Exeley Inc.

Halter Hipsky, Christine

da Costa, Daiane Cobianchi

Omoto, Ricardo

Zanette, Angela

Castilho, Lilian

Reid, Marion E.

Prevalence of RHD*DOL and RHCE*ce(818T)&nbsp; in two populations

Jackson, Pamela

Occurrence of antibodies to low-incidence antigens among a cohort of multiply transfused patients with sickle cell disease

Haywood, Jennifer R.

Grandstaff Moulds, Marilyn K.

Bryant, Barbara J.

Determination of optimal method for antibody identification in a reference laboratory

Mallory, Delores

In Memoriam: Marie Cutbush Crookston 1920&ndash;2009

From DNA to blood groups

ERRATUM: Vol. 24, No. 1, 2008; page 16

Johnson, Susan T.

I M M U N O H E M A T O L O G Y, VO L U M E 2 3 , N U M B E R 4 , 2 0 0 7 161
Clinical Case Presentation
Two units of leukocyte-reduced red blood cells
(LRBCs) were ordered for a 58-year-old Caucasian
woman whose hemoglobin was falling. She had under-
gone a cholecystectomy 2 weeks earlier. Her laboratory
test results are noted inTable 1.
Immunohematologic Evaluation and Results
Results of initial pretransfusion testing showed her
RBCs to be group O, D+. An antibody detection test
performed by gel technology was positive (2+) with
both reagent screening RBCs. An antibody identification
panel was also tested by gel and similar positive
reactivity (2+) was seen with all panel RBCs. The
hospital technologist performed a DAT by the gel test.
This was positive (4+) with anti-IgG while the saline
control was negative. The cards available at the hospital
did not include polyspecific antihuman globulin (AHG)
or anti-C3. At this point, the hospital technologist
performing the testing was convinced that this patient
had a warm autoantibody and sent a sample to the
Immunohematology Reference Laboratory (IRL) for
further evaluation to determine whether the patient had
any underlying alloantibodies. The clinician was
informed of the serologic results and that there would
be a delay in obtaining blood because the patient’s
sample was being sent out.
Panel positive reactions with equal reactivity are
most likely the result of an autoantibody or an alloanti-
body to a high-prevalence antigen present on all reagent
RBCs tested. An autologous control will help differen-
tiate an autoantibody from an alloantibody. If the
autologous control is negative,one is most likely dealing
with an antibody to a high-prevalence antigen, but if
positive with equal or stronger reactivity (2+ to 4+), an
autoantibody is most likely. Alternatively or in addition
to the autologous control,a DAT may be performed. This
may be the method chosen in laboratories routinely
using gel or solid phase for antibody detection and
identification. It is important to remember, however,
that the autologous control and DAT are different tests.
An autologous control includes patient serum whereas
a DAT only tests patient RBCs. In most cases, if the DAT
is positive, the autologous control will also be positive.
If the patient’s RBCs are sensitized with IgG or C3 in
vivo,the autologous control will be positive because this
occurred before incubation. However, there are
situations when only the autologous control will be
positive because the antibody reactivity is method-
dependent. The IgG coating of patient RBCs in this case
suggests a warm autoantibody.
Underlying alloantibodies are reported in 12 to 40
percent of patients with warm autoantibodies.1 Review
of the patient’s clinical history showed she had received
a transfusion of two units of RBCs 20 years earlier, and
she delivered three children.
The results obtained in the IRL using test tube
methods are inTable 2.
According to this IRL’s policy, an enzyme-treated
(ficin) antibody identification panel is first tested when
a warm autoantibody is suspected. Patient RBCs are
treated with ZZAP (ficin and DTT) before performing
Warm autoantibody or drug-
dependent antibody?That is the
question!
S.T. JOHNSON
E D U C A T I O N A L F O R U M
Table 1. Laboratory test results
Result Day 1 Day 2
Hb (g/dL) 7 5.1
Bilirubin, total (mg/dL) 1.2 4.3
LDH (IU/L) 847 2305
Reticulocyte count 6% NA
NA = not available
162 I M M U N O H E M A T O L O G Y, VO L U M E 2 3 , N U M B E R 4 , 2 0 0 7
S.T. JOHNSON
an autologous adsorption. Alternatively, if allogeneic
adsorptions are performed, these reagent RBCs are
pretreated with ficin. Initial screening of a ficin-treated
panel ensures that the autoantibody is directed against
an antigen present on ficin-treated RBCs,and, therefore,
adsorptions should be effective. The ficin panel was 3+
with all RBCs, and the autologous control was 4+
reactive. Enhanced reactivity is consistent with a warm
autoantibody.
Results of the test tube DATs performed in the IRL
are inTable 3. It is important to include a control to rule
out spontaneous agglutination when positive reactivity
is seen with all reagents.
The strongly positive DAT with IgG and comple-
ment (C3b,C3d) is most likely caused by a warm
autoantibody, especially in light of the serum results.
Approximately 50 percent of positive DATs in patients
with warm autoimmune hemolytic anemia (WAIHA)
show both IgG and complement coating the RBCs.1
In an effort to ensure that blood was available as
quickly as possible for this patient, the IRL technologist
started autologous adsorptions because the patient had
not been recently transfused. One milliliter of RBCs was
saved for the eluate. While the patient’s serum was
incubating at 37°C with the first set of the patient’s
ZZAP-treated RBCs, a rapid acid eluate (ELU-KIT II,
ImmucorGamma,Inc.,Norcross,GA) was performed. To
the technologist’s surprise, the eluate was negative!
The IRL technologist performing the work
immediately called the referring institution to notify
them of the results of the eluate. A negative eluate is
highly suggestive of a drug-dependent antibody. Drug-
dependent antibodies will not react,even if eluted from
patient RBCs,because the putative drug must be present
when testing the eluate with reagent RBCs. Were the
DAT positive because of a warm autoantibody, strongly
positive reactions (2+ to 4+) would be obtained when
testing the eluate.
In a recent report summarizing the experience of
this author’s laboratory, more than half of the cases of
drug-induced immune hemolytic anemia (DIIHA)
investigated demonstrated reactivity in initial antibody
detection tests.2 Serologic results in this case could
easily be misconstrued as a warm autoantibody. Positive
reactivity without adding drug to the test may have two
explanations. If the patient is on the drug at the time of
testing, the drug is likely circulating in the patient’s
plasma. Alternatively, a drug-independent warm auto-
antibody may be present. It is possible that drug-
independent autoantibody reactivity may resemble
reactivity seen when a patient experiences a delayed
transfusion reaction and is producing not only
Table 2. Antibody identification panel*: results of testing serum from the patient
Rh MNS Lu P Lewis Kell Duffy Kidd Saline
Cell D C E c e f M N S s Lua Lub P1 Lea Leb K k Fya Fyb Jka Jkb IS 37°C IAT†
1 + + 0 0 + 0 + + + + 0 + 0 + 0 0 + 0 + 0 + 0 0 2+
2 0 0 0 + + + + 0 + 0 0 + + 0 + + 0 + + + 0 0 0 1+
3 0 0 + + 0 0 0 + 0 + 0 + + 0 + + + + 0 0 + 0 0 1+
4 + + 0 0 + 0 + 0 + + 0 + 0 0 0 0 + + + + + 0 0 2+
5 0 0 + + + + + + 0 + 0 + 0 0 + 0 + 0 + 0 + 0 0 1+
6 0 + 0 0 + 0 + + 0 + 0 + + 0 + + + + + + + 0 0 2+
7 + 0 + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + + 0 0 0 1+
8 0 0 0 + + + + 0 + 0 0 + + 0 + 0 + + 0 0 + 0 0 1+
9 + + 0 + + + + + 0 + 0 + + + 0 0 + 0 + + 0 0 0 1+
10 + 0 + + 0 0 0 + + + 0 + + + 0 0 + + 0 + 0 0 0 1+
11 + 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 0 0 + + 0 0 1+
AC 0 0 4+
AC = autologous control
* In-house prepared RBC panel
†Anti-IgG, ImmucorGamma, Inc.,Norcross,GA
Table 3. DAT results
IS 10 min. RT incubation
Polyspecific AHG* 4+ NT
Anti-IgG† 4+ NT
Anti-C3b,C3d‡ 1+ 2+
Saline control 0 0
IS = initial spin,RT = room temperature,NT = not tested
*Polyspecific AHG,Ortho Clinical-Diagnostics, Inc., Raritan,NJ
†Anti-IgG, ImmucorGamma, Inc.
‡Anti-C3b,C3d, ImmucorGamma, Inc.
I M M U N O H E M A T O L O G Y, VO L U M E 2 3 , N U M B E R 4 , 2 0 0 7 163
Warm autoantibody or drug-dependent antibody?
alloantibody but also autoantibody. A careful drug
history is imperative when the eluate is negative.
This patient was receiving Zosyn (Wyeth
Pharmaceuticals, Philadelphia, PA) 2 g intravenously
every 6 hours. Zosyn is a broad-spectrum antibiotic
consisting of piperacillin sodium in combination with
the β-lactamase inhibitor tazobactam sodium. Arndt et
al.3 and Johnson et al.4 reported patients with DIIHA
caused primarily by piperacillin antibodies. Johnson et
al.5 later reported four cases associated with Zosyn.
Drug-dependent antibody was detected in the presence
of both Zosyn and piperacillin; however, reactivity was
greater with Zosyn.
Drug studies were performed testing the patient’s
serum in the presence of the drugs Zosyn, piperacillin,
and tazobactam because recent reports have shown that
Zosyn- and piperacillin-dependent antibodies react best
in this method.3–6 The results are inTable 4.
Although there are weakly positive reactions with
the patient’s serum and PBS, reactivity is significantly
increased in the presence of both Zosyn and pipera-
cillin, consistent with drug-dependent antibodies. In
addition,the eluate was positive in the presence of both
Zosyn and piperacillin.
Finally, autologous adsorptions removed the
antibody reactivity and no underlying alloantibodies
were detected in the adsorbed serum.
Conclusions
Zosyn- and piperacillin-dependent antibodies were
detected in the patient’s serum. Had all testing for drug-
dependent antibodies been negative,repeating the tests
using enzyme- (ficin or papain) treated reagent RBCs
may have enhanced the reaction with the drug-
dependent antibody. Had testing continued to yield
negative results, it may have been beneficial to treat
RBCs with each drug and then test the drug-coated
RBCs. If there is clear evidence of hemolysis as in this
case, testing by several methods may be required to
detect a drug-dependent antibody. As with common
RBC alloantibodies, drug-dependent antibodies do not
“read the book.” One can never be certain when the
next type of drug-dependent antibody may be identified!
The use of Zosyn was discontinued,and the patient’s
hemoglobin stabilized after two units of LRBCs were
administered. The patient was informed that it is impor-
tant to avoid Zosyn and piperacillin in the future to
prevent a repeated hemolytic event perhaps worse than
this episode.
In summary,this is a case of DIIHA caused by Zosyn-
and piperacillin-dependent antibodies. Initial serologic
results were identical to those seen in cases ofWAIHA.
The negative eluate was critical in differentiating this
case from WAIHA, emphasizing the importance of
performing an eluate on initial workup of apparent
cases ofWAIHA. This also demonstrates the importance
of a careful drug history in the face of significant RBC
hemolysis and serologic evidence ofWAIHA.
References
1. Petz LD,Garratty G.Immune hemolytic anemia.2nd
ed. Philadelphia:Churchill Livingstone, 2004.
2. Johnson ST, Fueger JT, Gottschall JL. One center’s
experience:the serology and drugs associated with
drug-induced immune hemolytic anemia—a new
paradigm.Transfusion 2007;47:697–702.
3. Arndt PA, Garratty G, Hill J, Kasper M,
Chandrasekaran V.Two cases of immune haemo-
lytic anaemia, associated with anti-piperacillin,
detected by the “immune complex” mechanism.
Vox Sang 2002;83:273–8.
4. Johnson ST,Weitekamp LA, Sauer DE, Fueger JT,
Aster RH. Piperacillin-dependent antibody with
relative e specificity reacting with drug treated red
cells and untreated red cells in the presence of
drug.Transfusion 1994;34(Suppl):S290.
5. Johnson ST, Fueger JT, Sauer DE, Bell JA. Drug-
induced hemolytic anemia associated with Zosyn,
Table 4. Results of testing patient’s serum with and without the presence of drugs
Serum PBS Zosyn Piperacillin Tazobactam
RT 37°C IAT RT 37°C IAT RT 37°C IAT RT 37°C IAT
Patient 0* 0 2/11† 8/24 8/22 8/17 4/18 4/18 4/9 0 0 1/8
Normal 0 0 0 0 0 0 0 0 0 0 0 0
Positive control NT NT NT 3+ 3+ 4+ 3+ 3+ 3+ 0 0 0
Eluate NT NT NT NT NT 3+ NT NT 2+ NT NT NT
RT = room temperature,NT = not tested
* agglutination strength (0 – 4+)
† titer/score
164 I M M U N O H E M A T O L O G Y, VO L U M E 2 3 , N U M B E R 4 , 2 0 0 7
S.T. JOHNSON
serologically presenting as warm autoimmune
hemolytic anemia and detected best in the
presence of drug. (abstract) Transfusion 2006;
46(Suppl):127A.
6. Leger RM,Arndt PA, Garratty G. Unlike penicillin,
the immune complex “method is the preferable
method for detecting piperacillin antibodies.”
(abstract)Transfusion 2006;46(Suppl):126A.
Susan T. Johnson, MSTM, MT(ASCP)SBB, Immuno-
hematology Services, BloodCenter of Wisconsin, P.O.
Box 2178, Milwaukee,WI 53201-2178.
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Prevalence of RHDDOL and RHCEce(818T)  in two populations

Occurrence of antibodies to low-incidence antigens among a cohort of multiply transfused patients with sickle cell disease

Determination of optimal method for antibody identification in a reference laboratory

In Memoriam: Marie Cutbush Crookston 1920–2009

From DNA to blood groups

ERRATUM: Vol. 24, No. 1, 2008; page 16

Warm autoantibody or drugdependent antibody? That is the question!

ERRATA: Vol.21,No.4,2005,p.146 Vol.21,No.4,2005,p.171

With many thanks Dedication to Marilyn K.Moulds

Special Notes When Using Anti-D Monoclonal Reagents

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