Serologic strategy in detecting RHD altered alleles in Brazilian blood donors

Rosangela Duarte de Medeiros Person Carine Prisco Arnoni Janaína Guilhem Muniz Tatiane Aparecida de Paula Vendrame Flavia Roche Moreira Latini Afonso José Pereira Cortez Jordão Pellegrino Jr Lilian Maria de Castilho About the authors

ABSTRACT

Background:

We evaluated different technological approaches and anti-D clones to propose the most appropriate serologic strategy in detecting the largest numbers of D variants in blood donors.

Methods:

We selected 101 samples from Brazilian blood donors with different expressions of D in our donor routine. The tests were performed in immediate spin (IS) with eleven commercially available anti-D reagents in a tube and microplate. The D confirmatory tests for the presence of weak D included the indirect antiglobulin test (IAT) in a tube, gel and solid-phase red blood cell adherence (SPRCA). All DNA samples were extracted from peripheral blood and the D variants were classified using different molecular assays.

Results:

The RHD variants identified by molecular analysis included weak D types (1, 2, 3, 11 and 38) and partial Ds (DAR1.2, DAR1, DAR3.1, DAU0, DAU2, DAU4, DAU5, DAU6, DMH and DVII). The monoclonal-monoclonal blend RUM-1/MS26 was the best anti-D reagent used in detecting the D antigen in the IS phase in a tube, reacting with 83.2% of the D variants, while the anti-D blend D175 + 415 was the best monoclonal antibody (MoAb) used in a microplate to minimize the need for an IAT, reacting with 83.2% of the D variants. The D confirmatory tests using SPRCA showed a reactivity (3 - 4+) with 100% of the D variant samples tested.

Conclusion:

Our results show that, even using sensitive methods and MoAbs to ensure the accurate assignment of the D antigen, at least 17% of our donor samples need a confirmatory D test in order to avoid alloimmunization in D-negative patients.

Keywords:
D variant; RHD alleles; Weak D; Partial D; Hemagglutination; RHD genotyping

Introduction

The Rh system is the most clinically important blood group system after the ABO.11 Avent ND, Reid ME. The Rh blood group system: a review. Blood. 2000;95:375-87. Two highly homologous genes, RHD and RHCE, which are located on Chromosome 1 encode the RhD and RhCE proteins, respectively.22 Cherif-Zahar B, Mattei MG, Le Van Kim C, Bailly P, Cartron JP, Colin Y. Localization of the humanRh blood group gene structure to chromosome 1p34.3-1p36.1 region by in situ hybridization. Hum Genet. 1991;86:398-400.,33 Cherif-Zahar B, Bloy C, Le Van Kim C, Blanchard D, Bailly P, Hermand P, et al. Molecular cloning and protein structure of a humanblood group Rh polypeptide. Proc Natl Acad Sci USA. 1990;87:6243-7. The D antigen is the most important antigen of the Rh system because of its implication in the hemolytic transfusion reaction (HTR) and hemolytic disease of the fetus and newborn (HDFN). The D-negative phenotype is observed in 15–17% of Caucasians compared to 5% of Africans and less than 3% in Asian populations.44 Daniels G. Human Blood Groups. 2nd ed. Oxford: Blackwell Science Ltd Editorial Offices; 2002. p. 209 p. In Caucasians, the D-negative phenotype results predominantly from the deletion of the RHD gene. In Africans, besides the RHD gene deletion and the inactive genes, the RHDΨ and the RHD-CE-Ds hybrid gene give rise to the loss of D expression.55 Daniels G. The molecular genetics of blood group polymorphism. Transplmmunol. 2005;14:143-53. In South East Asian populations, 15–30% of D-negative individuals carry an RHD allele that encodes a protein with very weak expression of the D antigen, leading to the DEL phenotype,66 Luettringhaus TA, Cho D, Ryang DW, Flegel WA. An easy RHD genotyping strategy for D- East Asian persons applied to Korean blood donors. Transfusion. 2006;46:2128-37.,77 MakKH KF, Cheng SS, Yuen MY. Rh phenotypes of Chinese blooddonors in Hong Kong, with special reference to weak D antigens. Transfusion. 1993;33:348-51. which can only be detected by adsorption-elution techniques.88 Wagner T, Körmöczi GF, Buchta C, Vadon M, Lanzer G, Mayr WR, et al. Anti-D immunization by DEL red blood cells. Transfusion. 2005;45:520-6.

Although most individuals are either RhD-positive or RhD-negative, a plethora of variants of D have been described.99 Flegel WA. Molecular genetics and clinical applications for RH. Transfus Apher Sci. 2011;44:81-91. The RHD gene is highly polymorphic and the D expression is caused by a large number of RHD alleles. A part of these alleles leads to a reduced or variable expression of D antigenic epitopes on the red cell surface.99 Flegel WA. Molecular genetics and clinical applications for RH. Transfus Apher Sci. 2011;44:81-91. These variations in the RhD antigen structure result either in a partial or a weak D phenotype, leading to qualitative or quantitative changes in Rh protein expression, respectively.

Despite the fact that more sensitive monoclonal reagents have been produced, not all anti-D reagents detect the same partial or weak expression of the D antigen.1010 Denomme GA, Dake LR, Vilensky D, Ramyar L, Judd WJ. Rh discrepancies caused by variable reactivity of partial and weak D types with different serologic techniques. Transfusion. 2008;48:473-7.1212 Lai M, Grasso C, Boschi I, D’Onofrio G, Pascali V, Leone G. Characterization of anti-D monoclonal antibody reagents based on their reactivity with weak D phenotype. Transfusion. 2009;49:937-42. Donors and patients with these atypical alleles may be mistyped by serology because many of these alleles do not react equally with all anti-D typing reagents.

Populations with African admixture, such as the Brazilian population, can present a high variety of RHD alleles.1313 Credidio DC, Pellegrino J, Castilho L. Serologic and molecular characterization of D variants in Brazilians: impact for typing and transfusion strategy. Immunohematology. 2011;27:6-11. Considering this fact, the best strategy of D typing in the donor routine would be a suitable combination of anti-D reagents to identify weak D variants that could induce anti-D formation in D-negative recipients.

In this report, we evaluated different technological approaches and clones of anti-D to propose the most appropriate serologic strategy to identify weak D variants in the donor routine. We also performed molecular analyses to characterize the RHD alleles in order to know the RHD repertoire in this Brazilian population and to identify the RHD alleles undetected in serologic tests that could be responsible for transfusion-induced anti-D formation.

Methods

Blood samples

From November 2013 to August 2014, we selected a total of 101 blood donor samples from 123,936 samples. In the first month of this study, all donors with weak D expression were selected, which corresponded to 62 samples from a total of 12,560 donors (0.49%). The other 39 samples were selected based on discrepant results with previous donations or samples with very low antigen density. The RhD was typed in a microplate with two commercial anti-D monoclonal antibodies (MoAbs), RUM-1 and D175 + 415, using an automatic immunoassay analyzer (NEO®, Immucor, Norcross, GA, USA) that showed atypical D serologic typing (reactivity pattern <4+). This study was conducted in accordance with an institutional ethical review.

Serologic analysis

Once a discrepancy was noted with the automated assay NEO®, the red blood cells (RBCs) were further analyzed in immediate spin (IS) in a tube and microplate with eleven commercially available anti-D reagents from different sources (Lorne Laboratories, Berkshire, UK; Fresenius, São Paulo, Brazil; Bio-Rad, Lagoa Santa, Belo Horizonte, Brazil and Immucor, Norcross, GA, USA). Fig. 1 shows a flowchart of the study. The D confirmatory tests for the presence of weak D included the indirect antiglobulin test (IAT) in a tube and gel test for RBC samples with reactivity <2+ at immediate spin and in solid phase red cell adherence (SPRCA) for all RBC samples tested in a microplate. The protocols were performed according to the manufacturer instructions. The serologic reactivity was graded according to the degree of hemagglutination from 0 to 4+.

Fig. 1
Flowchart of the study according to each methodology used.

Molecular analysis

The DNA was extracted from whole blood using the QIAmp DNA Blood Mini-Kit (Qiagen, Valencia, CA), according to the manufacturer recommendations. The DNA concentration and purity were calculated by the measurement of the optical density at 260 and 280 nm using a NanoDrop 2000 Spectrophotometer (Thermal Cycler, Uniscience Inc., São Paulo, SP, Brazil) and DNA samples were kept at −20 °C. The molecular tests performed on all 101 samples with atypical results in serology included: a multiplex PCR, PCR-RFLP and Sanger sequencing, according to our approach previously described.1414 WijkPA M, Faas BH, de Ruijter JA, von dem Borne AE, van Rhenen DJ, van der Schoot CE, et al. Genotyping of RHD by multiplex polymerase chain reaction analysis of six RHD-specific exons. Transfusion. 1998;38:1015-21.,1515 Arnoni CP, Latini FR, Muniz JG, Gazito D, Person RM, Vendrame TAP, et al. How do we identify RHD variants using a practical molecular approach?. Transfusion. 2014;54:962-9.

Results

Molecular findings

Molecular analyses confirmed that all RBC samples that showed atypical D serologic typing had altered RHD alleles, totalizing 15 different RHD alleles identified (Table 1). From 101 blood donor samples tested, 49 samples (48.5%) were molecularly characterized as partial D, 51 (50.5%), as weak D and 1, as weak/partial D (1%). The DAR1.2 was the most frequent allele found (29.7%), followed by weak D type 3 (20.8%), weak D type 38 (9.9%) and weak D type 1(8.9%). Less prevalent RHD alleles corresponded to 30.7% of all samples studied. Weak D types 1, 2 and 3 represented 37% of the RhD variants in this multiethnic population.

Table 1
Molecular characterization of samples from 101 blood donors with weak or discrepant results in RhD typing.

Serologic findings

Considering tube tests, our results showed a varied reactivity pattern among the several clones used, mainly in the IS phase, and these results are summarized in Table 2.

Table 2
RhD variants and agglutination degree in IS test with several anti-D reagents.

The Lorne IgM and blend clones were more sensitive, when compared with the other clones analyzed. The RUM-1 monoclonal IgM and the monoclonal blend RUM-1/MS26 were the best monoclonal (MoAb) anti-D reagents used to detect the D antigen in the IS phase in a tube, reacting with 84.1% and 83.2% of the D variants, while the anti-D blend D175 + 415 was the best MoAb used in a microplate to minimize the need of an IAT on donor samples, also reacting with 83.2% of the D variants. These results can be observed in Tables 3 and 4.

Table 3
Percentage of samples detected according to agglutination degree in IS tube method with IgM and blend anti-D clones.
Table 4
Percentage of samples detected according to agglutination degree and reaction score in IS microplate with blend anti-D clones.

The monoclonal anti-D IgM RUM-1 and the blend RUM-1/MS-26 reagents were reactive with all DAR1.2, DAR1 and weak D Type 3 samples in a tube and most of them reacted 1+ to 2+, while the other anti-D reagents showed variations in the reactivity.

Weak D types 1 and 2 showed heterogeneity in their reactivity with the anti-D reagents in IS. The monoclonal anti-D IgM RUM-1 was the only reagent to give positive reactions with all weak D type 1 samples analyzed, while weak D type 2 samples had at least one IS-negative result with all clones tested. Nevertheless, all of these samples were detected by an IAT with all anti-D reagents tested. Similar results were obtained in a microplate with the anti-D IgM RUM-1 and blend D175 + 415 reagents, but this method was less sensitive with the other anti-D MoAbs used. All blood samples classified as DAR1.2, DAR1, weak D types 1, 2 and 3 reacted with all the anti-D monoclonals tested by gel. The DAR3.1, DAU0, DAU2, DAU5, DAU6, DVII and DMH were detected in IS with all reagents and methods and reacted strongly in IAT in a tube, gel and SPRCA.

In relation to the tests performed with the gel technique, the D status was assigned as positive in 91 samples (90% of the total), with the blend clones TH28 + MS26 and MS201 + MS26 Bio-Rad in Liss/Coombs gel and 89 samples (88.1%) with the IgM TH28 Bio-Rad in NaCl/Enzyme gel. Of the 35 samples tested by ESD1, all of them showed positive results with scores similar to those of the other clones tested, but with improved performance in detection of the variant type 38.

Considering the clones tested with all samples: Blend TH28 + MS26, MS201 + MS26 and IgM TH28, only 30% of the weak D type 38 and none of the three Weak D type 11 samples were detected by an IAT in gel, while all these variants reacted strongly (3 - 4+) with the Immucor anti-D Blend D175 + 415 in SPRCA.

Overall, D confirmatory tests using gel cards showed agglutination reactions (3 - 4+) in 87.1% of the samples, detecting other variants, but serologic agglutination patterns were more consistent with the MoAbs in the solid phase, in which 100% of the samples showed strong positive reactivity (3 - 4+). The differences between clones and methodologies are demonstrated in Figs. 2 and 3.

Fig. 2
Comparison of reactivities using –D lgM clones with weak and partial D variants The degree of reactivity is reported according to the agglutination intensity.
Fig. 3
Comparison of reactivities using anti-D IgG clones with weak and partial D variants. The degree of reactivity is reported according to the agglutination intensity.

Discussion

Determining RhD antigen status in blood donors with reliable reagents and methods is essential to detect D variants in order to avoid alloimmunization in RhD-negative patients. However, the serologic distinction between D-positive and D-negative RBCs is not always straightforward in the case of D variants.1616 Fung Kee Fung K, Eason E, Crane J, Armson A, de la Ronde S, Farine Dan, et al. Maternal-Fetal Medicinal Committee, Genetics Committee. Prevention of Rh alloimmunisation. J Obstet Gynaecol Can. 2003;25:765-73. Monoclonal anti-D reagents are evaluated for their ability to identify some D variants and not react with other variants.

Herein we have demonstrated the serologic RhD typing complexity to identify D variants, showing the differences of reactivity obtained with different anti-D reagents and methods. In the donor samples studied, five different weak D types (1, 2, 3, 11 and 38) and eleven partial D types (DAR1.2, DAR1, DAR3.1, DAU0, DAU2, DAU4, DAU5, DAU6, DMH and DVII) were identified by molecular analyses.

The RHD alleles are grouped into D clusters, some of which comprise many alleles. Among the weak D type 4 cluster, partial D DAR1.2 was the most prevalent partial D in our donors, corresponding to 30% of our samples. This finding is in agreement with the previously reported frequency of the DAR1.2 allele in Brazilian blood donors.1717 Cruz BR, Chiba AK, Moritz E, Bordin JO. RHD alleles in Brazilian blood donors with weak D or D-negative phenotypes. Transfus Med. 2012;22:84-9. We observed that the variant alleles from the weak D type 4 cluster were better reactive with an IAT with all anti-D monoclonals and methods used.

Among the DAU cluster, we found a small number of samples classified as DAU0, DAU2, DAU4, DAU5 and DAU6, and all of them were detected in IS, except DAU4, which was not detected by the MoAb anti-D IgM RUM-1, nor by the blend RUM-1/MS-26 reagents. Of these, only DAU0 was found in Caucasians, while the other four DAU alleles were observed in Africans.1818 Wagner FF, Ladewig B, Angert KS, Heymann GA, Eicher NI, Flegel WA, et al. The DAU allele cluster of the RHD gene. Blood. 2002;100:306-11.

Among the most common weak D types, weak D types 1, 2 and 3 were found in 37% of our D variant blood donors. This finding is comparable to frequencies published for other donor populations from Southeast Brazil.1313 Credidio DC, Pellegrino J, Castilho L. Serologic and molecular characterization of D variants in Brazilians: impact for typing and transfusion strategy. Immunohematology. 2011;27:6-11.,1717 Cruz BR, Chiba AK, Moritz E, Bordin JO. RHD alleles in Brazilian blood donors with weak D or D-negative phenotypes. Transfus Med. 2012;22:84-9.,1919 Campos FC, Mota MA, Aravechia MG, Torres KB, Bub CB, Kutner JM, et al. Variant RHD Types in Brazilians With Discrepancies in RhD Typing. J Clin Lab Anal. 2016;30:845-8. but is in contrast with studies performed on Europeans, in whom these weak D types are common, encompassing more than 90% of all European weak D individuals.2020 Daniels G. Variants of RhD-current testing and clinical consequences. Br J Haematol. 2013;161:461-70.,2121 Ansart-Pirenne H, Asso-Bonnet M, Le Pennec PY, Roussel M, et al. RhD variants in Caucasians:consequences for checking clinically relevant alleles. Transfusion. 2004;44:1282-6. The weak D type 1 was better detected with the MoAb anti-D IgM RUM-1 and therefore, we can recommend this clone for detection of this weak D type in IS in a tube or microplate. The weak D type 2 showed a great variability in the reactivity in the IS phase with all the anti-D MoAbs used and, for this weak D type, we would recommend the indirect antiglobulin test in gel, or the SPRCA test. The weak D type 3 was the most prevalent weak D type found in our donor population, representing 21.7% of the samples and was reactive with most of the anti-D monoclonals employed.

By changing reagents and technological approaches, D antigen discrepancies between existing tests and historical records occur. When the automation using an immunoassay analyzer (NEO®, Immucor, Norcross, GA, USA) was introduced in our blood donor routine, D typing discrepancies were found with previous manually performed typing results. Most of them were related to weak D types 38 and 11, that have very low antigen density,2121 Ansart-Pirenne H, Asso-Bonnet M, Le Pennec PY, Roussel M, et al. RhD variants in Caucasians:consequences for checking clinically relevant alleles. Transfusion. 2004;44:1282-6.,2222 Costa S, Martin F, Chiba A, Langhi D, Chiattone C, Bordin J. RHD alleles and D antigen density among serologically D− C+Brazilian blood donors. Transfus Med. 2014;24:60-1. which had been classified as D negative in previous donations. Weak D type 38 is predominant in the Portuguese population,2323 Rodrigues MJ, Rodrigues F, Tilley L, Poole J, Chabert T, Souza G. Several new examples of weak D type 38 in the Portuguese population. Transfusion. 2006;46 Suppl, 141A–2A(abstract). but has also been found with a considerable frequency among Brazilians,1919 Campos FC, Mota MA, Aravechia MG, Torres KB, Bub CB, Kutner JM, et al. Variant RHD Types in Brazilians With Discrepancies in RhD Typing. J Clin Lab Anal. 2016;30:845-8.,2222 Costa S, Martin F, Chiba A, Langhi D, Chiattone C, Bordin J. RHD alleles and D antigen density among serologically D− C+Brazilian blood donors. Transfus Med. 2014;24:60-1. and corresponded to 10% of our D variant samples. Considering only the 101 study samples, when we performed a look back, we observed that 7 of 10 donors classified as weak D type 38 were mistyped as D negative in previous donations. Likewise, all donors with weak D type 11 identified in this study were previously typed as D negative. Considering all mistyped donors, a total of 37 blood units typed as D negative were uncorrected in the previous donations. Costa et al.2222 Costa S, Martin F, Chiba A, Langhi D, Chiattone C, Bordin J. RHD alleles and D antigen density among serologically D− C+Brazilian blood donors. Transfus Med. 2014;24:60-1. showed a similar scenario in 4 of 520 donors typed as RhD-negative, who were molecularly typed as weak D. Regarding these samples, no discrepancies involving other RhD variants were observed.

Serologic discrepancies caused by D variants are more likely to be resolved by genotyping, but this strategy cannot be applied routinely at all centers due to the cost and feasibility. The reactivity of the monoclonal anti-D reagents depends on the concentration and avidity of the antibodies. In fact, the number of antigenic sites, accessibility of an epitope, the presentation, the concentrations and the class of immunoglobulin can affect the reactions of weak and partial RhD antigens with monoclonal reagents.2424 Moulds MK. Review: monoclonal reagents and detection of unusual or rare phenotypes or antibodies. Immunohematology. 2006;22:52-7. The choice of the reagents and methodology requires special attention, mainly in a mixed population with the influence of Caucasian, Indian and African ethnicities, as are the Brazilians, leading to a diversity in D variants, in addition to an atypical heterozygous composition or the presence of new alleles.

In this study, the clones IgM RUM-1 and blend RUM-1/MS26 had better performance in detecting D variants in the IS tube tests, while the blend D175 + 415 showed better reactivity in microplates. Even considering the fact that the ESD1 was introduced later into the study, and we could not test it with all variants, including the variant weak D type 11, this clone was shown to be a good option for the IAT gel. When we compared ESD1 with the other Bio-Rad clones, we found a higher reactivity pattern, especially regarding the variants of low antigenic density. The proposed clones for each method described in this work are represented in Fig. 4.

Fig. 4
Anti-D clones more suitable for tube, microplate, gel and solid phase methodologies.

Regarding the technological approaches used, all of them have positive and negative points. Although the tube test has a short incubation, it was shown to be less sensitive in detecting D variants, requiring a greater RhD work-up. The gel test is more sensitive and could be used in an automated process. The solid phase is used as a confirmatory test in the automated analyzer NEO® and was the only methodology able to detect all the D variants identified, including the weak D types with very low density, such as the weak D types 38 and 11, that express less than 200 D antigen sites.

Success in donor D typing depends on the anti-D reagent used and on the exact conditions of the methods. Our results show a great variation in the methods and reagents in detecting D variants in Brazilian blood donors and, even using sensitive methods and different MoAb anti-D reagents to ensure the accurate assignment of the D antigen, at least 17% of the donor samples would need a confirmatory D test in order to avoid alloimmunization in D-negative patients. Sensitive confirmatory tests are indispensable in the detection of RhD variants in blood donors, particularly those with low antigenic density, and may not be detected, depending on the techniques and reagents used. This study allowed us to compare and determine the capacity and limitations of different technological approaches and reagents in identifying the most frequent D variants in a population with a high rate of miscegenation, particularly in individuals with weakened D expression.

References

  • 1
    Avent ND, Reid ME. The Rh blood group system: a review. Blood. 2000;95:375-87.
  • 2
    Cherif-Zahar B, Mattei MG, Le Van Kim C, Bailly P, Cartron JP, Colin Y. Localization of the humanRh blood group gene structure to chromosome 1p34.3-1p36.1 region by in situ hybridization. Hum Genet. 1991;86:398-400.
  • 3
    Cherif-Zahar B, Bloy C, Le Van Kim C, Blanchard D, Bailly P, Hermand P, et al. Molecular cloning and protein structure of a humanblood group Rh polypeptide. Proc Natl Acad Sci USA. 1990;87:6243-7.
  • 4
    Daniels G. Human Blood Groups. 2nd ed. Oxford: Blackwell Science Ltd Editorial Offices; 2002. p. 209 p.
  • 5
    Daniels G. The molecular genetics of blood group polymorphism. Transplmmunol. 2005;14:143-53.
  • 6
    Luettringhaus TA, Cho D, Ryang DW, Flegel WA. An easy RHD genotyping strategy for D- East Asian persons applied to Korean blood donors. Transfusion. 2006;46:2128-37.
  • 7
    MakKH KF, Cheng SS, Yuen MY. Rh phenotypes of Chinese blooddonors in Hong Kong, with special reference to weak D antigens. Transfusion. 1993;33:348-51.
  • 8
    Wagner T, Körmöczi GF, Buchta C, Vadon M, Lanzer G, Mayr WR, et al. Anti-D immunization by DEL red blood cells. Transfusion. 2005;45:520-6.
  • 9
    Flegel WA. Molecular genetics and clinical applications for RH. Transfus Apher Sci. 2011;44:81-91.
  • 10
    Denomme GA, Dake LR, Vilensky D, Ramyar L, Judd WJ. Rh discrepancies caused by variable reactivity of partial and weak D types with different serologic techniques. Transfusion. 2008;48:473-7.
  • 11
    Judd WJ, Moulds M, Schlanser G. Reactivity of FDA-approved anti-D reagents with partial D red blood cells. Immunohematology. 2005;21:146-8.
  • 12
    Lai M, Grasso C, Boschi I, D’Onofrio G, Pascali V, Leone G. Characterization of anti-D monoclonal antibody reagents based on their reactivity with weak D phenotype. Transfusion. 2009;49:937-42.
  • 13
    Credidio DC, Pellegrino J, Castilho L. Serologic and molecular characterization of D variants in Brazilians: impact for typing and transfusion strategy. Immunohematology. 2011;27:6-11.
  • 14
    WijkPA M, Faas BH, de Ruijter JA, von dem Borne AE, van Rhenen DJ, van der Schoot CE, et al. Genotyping of RHD by multiplex polymerase chain reaction analysis of six RHD-specific exons. Transfusion. 1998;38:1015-21.
  • 15
    Arnoni CP, Latini FR, Muniz JG, Gazito D, Person RM, Vendrame TAP, et al. How do we identify RHD variants using a practical molecular approach?. Transfusion. 2014;54:962-9.
  • 16
    Fung Kee Fung K, Eason E, Crane J, Armson A, de la Ronde S, Farine Dan, et al. Maternal-Fetal Medicinal Committee, Genetics Committee. Prevention of Rh alloimmunisation. J Obstet Gynaecol Can. 2003;25:765-73.
  • 17
    Cruz BR, Chiba AK, Moritz E, Bordin JO. RHD alleles in Brazilian blood donors with weak D or D-negative phenotypes. Transfus Med. 2012;22:84-9.
  • 18
    Wagner FF, Ladewig B, Angert KS, Heymann GA, Eicher NI, Flegel WA, et al. The DAU allele cluster of the RHD gene. Blood. 2002;100:306-11.
  • 19
    Campos FC, Mota MA, Aravechia MG, Torres KB, Bub CB, Kutner JM, et al. Variant RHD Types in Brazilians With Discrepancies in RhD Typing. J Clin Lab Anal. 2016;30:845-8.
  • 20
    Daniels G. Variants of RhD-current testing and clinical consequences. Br J Haematol. 2013;161:461-70.
  • 21
    Ansart-Pirenne H, Asso-Bonnet M, Le Pennec PY, Roussel M, et al. RhD variants in Caucasians:consequences for checking clinically relevant alleles. Transfusion. 2004;44:1282-6.
  • 22
    Costa S, Martin F, Chiba A, Langhi D, Chiattone C, Bordin J. RHD alleles and D antigen density among serologically D− C+Brazilian blood donors. Transfus Med. 2014;24:60-1.
  • 23
    Rodrigues MJ, Rodrigues F, Tilley L, Poole J, Chabert T, Souza G. Several new examples of weak D type 38 in the Portuguese population. Transfusion. 2006;46 Suppl, 141A–2A(abstract).
  • 24
    Moulds MK. Review: monoclonal reagents and detection of unusual or rare phenotypes or antibodies. Immunohematology. 2006;22:52-7.

Publication Dates

  • Publication in this collection
    07 Dec 2020
  • Date of issue
    Oct-Dec 2020

History

  • Received
    17 July 2018
  • Accepted
    15 Aug 2019
Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular (ABHH) R. Dr. Diogo de Faria, 775 cj 114, 04037-002, São Paulo / SP - Brasil - São Paulo - SP - Brazil
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