BHS training course 2017 Other rare red blood cells disorders Béatrice GULBIS, M.D., PhD 1
BHS training course 2017 Objectives of the training course • Rare red blood cells disorders: Inherited, haemolytic (anaemia) – Inheritance and epidemiology – Clinical diagnostics aspects – Useful diagnostic tests, algorithms – Therapeutics 2
Rare or very rare anaemias • About 90 rare types of anaemia have been described • Among them ± 80% are inherited • www.enerca.org • www.orpha.net 3
Rare or very rare anaemias • Haemoglobinopathies (Unstable haemoglobin variant) • Red blood cells membrane defects • Red blood cells enzymes defects • Rare iron metabolism related anaemia • Aplastic anaemia • Acquired haemolytic anaemia and erythropoietic defects 4
Rare or very rare anaemias • Prevalence – How much of condition at a certain period of time • Incidence – Rate of occurrence of new cases in a specified period 5
Rare haemolytic (anaemias) Epidemiology • Few international (European) registries (CDA) • Estimated incidence: C ongenital D yserythropoietic H ereditary A naemia(CDA) S pherocytosis (HS) /10 6 live births 1 2 3 4 5 6 7 10 100 1000 Rare RBC enzymopathies P aroxysmal N octurnal 6 H aemoglobinuria (PNH)
Rare haemolytic (anaemias) Epidemiology in your practice • Age of onset ? PNH Affects particularly young adults HS – CDA – RBC Enzyme disorders – Hbpathies From birth to adulthood 7
Rare haemolytic (anaemias) Epidemiology in your practice • Who is affected? • Influence of ethnic origin – No, but – effect of consanguinity (homogamy) 8
Rare haemolytic (anaemias) Epidemiology in your practice Relationship to Degree relatives Proportion genes each other in common Brothers and sisters, non-identical twins, First 50% parents and children Uncles and aunts, nephews and nieces, Second 25% grandparents and half brothers/sisters First cousins, half-uncles/aunts Third 12.5% half-nephews/nieces 9
Rare haemolytic (anaemias) Epidemiology in your practice • Who is affected? • Gender – No, but – Some X-linked (G6PD) 10
Very rare anaemia Inheritance and epidemiology • Mode of inheritance? 11
Very rare anaemias Inheritance and epidemiology Autosomal dominant inheritance Autosomal recessive inheritance 50% 25% Hereditary spherocytosis Hereditary spherocytosis And other membranopathies CDA III RBC enzymopathies (except G6PD) http://www.genetics.edu.au/ 12 CDA
Very rare anaemias Inheritance and epidemiology X-linked inheritance X-linked inheritance 25% G6PD Rare cases of CDA http://www.genetics.edu.au/ 13
Very rare anaemias Inheritance and epidemiology • X-linked inheritance – Random X chromosome inactivation: • a woman carrier can … Mutated 14
Very rare anaemias Inheritance and epidemiology X-linked inheritance X-linked inheritance 25% This is a girl… X r X r 15 http://www.genetics.edu.au/
Very rare anaemias Inheritance and epidemiology • Hardy-Weinberg law – two alleles 16
Very rare anaemia Inheritance and epidemiology • Examples – Haemophilia A: • Affected males: q= 1/10,000 >>> affected females q²= 1/100.10 6 – G6PD deficiency • Affected males: q= 1/50 >>> affected females q²= 1/2500 17
Rare haemolytic (anaemias) Inheritance • Selection (advantage) • Mutation rate – G6PD deficiency as a life long haemolytic anaemia is rare • Modulator genes – Spectrin alpha Low Expression Prague • … 18
BHS training course 2017 Rare haemolytic (anaemias) • Not only during childhood • If X-linked inheritance, not only boys • If autosomal recessive inheritance, importance of consanguinity (homogamy) 19
BHS training course 2017 Objectives of the training course • Rare red blood cells disorders: Inherited, haemolytic (anaemia) – Inheritance and epidemiology – Clinical diagnostics aspects – Useful diagnostic tests, algorithms – Therapeutics 20
Rare haemolytic (anaemias) In your practice • Only signs and symptoms of anaemia? 21
BHS training course 2017 Clinical diagnostics aspects Extravascular haemolysis +++ Chronic haemolytic (anaemia) of variable severity and/or Splenomegaly and/or Jaundice and/or Biliary lithiasis 22
Clinical diagnostics aspects Extravascular haemolysis +++ Signs and symptoms depend on the degree of decompensation of anaemia – Variable age at diagnosis, but • HS ± 65% with neonatal icterus • Each “stress” on the RBCs = decompensated anaemia – Birth – Infection (i.e. Parvovirus B19) – Pregnancy 23
Rare haemolytic (anaemias) • Only signs and symptoms related to red blood cell pathology? – Congenital dyserythropoietic anaemias 24
Rare chronic haemolytic (anaemias) RBC enzymopathies Acetylcholinesterase Lactate dehydrogenase Monophosphoglycerate mutase Adenosine deaminase Multiple inositol polyphosphate phosphatase Adenylate kinase NADPH diaphorase Aldolase (ALD) Phosphofructokinase (PFK) Bisphosphoglycerate mutase Nucleoside phosphorylase Catalase Phosphoglucomutase NADH-cytochrome b5 reductase 6-Phosphogluconate dehydrogenase Enolase Galactokinase 6-Phosphogluconolactonase Galactose-4-epimerase Phosphoglycerate kinase (PGK) -Glutamylcysteine synthetase Phosphoglycolate phosphatase Glucose phosphate isomerase (GPI) Phosphomannose isomerase Glucose-6-phosphate dehydrogenase Pyrimidine- 5’ -nucleotidase Gluthathione peroxidase Pyruvate kinase Gluthathione reductase Ribosephosphate isomerase Glutathione synthetase Superoxide dismutase Glutathione- S -transferase Transaldolase Glyceraldehyde 3-phosphate dehydrogenase Transketolase Hexokinase Triosephosphate isomerase (TPI) In bold= involved in haemolysis
Non-haematological manifestations Neuromuscular disease – TPI , GPI deficiency Myopathy (myoglobinuria) – PFK , PGK, ALD deficiency Impaired granulocyte function – TPI, GPI deficiency Mental retardation – ALD, PGK deficiency Rhabdomyolysis – ALD deficiency Psychomotor retardation – ALD deficiency Cardiomyopathy – TPI deficiency Increased susceptibility to infection – TPI deficiency .... Van Wijk and van Solinge, Blood 2005
BHS training course 2017 Rare haemolytic (anaemias) • First signs, symptoms: splenomegaly, jaundice, biliary lithiasis • Episode of haemolytic « crisis » (i.e. infection) • Not only haemolysis i.e. TPI deficiency, CDA I 27
BHS training course 2017 Objectives of the training course • Rare red blood cells disorders: Inherited, haemolytic (anaemia) – Inheritance and epidemiology – Clinical diagnostics aspects – Useful diagnostic tests, algorithms – Therapeutics 28
Haemolytic (anaemia) Algorithm Inherited or Erythropoiesis? Haemolysis? RBC indices Immunomediated? Acquired? Reticulocytes (IRF) WBC and Unconj. Bili. platelet counts? Haptoglobin LDH MCV MCH MCHC Coombs (DAT) test Family? Personal history? 29
BHS training course 2017 First Level Laboratory Testing General markers of haemolysis • ↑ Unconjugated bilirubin • ↑ Reticulocytes (absolute values) or unrelated with level of Hb • ↓ Haptoglobin Exclusion of acquired anaemias • Negative DAT Autoimmune haemolytic anaemia • Negative CD55 PNH • Negative CD59 30
BHS training course 2017 Red Blood Cell Morphology Distinct morphological Unremarkable /anisocytosis abnormalities RBC enzyme defect RBC membrane disorders CDA Unstable Hb Ask to the lab: RBC morpholgy 31
BHS training course 2017 RBC enzymopathies A red blood cell enzyme disorder should be assumed in cases of persistent normocytic haemolytic anaemia in which hemoglobin abnormalities and antiglobulin reactions have been excluded, spherocytes are absent, and osmotic fragility is normal RBC Enzyme activity DNA analysis 32
CDAs Retic. 700 10³ 600 500 400 Ineffective erythropoiesis 300 (reticulocytes < 150,000/mm³) 200 100 0 0 5 10 15 Hb g/dL Bone marrow C D A DNA analysis II Electrophoresis RBC 33 membrane proteins
BHS training course 2017 Unstable haemoglobin variant • Destabilization of Hb tetramers Heinz bodies Stability tests Isopropanol /heat Separation of Hb fractions/ DNA 34
BHS training course 2017 Red Blood Cell membrane disorders RBC Morphology Distinct morphological abnormalities Elliptocytes Elliptocytes Spherocytes Stomatocytes /Poikilocytes - Not always high % of abnormal cells - Spherocytes: AHAI, G6PD deficiency haemolytic crisis, etc. 35
BHS training course 2017 Hereditary spherocytosis (HS) and misdiagnosis of “ Stomatocytosis ” MCV MCHC Osmotic EMA Ektacytometry fragility binding test N or N or Hereditary (+*) Altered osmotic spherocytosis gradient curve Dehydrated N or / (-) Left-shifted osmotic ↑ Stomatocytosis gradient curve Overhydrated / (-) Right-shifted osmotic ↑↑ ↓ Stomatocytosis gradient curve * Might be negative DNA analysis 36
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