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Tech Com—Immunohematology
New Approach to Antibodies Directed
at High Frequency Kell and Cartwright
Antigens Using DTT-Modified RBCs
Ira A. Shulman, MD; Janice M. Nelson, MD; Hwai-Tai Lam, MT(ASCP)SBB;
Mitzi Okamoto, MT(ASCP); and Elisabeth Meyer, MT(NCA)
Red cells treated with 200 mM dithiothreitol (DTT) can be useful in the
identification of antibodies directed at certain high frequency red cell antigens. DTT denatures antigens of the Cartwright and Kell blood group systems. Whenever an antibody directed at a high frequency antigen fails to
react with DTT-modified RBCs, antibodies such as anti-Yta, anti-Js b , antiKp b , and anti-Ku should be considered. A patient whose serum contained
anti-Yt a is described. The antibody identification was greatly aided by the
use of DTT-modified red cells.
considered that the patient's serum
contained either anti-Yt a , anti-Js b ,
anti-Kp\ or some other antibody directed at a high frequency Kell blood
group antigen. The following reagent
red cells were then selected: Yt(a-),
Js(b-), Kp(b-), andKo.
Materials and Methods
T
he identification of antibodies directed at high frequency Kell and
Cartwright antigens may be difficult
because RBCs lacking these antigens
may not always be readily available
to blood bank laboratories. The ability to create RBCs lacking high frequency Kell and Cartwright blood
group antigens would therefore be a
valuable aid in the antibody identification process.
The Cartwright (Yta) and Kell blood
group antigens depend on disulfide
bonds for their integrity.1 Treatment
of RBCs with 200 mM dithiothreitol
(DTT) disrupts disulfide bonds and
renders Yta as well as K, k, Js a , Js b ,
Kpa, Kpb, and Ku completely nonreactive with corresponding antisera.1,2 These RBCs can thus aid in
the identification of anti-Yt", anti-Kpb,
anti-Js b , and anti-Ku. This article describes a patient with an antibody diFrom the Blood Bank. Los Angeles County-University
of Southern California Medical Center, Los Angeles,
CA 90033.
rected at a high frequency antigen,
anti-Yt", and demonstrates that the
lack of reactivity of a patient's serum
against DTT-modified RBCs can aid
in the identification of certain antibodies directed at high frequency antigens.
Case Report
The patient was a 23-year-old
woman in her third pregnancy (blood
group A, Rlr). Her first pregnancy resulted in a spontaneous abortion; her
second pregnancy was uncomplicated.
An antepartum antibody screen at 37
weeks' gestation revealed 2+ agglutination in the indirect antiglobulin
test. The initial antibody identification panel revealed 1 + to 2 + agglutination in the indirect antiglobulin
test of all unmodified RBCs tested.
DTT-modified RBCs were not agglutinated by the patient's serum in any
phase of testing. Because the patient's serum failed to react with DTTmodified RBCs, the possibility was
Serologic tests were performed using standard procedures. 3 All reagents were obtained from commercial
sources. The 200 mM DTT was prepared by dissolving 1 g desiccated
DTTa in 32 mL of 0.01 M phosphatebuffered saline, pH 8.0, as described
by Branch and colleagues,1 or in 32
mL normal saline. DTT-treated RBCs
were prepared as follows: 1 volume of
washed, packed RBCs was mixed with
4 volumes of 200 mM DTT solution
and incubated at 37° C for 30 minutes. The RBCs were then washed four
times with isotonic saline and resuspended to 3% to 5% in isotonic saline
for testing.1
The 200 mM DTT reagent can be
stored frozen in aliquots at - 20° C for
as long as one year. The reagent's activity can be assessed by testing Kpbpositive and Kell-positive RBCs with
anti-Kpb, and anti-K after DTT treatment. Failure of the DTT-modified
RBCs to react with these antisera indicates that the DTT reagent is functioning.
LABORATORY MEDICINE • VOL. 15, NO. 9, SEPTEMBER 1984 6 0 7
Results
The patient's serum reacted from 1 +
to 2 + in the indirect antiglobulin test
when tested against unmodified RBCs
including Js(b-), Kp(b-), and Ko.
The serum failed to react with eight
examples of unmodified Yt(a - ) RBCs
and with unmodified autologous RBCs.
All DTT-modified RBCs failed to react
with the patient's serum. The titer of
the antibody against Yta RBCs was
64. The patient was Yt(a-).
The patient's newborn had an uneventful neonatal course. Tests on the
cord blood showed a negative direct
antiglobulin test and a negative xylene eluate. Because Yta is weakly expressed on newborn RBCs, the Yta
typing of the newborn could not be
determined with certainty.4 The father's RBCs were not available for Yt°
typing. The patient's anti-Yt a was
presumably induced by pregnancy
since she had never been transfused.
Discussion
DTT-modified RBCs can be used
when attempting to identify antibod-*
ies directed at high frequency antigens. If a patient's serum contains an
antibody that appears to react with a
high frequency antigen, the failure of
that antibody to react with DTT-modified RBCs suggests that the antibody
might be anti-Yt", anti-Kpb, anti-Js b ,
or anti-Ku.
If a mixture of antibodies is suspected, an entire set of DTT-modified
RBCs in an antibody identification
panel could be employed. These RBCs
could assist in identifying antibodies
potentially masked by anti-Yta, antiKpb, anti-Js\ and anti-Ku.
The Table lists red cell antigens that
have been tested and appear to be unaffected by treatment with 200 mM
DTT. In addition to Yta and Kell blood
group antigens, other antigens that
have disulfide bonds might be affected, but published data are not yet
available. Those who use DTT-modified RBCs to tentatively identify blood
group antibodies must confirm the
antibody specificity by employing
RBCs that lack the antigen to which
the suspected antibody is directed. This
may require referring the sample to
a reference laboratory for confirmation of the antibody specificity.
In summary, DTT-modified RBCs
can be routinely used when attempting to identify certain antibodies directed at high frequency antigens.
Antigens Unaffected by
200 mM DTT Treatment*
A, A1, B, H
U
Le", Le"
Fy", Fyb
D, C, E, c, e, Cw
JK", JKb
M, N, S, s, U
P1,T|«
Lu«, Lub
Ml', Vw, Pr, Xg", By», JP, DP, Ch", Vel
'From Branch and colleagues1
Since DTT-modified RBCs are easy to
prepare, this test can be performed in
any blood bank laboratory.
References
1. Branch DR, Muensch HA, Sy Siok Hian AL, et
al: Disulfide bonds are requirement for Kell and
Cartwright (Yta) blood group integrity. Br J Haematol 1983;54:573-578.
2. Cleland WW: Dithiothreitol, a new protective reagent for SH groups. Biochemistry 1964;3:480482.
3. Widman F (ed): Technical Manual, ed 8. Washington, DC, American Association of Blood Banks,
1981.
4. Mollison PL: Blood Transfusion in Clinical Medicine, ed 7. Oxford, Blackwell Scientific Publications, 1983, p 204.
Supplier
a. Sigma Chemical Co, St. Louis, MO 63103.
Expansion of RARE Exchange Program
The Rare Antigen/Antibody Resource Exchange (RARE) is a service offered by the AABB
Committee on Reference Laboratories. Created in 1983 as a service to AABB-certified immunohematology reference laboratories, this exchange program has now been expanded to include
all blood bank laboratories.
RARE provides a central file of rare and unusual blood specimens, fluids, and other reagents
available in sufficient quantity to be offered for trade. (Samples in the RARE inventory are not
for sale.) Member laboratories receive a quarterly list of samples in the RARE trading inventory.
The program includes a mechanism for trading materials for samples listed in the inventory. In
most cases, a laboratory trades 2 to 5 mL of a sample in its inventory for 2 to 5 mL of sample
in another laboratory's inventory. A laboratory need not have a large inventory of samples to
participate.
Membership applications can be obtained by contacting the AABB National Office or the RARE
Coordinator. The membership application will request the type/specificity and available quantity
of samples available for trading, along with a nominal annual membership fee ($15 for AABB
institutional members and $20 for non-institutional members). The sample information submitted
for possible addition to the RARE trading inventory must be deemed rare or unusual to be
accepted. Should a sample be considered unsuitable, the institution's membership in RARE will
be continued, but the trading privileges will not be activated until a suitable sample can be added
to the RARE trading inventory.
RARE offers an excellent means to build an inventory of valuable blood specimens and share
samples with other laboratories. Applications and further information can be obtained by writing
the AABB National Office or W. Michael Tregellas, AABB RARE Coordinator, c/o Blood Systems
Central Laboratory, 6220 E Oak, Scottsdale, AZ 85257.
6 0 8 LABORATORY MEDICINE • VOL. 15, NO. 9, SEPTEMBER 1984