Rh Genotyping

Different PCR amplification protocols have been applied to determine the Rh phenotype in DNA obtained from normal donor peripheral blood, amniocytes, and trophoblastic cells.[12]

Analyses of RHCE single nucleotide polymorphism (SNP) of exons 5 and 2 have been used for E/e and c genotyping, respectively. A 109-bp insert in intron 2 of the RHCE gene, found only in C-positive individuals, was the target for the RHC allele typing[3] (Fig. 1).

RHD genotyping was more complex because of the large number of different RHD alleles described. Developed strategies varied from RHD single-region PCR tests (involving exon 10, intron 4, or exon 7) to multiplex PCR tests for scanning all RHD-specific exons. However, RHD genotyping by probing the presence of RHD-specific polymorphisms was not suitable for certain population groups, such as Africans, where the presence of RHDC can reach 40%. Generally, checking two sufficiently distant regions of the RHD gene together with the RHD C has proven to be safe for determination of the RhD phenotype[3,12] (Fig. 1).

CLINICAL ASPECTS OF Rh GENOTYPING

Routinely, Rh typing is performed by agglutination with polyclonal and immunoglobulin (Ig) M monoclonal antibodies, or blends of IgM and IgG monoclonal antibodies. However, there are some clinical situations in which serological techniques cannot determine the RBC pheno-type accurately:1-13-1

In fetuses at risk for hemolytic disease of the newborn because periumbilical blood sampling must be performed to obtain fetal erythrocytes. In cases of autoimmune hemolytic anemia because auto-antibody coating of RBCs may render serological typing impossible. After a massive transfusion, when agglutination methods would detect antigens on both the patient's and the donor's RBCs. In patients with altered expression of the D antigen and where serology is inconclusive for deciding transfusion therapy or anti-D prophylaxis.

RH genotyping with specificity and sensitivity comparable to serologic methods is of practical importance to overcome the limitations of classical hemagglutination and, in addition, to improve the currently possible resolution. Moreover, the application of molecular techniques to the identification of rare alleles is increasingly important, as many typing reagents on which serology is reliant are now either no longer available or in short supply.

Hemolytic Disease of the Newborn

The RhD hemolytic disease of the newborn continues to affect at least 1:1000 live births despite the use of prophylactic anti-D Ig. Current management strategies for RhD alloimmunization include invasive procedures such as serial amniocentesis (to predict fetal anemia) and cordocentesis (to directly determine fetal blood type serologically), but both alternatives involve a high risk of fetal loss and may result in an important increase in anti-D titers.[4]

Since the molecular cloning of the Rh system, efforts have been directed toward the development of methods for DNA-based Rh typing particularly for the prenatal assessment of the fetal RhD status. The extreme sensitivity of assays based on PCR made it possible to determine the presence of the RHD gene and also RHCE alleles in fetuses using DNA derived from a single amnio-centesis.[3,11,12,14] The high level of accuracy reached in RHD genotyping strategies allowed the incorporation of this prenatal diagnosis test into the management scheme applied to erythrocyte sensitization. If the fetus proves to be RhD-negative, the need for subsequent invasive procedures is obviated. If testing predicts that the unborn baby is RhD-positive, treatment can be planned with sufficient time.

However, to circumvent this risk associated with amniocentesis, several groups have investigated the possibility of determining the fetal RhD status through the use of fetal cells isolated from maternal blood.[15] The main problem is that the procedures needed to isolate sufficient numbers of fetal cells from maternal blood are time-consuming and technically demanding. Moreover, a cell subpopulation has been shown to persist for up to decades after delivery; thus it could interfere with fetal cell analysis in women who have had multiple pregnancies. An alternative approach based on the detection of RHD messenger RNA in fetal nucleated red cells has also been described, but the small number of subjects analyzed precludes any firm conclusion as to the reliability of this method.[16]

Recently, it has been found that during pregnancy, cellfree fetal DNA can be found in maternal plasma and may be utilized to determine fetal blood group status without invading the fetomaternal circulation. Lo[17] was able to demonstrate that fetal DNA is present at very high fractional concentrations in maternal plasma, constituting approximately 3% of total maternal plasma DNA during the second trimester of pregnancy and that fetal DNA is cleared very rapidly from the maternal plasma after delivery, with a half-life on the order of minutes. This high relative concentration suggests that fetal DNA could be robustly detected in maternal plasma using modern molecular technology and, unlike fetal cells in maternal blood, plasma DNA analysis is not complicated by the effect of persistence from prior pregnancies.

Both real-time PCR and conventional PCR technology have been applied to successfully determine the RhD status of unborn infants by analysis of plasma samples obtained from RhD-negative pregnant women. However, it is very likely that for future large-scale clinical usage, protocols based on real-time PCR may be preferred because of the high sensitivity and homogenous nature of the assays. In addition, genotyping of fetal DNA extracted from maternal plasma can potentially be used for the diagnosis of many disorders involving single genes.[17]

One of the latest developments in hemolytic disease of the newborn is the discrimination of RHD heterozygosity from homozygosity in RhD-positive fathers by the specific detection of RHD deletion through PCR amplification of the hybrid Rhesus box.[8] This determination is of considerable value in the prenatal assessment of the fetal RhD status.

Autoimmune Hemolytic Anemia

Rh polypeptides are the most common targets for pathogenic anti-RBC autoantibodies in patients with autoimmune hemolytic anemia.[4] In patients whose RBCs are heavily coated with IgG, testing with antiglobulin-reactive sera is difficult, whereas tests with high-protein agglutinin reagents are impractical. For RhD phenotyping of these patients, it is necessary to dissociate antibodies from erythrocytes by elution, without damaging RBC membrane integrity or altering antigen expression. Yet, when the affinity of the antibody is high, the dissociation procedure may be incomplete and lead to wrong results.[13] In some cases, the removal of autoantibodies coating the erythrocyte membrane is reduced after the elution procedure, but the direct antiglobulin test remains positive. If the sample is serologically typed as RhD-negative with a direct agglutination technique, doubts may arise when performing the indirect antiglobulin test. In these cases, DNA analysis can confirm the RhD-negative phenotype and discard a probable weak expression of D. When serological typing is inconclusive and cannot be accomplished with its usual ease, DNA-based phenotype prediction is superior to serotyping.

Transfusion

Patients requiring blood transfusion who present with pan-reactive antibodies pose great difficulty to blood banks. Appropriate antisera in sufficient volume are necessary to screen for compatible antigen-negative blood donors. Moreover, in polytransfused patients, persisting transfused red cells hamper a definitive RBC antigen profile and hence antibody identification.1-13-1 Genotyping is important in determining the true blood group of many polytrans-fused patients, can assist in the identification of suspected alloantibodies, and can help perform a more accurate selection of antigen-negative RBCs for transfusion.

Altered Expression of the D Antigen

Rh phenotyping may be inconclusive when erythrocytes possess a reduced expression of the D antigen. Limiting antibody sensitivity often hampers serological discrimination of such RhD phenotypes. Moreover, the use of monoclonal antibodies may fail to detect some weak D antigens.[13] In these cases with reduced antigen density, DNA-based analyses may be better than serological typing in inferring the true phenotype. Discrimination of RHD variants, such us partial D and weak D alleles, from the prevalent RHD allele would be advantageous to guide optimal RhD transfusion strategies or anti-D prophylaxis, considering the possibility of anti-D immunizations in these phenotypes that express aberrant RhD proteins.[6]

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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