경희대학교 의과대학·의학전문대학원

(1)

Diagnostic Tests in Leukemia,

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

(4)

Formation of a clone

Normal

tissue

Mutation

Clone

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

(5)

1. Morphology

PBS,

Bone Marrow aspirates &

biopsy

2. Cytochemical Stain

3. Immunophenotyping

1) Immunocytochemistry

2) Flow Cytometry

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

(9)

(10)

(11)

(12)

III. Immunophenotyping

• 세포의 특이한 항원결정인자 (epitope)

를 인지하는 항체를 이용하는 면역학적 방

법

– CD (Cluster designation/Cluster of

differentiation)

• Monoclonal antibody

• 세포를 인지하는 특이도가 높다

(13)

(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

(15)

Immunophenotyping by flow cytometry

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

(16)

AML

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

(17)

(18)

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

(19)

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

• Cytogenetic characteristics of

Hematologic malignancies

– Non random style

– Subgroup

• Conventional cytogenetics

(21)

Cytogenetics

(22)

(23)

(24)

(25)

(26)

(27)

*

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)