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Minimal Residual Disease(MRD) detections by Flow Cytometry in Acute Leukemia 2008. 4. 3 혈액학파트 안 미 숙.

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Presentation on theme: "Minimal Residual Disease(MRD) detections by Flow Cytometry in Acute Leukemia 2008. 4. 3 혈액학파트 안 미 숙."— Presentation transcript:

1 Minimal Residual Disease(MRD) detections by Flow Cytometry in Acute Leukemia
혈액학파트 안 미 숙

2 Introduction Morphologic remission in acute leukemia
: less than 5% blast in bone marrow The detection of MRD - evaluate the efficiency of therapy - predictive of a greater risk for relapse MRD assays - strongly associated with relapse. - increasingly important in the clinical management of patients with acute leukemia.

3 Methods for MRD detection
Detection method Target Sensitivity(%) Pathologic examination Cellular morphology 5 Cytogenetics Chromosome Structure 1-5 FISH Specific genetic marker 0.08-5 Flow cytometry Surface antigen expression 0.1-1 PCR DNA or RNA sequence Tissue antigen 2006;68:371

4 MRD 검사 방법들의 장.단점 검사방법 장점 단점 Pathologic examination 대량의 악성세포시 쉽게 판정
민감도가 낮다 Cytogenetics 염색체 이상의 악성세포의 출현 - clonal evolution 감별 가능 meterphase cell 만 분석 검사 시간 수일 소요. FISH 정량 분석 가능 각 세포의 형태학적 관찰과 함 께 판단 48hr내 결과 보고 Interphase cell 분석 가능 시약의 고가 사용 probe에 대한 결과만 알 수 있다. PCR 가장 민감하게 정량분석 소수의 악성 clone도 검색 가능 24hr 이내 결과 판정 가능 위양성 위험성 임상적 의미가 없는 세포의 양성 결과 Flow cytometry 직접적 정량 분석 가능 불필요한 세포(dying cells &celluar debris)의 분석 배제 검사방법이 쉽고 , 12hr이내결과 병의 진행과정에 따른 Marker 의 변화→ 위음성 초래

5 Exclude debris & non-viable cell

6 MRD detection by FCM The fundamental principle
-Identification of immunopheno-typic combinations expressed on leukemic cells, but not on normal hematopoietic cells aberrant expression by Multi-parameter FCM

7 Simulation test For simulation test - FC 500
- dilution for 20 /µl WBC count - 10/µl (pt’s WBC) + 100/µl(NL PB, WBC 8000/µl) + Mo ab 10/µl - 15 min incubation(RT) - add lysing solution (500µl ,10 min incubation) - washing of PBS - resuspention with 1% paraformaldehyde - FC 500

8 Simulation(l) WBC : 76,620 /µl (M/25)
-Intermediate SSC region was analyzed -Moderate to bright expression : CD33, CD13, CD34 & MPO -Consistent with acute myeloid leukemia

9

10 Simulation(ll)- Live gate

11 Simulation(ll) WBC : 39,190/ µl (F/9) - Low SSC region was analyzed
- Moderate to bright expression :CD10, CD19, CD79a - Aberrant expression CD13, CD33 - Consistent with precursor B ALL

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14 Markers of Leukemia KSLH concensus,2006

15 Immunophenotypic combinations of ALL( 4-color)

16 Cytomics FC500

17 Current items of Lab. 1. Lymphocyte Subset (T cell, T4, T8, B cell, NK cell)                  2. Immunophenotype (leukemic cell marker)                                  3. Stem cell count 4. Antiplatelet antibody & Platelet-associated IgG  5. DNA index                                                                                                

18 세포표지자-검사(나512)의 인정기준 <보건복지부-고시2007-92호>(2007.11.1시행)
세포표지자검사(Cell Marker Study)인정기준 - 급성백혈병 초기 진단시에는 18종 이내 - 치료효과판정을 위한 추적관찰 검사시 3-5종 이내로 인정함 만성골수성백혈병의 모세포기(blast crisis)의 경우는 급성백혈병에 준하여 인정함.( 시행)

19 의료수가 종목 보험수가 일반수가 Tdt를 제외한 모든 marker 27,610원 72,000원
Tdt (Terminal deoxynucleotidyl transferase) 43,070원 113,000원 Stem cell count (absolute count) 65,060원 170,000원

20 Conclusion Flow cytometry Vigilance in high sensitivity Simplicity
wide availability Vigilance in Design Acquisition Analysis


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