Haemoglobin (Hb) the appliance of science

Hb’opathy Screening Pilot

Dr Michael HamonConsultant Haematologist

Derriford Hospital Plymouth

[email protected]


Hb function O2 / CO2 transport

• Globin chains reconfigure according to O2 presence

• central pocket opens with O2 release

β chains move apart 2,3 DPG fits in pocket ’d O2 affinity

These interactions achieve “sigmoid” curve

Hb is packaged in the red blood cell640 x 106 molecules / cellsurvival 120 days


8 µ diameter ;

capillaries 3µ

flexible

biconcave discs


Hb geneticsChromosome 11 β (ε γ δ β)Chromosome 16 α (ζ α α)

Embryonic Gower 1 (ζ2 ε2), Portland (ζ2γ2), Gower 2 (α2ε2)

Fetal HbF (α2γ2)

Adult HbA (α2β2) 96%

HbA2 (α2δ2) ~2-3%, HbF <1%

Hb embryo adult

0

10

20

30

40

50

60

0 20 40 60 80

age (weeks)

% g

lob

in s

ynth

esis

ζ / ε

α

γ

β

δ

Birth20 40

Bone marrowLiverspleen

Yolksac

Site of haemopoiesis

HbF (α2γ2) HbA (α2β2)


• HbF promotes O2 passage across placenta

• Fetus has high Hb (20+ g/dl) successful “parasite”

• Neonatal period

HbF HbA

Left shift;Hb F has ’d O2

affinity


α globin genetics exceptional

x4 α / person ie x2 / chromosome 16;

1-2 deletions benign

• 3 deletions intermittent haemolysis• 4 deletions (hydrops foetalis) death in utero

α+ thal 1 gene present

α0 thal – chr 16 lacks functional α gene


α0 thalassaemia

Far East China

Thailand

Vietnam

Uncommon Greek

Italian

α+ thalassaemia Afro Caribbean

Arab


Initial blood making from 2/40’s onwards

HbF main form after 1st trimester (13/40)

α0α0 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


Hydrops foetalis

’ing anaemia

’ing red cell production

Liver ++ expansion “erythron”

impaired liver function


Hydrops foetalis

severe anaemia heart failure

liver failure low albumen

anasarca – whole body oedema, still birth of macerated fetus

Recurrent fetal loss; 14-18/40’s alpha thal

Hb Bart’s tetramer γ4


• 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/mHPLC High performance Liquid Chromatography


NH2

H

C COOH

R

R = CH2-CH2- COOH glutamic acid (glu)

R = CHCH3

CH3

valine (val)


• HbA HbS β6 glu val;

• with pH (less H+ in vitro) weak acid (glu) more (-ve) e on Hb A cf HbS

• with lower pH glu (COOH) not charged

• HbC β6 glu lys } Hb C / E same position

• HbE β26 glu lys } pH 8.9, separate pH 6.0


Hb electrophoresis

Alkali pH 8.9 Acid pH 6.0

Hb Bart’s

HbA

HbF

HbA2; HbE,HbC

HbS, HbD

Origin

+

-

-

+

OriginHbA, D, E

HbF

HbC

HbS


A

F

S

C

FA A

SC

A/SS/S

HbA2


Hardy Weinberg

Population genetics

p + q = 1; gene frequency p/q

p2 + 2pq + q2 = 1;

(p2 homoz p, 2pq heteroz, q2 homoz q)


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


• Over >10,000 years malaria => life cycle impaired in individuals with heterozygosity for α thal, β thal, HbS, Hb C, HbE, HbD

• Incidence within populations reflects previous malaria (common in Greece / Italy until 100 years ago)


Central Indochina85% either HbE, α thal +/or β thal

Human geneticists using Hardy Weinberg principle estimate where malaria holoendemic breeding advantage for HbAS

“selection factor” HbAA 0.9

HbAS 1.0

HbSS <0.1


α thal Far east, rarely Italy / Greece

β thal Greece, Italy, Ind./ Pak., Far east

Hb S sub Saharan Africa, India, Arab

Hb C Gambia

HbE Far east, Bangladesh

HbD India


HbAS with Plasmodium falciparum 2-8x ’d clearance of parasitised cells (cf HbAA)

Heterozygote at a BIG advantage


α & β Thal


Thalassaemia imbalance of α : β (1:1) ratio

Leads to moderate microcytosis (’d MCV) mimicking Fe deficiency (check ferritin)

αthal MCV ’s with no. of missing genes αNα+ MCV ~70

α0αN or α+α+ MCV ~62

α0α+ MCV ~56αN both α genes normal on chr 16


HbCHbS


HbEHbS

HbD


Natural Selection (Darwinian survival of fittest)

Malarial parasite disadvantaged

Invasion Ovalocytosis (membrane abnormality – Pacific), blood groups

Growth HbS, HbE, α thal, β thal, G6PD-

Release HbCC

All seen where malaria has been for generations

Eg β thal / HbS in Italy / Greece


World wide 150 million β thal carriers

18% Cypriots, 13% Sardinians are β thal carriers

Thalassaemia “blood from the sea” – severe / progressive anaemia

As with all chromosome 11 β globin abnormalities essentially silent until ~6/12’s of age (HbF HbA)


Sickle cell syndromes

SS SC SD SE Sβthal

Progressive haemolytic anaemia (shortened red cell survival) / failure to thrive from 6/12’s age

Thalassaemia “blood from the sea”

β thal major severe anaemia, growth failure

β thal-HbE severe anaemia

α thal as above


Screening

> 30 years UK all neonates PKU, T4-

Effective treatment when found, would be missed without screening

Guthrie card

Both irreversible developmental damage if missed

PKU phenylketonuria, T4- (hypothyroidism)


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) HPLC / Hb electrophoresis

All neonates Hb electrophoresis on Guthrie card (cystic fibrosis to follow)


Family Origin Questionnaire

1 page A4 – midwives capture parental ethnic background(s) to inform screening process

Terminology agreed with Bishop of York

FlowColumn

Sample B : weak Interaction, moves fast

PeakHeight

Retention Time

Assay Principle of G7 (1)

salt concentration

high

Sample Injection Column

Detector

－

－－－

－

low

１ 2 3

The detector only detects the red proteins in the sample by working at 415 and 510nm

Non-porous polymer (TSKgel G7 HSi)

Porous polymer (TSKgel GLYCO HS)

Hb A/S Hb S/S Hb S/C

Hb’opathy Screening Monthly Figures 2004 to 2006.

0

20

40

60

80

100

120

J04

M04

S04

J05

M05

S05

J06

M06

S06

J07

A/N

Total

Pre pilotANC 7 / month

Post pilotANC 60 / month


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


Cyprus p2 +2pq + q2=1

18% heterozygotes; p=0.9

81% normal, 18% carriers, 1% affected

~5% (1:2000) expected β thal majors ie 95% “prevention” – effective public health


β thal ∆ ’d MCV, raised HbA2 (>3.5%) embryo β 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% β thal

∆ diagnosis


Molecular / genetic anthropology

Standard haematology all β thal the same (MCV / HbA2)

PCR recalls migrations across Mediterranean over last 3000 years

β039 west Med (Sardinia / Spain / Portugal)

β+110 east Med (Turks / Cyprus / Lebanon)


β039 Phoenician civilisation 12-11C BC

β+ 110 Greek occupation 8-7 C BCWith 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)


Know ethnic origin test parents

define pcr system termination if +ve

In UK SW peninsula since Jan 2005 40 carriers, mostly Hb S or α thal in >12000 births

In Plymouth 60,000 births 14 years no disease

Beware I’m cynical; Asylum seekers

Documents

Haemoglobin (Hb) the appliance of science