Hb’opathy Screening Pilot

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Hbopathy Screening Pilot

• Dr Michael Hamon
• Consultant Haematologist
• Derriford Hospital Plymouth
• mike.hamon_at_phnt.swest.nhs.uk

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Hbopathy Screening Pilot

• Hb function O2 / CO2 transport
• Globin chains reconfigure according to O2
  presence
• central pocket opens with O2 release
• b chains move apart g 2,3 DPG
• fits in pocket g id O2 affinity
• These interactions achieve sigmoid curve

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Hb is packaged in the red blood cell 640 x 106
molecules / cell survival 120 days
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Hbopathy Screening Pilot

• 8 m diameter
• capillaries 3m
• flexible
• biconcave discs

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Hbopathy Screening Pilot

• Hb genetics
• Chromosome 11 b (e g d b)
• Chromosome 16 a (z a a)
• Embryonic Gower 1 (z2 e2), Portland (z2g2),
  Gower 2 (a2e2)
• Fetal HbF (a2g2)
• Adult HbA (a2b2) 96
• HbA2 (a2d2) 2-3, HbF lt1

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Site of haemopoiesis
Liver
Bone marrow
Yolk sac
spleen
Birth
20
40
HbF (a2g2)
HbA (a2b2)
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Hbopathy Screening Pilot

• HbF promotes O2 passage across placenta
• Fetus has high Hb (20 g/dl) successful
  parasite
• Neonatal period
• HbF g HbA

Left shift Hb F has hd O2 affinity
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Hbopathy Screening Pilot

• globin genetics exceptional
• x4 a / person ie x2 / chromosome 16
• 1-2 deletions benign
• 3 deletions intermittent haemolysis
• 4 deletions (hydrops foetalis) death in utero
• a thal 1 gene present
• a0 thal chr 16 lacks functional a gene

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Hbopathy Screening Pilot

• a0 thalassaemia
• Far East China
• Thailand
• Vietnam
• Uncommon Greek
• Italian
• a thalassaemia Afro Caribbean
• Arab

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Hbopathy Screening Pilot

• Initial blood making from 2/40s onwards
• HbF main form after 1st trimester (13/40)
• a0a0 can get by till 14/40s
• Hydrops foetalis alpha thal,
• severe HDN
• in utero blood failure (not fully understood)
• HDN Haemolytic Disease of the Newborn typically
  Rhesus anti D

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Hbopathy Screening Pilot

• Hydrops foetalis
• hing anaemia
• hing red cell production
• Liver g expansion erythron
• g impaired liver function

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Hbopathy Screening Pilot

• Hydrops foetalis
• severe anaemia g heart failure
• liver failure g low albumen
• anasarca whole body oedema, still birth of
  macerated fetus
• Recurrent fetal loss 14-18/40s alpha thal
• Hb Barts tetramer g4

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Hbopathy Screening Pilot

• Hb variants detection is by HPLC (screen) and
  electrophoresis
• Macromolecules separated by electric
• charge (e) / mass (m) (mobility e/m)
• pKa of different amino acids
• pH dependent differences in e/m
• HPLC High performance Liquid Chromatography

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Hbopathy Screening Pilot
H
NH2
C
COOH
R
R CH2-CH2- COOH glutamic acid (glu)
CH3
valine (val)
R CH
CH3
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Hbopathy Screening Pilot

• HbA g HbS b6 glu g val
• with hpH (less H in vitro) weak acid (glu) more
  (-ve) e on Hb A cf HbS
• with lower pH glu (COOH) not charged
• HbC b6 glu g lys Hb C / E same position
• HbE b26 glu g lys pH 8.9, separate pH 6.0

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Hbopathy Screening Pilot
Alkali pH 8.9
Acid pH 6.0
-

Hb Barts
HbF
HbA
HbA, D, E
Origin
HbF
HbS, HbD
HbS
HbA2 HbE,HbC
HbC

Origin
-
Hb electrophoresis
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Hbopathy Screening Pilot
F
A
A
A
F
S
S
C
C
HbA2
S/S
A/S
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Hbopathy Screening Pilot

• Hardy Weinberg
• Population genetics
• p q 1 gene frequency p/q
• p2 2pq q2 1
• (p2 homoz p, 2pq heteroz, q2 homoz q)

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Hbopathy Screening Pilot

• Hardy Weinberg defined in a population in
  equilibrium p2 2pq q2 1 provided
• Random mating
• No migration
• No selection

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Hbopathy Screening Pilot

• Over gt10,000 years malaria gt life cycle impaired
  in individuals with heterozygosity for a thal, b
  thal, HbS, Hb C, HbE, HbD
• Incidence within populations reflects previous
  malaria (common in Greece / Italy until 100 years
  ago)

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Hbopathy Screening Pilot

• Central Indochina
• 85 either HbE, a thal /or b thal
• Human geneticists using Hardy Weinberg principle
  estimate where malaria holoendemic g breeding
  advantage for HbAS
• selection factor HbAA 0.9
• HbAS 1.0
• HbSS lt0.1

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Hbopathy Screening Pilot

• a thal Far east, rarely Italy / Greece
• b thal Greece, Italy, Ind./ Pak., Far east
• Hb S sub Saharan Africa, India, Arab
• Hb C Gambia
• HbE Far east, Bangladesh
• HbD India

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Hbopathy Screening Pilot

• HbAS with Plasmodium falciparum 2-8x hd
  clearance of parasitised cells (cf HbAA)
• Heterozygote at a BIG advantage

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Hbopathy Screening Pilot
a b Thal
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Hbopathy Screening Pilot

• Thalassaemia imbalance of a b (11) ratio
• Leads to moderate microcytosis (id MCV)
  mimicking Fe deficiency (check ferritin)
• athal MCV is with no. of missing genes aNa
  MCV 70
• a0aN or aa MCV 62
• a0a MCV 56
• aN both a genes normal on chr 16

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Hbopathy Screening Pilot
HbS
HbC
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Hbopathy Screening Pilot
HbD
HbE
HbS
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Hbopathy Screening Pilot

• Natural Selection (Darwinian survival of fittest)
• Malarial parasite disadvantaged
• Invasion Ovalocytosis (membrane abnormality
  Pacific), blood groups
• Growth HbS, HbE, a thal, b thal, G6PD-
• Release HbCC
• All seen where malaria has been for generations
• Eg b thal / HbS in Italy / Greece

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Hbopathy Screening Pilot

• World wide 150 million b thal carriers
• 18 Cypriots, 13 Sardinians are b thal carriers
• Thalassaemia blood from the sea severe /
  progressive anaemia
• As with all chromosome 11 b globin abnormalities
  essentially silent until 6/12s of age (HbF g
  HbA)

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Hbopathy Screening Pilot

• Sickle cell syndromes
• SS SC SD SE Sbthal
• Progressive haemolytic anaemia (shortened red
  cell survival) / failure to thrive from 6/12s
  age
• Thalassaemia blood from the sea
• b thal major severe anaemia, growth failure
• b thal-HbE severe anaemia
• a thal as above

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Hbopathy Screening Pilot

• Screening
• gt 30 years UK all neonates PKU, T4-
• Effective treatment when found, would be missed
  without screening
• Guthrie card
• Both g irreversible developmental damage if
  missed
• PKU phenylketonuria, T4- (hypothyroidism)

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Hbopathy Screening Pilot

• 3 years ago NHS plan not much for paediatrics /
  ethnic minority
• Screen for all Hb variants / thalassaemia
• All mothers midwives document ethnic origin both
  parents blood count (esp MCH / ferritin) g HPLC
  / Hb electrophoresis
• All neonates g Hb electrophoresis on Guthrie card
  (cystic fibrosis to follow)

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Hbopathy Screening Pilot

• Family Origin Questionnaire
• 1 page A4 midwives capture parental ethnic
  background(s) to inform screening process
• Terminology agreed with Bishop of York

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Column
Flow
Sample B weak Interaction, moves fast
Peak Height
Retention Time
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Assay Principle of G7 (1)
The detector only detects the red proteins in
the sample by working at 415 and 510nm
Column
Sample Injection
-
-
-
Detector
-
-
1
2
3
low
high
salt concentration
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Hb A/S
Hb S/S
Hb S/C
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Hbopathy Screening Monthly Figures 2004 to 2006.
Pre pilot ANC 7 / month
Post pilot ANC 60 / month
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Hbopathy Screening Pilot

• Effective where high incidence of abnormalities
• Cyprus high awareness of cost
• consumes much of health care budget
• church / state / public motivated
• Pre marital, pre conceptual, early pregnancy

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Hbopathy Screening Pilot

• Cyprus p2 2pq q21
• 18 heterozygotes p0.9
• 81 normal, 18 carriers, 1 affected
• 5 (12000) expected b thal majors ie 95
  prevention effective public health

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Hbopathy Screening Pilot

• b thal D id MCV, raised HbA2 (gt3.5) embryo b
  gene silent
• chorionic villus sampling (cvs) 8-10/40s
• appropriate termination lt13/40s
• Polymerase chain reaction (PCR)
• 54 molecular variants (51 point mutations, 3
  deletions) 99.9 b thal
• D diagnosis

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Hbopathy Screening Pilot

• Molecular / genetic anthropology
• Standard haematology all b thal the same (iMCV /
  h HbA2)
• PCR recalls migrations across Mediterranean over
  last 3000 years
• b039 west Med (Sardinia / Spain / Portugal)
• b110 east Med (Turks / Cyprus / Lebanon)

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Hbopathy Screening Pilot

• b039 Phoenician civilisation 12-11C BC
• b 110 Greek occupation 8-7 C BC
• With any given ethnic origin up to 6 pcr primers
  gt98 detection using cvs within 2-3 days
• Asian Indians
• 5 alleles 90 mutations only 1 shared with
  Med top 6 in Asians molecular homozygosity
  common (consanguinity / distinct to given area)

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Hbopathy Screening Pilot

• Know ethnic origin test parents
• define pcr system termination if ve
• In UK SW peninsula since Jan 2005 40 carriers,
  mostly Hb S or a thal in gt12000 births
• In Plymouth 60,000 births 14 years no disease
• Beware Im cynical Asylum seekers