1. Major Histocompatibility Complex
Dept. of Lab Med
Eun Young Song

2. Contents Sturcutre and function of HLA molecule
HLA inheritance and polymorphism
HLA nomenclature
HLA tests

3. HLA (Human Leukocyte Antigen)
HLA - structrue and function
Class I molecule (HLA-A, B, C)
Most nucleated cells
Ag presentation to cytotoxic T-lymphocyte (CD8+)
rejection, kill virus-infected cells
Class II molecule (HLA-DR, DQ, DP)
B lymphocyte, activated T lymphocyte, monocyte, macrophage, dendritic cells…
Ag presentation to (helper) T-lymphocyte(CD4+)

4. HLA class I molecule cleft by α1 and α2 : Ag-binding pocket
α3 domain : interact with CD8 on T cells

5. HLA class II molecule
α2,  2: interact with CD4 on T cells

6. HLA genes
6p21.3

7. HLA polymorphism More than two kinds of molecules from same gene locus
Several tens ~ hundreds of alleles
Cause graft rejection due to variable HLA molecules in each persons         

8. HLA class I and class II polymorphismB C
DRB1
DQB1
DPB1
Allele No. (Dec. 2009)
(Mar, 2012)
(Jul, 2014)
965
1,757
2,884
1,543
2,338
3,589
626
1,304
2,375
762
1,166
1,540
107
162
664
138
152
422
Antigen No. 26 53 10 18 9 6
* HLA polymorphism :
class I : B > A > C
class II : DR > DQ, DP

9. The probability of agreement of HLA with one’s sibling : 1/4
HLA inheritance
HLA genes are located very closely each other and inherited as a haplotype.
Father
Mother A2
B44
DR13
A11
B54
DR9 A3
B27
DR8
A24
B40
DR7 
Child A2
B44
DR13 A3
B27
DR8
The probability of agreement
of HLA with one’s sibling : 1/4

10. HLA nomenclature (by WHO committee)
HLA Ag
ex) HLA-A2, B7, Cw1, DR4
HLA allele
ex) HLA-A*02:01, B*07:02, C*01:02, DRB1*04:05

11. subtypic or private specificity
HLA Ag nomenclature
named after the order in which the serologic specificty was defiend in International histocompatibility workshop.
subtypic or private specificity
subdivided Ag, when the original Ag was split and named
supertypic specificity
Original Ag before split
(e.g., B12 → B44, B45). B44 Ag-positive lymphocyte shows positive reaction also with B12 anti-sera.

12. Split antigens

13. HLA allele nomenclature
HLA-A*0201, Cw*0102, DRB1*0405 <2004 nomenclature>
HLA-A*01:01:01:02N <2010 nomenclature>
First region
agreement with serologic specificity
e.g.) A*0201 shows A2 serologic specificity
Disagreement with serologic specificity
when named after nucleotide sequence similarity
e.g.) B*15:18 didn’t show serologic B15 specificity, but shows B71 specificity.
Second region
The order in which alleles were defined.
A*02:01 was defined first, A*02:03 was defined third.
68년 WHO nomenclature committee구성
87년 처음 DNA nomenclature 도입
serologic 정보없이 allele 급증하면서 96년부터는 첫 두자리는 단순히 염기서열의 유사성
2010년 4월

14. HLA allele nomenclature
Third region
Silent or synonymous substitution.
Amino acids are same, though nucleotide sequence of coding region was different.
e.g.) B*27:05:02, B*27:05:03
Fourth region
nucleotide sequence of noncoding region was different.
DRB4*01:03:01:01, DRB4*01:03:01:02
Suffix, N, L, S, Q
N (null): No protein expression
L (low): Very low protein expression

15. 2010 HLA nomenclature
For A*02 and B*15 alleles, ‘92’ and ‘95’ had been used because the number of alleles were over 99.
Three digits are used in 2010 nomenclature.

16. The HLA Dictionary 2008 Tissue Antigens 2009:73:95-170
1997, 1999, 2001, and 2004
(5th update)

17. HLA typing Serologic test Molecular test Cellular detection method
Microlymphocytotoxicity Test
Molecular test
PCR-SSOP, Reverse SSOP
PCR-SSP
PCR-SBT
Cellular detection method
Mixed Lymphocyte Culture (MLC)
The polymorphism of the HLA molecules is determined by serological test using specific antibodies, cellular method such MLC and molecular analyses such as PCR-SSP (Sequence Specific Primers), PCR-SSOP (Sequence Specific Oligonucleotide Probes) and PCR-SBT (Sequence Base Typing). I am going to tell you more detail about PCR-SSOP.

18. Microlymphocytotoxicity test
Principle: complement-dependent cytotoxicity, CDC
Methods: microplate with various antisera (eg. anti-HLA-A2 Ab) +
lymphocytes of patients (HLA Ag) → adds rabbit complement → penetration of Eosin Y due to cell lysis → reading with inverted phase contrast microscopy .
+ complement
+ Cell lysis
Penetration of Eosin Y

19. Viable cells ≥ 90% Viable cells ≤ 10%

21. ASHI reading standard

23. HLA molecular test PCR-SSOP Sequence Specific Oligonucleotide Probes
PCR-SSP
Sequence Specific Primers
PCR-SBT
Sequence Based Typing

24. Classification of resolution of HLA tests
Low (low resolution)
serologic level (serologic/generic)
Intermediate
several allele groups (DRB1*0401/0421/0433/0434/0435/0438g)
High (high resolution)
allele level (allelic)

25. HLA molecular test Class I Class II Exon 2 Exon 3 Exon 1 Exon 4
Intron 1
Intron 2
Intron 3
Typing region
Class II
Exon 2
Exon 3
Exon 1
Exon 4
Intron 1
Intron 2
Intron 3
Typing region

26. Reverse SSOP reverse SSO – line blot SSO probes are attached on strip.
biotinylated primer are used in PCR.
Hybridization with probes on strip -> visualization -> reading
Kits used in Korea
Murex INNO-LIPA, Dynal RELI
reverse SSO – Luminex
SSO probes are attached on colored bead
Hybridization with Streptavidin-PE-labed PCR products
e.g.) LIFECODES HLA DNA (Tepnel Lifecodes)

27. Reverse-SSOP, line blot

28. Luminex corp. xMAP system

29. SSP (sequence specific primer)
PCR with sequence specific primers, typing using the existence of PCR products
The main kits in Korea Biotest, One Lambda, Biosewoom etc.,
B*44/B*55

30. SBT (sequence-based typing)
high resolution test
class I (HLA-A, -B, -C): exon 2, 3, (4) class II (HLA-DRB1): exon 2
The main kits in Korea Abott AlleleSEQR SBT kit, Biosewoom, In-house etc.,

32. Ambiguity of HLA typing
low resolution vs high resolution
- low resolution: DRB1* vs
- high resolution: B* vs
Heterozygote vs Allelic
- Heterozygote ambiguity: allele 1+2 = 3+4
A* = =
- Allelic ambiguity: allele 1(=2) + 3
A*0201/04/70, *2402/29/52
* Heterozygote ambiguity > Allelic amibiguity

33. Heterozygote ambiguity
International Histocompatibility Working Group (

34. HLA tests using cellular detection
MLC (mixed lymphocyte culture)
In vitro measure of class II disparity (mainly the response against HLA-DR molecule)
Replaced with molecular tests due to difficulties in standardization

35. Compatibility tests in organ transplantation
(1) ABO typing
(2) HLA-A, B, DR
(3) HLA crossmatch
(4) PRA
(5) MLC
solid organ : ABO typing, HLA crossmatch – critical
kidney, pancreas – consider HLA match
bone marrow (HSC) : HLA matching – critical
ABDR full match (1 antigen mismatch)
related : serologic (≒allele) level match
unrelated : serologic (≠allele) level match