Section 14.

9, References
14.9 Antibody identification
When an alloantibody is detected in the screening procedure, the specificity should
be determined and its likely clinical significance assessed. It is essential to determine
the specificity of all clinically significant antibodies present. It is important to employ a
systematic approach to antibody exclusion in the process of antibody identification.
Several reagent red cell panels may be needed to identify some combinations of
antibodies.
14.9.1 Principles of antibody identification
The patient's serum or plasma should be tested by an appropriate technique against
an identification panel of reagent red cells. As a starting point, the technique by
which the antibody was detected during screening should be used. Inclusion of the
patient's own red cells may be helpful, for example, in the recognition of an antibody
directed against a high frequency antigen or the presence of an autoantibody.
The specificity of the antibody should only be assigned when it is reactive with at
least two examples of reagent red cells carrying the antigen and non-reactive with at
least two examples of reagent red cells lacking the antigen. Note that, wherever
possible, the presence of anti-Jk
a
, -Jk
b
, -S, -s, -Fy
a
, and -Fy
b
should be excluded
using red cells having homozygous expressions of the relevant antigen.
A weak anti-D control serum or reagent (containing anti-D at a level of approximately
or less than 0.1 IU/mL) should be used on a regular basis to assure the efficacy of
the whole test procedure, including antigen expression on screening or panel cells.
When one antibody specificity has been identified, it is essential that the presence of
additional clinically significant antibodies has not been missed. Multiple antibodies
can only be confirmed by choosing cells antigen negative for the recognized
specificity, but positive for other antigens to which clinically significant antibodies
may arise. Knowing the phenotypes of the patient can aid in cell selection for the
identification and exclusion process. The requirements detailed above should be met
for each antibody detected but in some circumstances this may not be possible.
The use of a panel of enzyme (e.g. papain) treated cells is strongly recommended
for antibody identification, particularly when an antibody weakly reactive by the
antiglobulin technique, or a mixture of antibodies, is present. The use of
monospecific antiglobulin reagents in place of polyspecific reagent is beneficial when
determining the presence of IgG antibodies in serum samples containing
complement-binding antibodies. The use of room temperature saline techniques may
also be helpful.
14.9.2 Reagent red cells for use in antibody identification
See Section 12.2.3 for details.
If the patient is group A, B or AB and all reagent cells react with the test plasma, it
may be useful to include cells of the same ABO group as the patient to exclude the
possible presence of anti-HI.
When the phenotypes of the patient are known, it is not necessary to look for any
specificity where the patient is positive for the antigen. Where the patient is negative
there should be at least two examples of cells with phenotypes lacking, and at least
two examples of phenotypes carrying, expression of the corresponding antigen. The
panel should be able to resolve as many likely antibody mixtures as possible.
14.9.3 Red cell phenotyping in antibody identification
When an antibody has been identified, the patient's red cells should be phenotyped
using a reagent of the same specificity as the assigned specificity. Suitable controls
should be used, i.e. antigen positive (from a heterozygote) and antigen negative.
The patient's red cells, as determined during phenotyping, will normally be expected
to lack the antigen(s) corresponding to the antibody specificity (specificities)
assigned. If this is not the case:
(a) the antibody may be an autoantibody (in which case the patient's cells will
normally be DAT positive), and/or
(b) if an antiglobulin or potentiated test method has been used, the patient's cells
may be coated with globulin components (in which case the cells will be DAT
positive)
(c) the assignation of antibody specificity may be incorrect
(d) the patient may have been recently transfused.
The incorporation of a reagent control or AB serum control used by the same
technique as the phenotyping reagent is recommended, unless the reagent is known
to contain unpotentiated IgM antibody. A positive result in this control test will
invalidate the phenotyping test results.
When red cells taken from a blood donation unit are found to be positive in an
antiglobulin crossmatch against patient's plasma, but no activity is detected in the
plasma against red cells in an identification panel, it is likely that either:
(a) the plasma contains an antibody to a low frequency antigen
(b) the red cells in the donation are DAT positive
(c) blood of the wrong ABO group has been selected for crossmatching.
14.9.4 Extended red cell phenotyping
It may be useful to perform extended red cell phenotyping to identify the potential
antibodies that could be produced. This may not be possible to determine if the
patient has been recently transfused. The following antigens should be tested for:
C, c, D, E, e, M, N, S, s, K, k, Fy
a
, Fy
b
, Jk
a
, Jk
b
.
14.9.5 Investigation of a broadly reacting plasma sample
If all cells are positive, with reactions of equal strength:
(a) An antibody to a high incidence antigen should be considered. The use of an
enzyme (e.g. papain) treated panel is particularly useful as reactivity or non-reactivity
of the test plasma may exclude certain specificities immediately, and save time and
the unnecessary use of rare typing reagents.
(b) The cells of the patient should be typed for as many high incidence antigens as
possible and, if a negative is found, cells lacking that antigen should be matched
against the patient's plasma. Where possible the antigen negative cells should
include antigens that the patient lacks in order to exclude the presence of further,
underlying, alloantibodies.
If all cells are positive with varying degrees of strength:
(a) When cells give different strength reactions and a mixture of antibodies cannot be
confirmed antibodies to Ch, Rg, Cs
a
, Kn
a
/ McC
a
(CR1-related) should be
considered. Ch and Rg are antigens which reside on the C4d component of the
complement protein C4 and the serological activity in IAT tests is due to the small
amount of C4 adsorbed on to the surface of the cells in vivo. Coating reagent red
cells with C4 in vitro in a low ionic strength medium enhances the serological
reactivity of anti-Ch and anti-Rg. Plasma inhibition may also be used to confirm Ch
and Rg specificities but dilution of the test plasma may be needed for complete
inhibition to be observed.
(b) Because of the heterogeneity of the CR1 protein not all examples of Kn
a
-related
antibodies have the same specificity. Therefore, the cells of different antibody
makers may not be mutually compatible.
Use of an extended range of enzyme (trypsin or chymotrypsin) treated and
chemically (AET or DTT) modified cells can aid in the identification process if the
specificity cannot be determined or is in doubt. Reactivity or non-reactivity of the test
plasma may indicate a specificity within a certain blood group system.
An eluate prepared from group O, antigen matched, cells and sensitized with
antibody from the test plasma, may be helpful in antibody identification. The eluate
will isolate any antibody (present in the test plasma) to a high incidence antigen and
will enable the matching of rare phenotype cells, including null phenotype cells, of
any ABO group.
In a complex mixture of antibodies individual antibodies can be isolated by
adsorption of one antibody on to red cells positive for the corresponding antigen and
lacking the antigens for the other antibodies. An eluate may be prepared from the
first aliquot of cells to confirm the specificity of the adsorbed antibody. Both the
aliquots of absorbed sera and eluates can be tested for individual specificities. This
procedure is useful when there are insufficient fully typed reagent panel cells
available to detect each specificity individually in the raw serum.
References
1. Department of Health, The Caldicott Committee, Report on the Review of Patient-
Identifiable Information. December 1997 available at www.dh.gov.uk.
2. British Committee for Standards in Haematology (2004) 'Guidelines for
compatibility procedures in blood transfusion laboratories', Transfusion Medicine, 14,
pp5973.
3. Stainsby, D., MacLennan, S. and Hamilton, P.J. (2000) 'Management of massive
blood loss: a template guideline', British Journal of Anaesthesia, 85(3), pp48791.




UK Blood Transfusion Services. Use of this site implies your acceptance of our Conditions
of Use