July/August 2000. Vol. 2 . No. 4

Tandem mass spectrometry in newborn screening American College of Medical GeneticdAmerican Society of Hunzan Genetics Test and Technology Transfer Committee Working Group

Tandem mass spectrometry (MSIMS) has been used for sev- many times during analysis, so that one can detect and measure eral years to identify and measure carnitine esters in blood and butyl esters of acylcarnitines (by the signature ion at mlz 85) urine of children suspected of having inborn errors of metab- and the butyl esters of a-amino acids (by loss of a neutral 102 olism. Indeed, acylcarnitine analysis is a better diagnostic test fragment) in the same sample. for disorders of fatty acid oxidation than organic acid analysis MSIMS thus permits very rapid, sensitive and, with appro- because it can often detect these conditions when the patient is priate internal standards, accurate measurement of many dif- not acutely ill.' More recently, MSIMS has been used in pilot ferent types of metabolites with minimal sample preparation programs to screen newborns for these conditions and for dis- and without prior chromatographic separation. Because many orders of amino and organic acid metabolism as well. The pur- amino acidemias, organic acidemias, and disorders of fatty pose of this article is to describe MSIMS and discuss its poten- acid oxidation can be detected in 1 to 2 minutes, the system has tial role in newborn screening programs. adequate throughput to handle the large number of samples The mass spectrometer is a device that separates and quan- that are processed in newborn screening programs. Some con- tifies ions based on their masslcharge (mlz) ratios. In gas chro- ditions that can be diagnosed by MSIMS are listed in Table 1, matography-mass spectrometry of organic acids, for example, together with the compound(s) on which diagnosis is based. organic acid derivatives are first subjected to gas chromatogra- Amino acid quantitation by MSIMS is more accurate than phy and then enter the mass spectrometer, where each is ion- most methods now in use for newborn screening and would ized and fragmented and the abundance and m/z ratio of the thus provide more specific and sensitive screening for phenylk- various fragment ions are determined. etonuria,' maple syrup urine di~ease,~and homo~ystinuria.~ The modern tandem mass spectrometer usually consists of Analysis by MSIMS would also permit the screening menu to two quadrupole mass spectrometers separated by a reaction be expanded to include a number of disorders that are not chamber or collision cell; the latter is often another quadru- currently covered (Table l).5-h Among these are medium- pole. The mixture to be analyzed is subjected to a soft ioniza- tion procedure (e.g., fast atom bombardment or electrospray) chain acyl-CoA dehydrogenase (MCAD) deficiency and glu- to create quasimolecular ions, and is injected into the first taric acidemia type I (GAl),which are relatively common and quadrupole, which separates these parent ions from each other. difficult to detect before the onset of symptoms and whose These ions then pass (in order of mlz ratio) into the reaction outcome is substantially improved by early treatment. chamber, where they are fragmented; the mlz ratios ofthe frag- Infants with MCAD deficiency seem healthy in early infancy ments are then analyzed in the second quadrupole. Because but develop episodes of hypoketotic hypoglycemia during the separation of compounds in the mixture is by mass spectrom- first years of life; the first episode is fatal in 30% to 50% of etry instead of chromatography, the entire process, from ion- patients. Most of these deaths could be prevented if dietary ization and sample injection to data acquisition by computer, treatment and measures to prevent fasting were begun before takes only seconds. the onset of symptoms. Infants with GA1 develop normally The computer data can be analyzed in several ways. One can until they suddenly develop acute encephalopathy and irre- use aparent ion mode to obtain an array of all parent ions that versible striatal damage during the first 2 to 3 years of life. fragment to produce a particular daughter ion, or a neutral loss There is increasing evidence that striatal damage can usually be mode to obtain an array of all parent ions that lose a common prevented by L-carnitine and vigorous treatment of catabolic neutral fragment. Further, these scan functions can be changed episodes if begun before the onset of symptoms.

This guideline is designed primarily as an educational resource for inedical geneticists and other health care providers to help their1 provide quality medical genetic services. Adherence to this guideline does not izecessarily ensure n successfitl medical outcorne. This guideline should not be considered iizclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed toward obtaining the same results. In deterinitzing the propriety of any specific procedure or test, the geneticist should apply his or her own professional judgment to the speclfic clinical circzl~nstancespresented by the individual patient or specimen. It may be prudent, however, to docurnetlt in the patient's record the rationale for any signlficatzt deviation fronz tlzis guideline. ACMG/ASHG statement

Table 1 medical foods, and state laws and regulations regarding reim- Some disorders detectable by tandem mass spectrometry bursement vary. Disorder Diagnostic metabolite It has been argued that MSIMS analysis should not be used Amino asidemias in newborn screening until more is known about its sensitivity (false negatives) and specificity (false positives) for each of the Phenylketonuria Phenylalanine & tyrosine diagnosable disorders. Extensive experience with MSIMS, al- Maple syrup urine disease Leucine + isoleucine beit mostly with patients outside of the immediate newborn Homocystinuria (CBS deficiency) Methionine period, has shown that the number of false positives is very Citrullinemia Citrulline small. As was the case with all current screening methods, the number of false negatives will only be learned after newborn Hepatorenal tyrosinemia Methionine & tyrosine screening is implemented, and children that are not detected as Organic acidemias newborns are diagnosed later in life. Thus, as with all newborn Propionic acidemia C, acylcarnitine screening methods, screening should be accompanied by fol- Methylmalonic acidemia(s) C, acylcarnitine low-up sufficient to ensure that data on false negatives and false positives is collected. These considerations argue for pilot Isovaleric acidemia Isovalerylcarnitine demonstration programs with adequate resources to acquire Isolated 3-methyl~rotonylgl~cinemia 3-Hydroxyisovalerylcarnitine and report technical and clinical results. Glutaric acidemia (type I) Glutarylcarnitine Many conditions that can be detected by MSIMS, such as Hydroxymethylglutaric acidemia Hydroxymethylglutarylcarnitine citrullinemia, ~ropionicacidemia, and methylmalonic aci- demia, do not respond consistently to treatment. Nonetheless, Fatty acid oxidation disorders some patients do better with early diagnosis and treatment, SCAD deficiency C,,, acylcarnitines and early diagnosis can avoid trauma and expense to the family MCAD deficiency C,,,, , acylcarnitines and allow options for family planning to be considered before other affected siblings are born. VLCAD deficiency C ,,,,,,,,,,,, acylcarnitines The issue of informed consent for MS/MS screening is com- LCHAD and trifunctional protein C ,,,,, ,,,,,,, acyl- and 3-hydroxy deficiency acylcarnitines plicated, in part because uniformly effective therapies have not been developed for all the conditions the methodology can Glutaric acidemia type I1 Glutarylcarnitine detect and because it may detect previously unrecognized me- CPT-I1 deficiency C14,1~1,1~,1~I tabolites and/or disorders. An example is the detection of asymptomatic maternal 3-methylcrotonyl-CoA carboxylase deficiency by acylcarnitine screening of newborn blood spots.' It is important to note that MSIMS cannot replace current However, the computer parameters ofthe MSIMS can be set to programs to screen for biotinidase deficiency, hypothyroid- ignore certain molecular ions if a decision is made not to screen ism, hemoglobinopathies, virilizing adrenal hyperplasia, and for a particular disorder. galactosemia; these conditions cannot be identified by MSIMS In summary, MS/MS can provide substantial benefits to pa- at this time and must be detected by other means. tients and their families if thoughtfully integrated into new- Several issues must be considered before MSIMS is added to born screening programs, provided that sufficient funding is ongoing newborn screening programs. The instrument itself, made available to cover the costs of the additional and neces- including the computer and autosampler, is expensive, access sary personnel, medications, and medical foods. Indeed, the to alternate instruments is imperative in the event of break- expense and complexity of the instrumentation and the need down, and laboratory personnel must be trained extensively to for trained metabolic physicians to care for the additional pa- operate and maintain it. Nonetheless, if the cost of instrumen- tients could make it very difficult for states with small popula- tation is amortized over several years, MS/MS probably can be tions and/or few trained personnel to implement MSIMS, and added to existing newborn screening systems for an incremen- development of regional laboratories and services may well be tal cost on the order of $10 per sample. It is important to note necessary to address this need. that the cost of screening itselfwould be the same regardless of the number of tests added to the screening menu. Costs for References other screening components, however, e.g., patient retrieval, 1. Millington DS. Terada N, Kodo K. Chace DH. A review: carnitine and acylcarnitine verification of diagnosis, treatment, etc., would vary. analysis in the diagnosis of met~bolicd~seases: advantages of tandem mass spec- The inclusion of additional disorders in the newborn trometry In: Matsumoto I, editor. Advances in chemical diagnosis and treatment of screening menu could increase the number of patients identi- metabolic disorders. Vol I. New York: John Wiley & Sons, 199?:59-71. 2. Chace DH, Millington DS, Terada N, Kahler SG, Roe CR, Hofn~anLF. Rapid fied each year by 50% to loo%, and more physicians, nutri- diagnosis of phenylketonurla by quantitative analysls for phenylalanine and tionists, and genetic counselors will be needed to deal with tyroslne in neonatal blood spots by tandem mass spectrometry. Clin Chem 1993; their ongoing medical and nutritional care. Reimbursement 39;66-71. 3. Chace DH. Hillman SL. Millington DS, Kahler SG, Roe CR, Naylor EW. Rapid for the medical foods needed to treat these disorders must also diagnosis of maple syrup urlne disease in blood spots from newborns by tandem be addressed, because many third-party payers do not cover mass spectrometry. Clin Chem 1995;41;62-68.

Genety~Medicine ACMG/ASHG statement

4. Chace DH. Hillman SL, Millington DS. Rahler SG. Adam BM'. Levy HL. Rdpid America11 College of Medical Genetics/An~ericatzSociety of diagnosis of homocystinuria and other hypermethioninemias from newborns' blood spots by tandem mass spectrometry. CIin Chern 1996;42;349-355. Hutnan Genetics Test and 5. Van Hove IL, Zhang W, Kahler SG. Roe CR. Chen YT. Terada N. Chace DH, lafolla Techtlology Tratlsfer Cotiililittee IVorking Group: AR. Ding JH. Millington DS. Medrum-chain acyl-CoA dehydrogrnase Ih'lCAD) deficiency: diagnosis by acylc~rnitineanalysis in blood. Aln I Hlrrn Genct 1993;52: Joel Charrow 958-966. Children's Metnorial Hospital, Cllicago 6. Chace DH, Hillman SL. Van Hove ILK, Naylor EW. Rapid dragnosis of MCAD deficiency: quantitative analysis of octdnoylcarnrtine and other dcylcarnrtines in Stephen I. Goodman newborn blood spots by tandem mass spectrometry. Clin CI~etrl1997;43;2106- Uuiversity of Colorado Scl~oolof Medicine, Denver 2113. Edward R.G. McCabe 7. Gibson KM. Bennett MI. Naylor EW, Morton DH. 3-Methylcrotonyl-coenzyme A carboxylase deticiency rn AmlshIMennonite adults identrfied by detection of UCLA School of Medicine, Los Angeles increased acylcarnitines in blood spots of their chrldren. IPedlatr 1998;132;5 19- Piero Rinaldo 523. Mnyo Clitlic, Rocllester. Mitlrzesota