a brief on Newborn Screening and inborn errors of metabolism based on USA health policy protocols.

1. NEWBORN SCREENING
Presented by:
Dipesh Tamrakar
MSc. Clinical Biochemistry

2. OVERVIEW
• Newborn Screening
• Inborn errors of metabolism
• Parameters in NBS
• Sample collection techniques
• A brief on methods of analysis
• Summary

4. Demography of Nepal
In 2011,
• The birth rate of Nepal is 22
births/1000 people
• Infant mortality of 46 deaths/1000
people
In 2017,
• Birth Rate: 19.5 births/1,000
population
• Infant Mortality Rate:
• Total: 27.9 deaths/1,000 live
births
• Male: 29.2 deaths/1,000 live
births
• Female: 26.6 deaths/1,000
live births
Volume 01, NPHC 2011
National Population and Housing Census
2011
(National Report)
Government of Nepal
National Planning Commission Secretariat
Central Bureau of Statistics

5. Incidence of Different Conditions Screened in
India and China
S No Condition screened Incidence India Incidence China
1 Congenital Hypothyroidism 1: 1700 1:2020
2 G6PD Deficiency 1:2200 0.1%
3 Cong Adrenal Hyperplasia 1:2575 1:12000
4 Tyrosinemia 1:6234
5 Mapple Syrup Urine Disease 1:10215
6 Galactosema 1:10300
7 Phenylketouria 1:18300

6. What is Newborn Screening?
• Newborn Screening is a system for identifying genetic and other
health problems in newborns that leads to overall improvement
in the public health.
• NBS tests look for serious developmental, genetic, and
metabolic disorders so that important action can be taken during
the critical time before symptoms develop.
• Challenges for diagnosis are restrictions on blood volume,
difficulties in blood sampling, lack of appropriate reference values,
and disorders unique to the newborn.

7. NBS System

8. What is the purpose of newborn screening?
• The purpose of newborn screening is to detect potentially fatal or
disabling conditions in newborns as early as possible, often before
the infant displays any signs or symptoms of a disease or condition.
• Such early detection allows treatment to begin immediately, which
reduces or even eliminates the effects of the condition.
• Many of the conditions detectable in newborn screening, if left
untreated, have serious symptoms and effects, such as lifelong
nervous system damage; intellectual, developmental, and physical
disabilities; and even death.

9. Inborn Errors of Metabolism
Diverse heterogeneous group of hereditary biochemical disorders
with the versatile clinical manifestations mainly in children
Usually normal at birth
Non specific clinical manifestations, confounding with neonatal sepsis
Rapidly progressive, causes irreversible damage early in course.
Usually severe & lethal unless early diagnosis and proper therapy
promptly initiated.
Treatment can often be effective if commenced early and long term
outcome may be improved
Correct diagnosis helps in genetic counselling.

10. A high index of suspicion for IEM
• Parental consanguinity
• Positive family history of similar illness/death
• History of clinical deterioration in previously normal neonate
• Symptoms onset a few days after feeding (refusal to feed, vomiting,
lethargy, hypotonia, coma, seizures)
• Persistent vomiting, hypoglycemia, ketosis, metabolic acidosis
• Developmental delay or regression
• Unusual odour particularly during an acute illness
• Jaundice, visceromegaly, Dysmorphic features

11. What disorders are newborns screened for in the
United States?
• The mission of the Advisory Committee on Heritable Disorders in
Newborns and Children is to reduce morbidity and mortality in
newborns and children who have, or are at risk for, heritable disorders.
• The committee issues a Recommended Uniform Screening Panel
(RUSP) that identifies
1. A number of core conditions—those that states are highly
recommended to screen for—and
2. Secondary conditions, for which screening is optional.
• The committee’s recommendations are based on the Newborn
Screening: Towards a Uniform Screening Panel and System.
• As of November 2016, the RUSP included 34 core conditions and 26
secondary conditions.

12. 3 major NBS:
1. Hearing Test: Otoacoustic emissions (OAE) & Auditory brain stem
response ABR
2. Pulse Oximetry: to identify critical congenital heart disease (CCHD)
3. Other Blood Tests:
• The first screening is performed 24 to 48 hours after the infant is born,
ideally before the infant leaves the hospital. For some conditions, the
screening is not valid if the blood is taken within 24 hours of birth.
• The second screening is performed when the infant is 10 days to 2
weeks old to ensure that the health care provider has the most accurate
results possible.

17. Newborn screening programmes in Europe
• The conditions most screened are congenital hypothyroidism (37
countries) followed by phenylketonuria/hyperphenylalaninaemia (33
countries) and Congenital adrenal hyperplasia (15 countries).
• Total 39 conditions in NSB in various European Countries.

20. History of NBS in USA
• NICHD has been at the forefront of newborn screening efforts since the
1960s.
• One of the Institute's earliest research successes was validation of the mass
screening test developed by Dr. Robert Guthrie for the metabolic
disorder phenylketonuria (PKU).
• One of the Institute's early research efforts was a study of the effectiveness
of the Phe-restricted diet for children diagnosed with PKU.
• This research showed that children with PKU who followed a restricted diet
(one with limited amounts of the amino acid, Phe) were as healthy at age 7
years as their brothers and sisters who did not have PKU.
• In 2013, USA celebrated the 50th anniversary of NBS.
• An estimated 200 million newborns in the US have been screened during
that period for at least 1 disorder, phenylketonuria.
• Today’s screening effort detects >10,000 newborns with certain heritable
disorders from the 4 million screened annually.

21. • In 2002, the federal Health Resources and Services Administration's
Maternal and Child Health Bureau asked the American College of
Medical Genetics (ACMG) to develop guidelines for newborn
screening.
• The ACMG looked at 81 conditions and placed 29 of them in a core
screening panel, which made up the original Recommended
Universal Screening Panel (RUSP).
• ACMG assigned another 25 conditions to a secondary level of
screening because the conditions lacked an effective treatment or
because the disease was not well understood.
• The remaining conditions were deemed not appropriate for newborn
screening because no effective screening tool or treatment was
available for them.

22. • In 2003, the majority of U.S. states screened for only six disorders.
• In September 2005, Advisory Committee on Heritable Disorders in
Newborns and Children (ACHDNC) recommended the RUSP the
nation's newborn screening standard.
• It was adopted in May 2010 with the addition of severe combined
immunodeficiency (SCID).
• The list of screened disorders has since expanded to include critical
congenital cyanotic heart disease.
• By April 2011, all states were testing for at least 26 disorders.
• As of November 2016, the RUSP included 34 core conditions and 26
secondary conditions.

23. Is it necessary to perform NBS ?
• Many conditions included in today's U.S. newborn screening
programs no longer cause serious disability or illness because they
are detected early and treated immediately.
• The three examples that follow are conditions that cause serious
developmental and intellectual disabilities, or death, if they are not
detected and treated early.
• Successful newborn screening for these conditions and follow-up
treatment means that babies who might have died or needed
specialized long-term care, can now grow into healthy adulthood.

24. 1. Severe combined immune deficiency (SCID)
• One of the newest additions to the RUSP is an inherited condition,
called SCID , that makes a child’s body unable to fight off even mild
infections.
• The condition, also known as “bubble boy syndrome,” causes parts of
the immune system to not work properly.
• If untreated, infants with SCID are unlikely to live past the age of 2 years.
However, when SCID is detected and treated early, children can live
longer, healthier lives.
• SCID is rare, with between 40-100 infants diagnosed each year in the
United States.
• Because SCID is a newcomer to the RUSP, not all states screen for it yet,
meaning infants with the condition might be getting sick without being
diagnosed.

25. • Infants should be evaluated for SCID and other types of immune
system problems if they have:
1. A high number of infections
2. Infections that do not improve with antibiotic treatment for 2 or
more months
3. Diarrhea
4. Poor weight gain or growth (called “failure to thrive”)
5. Fungal infections in the mouth (called “oral thrush”) that will not go
away
6. An infant with any of these warning signs should be tested for SCID
as soon as possible.

26. 2. Phenylketonuria (PKU)
• PKU is a metabolic disorder of phenylalanine that is detected by newborn
screening.
• By 3 to 6 months of age, infants with untreated PKU begin to show
symptoms of intellectual and developmental disability. These disabilities
can become severe if Phe remains at high levels.
• Fortunately, PKU is treatable. Diet restriction containing little or no Phe and
higher levels of other amino acids as the research showed normal growth
with no health problems.
• Before newborn screening programs could detect PKU in the first few
hours after birth, PKU was one of the leading causes of intellectual and
developmental disabilities (IDD) in the United States.
• Today, as a result of newborn screening programs that allow for almost
immediate treatment of the condition, PKU has been virtually eliminated
as a cause of IDD in this country.

27. 3. Galactosemia
• Galactosemia which means being unable to use galactose.
• If someone with galactosemia consumes milk or milk products (human or
animal), the galactose builds up in their blood and causes serious damage to
their liver, brain, kidneys, and eyes.
• Infants with untreated galactosemia can die of a serious blood infection or of
liver failure. Those that may survive usually have IDD and other damage to the
brain and nervous system.
• Even milder forms of galactosemia still require treatment to prevent early
cataracts, an unsteady gait, and delays in learning, talking, and growth.
• The treatment for galactosemia is not to consume any milk or milk products
and to avoid other foods that contain this sugar.
• Before it could be detected either before birth or through a newborn
screening program, galactosemia was a frequent cause of IDD and early
death.
• Screening has identified more than 2,500 infants with the condition, many of
whom would have died without the screening.

28. How is newborn screening done?
• Newborn screening usually starts with a blood test, followed by a hearing
test and possibly other tests.
• First, hospital staff fill out a newborn screening card with the infant’s vital
information
—name, sex, weight, and date and time of birth—and the date and
time of the blood collection.
• Part of the card consists of special absorbent paper used to collect the
blood sample.
• "heel stick" a small puncture in the baby's heel and squeeze out a few
drops of blood. They put the absorbent part of the card in contact with
the blood drop.
• The card is then sent to a laboratory, where the blood is tested for the
various conditions as part of the newborn screening panel.

29. The filter papers, Whatman Grade 903 and Ahlstrom Grade 226, are made from
high-purity cotton linters and manufactured to yield accurate and reproducible blood
samples according to the Clinical and Laboratory Standards Institute’s specifications

30. Blood spot Collection
• Blood spot collection can be performed by trained personnel such as
hospital nursery staff, laboratory staff, or out-of-hospital birth
providers.
• Timing of Collection: blood spots drawn too early or too late may yield
false results
• Blood spots are best collected between 24 and 48 hours of age (at least
24 hours old).
• Blood spots collected before 24 hours generates an unsatisfactory result
that require another blood spot collection. (False Negative)
• Blood spots collected after 48 hours may not allow enough time for
results to be interpreted before serious symptoms occur in newborns
affected by certain disorders on the newborn screening panel.
• However, special circumstances may arise that require blood spot
collection at earlier or later time.

31. Proper specimen collection technique as outlined by the Clinical and
Laboratory Standards Institute include the following steps:
• Ensure that the expiration date of the newborn screening card has
not passed.
• Complete the required patient information fields requested on the
demographic data portion of the card.
• Avoid touching the area within the circles on the filter paper section
before, during, and after collection of the specimen, since oils and
other materials from the hands might affect or contaminate the card
or specimen.
• Do not allow water, feeding formulas, antiseptic solutions, glove
powder, hand lotion, or other materials to come into contact with the
specimen card before or after use.

32. • Blood collection from the heel is the standard for newborn screening.
• The medial and lateral parts of the underfoot are preferred.
• Blood should NEVER be collected from:
• the arch of the foot
• the fingers
• the earlobes
• a swollen or previously punctured site
• IV lines containing other substances (TPN, blood, drugs, etc.)

34. • Warm the heel with a warm damp cloth or commercially available heel
warmer and position the leg lower than the heart to increase venous
pressure before collecting the blood spots.
• The infant should be swaddled in a blanket with only one foot exposed.
• Powder-free gloves are best worn while collecting the blood spots.
• Lotion, Vaseline, and other substances, which can interfere with blood
spot analysis, should be kept off the infant’s skin.
• Wipe the skin clean with an alcohol wipe and allow to thoroughly air dry.
• Use a sterile lancet or heel incision device to make an incision 1 mm deep
and 2.5 mm long. (shallow incision is more safer).
• Wipe away the first drop of blood with a sterile gauze pad.
• Allow a large drop of blood to form using the thumb to intermittently apply
gentle pressure to the heel may be helpful in encouraging the drop to
coalesce.

35. • Touch the first circle on the newborn screening card gently against
the large blood drop, and in one step, allow the blood to soak
through the filter paper and fill the circle.
• Do not press the paper directly against the baby’s heel. Each of the
five/four circles need to be filled and saturated through.
• Apply the blood to only one side of the filter paper.
• Do not apply multiple layers of blood drops to the same circle.
• The circles are measured and should contain a set volume of blood.
Layering can interfere with the accuracy of the test by providing a
non-standard amount of blood or non-uniform analyte concentration.
• Excessive milking or squeezing of the puncture site can result in an
unsatisfactory specimen because of hemolysis breaking down the
blood cells to be analyzed or mixing tissue fluids in the specimen,
which can dilute the blood.

36. • Allow the specimens to dry flat at room temperature for at least 3
hours.
• This is essential to maintaining the integrity of the blood spots.
• Keep them out of direct sunlight and away from other heat sources.
• Do not allow them to touch other surfaces or specimens.
• Avoid stacking the cards.
• Do NOT close the biohazard flap over the spots until they are
completely dry.
• Once the blood spots have dried completely, the biohazard flap can be
closed and the newborn screening card can be sent to the Newborn
Screening Program.

39. Recommendedblooddraw volumes for pediatric patients

40. Techniques for blood tests
• MS/MS for metabolic diseases, IEF or HPLC, for hemoglobinopathies,
and all others).
• IEF, and HPLC for hemoglobin variants, DNA arrays used in
sequencing, and labeled bead technologies.

41. • Congenital adrenal hyperplasia (CAH)-primary marker used in
newbrn screening for 21-OH, 17-hydroxyprogesterone (17-OHP)
• Galactokinase deficiency (GALK)
• Glucose 6-phosphate dehydrogenase deficiency (G6PD)
• Hemoglobinopathies (Hb Pathies)- over 700 hemoglobin variants
identified by the methods used for screening, and 25-30 are
considered clinically significant.
• Homocystinuria (HCY)-screened for by detection of an elevated
concentration of methionine, a secondary biochemical marker of the
condition, homocysteine in blood spots

42. • Methylmalonic acidemia (MMA)-The biochemical marker of MMA is
propionylcarnitine
• 3-Methylcrotonyl-CoA carboxylase deficiency (3MCC)
• Tyrosinemia type I (TYR I)-concentration of tyrosine. There is
evidence of less than ideal sensitivity, measuring succinylacetone in
blood spots could improve the sensitivity and specificity
• Hyperbilirubinemia

43. Dried blood Spot DNA extraction
1. Erythrocyte lysis: cut dry blood spot & overnight in 1 ml of
0.5% saponin
2. Washing in phosphate buffered saline
3. DNA extraction with Chelex Resin
4. Avoid Chelex Resin Beads in final extraction
Dried blood spot protein extraction
The blood is eluted out in phosphate buffered saline containing
0.05% Tween 80 and 0.005% sodium azide, overnight at 4 °C.
Fully Automated procedures are available with 3 mm cut dry blood
spot.

44. Mass Spectrometry
1
• IONIZATION
2
• MASS
ANALYZER
3
• DETECTION
44

45. Tandem Mass Spectrometers
• 1st : used to select ions of particular m/z called either parent ion or
precursor ion
• 2nd: directed into collision cells, precursor ions collide with background gas
molecules & broken to form product ion
• Possible scan function involving 1st MS to select a given m/z and full
scanning through mass spectrum of product ion (structural
characterization)
• In constant neutral loss scan, 2MS are scanned synchronously with m/z
offset between parent and product ion
• Another scan function is multiple reaction monitoring (MRM)
45

46. High Performance Liquid Chromatography (HPLC)

47. Isoelectric Focusing (IEF) Electrophoresis

49. Immuno-Chromatography Technique (ICT)

50. In context of developing countries like Nepal
Point of Care Testing (POCT)
• Portable testing devices that are
easy to use.
• Small sample volume
• No sample preparation is required
• Provide rapid bed side results
• Can be connected to the hospital
LIS.
• Mainly used for: Bilirubin, glucose,
Hb, electrolyte and blood gases.
CHOICE of Analyzer
• Dead volume? The smaller, the
better
• Clot detector
• Sample cups & primary tubes

51. POCT limitations
• High cost, so its use should be rationalized for tests that needs short
TAT.
• Neither their performance nor their dynamic ranges are as good as
the tradition lab equipment.
• Should be properly validated using appropriate quality assurance
procedures.
• Very low or very high results should be checked in the main
laboratory.