면역학 검사의 해석
서울의대 검사의학교실 송은영
장기이식시 면역학 검사의 종류
ABO typing HLA typing HLA crossmatch (XM) PRA (panel reactive antibody)
HLA typing
혈청학적 검사(microlymphocytotoxicity test) 보체의존성 세포독성검사(complement-dependent
cytotoxicity, CDC) HLA 항원-항체반응 후 보체 첨가 → cell lysis →
Eosin Y로 염색
분자유전학적 검사(DNA test) SSO (sequence specific oligonucleotide) SSP (sequence specific primer) SBT (sequence based typing)
HLA Ab assay
CDC-XM (crossmatch) FCXM (Flowcytometric crossmatch) PRA (panel reactive antibody) Solid phase immunoassay (SPI) – ELISA, Luminex
DSA (donor HLA-specific Ab)
Sensitivity Luminex PRA > FCXM > CDC-XM
CDC-XM (crossmatch)
보체의존성 세포독성검사 (complement-dependent cytotoxicity, CDC) 공여자의 림프구 + 환자의 혈청 → 보체 첨가 → cell lysis → Eosin Y 염색 → 현미경 관찰
기본 - NIH법, 예민한 방법 – AHG (anti-human globulin) 방법
+ 보체
+ Cell lysis
염색약 침투
F
PE-anti-CD3
T세포 항원(CD3)
Laser
Flow
FCXM (Flowcytometric crossmatch)
Detector
HLA항원
F F
FITC-goat F(ab’)2-anti-human IgG
항-HLA 항체
FCXM (Flowcytometric crossmatch) More sensitive, IgG와 IgM 구별 가능
Cut-off – 음성대조혈청 이용 MCS (median channel shift) – linear scale MFI ratio – log scale
e.g.)
PRA (Panel Reactive Antibody)
환자 혈청 내 HLA 항체 선별 및 동정
종류 Screen (선별) Phenotype panel (동정) Single antigen assay (단일항원 동정검사)
Luminex technology
Multiplexed bead based flowcytometric assay
Luminex technology
PE-anti-IgG Allo Antibody A3
HLA Antigen
Negative Positive
BEAD 1
A1,A2,B7,B8
BEAD 2
A3,A29,B13,B27
No Antibody
MFI (mean fluorescence intensity)
PRA (Panel Reactive Antibody)
Screen (선별) HLA 항체 유무를 검사 림프구나 항원 pool (2-12개)을 이용
Phenotype Panel (동정) 수십 개의 림프구(purified Ag)로 구성된 패널을 이용 HLA 항체의 특이성 동정
Single Antigen assay (단일항원 동정검사) 재조합 항원(recombinant Ag)을 부착시킴. 장점: 특이성 동정이 용이함. 단점: distortion of molecule, 패널구성,
Ag density 차이(형광강도)에 대한 검증이 안 됨.
PRA - Screen – Luminex LIFECODES LifeScreen Deluxe (Immucor, USA)
Bead Specificity
1 Class I (300 donors)
2 A1, A19 CREG enriched
3 A2 CREG enriched
4 B5 CREG enriched
5 B7 CREG enriched
6 B8 CREG enriched
7 B12 CREG enriched
8 Class II (30 cell lines)
9 DR51 enriched
10 DR52 enriched
11 DR53 enriched
12 DQ enriched
PRA - ID LIFECODES Class I ID (Immucor, USA)
PRA - Single Ag LIFECODES LSA Class II (Immucor, USA)
Luminex PRA MFI vs. FCXM(+) Refs. Luminex Luminex MFI FXM (cut-off) Zachary AA, 2009
ID 6,000 (sen 86, spe 98) 6,000 (sen 91, spe 82)
T-FXM B-FXM
Morris GP, 2010
SAg 2,000 (PPV 79, spe 99) MFI ratio 0.2 (PPV 64, spe 93)
CDC-XM FXM (MCS 40/80)
Moreno C, 2012
SAg 6,500 (sen 85%, spe 82%) T-FXM (MCS 40)
Ellis TM, 2012
SAg 6,282 (sen 90, spe 95) 6,282 (sen 85, spe 56) 12,693 (sen 57, spe 100)
T-FXM (MCS 150) B-FXM (MCS 250) B-FXM (MCS 250)
Kim SY, 2012
ID 1,250 (sen 78, spe 99) 1,971 (sen 90, spe 98)
T-FXM (ratio 2.0) B-FXM (ratio 2.0)
MCS, median channel shift; sen, sensitivity; spe, specificity; PPV, positive predictive value
C1q assay
www.onelambda.com
보체와 결합하는 항체를 구별함 exogenous purified human C1q + PE-anti-human C1q Ab
Refs. Organ Outcome
Chen, Hum immunol 2011 Ht Kd
AMR TG, GF
Yabu, Tpl 2011 Kd TG, late GF
Chin, J Ht Lung Tpl 2011 Ht AMR
Sutherland, Ped Tpl 2012 Kd GF
Crespo, Tpl immunol 2013 Kd No association
Loupy, NEJM 2013 Kd GF
O’Leary JG, Am J Tpl 2015 Liver Pt survival↓ early rejection↑
C1q assay – clinical relevance
AMR, antibody mediated rejection; TG, transplant glomerulopathy; GF, graft failure
2723 LT cases Positive XM – 7.4%(T-cell), 10.1%(B-cell) Pt survival, GS, rejection → preservation injury↑ (P = 0.03 for T cells, P
= 0.003 for B cells) AKP, T.bil ↑ at POD#7
Ruiz R et al., Liver Transpl 2012
Positive XM in liver tpl.
Five strongly positive XM cases in 69 liver transplantation
HLA Ab screen, FCXM MCS (>200), DSA MFI level, and C1q assay
T-MCS = 383.5±38.9, B-MCS = 408.8±52.3 3/4 pts with T-FCXM(+) – cholestasis and/or AR
Leonard GR et al., Liver Transpl 2013
Leonard GR et al., Liver Transpl 2013
Leonard GR et al., Liver Transpl 2013
1270례 중 14% - preformed DSA positive Class II > MFI 5000-10000 – 23% persistent Class II - early rejection (HR = 1.58) Class I and/or Class II > MFI 5000 – ↑death (HR = 1.51)
O’Leary JG, Liver Tpl. 2013,19:973
Role of DSA in liver tpl.
86례 SLKT Class II DSA – renal AMR, liver rejection, graft loss, death
O’Leary JG, Am J Transplant. 2013;13:954
197례 중 19례(10%) - XM(+) 15/19 – 이식 후 XM(-) 4/19 – XM(+) 지속 – 3/4 AMR
Kozlowski T et al., Liver Transpl 2011
Paterno F et al, Am J Transplant 2012
Summary
DSA in liver tpl. CDC-XM titer
FCXM (MCS, MFI ratio)
PRA (MFI of DSA by Single Ag assay)
Preop high level DSA MFI 5,000(?)
Class II, IgG3 subclass, C1q assay
Postop F/U – 2 weeks(?), LFT abnormality
면역학 검사의 해석장기이식시 면역학 검사의 종류HLA typingHLA Ab assayCDC-XM (crossmatch) Slide Number 6FCXM (Flowcytometric crossmatch)�PRA (Panel Reactive Antibody) Luminex technologyLuminex technology PRA (Panel Reactive Antibody) PRA - Screen – Luminex PRA - IDPRA - Single AgLuminex PRA MFI vs. FCXM(+)C1q assayC1q assay – clinical relevanceSlide Number 18Slide Number 19Slide Number 20Slide Number 21 Role of DSA in liver tpl.Slide Number 23Slide Number 24Slide Number 25Summary