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Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2699-2708
RED CELLS
From Abteilung Transfusionsmedizin, Universitätsklinikum Ulm
and DRK-Blutspendedienst Baden-Württemberg, Institut Ulm, Ulm;
Biotest AG, Dreieich, Germany; ZLB Zentrallaboratorium,
Blutspendedienst SRK, Bern, Switzerland; Blutspendedienst des BRK,
München; DRK-Blutspendedienst Niedersachsen-Oldenburg, Institut
Oldenburg, Oldenburg, Germany; and DRK-Blutspendedienst
Sachsen, Institut Dresden, Dresden, Germany.
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 DIV
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)
The Rhesus D antigen (ISBT 004.001; RH1) is expressed
by the RhD protein. Anti-D is still the leading cause for the hemolytic disease of the newborn.1,2 Depending on the population, 3% to 25% of white individuals lack the antigen D. As anti-D
immunizations can occur readily in D-negative recipients,3
the antigen D is of critical importance for the blood transfusion
strategy, and the most important blood group antigen determined by a protein.
About 0.2% to 1% of white individuals carry red cells with a reduced
expression of the D antigen (weak D).4-6 For more than 45 years, anti-D immunizations are known to occur infrequently in
D-positives,7-10 often in individuals with such a low
antigen D density.7 Usually these cases could be traced to
a few partial D phenotypes,11,12 especially D category
VI.10 Most, but not all, partial D can be identified by the
lack of reactivity with certain monoclonal anti-D antibodies, which is
interpreted as lack of certain "D epitopes."13 The
increasingly elaborated D epitope schemes13-16 allowed the
identification and classification of many new partial D.
However, it had been impossible to obtain unequivocal evidence for
serologic differences in the majority of weak D. In such samples, the
lack of reactivity with anti-D may be attributed to the reduced
expression of the D antigen rather than the lack of any D
epitope.11,17,18 As no definitive serologic variation could
be established over the years, it became generally accepted that most
weak Ds possess a normal D antigen.19-23 As a consequence, the possibility of anti-D immunization in weak D was often
disregarded.20,22,23 It should be noted that a substantial
number of anti-D immunizations in weak D24 and in
unclassified samples25 remained unexplained in regard to a
serologic and molecular classification.
We showed that most, if not all, weak D phenotypes carried altered RhD
proteins.26 For example, in the weak D type 4, 2 transmembraneous amino acid residues are substituted, which was reminiscent of ARRO-1 reported as partial D.27 These
findings raised the possibility of qualitative changes in the D
antigen of some weak D types. To corroborate the safety of
the current D-positive transfusion strategy in weak D, a serologic
workup of a representative collection of different weak D types was timely.
With the use of numerous weak D samples of known genotype and more than
80 monoclonal anti-D, we investigated immunohematologic differences of
18 weak D types and 5 partial D, including 5 newly characterized
RHD alleles. The different weak D types presented distinct
immunohematologic features. We derived a Rhesus index, showed its use
as a rough estimate for an anti-D immunization rate, and analyzed 6 case reports of weak D with anti-D. On the basis of our results, we
proposed improved transfusion strategies in weak D patients.
Blood samples
Sequencing of the 10 RHD exons from genomic DNA
Immunohematology Monoclonal anti-D were provided by the Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens.29 Agglutination was tested in a gel matrix test (LISS-Coombs 37°C, DiaMed-ID Micro Typing System; DiaMed, Cressier sur Morat, Switzerland) using the following antibodies: B9A4B2 (number III-1-28), D-89/47 (29), HG/92 (30), D-90/7 (31), D-90/17 (32), D-90/12 (33), HeM-92 (34), 175-2 (35), 17 010C9 (36), NaTH28-3C11 (37), NaTH87-4A5 (38), NaTH53-2A7 (39), AUB-2F7/Fiss (41), CAZ7-4C5 (42), LOR11-2D9 (43), LOR12-E2 (44), LOR17-6C7 (45), LOR17-8D3 (46), LOR28-21D3 (47), LOR28-7E6 (48), LOR29-F7 (49), LORA (50), LORE (51), MAR-1F8 (52), NAU3-2E8 (53), NAU6-1G6 (54), NAU6-4D5 (55), NOI (56), NOU (57), SAL17-4E8 (58), SAL20-12D5 (59), SALSA-12 (60), VOL-3F6 (61), ZIG-189 (62), 822 (68), 819 (69), BTSN4 (71), BTSN6 (72), BTSN10 (73), LHM76/58 (74), LHM76/55 (75), LHM76/59 (76), LHM77/64 (77), LHM59/19 (78), LHM70/45 (79), LHM50/2B (80), LHM169/80 (81), LHM169/81 (82), LHM174/102 (83), LHM50/3.5 (84), LHM59/25 (85), LHM59/20 (86), T3D2F7 (87), C205-29 (88), CLAS1-126 (89), F5S (90), H2D5D2F5 (93), RAB.B15 (94), BIRMA-DG3 (95), BIRMA-D6 (96), BIRMA-D56 (97), P3187 (98), P3F17 (99), P3F20 (100), P3G6 (101), P3AF6 (102), BRAD3 (105), L87.1G7 (108), MS26 (112), MS201 (113), D10 (114), HIRO-1 (115), HIRO-6 (116), HIRO-3 (117), HIRO-4 (118), ID6-H8 (119), HIRO-7 (120), HIRO-8 (121), HIRO-2 (122), MCAD-6 (124), HS114 (134), BS87 (180).Flow cytometry Flow cytometry was performed using monoclonal IgG anti-D BS221, BS227, BS228, BS229, BS231, and H41 (Biotest, Dreieich, Germany); P3 × 35, P3 × 241, P3 × 249, P3 × 290, and HM16 (Diagast, Loos, France) and the following Workshop IgG anti-D: number 29, 30, 31, 32, 33, 36, 41, 43, 44, 45, 47, 49, 55, 56, 58, 59, 68, 71, 72, 73, 75, 76, 77, 80, 81, 82, 89, 90, 93, 94, 95, 96, 97, 101, 102, 105, 108, 112, 114, 117, 118, 119, 120, 121, 122, 124 plus BRAD5 (Workshop number 104), and D6D02 (123). The secondary antibody was goat antihuman IgG, Fab-fragment, FITC-conjugated (supplied by Dianova; Jackson Immunoresearch, Hamburg, Germany).Antigen density The RhD antigen density (RhD antigens per cell) of a red cell sample was estimated as the median of the epitope densities detected with all antibodies that resulted in epitope densities above a cutoff. This cutoff was defined as 0.1 of the 90 percentile of the epitope densities detected with all monoclonal anti-D used in flow cytometry.Rhesus D similarity index A Rhesus index was calculated as the ratio of the 10 percentile to the 90 percentile of the epitope densities detected with all anti-D. For 2 samples (weak D type 12 and type 17), no Rhesus index was given because the 90 percentile was less than 200 epitopes per cell.
Analysis of 6 weak D samples carrying anti-D We differentiated the antibodies in a gel matrix assay (LISS-Coombs 37°C, DiaMed-ID Micro Typing System; DiaMed). In addition, some antibodies were confirmed in a solid phase assay (Capture R; Immucor, Norcross, GA). Because some low-titer auto-anti-D may fail in weak D samples to cause positive direct antiglobulin tests, we performed antibody elutions to determine the presence of red cell-bound antibodies. For all 6 samples, the full-length coding sequence was determined by sequencing all 10 RHD exons from genomic DNA as described previously.Statistics For the interpretation of data, the epitope densities were assumed to have an approximate log-normal distribution; however, all inferences were derived using distribution-independent test methods. Different frequencies were compared using the 2 test
for a 2 × 2 contingency table.31 Antigen densities
of multiple types were compared using the 2-sided Wilcoxon rank sum test for each comparison31 and the Bonferroni-Holm
procedure32 to correct for multiple testing as indicated in
"Results."
Molecular characterization of RHD alleles The molecular bases of 6 RHD alleles were determined (Table 1). Weak D types 4.1, 4.2.1, and 4.2.2 shared both amino acid substitutions, T201R and F223V, that are typical of weak D type 4,26 but each carried 1 additional missense mutation. DIII type IV also displayed multiple dispersed missense mutations. Weak D type 17 and DIM had single missense mutations in the transmembraneous and exofacial protein segments, respectively.
Serology of 17 weak D types and 3 partial D An exhaustive collection of weak D phenotypes and, for comparison, 3 partial D were tested with 82 monoclonal anti-D (Table 2). As known for DIIIa and DIIIc,13,16 DIII type IV was agglutinated by all monoclonal anti-D. This finding corroborated the classification as DIII subtype, based on the negative crossmatch with DIIIc (see "Materials and methods"). DIV type III and DIM displayed antibody reactivity patterns as predicted by the known D epitopes. The reaction pattern of DIV type III differed from that reported for DIVb16 by negative results with 4 of 12 anti-D grouped in epitope 15/16, which may represent a serologic split of this epitope. The reaction pattern of DIM was unique and resembled an intermediate of D category VI18 and DFR.16,33
RhD antigen density of weak D is type-specific A large number of samples with the prevalent weak D types were analyzed for their RhD antigen densities (Table 3). The antigen density was type-specific. With the exception of weak D type 3 and 4, the antigen densities of all prevalent weak D types differed significantly for each pairwise comparison (P < .01; 2-sided Wilcoxon rank sum test with Bonferroni-Holm correction for multiple testing, n = 10).
Suppressive effect of C in trans The antigen densities of 2 CCDee weak D type 1 samples (deduced genotype based on the known CDe haplotype association26 of weak D type 1: CDe/Cde), 1 CcDEe weak D type 2 sample (deduced genotype: Cde/cDE), and 1 CcDee type 4 sample (deduced genotype: Cde/cDe) were determined (Table 4). These samples had considerably lower antigen densities than the controls with cde in trans. For comparison, a weak D type 3 sample with cdE in trans expressed an antigen density similar to its controls.
Epitope density profiles For antigen density determination, epitope density profiles were established with 59 IgG monoclonal anti-D as described previously.18,30 The regular D antigens of a CcDee control showed a single narrow peak (Figure 1, panel A).18 Two partial D, DHMi34 (panel B) and DIV type III26 (panel C), had considerably to extremely broadened peaks. Three representative weak D types (panels D to F) showed peaks ranging from single and narrow (weak D type 3), like that of the regular D antigen, to broadened (weak D type 4.0 and type 7), that was reminiscent of partial D.
Rh antigen densities of rare weak D types The epitope density profiles as shown in Figure 1 were determined for a larger number of control, partial D, and weak D phenotypes. These data were used to calculate the antigen density representing the quantitative expression of the D antigen. The antigen densities of controls and known partial D (Table 5) were consistent with previous reports.30,35,36 Of the 2 partial D defined in this study, DIII type IV had an enhanced antigen density compared with its appropriate control (ccDee). The second partial D had a very much diminished antigen density and, hence, was dubbed DIM. Its antigen density was lower than DVI type I and represented one of the lowest antigen densities ever reported for partial D.
Rhesus D similarity index A Rhesus D similarity index (Rhesus index) was defined as the ratio of the 10 percentile and 90 percentile of the epitope densities (Table 5). The Rhesus index measured a qualitative difference to the D antigen of the standard phenotype CcDee. Ideally, normal D antigens would have an index of 1, grossly aberrant partial D an index of 0. Because of the known assay variability, the Rhesus index for normal D antigens was expected to be approximately 0.845 (see "Materials and methods"). All normal D antigens tested showed Rhesus indices of 0.84 to 0.90 (Table 5). In contrast, all partial D tested had Rhesus indices of less than 0.7. The Rhesus index allowed us to discriminate DIII samples, which are agglutinated by all monoclonal anti-D, from normal D. Partial D lacking many D epitopes, like DVI or DIV, had a Rhesus index of 0.Distinct immunohematologic features of weak D types As shown in Table 3, weak D type 1 and type 2 can be distinguished from weak D type 3 and types 4 by their different antigen densities. To further differentiate weak D type 1 from type 2, we determined the ratios of epitopes detected by the 2 IgG monoclonal anti-D BS227 and BS229 (Figure 2), similar to our previous approach with the 3 DVI types.18 All 25 weak D type 1 samples had ratios greater than 1.2, whereas all 24 weak D type 2 samples had ratios equal to or less than 1.0, which allowed a ready discrimination by immunohematologic methods. Likewise, a discrimination of weak D type 3 from type 4 was possible by the ratio of epitopes detected by the 2 IgG monoclonal anti-D BS227 and H41 (data not shown).
Molecular structure and phenotype For 18 RHD alleles with single nucleotide substitutions, we correlated the involved amino acid positions with the antigen densities (Figure 3, panel A) and the Rhesus indices (Figure 3, panel B). Many of the weak D types with strongly reduced antigen densities of less than 500 RhD antigens per cell had amino acid exchanges in the transmembraneous part of the RhD protein. Most of these types also displayed moderately reduced Rhesus indices ranging from 0.2 to 0.6. Likewise, many weak D types with intracellular substitutions had antigen densities in the range of 500 to 2000 RhD antigens per cell, and most of them displayed almost normal RhD indices. Interestingly, weak D type 7, for which the substituted amino acid was predicted to be deeply buried in the transmembraneous helix 11, had a very low Rhesus index of 0.03 and a rather high antigen density of 2400 RhD antigens per cell. There was no simple relation of the type of substitution to the antigen density or the Rhesus index. For example, the seemingly conservative M295I substitution in weak D type 11 caused a very low antigen density.
Six case reports of weak D with anti-D The overlap of the Rhesus indices of weak D and partial D with documented anti-D production fostered a search for samples with weak D phenotype and anti-D.38 Six samples were referred to us.39 Four samples could be assigned to the previously described RHD alleles weak D type 1, type 2, and type 15. Two samples carried new alleles that belonged to the group of weak D type 4 alleles and were designated weak D type 4.2 (Table 6). The anti-D observed in the frequent weak D type 1 and type 2 were shown to represent auto-anti-D by antibody elution. In contrast, weak D type 15 and 1 sample of type 4.2 had allo-anti-D; it should be noted that these 2 types had Rhesus indices lower than 0.3, which was lower than all other weak D types (Table 3) with the single exception of weak D type 7.
The 17 analyzed weak D types expressed distinct phenotypes. Each weak D type was associated with a certain range of RhD antigen densities. In contrast to previous conjectures, weak D types possessed slightly to considerably altered D antigens. The extent of qualitative changes observed in weak D overlapped with that observed in partial D. We provided experimental evidence that the risk of allo-anti-D immunization in the frequent weak D types was, however, low. The data allowed us to formulate a rational framework for a transfusion strategy in weak D patients. Note added in proof. After the revised version of our manuscript was submitted, Hemker et al72 published the full coding sequence of the partial D DAR. It shares the 3 missense mutations found in weak D type 4.2.1 and type 4.2.2 but lacks their silent mutations. Hence, in "weak D parlance," DAR might be dubbed weak D type 4.2.0. But we acknowledge the priority of the DAR nomenclature.
We thank Hans-Hermann Sonneborn and Manfred Ernst, Biotest AG, Dreieich, Germany, for generously supplying us with their monoclonal anti-D. We are greatly indebted to all contributors of the Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens, who provided most other monoclonal anti-D. We thank Silvano Wendel, Sao Paulo, Brazil, and Anna Ribera, Barcelona, Spain, for red cells and antibodies of the rare DIIIc phenotype and Christoph Gassner and Diether Schönitzer, Innsbruck, Austria, for the weak D type 13 sample. We thank Anita Hacker, Marianne Lotsch, Katharina Schmid, Sabine Zahn, and Olga Zarupski for expert technical assistance.
Submitted August 13, 1999; accepted December 16, 1999.
Supported by the DRK-Blutspendedienst Baden-Württemberg, Stuttgart, Germany, and by the University of Ulm (Forschungsförderungsprojekt P 422 and P 531), Ulm, Germany.
T.H.M. is now at Franz-Volhard-Klinik, Max-Delbrück-Centrum für Molekulare Medizin, Berlin-Buch, Germany.
Reprints: Willy A. Flegel, Abteilung Transfusionsmedizin, Universitätsklinikum Ulm, and DRK-Blutspendedienst Baden-Württemberg, Institut Ulm, Helmholtzstrasse 10, D-89081 Ulm, Germany; e-mail: waf{at}ucsd.edu.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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F. F. Wagner, B. Ladewig, K. S. Angert, G. A. Heymann, N. I. Eicher, and W. A. Flegel The DAU allele cluster of the RHD gene Blood, June 17, 2002; 100(1): 306 - 311. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
([SDctrl × 
1.2816 × 0.0286/101.2816 × 0.0286 = 0.845
: weak D
type 1, n = 25; 
: weak D type 2, n = 24 (only 23 different
positions are discernible because 2 samples overlapped); ×: weak
D type 3, n = 12; 
: weak D type 4.0, n = 7; 
: weak D type 5, n = 6.
, antigen density less than 500 RhD
antigens/cell or Rhesus index less than 0.2, respectively), moderate
changes (
coordinator's report.
Transfus Clin Biol.
1996;3:333