Work placement Presentation

43 weeks in a biochemistry laboratory in Lyon, France

Connor Downing N0293299

The placement

• Placement situated in Lyon, France• A laboratory that deals with rare errors of

inborn metabolism• Given the title “stagiaire” roughly translated as

“intern”.• Duration of one year August 29th 2011-12• Large cultural differences, language used was

French

The organisation

• Service Maladies Héréditaires du Métabolisme• Part of Hospices Civil de lyon• Sectors on the floor comprised of biochemical,

radiological, enzymatic and biomolecular laboratories• Full time staff consists of a “cadre” of trained doctors

and management and also trained technicians.• Public sector service with samples received from all

over mid-region France.

My responsibilities

• Performing technician duties such as:– Carrying out the manipulations– Responding rapidly to occasional emergency

samples– Storage and cleaning of equipment– Basic maintenance of machinery – Removal of Hazardous waste– Basic calculation and quantification – Filing(paper) work

Classical organic acid disorders

• Manipulation consisted of dilution adjustment, urea removal, adding of known concentration standards and silylation.

• Diagnosed by gas chromatography coupled with mass spectrometry (enabling identification and quantification)

• Emergency cases in patients do occur (Hyperammonemia) so some samples are prioritised for expendenicy.

• Detected by marker molecules that are the substrates (and their toxic by-products) from a missing enzyme

• Examples of analytes included homogenisitic acid, methylcitrate and methymalonic acid.

• Amino acid, carnitine and acyl-carnitine profiles commonly ordered alongside.

Brand chained amino acid metabolism

Niemann-Pick C-1 Assay• Cholesterol transportation failure in cells• We made an attempt to functionalise a

biochemical assay for this disease from an article– Involved saponification of sample with KOH

(removal of FAs from cholesterol derivatives) – A step of solid phase extraction in addition to

the normal liquid/liquid extraction– Analysed by GC/MS

• Currently Niemann-Pick disorders (A,B,C and D) are screened by cholesterol studies on cultured fibroblasts using immunocytofluorescence

Article and Procedure

Thin Layer chromatography• Various Lysosomal storage disorders screened by TLC

• Oligosaccridosises (family of disorders)• Sialic acid pathologies• Adenylosuccinyl lyase deficiency

• Extremely old technique (60+ years)• Enables identification and crude quantisation• Consists of

– Ultra filtration of the sample– Deposition of sample– Migration of plate– Revelation of bands

• Rf values are calculated from length of bands against a control used

• Intensity of bands and depot judged by eye and recorded

• Finally Rf values are compared with a reference table to identify the bands.

Skills learned

• Laboratory conduct and expertise• Practical professional knowledge of biochemistry• Professional behaviour in a work-place• French language learned• Initiative when dealing with problems• Improved team skills • Perseverance

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