Transcript HealthyQuest Presentation - - Quest Diagnostics Nichols

Amino Acid Quantitation:
Diagnosis of Inborn Errors of
Metabolism and Nutritional Deficiencies
Julie Ann Neidich, MD
Medical Director
Biochemical Genetics and Cytogenetics
Quest Diagnostics Nichols Institute
www.nicholsinstitute.com
Amino Acids and Medicine
• 1902: Garrod: First application of Mendel’s
concept of a gene to a human disorder,
Alkaptonuria
• Clinical diagnosis: black matter in urine that
has been exposed to air
• Early 1900’s: Fischer: amino acids are bound
together to make polypeptides and proteins
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History of Amino Acid Assays 1
• 1952: Martin & Synge: Nobel Prize,
Chemistry: partition chromatography
• Synge then used chromatography columns
packed with starch to sequence amino acids
in peptides
• 1958: Stein & Moore: first automated amino
acid analyzer, partnered with Beckman
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History of Amino Acid Assays 2
• Time to analyze markedly reduced:
– 1946 months
– 1950 weeks
– 1958 a day with first Beckman automated
analyzer
– Improved resins in column: 4 hours
– Improved data output, increased sensitivity: ~2
hours
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History of Amino Acid Assays 3
• No real change, until now….
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Why Change Platforms?
• IMPETUS FOR CHANGE: Replace the
retiring Beckman 6300s for which Beckman
was no longer providing support
• Enable one platform for all sample types:
plasma, urine, CSF
• Decrease turn-around or anxiety time
• Retire qualitative amino acid assay
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Current Methods
• Ion Exchange Chromatography
– Beckman Amino Acid Analyzer
– Biochrom Amino Acid Analyzer
• HPLC
• LC/MS
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Amino Acid Analysis in the Nichols
Institute Biochemical Genetics
Laboratory
• All samples run on LC/MS
• Sample types include plasma, urine and CSF
• Tests vary from single amino acid assays like
urine cystine to 47 amino acid full panel
• No qualitative assays any more
• All amino acid tests are quantitative
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Plasma Amino Acids:
Current Methods vs. New Method (1)
LC/MS
– Run time: 25 min
80 min
Amino Acid Analyzers (no column switcher)
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21.5 min
HPLC
– Run time:
• Biochrom
– Run time: 165 min
(with column switcher)
– Single analyte: 5 min
• Beckman 6300
– Run time: 90-150
min
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Chromatogram from Old HPLC Method:
Part One (2 – 42 minutes)
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Chromatogram from Old HPLC Method:
Part Two (42 – 72 minutes)
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Myths of LC/MS
• “LC/MS is not routine”
• “Method only produces molecular weight
information”
• “LC/MS is not sensitive”
• “LC/MS is not quantitative”
• “LC/MS is not cost-effective”
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Interfacing HPLC to MS
• HPLC
• High pressure liquid phase
separation
• No mass range limitation
• Can use inorganic buffers
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MS
High vacuum required
Tolerates limited gas load
Elevated temperatures
Depends on m/z and analyzer
Prefers volatile buffers
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Benefits of LC/MS
For the chromatographer
• Complements existing LC detectors
• Does not depend on particular functional group
• Can be used as a mass-specific detector
• Provides both qualitative and quantitative information
For the mass spectrometrist
• Can analyze compounds not amenable to GC
(large, polar, thermally labile)
• Allows direct coupling of LC separation;
produces better information faster than "offline" LC/MS
• Automates probe analysis via flow injection
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Electrospray LC/MS
Advantages
• Softest ionization available
• LC/MS interface with best sensitivity
• Extends mass range for multiply charged analytes
• Works with a wide range of medium to high polarity
compounds
• Low maintenance
Disadvantages
• Solution chemistry influences ionization process
• Works less well with nonpolar analytes
• Adduct ions (other than M+H) possible with some analytes
• Some sensitivity loss at higher flow rates (~1 ml/min)
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API-Electrospray Ionization
Electrospray Ions
Nebulizer (gas shown
in red)
Heated nitrogen drying
gas
-5,000 V
Solvent spray
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Dielectric capillary
entrance
Evaporation
+ + +
+ -- - +
+- -+
+ - +
++
Rayleigh
Limit
Reached
+
++- +
+ -- ---+
+++++
Coulomb
Explosion
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++-- -++ + -+-+
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- -++ +-+- +
++-+
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++-- -++ + -++
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- - -+ --- +
+++
+ ++
+--+-+
-+
++
+--+-+
-+
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++++
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+++
Evaporation
+-+ +
+ - +
+ -- - +
+ - +
++
Analyte Ion
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++- +
+ -- ---+
++ ++
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++-- -++
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- - -+
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+--+-+
++- +
API Electrospray LC/MS:
Spray, Ionize, Evaporate
HPLC inlet
Skimmers
Nebulizer
gas inlet
Lenses
Quadrupole
Octopole
Capillary
HED detector
Nebulizer
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Neutral Molecules
Analyte Ions
Clusters
Salts
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heated N2
Waste
Fragmentation
zone (CID)
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What is CID (Collision Induced Dissociation)?
Skimmers
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Octopole
Capillary
Molecular fragmentation by ion
collisions with nitrogen
molecules in ion optics
Provides structural information
for qualitative analysis
Fragmentation
Zone (CID)
Lenses
Quadrupole
Provides confirmatory ions for
quantitative analysis
Controlled via a single ion optics
parameter -- "fragmentor"
M
DETAIL
[M ]1
n
+
[M ]2
+
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Single Quadrupole LC/MS
Limitations
• Lack of MS/MS specificity
(can use CID)
• Lack of accurate mass
information
• Slower scan speed than trap
Strengths
• Low cost, less than
MS/MS
• Robust, simple operation
• Tolerant of non-volatile
salts and background ions
• Easy to use
• High sensitivity in SIM
mode
Example applications: Combinatorial chemistry
Synthesis and purification
Assay and methods development
Process development and research
Stability and formulation studies
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Plasma Amino Acids:
Current Method vs. New Method
HPLC
– Column only good
for 200-250
injections.
– High cost of
columns.
– High cost of
mobile phase.
– Many interfering
substances.
LC/MS
– Column good for
>300 injections.
– Lower cost of columns.
– Lower cost of mobile
phase.
– More precision, few
interfering substances.
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Pre-Assay
2 Basic Steps:
• Drying step
• Coupling step
Makes the phenylthiocarbamyl (PITC)
derivatives that are actually analyzed
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Post-Assay
• Check each peak
• Create report
• Interpret report
– All interpretations for amino acid panels are
signed by ABMG certified biochemical
geneticists at Quest Diagnostics.
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Changes in Samples:
Newborn Screening and Follow-Up Studies
• Increased numbers of samples from infants at
risk for disorders now added to the expanded
newborn screen.
• Infant samples are often small volumes.
• Turn-around time is key!
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Screening Tests
• Population-based testing
• Used to identify neonates at risk for disorders
before they become ill
• Usually inexpensive compared to confirmatory
tests
• Higher level of false positives and potential for
false negatives
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Screening vs Confirmation
Screening: simple way to identify individuals in a large
group who may have an increased risk to have any
given condition.
• In general, genetic screening focuses on specific
populations at increase risk for a disease based on
family history, age, or geoethnic background.
• As knowledge and technology moved forward,
screening for specific genetic diseases has become
part of pediatric and obstetric practice.
Confirmatory: definitive and specific testing for a
condition.
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How to Choose What Disorders to Screen?
• 1994 Institute of Medicine Committee on Assessing
Genetic Risks:
• “Newborn screening should only take place
– 1) for conditions for which there are indications of clear
benefit to the newborn,
– 2) when a system is in place for confirmatory diagnosis,
and
– 3) when treatment and follow-up are available for
affected newborns…”
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OK, So How to Choose?
• Public health experts have a mathematical formula for
showing public health impact: Prevalence X Severity X
Effectiveness of intervention = Public Health Impact
• Need to demonstrate cost utility, showing benefit in
quality-adjusted years of life plus decreased public
health impact
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Really, HOW TO CHOOSE?
• Pick disorders for which early treatment is
clearly beneficial and
• For which you have a window of opportunity
for instituting treatment and
• Which have a significant prevalence among
the group tested
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Logistics of Newborn Screening
• Heel stick blood spots drawn before infant is
discharged from hospital
• Best done on day 2 of feeding, to make sure
there are abnormal metabolites
• With early discharge, should be repeated
• Primary care provider and referral center
contacted for abnormals to secure definitive
testing for confirmation of diagnosis before
treatment
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Results: Possible Outcomes
1) True Positives
Require continuing special
services
2) False Positives
No further follow-up (See 4)
3) False Negatives***
PCPs alert for unusual
presentations
4) True Negatives
Routine care for normal
babies
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National Newborn Screening Status Report
Updated 07/18/07
The U.S. National Screening Status Report lists the status of newborn screening in the United States.
Dot "" indicates that screening for the condition is universally required by Law or Rule and fully implemented
A = universally offered but not yet required, B = offered to select populations, or by request, C = testing required but not yet implemented
D = likely to be detected (and reported) as a by-product of MRM screening (MS/MS) targeted by Law or Rule
Core1 Conditions
STATE
Hearing
Endocrine
Hemoglobin
Additional Conditions Included in
Screening Panel (universally required
unless otherwise indicated)
Other
HEAR
CH
CAH
Hb S/S
Hb S/A
Hb S/C
BIO
GALT
CF
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B
Alabama
Alaska
Arizona
Arkansas
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California
HHH; PRO; EMA
Colorado
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Connecticut
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HHH; HIV 2 ; NKH
History of Newborn Screening
• Phenylketonuria (PKU)
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First newborn screening test in 1960’s
Promoted by father of affected child
1:10,000-25,000 individuals affected
Treatment with low phenylalanine diet prevents
mental retardation
– Screening done by Guthrie method
– Now screened in all 50 states
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History Part 2
• Congenital hypothyroidism
– 2 weeks to start replacement therapy to
prevent neurological damage
– 1:4000 affected
– Screened in all 50 states
• Galactosemia
– 1:60,000-80,000 affected
– Screened in 47 states and DC
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History Part 3
• Sickle Cell Disease
– 1987 NIH recommendation to screen all
newborns for Hgb SS
– 1:400 African Americans affected
– 1:10 African Americans carriers
– All newborns screened in 41 states and DC
– Only African Americans screened in 5 states, 4
states do not offer screening at all
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What Then Changed Newborn Screening?
• Advances in genetics and technology, like the
ability to use tandem mass spectroscopy to
quantitate a large number of analytes from a
blood spot
• Advances in genetic treatment for a variety of
disorders
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AAP State-by-State Survey - July 2000
• Survey of all 50 states and DC
• Web address:
www.aap.org/advocacy/archives/augscreenreport.htm
• Most states screen for 4 to 5 diseases only
• Most extensive-Massachusetts (11)
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AAP Press Release - August 7, 2000
• “Newborn Screening Report Addresses
Inconsistencies and Controversies” calls for
nationwide standards
• No states use the most modern methods for
screening
• State-to-state differences mean some
children with inborn errors will die due to
birth in the “wrong” state
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U.S. Newborn Screening Circa 2000
Pediatrics 2000;106:389-422
Copyright ©2000 American Academy of Pediatrics
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September 2000: First Newborn Screening
Awareness Month
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Promoted by 16 support groups for parents with
children who have or have died of metabolic diseases
and surviving patients
– Tyler for Life
www.TylerforLife.com, now savebabies.org
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Children's PKU Network, Florida PKU Parents, Kansas PKU
Network,, Mid Atlantic Connection for PKU & Allied Disorders,
National Coalition for PKU & Allied Disorders, New England
Connection for PKU & Allied Disorders, PKU Organization of
Wisconsin, PKU Parents of California, and Western NY PKU
Association.
Mitochondrial Disease Outreach Center, FOD Family Support Group
Magic Foundation
MSUD Family Support Group, The Organic Acidemia Association
Parents of Galactosemic Children
National Urea Cycle Disorder Foundation
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Newborn Tyler Mize
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New Trends in Newborn Screening:
Technology
• Tandem mass spectroscopy for 25-30 or more
diseases offers comprehensive screening for risk of
1:1200 to 1500 of true positives
– Commercial labs: Pediatrix
(formerly NeoGen) in Pennsylvania with license to Mayo
– Not-for-profit labs: Baylor in Dallas
(Drs. Roe and Sweetman)
• Instituted by individual states, from NJ to CA
• Done from dried blood spots
• Turn around time similar to regular screening
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Potential Disorders:
Amino Acid Abnormalities
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Argininemia
ASA lyase deficiency
Biopterin disorders (4)
Citrullinemia 1 and 2
Homocystinuria/CBS deficiency
Hypermethioninemia
PKU and variants
MSUD
Tyrosinemia 1-3 and transient
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Potential Disorders:
Organic Acid Abnormalities
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2-methylbutyryl-CoA dehydrogenase deficiency
3-OH-3-methylglutaryl-CoA lyase deficiency
3-methylcrotonyl-CoA carboxylase deficiency
3-methylglutaconic aciduria 1-4
Beta-ketothiolase deficiency
Glutaric aciduria 1
Isobutyryl-CoA dehydrogenase deficiency
Isovaleric acidemia
Methylmalonic acidemia/cobalamin defects
Propionic acidemia
Multiple carboxylase deficiency
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Potential Disorders: Fatty Acid
Oxidation Abnormalities
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Carnitine transporter deficiency
Carnitine-acylcarnitine translocase deficiency
Carnitine palmitoyl transferase deficiency 1 and 2
Long chain OH-acyl-CoA dehydrogenase deficiency
(LCHAD)
MCAD deficiency
Multiple acyl-CoA dehydrogenase deficiency
(MADD/glutaric acidemia 2)
SCAD deficiency
Trifunctional protein deficiency
VLCAD deficiency
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Potential Disorders: More
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2-methyl-3-OHbutyryl-CoA dehydrogenase deficiency
5-oxoprolinuria
Ethylmalonic encephalopathy
Homocitrullinuria-hyperornithinemia-hyperammonemia
(HHH)
Gyrate atrophy of the choroid and retina
Malonic aciduria
Non-ketotic hyperglycinemia
Prolinemia 1 and 2
Biotinidase deficiency
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Biochemical Genetics Laboratory Test Panel
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Plasma amino acid quantitation
Urine organic acid quantitation
Acylcarnitine profile
Carnitine levels
Lactate and pyruvate
Cellular-based enzymatic assays or DNA
analyses
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California Newborn Screening
Follow-Up Program
• Expanded newborn screening program to
begin in California on 7/1/2005
• ~600-700,000 births per year
• ~1:1500 infants with inborn errors
• Quest Diagnostics-Nichols Institute awarded
the contract to provide follow-up testing for all
abnormal screens
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Nutritional Disorders
• Many other conditions may cause abnormal
amino acids
• Primary problems due to malabsorption or
renal disorders
• Secondary problems due to poor intake,
including anorexia
• Monitoring of nutritional therapy
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Nutritional Disorders: More
• Gastrointestinal malabsorption
• Short gut syndromes
• Individuals on hyperalimentation or total
parenteral nutrition
– Infants, including prematures
– Elderly
• Cancer patients
• Renal Fanconi syndrome
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Other Nutritional Issues
• Naturipathic therapies
• Alternative medicine
• Treatment of adult disorders via amino acid
therapy, including depression and
hypertension
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Summary
• New LC/MS methodology developed for
amino acids analysis
• Decreased run time means decreased turn
around or anxiety time
• Improved accuracy of analysis
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Medical & Scientific Staff
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Senior Scientist: Scott Goldman
Medical Director of Genetics: Charles Strom, MD, PhD
Medical Director: Julie Neidich, MD
Scientific Director: Denise Salazar, PhD
Associate Scientific Director: Renius Owen, PhD
Associate Scientific Director: Rajesh Sharma, PhD
Genetic Counselor: Raynah Lobo, MS
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Contact Information
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Julie Neidich, MD
Medical Director
Biochemical Genetics and Cytogenetics
Quest Diagnostics-Nichols Institute
33608 Ortega Highway
San Juan Capistrano, CA 92675
949-728-4936
[email protected]
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Assay Information
• Amino Acids Analysis for MSUD, LC/MS, Plasma
– Includes: valine, isoleucine, leucine, alloisoleucine
• Test Code: 19779X
• Specimen Requirements:
– 2 mL sodium heparin (green-top) plasma, frozen
• Clinical use:
– Amino Acid analysis for MSUD is necessary for the
diagnosis of inborn errors of metabolism maple syrup
urine disease. The assay is also key for the continued
monitoring of treatment plans for these disorders and
useful for assessing nutritional status of patients.
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Assay Information
• 767X Amino Acid Analysis, LC/MS, Plasma
–
Includes: Aspartic Acid, Glutamic Acid, Hydroxyproline, Serine, Asparagine, Alpha-Amino Adipic
Acid, Glycine, Glutamine, Sarcosine, Beta-Alanine, Taurine, Histidine, Citrulline, Arginine,
Threonine, Alanine, 1-Methylhistidine, Gamma-Amino Butyric Acid, 3-Methylhistidine, BetaAmino Isobutyric Acid, Proline, Ethanolamine, Alpha-Amino Butyric Acid, Tyrosine, Valine,
Methionine, Cystathionine, Isoleucine, Leucine, Homocystine, Phenylalanine, Tryptophan,
Ornithine, Lysine
• 1776X Amino Acid Analysis, Limited, LC/MS, Plasma
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Includes: Tyrosine, Valine, Isoleucine, Leucine, Phenylalanine, Tryptophan
• 19779X Amino Acid Analysis for MSUD, LC/MS, Plasma
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Includes: Valine, Isoleucine, Leucine, Alloisoleucine
• 36183X Amino Acid Analysis, LC/MS, Urine
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Includes: Creatinine, Aspartic Acid, Glutamic Acid, Hydroxyproline, Serine, Asparagine, AlphaAmino Adipic Acid, Glycine, Glutamine, Sarcosine, Beta Alanine, Taurine, Histidine, Citrulline,
Arginine, Threonine, Alanine, 1-Methylhistidine, Gamma-Amino Butyric Acid, 3-Methylhistidine,
Beta-Amino Isobutyric Acid, Proline, Ethanolamine, Alpha-Amino Butyric Acid, Tyrosine, Valine,
Methionine, Cystathionine, Isoleucine, Leucine, Homocystine, Phenylalanine, Tryptophan,
Ornithine, Lysine, Cystine, Hydroxylysine
• 29881X Amino Acid Analysis, LC/MS, CSF
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Includes: Aspartic Acid, Glutamic Acid, Hydroxyproline, Serine, Asparagine, Alpha-Amino Adipic
Acid, Glycine, Glutamine, Sarcosine, Beta-Alanine, Taurine, Histidine, Citrulline, Arginine,
Threonine, Alanine, Gamma-Amino Butyric Acid, Beta-Amino Isobutyric Acid, Proline, AlphaAmino Butyric Acid, Tyrosine, Valine, Methionine, Isoleucine, Leucine, Homocystine,
Phenylalanine, Tryptophan, Ornithine, Lysine
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