Serologic And Molecular Characterization Of D Variants In Brazilians: Impact For Typing And Transfusion Strategy

Rh discrepancies are a problem during routine testing because of partial D or weak D phenotypes. Panels of monoclonal antibodies (MoAb) are being developed to identify D variants such as partial D and weak D when there are anomalous D typing results; however, molecular characterization offers a more specific classification of weak and partial D. The weak D and partial D phenotypes are caused by many different RHD alleles encoding aberrant D proteins, resulting in distinct serologic phenotypes and the possibility of anti-D immunization. We evaluated currently used serologic methods and reagents to detect and identify D variants and correlated the results with molecular analyses. A total of 306 blood samples from Brazilian blood donors and patients with discrepant results in routine D typing were analyzed. In total, 166 (54.2%) weak D, 136 (44.4%) partial D, 3 (1%) DEL, and 1 (0.3%) DHAR variants were identified. Among weak D samples, 76 weak D type 1 (45.8%), 75 weak D type 2 (45.2%), 13 weak D type 3 (7.8%), and 2 weak D type 5 (1.2%) alleles were found. Among the partial D samples, 49 type 4.0 weak partial D (36%), 9 DAR (6.6%), 24 DFR (17.6%), 6 DBT (4.4%), 1 DHMi (0.73%), 26 DVI (19%), 14 DVa (10.3%), 5 DIVb (3.7%), and 2 DVII (1.5%) were observed. Two samples identified as DEL by adsorption-elution were characterized by molecular analyses as RHD(IVS5-38DEL4) and one sample was characterized as RHD(K409K). One sample was characterized as DHAR, a CE variant positive with some monoclonal anti-D. Our results showed that the use of different methods and anti-D reagents in the serologic routine analysis revealed D variants that can be further investigated. Molecular methods can help to differentiate between partial D and weak D and to characterize the weak D types, providing additional information of value in the determind for the prevention of anti-D-related hemolytic disease of the fetus and newborn. Nation of D phenotypes. This distinction is important for optimized management of D- RBC units and for the prevention of anti-D-related hemolytic disease of the fetus and newborn.271611Avent, N.D., Reid, M.E., The Rh blood group system: a review [published correction appears in Blood 200095:2197] (2000) Blood, 95, pp. 375-387Westhoff, C.M., The structure and function of the Rh antigen complex (2007) Semin Hematol, 44, pp. 42-50Wagner, F.F., Gassner, C., Müller, T.H., Schönitzer, D., Schunter, F., Flegel, W., Molecular basis of weak D phenotypes (1999) Blood, 93, pp. 385-393Wagner, F.F., Frohmajer, A., Ladewig, B., Weak D alleles express distinct phenotypes (2000) Blood, 95, pp. 2699-2708Wagner, F.F., (1998) The RhesusBase. Department of Transfusion Medicine, , http://www.uni-ulm.de/~fwagner/RH/RB/, University Hospital, Ulm, Germany. Accessed March 11, 2011Müller, T.H., Wagner, F.F., Trockenbacher, A., PCR screening for common weak D types shows different distributions in three Central European populations (2001) Transfusion, 41, pp. 45-52Flegel, W.A., Wagner, F.F., Molecular biology of partial D and weak D. Implications for blood bank practice (2002) Clin Lab, 48, pp. 53-59Denomme, G.A., Wagner, F.F., Fernandes, B.J., Li, W., Flegel, W.A., Partial D, weak D types, and novel RHD alleles among 33 864 multiethnic patients: implications for anti-D alloimmunization and prevention (2005) Transfusion, 45, pp. 1554-1560Körmöczi, G.F., Gassner, C., Shao, C.P., Uchikawa, M., Legler, T.J., A comprehensive analysis of DEL types: partial DEL individuals are prone to anti-D alloimmunization (2005) Transfusion, 45, pp. 1561-1567Legler, T.J., Maas, J.H., Köhler, M., Sequencing: a new tool for decision making on transfusion therapy and provision of Rh prophylaxis (2001) Transfus Med, 11, pp. 383-388Denomme, G.A., Dake, L.R., Vilensky, D., Ramyar, L., Judd, W.J., Rh discrepancies caused by variable reactivity of partial and weak D types with different serologic techniques (2008) Transfusion, 48, pp. 473-478Flegel, W.A., Khul, S.R., Wagner, F.F., Primary anti-D immunization by weak D type 2 RBCs (2000) Transfusion, 40, pp. 428-434Mota, M., Fonseca, N.L., Rodrigues, A., Kutner, J.M., Castilho, L., Anti-D alloimmunization by weak D type 1 red blood cells with a very low antigen density (2005) Vox Sang, 88, pp. 130-135Flegel, W.A., How I manage donors and patients with a weak D phenotype (2006) Curr Opin Hematol, 13, pp. 476-483Flegel, W.A., Wagner, F.F., RHD epitope density profiles of RHD variant red cells analyzed by flow cytometry (1996) Transfus Clin Biol, 3, pp. 429-431Ansart-Pirenne, H., Asso-Bonnet, M., Le Pennec, P.-Y., Roussel, M., Patereau, C., Noizat-Pirenne, F., RhD variants in Caucasians: consequences for checking clinically relevant alleles (2004) Transfusion, 44, pp. 1282-1286Jones, J., Filbey, D., Selection of monoclonal antibodies for the identification of D variants: Ability to detect weak D and to split epD2, epD5 and epD6/7 (1996) Vox Sang, 70, pp. 173-179Castilho, L., Carvalho, T., Credidio, D., Pellegrino, J., RHD genotyping in blood donors with highly diverse ancestry phenotyped as D-negative (abstract) (2008) Transfusion, 48, pp. SP448188ASingleton, B.K., Green, C.A., Avent, N.D., The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in Africans with the Rh D-negative blood group phenotype (2000) Blood, 95, pp. 12-18Maaskant-van wijk, P.A., Faas, B.H., de Ruijter, J.A., Genotyping of RHD by multiplex polymerase chain reaction analysis of six RHD-specific exons [published correction appears in Transfusion 199939:546] (1998) Transfusion, 38, pp. 1015-1021Castilho, L., Rios, M., Rodrigues, A., Pellegrino Jr., J., Saad, S.T.O., Costa, F.F., High frequency of partial DIIIa and DAR alleles found in sickle cell disease patients suggests increased risk of alloimmunization to RhD (2005) Transfus Med, 15, pp. 49-55Garratty, G., Do we need to be more concerned about weak D antigens? (editorial) (2005) Transfusion, pp. 1547-155

Fetal RHD Genotyping by Analysis of Maternal Plasma in a Mixed Population

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Background: Maternal plasma analysis for the determination of the fetal RHD status is an exciting tool for the management of RhD-negative pregnant women, specially sensitized women. We assessed the accuracy of fetal RHD genotyping by analysis of maternal plasma in a multi-ethnic population. Methods: We analyzed plasma samples from 88 RhD-negative pregnant women between 11 and 39 weeks of gestation, median age of 28 years old to determine the fetal RHD genotype. This population was from Southeastern Brazil with high mixed ethnic background. Fourteen patients (16%) had anti-D alloantibody. We used Taqman primers and probes to detect by real-time PCR, exons 4, 5, and 10 of RHD. As internal controls we used primers/probes sets to SRY and CCR5. Peripheral or umbilical cord bloods from respective nenonates were collected during delivery and hemagglutination was performed. Results: Fifty-eight samples (66%) were genotyped as RHD+, 27 samples (31%) showed complete absence of RHD and 3 samples (3%) presented a D variant (RHD psi). All the results agreed with the neonatal typing, including the three fetuses with the RHD psi, phenotyped as RhD-negative. Thus, the accuracy of the fetal RHD genotyping in this mixed population was 100%. The earliest pregnancy in which fetal RHD was detected was 11 weeks. Conclusion: Our findings indicate that the accuracy of RHD gene using three regions (exons 4, 5, and 10) can be sufficient for clinical application in a multi-ethnic population. This knowledge helped us on the development of a feasible protocol for fetal RHD genotyping on DNA from maternal plasma for our population. J. Clin. Lab. Anal. 25:100-104, 2011. (C) 2011 Wiley-Liss, Inc.252100104Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2007/07151-3, 2009/05924-0

Atherosclerosis vascular damage in elderly athletes and sedentary people. Angiology. 1997 Jul;48(7):623-8

The objective of this study was to determine the degree of vascular atherosclerotic damage at the carotid and femoral levels in a group of subjects over sixty years old practicing endurance sports. Using high-resolution Doppler-color flow ultrasonography the authors carried out a vascular screening, comparing two distinct groups of subjects, the first consisting of 20 elderly male subjects (age 65.6 +/- 5.6 years) practicing endurance sports (runners) and the second of 20 subjects of the same gender and age (63.5 +/- 4.5 years), clinically healthy, but leading a sedentary life. All subjects were nonsmokers, nondiabetics, with normal lipid values and normal blood pressure. The authors examined the internal, external, and common carotid arteries, bilaterally, as well as the common femoral, deep femoral, and superficial femoral arteries of both legs. For each vessel they documented: (1) presence of plaques, (2) position and quantity of the plaques, (3) stenosis percentage produced by the plaques, (4) echographic structure of the plaques. In the group of sportsmen they calculated a global score of atherosclerotic damage of 5.58 +/- 2.21. This is statistically significant (P < 0.001) as compared with the global score observed in the sedentary group (9.24 +/- 3.9, range 6-14). The sportsmen exhibited small atherosclerotic plaques that were not hemodynamically significant; these plaques were present in 7 subjects (35%). In 4 of them (20%) the lesions were located in one carotid artery system. In the other 3 subjects (15%) the lesions were identified in one femoral artery system. In none of the sportsmen were they able to demonstrate simultaneous atherosclerosis of carotid and femoral arteries. In the sedentary subjects, atherosclerotic lesions were identified in 15 of them (75%). In 2 subjects hemodynamically significant plaques were located in one carotid artery system. In the other 13 subjects the plaques found were not hemodynamically significant; in 6 subjects this type of lesion was present in both femoral and carotid arteries; in 5 the lesions were located in one carotid artery system, and in 2 in one femoral artery system. In conclusion, endurance exercise appears to protect the elderly against atherosclerotic vascular damage

Atherosclerosis vascular damage in elderly athletes and sedentary people

The objective of this study was to determine the degree of vascular atherosclerotic damage at the carotid and femoral levels in a group of subjects over sixty years old practicing endurance sports. Using high-resolution Doppler-color flow ultrasonography the authors carried out a vascular screening, comparing two distinct groups of subjects, the first consisting of 20 elderly male subjects (age 65.6 +/-5.6 years) practicing endurance sports (runners) and the second of 20 subjects of the same gender and age (63.5 +/-4.5 years), clinically healthy, but leading a sedentary life. All subjects were nonsmokers, nondiabetics, with normal lipid values and normal blood pressure. The authors examined the internal, external, and common carotid arteries, bilaterally, as well as the common femoral, deep femoral, and superficial femoral arteries of both legs. For each vessel they documented: (1) presence of plaques, (2) position and quantity of the plaques, (3) stenosis percentage produced by the plaques, (4) echographic structure of the plaques. In the group of sportsmen they calculated a global score of atherosclerotic damage of 5.58 +/-2.21. This is statistically significant (P<0.001) as compared with the global score observed in the sedentary group (9.24 +/-3.9, range 6-14). The sportsmen exhibited small atherosclerotic plaques that were not hemodynamically significant; these plaques were present in 7 subjects (35%). In 4 of them (20%) the lesions were located in one carotid artery system. In the other 3 subjects (15%) the lesions were identified in one femoral artery system. In none of the sportsmen were they able to demonstrate simultaneous atherosclerosis of carotid and femoral arteries. In the sedentary subjects, atherosclerotic lesions were identified in 15 of them (75%). In 2 subjects hemodynamically significant plaques were located in one carotid artery system. In the other 13 subjects the plaques found were not hemodynamically significant; in 6 subjects this type of lesion was present in both femoral and carotid arteries; in 5 the lesions were located in one carotid artery system, and in 2 in one femoral artery system. In conclusion, endurance exercise appears to protect the elderly against atherosclerotic vascular damage