Pitfalls of Acylcarnitine Analysis Anthea Patterson Anthea - PowerPoint PPT Presentation

Pitfalls of Acylcarnitine Analysis Anthea Patterson Anthea Patterson Biochemical Genetics St James University Leeds

Complications Acylcarnitine Measurement and Interpretation – Analytical problems – Low C0 – Cord blood

Analytical Problems Analytical interference • Isobaric interference • Isobaric interference • General contamination

Baby AD 1.5 � mol/l Octanoyl Carnitine = 10 � mol/l Free carnitine = (C10:1 C6 not elevated) � � �� � � �� � � � D o lb y _ B L O O D 1 (1 .2 0 8 ) S b (3 0 ,3 0 .0 0 ); S m (S G , 1 0 x 1 .0 0 ) 2 : P a re n ts o f 8 5 E S + 4 0 3 .6 3 .9 5 e 6 1 0 0 % 3 8 1 .6 2 9 1 .5 4 0 0 .6 2 0 7 .5 1 7 1 .6 4 2 6 .5 2 5 5 .6 2 8 8 .5 2 0 4 .6 2 3 5 .5 4 2 4 .6 2 2 1 .6 3 1 6 .7 0 m /z 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0

Clinical Referral • Family contacted by local consultant • MCAD protocol • MCAD protocol followed • Samples taken • Information given

Follow Up Investigations ��������������������������� � � ����� � � • ������������������������������ � � � � ����� • • �������������������������� ! ��� ����������� 5538637_Dolby_1 1 (1.240) Sb (10,30.00 ); Sm (SG, 2x0.60) 2: Parents of 85ES+ 227.5 4.25e5 100 459.3 459.7 % 218.4 263.4 437.4 208.5 260.4 311.3 347.5 460.8 291.6 438.7 264.5 461.5 292.9 372.8 482.6 325.0 342.2 243.6 381.5 0 m/z 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

Screening Specimen: Derivatised Octanoyl carnitine = 0.09 � mol/l = 14.2 � mol/l Free carnitine ����������� 2235318_Dolby_2 1 (1.239) Sb (30,30.00 ); Sm (SG, 10x1.00) 2: Parents of 85ES+ 227.6 5.45e5 100 288 C8 459.6 peak % 263.5 437.6 260.5 347.5 208.4 311.5 218.5 291.5 456.5 277.5 482.5 274.5 288.6 403.5 480.7 0 m/z 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

? 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 C6 316 > 85 316 > 85 260 > 85 260 > 85 GA2 (MCAD) 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 •VF 14 years •Collapse, Coma •C0 = 2 � mol/l 1 Foster Victoria 1 1 (1.212) Sb (30,10.00 ); Sm (Mn, 2x1.00) 1: Parents of 85ES+ 459.5 2.25e6 100 Free carnitine 227.4 437.5 % 482.5 456.5 347.4 263.4 311.5 484.5 454.4 260.4 291.4 221.4 428.5 277.4 480.3 218.4 395.1 400.6 0 m/z 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 Deuterated internal standard

Post carntine supplementation 3 Foster Victoria 3 1 (1.212) Sb (30,10.00 ); Sm (Mn, 2x1.00) 1: Parents of 85ES+ 459.5 3.15e6 100 Free GA 2 carnitine 218.4 437.6 227.4 % Isovalery carnitine Decanoyl ����� ������� carnitine ��������� Butyryl ��������� 260.4 carnitine ��������� Dodecanoyl Hexanoyl 347.5 carnitine carnitine 263.4 ����� 372.5 311.5 482.5 456.5 344.5 288.4 291.4 426.5 316.4 277.4 201.3 400.5 395.3 0 m/z 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 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 C3 � mol/l � mol/l Corb Blood ��!� +��� ��!� No abnormality Study detected @3 day Diagnostic Diagnostic ��� ��� ��� ��� Pattern consistant Pattern consistant specimen/sib with PA @1day with PA � Leeds diagnostic ref : 0.3 – 2.6 CDC screening cut off, mean: 7

Early diagnosis GA2 C3 C4 C5 C6 C8 C10 C10:1 C5DC C12 C14 C16 ���������� CB ����� ��!�� ��+�� ���1� ��� � ���!� ����� ���� ���1� ����� ���� ���!� Day ����� ����� ����� 1�+1� 1���� ��1�� 1� �� ��+1� �� �� �� �� 1��+� !�� � ���4������ � �

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

The End