laboratory diagnosis in thalassemia and hemoglobinopathies ahmad shihada silmi msc, fibms staff...

Laboratory Diagnosisin

Thalassemia and Hemoglobinopathies

Ahmad Shihada Silmi Msc, FIBMS

Staff Specialist in Hematology

Medical Technology Department

Islamic University of Gaza

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Thalassemia and Thalassemia and hemoglobinopathieshemoglobinopathies

Disorders of globin chain production as a consequences of globin gene defects

As a results, hemoglobin productions are also affected.

Also, some properties of red blood cells are also affected.

Thus it can be recognized by various hematological laboratory tests.

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Making diagnosis of thalassemiaMaking diagnosis of thalassemia

History retrieve Physical examination Laboratory investigations

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Laboratory Thalassemia Diag Laboratory Thalassemia Diagnosisnosis

Red Cell Studies : CBC, One- Tube OF Test , DCIP Test

Hb Studies : Electrophoresis, Microcolum n chromatography, Alkali Denaturation Te

st, HPLC/ LPLC, Imnunologic Detection, Acid elution test

DNA studies : Gene mapping, PCR, Nt sequencing, RFLP analysis

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Blood sampleBlood sample

*Fresh venous blood sample stored in EDTA (3-5 ml) is enough.

*This blood sample is used for both RBC studies, Hb studies and DNA studies.

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RBC studiesRBC studies

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CBCCBC

CBC (automate is a must) for red blood parameters including Hb, Hct, RBC indicies and RBC morphology examination (already trained)

Hb H inclusion body test

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Hb and Hct Hb and Hct Low in thalassemia disease

– Hb < 6 g% in thal major– Hb 6-10 g% in thal intermedia– Hb 10-12 g% in thal minor or

thal trait Normal or slightly low in hete

rozygote – ( Male ~15 g%, Female ~ 13 g

%)

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MCV, MCH MCV, MCH

Cut-off level : MCV 80 fl, MCH 20 pg Low in thalassemia diseases and thal

assemia trait Normal or slightly low in - -thal2 trait

, HbE trait, Hb CS trait

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RBC Morphology RBC Morphology

Thalassemia disease : Hypochromia , Anisocytosis, Poikilocytosis, Polych

romasia, Target cells, Basophilic Sti ppling, NRBC

Thalassemia Trait and Homo E : Mo dest change in RBC morphology

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Normal or Normal or -thal 2 trait-thal 2 trait

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-thal 1 trait-thal 1 trait

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- thal trait- thal trait

x400

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Hb E traitHb E trait

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- thal/HbE disease- thal/HbE disease

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HbE/HbE/-thal-thal

400x 1000x

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Homozygous o-thalassemia

250x

)Hb6.3g/dl, Hct 20%, MCV62 fl ,Ret11.5%, NRBC10/100WBC(

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Hb H Disease Hb H Disease

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Hb Bart’s Hydrops Fetalis Hb Bart’s Hydrops Fetalis

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Hb H inclusion body Hb H inclusion body

testtest Principle Hb H (4) is an unstable hemoglobin

commonly seen in -thalassemia. On incubation with some oxidative chemicals such as brilliant cresyl blue (BCB), HbH is oxidised, denatured and precipitated in the erythrocytes and seen as small, evenly-distributed, intra-erythrocytic blue dots which termed HbH inclusion bodies.

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Hb H inclusion body testHb H inclusion body test

Reagents : As for reticulocyte countStep-by-step procedure : The stain and incubation are as for

reticulocyte countBut look for red blood cells containing HbH

inclusion bodies and report as numbers of those red blood cells in certain amount of total red blood cells examined.

If numerous HbH inclusion body containing eryhthrocytes are seen : Report in %

If few or rare HbH inclusion body containing eryhthrocytes are seen : Report in actual numbers of those rbc/30,000 rbc

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Hb H inclusion body testHb H inclusion body test

Homozygous -thalassemia IB : Negative HbE/-thalassemia IB : Negative AE Bart’s, EF Bart’s IB : 5-10% of total RBC Hb H disease, Hb H-CS disease IB : 50-100% of total RBC -thalassemia 1 heterozygote IB : 1/30,000

RBC -thalassemia 2 heterozygote IB : Rare

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Picture of Hb H inclusion bodiesPicture of Hb H inclusion bodies

-thal 1 trait

Hb H disease

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One-tube OF testOne-tube OF test

Principle At a constant hypotonic NaCl

solution of 0.36% (w/v), hypochromic red blood cells are able to uphold certain amount of water and remain intact whereas the normal erythrocytes cannot and explode.

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One-tube OF testOne-tube OF test

Reagent : 0.36% Buffered Saline Solution (BSS)Procedure Mix 20 ul EDTA blood with 5 ml 0.36% BSS Stand at RT for 5 min. Visualize for hemolysis -If clear red solution is observed : negative -If turbid red solution is observed : positive

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- One tube OF test (0.- One tube OF test (0.3636 % BSS) % BSS)

Negative Positive

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One Tube OF Test One Tube OF Test

Thalassemia diseases +- thal trait +- -thal1 trait +- -thal2 trait -+/

HbE trait -+/ Homo E -+/

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DCIP precipitation testDCIP precipitation test

Principle HbE (2E

2) has loose contact between and -globin chains. When it is incubated with dichlorophenol indophenol (DCIP); oxidizing agent, it will be denatured and precipitated. The reaction is stopped by adding ascorbic acid and the denatured HbE precipitates.

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DCIP testDCIP test

Reagent : DCIP reagentProcedure Mix 20 ul packed red cell with 5 ml DCIP

reagent in 13x100 test tube Incubate the mixture at 37C water bath

for 60 min. Look for precipitation before or after

addition of 5% ascorbic acid Report

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DCIP testDCIP test

Ways to report Negative : No precipitation Positive : Precipitation seen

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DCIP testDCIP test

Before adding 5% ascorbic acid

After adding 5% ascorbic acid

Pos Pos Neg

Blk Neg Pos Pos

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DCIP Tes DCIP Testt

Normal Negative Homo E 3+-4+

HbE trait 1+-2+

- thal/HbE disease 1+-2+

HbH disease 1+-2+

Report as positive or negative

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Hb studiesHb studies

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Hemolysate preparation

• Centrifuge EDTA blood at 3000-5000 rpm and remove plasma

• Wash packed red cell with NSS for three time and remove supernatant as much as possible at the last washing round

• Add DW 1.5 time the volume of PRC and mix vigorously

• Add CCl4 to the half of the volume of lysed red cells and mix vigorously

• Centrifuge 3000 -5000 rpm and collect the upper red portion which is “Hemolysate or Hemoglobin solution)

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HemoglobinHemoglobin e e lectrophoresis lectrophoresis at alkali at alkali pHpH

Hb: Amphoteric molecule• Molecular net charge depends on pH of t

he medium.• - pH > pI (Iso electric point) : Molecular ne

t charge is negative.• pH < pI : Molecular net charge is positive

.• - pI (Iso electric point) is the pH where mol

ecular net charge of hemoglobin is zero.

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HemoglobinHemoglobin e electrophoresislectrophoresis at at alkali pHalkali pH

Principle• In alkali medium, Hbs will gain negative

net charge.• Different Hbs have different molecular n

egative net charge.• Being placed between cathode and anod

e, Hbs will move away from the anode.• The velocity of the movement depends s

olely on the molecular net charge.

• Pattern from cathode to anode is : A2

/E, F , A, Bart’s, H

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HemoglobinHemoglobin e electrophoresislectrophoresis at at alkali pHalkali pH

Reagent :

- -Tris EDTA B orate (TBE)

pH 8.4-8.6

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Equipment

• 1. Power supply for 500 V• 2. Electrophoretic chambe

r • 3. Cellulose acetate plate• 4. Sample applicator• 5. Stain box• 6. Large filter paper or blotter

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Equipment

Sample preparation well Aligning base

Sample applicator

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Equipment

BlotterCellulose acetate plate

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Equipment

http://sun.science.wayne.edu/~hhmi/gifs/elec1.jpg

Power supply

Electrophoreticchamber

Cellulose acetate plate

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EquipmentEquipment

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Specimens

• Hb in the solution or “”.

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Procedure

• Hemolysate in wells• rrr um applicator dipped and applie

r rr rrrrrr rrrrrrrrr rrrrrrr rrrrr

• rrrrr rrrrrrrrr rrrrrrrr rrrr-rrr rr in electrophoretic chamber.

• rrr rrrrrrrrrrrrr rr 3 rrrrr r00rr 1 -02 0min.

• Stained with Ponceau S

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Ponceau S stainingPonceau S staining

Dip cellulose acetate plate in the stain and leave for 5 min

Wash with destaining solution (5% HOAc) twice and 5 min each time or until background becomes white

Read Hb bands

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During electrophoresisDuring electrophoresis

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Ponceau S and Destaining Ponceau S and Destaining solutionsolution

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Hb electrophoretic patternHb electrophoretic pattern

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Hb pattern on CAE with TBE pH 8.6

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Hb pattern on CAE with TBE pH 8.6

A2AA2FA

A2SF

EFFA

Carbonic anhydrase

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A2A

AE

AE

A2A

Carbonic anhydrase

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Portland Bart’s

Portland Bart’s

Portland Bart’s

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A2A

A2A

EE

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Microcolumn chromatography

Principle• In basic (alkali) solution, net charge of H

bs becomes negative.• Different Hbs have different negative net

charge.• On passing through a colume packed wit

-h DEAE cellulose or sephadex resin, diffe rent Hbs bind resin at different strength.

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Microcolumn chromatography

Principle• On passing Cl- ion through

column, it will elute Hb off the resin.

• Order of Hbs being eluted is dependent on negative

net charge.

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+

++

+

+

++

++

+

+

Hb A

HbA2

Hb F

Hb E

Hb H

Hb Bart’s

Cl-

Cl-Cl- Cl-

Cl-

Cl-Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Microcolumn chromatography Principle

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Microcolumn chromatographyMicrocolumn chromatography

Procedure Fill pre-swollened DEAE-Sephadex A-50 into

Pastuer pipette Dilute 200 ul hemolysate with 20 ml THK

buffer pH 8.5 Apply 10 ml diluted hemolysate into the

column, overlayer column with the resin Equilibrate with 5 ml THK pH 8.5 Move column to new tube and elute with 30 ml

THK pH 8.2 OD415 vs DW = ODA2

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Microcolumn chromatographyMicrocolumn chromatography

Procedure Mix 10 ml remaining diluted

hemolysate with 20 ml DW OD415 vs DW = ODTotal

Calculate HbA2=(ODA2 / ODTotal x 5) x 100

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Microcolumn chromatographyMicrocolumn chromatography

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Aim of Aim of mm icrocolumn icrocolumn cchromatographhromatograph

yy To quantify HbA2 or HbE Benefit: To make diagnosis of -

thalassemia heterozygote or HbE If < 10% : HbA2, If >10% : HbE

HbA2 6.32±0.88% = - thalassemia trait

HbE 25±5.5 % = HbE trait HbE 87.2±6.9 = Homo E HbE 41.3±7.9 = HbE/-thal

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Alkali denaturation testAlkali denaturation test

Principle Hb F is resistant to alkali

treatment while other Hbs are not and denatured.

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Alkali denaturation testAlkali denaturation test

Reagent: Cyanide solution, 1.2 N NaOH, Sat

Amm. SulfateEquipmet : Test tubes (13x100), Stop watch,

Funnel, Whatman No 1 filter paper, Spectrophotometer

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Alkali denaturation Alkali denaturation testtest

Procedure Mix 200 ul hemolysate with 3.8 ml cyanide

solution CyanHb Treat 2.8 ml cyanHb with 200 ul 1.2N NaCl for

2 min exactly Stop reaction with 2.0 ml Sat. Amm.Sulfate

and filter through Whatman No1 Read OD540 of filtrate = ODFiltrate Mix 400ul CyanHb with 6.75ml DW Read OD540 of total = ODTotal Calculate HbF(%) = ODFiltrate x 100/ODTotal x

10.01 OR HbF(%) =[ ODFiltrate/ODTotal] x 10

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Alkali Denaturation Test Alkali Denaturation Test

Normal 0.2-2.0% High in - thalassemia disease,

HPFH, -thalassemia N ormal or slightly high in - thal

trait : HbE trait and Homo E Hb Bart’s is also resistant to

alkali denaturation.

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Alkali denaturation testAlkali denaturation test

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Final product (OD540)Final product (OD540)

Filtrate

Total

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Acid Elution Test

• HbF also resists to acid treatment while others (except Hb Bart’s) do not.

• F cell : normal or slightly high in - thal trai t and heterocellular HPFH

• F cell :1 -01 00% in -thalassemia

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Make a thin blood film, let it air-dry Fix the smear in 80% EtOH for 2 min exactly Dip the fixed smear into 0.1% Amido Black

solution in 80% EtOH pH 1.5-2.0 for 2 min exactly

Wash the stained blood smear with flushing tap water

Let it air-dry Look for F cell (stained deep blue) under oil-

immersion power Report in % (in 1,000 rbc examined), but if

few F cells are seen, report as number per HPF

Acid Elution Test Acid Elution Test

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F Cells

Non-F cell

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Hb identification by HPLCHb identification by HPLCPrinciple Hb is amphoteric molecule and changes

net charge according to pH of medium. If pH < PI, net charge becomes positive

(cation ) and different Hbs have different positive charge.

HPLC separation of Hbs is based on cation exchange chromatography

Stationary phase is negatively charged by f unctional group, e.g. polyaspatic acid.

Mobile phase is buffer with pH lower than pI of Hbs

Order of Hbs : Bart’s, H, F, A, A2/E according

to RT

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Normal or -thal trait -thal trait

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Homo EHbE trait

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Hb H disease in newborn HbE/-thalassemia

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Immunologic Demonstration of Hemoglobin

• Polyclonal and monoclonal Abs for Hbs are produced aiming to detect Hbs in heterozygous state.

• Anti-Hb Bart’s for -thalassemia 1 heterozygote

• Anti-Hb Bart’s plus anti -globin chain for -thalassemia 1 heterozygote (SEA type)

• Anti HbE for HbE heterozygote• Anti HbA2 for -thalassemia heterozygote• Detection techniques : RIA, ELISA,

Immunochromatography (Strip test) Immunofluorescence staining and examine using fluoromicroscope or flow cytometer.

DNA Analysis

• DNA was released from nucleated cells ; white blood cells.

• Polymerase Chain Reaction (PCR) to amplify globin gene fragment

• Mutation detection by: electrophoresis, hybridization or gene sequencing

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