M. Letícia Ribeiro, CHC

Transcription

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

3 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, elasticity and deformability in circulation, under constant mechanical shear and stress forces, is attributed to its lipid layer and proteins Kightley Media CELLS alive

4 Vertical Interaction Erythrocyte Membrane Proteins Lux SE, Palek J: Disorders of the Red Cell Membrane Horizontal Interaction 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

5 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

6 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

7 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

8 Hereditary Elliptocytosis Prevalence 1:5000 among Caucasians (Protein 4.1 deficiency) 1:100 in certain African countries (Spectrin mutations) In the majority of HE individuals the anemia is very mild and often the elliptocytes are detected during a routine Hb 7-9 g/dl analysis Retic 11% Individuals with nonhemolytic HE Splenomegaly do notprotein have splenomegaly, and 4.1 deficiency their reticulocyte counts are slightly elevated to normal HE due to complete protein 4.1 deficiency is a severe hemolytic disease

9 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 SpαLELY

10 Pyropoikilocytosis DRS Hb g/dl MCV fl MCH pg MCHC % PBS Elliptocytes 3º day of life Hb g/dl 11.5 Bil mol/l 145 Anisopoikylocytosis Normal years old 37 Elliptocytes Normal Spectrin α 28 Arg-His (CGT-CAT) αlely αv/41 α(nt 1857 Leu-Val; CTA-GTA) Intron 45, nt 12 (C-T)

11 Southeast Asian Ovalocytosis Dominant inheritance SAO AE1 gene contains a deletion of 9 CD encoding amino acids , 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 MCV fl MCH 31 Hb g/dl pg MCHC RDW % %

12 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

13 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 SpαLEPRA allele (Low Expression Prague) is prevalent among nondominant HS (Boivin, et al 1993, Dhermy, et al 2000, Wichterle, et al 1996)

14 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 depending on the co-inherency of Gilbert syndrome Splenomegaly Anemia Post-infection hemolytic anemia Aplastic crises (Parvovirus B19 infection) Excellent response to splenectomy Family history

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16 HS Laboratory Characteristics Anemia Reticulocytes MCHC % Hyperdense cells Spherocytes Unconjugated DAT neg

17 Hyperchromic RBC Normal HS Methods: CBC+RET, CBC+RET,R CELL-DYN SAPPHIRE % hyperchromic RBC (CHC > 41g/dL) 120 fl 60 fl 28 g/dl 41 g/dl Samples: 21 HS 51 controls

18 HS - hyperchromic RBC Statistical significant correlation between HS and % HPR RBC (>2.5%) Test Mann Whitney (U) U=1071 p< Statistical significant association between HS and the typical scatter CHC Distribution (Test χ2) χ2 = 72 p<0.0001

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

20 Osmotic Fragility Parpart, et al 1947 Spherocytes take up less water in a HShypotonic solution before rupturing than entire curve erythrocytes shifted to the right, or do normal HS most of an it in the normal range with a OF gives indication of the volume-totail of fragile surface ratio cells curve withinof normal range in 10-20% Abnormal invariably indicates cases abnormal red cells of AIHH after incubation, OF 24h within the normalabnormalities range does notmore marked, butred stillcells with some falsemean normal negative Practical Haematology, Dacie and Lewis, 10th Edition, 2006 PK def.

21 Zanella, et al 1980 Acidified Glicerol Lyses-Time Test AGLT ctr Time taken for 50% hemolysis of a blood sample in a buffered hypotonic saline-glycerol mixture HS Cells with a high volume-to-surface area ratio resist swelling for a shorter time than normal cells AGLT50: HS ; normal >30 HS: 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 PINK TEST (Bucx, et al 1988) is a modified AGLT

22 Cryohemolysis Streichman and Gescheidt, 1998 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)

23 Flow Cytometric (Dye Binding) Test King, et al 2000 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 F Girodon at al 2007 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 For HS: sensitivity 92.7%; specificity 99.1% Gallagher PG, Ribeiro, Jarolim CHC P M. Letícia

24 Diagnosis of HS by Flow Cytometry Department of Haematology - Centro Hospitalar de Coimbra n=181 - routine samples with normal hematological parameters; 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 Conclusions: n= Racio 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 AIHA HMA nsha HS Controls Other HA HE

25 Osmotic gradient Ektacytometry Clark, et al 1983 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

26 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, (modified from Iolascon et al 1998))

27 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, ankyrin, band 3 and protein 4.2 Single ankyrin deficiency is not detectable in a nonsplenectomised HS patient with reticulocytosis Laemmli Fairbanks α-spectrin βankirin band 3 prot.4.1 prot.4.2 actin G3PD stomatin

28 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)

29 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 16.4 MCV 99 fl MCH pg 35 MCHC 34 % Spherocytes BandN3 Protein 4.2 N 13, Additive effects of two unequally expressed N AE1 mutant alleles can aggravate the N clinical features of an affected individual

30 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 in families at risk of having a child with a very severe form of HS

31 Family with dominant HS Heterozygous Band 3 Coimbra AE1488 (GTG ATG) Val Met Hb g/dl MCV fl MCHC % Ret % Spherocytes ? ? ++ Band 3 Prot % 17% 21% 18% 2 years before the couple had a stillborn baby (36 weeks of gestation)

32 Family with dominant HS Hydropsis Fetalis 36 weeks of gestation Laemmli Hb MCV fl MCH 49 g/dl pg MCHC % Erythroblasts x109/l Bil total mmol/l Bil unconj 79 Platelets x109/l mmol/l Metabolic acidosis NB 5% -15% ctr ctr Band 3 Prot 4.2 Homozygous Band 3 Coimbra AE1488 (GTG ATG) Val Met

33 Prenatal Diagnosis BSS - homozygous Father - heterozygous Band 3 Coimbra 488 (GTG ATG) Fetus - heterozygous

34 Flow chart for the diagnosis of HS Patient presenting Hemolytic Anemia Family History of HS; typical clinical & laboratory features Atypical blood film,? Recent infection; no family history of HS Dominant inheritance Screen proband family for abnormal RBCs No further investigations needed Variable clinical severity in different family members HS RBCs indicated in proband & sibling Search for co-inheriting hematological disorders β-thalassemia or Sickle Cell Disease None 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, (modified from Iolascon et alm.1998)) Letícia Ribeiro, CHC

35 References 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 Red Cell Membrane Disorders, Patrick G. Gallagher, Hematology 2005, The American Society of Hematology Practical Haematology, Dacie and Lewis, 10th ed Churchill Livingstone Hematology of Infancy and Childhood, Nathan and Oski's, Sixth Edition by David G. Nathan, Stuart H. Orkin, A. Thomas Look, David Ginsburg Cryohemolysis test as a diagnostic tool for hereditary spherocytosis, A. Iglauer D. Reinhardt W. Schröter A. Pekrun, Ann Hematol (1999) 78: Anaemia, a defective cytoskeleton and cation permeability, May-Jean King, International Blood Group, Reference Laboratory, Southmead, Bristol, Biomedical Science Congress 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 Nov;47(5): 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 Feb;140(4):468-70

36 Unidade de Anemias Congénitas Centro Hospitalar de Coimbra Celeste Bento Helena Almeida Elizabete Cunha Janet Pereira Umbelina Rebelo Luís Relvas