Serologic strategy in detecting RHD altered alleles in Brazilian blood donors

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.

Frecuencia de fenotipos del sistema Rh en donantes voluntarios de sangre / Frequency of Rh system phenotypes in voluntary blood donors / Frequência de fenótipos do sistema Rh em doadores de sangue voluntários

El sistema Rh es altamente polimórfico y está relacionado con la producción de aloanticuerpos y la enfermedad hemolítica del recién nacido. Los antígenos codificados por los genes RHD y RHCE forman el fenotipo Rh que es característico en cada población. Las variantes RHCE no han sido identificadas en la población ecuatoriana y así constituyen un riesgo de aloinmunización durante el embarazo o en transfusiones de componentes sanguíneos incompatibles. Prueba de ello es el estudio realizado en Ecuador que determinó una aloinmunización del 0,27%. Los anticuerpos con mayor frecuencia pertenecían al sistema Rh, resultados que motivaron la realización del presente estudio. Se analizaron un total de 1.298 muestras de donantes de sangre provenientes de 22 provincias ecuatorianas. Para la fenotipificación se utilizaron antisueros comerciales de la casa BIORAD y células de fenotipo conocido para el control de calidad interno, y se identificaron 20 fenotipos del sistema Rh distribuidos de forma heterogénea en las 22 provincias; el más frecuente fue Rz/R0. En donantes con fenotipo D débil el más común fue el R2/r, mientras que en los donantes Rh(D) negativo fue el fenotipo r/r. Estos datos demuestran la variedad de fenotipos en la población ecuatoriana y por ende la necesidad de su detección oportuna.

The Rh system is highly polymorphic. It is related to the production of alloantibodies and the hemolytic disease of the newborn. The antigens encoded by the RHD and RHCE genes form the Rh that is characteristic for each population. The RHCE variants have not been identified in the Ecuadorian population, constituting a risk of alloimmunization during pregnancy or in transfusions of incompatible blood components. Proof of this is the study carried out in Ecuador that determined an alloimmunization of 0.27% and the antibodies, more frequently belonged to the Rh system, results that motivated the realization of the present study. A total of 1298 samples from blood donors from 22 Ecuadorian provinces were analyzed. For the phenotyping, commercial antisera from the BIORAD house were used and cells of known phenotype for internal quality control. Identifying 20 phenotypes of the Rh system distributed heterogeneously in the 22 provinces, the most frequent was Rz/R0. In donors with weak D phenotype the most common was R2/r; whereas in Rh(D) negative donors was the r/r phenotype, these data demonstrate the variety of phenotypes in the Ecuadorian population and therefore the need for their timely detection.

O sistema Rh é altamente polimórfico e está relacionado com a produção de aloanticorpos e a doença hemolítica do recém-nascido. Os antígenos codificados pelos genes RHD e RHCE formam o fenótipo de Rh, que é característico para cada população. As variantes de RHCE não foram identificados na população equatoriana constituindo um risco de aloimunização durante a gravidez ou em transfusões de componentes sanguíneos incompatíveis. Prova disso é o estudo realizado no Equador que determinou aloimunização de 0,27%. Os anticorpos com maior frequência pertenciam ao sistema Rh, resultados que motivaram a realização do presente estudo. Um total de 1298 amostras de doadores de sangue de 22 estados equatorianos foram analisadas. Utilizou-se para a fenotipificação anti-soros comerciais BIORAD e células de fenótipo conhecido para controle de qualidade interno, identificando-se 20 fenótipos do sistema Rh heterogeneamente distribuídos nos 22 estados. O mais frequente foi Rz/R0. Em doadores com fenótipo D fraco, o mais comum foi o R2/r; ao passo que nos doadores Rh (D) negativo foi o fenótipo r/r. Esses dados demonstram a variedade de fenótipos na população do Equador, e portanto a necessidade da detecção precoce dos mesmos.

A Case of RhD with Anti-D / 대한수혈학회지

We report here on a case of a RhD blood group phenotype with anti-D. The RhD phenotype for partial D phenotyping with using six monoclonal anti-sera typed as normal RhD for this case. DNA sequencing analysis of the RhD gene covering intron 8 to exon 10 showed two AAATAAGATA insertion sites in intron 8 and a single nucleotide change in the exon 10 area as compared with the normal RhD gene. However, the functional role of the RhD antigen is unclear.

Analysis of Partial D Subtypes by Various Anti-D Reagents / 대한수혈학회지

BACKGROUND: There are some previous reports about partial D in Korea. However, the frequency of the partial D in Korea is still unknown. In this study, subtypes of partial D were analyzed by the use of various commercially available anti-D reagents. METHODS: We collected 273 cases determined as RhD negative by RhD typing using the tube method with monoclonal IgM/IgG anti-D reagent (Bioscot. Livingston, UK) from 80,062 cases that were screened between January 2004 and August 2007. The cases were divided into three periods (I, II, III), according to the manufacturers and numbers of anti-D reagents used. A weak D test was performed by using the tube method with various anti-D reagents. The cases with different reactivity between anti-D reagents were determined as partial D, and further analyzed the subtypes by reactivity patterns according to the target epitope of anti-D reagents. An additional test using the ID-Partial D Typing Card (DiaMed, Cressier, Switzerland) was conducted during period III. RESULTS: Five cases showed reactivity patterns of weak D and 16 cases showed reactivity patterns of partial D. Ten cases of partial D were typed as DVI and three cases were typed as DFR. During period III, five cases were typed as DVI and one case was typed as DFR. These results were different from the results obtained with the use of the ID-Partial D Typing Card. CONCLUSION: DVI, which is the most common subtype of partial D, is also common in Korea. Therefore, RhD typing and a weak D test should be performed using combined anti-D reagents that enable the differentiation of DVI from other subtypes.

Reactivity Patterns of Various Anti-D Reagents in 14 Cases with Partial D / 대한진단검사의학회지

BACKGROUND: A weak D type resulted from a quantitative reduction of the RhD antigen, whereas a partial D type resulted from a qualitatively altered RhD protein. Based on different serological properties from a weak D type, a partial D type was suspected in cases with anti-D in their serum or if nonreactive to some reagents. Most Red Cross Blood Centers pay attention to donors in determining RhD typing with a monoclonal anti-D reagent. This study examined the reactivity patterns of 4 different monoclonal anti-D reagents in RhD typing and a weak D test in 14 cases with partial D. MATERIALS AND METHODS: We collected a total of 201, 847 samples from blood donors and screened out 649 samples as Rh-negative in RhD typing with monoclonal anti-D (Bioscot) and bromelin treatment applied to an automatic analyzer between October 2002 and March 2003. Further, we performed RhD typing and weak D test using the tube method with 4 commercially available monoclonal anti-D reagents. In 14 cases with different reactivity patterns, we performed a confirming test for partial D using a `ID-partial RhD-typing' (Diamed, Switzerland) set consisting of 6 monoclonal antibodies. RESULTS: Partial D(DFR) was observed in 92.9% (13/14) and a partial D(indeterminate) was observed in 7.1% (1/14). The red blood cells from 14 cases with partial D were not agglutinated with 4 various commercially available anti-D reagents. However, in subsequently performed weak-D tests, different reactivity to their anti-D reagents were shown, namely irresponsiveness (Dade Behring, 14/14, 100%), trace-to-1+ responsiveness (Ortho-clinical diagnostics, 13/14, 92.9%), trace-to-3+ responsiveness (Bioscot, 14/14, 100%), and 1+-to-3+ responsiveness (GreenCross, Korea, 14/14, 100%). CONCLUSIONS: Considering that the most partial D discovered in the Southwestern area of Korea was partial D(DFR), it is recommended that RhD typing and/or weak D tests in blood donors should be done using more than two anti-D reagents from different clones.

Two cases of partial-D showing different reactivity to various anti-D reagents / 대한수혈학회지

The 19-year-old twin sisters donated their blood in 2000. Their blood had typical Rh D negative red cell phenotype in Rh typing and weak D test using an anti-D reagent (Dade Behring, USA). Twin sisters donated blood again in 2001. Both were negative in anti-D reagent (Bioscotte Ltd., UK) and weakly positive in additively performed weak-D test. So we have acquired blood samples from them for further study in 2002. The red blood cells from twin sisters were not agglutinated with 4 various commercially available anti-D reagents. But in subsequently performed weak-D test, different reactivity to their anti-D reagents were shown, namely negative (Dade Behring, USA) and weakly positive (Ortho-clinical diagnostics, USA; Greencross, Korea; Bioscotte Ltd., UK). The lack of reactivity with some anti-D as shown in these cases can indicate the presence of a partial D antigen. So we carried out a additional serologic test using 6 monoclonal anti-D antibodies in partial-D typing set (Diamed, Switzerland) on Rh D antigens of red cells from twin sisters. According to the different reactivity patterns, we confirmed elder sister was partial-D category DFR and younger sister was partial-D with indeterminate category.

A Case of Partial-D Category Va / 대한수혈학회지

The patient, a 65-year-old woman, was admitted for chronic subdural hematoma. ABO and Rh blood typing were performed as a pre-operation test. Her red blood cells were not agglutinated with anti-D reagent (Ortho Diagnostic System, USA). But they were positive in subsequently performed weak-D test and also agglutinated with three other anti-D reagents (Baxter Dade, USA; Biotest Diagnostics, Korea; Bioscot Ltd., UK). The patient s Rh phenotype was CcDe. Antibody screening test, direct and indirect antiglobulin tests showed negative results. Different reactivity to various anti-D reagents as shown in this case suggested that her cells have partial-D antigen which lack one or more components of the Rh D antigen. We considered that this case was category Va according to the reactivity patterns of monoclonal anti-D antibodies with various partial- D cells.

Study on the molecular genetic basis of weak D phenotypes in Han population of Northern China / 中国输血杂志

A mutation is predominant in weak D individuals,and DⅥⅢ mutation in partial D individuals.