DIAGNOSTIK MOLEKULER & ASPEK

GENETIK HEMATOLOGI
Dr. Tahono
Molecular biology
 Application
- Prenatal diagnostic
- Pre-implantation diagnostic
- Gene therapy
- Organ culture
- Genetic alteration
THE DIAGNOSTIC ALGORITHM FOR
PROLIFERATIVE DISEASE
Clinical features

Morphology

Histochemistry/Immunohistochemistry

Immunophenotyping

Molecular Genetic Studies

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 community
family

human being

family

organs

tissues New technology

cells
Protein
coded by
molecular
gene

atomic

DNA

RNA

PROTEIN

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 gangguan fungsi protein

Gejala klinis penyakit , Penanganan, Prognosis

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*temen2, karena gag ada slide dari dr tahono, kita kasi gambar
yang mirip aja ya....insyaALLAH materi yang dimaksud sama
:D smangadh!!!

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 DNA code:ACTG
intron

Transkripsi RNA code:ACTUG

splicing mRNA without introns,

(mRNA “messenger”-RNA)

translation
protein

*teman, yang grafik normal myeloid antigen exprassion sama

T lymphocyte development gag tau mau nyari kemana, soalnya
gag ada slide dr dr tahono T.T

A. CONGENITAL DISORDERS ASSOCIATED WITH

INCREASED RISK OF LEUKIMIA
- Down syndrome  ALL, AML
- Ataxia-telengiactasia  ALL
- Neurofibromatosis  AML, juvenile
myelomonocytic
leukemia
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 - Kostmann’s disease  AML
- Fanconi anemia  AML
- Bloom syndrome  ALL, AML
- Wiscott- Aldrich syndrome  AML

COMMON LEUKIMIA-ASSOCIATED
TRANSLOCATIONS AND RESULTANT FUSION GENES
AND MRNA SPECIES

1. Genetic abnormality : t(9;22)/ BCR-ABL1

Disease associations
- Chronic myeloid leukemia (100%)
- Ph+ adult acute lymphoblastic leukemia (ALL)
(20-25%)
- Ph+ pediatric acute lymphoblastic leukemia (3%)
Basic molecular

Chimeric fusion protein with deregulation of ABL

tyrosine kinase; effects on cell proliferation,
apoptosis, adhesion

Pathogenesis detection : RT-PCR; FISH

2. Genetic Abnormality: del 4q12/FIP1L1-PDGFRA

Disease associations: Hypereosinophilic

syndrome/chronic eosinophilic
Leukemia (subset of cases)

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 Basic molecular: Deregulation of PDGFRalfa
receptor tyrosine kinase

Pathogenesis detections : FISH > RT-PCR

3. Genetic Abnormality : t(15;17)/PML-RARalfa

Disease associations: acute promyelocyte leukemia

(100%)

Basic Molecular: chiremic fusion protein;

interference with myeloid maturation and
differentiation

Pathogenesis detection: RT-PCR; FISH

4. Genetic Abnormality: FLT3 MUTATIONS DNA

PCR

Disease association: acute myeloid (20-30%)

Basic molecular: internal tandem duplication (ITD)

or point mutation resulting in constitutive of FLT3
tyrosin kinase and cell proliferation

Pathogenesis detection: DNA , PCR

5. Genetic Abnormality : t(1;19)/E2A-PBX1RT-PCR

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 Disease associations: childhood pre-B cell ALL
(3%)

Basic Molecular: chiremic protein; interference with

normal early B-cell development

Pathogenesis detection : RT-PCR

GENETIC RISK STRATIFICATION IN PEDIATRIC B-

LINEAGE ACUTE LYMPHOBLASTIC LEUKIMIA

Abnormality Prognostic
Significance

- Highly hyperploidi (>52 chromosomes)

favorable
- t(12;21)/TEL –AML1
favorable
- t(1;19)/E2A-PBX1
unfavorable
- t(9;22)/BCR-ABL1
unfavorable
- 11q23/MLL gene rearrangements
unfavorable
- Hypoploidy (<40 chromosomes)
unfavorable

a. ETIOLOGY OF CML
- Philadelphia chromosome, ph1 (>95%)
- Non-philadephia chromosome (<5%)

The Philadelphia Chromosome and

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 Cronic Myelogeneus Leukimia (CML)

The translocated abl gene inserts into the bcr gene. The two
genes fuse. The alteredabl gene functions improperly, resulting
CML

b. WHO CLASSIFICATION OF AML

 AML with recurrent cytogenetic translocations
- AML-1 related: t(8;21)
- RARA related :t(15;17)
- ETO related : inv(16)(p13q22)
- MLL related: t(11;V)(q23;V)
 AML with multi-lineage dysplasia
- With prior MDS
- Without prior MDS
 Therapy related AML
- Alkylating agent-related
- Epipodophyllotoxin-related
- AML not otherwise categorized
- AML-M0 AML-M7
 Acute biphenotypic leukemia

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 c. WHO CLASSIFICATION OF ALL
 Precursor B-cell lymphoblastic leukemia
- BCR/ABL: t(9;22)
- AF4/MLL: t(4;11)
- PBX1/E2A:t(1;19)
- TEL/AML1:t(12;22)
- Hyperdiploid >50 chromosomes
- Hypodiploid
 Precursor T-cell lymphoblastic leukemia
- No further subclassification

Immunophenotyping Profile Study

Leukemic blasts

Lineage
A1  CD45 ± CD45 ± CD45 ± CD45 ± CD45 ±
B  CD19 ± CD19 - CD19 ± CD19 - CD19 -
B  cCD79a ± cCD79a - cCD79a - cCD79a - cCD79a -
T  CD7 - CD7 ± CD7 ± CD7 ± CD7 ±
T  cCD3 - cCD3 ± ± cCD3 - cCD3 - cCD3 -
My/Mo  MPO - MPO - MPO ± MPO - MPO -
My/Mo  CD13 ± CD13 ± CD13 ± CD13 ± CD13 ±
My/Mo  CD33 ± CD33 ± CD33 ± CD33 ± CD33 ±
My/Mo  CD117 - CD117 - CD117 ± CD117 ± CD117 ±
Meg  CD41a - CD41a - CD41a - CD41a ± CD41a -
Eryth  GPA - GPA - GPA - GPA - GPA ±

B-Lineage T-Lineage AML M1- AML-M7 AML-M5

M5

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B. ERITROSIT
 Struktur Eritrosit
- Membrane cytoskeleton
- Energy machinery
- ‘passenger’

 Protein transmembran (integral)

- Band 3 (ABO,I,i)
- Glycophorine A (M,N,P.falciparum)
B(S,s,U), C
- Protein Rh
 Fungsi transport, pompa, transduksi sinyal
 Protein cytoskeleton (peripheral)
- Spectrin alfa, beta (band 1,2), actin, ankyrin,
band 4.1m band 4.2
 Fungsi stabilitas sel
 Vertical : spectrin, band3, anykrin, protein 4.2
 Horizontal: spectrin, actin, protein 4.1

1. HEREDITARY HAEMOLYTIC ANEMIA

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 Metabolic defects, deficiency of:

- Glucose-6-phospate
- Pyruvate kinase
- Triose phosphate isomerase
- Pyrimidine-5-nucleotidase
- Glutathione synthetase

Hemoglobin defects:

- Defective synthesis
e.g thalasemia (alpha or betha)
- abnormal variants. E
e.g hb S, Hb C, unstable Hb

2. THE RED CELL MEMBRANE

- Hereditary spherocytosis
- Hereditary elliptocytosis
- Hereditary stomatocytosis
- Hydrocytosis (high MCV, low MCHC)
- Xerocytosis (low MCV, high MCHC, shrunken
RBCs due to K loss)
- Acanthocytosis- abeta-lipoproteinanemia
Sintesis Hb & Hemolisis

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 eritrosit

Hb 300 juta/sel

Heme + Globin

Fe + Porfirin Rantai α + Non α (β,γ,δ)

bilirubin

DIAGNOSIS

1. Analisis gen, kromosom, protein

2. Apusan darah tepi
3. Klinis anemia hemolitik
4. Riwayat keluarga

Laboratory test and findings

- Peripheral blood film  1-2% spherocytes in significant

- MCHC is increased or in the upper limit of normal
range ( due to decreased water content)
- Increased osmotic fragility (O.F)
Shift to the right of the entire curve or only part of it
Draw backs of this test:
1. Laborious test
2. Needs fresh defibrinated blood
3. Not specific for HS (can be increased in AIHA)
4. Insufficiently sensitive
(10-25%) of patients genetically proven to have HS
have normal O.F)

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 - Acidified glycerol lysis time. The rate haemolysis ( the
time required for 50% lysis) normal values>1800
seconds
- Auto hemolysis (screening test). 48 hours incubation
under sterile conditions

C. THALASSEMIA

- Gangguan genetic sintesis HB yang dturunkan

- Tidak adanya/berkurangnya sintesis rantai globin
- Kuantitas hb berkurang  eritrosit mudah rusak 
umur lebih pendek daya ikat O2 rendah

Hemoglobinopati

- Perubahan jenis asam amino rantai globin-α atauβ

tanpa ada penurunan sintesis rantai globin/kualitas
hemoglobinopati = hb abnormal
- Mis: HbC, Hb D, Hb E
- Lebih dari 300jt penduduk dunia sebagai pembawa gen
thalassemia dilahirkan setiap tahunnya

KOMPOSISI HB DEWASA

HbA (α2β2) 98%

HbA2(α2δ2) ~2,5%
α β
HbF (α2γ2) <1%
δ
γ

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 chromosomes
kilobases
0 50

ζ ζ α α2 α1
16 3’

ε Gγ Aγ β δ β
11 3’

α δ
Polypeptide γ β
chains
α2γ2 α2δ2 α2β2
hemoglobins F A2 A

Gen penyandi sintesis rantai globin α dan β

Gen α globin  ζ2 α2 α1
141 asam amino Kromosom 16

Gen β globin  ε Gγ Aγ δ β
Kromosom 11
146 asam
amino
Hb Gowers 1
hemoglobin
(ζ 2 ε 2)
Hb A (Mayor)
Hb Gowers 2 Hb F α2β2
(α 2 ε 2) (α2γ2)
Hb A2 (Minor)
Hb Portland α2δ2
(ζ 2 γ 2)
Masa EMBRIO JANIN DEWASA
Perkembangan
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 β Thalasemia-α
δ
Kelebihan: β
SSSSSSSS
Rantai globin –γ (fetus)
γ SSSSSSSSSSSSSSS
SSSSSSSSSSSSSSS
Rantai globin –β (adult)
SSSSSSSSSSSSSSS δ
α SSSSSSSSSSSSSSS α γ
γ4 (Hb Bart)
SSSSSSSSSSSSSSS
β4 (HbH)
SSSSSSSSSSSSSSS
SSSSSSSSSSSSSSS
SSSSS SSSSSS
Presipitasi terutama di sel darah merah matang
↓
Hemolisis di sirkulasi perifer (limpa)

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 Thalasemia-β

Excess of α-globin chains

β
α4 (very unstable) α
δ

γ
precipitates
Marrow
Peripheral circulation

RBC precursor Mature RBC

- Physical damage
- Metabolic changes
- Ineffective erythropoiesis Haemolysis in
- Intramedular destruction microcirculation (spleen)

MENGAPA DATA KLINIS DAN LABORATORIUM

PENTING ?

 Analisis DNA untuk mendeteksi mutasi thalassemia

dan hemoglobinopati.
 Spesifik gen ybs:
- Thalassemia beta- gen globin beta di kromosom 11
- Thalassemia alfa – gen globin alfa di kromosom 16
 Spesifik jenis mutasi-menentukan metode deteksi
mutasi:
- Thalassemia beta: lebih sering mutasi titik ( single
base substitution )
- Thalassemia alfa: lebih sering delesi besar ( delesi 1
gen atau 2 gen globin alpha )

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 Gene Deletions in Alpha Thalassemia

Normal
Alpha thalassemia
One gene trait, normal Hb,
deletion normal or slightly
(alpha/alpha+) reduced MCV
Two gene
deletion Normal Hb,
(alpha+/alpha+) microcytic
Two gene
deletion
(alpha/alpha 0) Hb disease, reduced Hb,
Three gene splenomegaly,
deletion (alpha transfusion dependent,
+/alpha 0) “golf ball” cellswith life
No alpha chain Three gene Incompatible
production deletion (alpha fetus dies in utero Hb
+/alpha 0) Barts

MACAM PEMERIKSAAN

1. Darah rutin:
- hgb, RBC, Hct
- Indeks eritrosit: MCV, MCH, MCHC
2. Morfologi darah tepi
3. Pengecatan supravital
4. Uji resistensi satu tabung
5. Dichlorophenol indophenols precipitation (DCIP) test
6. Fraksi HbA2
7. Fraksi HbF
8. Kadar feritin
9. Analisa Hb
10. Analisa DNA
11.

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 SUMBER DNA MANUSIA

Semua bagian tubuh manusia yg mengandung sel berarti

seperti darah, akar rambut,tulang, sperma,sel rongga
mulut dalam air kumuraan dll

SKRINING AWAL

1. Resistensi osmotik satu tabung

2. Dichlorophenol indophenols precipitation (DCIP) test

INDEKS ERITROSIT
MCV <= 80fl or MCH
<=26pg
Hb Hb
↓ normal

Iron status/ferritin&CRP
MDT & supravital

rendah N/ ↑ (+) (-)

Iron Analisa Analisa Hb

defficiency
DNA

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 DIFFERENTIAL DIAGNOSIS: Iron Defeciency Anemia

MCV Serum TIBC % ferritin

iron saturation
Iron Decreased Low High Low low
deficiency

Anemia of Normal to Low Low Low Normal to

chronic decreased increased
disease

thalasemia Decreased Normal Normal Normal to Normal or

to high high increased

D. HEMOFILIA
- Defek gen/protein FVIII(A), FIX (B)\
- Bleeding tendency
- Ringan, sedang, berat
- Herediter (x-linked) atau spontan (90%)

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 PLATELET ADHESION

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 DIAGNOSIS

1. Anamnesis (herediter,episode perdarahan berulang)

2. Klinis perdarahan
1. Sendi,otot,mukosa
2. Ringan, sedang berat
3. Laboratorium
1. aPTT, factor assay
2. analisis gen

Synthesis of factor VIII, the V Willebrand factor

and the factor VIII-vWF Complex

Factor VIII Von

Willebrand factor

Chromosome 12
X-
Chromosome

megakaryocyte

Endothelial cells
Liver sinusoidal cells Hepatocyte
Endothelial cells vWF subunit (Mr
Single chain form of ~220 kDa)
factor VIII transforms combines into
into two-chains form vWF protomer

vWF multimer
Factor VIII, two-chain
form, Mr ~280 kDa Mr~1-20 MDa

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 DEGREES OF SEVERITY IN HEMOFILIA

Coagulation activity Joint

VIII:C(%) IX:C bleeds
(%)
Hemophilia Severe <1 100 +++
A Moderate 1-4 100 +
mild 5-25 100 (+)
Hemophilia Severe 100 <1 +++
B Moderate 100 1-4 +
mild 100 5-25 (+)

Von willebrand factor

- the vWF is a large adhesive glycoprotein present in

both plasma, the α-granules of platelets, and the vessel
wall
- it is composed of a series of multimers ranging in
molecular mass from 800kDa to as much as 20.000
kDa (single subunit of 220 kDa)
- plays an important part in the earliest phase of
hemostasis by promoting the adhesion of platelets to
the subendothelium exposed after vessel injury
- serves as a carrier protein for factor VII , thereby
preventing the rapid removal of factor VII from the
circulation
- the high molecular weight multimers of vWF are the
most hemostatically efficient (interaction with platelets
and subendothelium)

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 MOLECULAR DIAGNOSTIC

- analysis of DNA
- analysis of RNA
- nucleated cells
- southern blot analysis
- in situ hybridization
- flouresennce in situ hybridization (FISH)
- PCR

WESTERN BLOT/PROTEIN BLOTTNG

 Pemindahan protein yg diuraikan secara

elektroforese dari gel poliakrilamid ke membrane
nitroselulose
 PENGGUNAAN:
1. Identifikasi protein antigen
2. Tes allergen
3. Identifikasi antibody dengan antigen
spesifik
4. Deteksi mikroelemen dari protein kompleks
5. Pewarnaan protein

SOUTHERN BLOT

 Pemindahan protein yang diuraikan secara

elektroforese dari gel poliakrilamid kemembran
nitroselulose
 Penggunaan:
1. Identifikasi protein antigen
2. Tes allergen
3. Identifikasi antibody dengan antigen
spesifik
4. Deteksi mikroelemen dari protein kompleks
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