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HLA 检测方法

上海交通大学医学院

彭奕冰 主任技师

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Major Histocompatibility Complex(MHC)

Major Histocompatibility Complex(MHC)

What is MHC?– HLA– H-2– Minor histocompatibility antigens

What is MHC?– HLA– H-2– Minor histocompatibility antigens

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Significance of the MHCSignificance of the MHCSignificance of the MHCSignificance of the MHC

role in immune responserole in organ transplantationrole in predisposition to disease

role in immune responserole in organ transplantationrole in predisposition to disease

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Genetic barriers to transplantationGenetic barriers to transplantation

• autologous: in the same individual (autograft)

• isologous: between genetically Identical individuals (isograft), i.e., identical twins (inbred animals)

• homologous: between individuals of the same species (allograft)

• heterologous: between individuals different species (xenograft)

• autologous: in the same individual (autograft)

• isologous: between genetically Identical individuals (isograft), i.e., identical twins (inbred animals)

• homologous: between individuals of the same species (allograft)

• heterologous: between individuals different species (xenograft)

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Principles of transplantationPrinciples of transplantation

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Minor histocompatibility antigensand graft survival

Minor histocompatibility antigensand graft survival

minor histocompatibility antigens also cause rejection

The rejection time is variable but longer than that for major histocompatibility antigen

They have additive effects

minor histocompatibility antigens also cause rejection

The rejection time is variable but longer than that for major histocompatibility antigen

They have additive effects

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Graft versus host (GVH) diseaseGraft versus host (GVH) disease

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GVH disease in humansGVH disease in humans

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The human MHC genesThe human MHC genes

class II c lass ID P D Q D R B C A

B C A

D P D Q D R

1 2 2 2 1 1 9 3 1 2 2 1 3

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The mouse MHC genesThe mouse MHC genes

I

DK

A E

class II c lass Iclass I class III(C4, factor-B ,TNF, etc.)

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Polymorphism of MHC antigens (based on phenotype)

Polymorphism of MHC antigens (based on phenotype)

HLA 基因系统的多态性及一些主要基因座位的等位基因数正式命名显示多态性的 HLA 基因座位有 31 个，共 2 320 个等位基因。 图中所示为常见基因座位的等位基因数。

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The inheritance of MHC genesThe inheritance of MHC genes

Jack Jill

D P

D Q

D R

B

C

A

1 2 3 4

Bo Kim M o Lee

1 3 41 2 3 42

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Crossing overresults in new haplotypes

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MHC products expressed on cellsMHC products expressed on cells

If Jack and Jill have four children; Bo, Kim, Mo and Lee

They’ll all inherit antigens of the parental MHC

Oft their haplotypes will be of the father or mother

Unless during meiosis, a crossover should occur

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Class-I expressed on all nucleated cells in man, and also on erythrocytes in mice.

Class-II expressed primarily on antigen presenting cells (dendritic cells, macrophages and B cells, etc.)

Differential expression of MHC antigens

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Alloreactivity of T cells:MLR and CTL generationAlloreactivity of T cells:

MLR and CTL generation

CD4+TH1

CD8+CTL

CD8+preCTL

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Alloreactivity of T cellsAlloreactivity of T cells

T CELLT CELL

DRW 11DRW 17

PositiveSelection

ThymusAlloreaction

(MLR)

Proliferation and

Differentiation

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Inflammation

lysis

ADCC

lysis

IL2, IFN

TNF, NO2

IL2, IL4, IL5

IL2, TNF, IFN

rejection

Mechanisms of graft rejectionMechanisms of graft rejection

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Tempo of rejection reactionTempo of rejection reaction

type of rejection

time taken cause

chronic months-years unclear causes: cross reactive Ab, immune complexes, slow cellular reaction, tolerance breakdown, disease recurrence

accelerated days reactivation of sensitized T cells (secondary response)

acute days-weeks

primary activation of T cells

hyperacute minutes- hours preformed anti-donor antibodies and complement

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A, B & DR matching and graft survivalA, B & DR matching and graft survival

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Removal of T cells from marrow graftRemoval of T cells from marrow graft

Magnet

Magnetic antibodies

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HLA and disease associationHLA and disease association

Disease Associated alleles Frequency in

Relative riskpatients control

Ankylsoing spondylitis

Reiter’s disease

Acute anterior uveitis

Psoriasis vulgaris

Dermatitis herpetiformis

B27

B27

B27

CW6

DR3

9

79

52

87

85

9

9

9

33

26

87.4

37.0

10.4

13.3

15.4

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HLA 抗原检测法

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微量细胞毒试验

原理　　　当特异性抗体与淋巴细胞表面 HLA 分

子结合，激活补体，使细胞溶解。在显微镜下可见细胞被活性染料着色。

　　　Ｔ淋巴细胞只表达 HLA I 分子，如欲测定 HLA II 类分子，需用Ｂ细胞。

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微量细胞毒试验 HLA I 类分型法

方法1. 加板： Terasaki 板中加入特异性抗 HLA I 类分

子抗体。2. 分离 PBMC 。制成浓度为 1106/ml 的细胞悬液。3. 每孔内加入 1l 细胞悬液。4. 室温培育 30min 。5. 1 l 兔补体。室温培育 60min 。6. 每孔内加入 5l 5% 伊红水溶液。室温 5min 。7. 每孔内加 37% 甲醛固定。8. 倒置相差显微镜下观察结果。

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微量细胞毒试验 HLA II 类分型法

与 HLA Ｉ类分型法区别1 ．须用富含Ｂ细胞的淋巴细胞悬液。2 ．抗体与细胞培育时间为 1h 。

3 ．加补体后培育时间为 2h 。

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微量细胞毒试验

应用实践 个体 HLA 分型可用于移植前供者和受者

配型，亲子鉴定，法医鉴定等。

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交叉配型

用于测定受者血清中受含有抗供者 HLA

的抗体。如受者血清中具有抗供者红细胞血型抗原抗体和 / 或抗白细胞抗原抗体，会引起超急排异反应。所以移植前应作红细胞血型测定，以及测定患者血清中是否具有抗供者 HLA 抗体。

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交叉配型方法

1. 分离供体 PBMC 。

2. 分离受者血清，作 1 ： 2 ， 1 ： 4 和 1 ： 8 稀释。加入 Terasaki 板，每孔 1l 。

其余步骤同微量细胞毒试验。

如结果为阳性，表明受者血清中具有抗供者 HLA

抗体。

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细胞学分型法

细胞学分型法已被血清学和分子生物学方法代替。单相混合淋巴细胞培养实验还在使用。

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分子生物学技术为基础的 HLA 分型法

原理

HLA 的多态性是由基因中核苷酸序列突变引起的。同一基因的不同等位基因之间核苷酸序列大多是相同的，变异集中在几个 DNA 片段中。在这些片断中，不同的等位基因具有特定的序列。测定这些片段的 DNA 序列就可以确定等位基因。

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变异片段 变异片段 变异片段

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a b c dac Aac ATCGGTCGGTCAAGGTCAAGGCCTATCCCTATCGATTGCGTAGGCGATTGCGTAGGC

bd ATCGGTbd ATCGGTCAAGCAAGGCCTATCGGCCTATCGATTGATTGCGTAGGCCGTAGGC

ac Aac ATCGGTCGGTCAAGGTCAAGGCCTATCCCTATCGATTGCGTAGGCGATTGCGTAGGC

ad Aad ATCGGTCGGTCAAGGCCTATCGTCAAGGCCTATCGATTGATTGCGTAGGCCGTAGGC

bc bc ATCGGTATCGGTCAAGCAAGGGCCTATCCCTATCGATTGCGTAGGCGATTGCGTAGGC

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根据片段组合指定等位基因

acac

acac

bcbc

ad ad

bdbd

acacefef

ghgh

efef

mlml

jkjk

oxox

rtrt

wqwq

svsv

zpzp