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Saudi Journal of Kidney Diseases and Transplantation
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Year : 2003  |  Volume : 14  |  Issue : 1  |  Page : 39-42
Comparison of HLA Class I and II Molecular and Serological Typing within Clinical Laboratory


Hospital Charles Nicolle - Immunology, Internal Medicine, Tunis, Tunisia

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   Abstract 

In order to develop an adequate tissue typing strategy, we performed human leukocyte antigen (HLA)-A, B and DR generic typing on 235 (120 HLA-A, B and 115 HLA-DR) routine clinical samples by polymerase -chain reaction (PCR)-SSP in parallel with conventional serological typing. At the A locus, there were two (1.7%) discrepancies between molecular and serological typing besides 25 (20.8%) serological blanks, which was defined by molecular typing. At the B locus, there were two (1.7%) discrepancies and 30 serological blanks, which were defined by molecular typing. At the DR locus there were two (1.8%) discrepancies and 44 serological blanks, which were defined by molecular typing. We conclude that molecular typing is of substantial benefit in the resolution of poorly defined serological antigens. In view of the low percentage of discrepancy between the serological and molecular typing besides the high cost of molecular typing, our policy is to perform HLA typing first by the serological method and to use PCR-SSP as an adjuvant tool.

Keywords: HLA-typing, PCR-SSP, Serology.

How to cite this article:
Ayed K, Jendoubi S A, Makhlouf M, Sfar I, Abdallah T B. Comparison of HLA Class I and II Molecular and Serological Typing within Clinical Laboratory. Saudi J Kidney Dis Transpl 2003;14:39-42

How to cite this URL:
Ayed K, Jendoubi S A, Makhlouf M, Sfar I, Abdallah T B. Comparison of HLA Class I and II Molecular and Serological Typing within Clinical Laboratory. Saudi J Kidney Dis Transpl [serial online] 2003 [cited 2020 Feb 20];14:39-42. Available from: http://www.sjkdt.org/text.asp?2003/14/1/39/33086

   Introduction Top


Histocompatibility testing plays an important role in the selection of donors for renal transplantation. [1] Although histocompatibility testing can be performed using various assays, human leukocyte antigen (HLA) typing by serology is the most commonly used method in the routine clinical setting. A correct assignment of HLA antigens is considered important due to the fact that inadequate HLA matching of patient-donors pairs is associated with rejection of the transplanted kidney. [2]

Recent progress in assigning HLA-class I and class II alleles by techniques involving DNA analysis indicates that serological typing may not be sufficiently reliable. [3] The limitations in HLA class I and class II typing by serology include: first, most of the commercial trays for HLA typing are designed for specificities more frequently found in Caucasians while the trays for typing different ethnic groups are usually expensive and not widely available; [4] second, both the quality and availability of antisera reactive with certain HLA class I and class II alleles are often inadequate to perform reliable HLA-typing. [5],6

These limitations of the serological HLA typing have raised the question of replacing it by more efficient techniques.

In order to develop an HLA typing strategy, we compared the results of the serological HLA class I and class II typing with those obtained with sequence specific primers in the polymerase chain reaction (PCR-SSP).


   Material and Methods Top


To compare the serological typing with PCR-SSP, 235 kidney transplant patients were HLA class I and class II typed by standard microlymphocytotoxicity test using well-standardized alloantisera. Only 120 of them were tested by PCR-SSP class I and 115 were tested by PCR-SSP class II.

HLA Typing:

1. HLA class I and HLA class II serotyping: whole blood was collected on citrate phosphate dextrose (CPD) and lymphocytes were isolated by centrifugation on Ficoll-Hypaque. For serological typing, T and B cells were separated on nylon wool columns. Purified cells populations were counted and viability was determined by trypan blue dye exclusion (viability > 80%). The isolated cells were counted and adjusted at 3x10 6 cells/ml. Serological typing was performed on purified T and B lymphocyte suspensions using the standard NIH microlymphocytotoxicity technique and local set of sera.

2. Molecular typing of HLA class I and class II alleles.

  • Genomic DNA was isolated from prote­inase-k treated peripheral blood leukocytes by salting out method (Miller 1988).
  • Low resolution typing of HLA-A, B and DRB1 loci was performed by means of PCR-SSP method using a micro SSP-HLA A-B-DRB generic DNA typing tray (one Lambda, Canoga Park CA, USA). According to the manufacture's instructions, the PCR was performed by using the Gene Amp PCR system 9700 (Applied Biosystems, Foster City CA, USA). The PCR products were separated on 2% agarose gel containing ethidium bromide and visualized under ultraviolet (UV) light.


The comparison between the serological and molecular typing was accomplished by defining five groups of samples: A - homozygotes (antigen/x), B - concordance for both antigens (antigen/antigen), C - confirmation for one uncertain antigen (antigen/antigen?), D - discrepancy (antigen1 = antigen 2) and E - missed antigen/allele assignment by PCR-SSP.


   Results Top


[Table - 1] shows that molecular typing was concordant with serological HLA-class I and II typing in 77% of the samples typed for (HLA-A), 73% typed for (HLA-B) and 61% typed for (HLA-DR), (defined groups, A + B + C). Discrepancies were noted in (1.7%) of the cases (group D). The samples of group E, where the second antigen was not identified (serological Blanks), were definable by the molecular typing.

At the locus A, there were 25 serological blanks (20.8%) for which the HLA-A allele was defined in 8 as (A80), 8 as (A33) and 5 as (A74).

At the B locus there were 30 serological blanks for which the HLA-B allele was defined in 14 as (B15), 5 as (B35 association with B53) and 6 as (B38). At the DR locus there were 44 serological blanks for which a HLA-DRB1 allele was defined in 22 as (DRB1 13), 6 as (DRB1 14) 8 as (DRB1 11) 8 as (DRB12).


   Discussion Top


This study demonstrates that the difference in results of PCR-SSP and serology was mainly due to an increase in doubtful results by serology, not technical failures or missed antigens. By serology, high percen­tages in incorrect or inconclusive assignments were obtained in HLA, DR 13, DR14, DR11, and DR12 for HLA class II and A80, A33, A74 and B38 for HLA class I. These results clearly show that in correct antigen assignment, which occurred in about (25%) of the HLA class I and 40% of HLA class II serological typing were all resolved by molecular typing.

At present, the serological techniques and reagents can not detect all currently known HLA alleles, for this reason the serological typing is often followed by the molecular typing. The molecular typing identifies nucleotide polymorphisms, which code for the different allelic variants, whereas the serological typing shows the molecules that are actually expressed on the cell and involved in the immune rejection of the mismatched grafts.

However, the use of antibodies to detect HLA specificities can support the molecular techniques and solve ambiguities occurring between the expressed and null alleles. Therefore, serology may still be an important tool for donor selection in transplantation. In view of the cost of HLA-class I and class II molecular typing versus serology (appro­ximately double) our policy now is to type all individuals first by serology. If the patient is homozygous or has a blank antigen, further HLA class I and/or class II typing by the molecular techniques will be performed.

 
   References Top

1.Methilinos J. HLA testing: the state of the art of genomic methods in 1996. Nephrol Dial Transplant 1996;11:2129-34.  Back to cited text no. 1    
2.Opelz G, Methilinos J, Scherer S, et al. Survival of DNA-DR typed and matched cadaver kidney transplants. Lancet 1991;338:461-3.  Back to cited text no. 2    
3.Otten HG, Tilanus MG, et al. Serology versus PCR-SSP in typing for HLA-DR and DQ: a practical evaluation. Tissue Antigens 1995;45:36-40.  Back to cited text no. 3  [PUBMED]  
4.Bozon MV, Delgado JC, Selvakuman A, et al. Error rate for HLA-B antigen assignment by serology: implications for proficiency testing and utilization of DNA-based typing methods. Tissue Antigen 1997;50:387-94.  Back to cited text no. 4    
5.Schrender GM, Hurley CK, March SG, et al. The HLA Dictionary: a summary of HLA-A, B, C DRB1 13, 14, 15, DQB1 alleles and their association with serologically defined HLA-A-B-C DR and DQ antigens. Tissue Antigens 1999;54:409-37.  Back to cited text no. 5    

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Correspondence Address:
Khaled Ayed
Department of Immunology, Internal Medicine, Hospital Charles Nicolle, Tunis
Tunisia
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PMID: 17657088

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    Abstract
    Introduction
    Material and Methods
    Results
    Discussion
    References
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