Immunohematology
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VO L U M E 1 9 , N U M B E R 2 , 2 0 0 3
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Review: biochemistry of carbohydrate blood group antigens
L.G. GILLIVER AND S.M. HENRY
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Neonatal alloimmune thrombocytopenia due to anti-HPA-2b (anti-Koa)
M. GOLDMAN, E.TRUDEL, L. RICHARD, S. KHALIFE, AND G.M. SPURLL
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Quantitation of red cell–bound IgG, IgA, and IgM in patients with autoimmune hemolytic anemia and
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Review: Biochemistry of
carbohydrate blood group
antigens
L.G. GILLIVER AND S.M. HENRY
This review presents the basics of the structural
chemistry of blood group glycoconjugates, with special
reference to red cell serology. Its aim is to create an
appreciation of the inherent subtleties of the
carbohydrate blood group antigens, which are
currently poorly understood within the field of blood
transfusion. It is hoped that a better understanding of
the intricacies of the carbohydrate blood group
systems will lead to further contributions to the body
of knowledge within this growing field.
Introduction
In 1900, Karl Landsteiner discovered the
carbohydrate blood group system of ABO after mixing
the cells and serum of his colleagues. Over the next
100 years many other blood group systems were
discovered and later resolved at both the molecular and
the genetic level. It is now clearly recognized that most
blood group systems are either carbohydrate, protein,
or protein-carbohydrate in nature, although they may
associate with other components and complexes to be
recognized as specific blood group determinants.
Essentially, a blood group antigen is any polymorphic
three-dimensional conformation expressed on the
outer membrane of a RBC against which an antibody is
available. The concept of the three-dimensional shape
is very important because this is what antibodies
recognize, and the number of potential shapes that can
be found in carbohydrate chains is enormous. The
variability in characteristics such as sugar type, linkage
type, branch positioning, shape, charge, ring size, and
epimeric (pairs of monosaccharides which differ only
in the configuration about one carbon atom are
epimers of each other e.g., D-glucose and D-mannose
are epimers with respect to the C2 carbon atom) and
anomeric configuration results in more than 1.05 × 1012
possible structural combinations from a six-sugar
glycan (the largest size expected for a carbohydrate
epitope). This is compared with only 46,656
combinations possible with a chain made up of six
amino acids, more than seven orders of magnitude
lower than the diversity possible in glycans.1
Fortunately, despite the vast potential for complexity in
carbohydrate chains, there is generally only a limited
range of structures representing the carbohydrate
blood group epitopes (glycotopes), although many of
the structural differences are subtle and some epitope
surfaces may be common. These subtleties may
represent different blood group antigens and in some
cases, specific antibodies may recognize the common
surfaces. For example, anti-A,B recognizes the
similarities between A and B antigens, not the subtleties
which distinguish them.2 Unfortunately, this potential
variation makes structural determination of carbohydrate antigens complex, requiring combinations of
powerful techniques such as nuclear magnetic
resonance spectroscopy and mass spectrometry to
elucidate the structures of carbohydrate antigens
without ambiguity.3,4 Progress in determining the
structures of carbohydrate blood group antigens has
been slow, hindered by a combination of factors, such
as the fact that carbohydrate blood group antigens
are not the primary product of a gene—being rather
secondary biosynthetic products—and that the
isolation and purification of carbohydrate blood group
antigens in requisite quantities is difficult. As a result,
we know only the basic structures of the major
glycotopes and antigens expressed in the carbohydrate
blood group systems. We have not yet characterized
many of the phenotypic subtleties, such as those that
determine ABO subgroups.
Because carbohydrate blood group antigens are
important to transfusion medicine and are becoming
more recognized in transplantation (especially xeno-
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
33
L.G. GILLIVER AND S.M. HENRY
transplantation5) and studies of micoorganism binding
and interactions,6 it is becoming more important for
serologists to understand the biochemistry of
carbohydrate blood group antigens.
We presently recognize several blood group
systems and collections7 where the blood group
glycotopes are comprised entirely of carbohydrate,
such as ABO, Lewis, H, Ii, P, Sd, Cad, etc.; or others, such
as MN, where carbohydrate moieties are essential to
the structure of the epitope; or even others, where
carbohydrates are associated with the character of the
antigen—e.g., GPI-linked membrane proteins such
as Cromer, Dombrock, Cartwright, and John Milton
Hagen, etc.
This review is not intended to cover the
carbohydrate blood group systems, rather it is intended
to provide a basic understanding of carbohydrate
blood group chemistry, and in this regard we will use
ABO antigens as examples.
Monosaccharides
The common blood group–related sugars, namely
β-D-glucose (Glc), N-acetyl-β-D-glucosamine (GlcNAc),
β-D-galactose
(Gal),
N-acetyl-β-D-galactosamine
(GalNAc), α-L-fucose (Fuc), and D-mannose (Man) are all
six-carbon sugars (hexoses). The carbonyl group
(C=O) of each residue is an aldehyde (HC=O), and thus
they are all classified as aldohexoses. Another special
monosaccharide which contains nine carbons is also
common in blood group antigens, with the most
common blood group variation being N-acetylα-D-neuraminic acid (also referred to as NeuAc or sialic
acid). There are various forms of sialic acid,8,9 but these
will not be reviewed here.
Sugar molecules can have configurational isomers
which differ in their arrangement in space even though
they have identical composition. The variation in the
configuration of individual carbohydrates was first
characterized in relation to the smallest chiral
carbohydrate, glyceraldehyde, by German chemist Emil
Fischer in the late 19th century. In extremely simple
terms, chirality is “handedness”—that is, the existence
of left/right apposition. For example, your left hand
and right hand are mirror images and therefore “chiral.”
The enantiomeric (mirror image) configurations
Fischer described were designated D and L by Rosanoff
in 1906.10 Today, all monosaccharides are classified
according to this principle, which depends on the
orientation of the hydroxyl group attached to the
highest numbered chiral carbon (C(n-1), where n is the
34
Carbon numbering
Fig. 1.
D-glucose
L-glucose
Carbon numbering of linear hexose sugars and the D- and Lstructures of glucose. The carbon numbering of a generic 6-carbon sugar is shown on the left (with the chiral center hydrogens and hydroxyl groups not shown). Carbons 2, 3, 4, and 5
are the chiral centers of 6-carbon sugars. The enantiomeric
form is designated by the direction of the hydroxyl group on
the highest numbered chiral carbon (arrows)—in the case of
6-carbon sugars, this is carbon 5. In D-glucose (middle), the
hydroxyl group of carbon 5 points to the right, and in L-glucose
(right), this hydroxyl group points to the left.
total number of carbons in the structure). If this
hydroxyl group points to the right when the carbonyl
group is at the top, the monosaccharide is said to be in
the D configuration. The mirror image of this structure,
with the hydroxyl group pointing to the left, is the L
configuration. The D and L configurations of glucose
are shown as open chain Fischer projection formulas
(Fig. 1). In blood group glycoconjugates, sugars exist
almost exclusively in the D configuration, except
fucose, which is found in the L configuration. The
chiral centers of saccharides can also be designated as
R or S under the Cahn-Ingold-Prelog system. This is a
more modern notation; however, it is not often used in
carbohydrate chemistry because it causes confusion in
the classification of some monosaccharides.
The seven common blood group sugars almost
exclusively form pyran rings—six atoms forming the
ring, compared with furan, which is a five-atom ring
(Fig. 2).
Numbering of the carbons in the pyranose ring
formation of hexose and nonose sugars is shown in
Figure 3.
The pyran ring comes about through the formation
of an oxygen bridge between a carbon (carbon 1 in the
common hexose blood group sugars, or carbon 2 in
sialic acid) and the oxygen of the hydroxyl group five
carbons away.10 In hexoses, carbon 5 rotates so that its
hydroxyl group apposes carbon 1. The double-bonded
oxygen on carbon 1 is reduced—taking the hydrogen
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
Review: carbohydrate blood group antigens
1
Generic furan ring
Generic pyran ring
D-galactose
β-D-galactofuranose
Fig. 2.
Open chain
D-galactose
2
β-D-galactopyranose
3
Furan and pyran rings of D-galactose. The open chain formation
of D-galactose is shown in the middle of the figure. The furanose
(lower left) and pyranose (lower right) forms of D-galactose (β is
used in this example) are formed from the open chain structure.
The generic furan (upper left) and pyran (upper right) ring formations are also shown.
β-D-galactopyranose
Fig. 4.
Scheme showing the formation of the pyran ring. Step 1—
Carbon 5 (square) of the open chain form of D-galactose rotates
so that its hydroxyl group apposes carbon 1 (circle). Step 2—The
double-bonded oxygen on carbon 1 is reduced and the hydroxyl
group of carbon 5 is oxidized, leaving carbon 1 and the oxygen
on carbon 5 each with one less than their preferred number of
bonds. Step 3—A bond forms between carbon 1 and the oxygen
of carbon 5 to yield β-D-galactopyranose.
hexose sugars and carbon 2 in sialic acid. This carbon
is termed the anomeric carbon, and the configurational
difference occurs in the orientation of its hydroxyl
group in relation to the oxygen on the anomericreference atom, which is carbon 5 in most hexoses.
Generic hexose
Fig. 3.
Nonose sugar, i.e., sialic acid
Carbon numbering in pyranose rings. Most of the common blood
group sugars are 6-carbon (hexoses), except for sialic acid, which
has 9 carbons (nonose). Carbons are numbered in a clockwise
direction from the oxygen bridge.
from the hydroxyl group of carbon 5, in the process
oxidizing the hydroxyl group to an oxygen radical with
only one bond. This leaves carbon 1 as a radical also,
with only three bonds. This is one less than the
preferred complement of bonds in the case of both
atoms, enabling the formation of a bond between them
(see Fig. 4 for the scheme of this reaction, using Dgalactose as an example). The scheme is basically the
same for sialic acid, except that the bridge is formed
between carbon 2 and the oxygen on carbon 6.
The two ring formations possible for each sugar are
either the α or β anomeric forms. These are two
isomeric forms that differ only in the configuration at
the carbon involved in the formation of the oxygen
bridge, which is carbon 1 in the common blood group
α-D-galactose
Fig. 5.
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
β-D-galactose
Fischer linear ring projection showing the configurational difference at the anomeric carbon hydroxyl group (top arrows) of α-Dgalactose (left) and β-D-galactose (right) in relation to the anomeric reference oxygen (bottom arrows). The anomeric carbon
hydroxyl group and the anomeric reference oxygen are on the
same side of the carbon chain in the α-form, and on opposite
sides of the carbon chain in the β-form.
35
L.G. GILLIVER AND S.M. HENRY
When in the Fischer linear representation of the ring
(Fig. 5), the α form occurs when the anomeric carbon
hydroxyl is on the same side of the carbon chain as the
oxygen on carbon 5. Accordingly, in the β form, the
anomeric carbon hydroxyl and the oxygen on carbon 5
are on opposite sides. Due to the rotation of carbon 5
in hexoses (carbon 6 in sialic acid) and hence its
hydroxyl group (Fig. 4), this rule cannot be applied to
the Haworth projection of the ring. However, the
anomeric form can be deduced in the Haworth
projection—it is α when the anomeric hydroxyl group
is down, and β when the anomeric hydroxyl is up.
The Haworth projections of the α and β forms of
mannose and galactose are shown in Figure 6. The two
cyclic α and β anomeric forms exist in equilibrium
with the open chain structure,11 however, the open
chain configuration is present only in minute amounts.
Because these anomers are interchangeable, the
anomerity of the sugar is really only of interest once it
is made permanent by bonding with another
saccharide.
Amino group
Fig. 7.
Acetyl group
Acetamido group
The acetamido group of N-acetyl-D-galactosamine, N-acetyl-Dglucosamine and sialic acid. The acetamido group is made up of
the amino group and the acetyl group.
hydroxyl group of carbon two is replaced by an
acetamido group, which is an amino group with an
acetyl group substituted for one of the hydrogens
(Fig. 7). This important substitution differentiates
blood group A from blood group B. Blood group A has
N-acetyl-α-D-galactosamine, while blood group B has
α-D-galactose. Furthermore, modification of the
blood group A sugar N-acetyl-α-D-galactosamine by
bacterial deacetylase enzymes can generate the
acquired B antigen (GalNAc to GalNH2—Fig 6). This is
where N-acetyl-α-D-galactosamine loses its acetyl
group but retains the nitrogen—thus, the acquired
B antigen is similar but not identical to the B
determinant α-D-galactose.
N-acetyl-D-glucosamine most
commonly adopts the β
form in blood group glycoconjugates, while N-acetyl-Dgalactosamine most commonly
adopts the α configuration—
α-L-fucose
α-D-galactose
N-acetyl-α-DAcquired B
β-D-galactose
although the β form is also
determinant
galactosamine
seen. Sialic acid is unlike
the other sugar residues
discussed here in that it
has nine carbons and the
anomeric carbon is carbon 2
(Fig. 6). It is substituted at
β-D-glucose
N-acetyl-β-Dα-D-mannose
β-D-mannose Sialic acid (N-acetyl-α-Dcarbon 5 with an acetamido
glucosamine
neuraminic acid)
Fig. 6.
Haworth projection formulae of the sugar residues commonly found in blood group–related group as described for carbon
glycoconjugates.
2 of N-acetyl-D-glucosamine
and N-acetyl-D-galactosamine.
The carboxyl group (COOH) of carbon 1 gives sialic
Figure 6 also shows the other saccharides
acid its acid characteristics, and because of this,
commonly found in blood group antigens. D-glucose
glycoconjugates incorporating it are known as
and D-galactose are most often found in the β form,
gangliosides, which are a subset of acidic glycowhile L-fucose, which is almost identical in structure to
sphingolipids. The acidic glycosphingolipid group also
galactose (except that fucose does not have a hydroxyl
encompasses uronoglycosphingolipids (containing
group on its sixth carbon, thus it is also known as
uronic acid residues), sulfoglycosphingolipids
6-deoxygalactose) is found in the α form. D-mannose is
(containing sulfate ester groups), phosphoglycoa sugar residue commonly found in glycoproteins
sphingolipids (containing phosphate mono- or
(exclusively N-linked oligosaccharides) and exists in
diester groups), and phosphonoglycosphingolipids
both the α and β configuration.
(containing
[2-aminoethyl]hydro-xyphosphoryl
N-acetyl-D-glucosamine and N-acetyl-D-galactosagroups).
mine are examples of amino sugars (Fig. 6). The
36
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
Review: carbohydrate blood group antigens
Up to this point, the pyranose-forming monosaccharides have all been represented as having planar
rings with functional groups projecting above or
below. In reality this is not the case—the electron
orbitals of the carbons and the oxygen in the ring and
the large functional groups attached to the carbons
prevent the rings from existing in a planar form. The
result is that the ring contorts into one of three threedimensional shapes, two of which are suggestive of a
chair and one of which looks like a boat (Fig. 8). The
Chair
Fig. 8.
Chair
Boat
Chair and boat conformations of pyranose rings. There are two
variants of the chair conformation, which differ mainly in the
axial versus equatorial orientations of the ring functional groups
(X and Y). In the boat conformation, the functional groups
labeled A are in an axial position, while those labeled E are
equatorial.
functional groups attached to the ring carbons are able
to adopt either an axial position—perpendicular to the
general plane of the ring—or an equatorial position—
in line with the general plane of the ring. In this way a
single saccharide can potentially assume three different
three-dimensional shapes, although in practice the
lowest energy conformation is preferred, i.e., the one
which minimizes the proximity of large functional
groups. The functional groups in the equatorial
orientation are less crowded together than those in the
axial position, and so the lowest energy conformation
occurs when as many as possible of the large hydroxyl
groups, rather than the relatively small hydrogens, are
situated in the equatorial orientation. Only saccharides
with alternating chirality, such as β-D-glucose (Fig. 1),
can position all the ring hydroxyl groups in the
equatorial position, because crowding in the axial
orientation is reduced due to the ring hydrogens being
able to alternate above and below the plane of the ring
(Fig. 9).
Fig. 9.
β-D-glucopyranose in its
preferred chair conformation. All the hydroxyl
groups are in the equatorial orientation. Note that
the hydrogens alternate
above and below the
plane of the ring in the
axial positions.
Bonds and Linkages
Sugar residues can be joined into chains by the
synthesis of glycosidic bonds.
As previously
mentioned, the two monosaccharide cyclic forms—α
and β—are in equilibrium with the open chain
formation when in aqueous solution. However, when
joined together by glycosidic linkages, the residues
become fixed in ring formation except for the last
sugar in the chain.12
Glycosidic bonds are the result of a condensation
reaction between the hydroxyl group of carbons 2, 3,
4, 6, or 8 of one residue and the anomeric carbon
hydroxyl group of a second sugar. The bond is
considered to belong to the sugar whose anomeric
carbon is involved, which for hexoses is carbon 1, and
is expressed as 1-2, 1-3, 1-4, or 1-6 depending on the
point of attachment. Sialic acid, whose anomeric
carbon is carbon 2, forms 2-3, 2-6, or 2-8 bonds.
Two types of glycosidic bond are possible; this
depends on the configuration of the anomeric carbon
hydroxyl group involved in the bond. The bond
Fig. 10.
Example of α and β linkages in the A type 1 molecule. This diagram shows A type 1 in two schematic forms; one structural and
the other alphanumeric—both a simple form (lower right) and
an expanded form associated with the structural diagram to identify the appropriate sugars and bonds. R = the remainder of the
molecule.
formed when the anomeric hydroxyl group is in the α
configuration is designated an α bond. Likewise, a β
bond will result when the anomeric hydroxyl is in the
β configuration. The A type 1 structure shown in
Figure 10 has two α bonds and three β bonds.
Gangliosides contain sialic acid residues. As
discussed above, the anomeric carbon of sialic acid is
carbon 2 (Figs. 3 and 6), and thus the glycosidic bond
occurs at the hydroxyl group of this carbon. Sialic acid
can be added to a hexose residue at carbons 3 or 6;
α2-3 and α2-6 bonds between sialic acid and galactose
are shown in Figure 11. Sialic acid usually occupies a
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
37
L.G. GILLIVER AND S.M. HENRY
(R. Oriol, personal communication). The actual structure
and size of the polysaccharide is determined by
interactions of various polymorphic blood group glycosyltransferases and other
monomorphic glycosyltransferases. Glycosyltransferases
α2-3) galactose
α2-6) galactose
α2-8) sialic acid
Sialic acid (α
Sialic acid (α
Sialic acid (α
are gene–encoded proteins
Fig. 11. Sialic acid linkages. Left: N-acetyl-α-D-neuraminic acid (left) bonded to β-D-galactose (right) through (enzymes), and are responan (α2-3) linkage. Center: N-acetyl-α-D-neuraminic acid (left) bonded to β-D-galactose (right) through an
for the stepwise
(α2-6) linkage. Right: two N-acetyl-α-D-neuraminic acid residues joined together through an (α2-8) link- sible
age. R = the remainder of the molecule.
addition of substrate residues
to acceptor structures. It is
terminal (and sometimes also a sub-terminal) position.
therefore clear that the biosynthesis of carbohydrate
A terminal sialic acid residue prevents further
blood group antigens is not a straightforward gene
elongation of the chain except for the addition of
equals antigen process. Instead, a gene must encode a
another sialic acid residue through an α2-8 linkage
functional protein (enzyme), which must be retained in
(Fig. 11).
the Golgi apparatus, and in the presence of appropriate
Different glycoconjugate precursor chain
precursors and substrates a carbohydrate blood group
structures have been identified and categorized into six
antigen can be synthesized (Fig. 12). Even this is overly
groups based on the identity of the terminal
Conceptual pathway
Biosynthesis of A antigen
disaccharide sugar residues and the type of glycosidic
genetic message (DNA, mRNA)
A gene
linkage that joins them together.13 These terminal
↓
↓
enzyme (glycosyltransferase)
N-acetyl-α-D-galactosaminyl
disaccharide structures of the six groups are shown in
transferase
Table 1.
↓
↓
precursor + activated sugar
H antigen + UDP-GalNAc
↓
↓
carbohydrate antigen
A antigen
■ membrane/antigen presentation ■
↑
↑
recognition by antibodies/lectins/receptors
Table 1. Six terminal disaccharide chain types.
Terminal Disaccharide Structure
Type 1
Galβ1-3GlcNAcβ1-R
Type 2
Galβ1-4GlcNAcβ1-R
Type 3
Galβ1-3GalNAcα1-R
Type 4
Galβ1-3GalNAcβ1-R
Type 5
Galβ1-3Galβ1-R
Type 6
Galβ1-4Glcβ1-R
Fig. 12.
Biosynthesis of Saccharide Chains
According to carbohydrate terminology, an
oligosaccharide consists of 3 to 10 sugar residues and a
polysaccharide contains more than 10 residues.11 The
blood group structures can exist in both linear and
branched forms and range from a few saccharide units
up to more than 60 sugar residues. Irrespective of the
size of any given polysaccharide, it is generally
accepted that the actual surface recognized by
antibody (glycotope) is usually no larger than 2–3
sugars. However, sometimes glycotopes may look
bigger (up to six sugars) because some extra sugars are
needed to stabilize the three-dimensional configuration
of the sugars actually recognized by the antibody
38
Conceptual scheme for the formation of carbohydrate blood
group antigens.
simplistic, because the formation of a carbohydrate
blood group antigen requires the interaction of several
glycosyltransferases unrelated to blood groups before
the blood group determinant(s) can be added.
Furthermore, competition and sometimes cooperation
occur between the various glycosyltransferases, some
of which show redundancy and degeneration.14
Redundancy is said to occur when the same
carbohydrate structure can be made by two separate
enzymes. For example, Se is capable of transferring a
fucose to the terminal β-D-galactose of both type 1 and
2 chains, while H can only utilize type 2 chains. Thus,
H type 2 can be made by two genetically different
enzymes. The presence of Se-formed H type 2 antigens
on Bombay RBCs is the basis of some para-Bombay
phenotypes. Degeneration refers to the situation
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
Review: carbohydrate blood group antigens
where one enzyme can make two different structures.
Using the same example, Se shows degeneration in that
it can make type 1 and type 2 H structures. Thus, it can
be seen that the occurrence of specific glycoconjugates is indirectly controlled by genes through
the occurrence and subsequent synthetic actions of
enzymes. After glycoconjugates are synthesized, they
are transported to various destinations in and out of the
cell, as well as being incorporated in the cell membrane
where they can be detected by various antibodies. A
more comprehensive review of the pathways leading
to the expression of the blood group–related glycosphingolipids can be found elsewhere.13
Glycolipids vs. Glycoproteins
Glycans can be conjugated to protein or lipid
molecules in biological systems; such molecules are
called glycoproteins or glycolipids, respectively. The
glycosylation pathways followed by glycoproteins and
glycolipids are not always identical. This possibly
reflects the different biological functions of
membrane–bound glycoconjugates (glycolipids and
glycoproteins) versus soluble structures (usually
glycoproteins).15,16
Fig. 13.
Structure of a sphingolipid (ceramide) and a monoglycosyl
sphigolipid. Top: the sphingolipid is composed of a FA (nervonic
acid C24:115—upper chain) and a LCB (4-sphingenine d18:14—
lower chain). Below: β-D-glucose is joined to the sphingolipid via
a β-linkage between carbon 1 of the saccharide and ceramide.
level of saturation (presence of double bonds), and the
number of hydroxyl groups—in fact, there are over 60
different variations of LCBs,17,18 each of which can be
combined with one of 20 common variants of FAs.10 It
is speculated that some of the variants may be
important for presentation of the antigen.
Glycoproteins
Glycans can be attached to proteins via nitrogen or
oxygen—N– or O-glycosidically linked, respectively.
N-glycosidically linked glycoproteins are found
primarily in serum and cell membranes, while Oglycosidically linked glycoproteins are located in
exocrine gland secretions and mucins, as well as in cell
membranes, although this is less frequent.7
Glycolipids
Glycosphingolipids are a class of glycolipids which
are composed of a carbohydrate chain and a
sphingolipid tail, the trivial name of which is ceramide.
Sphingolipids are common components of cell
membrane lipid bilayers and are composed of a longO-linked glycoproteins
chain base (LCB) amino linked to a fatty acid (FA). Due
An O-linkage occurs between the hydroxyl groups
to the long hydrophobic hydrocarbon chains present
of serine or threonine amino acid residues and the
in LCBs and the FAs, ceramides are able to insert into
anomeric carbon hydroxyl group of N-acetyl-αthe lipid bilayer of RBCs. The hydroxyl group on
D-galactosamine, with the linkage type being α1
carbon 1 of the sphingolipid is oxidized to form
(Fig. 14). Glycolipids are also O-linked, but the sugar
the β-linkage with the
Serine
Threonine
Asparagine
anomeric carbon of β-Dglucose to form a monoglycosyl sphingolipid (Fig.
13). This linkage is designated β1-1 because the
two hydroxyl groups
involved belong to carbon
1 of their respective
structures.
There is considerable
heterogeneity in ceramides, Fig. 14. Structure of glycoprotein O- and N-linkages. Left and center: O-linked glycoproteins where N-acetyl-Dgalactosamine is linked through an α bond to the oxygen of the amino acids serine or threonine.
mainly due to variations in
Right: N-linked glycoprotein where N-acetyl-D-glucosamine is linked through a β bond to the nitrogen of
hydrocarbon chain length,
the amino acid asparagine. R1 and R2 represent the remainder of the polypeptide chain.
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
39
L.G. GILLIVER AND S.M. HENRY
involved is β-D-glucose. Unlike the N-linked glycoproteins, there is no single common oligosaccharide
core structure for O-linked glycoproteins. Instead
there are six distinct di- and trisaccharide core
structure types and a variety of terminal structures.7
O-linked glycoproteins range from the very simple
mono-glycosylated structures to highly complex
molecules containing large amounts of galactose and
N-acetylglucosamine, along with lesser amounts of
N-acetylgalactosamine, fucose, and sialic acid. O-linked
glycoproteins do not contain mannose residues.
N-linked glycoproteins
An N-linkage occurs between the nitrogen of the
amino acid asparagine and the hydroxyl group of
N-acetyl-β-D-glucosamine, with the linkage type being
β1 (Fig. 14).
N-linked glycoproteins have a common
pentasaccharide structure consisting of Man3GlcNAc2.7
There are three main groups into which N-linked
glycoproteins can be divided: high mannose type,
complex type, and hybrid type. A comprehensive
description of these types can be found elsewhere.7
Red Cell Glycosylation and Serology
The outside of the human RBC is
highly glycosylated, incorporating
both glycoproteins and glycolipids.
The major function of the
glycoconjugate coat of the RBC is
probably to make them more
hydrophilic and to provide a “hard”
but flexible protective coating.
However, if this was the only
function of red cell glycosylation,
then it could be achieved by the
expression of large amounts of
simple carbohydrate chains, which
does not account for the complex
pattern of glycosylation that actually
exists. Some of the glycoconjugates
are very complex and bear
genetically defined polymorphisms,
for example ABH, Lewis, and P. Why
these polymorphisms exist is
unknown, but there is emerging
evidence of evolutionary and
biological function.15,16,19,20
The intention of this review is to
present
basic
concepts
of
40
carbohydrate chemistry in order to develop an
understanding of the complexities that exist within
carbohydrate blood group systems. Traditionally, the
antigens of these systems have been viewed
generically, without appreciation of the subtle
differences between them or the impact this may have
on phenotyping or biological function. Not only is
there a variety of glycotopes within an antigen
designation, but there also is extensive variation in the
carbohydrate molecules which carry the glycotopes
(Fig. 15).
This range of variants can be further expanded by
the polymorphic proteins and lipids which then
support them. The presentation of the carbohydrate
chain at the membrane surface may differ significantly
among the different types, as shown by molecular
modeling.21 The membrane orientation of saccharide
chains may vary from almost perpendicular (type 1), to
nearly parallel (types 2, 3, and 4). These different
conformations of the carbohydrate chains are
important for antibody binding and for the
immunogenic and microbial-interaction characteristics.
Some
may
actually
determine
phenotype
characteristics, although this has not yet been
determined. In addition, the carrier molecule may
influence the positioning of the carbohydrate chain on
A type 1; A-6-1
A type 2; A-6-2
ALeb; A-7-2
ALey; A-7-2
A type 4; Globo-A; A-7-4
A type 2; A-8-2
A type 3; Repetitive A; A-9-3
A-12-2
A-14-2
Fig. 15.
Structure of nine examples of A glycolipid structures in the group A erythrocyte membrane.
Shorthand nomenclature according to Holgersson.23
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
Review: carbohydrate blood group antigens
the cell membrane in relation to other membrane
components.22
From the perspective of blood transfusion, the
current lack of depth of understanding of carbohydrate
blood group antigens has been accepted as adequate.
However, as can be seen from other blood group
systems (e.g., Rh and HLA), there is value to be found in
unraveling their intricacies. It is hoped that by
investigating the subtleties of the carbohydrate blood
group systems, important new biological information,
in particular microbial interactions, will be discovered.
References
1. Laine RA. Invited commentary. Glycobiology
1994;4:759-67.
2. Oriol R, Samuelsson BE, Messeter L. Workshop 1.
ABO antibodies. Serological behaviour and
immuno-chemical characterization. J Immunogenet 1990;17:279-99.
3. Samuelsson BE, Pimlott W, Karlsson KA. Mass
spectrometry of mixtures of intact glycosphingolipids. Methods Enzymol 1990; 193:623-46.
4. Karlsson H, Johansson L, Miller-Podraza H, Karlsson
KA. Fingerprinting of large oligosaccharides linked
to ceramide by matrix-assisted laser desorptionionization time-of-flight mass spectrometry. Highly
heterogeneous polyglycosylceramides of human
erythrocytes with receptor activity for
Helicobacter pylori. Glycobiology 1999;9:765-78.
5. Oriol R, Barthod F, Bergemer AM, Ye Y, Koren E,
Cooper DK. Monomorphic and polymorphic
carbohydrate antigens on pig tissues: implications
for organ xenotransplantation in the pig-to-human
model. Transpl Int 1994;7:405-13.
6. Henry SM, Samuelsson BE. ABO polymorphisms
and their putative biological relationships with
disease. In: King M-J, ed. Human blood cells:
consequences of genetic polymorphisms and
variations. Imperial College Press, 2000:15-103.
7. Schenkel-Brunner H. Human blood groups:
chemical and biochemical basis of antigen
specificity. Revised Edition. New York: Springer
Wien, 2000.
8. Reuter G, Gabius HJ. Sialic acids structure-analysismetabolism-occurrence-recognition. Biol Chem
Hoppe Seyler 1996;377:325-42.
9. Traving C, Schauer R. Structure, function and
metabolism of sialic acids. Cell Mol Life Sci 1998;
54:1330-49.
10. Voet D,Voet JG. Biochemistry. New York:Wiley and
Sons, 1998.
11. Atkins RC, Carey FA. Organic chemistry: a brief
course. New York: McGraw-Hill, 1990.
12. Pazur JH. Neutral polysaccharides. In: Chaplin MF,
Kennedy JF, eds. Carbohydrate analysis: a practical
approach. 2nd ed. Oxford: IRL Press, 1994:100.
13. Oriol R. ABO, Hh, Lewis, and secretion: serology,
genetics, and tissue distribution. In: Cartron J,
Rouger P, eds. Blood cell biochemistry: molecular
basis of human blood cell antigens. New York:
Plenum Press, 1995:37-73.
14. Oriol R, Le Pendu J, Mollicone R. Genetics of ABO,
H, Lewis, X and related antigens. Vox Sang
1986;51:161-71.
15. Karlsson KA. Meaning and therapeutic potential of
microbial recognition of host glycosphingolipids.
Mol Microbiol 1998;29:1-11.
16. Karlsson KA. The human gastric colonizer
Helicobacter pylori: a challenge for host-parasite
glycobiology. Glycobiology 2000;10:761-71.
17. Karlsson KA. On the chemistry and occurrence of
sphingolipid long-chain bases. Chem Phys Lipids
1970;5:6-43.
18. Leray C. Cyberlipid Center:Your www site for fats
and oils. Available at: http://www.cyberlipid.org/
amines/amin0001.htm#1. Accessed September 26,
2001.
19. Henry SM. Review: phenotyping for Lewis and
secretor
histo-blood
group
antigens.
Immunohematology 1996;12:51-61.
20. Moulds JM, Moulds JJ. Blood group associations
with parasites, bacteria, and viruses. Transfus Med
Rev 2000;14:302-11.
21. Nyholm P-G, Samuelsson BE, Breimer M, Pascher I.
Conformational analysis of blood group A-active
glycosphingolipids using HSEA-calculations. The
possible significance of the core oligosaccharide
chain for the presentation and recognition of the Adeterminant. J Mol Recognit 1989;2:103-13.
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
41
L.G. GILLIVER AND S.M. HENRY
22. Hakomori S, Handa K, Iwabuchi K, Yamamura S,
Prinetti A. New insights in glycosphingolipid
function: “glycosignalling domain,” a cell surface
assembly of glycosphingolipids with signal
transducer molecules, involved in cell adhesion
coupled with signalling. Glycobiology 1998;8:11-9.
23. Holgersson J, Breimer ME, Samuelsson BE. Basic
biochemistry of cell surface carbohydrates and
aspects of the tissue distribution of histo-blood
group ABH and related glycosphingolipids. APMIS
Suppl 1992;27:18-27.
Lissa G. Gilliver, BSc, BAppSc(Hons), Project Leader,
Kiwi Ingenuity Ltd., Auckland, New Zealand, and
PhD student, Auckland University of Technology, New
Zealand. Stephen M. Henry, PhD, Director, Kiwi
Ingenuity Ltd., 19 Mount St., Auckland, New Zealand,
and Director, Glycoscience Research Centre, Auckland
University of Technology, Private Bag 92006,
Auckland 1020, New Zealand, email: [email protected]
(corresponding author).
Manuscripts: The editorial staff of Immunohematology welcomes manuscripts pertaining to blood group
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42
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
Neonatal alloimmune
thrombocytopenia due to
anti-HPA-2b (anti-Koa)
M. GOLDMAN, E.TRUDEL, L. RICHARD, S. KHALIFE, AND G.M. SPURLL
Most severe cases of neonatal alloimmune thrombocytopenia
(NAIT) are due to anti-HPA-1a (anti-PlA1) antibodies. We report a
case of NAIT due to anti-HPA-2b that resulted in in utero intracranial
hemorrhage.A 33-year-old G2P1A0 Caucasian woman had a routine
ultrasound at 34 weeks. The fetus appeared to have a left
hemispheric hematoma. IVIG, 1g/kg, was started immediately and
administered weekly until delivery. One day after receiving the first
dose of IVIG, fetal platelet count was 18 × 109/L, and Hb was 116
g/L. Eleven mL of matched platelets compatible by monoclonal
antibody immobilization of platelet antigens (MAIPA) assay were
transfused in utero, raising the platelet count to 62 × 109/L. Repeat
transfusions were done later that week and 1 week later, with
pretransfusion counts of 19 × 109/L and 16 × 109/L, respectively.
Delivery by C section was done at 35.5 weeks, after the third
platelet transfusion. Platelet count at birth was 77 × 109/L.
Drainage of the hematoma was performed after transfusion. Testing
with a solid phase ELISA revealed reactivity against GP1b/IX.
MAIPA testing after platelet treatment with the protease inhibitor
leupeptin demonstrated the presence of anti-HPA-2b. On PCR-SSP
the mother was HPA-2a homozygous, the father was HPA-2a/2b.
Antibodies against the HPA-2b antigen located on the GP1b/IX
complex have been reported in rare cases of NAIT. Testing is
complicated by proteolytic degradation of the antigen-bearing
fragment.
Compatible platelets are easily found since
approximately 85 percent of donors are HPA-2a/2a.
Immunohematology 2003;19:43–46.
Key Words: neonatal thrombocytopenia purpura,
NAIT, anti-HPA-2b, anti-Koa
Neonatal alloimmune thrombocytopenia (NAIT) is
caused by maternal antibodies directed against an
antigen on paternal and fetal platelets. NAIT can result
in intracranial hemorrhage and death or severe
neurologic sequelae.1 Unlike its RBC counterpart,
erythroblastosis fetalis, NAIT may affect the firstborn
infant. In the Caucasian population, most severe cases
of NAIT are due to anti-HPA-1a (anti-PlA1). Anti-HPA-5b
(anti-Bra) antibodies account for approximately 20
percent of serologically proven NAIT. They usually are
associated with milder thrombocytopenia and
therefore lead to less severe clinical manifestations.
HPA-2 (Ko, Sib) platelet alloantigens are located on
GPIbα.2 Anti-HPA-2b antibodies have been found in
multitransfused patients and in rare cases of NAIT.3–5
We report an additional case of severe NAIT due to
anti-HPA-2b. The case illustrates the importance of
performing sufficient laboratory investigations to
detect antibodies other than anti-HPA-1a in a clinical
setting suggestive of NAIT, in order to supply
appropriate phenotyped platelets for transfusion
support.
Case Report
The patient, a 33-year-old Caucasian woman of
Greek origin, G2P1A0, had an unremarkable first
pregnancy. During her second pregnancy, a routine
ultrasound of the fetus at 34 weeks’ gestation showed a
left hemispheric hematoma. She was taking no
medication and had no other medical problems. IVIG
(1g/kg/week) was started. Percutaneous umbilical
cord blood sampling (PUBS) done 1 day post IVIG
administration showed a fetal Hb of 116 g/L and a fetal
platelet count of 18 × 109/L. Microplate ELISA testing
demonstrated strong reactivity against GPIb/IX in the
Fig. 1.
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
Perinatal transfusions and the fetal platelet count are illustrated.
43
M. GOLDMAN ET AL.
maternal serum as well as against HLA Class I antigens.
Three intrauterine transfusions of 11 mL each were
given using platelets that were crossmatch compatible
in the monoclonal antibody immobilization of platelet
antigens (MAIPA) assay. Perinatal transfusions and the
fetal platelet count are illustrated in Figure 1. After
Cesarean delivery, a fourth crossmatch-compatible
transfusion was given and the hematoma, reconfirmed
on ultrasound, was evacuated. No petechiae or other
signs of bleeding were noted at delivery. The baby was
discharged home and is developmentally normal to
date, although a seizure disorder is present.
incubated 120 minutes at 4°C with 100 µL of goat antihuman IgG labeled with peroxydase (1 in 500). The
wells were then washed × 5 and filled with 100 µL of
orthophenylene diamine (OPD) substrate solution.
After a 15-minute incubation at room temperature in
the dark, the enzyme reaction was measured in a
spectrophotometer at 490 nm.
Allele-specific PCR (PCR-SSP)
Amplification was run using 25 µM each of the
specific primers, as described by Skogen et al.7 The
primers are designed as follows:
HPA-2a specific primer: 5'-CCCCCAGGGCTCCTGAC-3'
Material and Methods
HPA-2b specific primer: 5'-CCCCCAGGGCTCCTGAT-3'
GTI PAK™ 12 assay
In the ELISA assay, patient’s serum was added to the
microwells of the GTI PAK™ 12 platelet antibody
screening kit (GTI, Brookfield,WI) coated with platelet
glycoproteins and HLA class I antigens, allowing for
antibody to bind when present. After washing away
unbound immunoglobulins, an alkaline phosphatase–
labeled anti-human IgG was added. Unbound anti-IgG
was washed away and the enzyme substrate Pnitrophenyl phosphate was added. After incubation,
the reaction was stopped by a sodium hydroxide
solution and the optical density was measured in a
spectrophotometer at 405 nm.
MAIPA
The MAIPA assay was slightly modified from the
method described by Kiefel et al.6 Platelets (20 × 106)
isolated from EDTA-anticoagulated blood from known
donors were washed, suspended in 50 µL of PBS-BSA
2%, and incubated with 50 µL of human serum at 37°C
for 30 minutes. Platelets were then washed in PBS-BSA
2% and incubated with 0.2 µg of the glycoproteinspecific monoclonal antibody (anti-GP1b/IX,
Immunotech, Marseille, France) at 37°C for another 30
minutes. Platelets were washed in 100 µL of isotonic
saline and centrifuged × 3 at 12,000–14,000 g for 2
minutes. After the last centrifugation, the platelets
were suspended in 100 µL solubilization buffer
(containing 1% leupeptin) and allowed to lyse at 4°C
for 30 minutes. The lysates were then centrifuged at
12,000–14,000 g for 30 minutes at 4°C and 50 µL of the
supernatant was diluted in 200 µL of Tris buffer saline
(TBS). One hundred µL of the dilution was distributed
into wells of a microtiter plate coated with goat antimouse IgG (1 in 500). After overnight incubation at
4°C the microplate was washed × 5 with TBS and
44
HPA-2c common primer: 5'-GCAGCCAGCGACGAAAATA-3'
The cycling protocol used includes one cycle of
94°C for 5 minutes; 30 cycles of 94°C for 1 minute,
65°C for 2 minutes, and 72°C for 1 minute; and one
cycle of 72°C for 10 minutes. The mixture is made
using 1.5 mM MgCl2 10 X reaction buffer, 25 µM dNTPs
mix, 5 U/µL Taq polymerase (2.5 U/amplification), and
the human growth hormone gene as an internal
control.
Table 1. MAIPA testing of maternal serum: optical density
Platelet
Father Panel 1 Panel 2 Panel 3 Panel 4 Panel 5
Genotypes HPA-2ab HPA-2ab HPA-2ab HPA-2ab HPA-2aa HPA-2aa
Control
serum
0.121
0.009
0.026
0.009
0.028
0.009
Maternal
serum
0.365
0.269
0.298
0.319
0.029
0.019
Results
On GTI PAK™ 12 testing, strong reactivity was
demonstrated against GPIb/IX in the maternal serum
(OD = 0.919 vs. OD = 0.036 for negative control), as
well as against HLA Class I (OD = 1.741 vs. OD = 0.056
for negative control).
MAIPA testing using leupeptin-treated platelets and
monoclonal antibody anti-GPIb/IX demonstrated
reactivity against paternal and known HPA-2b(+)
platelets, as demonstrated in Table 1.
In PCR-SSP, the mother was found to be HPA-2a/2a,
while the father was HPA-2a/2b.
Discussion
The HPA-2 (Ko) alloantigen system was first
described by van der Weerdt et al. in 1961.8 The gene
frequency of HPA-2b is 0.09 in Caucasians in the United
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
NAIT due to anti-HPA-2b
States, 0.18 in African Americans, and 0.13 in Koreans.9
The GPIb/IX/V complex is the platelet receptor for von
Willebrand Factor (vWF). The HPA-2 alloantigens are
located in the N-terminal globular portion of the
molecule, as is the vWF binding site. In vitro, anti-HPA2b antibodies have been shown to inhibit vWF
binding.7 It is therefore possible that the antibody may
affect platelet function as well as platelet survival.
The N-terminal globular portion of the GPIb
molecule is sensitive to proteases.7 Therefore, it is
preferable to use a protease inhibitor, such as
leupeptin, during MAIPA testing to avoid degradation of
the antigen. In our protocols, 1% leupeptin is added to
platelet suspensions before testing, and also at the step
of solubilization in the lysing buffer.
The GTI PAK™ 12 microplate testing system
permits relatively rapid identification of reactivity
against a given platelet glycoprotein. However, we and
others have found that alloantibodies may be
undetectable in PAK™ 12 testing or may give
nonspecific reactions.10,11 Therefore, although the GTI
kit is useful as a screening test, it must be
complemented by other laboratory investigations. The
MAIPA assay, first described by Kiefel et al.,6 is used in
many platelet serology laboratories. At the 10th ISBT
International Platelet Genotyping and Serology
Workshop, in 2000, the MAIPA assay was reported to be
used alone, or in combination with other methods, by
28 of 38 laboratories.12 Although this assay may be
more sensitive and specific than the GTI kit, it also
requires a minimum of 10 hours of technical time and
an overnight incubation period. In addition, a
monoclonal antibody against the glycoprotein complex
of interest must be added. MAIPA testing using
monoclonal antibodies solely against GPIIb/IIIa and
GPIa/IIa in the expectation of finding an anti-HPA-1a or
anti-HPA-5b antibody, respectively, would have given a
negative result in this case. In addition, the use of a
frozen platelet panel or of platelets that have been
stored without protection from proteases may lead to
antigen degradation and a negative result. Finally, allelespecific PCR confirms the alloantigen incompatibility
between the two parents. However, it does not prove
that an alloantibody is actually present. Because
alloantisera are extremely rare, genotyping permits the
laboratory to develop a panel of platelets of known
HPA-2 specificities and to establish a bank of HPA-2a/2a
donors.
There have been several other reported cases of
NAIT due to anti-HPA-2b. Kroll et al.13 reported two
cases in Germany. In the first case, the third child of
healthy parents was found to have a postpartum
platelet count of 53 × 109/L at delivery, which
decreased to 11 × 109/L on day 4. Three maternal
platelet transfusions were necessary to prevent
bleeding. In the second case, a second pregnancy was
complicated by erythroblastosis fetalis due to anti-CD
treated by intrauterine transfusion. Fetal death
occurred, and a platelet count of 3 × 109/L was found.
Because of the small numbers of cases reported and
the laboratory diagnostic difficulties discussed, it is
difficult to know if these cases represent the true range
of severity of NAIT due to anti-HPA-2b.
The frequency of NAIT due to anti-HPA-2b is
difficult to determine. In a large prospective study by
de Moerloose et al.,14 of 8388 newborns in Switzerland,
40 newborns had NAIT; anti-HPA-1a and anti-HPA-5b
antibodies were detected in three and two mothers,
respectively, and no anti-HPA-2 antibodies were
present. In a study by Hohlfeld et al.,15 of 5194
neonates in France undergoing fetal blood sampling for
cytogenetic analysis and various infections and
hematologic disorders, anti-HPA-1a and anti-HPA-5b
were detected in 23 and 2 mothers, respectively, but no
anti-HPA-2 antibodies were present. In a series of 975
cases of NAIT reported by McFarland et al.16 spanning
11 years of testing by a tertiary referral center, only
three cases of anti-HPA-2b were detected. In all three
cases, anti-HPA-2b was found in the presence of other
alloantibodies (anti-HPA-3a in one, anti-HPA-3b in
another, and anti-HPA-1a and anti-HPA-3a in the third).
Although anti-HPA-2b antibodies are a rare cause of
NAIT, they should be considered in a clinical setting
that is suggestive of NAIT, when more commonly seen
antibodies have not been found. The detection of these
antibodies in the MAIPA assay requires fresh platelets,
treatment with leupeptin, and the use of an antiGPIb/IX monoclonal antibody. The GTI PAK™ 12 assay
may be useful in screening for these antibodies.
Acknowledgments
We would like to thank Mona Banville, Sonia GrandMaison, and Madeleine Malette for performing the
laboratory studies and Sylvain Chiroux for assistance
with the manuscript. We would also like to thank Dr. S.
Santoso and Dr. H. Kroll, Institute for Clinical
Immunology and Transfusion Medicine, Giessen,
Germany, for confirming the laboratory results.
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
45
M. GOLDMAN ET AL.
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platelet glycoprotein Ibα. Blood 1992;79:283-8.
3. Marcelli-Barge A, Poirier JC, Daussett J. Alloantigens
and alloantibodies of the Ko system. Serological and
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4. Bizarro N, Dianese G. Neonatal alloimmune
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Monoclonal antibody-specific immobilization of
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identification of platelet-reactive antibodies. Blood
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7. Skogen B, Bellissimo DB, Hessner MJ, et al. Rapid
determination of platelet alloantigen phenotypes
by polymerase chain reaction using allele-specific
primers. Transfusion 1994;34:169-76.
8. van der Weerdt CM, van der Wiel H, Engelfriet CP, et
al. A new platelet antigen. Proceedings of the 8th
Congress of the European Society of Hematology.
Basel, Switzerland: Karger, 1961:379.
9. Kim HO, Jin Y, Kickler TS, et al. Gene frequencies of
the five major human platelet antigens in African
American, white, and Korean populations.
Transfusion 1995;35:863-7.
10. Lucas GF, Rogers SE. Evaluation of an enzymelinked immunosorbent assay kit (GTI PakPlus‚) for
the detection of antibodies against human platelet
antigens. Transfus Med 1999;9:63-7.
46
11. Chiroux S, Malette M, Décary F, Roy G, Goldman M.
Evaluation of the GTI PAK™ 12 Elisa kit for platelet
alloantibody detection (abstract). Transfusion
1999;39(Suppl):37S.
12. Panzer S. Report on the Tenth International Platelet
Genotyping and Serology Workshop on behalf of
the International Society of Blood Transfusion. Vox
Sang 2001;80:72-8.
13. Kroll H, Kiefel V, Muntean W, Mueller-Eckhardt C.
Anti-Koa as the cause of neonatal alloimmune
thrombocytopenia (abstract). Vox Sang 1994;
(Suppl):12.
14. de Moerloose P, Boehlen F, Extermann P, Hohlfeld P.
Neonatal thrombocytopenia: incidence and
characterization of maternal antiplatelet antibodies
by MAIPA assay. Br J Haematol 1998;100:735-40.
15. Hohlfeld P, Forestier F, Kaplan C,Tissot J-D, Daffos F.
Fetal thrombocytopenia: A retrospective survey of
5,194 fetal blood samplings. Blood 1994;84:1851-6.
16. McFarland J, Curtis B, Friedman K, Aster H. Platelet
alloantigen incompatibilities implicated in 975
cases of suspected neonatal alloimmune
thrombocytopenia (abstract).
Blood 2001;
98(Suppl):449a.
Mindy Goldman, MD, Associate Senior Director,
Medical Affairs, Hematology, Héma-Québec, 4045
Côte-Vertu, Montreal, Quebec, Canada, H4R 2W7;
Élise Trudel, Service Chief, Hospital Services
Laboratory and Lucie Richard, PhD, Hospital Services
Laboratory Director, Héma–Québec, Quebec, Canada;
Samir Khalife, MD, Department of ObstetricGynecology, Royal Victoria Hospital, 687, Pine Ave
West Montreal, Quebec, Canada, H3A 1A1; and
Gwendoline M. Spurll, MD, Blood Bank Director,
Royal Victoria Hospital, Montreal, Quebec, Canada.
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
Quantitation of red cell–bound
IgG, IgA, and IgM in patients with
autoimmune hemolytic anemia
and blood donors by enzymelinked immunosorbent assay
A.A. BENCOMO, M. DIAZ,Y.ALFONSO, O.VALDÉS, AND M. E.ALFONSO
This paper describes an enzyme immunoassay for the quantitative
determination of IgG, IgA, and IgM immunoglobulins on RBCs.
Ether eluates made from RBCs were followed by an enzyme-linked
immunosorbent assay of immunoglobulin concentration.
Calibration curves were derived from immunoglobulin standards
and the number of molecules of each isotype per RBC was
calculated. The assay was carried out in 200 healthy blood donors
and 62 patients with warm autoimmune hemolytic anemia (AIHA),
two of them with a negative DAT. For healthy blood donors, mean
values were 58 IgG, 16 IgA, and 3 IgM molecules per RBC. For
patients with a positive DAT, the mean values were 3435 IgG, 157
IgA, and 69 IgM molecules per RBC. An increased level of IgA was
found in 12 patients without IgA autoantibodies demonstrable in
RBC eluates. Increased IgG levels were also observed in patients
with a negative DAT and, in one case, an increased level of IgA was
also found. The enzyme-linked immunosorbent assay using ether
eluates is a sensitive method for quantitating RBC autoantibodies in
patients with AIHA as well as immunoglobulins bound to RBCs in
healthy individuals. Immunohematology 2003;19:47–53.
Key Words: enzyme-linked immunosorbent assay,
autoimmune hemolytic anemia, IgG, IgA, and IgM
immunoglobulins, RBCs, quantitative method
Autoimmune hemolytic anemia (AIHA) is
characterized by immune-mediated hemolysis associated
with the presence of IgG, IgA, or IgM immunoglobulins
on the RBC membrane.1 The most common type of
AIHA is associated with warm-reactive antibodies and in
most cases they are detectable by the DAT.2 The
presence of immunoglobulins can also be revealed on
the surface of RBCs of DAT-negative healthy individuals
by more sensitive methods.3 Several methods to
quantitate RBC-bound immunoglobulins have been
reported, including an immunoradiometric assay,
autoanalyzer assays, antiglobulin consumption assays,
and enzyme-linked immunosorbent assay (ELISA).4–10
Quantitation of immunoglobulins in a patient with AIHA
would allow a more precise method of monitoring the
disease or defining the synergistic effect of the different
immunoproteins in causing RBC destruction.11
We have developed an enzyme immunoassay for
the quantitative determination of immunoglobulin
isotypes in ether eluates made from RBCs.This method
can be used to study RBC-associated immunoglobulins
in patients with AIHA and in healthy individuals.
Materials and Methods
Blood samples and ether elution
After informed consent had been obtained from
patients and blood donors, blood samples were
collected from venous blood in EDTA (Merck). The
samples were centrifuged at 270 × g for 8 minutes at
room temperature, and the plasma and buffy coat were
removed.
Ether eluates were prepared after washing 1 mL of
packed RBCs × 10 using 10 volumes of saline. Elution
was performed by adding 1 mL of PBS, pH 7.2,
containing 0.4% BSA (Sigma Chemical Co.) and 1 mL of
diethyl ether (Merck) to 1 mL of RBCs (an average of 1.1
× 1010 RBCs). The mixture was incubated in a 37°C
water bath for 10 minutes, with frequent mixing. After
centrifugation, the upper layer of ether was discarded
and the hemoglobin-stained eluate was transfered into a
test tube. The residual ether was evaporated at 37°C for
15 minutes.12 An average of 1 mL of eluate was
recovered. Eluates were kept frozen at –30°C until used.
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
47
A.A. BENCOMO ET AL.
ELISA for the quantitation of IgG, IgA, and IgM on
RBCs
IgG, IgA, and IgM standard curves were prepared
from reference sera (Nor-Partigen, Behring) in
concentration ranges from 0.0025 to 0.1 µg/mL in PBS,
0.05% Tween (Tw) 20 (Merck), and 0.4% BSA
(PBS/Tw/BSA).
Flat-bottomed 96-well plates
(Maxisorp, Nunce Immunoplates) were precoated with
100 µL per well of 5 µg/mL goat anti-human IgG, IgA, or
IgM (Sigma) in 0.05M carbonate buffer, pH 9.6, at 4°C
temperature overnight. After washing × 4 in PBS/Tw,
0.2%, 100 µL per well of standards or diluted eluates in
PBS/Tw/BSA were incubated at 37°C for 60 minutes.
The plates were washed × 4 in PBS/Tw, 0.05%, before
addition of 100 µL per well of peroxidase conjugated
(Sigma) goat anti-human IgG or IgM diluted 1 in 5000
or goat anti-human IgA diluted 1 in 1000. The plates
were incubated with the conjugate at 37°C for 60
minutes and then washed as above. A substrate
solution (100 µL per well) of 0.24 mg/mL of Ophenylenediamine (Merck) in 0.05M phosphate citrate
buffer, pH 5.0, with 0.024% of hydrogen peroxide
(Merck) was added. Color development was stopped
after 15 minutes by adding 100 µL per well of 3M
H2SO4 (BDH) and absorbance was read at 492 nm on
the Titertek Multiscan MC plate reader. Absorbances of
duplicate determinations were plotted against the
concentration of the standard points (linear scale). The
amount of IgG, IgA, and IgM was calculated and the
results were expressed as the approximate number of
molecules of immunoglobulin per RBC, using
Avogadro’s constant and the molecular weights of IgG
or IgA (160,000 daltons) and IgM (970,000 daltons).
According to Avogadro’s constant, 1 µg contains 376 ×
1010 molecules of IgG or IgA and 62 × 1010 molecules of
IgM. The number of molecules of immunoglobulins on
RBCs is equal to the concentration in µg/mL of Igs in
eluates, multiplied by the molecules of Igs in 1µg, and
divided among the number of RBCs in 1 mL.
Effect of test sample milieu on IgG, IgA, and IgM
quantitation by ELISA
We obtained free hemoglobin from human RBCs as
follows:To 6 mL × 10 washed RBCs an equal volume of
hypotonic lytic solution (5M sodium phosphate buffer,
pH 7.4) was added. We placed the mixture in a 37°C
water bath for 10 minutes. The hemoglobin solution
was collected by centrifugation at 370 × g for 30
minutes at room temperature and dialyzed overnight
against 20 volumes of PBS. IgG, IgA, and IgM standard
48
curves from reference serum (Nor-Partigen, Behring)
were prepared in the hemoglobin solution and one
sample without immunoglobulins was used as a blank.
To 1 volume of each point of standard curves prepared
in the hemoglobin solution and the blank, an equal
volume of diethyl ether was added and mixed. The
mixture was incubated at 37°C for 10 minutes. The
upper layer of ether was carefully discarded and the
residual evaporated at 37°C for 15 minutes. BSA and Tw
20 were added to each sample to obtain a concentration
of 0.4% and 0.05%, respectively. The ELISA was carried
out with standard curves with this treatment and with
standard curves prepared in PBS/Tw/BSA. The slopes of
the curves were compared with the t test.
Analytical evaluation of the ELISA
The limit of detection was calculated as the
concentration of the dilution of the first point of the
curves that gave an absorbance more than the mean
absorbance of the blank, plus 3 standard deviations
(SDs). The undiluted and diluted 1 in 5, 1 in 10, 1 in
100, and 1 in 500 in PBS/Tw/BSA of hemoglobin
solution treated with ether and the PBS/Tw/BSA were
used as blanks and also as a control for nonspecific
binding by the assay. The mean and SD were calculated
on 20 repeat estimation assays.
The working range of the assay was determined by
testing in replicates of 10 at each point of the standard
curves of IgG, IgA, and IgM and the coefficient of
variation (CV) was calculated. The working range
was accepted as that over which CV was no greater
than 10%.
Parallelism was investigated after serial dilution in
PBS/Tw/BSA of seven eluates made from RBCs with
reactions of 4+ (IgG, IgA) or 3+ (IgM), 1+, and negative
in the DAT with anti-IgG (BPL; Elstree, UK), -IgA, and
-IgM (CLB; Amsterdam).
The eluate of a pool of ten DAT-negative RBCs as
well as two eluates with IgG, IgA, and IgM
autoantibodies, and a standard human serum with
known
concentration
of
immunoglobulins
(Immunotrol, Bio-Merieux, France) adjusted to a
concentration of 0.052 µg/mL of IgG, IgA, and IgM,
were used to determine the precision of the ELISA. The
intra-assay and the inter-assay coefficient of variation on
separate days were calculated on 20 determinations.
Test samples
The ELISA was carried out in duplicate in ether
eluates from 200 healthy blood donors with a negative
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
Quantitation of red cell immunoglobulins
Results
Curves of IgG, IgA, and IgM represent the mean of
ten consecutive standard curves, each performed in
duplicate. Immunoglobulin standards made up in
hemoglobin and treated with ether did not significantly
affect the standard curves (p > 0.49) (Fig. 1). The
hemoglobin solution treated with ether used as a blank
gave values of optical density from 0.053 to 0.061
similar to the PBS/Tw/BSA (0.051). The working ranges
for the ELISA were found to be 0.0025 to 0.07 µg/mL
for IgG and IgM and 0.0025 to 0.06 µg/mL for IgA. The
limit of detection for IgG corresponded to 0.00062
Optical density
Immunohematologic studies
The immunoglobulin classes of the autoantibodies
were determined by the DAT13 using monospecific antiIgG (BPL), -IgM, and -IgA reagents (CLB, Amsterdam)
and in the eluates by a microplate test.
The microplate test was performed using
microtiter plates with V-shaped bottom wells (Greiner)
as follows: 10 µL of the mixture of three kinds of
packed group O RBCs (R1R1, R2R2, and rr), were
incubated with 100 µL of the eluate at 37°C for 1 hour
in a tube. The RBCs were washed × 4 and 0.5% of RBC
suspensions in saline were made. Anti-IgG, -IgA, and
-IgM were diluted 1 in 2 , 1 in 5, 1 in 10, and 1 in 20 in
PBS with 5% of fetal calf serum (Gibco, UK). Then 25
µL of the antiserum dilutions and the diluent (as
negative control) were transferred into appropriate
microtiter wells and an equal volume of the sensitized
RBC suspension was added. After overnight incubation
at 4°C, the microtiter plate was placed at a 60° angle for
10 minutes, then inverted for 5–10 minutes and read.
When the result was positive, the cells remained
together in a tight button, when negative they ran
down in a smear.
All the patients with a positive DAT had IgG
autoantibodies. In 11 and six patients, IgA and IgM
autoantibodies, respectively, were also present. Similar
patterns of immunoglobulin classes were found in the
eluates and with the DAT. Antibodies were not
detected in eluates from patients with AIHA and a
negative DAT.
IgG, µg/mL
Optical density
DAT, 60 DAT-positive patients with warm AIHA, and
two patients with AIHA and a negative DAT. Hemolytic
anemia was documented by a low hemoglobin
concentration, increased reticulocytes, increased
unconjugated bilirubin concentration, the presence of
marrow erythroid hyperplasia, and the exclusion of
other hemolytic disorders. Thirty-nine patients had
idiopathic AIHA; the hemolytic anemia in the
remainder was associated with other conditions: four
patients had systemic lupus erythematosus, four had
unspecified respiratory infections, four had chronic
lymphocytic leukemia, three were on methyldopa,
three had chronic active hepatitis, two had multiple
myeloma, two had acute lymphocytic leukemia, and
one had non-Hodgkin’s lymphoma. Patients’ ages
ranged from 1 to 84 years (median 45 years) and 41
were female and 21 were male. The eluates from blood
donors were tested undiluted and diluted 1 in 5; those
for patients were tested after serial dilution in
PBS/Tw/BSA to ensure that the tests were carried out
within the working ranges for the assay.
IgA, µg/mL
Fig. 1.
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
Curves for quantitating IgG (Fig. 1A) and IgA (Fig. 1B) with
immunoglobulin standards prepared in PBS,Tween 20 (0.05%),
and BSA (0.4%) (●) and in hemoglobin treated with ether (●).
The curve for IgM is similar to that for IgG.
49
A.A. BENCOMO ET AL.
µg/mL, for IgA to 0.0011 µg/ml, and for IgM to 0.0068
µg/ml (approximately 1 molecule of Igs per RBC). The
ELISA showed excellent parallelism for all the samples
diluted in the measuring ranges with interdilutional
coefficent of variation from 5.9% to 16% for IgG
samples, from 2.9% to 13% for IgA samples, and from
2.4% to 8.6% for IgM samples (Fig. 2). The intra-assay
variation ranged from 3.6% to 6.7% for IgG samples,
from 3.1% to 6.5% for IgA samples, and from 5.5% to
6.7% for IgM samples. The inter-assay variation ranged
from 9.2% to 10% for IgG samples, from 7.5% to 9.5%
for IgA samples, and from 8.2% to 9.3% for IgM
samples.
It was established that the immunoglobulins
present in the eluates were not contaminated by serum
immunoglobulins after ten washes of the RBCs (as
A
IgG,µg/mL
Strength
reaction
4+
described in Materials and Methods). The supernatant
of the last wash gave values of optical density similar to
those of the blank in the ELISA.
The ELISA on healthy blood donors gave mean and
standard deviation values of 58 ± 35 molecules of IgG
per RBC (range from 2 to 146), 16 ± 11 molecules of
IgA per RBC (range from 1 to 81), and 3 ± 2 molecules
of IgM per RBC (range from 1 to 11).
In AIHA patients with a positive DAT, the range of
results for IgG was from 206 to 20,000 molecules per
RBC, with a mean of 3435 molecules. In the group of
patients with IgA autoantibodies the range of results
was from 90 to 250 molecules per RBC, with a mean of
157 molecules. An increased level of IgA, with a range
from 94 to 385 molecules per RBC, was found in 12
patients with a negative DAT and in the test of eluates
by microplate with anti-IgA. In the remainder of cases,
the values of molecules of IgA per RBC were within the
range found in blood donors. A mean value of 69 with
a range from 26 to 109 molecules per RBC was
obtained in patients with IgM autoantibodies and
results were within the normal range in the rest of the
patients.The results are shown in Figure 3.
Increased IgG levels of 370 and 460 molecules per
RBC were observed in two patients with AIHA and a
negative DAT. In one case an increased level of IgA
(119 molecules per RBC) was also found.
Discussion
Dilutions
Strength
reaction
1+
IgG,µg/mL
B
Dilution factor
Fig. 2.
50
The relationship between the diluted samples and the amount of
IgG detected in the eluates with reactions of 4+ (●) (Fig. 2A), and
1+ (■) and negative reactions (▲) (Fig. 2B) in the DAT with antiIgG. Similar results were obtained in the assays for estimation of
IgA and IgM concentration after serial dilutions of the eluates.
The sandwich ELISA described provides a method
for the quantitation of RBC-bound IgG, IgA, and IgM
immunoglobulins. The assay was carried out in ether
eluates because our laboratory routinely performs
elution in the investigation of autoantibodies in AIHA
and because the testing of eluates is a more sensitive
method to detect immunoglobulins than direct testing
of the RBCs.14
Differences in the milieu in test samples and
immunoglobulin standards can produce nonspecific
effects, which modify the kinetics of antigen-antibody
reactions and could affect the estimation of Ig
concentration from the standard curve.11 However, the
presence of hemoglobin and the ether treatment of
immunoglobulins did not affect standard curves.
Therefore, all the test samples were investigated with
immunoglobulin standards prepared in PBS/Tw/BSA.
Hemoglobin has a peroxidase activity and obscures the
antibody assay if a peroxidase conjugate is used.15 In
this ELISA the hemoglobin in eluates was removed by
washing and did not interfere with the peroxidase
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
Quantitation of red cell immunoglobulins
IgG
Fig. 3.
C
Molecules per red cell
Molecules per red cell
B
Molecules per red cell
A
IgA
IgM
The number of IgG (A), IgA (B), and IgM (C) molecules per RBC in patients with AIHA. The increased values for IgA and IgM (●) were found in
patients with positive DATs with anti-IgA and -IgM, respectively. An increased level of IgA was also found in 12 patients without IgA autoantibodies demonstrable on their RBCs. A heavy horizontal line inside Figure 3 (A, B, and C) shows the highest value of molecule per red cell obtained in
healthy blood donors.
conjugate. The unaffected immunoglobulin detection
after ether treatment was not surprising in view of the
known use of this elution method for recovering
antibody attached to RBCs,16 although Dumaswala
et al.17 found decreased detectability of IgG after
treatment with another organic solvent such as xylene,
using an immunoblotting technique with peroxidaselabeled anti-IgG.
The ELISA showed a wide measuring range; the
lower limit corresponded approximately to one
molecule of immunoglobulin per RBC. Accordingly,
this assay is suitable for the quantitation of
immunoglobulins on RBC eluates from DAT-positive
patients and from normal subjects. Although the
working range had an upper limit of 0.07 µg/mL for
IgG and IgM, and 0.06 µg/mL for IgA, it could be
extended by dilution of high concentration samples
with PBS/Tw/BSA. This was in agreement with the
excellent dilution parallelism over the measuring range
showed by assay with all sample types tested.
We found the mean amount of 58 IgG, 16 IgA, and
3 IgM molecules per RBC by using the eluates from
healthy donors. Other methods gave similar results for
IgG and IgM.4,5,8–10 There has been only one previous
report of the number of RBC-associated IgA molecules
on normal human RBCs.10 That study reported a
median of < 29 molecules per RBC, which is
comparable to the amount quantitated by our assay.10
There was considerable variation in the number of
IgG, IgA, and IgM molecules per RBC in patients with
AIHA, demonstrating the ability of the assay to measure
widely different degrees of sensitization in clinical
samples. The results are comparable with previous
studies where the reported ranges in patients with
AIHA were from 230 to about 30,000 IgG molecules,
from 20 to 168 IgM molecules, and from < 29 to 4500
IgA molecules per RBC.8,9,18,19
We encountered IgA autoantibodies in 18 percent
of DAT-positive patients, which is similar to the 14
percent reported previously by Sokol et al.14 An
increase of RBC-bound IgA by ELISA was also found in
12 patients without IgA autoantibodies demonstrable
on RBCs by the DAT and in the eluates by a microplate
test. The clinical significance of antibodies only
detected by ELISA should be further elucidated
because an increased amount of cell-bound
immunoglobulins may also be due to immune
complexes and nonspecific adsorption of Igs onto
RBCs rather than as a result of autoantibodies.3
Similar considerations are applicable to the results
found in two patients with AIHA and a negative DAT
without antibodies demonstrated in eluates. However,
in these cases there were findings to suggest that IgG
and IgA (in one case) antibodies detected by ELISA
were responsible for the hemolysis. The hemolytic
anemia in these patients was associated with chronic
lymphocytic leukemia and non-Hodgkin’s lymphoma,
respectively. It is known that the incidence of AIHA
is higher in patients with these hematological
malignancies than in the general population.20
Previous investigators have demonstrated IgG and IgA
autoantibodies on RBCs with more sensitive methods
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
51
A.A. BENCOMO ET AL.
than the DAT in patients with a DAT-negative AIHA.21–25
The patients were diagnosed by clinical manifestations
including the exclusion of other hemolytic disorders
and a positive clinical course following administration
of corticosteroid.
These results showed that the quantitative
determination of immunoglobulins on eluates by ELISA
appears to be useful for the serologic diagnosis of DATnegative AIHA, although the assay should be evaluated
with a larger number of cases.
Previous investigation identified RBC autoantibodies of the IgM class in approximately 30 percent of
patients with warm AIHA by enzyme-linked DAT.26 We
found a frequency (around 10%) of warm IgM
autoantibodies that is similar to those found in earlier
studies20 in both the DAT and the ELISA. This
difference may be attributable to multiple washing
required by our assay, which carries the possibility for
loss of small quantities of IgM autoantibodies prior to
elution. In addition, it can be difficult to obtain IgM
autoantibodies in eluates. An investigation showed that
heat elution was the better method compared to acid
stromal, chloroform-trichlorethylene, and freeze-thaw
methods.27 We detected IgM antibodies in ether eluates
of only three of the six patients with IgM
autoantibodies, when a conventional IAT with anti-IgM
was used (data not shown). However, all the IgM
autoantibodies revealed by the DAT were detected in
the eluates by the microplate test and ELISA. As
described previously, ELISA also quantitated a very
small quantity of IgM on RBCs of healthy blood donors.
We have no explanation for our different findings from
a previous report.26
In essence, the ELISA, using ether eluates, is a
sensitive method to follow patients with RBC
autoantibodies as well as immunoglobulins bound to
RBCs in healthy individuals. The assay can be also
extended to measure IgG subclasses or C3 by using
appropriate specific antiserum.
52
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and autoimmune hemolysis. Transfusion 1990;
30:714-7.
27. Ellis JP, Sokol RJ. Detection of IgM autoantibodies in
eluates from red blood cells. Clin Lab Haematol
1990;6:99-104.
Antonio A. Bencomo, BSc, Senior Investigator, Head of
the Department of Immunohematology, Institute of
Hematology and Immunology, P.O. Box 8070, Ciudad
de la Habana, CP 10800, Cuba; and Martha Díaz,
MD;Yalile Alfonso, MS; Odalys Valdés, MD; and María
E. Alfonso, MD, Senior Investigator, Department of
Immunohematology, Ciudad de la Habana, Cuba.
Attention: State Blood Bank Meeting Organizers
If you are planning a state meeting and would like copies of Immunohematology for distribution, please
contact Mary McGinniss, Managing Editor, 4 months in advance, by phone or fax at (301) 299-7443.
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
53
ABO and Rh(D) blood typing on
the PK 7200 with ready-to-use kits
P. MONCHARMONT, A. PLANTIER,V. CHIRAT, AND D. RIGAL
The performance of ready-to-use kits was evaluated on the PK 7200
blood grouping system. The Olymp Group (kit 1) and Olymp
Group II (kit 2) containing anti-A, -B, -AB, and -D reagents were
tested for first and second determinations of A, B, and D antigens.
More than 500 RBC samples, including several variant ABO and D
phenotypes, were evaluated for specificity, repeatability,
reproducibility, and sensitivity. Specificity was tested with wellcharacterized reagent RBCs. Repeatability was established by at
least 12 assays per run with three reagent RBCs, and reproducibility
was established on one run per day for 5 days. No discrepancy
was observed in ABO and D determinations with either kit. In
repeatability, three discrepancies were found with group A and B
RBCs with kit 1. In reproducibility, no discrepancies were
observed. The kit 1 anti-A reagent detected A3 but not Ax RBCs and
anti-AB detected both. A B3 RBC was detected by both kits. Among
eight weak D phenotypes, six were positive with kit 1. With kit 2,
only one of five weak D phenotypes was detected.
Immunohematology 2003;19:54–56.
Key Words: ABO, Rh(D), PK 7200 automated blood
grouping system
Improvements in ABO and D typing may be related
to the introduction of new technologies1 or to changes
in reagents.2 New technologies include microplate
testing, i.e., the gel test,3 and solid phase systems.4
Blood typing reagents manufactured from human or
animal plasma or serum have been replaced with new
monoclonal reagents. With new reagents and
technologies, automation of blood typing using
computerized systems has been tested and introduced
into the laboratories. Recently, ready-to-use reagents
linked to one automated system were offered by the
manufacturer. We tested their ready-to-use kits for ABO
and D blood typing on a fully automated blood
grouping system, the PK 7200, and detected certain
problems regarding identification of weak D
phenotypes.
Materials and Methods
Samples
Whole blood was collected, in tubes containing
EDTA (Vacutainer, Becton Dickinson, Le Pont de Claix,
France), from healthy blood donors whose ABO and D
54
blood groups had been determined on at least two
prior donations. Samples were processed immediately
after collection (ABO and D, an IAT alloantibody
screening, anti-A and -B, and Hct). At a first donation
each sample had been evaluated on the automated PK
7200 (Olympus Biology, Rungis, France) for ABO and D
blood groups with monoclonal anti-A, -B, and -AB
reagents (first kit, AbiO+, Lyon, France) and one
monoclonal anti-D reagent (IgM like + IgG Bioclone®,
Ortho-Clinical Diagnostics, Issy, les Moulineaux,
France). In a second donation, the samples had been
tested with other monoclonal anti-A, -B, and -AB
reagents (AbiO+) and one other monoclonal anti-D
reagent (Totem®, IgG + IgM, Diagast Laboratories, Loos,
France). When a weak A or B phenotype was
suspected, a gel test (DiaMed, Cressier sur Morat,
Switzerland)3 was used, and if necessary, an elution was
performed using anti-A or -B reagents. For a weak D,
the sample was evaluated by an IAT in LISS and by a gel
test (DiaMed).
Methods
Two ready-to-use kits, the Olymp Group (kit 1) and
Olymp Group II (kit 2) (Diagast Laboratories, Loos,
France) were evaluated on the PK 7200 (Olympus
Biology, Rungis, France). Monoclonal anti-A, -B, -AB, and
-D reagents were used in these kits. Clones for each kit
were different (see Table 1). Specific microplates are
used on the PK 7200. In each well, hemagglutination
reading is enhanced by a charge-coupled device (CCD)
Table 1. Clones included in each ready-to-use kit
Reagent
Specificity
Anti-A
Anti-B
Anti-AB
Anti-D
Olymp group
(kit 1)
Clones
2521 B 8 + 16243 G 2
9621 A 8
2521 B 8 + 16243 G 2
+ 16247 E 10 + 7821 D 9
P3x61 + P3x21223 B 10
P3x290 + P3x35
Olymp group II
(kit 2)
Clones
9113 D 10
164 B 5 G 10 + 7821 D 9
152 D 12 + 9113 D 10
HM 10
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
ABO and D typing on the PK 7200
camera with a high-resolution image. Results are
reported as positive, negative, or indeterminate.
Control samples: group A, B, AB, O, D+, and D– were
tested before and after each run.
Four parameters were evaluated: specificity,
repeatability, reproducibility, and sensitivity. For each
antibody specificity, at least 100 RBC samples known to
be positive for the antigen and 100 RBC samples
known to be negative for the antigen were tested.
Repeatability was established on at least 12 assays per
run, with three well-characterized RBC samples per
reagent. Reproducibility was determined in one run
per day for 5 successive days with the same RBC
samples, which were stored at 4°C. For sensitivity, the
following weak phenotypes were tested: A3, Ax, and B3
for the ABO system and weak D RBCs. When a falsely
negative test was obtained with a reagent, the variant
RBCs were retested.
Results
Three hundred samples of A, B,AB, and O RBCs and
240 D+ and D– RBCs (122 D+, 118 D–) were tested
versus kit 1. Two hundred sixty-nine samples of A, B,
AB, and O RBCs and 329 samples of D+ and D– RBCs
(226 D+ and 103 D–) were tested versus kit 2. No
discrepancies were observed in A, B, or D
determinations. However, in repeatability testing, three
discrepancies were detected with group A and B RBCs
with kit 1. Two were group A and one was group B. No
discrepancy was found with kit 2. In reproducibility
testing, no discrepancy was observed with kit 1 or kit
2 with RBCs tested for 5 successive days (Table 2).
Table 2. ABO discrepancies (repeatability and reproducibility)
RBCs
Group A
Group B
Group AB
Repeatability*
Kit 1
Kit 2
2
1
0
0
0
0
Reproducibility†
Kit 1
Kit 2
0
0
0
0
0
0
*Established on ≥ 12 assays per run vs. three well-characterized RBCs per reagent
†
Determined by one run per day for 5 successive days with the same RBCs
Kit 1 anti-A reagent detected A3 RBCs but not the Ax
RBCs, and the anti-B reagent gave a positive result with
the B3 RBCs. These three variant RBCs were detected
also by the anti-AB reagent. Kit 2 detected the A3 and
B3 RBCs. Unfortunately, no fresh Ax RBCs were available
at the time we tested kit 2.
With kit 1, eight known weak D RBC samples were
tested. Four were positive, two gave indeterminate
results, and two gave a falsely negative result. With kit
Table 3. Detection of weak D phenotypes
Kit 1
(8)*
Kit 2
(5)*
positive indeterminate negative
4
2
2
positive indeterminate negative
1
0
4
*Number of samples tested
2, of the five weak D RBC samples tested, only one was
positive, none gave indeterminate results, but four gave
falsely negative reactions (Table 3).
Discussion
Our findings indicate that using the PK 7200 and
ready-to-use kits, difficulties remain, particularly in
typing blood donors with variant RBCs (i.e., A3 and
weak D). With the first-generation Olympus blood
grouping system, the PK 7100, sensitive monoclonal
anti-D reagents did not guarantee detection of weak D
phenotypes.5 Confirmation with another sensitive
technology (e.g., a gel test) was needed. The detection
system for the PK 7200 includes a CCD camera to
enhance the detection of hemagglutination in the
reaction wells and, therefore, increase the effectiveness
of the tests. Nevertheless, to avoid falsely positive or
falsely negative reactions, reagents must be adapted to
the automated system in use. Recently, some
manufacturers have offered ready-to-use reagents.
Our study shows that two ready-to-use kits failed to
detect weak D phenotypes. Despite improvements in
the PK 7200, kit 2 failed to detect four of five weak D
phenotypes. The anti-D reagents in kits 1 and 2 are
different. Kit 1 anti-D reagent contains a mix of four
monoclonal antibodies, while kit 2 has only one. The
anti-D reagents in both kits include an IgM monoclonal
antibody (P3x61 clone for kit 1, and HM 10 for kit 2).
In fact, Jones et al.6 recommend the use of an IgM
monoclonal anti-D with high avidity in saline for
detection of weak D phenotypes. Williams7 observed
good detection of weak D phenotypes with both the
IgM monoclonal antibody clone P3x61 (84%) and
clone HM 10 (74%). In our short study, kit 1 anti-D
(P3x61) detected 75 percent (6 of 8) of the weak D
phenotypes, whereas with kit 2 (HM 10) anti-D
reagent, which contains one monoclonal antibody, only
20 percent (1 of 5) were detected. Futhermore, Scott
and Voak8 state that, for detecting all weak D
phenotypes, a single monoclonal anti-D is not
adequate. To increase the efficiency in blood donor
typing, particularly for weak D phenotypes, kit 2 must
be used with another kit for the same automated
system or with another technology (e.g., a gel test).
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
55
P. MONCHARMONT ET AL.
Improvement in kit 2, i.e., addition of one or more antiD monoclonal antibodies, is needed. Lastly, detection of
Ax RBCs is increased by using an anti-AB reagent.
Several hypotheses could explain the discrepancies
observed with kit 1 anti-A and anti-B reagents with the
repeatability assay. First, a lower volume of RBCs
and/or reagent could have been dispensed by the PK
7200 in the wells of the microplate. Nevertheless, at
the time of the assay, no other grouping difficulty was
observed. Secondly, a wrong reading was possible, but
no problem was noted with the CCD camera. Even
though the assay was performed according to the
manufacturer’s directions, these discrepancies are not
clearly explained. Nevertheless, discrepancies with kit
1 anti-A and anti-B reagents were only observed with
the repeatability assay. The other tests for ABO blood
grouping gave good results with kit 1.
Conclusion
The ready-to-use kits for ABO and D blood typing
on the PK 7200 blood grouping system are efficient in
specificity, repeatability, and reproducibility for routine
testing of RBCs with normal ABO and D antigen
expression. However, numerous discrepancies, namely
falsely negative reactions, remain with kit 2 for weak D
phenotypes. We recommend the use of kit 2 in
association with kit 1 or with another ready-to-use
reagent or technology for weak D typing.
2. Le Pennec P Y. Evolution of reagents in erythrocyte
immunohematology. Transfus Clin Biol 2000;
7(Suppl 1):40-3.
3. Lapierre Y, Rigal D, Adam J, et al.The gel-test : a new
way to detect red cell antigen-antibody reactions.
Transfusion 1990;30:109-13.
4. Beck ML, Rachel JM, Sinor LT, Plapk FV. Semiautomated solid phase adherence assays for pretransfusion testing. Med Lab Sci 1984;41:374-81.
5. Moncharmont P, Giannoli C, Chirat V, et al. Weak
rhesus D (RH 1) grouping on the Olympus PK 7100
blood grouping system.Transfus Med 1997;7:66-7.
6. Jones J, Scott ML,Voak D. Monoclonal anti-D
specificity and RH D structure: criteria for selection
of monoclonal anti-D reagents for routine typing of
patients and donors.Transfus Med 1995;5:171-84.
7. Williams M. Monoclonal reagents for Rhesus-D
typing of Irish patients and donors: a review. Br J
Biomed Sci 2000;57:142-9.
8. Scott ML,Voak D. Monoclonal antibodies to Rh D—
development and uses. Vox Sang 2000;78
(Suppl 2):79-82.
Pierre Moncharmont, MD, PhD, Chief of office,
Department of Immunology, E.F.S. Rhône-Alpes Site de
Lyon, 1-3 rue du Vercors, 69364 Lyon cedex 07 France;
Annick Plantier, Technician, Véronique Chirat,
Technician, and Dominique Rigal, MD, PhD, Director,
E.F.S. Rhône-Alpes Site de Lyon, Lyon, France.
References
1. Chiaroni J, Mannessier L. Methodological evolutions
in immunohematology. Transfus Clin Biol 2000;
7(Suppl 1):44-50.
Notice to Readers: Immunohematology, Journal
of Blood Group Serology and Education, is
printed on acid-free paper.
56
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
Jk and Mi.III phenotype
frequencies in North Vietnam
NGUYEN THI HUYNH, DEREK S. FORD,TRAN THI DUYEN, AND MAI THANH HUONG
One hundred voluntary blood donors in Hanoi were typed for
antigens in the MNS, Rh, Kell, Duffy, and Kidd blood group systems.
They were also tested for the presence of the Mi.III (GP.Mur)
phenotype and Lewis system antigens. The Jk phenotype
frequencies were markedly different from those previously
reported. The frequency of the Mi.III phenotype was similar to that
reported in Chinese and Taiwanese. Immunohematology 2003;
19:57–58.
Key Words: blood groups, phenotype frequencies,
Vietnam, Jk, Miltenberger
There is minimal information on blood group
frequencies in Vietnam in English-language scientific
journals. The few reports have not included the
Miltenberger (Mi) phenotypes, of which Mi.III
(GP.Mur) is present with a relatively high frequency in
Chinese,Thai, and Taiwanese populations. There is also
a likelihood of erroneous information on Jk
phenotypes in previous publications.1,2
Materials and Methods
manufacturers’ instructions. Blood samples were
collected randomly from volunteer blood donors at the
blood bank at Thanh Nhan Hospital, Hanoi.
Results
Most of the blood group phenotype frequencies
were similar to those in other Asian populations (Table
1). However, the Jk phenotype frequencies were
markedly different from those previously reported in
Vietnam (Table 2). The Mi.III phenotype was present at
a level similar to that reported in the Chinese and the
Taiwanese (6%), but testing of an additional 60 donors
for Mi.III raised the frequency to 6.5 percent (Table 1).
Discussion
The phenotype frequencies reported here should
be considered to be approximate and some
amendments may be expected when larger numbers of
Vietnamese are tested.
For the majority of the phenotypes, the results
corresponded closely to those obtained on blood
donors in Ho Chi Minh City (South Vietnam) by Tran
Van Bé,1 but significant differences were noted in the
Jk system.
The discrepancies between our Jk typings and
those reported in earlier studies of the Vietnamese
population are striking.1,2 The earlier studies reported
a high frequency of the Jk(a–b–) phenotype, while the
To investigate the frequency of the Mi.III (GP.Mur)
phenotype and to reevaluate Jk phenotype
frequencies, 100 volunteer blood donors in Hanoi were
typed for antigens of the MNS, Rh, Kell, Duffy, and Kidd
blood group systems and antigens in the Lewis system.
ABO groups were not performed, as previous reports
from different areas of Vietnam were in general
agreement. A total of 160 donors were tested for Mi.III,
using anti-Mur and anti-Mur/Hop sera.
Blood group antisera were
donated by CSL Biosciences Table 1. Approximate phenotype frequencies of Hanoi donors based on testing 100 donors
MNSs
Rh
Fy
Jk
Kell
Le
(Australia) and Gamma Biologicals
Ms
58%
CDe 57%
Fy(a+b–) 88%
Jk(a+b–) 25%
K–k+ 100%
Le(a+b–)
a
b
(USA). Anti-Jk and anti-Jk were IgM
CcDe 13%
Fy(a+b+) 11%
Jk(a+b+) 53%
K+k+ 0%
Le(a–b+)
monoclonal reagents. Human anti- MSs 7%
MNs 18%
cDEe
6%
Fy(a–b+) 1%
Jk(a–b+) 22%
K+k–
0%
Le(a+b+)
Mur and anti-Mur/Hop sera were
Ns
17%
cDE
3%
Le(a–b–)
kindly supplied by Mr M.K. Mak of
CcDEe 19%
the Hong Kong Red Cross Blood
cDe
1%
Transfusion Service. All blood
CDEe 1%
typings were performed manually
Mi.III (GP.Mur) 6.0%*
by the tube method according to *Testing of an additional 60 donors raised the incidence to 6.5%
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
0%
62%
8%
30%
57
NGUYEN THI HUYNH ET AL.
References
Table 2. Different phenotype frequencies in the Jk system, according to
three studies
References
This study
1
Jk
(a+b–)
Jk
(a+b+)
Jk
(a–b+)
Jk
(a–b–)
25%
53%
22%
0%
Tran Van Bé
23.85%
0%
21.10%
55.05%
Do Trung Phan et al.2
13.68%
5.26%
62.12%
18.94%
Jk(a+b+) phenotype was completely absent in one
study1 and present at only 5.26 percent in the other.2
Our results show an absence of the Jk(a–b–)
phenotype, with frequencies much closer to those
reported in other Asian populations. It is possible that
the Jk typings in the earlier Vietnamese studies were
erroneous due to the use of low-potency antisera that
failed to detect the Jk(a+b+) phenotype.
The presence of the Mi.III phenotype in the
Vietnamese population had not been reported
previously, but its presence was expected as it had
been reported in Thailand,3 Hong Kong,4,5 China,6
Taiwan,7 and in Chinese in Australia.8 Mi.III was
present in 6.5 percent of 160 donors. Variation in
reactions with two reagents labeled as anti-Mi.III
suggests that Mi.VI (Bun) may also be present in North
Vietnam as it is in Thailand. Only those samples that
reacted with both reagents were classified as Mi.III in
the tables. King et al.9 have shown the value of
immunoblotting in determination of Mi phenotypes,
but molecular biological and immunoblotting
techniques could not be performed in this study. It is
hoped that such tests will be performed in the future
to more accurately identify the Mi phenotypes present.
Antibodies to the antigens represented in the Mi.III
phenotype have previously been reported as causing
both hemolytic transfusion reactions10 and hemolytic
disease of the newborn11 in Asians. Given a 6.5 percent
frequency of the Mi.III phenotype in the Vietnamese, it
is possible that its presence has resulted in some of the
cases of transfusion reactions and hemolytic disease of
the newborn that have been reported to be due to
unidentified antibodies. It becomes important that
screening panels used in Vietnam include RBCs with
the Mi.III phenotype, as do those used in Hong Kong.
Further studies to determine whether the
differences in JK phenotype frequencies between tests
on Hanoi (North Vietnam) donors and on donors from
Ho Chi Minh City (South Vietnam) represent a true
variation in the population are warranted. Testing of
ethnic minorities found in the mountain regions of
Northwest Vietnam would also be of anthropological
interest.
58
1. Tran Van Bé. Blood groups on red cells. J Haematol
Blood Tx Soc Viet Nam, 1994;3:1-4.
2. Do Trung Phan, Tran Hong Thuy, Bui Mai An, et al.
Haematology indexes in mature people of Viet
Nam. In: Memoirs of scientific researches of Ha Noi
Medical College 1998;1:162-9.
3. Chandanyingyong D, Pejrachandra S. Studies on the
Miltenberger complex frequency in Thailand and
family studies. Vox Sang 1975;28(2):152-5.
4. Lin CK, Mak KH, Cheng G, et al. Serologic
characteristics and clinical significance of
Miltenberger antibodies among Chinese patients in
Hong Kong. Vox Sang 1998;74:59-60.
5. Poole J. The Mi.III phenotype among Chinese
donors in Hong Kong: immunochemical and
serological studies. Transfus Med 1991;1:169-75.
6. Metaxas-Buehler M, Metaxas MN, Sturgeon P. MNS
and Miltenberger frequencies in 211 Chinese. Vox
Sang 1975;29:394-9.
7. Broadberry RE, Lin M. The distribution of the Mi.III
(GP.Mur) phenotype among the population of
Taiwan. Transfus Med 1996;6:145-8.
8. Winkler IG, Chi-Shen Wu, Woodland N, Flower R.
Detection of Miltenberger blood group
polymorphisms: should cells carrying Miltenberger
antigens be included in screening panels? Topics in
Transfus Med 1999;6(1):9-13.
9. King MJ, Poole J, Anstee DJ. An application of
immunoblotting in the classification of the
Miltenberger series of blood group antigens.
Transfusion 1989;29(2):106-12.
10. Lin M, Broadberry RE. An intravascular haemolytic
transfusion reaction due to anti-Mi(a) in Taiwan.
Vox Sang 1994;67:320.
11. Lin CK, Mak KH, Szeto SC, et al. First case of
haemolytic disease of the newborn due to anti-Mur
in Hong Kong. Clin Lab Haematol 1996;18:19-22.
Nguyen Thi Huynh, Tran Thi Duyen, Mai Thanh
Huong, Hematology/Blood Bank, Thanh Nhan
Hospital, Hanoi, Vietnam; and Derek S. Ford, BAppSci
(corresponding
author),
Immunohematology
Consultation and Education Services, PO Box 5436,
Port Macquarie, NSW 2444, Australia.
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
COMMUNICATIONS
Letter to the Editors
Vel– donors in Yugoslavia
The Vel antigen belongs to the high-frequency antigen series (901), ISBT number 901.001.1 It was described for
the first time in 1952 when it was discovered in the serum of a previously transfused patient,2 and the second
example was described in 1955.3 The Vel– phenotype appears to be inherited as a recessive character.4 Since the
frequency of the antigen is > 99 percent and the antibody usually causes hemolysis,4 Vel– donors have found a place
on many rare blood group donor lists. Anti-Vel is sometimes difficult to identify. In 1994, a misidentified anti-Vel,
which caused a fatal hemolytic transfusion reaction due to inappropriate use of serologic techniques, was described.5
Anti-Vel was detected in the serum of a group A patient during routine selection of blood for transfusion. She
had a history of two pregnancies and had been transfused with four units of blood 4 years before.
Serum prepared from the patient’s plasma, obtained by manual plasmapheresis, was used to type donors’ blood
for the Vel antigen. We typed 3108 group A donors and 3633 group O donors.
The serum demonstrated strongly positive reactions at room temperature, by IAT, and versus enzyme-treated
RBCs. Antibody specificity, anti-Vel, was determined by the IBGRL in Bristol. Further investigation of the patient’s
RBCs confirmed the following phenotype: group A, CcDEe, NNss, Lu(a–b+), kk, Le (a–b+), Fy(a–b+), Jk(a–b+), P1 +,
and Vel–. The antibody not only reacted in saline and by IAT, but also demonstrated hemolytic activity (IBGRL).
The titer of anti-Vel was 256 at room temperature and by IAT. The IAT was also performed using monospecific
reagents. Anti-IgG and -IgA reacted 2+ by IAT. Anti-IgM, -C3, and -C4 reacted 4+ by IAT.
Out of 6741 donors typed for Vel, two Vel– donors (0.02966%) were identified. The calculated frequency of
Vel was 0.9997, which is in accordance with the results obtained in other Caucasian populations.
This case points out the necessity of establishing a list of rare donors in our country and listing them with an
international blood exchange.
References
1. Reid ME, Lomas-Francis C. The blood group antigen facts book. San Diego, CA: Academic Press, 1997:373.
2. Sussman LN, Miller EB. Un nouveau facteur sanguin ‘Vel’. Rev Hématol 1952;7:368.
3. Levine P, Robinson EA, Herrington LB, Sussman LN. Second example of the antibody for the high-incidence
blood factor Vel. Am J Clin Pathol 1955;25:751.
4. Issitt PD,Austee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery Scientific Publications,
1998:801-4.
5. Storry JR, Mallory DM. Misidentification of anti-Vel due to inappropriate use of prewarming and adsorption
techniques. Immunohematology 1994;10:84-6.
Milan Djokic, MD, PhD
National Blood Transfusion Institute, Immunohematological Reference Laboratory,
St. Sava 39, PO Box 13, 11000, Belgrade, Yugoslavia
Andrija Kuzmanovic, MD
Clinical Center of Kraguzevac, Yugoslavia
Zivko Budisin, MD
National Blood Transfusion Institute, Belgrade
Snezana Srzentic, MD
National Blood Transfusion Institute, Belgrade
Joyce Poole, FIBMS, IBGRL, SW
Blood Transfusion Center, Bristol, UK
Radica Stevanovic
National Blood Transfusion Institute, Belgrade, Yugoslavia
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
59
BOOK REVIEW
Platelets in Thrombotic and Non-thrombotic
Disorders: Pathophysiology, Pharmacology, and
Therapeutics. Paolo Gresele, Clive P. Page, Valentin
Fuster, and Jos Vermylen, eds. Cambridge UK:
Cambridge University Press, 2002. 1124 pp. List Price:
$300.00 (£245.00). ISBN 052180261X. On-line
ordering: http://titles.cambridge.org/catalogue.asp
?isbn=052180261X
Platelets in Thrombotic and Non-thrombotic
Disorders is an informative and extensively referenced
textbook that is highly recommended to biomedical
scientists and clinical hematologists with strong
interests in platelet physiology and disease states. The
editors have assembled an outstanding group of
contributors to provide overviews of platelet
physiology, pathologic platelet disorders, and the
pharmacology of platelet-dependent disease. This
textbook will be most useful to scientists studying the
pathophysiology of platelet disorders and the basic
science of platelet function; many of the chapters,
especially the 29 physiology chapters, include the
relevant historical context and hard-to-find seminal
references that are essential for methodology,
manuscript preparation, and general background. The
textbook was published in 2002; therefore, the most
up-to-date references are from 2000–2001, including
several from 2001 that are listed as in press.
The scope of this textbook encompasses a large
number of separate chapters, necessitating relatively
concise presentation of data and discussions; this
enables the reader to quickly focus on a particular
topic, although more cross-referencing between
chapters would be helpful for future editions. The
chapters on platelet physiology are highly focused to
provide the reader with state-of-the-art knowledge on
distinct aspects of platelet function. For example,
platelet signaling is discussed over four separate
chapters, including the roles of cAMP and cGMP,
tyrosine kinases, protein kinase C, and calcium. These
briefs include important methodological detail that is
well referenced for benchwork. There is a strong group
of platelet pathology chapters that discuss
thrombocytopenia; a more comprehensive background
in earlier chapters on platelet circulation kinetics
might better serve these chapters. One novel aspect of
this textbook is the number of chapters that explore
60
the interactions of platelets with heterotypic cells in
both normal physiology and disease. These include
vascular control of platelet function, leukocyte-platelet
conjugate formation in vascular disease, the role of
platelets in tumor invasiveness and metastatic
potential, and the interaction of platelets with various
pathogens, both in vivo and during storage. Several
other chapters are highlighted below to provide a
snapshot of some of the specific strengths of this text.
A concise, highly relevant overview of the most
critical issues and controversies in platelet storage and
transfusion is provided by Scott Murphy. This chapter
is valuable for hematology and transfusion medicine
fellows and includes an excellent discussion of platelet
preparation and storage methodologies and
comprehensive tables for quick reference. The clinical
focus also encompasses the relevant indications for
platelet transfusion and includes a succinct outline of
alloimmunization and strategies for finding compatible
platelets. Thomas Kunicki authors a readable overview
of gene regulation of platelet function. This chapter
includes details on the regulation of megakaryocytespecific genes found in hematopoietic-derived cell
lines and of transcription factors, including the GATA
and Ets families. Recent studies that have examined
platelet polymorphisms as risk factors for clinically
relevant atherothrombosis are also discussed, including
clearly presented tables on α2β1 density and
polymorphisms of GPIbα,VNTR, and αIIbβ3.
The chapter on platelet procoagulant function by
Hemker is broadly written to give a concise overview
of the interactions between platelets and soluble/
plasma coagulation factors. There has been an
explosion of new information in this field since this
chapter was prepared, but the general principles
related to platelet-associated thrombin generation are
clearly written and serve as a firm background for
further reading and research. Several chapters are
included on the evaluation of trials of antiplatelet
therapy for cardiovascular, cerebrovascular, and
peripheral vascular disease. All of these include
comprehensive tables and detailed analyses of
antiplatelet efficacy and outcomes, as well as summary
recommendations on current therapeutic regimens.
Future scenarios for antiplatelet therapy are
discussed in the chapter on pharmacogenetics. This
chapter highlights some of the possible targets of
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
pharmacogenomic therapy, including those that are
applicable to general risk, e.g., high C-reactive protein
population subsets, and platelet-derived risk factors,
including polymorphisms of GPIIIa (PlA1/2),
differential thienopyridine metabolism, and the novel
P2Y12 receptor; the latter two are relevant to platelet
ADP-receptor antagonist therapy. There is also an
interesting discussion of the possibilities for future
screening of atherothrombotic risk factors, and the
subsequent use of targeted antiplatelet therapy.
Platelets in Thrombotic and Non-thrombotic
Disorders emphasizes strongly focused chapters and
comprehensive referencing on platelet physiology and
function; this extensive framework for up-to-date
platelet knowledge makes this text essential for
hematologists and blood bankers in training, and for
researchers and veteran clinicians with an interest in
hemostasis.
Henry M. Rinder, MD
Associate Professor of Laboratory Medicine
and Internal Medicine (Hematology)
Director, Coagulation Laboratory
Yale University School of Medicine
New Haven, CT 06520-8035
Masters (MSc) in Transfusion and Transplantation Sciences
At
The University of Bristol, England
Applications are invited from medical or science graduates for the Master of Science (MSc) degree in
Transfusion and Transplantation Sciences at the University of Bristol. The course starts in October 2003 and will
last for one year. A part-time option lasting three years is also available. There may also be opportunities to
continue studies for PhD or MD following MSc. The syllabus is organised jointly by The Bristol Institute for
Transfusion Sciences and the University of Bristol, Department of Transplantation Sciences. It includes:
•
•
•
•
•
Scientific principles underlying transfusion and transplantation
Clinical applications of these principles
Practical techniques in transfusion and transplantation
Principles of study design and biostatistics
An original research project
Applications can also be made for Diploma in Transfusion and Transplantation Science or a Certificate in
Transfusion and Transplantation Science.
The course is accredited by the Institute of Biomedical Sciences.
Further information can be obtained from the website:
http://www.blood.co.uk/ibgrl/MscHome.htm
For further details and application forms please contact:
Professor Ben Bradley
University of Bristol, Department of Transplantation Sciences
Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, England
FAX +44 1179 595 342, TELEPHONE +44 1779 595 455, E-MAIL: [email protected]
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
61
BOOK REVIEW
Cellular Therapy: New Frontiers in Transfusion
Medicine. Ronald A. Sacher, MD, ed. Bethesda, MD:
American Association of Blood Banks (AABB) Press,
2002. Stock #023030; softcover, 130 pages; list price:
$80.00; member price $50.00. To order: call (866) 2222498 or fax (301) 951-7150.
This reviewer has a research interest in adoptive
immunotherapy, and gladly agreed to review Cellular
Therapy: New Frontiers in Transfusion Medicine. I
was not disappointed. Although the book is relatively
short, the chapters are detailed and well-referenced and
present an appropriate spectrum of currently practiced
cellular therapy techniques and more esoteric
applications.
The first chapter is a concise yet thorough
historical perspective on cellular therapy written by
Harvey Klein, MD, and originally delivered as the 2001
Emily Cooley Memorial Lecture. Covering topics
including the development of the blood cell separator,
initial observations supporting the clinical feasibility of
adoptive immunotherapy, somatic cell gene therapy,
and the cellular vaccines, this chapter nicely covers the
continuing evolution of transfusion medicine in the
area of cellular therapy.
There are comprehensive chapters on established
cellular therapy methods that many blood banks are
already performing. Mobilization and collection of
peripheral blood progenitor cells, increasingly used for
hematopoietic cell transplantation, is discussed by
David Stroncek, MD, and Susan Leitman, MD. Included
are mobilization protocols, expected results, and donor
issues. As a companion chapter, Elizabeth Read, MD,
and Charles Carter describe methods for T-cell
depletion of hematopoietic progenitor cell products
prior to allogeneic transplantation, and the NIH clinical
experience with this procedure. While not quite “howto” guides, these chapters would nonetheless serve as
excellent introductions for practitioners whose
clinicians may request these services.
An evolving methodology with particularly
exciting potential is pancreatic islet cell transplantation
for type I diabetes, as discussed by Elizabeth Read, MD,
Janet Lee, MT(ASCP), and David Harlan, MD. The
authors cover pancreas procurement, processing to
isolate islets, and the current status of clinical trials.
After reading this chapter, it seems increasingly likely
that islet cell processing will become a technique
largely performed and overseen by transfusion
medicine specialists, and this chapter is a good primer
on the subject.
The remaining three chapters are more forwardlooking, covering areas that very few physicians and
technologists currently have experience with. For
example, David Williams, MD, discusses genetic
modification of stem cells, with an emphasis on
retroviral and other vectors currently in use. Ena Wang,
MD, and Francesco Marincola, MD, discuss immunotherapies for solid tumors. Finally, Catherine Verfaillie,
MD, and colleagues give an overview on tissue-resident
stem cells, including mesenchymal stem cells.
In summary, I recommend this well-written book
for anyone who wants an introduction to where
transfusion medicine may be heading, and what
components we may be asked to prepare in the future.
John D. Roback, MD PhD
Emory University School of Medicine
WMB 2307, 1639 Pierce Drive
Atlanta, GA 30322
Attention SBB and BB Students: You are eligible for a free 1-year subscription to Immunohematology. Ask
your education supervisor to submit the name and complete address for each student and the inclusive dates
of the training period to Immunohematology, P.O. Box 40325, Philadelphia, PA 19106.
62
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
LITERATURE REVIEW
General (2000–2003)
Blood group antigens
1. Cao Y, Merling A, Karsten U, Schwartz-Albiez R.
The fucosylated histo-blood group antigens H type
2 (blood group O CD173) and Lewis Y (CD174)
are expressed on CD34+ hematopoietic
progenitors but absent on mature lymphocytes.
Glycobiology 2001;11:677-83.
2. Cartron JP, Colin Y. Structural and functional
diversity of blood group antigens. Transfus Clin
Biol 2001;8:163-99.
3. Cooling LL, Kelly K. Inverse expression of Pk and
Luke blood group antigens on human RBCs.
Transfusion 2001;41:898-907.
4. Grahn A, Elmgren A, Akerg L, et al. Determination
of Lewis FUT3 gene mutations by PCR using
sequence-specific primers enables efficient
genotyping of clinical samples. Hum Mutat
2001;18:358-9.
5. Hosoi E, Hirose M, Hamano S. Expression levels of
H-type α (1,2)-fucosyltransferase gene and histoblood group ABO gene corresponding to
hematopoietic cell differentiation. Transfusion
2003;43:65-71.
6. Iwamoto S, Kamesaki T, Oyamada T, et al.
Reactivity of autoantibodies of autoimmune
hemolytic anemia with recombinant rhesus blood
group antigens or anion transporter band 3. Am J
Hematol 2001;68:106-14.
7. Jaskiewiez E, Gerwinski M, Culin Y, Lisowska E.
Recombinant forms of Gerbich blood group
antigens: expression and purification. Transfus
Clin Biol 2002;9:121-9.
8. Martine GHM Tax,Van der Schoot CE, van Doorn
R, et al. RHC and RHc genotyping in different
ethnic groups. Transfusion 2002;42:634-44.
9. Nathalang O, Kuvanout S, Punyaprasiddhi P,
Tasaniyanonda C, Sriphaisal T. A preliminary study
of the distribution of blood group systems in Thai
blood donors determined by the gel system. SE
Asian J Trop Med Pub Health 2001;32:204-7.
10. Noizat-Pirenne F, LePennec P-Y, Mouro I, et al.
Molecular background of D(c)(e) haplotypes
within the white population. Transfusion
2002;42:627-33.
11. Olsson ML, Irshaid NM, Hosseini-Maaf B, et al.
Genomic analysis of clinical samples with
serologic ABO blood grouping discrepancies:
identification of 15 novel A and B subgroup
alleles. Blood 2001;98:1585-93.
12. Reid ME. The Dombrock blood group system: a
review. Transfusion 2003;42:107-114.
13. Rios M, Hue-Roye K, Øyen R, Miller J, Reid ME.
Insights into the Holley– and Joseph– phenotypes.
Transfusion 2002;42:52-8.
14. Rios M, Hue-Roye K, Lee AH, Chiofolo JT, Miller JL,
Reid ME. DNA analysis for the Dombrock
polymorphism. Transfusion 2001;41:1143-6.
15. Roubinet F, Janvier D, Blancher A. A novel cis AB
allele derived from a B allele through a single
point mutation. Transfusion 2002;42:239-46.
16. Scott M. Section 1A: Rh serology. Coordinator’s
report. Transfus Clin Biol 2002;9:23-9.
17. Seltsam A, Hallensleben M, Eiz-Vesper B, Leuhard V,
Heymann G, Blasczyk R. A weak blood group A
phenotype caused by a new mutation at the ABO
locus. Transfusion 2002;42:294-301.
18. Wu GG, Jin SZ, Deng ZH, Zhao TM. Polymerase
chain reaction with sequence-specific primersbased genotyping of the human Dombrock blood
group DO1 and DO2 alleles and the DO gene
frequencies in Chinese blood donors. Vox Sang
2001;81:49-51.
19. Yahalom V, Ellis MH, Poole J, et al. The rare Lu:–6
phenotype in Israel and the clinical significance of
anti-Lu6. Transfusion 2002;42:247-50.
Blood group antibodies
1. Boctor FN, Ali NM, Mohandas K, Uehlinger J.
Absence of D-alloimmunization in AIDS patients
receiving D-mismatched RBCs. Transfusion
2003;43:173-6.
2. Burnette RE, Couter K. A positive antibody
screen—an encounter with the Augustine
antibody. J Natl Med Assoc 2002;94:166-70.
3. Collinet P, Subtil D, Puech F, et al. Successful
treatment of extreme fetal anemia due to Kell
alloimmunoization. Obstet Gynecol
2002;100:1102-5.
4. Daniels G, Hadley A, Green CA. Causes of fetal
anemia in hemolytic disease due to anti-K (letter).
Transfusion 2003;43:115.
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
63
5. Daniels G. Section 4: Antibodies to other blood
group antigens. Coordinator’s report. Transfus
Clin Biol 2002;9:75-80.
6. Fernandez-Jimenez MC, Jimenez-Marco MT,
Hernandez D, Gonzalez A, Omenaca F, de la
Camara C. Treatment with plasmapheresis and
intravenous immune globulin in pregnancies
complicated with anti-PP1Pk or anti-K
immunization: a report of two patients. Vox Sang
2001;80:117-20.
7. Hareuveni M, Merchav H, Austerlitz N, RahimiLeveen N, Ben-Tal O. Donor anti-Jka causing
hemolysis in a liver transplant recipient.
Transfusion 2002;42:363-7.
8. Joshi SR. Streptomycin-dependent panagglutinin
causing error in forward blood grouping. Vox
Sang 2001;81:55.
9. Levin M-D, de Veld JC, vander Holt B,Van’t Veer
MB. Screening for alloantibodies in the serum of
patients receiving platelet transfusions: a
comparison of the ELISA, lymphocytotoxicity, and
the indirect immunofluorescence method.
Transfusion 2003;42:72-7.
10. Lynen R, Krone O, Legler TJ, Köhler M, Mayr WR. A
newly developed gel centrifugation test for
quantification of RBC-bound IgG antibodies and
their subclasses IgG1 and IgG3: comparison with
flow cytometry. Transfusion 2002;42:612-8.
11. Maley M, Babb R, Chapman CE, Fitzgerald J,
Cavanagh G. Identification and quantification of
anti-D, -C, and -G in alloimmunized pregnant
women. Transfus Med 2001;11:443-6.
12. Sarci SU, Alpay F, Yesilkaya E, et al. Hemolytic
disease of the newborn due to isoimmunization
with anti-E antibodies: a case report.Turkish J
Pediatr 2002;44:248-50.
13. Strobel E. Comparison of monoclonal and
polyclonal anti-P1 reagents. Clin Lab 2001;47:
249-55.
14. Yahalom V, Ellis MH, Poole J, et al. The rare Lu:–6
phenotype in Israel and the clinical significance of
anti-Lu6. Transfusion 2002;42:247-50.
15. Yasuda H, Ohto H,Yamaguchi O, et al. Three
episodes of delayed hemolytic transfusion reaction
due to multiple red cell antibodies, anti-Di, anti-Jk,
and anti-E. Transfus Sci 2000;23:107-12.
Blood group genetics
1. Cartron JP, Colin Y. Structural and functional
diversity of blood group antigens. Transfus Clin
Biol 2001;8:163-99.
64
2. Darke C, Street J, Guttridge MG. Sequence,
genetics and serology of a new HLA-B allele
B*4903. Tissue Antigens 2001;57:478-80.
3. Grahn A, Elmgren A, Aberg L, et al. Determination
of Lewis FUT3 gene mutations by PCR using
sequence-specific primers enables efficient
genotyping of clinical samples. Hum Mutat
2001;18:358-9.
4. Hosoi E, Hirose M, Hamano S. Expression levels of
H-type α (1,2)-fucosyltransferase gene and histo
blood group ABO gene corresponding to
hematopoietic cell differentiation. Transfusion
2003;43:65-71.
5. Jaskiewiez E, Czerwinski M, Colin Y, Lisowska E.
Recombinant forms of Gerbich blood group
antigens: expression and purification. Transfus
Clin Biol 2002;9:121-9.
6. LePendu J, Henry S. Section 2: Immunochemical,
immunohistological and serological analysis of
monoclonal antibodies with carbohydrates.
Coordinator’s report. Transfus Clin Biol 2002;9:
55-60.
7. Lo YM. Fetal DNA in maternal plasma: application
to non-invasive blood group genotyping of the
fetus. Transfus Clin Biol 2001;8:306-10.
8. Martine GHM Tax,Van der Schoot CE, van Doorn
R, et al. RHC and RHc genotyping in different
ethnic groups. Transfusion 2002;42:634-44.
9. Noizat-Pirenne F, LePennec P-Y, Mouro I, et al.
Molecular background of D(c)(e) haplotypes
within the white population. Transfusion
2002;42:627-33.
10. Olsson ML, Irshaid NM, Hosseini-Maaf B, et al.
Genomic analysis of clinical samples with
serologic ABO blood grouping discrepancies:
identification of 15 novel A and B subgroup
alleles. Blood 2001;98:1585-93.
11. Reid ME, Lisowska E, Blanchard D. Section 3:
Epitope determination of monoclonal antibodies
to glycophorin A and glycophorin B.
Coordinator’s report. Antibodies to antigens
located on glycophorins and band 3. Transfus Clin
Biol 2002;9:63-72.
12. Rios M, Hue-Roye K, Lee AH, Chiofolo JT, Miller JL,
Reid ME. DNA analysis for the Dombrock
polymorphism. Transfusion 2001;41:1143-6.
13. Roubinet F, Janvier D, Blancher A. A novel cis AB
allele derived from a B allele through a single
point mutation. Transfusion 2002;42:239-46.
14. Seltsam A, Hallensleben M, Eiz-Vesper B, Lenhard V,
Heymann G, Blasczyk R. A weak blood group A
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
phenotype caused by a new mutation at the ABO
locus. Transfusion 2002;42:294-301.
15. Sobel E, Papp JC, Lange K. Detection and
integration of genotyping errors in statistical
genetics. Am J Hum Genet 2002;70:496-508.
16. Yan L, Zhu F, Fu Q. Jk(a–b–) and Kidd blood group
genotypes in Chinese people (letter). Transfusion
2003;42:289:90.
the indirect immunofluorescence method.
Transfusion 2003;42:72-7.
5. Lynen R, Krone O, Legler TJ, Köhler M, Mayr WR.
A newly developed gel centrifugation test for
quantification of RBC-bound IgG antibodies and
their subclasses IgG1 and IgG3: comparison with
flow cytometry. Transfusion 2002;42:612-8.
6. Scott M. Section 1A: Rh serology. Coordinator’s
report. Transfus Clin Biol 2002;9:23-9.
7. Shulman IA, Downes KA, Sazzma K, Maffei LM.
Pretransfusion compatibility testing for red blood
cell administration. Curr Opin Hematol 2001;
8:397-404.
8. Toyohiro T,Yasuyuk I,Toshio M. IgM alloantibody
detection by M-MPHA, a new solid-phase assay
system. Transfus Sc 2002;23:47-54.
9. Wu GG, Jin SZ, Deng ZH, Zhao TM. Polymerase
chain reaction with sequence-specific primersbased genotyping of the human Dombrock blood
group DO1 and DO2 alleles and the DO gene
frequencies in Chinese blood donors. Vox Sang
2001;81:49–51.
Blood group biochemistry
1. Cao Y, Merling A, Karsten U, Schwartz-Albiez R.
The fucosylated histo-blood group antigens H type
2 (blood group O CD173) and Lewis Y (CD174)
are expressed on CD34+ hematopoietic
progenitors but absent on mature lymphocytes.
Glycobiology 2001;11:677-83.
Monoclonal antibodies
1. Kumpel BM. In vivo studies of monoclonal anti-D
and the mechanisms of immune suppression.
Transfus Clin Biol 2002;9:9-14.
2. Reid ME, Lisowska E, Blanchard D. Section 3:
Epitope determination of monoclonal antibodies
to glycophorin A and glycophorin B.
Coordinator’s report. Antibodies to antigens
located on glycophorins and band 3. Transfus Clin
Biol 2002;9:63-72.
3. LePendu J, Henry S. Section 2: Immunochemical,
immunohistological and serological analysis of
monoclonal antibodies with carbohydrates.
Coordinator’s report. Transfus Clin Biol 2002;9:
55-60.
4. Strobel E. Comparison of monoclonal and
polyclonal anti-P1 reagents. Clin Lab 2001;47:
249-55.
Red cell serology/methods
1. Brumit MC, Stubbs JR, et al. Conventional tube
agglutination with polyethylene glycol versus red
cell affinity column technology (ReACT): a
comparison of antibody detection methods. Ann
Clin Lab Sci 2002;32:155-8.
2. Engelfreit CP, Reesink HW, Krusius T, et al. The use
of the computer cross-match.Vox Sang
2001;80:184-92.
3. Kopko PM, Paglieroni TG, Popovsky MA, Muto KN,
MacKenzie MR, Holland PV. TRALI: correlation of
antigen-antibody and monocyte activation in
donor-recipient pairs. Transfusion 2003;43:177-84.
4. Levin M-D, de Veld JC, vander Holt B,Van’t Veer
MB. Screening for alloantibodies in the serum of
patients receiving platelet transfusions: a
comparison of the ELISA, lymphocytotoxicity, and
White cell/platelet serology/methods
1. Blumberg N, Phipps RP, Kaufman J, Heal JM. The
causes and treatment of reactions to platelet
transfusions (letter). Transfusion 2003;43:291.
(Reply: Heddle N):292.
2. Darke C, Street J, Guttridge MG. Sequence,
genetics and serology of a new HLA-B allele
B*4903. Tissue Antigens 2001;57:478–80.
3. Kao GS,Wood IG, Dorfman DM, Milford EL,
Benjamin RI. Investigations into the role of antiHLA class II antibodies in TRALI. Transfusion
2003;42:185–91.
4. Levin M-D, de Veld JC, vander Holt B,Van’t Veer
MB. Screening for alloantibodies in the serum of
patients receiving platelet transfusions: a
comparison of the ELISA, lymphocytotoxicity, and
the indirect immunofluorescence method.
Transfusion 2003;42:72–7.
5. Lim J, Kim Y, Kyungja H, et al. Flow cytometric
monocyte phagocytic assay for predicting platelet
transfusion outcome.Transfusion 2002;42:309–16.
6. Torio A, Moya-Quiles MR, Muro M, et al.
Discrepancies in HLA-C typing in transplantation:
comparison of PCR-SSP and serology results.
Transplant Proc 2002;34:419–20.
Hemolytic anemias
1. Frohn C, Jabs WJ, Fricke L, Goerg S. Hemolytic
anemia after kidney transplantation: case report
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
65
and differential diagnosis. Ann Hematol
2002;81:158–60.
2. Iwamoto S, Kamesaki T, Oyamada T, et al.
Reactivity of autoantibodies of autoimmune
hemolytic anemia with recombinant rhesus blood
group antigens or anion transporter band 3. Am J
Hematol 2001;68:106–14.
3. Kondo H, Oyamada T, Mori A, et al. Directantiglobulin-test-negative immune hemolytic
anaemia and thombocytopenia in a patient with
Hodgkin’s disease. Acta Haematol 2001;105:233–6.
4. Maheshwari VD, Kumar R, Singh S. Coomb’s
negative autoimmune hemolytic anemia with
thrombocytopenia (Evan’s syndrome). J Assoc
Physicians India 2002;50:457–8.
5. Shnaider A, Busok A, Rogachev BV, Zlutnik M,
Tomer A. Severe Coombs-negative autoimmune
hemolytic anemia in a kidney transplant patient.
Am J Nephrol 2001;21:494–7.
Hemolytic disease of the newborn
1. Anderson AS, Praetorius L, Jorgensen HL, Lylloff K,
Larsen KT. Prognostic value of screening for
irregular antibodies late in pregnancy in rhesus
positive women. Acta Obstet Gynecol Scand
2002;81;407–11.
2. Chandrasekar A, Morris KG,Tubman TR,Tharma S,
McClelland WM. The clinical outcome of non-RhD
antibody affected pregnancies in Northern
Ireland. Ulster Med J 2001;70:89–94.
3. Collinet P, Subtil D, Puech F, et al. Successful
treatment of extreme fetal anemia due to Kell
alloimmunoization. Obstet Gynecol
2002;100:1102–5.
4. Cotorruelo C, Biondi C, Garcia BS. Early detection
of RhD status in pregnancies at risk of hemolytic
disease of the newborn. Clin Exp Med 2002;
2:77–81.
5. Daniele G, Hadley A, Green CA. Causes of fetal
anemia in hemolytic disease due to anti-K (letter).
Transfusion 2003;43:115.
6. Fernandez-Jimenez MC, Jimenez-Marco MT,
Hernandez D, Gonzalez A, Omenaca F, de la
Camara C. Treatment with plasmapheresis and
intravenous immune globulin in pregnancies
complicated with anti-PP1Pk or anti-K
immunization: a report of two patients. Vox Sang
2001;80:117–20.
7. Lo YM. Fetal DNA in maternal plasma: application
to non-invasive blood group genotyping of the
fetus. Transfus Clin Biol 2001;8:306–10.
66
8. Mair DC, Scofield TI. HDN in a mother
undergoing in vitro fertilization with donor ova
(letter). Transfusion 2003;43:288.
9. Maley M, Babb R, Chapman CE, Fitzgerald J,
Cavanagh G. Identification and quantification of
anti-D, -C, and -G in alloimmunized pregnant
women. Transfus Med 2001;11:443–6.
10. Patel RK, Nicolaides K, Mijovic A. Severe
hemolytic disease of the fetus following in vitro
fertilization with anonymously donated oocytes
(letter). Transfusion 2003;43:119–21.
11. Sarci SU, Alpay F,Yesilkaya E, et al. Hemolytic
disease of the newborn due to isoimmunization
with anti-E antibodies: a case report.Turkish J
Pediatr 2002;44:248–50.
12. Sarci SU,Yurdakok M, Serdar MA, et al. An early
(sixth hour) serum bilirubin measurement is
useful in predicting the development of
significant hyperbilirubinemia and severe ABO
hemolytic disease of the newborn with ABO
incompatibility. Pediatrics 2002;109:53.
13. Stanworth S, Fleetwood P, deSilva M. Severe
haemolytic disease of the newborn due to anti-Jsb.
Vox Sang 2001;81:134–5.
14. Stockman JA. Overview of the state of the art of
Rh disease: history, current clinical management,
and recent progress. J Pediatr Hematol/Oncol
2001;23:554–62.
Transfusion/transplantation
1. Atterbury C,Wilkinson J. Blood transfusion. Nurs
Stand 2000;14:47–52.
2. Blumberg N, Phipps RP, Kaufman J, Heal JM. The
causes and treatment of reactions to platelet
transfusions (letter). Transfusion 2003;43:291.
(Reply: Heddle N):292.
3. Brown M,Whalen PK. Red blood cell transfusion
in critically ill patients. Emerging risks and
alternatives. Crit Care Nurse 2000(Suppl):1–14.
4. Frohn C, Jabs WJ, Fricke L, Goerg S. Hemolytic
anemia after kidney transplantation: case report
and differential diagnosis. Ann Hematol 2002;
81:158–60.
5. Gryn J, Zeigler ZR, Shadduck RK,Thomas C.
Clearance of erythrocyte allo-antibodies using
Rituximab. Bone Marrow Transplant 2002;
29:631–2.
6. Hareuveni M, Merchav H, Austerlitz N, RahimiLeveen N, Ben-Tal O. Donor anti-Jka causing
hemolysis in a liver transplant recipient.
Transfusion 2002;42:363–7.
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
7. Howie JC,Tansey PJ, et al. Blood transfusion in
surgical practice—matching supply to demand. Br
J Anaesth 2002;89:214–6.
8. Krombach J, Kampe S, Gathof BS, Diefenbach C,
Kasper S-M. Human error: the persistent risk of
blood transfusion: a report of five cases. Anesth
Analg 2002;94:154–6.
9. Maxwell EL, Metz J, Haeusler MN, et al. Use of red
blood cell transfusions in surgery. ANZ J Surg
2002;72:561–6.
10. Reed W,Vichinsky EP. Transfusion therapy: a
coming-of-age treatment for patients with sickle
cell disease. J Pediatr Hematol/Oncol 2001;
23:197–202.
11. Shulman IA, Downes KA, Sazzman K, Maffei LM.
Pretransfusion compatibility testing for red blood
cell administration. Curr Opin Hematol
2001;8:397–404.
12. Torio A, Moya-Quiles MR, Muro M, et al.
Discrepancies in HLA-C typing in transplantation:
comparison of PCR-SSP and serology results.
Transplant Proc 2002;34:419–20.
13. Yasuda H, Ohto H,Yamaguchi O, et al. Three
episodes of delayed hemolytic transfusion reaction
due to multiple red cell antibodies, anti-Di, anti-Jk,
and anti-E. Transfus Sci 2000;23:107–12.
Disease associations
1. Boctor FN, Ali NM, Mohandes K, Uehlinger J.
Absence of D– alloimmunization in AIDS patients
receiving d-mismatched RBCs. Transfusion
2003;43:177–6.
2. Joshi SR. Streptomycin-dependent panagglutinin
causing error in forward blood grouping. Vox
Sang 2001;81:55.
3. Kao GS,Wood IG, Dorfman DM, Milford EL,
Benjamin RI. Investigations into the role of antiHLA class II antibodies in TRALI. Transfusion
2003;42:185–91.
4. Kondo H, Oyamada T, Mori A, et al. Directantiglobulin-test-negative immune hemolytic
anaemia and thombocytopenia in a patient with
Hodgkin’s disease. Acta Haematol 2001;105:233–6.
5. Kopko PM, Paglieroni TG, Popovsky MA, Muto KN,
MacKenzie MR, Holland PV. TRALI: correlation of
antigen-antibody and monocyte activation in
donor-recipient pairs.Transfusion 2003;43:177–84.
6. Maheshwari VD, Kumar R, Singh S. Coomb’s
negative autoimmune hemolytic anemia with
thrombocytopenia (Evan’s syndrome). J Assoc
Physicians India 2002;50:457–8.
7. Reed W,Vichinsky EP. Transfusion therapy: a
coming-of-age treatment for patients with sickle
cell disease. J Pediatr Hematol/Oncol 2001;
23:197–202.
8. Shnaider A, Busok A, Rogachev BV, Zlutnik M,
Tomer A. Severe Coombs-negative autoimmune
hemolytic anemia in a kidney transplant patient.
Am J Nephrol 2001;21:494–7.
Miscellaneous
1. Hoppe B, Stibbe W, Bielefeld A, Pruss A, Salama A.
Increased RBC autoantibody production in
pregnancy. Transfusion 2001;41:1559–61.
2. Krombach J, Kampe S, Gathof BS, Diefenbach C,
Kasper S-M. Human error: the persistent risk of
blood transfusion: a report of five cases. Anesth
Analg 2002;94:154–6.
3. Kumpel BM. On the mechanism of tolerance to
the RhD antigen medicated by passive anti-D
(RhD prophylaxis). Immunol Lett 2002;82:67–73.
4. Mair DC, Scofield TI. HDN in a mother
undergoing in vitro fertilization with donor ova
(letter). Transfusion 2003;43:288.
5. Nathalang O, Kuvanout S, Punyaprasiddhi P,
Tasaniyanonda C, Sriphaisal T. A preliminary study
of the distribution of blood group systems in Thai
blood donors determined by the gel system. SE
Asian J Trop Med Pub Health 2001;32:204–7.
6. Patel RK, Nicolaides K, Mijovic A. Severe
hemolytic disease of the fetus following in vitro
fertilization with anonymously donated oocytes
(letter). Transfusion 2003;43:119–21.
7. Stockman JA. Overview of the state of the art of
Rh disease: history, current clinical management,
and recent progress. J Pediatr Hematol/Oncol
2001;23:554–62.
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
67
ANNOUNCEMENTS
Monoclonal antibodies available. The New York
Blood Center has developed murine monoclonal
antibodies that are useful for donor screening and for
typing red cells with a positive DAT. Anti-Rh17 is a
direct agglutinating monoclonal antibody. Anti-Fya, antiK, anti-Jsb, and anti-Kpa are indirect agglutinating
antibodies that require anti-mouse IgG for detection.
These antibodies are available in limited quantities at
no charge to anyone who requests them. Contact:
Marion Reid, New York Blood Center, 310 E. 67th Street,
New York, NY 10021; e-mail: [email protected]
Annual Symposium. The National Institutes of
Health, Department of Transfusion Medicine, will hold
their annual symposium. Immunohematology and
Blood Transfusion, on October 2 and October 3,
2003. The symposium will be co-hosted by the
Greaater Chesapeake and Potomac Region of the
American Red Cross and is free of charge. Advance
registration is encouraged. For more information and
registration, Contact: Karen Byrne, NIH/CC/DTM,
Bldg. 10/Rm. 1C711, 10 Center Drive, MSC 1184,
Bethesda, MD 20892; e-mail: [email protected] or
visit our Web site: www.cc.nih.gov/dtm.
Workshop on Blood Group Genotyping. The
ISBT/ICSH Expert Panel in Molecular Biology has
recommended that a workshop be held on blood
group genotyping by molecular techniques. The results
of the meeting would culminate in a report at the ISBT
Congress in 2004 in Edinburgh. It was decided that
only laboratories that provide a reference service in
blood group genotyping would be included in the
workshop. One of the aims of the workshop would be
to establish an external quality assurance plan. If you
have any suggestions as to how the workshop should
be organized, we would be grateful for your opinions.
If you are interested in taking part in such
a workshop, please contact Geoff Daniels
([email protected]). Offer presented by Geoff
Daniels, Martin L. Olsson, and Ellen van der Schoot.
CLASSIFIED AD
SBB Program. The Department of Transfusion Medicine, National Institutes of Health, is accepting
applications for its 1-year Specialist in Blood Bank Technology Program. Students are federal employees
who work 32 hours per week. This program introduces students to all areas of transfusion medicine,
including reference serology, cell processing, and HLA and infectious disease testing. Students also design
and conduct a research project. NIH is an Equal Opportunity Employer. Application deadline is
June 30, 2003, for the January 2004 class. Contact: Karen M. Byrne, NIH/CC/DTM, Bldg. 10/Rm. 1C711,
10 Center Drive, MSC 1184, Bethesda, MD 20892-1184; phone: (301) 496-8335 or e-mail:
[email protected].
Phone, Fax, and Internet Information: If you have any questions concerning Immunohematology,
Journal of Blood Group Serology and Education, or the Immunohematology Methods and Procedures
manual, contact us by e-mail at [email protected]. For information concerning the National Reference
Laboratory for Blood Group Serology, including the American Rare Donor Program, please contact Sandra
Nance, by phone at (215) 451-4362, by fax at (215) 451-2538, or by e-mail at [email protected]
68
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
ADVERTISEMENTS
NATIONAL REFERENCE LABORATORY FOR
BLOOD GROUP SEROLOGY
National Platelet Serology Reference
Laboratory
Immunohematology Reference
Laboratory
Diagnostic testing for:
• Neonatal alloimmune thrombocytopenia (NAIT)
• Posttransfusion purpura (PTP)
• Refractoriness to platelet transfusion
• Heparin-induced thrombocytopenia (HIT)
• Alloimmune idiopathic thrombocytopenia
purpura (AITP)
• Medical consultation available
AABB, ARC, New York State, and CLIA licensed
(215) 451-4901—24-hr. phone number
(215) 451-2538—Fax
American Rare Donor Program
(215) 451-4900—24-hr. phone number
(215) 451-2538—Fax
[email protected]
Immunohematology
(215) 451-4902—Phone, business hours
(215) 451-2538—Fax
[email protected]
Quality Control of Cryoprecipitated-AHF
(215) 451-4903—Phone, business hours
(215) 451-2538—Fax
Granulocyte Antibody Detection and Typing
• Specializing in granulocyte antibody detection
and granulocyte antigen typing
• Patients with granulocytopenia can be classified
through the following tests for proper therapy
and monitoring:
—Granulocyte agglutination (GA)
—Granulocyte immunofluorescence (GIF)
—Monoclonal Antibody Immobilization of
Granulocyte Antigens (MAIGA)
For information regarding services, call Gail Eiber
at: (651) 291-6797, e-mail: [email protected],
or write to:
Neutrophil Serology Reference Laboratory
American Red Cross
St. Paul Regional Blood Services
100 South Robert Street
St. Paul, MN 55107
CLIA LICENSED
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
Test methods:
• GTI systems tests
—detection of glycoprotein-specific platelet
antibodies
—detection of heparin-induced antibodies (PF4
ELISA)
• Platelet suspension immunofluorescence test
(PSIFT)
• Solid phase red cell adherence (SPRCA) assay
• Monoclonal antibody immobilization of platelet
antigens (MAIPA)
For information, e-mail: [email protected]
or call:
Maryann Keashen-Schnell
(215) 451-4041 office
(215) 451-4205 laboratory
Sandra Nance
(215) 451-4362
Scott Murphy, MD
(215) 451-4877
American Red Cross Blood Services
Musser Blood Center
700 Spring Garden Street
Philadelphia, PA 19123-3594
CLIA LICENSED
69
ADVERTISEMENTS CONT’D
IgA/Anti-IgA Testing
Reference and Consultation Services
IgA and anti-IgA testing is available to do the
following:
• Monitor known IgA-deficient patients
• Investigate anaphylactic reactions
• Confirm IgA-deficient donors
Red cell antibody identification and problem
resolution
Our ELISA assay for IgA detects antigen to
0.05 mg/dL.
Paternity testing/DNA
HLA-A, B, C, and DR typing
HLA-disease association typing
For information regarding our services, contact
For information on charges and sample
requirements, call (215) 451-4351, e-mail:
[email protected],
or write to:
American Red Cross Blood Services
Musser Blood Center
700 Spring Garden Street
Philadelphia, PA 19123-3594
ATTN: Ann Church
Zahra Mehdizadehkashi at (503) 280-0210, or
write to:
Pacific Northwest Regional Blood Services
ATTENTION: Tissue Typing Laboratory
American Red Cross
3131 North Vancouver
Portland, OR 97227
CLIA LICENSED
CLIA LICENSED,ASHI ACCREDITED
Notice to Readers: All articles published,
including communications and book reviews,
reflect the opinions of the authors and do not
necessarily reflect the official policy of the
American Red Cross.
IMMUNOHEMATOLOGY IS ON THE WEB!
www.redcross.org/pubs/immuno
Password “2000”
For more information or to send an e-mail
message “To the editor”
[email protected]
IMPORTANT NOTICE ABOUT MANUSCRIPTS
FOR
IMMUNOHEMATOLOGY
Mail all manuscripts (original and 2 copies) to Mary H. McGinniss, Managing Editor, 10262 Arizona Circle,
Bethesda, MD 20817. In addition, please e-mail a copy to Marge Manigly at [email protected]
70
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
Immunohematology
JOURNAL OF BLOOD GROUP SEROLOGY AND EDUCATION
Instructions for Authors
SCIENTIFIC ARTICLES, REVIEWS, AND CASE REPORTS
Before submitting a manuscript, consult current issues of
Immunohematology for style. Type the manuscript on white bond
paper (8.5" × 11") and double-space throughout. Number the pages
consecutively in the upper right-hand corner, beginning with the
title page. Each component of the manuscript must start on a new
page in the following order:
1. Title page
2. Abstract
3. Text
4. Acknowledgments
5. References
6. Author information
7. Tables—see 6 under Preparation
8. Figures—see 7 under Preparation
Preparation
1. Title page
A. Full title of manuscript with only first letter of first word
capitalized
B. Initials and last name of each author (no degrees; all CAPS),
e.g., M.T. JONES
C.Running title of ≤ 40 characters, including spaces
D.3 to 10 key words
2. Abstract
A. 1 paragraph, no longer than 200 words
B. Purpose, methods, findings, and conclusions of study
C.Abstracts not required for reviews
3. Text (serial pages)
Most manuscripts can usually, but not necessarily, be divided into
sections (as described below). Results of surveys and review
papers are examples that may need individualized sections.
A. Introduction
Purpose and rationale for study, including pertinent background
references.
B. Case Report (if study calls for one)
Clinical and/or hematologic data and background serology.
C.Materials and Methods
Selection and number of subjects, samples, items, etc. studied and
description of appropriate controls, procedures, methods, equipment, reagents, etc. Equipment and reagents should be identified
in parentheses by model or lot and manufacturer’s name, city, and
state. Do not use patients’ names or hospital numbers.
D.Results
Presentation of concise and sequential results, referring to pertinent tables and/or figures, if applicable.
E. Discussion
Implications and limitations of the study, links to other studies;
if appropriate, link conclusions to purpose of study as stated in
introduction.
4. Acknowledgments
Acknowledge those who have made substantial contributions to
the study, including secretarial assistance.
5. References
A. In text, use superscript, arabic numbers.
B. Number references consecutively in the order they occur in
the text.
C.Use inclusive pages of cited references, e.g., 1431–7.
D.Refer to current issues of Immunohematology for style.
6. Tables
A. Number consecutively, head each with a brief title, capitalize
first letter of first word (e.g., Table 1. Results of ...), and use no
punctuation at the end.
B. Use short headings for each column, and capitalize first letter
of first word.
C.Place explanations in footnotes (sequence: *, †, ‡, §, ¶, **, ††).
7. Figures
A. Figures can be submitted either drawn or photographed
(5″ × 7″ glossy).
B. Place caption for a figure on a separate page (e.g., Fig. 1. Results
of ...), ending with a period. If figure is submitted as a glossy,
put title of paper and figure number on back of each glossy
submitted.
C.When plotting points on a figure, use the following symbols
when possible: ● ● ▲ ▲ ■ ■.
8. Author information
A. List first name, middle initial, last name, highest academic
degree, position held, institution and department, and
complete address (including zip code) for all authors. List
country when applicable.
SCIENTIFIC ARTICLES AND CASE REPORTS SUBMITTED
AS LETTERS TO THE EDITOR
Preparation
1. Heading—To the Editor:
2. Under heading—title with first letter capitalized.
3. Text—write in letter format (paragraphs).
4. Author(s)—type flush right; for first author: name, degree,
institution, address (including city, state, and zip code); for other
authors: name, degree, institution, city, and state.
5. References—limited to ten.
6. One table and/or figure allowed.
I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3
Send all submissions (original and two copies) to:
Mary H. McGinniss, Managing Editor, Immunohematology,
10262 Arizona Circle, Bethesda, MD 20817.
In addition, please e-mail your manuscript to Marge
Manigly at [email protected]
71
Becoming a Specialist in Blood Banking (SBB)
What is a certified Specialist in Blood Banking (SBB)?
• Someone with educational and work experience qualifications that successfully passes the American Society for
Clinical Pathology (ASCP) board of registry (BOR) examination for the Specialist in Blood Banking.
• This person will have advanced knowledge, skills and abilities in the field of transfusion medicine and blood banking.
Individuals who have an SBB certification serve in many areas of transfusion medicine:
• Serve as regulatory, technical, procedural and research advisors
• Perform and direct administrative functions
• Develop, validate, implement, and perform laboratory procedures
• Analyze quality issues preparing and implementing corrective actions to prevent and document issues
• Design and present educational programs
• Provide technical and scientific training in blood transfusion medicine
• Conduct research in transfusion medicine
Who are SBBs?
Supervisors of Transfusion Services
Supervisors of Reference Laboratories
Quality Assurance Officers
Why be an SBB?
Professional growth
Managers of Blood Centers
Research Scientists
Technical Representatives
Job placement
Job satisfaction
LIS Coordinators Educators
Consumer Safety Officers
Reference Lab Specialist
Career advancement
How does one become an SBB?
• Attend a CAAHEP-accredited Specialist in Blood Bank Technology Program OR
• Sit for the examination based on criteria established by ASCP for education and experience
Fact #1: In recent years, the average SBB exam pass rate is only 38%
Fact #2: In recent years, greater than 73% of people who graduate from CAAHEP-accredited programs pass the SBB
exam.
Conclusion:
The BEST route for obtaining an SBB certification is to attend a CAAHEP-accredited Specialist in Blood Bank
Technology Program
Contact the following programs for more information:
P ROGRAM
C ONTACT N AME
C ONTACT I NFORMATION
Walter Reed Army Medical Center
William Turcan
202-782-6210;
[email protected]
Transfusion Medicine Center at Florida Blood Services
Marjorie Doty
727-568-5433 x 1514; [email protected]
Univ. of Illinois at Chicago
Veronica Lewis
312-996-6721; [email protected]
Medical Center of Louisiana
Karen Kirkley
504-903-2466; [email protected]
NIH Clinical Center Department of Transfusion Medicine
Karen Byrne
301-496-8335; [email protected]
Johns Hopkins Hospital
Christine Beritela
410-955-6580; [email protected]
ARC-Central OH Region, OSU Medical Center
Joanne Kosanke
614-253-2740 x 2270; [email protected]
Hoxworth Blood Center/Univ. of Cincinnati Medical Center
Catherine Beiting
513-558-1275; [email protected]
Gulf Coast School of Blood Bank Technology
Clare Wong
713-791-6201; [email protected]
Univ. of Texas SW Medical Center
Barbara Laird-Fryer
214-648-1785; [email protected]
Univ. of Texas Medical Branch at Galveston
Janet Vincent
409-772-4866; [email protected]
Univ. of Texas Health Science Center at San Antonio
Bonnie Fodermaier
Linda Smith
SBB Program: 210-358-2807,
[email protected]
MS Program : 210-567-8869; [email protected]
Blood Center of Southeastern Wisconsin
Lynne LeMense
414-937-6403; [email protected]
Additional Information can be found by visiting the following Web sites: www.ascp.org, www.caahep.org, and www.aabb.org
Musser Blood Center
700 Spring Garden Street
Philadelphia, PA 19123-3594
(Place Label Here)