특수영역에서의진단검사의학 노인및소아과영역에서진단검사의학 임신관련검사 약물유전학과맞춤약물요법 세포유전학및분자생물학적검사기법 - 염색체검사 (conventional cytogenetics) - 형광동소보합결합 (fluorescence in situ hybridization, FISH) - 중합효소연쇄반응 (polymerase chain reaction, PCR) 및정량 PCR 개인식별분자진단검사 맞춤의학분자진단검사소개
노인및소아과영역에서진단검사의학 Biological influences-age 1. Newborn - Hemoglobin A (mature infant) vs. HbF (immature infant) - 출생직후혈관에서혈관외공간으로 fluid가이동하여혈장단백농도증가 - Activities of CK, GGT, and AST are high at birth. - bilirubin due to enhanced RBC destruction: peak at 3-5 days - Physiological jaundice: serum bilirubin< 5 mg/dl - Physiological hyperthyroidism gradually decline over the first year of life 2. Childhood to puberty - 어른에비해혈당이낮으며근골격계가성장하면서 ALP 와크레아티닌수치가증가
3. Adult: usually taken as the reference - Serum total cholesterol and TG conc. increase at a rate of 2 mg/dl per year to a maximum between ages 50 and 60 years. - The activity of most enzymes in serum greater during adolescence than during adult life 4. Elderly adult - The plasma conc. Of many constituents increase in women after menopause - Ccr may decline Thyroxine secretion and degradation thyroxine conc. PTH and aldosterone conc. cortisol secretion with aging 17-OHCS, 17-KS (U)
Changes in selected clinical chemistry analytes with aging Increased GGT Alkaline phosphatase, women only Alpha-1-antitrypsin Amylase AST BUN Creatine kinase, slight Gamma-globulin, slight Glucose fasting HDL Inorganic phosphate Lactate dehydrogenase Decreases Albumin Aldosterone Bilirubin Creatinine clearance DHEA Growth hormone p O 2 T 3 Total protein Transferrin pco 2 Potassium, slight Total cholesterol Triglycerides TSH, slight Uric acid
Biological influences-sex After puberty, the serum activities of ALP, ALT, AST, CK, and aldolase are greater in men than in women: due to greater muscle mass in men After menopause, ALP activity increases in women until it is higher than in men Higher conc. in men: albumin, calcium, magnesium, hemoglobin, iron, ferritin, cholesterol, LDL cholesterol Pregnancy - Increase in blood volume reduced protein conc. (P) - But, inc. of ceruloplasminand thyroxin-binding globulin lead to an increase of copper and thyroxin - Cholesterol, triglyceride, urine volume, GFR, ESR - iron, ferritin
임신관련검사 임신진단검사 Human chorionic gonadotropin (HCG) 1) serum, urine HCG 측정 임신의지표 2) serum HCG : 배란후 6~12 일째 4~6mIU/mL 이상증가 3) urine HCG : 정성검출 4) 임신이외증가원인 : trophoblastic tumor Peak! 10 weeks
산전선별검사 - 신경관결손 (neural tube defect) 1) Maternal serum α-fetoprotein (MSAFP) 2) MoM (multiples of the median) : 해당분만주수의정상임산부의 AFP 중앙값과비교 2.0~2.5 MoM 기준 3) 초음파소견및양수의 AFP, AChE(Acetylcholinesterase) - Down syndrome (trisomy 21) 1) 태아염색체선별검사 : 임신 20주이내 ( 미국산부인과학회 ) 2) Triple test: MSAFP & unconjugated estriol (ue3), HCG 3) inhibin A, pregnancy-associated plasma protein A - Edward syndrome (trisomy 18)
신경관결손 (Neural tube defect) - 혈청 α-fetoprotein (maternal serum AFP, MSAFP) 의증가 임신중기태아의신경관결손선별검사 - 결과를평가할때는아래와같은임상적인요소들을고려 1 임산부체중 : 임산부체중이증가할수록 MSAFP는감소 2 인종 : 미국에서는흑인이백인보다 MSAFP가 10~15% 높음 3 인슐린의존성당뇨병 (IDDM): 당뇨환자는건강인보다 MSAFP가 20% 낮음 4 다태아 : 쌍태아에서는 MSAFP가높음 5 임신주수결정 : 신뢰성있는임신주수계측이필수
기형아검사 - triple test: 임신중기 MSAFP, unconjugated estriol (ue3), HCG 측정 - 모체혈청에서측정한표지자혈청검사결과를행당표지자의임신주수별중앙값 (median value) 으로나누어서 MoM (multiples of median : 중앙값의몇배를의미함 ) 을구함 - 위험도산출 : 3가지표지자각각의 MoM과임신부체중, 당뇨병유무, 쌍태아유무를컴퓨터프로그램 (Alpha, AFP expert 및 Prenta 등 ) 에같이입력하여위험도산출
- Triple 검사에서모체혈청 AFP값이해당임신주수별중앙값의 2.0~2.5배 (MoM) 이상일경우개방성신경관결손 (open neural tube defect) 의위험성이있어양수 AFP 및양수 Acetylcholinesterase와같은추가검사 - 쌍태아일경우에는모체혈청 AFP가 4.0~4.5 MoM 이상일때고위험군으로 - 다운증후군태아를임신한경우 : MSAFP, ue3 감소하고, HCG 증가임산부연령과더불어세가지표지자를이용하면다운증후군의검출률약60%, 위양성률약 5% - inhibin A: 최근네번째표지자로등장. 다운증후군태아를임신한경우 inhibin A도증가 - 4가지표지자를이용하면다운증후군의검출률은거의75% 로증가
임신합병증관련검사 (I) - 태아적모구증 (erythroblastosis fetalis) 1) 태아의심한용혈성빈혈 2) Liley s test: 양수 bilirubin - 임신중독증 1) 임신 20주이후, 고혈압과단백뇨특징! 2) Thrombocytopenia, relative erythrocytosis 3) Increase : BUN/Cr, uric acid, AST, LD, D-dimer 4) HELLP syndrome : Hemolysis, elevated liver enzyme, low platelet
임신합병증관련검사 (II) 임신성당뇨병 - 임산부에서포도당불내성 태아폐성숙도 조기양막파수 - 질분비물 ph검사 ( 양수중성pH) - 양치형성 (fern pattern): 질분비물슬라이드 5분방치후관찰 - placental alpha-1 microglobulin - 태아 fibronectin 태아질식
태아폐성숙도 (Fetal lung maturity) - 임신 35주이후표면활성제의주성분 : dipalmitic lecithin -이후일주일후 phosphatidyl glycerol (PG) 나타나서임신말기까지증가꽈리의안정성유지 - 임신후기동안에양수의 sphingomyelin은일정 - 양수의표면활성제측정 참고방법 : 박층크로마토그래피에의한 L/S 비율 L/S 비율 > 2.0이면성숙, 1.5~2.0이면경계선, < 1.5이면미성숙 PG 신속검사 : 야간이나주말에크로마토그래피가안될경우이용신속라텍스응집방법 Microviscosity test( 형광편광 ): L/S 비율과상관성이좋아보편적으로사용
Cytogenetic Analysis (Amniotic fluid) - 양수천자 : 임신 16 주이후시행 양수세포 7~10 일정도배양후핵형분석
임신주수별검사 - 임신초기기본검사 CBC, UA, ABO typing, Ab screening, Rubella Ab, Screening test for syphilis, hepatitis and AIDS - 임신 14 주 ~21 주 Triple /Quad test, 양수검사 - 임신 24 주 ~28 주 임신성당뇨선별검사
약물유전학과맞춤약물요법 약물유전학 (pharmacogenetics) 개인및인구집단에서의약물및화학물질에대한대사와그반응의차이를유전학적으로분석하는분야 최근분자진단적분석방법의발전에따라유전형분석이선호 약물반응의차이를보이는유전자적특징은다유전자적양상으로발현되고, 이인자들은크게약물대사, 약물운반, 약물표적에관련된유전자다형성을등으로분류
약물유전학과맞춤약물요법 약물대사에관여하는효소들 20
인간에존재하는 cytochrome P450 약물대사에관여하는주요 cytochrome P450 대표적인약물대사 CYP450 와주요약물들 21
약물유전학과맞춤약물요법 Cytochrome P450 Inhibition: 약물간상호경쟁으로발생하거나효소활성이직접적으로억제되어나타남. CYP450 효소들은기질특이성이낮아약물을병용투여하면효소에대한약물의경쟁적결합때문에친화력이약한약물의대사가저해 Induction: 주로특정약물에의해효소생산이증가되어나타나게되며, 이로인해체내약물제거가너무빨리일어나혈중농도가전반적으로낮아짐 22
약물유전학과맞춤약물요법 약물유전학의활용 현재는후양적으로약물부작용을진단하는개인에특이적인검사에주로초점이맞추어져있음 향후에는대상인구집단에서선별된자료를바탕으로약물독성을예방하고치료를최적화하여치료제를선택하는전향적인검사로활용
염색체검사 (conventional cytogenetics)
염색체 DNA 와핵단백질 (nucleoprotein) 로구성
염색체검사활용분야 1) 소아과영역 : 선천성염색체이상, 다운증후군 말초혈액림프구 (T lymphocyte) PHA-stimulated 72 hr 배양후염색체획득 2) 산부인과영역 : 선천성염색체이상, 다운증후군 양수천자에서얻은소량의 Amniocyte 자극없이 10-14일배양후염색체획득 3) 혈액종양분야 : Ph + CML, t(8;21) AML, t(1;19) ALL 암세포만갖는염색체분석 암세포배양 (0 hr -48 hr ), 자극제없이 4) 기타 Chomosome breakage test (Fanconi anemia)
혈액질환에서염색체검사의유용성 1) 혈액종양의진단 t(8;21) AML; t(15;17) APL; inv(16) AML with eosinophilia; t(9;22)cml 2) 예후판정및치료방침결정 AML good; t(8;21), t(15;17), inv(16) poor; t(9;22) ALL good; hyperdiploidy poor; t(9;22) 포함한 hyperdiploidy MDS good; normal,del(5)(q), del(20)(q), -Y abnormal poor; complex karyotype, 7 번 MM good; hyperdiploidy, t(11;14) poor; hypodiploidy,t(4;14), t(14;16 ) 3) 치료효과판정및잔류병소측정 4) 조혈모세포이식후생착판정 5) 재발또는질환진행의진단
검체채취 검체채취 : 세포손상최소화, sterile Sodium-heparin, 진공채혈관이용시반드시 decaping, needle 제거 검체는절대얼려서는안되면검체접수가지연될경우 냉장보관할수있으나실온보관도추천 검체채취당일처리해야만세포배양성공확률높음
염색체분석을위한기본검사흐름도 Cell culture Harvest Slide preparation A,F : 10-12 days Blood :72hrs(PHA,PW) B.M : direct, 24hrs Colcemide : 50min Hypo.sol : 20min Fixation : methanol/acetic acid = 3/1 Air dry Warm dry Staining GTG-banding Observation & Analysis
염색체분류방법 (ISCN 표기법 ) Grouping 기준 size Large Medium rshort Short centromere metacentric submetacentri c meta or sub acro meta or sub meta acro group A (1-3) group B (4-5) group C (6-12, X) group D (13-15) group E (16-18) group F (19-20) group G (21-22, Y)
Chromosome Identification 1 2 3 4 5 6 7 X 8 9 1 0 1 3 1 4 1 5 1 1 16 1 7 12 18 1 9 20 2 1 22 Y
염색체밴드명명법 International System for Human Cytogenetic Nomenclature 표기법 염색체의밴드는 landmark 에의해 region 으로구분된다 번호는밴드와 region 은동원체로부터바깥으로가면서번호가할당된다 p terminal Band Numbering centromere 8q24.2 8 : 8 번염색체 q : 장완 2 : 영역 4 : 밴드 2 : 부밴드 q terminal
Numerical aberrations Tuner syndrome 45,X 45,X,-X 46,Xc,+X 44,-Xc 48,XY,+21c,+21 Down syndrome 47,XX,+21 48,XY,+21c,+21
Structural aberrations (1) Duplication ( 중복 ) Deletion ( 결실 ) Inversion ( 역위 ) 46,XY,dup(7)(q11.2q22) 46,XX,del(1)(q24q31) inv(3)(q24q27)
Isochromosome ( 동완염색체 ) Ring chromosome ( 환염색체 ) Structural aberrations (2) Translocation ( 전위 ) i(17)(q10) r(13) t(9;21)(q34;q11)
General Principles of Karyotype Designation (1) 1. Normal Karyotype : 46,XX or 46,XY 2. chromosome abnormalities 기술방법 * constitutional sex chromosome 은언제나기술한다 * autosomal chromosome 은비정상소견이있는경우에만기술한다. 일반기술순서 1 성염색체 (X Y) 2 상염색체번호빠른순서 3 개수변이가구조변이보다먼저 single chromosome 4 단완변이가장완변이보다먼저기술 5 proximal한변이가 distal 변이보다먼저 6 constitutional 변이가 acquired한변이보다먼저 7 구조변이약어의알파벳순으로 8 recipient가 donor 보다먼저기술 9 명확하지않은것은나중에적는다 (r, mar, dmin, inc) 10 mosaicism이관찰되는경우 clone 변이가단순할수록먼저 & 정상 clone은마지막 cf. constitutional 변이와 acquired 변이를구분위해 constitutional chromosome 에소문자 c 를뒤에붙인다. cf. homologous chromosome 의구분이필요한경우 single underlining 을한다.
General Principles of Karyotype Designation (2) 2. chromosome abnormalities 기술방법 1) 수적이상 (numerical aberrations) 기술방법 1 성염색체 constitutional 인경우숫자를표시하지않고있는대로써준다 acquired 경우개수변이는 +, - 로표시한다 2 상염색체 숫자가빠른순으로 +,- 로표시한다. 2) 구조이상 (structural aberrations) 기술방법 1 single chromosome: 46,XX,inv(2)(p21q31) rearrangement type (abbreviation) (chromosome number) (band) 2 two or more chromosomes: 46,X,t(X;18)(p11.1;q11.1) rearrangement type (abbreviation) (chromosome number; chromosome numer) (band;band)
염색체구조이상 (1) t(1;3)(p36.1;q21) t(8;21)(q22;q22) t(9;22)(q34;q11.2) t(15;17)(q22;q21)
형광동소보합결합 (fluorescence in situ hybridization, FISH)
형광동소보합검사원리 ( FISH; Fluorescence in situ Hybridization )
FISH 검사의유용성및장점 : 염색체분석법과비교 유용성 Aneuploide( 수적이상 ) identification Rapid analysis 장점 Microdeletion( 미세결손 ) identification Oncogene identification - Translocation( 전좌 ) - Deletion( 결실 ) - Amplification( 증폭 ) Intact cell morphology Multi - target Quantitative assay Both interphase and metaphase A variety of specimen : blood, paraffin tissue, frozen tissue, touch print, cytospin 골수이식후의생착여부
FISH 검사흐름도 A. 검체 (PB, BM, Tissue, 체액 ) FISH 전용슬라이드 B. 세포제단백및고정 C. 형광소식자부착 D. 변성후보합결합 E. 세척 F. 대조염색 제단백 : 20XSSC,NP-40 고정 : 에탄올 (70%,85%,100%) Probe 1ul + D.W 2ul + Buffer 7ul Hybridization - 73 2min ( 변성 ) - 37 overnight (16-18hrs) 0.4XSSC / 0.3%NP-40 2XSSC / 0.1%NP-40 DAPI II G. 검경
13.3 Prophase/Metaphase ish (4) D13S319 DXZ1 DNA Unique Segment Repetitive Segment Chromosome Number 1 ~ 22, X, Y 0 unknown XY homologous of X & Y Sequential Number --F- Small undefined families of homologous sequences found on multiple chromosomes
FISH positive pattern Normal pattern Abnormal pattern Dual Color, Single Fusion Probes flank breakpoints 2O2G 1O1G1F Estimated Cut Off ~ 10 % ES (Extra Signal) One large probe spans, 2O2G 2O1G1F other flanks breakpoint Estimated Cut Off ~ 1.5-3 % Dual Color, Break Apart 2F 1O1G1F Probes flank one breakpoint Estimated Cut Off ~ 1-3 % Dual Color, Dual Fusion Two large probes span breakpoints 2O2G 1O1G2F Estimated Cut Off < 1 %
중합효소연쇄반응 (polymerase chain reaction, PCR) 및정량 PCR
PCR 의종류 1. 역전사중합효소연쇄반응 (Reverse transcriptase PCR, RT-PCR) RNA 를증폭하기위하여고안된방법. RNA-> reverse transcriptase(rt) 효소를이용한 cdna 합성 -> PCR 2. 이중중합효소연쇄반응 (Nested PCR) 일차 PCR 후에두번째 primer set 로이차 PCR 을시행하는방법. 민감도를높이고특이도를증가시킨검사법. 3. 다중중합효소연쇄반응 (Multiplex PCR) 둘이상의 Target 부위를단일시험관내에서동시에증폭시키는방법. 4. 실시간중합효소연쇄반응 (Real-time PCR)
Hydrogen Bonds Cytosine (C) Adenine (A) Thymine (T) Guanine (G) Guanine (G) Thymine (T) Adenine (A) Cytosine (C) Deoxyribose Phosphoric Acid (Sugar molecule) (Phosphate molecule)
핵산추출방법 DNA 추출방법 RNA 추출방법 Sample Proteinase K Lysis buffer +ethanol Wash buffer AW1 Wash buffer AW2 Dry spin Elution buffer Pure viral nucleic acids
PCR 에필요한구성성분 - DNA 또는 mrna, DNA 중합효소및완충액, dntp, 시발체, MgCl2 등그외온도조절장비, 전기영동장치등이필요 - PCR 반응에가장중요한성분은시발체 (primer) - 증폭되는산물의크기 : 일반적으로 2,000 bp까지가능. 200~500 bp 정도가가장효율적으로증폭 1 주형 DNA 2 시발체 3 열저항 DNA 중합효소 ( 예 : Taq polymerase) 4 Deoxynucleoside triphosphates (dntp) : 동량의 datp, dttp, dctp, dgtp 로구성 5 이가양이온 : Mg 2+ 등 6 완충액 (Buffer): ph 8.3~8.8정도의 10 mm Tris-Cl 7 일가양이 : 50 mm KCl
HBV DNA HCV RNA Anti-HCV HBS Ag
No. of Cycles No. of Target Amplicon Copies 1 2 2 4 3 8 4 16 5 32 6 64 20 1,048,576 30 1,073,741,824
PCR 의이론과실제 Log Target DNA Linear phase 이론적증폭 Exponential Phase ( 지수기 ) 실제증폭곡선 Plateau Phase ( 평형기 ) Cycle #
정량 PCR 정량 (real time PCR) 은대상핵산증폭과검출과정을단일시험관내에서동시 에진행하면서 PCR 각주기마다실시간으로확인이가능한방법. Real time PCR 기법 1 SYBR Green Ⅰ 2 Taqman probe 3 Hybrization probe 4 Molecular beacon
5 exo-nuclease activity of Taq DNA Polymerase and FRET probe 5 3 5 Forward primer 3 Reverse primer 5 3 5 5 3 5 5 3 5 3 3 3 Excited quencher Taq DNA polymerase Reporter : FAM, VIC, TET, HEX Quencher : TAMRA Emitting reporter
Real time PCR 결과분석 endpoint측정이아닌 PCR의증폭이기하급수적으로증가하는지수기 (exponential phase ) 에서측정 Ct (Threshold Cycle) 값을기초로함 초기 template의양을알고있는 standard를이용하여 Ct 값에따른 standard 직선성을확보 미지샘플에서측정하고자하는 target의정량은 Ct 값을측정함으로써가능
Threshold Cycle,Ct (1) 초기의 background 를지나 fluorescence ( 형광 ) 의양이급격하게 증가하는시점의 PCR cycle 수를말한다 초기 template 의양과정확하게비례한다
Threshold Cycle, Ct (2) 연속적으로증가하는초기 template의양에대한 Ct 값의결과를 log로그리면정확하게직선을이룬다 초기 template의양이같은샘플을여러개반응시키더라도같은 Ct 값을보임 Fixed threshold
적응증 미세잔류백혈병 (MRD) 확인 TEL-AML1 gene in ALL ( 급성림프구성백혈병 ) TCR & IG gene in ALL ( 급성림프구성백혈병 ) AML1-ETO/CBFβ-MYH11 gene in AML ( 급성골수성백혈병 ) PML-RARa gene in APL ( 급성전골수성백혈병 ) BCR-ABL gene in CML ( 만성골수성백혈병 ) Viral load : CMV, EBV, HIV, HCV, HBV etc. 특정유전자발현 돌연변이확인 : drug resistances
핵산블롯부합법 (Nucleic acid blot hybridization) 블롯 : nitrocellulose 또는 nylon 막 (membrane) 에검체를고정시키는것 핵산블롯팅 : 전기영동을시행한핵산조각을겔에서막으로옮기는과정 세포에서핵산을추출하여크기에따라전기영동을시행한후 nitrocellulose 또는 nylon 에고정후핵산에특이적인탐색자와부합반응을통하여검출 탐색자에부착되는표지자에따라 방사선동위원소법, 효소를이용한화학발광법, 발색법등 PCR 개발후많은검사에서 PCR 로대체. 특정검사에서는여전히선호
1) 서던블롯부합법 (Southern blot hybridization) - 제한절편길이다형성 (restriction fragment length polymorphism, RFLP) 을이용하여개인의식별에이용될수있고, 유전자의결손이나증폭, 돌연변이, 염색체의전좌, 메틸화된DNA 등과관련된질환의진단에이용. - 유약엑스증후군 (Fragile X syndrome), 근육긴장퇴행위축 (myotonic dystrophy) 에이용 2) 노던블롯부합법 (Northern blot hybridization) - 기본원리와분석방법이서던블롯부합법과동일하지만, DNA가아니라 RNA를분석 - 신호의강도는특정유전자발현의정도에비례하기때문에정량적인분석이가능 - 비정상적인 RNA의발현은특정유전질환과관련 임상적진단과예후판정에이용
핵산부합원리를기본으로함 다량의부합반응을한번에시행할수있는기술이현실화된것 수천 ~ 수만개의유전자를한꺼번에분석
개인식별분자진단검사 개인식별검사 활용분야 친자감별 장기이식시조직적합성검사 조혈모세포이식후생착평가 임상검체에서의식별검사 법의학분야 유전표지자의종류 SNP (single nucleotide polymorphism) RFLP (restriction fargment length polymorphism) VNTRs (variable numbers of tandem repeat loci) or minisatellite STR (Short tandem repeat) or minisatellite
개인식별분자진단검사
맞춤의학분자진단검사소개 맞춤의학 : 맞춤종양의학의태동 분자적수준에서종양의원인규명 유전체염기서열분석및단백체분석 분자적표적약물개발 - HER2 양성유방암 : trastuzmab - BCR/ABL 만성골수성백혈병 : imatinib - EGFR유전변이비소세포폐암 : gefitinib, erlotinib - BRAF유전변이악성흑색종 : vemurafenib
Structure of Her-2/neu oncoprotein Extracellular domain (Ligand-binding site) Plasma membrane Plasma membrane Transmembrane domain Intracellular domain (Tyrosine kinase activity) Cytoplasm
Localization of Her-2/neu gene on chromosome 17 Normal Interphase Nucleus and Metaphase Spread HER2 Chromosome 17 Centromere 17 17
Immunohistochemistry of Her2 0 1+ 2+ 3+
Serum HER2 assay AE Anti-HER-2/neu Mab(TA-1)-AE Anti-HER-2/neu Mab(NB-3)-F ITC HER-2/neu Flurorescein Anti-Flurorescein Mab PMP