M. Letícia Ribeiro, CHC - Enerca

M. Letícia Ribeiro, CHC

LABORATORY DIAGNOSIS OF RARE
ANAEMIAS
Hereditary RBC membrane defects
3rd European Symposium on Rare Anaemias
M. Leticia Ribeiro
Madrid, Nov 2010
M. Letícia Ribeiro, CHC

Red Blood Cells
RBCs are biconcave under
physiological conditions
Their shape changes when navigating
narrow blood vessels or confined
spaces in tissues and organs
The ability of red cell to maintain its
discoid shape, shape,
elasticity and
deformability in circulation, under
constant mechanical shear and stress
forces, is attributed to its lipid layer
and proteins
Kightley Media
CELLS alive
M. Letícia Ribeiro, CHC

Erythrocyte Membrane Proteins
Vertical Interaction
Horizontal Interaction
Lux SE, Palek J:
Disorders of the Red Cell Membrane
A deficiency of, or a dysfunction in, any one of these membrane
proteins can weaken or destabilize the cytoskeleton, resulting
in abnormal red cell morphology and a shorter life span -
Hemolysis
M. Letícia Ribeiro, CHC

Red Blood Cell Membrane Disorders
congenital hemolytic anemias characterized by clinical,
laboratory and genetic heterogeneity
Hereditary Spherocytosis
Hereditary Elliptocytosis
Hereditary Pyropoikilocytosis
Hereditary Southeast Asian Ovalocytosis
Hereditary Stomatocytosis
M. Letícia Ribeiro, CHC

Hereditary Stomatocytosis
a group of dominantly inherited hemolytic anemias
with abnormal membrane permeability to
univalent cations
Hydrocytosis – Overhydrated stomatocytosis
Xerocytosis – Dehydrated stomatocytosis
Cryohydrocytosis
Familial Pseudohyperkaliemia
M. Letícia Ribeiro, CHC

Hereditary Elliptocytosis
By Rex Graham
Normal Pr 4.1 absent
Skeletal network
electron mycroscopy, by Yawata et al
The main defects in HE are protein 4.1 deficiency and spectrin
mutations, which can be detected as spectrin variants
The clinical severity in HE depends on the amount of spectrin
variant incorporated into the skeleton, and the closer the
mutation is to the junction where dimers associate, the less
stable is the tetramer
M. Letícia Ribeiro, CHC

Hereditary Elliptocytosis
Prevalence
1:5000 among Caucasians (Protein ( Protein 4.1 deficiency)
deficiency
1:100 in certain African countries (Spectrin ( Spectrin mutations)
mutations)
In the majority of HE individuals the anemia is very mild
and often the elliptocytes are detected during a routine
analysis
Hb 7-9 g/dL
Retic 11%
Individuals Splenomegaly with nonhemolytic HE
do not Protein
have splenomegaly, splenomegaly
4.1 deficiency
, and
their reticulocyte counts are slightly
elevated to normal
HE due to complete protein 4.1 deficiency is a severe
hemolytic disease
M. Letícia Ribeiro, CHC

Pyropoikilocytosis
HPP is a severe form of HE - patients often
present with severe hemolytic anemia during the
newborn period
These patients are either homozygous or
compound heterozygous for spectrin
mutations
HPP can also be due to the co-inheritance
of the low-expression allele Spα LELY
M. Letícia Ribeiro, CHC

Pyropoikilocytosis
D R S
Hb g/dL
MCV fL
MCH pg
MCHC %
PBS
14.1
92
29
31
Elliptocytes
3º day of life
Hb g/dL 11.5
Bil mol/L 145
Anisopoikylocytosis
13.3
13.4
3 years old
Normal
αLELY αV/41 Spectrin α 28 Arg-His (CGT-CAT)
α(nt 1857 Leu-Val; CTA-GTA) Intron 45, nt 12 (C-T)
80
30
37
96
27
29
Normal
Elliptocytes
M. Letícia Ribeiro, CHC

Southeast Asian Ovalocytosis
Dominant inheritance
SAO AE1 gene contains a deletion of 9 CD encoding amino
acids 400-408, at the boundary of the cytoplasmic and
membrane domains, in cis with Lys56Glu substitution
SAO red cells are rigid and hiperstable
Hemolysis is mild or absent
Fem, 27 years old
Pregnant
Origin: East Timor
Hb g/dL 11.1
MCV fL 89
MCH pg 31
MCHC % 34.5
RDW % 17
M. Letícia Ribeiro, CHC

Hereditary Spherocytosis
Membrane lesions involving the vertical interactions
between skeleton and lipid bilayer lead to vesiculation of the
unsupported surface components, causing a progressive
reduction in membrane surface area. area
The red cell shape changes from a flexible, deformable biconcave
disc to a spherical poorly deformable red cell – the
spherocyte
The severity of hemolysis depends on the contents of Spectrin
from Lux SE, Palek J: Disorders of the Red Cell Membrane, Blood Principles and Pratice of Hematology
M. Letícia Ribeiro, CHC

Hereditary Spherocytosis
The commonest cause of inherited chronic hemolysis in Northern
Europe and North America - Prevalence 1:5000 to 1:2000
Inheritance
Dominant ≈ 2/3
Non-dominant ≈ 1/3 de novo or recessive
In Italian population the occurency of de novo dominant mutations in HS
patients with normal parents is 6xs more common than recessive
mutations (Miraglia del G. et al, 2001)
The abnormal RBC morphology in HS is due to a deficiency of,
or a disfunction in, Spectrin, Ankyrin, Band 3 and/or Protein 4.2
The Spα LEPRA allele (Low Expression Prague) is prevalent among nondominant
HS (Boivin, et al 1993, Dhermy, et al 2000, Wichterle, et al
1996)
M. Letícia Ribeiro, CHC

HS – clinical features
Clinical severity of HS varies from symptom-free carrier to severe
hemolysis. Most individuals have mild to moderate disease
The diagnosis may be made at any time of life
Clinical manifestations:
Neonatal jaundice / Intermittent jaundice
Splenomegaly
depending on the co-inherency of Gilbert syndrome
Anemia
Post-infection hemolytic anemia
Aplastic crises (Parvovirus B19 infection)
Excellent response to splenectomy
Family history
M. Letícia Ribeiro, CHC

M. Letícia Ribeiro, CHC

HS Laboratory Characteristics
Anemia
Reticulocytes ↑
MCHC ↑
% Hyperdense cells ↑
Spherocytes
Unconjugated ↑
DAT neg
M. Letícia Ribeiro, CHC

120 fL
60 fL
Hyperchromic RBC
Normal HS
28 g/dL
41 g/dL
Methods: CBC+RET, CBC+RET,R
CELL-DYN ® SAPPHIRE
% hyperchromic RBC (CHC > 41g/dL)
Samples:
21 HS
51 controls
M. Letícia Ribeiro, CHC

HS - hyperchromic RBC
Statistical significant
correlation between
HS and % HPR RBC (>2.5%)
Test Mann Whitney (U)
Statistical significant association between
HS and the typical scatter CHC Distribution (Test χ2 )
U=1071
p

HS - Diagnosis
1. Screening tests
Osmotic fragility
AGLT (Pink test)
Cryohemolysis
Flow Cytometric (EMA)
Ektacytometry
2. Protein membrane
electrophoresis
3. Molecular studies
M. Letícia Ribeiro, CHC

Osmotic Fragility
Spherocytes take up less water in a
HS
hypotonic solution before rupturing than
entire do normal curve erythrocytes
“shifted to the right”, or
OF most gives of an it in indication the normal of the range volume-to- with a
surface tail of fragile ratio cells
curve Abnormal within OF normal invariably range indicates in 10-20% of
abnormal cases red cells
after OF 24h within incubation, the normal abnormalities range does not more
mean marked, normal but red still cells with some falsenegative
Practical Haematology, Dacie and Lewis, 10th Edition, 2006
Parpart, et al 1947
HS
AIHH
PK def.
M. Letícia Ribeiro, CHC

Acidified Glicerol Lyses-Time Test
AGLT
Time taken for 50%
hemolysis of a blood sample
in a buffered hypotonic
saline-glycerol mixture
Cells with a high volume-to-surface area ratio resist swelling
for a shorter time than normal cells
AGLT50:
: HS 25’’-150’’; normal >30’
AGLT 50
HS: sensitivity 98.3%; specificity 91.1% (Hoffman et al)
Short AGLT 50 in AIHA, HPFH, PK deficiency, severe G6PD,
pregnant women (1:3), CRF on dialysis (some), MDS
Special attention to the pH and osmolality
PINK TEST (Bucx, et al 1988) is a modified AGLT
Zanella, et al 1980
ctr
HS
M. Letícia Ribeiro, CHC

Cryohemolysis
Dependent on factors related to red cell
membrane molecular defects
Normal 3-15%, HS >20%
Increased hemolysis in HS and some CDAII
and SAO
For HS: sensitivity 95%; specificity 96% (Iglauer et
al, 1999)
Streichman and Gescheidt, 1998
M. Letícia Ribeiro, CHC

Flow Cytometric (Dye Binding) Test
Measures the fluorescent intensity of intact red cells labeled with
Eosin-5-Maleimide (EMA)
EMA binds to Band 3 Lys430 (80%), Rh blood group proteins, Rh
glycoprotein and CD47 (30%)
EMA binding Flow Cytometric test is efficient for HS
screening whatever the protein involved
Reduced fluorescence in CDAII,
cryohydrocytosis, SAO
Fluorescence intensity graded reduction
HPP< HS< HE≤ normal controls
Each lab should set the reference range
and cut-off values
King, et al 2000
F Girodon at al 2007
For HS: sensitivity 92.7%; specificity 99.1% Gallagher M. Letícia PG, Ribeiro, Jarolim CHC P

Racio
Diagnosis of HS by Flow Cytometry
Department of Haematology - Centro Hospitalar de Coimbra
n=181 - routine samples with normal hematological parameters;
n=183 n= 183 - samples from previously diagnosed patient with Hemolytic
Anemias (HA) of different types
Mean ratios and the 95% CI for each group
1.40
1.20
1.00
0.80
0.60
1.08
AIHA
1.10
nSHA
0.98
1.00
HMA
Controls
n=364
HS
0.71
1.07
0.96
Other HA
HE
Conclusions:
HS have significantly different
values from HA of other
aetiology, in special AIHA
HE values are quite similar to
controls
HS due to primary band 3
deficiency and HS due to
ankyrin/spectrin reduction have
no significant different values
M. Letícia Ribeiro, CHC

Osmotic gradient Ektacytometry
A laser diffraction viscometer that measures red
cell deformability at constant shear stress as
a continuous function of suspending
osmolality (hypotonic to hypertonic)
Results are plotted as a deformability curve,
which has a distinct shape for each type of
abnormal red cells tested
Distinct deformability curves for red cells from
patients with HS, HE, HPP, stomatocytosis
and sickle disease
Clark, et al 1983
M. Letícia Ribeiro, CHC

HS diagnosis - Screening tests
Take into account the :
sensitivity and specificity of the test
complexity of the protocol
cost of instruments and its maintenance
More specific tests:
Cryohemolysis – 95%
EMA binding - 99 %
(level IIa/III evidence, Grade B recommendation # )
Confirmation of diagnosis may be necessary if the
screening tests produce an equivocal or borderline
result
# Guidelines for the Diagnosis and Management of Hereditary Spherocytosis. General Haematology Task Force of the British Committee
for Standards in Haematology, 2004. (modified from Iolascon et al 1998))
M. Letícia Ribeiro, CHC

Protein Membrane Electrophoresis SDS-PAGE
SDS-PAGE Electrophoresis detects the qualitative and
quantitative membrane proteins alterations
Densitometry of the protein bands on the gel gives an overall
profile of spectrins, spectrins,
ankyrin, ankyrin,
band 3 and protein 4.2
Single ankyrin deficiency is not detectable in a non-
splenectomised HS patient with reticulocytosis
Laemmli
α-spectrin -spectrin
β-
ankirin
band 3
prot.4.1
prot.4.2
actin
G3PD
stomatin
Fairbanks
M. Letícia Ribeiro, CHC

Quantitation of membrane proteins
Quantitation of membrane proteins is not necessary for the
majority of HS cases
Very mild or HS carrier states (≈10% of HS patients) may
not have a detectable membrane protein deficiency
In CDAII - a more compact and faster migrating Band 3
In SAO - slower migrating Band 3
SDS-PAGE is recommended when:
the clinical phenotype is more severe than predicted from the
red cell morphology
the red cell morphology is more severe than predicted from
parental blood films where one parent is known to have HS
the diagnosis is not clear prior to splenectomy (MCV>100 fL)
M. Letícia Ribeiro, CHC

Hereditary Spherocytosis
SM
Normal
Band 3 Coimbra
488 (GTG - ATG) Val-Met
Band 3 Mondego
147 (CCT-TCT) Pro-Ser
Band 3 Montefiori
40(GAG-AAG) Glu-Lis
Hb. g/dL
MCV fL
16.4
99
MCH pg
MCHC %
35
34
Spherocytes -
Band N3
Protein 4.2 N
8.0
94
32
34
8.3
88
29
33
13,2
98
36
36
11.7
83
28
34
+++ +++
-
Additive effects of two unequally expressed
↓ 39 ↓ 40
N
AE1 mutant alleles can aggravate the
↓ 38 ↓ 36
N
clinical features of an affected individual
+
↓ 20
↓ 18
M. Letícia Ribeiro, CHC

Molecular studies
Almost 95% 95 of the HS-associated mutations identified were
private or sporadic occurrences
Knowledge of the gene mutation does not influence the
clinical management of the patient
Analysis of membrane protein genes
to establish the genetic basis of variable clinical
expressions among affected family members
to confirm recessive or de novo dominant mutations
when both parents are apparently normal
for Prenatal diagnosis
Prenatal diagnosis in families at risk of having a
child with a very severe form of HS
M. Letícia Ribeiro, CHC

Family with dominant HS
Hb g/dL 13.2 15.6
MCV fL 94 95
MCHC % 37 38
Ret % ? ?
Spherocytes ++ ++
Band 3 ↓ 20% ↓ 21%
Prot 4.2 ↓ 17% ↓ 18%
Heterozygous Band 3 Coimbra
AE1488 (GTG→ATG) (GTG ATG) Val→Met Val Met
2 years before the couple had a
stillborn baby (36 weeks of gestation)
M. Letícia Ribeiro, CHC

Family with dominant HS
Hb g/dL 5.2
MCV fL 147
MCH pg 49
MCHC % 49
Erythroblasts x10 9 /L 102
Platelets x10 9 /L 43
Bil total mmol/L 99
Bil unconj mmol/L 79
Metabolic acidosis
Hydropsis Fetalis
36 weeks of gestation
Laemmli 5% -15%
NB ctr
ctr
Band 3
Prot 4.2
Homozygous Band 3 Coimbra
AE1488 (GTG→ATG) (GTG ATG) Val→Met Val Met
M. Letícia Ribeiro, CHC

BSS - homozygous
↓
↓
Father - heterozygous
Prenatal Diagnosis
Band 3 Coimbra
488 (GTG→ATG)
(GTG ATG)
Fetus - heterozygous
↓
M. Letícia Ribeiro, CHC

Flow chart for the diagnosis of HS
Family History of HS;
typical clinical & laboratory features
No further
investigations needed
Dominant inheritance
Patient presenting Hemolytic Anemia
Variable clinical severity
in different family members
Search for co-inheriting
hematological disorders
β-Thalassemia or
Sickle Cell Disease
None
Atypical blood film, ?
Recent infection; no family history of HS
Screen proband family
for abnormal RBCs
HS RBCs indicated
in proband & sibling
Normal test
results
Not membrane
SDS-PAGE for
protein defect ? Thalassemia
? CDA ? MDS
DNA analysis for low-expression allele (mainly αSp)
Recessive or non-dominant inheritance
in proband with no family history
Guidelines for the Diagnosis and Management of Hereditary Spherocytosis. General Haematology Task Force of
the British Committee for Standards in Haematology, 2004. (modified from Iolascon et al M. 1998)) Letícia Ribeiro, CHC

References
1. Guidelines for the Diagnosis and Management of Hereditary Spherocytosis. P.H.B.
Bolton-Maggs, R. F. Stevens, N.J. Dodd, M-J. King, G. Lamont, Pl Tittensor, On behalf
of the General Haematology Task Force of the British Committee for Standards in
Haematology, 2004
2. Red Cell Membrane Disorders, Patrick G. Gallagher, Hematology 2005, The American
Society of Hematology
3. Practical Haematology, Dacie and Lewis, 10th ed. 2006 Churchill Livingstone
4. Hematology of Infancy and Childhood, Nathan and Oski's, Sixth Edition by David G.
Nathan, Stuart H. Orkin, A. Thomas Look, David Ginsburg
5. Cryohemolysis test as a diagnostic tool for hereditary spherocytosis, A. Iglauer D.
Reinhardt W. Schröter A. Pekrun, Ann Hematol (1999) 78: 555–557
6. Anaemia, a defective cytoskeleton and cation permeability, May-Jean King,
International Blood Group, Reference Laboratory, Southmead, Bristol, Biomedical
Science Congress
7. Diagnostic utility of the pre-incubated acidified glycerol lysis test in haemolytic and
non-haemolytic anaemias.Hoffmann JJ, Swaak-Lammers N, Breed WP, Strengers JL.
Eur J Haematol. 1991 Nov;47(5):367-70
8. Usefulness of the eosin-5'-maleimide cytometric method as a first-line screening test
for the diagnosis of hereditary spherocytosis: comparison with ektacytometry and
protein electrophoresis. Girodon F, Garçon L, Bergoin E, Largier M, Delaunay J,
Fénéant-Thibault M, Maynadié M, Couillaud G, Moreira S, Cynober. T. Br J Haematol.
2008 Feb;140(4):468-70
M. Letícia Ribeiro, CHC

Unidade de Anemias Congénitas
Centro Hospitalar de Coimbra
Celeste Bento
Helena Almeida
Elizabete Cunha
Janet Pereira
Umbelina Rebelo
Luís Relvas
www.chc-hematologia.org
M. Letícia Ribeiro, CHC