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INTERPRETASI ELEKTROFORESIS HEMOGLOBIN
TRI RATNANINGSIHBAG. PATOLOGI KLINIK FK UGM
SUB HEMATOLOGI INST. LABORATORIUM KLINIK RS DR SARJITO
WORKSHOP JOGLOSEMAR SOLO18 APRIL 2013
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KLASIFIKASI HB PATI2
PRODUKSI HEMOGLOBIN1
PEMERIKSAAN ELEKTROFORESIS PADA HB PATI3
STRATEGI DIAGNOSIS HB PATI 4
TEKNIK ANALISIS GLOBIN LAINNYA5
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KLASIFIKASI HB PATI2
PRODUKSI HEMOGLOBIN1
PEMERIKSAAN ELEKTROFORESIS PADA HB PATI3
STRATEGI DIAGNOSIS HB PATI 4
TEKNIK ANALISIS GLOBIN LAINNYA5
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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
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KLASIFIKASI HB PATI2
PRODUKSI HEMOGLOBIN1
PEMERIKSAAN ELEKTROFORESIS PADA HB PATI3
STRATEGI DIAGNOSIS HB PATI 4
TEKNIK ANALISIS GLOBIN LAINNYA5
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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.
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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
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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
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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
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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
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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 -
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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 -
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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
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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+
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KLASIFIKASI HB PATI2
PRODUKSI HEMOGLOBIN1
PEMERIKSAAN ELEKTROFORESIS PADA HB PATI3
STRATEGI DIAGNOSIS HB PATI 4
TEKNIK ANALISIS GLOBIN LAINNYA5
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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
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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)
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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.
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Flowchart diagnosis hemoglobinopati berdasarkan indeks eritrositSumber: www.mlo-online.com, 2009
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KLASIFIKASI HB PATI2
PRODUKSI HEMOGLOBIN1
PEMERIKSAAN ELEKTROFORESIS PADA HB PATI DAN THALASSEMIA
3
STRATEGI DIAGNOSIS HB PATI 4
TEKNIK ANALISIS GLOBIN LAINNYA5
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Retention time dari berbagai varian Hb Retention time A2 dan E sama Ditemukan beberapa kesamaan antara
retention time dan pola pada alkaline electrophoresis
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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.
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β o thalassemia major (b)β 1 thalassemia homozygous E (c)
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terimakasih
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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.
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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.