Pitfalls of Acylcarnitine Analysis Anthea Patterson Anthea - - PowerPoint PPT Presentation

Pitfalls of Acylcarnitine Analysis

Anthea Patterson Anthea Patterson Biochemical Genetics St James University Leeds

– Analytical problems – Low C0

Complications Acylcarnitine Measurement and Interpretation

– Cord blood

Analytical Problems

Analytical interference

• Isobaric interference
• Isobaric interference
• General contamination

Baby AD Octanoyl Carnitine = 1.5 mol/l Free carnitine = 10 mol/l (C10:1 C6 not elevated)

• 1

D

• lb

y _ B L O O D 1 (1 .2 8 ) S b (3 ,3 .0 ); S m (S G , 1 x 1 .0 ) 2 : P a re n ts

• f 8

5 E S + 3 .9 5 e 6

4 3 .6

1 6 1 8 2 2 2 2 4 2 6 2 8 3 3 2 3 4 3 6 3 8 4 4 2 4 4 m /z %

3 8 1 .6 2 9 1 .5 2 7 .5 1 7 1 .6 2 4 .6 2 5 5 .6 2 3 5 .5 2 2 1 .6 2 8 8 .5 3 1 6 .7 4 .6 4 2 6 .5 4 2 4 .6

Clinical Referral

• Family contacted by

local consultant

• MCAD protocol
• MCAD protocol

followed

• Samples taken
• Information given

Follow Up Investigations

• !
• 200

220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z 100 % 5538637_Dolby_1 1 (1.240) Sb (10,30.00 ); Sm (SG, 2x0.60) 2: Parents of 85ES+ 4.25e5

227.5 218.4 208.5 459.3 263.4 260.4 243.6 437.4 311.3 291.6 264.5 292.9 347.5 325.0 342.2 381.5 372.8 438.7 459.7 460.8 482.6 461.5

Screening Specimen: Derivatised

• 100

2235318_Dolby_2 1 (1.239) Sb (30,30.00 ); Sm (SG, 10x1.00) 2: Parents of 85ES+ 5.45e5

227.6

Octanoyl carnitine = 0.09 mol/l Free carnitine = 14.2 mol/l

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z %

208.4 218.5 459.6 263.5 260.5 437.6 347.5 311.5 291.5 277.5 274.5 288.6 403.5 456.5 482.5 480.7

C8 288 peak

? Source of Contamination

• 4 blank spots from various parts of the cards.
• 288 ion present on blank

C8 = 1.1, 1.0, 0.96, 0.5 C8 = 1.1, 1.0, 0.96, 0.5

Action

Derivatise all elevated C8 specimens prior to referral. .

Two Case From Sheffield

BR Male, 29/40, SCBU Day 5 dbs: C8 = 1.53 umol/l (1.6, 1.27/1.72) C8 = 1.53 umol/l (1.6, 1.27/1.72) C0 = 25 umol/l Acylcar full scan underiv: atypical, C5:1 increased in addition to C8, C6 and C10:1 normal

Sheffield

• Contamination on blank card
• All samples (7) from SCBU over 2 week

period reviewed – all samples and blank period reviewed – all samples and blank card normal

• SCBU discarded all stored cards and

improved storage conditions

Contamination

Problems

• Microtitre plates.

New batch of Greiner platesD Every specimen on run had C8 of ~1.3 mol/l. Changed to Corning plates.

• Transfer plates
• Transfer plates
• Pipette tips.
• Instrument failures – Backup essential

Isobaric Compounds

• "#"$%&'

"#"$&() C0 218 > 85 162 > 85 PCD * +,! 204 > 85 $-&' C3 274 > 85 218 > 85 MMA; PA C4 288 > 85 232 > 85 EMA;SCAD; GA2 C5:1 300 > 85 244 > 85 PA; BkT C5 302 > 85 246 > 85 IVA; GA2 *. 304 > 85 !,! (Ketosis) C6 316 > 85 260 > 85 GA2 (MCAD) C6 316 > 85 260 > 85 GA2 (MCAD) *. 318 > 85 +,! Biot;IVA;BkT;3HMG *! 344 > 85 288 > 85 MCAD /0) *1* 1+,! !,! Malonic Aciduria *!. 1+,! 304 > 85 (Metab Crisis) C10:1 370 > 85 314 > 85 MCAD C10 372 > 85 316 > 85 GA2 ** 374 > 85 +,! [MMA] ** 1!!,! 276 > 85 GA1 ; (GA2) *. 1!!,! 332 > 85 (Metab crisis) C12:1 398 > 85 342 > 85 [BDoxidn] C12 400 > 85 344 > 85 (BDoxidn]

Problems With Carnitine Depletion

Initial Specimen

100 1 Foster Victoria 1 1 (1.212) Sb (30,10.00 ); Sm (Mn, 2x1.00) 1: Parents of 85ES+ 2.25e6 459.5

Free carnitine

• VF 14 years
• Collapse, Coma
• C0 = 2 mol/l

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z % 227.4 221.4 218.4 437.5 347.4 263.4 260.4 311.5 291.4 277.4 428.5 400.6 395.1 456.5 454.4 482.5 480.3 484.5

Deuterated internal standard

Post carntine supplementation

100 3 Foster Victoria 3 1 (1.212) Sb (30,10.00 ); Sm (Mn, 2x1.00) 1: Parents of 85ES+ 3.15e6 459.5 218.4

Free carnitine

GA 2

• Hexanoyl

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z % 201.3 437.6 227.4 260.4 347.5 263.4 311.5 288.4 277.4 291.4 344.5 316.4 372.5 426.5 400.5 395.3 456.5 482.5

Butyryl carnitine

Isovalery carnitine Decanoyl carnitine Dodecanoyl carnitine

Deuterated internal standard

Low Free Carnitine

• Included in the MCADD protocol.

(To prevent erroneously low C8 due to carnitine depletion.)

• Free carnitine ≤ 2.0 (mean of

analysis in triplet ) )

Carnitine Transporter Deficiency

• 4 cases: Infant
• 4 cases : Maternal

Mean CO at screening: 1.05 mol/l

Clincal importance of CTD diagnosis

"*2.

• No cardiomyopathy
• All have myopathy ( aches and pains)
• Easy fatigability

3%

• All on supplements – all doing well
• One baby – withdraw from Rx. Wg loss

and FTT

• Recommenced symptomatic improvement

Cord Blood Analysis

• Joint project, Manchester and Leeds
• Cord blood was collected from births in

high IEM prevelance areas

• 25,000 samples over two years
• 25,000 samples over two years

– High C3s – Maternal diagnoses – False negatives

Problems with early C3s

CB Day 1 Day3 screening Organic acids C3 mol/l C3 mol/l Corb Blood Study ! + ! No abnormality detected @3 day Diagnostic

• Pattern consistant

Diagnostic specimen/sib with PA

• Pattern consistant

with PA @1day

• Leeds diagnostic ref :

0.3 – 2.6 CDC screening cut off, mean: 7

C3 C4 C5 C6 C8 C10 C10:1 C5DC C12 C14 C16 CB

• ! + 1 !

1

• !

Day

• 1+1 1 1 1 +1

1+ !

• Early diagnosis GA2
• 4

Maternal diagnosis

• 2,Carnitine Transporter Deficiency
• 1, MCAD
• 1, MCC Deficiency
• 1, MCC Deficiency

The End