BIRMINGHAM CHILDREN’S HOSPITAL NHS FOUNDATION TRUST PAEDIATRIC LABORATORY MEDICINE

Transcription

BIRMINGHAM CHILDREN’S HOSPITAL NHS FOUNDATION TRUST
PAEDIATRIC LABORATORY MEDICINE
DEPARTMENT OF NEWBORN SCREENING AND BIOCHEMICAL GENETICS
WEST MIDLANDS LABORATORY SERVICE FOR
INHERITED METABOLIC DISORDERS
BIRMINGHAM CHILDREN’S HOSPITAL
HANDBOOK FOR USERS
The Department of Newborn Screening and Biochemical Genetics at Birmingham
Children's Hospital NHS Foundation Trust (BCH) is a CPA Accredited Laboratory
which provides services for the diagnosis and monitoring of patients with Inherited
Metabolic Disorders (IMD) primarily for the West Midlands. The laboratory staff are
part of a wide multidisciplinary IMD team comprising clinical, nursing and dietetic
staff.
For general information about the Department of Newborn Screening and
Biochemical Genetics and Clinical IMD services please consult the Birmingham
Children's Hospital Website: http://www.bch.nhs.uk/
The purpose of this handbook is to provide information on the IMD laboratory service
including test repertoire, specimen requirements and details on accessing our
service. It also provides some guidance on investigating patients for suspected IMD.
IMD laboratory handbook
Document ID: DOC39
Version: 5
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Date of issue: July 2013
Final Authoriser: Mary Anne Preece
Contacts - “The Team”
Scientific Staff
Mrs Mary Anne Preece
Consultant Biochemist & Director of Laboratory IMD
Amino Acids, Organic Acids, Acylcarnitines
0121 333 9940
[email protected]
Dr Sarah Ball
Molecular Geneticist
Molecular Genetics & Antenatal
Diagnoses
0121 333 9877
[email protected]
Dr Tim Hutchin
Senior Biochemist
Storage Disorders, Cell Culture &
Antenatal Diagnoses
0121 333 9902
[email protected]
Mrs Linda Jenkinson
(until 31/07/13)
Chief Biomedical Scientist
0121 333 9942
[email protected]
Mrs Sue Alger
Principal Biochemist
0121 333 9941
[email protected]
Dr Rachel Webster
(until 02/08/13)
Principal Biochemist
0121 333 9941
[email protected]
Dr Adam Gerrard
Senior Biochemist
0121 333 9948
[email protected]
Mrs Glynis Kane
Laboratory Manager
0121 333 9920
(until 31/08/13)
[email protected]
Dr Alison Armitage
Senior Biochemist
0121 333 9948
[email protected]
Consultant Metabolic Paediatricians
Dr Anupam Chakrapani
0121 333 9899
[email protected]
Dr Saikat Santra
0121 333 9899
[email protected]
Dr Suresh Vijay
0121 333 9899
[email protected]
Consultants in Metabolic Disorders (adults)
Dr Tarek Hiwot
Queen Elizabeth Hospital
Birmingham
0121 627 1627 ext 51592
[email protected]
Dr Charlotte Dawson
Queen Elizabeth Hospital
Birmingham
0121 627 1627 ext 51592
[email protected]
Useful phone numbers:
Duty Metabolic Biochemist (general clinical queries) 0121 333 9864
Duty Senior Biomedical Scientist (results) 0121 333 9942
Fax Number
0121 333 9911
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GENERAL INFORMATION
Service Hours
Normal Working Hours
The laboratory is open for the receipt of routine samples from 09.00-17.10h Monday to
Friday (except Bank Holidays).
Out of Hours Service
Out-of-hours analytical services are available for plasma ammonia and CSF and plasma
lactate as part of the General Blood Sciences: Clinical Chemistry on-call service at BCH.
No other analytical service for inborn errors of metabolism is usually available out-of-hours
but, in emergency, specific tests may be provided if clinically justified and if staff are
available.
If analytical or advisory services are required out of hours please contact the on-call
Consultant Biochemist in the first instance (via the BCH switchboard, Telephone No. 0121333 9999).
Sending a Specimen
Information on sending specimens collected within the Birmingham Children’s Hospital can
be obtained from the Hospital Laboratory Handbook located on the Intranet.
Specimens collected at hospitals outside Birmingham Children’s Hospital should, where
possible, be sent via the clinical chemistry/blood sciences department in the originating
hospital. In some cases specimens for certain tests may require immediate transport by
courier or taxi. Specific needs are listed in the specimen requirement section of the table in
the Handbook. Tissue biopsies may require transport in special tubes in the frozen state.
Please contact the laboratory before sending such specimens. If using a courier or taxi
please request that the specimens are delivered to the Paediatric Laboratory Medicine Block
entrance at Whittall Street not to the main hospital post room.
Laboratory postal address
Department of Newborn Screening
and Biochemical Genetics
Paediatric Laboratory Medicine
Birmingham Children’s Hospital
Steelhouse Lane
Birmingham
B4 6NH
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Delivery address for specimens
sent by courier
Duty Scientist
Department of Newborn Screening
and Biochemical Genetics
Paediatric Laboratory Medicine
Laboratory Block
Whittall Street
Birmingham
B4 6DH
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Request forms and minimum data set
The request form must include a minimum of the following information:
1. Surname
2. Source of request ie originating hospital
3. Test requested
4. And two from
a) Forename
b) Date of birth
c) Registration number
The following information is desirable:
• Forename
• Date of birth
• Sex
• Postcode (requirement for molecular genetic tests)
• NHS number
• Registration number
• Name of requesting consultant
• Sample type
• Date and time of specimen collection
• Referring laboratory specimen number
• Clinical details (including fasting status and current drug therapy)
Specimen labelling and minimum data set
The specimen must be labelled with the following information:
1. Surname
2. And two from
a) Forename
b) Date of birth
c) Registration number
d) Referring laboratory specimen number
The following information is desirable:
• Date and time of specimen collection
• Forename
• Date of birth
• Sex
• Sample type
Note: for samples in small cryotubes, due to space constraints two patient identifiers are
acceptable: surname, and one from date of birth, registration number, referring laboratory
specimen number
A request for a urine “metabolic screen” will normally be interpreted by the laboratory as
requiring urine amino acids, organic acids and “spot tests”. However if the clinical
information supplied suggests other tests could be appropriate these may be added by the
Duty Metabolic Biochemist. Results will normally be returned via the local laboratory at the
requesting hospital.
Plasma and urine specimens are stored frozen for 3 months prior to disposal. If further tests
are required, please telephone to discuss whether enough specimen is remaining and
suitability for analysis.
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Urgent requests can often be dealt with more quickly if there is prior discussion with the
team. If your request is urgent please contact the Duty Metabolic Biochemist (0121 333
9864)
Key factors affecting test performance
Many factors can affect biochemical results; diurnal rhythm, exercise, fasting status, drug
therapy, method of specimen collection (e.g. venous or capillary), haemolysis and biological
variation. Some drugs interfere with certain analyses therefore it is helpful to provide a list of
current therapy on the request form accompanying the specimen. If specimens are not
stored and transported as indicated in the tables below, results of some analytes may be
affected. If urine specimens show signs of deterioration a repeat specimen is usually
requested because diagnostic abnormalities may not be apparent.
Specimen rejection
We do not analyse incorrectly or inadequately labelled specimens and/or request forms (see
above). Specimens that are inadequately labelled, in the incorrect anticoagulant or
preservative or are received too late after sampling will not usually be analysed and a report
will be issued to this effect. If appropriate, unsatisfactory specimens will be stored with
routine specimens for 3 months.
High Risk Specimens
We do not routinely analyse high-risk specimens without prior discussion. Please contact the
Duty Metabolic Biochemist before sending any such specimens.
Consent
It is the responsibility of the requesting doctor to obtain consent for the specimen collection
and the tests requested. It is implicit in the receipt of the request form that consent has been
obtained. We never take more sample than we need to but where there is material left over
after laboratory testing, it may be used for other purposes such as quality assurance or audit.
Specific research is regulated by the ethics committee. Consent for the use of tissue requires
that patients must be given the option to refuse permission for spare material to be used.
When this occurs, each request to the laboratory must be clearly marked so that specimens
are not used for other purposes.
There may be specific requirements for written consent for DNA tests sent to other countries,
please contact the molecular genetics laboratory for further information.
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Turnaround times
The turnaround times of all tests are monitored. Turnaround times quoted are the
anticipated times between specimen receipt in our laboratory and reporting under normal
operating conditions. Time taken for the specimen to reach the laboratory and for the report
to reach the requesting clinician are not included. Where appropriate, abnormal results will
be telephoned to the requesting physician.
The following are the targets that we aim to achieve for routine samples:Test
Amino acids (qualitative plasma)
Galactosaemia screen
Metabolites (3-hydroxybutyrate and free fatty acids)
Urine/faecal reducing substances
Acyl carnitines
Biotinidase
Chitotriosidase
Glucose-6-phosphate dehydrogenase
Plasma and white cell lysosomal enzymes
Tyrosinaemia screen
Urine metabolic screen (qualitative amino acids, spot tests,
organic acids)
White cell cystine
Simple DNA tests
Very long chain fatty acids, phytanate and pristanate
Galactose-1-phosphate
7-dehydrocholesterol
Methylmalonic acid
Orotate
Oxalate
Glycosaminoglycans
Oligosaccharides
Quantitative amino acids
Transferrin electrophoresis
Complex DNA tests
Tissue/fibroblast enzymes
Target Turnaround
90% in 1 week
90% in 2 weeks
90% in 3 weeks
90% in 4 weeks
90% in 8 weeks
Please enquire
For requests added on subsequent to receipt of the specimen, turnaround times from date of
test request will be as shown above.
Work referred away
The department regularly refers specimens to other specialist centres in order to provide a
comprehensive diagnostic service. Most UK laboratories to which samples are referred are
CPA accredited. However, many of those from Europe and around the world are either not
accredited or their accreditation status is unknown. The performance of referral laboratories
is routinely monitored.
Where work has been done in other centres it our policy to name the centre on our
laboratory report. A list of referral laboratories is given in appendix D.
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SERVICES PROVIDED
We offer an advisory service about which tests are appropriate for the investigation and
management of inherited metabolic disorders. We welcome discussion of patient
investigation with clinicians and laboratory staff prior to sending specimens.
Our test repertoire covers: 1) Postnatal Diagnosis
We provide a range of metabolic, enzymological and molecular genetic tests for the
diagnosis of inherited metabolic disorders. Most tests are done in house whilst some very
rare tests will be sent to other specialist laboratories in the UK and abroad.
2) Monitoring of Treatment
We provide a range of tests for the monitoring of treatment.
3) Antenatal Diagnosis
In liaison with the Regional Genetics Laboratory and Clinical Genetics Service we act as a
centre for advice on antenatal testing options for inherited metabolic disorders and coordinate sample handling and the reporting of results. We attempt the audit of all antenatal
diagnoses and record and report the results on an annual basis.
4) Newborn Screening
The West Midlands Newborn Screening Centre is housed within the Department of Newborn
Screening and Biochemical Genetics at Birmingham Children's Hospital and provides
newborn screening for congenital hypothyroidism, sickle cell disease, cystic fibrosis, medium
chain acyl CoA dehydrogenase deficiency and phenylketonuria including the investigation of
biopterin defects.
5) Cell Culture and Tissue Banking
We have facilities and are able to provide services for tissue banking and the culture and
storage of skin fibroblasts, transformed lymphoblasts, cultured chorionic villus cells and
cultured amniotic fluid cells.
We participate in multidisciplinary clinics and Ward Rounds:Birmingham Children’s Hospital
Clinical Inherited Metabolic Disorders (Dr Anupam Chakrapani, Dr Suresh Vijay, Dr Saikat
Santra).
Metabolic Neurology (Dr Evangeline Wassmer).
Metabolic Muscle Disorders (Dr Helen Roper, Dr Nick Davies).
Paediatric Liver Metabolic Disorders (Dr Pat McKiernan).
University NHS Trust
Adult Liver Metabolic Disorders (Dr Phil Newsome).
Adult Inherited Metabolic Disorders (Dr Tarek Hiwot, Dr Charlotte Dawson)
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TEST AVAILABILITY & SAMPLE REQUIREMENTS
Group Tests and Profiles
Some tests will detect a wide range of disorders that produce abnormalities in the particular
group of analytes eg amino acids, organic acids, acylcarnitines, mucopolysaccharides & very
long chain fatty acids. This type of Group Test is often used as a first line test.
In other cases we have combined tests into a profile that is aimed at detecting disorders with
a particular symptom or pattern of symptoms eg hepatosplenomegaly, hereditary optic
atrophy.
Individual Tests
We also offer tests designed to diagnose a specific disorder eg plasma 7-dehydrocholesterol
(Smith-Lemli-Opitz Syndrome), α-galactosidase A (Fabry’s Disease)
The volume & specimen type required and transport requirements are detailed below.
If the test you require is not included in our repertoire please visit the National Metabolic
Biochemistry Website:- http://www.metbio.net/metbioAssays.asp
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TESTS AVAILABLE
Code VB = Venous Blood, VP = Venous Plasma, S = Serum, U = Urine, BS = Blood Spot, L
= Liver, SB = Skin Biopsy or Cultured Fibroblasts.
METABOLITES
TEST
7-Dehydrocholesterol
(Smith-Lemli-Opitz
Syndrome)
Acyl carnitines
Amino Acids
(Quantitative)
Amino Acids (Qualitative)
Biopterins (total)
Carnitine
Free Fatty Acids & 3Hydroxybutyrate
Galactose-1-Phosphate
(Quantitative)
Glycosaminoglycans
(Mucopolysaccharides)
Homocystine (free)
SPECIMEN TYPE/VOLUME
VP (1ml Heparin) or
0.5ml plasma or serum
TRANSPORT
Room temperature
VB (Heparin 0.5ml) or VP Please send plasma AND blood
and a BS
spots
U (2ml), VB (Heparin
Store plasma and CSF frozen
0.5ml)
prior to shipment. Blood stained
CSF 0.5ml
CSF is unsuitable for analysis.
U (2ml), VP (Heparin
Preferably send a fasting sample.
0.5ml)
Store plasma and urine frozen
prior to shipment
6x10mm BS
None
Send at room temperature
U (0.5ml), VP (Heparin
0.5ml)
VP (Fluoride-Oxalate
0.5ml)
Room temperature
Store plasma frozen prior to
shipment
shipment. Please state fasting
status.
Whole VB (Heparin 1ml) The sample must be received
within 24 hours of collection.
U (5ml)
Store frozen prior to shipment
VP (Heparin 0.5ml)
Homocysteine (total)
VP 0.2 mL
Methylmalonic acid
(Quantitative)
Mono and Disaccharides
Oligosaccharides
U (1 ml)
VP (Heparin 1ml)
U (5ml)
Faeces (liquid)
U (1ml)
Organic Acid Analysis
Orotic Acid (Quantitative)
Oxalate (Oxalosis)
See sulphur-containing amino
acids quantitation for sample
preparation.
Separate within 30 minutes of
collection
Send frozen if possible
Room temperature
Room temperature
Room temperature
Room temperature
U (5ml)
U (5ml)
U (5ml)
Acidify to pH<2
None
Room temperature
shipment
None
Send at room temperature
Separate within 30 minutes of
collection and deproteinise
immediately (Deproteinising
solution available to referring
hospitals on request)
See above
Send frozen
Phenylalanine & tyrosine 2x10mm BS
(monitoring)
Phytanic & Pristanic acids VP (Heparin, FluorideOxalate or EDTA 1ml)
Porphobilinogen Synthase VB (Heparin 0.5ml). BS ,
Screen (Tyrosinaemia
red blood cells.
Screen)
Sulphur-containing Amino VB (Heparin 0.5ml)
Acid Quantitative (cystine,
homocystine and
methionine)
Sulphocysteine – please
request free and total
homocysteine
SPECIFIC NEEDS
shipment
See above
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Room temperature
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Transferrin Electrophoresis VP, VS 0.5 ml
(Congenital Disorders of
Glycosylation)
Very long chain fatty acids VP (Heparin, FluorideOxalate or EDTA 2ml)
White Cell Cystine
VB (Heparin 5ml –for
(Cystinosis)
monitoring, 10ml for
diagnosis)
None
Room temperature
shipment
Prior arrangement essential
Room temperature
Send whole blood to arrive
before 1pm on the day of
sampling
ENZYMES
Individual Lysosomal Enzyme Assays
(For sample requirements for enzyme profiles see below)
All samples should be received in our Department within 24 hours of collection
Assays are typically performed on leucocytes isolated from whole blood. For optimum yield
of cells the sample needs to reach us within 24 hours of collection. The volume of blood
required varies according to the number and type of tests requested. For these reasons it is
important to contact the laboratory prior to taking the sample. Please do not use first or
second-class post. Samples can be stored overnight at +4C - DO NOT FREEZE OR
SEPARATE. Some enzymes can be measured on plasma or blood spots as an initial screen
but any abnormal result would have to be confirmed on leucocytes isolated from whole
blood. Unless pre-agreed we will measure plasma or DBS in the first instance.
TEST
Acid Esterase (Wolman’s Disease)
α-N-Acetylgalactosaminidase (Schindler’s Disease)
α-Galactosidase A (Fabry’s Disease) (Males only)
WHOLE BLOOD
VB (EDTA 5ml)
-
VB (EDTA 5ml)
VB (EDTA 5ml) or
DBS (EDTA)*
-
α-Fucosidase (Fucosidosis)
VB (EDTA 5 ml)
α-Mannosidosis (Mannosidosis)
VB (EDTA 5 ml)
α-Glucosidase (Acarbose inhibited) (Pompe’s Disease)
Arylsulphatase A (Metachromatic Leucodystrophy)
PLASMA
VB (EDTA 5 ml) or
DBS (EDTA)*
VB (EDTA 5 ml)
VB (Heparin, EDTA or
clotted, 0.5ml) VP/VS
0.2ml
0.2ml
-
Aspartylglucosaminidase (Aspartylglucoaminuria)
-
β-Galactocerebrosidase (Krabbe’s Leucodystrophy)
β-Galactosidase (GM1 Gangliosidosis)
β-Glucosidase (Gaucher’s Disease)
VB (EDTA 5 ml)
0.2ml
-
VB (EDTA 5 ml)
-
VB (EDTA 5 ml)
-
β-Glucuronidase (Sly’s Disease, MPS VII)
VB (EDTA 5 ml)
β-Mannosidase (Mannosidosis)
VB (EDTA 5 ml)
Hexosaminidase A (Tay-Sach’s Disease)
VB (EDTA 5ml)
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0.2ml
0.2ml
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Hexosaminidase (Total) (Sandhoff’s Disease)
VB (EDTA 5 ml)
I-Cell Screen (Mucolipidoses II & III)
-
Palmitoyl Protein Thioesterase 1 (CLN1)
VB (EDTA 5 ml)
0.2ml
0.2ml
0.2ml
-
Sphingomyelinase (Niemann-Pick types A & B)
VB (EDTA 5 ml)
-
Tripeptidyl Peptidase 1 (CLN2)
VB (EDTA 5 ml)
-
*Samples should be taken in EDTA and either a dried blood spot (DBS) made or the whole
blood sent to our laboratory where we will make a blood spot (in these cases 1ml of sample
is sufficient).
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Cultured Fibroblast Enzymes
All the above enzymes can be assayed in cultured fibroblasts. Please see the requirements
for skin biopsies for culture.
Filipin Staining (Niemann-Pick Type C)
SB (Culture)
Group Tests of Lysosomal Enzymes
For each profile we require a minimum of 10 ml of venous blood in EDTA
Gangliosidosis Profile
Leucocyte β-galactosidase (GM1 Gangliosidosis)
Leucocyte total hexosaminidase (Sandhoff’s disease)
Leucocyte hexosaminidase A (Tay-Sach’s disease).
Hepatosplenomegaly Profile
Leucocyte sphingomyelinase (Niemann-Pick A & B)
Leucocyte β-glucosidase (Gaucher’s disease)
Leucocyte acid esterase (Wolman’s disease & Cholesterol Ester Storage disease)
Plasma chitotriosidase (Non specific screen for some lysosomal disorders such as Gaucher’s
disease and Niemann Pick C– screening test only).
Dysmorphic Profile
(See http://www.metbio.net/metbioGuidelines.asp for guidelines)
Plasma I-cell Screen (Mucolipidosis II & III)
Plasma aspartylglucosaminidase (Aspartyglucosaminuria)
Leucocyte arylsulphatase A (Multiple Sulphatase Deficiency)
Plasma /Leucocyte β-glucuronidase (MPS VII)
Leucocyte β-galactosidase (GM1 gangliosidosis & galactocerebrosidosis)
Plasma/ Leucocyte α- & β-mannosidase (α- & β-mannosidosis)
Plasma/ Leucocyte α-fucosidase (α-fucosidosis)
Leucodystrophy Profile
Leucocyte arylsulphatase A (Metachromatic leucodystrophy)
Leucocyte β-galactocerebrosidase (Krabbe’s leucodystrophy).
Battens Disease Profile
Leucocyte palmitoyl protein thioesterase 1 (CLN1)
Leucocyte tripeptidyl peptidase 1 (CLN2)
Foetal/Neonatal Hydrops Profile
(See http://www.metbio.net/metbioGuidelines.asp for guidelines).
Leucocyte β-glucosidase (Gaucher’s Disease)
Plasma/ Leucocyte β-glucuronidase (MPS VII)
Leucocyte β-galactosidase (GM1 gangliosidosis & galactosialidosis),
Leucocyte sphingomyelinase (Niemann-Pick A & B)
Leucocyte acid esterase (Wolman’s disease & Cholesterol Ester Storage disease)
Leucocyte arylsulphatase A (Metachromatic leucodystrophy)
Plasma chitotriosidase (Non specific screen for some lysosomal disorders such as Gaucher’s
disease and Niemann Pick C – screening test only).
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Non-Lysosomal Enzyme Assays
Leucocytes
Fructose 1,6 Bisphosphatase
Steroid Sulphatase (aryl sulphatase C)
VB (EDTA 10ml)
VB (EDTA 5 ml), SB (Culture)
Plasma
Samples can be sent as whole blood or as separated plasma. Please send via round robin,
courier or taxi. Do not send by first or second-class post.
Biotinidase
VB (Heparin 2ml)
VB (Heparin, EDTA or clotted, 0.5ml)
VP/VS 0.2ml
Chitotriosidase
Red Cells
Samples should be sent as whole blood. Please send via round robin, courier or taxi. Do not
send by first or second-class post.
Glucose-6-Phosphate
Dehydrogenase
VB (1 ml preferably
Heparin but EDTA is
acceptable)
Dihydropteridine
Reductase (DHPR)
2x10mmBS
Galactosaemia
Screen
VB (Heparin
0.5ml),2X10mm BS or
red cells
Tyrosinaemia screen
(PBG synthase
inhibition)
VB (Heparin 0.5ml)
2x10mmBS or red cells.
A blood transfusion within 6
weeks of sampling may
invalidate results
Cultured Fibroblasts
Citrulline Incorporation (Citrullinaemia & Argininosuccinic Aciduria) SB (Culture)
Cystine Incorporation (Cystinosis)
SB (Culture)
Ornithine Incorporation (Hyperornithinaemia with Gyrate Atrophy of
the Retina & Hyperammonaemia with Hyperornithinaemia &
SB (Culture)
Homocitrullinuria)
14
Leucine CO2 Release (Maple Syrup Urine Disease)
SB (Culture)
Fatty Acid (myristate/oleate) Oxidation Flux Assays
SB (Culture)
3
( H Release Assays)
14
Butyrate CO2 Release (Short Chain Fatty Acid Oxidation Defects) SB (Culture)
Isovaleric Acid Incorporation (Isovaleric Acidaemia &
SB (Culture)
Hydroxymethylglutaric Aciduria)
Fumarylacetoacetate Lyase (Tyrosinaemia Type 1)
SB (Culture)
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Tissues
All tissue samples should be collected in liquid nitrogen or dry ice and stored at –800C before
sending to our laboratory. All specimens should be sent frozen in dry ice (not in ice packs).
Please contact the laboratory before sending.
Fructose 1,6 Bisphosphatase
(Fructose Bisphosphatase
Deficiency)
Duodenal Disaccharidases
(Inherited Sucrase and Lactase
Deficiency)
Glycine Cleavage Enzyme (NonKetotic Hyperglycinaemia)
Fumarylacetoacetate Lyase
(Tyrosinaemia Type 1)
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Liver (Two Trucut Wedges) (at least 10 mg)
Duodenal Tissue - at least 10mg
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DNA ANALYSIS
Sample requirement: - 3ml whole blood in EDTA sent at room temperature as whole
blood or extracted DNA. The specimen can be sent by first class post. Analysis can
also be performed on other material eg muscle, saliva, urine - please contact the laboratory
concerning specimen requirements.
Nuclear DNA Encoded Genes
DISORDER
Medium Chain Acyl CoA
Dehydrogenase Deficiency
(MCADD)
Long Chain Acyl CoA
Dehydrogenase Deficiency
(LCHAD)
Galactosaemia
(Classical)
McArdle Disease (Glycogen
storage disease type V)
Hereditary Fructose Intolerance
(HFI)
Myoadenylate Deaminase
Deficiency (adenosine
monophosphate deaminase 1)
Carnitine Palmitoyl Transferase II
Deficiency
Pseudodeficiency State for
Arylsulphatase A
GENE
MUTATION(S)
METHOD
Medium Chain Acyl CoA
Dehydrogenase (ACADM)
c.985A>G (p.Lys329Glu)
PCR/RFLP
c.1528G>C (p.Glu510Gln)
PCR/RFLP
c.563A>G (p.Gln188Arg)
PCR/RFLP
c.148C>T (p.Arg 50X)
c.613G>A (p.Gly205Ser)
PCR/RFLP
Aldolase B (ALDOB)
c.448G>C (p.Ala150Pro)
PCR/RFLP
Myoadenylate Deaminase
(AMPD1)
c.34C>T (p.Gln12X)
Mitochondrial Trifunctional
Protein Alpha Subunit
(HADHA)
Galactose-1-Phosphate Uridyl
Transferase (GALT)
Muscle Glycogen
Phosphorylase (PYGM)
Carnitine Palmitoyl
Transferase 2 (CPT2)
c.338C>T (p.Ser113Leu)
PCR/RFLP
Arylsulphatase A (ARSA)
c.1524+95A>G
PCR/RFLP
Leigh Syndrome (Systemic
Surfeit 1 (SURF1)
Cytochrome Oxidase Deficiency)
Fumarylacetoacetate
hydrolase (FAH)
Glycogen Storage Disease Type I Glucose-6-Phosphate
Transporter (SLC37A4)
Non-A
Glycogen Storage Disease Type Glucose-6-phosphatase
(G6PC)
IA
Tyrosinaemia Type I
Menkes Disease
Non-Ketotic Hyperglycinaemia
(NKH) Glycine encephalopathy
(GCE)
Morquio Syndrome IVA
ATP7A
Glycine decarboxylase
(GLDC) P Protein
c.312_321delTCTGCCAGC
PCR/
C311_312insAT (Common
Fragment Analysis
Mutation)
Full gene sequencing
DNA sequencing
Common mutations
and sequencing.
Common mutations
Targeted mutation analysis
and sequencing.
MLPA for deletions &
Full gene sequencing and
sequencing for point
deletion/duplication analysis
mutations
Targeted mutation analysis
Full gene sequencing and
DNA sequencing and
deletion/duplication analysis MLPA
Amino methyl-transferase
Full gene sequencing
(AMT) Glycine cleavage
analysis
system T Protein (GCST)
Galactosamine-6-sulphatase
c.347G>T (p.Gly116Val)
(GALNS)
Cerebrotendinous xanthomatosis
CYP27A1
(CTX)
Juvenile neuronal ceroid
CLN3
lipofuscinosis (JNCL) Batten
disease type 3
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PCR/RFLP
DNA sequencing
PCR/RFLP
c.1184+1G>A
PCR/RFLP
1.02kb deletion
Deletion breakpoint
PCR
Page 15 of 26
Mitochondrial DNA Encoded Mutations
Mitochondrial DNA (TRNL1) m.3243A>G &
m.3271T>C
PCR/RFLP and ARMS
analysis
Mitochondrial DNA (TRNK) m.8344A>G
PCR/RFLP
Mitochondrial DNA (ATP6) m.8993 T>G/C
PCR/RFLP
Mitochondrial DNA (ATP6) m.9176T>C/G
PCR/RFLP
Mitochondrial DNA (ND5)
m.13513G>A
PCR/RFLP
DEAF (Maternally Inherited Mitochondrial DNA (RNR1)
Antibiotic Induced
Deafness)
LHON (Leber’s Hereditary Mitochondrial DNA (ND1)
Optic Neuropathy)
m.1555A>G
PCR/RFLP
m.11778G>A
PCR/RFLP
m.3460G>A
PCR/RFLP
m.14484T>C
PCR/RFLP
LDYT (Leber’s Hereditary Mitochondrial DNA (ND6)
Optic Neuropathy with
Dystonia & Leigh’s
Syndrome)
Mitochondrial DNA
Kearns-Sayre Syndrome
CPEO (Chronic Progressive
External Ophthalmoplegia)
Pearson Syndrome
m.14459G>A
PCR/RFLP
MELAS (Mitochondrial
Encephalomyopathy, Lactic
Acidosis and Stroke-like
Episodes)
MERRF (Myoclonic
Epilepsy with Ragged Red
Fibres)
NARP (Neurogenic Muscle
Weakness, Ataxia &
Retinitis Pigmentosa)
FBSN (Focal Bilateral
Striatal Necrosis & Leigh
Syndrome)
LS (Leigh’s Syndrome)
Rearrangements Long Range PCR
Southern Blotting
Group Tests of Mitochondrial DNA Mutations
Leber’s Hereditary Optic Atrophy
LHON m.11778G>A
LHON m.3460G>A
LHON m.14484T>C
Mitochondrial DNA Screen
MELAS m.3243A>G
MERRF m.8344A>G
NARP m.8993T>G or C
Leigh’s Syndrome Profile
FBSN m.9176C>T or G
LS m.13513G>A
LDYT m.14459G>A
Long Range PCR for mtDNA Rearrangements
The common mutation in the nuclear encoded SURF1 gene associated with systemic
cytochrome oxidase deficiency.
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CELL CULTURE,TISSUE AND SAMPLE STORAGE
Skin biopsies should be collected in a sterile manner (See Appendix) and transported
unfrozen in tissue culture medium to guarantee to reach us within 24 hours of being taken. A
guideline for sampling and storage is available. Skin biopsies will, in most cases, not be
cultured immediately but will be frozen in cryopreservative medium and stored at –800C. If
culture is required the biopsies can be thawed and in most cases cultured successfully up to
at least 12 months after banking. The cultured fibroblasts grown will be cryopreserved in
liquid nitrogen and stored unless disposal is specifically requested.
Similarly all cultured lymphoblasts, cultured chorionic villus cells and cultured amniotic fluid
cells will be cryopreserved and stored indefinitely. ??
Tissue specimens for eg liver, muscle, duodenum should be collected in small cryotubes
(which can be provided) and ideally, frozen at the bedside in liquid nitrogen or dry ice. If this
is not available the tubes should be put on water ice and immediately transferred to the
coldest freezer available.
Transport to our laboratory can then be arranged at a convenient date. Frozen tissue
samples should be transported on dry ice.
Where tissue specimens are stored a report is issued to this effect.
Extracted DNA is stored indefinitely, unless disposal is specifically requested.
GUIDELINES FOR INVESTIGATION
The following guidelines are available as separate documents. Please contact the Duty
Biochemist.
Investigation of hypoglycaemia.
Investigation of SUDI.
Stroke and Metabolic Disorder.
Hyperammonaemia.
Cardiomyopathy.
Developmental Delay.
Seizures.
Rhabdomyolysis
There is a National Metabolic Biochemistry Network, which provides information about
testing and guidelines for metabolic disorders.
http://www.metbio.net/metbioGuidelines.asp
TEACHING & EDUCATION
We provide in house training for clinical scientists, biomedical scientists, chemical
pathologists and other medical staff on the clinical and laboratory aspects of metabolic
disorders. This is suitable for those studying for the MRCPath and other professional and
academic examinations.
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If you wish one of our team to present a topic at your local Hospital please contact Mrs Mary
Anne Preece.
COSTS
Within the West Midlands the laboratory IMD services are provided as a contract with the
commissioners as part of the specialist services commissioning.
Services not covered by this arrangement are charged for on a cost per test basis. A price
list is available on request
APPENDICES
A.
Guidelines for taking skin biopsies for tissue culture.
B.
Guidelines for emergency specimen collection (inherited metabolic disorder
suspected)
C.
Investigation of Sudden Unexpected Death in Infancy.
D.
Laboratories to which tests are routinely referred.
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APPENDIX A: GUIDELINES ON TECHNIQUE OF SKIN BIOPSY FOR INHERITED
METABOLIC DISORDERS
When a sample of the skin is required for fibroblast culture for investigations for inherited
metabolic disorders, a minor surgical procedure is undertaken in order to obtain a suitable
biopsy sample.1 Skin biopsy taken via a punch device as detailed below is the preferred
method; other methods include a shave biopsy or surgical excision. Under normal
circumstances biopsies should not be taken without prior arrangement with the laboratory:
Department of Newborn Screening and Biochemical Genetics, Birmingham Children's
Hospital, Tel No. 0121 333 9942.
NOTE THAT A SKIN BIOPSY IS OFTEN COLLECTED FOR HISTOLOGICAL ANALYSIS
AND THAT THE SAMPLE HANDLING FOR THIS IS DIFFERENT. PLEASE CONTACT
YOUR LOCAL HISTOPATHOLOGY DEPARTMENT FOR ADVICE.
This procedure may be performed on the Ward, outpatients or in the operating theatre, by a
trained Health care professional (any training should include the practical procedure,
psychological aspects).2,3,4 A local anaesthetic is used when carrying out the procedure,
rarely; oral sedation may also be required.5,6 An aseptic non-touch technique should be
employed throughout the procedure.1,4,7,10 Consent must be obtained following BCH
policy.8,9,11 Ensure the Skin Biopsy Consent form is signed and filed in the patient notes
detailing consent to the procedure, analysis and storage of cells and asking whether
patient/parents agree to subsequent storage of cells for quality control, comparison purposes
and/or research. Information given should include the rationale for the biopsy, procedure
and risks involved, side effects such as healing and scarring and a time line to expected
results.4,12,13 The possibility of contamination or poor growth1 and repeat biopsy should be
discussed where possible. Should a repeat biopsy be required referral to the play specialists
may be appropriate.
Equipment
Plastic apron
Sterile gloves
Sterile dressing pack
25 Fg needle (orange)
23 Fg needle (blue)
2 ml syringe
A labelled skin biopsy cryotube (Obtainable from IMD laboratory Ext 9942)
4mm Punch Device
Lignocaine 1%
Local anaesthetic and appropriate covering/dressing (Ametop gel or Emla cream)
Normal saline sachet
ChloraPrep Sepp (Chlorhexidine 2% in 70% alcohol)
Steristrips
Small Mepore dressing.
Sterile scissors or scalpel blade
Sample requirements
A skin biopsy should be collected using a 4mm punch; smaller diameter punches may give
insufficient sample for reliable culture. The inner sides of the forearm or posterior aspect just
above the elbow are the preferred sites.1
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Technique
Apply local anaesthetic cream or gel to the biopsy site (following consultation with the family
re allergies) and cover with appropriate dressing. Leave for a minimum of 30 minutes.1,5,6,13
Position the child, continue to maintain the dignity of the child whilst placing them in
a comfortable position with the potential biopsy area exposed. Small children/infants
can lie or sit on an adult’s lap. Ensure a young child has his/her favourite cuddly toy
or comforter with him/her throughout.2,13
Prepare equipment and wash hands, put on apron and sterile gloves.4,7,10,14
Remove local anaesthetic cream and dressing and clean any residual cream from site.5
Sterilise the site with ChloraPrep Sepp, wait for the area to dry.7,11
Prepare local anaesthetic withdrawing from ampoule with blue needle, inject lignocane1%
using orange needle. Placement of lignocaine should be intradermally with the majority
subcutaneously such that an area 1.5 x1cm is affected.14
Wait 2-3 minutes.
Clean area vigorously with normal saline using non woven swabs from dressing pack, and
dry.
Pull the skin tight, introduce the punch and rotate 360 degrees with the cutting edge carrying
the punch down onto the tissue (the guard will prevent deep penetration).15
Withdraw the punch whilst applying pressure to site with a non-woven swab (found in
dressing pack).
Remove specimen using sterile forceps and scissors or scalpel. Without touching the
biopsy, transfer immediately into the skin biopsy cryotube. Replace the screw cap.
Apply pressure to wound with non woven swab until cessation of bleeding
Apply Steristrips over wound.4
Cover with Mepore or another suitable dressing.4
Provide family with after care instructions, including analgesic dose should this be
required.4,12,13
Dispose of equipment and waste per BCH policy.14
Send the sample immediately to the laboratory with a fully completed request form4,11 and
the lower part of the Skin Biopsy Consent form.
References
1. Olpin S. What are Biopsies, Cultures and Assays and what information do they provide? CLIMB
update 2008;3:11-14
2. NMC. Professional code of conduct. April 2008.
3. GMC. Good Medical Practice. November 2008; 2(a).
4. BCH. Procedure for the collection, handling and transport of specimens. October 2005.
5. Clarke S, Radford M. Topical anaesthesia for venepuncture. Archives of Disease in
Childhood.1986; 61: 1132-4.
6. BCH. Medicines policy. September 2008.
7. BCH. Guidance on the application of the aseptic technique and the aseptic non-touch technique
when undertaking clinical procedures. March 2007.
8. BCH. Consent, Tissue and Bodily fluids policy. 2006.
9. BCH. Seeking and obtaining consent to treatment, examination and research with Children, Young
People and those with “Parental Responsibility”. February 2006.
10. BCH. Policy for effective and appropriate hand hygiene. October 2006.
11. BCH. Procedure for the Collection handling and transport of specimens. October 2005.
12. GMC. Good Medical Practice. November 2008; 2(b).
13. BCH. Trust policy for the management of invasive/distressing procedures. February 2006.
14. BCH. Policy for the management of risks associated with needle stick injuries and mucous
membrane exposures to blood and body fluids. January 2007
15. Great Ormond Street Hospital. Skin biopsy: punch method clinical guidelines. July 2007.
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APPENDIX B: GUIDELINES FOR EMERGENCY SPECIMEN COLLECTION
(INHERITED METABOLIC DISORDER SUSPECTED)
In life-threatening situations, where an inherited disorder is thought to be likely (either from
family history, results of preliminary investigations or clinical presentation) appropriate
specimens should be collected.
At the earliest opportunity contact the metabolic laboratory duty biochemist (0121 333 9864)
to discuss appropriate investigations. If possible, urine and blood specimens should be
taken before death. Skin and tissue specimens, if not taken pre-mortem, should be taken as
soon as possible after death.
If any of the samples are taken after death it is extremely important to record accurately both
the time of death and when the samples were taken. Appropriate storage as detailed below
is essential.
Local laboratories should make arrangements for suitable storage and transport of tissues to
the specialist laboratory.
URINE
Urine, however little is extremely useful. Ideally 5-10ml should be stored. Collect into a
bottle with no preservative and store deep frozen (-20oC or lower). If the specimen is
contaminated with blood, centrifuge to remove cells before freezing the supernatant.
BLOOD
Collect 5-10ml in lithium heparin and 0.5ml in fluoride oxalate: separate the plasma as soon
as possible and store the plasma deep frozen (-20oC). Store the packed red cells at +4oC
(do not freeze). If DNA analysis is likely to be required, store a further 5-10ml of whole blood
(EDTA) in a plastic tube at +4oC.
SKIN (for fibroblast culture)
Skin taken up to 24 hours after death is likely to be viable provided it is not infected. Take a
skin biopsy and place it in tissue culture medium which is available from us or a suitable
transport medium (obtainable from most Virology or Cytogenetics Departments). In
emergency, sterile isotonic saline can be used, but do not use agar. The specimen should
be stored at +4oC before despatch. Do not freeze. See Appendix A for further
details.Transport to BCH by taxi or hospital transport to reach us within 24hours of collection.
Sterility is of paramount importance when taking skin biopsy specimens, especially at
necropsy. Tissue culture medium and a protocol are available on request.
TISSUE SPECIMENS
(Liver, heart muscle, skeletal muscle)
Please ensure that these specimens are labelled with the type of tissue as well as the patient
details.
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Label the tubes prior to taking the specimens. Biopsies should only be taken if there is a
strong clinical suspicion of a primary defect in one of these tissues. It is very important that
blood and urine specimens are also taken and not just tissue specimens. Necropsy tissue
specimens are suitable usually only for biochemical analysis if taken within two hours of
death.
Two or three needle biopsy specimens of tissue should be taken and placed in a small
plastic tube. Immediately place a small piece of plastic film over the top of the specimen to
prevent the biopsy drying out, cap the tube and snap freeze in liquid nitrogen (or solid CO2)
and store in the coldest freezer available. If liquid nitrogen or solid CO2 are not available
specimens must be stored immediately in the coldest freezer available. Please note that the
collection procedures for tissue for biochemical analyses are not necessarily appropriate for
histological tests and your local Histopathology Department should be contacted if
specimens are required for histological analysis. Please contact the IMD service before
sending samples and transport frozen specimens onsolid CO2 (dry ice)
CEREBROSPINAL FLUID
Sometimes a cerebrospinal fluid specimen may be useful. Collect a 1ml specimen. If blood
stained, centrifuge and separate the supernatant. Store deep frozen.
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APPENDIX C: SUDDEN UNEXPLAINED DEATHS IN INFANCY (SUDI)
SUDI Investigations should be carried out in all children less than 2 years of age who die
suddenly and whose death is unexplained. This covers the investigation for:- Inherited Metabolic Disorders
- Police Purposes (i.e. Forensic Laboratory and Radiology)
in children who die
- at home
- en route/shortly after arrival at hospital
- in hospital (Coroner’s case)
SUMMARY OF SAMPLES REQUIRED
Blood (heart stab) for;
Blood culture (Pedplus/F) – Pedplus/F bottles are designed to take 0.5 – 5.0ml blood.
The greater the sample volume, the more likely that bacteraemia can be detected.
Lithium heparin specimen (at least 0.5ml)
Guthrie card spots – These should ideally be prepared using fresh non-anticoagulated
blood
From the syringe. Place 4 drops of blood directly onto a labelled neonatal screening blood
test card provided in the SUDS kit.
Urine (bladder stab)
Place into a sterile plastic universal container. If urine is unobtainable, but the nappy is wet,
place the nappy in a plastic bag, seal and label.
Naso-pharyngeal swab for virology
The swab should be cut off, using scissors, into a bottle of viral transport medium.
Skin for fibroblast culture.
See the guidelines for skin biopsy protocol (Appendix A)
At BCH, medium for skin biopsies are located in the refrigerator outside the resuscitation
room in the emergency department.
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APPENDIX D: LABORATORIES TO WHICH TESTS ARE ROUTINELY REFERRED
Metabolites and enzymes
Addenbrookes Hospital,
Biochemical Genetics Unit
Box 247
Hills Road
Cambridge, CB2 2QQ
Great Ormond Street Hospital
Department of Chemical Pathology
Great Ormond Street
London
WC1N 3JH
University Hospital of Wales
Department of Medical Biochemistry
Heath Park
Cardiff
CF14 4XW
Sheffield Childrens Hospital
Department of Clinical Chemistry
Western Bank
Sheffield
S10 2TH
Guy's & St Thomas' Hospital, Purine Research Laboratory
4th Floor
North Wing
Westminster Bridge Road
London
SE1 7EH
Neurometabolic Unit
Box 105
National Hospital (UCLH Trust)
Queen Square
London
WC1N 3BG
Southmead Hospital
Department of Clinical Biochemistry
Blood Sciences Laboratories
Southmead Hospital
Bristol
BS10 5NB
University College London Hospitals
60 Whitfield St
London
W1T 4EU
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Bristol Royal Infirmary
Bristol
BS2 8HW
Kings College Hospital
Dept of Clinical Chemistry
Denmark Hill
London
SE5 9RS
Sahlgrenska University Hospital
Clinical Chemistry
Sahlgrenska
Gothenburg
Sweden
Pole Biologie-Pathologie-Pharmacie
Service de Virologie
Bâtiment Jean Dausset - 6 étage, Hopital Cochin
27 rue du Faubourg Saint Jacques
Paris
France
Academisch Medisch Centrum
Laboratorium Genetische Metabole Ziekten
Room FO-132A
Meibergdreef 9
Amsterdam
The Netherlands
Potsdam MVZ GbR
Institute fuer Medizinishe Diagnostik Berlin
Nicolaistrasse 22
Berlin
Germany
Molecular tests
West Midlands Regional Genetics Laboratory
Birmingham Womens Hospital NHS Trust
Metchley Park Road
Edgbaston
Birmingham
B15 2TG
Sheffield Diagnostic Genetics Service (Molecular)
Sheffield Diagnostics Genetics Service
Sheffield Children's NHS Foundation Trust
Western Bank
Sheffield
S10 2TH
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Oxford RGC
Oxford Radcliffe Hospitals NHS Trust
The Churchill
Old Road,
Headington
Oxford
OX3 7LJ
NEWCASTLE MITOCHONDRIAL NSCAG DIAGNOSTIC LABORATORY
Mitochondrial Research Group
4th Floor, The Medical School
Newcastle University,
Framlington Place
Newcastle upon Tyne, NE2 4HH.
West of Scotland Regional Molecular Genetics Laboratory (Glasgow)
West of Scotland Regional Molecular Genetics Laboratory
Duncan Guthrie Institue of Medical Genetics,
Yorkhill Hospital
Dalnair Road
Glasgow
G3 8SJ
London North East Thames RGC (Great Ormond Street Hospital)
NE Thames Regional Molecular Genetics Laboratory,
Great Ormond Street Hospital NHS Trust
Level 6 York House
37 Queen Square
London
WC1N 3BH
DNA Laboratory, GSTS Pathology, Guys Hospital
Genetics Centre
Guys Hospital
London
SE1 9RT
Department of Clinical Genetics - VU University Medical Center (Amsterdam)
Department of Clinical Genetics - VU University Medical Center (Amsterdam)
Laboratorium voor DNA en Eiwitdiagnostiek
De Boelelaan 1117
NL-1081 HV Amsterdam
NETHERLANDS
Yorkshire RGC (Leeds)
Yorkshire Regional DNA Laboratory
Ashley Wing
St James's University Hospital
Leeds
LS9 7TF
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BIRMINGHAM CHILDREN’S HOSPITAL NHS FOUNDATION TRUST PAEDIATRIC LABORATORY MEDICINE

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