Antihuman Globulin test / Coombs’ anti-gamma globulin activity, whereas non-

gamma globulin component was shown to be

test beta globulin and had specificity for
complement. Later studies, revealed that the
▪ Principle: AHG obtained from immunized complement activity was a result of C3 and C4.
nonhuman species bind to human
globulins such as IgG or complement, Antihuman globulin test Reagent
either free in serum or attached to
antigens on red blood cells (RBCs). REAGENT DEFINITION
▪ Importance: AHG test is an essential POLYSPECIFIC
testing methodology for transfusion Rabbit-Polyclonal Contains anti-IgG
and anti-C3 (may
medicine
contain other
There are two major types of blood group anticomplement and
antibodies: other anti-Ig
antibodies)
▪ IgM Rabbit/Murine Contains a blend of
- Large pentameric structure monoclonal blend rabbit polyclonal
- Bind to corresponding antigen and antihuman IgG and
directly agglutinate RBCs suspended murine monoclonal
in saline anti-C3b and anti
▪ IgG C3d
- Termed nonagglutinating, or Murine monoclonal Contains murine
incomplete antibodies, because their monoclonal anti-IgG,
single monomer structure is too small anti-C3b, and anti-
C3d
to directly agglutinate sensitized RBCs
MONOSPECIFIC ANTI-IgG
NOTE: Rabbit polyclonal Contains anti-IgG
with no
▪ Adding AHG reagent containing anti-IgG anticomplement
to RBCs sensitized with IgG antibodies activity (not
allows for hemagglutination of these necessarily gamma
sensitized cells chain specific)
▪ Anti-antibody: used to observed the IgG heavy-chain Contains only Ab
formation of an Ag/Ab complex that specific reactive against
would otherwise go undetected. human gamma
chains
History of AHG test Monoclonal IgG Contains murine
monoclonal anti-IgG
1945 – Coombs and associates described the use
ANTICOMPLEMENT
of the AHG test for the detection of weak and
Anti-C3d and Anti- Contains only Ab
non-agglutinating Rh antibodies in serum. C3b reactive against the
1946 – Coombs and coworkers described the use designated
of AHG to detect in vivo sensitization of the RBCs complement
(later called the DAT) of babies suffering from Anti-C3d, Anti-C4b, Component(s), with
Anti-C4d no anti-Ig activity
HDN
MURINE MONOCLONAL
1947 – Coombs and Mourant demonstrated that Anti-C3d Contains only Ab
the antibody activity that detected Rh antibodies reactive against the
was associated with the anti-gamma globulin designated
fraction in the reagents complement
Anti-C3b, Anti-C3d Component, with no
1951 – Dacie presented the first indication that anti-Ig activity
there might be another antibody activity present
that influence the final reaction
Polyclonal AHG
1957 – Dacie and coworkers published data
showing that the reactivity of AHG to cells ▪ Contain Ab to human IgG and to the C3d
sensitized with warm antibodies resulted from component of human complement.
 ▪ Other anticomplement antibodies: anti- - Monospecific anticomplement
C3b reagents: often blend of
▪ Can be made using polyclonal or monoclonal anti-C3b and C3d
monoclonal Ab. - Anticomplement activity in AHG to
▪ Can facilitate agglutination when RBCs allow the IAT to detect Ab, a
have been sensitized with IgG or C3d or polyspecific reagent was
both introduced.
▪ Commercially prepared polyspecific
ANTIBODIES CAPABLE OF BINDING
AHG: contains little, if any, activity against
COMPLEMENT
IgA and IgM heavy chains
MOST SOME RARE
▪ Polyspecific mixture: contain Ab activity a
ABO Xg D
to kappa and lambda light chains Le a LKE P1
common to all Ig classes, thus reacting Le b Lan Lua
with IgA or IgM molecules. Jk a Lub
Jkb Kell
Monospecfic AHG
Sc1 Fya
▪ Contains only one Ab specificity: either Co3 Fyb
anti-IgG or Ab to specific components of Ge2 Coa
complement, such as C3b or C3d Ge3 Cob
▪ Licensed monospecific AHG reagents in li Dia
common use are anti-IgG and anti-C3b,- P S
C3d P1PK s
▪ Anti-IgG Vel Yta
- labeled anti-IgG contain no
anticomplement activity. Preparation of AHG
- Contain Ab specific for the Fc
fragment of the gamma heavy ▪ Classic method of AHG production
chain of the IgG involves injecting human serum or
- Mixture of IgG1 and IgG3 purified globulin into rabbits
subclasses ▪ Human globulin: behaves as foreign Ag,
- Rarely, nonagglutinating IgM Ab the rabbit’s immune response is
may be found triggered, and an Ab to human globulin is
- Anti-Pr: the only RBC produced
alloantibodies that have been ▪ FOR EXAMPLE: human IgG injected into a
reported as being solely IgA. rabbit result in anti-IgG production;
- If not labeled “gamma heavy human complement components
chain-specific,” anti-IgG may injected into a rabbit result in
contain anti-light chain specificity anticomplement (produces polyclonal
and may therefore react with cells AHG serum)
sensitized with IgM, IgA, and IgG ▪ Polyclonal Ab: mixture of Ab from
- Human IgG classes: IgG1, IgG2, different plasma cell clones. The resulting
IgG3, IgG4 polyclonal Ab recognize different
- Gamma-clone anti-IgG: is the only antigenic determinants (epitopes), or the
FDA-licensed monoclonal IgG AHG same portion of the antigen but with
in the US; detects IgG1, IgG2, and different affinities.
IgG3, but as described in the ▪ Monoclonal Ab: derived from one clone of
package insert, it fails to react with plasma cells and recognize a single
IgG4 epitope.
▪ Anticomplement reagents
- Anti-C3b,-C3d reagents, are
reactive against only the
designated complement
components and contain no
activity against human Ig
Polyclonal AHG Production Preparation of monoclonal AHG reagents.
The production of Ab is longer lasting than the
polyclonal source, as the hybridoma can live
indefinitely. A monoclonal blend may be
manufactured by blending monoclonal anti-
C3b, monoclonal anti-C3d, and monoclonal
anti-IgG. Monospecific AHG reagents can be
manufactured by conventional or hybridoma
technology.

Use of Polyspecific vs. Monospecific AHG

in the IAT
▪ The most important function of
polyspecifc AHG is the detection of IgG
Ab.

Principle of the AHG test

The AHG test is based on the following simple
principles:
Preparation of polyclonal AHG reagents. ▪ Ab molecules and complement
Pooled donor antigen allows for a broader components are globulins
spectrum of reactivity, but the source of Ab is ▪ Injecting an animal with human globulin
limited to the life span of the inoculated animal. stimulates the animal to produce Ab to
Polyspecific antihuman globulin may be the foreign protein (i.e., AHG). Serologic
manufactured by combining polyclonal anti-IgG tests employ a variety of AHG reagents
with either polyclonal or monoclonal reactive with various human globulins,
anticomplement components. including anti-IgG antibody to the C3d
Monoclonal AHG Production component of human complement, and
polyspecific reagents that contain both
▪ Devised by Kohler and Milstein has been anti-IgG and anti-C3d activity.
used to produce AHG and has proved
NOTE: AHG reacts with human globulin
particularly useful in producing high-titer
molecules, either bound to RBCs or free in serum.
Ab with well-defined specificities to IgG
Washed RBCs coated with human globulin are
and to the fragments of C3
agglutinated by AHG.
The AHG Test ▪ In transfusion reaction work-up, DAT may
demonstrate IgG or C3d, or both,
A. IAT, with the sensitization occurring in
depending on the nature and specificity of
vitro, looking for unknown Ab in px
the px Ab.
serum/plasma. (Note: if the IAT was
identifying RBC Ag, the RBCs would be
▪ In the investigation of HDN, testing for
from the px and the Ab would be the
complement proteins is not necessary in
known reagent).
as much as the protein sensitizing the
newborn RBCs is presumed to be
B. DAT with sensitization already occurring
maternal IgG. Problems can arise in
in vivo (NOTE: the polyspecifc AHG
accurate D typing in the case of a
reagent is mixture of anti-IgG and anti-
newborn with a positive DAT.
C3d)

Direct AHG Test ▪ If the DAT is positive due to IgG and the
immediate spin for D typing is negative, a
Principle and Application test for weak D cannot be performed. The
same is true for px with AIHA due to warm
▪ DAT: detects in vivo sensitization of RBCs
with IgG or complement components. IgG Ab coating px cells. The Ab must be
▪ Clinical conditions that can result in in removed from the RBCs for accurate
phenotyping. Other techniques can be
vivo coating of RBCs with antibody or
used to remove Ab from px RBCs. These
complement are the following:
includes the following:
- HDFN
- HTR - Chloroquine diphosphate
- AIHA - EDTA-glycerine
- Murine monoclonal antibodies
DIRECT ANTIGLOBULIN TEST
Clinical Application In Vivo Sensitization DAT Panel: Patterns of Reactivity in
HDFN Maternal Ab coating Autoimmune Hemolytic Anemia
fetal RBCs Anti-IgG Anti-C3d Type of AIHA
HTR Recipient Ab coating + + WAIHA
donor RBCs + - WAIHA
AIHA Autoantibody coating - + CAS; PCH,
individual’s RBCs WAIHA
▪ DAT is not required test in routine + + Mixed-type
AIHA (cold
pretransfusion protocols.
and warm)
DAT Panel
▪ Initial DATs include testing one drop of 3% Evaluation of a Positive DAT
to 5% suspension of washed RBCs with
▪ Clinical consideration should dictate the
polyspecific (anti-IgG, anti-C3d) reagent.
extent to which a positive DAT is
evaluated.
▪ (+) results are monitored by a DAT panel
using monospecific anti-IgG and anti-C3d
▪ Interpreting the significance of a positive
to determine the specific type of protein
DAT requires knowledge of the px
sensitizing the cells.
diagnosis, drug therapy, and recent
transfusion history.
▪ The saline control serves to detects
spontaneous agglutination of cells or rxn
▪ A positive DAT may occur without clinical
occurring without the addition of AHG
manifestations of immune-mediated
reagents.
hemolysis. (TABLE 5-5)
▪ In warm IAHA, including drug-induced
▪ The AABB Technical Manual states that “a
hemolytic anemia, the RBCs may be
positive DAT result alone is not diagnostic
coated with IgG or C3d, or both.
of hemolytic anemia. Understanding the
 ▪ significance of this positive result requires blood products or components
knowledge of the px diagnosis; recent containing ABO-incompatible
drug, pregnancy, transfusion, and plasma?
hematopoietic transplantation history; 3. Does the px serum contain
and the presence of acquired or unexpected Ab?
unexplained hemolytic anemia. 4. Is the px receiving any drugs?
5. Is the px receiving antilymphocyte
▪ Answering the following questions before globulin or antithymocyte
investigating a positive DAT for px other globulin?
than neonates will help determine what 6. Is the px receiving intravenous
further testing is appropriate: immune globulin (IVIG) or
1. Is there evidence of in vivo intravenous Rh immune globulin
hemolysis? (IV RhIG)?
2. Has the px been transfused 7. Has the px received a marrow or
recently? If so, did the px receive other organ transplant?

TABLE 5-5 IN VIVO PHENOMENA ASSOCIATED WITH A POSITIVE DAT

CONDITION CAUSE
Transfusion 1. Recipient alloantibody and Alloantibodies in the recipient of
donor antigen a recent transfusion that react
with Ag on donor RBCs
2. Donor antibody and Antibodies present in donor
recipient antigen plasma that react with Ag on a
transfusion recipient’s RBCs
Drug induced 1. Type I (hapten-dependent Drug binds covalently to
Ab) membrane proteins and
stimulates hapten-dependent
Ab
2. Type II (autoantibody) Drug induces autoantibody
specific for RBC membrane
proteins through unknown
mechanism; Ab reacts with
normal RBCs in the absence of
drug.
3. Type III (drug-dependent Drug induces Ab that binds to
Ab) RBC only when drug is present
in soluble form, unknown
mechanism, Ab reacts with
normal RBCs when soluble drug
is present.
Autoimmune Hemolytic Anemia 1. WAIHA (IgG and/or C3) Autoantibody reacts with px
RBCs in vivo
2. CAS (C3) Cold-reactive IgM
autoagglutination binds to RBCs
in peripheral circulation (32C).
IgM binds complement as RBCs
return to warmer parts of
circulation; IgM dissociates,
leaving RBCs coated only with
complement.
3. PCH (IgG) The IgG autoantibody reacts
with RBCs in colder parts of
body, cause complement to be
bound irreversibly to RBCs, and
then elutes at warmer
temperature.
Hemolytic disease of fetus and Maternal alloantibody crosses Maternal (IgG) alloantibody,
newborn placenta (IgG) specific for fetal Ag, coats fetal
 RBCs, DAT is reactive with the
anti-IgG.
Miscellaneous 1. Absorbed proteins; Heterophile antibodies that are
administrations of present in ALG or ATG coat
antilymphocyte globulin recipient’s RBCs. High levels of
(ALG) and antithymocyte protein causing RBCs to
globulin (ATG) spontaneously agglutinate.
2. Administration of high- Nonantibody-mediated binding
dose IV gamma globulin of immunoglobulin to RBCs in px
and with hypergammaglobulinemia
hypergammaglobulinemia

Indirect AHG Test Add AHG reagent Forms RBC

agglutinates (RBC Ag
The IAT is performed to determine in vitro + Ab + anti-IgG)
sensitization of RBCs and is used in the following Centrifuge Accelerates
situations: agglutination by
bringing cells closer
▪ Detection of incomplete
together
(nonagglutinating) Ab to potential donor Examine for Interprets test as (+)
RBCs (compatibility testing) or to agglutination or (-)
screening cells (antibody screen) in Grade agglutination Determines the
serum reactions strength or rxn
▪ Determination of RBC phenotype using Add Ab-coated RBCs Checks for
known antisera (e.g., weak D, any other to (-) reactions neutralization of
Ag testing that requires IAT) antisera by free
▪ Titration of incomplete Ab. globulin molecules
(Coombs’ control
INDIRECT AHG TEST cells are D-positive
Application Tests In Vitro RBCs coated with
Sensitization anti-D)
Ab detection Compatibility Recipient Ab
testing reacting with
donor cells NOTE:
Ab screening Ab reacting
▪ The DAT can detect a level of 100 to 500
with
IgG molecules per RBC and 400 to 1,100
screening
cells molecules of C3d per RBC
Ab ID Ab panel Ab reacting ▪ For IAT, there must be between 100 and
with panel 200 IgG or C3 molecules on the cell to
cells obtain a positive rxn.
Ab titration Rh Ab titer Ab and
selected Rh
Factors Affecting the AHG Test
cells A. Ratio of serum to cells
RBC RBC Ag Specific - Increasing the ratio of serum to
phenotype detection (ex: antisera + cells increases the sensitivity of the
weak D, K, Fy) RBCs to
test system
detect Ag
- A minimum ration can be
achieved by using 2 drops of serum
TASKS AND PURPOSES OF THE IAT TEST and 1 drop of a 5% of vol. of solute
TASK PURPOSE per vol. of sol’n (v/v) suspension of
Incubate RBCs with Allows time for Ab cells.
antisera molecule attachment B. Reaction medium
to RBC Ag
Perform a minimum Removes free globulin B.1. Albumin:
of 3 saline washes molecules - Macromolecules of albumin allow
Ab-coated cells to come into closer
 contact with each other so that detection of clinically insignificant
agglutination occurs Ab
- Increased sensitivity of the IAT if - Barrett and associates reported
albumin was incorporated into the that since PEG has been used pre-
rxn medium (Stroup and MacIlroy) transfusion Ab screening.
- Reaction mixture: 2 drops of C. Temperature
serum, 2 drops of 22% bovine - Rate of rxn for the majority of IgG
albumin (w/v), and 1 drop of 3% to Ab is optimal at 37C
5% (v/v) cells - Also the optimum temperature for
- Petz and coworkers: albumin complement activation
technique may miss several D. Incubation time
clinically significant Ab. - Cells suspended in saline: 30-120
minutes
B.2. Low Ionic Strength Solutions
- LISS or PEG: 10-15 minutes
- Enhances Ab uptake and allow ▪ Temperature: 37C
incubation times to be decreased- ▪ Extended incubation in the
from 30 to 60 mins. to 10-15 mins.- LISS technique has been
by reducing the zeta potential shown to cause Ab to elute
surrounding the RBC. from the RBCs, decreasing
- Introduced by Low and Messeter: the sensitivity of the test
serum-to-cell ratio (CONFIRMED BY Voak and
- 2 drops of serum and 2 drops of a coworkers)
3% (v/v) suspension of cells in LISS E. Washing of RBCs
(Moore and Mollison) - RBCs must be saline-washed a
- Increasing the serum-to-cell ratio minimum of 3 times before adding
increased the ionic strength of the AHG reagent.
rxn mixture, leading to decrease in - Removes free unbound serum
sensitivity and counteracting the globulins
shortened incubation time of the - Inadequate washing results to
test. false-negative rxn because of
- LISS medium may be achieved by neutralization of the AHG rgt by
either suspending RBCs in LISS or residual unbound serum globulin
using a LISS additive rgt., with the - MOST IMPORTANT STEPS IN
latter being the more common TESTING
practice. - Can be controlled using check
cells, or group O cells sensitized
B.3. Polyethylene Glycol
with IgG.
- Is a water-soluble linear polymer - Residual saline dilutes the AHG rgt
and is used as an additive to and therefore decreased
increase Ab uptake. sensitivity of the test.
- Remove water molecules F. Saline for washing
surrounding the RBC (the water of - Should be fresh (expiration of 30
hydration theory), thereby days) and buffered to a pH of 7.2 to
effectively concentrating Ab. 7.4
- Anti-IgG is the AHG rgt of choice - If stored in a long period of time it
with PEG testing to avoid false- decreases the pH, which may
positive rxn. increase the rate of Ab elution
- PEG may cause aggregation of during the washing process,
RBCs, reading for agglutination yielding a false-negative result.
following 37C incubation in the IAT - Bacterial contamination: false-
is omitted. positive results
- Findings indicated that PEG G. Addition of AHG
increases the detection of clinically - Should be add to the cells
significant Ab while decreasing immediately to minimize the
chance of Ab eluting from the cell
 - and subsequently neutralizing the Sources of Error
AHG rgt
- Voak and associates: adding two ▪ EDTA should be used to collect blood
volumes of AHG may overcome samples for DAT to avoid the in vitro
washing problems when low levels complement attachment associated with
of serum contamination remain. refrigerated clotted spx.
- Residual serum does not contain ▪ All negative AHG test rxn must be
C3b and C3d to neutralize the checked by the addition of IgG-sensitized
anti-C3b and anti-C3d in the AHG cells
rgt. ▪ Adding IgG-coated RBCs to negative test
H. Centrifugation for reading rxn should demonstrate
- Crucial step in the technique hemagglutination of the RBCs with the
- CBER recommended method for anti-IgG in the AHG rgt.
the evaluation of AHG used 1,000 ▪ If no hemagglutination, result is invalid
RCFs for 20 sec (although it and the test must be repeated.
suggests 500 RCFs for 15-20 sec). ▪ Most common technical errors that result
- Higher RCFs yields more sensitive in failure to demonstrate
results hemagglutination:
- False-positive: inadequate a. Inadequate washing
resuspension b. Nonreactive AHG rgt
- Negative result: if resuspension is c. Failure to add AHG rgt
too vigorous ▪ Monospecific anti-C3d reactivity may be
verified with the addition of C3d-coated
RBCs to negative rxn.

Modified and Automated AHG Test ▪ Several different techniques have been
Techniques reported using either test tubes or
microplates.
A. Solid-Phase Technology ▪ Direct and indirect tests can be
▪ May be used for performing AHG tests. performed using solid-phase
methodology
 ▪ If Ab is specific for Ag on RBCs, the bottom ▪ If this is performed, a monospecific anti-
of the well will be covered with suspension IgG rgt must be used, because the low
▪ If no such specificity occurs, RBCs will ionic conditions cause considerable
settle to the bottom of the well. Known amounts of C3 and C4 to coat the cells
RBCs are bound to a well that has been and would give false-positive rxn if a
treated with glutaraldehyde or poly L- polyspecific rgt were used
lysine. ▪ It should be mentioned that the polybrene
has a low sensitivity to detection of anti-
B. Gel Test
Jka anf Jkb Ag
▪ Is a process that detects RBC Ag-Ab rxn ▪ The potential exists for a clinically
by means of a chamber filled with significant anti-Kidd Ab to be missed
polyacrylamide gel using the polybrene method of
▪ The gel acts as a trap; free unagglutinated enhancement for IAT
RBCs form buttons in the bottom of the
tube for hours D. Enzyme Linked-Antiglobulin Test (ELAT)
▪ Therefore, negative rxn appear as buttons ▪ An RBC suspension is added to
in the bottom of the microtube, and microliter well and washed with saline.
positive rxn are fixed in the gel AHG, which has been labeled with an
▪ Three different types of gel test: enzyme, is added. The enzyme-
1. Neutral: Ab screening and labeled AHG will be of IgG-sensitized
identification with enzyme-treated RBCs
or untreated RBCs and reverse ▪ Excess Ab is removed, and enzyme
ABO typing. substrate is added. The amount of
2. Specific: use a specific rgt color produced is measured
incorporated into the gel and are spectrophotometrically and is
useful for antigen determination. proportional to the amount of Ab rgt.
3. Antiglobulin: the Gel Low Ionic ▪ The optical density is usually
Antiglobulin Test (GLIAT) is a measured at 405nm. The number of
valuable of the gel test and may be IgG molecules per RBC can also be
used for the IAT or DAT. AHG is determined from this procedure.
incorporated into the gel. The
detection of unexpected Ab by
Comparison of AHG Methodologies
GLIAT compares favorably with COMPARISON OF AHG METHODOLOGIES
conventional AHG methods and Testing Advantages Disadvantages
provides a safe, reliable, and easy- Methodology
to-read AHG test. Saline-tube -No -Long
testing additives incubation
C. Low Ionic Polybrene Technique
▪ Lalezari and Jiang: adaption of the -Reduced -Least sensitive
automated LIP technique for use as a cost
manual procedure -Requires highly
-Avoids trained staff
▪ Relies on low ionic conditions to rapidly-
reactivity
sensitized cells with Ab.
with auto -Most
- Polybrene: a potent rouleaux- Abs procedural
formation rgt, is added to allow the steps
sensitized cells to approach each -Ability to
other and permit cross-linking by assess -Fewer
the attached Ab. multiple method-
- A high ionic strength is then added phases of dependent Abs
to reverse the rouleaux, however if reactivity detected
agglutination is present, it will LISS-tube -Reduced -Inability to be
remain testing cost automated
▪ Can be carried through to an AHG
technique if required -Avoids -Requires highly
reactivity trained staff
 with auto -Ability to
Abs -Many be
procedural automated
-Shortest steps Solid phase -No need for -Increased
incubation check cells sensitivity for all
time -Fewer Abs
method- -Stable
-increased dependent Abs endpoints -Detects
Ab uptake detected unwanted Abs
-Small test
-Most vol. -Warm auto
common Abs enhanced
tube -Enhanced
method anti-D -Increased
costs
-Ability to -Increased
assess sensitivity -Increased
multiple for all Abs need for
phases of additional
reactivity -Ability to instumentation
PEG-tube -Reduced -Requires highly be
testing cost trained staff automated

-Decreased -Many
incubation procedural
time steps

-Increased -Detects
Ab uptake unwanted Abs

-Enhances -Inability to be
most Abs automated

-Abilitiy to -Fewer
assess method-
multiple dependent Abs
phases of detected
reactivity
(not 37C)
Gel -More -Warm auto
sensitive Abs enhanced
DAT method
-Mixed-cell
-No washing agglutination
steps with cold Abs

-No need for -Increased

check cells costs

-Stable -Increased
endpoints need for
additional
-Small test instrumentation
vol.
-Increased
-Enhanced chances of
anti-D detected
detection unwanted Abs
 Hemolytic Disease of the Fetus and always limited to A or B infants of group O
mothers with potent anti-A,B Abs.
Newborn ▪ ABO HDFN can occur in the first
pregnancy and in any, but not necessarily
▪ Destruction of the RBCs of a fetus and
all, subsequent pregnancies because it
neonate by antibodies produced by the
does not depend on previous foreign RBC
mother.
stimulation
▪ The mother can be stimulated to form
▪ Tetanus toxoid administration and
RBC Ab naturally (ABO), by previous
helminth parasite infection: high-titered
pregnancy, or transfusion (RBC
IgG ABO Abs and severe HDFN
alloimmunization).
▪ Mild course of ABO HDFN: poor
▪ RhD continues to remain an important
development of ABO Ags on fetal RBCs
cause of incompatibility, although its
▪ Group A infant RBCs are serologically
frequency has been equaled or surpassed
more similar to A2 adult cells, with group
by other RBC Ab specificities.
A2 infant RBCs much weaker.
▪ Initial diagnosis of maternal RBC
- The weakened A Ag on fetal and
alloimmunization is SEROLOGIC.
neonatal RBCs is more readily
Etiology demonstrable with human than
with monoclonal anti-A rgt.
▪ Levine and Stetson: reported a
transfusion rxn from transfusing COMPARISON OF ABO VS. RhD HDFN
husband’s blood to a postpartum woman Characteristics ABO RhD
- Postulated that the mother has First Yes Rare
pregnancy can
been immunized to the father’s Ag
be affected
through fetomaternal
Disease No Yes
hemorrhage.
predicted by
▪ Most maternal alloimmunization: titers
- 83% due to previous pregnancy Causative Ab Yes (anti- Yes (anti-D,
- 4% due to previous transfusion IgG A,B) etc.)
- 14% unable to be determined Bilirubin level Normal Elevated
▪ Only Ab of the IgG class are actively at birth range
transported across the placenta via Fc Intrauterine None Sometimes
receptors. transfusion
- Most IgG Abs are directed against needed
bacterial, fungal, and viral Anemia at No Yes
antigens. birth
▪ In the case of HDFN, the Abs are directed Phototherapy Yes Yes
against the blood group Ags on the fetal beneficial
RBCs that were inherited from the father. Exchange Rare Uncommon
transfusion
Pathogenesis needed

A. HDFN Caused by ABO

▪ ABO incompatibility has become the ▪ Characteristics: microspherocytes and
most common cause of HDFN. increase RBC fragility.
▪ Maternal ABO Abs that are IgG can cross ▪ ABO HDFN causes a bilirubin peak at 1 to
placenta and attach to the ABO Ags of the 3 days, w/c is later than with HDFN caused
fetal RBCs by other Ab specificities.
▪ ABO Abs are present in the plasma of all - Phototherapy is usually sufficient
individuals whose RBCs lack the for slowly rising bilirubin levels.
corresponding Ag. - IVIG or exchange transfusion with
▪ This Abs, also called isohemagglutinins, group O RBCs: when there are
result from environmental stimulus in rapidly increasing bilirubin levels.
early life. ▪ Stillbirth hydrops fetalis and kernicterus:
▪ Group O: most likely to form high-titered rare in ABO induced HDFN
IgG anti-ABO Abs, ABO HDFN is nearly
 B. HDFN Caused by RBC Alloimmunization B.2. Maternal immune system factors
Factors affect maternal RBC alloimmunization ▪ In Rh-negative individuals who are
and severity of HDFN: transfused with 200 mL of RhD-positive
RBCs, approximately 85% respond and
B.1. Antigenic exposure
form anti-D.
▪ As little 1 mL of fetal RBC can immunize
the mother (usually happens when the ▪ If RhiG is not administered to an RhD-
placental separates from the uterus when negative mother, the risk of immunization
the mother is delivering the fetus; small is only about 16% after an RhD-positive
amount of fetal cell can escape from the pregnancy
placenta to the uterus thus stimulating
maternal circulation in producing an Ab ▪ Ig classes and subclasses of the maternal
(anti-D). Ab affects the severity of HDFN: IgG, IgM,
IgA, IgE, and IgD
▪ For this reason, the first-born infants - ONLY IgG IS TRANSPORTED TO
always unaffected in cases of Rh HDFN, PLACENTA.
however the second child is most likely to
be affected. ▪ Active transport of IgG begins in the 2nd
trimester and continues until birth.
▪ Fetomaternal Hemorrhage: previous
pregnancy with FMH is the leading cause ▪ IgG molecules are transported via the Fc
of maternal alloimmunization. portion of the Abs.
- Interventions such as
amniocentesis and chorionic villus ▪ IgG1 and IgG3: more efficient in RBC
sampling and trauma to the intravascular hemolysis.
abdomen can increase the risk of
B.3. Antibody specificity
FMH.
▪ Of all the RBC Ag, RhD is the most
antigenic

▪ For this reason, only Rh-negative blood is

transfused to a Rh-negative woman of
child bearing age

▪ Rh system (C, E, and c): also potent

immunogens and have been associated
with moderate to severe cases of HDFN

▪ Anti-E and anti-c: caused severe HDFN

that required intervention and treatment

▪ Anti-Kell: considered the most clinically

significant in its ability to cause HDFN
- Kell blood groups are present on
immature erythroid cells in the
fetal bone marrow
 ANTOBODIES INDENTIFIED IN PRENATAL SPX Anti-C Anti-N Anti-IH
THAT CAUSE OF HDFN
Anti-E Anti-S Anti-P1
Common Rare Never
Anti-c Anti-Jka
Anti-D Anti-Fya Anti-Lea
Anti-e
Anti-D+C Anti-s Anti-Leb
Anti-K
Anti-D+E Anti-M Anti-I

B.4. Influence of the ABO group ▪ When bone marrow fails to produce
enough RBCs to keep up with the rate of
▪ When the mother is ABO-incompatible
RBC destruction, erythropoiesis outside
with the fetus (major incompatibility), the
the bone marrow is increased in the
incidence of detectable fetomaternal
hematopoietic tissues of the fetal spleen
hemorrhage is decreased.
and liver.
Hemolysis, Anemia, and ▪ Organs become enlarged
(hepatosplenomegaly), resulting in portal
Erythropoiesis hypertension and hepatocellular
A. Hemolysis damage.
▪ Occurs when the maternal IgG attaches
to specific Ags of the fetal RBCs. B. Anemia
▪ The Ab-coated cells are removed from ▪ Severe anemia and hypoproteinemia
the circulation by the macrophages of the caused by decreased hepatic production
fetal spleen. of plasma proteins leads to the
▪ Destruction of fetal RBCs and the resulting development of high-output cardiac
anemia stimulate the fetal bone marrow failure with generalized edema, effusions,
to produce RBCs at an accelerated rate, and ascites, a condition known as
even to the point that immature RBCs are hydrops fetalis.
released into the circulation hence - Develop by 18-20 weeks’ gestation
causing erythroblastosis fetalis.
NEONATAL MANIFESTATIONS OF HDFN-INDUCED ANEMIA BY TIME OF ONSET
Early-Onset Anemia Late Hemolytic Anemia Late Hyporegenerative
Anemia
Onset Within 7 days of birth >2 weeks of age
Mechanism Ab-mediated 1.Ab-mediated 1.Ab destruction of RBC
hemolysis hemolysis precursors and RBCs

2. Natural decline of Hb 2. Marrow suppressions

levels by IUT and transfusions

3.Expanding 3.Erythropoietin
intravascular vol. of deficiency
growing infant
4.Expanding
intravascular vol. of
growing infant

Bilirubin Elevated Usually elevated Normal

Reticulocyte count Normal or high Normal or high Low or absent

NOTE: maternal Ab entering the infant’s

circulation through the placenta.
▪ The rate of destruction after birth
decreases because no additional
 - However, IgG is distributed both for several days to weeks after
extravascularly and delivery.
intravascularly and has a half-life
of 25 days, so Ab binding and
hemolysis of RBCs can continue

C. Bilirubin B. HDFN Caused by RBC Alloimmunization

▪ RBC destruction releases hbg, which is
metabolized to bilirubin in different
metabolic stages.
▪ Indirect bilirubin: is unconjugated and
does not dissolve in water. (Travels
through the bloodstream to the liver)
▪ Direct bilirubin: conjugated and rendered
water soluble. (Excreted through the GIT)
▪ During pregnancy, the indirect bilirubin
made by the fetus is transported across
the placenta and conjugated by the
maternal liver and safely excreted.
▪ After birth, the immature infant liver
cannot yet metabolize bilirubin
efficiently, and this leads to the
accumulation of unconjugated bilirubin B.1. ABO, RhD, and Ab Screen
and neonatal jaundice.
▪ When newborn infant affected with ▪ Prenatal spx must be typed for ABO and
HDFN: high levels of indirect bilirubin due RhD
to the pathologic RBC destruction. ▪ Ab detection method, or IAT, must be able
▪ Kernicterus: indirect bilirubin reaches to to detect clinically significant IgG
18-20 mg/dL alloantibodies that are reactive at 37C
and in the antiglobulin phase.
Diagnosis ▪ At least 2 separate reagents are used.
▪ For tube testing, an Ab-enhancing
A. HDFN Caused by ABO medium such as polyethylene glycol or
A.1. Postnatal Diagnosis LISS can increase sensitivity of the assay.
▪ Prenatal px may produce clinically
▪ No single serologic test is diagnostic for insignificant Abs, such as anti-Lea or anti-
ABO HDFN. Leb.
▪ DAT on the cord or neonatal RBC: most - Anti-IgG rgt is used.
important diagnostic test. - Immediate spin and room
- (+) to DAT: 100% ABO HDFN temperature incubation phases
requiring transfusion therapy can be omitted.
- (+) to DAT 90% with jaundice
- (+) absence of signs and B.2. Ab Identification
symptoms of clinical anemia in the ▪ Cold reactive IgM Abs can be ignored
newborn infant. (anti-I, anti-IH, anti-Lea, anti-Leb, and
▪ Collecting cord blood samples on all anti-P1
delivered infants is highly recommended. ▪ Lewis system Ab rather common in
- Collected by venipuncture (to pregnant women than in HDFN
avoid contamination and ▪ Anti-M and anti-N can cause mild to
Wharton’s jelly) moderate HDFN
- Should be anticoagulated for ▪ To establish the Ig class, the serum can be
storage. treated with sulfhydryl rgt, such as
dithiothreitol or 2-mercaptoethanol, and
the retested with appropriate controls.
 - J-chain of IgM will destroy by this ▪ Heterozygous: 50% chance.
treatment while IgG will remain ▪ If the mother has anti-D and the father is
reactive. RhD-positive, the paternal genotype
▪ A titer higher than 4 almost always determined by DNA methods is the only
indicates active immunization way to definitely determine how many
▪ Most common and most significant Abs: copies of the RHD gene and RhD-positive
anti-D, anti-K, anti-E, anti-c, anti-C, and person carriers.
anti-Fya ▪ Recommended test: RHD zygosity
genotype testing.
B.3. Ab Titers
B.5. Fetal DNA testing
▪ The relative concentration of all Abs
capable of crossing placenta and causing ▪ Risk stratification of the fetus can be
HDFN is determined by Ab titration. directly carried out by obtaining fetal cells
▪ The px serum or plasma is serially diluted through amniocentesis or chorionic
and tested against appropriate RBCs to villous sampling as early as 10 to 12 weeks’
determine the highest dilution at which a gestation.
reaction occurs. ▪ The fetal cells are grown in tissue cultures,
▪ Method use: IAT using anti-IgG rgt and then DNA is extracted to determine if
▪ Result is expressed by either the the fetus has genes coding for the blood
reciprocal of the titration endpoint or as group genes, including RHD and others,
titer score. (c, e, C, E, K, Fya, Fyb, Jka, Jkb, M)
▪ Recommended method: saline ▪ Fetal DNA can be isolated from maternal
antiglobulin tube test, with 60 mins. plasma from a PB sample to determine
Incubation at 37C and the use of the anti- RHD and KEL genotype.
IgG rgt. - Cell-free DNA method has
▪ RBCs used should have the same advanced fetal risk stratification
genotype, same storage time and for mothers with RBC
concentration. alloimmunization.
▪ First serum or plasma spx should be - Also used when the mother is RhD-
frozen and run in parallel. negative to determine if the fetus is
▪ Only a difference of >2 dilutions or a score predicted to be RhD-positive and
change of more than 10 is considered a therefore if RhIG should be
significant change in titer. administered.
▪ Considered critical titer: 16
- If the initial titer is 16 or >, a second Management of the Fetus
titer should be done at about 18 to A. Fetal Ultrasound
20 weeks’ gestation ▪ At about 16-20 weeks’ gestation,
▪ A titer reproducibly and repeatedly at >32 the clinical diagnosis of fetal
is an indication for color Doppler imaging anemia can be made using an
to assess middle cerebral artery peak ultrasound technique called fetal
systolic velocity after 16 weeks’ gestation middle cerebral artery peak
- <32, it should be repeated at 4- systolic velocity.
week intervals, beginning at 16-20 ▪ Measurement is based on the
weeks’ gestation and then every 2- reduced blood viscosity at lower
4 weeks during third trimester. hct and resulting in faster velocity
B.4. Paternal Phenotype and Genotype of the blood.
▪ Reading are done every 2 weeks to
▪ A spx of the father’s blood should be track the degree of fetal anemia;
obtained and tested for the presence and >1.5 MoM are sensitive enough to
zygosity (predict copy number of the predict significant fetal anemia in
gene) of the corresponding blood group which intervention may be
Ag to predict fetal risk of being affected by needed.
HDFN.
▪ Homozygous: 100% chance of passing
this gene to their offspring.
 Interpretation:
▪ Zone 1: mild or no disease
▪ Zone 2: moderate disease, require
intervention
▪ Zone 3: severe & often life-threatening
hemolysis, require intervention
B. Invasive Monitoring-Cordocentesis and
Amniocentesis ▪ If the fetus has not reached an acceptable
▪ Amniocentesis: fetal RBC can be gestational age for delivery, and the hct
tested for hbg, hct, blood type and level is <30%, intrauterine transfusion is
DAT usually indicated.
▪ Cordocentesis: umbilical vein is
visualized at the level of cord C. Intrauterine Transfusion
insertion into the placenta ▪ Intervention in the form of
- A needle is inserted into the intrauterine transfusion becomes
umbilical vessel and necessary when one or more of the
sample of the fetal blood is following conditions exists:
obtained a. MCA-PSV indicates anemia
▪ When fetal anemia becomes (>1.5 MoM)
moderate to severe as indicated b. Fetal hydrops is noted on
by Doppler MoM measurement ultrasound examination
exceeding 1.5, invasive testing via c. Cordocentesis blood
cordocentesis is done to determine sample has hbg level <10
fetal hct. g/dL
▪ For risk stratification of fetal d. Amniotic fluid optical
anemia, amniocentesis is to density 405 nm results are
monitor amniotic fluid bilirubin high and/or increasing.
levels has been replaced with ▪ Performed by accessing the fetal
MCA-PSV. umbilical vein and injecting donor
- amniotic fluid is tested by a RBCs directly into the vein.
spectrophotometric scan ▪ GOAL: maintain fetal hbg >10 g/dL
optical density at 405 nm. ▪ Initial intrauterine transfusion is
- Measurement is plotted on rarely performed after 36 weeks’
a Liley Curve Graph gestation.
according to gestational ▪ Blood cell products: Group O, RhD-
age. negative/RhD-positive, leukocyte
- Increasing or unchanging reduced, HgbS negative, CMV-
optical density at 405 nm safe, irradiated, and Ag-negative
as pregnancy proceeds for maternal RBC Abs.
predicts worsening of the
hemolysis (fetal hbg <8
g/dL) and require urgent
intervention.
Management of the Infant safe, irradiated, and Ag-negative
for maternal RBC Abs.
A. Cord Blood Testing ▪ RBCs units <7-10 days reduces the
▪ Serologic testing of the cord blood risk of hyperkalemia.
sample drawn at the time of birth ▪ Older blood units can be safe and
is used to confirm HDFN and effective for the newborn when
prepare for possible transfusion. infused slowly.
A.1. ABO Grouping - 90% of RBCs have been
replaced
▪ ABO Ag are not fully developed in - 50% bilirubin has been
newborn infants; their RBCs may show removed.
weaker rxn with Anti-A and anti-B
antisera than for older children and C. Simple Transfusions
adults. ▪ The infant may receive small-
▪ Infants do not have their own volume or “top-off” RBC
isohemagglutinins but may have those of transfusions to correct anemia
the mother. anytime from after birth to many
- Reverse typing cannot be used to weeks later.
confirm the ABO group. ▪ Hbg and Hct levels has been the
subject of a number of studies.
A.2. RhD Typing
▪ 1 unit dedicated to an infant with
▪ Rarely, the infant’s RBCs can be heavily HDFN and draw small aliquots
Ab-bound with maternal anti-D, causing from the parent RBC unit over time
a false-negative Rh type, or what has to decrease donor exposure over
been called block Rh. multiple transfusion episodes.
▪ An eluate from these RBCs will reveal
anti-D, and typing of the eluted RBCs will D. Phototherapy
show rxn with the anti-D. ▪ 460-490 nm is used to
metabolized the unconjugated
A.3. DAT
bilirubin to isomers that are less
▪ The most important serologic test for lipophilic, less toxic to the brain,
diagnosing HDFN with anti-IgG rgt. and able to be excreted through
▪ (+): indicates that there is Ab-coating the urine.
infant’s RBCs; evidence of hemolysis (e.g., ▪ Sufficient to adequately conjugate
mother received RhIG) the bilirubin and lessen the need
for transfusion in infants with mild
A.4. Elution
to moderate hemolysis.
▪ In cases of known HDFN and postnatal
ABO incompatibility is not needed, E. Intravenous Immune Globulin
because eluate results do not change ▪ Used to treat hyperbilirubinemia of
therapy. the newborn caused by HDFN
▪ Helpful when the cause of HDFN is in ▪ Competes with mother’s Abs for
question or suspected. the Fc receptors on the
macrophages in the infant’s
B. Exchange transfusion spleen, reducing the amount of
▪ Close observation of bilirubin hemolysis.
levels and hbg is warranted to ▪ Reduce the need for exchange
determine if neonatal exchange transfusions and phototherapy.
transfusion is needed to remove
bilirubin and maternal Ab. Prevention
▪ Needed when levels of bilirubin are A. Selection of RBCs for Females
critical ▪ Number of countries use of K-
▪ Blood cell products: Group O, RhD- negative RBC units for women
negative/RhD-positive, leukocyte younger than 45 to 50 years of
reduced, HgbS negative, CMV- age.
 ▪ 83% of maternal sensitization that ▪ Microdose can be used for abortions and
went on to cause severe HDFN was ectopic pregnancies before the 12th week
due to previous pregnancy and not of gestation.
transfusion itself. ▪ Total fetal blood volume is estimated to
be <5mL at 12 weeks.
B. Rh Immune Globulin ▪ A maternal sample should be obtained
▪ RhIG should be given to RhD- within 1 hr of delivery and screened using
negative mothers. a test such as the rosette technique for
- First dose: 28 weeks’ massive fetomaternal hemorrhage.
gestation, recommended - If positive, quantitation of the
by ACOG. hemorrhage must be done by
- Second dose: given after Kleihauer-Betke or by flow
delivery of an RhD-positive cytometry assays
infant (antenetal); 18 weeks ▪ Kleihauer-Betke test: a maternal blood
of gestation (3rd trimester) smear is treated with acid and then
▪ Recommended to give RhIG within stained with counterstain.
72 hours after delivery ▪ Fetal cells contain fetal hbg, which is
(Postpartum) resistant to acid and will remain pink.
▪ Half-life of IgG is about 25 days, so ▪ The maternal cells will appear as ghost
only about 10% of the antenatal
dose will be present at 40 weeks’
gestation.

B.2. Maternal Weak D

▪ Recently, authors have encouraged the
use of RhD genetic testing for px with a
weak D phenotype to provide accurate
and actionable results for RhD blood
typing and RhIG administration.

B.1. Dose and Administration

▪ Regular dose vial of RhiIG: contains
sufficient anti-D to protect against 15 mL
of packed RBCs or 30 mL of whole blood.