# Presentation: 498969

## Tandem MS Newborn Screening

- Larry Sweetman, Ph.D.Paula Ashcraft, M.T.Institute of Metabolic DiseaseBaylor University Medical CenterDallas, TX, USA

## Results Issues

- Markers
- Ratios
- Sub-population Reference Ranges
- Detection of disorders
- Diagnostic confirmation

## 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

## Markers

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

## 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

## Age and Reference RangesSub-Populations

## NB cut off6.84 uM

**C3 Acylcarnitine (uM) vs Age (days)**** **

- NB cut off6.84 uM

- Baby cut off4.12 uM

## Slide 8

## NB cut off9.53 uM

**C16 Acylcarnitine (uM) vs Age (days)**** **

- NB cut off9.53 uM

- Baby cut off6.47 uM

## Slide 10

## 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****	****	****  0.51****	****	****	****	****< 0.86**
**C8 carnitine****	****              2.84****	****VH****	****	****	****< 0.40**
**C10:1 carnitine****	****  0.49****	****H****	****	****	****< 0.40**
**C10 carnitine****	****	****  1.32****	****H****	****	****	****< 0.72**
**C8/C2 ratio****	****	****  0.09****	****VH****	****	****	****< 0.02****	**

## Repeat specimen: 13days old

_**Results**_
**Analyte(s)****	****	****Value****	****                  ****	****	****	****Newborn**
**	****                **(average of 3 extractions)	 		        **Reference Range**
**C6 carnitine****	****	****  0.57****	****	****      ****	****	****	****< 0.86**
**C8 carnitine****	****              0.53****	****H****	****      ****	****	****	****< 0.40**
**C10:1 carnitine****	****  0.37****	****	****      ****	****	****	****< 0.40**
**C10 carnitine****	****	****  0.47****	****	****      ****	****	****	****< 0.72**
**C8/C2 ratio****	****	****  0.02****	****	****      ****	****	****	****< 0.02****	**

**Baby**
**Reference Range**

**<0.95**
**<0.32**
**<0.34**
**<0.42**
**<0.03**

**H**
**H**

**Case Study 1: MCAD**

## 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  ****	****	****  2.23****	****VH****	****	****	****< 0.38**

## Repeat specimen: 6 days old

_**Results**_
**Analyte(s)****	****	****Value****	****                  ****	****	****Newborn**
**	****                **(average of 3 extractions)	                          **Reference Range**
**C5-DC   ****	****	****  0.87****	****H****	****      ****	****	****< 0.38**

**Case Study 3: GAI**

## Age and Reference RangesSub-Populations

## Slide 16

## Slide 17

## 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

## 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

## Detection of DisordersAcylcarnitines

- Detects disorders, fairly diagnostic	C6, C8, C10:1 C10 + ratios = MCADC14:1, C16, 18 +ratio C14:1/C2 = VLCADHigh free carnitine, free/C16, free/C18 	= CPT IC5-dicarboxylic = GA IC3-dicarboxylic = malonic

## 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?

## 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-3-	      hydroxybutyryl-CoA dehydrogenase	C3 + C5-OH; HCS vs biotinidase

## 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

## 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

## 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.