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Improving NBS Performance:The Mayo Clinic Experience
Dietrich MaternK. Raymond, S. Tortorelli, D. Gavrilov, D. Oglesbee, P. Rinaldo
Biochemical Genetics LaboratoryMayo Clinic College of Medicine, Rochester, MN
What influences NBS Performance?
• Pre-analytical
• Analytical‒ primary screen
‒ 2nd tier testing (molecular, biochemical)
• Result interpretation
• Follow up
• Feedback
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Pre-Analytical Factors• Sample is appropriate
‒ timing‒ treatment‒ volume‒ collection mode (capillary, venous/arterial, infusion)
• Sample originates from patient indicated on screening card
• Patient information is provided on screening card
• Correct demographics allow for prompt notification • Need baby’s primary provider for prompt follow-up• Accurate time and date of collection essential• Risk Factors, type of feeding, special circumstances helpful in
correct result interpretation
Minnesota NBS Card
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Analytical Factors• Know your reagents• Know your assay• Know your equipment• Participate in instrument training• Participate in assay training• Participate in QA/QC/PT programs
Result Interpretation• Know the detectable conditions
‒ what causes it / triggers a metabolic decompensation?‒ what markers should be abnormal‒ are the markers specific and sensitive‒ collection mode (capillary, venous/arterial, infusion)
• Review all available information
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X
C5 Acylcarnitine
C5 AC: 0.85 µM (normal <0.5)
2 8 5 0 3 6
X
0.5-1.0 µmol/L
C5-AC
≥ 1.0 µmol/L
BW >1,800 gGA >35 wks
ScreeningNEGATIVE
BW <1,800 g
ScreeningPOSITIVE
“sick”+/- ATB
ATB +/or “sick”
norisk factor
C5/C0, C5/C2, C5/C3ratios all H
NO YES
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2nd Tier Tests• A cost effective means to implement clinically
defined cutoffs when normal population and disease range overlap (poor specificity)
• Same specimen, no additional patient contact• Normal result overrules primary screening
2nd Tier Tests
• HCY/MMA/MCA/EMA (Met, C3, C4)
• Allo-ILE (BCAA)
• Steroids (CAH)
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Methionine
ScreeningNegative
NO
YES Met/Phe< 0.18
ScreeningPositive
8-11 µmol/L > 11 µmol/L< 8 µmol/L
2nd tier HCY>15 µmol/L
YES NO
Algorithm for Methionine
Algorithm for C3 Acylcarnitine
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Outcome of 2nd Tier Testing of Abnormal Met and C3 Acylcarnitine Results
Period 2007
Test volume 78,017
Abnormal C3/Met 2,902 (3.7%)
Reported abnormal 15
True positives 9 (1:8,669)
False positives 6 (0.008%)
Pos. predict. Value 60%
1 Propionic acidemia2 MMA1 Cbl C def.1 Remethylation defect1 Vit B12 def.3 Vit B12 def. (maternal)
2nd Tier Tests
• HCY/MMA/MCA/EMA (Met, C3, C4)
• Allo-ILE (BCAA)
• Steroids (CAH)
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Valine Isoleucine Leucine
isobutyryl-CoA
2-keto-isovaleric acid
methacrylyl-CoA
3-OHIsobutyric acid
3-OHisobutyryl-CoA
methylmalonatesemialdehyde
2-methylbutyryl-CoA
2-keto-3-methylvaleric acid
tiglyl-CoA
2-methyl-acetoacetyl-CoA
2-methyl 3-OHbutyryl-CoA
propionyl-CoA
isovaleryl-CoA
2-keto-isocaproic acid
3-methyl-crotonyl-CoA
3-OH 3-methyl-glutaryl-CoA
2-methylglutaconyl-CoA
acetyl-CoA acetoaceticacid
methylmalonyl-CoA succinyl-CoAKrebsCycle
SBCAD
Maple Syrup Urine Disease (MSUD) • Autosomal recessive Branched-
Chain Ketoacid Dehydrogenase (BCKD) Deficiency
• Clinical phenotypes– Classic (early onset; poor
feeding → coma → death)
– Intermediate (same as classic given sufficient stress)
– Thiamin responsive
• Diagnosis– Amino acids (allo-isoleucine)
– Urine organic acids
– Enzyme assay, molecular
• Included in NBS since 1964
BCKD
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Val - -(X)le/Phe 6.50 3.70(X)le/Ala 0.78 0.39
216.00 (n = 135)
066.00 (n = 106)
002.80 (n = 98)
000.31 (n = 73)
MSUD* Controls(1st %ile)* (1st – 99th %ile)
065 - 243
054 - 206
1.32 - 4.19
0.27 - 1.05
*www.region4genetics.org
TPN, total parenteral nutrition
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*Leu
/Ile/
Allo
-Ile/
OH
-Pro
line
*
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time, min
0.0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
8.0e5
9.0e5
1.0e6
1.1e6
1.2e6
1.3e6
1.4e6
Intensity, cps
3.95
Leu
Ile
Allo-d10
Val-d8
Val
Leu-d3
ControlVal 314 µM
Allo-Ile 0 µM
Ile 168 µM
Leu 249 µM
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time, min
0.00
5.00e4
1.50e5
2.50e5
3.50e5
4.50e5
5.50e5
6.50e5
7.50e5
8.50e5
9.50e5
1.05e6
1.14e6
3.95
Leu
Ile
Allo-d10
Val-d8
Val
Leu-d3
Allo
Val 246 µM
Allo-Ile 153 µM
Ile 161 µM
Leu 119 µM
MSUD
0.35%0.92%
0.10%
0.13%
0.64%
0.45%
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2nd Tier Tests
• HCY/MMA/MCA/EMA (Met, C3, C4)
• Allo-ILE (BCAA)
• Steroids (CAH)
17-OHP<12.5
ScreeningNegative
Y
ScreeningNegative
Y
NScreening
Positive
Ratio<3.5
N
Strategy for Multicomponent CAH Screening
* Ratio =Androstenedione + 17-OHP
Cortisol
C
A + BC
AB
17-OHP<12.5
17-OHP<12.5
ScreeningNegative
Y ScreeningNegative
Y
ScreeningNegative
Y ScreeningNegative
Y
NScreening
Positive
NScreening
Positive
Ratio<3.5
N
Ratio<3.5
Ratio<3.5
N
Strategy for Multicomponent CAH Screening
* Ratio =Androstenedione + 17-OHP
Cortisol
C
A + BC
AB
* Ratio =Androstenedione + 17-OHP
Cortisol
C
A + BC
AB
C
A + BC
AB
Clin Chem 50:621, 2004
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MayoBGL
MDHLaboratory
BirthingPlace
NewbornScreening
CHGALTCAHSCA
2nd TierTest
Message posted
on website
NormalResult (~90%)CAH
NORMAL
Changing CAH Screening in MN(2007)
• False positives 710 41• False (+) rate 0.97% 0.06%
Cost clinical F/U $847 per caseCost 2nd tier test $35 per test
w/o 2nd Tier w/ 2nd Tier
• Cost clinical F/U $601,370 $38,115• Cost 2nd tier test $0 $24,850• Total F/U cost $601,370 $62,965• Cost difference (savings) (89.5%)
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NBS Performance in the USA• False positive rate (MS/MS): 0.07% to 3.0%• True positive rate (MS/MS): 1:2,000-1:15,000
581.4Unnecessary evaluations WEEKLY (/100,000 births)
0.2%39%Positive predictive value
120,0002,800False positives / Year
2671,780True positives / Year
3.00%0.07%False positive rate
1:15,0001:2,249Detection rate
WORST caseBEST case
US scenarios (4 Millions births/year)
PER
FOR
MA
NC
E
Actual Case
Scenario
52*
*no repeat blood spot testing!!!
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Mayo NBS Performance (MS/MS)
2004 0.12% 27% SUAC (2nd tier)
2005 0.08% 37% MMA/HCY
2006 0.09% 47% Allo-Ile
2007 0.06% 47% SUAC (1st tier); MCA added to MMA/HCY
2008 0.06% 58% EMA added to MMA/HCY/MCA
YEAR FPR PPV ACTION
(Jan-Sep)
Partial List of Candidate Conditionsfor Expansion of Uniform Panel
(in alphabetical order)• ALD (X-linked)• CDG Ib• CMV• Creatine defects• Duchenne• G6PD• Gaucher (LSD)• HIV• MPS I/II/IIIa/VI (LSD)• Fabry (LSD)
• Fam. Hypercholesterol.• Fragile X• Friedreich ataxia• Krabbe (LSD) NY• Niemann-Pick (LSD)• Pompe (LSD) Taiwan• SCID WI• SLO• SMA• Wilson disease
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Late-Onset Dilemma is NOT Uniqueto LSD Screening
• Heterozygous FH:• Incidence: 1:500• Symptoms: xanthomata and corneal arcus may
occur already in childhood; premature ischemic heart disease
• Homozygous FH:• Incidence: 1:1,000,000• Symptoms:
• tendon and skin xanthomata and corneal arcus in early childhood; intermittent arthralgia; premature arteriosclerosis with evidence of cardiac dysfunction (e.g. angina) before 10 y/o
• fatal myocardial infarction by 2nd or 3rd decade
Familial Hypercholesterolemia
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NBS for Hypercholesterolemia- Impact on Medical Practice -
• Confirmation of diagnosis
• Treatment
• Counseling (family, patient)
• Affected family members:– (older) siblings?
– parents!
– aunts, uncles, cousins, etc.
– future children of patient
?
+NBS
?
CONCLUSIONSExpanded newborn screening may lead to improved health outcomes for affected children and lower stress for their parents. However, false-positive screening results may place families at risk for increased stress and parent-child dysfunction.
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• Performance affects the babies, their families, and health care costs
• Performance will become a bigger issue as more conditions are added to the screening panel
• Performance improvements can be made in all areas of the screening process
• 2nd tier testing improves specificity, false positive rate and positive predictive value
• 2nd tier testing can be regionalized
• Collaboration/harmonization is essential
Conclusions
Biochemical Genetics
Lab
