Click to add title

Size: px

Start display at page:

Download "Click to add title"

새론 채
6 years ago
Views:

1 Genetic and Conventional Tests of PNH 원자력병원진단검사의학과 장윤환

2 Agenda 1. Mutation analysis of the PIGA gene 2. Cytogenetic study 3. Ham test and sucrose hemolysis test 4. Gel microtyping system (Gel card test)

3 History 1882: Strü bing 수면후혈색뇨증을나타내는환자에대해기술함 1938: Ham PNH 세포가보체의용혈작용에대해민감함을밝힘 년대 (Rosse 등 ) - 보체방어단백들의결핍 - GPI anchor의결핍 1993: PIGA 유전자분리

4 PIGA gene Official Symbol: PIGA Name: phosphatidylinositol glycan anchor biosynthesis, class A [Homo sapiens] Other Aliases: GPI3, PIG-A Chromosome: X; Location: Xp22.1 consists of six exons PIGA mutation - always in somatic (specifically, hematopoietic) cells - affect the first step in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor

5 Glycosylphosphatidylinositol (GPI) anchor GPI-linked protein Complement regulators Decay accelerating factor (DAF, CD55) Membrane inhibitor of reactive lysis (MIRL, CD59) Enzymes Neutrophil alkaline phosphatase Blood 1995;86:3278 Acetylcholinesterase

6 GPI-linked protein GPI anchor Complement attack Block Cell Lysis Complement attack Deficiency of GPI anchor Cell

Polymerase Chain Reaction (PCR) Exons 1 2 3 4 5 6 Segments I II IV V

7 Polymerase Chain Reaction (PCR) Exons Segments I II IV V Marker VI I II IIIIV V I : 487 bp II : 497 bp III : 244 bp IV : 580 bp V : 361 bp

Dideoxy Fingerprinting (ddf) (1) 1 2 3 4 5

8 Dideoxy Fingerprinting (ddf) (1) Results of ddf for the screening of mutation in PIGA gene, showing abnormal band ( ) in lane 2 : Case 1, Segment II

9 Dideoxy Fingerprinting (ddf) (2)

10 DNA sequencing (1) G A T C G to A Sequencing result of PIGA gene, showing a point mutation (G to A) of 3 splice site (intron 5) : Case 5

Sequencing result of PIGA gene, showing a mutation of 25 bp deletion of CDs 409-417 : Case 7 G A T C T T A T T C A

11 Sequencing result of PIGA gene, showing a mutation of 25 bp deletion of CDs : Case 7 G A T C T T A T T C A G A C A G G T C A G C A A A C A G G T C G G A C A A T G A C C C G G C G T C A C T C T T A A C C DNA sequencing (2)

12 Summary of the 12 mutations in the PIGA gene seen in Korean PNH patients J Clin Pathol 2002;55:410-3.

13 J Clin Pathol 2002;55: Summary of the 12 mutations in the PIGA gene seen in Korean PNH patients Exon * * Point Mutations (n=6) = Missense (Substitution) (n=1) = Immediate Termination (n=2) = Altered Splicing (n=3) Frameshift Mutations (n=6) = Premature Termination (n=6) * Thrombosis symptom (+)

14 The spectrum of PIGA gene mutations in AA/PNH : a high incidence of multiple mutations and evidence of a mutational hot spot Blood 2003;101:

15 Cytogenetic abnormalities in PNH Br J Haematol 2001;115: In patients with PNH, generally, the karyotype of bone marrow cells has been reported to be normal. Retrospective review of 46 patients with a PNH clone : a karyotypic abnormality in 24% (11/46). In patients with PNH cytogenetically abnormal clones are not necessarily malignant and may not be predictive of evolution to leukemia.

16 Cytogenetic abnormalities in PNH Br J Haematol 2001;115:360-8.

17 Haematological data for subgroups of PNH patients based on karyotype Br J Haematol 2001;115:360-8.

18 PNH and abnormal karyotypes in the literature Br J Haematol 2001;115:360-8.

19 Conventional lytic tests 1. Sucrose hemolysis test ( 설탕용혈검사 ) 검사원리 : Sucrose는용액내이온강도를떨어뜨려보체결합을촉진시켜 PNH 세포의용혈이발생한다. 검체 : 항응고제 (Sodium citrate, EDTA) 를사용한전혈 2. Ham test (Acidified-serum test, 산성화혈청검사 ) 검사원리 : PNH 환자의적혈구를 ph 6.5~7.0의산성혈청과반응시키면보체의대체경로가활성화되어보체가적혈구막에부착되어 PNH 세포가용혈된다. 검체 : 항응고제 (Sodium citrate, EDTA) 를사용한전혈

20 Sucrose hemolysis test 검사방법 Tube % sucrose 액 환자의적혈구현탁액 정상인혈청 % NH 4 OH ( 단위 : ml) 실온에서 1시간 incubation 식염수 4 ml를전체 tube에넣어 1500 rpm에서 3분간원침하고상청액의흡광도측정 환자의용혈 % = 100 X (1-2-3)/(4-2) 결과판정 >10%: virtually diagnostic for PNH 5-10%: suspicious of PNH <5%: negative

21 Ham test 검사방법 Tube 1(4) 2(5) 3(6) 정상대조혈청 비동화된정상대조혈청 N HCl % 적혈구현탁액 * M MgCl 용혈 ( 양성에서 ) Trace * 1-3 : 환자의세포현탁액, 4-6 : 정상대조세포현탁액 ( 단위 : ml) 37ºC 에서 1 시간 incubation 3 분간원침 (1500 rpm) 후용혈유무확인기록 흡광도측정 Tube 2A : tube 2 의상청액 0.3 ml % NH 4 OH 5 ml Tube 7 : 정상대조혈청 0.5 ml % NH 4 OH 5 ml Tube 8 : 증류수 0.55 ml + 환자의 50% 적혈구현탁액 0.05 ml % NH 4 OH 5 ml 용혈 % = 100 X (2A-7)/(8-7)

22 Ham test 판독방법 2 번 tube 에만용혈 (+) : positive 모든 tube 에용혈 (-) : negative * 2번과 3번 tube에동시에용혈 (+) : false positive * 1번 tube에용혈 (+) : 환자와대조정상인의혈액형이다른경우 * 4번과 5번 tube에용혈 (+) : 대조정상인이비정상. 다른정상인으로재검요함. 임상적의의 : PNH 진단이목적이나, congenital dyserythropoietic anemia type II (hereditary erythroblastic multinuclearity associated with a positive acidified serum test, HEMPAS) 에서양성으로나타날수있다.

Gel microtyping system (Gel card test) 검사원리 : 보체방어단백인 CD55 (DAF) 와 CD59 (MIRL) 를확인하는검사로, gel 물질이들어있는마이크로튜브를이용하여단클론항체인 anti-daf 와 anti-mirl 을검체인적혈구와반응시켜응집반응을확인하여 PNH 를 선별하는검사이다.

23 Gel microtyping system (Gel card test) 검사원리 : 보체방어단백인 CD55 (DAF) 와 CD59 (MIRL) 를확인하는검사로, gel 물질이들어있는마이크로튜브를이용하여단클론항체인 anti-daf 와 anti-mirl 을검체인적혈구와반응시켜응집반응을확인하여 PNH 를 선별하는검사이다. 검체 : 항응고제 (Citrate, EDTA, CPDA) 가 처리된신선한혈액 시약 (DiaMed-ID Micro Typing System PNH Test; Diamed AG, Morat, Switzerland) ID-Card ID-PNH ID-PNH test reagent (anti-daf, anti-mirl, negative control) ID-Diluent II (LISS)

24 Gel card test 검사방법 Gel card에환자명을적은후알루미늄호일을제거한다. 적혈구부유액을만든다. : 시험관에 Diluent (LISS) 1,000 ul를넣은후환자의침사혈구 10 ul를넣어서잘혼합한다. Gel card의마이크로튜브에혈구부유액을 50 ul씩분주한다. Test reagent를각각 50 ul씩분주한다. Incubator에서 15분간항온한다. 10분간원심분리후육안으로결과를판독한다.

25 Gel card test 판독

26 Gel card test 판독방법 PNH 음성 : 4+, 3+ 일경우 (CD55, CD59 정상적으로존재 ) PNH 양성 : 음성반응일경우 (CD55, CD59 결핍 ) DCP (double cell population) 반응 : 양성반응과음성반응이같이나타나는경우 Ctl( 음성대조 ) 는반드시음성반응이어야한다. 단, 적혈구 IgG 가강하게 coating 되어있으면 Ctl 에서양성반응을보일수 있으며, 이러한경우 autoantibody 를제거한후재검사하여야한다.

27 A prospective comparison of four techniques for diagnosis of PNH Int J Lab Hematol 2007;29:

28 Results of flow cytometric and gel card tests obtained after serial dilution of erythrocytes in a case of PNH Int J Lab Hematol 2007;29:

29 Results of lytic tests after serial dilution of erythrocytes in a case of PNH with 50% CD59-deficient type III cells Int J Lab Hematol 2007;29:

30 PNH 관련검사 : 건강보험코드 (2010 년 ) 분류번호코드분류점수금액 나-120 B1200 산혈청 ( 햄 ) 검사 Acid serum (Ham s) test 나 -121 B1210 슈크로즈용혈검사 너 -42 Sucrose Hemolysis Test PNH 검사 , ,770 BY421 가. Gel 검사 ,210 BY422 나. 유세포분석법 Flow Cytometry ,130

complement Clinical manifestations Available

31 Summary Pathogenesis? Stem cell : PIGA gene mutation Hematopoietic cell membrane : GPI anchor deficiency Complement defense proteins Sensitivity to complement Clinical manifestations Available tests PIGA analysis Flow cytometry, Gel card test (CD59, CD55) Ham test Sucrose hemolysis test

혈액은행 자동화장비 IH-1000소개와 Autovue Innova와의 결과비교

혈액은행 자동화장비 IH-1000소개와 Autovue Innova와의 결과비교 혈액은행자동화장비도입을 위한 IH-1000 평가 혈액은행파트 김화영 1. 배경 (Background) 2. 목적 (Purpose) 3. IH-1000 소개 4. 실험방법 5. 실험결과 6. 결론 7. 검사단가및보험수가 1. 배경 ABO-RhD grouping과항체선별검사는혈액은행에서가장중요하고필수적인검사이고사소핚 Error로도 Acute hemolytic transfusion