guide to diagnosis ofinborn errors of metabolism district ... · glucose(clinistix, orbmtest strip)...

, Association of Clinical Pathologists

Broadsheet 120: January 1989

Guide to diagnosis of inborn errors of metabolism indistrict general hospitals

Clinical and laboratory awareness of inheritedmetabolic disorders has increased due to advances indiagnostic tests and treatment. In the United King-dom the only inherited disorders which are routinelyscreened for in the neonatal period are phenylketo-nuria and congenital hypothyroidism. For the rest,correct diagnosis depends firstly on the appropriatetests(s) being requested, and secondly, on the analysisbeing carried out reliably and with correct interpreta-tion and action. The laboratory has a responsibility toboth patient and clinician to ensure that this secondstep is provided.

Because there are large numbers of different, al-though rare disorders, paediatricians in district gen-eral hospitals have little experience of them and mayrequire advice on which tests to request. It is in thisrespect that the local laboratory is so importantbecause of the way in which it can guide investigations.If the simpler more easily available tests are done firstuseful clues are often given as to which of the morespecialist (and often more expensive) investigationswould then be most appropriate.Each laboratory must decide ifparticular tests are to

be carried out locally or referred to a specialistlaboratory. This will vary, depending on availablemanpower and expertise and workload, proximity andease of transport to a more specialist laboratory.Another important consideration is the volume ofwork; laboratories with small workloads are not likelyto have gained the necessary experience of these "rare"disorders.

It is also important to remember that some testsmust be instantly available and often out of normallaboratory hours. In the case ofan acutely sick baby orchild first-line investigations should be completedwithin 24 hours.The most important role for the local laboratory is

good communication with the clinical team andspecialist laboratories; without this the latter maywaste time and effort, not to mention money oninappropriate tests, added to which the diagnosis maybe missed.

Accepted for publication 4 August 1988

Definitive diagnoses of inborn errors ofmetabolismare vital if families are to benefit from the advancingtechnology of molecular biology, and in particular, ifthey are to take advantage of new treatments andearlier prenatal diagnosis by chorionic villus sampling.

Clinical presentation(s) and investigation

The clinical presentation of these disorders is variableand diagnosis is not easy because most of the presenta-tions are non-specific and the disorders are rare. Mostbabies with inborn errors of metabolism seem to benormal at birth. Symptoms that may develop duringthe first week of life include lethargy, poor feeding,vomiting, hypotonia, excessive weight loss andtachypnoea. Severe hypotonia is particularly suggest-ive of non-ketotic hyperglycinaemia or a peroxisomaldisorder, especially if associated with liver dysfunc-tion. Hyperventilation secondary to an unexplainedmetabolic acidosis suggests an organic acid disorder:unheralded and early onset of fits may be the first sign.The onset of symptoms in relation to feeding isrelevant. Dysmorphic features, although moresuggestive of a chromosome abnormality, are,however, characteristic of some inborn errors. Todecide on the most appropriate investigations it isuseful to consider the clinical presentation under thefollowing categories, depending on whether it is acuteor chronic.

ACUTELY ILL NEONATES OR INFANTSThe possibility of an inborn error in a neonate orinfant with an unexplained acute illness should alwaysbe considered, particularly if there are previous unex-plained deaths or illnesses in the family. Such an eventmay occur in the neonatal period or after severalmonths ofapparently healthy childhood. It is essentialto investigate patients with a "Reye-like" ence-phalopathy as amino acid, urea cycle, organic acid orfatty acid oxidation defects can present in this way.

Consanguinity should increase the index of sus-picion and a history of "male deaths" in maternalrelatives suggests an X-linked disorder. In many casesthere is rapid deterioration, and treatment must bestarted quickly. Even if there is no effective treatmentit is important to make a diagnosis to enable genetic

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Association of Clinical Pathologists; Broadsheet 120, January 1989counselling and possible prenatal diagnosis.

Clinical signs which can be important clues in theacute situation include: abnormal smell-sweet,sweaty, cabbage-like (amino acid organic acid dis-orders); cataracts with jaundice (galactosaemia);neurological depression with respiratory alkalosis(urea cycle disorders).

It is particularly important to remember that diag-nosis of an infection does not preclude an inheritedmetabolic disorder; Escherichia coli septicaemia isoften associated with classic galactosaemia.For further details on inherited metabolic disorders

which present in the neonatal period and the morecommon clinical presentations, the reader is referredto more comprehensive texts.'Whenever possible, investigations should proceed

in a logical manner; the initial tests as detailed belowshould be carried out before proceeding to specifictests for metabolic disorders. It is, however, essentialto collect both blood and urine specimens during anattack and store for further investigation. If, however,the baby deteriorates rapidly and is likely to succumb(or has already died) before results of these initial testsare available, more rigorous sampling should proceed(see Appendix: guide for emergency specimen collec-tion).

Initial biochemical investigationsThe following investigations should be undertakenlocally before proceeding to more specific metabolictests.

Urine:Reducing substances (Clinitest or Benedict's test)Glucose (Clinistix, or BM test strip)Ketones (Acetest, Ketostix or Boehringer Mannheimtest strip)

Blood:Hydrogen ion, Pco2, bicarbonate, base excessSodium, potassium, chloride (calculate Anion gap):(Na + K) - (HCO3 + Cl) = (normally less than

20 mmol/l)Glucose (fasting, if hypoglycaemia not a presenting

feature)Calcium, magnesiumBilirubin, alkaline phosphatase, alanine amino trans-

ferase or aspartate amino transferaseCreatinine (or urea)

Further investigations should then proceed accord-ing to the clinical problems and biochemical abnor-malities identified. These can be categorised into fivegroups (table 1). The investigations listed are thosewhich should be readily available and would either bediagnostic or would provide a useful guide to otherinvestigations. Primary disorders of the immune sys-

tem, gastrointestinal tract, and haemolytic disordershave not been included.

If more than one of the problems listed (table 1) ispresent then a metabolic disorder is more likely.

Specimens for more specific investigations (such asenzymes) usually need not be taken until results of theinvestigations suggested are available (table 1)-theexception is that if the child deteriorates rapidly or dies(see Appendix) or unless a particular disorder issuspected on clinical grounds or family history, inwhich case investigations should be discussed withworkers in a specialist laboratory before taking anyspecimens.

NON-ACUTE PRESENTATIONInvestigation of a patient with a chronic problem isusually more difficult as clinical presentation may beeven more variable and many of the investigationsmore complex. A brief guide to the initial biochemicalinvestigation of patients with these types of chronicpresentations is provided below:Liver diseaseMetabolic disorders which may present in this wayinclude: galactosaemia; tyrosinaemia (type I); fruc-tosaemia/hereditary fructose intolerance; Wilson'sdisease; a-l-antitrypsin deficiency; glycogen storagedisorders; Niemann Pick type C; cystic fibrosis.Biochemical investigations which should be con-sidered include: fasting glucose (plasma); amino acids(urine and plasma); sugars (urine); a-l-antitrypsin(plasma); a-fetoprotein (plasma); urate (plasma);cholesterol (plasma); lactate (plasma); sweat test. Ifneonate: galactose-l-phosphate uridyl transferase(blood). Ifmore than 5 years old: copper and caerulo-plasmin (plasma).

Unexplainedfailure to thriveDisorders of amino acid, urea cycle, and organic acidmetabolism can present in this way. Investigationswhich should be considered are amino acids (urine andplasma), plasma ammonia, and urine organic acids.Decision to investigate will be influenced by familyhistory and presence of any suggestive clinical ormetabolic abnormalities.

Mental retardation-with no specific signsDecision to investigative patients in this group formetabolic disorders will be influenced by familyhistory and results of other investigations. The readeris referred to other discussions on this topic.45

Neurological degenerative disordersThese include the lysosomal storage and peroxisomaldisorders. Most patients present non-acutely withneurological dysfunction, often with associated liver

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86 Association of Clinical Pathologists; Broadsheet 120, January 1989Table 1 Guide tofurther investigations in acutely ill neonates or infants

Possible metabolic SuggestedPresentation disorders investigations

Unexplained hypoglycaemia: Organic acid disorders Organic acids (U)Amino acid disorders Amino acids (U, P)Glycogen storage disease (type I) Lactate (P)Disorders of gluconeogenesis Insulin (P)Congenital adrenal hyperplasia Cortisol (P)

17-hydroxyprogesterone (P)Acid base imbalance:-metabolic acidosis Organic acid disorders Organic acids (U)exclude primary cardiac and respiratory disorders Congenital lactic acidosis Lactate (P)

Amino acids (U, P)-respiratory alkalosis Urea cycle disorders Ammonia (P), Orotic acid (U),

Amino acids (U, P)Liver dysfunction: Galactosaemia Galactosaemia screen (B)

often associated with hypoglycaemia and Fructose 1 6, Sugars (U)galactosuria diphosphatase deficiency Amino acids (U, P)

Fructose intolerance Succinyl acetone (U)Tyrosinaemia (type) Alpha-fetoprotein (P)Glycogen storage (type I) Lactate (P)Disorders of gluconeogenesis Oligosaccharides (U)

Organic acids (U)Alpha-l-antitrypsin (P)

Neurological dysfunction: Non-ketotic hyperglycinaemia Amino acids (U, P)-seizures Glyceric acidaemia Organic acids (U)-depressed consciousness Urea cycle disorder Orotic acid (U)

Xanthine/sulphite oxidase deficiency Ammonia (P)-hypotonia Urate (P, U)

Sulphite (U) Lactate (P)Cardiomyopathy: Glycogen storage type II (Pompe's) Lactate (P)

Fatty acid oxidation disorders Oligosaccharides (U)Tyrosinaemia (type 1) Organic acids (U)

Carnitine (P)Amino acids (U, P)

U = urine; B = whole blood; P = plasma

disease, eye abnormalities, and sometimes dysmorphicfeatures.

Other non-biochemical investigations, especiallyradiological and testing for chromosomal abnor-malities are important in this group. Although thediagnosis of these disorders is usually outside thescope of the district general hospital laboratory,investigations which should be considered are: aminoacids (plasma and urine); vacuolated lymphocytes onperipheral blood film, oligosaccharides/sialic acid(urine); glycosaminoglycans (urine); organic acids(urine); bile acids (urine).For all types of chronic presentation, specific

leucocyte or tissue enzymes may be indicated eitherfrom the results of these initial tests or on clinicalpresentation alone. Before embarking on these,detailed discussion with the clinician and specialistbiochemist is required.

Laboratory methods for district general hospitals

SPECIMEN REQUIREMENTSAppropriate specimens are crucial ifdiagnose§ ares notto be missed. The following information should beprovided with all specimens:

1 Details of feeding regimen at the time ofand beforesampling (especially if amino acids, organic acids,or sugars are requested).

2 Details of all drugs or other treatment (includingblood transfusion).

3 Full clinical details (including information on anyprevious sibs and results of any preliminaryinvestigations). Indication ofthe degree ofurgencyis required.

The acute attackIn some cases biochemical "markers" of the diseasemay be detected only during an acute attack.Therefore, the diagnosis can be missed ifspecimens arenot obtained at that time. The following specimensshould be collected as a minimum. If the child isseverely ill, more rigorous specimen collection isindicated (see Appendix, guide for emergencyspecimen collection).I Urine: 5 ml (smaller quantities are useful if there is

difficulty). Store deep frozen (- 20°C or lower)immediately with no preservative.

2 Blood: Collect at least 1 ml blood (preferably 5 ml)into heparin if possible before the child is givenglucose or saline or any other treatment. Storeplasma deep frozen (- 20'C or lower) and red cells


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Association of Clinical Pathologists; Broadsheet 120, January 1989at + 4'C. If there is sufficient specimen deprotein-ise 1 ml whole blood with 1 ml 1M perchloric acidand store supernatant deep frozen.As most investigations cannot be done locally it is

important that contact with a specialist laboratory ismade at the earliest opportunity. For details onavailability of and specimen requirements for special-ist investigations, the reader is referred to UK Direc-tory of laboratories diagnosing inborn errors ofmetabolism.6

METHODSAs a minimum, every laboratory supporting neonataland paediatric patients should have the followinginvestigations available: urine screening tests forreducing substances, glucose, and ketones; plasma (orwhole blood) lactate; plasma ammonia (quantitative).The following may also be appropriate for somelaboratories: plasma amino acids (qualitative); urineamino acids (qualitative); urine sugars (TLC); urinemethylmalonic acid (TLC).Chromatography for methylmalonic acid is not a

substitute for a full organic acid profile by gaschromatography-mass spectrometry. As the methyl-malonic acidaemias are one of the commoner groupsof organic acid disorders, however, it can be useful toinclude this as an extension to amino acidchromatography.A decision to provide these optional tests must be a

local one, depending on expertise, workload, andavailability of assays being provided elsewhere. Wherespeed is essential, expert technique and interpretationis necessary and local laboratories with insufficientexperience should not be tempted to provide theseadditional investigations.

Tests outside the remit of most district generalhospital laboratories are: urinary organic acids (gaschromatography-mass specrometry); urinaryglycosaminoglycans (mucopolysaccharides-electro-phoresis and quantitation); urinary oligosaccharides(chromatography). Although the techniques may notbe particularly difficult and equipment may already beavailable, interpretation is difficult. Sufficient expertiseis unlikely to be available in a local laboratory and it istherefore inappropriate that these tests be carried out.

URINE SCREENING TESTS

Most ofthese tests are non-specific but a positive resultmay focus attention on a particular area for investiga-tion (table 2). For more details the reader is referred toSelected screening testsfor genetic metabolic diseases.!

pH and nitriteThe quality of urine specimens can be assessed bychecking the pH and using the "nitrite" pad on N-

Table 2 Some causes ofpositive urine screening tests

Metabolic disorder/cause Abnormalcolour

Ferric chloride:Phenylketonuria (Phenylpyruvic acid) Dark greenTyrosinaemia Light greenMaple syrup urine disease GreyHistidinaemia (imidazole pyruvic acid) Blue-greenAlcaptonuria (homogentisic acid) Green/brownAzide RedSalicylate PurpleBilirubin GreenAcetoacetate Red

Cyanide nitroprusside:CystinuriaHomocystinuriaBeta-mercaptolactate cysteine disulphiduriaConcentrated urine

Dinitrophenylhydrazine:Maple syrup urine diseaseMethioninaemiaValproic acid treatment

labstix. An alkaline pH (greater than 7 0) suggestsbacterial contamination. The nitrite test will bepositive only ifthe contaminating bacteria are "nitriteproducers".

Ferric chloride testThis comprises 1 g ferric chloride and 1 g ferrousammonium sulphate (Fe(NH4)2 (SO4)2 6H20) in100 ml of 0-02 M hydrochloric acid.Add 10 drops of urine to 1 ml of ferric chloride

reagent. Ferric chloride gives a characteristic greencolour with phenylpyruvic acid and suggests a raisedphenylalanine. It is useful for the detection of missedor maternal phenylketonuria. It is unreliable withalkaline urine specimens and may also be subject tointerference from drugs and phosphates.

Cyanide/nitroprusside (CNP) test for cystine/homo-cystineTo 1 ml urine add 2 drops concentrated ammoniasolution followed by 0 5 ml of 10% (w/v) sodiumcyanide (care needed). Leave for at least five minutesand then add 5 drops of 5% (w/v) sodium nitroprus-side. Positive samples give a distinct magenta colour.It is advisable to run a positive control in parallel. If anauthentic urine is not available a normal urine spikedwith cystine (0 3 mmol/l) is suitable; aliquots can bestored at - 20°C. Any compound that contains sul-phydryl groups or yields sulphydryl groups on reduc-tion will give a positive reaction. This test is sensitive to0-25 mmol/l cystine and 0-25 mmol/l homocystine.Concentrated- normal urine samples (creatininegreater than 7 mmol/l) may give a misleading positiveresult.

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Association of Clinical Pathologists; Broadsheet 120, January 1989Dinitrophenylhydrazine (DNPH) test for keto com-poundsThe reagent comprises 0-7 g 2,4-dinitrophenyl-hydrazine in 250 ml I m hydrochloric acid. Warm todissolve. When cool, filter reagent and store at roomtemperature. Cloudy urine samples should be cen-trifuged before analysis. Mix an equal volume ofurineand DNPH reagent. Observe after five minutes.DNPH forms insoluble hydrazones with keto com-pounds (such as a-ketoisocaproic acid) which result inthe formation ofa heavy yellow-white precipitate. Thepresence of these keto compounds may indicate anunderlying metabolic defect such as maple syrup urinedisease. Patients receiving valproic acid may give apositive result.

Other useful tests to include are Clinitest, Acetest,Clinistix and Albustix or the Boehringer Mannheimtest strip range. A false positive Albustix result may beobtained in a urine with an alkaline pH. These resultsshould be checked with the sulphosalicylic acid test forurine protein.

PLASMA LACTATELactate can be reliably measured using the BoehringerKit assay (No 149993) without deproteinisation). Theprinciple of the method is oxidation of L-lactate to L-pyruvate using lactate dehydrogenase. The pyruvateformed is then removed by reaction with c-glutamateusing alanine amino transferase.The method is performed manually according to the

manufacturer's instructions or can be adapted forautomation-for example, by using a centrifugalanalyser.8

Specimen collection for lactateCollection tubes are prepared by placing 20 pl of the(Boehringer Corporation, London) anticoagulant(fluoride/edetic acid solution)) in each tube andevaporating to dryness. Small plastic tubes, such asSarstedt bullet tubes 72 690PP, 1.5 ml capacity, aresuitable for this. Alternatively, commercially availablefluoride/oxalate tubes used for the measurement ofblood glucose can be used.For most purposes fasting blood (four hours or

overnight) should be collected. Venous blood shouldbe drawn without stasis and about 500 p1 placed intothe special tube. Plasma should be separated as soon aspossible and always within two hours. If analysiscannot be undertaken immediately the plasma shouldbe stored at - 20'C.For quality assurance there are several lyophylised

plasmas commercially available: BCL Precinorm andPrecipath or Merz and Dade QAP Chemistry Controlslevel I and 2.For automated methods using a Cobas Bio, inter-

batch precision ofabout 4% can be achieved at lactateconcentrations of 1-2 5 mmol/l.

PLASMA AMMONIAAmmonia can be quantitated either by a specificflowthrough electrode system or by one of the com-mercially available enzymatic kit methods (spectro-photometric or fluorimetric).The ion specific electrode is best used as a flow-

through system.9 Since this method was published,further modifications have been introduced (Hjelm M,Jenkins P, personal communication). In the author'sexperience this provides an undemanding and robustmethod suitable for routine use. Further details can beobtained from the author.The Swiftest screening test (DIC blood ammonia

test kit, Clandon Scientific Instruments, Aldershot,Hampshire), although useful as a screening method isnot a substitute for a quantitative method.The specimen required is 500 pI venous (or arterial)

plasma anticoagulated with lithium heparin (beware ofammonium heparin tubes). Each batch of specimentubes should be checked for ammonia contaminationbefore use. The blood must be centrifuged within 15minutes of collection. Whole blood should not bestored, even at + 4'C. Centrifuge at + 4'C for fiveminutes at 2000 g and separate plasma, avoiding thebuffy coat. Haemolysed samples are unsuitable ashigher concentrations of ammonia occur in erythro-cytes. Plasma ammonia should be assayed at once, butif storage is unavoidable then it should be a - 70°C orlower. Storage of plasma at - 20'C is unreliable.

AMINO ACIDS (PLASMA AND URINE)The reader is referred to a discussion of the technicalaspects and interpretation of amino acid investiga-tions before deciding to provide this service.'°

URINARY METHYLMALONIC ACID BY THIN-LAYERCHROMATOGRAPHYMethylmalonic acid is separated by thin-layerchromatography on cellulose and stained with fastblue B salt." 12The specimen should comprise fresh random urine

with no preservative. Store at - 20°C until analysis. Astandard should be prepared by dissolving 100 mgmethylmalonic acid (Sigma Chemicals) in 100 ml0-5M hydrochloric acid. Store at + 4'C. The thin-layerplates should be DC-alufolien cellulose 20 x 20 cm(Merck, product No 5552). These are cut to form10 cm x 20 cm plates. The Solvent should comprisen-butanol/acetic acid/water (5:1 :2v/v). This isprepared freshly for each run. Fast blue B (0 25 g).BDH, product No 341162N) is "dissolved" in amixture of 38 ml absolute ethanol and 12 ml water(note that the dye will not completely dissolve).

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Association of Clinical Pathologists; Broadsheet 120, January 1989

Glacial acetic acid (2 ml) is added and mixed. Thestain is not stable and must be prepared freshly foreach run.

ChromatographySamples (2 p1 neat urine) and standard (2up1) areapplied as a series of spots 1 55 cm apart and 1U5 cmfrom the bottom (20 cm edge). Standard applicationsshould be included at both edges. Chromatographuntil solvent is 1 cm from top edge. Dry the plate atroom temperature, spray with freshly prepared stain,and develop in an oven at 100°C for one minute.Methylmalonic acid is fast running (Rf 0 83) andstains as a bright magenta spot within a minute. The

chromatogram should be viewed immediately as thereis rapid colour change and fading. In normal urineseveral minor spots appear (figure). Beware of excessacetoacetate which stains an orange colour and maymask methylamalonic acid. The detection limit by thistechnique is 25 pg. A detectable spot is clinicallyimportant.

Urine sugars (mono and disaccharides) by thin-layerchromatography

It is useful to "screen" urines for reducing substancesand glucose with Clinitest and Clinistix before perfor-ming sugar chromatography. The most importanturine sugars to identify accurately are galactose andfructose. Several suitable one-dimensional thin-layerchromatographic techniques have been described.'3

It is essential that glucose and galactose separatereliably. It is useful to set up duplicate plates so that aspecific stain for fructose, sucrose, lactose andraffinose-for example, naphthoresorcinol-can beused in addition to p-aminobenzoic acid. Other stains,such as aniline-diphenylalanine phosphate reagentmay be used as a further aid to identification.

Interpretation and further investigationsLACTATE

A plasma lactate concentration greater than2-5 mmol/l is clinically important provided thespecimen has been collected without stasis. Increasedconcentrations occur in some types of glycogenstorage disease, defects ofpyruvate metabolism, mito-

1* chondrial myopathies and some organic acid dis-Figure One-dimensional chromatography for urine orders, as well as liver or heart disease not associatedmethylmalonic acid. with a metabolic disorder. In some disorders the

Urine Spot Compound R, Comments

1 Normal urine Normal urines with a highcreatinine concentrationmay show several grey-brown spots

2 Normal urine

3 Patient with tyrosinaemia type I A P-hydroxphenyl pyruvate 0-91 Purple spot running justahead of methylmalonicacid

4 Ketotic patient B Acetoacetate 0-85 Orange spot running in thesame position asmethylmalonic acid

5 Patient with methylmalonic aciduria C

6 100 mg% methylmalonic acid D Methylmalonic acid 0|85 Intense magenta spotstandardII II

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Association of Clinical Pathologists; Broadsheet 120, January 1989increase may be intermittent or exacerbated by acarbohydrate meal or by exercise. A chronic increaseof plasma lactate concentration may be associatedwith increased plasma and urine alanine, plasmaproline, and urate concentrations. Plasma pyruvateis usually increased but does not aid differentialdiagnosis.

Increased urinary lactate only occurs if the renalthreshold (about 7-0 mmol/1) is exceeded. Furtherinvestigation of a clinically important lactic acidosisincludes fasting glucose, urine and plasma aminoacids, and urine organic acids.

AMMONIAPlasma ammonia in healthy adults and children (1month to 14 years) is less than 40 ymol/l and is notaffected by fasting. Healthy term infants in the firstfew days of life may have concentrations up to 100umol/l. In preterm infants and sick neonates concen-trations may reach 200 pmol/l. Hyperammonaemia isnot a diagnosis and requires further biochemicalinvestigation. 14

URINARY METHYLMALONIC ACIDExcess urinary methylmalonic acid occurs as a resultofcobalamin deficiency (including a vegan diet) or oneof the several inherited defects of methylmalonic acidmetabolism. The finding of methylmalonic aciduriatogether with homocystinuria occurs in some of thecobalamin metabolic defects. This is a complex areaand the finding of methylmalonic acid in the urinemust be fully investigated.URINARY SUGARSClinically important galactosuria-that is, greaterthan g/100 ml-in a neonate suggests galactosaemia,but may also occur in patients with other causes ofliver disease-for example, tyrosinaemia type I. Thebaby should be taken off lactose immediately whilefurther investigations (particularly erythrocyte galac-tose-l-phosphate uridyl transferase and urine/plasmaamino acids) are undertaken.

In older children without liver disease galactosuriasuggests the possibility of glalactokinase deficiency.Fructosuria requires investigation for the possibilityof fructose intolerance or fructosaemia.Beware of xylose following tolerance tests. Several

minor bands-for example, lactulose, raffinose-canresult from dietary sources. Heavily staining bandsat or near the origin require further investigationof urinary oligosaccharides as they may indicatemucolipidoses or possible glycogen storage disease.

More detailed interpretation of these tests and aguide to further investigations is beyond the scope ofthis paper, and advice from a specialist centre must besought.

Appendix

GUIDE FOR EMERGENCY SPECIMEN COLLECTIONIn life-threatening situations, where an inheritedmetabolic disorder is thought to be likely (either fromfamily history, results ofpreliminary investigations, orclinical presentation), the following specimens shouldbe taken. At the earliest opportunity contact a special-ist laboratory to discuss appropriate investigations. Ifpossible, urine and blood specimens should be takenbefore death. Skin and tissue specimens should betaken as soon as possible after death.

If any of the samples are taken after death it isextremely important to record accurately both thetime of death and when the samples were taken.Appropriate storage as detailed below is essential.

1 UrineIdeally at least 120 ml of random urine. Collectinto a bottle with no preservative and store deepfrozen (-20°C or lower). If the sample is con-taminated with blood centrifuge to remove cellsbefore freezing supernatant.

2 BloodCollect 10-20 ml of heparinised blood, separateplasma as soon as possible and store plasma deep-frozen (- 20°C). Store the packed red cells at + 4°C(do not freeze). If DNA analysis is likely to berequired, store a further 120 ml whole blood(edetic acid) in a plastic tube deep frozen (at least- 20°C).

3 Skin (for fibroblast culture)Skin taken up to 24 hours after death is likely to beviable provided it is not infected. Take a skin sampleand place it in suitable transport medium* (obtain-able from most virology or cytogenetics depart-ments). In an emergency sterile isotronic saline canbe used, but do not use agar. The specimen shouldbe stored at + 4'C before despatch. Do notfreeze.

Sterility is ofparamount importance when taking skinbiopsy specimens, especially at necropsy.

If indicated:4 Tissue samples (liver, heart muscle, skeletal muscle)

These should only be taken if there is a strongclinical suspicion of a primary defect in one ofthese tissues. It is very important that blood andurine specimens are also taken and not just tissuespecimens. Necropsy tissue samples are only suit-able for biochemical analysis if taken within twohours of death. Two or three needle biopsyspecimens of tissue should be taken, wrapped inaluminium foil, and snap frozen in liquid nitrogen(or solid Co,). Store the sample deep frozen, as coldas possible.

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Association of Clinical Pathologists; Broadsheet 120, January 1989 91

Please note that these samples are required forbiochemical analyses only. Appropriate fixed sam-ples may also be required for histological investiga-tion.

5 Cerebrospinal fluid Sometimes a cerebrospinalsample may be useful. Collect a sample and storedeep frozen (- 20°C).

I am grateful to Sheena Grant, Steven Moore, MaryAnne Preece and Ian Sewell for their help withtechnical details and to Edith Green for preparation ofthe manuscript.

ANNE GREENDepartment of Clinical Chemistry,

The Children's Hospital, Birmingham.

References

1 Leonard JV. The early detection and management ofinborn errorspresenting acutely in the neonatal period. Eur J Paediatr1985;143:253-7.

2 Holton JB. Diagnosis of inherited metabolic diseases in severely illchildren. Ann Clin Biochem 1982;19:389-95.

3 Haan EA, Danks DM. Clinical investigations of suspectedmetabolic disease. In: Barson AJ, ed. Laboratory investigation of

fetal disease. Bristol: John Wright, 1981:410-28.4 Green A, Insley J. A paediatric vade-mecum. I1th Edition.

London: Lloyd-Luke, 1986:269-72.5 Green SH, Newell SJ. Diagnostic classification of the aetiology of

mental retardation in children. Br Med J 1987;294:163-6.6 Holton JB. Directory of laboratories diagnosing inborn errors of

metabolism. 3rd Edition. Bristol: Research Trust for MetabolicDiseases in Children, 1988.

7 Thomas GH, Howell RR. Selected screening tests for geneticmetabolic diseases. London: Year Book Medical Publishers,1973.

8 Hansen JL, Freier EF. Direct assays of lactate, pyruvate, beta-hydroxybutyrate and acetoacetate with a centrifugal analyser.Clin Chem 1978;24:475-9.

9 Park NH, Fenton JCB. A simple method for the estimation ofplasma ammonia using an ion-specific electrode. J Clin Path1973;26:802-4.

10 Edwards MA, Grant S, Green A. A practical approach to theinvestigation of amino acid disorders. Ann Clin Biochem1988;25:12941.

11 Bhatt HR, Green A, Linnell JC. A sensitive micromethod for theroutine estimation of methylmalonic acid in body fluids andtissues using thin-layer chromatography. Clin Chim Acta1982;118:31 1-12.

12 Gutteridge JMC, Wright EB. A simple and rapid thin-layerchromatographic technique for the detection of methylmalonicacid in urine. Clin Chim Acta 1970;27:289-91.

13 Ersser RS, Andrew BC. Rapid thin-layer chromatography ofclinically important sugars. Med Lab Technol 1971;28:355-9.

14 Green A. When and how should we measure plasma ammonia?Ann Clin Biochem 1988;25:199-209.


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