Neonatal Screening Tests

Symposium on The Laboratory in Pediatric Practice

Neonatal Screening Tests

Peter Mamunes, M.D.*

In the upcoming decade the pediatrician will be faced with an ever increasing number of patients with positive mass neonatal screening tests. This is so because the success of phenylketonuria (PKU) screening has spurred the development of similar procedures for the detection of more than 20 additional disorders. After briefly reviewing the rationale and extent of such programs, this presentation will outline a logical approach for the evaluation of positive test results in each of the more commonly screened conditions. Many factors must be considered in determining the feasibility of including any given disorder in a mass neonatal screening program (Table 1). (1) The major basis for detection is that early treatment can prevent or ameliorate the severe mental retardation that would otherwise occur. This is usually accomplished in the aminoacidopathies by dietary restriction of the accumulating substrate, in galactose disorders by strict dietary elimination of the offending sugar, and in hypothyroidism by oral hormonal replacement. In almost all neonatal screening procedures, the specimen presently utilized is a dried blood spot on filter paper collected prior to the neonate's discharge from the nursery; because of its ease and safety of acquisition, it has received wide acceptance by parents and the health care team. Tests requiring cord blood, neonatal capillary blood, or urine are less desirous because they are more difficult to obtain, thus causing a large number of infants to escape the screening process. (2) The cost of the tests is considerably dependent on the volume and variety performed by the laboratory. Ample analyses have been reported, however, to demonstrate that the cost for case finding is considerably lower than that which results from the special education and residential care needed for the undetected and subsequently retarded child. 7 It might be difficult to appreciate how this might be the case for disorders with low occurrence rates (such as the approximate 1:225,000 incidence for both homocystinuria and maple syrup urine disease). Table 2 therefore compares the cost of mental retardation with that for screen"Professor of Pediatrics and Human Genetics, Medical College of Virginia, Richmond, Virginia Pediatric Clinics of North America-VoL 27, No.4, November 1980

733

734

PETER MAMUNES

Table 1. Factors to Consider in Determining which Disorders to

Screen in the Neonatal Period 1. Availability of treatment to substantially improve the outcome. 2. Acceptance of the procedure to acquire the necessary specimen. 3. Cost of the test. 4. Frequency and severity of the disorder. 5. Availability of facilities and funds to effect necessary follow-up and treatment. 6. Reliability of the test.

ing either of the above two disorders. By using an automated punch index machine, it has been estimated that the cost per test (when "added on" to an already existent PKU and hypothyroid screening program) is only $0.15, and the case-finding cost thus $33,750. This latter figure compares favorably to the cost of caring for a mentally retarded child, whether moderately or severely retarded. There are a considerable number of intangible costs and benefits of mass neonatal screening; they recently have been presented elsewhere33 , 50, 57 and are summarized in Table 3. Because of the rarity and complexity of treatment of the majority of the disorders screened, most patients with suspected or confirmed diagnoses require consultation at a metabolic or endocrine center. The unavailability of appropriate referral procedures or facilities can adversely affect the credibility of a neonatal screening program. Such was the case with the recent experience in New York City, where there was a poor follow-up rate for neonates positively screened for a hemoglobinopathy.I9 The reliability of the test is determined mainly by its sensitivity and specificity. The former is defined as the accuracy in correctly identifying all of the at-risk patients, and the latter as that per cent of healthy individuals excluded from detection. For a screening procedure to be acTable 2. Cost Considerations in Screening for Rare Inborn Errors

of Metabolism Cost of detecting one case, where incidence of the disorder is 1 :225,000 $0.15 (cost per test) x 225,000

$33,750

vs. Cost of care of one child with mental retardation that results as a consequence of absence of screening: Preschool training (4 years @ $2,000/yr. 8,000 Education of the moderately retarded child for 12 years @ 42,000 $3500/yr. Total $50,000 ±*

Yearly cost for residential care of the severely retarded for 40 years @ $7500/yr.

$300,000

"The need for residential care is dependent on the type of disorder, age of diagnosis, and success of treatment.

NEONATAL SCREENING TESTS

735

Table 3. Intangible Costs and Benefits of Mass Neonatal Screening Programs BE1'IEFI'fS

1. Prevention of familial unheaval resulting from the development of moderate to severe retardation. 2. Achievement of a better understanding of frequency and heterogeneity of disorders screened. 3. Stimulation of interdisciplinary cooperation of and cross fertilization by physicians, nurses, public health workers, psychologists, nutritionists, and so forth. 4. Increased productivity by healthy, non-retarded adults. 5. A successful program encourages consideration of expansion to include additional disorders. COSTS

1. Time expenditure and expense in collecting and transporting specimens. 2. Inappropriate interpretation of false positive test results and consequent unnecessary (and at times harmful) treatment. 3. Anxiety and inconvenience attendant upon need for follow-up testing of false-positive results. 4. Treatment of the condition requires continual and considerable time input of parents and the health care team. 5. Loss of time of health care team which could be better utilized to attend to more important societal ills. 6. Cost of screening is borne by all, whereas it benefits only a very few. 7. A false-negative screening test gives a false sense of security that a given disorder is not the cause of psychomotor retardation.

ceptable, there clearly must not be too many false-negative (low sensitivity) or false-positive (low specificity) results. In the United States presently there is no uniform policy regarding the regulations which enable the screening process, the laboratory testing site, or the extent or type of testing indicated. Because each state determines its own procedures, there is very wide variance in the existent programs. It is therefore incumbent on the practicing physician to acquaint himself with the status of the screening program in his state and to actively work for its improvement. Almost all states mandate PKU screening by statute. In the majority of those states offering other tests, the additional screening is generally performed either by a variable degree of informed consent or by broad powers of genetic screening granted to the Commissioner of Health by state law. The State of Maryland has recently rescinded its mandatory screening law and replaced it with a voluntary testing program with a meaningful informed consent procedure. This makes education of the parents a more integral part of the screening process, and surprisingly has not significantly reduced the proportion of neonates screened. An advisory commission continually oversees the quality of the program and recommends additions and changes to state health officials. In an extensive review of genetic screening published in 1975, the National Academy of Sciences strongly recommended centralization of laboratory testing to improve reliability and cost, provide flexibility in the testing system, and improve the efficiency of sample collection and follow-up.5o However, presently only approximately 60 per cent of the

736

PETER MAMUNES

states conduct all of their screening in a centralized laboratory. In the remainder, while the state may offer the service, testing is also dispersed among many hospital and private laboratories. Recognizing the virtue of centralizing laboratory efforts, fourteen states have pooled their resources to form three regional centers in the New England, Northwest, and Rocky Mountain areas. * When multiple testing is performed on the same dried blood spot, cost and reliability considerations make it almost mandatory that the screening be conducted in a laboratory that processes a large volume of specimens yearly. Since the Quebec experience demonstrated its feasibility in 1975,14 approximately one half the states have added hypothyroid screening to the testing performed from the neonatal dried blood spot. Additionally, approximately 20 states are utilizing the same specimen to screen for one or more of the following conditions: galactosemia, homocystinuria, maple syrup urine disease, histidinemia, tyrosinemia, adenosine deaminase deficiency, and sickle cell trait and anemia. As further experience becomes available it is likely that a consensus will be reached regarding which disorders are appropriate for mass neonatal screening and there will be more uniformity in such programs from state to state. Presently the majority of those most intimately involved in the development and practical application of neonatal screening tests would probably agree that the minimum program should include the five disorders listed in Table 4. Therefore each of them (except for hypothyroidism which is presented in a separate article) will now be discussed in some detail, with the main emphasis being the method of evaluation of a positive screening test. Subsequently, the many other candidate disorders will be discussed more briefly.

PHENYLKETONURIA This disorder is caused by a lack of activity of the enzyme phenylalanine hydroxylase (Fig. 1). The consequent build-up of phenylalanine and its metabolites causes irreparable damage to the developing central nervous system unless the condition is diagnosed and treated within the first 3 to 6 weeks of life. As is the case for almost all screened aminoacidopathies, the detection is based on finding substrate amino acid accumulation by the use of a bacterial inhibition assay (BIA). This test procedure, first developed by Guthrie for phenylalanine in 1961,21 utilizes a 114 or 118 inch disc punched from filter paper onto which the neonate's blood has been spotted and mailed to the laboratory. The disc is placed on an agar plate with other unknowns and one row of control discs containing varying concentrations of the substance being tested. B. subtilis spores in the agar grow around the disc after overnight incubation at 37° C if its blood concentration is above normal, and the diameter of the growth ring correlates well with the degree of elevation present. The amino acid being "In the New England consortium Massachusetts screens for Maine, New Hampshire, Rhode Island, and Connecticut; in the Northwest region, Oregon screens for Montana, Idaho, Alaska, and Nevada; and in the Rocky Mountain area Colorado screens for New Mexico, Arizona, and Wyoming.

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Table 4. Suggested Minimum Panel of Disorders for Mass Neonatal Screening and Some of Their Characteristics

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MAPLE SYRUP PHENYLKETONURIA

HYPOTHYROIDISM

GALACTOSEMIA

URINE DISEASE

HOMOCYSTINURIA

Incidence

1:14,000

1:4,250

1:62,000

1:224,000

1:230,000

.\Ilost commonly used test

Guthrie BIA'" for phenylalanine

T. radioimmunoassay + TSH follow-up

Paigen bacterial assay

BIA for leucine

BIA for methiontine

Cost per test

$0.50t

$1.00

$0.35

$0.15!

$0.15!

Case-finding cost

$7,000

$4,250

$21,700

$33,600

$34,500

*Bacterial Inhibition Assay tCost when done as an isolated screening test tCost when the screening is automated and "added on" to an existent PKU program

'-J ~

'-J

738

PETER MAMUNES

measured and the size of the ring are determined respectively by the type and concentration of a growth inhibitor which has been added to the agar. When the inhibitor used is beta-2-thienylalanine, the test procedure measures phenylalanine and is called the Guthrie test. Another screening technique is to determine the phenylalanine level in the eluate of the dried blood by an automated fiuorometric procedure. 16 However, although it may be more sensitive and specific than the Guthrie test, it is used infrequently because the cost is higher and more technical expertise is required. 22 Most states set 4 mg/dl or above as that level of phenylalanine which is "positive" - Le., the infant is determined to be in need of further investigation to rule out PKU (Fig. 2). However, in one large survey of infants with PKU, 7 per cent had Guthrie readings of between 2 and 4 mg/dl when tested at about 3 days of age. 24 Therefore in some state programs, any infant with a phenylalanine level of >2 mg/dl receives follow-up testing; this procedure causes more false-positive tests (decreased specificity) but also reduces the incidence of false-negative screening (Le., increases sensitivity). Only 5 to 10 per cent of infants with positive Guthrie tests will prove subsequently to have classical PKU. The two major causes for these false-positive tests are transient neonatal tyrosinemia (TNT) and transient hyperphenylalaninemia (hyperPHE). The former is due to decreased activity of the enzyme p-OH phenylpyruvic acid oxidase (Fig. 1) which occurs as the consequence of either prematurity :J9 or an excessive protein intake (producing an excess of substrate, which paradoxically inhibits its own enzyme).29 Therefore the first step in evaluating a positive Guthrie test is to determine the tyrosin~ (TYR) level of the patient, either by a BIA procedure for TYR using the original specimen, or by microfiuorometric quantitation of a blood specimen collected in capillary tubes from a heel puncture. If the tyrosine level is elevated (> 4 mg/ dl), the infant most likely has dihydropteridine reductase (DHPR)

TETRAHYDROBIOPTERIN

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TYROSINE

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PARA-OH PHENYLPYRUVIC AC ID (P-OH PPA)

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PHENYLACETIC ACID

ORTHO-HYDROXYPHENYLACETIC ACID

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Figure 1. Metabolic pathways of phenylalanine and tyrosine as they relate to newborn sc
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repeat serum PA level Transient Neonatal Tyrosinemia

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Hepatorenal type

Permanent HyperphenYlalaninem~J~·a ________~______,.

"""'lr' Classical PKU (PA ~20 mg/dl)

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Diagnostic-considerations with a positive Guthrie test (blood PA level

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Malignant HyperPHE (biopterin defect)

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740

PETER MAMUNES

a transient oxidase deficiency. The activity of this enzyme is almost always easily normalized within a week by the oral administration of 100 mg of vitamin C orally and/or the reduction of a high protein intake that usually is the consequence of the use of an evaporated milk formula. Blood TYR levels in these conditions may be as high as 40 to 50 mg/dl, but those associated with prematurity usually resolve spontaneously within 2 to 4 weeks of life and long term follow-up has not disclosed any adverse effect on the developing central nervous system. 39 However, elevated levels may persist longer when the etiology is a high protein intake, and Mamunes et al. 41 found significantly low intelligence quotients when this TNT subgroup was studied at age 5. Therefore, in order to assure that the tyrosinemia is not prolonged or permanent, serial TYR levels should be obtained until it resolves. Permanent tyrosinemia of either the Oregon type (mental retardation, microcephaly, skin, and eye lesions)27 or hepatorenal type 30 are very rare conditions but these must be considered if the tyrosine elevation is persistent and not associated with prematurity or a high protein intake or if the infant is symptomatically ill. If the initial tyrosine level is normal, the phenylalanine level should be re-evaluated on a freshly submitted specimen (either by the Guthrie method or preferably by a quantitative microfluorometric assay) as quickly as possible. Serum phenylalanine values 2::20 Ing/dl are characteristic of classical PKU and demand immediate referral to a metabolic center for institution of a restrictive diet. It has been definitively shown that when the PKU infant is placed on dietary treatment before age 1 month, good control of the serum phenylalanine level will effect normal mental and physical growth. 9• 63 However, despite experience with the restrictive diet for over 25 years,2. 64 the age at which it can be safely discontinued is still unclear. At the present time, data from the PKU Collaborative Study suggest that discontinuation at age 6 may be associated with a decrement in the rate of learning over the subsequent 2 years. 9 For this reason it is the present consensus that the diet should be maintained until at least age 8. Infants whose phenylalanine levels remain in the 4 to 20 mg/dl range beyond age 3 months have a condition termed hyperPHE. Opinion differs regarding the precise phenylalanine level at which dietary restriction of such patients is indicated to avert central nervous system damage, but most metabolic centers would seriously consider treatment when it reaches 15 mg/dl. Recently it has been shown that 1 to 2 per cent of permanently hyperphenylalaninemic infants do not have a deficiency of phenylalanine hydroxylase activity. Rather, their primary defect is either reduced activity of dihydropteridine reductase (DHPR),43 or deficient synthesis of biopterin. 53 Tetrahydrobiopterin (BH4) is the cofactor necessary for adequate phenylalanine hydroxylase activity, and DHPR is an enzyme which catalyzes the reduction of quinonoid dihydrobiopterin (formed in the conversion of phenylalanine to TYR) back to BH4 (Fig. 1). Infants with these defects are said to have "malignant" hyperphenylalaninemia because severe, progressive central nervous system dysfunction develops, probably as a result of deficient synthesis of 5-hydroxytryptophan and L-dopa (neurotransmitters whose formation is dependent on other biotin-dependent hydroxylases).8. 11 Early recognition ofthese conditions

741

NEONATAL SCREENING TESTS

is most important because administration of these substances appears to improve the clinical status of some of the affected patients. Screening of all infants with hyperphenylalaninemia can be accomplished by demonstrating decreased DPHR activity in fibroblasts,43 elevated54 (in DPHR deficiency) or decreased 12 (in defective biopterin synthesis) urinary pteridines, or a fall in the serum phenylalanine level 6 hours after the oral administration of BH4. 12 Because such evaluation is only available at metabolic centers it should be clear that every infant with permanent hyperPHE should be referred for further evaluation. There has been considerable disagreement regarding the necessity of repeat testing at 2 to 4 weeks of age. 55 The major evidence to support such a procedure came from a review by Holtzman et al. 23 who found that approximately 10 per cent of patients with PKU had negative Guthrie tests when tested before 72 hours of age. While technical and administrative errors could contribute to a false-negative test, biological variability in the rate of serum phenylalanine rise has been thought to be the main causeY' 42 However, more recent evidence refutes this contention. In two studies a total of 17 siblings of patients known to have PKU who were subsequently shown to also have the disorder, all had phenylalanine levels of 4 mg/dl or greater by age 48 hours. 17, 42 In a recent review of 2.38 million repeat Guthrie tests only two new PKU patients were uncovered where their initial result was normal, and the authors concluded that a second test was not indicated. 56 Therefore the current feeling is that when properly performed by the laboratory the initial Guthrie test will detect almost all infants with PKU by 48 hours of age if there has been adequate milk intake, and repeat testing solely for PKU is not warranted.

GALACTOSEMIA This is the second most common metabolic disorder for which neonatal screening is indicated. In a recent worldwide survey of almost 6 million tests, the incidence of the classical form of galactosemia was found to be 1:62,000. 34 The incidence may be as high as 1:40,000 because that was the experience when the New England Regional Screening Program recently used the more sensitive Paigen assay in over 400,000 neonates. 34 In comparison to PKU, there is considerably more urgency in establishing the diagnosis of classical galactosemia. The majority of patients with this disorder become symptomatic within the first week of life with jaundice, lethargy, and poor weight gain, and E. coli sepsis has developed in approximately half. 36 If they survive the neonatal period and are not treated, cataracts, hepatic cirrhosis, and behavioral and learning disorders develop in most. At least four different procedures, all using the dried blood spot, have been used in screening for galactosemia.

Bacterial Inhibition Assay This auxotrophic test employs a mutant strain ofE. coli which lacks galactose-I-phosphate uridyl transferase activity,20 the same enzyme deficient in galactosemic patients. Elevated levels of galactose in blood

742

PETER MAMUNES

discs placed on agar are therefore toxic to these bacteria and prevent growth that normally occurs with normal blood galactose levels. In comparison to the BIA for amino acids, a positive test exists when there is no bacterial growth. This assay has the disadvantage that the mutant bacteria gradually lose their sensitivity to galactose, and therefore the test is presently less frequently used than the others.

Beutler Test Whereas the other three screening procedures detect elevated levels of galactose, this one qualitatively measures the activity of the transferase enzyme in the red blood cells of the specimen. 4 This is accomplished by incubating the blood disc with a solution of the enzyme's substrate, galactose-I-phosphate, and NADP. In the presence of normal transferase activity, fluorescence of the solution develops because of the generation of NADPH by the action of two enzymes further along the pathway of galactose metabolism in the red blood cell. Because the test does not require prior feeding and because of the need for early diagnosis it has also been performed on umbilical cord blood spotted on filter paper. 32 Until recently, this assay was the most popular method of screening for galactosemia, but many false positive tests occur because of the detection of benign low-activity variants and a loss of enzyme activity with excessive exposure of the blood disc to heat or humidity.34 Additionally, other disorders of galactose metabolism (e.g., galactokinase deficiency) are not detected.

Automated Spectrophotometric Assay The galactose level can be quantitatively determined using a galactose dehydrogenase enzyme assay of an eluate from the blood disc,18 but the procedure is not widely used because it is more costly and technically difficult than others. Paigen Bacterial Assay This test differs from the previous bacterial assay in that it utilizes a mutant E. coli strain which is deficient in the epimerase enzyme, and in the presence of elevated blood galactose they resist destruction of C2I bacteriophage. 34 Three characteristics presently make the Paigen assay the preferred procedure for screening disorders of galactose metabolism: (1) the test system is stable, (2) a positive test occurs with growth rather than inhibition of growth, and therefore is easier to interpret, and (3) the procedure can be modified to also detect elevated levels of galactose-Iphosphate. 16 This metabolite becomes elevated sooner than galactose and thus makes the assay more sensitive. While good data on this subject are not yet available, it appears that false negatives are very rare and the false positive rate is low. An infant with a positive screening test should immediately be placed on a lactose elimination diet (Nutramigen, Prosobee) until confirmatory testing has been done. A definitive diagnosis is established by noting diminished activity of galactose-I-phosphate uridyl transferase in a specially prepared red blood cell pellet or in fibroblasts cultured.from a skin biopsy. Because of the existence of multiple variant forms of the

j

NEONATAL SCREENING TESTS

743

transferase enzyme (e.g., Duarte, Indiana, Rennes), more than half of the patients with either decreased enzyme activity or elevated galactose detected by neonatal screening do not have classical galactosemia, and many do not require dietary treatment. 3, 4. 34. 58 In general, transferase activity of greater than 20 per cent of normal is sufficient to prevent the accumulation of toxic levels of galactose and galactose-1-phosphate. 34 If the transferase activity is normal and the blood galactose level elevated, two other disorders of galactose metabolism should be considered-galactokinase 15 and 4-epimerase 5 deficiencies. The former causes only cataracts which are preventable by a galactose elimination diet, but the latter probably does not induce any clinically significant disease. Both disorders are also confirmed by measuring the enzyme activity in red blood cell preparations. A lactose elimination diet substantially improves the outcome of galactoseInia 10 and if started early enough may prevent the development of neonatal sepsis. 36 However, because cataracts have been present in the young neonate with galactoseInia,26 and psychomotor development, despite early treatment, is often suboptimal,13. 28. 46 it is likely that the fetus is adversely affected. Further long-term follow-up is needed to determine if early diagnosis and treatment can completely prevent damage to the central nervous system and at what age the strict eliInination diet may be relaxed or discontinued.

MAPLE SYRUP URINE DISEASE There are at least four different forms of this disease, all of which are due to deficient oxidative decarboxylation of the alpha-keto acids of the branched-chain amino acids isoleucine, valine, and leucine. Neonatal detection of the two more common and severe forms, the classic and intermediate variants, is possible by finding elevated (>4 mg/dl) levels of leucine by BIA of the dried blood spot. The two Inilder variant forms, intermittent and thiaInine-responsive maple syrup urine disease, are usually not detectable because they are associated with slower and milder rises in the serum leucine level. In a recent review of over nine million neonatal screening tests performed over a 12 year period, the combined incidence of the classical and intermediate variant forms was found to be 1:224,000. 48 The classical form is most important to detect early because of the severity of the acid-base, gastrointestinal, and neurological complications which usually begin by the first week of age; the majority of such infants succumb to their disorder if there is no screening. The intermediate form, representing approximately 25 per cent of all cases of maple syrup urine disease detected by neonatal screening, has a slower and more variable onset, but like the classical form, surviving infants are almost invariably severely mentally retarded without early dietary treatment. This BIA is even more sensitive than the Guthrie PKU test, apparently because of a more prompt rise in the serum leucine level after milk feeding. In fact, no cases of the classical or intermediate forms were

744

PETER MAMUNE S

known to have been missed in the more than nine million screens previously mentioned. 48 In addition the specificity was in the same range as that for PKU, with only 0.05 per cent false-positive tests. While the test is highly sensitive and specific, case detection often occurs only after the infant has become severely symptomatic. Treatment at this time may prevent death but usually irreparable central nervous system damage has already occurred. Therefore, if maple syrup urine disease and galactosemia testing is included, the blood specimen for newborn screening of metabolic disorders should preferably be obtained at 48 to 60 hours of age, rather than awaiting nursery discharge. Additionally, hospitals must mail their collections daily and the laboratory remain functional 7 days a week and promptly report positive results. Such earlier specimen acquisition will probably decrease the specificity but not the sensitivity of PKU testing,17.42 but would not significantly affect hypothyroid screening. If a positive screening test is reported, the infant should be examined immediately for early signs or symptoms of maple syrup urine disease, and the urine screened for excess alpha-keto acid. If the disorder is present, 10 drops of a 10 per cent solution of freshly prepared ferric chloride added to 1 ml of acidified urine will usually cause a gray-blue or bluegreen color to develop. An alternative test for keto aciduria is to mix equal amounts of urine and 2,4 dinitrophenylhydrazine, 0.5 per cent (wi v) in 2N Hel; a yellow precipitate within a minute indicates a large quantity of keto acid. Also the urine might have the odor of maple syrup, carmel or curry. If the infant is symptomatic or any of the above urinary tests are positive, he or she should be referred to a metabolic center for confirmatory testing (quantitation of serum amino acids and enzyme measurement in white blood cells or fibroblasts), and the institution of a diet which limits the branched-chain amino acid intake to the minimum required for adequate growth. 59 Also, if there is already significant neurologic involvement, peritoneal dialysis and/or exchange transfusion may be indicated to remove some of the accumulating toxic branchedchain alpha-keto acids. 59 While awaiting the transfer of an infant with suspected maple syrup urine disease, protein intake should be discontinued but adequate calories given to minimize endogenous protein catabolism. If the infant with the positive screen is asymptomatic and the urine examination negative, blood should be obtained 3 or 4 hours postprandially for the quantitation of serum amino acids, and the infant closely followed and maintained on a low protein milk formula while awaiting the results. Because the branched-chain alpha-keto acids are neurotoxic, the restrictive diet must be maintained throughout life. Experience with this diet over the past 18 years 60 indicates that normal growth can occur and ketoacidosis and neurotoxicity can be minimized or eliminated, but the mental retardation is preventable probably only if the treatment is begun within the first week of life.

HOMOCYSTINURIA The frequency of this disorder, as judged from mass neonatal screening, is approximately 1 :230,000. 32 Because it is caused by a deficiency

NEONATAL SCREENING TESTS

745

in the activity of the enzyme cystathionine synthetase, homocystine and its precursor, methionine, accumulate in the blood. In approximately half the cases of homocystinuria this deficient enzyme is "activated" by the provision of pharmacologic doses of its cofactor, pyridoxine (i.e., they have pyridoxine-responsive homocystinuria), and the biocheInical abnormalities are reversed. Such patients are at a low risk for mental retardation, but frequently the other complications of this disorder develop-ectopic lenses, thromboembolic episodes, osteoporosis, arachnodactyly, and loose ligaments. The BIA to screen for this disorder is coneidered positive when the methionine level is 2::2 mg/dl, but pyridoxine-responsive cases are not detected, presumably because of the slower rise in this form of the disease. The true incidence of both forms of the disease is therefore approximately twice that determined from the newborn screening experience discussed previously. The sensitivity of this assay is not as good as that for PKU or maple syrup urine disease, but because of the rarity of the disorder and its subtlety in presentation during infancy, a precise false-negative rate is presently not available. The specificity, however, is very good, with the false-positive rate slightly above that for PKU and maple syrup urine disease. When a positive screening test for homocystinuria is received, the physician should exaInine the patient, but it is unlikely that any abnormalities will be found, as is the case with PKU in the first month. While the urinary cyanide-nitroprusside test is a useful screening procedure for detecting homocystinuria in the older patient, it often is negative in the infant with the disorder in the first two weeks. 37 Nevertheless, it should be performed in the following manner: Mix: an equal volume of urine with sodium cyanide reagent, 5 per cent Na eN (w/v) in water; after allowing to stand for 10 minutes, add 3 or 4 drops of nitroprusside reagent (freshly made solution containing a few crystals in water); a magenta color indicates a positive test, but care should be taken that another disulfide compound (e.g., cystine) is not causing a false-positive result. If the test is positive, referral to a metabolic center for confirmatory testing is indicated. If the test is negative, the significance of the positive BIA for methionine can next most simply be determined by quantitating the serum amino acids after a 3 to 4 hour fast. Elevated levels of methionine and homocystine would dictate the need for referral for treatment. For the pyridoxine-resistant cases positively identified in neonatal screening programs, low methionine, cystine-supplemented diets have been tried with variable success; mental retardation has been minimized or prevented in several cases. 45 However, considerably more experience with the diet is needed to determine its protective effect, how early it must be initiated to avert permanent central nervous system damage, and the degree to which the serum methionine and homocystine levels must be controlled. It should be obvious that because all four of the above-discussed inborn errors of metabolism are inherited in an autosomal recessive fashion, any infant with a positive screening test and a sibling previously affected by the disorder should be referred immediately for confirmatory testing and probable treatment pending results. Additionally, the like-

746

PETER MAMUNES

lihood that the positive screening report is not a false one would rise considerably should the family history reveal that the parents are consanguineous. In the absence of a positive family history or symptoms, and where the level of the metabolite is barely above the normal range, it is more likely that the screening result is falsely positive. Therefore in this circumstance the physician should make a concerted effort to allay anxiety of the parents while awaiting follow-up testing.

OTHER TESTS USED BY SOME STATES OR REGIONS Histidinemia As with the other aminoacidopathies previously discussed, a BIA is available to detect elevated levels of histidine. With the frequency of the disorder at approximately 1 :22,000,32,38 one would think that this disorder is well-suited to mass neonatal screening. However, such is not the case because it apparently is not a severe disease, the effectiveness of therapy has not been well demonstrated, and there are a substantial number of false-negative tests because at times there is a slow rise in the serum histidine. In a prospective study of 20 consecutive histidinemic children detected in a neonatal screening program, Levy et al. 38 could detect no mental retardation when they were tested at a mean age of 4 to 7 years. On the other hand, Popkin et al. 51 concluded from their review of the literature that approximately 40 per cent are mentally subnormal as a result of their disease. The most prominent central nervous system dysfunction is a speech defect more severe than the degree of intellectual impairment. Because of the uncertainty regarding the consequence of the disorder, the value of the histidine-restricted diet is difficult to determine. While the serum level can usually be well controlled,62 the clinical course is usually not significantly altered, and slow growth results. Further experience with this disorder, including perhaps a controlled study in which neonatal cases are randomized to treatment and non-treatment groups, is needed before it can be strongly recommended for mass neonatal screening.

Tyrosinemia Except in certain genetic isolates, hereditary tyrosinemia is extremely rare (probably less than 1:500,000) and TNT (as previously discussed) is probably not clinically significant unless severe and discovered after 4 weeks of age. 41 Despite the availability of a BIA for tyrosine, mass neonatal screening for this disorder therefore is not indicated. Adenosine Deaminase (ADA) Deficiency A deficiency ofthis enzyme is present in 10 to 50 per cent of patients with combined immune deficiency disease. Early diagnosis and treatment with bone marrow transplantation might cure such patients, making this disorder a potential candidate for screening because ADA is present in the red cells of the dried blood spot. However, Naylor et

NEONATAL SCREENING TESTS

747

al. 49 recently detected only one case in over 400,000 screened neonates utilizing the liberation of ammonia from adenosine as a measurement of enzyme activity.44 While the test can be automated and is very sensitive and specific, ADA screening is presently not indicated because of the rarity of the disorder, and the treatment is very difficult and often not successful.

Sickle Cell Trait and Anemia These conditions are included in the present discussion because one state (New York) mandates their screening by law. The principal justifications for such testing are that (1) the early detection of sickle cell aneInia might reduce its mortality and morbidity, (2) establishing a diagnosis of sickle cell aneInia forewarns the parents of their 1:4 risk for a siInilar occurrence in subsequent pregnancies, and (3) sickle trait detection identifies which families are at risk to also carry this gene abnormality and thereby aids in detection of pairs of parents at risk for bearing children with sickle cell disease. However, it has not been proved that early detection of sickle cell aneInia significantly improves morbidity or mortality, and at-risk couples are more efficiently detected by carrier detection programs directed at teen-agers and young adults. Several recent reports 19, 52 have also disclosed the consequences of such a program when proper follow-up procedures are not established. Until the value of neonatal sickle cell aneInia detection is ascertained and pilot programs better define the impact of carrier detection on the family unit and the proper mechanisms for follow-up of positive tests, mass screening for hemoglobinopathies from neonatal blood spots is probably not warranted.

OTHER DISORDERS POTENTIALLY DETECTABLE USING DRIED BLOOD Many other test procedures have been developed to screen for inborn errors of metabolism utilizing the same dried blood spot subInitted from the neonatal nursery (e.g., argininosuccinic acideInia, orotic aciduria, alpha1-antitrypsin deficiency, hereditary angioneurotic edema, muscular dystrophy, and other aminoacidopathies via paper chromatographic separation). They are presently not included in any mass screening programs in the United States because the disorders are either (1) too rare (2) or not treatable, or (3) the test procedure is too technically difficult or costly. For a brief discussion of these entities the interested reader is referred to a previous review on newborn screening by the author. 40

URINE SCREENING An alternative to the use of a repeat dried blood specimen for followup screening (to uncover prior false negative tests) is the testing of urine obtained from the neonate at age 3 to 4 weeks. This approach has several advantages: (1) the specimen can be easily collected by the parent utilizing filter paper positioned between the diaper and the perineum, (2)

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it can detect renal tubular disorders as well as aminoacidopathies, (3) it might better detect those disorders where the urinary concentration of the metabolite is higher than that in the blood (e.g., histidinemia), and (4) it can detect organic acidurias. The largest experience with this technique was reported by Levy et al. 35 who screened the urine of over 200,000 infants. Approximately 85 per cent of parents complied with the request given them at the time of nursery discharge to mail the specimen obtained between the ages of 3 and 4 weeks. Although the incidence of detectable defects was quite high at 1 :3,500, most were benign tubular disorders, and no infants were found with organic acid urias utilizing an aniline-xylose stain. More recently Levy 12 reported an incidence of methylmalonic aciduria of approximately 1 :80,000 when the staining of the urine chromatogram was changed to Fast Blue B. The major drawback to such a urine screening program is that many spurious spots are identified by chromatography and at least 1 per cent require follow-up testing. This causes considerable undue anxiety to both parents and physicians. An alternative to chromatography for detecting aminoaciduria in the dried urine specimen is the use of the BIA previously described for blood spots. Recently the variety of such assays has been expanded to permit the detection of almost any amino acid, and automation could minimize the cost of performing eight or ten such tests for individual amino acid excessesY These procedures are more sensitive and specific than chromatography, thus reducing the number of false-negative and false-positive results. If the incidence of methylmalonic acidemia is as high as that recently reported by Levy and BIA procedures can be adapted inexpensively, the dried urine specimen may prove to be an important addition to the neonatal screening process.

CONCLUSION The concept of mass neonatal screening for inherited metabolic disorders has finally been recognized as a necessary and desirous component of preventive health care. However, support for its continuation is predicated upon careful attention to the many complex facets of such a program, some of which have been discussed in this presentation. The extent and form of the modern day neonatal screening program will change as new technologies are developed (e.g., automated high pressure liquid chromatography, mass spectroscopy, BIA of urine), the etiology and treatment of metabolic disorders are further elucidated, and legal and ethical issues become clarified. Clearly, those responsible for the content and operation of such services must continuously assess these developments and restructure their programs accordingly.

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33. Levy, H. L.: Newborn screening for metabolic disorders. New Engl. J. Med., 288:1299, 1973. 34. Levy, H. L., and Hammersen, G.: Newborn screening for galactosemia and other galactose metabolic defects. J. Pediatr., 92:871, 1978. 35. Levy, H. L., Madigan, P. ,\1., and Shih, V. E.: Massachusetts metabolic disorders screening program. 1. Technics and results of urine screening. Pediatrics, 49 :825, 1972. 36. Levy, H. L., Sepe, S. J., Shih, V. E., et al.: Sepsis due to Escherichia coli in neonates with galactosemia. New Engl. J. Med., 297:823, 1977. 37. Levy, H. L., Shih, V. E., and MacCready, R. A.: Screening for homocystinuria in the newborn and mentally retarded population. In Carson, N. A., and Raine, D. N., eds. Inherited Disorders of Sulfur Metabolism. London, Livingstone, 1971. 38. Levy, H. L., Shih, V. E., and Madigan, P. M.: Routine newborn screening for histidinemia. New Eng!. J. Med., 291 :1213, 1974. 39. Light, 1. J., Sutherland, J. M., and Berry, H. K.: Clinical significance of tyrosinemia of prematurity. Amer. J. Dis. Child., 125:243, 1973. 40. Mamunes, P.: Newborn screening for metabolic disorders. Clinics in Perinato!., 3:231, 1976. 41. Mamunes, P., Prince, P. E., Thornton, N. H., et al.: Intellectual deficits after transient tyrosinemia in the term neonate. Pediatrics, 57:675, 1976. 42. Meryash, D. L., Carr, J. R, and Levy, H. L.: A prospective study of early newborn screening for PKU. Pediat. Res., 14:525, 1980. 43. Milstien, S., Holtzman, N. A., O'Flynn, M. E., et al.: Hyperphenylalaninemia due to the dihydropteridine reductase deficiency. J. Pediatr., 89:763, 1976. 44. Moore, E. C., and Meuwissen, H. J.: Screening for ADA deficiency. J. Pediatr., 85:802, 1974. 45. Mudd, S. H., and Levy, H. L.: Disorders of transsulfuration. In Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, P. S., eds.: Metabolic Basis of Inherited Diseases. New York, McGraw Hill, 1978, pp. 458-503. 46. Nadler, H. L., Inouye, T., and Hsia, D. Y. Y.: Clinical galactosemia: A study of fifty-five cases. In Hsia, D. Y. Y., ed.: Galactosemia. Springfield, Illinois, Charles C Thomas. 1969, p. 247. 47. Naylor, E., and Guthrie, R: Conference on Newborn Screening. Los Angeles Children's Hospital, April 1, 1977. 48. Naylor, E. W., and Guthrie, R: Newborn screening for maple syrup urine disease (branched-chain keto aciduria). Pediatrics, 61 :262, 1978. 49. Naylor, E. W., Orfanos, A. P., and Guthrie, R: An improved screening test for adenosine deaminase deficiency. J. Pediatr., 93:473-476, 1978. 50. National Research Council: Committee for the Study of Inborn Errors of Metabolism: Genetic screening: programs, principles, and research. Washington, D.C. National Academy of Sciences, 1975. 51. Popkin, J. S., Clow, D. L., Scriver, C. R, et al.: Is hereditary histidinaemia harmful? Lancet, 1 :721, 1974. 52. Rubin, E. M., and Rowley, P. T.: Sickle cell trait/hereditary persistence of fetal hemoglobin trait. Misdiagnosis as sickle cell anemia by newborn screening. Amer. J. Dis. Child., 133 :1248, 1979. 53. Schaub, J., Dauming, S., Curtis, H-Ch, et al.: Tetrahydrobiopterin therapy of atypical phenylketonuria due to defective dihydrobiopterin biosynthesis. Arch. Dis. Child., 53:674, 1978. 54. Schlesinger, P., Watson, B., Cotton, R. G. H., et al.: Urinary dihydroxanthopterin in the diagnosis of malignant hyperphenylalaninaemia and phenylketonuria. Clin. Chim. Acta, 9'2: 187, 1979. 55. Scriver, C. R, Mamunes, P., Feingold, M., et al.: Screening for congenital metabolic disorders in the newborn infant. Congenital deficiency of thyroid hormone and hyperphenylalaninemia. Pediatrics, 60:389, 1977. 56. Sepe, S. J., Harvey, M. P. H., Levy, H. L., et al.: An evaluation of routine follow-up blood screening of infants for phenylketonuria. New Eng!. J. Med., 300:606, 1979. 57. Scriver, C. R: PKU and beyond: When do costs exceed benefits? Pediatrics, 54 :616, 1974. 58. Shih, V. E., Levy, H. L., Karolkewiez, V., et al.: Galactosemia screening of newborns in Massachusetts. New Eng!. J. Med., 284:753, 1971. 59. Snyderman, S. E.: Branched chain ketoaciduria (maple syrup urine disease). Clinics in Perinatol., 3:41, 1976. 60. Snyderman, S. E., Norton, P. M., Roitman, E., et al.: Maple syrup urine disease with particular reference to dietotherapy. Pediatrics, 34 :454, 1964. 61. Van Pelt, A., and Levy, H. L.: Cost-benefit analysis of newborn screening for metabolic disorders. New Eng!. J. Med., 291 :1414, 1974.

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62. VanSprang, F. J., and Wadman, S. K.: Treatment of a patient with histidinemia. Acta Paediat. Scand., 56:493, 1967. 63. Williamson, VI., Koch, R., and Dobson, J. C.: PKU Collaborative Study-Current Status. A report presented at the 3rd IASSMD Congress, the Hague, the Netherlands, 1973. 64. Woolf, L. I., Griffiths, R., and Moncrieff, A.: Treatment of phenylketonuria with a diet low in phenylalanine. Brit..VIed. J., 1 :57, 1955. Box 140 VIedical College of Virginia Rich ,nond, Virginia 23298