Application Note
PN 33569
Diagnostic Determination of Biotinidase Activity
Using AcroPrep™ Advance Filter Plates
Emily Berlin, Sr. Marketing Manager, Pall Life Sciences
Alana Lerch, Product Manager, Astoria-Pacific International
Todd Allen, Sr. Scientist, Astoria-Pacific International
Introduction
Newborn screening is the process of testing
newborn babies for treatable diseases that,
if undiagnosed, can be harmful or potentially
fatal. In 1963, voluntary newborn screening
was introduced in the United States. Today, in
the U.S. alone, 97% of more than four million
babies are tested for genetic disorders that
can affect the physical and mental development of a newborn baby.1 The expansion of
newborn screening programs is viewed as
one of the most successful public health and
disease prevention programs in the country.2
In 2005, only 38% of infants born underwent
the required screening for 21 of the 29 more
serious functional or genetic disorders recommended by the American College of Medical
Genetics. In 2009, all 50 states (including
the District of Columbia), mandated that all
newborns be screened for 21 of the 29 conditions. Presently, 46 states and the District of
Columbia screen for 26 of the conditions;
and 24 states have mandated testing for
all 29 disorders.3
Today, newborn screening is performed
worldwide and is continuing to expand in
prevalence. The screening performed by dedicated professionals has significantly improved
the quality of life for thousands of babies and
their families. The increased attention over the
past few years has led to early detection and
diagnosis of metabolic birth defects, which
can make the difference between healthy
development or lifelong disabilities. New
diagnostic techniques allow earlier diagnosis
and, as a result, effective treatment can be
instituted without delay.
Within 48 hours of birth, a blood sample is
collected from the newborn via a heel stick.
Filter paper is used to collect the newborn
blood sample. The blood collected on the
filter paper is allowed to dry and then sent
to laboratories to be screened for a panel
of conditions, such as Biotinidase Deficiency
(BIOT) and Galactosemia. Testing can be performed either at state public health laboratories
or designated contract testing laboratories.
According to the Centers for Disease Control,
approximately 5,000 babies with severe
disorders are identified in the U.S. each
year using newborn screening programs.4
Biotinidase Deficiency
Biotinidase Deficiency is an autosomal recessive
disorder caused by the lack of an enzyme
called biotinidase. This deficiency leads to the
inability to metabolize biotin, a water-soluble
B-complex vitamin. Biotin is required for
several metabolic processes including the
metabolism of fats, proteins, and amino acids;
cell growth and differentiation; and participation
in the citric acid cycle. BIOT is easily treated
with vitamin supplementation to alleviate or
even stop symptoms. Without treatment, this
disorder can lead to seizures, developmental
delay, eczema, and hearing loss.
AcroPrep™ Advance filter plates have been optimized
for use in neonatal screening assays and play an
integral role in the sample preparation procedure
for the diagnostic determination of BIOT. AcroPrep
Advance plates can be used in conjunction with the
two 510(k) approved kits for the semi-quantitative determination of biotinidase activity in dried whole blood
spots available from Astoria-Pacific International. Both
of Astoria-Pacific’s SPOTCHECK◆ Biotinidase Reagent
Kits are intended for in vitro screening of decreased
biotinidase activity, primarily for the diagnosis and
treatment of BIOT in newborns. One reagent kit is
used solely on the SPOTCHECK Continuous Flow
Analyzer while the other kit utilizes only standard
microplates. The results can be collected with a
colorimetric plate reader.
Materials
• AcroPrep Advance filter plate for neonatal screening,
PN 8079
• AcroPrep Advance filter plate for biotinidase assays,
PN 8060
• Vacuum manifold, PN 5017
• Vacuum/pressure pump, PN 13157
• SPOTCHECK Biotinidase Continuous Flow Kit,
PN 80-8000-13K
• SPOTCHECK Biotinidase Microplate Kit,
PN 81-8000-13K
• SPOTCHECK Continuous Flow Analyzer
• Plate reader
• Optimized well design provides increased consistency
in filtration times ensuring reproducible results lot
after lot.
• New outlet tip geometry minimizes sample leakage
and loss during incubation steps, decreasing the time
required for sample retesting and second screens.
• Redesigned outlet tips minimize the formation
of hanging drops, reducing the chance of cross
contamination following filtration (Figure 1).
• High performance membrane effectively retains
fibers from dried blood spots, preventing optical
disturbances, banishing clogged lines, and ensuring
optimal performance of the SPOTCHECK Continuous
Flow instrumentation. In the microplate assay, the
effectiveness of the membrane prevents optical
disturbances which are caused by the presence of
fibers during reading.
• Features of the membrane include low non-specific
binding and no added surfactants to bind samples
or interfere with assay performance. Use of the
AcroPrep Advance filter plate provides consistent
results in biotinidase response (Figure 3).
• Unique membrane configuration combines the
incubation and precipitation steps in one plate
when used with the SPOTCHECK Microplate Assay
Kit. This decreases sample handling, as well as
minimizes operator intervention and consumable
use, resulting in a time and cost savings.
Figure 1
AcroPrep Advance Filter Plate Minimizes Sample
Cross Contamination
• Incubator/shaker
Comparison of Hanging Drops Following Filtration
Methodology
Biotinidase activity is determined colorimetrically by
measuring the amount of p-aminobenzoic acid (PABA)
released from biotinyl-p-aminobenzoate (Biotin-PAB).
Samples with biotinidase activity develop a purple
color, whereas samples with low biotinidase activity
remain straw-colored (read at 550 nm). The intensity of
color produced in each sample is directly proportional
to the amount of biotinidase activity.
AcroPrep Advance filter plates provide an optimized
platform for the upstream sample preparation procedure
in the diagnostic determination of biotinidase activity.
The design of the plate and membrane selection offers
the following performance advantages during the
critical sample preparation procedure:
2
Hanging Drop (mg/plate)
150
100
50
0
AcroPrep
Advance
Competitor M
The new design of the AcroPrep Advance filter plate has been optimized to minimize the number of hanging drops on the well outlet
tips after filtration. Compared to competitive plates, the AcroPrep
Advance filter plate reduces the presence of hanging drops, thus
increasing automation compatibility and reducing the concern over
sample cross contamination due to falling droplets.
The use of the AcroPrep Advance filter plates in
conjunction with Astoria-Pacific’s Biotinidase Kits
provide several benefits over the manual Wolf method.
The advantages include:
Sample Preparation Procedure
1. Punch one 3/16 in. spot or two 1/8 in. spots from
the dried blood spot and place into a well of the
AcroPrep Advance filter plate (PN 8079).
• Reduced incubation times and same day results.
Ninety minute (continuous flow) or 4 hour (microplate
assay) incubations versus 16 hour incubation with
the Wolf method.
2. Add water to the wells of the filter plate and shake
for 30 minutes.
3. Filter the liquid from the wells into a solid bottom
collection plate by applying vacuum until the wells
have emptied. Then release the vacuum.
• Semi-quantitative results with the filter plate and
biotinidase kits versus a qualitative subjective visual
inspection with the Wolf method.
4. Place the collection plate on the autosampler and
start the automated process on the instrument.
• The plates and samples can be processed in
manual, semi-automated, and fully automated
modes versus manually with the Wolf method.
Automated Continuous Flow Procedure
1. The autosampler delivers calibrants, controls, and
samples to the cartridges.
• The use of optimized filtration plates helps remove
the precipitate whereas the blood spots stay in the
well with the Wolf method.
2. The samples are incubated online for approximately
90 minutes.
• Pre-weighed and color coded reagents in the
SPOTCHECK kits versus manual reagent preparation with the Wolf method.
3. Online dialysis separates the released PABA from
other proteins and undesirable components in
the sample.
The sample preparation procedure for biotinidase
testing for both the Continuous Flow System and Microplate Kit available from Astoria-Pacific require the use
of a filter plate for proper upstream sample preparation.
Two versions of the AcroPrep Advance filter plate are
available and have been optimized for use with these
kits. Each method and filter plate is outlined below.
4. The online addition of three color reagents produces
an azo dye.
5. A colorimetric measurement is read at 550 nm.
6. A standard curve prepared from a stock PABA
solution is used to quantitate the results.
Results
Specimen responses (peaks) are compared with the
calibration curve to assign the numeric values to the
test results. These values are then evaluated by their
relationship to the predetermined cut-off of the assay.
Results above the cut-off are presumptive negative for
BIOT (normal enzyme activity), and results below the
cut-off are presumptive positive for BIOT (deficient
enzyme activity).
Methodology
Instrumentation-Based
Microplate-Based
Continuous Flow System
Manual, Semi-automated,
Automated Processing
AcroPrep Advance for
Neonatal Screening
PN 8079
AcroPrep Advance for
Biotinidase Assays
PN 8060
An example of calibrant and sample peaks is shown
in Figure 2 on the following page.
Method I
AcroPrep Advance Filter Plate for Neonatal
Screening (PN 8079) and the SPOTCHECK
Biotinidase Continuous Flow Kit (PN 80-8000-13K)
The Biotinidase Continuous Flow System allows for
the semi-quantitative determination of biotinidase
activity. The system can be run simultaneously with
GALT, Phenylalanine, and/or G6PD and requires
minimal operator involvement following the sample
preparation procedure.
3
Figure 2
Calibration for S4C1 (Biotinidase)
Method II
The AcroPrep Advance filter plate is a critical component in the success of the biotinidase assay. Filter
plates from another manufacturer have been found
to decrease the biotinidase enzyme activity simply by
being in contact with the specimens. This effect varies
from lot-to-lot and can lead to an increased number of
false positives (Figure 3). The resultant effect of false
positive results causes an emotional burden on families,
a labor burden on the laboratories, and a financial
burden on the testing and follow-up process.
120
100
Sample Preparation Procedure
1. Punch two 1/8 in. spots and place into the well
of the AcroPrep Advance filter plate (PN 8060).
2. Add substrate and incubate/shake for 4 hours.
Operating Procedure
1. Add supplied reagent to precipitate proteins.
2. Filter samples into microtiter plate by applying
vacuum until the wells have emptied. Then release
the vacuum. An optimized membrane configuration
in the AcroPrep Advance filter plate provides efficient
processing times, as well as ensures clear filtrate
for downstream analysis (Figure 4). This is important
since the assay causes a precipitation of proteins
that must be removed. An inadequate filter plate
may clog and the sample may not fully transfer.
80
3. Add supplied three color reagents.
60
4. Read the plate with any commercially available
plate reader at 550 nm. A reference reading is
also taken at 690 nm. The intensity of the color
produced is directly proportional to the amount
of biotinidase activity.
40
Pall
Lot O
Lot N
Lot M
Lot K
Lot L
Lot I
Lot J
Lot H
Lot E
Lot F
Lot G
Lot D
0
Lot C
20
Lot A
Lot B
Expected Biotinidase Response (%)
Figure 3
AcroPrep Advance Filter Plates Provide Consistent
Biotinidase Response
AcroPrep Advance Filter Plate for Biotinidase
Assays (PN 8060) and the SPOTCHECK
Biotinidase Microplate Kit (PN 81-8000-13K)
The AcroPrep Advance filter plate for biotinidase
assays has been specifically optimized for use with the
Biotinidase Microplate Kit. The Microplate Kit provides
semi-quantitative results for the diagnostic determination
of biotinidase activity. The kit and AcroPrep Advance
filter plate can be used with three levels of automation:
manual, using an 8 or 12 channel pipette; semi-automated using a benchtop liquid handling station for
pipetting; or with a fully automated robotic system,
such as the Tecan Freedom EVO◆, which does all of
the pipetting, incubating, shaking, transferring, filtering,
and independent reading.
Plate
Competitor M
Fifteen lots tested of a competitor’s filter plates (shown in blue) were
found to decrease the expected biotinidase activity in the specimens.
Six lots (data pooled) of the AcroPrep Advance filter plate media
(orange) were evaluated for biotinidase response and consistently
preserved the full measure of enzyme activity in the specimens. All
six lots demonstrated 100% biotinidase response. The AcroPrep
Advance filter plates provide the consistency needed for critical
diagnostic screening assays for newborns. Data generated in
conjunction with Astoria-Pacific International.
www.pall.com/lab
4
be easily treated through dietary supplementation.
The methodology for detecting biotinidase activity is
done through an assay process in which the AcroPrep
Advance filter plate plays an integral role. Robust and
consistent performance of the filter plates is a key element to providing accurate screening results. Variability
in plate performance introduces the need for repeat
testing, added costs, and delays for a diagnosis for
the newborn. AcroPrep Advance filter plates have
been optimized for use in newborn screening tests
and demonstrate reproducible performance ensuring
results that can be trusted.
Evacuation Time (seconds)
Figure 4
AcroPrep Advance Filter Plates Provide Faster
Processing Times
50
45
40
35
30
25
20
15
10
5
In summary, the benefits of the AcroPrep Advance
filter plate with the 510(k) approved kits available
from Astoria-Pacific include:
0
AcroPrep
Advance
Biotinidase Plates
AcroPrep
Advance
Neonatal Plates
Competitor M
Plates
• New well design for consistency in filtration times.
Evacuation times of different filter plates (n=2) were tested for use
in the SPOTCHECK Biotinidase Microplate assay. Both AcroPrep
Advance filter plates showed significantly decreased processing
times as compared to Competitor M, allowing for increased
throughput, especially on automated instrument platforms.
Results
Specimen responses are compared with a calibration
curve (Figure 5) to assign numeric values to the specimens. These values are evaluated by their relationship
to the predetermined cut-off of the assay. Results
above the cut-off are presumptive negative for BIOT
(normal enzyme activity), and results below the cut-off
are presumptive positive for BIOT (deficient
enzyme activity).
Abs 550-690
• A high performance membrane to effectively retain
fibers from dried blood spots and prevent the lines
from becoming clogged.
• Low non-specific binding and no added surfactants
to bind samples or interfere with assay performance.
• A unique membrane configuration that combines the
incubation and precipitation steps in one plate when
used with the SPOTCHECK Microplate Assay kit.
1. Association of Public Health Laboratories – Public Health
Laboratories and Newborn Screening. http://www.aphl.org/
aphlprograms/nsg/Pages/PublicHealthLaboratoriesandNewbornScreening.aspx
Curve
0.250
0.200
2. American Academy of Pediatrics. Serving the family from birth to
medical home: a report from the Newborn Screening Task Force:
Pediatrics 2000, (6 Suppl 2):383-427.
0.150
0.100
3. States expand newborn screening for life-threatening disorders.
March of Dimes. February 2009. http://www.marchofdimes.com/
printableArticles/22684_51920.asp
0.050
0.000
-0.050
0
• Redesigned outlet tips minimize the formation
of hanging drops, reducing chances for cross
contamination.
References
Figure 5
Calibration Curve for Biotinidase Microplate Kit
0.300
• New outlet tip geometry to minimize sample leakage
and loss and the need for second screens.
20
40
60
80
100
120
140
160
180
200
Plate Layout Settings
4. http://www.cdc.gov/nceh/dls/newborn.htm
Conclusions
It is widely recognized that the practice of screening
newborn infants for a panel of genetic or functional
disorders has been instrumental in early detection
and prevention of severe disabilities or death. BIOT is
just one disorder that, when detected early, can
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5
Acknowledgements
Article written in partnership with:
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2/10, 1k, AA GN09.3338
PN33569