m. letícia ribeiro, chc - enerca · 2017-09-13 · m. letícia ribeiro, chc red blood cells...

M. Letícia Ribeiro, CHC


3rd European Symposium on Rare Anaemias

Madrid, Nov 2010Madrid, Nov 2010

LABORATORY DIAGNOSIS OF RARE LABORATORY DIAGNOSIS OF RARE

ANAEMIASANAEMIAS

Hereditary RBC membrane defectsHereditary RBC membrane defects

M. Leticia RibeiroM. Leticia Ribeiro


Red Blood CellsRed Blood Cells

Kightley Media

CELLS alive

RBCs are RBCs are biconcavebiconcave under under physiological conditionsphysiological conditions

Their shape changes when navigating Their shape changes when navigating narrow blood vessels or confined narrow blood vessels or confined spaces in tissues and organsspaces in tissues and organs

The ability of red cell to maintain its The ability of red cell to maintain its discoid shapediscoid shape, , elasticityelasticity and and deformabilitydeformability in circulation, under in circulation, under constant mechanical shear and stress constant mechanical shear and stress forces, is attributed to its forces, is attributed to its lipid layerlipid layer and and proteinsproteins


Erythrocyte Membrane ProteinsErythrocyte Membrane Proteins

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 -

HemolysisHemolysis

Lux SE, Palek J:Disorders of the Red Cell Membrane

Horizontal Interaction

Ver

tica

l In

tera

ctio

n


Red Blood Cell Membrane DisordersRed Blood Cell Membrane Disorders

congenital hemolytic anemias characterized by clinical, laboratory and genetic heterogeneity

Hereditary SpherocytosisHereditary Spherocytosis

Hereditary ElliptocytosisHereditary Elliptocytosis

Hereditary PyropoikilocytosisHereditary Pyropoikilocytosis

Hereditary Southeast Asian OvalocytosisHereditary Southeast Asian Ovalocytosis

Hereditary StomatocytosisHereditary Stomatocytosis


Hereditary StomatocytosisHereditary Stomatocytosis

a group of dominantly inherited hemolytic anemias with abnormal membrane permeability to univalent cations

HydrocytosisHydrocytosis – Overhydrated stomatocytosis

XerocytosisXerocytosis – Dehydrated stomatocytosis

CryohydrocytosisCryohydrocytosis

Familial PseudohyperkaliemiaFamilial Pseudohyperkaliemia



By Rex Graham

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

Skeletal network electron mycroscopy, by Yawata et al

Normal Pr 4.1 absent



PrevalencePrevalence

1:5000 among Caucasians (1:5000 among Caucasians (Protein 4.1 deficiencyProtein 4.1 deficiency))

1:100 in certain African countries (1:100 in certain African countries (Spectrin mutationsSpectrin mutations) )

In the majority of HE individuals the anemia is very mild In the majority of HE individuals the anemia is very mild and often the elliptocytes are detected during a and often the elliptocytes are detected during a routine routine analysisanalysis

Individuals with Individuals with nonhemolytic HEnonhemolytic HE do do not have splenomegalynot have splenomegaly, and , and their reticulocyte counts are slightly their reticulocyte counts are slightly elevated to normalelevated to normal

HE due to HE due to complete protein 4.1 deficiency is a severe complete protein 4.1 deficiency is a severe hemolytic diseasehemolytic disease

Hb 7-9 g/dL

Retic 11%

SplenomegalyProtein 4.1 deficiency


PyropoikilocytosisPyropoikilocytosis

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 SpαLELY


D R S

Normal

αLELY αV/41 α(nt 1857 Leu-Val; CTA-GTA) Intron 45, nt 12 (C-T)Spectrin α 28 Arg-His (CGT-CAT)

Pyropoikilocytosis Pyropoikilocytosis

3º day of life3º day of life

3 years old3 years old

Hb g/dL MCV fL MCH pg MCHC %PBS

14.1 9229 31Elliptocytes

13.4 9627 29Normal

13.3 8030 37Elliptocytes

Hb g/dL 11.5

Bil mol/L 145

Anisopoikylocytosis


Southeast Asian OvalocytosisSoutheast Asian Ovalocytosis

Dominant inheritance

SAO AE1 gene contains a deletion of 9 CDdeletion of 9 CD encoding amino acids 400-408, at the boundary of the cytoplasmic and membrane domains, in cis with Lys56GluLys56Glu substitution

SAO red cells are rigid and hiperstablerigid and hiperstable

Hemolysis is mild or absent

Fem, 27 years oldFem, 27 years old

PregnantPregnant

Origin: East TimorOrigin: East Timor

Hb Hb g/dLg/dL 11.111.1

MCVMCV fL fL 8989

MCH MCH pgpg 3131

MCHC MCHC %% 34.534.5

RDW RDW %% 1717


Hereditary SpherocytosisHereditary SpherocytosisMembrane lesions involving the vertical interactionsvertical interactions between skeleton and lipid bilayer lead to vesiculationvesiculation of the unsupported surface components, causing a progressive reduction in membrane surface areareduction in membrane surface area.

from Lux SE, Palek J: Disorders of the Red Cell Membrane, Blood Principles and Pratice of Hematology

The red cell shape changes from a flexible, deformable bi-concave disc to a spherical poorly deformable red cell – the

spherocytespherocyteThe severity of hemolysis depends on the contents of Spectrin



The commonest cause of inherited chronic hemolysis in Northern The commonest cause of inherited chronic hemolysis in Northern Europe and North America - Prevalence 1:5000 to 1:2000Europe 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 novode novo dominant mutations dominant mutations in HS patients with normal parents is 6xs more common6xs 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 SpαSpαLEPRALEPRA allele allele (Low Expression Prague) is prevalent among non-dominant HS (Boivin, et al 1993, Dhermy, et al 2000, Wichterle, et al 1996)


HS – clinical featuresHS – clinical featuresClinical 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 Neonatal jaundice / Intermittent jaundice

depending on the co-inherency of Gilbert syndrome Splenomegaly Splenomegaly

AnemiaAnemia Post-infection hemolytic anemiaPost-infection hemolytic anemia Aplastic crises Aplastic crises (Parvovirus B19 infection)

Excellent response to splenectomyExcellent response to splenectomy

Family historyFamily history


HS Laboratory CharacteristicsHS Laboratory Characteristics

Anemia Reticulocytes ↑ MCHC ↑ % Hyperdense cells ↑ Spherocytes Unconjugated ↑

DAT neg


Hyperchromic RBC Hyperchromic RBC

Samples:Samples:

21 HS 21 HS

51 controls51 controls

28 g/dL

120 fL

60 fL

41 g/dL

Methods: CBC+RET, CBC+RET,RMethods: CBC+RET, CBC+RET,RCELL-DYN ® SAPPHIRE CELL-DYN ® SAPPHIRE % hyperchromic RBC (CHC > 41g/dL)% hyperchromic RBC (CHC > 41g/dL)

NormalNormal HSHS


Statistical significant Statistical significant

correlation between correlation between

HS and % HPR RBCHS and % HPR RBC (>2.5%) (>2.5%)

Test Mann Whitney (U) Test Mann Whitney (U)

Statistical significant association between Statistical significant association between HS and the typical scatterHS and the typical scatter CHC Distribution (Test CHC Distribution (Test χ2))

χ2 = 72 p<0.0001

U=1071p<0.0001

HS - hyperchromic RBC HS - hyperchromic RBC


HS - DiagnosisHS - Diagnosis

1.1. Screening testsScreening tests

Osmotic fragilityOsmotic fragility

AGLT (Pink test)AGLT (Pink test)

CryohemolysisCryohemolysis

Flow Cytometric (EMA)Flow Cytometric (EMA)

Ektacytometry Ektacytometry

2.2. Protein membrane Protein membrane electrophoresiselectrophoresis

3.3. Molecular studiesMolecular studies


Osmotic FragilityOsmotic Fragility

Practical Haematology, Dacie and Lewis, 10th Edition, 2006

Parpart, et al 1947

PK def.PK def.

AIHHAIHH

HSHSSpherocytes take up less water in a hypotonic solution before rupturing than do normal erythrocytes

OF gives an indication of the volume-to-surface ratio

Abnormal OF invariably indicates abnormal red cells

OF within the normal range does not mean normal red cells

HSHS entire curve “shifted to the right”, or

most of it in the normal range with a tail of fragile cells

curve within normal range in 10-20% of cases

after 24h incubation, abnormalities more marked, but still with some false-negative


Acidified Glicerol Lyses-Time TestAcidified Glicerol Lyses-Time Test

Cells with a high volume-to-surface area ratio resist swelling for a shorter time than normal cells

AGLTAGLT5050: HS 25’’-150’’; normal >30’: HS 25’’-150’’; normal >30’ HS: sensitivity 98.3%; specificity 91.1%sensitivity 98.3%; specificity 91.1% (Hoffman et al)

Short AGLT50 in AIHA, HPFH, PK deficiency, severe G6PD,

pregnant women (1:3), CRF on dialysis (some), MDS

Special attention to the pH and osmolality

HSHS

ctrctr

Zanella, et al 1980

PINK TESTPINK TEST (Bucx, et al 1988) is a modified AGLT

AGLTAGLT Time taken for 50%

hemolysis of a blood sample in a buffered hypotonic saline-glycerol mixture


CryohemolysisCryohemolysis

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


Flow Cytometric (Dye Binding) TestFlow 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 EMA binding Flow Cytometric test is efficient for HS

screening whatever the protein involvedscreening 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

For HS: sensitivity 92.7%; specificity 99.1%For HS: sensitivity 92.7%; specificity 99.1%Gallagher PG, Jarolim P

F Girodon at al 2007


n=181n=181 - routine samples with normal hematological parameters;

n=n=183183 - samples from previously diagnosed patient with Hemolytic Anemias (HA) of different types

Diagnosis of HS by Flow CytometryDiagnosis of HS by Flow Cytometry

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

Department of Haematology - Centro Hospitalar de Coimbra

Error Bars show Mean +/- 2.0 SD

AIHAnSHA

HMAControls

HSHE

Other HA

Groups

0.60

0.80

1.00

1.20

1.40

Raci

o

1.08 1.10

0.98 1.00

0.71

0.96

1.07

Mean ratios and the 95% CI for each group

n=364n=364


Osmotic gradient EktacytometryOsmotic gradient Ektacytometry

A laser diffraction viscometer that A laser diffraction viscometer that measures red measures red cell deformability at constant shear stresscell deformability at constant shear stress as as a continuous function of suspending a continuous function of suspending osmolality (hypotonic to hypertonic)osmolality (hypotonic to hypertonic)

Results are plotted as a deformability curve, Results are plotted as a deformability curve, which has a distinct shape for each type of which has a distinct shape for each type of abnormal red cells testedabnormal red cells tested

Distinct deformability curvesDistinct deformability curves for red cells from for red cells from patients with HS, HE, HPP, stomatocytosis patients with HS, HE, HPP, stomatocytosis and sickle diseaseand sickle disease

Clark, et al 1983


HS diagnosis - HS diagnosis - Screening testsScreening tests

Take into account the :Take into account the : sensitivity and specificity of the testsensitivity and specificity of the test complexity of the protocolcomplexity of the protocol cost of instruments and its maintenancecost of instruments and its maintenance

More specific tests: More specific tests: Cryohemolysis – 95% Cryohemolysis – 95% EMA binding - 99 %EMA binding - 99 %(level IIa/III evidence, Grade B recommendation#)

Confirmation of diagnosis may be necessary if the Confirmation of diagnosis may be necessary if the screening tests produce an equivocal or borderline screening tests produce an equivocal or borderline resultresult

#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))


Protein Membrane Electrophoresis SDS-PAGE Protein Membrane Electrophoresis SDS-PAGE

SDS-PAGE Electrophoresis detects the SDS-PAGE Electrophoresis detects the qualitativequalitative and and quantitativequantitative membrane proteins alterations membrane proteins alterations

Densitometry of the protein bands on the gel gives an overall Densitometry of the protein bands on the gel gives an overall profile of profile of spectrinsspectrins, , ankyrinankyrin, , band 3band 3 and and protein 4.2protein 4.2

Single Single ankyrin deficiencyankyrin deficiency is not detectable in a non- is not detectable in a non-splenectomised HS patient with reticulocytosissplenectomised HS patient with reticulocytosis

FairbanksLaemmli

G3PDG3PDactinactin

αα-spectrin-spectrinββ--

ankirinankirin

band 3band 3

prot.4.1prot.4.1prot.4.2prot.4.2

stomatinstomatin


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)



Hb. g/dL MCV fL MCH pg MCHC %

SpherocytesBand 3

Protein 4.2

16.4 9935 34 -NN

8.0 94 32 34+++↓ 39↓ 38

8.3 88 29 33+++↓ 40↓ 36

11.7 83 28 34 -NN

13,2 98 36 36 +↓ 20↓ 18

Normal

Band 3 Mondego 147 (CCT-TCT) Pro-Ser

Band 3 Montefiori 40(GAG-AAG) Glu-Lis

Band 3 Coimbra 488 (GTG - ATG) Val-Met

SMSM

Additive effects of Additive effects of two unequally expressedtwo unequally expressedAE1 mutant allelesAE1 mutant alleles can aggravate the can aggravate the clinical features of an affected individualclinical features of an affected individual


Molecular studiesMolecular studies

Almost 9595% 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 genesAnalysis of membrane protein genes

to establish the genetic basis of variable clinical variable clinical expressionsexpressions among affected family members

to confirm recessive or de novo dominant mutations when both parents are apparently normal

for Prenatal diagnosisPrenatal diagnosis in families at risk of having a child with a very severe form of HS


Family with dominant HS

Hb g/dL 13.2 15.6MCV fL 94 95MCHC % 37 38Ret % ? ?Spherocytes ++ ++

2 years before the couple had a stillborn baby (36 weeks of gestation)

HeterozygousHeterozygous Band 3 CoimbraBand 3 CoimbraAE1488 (GTGAE1488 (GTG→→ATG) ValATG) Val→→MetMet

Band 3 ↓ 20% ↓ 21%Prot 4.2 ↓ 17% ↓ 18%


Family with dominant HS

Hb Hb g/dLg/dL 5.25.2MCVMCV fL fL 147147MCH MCH pgpg 4949

MCHC MCHC %% 4949Erythroblasts Erythroblasts x10x1099//L 102102Platelets Platelets x10x1099//L 4343Bil total Bil total mmol/Lmmol/L 9999Bil unconj Bil unconj mmol/Lmmol/L 7979Metabolic acidosisMetabolic acidosis

Laemmli 5% -15%

Band 3Band 3

Prot 4.2Prot 4.2

NBNB ctrctrctrctr

Homozygous Band 3 CoimbraHomozygous Band 3 CoimbraAE1488 (GTGAE1488 (GTG→→ATG) ValATG) Val→→MetMet

Hydropsis Fetalis 36 weeks of gestation


Band 3 Coimbra Band 3 Coimbra 488 (GTG488 (GTG→→ATG)ATG)

↓

FatherFather - heterozygous - heterozygous

↓

FetusFetus - heterozygous - heterozygous

BSSBSS - homozygous - homozygous

↓

Prenatal DiagnosisPrenatal Diagnosis


Patient presenting Hemolytic AnemiaPatient presenting Hemolytic Anemia

Family History of HS; typical clinical & laboratory features

Family History of HS; typical clinical & laboratory features

Atypical blood film, ? Recent infection; no family history of HS

Atypical blood film, ? Recent infection; no family history of HS

No further investigations needed

No further investigations needed

Variable clinical severity in different family membersVariable clinical severity

in different family members

Search for co-inheritinghematological disordersSearch for co-inheritinghematological disorders

β-Thalassemia or Sickle Cell Diseaseβ-Thalassemia or Sickle Cell Disease NoneNone

Screen proband family for abnormal RBCs

Screen proband family for abnormal RBCs

Normal test results

Normal test results

HS RBCs indicated in proband & siblingHS RBCs indicated in proband & sibling

SDS-PAGE forprotein defectSDS-PAGE forprotein defect ? Thalassemia

? CDA ? MDS? Thalassemia? CDA ? MDS

DNA analysis for low-expression allele (mainly αSp)Recessive or non-dominant inheritance

in proband with no family history

DNA analysis for low-expression allele (mainly αSp)Recessive or non-dominant inheritance

in proband with no family history

Dominant inheritanceDominant inheritance

Not membraneNot membrane

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))

Flow chart for the diagnosis of HS


References 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 Livingstone4. Hematology of Infancy and Childhood, Nathan and Oski's, Sixth Edition by David G.

Nathan, Stuart H. Orkin, A. Thomas Look, David Ginsburg5. Cryohemolysis test as a diagnostic tool for hereditary spherocytosis, A. Iglauer D.

Reinhardt W. Schröter A. Pekrun, Ann Hematol (1999) 78: 555–5576. 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


Unidade de Anemias CongénitasUnidade de Anemias CongénitasCentro Hospitalar de CoimbraCentro Hospitalar de Coimbra

Celeste BentoHelena Almeida Elizabete CunhaJanet PereiraUmbelina Rebelo Luís Relvas

www.chc-hematologia.org

m. letícia ribeiro, chc - enerca · 2017-09-13 · m. letícia ribeiro, chc red blood cells...

Documents