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Aim: The use of UNOS calculated panel reactive antibodies (cPRA) calculator to calculate sensitization for Saudi patients may not be relevant due to different donor pools. In this study, we compared HLA frequency of deceased donors at King Fahad Specialist Hospital-Dammam (KFSH-D) with UNOS frequency to evaluate the applicability of implementing the UNOS cPRA temporarily among Saudi patients. Methods: Medium resolution HLA class I and II genotypes were obtained from 148 deceased donors. Separate allele frequency was calculated for KFSH-D deceased donors belonging to West Asia, East Asia, South Asia and Africans. Phylip (v5) was used to calculate Nei’s genetic distance and generate Neighbor-joining (NJ) trees. Results: Majority of deceased donors at KFSH-D were of Asian descent.[figure1]In comparison to the antigen frequency in UNOS donors, some antigens were more frequent (eg, A24, B7, B8, B13, B27, B35, B44, DR10 and DQ5) while others were less frequent (eg, A3, DR1, DR4, DR7, DR13 and DR15). Genetically, South Asians and East Asians were closure to UNOS Asian donors while West Asians were closure to Caucasians.[figure2] Conclusions: Deceased donors analyzed at KFSHD have genetic similarity with Caucasian and Asian donors of UNOS. Adequate method for measuring the level of HLA sensitization based on national Saudi deceased donors HLA frequency is needed to establish a Saudi Arabian cPRA. [Copyright &y& Elsevier]

Aim: High-resolution HLA typing plays a central role in medical decisions concerning the matching of stem cell transplant recipients to unrelated donors. However, DNA sequencing based typing (SBT) may result with three or more allele combination. We undertook this study to validate whether luminex based PCR-SSO method can resolve these SBT ambiguities. Methods: HLA medium and high resolution genotyping was performed using Luminex-based sequence-specific oligonucleotide (SSO) (Thermofiser, LabtypeSSO) and Sequencing based typing (Atria Genetics HLA SBT kits) respectively. Fluorochrome-labeled DNA fragments were electrophoresed by capillary electrophoresis on an ABI 3130 Genetic Analyzer. Heterozygous Ambiguity Resolving Primers (HARPs) were used as the gold standard method to resolve ambiguity. Results: A total of 64 DNA samples typed by SSO and SBT (15 A, 14 B, 19 C and 16 DRB1) showed HLA assignments that have more than four possible antigen/allele combinations. SSO based typing resolved 57% of ambiguities for HLA-A; 51% for HLA-B, 60% for HLA-C and 65% for HLA-DRB1 locus. On the contrary, HARPS resolved 94% of ambiguities for HLA-A and -B, 90% for HLA-C and 83% for HLA-DRB1. Overall, SSO significantly resolve ambiguities for Class I (p<0.0001, 95% CI=1.99 - 4.38) and Class II (p<0.0001 95% CI=2.08-4.78). HARPs analysis confirmed the SBT ambiguity resolution by SSO. Conclusions: Significant ambiguity resolution of SBT results can be achieved by SSO, which would significantly reduce the need of additional amplifications and financial cost involved in HARP analysis. Therefore, we recommend the combination of SSO and SBT for the generation of high resolution HLA typing and advocate the use of HARPS only in cases of unresolved HLA typing by both SSO and SBT. Also, it is important that SSO probe reaction patterns be reviewed thoroughly to assure the right allele combinations. [Copyright &y& Elsevier]

Aim: The Human Leukocyte Antigen-DQB1∗03:19 allele was identified by SSO and sequencing-based typing in prospective solid organ and bone marrow transplant recipients and donors in Kingdom of Saudi Arabia. HLA-DQB1∗03:19 is identical to DQB1∗03:01except for a single nucleotide substitution at position 554 (C/T) in Exon 2, which results in threonine to isoleucine amino acid change. Methods: Medium resolution genotyping for HLA-DRB1 and -DQB1 was performed by Luminex-based sequence-specific oligonucleotide (SSO) method. Sequencing based typing (SBT) was performed for high resolution HLA-DQB1 genotyping by Atria Genetics HLA SBT kits. The fluorochrome-labeled DNA fragments were electrophorased by capillary electrophoresis on an ABI 3130 Genetic Analyzer. The sequence analysis was performed using ASSIGN 3.6 software (Conexio Genomics). Results: HLA-DQB1∗03:19 allele was observed frequently in Saudi Arabian deceased donors. Majority of HLA-DQB1∗03:19 positive samples (∼72%) also carried DR11 allele. Based on the known HLA linkage disequilibrium, our data suggest that the haplotype DR11-DQB1∗03:19 is frequent in Saudi Arabian populations. Conclusions: Our data suggests that DQB1∗03:19 was frequently observed in Saudi Arabian deceased donors and majority of time it occurs as DR11-DQB1∗03:19 haplotype. [Copyright &y& Elsevier]

Aim: HLA-B∗15 is the largest HLA B allele group with more than 200 different alleles that corresponds to various serological equivalents including B62, −63, −70, −75, −76 and −77. However, not all the B∗15 group alleles have been tested and characterized by serological methods and thus serological equivalents and cross reactivity has not been determined for many HLA-B∗15 alleles. One such allele of this group is B∗15:151 which was first reported in 2008 in French population and later on in Brazilian population. Here, we report the serological equivalent and cross-reactive antigens for HLA-B∗15:151 allele. Methods: One of the DNA samples tested by medium resolution SSO and high resolution SBT in our laboratory was genotyped as HLA-B∗15:151. A blood sample then was obtained and tested by Microcytotoxicity typing method. IMGT database alignment for both nucleotide and amino acid sequences was done for HLA-B∗15:151, B∗15:03, B∗41 and B∗42 alleles. Results: HLA-B genotyping results by microcytotoxicity method was in concordance with molecular typing. However, cells were found reacting strongly with B70 sera and weaker reactions were also observed with B41 and B42 sera. Nucleotide and amino acid alignment of B∗15:151 to B∗15:03:01 showed great similarity between B∗15:03:01 and B∗15:151 in exon 2 and exon 3 with only single mismatch at codon 152. This mismatch was an adenine in allele B∗15:03 and a Thymine in B∗15:151 resulting in a change of amino acid Glutamic acid to Valine respectively. Further, alignment with, B∗41 and B∗42 alleles showed the same mismatch being shared with all B∗41 and B∗42 alleles. Conclusions: Allele HLA-B∗15:151 is most likely a B70 antigen which shares a cross reactive epitope with B41 and B42 antigens. [Copyright &y& Elsevier]

Definition of sensitization to class II antigens based on its β chain may be problematic for DQ due to the polymorphism of both α & β chains in DQ antigens and can wrongly assigned DSA that may decline immunologically compatible transplant. The patient presented here highlights this problem A 23-year-old male referred for kidney transplantation. HLA-typing for him and three living related potential donors are summarized below. A B C DRB1 DQA1 DQB1 Pt 02, 33 49, 51 07, 15 17, 16 05:01, 01:02 02:01, 05:02 D1 11, 68 35, 51 04, 15 04, 11 03:02, 05:05 8, 7 D2 01, 03 51 06, 16 04 03:01 8 D3 01, 31 35, 51 06, 07 17, 04 03:01, 05:01 8, 02:01 HLA-antibodies by SAB were positive for DQ7–9. Flow XM with donor 1&2 (D1, D2) were positive for B-cells but negative with donor 3 (D3). A closer analysis of SAB data (Fig. 1) revealed positive reactivity of all DQ8 coated beads except bead no. 82. DQA1 ∗ 03:02 was thought then contributing to the positivity. High resolution typing (HR) for DQ8 confirmed sharing of DQA1 ∗ 03:02, DQB1 ∗ 03:02 between D1&2. Reactivity analysis for bead no. 42 and 43 could not exclude positivity to DQ8 β (B ∗ 03:02) component due to negative reaction of the corresponding α components. Furthermore, the combination DQA ∗ 03:01, DQB ∗ 03:02 coated beads (present in D3) reacted strongly. Flow PRA class II bead on BD analyzer, showed positive DQA1 ∗ 03:02 homozygous bead only. DQA1 ∗ 03:02 homozygous present in D2 & a single copy of this allele present in D1, while D3 does not carry this allele, so we concluded that actual antigenic epitopes is located in DQA1 ∗ 03:02, possibly the homozygous conformation increased its expression & contributed to the higher channel shift of the FXM with D2. Due to uncertainty in assessing the immunological risk the transplant was denied several times before the patient underwent uneventual transplant from D3 and continued enjoying good kidney function up to one month post transplant. [ABSTRACT FROM AUTHOR]