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French Researchers Develop Automated, Array-based Molecular Blood Group Typing Technique

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A team of researchers in France has designed a microarray-based approach for blood group typing that it believes is more comprehensive than conventional methods, while less expensive and more scalable than other chip-based techniques.

Scientists at the Pyrénées Méditerranée site of the French Blood Service, the Etablissement Français du Sang (EFS), led the development of the system and carried out with other EFS sites a pilot study of 960 blood donor samples. The results of their work were described in a new paper published this week in the Journal of Molecular Diagnostics.

According to Jean-Charles Brès, the lead investigator on the project, the EFS several years ago initiated an effort to develop molecular-based blood group typing techniques for use in its immunohematology qualification laboratories with the objective of overcoming the limitations of conventional hemagglutination.

In antibody-based agglutination, red blood cells suspended in liquid collect into clumps when bound by the blood group-specific antigen. While the method is currently the only blood group typing approach used in France today, it is lacking in terms of comprehensiveness and scalability, Brès told BioArray News.

He cited its "limited range of testing" as one disadvantage. "In the French Blood Service, Blood Donation Qualification Laboratories test all blood donations for ABO, Rhesus on a first donation, confirm the RH, and KEL phenotype on a second donation, but testing for other clinically significant antigens, including FY1, FY2, JK1, JK2, MNS3, MNS4, is performed on only a fraction of donations, between 5 and 10 percent," he said.

The second disadvantage of antibody-based agglutination involves the long duration of the procedure, Brès said. "For these reasons, conventional hemagglutination is ill-suited to high-throughput blood group phenotyping."

In their effort to find a new approach to type blood groups, the researchers evaluated several array platforms, including Progenika's BloodChip and Immucor's BeadChip HEA, as well as real-time PCR, but decided against implementing them, citing the platforms' semi-automated formats and high cost.

The EFS investigators ultimately established the feasibility of genotyping human platelet antigens by combining single PCR and DNA microarray hybridization, and designed an automated robotic system consisting of Hamilton Robotics and Roche instruments for performing the assay.

The resulting method relies on an automated genotyping system capable of processing 96-well microarrays.

According to the paper, Berlin-based Scienion used its SciFlexarrayer S11 to spot the arrays on Greiner Bio-One 3D epoxy-coated microarray plates. Each array consisted of eight SNPs used to identify 16 alleles in four blood group systems – Kell, Kidd, Duffy, and MNS.

"Different manufacturers were tested for the 96-well DNA microarrays," said Brès. "Some arrays were manufactured in glass and some were in plastic, such as Greiner's microarrays," he said. "After many developments and evaluation of different 96-well microarrays, the best results were obtained by using Greiner Bio-One's 96-well microarrays."

To spot the arrays, the EFS decided to work with Scienion "because [it] can offer a high-quality service for probes arraying on microarrays," Brès added. "Scienion can provide a quality control and QC reports for printing probes on arrays, and these points were very important for our evaluation, to get quality controlled DNA microarrays."

The choice of using a 96-well microarray platform, rather than a slide-based array, had to do with its scalability.

"We decided to use a 96-well microarray platform because these platforms are mainly used for high-throughput screening and most commercial automated platforms, such as the Starlet from Hamilton Robotics, are well-adapted to this format and they can be easily adapted to 384-well format," said Brès.

EFS' platform decision also provided some cost advantages. As noted in the paper, for small-batch production, the cost of genotyping, including genomic DNA extraction, labor, and equipment, was less than $2.60 per SNP for a multiplex set of eight SNPs – four times lower than the per-antigen cost using serologic methods.

To round out the system, EFS researchers selected a Tecan LS200 system to scan the arrays, and carried out data analysis using Molecular Devices' GenePix Pro 6.0 software.

Pilot study and future applications

After the EFS researchers set up the new platform, they embarked on a pilot study to assess its utility.

A total of 1,132 blood samples were collected by the EFS site in Rhône Alpes, and the random donors, most of them Europeans, were phenotyped using conventional hemagglutination techniques in the Blood Donation Qualification Laboratory in Metz-Tessy.

One hundred seventy-two of the samples were used to determine scoring criteria for predicting phenotype, and the remaining 960 samples were used for validation of the 96-well microarray system.

According to the paper, when the phenotypes predicted from genotypes were compared with those obtained by serologic typing, the concordance rate between the DNA-based and standard hemagglutination assays was high for all four blood group systems. Just three predicted phenotypes were discordant.

"These findings demonstrate that our assay using a simple protocol allows accurate, relatively low-cost phenotype prediction at the DNA level," the authors concluded in the paper. "This system could easily be configured with other blood group markers for identification of donors with rare blood types," they wrote.

Brès said that the new system could be used not only to identify blood donors to obtain rare blood units, but also to type certain kinds of patients.

"There are several clinical situations, such as polytransfused patients [and] patients with a positive direct antiglobulin test, where making diagnosis is difficult," Brès said. "In such cases, antibody-based agglutination techniques are limited and they are not able to give information on patient's red blood cells," he said. "Molecular methods can overcome this limitation by providing a predicted phenotype based on the determined genotype."

Ultimately, said Brès, the implementation of the array platform could provide for "better patient blood management, and it will increase blood transfusion safety by providing antigen-matched blood components and preventing alloimmunization."

While the new array platform has such potential, Brès cautioned that it cannot be put into immediate use as a routine DNA-based method for typing donors, as it will require various approvals before it becomes a standard test.

"The EFS' primary mission consists of guaranteeing France's self-sufficiency in blood supply while maintaining optimum safety and quality conditions," he said, adding, "We are confident that blood-group genotyping will be integrated in the future as a new routine test into blood donor screening platforms."

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