october 2007 final
TRANSCRIPT
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Laboratory Diagnosis of
Haemoglobinopathies
Yvonne Daniel
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Geneti
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Haemoglobin Production during
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Haemoglobin Types and
l in h in m iti n
Haemoglobin F alpha and gamma
Haemoglobin A alpha and beta
Haemoglobin A2 alpha and delta
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At birth cord blood contains approx 70%Hb F
3 months of age approx 20%
6 months of age approx 7.5%
Expected quantities of Hb F in Full Term
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Haemoglobin A - 95%
Haemoglobin A2 - 1.8 3.3%
Haemoglobin F - approx 1%
Adult Haemoglobin
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Categories of
Haemoglobinopathies Heterogenous group of disorders which can be
classified into three main groups
Variant Haemoglobins
Thalassaemia
Hereditary Persistance of Foetal Haemoglobin
In reality form a continuum
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Variant
Usually caused by single point mutations
May result in a change in the electricalcharge
All chains can be affected
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Thalassaemia
Genetic cause may be due to pointmutations or insertion/deletions.
Affects all chains alpha and betasignificant
Fewer chains means there are notenough to combine with the normalchains excess globin causes
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Hereditary Persistance of Foetal
HaemoglobinBenign group of conditions
Synthesis of foetal haemoglobin remainsraisedthroughout life
Molecular mechanism may be deletionalor nondeletional
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Screening Versus
Wilson and Jungner 1986
-knowledge of the disease
-knowledge of the test-Treatment for disease
-Cost considerations
UK National Screening Committee
www.nsc.nhs.uk
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Newborn Screening (UK)Objective detect infants at risk of
sickle cell disorders within the
newborn period, in order to allowearly detection and to improveoutcomes through early treatment
and care.
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Variants to be detected
Sickle Cell Disorders:
Other clinically significant haemoglobins(thalassamia major/intermedia)
Limitations:
Low level of beta chain expression
Blood Transfusion
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Antenatal Screening (UK)Aim offer sickle cell and
thalassaemia screening to all
eligible women and couples in atimely manner in pregnancy.
Two approaches - high prevalence- low prevalence
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Significant maternal
Hb AS
Hb AC
Hb ADPunjab
Hb AE
Hb AOArab
Hb ALepore
Delta beta thalassaemia trait
Beta thalassaemia traitAlphazero thalassaemia trait
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Programme Features
Risk Assessment conditions whichmay be missed
Interpretation and reportingalgorithms / guidelines
Referral guidelines
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Diagnosis - Variant
Protein based HPLC
Electrophoretic
Protein sequencing massspectrometry
Molecular
PCR based techniques
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Beta thalassaemiaRed cell indices
Raised Haemoglobin A2 HPLC,
microcolumn, elution
Globin chain synthesis
Molecular detection of mutation
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Alpha thalassaemia
Red cell indices
Haemoglobin H preparation
Globin chain synthesis
Molecular analysis
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Alpha gene inheritance patterns
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HPFH/delta beta
Red cell indices
Presence of Haemoglobin F quantitate using HPLC or 2 mindenaturation
Molecular analysis
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Full Blood Count/red cellindices
Red Cell Count
Haemoglobin
Mean Cell Volume (MCV)
Mean Cell Haemoglobin (MCH)
(review reference ranges which vary withage and sex (RBC and Hb))
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Blood film showing sickle cells
HPLC
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HPLCHigh Performance Liquid
Chromatography
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Separation by Cation Exchangechromatography:
Hemolysate contains positivelycharged haemoglobins
Functional groups on the resin arenegatively charged
Haemoglobins are separated based
on ionic interaction with resinElution using a continuous buffergradient of increasing ionic strength
Principl
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Application to
Identify variant haemoglobins bychange in electrical charge
Change in gradient of buffer meanshaemoglobins attached to columnwill elute at different times
Enables provisional identification
Quantitates haemoglobins
HPLC Plot
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HPLC Plot -
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HPLC Plot Sickle Cell
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HPLC Plot Hb
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HPLC Plot Hb SC
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HPLC Plot Beta Thal Trait
l l h h i
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HPLC Plot Alpha chain
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HPLC Plot Beta Thal +
HPLC Pl t H l bi
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HPLC Plot Haemoglobin
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confirmatory tests
- Sickle Solubility
-Acid/ Alkali Gels
-Iso electric Focusing
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Sickle Solubilit
Add blood to phosphate buffer containingreducing and lysis agents (saponin and
sodium hydrosulphite)Sickle haemoglobin is induced to sickle(reducing agent) this traps the haemoglobinin the red cells.
Normal haemoglobin is lysed.
Centrifuge for 10 minutes and read results.
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Cannot reliably differentiate between AS, SS, SCor
other sickle haemoglobins.
Other rare sickling haemoglobins existEg. Hb C Harlem, S Antilles, C Ziquinchor
created by having more than one substitution inthebeta chain one of which is the Hb S mutation.
Cannot reliably detected the presence of Hb Sless than15% (not suitable for babies)
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Alkaline ElectrophoresispH 8.2-8.6
Either cellulose acetate or agarose gel.
Separation largely determined by electricalcharge on
Hb molecule at this pH Hb is negativelycharged andPermits provisional id of A, F ,S/G/D, A2/C/E/O-
Arab,
H and many less common variants.
Quantitation can be performed using elution
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S/D/
F
C/E/O/A
Example - Alkali
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Acid Gel Electrophoresis pH 6.0-6.2
Separation depends not only on electricalcharge but also on interaction with variouscomponents of agar and agarose gel.
Separates S and D/G but not most types of Dor G
Also separates C and E and C-Harlem and O-Arab.Haemoglobins A/D/G/E and A2 elute together.
Not suitable as screening technique.
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Example - Acid Gel
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Iso electric focusing
Depends on the fact that the net charge of aproteinis dependent on the pH of the surroundingsolution.
Haemoglobins are separated in the gel on the
Commercially available plates of
polyacrylamide orcellulose acetate contain carrier amphotericmoleculesthat have various pI values thus establishing a
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When a haemolysate is applied in a strong
electricalfield the haemoglobin molecules migratethrough theplate until they reach the point which
More expensive procedure than acid oralkalineelectrophoresis but separates more
variants.
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Iso electric
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Other Protein Based
Haemoglobin Hbodies
Globin ChainSeparation
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Electrospray ionization Mass Spectrometry
Depends on the measurement of the mass tocharge
Initially the mass of the alpha and betachains isassessed in relation to normal, using thisapproachtwo globin chains differing by 6 daltons (Da)can be easily detected
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+Q3: 12 MCA scans from Sample 10 (AA431) of Haem Full scan 2004-Sep-28.wiff (Turbo Spray) Max. 1.1e7 cps.
600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100m/z, amu
1.00e6
2.00e6
3.00e6
4.00e6
5.00e66.00e6
7.00e6
8.00e6
9.00e6
1.00e7
1.10e7I
n
t
e
n
s
it
y
,
c
p
s
890.8
616.3946.5
1009.3992.7841.5
797.01058.8934.3
1081.4
882.51164.41221.3836.3
721.5 1323.1 1375.8
1231.3 1443.3740.0 1513.6688.5 862.3 893.2
951.2 1084.2 1587.71135.9 1268.8787.7 1377.9 1681.6 1763.81499.3703.7 1445.3 1891.71363.7 1983.9682.5 1554.9 1832.1 2061.3
Acq. File: Haem Full scan 2004-Sep-28.wiff Sample Name: AA431
Sample Number: N/A
Mass reconstruct ion of +Q3: 12 MCA scans from Sample 10 (AA431) of Haem Full scan 2004-Sep-28.wiff (Turbo Spray) Max. 8.0e7 cps.
1.50e4 1. 51e4 1. 52e4 1.53e4 1. 54e4 1.55e4 1.56e4 1.57e4 1.58e4 1.59e4 1.60e4 1.61e4 1. 62e4 1.63e4 1.64e4 1.65e4
Mass, amu
1.0e7
2.0e7
3.0e7
4.0e7
5.0e7
6.0e7
7.0e7
8.0e7I
n
t
e
n
s
it
y
,
c
p
s
15126.0
15866.0
15863.0
15147.0 15888.015905.015166.0 16481.015740.0 15993.0
16172.0 16228.015473.015382.0 15784.0 16355.015671.015500.015289.015098.015030.0
Acq. File: Haem Full scan 2004-Sep-28.wiff Sample Name: AA431
Sample Number: N/A
Whole blood scan with deconvolutionalanalysis (Biotools) AA sample
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+Q3: 12 MCA scans from Sample 6 (AS393) of Haem Full scan 2004-Sep-28.wiff (Turbo Spray) Max. 9.7e6 cps.
600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100m/z, amu
1.0e6
2.0e6
3.0e6
4.0e6
5.0e6
6.0e6
7.0e6
8.0e6
9.0e6
9.7e6In
t
e
n
s
i
t
y
,
c
p
s
890.8
616.3 946.5797.2 841.5
1009.3
757.3 992.7 1081.3934.3
882.5721.3 1134.3 1164.41375.8836.2 1221.4
990.8740.0 932.51443.3794.3 1513.71010.8688.7 862.3
1268.8 1587.81031.2 1681.71099.8900.5 1166.2 1312.6911.5 1431.9850.3 1037.4 1891.41763.7714.5 1499.4764.5697.8 1984.3647.5 1831.9 2020.2
Acq. Fil e: Haem Full scan 2 004-Sep-28.w iff Samp le N ame: AS 393
Sample Number: N/A
Mass reconstruct ion of +Q3: 12 MCA scans from Sample 6 (AS393) of Haem Ful l scan 2004-Sep-28.wiff (Turbo Spray) Max. 6.6e7 cps.
1.50e4 1.51e4 1.52e4 1.53e4 1.54e4 1.55e4 1.56e4 1.57e4 1.58e4 1.59e4 1.60e4 1.61e4 1.62e4 1.63e4 1.64e4 1.65e4
Mass, amu
1.0e7
2.0e7
3.0e7
4.0e7
5.0e7
6.0e7
6.6e7In
t
e
n
s
i
t
y
,
c
p
s
15126.0
15866.0
15863.0
15836.0
15147.015888.0 15993.015740.015166.0 16481.016028.0 16228.016213.015471.0 16254.015500.015382.015288.0 15672.015030.0 15116.0 15765.0 16413.0
Acq. Fil e: Haem Full scan 2 004-Sep-28.w iff Samp le N ame: AS 393
Sample Number: N/A
Whole blood scan with deconvolutionalanalysis (Biotools) AS sample
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Sufficient information in an MS scan todifferentiate wild type beta chain from
sickle beta chain not specific
Clinical ProteomicsWhole Blood Scan
Mass change of 30 DaVal GluArg Trp
Thr Met
Gly SerAla Thr
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Clinical proteomicsScreening for clinically significanthaemoglobinopathies by MSMS
AimsHigh sensitivity sickle screeningDetection of other clinically significanthaemoglobinopathies required in
screening programme
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Require:Sensitivity MSMS?
Specificity - Mass resolution required notachievable on whole moleculeTryptic digestionProteomic sequence targeting
Clinical proteomicsScreening for clinically significant
haemoglobinopathies by MSMS
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Tryptic digestion predictable
Human b-globin15 peptides, T1-T15Position of mutations knownSickle mutation in T1
Clinical proteomicsScreening for clinically significant
haemoglobinopathies by MSMS
Clinical proteomics
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Screening for clinically significant haemoglobinopathiesby MSMS
Wild type T1 Isolation
Clinical proteomics
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Screening for clinically significant haemoglobinopathiesby MSMS
Sickle T1 Isolation
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CID of a selected peptide produces a
reproducible peptide ion series, termed y ionsand b ions, as amino acids are removedThe y ion retains a positive charge at the C-terminal end
The b ion retains a positive charge at the N-terminal end
Provides sequence data
Clinical proteomicsScreening for clinically significant
haemoglobinopathies by MSMS
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Clinical proteomicsScreening for clinically significant
haemoglobinopathies by MSMS
Sickle T1fragmentationWild-Type T1fragmentation
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Possible to detect informative sequencesusing specific MRM transitionsProteomic sequence targeting
(Daniel et al, British Journal of Haematology, 2005 130,635-643)
Clinical proteomics
Screening for clinically significanthaemoglobinopathies by MSMS
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ir r l i r r
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time,min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
3.4e4
3.6e4
3.8e40.35
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time,min
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
1.5e4
1.6e4
1.7e4
1.8e4
1.9e4
2.0e4
2.1e4
2.2e4
0.35
ir r l i r r
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time,min
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
1.5e4
1.6e4
1.7e4
1.8e4
1.9e4
2.0e4
2.1e4
0.36
Clinical proteomicsScreening for clinically significant
haemoglobinopathies by MSMS
AA AS SS
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ir r l i r r
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time,min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
3.4e4
3.6e4
3.8e4
0.33
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time,min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.4e4
0.36
ir r l r i r r
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time,min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
6.0e4
6.5e4
7.0e4
7.5e4
8.0e4
8.5e4
9.0e4
9.5e41.0e5
1.1e5
1.1e5
1.2e5
1.2e5
1.3e5
1.3e51.3e5
0.37
Clinical proteomicsScreening for clinically significant
haemoglobinopathies by MSMS
AA AC CC
li i l i
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ir r l i r r
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time,min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
3.4e4
3.6e4
3.8e4
0.33
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time min
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
1.5e4
1.6e4
1.7e4
1.8e4
1.9e4
2.0e4
2.1e4
2.2e4
0.35
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time,min
0.00
5000.00
1.00e4
1.50e4
2.00e4
2.50e4
3.00e4
3.50e4
4.00e4
4.50e4
5.00e4
5.50e4
6.00e4
6.50e4
7.00e4
7.50e4
8.00e4
8.50e4
9.00e4
9.50e4
1.00e5
1.05e5
1.10e5
1.15e5
1.18e5
0.37
Clinical proteomicsScreening for clinically significant
haemoglobinopathies by MSMS
AA AS SC
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Area Ratio vs Index (T1 sickle MRM)
0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850Index
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
Electronic mutation recognitionWith a linear ion trap could trigger product ion scansequence data during MRMSingle-step mutation detection no follow up withmultiplex mutation analysis in 1min
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Thalassaemia
Target delta chain peptides and calculatea delta/delta + beta chain ratio as a surrogate
HbA2for thalassaemia detection
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Mutation Analysis and Gene Sequencing
Knowing the mutation with thalassaemiagivessome ability to predict severity of
phenotype.
Required to diagnose alpha thalassaemia no
diagnostic phenotypic test.
Results must be interpreted in
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PCR
Advantages
DNA from 1 cell
Any source
Qualitatively
Detect any mutation
Faster than othertechniques
More sensitiveEasy to interpret
Cheap
Disadvantages
Sensitive
Conditions worked out
Primer design
Cross reactivity of nontarget DNA
Contamination
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Alpha Thalassaemia Gel
Lane 1 100bp ladderLane 2 2
Lane 3 3.7kb
Lane 4 4.2kbLane 5 20.5kb
Lane 6 FIL
Lane 7 SEA
Lane 8 MEDLane 9 THAI
Lane 10 100bp ladder
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Alpha Thalassaemia Gel
Lane 1 ladder
Lane 2,3&4control
Lane 5,6&7homozygous3.7kb
Lane 8 ladder
Lane 9,10&11heterozygous SEA
Lane 12,13&14heteroz ous FIL
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Sequencing
Highly sensitive diagnostic tool
Accurate method
Specialist equipment
Time consuming
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Sequencing
Primer initiated DNA synthesis-determines direction and sequence
3 hydroxyl groups are required forsynthesis
Dideoxynucleotides terminate synthesis
Fragments separated by size -
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Automated Sequencing
Dideoxynucleotides flourescently tagged
Polymer filled capillary electrophoresis
Fragments migrate in order of size
Pass over laser fluorescent dyes
Emissions collected by scanning assembly
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Sequencing
Computer software converts informationinto a series of peaks
One colour for each base
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Summary
Complex subject - many factors mayinfluence results
No single test can provide a diagnosis
Investigations must be interpreted with
appropriate clinical data