The weak D phenotype is the most common D variant, with a frequency of 0.2-1% in Caucasian individuals. There are several weak D types, with different frequencies in European countries, which may pose serologic problems and have the potential for alloimmunization. Samples from Portuguese individuals were tested for RhD by two or three distinct monoclonal and oligoclonal antisera, in direct agglutination tests. When discrepant results were observed, samples were tested with panels of monoclonal anti-D by LISS-indirect antiglobulin test. Cases that reacted weakly with IgM but positive with IgG anti-D were analysed by PCR-sequence-specific primers and real-time PCR. Ninety-nine samples were referred after being characterized as weak D. This genotype was recognized, with a preponderance of weak D type 2 (63.6%) over type 1 (16.2%) and 3 (14.1%). The high incidence of weak D type 2 in our population is in marked contrast to studies performed in other European populations and might be due to our sample selection criteria or ethnic variation. There are advantages in genotyping serologically depressed D samples to avoid the waste of D-negative RBC units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization.

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Background Until the 1990s, blood screening, typing and diagnostics depended entirely on serological techniques. For over a century, agglutination has been the gold standard for red blood cell (RBC) antigen detection used in all blood services. However, haemagglutination has certain limitations, such as, the difficulty to phenotype recently and multi-transfused patients or direct antiglobulin test (DAT)-positive patients. The haemagglutination test provides only indirect indications of risk or severity of haemolytic disease of the new born. In part, to overcome these limitations, nucleic acid-based technologies have been used in immunohematology reference laboratories.Methods There are many molecular methods available for red cell genotyping: PCR, PCR-RFLP, PCR-SSP or PCR-ASP, real time PCR, DNA sequencing and pirosequencing and methods with microarrays-based systems.Other molecular techniques that are under development and may be available for red cell genotyping in the next decade include fluidic or open microarrays; matrixassisted laser desorption ⁄ ionization time-of-flight mass spectrometry (MALDI-TOF MS); and mini-sequencing.Conclusions Although serology may be superior for some blood group typing, genotyping assays offer a good alternative for problems encountered by serology. In many laboratories, blood group genotyping is already used at a low-throughput level for diagnostics in cases of problematic serology. Especially in case of weak expression of antigens, the presence of rare antigens or auto-antibodies or after multiple transfusions, genotyping is superior. The non-invasive determination of the foetal RHD analysis in maternal plasma by real-time PCR is well established and already offered as a clinical service in a number of countries.The recent availability of automated, high throughput, DNA-array platforms, allows to introduce into the hospital and donor centres this DNA-based typing methodology. The evolution of molecular methods combined with automation and high-quality standards will make large-scale screening a cost-effective reality.

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