Pathologies constitutionnelles de la membrane érythrocytaire
Loïc Garçon
La déformabilité : qualité essen4elle
Produc'on énergé'que
Viscosité interne du globule rouge
Viscoélasticité membranaire
Chen L and Weiss L, Blood, 1973, 41 (4)
Ratio Surface/volume
Le réseau protéique sous-membranaire
• Recouvre 65% de la surface cytoplasmique de ladouble couche lipidique
• Est relié à la double couche lipidique par descomplexes protéiques transmembranaires
• Structure complexe multiprotéique composée deprotéines transmembranaires et de protéines sous-membranaires
Bijleveld R et al , NJM, 2015
Les pathologies membranaires cons2tu2onnelles
• Altération des interactions horizontales: doncde l’élasticité érythrocytaire: Elliptocytoseshéréditaires
• Altération des interactions verticales, donc dela STABILITE membranaire: diminution durapport surface/volume: sphérocytoseshéréditaires
• Altération de l’hydratation érythrocytaire:Stomatocytoses héréditaires (hyperhydratéeset deshydratées)
- 1/2000 naissances en Europe et Amérique du Nord
- 1% des explorations d’INN (V. Saada et al, Pediatr Hematol Oncol. 2006)
- Dominant 75% des cas
AnkyrineGène ANK1 (8p)
40-65% population caucasienneDominant (80%) ou de novo (20%)
Déficit combiné en spectrine
Bande 3Gène SLC4AE1(17q)
30% population caucasienneDominant
Spectrine βGène SPTB(14q)
20% population caucasienneDominant ou de novo
Spectrine αGène SPTA(1q)
5% population caucasienneRécessive, sévère
Peut-être associée à des polymorphismes α-LEPRA
Protéine 4.2Gène EPB42
50 % au JaponRécessive
Perro]a S et al., Lancet, 2008, 372:1411-26
Anomalies des interactions verticales: sphérocytose héréditaire
Seminar
www.thelancet.com Vol 372 October 18, 2008 1413
apparent in the homozygous or compound heterozygous state. These patients have severe disease. Homozygous and compound heterozygous defects have been associated with null mutations and variants are associated with low-expression alleles.33–36 For example, the α-LEPRA (low-expression allele Prague) allele produces about six-fold less of the correctly spliced α-spectrin transcript than does the healthy allele.33 The presence of two null α-spectrin alleles is speculated to be lethal. Blood smears from patients with severe α-spectrin defi ciency contain many microspherocytes, contracted erythrocytes, and abnormal poikilocytes (table 2).24,33
The biochemical phenotype of combined spectrin and ankyrin defi ciency is the most common abnormality noted in about 40–65% of patients with hereditary spherocytosis in northern European populations,36–38 but in only 5–10% of cases in Japan (table 2).12 Patients with ankyrin defects have prominent spherocytosis without other morphological abnormalities (fi gure 3). Ankyrin mutations (OMIM +182900) cause both dominant and recessive disease that can range from clinically mild to severe.1,20,25,38–41 About 15–20% of ankyrin-1 (ANK1) gene mutations reported are de novo;25,32,38,42 recurrent mutations have been described.43
Mutations—eg, nucleotide −108T to C and −153G to A, and deletion of nucleotides −72/73 of the ankyrin promoter, leading to decreased ankyrin-1 expression have been identifi ed in some patients.44,45 A few patients with atypical hereditary spherocytosis associated with karyo-typic abnormalities—ie, deletions or translocations of the ankyrin gene locus on chromosome 8p—have been
described.46,47 ANK1 gene deletion might be part of a contiguous gene syndrome with manifestations of spherocytosis, mental retardation, typical faces, and hypogonadism.
Ankyrin-1 plays a pivotal role in the stabilisation of the membrane, providing the main membrane binding site for the spectrin-based membrane skeleton. Since it links β spectrin to band 3, ankyrin defi ciency leads to a proportional reduction in spectrin assembly on the membrane despite normal spectrin synthesis.48 The defi ciency of one protein is strictly associated with the defi ciency of the other and the extent of protein defi ciency is related to the clinical severity. A high reticulocyte count might mask a reduction in ankyrin-1 in biochemical studies.49
Defi ciency of band-3 protein arises in about 33% of patients, presenting with mild to moderate, dominantly inherited disease (table 2). Mushroom-shaped or pincered erythrocytes might be seen on peripheral blood smear (fi gure 3).1,50 SDS-PAGE analysis shows a reduction in band 3 in about 20–30% of patients, as the wild-type allele in trans partly compensates for the mutated allele.51 Erythrocyte membranes from these patients also have defi ciency of protein 4.2.52 A range of band-3 mutations (OMIM+109270) associated with hereditary spherocytosis have been reported; these mutations are spread out throughout the band-3 (SLC4A1) gene.1,20,25,53–55
Alleles that aff ect band-3 expression when inherited in trans to a band-3 mutation, aggravate band-3 defi ciency and worsen clinical severity.56–58 Severe disease has been reported in patients who are compound heterozygotes or homozygotes for band-3 defects.59–62
Release of microvesiclesHaemolysis
Erythrostasis
Low pHHigh macrophagecontact
Low glucoseconcentrationHigh oxidantsconcentration
Reducedcellular deformability
Splenictrapping
Further lossof membrane
Tail of osmoticfragility curve
Splenicconditioning
Reducedsurface-to-volume ratio
Release of microvesicles
Band-3 deficiency
Spectrin, ankyrin, orprotein 4.2 deficiency
Lipid bilayerSpectrinAnkyrinBand 3
Figure 2: Pathophysiological eff ects of hereditary spherocytosisModifi ed from Gallagher and colleagues21 with permission.
Seminar
www.thelancet.com Vol 372 October 18, 2008 1413
apparent in the homozygous or compound heterozygous state. These patients have severe disease. Homozygous and compound heterozygous defects have been associated with null mutations and variants are associated with low-expression alleles.33–36 For example, the α-LEPRA (low-expression allele Prague) allele produces about six-fold less of the correctly spliced α-spectrin transcript than does the healthy allele.33 The presence of two null α-spectrin alleles is speculated to be lethal. Blood smears from patients with severe α-spectrin defi ciency contain many microspherocytes, contracted erythrocytes, and abnormal poikilocytes (table 2).24,33
The biochemical phenotype of combined spectrin and ankyrin defi ciency is the most common abnormality noted in about 40–65% of patients with hereditary spherocytosis in northern European populations,36–38 but in only 5–10% of cases in Japan (table 2).12 Patients with ankyrin defects have prominent spherocytosis without other morphological abnormalities (fi gure 3). Ankyrin mutations (OMIM +182900) cause both dominant and recessive disease that can range from clinically mild to severe.1,20,25,38–41 About 15–20% of ankyrin-1 (ANK1) gene mutations reported are de novo;25,32,38,42 recurrent mutations have been described.43
Mutations—eg, nucleotide −108T to C and −153G to A, and deletion of nucleotides −72/73 of the ankyrin promoter, leading to decreased ankyrin-1 expression have been identifi ed in some patients.44,45 A few patients with atypical hereditary spherocytosis associated with karyo-typic abnormalities—ie, deletions or translocations of the ankyrin gene locus on chromosome 8p—have been
described.46,47 ANK1 gene deletion might be part of a contiguous gene syndrome with manifestations of spherocytosis, mental retardation, typical faces, and hypogonadism.
Ankyrin-1 plays a pivotal role in the stabilisation of the membrane, providing the main membrane binding site for the spectrin-based membrane skeleton. Since it links β spectrin to band 3, ankyrin defi ciency leads to a proportional reduction in spectrin assembly on the membrane despite normal spectrin synthesis.48 The defi ciency of one protein is strictly associated with the defi ciency of the other and the extent of protein defi ciency is related to the clinical severity. A high reticulocyte count might mask a reduction in ankyrin-1 in biochemical studies.49
Defi ciency of band-3 protein arises in about 33% of patients, presenting with mild to moderate, dominantly inherited disease (table 2). Mushroom-shaped or pincered erythrocytes might be seen on peripheral blood smear (fi gure 3).1,50 SDS-PAGE analysis shows a reduction in band 3 in about 20–30% of patients, as the wild-type allele in trans partly compensates for the mutated allele.51 Erythrocyte membranes from these patients also have defi ciency of protein 4.2.52 A range of band-3 mutations (OMIM+109270) associated with hereditary spherocytosis have been reported; these mutations are spread out throughout the band-3 (SLC4A1) gene.1,20,25,53–55
Alleles that aff ect band-3 expression when inherited in trans to a band-3 mutation, aggravate band-3 defi ciency and worsen clinical severity.56–58 Severe disease has been reported in patients who are compound heterozygotes or homozygotes for band-3 defects.59–62
Release of microvesiclesHaemolysis
Erythrostasis
Low pHHigh macrophagecontact
Low glucoseconcentrationHigh oxidantsconcentration
Reducedcellular deformability
Splenictrapping
Further lossof membrane
Tail of osmoticfragility curve
Splenicconditioning
Reducedsurface-to-volume ratio
Release of microvesicles
Band-3 deficiency
Spectrin, ankyrin, orprotein 4.2 deficiency
Lipid bilayerSpectrinAnkyrinBand 3
Figure 2: Pathophysiological eff ects of hereditary spherocytosisModifi ed from Gallagher and colleagues21 with permission.
Défaut d’interactions verticales membrane - cytosquelette
Perte de surface, volume constantDiminution ratio S/V
è Sphérisation du GRMoindre déformabilité
Seminar
www.thelancet.com Vol 372 October 18, 2008 1413
apparent in the homozygous or compound heterozygous state. These patients have severe disease. Homozygous and compound heterozygous defects have been associated with null mutations and variants are associated with low-expression alleles.33–36 For example, the α-LEPRA (low-expression allele Prague) allele produces about six-fold less of the correctly spliced α-spectrin transcript than does the healthy allele.33 The presence of two null α-spectrin alleles is speculated to be lethal. Blood smears from patients with severe α-spectrin defi ciency contain many microspherocytes, contracted erythrocytes, and abnormal poikilocytes (table 2).24,33
The biochemical phenotype of combined spectrin and ankyrin defi ciency is the most common abnormality noted in about 40–65% of patients with hereditary spherocytosis in northern European populations,36–38 but in only 5–10% of cases in Japan (table 2).12 Patients with ankyrin defects have prominent spherocytosis without other morphological abnormalities (fi gure 3). Ankyrin mutations (OMIM +182900) cause both dominant and recessive disease that can range from clinically mild to severe.1,20,25,38–41 About 15–20% of ankyrin-1 (ANK1) gene mutations reported are de novo;25,32,38,42 recurrent mutations have been described.43
Mutations—eg, nucleotide −108T to C and −153G to A, and deletion of nucleotides −72/73 of the ankyrin promoter, leading to decreased ankyrin-1 expression have been identifi ed in some patients.44,45 A few patients with atypical hereditary spherocytosis associated with karyo-typic abnormalities—ie, deletions or translocations of the ankyrin gene locus on chromosome 8p—have been
described.46,47 ANK1 gene deletion might be part of a contiguous gene syndrome with manifestations of spherocytosis, mental retardation, typical faces, and hypogonadism.
Ankyrin-1 plays a pivotal role in the stabilisation of the membrane, providing the main membrane binding site for the spectrin-based membrane skeleton. Since it links β spectrin to band 3, ankyrin defi ciency leads to a proportional reduction in spectrin assembly on the membrane despite normal spectrin synthesis.48 The defi ciency of one protein is strictly associated with the defi ciency of the other and the extent of protein defi ciency is related to the clinical severity. A high reticulocyte count might mask a reduction in ankyrin-1 in biochemical studies.49
Defi ciency of band-3 protein arises in about 33% of patients, presenting with mild to moderate, dominantly inherited disease (table 2). Mushroom-shaped or pincered erythrocytes might be seen on peripheral blood smear (fi gure 3).1,50 SDS-PAGE analysis shows a reduction in band 3 in about 20–30% of patients, as the wild-type allele in trans partly compensates for the mutated allele.51 Erythrocyte membranes from these patients also have defi ciency of protein 4.2.52 A range of band-3 mutations (OMIM+109270) associated with hereditary spherocytosis have been reported; these mutations are spread out throughout the band-3 (SLC4A1) gene.1,20,25,53–55
Alleles that aff ect band-3 expression when inherited in trans to a band-3 mutation, aggravate band-3 defi ciency and worsen clinical severity.56–58 Severe disease has been reported in patients who are compound heterozygotes or homozygotes for band-3 defects.59–62
Release of microvesiclesHaemolysis
Erythrostasis
Low pHHigh macrophagecontact
Low glucoseconcentrationHigh oxidantsconcentration
Reducedcellular deformability
Splenictrapping
Further lossof membrane
Tail of osmoticfragility curve
Splenicconditioning
Reducedsurface-to-volume ratio
Release of microvesicles
Band-3 deficiency
Spectrin, ankyrin, orprotein 4.2 deficiency
Lipid bilayerSpectrinAnkyrinBand 3
Figure 2: Pathophysiological eff ects of hereditary spherocytosisModifi ed from Gallagher and colleagues21 with permission.
Physiopathologie
Déficit en Bande 3
Déficit en Spectrine, Ankyrine ou 4.2
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www.thelancet.com Vol 372 October 18, 2008 1413
apparent in the homozygous or compound heterozygous state. These patients have severe disease. Homozygous and compound heterozygous defects have been associated with null mutations and variants are associated with low-expression alleles.33–36 For example, the α-LEPRA (low-expression allele Prague) allele produces about six-fold less of the correctly spliced α-spectrin transcript than does the healthy allele.33 The presence of two null α-spectrin alleles is speculated to be lethal. Blood smears from patients with severe α-spectrin defi ciency contain many microspherocytes, contracted erythrocytes, and abnormal poikilocytes (table 2).24,33
The biochemical phenotype of combined spectrin and ankyrin defi ciency is the most common abnormality noted in about 40–65% of patients with hereditary spherocytosis in northern European populations,36–38 but in only 5–10% of cases in Japan (table 2).12 Patients with ankyrin defects have prominent spherocytosis without other morphological abnormalities (fi gure 3). Ankyrin mutations (OMIM +182900) cause both dominant and recessive disease that can range from clinically mild to severe.1,20,25,38–41 About 15–20% of ankyrin-1 (ANK1) gene mutations reported are de novo;25,32,38,42 recurrent mutations have been described.43
Mutations—eg, nucleotide −108T to C and −153G to A, and deletion of nucleotides −72/73 of the ankyrin promoter, leading to decreased ankyrin-1 expression have been identifi ed in some patients.44,45 A few patients with atypical hereditary spherocytosis associated with karyo-typic abnormalities—ie, deletions or translocations of the ankyrin gene locus on chromosome 8p—have been
described.46,47 ANK1 gene deletion might be part of a contiguous gene syndrome with manifestations of spherocytosis, mental retardation, typical faces, and hypogonadism.
Ankyrin-1 plays a pivotal role in the stabilisation of the membrane, providing the main membrane binding site for the spectrin-based membrane skeleton. Since it links β spectrin to band 3, ankyrin defi ciency leads to a proportional reduction in spectrin assembly on the membrane despite normal spectrin synthesis.48 The defi ciency of one protein is strictly associated with the defi ciency of the other and the extent of protein defi ciency is related to the clinical severity. A high reticulocyte count might mask a reduction in ankyrin-1 in biochemical studies.49
Defi ciency of band-3 protein arises in about 33% of patients, presenting with mild to moderate, dominantly inherited disease (table 2). Mushroom-shaped or pincered erythrocytes might be seen on peripheral blood smear (fi gure 3).1,50 SDS-PAGE analysis shows a reduction in band 3 in about 20–30% of patients, as the wild-type allele in trans partly compensates for the mutated allele.51 Erythrocyte membranes from these patients also have defi ciency of protein 4.2.52 A range of band-3 mutations (OMIM+109270) associated with hereditary spherocytosis have been reported; these mutations are spread out throughout the band-3 (SLC4A1) gene.1,20,25,53–55
Alleles that aff ect band-3 expression when inherited in trans to a band-3 mutation, aggravate band-3 defi ciency and worsen clinical severity.56–58 Severe disease has been reported in patients who are compound heterozygotes or homozygotes for band-3 defects.59–62
Release of microvesiclesHaemolysis
Erythrostasis
Low pHHigh macrophagecontact
Low glucoseconcentrationHigh oxidantsconcentration
Reducedcellular deformability
Splenictrapping
Further lossof membrane
Tail of osmoticfragility curve
Splenicconditioning
Reducedsurface-to-volume ratio
Release of microvesicles
Band-3 deficiency
Spectrin, ankyrin, orprotein 4.2 deficiency
Lipid bilayerSpectrinAnkyrinBand 3
Figure 2: Pathophysiological eff ects of hereditary spherocytosisModifi ed from Gallagher and colleagues21 with permission.
Déstabilisation de la bi-couche lipidiqueVésiculation de la membrane
D’après S. Pero.a, Lancet 2008
Liu, Derick, Agre and Palek, 1990
D’après S. Perotta, Lancet 2008
Séquestration cordons spléniques
ê pH
êGlucoseêATP
é contact avec macrophages
é Oxydants
Seminar
www.thelancet.com Vol 372 October 18, 2008 1413
apparent in the homozygous or compound heterozygous state. These patients have severe disease. Homozygous and compound heterozygous defects have been associated with null mutations and variants are associated with low-expression alleles.33–36 For example, the α-LEPRA (low-expression allele Prague) allele produces about six-fold less of the correctly spliced α-spectrin transcript than does the healthy allele.33 The presence of two null α-spectrin alleles is speculated to be lethal. Blood smears from patients with severe α-spectrin defi ciency contain many microspherocytes, contracted erythrocytes, and abnormal poikilocytes (table 2).24,33
The biochemical phenotype of combined spectrin and ankyrin defi ciency is the most common abnormality noted in about 40–65% of patients with hereditary spherocytosis in northern European populations,36–38 but in only 5–10% of cases in Japan (table 2).12 Patients with ankyrin defects have prominent spherocytosis without other morphological abnormalities (fi gure 3). Ankyrin mutations (OMIM +182900) cause both dominant and recessive disease that can range from clinically mild to severe.1,20,25,38–41 About 15–20% of ankyrin-1 (ANK1) gene mutations reported are de novo;25,32,38,42 recurrent mutations have been described.43
Mutations—eg, nucleotide −108T to C and −153G to A, and deletion of nucleotides −72/73 of the ankyrin promoter, leading to decreased ankyrin-1 expression have been identifi ed in some patients.44,45 A few patients with atypical hereditary spherocytosis associated with karyo-typic abnormalities—ie, deletions or translocations of the ankyrin gene locus on chromosome 8p—have been
described.46,47 ANK1 gene deletion might be part of a contiguous gene syndrome with manifestations of spherocytosis, mental retardation, typical faces, and hypogonadism.
Ankyrin-1 plays a pivotal role in the stabilisation of the membrane, providing the main membrane binding site for the spectrin-based membrane skeleton. Since it links β spectrin to band 3, ankyrin defi ciency leads to a proportional reduction in spectrin assembly on the membrane despite normal spectrin synthesis.48 The defi ciency of one protein is strictly associated with the defi ciency of the other and the extent of protein defi ciency is related to the clinical severity. A high reticulocyte count might mask a reduction in ankyrin-1 in biochemical studies.49
Defi ciency of band-3 protein arises in about 33% of patients, presenting with mild to moderate, dominantly inherited disease (table 2). Mushroom-shaped or pincered erythrocytes might be seen on peripheral blood smear (fi gure 3).1,50 SDS-PAGE analysis shows a reduction in band 3 in about 20–30% of patients, as the wild-type allele in trans partly compensates for the mutated allele.51 Erythrocyte membranes from these patients also have defi ciency of protein 4.2.52 A range of band-3 mutations (OMIM+109270) associated with hereditary spherocytosis have been reported; these mutations are spread out throughout the band-3 (SLC4A1) gene.1,20,25,53–55
Alleles that aff ect band-3 expression when inherited in trans to a band-3 mutation, aggravate band-3 defi ciency and worsen clinical severity.56–58 Severe disease has been reported in patients who are compound heterozygotes or homozygotes for band-3 defects.59–62
Release of microvesiclesHaemolysis
Erythrostasis
Low pHHigh macrophagecontact
Low glucoseconcentrationHigh oxidantsconcentration
Reducedcellular deformability
Splenictrapping
Further lossof membrane
Tail of osmoticfragility curve
Splenicconditioning
Reducedsurface-to-volume ratio
Release of microvesicles
Band-3 deficiency
Spectrin, ankyrin, orprotein 4.2 deficiency
Lipid bilayerSpectrinAnkyrinBand 3
Figure 2: Pathophysiological eff ects of hereditary spherocytosisModifi ed from Gallagher and colleagues21 with permission.
« Conditionnement splénique »
Molnar Z. and Rappaport H, Blood, 1972, 39 (81-98)
α-spectrine
Bande 3 formes hétérozygotes formes homozygotes
Ankyrine
β-spectrine
D’après S. Perotta, Lancet 2008
Hb N > 8 g/dl 6 – 8 g/dl < 6 g/dl
Réticulocytes < 6 % > 6 % > 10 %
Bilirubine 17 - 34 μmol/l 34-51 μmol/l > 51 μmol/l
Frottis sanguin Sphérocytes Microsphérocytes
et poikilocytose
Transfusions 0-1 2 >2 Dépendance transfusionnelle
Transmission AD AD, de novo AR
Mineures30%
Modérées60-70%
Modérément sévères 10%
Sévères3-5%
Sévérité et fréquence
Sphérocytose HéréditaireFormes particulières
- Formes néonatales
-Formes sévères-Bande 3 homozygote
- Mutations nulles- Parfois associées à des acidoses tubulaires distales
-Formes récessives- Homozygotie/hétérozygote composites pour des mutations SPTA
-Facteurs associés modulant le phénotype clinique-AssociaBon trait AS et de SH: risque d’infarctus splénique.
Delhommeau F et al, Blood, 2000
Frottis cytologique
Non spécifiques- AH immulogiques- CDA II- Hémolyses mécaniques- PPH- Choc thermique-Toxiques (venins), stress oxydatif aigu-Hypophosphatémie, intoxication au zinc-In vitro, sang vieilli
Mariani et al, Haematologica, 2008
Indices érythrocytaires Valeur diagnostique dans les SHCynober T et al, Int J of Lab Hematol, 1996,
60fL 120fL 28g/dL 41g/dL
Sévérité
>4% CHD:Meilleur discriminateur:-100% SH-0% des contrôles
Tests de confirmation
-Test de résistance osmotique (Parpart et al, 1947)
- Avantages: Large utilisation
-Inconvénients: manque de sensibilité, nécessité de témoins adaptés
-Pink Test (Test de lyse in vitro)/AGLT (Zanella et al, 1980; Bucx et al, 1988)
- Avantages: Simplicité, peu de volume sanguin, sensibilité (96%)
-Inconvénients: spécificité variable
-Test de cryohémolyse (Streichman et Gescheidt, 1998)
-Explore les propriétés mécaniques des SH
-Sensibilité variable : seulement 53% dans une étude (Mariani et al, 2008)
-Cytométrie de flux: marquage au 3’EMA (King et al, 2000)
- Ektacytométrie (Bessis et al, 1975)
- SDS-PAGE
Dépendent
S:V
Cytométrie en flux:Marquage à l’éosine 5-maleimide (EMA)
M.J. King et al, Bri0sh Journal of Haematology, 2000, 111, 924–933.
Interac0on prédominante entre EMA et certaines protéines de la membrane érythrocytaire: Bande 3 (80%) et protéines du groupe Rhésus (CD47, RhAG, Rh proteins)
-Kar R et al, Int Journal of Lab Hematol, 2008 Série de 200 pa0ents dont 20 HS, 20 probables, 20 AHAI, 20 contrôles
- Sensibilité: 96.4%- Faux posi0fs dans 3 AHAI et un CDA II- Spécificité:94.2%
-Stoya et al, 2006- Sensibilité 96%- Spécificité 99%
-King MJ et al, Bri0sh Journal of Haematology, Série de 174 pa0ents
- Sensibilité 93%- Spécificité 99%- Faux posi0fs
- CDAII- Pas dans 8 AHAI
Laser
Echantillon
Ektacytométrie(M Bessis et N Mohandas, Blood cells 1975 ; 1 : 307-13)
Index de déformabilité (ID) = grand axe - petit axegrand axe + petit axe
Osmolarité croissante
Point hypo: déformabilité nulle en milieu hypoosmolaire
Correspond à la fragilité osmo;ique DEPEND DU RAPPORT S:V
ID maxMilieu isoosmolaire
DEPEND de SCONSTANT DANS HS
Point hyper: mesuré en milieu hyperosmolaire
Reflet de l’état d’hydratation érythrocytaire
SDS PAGE: électrophorèse des protéines de membrane
TP
TP P
Diagnos;c posi;f
Sensibilité variable+++,En général 70-80%
Spécificité
Dr Ghazal, CHU Bicêtre
Diagnostic differentiel
Aspect qualitatif :anomalie de migration de la Band 3
dans les CDA II
TP
Madame M.
Adressée à 21 ans pour anémie:• Splenomégalie
• Ictère
• Lthiase biliaire asymptoma?que• Transfusée à deux reprises:
• FCS
• Grossesse
Adressée à 21 ans pour anémie:• Hb= 9g/dL
• Réticulocytes= 160 G/L
• Plaquettes et leucocytes normaux• LDH élevée, haptoglobine effondrée,
bilirubine libre à 25 µmol/L
- Test de Coombs négatif- Pas d’autres causes extra-
corpusculaires- Pas d’HPN- Pas d’hémoglobinopathie, de
déficit enzymatique…• Ektacytométrie: « courbe
subnormale, mais diminution de l’index de déformabilité et résistance globulaire un peu diminuée »
Sphérocytose héréditaire?
Sphérocytes
Un exemple de diagnostic différentiel
Myélogramme
Prise en charge thérapeu0que
Recommendations regarding splenectomy in hereditary hemolytic anemias, Haematologica, Aug 2017, Working Group on Red Cells and iron,
Indications de la splénectomieFORMELLE : formes sévères et modérément sévèresDépendance transfusionnelle
POSSIBLE : formes modéréesRetentissement sur qualité de vie :ê performances scolairesSplénomégalie douloureuseIctère marqué
NON INDIQUEE : formes mineures Asymptomatiques
Après 5-6 ans ++Risque infectieux
� Place de la splénectomie subtotale avant 5 ans
Laparoscopie > LaparotomieComplications post-opératoiresDurée d’hospitalisationDurée de convalescence
Splénectomie subtotale• But :– Surseoir à splénectomie totale– Moindre risque infectieux
• Mais :– « Repousse » splénique– Persistance hémolyse de fond
Suivi à long terme après splénectomie subtotalePincez, Guitton et al. Blood 2016
Formes sévères Formes modérées
Pincez, Guitton et al. Blood 2016
Réponse hématologique•90% réponse•8 rechutes
îî besoins transfusionnels
Rate fonctionnelle ≥ 1 an : 96%
Pincez, Guitton et al. Blood 2016
Totalisation splénectomie •20 patients (25%)•8.4 � 1,2 ans après SST
Groupe A : •17 patients (47%) •dont 8 rechutes
Groupe B : •3 patients (8%)
Repousse splénique•volume x 5
Intérêt de la Splenectomie Sub-TotaleGroupe A (formes sévères)
•îîî besoins transfusionnels•8 rechutes nécessitant totalisation•Mais âge > 5-6ans (hors risque infectieux)
è Candidats idéaux à SST
Groupe B (formes modérées)
•Evite splénectomie totale pour formes peu sévères
MAIS :
•Persistance hémolyse à bas bruit•Lithiases chez 16/46 patients non cholecystectomisés•43% groupe A vs. 15% groupe B ; p=0.04
Anomalies de l’hydratation« Stomatocytoses »
Régulation de l’hydratation érythocytaire (dire primitif ou secondaire: F
Regula'on du volume érythocytaie et CCMH: essen'el- Déformabilité- viscosité interne
Teneur en hémoglobine Eau et contenu soluble
Pseudo-hyperkaliémie familiale(PHF)
Andolfo et al Haematologica, 2016
Mutations HTZ le plus souvent du gène ABCB6 (Antigène Lan)
Screening de 327 dons de sang:
La mutation la plus fréquente R276W: 0,3%
Stomatocytoses à cellules hyperhydratées: OHSt
Entrée ++++ du Na+, sor0e plus modérée du K+
Entrée d’H2O: HYPERHYDRATATION
Autosomique dominante ou mutations de novoRares++
Petite série de 4 cas (CHU Bicêtre):
-Hb: 10.7 g/dL
-VGM 129 fL
-CCMH 24%,
-Réticulocytose 11%
60fL 120 fl 28 41
Indices érythocytaires (ADVIA 2120)
Volume Chromie
Fro$s sanguin: très nombreux stomatocytes
Control
200 300 400
DI
0.40
0.20
0.40
0.20
Omin O’
OHSt
OSMOTIC GRADIENT EKTACYTOMETRYDr Picard
(CHU Bicêtre)
SDS-PAGEDr Fénéant-Thibaut
(CHU Bicêtre)
Perrotta S et al., Lancet, 2008, 372:1411-26
Autosomal dominantK+ leak, barely balanced and Na+ entrance
31 Pa:ents (CHU Bicêtre) -Hb: 137 g/L-MCV 99 fL
-CCMH 36.7%-Ré:culocytes 7%
-Stomatocytes <10%
60 120 fl
Dehydrated Hereditary Stomatocytosis DHSt (OMIM #194380 )
Red Cell Indices
28 41
Volume
Chromia
DI
0.4
0.2
Patient
Control
100 200 300 400Omin O’
EKTACYTOMETRY++
Pleiotropic syndrom associated with DHStS. Grootenboer et al., Blood 2000 ; 96 : 2599-2605
DHSt Pseudohyperkalemia
PerinatalEdema
Hyperferritinemia
Not transfusion relatedLead to 0ssular complica0ons
Can reveal the disease
Indications of Ektacytometryn= 103, 49 families
Familial screening
52%
Chronic hemolysis
29%
Thrombotic events(8%)
Perinatal oedema6%
Iron overload5%
% d
es p
atie
nts
Clinical and Biological features Familial tes6ng
N=54
0
10
20
30
40
50
60
70
80
NSCH Thrombosis PO HyperferritinemiaNSCH Thrombosis Perinatal Oedema
Hyperferritinemia
Age at HX diagnosis
14
8
14 1513
11
58
02468
10121416
1 2 3 4 5 6 7 8 9
Num
ber o
f pat
ient
s
<1 1-10 10-20 20-30 30-40 40-50 50-60 >60
0
10
20
30
40
50
60
70
80
90
1
Mea
n Ag
e
NSCH PO Hyperferritinemia Thrombosis
Hematological features at diagnosis
13,5
0
2
4
6
8
10
12
14
16
18
1
98,9
0
20
40
60
80
100
120
1
252
0
50
100
150
200
250
300
350
400
450
1Hemoglobin (g/L)
MCV(fL)
Reticulocytes(G/L)
Most of the times, HX induces a compensated
hemolysis
Absent 53%
Present47%
Hyperferritinemian=55
14 by phlebotomy6 by DFO6 by DFX1 by DFP
No transfusion dependant anemia
Other biological / clinical features
P50 evaluation (Dr Kiger- Dr Picard, e-poster 1082)
Perinatal edema17 (13 families)
Transfusion in Utéro : 30 % of patients (Hb 7,1 g/dL – 19,1 g/dL)
Mostly from the 2nd trimester
Hydrops fetalis: 66% of patients 2 MFIU - 1 IMG – 1 death early after birth
40 % prematurity (half before 32 SA)
Favorable outcome in less than 1 month in 50 % of patients NO RELAPSE except 1 patient with lymphoedema at adult age
4
2
4
3
2
2
Embolie pulmonaire
TVP
Thrombose porte
AVC
HTAP
Infarctus splénique
0
20
40
60
80
100
120
1
Nonsplenectomized
Splenectomized83%
12 patients with thrombotic events17 thrombotic events
Pulmonary embolism
PVT
Portal thrombosis
Cerebral stroke
Pulmonary hypertension
Spleen infarct
11 Patients were splenectomized
-Mean Age at splenectomy: 23,6 (10-41 yo)
-10/11 Patients experienced thrombotic events
-Mean delay of thrombotic events after splenectomy:
9 years (14 days-27 years)
Force mécanique
. Bagriantsev et al.. 2014
Piezo1 est exprimé à la membrane du GR(Andolfo et al, Blood, 2012)
Mutations « gain de fonction » (Albuisson et al, Nat Com, 2013)
ìCa2+
Gardos channel KcL cotransporteur
Fuite cationiqueDeshydratation
Rôle dans d’autres pathologies érythrocytaires?PSickle? (Gallagher et al, 2014)
CONCLUSION
-Pathologies rares
-Y penser à tout âge!
-Autosomique dominante
-Modes de présentation inhabituelle (hémochromatose...)
-Hémolyse compensée fréquente
-Diagnostic à éliminer avant toute splénectomie-Frottis
-Indices érythrocytaires-Ektacytométrie
-Biologie moléculaire
RISQUE TRHOMBOTIQUE MAJEUR APRES SPLENECTOMIE
Bijleveld R et al , NJM, 2015
Anomalies des interactions horizontales: Elliptocytose héréditaire
-Mutations SPTA1: 75% des cas-La plupart des mutations sont dans la partie N-ter de la spectrine alpha, -Altérant les sites d’oligomérisation
-Mutations 4.1: 4.1 (-) HE-- Nombreux elliptocytes (100%)-Asymtomatique à l’état hétérozygote
-Mutations SPTB-Rares -- Parfois très symptomatiques meme à l’état HTZ
Localisées en général dans la région C-terminale
� Anomalie membranaire avec présence de 20 à 100% d’elliptocytes �Transmission autosomique dominante� Prévalence : 1-2% dans certaines régions d’Afrique et aux Antilles, 15 à 20 fois plus rare en Europe.
Site d’autoassociation Site joncJonel
HE
N-ter C-ter
HPP
Anomalies des interactions horizontales: Elliptocytose héréditaire
Elliptocytose héréditaire simple (HE)
-90% des cas-Asymptomatique jusqu’à hémolyse très modérée-Ex: (4.1)- HE; mutations SPTA1-Diagnostic sur frottis sanguin
Témoin
Pyropoïkilocytose héréditaire (PPH)-Tableau d’anémie hémolytique parfois sévère, transfusion dépendant
PPH
- Mutations SPTB- Forme homozygote (4.1R)-Hétérozygote composite SPTA1
King MJ et al, ICSH Guidelines, 2015)
Le métabolisme du globule est anaérobie:-Absence de noyaux
-Absence de mitochondrie
-Shunt de Rappaport (1):
Production de 2.3 DPG èlibère O2 aux tissus
- Glycolyse anaérobie (2):
Production d’énergie (90% de l’ATP de la cellule)
et du NADH
- Voie des Pentoses-Phosphates (3):
Production du pouvoir réducteur NADPH (1)
(2)
(3)
Enzymes érythrocytaires
Le déficit en G6PD: épidémiologie
Gène sur le chromosome XTouche les hommes hémizygotes Femmes conductrices400 millions de porteurs dans le mondeSe rencontre partout Peut se révéler tard
Répartition proche de celle du paludisme:Protection des infestations graves des sujets déficitaires- Variant Africain A-, sévérité modérée
- Variant Méditerranéen B-, sévère: FAVISME
- 8% de la population SE asiatique
Seule voie de production
de NADPH pour l’érythrocyte
(pas de mitochondries)
GSH réduit GSH oxydé
NADPH: pouvoir réducteur qui protège le GR du stress oxydatif
STRESS OXYDATIF- Infection- Médicaments
Voie des pentoses phosphates
Stress oxydatif état basal
Stress oxydatif aigu
- Fièvre
-Infection
-Médicaments
-Aliments (fèves)
GR normaux Déficit
Pas d’hémolysePas d’hémolyse
GR normaux Déficit
Le déficit en G6PD:physiopathologie
Pouvoir réducteur dépasséTableau d’hémolyse aigue déclenchée
Par une exposition à un stress oxydatif
- Infection bactérienne, virale
- Prise médicamenteuse: Quinine, sulfamides, acide nalidixique, Vitamine C à forte dose...
- Alimentation : Fèves (formes méditérannéennes: FAVISME)
- Sodas à base de quinine
Tableau clinique• Différentes types de classes
– - I: AH chronique:sporadiques+++– Classe II: déficit sévère (1-10% d’activité):Med– Classe III: déficit modéré (10-60%):A-– Classe IV: Activité normale (60-100%)– Classe V: augmentation d’activité (150%).
• Classiquement: poussée d’hémolyse intravasculaires déclenchées par:– -Médicaments oxydants: Primaquine, sulfamides, sulfones (AFSSAPS)– Infections (hépatite++, CMV, typhoïde…)– Favisme+++++
• Fièvre, urines noires, douleurs lombaires, malaise, choc, IRA
• Anémie d’abord arégénérative. Hémoglobinémie, hémoglobinurie, haptoglobine effondrée, bili svt secondaire, LDH haute, corps de HEINZ
• Le dosage doit être contrôlé à distance+++ (Enzyme DE REFERENCE, Hexokinase++++++)
• Biologie moléculaire
Un exemple de crise hémolytique
Homme de 20 ansNé en FranceAucun antécédent particulier( a posteriori INN sans incompatibilité materno-fœtale,Ayant nécessité une photothérapie pendant qqs jours)
SURVENUE BRUTALE : •Céphalées•Vomissements
•Asthénie
AGGRAVATIONURINES ROUGES
URGENCES
HB : 5g/dL ; Réticulocytes : 60G/LHémoglobinurie , hémoglobinémie
Ascendance siciliennelointaine.Ingestion de fèves
Heinz
Le métabolisme du globule est anaérobie:-Absence de noyaux
-Absence de mitochondrie
Métabolisme énergétique du globule rouge
Glycolyse: 90% du glucose est utilisé dans cette voie: Embden-Meyerhof Production d’ATP
Déficit enzymatique dans la voie de la glycolyse:-Pyruvate Kinase +++-G6P isomérase-Hexokinase -Phosphofructokinase -Triose phosphate isomérase-Aldolase-Diphosphoglycérate mutase- Phosphoglycérate kinase
Glucose
Glucose 6 P
Pyruvate
ATP NADH
Fe 2+Pompe
Na/K-ATP dépendante
Transmission autosomale récessive.Hétérozygotes composites.Hétérozygotes : 1-2%
Déficit en Pyruvate –Kinase
Tableau d’hémolyse chronique à prédominance spléniqueDiagnostic par dosage enzymatique en dehors d’une poussée d’hémolyse +/- biologie moléculaire
Métabolisme énergétique du globule rouge:
5’ pyrimidine nucléotidase
-Enzyme de dégradation des bases pyrimisiques
-3 ème cause d’enzympathie dans le monde
-Inhibée par le plomb!
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