680 Am J Clin Pathol 2004;122:680-685

680 DOI: 10.1309/CJAW6N8J6H0HR2WM

American Society for Clinical Pathology
Coagulation and Transfusion Medicine / CHARACTERISTICS OF WARM AUTOANTIBODIES
Warm Reactive Autoantibodies
Clinical and Serologic Correlations
Christine A. Wheeler, MD,
*
Loni Calhoun, MT(ASCP)SBB, and Douglas P. Blackall, MD
Key Words: Autoantibodies; Hemolysis; RBCs
DOI: 10.1309/CJAW6N8J6H0HR2WM
A b s t r a c t
Warm reactive autoantibodies are encountered
relatively frequently in tertiary care hospitals. We
studied 100 consecutive patients with warm
autoantibodies to correlate their clinical and serologic
features. Study patients (56 male, 44 female) had
various diagnoses and a mean age of 53.5 years (range,
3-90 years). Autoimmune hemolysis was documented in
29 patients; 20 patients (69%) in this subset had
diseases classically associated with warm autoimmune
hemolytic anemia (hematologic and autoimmune
disorders). All study patients demonstrated IgG on their
RBCs (direct antiglobulin test [DAT] reactivity range,
microscopic to 4+); 49 also demonstrated C3
(reactivity range, microscopic to 3+). The DAT for IgG
was 2+ or more in 25 (86%) of 29 patients with
hemolysis; the DAT for IgG was 1+ or less in 45 (63%)
of 71 patients without hemolysis. In patients with
hemolysis, 21 (72%) of 29 had a DAT reactive for C3.
These findings may be useful in determining the clinical
significance of warm autoantibodies and the extent to
which patients should be followed up for hemolysis.
Warm reactive autoantibodies are RBC-directed immune
responses that are maximally reactive at 37C. These antibod-
ies react with the patients own RBCs and typically bind to all
reagent or donor RBCs against which they are tested.
However, they sometimes have specificity (absolute or rela-
tive) for a blood group antigen, most commonly a member of
the Rh blood group system. Warm reactive autoantibodies are
usually polyclonal IgG antibodies, although IgA or monomer-
ic IgM antibodies are seen occasionally. Complement (C3)
also may accompany these antibodies, as detected by the
direct antiglobulin test (DAT). The clinical significance of
warm reactive autoantibodies relates to their hemolytic poten-
tial and the interference that they can cause with routine pre-
transfusion compatibility testing (eg, the detection of blood
group antibodies and the provision of crossmatch-compatible
units of blood).
1
Warm reactive autoantibodies are encountered relatively
frequently at our institution. During the years 2001 and 2002,
warm autoantibodies constituted approximately 7% of all anti-
body identification evaluations. However, it was our impres-
sion that hemolytic anemia occurred in only a minority of
patients with warm autoantibodies. This study was undertak-
en to correlate the clinical and serologic features of patients
with warm autoantibodies and to determine whether clinical
assessment practices could or should be improved.
Materials and Methods
The study patients were selected from a daily log of all
patients who had antibody (alloantibody, autoantibody, or
both) detected in their serum or plasma or on their RBCs. In
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Coagulation and Transfusion Medicine / ORIGINAL ARTICLE
this retrospective study, 100 consecutive patients with warm
autoantibodies were evaluated. Patients with insufficient clin-
ical or laboratory data to evaluate for the presence or absence
of hemolysis due to warm autoantibodies were excluded.
Age, sex, diagnosis, and serologic reactivity were record-
ed. EDTA-anticoagulated blood samples were used for indi-
rect antiglobulin testing and the DAT. Serologic testing con-
formed with methods provided in the Technical Manual of the
American Association of Blood Banks.
1
Antibody detection
and identification were performed by gel testing according to
the manufacturers specifications (Ortho-Clinical Diagnostics,
Raritan, NJ). Autologous or allogeneic adsorption was per-
formed, as needed, to detect underlying alloantibody and to
confirm autoantibody reactivity.
DATs were performed in the following situations: positive
autocontrol in antibody identification studies, suspicion of
hemolysis, or request by a clinician. DATs were performed by
test tube methods using a licensed, commercial, polyspecific
antiglobulin reagent (Ortho-Clinical Diagnostics). Monospecific
antiglobulin reagents for IgG and C3 (Ortho-Clinical
Diagnostics) were used to test samples with positive DAT
results. In some cases, antibody was eluted to help establish
the presence of a warm autoantibody or to evaluate for the
presence of an alloantibody on RBCs. Elutions were per-
formed using a commercially available acid elution kit (Elu-
Kit II, Gamma Biologicals, Houston, TX). The eluates were
tested using the gel-IgG method with a last wash saline con-
trol. The presence of a warm autoantibody was established by
pan-reactivity of an acid eluate prepared from patient RBCs or
by pan-reactivity of patient plasma or serum tested against a
panel of reagent RBCs.
Clinical and laboratory data were reviewed for all patients
using a computer database. The patients were placed into 1 of
8 disease categories based on their predominant diagnosis:
autoimmune disorder, hematologic malignancy, other hemato-
logic disorder, nonhematologic malignancy, end-stage renal or
liver disease, idiopathic autoimmune hemolytic anemia, car-
diac disease, or other disease. Clinical records, transfusion
requirements, and the following laboratory parameters were
used as a basis for determining whether an individual patient
was likely to be experiencing hemolysis: hematocrit, less than
37.4% (<0.37); hemoglobin, less than 12.3 g/dL (<123 g/L);
reticulocyte count, more than 2.06% (>0.021) or an absolute
count of more than 107.2 10
3
/L (>107.2 10
9
/L); total
bilirubin concentration, more than 1.5 mg/dL (>26 mol/L;
majority unconjugated); lactate dehydrogenase level, more
than 220 U/L (>220 U/L); and haptoglobin, less than 6 mg/dL
(<0.06 g/L).
Responses to transfusion also were reviewed for patients
who experienced hemolysis due to warm autoantibodies. A
full response to transfusion of 1 U of RBCs was considered to
be a hematocrit increase of 2% to 3% (0.02-0.03) or an
increase of 1 g/dL (10 g/L) in the hemoglobin concentration.
Other therapies (eg, steroids, intravenous immune globulin,
and splenectomy) also were reviewed.
Results
There were 56 male and 44 female study patients. Their
ages ranged from 3 to 90 years (mean age, 53.5 years). Table
1 shows the disease categories of all patients with warm
autoantibodies, the number of patients with warm autoanti-
bodies who experienced hemolysis due to these antibodies,
and the number of patients with warm autoantibodies who did
not experience hemolysis.
Autoimmune disorders included systemic lupus erythe-
matosus (6), rheumatoid arthritis (1), polyglandular autoim-
mune disorder (1), Graves disease (1), ankylosing spondyli-
tis (1), ulcerative colitis (4), autoimmune liver disease (2),
antiphospholipid syndrome (2), and an unspecified catego-
ry (3). Hematologic malignant neoplasms included lym-
phoma (7), mycosis fungoides (1), leukemia (6), and
Castleman disease (1). Other hematologic disorders includ-
ed idiopathic thrombocytopenic purpura (3), congenital
dyserythropoietic anemia (1), myelodysplastic syndrome
(4), lymphadenopathy (1), and sickle cell disease (2).
Nonhematologic malignant neoplasms (1 or 2 cases of each
entity) included malignant mesothelioma, adenocarcinoma,
nonsmall cell carcinoma (lung), transitional cell carcino-
ma, hepatocellular carcinoma, and malignant melanoma.
Other diseases (1 or 2 cases of each entity) were subarach-
noid hemorrhage with cerebral aneurysm, moyamoya dis-
ease, cerebrovascular accident, chronic lung disease,
abdominal aortic aneurysm, gastrointestinal bleeding, hepa-
titis or liver disease, and interstitial cystitis. One patient
who was given methyldopa and then developed warm
autoantibodies, without hemolysis, is included in the other
diseases category.
Table 1
Disease Categories in Patients With and Without Autoimmune
Hemolysis
*
Disease Category Hemolysis No Hemolysis
Autoimmune disorder (n = 21) 9

(43) 12 (57)
Cardiac disease (n = 19) 1 (5) 18 (95)
Hematologic malignancy (n = 15) 8 (53) 7 (47)
End-stage renal or liver disease (n = 12) 3 (25) 9 (75)
Other diseases (n = 12) 3 (25) 9 (75)
Other hematologic disorders (n = 11) 3 (27) 8 (73)
Nonhematologic malignancy (n = 8) 0 (0) 8 (100)
Idiopathic hemolysis (n = 2) 2 (100) 0 (0)
Total (n = 100) 29 (29) 71 (71)
*
Data are given as number (percentage).

Includes 1 patient who also experienced a delayed hemolytic transfusion reaction.

682 Am J Clin Pathol 2004;122:680-685
682 DOI: 10.1309/CJAW6N8J6H0HR2WM
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Wheeler et al / CHARACTERISTICS OF WARM AUTOANTIBODIES
Autoimmune hemolysis occurred in 29 patients. Three of
these patients experienced hemolysis at an outside institution
but no longer had evidence of hemolysis when evaluated at
our institution. One patient died as a direct result of autoim-
mune hemolysis, which was intravascular. Three patients
experienced hemolysis not thought to be attributable to their
warm reactive autoantibodies: 1 had passenger lymphocyte
syndrome after allogeneic stem cell transplantation, 1 had
hemolysis secondary to aortic valve stenosis, and 1 had sys-
temic lupus erythematosus and Kasabach-Merritt syndrome
with chronic, low-level disseminated intravascular coagula-
tion. One patient with systemic lupus erythematosus had a
warm reactive autoantibody and evidence of a delayed
hemolytic transfusion reaction due to anti-Jk
a
(as evidenced
by rapid emergence of the alloantibody shortly after transfu-
sion). Based on persistent laboratory evidence of hemolysis
and continued transfusion requirements, it seemed that
autoimmune hemolysis was also present. Of note, 20 (69%) of
29 patients with warm autoantibodies who had autoimmune
hemolysis had diseases classically associated with warm
autoimmune hemolytic anemia: autoimmune disorders
(including idiopathic thrombocytopenic purpura) and hemato-
logic malignant neoplasms.
The DAT was reactive for IgG in all patients with warm
autoantibodies and ranged in strength from microscopically
reactive to 4+. In Table 2, DAT results (for IgG) are correlat-
ed with the presence or absence of autoimmune hemolysis,
and the predictive value of individual DAT reaction strengths
is shown. The DAT was 2+ or greater in strength in 25 (86%)
of the 29 patients with warm autoantibodies who experienced
autoimmune hemolysis, and the predictive value of a strongly
reactive DAT (2+) was 49%. The DAT was 1+ or weaker in
strength in 45 (63%) of 71 patients who did not experience
hemolysis. The relationship between the presence or absence
of hemolysis and the DAT strength was highly statistically sig-
nificant by
2
analysis (P < .005).
The DAT for C3 was reactive in 49 patients with warm
autoantibodies and ranged in strength from microscopically
reactive to 3+. Of these patients, 21 (43%) experienced hemol-
ysis, and 28 (57%) were unaffected. However, closer evalua-
tion of the 29 patients who experienced hemolysis revealed
that 21 (72%) had a positive DAT for C3. In Table 3, DAT
results (for C3) are correlated with the presence or absence of
autoimmune hemolysis, and the predictive value of individual
DAT reaction strengths is shown. The relationship between
hemolysis and the presence of complement was highly statis-
tically significant (P < .005).
Autoantibody only was detected on the RBCs of 30
patients but also was detected in the serum or plasma of the
other 70 patients. In 12 patients, the autoantibody mimicked
the specificity of an alloantibody as follows: anti-e, 6; anti-E,
2; anti-C and anti-e, 1; anti-D, 1; anti-f, 1; and anti-Jk
b
, 1. In
34 patients, autoadsorptions were performed; in 30 patients,
alloadsorptions were performed. Alloantibodies were detected
in 53 patients. Of these, 24 had single specificities, and 29 had
multiple antibodies (range, 2-7). The specificity of the alloan-
tibodies detected by blood group system is shown in Table 4.
Cold agglutinins (all reactive at room temperature or lower)
also were present in 8 patients.
Of the 29 patients who had autoimmune hemolytic ane-
mia, 23 received transfusions of RBCs. Of these, 16 (70%)
had at least a partial response to transfusion. In 7 patients, the
responses to some or all of the transfusions could not be deter-
mined because hemoglobin and hematocrit values were not
Table 4
Patients With Alloantibodies by Blood Group
Blood Group No. of Patients
*
Rh 40
Kell 21
Kidd 5
Duffy 6
MNS 7
Lutheran 2
High-titer, low avidity 6
*
A total of 53 patients had alloantibodies, some multiple.
Table 3
DAT Results (for C3) Correlated With Hemolysis
DAT No. of Patients No. of Patients Predictive Value
*
Strength With Hemolysis Without Hemolysis (%)
0 8 43 16
m+ 6 15 29
1+ 8 12 40
2+ 6 1 86
3+ 1 0 100
4+ 0 0 0
Total 29 71
DAT, direct antiglobulin test; m+, microscopically reactive.
*
Likelihood that a patient with a given DAT result will experience hemolysis,
calculated as follows: predictive value = TP/(TP + FP), where TP indicates patients
with a positive DAT result who experience hemolysis and FP patients with the same
positive DAT result who do not experience hemolysis.
Table 2
DAT Results (for IgG) Correlated With Hemolysis
DAT No. of Patients No. of Patients Predictive Value
*
Strength With Hemolysis Without Hemolysis (%)
m+ 2 14 13
1+ 2 31 6
2+ 13 16 45
3+ 7 9 44
4+ 5 1 83
Total 29 71
DAT, direct antiglobulin test; m+, microscopically reactive.
*
Likelihood that a patient with a given DAT result will experience hemolysis,
calculated as follows: predictive value = TP/(TP + FP), where TP indicates patients
with a positive DAT result who experience hemolysis and FP patients with the same
positive DAT result who do not experience hemolysis.
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Coagulation and Transfusion Medicine / ORIGINAL ARTICLE
available or transfusions were given elsewhere. Twenty-five
patients received other therapy for their hemolytic anemia,
including steroids, intravenous immune globulin, rituximab
(anti-CD20 monoclonal antibody), 6-mercaptopurine, chlor-
ambucil, cyclosporine, vincristine, methotrexate, mycopheno-
late, pentostatin, thalidomide, and splenectomy.
Discussion
During the last 15 years, the medical literature has con-
tained little new information focused on correlating the clini-
cal significance of warm reactive autoantibodies with associ-
ated serologic findings. This is an important concern because
this time frame has borne witness to a variety of novel
immune modulating agents and therapies, a consequence of
which might be autoantibody development. When RBC
autoantibodies are detected, it would be useful to be able to
predict the biologic behavior of these antibodies to plan ther-
apy and provide appropriate patient follow-up. A better under-
standing of the serologic features of warm reactive autoanti-
bodies might prove helpful in this regard.
In the present study, only 29% of the patients with warm
autoantibodies actually experienced a hemolytic anemia.
However, 20 (69%) of 29 patients with hemolysis had diseases
classically associated with warm autoimmune hemolytic ane-
mia: autoimmune disorders, including idiopathic thrombocy-
topenic purpura, and hematologic malignant neoplasms. A few
studies of patients with a specific disease entity who also had
warm autoantibodies address the issue of the proportion of
patients who experienced hemolysis. In one study of patients
with carcinoma and warm reactive autoantibodies (not associ-
ated with cold agglutinins), 49 of 99 patients experienced
autoimmune hemolytic anemia.
2
In a second study, 46 patients
with myelodysplastic syndrome had erythrocyte autoantibod-
ies (warm, mixed warm and cold, or cold); 15 of these patients
had autoimmune hemolysis, with 7 patients experiencing
hemolysis secondary to warm autoantibodies alone.
3
In anoth-
er study, 14 pediatric patients with sickle cell disease devel-
oped warm autoantibodies after receiving multiple erythrocyte
transfusions; 4 of these patients experienced autoimmune
hemolysis.
4
Issitt et al
5
describe 3 groups of a total of 150 sub-
jects with warm autoantibodies: 87 had autoimmune hemolyt-
ic anemia, 33 had taken -methyldopa but most did not expe-
rience hemolysis, and 30 were hematologically healthy with-
out evidence of hemolysis (25 healthy blood donors and 5
additional patients without autoimmune disorders).
All patients in our study had warm reactive autoantibod-
ies and a DAT reactive for IgG. Of these patients, 49% also
had a DAT reactive for complement. Several research reports
in the medical literature describe the nature of DAT results in
patients with warm autoimmune hemolytic anemia.
5-11
These
studies reported the DAT to be reactive for IgG alone in 21%
to 56% of patients. The DAT was reactive for both IgG and C3
in 31% to 64% of patients, and the DAT was reactive for C3
alone in 0% to 45% of patients. Some of these studies also
demonstrated the DAT to be reactive for IgA, without IgG or
C3. In the study by Issitt et al,
5
all 150 subjects had warm
autoantibodies and a DAT reactive for IgG; 91 had a DAT
reactive for IgG alone; 49 for IgG and complement; 5 for IgG,
IgM, and complement; 2 for IgG, IgA, IgM, and complement;
2 for IgG and IgA; and 1 for IgG, IgA, and complement.
In the present study, 25 (86%) of the 29 patients with
warm autoantibodies who experienced hemolysis had a DAT
for IgG that was 2+ or greater in reaction strength. Of 71
patients with warm autoantibodies who did not experience
hemolysis, 45 (63%) had a DAT for IgG that was 1+ or weak-
er. The medical literature addresses the strength of reactivity
of the DAT in patients with warm autoimmune hemolytic ane-
mia but does not focus on individuals with warm autoantibod-
ies without hemolysis. Issitt and Anstee
11
commented that in
the majority of cases of warm autoimmune hemolytic anemia,
the DAT usually is strongly reactive. In one study of 23
patients with warm autoimmune hemolytic anemia due to IgG
autoantibodies, 21 patients had a DAT that was 4+ and 2
patients had a DAT that was 3+.
12
In another study, 19 (61%)
of 31 patients with warm autoimmune hemolytic anemia had
a DAT for IgG of 2+ or greater.
13
A study evaluating erythro-
cyte autoantibody behavior in the monocyte monolayer assay
revealed that the level of RBC sensitization with IgG was an
important positive predictor of in vivo hemolysis.
14
Furthermore, in patients with warm reactive autoantibodies
and positive monocyte monolayer assay results, testing indi-
cated that this type of autoantibody was more likely to be
associated with phagocytosis than simple RBC adherence
(identified more commonly in patients with cold and mixed-
type RBC autoantibodies). Thus, it seems that in patients with
warm reactive autoantibodies, there is an important correla-
tion between DAT reaction strength and the probability of
hemolysis. However, when heterogeneous groups of individu-
als with positive DAT results have been evaluated for hemol-
ysis, there is not a good correlation between DAT reaction
strength and the likelihood of hemolysis.
15
In our study, there also was a correlation between autoim-
mune hemolysis and the presence of complement on RBCs. In
addition, the strength of reactivity of the DAT for complement
also was important. Although the relationship between the C3
DAT and hemolysis is not as well studied as that of the IgG
DAThemolysis relationship, there is evidence that the presence
and amount of C3 is an important predictor of hemolysis and
might act synergistically with IgG.
14
In contrast, several studies
have shown that approximately 8% of hospitalized patients had
a positive DAT with no evidence of hemolysis.
7,9,16-18
The pos-
itive DAT results usually were weakly reactive, 1+ or less, and
684 Am J Clin Pathol 2004;122:680-685
684 DOI: 10.1309/CJAW6N8J6H0HR2WM
American Society for Clinical Pathology
Wheeler et al / CHARACTERISTICS OF WARM AUTOANTIBODIES
80% or more of these positive DAT results represented the
presence of complement without immunoglobulin on the
RBCs. As Issitt and Anstee
11
describe, it follows that the
remaining 20% of these hospitalized patients (with a positive
DAT) and no hemolysis must have IgG (with or without com-
plement) on their RBCs. Thus, between 1% and 2% of hospi-
talized patients have a weakly reactive DAT for IgG but do not
experience hemolysis. These same studies also demonstrated
that 1% of hospitalized patients without hemolytic anemia had
a DAT reactive for complement at a strength of 2+ to 4+. In
the series by Issitt et al,
5
in most of the 30 hematologically
healthy subjects with positive DAT results (25 healthy donors
and 5 patients with diseases other than autoimmune disor-
ders), the strength of the DAT was 3+ to 4+: 21 subjects had a
DAT reactive for IgG alone; 7 had a DAT reactive for IgG and
complement; 1 had a DAT reactive for IgG and IgA; and 1 had
a DAT reactive for IgG, IgM, and complement.
In our series, autoantibodies were identified in the serum
or plasma of 70% of patients, with 30% of patients having
autoantibodies detected on their RBCs alone. These data are
comparable to other data in the medical literature. In the study
of 150 subjects with warm autoantibodies by Issitt et al,
5
62%
of patients had autoantibodies in their serum samples (78% of
patients with warm autoimmune hemolytic anemia, 52% of
subjects receiving -methyldopa, and 27% of the hematolog-
ically healthy subjects). Petz and Garratty
7
identified warm
autoantibodies in serum samples of 57% of patients with
warm autoimmune hemolytic anemia.
Of the patients studied in the present series, 53% had
alloantibodies in addition to their warm reactive autoantibod-
ies. The alloantibodies were defined as such by demonstrating
that patients lacked the corresponding blood group antigens
on their own RBCs. However, because more demanding
adsorption and elution studies were not performed, we cannot
exclude the possibility that some of these antibodies actually
were mimicking autoantibodies. A review of the literature
shows differing incidences of accompanying alloantibodies.
Laine and Beattie
19
detected serum alloantibodies in 37.6% of
109 serum samples containing unbound warm reactive
autoantibodies. James et al
20
identified alloantibodies in
31.7% of such serum samples. Morel et al
21
identified alloan-
tibodies in 40% of serum samples studied. However,
Wallhermfechtel et al
22
demonstrated alloantibodies in only
15.2% of serum samples, and Issitt et al,
23
in a study of 138
patients, identified alloantibodies in only 23%.
One explanation offered by Mollison et al
24
and Issitt et
al
23
for the higher incidence of alloantibodies in some studies
is that the primary or exclusive use of allogeneic adsorption
may leave behind alloantibodies that really are autoantibod-
ies with mimicking specificities. In our study, allogeneic
adsorptions were performed in 30 cases; in only 8 patients
were underlying alloantibodies detected. Therefore, the use of
allogeneic adsorption is unlikely to account for the high per-
centage of alloantibodies in this series. Instead, this may relate
to the heavily transfused nature of our study patients.
Alternatively, by selecting patients for study with demonstra-
ble warm autoantibodies, we also may have selected for
patients with alloantibodies. There is a recognized relationship
between the development of RBC alloantibodies and the later
development of autoantibodies.
25-27
Our study demonstrates that the majority of patients with
serologically detectable warm reactive autoantibodies do not
experience hemolysis, that the vast majority of patients who
experience hemolysis have primary clinical conditions classi-
cally associated with autoimmune hemolysis, and that the
strength of a patients DAT (for IgG and C3) correlates with
the presence or absence of hemolysis. These findings may
help in determining the hemolytic potential of warm reactive
autoantibodies and the subsequent follow-up required to mon-
itor patients for evidence of ongoing hemolysis.
From the Department of Pathology and Laboratory Medicine,
University of California, Los Angeles.
Address reprint requests to Dr Blackall: Dept of Pathology,
Arkansas Childrens Hospital, Mail Slot 820, 800 Marshall St,
Little Rock, AR 72202.
Acknowledgments: We thank the technical staff of the UCLA
blood bank for excellence in performing the serologic studies
described in this article, and the Fenwal Division, Baxter
Healthcare, Deerfield, IL, for continued support of the American
Association of Blood BanksFenwal Scholarship Awards for
transfusion medicine fellows.
*
Dr Wheeler was a 2003 recipient of an American
Association of Blood BanksFenwal Scholarship award. This
article resulted from work recognized by the fellowship award
program.
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