How To Assure Qualified

Dried Blood Spot for

Newborn Screening Test

Dr Thyrza L Darmadi, SpPK

Screening vs Diagnostic
• Screen
– Can not determine the
presence or absence of a
disorder
• Diagnostic
– Confirm the diagnosis of a
diseases
• The value of newborn screening is
helping to rescue the lives of
children
• The success of newborn screening
depends to a great extent on the
quality of the baby’s blood
specimen  must be properly
collected
Scope of Laboratory Responsibilities
in Newborn Screening

Communication
Specimen
of abnormal
Collection
or unsatisfactory
and Receipt results
QC
Analysis
documentation
Report theresults
the specimen
COLLECTION OF DRIED BLOOD SPOT
Time
• Ideal time: 48-72 hours
• Can not be collected less than first
24 hours  false positive in TSH
Required supplies

Filter paper / blood

Lancet Safety box Sterile Gauze Pad
spot card

70% alcohol swab Bandage Sealable plastic bag Desiccant

SPECIMEN COLLECTION

• Universal Safety Precautions

– Treat all blood samples as though they are infectious
– Wash hands
– Wear gloves and apron / lab coat
– Take precaution to avoid needle injury
– Dispose of contaminated sharps and waste appropriately
• Ensure the expiration date of the filter paper has not passed
Data in DBS
• Hospital’s name
• Baby’s name
• Sex
• Baby’s weight
• DOB (date of Birth)
• Sampling date
• Gestational Age
• Blood transfusion history
• History of anti thyroid drugs (mother)
• Parent’s name, address and phone number
• Clinician’s name

Sardi, F. Skrining Hipotiroid Kongenital

SPECIMEN COLLECTION

• Clearly label each card with appropriate identification number

• Avoid touching the areas within the circles on the filter paper section  oils
and other materials from the hands might affect or contaminate the card or
specimen
SPECIMEN COLLECTION
• Parent swaddle baby (breastfeeding
or sucking is best) for remainder of
procedure
• Skin to skin contact during specimen
colletion will decreases stress
response in newborn
• Position the infant’s leg lower than
the heart
– Increases venous pressure  increase
blood flow
SPECIMEN COLLECTION

Prepare the Site

Warm the heel with soft cloth, moistened
with warm water (40◦C) for 3 to 5 minutes

Warmth leads to vasodilation, which

increases blood flow and chance of
collection success
SPECIMEN COLLECTION
Lancet Specification

• Full Term Infants (GA ≥ 37 wks)

– Standard incision is 2.5 mm in length & 1 mm in depth
• The vascular bed of an infant’s heel is about 0.35 to 0.82
mm beneath the skin; the 1 mm length of the lancet incises
across this capillary-rich area for optimum blood flow. Pain
fibers in abundance below 2.4 mm; the 1 mm incision depth
 reduce the pain experience
• Premature Infants (GA < 37 wks)
– Incision specification are 1.75 mm in length & 0.85 mm in
depth
Hatched area indicates safe areas for puncture site.
Damage to nerves and/or the heel bone may occur • About 40% smaller than the incision for a full term infant
for punctures outside of the hatched region
SPECIMEN COLLECTION
Clean site with
alcohol swab.
Allow the heel to
air dry.

Residual alcohol
can affect NBS
results &/or lead
to unsatisfactory
specimens.
SPECIMEN COLLECTION
Apply gentle pressure
with thumb and
around heel but not
near the puncture site.
Puncture heel. Wipe
away first blood drop
with sterile gauze pad.
Allow another LARGE
blood drop to form
Avoid “milking” the
puncture site
HOW TO COLLECT DRIED BLOOD SPOT

Lightly touch filter paper to LARGE blood drop. Allow

blood to soak through and completely fill circle with
SINGLE application to LARGE blood drop
If enhanced blood flow is needed, apply VERY
GENTLE, intermittent pressure to area surrounding
puncture site.
Apply blood to one side of filter paper only
SPECIMEN COLLECTION
Fill remaining circles in the same manner as
previous step, with successive blood drops.
If blood flow is diminished, repeat 3 previous
steps
Dry blood spots on a dry, clean, flat, non
absorbant surface for a minimum of three
hours.
Keep blood spots away from direct sunlight and
other sources of heat
SPECIMEN COLLECTION: Alternative samples

• Capillary tubes
– Not preferred: higher risk for collection error
– Anticoagulants – EDTA, citrate & heparin – interfere with results
– Risk of scratching the filer paper
• Venous samples
– Discouraged
– More invasive than a heel stick
• Umbilical cord blood
– Discouraged, only for sick baby or extremely low birth weight baby
– Risk for maternal blood contamination
SPECIMEN TRANSPORTATION
• After the blood card has dried, put the card into the zip
lock plastic.
– Each blood card into each plastic, OR
– Stack the blood card alternately to avoid the blood
spot touching each other, OR
– Place the paper between the blood card
• Put the zip lock plastic into the envelope
• Put the envelope in to the plastic
• Send the specimens to the referral laboratory
• Delivery should not be more than 7 days form the time
the specimen was taken. Shipping should not be more
than 3 days
SPECIMEN COLLECTION: What not to do

• Do NOT dab or”color in” the filter paper circles

• Do NOT apply multiple drops of blood per circle
• Do NOT scratch the filter paper
• Do NOT contaminate specimens
– Insufficient drying of alcohol, oils on hands, lotions, compressing the circles, spills, etc
• Do NOT stack specimens
– Risk for leaching and cross contamination between specimens
• Do NOT submit wet specimens
• Do NOT place specimens in direct sunlight or in front of air vents or other sources of moving
air
• Do NOT place wet specimens in plastic bags
HOW TO COLLECT DRIED BLOOD SPOT
Why a repeat screen may be needed ?

• Unsatisfactory specimen
• Out of range results
– Possible disorder identified
– Hemoglobinopathy trait condition
• Specimens collected less than 24 hours
– Risk for missing some disorders
• Premature or Sick Infants (TPN and antibiotics could affect results)
• Not collected prior to a blood transfusion
Acceptable filter paper
• Pre-printed circles are
completely filled with
blood
• Blood has soaked all the
way through the filter
paper
• Absence of clots blood
• Absence of serum rings
Multiple Application
• It’s easier to identify multiple
application of blood drops by
observing the back side of
the filter paper
• When bloodspots overlaps, it
creates an uneven absorption
of blood  analyte levels can
not be accurately measured
 inaccurate results
Multiple Application- Prevention
• Apply one large drop blood
to fill each pre-printed circle
• Patience !!!
– Wait for a full, healthy drop
of blood before applying to
the filter paper
• Avoid overlapping blood
spots
Clotted Blood

• Clots can occur using

capillary tubes or if too
much blood is applied
to the pre-printed
circles
• Samples with clots are
not suitable for testing
Clotted Blood – Prevention

• Avoid using capillary

tubes for collection
• Do not wait too long t o
apply the blood to the
filter paper
• It is easier to identify
clots when the specimen
is dry
Serum Rings
• Notice the halos around the periphery
of most of the pre-printed circles. This
can occur due to the following:
– Insufficient drying of alcohol on the
baby’s heel prior to heelstick
– Drying the specimen vertically instead of
horizontally
– Closing the flap of the filter paper on top
of the circles while the specimen is still
wet
– Placing wet specimen in plastic bags
– Milking or squeezing the puncture site
Serum Rings - Prevention
• Ensure the alcohol dries on the
newborn’s heel prior to the puncture
• Avoid milking the heel site. Work your
way around the infant’s heel, using
intermittent pressure as the drop of
blood forms
• Allow specimen to dry appropriately after
collection:
– Lay horizontally for 3-4 hours
– Do not place wet specimen in plastic bags
– Do not close the flap over the pre
printed-circles until the specimen is dry
– Avoid contamination, including alcohol
and hand lotion
Inadequate Amount of Blood
• The filter paper circles
are not sufficiently
filled with blood for
testing
Inadequate Amount of Blood - Prevention

• Prior to specimen collection, ensure:

– Baby’s heel has been pre-warmed via
warm cloth or heel warmer for up to 3-5
minutes
– Baby’s heel is lower than the heart
• Patience !
– Wait for a full, healthy drop of blood
before applying to the filter paper
• After each blood drop is applied,
check to ensure that the blood has
soaked completely through the filter
paper
Under-Saturation
• Notice how the blood
has not soaked all the
way through the filter
paper.
• Causes:
– Not enough blood in this
sample for testing
– High hematocrit
Under-Saturation - Prevention
• After each blood drop is
applied, check to ensure
that the blood has soaked
completely through the
filter paper
• Patience !
– Wait for a full, healthy drop
of blood before applying to
the filter paper
Smeared blood – Pressed too hard against the heel
Smeared blood – Prevention

• Patience !
• Wait for a full, healthy drop of blood before applying to the filter paper
• The filter paper should not contact with the baby’s heel
Folds and creases through the blood circles – Prevention

• Lay horizontally for 3-4 hours

• Do not fold the filter paper
Why the lab need all 5 circles ?

• If a result is flagging out of range, the specimen will be retested and the final
result with be an average of three results. Each test requires an additional
punch to be taken from the pre-printed circles.
• The specialist and family may request for the specimen to be sent to another
laboratory for additional testing to assist in determining diagnosis
Babies with Special Condition Considerations
Factors that can interfere with screening results:
• Prematurity,LBW and sick baby may affect TSH & 17 OHP results
• Hemolytic may affect the G6PD results
Infant • Carrier status may affect all NBS results

• Steroids may affect 17 PHP results

Treatment • ECLS & blood transfusions may affect all NBS results.

• PTU therapy or radioactive iodine may affect infant TSH results

Maternal • Steroids may affect infant 17 OHP results

• Contamination: oils / lotion from hands, spills, residual alcohol, heat / humidity
• Early / delayed specimen collection
Collection • Transit time delays
issues
• Unsatisfactory specimens
Babies with Special Condition Considerations

MELEV (Multiple Elevations of Analytes)

• This results is often due to additives to feedings, such as TPN, carnitines,

MCT oil
• A repeat screen for an MELEV result should be collected after TPN is discontinued

Transfusions

• Can affect all NBS results. Obtain NBS specimen prior to blood
transfusion, if possible.
• If baby is transfused prior to initial screen, obtain two repeat screens:
• 7 days post transfusion &
• 90 – 120 days post transfusion
SPECIMEN COLLECTION: Reminders
• Pre collection:
– Check the ED of the filter paper. Discard the expired paper
• Post collection:
– Air dry specimen horizontally for 3-4 hours
• Transporting wet specimens can make them unsatisfactory for testing
– Send specimen with courier within 24 hours of collection
• Maintain specimen collection log & ensure screening results are received &
recorded
• Ensure that everybody who handles the filter paper or is involved in the newborn
bloodspot collection process is trained
PROFICIENCY TESTING OF DBS
• In order to asses analytical performance, external PT is essential.
• Results and corrective actions should be documented
• Newborn Screening Programs: US NSQAP CDC, UKNEQAS,
France, Germany, Japan, Korea, New Zealand, China, Thailand
• Unknown specimens distributed to program participants  PT
specimens (dried blood on filter paper) should be analyzed in a
manner identical to that used for patient specimens results are
reported back for comparison with participant results
UK NEQAS
NSQAP CDC
• NSQAP creates and distributes two
types of certified laboratory dried
blood spot materials in support of
regulatory compliance and
accreditation for testing: quality
control materials and proficiency
testing materials
NSQAP QC Materials
• Quality control materials are sent twice per year for
periodic use
• DBS QC lots were prepared from whole blood of
50% hematocrit.
• The QC materials were enriched with
predetermined quantities of selected analytes and
dispensed in 100 μL aliquots on Grade 903 filter
paper
• NSQAP publishes annual and midyear reports that
summarize quality control results for all reported
methods used for testing.
NSQAP PT Materials
• NSQAP distributes PT materials
three times per year. PT panels
consist of five blind-coded
specimens.
Critical points for developing a high quality screening INSIDE
laboratory

• A manual of procedures includes quality assurance guidelines

• Testing should include internal specimen tracking and monitoring, and
documenting quality assurance
• Adequate instrumentation, appropriate consumable supplies, instrument
maintenance and calibration
• Adequate trained personnel
• Filter paper should be the same as those used for QC and kit calibration
materials
• Time of specimen collection, time of receipt, time of reporting must be
monitored
• Quality of assay performance should include: monitoring reagent quality,
internal and external assay quality assessment, result validation before release
• Rapid reporting of presumptive positive tests
• A proper transfer of report to the appropriate person to ensure rapid follow up
Critical points for developing a program of high quality
OUTSIDE laboratory

• A follow up procedure manual

• Follow up staffing adequately trained personnel
• Tracking should include defined follow up actions before considering a case
“lost to follow up”
• Educational materials explaining the program should be available to parents
before and after delivery as needed, to explain the condition (for positive)
tests
• In cases where testing results are positive, educational materials should be
available to physicians that describe what immediate actions need to be taken
and provide additional resources for further reference.
• A system for providing follow-up patient information (outcome progress) from
the program should be established and used to evaluate program activities.
Laboratory Quality Indicators

• Analytical / reporting TAT

• False positive rate
• False negative rate
• Performance in external PT programs
• Available / updated operating manual
• Trained personnel
• Record keeping:
– Maintenance
– Analytical performance
– Patient records
– Corrective Actions
Non Laboratory Quality Indicators

• Number of reason for rejected specimens

• Number of reason for parents opting out (in)
• Recalled patients “lost” to follow up
• Educational materials available / provided
• Time in transit for specimens / reports
• Time from birth to diagnosis / treatment
• Extended follow up:
– Compliance with medication / management
– Morbidity / mortality data
– Overall health outcome
– Costs (for cost – benefit comparison)