Thalassaemia Thalassaemia
§ Quantitative defect → Imbalance of globin chains → § Reduced Hb synthesis and anaemia § Precipitation of abnormal Hb → haemolysis and ineffective erythropoiesis § Alpha § Beta § Delta-beta § Gamma-delta-beta
Thalassaemia major § Transfusion dependant Thalassaemia intermedia § Anaemia and splenomegaly Thalassaemia trait § Symptomless
Beta thalassaemia § Mediterranean, Middle East and South Asia (Africa as well but less common) § Chromosome 11; one gene per chromosome § >200 gene defects – categorised into B0 and B+ depending on whether any beta chain is synthesised (B+ is therefore a very broad category) § Large variety in phenotype – relates to primary, secondary and tertiary factors: § Primary (different gene mutations cause a variety of reduction in alpha chain synthesis) § Transcription (deletions / promoter defects) § Processing of mRNA (splice junction, consensus sequence, internal IVS, cryptic spice sites in exons) § Translation (nonsense, frame shift, initiation site) § Post translational instability (exon 3 mutations, other unstable beta chains) § Secondary § Coinheritance of alpha thal reduces excess alpha chain production improving symptoms § Genetic modifiers of gamma chain production § Tertiary § Factors that affect the complication of disease (eg. iron metabolism / bone disease)
Diagnosis § Excess of alpha chains § Some will bind with delta = A2 § Some will bind with gamma = F (heterogeneously distributed cf. HPFH) § Some precipitate in red cell precursors = intracellular inclusions = ineffective erythropoiesis § Some with inclusions enter circulation = haemolysed § Increased erythropoieitin – bone marrow expansion and bony abnormalities
Bthal trait § Heterozygosity for b thalassaemia § Usually asymptomatic – may need transfusion at times of haematopoietic stress e.g. pregnancy or intercurrent illness. § FBC § Normal or slightly reduced Hb § MCV / MCH low § MCHC normal § RDW normal § Blood film § Normal ranging to markedly abnormal with: § Microcytosis § Anisocytosis, poikilocytosis § Hypochromia § Basophilic stippling § Target cells § Irregularly contracted cells § Elliptocytes move commonly found in iron deficiency § HPLC § Raised A2 § NB 4% of A2 is A’2 which is found in the HbS band § 4-5% usually indicates heterozygosity for B0 or severe B+ thal (usually 3.6-4.2% if heterozygous for B+ thal) § Some cases are silent with normal red cell indices and normal A2 § Some cases are almost silent – with abnormal indices and a slightly raise A2 (referred to as ‘normal A2 Bthal’) § Both can be detected using genetic studies and studies of b chain synthesis § Rarely patients have normal red cell indices and a raised A2
Dominant Bthalassaemia Rarely mutations cause symptoms in heterozygotes (mainly splenomegaly / gall stones) May need blood transfusions Iron overload can occur
Bthal intermedia § Very variable phenotype – results from many different mutations § Anaemic but not transfusion dependant § Splenomegaly – may develop hypersplenism § Leg ulcers § Bony deformity § Transfusions may be needed at times of stress and can become necessary in later life § Gall stones § Iron overload § Gonadal failure § Extramedullary tissue can form tumour like masses in the mediastinum or pleura. Occasionally affect the spinal cord § Congestive cardiac failure may occur
§ Blood film § Similar to Bthal trait but more severe: § Hypochromia § Microcytosis, anisocytosis, poikilocytosis § Basophilic stippling § Polychromasia § Possibly circulating erythroblasts § Raised HbA2 (more than Bthal trait) § Raised HbF
Bthal Major § Severe disease - presents in first year of life (as HbF falls) and transfusion is required to sustain life beyond early childhood (by definition) § Usually due to Bthalo homozygote or compound heterozygotes (very rarely heterozygotes have the same clinical phenotype) § Can result from compound heterozygosity with a thalassaemic haemoglobinopathy – e.g. HbEBthal0 (less commonly Hb Malay) Clinical features § Pallor § Failure to thrive § Splenomegaly - later hypersplenism (worsens anaemia / can cause thrombocytopenia) § Hepatomegaly § Bone pain and tenderness - bone fractures § Skull bossing § Jaundice, gallstones § Stunting of growth § Without iron chelation, develop progressive tissue siderosis (hepatic, endocrine, cardiac) § Delayed sexual development § Commonest cause of death – cardiac failure in third decade § Death by 4 years in homozygous B0 / late childhood for homozygous B+ thal
Lab features § Hb 2-8 § Blood film § Hypochromia / hypochromic microcytes § Marked anisocytosis § Poikilocytosis including fragments, teardrops § Target cells § Basophilic stippling § Pappenheimer bodies § NRBCs with dyserythropoietic features § Increased white count (but may have neutropenia and thrombocytopenia if massive splenomegaly) § Hyposplenic features if previous splenectomy (Howell-Jolly bodies, target cells, lymphocytosis, thrombocytosis, giant platelets) § Post splenectomy may contain alpha chain precipitates (can be seen pre splenectomy but less frequent) § Leptocytes seen post splenectomy – very flat cells with striking hypochromia § Transfused cells § Bone marrow § Severe dyserythropoiesis with nuclear lobulatoin and fragmentation, basophilic stippling § Phagocytic macrophages § Pseudo-Gaucher cells § Increased iron stores § HPLC § B0B0 = No HbA, Raised HbF, HbA2 variable § B+B0 HbA may be present up to 35% § Markers of haemolysis – LDH, bilirubin, haptoglobins
Bthal + other B globin chain abnormality Sickle cell B-thal § Phenotype depends on severity of the B-thal § HbS-Bthal+ § African populations which have mild Bthal – normal survival with only occasional crises § HbS and HbA (5-30%) with elevated HbA2 § HbS-Bthalo § Mediterranean population – phenotype often indistinguishable from HbSS § HbS with elevated HbA2 and HbF
HbC B-thal § Mild haemolytic anaemia § Target cells and thalassaemic red cells § Diagnosis – HbC predominantly on electrophoresis (also A / A2 depending on severity of Bthal)
HbE B-thal § Commonest severe form of thal in SE Asia and Indian subcontinent § HbE is inefficiently produced, hence if inherited with Bo = marked reduction in beta globin chain synthesis § Can cause severe thalasaemia phenotype (anaemia / bone changes) § Very high mortality in early life § Diagnosis = HbE and HbF on electrophoresis (may be small amount of HbA if Bthal+)
Delta-beta thalassaemia § Deletions of both delta and beta genes (dB-thalo) or creation of a delta beta fusion gene (dB-thal+) § Fusion gene produces a delta beta fusion chain which can bind with alpha chains to produce Hb lepore § dB-thalo § homozygotes - mild anaemia (Hb 8-10) with 100% HbF (can’t make A or A2) § thal intermedia § heterozygotes – thalassaemia blood picture, with elevated HbF but normal A2 § HbF is heterogenous distribution cf HPFH which is homogenous § Hb lepore (Fusion gene of delta and beta) § Homozygotes – § Clinical picture Bthal major or thal intermedia § HbF and Hb lepore only § Heterozygotes (Hb Lepore trait) § Blood film and blood count indistinguishable from BThal trait § 10-15% Hb lepore § HbA2 reduced
Gamma-delta-beta thal § Actually entire beta cluster is deleted – only found in heterozygotes as homozygosity would be incompatible with fetal life § Micreocytosis, basophilic stippling (resembles bthal trait without any F or A2)
Delta thalassaemia § Only affects synthesis of A2 and is of no clinical significance § However, can cause a falsely normal A2 with Bthal trait – so it will be missed
Hereditary persistence of foetal haemoglobin (HPFH) § Persistent fetal haemoglobin in adult life – variety of different definitions (>2% or >8% of cells) § Can modify severity of beta-haemoglobinopathies § May be mildly polycythaemic due to higher oxygen affinity of HbF
HPFH – sickle § 70% sickle 30% HbF – mild sickle syndrome
HPFH – Bthal § 70% HbF – variable phenotype – mild or resembling Bthal intermedia
Alpha-thalassaemia § Either the alpha 2 (aTa) or alpha 1 (aaT) gene can be involved (order they appear on the chromosome 16) § a2 gene makes about 70% of the alpha chains and deletions have a worse phenotype than a1 § Deletions and mutations fall into several categories § Deletions § Part of the gene or the whole of one of the genes = a+ § Deletion of both genes (a0) § Deletion of part of an a2 and an a1 gene with formation of a fusion a gene (a+ phenotype – the two most common mutations a3.7 and a4.2 occur by this mechanism) § Deletion of a major upstream regulatory element – a0 § RNA splice variants § Polyadenylation (eg aTSaudia) common in the Mediterranean gives severe a+ phenotype sometimes regarded as a0 § Mutations involving RNA translation § Initiation / frame shift result in inactivation of the gene § Termination sequence mutations result in an elongated alpha chain that is synthesised at a slower rate (e.g. Hb Constance Spring – found in SE Asia = nondeletional alpha+ thal) § Mutations causing marked post translational instability of a highly abnormal alpha chain § Transactivating abrnormality resulting from mutation in the ATRX gene on the X chromosome which encodes a DNA helicase (results in a syndrome with alpha thal and severe mental retardation)
§ a0 § Mediterranean (__MED) [1.5% of population] § Middle East (__MED) [rare] § S Asia (__ SEA, __FIL, __THAI) [3-4% of population] § a+ § Mediterranean (-a3.7 and aTSaudia) [10%] § Middle East (-a3.7 and aTSaudia) [variable – up to 80% in Oman] § South East Asia (-a3.7, -a4.2 and aCSa) [around 10%] § India / Sri Lanka (-a3.7 and -a4.2) [around 10%] § Africa (-a3.7) [around 25% are heterozygous for this mutation / 1-2% homozygous] § Melanesia / Polynesia (-a3.7) [10%]
§ Cis = --/α α = S Asian = α o § Trans = α-/ α- = African = α+
Heterozygous a+ § May have completely normal FBC or trivial anaemia and microcytosis (Hb 1g less on average than normal)
Alpha-thal trait (aa/-- or a-/a-) § Mild hypochromic microcytic anaemia § Confirmatory diagnosis requires DNA analysis § Similar to beta thalassaemia trait (hypochromic microcytes, target cells) but HbA2 is normal
Homozygous a+
Heterozygous a0 thal § Very mild anaemia with Hb concentration overlapping the normal range § RBC increased, MCV and MCH reduced § Reticulocyte count 2-3% § 5-10% HbBarts at birth (higher but overlapping with a+ homozygotes) § Will have occasional cells with HbH but this is not a good diagnostic test § Definitive diagnosis requires DNA analysis – this is normally done after excluding iron deficiency (with serum ferritin – TF receptor often increased in alpha thal) and beta and delta-beta thalassaemia trait
HbH disease § Clinical syndrome arising from a number of different defects (a-/--, --/aTa, aTa/ aTa, --/ aCSa....) § Homozygotes for severe a+ thal may have the clinical features of haemoglobin H § Only occurs where ao-thal is common § Greatly reduced alpha chain production leading to the formation of HbH, which has very high oxygen affinity § HbH precipitates in bone marrow cells leading to intramedullary death and older red cells leading to haemolysis § Haemolysis is more important than ineffective erythropoiesis as the cause of anaemia Clinical features § Variable anaemia (7-10) and splenomegaly § Jaundice may be present § Survive into adult hood § Some patients may have marrow expansion and bony deformity similar to beta thal major § Severe haemolytic episodes caused by infection, oxidant drugs, hypersplenism, folate deficiency Laboratory features § Hb 7-10 MCV 50-65 MCH 15-20 MCHC 25-30 § Film § Anisocytosis, poikilocytosis, hypochromia, microcytosis § Basophilic stippling may be present § Target cells § Fragments and tear drops § Nucleated red cells may or may not be present § HbH 5-40% (normally around 10%) § HbA2 normally reduced (1-2%); HbF increased (1-3%) § Cresyl blue staining shows golf ball inclusions in 35-90% of cells and Heinz bodies in patients with previous splenectomy § Neonates have Hb Barts 10-40%, with HbH at a low percentage
Hb barts (Homozygous inheritance of ao-thal (--/--)) § Only occurs where ao-thal is common = Se Asia and Mediterranean § Very high oxygen affinity resulting in tissue hypoxia § Hydrops fetalis § Usually still born between 28-40 weeks § High incidence of obstetric complications due to the large placenta
Haemoglobin Constance Spring § More anaemia than would be expected with a+ thal heterozygote but tends to be less microcytic § Basophilic stippling often prominent § Homozygotes – anaemic (10g) with low MCH but only a mild reduction in MCV § Reiculocytosis due to shortened red cell survival (only 2-11% HbCS seen)
Alpha thalassaemia associated with mental retardation § Chromosome 16 or X chromosome § Alpha-thalassaemia associated with MDS § X chromosome
Ante-natal screening programs
§ FBC and HPLC § Variant Hb – confirmatory testing § Test partner § Raised HbF – quantitate § Kleihauer § Homogenous = HPFH § Heterogenous – dB-thal – test partner § MCH <27 § Quantitate A2 § Normal § IDA – check ferritin § alpha-thal trait § normal A2 Bthal trait ¨ ethnic origin Ø N. European, African, Afrocaribbean = no action Ø Chinese, SE Asian, Greek, Turkish, Cypriot and MCH <25 = risk of ao trait § MCH partner – if <25 and same ethnic group · DNA analysis Ø Mediterranean, Saudi Arabian, S Asian § Normal A2 Bthal trait § Co-existing B and delta thal trait · Test partner · Normal = no action · Abnormal = DNA analysis § Borderline § Beta and alpha thal trait § Bthal trait and iron deficiency § Mild B thal trait ¨ Test partner Ø Abnormal = DNA analysis § Raised § Bthal trait – test partner
Treatment § BMT § 90% survival if good match and no iron loading § Symptomatic § Transfusion to suppress erythropoiesis § Iron chelation § Splenectomy if hyperspleinsm
Diagnosis of haemoglobinopathies § FBC § Film § Sickle solubility (only S) § Blood + phosphate buffer (lysing agent and reducing agent) + centrifuge § Sickle Hb sickles and is trapped within rbcs § Normal Hb is lysed § Affected by HbF and transfused blood § HPLC § Identifies variant Hb by change in electrical charge § Quantitates § Beware: HIV mimics Bthal trait with an elevated A2, but the meds usually cause macrocytosis § HbH and barts elute very quickly and may be missed § Confirmatory tests § Alkaline electrophoresis (pH 8.2-8.6) § A, F, S/G/D, A2/C/E/O-arab § Quantitate using elution or spectrophotometry § Previously used as screening test § Acid gel (pH 6.0-6.2) § Do to tell S apart and C apart § S and D/G, C and E, C-harlem and O-arab § A/D/G/E/A2 run together § Iso-electric focusing § Suitable for neonates § More expensive, but separates more variants than electrophoresis § Mass spectrometry § Identifies many variants including those silent by other methods § Requires small sample volumes § Quick § Frozen samples can be used § Reagents not expensive § DNA analysis § Definite diagnosis § Knowing the mutation with thalassaemia enables some prediction over the severity of the phenotype § Required for alpha-thalassaemia
Alpha-chain variants § Eg. G-philadelphia § Not normally clinically significant as there are 4 alpha genes § On HPLC – normally get a peak for A and A2, therefore with a variant – 4peaks – A, Av, A2, A2v
Unstable haemoglobins § Easily confused with alpha-chain variants § If not detected by HPLC or electrophoresis § Extremely unstable or electrophoretically silent § Stability tests (hetor isopropanolol) § Molecular analysis/ mass spectrometry
High affinity haemoglobins § Many are electrophoretically silent § Assess affinity using P50 § Molecular or mass spectrometry
Stem cell transplant in haemoglobinopathies
Beta thalassaemia major § Survival at 35 years estimated to be about 90% § Indication for transplant § <16 years with transfusion dependence § HLA identical family donor § Consider if >16 years or previously failed transplant
Sickle cell disease § Median survival 45 years § 25% will have stroke by 30 years § Indication for transplant § Stroke § Recurrent chest syndrome § Recurrent vaso-occlusive crisis if HU fails (>4 admissions/ year) § Transfusion program § Emerging – CNS disease
Conditioning § Hypertransfuse for 6 weeks to suppress erythropoiesis § Busulphan/ cyclophosphamide (reduced dose Bu to reduce long-term toxicity) § Campath § Stem cells – bone marrow rather than PBSCs due to less GvHD § Cyclosporin and methtrexare for GvHD prophylaxis
Improved outcome if limit to those not iron overloaded – can intensely chelate them first Can venesect once engrafted, see reversal of liver iron and fibrosis 30% mixed chimerism, but all remained transfusion independent
Limitations § Lack of donor § Length of treatment § Transplant related mortality § Log term effects § Infertility § Pubertal failure (50% = the same for Bthal major) § Chronic GvHD 5% § Organ toxicity – rare § Secondary malignancy <1% §
HbD similar to Hb E
Microcytosis with normal A2 and F § Alpha thal trait § Delta / beta thal coinheritance § gdb thalassamia § PV complicated by iron deficiency |
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