DIFETTI CONGENITI DELLA MEMBRANA ERITROCITARIA - Aspetti fisiopatologici, clinici e diagnostici
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Milano, 1 marzo 2018
DIFETTI CONGENITI DELLA MEMBRANA
ERITROCITARIA
Aspetti fisiopatologici, clinici e diagnostici
A. Zanella
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milanoµm
0.2µ
Milano 1 marzo 2018SPECTRIN
α-CHAIN (280 kd) 21 REPEAT UNITS
β-CHAIN (246 kd) 17 REPEAT UNITS
Milano 1 marzo 2018REPEAT UNIT OF SPECTRIN (106 aa)
CONNECTING REGION TURN REGION TURN REGION
HELIX 3 HELIX 1 HELIX 2
NH2
From MARCHESI V.T. ANN.REV.CELL.BIOL. 1985
Milano 1 marzo 2018Spectrin domains after controlled tryptic digestion
21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1
α spectrin
C 41 kD 52 kD 52 kD 46 kD 80 kD N
V IV III II I
interchain dimer self-
association association
IV III II I
N 74 kD 33 kD 65 kD 17 kD C
β spectrin
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Milano 1 marzo 2018RED CELL MEMBRANE
From: Bianchi P, M Narla, Post Graduate Hematology 7 eds. , 2015
Milano 1 marzo 2018A. Defects of RBC membrane structural proteins
B. Defects of RBC ion transport
M.J. King & A. Zanella, Int J Lab Hematol, 2013;35:237-43
Milano 1 marzo 2018HEREDITARY SPHEROCYTOSIS (HS)
Affects about 1:2000 (Gallagher & Lux, 2003)
Dominant transmission in 75% of cases (Tse&Lux,1999)
Compensated to very severe hemolysis
Excellent response to splenectomy
GPI
“Vertical interactions”
RhAG glycophorin C
Band 3
4.2 p55 4.1R
ankyrin spectrin
spectrin
Milano 1 marzo 2018
.Perrotta S, Lancet 2008; 372:1411-26
• Two factors are implicated in the pathophysiology of HS: an intrinsic red cell membrane
defect and an intact spleen that selectively retains, damages and removes defective RBCs
• The diversity of membrane imbalance is likely to result in different red cell clearance
mechanisms.
Milano 1 marzo 2018OSMOTIC GRADIENT EKTACYTOMETRY
0.8 NC
Band 3 defect, LR1
Elongation index
0.6
0.4
Spectrin/ankyrin defect, AZ15
0.2
122 188 224 253 317 386 430 482 553
Osmolality, mOsm/kg
Reliene R, Mariani MG, Zanella A, Reinhart W, Ribeiro L, Miraglia del Giudice E, Iolascon A, Eber S, Lutz HU
Blood 2002;100:2208-15
Milano 1 marzo 2018Osmoscans from RBC populations from unsplenectomized
and splenectomized HS patients
0.8
NC
Sp defect,AZ16 splenect.
Elongation index
0.6 B3 defect,LR2, splenect.
B3 defect,LR1, unsplenect.
0.4
Sp defect,AZ15, unsplenect.
0.2
122 188 224 253 317 386 430 482 553
Osmolality, mOsm/kg
Reliene et al, Blood 2002;100:2208-15 Milano 1 marzo 2018Reliene R, Mariani M, Zanella A, Reinhart WH, Ribeiro ML, Del Iudice EM, Perrotta S, Iolascon A, Eber
S, Lutz HU. Splenectomy prolongs in vivo survival of erythrocytes differently in spectrin/ankyrin-
and band 3-deficient hereditary spherocytosis. Blood 2002; 100(6):2208-15
Sp/Ank deficient cells escape opsonization
by releasing Band-3 containing vescicles
Splenectomy prevents early loss of immature cells
in both types of defect, but it has an additional
beneficial effect on mature Sp/Ank deficient cells,
prolonging their survival as assessed by 4.1a/4.1b
Milano 1 marzo 2018Haematologica, 2008; 93(9), 1310 300 HS pts from 212 families , 141 M and 159 F Age at diagnosis 20 yrs (1- 80 yrs):40%:
CLINICAL DATA IN 259
NOT SPLENECTOMIZED HS PATIENTS
splenomegaly 72%
anemia 62% #
jaundice 40%
neonatal jaundice 33% $
gallstones 32%
cholecystectomy
aplastic crises
transfusion support
0 10 20 30 40 50 60 70 80 90 100
% of patients
# ANEMIA: severe 6%, moderate 16%, mild 40%, compensated 38%
$ EXCHANGE TRANSFUSION: 14/82 cases
Mariani et al, Haematologica, 2008 Milano 1 marzo 2018Clinical features of 259 not splenectomised HS patients divided
according to the type of membrane defect (90 Sp/Ank; 139 Band 3)
• splenomegaly and gallstones more
frequent in B 3 deficient pts
• anaemia, neonatal jaundice and Tx
requirement more common in Sp/Ank
deficiency.
• ExTx similar in the two groups
• isolated Sp or combined Sp/Ank defic.
more frequently diagnosed in childhood
than in adulthood (55% vs. 45%),
contrary to B 3 (60% in adults vs. 40%
in children).
Mariani et al, Haematologica, 2008
Milano 1 marzo 2018EFFECTS OF SPLENECTOMY IN HS PATIENTS
Hematologic and biochemical data of 21 HS patients before and after
splenectomy
Hematologic Pre-splenectomy Post-splenectomy
Hb (g/dL) 10.8 (7.6-15.1) 13.9 (12.6-18.8)
MCV (fL) 84 (68-106) 84 (73-95)
MCHC (g/dL) 35.4 (28.3-38.8) 34.8 (33.3-37.1)
Spherocytes (%) 9 (1-32) 4 (3-16)
Reticulocytes (x109/L) 337 (96-640) 51 (11-118)
Unc. bilirubin (mg/dL) 1.9 (0.7-8.9) 0.7 (0.35-1.83)
Hemoglobin levels 10.8 g/dL 13.9 g/dL
Reticulocytes 337x109/L 51x109/L
Unconjugated bilirubin 1.9 mg/dL 0.7mg/dL
Effects more evident in Band 3 vs Spectrin
Effects of splenectomy comparable in young (n=6) and adult patients
Milano 1 marzo 2018Br J Haematol 126:455-474, 2011
The laboratory diagnosis of HS is usually straightforward and it is based upon a combination of
clinical history, family history, physical examination (splenomegaly, jaundice) and laboratory data
(full blood count, especially red cell indices and morphology, and reticulocyte count)
Milano 1 marzo 2018HS due to spectrin deficiency
Residual Spectrin
81% 53% 28% (+Ank)
Milano 1 marzo 2018HS (B3 deficiency) HS (4.2 deficiency)
Milano 1 marzo 2018- MCV was decreased in 22% MCHC was increased overall in 38% of cases - Hb levels were normal in 38% pts (50% of band 3 and 42% in Sp deficiency) - Reticulocyte count was normal in 16/259 (6%) - Median n. spherocytes was 7% (range 0-56); 22/259 (8%) pts had
DIAGNOSIS OF RBC MEMBRANE DEFECTS
Int J Lab Hematol. 2015 Jun;37(3):304-25.
Milano 1 marzo 2018Int J Lab Hematol 2015;37:304-25
Milano 1 marzo 2018Screening tests for the diagnosis of Hereditary Spherocytosis
Osmotic fragility (OF) test Measure absorbance at 540 nm for fresh Affected by elevated reticulocyte counts
(Parpart et al, 1947) blood and after 24 h incubation. Plot a Also increased in AIHA
graph of % haemolysis versus NaCl
concentration
Acidified glycerol lysis test (AGLT) Measure the time taken for absorbance of
(Zanella et al, 1980) red cell suspension at Also positive in AIHA, enzyme deficiency,
625 nm in glycerol to fall to half of its pregnant women, chronic renal failure
The Pink test (Vettore & Zanella, 1984) original value before glycerol addition and myelodysplastic syndrome.
is a modified AGLT (AGLT50)
Osmotic gradient ektacytometry A laser diffraction viscometer that Distinct deformability curves for red cells
(Clark et al, 1983 measures red cell deformability at constant from patients with HS, hereditary
shear stress as a continuous function of elliptocytosis, hereditary
Laser-assisted Optical Rotational Cell suspending osmolality (hypotonic to pyropoikilocytosis, stomatocytosis and
Analyser (LORCA) hypertonic) sickle disease (Mohandas et al, 1980)
Hypertonic cryohaemolysis test % cryohaemolysis at 540 nm after transfer Positive results for HS, some CDAII and
(Streichman & Gescheidt, 1998) of red cells from 37C to Melanesian elliptocytosis
0C for 10 min
Eosin-5-maleimide (EMA) binding Reduced fluorescence (green) intensity of Distinct histograms for red cells of HS.
(King et al, 2000) EMA-labelled red cells by flow cytometry Reduced in CDAII, cryohydrocytosis,
SAO.
Milano 1 marzo 2018Hereditary Spherocytosis Dehydrated Stomatocytosis
Hereditary Elliptocytosis
L. Da Costa, 2013 Milano 1 marzo 2018Screening tests for the diagnosis of Hereditary Spherocytosis
Osmotic fragility (OF) test Measure absorbance at 540 nm for fresh Affected by elevated reticulocyte counts
(Parpart et al, 1947) blood and after 24 h incubation. Plot a Also increased in AIHA
graph of % haemolysis versus NaCl
concentration
Acidified glycerol lysis test (AGLT) Measure the time taken for absorbance of
(Zanella et al, 1980) red cell suspension at Also positive in AIHA, enzyme deficiency,
625 nm in glycerol to fall to half of its pregnant women, chronic renal failure
The Pink test (Vettore & Zanella, 1984) original value before glycerol addition and myelodysplastic syndrome.
is a modified AGLT (AGLT50)
Osmotic gradient ektacytometry A laser diffraction viscometer that Distinct deformability curves for red cells
(Clark et al, 1983) measures red cell deformability at constant from patients with HS, hereditary
shear stress as a continuous function of elliptocytosis, hereditary
Laser-assisted Optical Rotational Cell suspending osmolality (hypotonic to pyropoikilocytosis, stomatocytosis and
Analyser (LORCA) hypertonic) sickle disease (Mohandas et al, 1980)
Hypertonic cryohaemolysis test % cryohaemolysis at 540 nm after transfer Positive results for HS, some CDAII and
(Streichman & Gescheidt, 1998) of red cells from 37C to Melanesian elliptocytosis
0C for 10 min
Eosin-5-maleimide (EMA) binding Reduced fluorescence (green) intensity of Distinct histograms for red cells of HS.
(King et al, 2000) EMA-labelled red cells by flow cytometry Reduced in CDAII, cryohydrocytosis,
SAO.
Milano 1 marzo 2018EMA-binding test
Flow cytometric test that measures the fluorescence intensity of intact red cells
labelled with the dye eosin- 5-maleimide, which interacts with Lys-430 on the first
extracellular loop of band 3.
EMA-binding test is a sensitive (92%) and specific(99%) tool for HS directly
targeting the structural lesion of this disease.
Bianchi et al, Haematologica 2012
Normal control HS
Milano 1 marzo 2018Use of diagnostic tests for red cell membrane defects
% of centers
Always performed ENERCA White Book 2015
Performed in particular cases Milano 1 marzo 2018Method with best specificity and sensitivity
Combination of tests No answer (4)/Not known (4)
RBC Morphology +EMA 8
EMA+AGLT
AGLT+ Cryo 11
EMA+AGLT+RIA
OF +EMA+Cryo
Cryo+EMA+SDS 2
EMA+pink+OF 1
RBC Morphology + Pink 1 1
AGLT+OF+RIA EMA-binding
EMA+AGLT+SDS
OF
Pink test
Ectacytometer
ENERCA White Book 2015
Milano 1 marzo 2018Number of different diagnostic tests * performed per Center
Number of Centers
Most centres use a
“battery of tests”
(3-6 different tests)
*Tests considered: NaCl on
fresh and incubated blood
(2 concentrations or curve);
glycerol lysis tests; Ema-
binding; Ecktacytometry
SDS-PAGE
Number of diagnostic tests/ Center
ENERCA White Book 2015
Milano 1 marzo 2018Sensitivity of single tests in 150 HS patients grouped according to the biochemical
defect. Number represents the ratio of positive cases/total cases; in brackets percent
values.
Milano 1 marzo 2018Sensitivity of various diagnostic tests in 22 splenectomised and 128 not splenectomised HS patients. Not splenectomised HS were divided according to the clinical phenotype. Bianchi et al, Haematologica 2012 Milano 1 marzo 2018
Combined tests’ sensitivity in total HS cases. Number represents the ratio of
positive cases/total cases; in brackets percent values.
_______________________________________________________________________________________________________
EMA + AGLT EMA + OF NaCl fresh EMA + OF NaCl inc. EMA + Pink OF NaCl inc. + AGLT
Total HS pts. 150/150 (100%) 143/150 (95%) 143/150 (95%) 149/150 (99%) 146/150 (97%)
_______________________________________________________________________________________________________
• The combination of EMA & AGLT enabled to identify the totality of HS patients
In particular, 133/150 (88%) of HS tested EMA+AGLT+, 7/150 (5%) EMA+AGLT-, and
10/150 (7%) EMA-AGLT+
• The combination of EMA & Pink test unabled identification of 99% of HS cases
• In case of non-availability of flow cytometry, the association of AGLT & incubated NaCl
OF had a sensitivity of 97%
Bianchi et al, Haematologica 2012 Milano 1 marzo 2018Results of individual diagnostic tests in patients with haemolytic anaemias other then HS, compared with HS. The shadowed area represents normal reference intervals. Bianchi et al, Haematologica 2012 Milano 1 marzo 2018
DIFFERENTIAL DIAGNOSIS OF HS AND CDAII
Anemia
SDS-PAGE analysis of RBC membrane proteins
Spherocytes
Jaundice
CDAII CDAII Ctr Hyperbilirubinemia
Splenomegaly Band 3
Undetected hypoglycosylation
Band3/ 4.2
Spectrin
Band 3 Ankyrin
13% of patients referred with a suspect of HS were CDAII
SDS-PAGE displayed a sensitivity of 97%, being repeatedly normal in 2/101
Mariani, 2008 ; Fermo, 2010 Milano 1 marzo 2018Milano 1 marzo 2018
CASE REPORT
62 years old female, no consanguinity
- Moderate haemolytic anemia since childhood
- HS diagnosed at 19 years (Hb 9 g/dL, Unconjugated bilirubin 1.9-4 mg/dL)
- Cholecystectomy and splenectomy at 39 yrs , Hb post splenectomy 12.3 g/dL
- 50 years: diabetes type I
32 yrs 28 yrs
Mild neonatal jaundice, mild anemia and splenomegaly in childhood, HS diagnosed at 4 yrs
15 years: Hb 13.2 g/dL 11 years: Hb 10.9 g/dL
Mild reticulocytosis 3% Mild reticulocytosis 3.5%
Unconj.bilirubin 3.7 mg/dL Unconj.bilirubin 2.5 mg/dL
Cr51 RBC survival: 21 d (25-35) Cr51 RBC survival: 24 d
Milano 1 marzo 2018Hb 11.7 g/dL
MCV 95fl
MCH 32 pg
MCHC 33.7 g/dL
Retics 63 x 109/L
Unconj Bilirubin 1.8 mg/dL
Serum iron 139 mcg/dL
Ferritin 697 ng/mL
Transferrin sat. 63 %
Pt Pt Ctr
AGLT 50 >900 sec
hypoglycosylated
NaCl osmotic fragility normal Band 3
Ema binding test 12%
HFE Cys282/Tyr wt
HFE His63/Asp wt
Gilbert UGT-1A 6/7
Milano 1 marzo 2018SEC23B gene: SEC23B gene:
c.325G>A/wt c.325G>A/c.325G>A
SEC23B gene: SEC23B gene:
c.325G>A/c.325G>A c.325G>A/c.325G>A
Hb 11.3 g/dL Hb 9.2 g/dL
Reticulocytes 115000/µL Reticulocytes 121000/µL
Unconj.bilirubin 3.3 mg/dL Unconj.bilirubin 1.8 mg/dL
RBC morphology: anisopoikilocytosis, RBC morphology: anisopoikilocytosis,
elliptocytes 4%, spherocytes 2%, spherocytes 3%, schistocytes 2%,
schistocytes 2%, dacriocytes 3%
Ferritin 792 ng/mL (19-87) Ferritin 501 ng/mL (19-87)
Transferrin sat. 97 % (17-52) Transferrin sat. 97 % (17-52)
OF : normal OF : normal
AGLT50: 210 AGLT50: 106
Band 3 hypoglycosylation: YES Band 3 hypoglycosylation: YES
Milano 1 marzo 2018HEREDITARY ELLIPTOCYTOSIS (HE)
GPI HE (4.1 deficiency)
RhAG glycophorin C
band
3AE1
4.2 p55 4.1R
ankyrin spectrin
spectrin
“Horizontal
HE (Sp αI/74 )
interactions”
.
HPP (Sp αI/74/ αLELY)
Milano 1 marzo 2018Hereditary Elliptocytosis
• The majority of HE are caused by Sp mutation (spectrin structural
variants), Clinical severity depends on the amount of spectrin variant
incorporated into the membrane,
• The closed the mutation is to
the spectrin dimer interaction,
the less stable is the tetramer
N Mohandas, P Gallagher, 2008
Typically, heterozygous are asymptomatic, have a very mild
anemia and often the elliptocytes are detected during routine analysis.
No splenomegaly, normal or slightly elevated reticulocyte count.
Homozygous or compound heterozygous may have moderate to
severe hemolytic anemia (10-12% of the cases)
The spleen is enlarged and the response to splenectomy is good
Milano 1 marzo 2018Hereditary Pyropoikilocytosis
HPP is a severe form of HE
• Severe hemolytic anemia during the neonatal period
• Blood film with poikilocytes, elliptocytes,
microspherocytes and many fragments and bizarre-
shaped RBCs
In HPP the red cell membrane has two defects:
1) Homozygous or compound heterozygous for structural Sp
variants (in the region of the dimer Sp self-association site)
2) Heterozygous for a single structural Sp variant (located in
the Sp association site) in trans with a low expression α-Sp
allele (Sp α Lely, Sp α V/41 )
Milano 1 marzo 2018Hereditary Elliptocytosis – Diagnosis
Blood film examination
SDS- PAGE analysis ( may detect Truncated α and β chains, 4.1 deficiency)
Sp functional study Tryptic Sp peptide mapping
T
D
α I/80
α I/74 variant
TG Co TG Co
30° 4°
decreased Sp tetramerization
DNA studies
Ektacytometry TG Co
Milano 1 marzo 2018HEREDITARY STOMATOCYTOSES (HSt)
Dehydrated Hereditary Stomatocytosis (DHSt)
Overhydrated Hereditary Stomatocytosis (OHSt)
Cryohydrocytosis (CHC, type I-II)
Familial Pseudohyperkaliemia
• Dominantly transmitted disease
• Monovalent cation leak
• Mild to severe hemolytic anemia (DHSt, OHSt, CHC-II)
• Splenectomy is controindicated for thrombotic or
thromboembolic complications (DHSt, OHSt)
Milano 1 marzo 2018HEREDITARY STOMATOCYTOSES
Dehydrated stomatocytosis Overhydrated stomatocytosis
(DHSt) (OHSt)
Xerocytosis Hydrocytosis
Frequency 1:10,000 1-100,000:1,000,.000
Clinical features Mild-compensated anemia Moderate-severe anemia
MCV Normal- Slighlty increased Markedly increased (110-140)
MCHC increased Decreased
RBC morphology Stomatocytes 30%
Osmotic fragility normal-decreased Increased
[Na+]i > >>
[K+]i [Na+]i +[K+]i
< intracellular water > intracellular water
Ektacytometer curve Shift to the left Shift to the right
Gene FSM38A RhAG
Milano 1 marzo 2018OSMOTIC GRADIENT EKTACYTOMETRY
DHSt
C
OHSt
Elongation index (0.2)
210 247 314 335 407 471 550
Osmolality (mosM)
Osmoscans from filtered whole blood
Milano 1 marzo 2018Molecular basis of defects of red cell permeability
Piezo1 RhAG Band3 GLUT1 KCNN4
DHSt Mutations of FAM38A coding for PIEZO1 protein, a mechanosensitive
ion channel protein that regulates the RBC response to ion fluxes accompanying
mechanical stresses when circulating through narrow passages in capillaries The
mutations cause a gain-of-function (or increased permeability) in mechanically
activated PIEZO1 ion channels (Zarychanski R. et al Blood 2012; Andolfo I. et al Blood
2013; Bae C. et al, PNASS 2013; Albuisson J.et al, Nat Commun 2013).
Mutations of Gardos Channel protein (KCNN4) (Rapetti-Mauss et al, Blood 2015;
Glogowska et al, Blood 2015)
OHSt Mutations (Phe65Ser or Ile61Arg) in RhAG gene resulting in increased
pore size of the mutated Rh-associated Glycoprotein that facilitates a more
rapid rate of cation leak through the RBC membrane (Bruce LJ et al, Blood 2009).
Other «leaky” red cell syndromes Band 3 ( AE1) mutations (Barneaud-Rocca, 2011),
GLUT1 mutations (Flatt JF et al, Blood 2015).
Milano 1 marzo 2018Next Generation Sequencing (NGS) in the diagnosis of RBC
membrane disorders
Feasible and reliable method to detect mutations and diagnose patients with
red blood cell membrane disorders.
Particularly useful for diagnosis of atypical presenting patients
Knowledge of the genetic background
understanding of the complex genotype-to-phenotype correlationsNext Generation Sequencing
1980
I.1 I.2
II.1 II.2 II.3 II.4
2014
III.1
Fermo et al, 57°ASH meeting, Orlando 2015 Milano 1 marzo 2018Whole Exome sequencing
Only one candidate gene identified: KCNN4, coding for
Gardos channel.
Heterozygous missense mutation was detected as a de
novo mutation in the proband, and dominantly
transmitted to the daughter.
c.1055G>A,
p.R352H
Fermo et al, 57°ASH meeting, Orlando 2015 Milano 1 marzo 2018Conclusions
Both structural protein defects of the erythrocyte membrane and
functional abnormalities of cation permeability may result in congenital
hemolytic anemias, which are not single-gene diseases. In some cases,
mutations in different positions of the same gene (i.e. Spectrin or Band 3)
may result in different diseases (HS, HE, HSt).
Although a majority of patients present with adequate
characteristic red cell morphology, clinical features and routine
laboratory testing for making an appropriate diagnosis, in less typical
cases additional specialized tests may be necessary, in particular to
differentiate HS from HSt and from CDA-II, and to correctly identify the
mild-compensated forms.
Milano 1 marzo 2018Paola Bianchi Elisa Fermo Cristina Vercellati Anna Marcello Anna Zaninoni Wilma Barcellini
Conclusions
• The effects of defective structural proteins and anomalous cation
permeability on red blood cells are manifested as hereditary hemolytic
anemias, which are not single-gene diseases. In some cases, mutations in
different positions of the same gene (i.e. Spectrin or Band 3) may result
in different diseases (HS, HE, HSt).
• Although a majority of patients present with adequate characteristic red
cell morphology and clinical features for making an appropriate
diagnosis without resorting to additional laboratory testing, occasional
overlapping clinical features of HS and hereditary stomatocytoses (and
CDA II) can occur.
• A diagnosis of hereditary stomatocytosis should be considered when a
patient has a negative direct antiglobulin test, a compensated hemolytic
anemia, high MCV, and possibly elevated plasma [K+]. The proviso is
that HS, enzymopathy and macrocytic anemia are already excluded.
Milano 1 marzo 2018You can also read
