1

Laboratory Diagnosis of Laboratory Diagnosis of ThalassaemiaThalassaemiaand Abnormal and Abnormal HaemoglobinsHaemoglobins

Pranee Pranee WinichagoonWinichagoon FucharoenFucharoenThalassemia Research CenterThalassemia Research Center

Institute of Institute of Molecular BiosciencesMolecular BiosciencesMahidol UniversityMahidol University

Disorders of Hemoglobin Synthesis

Hereditary Persistence of Fetal

Hemoglobin (HPFH)

Persistence in the synthesis of γ-globin chain

4.

Thalassemia-hemoglobinopathy

Synthesis of an abnormal globinchain with reduced synthesis of the Hb variant: quantitative & qualitative disorder

3.

Abnormal Hb(Hb Variant)

Synthesis of an abnormal globinchain (amino acid change): qualitative disorder

2.

ThalassemiaDecreased or absence of globinchain synthesis of hemoglobin: quantitative disorder

1.

Laboratory Diagnosis of ThalassemiaRBC IndicesRBC Osmotic FragilityRBC MorphologyIntraerythrocytic Inclusion BodiesHb TypingHb A2 QuantitationHb F QuantitationAcid Elution StainingSerum FerritinGlobin Chain SynthesisDNA AnalysisFamily Study

@ One tube osmotic fragility (OF) testor MCV/MCH

@ Dichlorophenolindophenol (DCIP) precipitation test

@ Detection of Hb Bart’s

SCREENING FOR THALASSEMIA

0.36% buffered saline 0.36% buffered saline

Normal β-thalassemia/HbE disease

2

Red Blood Cell IndicesRed Blood Cell Indices

• Mean Corpuscular Volume (MCV) 87+5 fL

• Mean Corpuscular Hemoglobin (MCH) 29+2 pg

• Mean Corpuscular Hemoglobin Concentration (MCHC) 34+2 %

Mean Corpuscular Volume (MCV)Mean Corpuscular Volume (MCV)

65+366+1081+564+574+368+583+788+6

MCV (fL)

23+9Hb E with α-Thal 1 trait

23+5α-Thal 1 trait22+1Homozygous α-Thal 2 20+3β-Thal trait

27+3α-Thal 2 trait

20+1Homozygous HbE

26+2Hb E trait

29+2NormalMCH (pg)

DichlorophenolindophenolDichlorophenolindophenol (DCIP) (DCIP) precipitation testprecipitation test

Hb E (α2β2Glu->Lys) เปนฮีโมโกลบินผิดปกติ เกิดจาก

กรดอะมิโนตําแหนง 26 ในสาย β-globin เปล่ียนจาก glutamic เปน lysine เปนผลใหการรวมกันของสาย α-globin และ β-globin ไมเสถียรและเผยหมู sulfhydryl(-SH) อิสระออกมา จึงถูก oxidized ดวยสาร DCIP ไดงายและเร็วกวาฮีโมโกลบินชนิดอ่ืนและตกตะกอนลงมา

α β

δγ

NormalHb A=α2β2HbA2=α2δ2Hb F=α2γ2

α β

δγ

α-Thalassemiaβ4=HbH

γ4=HbBart’s

α β

δγ

β-ThalassemiaIncreased HbA2

(4-6%)

ImmunodetectionImmunodetection of of HbHb BartBart’’ssImmunodetection of Hb Bart’s

1 2 3

4 5 6

Screening for α-thalassemia

Positive for: α-thalassemia 1 heterozygote, α-thalassemia 2 homozygote, Hb H disease, EA Bart’s disease and Hb Bart’s hydrops fetalis

3

iron supplementation x 3mo.Repeated study after

Dx : Hb E trait

Normal

Dx : Normalα-Thal 2 traitHb CS trait

Negative Positive

DCIP

Hb Bart’s

Dx : α-Thal 1 traitβ-Thal trait

Fe deficiency

Decrease

PositiveNegative

DCIP

- Clinical- Rbc morphology- Inclusion body

(Hb H disease)- F cell stain

(β-thal/Hb E)

Dx : Hb E traitHb E Homozygoteβ-Thal/Hb EHb H disease

Negative PositiveDx : β-Thal trait

Fe deficiencyDx : α-Thal 1

trait

MCV or One tube osmotic fragility

Routine Laboratory Diagnosis of Thalassemia

@ Hb Concentration@ RBC Indices and Morphology@ Hb Typing@ Hb F and A2 Quantitation@ RBC Inclusion Bodies

Hb Bart’s in Bart’s hydrops fetalis

Orig

in

A2(

E)

F A Bar

t’sH

+-

B/E diseaseHomo EE trait

B traitE traitB/E disease

Homo EHomo E

Cellulose acetate electrophoresis

- Cellulose acetate electrophoresis and elution

- Microcolumnchromatography

- Automated HPLC, LPLC

- Capillary electrophoresis

Hb analysis and quantification Cellulose Acetate Electrophoresis

4

Hemoglobin Separation by Ion Exchange Chromatography Variant Variant HbHb testing systemtesting system

• Automated high performance liquid chromatography system (HPLC) improves resolution quality (sharp peaks)

• Cation exchange analytical cartridge (30 mm, ID 4.6mm) / particle size ~ 6-9 microns

• 5 μL of blood only. Blood stable for testing ~30 days

• Fast analysis intervals of 6.5 minutes

Principle: HPLC as a Hb testing system

2. Phosphate buffer at different concentration (mobile phase) pass under pressure (either high or low) through the ionic exchange column.

3. Due to the buffer gradient of increasing ionic strength and pH, Hbs are separated according to their ionic interaction with the stationary phase.

1

2

3 4 1. Hemoglobins in the hemolysate are adsorbed on a resin of anionic or cationic particles column (stationary phase).

Principle: HPLC as a Hb testing system

4. Separated Hbs pass through the flow cell of photometer where changes in the absorbance at 415 nm are measured.

5. The assays provide quantitative results for percent HbA2 and HbF, while detecting the most commonly occurring abnormal hemoglobins.

Windows are the time ranges in which Hbs are eluted.

- Common Hbs: F, A, A2

- Abnormal Hbs: S, C, D

Retention time is measured from the time of sample injection to maximum point of each elution peak.

RT (retention time) values Interpretation of results : RT

RT Window

• F 1.15 (1.00-1.30) mins• P2 1.45 (1.30-1.60) mins• P3 1.75 (1.60-1.90) mins• A0 2.60 (2.20-3.30) mins• A2 (E) 3.83 (3.68-3.98) mins• D-window 4.05 (3.98-4.12) mins• S-window 4.27 (4.12-4.42) mins• C-window 5.03 (4.88-5.18) mins

Hb variants have specific RT values

5

Capillary Electrophoresis Principle

The Capillarys® system uses the principle of capillary electrophoresis in free solution, charged molecules are separated by their electrophoreticmobility in an alkaline buffer with a specific pH.

- Separation occurs according to the electrolyte pH and electro-osmotic flow, from cathode to anode.- Hemoglobins were separated within 4 minutes, according to isoelectricpoint, by migration in an electric field in the liquid phase. Direct detection of the hemoglobin is made at an absorbance wavelength of 415 nm at their cathodic end

Positives charges from buffer solution

Negatives charges from capillary wall

Electro-osmotic flow carries allproteins towards the cathodic side

This interaction leads to electro-osmotic flow (EOF) from theanode to the cathode

Control AFSC

Capillarys Electrophoresis PrincipleThermic bridgeTemperatureControlled byPeltier device

Anode +Cathode -

Detector Deuterium lamp

MigrationMigration

High High VoltageVoltage

A

FS

A2C

Normal Hb Type

α β

δγ

NormalHb A=α2β2HbA2=α2δ2Hb F=α2γ2

α β

δγ

α-Thalassemiaβ4=HbH

γ4=HbBart’s

α β

δγ

β-ThalassemiaIncreased HbA2

(4-6%)

α-Thalassemia

α-globin Gene Deletions

ζ2 α1α2 θ1

0kb 10kb 20kb 30kb

ψζ1ψα2 ψα1inter- ζ HVR 3'HVR

--MED

--20.5

--SEA

-α3.7

-α4.2

α-Globin Genotype

Hb Bart’s hydropsfetalis

Hb H diesease

Normal α-Thalassemia 2 α-Thalassemia 1

Hb ConstantSpring

Hb H-CS disease HomozygousHb CS

−CS −CSCS− CS−

6

αα -- Thalassemia 1 TraitThalassemia 1 TraitNormal Normal HbHb levellevelLow MCV (Low MCV (~~ 70 fl)70 fl)HbHb type Atype A22+A+ANormal or Normal or slsl. decreased . decreased HbHb AA22DNA analysisDNA analysisObligatory cases of Obligatory cases of HbHb BartBart’’s s hydropshydrops fetalisfetalis

α β

δγ

NormalHb A=α2β2HbA2=α2δ2Hb F=α2γ2

α β

δγ

α-Thalassemiaβ4=HbH

γ4=HbBart’s

α β

δγ

β-ThalassemiaIncreased HbA2

(4-6%)

β-Thalassemia

Beta thal heterozygote

Hb E heterozygoye Homozygous Hb E

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56.3+13.659.4+12.950.3+13.84.9+1.6β-Thal/Hb E

87.7+5.990.2+4.992.9+3.34.1+0.8Hb E homozygote

20.7+1.221.9+0.616.3+0.84.0+0.3Hb E trait with α-thal 1

29.4+2.327.8+7.525.6+1.43.5+0.4Hb E trait

Hb A2+Hb E /Elution

Hb A2+Hb E /HPLC

Hb E/CEHb A2 / CE

Comparison of hemoglobins A2 and E levels among capillary electrophoresis, automated HPLC and

elution techniques

Hemoglobin H Disease

Hb H –Cs Disease Intraerythrocytic Inclusion Bodies

reticulocyte

inclusion bodies

Hb Q (α 74 Asp-His)-H Disease• Hb: 9.5 g/dl• RBC: 4.83X1012/L• HCT: 30.5 %• MCV: 63 fl• MCH: 19.6 pg• MCHC: 31 g/dl• Hb A2: 1.6%• Hb Q (RT 4.66): 96.4%• Hb Bart’s: positive• DNA studies

Bart’s+H

Hb Q (α 74 Asp-His) trait

• Hb: 11.9 g/dl• RBC: 4.99X1012/L• HCT: 35.5 %• MCV: 71 fl• MCH: 23.8 pg• MCHC: 33 g/dl• Hb A2: 2.5%• Hb Q (RT 4.65): 30.9%• Hb A2 2.5%• DNA studies: -α4.2/αα

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β-Thalassemia / Hb E

• Hb: 4-10 g/dl• RBC indices MCV: 75 fl

MCH: 25 pgMCHC: < 30 g/dl

• RDW: increased• Reticulocyte: increased• S.ferritin: normal or increased• Dichlorophenol- indophenol

(DCIP) test: positive• Hb E: 30-70%

E=55%F+40%

Beta thal/E Disease

Hb CS E A Bart’s disease Effects of α-thalassemia to Hb E levels

• Hb E trait 25-30%• Hb E α-thal 2 25-30%• Hb E α-thal 1 19-23%• Hb E + Hb H 13-18%

(EA Bart’s disease)

56.3+13.659.4+12.950.3+13.84.9+1.6β-Thal/Hb E

87.7+5.990.2+4.992.9+3.34.1+0.8Hb E homozygote

13.9+1.816.7+0.812.6+0.82.2+0.2CS EA Bart’s

13.0+2.114.9+1.611.8+0.73.7+0.2EA Bart’s

20.7+1.221.9+0.616.3+0.84.0+0.3Hb E trait with α-thal 1

29.4+2.327.8+7.525.6+1.43.5+0.4Hb E trait

Hb A2+Hb E /Elution

Hb A2+Hb E /HPLC

Hb E/CEHb A2 / CE

Comparison of hemoglobins A2 and E levels among capillary electrophoresis, automated HPLC and

elution techniques

CS EF Bart’s disease or

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Hb E/β+-thalassemia vsHb E/Hb Malay

• Hb: 10 g/dl• RBC: 5.3X1012/L• Hct: 31 %• MCV: 62 fl• MCH: 19 pg• MCHC: 32 g/dl• Hb E+ Hb A2: 58.9 %• Hb F: 5.4 %• Hb A (Hb Malay): 26.5 %• Parents: Hb Malay and Hb E

trait• DNA studies

Hb E/Hb Dhonburi (β126 Val-Gly)• Hb: 12.1 g/dl• RBC: 5.8X1012/L• Hct: 44 %• MCV: 77 fl• MCH: 21 pg• MCHC: 27 g/dl

Triton urea gel: the abnormal fraction migrates more rapidly than βA-chain

Hb type E +“A”Hb E+ Hb A2: 38.3 %Hb F: 0.8 %Hb Dhonburi (“A”) 42.6 %DNA studies

α2γ2(HbF)α2γ2 (HbF)

α2δ2 (HbA2)α2β2 (HbA)

Hb Gower II α2 ε2Hb Portland ζ2γ2Hb Gower I ζ2ε2

Embryo Fetus Adult

δ

εε GGγ AAγ δ βψβψβ

ζ2

α-like globin gene (Ch16)

β-like globin gene(Ch11)

α2 α1

γ γ δ β

Postnatal Age (Weeks )Postconceptual Age (Weeks )

Site oferythropoisis

Cell

6 12 18 24 30 36 0 6 12 18 24 30 36 42 48

Postnatal Age (Weeks )Postconceptual Age (Weeks )

0

10

20

30

40

50

60

% o

f tot

al g

lobi

n syn

thes

is

Bone marrowspleenliver

Yol

k sa

c

Site oferythropoisis

type megaloblast macrocyte normocyte

αα

γγ

ββεε

ζζ

αα

γγ

ββ

δ

* Acquired :- Physiology : pregnancy (HbF ~ 3-7%)- Recovery from marrow hypoplasia :

after BM transplant,chemotherapy

- Fetal erythropoiesis : juvenile chronic myeloid leukemia

- Miscellaneous : aplastic anemia, PNH, leukemia, hepatoma

Increased hemoglobin FIncreased hemoglobin F

Normal adult: Hb F ≤ 1%Increased Hemoglobin F

* Genetic : Thalassemia: Hereditary persistence of

fetal hemoglobin (HPFH)- pancellular- heterocellular

Hemoglobin FHemoglobin F

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Hb F-like Variants (by LPLC): Hb Hope

AFS

C

N(NTT)

βHope/βE

β0/βE

31

A2

0

A

65.322.66733.411.39Proband

F

Hb typing (%)MCH

(pg)

MCV (fL)

Hct(%)

Hb(g/dL)

Age (yrs)Cases

25

Viprakasit V.

Hb F-like Variants (by LPLC): Hb Hope

2.647.849.628.08830.010.030Mother

2.7

A2

48.7

A

48.624.67532.110.58Proband

F

Hb typing (%)MCH

(pg)

MCV (fL)

Hct(%)

Hb(g/dL)

Age (yrs)Cases

HPFH? Negative for F-cell analysis by Flow cytometryand alkaline F = 1.1%

(SPW)

N

AF

S

C

Hope,Pyrgos,New york, Kodaira

F A

Viprakasit V.

Red Cell Indices

Normocyte MicrocyteHb Analysis Hb Analysis

A2 + A A+CS A+E EE A2 + ANormalα-thal 2

Thalassemic disease(specified by Hb types)

Hb ConstrantSpring

Hb E Determination Hb A2 DeterminationHb E Homozygote

E ~ 25-30% E 21% or less A2 < 3.5%A2 > 3.5%

Hb E traitα-thal 2/Hb E

β-thal trait

Hb < 10 g/dl Hb > 10 g/dl Hb < 10 g/dl Hb > 10 g/dl

Hb E withFe deficiency

α-thal 1/Hb E Fe deficiency orFe deficiencyon top of thalassemia trait

α-thal 1 traitHomo α-thal 2

Hb Bart’sHb Bart’s

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THALASSEMIA DIAGNOSIS

PRENATALBlood or fetal tissues

- NEONATALCord blood

- POSTNATALPeripheral blood

Normal Cord Blood

Cord blood:Homozygous Hb E

Cord blood: β-Thalassemia/Hb E

Hb E in Cord Blood

%Hb E %Hb A

Hb E trait 4.5+1.56 10.1+3.52Homozygous E 8.0+3.55 0β-Thal/Hb E 2.0 0

Hb Bart’s Hydrops Fetalis

12

α-Thalassemia%Hb Bart’s

α-Thalassemia 2 trait 1.9+0.71Homozygous α-thalassemia 2 8.0+2.01 α-Thalassemia 1 trait 9.2+1.09Hb CS trait 2.4 + 0.31Homozygous Hb CS 15Hb H disease (include EABart’s

and EFBart’s disease) 20-30

Laboratory Investigation*Subjects:1.Adults: Child

AdolescentReproductivePregnancy

2. Neonatal (Cord blood)3. Prenatal (Fetal blood)4. Family members

INTERPRETATIONINTERPRETATION

Clinical dataLaboratory dataFamily studyHistory : Previous blood transfusion

Type of specimens (cord/fetal blood, child/adult blood)

HEMOGLOBINOPATHIES IN THAILAND (1)

1. HEMOGLOBIN VARIANTS

1.1 COMMON- Hb E: β 26 Glu-Lys- Hb Constant Spring: elongated α-chain1.2 RARE ALPHA MUTANTS- Anantharaj: α 11 Lys-Gln - Siam: α 15 Gly-Arg- Queens: α 34 Leu-Arg - Thailand: α 56 Lys-Thr- J-Buda: α 61 Lys-Asn - Mahidol: α 74 Asp-His- Suan Dok: α 109 Leu-Arg

1.3 RARE BETA MUTANTS

- Hb C: β 6 Glu-Lys - Siriraj: α 7 Glu-Lys- Malay: β 19 Asn-Ser - G-Hsin Chu: β 22 Glu-Ala- J-Bangkok: β 56 Gly-Asp - Pyrgos: β 83 Gly-Asp

- New York: β 113 Val-Glu - D-Punjab: β 121 Glu-Gln- Dhonburi: β 126 Val-Gly - Tak: elongated β-chain- Lepore (Boston)

HEMOGLOBINOPATHIES IN THAILAND (2)

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RT Values for Hb VariantspH 8.5

+_

A2 (3.6)

Tak (4.3)D Punjab (4.2)

S (4.4)Montgomery (4.6)Q-Mahidol (4.6)

C (5.0)O-Arab (4.8)

E (3.6)Lepore (3.6)D-Iran (3.6)

Hb Constant Spring (5.1)

A (2.4)

Hb Malay (2.4)Hb Dhonburi

H (0.5)

J Bangkok (1.9)

New York (1.4)Hope (1.38)

Pyrgos (1.38)Kodaira (1.4)

F (1.2)

Bart’s (0.2)

Abnormal Hbs

Hb G-Coushattaα 22 Glu-Ala

Hb Siamα 15 Gly-Arg

Hb J-Budaα 61 Lys-Asn

Hb Hope(β136 Gly->Asp)

Hope,Pyrgos,New york, Kodaira

Abnormal Hb at P2 Hb D/Hb E

HbTak

Hb Tak / Hb E

Hb Tak (β147 +AC) trait• Hb: 16.6 g/dl• RBC: 5.6X1012/L• Hct: 48 %• MCV: 87 fl• MCH: 30 pg• MCHC: 34 g/dl• RDW: 12.3• Hb F 0.4 % RT 1.11• Hb A 55.7% 2.46• Hb A2 4.0% 3.6• Hb Tak 34.4% 4.23

Hb E/Hb Tak• Hb: 13.7 g/dl• RBC: 5.7X1012/L• Hct: 40 %• MCV: 69 fl• MCH: 24 pg• MCHC: 33 g/dl• RDW: 15.8 %• Hb E+ Hb A2: 47.3%• Hb F: 1.9 %• Hb Tak: 46.4%• DNA studies

HbTak

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β0-thalassemia / Hb Tak

• Hb: 20.4 g/dl• RBC: 8.2X1012/L• Hct: 59 %• MCV: 72 fl• MCH: 25 pg• MCHC: 35 g/dl• RDW: %• Hb A2: 5.2%• Hb F: 5.5%• Hb Tak: 85.1%• DNA studies

Erythrocytosis

Problem: Problem: HbHb EE vsvs HbHb LeporeLepore or or HbHb DD--IranIran

HPLC: Hb Lepore has same RT value as Hb A2& Hb E, Hb D-Iran

In heterozygote:

- Hb Lepore + HbA2 8-15%- Hb E α-thalassemia: Hb E + HbA2 <25% - Hb E + Hb A2 25-30%- Hb D-Iran + Hb A2 35-45%

• Hb: 14.0 g/dl• RBC: 6.48 X1012/L• Hct: 39%• MCV: 72 fl• MCH: 22 pg• MCHC: 30 g/dl• RDW: 13.7%• Mild hypochromia,

microcytosis, target cells• Phenotype: thalassemia

trait

Hb Lepore trait• Analyte % Time

• F 2.2 1.11• A0 75.4 2.49• Hb Lepore+A2 13.2 3.6

S,F,A

Pt

Father

N

E. George

Homozygous Hb Lepore

Hb : 8.0 g/dlRBC 3.05X1012/LMCV : 78 flMCH : 26 pgMCHC 33 g/dlRDW 29Hb F: 86%Hb Lepore: 10%No Hb AHPLC:Hb Lepore = 10.7%

(RT 3.5)

S,F,A

Pt

Father

N

Lepore is a δβ fusion gene

Hb E/β0-thalassemia vs Hb E/Hb Lepore• Hb: 11.4 g/dl• RBC: 4.91 X1012/L• Hct: 34 %• MCV: 70 fl• MCH: 23 pg• MCHC: 33 g/dl• Reticulocyte:2.4%• HPLC Hb E+Lepore: 67.6

Hb F: 26.3% No Hb A

• Parents: Hb E and HbLepore trait

• DNA studies

Hb electrophoresis, pH 9.1E(A2) +F + Lepore

DEAE cellulose chromatography- Hb E+A2 53%- Hb F 30%- Hb Lepore 12.7%