Laboratory Guidelines For Screening And Detection Of Inborn Errors Of Metabolism

Dr. Fayza Abdel-Hamid Hassan Prof. Clinical and Chemical pathology Faculty of Medicine ,Cairo University

The first few days of a newborn’s life can be critical to his future well-being.

Clinical manifestations of IEM are non specific & overlap with common illnesses
Frequently remain undiagnosed until late  Delay in recognition & ttt tragic consequences -5 IQ units lost /month  20% of infants presenting with a “sepsis” picture in absence of risk factors (prematurity, chorioamnionitis …. Etc) have IEM

Two types of presentations :

Apparently healthy at birth after symptom-free interval . Placental protection: placenta provides an effective dialysis system for removal of toxic metabolites, most babies with IEM are born in good condition & normal birth wt .


acidosis aciduria II

Glutaric Non

ketotic hyperglycinuria


Neonate with over whelming neurological illness with unconsciousness, convulsions, apnea with no apparent symptom-free interval.

:Pathogenesis of IEM
Enzyme or structural protein deficiency .1 . accumulation of toxic metabolites 2. Defect in membrane transport cystinuria. . Deficiency of co-factors or other substrates .3

ati ve me t. pat hw

eg: albinism Collagen defects




Toxic metabolites

Classical Phenylketonuria (PKU) Classical Phenylketonuria


Hydroxylase Phenylalanine





Detection of IEM
 Mass

neonatal screening.  Screening the at risk.  Detection of suspected IEM.

Requirements for newborn screening

Rapid and accurate analysis and reporting. Screen a large number of newborns with: * Minimum false positives * Eliminates false negatives

Cost effective. * Multiple tests from one sample for many disorders. * Treatable disorders included will reduce cost of special care for mentally and physically disabled. Testing should be doable utilizing existing personnel. * Result reporting must be straightforward & not cumbersome.

tives/False Negatives

There are two possible scenarios when the sample, the analysis, or the interpretation of the data give erroneous results:

False Positives  A laboratory test detects an abnormally high metabolite  The sample is retested and is still high  A new sample is taken from the child  The new sample is normal

Think of the parental anxiety when the new sample is taken!

False Negatives  A laboratory test DOES NOT detect an abnormally high metabolite  The sample is reported as being normal  Nothing is known until the child presents clinically

The screening has failed!

History of NBS
 1957

- Dr. Centerwall (father of a child with mental retardation (Ferric chloride) - Dr. Guthrie (BIA)

 1961  1963

- MA and OR implement legislation

Early screening methods
 Bacterial  Paper

inhibition assays.

Single test for single disorder (BIA)

and thin layer chromatography.

few disorders of single type

These methods are:
Tedious  Time consuming  False positive and negative rates (high)  Few disorders ,difficult interpretation (TL,&PC)

Technical challenges have limited their applications

PKU by Bacterial Inhibition Assay

 Recent

use of the tandem mass spectrometer has allowed for changes in traditional newborn screening services leading to expansion and improvement of testing

History of NBS
 1997  1999

- NC begins piloting MS/MS

- NC and MA begin newborn screening with MS/MS - WI begins MS/MS NBS

 2000

 It

requires a for

very small sample size

to screen

a large number of rare disorders
in a very quick amount of time, about 2 minutes

Expanded neonatal screening by MS/MS

individually rare their cumulative effect can be devastating
USA Germany KSA 1 in 3500 1 in 4000 1 in 750

?What tests and when to order
 Lab

abnormalities can be transient.  Tests need to be repeated during an episode of acute illness,or require provocation in a specialized center.  Most IEMs with acute life threatening episodes can be categorized based on initial lab findings as one of the following:  Metabolic acidosis  Hypoglycemia  Hyper-ammonemia

Investigations of suspected hypoglycemia in children
Obtain critical blood sample
12h fast if< 6m 24h fast if>12m

Measure blood glucose


Normal glucose

No hepatomegaly
Measure: Insulin, GH, Cortisol, Lactate, B(OH)B

Ketotic hypoglycemia


No in B(OH)B FAO defects

GH Or Cortisol


All Mass Spectrometers

• Must generate ions • separate ions • detect ions • compute ion intensities • interpret Data

1.Quadrupole Mass Analyzer

Resonant Ion Nonresonant Ion


rf and -dc


dc and Rf voltages

rf and +dc

What is a tandem mass spectrometer

Two mass spectrometers in series connected by a chamber that breaks a molecule into pieces (collision cell). A sample is sorted and weighed in the first MS, then broken into pieces in the collision cell& a piece or pieces sorted and weighed in the second MS.

Why tandem mass
Can improve laboratory analysis because:
Very specific in identification of compounds  Very accurate and sensitive.  More than one compound simultaneously in a single two minute analysis.  Reduces false positive rates by more than ten fold.

Ion Fragmentation

Disorders Screened Using MS
 

Amino Acid Disorders Fatty Acid Oxidation Disorders d -Leu Organic Acid Disorders


Al a


Le u Me t

Phe d3-Met

d5-Phe Ty r


0 100


Elevated Phenylalanine



0 140 160 180 200 220 240 260

m /z 280

Selective Screening by Tandem MS
Amino Acids Disorders:
         

1.Phenylketonuria (PKU)/ Hyperphenylalaninemia 2. Maple syrup urine disease (MSUD) 3. Homocystinuria/ Hypermethioninemia 4. Hypemethioninemia associated with abnormal B12 metabolism 5. Pyroglutamic aciduria 6. Citrullinemia 7. Argininosuccinase Def. (Argininosuccinic Aciduria) 8. Tyrosinemia type 1 9.Non-ketotic Hyperglycinemia 10. Argininemia

:Organic Acids Disorders
        

1. Methylmalonic Acidemia (MMA; different types) 2. Propionic Acidemia (PA) 3. Isovaleric Acidemia (IVA) 4. Glutaric Acidemia type I (GA-I) 5. Multiple CoA Carboxylase def. (MCD) 6. 3-Hydroxy-3-methylglutaryl- CoA Lyase def. (HMG) 7. 3-ketothiolase def. (BKT) . Methylcrotonyl-CoA carboxylase def. (MCC) .Malonic acidemia

:Fatty Acid Oxidation Defects
       

. Short-Chain acyl-CoA dehydrogenase def. (SCAD) . Medium-Chain Acyl-CoA Dehydrogenase Deficiency (MCAD) . Very long-Chain Acyl-CoA Dehydrogenase Deficiency (VLCAD) Hydroxy-long-Chain Acyl-CoA Dehydrogenase Deficiency (LCHAD) . Glutaric acidemia Type-II (GA-II) .Carnitine transport defect (CTD) . Carnitine palmitoyltransferase def. type I (CPTI) . Carnitine-acylcarnitine translocase def.

Decision Criteria, Interpretation, and Newborn Screening Protocol
1. Technical interpretation of acquired data. 2. Clinical interpretation and decisionmaking.

Test Limitations & Interfering :Substances
MS/MS screening and other screening tests should be done at the appropriate time after birth, which is 24-72 hours. Collection of sample before 24 hours may lead to falsenegative results. The results of this test do not include values for acylcarnitines. Antibiotics that are used as pivalate esters give interfering signal with that for the diagnosis of isovaleric acidemia In this case, confirmation by urine GC/MS analysis for organic acids is necessary.

Reporting of results
 

The interpretation is by pattern recognition. Most newborn screening laboratories are used to provide quantitative results without interpretation. A decision limit (cutoff) for each analyte or analyte ratio should be set on the 99.5th (0.05th) percentile, based on

Data collected and analyzed from a large number of healthy babies. Samples, flagged for one or more analyte must be repeated from the same blood spot.

Metabolite level


Metabolite level

Laboratories can elect to establish two levels of abnormal .results
Flagged indicative of a particular disorder for immediate referral to the clinical .management team for follow-up Borderline that require re-sampling and retesting. :Decision to use two-level system might be dependent on .the availability of follow-up resources

Reference ranges
 Phe:

37 - 85 μmol/L  Leu/Ile: 107 - 286 μmol/L  Met: 12 - 43 μmol/L  Tyr: 51 - 275 μmol/L  Phe/Tyr ratio: 0.20 - 1.00 (From 3000 normal neonates)

AIM A pilot prospective neonatal screening using MS/MS will be initiated with a view to subsequent introduction into general use &/or high risk groups :Screening will be directed to a limited range of clearly defined diseases with  Adequate specificity no need for repeat sampling  Available confirmatory tests :Aims Technical validation of the method in Egypt:Collection of data onReference ranges Cut off values in different pediatric age PPV of different metabolites groups development of EQAS-

Important points for sampling for :screening of IEM
Obtain samples before giving supportive therapy to avoid false negative tests . For amino acid disorders take samples after 2 – 3 feedings with protein meal. to allow abnormal a a to accumulate to avoid false negative results.

Special problems of obtaining proper specimens from newborns

Skin puncture → least hazardous
 

Depth < 2,4 mm Respect Site allowed for heel puncture

 

Venepuncture → avoid prolonged stasis Arterial puncture → best for BG
Lactate Ammonia

 

Umbilical Artery Venous catheters → avoid using first 2 drops of blood

Sample size:
Smallest possible for neonates but sufficient for reliable results


Time: 3rd -7th day after 4-5 milk feeding to allow accumulation of metabolites. Site: Heel prick – free flowing blood on filter paper. (Air dry-send by mail)

Dried blood spot samples (Guthrie Card)

A punch is taken out of the filter card. In most laboratories a 3mm punch is used, equal to approximately 3µL of whole blood

Lab request form for suspected IEM
            

NAME: HOSP NO: DOB: SEX: WARD: HOSPITAL: CONSULTANT: Tests required: Date of specimen Time of specimen Date & time of admission/acute symptoms History Family history:
 

 

Nutrition Feeding: Breast fed Formula fed Normal diet TPN Confirm protein 2 g/kg yes / no Any supplements

Consanguinous yes/No Previous sibling death or affected case?

Positive MS/MS NBS Result
PMD called and faxed results  Consultant names provided  Medical management recommended  F-up by PMD or Genetic Center

For the critically ill
Important samples to be taken before giving any supportive treatment

 2-3

ml heparinized & EDTA blood . blood spots on screening cards.

 Dried  5-10

ml urine in sterile container save &

freeze all urine passed for future analysis.
 For

lactate & ammonia, arterial samples are

preferable, contact lab for precautions.

Special precautions for certain analytes for the critically ill

Arterial samples are preferable, best during attack do not use tourniquet. Sample after exercise or struggling during vene-puncture (false increase) Anticoagulant : Fluoride / oxalate. Arterial sample is better, (avoid smoking in the sampling area) . Fill tube (no air bubbles). Place sample tube immediately on ice centrifuge at -4oC rapidly .


 

Prolonged crying

↑ Lactate



use smallest syringe

completely sealed → no air send immediately

 

Same precautions as lactate except : Pyruvate is extremely unstable, 2 ml blood immediately added to 4.0 ml perchloric acid (8%) kept on ice & delivered to lab for rapid separation.

When death is inevitable establishing a diagnosis is very important
counseling For  Later prenatal diagnosis
 genetic

Collect relevant specimens & biopsy before or shortly after death
 Blood

spots on screening cards  Lithium heparin & EDTA blood  All possible urine  Skin, liver biopsy

Close co-operation between attending clinician & lab supervisors is important to avoid unnecessary and expensive lab investigations

Newborn Screening Summary Neonatal Screening Summary

Reproduced with permission from American Scientist vol. 90 (2002)

tives/False Negatives

There are two possible scenarios when the sample, the analysis, or the interpretation of the data give erroneous results:

False Positives  A laboratory test detects an abnormally high metabolite  The sample is retested and is still high  A new sample is taken from the child  The new sample is normal

Think of the parental anxiety when the new sample is taken!

False Negatives  A laboratory test DOES NOT detect an abnormally high metabolite  The sample is reported as being normal  Nothing is known until the child presents clinically

The screening has failed!