Performance of a microarray-based genotyping system for red cell and platelet antigens in China.

Dear Sir, Accuracy of pre-transfusion testing for the identification of antigen profiles is crucial for the safety of patients. In their commitment to reduce the risks associated with transfusion1, blood banks and hospitals are progressively adopting genotyping-based molecular approaches to complement or even replace immunohaematological techniques2–4. It is, therefore, critical that blood genotyping arrays account for clinical relevant polymorphic variations present in every racial and ethnic population. Several recent studies performed with the BLOODchip® Reference (Progenika, a Grifols company, Bilbao, Spain) and other molecular techniques have explored the performance of this genotyping tool to identify allele variants and frequencies in patients and donors of different populations. In this study, the BLOODchip® Reference genotyping system was used for the first time in China to analyse the molecular background of red blood cell (RBC) and platelet antigens (HPA) of blood donors at the Shanghai Blood Center. Specimens of 403 whole blood samples obtained from random, routine blood donors and voluntary employees of the Shanghai Blood Center were collected in EDTA and stored at 4 °C. Genomic DNA was manually extracted with a QIAamp™ DNA Blood Mini Kit (Qiagen, Hilden, Germany) and samples were stored at 20 °C until testing with BLOODchip® Reference, usually 1 to 2 weeks after extraction. The minimum concentration of DNA was 20 ng/μL and the A260/A280 ratio was in the range of 1.6–1.95. The percentage of RBC antigens and HPA alleles in the blood groups identified with the BLOODchip® Reference were calculated. If a found group had a frequency known to be extremely rare in Asians (<0.01%) the result was confirmed by sequencing (polymerase chain reaction [PCR] carried out by in-house methods developed by the Shanghai Blood Center). The results for the following RBC antigens were compared with those yielded by routine serological methods using monoclonal or polyclonal antibodies (Grifols, Barcelona, Spain): A, B, D, C, c, E, e, Cw, Fya, Fyb, Jka, Jkb, Jsb, K, k, Kpa, Kpb, Lua, Lub, S, s, M, N. The “tube method” was used to detect IgM antibodies, and DG Gel® cards (Grifols, Barcelona, Spain) to detect IgG antibodies. The results for the following RBC antigens were compared with the results of sequencing the genetic region including the single nucleotide polymorphic sites determining the antigen polymorphism: Cx, Kpc, Jsa, Dia, Dib, Doa, Dob, Coa, Cob, Fyx. Sequencing was also used when there was a “no call” (NC), i.e. when the BLOODchip® software was not able to assign a result, or a not valid result that could not be resolved by repeating the BLOODchip® Reference test. Non-concordant results between serology and the BLOODchip® Reference due to a discrepancy or to a NC were investigated. RBC rare phenotypes for which reference techniques are not available were not compared but confirmed by sequencing. The NC results of HPA systems were confirmed by PCR with sequence-specific amplification (PCR-SSP) (SSPTyper™ Genotyping System [G&T Biotech, Rockville, MD, USA]) only in NC cases. All of the 403 collected samples came from donors of Han ethnicity. All samples were tested with BLOODchip®, although the comparison study was performed on only 387 samples (96.0%) since in 16 samples (4.0%) no additional blood or DNA sample was available to perform the validation experiments. Frequencies of the different RBC antigens and HPA alleles identified by BLOODchip® Reference are shown in Table Ia and ​andIb.Ib. RBC group frequencies observed in the Shanghai Blood Center were mostly in line with those reported in the Chinese Han population. Rare groups identified were: one VS+ (0.3%), one s– (0.3%) and three Do(a+b−) (0.8%). Table Ia Frequencies of the different red blood cell antigens identified by the BLOODchip® Reference in the 403 samples tested. Table Ib Frequencies of the different HPA alleles identified by the BLOODchip® Reference in the 403 samples tested. Of the 12,005 RBC antigens compared with the reference method, there were 11,967 concordances (99.68%) and 38 non-concordances (including NC) (0.32%) between the BLOODchip® Reference and the reference method (Table II). Table II Distribution of the concordances and non-concordances observed in the 22,833 tests performed on 387 samples for red blood cell and platelet antigen systems. There were four cases (1 RhCE and 3 Kell) that were initially non-concordant but were subsequently concordant after further testing. The RhCE sample was initially identified as CCEE by the BLOODchip® Reference, which was inconsistent with the serology result (CcEe) and was probably caused by a DNA sample error. After retesting the sample with the BLOODchip® Reference, the result was consistent with that of serology. The three K samples corresponded to Jsa antigen identified by the BLOODchip® Reference; however, sequencing results could not be distinguished due to hybrid peaks (heterozygous sequence). The 13 NC found in the ABO system (Table II) were due to a new combination of single nucleotide polymorphisms (A101/A102 in sequencing) that can be corrected in the future by including them in the BLOODchip® matrix of the software. NC results of the RhD (1 D+), RhCE (3 C, 3 c) and the MNS (2 M, 2 N) (Table II) were due to low signals. Both serology and sequencing confirmed RhD+, three C+c+, one M+N+, and one M−N+. The M+N+ sample was retested with BLOODchip® Reference and the M+N+ result was obtained, but no additional DNA was available to confirm the three C+c+, and M−N+ result. The four samples NC in the HPA6 system were HPA6 (HPA6a/6wb) in PCR-SSP and sequencing, which may have been caused by a silent polymorphism next to the polymorphism of HPA-6a/b, ITGB3:c, 1545G>A which has been frequently found in Caucasians5 and can affect probe hybridisation. The five results for the N antigen (Table II) were identified by serology as weak N+. When tested by anti-N monoclonal antibody (LN3), the result was also weak N+. However, the BLOODchip® Reference and sequencing result (IVS1-79delA, IVS1-9G, 59C, 71G, 71T) were both N–, possibly indicating a serological false positive caused by a non-specific signal of the LN3 clone. Discrepancies for the Dombrock antigen (Table II) were found in one sample: Do(a−b+) by the BLOODchip® Reference and Do(a+b+) by sequencing. The Do(a+b+) result was confirmed after repeating sequencing (heterozygous silent polymorphism at position DO*825G>A; codon: CTG>CTA; non-amino acid change: Leu>Leu). This polymorphism is an unreported silent single nucleotide polymorphism that should not affect Dombrock antigen expression but prevents Dombrock exon 19 amplification by BLOODchip® Reference. The frequency of this polymorphism in the population of the present study was 0.2%, so the incidence in the population appears to be very low. In summary, the results of this first implementation of the BLOODchip® Reference in China indicate that this microarray-based genotyping system performed consistently. Improved genotyping results will be obtained after software adjustments aiming at polymorphisms and other rare alleles in Chinese people.