Malcolm Needs

Has recent work challenged our traditional views on weak D and partial D, how

we test for these and their clinical relevance in patients and donors?

Introduction.

In order to fully understand the differences between the wild-type D, weak D and

partial D, (and, indeed, exalted D) and how we should test for these, and their clinical

relevance in patients and donors, it is necessary to explore some of the history

surrounding these antigens. Indeed, a certain amount of debate has surrounded this

antigen ever since its first description.

The early history of D.

In 1939, Levine and Stetson1 described an antibody they had found in a sample of

blood in July1937, from a 25-year-old secundipara female, with an intrauterine death

and peri- and post-partum haemorrhage. Both the mother and father grouped as O,

and so the mother was transfused with blood from the father. This resulted in a

haemolytic transfusion reaction. The antibody, which was said to react equally well

at 20oC and 37oC, reacted with approximately 80% of random group O blood

samples. A sample taken two months later showed diminished reactions, and, in a

sample taken a year later, no atypical alloantibodies were detected. This antibody

was the first example of anti-D to be described.

It would seem reasonable, with today’s knowledge, to assume that, as the antibody

was initially detected at both 20oC and 37oC, and caused an intrauterine death, the

antibody was a mixture of IgM and IgG, and that, after a year, the antibody

production had switched, almost exclusively, to IgG. At this time, only the presence

of IgM antibodies could be demonstrated by agglutination, and ABO, M, N, and P

were the only “blood factors” known.

In 1940, Landsteiner and Wiener2 published a preliminary paper describing an

antibody (that was originally named anti-Rh, but was later re-named anti-LW)

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produced in rabbits that had been immunised with blood from Rhesus monkeys

(Macacus rhesus). This antibody gave reactions that were almost identical to the

anti-D found in the patient described by Levine and Stetson1. In a much more

comprehensive publication in 1941, Landsteiner and Wiener3 reported the use of

guinea pigs, rather than rabbits, to produce a more avid antibody, and tested 561

individuals with both this, and human-derived antibody. They reported that, of the

448 White individuals tested, 15.4% were negative, whilst of the Black individuals

tested, only 7.9% were negative. They suggested a racial difference.

Both Levine and Stetson1 and Landsteiner and Wiener3 noted that the antibodies that

had caused haemolytic transfusion reactions (other than ABO antibodies), including

fatal reactions, had, almost exclusively, occurred in women that had been pregnant,

and suggested that a factor inherited from the father had caused this immunisation.

Interestingly, Landsteiner and Wiener3 noted some differences in reaction between

the antibody derived from animals, and that derived from humans, but put this down

to experimental differences, rather than a difference in specificity.

In addition, Wiener and Peters4 had published a paper in 1940 concerning three

antibodies that had caused haemolytic disease when blood of the same group had

been given (probably anti-D), but did not provide sufficient evidence to challenge

Levine and Stetson’s claim to primacy.

These finding of antibodies, assumed to be of identical specificity, fuelled the first

“debate”, as it was not until 1963 that Levine et al5 conclusively proved that, not only

was the D antigen different from the LW antigen, but that LW was an independent

Blood Group System from Rh. For a long time, there had been a somewhat lively

debate between Levine and Wiener as to which of them had discovered anti-D!

By 1944, the C, c and E antigens had also been described, and this resulted in a

further debate as to the inheritance and nomenclature of the Rh Blood Group

System, as described by Wiener6 and Race7, who cited the work of Fisher. Briefly,

Wiener suggested that the Rh genes are inherited as a block (resulting in the

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“normal” haplotypes, R1, R2, Ro, Rz, r, r’, r” and ry), whilst Fisher thought that there

were three sets of allelic genes (resulting in the “normal” haplotypes, CDe, cDE, cDe,

CDE, cde, Cde, cdE and CdE). It is now known that neither of these ideas is correct,

but they are still used almost universally for convenience.

In 1948, Race8 presented a paper at the International Haematology and Rh

Conference in Dallas, Texas explaining Fisher’s Rh inheritance theory, and, although

Dr.Strandskow, a geneticist, said that he found trouble believing that there would be

three alleles so close on a chromosome, welcomed the theory. Amongst other

things, Race stated that Fisher thought that, although the custom had already

become established to order the genes as C, then D, and then E, the actual order

was D, then C, and then E.

Also in 1951, Wiener9 published a paper comparing his own “International

Nomenclature”, with that of the British (Fisher and Race). He disparaged the

theories of Fisher and Race, writing that they had invented some of their findings,

describing them as “armchair philosophers”, and stating that the C, D, E, notation

(“one can hardly dignify the British notations by calling them a nomenclature,

considering that they make no adequate provision for designating phenotypes”) was

already falling into disrepute, as the genes were, in his opinion, inseparable. It was

one of the most vitriolic, and ill-argued papers ever to have been published about

blood groups.

The above paper was written despite the fact that a year earlier Race et al10 had

described the first example of –D-/-D- in late 1950. They ended their short paper

with the following prophetic words, “Whatever the exact genetic mechanism, the fact

that C and E are involved supports from an unexpected angle Fisher’s tentative

suggestion that the order of the genes on the chromosome would be found to be D C

E. It also seems that the very controversial question whether the genes are

separable or not is settled in the most convincing way of all – by their separation.”

This, incidentally, was the first description of the exalted D phenotype, as they noted

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that some “incomplete” (IgG) anti-D reagents agglutinated the saline-suspended red

cells.

In 1962, Rosenfield et al11 put forward a suggestion that a numerical system,

“divorced of speculative implications” concerning gene structure, but describing

phenotype, should be used for each antigen, and, to a certain extent, this has been

adopted.

The early history of Du.

In 1946, Stratton12 described Du (now called weak D) as a new Rh allelomorph. He

originally found this in an R2r donor, whose D antigen gave varying reactions with an

assortment of anti-D reagents. With some, the reaction gave normal agglutination,

but with others, the agglutination was either very weak, or there was no agglutination

at all. He showed that this “new allelomorph” was an inherited factor, and described,

albeit briefly, three other examples (two R1ur and one R1

ur’). He noted that, at the

time when he wrote the paper, all anti-Du sera were a mixture of anti-D+Du, and

attempts to produce anti-Du, by immunisation of two individuals (a rr who had already

produced “anti-D+Du”, and a rr who had produced no atypical alloantibodies) failed to

either increase the titre of anti-Du, or produce de novo anti-Du. He suggested that the

relationship between D, d and Du was rather like the relationship between C, c and

Cw (that, at the time were also thought to be allelomorphs13).

By 1948, Race et al14 had completed over 20,000 tests involving samples of Du blood.

They showed that “normal D-positive” red cells could absorb out the “anti-Du” element

of anti-D+Du, and that Du cells could absorb out the anti-D element of

anti-D+Du. They also showed that anti-D could be eluted from Du cells sensitised

with anti-D+Du, but that anti-Du could not be eluted from “normal D-positive” cells

sensitised with anti-D+Du. Although they did not overtly suggest that D and Du were

products from the same D gene, they did hint that this was a possibility, when they

wrote, “Blood-group genes have always been supposed to be free from the effect of

the external and internal environment, and therefore it is with considerable hesitation

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that we put this forward as an example of variable expressivity of a blood group

gene. No other explanation suggests itself. Of the nature of the modifying factor

there is no clue. It clearly is not sex, nor the A1A2BO, MN nor P blood groups.” In the

summary at the end of this paper, they write, “Evidence is presented strongly

suggesting that these minor differences are inherited ones, and that there exists a

string of allelomorphs which behave as if they had originated as ‘stepped’ mutations

from D.”

They stated that there were many forms of Du, but divided them in into two main

groups; “high grade”, that could be detected by some saline-reacting anti-D reagents,

and “low grade”, that could only be detected by use of the antiglobulin test.

In 1955, Ceppellini et al15 described a different Du, of the high grade type, that was

not due to an “allele Du”, but to a positional effect exerted by Cde on an ordinary D in

the opposite gene complex; in other words, the C gene in the trans position, as

opposed to the cis position, weakened the expression of the D antigen. This gene

interaction is not always evident, as not all CDe/Cde individuals exhibit the weakened

D antigen.

Race and Sanger16 produced the (slightly modified) family tree showing the

“Ceppellini effect” on page 234 of the 6th Edtion of Blood Groups in Man (see Figure

1 on page 7). They also stated that the same phenomenon can sometimes be with E

in trans to D (e.g. CDue/cdE).

In 1960, Sanger et al17 reported a new antibody within the Rh Blood Group System,

anti-VS. This paper, concerning an antibody made against a variant at the e locus

may seem inappropriate to an essay concerning the D antigen, but its very real

relevance will become clear later.

The early history of D variants.

In 1953, Argall et al18 reported a case of a D-positive individual, who had themselves

an allo-anti-D in their circulation. They noted that there was no evidence of an

acquired haemolytic anaemia, and theorised that the anti-D had been made against a

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“missing-part” of the wild-type D. This was the first reported case of a D variant (or,

as it is now called, a partial D). Many other reports followed, and it was eventually

possible to categorise the different types. Once again, however, there was

considerable debate concerning the nomenclature to be employed, and, once again

Wiener was involved.

In 1957, Wiener et al19 discovered another case of a D variant with anti-D, this time

causing a mild form of haemolytic disease of the newborn. In the abstract to this

paper, there is a passage that, to a certain extent, has proved true, viz, “As one of us

(W.) has pointed out, indeed, the number of blood factors characterizing each Rh-Hr

agglutinogen appears to be limited mainly by one’s enterprise and ingenuity in

searching for and identifying new antibodies.” The summary, at the end of the paper,

however, gives an idea both of the proposed nomenclature, and of the way Wiener

was thinking. It bears to be quoted, almost in full, “A case of typical erythroblastosis

fetalis is described where the blood cells of the mother were Rho positive yet her

serum contained a potent Rh antibody, seemingly anti-Rho. The paradox was

explained when comparative serological tests and titrations showed that the Rho

factor is serologically complex and comprises in addition to Rho multiple factors RhA,

RhB, RhC, etc. While the blood cells of Rho-positive individuals ordinarily possess all

these components, rare instances are encountered where one of them is missing,

leaving open the possibility of isosensitization to the missing factor. The resulting

antibodies, namely, anti-RhA, anti-RhB, anti-RhC, etc, are indistinguishable in their

specificities from ordinary anti-Rho except in tests made on blood cells from those

rare Rho-positive individuals whose blood lacks one of the components of Rho.” (sic).

In 1962, Tippett and Sanger20 published a paper that split the D variants into six

categories (I to VI) by testing the red cells of 18 group D-positive samples from

individuals who had also made anti-D, against the anti-D produced by the other 17.

Interestingly, they state that, in their (present) view, people of category VI have no D,

and are more properly thought of as Cde/cde. They stated that, as a result, the

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Cde/CDe215

Cde/cde0

Cde/cde0

CDe/cde440

Cde/cde0

CDe/cde444

Cde/cde0

Cde/CDe219

Cde/cde0

CDe/cde450

Proposita

Figure 1. Gold represents a “normal” D-positive individual. Orange represents a “normal” D- individual. Red represents individuals with a weakened expression of the D antigen, due to the effect of a C gene in the trans position. The figures represent the total scores for titrations with 6 anti-D reagents.

production of anti-D by these individuals presented no problem, but noted that their

reaction with anti-G was “somewhat different from that of the ordinary Cde/cde”.

This paper formed the basis of partial D classification for many years, and still

influences the classification to this day.

Unfortunately, the relationship between Wiener’s group, and Race’s group had

reached such a state of discord, that there was no longer any exchange of samples

amongst the two, and for some time this had the effect of holding up any progress in

the understanding of this phenomenon.

Either way, at this stage, the usual way of explaining a partial D was to imagine a

block of four to six squares as the wild-type D, and the partial D as an identical

diagram, but with one square, or more, missing (see Figure 2 on page 9). At a very

basic level, this kind of diagram, albeit somewhat fanciful and naive, served well to

explain to the notion of the partial D to all but experts in the field of blood group

serology.

Biochemical breakthroughs.

In 1972, Green21 decribed a way to solubilise the Rh protein in such a way that it

retained its serological activity. Prior to this, the red cell membrane studies had

shown that the Rh antigens were probably proteins, by the effect of sulphydryl

reagents on insoluble erythrocyte membrane Rh antigen activity. Green stated that

when “using iodoacetamide and both organic and inorganic mercurial sulhydryl

reagents, it was found that the membrane Rh activity was lost, but the activity could

be regenerated, in the case of the mercurials, after incubation of the preparation with

an excess of thiol.” In his paper, Green showed a graded regeneration of the Rh

antigen activity of human Rh-positive lyophilised red cell membranes that had been

abolished by 1-butanol extraction. He showed that “the Rh activity is dependent on

the presence of bound phospholipid, containing at least one unsaturated fatty acid,

with neither the polar, nor non-polar portion of the molecule alone satisfying the

requirement.” Thus, workers were then able to isolate and work upon solublised Rh

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9

YFigure 2. An example of diagrams used to show the difference between a “normal D” antigen (on the left), and a “D variant” (on the right). Antibodies can be made against the “missing block”.

antigen.

In 1975, Köhler and Milstein22 published their paper on the production of monoclonal

antibodies, which resulted in a huge explosion in the production of avid, specific and

safe antibodies of various specificities. This paper is not, perhaps, the easiest to

read for the average blood transfusion laboratory worker, but a splendid review paper

by Blann23 in 1979, makes this technology open to all.

In 1985, Giorno24 described a model for the Rh blood group system based on a

discontinuous gene structure. Although he thought that the D subregion (or R1

region, as he named it) existed as a seven exon structure, alternating with introns,

which is now known to be mistaken, substantially, the underlying theory was correct.

In 1985, Saiki et al25 described PCR. They described a primer-mediated enzymic

amplification of specific target sequences in genomic DNA, resulting in an

exponential increase (220 000) of target DNA copies. Although they were studying a

way of predicting sickle cell anaemia in the foetus, their “Abstract” to the paper was

prophetic, if somewhat understated, when they wrote, “(the tests) may also be

generally applicable to the diagnosis of other genetic diseases and in the use of DNA

probes for infectious disease diagnosis.” The techniques described revolutionised

the way that the wild-type D, weak D, partial D and exulted D were studied.

Early evidence from monoclonal anti-D reagents.

By 1987, the monoclonal anti-D MAD-2, produced by the fusion of an Epstein-Barr

virus transformed human peripheral lymphocyte with the mouse myeloma X63-

ag8.653, which became the mainstay of D grouping at the hospital blood bank level

for a few years, was in mass production (Warden26).

By 1989, sufficient monoclonal anti-D reagents were available for closer classification

of partial D phenotypes. Lomas et al27 had shown, by use of monoclonal anti-D

reagents, the distribution of epitopes on the cells of those partial D categories

investigated (see Figure 3 on page 11), and Storry and Mallory28 were able to classify

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36 partial D samples, some of which had previously only been classified under the

RhA, etc classification, into categories III (4), IV (6), V (6), VI (18) and VII (2) using

almost exclusively monoclonal anti-D reagents (with the use of a polyclonal anti-Tar

[Rh40] to differentiate between categories III and VII, and polyclonal anti-Goa [Rh30]

to categorise all DIV samples to DIVa).

Hughes-Jones29 noted that “the demonstration of multiple epitopes through the use of

category cells is complementary to the results obtained on the variation in the

number of D sites present on normal cells when using different monoclonal anti-D

antibodies.” He went on to say, “There is no doubt that the seven antibodies

recognize epitopes situated on the same molecule as all the antibodies were mutally

inhibitory. It was found that antibodies that recognize many sites would completely

inhibit uptake of those that recognize few sites and conversely, as was to be

expected, those antibodies recognizing few sites only partially inhibited those

recognizing many sites.”

By 1993, RUM-1, another monoclonal anti-D used extensively in blood transfusion,

had been described by Scott et al30. This antibody detected partial D categories II,

IIIc, IVa, IVb, Va and VII by saline technique to a titre of 256, as well, of course, as

wild-type D. It also reacted with seven partial D cells that had yet to be categorised.

Importantly, it did not detect DVI. In addition, it detected 60 of 65 low grade

examples of Du, compared with only 9 detected by MAD-2.

Voak et al31 pointed out that many low grade Du phenotypes would now be re-

categorised at the wild-type D, and that there would now be no requirement to test

established apparent D-negative donors by the indirect antiglobulin test (IAT), and

only D-, C+ and/or E+ new donors by IAT.

More and more partial D types were being reported with the use of various

monoclonal anti-D reagents. Jones32 reported DHMi and DHMii that only gave negative

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Category Epitopes present.

III epD1 epD2 epD3 epD4 epD5 epD6 epD7

IVa - - - epD4 epD5 epD6 epD7

IVb - - - - epD5 epD6 epD7

Va - epD2 epD3 epD4 - epD6 epD7

Vc - - - - epD5 epD6 epD7

VI - - epD3 epD4 - - -

VII epD1 epD2 epD3 epD4 epD5 epD6 epD7

Figure 3. The distribution of epitopes on the cells of those D categories

investigated (Lomas et al27).

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results with a monoclonal HAM-A, whilst giving positive results with 11 other IgM and

24 other IgG monoclonal anti-D reagents. Wallace et al33 reported a DVa phenotype

that was Dw-.

Fortuitously, the original category DII propositus became available again after many

years, and Lomas et al34 were able to test the red cells with various monoclonal anti-

D reagents. They were able to show that the DII cells express epD1, epD2, epD3,

epD5, epD6/7 and epD8, but lacked epD4 and a new epitope epD9, and for some

time the 9 epitope model was accepted.

In 1994, Scott et al35 reported that epitope D6/7 had been split by the reactions of

monoclonal antibodies against an individual Li.

PCR “joins the party”.

In 1994, Wolter et al36 reported the use of PCR to determine genotypes using two

reverse primers that were designed to a region of exon 7 that contains a number of

allele specific sequence differences. They claimed that, after electrophoresis, a

comparison of the RhCcEe and RhD DNA band intensities give an exact measure of

RhD gene zygosity, and that this could be used for foetal genotyping. In 1995,

however, Pailing37 showed that the available primers were not suitable for a

diagnostic test without further study, as 3 of 100 samples tested gave aberrant

results. One of these was typed by PCR as D positive, but serologically as Cde/cde.

On the other hand, Avent et al38 reported a multiplex assay, performed in one tube,

that relied upon the fact that intron 4 of the RHD is smaller than that of the RHCE

gene (600bp cf 1200bp), and exon 10 of the RHD gene is larger than the RHCE gene

exon 10. By using an antisense PCR primer, which straddles the site of sequence

divergence of the RHCE and RHD introns, RHD-specificity was obtained, and when

combined with RHD exon 10 specific primers, two RHD-specific bands were obtained

in D-positive samples.

At the same time, Scott et al39 showed that 10 of 13 IgM monoclonal anti-D reagents

showed a cold-agglutinin-like reaction with D-negative red cells. This was shown to

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be connected with the monoclonal antibodies preferentially using the VH4.21 gene.

Other antibodies using this gene had been associated with auto-immune cold

agglutinin disease. Of those reagents tested, RUM-1 gave the strongest cold

agglutinin reactions. They pointed out that, in view of their potential cold reactivity,

IgM monoclonal anti-D typing reagents should be brought to room temperature for

routine use, rather than taken straight from 4oC storage.

This notwithstanding, monoclonal anti-D reagents, used at optimal temperature,

continued to reveal more partial D phenotypes. Jones and Filbey40 tested an

extensive range of partial D phenotypes with 26 IgG and 15 IgM monoclonal anti-D

reagents and with these subsplit epD2, epD5, epD6/7 and epD9.

This led Scott41 to wonder how many D epitopes existed. She reported on the work

of the ICSH/ISBT working party on blood grouping reagents, which had studied the

reactivity of 38 monoclonal anti-D reagents with 80 different D category and variant

red cells. This work showed at least 20 different reaction patterns, including,

surprisingly, an enzyme sensitive epitope within the D mosaic.

In a review article, published in 1995, Anstee42 reported that the structure of the D

carrier molecule had six extracellular loops, with the NH2 and COOH termini both

being intracellular, and spans the membrane 12 times. This was already known, but

he deduced that, because there is an extracellular sulphydryl group that is a

requirement for expression, the amino acid residues critical for Rh antigen

expression are not contained within a linear sequence, but are dependent upon the

correct juxtaposition of critical residues when the proteins are assembled in the

membrane (see Appendix 3).

By 1996, many of the genetic backgrounds of partial D phenotypes had been

elucidated (see Issitt43 for a review), but more were being described, whilst still

others, the serological reactions of which had been previously described, were now

being revisited on a molecular level44, 45.

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Also in 1996, Scott46 reported on the use of genetically engineered antibodies that

can be produced in vivo against human “self antigens”, by the use of cells from

individuals who have not been immunised, and thus very rare antisera can be

produced, including antibodies to certain epitopes of the D antigen. In addition,

Avent47 showed that certain D-negative individuals, who are serologically D-negative

who are serologically cdE or Cde, can produce false positives using PCR because

they have partially deleted or intact (but dysfunctional) RHD genes. At the same

time, however, he speculated that there would be a possibility of harevesting foetally-

derived cells from the maternal circulation, and this would circumvent the

requirement for amniocentesis as a source of foetal material prior to PCR analysis.

In 1997, Fukumori et al48 demonstrated that DEL red cells, which only demonstrated

sensitisation with anti-D by elution techniques, have some form of the RHD gene

present, as exons 4, 7 and 10 could be demonstrated. This form of partial D was

only found with CCee, CcEe and Ccee, but not with CCEe, CcEE, ccEE or ccee

phenotypes amongst 102 examples.

Wallace et al49 described DBT, a partial D phenotype associated with the low

incidence antigen Rh32. This partial D gave varying reactions with both monoclonal

and polyclonal anti-D, but of the 8 probands studied, 3 had produced anti-D. As all

the probands were female, it may well be that, at least some, were immunised during

pregnancy, although no information is given in this paper. The authors speculate,

however, that DBT individuals are more easily immunised than, for example,

individuals who are DFR.

In 1998, Flegel et al50 published a review paper in which they propose that all weak

and partial D phenotypes should be brought under the umbrella term of an “aberrant

RHD” allele when referring to all RHD alleles coding for one or more amino acid

substitutions compared to the wild-type RHD. They stated that “the name reflects the

sporadic occurrence of these alleles and, hence, of their “aberrant RhD” phenotypes,

including, but not limited to, partial D. The proposed nomenclature has the

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advantage of covering almost all known RHD alleles, including all known clinically

relevant partial D and molecular weak D types. The nomenclature is unequivically

defined, and does not depend on the availability of suitable anti-D.” They went on to

state, “Null alleles of the RHD gene, which are associated with ineffective RhD

protein expression, like RHD(Q41X) may be referred to as nonfunctional RHD.” This

excellent paper describes how the different analyses are performed, but, most

importantly for this particular piece of work, which of the abberant D phenotypes are

capable of producing clinically significant anti-D.

In January 1999, Wagner et al51 published a paper on the molecular basis of weak D

phenotypes. They tested 161 samples of weak D, and found a total of 16 different

molecular weak D types, together with two alleles characteristic of partial D. “The

amino acid substitutions of weak D types were located in intracellular and

transmembraneous protein segments and clustered in four regions of the protein

(amino acid positions 2 to 13, around 149, 179 to 225, and 267 to 397). Based on

sequencing, polymerase chain reaction-restriction fragment length polymorphism and

polymerase chain reaction using sequence-specific priming, none of the 161 weak D

samples investigated showed a normal RHD exon sequence. We concluded, that

in contrast to the current published dogma most, if not all, weak D phenotypes

carry altered RhD proteins, suggesting a causal relationship. Our results

showed means to specifically detect and to classify weak D. The genotyping of weak

D may guide Rhesus (sic) negative transfusion policy for such molecular weak D

types that are prone to develop anti-D.” (bold type entered by the present author).

(see also Appendix 4).

Singleton et al52 described an RHD pseudogene that is present in most Africans with

the RhD –negative blood group phenotype. They stated, “The intact RHD is a

pseudogene (RHDψ) that contains a 37 bp duplication of the intron 3/exon 4

boundary and may introduce a premature stp codon at position 210. A second stop

codon is present in exon 6. RHDψ is present in about 25% of D-negative African

16

Americans and 15% of Coloured South Africans. No RhD transcript could be

detected in D-negative individuals with RHDψ.” This allowed Martin et al53 to devise

a new PCR test that is appropriate for the genotyping of populations containing a

substantial portion of individuals of African ethnicity.

In the same year, the repertoire of partial D/aberrant D types increased with the

description of DMH54, DOL54 and DAR55. In each case, the individual had,

themselves, either produced alloanti-D, or had stimulated alloanti-D in another RhD-

negative individual.

Avent56 published a major review of the then situation in 1999. Rather surprisingly,

perhaps, considering the fact that many of the techniques available to him and his

group are unavailable at the hospital, or even the Blood Centre level, as a matter of

routine, he wrote, “Three main areas of Rh research remain to be fully explored.

First, a greater understanding of the structure of the D antigen is needed. Moreover,

some indication of how anti-D interacts with the protein would provide considerable

insight into how immunologic responses to Rh antigens vary between individuals.

Molecular diagnostics is also beginning to have an impact on the routine serotyping

laboratory. Secondly, it is now almost easier to define most partial D phenotypes by

PCR rather than using large numbers of monoclonal anti-D or rare human

alloantisera to low-frequency antigens. PCR-based typing methods for partial D

phenotypes include multiple sequence-specific primer PCRs and multiplex RHD PCR

involving primers directed toward the 6 RHD-specific exons. Finally, although there

has been extensive research into the molecular basis of Rh antigen expression,

much has still to be learned about not only the function of the Rh protein family

members, but also how they interact with their neibours in the red cell membrane in

its mature and developing states. Such studies may reveal why members of the Rh

protein family have survived such genetic diversity.”

There is the possibility that, with bespoke monoclonal antibodies, and/or bespoke

RhD protein fragments, that another way of easily defining RhD phenotypes may be

17

through the microarray system, although microarray systems are still at the research

and development stage.

The situation from 2000.

For many years, there had been sporadic, but rare reports of D-negative individuals

producing an alloanti-D following the transfusion of blood from a donor with the weak

D phenotype57. In 2000, Flegel et al58 suggested that many of these reports had

emanated from cases in the early 1960s, when anti-D reagents were much less

potent than those available today. They stated that many of the causative donations

would now, using the potent anti-D reagents commonly available, be typed as D-

positive. They reported, however, a case of a donation of weak D type 2 (see

appendix 4) that had unequivocally caused the recipient to produce an alloanti-D by

primary immunisation. Indeed, the alloanti-D produced by the recipient reached a

titre of 128. Tests showed that the donor’s blood expressed only about 450 D

antigens per red cell.

The donor had previously been grouped as D-negative on three occasions (once as

a result of a technical error, once as a result of a clerical omission and once as a

result of the lack of sensitivity of the D typing in the confirmatory blood donor

grouping), but they found that, if tests are carried out strictly following the standard

operating procedures (SOP) and good manufacturing practice (GMP), weak D type 2

should have been detected. They suggested, however, that there is an urgent need

to establish the lower threshold for the number of D antigen sites per red cell that can

cause primary (or secondary) immunisation.

Wagner and Flegel59 reported that RHD and RHCE face each other by their 3’ tail

ends, and a third gene, SMP1, was found to be interspersed between the two Rh

genes. Two 9, 000 bp DNA segments, dubbed “rhesus boxes”, of identical

orientation fringed the RHD gene.

In 2001, Wagner et al60 described the partial D type DBS, which carries an RHD(4)-

CE(5)-D(6-7) coding. A year later, Omi et al61 described the partial D type DTI, which

18

appeared to be identical to DBS, and these types are now twinned. This shows,

therefore, that even in the time of monoclonal antibodies, multiple epitopes and

molecular characterisation, things are never simple!

For some time it has been possible to express the D antigen on K562

erythroleukaemia cells (Smythe et al62). In 2001, Kamesaki et al63 demonstrated that

weak D phenotypes could be characterised at the molecular level by site-directed

mutagenesis and expression of mutant Rh-green fluorescence protein fusions in

K562 cells. These workers showed that the intracellular and transmembranous

amino acid substitutions that are found in the various weak D phenotypes had

obvious effects on the D epitopes recognised by monoclonal antibodies. For the first

time, they provided direct evidence that these mutations can account for weak D

phenotypes; something that had been suspected for some time.

Wagner and Flegel64 reported that their work with human and mouse DNA had shown

that RHD arose as a duplication of RHCE. It is likely that the orientation of the RHD

gene (i.e. in the opposite direction to that of RHCE) was inverted during this event.

In 2002, Wagner et al65 reported on an entirely newly discovered cluster within the

RHD gene, DAU. The first of these, DAU-0, was found only in Europeans, and these

expressed a normal D antigen. The others, DAU-1 to DAU-4, were found in Africans

with partial D phenotypes, with alloanti-D found in the DAU-3 individual. All five of

these clusters demonstrated a T379M substitution, with DAU-1 to DAU-4 also

exhibiting one or two additional mis-sense mutations. Their studies resulted in them

making the following statement:

“In populations without African admixture, including whites, Asians, Arabs, and

probably American Indians, partial D phenotypes are likely to be rare and to derive

from the limited and serologically well-characterized set of alleles of the Eurasian D

cluster. For these populations, the current D-typing strategies applied in Europe

appear to be appropriate and sufficient. Typing strategies for African populations and

those with African admixture may take account of the various frequently occurring

19

alleles of the “African” cluster. Several of these alleles characterized by multiple

dispersed amino acid substitutions are difficult to discern by serological means and

may in the future warrant genotyping approaches for detection in patients and

donors.”

Also in 2002, Wagner et al66 described the partial D type DNB. This category types

as normal D in routine typing, but 5 of the individuals studied had formed alloanti-D,

one with a titre of 128. They noted that in Germany and Switzerland, although not in

Denmark, DNB was the most frequent partial D recognised so far in the White

population.

In 2003, Hemker et al67 reported that the Rh complex exports ammonium from human

red blood cells. They measured ammonium import during the incubation of red cells

in a solution containing a radio-labelled analogue of NH4Cl (14C-methyl-NH3Cl). Rhnull

cells of the regulator type (expressing no Rh complex proteins) accumulated

significantly higher levels (P=0.05) of radio-labelled methyl-ammonium ions than

normal red cells at room temperature. Rhnull cells of the amorph type (expressing

limited amounts of Rh complex proteins) accumulated an intermediate amount of

methyl-ammonium ions. To show that decreased ammonium export contributes to its

accumulation, the release of intracellular methyl-ammonium from the cells was

measured over time. In 30 seconds, normal red cells released 87% of the

intracellular methyl-ammonium ions, whereas Rhnull cells of the regulator type

released only 46%. It could not be concluded whether this was a function of RhAG

or the serologically active Rh proteins. It was suggested that the ammonium

transport function of the Rh proteins could serve as a protection for the red cells in an

environment with high ammonium levels, as may be found in the kidney during the

excretion of acids. If Rh proteins are absent, haemolysis might then occur, as

noticed in Rhnull donors.

In 2004, Körmöczi et al68 described another partial D, DWI, in an individual typing as

D-positive, but with alloanti-D in her circulation, and this 25 years after the last

20

possible time that she could have been immunised. Once again, the authors point

out the need to develop a fast and economic DNA-based method for large-scale

routine use in DNA typing (although, in this case, no additional DWI individuals were

identified among 1, 377 and 911 D+ blood donors from Eastern and Western Austria,

respectively.

In 2004, Ansart-Pirenne et al69-70 published a study that suggested there is a need to

develop a routine genotyping test for weak D types 1 and 2, as these have sufficient

numbers of the D antigen (400 or above) to prevent them being stimulated to

produce alloanti-D when transfused with wild-type D-positive blood (except when

found in the trans position with C, when the number of D antigen sites are said to

drop dramatically, down to about 500 – which, in comparative terms, is too close to

400 for comfort). They state that weak D types 1 and 2 are the most prevalent

amongst Caucasians (a statement confirmed be Araújo et al71), but that such

individuals have yet to be reported to have produced alloanti-D when stimulated with

wild-type D-positive blood. They hypothesised, therefore, that if such individuals

could be recognised by a routine genotyping test, they could routinely receive wild-

type D-positive blood and that, as a consequence, there would be less wastage of D-

negative blood. In their study, they confirmed the findings of Wagner et al66, that

individuals with the DNB phenotype are prone to produce alloanti-D, as all six DNB

carriers in their study had produced this antibody.

In 2004, Westhoff72 published a review article entitled, “The Rh blood group system in

review: a new face for the next decade”. This is a paper that is eminently readable

and informative, but does contain some errors about which any prospective reader,

who has not themselves studied the subject, should be made aware. For example,

she states that, “The antigens Cw and Cx, which were thought to be antithetical to C,

are the result of single amino acid changes on the first extracellular loop of RhCE.”

All of this is true, but what she does not say, explicitly, is that they are allelomorphs of

MAR (or Rh51)73. In addition, she states that there are three types of partial DVI

21

(which is incorrect, as there are four), and then goes on to say that DVI red cells carry

the BARC (Rh52) antigen (which, again, is incorrect, as type 1 partial DVI red cells

are BARC-negative). This information can easily be found in Reid and Lomas-

Francis74, a reference that is given by Westhoff in her review! Lastly, she published a

correction herself in a later edition of Transfusion75, as she had neglected to mention

the early work of Gorman and Pollack in the treatment and prevention of Rh

sensitisation.

In 2004, the British Blood Transfusion Society (BBTS) hosted the Annual Scientific

Meeting of the International Society of Blood transfusion (ISBT). Several papers

came out concerning the Rh Blood Group System, many with particular reference to

the D antigen. Amongst those was a short educational paper published by Scott76,

which, in the opinion of this author, was far superior to that published by Westhoff72,

and should be recommended reading. That notwithstanding, there are some

comments, even in this paper, that challenge logical thinking, (although, may well be

correct!). For example, when writing about enhanced D, as seen in the phenotype D

- -, she writes, “These cells totally lack the RhCE protein, and it is most likely that

normally the RhD and RhCE proteins compete for binding to the Rh-associated

glycoprotein, thus absence of the RhCE proteins will result in more D antigen being

expressed Cells of these type can be readily agglutinated directly by IgG anti-D,

whereas cells with normal numbers of the D antigen will only be agglutinated by IgG

anti-D if the cells are enzyme-treated, or an antiglobulin reagent is used.” Logical

thought, however, would be that, in most cases of Caucasian rr, where the entire D

protein is missing, the RhCE protein would not have compete with the RhAG, and

this would result in more c and e antigens being expressed. It would be expected,

therefore, that these cells would, in turn, be agglutinated directly by IgG anti-c and

IgG anti-e, but this, of course, is not the case.

Three other excellent educational papers, relevant to readers of this essay, were also

read at this meeting; Ridgwell77 (Genetic tools: PCR and sequencing), Daniels78

22

(Molecular blood grouping) and van der Schoot79 (Molecular diagnostics in

immunohaematology).

In 2004, Patients with weak D type 1 were reported to have the capability to produce

either auto-anti-D (Beckers et al80) and alloanti-D (Roxby et al81) themselves, but also

to be capable of stimulating alloanti-D in others, with a more comprehensive paper in

2005, (Mota et al82-83), and this had not been previously described. This suggests

that, like partial DVI individuals, they should, perhaps, be treated as D-negative when

in receipt of a transfusion, but as D-positive when donating blood. Meanwhile,

Döscher et al84 described four more RHD-alleles with previously unknown

polymorphisms, but in their cases, none had produced an alloanti-D.

Also in 2004, Wagner et al85 described a weak D that was mis-typed as weak D type

1 by PCR, because the serological reactions had been ignored, whilst the PCR

results had been taken in isolation. For workers in blood group serology, with no

particular experience in PCR, this came as remarkably good news, following the

papers by, for example, Anstee86, predicting the early demise of agglutination tests!

By 2005, however, Avent87, in an Editorial in Transfusion, was suggesting that

widespread RHD genotyping to eliminate weak D and partial D phenotype red cells

from the D-negative donor pool may soon be a viable option, possibly by genotyping

all recruited blood donors. He pointed out that mass scale programs already exist to

detect viral contamination of blood products via nucleic acid technology, and that the

infrastructure already exists within blood centres to extract DNA from individual blood

donations and to apply automated systems to the detection of viral genomes. He

went on to write, “Technology capable of performing such analysis is being

developed both in Europe (for example the EU Bloodgen project,

http://www.bloodgen.com) and in North America.”

In another Transfusion Editorial, Flegel88 suggested that “Transfusion medicine

specialists should no longer be satisfied when a patient produces anti-D while

reported to have received D- RBC units only. We should rather check the involved

23

donors for undiscovered weak D or DEL; most of us have not been doing that but are

usually looking for other reasons like RBC contamination of plasma products.” He

also stated that, “At the blood centre in Ulm, Germany, [his Centre] RHD genotyping

of D- first-time donors (among 5000,000 donations/year) has been routine procedure

since January 2001. Extending RHD genotyping to repeat donors would allow

lookbacks [sic] to monitor effects of previous donations. Thus, any possible adverse

effects of donations from weakly D+ donors in our D- donor pool would be rapidly

detected.”

In the same edition of Transfusion were two other important papers. The first, by

Wagner et al89, described the fact that a donor with a DEL phenotype, with probably

less than 30 D antigen sites per red cell, had caused primary immunisation in a D-

negative recipient. The anti-D titre was determined as between 64 and 128 one year

after the transfusion. Prior to the transfusion, she had received no other transfusions

or transplants, and had only borne one D-negative child. All other forms of

stimulation, such as a concealed pregnancy, or transfusion of a known D-positive

unit, were ruled out.

Until this report, “The lowest known D variants with absolute D density numbers

[determined by the method employed by these workers] are weak D type 12, weak D

Type 17, weak D Type 11, and weak D Type 38, with as few as 96, 66, 57 and 53 D

antigens per RBC, respectively.”

It is known that some recipients form antibodies more readily than others90, and that

there may be an association between the HLA group of the recipient and the

production of anti-D91, in that 18% of women with high anti-D titres have the HLA-

DQB1 allele *0201, and it may be that this woman was within this group (no

information is given), but this particular episode of immunisation is worrying.

Given the evidence from the above paper89, the second of the two papers from this

edition of Transfusion is equally disturbing. Gassner et al92 published the findings of

a European multi-centre study looking for the presence of RHD in serologically D-

24

negative, C/E-positive individuals. They studied a total of 1, 700 serologically D-

negative samples that were positive for the C and/or E antigens. Of these, 89 were

found to have either a complete or partial RHD gene (just over 5% of the

serologically D-negative samples). The worry thing was that some of these 89 were

DEL phenotypes, whilst others were weak D, and others partial D. In other words,

when the information from the two papers is combined, it will be seen that many of

these supposedly D-negative samples could be capable of sensitising a D-negative

recipient.

Much earlier in this essay, (page 5) it was noted that anti-VS was described in

196017. The relevance of this is that the amino acid residue substitution giving rise to

the VS antigen is transmembraneous. This, in turn, gives rise to a weakened form of

the e antigen; in other words, a conformational change to the external structure of the

RhCE protein. This conformational change, when introduced into a VS-negative

individual can stimulate anti-VS. This is an exact analogy with the weak D situation,

where a transmembraneous or intracellular change can influence the extracellular

conformation of the RhD protein.

Although just over 5% of the serologically D-negative samples in this study were

found to be D-positive, the authors point out that the findings varied widely with

geographical origin. It must not be assumed, therefore, that 5% of the D-negative,

C/E-positive pool of donors within the NBS, for example, would also be found to be

D-positive. It may be of concern, however, that C+ and/or E+, D-negative units of

blood are suitable for transfusion to, for example, rr recipients, on the grounds that

the C and E antigens are not particularly immunogenic. The outcome of the class

action under product liability, brought by UK recipients infected by hepatitis C, could

well have a bearing on whether this practise is continued.

In an Editorial for Transfusion in 2005, Garratty93 reviewed the situation vis-à-vis the

weak D and partial D situation, and came to no definitive conclusions as to the best

way to test for these individuals. He did mention that Europe has the advantage over

25

the USA in that only four monoclonal anti-D reagents are licensed for routine use by

the FDA, but said that he fervently hoped that blood group antigen DNA technology

would become more, if not universally available.

A study published by Denomme et al94 showed that a disturbing number of females of

child-bearing age are being grouped serologically as D-positive, when they actually

express a D variant that known to permit anti-D immunisation. There were 55

discrepancies (0.96% of D-negative) noted among 33, 864 (5, 672 D-negative)

ethnically diverse patients (women of child-bearing age, and new-born babies) over

the 18 months of the study, of which 54 represented mutated RHD alleles. They

suggested a typing regime not unlike that in use by English hospitals, the NBS and

the IBGRL for patient samples. They suggested the use of two anti-D reagents, one

detecting a broad spectrum of D phenotypes, and one detecting a much narrower

spectrum of D phenotypes. If there is a discrepancy between the two, a sample is

sent to the Reference Laboratory for elucidation. If the Reference Laboratory is able

to identify the D antigen type, using a battery of monoclonal anti-D reagents,

designed to detect certain epitopes, the tests will go no further. If the Reference

Laboratory is unable to identify the D type, then the sample will be tested with more

monoclonal anti-D reagents, and DNA typing will also be undertaken. A novel partial

D, named DTO, together with two more DAU types and a novel weak D were

detected during this study.

Körmöczi et al95 described two novel weak D types (31 and 32), which were only

detected serologically by use of adsorption and elution. These gave single

nucleotide substitutions predicted to lie in the intracellular domains of the RhD

protein, resulting in antigen site densities of only 130 and 50 per red cell. Obviously,

from what has been written before, these individuals could potentially produce

alloanti-D themselves, if transfused with wild-type D-positive blood, or could stimulate

alloanti-D in a true D-negative individual, if their blood was transfused to such an

individual.

26

Körmöczi et al96 reported on a comprehensive study of five of the six known DEL

types, and found that one (RHD[IVS3+1g>a]) was a partial D, whilst the others were

weak D. Two of the RHD(IVS3+1g>a) individuals had produced alloanti-D. This was

the first definitive report of alloanti-D in a DEL individual. The authors point out,

however, that there may be more cases that have gone unidentified, as, unless

adsorption-elution studies and/or molecular studies are carried out, such individuals

could be assumed to be wild-type D-negative individuals, in which alloimmunisation

against the D antigen would not be seen as out of the ordinary.

Yasuda et al97 described the case of a 67-year-old women with anti-D (titre 8). She

received blood from 19 apparently D-negative donors, after which her anti-D titre

rose to 128. A subsequent look-back at the donors revealed that two of them were of

the DEL type (RHD[K409K]), and that a secondary anti-D immunisation had taken

place. It was thought that this was the first such example, but a note added at proof

recognised the primacy of Wagner et al89. What they did mention, however, is that

the DEL phenotype is much more common in the Far East, than in Europe (1:110

amongst apparently D-negative individuals in China, against 1:9091 amongst

apparently D-negative individuals in Europe).

Doescher et al98 revealed that 7 of 23 donors thought to be weak D type 1 in Northern

Germany had, in fact, been initially mis-typed by PCR, and that further serological

and molecular studies had revealed a second amino acid substitution. Serologically,

direct typing was extremely weak, but was easily detected by use of the IAT. The

study demonstrated a subtype of weak D type 1, designated weak D type 1.1, and

again demonstrated the necessity to use both serological and molecular techniques.

Another point made in the report was the restricted area of some of the weak D

types.

In 2005, Castilho et al99 published a study that had been undertaken with sickle cell

disease patients, to ascertain the frequency amongst them of the two partial D alleles

DIIIa and DAR. Both these alleles have an increase risk of alloimmunisation to the D

27

antigen, but both also give positive results with monoclonal anti-D grouping reagents.

Of the 130 patients studied, 12 (9.2%) were shown to carry either the DIIIa or DAR

allele, or, in a minority of cases, both. They suggested that sickle cell disease

patients, who are candidates for a chronic transfusion regime, might benefit from

genotyping for DIIIa and DAR to prevent alloimmunisation. It may also be important

from another point of view. These patients are at a higher risk than are Caucasian

patients of producing anti-hrB, and even anti-HrB. If they produce both anti-D and

anti-hrB, they would have to be supported with the rare phenotype r”r”, and if they

produce anti-D as well as anti-HrB, the only transfusion support available may be the

even rarer Rhnull cells.

Laycock et al100, described a case of severe HDFN caused by an anti-D produced in

a DEL woman. The woman, who had no previous obstetric history, was found at

booking to have anti-D in her circulation. The anti-D rose from 22.7IumL-1 to

208.4IumL-1 during the pregnancy. The foetus required four intrauterine transfusions,

the baby two exchange transfusions, and double phototherapy, and yet the mother

was found to have an RHD allele.

In the same year, Tilley et al101 described another novel RhD variant associated with

an extremely low serological expression of the D antigen they named DIT. This RHD

expressed a mutation at position 288, which is predicted to lie within the fifth

extracellular loop. It is thought that this patient, originally grouped a D-positive when

a donor, but grouped as D-negative as a patient (she was pregnant), was capable of

producing an alloanti-D.

Rodrigues et al102, showed that partial D antigen DBS, first described by Wagner et

al60 (see above), could only be distinguished from DFR by use of a molecular

biological method.

Bruce et al103 described yet another novel RHD mutation that had resulted in immune

alloanti-D being produced following the transfusion of D-positive blood. This

mutation was named DNAK.

28

The challenge to our traditional views on weak D and partial D, testing in the

future, and the clinical relevance in patients and donors – where do we stand?

It is obvious, from all of the above, that the traditional views on weak D and partial D

are no longer tenable. Appendicies 4 to 9104 show quite clearly that the dogma of

weak D being a quantitative difference and partial D being a qualitative difference

(with, or without an accompanying quantitative difference) from the wild-type D is

totally incorrect. It is possibly more correct to state that weak D is a product of one or

more point mutations that give rise to amino acid substitutions that are

intramembraneous and/or transmembraneous, and that partial D is a product of exon

substitution (with or without accompanying point mutation(s)), or one or more point

mutations that give rise to amino acid substitutions that are extramembraneous (with

or without accompanying intramembraneous and/or transmembraneous amino acid

substitutions), but even this is a crass simplification of the real situation.

Many of the samples that would previously have been typed as weak D are now

being typed as an “ordinary D”, and some of those that were previously typed as D-

negative are now being typed as weak D or DEL.

In some ways, the introduction of both monoclonal antibodies and molecular typing

has muddied the waters, rather than made the situation clearer. It will be noted, for

example, that, whereas it was always known that there were different types of weak

D (from the strength of the reactions given with different anti-D grouping reagents), it

is probably unlikely that anyone would have predicted the discovery of the 57

different types of weak D quoted in Appendix 4, together with those in Appendix 7

awaiting formal classification, and the DEL types quoted in Appendix 8, (all with

“power to add”) before monoclonal antibodies and molecular charaterisation became

available. The same can probably be said of the partial D classification (see

Appendicies 5, 6 and 7), and almost certainly be said concerning D-negatives (see

Appendix 8), and D-positives (see Appendix 9). The “clock”, however, cannot be

29

“turned back”, and the advantages of these technologies far outweigh their

disadvantages.

Molecular typing was originally used as a tool for foetal genotyping78, in an effort to

diagnose those pregnancies that did, or did not, require intervention, when there was

an alloantibody in the maternal circulation that could potentially cause Haemolytic

Disease of the Foetus (HDF), in particular, and Haemolytic Disease of the Newborn

(HDN), in general. Its use has since been expanded to include the molecular

“grouping” of transfused patients, those with a positive Direct Antiglobulin Test (DAT),

donor screening (e.g. for typing within the Dombrock Blood Group System), and as a

general aid to serological reference work in “sorting out awkward problems”.

In parallel with the use of these new techniques, as stated above, has been the

development of ever more sensitive monoclonal antibodies (see the DEL

phenotypes).

With the discovery of several different types of “weak D” that have been sensitised to

produce alloanti-D (albeit, that they are, in the bigger picture, rare), and, conversely,

the DEL phenotypes that have themselves caused both primary and secondary

immunisation against the D antigen, together with the ever more litigious nature of

the public at large (see the section on hepatitis C in reference 105), there is a danger

that we may be forced into a situation where pragmatism is no longer an option.

At present, the working “rule of thumb” for patient samples is that, if the red cells

react with two anti-D reagents blended to detect all of the different D exons (although

the two should detect a different range of exons), and the reactions are noticably

weaker than those obtained with the control cells, the patient is defined as having a

weak D. If, on the other hand, one of the anti-D reagents gives a positive reaction,

whilst the other gives a negative reaction, the patient is defined as having a partial D.

This may soon no longer be sufficient, even though some of the weak D and partial D

types are extremely restricted in a geographical sense98. It would seem likely that,

with the legal decision made concerning the testing for hepatitis C, it could only take

30

one test case where, for example, a person with weak D has produced alloanti-D,

following transfusion of a D-positive unit of blood, or, possibly worse, where a female

is potentially child-bearing, who is D-negative, produces an alloanti-D as the result of

being transfused with a unit of DEL blood, for our hands to be forced, resulting in a

complete change of practice.

At present, even with pooling donor samples of genomic DNA, it may be impossible

to test all donors by molecular means106, as it would be a huge task to test all existing

donors, as well as new donors. It may also transpire that it would be sufficient,

bearing in mind the geographical restrictions of some weak D types, to test only

certain of the D exons.

The mass screening of D-negative pregnant women is, at present, undergoing trial,

and the outcome of this trial may well influence the decision as to the practicality of

screening, at least at the beginning, all new donors that are serologically D-negative.

The blood from these “genotyped donors” could then be reserved for females who

are potentially child-bearing, those patients who are on a chronic transfusion regime,

and others with antibodies, such as anti-hrB, who are prone to have a DIIIa type in

trans, simply because of the ethnic origin of those able to make anti-hrB, and who

can, therefore, be stimulated to produce alloanti-D. Approaching the problem in such

a way would mean that those patients most vunerable to alloimmunisation, in terms

of possible sequalae, would be the best protected.

Those units that are from “ungenotyped donors” could then be used for males, who

are not on a chronic transfusion regime, and females who are not potentially child-

bearing, and who are not on a chronic transfusion regime.

Such an approach, should any such approach be deemed necessary, would allow

time for a “catching-up period”, with reference to established donors. It will, however,

be a massive undertaking even to genotype new donors, let alone established

donors, and it may well be that a decision so to do will be deferred for as long as

31

possible, or until there is a test case in court. A cost-benefit analysis could help

make this decision, but whether this would be accepted in court is another matter.

Turning to patients, this group presents a much bigger problem. For all potential

recipients of blood to be “grouped” at the molecular level would require several things

to come together. Firstly, either all hospitals would have to have the ability to

perform “molecular grouping”, or all patients’ samples would have to be sent to a

centralised laboratory capable of carrying out these tests. Secondly, a sample would

have to be provided early enough, prior to transfusion, for the tests to be carried out,

and this could prove impossible (it is sometimes difficult to get patient samples early

enough to carry out full serological testing prior to a “cold” transfusion, let alone

“molecular grouping”). Thirdly, it would have to be established which of the weak D

and partial D types are susceptible to alloimmunisation (otherwise valuable D-

negative blood could be wasted on those patients who are most unlikely to produce

alloanti-D), and there is no concensus as yet, and may never be, as to the minimum

D sites required per red cell to avoid the recipient becoming alloimmunised. Fourthly,

it is almost certain that the patient would have to give permission for such tests to be

carried out, as the tests would not necessarily be seen as clinically essential. If such

tests were carried out without permission, those wishing to preserve civil liberties

could vigorously protest.

It would be pertinent, therefore, to stage such a move, as for donors samples.

Initially, there would have to be a wide-ranging publicity exercise, to ensure that

recipients realise that such tests, whilst not necessarily clinically essential, could

prove beneficial.

Secondly, it would almost certainly be necessary to stage the testing of patients in

either the following way, or something very similar. Those at most risk of being

alloimmunised should be tested first. These must include, initially, pregnant women

who group serologically as D-negative. Eventually, this should be extended, if

possible, to all females who are potentially child-bearing (but see the caveats above),

32

and to all patients who are, or who will potentially be, chronically transfused. Patients

with sickle cell anaemia will lie within this group, and it should be remembered that

there is a high frequency of partial DIIIa and DAR amongst such patients99. It would

be prudent, therefore, to also test D-positive females who are potentially child-

bearing within this group, as patients with partial DIIIa and/or DAR have an increased

risk of alloimmunisation to the D antigen.

It should also be remembered that alloanti-D can be stimulated in ways that are not

directly through a transfusion, per se, in the narrow view.

Mijovic107, found that recipients of haemopoietic stem cell transplants (D-negative

donation into a D-positive recipient) are more likely to be stimulated to produce

alloanti-D if there is no myeloablative conditioning prior to the transplant, and that

such patients should receive D-negative transfusions.

Patel et al108 reported a case of fatal haemolytic disease of the foetus in a lady who

had already produced anti-D. This case was unusual, however, in that the oocytes

had been donated anonymously, and had been fertilised in vitro by her partner’s

sperm. As a result, one of two twins (the index IUD) inherited the R1R1 phenotype.

As a result of this report, all such donors are now fuly blood typed, so that no such

problem should happen in the future.

“Molecular grouping” as a routine technique is for the future. What should we be

doing at present? Even the newer commerically available partial D kits are unable to

distinquish all of the partial D types known. In many kits, for example, DFR gives the

same reaction pattern as DOL. It would be sensible, therefore, for all partial D

patient’s samples to be sent to a centre of excellence, such as the IBGRL, for typing

at the molecular level. In this way, those most likely to be alloimmunised to the D

antigen can be transfused with D-negative blood. Indeed, individuals with a partial D

are still comparatively rare, and it may be that all should be transfused with D-

negative blood. The problem really is the weak D types. This author found no lists

available that show which weak D types have been known to produce alloanti-D, and

33

which do not. Even if such a list were in existence, without knowing the molecular

“make-up” of any particular weak D, it would be impossible for the hospitals, or the

NBS Centres, to know which should be given D-negative blood. On the other hand,

not all weak D types require D-negative blood, and so would be a waste of this

precious commodity to give D-negative blood to all weak D transfusion recipients. It

may be that it would be prudent to give D-negative blood only to individuals whose

red cells give 1+ reactions with potent monoclonal anti-D reagents. This is far from a

pragmatic approach, as alloanti-D in a weak D individual is both rare and, often,

weak, but such an approach would protect both hospital and NBS laboratories

(particularly when advice, rather than a product is given) from the possibility of being

taken to court, should such an individual be persuaded to go down this route.

34

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37

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38

64. Wagner FF, Flegel WA. RHCE represents the ancestral RH position, while RHD is the duplicated gene. Blood 2002; 99 (letter): 2272-2273.

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77. Ridgwell K. Genetics tools: PCR and sequencing. Vox Sang 2004; 87 (suppl 1): S6-S12.

78. Daniels G. Molecular blood grouping. Vox Sang 2004; 87 (suppl 1): S63-S66.

79. van der Schoot CE. Molecular diagnostics in immunohaematology. Vox Sang 2004; 87 (suppl 1): S189-S192.

39

80. Beckers EAM, Ligthart PC, Overbeeke MAM, Maaskant PA, van Rhenen DJ. A patient with weak D type 1 and auto-anti-D: auto or allo? Vox Sang 2004; 87 (suppl 3): 75.

81. Roxby D, Coloma M, Flegel WA, Poole J, Martin P, Abbott R. Observations of an anti-D after D-positive transfusion in an individual with weak D type-1 phenotype. Vox Sang 2004; 87 (suppl 3): 77-78.

82. Mota M, Fonseca N, Kutner, Rosenblit J, Castilho L. Alloanti-D immunization by weak D type 1 RBCs. Vox Sang 2004; 87 (suppl 3): 132.

83. Mota M, Fonseca NL, Rodrigues Á, Kutner JM, Castilho L. Anti-D alloimmunization by weak D type 1 red cells with a very low antigen density. Vox Sang 2005; 88: 130-135.

84. Döscher A, Ladewig B, Gerdes J, Das Gupta C, Gnoth S, Wagner FF, Schunter F, Petershofen EK. Four new RHD-alleles with previously unknown polymorphisms. Vox Sang 2004; 87 (suppl 3): 81.

85. Wagner FF, Döscher A, Bauerfeind U, Peterschofen EK. Mistyping of a new weak D allele as weak D type 1 by PCR. Vox Sang 2004; 87 (suppl 3): 84.

86. Anstee DJ. Goodbye to agglutination and all that? Transfusion 2005; 45: 652-653.

87. Avent ND. High variability of the RH locus in different ethnic backgrounds (Editorial). Transfusion 2005; 45: 293-294.

88. Flegel WA. Homing in on D antigen immunogenicity (Editorial). Transfusion 2005; 45: 466-468.

89. Wagner T, Buchta C, Vadon M, Lanzer G, Mayr WR, Legler TJ. Anti-D immunization by DEL red blood cells. Transfusion 2005; 45: 520-526.

90. Mollison PL, Englefriet CP, Contreras M. Blood Transfusion in Clinical Medicine, 10th edn. Oxford: Blackwell Science, 1997.

91. Hilden JO, Gottvall T, Lindblom B. HLA phenotypes and severe Rh(D) immunization. Tissue Antigens 1995; 46: 313-315. Cited in Hadley A, Soothill P. Alloimmune disorders of pregnancy, 1st edn. Cambridge University Press, 2002.

92. Gassner C, Doescher A, Drnovsek TD, Rozman P, Eicher N, Legler TJ, Lukin S, Garritsen H, Kleinrath T, Egger B, Ehling R, Körmöczi GF, Kilga-Nogler S, Schoenitzer D, Petershofen EK. Presence of RHD in serologically D-, C/E+ individuals: a European multicenter study. Transfusion 2005; 45: 527-538.

93. Garratty G. Do we need to be more concerned about weak D antigens? (Editorial). Transfusion 2005; 45: 1547-1551.

94. Denomme GA, Wagner FF, Fernandes BJ, Li W, Flegel WA. Partial D, weak D types, and novel RHD alleles among 33, 864 multiethnic patients: implications for anti-D alloimmunization and prevention. Transfusion 2005; 45: 1554-1560.

40

95. Körmöczi GF, Förstemann E, Gabriel C, Mayr WR, Schönitzer D, Gassner C. Novel weak D types 31 and 32: adsorption-elution-supported D antigen analysis and comparison to prevalent weak D types. Transfusion 2005; 45: 1574-1580.

96. Körmöczi GF, Gassner C, Shao C-P, Uchikawa M, Legler TJ. A comprehensive analysis of DEL types: partial DEL individuals are prone to anti-D alloimmunization. Transfusion 2005; 45: 1561-1567.

97. Yasuda H, Ohto H, Sakuma S, Ishikawa Y. Secondary anti-D immunization by Del red blood cells. Transfusion 2005; 45: 1581-1584.

98. Doescher A, Flegel WA, Petershofen EK, bauerfeind U, Wagner FF. Weak D type 1.1 exemplifies another complexity in weak D genotyping. Transfusion 2005; 45: 1568-1573.

99. Castilho L, Rios M, Rodrigues A, Pellegrino jnr J, Saad STO, Costa FF. High frequency of partial DIIIa and DAR alleles found in sickle cell disease suggests increased risk of alloimmunization to RhD. Transf Med 2005; 15: 49-55.

100. Laycock RA, Makar Y, Elhanash S, Poole J, Martin P, Tilley L, Bullen P. A case of a DEL woman being immunised to produce anti-D which caused severe HDFN. Transf Med 2005; 15 (suppl 1): 48.

101. Tilley, L, Bullock T, Kingdom S, Poole J, Daniels G. A novel RhD variant associated with extremely low serological expression of D-antigen. Transf Med 2005; 15 (suppl 1): 48-49.

102. Rodrigues MJ, Tilley L, Tomás C, Araújo F, Poole J, Fernandes S, Chabert T. Identification of partial antigen DBS. Transf Med 2005; 15 (suppl 1): 51.

103. Bruce DG, Poole J, Tilley L, Wallis JP, Todd A. Immune alloanti-D in a patient with a novel RHD mutation. Transf Med 2005; 15 (suppl 1): 52.

104. Wagner FF: The Rhesus Base. Ulm, Germany, University Hospital, Department of Transfusion Medicine, 1998 [http://www.uni-ulm.de/~fwagner/RH/RB/].

105. Kitchen AD, Barbara JAJ. Transfusion-transmitted infections (Chapter 19) in: Practical Transfusion Medicine, 2nd edn. Editors Murphy MF and Pamphilon DH. Oxford: Blackwell Publishing, 2005.

106. Scott ML. Personal communication (2006).

107. Mijovic A. Alloimmunization to RhD antigen in RhD-incompatible haemopoietic cell transplants with non-myeloablative conditioning. Vox Sang 2002; 83: 358-362.

108. Patel RK, Nicolaides K, Mijovic A. Severe hemolytic disease of the fetus following in vitro fertilization with anonymously donated oocytes. Transfusion 2003; 43 (Letter): 119-120.

109. Scott ML, Voak D, Jones JW, Liu W, Avent ND, Hughes-jones N, Sonneborn H-H. A model for RhD – the relationship of 30 serologically defines epitopes to predicted structure. Biotest Bulletin 1997; 5: 459-466.

41

110. Daniels G. Human Blood Groups, 2nd edn. Oxford: Blackwell Science, 2002.

42

Appendix 1.

Amino Acid Three-letter Code Single-letter Code

Properties

Alanine Ala A Non-polarArginine Arg R Polar, Positively

ChargedAsparagine Asn N Polar, Uncharged

Aspartic Acid Asp D Polar, Negatively Charged

Cysteine Cys C Polar, UnchargedGlutamine Gln Q Polar, Uncharged

Glutamic Acid Glu E Polar, Negatively Charged

Glycine Gly G Polar, UnchargedHistidine His H Polar, Positively

ChargedIsoleucine Ile I Non-polar

Leucine Leu L Non-polarLysine Lys K Polar, Positively

ChargedMethionine Met M Non-polar

Phenylalanine Phe F Non-polarProline Pro P Non-polarSerine Ser S Polar, Uncharged

Threonine Thr T Polar, UnchargedTryptophan Trp W Non-polar

Tyrosine Tyr Y Polar, UnchargedValine Val V Non-polar

Table 1. Three letter and single letter amino acid codes, together with the amino acid properties (modified from Reid and Lomas-Francis74).

43

Appendix 2.

Red Cell Membrane

LipidBilayer

Cytosol

E1 E2 E3 E4 E5 E6 E7

E8

E9

E10

Exon Boundary

1NH2 417

COOH

49 112162

212

267 313

358

398

409

Figure A simplified model of the wild-type D carrier molecule, showing the epitope boundaries ( = ) (modified from Scott et al109).

44

Appendix 3.

2

34

56

7

8 9

1011

12

112

3

4

5

6

NH2

COOH

45

Figure A model of the proposed correct juxtaposition of the D carrier molecule (blue arrows = external loops, pink lines = internal loops and termini, and the multicolour circles = transmembranous prtions of the molecule (the colours refer to the exons shown in appendix 2) (after Daniels110).

46

Appendicies 4 to 9 are taken from Wagner FF: The Rhesus Base. Ulm, Germany, University Hospital, Department of Transfusion Medicine, 1998 [http://www.uni-ulm.de/~fwagner/RH/RB/]104 (correct as of 9.5.2006).

47

Appendix 4: Weak D Types and Amino Acid Substitutions (for colour code, refer to Appendix 1).

Weak D Type 1 V270GWeak D Type 1.1 L18V, V270GWeak D Type 2 G385AWeak D Type 3 S3C

Weak D Type 4.0 T201R, F223VWeak D Type 4.1 W16C, T201R, F223VWeak D Type 4.2 T201R, F223V, I342TWeak D Type 4.3 T201R, F223V, P291RWeak D Type 5 A149DWeak D Type 6 R10QWeak D Type 7 G339EWeak D Type 8 G307RWeak D Type 9 A294P

Weak D Type 10 W393RWeak D Type 11 M295IWeak D Type 12 G277EWeak D Type 13 A276PWeak D Type 14 S182T, K198N, T201RWeak D Type 15 G282DWeak D Type 16 W220RWeak D Type 17 R114WWeak D Type 18 R7WWeak D Type 19 I204TWeak D Type 20 F417SWeak D Type 21 P313LWeak D Type 22 G408CWeak D Type 23 G212CWeak D Type 24 L338PWeak D Type 25 R114QWeak D Type 26 V9DWeak D Type 27 P221SWeak D Type 28 T384TWeak D Type 29 I60L, S68N, K198N, F223V, I342TWeak D Type 30 E340MWeak D Type 31 L6PWeak D Type 32 I374NWeak D Type 33 V174MWeak D Type 34 V270EWeak D Type 35 G87DWeak D Type 36 V281GWeak D Type 37 K133NWeak D Type 38 G278DWeak D Type 39 G339RWeak D Type 40 T201RWeak D Type 41 E398VWeak D Type 42 K409MWeak D Type 43 A202VWeak D Type 44 Y243CWeak D Type 45 A399TWeak D Type 46 F407L

48

Weak D Type 47 R114GWeak D Type 48 G61VWeak D Type 49 S257FWeak D Type 50 Y243NWeak D Type 51 K198N, T201RWeak D Type 52 F31SWeak D Type 53 V247G

49

Appendix 5. Partial D Categories Showing Amino Acid Substitutions and/or Exon Substitutions (For colour code of amino acids, see Appendix 1. Exon

Substitutions shown in pink).

Partial D Category I RedundantPartial D Category II A354D

Partial D Category IIIa N152T, T201R, F223VPartial D Category IIIb RHD1-RHCE2-RHD3-10Partial D Category IIIc RHD1-2-RHCE3-RHD4-10

Partial D Category III Type 4 L62F, A137V, N152TPartial D Category III Type 5 L62F, A137V, N152T, T201R, F223VPartial D Category III Type 6 A137V, N152T, T201R, F223V

Partial D Category III Type 7I60L, S68N, S103P, A137V, N152T,

T201R, F223VPartial D Category IVa L62F, N152T, D350H

Partial D Category IVa-2 L62F, A137V, N152T, D350HPartial D Category IVb RHD-CE(7:D350H-9)-D

Partial D Category IV Type 3 RHD-CE(6-9)-DPartial D Category IV Type 4 RHD-CE(7:D350H-354N)-DPartial D Category IV Type 5 RHD-CE(7-9)-D

Partial D Category V Type 1/DV (FK)/DV (Kou.)

RHD-CE(5:F223V, E233Q)-D

Partial D Category DV Type 2/DV (Hus.)

RHD-CE(5)-D

Partial D Category DV Type 3/DBS-1 F223V, A226P, E233Q, V238MPartial D Category DV Type 4/DV (SM) E233Q

Partial D Category Partial DV Type 5/DHK

E233K

Partial D Category DV Type 6/ DV (Jpn)

RHD-CE(5:L223V, V238M)-D

Partial D Category DV Type 7 RHD-CE(5:F223V, G263R)-DPartial D Category DV Type 8/DV (TT) RHD-CE(5:F233V, V245L)-RHDPartial D Category DV Type 9/DV (TO) RHD-CE(5:E233Q, V238M)-D

Partial D Category DVI Type 1 RHD-CE(4-5)-DPartial D Category DVI Type 2 RHD-CE(4-6)-DPartial D Category DVI Type 3 RHD-CE(3-6)-DPartial D Category DVI Type 4 RHD-CE(2-5)-D

Partial D Category VII L110PPartial D Category VII Type 2 S103P, L110P

50

Appendix 6: Other Partial D Types Showing Amino Acid Substitutions and/or Exon Substitutions (For colour code of amino acids, see Appendix 1. Exon

Substitutions shown in pink).

D667 F223VDAR T201R, F223V, I342T

DAR-E T201R, F223V, E233Q, I342QDAU-1 S230I, T379MDAU-2 R70Q, S333N, T379MDAU-3 E279M, T379MDAU-4 E233K, T379MDAU-5 F223V, E233Q, T379MDAU-6 S333N, T379M

DBS-1/DTI RHD-cE(5)-DDBT-1 RHD-CE(5-7)-DDBT-2 RHD-CE(5-9)-DDCS RHD-CE(5:F223V, A226P)-DDDE D40EDFL Y165CDFR RHD-CE(4:M169L, I172F)-D

DFR-2 RHD-CE(4)-DDFW H166P

DHAR RHCE-D(5)-CEDHK/DYO E233K

DHMi T283IDHMii RHD-CE(3-5(part))-DDHO K235TDHR R229KDIM C285YDMH L54P

DNAK G357DDNB G355SDNU G353RDOL M170T, F223V

DOL-2 M170T, F223V, L378VDOL-3 A137V, M170T, F223V

DQC/DYU R234WDTO F223V, S225FDWI M358T

Unnamed RHD(229delR) 229 delR

51

Appendix 7: Incompletely Characterised Weak and Partial D Types Showing Amino Acid Substitutions or Base Substitutions (For colour code of amino

acids, see Appendix 1).

D674 S225FDBA L227PDHQ H171QDIT I288TDLO S284LDMA L207F

Unnamed RHD(Q49R) Q49RUnnamed RHD(G96S) G96SUnnamed RHD(S103P) S103P

Weak RHD 165C>T RHD(165C>T)

52

Appendix 8: D-negative and DEL Types (For colour code of amino acids, see Appendix 1. Exon Substitutions shown in pink).

Structure Allele Mechanism PhenotypeRHD deletion RHD deletion Large deletion D-negative

RHCE(1-3)-D(10) RHCE(1-3)-D(10) Large hybrid? D-negativeRHCE(1-9)-D(10) RHCE(1-9)-D(10) Large hybrid D-negativeRHD-CE(2-9)-D RHD-CE(2-9)-D Large hybrid D-negative

RHD-CE(3-9)-DRHD-CE(3-9)-D

InnsbruckRHD-CE(3-9)-D

Large hybrid D-negative

RHD-CE(2-7)-D RHD-CE(2-7)-D Large hybrid D-negativeRHD-CE(3-7)-D RHD-CE(3-7)-D Large hybrid D-negativeRHD-CE(4-7)-D RHD-CE(4-7)-D Large hybrid D-negative

RHCE(1-5)-D(6)-CE(7-10)?

RHCE-D(6)-CETemplated

missense in RHCE

D-negative

RHD-CE(8-9)-D RHD-CE(8-9)-D Unknown D-negativeRHD psi RHDψ Not transcribed D-negative

RHD(M1I, L84P) RHD(M1I, L84P) Start codon lost DEL

RHD(W16x) RHD(W16x)Premature stop-

codonD-negative

RHD(Q41x) RHD(Q41x)Premature stop-

codonD-negative

RHD(147delA) RHD(147delA) Frameshift DEL

RHD(IVS1+1G>A)DelRHD(IVS1+1G

>A)Splice site mutation

DEL

RHD270G>A RHD(W90x)Premature stop-

codonD-negative

RHD(325delA) RHD(325delA) Frameshift D-negative

RHD(IVS2+1G.A) RHD(IVS2+1G.A)Splice site mutation

D-negative

RHD(IVS2-1G>A) RHD(IVS2-1G>A)Splice site mutation

D-negative

RHD(343delC) RHD(343delC) Frameshift D-negativeRHD(449delT) RHD(449delT) Frameshift D-negative

RHD(L153P) RHD(L153P)Missense mutation

DEL

RHD(IVS3+1G>A) RHD(IVS3+1G>A)Splice site mutation

DEL

RHD(IVS3+2T>A) RHD(IVS3+2T>A)Splice site mutation

D-negative or DEL

RHD(488del4) RHD(488del4) Frameshift D-negative

RHD(W185x) RHD(W185x)Premature stop-

codonD-negative

RHD(G212V) RHD(G212V)Splice site affected?

D-negative

RHD(del711C) RHD(711delC) Frameshift D-negative

RHD(785delC) RHD(785delC) FrameshiftD-negative or

DELRHD(IVS5-

38del4)DELRHD(IVS5-

38del4)Unknown

(missplicing?)DEL

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RHD(Y269x) RHD(Y269x)Premature stop-

codonD-negative

885G>T RHD(M295I)Missense mutation

DEL

RHD(908 ins tggct,

IVS6+1del4)

RHD(906 ins tggct,

IVS6+1del4)

Frameshift + splice site mutation

D-negative

RHD(Y311x) RHD(Y311x)Premature stop-

codonD-negative

RHD(G314V) RHD(G314V) Unknown D-negative

RHD(Y330x) RHD(Y330x)Premature stop-

codonD-negative

RHD(IVS8+1G>A) RHD(IVS8+1G>A)Splice site mutation

D-negative

RHD(delEx9) RHDel Large deletion DEL

RHD(Y401x) RHD(Y401x)Premature stop-

codonD-negative

RHD(W408R) RHD(W408R)Missense mutation

DEL

RHD(K409K) RHD(1227G>A)Splice site affected?

DEL

RHD(exon 5 variant)

D negative Exon 5 variant

Premature stop-codon

D-negative

RHD(x418L) RHD(x418L) Elongation DEL

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Appendix 9: D-positive Types (For colour code of amino acids, see Appendix 1.).

Phenotype Allele MutationStandard RHD RHD -

DAU-0 RHD(T379M) 1136 C>TDAU-0.1 RHD(T379M) 579 G>A, 1136 C>TDUC-1 RHD 636 C>TDUC-2 RHD(V245L) 733 G>C

Unnamed RHD (384 T>C) RHD 384 T>C

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