MICO Maggio 2016 Laboratory Diagnosis of Thalassemia

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1 MICO Maggio 2016 Laboratory Diagnosis of Thalassemia Maria Domenica Cappellini Fondazione Ca Granda Policlinico IRCCS University of Milan

2 Disclosure Member of Advisory Board: - Novartis - Genzyme/Sanofi - Celgene - Shire - Merganser Biotech - Isis

3 Agenda Definition and pathophysiology of Thalassemias Classification Carrier diagnosis NTDT diagnosis TDT diagnosis

4 Classification of the thalassaemias Group of inherited haemoglobin disorders Absence or reduced synthesis of α chains of Hb α-thalassaemias Absence or reduced synthesis of β chains of Hb β-thalassaemias Muncie HL, Campbell JS. Am Fam Physician. 2009;80:

5 Globin genes cluster Chromosome 16 ζ α 2 α 1 Chromosome 11 ε Gγ Aγ δ β Embryonics: ζ 2 ε 2 Gower 1 ζ 2 γ 2 Portland α 2 ε 2 Gower 2 Fetals: α G o A 2 γ 2 HbF Adults α 2 β 2 HbA α 2 δ 2 HbA2

6 Globin synthesis at various stages of embryonic and fetal development Megaloblast Macrocyte Normocyte S ite of cell erythro-type poies is Liver Bone Marrow 50 Yolk sac α Spleen α 40 γ β ζ ε β δ γ B irth Post-conceptual age (weeks) Postnatal age (weeks)

7 Thalassemias are a group of inherited hemoglobinopathies Absence or reduced synthesis of α chains of Hb Absence or reduced synthesis of β chains of Hb α thalassemias 1 α thalassemia silent carrier (single α gene deletion) α thalassemia trait minor (double α gene deletion) Hb constant spring (reduced output of α globin) Hb H disease (triple α gene deletion) Hb Barts Hydrops (absence of α genes) 1. Muncie HL and Campbell JS. Am Fam Physician 2009;80: ; 2. Galanello R and Origa R. OrphanetJ Rare Dis 2010;5:11. β thalassemias 2 β thalassemia minor (silent or carrier ) β thalassemia intermedia β thalassemia minor β thalassemia with Hb anomalies Hb C/β thalassemia Hb E/β thalassemia Hb S/β thalassemia Hereditary Hb F and β thalassemia β-thalassemia associated with Trichothiodystrophy X-linked thrombocytopenia

8 Thalassemia has a broad clinical spectrum, complicating diagnosis and management NTDT patients do not require regular red cell transfusions but may require occasional transfusions for growth failure, pregnancy, infections and other specific situations 1 4 NTDT β thalassemia intermedia Mild/moderate Hb E/β thalassemia Hb H disease (α thalassemia) Hb S β thalassemia Hb C thalassemia Transfusions seldom required Occasional transfusions required (eg surgery, pregnancy, infection) Intermittent transfusions required (eg poor growth and development, specific morbidities) Regular, lifelong transfusions required for survival Transfusions not required α thalassemia trait β thalassemia minor 1.Taher AT et al. Br J Haematol 2011;152: ; 2. Galanello R and Origa R. Orphanet Journal of Rare Diseases 2010;5:11; 3. Vichinsky E. Hematology Am Soc Hematol Educ Program 2007;79 83; 4. Muncie HL and Campbell JS. Am Fam Physician 2009;80: ; 5. Figure adapted from Musallam KM et al. Haematologica 2013;98: Transfusion-dependent thalassemia (TDT) β thalassemia major Severe Hb E/β thalassemia Hb Barts hydrops (α thalassemia major).

9 Agenda Definition and patophysiology of Thalassemias Classification Carrier diagnosis TDT diagnosis NTDT diagnosis

10 Epidemiology of thalassemia: Carrier frequencies of thalassemia alleles (%) Region b-thalassemia a 0 -thalassemia a + -thalassemia Americas Eastern Mediterranean Europe Southeast Asia Sub-Saharan Africa Western Pacific Weatherall D et al. Inherited Disorders of Hemoglobin Disease Control Priorities in Developing Countries (2nd Edition).

11 Several assessments can assist diagnosis Family history Laboratory exams Diagnosis should incorporate as much information as possible, utilizing family history, and laboratory examinations 1. Taher A et al. Guidelines for the management of NTDT. 2013;TIF Publication No. 19; 2. Weatherall DJ. Blood Rev 2012;26S:S3 S6.

12 Primary haematology tests Full medical history and family history Complete blood cell count with erythrocyte indices Blood smear/ BCB staining _ Other causes of anaemia? Low MCV (<80 fl) + Low MCH (<27 pg) Serum ferritin <12 ng/ml Microcytosis Hypochromia Consider iron deficiency anaemia

13 Prevalence of anaemia by aetiology Females Males Kassebaum NJ, et al. Blood 2014;123:615-24

14 ID and ID(A): Definition Absolute Iron Deficiency (no iron stores) Low Transferrin Saturation (TSAT<20%) Serum ferritin low (<20ng/ml or <100ng/ml in case of inflammation) Elevated Serum Transferrin Receptor (stfr) Iron Deficiency Depleted iron stores Functional Iron Deficiency (full iron stores but ID in erythroid bone marrow) Low TSAT (TSAT<20%) Serum ferritin normal or elevated (>20 ng/ml or >100ng/ml in case of inflammation) Normal stfr Iron Deficiency Anaemia Depleted iron stores Haemoglobin (Hb) concentration falls below defined lower limit (12g/dl for women, 13 g/dl for men) Adapted from Hush R. and Schaefer R. Pocket Atlas Special. Thiene 2006 and Prof. IY. Beguinoral communication. Parallel Symposia at EHA congress 2014, Milan, Italy

15 Primary haematology tests Full medical history and family history Complete blood cell count with erythrocyte indices Blood smear/ BCB staining _ Other causes of anaemia? Low MCV (<80 fl) + Low MCH (<27 pg) Serum ferritin <12 ng/ml Microcytosis Hypochromia Adequate iron supplement for 3 months Consider iron deficiency anemia

16 Primary haematology tests Full medical history and family history Complete blood cell count with erythrocyte indices Blood smear/ BCB staining _ Other causes of anaemia? Low MCV (<80 fl) + Low MCH (<27 pg) Serum ferritin > 12 ng/ml Microcytosis Hypochromia Target cells +/- Inclusion bodies (Hb H)

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18 Formulas based on parameters from the CBC The most commonly used formulas are the following: Mentzer index (MI) = MCV/RBC Discriminant factor (DF) = MCV x (RDW/Hb 9 x100) Shine and Lal Index (S&L) = MCV x (MCH/100) Srivastava Index (SI) = MCH/RBC; RDW index (RDWI) = (MCV 9 RDW)/RBC Chatterjee T, Chakravarty A, Chakravarty S. Hemoglobin 2015;39:384 8.

19 Full medical history and family history Complete blood cell count with erythrocyte indices Blood smear/ BCB staining _ + I. Primary haematolog y tests II. Type of thalassaemia Low MCV (<80 fl) + Low MCH (<27 pg) Microcytosis Hypochromia Target cells +/- Inclusion bodies (Hb H) Serum ferritin >12 ng/ml Hb electrophoresis & HPLC Other causes of anemia? Serum ferritin <12 ng/ml Consider iron deficiency anemia Adequate iron supplement or 3 months No improvement HbA 2 4% HbF 0.1-5% HbA 2 < 4% HbF < 1% β-thalassaemia trait α-thalassaemia traits and related disorders*

20 HPLC and electrophoresis examples Hb electrophoresis of Hb H disease Vip Viprakasit. EHA Educational Book 2013.

21 Agenda Definition and patophysiology of Thalassemias Classification Carrier diagnosis NTDT diagnosis TDT diagnosis

22 Thalassemia has a broad clinical spectrum, complicating diagnosis and management NTDT patients do not require regular red cell transfusions but may require occasional transfusions for growth failure, pregnancy, infections and other specific situations 1 4 NTDT β thalassemia intermedia Mild/moderate Hb E/β thalassemia Hb H disease (α thalassemia) Hb S β thalassemia Hb C thalassemia Transfusions seldom required Occasional transfusions required (eg surgery, pregnancy, infection) Intermittent transfusions required (eg poor growth and development, specific morbidities) Regular, lifelong transfusions required for survival Transfusions not required α thalassemia trait β thalassemia minor 1.Taher AT et al. Br J Haematol 2011;152: ; 2. Galanello R and Origa R. Orphanet Journal of Rare Diseases 2010;5:11; 3. Vichinsky E. Hematology Am Soc Hematol Educ Program 2007;79 83; 4. Muncie HL and Campbell JS. Am Fam Physician 2009;80: ; 5. Figure adapted from Musallam KM et al. Haematologica 2013;98: Transfusion-dependent thalassemia (TDT) β thalassemia major Severe Hb E/β thalassemia Hb Barts hydrops (α thalassemia major).

23 Diagnosis of NTDT is challenging due to the diversity of genotype interactions and limited understanding of the disease Weaker understanding of milder forms Diagnosis can be more difficult NTDT Excellent understanding of severe forms such as β thalassemia major Diagnosis is clear TDT Weaker Moderate Strong Early identification is key to ensure the most appropriate monitoring and treatment 1. Musallam KM. Haematologica 2013;98:

24 Several laboratory tests are available which together contribute to an NTDT diagnosis Hb levels Blood smear Other RBC findings HPLC electrophoresis DNA analysis (not always available) Diagnosis should incorporate as much information as possible, utilizing patient history, physical and laboratory examinations HPLC, high-performance liquid chromatography; RBC, red blood cells 1. Taher AT et al. Blood Cells Mol Dis 2006;37:12 20; 2. Galanello R and Origa R. Orphanet J Rare Dis 2010;5:11; 3. Harteveld C and Higgs D. Orphanet J Rare Dis 2010;5:13.

25 Physical examination: common symptoms that can help distinguish and diagnose different forms of NTDT β TI Hb E/β thalassemia Hb H Splenomegaly Varying degree of enlargement 1 Common 2 Common 3,4 Hepatobiliary Skeletal Infections Vascular/heart Gallstones 4 Moderate to severe liver enlargement 4 Gallstones 2 Growth retardation 1 Expansion of facial bones 1 Obliteration of maxillary sinuses 1 Protrusion of upper jaw 1 Extramedullary hematopoiesis 1 Growth retardation 2 Extramedullary hematopoiesis 2 Gallstones 3,5 Variable jaundice 3,5 Enlarged liver 6 Increased susceptibility 1 Septicemia 2 Infections 5 Leg ulcers 1 Leg ulcers 2 Leg ulcers 5 Pulmonary hypertension 1 Cardiovascular disease 1 Congestive heart failure 2 Growth retardation3,5 Dysmorphic facial features 6 Hb, hemoglobin 1. Taher AT et al. Blood Cells Mol Dis 2006;37:12 20; 2. Fucharoen S et al. J Pediatr Hematol Oncol 2000;22: ; 3. Fucharoen S and Viprakasit V. Hematology Am Soc Hematol Educ Program 2009:26 34; 4. Thalassaemia International Federation. Guidelines for the Clinical Management of thalassaemia, 2nd edition revised 2008; 5. Harteveld C and Higgs D. Orphanet J Rare Dis 2010;5:13 34; 6. Laosombat V et al. Ann Hem 2009;88:

26 Laboratory examination: parameters that can help distinguish and diagnose different forms of NTDT β TI Hb E/β thalassemia Hb H Hb levels ~7 10 g/dl 1 Blood smear HPLC electrophoresis DNA analysis Basophilic stippling 6 Nucleated RBC 6 Hb F 10 50% (up to 100%) 5 Hb A 2 >4% 5 Genetic analysis should be performed in event of abnormal hematology findings 2 Mild 2 Moderately Severe 2 Severe g/dl 6 7 g/dl 4 5 g/dl Target cells 7 Red cell hypochromia 7 Microcytes 7 Nucleated RBC 7 hemolysis 4 Hb E and F 6 Hb A 2 9 To distinguish between different Hb E disorders g/dl 3 Microcytosis 8 Hypochromia 8 Target cells 8 Inclusion bodies 8 Irregularly crenated RBC 8 Increased reticulocytes (5 10%) 8 α/β-globin chain synthesis ratio measurement 3 RBC indices 3 Hb A 2 3 Variable Hb H (0.8 40%) and occasional Hb Barts hydrops 3 Gap-PCR developed for seven common α thalassemia 3 For unknown rearrangements, Southern Blotting or MLPA analysis required 3 MLPA, multiplex ligation-dependent probe amplification PCR, polymerase chain reaction 1. Taher AT et al. Blood Cells Mol Dis 2006;37:12 20; 2. Galanello R et al. Orphanet J Rare Dis 2010;5:11; 3. Harteveld C et al. Orphanet J Rare Dis 2010;5:13; 4.Vichinsky E. Hematology Am Soc Hematol Educ Program 2007;79 83; 5. Thalassaemia International Federation. Guidelines for the Clinical Management of thalassaemia, 2nd edition revised 2008; 6. Yaish HM et al Fucharoen S et al. Clin Chem 1998;44:

27 Different analysis platforms can help distinguish between Hb abnormalities in NTDT Percentage F HPLC: Biorad Time (minutes) A Isoelectric focusing N A F S C Capillary electrophoresis LPLC: Hb Gold Peak RT(s) % of Hb A % of Hb 1 Unknown F Window Unknown Cellulose acetate electrophoresis Name Rack: SEBIA Pos.: 5 Sample: num.: 29 Date: 8/28/2013 ID: Z15 Z14Z13 Z12 Z11Z10 Z9 Z8 Z7 Z6Z5 Z4 Z3 Z2 Z1 Hb A 4 A0 Window A2 Window s Area=797 Base line=1907 Hc F Hb A LPLC, low-pressure liquid chromatography Viprakasit V, unpublished data 2013.

28 Diagnostic work-up of NTDT Primary hematology tests Full medical history Family history (screen families in high risk areas) Complete blood cell count with erythrocyte indices (use an automated blood cell counter) Blood smear/bcb staining + Low MCV (<80 fl) ± Low MCH (<27 pg) Microcytosis Hypochromia Target cells ± Inclusion bodies (Hb H) Serum ferritin >12 ng/ml _ Consider other causes of anemia Serum ferritin 12 ng/ml Consider iron deficiency anemia Adequate iron supplement for 3 months Screen for type of thalassemia Hb A 2 4% Hb F 0.1 5% Hb electrophoresis and HPLC Hb A 2 <4% Hb F <1% Hb A 2 4% Hb F >5 50% Hb H <5 25% Hb A 2 <4% ±Hb CS/PS No improvement + Other normal Hb variant Genetic testing β thalassemia trait α thalassemia traits and related disorders β thalassemia intermedia Hb H disease Hb E disorders Hb S disorders Hb C disorders Others DNA analysis for α and β globin mutations BCB, brilliant cresyl blue; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin Viprakasit V et al. Personal communication.

29 β thalassemia intermedia In β TI, red blood cell abnormalities can help distinguish the disease; however, diagnosis remains largely a clinical decision Basophilic stippling 2 Diagnoses remains largely clinical Characterized by: Hb levels maintained ~7 10 g/dl without need for regular transfusions 1 More severe red blood cell abnormalities than thalassemia minor Varying degree of spleen enlargement Increased susceptibility to infections Skeletal changes 1 Nucleated RBC 2 1. Taher AT et al. Blood Cells Mol Dis 2006;37:12 20; 2. Yaish HM et al.

30 β thalassemias Both clinical and laboratory parameters can help distinguish the diverse spectrum of β thalassemias Clinical β TM more likely β TI more likely Presentation (years) <2 >2 Hb levels (g/dl) Liver/spleen enlargement Severe Moderate to severe Hematologic Hb F (%) > (may be up to 100%) Hb A 2 (%) <4 >4 Genetic Parents Both carriers of high Hb A 2 β thalassemia Molecular 1 or both atypical carriers: High Hb F β thalassemia Borderline Hb A 2 Type of mutation Severe Mild/silent Co-inheritance of α thalassemia No Yes Hereditary persistence of Hb F No Yes δβ thalassemia No Yes Gγ XMN1 polymorphism No Yes 1. Thalassaemia International Federation. Guidelines for the clinical management of thalassaemia, 2nd Edition revised 2008.

31 Hb E/β thalassemia Diagnosis of Hb E/β thalassemia is based on a variable anemia and laboratory profile Blood smear Target cells, microspherocytes, red cell hypochromia, red blood cell fragments, nucleated red blood cells 1 Number of cases (%) Level of hemoglobin (g/dl) 2 60% F E 40% Hb electrophoresis and HPLC Hb E and F 3 Hb A 2 Hemolysis 2 DNA analysis to distinguish syndromes 20% 0% Time (minutes) 1. ta_thalassaemia.aspx; 2. Vichinsky E. Hematology 2007;79 83; 3. Fucharoen S et al. Clin Chem 1998;44:

32 Blood smear: severe Hb E/ β thalassemia Homozygous state for Hb E showing large numbers of target cells 1 Nucleated red cells and platelets in Hb E/β thalassemia after splenectomy 1 The main blood smear features for severe Hb E/β thalassemia include: Target cells 2 Nucleated red blood cells 1,2 Microcytes 2 Irregularly contracted RBC 2 1. Fuchareon S and Weatherall DJ. Cold Spring Harb Perspect Med 2012;2:a011734; 2. Vichinsky E. Hematology 2007:79 83.

33 There are different severities of Hb E/β thalassemia, and laboratory and clinical parameters can help distinguish between them Baseline hematology and demographic data in Hb E/β thalassemia patients with different clinical severity Hb E/β thalassemia, n=116 Hb E/β thalassemia Parameters Mild, n=33 Moderate, n=18 Severe, n=65 No. of males (%) 17 (51) 11 (61) 26 (40) Age, y 9.46 ± ± ± 4.36 Hb, g/dl 8.69 ± ± ± 0.98 Hct, % ± ± ± 2.94 MCV, fl ± ± ± 8.23 MCH, pg 18.9 ± ± ± 2.97 Retic, % 5.7 ± ± ± 5.13 RDW, % ± ± ± 4.44 Hb F, g 4.42 ± ± ± 0.85 Hepatomegaly (%) 7 (21) 9 (50) 33 (51) Splenomegaly (%) 14 (42) 12 (66.7) 51 (78.5) Hx transfusion (%) 5 (15) 9 (50) 60 (92) Splenectomy (%) 2 (6) 1 (5.5) 14 (21.5) Hct, hemocrit; Hx, hemopexin; Retic, reticulocytes; RDW, red blood cell distribution width 1. Viprakasit V et al. Blood 2004;103:

34 Blood smear: β thalassemia major β thalassemia major is characterized by severe degree of: Microcytosis Hypochromia Hypochromic macrocytes Poikilocytosis Nucleated red blood cells Basophilic stippling Basophilic stippling Nucleated red blood cell M, microcytosis; P, anisopoikilocytosis; T, target cells, 1. Steinberg MH et al. Disorders of Hemoglobin; 2nd edition 2009; Cambridge University Press.

35 Hb H disease (α thalassemia) Clinical features and laboratory abnormalities may contribute to a diagnosis of Hb H disease Characterized by: 1 Hb levels ~3 13 g/dl, variable amounts of Hb H (0.8-40%), occasionally Hb Barts hydrops Splenomegaly (may be severe), occasionally complicated by hypersplenism Jaundice in variable degrees Children may show growth retardation Severity of clinical features related to molecular bass of disease 1 Blood smear 2 Microcytosis, hypochromia Target cells Inclusion bodies Hemolysis Hb Barts hydrops (γ tetramers) Hb H (β tetramers) 1. Harteveld C and Higgs D. Orphanet J Rare Dis 2010;5:13; 2. thalassaemias.

36 Hb H disease (α thalassemia) Diagnosis of Hb H may require HPLC or electrophoresis to help distinguish Hb abnormalities Hb electrophoresis or HPLC (Hb A 2, Hb Barts hydrops, Hb H) DNA analysis Gap-PCR MLPA/Southern blot Sequencing Capillary electrophoresis Z15 Z14 Z13 Z12 Z11 Z10 Z9 Z8 Z7 Z6 Z5 Z4 Z3 Z2 Hb A HPLC 30.0 % 22.5 Hb H Hb H 7.5 Hb F F 2.41 Hb A 2 A2 Hb X Hb F Hb A Time (minutes) 1. Harteveld C and Higgs D. Orphanet J Rare Dis 2010;5:13.

37 Blood smear: Hb Barts hydrops Hb Barts hydrops shows high degree of: 1 Microcytosis Hypochromia Target cells Increased reticulocytes (5 10%) A peripheral blood film for Hb Barts with immature red-cell precursors and hypochromic, microcytic, red cells showing anisocytosis and poikilocytosis thalassaemias; 2. Harteveld C and Higgs DR. Orphanet J Rare Dis 2010;5:13.

38 Genotype-phenotype association in the β thalassemias lead to a varying clinical severity NTDT Phenotype Genotype Clinical severity Major β 0 /β 0, β + /β +, or β 0 /β + Early presentation Severe anemia Transfusion dependent Intermedia β 0 /mild β +, β + /mild β +, or mild β + /mild β + β 0 /silent β, β + /silent β, mild β + /silent β, or silent β/silent β β 0 /β 0, β + /β +, or β 0 /β + and deletion or nondeletion α-thalassemia β 0 /β 0, β + /β +, or β 0 /β + and increased capacity for γ chain synthesis Deletion forms of δβ thalassemia and HPFH β 0 /β or β + /β and ααα or αααα duplications Dominant β thalassemia (inclusion body) Late presentation Mild-to-moderate anemia Transfusion independent Clinical severity is variable and ranges between minor to major Trait/minor β 0 /β, β + /β, or mild β + /β Borderline asymptomatic anemia Microcytosis and hypochromia Silent carrier silent β/β Asymptomatic No hematological abnormalities Severity of disease 1. Musallam KM et al. Haematologica 2013;98:

39 Ringraziamenti Centro Anemie Congenite e Medicina Interna 1A Policlinico di Milano Maria.cappellini@unimi.it