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Hb’opathy Screening Pilot

Hb’opathy Screening Pilot. Dr Michael Hamon Consultant Haematologist Derriford Hospital Plymouth mike.hamon@phnt.swest.nhs.uk. 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

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  1. Hb’opathy Screening Pilot Dr Michael Hamon Consultant Haematologist Derriford Hospital Plymouth mike.hamon@phnt.swest.nhs.uk

  2. 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

  3. Hb is packaged in the red blood cell 640 x 106 molecules / cell survival 120 days

  4. Hb’opathy Screening Pilot 8 m diameter ; capillaries 3m flexible biconcave discs

  5. 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%

  6. Site of haemopoiesis Liver Yolk sac Bone marrow spleen Birth 20 40 HbF (a2g2) HbA (a2b2)

  7. 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

  8. 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

  9. Hb’opathy Screening Pilot a0 thalassaemia Far East China Thailand Vietnam Uncommon Greek Italian a+ thalassaemia Afro Caribbean Arab

  10. 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

  11. Hb’opathy Screening Pilot Hydrops foetalis h’ing anaemia h’ing red cell production Liver g ++ expansion “erythron” g impaired liver function

  12. 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

  13. 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

  14. Hb’opathy Screening Pilot H NH2 C COOH R R = CH2-CH2- COOH glutamic acid (glu) CH3 valine (val) R = CH CH3

  15. 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

  16. 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

  17. Hb’opathy Screening Pilot F A A A F S S C C HbA2 S/S A/S

  18. 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)

  19. Hb’opathy Screening Pilot • Hardy Weinberg defined in a population in equilibrium p2 + 2pq + q2 = 1 provided • Random mating • No migration • No selection

  20. 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)

  21. 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

  22. 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

  23. Hb’opathy Screening Pilot HbAS with Plasmodium falciparum 2-8x h’d clearance of parasitised cells (cf HbAA) Heterozygote at a BIG advantage

  24. Hb’opathy Screening Pilot a & b Thal

  25. 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

  26. Hb’opathy Screening Pilot HbS HbC

  27. Hb’opathy Screening Pilot HbD HbE HbS

  28. 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

  29. 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)

  30. 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

  31. 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)

  32. 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)

  33. 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

  34. Column Flow Sample B : weak Interaction, moves fast Peak Height Retention Time

  35. 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

  36. Non-porous polymer Porous polymer (TSKgel G7 HSi) (TSKgel GLYCO HS)

  37. Hb A/S Hb S/S Hb S/C

  38. Hb’opathy Screening Monthly Figures 2004 to 2006. Pre pilot ANC 7 / month Post pilot ANC 60 / month

  39. 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

  40. 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

  41. 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

  42. 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)

  43. 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)

  44. 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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