5. dr. triratnaningsih - interpretasi elektroforesisfinal

INTERPRETASI ELEKTROFORESIS HEMOGLOBIN

TRI RATNANINGSIHBAG. PATOLOGI KLINIK FK UGM

SUB HEMATOLOGI INST. LABORATORIUM KLINIK RS DR SARJITO

WORKSHOP JOGLOSEMAR SOLO18 APRIL 2013

KLASIFIKASI HB PATI2

PRODUKSI HEMOGLOBIN1

PEMERIKSAAN ELEKTROFORESIS PADA HB PATI3

STRATEGI DIAGNOSIS HB PATI 4

TEKNIK ANALISIS GLOBIN LAINNYA5

Hb pati

SindromThalassemia

Thalassemiaalfa

Silent carrier,trait,

HbH disease, hydrops

Thalassemiabeta

Minor, Mayor, HPFH

Hb varian

Hb S

AS, SS, SC

Hb C

AC, CC

Hb E

AE, EE, Trait β/ βE, β E/βE

homozigot

Hb D

AD, DD

alkaline acid

Pada pH alkali: Hb bermigrasi pada cellulose acetate membrane dari cathode anode denganurutan sbb:

HbA2, Hb C, Hb E, Hb S, Hb D, Hb Lepore, Hb F, Hb A dan fast moving Hb Bart's dan Hb H

HbA2, Hb C, Hb E, dan O migrasi bersama membentuk single band. Hb S, D dan G juga komigrasi.

Pada pH asam:

Hb C terpisah dari E dan O. Hb S terpisah dari D dan G.

Hb D dan G tetap tidak dapat terpisah.

Hemoglobin (%)

A H (β4) B(4)

pada saat lahir

Constant

spring

Normal 97 0 0 0

α+-Thalassemia silent

-α/αα 98-100 0 (0-2) 0

α0-Thalassemia trait

-α/-α (α+homozigot) 85-95 inklusi (5-15) 0

atau--/αα(α0heterozigot)

ααCS/ααCS

85-95 0 (5-15) 5

Hemoglobin H disease

--/-α (α0/α+) 70-95 5-30 Trace Trace

--/ααCS (α0/αCS) 60-90 5-30 Trace 5-10

Hydrops fetalis

--/-- (α0/α0) 0 5-10 90-95 0

ClinicalSyndrome

Genotype Hb(g/dl)

A F A2

Minor (Trait)

/ +

or / ° 10-13 90-95 1-5 4-7a

Intermedia +/+ 7-10 30-50 50-70 0-5

Major +/° < 7 2-10 >85 1-6

or °/° 0 95-100 0-5

Hb Minor Intermedia Major

Hb A Dec Dec Dec

Hb A2 N to Inc N to Inc Usually Inc

Hb F N to Inc Increased Increased

Hb Minor Intermedia Major

Hb A Dec Dec Dec

Hb A2 N to Inc N to Inc Usually Inc

Hb F N to Inc Increased Increased

ClinicalSyndrome

Genotype Hb (g/dl) A F A2

Minor (Trait) / +

or / ° 10-13 90-95 1-5 4-7a

Intermedia +/+ 7-10 30-50 50-70 0-5

Major +/° < 7 2-10 >85 1-6

or °/° 0 95-100 0-5

Hemoglobinopati

Hemoglobin

A%

F%

S%

C%

D%

E%

Kelainan klinis

Hb CC 1-7 >90 ringan

Hb AC 50-60 <2 40-50 -

Hb SC 1-7 50 50 Sedang-berat

Hb SS 1-10 80-90 berat

Hb AS 55-70 <2 30-45 ringan

Hb DD <2 95 -

Hb AD 50-65 <2 35-50 -

Hb EE 1-5 95 ringan

Hb AE 60-80 20-40 -

Hbpati

HemoglobinA%

F%

S%

C%

D%

E%

Kelainan klinis

Hb CC 1-7 >90 ringanHb AC 50-60 <2 40-50 -Hb SC 1-7 50 50 Sedang-berat

Hb SS 1-10 80-90 beratHb AS 55-70 <2 30-45 ringanHb DD <2 95 -Hb AD 50-65 <2 35-50 -Hb EE 1-5 95 ringanHb AE 60-80 20-40 -

Hemoglobin

A F A2 E/S/C

Normal 97 <1 2-3 0

Hemoglobin E E

Trait β/ βE 60-65 1-2 2-3 30-35

β E/βE homozigot 0 0 5 95

E/ β Thal βE/β0 0 45 1-5 40-50

E/ α Thal 70 0 2-3 30

Hemoglobin S S

Trait β/ βS 60-70 1-2 2-3 30-40

β S/βS homozigot 0 5-10 - 90-95

S/ β Thal βS/β0 0 5-10 4-7 80-90

βS/β+ 20-30 5-10 4-7 50-70

Hemoglobin C C

Trait β/ βC 55-65 3-5 30-40

β C/βC homozigot 0 5-10 - 90-95

C/ β Thal βC/β0 0 5-10 - 90-95

βC/β+ 20-30 5-10 - 60-75

Pola hemoglobin pada hemoglobinopati kombinasi

Hemoglobin

A F A2 E/S/C

Normal 97 <1 2-3 0

Hemoglobin E E

Trait β/ βE 60-65 1-2 2-3 30-35

β E/βE homozigot 0 0 5 95

E/ β Thal βE/β0 0 45 1-5 40-50

E/ α Thal 70 0 2-3 30

E/ β Thal βE/β0E/ β Thal βE/β+

ANALISIS GLOBIN

Data hematologi

Manifestasi klinis

Informasiras/etnik

Elektroforesis

HPLC

Capillary electrophoresis

IdentifikasiHb pati dg

Probabilitasterbesar

Mendukung kecurigaanjenis varian yg prevalen

Analisis Globin

Analisis Gena

Darah vena PemeriksaanCBC

MCV <80 fl dan MCH <27 pg MCV >80 fl dan atau MCH >27 pg

SELESAIReview morfologi eritrosit

Eritrosit normositik normokrom, tidak tampak kelainan morfologi eritrosit

Eritrosit mikrositik hipokrom dan/ atau ditemukan kelainan

morfologi eritrosit

Analisis hemoglobin

HbA2 > 8 %

Thalassemia beta trait

HbA2< 3,5% HbF < 1,0%

5%> HbF > 1,0%Thalassemiaheterozigot

ganda

8%> HbA2 >3,5%

Analisis lebihlanjut untuk Hb

varian

HbF >5%

Analisis lebih lanjut untuk HPFH, thalassemia beta intermedia atau

atau Hb varian lain

(PDS Patklin)

Lakukan pemeriksaan analisis hemoglobin dengan metodeHPLC atau capillary electrophoresis sesuai dengan petunjukpemeriksaan dengan masing-masing alat.

Pada setiap run, lakukan pemeriksaan bahan kontrol untukmenilai validitas pemeriksaan.

Untuk metode HPLC, syarat-syarat untuk kalibrasi harusterpenuhi, dan hasil pemeriksaan bahan kontrol masuk dalamrentang nilai kontrol.

Selain itu, total area di bawah kurva harus berkisar antara1.000.000-4.000.000.

Lakukan interpretasi hasil dengan menilai kadar HbA2, HbFdan melihat adanya Hb varian yang mungkin terdeteksi.

Flowchart diagnosis hemoglobinopati berdasarkan indeks eritrositSumber: www.mlo-online.com, 2009

http://www.mlo-online.com/





PEMERIKSAAN ELEKTROFORESIS PADA HB PATI DAN THALASSEMIA

3



Retention time dari berbagai varian Hb Retention time A2 dan E sama Ditemukan beberapa kesamaan antara

retention time dan pola pada alkaline electrophoresis

HPLC and Capillary Hb electrophoresis patterns of an adult with HbH disease. The HbH (β4 tetramers) peak elutes from the column as a compressed fraction, and as a fast moving fraction in electrophoresis.

β o thalassemia major (b)β 1 thalassemia homozygous E (c)

terimakasih

HbA2 range Interpretation> 7.0 % Rare, repeat to verify test.

Exclude a structural variant.

Can be due to rare β thal mutations.

3.8 – 7.0 % Beta thal trait or unstable Haemoglobin.

3.4 – 3.7 % Fe deficiency in β thal trait; Δ chain variant with β thal trait.

Interaction of α and β thal traits; rare β thal mutations.

HbS making measurement inaccurate; interaction of α - Hb S.

2.0 – 3.3 % Normal.

Δ and β thal (but HbF should be elevated); alpha thal trait.

Rare cases of β thal trait coexisting with either Δ or α thal trait.

< 2.0 % Δ β thal (but HbF should be elevated).

Alpha thal trait; Hb H disease; Δ variant or delta Thalassemia.

Iron deficiency.

HbA2 range Interpretation

> 7.0 % Rare, repeat to verify test.

Exclude a structural variant.

Can be due to rare β thal mutations.

3.8 – 7.0 % Beta thal trait or unstable Haemoglobin.

3.4 – 3.7 % Fe deficiency in β thal trait; δ chain variant with β thal trait.

Interaction of α and β thal traits; rare β thal mutations.

HbS making measurement inaccurate; interaction of α - Hb S.

2.0 – 3.3 % Normal.

δ and β thal (but HbF should be elevated); alpha thal trait.

Rare cases of β thal trait coexisting with either δ or α thal trait.

< 2.0 % δ β thal (but HbF should be elevated).

Alpha thal trait; Hb H disease; δvariant or delta Thalassemia.

Iron deficiency.

5. dr. triratnaningsih - interpretasi elektroforesisfinal

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