HLA MHCMajor Histocompatibility Complex Highly polymorphic genes Recognition

HLA 검사 • MHC(Major Histocompatibility Complex) – Highly polymorphic genes – 항원인식 • Recognition by T lymphocytes • HLA(Human Leukocyte Antigen) – Human MHC – 면역성이 강해 타인에게 이식될 경우 강한 면역 반응 유도 – HLA 유전자 • Class I: HLA-A, -B, -C • Class II: HLA-DR, -DQ, -DP • Class III 2

HLA Nomenclature • • A 1, A 2, A 3, A 9, A 10, A 11. . . B 5, B 7, B 8, B 12. . . Cw 1, Cw 2 … DR 1, DR 2 … A*01: 01 B*51: 02 Cw*01: 02 DRB 1*04: 05

Example

HLA typing • Serological method – Microcytotoxicity • Cellular typing – Mixed Lymphocyte Culture (MLC) • Molecular Analysis – PCR-SSP – PCR-SSOP, Reverse SSOP – PCR-SBT – PCR-RFLP, PCR-RSCA, PCR-SSCP etc. 6

HLA typing – Serologic test • 검사원리 – 보체의존성 세포독성검사 (complement-dependent cytotoxicity) – microcytotoxicity test

HLA typing – Serologic test - 현미경 판독 위상차현미경 X 100배 시야에서 판정한다 죽은 세포의 %에 의해 1+-8+의 score를 기록한다. % Dead Cells 0 - 10% 11 - 20% 21 - 50% 51 - 80% 81 - 100% Score 1 2 4 6 8 Interpretation Negative Doubtful negative Weak positive Positive Strong positive

8 (strong positive, 81 -100%) 6 (positive, 51 -80%) 1 (negative, 1 -10%) 4 (weak positive, 21 -50%)

HLA typing – DNA test 1. SSO (sequence-specific oligonucleotide) typing 2. Reverse-SSO (sequence-specific oligonucleotide) 3. PCR-RFLP typing 4. PCR-SSCP (Single-Stranded Conformation Polymorphism) 5. Direct sequencing 6. PCR-SSP (sequence-specific primers)

Sequence-Specific Oligonucleotide Hybridization 12

SSO probe

Reverse SSO HLA DNA typing

SSP (sequence-specific priming) HLA-DR SSP typing • 24 SSP PCR reaction

HLA high resolution typing(SBT) • 각 locus specific PCR primer → polymorphism 갖는 부위의 DNA증폭 → sequencing primer 이용하여 sequencing • 가장 정확한 고해상도 검사방법 16

FCM HLA X-match • 검사원리 – Indirect cross-match technique • Direct XM: CDC – Indirect immunoflourescence • Donor cell과 patient's serum을 incubation하여 binding 단 계를 거친 후, • FITC-conjugated anti-human Ig. G를 첨가하여 target cell 에 반응된 patient's Ab를 detection • Flow cytometry로 그 정도를 측정하여 항체의 유, 무를 판정 • 낮은 농도의 Ab까지도 측정 가능

• Fluorochrome – Fluoresceinated (FITC) – Phycoerythrin (PE) – Perdinin chlorophyll protein (Per. CP) • CD 3 monoclonal with fluorochrome conjugates – Specifically detect T cell reactivity • CD 19 or CD 20 monoclonal with fluorochrome conjugates – Specifically detect B cell reactivity

면역기능 검사 1. Immune system Cellular immune system – pph lymphocyte Humoral immune system – serum 2. 검사 분류 1) 선별단계 : CBC/DC, lymphocyte subset, lymphocyte stimulation test, Ig 정량 2) 확진단계 : 재검, 병력 및 가족력 피부반응검사, NK cell 활성도 검사, cytokine 생성검사, MLC 연령에 따른 특이항체 존재유무, ABO 항체검사

세포면역기능의 검사법 - lymphocyte subset test 1) cluster of differentiation (CD) 2) 단클론성 항체를 이용한 면역형광법 : 형광현미경, FCM 3) CD 4/CD 8 ratio 림프구 분포 이용되는 단클론성 항체 T cell 50 -70 % CD 3, CD 2, CD 5 Helper T cell 30 -50 % CD 4 Cytotoxic T cell 20 -40 % CD 8 B cell 7 -25 % CD 19, CD 20 NK cell 8 -35 % CD 16, CD 56

Flow cytometer (FCM)

체액면역 검사법 1) protein - albumin 2) Ig 정량검사 ; Ig. G, Ig. M, Ig. A, Ig. E 3) serum protein electrophoresis & IFE