The Role of Lectins: Blood Group Serology

August 1980 171
THE ROLE OF LECTINS I N BLOOD GROUP SEROLOGY
Author: W. John Judd

Department of Pathology
University of Michigan Medical School
Ann Arbor, Michigan
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/06/14
Referee: Peter D. lssirt

Paul I . Hoxworth Blood Center
University of Cincinnati
Cincinnati. Ohio
INTRODUCTION
Terminology and Definitions

The word lectin comes from the Latin fegere, meaning to pick out or to choose.
Boyd and Shapleighl originally applied the term to define those saline extracts of seeds
that preferentially agglutinate red cells of a particular blood group phenotype. The
term “phytohemagglutinin” has been used to describe those plant seed agglutinins that
display no blood group specificity, and Prokop, Uhlenbruck, and Kohler’ have sug-
gested that the word “protectin” be used to distinguish the blood group reactive hem-
agglutinins found in molluscs and lower vertebrates from those of plant origin.
For personal use only.
While most blood group serologists tend to think of lectins in terms of Boyd and
Shapleigh’s definition,’ other scientists would define a lectin as any protein, regardless
of its origin, whose activity can be neutralized by simple sugars. This definition not
only includes those proteins that act as hemagglutinins, but certain mitogens and en-
zymes. Perhaps a more descriptive designation to this generalized definition has been
made by Gold and Balding3 in the title of their book Receptor Specific Proteins -
Planr and Animal Lectins. Thus, this review will not be confined to the blood-group-
reactive plant seed agglutinins, but will by necessity include lectins from plant roots
and leaves, and lectins from such diverse sources as bacteria, fish, marine sponges,
molluscs, snakes, and fungi.
Few lectins possess specificity for a single blood group determinant. Some lectin
preparations contain a single lectin protein that cross-reacts with two or more red cell
surface receptors, while others contain separable agglutinins, each with a different
blood group specificity. It should be noted that in this review, lectins designated as
having anti-A/B or anti-T/Tk activity, etc., contain only a single lectin protein,
whereas designations such as anti-Tn + Cad activity imply that the lectin contains ag-
glutinins that are separable by adsorption and elution experiments.
Historical Aspects
The first description of what we now know as a lectin was made in 1888 when Still-
mark,4 studying the toxic effects of the castor bean (Ricinus cornmunis) on blood,
noted that red cells were agglutinated by saline extracts of these seeds. Subsequently,
many other seed extracts were studied, and some were shown topreferentially agglutin-
ate red cells of a particular species. However, it was not until 1945 that Boyd made
the startling discovery that lectins could be blood group specific, when he demon-
strated anti-A activity in saline extracts of the lima bean (Phaseolus funatus, syn. li-
rnensis). Boyd’s finding was not reported until 1949 when, in conjunction with Re-
guera, he reported the results of investigations on some 262 varieties of plant seeds.s
In the year prior to the appearance of this report, Renkoned published his findings
of anti-A activity in the seeds of Vicia cracca, and anti-H lectins from Laburnum
172 CRC Critical Reviews in Clinical Laboratory Sciences
alpinurn. Cytisus sessilofolius, and Lotus tetragonolobus seeds. Since then, other re-
ports of studies o n large numbers of seeds have been published, but such data has
invariably resulted in little practical application. Perhaps some notable exceptions to
this would be the findings by Cazal and Laularie’ of anti-H in three species of Ulex,
the discovery of anti-N in the seeds of Vicia graminea by Ottensooser and Silbersch-
midt,’ and Bird’s finding of anti-A, activity in extracts of Dolichos biflorirs seeds.P
The latter lectin is without doubt the most widely used plant seed agglutinin in blood
group serology.
In recent years, much interest has been aroused by the discovery of blood group
reactive lectins in the eggs and albumin glands of snails and lower vertebrates. These
lectins provide an abundant source of reagents for blood typing purposes. Lectins from
the peanut (Arachis hypogaea), soy bean (Glycine max, syn. soja), and Salvia seeds
are used extensively by investigative immunohematologists in the classification of red
cell polyagglutination. Furthermore, by virtue of their specific carbohydrate-binding
properties, lectins have been used to define the structure of certain red cell surface
antigens. These and other applications of lectins in blood group serology will be dis-
cussed in this review.
This review will primarily be confined to those lectins that have been extensively
investigated, or appear to have some practical application. For readers seeking a com-
prehensive compilation of all the plant and animal lectins that have been studied for
blood group reactivity, the review by Gold and Balding’ is recommended.
Biological and Physical Properties of Lectins

Lectins may act as hemagglutinins or mitogens, and some display both hemagglutin-
ating and mitogenic activity. The hemagglutinating lectins also precipitate soluble
blood group substances in agar diffusion tests. Other proteins which may be classified
as lectins, including those accompanying toxins in bacteria, fungi, and snake venom,
act as hemolysins. These have been identified as enzymes and d o not, as a rule, require
complement in order to express their hemolytic activity.’
Characteristically, the biologic activity of a lectin can be inhibited by weak solutions
of low molecular weight sugars, a property which distinguishes them from most im-
munoglobulins. In this respect. lectins are highly specific. Such specificity may not be
apparent from the results of hemagglutination tests, in particular with those proteins
that some would classify as phytohemagglutinins. However, even these serological re-
actions of no apparent specificity can be abolished by the addition of a single sugar
or a few structurally similar carbohydrates, thereby demonstrating their ability to
“pick out or to choose”.
Nearly 50 lectins have been purified by affinity chromatography. A detailed account
of these has been given by Goldstein and Hayes.” The amino acid sequence, molecular
weight, and preferential carbohydrate-binding activity of some of the purified blood
group reactive lectins have been determined. Lectins may be comprised of two, four,
six, or eight subunits linked by disulfide bonds. Not all subunits may possess carbo-
hydrate-binding sites. Some lectins require calcium and/or manganese ions in order to
express their biologic activity. Where pertinent. such data will be discussed in the ap-
propriate sections of this review.
Function of Lectins in Nature

The function of lectins in nature is not fully understood. Recent evidence“ suggests
that their presence in leguminous plants is related to the plants’ ability to fix nitrogen.
It has also been suggested that lectins function as protective agents by specific: interac-
tion with polysaccharides of viral and fungal origin. I’ Such protective capabilities were
undoubtedly considered by Prokop, Uhlenbruck, and Kohler’ in using the word “pro-
August 1980 173
tectin”, and have been reported for the lectins obtained from wheat germ and barley
seed.13.14It is also tempting to speculate that lectins are responsible for sugar transpor-
tation within the host, but there is little evidence to support this suggestion.
THE ABH-REACTIVE LECTINS

Introduction
Lectins displaying preferential specificity towards the ABH blood group antigens
have been found not only in plant seeds, but in bacteria, fish (gonads, serum, and
ova), molluscs (albumin glands and ova), fungi, and snakes. Some of the better known
and more extensively investigated lectins with ABH blood group reactivity are shown
in Table 1. Many others have been reported (for a review, see Reference 3). In this
section, the use of some of these lectins as an alternative to human sera for blood
typing purposes will be discussed. In addition, the application of these lectins in the
classification, and elucidation of the genetic basis, of certain uncommon ABH pheno-
types, and in determining the biochemical structure of the A, B, and H antigens, will
be reviewed.
Blood Typing Reagents

Not all the lectins shown in Table 1 have been employed as reagents for routine
blood typing purposes. This is due t o a number of reasons, such as the difficulty in
obtaining, or scarcity, of the raw material, the instability of some lectin reagents, and
the need to potentiate the serological reactivity of certain lectins with high-protein
media or by the use of protease pretreated red cells. Another limiting factor is that
many lectins displaying a primary specificity towards the ABH blood group antigens
also cross-react with antigens of other blood group systems and with receptors exposed
on polyagglutinable red cells. Furthermore, anti-A and anti-B lectins invariably react
poorly with group AB red cells, and the weak expression of the ABH antigens on cord-
blood red cells restricts the use of lectins for ABH blood typing purposes to adult
blood samples. A list of anti-A and anti-B lectins that have been used for routine blood
typing purposes is shown in Table 2.
Anti-A Reagents
As mentioned previously, Dolichos biflorus is without doubt the most widely used
lectin in blood group serology. It is primarily used in the diluted state as an anti-A,
blood typing reagent by both manual and automated techniques. However, it should
be noted that undiluted D. biflorus extracts display anti-A (not anti-A,) activity, for
they react with.both A, and A, red cells. D. biflorus lectin is also used in investigative
immunohematology, t o resolve discrepant ABH typing results, and to define certain
types of polyagglutinable red cells (see later). Its acceptance as the preferred source of
an anti-A, reagent has, of course, been enhanced due to the paucity of avid anti-Ar
agglutinins of human origin that are free of other isohemagglutinins. The anti-A from
Vicia cracca may also be used as an anti-Al reagent,6 as may extracts from other vari-
eties of Vicia and D o l i c h o ~ s e e d s . ~ ~
Extracts of the lima bean (Phaseolus lunatus) possess potent anti-A a ~ t i v i t y Some
.~
lima bean extracts also react weakly with group B red cells, -a problem that can be
avoided by simple dilution of the lectin to a point at which it displays specific anti-A
activity. At least three isomeric forms of lima bean lectin have been recognized (for a
review, see Reference 10). Judd’O found the purified lima bean lectin fraction 111 (LB
111) to be the most selective of all the anti-A lectins, for unlike D. biflorus and the
Helix agglutinins, it does not react with Tn- or Cad-polyagglutinable red cells (see
later).
Table 1
SOME POTENTIAL SOURCES O F ABH-REACTIVE LECTINS
Primary
specificity source Common name
Anti-A, (anti-A) Dolichos binorus (seeds) Horse gram

Saxidomus giganteus (whole body) Butter clam

Hyptis suaveolens (seeds)
Anti-A Phaseolus lunatus (seeds) Lima beans
Vicia cracca (xeds) Tufted vetch
Helix horrensis (albumin gland or eggs)
Helix pomatia (albumin gland or eggs) Edible snail
Helix aspersa (albumin gland or eggs) Garden snail
Anti-B Fomes fomenrarius (fungus)
Marasmus oreades (fungus)
Perca fluvatilis (female gonads) Perch
Salmo irideus (ova)
Salmo trutta (ova) Trout
Salmo salar (ova) Salmon
Clupea harengus (ova) Herring
Ptilota plumosa (algae)
Strcptomycc527 S5 (bacterium)
Aristolachia galeata (=W
Bandeiraea simplicifolia (stored seeds)
Anti-B + H Evonymus europaeus (seeds)
Anti-A/B Sophora japonica (=W Japanese pagoda tree
Bandeiraea simplicifolia (fresh seeds)

Python sebae (serum)
Anti-H Ulex europaeus (seeds) Common gorse
Lotus tetragonolobus (suds) Asparagus pea
Laburnum alpinum (seeds)
Cerastium tomentosum (seeds) Snow-in-summer
Cytisus sessilofolius (seeds) Broom (var.)
Anguilla anguilla (serum) Eel
Data compiled. in part, from References 3, 15, and 16.
Potent anti-A lectins have been obtained from the albumin glands and eggs of snails.
notably Helix pomatia, H.hortensis, and H.aspersa.'9.'o The hemagglutinating activ-
ity of these lectins is greatly enhanced in tests employing bovine albumin and protease-
treated red cells. They have been used extensively by some European workers for the
automated typing of donor blood since suitable human group B serum is
often in short supply. A single egg or albumin gland, homogenized in isotonic saline,
can provide several liters of a potent anti-A reagent. Beck"' considers that this abun-
dant source of anti-A warrants investigation in the U.S.,as a n alternative to obtaining
anti-A from hyperimmunized donors.
Anti-BReagents
These have not been routinely employed. One reason fo this is that few seed. extracts
contain adequate anti-B activity (see Table 1). Also, many of the anti-B lectins (notably
those from Fomes fomentarius and the Salmonidaeroe) are unstable and are essentially
incomplete agglutinins inasmuch as they react preferentially-with proteases-treated red
cells. In addition, the Salmonidae roe agglutinins cross-react with antigens of the P
blood group system.*' However, Rees, Cotton, and Holt et al.24showed that the sta-
bility of the trout ova lectin (Salrno rrutra) was greatly improved by prior extraction
of the roe with ether. Even so, Downie, Madin, and VoakZ5found saline homogenates
of ether extracted S. salar t o be quite unsuitable in automated tests employing albumin
August 1980 175
Table 2
ANTI-A AND ANTI-B BLOOD
TYPING REAGENTS PREPARED
FROM LECTIN SOURCES
Primary
specificity Source Ref.
Anti-A, (anti-A) Dolichos biflorus 9

Anti-A Phaseolus lunatus 28
Helix pomatia 21
Helix hortensis 21
Helix aspersa 22
Anti-8 Fomes fomentarius 26
Salmo trutta 24
Salmo salar 25
Sophora japonica 27
Ptilota plumosa 29
Bandeiraea simplicifolia’ 37
Modified with privine hydrochloride@.

’ Purified lectin + Nacetyl-D-galactosamine.
or protease-treated red cells. In such test systems, they found albumin to inhibit hem-
agglutination, and protease treatment of red cells accentuated the anti-P,/P/Pk activity
of the lectin. Downie and colleagues modified the automated procedure by using a
mixture of methyl cellulose and PoIybreneG3 as both a diluent and potentiator of hem-
agglutination. This permitted accurate typing of group B donor samples, but was
found unsatisfactory for typing group AB blood samples.
Pardoe, Uhlenbruck, and Anstee et a1.I6 prepared a useful, but somewhat unstable,
anti-B lectin from mycellium cultures of the fungus F. fomentarius. Also, Chattoraj
and Boyd” modified the anti-A/B lectin from Sophora japonica with Privine hydro-
chloride@ (naphazoline hydrochloride, a nasal decongestant and ocular
vasoconstrictor!!). This bizarre concoction was shown to have anti-B specificity, and
in conjunction with P. lunafusanti-A lectin, it was impregnated into cards by Hynson,
Westcott, and Dunning for routine blood typing purposes.** Recently, Rogers, Blun-
den, and Evansz9isolated an anti-B lectin from the red marine algae Pfilota plumosa.
Makela and MPkeW0 reported that extracts from stored Bandeiraea simplicifolia
seeds specifically agglutinated group B red cells. Fresh seeds were found to contain
anti-A/B activity. Gold-” was unable t o confirm the anti-B specificity in extracts from
stored seeds. Moreover, Murphyt6 found anti-A/B activity in purified preparations
from both stored and fresh B. simplicifolia seeds. These equivocal findings account
for the fact that B. simplicifolia seeds are listed in Table 1 as having both anti-B and
anti-A/ B activity.
Hayes and Goldstein3’ isolated an a-D-galactopyranosyl-bindinglectin from B. sim-
plicifolia seeds by affinity chromatography. Although this highly purified lectin pref-
erentially agglutinated group B red cells, group A red cells were agglutinated (titer one
fourth that obtained against group B red cells). Following an extensive serological
investigation, Judd, Steiner, and Friedman et aLJ’ concluded that the purified lectin
was unsuitable as an anti-B reagent for blood typing purposes since they were unable
to separate the anti-A activity from that against group B red cells by dilution, absorp-
tion, elution, or neutralization procedures.
It was largely as a result of studies b y Murphy and Goldstein’* that a highly-specific
anti-B reagent was prepared by modification of the a-D-galactopyranosyl-bindinglec-
tin from B. simplicifolia seeds. However, before discussing these developments, it
176 CRC Critical Reviews in Clinical L a h a t o r y Sciences
should be noted that two other lectins have been isolated from these same: seeds in
addition to that described by Hayes and Goldstein.” The latter has been designated
BS I lectin. Iyer, Wilkinson, and Goldstein et al.15 isolated an N -acetyl-D-glucosami-
nyl-binding lectin from B. simplicifolia seeds by affinity chromatography on chitin.
This lectin, designated BS 11, has been shown to have anti-Tk ~ p e c i f i c i t y A . ~ ~third
lectin (BS 111) has no apparent blood group specificity.’6
The studies of Murphy and Goldstein” revealed that the BS I lectin of Hayes and
G o l d ~ t e i nis,~ ~in fact, comprised of five isolectins; BS UA.), BS I(A,B), BS I(A2B2),
BS I(AB,), and BS 1(B4). The figures in parenthesis indicate the number of A and B
subunits and antigen combining sites of each isolectin. Figure 1 is a diagrammatic
representation of the tetrameric structure of the five BS I isolectins. BS &B4) was
shown to have anti-B specificity whereas BS I(A4) reacted strongly with group A red
cells and less readily with group B red cells. BS I(A,) also displayed some anti-N activ-
ity in tests at 4OC.”
The isolation of BS I(B4) is both time consuming and costly, and is therefore not a
practical method of preparing large quantities of anti-B for blood typing purposes.
However, following observations from data obtained in studies on the inhibition of
precipitation between hog A substance and BS I isolectins by N-acetyl-
D-galactosamine (D-GalNAc), Judd, Murphy, and Goldstein et al.” predicted that an
anti-B reagent could be prepared from a mixture of BS I isolectins by the simple addi-
tion of a small quantity of D-GalNAc. This prediction proved true and resulted in the
production of a highly specific anti-B reagent (BS I + GalNAc anti-B). The storage
potential and hemagglutinating properties of this reagent were enhanced by using bo-
vine albumin as a diluent. In addition, since highly purified proteins

(and even some crude saline extracts of seeds) have the propensity to adhere to the
surface of glass test tubes, Tween 20@ (a wetting agent) was added to the diluent to
facilitate the reading of hemagglutination reactions.
BS I + GalNAc anti-B was used for both manual and automated typing o f patient
and donor blood samples. Table 3 summarizes the data obtained by Judd, Murphy,
and Goldstein et al.37 in tests on some 500 patient and 2000 donor bloods. As with
other studies on anti-A and anti-B lectins, cord bloods reacted poorly, as did group
A,B donor blood samples. The proclivity of the reagent to give false positive results
was apparent in tests on a blood sample from a patient with serum protein abnormal-
it ies.
Anti-H Reagents
Anti-H reagents of human origin are rare and invariably contain contaminating iso-
hemagglutinins. Thus, anti-H lectins readily fill the void and provide a valuable source
of material for use in investigative immunohematology.
Large-scale typing of blood samples with anti-H reagents is not a common practice.
Rather, anti-H lectins are primarily used to resolve anomalous ABH blood typing re-
sults and for the detection of water-soluble H substances in secretions. Among the
anti-H lectins shown in Table 1, Ulex europaeus is probably the most commonly em-
ployed. Voak, Lodge, and Reed et a1.l8 have shown that the hemagglutinating activity
of U.europaeusis greatly enhanced if human group AB serum is used as a diluent.
In tests on human red cells with anti-H lectins, group 0 red cells give the strongest
reactions, and group A,B the weakest. The precise arrangement of the comnion ABO
phenotypes according to their strength of reaction is: 0 > A2 > AzB > B > A, > A,B.39
In the investigation of blood samples with a n uncommon ABH phenotype, anti-H
lectins are used to determine not only the H antigen strength of the red cells, but also
the presence of H substances in secretions. Table 4 summarizes the classification of
rare ABH phenotypes that have been reported based on the results of such studies.
August 1980 177
BS I(A,Bl Bs I(A,B:)
BS I(AB,) Bs I(B.1
FIGURE 1. Diagramatic representation of the five BS I isolectins. A = A antigen binding site; B = B

antigen binding site. (From Murphy, L. A. and Goldstein, I. J., J . Biol. Chem., 252. 4739, 1977. With
permission.)
Table 3
RESULTS OF MANUAL AND AUTOMATED TESTS
WITH BS I + GalNAc ANTI-B
Total Strongly Weakly

Blood type tested reactive reactive Nonreactive
0 215 215
Manual A 198 I' I97
tests B 83 83
AB 23 21 2'
0 881 881
Automated A 747 747
tests B 282 28I 1
AB 100 83 12 5
Sample from patient with elevated cryoglobulins.

' Cord-blood samples.
From Judd, W. J., Murphy, L. A., Goldstein, I. J., Campbell, L.. and Ni-
chols. M.E.. Transfusion,18. 274.1978. With permission.
Voak and Lodge" consider the Lotus tetragonolobuslectin to be anti-HI, for it fails
to agglutinate adult group 0 I-negative red cells and Bombay (0,) red cells. Similarly,
they consider the anti-H from Cytisus sessilofolius to be anti-H/HI, for C. sessilofolius
reacts less readily with group 0 I-negative red cells than it does with group-0 red cells
that are I-positive. Bird" views these differences as resulting from the steric interrela-
tionship between the H, I, and i determinants rather than subtle variations in anti-H
lectin specificity. This opinion is supported by Bird's finding that following protease
or neuraminidase treatment (which presumably removes sterically hindering adjacent
antigen structures), group 0 I-negative red cells react as strongly as I-positive red cells
in tests with C. sessilofolius anti-H lectin.
Table 4
CLASSIFICATION OF THE ABH VARIANT PHENOTYPES
Anti- Isohemagglutinins ABH substances
Phenotype Anti-A hti-B A.B Anti-H present in serum present fin saliva
A, 2+mP 0 3+mf 3+ Anti-B A+H

A, W 0 W 4+ Anti-B A+H
A. 0 0 2+ 4+ Anti-B + A , H
A., 0 0 0 4+ Anti-B H
A,. +(I%) 0 +(1%) 4+ Anti-B H
A, 1 +mf 0 l+mf 4+ Anti-B A+ H
A* W 0 W 0 Anti-B + H nont:
A: 0 0 O/ + 0 Anti-B A+ H
B, 0 l+mf 2+mf 3+ Anti-A B + I-!

B. 0 0 2+ 4+ Anti-A H
B, 0 W W 0 Anti-A + H nonc:
B: 0 0 0 a Anti-A B+H
6-1' 0 I+ 1+ 3+ Anti-A + B B + 13
B 11'- 0 I+ 1+ 3+ Anti-A B + 13
B - 111' 0 I+ 1+ 3+ Anti-A H
0. 0 0 0 0 Anti-A + B + H none
0: 0 0 0 0 Anti-A + B H
The symbol mf denotes mixed-field agglutination. H reactions of red cells and saliva obtained
primarily from studies with U. europaeus
* Notation of Race and Sanger."
Data compiled from References 15, 40. and 41.
Biochemical Specificity
The carbohydrate-binding specificity of the ABH-reactive lectins has confirmed
other biochemical data concerning the structure of the A , B. and H antigens. In 1953,
Morgan and Watkins.. found that the agglutination of group A red cells by P. limensis
was preferentially inhibited by a-D-GalNAc. Also, they found that the agglutination
of group 0 red cells by the anti-H lectin from L. tetragonofobus seeds was abolished
by the addition of u-L-fucose (U-L-Fuc). They interpreted these findings as indicating
that a-D-GalNAc and a-L-FUC are the immunodominant sugars conferring A and H
blood group specificity, respectively. Their conclusions were substantiated some 12
years later by studies on the sequential enzymatic degradation of water-soluble ABH
blood group substances."
a-GalNAc preferentially inhibits the majority of anti-A lectins, and a-D-galactose
(0-D-Gal), the immunodominant sugar conferring blood group B ~pecificity,'~ prefer-
entially inhibits most anti-B lectins. Some anti-B lectins are also readily inhibited by
L-rhamnose. U. europaeus extracts are known to contain two anti-H lectins: UE I,
which is inhibited by U-L-FUC,and UE 11, which is preferentially inhibited by salicin
(a glucose derivative) and by di- N-acetylchitobiose (D-G~cNAc)~ (a derivative of N-
acetyl-D-glucosamine).U Various investigators, notably Bird and Wingham,'' have
found the anti-H lectins from the eel Anguifla anguifla and L. tetragonofobus are pref-
erentially inhibited by U-L-FUC, whereas anti-H lectins from Cerastium tomentosum.
Laburnum alpinum, and C. sessifofoliusare preferentially inhibited by salicin. Table
5 summarizes these findings.
Further Applications
As mentioned above, the weak reactions displayed by anti-A and anti-B lectins with
August 1980 I79
Table 5
PREFERENTIAL CARBOHYDRATE-BINDING ACTIVITY
OF THE ABH-REACTIVE LECTINS
Primary carbohydratc- Primary blood
binding specificity group specificity
u-D-GalNAc A1 Dolichos biflorus

A Phaseoulus limensis
A Vicia cracca
A Helix agglutinins
a-D-Gal > u-D-GalNAc B (A/B) Bandeiraea simplicifolia (BS I)
u-D-Gal B Ptilota plumosa
B Fomes fomentarius
u-D-Cal/L-Rha B Salmonidae roe agglutinins
U-L-FUC H Ulex europaeus I
H Lotus tetragonolobus
H Anguilla anguilla
H Ulex europaeus I I
H Cytisus sessilofolius
H Laburnum alpinum
H Cerastium tomentosum
Note: 0-D-GalNAc = u-Nacetyl-D-Gal = u-D-galactose; L-Rha = L-rhamnose;

U-L-Fuc= u-L-fucosc;(D-GIcNAc), = di-Nacetylchitobiose.
cord-blood red cells and group AB red cells imposes a severe restriction on their use
as routine blood typing reagents. Rogers, Topliss, and B l ~ n d e n 'and

~ Judd, Murphy,
and Coldstein et a1.l' conclude that P. pfumosa and BS I + GalNAc anti-B prepara-
tions are better suited for use in investigative immunohematology, rather than as alter-
natives to human group A serum as a source of anti-B for blood typing purposes. In
contrast to human anti-B sera, both lectin reagents fail to agglutinate red cells that
have acquired B-like antigens in vivo. Also, both lectins d o not agglutinate red cells
sensitized with a B-antigen-containing lipopolysaccharide from Escherichia coli OS6.
They, therefore, make the extremely useful distinction between genetically determined
B antigens and those that may be acquired, either in vivo following deacetylation of
A antigen into a B-like structure by bacterial enzyme^,'^ or in vitro by adsorption of
B antigen from bacterial p o l y s a c ~ h a r i d e s . ~ ~
BS I + GalNAc anti-B, D. biflorus anti-A,, and the Helix agglutinins react with
certain types of polyagglutinable red cells. Moreover, some types of red cell polyagglu-
tination give weaker than normal reactions with selected anti-H lectins. These aspects
will be discussed more fully below.
The ABH-reactive lectins have been used to separate mixed red cell populations such
as may occur in blood group mosaicism resulting from chimerism, dispermy, somatic
mutation, or a bone marrow transplant. Lectins are particularly suitable for this pur-
pose since dispersal of the agglutinated red cells can readily be accomplished by the
addition of the appropriate sugar. The two populations of red cells can then be tested
for other blood group factors.
An affinity chromatography column of P. funatus anti-A lectin was used by Oguchi,
Kawaguchi, and Suzata et al.5' to separate two populations of red cells from a group
A, individual. The two populations were then subjected to binding studies with "C
labeled P. funatus, and I Z 5 I labeled eel (Anguillajaponica) lectin. The agglutinable (P.
funatus binding) A, red cells were shown to have small numbers of A antigens similar
to the A antigen on A, red cells. However, the agglutinabIe A, red cells were shown
to have an H-antigen content identical to that of free (nonreactive with P. funatus) A,
red cells. Affinity chromatography columns have also been used in attempts to separate
stem cells from normoblasts (Murphy’”). Such a n approach seems reasonable, since
the ABH antigens are most likely formed at, o r just prior to, the normoblast stage.”
Bird” used purified D. biflorus lectin in agar diffusion tests to show that the lectin
reacted with a common component of the A antigen present in human A, and A, blood
group substances, and in hog A substance. These findings differ from those of Boyd
and Shapleigh,’ who found D. biflorus t o precipitate with human A1 substilnce, but
not with A2. BirdSZattributes these conflicting results to quantitative rather than qual-
itative differences between A l and A2.
Because of their inability t o react strongly with red cells containing less than normal
quantities of A, B, and H antigens, lectins are suitable reagents for studying the vari-
ations in expression of the ABH antigens among the various common ABO phenotypes
and in certain disease processes, particularly those resulting in polyagglutination (see
later). Gillespie and and Reid and Judd,” used F. fomentarius a n t i 4 lectin to
demonstrate the interaction between the A and B blood group genes that is responsible
for the weak expression of the B antigen in A I B individuals. Also, ferritin Iilbeled or
radioiodinated lectins have been used to determine the number of A and €3 antigen
sites on red cells (Hayes and G o l d ~ t e i nWilliams
,~~ and V ~ a k , and
~ ” Boyd, Bhatia, and
Diamond et al.56).While the data obtained by such studies indicate that a significantly
greater number of sites are present than revealed by tests with human sera,57the ratios
of the number of A and B sites among the various ABO phenotypes are constant.
Studies on Cord and H-Deficient Bloods

Voak and Lodges8 studied group A cord-blood red cells with D. biflorus and U.
europaeus Iectins. In contrast to adult A, red cells, A, cord-blood red cells were non-
reactive with a potent preparation of D. biflorus. However, A2 red cells from cord-
blood samples reacted as strongly as group 0 cord-blood red cells in tests with U.
europaeus. Voak and Lodge consider their findings to indicate that A antigens are
formed independently from H. This proposal conflicts with the widely accepted hy-
pothesis that H substance is the precursor material from which the A and B antigens
develop.45 Additional studies by Voak, Stapleton, and B o ~ l e y and , ~ ~Voak, Lodge,
and Stapleton et al. provide yet further evidence for the independent development
of A. These workers found one in 139 A, and one in 13 AzB bloods t o be deficient in
H antigen. However, from the results of family studies, Voak and colleagues suggest
their findings may be explained by the presence of weak alleles a t the H / h 10c:us. Con-
sequently, in some family members, there is insufficient precursor H material for the
production of normal A antigens.
Bird 43 found L. tetragonolobus anti-H lectin, in contrast to U.europaeus, L. al-
pinum, and C. sessilofolius extracts, t o be nonreactive with neuraminadase-treated 0,
(Bombay) red cells. That some anti-H lectins d o react with 0, red cells following neur-
aminidase treatment may indicate that H-receptors are present as cryptantigens on
these red cells. This finding causes one to question whether the 0, (Bombay) phenotype
is due t o a “double-dose” of the amorphic gene h, or, as indicated by these results, is
simply due to a quantitative defect in the expression of H antigen resulting from the
inheritance of weak H alleles or modifying genes at the H / h locus. Both possibilities
are discussed by Gallasch,61 and the latter used to explain the genetic background of
the para-Bombay phenotypes (for a review, see Reference 15). Perhaps it is pure coin-
cidence, but it has struck this author that those anti-H lectins that react with neuramin-
idase-treated red cells are all inhibited by salicin and by (D-GICNAC)~. Perhaps, there-
fore, these lectins are reacting with D-GIcNAc structures that are exposed -when red
cells are treated with neuraminindase, and not with H g e n e products at all. Clearly,
further studies are warranted to elucidate the nature of these reactions.
August 1980 181
The results of hemagglutination and saliva inhibition studies using lectin anti-H re-
agents on individuals with an uncommon ABH phenotype (Table 4). have provided
insight into the nature of the genetic aberrations associated with some of these rare
entities. The lack of cellular A and H antigens on the red cells, but their presence in
the secretions of individuals of the A, phenotype, led Weiner, Lewis, and Moores et
aL6' to suggest that the A, phenotype arises from the lack of a common gene Y. This
Y gene is considered essential for the development of A and H antigens on red cells,
but does not control their development in the secretions. In contrast, while the precise
genetical background is not always clear, the strong reactivity of A, red cells with anti-
H lectins, and the presence of H, but not A, blood group substance in the secretions,
indicates that the genetic defect responsible for the A, phenotype lies not in the pro-
duction of H, but rather in the development of A from precursor H material at both
cellular and secretory levels.
Conclusions
Lectins with a primary specificity towards the ABH blood group antigens provide
an alternative to human sera as a source of blood typing reagents. With the exception
of Ulex europaeus (anti-H). and Dofichos biflorus (anti-A,), few are used by more
than a handful of investigators on a routine basis. In part, this is due to the poor
reactions given with cord-blood red cells, and with red cells from group AB individu-
als. Nonetheless, these reagents have considerable application in studies on red cells
of an uncommon ABH phenotype.
LECTINS THAT BIND TO THE MN AND Ss SIALOGLYCOPROTEINS
Introduction
Two red-cell-membrane sialoglycoprotein (SGP)structures that transverse the bili-
pid layer are known to be intimately associated with the MN system antigens, the latent
T- and Tn-polyagglutinable receptors, and the En. and Pr-Sp determinants. These
structures, also referred to as glycophorin A and B, are comprised of a series of amino
acids to which alkali-labile tetrasaccharides are attached to serine or threonine residues
at various points along the protein m01ecule.~~.~' The trypsin-sensitive structure, gly-
cophorin A (MN SGP), carries the M and N blood group antigens, and the S and s
antigens are found on glycophorin B (Ss SGP). It is to the attached alkali-labile tetra-
saccharides that certain lectins bind. Some lectins display a preferential specificity for
red cells that possess either M or N blood group antigens, while some react more
strongly, and yet others less readily, with red cells that are deficient in either MN SGP
or Ss SGP. In this section, the use of such lectins as anti-M and anti-N blood typing
reagents, and their application to the study of the MN and related red cell surface
antigens and in defining the SGP deficiencies associated with certain MN system var-
iant phenotypes, will be reviewed.
Blood Typing Reagents

Anti-M and anti-N reagents for routine blood typing purposes are usually obtained
by injecting rabbits with the appropriate human red cells, since potent antibodies of
human origin are not frequently encountered. Some of the lectins shown in Table 6
provide possible alternatives to rabbit reagents as sources of anti-M or anti-N for blood
typing purposes.
Anti-M Reagents
Allen and Brilliantine65reported anti-M activity in saline extracts of three varieties
of seeds of the genus Iberis. These are the only anti-M lectins mentioned in the review
I82 CRC Critical Reviews in Clinical Laboratory Sciences
Table 6
POTENTIAL SOURCES OF LECTMS
DISPLAYING PREFERENTIAL
SPECIFICITY FOR M OR N ANTIGENS
Primary
specificity source Ref.
Anti-M Iberis amara 65

Iberis umbellata 65
lberis semperivens 65
Japanese radish/turnip 66.67
Anti-N Vicia graminea 8
Vicia leganaya 71
Vicia picta 71
Vicia unijuga 72
Ba uhinia candicans 71
Bauhinia variegata 71
Bauhinia bonatiana 71
Bauhinia purpurea 71
Anti-A/N Bandeiraea simplicifolia [BSI(A.)] 37
Moluccefla laevis 73
by Gold and Balding,’ although Race and Sanger” cite references t o Nakajima and
colleague^^^.^^ who found anti-M activity in extracts of seeds from four varieties of
Japanese radish and one variety of turnip (genus and species not known, papers not
seen by this author). An anti-M blood typing reagent from I . arnara (candy tuft) was
marketed for a brief period of time by Hyland Laboratories, Los Angeles, Calif., but
was soon withdrawn, presumably due to production difficulties. Other workers have
attempted to prepare anti-M reagents from these seeds, but with little success. Roma-
nowska6’ observed weak anti-M activity in concentrated ammonium sulfate precipi-
tates of extracts from I . amara. By preparing the extracts in either glycine or bovine
albumin, Moulds and Juddna6demonstrated a weak anti-M like agglutinin in 1. umbel-
lata seeds. These preparations were quite unsuitable for blood typing purposes, their
reactivity being influenced by the Ss antigen status of the test red cells (most reactive
with MS red cells, but causing weak agglutination of Ns and Nu red cells). These
reactions were abolished by treatment of red cells with proteolytic enzymes and en-
hanced following treatment of red cells with neuraminidase.
An ti-N Reagents
Anti-N from the seeds of Vicia graminea is available commercially as a blood typing
reagent and can be readily prepared from crude saline extracts of the seeds. Such re-
agents cause avid agglutination of N and MN red cells, but also react weakly with red
cells from individuals that have inherited two Mgenes. This weak agglutination is due
to the presence of ‘N’, a n N-like antigen present on the Ss SGP, glycophorin B (see
later).”.69 In order to avoid discrepant blood typing results due t o ‘N’, V. graminea
lectin is employed in tests a t 37°C.
Other sources of anti-N have been reported in several varieties of Vicia seeds, and
in seeds of the genus Bauhinia.’O Extracts of the latter may require the addition of
D-Gal or D-glucose if they are to be used for blood typing purposes, otherwise they
display no serological specificity.” Potent anti-N activity has also been demonstrated
in extracts of leaves from the Korean V. uniguga.” In addition, anti-A/N activity,
apparently inseparable as judged by the results of inhibition, absorption, arid elution
experiments, has been reported for the BS I(AJ isolectin from Bandeiraea sintplicifofia
seeds3’ and in extracts of Molucceffafaevis seeds (Bells of I ~ e l a n d ) . ’Of
~ all the anti-N
lectins shown in Table 6, V. graminea is probably the most widely employed.
August 1980 183
Specificity of the MN-Reactive Lectins

The MN-reactive lectins V . graminea, B. variegata, B. purpurea, and 1. amara, do
not react with the same portion of the M and N antigens defined by human or rabbit
reagents. The reactivity of the latter is often destroyed by prior treatment of red cells
with neuraminadase, whereas the lectin reagents give enhanced reactions with neura-
minidase-treated red cell^.^*-'^ The studies of Romanowska6* also demonstate subtle
differences in the specificity of V. graminea and B. variegata anti-N lectins. In partic-

ular, the V. graminea receptor, in contrast to that defined by B. variegata, is destroyed
by enzymes from Trichomonas foetus. Also, the inhibitory capacity of N substance in
tests with V. graminea is increased 100-fold by pretreatment with neuraminadase, but
only a threefold increase in inhibition is obtained in tests with B. variegata. Moreover,
neuraminadase-treated M substance is a poor inhibitor of V. graminea, but is only
slightly less effective than neuraminidase-treated N substance in inhibiting B. varie-
ga ta.
Based on this data, Uhlenbruck7’ proposed that a three-component precursor sub-
stance exists from which the MN (and SsU) antigens defined by human and rabbit
reagents develop: a poorly differentiated receptor detected by the Bauhinia agglutinins,
and more well defined M and N precursor material reacting with I . amara and V.
graminea lectins, respectively. In an extensive investigation, Uhlenbruck and Dahr7*
demonstrated inhibition of B. purpurea anti-N lectin with simple sugars: D-GalNAc
being twice as inhibitory as D-Gal. However, I. amara and V. graminea lectins were
not inhibited by simple sugars, only by M- or N-active glycoproteins. From this data,
Uhlenbruck and Dahr conclude that B. purpurea reacts with subterminal D-GalNAc
residues of both M and N active glycoproteins. They consider the binding sites of I.
amara anti-M and V. graminea anti-N to be more complex, involving primarily D-
GalNAc and D-Gal residues, respectively, but incorporating adjacent carbohydrates
present in the MN-active glycoproteins.
Studies on MN Antigen Structure

According to Springer and H ~ p r i k a r ’and
~ Springer, Tegtmeyer, and Huprikar,80
blood group M and N specificities are determined by the carbohydrate structures pres-
ent in oligosaccharide chains that form part of the MN SGP. This conclusion is based
on the results of studies with human and rabbit antisera and with V. graminea anti-N
lectin. In contrast to some human and rabbit reagents, V. graminea gives enhanced
reactions with neuraminidase-treated N-positive red cells (but see below) and reacts
weakly with untreated red cells from individuals that are genetically MM. These reac-
tions also enhanced by neuraminidase. Springer and colleagues conclude that the V.
graminea receptor is the precursor material from which the M and N antigens defined
by human and rabbit reagents develop. They claim that blood group N activity arises
from the attachment of an u-Nacetylneuraminic acid (NANA) residue linked glycos-
idically to one of two P-D-Gal residues that are both linked to a single a-D-GalNAc
residue. The product of the M g e n e is considered to be a specific sialyl-transferase
which attaches an additional NANA to the vacant P-D-Gal residue present on pre-
formed N substance.
There is now considerable evidence to refute the precursor transferase theory out-
lined above. This evidence includes:
1. M-positive red cells are agglutinated by V. gramiflea anti-N lectin following treat-
ment with acetic anhydride, which does not remove terminal NANA residues,
but blocks the r-amino groups of lysine and arginine.8’ This modification results
in reorientation of the amino-acids that form the “backbone” of the MN SGP,
making P:D-Gal residues present in the attached oligosaccharides more accessible
to V. graminea lectin.
2. There is no detectable difference in the amount of NANA present in M (N-) and

N (M-) red cells. Such a difference would be anticipated if M and N antigens
contain two and one NANA residues each, respectively. That the MN SGP con-
tains a large proportion of the total red cell surface NANA is evident by the fact
that in one hemizygote (M/-) the red cell NANA level was markedly reciuced.Oa
3. Lisowska and Kordowic~’~ have prepared antibodies (anti-M‘ and anti-N‘) by
injecting desialyzed M and N substances into rabbits. As expected, these antibod-

ies give enhanced reactions with neuraminadase-treated red cells of the appropri-
ate MN phenotype.
4. Judd, Issitt, and Pavone et have shown, in contrast to the results of earlier
investigator^,^^“^ that not all anti-M or anti-N reagents of human or rabbit origin
fail to agglutinate neuraminadase-treated red cells. Indeed, they found some MN-
system antibodies to give enhanced reactions with red cells containing less than
5% of their original NANA.
5. Two independent groups of investigators, Dahr, Uhlenbruck, and Janssen et a1.”
and Whniowska et a1.,86 found the amino acids at positions one and five from
the NH2-terminus of the MN SGP to differ in M red cells from that found in N
red cells. In red cells from MM individuals the sequence is serine-serine-threo-
nine-threonine-glycine, and in red cells from N N individuals it is leucine-serine-
threonine-threonine-glutamicacid. The second, third, and fourth amino acids,
which are identical in each case, have attached alkali-labile oligosaccharides.
The above observations support the hypothesis that the M and Ngenes code for the
amino acid sequence of the polypeptide “backbone” of the MN SGP, and not for the
sialyl-transferase as proposed by Springer and H ~ p r i k a r ’and
~ Springer, Tegtmeyer,
and Huprikar.m The reactions of the apparent MN-specific plant lectins would appear
to be dependent on the steric configuration (which is determined by the amino acid
sequence of the polypeptide “backbone”) of the attached oligosaccharides that are
linked to either serine or threonine residues at 15 positions on the portion of the MN
SGP that extends from the red cell membrane.64.’4These oligosaccharides are identical
to the alkali-labile tetrasaccharides of Thomas and W i n ~ l e r , ’and
~ are shown in Figure
2A.
In attempting to integrate the two conflicting theories regarding the nature of the
M and N blood group antigens, Steane, Flanders, and Steane et aL8*proposed that
there are six different N specificities (Nn, NP, “N”IV, “N”‘, Nv,, and N3, arid two M
specificities (Mn and MP). Antibodies to NN, “N’”, and M”, define structures deter-
mined by NANA. V. graminea lectin defines the NvGreceptor, and NF receptors are
defined by those anti-N antibodies produced by patients undergoing hemodialysis on
instruments cleansed with formaldehyde. Anti-NP, anti-”N”P, and anti-MP, which may
be identical to the NANA-independent antibodies described by Judd et a1.,84 or the
M‘ and N‘ antibodies described by Lisowska and Kordowic~,’~ are directecl towards
the M or N antigens determined by the sequence of the NH2-terminal amino acids of
the MN SGP. However, Judd. Issitt, and Pavone et al.” consider both NANA-inde-
pendent and NANA-dependent MN-system antibodies to be directed towards the NH,-
terminal amino acids of the MN SGP. They consider the difference between the two
types of antibodies to be that those defining NANA-dependent antigens, in contrast
to antibodies defining NANA-independent antigens, require a binding site that in-
cludes NANA residues in addition to the NHz-terminal amino acids of the lLlN SGP.
Studies on Sialoglycoprotein Deficient Bloods

Introduction
The sialoglycoproteins on which the M, N, S, and s antigens are located, can be
August 1980 I85
( A ) Alkali-labile tetrasaccharide:
Serine or
NANA--a (2-3)-D-Gal-p (1-3)-D -GalNAc---(I---
I Threonine
(I(216)
I
NANA
(5)T antigen:
D-Gal-p (1-3)-D-GalNAc---a-- Serine or
Threonine
(C) Tn antigen:
Serine or
D -GalNAc---a---
Threonine
FIGURE 2. Structure of the alkali-labile tetrasaccaride and precursor T- and Tn-receptors present on
human red cell membranes. N o k T and Tn antigens are latent receptors on normal red cells. NANA =
N-acetylneuraminic acid; D-Gal = D-galactose: D-GalNAc = N -acetyl-D-galactosamine; u/p = ano-
rneric configuration. (A) After Thomas, D. B. and Winzler, R . J.. J . Biol. Chem., 244, 5943, 1969; (B)
(C) Modified from Springer, G . F. and Desai, P. R . , Carbohydr. Res.,QO, 183, 1975.
recognized following periodic-acid Schiff’s Base (PAS) staining of human red cell
membrane components separated by sodium dodecyl-sulfate polyacrylamide gel elec-
trophoresis (SDS-PAGE), as shown in Table 7. The PAS staining bands revealed by

this procedure are u (syn. PAS 2, monomeric MN SGP), u2 (syn. PAS 1, dimeric MN
SGP), 0 (syn. PAS 2’). d (PAS 3, monomeric Ss SGP), d2 (syn. component C, dimeric
Ss SGP), and ad (syn. PAS 4, an aggregate of MN and Ss SGP). A minor component
(y) is also apparent, but this has not been well characterized. In addition, the red cell
membrane contains another heavily glycosylated glycoprotein that contains but low
levels of NANA. This component is referred to as band 3, or Coomassie 3.89.90
PAS 2’ and the Ss SGP, like the MN SGP, have attached alkali-labile tetrasacchar-
ides at serine or threonine residues along their polypeptide chains. These carbohydrates
are the receptors to which lectins such as V. graminea, Maclura aurantiaca (syn. pom-
ifera, the Osage orange) and Arachis hypogaea (peanut lectin) bind. The MN SGP, in
contrast to the Ss SGP, has (at amino acid residue 26) a n attached alkali-stable tetra-
saccharide which serves as a binding site for Phaeolus vulgaris and Ricinus communis
lectins. 64.*9.9 Receptors for P. vulgarislectin are also present on PAS 2‘.
Table 8 summarizes some of the SGP deficiencies and abberations associated with
the inheritance of certain uncommon blood group genes. The genetic background of
some SGP deficient bloods is shown in Table 9. For further information, the interested
reader is referred to the recent review by Amtee.*’ In this section, an attempt will be
made to relate the data shown in Tables 8 and 9 with the results of tests on the behavior
of intact SGP-deficient red cells and their isolated membrane components with certain
lectins.
The S-s- Phenotypes and the “”’Antigen

Dahr, Issitt, and Moulds et aL9’ have shown that S-s-U- red cells are devoid of Ss
SGP, and S-s-U+ red cells have only 5 % of normal Ss SGP. Both phenotypes have
normal MN SGP. Using purified radioiodinated M. aurantiaca and A. hypogaea lec-
tins (the latter was tested against membrane components obtained from red cells that
had been previously incubated with neuraminidase to expose latent T-receptors), Tan-
ner, Anstee, and Judson’’ observed no binding of the lectins to the SDS-PAGE com-
Table 7
THE PAS-STAINING, COMPOSITION, AND ANTIGEN CONTENT OF HUMAN
RED CELL MEMBRANE SIALOGYCOPROTEINS
Coomassic
SMoJycoprotein PAS 2’ M N SOP M N + Ss SOP ss SOP Ill
PAS staining band P U 0 2 ud d d,

Alternative notation PAS 2’ PAS2 PAS1 PAS 4 PAS 3 UandC Band3
Composition Monomer Dimer Aggregate Monomer Dimer
Antigen content IT, Tnl’ [T, Tnl. [T. Tnl. IT, Tnl. ?
M, N, Pr M. N, Pr. IN’,Pr.
‘N’,S , s s, s,
U’ U‘
a
[ 1 = Latent receptors exposed only on T-activated or Tn-polyagglutinable red cells.
’ U antigen probably associated with Ss SGP.
August 1980 I87
Table 8
THE EFFECT OF SOME BLOOD GROUP GENES ON THE PRODUCTION OF
RED-CELL-MEMBRANE SIALOGLYCOPROTEINS AND ASSOCIATED
UNCOMMON MN-SYSTEM ANTIGENS
Normal M N SBP Normal Ss SGP
Gene production production Associated SGP aberrations
s' Normal None/partial None/abnormal ud

En. UK None Probably none Probable hybrid M-S SGP,"M" antigen'
En.Fin None Normal None'
M' None None None
Mi' Normal Normal Abnormal PAS 2'
Mi'' Normal Normal Abnormal PAS 2'
Mi''/ Normal Abnormal (dYi."') None
Mi'" Normal Abnormal (d"'.'") None
MiV None None Hybrid N-s-Hi1 SGP (a-d)yi.vb
M i "' Normal Abnormal (d"' "') None
Ph None None' Hybrid (d-u)"
M' Altered Normal None
M' Normal Abnormal (d"') None
. Membranes from both English and Finnish individuals show increased glycosylation of band 3.
' Membranes from Mi.V individuals appear to have elevated levels of PAS 2'."'
' Assumed from studies on heterozygote.
Adapted from Anstee. D. J.. Immunobiology of the Red Cell, Liss. New York. in preparation, 1980.
Table 9
THE GENETIC BACKGROUND OF SOME
SIALOGLYCOPROTEIN-DEFICIENTBLOODS
Donor Genotype M N SGP ss SGP Hybrid SGP
M.W. S'/S' Normal Altered/absent Absent

M.P. SVPh 50% of normal Altered/absent Present
R.L. En.Fin/En. Fin Absent Normal Absent
M.E-P. En. UK/M' Absent Probably none Probable
F.M. M i "/Mi " Absent Absent Present
H.T. M '/M' Absent Absent Absent
J.R. ? Absent Absent Present
L.R. M i "/Mifv 50% of normal Altered Present
Adpated from Anstee, D. J., Immunobiology of the Red Cell, Liss, New York, in
preparation, 1980.
ponents corresponding to the Ss SGP in red ceIl membranes isolated from S-s-U- indi-
viduals. They did, however, observe substantial reduction, but not total absence, of
PAS staining when the electrophoresis was carried out using the buffer system de-
scribed by Fairbanks, Steck, and Wallach." By employing the-buffer system of Lae-
mrnke5PAS staining was observed in the region corresponding to normal ad (aggre-
gate of MN and Ss SGP), but not to components d and d2 (monomeric and dimeric
SGP, respectively). From these results, which do not correlate entirely with those of
Dahr and associate^,^' AnsteeE9considers that S-s- red cells do not contain normal Ss
SGP, but do contain an abnormal component, which like normal Ss SGP complexes
with the a component of the MN SGP to form an aggregate that has the same electro-
phoretic mobility as normal ad.
Allen, Corocan, and EllisP6found MU-red cells (genotype MS”/MSS to be nonreac-

tive with V. graminea lectin. Further studies by Allen, Maddan, and King,97by Dahr,
Uhlenbruck, and K n ~ t t , ~and
’ by Rolih and I s ~ i t thave
, ~ ~ shown that following trypsin
treatment (which cleaves the MN SGP, but not the Ss SGP) all S-s-U-and S-s-U +- red
cells, regardless of their MN phenotype, are nonreactive with V . graminea anti-N. In
contrast, U-positive red cells that are either S + or s + , or both S + and s + , react
with V. graminea lectin after they have been treated with trypsin. Furthermore, the
studies of FurthmayrU indicate that the polypeptide “backbone” of the Ss 5GP ob-
tained from all individuals of a common MNSsU phenotype has the same sequence of
NH2-terminal amino acids as are found in the MN SGP isolated from red cell mem-
brane components of NN-individuals. These findings indicate that an N-like antigen,
“N” (sometimes referred to as “intrinsic N”), is present on the Ss SGP of all normal
S + and/or s + red cells.69 This hypothesis seems a more tenable explanation for the
reactivity of MM red cells with V. graminea lectin than that suggested by Springer and
~ Springer, Tegtmeyer, and Huprikar’O who consider such reactions to
H ~ p r i k a r ’and
be due to the presence of N precursor material (Nv,receptor) from which the M anti-
gen develops.
Red cells from donor H. T. (Mk/M? and donor M. P. (S”/Ph)(see Table 9), also
lack S, s, and U Unlike other S-s-U- red cells, the M. P. red cells react
strongly with V. graminea lectin after they have been treated with trypsin. These red
cells have an abnormal SGP which forms complexes with itself and with normal MN
and Ss SGP. This abnormal component has receptors for M. auranriaca lectin, but
not for P. vulgaris lectin. Anstee re suggests that this abnormal component is a hybrid
molecule (d-u)“”, which has Si SGP at its NH1-terminus and MN SGP at its C-termi-
nus.
The En(a-) Phenotypes

This phenotype, which is undoubtedly heterogenous, has been found in a Finnish
family’03where the propositus is considered to be genetically En.Fin/En. Fin,’9 and in
an English familyao3where the genetic background appears to be En.UK/Mk.69.’9 A
further En(a-) individual whose red cells appear to be indistinguishable from those of
type En.Fin/En.Fin, but who does not appear to be related to the Finnish En(a-)
propositus, has been found in Canada.’” Furthermore, donor H.T. (Mk/kf.?,in ad-
dition to being s-s-&, must also be considered to be En(a-).09
En(a-) red cells are characterized by a marked reduction in NANA, lack the MN
SGP,and type as Wr(a-b-) (for review, see Reference 89). Evidence for lack of the
MN SGP was obtained by Anstee, Barker, and Judson et al.’OS and by Tanner and
Anstee.’OC Using radioiodinated M. auranfiaca and A . hypogaea lectins (the latter was
again tested on membranes from neuraminidase-treated red cells), they found the lec-
tins to bind to both the MN and Sd SGP of normal red cells, but only to PA4S2‘ and
the Ss SGP obtained from En.Fin/En.Fin and En.UK/M’red cells. Binding of M.
auranriaca and P. vulgaris lectins to band 3 of En(a-) red cells was stronger than that
obtained with normal red cells. This finding correlates with the increased glycosylation
of band 3 in the En(a-) phenotype,w which appears to be a consistent feature of red
cells that lack the MN SGP.r9 Furthermore, these findings clearly indicate that alkali-
labile oligosaccharides are present on PAS 2‘ and band 3, and that the latter also has
attached alkali-labile tetra saccharide^.'^
En(a-) red cells (type En.UK/M’) were studied with lectins by Bird and Wingham.’07
They found the reactivity of these red cells to be enhanced in tests with anti-H lectins
(presumably due to the reduced <-potential of the red cells), to be more strongly agglu-
tinated than normal red cells by a variety of “non-blood-group-specific” lectins (no-
August 1980 189
tably Sophora japonica and Glycine m a ) , and to give weaker reactions than normal
with M . aurantiaca lectin. This latter observation was confirmed by Tanner and An-
steelo6 who also found En.Fin/En.Fin red cells to react weaker than normal with M.
aurantiaca lectin. Tanner and Anstee further ,observed enhanced reactions with both
forms of En(a-) red cells in tests with Lens culinaris, R. communis, Triticum vulgaris,
Canavalia ensiforms, and P. vulgaris lectins. Furuhjelm, Myllyla, and Nevanlinna et
a1.'02 tested the original (En.Fin/En. Fin propositus (Mr. V. B.) with lectins and noted
stronger than normal reactions with S. japonica and U.europaeus lectins. These red
cells were nonreactive with V. graminea anti-N lectin, but gave enhanced reactions
with a n extract from B. purpurea seeds. The results of absorption studies using En(a-)
red cells on the B. purpurea extract indicated that this lectin contains two agglutinins:
one anti-En' and the other anti-N.
Dahr, Uhlenbruck, and Leikola et al.lO"have shown that the Ss SGP of the English
En(a-) proposita (Mrs. M.E-P. of Table 9), has serine and not leucine as the NH,-
terminal amino acid. This finding correlates with the results of anti-M binding studies
by Anstee, Barker, and Judson et al.,lo5 who demonstrated an M-like receptor ('M',
analogous to the 'N' antigen) on the Ss SGP of En.U K / M k red cells, but not on the
Ss SGP of En.Fin/En.Fin red cells. Dahr Unleribruck, and Leikola et al.,loS consider
the Ss SGP from Mrs. M. E-P. to be a hybrid molecule with its NHI-terminus derived
from MN SGP and its C-terminus from Ss SGP.Ansteeo9reports that the site of this
hybridization does not go beyond the asparagine residue at position 26 of the MN SGP
to which an alkali-stable oligosaccharide is attached, for the hybrid molecule does not
bind t o P. vulgaris lectin. Since 'M' does not appear to be present on the Ss SGP of
the Finnish En(a-) individual, Mr. G. W., the En.Fin and En.UK genes clearly have
different polymorphic effects. The red cells of Mrs. M. E-P. also have reduced levels
of Ss SGP,which correlates with the suggestion of Dahr et aLS9that the genetic back-
ground of the En(a-) phenotype in the English family is En/M*. This notation was
later modified by Ansteeo9 to En. U K / M t to account for the 'My antigen produced by
En.UK, but not by En.Fin.
En(a +), Wr(a-b-) Red Cells

The red cells of F. M. and J. R. of Table 9, like En.UK/Mk, En.Fin/En.Fin, and
M'/M* red cells, also type as Wr(a-b-), but are considered to be En(a+).lo9 The
red cells of F. M., considered to be genetically homozygous for the rare Miltenberger
gene Mi",since the proposita and both her parents are positive for the rare Hi1 antigen
and carry (in addition to Hil) a depressed N antigen and a greatly exalted s antigen.
In contrast, the genetic background responsible for the unique phenotype of J. R. is
not, at present, fully understood. It is, however, clearly distinct from that involved
with F. M., for J. R.'s red cells are nonreactive with Miltenberger system antibodies
(including Hil), and carry a weak M antigen and a slightly exalted S antigen.
SDS-PAGE analyses of membranes from the red cells of F. M. and J. R. reveal that
both red cell types have hybrid MNSs SGP molecules designated (a-d)" " and (a-d)' ,
respectively. Bath hybrids appear to have an outer portion (NHderminal) derived
from MN SGP and an inner po.Cion, part of which extends outside the red cell mem-
brane, derived from Ss SGP.'09 110 The nature of such hybridization has, in part, been
ascertained by studies on intact red cells with V. graminea lectin. The J. R. red cells,
which in the untreated state are nonreactive with V . graminea, are strongly reactive
with this lectin after they have been treated with neuraminidase, but are nonreactive
following trypsinization. These findings suggest that all of the alkali-labile oligosac-
charides attached to the hybrid SGP of J. R. are between the trypsin-sensitive site
(presumably present on the MN SGP-derived portion of the hybrid molecule) and the
NH,-terminus. (Note: normal red cells react with V. graminea after they have been
treated with trypsin because normal Ss SGP does not carry a trypsin-sensitive site.)
The red cells of F. M.. either untreated or neuraminidase treated, are strongly reactive
with V. graminea lectin and are also nonreactive with V. graminea lectin following
trypsinization. This latter finding indicates (as with J. R.) that the attached alkali-labile
oligosaccharides are confined to that portion of the hybrid ( L I - ~ ) ~ ' molecule
." that re-
sembles the MN SGP, and are nearer to the NHI-terminus than is the trypsin-sensitive
site.
Tbe M* Phenotype
The M'gene produces no M. N, S, s, 'N', or U antigens (for review, see References
IS and 40).This is consistent with the observation that red cells heterozygous for this
condition contain only 50% of apparently normal MN and Ss SGP.IILThe MN SGP
from these red cells binds, but weakly, with both M. aurantiaca and A. hypogaea
lectinsa9(the latter lectin reacting only with MN SGP from neuraminadase-treated M'
red cells). Recently, two related Japanese individuals considered to be honiozygous
M*/M*have been reported.'"'' These red cells appear to be devoid of MN and Ss SGP.
As previously mentioned, they can be considered as being both En(a-) and S-s-U-.
Sturgeon, Metaxas-Buhler, and Metaxas et al."' found a rather unique donor with
the genotype Mi'"/M'. SDS-PAGE analysis of these membranes revealed that they
lack PAS staining bands d and&, corresponding to normal Ss SGP.89
As yet, no data are available on the results of tests on intact M'M' red cells with
lectins. Red cells heterozygous for M' react strongly with S. japonica and G. max
lectins. Like En(a-) red cells, they give enhanced reactions with anti-H lectins due to
the reduced C-potential of the red Also, they react weaker than normal with
M. aurantiaca lectin.L87
MI Red Cells
The M* antigen would appear to be the product of a rare allele at the MN locus and
is defined by a specific anti-Ma antibody."' The MI antigen is denatured by trypsin
and is carried on a n altered MN SGP.'I5 This altered MN SGP binds weakly to M.
aurantiaca lectin in studies on the only known homozygous Mr/Maindividual. Leucine,
and not serine, appears to be the NH1-terminal amino acid of this altered MN SGP.Il6
As discussed by Anstee," it is possible that Mr/Mr red cells lack the three alkali-labile
sialotttrasaccharides which are normally attached to the three inner serine and threo-
nine residues of the first five amino acids at the NHa-terminus of the MN SGP.This
is consistent with the observation of Dahr, Uhlenbruck. and Knott'" which revealed
that Mr/Mrred cells contain 12% lower than normal levels of NANA, for each at-
tached alkali-labile sialotetrasaccharide contributes to some 4% of the total red-cell-
surface NANA. This lack of such carbohydrate moieties would account for the reduced
binding of the MN SGP t o M. aurantica lectin. The presence of leucine as the NH,-
terminal amino acid probably accounts for the weak N activity of MVMn red cells.89
In tests with lectins, Nordling et al."' reported that M* red cells were agglutinated
by S. japonica lectin, but not to the degree observed with M' red cells. Judd" reported
Mr red cells to be nonreactive in tests with G. max. It is likely that the small reduction
in surface NANA of Ma heterozygotes is insufficient to render them agglutinable by
this lectin which is routinely used in studies on polyagglutinable red cells (see later) to
detect reduced red cell surface NANA levels.
Other MN-System Variants

The unique red cells of donor L. R. (Table 9 ) . considered to be Mi"/M", appear to
have only 25% of the monomeric form of the Ss SGP.'' They d o not react with V.
graminea following treatment with trypsin. ' I ' It would, therefore, appear that these
red cells have a reduced level of an abnormal trypsin-sensitive Ss SGP.
August 1980 191
The aberrations to PAS 2' of Miltenberger class I and I1 red cells, and the abnormal
Ss SGP of classes 111, IV, and VI, are not known at present to result in any abnormal
behavior in direct agglutination tests on intact red cells with lectins. Furthermore, since
the data regarding the SGP aberrations and deficiencies of these red cells has not been
achieved or supported by lectin-binding studies on red cell membrane components,
discussion on the alterations to the SGP in these red cells is inappropriate in this re-
view. The interested reader is referred to Reference 89 for further information.
Additional Comments
It should be noted that simple lack of binding of M. aurantiaca, A. bypogaea, or
S. japonica lectins to the red cell membrane MN or Ss components following separa-
tion by SDS-PAGE does not in itself prove that red cells entirely lack that particular
SGP. Rather, such results imply that only the attached oligosaccharides are absent.
PAS staining of components separated by SDS-PAGE also only indicates the presence
of attached oligosaccharides, for this technique relies on the interaction between the
PAS reagent and NANA. The methods which have been used to detect the polypeptide
moiety of the MN and Ss SGP are reviewed by A n ~ t e e . ' ~
When interpreting the data revealed by lectin studies on intact red cells from SGP-
deficient bloods, the following should be considered. Firstly, enhanced reactions may
be due to the decreased [-potential of the red cells. This is most likely responsible for
the behavior of En(a-) and Mk red cells with anti-H lectins. Secondly, enhanced reac-
tions may result from lack of membrane components that would normally sterically
hinder the association between the red cell surface receptor and lectin protein. Such
absence of sterically hindering components would account for the agglutination of

En(a-) and Mk red cells with S. japonica and G. max lectins. Finally, absence of the
MN SGP in En(a-) red cells, accounts for the weak agglutination of intact En(a-) red
cells in tests with M. aurantiaca lectin.
Conclusions
Lectins that bind to the red cell membrane sialoglycoproteins have been used, not
only as an alternative to human and rabbit anti-N reagents for blood typing purposes,
but have been applied to define the nature of the M and N blood group antigens.
Furthermore, the use of radioiodinated lectins in tests on isolated red cell membrane
sialoglycoprotein components has resulted in confirmation of other data regarding the
defects associated with certain uncommon MN-system variant phenotypes, in particu-
lar S-s-U-, En(a-), and M' red cells. The wealth of information revealed by such stud-
ies has provided considerable insight into the genetics, biochemistry, and polymorph-
ism of the MN-system antigens. It is to be expected that further information will be
revealed by such studies in the future.
THE ROLE OF LECTINS IN THE STUDY OF RED CELL

POLYAGGLUTINATION
Introduction
Polyagglutination is defined as that condition wherein red cells are agglutinated by
a high-proportion of ABO blood group compatible, normal adult human sera. The
serum proteins responsible for this agglutination are IgM immunoglobulins, react best
in saline tests at ambient or below ambient temperatures, and are not usually present
in cord-blood samples.
In vitro contamination of blood bank reagents or blood samples may result in po-
lyagglutination. Indeed, knowledge of this phenomenon was first gained through stud-
ies on red cells accidentally infected in vitro.ll*-lzoIn honor of the early investigators,
the condition has been referred to as the Thomsen-Hiibener-Freidenreich phenome-

non, o r simply the Thomsen phenomenon. The first example of in vivo-induced po-
lyagglutination was described in 1938 by Levine and Katzin.'*' To date, nine forms of
polyagglutination have been described, although some may be weak or variant forms
of each other.
Characteristics and Classification

Lectins are used extensively by investigative immunohematologists in the recognition
and classification of polyagglutination. The principal lectins employed are Arachis hy-
pogaea (anti-T/Tk), Dolichos biflorus (anti-A,/Tn/Cad), Salvia scfarea (anti-Tn), Sal-
via horminum (anti-Tn + Cad), and Glycine max (syn. soja). The latter lectin has no
particular polyagglutinable red cell specificity. but reacts with red cells that are defi-
cient in NANA, including red cells that have been treated with proteelytic
In addition, some workers have used BS I1 lectin. from Bandeiraea simplicifolia seeds,
which appears to be a specific anti-Tk reagent.36 Anti-H lectins have been used to
determine the H antigen content of polyagglutinable red cells. The behavior of these
and other lectins with the various types of polyagglutinable red cells will be discussed
in this section.
In addition to studies with lectins, other parameters are frequently studied when
attempting to classify polyagglutinable red cells. These include red cell electrophoretic
mobility studies using apparatus similar to that described by Bangham, Flemans, and
Heard et a1.lz2 or the PolybreneO aggregation Like tests with G. max lectin,
both procedures are indirect methods for determining the red cell NANA content,
which may also be measured directly by biochemical means."' The effect of proteolytic
enzymes on the polyagglutination observed with AB serum, o r on the reactivity of the
red cells with the appropriate lectin, is another parameter that is frequently studied.
Thus, tests with lectins usually form but a part of the investigative procedures under-
taken when classifying polyagglutinable red cell types. The results of such studies on
the various types of polyagglutinable red cells, are summarized in Table 10.
T-Activation
T-activation of red cells, either in vivo or in vitro, is caused by those organisms that
produce the enzyme neuraminidase (receptor-destroying enzyme, RDE). These include
Vibrio cholerae, Clostridium perfringens, pneumococci, and influenza viruses. Neu-
raminadase cleaves NANA from red cells and exposes latent T-receptors which are
recognized by a specific anti-T polyagglutinin present in most normal adult human
sera. The T-receptor is destroyed by proteolytic enzymes.'25
Anti-T in extracts of A. hypogaea (peanut lectin) was first reported by Bird.'16 These
extracts give avid reactions with red cells T-activated in vivo and with red cells T-
activated by neuraminidase in vitro. Issitt, Moulds, and BermanlZ7found the T-recep-
tor defined by A . hypogaea to be destroyed by ficin, but not by papain, unless the
latter enzyme was used at concentrations greater than 3 g / d l . In contrast, Gunson,
Stratton, and Mullard1z8found papain and ficin to be equally as effective in destroying
the T-receptor. Juddtn9found only activated papain preparations (activated with L-
cysteine hydrochloride according t o the method of L'dwLZ9 or by the addition of EDTA)
to depress the reactivity of neuraminidase-treated red cells with A.+hypogaealectin. It
is likely that variations in the degree of T-activation of the red cells studied by different
investigatars is, in part, responsible for the conflicting data.190
Associated with T-activation due to loss of NANA is a decrease in the electropho-
retic mobility of the red cells, loss of M and N antigens defined by some, but not all,
rabbit and human anti-sera, enhancement of agglutination by the MN-active lectins
from Vicia graminea and Iberis amara, and loss of the Sp, blood group antigen.84.1z5.130
Table 10
PRINCIPAL CHARACTERISTICS OF THE VARIOUS TYPES OF POLYAGGLUTINABLE RED
CELLS
NANA Effect of H antigen Arachis DoIichos Glycine Salvia Salvia Helix BS 11
Type content protcasa content hypogaea biflorusJ max scIarea horminum pomatia' lectin
T Red' Desb Nor',' i W + 0 0 i 0

Th Nor Des Nor + 0 0 0 0 ? 0
Tn(Tcr) Red Des Nor' 0 + + + + + 0
Tk Nor Enha Red i 0 0 0 0 ? +
Cad Nor* None' Nor 0 + + 0 + + 0
HEMPAS SI. red' Enh Red 0 0 + 0 0 + 0
VA SI. red' None' Red 0 0 0 0 0 + 0
Acquired-8 Nor None' Nor 0 Red^ 0 0 0 +' 0
Red = reduced.
Des = destroyed.
* Nor = normal.
' May be slightly enhanced due to reduced [-potential.
' + Denotes agglutination.
0 = Noreaction.
Enh = enhanced.
a May be slightly reduced.
' Slightly reduced.
Reactions of groups 0 and B red cells.
' Agglutination due to presence of A antigen.
Datacompiled from References 15. 18. 125, 127, 138, 146, 156, 187, and 197.
c
W
Consistent with their reduced NANA content, T-activated red cells are strongly reac-
tive with G. max lectin. 1 3 ’
T-activation in vivo is a transient phenomenon. As with all cases of bacterial-en-
zyme-induced changes that may occur to red cells in vivo, the patient may present with
septicemia or wound infection. In other instances, it can be shown the patient has an
ulcerative or obstructing lesion of the G.I. tract, but is not necessarily septic. In these
cases, it is likely that the enzymes produced by the intestinal flora penetrate the gastric
mucosa. Once the septicemia has been remedied, or the lesion repaired, the T-activa-
tion progressively disappears.
Th-Activation
This form of polyagglutination has only been recently described.”’ It appears to be
another type of bacterial-enzyme-induced polyagglutination. In the opinion of‘this au-
thor, the results of lectin studies on Th-activated red cells (see Table lo), including
their nonreactivity with G. max lectin, d o not readily distinguish this form of polyag-
glutination from what might be anticipated with red cells that are only mildly T-acti-
vated.
Tk-Activation
Several similarities exist between T-activation and Tk-activation. Both fornis of PO-
lyagglutination are caused by bacterial enzymes, and Tk-activated red are also agglu-
tinated by A. hypogaea lectin. Sometimes, blood samples are encountered that are
both T- and Tk-activated.”’ However, Tk-activation does appear t o be a distinct en-
tity, rather than a variant form of T-activation. In vivo and in vitro, it has been asso-
ciated with infection by certain strains of Eacteroides fragiljs36.133.134 Tk-activated red
cells show no loss of surface NANA (they are nonreactive with G. max lectin), and
MN antigen content and electrophoretic mobility are normal. 132.1’3 Moreover, whereas
the T-receptor is destroyed by proteases. the reactivity of Tk-activated red cells with
both AB serum and A. hypogaea lectin is greatly enhanced following treatment of the
red cells with either papain or f i ~ i n . ~ ~In
. ’ ’addition,
~ Tk-activated red cells have de-
pressed I and i antigens, 1 1 5 but neuraminadase-treated red cells give slightly enhanced
reactions in tests with anti-I and anti-i reagents.
Specific anti-Tk activity is present in the highly-purified Nacetyl-D-glucosaminyl-
binding lectin (BS 11) from B. simplicifolia ~ e e d s . ’BS ~ I1 lectin agglutinates red cells
T-activated in vivo, but not red cells T-activated by purified neuraminadase in vitro.
It also reacts with a high proportion of blood samples that have acquired B-like antigen
in vivo. Thus, it would appear that in vivo Tk-activation quite frequently occurs con-
comitantly with other bacterial-enzyme-induced changes to the red cell membrane (see
below).
Lectins from Eryfbrina indica seeds and Sambacus nigra (elderberry juice) can also
be used to distinguish between T- and Tk-activation. S. nigra gives enhanced reactions
with neuraminidase-treated red cells and reacts more weakly with Tk-activated red cells
than it does with normal red cells. E. indica, which also appears to be a “nonspecific”
lectin, gives enhanced reactions with neuraminidase-treated red cells, but reacts equally
well with Tk-activated and normal red cells.’’*
Acq uired-E Polyagglutination

According to Gerbal, Maslet, and Salmon‘’ bacterial enzymes produced hy certain
strains of Escherichia cofihave the capacity to deacetylate a-D-GalNAc (a-N-acetyl-D-
galactosamine), the immunodominant sugar conferring blood group A specificity. The
resultant structure (a-D-galactosamine) is sufficiently similar t o a-D-Gal (a-D-Galac-
tose, B antigen specificity) as to cross-react with human anti-B reagents. This acquisi-
August 1980 195
tion of B-like antigens occurs at the expense of the A antigen (as revealed by binding
studies with D. biflorus lectin) and occurs only in group Al individuals in vivo. How-
ever, in vitro, B-antigen-containing bacterial polysaccharides may be incorporated into
the membranes of groups 0 and Af red cells.50~136 A a individuals seem t o be protected
against the acquisition of B-like antigens in vivo by the presence of an anti-B compo-
nent in their sera that specifically reacts with acquired-B antigens. Approximately 95 '70
~ inference of this find-

of A l individuals have this "anti-acquired-8" c ~ m p o n e n t . 'The
ing is that only the remaining 5% of A, individuals have the potential for developing
acquired-B antigens.
Beck, Walker, and Oberman13' observed polyagglutination in association with ac-
quired-B antigens. They used the term "type I11 polyagglutination" to distinguish it
from other forms (according to their notation, T = type I, Tn = type 11, and Cad =
type IV). Using BS I1 lectin, Judd et found exposed Tk-receptors in a high pro-
portion of acquired-B blood samples. These workers suggested that the term type I11
polyagglutination was synonymous with Tk-polyagglutination. However, Gerbal,
Maslet, and Salmon*9and Andreu, Mativet, and Doinel et al.13shave shown acquired-
B red cells to have a form of polyagglutination that is not T or Tk, but rather would
appear to be another change to the red cell membrane produced by the deacetylase
that is responsible for the development of acquired-B antigens. This form of polyag-
glutination is simply referred to as "acquired-B polyagglutination' ', and can best be
recognized using AB serum from which the anti-T and anti-Tk have been removed by
absorption. That this form of polyagglutination is not identical to Tk has further been
established by the study of R o p a r ~ which
' ~ ~ reveals that following reacetylation of Tk-
activated acquired-B red cells with acetic anhydride (which results in loss of acquired-
B antigens and acquired-B polyagglutination with a concomitant gain in A, reactivity),
such red cells remain reactive with BS I1 lectin.
The presence of both exposed T- and Tk-receptors on acquired-B red cells has been
recently demonstrated by Judd, McGuire-Mallory, and Anderson et al.I3" These work-
ers used purified A. hypogaea and BS I1 lectins in tests with red cells treated with
dilute solutions of neuraminidase and/or with a Tk-activating culture broth of B. fra-
gilis. Red cells exposed only to neuraminidase were strongly reactive with A. hypogaea
Iectin (titer = 2000) but were not agglutinated by BS I1 lectin. Following further treat-
ment with ficin, these red cells were only weakly reactive with A. hypogaea lectin (titer
= 4). as were normal ficin-pretreated red cells, and remained inactive in tests with BS
I1 lectin. In contrast, the titration end-points obtained with both lectins in tests against
red cells Tk-activated by B. fragilis in vitro were increased fourfold following treat-
ment with ficin. When red cells were sequentially treated with neuraminidase and then
by the B. fragilis enzyme, they were agglutinated to the same degree (titer = 2000) by
A . hypogaea, as were red cells treated only with neuraminidase. However, only a minor
reduction in the titration end-point value was observed when these T + Tk-activated
red cells were tested with A. hypogaea after they had been treated with ficin. The
titration end-point observed was, in fact, identical to that obtained with ficin-treated
Tk-activated red cells. Comparable findings were encountered with three acquired-B
samples in tests with A. hypogaea and BS I1 lectins both before and after treatment
with ficin (see Table 11). Judd and colleagues concluded that while Tk-activation is
often associated with acquired-B red cells, these blood samples may, on occasion, also
be T-activated'. They further suggest that all three recognizable changes (T-activation,
Tk-activation, and acquisition of B-like antigens) that may occur to red cells modified
by bacterial enzymes in vivo are each due to the action of different bacterial enzymes.
Tn-Polyagglutination
Tn-polyagglutination was first described by Moreau, Dausset, and Bernard et al.139
1% CRC Critical Reviews in Clinical Laboratory Sciences
This class of polyagglutinable red cells is most likely heterogenous, as disciissed by

Berman, Smarto, and Issitt et al..Iz5 and probably includes the single case of Tcr-po-
lyagglutination studied by Lalezari and a l - M ~ n d h i r i . 'It~ ~is not thought to arise by
the direct action of bacterial enzymes. Rather it has been suggested that bacterial phage
viruses, or other agents, alter the DNA synthesis of primitive red cell precursors result-
ing in the incomplete production of red cell membrane components.4o Recently, and
consistent with such a somatic mutation, it has been shown that Tn-polyagglutinable
red cells lack a red cell membrane galactosyl-transferase. 14' The significance of this
finding will be discussed more fully below.
Once acquired, Tn-polyagglutination is usually a permanent state. Not all the red
cells may be polyagglutinable. The condition is therefore sometimes referred to as per-
manent mixed-field polyagglutination (PMFP). 142 The Tn-receptor has the same ter-
minal sugar (a-D-GalNAc) that confers blood-group-A specificity.14' Because of this
A-like nature of the Tn-receptor and the mixed red cell population, the condvtion has
been mistaken for an example of blood group chimeri~m.~.~ Often, though not. always,
associated with Tn-polyagglutination, are a mild leukopenia, thrombocytopenia, and
anemia. There is also often a history of bacterial or viral infection. The condition has
been known to precede leukemia,'U but several examples have been found among ap-
parently healthy blood donors.
Like T-activated red cells, Tn-polyagglutinable red cells have a reduced NANA con-
tent, and the Tn-receptor is destroyed by treatment of the red cells with proteolytic
enzymes. Iz5 However, in contrast, Tn-polyagglutinable red cells contain no detectable
Nv, receptors defined by V. grarninea le~tin.'~'The M and N antigens, defined by
human and rabbit reagents, also appear to be depressed. Such apparent depression of
M and N antigens is most likely a reflection of the known mixed cell population asso-
ciated with Tn-polyagglutinable blood samples. It is possible that while the Tn-acti-
vated red cells fail to react with anti-M and anti-N antibodies, and while the non-Tn-
activated red cells react normally, the net effect of a mixture of these two cell popula-
tions is an apparent depression, not absence or normal levels, of reactivity with anti-
M and anti-N antibodies.
Group 0 Tn-polyagglutinable red cells are agglutinated by D. bifforus (anti-Al) lec-
tin, and by the BS I isolectins from B. simplicifolia ~ e e d s . " Consistent
~~~~ with their
reduced NANA content, they are strongly reactive with G. max l e ~ t i n ' ~and . ' ~ react
~
more strongly than normal with anti-H lectins.
Bird and Wingham,147-'49 have published a series of papers reporting anti-Tn or
separable anti-Tn and anti-Cad reactivity in saline extracts of seeds from many vari-
eties of seeds of the genus Saliva. Those with specific anti-Tn or anti-Tn + Cad reactiv-
ity are shown in Table 12. Other Salvia lectins have been found to react preferentially
with neuraminindase- or protease-treated red cells and are not included in this table.
The Salvia lectins with anti-Tn or anti-Tn + Cad reactivity provide yet further reagents
for the classification of red cell polyagglutinable types. Perhaps the most widely em-
ployed are s. scfarea(anti-Tn) and s. horminum (anti-Tn + Cad).
Extracts of S. scfarea seeds were used by Beck, Hicklin, and Pierce et al.lS0in studies
on buffy-coat preparations obtained from Tn-polyagglutinable blood samples. Buffy-
coat preparations from all six samples studied, in contrast to normal bloods, absorbed
anti-Tn from S. scfarea extracts. Using separated cellular components, they demon-
strated exposed Tn-receptors on granulocytes, lymphocytes, and platelets from Tn-
polyagglutinable blood samples, but not from normal samples. Since three of the Tn-
polyagglutinable samples studied were from individuals with a normal leukocyte and
platelet count, Beck and colleagues concluded that the presence of Tn-receptors on
these cellular components does not contribute to the leukopenia or thrombocytopenia
that is more often than not associated with Tn-polyagglutination.
August 1980 197
Table 11
EFFECT OF FICIN ON THE REACTIONS
BETWEEN A.HYPOGAEA AND BS I1
LECTINS AND T-ACTIVATED, Tk-
ACTIVATED, AND 3 ACQUIRED-B BLOOD
SAMPLES
Titer vs. Titer vs.

Red blood cells A. hypogaea BS I1 lcctin
T-active 2.000 0
-
T-active ficinized 4 0
Tk-active 64 I28
-
Tk-active ficinized 512 .%000
T + Tk-active 2,000 I28
-
T + Tk-active ficinized 512 8.000
Acquired-B # I 512 32
-
Acquired-B 1 1 ficinized 64 256
Acquired-B #2 128 8
-
Acquired-B #2 ficinized 16 64
Acquired-B 13 1,000 64
-
Acquired-B #3 ficinized 64 1,m
Normal 0 0
-
Normal ficinized 4 0
Modified from Judd, W. J., McGuire-Mallory, D., Anderson,

K., Heath, E. J., Swanson, J.. Gray, J. M., and Oberman. H.

A., Transfusion, 19,293, 1979. With permission.
Table 12
THE ANTI-Tn AND ANTI-CAD SPECIFICITY
OF CERTAIN S M VL4 AGGLUTININS
Species Specificity
S. aethiopsis Anti-Tn
S. argentea Anti-Tn
S. aurea Anti-Tn
S. candelabrum Anti-Tn
S. farinacea Anti-Tn + Cad
S. forskohlei Anti-Tn
S. grandiflora Anti-Tn
S. haematodes Anti-Tn
S. horminum Anti-Tn + Cad
S. japonica Anti-Tn + T (weak anti-cad)
S. nemcrose Anti-Tn
S.nilotica Anti-Tn (weak anti-T and anti-Cad)
S. pratensis Anti-Tn
S. sclarea Anti-Tn
S. sclarea turkestanica Anti-Tn
S. sclareoides Anti-Tn
S. superb# Anti-Tn
S. tarasacifolia Anti-Tn
S. transylvanica Anti-Tn
S. vcrbenaca Anti-Tn
S. verticilla ta Anti-Tn (weak anti-T and anti-Cad)
May be the same variety.
Data compiled from References 147, 148. and 149.

Cad-Polyagglu tination
This form of polyagglutination is not associated with any disease process. It was
first described by Cazal, Monis, and Caubel et al.'" in 1968. The genes responsible
show a varying degree of expression, and three phenotypes (Cad 1, Cad 2, and Cad 3)
can be recognized. Only the strong Cad-positive (Cad 1) samples are polyagglutinable.
There is a strong association between the Cad-receptor and the Sda (Sid) blood group
antigen. Cad-positive red cells appear to have elevated levels of Sda antigen, since they
react more strongly with anti-Sd. reagents than d o normal Sd(a + ) red cells. Also, both
Cad-positive and strong Sd(a + ) red cells that are group 0 or group B are agglutinated
by D. bifloruslectin.
Cad-positive red cells d o not have a markedly reduced NANA ~ 0 n t e n t . l ~Nonethe-
'
less, they are agglutinated by G. max lectin."6 which is most likely due to the presence
of exposed D - G ~ N A cresidues,153since G. max is preferentially inhibited by both a
and 0 anomers of this sugar.'O In addition, Cad-positive red cells are agglutinated by
saline extracts from S. horminum and S. farinacea seeds.14' Also, Cad-positive red
cells appear to have less H antigen than normal red cells.153
In spite of a virtually normal NANA content, Cad-positive red cells have a depressed
M and N antigen content, whether measured by human, rabbit, or lectin reagent^.'^
The T antigen of Cad-positive red cells (exposed after neuraminidase treatment) as
measured with A. hypogaea lectin is weaker than that of control samples. In contrast
to T-activated and Tn-polyagglutinable red cells, the Cad-receptor is not destroyed by
proteolytic enzymes.12s
HEMPAS
HEMPAS is the acronym for hereditary erythroblastic multinuclearity with a posi-
tive acidified serum test.lS4 Also known as hereditary dyserythroblastic anemia type
11, the condition is extremely rare. HEMPAS red cells react poorly with a variety of
seed anti-H reagents and have an affinity for anti4 sera. They have a slightly reduced
NANA and react weakly with G. rnax lectin" and with H. pornatia l e ~ t i n . ' ~ '
Also, group 0 HEMPAS red cells are agglutinated more strongly than normal group-
0 red cells in tests with the BS I(A,) isolectin from B. sirnplicifolia seeds.''
VA-Polyagglutination
VA (Vienna) polyagglutinable red cells show some similarities to HEMPAS red cells.
They have a slightly decreased electrophoretic mobility and react poorly with anti-H
lectins. However, there is no abnormality with the I and i antigens, and acid hemolysis
tests are negative. Characteristically, they give a stippled appearance in tests with H.
pornatia lectin when examined by electron microscopy. 1s7.158
Only two examples of VA-polyagglutination have been reported, one associated with
T k - a c t i v a t i ~ n . 'Thus,
~ ~ VA-polyagglutination may be another example of a bacterial-
enzyme-induced form of polyagglutination, although the authors of the first reported
example could not rule out a congenital a b n ~ r m a l i t y . ' ~ '
NOR-Polyagglu tina lion
This recently discovered form of polyagglutination is not known to react with any
of the lectins routinely employed in the identification of polyagglutinable red cells. It
appears to be another form of inherited polyagglutination. Agglutination of the red
cells by human group AB sera is inhibited by hydatid cyst fluid containing soluble PI
blood group substance.'92
Studies on the Nature of the Polyagglutinable Receptors

Lectin Specificity
By virtue of their specific carbohydrate-binding properties. the aforementioned lec-
August 1980 199
tins have been applied to the characterization of some of the polyagglutinable recep-
tors, notably the T and Tn antigens, and to a lesser degree the Cad- and Tk-receptors.
The preferential carbohydrate-binding specificity of these and other relevant lectins is
shown in Table 13.
When attempting to define the nature of red cell surface receptors by inhibition of
agglutination by simple sugars, it is essential to consider the biochemical purity, hom-
ogeneity, and serological specificity of the lectins employed in such studies. Some of
the factors to be considered have been reviewed by Gold and Balding,' and by Bird.'46
They are particularly applicable to the interpretation of results obtained in tests using
impure lectin preparations. They include: the presence of more than one lectin protein
in a seed extract (e.g., the three lectins present in B. simplicifolia seed^),^' the presence
of polymeric forms of the same lectin protein (e.g., P. lunatus lectin),'O the presence
of separable lectins active against a different facet of the same receptor (e.g., the two
anti-H lectins from Ulex europaeus seed^),'^ the presence of two separable lectin pro-
teins in the same seed extract with affinity for different receptors having the same
terminal sugar (e.g., S. horminurn, which contains separable anti-Tn and anti-Cad),I4'
and lectin proteins with more than one blood group specificity carried on the same
protein molecule (e.g., the anti-A/N lectin from Moluccella seed^).'^
As discussed by Gold and Balding,' it is, perhaps, unfortunate that much of what
is known regarding the nature of the polyagglutinable receptors has been gained using
impure lectin preparations (crude saline extracts of seeds). Recently, therefore, JuddI8
studied the various types of polyagglutinable red cells with purified lectin preparations.
The data obtained by such studies are included in Table 13. Since the purity of these
preparations was established by biochemical means, it is unlikely that the agglutination

of more than one type of polyagglutinable red cells by a particular lectin is due to the
presence of two or more proteins having a different carbohydrate-binding specificity.
Thus, the agglutination of A,, Tn, and Cad red cells by D. biflorus lectin, and the
agglutination of both T- and Tk-polyagglutinable red cells by A. hypogaea, is due to
the reactivity, in each case, of a single lectin protein.
Nonetheless, some further considerations must be made when attempting to eluci-
date the nature of red cell surface re'ceptors using lectins. As shown in Table 13, the
majority of lectins employed in the investigation of red cell polyagglutination are pref-
erentially inhibited by a-D-GalNAc. It is, therefore, obvious that this sugar plays an
important role in the structure of some of the polyagglutinable receptors, in particular
the Tn and Cad antigens. However, not all the a-D-GaINAc binding lectins agglutinate .
Tn- and Cad-polyagglutinable red cells to the same degree. Moreover, the Salvia lectins
agglutinate Tn- and Cad-polyagglutinable red cells, but not group A red cells, while
purified lima bean lectin fraction I11 (Phaseolus lunatus 111) reacts only with group A
red cells.'8 One plausible explanation for this is that the a-D-GalNAc residues that
define blood group A specificity are readily accessible to P. lunatus 111 lectin, but P.
lunatus 111. lectin is unable to bind t o the Tn- and Cad-receptors because either the
configuration of the lectin protein or the size of the lectin molecule, or perhaps mem-
brane structures adjacent to the Tn- and Cad-receptors, sterically hinder the associa-
tion between P . lunatus I11 lectin and the a-D-GalNAc residues that determine the Tn-
and Cad-receptors. The converse situation can. be applied to explain the ability of the
Salvia lectins to agglutinate Tn- and Cad-polyagglutinable red cells, but not group A
red cells.
Alternatively, some lectins may require a binding site of several sugars.' It is possible
that in the Cad-receptor the subterminal sugars may be quite different from those
present in the A blood group active red cell surface structures. Thus, P. lunatus 111
lectin may require the presence of subterminal D-Gal and/or an adjacent L-fucose (L-
Fuc) group (both present in H-active structures from which blood group A antigen
Table 13
THE SPECIFIC CARBOHYDRATE-BINDING PROPERTIES AND COMPARATIVE
AGGLUTINATION TITERS OF CERTAIN LECTINS IN TESTS AGAINST NORMAL AND
POLYAGGLUTINABLE RED CELLS' 2
0
Red Cells
Lectin' Carbohydrate-bindingspecificity' A, A1 B 0 T Tk Tn Cad I HEMPAS
D. biflorus a-D-GalNAc' 10 3 ' 8 13

P . lunarus(LB Ill) a-D-GalNAc 10 8
H.pomaria a-D-GalNAc Pa-D-Glc NAc' 12 10 10 I1 8 6
S. sclarea o-D-GalNAc 6 2
S. horminum o-D-GalNac 6 6
G. man u//?-D-GalNac B o/&D-Gal' 6 5 6 5 1 1 5 1 2 9 7
S.japonica u/p-D-GalNac > a/p-D-Gal 8 5 12 + + a 7 0
BS I(A.1 a-D-GalNAc > a-D-Gal 9 5 4 0 6 13 6
BS I(B,) a-D-Gal &- u-D-GalNAc a 2 6
BS I I o//~-D-GIcNAc 10
A. hypogaea D-Gal-/3-(1-3)-D-GalNAc 2 14 7 3
Noie:All lectins used at an initial concentration of 0.2 mg/ml and diluted in I I % bovine albumin
Figures indicate the log, of titration endpoints obtained in tests at room temperature.
' All lectins purified with the exception of S.sclareeand S.horminum.
' Data from Reference 10.
' D-GalNAc = Nacetyl-D-gaiactosamine.
' No result recorded indicates no agglutination observed.
D-GlcNAc = Nacetyl-D-ghcosamine.
4 = Not tested.
A D-Gal = D-galactose.
Adapted from Judd, W. J.. Transfusion, 19,768, 1979. With permission.

August 1980 201
develop^).'^ These D-Gal and L-FUCresidues are not part of the Tn-receptor (see be-
low), and the subterminal sugars of the Cad-receptor have yet to be defined.
Yet another possible explanation is that some lectins react preferentially, or only
with, carbohydrate residues linked to an adjacent sugar in a particular configuration
(e.g., u(l-4), but not u(1-3)).' This may explain why the BS I isolectins from B. simpli-
cifolia seeds agglutinate group B red cells, where the D-Gal residue is linked to its
subterminal sugar in the u(1-3) c~nfiguration,~' but not P I red cells" which have a
terminal D-Gal residue linked to the same subterminal sugar as is present in B active
structures, but in the u(1-4) configuration.'60
All of the above considerations should be borne in mind when attempting to inter-
pret data such as those shown in Table 13. What follows is an interpretation of these
data relative to the nature of the polyagglutinable receptors, in particular the T and
Tn antigens, and some observations on the nature of the Tk-receptor.
T- and Tn-Receptors
The T- and Tn-receptors reside within, but are not restricted to, the alkali-labile
tetrasaccharide structures of Thomas and Win~ler,'~ as shown in Figure 2. Both are
considered to be cryptantigens or latent receptors since they are not exposed on normal
red cells. The action of neuraminidase on these structures is to cleave NANA residues
and expose a 0-linked D-Gal residue that is linked to a-D-GalNAc.161The structure
thus formed (T antigen) is recognized by A. hypogaea lectin.162Consistent with the
structure of the T antigen is the observation of Lotan, Shutelsky, and Danon et al.163
that among all the sugars studied, the disaccharide D-Gal-p-( 1-3)-D-GalNAc is by far
the most potent inhibitor of the agglutination reaction between T-activated red cells
and A. hypogaea lectin.
Treatment of T antigen with P-galactosidase has been shown by Springer and De-
sail6*to result in the formation of Tn-receptors. Confirmation of the structure of the
Tn antigen has been made by showing that many of the anti-Tn lectins (D.biflorus,
S. sclarea and BS I isolectins) precipitate with neuraminidase-treated ovine submaxil-
lary mucin which prior to treatment has the structure: NANA-u-(~-~)-D-G~~NAc linked
in the u-configuration to either serine or t h r e ~ n i n e . ' ~ These
~ . ~ ~ 'structures are consid-
ered by Springer et al.79~s0~161to be the precursor material from which the M and N
antigens develop (see the section on the nature of the M and N antigens). However, as
stated previously, such a hypothesis no longer seems tenable, particularly in light of
the serine/leucine and glycine/glutamic acid polymorphisms now known to determine
M and N blood group specificities.'5.86
As mentioned previously, the alkali-labile tetrasaccharides of Thomas and Winzlers7
are attached either to serine or to threonine residues of the MN and Ss sialoglycopro-
teins (SGP).63 Consistent with the fact that Tn-polyagglutinable red cells contain no
detectable Nv, receptors (defined by V . graminea lectin, which reacts with the internal
/3-D-Gal residues of these alkali-labile tetrasac~harides)'~~ is the finding of Dahr, Uhl-
enbruck, and Gunson et a1.164that the MN and Ss SGP, and band 3, of Tn-polyagglu-
tinable red cells are incompieteiy giycosylated. Furthermore, Cartron, Andreu, and
Cartron et al.I4l have shown that Tn-polyagglutinable red cells lack a membrane-bound
~(1-3)-galactosyl-transferase which transforms Tn antigen into T-active structures.
Thus, while many workers would dispute the concepts of Springer and H ~ p r i k a r and '~
Springer, Tegtmeyer, and HuprikarOO regarding the nature of the M and N blood group
antigens, it doesappear that Tn is a precursor of T, at least as far as the alkali-labile
tetrasaccharides are concerned (see Figure 2). However, T-receptors may also be pres-
ent on red cell membrane gly~olipids,'~~ which would explain why it is possible to
render Tn-polyagglutinable red cells T-active by treatment with neuraminidase. In ad-
dition, it should be noted that NANA residues may be linked, not to D-Gal residues
as in the alkali-labile tetrasaccharides, but to D-GalNAc residues.162This might account

for the agglutination of T-activated red cells by H.pomatja lectin (see Table 13).
It is also possible that the a-D-Gal residue that defines the T-receptor may be linked
not only to a-D-GalNAc, but also to a-D-Gal r e s i d ~ e s . ' ~This
. ~ ~possibility is discussed
by Judd, Murphy, and Goldstein et al." who found the BS I isolectin, BS I(B.), which
preferentially binds t o a-D-Gal, to agglutinate Tn-polyagglutinable red cells. However,
this isolectin also binds to a-D-GalNAc residues, albeit 100 times less readily than it
binds to a-D-Gal. l 6 Furthermore, has found group-0 Tn-polyagglutinable
red cells to absorb and elute anti-B, although not as readily as they absorb and elute
anti-A. lZs
The Tk-Receptor
There are few data regarding the nature of the Tk-receptor, apart from the rather
conflicting information that can be deduced from Table 13. Tk-polyagglutinable red
cells are agglutinated by both A. hypogaea lectin, which is inhibited by D-Gal,'46 and
by BS 11 lectin, which is inhibited by N-acetyl-D-glucosamine ( D - G ~ C N A CThe
) . ~ ~reac-
tivity of A. hypogaea lectin with both T- and Tk-activated red cells is inhibited by D-
Gal,'46 and Tk-activated red cells fail to react with A. hypogaea after it has been ab-
sorbed with T-activated red Moreover, purified A. hypogaea reacts with both
T- and Tk-activated red cells (see Reference 18 and Table 13). Thus, the agglutination
of both red cell types by A. hypogaea is probably a function of a single lectin protein.
There is some evidence to suggest that A. hypogaea and BS I1 lectins are not reacting
with the same determinant on the surface of Tk-activated red cells. This evidence in-
cludes Tk-activated red cells that, even after protease treatment, fail to react with A.
hypogaea lectin, yet in the untreated state they react strongly with BS I1 1 e ~ t i n . Fur-
I~~
thermore, red cells exposed to the action of @-galactosidasesreact strongly with BS I1
lectin, but not with A. h~p.0gaea.l~' Moreover, among the enzymes present in broth
cultures of B. fragilis that this author has studied, there is always not only a 0-galac-
tosidase, but a hexosaminidase ( N -acetyl-D-galactosaminidase). This latter enzyme
renders red cells agglutinable by A. hypogaea lectin, but not by BS I1 lectin. The prob-
lem is further compounded by the fact that Tk-activated red cells have a reduced I and
i antigen content. '15 The terminal sugars of these antigens include D-Gal residues linked
in the 0-configuration to D-GIcNAc.'~~ Thus, the action of P-galactosidase would ac-
count for both the BS I1 reactivity and reduced I and i antigen content of Tk-activated
red cells. Evidently, the alterations to red cell membrane structures associated with
Tk-activation are more complex than those produced by neuraminidase. However, one
fact is quite certain, and that is the Tk-receptor is unlikely to be a latent determinant
within the alkali-labile tetrasaccharides attached to the MN SGP that are implicated
in both T- and Tn-polyagglutination, since while the T- and Tn-receptors are destroyed
by proteases, the ag lutination of Tk-activated red cells by A. hypogaea and BS I1
lectins isgreatly rnhirnccrl hy treatment af the red cells with pratealytic
The Cad-Receptor
The agglutination of Cad-positive red cells by D. biflorus lectin and the inhibition
of this agglutination by a-D-GalNAc clearly indicates that a-D-GalNAc is an integral
part of the Cad-receptor and may well be the terminal nonreduting s u g a ~ . ' ~This '
would account for the agglutination of Cad-positive red cells by H. pomatia (see Table
13). However, the nonreactivity of Cad-positive red cells with P. lunatus 111 lectin,
which also binds to u-D-GalNAc, remains an enigma. It is also apparent, that the a-D-
GalNAc residue defining the Cad-receptor differs in either its linkage or orientation
within the red cell membrane from that which defines the Tn-receptor, since Cad-pos-
itive red cells, in contrast to Tn-polyagglutinable red cells, are nonreactive in tests with
human anti-A reagents.
August 1980 203
As discussed previously, Cad-positive red cells have elevated levels of the Sdn blood
group antigen.Isz Like ABH blood group substances, the Sd. antigen can be found in
the body fluids of man and other specie^.'^' However, purified ABH blood group
substances do not possess Sd’ activity. Nonetheless, it is likely that the Sd- antigen is
comprised of an oligosaccharide linked to a protein or to a lipid “backbone”.168
HEMPAS-Receptor
There is too little information available at present to form any conclusions regarding
the nature of the HEMPAS receptor. Whatever the defects are in the membranes of
HEMPAS red cells, they result in a reduced H-antigen content and an abnormal
expression of the I and i antigens.’s4.1s6The reactvitiy of these red cells with H.pomafia
lectin, and the BS I(A4) isolectin from B. simplicifolia seeds, suggests that terminal a-
D-GalNAc residues are exposed.
VA-Receptor
Again, there is insufficient data available regarding the nature of this receptor. Like
HEMPAS red cells, VA-polyagglutinable red cells have a reduced H-antigen content.
This may well result from the action of a bacterial a-L-fucosidase, at least in the case
described by Beck, Myers, and Moulds et al.Is9
Conclusions
Lectins, notably those from A . hypogaea, D. biflorus, B. simplicifolia, G . m a , and
Salvia seeds, provide an invaluable source of reagents for use in the recognition and
classification of red cell polyagglutinable states. Because of their specific carbohydrate-
binding properties, these and other lectins have been used to define the membrane
alterations that may occur to red cells that render them polyagglutinable.
OMNIUM GATHERUM
Introduction
It is hoped that this author will not be admonished for failure to restrain this flip-
pancy, but the somewhat botanical-sounding title to the final section of this review
seemed too appropriate to overlook! Suffice it to say, Omnium garherum is not a
lectin! ! There are, however, yet further lectins which display blood group polymorph-
ism that do not readiiy fit into any of the three preceding sections. Hence, the title
“Omnium-gatherum”, meaning miscellany or potpourri ( Webster’s Collegiate Thesau-
rus, 1976).
Anti-Gy in Peanuts*
In 1959, Boyd, Green, and Fujinaga et al.169described a new blood group factor
(Gy) that was detected by extracts of the peanut Arachis hypogaea. This report pre-
cedes that of Bird”‘ who demonstrated anti-T activity in such extracts by some 6 years.
Boyd and associates found that saline extracts of A . hypogaea would agglutinate al-
bumin suspended red cells from certain individuals, but not others, in tests at room
temperature and at 37OC. This agglutination appears to be independent of the ABH.
Rhesus, Duffy, Kell, Kidd, Lewis, Lutheran, MNSs, and P blood group systems, al-
though certain batches of A . hypogaea lectin agglutinated a higher proportion of red
cell samples that were group A , B, or AB than they did group 0 red cell samples. This
It should be noted that Gy (as discussed here) is not the same as, nor is it associated with, the much
later named Gy’ or Gregory antigen (Forreview, see References IS and 40.)
latter observation was confirmed by Schwarzfischer and Liebrich”’ (for an English

translation of their data see Reference 171).
Boyd and colleagues found the frequency of Gy-positive individuals to be in the
order of 23%. However, not all samples gave “clear-cut” positive or negative reac-
tions, as indicated by the results of multiple repeat testings on selected samples. Family
studies revealed that the Gy factor is inherited as a simple Mendelian trait.
The Gy factor has not been subjected to anthropological investigation. Also, this
author is unaware of any further publications on this topic. Thus, it is not known if
the anti-Gy and anti-T activity of A. hypogaea extracts is a function of the same lectin
protein, or if two distinct agglutinins are present in these seeds. However, agglutination
of both Gy-positive and T-activated-red cells by A. hypogaea lectin can be inhibited
by the addition of lactose.’26~169 The reason for drawing attention to the Gy factor in
this review is to point out that there is now a tendency by some workers, including
this author, to potentiate the activity of lectin reagents by preparing seed extracts in
bovine albumin. As may be seen in Table 13, group A and group 0 Tn-polyagglutina-
bie red cells are agglutinated by purified A. hypogaea lectin a t concentrations exceed-
ing 0.05 mg/ml of lectin protein. It is not known if this agglutination is due to the
Gy factor or not. Nonetheless, it is important to standardize A. hypogaea preparations
containing bovine albumin against non-T-activated red cells of all common ABH phen-
otypes and to select a dilution for use in the investigation of red cell polyagglutination
at which they display the desired anti-T/Tk specificity.
Anti-Rh Lectins
Prokop and Uhlenbruck”’ cite the observations of the Yugoslav workers Mitrovic
and Simonovic (Bilten Transfuzije, 8: 8 I , 1959, paper not seen by this author) regard-
ing the presence of anti-Rh. (D) activity in extracts of the stems and tubers from im-
munized Begonia punctata. Had this finding been confirmed, it would have had far-
reaching implications and would not only have provided an abundant source of anti-
Rho for blood typing purposes, but might also have permitted characterization of the
Rhesus blood group antigens. However, Jenkins, Mollison and Race’’’ found the orig-
inal Yugoslavian extract to react by the indirect antiglobulin test and to give identical
precipitin lines to whole human serum o n immunoelectrophoretic analysis! Jenkins
and colleagues concluded: “It seems impossible to escape the conclusion that by some
accident the extract became contaminated with human serum containing anti-D”.
While there are n o anti-Rh specific lectins known at the present time, Bird and
Wingham109in the course of studying the reactivity of En(a-) red cells with lectins
noted that group A Rh..,, and group A -D-/-D- red cells were less readily agglutinated
than red cells with a normal Rh phenotype by extracts from Clerodendron trichorno-
turn seeds. Moreover, group 0 RhmMl, or group-0 -D-/-D- red cells were agglutinated
to the same as normal Rh-positive or Rh-negative red cells. Thus, C. trichomotum
extracts seem to demonstrate some ABH-Rh interaction akin to the unusual anti-Rh.
serum reported by Ikin et al.,173which in saline reacted only with group A Rh-positive
red cells. The precise significance of this finding, relative t o the nature of the Rh anti-
gens (which appear to be lipoproteins, see References 15 and 174 for a review:)remains
unresolved. Furthermore, while all Rh antigenicity appears t o involve a protein moe-
ity,I7’ it is not known a t present if these proteins are glycosylated.
.
Lectins that React with the P System Antigens

As mentioned in the section on ABH-reactive lectins, certain extracts displaying a
preferential specificity for group B red cells also cross-react with the P I , P, and Pk
antigens of the P blood group system. These include primarily extracts from Salmoni-
dae roe and the more recently described anti-A/B/P lectin from Python ~ e b a e . ~ ~In. ’ ’ ~
August 1980 205
contrast, the anti-B reagent prepared from Bandeiraea simplicifolia seeds shows no
affinity for P system antigen^.'^
Schnitzler, Muller, and P r ~ k o p studied
’ ~ ~ extracts from the roe of Rutilus rutilus
(Roach) and noted that such extracts agglutinated PI red cells more strongly than P,
red cells. These reactions were stronger in tests incubated at 4”C, and were enhanced
by the addition of gelatin to the reaction medium. In such a test system, group A, or
group 0 P, positive red cells were agglutinated by R. rutilus lectin at a dilution of

1:16, whereas groups A P, and 0 P, red cells were only weakly reactive (titer = 1).
However, this extract was strongly reactive with group B red cell samples regardless
of their P phenotype. The authors concluded that the lectin had inseparable (as judged
by the results of adsorption and elution experiments) anti-B and anti-PI activity. None-
theless, they considered the extracts to be useful reagents in determining the P, status
of groups A and 0 red cells. However, it should be noted that Anstee, Holt, and
~ unable to confirm the presence of anti-P, activity in the R. rutilus
P a r d ~ e ”were
extracts that they studied.
The Salmonidae roe agglutinins react with P system antigens preferentially in tests
employing protease-treated red cells. In an extensive investigation, Voak, Todd, and
Pardoe” examined the serological behavior of Salmo eriox, S. gairdeneri, S. salar, and
S. trutta in tests with red cells of various ABH and P blood group phenotypes. Only
red cells lacking B, P,, P, and P‘ antigens (i.e., group A, pp red cells] were nonreactive.
In addition, the agglutination of groups B pp, 0 PI, 0 P,, and Al PIkred cells by
these lectins was abolished by the addition of a-D-galactose ( u - D - G ~ )indicating
, that
a-D-Gal is the immunodominant sugar of the B, PI, P, and P‘ antigens. In an earlier

publication, these same authors had shown that a-D-Gal plays an important role in
determining the structure of the P‘ determinant by studies, not only with the afore-
mentioned lectins, but also in tests with human sera containing anti-P‘ agg1~tinins.l’~
It is now known that a-D-Gal is the terminal nonreducing sugar of the B, PI, and
Pk antigens.160,1mRed cell membrane B and PI antigens are formed by the action of
specific galactosyl-transferases (B = a( 1-3)-galactosyl-transferase;P, = a( 1-4)-galac-
tosyl-transferase) on a preformed glycosphingolipid, referred to as paragloboside,
which must be modified into H antigen by the addition of an a-L-fucose residue before
the B-specific-transferase can The Pk antigen (ceramide trihexoside), which also
has a terminal nonreducing a-D-Gal residue, appears to be an intermediate structure
formed in the production of globoside. This latter glycosphingolipid has a terminal
nonreducing a-N-acetyl-D-galactosamineresidue and has been identified as the P de-
terminant. Thus, while Voak, Todd, and Pardoe” were correct in their conclusions
regarding the nature of the PI and P k determinants, a-D-Gal is not the terminal non-
reducing. sugar of the P antigen. Perhaps, therefore, the specificity of the Salmonidae
roe agglutinins should be regarded as anti-B/P,/Pk, rather than as anti-B/P,/P/P*.
This would not necessarily preclude the agglutination of P2 red cells by these lectins,
for although P, (and P I ) red cells are nonreactive with human anti-P* Sera, theP*-
receptor must (as it is a precursor of the P antigen) be present on P, red cells as a
cryptantigen. Treatment of P, red cells with proteolytic enzymes, which is essential to
obtain avid agglutination of nongroup-B red cells with the Salmonidae roe agglutinins,
might expose unconverted Pk substance which can then be recognized by the lectin
reagents. The failure of human anti-P’ sera to react with protease-treated PI and PI
red cells is probably a reflection on the size of the antigen combining site of the im-
munoglobulin molecules, which are undoubtedly larger than those of the Salmonidae
roe agglutinins. This latter assumption is supported by Voak, Todd, and P a ~ d o who e~~
consider the Salmonidae roe agglutinins to require a binding site comprised of a single
sugar, whereas human IgG immunoglobulins have binding sites that can accommodate
at least five pyranosyl residues.
Lectins with a Preference for I-Positive Red Cells

I t should be noted that there are no lectins that react solely with the I determinant
of intact human red cells. However, as mentioned previously, certain anti-H lectins
appear to have anti-HI or anti-I/HI activity when tested against untreated red cells,
but show no preference for either I-positive o r I-negative red cells that have been
treated with neuraminidase or proteolytic enzymes.
Chien, Lemanski, and Poretz'" studied the reactivity of the anti-A/B agglutinins
from Sopbora japonica seeds. Using a purified S. japonica preparation, they found
cord-blood red cells and adult I-negative red cells that were either group A or group B
to be less readily agglutinated than groups A or B I-positive red cells. Between 10 and
40 times greater concentrations of S. japonica lectin were required to agglutinate adult
and cord-blood I-negative red cells to the same degree as I-positive red cells. Group 0
red cells, regardless of their Ii phenotype, were nonreactive with S. japonica lectin,
even at lectin protein concentrations of 3.0 m g l m f . Thus, the specificity of S. japonica
lectin can be considered as anti-AI/BI, reacting with joint products of the A and I.
and B and I, determinants. While such specificity was apparent from the results of
tests on intact red cells, S. japonica lectin was shown to precipitate with a group 0
preparation of human I-blood-group substance. This is, perhaps, not surprising, for
S. japonica lectin is known to be preferentially inhibited by P-anomers of D-Gal, which
also are most likely the terminal residues of the I-blood-group antigen.166
The preference of some lectins with anti-A, anti-B, or anti-H activity for red cells
that are I-positive may in part account for the weak reactions of cord-blood red cells
with ABH blood-typing reagents prepared from lectin sources. However, as discussed
previously, cord-blood red cells also have less ABH antigens than d o adult red cells.
The latter must be responsible for the poor reactions of the BS I + GalNAc anti-B
reagent prepared from Bandeiraea simplicifolia seeds with group B cord-blood red
cells, for BS I + GalNAc anti-B reacts to the same degree with both adult group B I-
negative and group-B adult I-positive red cells." In contrast, Rogers1aZfound a n anti-
B lectin from the ova of the Black Sea bream, Spondyfiosoma cantbarus, to have a
definite preference for adult group B I-positive red cells.
Conclusions
Despite over some 35 years of extensive investigation, there are but few blood group
lectins that display serological activity outside of the ABH and MN blood group sys-
tems. This must, in part, reflect the fact that not all blood group antigens are com-
prised of carbohydrate moieties with which lectins bind. Moreover, the terminal non-
reducing portion of the ABH, P, and 1 blood-group-system antigens essentially involve
either D-galactose or N -acetyl-o-galactosamine residues. This fact accounts not only
for the enormous cross-reactivity that lectins display with the various blood-group an-
tigens, but must also contribute to the notable absence of specific anti-PI and anti-I
agglutinins of lectin origin.
SUMMARY
Many lectins display blood group activity, and.extracts from Dolicbos bifforus(anti-
A,), Ufex europaeus (anti-H). and Vicia grarninea (anti-N) seeds provide an alternative
to human sera as a source of blood typing reagents. However, the major application
of lectins in blood-group serology undoubtedly lies in the recognition and elucidation
of red cell polyagglutinable states. In this respect, lectins from Aracbis bypogaea (anti-
TITk), Safvia scfarea (anti-Tn), Safvia horrninurn (anti-Tn + Cad), Gfycine max (syn.
s o h and the Nacetyl-D-glucosaminyl-binding lectin (BS 11) from Bandeiraea simpfi-
cifofia seeds provide invaluable reagents for use in investigative immunohematology.
August 1980 207
Because of their specific carbohydrate-binding properties, lectins have also been used
as probes in studies on the topography of the red cell surface. This latter application
has provided much information on the structure of the MN, T, and Tn red cell surface
receptors and has aided in defining the red cell membrane abnormalities associated
with certain uncommon phenotypes within the MN blood-group system.
ADDENDUM
During the preparation of this manuscript, this author’s attention was drawn to a
recent publication by Bird and Wingham,’83 concerning their discovery of a predomi-
nantly Cad-specific lectin in extracts of Leonurus cardiaca seeds.’ This lectin promises
to be a useful adjunct to the reagents already employed in the elucidation of red cell
polyagglutinable states, and it may aid in further defining the nature of the Cad-recep-
tor. While extracts from L. cardiaca seeds also react weakly with T- and Tn-polyagglu-
tinable red cells, a specific anti-Cad reagent can be prepared at an appropriate dilution.
Agglutination of Cad-positive red cells.by L. cardiaca lectin is inhibited by D-GalNAc
and by guinea pig urine, which is rich in Sd. (Sid) blood group substance. These results
further establish the involvement of Cad with Sd’, and again indicate that D-GalNAc
is the immunodominant sugar of the Cad-receptor. However, L-fucose also inhibits
this agglutination, a fact which obviously warrants further investigation.
L. cardiaca is of the natural order Labiatae (mint family) and is more commonly
known as mother wort. It is a native of Asia and Europe, but has been naturalized in
North America. Hopefully, therefore, these seeds will become readily available to in-
vestigators in the U.S.
REFERENCES
1. Boyd, W.C. and Shapleigh, E.. Specific precipitating activity of plant agglutinins (lectins). Science,
119,419. 1954.
2. Prokop, 0.. Uhlenbruck. G.,and Kohler, W.. A new source of antibody-like substances having anti-
blood group specificity. A discussion of the specificity of the Helix agglutinins, Vox Sang., 14. 321,
1968.
3. Gold. E. R. and Balding, P., Receptor Specific Proteins - Plant and Animal Lectins. Excerpta
Med.. Amsterd?m, 1975.
4. Stillmark, H.. Uber Rizin. ein giftiges Ferment aus Samen von Ricinus cornrnunis L.. und einigen
anderen Euphorbiaceen, Inaugaral Dissertation. Dorpat, 1888.
5. Boyd, W. C. aud Reguera, R. M.. Hemagglutinating substances for human cells in various plants,
J. Immunol.. 62,333. 1949.
6. Renkonen, K. 0..Studies of hemagglutinins present in some representatives of the family of Leg-
uminosae, Ann. Med. Exp. Biol. Fenn.. 26,66. 1948.
7. C d . P. and Laularie, M.. Reserches sur quelques phyto-agglutinins specifiques des groupes san-
guins ABO. Acta Haematol., 8,73. 1952.
8. Ottearooser, F. and Silbcrschmidt. K.. Hemagglutinin anti-N in plant seeds, Nature (London), 172,
914, 1953.
9. Bird, 0. W.G., Specific agglutinating activity for human red blood corpuscles in extracts of Dolichos
binorus. Cum. Sci., 20.298, 1951.
10. Goldstein, I. J. and Hayes. C. E., in Advances in Carbohydrate Chemistry and Biochemistry,Tipton,
R. S . and Horton, D., Eds., Academic Press, New York, 35. 1978, 127.
11. Wolpat. J. S. and Albersheim. P., Host-symbiont interactions. 1. The lectins of legumes interact
with the 0-antigen-containing lipopolysaccharides of their symbiont Rhizobia, Biochern. Biophys.
Res. Commun., 70,729, 1976.
12. Fountain. D. W.,Foard. D. E., Replogle, W. D., and Yang, W.K., Lectin release by soybean seeds,
Science, 197. 1185. 1977.
13. Sharon, N., Lectins, Sci. A m . . 236. 108, 1977.

14. Partridge, J., Shannon, L.. and Gumpf. D., A barley lectin that binds free amino sugars. I. Purifi-
cation and characterization, Biochim. Biophys. Acta, 451,470. 1976.
15. Race. R. R. and Sanger. R., BloodGroupsin Man. 6th ed., Blackwell Scientific, Oxford, 1975.
16. Murphy, L. A., Ph.D. dissertation, University of Michigan, 1978.
17. Boyd, W. C., W a s z c z c n k o - Z a c h o , E.. and Goldwasser. S.. List of plants tested for hemag-
gfutinating activity, Transfusion (Philadelphia), I , 374. 1961.
18. Judd, W.J., The use of purified lectins in immunohematology. Transfusion (Philadelphhl), 19, 768,
1979.
..
19. Prokop. O., Schlessinger. D., and Rackwitz. A.. Uber einc thermostabile “antibody-like substance”
(anti-A,,) bei Helix pomatia und deren Herkunft, 2. Immuniraefsforsch. Allerg. Klin. .Immunol.,
129.402, 1965.
20. Gold, E. R. and Thompson, T. E.. Serological differences between related species of snails. 1. Re-
vealed by passive agglutination tests, Vox Sang., 16.63, 1969.
21. Tovey. G.H. and Lockyer. W.J., Valuable new sources of anti-A and anti-B, 1. Med. Lab. Technol.,
26,264. 1969.
22. Cann, G. B.. A laboratory evaluation of the hemagglutinating substance from the snail Helix aspersa,
J. Med. Lab. Technol.. 3 I , 1 I 1974.
~
23. Voak. D..Todd, G. M.. and Pardoe, G. I.. A study of the serological behaviour and nature of the
anti-B/P/P‘ activity of the Salmonidaeroe protectins. Vox Sang., 26, 176, 1974.
24. Reer, P.. Cotton, R.. Holt, P. D. J.. and Anstce. D. J., Trout ova, an alternative source of anti-B,
Med. Lab. Sci.,33. 13, 1976.
25. Downie, D.M., Madin. D. F.. and Voak, D.. An evaluation of the salmon anti-B reagent in manual
and automated blood grouping, Med.. Lab. Sci.. 34,3 19, 1977.
26. Pardoc. G. I.. Uhlenbruck. G., Anstce. D. J.. and Reifenberg. U.. On the specificity of the broad
spectrum agglutinins. 111. Heterophilc agglutinins associated with anti-B specificity, 2. Immunitaets-
forsch. Allerg. Klin. Immunol., 117,220, 1970.
27. Chattoraj, A. and Boyd, W.C., A specific anti-B lectin for routine diagnostic purposes, J. Immunol.,
96,898,1966.
28. Boyd. W.C..Lectins, Ann. N . Y . Acad. Sci., 169, 168, 1970.
29. Rogers. D. J., Blunden, G.. and Evans, P. R.. Ptilora plumosa, a new source of an anti-B blood
group specific lectin. Med. Lab. Sci., 34, 193, 1977.
30. Ma;kelX, 0.and h€Akela. P., Some new blood group specific phytagglutinins, Ann. Med. Exp. Biol.
Fenn., 34,402, 1956.
31. Gold, E.R.,unpublished observations, cited in Reference 3.
32. Hayes. C. E. and Goldstein. I. J.. An u-D-galactosyl-binding lectin from Bandeiraea simplicifolia
seeds, J . Biol. Chem., 249. 1904. 1974.
33. Judd. W.J., Stcina. E. A.. Friedman, B. A., Hayes. C. E.,and Goldstein. I. J.. Serological studies
on an 0-D-galactosyl-binding lectin from Bandeiraea simplicifolia seeds, Vox Sang., 30, 261, 1976.
34. Murphy, L. A. and Goldstein, I. J., Five u-D-galactopyranosyl-bindingisolectins from Eandeiraea
simplicifoliasnds, J . Biol. Chem.. 252,4739, 1977.
35. Iycr. P. N.. Wilkinson, K. D., and Goldstein,1. J., An N-Acetyl-D-glucosamine-bindingIcctin from
Bandeiraea simplicifoliaseeds, Arch. Biochem. Biophys., 177, 330, 1976.
36. Judd. W. J., Beck, M. L.. Hicklin. B. L.. Iyer. P. N., and Goldstein, I. J., BS I 1 Lectin: a second
hemagglutinin isolated from Bandeiraea simplicifolia seeds with affinity for type 11I polyagglutinable
red cells, Vox Sang.,33. 246, 1977.
37. Judd, W.J.. Murphy, L. A., Goldstein, I. J.. Campbell. L.. and Nichols, M. E.. An anti-B reagent
prepared from the o-D-galactopyranosyl-binding isolectins from Bandeiraea simplicifolia seeds,
Transfusion. 18, 274, 1978.
38. Voak. D.. Lodge, T. W..and Reed. J. V., The enhancement of Ulex europaeus anti-H activity by
human sera, Vox Sang.,17, 134. I %9.
39. Mollison. P. L., Blood Transfusion in Clinical Medicine, 5th ed.. Blackwell Scientific, Oxford, 1972.
40. Issitt, P. D. and Issitt, C. H..Applied Blood Group Serology, 2nd ed.. Spectra Biologicals, Oxnard.
1975.
41. Beattie. K. M.. in A Seminar on ProblemsEncountered in Pretransfusion Tests, Walker, R . H.. Ed.,
American Association of Blood Banks, Washington, D.C., 1972. 129.
42. Voak. D. and Lodge, T.W.,The demonstration of anti-I/HI-H activity in seed anti-H reagimts. Vox
Sang.,20. 36, 1971.
43. Bird, G.W.G., Heterogeneity of anti-H lectin, Rev. Fr. Transfus. Immuno-hemarol., 19, 175, 1976.
44. Morgan, W. J. and Watkins. W. M.. The inhibition of hemagglutinins in plant seeds by human
blood group substances and simple sugars, Br. J. Exp. Parhol.. 34, 94, 195Z.
45. Watkins, W.M..Blood group substances, Science, 152. 172, 1955.
August 1980 209
46. Mauumoto, I. and Osawa. T.. On the specificity of various heterologous anti-H hemagglutinins.
VoxSang.. 21,584, 1976.
47. Bird, G. W. G. and Wingham, J., Anti-H from Cerasrium tomentosum seeds. A comparison with
other seed anti-H agglutinins, VoxSang., 19, 132, 1970.
48. Rogers, D.J.. Topliss, J. A., and Blunden. G., Reactions of anti-B lectins with erythrocytes sensitized
with lipopolysaccharides from Escherichia coli OM,Med. Lab. Sci., 36,79, 1979.
49. Gerbal, A.. Maslet. C.. and Salmon. C., Immunological aspects of the acquired-B antigen. Vox
Sang., 28,398. 1975.

50. Anderson. J., Production of a B character on human red cells by hemosensitization with purified
Escherichia coli 0, substance, Nature (London),190,730. 1961.
51. Oguchi. Y.,Kawaguchi. T., Suzata, T., and Osawa, T., The nature of human blood group A, eryth-
rocytes, Vox Sang., 34. 32, 1978.
52. Bird, G. W. G., Agar gel studies of blood group specific substances in precipitins of plant origin. 1.
The precipitins of Dolichos biflorus, Vox Sang., 4.307. 1959.
53. Gillespie, E. M. and Gold, E. R.. Weakening of the B antigen by the presence of A, as shown by
ieactions with Fomes fomenrarius(anti-B) extract, VOX Sang., 5,497. 1960.
54. Reid, M. E. and Judd, W. J., The effect of gene interaction on the A and B blood group antigens,
J. Med. Lab. Techno/., 26, 136, 1969.
55. Williams. M. A. and Voak, D.. Studies with ferritin labelled Dolichos bifloruslectin on the numbers
and distribution of A sites on A, and AXerythrocytes, and on the nature of its specificity and enhance-
ment by enzymes. Vox Sang., 23.427, 1972.
56. Boyd, W. C.. Bhatia. H. M.,Diamond, M. A.. and Matsubara. S., Quantitative study of the com-
bination of Lima bean lectin with human erythrocytes, J. Immunol., 89,463. 1962.
57. Economidou, J.. Hughes-Jones. N. C.. and Gardner. B.. Quantitative measurements concerning A
and B antigen sites, VoxSsng., 12,321,1967.
58. Voak, K. and Lodge, T. W., The role of H in the development of A. VoxSang., 15,345,1968.
59. Voak. D.,Stapleton. R. R., and Bowley, C. C., A I A A la, new variant of A, in two group A members
of an English family, VoxSang., 14. 18, 1968.

60. Voak. D.. Lodge, T. W., Stapleton. R. R., Fogg. H., and Roberts, H. E.. The incidence of H
deficient Az and AzB bloods and family studies on the AH/ABH status of an A,., and some new
variant blood types A~?H--’I. AltBwwAl’and A21B.w*1’). VoxSang., 19. 73, 1970.
61. Gallasch, E.. Bombay phenotypes (Letter to the Editor), Vox Sang., 35,436, 1978.
62. Weiner. W., Lewis, H. B. M.,Moores, P.. Sanger. R.. and Race, R. R.. A new gene, y, modifying
the blood group antigen A. Vox Sang., 2,25, 1957.
63. Tornita, M. and March&, V. T.,Amino-acid sequence and oligosaccharide attachment sites of hu-
man erythrocyte glycophorin, Proc. Narl. Acad. Sci., 72,2964. 1975.
64. Furthmayr, H.. Structural comparisons of glycophorins and immunochemical analysis of genetical
variants. Narure(London). 271,519. 1978.
65. Allen. N. K. and Brilliantine, L.. A survey of hemagglutinins in various seeds, 1. Immunol.. 102,
1295, 1969.
66. Nakajima, H. and Nakayama, T., Anti-M lectin of lberis amara L., Rep. Jpn. Narl. Res. lnsrirure
of Police Science, 2 1, 1 11, 1968.
67. Okada. T. and Nakajima. H.. Anti-M lectin of Japanese radish, Rep. Jpn. Narl. Res. Insrirure of
Police Science. 23.207, 1970.
68. Romanawska, E., Reaction of M and N blood group substances natural and degraded with specific
reagents of human and plant origin, Vox Sang., 9,578. 1964.
69. Dahr. W., Issitt. P. D., and Uhlmbruck. 0..New concepts of the MNSs blood group system, in
Human Blood Groups, Mohn, J. F., Plunkett, R. W., Cunningham, R. K., and Lambert. R. M.,
Eds., S. Karger, Basel, 1977.197.
70. Boyd, W. C. and Waszczenko-Zacharczenko. E.,Erythrocyte receptors for t w o “non-specific” lec-
tins, Transfusion. 1,223, 1961.
71. Makela. O., Studies on hemagglutinins in Leguminosae seeds. Ann. Med. Exp. Biol. Fenn.. 35
(Suppl. 11). I , 1957.
72. Moon. 0. J. and Wiener, A. S.. A new source of anti-N lectin: leaves of the Korean Vicia unijuga,
VoxSang., 26, 167. 1974.
73. Bird. 0. W. G. and Wingham, J.. Agglutinins for antigens of two different human blood group
systems in the seeds of Moluccella laevis, Vox Sang., 18, 235, 1970.
74. Makela. 0. and Cantell. K., Destruction of M and N blood group receptors of human red cells by
some influenza viruses, Ann. Med. Exp. Biol. Fenn., 36.366, 1958.
75. Bird, 0. W. 0.and Wingham, J., Nacetylneuraminic (sialic) acid and human group antigen struc-
ture, VoxSang., IS, 240. 1970.
76. springer, G . F. and Ansell. N. J.. Inactivation of human erythrocyte agglutinogens M and N by
influenza viruses and receptor destroying enzymes, Proc. Natf. Acad. Sci., 44,182, 1958.
7 7 . Uhlenbruck. G., Possible genetical pathways in the MNSsUu system, VoxSang.. 16,200, 1969.
'8. Uhlrnbruck. G. and Dahr, W., Studies on lectins with a broad agglutination spectrum. XII. N -
acetyl-D-galactosamine specific lectins from the seeds of Soja hispida. Bauhinia purpurea, Iberis
srnara. Moluccella laevisand Viciagraminea Vox Sang., 21, 338. 1971.
79. Springer, G. F. and Huprikar. S. V., On the biochemical and genetic basis of the blood group M N
specificities, Haematologia. 6. 81, 1972.

80. springer. G. F., Tegtmeyer, H.. and Huprikar. S. V., Anti-N reagents in the elucidataon of the
genetical basis of human blood group MN specificities, Vox Sang., 22, 325, 1972.
81. L k w s k a , E. and Duk. M.. Effect of modification of amino groups of human erythrocytes M, N
and N,, blood group specificities, Vox Sang., 28, 392. 1975.
82. Walker. M. E., Rubinstein, P.. and Allen. F. H..Jr.. Biochemical genetics of MN, Vox Sang., 32,
I l l , 1977.
83. Lisowska. E. and Kordowiu. M.. Specific antibodies for desialyted M and N blood group antigens,
Voxsang., 164, 1977.
84. Judd, W. J., Issitt, P. D., Pavone. 8.G.,Anderson. J., and Aminoff, D..Antibodies that define
NANA-independent MN-system antigens, Transfusion. 19. 12. 1979.
85. Dahr, W., Uhlenbruck. G.,Janssen. E., and Schmatisch, R.. Different Nterminal amino acids in
the MN glycoprotein from MMand NNerythrocytcs, Humangenetik, 35.335, 1977.
86. W k i o w s k a . K., D m n i e k , Z., and Lisowska. E., The amino acids of M and N blood group glyco-
peptides are different, Biochem. Biophys. Res. Commun..76.385, 1977.
8 7 . nomas, D. B. and Winzler. R. J.. Structural studies on human erythrocyte glycoproteins. Alkali-
labile tetrasaccharides, 1.Biol. Chem.. 244. 5943, 1969.
88. Steane. E. A.. Flanders. J. Y.,Steane. S. M.,and Grecnwalt. T.J., The complexity of N specificity:
a hypothesis correlating serology and biochemistry, preliminary data in support, and a proposal for
nomenclature for the various M and N antigenic specificities. Transfusion, 18, 645, 1978.
89. Anstee. D. J., Blood group MNSs-active siaolglycoproteins of the human erythrocyte membrane, in
Immunobiology of the Red Cell, Liss. New York. in preparation.
90. Gahmberg, C. G.,MyllyG. G., Leikola. J., Pirkola, A., and Nordling. S.. Absence of ,:he major
sialoglycoprotein in the membrane of human En(a-) erythrocytes and increased glycosylation of band
3. 1.Biol. Chem..251, 6108. 1976.
91. Kornfeld. R. and Kornfeld, S.. The structure of a phytohemaggultinin receptor site from human
erythrocytes, J. Biol. Chem..245.2536. 1970.
92. Dahr. W.,Issitt. P. D.. Moulds, J.. and Pavone. B. G.,Further studies on the membrane glycopro-
tein defects of S-s- and En(a-) erythrocytes, Hoppe-Seyler'sZ. Physiol. Chem., 359, 1217, 1978.
93. Tanner, M. J. A., Anstet. D. J., and Judson. P. A., A carbohydrate deficient membrane glycopro-
tein in human erythrocytes of phenotype S-s-, Biochem. 1..165, 157, 1977.
94. Fairbanks, G., Steck. T.L.. and Wallach, D.F. H..Electrophoretic analysis of the major polypeptide
of the human erythrocyte membrane, Biochemistry, 10,2606, 1971.
95. Laemmli. U. K., Cleavage of structural proteins during the assembly of the head of the bacteriophage
T 4 . Nature (London). 227,680, 1970.
96. Allen, F. H..Jr., C o r m . P. A., and Ellis. F. R.. Some new observations on the MN system, Vox
Sang., 5 , 224, 1960.
9 7 . Allen, F. H., Jr.. Madden, H.J.. and King. R. W., The M gene MUwhich produces M and U but
no N, S or s , Vox Sang., 8,549, 1963.
98. Dahr. W.,Uhlmbruck. G.. and Knott. H.,lmmunochernical aspects of the MNSs blood group sys-
tem, J . Immunogenet., 2.87, 1975.
99. Rolih. S. D. and Issitt, P. D.. Effects of trypsin on the Vicia graminea receptors of glycciphorin A
and 8, Transfusion, 18,637, 1978.
100. Tokunaga, E.. Sasakawa. S.. Tamaka. K., Kawamata. H..Gila. C. M.. Ikin, E. W..Poole. J..
A n s t e , D, J.. Mawby. W. J.. and Tanner, M. J. A.. Two apparently healthy Japanese individuals
of type M'M' have erythrocytes which lack the MN and Ss-active sialoglycoproteins, J . Irnmunoge-
net., in press.
101. Anstee. D. J.. Tanner, M. J. A.. Mawby. W. J.. and Giles. C. M.. A new efythrocyte variant (Ph)
containing an abnormal membrane sialoglycoprotein. in preparation.
102. Furuhjelm. U.. Myllyla. G., Ncvanlinna, H.R.. Nordling. S.. Pirkola. A., Gavin, J., Gooch, A..
SZUIger. R.. and Tippett, P., The red cell phenotype En(a-) and anti-En': serological and physico-
chemical aspects. Vox Sang., 17,256, 1969.
103. Darnborough, J.. Dunsford. I., and Wallace, J. A.. The En' antigen and antibody. A genetical
modification of human red cells affecting their blood group reactions, VoxSang.. 17. 241, 1!369.
August 1980 21 1
104. Taliano. V., Gu&in, R. M.. Hebrt, D., Daniels, G. L., Tippett, P., Anstee, D. J., Mawby, W. J.,
and Tanner, M. J. A., The rare phenotype En(a-) in a French-Canadian family, Vox Sang., in press.
105. Anstce. D. J.. Barker, D. M.. Judson. P. A., and Tanner, M. J. A.. Inherited sialoglycoprotein
deficiencies in erythrocytes of type En(a-). Br. 1.Haernarol.,35.309, 1977.
106. Tanner, M. J. A. and Anstn. D. J., The membrane changes in En(a-) erythrocytes. Biochem. J.,
153.271, 1976.
107. Bird, G. W. G. and Wingham. J.. The action of seed and other reagents on En(a-) erythrocytes,
VoxSang., 24.48, 1973.

108. Dahr. W.,Uhlenbruck, G., Leikola, J., and Wagstaff. W., Studies on the membrane glycoprotein
defect on En(a-) erythrocytes. Ill. Nterminal amino-acids of sialoglycoproteins from normal and
En(a-) red cells. J. Immunogenet.. 5, 117, 1978.
109. Vengelen-Tyler, V., Anstec. D. J., Issitt. P. D., Pavone. B. G.. Ferguson. S. J.. Mawby. W. J..
Tanner, M. J. A., Blajchman. M. A.. and Lorque. P.. Studies on the blood of an Mi”homotygote,
Transfusion,submitted, 1979.
110. Langlcy. J. W., Issitt, P. D.. Anstee, D. J.. McMahan. M.. Smith, N.. Pavonc. B. 0.. Tesscl. J.
A.. and Carlin. M. A.. Another individual (J. R.) whose red cells appear to carry a hybrid sialogly-
coprotein, Transfusion, submitted. 1979.
1 1 1 . Dahr. W.,Uhlenbruck, G., and Knott. H., The defect of M‘ erythrocytes as revealed by sodium
dodecysulphate-polyacrylamidegel electrophoresis. 1.Immunogenet.. 4. 191, 1977.
112. Sturgeon. P., Metaxas-Buhler, M., Metaxas, M. N.. Tippet. P., and Ikin, E. W.,An erroneous
exclusion of paternity in a Chinese family exhibiting the rare MNSs complexes M‘ and MsY‘.”‘.Vox
Sang., 18,395, 1970.
113. Nordliig, S.. Sanga. R.. Gavin. J., Furuhjelm, U.. MyUyri, G.. and Metaxas. M. N., Mk and MI:
some serological and physicochemical observations, Vox Sang., 17, 300, 1969.
114. Allen. F. H., Jr., Corocan, P. A.. Kcnton. H. B.. and Brcare, N., MI. a new blood group antigen in
the MNS system, Vox Sang., 3.81, 1958.
I IS. Ansta. D. J. and Tanner, M. J. A., Genetic variants involving the major membrane sialoglycopro-
teins of human erythrocytes: studies on erythrocytes of type M‘, Miltenberger class V. and MS.
Biochem. 1.. 175, 149, 1978.
116. Dahr. W.and Uhlenbruck, G., Structural properties of the human MN blood group antigen receptor
sites, Hoppe-Seyler’s Z . Physiol. Chem., 359,835. 1978.
117. Dahr, W.. Longster, G.. Uhlenbruck. G.. and Schumacher. K., Studies on Miltenberger class Ill.
V, M’ and M‘ red cells. I. Sodium dodecylsulphate polyacrylamide gel electrophoresis investigations,
Blur, 37, 129. 1978.
I 18. Thornsen. 0.. Ein vermehrungesfahiges Agens als Veranderer des isoagglutinatorischen Verhaitenes
der roten Blutoperchen, cine bishe unbekannte Quelle der Fehlbestimmung. 2. Imrnunitaetsforsch
Allerg. Klin. Immunol.,52, 85, 1927.
119. Hiikncr. 0.. Unterschunger iiber lsoagglutination mit besonderer Berlicksichtigung scheibarer Ab-
weichungen vom Gruppenschema, Z.Immuniraetsforsch Allerg. Klin. Immunol., 45.223. 1925.
120. Freidenreich, V.. The Thornsen Hemagglutinating Phenomenon, Levin and Mrinksgaard. Copen-
hagen. 1930.
121. Lcvinc, P. and Katzin. E. M.. Temporary agglutinability of red blood cells, Proc. SOC.Exp. Biol.
N . Y.,39. 167. 1938.
122. h i g h a m . A. D.. Flemans. R.. Heard, D. H..and Seaman, G. V. F.. An apparatus for microelectro-
phoresis of small particles, Nature (London), 183.642. 1958.
123. Stcane. E. A.. Unpublished observations cited in Reference 40.262.
124. Aminoff. D.. Methods for the quantitative estimation of N -acetylneuraminic acid and their appli-
cation to hydrolysates of sialomucoids, Biochem. J., 81. 384, 1961.
125. h a n . H. J.. Smarto, J., Issitt. C. H., Issitt, P. D.. Marsh, W. L.. and Jensen. L.. Tn-activation
with acquired A-like antigen, Transfusion. 12. 35, 1972.
126. Bird, G. W. 0.. Anti-T in peanuts, Vox Sang.,9,748,1964.
127. Issitt, P. D.. Moulds, J., and Berman, H.,Some observations on the T,Tn and Sd’ antigens and
the antibodies that define them, Transfusion, 12,217. 1972.
128. Gunson. H.H..Stratton. F., and Mullard. G.W.., An example of polyagglutinability due to the Tn
antigen. Br. J . Haemarol.. 18.309, 1970.
129. LOW, B.. A practical method using papain and incomplete Rh antibodies in routine Rh blood-group-
ing. VoxSang..5 (0,s.).1955.
130. Beck, M. L. and Marsh, W. L.. Red cell Sp, antigen change associated with in vivo polyagglutinabil-
ity, Voxsang., 19. 91, 1970.
131. Bird, G. W. G.. Wingham. J., Beck. M. L.. Pierce. S. R.. Oates. G. D.. and Pollack, A., Th, a
“new” form of erythrocyte polyagglutination. Lancer. 1, 1215, 1978.
21 2 CRC Critical Reviews in Clinical Laboratory Sciences
132. Bird, G. W. G. and Wingham, J., Tk: a new form of red cell polyagglutination, Br. J . Haematof.,
23,759. 1972.
133. Inglis, G., Bird, G . W. G.. Mitchell. A. A. B.. M h e . G. R.. and Wingham. J., Erythrocyte polyag-
glutination showing properties of both T and T k , probably induced by Bacteroides fragilis infection,
VoxSang.,28,314,1975.
134. Inglis, G..Bird. G. W. G.. Mitchel, A. A. B.. Milne, G.R.. and Wingham,J., Effect of Dacieroides
iragilison the human erythrocyte membrane; pathogenesis of Tk polyagglutination, J . Clm. Pathol.,
28,964, 1975.
135. Andreu, G., Mativet, S.. Doinel. C.. Cartron. J. P.. and Salmon. C., In virroTk-polyagglutination
- relations with acquired-B. I and i antigens. Abstr. X V Congr. Inr. SOC.Blood Transf., Librarie-
Arnette. Paris, 1978. 551.
136. Marsh, W.L., The pseudo-B antigen, a study of its development, Vox Sang., 5.387,1960.
137. Beck. M. L.. Walker, R. H.. and Oberman. H. A., Atypical polyagglutination associated with an
acquired-B antigen, Transfusion. I I , 2%. 1971.
138. Judd. W. J.. Mffiuire-Mallory, D.,Anderson. K.. Heath. E. J.. Swanson. J., Gray, J. M..and
Obcrman, H. A.. Concomitant T-and Tk-activation associated with acquired-B antigens, Transfu-
sion. 19. 293, 1979.
139. Moreau, R., Daussct. J., Bernard, J.. and Moullec. J.. A n h i e he’molytique acquise avec polyagglu-
tinabilite des hemaites par un noveau facteur prgsent dans le sgrum humain normal (anti-Tn). Bull.
SOC. Med. Hop. ParisSceance.May 17th. 1971.
140. Lalaari. P. and al-Mondhira. H.. Sialic acid deficiency of human red blood cells associated with
persistent red cells, leukocyte and platelet polyagglutinability, Br. 1. Haematol.. 25,399.408.
141. Cartron, J. P.. Andreu. G.,Cartron, J.. Bird, 0. W. G.. and Salmon, C., Demonstration of T-
transferase deficiency in Tn-polyagglutinable blood samples, Eur. J . Biochem., 92. I 1 1 , 1978.
142. Sturgeon. P.. McQuiston, D. T., faswell. H. F.. and Allan. C. J., Permanent mixed-field polyagglu-
tinability (PMFP). 1. Serological observations, VoxSang..25,481, 1973.
143. Myllylji. G.,F u r u h je e . U..Nordling. S., Pirkola, A., Tippat, P., Gavin. J.. and Sanger, R., Per-
sitent mixed field polyagglutinability. Electrokinetic and serological aspects, Vox Sang.,20,7. 1971.
144. Ness. P. M.,Garratty. G., Morel, P. A., and Perkins. H.A., Tn-polyagglutination preceding acute
leukemia, Blood. 54, 30, 1979.
145. Bird, G. W.G. and Wingham, J., The M, N, and N,, receptors of Tn erythrocytes, Vox Sang., 26,
171, 1974.
146. Bird, G. W. G.. Significant advances in lectins and polyagglutinable red cells, Plenary Sessions X V
Congr. Inr. SOC.Blood Transf., Paris, 1978, Librarie-Arnette, Paris. 1978.
147. Bud, G.W.G. and Wingham,J., Hemagglutinins from Safvia, VoxSang..25, 163. 1974.
148. Bird, G.W. G. and Wingham,1.. More Salivaagglutinins, VoxSang., 30.217, 1976.
149. Bird, G.W. G. and Wingham. J., Yet more Salviaagglutinins, VoxSang.,32. 121. 1977.
150. Beck. M. L.. Hicklin. B. L., Pierce. S. R., and Edwards. R. L.. Observations on leukocytes and
platelets in six cases of Tn-polyagglutination. Med. Lab. Sci.. 34.325, 1977.
151. Cazal. P.,Mods, M.,Caubel. J.. and Brives. J.. Polyagglutinabiliti;h&ditaire dominante: antig‘ene
privh(Cad) correspondent un anticorps public et-a une lectin Dolichos biflorus, Rev. Fr. Transfus.
lmrnuno-hematol., 11,209, 1968.
152. Sanger, R., Gavin. J., Tippctt. P., Teesdale. P., and Eldon. K.. Plant agglutinin for another blood
group factor, Lancet. 1. 1130, 1970.
153. Bird, G. W.G. and Wingham, J.. Some serological properties of the Cad receptor, Vox Sang., 20.
5 5 . 1974.
154. Crookston. J. H.,Crookston. M.C.. and Rosse. W.. Red cell abnormalities in HEMPAS (hereditary
erythroblastic multinuclearity with a positive acidified serum test), Br. J . Haematol..23.81, 1972.
155. Gockerman. J. P.. Duracbu, J. R.. and Conrad. M. E.. The abnormal surface characteristics of the
red cell membrane in congenital dyserythropoetic anemia type I1 (HEMPAS), Br. J. Hacniatol., 30,
383. 1975.
156. Bird, G. W. G.and Wingham. J., The action of seed and other reagents on HEMPAS erythrocytes,
Acta Haematol..55, 174,1976.
157. Graninger. W., Ramcis. H..Fisher, K., Poschmann. A., Bird, G. W. G.. Wingham, J., and Neu-
mann, E., ‘VA’, a new type of polyagglutination characterized by depressed I4 receptors aad associ-
ated with hemolytic anemia. 1. Serological and hematological observations, Vox Sang., 32. 195. 1977.
158. Graninger, W., Poschmann. A.. Fiscber. K.,Schedl-Giovannoni, I., Horander, H..and Klaushofer,
K., ‘VA’. a new type of polyagglutination characterized by depressed H receptors and associated
with hemolytic anemia. 11. Observations by immunofluorescence, electron microscope and biochem-
istry. VoxSang.,32.201. 1977.
159. k k . M. L., Myers. M. A., Moulds, J.. Pierce, S. R., Hardman. J.. Wingham, J., and Bird. G. W.
G . , Coexistent Tk and VA polyagglutinability. Transfusion, 18,680,1978.
August 1980 213
160. Naiki. M. and Marcus, D. M., An immunochemical study of the human blood P I , P and P’ glyco-
sphingolipid antigens, Biochemistry, 14,4837,1975.
161. Springer, G. F. and Desai. P. R.. Human blood group MN and precursor specificities: structural
and biological aspects, Carbohydr. Res., 40, 183,1975.
162. Uhlcnbruck, G., Pardoe, G. I.. and Bird, G. W. G., On the‘specificity of lectins with a broad hem-
agglutination spectrum. 11. Studies on the nature of the T antigen and the specific receptors of Arachis
hypogaea(ground nut), Z.Immunitaersforsch. Allerg. Klin. Immunol., 138.423, 1969.
163. Lotan, R.. Skutelsky, E.. Danon. D.. and Sharon, N.. The purification, composition, and specificity
of the anti-T lectin from the peanut (Arachis hypogaea), 1. Biol. Chem.,250,8518,1975.
164. Dahr. W., Uhlmbruck. 0.. Gunson. H.H.,and van der Hart,M.. Molecular basis of Tn polyagglu-
tinability, VoxSang.. 29,36, 1975.
165. Mullard, G. W., Haworth, C., and Lee. D.,A case of atypical polyagglutinability due to Tk-trans-
formation, Br. J. Haematol.,40,571,1978.
166. Zopf, D. A.. Tsai, C. M.. and Ginsburg, V.. Studies on the carbohydrate receptors of cold agglutinins
using synthetic antigens, in Human Blood Groups. Mohn, J. G.. Plunkett, R. W., Cunningham, R.
‘
K., and Lambert, R. M..Eds.. S. Karger, Basel, 1977,172.
167. Mackvie. S. E.. Morton, J., and Pickles, M., The reactions and inheritance of a new blood group
antigen, Sd’, Vox Sang., 13,485,1967.
168. Morton. J. A. and Tmy, A. M.. The Sd’ blood group antigen. Biochemical properties of urinary
Sd‘, VoxSang., 19, 151, 1970.
169. Boyd, W. C., Green. D. M., Fujinaga. D. M.. Drabik. J. S., and Waszczenko-Zacharczenko,E., A
blood factor, possibly new, detected by extracts of Arachis hypogaca. Vox Sang., 6,456,1959.
170. Schwarzfiiher. F. and Liebrich. K., Populationgenetische Unterschungen uber das Blutkorperchen-
merkmal Gy, Blur, 8, 161. 1962.
171. Prokop. 0.and Uhlenbruck, G., Human Bfood and Serum Groups, McClaren and Sons, London,
1969.
172. Jenkins, 0.C., Mollison, P. L.. and Race, R. R., Anti-D in Begonia puncrara, Lancet, 1, 1347,
1961.
173. Ikin, E. W.,Mourant. A. E.. and Pugh, V. W., An anti-Rh serum reacting differently with A and
0 red cells, Vox Sang., 3(0.S.), 74, 1953.
174. Issitt, P. D.. Serofogyand Generics of Rh, Montgomery Scientific Publications, Cincinnati, 1979.
175. Lockyer, W. J. and Gold, E. R.. ‘Natural’ anti-A and anti-B with small combining sites, together
with anti?, in t h e serum of the South African snake, Pytbon sebae, Med. Lab. Sci., 34. 303, 1977.
176. Plapp. F. V.. Kowalski. M. A., Tilzer, L., Brown, P. J.. Evans, J.. and Cluger. M.. Partial purifi-
cation of Rh. (D) antigen from Rh positive and negative erythrocytes, Proc. Narl. Acad. Sci., 76,
2964, 1979.
177. Schnitzler. S.. Muller. G., and Prokop, 0.. Ein “neur” Antikorper: anti-P.., aufgefunden im Rogen
von Rutilus rutilus, 2. Immun-Forsch. Allerg. Klin.Immunol., 134.45, 1968.
178. Anstce, D.J., Holt, P. D. J., and Pardoe. 0. I., Agglutinins from fish ova defining blood groups B
and P. Vox Sang., 25,347,1973.
179. Voak, D. J., Anstee. D. J., and Pardoe, D. J.. The u-galactose specificity of anti-PL, Vox Sang.,
25,253, 1975.
180. Schwarting. G. A., Marcus, D. M., and Metaxas, M., Identification of sialoparagloboside as the
erythrocyte receptor for an ‘anti-p’ antibody. Vox Sang.,32,257,1977.
181. Chien..S. M.. Lrmanski, T.. and Poretz. R. D., The anti-I activity of the Sophora japonica lectin,
Immunochemistry. 11.501, 1974.
182. Rogers, D. J., Lectin type agglutinins, with anti-BI and anti-HI activity, from the ova of the black
sea-bream Spondliosoma cantharus, Med. Lab. Sci., 35,239.1978.
183. Bird. 0. W. G. and Wingham. J., Anti-Cad lectin from the seeds of Leonurus cardiaca, Clin. Lab.
Haematol., 1,57,1979.
184. Beck, M. L.. personal communication, 1979.
185. Murphy, L.A.. personal communication, 1978.
186. Moulds. J. J. and Judd, W. J., unpublished observations, 1977.
187. Judd, W. J.. unpublished observations, 1978.
168. Beck, M.L., personal communication, 1976.
189. Judd, W. J., unpublished observations, 1972.
190. Beck. M. L.. cited in Issitt, P. D.. Moulds, J.. and Beman, H.,Transfusion, 12,217,1972.
191. Ropan. C.. personal communication, 1978.
192. Harris,P.. personal communication, 1979.
193. Murphy, L. A., personal communication. 1977.
194. Anst-. D. J.. personal communication. 1977.
195. Moulds. J. J., personal communication, 1979.
214
197. Judd, W. J., unpublished observations, 1979.

196. Bird. G. W. G . , personal communication, 1978.
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The Role of Lectins: Blood Group Serology

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August 1980 171

THE ROLE OF LECTINS I N BLOOD GROUP SEROLOGY

Author: W. John Judd

Referee: Peter D. lssirt

Terminology and Definitions

Biological and Physical Properties of Lectins

Function of Lectins in Nature

THE ABH-REACTIVE LECTINS

Blood Typing Reagents

Anti-A, (anti-A) Dolichos binorus (seeds) Horse gram

Saxidomus giganteus (whole body) Butter clam

Bandeiraea simplicifolia (fresh seeds)

Data compiled. in part, from References 3, 15, and 16.

specificity Source Ref.

Anti-A, (anti-A) Dolichos biflorus 9

Modified with privine hydrochloride@.

vine albumin as a diluent. In addition, since highly purified proteins

FIGURE 1. Diagramatic representation of the five BS I isolectins. A = A antigen binding site; B = B

Total Strongly Weakly

Sample from patient with elevated cryoglobulins.

A, 2+mP 0 3+mf 3+ Anti-B A+H

B, 0 l+mf 2+mf 3+ Anti-A B + I-!

Data compiled from References 15, 40. and 41.

u-D-GalNAc A1 Dolichos biflorus

Note: 0-D-GalNAc = u-Nacetyl-D-Gal = u-D-galactose; L-Rha = L-rhamnose;

as routine blood typing reagents. Rogers, Topliss, and B l ~ n d e n 'and

Studies on Cord and H-Deficient Bloods

LECTINS THAT BIND TO THE MN AND Ss SIALOGLYCOPROTEINS

Blood Typing Reagents

specificity source Ref.

Anti-M Iberis amara 65

Specificity of the MN-Reactive Lectins

differences in the specificity of V. graminea and B. variegata anti-N lectins. In partic-

Studies on MN Antigen Structure

2. There is no detectable difference in the amount of NANA present in M (N-) and

injecting desialyzed M and N substances into rabbits. As expected, these antibod-

Studies on Sialoglycoprotein Deficient Bloods

trophoresis (SDS-PAGE), as shown in Table 7. The PAS staining bands revealed by

The S-s- Phenotypes and the “”’Antigen

PAS staining band P U 0 2 ud d d,

Gene production production Associated SGP aberrations

s' Normal None/partial None/abnormal ud

M.W. S'/S' Normal Altered/absent Absent

Allen, Corocan, and EllisP6found MU-red cells (genotype MS”/MSS to be nonreac-

The En(a-) Phenotypes

En(a +), Wr(a-b-) Red Cells

Other MN-System Variants

view. The interested reader is referred to Reference 89 for further information.

absence of sterically hindering components would account for the agglutination of

THE ROLE OF LECTINS IN THE STUDY OF RED CELL

the condition has been referred to as the Thomsen-Hiibener-Freidenreich phenome-

Characteristics and Classification

T Red' Desb Nor',' i W + 0 0 i 0

Acq uired-E Polyagglutination

~ inference of this find-

This class of polyagglutinable red cells is most likely heterogenous, as disciissed by

Titer vs. Titer vs.

Modified from Judd, W. J., McGuire-Mallory, D., Anderson,

K., Heath, E. J., Swanson, J.. Gray, J. M., and Oberman. H.

May be the same variety.

Data compiled from References 147, 148. and 149.

Studies on the Nature of the Polyagglutinable Receptors