY, VOLUME 23, DECEMBER 1990
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18.
VI. Experience with 20,000 pregnancies. JAMA 1980; 244: 2731-35. Crandall BF, Matsumoto M. Routine amniotic fluid alphafetoprotein assay: experience with 40,000 pregnancies. A m J Med Genet 1986; 24: 143-49. Seller MJ, Singer JD, Coltart TM, Campbell S. Maternal serum alpha-fetoprotein levels and prenatal diagnosis of neural tube defects. Lancet 1974; 1: 42829. Estimating an individual's risk of having a fetus with open spina bifida and the value of repeat alphafetoprotein testing; Fourth Report of the UK Collaborative Study on Alpha-fetoprotein in Relation to Neural Tube Defects. J Epidemiol Commun Health 1982; 36: 87-95. Burton BK. Outcome of pregnancy in patients with unexplained elevated or low levels of maternal serum alpha-fetoprotein. Obstet Gynecol 1988; 72: 709-13. Merkatz IR, Nitowsky HM, Macri JN, Johnson WE. An association between low maternal serum a-fetoprotein and fetal chromosome abnormalities. A m J Obstet Gynecol 1984; 148: 886-94. Cuckle HS, Wald NJ, Lindenbaum RH. Maternal serum alpha-fetoprotein measurement: a screening test for Down syndrome. Lancet 1984; 1: 926-29. Cuckle HS, Wald NJ, Thompson SG. Estimating a woman's risk of having a pregnancy associated with Down's syndrome using her age and serum alphafetoprotein level. Br J Obstet Gynaecol 1987; 94: 387-402. Crandall BF, Lebherz TB, Schroth PC, Matsumoto M. Alpha-fetoprotein concentrations in maternal serum: relation to race and body weight. Clin Chem 1983; 29: 531-33. Johnson AM, Palomaki GE, Haddow JE. Maternal serum a-fetoprotein levels in black and white women with fetal open spina bifida: a United States Collaborative Study. Am J Obstet Gynecol 1990; 162: 328-31. Milunsky A, Alpert E, Kitzmiller JL, Younger MD, Neff RK. Prenatal diagnosis of neural tube defects VIII. The importance of alpha-fetoprotein screening in diabetic women. Am J Obstet Gy~zecol 1982; 142: 1030-33. Cuckle H, Wald N, Stevenson JD, et al. Maternal serum alpha-fetoprotein screening for open neural tube defects in twin pregnancies. Prerzat Diagn 1990; 10: 71-77. Johnson AM, Palomaki GE, Haddow JE. The effect of adjusting maternal serum ~-fetoprotein levels for maternal weight in pregnancies with fetal open spina bifida. A United States Collaborative Study. A m J Obstet Gynecol 1990; 163: 9-11. Hall JG, Friedman JM, Kenna BA, Popkin J, Jawanda M, Arnold W. Clinical, genetic and epidemiological factors in neural tube defects. A m J Hum Genet 1988; 43: 827-37. Evans MI, Belsky R, Greb A, Dvorin E, Drugan A. Wide variation in maternal serum alpha-fetoprotein reports in one metropolitan area: concerns for the quality of prenatal testing. Obstet Gynecol 1988; 72: 342-45. Cuckle HS, Wald NJ, Nanchahal K, Densem J. Repeat maternal serum alpha-fetoprotein testing in antenatal screening programmes for Down's syndrome. Br J Obstet Gynaecol 1989; 96: 5 2 ~ 0 . Wald N, Cuckle H, Nanchahal K. Amniotic fluid acetylcholinesterase measurement in the prenatal diagnosis of open neural tube defects; Second Report of 475
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19.
20.
21.
22.
23.
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the Collaborative Acetylcholinesterase Study. Prenat Diagn 1989; 9: 813-29. Richards DS, Seeds JW, Katz VL, Lingley LH, A1bright SG, Cefalo RC. Elevated maternal serum alpha-fetoprotein with normal ultrasound: is amniocentesis always appropriate? A review of 26,069 screened patients. Obstet Gynecol 1988; 71: 203-207. Allen LC, Doran TA, Miskin M, Rudd NL, Benzie RJ, Sheffield I.J. Ultrasound and amniotic fluid alphafetoprotein in the prenatal diagnosis of spina bifida. Obstet Gynecol 1982; 60: 169-73. Canick JA, Knight GJ, Palomaki GE, Haddow JE, Cuckle HS, Wald NJ. Low second trimester maternal serum unconjugated oestriol in pregnancies with Down's syndrome. Br J Obstet Gynaecol 1988; 95: 330-33. Bogart MH, Pandian MR, Jones OW. Abnormal maternal serum chorionic gonadotrophin levels in pregnancies with fetal chromosome abnormalities. Prenat Diagn 1987; 7: 623-30. Wald NJ, Cuckle HS, Densem JW, et al. Maternal serum screening for Down's syndrome in early pregnancy. Br Med J 1988; 297: 883-87.
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T h e C a n a d i a n S o c i e t y o f Clinical C h e m i s t s ' T a s k F o r c e on M S A F P S c r e e n i n g
Chairman A. R. MacRae, PhD, FCACB (Oshawa)
Committee Members B. J. Cadeau, PhD, FCACB (Saint John) A. Drobnies, PhD, FCACB (Vancouver) T. E. England, PhD, FCACB (Etobicoke) M. L. Givner, PhD, FCACB (Halifax) A. W. Luxton, MD, FRCPC, FCACB (Hamilton) M. L. Salkie, MB, BChir, FRCPC, FCACB (Edmonton)
Associates K. L. Massey, MD, FRCPC, FCACB (Saskatoon) L. E. Seargeant, PhD, FCACB (Winnipeg)
CLINICALBIOCHEMISTRY,VOLUME 23, DECEMBER 1990
0009-9120/90 $3.00 * .00 Copyright e 1990 The.C,,a,~dian Society of Clinical Chemists.
Position Paper: Screening for High-Risk Pregnancies with Maternal Serum Alpha-fetoprotein MSAFP ANDREW R. MACRAE AND THE MEMBERS OF THE CANADIAN SOCIETY OF CLINICAL CHEMISTS' TASK FORCE ON MSAFP SCREENING Introduction
A
positive association between the risk of open neural tube defects (ONTD) and increased concentrations ofammotic fluid alpha-fetoprotein (AFAFP) was first reported in 1972 (1). An elevated concentration of AFAFP, when combined with an abnormal result of a second amniotic fluid marker, the acetylcholinesterase (ACHE) activity, is highly diagnostic of ONTD (2,3). Since these diagnostic tests require amniocentesis which is itself associated with a risk of fetal loss, the tests on amniotic fluid are only performed in pregnancies where the risk of ONTD is sufficiently high to warrant amniocentesis. Increased maternal serum alpha-fetoprotein (MSAFP) concentrations are also predictive ofONTD, but with a lesser diagnostic accuracy than A F A F P (4). However, MSAFP concentrations, when applied in a carefully structured screening program, will identify those patients who are at sufficient risk of ONTD to support counselling for amniocentesis (5). Depending on the screening cut-off selected, most MSAFP screening programs will identify between 70 and 85% of all ONTD by selecting between 2.5 and 5.0% of the pregnant population for follow-up testing and possible amniocentesis. In the past decade, evidence has accumulated that increased concentrations of MSAFP are also predictive of other adverse pregnancy outcomes and complications during the course of the pregnancy, including ventral wall defects, recent fetal demise, imminent miscarriage, premature delivery and low birth weight (6). It has long been known that advanced maternal age is associated with an increased risk of Down's syndrome. On the basis of this association, it is common practice to offer amniocentesis to all patients with a maternal age greater than 34 years at expected date of confinement. This screening by maternal age detects approximately 15-25% of all
Correspondence: Dr. A.R. MacRae, Department of Laboratory Medicine, Oshawa General Hospital, 24 Alma Street, Oshawa, Ontario, Canada L1G 2B9. Manuscript received July 26, 1990; revised October 10, 1990; accepted October 11, 1990. CLINICAL BIOCHEMISTRY,VOLUME 23, DECEMBER 1990
Down's syndrome pregnancies, depending on the proportion of the total pregnant population aged 35 or greater. Recently, low concentrations of MSAFP have been found to be associated with an increased risk of Down's syndrome (7). The risk of Down's syndrome associated with low MSAFP concentrations can be combined with the risk associated with the maternal age (8). If the combined age/MSAFP risk approaches that of the amniocentesis procedure, amniocentesis may be counselled. The combined age/ MSAFP screen typically identifies 5% of the pregnant population under the age of 35 as candidates for amniocentesis. This high-risk sub-population includes approximately 30% of all Down's syndrome pregnancies (9). The end-product of MSAFP screening is information concerning the risk of an adverse pregnancy outcome. A screening procedure, by design, selects patients at increased risk; however, it must be clearly understood by physicians and the public that the majority of patients with a positive MSAFP screen will have a normal pregnancy outcome. The power of the MSAFP screen in predicting the risk of having an affected pregnancy is dependent on rigorous attention to a wide variety of factors which alter the concentration of MSAFP, and hence the assessment of risk and the appropriate counselling of patients on follow-up procedures. The success of an MSAFP screening program is dependent on the education of, and communication between the various medical specialties that provide care to pregnant patients. The Canadian Society of Clinical Chemists has prepared this Position Paper to support the establishment of MSAFP screening programs of the highest quality. P r e r e q u i s i t e s f o r t h e e s t a b l i s h m e n t of a n MSAFP screening program A screening program can be defined as follows: "The process of identification, among apparently healthy individuals, of those who are sufficiently at risk of a specific disorder to justify a subsequent diagnostic test or procedure."
Screening procedures provide patients with information concerning their risk of a specific disorder, 469
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and a positive screening result is almost invariably a source of anxiety for patients. It is therefore mandatory that patients make an informed choice to enter an MSAFP screening program, and be free to alter their choice at any stage in the process. Some programs record, by means of a signature, the process of informing the patient of the intent and possible outcome of the screen; however, many do not, since the signature does not bind the patient to any consent or refusal to be screened. It is the role of the ordering physician to inform the patient, and it is the responsibility of the local director of the screening program to supply physicians with appropriate educational material concerning the screening protocol, the detection rate and the false-positive rate of the screen. The educational material for both the physician and the public must be furnished in advance of establishing a comprehensive screening program. The false-positive rate of the screen will be determined by the screening cut-off value. The selection of the cut-off value represents a balance between the desire to detect a high proportion of affected individuals and to limit the false-positive rate. In establishing this cut-off value, the availability of resources for follow-up procedures must be considered. Representatives of all participating health care professions should be involved in establishing the cut-off value (clinical biochemists, geneticists, ultrasonographers, obstetricians and family practitioners). The need for frequent interaction between the providers and consumers of screening information requires close communication with a laboratory specifically qualified to provide the screening procedures. The screening laboratory must be prepared to commit substantial resources in support of the screening procedure; in addition to analytical materials and registered technologists specifically trained in the procedures, the laboratory requires a director to oversee the entire laboratory program, a coordinator to provide communication with ordering physicians and referral centres, and clerical support staff skilled in the use of a computer keyboard.
Location of service
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tumour marker does not provide a sufficient basis of knowledge for an MSAFP screening service. The structures for health care delivery in Canada vary from province to province. The standards outlined in this Position Paper should be implemented in the context of the provincial and regional health care systems in existence. The requirements for effective communication with ordering physicians, rapid turnaround time (not to exceed three working days), liaison with genetic counselling services and follow-up testing should define the appropriate locations of service in any provincial setting.
Scope of responsibility of the clinical biochemistry laboratory The scope of laboratory practice for which the laboratory director is responsible includes all activities associated with the laboratory analysis and the provision of the laboratory report. The laboratory report should contain, in addition to other information detailed below, an assessment of the risk of the patient for both ONTD and Down's syndrome, together with interpretive text designed to educate and assist the physician in counselling the patient. The report should represent the current expertise based on the population being screened. It is the responsibility of the physician to interpret this information in light of the patient's individual presentation, history and predisposition. In accepting the responsibility for maintaining expertise in the interpretation of MSAFP results, the laboratory director must also accept a role in the education, where necessary, of physicians, allied medical professionals and the public. The laboratory director should emphasize the factors that affect the laboratory service and the interpretive report, such as the optimum and acceptable gestational ages, the necessity for complete clinical information, the limitation of this test as a screening tool, and the method by which the risk assessment in the laboratory report is calculated.
Required information for the a s s e s s m e n t of risk
The provision of an MSAFP testing service requires a foundation of analytical expertise, implicit in the profession of the clinical biochemist. The advent of new markers for antenatal assessment will further enhance the requirement for a clinical biochemist to direct the analytical component of such a program. Of no less importance is the need for interpretive expertise, efficient transportation systems and good communication with the ordering physicians and local geneticists. A deficiency in any of these components would significantly diminish the overall quality of service. Expertise in the use of AFP as a
An optimal assessment of risk requires that all of the information listed in Table 1 be supplied with the sample. A requisition specifically designed to gather this information should be distributed throughout the catchment area. It is preferable that the requisition should also contain information on the appropriate sample requirements for the assay, together with transportation instructions and the address and telephone number of the laboratory performing the assay. Where more than one screening centre exists within a province, a common provincial requisition should be designed for patient-specific information, with the laboratory-specific information added by each assaying laboratory.
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• • • • • • • • • • • • • • •
TABLE 1 Required Patient Information patient demographic data, sufficient to uniquely identify the patient; date of birth; racial origin (10,11); diabetic status (12); multiple gestation status of the current pregnancy (13); last menstrual period (LMP) date; gestational age as determined by the ordering physician; date for which the gestational age applies; method by which the gestational age has been determined; specimen date and specimen type (serum or amniotic fluid); patient's weight in specified units 110,14); any specimen numbers assigned; existence and nature (high or low) of any previous abnormality in AFP concentration in the current pregnancy; family history of any affected pregnancies (15); results of any chromosome studies in the current pregnancy.
A n a l y t i c a l considerations Monoclonal antibodies, employed in non-radioisotopic assays, provide the analyst with methods ensuring stable results, and enable comparable results to be obtained in separate laboratories across wide geographical areas. These assay characteristics allow the individual laboratory directors to adopt a method t h a t will reduce inconsistencies between laboratories. Polyclonal assays may also be considered if they are accompanied by rigorous attention to the consistency of reagent lots over an extended period of time. Similarly, the major components of the assay procedure, such as the number of replicate assays on each specimen, should be consistent between laboratories. It is strongly preferred t h a t any centre performing amniocentesis should perform both the MSAFP and AFAFP assays, and t h a t a single assay method should be employed for both these analyses. The selected assay must be sufficiently precise to support the decisions made on the basis of MSAFP results. In practice, this means t h a t a value of 10 ~g/L should be significantly distinct from both 9 ~g/L and 11 ~g/L. Laboratories must attain a between-assay coefficient of variation (precision) of 5% or less at all levels greater than 10 ~g/L, or they must attain the same precision through replication of critical samples. AFP concentrations are expressed in the SI units of ~g/L (serum) or mg/L (amniotic fluid), and also as the multiple of the median (MoM) value of the reference population for a given gestational age, If the correct gestational age (and hence the correct
CLINICALBIOCHEMISTRY,VOLUME23, DECEMBER 1990
median) is assigned to a sample, the MoM result will be independent of the gestational age. The risk assessments in the literature for both open spina bifida (OSB) and Down's syndrome are based on MoM values for reference populations comprising the initial samples from the Caucasian, non-diabetic, singleton-pregnancy population. The median value is used rather than the mean, in order to obviate the skew to elevated levels t h a t would otherwise be present. Medians should be recalculated with reasonable frequency in each laboratory. A common set oi median values (e.g., one for each week of gestational age) may be adopted by laboratories within a region that use the same method, provided t h a t this practice enhances the consistency of the reported MoM results. Since the confidence of the median calculations increases with the number of samples in the population, a central data base will be required for most regions or provinces. In order to monitor the consistency of service, a quality control program is required t h a t entails frequent assessment of proficiency, not only of analytical procedures, but also of the assessment of risk in the final reports issued. A m i n i m u m bimonthly challenge of a number of specimens, including both maternal serum and amniotic fluid, is required. The enrollment of all laboratories in a national or international proficiency survey is strongly recommended.
Factors to be employed in the interpretation MSAFP concentrations are affected by a number of factors that are population specific. It is essential t h a t these factors be applied uniformly in the assessment of risk (16). There must also be agreement across medical disciplines on the factors to be used in assigning risk. A consensus process is suggested as the method of attaining this degree of consistency, and the expertise of all appropriate medical disciplines should be sought in the process. The factors that must be common include: • method of expressing gestational age, such as completed weeks, derived from the gestational age assigned by the ordering physician; • r a n g e of gestational ages for which an interpretation is made; • median correction factors for different patient racial origins, if separate log-Gaussian population distributions are not determined; • median correction factor for the insulin-dependent diabetic patient population, if a separate log-Gaussian population distribution is not determined; • maternal weight correction formula for the MoM value; • log-Gaussian population curve parameters for the unaffected, OSB, anencephalic, twin gestation and Down's syndrome populations;
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• b a c k g r o u n d p r e v a l e n c e of ONTD and Down's syndrome in the population; • ONTD r a c i a l prevalence factors for adjusting the background prevalence to that of the different patient racial origins; • ONTD insulin-dependent diabetes mellitus prevalence factor for adjusting the background prevalence to that of the insulin-dependent diabetic population; • any other a priori r i s k f a c t o r , such as previous family history; • OSB r i s k s t h a t discriminate between normal, moderately elevated and high-risk patients, for both initial samples and repeat samples, when appropriate; • method of e x p r e s s i n g D o w n ' s s y n d r o m e risk, as either the risk at term or the risk at midtrimester; • range of maternal ages and MSAFP values for which a Down's syndrome risk will be calculated; • m e t h o d of a s s e s s i n g r i s k in m u l t i p l e gestation p r e g n a n c i e s , and reporting the risk of OSB and Down's syndrome in these patients; • p r o t o c o l for recommending non-directive consideration of further assessment of risk through subsequent procedures.
Screening cut-off values A risk of Down's syndrome at term of 1:385 (that of a 35.5-year-old woman) or greater is a widely accepted standard of practice for recommending amniocentesis. In contrast, the selection of the screening cut-off values for OSB must be appropriate for the local setting. (It is appropriate to consider only the risk of OSB in setting the cut-off value, since anencephaly causes substantial elevations in MSAFP that are easily detected.) The screening cut-off for OSB establishes a balance between the desired detection rate for OSB and the burden of the false-positive rate t h a t is associ-
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ated with t h a t detection rate. Therefore, the falsepositive rate should be selected bearing in mind its effect on available resources, namely, repeat assays, genetic consultations, level II ultrasound examinations and amniocentesis. The detection/'ate should be sufficiently high to support the public expectation of a screening program, and the a t t e n d a n t reduction of health care expenditures t h a t are associated with screening protocols. Examples of wellestablished perinatal screening programs are those for phenylketonuria (PKU) and hypothyroidism. For MSAFP screening, the MoM values of 2.00 and 2.50 are common cut-off values. Any value between these limits can be justified based on the considerations indicated above. The local prevalence of OSB will not affect the false-positive rate, or the detection rate (the fraction of the affected pregnancies detected). However, the prevalence will determine the odds of being affected, given a positive screening result. Before a cut-off is implemented, these odds at the cut-off (as distinct from the odds at all levels above the cut-off) should be calculated, and they should be appropriate for the nature of the recommendations for follow-up procedures. As an example, i f a repeat testing protocol is used, a significant fraction of those initially determined as screen positive will revert to negative. Similarly, the local reliance on the noninvasive procedure of level II ultrasound will influence the acceptability of the odds at the cut-off. For these reasons, the risk at the cut-off for OSB need not be the same as t h a t used for Down's syndrome. As a guide for the selection of a cut-off, Table 2 provides the approximate projected relationships between MoM cut-offs, the predicted false-positive rate and detection rate, and the odds of being affected given a positive screening result (OAPR) for three levels of OSB prevalence.
Contents of the patient report The patient report should contain all of the information used in the assessments of risk, such t h a t
TABLE 2 Projected MSAFP Screening Parameters at Various OSB Cut-offs MoM Cut-off Value 2.00 2.10 2.20 2.30 2.40 2.50
Prevalence of OSB 1:2000 1:1000 1:500
FalsePositive Rate (c2)
Detection Rate (%}
4.5 3.9 3.4 2.9 2.6 2.5
85 83 80 77 74 70
OPAR* at the Cut-off 1:1500 1:1180 1:940 1:750 1:600 1:480
1:750 1:590 1:470 1:375 1:300 1:240
1:375 1:295 1:235 1:185 1:150 1:120
*Odds of being affected, given a positive result.
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SCREENING FOR HIGH-RISKPREGNANCIESWITH MSAFP the ordering physician will be aware of the data elements used in the interpretation. This requires t h a t the patient report must contain all of the information listed in Table 1. In addition, the report must contain all calculated values and limits: • patient's calculated age at expected date of confinement; • gestational age used in the calculations, and the method by which it was derived; • AFP result, in SI units (~g/L for serum; mg/L for amniotic fluid) expressed to one decimal place, and in MoM units expressed to two decimal places; • an indication if the MoM unit result has been adjusted for patient weight, racial origin and diabetic status; • OSB screen cut-off value in MoM units; • calculated patient-specific risk of OSB; • absolute risk of Down's syndrome, and the ageequivalent risk expressed in years; • date of the report, the identity of the individual responsible for the report, and the identity and telephone number of the assaying laboratory. If assumptions are made in instances where date elements have not been obtained, then both the data used, and the fact t h a t these data were not specified should be noted in the report; (e.g., a singleton pregnancy should be assumed if not otherwise specified, and this assumption must be indicated). There should be agreement on the factor values to be employed in the absence of patientspecific data, for example, the general population prevalence should be assumed in the absence of information on the racial origin and/or the patient's diabetic status. If the gestational age as of the sample date, and the patient's date of birth are not provided, then the report should withhold the MSAFP screening interpretation until these essential data are supplied.
Central data base Where more t h a n one laboratory is offering an MSAFP service within a province, each laboratory should make their data available to a central data base in the province (16). By inspection of these data, consistency of service between laboratories will be enhanced, and the collective data will assist in updating the factors t h a t are used in interpretation. The data base should include the results of follow-up procedures and pregnancy outcome data which can be supplied by the genetic centres and physicians involved. To enable tracking of patients within the province, an identical method should be used for assigning unique patient identifications. The use of a common interpretive computer program is required for maintaining a consistent service and creating the central data base, which could encompass the data from several or all provinces.
CLINICALBIOCHEMISTRY,VOLUME23, DECEMBER 1990
Protocol for r e c o m m e n d a t i o n of confirmatory procedures As indicated in the preceding sections, a common protocol for the recommendation of confirmatory tests should be established. This protocol should be based on a consensus of the laboratory and medical professionals involved in the care of pregnant patients within the region. As a guide to such a consensus, several MSAFP screening programs for ONTD recommend a repeat MSAFP sample in situations where: (i)the risk of OSB on the basis of MSAFP is moderate (for example, above the normal risk cut-off but less than 1:120); and (ii) there is sufficient time for a second sample, preferably one week later (i.e., the gestational age at the time of the initial sample is 18 completed weeks or less}. The benefits of a repeat MSAFP sample one week following a moderately elevated result include: a stimulus and opportunity to reassess the gestational age of the patient; an enhanced separation of the affected and unaffected populations through regression towards their respective medians; an opportunity to counsel the patient prior to offering more invasive follow-up procedures; a reduction in health care expenses through enhanced exclusion of the unaffected population (up to one third of patients with first sample elevations will have a normal second sample as a result of the above factors). A repeat MSAFP sample is not recommended for: (i) low MSAFP values t h a t indicate an increased risk of Down's syndrome (17}; (ii) very elevated samples t h a t indicate a risk of OSB that exceeds 1:120; and (iii)elevated MSAFP levels at a gestational age greater t h a n 18 weeks. In these instances, level II ultrasound and amniocentesis are recommended. A level I ultrasound examination, to confirm gestational age, is frequently recommended for any patient with an abnormality in MSAFP levels, where an ultrasound has not been performed at a gestational age greater than five weeks. A level II ultrasound, to identify fetal anomalies, is often recommended for any patient with a persistent moderately elevated MSAFP, and any patient considering amniocentesis on the basis of an elevated MSAFP result.
Confirmatory procedures for ONTD following a high M S A F P The confirmatory tests available for detecting ONTD are the combination of level II ultrasound with the analyses of AFAFP and amniotic fluid AChE activity. Collaborative studies recommend t h a t the combination of level II ultrasonography with amniocentesis optimizes the diagnosis of ONTD (18). A negative level II ultrasound in the presence of only a moderate (< 1:400) risk of OSB on the basis
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of MSAFP may be considered by the physician as sufficient evidence against the existence of an ONTD to forego further assessment (191. Ultrasonographers should be aware t h a t physicians and patients might substitute this procedure for the more definitive amniotic fluid assays; therefore, ultrasonographers might be advised to indicate the relative certainty of their findings. If amniocentesis is selected, a normal AFAFP concentration (<2.00 MoM) essentially rules out the presence of ONTD (18). The calculation of an assessment of risk for OSB on the basis of AFAFP concentrations requires the same attention to analytical procedures, clinical information and interpretation as for MSAFP measurement. Because sufficient data are not available at present, an adjustment of the median value and prevalence of ONTD is not performed on the basis of racial origin or diabetic status. Maternal weight and multiple gestation status have no established effect on AFAFP concentrations. With these exceptions, the interpretation of AFAFP concentrations requires the same consistency of factors between assaying laboratories as outlined for MSAFP screening. Both of these procedures should be performed in each screening laboratory. In the presence of a moderately elevated AFAFP result (->2.00 MoM), AChE measurement significantly adds to the diagnostic certainty of the AFAFP test (18). As with any specialized diagnostic laboratory technique, AChE analysis requires constant attention. Since it is reserved for the rare instances of an elevated AFAFP level, it is strongly recommended t h a t the AChE assay be performed at one centre for a province. It follows that efficient means of transportation and communication be established between the AFAFP laboratories and the single AChE laboratory. The laboratories that refer specimens to a common AChE testing centre should adopt a common protocol for selection of samples for AChE testing.
Confirmatory procedures for D o w n ' s syndrome following a l o w M S A F P The confirmatory test for Down's syndrome is amniocentesis followed by chromosome analysis. There is no association between ONTD and Down's syndrome. Furthermore, since these two disorders have opposite effects on MSAFP concentrations, a patient determined to be at risk of Down's syndrome on the basis of a low MSAFP will have a risk of ONTD t h a t is far below the background population risk. The question arises, should AFAFP analyses be performed on samples from patients at risk of Down's syndrome either on the basis of age alone, or age in combination with MSAFP? The prevalence of ONTD in these patients will be quite low, and the diagnostic potential of any test is affected by the prevalence of the disorder in the population tested. The lower the prevalence, the less the likelihood of the screenpositive patient having ONTD; hence the diagnostic potential of the test is diminished (20). For this 474
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reason it is recommended that, if AFAFP measurements are performed in these patients, a positive AFAFP result must be combined with an AChE analysis before counselling on the risk of ONTD. Laboratories may initially choose to accept amniotic fluid samples from patients at risk for Down's syndrome on the basis of advanced maternal age only, for the purpose of establishing median values. Samples from patients at risk of Down's syndrome on the basis of a low MSAFP must not be included in any amniotic fluid median study, since the median values will be slightly negatively biased.
N e w markers for Down's s y n d r o m e In recent years, two additional maternal serum markers for the risk of Down's syndrome have been reported: unconjugated estriol (uE3) (21), and human chorionic gonadotrophin (hCG) (22}. Preliminary indications are t h a t these mid-trimester markers, when combined with maternal age and MSAFP, will enable the detection of approximately 60% of Down's syndrome pregnancies with a falsepositive rate of less t h a n 5~ (23). This detection rate is approximately twice t h a t seen with maternal age and MSAFP in combination, and approximately triple the rate with age as the only screening variable. Of these four risk indices, an increased concentration of mid-trimester maternal serum hCG is the most powerful single indicator of Down's syndrome risk. Conscientious application of current methods for serum hCG would enable adequate measurement of this marker. In contrast, uE3 levels in mid-trimester are low, and assays currently available for third-trimester estriol concentrations are not sufficiently precise at the mid-trimester levels. The addition of uE3 to a screening protocol has only a small beneficial effect on the detection rate; however, it has a significant effect on reducing the false-positive rate of the combined "triple marker" screen (e.g., from 6.7c~ to 4.7~1 (23). Whereas the additional markers for Down's syndrome risk have great promise, it is not recommended t h a t they be incorporated into a newly established MSAFP screening program. The major reasons for this are:
The lack of sufficiently precise methods for uE3; Because of its beneficial effect on the false-positive rate, uE3 should be incorporated into a screening program at the same time as hCG. The currently available uE3 methods must be optimized for the mid-trimester levels. As with AFP, the methods for uE3 (and hCG) must be comparable between laboratories in order to achieve a consistent performance.
The concept of risk volatility; Risk is a difficult concept for physicians to convey to their patients. For each of the serum markers, CLINICAL BIOCHEMISTRY, VOLUME 23, DECEMBER 1990
SCREENING FOR HIGH-RISK PREGNANCIES WITH MSAFP the c a l c u l a t i o n of r i s k is u l t i m a t e l y d e p e n d e n t on the g e s t a t i o n a l age of t h e p a t i e n t . Since g e s t a t i o n a l age is a c o m m o n factor in all t h r e e r i s k d e t e r m i n a n t s , a l t e r a t i o n s in t h e assessed g e s t a t i o n a l age will s u b s t a n t i a l l y c h a n g e t h e c a l c u l a t e d c o m b i n e d risk. W i t h t h r e e m a r k e r s , a difference of two w e e k s in g e s t a t i o n a l a g e could c h a n g e the r i s k e s t i m a t e tenfold. W i t h the single m a r k e r of M S A F P , t h e effect of g e s t a t i o n a l age on r i s k is less t h a n twofold. T h e c o m p o u n d e d effect of g e s t a t i o n a l age w i t h t h r e e m a r k e r s is r e f e r r e d to as " r i s k v o l a t i l i t y . " In fact, the p o t e n t i a l for s u b s t a n t i a l c h a n g e in the r i s k e s t i m a t e b a s e d on the i n f o r m a t i o n supplied is an indication of the p o w e r of t h e screen, if the i n f o r m a tion supplied is correct. P h y s i c i a n s m u s t be a w a r e of t h e s e concepts before the triple m a r k e r s are employed. T h e s e issues which c u r r e n t l y i m p e d e the use of the new m a r k e r s can be a d d r e s s e d t h r o u g h r e s e a r c h a n d education. In t h e i n t e r i m , new studies will be p u b l i s h e d w h i c h will aid in p r e d i c t i n g t h e economic b e n e f i t s of triple m a r k e r s c r e e n i n g for D o w n ' s syndrome.
3. 4.
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8. 9.
Quality assurance It is m a n d a t o r y t h a t a s c r e e n i n g p r o g r a m , which by design is offered to a l a r g e p o p u l a t i o n , should be subjected to a n ongoing q u a l i t y a s s u r a n c e e v a l u a tion. T h e r e are s e v e r a l p a r a m e t e r s of the s c r e e n i n g p r o g r a m t h a t should be assessed: the false-positive rate; p r e g n a n c y o u t c o m e s in t h e s c r e e n e d a n d uns c r e e n e d population; t h e u t i l i z a t i o n of follow-up resources; t h e level of c o m p r e h e n s i o n of the s c r e e n i n g p r o g r a m a m o n g its c o n s u m e r s ; the d e g r e e to which new p a r a m e t e r s are successfully i n t e g r a t e d into the s c r e e n i n g protocol. An M S A F P s c r e e n i n g p r o g r a m r e q u i r e s a subs t a n t i a l a n d c o n t i n u o u s c o m m i t m e n t by t h e laboratory director. T h e q u a l i t y a s s u r a n c e of the p r o g r a m r e q u i r e s t h a t the l a b o r a t o r y director b e c o m e a fully p a r t i c i p a t i n g m e m b e r of the h e a l t h care d e l i v e r y team.
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Acknowledgements T h e C a n a d i a n Society of Clinical C h e m i s t s g r a t e fully a c k n o w l e d g e s the a s s i s t a n c e of Dr. J. E. H a d dow, Mr. G. E. P a l o m a k i a n d Dr. G.J. K n i g h t f r o m the F o u n d a t i o n for Blood R e s e a r c h , S c a r b o r o u g h , Maine, in r e v i e w i n g this paper.
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ET AL.
T h e C a n a d i a n S o c i e t y o f Clinical C h e m i s t s ' T a s k F o r c e on M S A F P S c r e e n i n g
Chairman A. R. MacRae, PhD, FCACB (Oshawa)
Committee Members B. J. Cadeau, PhD, FCACB (Saint John) A. Drobnies, PhD, FCACB (Vancouver) T. E. England, PhD, FCACB (Etobicoke) M. L. Givner, PhD, FCACB (Halifax) A. W. Luxton, MD, FRCPC, FCACB (Hamilton) M. L. Salkie, MB, BChir, FRCPC, FCACB (Edmonton)
Associates K. L. Massey, MD, FRCPC, FCACB (Saskatoon) L. E. Seargeant, PhD, FCACB (Winnipeg)
CLINICALBIOCHEMISTRY,VOLUME 23, DECEMBER 1990