Review

A 13-Question Approach to Resolving Serological

Discrepancies in the Transfusion Medicine

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Laboratory
Jovana Yudin, MD, BSc,1 Nancy M. Heddle, MSc, FCSMLS(D)1,2*
Lab Med Summer 2014;45:000

DOI: 10.1309/LMEWVSNT2F3O5JDN

ABSTRACT question-based problem-solving approach considers patient factors

including diagnosis, transfusion history, previous pregnancies, and
Laboratory professionals, consultants, and treating physicians may medication history, along with serological test results: ABO and Rh
encounter discrepancies in serological testing results for numerous groups, direct and indirect antiglobulin tests, reacting temperature of
reasons; identifying the reason(s) for the presence of an unexpected the antibody, effect of enzyme treatment of cells, strength of reactivity,
antibody or antigen can be challenging. A question-based approach and antibody reactivity with umbilical cord cells. We also demonstrate
can be useful in identifying the underlying cause of the discrepancy. the usefulness of this approach through a case scenario.
We describe a new approach to serological problems in a transfusion-
service laboratory. The approach we outline herein is targeted towards Keywords: serological testing, antibody discrepancy, antigen
a general transfusion medicine service, rather than a center that offers discrepancy, transfusion
complex antibody investigations using specialized techniques. This

Discrepancies in serological testing may occur for a Unresolved serological antibody discrepancies can delay
variety of reasons; these data have often been categorized the identification of compatible units for transfusion, the
according to a lack of expected antigens or antibodies or availability of compatible blood, or identification of an
by the presence of unexpected antigens or antibodies. underlying abnormality.
Resolving them, however, requires a systematic approach
for gathering relevant information that will assist the A question-based approach was developed for serological
laboratory professional, consultant, and/or physician problems in which the response to each question provides
to understand the significance of the findings and to a piece of the puzzle that will eventually resolve the
identify additional tests that may resolve discrepancies. clinical picture. This approach has been taught to many
physician trainees and can be applied to serological
discrepancies encountered in transfusion services; it
is designed for physicians who serve as directors of
transfusion laboratories, trainees who consult for the
Abbreviations transfusion medicine (TM) laboratory as part of their
TM, transfusion medicine; IgG, immunoglobulin G; DAT, direct anti- training, and laboratory technologists. It is meant to be a
globulin test; IgM, immunoglobulin M; EBV, Epstein-Barr virus; PCH, tool to aid in the investigation and resolution of serological
paroxysmal cold hemoglobinuria; RBC, red blood cell; IVIG, intravenous
immunoglobulin; DSTR, delayed serological transfusion reaction; RHIG, discrepancies in the routine hospital setting. The approach
Rh immunoglobulin; ITP, idiopathic thrombocytopenic purpura; AIHA, presented herein is targeted towards a general TM service
autoimmune hemolytic anemia; IV, intravenous; PVP, polyvinylpyrrol- rather than toward centers that undertake complex
idone; HTLA, high titer low avidity; NSAIDs, nonsteroidal anti-inflam- antibody investigations using specialized techniques.
matory drugs; NA, not applicable

Division of Hematology, Department of Medicine, McMaster

1 In this article, we identify and discuss 13 essential
University, Hamilton, Ontario, Canada; 2Centre for Innovation, Canadian questions that laboratory professionals, consultants, and
Blood Services, Hamilton, Ontario, Canada treating physicians should ask (Figure 1). The order in
*To whom correspondence should be addressed. which the questions should be asked varies according
E-mail: [email protected] to the serological problem encountered and the answers

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Review

1) What is the patient’s diagnosis? Consider differential for an extra antigen,

extra antibody, and false agglutination.

2) What is the transfusion history? Consider alloimmunization versus a passive

antibody, or mixed field due to recent transfusion.
Clinical

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3) What is the medication history? Consider drug associated mechanisms.

Consider alloimmunization, insignificant cold

4) What is the pregnancy history? antibodies, atypical agglutination (rouleaux/mixed
field), or passive anti-D.

5) What is the ABO group? Consider anti-A1, anti-H, or passive

Questions anti-A/anti-B causing a positive DAT result.

6) What is the Rh type? If Rh negative, consider alloimmunization,

or passive anti-D.

7) Is the DAT positive? Consider six differential causes of a

positive DAT.

8) At what temperature does the Consider if the antibody is warm (IgG)

antibody react? or cold reating (IgM).

Enzymes enhance reactivity of the Rh, Kidd, Lewis,

Laboratory 9) Does the antibody react with P, and I system antibodies and warm-reacting antibodies.
enzyme treated cells? Enzymes destroy M, N, S, Duffy and Xga antigens.

10) Is the antibody still reactive An IgM antibody detected because of complement
when monospecific anti-IgG activation will be negative with monospecific anti-IgG.
is used in the DAT?

11) Is the agglutination unusual Consider causes of rouleaux or a mixed field reaction.
in appearance?

12) Does the agglutination show Consider multiple antibodies, an antibody showing
variable strength in activity? dosage, or variable antigen expression.

13) Does the antibody react with I, Lewis, Lua, Cha, Rga antibodies will have weak or
cord cells? negative reactions with cord cells as antigens are not
developed at birth.

Figure 1
Thirteen questions (clinical- and laboratory-based) that laboratory profesionals, consultants, and treating physicians can ask themselves
to guide their investigations of the serological discrepancies and atypical findings they encounter in the transfusion medicine laboratory.
DAT indicates direct antiglobulin test; IgG, immunoglobulin G; IgM, immunoglobulin M; RBCs, red blood cells.

revealed through the process. In some situations, the Question #1: What is the
problem may be resolved without addressing all 13 diagnosis for the patient?
questions. It is important not only to ask the questions but
also to understand the relevance and potential implications It is helpful to know the diagnosis for the patient because
of the answers because this may reveal which question is certain serological problems are more common with
most relevant to ask next. particular diseases. Diseases associated with warm or

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Diagnoses/treatments associated with

autoantibodies and rouleaux

Warm autoantibodies (IgG) Cold autoantibodies (IgM) Biphasic autoantibody (IgG) False agglutination (Rouleaux)

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Reticuloendothelial neoplasms Cold hemagglutinin disease Paroxysmal cold Paraproteinemias
• Chronic lymphocytic infections hemoglobinura (PCH) • Multiple myeloma,
leukemia • Mycoplasma • Waldenstrom’s
• Hodgkin’s/Non-Hodgkin’s pneumonia macroglobulinemia
lymphoma • Mononucleosis Characteristics • Amyloidosis
• Thymomas Pregnancy • Positive DAT result Hyperfibrinogenemia
• Multiple myeloma • Positive DLc test
• Waldenstrom’s Intravenous solutions
macroglobulinemia Characteristics • Dextran
• ABO and Rh typing • Polyvinylpyrrolidone (PVP)
Collagen vascular diseases discrepancy
• Systemic lupus • Positive DATa
• Sclerdoderma Characteristics
result (C3) • See question 11
• Rheumatoid arthritis • Positive antibody
Infectious diseases screen (depends on
• Childhood viral AHGb reagent)
syndromes
Immunologic disease
• Hypogammaglobulinemia
• Dysglobulinemia
• Other immunodeficiency
Gastrointestinal disease
• Ulcerative colitis
Benign tumors
a
DAT = Direct antiglobulin test
Ovarian dermoid cyst
b
AHG = Antihuman globulin
c
DL = Donath Landsteiner
Characteristics
• Positive DAT result
• Positive Rh control
• Panagglutinin

Figure 2
Summary of conditions that may be associated with autoantibody formation and conditions in which pseudoagglutination (rouleaux)
can be present. IgG indicates immunoglobulin G; IgM, immunoglobulin M; DAT, direct antiglobulin test; DL, Donath-Landsteiner.

cold autoantibody formation are summarized in Figure positive results in the antibody screen and cross-match if
2. Warm autoantibodies are typically immunoglobulin G the antibody has high thermal activity and/or if a polyspecific
(IgG) antibodies that may be primary or can be secondary antiglobulin reagent is being used.4 The biphasic IgG
to underlying disease states, such as lymphoproliferative antibody found in paroxysmal cold hemoglobinuria (PCH),
diseases or in collagen vascular diseases.1,2 Typical features which occurs primarily in children after a viral infection,5
of warm autoantibodies include a positive direct antiglobulin is unlikely to be detected in routine serological testing
test (DAT) result; a positive Rh control (depending on the because biphasic testing would be required as part of the
reagent being used); and, in most cases, a positive antibody investigation for an IgG that is associated with PCH. In
screening (panagglutinin) result. Warm autoantibodies some cases, however, the DAT result may be positive with
do not typically cause an ABO-grouping discrepancy. complement present on the red cell surface.
Cold autoantibodies, which usually are immunoglobulin
M (IgM) type, are produced in primary or secondary cold Some diagnoses are associated with abnormal plasma
hemagglutinin disease.1,3 Mycoplasma pneumoniae infection proteins that can cause artifactual agglutination, or
is associated with production of anti-I; Epstein-Barr virus rouleaux. Rouleaux is also observed in the laboratory when
(EBV) is associated with production of anti-i.3 blood specimens are collected after infusion of certain
intravenous fluids (eg, high-molecular-weight dextran)
Cold autoantibodies typically cause discrepancies in ABO and in medical situations in which plasma protein levels
grouping and Rh typing, a positive DAT (usually C3), and are elevated, such as multiple myeloma, inflammatory

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Table 1. Medical Conditions and Mechanisms of Polyagglutination That Can Cause Serological
Discrepancies

Extra Antigen Diagnosis/Comorbidities Mechanism

T activation7 Clostridia Unmasking of the cryptic T antigen by bacterial sialidases, which cleaves sialic acid
Corynebacteria residues from the red cell membrane glycoproteins and glycolipids leaving a terminal

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Escherichia coli D-galactose.
Streptococcus
Staphylococcus
Vibrio cholera
Influenza virus
Primary and metastatic carcinoma
(breast, bladder, lung and pancreas)

Tn activation7 Leukemia The Tn antigen is similar to the T antigen but lacks the terminal galactose due to a
Breast carcinoma somatic mutation.

Tk activation7 Bacteroides fragilis The Tk receptor is associated with normal cellular sialic acid content, but is formed with
Serratia marcescens microbial endo- or exo-B-galactosidases act on the red cell surface.
Candida albacans
Clostridia
Pneumococci

Acquired B8 Escherichia coli The acquired B phenomenon refers to the deacetylation of group A receptors (N-acetyl-
Clostridium tertium D-galactosamine), via microbial activity leaving a terminal D-galactosamine that minim-
Gastric or colonic malignancy ics the terminal D-galactose of a normal B antigen.
Bowel disorders

and connective tissue disorders, cancers, and pregnancy disappear, and the group A RBCs of the patient may show
(Figure 2). Rouleaux is discussed further under question 11: a weak or negative reaction with anti-A. The suspected
Is the agglutination unusual in appearance?” mechanism for a weak or absent A antigen is a decrease
in the production of N-acetyl-D -galactosaminyltransferase,
Some diseases are associated with production of an the enzyme responsible for adding the terminal N-acetyl-
extra antigen. An extra antigen can be suspected when D -galactosamine to the H subunit to form the A antigen.
a discrepancy between the forward and reverse ABO Cases of missing antibodies may be observed in neonates,
grouping occurs; our experience has been that the elderly individuals (greater than 70 years of age), patients
discrepant result occurs within the forward grouping. who have just undergone bone marrow transplantation,
These discrepancies are often categorized under the or in association with hypo- or agammaglobulinemia.10
term polyagglutination (red blood cells [RBCs]) that Neonates do not produce anti-A or anti-B antibodies
agglutinate in the presence of almost all adult human sera until they reach the age of 3 to 6 months; elderly patients
but not with autologous serum or the sera of neonates6). may have reduced levels of anti-A or anti-B along with
Polyagglutination is a rare event in which cryptic antigens generally reduced levels of immunoglobulins.11 It is useful
become unmasked (T antigen) or antigen structures on the to know the common diagnoses that can be associated
RBC membrane become modified (to become Tn, Tk, or with serological anomalies because this information may
acquired B antigens). Diagnoses and comorbidities, as well provide clues to possible reasons for the results.
as the mechanisms by which these extra antigens appear,
are summarized in Table 1. Most of these extra antigens
are rarely encountered; in some cases, they would not be
detected in a modern laboratory because of the switch Question 2: What is the
from human to monoclonal ABO typing reagents.7-9
transfusion history?
Knowing the diagnosis of the patient can also be useful in Has the patient ever received a transfusion? Has he or she
cases of a suppressed antigen or missing antibodies. In received a transfusion within the previous 3 months? If so,
acute leukemia, the A antigen may decrease in strength or which blood products were administered?

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A history of previous transfusion may reveal a patient that of additional antibodies because they always contain
has been alloimmunized to RBC antigens with formation ABO antibodies and sometimes also contain antibodies
of a potentially clinically relevant IgG antibody. Clinically against other blood group antigens.
significant alloantibodies occur most commonly after
RBC transfusion; however, active alloimmunization can If the patient has received a transfusion recently, the
be induced by RBC contamination of platelet products; laboratory should consider the possibility of passive

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also, in less common situations, the presence of passive antibodies; however, if the transfusion history is remote,
antibody can be detected in the patient’s blood after alloimmunization should be considered. Formation of
the transfusion of large volumes of plasma. Hence, it is an RBC alloantibody can occur in the days or weeks
important to ask not only about RBC transfusions but after transfusion. Typically, alloimmunization by this
also about transfusions with other blood products. The mechanism is clinically silent, and the alloantibody will
frequency of RBC alloimmunization varies according to only be detected during subsequent pretransfusion
the antigen distribution among patients and donors in testing. If antibody testing is performed within the
a given geographic area, with estimates of 1% to 2% in first few weeks after transfusion and a new antibody
the general hospital population12 and 15% or greater in is identified, this might be accompanied by a positive
patients with transfusion-dependent diseases such as DAT result in which antibody is bound to the transfused
sickle cell disease or hemoglobinopathies.13-15 If a patient antigen-positive RBCs. This phenomenon is called a
has never received a transfusion or been pregnant, IgG delayed serological transfusion reaction (DSTR) and
RBC alloantibodies should not be present; however, a occurs when there are no additional laboratory signs and/
patient of this type can possess IgM alloantibodies to or clinical symptoms of hemolysis.
RBCs because these can be environmentally stimulated.
Environmentally stimulated IgM antibodies are not If the patient has no history of recent or remote
usually clinically relevant; however, this will depend transfusion and no history of pregnancy, it is rare for
on the thermal range of the antibody and the ability of this individual to possess IgG RBC alloantibodies. If an
the antibody to activate complement. Other than ABO antibody is detected in the serum of such an individual, it
antibodies, the typical IgM alloantibodies detected in the is likely that this entity is of the IgM (allo- or autoantibody)
transfusion laboratory are directed against the following or IgG autoantibody type.
antigens: M, N, S (some are of IgM and some of IgG
type), Lea, Leb, P1, Lua, and anti-A1.

If the patient has received a transfusion, it is important

to know whether the transfusion occurred within the past Question 3: What is the
3 months and, if so, which blood product(s) was (were)
administered. When a patient has recently received a
pregnancy history of the
transfusion, certain factors should be considered. If patient?
the recent transfusion consisted of RBCs, the patient
could be having a delayed serological or delayed In the serum of a female patient with a history of previous
hemolytic transfusion reaction. If the recent transfusion pregnancy, IgG RBC antibodies can be detected due to
was a plasma product or intravenous immunoglobulin alloimmunization caused by fetal-maternal bleeds. If the
(IVIG), passive antibody might be causing the problem. patient is currently pregnant, a number of serological
If phenotyping of the RBCs is required, transfused problems can occur and should be considered, including
donor cells may still be present in the circulatory passive anti-D, clinically insignificant cold agglutinins, and
system of the patient and cause erroneous results. atypical agglutination (as discussed in question 11).
If a patient has received a transfusion or has been
pregnant within the past 3 months, the laboratory should
perform phenotyping using a pretransfusion specimen, Passive Anti-D
reticulocyte separation, or RBC genotyping. Packed Identification of anti-D in the serum of a pregnant patient
RBCs, plasma, platelets, and cryoprecipitate may contain may occur because of active production of the antibody
immunoglobulin contaminants; hence, the laboratory due to alloimmunization or, more commonly, due to
must consider the possibility of passive antibody transfer. passive antibody transfer following injection of Rh
Immunoglobulin concentrates, such as IVIG, are a source immunoglobulin (RHIG). RHIG is typically administered,

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Table 2. Drugs Associated With Immune Hemolytic Anemia or a Positive Direct Antiglobulin Test20, 21

Drug Category Drugs Implicated

Analgesic and NSAID

Acetaminophena Glafeninea Naproxena
Azapropazonea Ibuprofena Phenacetina

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Diclofenaca Methadonea Sulindaca
Dipyronea Mefenamic acidb Tolmetina
Antiarrhythmic Procainamideb
Antidepressant Nomifensinea
Antidiabetic Chlorpropamidea Insulina Tolbutamidea
Antidiarrheal Catechina
Antiemetic Chlorpromazinea
Antihistamine Antazolinea
Antihypertensive Captoprilb Methyldopab
Antimicrobial and β-lactamase inhibitor
Amphotericin Ba Chloramphenicola Quininea
Amoxicillina Cloxacillina Quinidinea
Cefalothinc Clavulinic acidc Rifampina
Cefazolina Erythromycina Stibophena
Cefotaximea Isoniazida Sulbactamc
Cefotetana,c Levofloxacina Streptomycina
Cefoxitina Mefloquine hydrochloridea Tazobactamc
Ceftazidimea Nafcillina Temafloxacina
Ceftizoximea Nalidixic acidb Tetracyclinea
Ceftriaxonea P-aminosalicylic acida Ticarcillina
Cefuroximea Penicillin Ga Trimethoprim-sulfamethoxazolea
Cephalexina Piperacillina
Cephalothina Pyrimethaminea
Antineoplastic Carboplatina Elliptinium acetatea Interleukin-2b
Cisplatina Fludarabine phosphateb Methotrexatea
Cisplatinc Fluorouracila Oxaliplatina,c
Cladribineb Imatinib mesylatea
Diglycoaldehydec Interferonb
Antiparkinsonian Levodopab
Antithyroid Carbimazolea
Diuretic Furosemidea Hydrochlorothiazide/Triamterenea
Estrogen Diethylstillbestrola
H2 blocker Ranitidine Hydrocloridea Cimetidineb
Immunomodulators Cyclosporinea Tacrolimusb
Sulfasalazinea
Uricosuric Probenecida
a
Drug dependent mechanisms–Drug Adsorption and Immune-Complex Mechanisms
b
Drug Independent–Autoimmune Mechanism
c
Drug Independent–Nonimmunologic Protein Adsorption

at 28 weeks’ gestational age, to nonalloimmunized Clinically Insignificant IgM Antibodies

obstetrical patients with Rh negativity to prevent Cold agglutinins that are not clinically significant (anti-I,
sensitization to fetal D antigen, which may enter the anti-H, anti-HI, and Lewis antibodies) may be detected
circulation of the mother during pregnancy.16 It is also in the serum of a pregnant patient, depending on the
routinely administered after delivery if the patient with Rh screening method used. These antibodies are not
negativity has an infant with Rh positivity. RHIG should be clinically significant; however, when detected, they
administered in other situations that involve procedures generate additional investigative work for the laboratory.
or trauma during pregnancy. RHIG has a half-life of
approximately 3 weeks; using a sensitive antiglobulin test, Atypical Agglutination
the passive antibody can be detected in the serum at An increase in fibrinogen is a normal physiologic change
least 8 to 10 weeks after injection. during pregnancy17 and can result in rouleaux.18 Mixed-field

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Table 3. Typical Serological Findings Associated With a Drug-Induced Positive DAT Result20,21

Drug Dependent

Monospecific DAT Results

Mechanism Mechanistic Details (IgG, C3, or Both) IAT Eluate

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Drug adsorption IgG antibodies bind to drugs that coat IgG Negativea Nonreactive with
the RBCs; antibodies interact with Fc normal RBCs but
receptors of macrophages to cause positive with drug-
extravascular hemolysis coated RBCs

Immune complex Antibody reacts with the drug and the C3 Typically negativeb Usually nonreactive
RBC membrane; complement is activated,
causing predominately intravascular hemolysis

Drug Independent

Nonimmunologic Drug causes nonimmunologic binding of proteins IgG or C3 Negative Nonreactive

adsorption of proteins to the RBC membrane (IgG, complement, albumin,
etc). Typically, hemolysis has not been observed;
however, more recently, some drugs have been
reported to cause hemolysis via this mechanism
Autoimmune The drug causes dysregulation of the immune
system, resulting in autoantibody production IgG Positive or negativec Positivec

DAT, direct antiglobulin test; IgG, immunoglobulin G; IAT, indirect antiglobulin test; RBCs, red blood cells.
a
IAT results are negative unless RBCs are coated with the drug.
b
Negative unless drug and fresh normal serum (as a source of complement) are added to the test.
c
Antibody typically reacts as a panagglutinin.

reactions have also been reported19 after a fetomaternal antibodies require that a certain drug be present to
hemorrhage of a large volume of blood19 (see question 11). detect the antibody; the drug is bound covalently to the
RBC membrane or exists freely in the plasma. Drug-
independent antibodies do not require the presence
of any drug to be detectable in the serum (ie, the
Question 4: What is the antibody reacts similar to an autoantibody observed
medication history of the patient? in patients with warm autoimmune hemolytic anemia).
Drug-dependent antibodies are more common than
A detailed drug history is helpful to determine whether drug-independent antibodies. 21 Table 3 shows typical
serological and clinical findings are consistent with drug- serological findings for different mechanisms of drug-
induced hemolytic anemia. Although it is not a complete induced hemolytic anemias. Laboratory professionals
list, Table 2 indicates many of the drugs that have caused should consider the possibility of drug-induced hemolytic
a positive direct antiglobulin test result and/or hemolysis. anemia if the patient has hemolysis and/or a positive
Use of several other medications by patients with drug- DAT result, with or without a positive antibody screening
induced hemolytic anemia has been reported in the result. 21 A comprehensive drug history (current and
literature; a more comprehensive list by Garratty and within the past 6 months) of the patient is essential when
Arndt20 and another by Garratty21 includes drugs implicated investigating serological problems.
in single instances of this condition. Contrast material
and dyes have also been implicated. Three categories of
medications are responsible for most positive serological-
test results: antimicrobials, 42%; anti-inflammatory drugs, Question 5: What is the ABO
15%; and antineoplastic agents, 11%.20
group for the patient’s blood?
There are 2 types of drug-induced antibodies: drug Some antibodies that cause serological puzzles are
dependent and drug independent. Drug-dependent directed against antigens in the ABO blood groups. If

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Table 4. Summary of Serological Reactions to Differentiate Cold Autoantibodies (Anti-A1 and

Antibodies in the I and H Systems)

Reactions with RBCs by Group

Antibody ABO Group of Individuals Who

Can Form Antibody OI (adult) A1I (adult) A2I (adult) Oi (cord) A1 (cord)

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Anti-HI A1, A1B ++++ + ++++ +/– –

Anti-H A1, A1B ++++ +/– ++ ++++ ++++

Anti-I All groups ++++ ++++ ++++ +/– –

Anti-i All groups +/– +/– +/– ++++ ++++

Anti-A1 A2, A2B – ++ or +++ – – +/–

RBCs, red blood cells.

Grading system for agglutination typically used in immunohematology: ++++, One solid clump of cells; +++, Several large clumps of cells; ++, moderate size clumps of cells;
+, very small clumps of cells; +/–, very weak with red cell clumping only visible using a microscope; –, Negative.

the patient is blood group A or AB, the possibility that reactions occur with the group O screening cells, which
anti-A1 or anti-H is present should be considered. Anti-A1 strongly express H-antigen, whereas the cross-matched
is an IgM-type, environmentally stimulated antibody that A1 or A1B units yield weak or negative results since H
typically reacts at colder temperatures; its presence is antigen is weakly expressed on A1 and A1B RBCs. Anti-H
usually not clinically significant. It is found in the plasma antibodies may be detected when using an immediate
of 1% to 3% of individuals with A 2 and may be present spin cross-match if the antibody has activity at room
in the plasma of approximately 25% of individuals who temperature and if A 2 donor units are selected for cross-
are the A 2B phenotype. 22 The ABO serotyping result match (A 2 RBCs have greater H-antigen expression).
can sometimes reveal that the patient may be an A 2B There is also an antibody that reacts with RBCs that
phenotype because the anti-A typing result may not give express the H and I antigens. Table 4 summarizes the
a typical 4+ reaction (it often gives a 2+ or 3+ result). typical reactivity of anti-A1, anti-H, and anti-HI, and
Anti-A1 typically appears as a discrepancy between the compares this reactivity to IgM autoantibodies in the I–
results of ABO forward and reverse testing: the RBCs blood-group system. 22
of the patient appear as type as A or AB, but the serum
reacts with the reagent A cells. Unexpected reactions in
the reverse grouping may also be caused by the presence
of other IgM alloantibodies (anti-M, N, S Lea, Leb, P1, or Question 6: What is the Rh
Lua); however, when the blood of the patient is of the
group A or AB designation, the laboratorian should also
type of the patient’s blood?
consider the possible presence of anti-A1. In Rh-negative patients, anti-D is always a possibility.
With routine prophylactic use of RHIG in the obstetrical
Antibodies against the H antigen can be detected in the population, the frequency of anti-D alloimmunization
plasma of individuals who red cells group as A1 and has decreased dramatically; however, anti-D formation
A1B. 22 These are autoantibodies (A1 and A1B RBCs contain can still occur. More commonly, passive anti-D may be
small amounts of H antigen in their surface); however, the detected in the plasma of Rh-negative pregnant women
autocontrol typically tests negative. Anti-H is usually not because they have been injected with RHIG at 28 weeks’
detected in routine serological testing because it typically gestation or have received RHIG after delivery or for
reacts only at cold temperatures; however, sometimes the other indications throughout their pregnancy. If a female
thermal range of this reaction extends to 22°C or higher, patient is Rh negative with a positive antibody screening
which causes interference with serological testing. Anti-H result when tested during pregnancy or after delivery,
may be detected via the antibody screen if a polyspecific it should be verified whether the patient has recently
antiglobulin reagent is being used. The strongest received RHIG.

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can also be informative in suspected cases of warm

Question 7: Is the DAT result autoimmune hemolytic anemia (AIHA) because it may be
possible to elute IgG from the RBCs.
positive? If yes, what were the
results of eluate testing?

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A positive DAT result reveals that circulating RBCs of Question 8: At what
the patient are coated with IgG and/or C3.22 However,
a positive DAT result may or may not be associated
temperature does the antibody
with hemolysis and should be correlated with clinical react?
and laboratory findings. Likewise, a negative DAT result
does not exclude the possibility of immune-mediated RBC antibodies fall into 2 categories, namely, cold
hemolysis. The differential diagnosis for a positive DAT reacting (optimal temperature around 4°C) and warm
result includes a delayed serological or delayed hemolytic reacting (37°C). Cold-reacting antibodies are usually IgM
transfusion reaction; warm autoimmune hemolytic and can be alloantibodies or autoantibodies. The most
anemia; cold autoimmune hemolytic anemia; immune common IgM alloantibodies are directed against antigens
hemolysis due to the presence of a certain drug or drugs M, N, S, Lea, Leb, P1, and A1. Antibodies to Lua are also of
in the plasma; hemolytic disease of the newborn; and IgM type but are far less common.26,27 IgM autoantibodies
hypergammaglobulinemia.23 The plasma of as many as usually are specific to the I antigens (I or i) but may be
15% of hospitalized patients may yield a positive DAT directed against the HI or Pr antigens.28. IgM antibodies
result.24 When a patient has a positive DAT result, it is do not cross the placenta and therefore do not cause
important to consider the information received from the hemolytic disease of the newborn; however, they can
previous questions asked. For example, if a patient has occasionally cause RBC hemolysis after transfusion if
idiopathic thrombocytopenic purpura (ITP) and received the antibody has thermal activity above 30°C and can
IVIG a week ago, the most likely causes for the positive DAT activate complement. In most situations, the presence of
result is a delayed serological and/or hemolytic transfusion these antibodies is not clinically relevant; however, these
reaction due to passive antibody in the IVIG, or treatment- antibodies may cause serological discrepancies in tests
induced hypergammaglobulinemia. A positive DAT result that are performed at room temperature, such as the
associated with hypergammaglobulinemia occurs in ABO-grouping or Rh-typing tests, or in the antiglobulin
patients whose disease process or treatment results in phase if a polyspecific antiglobulin reagent is used.
elevated immunoglobulin levels. IgG binds nonspecifically
to the RBC surface; however, typically, hemolysis does IgG antibodies react optimally at 37°C and frequently
not occur.25 The use of monospecific antiglobulin reagents display specificity for Rh, Kell, Duffy, Kidd, and Ss
(Anti-IgG and Anti-C3d) and elution studies, combined with antigens. Antibodies that are specific for Lub and Xga
a comprehensive clinical history, usually helps to identify are rare; these antibodies typically are detectable by
the cause of the positive DAT result. the antiglobulin test. Because these antibodies react at
the normal body temperature of 37°C and can cross the
There are a variety of techniques to elute IgG from the placenta, their presence is usually clinically significant
RBC surface: namely, chemical methods that disrupt with regard to hemolytic transfusion reactions and can
the RBC membrane, causing antibodies bound to the cause hemolytic disease in newborns.
RBC surface to be released, and the use of heat (56°C)
to dissociate antibodies from the membrane—this latter
approach works well when ABO antibodies are on
the RBC surface. A negative eluate result when IgG is Question 9: Does the antibody
detected on the RBC surface is consistent with a positive
DAT result due to the presence of certain drugs in the
react with enzyme-treated
plasma, hypergammaglobulinemia, or passive anti-A and RBCs?
anti-B if elution studies are only carried out using group-O
screening or panel cells. Whenever IgG is present on Proteolytic enzymes (eg, papain, ficin, trypsin, and bromelin)
RBCs, it is useful to perform elution studies; however, have been used in serological testing for many years.
creating an eluate when only C3 is detected by the DAT Papain and ficin are used most frequently. Proteolytic

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Review

enzymes cleave amino acids at specific sites, removing of a stack of coins. Rouleaux formation typically occurs
protein fragments on the RBC surface. If blood group when patients have an altered albumin:globulin ratio.
antigens are located on the cleaved protein, antibodies Conditions in which this phenomenon occurs include
directed against these antigens will no longer react. multiple myeloma, cryoglobulinemia, macroglobulinemia,
Antigens that are destroyed by proteolytic enzymes include cirrhosis, and hyperfibrinogenemia (the latter occurs in
Fya, Fyb, M, N, S, Xga, Cha, Rga, and JMH.29 Proteolysis can patients with acute infections and during pregnancy).17,18,30

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also make other antigen sites more accessible to antibodies, Rouleaux has also been observed following infusion of
resulting in enhanced reactivity of antibodies toward Rh, intravenous (IV) solutions containing high-molecular-
Kidd, Lewis, P, and I antigens, including enhancement of weight dextran or polyvinylpyrrolidone (PVP). When
warm-reacting autoantibodies. Enzyme treatment is not rouleaux is suspected, phenotyping may be done with
a routine procedure but it can be useful as an additional washed RBCs and a saline-replacement technique can
test, especially when multiple antibodies are present in the be used for tests involving direct agglutination.31 Rouleaux
plasma and 1 or more of the antibodies are directed against is not observable in antiglobulin test results because
an antigen that is destroyed by enzymes. the washing phase that is performed before adding the
antiglobulin reagent typically removes the plasma proteins
that cause the phenomenon.

Question 10: Is the The second unusual pattern that may be observed
is a mixed-field reaction in which some of the RBCs
antibody still reactive when are agglutinated, whereas others are not. This pattern
monospecific Anti-IgG is used may be observed macroscopically if the test is
in the DAT? performed in tubes: visible agglutination is present,
whereas the background appears pink because of
Many laboratories routinely use Anti-IgG to detect the the nonagglutinated RBCs. Mixed-field reactions are
presence of clinically significant antibodies; however, in easier to detect microscopically or when using column-
laboratories that use a polyspecific antiglobulin reagent, agglutination methods (2 RBC bands are visible).
it is important to consider whether the reactivity occurs The differential diagnosis for mixed-field reactions
due to IgG or C3 on the RBC surface. When a room depends on whether the pattern is observed when
temperature–reacting IgM antibody binds to RBCs, phenotyping or when performing antibody-screening
complement may be activated and bind to the RBC procedures. When a mixed-field reaction is observed
membrane when the serological test is set up at room with phenotyping, the following possibilities should be
temperature. Although the IgM antibody may elute from considered: recent transfusion including intrauterine and
the RBC when the test is incubated at 37°C, complement exchange transfusions; recent allogeneic hematopoietic
(C3b) may remain on the RBC surface and be detectable stem-cell transplantation; the A3 phenotype; extensive
via the polyspecific antiglobulin reagent. Hence, if fetomaternal or maternal-fetal bleeds; in utero exchange
complement activation by an IgM antibody is suspected, it of hematopoietic stem-cell tissue in twins (ie, the chimera
may be helpful to repeat the test with monospecific Anti- phenomenon); and polyagglutination.10 Antibodies that
IgG reagent or using a prewarming technique. typically cause mixed-field reactions include anti-Lua,
anti-Sda (the appearance of agglutinates is spherical and
refractory); and, some antibodies that exhibit high titer and
low avidity (HTLA) characteristics.32
Question 11: Is the
agglutination unusual in
appearance? Question 12: Does the
Agglutination typically results in clumps of RBCs that
agglutination show variable
appear somewhat homogeneous; however, 2 unusual reactivity?
patterns of RBC clumping can occur in serological testing.
Rouleaux formation can mimic agglutination; however, on When panel cells, screening cells, or cross-matched
microscopic assessment, the cells have the appearance units show variable reactivity, there are 3 possibilities

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 Review

Table 5. Useful Reference Lists for Solving Serological Problems

Variable Antibodies/Antigens

Common IgM alloantibodies (react at room temperature or lower) Have specificity for:
M
N

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S (rarely—usually IgG)
Lewis (Lea, Leb)
P1
Lua
A1

IgM autoantibodies (typically react at room temperature or lower but may Have specificity for:
have higher thermal activity in autoimmune hemolytic anemia). I
I
H
HI
Pr (uncommon)

Antigens destroyed by proteolytic enzymes M

N
S
Duffy (Fya Fyb)
Xga

Antibodies enhanced by RBC enzyme treatment Antibodies of the following types:

Rh
Kidd
Lewis
P1
I-system

Antibodies that can show dosage Usually have specificity for:

Rh antigens
MNS antigens

Antigens not developed or weakly developed in umbilical-cord RBCs Lewis (Lea, Leb)
P1
I
Lua

Causes of variable reaction strength Dosage

Multiple antibodies
Variable antigen expression unrelated to dosage (eg, P1, I, Lewis
antigens)

IgM, immunoglobulin M; RBCs, red blood cells.

to consider: 1) multiple antibodies, 2) stronger reactions Question 13: Does the antibody
with double-dose (homozygous) versus single-dose
(heterozygous) cells, suggesting an antibody that shows
react with umbilical cord cells?
dosage, and 3) variable antigen expression unrelated to Antibody panel manufacturers do not supply umbilical cord
dosage (such as P1, Lewis, and I-system antigens). cells on their panels (hereafter, cord cells); however, when
certain types of antibodies are suspected it may be useful

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Review

Question Answer Think about

What is the Admitted for GI Treatments for ITP that

patient’s diagnosis? bleed and ITP could affect serology:
• IVIG

Alloimmunization from

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Has the patient
been transfused RBC 3 years ago previous RBC transfusion.
any blood product? IVIG days ago Positive DAT result from
hypergammaglobulinemia.
Passive antibody causing
a positive DAT result
What is the patient’s Group A Rh and/or antibody screen:
ABO and Rh? negative
• ABO
• Other IgG antibodies

Yes 1+
Is the DAT (IgG detected Differential diagnosis for a
positive? on the RBCs) positive DAT result (IgG):
• Warm AIHA
• Drug-induced hemolytic
anemia
• Passive antibody from
IVIG
What medication is • Hypergammaglobulemia
None Rules out (from the IVIG)
patient taking?

Unlikely but cannot

be ruled out
Is the eluate
reactive with:
• A Rh negative Group O RBCs - negative
RBCs Group A RBCs - positive Consistent with
• O Rh positive
RBCs

Reasons why anti-D is present:

Is the antibody
screening result Yes–anti-D detected • Previous RBC transfusion
positive? if Rh-positive blood was
administered
• Pregnancy
Has the patient • Passive anti-D in the IVIG
been/is pregnant? No

Is the anti-D detectable Yes Consistent with

in the IVIG?

Check previous
transfusion history?

Figure 3
Data from a case scenario, examined using the question-based approach. A 40-year-old woman is admitted to the hospital for gastro-
intestinal bleeding, and the transfusion laboratory is called on to provide compatible units. A positive direct antiglobulin test (DAT) result
is identified, and the laboratorian is asked to consider the most likely cause of these findings. Depending on the case, it may not be
necessary to ask every question; the order in which the questions are asked may vary depending on the clinical and laboratory findings.
GI indicates gastrointestinal; ITP, idiopathic thrombocytopenic purpura; IVIG, intravenous immunoglobulin; DAT, direct antiglobulin test;
IgG, immunoglobulin G; RBCs, red blood cells; AIHA, autoimmune hemolytic anemia.

204 Lab Medicine Summer 2014 | Volume 45, Number 3 www.labmedicine.com

 Review

to select and/or pool several group-O cord cells for use

in serological testing. Some antibodies do not react with References
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