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Optimization of Biotinidase Assay: Dr. Faryal Husnain Resident Clinical Chemistry

This document discusses optimizing the biotinidase assay. It begins by introducing biotinidase deficiency and the importance of biotinidase in recycling biotin. It then discusses current reference ranges for biotinidase activity and categories of deficiency based on activity levels. The objective is stated as validating a quantitative colorimetric biotinidase assay and establishing reference ranges in adults and children. Methods that will be used include assessing analytical measurement range, precision, accuracy, sensitivity, specificity, and establishing reference ranges.

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Saad Khan
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63 views

Optimization of Biotinidase Assay: Dr. Faryal Husnain Resident Clinical Chemistry

This document discusses optimizing the biotinidase assay. It begins by introducing biotinidase deficiency and the importance of biotinidase in recycling biotin. It then discusses current reference ranges for biotinidase activity and categories of deficiency based on activity levels. The objective is stated as validating a quantitative colorimetric biotinidase assay and establishing reference ranges in adults and children. Methods that will be used include assessing analytical measurement range, precision, accuracy, sensitivity, specificity, and establishing reference ranges.

Uploaded by

Saad Khan
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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Optimization of Biotinidase

Assay
Dr. Faryal Husnain
Resident Clinical Chemistry
Introduction
• Biotinidase deficiency (OMIM #253260) also called Multiple
Carboxylase Deficiency is a rare autosomal recessive disorder of
biotin metabolism.
• Biotinidase is required for the endogenous recycling and release
of biotin from dietary protein.
•  Biotin (vitamin H or B8) is an essential co-factor for carboxylase
enzymes involved in fatty-acid synthesis, amino-acid catabolism
and gluconeogenesis.
•  Biotinidase deficiency can cause insufficient biotin to be made
available for the maintenance of these processes; which can
lead to life-threatening metabolic derangements eliciting
organic aciduria and neurological derangements.
Biocytin(N-
Biotinyl-L-
Lysine)
Biotinidase Deficiency
• Mayo Clinic reference range=3.5 U/L to 13.8 U/L
• Partial deficiencies and carriers may occur at the low
end of the reference range.
• Profound biotinidase deficiency results when the
activity of biotinidase is reduced to less than 10
percent of normal.
• Partial biotinidase deficiency occurs when biotinidase
activity is reduced to between 10 percent and 30
percent of normal.
• 50% of normal activity is seen in heterozygotes.
Category Mean Biotinidase activity ± SD
(nmol/min/ml serum) (n)
Normal individuals 7.57 ± 1.41 (100)
Obligate heterozygotes 3.49 ± 0.72 (21)
Affected individuals (symptomatic) 0.12 ± 0.18 (23)
Affected individuals (newborn screening) 0.19 ± 0.16 (41)
Individuals with partial biotinidase
1.47 ± 0.41 (23)
deficiency
Biotinidase Deficiency
• Patient's urinary organic acids (GC/MS) analysis shows an excretion of 3-
hydroxyisovaleric acid and 3-hydroxypropionic acid. Their plasma biotin
concentration is decreased, however, it can be normal in early forms
• Ketolactic acidosis and mild hyperammonemia may also be present.
Patients with partial BTD (10% to 30% of mean normal BTD activity) may
be asymptomatic.
• Urinary excretion of 3-hydroxyisovaleric acid can be used for monitoring
response to treatment.
• Other metabolic abnormalities are more variable and may include elevated
excretion of 3-hydroxyisovaleric, lactic and 3-hydroxypropionic acids, and
3-methylcrotonylglycine by urine organic acid analysis, as well as mildly
elevated 3-hydroxyisovalerylcarnitine (C5–OH) by plasma acylcarnitine
analysis.30 These metabolic abnormalities are variable
Objective
• A quantitative colourimetric biotinidase assay
will be validated using a quantitative
validation method and it’s reference range
will be calculated in adults(older than 18
years) and children(younger than 18 years)
SUBJECTS AND METHODS:

• STUDY DESIGN: case control, descriptive


• STUDY SETTING: An interdisciplinary study will be
conducted at the Section of Chemical Pathology,
Department of Pathology & Lab Medicine, Aga
Khan University. All biochemical analysis will be
done at Section of Chemical Pathology.
• STUDY DURATION: One year after the approval
of ERC.

• SAMPLE SIZE: 120+120+no. of pateints with


Biotinidase Deficiency(not yet known)
SUBJECTS AND METHODS

SAMPLING TECHNIQUE: Purposive, non-Probability

SAMPLE SELECTION CRITERIA:


Inclusion criteria:3 groups
a)Newborns with biotinidase Deficiency
b)Serum Samples from healthy adults above 18 years
c)Serum Samples from healthy children under 18 years
SUBJECTS AND METHODS

DATA COLLECTION PROCEDURE: Subjects will be


recruited from Paediatrics and Child Health
unit,healthy adults and children serum samples
from other studies or fresh recruits. Informed
consent will be taken after explaining procedure.
Subject’s demographic details along with their
Biotinidase levels AND OTHER AVAILABLE
biochemical parameters will be filled in
performa, at the time of sample collection.
Ethical review committees’ approval will be
SUBJECTS AND METHODS

DATA ANALYSIS: Data will be entered into SPSS version


23.0 and analysed.Other parameters will be calculated 
Parameters to be calculated
• Reportable Range/Analytical Measurement
Range (Linearity)
• Precision
• Accuracy
• Sensitivity and Specificity
• Reference Range
Accuracy

•Compare the results from the two methods for the same
samples.it is recommended that a minimum 20 samples are
compared, however, CLSI consider 40 samples to be for a good
method comparison, increasing numbers improve the quality up
to a point, but a good distribution of data is much more important
than a large number.
•Enter the data in Alternate Method Comparison Module of EP
(statistical software) the statistical tool for alternate method
comparison is a linear regression.
•A specimen passes accuracy if all measured results fall within the
target range.
AMR
• Analytical Measurement Range
• Range of analyte values that a method can directly measure
w/o modification (no dilutions, concentrations, other
pretreatments that are not part of the usual assay process)
• When initially introducing a new method, it is necessary to
verify the AMR independently from the calibration process
• For assays that have that have a minimum three calibrators
with low, high & mid-point values covering most of the AMR
and where calibration confirmation also covers most of the
AMR confirmation of linearity is not required.
AMR
• Material for linearity checking needs to have a level
of the analyte at the upper limit of the AMR. Choice
of material may comprise the following:
• Calibrators from a different lot number.
• Material manufactured specially for linearity
checking. Or CAP linearity survey or validation
materials can be used to verify performance claims.
Compare to peer group statistics appearing in the
survey.
Precision
• Measure the same analyte for 20 consecutive days, do not
include the outliers in your calculation (Outliers are results
outside the QC range)
OR
• Twice a day in duplicate for 10 days.
OR
• Once a day for 20 days
OR
• Calculation of Precision assess using EP evaluator software
Reference Range

• Reference range to be verified may be for:


– Current laboratory ranges.
– Manufacturer’s ranges.
– Published reference ranges.
– Locally established reference range
• Appropriate samples are collected from the 20 healthy subjects
(Healthy subjects will be identified using the sample questionnaire as
per CLSI recommendation. Annexure E Questionnaire for Verification
of Reference Intervals in Healthy Subjects (LAB-QM-005GF6)
• Staff members/ blood donors are typically used as healthy subjects
and handled in the usual manner, so they are subjected to the pre-
analytical variables as patient’s samples.
Reference Range
 If any reference range has partition like gender, age, smoking,
pregnancy etc. and then reference range will be validated in each
group/partition .separately taking 20 samples.
 The sample is analyzed in the same way as patient samples.
 The data is examined and if there are obvious outliers these should
be eliminated and additional reference individuals added so that the
total number of 20 is maintained.
 EP evaluator can be used for statistically analyze the “reference
interval.
Analytical Sensitivity (Limit of
detection):
• The term “Analytical Sensitivity” is sometimes used
interchangeably with LOD in many laboratories.
• The limit of detection need to be defined for each
qualitative assay(manufacturer’s lod ,maybe used)
• Replicate response measurements are made for two
samples: a blank (zero-concentration) sample, and second
sample with a known, low concentration. The experiment
requires a minimum of 10 replicate measurements for the
blank and 3 replicates for the low-concentration sample.
Use EP evaluator to assess the sensitivity
Analytical Specificity
(Interferences)
• The interfering substances for all assays need
to be assessed. For FDA/CE –IVD approved
assay it is acceptable to take this information
from the manufacturer’s and or published
literature and independent verification is not
required.
• Interfering substances should be noted in the
assay procedure.
• Successful approval for the US and the CE-IVD market
• Quality system in compliance ISO 13485 and 21CFR part 820
• Electronic records and signatures according to 21CFR part 11
• Software Development Lifecycle compliant with ISO 62304
• Usability Engineering for Medical Devies according to ISO
62366
• Risk Management in compliance with ISO 14971
• All steps completely traceable

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