BRIEF REPORT
A case series of pediatric patients with direct antiglobulin test
negative autoimmune hemolytic anemia
Jonathan Miller,1 Wei Cai,2 Jennifer Andrews,3,* and Anupama Narla
BACKGROUND: The diagnosis of autoimmune
hemolytic anemia (AIHA) can be challenging since the
direct antiglobulin test (DAT) has been reported to be
falsely negative in 3%-11% of cases. In children with
anemia, laboratory and/or clinical evidence of hemolysis
and a negative DAT, clinicians should consider further
specialized testing to confirm AIHA to accurately
diagnose and treat this uncommon pediatric entity.
STUDY DESIGN AND METHODS: A retrospective
chart review was undertaken at a large tertiary care
academic pediatric hematology practice to describe our
experience with DAT-negative AIHA.
RESULTS: From January 1, 2010 through August 1,
2016, 10 children were described who had clinical and
laboratory evidence of AIHA, a negative DAT, and further
specialized serologic testing confirming this diagnosis.
CONCLUSION: This case series highlights the need for
further serologic workup when a child’s clinical
presentation is highly consistent with AIHA despite a
negative DAT.
2528 TRANSFUSION Volume 59, August 2019
4,*
T
he diagnosis of autoimmune hemolytic anemia
(AIHA) requires clinical suspicion in conjunction
with evidence of anemia, hemolysis, and the pres-
ence of an autoantibody. An array of laboratory test-
ing can be suggestive of hemolysis, with low haptoglobin and
elevated lactate dehydrogenase, often accompanied by an
indirect hyperbilirubinemia, elevated reticulocyte count, uri-
nalysis with free hemoglobin, and spherocytes on the periph-
eral blood smear.
AIHA can be due to warm, cold, or mixed autoantibody
types.1,2 A warm autoantibody, the most common cause of
AIHA, is detected through the direct antiglobulin test (DAT).1
The DAT detects IgG antibodies and/or C3 bound to the
patient’s red blood cells (RBCs) upon addition of the antihu-
man globulin (AHG) reagent. Warm autoantibodies are typi-
cally a member of the IgG immunoglobulin class and can be
directly detected by the AHG reagent. Cold autoantibodies
are usually of the IgM class and are evidenced by comple-
ment (usually C3) found on the RBC membrane. Despite
being a routine and effective diagnostic tool for AIHA, the
DAT is falsely negative in 3%-11% of patients with AIHA.3–6
There are four possible explanations for DAT-negative
AIHA: 1) a quantitatively low level of IgG bound to the RBC
such that it is below the threshold of detectability by the stan-
dard assay; 2) a low affinity IgG such that it becomes unbound
from the RBC membrane easily; 3) the antibody is of the IgA
or IgM subtype, or an IgG isoallotype which are not detected
From the 1Department of Oncology, St. Jude Children’s Research
Hospital, Memphis, 3Department of Pediatrics and Department of
Pathology, Microbiology and Immunology, Vanderbilt University
School of Medicine, Nashville, Tennessee; the 2Department of
Pathology, Division of Transfusion Medicine, and the 4Department
of Pediatrics, Division of Hematology & Oncology, Stanford
University School of Medicine, Stanford, California.
Address reprint requests to: Anupama Narla, MD, CCSR-1215b,
269 Campus Drive, Stanford, CA 94305-5162; e-mail: anunarla@
stanford.edu.
*Co-senior authors.
Received for publication January 23, 2019; revision received
May 2, 2019, and accepted May 3, 2019.
doi:10.1111/trf.15350
© 2019 AABB
TRANSFUSION 2019;59;2528–2531
 DAT NEGATIVE AIHA IN CHILDREN

by routine DAT; or 4) natural killer (NK) cell-mediated
hemolysis, i.e., antibody-independent cytotoxic events.7,8
When clinical suspicion for AIHA remains high despite a
negative DAT, clinicians should consider further serologic test-
ing performed by specialized laboratories as described below.
MATERIALS AND METHODS
After obtaining Institutional Review Board approval from
Stanford University (IRB 38570), a retrospective chart review
of children with DAT-negative AIHA between January 1, 2010
and August 1, 2016 was performed. Information collected
included demographic information, concurrent diagnoses
(if any), relevant laboratory testing including specialized labo-
ratory serologic testing, and treatment (if any). Descriptive
statistics were used to describe patients’ specialized DAT test
results. Stanford University employs a two-step procedure for
the DAT which remained consistent from 2010 through 2016.
The DAT reagent used is a polyspecific murine monoclonal
antihuman globulin (Gamma-clone, Immucor, Inc.) for anti-
IgG and -C3d. Further testing is done for a positive DAT. If
DAT was negative, no further internal testing is employed
and a physician trained in transfusion medicine alongside
the clinical hematologist determines utility of specialized
external testing on a case-by-case basis. In this case report,
since all the cases were DAT negative at Stanford University,
only the polyspecific reagent was used and all cases under-
went external testing by the Immunohematology Research
Laboratory (IRL) at the American Red Cross Blood Services
in the Southern California Region (Pomona, CA).
RESULTS
Ten patients, aged 4 months to 25 years, were found to have
DAT-negative AIHA at our institution during a 6-year period
(Table 1). Some children were previously healthy, and others
were medically complex with concurrent diagnoses of acute
lymphoblastic leukemia (ALL), Diamond Blackfan anemia
(DBA), or Fraser Syndrome. Concurrent laboratory markers
supported hemolysis in all patients. Six patients had concur-
rent thrombocytopenia. All patients had negative DAT in our
hospital transfusion laboratory at diagnosis.
In all cases, because of a high index of suspicion for the
diagnosis of AIHA, specialized serologic testing was per-
formed by the IRL.7 This testing included: 1) two polyclonal
anti-IgG reagents (Ortho Clinical Diagnostics and Immucor)
are used to detect anti-IgG or -C3 antibodies on the RBC
membrane; 2) anti-IgA and anti-IgM that have been stan-
dardized for use with RBCs, as well as an anti-C3 enriched
with anti-C3d (C3dg) to detect anti-IgA, IgM, or C3d on the
RBC membrane5; 3) specialized IgG testing with cold saline
or low ionic strength saline (LISS) at room temperature in
addition to standard room temperature washes to identify
low-affinity RBC-bound antibodies; and 4) specialized
Polybrene (Sigma-Aldrich) testing, which as a quaternary
ammonium compound aggregates RBCs, with subsequent
addition of sodium citrate to disperse non-immunoglobulin
bound RBCs, leaving only IgG bound RBCs for detection by
anti-IgG reagents listed above.7,9 DAT performed with use of
a LISS wash followed by anti-IgG testing is performed only if
standard IgG testing is negative. IgG testing with Polybrene is
performed only if standard DAT with anti-IgG with and with-
out LISS wash are both negative.
Eight cases (73%) were found to have very weak, weak,
or moderate positivity for C3 bound to their RBCs at the IRL
(Table 2). Two patients had IgG antibodies detected on their
RBCs at the IRL (one weak, defined as 1+, and two moderate,
defined as 2+), and another had IgA antibodies detected. One
patient had IgG antibodies detected only with the LISS wash
DAT. Treatment ranged from watchful waiting to various

TABLE 1. Ten children with DAT-negative AIHA with associated demographics, medical history, and laboratory studies
at initial diagnosis
Hgb Platelets Retic LDH Haptoglobin T Bili
Case Age Co-morbidities (g/dL) (K/uL) Abs Retic % (U/L) (mg/dL) (mg/dL)
1 4 mo Splenomegaly 8.3 28 270.9 8.87 331 –* 0.3
2 9 mo None 5.0 305 304.7 19.39 584 <8 2.2
3 1 yr Bronchiolitis 5.0 407 21.1 0.92 444 <8 2.4
4 4 yr DBA 8.9 277 – <0.45 364 <8 1.6
5 5 yr HLH, immunodeficiency, Fraser Syndrome 7.1 87 63.1 2.14 – <8 0.6
6 11 yr Aplastic Anemia, s/p SCT 11.9 85 131.8 4.26 915 <8 0.2
7 12 yr Evan’s Syndrome 9.8 56 62.0 1.73 744 <8 0.3
8 13 yr ALL 8.1 63 62.5 2.7 239 <8 0.7
9 14 yr None 6.3 272 646.0 29.98 693 <8 2.5
10 25 yr† DBA, s/p SCT 10.3 51 37.0 1.30 632 <8 2.5
All cases had negative DAT recorded at time of lab testing (data not shown).
* Testing not sent or not reported.
† Patient was initially seen at our institution at age 20 because of a suspected diagnosis of Diamond Blackfan anemia (DBA), an inherited bone
marrow failure syndrome. Given our expertise in DBA, we elected to continue to follow and treat the patient at our pediatric institution.
mo = months old; yr = years old; DBA = Diamond Blackfan anemia; HLH = hemophagocytic lymphohistiocytosis; SCT = stem cell transplant;
ALL = acute lymphoblastic leukemia; ANA = anti-nuclear antibody; Hgb = hemoglobin; Retic Abs, % = reticulocyte count absolute & percentage;
LDH = lactate dehydrogenase; T Bili = total bilirubin.

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MILLER ET AL.

TABLE 2. Results of in-depth serologic direct antiglobulin testing and subsequent medical treatment
Anti-IgG Anti-IgG Anti-C3
Case Anti-IgG LISS wash Polybrene enriched Anti-IgA Anti-IgM Treatment & outcome
1 − − − + − − None, spontaneous resolution
2 +* + − + − − s/p steroids with good response
3 − − − + − − Transfusion, spontaneous resolution
4 +* NA NA − NA NA Transfusion, spontaneous resolution
5 − − − + − − Steroid dependent; etoposide + steroids
6 − − − + − − Transfusions, failed steroids, failed rituximab,
plasmapheresis/bortezomib
7 − − − + − − Steroid dependent; failed rituximab; transaminitis with sirolimus
8 − − − + − − s/p steroids with good response
9 − − + − + − Steroid dependent; refractive to rituximab, MMF
10 +* NA NA − − − s/p steroids with good response, SCT
* Initial testing for standard IgG anti-RBC antibodies was negative; only positive with in-depth testing.
NA = not applicable; s/p = status post; MMF = mycophenolate mofetil; SCT = stem cell transplant.

pharmacologic therapies including steroids and rituximab to
plasma exchange.
DISCUSSION
We describe 10 pediatric cases where DAT-negative AIHA
was diagnosed with specialized serologic testing at an exter-
nal IRL. Although several case reports of DAT-negative
AIHAs have been reported, this represents one of the largest
case series in pediatrics to date. A 2013 article by Segel and
Lichtman reviewed 25 clinical case reports since 1971,
which described 55 patients with DAT-negative AIHA over
4 decades.10 Of those 55 cases, only 11 occurred in children.
We hypothesize that the infrequency of reported cases of
pediatric DAT-negative AIHA is likely related to mis-
diagnosis since many hospital blood banks and research
laboratories do not perform this specialized testing. In addi-
tion, clinicians may be unaware of such specialized testing
as described and such do not request referral testing. In a
case series of 15 children with AIHA following stem cell
transplant, one third of the patients were DAT-negative.11
Perhaps this testing algorithm is more widely known and
used in tertiary care academic hospitals where pediatric
patients generally receive stem cell transplantation.
Garratty et al offer a technical explanation for a nega-
tive DAT in patients with AIHA. One hundred seventy-five
of 398 DATs (44%) referred to their reference laboratory as
DAT-negative were found to be positive.12 Possible explana-
tions for this included the loss of low-affinity autoantibodies
due to improper washing of RBCs at high temperatures,
lower potency of commercial anti-C3 activity, and superior
ability of reference laboratory technologists in reading anti-
globulin tests.12 The use of commercial antiglobulin sera
with low potency anti-C3d common in many mainstream
hospital laboratories may explain the negative standard
DAT in the majority of our patients, as 8 out of 10 cases
tested positive for C3 at the IRL (Table 2). Development of
improved commercial anti-C3 antibodies may prove impor-
tant in identifying more patients with AIHA via the upfront
standard DAT and avoid the need for additional “Super
Coombs” testing with enriched antibodies currently avail-
able only in these specialized reference laboratories. Addi-
tionally, Howie et al identified limitations in the murine
monoclonal anti-IgG antibody gamma-clone and 3 out of
10 cases tested positive for IgG at the IRL with use of two
polyclonal anti-IgG antibodies, which raises the possibility
of incorporating polyclonal anti-IgG antibody testing into
mainstream laboratory upfront testing.8
This study effectively highlights the opportunity for cli-
nicians to identify a definitive diagnosis of AIHA when the
standard DAT is negative, and is meant to educate and raise
awareness about the availability and efficacy of specialized
testing through the presentation of a small case-series. Limi-
tations of this study include sample size (small patient
cohort from a single institution) and failure to fully address
the indications for specialized testing as this information is
largely anecdotal. Better prediction models and risk stratifi-
cation tools that incorporate laboratory data or other clinical
information to better identifying patients with AIHA are
needed, especially when initial DAT is negative.
CONCLUSION
AIHA is a serious medical condition that can be life-threat-
ening. Laboratory testing consistent with hemolysis should
trigger testing with DAT. When clinical suspicion remains
high, the clinician must consider in-depth serologic testing
in consultation with the Transfusion Medicine division at
their hospital and external IRLs to successfully diagnose
DAT-negative AIHA. Lab testing does not significantly delay
treatment, as testing sent on patients described here was
expeditious, taking on average 3 days to process, although
next day results were provided in two cases where a more
urgent response was necessary.
The definitive diagnosis of AIHA is critical in establishing
an appropriate treatment plan. Our patients received a vari-
ety of treatments directed at their AIHA (see Table 2), rang-
ing from observation only to steroids to immunosuppressive

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therapy. With a confirmatory diagnosis, appropriate therapy
can be initiated promptly. Given the occasional refractory
nature of AIHA, confirmatory testing empowers clinicians to
continue with second- or third-line therapy as needed.
CONFLICT OF INTEREST
The authors have disclosed no conflicts of interest.
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