Vol. 26, No. 1, 2010

Transcription

Immunohematology
Journal of Blood Group Serology and Education
Volume 26, Number 1, 2010
Immunohematology
Volume 26, Number 1, 2010
Contents
1
Letter to the Readers
G.M. Meny
2
Review
The Knops blood group system: a review
J.M. Moulds
8
Case Report
Role for serial prenatal anti-Vel quantitative serologic monitoring
with 2-ME serum treatment
W.J. Linz, J.T. Fueger, S. Allen, and S.T. Johnson
11
Review
Granulocyte serology: current concepts and clinical significance
M.E. Clay, R.M. Schuller, and G.J. Bachowski
22
Review
A review of the Colton blood group system
G.R. Halverson and T. Peyrard
27
Case Report
Alloimmunization to the D antigen by a patient with weak D type 21
H. McGann and R.E. Wenk
30
39
40
41
Review
A review of the Chido/Rodgers blood group
R. Mougey
In Memoriam
Marie Cutbush Crookston (1920–2009)
D. Mallory
In Memoriam
Eloise Giblett (1921–2009)
D. Mallory
Announcements
42
Advertisements
46
Instructions for Authors
Editors-in-Chief
Managing Editor
Sandra Nance, MS, MT(ASCP)SBB
Philadelphia, Pennsylvania
Cynthia Flickinger, MT(ASCP)SBB
Connie M. Westhoff, PhD, MT(ASCP)SBB
Technical Editors
Christine Lomas-Francis, MSc
New York City, New York
Senior Medical Editor
Geralyn M. Meny, MD
Dawn M. Rumsey, ART (CSMLT)
Glen Allen, Virginia
Associate Medical Editors
David Moolten, MD
Ralph R. Vassallo, MD
Editorial Board
Patricia Arndt, MT(ASCP)SBB
Pomona, California
Brenda J. Grossman, MD
St. Louis, Missouri
James P. AuBuchon, MD
Seattle, Washington
Christine Lomas-Francis, MSc
Martha R. Combs, MT(ASCP)SBB
Durham, North Carolina
Gary Moroff, PhD
Rockville, Maryland
Geoffrey Daniels, PhD
Bristol, United Kingdom
John J. Moulds, MT(ASCP)SBB
Shreveport, Louisiana
Anne F. Eder, MD
Washington, District of Columbia
Paul M. Ness, MD
Baltimore, Maryland
George Garratty, PhD, FRCPath
Pomona, California
Joyce Poole, FIBMS
Bristol, United Kingdom
Mark Popovsky, MD
Braintree, Massachusetts
Marion E. Reid, PhD, FIBMS
S. Gerald Sandler, MD
Washington, District of Columbia
Jill R. Storry, PhD
Lund, Sweden
David F. Stroncek, MD
Bethesda, Maryland
Emeritus Editorial Board
Delores Mallory, MT(ASCP)SBB
Supply, North Carolina
Production Assistant
Editorial Assistant
Patti A. Brenner
Copy Editor
Proofreader
Lucy Oppenheim
Mary L. Tod
Marge Manigly
Electronic Publisher
Wilson Tang
Immunohematology is published quarterly (March, June, September, and December) by the American Red Cross, National
Headquarters, Washington, DC 20006.
Immunohematology is indexed and included in Index Medicus and MEDLINE on the MEDLARS system. The contents are also cited in
the EBASE/Excerpta Medica and Elsevier BIOBASE/Current Awareness in Biological Sciences (CABS) databases.
The subscription price is $40.00 (U.S.) and $50.00 (foreign) per year.
Subscriptions, Change of Address, and Extra Copies:
Immunohematology, P.O. BOX 40325, Philadelphia, PA 19106
Or call (215) 451-4902
Web site: www.redcross.org/immunohematology
Copyright 2010 by The American National Red Cross
ISSN 0894-203X
Communication
Letter to the Readers
G.M. Meny
This issue introduces an exciting change to the cover of Immunohematology. Images depicting a visual interpretation of
a journal article or another timely aspect of blood banking will grace the cover of selected issues. I am pleased to announce
that David Moolten, MD, award-winning poet and associate medical editor of Immunohematology, will provide a description of the cover art from both an artistic and a scientific perspective. I hope these images remind us of the art as well as the
science of transfusion medicine. Your feedback is welcome.
Geralyn M. Meny, MD
Senior Medical Editor
Immunohematology
On Our Cover
El Acueducto de las Ferreras, an aqueduct also known as Puente del Diablo (Devil’s Bridge), spans a ravine
approximately 4 km north of the town to Tarragona in the Autonomous Community of Catalonia, Spain. The aqueduct
has a maximum height of 26 m and a length of 249 m. Built by the Roman Empire to supply water to the ancient city
of Tarraco, the aqueduct features a superimposed series of arches, and may represent the first aqueduct of its kind.
The Colton blood group antigen (discussed in this issue) is of course found on aquaporin-1, a widely expressed water
channel protein.
——David Moolten, MD
IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010
1
Review
The Knops blood group system: a review
J.M. Moulds
The Knops blood group system finds its roots in the
group of antibodies that were previously called high-titer,
low-avidity, or HTLA, antibodies. This term was used to describe a group of alloantibodies that appeared to have many
of the same characteristics, including very weak reactions
at the anti-human globulin (AHG) phase of testing. However, as their specificities were more clearly defined, each was
placed into a blood group system, and Knops became the
22nd system recognized by the ISBT Committee on Terminology for Red Cell Surface Antigens.1 Since that time, the
protein bearing the Knops antigens has been identified, the
gene has been cloned, and all of the known Knops antigens
have been characterized at the molecular level. This has allowed investigators to more precisely study the role of the
Knops blood group system in disease.
The Knops blood group system began to expand when
Molthan and Moulds6 described a new antigen, McCa, which
seemed to be related to Kna. They reported that 53 percent
of McC(a–) samples were also Kn(a–). Interestingly, a majority of McCa antibody producers were Black whereas most
of those making anti-Kna were Caucasian, thus suggesting
that ethnic differences might exist in their respective gene
frequencies.
The next pair of alleles, Sla and Vil, was reported in
separate abstracts with one author using the term McCc for
Sla and McCd for Vil.7,8 The Sl terminology came from the
names of the first two antibody producers, i.e., Swain and
Langley. It was preferred by these authors because they believed that this antigen was independent from McCoy. After
identification of the protein bearing the Knops antigens, Sla
was renamed S11 and Vil became S12.9
The last high-incidence antigen identified in the Knops
system was KAM,10 later renamed KCAM by the ISBT Committee on Terminology for Red Cell Surface Antigens.11 The
antibody producer was a Caucasian man who exhibited the
Helgeson phenotype, i.e., serologic Kn null. Like Sla, this
antigen showed a widely diverse frequency in Blacks vs.
Caucasians (Table 1). Although KCAM is a high-frequency
antigen in Caucasians, only 20 percent of African Blacks
were KCAM+.
History
In 1965, the sera from three unrelated patients (Copeland, Stirling, and Wainright) were reported as having an
antibody with similar specificity.2 The antibody was named
after the first two antibody producers, Co and St (Cost),
and given the designation Csa. Ten years later a new antigen, York (Yka) was reported.3 The York serum was initially
believed to be an example of anti-Csa until the RBCs of the
donor were found to be Cs(a+). This same study found that
12 of 1,246 Caucasian donors were both
Cs(a–) and Yk(a–), suggesting that these
antigens were at least phenotypically related. Neither, however, was given blood Table 1. Frequency of the Knops antigens in several ethnic groups
Kn b
McC a
McC b
S11
S12
group status, and they became part of the Population Kn a
98%
4%
98%
1%
99%
<1%
HTLA group of antibodies. Even though Caucasians
Yka was thought to be associated with Csa
100%
NT
89–92% 49–54% 30–38% 95%
at this time, it would later be assigned to West
Africans
the Knops system.
99% <1%*
90%
44%
51–61% 80%*
Another similar antibody being inves- African
tigated in the 1960s was anti-Kna. Anti-Kna Americans
98%
2%
93%
42%
70%
86%
was described in a transfused Caucasian African
woman who had a saline-reactive anti-K Brazilians
100%
0%
100%
0%
100%
0%
plus an unidentified antiglobulin-reactive Asian
antibody to a high-frequency antigen.4 Brazilians
The antithetical Knb was later reported in *Marilyn Moulds, personal communication.
12
the Hall serum, which contained a potent Note: The publication by Covas et al. incorrectly lists 4870A
(isoleucine)
as
being
KCAM
negative.
anti-Kpb and was being used to screen
NT = not tested.
Australian blood donors.5
2
Yka
KCAM
Refs.
90%
98%
3, 5, 8,
12, 13
NT
20%
10, 14
98%
NT
8, 13,
14
NT
53%
12
NT
95%
12
Knops blood group system
Knops Protein
In 1991, two groups identified complement receptor one
(CR1) as the protein carrying the Kna, McCa, and Sla blood
group antigens.15,16 In addition, Moulds et al.15 also located
Yka to CR1 and suggested that the Helgeson phenotype was
attributable to low CR1 copy numbers on the erythrocytes
(E-CR1). Several years later, Petty et al.17 confirmed these
reports using monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) analyses and also
confirmed that Csa was not on CR1. Thus, Csa and Csb have
remained a collection as defined by ISBT.
CR1 is a membrane-bound glycoprotein and, with the
exception of platelets, is found on most human peripheral blood cells. Depending on the methods used, erythrocytes display approximately 300 to 800 CR1 molecules per
cell whereas leukocytes display approximately 10,000 to
30,000 molecules per cell. Because erythrocytes are present in the peripheral circulation at concentrations 103-fold
higher than the peripheral blood mononuclear cells (PBMCs), they account for greater than 85 percent of CR1 in
the blood. E-CR1 binds immune complexes (ICs) that are
shuttled to the liver or spleen for transfer to and ingestion
by macrophages, leading to their elimination. IC-free erythrocytes return to the circulation, where they may continue
participating in IC clearance. We have noted that individuals with high CR1 copy numbers may exhibit a weak falsepositive DAT in the gel technique, most likely as a result of
increased binding of ICs.
Knops Antigen Characteristics
The Knops antigens can vary greatly in strength (Table
2), and weakly reactive cells can be falsely phenotyped as
negative if the cells are stored for some duration or have
low E-CR1 numbers. Chemicals that can disrupt disulfide
bonds, i.e., DTT and AET, can destroy Knops, McCoy,
Swain-Langley, and York antigens because they destroy the
conformational structure of the short consensus repeats
(SCRs). There are no examples of these antibodies that are
enhanced by enzyme treatment of RBCs, and most are still
reactive (sometimes weaker) with either ficin- or papaintreated cells.18,19 However, all Knops system antibodies currently identified are nonreactive with trypsin-treated RBCs.
It is known that a trypsin cleavage site exists in SCR 28 of
the CR1 protein. Because the blood group antigens identified to date have been found in SCR 25, they are lost on
trypsin treatment of the cells. This fact can be a useful tool
not only in antibody identification but also for absorption to
remove other antibodies from a sample.
Although the variability in antigen strength can be inherited, it may also be acquired. Diseases that have high
levels of ICs being processed, e.g., systemic lupus erythematosus or HIV infection, can cause an increased loss of
E-CR1, resulting in weakened Knops antigen strength.20 In
addition, when RBCs are stored, vesicles are budded from
the membrane, and these vesicles contain CR1.21 Therefore,
the longer RBCs are stored, the weaker the Knops antigens
become. This may explain why In(Lu) RBCs were initially
reported to have weakened Knops antigens.22
Table 2. Characteristics of the Knops antigens
Inherited as Mendelian-dominant traits
High-frequency RBC antigens (except Knb and McCb)
Developed on cord blood cells but may be weaker
Weaker on stored RBCs
Not destroyed by ficin or papain
Destroyed by trypsin treatment
Not found on platelets
Found in low levels in plasma but not in urine or saliva
Knops Antibodies
The Knops antibody characteristics are shown in Table
3. Two characteristics that were often attributed to Knops
antibodies were (1) they were not neutralized with plasma,
saliva, or urine, and (2) they were difficult to adsorb and
elute. The latter most likely reflects the low density of the
CR1 protein on the RBC membrane. However, Race and
Sanger23 reported that adsorption performed with buffy
coats (WBCs) were able to remove anti-Kna from serum.
This led to the speculation that anti-Kna and related specificities were WBC antibodies. The ability of antigen-positive
WBCs to adsorb Knops antibodies most likely relates to the
fact that the CR1 copy number on WBCs is in the tens of
thousands compared with a few hundred on RBCs.
Table 3. Characteristics of Knops system antibodies
Titer is dependent on E-CR1 levels
Can demonstrate variable reactions
Not neutralized by pooled serum or other body fluids
Difficult to adsorb and elute from RBCs
IgG reacting by AHG technique
Do not bind complement
Usually not clinically significant
Although CR1 has not been found in the saliva, low
levels have been found in both urine24 and plasma.25 These
are believed to be the result of proteolytic cleavage of CR1
from leukocytes.26 Serum CR1 is present only in nanogram amounts,27 and therefore the levels are insufficient to
neutralize Knops antibodies using routine serologic techniques. Hence, Moulds and Rowe28 developed an inhibition
technique using recombinant, soluble CR1 (sCR1). Because
their source of sCR1 was genetically positive for Kna, McCa,
Sla, and Yka, it would only inhibit these antibodies and not
inhibit anti-Knb or McCb. It must be remembered that the
Knops phenotype of the sCR1 will be dependent on the gene
chosen for its production. More recently, these investigators
3
J.M. Moulds
have used mutated CR1 constructs to produce peptides capable of inhibiting anti-McCb and S12 (Vil).29
Clinical Importance
Because the Knops antibodies are found in the serum
of multiply transfused individuals, these sera often contain
additional alloantibodies directed at (for example) K, E,
and Duffy antigens. Most of the Knops antibodies are not
considered clinically significant because they do not cause
overt hemolytic transfusion reactions or hemolytic disease
of the fetus and newborn. Some examples of Knops system antibodies have been studied using in vitro tests such
as the monocyte monolayer assay (MMA). Arndt and Garratty30 studied three anti-Kna, three anti-Yka, and five antiKn/McC and found that only one had a macrophage index
of greater than 20, suggesting increased RBC destruction.
Other MMA studies of two anti-Kna, two anti-Sla, one antiKCAM, and one anti-Yk/Cs all gave indexes of less than
0, indicating no enhanced RBC destruction (J.J. Moulds,
unpublished data).
The third commonly recognized polymorphism is based
on quantitative differences in E-CR1. A HindIII RFLP is
detected by two allelic fragments of 7.4 or 6.9 kb on Southern blots. Homozygotes for the 7.4-kb fragment are high
CR1 expressors (H), and homozygotes for the 6.9-kb fragment are low expressors (L) of CR1.32 Alternatively, a PCRRFLP can be used that results in a band of 1.8 kb for H or
1.3 and 0.5 kb for L alleles.33 Although this RFLP correlates
with RBC expression in Caucasian and Chinese individuals,34 there is no relationship between this polymorphism
and CR1 expression in African Americans35 or West Africans.36 The exact molecular mechanism regulating CR1
RBC expression has been elusive but does not appear to be
part of the promoter or 3′ untranslated portions of the CR1
gene.37
Molecular Basis of Knops Antigens
Using CR1 deletion constructs, Moulds et al.29 first localized the McCoy and S11 (Sla) antigens to LHR-D of CR1.
By direct DNA sequencing they were then able to identify
two separate mutations in SCR 25 that correlated with these
two blood group antigens. The McCa/McCb polymorphism
is at base pair 4795, where an A encodes proline (McCa)
and a G encodes aspartic acid (McCb). The S11/S12 mutation is only 11 amino acids away; at base pair 4828, an A
encodes arginine and a G encodes glycine. Accordingly, the
ISBT has now assigned these antigens to the Knops system
with the numbers shown in Table 4.38
The CR1 Gene
The CR1 gene resides on chromosome 1 (1q32) and
comprises 39 exons spread out over approximately 133
kilobase (kb) pairs of DNA.31 These exons encode SCRs
of approximately 60 amino acids in the functional CR1
protein. Seven SCRs are organized into larger units called
long homologous repeats (LHRs). The most
common size protein product, CR1-1, is made
Table 4. Molecular basis of the Knops antigens
up of four LHRs (A, B, C, D), a transmembrane
ISBT
region (TM), and a cytoplasmic tail (CYT), as
number
Antigen
Nucleotide
Nucleotide†
shown in Figure 1. The binding sites for C3b
a
4708G
4681G
KN1
Kn
and C4b have been localized to SCRs 8-9 and
KN2
Knb
4708A
4681A
15-16 (LHRs B and C) and to SCRs 1-2 (LHR-A),
a
KN3
McC
4795A
4768A
respectively.
LHR-A LHR-B LHR-C LHR-D TM CYT
●●●●●●●●●●●●●●●●●●●●●●●●●●●● ╫□
SCR 1–7 SCR 8–14 SCR 15–21 SCR 22–27
Fig. 1. Schematic of the CR1-1 protein bearing the
Knops antigens in SCR 25.
Amino acid
Va11561
Met1561
Lys1590
KN4
S11
4828A
4801A
Arg1601
KN5
Yka
Unknown
Unknown
Unknown
KN6
McCb
4795G
4768G
Glu1590
KN7
S12
4828G
4801G
Gly1601
KN8
S13 (provisional)
4828A, 4855T*
4801A, 4828T*
Arg1601, Ser1610
KN9
KCAM
4870A
4843A
Ile1615
The CR1 gene also exhibits two other poly*Note: The publication by Moulds et al.39 incorrectly lists the 4855 mutation as
A>G. The correct substitution is T>A.
morphisms besides the Knops blood group. A
†Nucleotide number when nucleotides are counted beginning with the A of the
structural, or size, polymorphism results from
AUG start codon
four different genes encoding four different
molecular weight proteins: 190 kDa (CR1*3), 220 kDa
Two other mutations have been identified in SCR 25,
(CR1*1), 250 kDa (CR1*2), and 280 kDa (CR1*4). Owing to
one of which was found in a Caucasian and is related to
the looping structure imparted by multiple disulfide bonds,
S11. KMW was initially believed to have produced anti-Sla.
the molecular weight shifts downward by approximately 30
However, after gene sequencing it was found that she was
kDa when the proteins are separated on SDS-PAGE using
not only homozygous for S11 but was homozygous for annonreducing conditions. It is now known that the molecuother SNP at position 4855 that substituted threonine for
lar weight polymorphism is independent from the blood
serine at amino acid 1610. After extensive molecular studies, Moulds et al.39 proposed a model in which S13 was
group polymorphism.
4
a conformational epitope needing S11 (1601R) and S14
(1610S) to react. S14 and the antithetical antigen S15 are
proposed epitopes awaiting the identification of the appropriate antibodies before they can be officially recognized.
However, the related SNPs can be identified molecularly.
Limited population studies of Caucasians, Blacks, and
Asians predict that S14 may be a high-frequency antigen in
all populations and that S15 predominantly occurs in Caucasians.12,39
The final antigen assigned to the Knops system is KCAM
(initially reported as KAM). Interestingly, the SNP producing KCAM was already known but had not been associated
with a blood group specificity until a McCoy-like antibody
was found in a Caucasian blood donor. On DNA sequencing, it was discovered that he was homozygous for an SNP
at bp 4870 that substituted valine for isoleucine.10 Hence,
it was assigned the number KN9, and the antigen was renamed KCAM (KC for the city where the donor was found
and AM for the donor’s initials).
Disease Associations
In 1997, Rowe et al.40 identified CR1 as a ligand for
the rosetting of Plasmodium falciparum–infected RBCs
with uninfected cells. The ability of erythrocytes infected
with P. falciparum to form rosettes is a property shown by
only some parasite isolates, but is of importance because
it has been associated with severe malaria.41 These authors
showed that CR1 on uninfected erythrocytes was required
for the formation of rosettes by demonstrating that CR1deficient erythrocytes (Helgeson phenotype) had reduced
rosetting and soluble recombinant CR1 could inhibit rosetting. RBCs having the Sl:–1 phenotype showed reduced
binding to the parasite rosetting ligand PfEMP1.14 Thus, the
authors hypothesized that this polymorphism may have
been selected for in malaria-endemic regions by providing protection against severe malaria. The hypothesis was
then tested by studies of Africans in Mali and Kenya, where
it was found that the combined Knops haplotype Kn(a+)/
McC(a–)/Sl:–1 appeared to be protective.
CR1, as well as other complement receptors, has been
identified as a receptor facilitating cell entry for a variety
of pathogenic organisms. Pathogens using CR1 include
Leishmania major (monocyte-macrophage),42 Legionella pneumophila (monocyte-macrophage),43 Leishmania
panamensis,44 and Mycobacterium tuberculosis (monocyte-macrophage).45 In an AABB abstract, Noumsi et al.46
reported that individuals heterozygous for McCa and McCb
were less likely to be infected by M. tuberculosis (odds ratio, 0.42; 95% confidence interval, 0.22 to 0.81; P = 0.007).
These results suggested that McCb may have evolved among
African populations in the context of M. tuberculosis pressure, and conferred a survival advantage in its heterozygous
form.
Conclusions
The molecular identification of the Knops blood group
polymorphisms holds the promise for a better means of typing for these antigens, the only exception to this being the
yet unidentified Yka. Because of the inherited or acquired
changes in RBC expression of CR1, i.e., Knops antigens,
genotyping may become the method of choice for identifying these antigens, as it is for those of the Dombrock system. It is interesting to note that all of the known Knops
polymorphisms are in SCR 25 of the CR1 protein. However,
additional SNPs have been found in the CR1 gene, which
suggests that the identification of new Knops blood group
antigens is not yet at an end and will surely provide a challenge to serologists in the future.
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with the number of CR1 on erythrocytes. J Exp Med
1986;164:50–9.
33. Cornillet P, Philbert F, Kazatchkine MD, Cohen JHM.
Genomic determination of the CR1 (CD35) density
polymorphism on erythrocytes using polymerase chain
reaction amplification and HindIII restriction enzyme
digestion. J Immunol Methods 1991;136:193–7.
34. Moulds JM, Brai M, Cohen JHM, et al. Reference typing report for complement receptor 1 (CR1). Exp Clin
Immunogenet 1998;15:291–4.
35. Herrera AH, Xiang L, Martin SG, Lewis J, Wilson JG.
Analysis of complement receptor type 1 (CR1)
expression on erythrocytes and of CR1 allelic markers
in Caucasian and African American populations. Clin
Immunol Immunopathol 1998;87:176–83.
36. Rowe JA, Plowe CV, Diallo DA, et al. Erythrocyte complement receptor 1 expression level does not correlate
with HindIII restriction fragment length polymorphism
in ; implications for studies on malaria susceptibility.
Genes Immun 2002;8:497–500.
37. Cockburn IA, Rowe JA. Erythrocyte complement receptor 1 (CR1) expression level is not associated with polymorphisms in the promoter or 3′ untranslated regions
of the CR1 gene. Int J Immunogenet 2006;33:17–20.
38. Daniels GL, Anstee DJ, Cartron JP, et al. International Society of Blood Transfusion working party on
terminology for red cell surface antigens. Vox Sang
2001;80:193–6.
39. Moulds JM, Zimmerman PA, Doumbo OK, et al. Expansion of the Knops blood group system and subdivision of Sla. Transfusion 2002;42:251–6.
40. Rowe JA, Moulds JM, Newbold CI, Miller LH. P. falciparum rosetting mediated by a parasite-variant erythrocyte protein and complement-receptor 1. Nature
1997;388:292–5.
41. Rowe JA, Obeiro J, Newbold CI, Marsh K. Plasmodium
falciparum rosetting is associated with malaria severity
in Kenya. Infect Immun 1995;63:2323–6.
42. Da Silva RP, Hall BF, Joiner KA, Sacks DL. CR1, the
C3b receptor, mediates binding of infective Leishmania
major metacyclic promastigotes to human macrophages. J Immunol 1989;143:617–22.
43. Payne NR, Horwitz MA. Phagocytosis of legionella
pneumophila is mediated by human monocyte complement receptors. J Exp Med 1987;166:1377–89.
44. Robledo S, Wozencraft A, Valencia AZ, Saravia N. Human monocyte infection by Leishmania (viannia) panamensis. J Immunol 1994;152:1265–75.
45. Schlesinger LS, Bellinger-Kawahara CG, Payne NR,
Horwitz MA. Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement
receptors and complement component C3. J Immunol
1990;144:2771–80.
46. Noumsi GT, Tounkara A, Diallo D, Moulds JM. Knops
blood group polymorphism and protection from Mycobacterium tuberculosis (abstract). Transfusion
2006;46(Suppl):19A.
Joann M. Moulds, PhD, MT(ASCP)SBB, Director, Clinical
Immunogenetics, LifeShare Blood Centers, 8910 Linwood
Avenue, Shreveport, LA 71106.
Manuscripts
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7
Case Report
Role for serial prenatal anti-Vel quantitative
serologic monitoring with 2-ME serum
treatment during pregnancy: case report
W.J. Linz, J.T. Fueger, S. Allen, and S.T. Johnson
Anti-Vel is an uncommon antibody to a high-prevalence antigen.
Its clinical significance and management in the prenatal setting are
not well characterized. We present a case that demonstrates the
utility of serial prenatal anti-Vel quantitative serologic monitoring
with 2-ME serum treatment during pregnancy. The patient is a
23-year-old Hispanic woman with history of prior pregnancy and
prior transfusion who was discovered to have an antibody to the
high-prevalence Vel antigen in the first trimester (week 7) of her
second pregnancy. Interval measurements of the serologic antibody
titers were performed during the next 26 weeks. The untreated serum (IgM and IgG) titer increased from a baseline of 4 to 16 during
that interval, while the 2-ME (presumed IgG component) titer remained stable at 4. Responding to ultrasound findings suspicious
for fetal anemia, the child was delivered without complications at
34 weeks’ gestation. At birth, the DAT was negative and there was
no evidence of HDN. Placed in the context of other similar reports,
this case demonstrates the importance of separately reporting the
IgG fraction (after either DTT treatment or 2-ME treatment) from
the untreated (IgM and IgG) fraction and the importance of correlating the treated serum titer with potential clinical significance.
Immunohematology 2010;26:08–10.
Key Words: Vel antigen collection, anti-Vel, prenatal serologic
monitoring, 2-ME-treated serum, pregnancy
V
el is a high-prevalence antigen first described by
Sussman et al. in 1952 in association with a transfusion reaction.1 Although several additional cases
quickly confirmed these authors’ findings,2 our knowledge
of the Vel antigen (collection) remains incomplete. The
presence of the Vel-negative phenotype in the general population is estimated to be 1 in 4000 individuals.3 Vel alloimmunization can be mediated through IgG or IgM. Anti-Vel
is thought to be associated rarely with hemolytic disease of
the fetus and newborn (HDFN) because many examples of
anti-Vel are mostly IgM and Vel is weakly expressed on the
surface of fetal RBCs. Although the risk may be low, it is reasonable to monitor prenatal serologic titers serially during
pregnancy.4 The findings published in a recent case report
by van Gammeren et al.,5 of severe HDFN from anti-Vel,
support this approach. The van Gammeren case report describes discovery in a prenatal patient of an anti-Vel, which
at initial presentation was determined to be composed exclusively of IgM. At the end of the pregnancy, an anti-Vel
titer of 64 was demonstrated in untreated serum, whereas
the DTT-treated serum (presumed IgG content) titer rose
to 16. In their case, the infant exhibited severe jaundice and
8
reticulocytosis. As a corollary to the van Gammeren case report, we present a case of an anti-Vel for which the IgG titer
as determined by 2-ME treatment was stable through pregnancy despite a significant rise in the titer of the anti-Vel in
the untreated serum. At birth, the child showed no evidence
of HDFN. This case report supports the importance of serial
determination of the prenatal IgG titer to help assess risk
of HDFN.
Case Report
A 23-year-old Hispanic woman, with a history of prior
blood transfusion and one successful pregnancy, presented
initially early in the first trimester of the index pregnancy.
Her RBCs were typed as group O, D+. Routine serologic
testing demonstrated variable reactivity using gel technology. The autocontrol was negative. Further testing using tube
technology with PEG as enhancement also demonstrated
variable weak reactivity, again with no specificity. No reactivity was demonstrated when using tube technology with
LISS. A repeat serologic evaluation was performed 4 weeks
later to help clarify the nature of this reactivity. The subsequent serologic evaluation demonstrated reactivity with all
RBCs tested whether using gel or test tube technology with
LISS or PEG methods. The autocontrol was consistently
negative. As an antibody to a high-prevalence antigen was
suspected, the sample was referred to a large nationally recognized immunohematology reference laboratory. With use
of rare RBCs lacking various high-prevalence antigens, reactivity was determined to be that of anti-Vel. The patient’s
RBCs were shown to lack the Vel antigen. The initial quantitative serologic titer was 4 when tested against Vel+ RBCs
using both 2-ME-treated and untreated serum. The Marsh
score ranged from 14 with untreated serum to 4 with 2-MEtreated serum. The developing baby’s RBCs were presumed
to be Vel+ as the father’s RBCs were demonstrated to express the Vel antigen. Subsequent quantitative serologic
monitoring was performed in approximately 4- to 6-week
intervals for the next 20 weeks of pregnancy (Table 1).
At 33 2/7 weeks’ gestation, Doppler ultrasonography
showed an increase in middle cerebral artery velocity (>1.5
multiples of the median [MoM] for gestational age) suspicious for fetal anemia. This finding was a significant change
from previous Doppler ultrasound studies and corresponded with the change in quantitative serologic titer of the
Role for anti-Vel prenatal monitoring
untreated serum from 4 at presentation to 16 by week 26
(Table 1). A multidisciplinary risk-benefit analysis, including consideration of potential maternal and fetal need for
transfusion with an extremely limited blood supply (two
available units), the unpredictable timing issues encountered with induction, and the limited time-availability for
transfusion after thawing the available units, prompted
the decision to move toward delivery in a manner that optimized the availability of blood for the newborn, if it was
indeed anemic. The patient underwent cesarean delivery at
34 3/7 weeks’ gestational age, 2 days after the initial administration of Celestone to promote fetal pulmonary maturity.
A 3088-g male newborn with Apgar scores of 7 at 1 minute and 9 at 5 minutes was born without complication. The
child’s RBCs typed as group A, D+, and the antibody screen
was negative. Initial laboratory data were also remarkable
for a Hb level of 16.6 g/dL (normal range), reticulocyte count
of 3.5% (normal range), and negative DAT. Approximately
1 week after delivery, a mild rise in bilirubin was observed,
reaching a peak of 10 mg/dL. Given that the DAT and the
initial screen were negative, the mild hyperbilirubinemia
was thought not likely to have resulted from transplacental anti-Vel or a possible anti-A from a group O mother.
Moreover, although laboratory data are a bit limited, there
was no evidence of anemia or hemolysis. Importantly, the
mild hyperbilirubinemia quickly resolved, and the newborn
was released to home and is doing well approximately 10
months after birth.
Table 1. Quantitative serologic monitoring
Approximate gestational
age (wk)
Serologic
findings
Untreated
serum
titer
Untreated
serum
score
2-MEtreated
serum
titer
2-MEtreated
serum
score
7
Weak,
variable
reactivity
N/A
N/A
N/A
N/A
14
Anti-Vel
4
24
4
24
18
Anti-Vel
4
27
4
14
22
Anti-Vel
8
N/T
N/T
N/T
26
Anti-Vel
16
25
4
18
34
Anti-Vel
16
30
4
14
N/A = not applicable; N/T = not tested.
Methods
ABO and D testing and DAT were performed using
commercially available reagents according to manufacturers’ protocols using tube method (Immucor Gamma Inc.,
Norcross, GA). Antibody detection and identification were
performed using either gel technology according to the manufacturer’s protocol using reagent cells (RBCs 0.8% Surgiscreen and reagent RBCs 0.8% Resolve Antigram, ID-MTS
Gel Test; Ortho-Clinical Diagnostics, Raritan, NJ) or tube
method with commercially available cells and reagents
(PEG and LISS, Immucor Gamma Inc.). Titration studies
performed at the immunohematology reference laboratory
used tube method. The patient’s serum was diluted in 6%
albumin. A 30-minute 37°C incubation was performed, followed by a saline IAT (Anti-IgG, ImmucorGamma Inc.).6
Reaction scoring was performed using the system originally
described by Marsh.7 The patient’s serum was treated with
2-ME (Fisher Scientific Co., Hanover Park, IL) and dialyzed
overnight using dialysis membranes (Spectra/Por2, 12,000
to 14,000 dalton molecular weight cut-off [MWCO], Spectrum Laboratories, Inc., Rancho Domingtuez, CA) in PBS,
pH 7.3. Dialyzed serum was tested using the methods previously described.
Discussion
This case adds to the limited published information
regarding the natural history of anti-Vel in the prenatal
setting, and it supports the value of sorting immunoglobulin classes as an aid to proper prenatal management. It is
worth noting that at initial presentation, weak, variable serologic reactivity was demonstrated. Despite use of several
standard methods, specificity could not be assigned to the
serologic reactivity. In the initial serologic evaluation, reactivity was present using gel technology and tube technology
with PEG as enhancement. Tube testing with LISS showed
no reactivity. Although a LISS tube method is considered
an appropriate antibody detection method in the prenatal
setting,8 this method would not have detected the antibody.
A repeat study approximately 7 weeks after the initial study
did demonstrate the presence of strong serologic reactivity
using tube testing with LISS and PEG as well as with gel.
The titer of the untreated serum and 2-ME–treated serum
was 4. Presumably, late in the first trimester of pregnancy
the mother was exposed to fetal RBCs expressing the Vel
antigen because by week 14 she demonstrated a specific serologic immune response. Interestingly, for the remainder
of the pregnancy, the titer of the anti-Vel in the 2-ME (presumed IgG component)–treated serum remained stable although the titer in the untreated serum (presumed IgM and
IgG) increased by two dilutions. This observation may be
useful to laboratories without the capacity to treat patient
serum with 2-ME or DTT. Namely, in this case anti-Vel titer
in the untreated serum appeared to rise before the anti-Vel
IgG titer in the 2-ME–treated serum.
The rise in antibody titer in the untreated serum combined with the abnormal Doppler velocimetry prompted
the preterm delivery. At birth, the DAT and antibody screen
were negative, and the hemoglobin and reticulocyte count
were in the normal range. As the anti-Vel present in the maternal serum contained an IgG component, the negative DAT
suggests that Vel antigen on the fetal RBCs may either be
weakly or partially expressed9 or the antibody avidity to the
antigen was poor. Regardless, in this case, an anti-Vel titer of
4 does not appear to be independently sufficient for HDFN,
whereas a titer of 16 in the case reported by van Gammeren
et al.5 was associated with HDFN. The cause of the rise in
9
W.J. Linz et al.
the bilirubin level after birth remains uncertain, although
liver immaturity is the most likely explanation. Finally, the
abnormal Doppler velocimetry in the absence of true fetal
anemia reflects the inherent limitations of a screening test
that is known to have a 12 percent rate of false-positive results.10
The Vel antigen (collection) and anti-Vel remain enigmatic and are in need of further study. To date, it is known
that the Vel-negative phenotype is universally uncommon.
The incidence of the Vel-negative phenotype in the Hispanic
population is not known with certainty. Anti-Vel is famously associated with in vitro hemolysis, severe transfusion reactions, and shortened RBC survival,11 although there is at
least one case report of Vel+ blood being successfully transfused to a recipient possessing anti-Vel.12 An auto-anti-Vel13
has been described, although it is uncertain to what degree
the autoantibody contributed to the patient’s RBC destruction. Anti-Vel can be difficult to detect, and it can simulate
the serologic profile of a cold-reactive autoantibody, with
disastrous consequences if improper analytic methods are
used.14 The Vel antigen appears to have a variable antigenic
expression and appears to be best expressed on adult cells.
When viewed in concert with the recent case report by
van Gammeren et al.,5 our case report further supports the
need not only to properly identify anti-Vel in the prenatal
setting but also to perform serial quantitative serologic
monitoring using methods to clearly separate the IgG fraction (DTT treatment or 2-ME treatment) from the untreated (IgM and IgG) fraction. Although both the untreated
and treated serum titer and score results may be reported,
changes in the treated serum fraction, representing the IgG
component, may be a better trigger to initiate additional
clinical investigation. Additional cases may help identify
the most appropriate critical 2-ME or DTT titer for antiVel.
References
1. Sussman LN, Miller EB. New blood factor: Vel. Rev Hematol 1952;7:368–71.
2. Levine P, Robinson EA, Herrington LB, Sussman LN.
Second example of the antibody for the high-incidence
blood factor Vel. Am J Clin Pathol 1955;25:751–4.
3. Issitt PD, Oyen R, Reihart JK, Adebahr ME, Allen
FH, Kuhns WJ. Anti-Vel 2, a new antibody showing heterogeneity of Vel system antibodies. Vox Sang
1968;15:125–32.
4. Tarsa M, Kelly TF. Managing a pregnancy with antibodies: a clinician’s perspective. Immunohematology
2008;24:52–7.
5. van Gammeren AJ, Overbeeke MA, Idema RN, van
Beek RH, Ten Kate-Booij MJ, Ermens AA. Haemolytic
disease of the newborn because of rare anti-Vel. Transfus Med 2008;18:197–8.
6. Roback JD. Technical manual. 16th ed. Bethesda, MD:
AABB 2008: 900.
10
7. Marsh WL. Scoring of hemagglutination reactions.
8. Judd WJ; Scientific Section Coordinating Committee of the AABB. Practice guidelines for prenatal and
perinatal immunohematology, revisited. Transfusion
2001;41:1445–52.
9. Reid ME, Lomas-Francis C. The blood group antigen
factsbook. 2nd ed. San Diego, CA: Academia Press,
2004:503-4.
10. Giancarlo M. Noninvasive diagnosis by Doppler ultrasonography of fetal anemia due to maternal red cell alloimmunization. N Engl J Med 2000;42:9–14.
11. Tilley CA, Crookston MC, Haddad SA, Shumak KN. Red
blood cell survival studies in patients with anti-Cha,
anti-Yka, anti-Ge and anti-Vel. Transfusion 1977;17:169–
72.
12. Issitt PD, Anstee DJ. Applied blood group serology. 4th
ed. Durham, NC: Montgomery Scientific Publications,
1998: 802.
13. Becton DL, Kinney TR. An infant girl with severe autoimmune hemolytic anemia: apparent anti-Vel specificity. Vox Sang 1986;51:108–11.
14. Storry JR, Mallory D. Misidentification of anti-Vel due
to inappropriate use of prewarming and adsorption
techniques. Immunohematology 1994;10:83–6.
Walter J. Linz, MD, MBA, Medical Director, Scott and
White Transfusion Service and Donor Center, Assistant
Professor of Pathology, Texas A&M UHSC, 2401 South 31st
Street, Temple, TX 76508; Judith T. Fueger, MT(ASCP)SBB,
Immunohematology Reference Laboratory, Diagnostic
Laboratories, BloodCenter of Wisconsin, Milwaukee, WI;
Steven Allen, MD, Associate Professor, Texas A&M UHSC/
Scott and White Memorial Hospital, Chair, Department
of Ob/Gyn, Director, Division of Obstetrics and Gynecology, Scott & White Healthcare and Texas A&M University
Health Science Center College of Medicine, Temple, TX;
and Susan T. Johnson, MSTM, MT(ASCP)SBB, Immunohematology Reference Laboratory, Diagnostic Laboratories, BloodCenter of Wisconsin, Milwaukee, WI.
Notice to Readers
Immunohematology, Journal of Blood Group
Serology and Education, is printed on acid-free
paper.
Review
Granulocyte serology: current concepts and
clinical signifcance
M.E. Clay, R.M. Schuller, and G.J. Bachowski
Applying serologic procedures to the detection of RBC and lymphocyte antigens has facilitated the identification of granulocyte antigens with established clinical significance, which are
now classified in the human neutrophil antigen system. Granulocyte alloantibodies and autoantibodies have been implicated
in a variety of clinical conditions including alloimmune neutropenia, autoimmune neutropenia, febrile and severe pulmonary
transfusion reactions, drug-induced neutropenia, refractoriness to granulocyte transfusions, and immune neutropenia
after hematopoietic stem cell transplantation. Although the
intrinsically fragile nature of granulocytes contributes to the inherent challenges of granulocyte serology, several advances in
laboratory procedures have improved detection of granulocyte
antibodies. This review will provide a current perspective about
the importance and use of granulocyte serology for detection
of granulocyte antibodies that have significant medical effects.
Key Words: granulocyte, neutrophil, antigens, antibodies, HNA,
alloimmune neonatal neutropenia, autoimmune neutropenia,
transfusion-related acute lung injury, TRALI
I
t has now been half a century since Lalezari1 described
the first case of neonatal neutropenia attributable to
transplacental transfer of granulocyte-specific antibodies, thus initiating the field of granulocyte serology. This
event fostered several decades of work directed at the identification of granulocyte-specific antigens (and antibodies),
methods for their detection, and an appreciation for their
clinical significance. In many ways, granulocyte serology
developed along the laboratory footprints of RBC and human lymphocyte antigen (HLA) serology with one major
exception—granulocytes could not be preserved for testing
purposes. The requirement for fresh cells was and continues to be a major impediment for wide-scale implementation of granulocyte immunobiology studies. Although the
number of laboratories worldwide that specialize in this
technology is limited, the clinical significance of granulocyte antibodies has not diminished and is now supporting
the development of new methodologies that have the potential to enhance the utility of testing and growth of this field.
Therefore, as we move ahead, it is important to (1) focus
on the clinical events that support the need for granulocyte
serology, (2) understand the benefits and limitations of the
current serologic assays, and (3) evaluate the application of
new technologies for the detection of granulocyte antigens
and antibodies.
Clinical Significance
Alloimmune Neonatal Neutropenia
Neutropenia observed in the neonate is primarily the
result of neutrophil-specific alloantibodies transplacentally
transferred to the fetus from the maternal circulation. The
presence of these human neutrophil antigen (HNA) antibodies most likely is the result of prenatal maternal sensitization by fetal neutrophils crossing the placental barrier,
although the antibodies may have also arisen in association
with autoimmune diseases such as systemic lupus
erythematosus or rheumatoid arthritis. 2,3 Maternal alloimmunization can occur anytime after the first
trimester of pregnancy. Once present in the fetal circulation, maternal HNA antibodies will bind to the mature fetal
cells expressing the corresponding neutrophil alloantigens,
as the neutrophil precursor cells are spared.4 Neonates affected by alloimmune neonatal neutropenia (ANN) are almost always neutropenic at birth, although cases have been
reported in which the neutropenia has been delayed by 1 to
3 days.4 ANN is often asymptomatic and goes undiscovered
unless the deficit of the neutrophils is profound enough
(absolute neutrophil count < 1.5 × 109/L) that the newborn
becomes susceptible and succumbs to infection caused by
bacterial or fungal pathogens. These children commonly
present with fever, malaise, lethargy, skin infections such
as cellulitis and omphalitis, mucosal and respiratory infections (stomatitis, otitis media, and pneumonia), urinary
infections, and very rarely septicemia. These infections are
usually mild, but fatalities have been reported in 5 percent
of cases.5 As there are numerous reasons for neonatal neutropenia (i.e., congenital, antibody-mediated destruction,
or infection that decreases hemopoietic cell production),
detection of these maternal neutrophil antibodies is very
important for determining the mechanism causing neutropenia. This allows the physician to focus on the appropriate
treatment. The presence of maternal antibodies can persist
up to 6 months after birth although antibodies and clinical effects usually dissipate more quickly. The incidence of
ANN has been estimated to be 0.1 to 0.2 percent.6,7 Currently, neutrophil antibodies with the following specificities
have been implicated in cases of ANN: HNA-1a,8–10 HNA1b,11 HNA-1c,12 FcγRIIIb (CD16),13,14 HNA-2a,15 HNA-3a,16
and HNA-4a.17
11
M.E. Clay et al.
Autoimmune Neutropenia
Autoimmune neutropenia (AIN) occurs in both infants and adults. It is the most common form of chronic
neutropenia in infants. Severe neutropenia is usually recognized 4 to 7 months after birth.4 In the vast majority of AIN
cases infants exhibit relatively mild clinical issues such as
stomatitis, otitis, and respiratory infections. The presence
of clinical symptoms lessens as the child becomes older.
Neutropenia in infants is self-limited with complete resolution commonly observed within 7 to 24 months.18 However, this condition necessitates aggressive hygienic care of
infants to prevent infection and vigorous therapy that may
include severe antibiotic therapy along with treatment with
recombinant G-CSF and IVIG when infection occurs. Cell
destruction can occur in the peripheral circulation in addition to interference with myelopoiesis in the bone marrow.
Complete blood counts demonstrate absolute neutropenia,
although random fluctuations in the neutrophil count can
range from zero to near normal.
AIN is also observed in adults. Monocytosis with or
without lymphopenia may also be present.19 As in infants,
the bone marrow commonly demonstrates an absence of
mature cells with an increase in myeloid precursors. This
apparent imbalance of precursors to mature segmented
neutrophils is distinct in cause from the maturation arrest
that occurs in congenital defects of neutrophils or hematologic malignancy. Unlike RBC autoantibodies, antibodies in
AIN are often reported to have specificity that can include
HNA-1a, HNA-1b, and HNA-2a.18–20
Drug-Induced Neutropenia
Although neutrophil antibodies are believed to be involved in drug-induced neutropenia, neither the precise
mechanisms nor the particular antigens on the cell surface
have yet been clarified. Some cases of drug-induced granulocytopenias probably result from direct marrow toxicity,
although immune-mediated processes also occur. Quinine,
quinidine, ibuprofen, and psychotropic medications such
as clozapine are recognized as potential causes of neutropenia.21–23 Drug-dependent antibodies have been found to react with both granulocytes and granulocyte precursors. The
FcγRIIIb and CD177 neutrophil glycoproteins have been
reported to form neoantigens with drugs or their metabolites, which are recognized by drug-dependent neutrophil
antibodies.
Transfusion Reactions
Antibodies to neutrophil and HLA antigens can result
in a variety of transfusion reactions. The spectrum of severity can range from mild febrile reactions to death in the case
of severe transfusion-related acute lung injury (TRALI). Recently TRALI has been shown to be the most common cause
of transfusion-related fatalities in the United States.24
Febrile nonhemolytic transfusion reactions are common and can occur when blood products containing WBCs
are transfused into patients who have leukocyte antibodies.25
12
The occurrence of febrile reactions has been mitigated with
the use of leukocyte-reduced RBCs and platelet products.
As early as 1951 blood transfusions were implicated in
noncardiogenic lung edema.26 The term TRALI was conceived in 1985 when Popovsky and Moore27 investigated a
series of 36 patients with well-defined transfusion-related
acute lung injury. They detected leukocyte (HLA and HNA)
antibodies in 89 percent of the implicated donors. Seventytwo percent of these patients were treated with mechanical
ventilation, and 6 percent died. The leading theories for the
cause of TRALI involve the priming or activation of neutrophils with antigen-antibody interactions, inducing an
acute inflammatory response.28 Antibodies to HLA class I
and class II as well as HNAs have all been implicated in the
development of TRALI.28
Even though HLA class I alloantibodies are the most
frequently encountered WBC antibodies in implicated blood
donors, they may act as much weaker triggers of acute lung
injury when compared with HLA class II and HNA antibodies. In a study designed to investigate leukocyte antibody
specificities in severe TRALI reactions, Reil et al.29 reported
that even though 73 percent of leukocyte antibodies identified in their donor population were HLA class I, HLA class
II and HNA antibodies were associated with 81 percent of
the antibody-mediated TRALI cases.29 Specifically, in their
36 reported cases, 17 were elicited by blood products containing HLA class II antibodies, and HNA antibodies were
implicated in 10 cases. Of the 10 fatal outcomes, 6 were
linked to HNA-3a antibodies in the transfused blood products, and HLA class II antibodies were associated with 3 fatalities.
Although leukocyte agglutinating (aggregating) antibodies have been frequently associated with TRALI,30–34
the ability of HNA-3a antibodies to also prime and activate
neutrophils in the pulmonary vasculature is thought to be
central to severe and fatal TRALI.35 Studies have now shown
that in addition to HNA-3a antibodies, HNA-2a and HNA4a antibodies also have the ability to prime neutrophils in
vitro.36–38
Although the pathophysiology of TRALI appears complex and remains under investigation,39 substantial clinical
and scientific information suggests that HLA (especially
class II) and HNA antibodies do play a significant role in
this disorder.28 Efforts are now being focused on detecting
HLA and HNA antibodies in donors both to retrospectively
investigate the cause of acute lung injury in blood recipients
and to serve as a valuable strategy in preventing TRALI by
identifying donors who may be responsible for the transfusion reaction.
Granulocyte Antigens
Granulocyte-specific antigens are those with a tissue
distribution restricted to granulocytes (neutrophils, eosinophils, and basophils), whereas neutrophil-specific antigens
are only present on neutrophils. Because of the difficulty in
characterizing antigens on basophils and eosinophils, many
Granuloycte serology: concepts and significance
antigens described as neutrophil-specific have not been
tested to determine whether they are present only on neutrophils or on all granulocytes. Although granulocyte (neutrophil) antigen systems were initially identified through
studies of ANN and AIN, both terms, granulocyte and neutrophil, have been used to describe the alloantigens.
of detection. The HNA system currently includes seven antigens, which are restricted to five antigen groups. The key
features of the HNA system are summarized in Table 1. An
extensive amount of information is now known about these
antigens, and the reader is referred to Bux’s recent and
comprehensive review about the antigens that constitute
this system.58
HNA-1 is the best-characterized group, and it contains
three antigens: HNA-1a, HNA-1b, and HNA-1c. The HNA-1
antigens are expressed only on neutrophils, and antibodies
to these antigens are frequently implicated in cases of alloimmune or autoimmune neutropenia.18,58 The antigens are
epitopes that occur on the neutrophil low-affinity FcγRIIIb
receptor (CD16). This molecule binds the Fc region of IgG
antibodies complexed to other antigens or immunoglobulins. The gene frequencies of HNA-1a, HNA-1b, and HNA1c vary among different ethnic populations.58 Furthermore,
individuals lacking FcγRIIIb have been identified. These individuals do not express the HNA-1 antigens on their neutrophils, thus presenting as an HNA-1 null phenotype.54,62
The HNA-2 group has one characterized antigen—
HNA-2a—which is located on a 58- to 64-kDa glycoprotein
that is expressed only on neutrophils.63 Although HNA-2a is
a high-frequency antigen phenotype (i.e., greater than 90%
for most populations), it is unique in that it is expressed
on subpopulations of neutrophils among antigen-positive
people. The expression of HNA-2a is greater on neutrophils
from women than men, and two or three subpopulations
of expression are often detected: one population that lacks
HNA-2a expression and one or two populations that express
the antigen but with different intensities.64 The functional
Nomenclature
Lalezari and colleagues8 identified the first granulocyte antigen during their investigation of a neonate with
transient ANN and subsequently proposed the first nomenclature system for these antigens. Because their studies indicated that the antigen was neutrophil-specific, they
designated it the N system and the antigens were named in
chronologic order of discovery. The antigens were labeled
alphabetically and the alleles were described numerically.
The system eventually classified seven antigens: NA1,8
NA2,40 NB1,41 NB2,42,43 NC1,44 ND1,45 and NE1,46 which became the foundation for granulocyte serology.
During this same period and subsequently, several different granulocyte antigens were identified. Some were
specific for granulocytes (or neutrophils): HGA-3, GA,
GB, GC, Gr1, Gr247–49; some were shared with other cells
or tissues: HGA-1, 5a, 5b, 9, MART47,50–52; and some had
unclassified distribution: CN1, KEN, LAN, LEA, SL.53–56
All of these granulocyte antigens were detected with sera
from patients with autoantibodies or alloantibodies directed against granulocytes, using traditional blood group
serologic techniques such as agglutination, immunofluorescence, and cytotoxicity. Unfortunately, most of these
antigens have not been further classified owing to the lack
of sufficient biologic material for study.
Table 1. Human neutrophil antigens†
Although this led to a consolidation of
Antigen location/
individuals working in this field, advances
Antigen
Former
glycoprotein CD
Coding gene
Clinical
were made and efforts were directed at imgroups Antigens
name
number
alleles‡
significance
munobiochemical, genetic, and structural
HNA-1
HNA-1a
NA1
FcγRIIIb/CD16b
FCGR3B*01
ANN, AIN,
or functional studies for a few of the wellHNA-1b
NA2
FcγRIIIb/CD16b
FCGR3B*02
TRALI,§
classified granulocyte antigens. With the
HNA-1c
SH
FcγRIIIb/CD16b
FCGR3B*03
advent of this information came the need
HNA-2
HNA-2a
NB1
58–64 kDa/CD177
Unknown
ANN, AIN, TRALI,
febrile transfufor a standardized granulocyte antigen nosion reactions,
menclature system based on the molecules
drug-dependent
carrying the antigens and the genes encodneutropenia
ing each allele. In 1999, the Granulocyte
HNA-3
HNA-3a
5b
CTL2/Unknown
ANN, TRALI ,
Antigen Working Party of the International
HNA-3b
5a
CTL2/Unknown
febrile transfusion
Society of Blood Transfusion (ISBT) estabreactions
lished a new nomenclature system for the
HNA-4
HNA-4a
MART
CR3/CD11b
ITGAM*01
ANN
well-characterized granulocyte antigens,
(α M subunit)
(230G)
which was based on the glycoprotein locaHNA-5
HNA-5a
OND
LFA-1/CD11a
ITGAL*01
Unknown
tion of the antigens.57 In this system the
(α L subunit)
(2372G)
†Information from references 20, 58, 59, 60.
granulocyte antigens are called human neu‡Alleles of the coding genes are named according to the ISGN Guidelines for Hutrophil alloantigens to indicate they are exman Gene Nomenclature.61
pressed on neutrophils; however, this does
§Except HNA-1c.
AIN = autoimmune neutropenia; ANN = alloimmune neonatal neutropenia; CD =
not imply that they are neutrophil-specific.
clusters of differentiation; CR3 = complement receptor 3; CTL2 = choline transEach antigen group is assigned a number,
porter-like protein 2; FcγRIIIb = Fc gamma receptor IIIb; HNA = human neutrophil
and polymorphisms within the group are
antigen; LFA-1 = leukocyte function antigen-1; TRALI = transfusion-related acute
designated alphabetically in sequential order
lung injury.
13
M.E. Clay et al.
role of HNA-2a is not known, and antibodies to this antigen
have been associated with AIN,20 ANN,65 febrile transfusion
reactions,66 TRALI,67 and drug-dependent neutropenia.68
HNA-3a is expressed on granulocytes, lymphocytes,
platelets, endothelial cells, kidney, spleen, and placental
cells.50 It is a high-frequency antigen located on choline
transporter-like protein 2 (CTL2), and its function is not
known.59,60 Alloantibodies to HNA-3a have been associated
with occasional cases of febrile transfusion reactions69 and
one case of ANN.70 Although antibodies to HNA-3a are rare,
their major clinical significance has been their association
with serious and fatal TRALI events.32–34
The HNA-4 and HNA-5 antigens are located on subunits
of the β2 integrin family (CD11a and CD11b molecules). Integrins are a large family of adhesive receptors that are essential for cell-cell interactions and cell trafficking. HNA-4a
is an epitope on the α M (CD11b) chain of CD11b/CD18 (the
CR3 molecule), and HNA-5a is an epitope on the α L chain
(CD11a) of CD11a/CD18 (the leukocyte function antigen-1
[LFA-1] molecule).71 HNA-4a is expressed on granulocytes,
monocytes, and natural killer cells, whereas HNA-5a is expressed on all leukocytes.58 Alloantibodies to HNA-4a can
cause ANN,72 and the CD11b/CD18 complex has been reported to be the target of autoantibodies.18,73 To date, HNA5a antibodies have not been clinically associated with
neutropenia.
A general consensus exists that granulocytes do not express ABO antigens74 or HLA class II antigens.75 Although
the density of HLA class I antigens on granulocytes is fairly
low, it can vary among individuals.76 Therefore, these antigens must be taken into consideration when patient samples
are tested for granulocyte antibodies. HLA class I antigens
may be of particular interest during granulocyte transfusion
for neutropenic patients as these antigens, in granulocyte
concentrates, may be introduced in high amounts to recipients with preformed class I antibodies. Transfusion reactions including TRALI have been observed in this situation,
and the antigen-antibody interaction has been proposed to
limit effectiveness of the transfusion.77–79
Serologic Testing
The detection of HNA antibodies is labor intensive and
technically challenging. Unlike HLA testing, commercial
test kits are not readily available for the detection of granulocyte antibodies by solid-phase methodologies. Therefore,
intact viable granulocytes are required for granulocyte antibody screening. Granulocytes are intrinsically designed
to respond to physiologic priming signals, so they must be
handled very carefully to prevent activation. Once activated
it is impossible to interpret any serologic test results involving these cells, as false-positive test results abound. Granulocytes are also extremely labile and must be used within
24 hours of collection, necessitating ready access to panel
14
cell donors with the needed antigen phenotypes. Although
many have tried, attempts to develop short-term or longterm granulocyte preservation procedures have been unsuccessful, thus requiring that fresh suspensions of granulocytes be prepared daily for testing procedures. Finally, the
presence of HLA antibodies in test sera makes identifying
HNA antibodies difficult because granulocytes also express
HLA class I antigens on their cell surface.
Preparation of Pure Granulocyte Suspensions
The isolation of pure granulocyte suspensions from peripheral blood was greatly facilitated and simplified with
Bøyum’s introduction in 1968 of the Ficoll-Isopaque gradient technique.80 Double-density gradient centrifugation is
now the most widely used approach for the isolation and
purification of granulocytes for serologic testing. The procedure makes use of discontinuous Ficoll gradients, and numerous publications exist describing the methodology and
the recovery, purity, functional integrity, and activity of the
isolated granulocytes.81–84
Granulocyte Antibody Detection and Antigen Typing
Laboratory approaches for the detection of granulocyte
antibodies require procedures that are accurate, reproducible, and practical. Although numerous granulocyte serologic assays have been described, not all meet these criteria.
In addition to the assay used, the ability to detect antibodies
in test specimens or antigens on test cells may also depend
on target cell antigen density and the concentration and
immunoglobulin type of the antibody in the test reagent.
Therefore, the successful detection of granulocyte antibodies often requires that a combination of methods be used.
Although several methods for the detection of granulocyte-specific antibodies are used by the few laboratories that perform this testing, the ISBT Working Party on
Granulocyte Immunobiology, a worldwide consortium of
16 laboratories that participate in the annual International
Granulocyte Immunology Workshop (IGIW), have recommended that granulocyte antibodies should be investigated
using a minimum of two methods: the granulocyte immunofluorescence test (GIFT) and the granulocyte agglutination
test (GAT).85 Multiple workshops have shown the sensitivity of GIFT to far exceed that of GAT; however, agglutinating antibodies such as HNA-1c, HNA-3a, and HNA-4a are
much more readily detectable in GAT and in many cases it
is the only reliable method for detecting these specificities.
For example, the seventh IGIW included a test specimen
that contained an HNA-3a antibody that could only be detected by GAT and the granulocyte chemiluminescence test
(GCLT). Although GCLT has been useful in selective laboratory settings, it is not routinely used for clinical granulocyte
antibody detection and is not discussed.
Antibody Detection Methods
Granulocyte Agglutination Test
Lalezari86 developed the basis of this assay in the early
1960s. Since that time, micro techniques and the use of
pure granulocyte suspensions with optimized concentrations of EDTA have vastly improved the performance of
this test. Typically, a panel of three to five donors is selected
to include all known neutrophil antigens currently defined
by the ISBT consortium. Purified granulocytes are isolated
from EDTA-anticoagulated peripheral whole blood using
a double-density gradient. These granulocytes are then
washed in PBS before use. The test is biphasic and is based
on the intrinsic response of granulocytes to aggregate when
stimulated by antibodies reacting to corresponding cell
surface antigens. The resulting agglutination is the consequence of the granulocyte activation that occurs during the
sensitization phase, which induces the cells to form pseudopods and slowly migrate toward one another during the
aggregation phase, until membrane contact is established.
Typically, the serum or plasma from the specimen being
investigated is incubated with the granulocyte suspension
for 4.5 to 6 hours at 30ºC.87 The reactions are evaluated using an inverted-phase microscope and graded from negative to 4+ on the basis of the percentage of cells that are
agglutinated. Our laboratory has determined cutoff scores
for both adult and pediatric patients (<6 years old) on the
basis of the reaction grades established during microscopic
evaluation. Both IgG and IgM antibodies are detected by
this method.
Granulocyte Immunofluorescence Test
Developed by Verheugt et al.88 in the late 1970s, this
fluorescent “antiglobulin” technique is used for the detection of granulocyte alloantibodies and autoantibodies that
are circulating and cell bound. As with the GAT, an antibody screening panel of three to five donors is selected
to include all currently defined HNA antigens. A purified
granulocyte suspension is prepared and treated with 1%
paraformaldehyde for a short time to prevent nonspecific
immunoglobulin binding to the neutrophil Fc receptors and
to stabilize the cell membrane. Serum or plasma from the
patient is then incubated with an optimized concentration
of granulocytes for 30 minutes at 37ºC. After a wash step
that removes unbound antibodies, the granulocytes are
then incubated with F(ab′)2 fragments of a fluorescent conjugated anti-human antibody for approximately 30 minutes at room temperature in a dark environment. The assay’s performance is optimized with the use of a fluorescent
secondary probe that can detect both IgM and IgG antibodies (to ensure the detection of both primary and secondary
immune responses) and the use of F(ab′)2 Ig fragments to
prevent the probe from binding to the high concentrations
of Fc receptors on granulocyte surface membranes. The cells
undergo another wash cycle, are resuspended, and then are
analyzed. Detection of the immunofluorescence reactions
can be accomplished by using either a fluorescence-detecting microscope or a flow cytometer. Evaluation by a flow
cytometer has in most cases replaced the fluorescence microscope as more cells can be analyzed in a shorter time,
resulting in improved assay sensitivity and reproducibility,
and it does not require the expertise needed to evaluate the
characteristic staining pattern seen with specific granulocyte antibodies by microscopic analysis.
Monoclonal Antibody Immobilization of Granulocyte Antigens
The monoclonal antibody immobilization of granulocyte antigens assay (MAIGA) is based on the platelet version (MAIPA) developed by Kiefel et al. in 1987.89 MAIGA
relies on the capture of neutrophil-specific antigen-antibody
complexes by a murine monoclonal antibody onto a solidphase surface.90 The benefits of this test are twofold: first,
this is currently the most sensitive assay for the detection of
granulocyte antibodies, and second, the assay is designed to
detect only HNA antibodies even when HLA antibodies are
present in the test specimen. The disadvantage of this procedure is that it is very complex and requires highly skilled
staff to perform the detailed techniques.
Granulocytes are incubated with the test serum or plasma for 30 minutes at 37ºC. This granulocyte suspension is
then washed to remove any unbound immunoglobulins and
incubated with a murine monoclonal antibody to a specific
neutrophil glycoprotein for an additional 30 minutes. After another wash step, the granulocyte membranes are disrupted in a mild detergent and centrifuged. The resulting
lysate is then transferred to polystyrene microwells coated
with anti-mouse immunoglobulins for incubation. The
trimolecular neutrophil antigen–patient HNA antibody–
murine monoclonal antibody complex present in the lysate
is captured on the solid phase, whereas any HLA
antibody-antigen complexes (if present) are removed by
a subsequent wash step. Remaining complexes are then
detected by the addition of anti-human IgG conjugated to
horseradish peroxidase followed by a substrate (OPD dissolved in 30% H2O2), and the reaction is analyzed with a
spectrophotometer.
MAIGA can be used to detect antibodies specific
to HNA-1a, HNA-1b, and HNA-1c antigens located on
FcγRIIIb (CD16); HNA-2a present on gp 58- to 64-kDa
(CD177); HNA-4a on the complement component receptor
C3bi (CD11b); and HNA-5a on the LFA-1 receptor (CD11a).
HNA-4a and HNA-5a antibodies can also be detected by a
common CD18 monoclonal antibody.
Antigen Detection and Typing Methods
The same methodology that is used to detect antibodies
in serum and plasma can be used to characterize the antigen
phenotype of an individual’s granulocytes. Just as the short
lifespan of granulocytes is a limitation to obtaining panel
cells for antibody testing, it also restricts the ability to do
antigen typing because granulocyte specimens must be tested
15
M.E. Clay et al.
within 24 hours of collection to obtain reliable results. A
sufficient volume of whole blood must be collected to obtain
an adequate number of granulocytes for phenotyping. This
can be a dilemma in pediatric patients or patients who are
neutropenic. Because ample volume, freshness, and availability of granulocytes is critical, this also acts as a practical
barrier to doing donor-recipient crossmatch testing during
investigations of suspected granulocyte-antibody–mediated transfusion reactions such as TRALI. The highly characterized antisera used to phenotype granulocytes should be
free of HLA class I antibodies as their presence can result in
a false-positive HNA typing. At a minimum, the titer of the
HLA antibody should be weaker than the HNA antibody
as it can then be readily diluted to a nonreactive level. Another constraint to phenotyping granulocytes is the limited
availability of qualified antisera for the currently defined
HNA antigens.
Monoclonal antibodies specific to several HNA antigens are commercially available and have been used to type
granulocytes by GIFT using the flow cytometer. An advantage to phenotyping by this method is that it can be done
with whole blood instead of with isolated granulocytes.
Because phenotyping granulocytes cannot often be performed or can be unreliable if samples are not handled in a
careful and timely manner, genotyping by PCR, which has
less stringent sample age and handling restraints, can serve
either as an alternative to or as confirmation of serologic
results.
splicing defects, and no mutations have been detected in
the CD177 introns or exons in these individuals.93 Because
the gene encoding HNA-3a has only recently been identified, there has not been a genotyping procedure for this
antigen, but the clinical importance of this antigen is supporting efforts to get a molecular method developed.
Genomic DNA (gDNA) can be isolated and purified
from anticoagulated blood using any number of published
methods. A unique set of sense and antisense oligonucleotide primers that border the DNA fragment to be amplified
are added to a master mix of reagents that includes deoxynucleoside triphosphates (dNTPs), Taq polymerase, a buffer solution containing Mg2+, and the individual’s gDNA.
The master mix is subjected to a series of 30 to 40 temperature changes that define the amplification cycles. Each cycle
consists of three temperature steps that are critical in the
amplification of the DNA target strands. The first step in
the cycle typically involves heating the master mix to a temperature of 95ºC. This denaturation step results in doublestranded DNA separating into single strands by disrupting
the hydrogen bonds between each nucleotide base pair. The
annealing step follows in which the reaction temperature is
lowered to 50ºC to 65ºC. This allows the primers to hybridize to the opposing strands of the target DNA. The final extension step heats the master mix from 71ºC to 72ºC, which
results in DNA synthesis in the presence of Taq polymerase
and dNTPs complementary to the oligonucleotide primers.
A final extension step is added after all cycles have been
completed to ensure that any remaining single-stranded
DNA is fully elongated. The amplified double-stranded
DNA (amplicon) is separated by size using agarose gel
electrophoresis, stained with ethidium bromide, and visualized by fluorescence using ultraviolet light. The sizes of
the amplicons are determined by comparison with a DNA
ladder, which contains DNA fragments of known sizes that
were run alongside the PCR products during the agarose gel
electrophoresis step.
Granulocyte Genotyping
The discovery of the PCR in the 1980s facilitated the
development of molecular methods that have now been applied to delineate the genetics and allelic variations of the
HNAs. Initially, the characterization of the genes encoding
the HNA-1 antigen system (HNA-1a, HNA-1b, and HNA-1c)
allowed for the development of PCR assays using sequencespecific primers (SSP) to differentiate the alleles. To complicate matters there is a high degree of homology between
Future Directions
the FCGR3A gene that encodes the FcγRIIIa granulocyte
Solid-Phase Technology
receptor and the FCGR3B gene where the three different
The complement-dependent lymphocyte cytotoxicity
HNA-1 polymorphisms reside. HNA-1a, HNA-1b, and HNAassay was the first platform, developed nearly 50 years ago,
1c are encoded by FCGR3B*1, FCGR3B*2, and FCGR3B*3
for identifying HLA antibodies and remains the standard
genes, respectively. FCGR3B*1 differs from FCGR3B*2
for the development of new methodologies in this field.
by five nucleotide bases, and a single nucleotide polymorphism (SNP) differentiates FCGR3B*2 from
Table 2. Nucleotide differences for the three HNA-1 alleles (1a, 1b, and 1c) on
FCGR3B*3 (Table 2). FCGR3A differs from FcγRIIIb and among the FcγRIII genes†
FCGR3B at five different nucleotide locations
HNA
Gene
Polymorphic nucleotides
(Table 2).
141
147 227 266 277 349 473 505 559 641 733
Methods are also available to genotype
1a
FCGR3B*1
G
C
A
C
G
G
A
C
G
C
T
the HNA-4a and HNA-5a alleles.71 Both of
these polymorphisms are the result of SNPs.
1b
FCGR3B*2
C
T
G
C
A
A
A
C
G
C
T
Even though the molecular sequence of HNA1c
FCGR3B*3
C
T
G
A
A
A
A
C
G
C
T
2a has been identified, genotyping methods
FCGR3A
G
C
G
C
G
A
G
T
T
T
C
to detect the gene that encodes this alloanti†Information from references 91 and 92.
gen are not available. The HNA-2a negative
NOTE: Boldface indicates substituted nucleotides.
FcγRIIIb = Fc gamma receptor IIIb; HNA = human neutrophil antigen.
phenotype is attributable to CD177 mRNA
16
Multiple new technologies are now being used by HLA
laboratories, and in the past few years solid-phase assays
have been developed in which purified HLA molecules are
bound to a well in a microtiter plate or to a bead. HLA antibody is then detected either using an enzyme-linked immunosorbent assay (ELISA) technique or by analyzing the
beads with a flow cytometer, which uses a new technology
developed by Luminex (Austin, TX).94 A critical advantage
of this technology is its ability to measure multiple analytes
simultaneously in a single reaction system. The commercially available test kits use purified HLA antigens bound
to microbeads, which can be individually differentiated by
varying ratios of internal fluorescent dye prepared during
the manufacturing process. The test involves incubation of
test sera with the panel of microbeads, and the bound HLA
antibody is detected by a secondary R-phycoerythrin (PE)
conjugated IgG (or IgM)–specific antibody.
Analysis is done with the use of a Luminex flow analyzer in which the beads pass through two lasers, similarly to
classic flow cytometry. One laser identifies the specific bead
being analyzed, thereby identifying the unique HLA antigen. The other laser detects the presence of bound Ig on the
surface of the microbead. The color signals are then detected and processed into data for each reaction. A significant
fluorescence shift from the negative control range indicates
the presence of specific antibody in the test sample.
The widespread use of Luminex technology by HLA
laboratories has fostered commercial activity directed at
using this technology for the detection of HNA antibodies
that have been related to febrile and pulmonary transfusion
reactions. One Lambda, Inc. (Canoga Park, CA), has developed recombinant cell lines for HNA-1a, -1b, -1c, -2a, and
-4a, and these cell lines have allowed the production of purified proteins that have been individually immobilized on
Luminex microbeads for use in their LABScreen Multiflow
bead assay. Stroncek and colleagues95 evaluated the specificity and sensitivity of this new procedure using 22 sera that
had known HNA antibodies detected with the GA assay and
140 samples from nontransfused men. All HNA antibody
specificities (except 4a) recognized by GAT were detected
in the solid-phase flow bead system (8 HNA-1a, 6 HNA-1b,
1 HNA-1c, and 8 HNA-2a positive sera), and HNA-1c reactivity was detected in four specimens that were negative by
GAT. In addition, all of the 140 negative control samples
had negative reactions when tested by LABSreen Multi, and
the system was able to detect both HNA and HLA antibodies (both class I and class II) in some individual specimens.95
Although preliminary, these results suggest that this solidphase technology may emerge as a major achievement in
the development of a new sensitive and specific platform
for the detection of granulocyte antibodies in an automated
manner with limited complexity and high cost efficiency.
Cell Lines Expressing HNA Antigens
Another approach to eliminate the need for freshly
isolated granulocytes for granulocyte antibody screening
has been the establishment of cell lines that express HNA
antigens. In 1999 Bux’s group transfected Chinese hamster ovary (CHO) cells with HNA-1a, HNA-1b, and HNA1c cDNA and established stable cell lines expressing these
antigens.96 Although the cell lines were stable for 1 month
at 4°C, they demonstrated high background fluorescence in
the GIF assay and could only be used for the MAIGA, which
limited their overall utility. More recently, Yasui et al.97,98
transduced HNA genes into the human erythroleukemia
cell line K562 and established a panel of K562 cell lines that
stably expressed HNA-1a, -1b, -1c, -2a, -4a, -4b, -5a, and -5b
for at least 1 year while being maintained in culture. They
were not able to establish a cell line expressing HNA-3a because of the previous lack of biochemical and genetic information for this antigen. The group’s report demonstrated
the utility of using the cultured cells in the GIF assay to detect HNA antibodies in serum samples from patients with
HNA antibody–related disorders, but experience with this
new cell line technology is very limited and it remains to be
seen whether the cell lines can be maintained or preserved
in a manner that would support the use of these cells for
routine (nonresearch) granulocyte serology applications.
ISBT Working Party on Granulocyte Immunobiology
In 1989, Professor Alan Waters organized the UK Platelet and Granulocyte Serology Working Group, and 11 international laboratories participated in the First International
Workshop on Granulocyte Serology. The objectives of this
first workshop were as follows: (1) to initiate an exchange
of sera among laboratories to evaluate the techniques being used for the detection of granulocyte antibodies, (2) to
foster working relationships among laboratories involved
in this field, and (3) to support the development of the science and technology for the detection of granulocyte antibodies.99
The Working Party was initially affiliated with the British Blood Transfusion Society but is now affiliated with the
ISBT. The goals of the Second Workshop, in 1996, were
the (1) establishment of typed granulocyte panels by welldefined typing sera, (2) proficiency testing of granulocyte
antibody detection, (3) investigation of uncharacterized
sera, (4) proficiency testing of HNA genotyping, and (5) exchange of information to establish standards in granulocyte
serology.100 Fifteen international laboratories participated
in the Fourth Workshop, in 2001, with the main objective being the establishment of a formal quality assurance
(QA) scheme for granulocyte serology and molecular typing
methods.85
To date, the Working Party has conducted nine Granulocyte Serology Workshops and comprises 16 international
member laboratories, which are listed in Table 3. Also, the
American Red Cross Mid-America Blood Services Neutrophil & Platelet Immunology Laboratory has been a member
of this consortium since its inception in 1989. The workshops
continue to focus on the QA and standardization of the test
systems, and the Working Party recently published its
17
M.E. Clay et al.
serologic screening recommendations for the investigation
and prevention of leukocyte antibody-mediated TRALI.101
Conclusions
Granulocyte antibody and antigen testing has played
and continues to play a critical role for diagnosing and
investigating relatively rare but potentially lethal clinical conditions such as TRALI and immune neutropenias.
Although mitigation strategies are already being implemented nationwide, TRALI still appears to be a leading and
preventable cause of transfusion fatalities. As advancing
technology allows granulocyte antibody testing to be done
on a less manual and larger scale with more standardized
and reproducible results, it now has the potential to become
Table 3. Members of the ISBT Working Party on Granulocyte
Immunobiology
· Platelet and Leucocyte Immunology Laboratory, EFS Ile de France,
Hopital Henri Mondor, Creteil, France
· Leucocyte and Platelet Immunology Laboratory, Blood Service West of
the German Red Cross, Hagen, Germany
· The BloodCenter of Wisconsin, Platelet and Neutrophil Immunology
Laboratory, Milwaukee, WI, USA
· Institute of Transfusion Medicine, University of Schleswig-Holstein, Kiel,
Germany
· Australian Red Cross Blood Service-Queensland, Brisbane, Australia
· Platelet and Granulocyte Immunology Laboratory, National Blood Se
vice, Bristol, UK
· Blood Transfusion Center of Slovenia, Ljubljana, Slovenia
· Centre de Transfusio I Banc de Teixitis, Hospital Vall d’Hebron, P. Vall
d’Hebron, Barcelona, Spain
· Sanguin Diagnostics, Immunohaematology Diagnostic Department,
Amsterdam, The Netherlands
· Institute for Clinical Immunology and Transfusion Medicine, Justus
Liebig University, Giessen, Germany
· American Red Cross Mid-America Blood Services, Neutrophil & Platelet
Immunology Laboratory, St. Paul, MN, USA
· Department of Transfusion Medicine, Graduate School of Medical Sc
ences, University of Tokyo, Tokyo, Japan
· Department of Immunohaematology and Transfusion Medicine, Institute
of Haematology and Blood Transfusion, Warsaw, Poland
· Aberdeen and North East Scotland Blood Centre, Scottish National
Blood Transfusion Service, Aberdeen, UK
· Platelet and Leucocyte Immunology Laboratory, Institute of Biology,
Nantes, France
· Department of Transfusion Medicine, Karolinska University Hospital,
Stockholm, Sweden
even more valuable as a tool to increase blood safety by actually preventing transfusion reactions without compromising an adequate supply of blood.
Acknowledgments
The authors would like to thank Penny Milne and Bobbie Gibson for their helpful assistance with the preparation
of the manuscript.
18
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15. Stroncek DF, Sharpiro RS, Filipovich AH, et al.
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30. Ward HN. Pulmonary infiltrates associated with leukoagglutinin transfusion reactions. Ann Intern Med
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31. Thompson JS, Severson CD, Parmely MJ, et al. Pulmonary ‘hypersensitivity’ reactions induced by transfusion of non-HLA lekoagglutinins. N Engl J Med
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35. Silliman CC, Curtis BR, Kopko PM, et al. Donor antibodies to HNA-3a implicated in TRALI reactions prime
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45. Verheugt FWA, von dem Borne AEGKr, van NoordBokhorst JC, et al. ND1, a new neutrophil granulocyte
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46. Claas FHJ, Langerak J, Sabbe LJM, et al. NE1, a
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1979;13:129–34.
47. Thompson JS, Overlin VL, Herbick JM, et al. New
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48. Hasegawa T, Bergh OJ, Mickay MR, et al. Preliminary human granulocyte specificities. Transplant Pro
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49. Caplan SN, Berkman EM, Babior BM. Cytotoxins
against a granulocyte antigen system: detection by a
new method employing cytochalasin-B-treated cells.
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50. Van Leeuwen A, Eerrise JG, van Room JJ. A new leukocyte group with two alleles: leukocyte group five. Vox
Sang 1964;9:431–7.
51. Van Rood JJ, van Leeuwen A, Schippers AMJ, et al.
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1967:203–19.
52. Kline WE, Press C, Clay M, et al. Three sera defining a
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Sang 1986;50:181–6.
53. Madyastha PR, Glassman A, Levine D, et al. Identification of a new neutrophil antigen (CN1) more prevalent among American blacks (abstract). Transfusion
1983;23:426.
54. Schnell M, Halligan G, Herman J. A new granulocyte
antibody directed at a high frequency antigen causing
neonatal alloimmune neutropenia (abstract). Transfusion 1989;29(Suppl):46S.
55. Rodwell RL, Tudehope DI, O’Regan P, et al. Alloimmune
neonatal neutropenia in Australian aboriginals: an unrecognized disorder? Transfusion Med 1991;1:63–7.
56. Stroncek DF, Ramsey G, Herr GP, et al. Identification of
a new white cell antigen. Transfusion 1994;34:706–11.
57. Bux J, Bierling P, von dem Borne AE, et al. ISBT Granulocyte Antigen Working Party. Nomenclature of granulocyte alloantigens. (letter).Vox Sang 1999;77:251.
58. Bux J. Human neutrophil alloantigens. Vox Sang
2008;94:277–85.
59. Greinacher A, Wesche J, Hammer E, et al. Characterization of the human neutrophil alloantigen-3a. Nature
Med 2010;16:45–8.
60. Curtis BR, Cox NJ, Sullivan MJ, et al. The neutrophil
alloantigen HNA-3a (5b) is located on choline transporter-like protein 2 (CTL2) and appears to be encoded by an R>Q 154 amino acid substitution. Blood
2010;115:2073-6.
61. Shows TB, McAlpine PJ, Boucheix C, et al. Guidelines
for human gene nomenclature. An international system
for human gene nomenclature (ISGN, 1987). Cytogenet
Cell Genet 1987;46:11–28.
62. Stroncek DF, Skubitz KM, Plachta LB, et al. Alloimmune neonatal neutropenia due to an antibody to the
neutrophil Fc-gamma receptor III with maternal deficiency of CD16 antigen. Blood 1991;77:1572–80.
63. Stroncek DF, Skubitz K, McCullough JJ. Biochemical
nature of the neutrophil-specific antigen NB1. Blood
1990;75:744–55.
64. Matsuo K, Lin A, Procter JL, et al. Variations in the
expression of granulocyte antigen NB1. Transfusion
2000;40:654–62.
20
65. Bux J, Jung KD, Kauth T, et al. Serological and clinical
aspects of granulocyte antibodies leading to alloimmune
neonatal neutropenia. Transfusion Med 1992;2:143–9.
66. Bux J. Granulocyte antibody mediated neutropenias
and transfusion reactions. Infusions Ther Transfusion
Med 1999;26:152–7.
67. Bux J, Becker F, Seeger W, et al. Transfusion-related
acute lung injury due to HLR-A2-specific antibodies in
recipient and NB1-specific antibodies in donor blood.
Br J Haematol 1996;93:707–13.
68.Stroncek DF, Shankar RH, Herr GP. Quininedependent antibodies to neutrophils react with a 60
kD glycoprotein on which neutrophil-specific antigen
NB1 is located and an 85 kD glycosylphosphatidylinositol–linked N-glycosylated plasma membrane protein.
Blood 1993;81:2758–66.
69. Lalezari P, Bernard GE. Identification of a specific
leukocyte antigen: another presumed example of 5b.
70. de Haas M, Muniz-Diaz E, Alonso LG, et al. Neutrophil
5b is carried by a protein, migrating from 70 to 95 kDa,
and may be involved in neonatal alloimmune neutropenia. Transfusion 2000;40:222–7.
71. Simsek S, van der Schoot CE, Daams M, et al. Molecular characterization of antigenic polymorphisms (ONDa
and MARTa) of the ß2 family recognized by human leukocyte alloantisera. Blood 1996;88:1350–8.
72. Fung L, Pitcher LA, Willett JE, et al. Alloimmune neonatal neutropenia linked to anti-HNA-4a. Transfusion
Med 2003;13:49–52.
73. Hartman KR, Wright DG. Identification of autoantibodies specific for the neutrophil adhesion glycoproteins
CD11b/CD18 in patients with autoimmune neutropenia. Blood 1991;78:1096–104.
74. Kelton JG, Bebenek G. Granulocytes do not have surface ABO antigens. Transfusion 1985;25:567–9.
75. Dunstan RA, Simpson MB, Sanfilippo FP. Absence of
specific HLA-DR antigens on human platelets and neutrophils (abstract). Blood 1984;64:85a.
76. Thorsby E. HLA antigens on human granulocytes studied with cytotoxic iso-antisera obtained by skin grafting. Scan J Haematol 1969;6:119–27.
77. Sachs UJ, Bux J. TRALI after transfusion of cross-matchpositive granulocytes. Transfusion 2003;43:1683–6.
78. Stroncek DF, Leonard K, Eiber G, et al. Alloimmunization after granulocyte transfusions. Transfusion
1996;36:1009–15.
79. McCullough J, Clay M, Hurd D, et al. Effect of leukocyte antibodies and HLA matching on the intravascular
recovery, survival, and tissue localization of 111-indium
granulocytes. Blood 1986;67:522–8.
80. Bøyum A. Isolation of mononuclear cells and granulocytes from blood. Isolation of mononuclear cells by
one centrifugation and of granulocytes by combining
centrifugation and sedimentation at 1g. Scan J Clin Lab
Invest 1968;21(Suppl 97):77–89.
81. English D, Anderson BR. Single-strep separation of red
blood cells, granulocytes, and mononuclear leukocytes
on discontinuous density gradients of Ficoll-Hypaque.
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82. Ferrante A, Thong YH. A rapid one-step procedure for
purification of mononuclear and polymorphonuclear
leukocytes from human blood using a modification of
the Hypaque-Ficoll technique. J Immunol Methods
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83. Ferrante A, Thong HY. Optimal conditions for simultaneous
purification of mononuclear and polymorphonuclear
leukocytes from human blood by the Hypaque-Ficoll
method. J Immunol Methods 1980;36:109–17.
84. Madyastha P, Madyastha KR, Wade T, et al. An improved method for rapid layering of Ficoll-Hypaque
double density gradients suitable for granulocyte separation. J Immunol Methods 1982;42:281–6.
85. Lucas G, Rogers S, de Haas M, et al. Report on the
Fourth International Granulocyte Immunology Workshop: progress toward quality assessment. Transfusion
2002;42:462–8.
86. Lalezari P, Pryce S. Detection of neutrophil and platelet antibodies in immunologically induced neutropenia and thrombocytopenia. In: Rose NR, Friedman H,
eds. Manual of clinical immunology. Washington, DC:
American Society of Microbiology 1980;744–9.
87. McCullough J, Clay ME, Press C, et al. Granulocyte serology: a clinical and laboratory guide. Chicago: ASCP
Press, 1988:12–14, 168–71.
88. Verheugt FWA, Von dern Borne AEGKr, Decary S, et
al. The detection of granulocyte alloantibodies with
an indirect immunofluorescence test. Br J Haematol
1977;36:533–44.
89. Kiefel V, Santoso S, Weisheit M, et al. Monoclonal antibody-specific immobilization of platelet antigens (MAIPA): a new tool for the identification of platelet reactive
antibodies. Blood 1987;70:1722–6.
90. Minchinton R, Noonan K, Johnson TJ. Examining
technical aspects of the monoclonal antibody immobilization of granulocyte antigen assay. Vox Sang
1997;73:87–92.
91. Stroncek D. Neutrophil alloantigens. Transfusion Med
Rev 2002;16:67–75.
92. Wang E, Marincola FM, Stroncek D. Human leukocyte
antigens and human neutrophil antigens systems. In:
Hoffman R, Benz E, Shattil Sj, et al., eds. Hematology:
basic principel and practices 4th ed. Philadelphia: Elsevier, 2005:2401–22.
93. Kissel K, Scheffler S, Kerowgan M, et al. Molecular basis of NB1 (HNA-2a, CD177) deficiency. Blood
2002;99:4231–3.
94. Vigrali DA. Multiplexed particle-based flow cytometric
assays. J Immunol Methods 2000;243:243–55.
95. Stroncek DF, Adams S, Lee JH, et al. Neutrophil antibody testing. ASHI Q 2008;32:68–72.
96. Bux J, Kissel K, Hofmann C, et al. The use of allele-specific recombinant Fcγ receptor IIIb antigens for the detection of granulocyte antibodies. Blood 1999;93:357–
62.
97. Yasui K, Miyazaki T, Matsuyama N, et al. Establishment of cell lines stably expressing HNA-1a, 1b, and
-2a antigen with low background reactivity in flow cytometric analysis. Transfusion 2007;47:478–86.
98.Yasui K, Hirayama F, Matsuyama N, et al. New cell lines
express HNA-1c, -4a, -4b, -5a, or -5b for identification
of HNA antibodies. Transfusion 2008;48:1037–9.
99. Lucas GF, Carrington PA. Results of the First International Granulocyte Serology Workshop. Vox Sang
1990;59:251–6.
100. Bux J, Chapman J. Report on the Second International Granulocyte Serology Workshop. Transfusion
1997;37:977–83.
101. ISBT Working Party on Granulocyte Immunobiology. Recommendations of the ISBT Working Party on
Granulocyte Immunobiology for leukocyte antibody
screening in the investigation and prevention of
antibody-mediated transfusion-related acute lung injury. Vox Sang 2009;96:266–7.
Mary E. Clay, MS, MT(ASCP), (corresponding author) Director, Transfusion Medicine, Research and Development
Program, University of Minnesota Medical School, Department of Laboratory Medicine and Pathology, MMC
198, D-251 Mayo Building, 420 Delaware Street, SE, Minneapolis, MN 55455, and Consultant, Neutrophil & Platelet Immunology Laboratory, American Red Cross, MidAmerica Blood Services Region, 100 South Roberts Street,
St. Paul, MN 55107, Randy M. Schuller, BS, MT(ASCP),
Supervisor, and Gary J. Bachowski, MD, PhD, Assistant
Medical Director, Neutrophil and Immunobiology Laboratory, American Red Cross, Mid-America Blood Services
Region, St. Paul, MN.
For information concerning the National
Reference Laboratory for Blood Group
Serology, including the American Rare
Donor Program, please contact Sandra
Nance, by phone at (215) 451-4362, by fax at
(215) 451-2538, or by e-mail at snance@usa.
redcross.org
21
Review
A review of the Colton blood group system
History
The idea that there must be openings in the cell membrane to permit the flow of water and salts to maintain cellular function and to eliminate the byproducts of cellular
metabolism was recognized by the middle of the 19th century. Water is the major component of all living cells, including those of vertebrates, invertebrates, and unicellular
organisms and plants. Salt water comprises approximately
70 percent of the human body. After debating for decades
about the process of water transfer across cell membranes,
most scientists finally decided that water must pass freely through biologic membranes by simple diffusion. Only
a small number of scientists disagreed, and they had only
indirect evidence that there were special channels for the
transport of water. After all, there must be something to
explain the high permeability of RBCs and renal tubules.
Moreover, the water permeability of these tissues can be reversibly inhibited by mercuric ions.1
By 1950 it had been shown that water moves quickly
into and out of cells through pores that are selective for
water molecules only. Every second, billions of water molecules pass through a single channel. By the mid-1980s
scientists had found an RBC membrane protein whose 28kDa N-terminal amino acid sequence was related to the
26-kDa major intrinsic protein (MIP26) of bovine lens fiber cells.2,3 The 28-kDa protein was shown to be a unique
molecule that is abundant on erythrocytes and on renal tubules. Preston and Agre4 succeeded in 1991 in isolating the
same membrane protein from RBCs, which in 1997, was officially named aquaporin-1 (AQP1) by the Human Genome
Organization, for the water channel. AQP1 has been found
in many tissues including several parts of the kidney, liver,
gall bladder, eye, choroid plexus, hepatobiliary epithelium,
and capillary endothelium. AQP1 turned out to be the first
of the family of major intrinsic proteins (MIP), which are
highly conserved membrane proteins that regulate small
molecule transport. The MIP family of proteins is believed
to date back 2.5 to 3 billion years in evolutionary time.5 It is
believed that the MIP family evolved from two basal lineages: AqpZ-like water channels and GlpF-like glycerol facilitators. These divergent lineages probably originated from
an archaeal (AqpM-like) aquaporin.5 The MIP family is now
divided into two groups: aquaporins (AqpZ, AQP0, AQP1,
AQP2, AQP4, AQP5, AQP6, and AQP8) and aquaglycerolporins (GlpF, AQP3, AQP7, AQP9, and AQP10).6–10 In 1994,
AQP1 protein was characterized as carrying the Colton
blood group antigens.11
Genetics and Inheritance
In 1965 in Oslo, Norway, the discovery of an antibody
that detected a “public” (or high prevalence) antigen was
22
linked to two other cases discovered earlier by workers in
Minneapolis, Oxford, and London. The antigen was named
Coa (Coltona) in 1967 for the first of the three producers of
anti-Coa, who was from Minneapolis; it should actually have
been named “Calton,” but the handwriting on the tube apparently was misread.12,13 Coa was shown to be an inherited
characteristic.14 In 1970, the antithetical antigen, Cob, was
reported by Giles et al (Table 1).15 In analysis of unrelated
people of European extraction, the occurences of the three
major Colton phenotypes were as follows: Co(a+b–) 0.914,
Co(a+b+) 0.084, and Co(a–b+) 0.002 (Table 2).16 In 1971,
the genetic independence of the Colton blood group system
from Kell and Lutheran was reported, and it was confirmed
that it is independent from MNSs, P, Rh, Duffy, Dombrock,
and sex.17 Coa and Cob are codominant alleles that respectively encode the Coa and Cob antigens at the RBC surface. In
1974 the Co(a–b–) phenotype of three persons in a French
Canadian family was reported.18 The serum from a patient
in 1964 (Swarts) was eventually found to be negative only
with these Co(a–b–) family members.16 Colton is the 15th
human blood group system recognized by the ISBT (ISBT
015).
Molecular Basis
The AQP1 locus was mapped to human chromosome
7p14 by fluorescent in situ hybridization.19 The Colton (Co)
blood group antigens had been previously linked to the
short arm of chromosome 7.20 Immunoprecipitation studies
Table 1. Antigens of the Colton blood group system: their prevalence, molecular basis, and sensitivity to enzyme and DTT treatment
Name
ISBT
symbol
ISBT
number
Prevalence
Molecular
basis
Effect of
enzymes
Coa
CO1
015001
99.8%
Ala 45
PR TR CR
NR DTTR
Cob
CO2
015002
8.6%
Val 45
PR TR CR
NR DTTR
Co3
CO3
015003
>99.99%
unknown
PR TR CR
NR DTTR
CR = α1-chymotrypsin resistant; DTTR = dithiothreitol resistant;
NR = neuraminidase resistant; PR = papain resistant; TR = trypsin
resistant.
Table 2. Phenotypes (% occurrence)
Phenotype
% Occurrence (most
populations)16
Co(a+b–)
91.4
Co(a–b+)
0.2
Co(a+b+)
8.4
Co(a–b–)
<0.01
Colton blood group system review
and DNA sequencing showed that the Colton antigens are
the result of a polymorphism in AQP1 at the extracellular
site of loop A, which connects the first and second spanning
domains. Figure 1 depicts the structure of the protein as it
passes through the membrane, and indicated are the Coa/
Cob polymorphic site at position 45 and the only N-glycan
site at position 42. The Coa/Cob polymorphism arises from
a single amino acid change of Ala45Val (alanine for Coa, valine for Cob) caused by a nucleotide change of C>T at position 134 in exon 1 of AQP1 gene, named CO according to
the ISBT nomenclature and AQP1 according to the Human
Genome Organization (HUGO).11 The Colton gene consists
of four exons distributed over 11.6 kbp of genomic DNA.
The amino acid sequence of the wild-type AQP1 protein is
shown in Table 3. Also known as the channel-forming integral protein (CHIP), the Colton glycoprotein is a multipass
protein consisting of three external loops and six membrane-spanning regions, as well as 2 cytoplasmic loops,
with both the NH2 and COOH terminus located on the cytoplasmic side of the membrane.22 Common to all MIP superfamily proteins is the occurrence of the asparagine-prolinealanine (NPA) motifs, two being present in each half of the
protein. An hourglass structural model has been proposed
by Agre and colleagues.23,24
N-glycosylation site
N
42
Out
A
A = Coa
V = Cob
A/V 45
E
C
192
RBC
Membrane
1
2
3
5
N
194
P
A
6
N PA
76
In
4
78
B
D
NH2
the Co protein have been reported to cause decreased or no
expression of the Colton antigens. An amino acid change
of Gln47Arg was observed to cause a Co(a–b+w) typing.29
The same amino acid substitution was recently fully investigated in a Turkish female with the Co(a–b–) phenotype.30
Her RBCs were found by molecular studies to have normal
water permeability by stopped-flow analysis and a normal
amount of AQP1 protein in the RBC membrane. (See section on clinical significance). By PCR-RFLP the specimen
was genotyped as Coa/Coa, but full sequencing revealed a
140A>G mutation leading to a Gln47Arg amino acid substitution. This change in amino acids is close to the Coa/Cob
polymorphism, and thus, the probable cause of the apparent discrepancy between phenotype and genotype. When
using a specifically designed K-562 cell expression system
for Colton antigens, the Gln47Arg substitution was
shown to inhibit both Coa and Cob antigen expression.29
The antibody produced by these Co(a–b–) individuals
with a Gln47Arg substitution is not a mixture of anti-Coa
and -Cob, rather it is “anti-Co3–like,” reacting with both
Co(a+b–) and Co(a–b+) RBCs, but more weakly than antiCo3 produced by the true null phenotype. This suggests the
existence of a new high-prevalence antigen in the Colton
blood group system, with a corresponding antibody that
Arnaud et al. proposed to name anti-Co4.30 Therefore, it is
possible that some of the Co(a–b–) people known throughout the world, who would have not been fully investigated
by molecular techniques, may actually not be “true” Conull
but “CO:–1,–2,3,–4.” This may also be why some antibodies directed against a high-prevalence Colton antigen, mistakenly considered as anti-Co3, were unexpectedly weakly
reactive with Co:3 RBCs.31
COOH
Biochemical Physiology of the Colton Glycoprotein
Protons are necessary for the bioenergetics of the living
Fig. 1 Linear diagram depicting the Colton protein with the six
cell. The proton gradient drives many transport functions,
membrane-spanning domains with both the N terminus and C
membrane fusions, and the synthesis of ATP. Maintaining
terminus inside the cell membrane. The Coa/Cob polymorphism is
the proton gradient is essential, and indiscriminate proindicated at position 45, as is the glycosylation site at N42. The
21
ton leakage across cellular membranes would be fatal to
signature NPA motifs are depicted on the internal loops B and D.
the living cell. At the same time, cells must maintain the
balance of water’s ability to permeate the membrane biTable 3. Amino acid sequence of wild-type AQP1 protein13
layer. Aquaporins are strictly selective water chanMASEFKKKLF WRAVVAEFLA TTLFVFISIG SALGFKYPVG NNQTAVQDNV 50
nels that have evolved for that purpose. AQP1 was
found to have a configuration with intracellular N
KVSLAFGLSI ATLAQSVGHI SGAHLNPAVT LGLLLSCQIS IFRALMYIIA
100
and C terminals, similar to that of other ion chanQCVGAIVATA ILSGITSSLT GNSLGRNDLA DGVNSGQGLG IEIIGTLQLV
150
nel proteins. Protein modeling from the cDNA
LCVLATTDRR RRDLGGSAPL AIGLSVALGH LLAIDYTGCG INPARSFGSA 200
sequences predicts a two-tandem repeat of three
VITHNFSNHW IFWVGPFIGG ALAVLIYDFI LAPRSSDLTD RKVWTSGQV
250
membrane-spanning helices that exist as tetramEEYDLDADDI NSRVEMKPK
269
ers within the cell membrane (Fig. 2). An electroAmino acid sequence taken from GenBank, accession # M77829
static field created by the highly conserved amino
Also see the Blood Group Antigen Gene Mutation Database (dbRBC)
acid sequence NPA forms the water channel, which
at http://www.ncbi.nlm.nih.gov/gv/mhc/xslcgi.cgi?cmd=bgmut/home
prevents proton loss while allowing water to perme(accessed April 13, 2010).
ate the membrane. The most prominent feature is
the association of loop B with loop E driven by their
positive N-terminal ends that brings together the
The molecular basis of the null Co(a–b–) phenotype
NPA motifs at the center of the pore. This causes
has been attributed to several different molecular backthe
formation of strong electrostatic fields that block
grounds; these are depicted in Table 4. Other changes in
23
Table 4. Molecular bases of the Colton-null [Co(a–b–)] phenotype: effect of the change at the DNA and protein level, and ancestry
of the probands
Effect
Observed result on
AQP1 protein
Presence
of anti-Co3
HDFN
reported
Large deletion comprising most of exon 1
Absent or aberrant
transcript
AQP1 not detected in
RBC membrane extracts
Not reported
c.309insT
Frameshift mutation
AQP1 not detected in
c.113C>T
Missense mutation
(p.Pro38Leu)
c.576C>A
Molecular alteration
Ancestry
Reference
No
Northern
European
Proband 1 in Preston
et al.25
Yes
No
French
et al.25
<1% AQP1 expression in
Not reported
No
Northern
European
et al.25
Missense mutation
(p.Asn192Lys)
Not tested
Yes
No
Portuguese
Chrétien et al.26
c.232delG
Frameshift mutation
Not tested
Yes
Yes
Indian
Joshi et al.27
c.601delC
Frameshift mutation
Not tested
Yes
No
Caucasian
Nance et al.28; Reid
and Lomas-Francis13
c.140A>G
Missense mutation
(p.Gln47Arg)
Not tested
No
No
Not reported,
presumably
Caucasian
Wagner and Flegel29
c.140A>G
Missense mutation
(p.Gln47Arg)
Normal AQP1 expression
in RBC membrane
No
No
Turkish
Arnaud et al.30
Conull phenotypes
Altered Co phenotypes
AQP1 = aquaporin 1; HDFN = hemolytic disease of the fetus and newborn.
According to the recommendations of the Human Genome Variation Society and National Institutes of Health Single Nucleotide Polymorphism database, “c.” means that the position of the polymorphism refers to the complementary DNA, “p” means that the position
of the polymorphism refers to the protein.
the permeation of protons as well as H3O+ and other cations. When Xenopus laevis oocytes were transfected with
cRNA, they were found to have markedly increased water
permeability, causing the oocytes to swell and burst.32 This
permeability could be reversibly inhibited by Hg2+.33 When
the same cRNA transcript was reconstituted into lipid vesicles, the same observations were made. This confirmed that
membranes exhibit water permeability and showed that the
water permeability of membranes with AQP1 was up to 100
times greater than that of those without it.
Out
RBC
Membrane
1 6 2
N
P A
H2O
4 3 5
A P N
In
NH2
COOH
Fig. 2 The AQP1 water channel is shown as an hourglass configuration created by the association of the six transmembrane domains
and the crossing of loops B and E with the electrostatic field produced by the NPA motifs forming the water channel.21
24
Colton Antigen Typing
To date, anti-Coa and anti-Co3 reagents, either monoclonal or polyclonal, are not commercialized. As a result,
human polyclonal reagents are only available in immunohematology reference laboratories, but usually in small
amounts. However, a human polyclonal anti-Cob is available in Europe as a CE-marked in vitro diagnostic reagent
(DiaMed, a division of Bio-Rad, Cressier sur Morat,
Switzerland). The two major high-throughput molecular genotyping platforms, a commercialized DNA glass array
by Progenika (Barcelona, Spain) and a DNA bead array by
BioArray/Immucor (Norcross, GA) allow Coa and Cob allele typing by searching for the 134C>T polymorphism in
exon 1, with subsequent prediction of Colton phenotype. It
is important, however, to be aware that prediction of the Co
phenotype in Conull and Covariant people would be a source of
misinterpretation with these DNA-based techniques.
Clinical Significance
Antibodies to Colton blood group antigens are generally IgG and react best by the antiglobulin test, especially
when protease-treated RBCs are used.34,35 Some anti-Coa,
-Cob and -Co3 may bind complement.36 Few reports of significant delayed or acute transfusion reactions or hemolytic
disease of the fetus and newborn (HDFN) attributable to
anti-Coa have been reported, although both are known to
occur with severe morbidity.27,37–40
Anti-Cob is relatively rare and often found in patient’s
sera containing other alloantibodies.34,35 Two reports of in
Colton blood group system review
vivo survival studies of 51Cr-labeled Co(b+) RBCs in patients with anti-Cob showed survival at 94 percent and 85
percent, respectively, at 1 hour, decreasing to 51 percent at
24 hours and 10 percent at 96 hours.41,42 Cob is fully developed at birth, but no significant HDFN has been reported to
date.34
Anti-Co3 was reported to cause severe HDFN requiring neonatal transfusion.43 Transfusion of incompatible
Co(a+b–) RBCs in a patient with anti-Co3 was reported to
be responsible for a mild hemolytic transfusion reaction.27
There are also reports of autoantibodies mimicking Colton
specificities.44
AQP1-deficient individuals were studied for their renal
function and capillary permeability before and after water
deprivation.45 Baseline studies did not reveal any abnormalities; however, after water deprivation this study revealed an
inability to concentrate urine. Thus, Colton-null individuals
may be subject to serious hydroelectrolytic and metabolic
disorders should they become severely dehydrated.
Acknowledgments
The authors thank Robert Ratner for preparation of the
manuscript and figures, and Hallie Lee-Stroka for her critical review and helpful comments. This work was funded in
part by a grant from the MetLife Foundation (GH).
References
1. Macey RI. Transport of water and urea in red blood
cells. Am J Physiol 1984;246:C195–203.
2. Agre P, Saboori AM, Asimos A, Smith BL. Purification
and partial characterization of the Mr 30,000 integral
membrane protein associated with the erythrocyte
Rh(D) antigen. J Biol Chem 1987;262:17497–503.
3. Saboori AM, Smith BL, Agre P. Polymorphism in the
Mr 32,000 Rh protein purified from Rh(D)-positive
and -negative erythrocytes. Proc Natl Acad Sci U S A
1988;85:4042–5.
4. Preston GM, Agre P. Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons:
member of an ancient channel family. Proc Natl Acad
Sci U S A 1991;88:11110–4.
5. Kozono D, Ding X, Iwasaki I, et al. Functional expression and characterization of an archaeal aquaporin.
AqpM from Methanothermobacter marburgensis. J
Biol Chem 2003;278:10649–56.
6. Roudier N, Ripoche P, Gane P, et al. AQP3 deficiency in
humans and the molecular basis of a novel blood group
system, GIL. J Biol Chem 2002;277:45854–9.
7. Vajda Z, Pedersen M, Füchtbauer EM, et al. Delayed onset of brain edema and mislocalization of aquaporin-4
in dystrophin-null transgenic mice. Proc Natl Acad Sci
U S A 2002;99:13131–6.
8. Ikeda M, Beitz E, Kozono D, Guggino WB, Agre P, Yasui
M. Characterization of aquaporin-6 as a nitrate channel
in mammalian cells. Requirement of pore-lining residue threonine 63. J Biol Chem 2002;277:39873–9.
9. Carbrey JM, Gorelick-Feldman DA, Kozono D, Praetorius J, Nielsen S, Agre P. Aquaglyceroporin AQP9: solute permeation and metabolic control of expression in
liver. Proc Natl Acad Sci U S A 2003;100:2945–50.
10. Endeward V, Musa-Aziz R, Cooper GJ, et al. Evidence
that aquaporin 1 is a major pathway for CO2 transport
across the human erythrocyte membrane. FASEB J
2006;20:1974–81.
11. Smith BL, Preston GM, Spring FA, Anstee DJ, Agre P.
Human red cell aquaporin CHIP. I. Molecular characterization of ABH and Colton blood group antigens. J
Clin Invest 1994;94:1043–9.
12. Heistö H, van der Hart M, Madsen G, et al. Three examples of a new red cell antibody, anti-Coa. Vox Sang
1967;12:18–24.
13. Reid ME, Lomas-Francis C. The blood group antigen
factsbook. 2nd ed. San Diego, CA: Academic Press,
2004.
14. Wray E, Simpson S. A further example of anti-Coa and
two informative families with Co(a–) members. Vox
Sang 1968;14:130–2.
15. Giles CM, Darnborough J, Aspinall P, Fletton MW.
Identification of the first example of anti-Cob. Br J Haematol 1970;19:267–9.
16. Race RR, Sanger R. Blood groups in man. 6th ed. Oxford, England: Blackwell Scientific Publications, 1975.
17. Lewis M, Kaita H, Anderson C, Chown B. Independence
of Colton blood group. Transfusion 1971;11:223–4.
18. Rogers MJ, Stiles PA, Wright J. A new minus-minus
phenotype: three Co(a–b–) individuals in one family
(abstract). Transfusion 1974;14:508.
19. Moon C, Preston GM, Griffin CA, Jabs EW, Agre P.
The human aquaporin-CHIP gene. Structure, organization, and chromosomal localization. J Biol Chem
1993;268:15772–8.
20. Zelinski T, Kaita H, Gilson T, Coghlan G, Philipps S,
Lewis M. Linkage between the Colton blood group locus
and ASSP11 on chromosome 7. Genomics 1990;6:623–5.
21. Agre P, King L, Yasui M, et.al. Topical Review: Aquaporin water channels—from atomic structure to clinical
medicine. J Physiol. 2002;542:3–16.
22. King LS, Agre P. Pathophysiology of the aquaporin water channels. Annu Rev Physiol 1996;58:619–48.
23. Mathai JC, Agre P. Hourglass pore-forming domains
restrict aquaporin-1 tetramer assembly. Biochemistry
1999;38:923–8.
24. Jung JS, Preston GM, Smith BL, Guggino WB, Agre
P. Molecular structure of the water channel through
aquaporin CHIP. The hourglass model. J Biol Chem
1994;269:14648–54.
25. Preston GM, Smith BL, Zeidel ML, Moulds JJ, Agre P.
Mutations in aquaporin-1 in phenotypically normal humans without functional CHIP water channels. Science
1994;265:1585–7.
25
26. Chrétien S, Catron JP, de Figueiredo M. A single mutation inside the NPA motif of aquaporin-1 found in a
Colton-null phenotype (letter). Blood 1999;93:4021–3.
27. Joshi SR, Wagner FF, Vasantha K, Panjwani SR, Flegel WA. An AQP1 null allele in an Indian woman with
Co(a–b–) phenotype and high-titer anti-Co3 associated
with mild HDN. Transfusion 2001;41:1273–8.
28. Nance S, Kavitsky DL, Meny G, Vege, S, Westhoff CM.
An example of anti-Co3 not causing hemolytic disease
of the newborn. Transfusion 2002;42(Suppl):105S.
29. Wagner FF, Flegel WA. A clinically relevant Co(a)-like
allele encoded by AQP1 (Q47R) (abstract). Transfusion
2002;42(Suppl):24S–5S.
30. Arnaud L, Helias V, Menanteau C, Peyrard T, et al. A
functional AQP1 allele producing a Co(a–b–) phenotype revises and extends the Colton blood group system. Transfusion 2010. In press.
31. Lacey PA, Robinson J, Collins ML, et al. Studies on the
blood of a Co (a–b–) proposita and her family. Transfusion 1987;27:268–71.
32. Preston GM, Carroll TP, Guggino WB, Agre P. Appearance of water channels in Xenopus oocytes expressing
red cell CHIP28 protein. Science 1992;256:385–7.
33. Kozono D, Yasui M, King LS, Agre P. Aquaporin water channels: atomic structure and molecular dynamics
meet clinical medicine. J Clin Invest 2002;109:1395–9.
34. Daniels G. Human blood groups. 2nd ed. Malden, MA:
Blackwell Science, 2002.
35. Issitt PD, Anstee DJ. Applied blood group serology. 4th
ed. Durham, NC: Montgomery Scientific Publications,
1998.
36. Covin RB, Evans KS, Olshock R, Thompson HW. Acute
hemolytic transfusion reaction caused by anti-Coa. Immunohematology 2001;17:45–9.
37. Simpson WKH. Anti-Coa and severe haemolytic disease
of the newborn. S Afr Med J 1973;47:1302–4.
38. Agre P, Smith BL, Baumgarten R, et al. Human red cell
aquaporin CHIP. II. Expression during normal fetal development and in a novel form of congenital dyserythropoietic anemia. J Clin Invest 1994;94:1050–8.
39. Kurtz SR, Kuszaj T, Ouellet R, Valeri CR. Survival of homozygous Coa (Colton) red cells in a patient with antiCoa. Vox Sang 1982;43:28–30.
40. Michalewska B, Wielgos M, Zupanska B, Bartkowiak R.
Anti-Coa implicated in severe haemolytic disease of the
foetus and newborn. Transfus Med 2008;18:71–3.
41. Dzik WH, Blank J. Accelerated destruction of radiolabeled red cells due to anti-Coltonb. Transfusion
1986;26:246–8.
42. Hoffmann JJML, Overbeeke MAM. Characteristics of
anti-Cob in vitro and in vivo: a case study. Immunohematology 1996;12:11–13.
43. Savona-Ventura C, Grech ES, Zieba A. Anti-Co3 and severe hemolytic disease of the newborn. Obstet Gynecol
1989;73:870–2.
44. Moulds M, Strohm P, McDowell MA, Moulds J. Autoantibody mimicking alloantibody in the Colton blood group
system (abstract). Transfusion 1988;28(Suppl):36S.
45. King LS, Choi M, Fernandez PC, Cartron JP, Agre P. Defective urinary-concentrating ability due to a complete
deficiency of aquaporin-1. N Engl J Med 2001;345:175–9.
Gregory R. Halverson, BS, MT(ASCP)SBB, DLM (corresponding author), Laboratory of Immunochemistry,
New York Blood Center, 310 East 67th Street, New York,
NY 10065, and Thierry Peyrard, PharmD, MS, European
Specialist in Clinical Chemistry and Laboratory Medicine,
National Immunohematology Reference Laboratory, National Institute of Blood Transfusion, 20 rue Bouvier 75011
Paris, France.
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26
Case Report
Alloimmunization to the D antigen by a
patient with weak D type 21
Antibodies of apparent D specificity may be found in D+ patients. We report a D+, multi-transfused Caucasian woman
with myelodysplasia who exhibited several alloantibodies.
One antibody was a moderately strong (2+) anti-D that persisted for 9 months, until the woman died. Molecular analysis of the patient’s RHD gene identified the rare weak D type
21 (938C>T) allele. D alloantibodies do not occur in patients
with most weak D types, but some patients with a weak D
phenotype, including those with type 21, can produce antibodies to nonself epitopes of the wild-type D antigen.
Key Words: alloimmunization, anti-D, RHD gene, weak D
A
ntibodies with D specificity are unusual in D+ patients. Alloantibodies to D may be (1) passively
transfused in donor plasma products, (2) passively
transferred but actively produced by lymphocytes carried
in transplanted organs, and (3) actively produced by an alloimmunized patient. Autoantibodies to D may be warm- or
cold-reactive antibodies with a preference for D+ RBCs or
may be antibodies to LW that mimic anti-D specificity.1
In practice, the combination of clinical, serologic, and
molecular investigations helps to determine why a D+ patient has anti-D. Establishing the source of a D+ patient’s
alloantibody or autoantibody with D specificity is important
in deciding whether D+ or D– units should be transfused to
optimize donor RBC survival (Table 1).
In this case report, we describe a woman whose RBCs
were strongly D+ at the phenotypic level, but had an RHD
genotype consistent with weak D, and an alloanti-D.
Case Report
A 72-year-old, gravida 3, Caucasian woman with myelodysplasia and pancytopenia was transferred from another
hospital, where in 6 months of treatment, she had received
an unknown number of platelet transfusions, but no RBC
transfusions.
In our hospital and before any RBC transfusion, the
patient’s RBCs serologically typed as D+ (4+) by tube agglutination at immediate spin phase, using a monoclonalpolyclonal blended anti-D (Ortho Clinical Diagnostics Co.,
Raritan, NJ). During a disease course of 13 months, the
patient received 37 apheresis-processed platelet units and
108 leukocyte-reduced units of RBCs, which produced alloimmunization to K, E, and Cw. After 9 months, her serum
also demonstrated a 2+ anti-D in a gel-based, antibody detection system (Micro Typing Systems, Inc., Ortho Clinical
Diagnostics, Pompano Beach, FL). The initial DAT (tube,
Table 1. Evaluation and transfusion of patients with D+ RBCs and
serum anti-D
Etiology/
pathogenesis
Diagnostic evidence
Preferred RBC
transfusion
Passive alloantibody
History of transfusion
with D– RBC, plasma,
IVIG, RhIG, other product
with anti-D
D– until anti-D
wanes
Alloantibodies from
passenger lymphocytes
in donor organ
Organ transplant from D–
donor to D+ recipient
D– until anti-D
wanes
Autoantibody to D
antigen
Positive direct/indirect
antiglobulin test
(anti-IgG)
D– over D+
Anti-D specificity in
eluate or serum
History: pregnancy, warm
autoimmune hemolytic
anemia, malignancy
Autoantibodies or
alloantibodies to LW
Antibody fails to react
with RBC treated with
dithiothreitol
D– over D+
Antibody is absorbed by
D– RBC
Alloantibody to epitope
absent from normal D
protein
Anti-D fails to react with
RBC of person who
produced it
D– only
Anti-D fails to react with
D– RBC
anti-IgG) and all but one subsequent DAT were negative as
was the auto-control. On the one occasion, the DAT became
weakly positive in a mixed-field reaction. Acid eluates prepared from the patient’s RBCs contained only anti-E. There
was no history of organ transplantation or administration
of a plasma product that might contain anti-D. The anti-D
persisted throughout the patient’s course. Blood samples
were collected and referred for RHD and RHCE gene analyses.
Although continued transfusions with D– blood were
prescribed, the patient refused all further medical treatment. Her myelodysplasia progressed to early acute leukemia before she expired at home.
Molecular Test Results
Molecular tests were performed at the Molecular Red
Cell and Platelet Testing Laboratory of the American Red
Cross, Penn-Jersey Region, and the University of Pennsylvania DNA Sequencing Facility, both in Philadelphia,
27
Pennsylvania. The patient was found to be heterozygous
for RHD. No changes associated with any of the known
partial D phenotypes were identified. A change (938C>T)
was identified that is associated with weak D type 21 that is
predicted to encode an intracellular amino acid change of
Pro313Leu. The patient’s RH genotype could be interpreted
to be RHD*weak D type 21, RHCE*Ce/RHCE*ce.
Discussion
Initially, the finding of anti-D in a D+ patient was puzzling and the several causes listed in Table 1 were considered. Patients whose anti-D was passively transferred in
plasma products (IVIG, RhIG, etc.) or whose serum antibodies were produced by a donor’s passenger lymphocytes
may require D– RBCs until the acquired anti-D has weakened, usually in a matter of weeks to months.
Passive anti-D, however, was ruled out by history. Before hospital admission at our institution, the patient had
been transfused only with apheresis platelets, but she had
not been transfused with plasma or cellular products from
D– donors, nor had she been infused with IVIG or injected
with RhIG. Persistence of the anti-D was further evidence
that the anti-D did not involve passive immunization.
The transfer of passenger lymphocytes could be excluded by history as well. The patient had never received a
transplant. Passenger lymphocytes are alloantibody-secreting
B cells that are carried in a transplanted donor organ to a
recipient. An anti-D produced by passenger lymphocytes
arises in the donor by alloimmunization to the D antigen
before transplantation. Donor lymphocytes continue to
manufacture detectable anti-D for about 3 months after
transplantation.
Many patients with autoimmune hemolytic anemia and
mimicking antibodies to Rh antigens are treated medically
and transfusion is avoided altogether. In other patients,
transfusion can be delayed until the concentration of antibodies is reduced by medical treatment. Many mimicking
warm autoantibodies have apparent Rh specificity, especially
anti-e. Only rare D+ patients will possess warm autoantibodies that mimic anti-D. If a patient with autoantibodies
requires transfusion, antigen-negative donor RBCs may be
preferable because they may survive longer than antigenpositive RBCs.2
Antibodies to LW are almost always autoantibodies;
those that are less potent appear to have anti-D specificity because the number of D antigen sites per RBC correlates with the number of LW antigen sites.3 Thus, D– RBCs,
which have fewer LW antigen sites than D+ RBCs, may not
be agglutinated by an anti-LW. However, anti-LW may be
distinguished from anti-D by its failure to agglutinate RBCs
treated with DTT. If transfusion is required for a patient
with autoanti-LW, D– RBCs are preferable to D+ RBCs providing such a patient is c+.
Patient autoantibodies, including anti-LW, are often
transient, but our patient’s anti-D persisted for 4 months
at constant serologic strength (2+ by the IgG gel test).
28
Alloantibodies to Rh antigens are often durable (85%).
Negative DATs and absence of RBC agglutination in autocontrol tests also indicated the presence of D alloantibody.
LW alloantibodies, which can mimic anti-D, arise
only in very rare individuals whose RBCs are LW(a–b–)
or LW(a–b+). Neither autoantibodies nor alloantibodies
to LW were specifically evaluated in the patient described
here.
Alloantibodies to the D antigen occur in people who are
known to lack one or more epitopes of D (partial D phenotype). On the other hand, anti-D alloimmune responses in
patients with other weak D types are unusual because their
RBCs do not appear to lack D epitopes when tested with a
standard battery of anti-D monoclonal reagents.4
Suspicion that the patient had been alloimmunized to
D antigen was raised by the serologic finding of negative
DATs and the antibody’s persistence. In addition, people
who carry variants of the D antigen that are detectable only
by molecular means are capable of producing antibodies
to wild-type protein. In the present case, finding a variant
RHD gene supported the clinical and serologic evidence of
D alloimmunization.
Historically, some D+ individuals who made anti-D were
initially considered to express a weak D phenotype (first
termed “DU”) when tested with human polyclonal reagents.
Serologic evidence of alloimmunization to D in a D+ person
included a negative DAT, a serum antibody, and, for some
partial D phenotypes, the patient’s RBCs also expressed a
low-prevalence Rh antigen.5 A D+ patient with alloanti-D
was thought to lack part(s) of the D antigen, i.e., a “partial
D” type. A person with a partial D phenotype would only
produce antibody to those epitopes of the D antigen missing
from his or her own RBCs. Investigative use of monoclonal
antibody revealed many epitopes of the D antigen and supported the concept that partial D types lacked one or more
epitopes of their D antigen. The monoclonal antibodies also
extended the identification and classification of the various
partial D types initially defined by polyclonal anti-D.
Many individuals, however, were found to have RBCs
that agglutinated poorly with common anti-D reagents, but
demonstrated the presence of all known epitopes. These D+
RBCs were called “weak D” in preference to DU. Weak D
RBCs expressed fewer D molecules per RBC, a finding that
accounted for their weak agglutination with reagent antibodies. Theoretically, individuals who carried these weak
D types should not be able to develop alloantibodies to D.
However, it is virtually impossible to distinguish a person
with a weak D phenotype from a person with a weak partial D phenotype by serologic tests; only the production
of alloanti-D will reveal someone to have a weak partial D
phenotype.
When molecular analysis became available, the RHD
(and RHCE) gene(s) revealed far more extensive and complex polymorphisms than were evident by serologic methods. Weakly agglutinating D serotypes could be related to
point mutations, nonsense mutations, deletions, and tandem
Anti-D in weak D type 21
rearrangements in the RHD and RHCE DNA. The cause of
weak RBC agglutination was confirmed to be an absence of
epitopes in partial D types, a decreased number of protein
molecules in weak D types, or both. In addition, partial D
phenotypes are expressions of genotypes that encode for
changes in the amino acids on the outer surface of the RBC
where the D protein loops outside the RBC membrane.
Patients whose RBCs express partial D types are often immunized to D+ RBCs by transfusion or pregnancy. On the
other hand, genotypes that encode weak D phenotypes are
predicted to result in intracellular or intramembranous
changes in amino acids that, in theory, express all epitopes
of D so that patients whose RBCs express weak D types
are not expected to produce anti-D after immunization by
transfusion or pregnancy. Theory is partly supported by
evidence—the most common weak D variants (types 1–3),
which constitute 70 to 90 percent of all weak D variants, do
not produce alloanti-D.6
Molecular examination of the various partial and weak
D types indicated that many serologic phenotypes had more
than one basis, but did not suggest that new epitopes could
be identified.7 However, serologic evidence suggested otherwise. Anti-D has been found in some weak D patients
whose D protein is predicted by their DNA sequences to
have altered intramembranous and intracellular amino acids of the D protein. Thus, some weak D molecular alleles
contain missense mutations that not only reduce the number of antigens per RBC and cause weak RBC agglutination
by reagent antibodies but also cause a loss of D epitopes
and produce changes in the three-dimensional antigenic
structure of the D protein.
The case presented here indicates that the rare weak
D type 21 is an allelic variant whose patient-carriers are
susceptible to transfusion alloimmunization by wild-type D
antigen. Weak D type 21 is the result of a point mutation
that alters an amino acid thought to be in an intracellular
region of the D protein.8 The rare allele was first discovered
in one individual during investigation of 270 known weak
D samples in people from northern Germany,9 and there
has been one subsequent independent observation of it in
Austria.10 In both cases, the allele has been found on the
same chromosome (cis) as a RHCE*Ce allele in the haplotype RHDweak D type 2-RHCE*Ce. The Pro313Leu change in the
translated D protein is in its tenth intracellular helix, and all
37 tested epitopes of the D antigen appear to be present.9 As
expected, the number of D antigen sites per RBC is reduced
(5.2 × 103) when compared with the normal number (104
to 2.5 × 104) of sites.11 This antigen density is the highest
among all known weak D alleles. The high density probably
accounts for the patient’s 4+ serotype and explains why she
was not identified as a weak D carrier.
Summary
We evaluated a patient with a strongly expressed D antigen with a posttransfusion antibody to the D antigen. The
antibody had clinical and serologic characteristics of an
alloantibody. After RHD and RHCE genotyping, the patient
was found to have the RHDweakD type 21RHCE*Ce/RHCE*ce
genotype (R1weak D type 21r phenotype). Although most patients
with a weak D phenotype do not produce alloanti-D, patients who carry the rare weak D type 21 in the absence of a
normal RHD allele appear susceptible to the production of
D alloantibodies.
Acknowledgments
The authors thank Connie Westhoff, PhD, and Sunitha
Vege for the molecular analyses.
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Heather McGann, MT(ASCP) SBB (corresponding author),
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Robert E. Wenk, MD, Consulting Pathologist, Department
of Pathology, Sinai Hospital, 2401 West Belvedere Avenue,
Baltimore, MD 21215.
29
Review
A review of the Chido/Rodgers blood group
R. Mougey
. . . and the blind, wise blood bankers said, “These antibodies are high-titered and have low avidity, they are red
cell, white cell antibodies, they are clinically insignificant,
are not reactive with enzyme-treated red cells, and can be
absorbed with white cells!” Then the chorus of other blind,
wise blood bankers said, “NO, THEY ARE NOT!”
based on memories of discussions about these antibodies
back in the 1970s and the fable of the elephant and blind wise
men—r. mougey
It was a delight, an honor, and also a little intimidating
to be asked to write a review of the Chido/Rodgers blood
group system, especially considering that the last author to
pen a review for Immunohematology on this subject was
Carolyn M. Giles, the author of so many important publications on Chido and Rodgers. Moreover, the recent discoveries about Chido and Rodgers have moved well beyond
the field of blood group serology to an understanding of the
functional protein and genetics at a molecular level.
My delight was in the opportunity to revisit my early
days in performing antibody identification and think again
about the challenges that blood bankers faced then in working with a group of antibodies that no one could agree about.
Chido and Rodgers were usually lumped in with this group,
which in light of current knowledge is quite understandable.
As I recall even the pronunciation for Chido was a matter of
some dispute, with adherents claiming that “Cheedo” with
a short i vowel, not Chido with a long i sound, was correct.
It occurred to me that this review should cover a historical
perspective, which might be helpful to those blood bankers
whose testing experience is derived from the newer methods of gel column or solid-phase technology and who might
be wondering what all the fuss was about. The review will
then cover the current understanding of the serology, function, and biochemistry, and then the genetics of C4 and the
Chido/Rodgers blood group system. The nomenclature for
the complement system is very confusing even for blood
bankers who deal routinely with a nomenclature for a blood
group that is just as confusing.
Historical Perspective
In the beginning there was confusion and chaos. During the 1960s and ’70s, the process for antibody identification was usually a straightforward and consistent exercise
with good consensus between individual technical staff and
among laboratories, except there was this group of antibodies that “would not cooperate.” In the reference laboratory where I worked, we called them “the grubbies,” but
we were too frustrated by working with them to go much
30
further than that. The usual experience was to spend an
inordinate amount of time performing every possible test,
which led only to excluding what the antibody could not
be. Then when additional samples were obtained to continue the investigation, it was not uncommon to find that
tests that had appeared to be nonreactive were now reactive
with the new antibody sample or with a freshly collected
sample of the test cell. It took me a while to recognize that
with these antibodies, if you wanted to test for specificity
by typing the patient’s RBCs or testing known antigen positive or negative RBCs, it was best to use the freshest RBCs.
If you wanted to find compatible blood, older donor units
were more likely to be nonreactive. That is, it was generally agreed that there were some patients whose antibodies could not be identified as any of the then known antibodies and whose reactivity was so weak there was a lot of
disagreement as to which test RBCs were truly nonreactive.
This made it very difficult to define and describe the true
characteristics of the antigens and the antibodies and show
that the antigens were expressed on the RBC as an inherited
trait. This variability was partly a result of testing methods,
which relied heavily on a multiphasic set of incubation temperatures and the use of a polyclonal antiglobulin reagent.
In that period, blood bankers went to great lengths to detect and identify room temperature–reactive antibodies as
well as antibodies active at 37°C. The use of fresh serum for
testing was usually considered essential, with the end result
that blood bankers were pretty good at detecting clinically
significant antibodies but even better at finding clinically
insignificant antibodies. Blood bankers got lots of practice at identifying these clinically insignificant antibodies,
and neutralizing them with human breast milk, avian egg
albumin, hydatid cyst fluid, saliva, and human and guinea
pig urine. The grubbies defied these efforts, and there was
much discussion, some of it acrimonious, on how patients
with these antibodies ought to be managed. The term high
titer, low avidity was preferred by many if only for its acronym HTLA, even though everyone knew that these antibodies were not always of high titer or of low avidity. When
such weak antibodies were shown to be of high titer, it defied logic that the antibody should show such poor avidity.
Obviously, the fault could lie with the antigen, either its orientation or its number of sites, or some other property not
yet realized. Again, there was a contradiction in the patient
making a high-titered antibody if there was so little antigen
to stimulate the production of antibodies. The first glimmer
of understanding in these contradictory findings was the reports on anti-Chido.
Chido/Rodgers review
Discovery and Serology of Chido/Rodgers
The Chido antibody was first studied in a patient in
1962, and in a collaborative effort between laboratories in
Minneapolis, Minnesota, and London, United Kingdom, it
was found to be reactive with nearly all cells tested but was
consistently nonreactive with the RBCs of a London panel
donor. Six additional examples were found, and the antibody was named Chido after one of the patients and the
combined results were published by Harris et al. in 1967.1
Their title is as good a description of the grubbies as any, “A
nebulous antibody responsible for crossmatching difficulties.” The authors reported that the antibodies could not be
inhibited by saliva, urine, or hydatid cyst fluid.
During this time, there was much speculation about
these grubby antibodies being RBC-WBC antibodies, that
came from some successful experiments using WBCs to adsorb these antibodies.2 This whole issue of mistaken identity
was very understandable because of the not uncommon antibody problem known as anti-Bgs. Bg antibodies had been
shown to be hemagglutinins with anti-HLA specificity, and
the Bg antigens were thought to be remnants of those HLA
antigens expressed on the RBC before the loss of its nucleus. Because Bgs were antigens of lower frequency, it would
have been so tidy to have them be the antithetical allele to
Chido as the variable strength of reactivity seemed so similar. Eventually it was realized that the “grubby” antibodies
had nothing to do with WBC antigens. There may have been
sufficient residual plasma in the WBC preparations used to
adsorb the antibodies, now known to be capable of inhibiting anti-Chido, to lead to this mistaken conclusion. The
speculation that the Chido antigen was a WBC antigen did
lead to studies for possible linkage with HLA, an important
advance as linkage studies had shown no association with
any other blood groups.
Many additional examples of anti-Chido were found,
and it was thought that these antibodies did not seem to
cause obvious destruction of Chido-positive donor RBCs.
The work of Middleton and Crookston3 published in 1972
provided the clue that led to recognizing the true nature of
the blood group. In an attempt to enhance the reactivity
of the anti-Chido they were studying, inert human plasma
was added to the tests; a common practice of the time was
to add a source of protein to enhance antibody binding for
antihuman globulin (AHG) reactions when using polyspecific AHG. The authors recognized that when the antibody
reactivity disappeared after the addition of plasma the
Chido antigen must also be in plasma. They then found
that performing Chido antigen typings using the plasma of
the person being typed in a neutralization test was a much
more reliable way to type individuals for the Chido antigen
than testing their RBCs. They also noted that weak Chido
antigen reactivity on an individual’s RBCs did not appear
to correlate with the amount of antigen in that person’s
plasma. The ability to neutralize anti-Chido was also a useful tool for antibody identification, especially in sera with
multiple antibodies. This was the first way in which Chido
was distinguished from the other so-called HTLA specificities Knops, Csa, and Yka, although some workers continued
to think of anti-Yka as also showing some inhibition with
plasma.4
At this point, the Chido antigen was thought to be a
plasma antigen that was adsorbed onto the RBC much like
the Lewis antigens. Once accurate, reproducible antigen
typings were possible, family studies on the inheritance of
the Chido gene led to the discovery that Chido was linked
to HLA.5 This potential relationship to HLA prolonged the
use of the term RBC-WBC antibodies, but the recognition of
HLA linkage to Chido led to the studies to see whether other
genes known to be linked to HLA would also show linkage
to Chido.
The description of anti-Rodgers, a second antibody reactive with an RBC antigen of high prevalence that could be
neutralized by plasma, followed in 1976.6 The antibody was
formed by a transfused patient whose RBCs were Chidopositive who also made an anti-E. The anti-Rga (as anti-Rg
was referred to at that time) gave weak to 4+ reactions with
E– RBCs (not your typical low-avidity antibody). Using
plasma neutralization tests, Longster and Giles found that
3.1 percent of donors could not inhibit the antibody, i.e.,
were Rodgers-negative, although they found some donors
who were only partial inhibitors. Nordhagen et al.7 were
also able to demonstrate partial inhibition of anti-Chido by
the plasma of Chido-positive subjects, and Giles8 showed
that partial inhibition was an inherited trait. These findings
would explain conflicting results in tests on Chido/Rodgers
antibodies among laboratories if a single source of plasma
was used in inhibition tests. The common practice now is to
use a pool of inert plasmas when attempting to determine
whether an unknown antibody is neutralizable by human
plasma.
Rodgers was also shown to be linked to HLA in two
studies in 1976 that set the stage for the discovery of the
relationship of Chido and Rodgers to the C4 protein.9,10 In
1978, O’Neill et al.11 showed that Chido and Rodgers are antigenic determinants expressed on the C4 complement protein. Then Tilley et al.12 showed that the Chido and Rodgers
determinants are on the C4d fragment of the C4 molecule.
The small amount of C4d normally found on the RBC membrane is the limiting factor that explains the low avidity of
anti-Chido or anti-Rodgers. In fact, if large amounts of C4d
are bound to the RBC under low ionic conditions, anti-Ch
or anti-Rg can be turned into a strongly reactive, direct agglutinin.13 The puzzle of incomplete or partial inhibition of
the antibodies is caused by the extreme polymorphism of
the C4 genes with multiple alleles that cause variations in
the Ch or Rg determinants.
All this progress made Chido and Rodgers antibodies
easier to recognize with the exception of Chido-positive
variant individuals who have made anti-Chido to the part
of the Chido antigen that they lack.14 The further serologic
complexities of Chido/Rodgers specificity will be discussed
in the section on genetics.
31
R. Mougey
The inexorable unraveling of the puzzle of the true nature of the blood group Chido/Rodgers is an excellent example of the importance of recognizing anomalous findings
in the study of antibodies that most blood bankers wished
could just be ignored! The final refutation of the term HTLA
came from the revelation that the Knops, McCoy, SwainLangley, and York blood group sera recognize antigens that
are carried on the RBC complement receptor CR1.15,16 However, this finding seemed to have brought a level of kinship
to these antibodies with Chido and Rodgers that must have
been satisfying to those who wanted to believe that Chido,
Knops, etc. must be related. Again the low avidity of most
Knops antibodies is probably related to the low copy number of the individual’s CR1 receptor. It also seems likely that
these blood groups will be forever linked in the literature as
those HTLAs when they are studied by aspiring blood bankers.
Clinical Significance of Chido/Rodgers
It is well documented that many patients with Chido/
Rodgers antibodies have been safely transfused with “random” RBC units, but there are also reports of significant reactions to plasma or platelet transfusions in patients with
anti-Chido or -Rodgers,17,18 a cautionary incident reminding blood bankers not to ignore the potential significance of
an antibody based on specificity alone. Strupp et al.19 also
pointed out the danger in assuming that antibodies that
appear to be of low avidity may be safely ignored without
further investigation. They reported 4 patients with sickle
cell disease for whom the provision of compatible blood and
benefit of transfusion were delayed because Dombrock antibodies were not initially recognized and identified. However, there is a need to determine the best way to detect and
identify these antibodies without incurring unnecessary
delays and expense to the patient, but how much testing is
enough? It sometimes seems that when we had the time,
resources, and people to detect and identify these antibodies, we did not have a clue what they were. Now we know all
about them and do not have the time, people, or resources
to do much with them. In this area as with so many other
blood group issues, perhaps molecular methods will identify
good patient practices, and with the use of microarray technology, definitive answers to some of these thorny issues
of cost versus benefit will be found. Until that time, when
patients with known anti-Chido or anti-Rodgers require
RBC transfusions, antibody detection, identification, and
crossmatching methods should include plasma-neutralization studies. After all, these patients are known antibody
formers. If transfusion of plasma components is required,
there should be some consideration given to the possible
effects on the patient of receiving a large volume of plasma,
such as the formation of precipitating immune complexes
or anaphalaxis.17,18 Finally, the presence of antibodies to C4
proteins may indicate a potential disease association such
as is seen with the patients who have anti-Ch and anti-Rg
and are C4-deficient.
32
The story of Chido and Rodgers blood groups has moved
well beyond the “crossmatching difficulties,” and there is
now a wealth of new material that shows the C4 genes and
proteins to be a fascinating model for the evolution of the
genes for critical biologic processes that can both protect
and attack their host organism.
Structure and Function of the C4 Protein
The C4 protein expressing the Chido or Rodgers
epitopes occurs in two isoforms, called C4A for acidic (formerly C4F) and C4B for basic (formerly C4S).20 Awdeh and
Alper20 proposed the change from F for fast and S for slow
to conform with the then new International System for Human Gene nomenclature and because in their experiments
sialic acid was removed from plasma proteins before electrophoresis, resulting in distinct but different bands from
those in the experiments of O’Neill et al.,11 which were the
first to link the Chido/Rodgers antigen to C4 proteins. C4A
proteins usually carry the Rodgers antigens and C4B, the
Chido antigens, although a number of haplotypes have been
discovered in which these associations were switched.21,22
C4A or C4B protein is expressed as a single-chain precursor of 1744 amino acid residues, and the C4A or C4B
amino acid sequences are nearly identical (99%) except near
residues 1000 to 1200.23 The amino acid substitutions that
occur at residues 1101 to 1106 distinguish C4A from C4B.
Single-nucleotide polymorphisms (SNP) are also found in
the region that distinguishes the alleles of C4A from each
other and from C4B and its alleles. The Chido and Rodgers
antigens are thought to differ by both amino acid substitution as well as conformational binding sites.24
After translation, the pro-C4 protein is cleaved into alpha, beta, and gamma polypeptide chains that are linked
by disulfide bonds.25 However, incomplete processing actually leads to multiple structural forms of the peptide being secreted in the plasma.26 The activated complement
component C1 initiates cleavage of 77 amino acids from
the N-terminus of the alpha chain, releasing C4a, and the
remainder of the molecule C4b changes shape, exposing
a thioester bond between the sulfhydryl group of Cys-991
and the carbonyl group of Gln-994. This binding site that
is exposed on activation will allow the formation of either
a covalent ester or an amide linkage.27,28 Activated C4B is
more efficient than C4A at pathogen cell lysis because of its
histidine residue at position 1106 and its serine residue at
position 1102.29
The liver is the primary site of C4 production, with
smaller amounts produced by macrophages in other tissues.30 There is a wide variation in the amount of C4 produced among individuals. The concentration of C4 in the
plasma varies from 80 to 1000 µg/mL with variable concentrations of C4 isotypes.31 Moulds et al.32 found variation
in patterns of C4 protein levels in individuals that showed
either a complete absence of C4A or C4B, more C4A than
C4B, more C4B than C4A, or equal amounts of both. There
is some correlation between C4 gene copy number variations and certain polymorphisms with the level of C4 protein produced, but the effect at the haplotype level can be
lost in the diploid gene expression.33
C4 can be activated via the classical pathway or the
mannan-binding lectin pathway, although this may be a
simplistic view as a recent paper demonstrated the potential for localized release of anaphylatoxins C3a and C4a by
tryptase under certain conditions such as those found in the
airways of asthmatics.34 Although it is simpler to consider
organized pathways in which complement proteins perform
their complex dance with the other parts of the innate and
humoral immune response, it is likely that the protection
or attack effect is far more chaotic and layered in multiple
mechanisms that activate, block, or break down these proteins (see discussion on disease association).
In the classical pathway, C4 is processed by activated
C1, which removes C4a anaphylatoxin from the alpha chain.
C4a is a mediator of local inflammation and can induce the
contraction of smooth muscle, increase vascular permeability, and cause histamine release from mast and basophilic
WBCs. C4a levels along with the anaphylatoxins C3a and
C5a in plasma were shown to be elevated during normal
pregnancy as an indication of the increased activation of the
complement system, possibly as a protection against infection.35
The remaining C4b fragment forms the enzymes C3
convertase (C4bC2a) and C5 convertase (C3bC4bC2a).
These enzymes catalyze the membrane attack proteins,
which open a channel into the cell membrane. C4 binding
protein (C4bp) is another plasma protein that regulates C4
activity by blocking the formation of and promoting the
decay of the classical pathway C3 convertase. Deficiency of
C4bp would be expected to result in increased activation of
C3 and complement activity in response to classical pathway or lectin pathway activation by immune complex formation, bacterial infections, apoptosis, and other triggering
mechanisms.36
Activated C4B (C4b-B) binds to its target rapidly but
has a short half-life of less than 1 second and has greater
hemolytic activity than C4A.28 Activated C4A (C4b-A) is
10 times slower than C4B, but it forms amide bonds with
amino group targets of immune complexes (IC) or protein
antigens. C4B more efficiently forms ester bonds with carbohydrate antigens, resulting in better clearance of pathogens.
Besides activation of the complement cascade via the
classical or lectin pathways, the covalent binding of C4 to
immunoglobulins and to IC enhances the solubilization of
immune aggregates and the clearance of IC by binding to
complement receptor on RBCs (CR-1). This IC binding on
the RBC then acts as a shuttle and delivers the IC for phagocytosis by macrophages. Thus C4 plays multiple roles for
both the innate and adaptive immune systems in vertebrate
animals.37
Part of this multiple role is the normal constant low
level of activation of C3 via the classical pathway and the
alternate pathway. In the classical pathway, it was thought
that specific antigen binding by an antibody capable of activating C1 would initiate the pathway, but Manderson et
al.38 have shown that C3 is also being activated by C1–C4 in
the absence of antibody-antigen complexes. They propose
that although circulating constant low levels of IC could
account for their findings, the continual circulation of the
normal levels of IgG antibodies may form “spontaneous unstable Ig aggregates that would provide for a C3 tickover” as
well. In their experiments, vascular stasis would encourage
or initiate complement activation and would therefore be
augmented at sites of inflammation. This would facilitate
pathogen recognition by allowing the deposition of activated
C3 fragments onto foreign surfaces (host cell surfaces with
complement regulatory proteins would be unaffected).
Thus the various polymorphisms of C4 affect the normal
functioning of C4 and can be affected by the individual’s
ability to produce suitable levels of C4 proteins. Deficiencies
of C4 proteins whether acquired or inherited can increase
the risk of severe viral and bacterial infections.33 How high
levels of C4 may also contribute to the risk of diseases is
just beginning to be understood (disease association will be
discussed later).
Genetics of C4 and Chido/Rodgers
The original notation of Cha and Rga implied the expectation that there would be additional antithetical alleles Chb and Rgb.1,6 However, it was soon realized that this
blood group did not fit this pattern of inheritance. Chido,
or Ch, and Rodgers, or Rg, are used to describe the cluster
of determinants that are expressed on the C4A or C4B proteins that can neutralize the antibodies formed by Chido- or
Rodgers-negative individuals. Also individuals can type as
positive with anti-Chido, but form anti-Chido to the Chido
epitopes they lack. Currently nine antigens have been described: six of high prevalence for Chido, two of high prevalence for Rodgers, and one of low prevalence, WH.24 Eight
phenotypes for the CH/RG system, with their respective occurrences, are shown in Table 1 as the prevalence for the
other antigen combinations has not been established (Table
1).39 The polymorphisms for the Ch and Rg epitopes as well
Table 1. Prevalence of Chido/Rodgers phenotypes in random samples
Chido
phenotype
Prevalence (%)
Rodgers
phenotype
Prevalence (%)
Ch:1,2,3
Ch:1,–2,3
88.2
Rg:1,2
95.0
4.9
Rg:1,–2
2.5
Ch:1,2,–3
3.1
Rg:–1,–2
2.5
Ch:–1,–2,–3
3.8
Rg:–1,2
(proposed but
Ch:–1,2,–3
Rare
Ch:–1,2,3
(proposed but
Ch:–1,–2,3
may not exist)
may not exist)
33
R. Mougey
as the polymorphisms for the electrophoretic behavior of
the C4 protein confounded the early investigations on the
inheritance of the C4 genes. No attempt is going to be made
by this author to explain the reported phenotypic frequencies of Ch and Rg antigens with the current understanding
of the multiple copy number variation of the C4 gene.
In early studies of the inheritance of Chido/Rodgers,
it appeared that the genes must be encoded by two closely
linked loci. The majority of persons were Chido-positive and
Rodgers-positive. Approximately 1.7 percent of those studied were Chido-negative, Rodgers-positive, and 3 percent
were Chido-positive, Rodgers-negative.3,6 After the linkage
of Chido/Rodgers with HLA was established and the discovery of C4 as the plasma protein carrier was made, family
studies detecting both serologic allelic forms and electrophoretic alleles seemed initially to be in agreement with
this model. It was proposed that null genes or duplicated
genes for C4A or C4B led to lack of Rodgers or Chido.40
As methods and antisera improved, numerous C4 alleles were discovered and the results of a workshop to
define and standardize nomenclature were published in
a joint paper by Mauff et al.41 They described 14 determinants for the C4A gene and 17 for the C4B gene. Genes that
express no C4A or C4B are designated with a *Q0 (meaning quantity naught). In patients with complete deficiency
of C4 (C4A*Q0/B*Q0), null alleles were thought to be attributable to gene deletions or point mutations that cause
a frameshift in the DNA sequence, leading to defective or
no expression of the protein. One such example is a 2-bp
insertion into the sequence for codon 1213 at exon 29 of the
mutant C4A gene seen in both Caucasians and those of African descent, but very rarely in those of Asian descent.42
The origin of the complement system C3 and C4 proteins predates the ability to make lymphocytes or antibodies; the latter did not arise on the evolutionary scene until
cartilaginous fish and sharks.43 Homology between C3 and
C4 proteins indicates that the C4 gene may have evolved
from C3 as a duplicated gene.44 The C4A gene is thought
to have arisen by gene duplication and mutation from C4B
because of its efficiency in facilitating the clearance of IC.28
The C4A or C4B gene consists of 41 exons that in addition to encoding for an acidic or basic isotype can also
exist as a long form of 20.6 kb or a short form of 14.2 kb.45
The long form has an insertion of a human endogenous retrovirus (HERV-K[C4]) into intron 9. The insertion of this
retroviral material is thought to have occurred after duplication of the ancestral mammalian C4A gene but before the
divergence of humans from the ancestors of other primates.
The reading frames of the retrovirus are all closed, but
there is still promoter activity and the HERV-K(C4) insert
is thought to be able to modulate the expression of the C4
gene.46 The long form was originally thought to be a C4A
gene, but C4 genes can be C4A (long or short) or C4B (long
or short). However, most C4A genes have the HERV-K(C4)
insert and a greater proportion of C4B genes do not.
34
The genes for C4A and C4B were shown to be located
between HLA-DR and HLA-B loci on the short arm of chromosome 6 along with C2 and factor B.40,47 C4A and C4B
proteins are distinguished phenotypically by their migration patterns in electrophoretic gels and genetically by four
nucleotide substitutions that encoded changes at residues
1101 to 1106 where C4B encodes for LSPVIH and C4A encodes for PCPVLD at the protein level. C4A and C4B are
also distinguished phenotypically by their expression of either the Chido or the Rodgers antigen, which are the result
of two amino acid substitutions at residues 1188 to 1191.
The Ch1 epitope has ADLR and the Rg1 has VDLL. As data
from family studies began to contradict a simple two-gene
model, it was realized that a haplotype could have one, two,
three, or four C4 genes and that the C4 genes were inherited as part of a complex of genes.48 The two-gene theory had
to give way to the current hypothesis of the inheritance of a
haplotype modular gene complex that can vary by C4 copy
number and is part of a larger gene complex in the MHC
locus. This model explains both the qualitative and quantitative nature of the C4 gene expression as regards epitopes,
concentration of the C4 protein in the plasma, the frequency
of certain haplotypes, and the association of C4 copy number
variation with certain diseases. In fact, the C4 gene as part of
the MHC complex can serve as an example of how the genes
of an important biologic pathway evolve via the mechanics of
meiosis and through the pressure of natural selection to conserve important genes and to maximize the utility of polymorphism potential. Gene duplication, translocation, singlenucleotide polymorphisms, insertions, and deletions all have
played a role in the plastic nature of the C4 gene.
Both forms of C4 are highly homologous but either
by SNP or by insertions or deletions display great qualitative and quantitative diversity. The copy number variation
comes from whether an individual has inherited haplotypes
that have one, two, three, or four C4 genes.49 This gene cluster occurs as a complex in the MHC locus near the centromere
with flanking genes in the following order: first an intact
RP gene (a serine/threonine kinase gene), an intact C4
gene (either C4A or C4B), then an intact CYP21B gene (cytochrome P450 21-hydroxylase gene), and an intact TNXB
(tenascin-X protein). Approximately 17 percent of persons
of Northern European ancestry have this haplotype, termed
monomodular.50 In 83 percent of those of Northern European ancestry the C4 gene complex can contain one, two,
or three duplicated gene clusters made of fragments or mutations of the above flanking genes and an intact C4 gene.
The duplicated gene complex, called RCCX, occurs between
the C4 gene and the intact CYP21B gene and consists of a
CYP21 (usually a mutated form) fused to a 4.9-kb fragment
of TNXA fused to an RP2 0.91-kb fragment and an intact
C4 (A or B and long or short). Depending on the number of
RCCX modules duplicated, the complete C4 gene may be a
monomodular (no RCCX duplication), bimodular, trimodular, or quadrimodular form.
This modular gene duplication maximizes opportunities to conserve the C4 gene, whereas the opportunity for
recombination allows greater diversity of polymorphisms.
This great diversity should allow for a better response to
pathogens but can create opportunities for unequal crossing over during meiosis. Thus low or high copy number
variations could be as important a risk factor for disease as
the actual C4 polymorphism.
C4 gene copy number studies done on healthy subjects
of different ages indicate that those individuals with a low
copy number for C4B may be less healthy or have a shorter
lifespan than those having more than two copies of the C4B
gene.51 These studies found that in healthy individuals older than 50 years, there was a lower incidence of low copy
number for the C4B gene when compared with the low copy
number gene frequency in younger healthy individuals.
C4 and Disease Associations
The association of C4 and a number of diseases has been
a fruitful area of investigation, and at the genetic level, the
frequency of C4 nulls or low copy variation shows a solid
association with autoimmune disease, particularly systemic
lupus erythematosus (SLE).52 Although the varying efficiencies of complement function that result from partial or complete deficiency may have a profound effect on autoimmune
susceptibility, above-average C4 protein levels may also increase risk. Once a loss of self-tolerance occurs, having high
levels of C4 may exacerbate the disease. Moreover, it cannot
be overstated that association is not the same as causation.
SLE can be thought of as a prototype for the intricacies of
the interaction of C4 proteins both in the development of
autoimmunity and in disease progression. Inheritance of a
C4A*Q0 null gene shows a greater risk in Caucasian and
African populations, but C4B*Q0 nulls are more frequent
in SLE patients of Asian descent.53 SLE is a heterogeneous
disease that is characterized by the presence of multiple autoantibody specificities that attack various tissues, resulting in tissue damage, which then presents more antigen to
the host’s immune system. It has become increasingly clear
that multiple genetic pathways are likely involved and that
C4 polymorphism, especially copy number variation, can
be a negative or a positive risk factor in the development
of autoimmune diseases.54 This postulates that the function
of C4 in the initiation of autoimmunity may be related to
its role in presenting antigen to the host immune system,
which is separate from the role of complement in the inflammatory process once autoantibodies have been formed.
The current understanding of SLE genetics is that there are
distinctions in severity, risk, and clinical manifestation that
vary by ethnicity, geography, and sex with a greater prevalence in women and some non-Caucasian ethnic groups.
Although genetics undoubtedly plays a role, SLE may also
be associated with poverty and exposure to infectious diseases.55,56
Progress both in human genetics, particularly by
genome-wide association studies, and in the understanding of biologic pathways has led to the discovery of more
than 20 different loci that show strong correlation to SLE.54
Most of the genes identified are involved in three kinds of
biologic processes. Minor variations in the genes encoding
the proteins of IC processing, production of interferon, or
signal transduction of immune cells can greatly increase the
susceptibility of an individual to SLE. It is to be expected
that more than one kind of trigger, such as pathogens, allergens, or trauma, could then lead to disease.
Inasmuch as a major function of the C4 protein is participation in innate immunity, there is a strong
association between deficiencies of C4 and recurrent infections; although this is less frequent than other complement deficiencies.57 Table 2 lists other reported diseases
and their possible association with C4 or complement.
Table 2. Disease associations with low levels of C4/complement
Date
Disease
Citation
2009
Schizophrenia
Shi58
2008
Role of complement in schizophrenia
Mayilyan59
2009
Chronic fatigue syndrome
Sorenson60
2008
Autism and missing or nonfunctional C4B
gene
Sweetehn61
2008
Alzheimer’s and increased C4 deposition
Zhou62
2008
Increased morbidity/mortality associated
with low expression of C4B, smokers, and
stroke
Szilagyi63
2007
Cardiovascular disease, smokers, and low
expression of C4B
Arason64
2006
C4 nulls and adult rhinosinusitis
Seppänen65
2002
Systemic sclerosis
Arason66
1996
Insulin-dependent diabetes and C4 polymorphisms
Lhotta67
1993
Graves’ disease
Ratanachaiyavong68
1991
C4A deficiency and IgA nephropathy
Wyatt69
1990
Felty’s syndrome
Hillarby70
1989
Psoriasis
Wyatt 71
1989
Rheumatoid arthritis
Fielder72
1988
C4 allotypes in juvenile dermatomyositis
Robb73
1987
C4B2 and primary biliary cirrhosis
Briggs74
1984
Glomerulonephritis, Henoch-Schönlein
purpura
McLean75
In their paper, Atkinson and Frank43 also warn that patients with complement C2- or C4-deficient states (either
acquired or inherited) have backup complement activation
mechanisms that must be considered in defining the role of
complement and a disease. For example, the immune response of specific antibody produced to a pathogen (antibody
35
R. Mougey
excess) may be all that is needed to trigger more ancient
pathways to perform cell lysis in the absence of sufficient
complement proteins.
Summary
The C4 protein plays an important role in maintaining
health and, in some situations complicated by poor expression of the C4 protein, may lead to or exacerbate certain
diseases. The blood groups Chido and Rodgers are epitopes
on the C4 protein, and polymorphisms associated with
these epitopes may lead to the formation of antibodies to
the Chido or Rodgers antigens in transfused patients. Identification of anti-Ch or anti-Rg is still based on the antibody
neutralization with plasma from Ch-positive or Rg-positive
individuals and lack of reactivity with qualified Ch-negative
or Rg-negative RBCs. These antibodies may be useful in
genetic studies of C4 polymorphisms or, in the case of C4deficient patients, a signal of the potential for serious illnesses. The recognition of the extreme polymorphism of the
C4 gene and the gene complex RCCX should lead to more
insights in the understanding of disease risk and potential
treatment.
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60.
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Ruth Mougey MT(ASCP)SBB, President, Mougey, Incorporated, 1705 Highland Avenue, Carrollton KY 41008.
In Memoriam: Marie Cutbush Crookston
1920–2009
D. Mallory
Marie Cutbush Crookston, a dynamic person and exceptional scientist, was born in 1920 in Victoria, Australia.
She received a Bachelor of Science degree from the University of Melbourne, and, in 1947, she set sail for England.
There, she worked for 10 years with Dr. P. L. Mollison at
the Medical Research Council Blood Transfusion Research
Unit and contributed to the writing of the first edition of Dr.
Mollison’s Blood Transfusion in Clinical Medicine.
In 1957, she married Dr. John Crookston, a hematologist from Toronto, who was in London working for Dr. J. V.
Dacie. Giving up her studies for a PhD, she moved to Toronto to raise two sons and to continue to work in the field
of immunohematology. In 1964 she joined the University
of Toronto, Department of Pathology, where she directed
research, and in 1978 she became an associate professor.
She also served as immunohematologist in the Department
of Hematology at the Toronto General Hospital and acted
as a consultant for the Blood Transfusion Laboratory until
she retired in 1986.
Marie’s contributions to immunohematology are outstanding and have made a permanent impact on the field.
She and her late husband John helped transform the educational level and direction of immunohematology in the
1960s when they returned to Toronto from London. There,
they helped to organize the Ontario Antibody Club, which
was active for 25 years.
Marie’s studies on conversion of incomplete to complete
antibodies by chemical modification were used by commercial companies to make chemically modified Rh antiserum.
In addition, she was known for her discoveries of anti-Fya
and anti-Lub and her early work in exchange transfusion
of the newborn, long-term preservation of RBCs by freezing, and linkage of Chido and HLA. She and John Crookston also coined the term HEMPAS to describe an inherited
chronic hemolytic anemia with multinucleated cells in the
bone marrow associated with a positive acidified serum test
(modified Ham’s solution).
Everyone who knew Marie and read any of her 65 published papers remembers her wonderful ability to write in
plain intelligible English, or as she would say, “deathless
prose,” and how she kept strictly to proper blood group terminology. Her papers were always accepted as submitted.
She was on the editorial boards of Immunohematology,
Journal of Blood Group Serology and Education, Transfusion, and Vox Sanguinis.
These are some of her awards:
• The Karl Landsteiner Memorial Award in 1976 with Dr.
Eloise Giblett from the American Association of Blood
Banks, awarded “for her discoveries of anti-Fya, antiLub, and anti-HEMPAS: for her studies of the relationship between blood-group antibody activity in vitro and
in vivo: for her description of red cell uptake of Lea and
Leb antigens from plasma; for her investigation of the
Chido antigen in plasma; and for the detection of the
linkage between Ch and HLA.” (quotation from citation
on award)
• The Nuffield Foundation Award in 1952 with Dr. Hermann Lehmann for studying the blood groups of tribes
in the Nilgiri Hills of South India.
• The Buchanan Award in 1980 from the Canadian Society of Transfusion Medicine.
• The Canadian Blood Services Award in 2002 for “Lifetime Achievement.”
Marie always found the time to help students and scientists who needed directions with their studies or help with
their manuscripts. She was a wonderful, caring person who
was an enthusiastic, intelligent, and brilliant scientist and
one who will be missed by her many friends and colleagues
around the world.
39
In Memoriam: Eloise Giblett 1921–2009
D. Mallory
Eloise “Elo” Giblett was born in Tacoma, Washington,
in 1921 and, in 1931, the family moved to Spokane. Elo developed a keen interest in music at an early age, taking piano, dance, and violin, the instrument on which she would
focus, becoming concertmaster of her high school orchestra. She put aside music, turned to science, and received a
BS in bacteriology from the University of Washington (U
of W) in 1942. World War II had started, so Elo joined the
WAVES in 1944, became a medical laboratory technician,
and served until 1946. She returned to the U of W and received her MS degree in microbiology in 1947 and then, in
1951, graduated first in her class in the second class to graduate from the new medical school at the U of W.
During the medical internship and hematology fellowship at U of W, her interest in human genetics was encouraged. In 1955, she became the first full-time physician at
King County Blood Bank (now the Puget Sound Blood Center) as the Associate Director of the Typing and Crossmatch
Laboratory and was given a 6-month sabbatical with Dr.
Patrick Mollison at the Blood Transfusion Research Unit in
London, England. There, she studied the Lewis blood group
system, measured the rate of tagged RBC destruction, became acquainted with the clinical potential of the Coombs
antiglobulin test, and mastered the tests used at that time
in Dr. Mollison’s laboratory. When she returned to Seattle,
she began to use the antiglobulin test to find RBC antibodies and discovered anti-V and anti-Jsa.
Her numerous studies of the polymorphisms of haptoglobin and transferrins and her interest in RBC, plasma,
and protein polymorphisms resulted in the renowned 1969
publication of her book, Genetic Markers in Human Blood.
In addition, she discovered that two forms of inherited immunodeficiency disease were caused by deficiencies of the
enzymes adenosine deaminase and purine nucleoside phosphorylase.
In 1979, Dr. Giblett became the Director of the Puget
Sound Blood Center and remained so until she retired in
1987. Upon her retirement, she became Director Emeritus
of the Blood Center and Professor Emeritus of the U of W
Medical School. After 40 years, she returned to the violin
and even helped found a new music school.
Dr. Giblett was president of the American Society of
Human Genetics (1973); a member of the Advisory Board of
the American Society of Hematology (1980–1986); a member of the Editorial Board of Blood, Transfusion, and The
American Journal of Human Genetics (among a few); and
40
chairperson of the Enzyme Nomenclature Committee of the
International Workshop on Human Gene Mapping.
She received many honors during her lengthy career, including two from the American Association of Blood Banks:
• 1975—Emily Cooley Lecture Award (American Association of Blood Banks)
• 1976—Karl Landsteiner Memorial Award (American
Association of Blood Banks)
• 1978—The Philip S. Levine Award (American Society of
Clinical Pathology)
• 1980—Election, National Academy of Science
• 1987—Distinguished Alumna Award (University of
Washington)
Dr. Giblett has left a legacy of original scientific discoveries, a book that was groundbreaking for the field of immunohematology, and colleagues who were scientifically
mentored and generously helped by her excellent editorial
skills. Those who knew her will remember a very interesting
and giving human being.
Announcements
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Applications are invited from medical or science graduates for the Master of Science (MSc)
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Fax +44 1179 595 342, Telephone +44 1779 595 455, e-mail: p.a.denning-kendall@bristol.
ac.uk.
41
Announcements, cont., and advertisements
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The Department of Transfusion Medicine, National Institutes of Health, is accepting applications for its 1-year Specialist
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The New York Blood Center has developed a wide range of monoclonal antibodies (both murine and humanized) that are
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Diagnostic testing for:
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Medical consultation available
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• Granulocyte immunofluorescence by flow cytometry (GIF)
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For further information contact:
Neutrophil Serology Laboratory (651) 291-6797
Randy Schuller(651) 291-6758
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Musser Blood Center
700 Spring Garden Street
Philadelphia, PA 19123-3594
CLIA licensed
42
Neutrophil Serology Laboratory
100 South Robert Street
St. Paul, MN 55107
CLIA licensed
Advertisements, cont.
Reference and Consultation Services
Antibody identification and problem resolution
HLA-A, B, C, and DR typing
HLA-disease association typing
IgA/Anti-IgA Testing
IgA and anti-IgA testing is available to do the
following:
Paternity testing/DNA
• Identify IgA-deficient patients
• Investigate anaphylactic reactions
• Confirm IgA-deficient donors
For information, contact
Our ELISA for IgA detects protein to 0.05 mg/dL.
Mehdizadeh Kashi at (503) 280-0210, or write to:
Pacific Northwest Regional Blood Services
For additional information contact Cindy Flickinger at
(215) 451-4909, or e-mail: [email protected],
ATTENTION: Tissue Typing Laboratory
or write to:
American Red Cross
3131 North Vancouver
Portland, OR 97227
CLIA licensed, ASHI accredited
Musser Blood Center
ATTN: Cindy Flickinger
National Reference Laboratory
for Blood Group Serology
Immunohematology Reference Laboratory
AABB, ARC, New York State, and CLIA licensed
(215) 451-4901— 24-hr. phone number
(215) 451-2538— Fax
American Rare Donor Program
(215) 451-4900— 24-hr. phone number
(215) 451-2538— Fax
[email protected]
Immunohematology
CLIA licensed
Donor IgA Screening
• Effective tool for screening large volumes of donors
• Gel diffusion test that has a 15-year proven track record:
Approximately 90 percent of all donors identified as IgA deficient by are confirmed by the more sensitive testing methods
For additional information, call Kathy Kaherl at:
(860)678-2764, e-mail: [email protected]
(215) 451-4902— Phone, business hours
(215) 451-2538— Fax
[email protected]
or write to:
Quality Control of Cryoprecipitated-AHF
(215) 451-4903— Phone, business hours
(215) 451-2538— Fax
Reference Laboratory
American Red CrossBiomedical Services
Connecticut Region
209 Farmington Ave.
Farmington, CT 06032
CLIA licensed
43
Blood Group
Antigens &
Antibodies
A guide to clinical relevance
& technical tips
by Marion E. Reid & Christine Lomas-Francis
This compact “pocketbook” from the authors of the Blood
Group Antigen FactsBook is a must for anyone who is involved
in the laboratory or bedside care of patients with blood
group alloantibodies.
The book contains clinical and technical information about
the nearly 300 ISBT recognized blood group antigens and
their corresponding antibodies. The information is listed in
alphabetical order for ease of finding—even in the middle of
the night. Included in the book is information relating to:
•Clinical significance of antibodies in transfusion and HDN.
•Number of compatible donors that would be expected
to be found in testing 100 donors. Variations in different
ethnic groups are given.
•Characteristics of the antibodies and optimal technique(s)
for their detection.
•Technical tips to aid their identification.
•Whether the antibody has been found as an autoantibody.
Pocketbook Education Fund
The authors are using royalties generated from the sale of
this pocketbook for educational purposes to mentor people
in the joys of immunohematology as a career. They will
accomplish this in the following ways:
•Sponsor workshops, seminars, and lectures
•Sponsor students to attend a meeting
•Provide copies of the pocketbook
(See www.sbbpocketbook.com for details to apply for funds)
44
Ordering Information
The book, which costs $25, can be
ordered in two ways:
•Order online from the publisher
at: www.sbbpocketbook.com
•Order from the authors, who
will sign the book. Send a check,
made payable to “New York Blood
Center” and indicate “Pocket
book” on the memo line, to:
Marion Reid
Laboratory of Immunochemistry
New York Blood Center
310 East 67th Street
New York, NY 10065
Please include the recipient’s
complete mailing address.
Becoming a Specialist in Blood Banking (SBB)
What is a certified Specialist in Blood Banking (SBB)?
• Someone with educational and work experience qualifications who successfully passes the American Society for Clinical Pathology (ASCP)
board of registry (BOR) examination for the Specialist in Blood Banking.
• This person will have advanced knowledge, skills, and abilities in the field of transfusion medicine and blood banking.
Individuals who have an SBB certification serve in many areas of transfusion medicine:
• Serve as regulatory, technical, procedural and research advisors
• Perform and direct administrative functions
• Develop, validate, implement, and perform laboratory procedures
• Analyze quality issues preparing and implementing corrective actions to prevent and document issues
• Design and present educational programs
• Provide technical and scientific training in blood transfusion medicine
• Conduct research in transfusion medicine
Who are SBBs?
Supervisors of Transfusion Services
Supervisors of Reference Laboratories
Quality Assurance Officers
Why be an SBB?
Professional growth
Managers of Blood Centers
Research Scientists
Technical Representatives
Job placement
LIS Coordinators
Consumer Safety Officers
Reference Lab Specialist
Job satisfaction
Educators
Career advancement
How does one become an SBB?
• Attend a CAAHEP-accredited Specialist in Blood Bank Technology Program OR
• Sit for the examination based on criteria established by ASCP for education and experience
However: In recent years, a greater percentage of individuals who graduate from CAAHEP-accredited programs pass the SBB exam.
Conclusion:
The BEST route for obtaining an SBB certification is …
to attend a CAAHEP-accredited Specialist in Blood Bank Technology Program
Contact the following programs for more information:
Program
Contact Name
Phone Contact
Email Contact
Website
On site
or On line
Program
Walter Reed Army Medical Center
William Turcan
202-782-6210
[email protected].
MIL
www.militaryblood.dod.mil
On site
American Red Cross, Southern California Region
Michael Coover
909-859-7496
[email protected]
none
On site
ARC-Central OH Region
Joanne Kosanke
614-253-2740 x 2270
[email protected]
none
On site
Blood Center of Southeastern Wisconsin
Lynne LeMense
414-937-6403
[email protected]
www.bcw.edu
On site
Community Blood Center/CTS Dayton, Ohio
Nancy Lang
937-461-3293
[email protected]
http://www.cbccts.org/education/
sbb.htm
On line
Gulf Coast School of Blood Bank Technology
Clare Wong
713-791-6201
[email protected]
www.giveblood.org/education/
distance/htm
On line
Hoxworth Blood Center, Univ. of Cincinnati
Susan Wilkinson
513-558-1275
[email protected]
www.hoxworth.org
On site
Indiana Blood Center
Jayanna Slayten
317-916-5186
[email protected]
www.indianablood.org
On line
Johns Hopkins Hospital
Jan Light
410-955-6580
[email protected]
http://pathology2.jhu/
department/divisions/trnafusion/
sbb.cfm
On site
Medical Center of Louisiana
Karen Kirkley
504-903-3954
[email protected]
none
On site
NIH Clinical Center Dept.. of Transfusion Medicine
Karen Byrne
301-496-8335
[email protected]
www.cc.nih.gov/dtm
On site
Rush University
Veronica Lewis
312-942-2402
[email protected]
www.rushu.rush.edu/health/dept.
html
On line
Transfusion Medicine Center at Florida Blood
Services
Marjorie Doty
727-568-5433 x 1514
[email protected]
www.fbsblood.org
On line
Univ. of Texas Health Science Center at San Antonio
Linda Myers
210-731-5526
[email protected]
www.uthscsa.edu
On site
Univ. of Texas Medical Branch at Galveston
Janet Vincent
409-772-3055
[email protected]
www.utmb.edu/sbb
On line
Univ. of Texas SW Medical Center
Barbara LairdFryer
214-648-1785
[email protected]
http://telecampus.utsystem.edu
On line
Additional Information can be found by visiting the following Web sites: www.ascp.org, www.caahep.org and www.aabb.org
Revised August 2007
45
Immunohematology
Immunohematology
Journal
SerologyAND
andEDUCATION
Education
JOURNAL of
OF Blood
BLOOD Group
GROUP SEROLOGY
Instructions tofor
theAuthors
Authors
Instructions
I. GENERAL INSTRUCTIONS
Before submitting a manuscript, consult current issues of
Immunohematology for style. Double-space throughout the manuscript.
Number the pages consecutively in the upper right-hand corner, beginning
with the title page.
II. SCIENTIFIC ARTICLE, REVIEW, OR CASE REPORT WITH
LITERATURE REVIEW
A. Each component of the manuscript must start on a new page in the
following order:
1. Title page
2. Abstract
3. Text
4. Acknowledgments
5. References
6. Author information
7. Tables
8. Figures
B. Preparation of manuscript
1. Title page
a. Full title of manuscript with only first letter of first word capitalized
(bold title)
b. Initials and last name of each author (no degrees; all CAPS), e.g., M.T.
JONES, J.H. BROWN, AND S.R. SMITH
c. Running title of ≤40 characters, including spaces
d. Three to ten key words
2. Abstract
a. One paragraph, no longer than 300 words
b. Purpose, methods, findings, and conclusion of study
3. Key words
a. List under abstract
4. Text (serial pages): Most manuscripts can usually, but not necessarily,
be divided into sections (as described below). Survey results and
review papers may need individualized sections
a. Introduction
Purpose and rationale for study, including pertinent background
references
b. Case Report (if indicated by study)
Clinical and/or hematologic data and background serology/molecular
c. Materials and Methods
Selection and number of subjects, samples, items, etc. studied and
description of appropriate controls, procedures, methods, equipment,
reagents, etc. Equipment and reagents should be identified in
parentheses by model or lot and manufacturer’s name, city, and state.
Do not use patient’s names or hospital numbers.
d. Results
Presentation of concise and sequential results, referring to pertinent
tables and/or figures, if applicable
e. Discussion
Implication and limitations of the study, links to other studies; if
appropriate, link conclusions to purpose of study as stated in
introduction
5. Acknowledgments: Acknowledge those who have made substantial
contributions to the study, including secretarial assistance; list any grants.
6. References
a. In text, use superscript, Arabic numbers.
b. Number references consecutively in the order they occur in the text.
7. Tables
a. Head each with a brief title; capitalize the first letter of first word (e.g.,
Table 1. Results of . . .) use no punctuation at the end of the title.
b. Use short headings for each column needed and capitalize first letter
of first word. Omit vertical lines.
c. Place explanation in footnotes (sequence: *, †, ‡, §, ¶, **, ††).
8. Figures
a. Figures can be submitted either by e-mail or as photographs (5″ × 7″
glossy).
b. Place caption for a figure on a separate page (e.g. Fig. 1 Results of...),
ending with a period. If figure is submitted as a glossy, place first
author’s name and figure number on back of each glossy submitted.
c. When plotting points on a figure, use the following symbols if
possible: � � � � � �.
9. Author information
a. List first name, middle initial, last name, highest degree, position held,
institution and department, and complete address (including ZIP
code) for all authors. List country when applicable.
III. EDUCATIONAL FORUM
A. All submitted manuscripts should be approximately 2000 to 2500
words with pertinent references. Submissions may include:
1. An immunohematologic case that illustrates a sound investigative
approach with clinical correlation, reflecting appropriate collaboration
to sharpen problem solving skills
2. Annotated conference proceedings
B. Preparation of manuscript
1. Title page
a. Capitalize first word of title.
b. Initials and last name of each author (no degrees; all CAPs)
2. Text
a. Case should be written as progressive disclosure and may include the
following headings, as appropriate
i. Clinical Case Presentation: Clinical information and differential
diagnosis
ii. Immunohematologic Evaluation and Results: Serology and
molecular testing
iii. Interpretation: Include interpretation of laboratory results,
correlating with clinical findings
iv. Recommended Therapy: Include both transfusion and
nontransfusion-based therapies
v. Discussion: Brief review of literature with unique features of this
case
vi. Reference: Limited to those directly pertinent
vii. Author information (see II.B.9.)
viii. Tables (see II.B.7.)
IV. LETTER TO THE EDITOR
A. Preparation
1. Heading (To the Editor)
2. Title (first word capitalized)
3. Text (written in letter [paragraph] format)
4. Author(s) (type flush right; for first author: name, degree, institution,
address [including city, state, Zip code and country]; for other authors:
name, degree, institution, city and state)
5. References (limited to ten)
6. Table or figure (limited to one)
Send all manuscripts by e-mail to [email protected]
I M M U N O H E M A T O L O G Y, V O L U M E 2 2 , N U M B E R 4 , 2 0 0 6
46
215
Musser Blood Center
FIRST CLASS
U.S. POSTAGE
PAID
SOUTHERN MD
PERMIT NO. 4144

Vol. 26, No. 1, 2010

Transcription

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