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

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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.

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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.

Défaut d’interactions verticales membrane - cytosquelette

Perte de surface, volume constantDiminution ratio S/V

è Sphérisation du GRMoindre déformabilité

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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.

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

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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.

« 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!