Researchers develop novel molecular blood group typing technique

Philadelphia, PA, April 10, 2014 – Scientists in France have designed a new system for molecular blood group typing that offers blood banks the possibility of extensive screening of blood donors at a relatively low cost. Their approach is described in the current issue of The Journal of Molecular Diagnostics.

Although blood transfusion is generally safe, alloimmunization (when an antibody is formed in response to an antigen that is not present on a person's own red blood cells [RBCs]) remains a dreaded complication, particularly in patients with sickle cell diseases.

"This may cause problems, ranging from delayed hemolytic transfusion reaction to difficulty in obtaining matched RBCs. Where patients have alloantibodies, producing a sufficient quantity of extensively typed blood units will never be feasible using conventional serologic donor screening methods," explains lead investigator Jean-Charles Brès, PhD, of the Etablissement Français du Sang Pyrénées Méditerranée, Montpellier.

The standard technique, conventional hemagglutination, is a lengthy procedure and involves only a limited range of antigen testing. In this antibody-based agglutination, RBCs suspended in liquid collect into clumps when bound by the antigen-specific antibody. Dr. Brès adds, "In the French Blood Service, the Etablissement Français du Sang (EFS), blood donation qualification laboratories test all blood donations for A, B, O, Rhesus (RH1), and KEL (KEL1) blood groups, but only 5% to 10% of donations are tested for other clinically significant antigens."

The investigators therefore developed a new flexible DNA microarray platform for molecular blood group typing. This includes two robotic workstations that allow processing from blood sample to the genotype. A pilot study shows promising results for responding to blood donor laboratories' requirements for simple, low-cost screening.

For small batch production, the cost of genotyping, including genomic DNA extraction, labor, and equipment, was less than $2.60 per single-nucleotide polymorphism (SNP) for a multiplex set of eight SNPs – four times lower than the per-antigen cost using serologic methods.

"High-throughput DNA typing could facilitate support for patients undergoing long-term transfusion who are at high risk of alloantibody production, such as patients with sickle cell disease, thalassemia, or autoimmune hemolytic anemia. Another application would be donor identification to obtain rare blood units for specific patients and improve the ability to supply rare blood types," says Dr. Brès. "The availability of high throughput DNA-based blood group genotyping would be a great boon for transfusion medicine." He continues, "In addition to providing more fully antigen-matched RBCs and allowing better identification of rare donor blood types, this technology will reduce adverse reactions and decrease the relative cost of analysis."

TECHNICAL DETAILS OF THE STUDY

The purpose of this study was to set up and validate a flexible robotic platform using a 96-well DNA microarray for multiplex blood group genotyping.

A total of 1,132 EDTA-anticoagulated blood samples were collected by the EFS in Rhône Alpes, France. Random donors, mostly Caucasian, were extensively phenotyped using standard serologic hemagglutination techniques in the Blood Donation Qualification Laboratory (Metz-Tessy, France). One hundred seventy-two samples were used to determine scoring criteria for predicting phenotype. The remaining 960 samples were used for validation of the 96-well DNA microarray system.

Genomic DNA extraction from whole blood samples (200 mL) was performed using a MagNA Pure 96 system (Roche Diagnostics, Rotkreuz, Switzerland) and Viral NA Small Volume Kit (Roche Diagnostics) in a 96-well microarray plate according to the manufacturers' instructions. After extraction, DNA was eluted in 50 µL of buffer solution and quantified using a NanoVue spectrophotometer (GE Healthcare, Little Chalfont, UK).

A total of 938 samples were considered as valid and assigned genotypes based on the scoring criteria determined for the eight SNPs. 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. Only three predicted phenotypes that involved the KEL, JK, and MNS systems were discordant.

This version allows simultaneous multiplex assay of up to 96 samples in a single reaction run, but the system allows other DNA microarray formats with a lower number of wells to be easily adapted and processed on this platform.