Dr. dr.

Agus Susanto Kosasih, SpPK, MARS

Birth Place and Date : Jakarta, Feb 1st 1961

Current Position : Medical Staff Clinical Pathology Department
Dharmais Cancer Hospital, Jakarta, Indonesia

EDUCATION
2015 Graduated Doctoral in Medicine, Medical Faculty University of Indonesia, Jakarta
2001 Graduated as MARS (Magister of Hospital Administration), University of Indonesia, Jakarta
1991 Graduated as Clinical Pathologist, Medical Faculty University of Indonesia, Jakarta
1987 Crash Program doctor type C hospital laboratory, Medical Faculty University of Indonesia, Jakarta
1985 Graduated as Medical Doctor, Medical Faculty University of Indonesia, Jakarta

PROFESSIONAL EXPERIENCE
1992 - present Clinical Pathology Consultant in Laboratory Medicine, Metropolitan Medical Centre Hospital, Jakarta
2002 – present Clinical Pathology Consultant in Laboratory medicine, Hermina Jatinegara Hospital, Jakarta
2011 – present Clinical Pathology Consultant in Laboratory Medicine, Sioam Semanggi, MRCCC Hospital, Jakarta
PEMERIKSAAN SUMSUM TULANG &
IMMUNOPHENOTYPING PADA
LEUKEMIA
Dr. dr. Agus S. Kosasih, SpPK, MARS
DHARMAIS CANCER HOSPITAL, JAKARTA
NATIONAL CANCER CENTRE
EPIDEMIOLOGI LEUKEMIA DI INDONESIA
10 Keganasan terbanyak di RS Kanker Dharmais – Pusat Kanker Nasional
sepanjang 2011 – 2013
Male Female
Leukemia 13.5 Breast 42.72

Bronchus and Lung 12.0 Cervix 14.16 34.53

Nasopharynx 10.5 Ovary 6.48

65.47
Colon 6.2 Leukemia 4.23

Lymph node 5.9 Thyroid Gland 3.99

Male
Rectum 5.4 Colon 2.69 Female

Liver 5.0 Bronchus and Lung 2.48 1000

Male
800 Female
Prostate Gland 3.9 Corpus Uteri 2.41
600
Skin 3.4 Nasopharynx 2.28 400

Thyroid Gland 200

2.8 Lymph node 2.18
0
0.0 5.0 10.0 15.0 0.00 10.00 20.00 30.00 40.00 50.00 0- 10-20-30-40-50-60-70-80-90-
 ALL CLL Limphoma MM

KLASIFIKASI LEUKEMIA naïve

B-lymphocytes

Plasm
a
Lymphoid
• FAB (French progenitor T-lymphocytes
cells

American British)
AML Mieloproliferative Disorders
stem cell Myeloid Neutrophils
• WHO (World progenitor

Health Eosinophils

Organization)
Basophils

Monocytes

Platelets

Red cells
 ACUTE LEUKEMIA

ACUTE MYELOID LEUKEMIAS (AML)

ACUTE LYMPHOBLASTIC LEUKEMIAS (ALL)

ACUTE LEUKEMIA OF AMBIGUOUS LINEAGE

KLASIFIKASI LEUKEMIA MIELOBLAS
AKUT
30%
• Blasts must comprise at least of nucleated cells in bone marrow or blood to
establish a diagnosis of AML
FAB • Morphology, Cytochemistry, Immunophenotyping

• Newer version
• Blasts must comprise at least 20% of nucleated cells in bone marrow or blood to establish a
diagnosis of AML
WHO • Morphology, Immunophenotyping, Cytogenetic and molecular biology.
DIAGNOSIS OF HEMATOLOGICAL MALIGNANCIES
Clinical Symptoms Laboratory
and signs findings

Morphology + cytochemistry

Cytogenetics

Immunophenotyping

Molecular biology / FISH

PEMERIKSAAN SUMSUM TULANG
PADA LEUKEMIA
PENDAHULUAN
International working party for standarization of bone marrow specimens yang dulunya ICSH
membuat konsensus tahun 2008
TRANSPORT SPESIMEN SUMSUM TULANG
Sumsum tulang harus sesegera mungkin dikirim ke laboratorium
( max 6 jam)

Bila tidak dapat segera dikirim harus dibuatkan sediaan hapus

tanpa fiksasi.

Harus disertai formulir permintaan yang dilengkapi oleh :

• Data darah tepi (Hemoglobin, Leukosit, trombosit , Retikulosit)
• Riwayat penyakit dan diagnosis
• Hepar, Lien dan KGB
• Sediaan hapus darah tepi
SEDIAAN SUMSUM TULANG
Sumsum Tulang
• Sediaan langsung (tanpa antikoagulan)
• Antikoagulan K3EDTA
Hapusan dibuat dari bahan sumsum tulang yang ada partikelnya
• Smear /hapusan sebanyak 5 – 6 slide
• Squash (crush) sebanyak 2 – 3 slide
Hapusan Ular (Ganda Soebrata)
 PEMBUATAN SEDIAAN SUMSUM
CaraTULANG
Membuat
Siapkan objek glass. Kaca objek harus bersih dan
kering. Homogenkan Sumsum tulang

Tuangkan sumsum tulang ke objek glass, hingga

ditemukan partikel. Kelebihan darah dikembalikan
ke wadah penampung
 PEMBUATAN SEDIAAN SUMSUM
CaraTULANG
Membuat
Ambil partikel dengan kaca penghapus

Dengan gerak yang mantap, dorong kaca penghapus

sehingga terbentuk hapusan sumsum tulang

Biarkan hapusan sumsum tulang mengering di udara.

Kemudian fiksasi sediaan dengan metanol absolut.
PEMBUATAN SEDIAAN SUMSUM TULANG
Aspirat yang mengandung partikel, digeser
sehingga membentuk gambaran seperti
“ular”

Aspirat yang mengandung partikel, Ditekan

dan dorong dengan menggunakan 2 kaca
slide

Crush
PEWARNAAN SEDIAAN SUMSUM TULANG
Pewarnaan yang dianjurkan oleh WHO :

Pewarnaan Wright
Pewarnaan Giemsa
tidak dianjurkan untuk
Pewarnaan Wright-Giemsa sediaan hapus sumsum
tulang

Pewarnaan MGG
PEWARNAAN BESI SUMSUM TULANG
Pewarnaan Prusian Blue atau Pearl’s Stain

Pewarnaan tanding dengan Safranin

Menilai cadangan besi dan ring sideroblast pada MDS

Mengunakan alat bebas zat besi.

Menggunakan kontrol
 PELAPORAN SUMSUM TULANG
Penilaian kualitatif dan kuantitatif

Pembacaan oleh Spesialis Patologi Klinik terlatih

Mengunakan slide smear dan crush

Hitung jenis sumsum tulang 200 – 500 sel

Lihat dengan pembesaran lemah (10 x 10 )

• Mendapatkan gambaran yang menyeluruh
• Perhatikan penyebaran sel, kepadatan sel dan adanya megakariosit
Pembesaran 10 x 40 dan 10 x 100
• Identifikasi maturasi eritropoeisis, granulopoeisis dan Trombopoeisis
• Menentukan M:E ratio. Normal 2–4 :1
• Melakukan differential count (200–500 sel)
• Mencari sel non hemopoetik , parasit dalam makrofag
• Menentukan cadangan Besi iron
 Selularitas Sumsum Tulang

Hiposeluler Normoseluer Hiperseluler

Seluleritas sumsum tulang tergantung usia, normal dewasa 50%, pada anak 80%, pada usia >70
tahun hanya 30%

Wirawan R. Pemeriksaan sumsum tulang. Pemeriksaan laboratorium hematologi 1st ed. Jakarta . Badan penerbit FKUI. 2011 : 233-51
 Penilaian Megakariosit
Banyak pada ujung dan tepi sediaan hapus
Atau pada dekat Partikel
Dinilai maturasi inti dan sitoplasma
• Non budding megakarocyte
• Hypolobulated megakaryocyte
• Abnormal maturation
• Nuclear cytoplasmic asyncrony
( A ) Normal megakariosit, nukleus lobus multipel
( B ) Mature megakariosit, lobulasi nukleus prominent
( C ) emperipolesis (ditemukan sel intak dalam
megakariosit)
( D ) Stripped megakaryocyte nucleus (panah)
( E ) Megakarioblast
( F ) Mikromegakariosit dengan nukleus unilobular
( G ) Megakariosit dengan lobulasi nukleus terpisah
Sel Non Hemopoetik, sel Ganas Histoplasma Capsulatum
 SITOKIMIA (AML vs ALL)
Cytochemistry Method AML ALL

Myeloperoxidase (MPO) + -
Sudan Black B (SBB) + -
Non-specific esterase (NSE) + (M4,5) -

PAS + (M6) +
Acid phospatase + (M6) +

The limitation of cytochemistry method:

• Can’t identify M0 and M7
• Can’t distinguish ALL-B, ALL-T and MPAL
 Mieloperoksidase (MPO)
INTERPRETASI HASIL
Membedakan blast antara
myeloblast dan lymphoblast
Defisiensi MPO kongenital →
neutrofil dan prekursornya (-)
Neutrofil displastik → hasil (-)
Auer rod → ikut terwarnai

Myeloperoxidase (MPO). Myeloperoxidase staining shows

Auer rods and cytoplasmic granular staining

Swirsky D, Bain BJ. Erythrocyte and leucocyte cytochemistry-leukaemia classification. In: Bain BJ, Bates I, Laffan MA, Lewis SM, editors.
Dacie and Lewis practical haematology. 9th ed. London: Elsevier Churchill Livingstone; 2001. p. 269-95.
 Sudan Black B (SBB)
INTERPRETASI HASIL
Hasil reaksi:
Hitam, granula abu-abu hitam

Interpretasi hasil mirip dgn hasil MPO

Kecuali:
◦ Granula eosinofil: inti lebih jernih
◦ 1-2% ALL: non-granular smudgy positivity Sudan Black B (SBB).
SBB localized positive reaction in the blast cells is
◦ Basofil: umumnya negatif, tetapi mungkin more definite and Auer rods are prominent.
menunjukkan metakromatik merah/ungu pada
granulanya

Swirsky D, Bain BJ. Erythrocyte and leucocyte cytochemistry-leukaemia classification. In: Bain BJ, Bates I, Laffan MA, Lewis SM, editors.
Dacie and Lewis practical haematology. 9th ed. London: Elsevier Churchill Livingstone; 2001. p. 269-95.
 PELAPORAN SUMSUM TULANG
Kepadatan Sel
Jumlah dan morfologi megakariosit
Maturasi sistem eritropoeisis, granulopoeisis dan trombopoeisis.
Hitung jenis sel (200 sel)
Meiloid  Eritropoeisis
Blast  Rubriblas
 Promilosit  Prorubrisit
 Mielosit
 Rubrisit
 Meta mielosit
 Batang  Meta rubrisit
M : E ratio
 Segmen
 Eosinofil  Limfoid
 Basofil  Limfoblas
 Monosit  Prolimfosit
 Limfosit
 Plasmosit
PELAPORAN SUMSUM TULANG DI RS KANKER DHARMAIS
IMMUNOPHENOTYPING
PADA LEUKEMIA
FLOWCYTOMETRY PRINCIPLE
 IMMUNOPHENOTYPING
CD # = cluster designation number Granulocytes

Lymphocytes

SSC
Monocytes

RBCs, Debris,
Dead Cells
FSC

Why Look at FSC v. SSC

Since FSC ≈ size and SSC ≈ internal structure, a correlated measurement between them can allow
for differentiation of cell types in a heterogeneous cell population
CD45 GATING
 GOAL: to identify the leukemic population
Normal Abnormal

SSC Height
SSC Height

CD45 PerCP-Cy5.5 CD45 PerCP-Cy5.5

The abnormal sample contains a prominent population of blast cells (red) with
low SSC and less expression of CD45 than normal lymphocytes (green)
Immunophenotyping
Identifikasi dan kuantifikasi sel blas

Menentukan lini sel blas

Identifikasi sel blas abnormal

Sub klasifikasi
IDENTIFIKASI POPULASI ABNORMAL
Peningkatan atau penurunan ekspresi antigen

Ekspresi antigen yang tidak sinkron (Asynchonous)

Ekspresi Aberran

Ekspresi antigen homogen

INDIKASI PEMERIKSAAN IMMUNOPHENOTYPING

Aalisis Immunophenotyping harus dilakukan sistematik pada

semua kasus berikut :
1. Acute leukemia AML VS ALL
2. Transformasi blas pada CML
3. Transformasi blas pada keganasan myeloproliferative dan myeloid
dysplasia.
4. Mixed Lineage Acute Leukemia (MPAL)

Minimal residual disease (MRD) for ALL.

 Phenotyping Pada Leukemia Akut

Stem Cells

cMPO

cCD79a
Hoffbrand AV, Moss PAH. Essential Haematology, 6th ed. London: Wiley BlackWell, 2011.
PENANDA IMMUNOPHENOTHYPING
PENANDA IMMUNOPHENOTHYPING
 PENANDA SEL PREKURSOR

PREKURSOR SEL MIELOID

• CD34,anti HLA-DR,CD38,CD117,CD133

PREKURSOR SEL LIMFOID

• CD34, anti HLA-DR, CD38, Anti TdT,
LEUKEMIA AKUT
Precursor B-cell acute lymphoblastic
leukemia

Precursor T-cell acute lymphoblastic

leukemia

Precursor Mieloid Cell acute

Myeloblastic leukemia
Precursor B-cell ALL

IMMUNOPHENOTYPING :
CD 19 plus cCD79a positive

cCD3 negative, cMPO negative

One or more myeloid antigen positive eg CD13,CD15,CD33or

CD65, not mixed leukemia
ALL B with aberrant exp CD
13
Precursor T-cell ALL
IMMUNOPHENOTYPING :
cCD3 and CD7 positive

cCD79a negative, cMPO negative

One or more myeloid antigen positive eg CD13,CD15,CD33or

CD65, not mixed leukemia
Lymfoid differentiation
AML
cMPO positif atau NSE jika monositik
Myeloid: CD13, CD33 CD15,CD16,CD65 positif
Monositik: CD14,CD64,CD11b,CD11c CD36,CD4 positif
Megakaryocytic: CD61, CD41, CD42
Erythroid marker: CD 235a (glycophorin A)
cCD3 negatif, cCD79a negatif
Satu atau lebih antigen limfoid positif, contoh CD2,CD5,CD7,CD19,CD22,
CD56, atau ekspresi lemah CD79a. Bukan termasuk mixed leukemia
 Myeloid
differentiation
MIXED PHENOTYPE ACUTE
LEUKEMIA

(WHO 2016)
 Mixed Phenotype
Acute Leukemia T - Mieloid
 DHARMAIS PANEL ANTIBODI MONOCLONAL UNTUK
DIAGNOSIS LEUKAEMIA AKUT DI RS KANKER DHARMAIS
Screening Panel 4 Color: Maturation
CD11b FITC/ CD13 PE/ CD45 PerCp / CD34 APC
1.CD36 FITC/ CD33 PE/ CD45 PerCp/ CD34 APC
Intra cells
2.HLA-DR FITC/ CD117 PE/ CD45 PerCp/---
MPO FITC/ CD3 PE/CD45 PerCp/CD34 APC
3.CD7 FITC/ CD3 PE/ CD45 PerCp/ CD5 APC
CD19 FITC/ CD79a/CD45 PerCp/CD34 APC
4.CD10 FITC/ CD19 PE/ CD45 PerCp / CD20 APC
5. --- /CD 13 PE/ CD45 PerCp/CD14 APC

NK: CD2 FITC/ CD 56 PE/ CD45 PerCp/ CD34 APC

M6: CD71 FITC/ GLY A PE/ CD45 PerCp/ CD34 APC
M7: CD61 FITC/ CD3 PE/ CD45 PerCp/ CD34 APC
Patient 1
MA, male, 2 years old
What is the blast morphology in this patient?
Patient 1
Gating at blast area was positive with:
◦ CD34
◦ HLA-DR
◦ CD19
◦ CD10
◦ CD13

Conclusion: B lineage with aberrant expression of CD13

Patient 2
KAS, female, 10 years old
What is the blast morphology in this
patient?
Patient 2
Gating at blast area was positive with:
◦ CD34
◦ CD33
◦ HLA-DR
◦ CD117
◦ CD13

Conclusion: MYELOID lineage

Patient 3
AM, male, 24 years old
What is the blast morphology in this
patient?
Patient 3
Gating at blast area was positive with:
◦ CD33 • CD19
◦ CD34 • CD10
◦ HLA-DR • CD20/weak positive
◦ CD13 • cyCD79a
Conclusion: B-lineage with aberrant expression of CD33 and CD13
Patient 4
SEI, female, 10 days old
What is the blast morphology in this
patient?

Sudan Black B
 Patient 4
Gating at blast area was positive with:
◦ CD33 • CD117 • CD61
◦ CD34 • CD7
◦ HLA-DR • CD13

And negative with cyMPO

Conclusion: MEGAKARYOCYTIC with abberant CD7
Patient 5
GGG, male, 37 years old
What is the blast morphology in this
patient?
Patient 5
Gating at blast area was positive with:
◦ CD33
◦ CD36
◦ HLA-DR
◦ CD117
◦ CD13

Conclusion: MYELOID lineage (monocytic)