guidelines for lab detection of iem fayza20febr2008final

8/14/2019 Guidelines for Lab Detection of IEM Fayza20febr2008final

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Laboratory Guidelines For

Screening And Detection OfInborn Errors Of Metabolism

Dr. Fayza Abdel-Hamid HassanProf. Clinical and Chemical pathology

Faculty of Medicine ,Cairo University


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The first few days of a newborns life

can be critical to his future well-being.


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Clinical manifestations of IEM are non

specific & overlap with common illnesses

Frequently remainundiagnosed 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


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Apparently healthy at birth after symptom-free interval .

Placental protection: placenta provides an effective dialysissystem for removal of toxic metabolites, most babies with IEM

are born in good condition & normal birth wt .

exceptions

Lactic acidosis

Glutaric aciduria II

Non ketotic hyperglycinuria

Two types of presentations :

2. Neonate with over whelming neurological illness withunconsciousness, convulsions, apnea with no apparent

symptom-free interval.


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:Pathogenesis of IEM

SubstrateE

Product

activityeg: albinism

Collagen defectsPKUMSUD

Toxic metabolites

galactosemia

Alternativemet.pathway

.1Enzyme or structural protein deficiency

. accumulation of toxic metabolites

2. Defect in membrane transport

cystinuria.

.3Deficiency of co-factors or other substrates.


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Hydroxylase

X

Phenylalanine

Hydroxylase

O

NH2

OH

O

NH2OH

OH

[Phenylalanine][Phenylalanine] [Tyrosine][Tyrosine]

Classical Phenylketonuria (PKU)

Classical Phenylketonuria


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Detection of IEM

Mass neonatal screening.

Screening the at risk.

Detection of suspected IEM.


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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.


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ives/False Negatives

There are two possible scenarios when the sample, the analysis, or theinterpretation 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!

RETURN


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History of NBS

1957 - Dr. Centerwall (father of a child

with mental retardation (Ferric chloride)

1961 - Dr. Guthrie (BIA)

1963 - MA and OR implementlegislation


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Early screening methods

Bacterial inhibition assays. Single test for single disorder (BIA)Paper 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


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PKU by Bacterial InhibitionPKU by Bacterial Inhibition

AssayAssay


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Recent use of the tandem massRecent use of the tandem mass

spectrometer has allowed for changes inspectrometer has allowed for changes in

traditional newborn screening servicestraditional newborn screening services

leading to expansion and improvement ofleading to expansion and improvement of

testingtesting


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History of NBS

1997 - NC begins piloting MS/MS

1999 - NC and MA begin newbornscreening with MS/MS

2000 - WI begins MS/MS NBS


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It requires aIt requires a very small sample sizevery small sample size to screento screen

forfor

a large number of rarea large number of rare

disordersdisordersinin

a very quicka very quick amount of time, about 2amount of time, about 2minutesminutes


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Expanded neonatal

screening by MS/MS

Although individually rare their

cumulative effect can be

devastatingUSA 1 in 3500

Germany 1 in 4000

KSA 1 in 750


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?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


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Investigations of suspected hypoglycemia

in children

12h fast if< 6m

24h fast if>12m

glucose

Normal

Ketotic

hypoglycemia

insulinNo in B(OH)B

FAO defects

GH Or

Cortisollactate

Normal glucose

Measure blood

glucose

Measure:

Insulin,GH,Cortisol,

Lactate,B(OH)B

No hepatomegaly

Obtain critical blood sample


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All Mass Spectrometers

Must generate ions

separate ions detect ions

compute ion intensities interpret Data


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1.Quadrupole Mass Analyzer

Source

Detector

Nonresonant Ion

Resonant Ion

dc and Rf voltages

rf and -dc

rf and +dc


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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.


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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 singletwo minute analysis.

Reduces false positive rates by more than ten fold.


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IonFragmentation


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Disorders Screened Using MS

Amino Acid Disorders Fatty Acid Oxidation Disorders

Organic Acid Disorders

140 160 180 200 220 240 260 280m/z0

100

%

0

100

%

Normal

PKU

Le

u

d3-Leu

d4-AlaAla

PheTyr

Met

d3-Met

d5-Phe

d6-Tyr

Phe

Elevated Phenylalanine


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Selective Screening by TandemSelective Screening by Tandem

MSMSAmino Acids Disorders:Amino Acids Disorders:

1.Phenylketonuria (PKU)/ Hyperphenylalaninemia1.Phenylketonuria (PKU)/ Hyperphenylalaninemia 2.2. Maple syrup urine disease (MSUD)Maple syrup urine disease (MSUD) 3.3. Homocystinuria/ HypermethioninemiaHomocystinuria/ Hypermethioninemia

4.4. Hypemethioninemia associated with abnormal B12 metabolismHypemethioninemia associated with abnormal B12 metabolism 5.5. Pyroglutamic aciduriaPyroglutamic aciduria 6.6. CitrullinemiaCitrullinemia 7.7. Argininosuccinase Def. (Argininosuccinic Aciduria)Argininosuccinase Def. (Argininosuccinic Aciduria) 8.8. Tyrosinemia type 1Tyrosinemia type 1

99..Non-ketotic HyperglycinemiaNon-ketotic Hyperglycinemia 1100.. ArgininemiaArgininemia


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:Organic Acids Disorders:Organic Acids Disorders

1.1. Methylmalonic Acidemia (MMA; different types)Methylmalonic Acidemia (MMA; different types) 2.2. Propionic Acidemia (PA)Propionic Acidemia (PA) 3.3. Isovaleric Acidemia (IVA)Isovaleric Acidemia (IVA) 4.4. Glutaric Acidemia type I (GA-I)Glutaric Acidemia type I (GA-I) 5.5. Multiple CoA Carboxylase def. (MCD)Multiple CoA Carboxylase def. (MCD) 6. 3-6. 3-Hydroxy-3-methylglutaryl- CoA Lyase def. (HMG)Hydroxy-3-methylglutaryl- CoA Lyase def. (HMG) 7. 3-7. 3-ketothiolase def. (BKT)ketothiolase def. (BKT) .. Methylcrotonyl-CoA carboxylase def. (MCC)Methylcrotonyl-CoA carboxylase def. (MCC) ..Malonic acidemiaMalonic acidemia


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:Fatty Acid Oxidation Defects:Fatty Acid Oxidation Defects

.. Short-Chain acyl-CoA dehydrogenase def. (SCAD)Short-Chain acyl-CoA dehydrogenase def. (SCAD) .. Medium-Chain Acyl-CoA Dehydrogenase DeficiencyMedium-Chain Acyl-CoA Dehydrogenase Deficiency

(MCAD)(MCAD) .. Very long-Chain Acyl-CoA Dehydrogenase DeficiencyVery long-Chain Acyl-CoA Dehydrogenase Deficiency

(VLCAD)(VLCAD) Hydroxy-long-Chain Acyl-CoA DehydrogenaseHydroxy-long-Chain Acyl-CoA Dehydrogenase

Deficiency (LCHAD)Deficiency (LCHAD) .. Glutaric acidemia Type-II (GA-II)Glutaric acidemia Type-II (GA-II)

.Carnitine transport defect (CTD).Carnitine transport defect (CTD) .. Carnitine palmitoyltransferase def. type I (CPTI)Carnitine palmitoyltransferase def. type I (CPTI) .. Carnitine-acylcarnitine translocase def.Carnitine-acylcarnitine translocase def.


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Decision Criteria, Interpretation,

and Newborn Screening

Protocol

1. Technical interpretation of acquired

data.2. Clinical interpretation and decision-

making.

Test Limitations & InterferingTest Limitations & Interfering


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Test Limitations & InterferingTest Limitations & Interfering

:Substances:Substances

MS/MS screening and other screening tests should beMS/MS screening and other screening tests should bedone at the appropriate time after birth, which isdone at the appropriate time after birth, which is

24-72 hours.24-72 hours.

Collection of sample before 24 hours may lead to false-Collection of sample before 24 hours may lead to false-

negative results.negative results.The results of this test do not include values forThe results of this test do not include values for

acylcarnitines.acylcarnitines.

Antibiotics that are used as pivalate esters give interferingAntibiotics that are used as pivalate esters give interfering

signal with that for the diagnosis of isovaleric acidemiasignal with that for the diagnosis of isovaleric acidemiaIn this case, confirmation by urine GC/MS analysis forIn this case, confirmation by urine GC/MS analysis for

organic acids is necessary.organic acids is necessary.

R ti f lt


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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.


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Metabolite level

frequencies

frequenci

es

Metabolite level


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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


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Reference ranges

Phe: 37 - 85 mol/L

Leu/Ile: 107 - 286 mol/L

Met: 12 - 43 mol/LTyr: 51 - 275 mol/L

Phe/Tyr ratio: 0.20 - 1.00

(From 3000 normal neonates)


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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 specificityno need for repeat sampling Available confirmatory tests

:Aims

Technical validation of the method in Egypt-

:Collection of data on-

Reference ranges

Cut off values

PPV of different metabolites

development of EQAS-

in different pediatric agegroups


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Important points for sampling for

:screening of IEM

-Obtain samples before giving supportive therapyto avoid false negative tests .

- For amino acid disorders take samples after 2 3feedings with protein meal.

to allow abnormal a a to accumulate to avoid

false negative results.

S i l bl f bt i i


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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 ofblood

Sample size:

Smallest possible for neonates but sufficient for reliableresults


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Sampling

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)


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Dried blood spot samples


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A punch is taken out of the filter card.

In most laboratories a 3mm punch is used, equal to

approximately 3L of whole blood

Dried blood spot samples

(Guthrie Card)

Lab request form for suspected IEM


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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 fedNormal dietTPNConfirm protein 2 g/kg yes / noAny supplements

Consanguinous yes/No Previous sibling death or affected case?


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Follow-up

Positive MS/MS NBS Result

PMD called and faxed results

Consultant names provided

Medical management recommended

F-up by PMD or Genetic Center


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For the critically illFor the critically ill

Important samples to be taken before giving any supportive

treatment

2-3 ml heparinized & EDTA blood .

Dried blood spots on screening cards.

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.


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Special precautions for certain

analytes for the critically ill

Lactate Arterial samples are preferable, best during attack do not

use tourniquet.

Sample after exercise or struggling during vene-puncture

(false increase)

Anticoagulant : Fluoride / oxalate.

Ammonia:

Arterial sample is better, (avoid smoking in the samplingarea) .

Fill tube (no air bubbles).

Place sample tube immediately on ice centrifuge at -4oC

rapidly .


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Prolonged crying Lactate glucose


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se smallest syringe

completely sealed no air send immediately


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Pyruvate

Same precautions as lactate except : Pyruvate is extremely unstable, 2 ml blood

immediately added to 4.0 ml perchloricacid (8%) kept on ice & delivered to labfor rapid separation.

Wh d th i i it bl t bli hi


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When death is inevitable establishing

a diagnosis is very important

Collect relevant specimens & biopsybefore or shortly after death

Blood spots on screening cards

Lithium heparin & EDTA bloodAll possible urine

Skin, liver biopsy

genetic counseling Later prenatal diagnosis

For


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Close co-operation between

attending clinician & lab

supervisors is important to

avoid unnecessary andexpensive lab investigations


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ives/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!

RETURN

guidelines for lab detection of iem fayza20febr2008final

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