Hematology — ASCP Exam Prep Alpha-Thalassemia

Classify alpha-thalassemia by gene deletion. Identify HbH and Hb Bart’s and their clinical significance for ASCP.

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Chapter 17 — THALASSEMIAS

ASCP Board Exam · Lab-Focused Study Guide

Definitions & Epidemiology · Globin Gene Clusters & Designations · Pathophysiology (α vs β) · β-Thalassemia Syndromes · HPFH, δβ⁰ & Hb Lepore · α-Thalassemia Syndromes (Silent → Hb Bart Hydrops) · Hb S/C/E with Thalassemia · Laboratory Diagnosis (CBC, Supravital Stain, HPLC, CZE, Molecular) · Thalassemia Minor vs Iron Deficiency · Practice Questions

1 · Priority Table & Quick Foundation

Topic	Priority	Why It’s Tested
Thalassemia = reduced/absent synthesis of a normal globin chain (quantitative)	HIGH	Definition (Rodak Q1)
Chain imbalance → hemolysis and ineffective erythropoiesis	HIGH	Mechanism of anemia (Rodak Q3)
β-thal minor: ↑ Hb A₂ (3.5–7%), MCV <75, RBC count high, target cells, stippling	HIGH	Most common exam case (Rodak case study, Q4, Q14)
β-thal major: Hb F 70–>90%, severe anemia from 6 months, transfusions, iron overload	HIGH	Rodak Q5, Q6; treatment and chelation
α-thal: 4 syndromes by genes deleted; Hb Bart (γ₄) vs Hb H (β₄)	HIGH	Gene count ↔ syndrome (Rodak Q8–Q11)
Hb H inclusions (golf ball) vs Heinz bodies on supravital stain	HIGH	Morphology (Rodak Q13)
Thalassemia minor vs iron deficiency anemia (ferritin, A₂, RBC count)	HIGH	Differential diagnosis — iron deficiency lowers A₂
HPLC / CZE / electrophoresis limitations (A₂–E, A₂–C overlap)	HIGH	Method selection
HPFH, δβ⁰-thal, Hb Lepore (Table 28-3)	MEDIUM	Hb F patterns; Kleihauer-Betke pancellular vs heterocellular (Rodak Q7)
Thalassemia with Hb S, C, E (Table 28-5)	MEDIUM	Hb S-β⁰ resembles sickle cell anemia (Rodak Q12)
Genetic designations (β⁰, β⁺, β^silent, α⁰, α⁺, α^T, Constant Spring)	MEDIUM	Terminology
Molecular testing strategy; prenatal diagnosis	LOW	Table 28-6 step approach
Epidemiology numbers, history (Cooley, Whipple)	LOW	Background

🎯 KEY POINT — Where to spend your time

Focus: the chain-imbalance mechanism, β-thal minor lab pattern (↑ RBC count, MCV <75, ↑ A₂, normal ferritin), β-thal major (↑↑ F, transfusion, iron overload), α-thal gene count ↔ syndrome, Hb Bart vs Hb H, golf-ball inclusions, and the IDA differential. Skim: carrier percentages by country, gene-therapy details, discrimination-index names.

Quick Foundation — from zero

A thalassemia is a genetic disorder where one globin chain is made in reduced amounts or not at all. The chain itself is normal — the problem is quantity (hemoglobinopathies were about quality).
Less globin → less hemoglobin per cell → microcytic, hypochromic RBCs.
The partner chain keeps being made normally, so it is left unpaired. Excess chains are the real villain: excess α chains (β-thal) precipitate in marrow precursors and kill them; excess γ or β chains (α-thal) form odd tetramers (Hb Bart, Hb H).
Named for the chain that is deficient: β-thalassemia (chromosome 11 genes) and α-thalassemia (chromosome 16 genes).
Severity depends on how many genes are affected and how completely — from no symptoms (silent carrier) to death before birth (Hb Bart hydrops fetalis).

💡 Analogy

Hemoglobin is built like pairs of dance partners — each α must pair with a β. In β-thalassemia, there aren’t enough β partners; the lonely α chains clump together on the dance floor and wreck the hall (precipitate in marrow → ineffective erythropoiesis). In α-thalassemia, the lonely β (or γ in babies) chains just dance in groups of four (Hb H, Hb Bart) — they don’t wreck the hall early, but they can’t do the job (very high O₂ affinity) and Hb H groups eventually fall apart in older cells.

🏥 Why this matters clinically

β-Thal minor is often mistaken for iron deficiency → unnecessary iron (adding to iron load) or needless colonoscopy. Carrier detection allows genetic counseling: two carrier parents have a 25% risk per pregnancy of a child with thalassemia major. Mass screening and prenatal diagnosis in Italy and Greece sharply reduced births with β-thal major.

Concept	Plain-English Explanation
Thalassemia	Reduced or absent synthesis of a structurally normal globin chain
β⁰ / β⁺	No β chain made / β chain made at 5–30% of normal
α⁰ (– –) / α⁺ (– α)	Both α genes on one chromosome deleted / one of the two deleted
Ineffective erythropoiesis	Precursors die in the marrow before release
Hb Bart (γ₄)	Tetramer of 4 γ chains — fetal/newborn α-thal
Hb H (β₄)	Tetramer of 4 β chains — adult α-thal (3 genes deleted)
Transfusion-dependent	Needs regular transfusions to survive (thal major)
Cis vs trans	Deletions on the same chromosome (– –/αα) vs one on each (– α/– α)

2 · Core Content Tables

2.1 Definitions & History

Feature	Details
Definition	Heterogeneous inherited disorders from mutations that reduce or prevent synthesis of one or more globin chains
Consequences	↓ Hb production + α/β chain imbalance; accumulated unpaired chains damage RBCs or precursors → premature destruction
1925	Cooley and Lee: 4 children with anemia, splenomegaly, hepatomegaly, mongoloid facies — β-thal major (Cooley’s anemia)
1932	Whipple and Bradford named it Thalassic anemia (Greek ‘great sea’ — Mediterranean) → thalassemia
1940s–1950s	Genetic basis (homozygous = major, heterozygous = minor); α-chain defects described in the 1950s

2.2 Epidemiology & Malaria

Feature	Details
Carriers	~5% of the world carries a significant hemoglobinopathy/thalassemia mutation; ~56,000 infants/year born with thalassemia major
Thalassemia belt	Mediterranean → Middle East → India → Southeast Asia, and North Africa
β-thal carriers	Europe highest in Sardinia, Cyprus, Greece (6–19%); Asia: India, Thailand, Indonesia (0.3–15%)
α-thal carriers	Cyprus 14% (Europe’s highest); 50–60% in eastern Saudi Arabia and parts of Asia/Africa; up to 75–80% in groups in Nepal, India, Thailand, Papua New Guinea
Malaria protection	Thalassemia minor protects against malaria. Death risk from malaria 40% lower in αα/–α and >60% lower in –α/–α. Proposed: defective parasite growth, ↑ phagocytosis of infected cells

2.3 Reference Intervals for Adult Hemoglobins (Rodak Table 28-1)

Hemoglobin	Chains	Adult Reference
Hb A	α₂β₂	95–100%
Hb A₂	α₂δ₂	0–3.5%
Hb F	α₂γ₂	0–2%

2.4 Genetics of Globin Synthesis

Feature	Details
α-like cluster (chr 16)	HBZ (ζ), HBA2 (α₂), HBA1 (α₁) + pseudogenes (ψ)
β-like cluster (chr 11)	HBE (ε), HBG2 (^Gγ), HBG1 (^Aγ), HBD (δ), HBB (β) — in order of developmental expression
γ chains	^Gγ and ^Aγ differ at position 136 (glycine vs alanine); no functional difference
Inheritance	One chr 11 cluster from each parent → normal β/β. Two α genes per chr 16 → normal αα/αα (4 α genes)
Switches	By 10 weeks: ζ → α (chr 16) and ε → γ (chr 11) → Hb F. γ → β switch around birth; Hb A predominates by 6 months. δ activated shortly before birth (weak promoter → low Hb A₂)

2.5 Genetic Designations (Rodak Table 28-2)

Designation	Meaning
β	Normal β gene (one on each chr 11)
αα	Normal α₁ + α₂ genes on one chr 16 (normal genotype αα/αα)
β⁰	No β chains produced
β⁺	β chain production decreased (5–30% of normal)
β^silent	Mildly decreased β chain production
δβ⁰	Deletional/nondeletional mutation — no δ or β chains; some ↑ γ
δβ^Lepore	δβ fusion → small amount of Hb Lepore; no δ or β chains; some ↑ γ
HPFH	δβ deletion or γ-promoter mutation — no δ/β but ↑ γ (Hb F)
α⁰ (– –)	Both α genes on one chromosome deleted → no α from that chromosome
α⁺ (– α)	One α gene deleted → decreased α
α^T (α^Tα)	Nondeletional (point) mutation in one α gene → decreased α

2.6 Genetic Defects Causing Thalassemia

Feature	Details
Mutation types	Single nucleotide (point) mutations, small insertions/deletions, large deletions
Transcription	Promoter or initiation codon mutations; polyadenylation site mutations ↓ mRNA stability
mRNA processing	Splice-site mutations → no chain or altered chain
Translation	Frameshift, missense, nonsense (premature stop), or loss of stop codon → elongated unstable mRNA/chain
Gene deletion	One or more globin genes lost → no chain (typical of α-thal)
β vs α	β-thal: mostly point mutations (>300 known) · α-thal: mostly large deletions

2.7 Pathophysiology — β-Thalassemia

Feature	Details
Two roots	(1) ↓ chain → ↓ Hb synthesis → microcytic hypochromic RBCs · (2) α/β imbalance → ↓ survival of RBCs and precursors (the main determinant of severity)
Excess α chains	Precipitate in developing RBCs → inclusion bodies → oxidative membrane damage → apoptosis → phagocytosis in marrow = ineffective erythropoiesis (also iron accumulation and cytokines)
Surviving cells	Laden with inclusions → trapped and destroyed in the spleen (extravascular hemolysis)
Onset	Asymptomatic in fetal life to ~6 months (Hb F); symptoms at 6–24 months after the γ→β switch
Compensation	γ and/or δ upregulated but insufficient in β-thal major
Marrow expansion	Profound anemia → ↑ EPO → massive ineffective erythroid hyperplasia → bone thinning, fractures; lacy long bones; skull ‘hair on end’; frontal bossing, prominent cheekbones and upper jaw
Extramedullary	Hepatosplenomegaly; foci of hematopoiesis; hypersplenism → neutropenia, thrombocytopenia
Hemolysis products	↑ Indirect bilirubin → jaundice, gallstones, leg ulcers; ↑ thrombosis risk
Iron overload	Mainly from transfusions; plus ineffective erythropoiesis suppresses hepcidin → ↑ intestinal iron absorption. Children: growth retardation, no sexual maturity; adults: cardiomyopathy, liver fibrosis/cirrhosis, endocrine dysfunction. Organ damage risk rises after age 10–11

2.8 Pathophysiology — α-Thalassemia

Feature	Details
Onset	Can manifest in utero — α chains are in both fetal (Hb F) and adult (Hb A) Hbs
Fetus/newborn	Excess γ chains → Hb Bart (γ₄), in proportion to the number of defective α genes
After 6 months	Excess β chains → Hb H (β₄)
Marrow	Hb Bart and Hb H are relatively stable — don’t precipitate significantly in precursors → no severe ineffective erythropoiesis
Circulation	Hb H precipitates as RBCs age → inclusion bodies → splenic removal → moderate hemolytic anemia
O₂ delivery	Hb Bart and Hb H have very high O₂ affinity → poor tissue delivery. Fetus with only Hb Bart (4-gene deletion) → hypoxia, heart failure, hydrops fetalis → dies in utero/shortly after birth

2.9 Clinical Syndromes of β-Thalassemia (Rodak Table 28-3)

Syndrome / Genotype	Hb A	Hb A₂	Hb F	Hb Lepore
Normal β/β	N	N	N	0
Silent carrier β^silent/β	N	N	N	0
Minor β⁺/β	↓	↑	N to sl ↑	0
Minor β⁰/β	↓	↑	N to sl ↑	0
Minor δβ⁰/β	↓	N to ↓	5–20%	0
Minor δβ^Lepore/β	↓	↓	↑	5–15%
Major β⁺/β⁺	↓↓	V	↑↑	0
Major β⁺/β⁰	↓↓↓	V	↑↑	0
Major β⁰/β⁰	0	V	↑↑	0
Major δβ^Lepore/δβ^Lepore	0	0	80%	20%
Intermedia β^silent/β^silent	↓	↑	↑	0
Intermedia β⁺/β^silent or β⁰/β^silent	↓	↑	↑	0
Intermedia δβ⁰/δβ⁰	0	0	100%	0
Intermedia β⁰/δβ⁰	0	N	↑↑	0

N, normal; sl, slight; V, variable. Intermedia also includes dominantly inherited β-thal and β-thal minor with a triplicated α gene (ααα/αα).

2.10 β-Thalassemia — Four Clinical Categories

Feature	Details
Silent carrier (β^silent/β)	No hematologic abnormalities; near-normal α/β ratio. Found through families where children had worse disease than the parent with β-thal minor; parents had normal A₂ and slight microcytosis. β^silent/β^silent → intermedia with ↑ F and A₂
Minor / trait (heterozygous)	Mild asymptomatic anemia, microcytic/hypochromic
Major (homozygous or compound heterozygous)	Severe hemolytic anemia, transfusion-dependent
Intermedia	Mild–moderate anemia, transfusion-independent
Inheritance	Autosomal recessive: two carrier parents → 25% chance per pregnancy of major or intermedia

2.11 β-Thalassemia Minor (Trait)

Feature	Details
Genetics	One β gene affected (β⁰/β or β⁺/β most common)
Hemoglobin	Men 12.4–14.2 g/dL; women 10.8–12.8 g/dL — mild, asymptomatic
RBC count	Within reference interval or slightly elevated
Indices	MCV <75 fL, MCH <26 pg; RDW often normal
Retics	Normal or slightly ↑
Film	Microcytes, hypochromia, target cells, elliptocytes, basophilic stippling
Marrow / organs	Mild–moderate erythroid hyperplasia, minimal ineffective erythropoiesis; hepatosplenomegaly in a few
Hb fractions	Hb A 92–95% · Hb A₂ 3.5–7.0% (characteristically ↑) · Hb F 1–5%
Atypical	δβ⁰/β and δβ^Lepore/β (A₂ not ↑); Dutch β⁰ (↑ A₂ + F 5–20%); a Sardinian mutant with normal A₂

2.12 β-Thalassemia Major (Cooley’s Anemia)

Feature	Details
Diagnosis	Severe anemia requiring regular transfusion; usually diagnosed at 6 months to 2 years when Hb A fails to rise
Untreated	Hb as low as 3–4 g/dL; MCV 50–70 fL
Film	Marked microcytosis, hypochromia, anisocytosis, poikilocytosis (target cells, teardrop cells, elliptocytes); polychromasia, nRBCs; basophilic stippling, Howell-Jolly and Pappenheimer bodies (excess nonheme iron)
Retics	Only mildly–moderately ↑ — inappropriately low (ineffective erythropoiesis)
Hb fractions	Hb A absent (β⁰/β⁰) or 10–30% (β⁺); Hb F 70% to >90%; Hb A₂ variable
Marrow	Marked erythroid hyperplasia; M:E ratio 1:20 (normal 1.5:1 to 3.3:1)
Chemistry	Haptoglobin ↓/absent; LDH markedly ↑; ↑ indirect bilirubin

2.13 β-Thalassemia Major — Treatment

Feature	Details
Transfusion	Start when Hb <7 g/dL with symptoms; 10–15 mL/kg every 2–5 weeks; RBCs <7–10 days old; antigen-negative donor RBCs after extended typing to reduce alloimmunization
Hypertransfusion	Since mid-1970s: pretransfusion Hb kept at 9–10.5 g/dL → suppresses erythropoiesis → no marrow expansion or bone changes, ↓ iron absorption, better growth, no hepatosplenomegaly
Iron overload	No excretory pathway → iron in liver (cirrhosis), heart (dysfunction, arrhythmia — former cause of death in teens), pancreas
Chelation	Deferoxamine subcutaneous infusion over 8–12 h daily (standard; compliance problems) · oral deferasirox and deferiprone (FDA-approved). Survival now into 4th–5th decade
HSCT	Only cure. Good-risk patients: >90% overall and 80% thalassemia-free survival; best with HLA-identical sibling (25% chance per sibling)
Hb F inducers	Hydroxyurea, 5-azacytidine, short-chain fatty acids, ESAs, thalidomide derivatives → switch γ back on; hydroxyurea helped a few
Gene therapy	2010: lentiviral β-globin gene transfer made an adult with severe Hb E-β⁰-thal transfusion-independent; trials ongoing

2.14 β-Thalassemia Intermedia

Feature	Details
Definition	More severe than minor but transfusion-independent; Hb usually >7 g/dL — clinical features, not the Hb level, decide the diagnosis
Genotypes	Homozygous mild mutations; compound heterozygous mild + severe; rarely one severe gene (dominantly inherited β-thal)
Milder modifiers	Coinherited α-thalassemia (fewer free α chains) or ↑ γ-chain production (δβ⁰ forms)
Worse modifier	Coinherited triplicated α gene (ααα) with β-thal minor → more α excess
Clinical	Variable anemia/jaundice; splenomegaly (↓ platelets/neutrophils); exercise intolerance, fractures; iron overload without transfusion (hepcidin suppression) — complications 10–20 years later than in major

2.15 Other β-Gene Cluster Thalassemias

Condition	Genetics	Heterozygote	Homozygote
HPFH (hereditary persistence of fetal Hb)	Deletion in δβ region, or nondeletional γ-promoter mutations	Asymptomatic, normal MCV, Hb F 10–35%	Asymptomatic; 100% Hb F; normal to slightly ↑ Hb (higher O₂ affinity); slightly hypochromic, microcytic
δβ⁰-thalassemia	Deletion of δ and β (nondeletional forms exist); γ ↑ but not enough	↓ A, N or ↓ A₂, F 5–20%; β-thal minor phenotype	100% F; hypochromic microcytic; β-thal intermedia
Hb Lepore	δβ fusion from nonhomologous crossover in meiosis; N-terminal δ (22–87 aa) + C-terminal β (31–97 aa); under weak δ promoter → low output. Reciprocal = anti-Lepore	↓ A and A₂, ↑ F, Lepore 5–15%; like β-thal minor	No A or A₂; ~80% F, ~20% Lepore; like β-thal major

2.16 Kleihauer-Betke & Hb F Distribution

Feature	Details
Principle	Ethanol-fixed film in citrate buffer pH 3.3: adult Hbs eluted (ghost cells); Hb F resists elution and stains
Pancellular	Hb F in all RBCs — deletional HPFH
Heterocellular	Hb F in some cells — nondeletional HPFH and all other β-cluster thalassemias
Other use	Estimate fetal–maternal hemorrhage for Rh immune globulin dosing (flow cytometry now standard)

2.17 Screening for β-Thalassemia Minor

Feature	Details
Why	High carrier frequency; carrier parents have 25% risk of thal major/intermedia per pregnancy
Initial tests	Hemoglobin, MCV, Hb A₂ and Hb F; rule out iron deficiency
Programs	Mass screening + prenatal diagnosis in Italy and Greece markedly reduced affected births
Molecular	HBB gene testing for couples seeking preconception counseling or prenatal testing

2.18 α-Thalassemia — Genetics

Feature	Details
Main defect	Large deletions of α₁ and/or α₂; nondeletional (point) mutations uncommon
α₂ dominance	α₂ produces ~75% of α chains → α₂ mutations cause more severe anemia
α⁰ haplotype (– –)	Formerly α-thal-1; both genes deleted (21 known deletions, some including ζ). ~4% in Southeast Asia; less in Mediterranean; rare elsewhere
α⁺ haplotype (– α)	Formerly α-thal-2; one gene deleted — most common α-thal haplotype; carrier frequency 50–80% in parts of Saudi Arabia, India, SE Asia, Africa; ~30% of African Americans
α^Tα (nondeletional)	>40 point mutations mostly in α₂; unstable/fewer chains → more severe than – α
Hb Constant Spring	α₂ 142 Stop→Gln: stop codon lost → mRNA read on → α chain with 31 extra amino acids; unstable mRNA and tetramer → very low levels. Faint, slow band near the origin on alkaline electrophoresis

2.19 Clinical Syndromes of α-Thalassemia (Rodak Table 28-4)

Syndrome	Genotype	Hb A	Hb Bart (Newborn)	Hb H (Adult)	Hb Constant Spring
Normal	αα/αα	N	0	0	0
Silent carrier (asymptomatic, normal hematology)	– α/αα	N	1–2%	0	0
	α^CSα/αα	N	1–3%	0	<1%
α-Thal minor (mild anemia, microcytic, hypochromic)	– –/αα (cis)	sl ↓	5–15%	0	0
	– α/– α (trans)	sl ↓	5–15%	0	0
	α^CSα/α^CSα	sl ↓	5–15%	0	<6%
Hb H disease (mild–moderate hemolytic anemia, transfusion-independent)	– –/– α	↓	10–40%	1–40%	0
	– –/α^CSα (Hb H Constant Spring)	↓	↑↑	↑↑	<1%
Hb Bart hydrops fetalis (stillborn or dies shortly after birth)	– –/– –	0	80–90% (rest Hb Portland)	NA	0

2.20 α-Thalassemia Syndromes — Details

Feature	Details
Silent carrier (– α/αα)	Near-normal α/β ratio; no hematologic abnormality; slight ↑ γ at birth → Hb Bart 1–2%. No reliable diagnosis except genetic analysis. α^Tα/αα also silent; α^CSα/αα: Constant Spring <1%
α-Thal minor (2 genes)	Homozygous α⁺ (– α/– α) or heterozygous α⁰ (– –/αα). Hb 12–13 g/dL, microcytic hypochromic; Bart 5–15% at birth; no Hb H in adults; Hb A₂ not increased. α^Tα/α^Tα → mild–moderate hemolysis; α^CSα/α^CSα: CS 5–6%, Hb 9–11 g/dL
Hb H disease (3 genes: – –/– α)	Common in Asians (α⁰ haplotype). Newborn Bart 10–40%; adult Hb H 1–40%, ↓ A₂, traces of Bart. Nondeletional (– –/α^Tα, e.g., Hb H-Constant Spring) → more severe, more Hb H
Hb H — clinical	Hb 7–10 g/dL, retics 5–10%; splenomegaly; marrow erythroid hyperplasia; transfusion-independent but hemolytic crises with infection, pregnancy, oxidant drugs
Hb H — film	Microcytic, hypochromic, marked poikilocytosis (target cells, bizarre shapes); Hb H precipitates in older circulating cells
Hb H inclusions	Brilliant cresyl blue or new methylene blue → fine, evenly distributed granules — ‘golf balls’ / ‘raspberries’. Pitted by spleen; after splenectomy most cells contain them. Splenectomy helps markedly enlarged spleens
ATR-16	Large deletion on the short arm of chr 16 (ζ, α and flanking genes) → deformities, intellectual disability, Hb H disease
ATRX syndrome	ATRX gene mutation on the X chromosome (regulates α-globin expression) → males with intellectual disability, deformities, developmental delay, Hb H disease. Acquired ATRX mutations in MDS (acquired Hb H)
Hb Bart hydrops fetalis (– –/– –)	No α chains. Hb Bart predominant + small Hb Portland (ζ₂γ₂, lets fetus survive to 3rd trimester) + traces of H. Hb 3–8 g/dL, many nRBCs, hepatosplenomegaly, cardiomegaly, edema, ascites; stillborn or dies shortly after birth. Mother: toxemia, postpartum hemorrhage
Hydrops — prenatal	Ultrasound at midgestation; if both parents carry α⁰ (– –/αα), molecular testing of chorionic villi or amniotic fluid; early termination prevents maternal complications

2.21 Thalassemia with Structural Hb Variants

Feature	Details
Hb SS + α-thal	Common (both frequent in African ancestry). Milder anemia, higher Hb, lower retics: αα/αα Hb 8.4 (retics 10.8%) · – α/αα 9.0 (8.8%) · – α/– α 9.5 (6.9%)
Hb S-β⁺-thal	Mostly Hb S, slightly ↑ A₂, variable F and A; S > A; microcytosis, splenomegaly (vs trait: A > S)
Hb S + β^silent	Slightly more severe than sickle trait; mild hemolysis, splenomegaly, microcytosis; S > A
Hb S-β⁰-thal	No Hb A → resembles sickle cell anemia (stroke, life expectancy, painful crises); microcytosis and ↑ A₂ distinguish it
Hb C-β-thal	Moderately severe hemolysis, splenomegaly, microcytosis, many target cells; more C with less β production
Hb E-β-thal	Major concern in Southeast Asia and eastern India; Hb E splice mutation ↓ production; clinical picture like β-thal intermedia or major

2.22 β-Thalassemia with Structural β-Globin Variants (Rodak Table 28-5)

Genotype	Hb A	Hb A₂	Hb F	Other Hb	RBC Morphology	Clinical / Treatment
Hb S-β⁺-thal	↓↓	↑	N to ↑	S > A	Microcytes, sickle cells, target cells	Mild–severe, recurrent VOC; none → transfusion and pain control
Hb S-β⁰-thal	0	↑	N to ↑	S	Same	Same (more severe)
Hb C-β⁺-thal	↓↓	†	↑	C > A	Microcytes, Hb C crystals, target cells	Moderate–severe anemia; usually no treatment
Hb C-β⁰-thal	0	†	↑	C	Same	Same
Hb E-β⁺-thal	↓↓	†	↑↑	E > A	Microcytes, target cells	Mild–severe, may need transfusions
Hb E-β⁰-thal	0	†	↑↑	E	Same	Same

†Not all methods can quantitate A₂ with the abnormal Hb: HPLC separates A₂ from Hb C; capillary zone electrophoresis separates A₂ from Hb E.

2.23 Diagnosis — History & CBC

Feature	Details
History	Individual and family history are paramount; ethnic background
Physical	Pallor, jaundice, splenomegaly, skeletal deformities (untreated β-thal major)
CBC	↓ Hb, Hct; RBC count disproportionately high for the anemia → very low MCV and MCH; ↓ MCHC. RDW ↑ in untreated major; often normal in minor
Film	Silent carriers normal. Minor/Hb H: microcytes, target cells, slight–moderate poikilocytosis. Major: extreme poikilocytosis, target cells, elliptocytes, polychromasia, stippling, HJ and Pappenheimer bodies, nRBCs
Retics	Hb H 5–10%; homozygous β-thal 2–8% (inappropriately low)
Supravital stain	Brilliant cresyl blue / new methylene blue → Hb H inclusions (small, multiple, greenish-blue, evenly distributed, golf ball). Almost all cells in Hb H disease; few in α-thal minor; rare in silent carrier. Heinz bodies are larger, fewer, eccentric along the membrane

2.24 Laboratory Diagnosis of Thalassemias (Rodak Table 28-6)

Level	Test	Expected Findings
Screening	CBC · film · iron studies (rule out IDA)	↓ Hb, Hct, MCV, MCH, MCHC; retics sl–mod ↑; microcytosis, hypochromia, target cells, poikilocytosis, inclusions, nRBCs; ferritin and iron N or ↑, TIBC N
Presumptive	Supravital stain · Hb fractions by electrophoresis, HPLC and/or CZE	α-thal: Hb H inclusions · β-thal: A ↓ or 0, A₂ ↑ (carriers), F usually ↑, Hb Lepore/other · α-thal: A ↓ or 0 (hydrops), A₂ ↓, Hb Bart, Hb H, Hb Constant Spring
Definitive	Molecular genetics: β-thal >250 mutations in HBB; α-thal >100 in HBA1/HBA2	Step 1 targeted mutation analysis (4–6 common β mutations; 7 common α deletions) → Step 2 DNA sequencing → Step 3 deletion/duplication analysis (MLPA, aCGH)

2.25 Hemoglobin Fraction Methods

Feature	Details
Principle	No single method identifies and quantifies all Hbs → confirm with at least two methods; molecular testing for prenatal, preconception, rare/complex mutations, prognosis
Alkaline electrophoresis	Lysate on agarose in alkaline buffer; bands quantified by densitometry. Distinguishes A, F, S, C and fast Hbs (H, Bart). Can’t accurately quantitate A₂ and F; D and G comigrate with S; E and O-Arab with C
Cation-exchange HPLC	Elution gradient; detector at 415 nm; area under peak = %. Replaced electrophoresis as routine screen; ideal for thal screening — quantitates A, A₂ and F with 100% sensitivity and 90% specificity without variants
HPLC limitations	A₂ and E same retention time; A₂ overestimated with Hb S and underestimated with Hb D-Punjab; can’t identify all variants; needs trained staff
Capillary zone electrophoresis	Silica capillary, alkaline buffer, migration toward cathode by electro-endosmotic flow; 415 nm. Advantage: separates and quantifies A₂ with Hb E; limitation: A₂ overlaps with Hb C
Newborn screening	IEF and mass spectrometry also used
Hb A₂ microcolumn	Manual method for A₂ quantitation

2.26 Molecular & Other Procedures

Feature	Details
HBB targeted PCR	4–6 common mutations per ethnic group → 91–95% detection (Mediterranean, Middle East, Thai, Chinese); 75–80% (African/African American)
HBB sequencing	Exons, introns, splice sites, 5′ and 3′ UTRs → ~95% of known mutants; then deletion/duplication analysis (MLPA, aCGH)
HBA1/HBA2	PCR for 7 common deletions (~90% of alleles) → sequencing → deletion/duplication
Prenatal	Amniocentesis (15–18 weeks), chorionic villus sampling (10–12 weeks), preimplantation genetic diagnosis (3-day embryo, IVF)
Alkali denaturation	Hb F resists strong alkali; accurate for Hb F 0.2–50%; HPLC now often used
Single-tube osmotic fragility	Screening in low-resource settings: blood in 0.375% saline for 5 min. Normal cells lyse (clear); hypochromic carrier cells resist (turbid). Not specific — positive in iron deficiency too

2.27 Thalassemia Minor vs Iron Deficiency Anemia

Feature	β-Thalassemia Minor	Iron Deficiency Anemia
History	Family history of thalassemia; ethnicity	Previously normal Hb/indices, bleeding, pica (pagophagia — ice craving)
RBC count	Normal to ↑ (mild erythrocytosis)	Normal or ↓
MCV	Markedly ↓ for the degree of anemia	Normal to ↓ (depends on stage)
RDW	Normal or ↑ (overlaps)	↑ (overlaps)
Serum ferritin	Normal or ↑	↓
Serum iron / TIBC / saturation	N / N / N	↓ / ↑ / ↓
Hb A₂	↑ (3.5–7%)	Normal or ↓ (iron deficiency lowers A₂)
Film	Basophilic stippling favors thal; target cells in both	Target cells possible
Other	—	↑ soluble transferrin receptor, ↑ zinc protoporphyrin*
Key rule	Rule out/replete iron deficiency before measuring A₂ — coexisting IDA can mask ↑ A₂

*Rodak lists sTfR and ZPP as additional tests (Chapter 20); the direction of change is from that chapter. Discrimination indices (Mentzer, Green and King, England and Fraser, Shine and Lal, Srivastava) have 60–96% sensitivity — not appropriate for screening.

3 · Visual Memory Hooks

🧠 Qualitative vs quantitative
“Thalassemia = Too Little; Hemoglobinopathy = Wrong Kind”.

🧠 The real villain
“It’s the leftover chains that kill” — imbalance, not just deficiency, drives severity.

🧠 β vs α mechanism
“β-thal: α’s Attack the marrow (ineffective); α-thal: β’s/γ’s form Bands of four (H, Bart)”.

🧠 Bart vs H
“Bart is a Baby (γ₄); H is for Hulk-grown adult (β₄)”.

🧠 Gene count
“1 gene = Silent, 2 = Trait, 3 = H, 4 = Hydrops” (α-thal).

🧠 α₂ gene
“α₂ does 75% of the Work”.

🧠 Constant Spring
“Constant Spring never Stops” — lost stop codon, +31 amino acids.

🧠 β-thal minor
“High RBC, Low MCV, High A₂, Normal ferritin”.

🧠 Mentzer*
“MCV/RBC <13 → Thal; >13 → Iron deficiency” — but not for screening.

🧠 β-thal major labs
“F is Fabulous (70–90%), A is Absent, Retics are Retiring (low)”.

🧠 Skull
“Hair on end = marrow on overdrive”.

🧠 Iron overload
“Blood in, iron stays” — no excretion pathway; chelate (deferoxamine).

🧠 Transfusion plan
“10–15 mL/kg every 2–5 weeks, keep Hb 9–10.5”.

🧠 Hb H inclusions
“H = Hundreds of tiny golf-ball dimples; Heinz = Handful of big dots at the edge”.

🧠 HPFH
“HPFH = Happy People with Fetal Hb” — asymptomatic, pancellular (deletional).

🧠 Lepore
“Lepore = δ head + β tail, driven by the lazy δ promoter”.

🧠 HPLC vs CZE
“HPLC can’t split A₂ from E; CZE can’t split A₂ from C”.

🧠 A₂ pitfalls on HPLC
“S Swells A₂; D-Punjab Diminishes A₂”.

🧠 IDA and A₂
“Iron deficiency Irons out (lowers) A₂” — replete iron first.

🧠 Kleihauer-Betke
“Acid washes out Adult Hb; Fetal Hb stays Fast”.

*Rodak names the Mentzer index but doesn’t give the cutoff; <13 favoring thalassemia is the standard general-reference value.

Pattern Triggers

If you see…	Think…	Because…
RBC 5.7, Hb 10.2, MCV 61, A₂ 4.9%, ferritin normal, Mediterranean family	β-thalassemia minor (Rodak case study)	Heterozygous β mutation
Microcytosis with normal A₂, normal ferritin, African American	α-thal minor (– α/– α)	A₂ not increased in α-thal
Child 6–24 months, Hb 4, MCV 60, many nRBCs, target/teardrop cells, Hb F 90%	β-thalassemia major	γ→β switch reveals the defect
Golf-ball inclusions in nearly all cells, Hb 8, retics 7%, Asian	Hb H disease (– –/– α)	β₄ precipitates
Hydropic stillborn, Hb 5, many nRBCs, Hb Bart 85%	Hb Bart hydrops fetalis (– –/– –)	No α chains
Newborn with Hb Bart 1–2%, normal CBC	α-thal silent carrier	One α gene deleted
Hb F 25%, normal MCV, well adult, pancellular K-B stain	Heterozygous deletional HPFH	γ production persists
Hb F 10%, low A₂, microcytosis	δβ⁰-thal trait	δ and β deleted
Extra band 5–15%, ↓ A₂, microcytosis	Hb Lepore trait	δβ fusion
Sickle cells + microcytes + ↑ A₂, no Hb A	Hb S-β⁰-thal	Resembles SCA
S > A with microcytosis and splenomegaly	Hb S-β⁺-thal	Trait would show A > S
Southeast Asian child needing transfusions, Hb E + F	Hb E-β-thal	E splice defect + β-thal
Intellectual disability, Hb H disease, male	ATRX syndrome	X-linked α-globin regulator
Elderly MDS patient with new Hb H inclusions	Acquired Hb H (ATRX mutation)	Somatic ATRX
Microcytosis, ferritin 6, A₂ 2.8%, family history of thal	IDA — possibly masking β-thal trait	Iron deficiency lowers A₂; recheck after iron

Commonly Confused Pairs

Feature	A	B
Thalassemia vs hemoglobinopathy	↓ amount of normal chain	Abnormal chain structure
β⁰ vs β⁺	No β chain	5–30% of normal β
α⁰ vs α⁺	Both α genes on one chr deleted (– –)	One α gene deleted (– α)
Cis vs trans α-thal minor	– –/αα (Asian; risk of hydrops offspring)	– α/– α (African)
Hb Bart vs Hb H	γ₄, fetus/newborn	β₄, adults, precipitates
β-thal minor vs α-thal minor	↑ A₂	Normal A₂; diagnosis often by exclusion/DNA
β-thal major vs intermedia	Transfusion-dependent	Transfusion-independent (Hb >7)
Hb H inclusions vs Heinz bodies	Many, small, even, golf ball	Few, large, eccentric at membrane
HPFH vs δβ⁰-thal	Normal MCV, no anemia, F 10–35% (het)	Microcytic, β-thal minor phenotype, F 5–20%
Pancellular vs heterocellular Hb F	Deletional HPFH	Nondeletional HPFH, other thals
HPLC vs CZE	A₂ = E overlap; separates A₂ from C	Separates A₂ from E; A₂ = C overlap
β-thal minor vs IDA	↑ RBC, N ferritin, ↑ A₂, stippling	↓ ferritin, ↓ A₂, history of bleeding/pica

Timeline Table

Event	Timing	Key Point	Best Exam Use
ζ→α and ε→γ switches	By 10 weeks’ gestation	Hb F becomes dominant	α-thal affects fetus
Hb Portland protects hydrops fetus	Until 3rd trimester	ζ₂γ₂ carries some O₂	Why hydrops fetus survives to late pregnancy
γ→β switch	Around birth; Hb A dominant by 6 months	β-thal symptoms start 6–24 months	Onset of β-thal major
β-thal major diagnosis	6 months–2 years	Hb A fails to rise	Age clue
Transfusions	Every 2–5 weeks	10–15 mL/kg; RBCs <7–10 days old	Regimen
Organ iron damage	Rises after age 10–11	Heart, liver, endocrine	Chelation importance
Intermedia complications	10–20 years later than major	Iron overload w/o transfusion	Hepcidin suppression
CVS / amniocentesis	10–12 weeks / 15–18 weeks	Prenatal diagnosis	Counseling
Hydrops detection	Midgestation ultrasound	Hydropic changes	Prenatal screening

4 · Diagnostic Flowcharts

Flowchart 1 — Microcytic hypochromic anemia: thal trait or iron deficiency?

Low MCV/MCH	→	History (family, ethnicity, bleeding, pica) + CBC + film
Iron studies	→	Ferritin ↓ → iron deficiency → treat, then re-evaluate if microcytosis persists
Ferritin normal/↑	→	Hb fractions by HPLC or CZE
Hb A₂ >3.5%	→	β-thalassemia minor
Hb A₂ normal, F normal	→	Consider α-thal (supravital stain; DNA) or δβ/Lepore variants
Hb F ↑ with normal/↓ A₂	→	δβ⁰-thal or HPFH; extra band → Hb Lepore
⚠️ Common mistake: measuring A₂ while the patient is iron deficient — IDA lowers A₂ and can hide β-thal trait.

Flowchart 2 — Classifying β-thalassemia

Asymptomatic, normal indices, normal A₂	→	Silent carrier (β^silent/β) — DNA only
Mild anemia, MCV <75, A₂ 3.5–7%	→	β-thal minor
Severe anemia 6–24 months, F 70–>90%, needs transfusion	→	β-thal major
Moderate anemia (Hb >7), no regular transfusion, splenomegaly	→	β-thal intermedia
Modifiers	→	α-thal coinherited → milder; ααα triplication → worse
⚠️ Common mistake: labeling intermedia by the Hb level alone — the clinical course (transfusion need) defines it.

Flowchart 3 — α-thalassemia by number of genes deleted

1 gene (– α/αα)	→	Silent carrier: normal CBC; Hb Bart 1–2% at birth
2 genes (– –/αα or – α/– α)	→	α-thal minor: mild microcytic anemia; Bart 5–15% at birth; normal A₂
3 genes (– –/– α)	→	Hb H disease: Hb 7–10, retics 5–10%, golf-ball inclusions, Hb H 1–40%
4 genes (– –/– –)	→	Hb Bart hydrops fetalis: Bart 80–90%, fatal
Counseling	→	Two – –/αα (cis) parents → 25% risk of hydrops
⚠️ Common mistake: forgetting that cis (– –/αα) carriers, common in Southeast Asia, can have a hydrops baby while trans (– α/– α) carriers cannot.

Flowchart 4 — Supravital stain (brilliant cresyl blue) interpretation

Incubate blood with BCB / new methylene blue	→	Examine for inclusions
Many small, evenly spread greenish-blue granules; golf-ball look	→	Hb H inclusions → Hb H disease (almost all cells)
Occasional cells with golf-ball inclusions	→	α-thal minor (few) or silent carrier (rare)
Few large inclusions at the membrane edge	→	Heinz bodies → G6PD, unstable Hb, oxidant drugs
Mesh-like precipitated RNA	→	Reticulocytes (don’t confuse)*
⚠️ Common mistake: calling Heinz bodies Hb H inclusions — size, number and position differ.

*Reticulocyte reticulum on the same stain is general knowledge, not from Rodak Ch. 28.

Flowchart 5 — Choosing and interpreting Hb fraction methods

Screen for thal carriers	→	HPLC: quantitates A, A₂, F
Peak at A₂ retention time >10%*	→	Suspect Hb E (same retention as A₂) → CZE separates
Hb S present	→	A₂ may be overestimated on HPLC
Hb C present	→	CZE can’t quantify A₂ (overlap) → use HPLC
Unresolved/complex	→	Molecular genetics (targeted → sequencing → MLPA/aCGH)
⚠️ Common mistake: trusting a single method — confirm variants with at least two methods.

*A ‘>10% at the A₂ window suggests Hb E’ threshold is a common laboratory rule of thumb, not from Rodak.

Flowchart 6 — Managing β-thalassemia major

Hb <7 g/dL + symptoms	→	Start regular transfusion (extended antigen-matched, fresh RBCs)
Maintain pretransfusion Hb 9–10.5	→	Suppresses erythropoiesis and bone changes
Monitor ferritin / iron load	→	Chelation: deferoxamine SC 8–12 h/day or oral deferasirox, deferiprone
HLA-identical sibling available	→	HSCT — only cure
Investigational	→	Hb F inducers (hydroxyurea), gene therapy
⚠️ Common mistake: stopping chelation because the patient ‘feels fine’ — iron damages the heart silently.

5 · 25 High-Yield Facts

#	Fact
1	Thalassemias are caused by absent or defective synthesis of a polypeptide (globin) chain.
2	Thalassemia is common in malaria regions because heterozygotes are protected against malaria.
3	The hemolytic anemia in thalassemia is due to imbalance of globin chain synthesis.
4	α and ζ genes are on chromosome 16; ε, γ, δ and β genes are on chromosome 11.
5	β⁰ means no β chains are produced; β⁺ means 5–30% of normal production.
6	In β-thalassemia, excess α chains precipitate in precursors and cause ineffective erythropoiesis.
7	β-thalassemia symptoms start at 6–24 months, after the γ→β switch.
8	β-thalassemia minor shows MCV <75 fL, a normal to slightly high RBC count and Hb A₂ 3.5–7%.
9	Microcytes, hypochromia, target cells, elliptocytes and stippled cells are typical of β-thalassemia.
10	In β⁰-thalassemia major, the predominant hemoglobin is Hb F (70% to >90%).
11	β-thalassemia major has an M:E ratio around 1:20 and an inappropriately low reticulocyte count.
12	Transfusions of 10–15 mL/kg every 2–5 weeks keep pretransfusion Hb at 9–10.5 g/dL.
13	Iron overload is the major complication of transfusion therapy; deferoxamine is the standard chelator.
14	HSCT is the only cure for thalassemia major.
15	Thalassemia intermedia is transfusion-independent; iron overload still occurs from increased absorption.
16	Heterozygous HPFH has 10–35% Hb F with normal RBC morphology and MCV.
17	Hb Lepore is a δβ fusion; heterozygotes have 5–15% Hb Lepore.
18	Hb Bart is γ₄ and Hb H is β₄.
19	Deletion of one α gene (–α/αα) gives a silent carrier with normal hemoglobin.
20	Hb H disease results from deletion of three α genes (– –/–α).
21	Hb H inclusions on supravital stain look like small, evenly distributed golf-ball granules.
22	Hb Bart hydrops fetalis (– –/– –) is most common in Southeast Asia, where the α⁰ haplotype occurs.
23	Hb S-β⁰-thalassemia has a clinical course resembling sickle cell anemia.
24	HPLC cannot separate Hb A₂ from Hb E; capillary zone electrophoresis cannot separate A₂ from Hb C.
25	Iron deficiency must be ruled out before Hb A₂ is used to diagnose β-thalassemia minor.

⭐ EXAM TIP — The single most testable pattern

Microcytosis out of proportion to anemia + high RBC count + normal ferritin + Hb A₂ >3.5% = β-thal minor. Same picture with normal A₂ → think α-thal (count the genes). Hb F 70–90% in an infant needing transfusion → β-thal major. Golf-ball inclusions → Hb H disease.

Quick Interpretation Table

Lab Finding	Clinical Interpretation
Sicilian student: RBC 5.74, Hb 10.2, MCV 61, MCH 17.8, MCHC 29.1, stippling, A₂ 4.9%, ferritin 320	β-thal minor (Rodak case study) — partner screening; if partner is a carrier, 25% risk of thal major per pregnancy
Mother: always anemic, repeatedly given iron, gallbladder attacks	Probable undiagnosed β-thal minor treated as IDA; gallstones from chronic hemolysis
Hb 7.5, retics 6%, Hb H 15%, golf-ball inclusions	Hb H disease
Hb 12.5, MCV 72, A₂ 2.5%, F 1%, ferritin 90, African American	Probable α-thal minor (– α/– α) — confirm by DNA
Hb F 100%, well adult, slightly microcytic	Homozygous deletional HPFH
Hb A 0, S 85%, A₂ 5.5%, F 8%, MCV 68	Hb S-β⁰-thalassemia
4-month-old Asian infant: Hb 10, MCV 77, microcytosis, poikilocytosis, polychromasia	Suspect β-thal major (onset after γ→β switch) — Rodak Q15*
Ferritin 5, A₂ 2.9%, strong family history of thal trait	IDA; recheck A₂ after iron repletion

*My reasoning for Rodak Q15 (answer key not in the upload): hydrops (– –/– –) is incompatible with this picture, silent carriers are hematologically normal, and IDA is unusual at 4 months — leaving early β-thal major.

🎴 Flashcards — Active Recall Practice

How it works: tap the card to flip it. Then mark it ✓ Knew It or ✗ Review Again (buttons unlock after flipping). Use 🔀 Shuffle to randomize and 🏁 Finish anytime — your summary appears at the end, and you can drill only the cards you missed.

📝 Practice Quiz — Test Your Knowledge

How it works: tap an answer for each case. You’ll get instant feedback with the full explanation and the examiner’s trap. Your final score appears once all 10 are answered.

🪤 Trap Questions — Beat the Examiner

Harder on purpose. Each question targets a real misconception. After answering you’ll see the correct reasoning, the trap explained, why the examiner wrote it, and the wrong answer most students choose.

8 · Rapid Revision Sheet

📖 READ THIS 2 HOURS BEFORE YOUR EXAM

Basics

Thal = ↓ amount of normal chain
Microcytic hypochromic + chain imbalance
α/ζ chr 16 · ε, γ, δ, β chr 11
Adult: A 95–100, A₂ 0–3.5, F 0–2%
Malaria protection; 5% of world carriers

Designations

β⁰ none · β⁺ 5–30% · β^silent mild
α⁰ (– –) · α⁺ (– α) · α^T point
δβ⁰ · δβ^Lepore · HPFH
β-thal: point mutations (>300)
α-thal: deletions

β-Thal Mechanism

Excess α precipitates in precursors
Ineffective erythropoiesis + splenic hemolysis
Onset 6–24 mo (γ→β switch)
Marrow expansion: hair-on-end, frontal bossing
Iron overload: transfusion + ↓ hepcidin

β-Thal Minor

Hb M 12.4–14.2, F 10.8–12.8
RBC N–↑ · MCV <75 · MCH <26
Target cells, elliptocytes, stippling
A 92–95% · A₂ 3.5–7% · F 1–5%
AR: two carriers → 25% major

β-Thal Major

Dx 6 mo–2 yr · Hb 3–4 untreated · MCV 50–70
F 70–>90% · A 0 (β⁰) or 10–30% (β⁺)
nRBCs, teardrops, HJ, Pappenheimer
M:E 1:20 · retics inappropriately low
10–15 mL/kg q2–5 wk · Hb 9–10.5 · chelate · HSCT

Intermedia & Others

Intermedia: transfusion-independent, Hb >7
α-thal coinherited → milder; ααα → worse
HPFH het: F 10–35%, normal MCV
δβ⁰ het: F 5–20% · homo: F 100%
Lepore het 5–15% · homo 80% F / 20% Lepore

α-Thal Genetics

Mostly deletions · α₂ = 75% of α
α⁰ (– –) ~4% SE Asia
– α most common; ~30% African Americans
α^Tα worse than – α
Constant Spring: stop 142 lost, +31 aa

α-Thal Syndromes

1 gene: silent, Bart 1–2%, DNA only
2 genes: minor, Hb 12–13, Bart 5–15%, A₂ normal
3 genes: Hb H, Hb 7–10, retics 5–10%, H 1–40%
4 genes: hydrops, Bart 80–90% + Portland
Golf-ball inclusions (BCB) · ATR-16 · ATRX

Lab Methods

HPLC 415 nm: A₂ = E; S ↑A₂, D-Punjab ↓A₂
CZE: separates A₂/E; A₂ = C overlap
Electrophoresis: poor A₂/F quantitation
K-B pH 3.3: pancellular (del HPFH)
DNA: targeted → sequencing → MLPA/aCGH

Thal vs IDA

Thal: ↑ RBC, N ferritin, ↑ A₂, stippling
IDA: ↓ ferritin, ↓ iron, ↑ TIBC, pica
IDA lowers A₂ → replete iron first
RDW overlaps; indices not for screening
OFT 0.375% saline: turbid = carrier (nonspecific)

9 · Final Memory Map

⚖️ THALASSEMIAS
- Foundations
  - Definition, history, epidemiology, malaria
  - Gene clusters chr 16 & 11 · designations
  - Mutation mechanisms (transcription, splicing, translation, deletion)
- Pathophysiology
  - β-thal: α excess → ineffective erythropoiesis, iron overload
  - α-thal: Bart (γ₄), H (β₄), high O₂ affinity
- β-Thalassemia
  - Silent carrier · minor (↑ A₂) · intermedia · major (↑↑ F)
  - Transfusion, chelation, HSCT, Hb F inducers, gene therapy
  - HPFH · δβ⁰ · Hb Lepore
- α-Thalassemia
  - Silent (1) · minor (2) · Hb H (3) · hydrops (4)
  - Constant Spring · ATR-16 · ATRX
- With Structural Variants
  - Hb SS + α-thal (milder)
  - S-β⁺ / S-β⁰ (like SCA)
  - C-β-thal · E-β-thal
- Diagnosis
  - CBC, film, retics, supravital stain
  - HPLC, CZE, electrophoresis, molecular
  - Kleihauer-Betke, alkali denaturation, OFT screen
  - Differential from iron deficiency