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Methods – In this study, serological HLA-A, -B and -C typing results were compared to typing results obtained using sequence-specific primers in the polymerase ...
Arch Iranian Med 2003; 6 (1): 23 – 28

ORIGINAL ARTICLE POLYMERASE CHAIN REACTION-SEQUENCE SPECIFIC PRIMER (PCR-SSP) VERSUS SEROLOGY IN TYPING OF HLA- A, -B AND -C IN IRANIAN PATIENTS •

Mehrnaz Narouei-Nejad MSc , Farideh Khosravi MSc, Abdolali Danesh MSc, Behrooz Nikbin PhD Immunogenetics Laboratory, Immunology Department, Medical School, Tehran University of Medical Sciences, Tehran, Iran Background – Histocompatibility locus antigen (HLA) class I typing by serology is the most common method in the routine clinical setting. With DNA sequence information available for alleles of the HLA system and the development of molecular biological techniques, it is possible to tissue type for allelic differences in HLA genes. Methods – In this study, serological HLA-A, -B and -C typing results were compared to typing results obtained using sequence-specific primers in the polymerase chain reaction (PCRSSP). HLA-A, -B and -C typing was performed on a random Iranian population consisting of 40 healthy individuals. Results – There were 16 blank antigens (antigen not detected) on serological testing, 9 in the A locus and 7 in the B locus. PCR-SSP allowed assignment of 2 of the 9 blanks in the A locus and 3 of the 7 blanks in the B locus. In this study, there was a 31% difference in the two typing methods. Conclusion – DNA typing is necessary, particularly in individuals with uncommon alleles and immunosuppression. Keywords

HLA typing

lymphocytotoxicity test

Introduction

H

istocompatibility testing plays an important role in selection of bone marrow and kidney donors for transplantation. Although histocompatibility testing can be performed using various assays, histocompatibility locus antigen (HLA) class I polymorphisms have traditionally been detected at the cell surface using serology with allo antisera or monoclonal antibodies. HLA typing by serology is the most commonly used method in routine clinical settings.1, 2 A correct assignment of HLA antigens is considered important given that inadequate HLA matching of patient-donor pairs is associated with rejection in kidney transplantation and rejection or graft-versus-host disease (GVHD) in bone marrow • Correspondence: M. Narouei-Nejad MSc, Immunogenetics Laboratory, Ali-Asghar Hospital, Zahedan, Iran. Fax: +98-5413232082, E-mail: [email protected].

PCR

transpla ntation. 3, 4 Several DNA-based typing methods have been applied to HLA-class II typing, such as restriction fragment length polymorphism,5,6 polymerase chain reaction followed by sequence-specific oligonucleotide probing,7, 8 direct sequencing,9 and PCR using sequence-specific primers (PCR-SSP).1, 10 – 13 Only recently sufficient numbers of DNA sequences have been available to enable similar development of DNA-based typing for HLA class I genes. Amplification of HLA loci with PCR-SSP has proved to be a rapid and accurate method for genotyping HLA-A, -B and -C alleles14–17 and indicates that HLA typing by serology may not be sufficiently reliable.18 – 20 Serology is a quick and convenient method of HLA class I detection, but it is hindered in many cases by serological cross reactivity and decrease in expression of HLA antigens, particularly in Archives of Iranian Medicine, Vol 6, No 1, January 2003

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PCR-SSP Versus Serology in HLA Typing in an Iranian Population

immunosuppressed patients or in patients with different hematological tumors. The present study extends the low-resolution system into a serologically equivalent system. The principle of PCR-SSP is that each individual allele (making up a serological specificity) is amplified by a primer pair exactly matched to that region. The low-resolution typing panel consists of a combination to produce 32 allele -specific reactions for HLA-A, 27 for HLA-B and 23 for HLA-C.

Patients and Methods DNA samples Forty randomly selected individuals were investigated in this study. DNA was prepared from anticoagulated whole blood using the modified salting out method (with sodium perchlorate).10, 11 These samples were serologically HLA-typed as the first step of this study. Amplification primers PCR primers used for the low-resolution panel included 82 primers prepared according to the sequence of the 12th International Histocompatibility Workshop (Table 1).21 All amplification primers were designed to give a melting point of between 55 C and 70 C, to ensure that all alleles would amplify under a standard set of conditions. In order to verify the success of each reaction, positive internal control primers amplifying a 256-bp fragment of exon 15 of the adenamoutous polyposis coli (APC) gene were included in each reaction mixture. A 14.5- ìL amplification reaction mixture consisted of 100 ng of genomic DNA, 10X buffer (67 mM Tris base pH 8.8, 16.6 mM ammonium sulfate, 0.01% [v/v] Tween 20), 2.5 mM magnesium chloride, 250 mM of each dNTP , 0.2–1 ìM allele -specific primers, 0.1–0.8 ìM positive control primers and 0.5 unit Taq DNA polymerase. PCR amplifications were carried out in a Hybaid thermal cycler (OmniGene, England). In total, 30 cycles were used, comprising 1 minute at 96 C followedby 5 cycles of 25 seconds at 96 C, 45 seconds at 70 C and 30 seconds at 72 C, then 21 cycles of 25 seconds at 96 C, 45 seconds at 65 C and 30 seconds at 72 C, followed by 4 cycles of 25 seconds at 96 C, 60 seconds at 55 C and 120 seconds at 72 C, with a final cycle of 10 minutes at 72 C. Gel electrophoresis PCR products were separated using electro24

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phoresis in 2.0% agarose gel containing 0.5 ìg/mL ethidium bromide. After the addition of 3 ìL loading buffer (0.25% bromophenol blue, 0.25% xylon cyanol and 15% ficoll), 10 ìL of each reaction mix was loaded onto the agarose gel. Minigels were run for 30 minutes at 10 v/cm in 1X TBE (89 mM Tris Base, 89 mM Boric acid, 2 mM EDTA, pH 8.0). PCR products were visualized under ultraviolet illumination. The HLA-A, -B and -C allele s low-resolution panel was designed to cover all serologically defined antigens in 82 separate alleles. Specific amplified fragments range in size between 180 and 1,062 bp, with a positive internal control product of 265 bp. Identification of alleles is based on the presence or absence of amplified product of the predicted size after agarose gel electrophoresis. A total of 40 DNA samples were typed using the low-resolution HLA-A, B, C typing panel.

Results All samples were typed successfully, irrespective of DNA extraction method. Where there were no specific bands, the presence of the positive internal control band verified the success of the PCR reaction, thus avoiding assignment of false negatives. In our study, we did not find complete concordance between DNA typing and serological typing. We used 32 primers for A, 27 for B and 23 for C loci. HLA-A, -B and -C typing by serology and PCR-SSP were compared at these loci (Table 2). We found 16 blank antigens (antigen not detected) with serology. Nine blank antigens belonged to the A locus and 7 to the B locus. In the A locus, we were able to assign different alleles and also subtypes by PCR-SSP. With this technique, there were fewer cross reactions than in the serological method. There were very few cross reactions in A11, A3, A2 and A28 alleles by PCR-SSP, but there were some cross reactions in alleles such as A1, A11 and Aw19 splits. We found an extra band in some A2 alleles. Of the 9 blank antigens in the A locus on serological typing, 2 were assigned on PCR-SSP (Table 3). Due to extensive polymorphism in the B locus, we had more difficulty typing B alleles. Some samples produced more than 2 specific bands. This was because some alleles were amplified by more than one primer combination. Extra reactions were found for B14 alleles and less frequently for B40,

M. Narouei-Nejad, F. Khosravi, A. Danesh, et al

Table 1. List of class-I HLA typing anti-sera used in serological typing. HLA-A

HLA-B

HLA-C

A*01 (0l, 02)

B*27 (02, 03, 05, 06, 07, 09)

Cw*01 (01, 02)

A*3601

B*2708, 7301

Cw*02 (01, 02)

A*0202

B*40011, 40012

Cw*04 (01, 02)

A*0203

B*4007

Cw*0501

A*02 (12, 13)

B *4003

Cw*0602

A*0207

B*4701

Cw*07 (01, 02, 03)

A*0214

B*40 (02, 04, 05, 06)

Cw*08 (01, 02, 03)

A*02(0508)

B*41 (01, 02)

Cw*0302

A*02 (01, 0406, 09, 10, 11)

B*13 (01, 02)

Cw*0303

A*03(01 ,02)

B*44 (02, 03, 04)

Cw*0304

A*2301

B*4501

Cw*12 (01, 02)

A*24(02,03)

B*1514

Cw*1203

A*2501

B*4901

Cw*1 301

A*26 (01, 02, 04)

B*5001

Cw*14 (01, 02, 03)

A*2603

B*5401

Cw*15 (01, 02, 03, 05)

A*4301

B*5901

Cw*1504

A*3402

B*55 (01, 02), 56(01, 02)

Cw*1601

A*3401

B*38 (01, 02)

Cw*1602

A*66 (01, 02)

B*39 (011, 013, 121, 3, 03, 05, 06, 07)

Cw*0704

A*11 (01, 02)

B*6701

Cw*1701

A*6801

B*14 (01, 02)

A*6802

B*07 (02, 03, 04, 05)

A*6901

B*0802

A*29 (01, 02)

B*0801

A*30 (01, 02, 03)

B*16 (01, 02)

A*3101

B*3701

A*3201

B*5301

A*33 (01, 02)

B*35 (01, 02, 03, 04, 05, 06, 07, 08)

A*7401

B*51 (01, 02, 03, 04, 05)

A*8001

B*52 (11, 12) B*7801 B*57 (01, 02) B*5801 B*15 (01, 04, 05, 06, 07, 12, 19, 20) B*1503, 4802 B*1521 B*15 (13, 16, 17) B*15 (02, 08, 11, 15), 4601 B*4201 B*4801 B*15 (09, 10, 18) Archives of Iranian Medicine, Vol 6, No 1, January 2003

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B7 and B54 alleles and cross reactions between B18 and B15. PCR-SSP was able to assign 3 of the 7 blank antigens on serologic al typing (Table 3). Thus, we found a significant difference between these 2 techniques (31% of antigens that were not detected by serological typing were identified by the DNA technique).

The results for the C locus were disappointing because the many extra reactions made it impossible to assign even one allele correctly.

Discussion Traditionally, serological methods for the and II

Table 2. Results of serological and PCR-SSP typing. Serological

PCR-SSP

A2, A24, B35, B38, Bw4, Bw6, Cw4

A2, A24, B35, B16, Bw4, Bw6

Al, A29, B39, Bw63, Bw4, Bw6, Cw7

Al, A29, B16, B15, Bw4, Bw6

A11.1, —, B35, Bw47, Bw6, Cw4

All, —, B35, B4701, Bw6

A2, A26, B27, B49, Bw4, Cw2, Cw7

A2, A26, B27, B4901, Bw4

A2, A11.l, B35, Bw50, Bw6, Cw6

A2, All, B35, B5001, Bw6

Al, A3, B35, B7, Bw6, Cw4, Cw7

Al, A3, B35, B7, Bw6

Al, A24, Bw57, B27, Bw4, Cw6

Al, A24, B57, B27, Bw4

A2, A3, B51, B27, Bw4, Cw61

A2, A3, B5, B27, Bw4

Al, A2, Bw41, B51, Bw4, Bw6

Al, A2, B4101, B5, Bw4, Bw6

A3, A24, B8, B51, Bw4, Bw6, Cw2

A3, A24, B8, B5, Bw4, Bw6

A68, A24, B44, B49, Bw4, Cw7

A68, A24, B44, B4901, Bw4

A1l.1, A26, B27, B35, Bw6, Cw2, Cw4

All, A26, B27, B35, Bw6

A29, —, B14, B44, Bw4, Bw6

A29, —, B14, B44, Bw4, Bw6

Al, A23, B49, B55, Bw4 , Bw6, Cwl

Al, A23, B4901, B22, Bw4, Bw6

A11.1, A24, B44, B55, Bw4, Bw6, Cw4, Cw9

All, A24, B44, B22, Bw4 ,Bw6

A2, —, B5(35), B36, Bw4 , Cw4, Cwl0

A2, —, B5, B16, Bw4

Al, A24, B6, Bw63, Bw4 , Bw6

Al, A24, B6, B15, Bw4, Bw6

A28, A29, B14, Bw60, Bw6 , Cwl0

A69, A29, B14, B4001, Bw6

Al, A33, Bl4, —, Bw6

Al, A33, B14, —, Bw6

A24, Awl9, B14, B40, Bw4

A24, A30, Bl4, B40, Bw4

A2, A26, B5, B22

A2, A26, B5, B22

A2, —, B15, B55(?B), Bw6, Cw1

A2, —, B15, B22, Bw6

All, A24, B35, —, Cw4

A11, A24, B35, —

Al1, —, B39, B44, Bw4, Bw6

All, —, B16, B12, Bw4, Bw6

A26, —, B35, —, Bw6, Cw4

A26, —, B35, —, Bw6

Al, A32, B21, B51, Cw3, Cw4

Al, A3201, B21, B5

Al, All.1, B35, Bw55, Bw6, Cw4, Cw9

Al, All ,B35, B22, Bw6

Al, A30, B13, B14, Bw4, Bw6, Cw6, Cw7

Al, A30, B13, B14, Bw4, Bw6

A3, A26, B51, —, Bw4, Bw6, Cw6

A3, A68, B5,—, Bw4, Bw6

A2, A3, B35, Bw41, Bw6, Cw4

A2, A3, B35, B4101, Bw6

Al, A2, B5, Bw41, Bw4, Bw6, Cw4

Al, A2, B5, B4101, Bw4, Bw6

A2, A30, B13, Bw5O, Bw4, Bw6, Cw6

A2, A30, B13, B21, Bw4, Bw6

A2, A24, B51,B35, Bw4, Bw6, Cw4

A2, A24, B5, B35, Bw4, Bw6

A26, A28, B5, B27, Bw4, Cw2

A26, A68, B5, B27, BW4

A24, A28, B38, B49, Bw4, Cw7

A24, A68 , B16, B4901, Bw4

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M. Narouei-Nejad, F. Khosravi, A. Danesh, et al

Table 3. Differences between the results of serological and PCR-SSP typing. Serology

PCR

A23, A26, B7, —

A23, A26, B7, Bl8

A26, A30, B35, —

A26.A30, B35, B16

A26, A24, B18, —

A26, A24, B18, B48

A3, —, B8, B51 A28, —, B16, B51

A3, A28 ,B8, B51 A28, A24, B16, B51

detection of HLA polymorphism of both class I gene products have been the mainstay of tissue typing. 2, 22 For class-I, biochemical methods such as one-dimensional isoelectric focusing23 were developed and although it was possible using this test to discriminate many more antigenic variants than by serological assays, it was never a realistic routine typing tool. However, the advent of DNA sequencing revealed the existence of new class I allele sequences that could not be identified by either serological or biochemical techniques.17 In this study, the results of HLA-A, -B and -C typing using serology in 40 normal individuals were compared to the results of typing with PCRSSP. In serological HLA-typing, we used antisera from the third Asia -Oceania Histocompatibility Workshop. 22 This large antisera panel consists of 23 different alleles for the A locus, 49 for the B locus and 8 for the C locus.21 In the PCR-SSP low-resolution method, we used 32 different primer pairs for the A locus, 27 for the B locus and 23 for the C locus. In spite of using a very large panel of antisera in the serological method, there were at least 16 blank or undefined antigens (9 in the A locus and 7 in the B locus). The PCR-SSP low-resolution method allowed identification of 2 blanks in the A locus and 3 blanks in the B locus; hence, 31% of blanks were identified by PCR-SSP. 5, 17 The resolution of our HLA-A PCR-SSP method was largely unaffected by cross-reactivity and we were able to obtain correct and exact results in this locus. In the B locus, there were more problems with cross-reactions, extra reactions and the presence of mixed primers. With our primers, it was impossible in some alleles (such as HLA-B15) to determine the exact allele. Thus, the highresolution method is recommended to define expected alleles. In this study, we were not able to determine the HLA-C alleles. We need more experience in designing and synthesizing HLA-C locus primers.

From the results of this and other studies,1,13,17,19 it can be concluded that, unlike serology, PCR-SSP is a reliable technique for HLA-typing in patients as well as in healthy individuals. In different situations, such as aplastic anemia and leukemia, when the expression of HLA antigens on the cell surface is down-regulated, it is impossible to type by serological methods and it is advisable to use molecular typing such as PCR-SSP. In addition, the PCR-SSP technique is fast and easy to perform and it is easy to handle specimens, because the viable cells necessary for serological typing are not needed. 1,13,18 PCR-SSP allowed determination of the subtypes of HLA antigens very clearly. We now use PCR-SSP in parallel with serological typing, but it can be envisaged that in the near future HLA-typing by serology will be replaced by PCR-SSP in routine clinical practice.

Acknowledgment We wish to express our thanks to Dr. Ali-Akbar Amir-Zargar for his expert assistance.

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