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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

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

slide3

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%

slide6

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

slide34

Column

Flow

Sample B : weak

Interaction, moves fast

Peak

Height

Retention Time

slide35

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

slide36

Non-porous polymer

Porous polymer

(TSKgel G7 HSi)

(TSKgel GLYCO HS)

slide39

Hb A/S

Hb S/S

Hb S/C

hb opathy screening monthly figures 2004 to 2006
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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