Hb’opathy Screening Pilot
Dr Michael Hamon
Consultant Haematologist
Derriford Hospital Plymouth
mike.hamon@phnt.swest.nhs.uk
Hb’opathy 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 i’d O2 affinity
These interactions achieve “sigmoid” curve
Hb is packaged in the red blood cell
640 x 106 molecules / cell
survival 120 days
Hb’opathy Screening Pilot
8 m diameter ;
capillaries 3m
flexible
biconcave discs
Hb’opathy 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 <1%
Site of
haemopoiesis
Yolk
sac
Liver
spleen
Bone marrow
Hb embryo g adult
% globin synthesis
Birth
60
HbF (a2g2)
20
HbA (a2b2)
40
50
z/ e
40
a
30
g
20
b
10
d
0
0
20
40
age (weeks)
60
80
Hb’opathy Screening Pilot
100
90
80
70
Left shift;
Hb F has
h’d O2
affinity
60
%
• HbF promotes O2
passage across
placenta
• Fetus has high Hb
(20+ g/dl) successful
“parasite”
• Neonatal period
HbF g HbA
50
40
30
20
10
0
0
20
60
40
mmHg
80
100
Hb’opathy Screening Pilot
a 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
Hb’opathy Screening Pilot
a0 thalassaemia
Far East
Uncommon
a+ thalassaemia
China
Thailand
Vietnam
Greek
Italian
Afro Caribbean
Arab
Hb’opathy Screening Pilot
Initial blood making from 2/40’s onwards
HbF main form after 1st trimester (13/40)
a0a0 can get by till ~14/40’s
Hydrops foetalis
alpha thal,
severe HDN
in utero blood failure (not fully understood)
HDN Haemolytic Disease of the Newborn; typically Rhesus anti D
Hb’opathy Screening Pilot
Hydrops foetalis
h’ing anaemia
h’ing red cell production
Liver g ++ expansion “erythron”
g impaired liver function
Hb’opathy 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; 14-18/40’s alpha thal
Hb Bart’s tetramer g4
Hb’opathy 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
Hb’opathy Screening Pilot
H
NH2
C
COOH
R
R = CH2-CH2- COOH
glutamic acid (glu)
CH3
valine (val)
R = CH
CH3
Hb’opathy 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
• HbE
b6 glu
b26 glu
g lys } Hb C / E same position
g lys } pH 8.9, separate pH 6.0
Hb’opathy Screening Pilot
Alkali pH 8.9
+
Acid pH 6.0
Hb Bart’s
HbF
-
HbA
HbF
HbA, D, E
Origin
HbS, HbD
HbS
HbA2; HbE,HbC
-
Origin
HbC
+
Hb electrophoresis
Hb’opathy Screening Pilot
F
A
F
S
A A
S
C
C
HbA
2
A/S
S/S
Hb’opathy 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)
Hb’opathy Screening Pilot
• Hardy Weinberg defined in a population in
equilibrium p2 + 2pq + q2 = 1 provided
– Random mating
– No migration
– No selection
Hb’opathy Screening Pilot
• Over >10,000 years malaria => 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)
Hb’opathy 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
<0.1
Hb’opathy Screening Pilot
a thal
b thal
Hb S
Hb C
HbE
HbD
Far east, rarely Italy / Greece
Greece, Italy, Ind./ Pak., Far east
sub Saharan Africa, India, Arab
Gambia
Far east, Bangladesh
India
Hb’opathy Screening Pilot
HbAS
with Plasmodium falciparum 2-8x
h’d clearance of parasitised cells (cf
HbAA)
Heterozygote at a BIG advantage
Hb’opathy Screening Pilot
a & b Thal
Hb’opathy Screening Pilot
Thalassaemia imbalance of a : b (1:1) ratio
Leads to moderate microcytosis (i’d MCV)
mimicking Fe deficiency (check ferritin)
athal MCV i’s with no. of missing genes
aNa+
MCV ~70
a0aN or a+a+ MCV ~62
a0a+
MCV ~56
aN both a genes normal on chr 16
Hb’opathy Screening Pilot
HbS
HbC
Hb’opathy Screening Pilot
HbD
HbS
HbE
Hb’opathy 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, G6PDRelease
HbCC
All seen where malaria has been for generations
Eg b thal / HbS in Italy / Greece
Hb’opathy 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
~6/12’s of age (HbF g HbA)
Hb’opathy Screening Pilot
Sickle cell syndromes
SS SC
SD
SE
Sbthal
Progressive haemolytic anaemia (shortened red cell
survival) / failure to thrive from 6/12’s age
Thalassaemia “blood from the sea”
b thal major severe anaemia, growth failure
b thal-HbE severe anaemia
a thal as above
Hb’opathy Screening Pilot
Screening
> 30 years UK all neonates PKU, T4Effective treatment when found, would be
missed without screening
Guthrie card
Both g irreversible developmental damage
if missed
PKU phenylketonuria, T4- (hypothyroidism)
Hb’opathy 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)
Hb’opathy Screening Pilot
Family Origin Questionnaire
1 page A4 – midwives capture parental
ethnic background(s) to inform screening
process
Terminology agreed with Bishop of York
Column
Flow
Sample B : weak
Interaction, moves fast
Peak
Height
Retention Time
Assay Principle of G7 (1)
Sample Injection
Column
－
－
－
－
１
low
2
3
high
salt concentration
The detector only detects the
red proteins in the sample by
working at 415 and 510nm
Detector
－
Non-porous polymer
Porous polymer
(TSKgel G7 HSi)
(TSKgel GLYCO HS)
Hb
A/S
Hb S/S
Hb S/C
Hb’opathy Screening Monthly
Figures 2004 to 2006.
120
100
80
60
A/N
40
Total
20
0
J
M
S
J
M
S
J
M
S
J
04
04
04
05
05
05
06
06
06
07
Pre pilot
ANC 7 / month
Post pilot
ANC 60 / month
Hb’opathy 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
Hb’opathy Screening Pilot
Cyprus p2 +2pq + q2=1
18% heterozygotes; p=0.9
81% normal, 18% carriers, 1% affected
~5% (1:2000) expected b thal majors ie 95%
“prevention” – effective public health
Hb’opathy Screening Pilot
b thal
D i’d MCV, raised HbA2 (>3.5%)
embryo b gene silent
chorionic villus sampling (cvs) ~ 8-10/40’s
“appropriate” termination <13/40’s
Polymerase chain reaction (PCR)
54 molecular variants (51 point mutations,
3 deletions) = 99.9% b thal
D diagnosis
Hb’opathy 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)
b+110 east Med (Turks / Cyprus / Lebanon)
Hb’opathy Screening Pilot
b039 Phoenician civilisation
b+ 110 Greek occupation
12-11C BC
8-7 C BC
With any given ethnic origin up to 6 pcr primers
>98% 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)
Hb’opathy 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 >12000
births
In Plymouth 60,000 births 14 years no disease
Beware
I’m cynical;
Asylum seekers