– Sample collection

– Assay

– Data evaluation

– Reporting

Dried Blood Spot (DBS) collection for

newborn screening

– Timing

– Blood collection

– Package and transfer

PONZONE ET AL. J Pediatr Gastroenterol Nutr 46(5), 2008

Newborn with PKU on a diet without phenylalanine

• Etiology:

– Birth stress– Postnatalis éhezés– Low energy intake– Immature enzyme systems

• Clinical symptoms:

– Weight loss– Increased nitrogen excretion (urine)

Catabolism

• Metabolite levels change related to time

• Maximal sensitivity and specificity:

2-6 days after birth

Time of blood collection

0. 1. 3. 4. 2. nap

24 48 72 96 120 óra

Limited Acceptable Optimal

(Newborn Screening in New York State, 2003)

• Hypothyroidism:

2005-6: 23/126 = 18,2%

2009-2010: 28/129 = 21,7%

• Classic PKU:

< 2007 ~1 / 9 000

2008-2010 1 / 8 333

Incidence

Criteria: 60 ml milk/formula intake

?

Screening test for galactosemia – blood galactose level

Repeated sample is needed 2 weeks and 60days after

blood transfusion

– Feeding – parenteral feeding

– Blood transfusion

– Gestational age

– Drugs

Factors with influence on metabolite

levels

Beküldő intézet (pecsét):

Orvos (pecsétszám):Gyermek családi neve: ♂ ♀❏ ❏

Születési súly:Utóneve: └┴┴┴┘g

Terhességi hét:└┴┘Születési dátum: Vérvétel időpontja└┴┴┴┴┴┴┴┘ └┴┴┴┴┴┴┴┘

A vérvétel időpontjában a baba:□ 48 óránál idősebb

□ 48 óránál fiatalabb

□ koraszülött Figyelem! Kötelező

□ transzfúzió a szűrővizsgálat

□ 2. vérvétel megismétlése!

Táplálás: □ anyatej □ tápszer □ parenter. Gyógyszerek:Anya családi neve: Utóneve:TAJ: Telefon:Irányítószám: Település:Utca, házszám:Szegedi Tudományegyetem, Gyermekgyógyászati KlinikaAnyagcsere laboratórium • Újszülöttkori szűrés6720 Szeged, Korányi fasor 14-15. Telefon: (06 62) 54 59 51 Fax: (06 62) 54 53 29

• Correctly complete all the

information on the card

Name:

Birth date:

Metabolic Laboratory, Dept. Pediatrics, Univ Szeged

Repeated testing

– Gestational age < 32 weeks

– Blood collection < 48 h after birth

– Food intake < 60 ml

– Blood tansfusion/parenteral feeding

• Newborns, small infants (<9kg)– Heel – Do not puncture the fingers

• Larger infants (> 9kg) – Heel or lateral aspect of the big toe – Do not puncture the fingers– Stick fingers in children > 2 years of age

Site of puncture

Safe

Safe

• Clean site with alcohol

• Allow to air dry for 30 seconds

• Puncture to a depth less than 2 mm using a sterile

lancet

• Wipe away first blood with sterile gauze pad

• The initial drop contains tissue fluid that may

dilute the specimen

• Allow another large blood drop to form

• Lightly touch the filter paper to the large drop of blood

• Avoid touching the spots

• Allow to soak through and completely fill the circle

• Apply blood to one side of the filter paper only

• Do not layer successive drops of blood or apply

blood more than once to the same collection circle

• Fill remaining circles in the same manner with

successive drops of blood

• If blood flow is diminished repeated puncturing

may be necessary

• Inspect card to ensure you have collected

enough blood, and the specimen is valid

Valid Specimen

• Place filter paper on a

horizontal surface and

AIR DRY for at least 3

hours

• Keep away from direct

sunlight, heat and dust

• Do not allow blood spots

to come into contact with

any surface or each other

• Do not store, transport

cards every day

Valid and invalid DBS

Invalid DBS

Assay

QC

• CDC: amino acids (Phe, Tyr), galactose,

TSH17OH-progesterone

• Interlaboratory Quality Assurance for Newborn Screening (Geesthacht, Germany):

Phe, galactose

• ERNDIM:

Evaluation of data

Cut-off levels

Sahai I, Marsden D: Crit Rev Clin Lab Sci 46(2):55-82,2009

Analyte concentration

Mean

(Normal)

Mean

(Affected)Cut-Off

Affected

Normal Population

Mean

(Normal)

Normal Population

AffectedFals

Positives

Fals Negatives

True positives

Nu

mb

er o

f n

eon

ates

True Negatives

Screening test performance

• Sensitivity

• Specificity

• False negative rate

• False positive rate

• Positive predictive value

• Negative predictive value

Problems

• Incorrect blood sampling

• Repeated sample

- parents not found

• Screening assay

• Does not exclude all metabolic

disorders

Keep in mind

Second-tier tests

• Beutler-test

• BH4-assay

• T3, T4

• Steroid profile

Mutation analysis

• Diagnosis

• Prognostic value

• Prenatal diagnosis

Protocols

Analytical side - Clinical side

screening therapy

follow up follow up

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