경희대학교 의과대학·의학전문대학원

전체 글

(1)

Diagnostic Tests in Leukemia,

Diagnostic Tests in Leukemia,

Lymphoma & Myeloma

Lymphoma & Myeloma

Hematology 23

Lee Woo In, MD, PhD

(2)

Definition

• Blast

– 정상 골수 <5% of ANC(all nucleated cell)

– Acute leukemia ≥ 20% of ANC

• FAB classification vs. WHO classification

– MDS < 20%

• Multiple myeloma – plasma cell

• MPD-lineage proliferation

(3)

Characteristics of Hematological

Malignancies

• Proliferation of malignant clone

– * Clonality ?

• Proliferation of daughter cells originating from a single precursor cell,

• Identical phenotypes, growth characteristics as that originating precursor cells

• Frozen of the maturation stage

– Except :CML

– Lymphoid

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Formation of a clone

Normal

tissue

Mutation

Clone

Possible outcomes .Apoptosis .Neoplastic growth .Chromosome evolution .Metastatic potential

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1. Morphology

PBS,

Bone Marrow aspirates &

biopsy

2. Cytochemical Stain

3. Immunophenotyping

1) Immunocytochemistry

2) Flow Cytometry

Diagnostic Tests

Diagnostic Tests

(6)

4. Cytogenetics

1) Karyotyping

2) Fluorescent In Situ Hybridization(FISH)

5. Molecular Methods

1) Southern blotting

2) PCR, RT-PCR, Multiplex PCR,

Competitive PCR

3) Quantitative Real-Time PCR

4) Gene Scan

(7)

Blast Myeloblast (Auer rod) Lymphoblast

I.

Morphology

PBS and BM

(8)

-II.

II.

급성 백혈병의 세포 화학적 염색 양상

급성 백혈병의 세포 화학적 염색 양상

Acute Leuk Peroxidase/SBB ANBE PAS AP

ALL - - +/++ -/+ AML, M0 - - - -AML, M1 + - + -AML, M2 ++ - + + AML, M3 +++ - -/+ ++ AML, M4 ++ ++ -/++ ++ AML, M5 - +++ ++ + AML, M6 - + ++ -AML, M7 - - ++ +

*sudan black B /MPO..myeloperoxidase Alpha-naphtylbutyrate esterase

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(10)
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III. Immunophenotyping

• 세포의 특이한 항원결정인자 (epitope)

를 인지하는 항체를 이용하는 면역학적 방

– CD (Cluster designation/Cluster of

differentiation)

• Monoclonal antibody

• 세포를 인지하는 특이도가 높다

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(14)

각 세포별

CD 표지자

• T cell – CD2, CD7, CD3( 이상 pan-T cell), CD4, CD8 • B cell – CD10(CALLA), CD19, CD20, CD21, CD22, CD23, CD40 • NK cell – CD16, CD56, CD57 • Granulocytic lineage

– HLA-DR, CD34, CD38(non-comm. Stem cell) – Myeloblast : HLA-DR, CD13, CD33, CD11b • Monoblast : CD14, CD15, CD11b • Platelet – CD41, CD42, CD61, CD62, CD63 • RBCs – CD45, CD71, glycophorin A

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Immunophenotyping by flow cytometry

세포의 크기 (forward scatter) 세포내의 복잡성 (side scatter) 형광염색

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AML

M0-* M1- M2- M3- M4- M5-M6-* M7-*

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Scoring system for Dx of

Acute Leukemia

Score B cell lineage T cell lineage Myeloid lineage

2 m/cyt CD22, cyt CD79a, cyt IgM, surf Ig m/cyt CD3, m/cyt TCR MPO, cyt CD13 1 CD19, CD20, CD10 CD2,CD5,CD8 CD13,CD33, CD117, CD65 0.5 TdT, CD24 TdT, CD7, CD1a CD14, CD15, CD64 Dx … Lineage score > 2.5

1) Acute biphenotypic leukemia, 2) aberrant antigen axpression *m: membranous, cyt :cytoplasmic

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Immunophenotyping of B lineage ALL

Marker Null ALL Common ALL Pre-B ALL B-ALL

CD34 + + + -Tdt ++ ++ ++ -HLA-DR ++ ++ ++ ++ CD10 - ++ ++ + CD19 ++ ++ ++ ++ CD20 - + + ++ CD22 ++ ++ ++ ++ Cyt CD79a ++ ++ ++ ++

Pre-B cyt Ig - - +

(20)

IV. Cytogenetic tests

IV. Cytogenetic tests

• Cytogenetic characteristics of

Hematologic malignancies

– Non random style

– Subgroup

• Conventional cytogenetics

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Cytogenetics

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(25)
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*

급성 백혈병에서 흔히 관찰되는 염색체 이상 소견

급성 백혈병에서 흔히 관찰되는 염색체 이상 소견

Disease

Disease TranslocationTranslocation Genes Genes involved

involved

AML t(6;9)(p23;q34) DEK-CAN AML-M2 t(8;21)(q22;q22) AML1-ETO AML-M3 t(15;17)(q22;q21) PML-RARa

AML-M4Eo inv(16)(p13;q22) CBFbeta-MYH AML-M4/M5 t(11;19)(q23;p13) MLL-ENL

AML-M5 t(9;11)(p21;q23) MLL-AF9 CML t(9;22)(q34;q11) BCR-ABL Precursor B-ALL t(9;22)(q34;q11) BCR-ABL Pre-B-ALL t(1;19)(q23;p13) E2A-PBX1 B-ALL t(8;14)(q24;q32) MYC ㅡ IgH

(28)

* Myelodysplastic syndrome

+8, 5, 7

번 염색체 이상

*

비호즈킨 림프종에서 흔히 관찰되는 염색체 이상

Disease

Disease TranslocationTranslocation Genes Genes

SLL t(14;19)(q32;q13) BLC-3 Follicular center cell t(14;18)(q32;q31) BCL-2 Burkitt t(8;14)(q24;q32) C- MYC

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Fluorescent

Fluorescent

In Situ

In Situ

Hybridization(FISH)

Hybridization(FISH)

Metaphase, interphase, paraffin,

Metaphase, interphase, paraffin,

frozen, cytospin, touch print,

frozen, cytospin, touch print,

BM aspiration smears, etc

(30)

ETO

AML1

t(8;21)… AML

(31)

2O2G : no rearrangement of AML 1/ETO

(32)

Chromosome Enumeration Probe: X/Y probe

(33)

V. Molecular study

V. Molecular study

• Clonality

– Clonal process … a proliferation of

daughter cells originating from a

single precursor cell , each having

identical phenotypes & growth

characteristics

• Normal B & T cell

(34)

혈액질환에서 분자생물학적 검사의

적응증과 검사방법

• 유전성혈액질환

– 원인유전자의 돌연변이

• 혈액종양 , 백혈병 , 림프종

– 진단과 분류 , 최소잔존질병추적 • 유전자재배열 , 클론성 , 돌연변이

• RT-PCR, DNA-PCR, Southern blot, real-time PCR

• 조혈모세포이식

– 이식 전 평가 , 이식 후 골수착상 추적

• HLA, DNA typing

• PCR based HLA typing, STR/VNTR

• 혈액질환과 관련된 감염성 질환

– 혈액종양 , 면역저하 , 혈액질환 유발성

• 감염원

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Monoclonal protein confirm

& quantitation

• Multiple myeloma 99%

– M protein (+) in serum, urine

– Dx:

M protein

, bone study, BM study

• Protein electrophoresis( 단백전기영동 )

• Immunofixation electrophoresis( 면역고정

전기영동

)… 확진

• Quantitation of immunoglobulin

• Free light chain quantitation

(37)

Photo - PEP &

densitometry

(38)

Photo -osteolytic

lesion

(39)

Photo - BM plasma

cell

(40)

MPD

M

yeloproliferative disorders

• Polycythemia Vera

Jak2V617F or JAK2

• Chronic myelocytic leukemia

– t(9;22)(q34;q11.2)

• Essential thrombocythemia

• Idiopathic myelofibrosis

(41)

Myelodysplastic syndrome(MDS)

• Ineffective Hemopoiesis

• Apoptosis 증가

• Peripheral cytopenia

• BM blast <20%

• BM morphology : Daysplasia

• Old age

• Chromosomal abnormality 동반

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