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