PROJECT CAH NEWBORN SCREENING DBS SPECIMEN COLLECTION AND HANDLING dr saras to HTA , HPJ and HOSP SELAYANG.pptx

A PILOT STUDY FOR
NEWBORN SCREENING
OF CLASSICAL
CONGENITAL ADRENAL
HYPERPLASIA (CAH)
NMRR-21-1283-60298
PRESENTER : DR SARASWATHY APPAROW
DONE BY ENDOCRINE AND BIOCHEMISTRY UNIT , SPECIALISED DIAGNOSTIC CENTRE , IMR

Congenital
Adrenal
Hyperplasia
• CAH: an inherited autosomal recessive
disorder.
• Most common gene involved: CYP21A2
• The disorder make loss / severely impaired
activity of steroidogenic enzymes.
Source: Pathologic Basis of Disease,
Robbins and Cotran, 7th Edition, 2010.
DONE BY ENDOCRINE AND BIOCHEMISTRY UNIT , SPECIALISED DIAGNOSTIC CENTRE , IMR

Clinical forms of CAH
SOURCE: Dessinioti, Clio & Katsambas, Andreas. (2009). Congenital adrenal
hyperplasia. Dermato-endocrinology. 1. 87-91. 10.4161/derm.1.2.7818.
Could be detected
through massive
newborn screening

SOURCE: Congenital Adrenal Hyperplasia : Diagnosis, Evaluation, and Management, Zoltan Antal and Ping Zhou, Pediatrics in Review
2009;30;e49
Characteristic of Different Forms of Congenital Adrenal Hyperplasia (CAH)
∼95% of the CAH cases: 21-
hydroxylase deficiency (21OHD)
due to mutations in CYP21A2.

Clinical presentation: Classical type CAH
• Decreased
activity/ fatigue
• Altered
sensorium/
unresponsiveness
• Poor feeding/
weak suck
• Dry mucous
membranes
• Hyperpigmentatio
n
• Abdominal pain
• Vomiting
• Hyponatremia
• Hyperkalaemia
• Hypoglycaemia
• Metabolic acidosis
• Hypothermia
• Hypotension
• Dehydration
• Lack of weight
gain
Salt wasting/ Acute
adrenal crisis (Life
threatening)
SOURCE: Congenital Adrenal Hyperplasia : Diagnosis, Evaluation, and
Management, Zoltan Antal and Ping Zhou, Pediatrics in Review
2009;30;e49
1. SW form (most lethal form)
Milder defect with mineralocorticoids
secretion – prevent SW
Girl- simple virilised
Boy: peripheral precocious puberty
SOURCE: The impact of CYP21A2 (P30L/I172N) genotype on female
fertility in one family, Kocova et al, 2019. European Journal of Medical
Research.
2. SV form
Female infants with
ambiguous genitalia –in
utero exposure to
androgen.
Scrotal hyperpigmentation

Prevalence of Disease Singapore:1 : 22321
(Loke et al, 2002)
**Indonesia 1 : 594
(Aman et al. 2020)
Japan 1 : 19,859
(Tsuji et al. 2013)
Laos 1 : 11,362
(Hoehn et al. 2013)
Saudi Arabia 1 : 7908
(Alfadhel et al. 2017)
**Malaysia 1:3000
(Wu et al, 1994) – case survey
**Thailand 1 : 5,771
(Somboonnithiphol et al. 2011)
** prevalence based on selected center/ population
Worldwide incidence of 1 in 15,000–20,000 livebirths

How Frequent Is CAH Disease In Malaysia?
IMR STUDY 1
A retrospective study
through laboratory
electronic patient
records during the
period 2014-2018.
17 OHP results for
baseline pre-synacthen
test were analysed.
**17OHP concentration was
measured by ELISA (IBL
International).
Result:
Refer to JCEM CAH Guideline
2010 - basal 17-OH
progesterone >300 nmol/L
are consistent with Classical
CAH diagnosis.
24/ 177 (13.6%): consistent
with Classic CAH.
**This data was presented in MACB conference 2021

• CAH IS A POTENTIALLY LIFE-THREATENING CONDITION THAT REQUIRES
ACCURATE DIAGNOSIS AND URGENT TREATMENT WITH GLUCOCORTICOID AND
MINERALOCORTICOID REPLACEMENT.
• SYMPTOMS AND SIGNS MAY EASILY BE OVERLOOKED, PARTICULARLY IN MALE
INFANTS WHO DO NOT HAVE A GENITAL AMBIGUITY. FURTHERMORE,
DIAGNOSIS OF AMBIGUOUS GENITALIA DUE TO CAH IN THE NEWBORNS ARE
OFTEN LATE AND SOMETIMES TOO LATE INTO THE ADULTHOOD WHEN SEVERE
VIRILISATION OF FEMALE INFANTS BY ADRENAL ANDROGENS MAY RESULT IN
INCORRECT GENDER ASSIGNMENT TO MALE.
• INCORRECT GENDER ASSIGNMENT WILL LEAD TO CATASTROPHIC
PSYCHOLOGICAL AND SOCIAL CONSEQUENCES TO THE AFFECTED PATIENTS.

Newborn Screening
- What is ‘Newborn Screening’?
• Newborn screening (NBS) allows
babies to be identified early for
inherited medical conditions
before symptoms manifest
• NBS allows timely treatment and
intervention
• Prevent death and irreversible
permanent disability.

2023/2/15
• Malaysia’s newborn screening only consists of Congenital
hypothyroidism and G6PD.
• NBS for CAH allows early detection of severe 21-OHD (Salt Wasting-
CAH) and simple virilising CAH within the 1st week of life.
Newborn Screening for CAH
• NBS for congenital adrenal hyperplasia (CAH)
use 17-hydroxyprogesterone (17-OHP) as
the primary marker for 21-Hydroxylase
deficiency.
• Cut off determination for specific population is

Source: Consolidating newborn screening efforts in the Asia Pacific region. (Padilla et al, 2012)
2007
CAH Newborn Screening worldwide & Asian
country
They already started…..
MONGOLIA 2000
1996
1998
1989
2006
2019
1990’
s
1996
More than 30
countries
(worldwide)
already running
NBS for CAH.
JUSTIFICATION OF THE PROJECT
Singapore:1:22321
(Loke et al, 2002)
**Indonesia 1:594
(Aman et al. 2020)
CAH Newborn
Prevalence data..
Japan 1:19,859
(Tsuji et al. 2013)
Laos 1:11,362
(Hoehn et al. 2013)
Saudi Arabia
1:7908 (Alfadhel et
al. 2017)
**Malaysia 1:3000
(Wu et al, 1994) –
case survey
**Thailand 1:5,771
(Somboonnithiphol et al.
2011)
** prevalence based on selected
center/ population
Malaysia –
? 2022

EXPANDED NEWBORN SCREENING : CAH
• EXPANDED NEWBORN SCREENING AIMS FOR EARLY DETECTION OF METABOLIC
DISEASE BEFORE THE DISEASE MANIFEST. THUS IT AIMS AT THE PREVENTION OF
EARLY DEATH AND REDUCTION OF MORBIDITIES AND HOSPITALISATION COSTS.
• A UNIVERSAL NEWBORN SCREENING PROGRAM FOR 21-OHD BASED ON 17-
HYDROXYPROGESTERONE (17-OHP) ASSAY ON THE DRIED BLOOD SPOT (DBS)
FILTER PAPER WAS INITIATED IN ORDER TO AVOID POTENTIAL EARLY LIFE-
THREATENING SHOCK AND DEATH DUE TO SALT WASTING CRISIS.
• AS YET, THERE IS NO NEWBORN SCREENING PROGRAM FOR CAH IN MALAYSIA.
THUS PATIENTS OFTEN PRESENT LATE WHEN THEY ARE ALREADY BECOME
SYMPTOMATIC. INFANTS WITH CAH HAVE IMPAIRMENT OF MINERALOCORTICOID
SYNTHESIS WHICH LED TO LIFE-THREATENING, SALT-WASTING CRISIS IN THE FIRST
FEW WEEKS OF LIFE.

• Early gender assignment is vital and
prevents the stigma and uncertainties faced
by the affected patients.
• The diagnostic test for 17-
Hydroxyprogesterone (17-OHP) is available
in IMR by using immunoassay ELISA
method either directly measures the 17-
Hydroxyprogesterone (17-OHP) or upon
stimulation after a synthetic ACTH to the
suspected children.
• It uses serum sample and is not automated.
Fluoroimmunoassay has supplanted
radioimmunoassay and ELISA in most NBS
programs due to the improved diagnostic
sensitivities. The current ELISA method is
not suitable for universal newborn
screening.
• Whereas, Dried blood spot (DBS) sample
for newborn screening has many
advantages; with minimally invasive
technique with, no centrifugation required
for sample separation.
• Thus the processing time, cost for
equipment and human resource are
reduced. The DBS only requires a few
drops of blood (50µL of blood), can be
transported at ambient temperature and has
long storage stability at room temperature
and longer at 4-8ºC

Early diagnosis
Early diagnosis is possible
with
Newborn screening
NBS for CAH allows early detection of severe 21-OHD (Salt Wasting-CAH) and
simple virilising CAH within the 1st week of life.

Timeline of worldwide
CAH newborn screening implementation
1977 1984 1989 1990 1996 1999 2000
2006 2007
Alaska
Washington
>40 states in US implement CAH NBS
Japan China
Philippines
Vietnam
Italy
South Korea **India
2019 2020
1986
2008 2014
1997 2003
2002
Mongolia
**Singapore **Indonesia
New Zealand Sweden **Thailand
Switzerland Brazil
Belgium Netherlands
2022
France
? Malaysia
**only implement at selected centre

Newborn Screening in Malaysia
• Malaysia’s newborn screening only consists of Congenital
hypothyroidism and G6PD.

Newborn Screening in Malaysia
• Institute Medical Research, Specialised Diagnostic Centre,
Endocrine Laboratory:
- On-going planning to start a pilot study for CAH newborn screening in
Malaysia.
- The screening program will use dried blood spot for 17-hydroxyprogesterone
(17-OHP) measurement as the primary marker for 21-Hydroxylase deficiency.
- Objectives of this study:
a) To evaluate the reliable method for CAH NBS.
b) To set the local population cut off in differentiating CAH and non-CAH.

Looking towards Paediatric Endocrine Newborn
Screening in Malaysia
• Expanded Paediatric Endocrine Newborn screening aims for early
detection of endocrine metabolic disease before the disease
manifest.
• Thus, it aims at the prevention of early neonatal death and
reduction of morbidities and hospitalisation costs.
• A universal newborn screening program for 21-OHD based on
17-hydroxyprogesterone (17-OHP) assay on the dried blood spot
(DBS) filter paper should be initiated in order to avoid potential
early life-threatening shock and death due to salt wasting crisis.

Getting consent
from parent or
guardian

Obtaining consent procedure:
Step 1 The competent clinical personnel from the clinical team, will explain the study to the potential subject
verbally, providing all pertinent information (purpose, procedures, risks, benefits, alternatives to participation,
etc.), and will allow the potential subject ample opportunity to ask questions.
Step 2 Following this verbal explanation, the potential subject will be provided with a written consent form and
afforded sufficient time to consider whether or not to participate in the research. "Sufficient time" can range from
hours to days, depending on how long it reasonably takes to evaluate the procedures, risks, potential benefits, and
alternative treatments.
Step 3 If the potential subject have any additional questions, they may contact any of the Investigators listed on
the patient information sheet/ consent form.
Step 4 If the subject’s representative is satisfied with the explanation, they may sign the consent form at that time.

3.4.1 Inclusion Criteria (Please remove this section if it is not applicable)
1. All healthy eligible Malaysian newborns that were born in the hospitals
2. Sample taken 48-168 hours of life.
3. Consented participants.
3.4.2 Exclusion Criteria (Please remove this section if it is not applicable)
1. Sample receive with conditions, which are known to cause anomalous analytical assay results:
a) DBS spot not uniformly saturated with blood
b) DBS spots punched too close to the edge of the blood spot.
c) Poorly collected and improperly dried DBS
d) Non-eluting blood spot due to deterioration of sample caused by exposure to heat and humidity.
e) Contamination of blood spot filter paper with fungus material
f) Sample applied to the filter paper from EDTA or citrate or capillaries whole blood, as these
anticoagulation reagents will affect the assay by chelating the europium label.
g) Use of filter paper other than 903 grade newborn screening filter paper.
2. Mother or newborn with dexamethasone treatment (< 6 days)
3. Sample was not within study age.
4. Dead newborns.

Dried
blood
spot card
Surgical
Mask
Alcohol
swab
Newborn
lancet
Gloves
Medical
tape
Cotton ball
Drying rack
with velcro
Desiccant
silica gel
samples
transportation
boxes
Biohazard plastic
bag
Printed consent
form & Brochure
COLLECTION KIT
TO BE
DISTRIBUTED TO
THE COLLECTION
SITE

DBS SPECIMEN
COLLECTION AND
HANDLING
DBS SPECIMEN COLLECTION & HANDLING : This slide is prepared by Biochemistry Unit, Institute For Medical Research
Malaysia (IMR)

WHAT ARE OUR
RESPONSIBILITIES?
• Collect valid specimens
• Label and store appropriately until transported for testing
• Ensure records are properly maintained and organized
• Avoid transcription errors
A test result is only as good as the specimen collected

WHAT IS DRIED BLOOD SPOT
(DBS)
• DBS is whole blood collected on filter paper and dried
• It was made directly from the patient’s whole blood.
• Dried blood spot specimens are collected by applying a few drops of blood,
drawn by lancet from the finger, heel or toe, onto specially manufactured
absorbent filter paper.
• Dried blood spot testing (DBS) is a form of biosampling where
blood samples are blotted and dried on filter paper.

COLLECTION SPECIMENS:
DRIED BLOOD SPOTS (DBS)
IN NEW BORN SCREENING
(NBS)

STEP 1
• Complete the required patient information fields requested on the card.
• Avoid touching the area within the circles on the filter paper
section before, during, and after collection of the specimen
• Do not allow water, antiseptic solutions, glove powder, hand lotion,
or other materials to come into contact with the specimen card
before or after use.

13 mm circle = 75 ul blood
2 consent form
Link to DBS card pdf

STEP 2
DBS SPECIMEN COLLECTION & HANDLING : This slide is prepared by Biochemistry Unit, Institute For Medical
Research Malaysia (IMR)
• 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

DBS COLLECTION – PREPARING
FOR THE HEEL-PRICK
1. Hatched area indicates safe areas
for puncture site.
2. Warm heel with warm pack or soft
cloth that has been moistened
with warm water for 3 to 5 minutes
3. Clean site with alcohol prep.
Wipe DRY with sterile gauze pad.

DBS COLLECTION – PREPARING
FOR THE HEEL-PRICK
4. Puncture heel. Wipe away first
blood drop with sterile gauze pad.
Allow another LARGE blood drop
to form.
5. Lightly touch filter paper to LARGE
blood drop. Allow blood to soak through
and completely fill circle with SINGLE
application to LARGE blood drop.
6. 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

DBS COLLECTION TIPS
• Use universal Safety Precautions
• Clearly and completely label card with subject information data
• Follow finger prick procedure
• Uniformly saturate the entire circle
• Do not touch the areas of the card that will be used to collect specimens
whether gloved or ungloved.
• Do not apply blood to both sides of the filter paper.
• Do not apply blood more than once in the same collection circle.
• Do not apply blood from more than one patient on the
same collection card.

STEP 3: HOW TO DRY
DBS
• Avoid touching or smearing the blood spots
• Allow the specimen to fully air dry horizontally (at least 3 hours)
at room temperature
• Keep away from direct sunlight and away from other heat sources.
(STRICTLY NO HAIR DRYER)
• Do not allow them to touch other surfaces or specimens.
Avoid stacking the cards.

STEP 4 : HOW TO PACKAGING AND
STORE DBS
• Appropriately stack DBS
• Insert into sealable plastic bag
• Add desiccant packets
• Label contents of bag and seal
• Keep packaged DBS (in sealable plastic bags) cool and dry
until transported to reference laboratory
• Please include appropriate documentation (request form) with
the samples
• Avoid leaving in vehicle, as sun and heat will deteriorate DBS

VALID & INVALID
DBS SPECIMEN

VALID DBS SPECIMEN
BACK
FRONT

VALID DBS SPECIMEN
FRONT
BACK
SEPARATION
• Not wiping alcohol from the puncture site
• Allowing filter paper to come into contact with
alcohol, hand lotion etc.
• Drying specimen improperly

INVALID DBS SPECIMEN
WET
• Drying specimen improperly. Minimum dry DBS
three hours at room temperature

FRONT
BACK
DILUTED
• Squeezing or milking of area surrounding the
puncture site. Not wiping alcohol from the
puncture site.
• Allowing filter paper to come in contact with
gloved or ungloved hands, or substances such as
alcohol, antiseptic solutions, water, etc.
• Exposing blood spots to direct heat.
• Allowing blood spots to come in contact with
tabletop, etc. (while drying the sample).

SCRATCHED OR ABRADED
• Filter scratched, torn or abraded. Possible causes:
Improper use of capillary tubes.
• To avoid damaging the filter paper fibers, do not
allow the capillary tube to touch the filter paper.
• Actions such as “coloring in” the circle, repeated
dabbing around the circle, or any technique that
may scratch, compress, or indent the paper should
not be used.
scratch / abraded
area

LAYERING, CLOTTED OR SUPERSATURATED
• Possible causes: Touching the same circle on filter
paper to blood drop several times.
• Filling circle on both sides of filter paper.
• Application of excess blood.
• Clotted swirl marks from improper capillary
application.

LACK OF BLOOD COVERAGE (Insufficient for testing,
blood spot’s diameter is too small)
• Removing filter paper before blood has completely
filled circle
• Not allowing an ample sized blood drop to form
before applying to filter paper.
• Inadequate heel stick procedure.
• Improper applying blood to filter paper with a
capillary tube

INCOMPLETE BLOOD SATURATION
(blood did not soak through the filter paper)
• Removing filter paper before blood has completely
filled circle or before blood has soaked through to
opposite side
• Improper capillary tube application
• Allowing filter paper to come into contact with
gloved or ungloved hands or substance such as
hand lotion etc.
FRONT

CONTAMINATED
• Allowing filter paper to come in contact with
substances such as water, alcohol, antiseptic
solutions, etc. either before or after blood
specimen collection.
• Improper packaging during transportation sample.
• Drying specimen improperly.
Fungus

Step 5. Send
to the lab
Lab staff will come to the peadiatric
department to collect the sample
once/ weekly.
Stability of samples:
17-OHP is known to be quite stable in dried blood spot specimens. At
room temperature (20 °C and 25 °C with humidity levels somewhere
between 30% and 50%) 17-OHP has been shown to be stable for 7
months

Step 6.
Samples
processing in
the lab

Step 7.
Results
management
To inform
Paediatricia
n

Step 8.
UPDATE
MM/YY: <2000 g (min
350)
2000-2500 g (min
350)
>2500 g (min
350)
HTAzizah
HPutrajaya
HSelayang
Jumlah
Bilangan yang
perlu

STAKEHOLDERS BENEFITS
1. GENERAL POPULATION
IMPROVE QUALITY OF CAH’S
PATIENT LIFE. 2. Clinician
Earlier detection will help
improving the diagnosis,
management and treatment.
3. MOH
Change in National Policy
health planning
Increase the quality of
healthcare service to the
nation.

SUMMARY
• CAH WITH LATE DIAGNOSIS AND IMPROPER TREATMENT MAY LEAD TO
NEONATAL DEATH (SALT WASTING CRISIS), DISCREPANCY IN GENDER
ASSIGNMENT, LOW QUALITY OF LIFE FOR CAH PATIENT.
• IMPLEMENTATION OF CAH AS PART OF NEWBORN SCREENING WILL GIVE
EARLIER DIAGNOSIS FOR EARLY TREATMENT AND HELP TO OVERCOME
THE LIFE THREATENING COMPLICATIONS.
• THE CONSEQUENCES OF THE DISEASE AND COMPLICATIONS OF THE
TREATMENT STILL REMAIN A HUGE CHALLENGE AND REQUIRE THE
INVOLVEMENT OF MULTIPLE SUBSPECIALTIES AND STAKE HOLDERS AND
CHANGING THE NATIONAL POLICY PLAN.

REFERENCE
• https://health.mo.gov/
• https://www.akronchildrens.org
• https://www.health.state.mn.us/index.html
• https://acutecaretesting.org/
• https://clsi.org/
SCAN ME

THANK YOU
BIOCHEMISTRY UNIT/ENDOCRINE UNIT
INSTITUTE FOR MEDICAL RESEARCH

PROJECT CAH NEWBORN SCREENING DBS SPECIMEN COLLECTION AND HANDLING dr saras to HTA , HPJ and HOSP SELAYANG.pptx

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