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Laboratory Investigation of Thalassemia and Hemoglobinopathies, Hb Typing,…
Laboratory Investigation of Thalassemia and Hemoglobinopathies
Hb Bart's Hydrops Fetalis (--/--, βN/βN))
Homozygous α-thalassemia 1
β-thalassemia trait
β0/βA
β+/βE
Homo β-thalassemia disease
β0/βE
β+/βA
β0/β+
β0/β0
β0/βN
Hb E
βE/βA (trait)
βE/βE (Homo HbE)
β+/βE (β+-thal/HbE disease)
β0/βE (β0-thal/HbE disease)
βE/βN (trait)
Laboratory Diagnosis
Sickle Cell Disease (SCD)
Sickling test
sodium metabisulphite reduces O2 tension >> induce sickle-shape
solubility test
Hb S is insoluble in reduced state in phosphate buffer soln
HbS
low molarity: no precipitate
high molarity: precipitate
HbD or HbG
low molarity: no recipitate
high molarity: no precipitate
inconclusive
low molarity: precipitate
high molarity: precipitate
α-thalassemia
1 trait (--/αα, βN/βN)
Hb H disease (--/-α, βN/βN)
Hb CS
trait (αCSα/αα, βN/βN)
Homozygous Hb CS (αCSα/αCSα, βN/βN)
Hb H-CS disease (--/αCSα, βN/βN)
α- and β-thalassemia
Hb E trait + α-thalassemia 2 trait (-α/αα, βE/βN)
Hb F = acid and alkaline resistance
Acid Elution Test (pH 1.5 [6 N HCl])
Alkaline Denaturation Test (1.2 N NaOH)
Hb Typing
Interpretation
A2A
normal
α-thalassemia 1 trait
α-thalassemia 2 trait
β-thalassemia
EA (Hetero HbE)
EE (Homo HbE)
EF (β-thalassemia/HbE disease)
Bart's
A2A Bart's H (HbH disease)
EA Bart's (disease)
EF Bart's (disease)
cut-off values
3.5
Hb A2
VS < 3.5%
10
Hb A2/E
Hb A2 <10%
Hb A2 + E >10%
25
Hb A2/E
less 25% (pure HbE trait)
<25% (HbE trait w/ or w/o α-thalassemia)
27
MCH: 27 pg
less 27 (negative)
<27 (positive)
80
MCV: 80 fL
less 80 (negative)
<80 positive)
Quantitative measurement
Hb A2 (E)
Hb F
HbA
CZE
from anode to cathode
zones:
Z2: Hb CS
Z3 : Hb A2
Z4 : Hb E
Z7 : Hb F
Z9 : Hb A
Z12 : Hb Bart's
Z15 : Hb H
Qualitative measurement
Hb Bart's
Hb CS/PS
cellulose acetate electrophoresis (CEA)
from cathode to anode
HPLC
cation exchanger
Screening test
OF/DCIP interpretation
Group I: -/-
non-thalassemia or non-clinically significant thalassemia
Group II: +/-
suspected α-thalassemia and/or β-thalassemia
Group III: -/+
suspected Hb E trait
Group IV: +/+
suspected Hb E either w/ or w/o α-thalassemia and/or β-thalassemia
OF test
screen for α-thalassemia and/or β-thalassemia
DCIP test
screen for HbE-related syndromes
IC strip test
detect Hb Bart's
MCV/MCH
Risk Assessment of Severe Thalassemia
Group A
Homo α-thalassemia 1 (--/--, Hb Bart's Hydrops Fetalis)
Group B
Homo β-thalassemia
Group C
suspected β+/β0 -thalassemia / Hb E (β+/β0 /βE)
Group D
No risk
DNA analysis (PCR methods)
α-thalassemia
deletion type
Gap-PCR/Agarose gel electrophoresis
Relative Quantitative PCR
β-thalassemia
reverse dot blot hybridization (RDB) (point mutation)