Acylcarnitine measurement in blood spots: methodological aspects, problems and pitfalls with reference to the ERNDIM QA scheme Charles Turner Laboratory Guy s Hospital (Evelina Childrens Hospital, St Thomas )
Acylcarnitine measurement in blood spots ERNDIM QA scheme Acylcarnitine analysis Methodological aspects Overview of ERNDIM QA Scheme Problems and Pitfalls
Acylcarnitine analysis Electrospray MSMS Precursor ion scan, m/z 85 Blood spots/plasma/serum Rapid analysis Flow injection analysis Simultaneous acquisitions for aminoacids etc Range of disorders Organic acidaemias, fatty acid oxidation disorders Particularly useful for long chain fat ox LCHADD, VLCADD Chace, D. H., T. A. Kalas, et al. (2003). "Use of tandem mass spectrometry for multianalyte screening of dried blood specimens from newborns." Clinical Chemistry. 49(11): 1797-817
Acylcarnitine analysis Quality control In house stable isotope internal standards quantitation - MRMs
Octanoyl carnitine within run precision profile: MRM v Scan 80 70 60 MRM data Scan Data 50 CV% 40 30 20 10 0 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Blood spot octanoyl carnitine µmol/l
Acylcarnitine measurement in blood spots ERNDIM QA scheme Methodological aspects Butylation v direct assay Questionnaire on 1 st circulation 34/35 butylation 1/35 - direct
Acylcarnitines butylation
Acylcarnitines butylated fragmentation
Acylcarnitines underivatised fragmentation
Acylcarnitine measurement in blood spots ERNDIM QA scheme Need for scheme? CDC excellent quantitative scheme Spiked blood samples Not specific for acylcarnitines More suitable for control of neonatal screening programmes No free carnitine
Acylcarnitine measurement in blood spots ERNDIM QA scheme Need for scheme? Personal Methodological Butylated v direct Offered to set-up pilot for 1 year if sufficient interest Questionnaire to see what users wanted
Acylcarnitine analysis Quality control ERNDIM Clinical material (blood spot/serum/plasma) Interpretative (qualitative/quantitative) Learning exercise Accreditation Model: urine organic acid scheme
Acylcarnitine analysis ERNDIM QA scheme ERNDIM users canvassed Over 30 laboratories expressed an interest By the dispatch date, 4 th April 2003 45 laboratories registered Problems of blood volume and sample transport scheme for plasma/serum excluded Blood spots from real clinical cases Quantitative special assays serum/urine includes free carnitine
Acylcarnitine measurement in blood spots ERNDIM QA scheme Initially 30 laboratories expressed an interest 2003 45 laboratories registered 2004 59 laboratories registered 2005 67 laboratories registered
Acylcarnitine measurement in blood spots ERNDIM QA scheme Patients Presenting to the the metabolic service at Guy s Hospital (now Evelina Childrens Hospital, St Thomas s) Diagnoses confirmed by enzymology or DNA sequencing Routine blood sampling written informed consent (patient/parent) obtained for use of excess blood for quality control purposes authorised by the chairman of the Guy s Hospital Ethical Committee Scheme participants encouraged to submit samples 1 donor so far + 2 recent offers
Acylcarnitine measurement in blood spots ERNDIM QA scheme Samples Lithium heparin anti-coagulated whole blood 40µl aliquots spotted onto Schleicher & Schuell 903 paper Dried for 24h at RT before packing and despatch Any delay stored at 80 o C Current requirement for 1 circulation approximately 4ml liquid whole blood 70+ dried blood spots
Acylcarnitine measurement in blood spots ERNDIM QA scheme 6 circulations to date April 2003 45 sent, 35 returns (19 by due date) January 2004 45 sent, 32 returns (25 by due date) July 2004 59 sent, 41 returns (38 by due date) Nov 2004-60 sent, 42 returns (38 by due date) May 2005-67 sent, 53 returns (45 by due date) Nov 2005-67 sent, due date not yet passed
ERNDIM QA Scheme Normal Samples 1a, 3b, 4c, 5b 5b was from diabetic patient hypoglycaemic due to insulin overdose Others from healthy subjects Most respondents reported acylcarnitine profile as normal Many suggested further tests
ERNDIM QA Scheme MCADD Samples 3a, 5a 3a 3y old female, seizures and developmental delay 39/41 respondents noted a high excretion of octanoylcarnitine 27/39 respondents commented on raise C 6, C 10, and/or C 10:1. 24 noted a low or low normal free carnitine 32 labs quantitative C 8 median 1.00µmol/l (0.51-2.26) 39 labs suggested medium chain acylcoa dehydrogenase deficiency as the diagnosis. 1 normal and 1 carnitine transporter defect. 5a 3y old male, left side unresponsive to stimuli 53/53 respondents reported raised octanoyl carnitine and suggested MCADD as the probable diagnosis. 42 reported quantitative values (median 1.01, range 0.52-2.43micromol/l). 35 respondents commented upon the low free carnitine (31 gave quantitative results: median 8.4, range 4.4-32.4micromol/l), 24 reported a raised C10:1 carnitine and 16 a raised C6 carnitine.
Acylcarnitine analysis ERNDIM QA scheme Sample 3a +Prec (85.00): 0.434 to 0.869 min from Sample 1 (3a) of Carn 2004-July-13.wiff (Turbo Spray) Max. 2.6e4 cps. 2.6e4 2.5e4 2.4e4 2.3e4 2.2e4 2.1e4 2.0e4 1.9e4 1.8e4 1.7e4 1.6e4 1.5e4 1.4e4 1.3e4 1.2e4 1.1e4 1.0e4 9000.0 8000.0 7000.0 6000.0 171.4 5000.0 165.6 4000.0 207.4 204.4 221.4 235.4 255.4 263.3 282.4 291.5 288.4 319.4 3000.0 172.4 264.5 218.6 244.4 270.5 314.6 320.4 2000.0 310.6 335.5 168.4 185.3 198.1 246.4 330.1 1000.0 214.3 219.3 280.1 286.5 386.5 252.4 298.5 315.5 357.5 371.8 400.4 414.6 426.8 195.1 352.5 378.5 447.6 479.0 486.5 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z, amu 338.5 347.5 348.5 375.6 403.5 431.5
Acylcarnitine analysis ERNDIM QA scheme Sample 5a +Prec (85.00): 0.668 to 1.303 min from Sample 22 (5a Mcadd BS) of Carnscan 2005-May-13.wiff (Turbo Spray), Smoothed Max. 4.8e4 cps. Intensity, cps 4.8e4 4.6e4 4.4e4 4.2e4 4.0e4 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e4 8000.0 6000.0 4000.0 2000.0 171.4 162.4 207.3 204.3 221.4 235.3 255.4 263.4 282.5 291.3 319.5 288.3 338.5 331.5 218.4 256.5 314.5 400.5 426.5 310.4 281.4 296.5 341.3 167.3 228.4 244.4 267.4 312.5 357.5 369.5 377.5 173.4 187.3 223.4 405.5 433.5 447.4 471.5 499.0 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z, amu 347.5 359.5 375.5 403.5 431.5
ERNDIM QA Scheme Glutaric Aciduria Type 1 Samples 2a, 5c 2a 11month old male, collapse following intercurrent illness, large head with frontal bossing GlutarylCoA dehydrogenase deficiency (type 1 glutaric aciduria) enzyme confirmed Bblood spot/plasma acylcarnitines consistently normal Urine organic acids - only once was 3OHglutarate suspicious 32/32 labs reported an essentially normal profile Only 8 respondents suggested further investigation to exclude type 1 glutaric aciduria A normal acylcarnitine result does not exclude type 1 glutaric aciduria
Acylcarnitine analysis ERNDIM QA scheme Sample 2a +Prec (85.00): 0.468 to 0.835 min from Sample 39 (BS 2a) of Carn 2004-Jan-12.wiff (Turbo Spray) Max. 3.4e4 cps. 3.4e4 3.2e4 204.1 347.3 3.0e4 2.8e4 2.6e4 375.4 2.4e4 403.5 2.2e4 2.0e4 1.8e4 162.4 291.3 319.0 338.5 1.6e4 171.1 207.1 255.3 263.1 1.4e4 1.2e4 431.5 1.0e4 8000.0 221.1 235.1 6000.0 173.1 282.5 359.5 4000.0 218.1 261.1 213.9 256.4 331.5 400.3 428.3 2000.0 165.1 236.1 296.4 310.1 228.3 264.3 288.3 349.1 405.6 201.9 369.3 241.4 316.3 344.3 377.4 398.4 419.4 433.5 450.5 176.1 189.9 223.9 286.3 350.5 470.6 483.0 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
ERNDIM QA Scheme Glutaric Aciduria Type 1 Samples 2a, 5c 5c 3y old male, intercurrent infection, altered consciousness, hypoglycaemia 52/53 ^ C5-dicarboxyl-carnitine and suggested a diagnosis of glutaryl- CoA-dehydrogenase deficiency. 40 quantitative results (median 1.2, range 0.39-3.1micromol/l) 34 quoted an upper limit of normal (median 0.15, range 0.03-0.5micromol/l). 42 suggested followup tests, mainly urine organic acids & enzyme assay 17 respondents made suggestions for clinical management 1 respondent reported the sample as normal. High awareness of GA1, unlikely to be missed if metabolite present
Acylcarnitine analysis ERNDIM QA scheme Sample 5c +Prec (85.00): 0.668 to 1.270 min from Sample 24 (5c GA1 BS) of Carnscan 2005-May-13.wiff (Turbo Spray), Smoothed Max. 7.7e4 cps. 7.5e4 7.0e4 204.3 282.5 6.5e4 6.0e4 276.3 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4 5000.0 171.4 162.4 207.3 221.4 235.4 255.4 263.4 282.5 291.3 165.4 276.3 218.3 400.5 426.5 264.5 232.4 296.5 310.5 341.5 167.1 202.4 228.4 246.3 267.3 316.4 357.5 377.5 398.4 405.5 424.3 447.5 319.4 331.5 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z, amu 338.6 347.5 359.5 375.5 272.6 403.5 275 280 285 m/z, amu 431.5 283.4 280.5 284.5 286.5
Acylcarnitine analysis ERNDIM QA scheme Sample 2c 2month old female, Reye-like illness Very long chain acylcoa dehydrogenase deficiency (VLCADD) enzyme and mutation analysis confirmed 16/32 respondents reported increased tetradecenoylcarnitine (C 14:1 ) and 12/16 provided quantitation (median 0.35µmol/l, 0.19-0.55), laboratory normal ranges, UL median 0.22, 0.07-0.55 All 16 suggested VLCADD as the diagnosis 16 did not suggest VLCADD or comment on C 14:1 but 5/16 supplied scans in which peaks at m/z 370 (direct) and m/z 426 (butylated) were apparent 7/16 provided quantitation (median 0.27µmol/l, 0.09-0.38) within the laboratory normal ranges, UL median 0.80, 0.54-1.07 Patient in recovery phase may not show gross elevation of C14:1 Variation in normal ranges
Acylcarnitine analysis ERNDIM QA scheme Sample 2c +Prec (85.00): 0.468 to 0.835 min from Sample 41 (BS 2c) of Carn 2004-Jan-12.wiff (Turbo Spray) Max. 3.3e4 cps. 3.3e4 3.2e4 347.5 3.0e4 2.8e4 2.6e4 2.4e4 375.5 2.2e4 403.5 2.0e4 171.1 319.4 1.8e4 1.6e4 207.1 255.3 263.0 291.3 338.5 1.4e4 1.2e4 1.0e4 204.1 221.3 235.3 C14:1 431.4 8000.0 6000.0 173.1 282.5 162.3 359.5 4000.0 213.9 320.3331.4 370.4 218.1 296.3 310.1 400.5 2000.0 222.1 261.0 341.3 349.4 164.9 288.0 426.1 198.9 202.9 253.9 335.0 377.5 433.5 184.6 217.1 397.4 419.3 447.3 226.9 258.4 300.0 337.3 363.0 461.1 482.5 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z, amu
VLCADD when unwell Intensity, cps 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 171.3 207.2 221.3 235.3 347.4 375.5 255.4 319.4 331.4 403.6 263.3 291.3 431.5 1.0e4 162.2 204.2 282.4 341.5 370.4 400.5 426.5 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z, amu
Acylcarnitine analysis ERNDIM QA scheme Sample 4b 6month old female, currently well. History of acute collapse at 4 days of age with apnoea, profound metabolic acidosis, hypoglycaemia, left ventricular hypertrophy MalonylCoA decarboxylase deficiency Increased malonylcarnitine
Acylcarnitine analysis ERNDIM QA scheme Sample 4b 20/42 malonic aciduria (malonyl CoA carboxylase deficiency) ^C3DC, m/z 248 underivatised, m/z 360 butylated). 15 quantitative values (median 1.1, range 0.35-2.2micromol/l), 12 gave reference ranges (upper limit of normal: median 0.11, range 0.05-0.8micromol/l). 22 respondents did not suggest malonic aciduria 11 supplied spectra of butylated sample in which the peak at m/z 360 was clearly visible. 3 supplied MRM acquisition data which did not include malonyl carnitine. 4 described the sample as normal
Acylcarnitine analysis ERNDIM QA scheme Sample 4b +Prec (85.00): 0.368 to 0.835 min from Sample 3 (Malonic) of EQC BS 2004-Nov-23.wiff (Turbo Spray) Ma 3.1e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 347.5 2.0e4 1.8e4 1.6e4 1.4e4 255.3 263.3 319.3 375.4 1.2e4 171.4 291.3 338.5 403.5 1.0e4 8000.0 6000.0 207.1 218.3 221.4 235.4 282.5 431.8 4000.0 227.3 232.3 260.5 165.4 205.1 359.3 2000.0 246.1 283.5 288.4 320.6 331.6 219.1 228.4 264.5 316.3 341.5 372.5 426.4 172.6 187.3 199.1 275.1 304.5 315.5 368.5 398.6 433.6 446.1 469.3 4 223.6 233.1 258.5 277.5 344.5 383.4 413.3 416.5 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 48 m/z, amu 400.4
Other Diagnoses Propionic acidaemia Isovalericacidaemia Carnitine transporter defect Clear acylcarnitine profiles Majority of laboratories made diagnoses
Normal Summary of ERNDIM QA results Most labs report normal scans Demonstrates difficulties of ruling out metabolic disease with single test MCADD, IVA, Propionic, Carnitine transporter Clear diagnoses, no difficulty for most labs Methylmalonic All labs pick up raised C3, C4DC less easy
Malonic Summary of ERNDIM QA results Rare disorder not recognised by some Some labs using MRM acquisition only did not include malonyl carnitine in panel VLCADD Minor elevation of C14:1 in treated patients Issues with normal ranges GA1 Some patients do not generate metabolites When C5DC present well detected No difference between butylators & non-butylators
Acylcarnitine analysis Future perspectives Technology Greater sensitivity Routine product ion scanning Consolidation not revolution
Acylcarnitine analysis Future perspectives Use of electrospray MSMS more effectively and efficiently for diagnosis Clinically targeted Hyperammonaemia Hypoglycaemia Storage disorders Liver failure etc
Conclusions Acylcarnitine analysis integral part of acute biochemical genetics laboratory investigation Butylation vs non-butylation No clear diagnostic advantages either way Underivatised simpler and faster for acute More robust for population screening ERNDIM QA scheme Real samples allow labs to build up experience of clinical variation Useful forum to pool knowledge and share insight
Acknowledgements Neil Dalton The patients and their families, other labs who have supplied samples The clinical and laboratory staff at Guy s Dr Mike Champion and Jane Gick The WellChild Trust Charitable Foundation Guy s & St Thomas Hospitals Guy s & St Thomas NHS Trust
Acylcarnitine analysis ERNDIM QA scheme Normal Samples 1a 49y old male, fatigue & muscle cramps: normal 34/36 labs reported normal, 27 labs suggested no further action 3b 50y old male, mild peripheral neuropathy, increased CK Otherwise healthy subject with Charcot-Marie Tooth syndrome 31/41 respondents considered the profile normal 9/41 commented on elevated long chain acylcarnitines 5 concluding probably normal, with 2 offering other tests. 2 LCHAD, 1 CPT-2 and 1 VLCADD, 1 ^ C3 & C4DC - MMA 4c 16y male, vacant episodes: no known metabolic disease 38/42 normal, 2 MADD, 1^ C3, 1 low carnitine 5b 31y old female, episodes of hypoglycaemia 44/53 normal, 7 mentioned LCFAO disorders not excluded if well 1 respondent suggested paired plasma insulin and glucose. 5 ^ C5OH carnitine 1^ C3 and 1 reduced C12 and C16 carnitines, both suggested repeating acylcarnitine analysis on plasma.