Clin. lab. Harmut.

1989, 11, 317-322

The Rh-negative donor

MARCELA CONTRERAS, MD, MRCPath &
ROBIN C. KNIGHT, FIMLS
North London Blood Transfusion Centre, Colindale Avenue,
Colindale, London N W9 5BG

Accepted for publication 21 July 1989

Summury By typing fully for Rh, donor samples found to be D-negative but
C-positive and/or E-positive on the Kontron Groupamatic G2000, the incidence
of Ccddee (r’r) was found to be 0.44%, ccddEe (r”r) 0.50% and Du 0.30%. A
total of 15 000 samples typed on the Groupamatic as D-, C- and E-negative were
tested by an antiglobulin technique for D”, but none was found to be positive. A
new strategy was therefore adopted for routine Rh typing of donations that
includes typing first-time donors on the Groupamatic for C, D and E antigens;
those that type as D-negative, C-positive and/or E-positive are further tested but
no routine D” testing of D-, C- and E-negative donations is performed.
Donations are labelled according to their D type, and the once used terminology
‘Rh-positive donor, Rh-negative recipient’ is no longer used except for those rare
Rh D-positive donors belonging to the D category VI of Tippett & Sanger (1962).

Keywords: Rh negative, Rh typing, D”, D category

In the U K there is a tradition that a Rh-negative donor is one whose red cells lack
not only D but also C and E. With this method of selecting Rh-negative donors,
those subjects who are D-negative but are C- or E-positive, e.g. Ccddee or
ccddEe, are classed as Rh-positive if they are donors but as Rh-negative if they
are recipients. However, the case for trying to avoid giving C- or E-positive blood
to subjects lacking these antigens is very poor, since immune responses to C and E
are very rare except when an immune response to D is induced by the same red
cells. Moreover, no steps are taken to avoid exposing ccDdEe (e.g. R,r) recipients
to the risk of immunization by C in, for example, CcDdee (R,r) cells or to avoid
exposing CcDdee (R,r) recipients to the risk of immunization by E in, for
example, ccDdEe (R,r) cells, although these risks are relatively common: thus, in
the UK, some 82% of D-positive units are C-positive and 32% E-positive,
whereas of D-negative units some 2.7% are C-positive and 3% E-positive. If no
steps are taken to avoid exposing CCDDee (R,R,) subjects to immunization by
the c antigen, the risk is substantially greater than the risks just considered. In the
UK, anti-c, after anti-D, is currently second in importance as a cause of moderate
and severe haemolytic disease of the newborn. In the USA and some European

Correspondence: Dr M. Contreras, Director, North London Blood Transfusion Centre, Colindale

Avenue, London NW9 5BG.

317
318 M . Contrerus and R.C. Knight

countries the term Rh-negative, for both donors and recipients, means D-
negative; C and E antigens are not tested for.
In selecting Rh-negative donors, because D is far more important than other
Rh antigens, it is traditional to confirm the D status of all subjects initially typed
as D-negative. For this confirmation, the indirect antiglobulin test (IAT) is used
and donors whose red cells react in this test, although they have not reacted in the
preliminary test, are regarded as D": these subjects used to be considered as Rh-
positive when they were donors but as Rh-negative when they were recipients but
nowadays they are regarded simply as Rh-positive.
The performance of an indirect antiglobulin test to detect D" donors is
expensive and its value has been questioned mainly because D" cells have been
shown to be very poorly immunogenic (Schmidt, Morrison & Shohl 1962; Moore
1984). In addition, modern blood grouping reagents will detect the vast majority
of cells with reduced numbers of D antigen sites; i.e. red cell samples which used
to be classified as 'high grade' or 'moderate grade' D" will now be classified as
straightforward D-positive. On the other hand, there will always be a small
proportion of samples with red cells carrying such a low number of D sites that
they will not be detected by IAT but only by absorption/elution techniques.
In the past, the specificity of the anti-D reagents used to type donor red cells
has not been standardized and the ability to detect D variant cells must therefore
be variable. However, it is possible, if a selection of immunized donors is
available, to produce two different routine anti-D reagents, one that detects and
another one that misses cells that belong to the D category VI (D"' cells) of
Tippett & Sanger (1962), as this is considered the type of D variant cells of
greatest clinical importance.
The purpose of this paper is to present data supporting some of the foregoing
assertions and to describe the implementation of a more rational policy for
defining Rh-negative donors than has prevailed in the past.

Materials and methods

Approximately 800 donations are typed daily at the North London Blood
Transfusion Centre. Previously grouped donor samples from established donors
are tested on a Kontron Groupamatic G2000 and first-time donors on the
Kontron LHS-Microgroupamatic using Rh typing reagents prepared from sera
obtained locally from immunized donors. In these blood grouping machines, the
red cells under test are suspended in a bromelin solution and mixed with typing
sera at room temperature.
The following Rh typing reagents were used on first-time donors: anti-D (two
examples), anti-C + D, anti-E and anti-c. Red cell samples found to be negative
+
with the anti-D sera as well as with anti-C D and anti-E used to be tested with
one anti-D by IAT; if negative they were classified as Rh-negative and not tested
any further.
Samples found to be D-negative but C- or E-positive and those that gave
 The Rh-negative donor 319

variable reactions with the anti-D reagents were typed manually using two
examples of anti-D, anti-C, anti-E, anti-c and anti-e in microplates using
bromelin-suspended red cells by a technique described by Knight & Redman
(1987). These cells were also tested by an indirect antiglobulin technique with
these two anti-D reagents plus one other, one of which was selected for failing to
react with D"' red cells. Those cells that only react with anti-D sera by IAT are
termed D". Most Dv' red cells react weakly by the microplate technique but give
variable reactions with the relevant anti-D on the G2000 grouping machine.
Samples from previously grouped donors were tested with anti-D, anti-C D +
and anti-E; any samples giving results at variance with those from previous
typings were retested manually as above.

Results

Table 1 shows the data obtained from typing first-time (new) donors on the
Kontron Groupamatic G2000 over 6 months.
In a 3-year period 15 000 red cell samples from new donors found to be D-, C-
and E-negative on the G2000 were tested with one strong anti-D reagent by the
indirect antiglobulin technique, and found negative.

RATIONALIZATION OF Rh TESTING

Known donors are now tested with one example of anti-D and one of anti-C + D.
Donors whose red cells type as C-positive but D-negative Ccdee (r'r) or CCdee
(r'r') are labelled as Rh-negative, with auxiliary label attached so that they are not
selected when cross-matching for a patient with anti-C + D. This simple solution
overcomes the most often cited objection to labelling r' donations as Rh-negative.
Those donations negative with both antisera are labelled as Rh D-negative
without any further testing.
First-time donors (approximately 15% of total donations) are tested twice on
the LHS-microgroupamatic with a total of two different anti-D reagents, in
addition to anti-C+D, anti-E and anti-c. Those that type as D-, C- and E-
negative are regarded as Rh-negative without any further testing; they are not

Table 1. Data obtained from typing first-time donors on the Kontron Groupamatic G2000 over 6
months

Total samples tested 16 484

Typed as D- C- E-
Typed as D- C + E-
Typed as D- C - E +
Typed as 'probable D"' on the
Groupamatic
Total D" examples
2562; confirmed manually 2562 (15.5% of total)
87; confirmed manually 73 (0.44% of total) R I U r =14
90; confirmed manually 82 (0.50% of total) R;r=8
27; confirmed manually 27
49 (0.30% of total)
320 M . Contrerus and R.C. Knight

now tested with anti-D by the indirect antiglobulin technique. Those few samples
that type as D-negative but C- or E-positive or D" (i.e. weak or variable reactions
with the anti-D reagents in the Groupamatic) are fully typed for Rh manually,
with the inclusion of three different anti-D sera, one of which fails to react with
D"' cells, by the indirect antiglobulin technique. Those that are confirmed as C- or
E-positive but D-negative are labelled Rh D-negative.
The reasons for continuing to type for the C and E antigens are threefold: (i)
Using these sera, plus anti-c, makes it possible to select donor units, for example,
that are E- and c-negative, for manual confirmation, in order to meet the
increasing demand for Rh typed blood for patients with Rh alloantibodies and for
cells used in antibody screening and identification panels supplied within the
centre and to hospitals in the region. (ii) It allows for C-positive, D-negative
donations to be labelled with an auxiliary label as explained above. (iii) The
further manual testing of donations typed on the machine as Ccdee, ccdEe, and
D" is a cost-effective way of recognizing the small number that are in fact D".

Discussion

The major advantages of the changes have been a 6% increase in Rh-negative

blood stocks, previously always in short supply, in addition to a saving in time
and expense as 'D" testing' has been discontinued and anti-E is no longer used for
testing previously grouped donors.
In this series, the overall detection rate for D" of 0.30% was lower than other
published figures of 0.56% (Garretta, Muller & Gener 1974) and significantly less
than the 3% reported using potentiated anti-D reagents, presumably by an
immediate spin technique (Stroup Waters 1988). It is known that many examples
of D" will react with potent anti-D reagents by enzyme techniques. Therefore the
use of bromelin, both in the Groupamatic and on microplates, results in the so-
called 'high grade' and 'moderate grade' D" examples being typed as straight-
forward D-positive, and only the 'low grade' examples being classified as D".
Although the potential risk to recipients is considered to be very low if 'low
grade D"' or D" category donations are labelled as Rh-negative, most should be
detected by the testing procedures which have been adopted. The purpose of these
procedures is to provide typed blood for transfusion of immunized patients and
for red cell panels. In the absence of these requirements, the testing of donors with
reagents other than anti-D or anti-D plus anti-C+D would not be necessary or
cost-effective.
The strength of the D antigen on red cells of D categories varies and it is
probable that some are not recognized by routine techniques as they may give
normal strength reactions with most anti-D reagents. Hence, those samples that
we classify as D"' are likely to be those that show a weaker expression of the D
antigen. Since we produce our own anti-D reagents from immunized donors we
are able to select sera so that one of the two anti-D reagents used will react with
Dv' cells. Donors who type as D"' are given cards as 'Rh-positive donors, Rh-
 The Rh-negative donor 321

negative recipients’ as they are at an unknown risk of producing anti-D if

transfused with Rh D-positive blood. However, the risk is probably very small.
Using monoclonal anti-D reagents to type donors, the incidence of D”’ is reported
to be 0.02’/0 (K.A. Leader et al., submitted for publication).
Before the new policy was implemented, a letter was sent to all consultant
haematologists in charge of hospital blood banks supplied from our Centre
explaining the reasons for, and the consequences of, these changes. An explana-
tion was also given to all our collection teams and their Standard Operating
Procedures (SOPS) were updated.
Computer programs on the grouping machines and the main-frame computer
were changed so that donations typed as C-positive and/or E-positive but D-
negative were designated Rh-negative.
An explanatory leaflet for C-positive and/or E-positive but D-negative donors
(i.e. r’ and r” donors) affected by these changes was prepared to explain to them
that the terminology used had been changed and not their blood groups. Their
value as Rh-negative donors was stressed. One problem arising from this policy
change is that existing donor records and certificate books that still carry the ‘Rh-
positive donor, Rh-negative recipient’ label have to be updated, and this can only
be done after a current donation has been tested and fully phenotyped according
to present policies.
The elimination of the obsolete and confusing terminology of ‘Rh-positive
donor, Rh-negative recipient’ has probably led to an increase in donor confidence
(staff at the blood centre know what they were talking about!). This should not be
overlooked.
Hospital blood banks must request E-negative blood, or type units with anti-E
(supplied by the transfusion centre) rather than waste precious Rh-negative blood
(that was previously typed as D-, C- and E-negative) for Rh-positive patients with
anti-E. The Centre maintains stocks of Rh phenotyped blood for patients with Rh
antibodies or for those requiring long-term transfusion support with ‘Rh compat-
ible blood’.
The policy has been fully implemented for over 18 months and no problems
have been reported to the Centre apart from the inconvenience to hospital blood
banks of having to type the occasional unit with anti-E for patients with allo-anti-
E. None of the hospitals supplied with ‘Rh-negative blood’ reported anti-G in any
transfused Rh-negative patient.

References

GARRETTA M., MULLERA. & GENER J. (1974) Determination du facteur Dusur Groupamatic. Etude
comparee avec les techniques manuelles. Bilan sur 203, 240 examens. Rev. Fr. Transfus. 17,
21 1-215
KNIGHTR.C. & REDMAN M. (1987) Basic microplate techniques. In: The Use of Micrupiates in Blood
Croup Serology (eds R. Knight & G . Poole) pp 14-19. British Blood Transfusion Society,
London
322 M . Contreras and R.C. Knight
MOOREB.P.L. (1984) Does knowledge of the D" status serve a useful purpose? Vox Sang. 46
(Suppl.), 95-97
SCHMIDT P.J., MORRISON E.G. & SHOHLJ. (1962) The antigenicity of the Rh,(D") blood factor.
Blood 20, 196-202
STROUPWATERSM. (1988) Letter to the editor. Zmmunohaematology 4, 16-17
TIPPETTP. & SANGER R. (1962) Observations on subdivisions of the Rh antigen D. Vox Sung. 7,
9-1 3