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Distribution of weak D types in Croatian population

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Vesna Dogic at Croatian Institute of Transfusion Medicine
Vesna Dogic
Jasna Bingulac-Popovic at Croatian Institute of Transfusion Medicine
Jasna Bingulac-Popovic
Ivana Babic at Croatian Institute for Transfusion Medicine
Ivana Babic
Zeljka Hundric-Haspl
Zeljka Hundric-Haspl
Zeljka Hundric-Haspl
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Transfusion Medicine, 2011, 21, 278– 279 doi: 10.1111/j.1365-3148.2011.01071.x
LETTER TO THE EDITOR
Distribution of weak D types in the Croatian population
Dear Sir,
The Rh blood group system is determined by the
highly homologous RHD and RHCE genes located on
the first chromosome, which encode for the RhD and
RhCE polypeptides. D antigen is of special clinical rele-
vance in the fields of transfusion medicine and obstetrics.
Owing to its high immunogenicity, D antigen can induce
the production of alloantibodies and thus cause post-
transfusion haemolytic reaction and haemolytic disease
of the newborn (Wagner et al., 2000).
About 0·2–1% of the European population are car-
riers of structurally altered RHD alleles encoding for
various types of weak D proteins. At the molecular
level, point mutations resulting in amino acid substi-
tutions in the intracellular or transmembranous seg-
ments of RhD protein are causing weak D phenotypes.
More than 170 different RHD alleles closely related to
the expression of the respective D phenotype, includ-
ing more than 70 weak D types, have been discov-
ered to date (Flegel, 2007). Some weak D types (types
1, 2 and 3) are not associated with the development
of alloantibodies; however, alloimmunisation in weak
D types 4·2, 11 and 15 carriers have been reported
(Flegel, 2006). Owing to the extremely small pheno-
typic variation, particular weak D types are very difficult
to differentiate by serology and can only be identified
by molecular methods, thus enabling definitive deci-
sion on the mode of transfusion treatment and the need
of anti-D prophylaxis in pregnant women. The indi-
viduals who are carriers of weak D types 1, 2 and 3
can receive transfusion of D+red blood cell (RBC)
units, although such pregnant women do not require
anti-D prophylaxis. Thus, the unnecessary utilisation of
D−RBC units and RhIg is avoided (Flegel and Wagner,
2002).
Particular segments of the RHD gene sequence are
multiplied by RHD genotyping using primers specific
for the known mutations characterising particular weak
D types by use of the polymerase chain reaction with
sequence-specific priming (PCR-SSP). This procedure
is employed to determine polymorphism of the weak
D types.
Correspondence: Vesna Dogic, MD, Head, Department of Molecular
Immunogenetics, CITM, Petrova 3, 10000 Zagreb, Croatia.
Tel .: +385 1 4600328; fax: +385 1 4600307;
e-mail: vesna.dogic@hztm.hr
Genetic RHD gene variations differ among ethnic
groups and populations. The distribution of weak D
types in three Central European regions (northern and
southwestern Germany and Tyrol in Austria) greatly
varies. In the population of Tyrol, type 3 is most
common (50%), whereas type 1 is found in 33% and
type 2 in 8% of the population. The populations of
northern and southwestern Germany have the highest
frequency of type 1 (65 and 69%) and lower frequency
of type 3 (17 and 4%), respectively (M¨
uller et al.,
2001). Similar figures have been recorded in Denmark,
where weak D type 1 accounts for 67% of all weak D
types (Christiansen et al., 2008). Considerable deviation
of weak D type distribution has been recorded in the
population of Portugal, with the highest frequency of
weak D type 2 accounting for 63·6% of all weak D
types, whereas types 1 and 3 are present in 16·2and
14·1%, respectively (Araujo et al., 2006).
As there are no literature data on the molecular basis
of weak D types in the Croatian population, the aim
of the study was to assess the distribution of weak D
types in Croatia and to compare it with the respec-
tive data from other European countries. In Zagreb and
Split, the two Croatian transfusion centres collecting
approximately 51% of all blood units in Croatia, 167
samples were selected after discrepant results of test-
ing for D with monoclonal anti-D reagents between
2002 and 2009. Zagreb represented the population of
central Croatia and Split represented the Mediterranean
part of Croatia. DNA extraction from ethylenediaminete-
traacetic blood samples was done by QIAamp DNA
Blood Mini kit (Qiaqen, Hilden, Germany) or MagNA
Pure LC (Roche Molecular Biochemicals, Mannheim,
Germany). Molecular typing of weak D types was per-
formed by PCR-SSP using a genotyping kit (Weak
D-SSP, Inno-Train, Kronberg, Germany) that enables
genotyping to 11 different weak D types (1, 2, 3, 4·0, 4·1,
4·2, 5, 11, 14, 15 and 17) frequently found in Europe.
Additional RHD typing was performed by Ready Gene
CDE and Ready Gene D neg kits (Inno-Train, Kron-
berg, Germany).
Seven different weak D types (1, 2, 3, 4·2, 11/RHD
(M295I), 14 and 15) were determined in the Croatian
population. None of the 167 samples remained unre-
solved by the assay and none harboured two weak D
types simultaneously. Type 3 was the most prevalent
©2011 The Authors
278 Transfusion Medicine ©2011 British Blood Transfusion Society
Letter to the Editor 279
weak D type (46·1%), followed by type 1 (37·7%), type
14 (8·4%), type 2 (3·6%), type 11/RHD(M295I) (3·0%)
and types 4·2 and 15 (0·6% each). All five weak D
type 11 cases were found to have CcDdee genotype
and were considered as DEL phenotype associated with
RHD(M295I) (K¨
orm¨
oczi et al., 2005; Polin et al., 2009).
Statistical analysis of data between central and Mediter-
ranean regions of Croatia showed significant differences
in the frequency of weak D types 1, 3, 11/RHD(M295I)
and 14 (Pearson’s χ2test; χ2=24·75, P<0·001). In
central Croatia, type 3 (49·6%) predominated, followed
by type 1 (34·2%), type 14 (10·6%) and no case of type
11/RHD(M295I). In the Mediterranean part of Croatia,
weak D type 1 (47·7%) prevailed, followed by type 3
(36·4%), DEL RHD(M295I) (11·4%) and no case of
type 14. The distribution of weak D types in Croatia
showed some specificity possibly connected with intra-
ethnic variation. The high prevalence of weak D type
3 was only comparable to data from Tyrol. There are a
few possible explanations for this frequency similarity.
First, it could be postulated that populations from cen-
tral and southeast Europe have a higher prevalence of
weak D type 3 as compared to other European regions.
However, data on the distribution of weak D types in
other countries of southeast Europe (especially former
Yugoslavia countries) are missing. Second, it might be
due to historical reasons as Croatia had been part of
the Austro-Hungary kingdom in the past. Third, eco-
nomic migration of Croats and other southeast European
nationalities to Austria in the middle of the last century
may have influenced the frequency of weak D type 3
in Austrian population. The high frequency of weak D
type 14 is peculiar for the population of Croatia. Weak D
type 4·2, rarely present in Europe but common in Africa,
was found in our Mediterranean region, which might be
ascribed to the geographical position of Croatia in the
international maritime traffic.
Study results revealed geographical variation in the
distribution of weak D types between Croatian popu-
lation and other European populations. This study also
indicated that the use of RHD gene molecular genotyp-
ing in cases of ambiguous serologic interpretation of D
antigen would allow for the use of D+RBC units in
87·4% of cases (weak D types 1–3), thus obviating the
need of RhIg (Flegel, 2006). Such an approach would
lead to a more conservative and rational management of
D−RBC unit supply in Croatian transfusion medicine.
ACKNOWLEDGMENTS
V. D. designed the research study, performed the
research, analysed the data and wrote the paper. J. B.-P.
designed the research study, performed the research,
analysed the data and wrote the paper. I. B. performed
the research. Z. H.-H. designed the research study and
revised the paper critically. N. J.-L. and J. M.-M. con-
tributed essential reagents or tools. T. V. analysed the
data and revised the paper critically. M. B. and I. J.
approved the submitted and final versions.
CONFLICT OF INTEREST
All authors declare no conflict of interest.
V. Dogic,1J. Bingulac-Popovic,1I. Babic,1
Z. Hundric-Haspl,2N. Jurakovic-Loncar,2
J. Mratinovic-Mikulandra,3T. Vuk,4M. Balija5
&I.Jukic6
1Department of Molecular Immunogenetics,
2Department of Immunohematology, CITM, Zagreb, and
3Department of Transfusion Medicine, Split University
Hospital Center, Split, and 4Department of Quality
Control and Quality Assurance, 5Medical Department,
and 6CITM, Zagreb, Croatia
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©2011 The Authors
Transfusion Medicine ©2011 British Blood Transfusion Society, Transfusion Medicine,21, 278–279
... 22 In fact, in some large cohorts of European individuals screened for weak D phenotype, this variant was not encountered. 5,23 Referring to weak D type 11, the frequencies in literature vary because of an increase in the sensitivity of serologic methods for D testing, and, consequently, the ability to detect this variant has also increased over time. 23,24 The population frequency of weak D type 11 associated with the CDe haplotype in southwestern Germany was 0.015 percent, higher than that found in our cohort (0.002 percent). ...
... 5,23 Referring to weak D type 11, the frequencies in literature vary because of an increase in the sensitivity of serologic methods for D testing, and, consequently, the ability to detect this variant has also increased over time. 23,24 The population frequency of weak D type 11 associated with the CDe haplotype in southwestern Germany was 0.015 percent, higher than that found in our cohort (0.002 percent). 24 Weak D type 38 was first observed among white individuals with weak D phenotype. ...
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Full-text available
  • Jun 2020
  • Immunohematol
Conclusions: Recent evidence shows that, among Brazilians, the distribution of weak D types significantly differs from that represented in people of European descent, with a high percentage of weak D types 38 and 11. Our goal was to determine the population frequencies of weak D types 38 and 11 in a Brazilian population and to validate a molecular approach to identify these two variants. Blood donors were sequentially enrolled in the study in a 5-year period. Donors with serologic weak D phenotype had the RHD coding region sequenced. The frequencies of weak D type 38 and weak D type 11 (CDe-associated) were calculated. Two allele-specific-polymerase chain reaction (AS-PCR) assays were designed to detect RHD*weak D type 38 and RHD*weak partial 11 and were validated with samples positive and negative for these two variants, respectively. A total of 618,542 donors were enrolled, of which 265 presented with a serologic weak D phenotype. When considering all donors evaluated, the frequencies of weak D types 38 and 11 were 0.013 and 0.002 percent, respectively. In the subgroup of donors with a serologic weak D phenotype, the frequencies of weak D types 38 and 11 were 30.2 and 4.9 percent, respectively. The two proposed AS-PCR assays for detection of RHD*weak D type 38 and RHD*weak partial 11 showed 100 percent accuracy. The frequencies of weak D types 38 and 11 among Brazilians are high compared to that previously described for other populations. The AS-PCR assays to detect RHD*weak D type 38 and RHD*weak partial 11 represent potentially helpful tools for investigating Brazilian individuals with these weak D phenotypes.
View
... The heterogeneity of erythrocyte antigen distribution 41 along with the AB phenotype may have caused the mixed-field agglutination, not typically expected for the A 2 phenotype, and erroneously pointing to the possibility of two RBC populations. [32][33][34][35] The RHD genotyping kit applied in our routine clinical protocol incorrectly determined an RHD*01W.14 allele 26,42 (Table 2). This molecular result was discordant with the African American ethnicity of the patient and the serologic Rh phenotype CcDe, because RHD*01W.14 has previously only been observed in the white population and is associated with the RHCE*cE allele, 26 whereas this patient was E-. ...
Transfusion support for a woman with RHD*09.01.02 and the novel RHD*01W.161 allele in trans
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According to recent work group recommendations, individuals with the serologic weak D phenotypes should be RHD genotyped and individuals with molecular weak D types 1, 2, 3, 4.0, or 4.1 should be treated as D+. We report an African American woman with a long-standing history of metrorrhagia, who presented for infertility evaluation. Blood grouping showed AB with a possible subgroup of A, based on mixed-field agglutination, and a serologic weak D phenotype. Results from routine red cell genotyping for the RHD gene was incongruent with the serologic RhCE phenotype. For the surgical procedure, the patient was hence scheduled to receive group AB, D- RBC transfusions. Subsequent molecular analysis identified the ABO*A2.01 and ABO*B.01 alleles for the ABO genotype and the novel RHD allele [NG_007494.1(RHD):c.611T>A] along with an RHD*09.01.02 allele for the RHD genotype. Using a panel of monoclonal anti-D reagents, we showed the novel RHD(I204K) allele to represent a serologic weak D phenotype, despite occurring as a compound heterozygote, designated RHD*weak D type 161 (RHD*01W.161). Individuals with a weak D type 4.2 allele are prone to anti-D immunization, while the immunization potential of novel RHD alleles is difficult to predict. For now, patients should be treated as D- in transfusion and pregnancy management, when they harbor a novel RHD allele along with any weak D allele other than weak D types 1, 2, 3, 4.0, or 4.1. This study exemplifies strategies for how and when a laboratory should proceed from routine genotyping to nucleotide sequencing before any decisions on transfusion practice is made.
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... Most serological weak D phenotypes (>95 % in Northern Europeans) are the expression of weak D types 1, 2, and 3 [2]. Weak type 4.0, 4.1, 4.2, and 4.3 [2,42,43], type 11 [2,43], type 15 [2,34,43,44], and type 5 [43] are rare weak D variants in Caucasians.Among a different ethnic population, e.g., most frequent weak D alleles in southern Egypt were found to be type 4 [45]. In the northeastern Chinese population, type 15, DVI type 3, and R8HD1227A were reported as the most prevalent D variant alleles [46]. ...
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View
... Most serological weak D phenotypes (>95 % in Northern Europeans) are the expression of weak D types 1, 2, and 3 [2]. Weak type 4.0, 4.1, 4.2, and 4.3 [2,42,43], type 11 [2,43], type 15 [2,34,43,44], and type 5 [43] are rare weak D variants in Caucasians.Among a different ethnic population, e.g., most frequent weak D alleles in southern Egypt were found to be type 4 [45]. In the northeastern Chinese population, type 15, DVI type 3, and R8HD1227A were reported as the most prevalent D variant alleles [46]. ...
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Serological and molecular characterisation of the most prevalent weak D variants in Croatian population
Article
  • Dec 2022
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Introduction: Existence of hundreds of RHD gene variants contributes to variable D antigen expression and inconsistencies in reporting the RHD results. The aim of the study was to determine the serological and molecular characteristics of the most prevalent RHD alleles encoding serologically weak D variants. Material and methods: Blood donors (n = 145 924) were typed for D antigen using the direct serologic micromethod. Nonreactive samples were analysed in IAT method with the IgM/IgG anti-D monoclonal blend, and 0,2% (n = 263) confirmed weak D antigen expression. After genomic DNA extraction (Qiaqen, Germany), RHD genotyping was performed using in house reagents and PCR-SSP kits (Inno-Train, Germany). Results: The prevalence of serologically weak D in blood donor population was 0.2% (n = 263). RHD genotyping confirmed weak D allele in 92.4% and partial D allele in 7.6%. The most common was weak D type 1 (49.7%) followed by weak D type 3 (24.7%) and type 2 (9.5%). Relatively high frequency was detected for weak D type 14 (4.6%) and type 64 (2.3%). In the category of partial D phenotypes, only DVI variant was found. Direct typing has shown great variability in the strength of reactions with different clones of anti-D reagents. Conclusion: Weak D type 1 is the most common weak D variant in Croatian blood donor population. The frequency of D variants and distribution of Rh phenotypes in our study was in concordance with other studies. It has been shown that serological methods and the combination of clones used, cannot distinguish variant D types, which justifies the use of molecular methods.
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Objective: This work proposes a review of the antibodies which have been associated with variant RHD and RHCE alleles, except null alleles. Background: The data on this topic is dispersed in the literature. Methods: A review of the articles referenced in PubMed and of abstract books from major conferences was performed. Most antibodies have been published in full-length articles, and several more have been reported in conference abstracts. The anti-D antibodies reported in carriers of D variants and the antibodies to CE antigens reported in carriers of CE variants were listed, including antibodies to low prevalence antigens. The RHCE alleles for which the RH10 (V) and RH20 (VS) phenotypes have been reported were also collected. The reports of antibody formation were compared to the prevalence evaluated by the Erythrogene database in the 1000 Genomes dataset. Conclusions: It is noted in this review that studies reporting anti-D or antibodies to CE antigens associated with Rh variants only rarely include detailed serological descriptions of the findings. This review lists several alleles which are not exceptional, and for which no carrier has been reported to form the antibody to the expressed antigen(s) (e.g., no allo-anti-D has been reported so far in carriers of RHD*01EL.01, c.1227A). Considering the antibody reports in carriers or absence thereof and the prevalence for each RH allele, it may become possible to propose case-by-case recommendations for more RH alleles in the near future.
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Frequency and characterization of RHD variant alleles in a population of blood donors from southeastern Brazil: Comparison with other populations
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BACKGROUND The correct determination of D antigen could help to avoid alloimmunization in pregnant women and patients receiving blood transfusions. However, there are limitations in the identification of D variants as the partial and weak D phenotypes make the determination of D antigen a great challenge in the transfusion routine.’ STUDY DESIGN AND METHODS The molecular characterization of D variants was performed on blood donors from southeastern Brazil with atypical D typing. Furthermore, the serological profile of all RHD variant alleles identified was analyzed using different Anti-D clones. The prevalence of RHD alleles and genotypes found was compared with those described in other countries and in other regions from Brazil. RESULTS Atypical serologic D typing occurred in 0.79% of blood donors. The majority of RHD variant alleles (88%) were first characterized by multiplex PCR and PCR-SSP as RHD*weak partial 4 (47%), followed by RHD*weak D type 3 (29.9%), RHD*weak D type 2 (3.9%) and RHD*weak D type 1 (3.1%). Genomic DNA sequencing characterized the RHD*weak partial 4 variants found in RHD*DAR1.2 (weak 4.2.2) (22%), RHD*DAR3 (weak 4.0.1) (2.4%), RHD*DAR3.1 (weak 4.0) (22%) and RHD*DAR4 (weak 4.1) (0.8%). RHD variant alleles associated with partial D, such as, RHD*DAU-4 (1.6%), RHD*DAU-5 (2.4%), RHD*DAU-6 (1.6%), RHD* DIII type 8 (1.6%), RHD*DVII (3.9%) and RHD* DMH (0.8%) were also observed. CONCLUSION The prevalence of RHD variant alleles observed in this cohort differ from those found in other populations, including Brazilians from other regions. RHD allele distribution in specific regions should be considered for implementation of algorithms and genotyping strategies aiming at a more effective and safe transfusion.
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RHD and RHCE genes polymorphism: literature review
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The article provides a literature review about RHD and RHCE polymorphisms which encode different RhD and RhC antigen variants. The data about genes RHD and RHCE polymorphisms, RhD weak types, RhD partial types and RhC variants in Russians is presented for the first time. The molecular and serological characteristics of rare RhD and RhC antigens are summarized. The role of serological and molecular methods in Rhesus system antigens identifying is shown.
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Article
  • Nov 2020
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The genetics of the Rhesus Blood Group system
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Full-text available
  • May 2007
Introduction: Rhesus blood group antigens are expressed by the RhD and the RhCE proteins. Rhesus is the most important protein based blood group system, clinically. It comprises the largest number of antigens and the most complex genetics of all 29 known blood group systems. The molecular genetic basis has been elaborated in detail over the last 15 years. Some serological characteristics have been confirmed during this period, where others have been recognized for the first time, using molecular genetic techniques. Methods: Selective literature review. Results: The RHD and the RHCE gene are strongly homologous and located adjacent to one another at chromosome position 1p36.11. Part of the genetic complexity is explained by the unusual orientation of both genes on chromosome 1 with their tail ends facing each other. In the case of Rhesus, molecular polymorphisms can be compared with antigens which have been precisely characterized. Discussion: Rhesus exemplifies the correlation of genotype and phenotype, facilitating the understanding of underlying genetic mechanisms. For clinical purposes, genetic diagnostics of blood group antigens will improve the cost effective development of transfusion medicine.
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PCR screening for common weak D types shows different distributions in three Central European populations
Article
Full-text available
  • Jan 2001
DNA sequencing showed RHD mutations for all weak D phenotypes investigated in a study from Southwestern Germany. Molecular classification of weak D offers a more reliable basis than serotyping and is relevant for optimal D transfusion strategies. Sequence-specific primers were designed to detect weak D types 1 to 5 and the partial D phenotype HMi in a modular set for conventional PCR analysis. Alternatively, all reactions were multiplexed into a single tube, and the products were identified after automated capillary electrophoresis by their size and fluorescence. Weak D phenotype samples from 436 donors in the Tyrol (Austria) and Northern Germany were investigated by PCR. More than 90 percent of the weak D types identified by PCR represented type 1, 2, or 3. The distribution among the common types varied between the Tyrol and Northern Germany (p<0.0001). Three new RHD alleles were identified. A PCR method of detecting the common weak D types was validated. This PCR system introduces a simple and rapid tool for routine DNA typing of weak D samples. The results confirmed that all weak D phenotype samples identified by current serologic criteria carry altered D proteins.
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Molecular biology of partial D and weak D: Implications for blood bank practice
Article
Full-text available
  • Feb 2002
  • CLIN LAB
Two genes, RHD and RHCE, encode the antigens of the RH blood group system. The clinically most important antigen D is determined by the presence of a functional and grossly normal RHD gene. About 18% of Europeans do not express an antigen D, most often but not always caused by the RHD gene deletion. Rhesus negative phenotypes in Africans are caused by the RHD gene deletion, the RHD pseudogene RHD psi, and the Cde(s) allele. About 1% of Europeans carry RHD alleles with aberrant structures encoding for diminished D-immunoreactivity. In Africans the frequency of aberrant RHD alleles is much higher. Aberrant RHD alleles encode partial D, some of which were dubbed D categories, and weak D. Since we defined the molecular basis of the RHD deletion, a specific detection of heterozygous carriers became feasible.
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A comprehensive analysis of DEL types: partial DEL individuals are prone to anti-D alloimmunization
Article
Full-text available
  • Oct 2005
The D antigen of the polymorphic Rh blood group system is of particular clinical importance regarding transfusion- and pregnancy-induced alloimmunization. Different RhD variants with specific clinical implications have been characterized. The least expressed D variants collectively called DEL are serologically detectable only by adsorption-elution techniques, with so far only poorly defined antigenic properties. A comprehensive immunohematologic analysis of five of the six currently known DEL genotypes was performed. DEL phenotypes associated with the RHD(M295I), RHD(IVS3+1g>a), RHD(K409K), RHD(X418L), or RHD(IVS5-38del4) allele were characterized with extended serology and flow cytometry. Epitope mapping with adsorption-elution revealed a prominent D epitope loss in the RHD(IVS3+1g>a)-associated DEL phenotype, whereas in the other four DEL types no signs of qualitative D antigen alteration were detected. The observation of alloanti-D in two RHD(IVS3+1g>a) cases confirmed the partial nature of this DEL phenotype. The RHD(M295I) phenotype exhibited the highest D antigen expression among all investigated DEL types, as determined by a semiquantitative adsorption-elution approach and flow cytometry. In conclusion, evidence is provided that different DEL genotypes code either for partial or complete D antigen expression and that this finding is clinically relevant.
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Weak D type 2 is the most prevalent weak D type in Portugal
Article
Full-text available
  • Mar 2006
summary. The weak D phenotype is the most common D variant, with a frequency of 0·2–1% in Caucasian individuals. There are several weak D types, with different frequencies in European countries, which may pose serologic problems and have the potential for alloimmunization. Samples from Portuguese individuals were tested for RhD by two or three distinct monoclonal and oligoclonal antisera, in direct agglutination tests. When discrepant results were observed, samples were tested with panels of monoclonal anti-D by LISS-indirect antigobulin test. Cases that reacted weakly with IgM but positive with IgG anti-D were analysed by PCR-sequence-specific primers and real-time PCR. Ninety-nine samples were referred after being characterized as weak D. This genotype was recognized, with a preponderance of weak D type 2 (63·6%) over type 1 (16·2%) and 3 (14·1%). The high incidence of weak D type 2 in our population is in marked contrast to studies performed in other European populations and might be due to our sample selection criteria or ethnic variation. There are advantages in genotyping serologically depressed D samples to avoid the waste of D-negative RBC units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization.
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Weak D alleles express distinct phenotypes
Article
  • Apr 2000
The weak D phenotype is caused by many different RHD alleles encoding aberrant RhD proteins, raising the possibility of distinct serologic phenotypes and of anti-D immunizations in weak D. We reported 6 new RHD alleles, D category III type IV, DIM, and the weak D types 4.1, 4.2.1, 4.2.2, and 17. The immunohematologic features of 18 weak D types were examined by agglutination and flow cytometry with more than 50 monoclonal anti-D. The agglutination patterns of the partial D phenotypes DIM, DIII type IV, and DIVtype III correlated well with the D epitope models, those of the weak D types showed no correlation. In flow cytometry, the weak D types displayed type-specific antigen densities between 70 and 4000 RhD antigens per cell and qualitatively distinct D antigens. A Rhesus D similarity index was devised to characterize the extent of qualitative changes in aberrant D antigens and discriminated normal D from all tested partial D, including D category III. In some rare weak D types, the extent of the alterations was comparable to that found in partial Ds that were prone to anti-D immunization. Four of 6 case reports with anti-D in weak D represented auto-anti-D. We concluded that, in contrast to previous assumptions, most weak D types, including prevalent ones, carry altered D antigens. These observations are suggestive of a clinically relevant potential for anti-D immunizations in some, but not in the prevalent weak D types, and were used to derive an improved transfusion strategy in weak D patients.
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Identification of RHD alleles with the potential of anti-D immunization among seemingly D- blood donors in Upper Austria
Article
  • Feb 2009
  • TRANSFUSION
Aberrant RHD alleles leading to a reduced expression of D antigen on the red blood cell (RBC) surface may be mistyped as D- by serology. To quantify the occurrence of weak D, DEL, and D+/- chimera among apparent D- first-time blood donors, polymerase chain reaction (PCR) screening was implemented as a routine service. A total of 23,330 pretyped D- samples were tested for RHD markers in Exons 4, 7, and 10 in pools of 20 by PCR. Samples with positive results in PCR were reevaluated by exon-specific PCRs, DNA sequencing, and serologic methods. Among 94 PCR-positive samples, 74 exhibited a weak D or DEL phenotype, dubbed weak D type 1, weak D type 2, weak D type 5, weak D type 32, weak D type 4.3, RHD(M295I), RHD(del147), and RHD(1227G>A). The most prevalent alleles were weak D type 4.3 (n = 31) and RHD(IVS3+1G>A) (n = 24). As a clinical consequence, 74 blood donor samples carrying weak D and DEL phenotypes with the potential of causing secondary immunizations in recipients were reclassified as D+. Those samples were reliably amplified by RHD Exon 7 PCR; therefore, its usage in the Upper Austrian population is recommended. The association of the weak D type 4.3 samples with a ce leads to the policy that all apparently D- donors should be tested with genotyping methods; otherwise, potentially immunogenic RHD alleles may be overseen.
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Weak D alleles express distinct phenotypes
Article
  • May 2000
The weak D phenotype is caused by many different RHD alleles encoding aberrant RhD proteins, raising the possibility of distinct serologic phenotypes and of anti-D immunizations in weak D. We reported 6 new RHD alleles, D category III type IV, DIM, and the weak D types 4.1, 4.2.1, 4.2.2, and 17. The immunohematologic features of 18 weak D types were examined by agglutination and flow cytometry with more than 50 monoclonal anti-D. The agglutination patterns of the partial D phenotypes DIM, D(III) type IV, and D(IV) type III correlated well with the D epitope models, those of the weak D types showed no correlation. In flow cytometry, the weak D types displayed type-specific antigen densities between 70 and 4000 RhD antigens per cell and qualitatively distinct D antigens. A Rhesus D similarity index was devised to characterize the extent of qualitative changes in aberrant D antigens and discriminated normal D from all tested partial D, including D category III. In some rare weak D types, the extent of the alterations was comparable to that found in partial Ds that were prone to anti-D immunization. Four of 6 case reports with anti-D in weak D represented auto-anti-D. We concluded that, in contrast to previous assumptions, most weak D types, including prevalent ones, carry altered D antigens. These observations are suggestive of a clinically relevant potential for anti-D immunizations in some, but not in the prevalent weak D types, and were used to derive an improved transfusion strategy in weak D patients. (Blood. 2000;95:2699-2708)
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How I manage donors and patients with a weak D phenotype
Article
  • Dec 2006
Since the adoption of molecular blood-group typing, the considerable heterogeneity of the serologic entities weak D and DEL at the molecular level has come to light. I offer an approach to the management of donors and patients expressing D antigen weakly and carrying any of the various molecular types of weak D and DEL. More than 50 distinct weak D alleles have been described. An internet-based survey of anti-D immunizations occurring in D-positive transfusion recipients reveals that no allo-anti-D has been observed in patients carrying prevalent weak D types. Allo-immunizations are documented for weak D types 4.2 (also known as DAR), 11 and 15. Anti-D immunizations have been reported in D-negative persons transfused with weak D and DEL red blood cells. Patients carrying any of the prevalent weak D types 1, 2, 3 or 4.1 are not prone to allo-anti-D immunization and may safely be transfused with D-positive red blood cells. Pregnant women with these weak D types need not receive RhIg. We should pay attention to weak D- or DEL-positive blood units that are labelled D-negative. The clinical benefit of removing DEL blood units from our supply of D-negative red blood cell units should be determined.
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Correlation between serology and genetics of weak D types in Denmark
Article
  • Feb 2008
To date more than 100 variant D types have been reported and the frequencies vary among populations. Blood donor typing should reveal all donors expressing D antigens, while patient typing should prevent the development of anti-D in patients with a D- or variant D blood type. Serotyping is the standard method to assign transfusion strategies, whereas molecular classification offers a more specific grouping of weak and partial D. Blood donor and patient samples with discrepant results of D phenotyping were collected to investigate the frequency of weak D subtypes in Denmark and to evaluate currently used serologic methods. Nine different weak D types were identified among the 101 samples. Weak D Types 1, 2, and 3 constituted 80 percent of the analyzed samples and 10 percent of the samples identified as weak D from serology were actually partial D. The distribution of weak D types in Denmark was found to resemble the distribution in Northern Germany in respect to the three most prevalent weak D types. Correctly defining all samples that show weak reactions in D typing as weak D or partial D is not possible with serotyping alone; genotyping offers the only exact categorization. Serology is superior for routine blood typing, however.
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