Traditionally, erythrocyte immunohematology is based on the study of the phenotype, determined by the search for the presence on the surface of the red blood cells of antigens belonging to blood group systems using appropriate antisera. Direct genotype determination is now routinely applied in immunohematology.

DNA extraction usually takes place from whole blood collected in EDTA, using automated methods that can lighten the workload and at the same time ensure good quality of the extracted nucleic acids. After the extraction phase, we move on to the amplification phases of the target with a multiplex method, which allows the simultaneous and allele-specific amplification of multiple target sequences.

In the routine of our immunohematology laboratory, commercial gene-chips are usually used for the detection of amplified products, which allow to study polymorphisms of different blood group systems at the same time. The Blood Chip Reference system, in routine use in our laboratory, allows to determine the genotype of antigens related to ABO, RH, Kell, Duffy, Dombrock, Colton, Lutheran, Diego Kidd and MNS blood group systems. The system foresees that the amplicons, labelled with a fluorescent substrate, are detected by binding with allele-specific probes immobilized on a slide. The slide is then scanned by a laser, and for each probe the positivity for the single allele is assessed, based on the bound fluorescence, using specific software.

Figure 1 shows genotyping in immunohematology; the Blood Chip Reference system adopted in our Laboratory.

Fig1. Genotyping in immune hematology; the Blood Chip Reference system adopted in our laboratory

Another widespread system is the one called HEA BeadChip, capable of determining the genotype of antigens related to the blood group systems ABO, RHD, RHCE, Kell, Duffy, Dombrock, Lewis, Colton, Scianna, Lutheran, Diego, Kidd, and MNS. In this system, the amplified products are denatured and linked, through the use of specific allele probes, to color-coded microspheres. Upon successful bonding, the probe elongates and produces a fluorescent signal which is detected by an image capture system using a fluorescence microscope. The intensity of the signal and the probe related to it allow to trace the genotype.

At present, genotyping methods find routine applications in the extended typing of donors for the establishment of “banks of rare groups”, which can only be computerized or provide for the storage of frozen erythrocyte concentrates. They are widely used in the study of transfusion-dependent subjects (i.e., patients with hemoglobinopathies), in which the determinable phenotype no longer corresponds to the genotype, as well as in the study of rare groups or carriers of weak antigenic variants. The applications in the field of prevention of MEN appear to be very interesting, with the possibility of carrying out foetal genotyping both from foetal cell material obtained with an invasive method and from free DNA in maternal plasma.

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مواضيع ذات صلة

phosphate (PO4 , Phosphorus [P])

pheochromocytoma suppression and provocative testing (Clonidine suppression test [CST], Glucagon stimulation test)

parvovirus B19 antibody

Direct Antiglobulin Test (Direct Coombs Test)

Indirect Antiglobulin Test (Indirect Coombs Test)

Partial thromboplastin time, activated (APTT, Partial thromboplastin time [PTT])

parathyroid scan (Parathyroid scintigraphy)

parathyroid hormone (PTH, Parathormone)

Paracentesis (Peritoneal fluid analysis, Ascitic fluid cytology, Peritoneal tap)

Clinical Immunology Laboratory (Diagnostic Testing)

Determination of Type D and Rh Phenotype

Determination of the ABO Group