1. Tandem MS Newborn Screening
Larry Sweetman, Ph.D. Paula Ashcraft, M.T. Institute of Metabolic Disease Baylor University Medical Center Dallas, TX, USA

2. Results Issues Markers Ratios Sub-population Reference Ranges
Detection of disorders
Diagnostic confirmation

3. Markers
Consider pattern of multiple markers, rather than primary and secondary markers
Remember MS/MS doesn’t distinguish isobaric compounds
Remember MS/MS doesn’t distinguish structural isomers ex. C5 = isovaleryl plus 2-methylbutyryl

4. Markers
Consider pattern of multiple markers, rather than primary and secondary markers ex. MCAD: C6, C8, C10:1, and C10 acylcarnitines ex. MSUD: Leu (+Ile, allo-Ile, OH-Pro) and Val and Leu/Ala

5. Ratios as Markers
Ratios of biochemical precursor/product ex. Phe / Tyr ex. MCAD: C8 / C2, C10:1 / C2 ex. VLCAD: C14:1 / C2 ex. CPT II or CACT: C16 / C2
Other ratios based on altered metabolism ex. MSUD: Ile/Ala ex. CPT I: Free Carnitine / C16 Free carnitine / C18

6. Age and Reference Ranges Sub-Populations

7. C3 Acylcarnitine (uM) vs Age (days)
NB cut off 6.84 uM
Baby cut off 4.12 uM

9. C16 Acylcarnitine (uM) vs Age (days)
NB cut off 9.53 uM
Baby cut off 6.47 uM

11. First specimen: Two days old, 7lbs 4oz
Age Related Reference Ranges
Case Study 1: MCAD
First specimen: Two days old, 7lbs 4oz
Results
Analyte Value Newborn Reference Range
(average of 3 extractions) (0 - 7 days old and >2000grams)
C6 carnitine < 0.86
C8 carnitine VH < 0.40
C10:1 carnitine H < 0.40
C10 carnitine H < 0.72
C8/C2 ratio VH < 0.02

12. Repeat specimen: 13days old
Case Study 1: MCAD
Repeat specimen: 13days old
Results
Analyte(s) Value Newborn
(average of 3 extractions) Reference Range
C6 carnitine < 0.86
C8 carnitine H < 0.40
C10:1 carnitine < 0.40
C10 carnitine < 0.72
C8/C2 ratio < 0.02
Baby
Reference Range
<0.95
<0.32
<0.34
<0.42
<0.03 H

13. First specimen: Two days old, 3540grams
Age Related Reference Ranges
Case Study 3: GAI
First specimen: Two days old, 3540grams
Results
Analyte Value Newborn Reference Range
(average of 3 extractions) (0 - 7 days old and >2000grams)
C5-DC VH < 0.38

14. Repeat specimen: 6 days old
Case Study 3: GAI
Repeat specimen: 6 days old
Results
Analyte(s) Value Newborn
(average of 3 extractions) Reference Range
C5-DC H < 0.38

15. Age and Reference Ranges Sub-Populations

18. Detection of Disorders Amino Acids
Detects disorders, fairly diagnostic Phe, Phe/Tyr = PKU or hyperPhe Ile and Val, Ile/Ala = MSUD Arg = Argininemia Orn = HHH syndrome 5-Oxoproline = 5-Oxoprolinuria

19. Detection of Disorders Amino Acids
Detects disorders, needs differential Met: homocystinuria (cystathionine synthetase) vs Met adenosyltransferase Cit: Citrullinemia vs Argininosuccinic aciduria Tyr: Tyrosinemia I, II, III, transient neonatal, liver disease

20. Detection of Disorders Acylcarnitines
Detects disorders, fairly diagnostic C6, C8, C10:1 C10 + ratios = MCAD C14:1, C16, 18 +ratio C14:1/C2 = VLCAD High free carnitine, free/C16, free/C18 = CPT I C5-dicarboxylic = GA I C3-dicarboxylic = malonic

21. Detection of Disorders Acylcarnitines
Detects disorders, needs differential C16, C18:1 , C18; CPT II vs CACT C14-OH, C16-OH, C18:1-OH, C18-OH; LCHAD vs MTP C5; IVA vs 2MBCD C4: SCAD vs IBCD C3: PPA vs MMA vs cbl (B12) C4, C5 and/or longer chains = MADD?

22. Detection of Disorders Acylcarnitines
Detects disorders, needs differential C5-OH; MCC vs maternal MCC C5-OH, C6-dicarboxylic; HMG vs 3-methylglutaconic C5:1; thiolase vs 2-methy hydroxybutyryl-CoA dehydrogenase C3 + C5-OH; HCS vs biotinidase

23. Confirmation of Screening Abnormalities
Repeat card for screen Requires age related cut offs Appropriate for Tyr (transient neonatal)
Recommend diagnostic testing Required when markers can suggest different disorders Recommended for most disorders

24. Confirmation of Diagnoses Depends on the Disorder
Quantitative Amino Acids in Plasma
Quantitative Organic Acids in Urine
Acylcarnitine profile in plasma or DBS (DBS preferred for long-chain disorders)
Intact fibroblast metabolism studies, assay of enzyme activities in fibroblasts or lymphocytes
Mutations analysis of DNA

25. Conclusion
Although interpretation and follow up of tandem MS newborn screening results is complex,
It can be done, and is of benefit to babies with a wide variety of inherited metabolic disorders.