October 2007 Final

8/14/2019 October 2007 Final

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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 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




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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



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



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



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

October 2007 Final

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