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Hb’opathy Screening Pilot. Dr Michael Hamon Consultant Haematologist Derriford Hospital Plymouth [email protected] Hb’opathy Screening Pilot. Hb function O 2 / CO 2 transport Globin chains reconfigure according to O 2 presence central pocket opens with O 2 release

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Hb’opathy Screening Pilot

Dr Michael Hamon

Consultant Haematologist

Derriford Hospital Plymouth

[email protected]


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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


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Hb is packaged in the red blood cell

640 x 106 molecules / cell

survival 120 days


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Hb’opathy Screening Pilot

8 m diameter ;

capillaries 3m

flexible

biconcave discs


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Hb’opathy Screening Pilot

Hb genetics

Chromosome 11 b (e g d b)

Chromosome 16 a (z a a)

Embryonic Gower 1 (z2e2), Portland (z2g2), Gower 2 (a2e2)

Fetal HbF (a2g2)

Adult HbA (a2b2) 96%

HbA2 (a2d2) ~2-3%, HbF <1%


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Site of

haemopoiesis

Liver

Yolk

sac

Bone marrow

spleen

Birth

20

40

HbF (a2g2)

HbA (a2b2)


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Hb’opathy 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

h’d O2

affinity


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Hb’opathy 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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Hb’opathy Screening Pilot

a0 thalassaemia

Far East China

Thailand

Vietnam

Uncommon Greek

Italian

a+ thalassaemia Afro Caribbean

Arab


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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


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Hb’opathy Screening Pilot

Hydrops foetalis

h’ing anaemia

h’ing red cell production

Liver g ++ expansion “erythron”

g impaired liver function


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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


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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


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Hb’opathy Screening Pilot

H

NH2

C

COOH

R

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

CH3

valine (val)

R = CH

CH3


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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 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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Hb’opathy Screening Pilot

Alkali pH 8.9

Acid pH 6.0

-

+

Hb Bart’s

HbF

HbA

HbA, D, E

Origin

HbF

HbS, HbD

HbS

HbA2; HbE,HbC

HbC

+

Origin

-

Hb electrophoresis


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Hb’opathy Screening Pilot

F

A

A

A

F

S

S

C

C

HbA2

S/S

A/S


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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)


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Hb’opathy 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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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 previousmalaria (common in Greece / Italy until 100 years ago)


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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


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Hb’opathy 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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Hb’opathy Screening Pilot

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

Heterozygote at a BIG advantage



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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




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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, G6PD-

Release HbCC

All seen where malaria has been for generations

Eg b thal / HbS in Italy / Greece


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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)


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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


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Hb’opathy Screening Pilot

Screening

> 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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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 neonatesg Hb electrophoresis on Guthrie card (cystic fibrosis to follow)


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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


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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

2

3

low

high

salt concentration


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Non-porous polymer

Porous polymer

(TSKgel G7 HSi)

(TSKgel GLYCO HS)


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Hb A/S

Hb S/S

Hb S/C


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Hb’opathy Screening Monthly Figures 2004 to 2006.

Pre pilot

ANC 7 / month

Post pilot

ANC 60 / month


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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


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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


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Hb’opathy Screening Pilot

b thal Di’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


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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)


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Hb’opathy 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 >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)


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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


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