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Laboratory analysis of amino acids, 2018 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG)

      Abstract

      Amino acid abnormalities are observed in a broad spectrum of inheritedmetabolic diseases, such as disorders of amino acid metabolism and transport,organic acidemias, and ureagenesis defects. Comprehensive analysis of physiologicamino acids in blood, urine, and cerebrospinal fluid is typically performed in thefollowing clinical settings: evaluation of symptomatic patients in whom a diagnosisis not known; evaluation of previously diagnosed patients to monitor treatmentefficacy; evaluation of asymptomatic or presymptomatic (at-risk) relatives of knownpatients; follow-up testing for an abnormal newborn screen; and assessment ofdietary protein adequacy or renal function in general patient populations.Currently, the most common analytical method to quantify amino acids is based on ionexchange chromatography using post-column derivatization with ninhydrin andspectrophotometric detection. Newer methodologies are based on liquidchromatographic separation with detection by mass spectrometry or spectrophotometry.Amino acid analysis by nonseparation methods, such as the flow injection–tandem massspectrometric (MS/MS) method used for newborn screening, is considered inadequatefor the diagnosis of at-risk patients. The purpose of this document is to provide atechnical standard for amino acid analysis as applied to the diagnosis andmanagement of inborn errors of metabolism.

      Keywords

      Background

      Amino acids and proteins

      Amino acids serve as protein building blocks, metabolicintermediates, and substrates for energy production. By definition, amino acidscontain an amino group and a carboxyl group, and often contain anotherfunctional group (e.g., sulfhydryl, hydroxyl, or secondary amino- orcarboxyl-group). Proteins consist of 20 different amino acids, half of which aresynthesized endogenously (nonessential), while the remaining amino acids areobtained from dietary sources (essential). For almost a century, the detectionof amino acids depended on ninhydrin, a chemical that reacts specifically withprimary and secondary amines to produce a purple color that can be measuredspectrophotometrically. The development of the amino acid analyzer (based on ionexchange chromatographic separation of amino acids coupled with post-columnninhydrin derivatization) in the 1950s was an important advance that startedlarge-scale investigations into inborn errors of metabolism. While only 20 aminoacids are found in proteins, more than 100 amino acids occur in bodyfluids

      Bremer HJ. Disturbances of amino acid metabolism: clinicalchemistry and diagnosis. Urban & Schwarzenberg: Munich, Germany,1981.

      and at least 76 of these are of biologicalinterest.

      Thöny B, Duran M, Gibson KM. Laboratory guide to the methods inbiochemical genetics. Berlin: Springer, 2008.

      Amino acids are essential for humans, anddaily urinary losses are very small due to renal tubular reabsorption. Defectsin amino acid metabolism and transport cause inborn errors ofmetabolism
      • Camargo S.M.
      • Bockenhauer D.
      • Kleta R.
      Aminoacidurias: clinical and molecularaspects.
      and generalized aminoaciduria can be seen inpatients with impaired kidney function. In addition, several amino acids act asneurotransmitters, and abnormal levels are significant for certainneurometabolic disorders. Because the normal concentrations of most amino acidsin cerebrospinal fluid are quite low, analytical methods must be sensitive andaccurate. As amino acid disorders include both catabolic and anabolic pathways,analysis should be capable of detecting pathophysiological elevations andreductions of amino acid levels.

      Thöny B, Duran M, Gibson KM. Laboratory guide to the methods inbiochemical genetics. Berlin: Springer, 2008.

      ,
      • Grier R.E.
      • Gahl W.A.
      • Cowan T.
      • Bernardini I.
      • McDowell G.A.
      • Rinaldo P.
      Revised sections F7.5 (quantitative amino acidanalysis) and F7.6 (qualitative amino acid analysis): American College ofMedical Genetics Standards and Guidelines for Clinical GeneticsLaboratories, 2003.

      General description of amino acidemias and acidurias

      Inherited defects of amino acid catabolism, biosynthesis, ortransport have been known for many years, and new defects continue to beidentified.
      • Scriver C.R.
      • Lamm P.
      • Clow C.L.
      Plasma amino acids: screening, quantitation, andinterpretation.

      Scriver CR, Blau N, Duran M, Blaskovics ME, Gibson KM. Physician’sguide to the laboratory diagnosis of metabolic diseases. Berlin: Springer,2012.

      Clarke JTR. A clinical guide to inherited metabolic diseases.Cambridge University Press: Cambridge, UK, 2005.

      Zschocke J, Hoffmann GF. Vademecum metabolicum: diagnosis andtreatment of inborn errors of metabolism. Milupa Metabolics: Friedrichsdorf,Germany, 2011.

      Saudubray JM, van den Berghe G, Walter JH. Inborn metabolicdiseases: diagnosis and treatment. Berlin: Springer, 2014.

      Cystinuria (MIM 220100) and alkaptonuria(MIM 203500) were among the first inherited metabolic disorders described byArchibald Garrod.
      • Garrod A.E.
      The Lancet. The incidence of alkaptonuria: a studyin chemical individuality.
      Phenylketonuria (PKU) (MIM 261600) (ref.
      • Williams R.A.
      • Mamotte C.D.
      • Burnett J.R.
      Phenylketonuria: an inborn error of phenylalaninemetabolism.
      ), the inherited amino acid disorder with thehighest impact on public health, was first described in 1934 (ref.
      • Fölling A.
      Uber ausscheidung von phenylbrenztraubensaure in denharn als stoffwechselanomalie in verbindung mit imbezillitat.
      ) and prompted the initiation of routinenewborn screening in the early 1960s (ref.
      • Guthrie R.
      • Susi A.
      A simple phenylalanine method for detectingphenylketonuria in large populations of newborn infants.
      ).
      Amino acid disorders are clinically and biochemically heterogeneous.They are characterized biochemically by the accumulation or dissipation ofpathological amounts of normal metabolites, or metabolites that are not presentunder normal physiological conditions, but are produced from alternativepathways in response to loss of a specific gene product’s function (typically anenzyme or transporter protein). Primary disorders of amino acid metabolisminclude defects in the catabolic pathways of aromatic amino acids (e.g.,phenylketonuria and tyrosinemia), sulfur-containing amino acids (classichomocystinuria, MIM 236200), branched-chain amino acids (maple syrup urinedisease, MIM 248600), urea cycle intermediates (including ornithinetranscarbamylase deficiency, MIM 311250; classic citrullinemia, MIM 215700; andargininosuccinic aciduria, MIM 207900), and several others. The broad categoryof amino acid disorders also includes transport defects such as cystinuria (MIM220100), lysinuric protein intolerance (LPI, MIM 22700), citrin deficiency (MIM605814 and 603471), and Hartnup disorder (MIM 234500) (ref.
      • Camargo S.M.
      • Bockenhauer D.
      • Kleta R.
      Aminoacidurias: clinical and molecularaspects.
      ). Several other amino acid disorders arecharacterized by abnormally low amino acid concentrations caused by theinability to synthesize a nonessential amino acid. Examples include asparaginesynthetase deficiency (MIM 615574) (ref.
      • Ruzzo E.K.
      • Capo-Chichi J.M.
      • Ben-Zeev B.
      • et al.
      Deficiency of asparagine synthetase causescongenital microcephaly and a progressive form ofencephalopathy.
      ) and glutaminesynthetase deficiency (MIM 610015) (ref.
      • Haberle J.
      • Gorg B.
      • Rutsch F.
      • et al.
      Congenital glutamine deficiency with glutaminesynthetase mutations.
      ), as well asremethylation
      • Iacobazzi V.
      • Infantino V.
      • Castegna A.
      • Andria G.
      Hyperhomocysteinemia: related genetic diseases andcongenital defects, abnormal DNA methylation and newborn screeningissues.
      and serine deficiencydisorders.
      • van der Crabben S.N.
      • Verhoeven-Duif N.M.
      • Brilstra E.H.
      • et al.
      An update on serine deficiencydisorders.
      Recently, a defect in branched-chainketoacid dehydrogenase kinase
      • Novarino G.
      • El-Fishawy P.
      • Kayserili H.
      • et al.
      Mutations in BCKD-kinase lead to a potentiallytreatable form of autism with epilepsy.
      has been reported that is characterizedby increased catabolism leading to abnormally low concentrations ofbranched-chain amino acids.
      Clinical findings associated with amino acid disorders are diversebut are often disease-specific. Signs and symptoms include overwhelming illnessresulting from systemic biochemical derangements such as hyperammonemia,hypoglycemia, metabolic acidosis, or respiratory alkalosis. Several amino aciddisorders also cause neurologic abnormalities including seizures, hypotonia,lethargy, coma, developmental delay, unexplained intellectual disability,failure to thrive, or autistic behaviors. Depending on the particular disorder,patients may present from birth to adulthood with one or more organ systemaffected.
      It is important to take into consideration that certain amino acidconditions (e.g., aminoacylase 1 deficiency [MIM 609924] and aromatic L-aminoacid decarboxylase [AADC] deficiency [MIM 608643]) may not be amenable todetection by amino acid analysis due to the nature of the intermediatesproduced. In the case of aminoacylase 1 deficiency, abnormal urinary excretionof N-acetylated L-amino acids can be detected by organic acid analysis using gaschromatography–mass spectrometry. Therefore, a comprehensive metabolic workupideally should include at a minimum urine organic acid and plasma acylcarnitineanalysis, in addition to amino acid analysis.

      Secondary amino acid abnormalities

      Amino acid abnormalities are not only associated with inborn errorsof metabolism but are also sensitive markers of an individual’s nutritionalstate and can indicate dysfunction of various organs including liver, kidney,and muscle. For example, liver is the primary organ for the metabolism ofmethionine and tyrosine, leading to their elevations in metabolic andnonmetabolic causes of hepatic dysfunction. Because acquired changes in aminoacid concentrations may be subtle during the malfunctioning of a particularorgan, amino acid analysis should have high accuracy and reproducibility toenable interpretation of these changes.

      Thöny B, Duran M, Gibson KM. Laboratory guide to the methods inbiochemical genetics. Berlin: Springer, 2008.

      Incidence

      The combined incidence for all amino acidopathies is more frequentthan 1 in 10,000 (ref.

      Saudubray JM, van den Berghe G, Walter JH. Inborn metabolicdiseases: diagnosis and treatment. Berlin: Springer, 2014.

      ). This estimate does not include otherinborn errors of metabolism (e.g., organic acidemias or congenital lacticacidemias) that may need amino acid analysis for comprehensive diagnosis andtreatment monitoring. The incidence of PKU in Caucasian populations is between 1in 10,000 and 1 in 15,000 live births.
      • National Institutes of Health Consensus DevelopmentPanel.
      National Institutes of Health Consensus DevelopmentConference Statement: phenylketonuria: screening and management, October16-18, 2000.
      The incidence of ureacycle disorders is estimated to be at least 1:35,000births,

      Ah Mew N, Lanpher BC, Gropman A, Chapman KA, Simpson KL, Summar ML.Urea cycle disorders overview. In: Pagon RA, Adam MP, Ardinger HH, et al., eds.GeneReviews. University of Washington (Seattle) and the NCBI (Washington, DC)1993–2016.

      with ornithine transcarbamylase deficiencybeing the most common urea cycle defect.

      Lichter-Konecki U, Caldovic L, Morizono H, Simpson K Ornithinetranscarbamylase deficiency. In: Pagon RA, Adam MP, Ardinger HH, et al., eds.GeneReviews. University of Washington (Seattle) and the NCBI (Washington, DC)1993–2016.

      Mode of inheritance

      The majority of disorders of amino acid metabolism or transport areinherited as autosomal recessive traits. Exceptions include disorders such asX-linked ornithine transcarbamylase deficiency (MIM 311250), methionineadenosyltransferase deficiency (MIM 250850, an autosomal dominant form ofhypermethioninemia caused by heterozygosity for the p.R264H variant in theMAT1A gene),
      • Marcao A.
      • Couce M.L.
      • Nogueira C.
      • et al.
      Newborn screening for homocystinuria revealed a highfrequency of MAT I/III deficiency in Iberian Peninsula.
      and hawkinsinuria (MIM140350, an autosomal dominant disorder of tyrosine metabolism caused bydeficiency of 4-hydroxyphenylpyruvic acid dioxygenase).
      • Tomoeda K.
      • Awata H.
      • Matsuura T.
      • et al.
      Mutations in the 4-hydroxyphenylpyruvic aciddioxygenase gene are responsible for tyrosinemia type III andhawkinsinuria.

      Methods

      The laboratory technical standard was informed by a review of theliterature, including any current guidelines, and expert opinion. Resourcesconsulted included PubMed (search terms: inborn errors of metabolism, aminoacidopathies, amino acid analysis methods, ion exchange chromatography [& aminoacid], ultra-performance liquid chromatography [& amino acid], tandem massspectrometry [& amino acid & liquid OR gas chromatography]), the AmericanCollege of Medical Genetics and Genomics (ACMG) Standards and Guidelines forClinical Genetics Laboratories, Clinical and Laboratory Standards Institute (CLSI)guidelines, CLIA regulations, and the Centers for Disease Control and PreventionMorbidity and Mortality Weekly Report (MMWR) on Good Laboratory Practices forBiochemical Genetics Testing and Newborn Screening for Inherited MetabolicDisorders. When the literature provided conflicting evidence about a topic or whenthere was insufficient evidence, the authors used expert opinion to inform therecommendations. Expert opinion included the coauthors of the document, members ofthe ACMG Laboratory Quality Assurance Committee, as well as the experts consultedoutside of the Committee but acknowledged in this document. Any conflicts ofinterest for workgroup members or consultants are listed. A draft was delivered tothe ACMG Board of Directors for review and member comment. The draft document wasposted on the ACMG website and an email link was sent inviting ACMG members toprovide comment. All comments were assessed by the authors. When appropriate,additional evidence was included to address member comments and the draft wasamended. Both member comments and author responses were reviewed by a representativeof the ACMG Laboratory Quality Assurance Committee and by the ACMG Board ofDirectors. The final document was approved by the ACMG Board of Directors. Thisupdated standard replaces the previous version in Section F: Clinical BiochemicalGenetics, American College of Medical Genetics and Genomics Standards and Guidelinesfor Clinical Genetics Laboratories (2008 Edition, Revised 02/2007), section F7.5(ref.

      American College of Medical Genetics and Genomics. Standards andguidelines for clinical genetics laboratories. 2008. http://www.acmg.net/PDFLibrary/Standards-Guidelines-Clinical-Biochemical-Genetics.pdf.

      ).

      Preanalytical requirements

      Specimen requirements

      Plasma is the preferred specimen for the evaluation of most primarydisorders of amino acid metabolism. Lithium, or sodium, heparin is the preferredanticoagulant. Ethylenediaminetetraacetate (EDTA) plasma yields similar resultsto heparinized plasma with minor differences that are typically notdiagnostically significant.

      Hommes FA. Techniques in diagnostic human biochemical genetics: alaboratory manual. Wiley: New York, US, 1990.

      • Parvy P.R.
      • Bardet J.I.
      • Kamoun P.P.
      EDTA in vacutainer tubes can interfere with plasmaamino acid analysis.
      • Chuang C.K.
      • Lin S.P.
      • Lin Y.T.
      • Huang F.Y.
      Effects of anticoagulants in amino acid analysis:comparisons of heparin, EDTA, and sodium citrate in vacutainer tubes forplasma preparation.
      Yet, it should be noted that EDTA can reactwith ninhydrin and produce a ninhydrin positive contaminant that may lead tointerference.
      • Chuang C.K.
      • Lin S.P.
      • Lin Y.T.
      • Huang F.Y.
      Effects of anticoagulants in amino acid analysis:comparisons of heparin, EDTA, and sodium citrate in vacutainer tubes forplasma preparation.
      Sodium citrate–treated blood yieldslower values for most amino acids. Laboratories should have establishedprocedures for procuring specimens using their preferred anticoagulant. Fastingplasma samples are recommended to avoid erroneous interpretation of severaldisorders, particularly those involving amino acid synthesis that arecharacterized by low concentrations of certain aminoacids.

      Thöny B, Duran M, Gibson KM. Laboratory guide to the methods inbiochemical genetics. Berlin: Springer, 2008.

      Serum should not be used because clottingtimes may lead to analyte-specific artifacts.
      • Chuang C.K.
      • Lin S.P.
      • Lin Y.T.
      • Huang F.Y.
      Effects of anticoagulants in amino acid analysis:comparisons of heparin, EDTA, and sodium citrate in vacutainer tubes forplasma preparation.
      Testing can also beperformed on amino acids eluted from dried-blood spots (DBS).
      Urine is the appropriate specimen for the identification ofcompounds that are efficiently cleared by the kidney (e.g., argininosuccinicacid [ASA]) or specific disorders of renal amino acid transport (e.g., renalFanconi syndrome or cystinuria). However, urine is generally less reliable forfirst-tier investigations of primary amino acid disorders due to effectivetubular reabsorption of a majority of amino acids, and susceptibility tointerference from medications. Urine amino acid analyses may often be orderederroneously in place of, or unnecessarily in addition to, plasma amino acidtesting; therefore clinical indications should be preemptively reviewed (ifavailable). When urine is analyzed, it should be collected without preservativesor fecal contamination.
      The analysis of cerebrospinal fluid (CSF) is useful for diagnosingseveral biochemical genetic disorders, including asparagine and glutaminesynthetase deficiencies, serine deficiency disorders, and most notably, glycineencephalopathy (nonketotic hyperglycinemia [NKH; MIM 605899]). In NKH, CSFshould be collected and analyzed along with a simultaneously collected plasmasample, so that the CSF:plasma glycine ratio can becalculated.
      • Applegarth D.A.
      • Edelstein A.D.
      • Wong L.T.
      • Morrison B.J.
      Observed range of assay values for plasma andcerebrospinal fluid amino acid levels in infants and children aged 3 monthsto 10 years.
      • Steiner R.D.
      • Sweetser D.A.
      • Rohrbaugh J.R.
      • Dowton S.B.
      • Toone J.R.
      • Applegarth D.A.
      Nonketotic hyperglycinemia: atypical clinical andbiochemical manifestations.
      • Applegarth D.A.
      • Toone J.R.
      Nonketotic hyperglycinemia (glycine encephalopathy):laboratory diagnosis.
      It is noteworthy that CSF should becollected into preservative-free collection tubes; however, fluoride oxalate andlithium heparin tubes also suffice. CSF should be stored frozen if notimmediately analyzed. Specimens contaminated with blood should be interpretedwith caution as most amino acids are present in blood at much higherconcentrations than in CSF. In such cases, a repeat CSF specimen may beadvised.

      Sample handling, shipping, and storage

      Plasma should be centrifuged immediately to reduce the influence ofother blood constituents on the free amino acid concentrations. Glutamate,aspartate, and taurine have high intracellular levels and increase by hemolysis.Plasma should be kept at –20 °C (for long-term storage, –70 °C) until analysisto slow the decomposition of glutamine.

      Thöny B, Duran M, Gibson KM. Laboratory guide to the methods inbiochemical genetics. Berlin: Springer, 2008.

      Serum, if used, should becentrifuged after the standard time allowed for clotting (usually 30 minutes).However, the added time for clotting can lead to artifacts from the deaminationof arginine to ornithine by red blood cell arginase, and by the release ofoligopeptides. Postprandial samples should be avoided and information aboutcurrent medications (e.g., antibiotics or antiseizure medications) or dietarystatus may be informative. Following centrifugation, samples should be keptfrozen (–20 °C; for long-term storage, –70 °C) until the time of analysis. CSFand urine specimens should be frozen as soon as possible after collection andstored frozen prior to analysis. Samples sent to the laboratory from an outsidereferral source should be shipped on dry ice via an overnight courier.
      Finally, it is important to note that amino acids with terminalsulfhydryl groups (e.g., cysteine and homocysteine) readily form disulfide bondsleading to their association with plasma proteins,
      • Schaefer A.
      • Piquard F.
      • Haberey P.
      Plasma amino-acids analysis: effects of delayedsamples preparation and of storage.
      ,
      • Sahai S.
      • Uhlhaas S.
      Stability of amino acids in humanplasma.
      particularlyalbumin.
      • Refsum H.
      • Ueland P.M.
      • Svardal A.M.
      Fully automated fluorescence assay for determiningtotal homocysteine in plasma.
      Therefore, the accurate quantification ofthese amino acids necessitates separate techniques utilizing strong reducingagents.
      • Demaster E.G.
      • Shirota F.N.
      • Redfern B.
      • Goon D.J.
      • Nagasawa H.T.
      Analysis of hepatic reduced glutathione, cysteineand homocysteine by cation exchange high-performance liquid chromatographywith electrochemical detection.
      ,
      • Magera M.J.
      • Lacey J.M.
      • Casetta B.
      • Rinaldo P.
      Method for the determination of total homocysteinein plasma and urine by stable isotope dilution and electrospray tandem massspectrometry.
      This is particularly true for plasmahomocysteine, an important biomarker for disorders of the methionine cycle,folate and vitamin B12 metabolism, and a risk factor forcardiovascular disease.

      Preanalytical variables

      Sample collection and handling are discussed in the previoussection. Interference from coeluting, ninhydrin-positive compounds occur withcertain antibiotics (e.g., ampicillin), contrast dyes, and other medications.Amino acid concentrations can also be influenced by anticonvulsants (e.g.,hyperglycinemia with valproate use), nutritional status (e.g., intravenous [IV]nutrition or starvation), clinical status (e.g., fever, infections, and liver orkidney dysfunction), treatment of acute lymphoblastic leukemia (ALL) usingL-asparaginase,
      • McCredie K.B.
      • Ho D.H.
      • Freireich E.J.
      L-asparaginase for the treatment ofcancer.
      ,
      • Shrivastava A.
      • Khan A.A.
      • Khurshid M.
      • Kalam M.A.
      • Jain S.K.
      • Singhal P.K.
      Recent developments in L-asparaginase discovery andits potential as anticancer agent.
      as well as bacterial contamination. Thesefactors may affect results and should be taken into account when interpretingresults.

      Clinical indications for testing

      Clinical presentations of different disorders of amino acidmetabolism are variable and often nonspecific. Onset of symptoms may occur inthe neonatal period or as late as adulthood. Amino acid analysis should beconsidered in a wide variety of clinical situations, such as:
      • 1.
        Lethargy, coma, seizures, or vomiting in aneonate
      • 2.
        Hyperammonemia
      • 3.
        Failure to thrive
      • 4.
        Electrolyte abnormalities, including metabolicacidosis or respiratory alkalosis
      • 5.
        Lactic acidemia
      • 6.
        Unexplained developmental delay or intellectualdisability
      • 7.
        Abnormal newborn screening results
      • 8.
        A previously diagnosed sibling
      • 9.
        Clinical presentation suggestive of a specificamino acid disorder
      • 10.
        Monitoring treatment efficacy (e.g., dietary) ofknown patients
      Amino acid analysis can also be used for dietary monitoring(including assessment of dietary protein adequacy or renal function) ofmetabolic patients on protein restriction and/or special formulas, irrespectiveof pathway affected, or in the general patient population not affected with aninherited metabolic disorder. Indeed, specific amino acid analytes can be usedas biomarkers to assess the risk of developing more common medical conditions,including diabetes
      • Knebel B.
      • Strassburger K.
      • Szendroedi J.
      • et al.
      Specific metabolic profiles and their relationshipto insulin resistance in recent-onset type 1 and type 2diabetes.
      and cardiovasculardisease,
      • Wurtz P.
      • Havulinna A.S.
      • Soininen P.
      • et al.
      Metabolite profiling and cardiovascular event risk:a prospective study of 3 population-based cohorts.
      although these applications are outside thescope of these Standards.
      The laboratory should be aware of the clinical indication(s) fortesting, including the need for immediate analysis in a critically ill patient,so that testing can be triaged, results interpreted appropriately, andadditional testing recommended as needed. Depending on the clinical situation,amino acid analysis is often performed together with urine organic acids, plasmacarnitine (free and total), and plasma acylcarnitine profile analysis as part ofa comprehensive metabolic evaluation. Ideally, these tests should all beperformed by the same laboratory, and results integrated into a completeinterpretation.

      Method validation

      Calibration and quantitation

      The quantitation of amino acid concentrations should be performedin relation to known reference or calibration standards. Amino acid calibrationmixtures are available from several commercial sources. Some manufacturers haveomitted certain relatively unstable amino acids (e.g., asparagine) from thesemixtures; in these cases, freshly prepared solutions of the missing compoundscan be added to the commercial standards to form a complete set. Performance ofcalibration material should be verified either with weighed standards, or withamino acid calibrators obtained from an independent source (e.g., NationalInstitute of Standards and Technology [NIST]). Instrument calibration should beverified, and/or the instrument should be recalibrated, at regular intervals asestablished by the laboratory per manufacturer’s recommendations, and asrequired by CLIA ’88. This should also occur with the introduction of newcolumns, reagent lots, or following service to the instrument or itscomponents.
      Quantitation using ion exchange chromatography (IEC) orultraperformance or high-performance liquid chromatography (U/HPLC) coupled withspectrophotometric
      • Narayan S.B.
      • Ditewig-Meyers G.
      • Graham K.S.
      • Scott R.
      • Bennett M.J.
      Measurement of plasma amino acids byultraperformance® liquid chromatography.
      or fluorometric
      • Pappa-Louisi A.
      • Nikitas P.
      • Agrafiotou P.
      • Papageorgiou A.
      Optimization of separation and detection of6-aminoquinolyl derivatives of amino acids by using reversed-phase liquidchromatography with on line UV, fluorescence and electrochemicaldetection.
      detection should beperformed using at least one internal standard (IS). The selected standardshould elute at a unique position in the chromatogram and not interfere with theanalysis of other nearby compounds. Quantitative results should be calculatedusing an IS method that adjusts for the amount of IS in the patient samplerelative to that in the calibration mixture.

      Hommes FA. Techniques in diagnostic human biochemical genetics: alaboratory manual. Wiley: New York, US, 1990.

      For methods that employmass spectrometric detection, stable isotope analogs for each amino acid shouldbe used as ISs when available. The lack of ISs for specific analytes may reducethe performance of stable isotope dilution, mass-spectrometric methods for thoseanalytes.
      • Vogeser M.
      • Seger C.
      Quality management in clinical application of massspectrometry measurement systems.
      For the majority of amino acids withclinical significance, stable isotope standards are commercially available. Thelaboratory should establish protocols to determine and periodically verify theirmethod’s linear range, analytical measurement range, and lower limit ofdetection (see CLSI document C24-A3: Clinical and Laboratory StandardsInstitute. Statistical Quality Control for Quantitative Measurement Procedures:Principles and Definitions; Approved Guideline—Third Edition). Procedures shouldbe in place for analytical values that fall outside of an assay’s performancelimits.

      Reference ranges

      Laboratory-specific reference ranges should be determined for eachsample type (plasma, DBS, urine, and CSF), using guidelines as defined by alaboratory’s policy and procedures for test validation (e.g., CLSI documentEP28-A3c: Defining, Establishing, and Verifying Reference Intervals in theClinical Laboratory; Approved Guideline—Third Edition). It is important to notethat amino acid levels obtained from different sample types (e.g., plasma versusDBS) are not comparable and should not be used interchangeably for monitoringpurposes (e.g., phenylalanine). Anonymized samples from a general patientpopulation are acceptable for generating a laboratory-specific reference rangewhen patients with an identified diagnosis are excluded. These ranges should becategorized by age and sex when appropriate and verified on a regular intervalas required by CLIA ’88. When literature-based ranges are used, they should beverified using laboratory-specific methods. Because amino acid levels varysignificantly with age, reference intervals should be age-specific.

      Testing personnel

      Laboratory personnel performing quantitative amino acid analysisshould be documented to have received appropriate training and demonstratedcompetency in the performance of the test. In addition, laboratory personnelshould satisfy CLIA ’88 requirements for high-complexity testing and have, at aminimum, an associate degree in a laboratory science or medical laboratorytechnology from an accredited institution. More comprehensive requirements mayapply to laboratories in some US states (see General Policies B: PersonnelPolicies of the ACMG Standards and Guidelines for Clinical GeneticsLaboratories, 2008 Edition, Revised 02/2007 (https://www.acmg.net/acmg/Publications/Standards___Guidelines/Personnel_Policies.aspx).

      Analysis of amino acids

      Analytical methods

      Overview of methods

      To analyze free amino acids in physiological specimens,de-proteinization of the sample is necessary. For comprehensive,quantitative amino acid analysis, this is commonly achieved by proteinprecipitation using sulfosalicylic acid (SSA) or trichloroacetic acid (TCA),followed by centrifugation, and/or filtration. Supernatants or filtrates arediluted with buffer of appropriate pH prior to analysis. For limited aminoacid analysis such as for newborn screening by flow injection tandem massspectrometry, extraction of amino acids from DBS is achieved usingacetonitrile, methanol, or an admixture of these solvents. Samplepreparation should include the addition of at least one IS to control forrun-to-run variation in sample extraction and analysis. For urine, thecreatinine concentration is measured and useful for adjusting the samplevolume prior to analysis. Postanalysis, amino acid concentrations are alsooften normalized to creatinine molality (e.g., mmol/mol creatinine). It isimportant to note that the Jaffé method, in which creatinine reacts withalkaline picrate to form a complex absorbing at 480–520 nm, is stillcommonly used to determine creatinine, despite well-recognized interferenceby bilirubin, protein, ketones, ketoacids, fatty acids, and othercompounds.
      • Spencer K.
      Analytical reviews in clinical biochemistry: theestimation of creatinine.
      HPLC
      • Tsikas D.
      • Wolf A.
      • Frolich J.C.
      Simplified HPLC method for urinary and circulatingcreatinine.
      and particularlyliquid chromatography–tandem mass spectrometry(LC-MS/MS)
      • Takahashi N.
      • Boysen G.
      • Li F.
      • Li Y.
      • Swenberg J.A.
      Tandem mass spectrometry measurements of creatininein mouse plasma and urine for determining glomerular filtrationrate.
      methods for measuring creatinineoffer significant improvements in sensitivity and specificity over the Jaffémethod. Urine amino acid levels can also be reported from a 24-hour specimencollection (e.g., μmol/24 hours).
      Analysis of the full physiologic amino acid profile can beachieved using several methods.
      • Kaspar H.
      • Dettmer K.
      • Gronwald W.
      • Oefner P.J.
      Advances in amino acid analysis.
      IEC with post-column derivatizationusing ninhydrin and spectrophotometric detection has been the most widelyperformed method for several decades and is still a commonly used clinicallaboratory method. Alternative methods have been developed that takeadvantage of the selectivity of mass spectrometry and the reduced analyticaltime of ultraperformance liquid chromatography. When coupled with techniquessuch as ultraperformance liquid chromatography or gas chromatography, higherthroughput and enhanced performance of amino acid analysis has beenreported.
      • Narayan S.B.
      • Ditewig-Meyers G.
      • Graham K.S.
      • Scott R.
      • Bennett M.J.
      Measurement of plasma amino acids byultraperformance® liquid chromatography.
      ,
      • Piraud M.
      • Vianey-Saban C.
      • Petritis K.
      • Elfakir C.
      • Steghens J.P.
      • Bouchu D.
      Ion-pairing reversed-phase liquidchromatography/electrospray ionization mass spectrometric analysis of 76underivatized amino acids of biological interest: a new tool for thediagnosis of inherited disorders of amino acid metabolism.
      • Kaspar H.
      • Dettmer K.
      • Chan Q.
      • et al.
      Urinary amino acid analysis: a comparison ofiTRAQ-LC-MS/MS, GC-MS, and amino acid analyzer.
      • Waterval W.A.
      • Scheijen J.L.
      • Ortmans-Ploemen M.M.
      • Habets-van der Poel C.D.
      • Bierau J.
      Quantitative UPLC-MS/MS analysis of underivatisedamino acids in body fluids is a reliable tool for the diagnosis andfollow-up of patients with inborn errors of metabolism.
      • Le A.
      • Ng A.
      • Kwan T.
      • Cusmano-Ozog K.
      • Cowan T.M.
      A rapid, sensitive method for quantitative analysisof underivatized amino acids by liquid chromatography-tandem massspectrometry (LC-MS/MS).
      Such methods also require lesssample volume when compared with IEC. However, it is recognized that massspectrometric methods with additional complexities present relatively morechallenges to laboratories that do not have the necessary expertise.Furthermore, each methodology may present a different source of variance(e.g., recovery of amino acids may be affected by the solvent used toprecipitate protein in plasma samples). Thus, it is the laboratories’responsibility to determine the possible pitfalls of their specific test andto validate their methodology to address possible challenges andlimitations.
      A number of different instrument configurations arecommercially available for amino acid analysis; these instruments havepublished methodologies that should be validated in the individuallaboratory. Also see General Policies C7: Levels of Development of aDiagnostic Test, and C8: Test Validation of the ACMG Standards andGuidelines for Clinical Genetics Laboratories, 2008 Edition, revised 02/2007 (https://www.acmg.net/acmg/Publications/Standards___Guidelines/General_Policies.aspx).

      Ion exchange chromatography (IEC)

      IEC is the most commonly used method of amino acid analysis andrecent method updates have improved observed matrixeffects.

      Pickering Laboratories. Amino acid analysis part 1. https://www.pickeringlabs.com/library/primers/amino-acid-analysis-part-1/. Accessed 26 December 2016.

      IEC requires derivatization of aminoacids for detection. This can be accomplished prior to separation of aminoacids (pre-column derivatization) using o-phthalaldehyde (OPA) orphenylisothiocyanate (PITC), or following separation (post-columnderivatization) using ninhydrin.
      Post-column ninhydrin derivatization has been the preferredmethod for many years by a majority of laboratories, although methods basedon newer technologies are now gaining in popularity. In this method,compounds are simultaneously detected at wavelengths of 570 nm and 440 nm using a dual-wavelength spectrophotometer. Laboratories may report valuesfrom the 570 nm channel only, from the sum of the two channels, or from acombination of both (e.g., hydroxyproline and proline from the 440 nm channel, and the remainder of the amino acids from the 570 nm channel).Identification of amino acids following chromatographic separation is basedon retention time. Most systems are capable of resolving and quantifyingroughly 40 amino acid peaks in a typical patient sample (the exact numbervaries as some systems do not resolve all amino acids from neighboringpeaks). Laboratories, however, may elect to report a smaller subset ofclinically relevant compounds.
      Each chromatogram should be visually inspected for runperformance, as well as for atypical peaks that may not be included in peakidentification tables, including Δ-1-pyrroline-5-carboxylate,homocitrulline, ASA, and alloisoleucine (especially when the ratio ofbranched-chain amino acids is disturbed). For ninhydrin-based systems withspectrophotometric detection, an identification based on retention timecomparison can be supported by standard spiking and by calculating the ratioof response at 570 nm to that at 440 nm. The 570/440 ratio can beestablished for all standard compounds, and compared with patient values toconfirm peaks where coeluting interfering substances are suspected. Indetermining these ratios, peak baselines should be carefully examined suchthat potential artifacts from baseline fluctuations areeliminated.
      • Davies M.
      Protein and peptide analysis. Using a dedicated amino acid analyser.The applications book..
      Alternatively, where coelution isknown to occur for amino acids that are diagnostically significant (e.g.,homocitrulline and methionine on certain IEC systems), reinjection of thesample and analysis using an alternative gradient method may be useful toverify identity. Additionally, ASA identification can be confirmed byheating the sample at 90 °C and observing the change of retention time withanhydrous ASA formation. Quantitation should be based on the recovery of theIS in each specimen compared with the recovery of the IS in the calibrationmixture.

      Ultra Performance Liquid Chromatography (UPLC)

      UPLC is increasingly being utilized due to its rapid separationof amino acids in approximately 35 minutes compared with 2 hours for atypical IEC analysis. One example of an UPLC application for plasma, serum,CSF, and urine amino acids analysis is a method that utilizes pre-columnderivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Thisreagent rapidly reacts with both primary and secondary amines to producestable chromophores that can be detected by ultraviolet spectrophotometry at260 nm. Derivatized samples have a stable baseline, superb peak separation,and excellent peak symmetry for all amino acids including those that eluteat the end of analysis. Using norvaline as an IS, correlation of this assaywith IEC was satisfactory with adequate detection of most significant aminoacids.
      • Narayan S.B.
      • Ditewig-Meyers G.
      • Graham K.S.
      • Scott R.
      • Bennett M.J.
      Measurement of plasma amino acids byultraperformance® liquid chromatography.
      The superior resolution of UPLC comparedwith IEC confers better sensitivity for this method. Currently, the onlyknown negative characteristic of this technique is the inability to separateASA from ethanolamine with published methods. Laboratories using UPLC shouldconsider validating a secondary analysis for specifically measuring ASA toconfirm a suspected elevation. Noteworthy is the observation thatalloisoleucine is separated from other branched-chain amino acids, negatingthe need for a special analysis to confirm the potential of maple syrupurine disease.

      Tandem mass spectrometry methods

      Within the last 15 years, methods utilizing liquidchromatography–electrospray–tandem mass spectrometry (LC-ESI-MS/MS) havebeen developed for amino acid analysis in plasma, urine, and CSF and appliedto clinical practice.
      • Piraud M.
      • Vianey-Saban C.
      • Petritis K.
      • Elfakir C.
      • Steghens J.P.
      • Bouchu D.
      Ion-pairing reversed-phase liquidchromatography/electrospray ionization mass spectrometric analysis of 76underivatized amino acids of biological interest: a new tool for thediagnosis of inherited disorders of amino acid metabolism.
      ,
      • Waterval W.A.
      • Scheijen J.L.
      • Ortmans-Ploemen M.M.
      • Habets-van der Poel C.D.
      • Bierau J.
      Quantitative UPLC-MS/MS analysis of underivatisedamino acids in body fluids is a reliable tool for the diagnosis andfollow-up of patients with inborn errors of metabolism.
      ,
      • Le A.
      • Ng A.
      • Kwan T.
      • Cusmano-Ozog K.
      • Cowan T.M.
      A rapid, sensitive method for quantitative analysisof underivatized amino acids by liquid chromatography-tandem massspectrometry (LC-MS/MS).
      ,
      • Ross P.L.
      • Huang Y.N.
      • Marchese J.N.
      • et al.
      Multiplexed protein quantitation in Saccharomycescerevisiae using amine-reactive isobaric tagging reagents.
      • Dietzen D.J.
      • Weindel A.L.
      • Carayannopoulos M.O.
      • et al.
      Rapid comprehensive amino acid analysis by liquidchromatography/tandem mass spectrometry: comparison to cation exchange withpost-column ninhydrin detection.
      • Held P.K.
      • White L.
      • Pasquali M.
      Quantitative urine amino acid analysis using liquidchromatography tandem mass spectrometry and aTRAQ reagents.
      • Filee R.
      • Schoos R.
      • Boemer F.
      Evaluation of physiological amino acids profiling bytandem mass spectrometry.
      • Prinsen H.C.
      • Schiebergen-Bronkhorst B.G.
      • Roeleveld M.W.
      • et al.
      Rapid quantification of underivatized amino acids inplasma by hydrophilic interaction liquid chromatography (HILIC) coupled withtandem mass-spectrometry.
      LC-ESI-MS/MS methods offer theadvantages of increased specificity and throughput as compared withtraditional IEC-UV/VIS methods.
      • Kaspar H.
      • Dettmer K.
      • Chan Q.
      • et al.
      Urinary amino acid analysis: a comparison ofiTRAQ-LC-MS/MS, GC-MS, and amino acid analyzer.
      The main challenges for developing arobust LC-ESI-MS/MS amino acid test include the necessity to control for ionsuppression or enhancement of the numerous analytes included in theanalysis, and the separation of isobaric amino acid species. Additionally,these instruments usually require more frequent calibration thanIEC-UV/VIS
      • Prinsen H.C.
      • Schiebergen-Bronkhorst B.G.
      • Roeleveld M.W.
      • et al.
      Rapid quantification of underivatized amino acids inplasma by hydrophilic interaction liquid chromatography (HILIC) coupled withtandem mass-spectrometry.
      and data review and processing maybe cumbersome.
      • Filee R.
      • Schoos R.
      • Boemer F.
      Evaluation of physiological amino acids profiling bytandem mass spectrometry.
      LC-ESI-MS/MS methods have been published for the analysis ofunderivatized or derivatized amino acids. These methods used UPLC or HPLCwith one or more column chemistries, stable isotope dilution or isobarictagging, and detection by selected reaction monitoring(SRM).
      • Kaspar H.
      • Dettmer K.
      • Gronwald W.
      • Oefner P.J.
      Advances in amino acid analysis.
      • Piraud M.
      • Vianey-Saban C.
      • Petritis K.
      • Elfakir C.
      • Steghens J.P.
      • Bouchu D.
      Ion-pairing reversed-phase liquidchromatography/electrospray ionization mass spectrometric analysis of 76underivatized amino acids of biological interest: a new tool for thediagnosis of inherited disorders of amino acid metabolism.
      • Kaspar H.
      • Dettmer K.
      • Chan Q.
      • et al.
      Urinary amino acid analysis: a comparison ofiTRAQ-LC-MS/MS, GC-MS, and amino acid analyzer.
      • Waterval W.A.
      • Scheijen J.L.
      • Ortmans-Ploemen M.M.
      • Habets-van der Poel C.D.
      • Bierau J.
      Quantitative UPLC-MS/MS analysis of underivatisedamino acids in body fluids is a reliable tool for the diagnosis andfollow-up of patients with inborn errors of metabolism.
      • Le A.
      • Ng A.
      • Kwan T.
      • Cusmano-Ozog K.
      • Cowan T.M.
      A rapid, sensitive method for quantitative analysisof underivatized amino acids by liquid chromatography-tandem massspectrometry (LC-MS/MS).
      ,
      • Dietzen D.J.
      • Weindel A.L.
      • Carayannopoulos M.O.
      • et al.
      Rapid comprehensive amino acid analysis by liquidchromatography/tandem mass spectrometry: comparison to cation exchange withpost-column ninhydrin detection.
      • Held P.K.
      • White L.
      • Pasquali M.
      Quantitative urine amino acid analysis using liquidchromatography tandem mass spectrometry and aTRAQ reagents.
      • Filee R.
      • Schoos R.
      • Boemer F.
      Evaluation of physiological amino acids profiling bytandem mass spectrometry.
      Sensitivities as low as 1–2 μm andlinearity of up to 1000–2000 μm have been reported.
      • Piraud M.
      • Vianey-Saban C.
      • Petritis K.
      • Elfakir C.
      • Steghens J.P.
      • Bouchu D.
      Ion-pairing reversed-phase liquidchromatography/electrospray ionization mass spectrometric analysis of 76underivatized amino acids of biological interest: a new tool for thediagnosis of inherited disorders of amino acid metabolism.
      ,
      • Waterval W.A.
      • Scheijen J.L.
      • Ortmans-Ploemen M.M.
      • Habets-van der Poel C.D.
      • Bierau J.
      Quantitative UPLC-MS/MS analysis of underivatisedamino acids in body fluids is a reliable tool for the diagnosis andfollow-up of patients with inborn errors of metabolism.
      ,
      • Le A.
      • Ng A.
      • Kwan T.
      • Cusmano-Ozog K.
      • Cowan T.M.
      A rapid, sensitive method for quantitative analysisof underivatized amino acids by liquid chromatography-tandem massspectrometry (LC-MS/MS).
      ,
      • Held P.K.
      • White L.
      • Pasquali M.
      Quantitative urine amino acid analysis using liquidchromatography tandem mass spectrometry and aTRAQ reagents.
      • Filee R.
      • Schoos R.
      • Boemer F.
      Evaluation of physiological amino acids profiling bytandem mass spectrometry.
      • Prinsen H.C.
      • Schiebergen-Bronkhorst B.G.
      • Roeleveld M.W.
      • et al.
      Rapid quantification of underivatized amino acids inplasma by hydrophilic interaction liquid chromatography (HILIC) coupled withtandem mass-spectrometry.
      Unlike IEC, LC-ESI-MS/MS methods aresubject to matrix effects, and thus require the use of stable isotope,internal standards (IS) to compensate for variable recoveries and ionizationefficiencies.
      • Vogeser M.
      • Seger C.
      Quality management in clinical application of massspectrometry measurement systems.
      ,

      William C, Molinaro RJ, Bachmann LM, et al. Clinical and LaboratoryStandards Institute (CLSI): Wayne, PA, 2014. https://clsi.org/media/1346/c62a_sample.pdf.

      A stable isotope IS for each analyte ispreferred; however, depending on the method, this may not be feasible. Thelack of an IS may affect accuracy and precision for a specific analyte andis an important consideration for each laboratory. Published methods thatemploy stable isotope dilution often utilize commercially available[2H2] to[2H8] labeled aminoacids for IS.
      • Piraud M.
      • Vianey-Saban C.
      • Petritis K.
      • Elfakir C.
      • Steghens J.P.
      • Bouchu D.
      Ion-pairing reversed-phase liquidchromatography/electrospray ionization mass spectrometric analysis of 76underivatized amino acids of biological interest: a new tool for thediagnosis of inherited disorders of amino acid metabolism.
      ,
      • Waterval W.A.
      • Scheijen J.L.
      • Ortmans-Ploemen M.M.
      • Habets-van der Poel C.D.
      • Bierau J.
      Quantitative UPLC-MS/MS analysis of underivatisedamino acids in body fluids is a reliable tool for the diagnosis andfollow-up of patients with inborn errors of metabolism.
      ,
      • Dietzen D.J.
      • Weindel A.L.
      • Carayannopoulos M.O.
      • et al.
      Rapid comprehensive amino acid analysis by liquidchromatography/tandem mass spectrometry: comparison to cation exchange withpost-column ninhydrin detection.
      It is generally accepted that IS with aconsiderable degree of deuterium label may not reliably reduce all matrixeffects in every sample due to the slight alterations in physicochemicalproperties, leading to small shifts in retention time and differences inionization.

      William C, Molinaro RJ, Bachmann LM, et al. Clinical and LaboratoryStandards Institute (CLSI): Wayne, PA, 2014. https://clsi.org/media/1346/c62a_sample.pdf.

      ,
      • Wang S.
      • Cyronak M.
      • Yang E.
      Does a stable isotopically labeled internal standardalways correct analyte response? A matrix effect study on a LC/MS/MS methodfor the determination of carvedilol enantiomers in humanplasma.
      For several published LC-ESI-MS/MSmethods, stable isotope–labeled analogs were not utilized for every aminoacid measured, necessitating the application of surrogate IS for a subset ofanalytes. For these reasons, while LC-ESI- MS/MS methods have compared wellwith established amino acid analytical methods, there are limitations withthe measurement of some target analytes.
      The sample preparation procedure for underivatized, LC-MS/MSmethods
      • Piraud M.
      • Vianey-Saban C.
      • Petritis K.
      • Elfakir C.
      • Steghens J.P.
      • Bouchu D.
      Ion-pairing reversed-phase liquidchromatography/electrospray ionization mass spectrometric analysis of 76underivatized amino acids of biological interest: a new tool for thediagnosis of inherited disorders of amino acid metabolism.
      ,
      • Waterval W.A.
      • Scheijen J.L.
      • Ortmans-Ploemen M.M.
      • Habets-van der Poel C.D.
      • Bierau J.
      Quantitative UPLC-MS/MS analysis of underivatisedamino acids in body fluids is a reliable tool for the diagnosis andfollow-up of patients with inborn errors of metabolism.
      ,
      • Le A.
      • Ng A.
      • Kwan T.
      • Cusmano-Ozog K.
      • Cowan T.M.
      A rapid, sensitive method for quantitative analysisof underivatized amino acids by liquid chromatography-tandem massspectrometry (LC-MS/MS).
      ,
      • Prinsen H.C.
      • Schiebergen-Bronkhorst B.G.
      • Roeleveld M.W.
      • et al.
      Rapid quantification of underivatized amino acids inplasma by hydrophilic interaction liquid chromatography (HILIC) coupled withtandem mass-spectrometry.
      ,
      • Piraud M.
      • Vianey-Saban C.
      • Petritis K.
      • et al.
      ESI-MS/MS analysis of underivatised amino acids: anew tool for the diagnosis of inherited disorders of amino acid metabolism.Fragmentation study of 79 molecules of biological interest in positive andnegative ionisation mode.
      is similar to IEC post-columnderivatization methods in most cases: samples are combined with ISs andplasma proteins are precipitated using sulfosalicylic acid or organicsolvent. Separation of underivatized amino acids has been achieved usingreverse-phase chromatography with ion pairingreagents,
      • Piraud M.
      • Vianey-Saban C.
      • Petritis K.
      • Elfakir C.
      • Steghens J.P.
      • Bouchu D.
      Ion-pairing reversed-phase liquidchromatography/electrospray ionization mass spectrometric analysis of 76underivatized amino acids of biological interest: a new tool for thediagnosis of inherited disorders of amino acid metabolism.
      ,
      • Waterval W.A.
      • Scheijen J.L.
      • Ortmans-Ploemen M.M.
      • Habets-van der Poel C.D.
      • Bierau J.
      Quantitative UPLC-MS/MS analysis of underivatisedamino acids in body fluids is a reliable tool for the diagnosis andfollow-up of patients with inborn errors of metabolism.
      ,
      • Le A.
      • Ng A.
      • Kwan T.
      • Cusmano-Ozog K.
      • Cowan T.M.
      A rapid, sensitive method for quantitative analysisof underivatized amino acids by liquid chromatography-tandem massspectrometry (LC-MS/MS).
      ,
      • Piraud M.
      • Vianey-Saban C.
      • Petritis K.
      • et al.
      ESI-MS/MS analysis of underivatised amino acids: anew tool for the diagnosis of inherited disorders of amino acid metabolism.Fragmentation study of 79 molecules of biological interest in positive andnegative ionisation mode.
      and with hydrophilic interactionchromatography using an amide UPLC column
      • Prinsen H.C.
      • Schiebergen-Bronkhorst B.G.
      • Roeleveld M.W.
      • et al.
      Rapid quantification of underivatized amino acids inplasma by hydrophilic interaction liquid chromatography (HILIC) coupled withtandem mass-spectrometry.
      for the analysis of26 to 52 amino acids with 18- to 30-minute run times. As noted above, fullquantification using stable isotope dilution was performed for only a subsetof clinically significant amino acids, with reduced performance of severalanalytes without a corresponding IS. Other challenges with theseunderivatized methods included inferior performance of highly polar aminoacids that are poorly retained on a reverse-phasecolumn,
      • Waterval W.A.
      • Scheijen J.L.
      • Ortmans-Ploemen M.M.
      • Habets-van der Poel C.D.
      • Bierau J.
      Quantitative UPLC-MS/MS analysis of underivatisedamino acids in body fluids is a reliable tool for the diagnosis andfollow-up of patients with inborn errors of metabolism.
      the requirement for extensivereconditioning of ion-pairing reverse-phasesystems,
      • Piraud M.
      • Vianey-Saban C.
      • Petritis K.
      • Elfakir C.
      • Steghens J.P.
      • Bouchu D.
      Ion-pairing reversed-phase liquidchromatography/electrospray ionization mass spectrometric analysis of 76underivatized amino acids of biological interest: a new tool for thediagnosis of inherited disorders of amino acid metabolism.
      and the lack of separation ofD-alloisoleucine from isoleucine with methods utilizing hydrophilicchromatography.
      • Prinsen H.C.
      • Schiebergen-Bronkhorst B.G.
      • Roeleveld M.W.
      • et al.
      Rapid quantification of underivatized amino acids inplasma by hydrophilic interaction liquid chromatography (HILIC) coupled withtandem mass-spectrometry.
      A published LC-ESI-MS/MS method that utilizes amine-reactiveisobaric tagging reagents (iTRAQ®; later aTRAQTMReagent 121) is an alternative approach to using stable isotopeIS.
      • Held P.K.
      • White L.
      • Pasquali M.
      Quantitative urine amino acid analysis using liquidchromatography tandem mass spectrometry and aTRAQ reagents.
      ,
      • Filee R.
      • Schoos R.
      • Boemer F.
      Evaluation of physiological amino acids profiling bytandem mass spectrometry.
      These reagents were initially used formultiplexed quantification of peptides and have been applied to measuringphysiological amino acid concentrations.
      • Ross P.L.
      • Huang Y.N.
      • Marchese J.N.
      • et al.
      Multiplexed protein quantitation in Saccharomycescerevisiae using amine-reactive isobaric tagging reagents.
      These methods arebased on the differential derivatization of IS with an isotopicallyunlabeled reagent(2,5-dioxopyrrolidin-1-yl-2(4-methylpiperazin-1-yl)acetate) and thederivatization of sample with13C6,15N2- stable isotopelabeled reagent to generate isobaric tags. The differentially labeled sampleand IS have identical chromatographic retention, and can be separated byMS/MS by the generation of product ions with an m/z difference of 8 amu.Pre-derivatized standards are combined with the sample after derivatizationand serve to control for differences in injection volume and ionizationefficiency. Two nonphysiologic amino acids are added to the sample beforederivatization for monitoring sample extraction and derivatizationefficiency. Analyte quantification can be obtained by comparison with IS asprovided in specified amounts in each commercial kit. Alternatively,quantification using calibrators extracted with each run, and normalizedusing the ISs, improves accuracy by reducing variability due to different ISlots or tagging efficacy.
      • Held P.K.
      • White L.
      • Pasquali M.
      Quantitative urine amino acid analysis using liquidchromatography tandem mass spectrometry and aTRAQ reagents.
      ,
      • Filee R.
      • Schoos R.
      • Boemer F.
      Evaluation of physiological amino acids profiling bytandem mass spectrometry.
      Chromatography is performed using a C18(5 µm, 4.6 mm × 150 mm) column with a total analysis time of18 minutes
      • Held P.K.
      • White L.
      • Pasquali M.
      Quantitative urine amino acid analysis using liquidchromatography tandem mass spectrometry and aTRAQ reagents.
      ,
      • Filee R.
      • Schoos R.
      • Boemer F.
      Evaluation of physiological amino acids profiling bytandem mass spectrometry.
      with efficient separation of isobaricamino acids with the exception of alloisoleucine and isoleucine; for theaccurate quantification of these branched-chain amino acids, it is necessaryto modify the method to include the detection of the underivatizedalloisoleucine and isoleucine, and the use of an underivatized stableisotope–labeled IS.
      • Kaspar H.
      • Dettmer K.
      • Gronwald W.
      • Oefner P.J.
      Advances in amino acid analysis.
      ,
      • Held P.K.
      • White L.
      • Pasquali M.
      Quantitative urine amino acid analysis using liquidchromatography tandem mass spectrometry and aTRAQ reagents.
      ,
      • Filee R.
      • Schoos R.
      • Boemer F.
      Evaluation of physiological amino acids profiling bytandem mass spectrometry.
      Thus, an aliquot is removed from thesample prior to the labeling (derivatization) reaction, mixed with IS, andrecombined with the derivatized extract prior to injection on the column. Anadvantage of isobaric tagging compared with isotope dilution is theavailability of a commercial kit that contains IS for each of the 42analytes.
      • Held P.K.
      • White L.
      • Pasquali M.
      Quantitative urine amino acid analysis using liquidchromatography tandem mass spectrometry and aTRAQ reagents.
      ,
      • Filee R.
      • Schoos R.
      • Boemer F.
      Evaluation of physiological amino acids profiling bytandem mass spectrometry.
      The possible pitfalls of a specific LC-ESI-MS/MS method ofchoice should be determined by the laboratory and the assay should undergo athorough and systematic validation before use in a clinicallaboratory.

      Quality control

      Quality control (QC) material should be evaluated at two differentconcentrations for all reported amino acids, and should be derived with the samematrix as patient specimens. QC material should be analyzed along with eachpatient batch. In situations where samples are run continuously (e.g., addingsamples as they arrive to an ongoing queue, rather than in discrete batches), QCsamples should be run daily (every 24 hours) per College of AmericanPathologists (CAP, Inspector Checklist CBG 12800). Thresholds for acceptance orrejection of a QC sample result, and remedial actions in the event of a QCfailure, should be established and documented by the laboratory. QC data shouldbe regularly monitored for trends that may affect test performance, and problemsshould be documented and remediated as appropriate. Thresholds for appropriateIS response should be established, and IS responses for each specimen should bemonitored as another level of quality control. It is recommended to considerbasic statistical QC analyses for clinical testing to enhance the performance oflaboratory methods

      Burtis CA, Ashwood ER, Bruns DE. Tietz textbook of clinicalchemistry and molecular diagnostics. Elsevier Health Sciences: Philadelphia, US,2012.

      (see also CLSI document EP28-A3c: Defining,Establishing, and Verifying Reference Intervals in the Clinical Laboratory;Approved Guideline—Third Edition).

      Proficiency testing

      Participation in biannual proficiency testing (PT) activities is animportant element of any laboratory quality assurance program. A PT program thatevaluates both analytical and interpretive/diagnostic proficiency of amino acidanalysis is offered by CAP and supervised by the CAP/ACMG Genetic Biochemicaland Molecular Genetic Resource Committee. Another excellent program is offeredby the European Research Network for evaluation and improvement of screening,Diagnosis and treatment of Inherited disorders of Metabolism (ERNDIM; http://www.erndimqa.nl), in which analytical and interpretive (or clinical) proficiencyis monitored through the regular distribution of PT.

      Test interpretation and reporting

      Interpretation

      Clinical amino acid analyses should be interpreted by an AmericanBoard of Medical Genetics and Genomics (ABMGG)-certified clinical biochemicalgeneticist. Because normal amino acid concentrations vary with age, quantitativeresults should be compared with a properly defined reference age group.Interpretations of amino acid results are based upon relative amino acid levels,pattern recognition, and correlation of positive and negative findings, ratherthan on individual amino acids levels alone. Amino acid abnormalities or overallprofiles should also be considered in the context of clinical findings and/oradditional test results.

      Reporting

      Reports should contain appropriate patient and specimen informationwhenever available as described in the American College of Medical GeneticsStandards and Guidelines for Clinical Genetics Laboratories, Sections 2.4, 2.41,and 2.42 (https://www.acmg.net/acmg/Publications/Standards_Guidelines/General_Policies.aspx) and as specified by CLIA ’88. Written reports should provideunits of measure, age-dependent, laboratory-specific reference ranges, and aninterpretation of findings. When abnormal results are detected, theinterpretation should include an overview of significant results, correlation toany available clinical information, elements of differential diagnosis,recommendations for additional testing or confirmatory studies (e.g., enzymeassay, molecular analysis), and a phone number to reach the reporting laboratoryfor any additional questions. Recommendations for follow-up evaluation,including referral to a metabolic specialist, should also be included whenappropriate.

      Ethics declarations

      Disclosure

      The following authors direct laboratories that perform amino acid analysis as a fee-for service: J.D.S., I.D.B., D.M., S.Y., M.J.B., and A.A.T.

      Acknowledgements

      The authors gratefully acknowledge Russell Grant for critical reading of themanuscript. Members of the ACMG Biochemical Genetics Subcommittee and the LaboratoryQuality Assurance Committee reviewed and endorsed the final document.

      Additional information

      Disclaimer

      This laboratory standard is designed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to this standard is voluntary and does not necessarily assure a successful medical outcome. This standard should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the patient or specimen.
      Clinical laboratory geneticists are encouraged to document in the patient’s record the rationale for the use of a particular procedure or test, whether or not it is in conformance with this standard. They also are advised to take notice of the date any particular standard was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.
      Disclosure: The following authors direct laboratories that perform amino acid analysis as a fee-for service: J.D.S., I.D.B., D.M., S.Y., M.J.B., and A.A.T. The other authors declare no conflicts of interest.
      Board Approval: The Board of Directors of the American College of Medical Genetics and Genomics approved this technical laboratory standard on 21 May 2018.

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