Escolar Documentos
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25
Celebrating
Years
Volume 25, Number 2, 2009
Immunohematology
Journal of Blood Group Serology and Education
Volume 25, Number 2, 2009
Contents
39 Invited Review: Milestones Series
Milestones in laboratory procedures and techniques
M.E. Reid
44 Case Report
Anti-Kpa–induced severe delayed hemolytic transfusion reaction
R. Koshy, B. Patel, and J.S. Harrison
48 Review
The ABO blood group system revisited: a review and update
J.R. Storry and M.L. Olsson
60 Case Report
Clinical evaluation for lymphoproliferative disease prompted by
finding of IgM warm autoanti-IT in two cases
R.M. Leger, F. Lowder, M.C. Dungo, W. Chen, H.M. Mason, and G. Garratty
63 Original Report
Southeast Asian ovalocytosis is associated with increased expression
of Duffy antigen receptor for chemokines (DARC)
I.J. Woolley, P. Hutchinson, J.C. Reeder, J.W. Kazura, and A. Cortés
67 Review
Sickle cell disease: a review
S.D. Roseff
75 Report
Consortium for Blood Group Genes (CBGG): 2008 report
G.A. Denomme, C.M. Westhoff, L. Castilho, and M.E. Reid for the CBGG
81 Special Dedication
W. John Judd, FIBMS, MIBiol
D. Mallory
83 Announcements
84 Advertisements
Editorial Board
Martha R. Combs, MT(ASCP)SBB Christine Lomas-Francis, MSc Marion E. Reid, PhD, FIBMS
Durham, North Carolina New York City, New York New York City, New York
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
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W
hen I left England in 1969, hemagglutination was tory used plasma-suspended RBCs, which neutralized most
done by sedimentation. In New York, I was intro- antibodies to Lewis and Ch/Rg antigens and thus they did
duced to centrifugation as a method to speed the not spend time identifying these clinically insignificant an-
process. Both approaches accomplished similar goals, but tibodies. Abandonment of unnecessary testing dramatically
one was more time efficient. Use of other techniques, such improved the efficiency of testing by manual hemagglutina-
as Western immunoblotting, cloning genes, and polymerase tion and did not have a noticeable negative impact on the
chain reaction, made it possible to gain much knowledge efficacy of transfusion. Other changes, which occurred with
about blood group antigens. the same goal, include using anti-IgG instead of the broad-
spectrum reagent in routine antiglobulin tests, eliminating
Immunohematology Beginnings the routine direct antiglobulin test (DAT), and not prepar-
In 1975, Sandy Ellisor and I from Central California ing eluates from all samples whose RBCs were positive in
Region of the American Red Cross (ARC) and Helen Glid- the DAT.
den from ARC Headquarters volunteered to co-edit the first Also in the late 1970s, an extraordinary number of
American Red Cross Reference Laboratory Newsletter. crossmatches were performed. Indeed, the number was ex-
The purpose of this newsletter, which was for the technolo- cessive. The concepts of a “type and screen”2 and a “maxi-
gists and by the technologists, was to share techniques and mum surgical blood order schedule”3 were introduced.
technical tips, educate, and mentor in technical writing. The These approaches were quickly adopted, and they reduced
first year (1976), the newsletter was low-tech; it was simply not only the number of crossmatches but also the number
photocopied. That year, the ARC Reference Laboratory of RBC components reserved for a particular patient. Soon
Committee ran a competition and by popular vote the news- after, the suggestion to drop the antiglobulin crossmatch
letter was renamed the Red Cell Free Press. And to improve for patients who had a negative antibody screen was un-
its appearance it was printed. This informal newsletter was leashed.4–6 Despite concerns that antibodies to antigens
published for 8 years, until in 1984 it evolved into Immuno- not expressed on screening RBCs (e.g., anti-Wra) would
hematology under the able editorship of Delores Mallory.1 be missed, this practice is now commonplace. Indeed, the
During the 25-year life of Immunohematology, techniques physical immediate-spin crossmatch is now frequently re-
evolved that allowed the gathering of an amazing amount placed by a computer crossmatch.7
of knowledge about blood groups and the membrane com-
ponents on which they are carried. Despite this evolution, Use of Enhancement Reagents and Nonserologic
to this day we still mainly rely on hemagglutination (direct Techniques
and indirect) for detection and identification of antibodies Old standby techniques such as saline and albumin fell
to blood group antigens and for testing for incompatibility. from favor and were largely replaced by low-ionic-strength
This amazing technique is hard to beat in terms of sensitivi- solution (LISS) methods in the mid-1970s8 and by LISS-ad-
ty and specificity that are appropriate for safe transfusions. ditive solutions shortly thereafter. LISS rapidly gained popu-
larity because it simultaneously reduced incubation time and
Early Immunohematology Testing Practices increased sensitivity. In 1984, the same year that the first vol-
In the era before the ARC Newsletter, it was common- ume of Immunohematology appeared, a solid-phase adher-
place to perform excessive testing (e.g., testing at room ence assay for detection of antibody-antigen reactions was
temperature, use of enzyme-treated RBCs at temperatures introduced. For a review of this technology, see Beck et al.9
below 37ºC, absorbing eluates from autoimmune hemo- Subsequently, other techniques were reported and quickly
lytic anemia cases, and performing minor crossmatches) became popular; two examples are the use of polyethylene
on a single blood sample. Fortunately, Eloise Giblett in the glycol (PEG) as a potentiator of antibody-antigen reactions
early 1960s advocated stopping unnecessary testing; she in 198710 and the column agglutination technology, using
believed testing should be done as close to physiologic con- a gel as the support medium, in 1990.11 Automated assays
ditions as possible, i.e., testing at 37ºC using plasma-sus- based on hemagglutination and manual or automated solid-
pended RBCs. Her reasoning was scholarly and logical and phase adherence assays are now in use, but they have not
changed the way testing was (and is) done. Giblett’s labora- replaced hemagglutination tests in tubes in many settings.
Many reagents became commercially available, and as biology techniques have made it possible to convert murine
compliance requirements became more stringent, labora- IgG monoclonal antibodies to human IgG or directly agglu-
tories reduced the number of reagents they prepared in- tinating IgM.20
house. Safety concerns prompted many changes. Favorite
methods fell from grace, such as the use of ether—as a re- Techniques to Predict Clinical Significance of Anti-
sult of concerns about its low flash point. Also, teratogenic, bodies
carcinogenic, and neurotoxic chemicals became less read- Hemagglutination detects antibodies, but in itself does
ily available. Awareness of the potential hazards of work- not predict their clinical significance. Criteria such as titer,
ing with certain chemicals and with blood samples that may phase of reactivity, Ig class, IgG subclass, and specificity
contain various pathogens led to the strict requirement to (and the historic knowledge of the clinical significance of an
wear personal protective equipment such as gloves, labora- antibody in other patients) are useful indicators but are not
tory coats, eye protection, and closed-toed shoes. The long- unequivocally good predictors of clinical significance. The
standing practice of drinking, eating, and smoking in the monocyte monolayer assay (MMA) is as close to conditions
laboratory was abolished. in vivo as is possible to establish in vitro and provides in-
The value of proteolytic enzymes, notably ficin, for en- sight into the potential clinical significance of an antibody.
hancing the reactivity of some antibody-antigen reactions It has been applied to predicting the clinical relevance of
while weakening or ablating others has been appreciated antibodies in transfusion21 and hemolytic disease of the
for many years.12 Other red cell modifications such as di- newborn,22–24 but is a highly specialized and technically dif-
thiothreitol (DTT), trypsin, and α-chymotrypsin13 were in- ficult test that belongs in a small number of laboratories.
troduced. However, it took some time before the full power Another technique, flow cytometry, has been used to detect
of using the combination (i.e., testing RBCs treated with minor RBC populations in a fetal maternal hemorrhage and
ficin or papain, trypsin, α-chymotrypsin, or DTT) of these to follow the survival of transfused RBCs,25,26 to determine
methods to aid in antibody identification was appreciated. the dosage of a blood group antigen on RBCs,27 and quanti-
Although classic antibody identification approaches (and tation of RBC-bound IgG.28
appropriate controls) are still necessary to name an anti- Numerous techniques have been used to identify al-
body specificity positively, a system of parallel testing of the loantibodies underlying autoantibodies.29–32 All are time-
same RBCs untreated and treated with different reagents consuming, as are methods to separate patient RBCs from
provides a valuable tool in helping to guide us in the right di- those of transfused donor RBCs so that the patient’s RBCs
rection, without requiring a vast library of rare reagents. 14,15 can be phenotyped.33 Washing peripheral blood RBCs from
When using enzymes for this purpose, it is important transfused patients with sickle cell disease with a hypoton-
to include a negative (or auto) control, as a proportion of ic solution is a simple and clever technique to obtain the
human sera contain antibodies that attach to RBCs treated patient’s RBCs for typing by hemagglutination.34 Although
with a proteolytic enzyme. these techniques have value, they also have limitations.
presence of donor RBCs in a patient’s blood sample or IgG Table 2. Potential uses of DNA-based assays
on RBCs (positive DAT) does not interfere with the tests. To type patients who have been recently transfused
RBCs are not required to type a fetus at risk for hemolytic To identify a fetus at risk for hemolytic disease of the newborn
disease of the newborn (Table 2). Thus, during the life of
To type patients whose RBCs are coated with immunoglobulin (positive
Immunohematology, we have had a reversal of phenotype DAT)
and genotype. We used to phenotype RBCs to predict the To type patients with AIHA* to select antigen-negative RBCs for absorp-
genotype; now we test DNA to predict the phenotype, both tion of autoantibodies when searching for underlying alloantibodies
with the understanding that variant and null alleles exist. To type donors, including mass screening for antigen-negative donors,
If high-throughput platforms were inexpensive enough, when appropriate antisera are not readily available
DNA testing and prediction of antigen types could be used To type donors for use on antibody identification panels when antisera are
as a screening method to increase antigen-negative inven- not available
tories. Where antisera are available, negative DNA screening To type patients who have an antigen that is expressed weakly on RBCs
results could be confirmed by hemagglutination methods,
To resolve blood group A, B, and D discrepancies
thereby conserving reagents. When antisera are not avail-
To study unusual and novel serologic reactions
able, (e.g., anti-Dob, anti-Hy) the predicted type is often
more reliable than the crossmatch. The availability of *AIHA = autoimmune hemolytic anemia
increased inventories of RBC components with various
combinations of antigen-negativity would make it possible Modifications of classic hemagglutination, which in-
to more precisely match a patient’s phenotype, before the creased its sensitivity and specificity and reduced the time
patient was immunized. The technique could be used to es- it takes to perform the test, have contributed to the safe
sentially eliminate repetitive time-consuming techniques transfusion practice as we know it today. Although West-
needed to identify underlying alloantibodies in cases of ern immunoblotting and PCR-based assays have led to an
warm autoimmune hemolytic anemia and antibodies to understanding of the function and structure of RBC membrane
high-prevalence antigens. components carrying blood groups, hemagglutination
remains the workhorse of immunohematology testing.
However, despite all our advances, errors in identification
and clerical errors are still the big problem,
Table 1. Structure and function of RBC membrance components and ABO incompatibility remains a primary
carrying blood group antigens
cause of preventable transfusion-related fatali-
Function Carbohydrate Single-pass Multi-pass GPI-linked Adsorbed ties.38–40 Approximately half of the errors occur
Glycocalyx ABO MNS LE when blood is transfused to the wrong patient.
P Sample collection error, i.e., drawing blood
H
I from the wrong patient or mislabeling the
GLOB sample, is also responsible for the blood being
Structural GE DI transfused to the wrong patient.41 Hemaggluti-
RH nation in tubes does not require sophisticated
XK equipment. It is simple, inexpensive (although
Transport CO antibodies are becoming expensive and tech-
DI nologist costs constantly rise), sensitive, spe-
GIL
cific, and reproducible. It remains the basic
JK
RH method for detecting reactions between RBCs
RHAG and antibodies.
XK
Adhesion/ IN FY JMH Acknowledgments
receptor LU RAPH I thank Steve Pierce for helpful sugges-
LW
MNS
tions and Robert Ratner for help in preparing
OK the manuscript.
SC
XG
Enzyme KEL DO
YT
Complement KN CR CH/RG
regulator MSN
Kpa is a low-frequency antigen occurring in less than 2 percent rectal bleeding, nausea, weakness, and dizziness. Rectal ex-
of the Caucasian population. Mild to moderate delayed hemolytic amination confirmed the rectal fissures and bleeding. There
transfusion reactions (DHTR) and hemolytic disease of the fetus were no other remarkable findings noted on physical exam-
and newborn attributable to anti-Kpa have been reported. Severe ination. ECG showed sinus tachycardia at 106 beats/min.
overt DHTR has not been reported with anti-Kpa. A case of a se-
She was admitted for further workup and for RBC transfu-
vere DHTR attributed to anti-Kpa after multiple RBC transfusions
is being reported. A 52-year-old Caucasian woman received mul- sions.
tiple units of RBCs for a lower gastrointestinal bleed. She was re- Past history was negative for prior transfusions. Patient
ferred to our institution for hepatic and renal failure, which was has an adult son who was listed as a contact person, and no
supported by laboratory findings of peak LDH, bilirubin, BUN, other pregnancy history was available. She had left renal
and creatinine elevations. Hemoglobin had dropped on Day 10 angioplasty several years ago for left renal artery stenosis.
after transfusion. The DAT and antibody screen (ABS) were nega- She had a history of rectal fissures. She is a smoker (2 packs
tive. Initial workup and subsequent ABS were negative. Anti-Kpa per day) and drinks occasionally. She has a history of al-
was identified when an additional RBC panel was tested. One of lergy to amitriptyline.
the RBC units transfused was incompatible by antihuman globu-
lin (AHG) crossmatch with the patient’s plasma and typed positive
for Kpa. DHTR was confirmed after extensive workup. The patient
responded to supportive therapy and experienced an uneventful
recovery. DHTR may not be considered when DAT and ABS are
negative. However, correlation of recent transfusion with signs
and symptoms should alert the clinician to entertain and investi-
gate a DHTR that should include the AHG crossmatch of all impli-
cated RBC units. The severity of the reaction also raises concerns
as to when and what antigen specificity should be considered for
inclusion in the antibody screening cells.
Immunohematology 2009;25:44–47.
K
pa (KEL 3, Penney) is one of 31 antigens in the Kell
(ISBT symbol, KEL) blood group system. The Kell
antigens, encoded by KEL, located on chromosome
7q33, appear to be erythroid specific and are found in the
Fig. 1 Hemoglobin (Hb; g/dL) and hematocrit (Hct; %) values for day
fetal liver and in bone marrow cells.1 Kpa is a low-incidence
0 to day 13 are shown.
antigen found in less than 2 percent of Caucasians.1,2 The
antigen is resistant to the effects of enzyme treatment but is
sensitive to treatment with dithiothreitol and acid. Mild to
moderate delayed hemolytic transfusion reaction (DHTR)
and mild to moderate hemolytic disease of the fetus or
newborn (HDFN) have been reported as well as a case of
hydrops fetalis attributed to anti-Kpa.3 The antibody is an
IgG.4–6 Suppression of erythropoiesis by anti-Kpa has been
reported as the cause of decreased hemoglobin in HDFN.7
Reports of an overt DHTR attributable to several antibodies
missed in the antibody screen and immediate-spin cross-
match mention anti-Kpa as one of the antibodies.8 However,
there have been no reported cases of severe overt DHTR as
a result of anti-Kpa as per MEDLINE search.
Case Report Fig. 2 Alkaline phosphatase (Alk Phos), lactate dehydrogenase (LDH),
A 52-year-old Caucasian woman presented to the emer- and creatine phosphokinase (CPK) values for day 0 to day 14 are
gency room complaining of being light-headed and having shown.
Table 1. Laboratory data from the referring hospital transfused. Urinalysis on Day 11
Tests Normal range Patient results showed evidence of increased free
Admission Day 2 Day 7 Day 10 Day 11 Day 12 hemoglobin, bilirubin, and uro-
Hemoglobin 4.5–11 g/dL 6.1 12 — 8.7 10.2 9.9
bilinogen. Computed tomographic
scan and magnetic resonance im-
Hematocrit 36%–48% 19.6 — — 26.9 31.2 29.3
aging to rule out acute cholecystitis
WBC 4.5–11.0 x 9.9 — — — — — revealed an enlarged gallbladder
109/dL
with thickened wall and inflamma-
Platelets 120,000– 628,000 — — — — —
tion of adjacent areas, reported as
450,000/µL
suspicious for cholecystitis. Liver,
Reticulocytes 0.9%–1.9% — — — — 4.1 —
spleen, and pancreas were within
PT 11.5–14.7 s 13.8 — — 13.5 15.2 18.1 normal limits. There was also evi-
Glucose 65–100 mg/dL 125 — — — dence of atrophic left kidney and
Alkaline phosphatase 30–133 U/L 138 142 203 211 422 393 hypertrophic right kidney with se-
BUN 7–21 mg/dL 18 — 18 36 46 51 vere stenosis of a segment of the
right renal artery close to its origin.
Creatinine 0.5–1.4 mg/dL 0.8 — 1.0 4.2 5.6 6.2
The patient received 3 additional
AST 5–39 U/L 31 — — 161 529 303
units of RBCs between Days 10 and
ALT 7–56 U/L 19 — — 113 168 120 11 for the drop in hemoglobin.
LDH 333–699 U/L — 521 3983 — 7905 2215 On the 13th day of her hospital
7637 course, she was admitted to the re-
CPK 35–230 U/L 87 425 — — — — ferral hospital, our institution, for
Total bilirubin 0.2–1.3 mg/dL 0.3 — — 1.8 23.5 18.3 investigation of acute liver and re-
Direct bilirubin 0.0–0.4 mg/dL — — — — 24.7 16.1 nal failure. DHTR was suspected,
ABO, D — O — — O O —
and a workup was ordered by the
D positive D positive D positive hematologist. Pigment nephropa-
Antibody screen — Negative — — Negative Negative —
thy attributable to intravascular
hemolysis was considered as the
DAT — — — — — Negative —
cause for the acute renal failure.
HBsAg, HBc antibody, HBs antibody, and hepatitis C antibody—all nonreactive The cause of the severe hepatotox-
Urinalysis — — — — — icity could not be ascertained.
Bilirubin Negative Moderate
Urobilinogen <1 mg/dL 4 mg/dL
Free hemoglobin Negative Large Materials and Methods
At the referral hospital, ABO and
ALT = alanine aminotransferase; AST = aspartate aminotransferase; BUN = blood urea nitrogen; D typings were performed using the
CPK = creatine phosphokinase; HBc antibody = hepatitis B core antibody; HBs antibody = Ortho A/B/D Monoclonal and Re-
hepatitis B surface antibody; HBsAg = hepatitis B surface antigen; PT = prothrombin time; WBC verse Grouping Card (Ortho-Clinical
= white blood cell count. Diagnostics, Inc., Raritan, NJ). Affir-
magen reagent RBCs (pooled cells),
Admission Hb was 6.1 g/dL. She was transfused with 0.8%, were used for reverse typing, and antibody screen was
5 units of type-specific, leukoreduced or washed (as per done by IgG gel card, 0.8% Surgiscreen (Johnson & Johnson,
hospital policy), immediate-spin, crossmatch-compatible New Brunswick, NJ). Antibody panel was performed using
RBC during the course of 24 hours of admission, which 0.8% Resolve Panel A (Ortho-Clinical Diagnostics, Inc.) and
raised her Hb to 12 g/dL. She remained hospitalized for Panocell 16, by Immucor, Inc. (Norcross, GA). The DAT was
further evaluation for gastrointestinal bleed. On the 10th performed using anti-IgG-C3d with check cells, anti-IgG and
posttransfusion day, her Hb dropped to 8.7 g/dL and her check cells, and anti-C3b/C3d with complement check cells
Hct was 26.9%. Her reticulocyte count was 4.1% (reference (Immucor, Inc.). Reagents used for phenotyping were from
range, 0.9 to 1.9%) on Day 11. There was no evidence of con- Immucor, Inc. Elution was performed with Gamma ELU-KIT
tinued rectal bleed. There was a gradual increase of several II (Gamma Biologicals, Inc., Houston, TX) following initial sa-
chemistry results, which peaked on the 10th and 11th post- line wash (Fisher Diagnostics, Middleton, VA) of patient cells.
transfusion days (Table 1; Figs. 1–4). The patient appeared ABO and D typings and a three-cell antibody screen were
severely jaundiced with signs of acute liver and renal fail- performed by gel technique and read through the MTS reader
ure, which was supported by her laboratory values. Investi- SA (Ortho-Clinical Diagnostics, Inc.). DAT was performed
gation for a suspected DHTR revealed a negative DAT and with polyclonal AHG, anti-IgG, and anti-C3d by tube method
a negative antibody screen. Investigation did not proceed and eluate panel by gel method using Ortho and Immucor
to antihuman globulin (AHG) crossmatch for all RBC units panel cells.
Results
The DHTR investigation on the day of admission at the
referral hospital revealed a negative antibody screen with
the three-cell antibody screen. The DAT was negative (in-
cluding a 15-minute incubation) with polyspecific, IgG, and
C3b/C3d AHG. At the referral hospital, tests with a 16-cell
reagent RBC panel (Immucor, Inc.) revealed the presence
of anti-Kpa. All other clinically significant antibodies were
ruled out. Eluate prepared from DAT-negative RBCs was
negative against Kp(a+) RBC on Immucor Panocell 16. The
patient typed negative for Kpa. AHG crossmatch was done
Fig. 3 Total and direct bilirubin values for day 0 to day 20 are shown.
from samples obtained from donor segments of the trans-
fused RBC units received from the blood supplier. One of
the five RBC units transfused on Day 1 of admission was in-
compatible with the patient’s plasma and typed positive for
Kpa. Haptoglobin was 1 mg/dL (reference range, 34–200
mg/dL). A diagnosis of severe DHTR with intravascular
hemolysis attributable to anti-Kpa was confirmed. As the
patient’s renal and liver functions improved, she did not
undergo dialysis or consideration for further liver or renal
evaluation.
The patient was monitored and maintained on support-
ive therapy. Her hospital course was uneventful. All labora-
tory results returned to normal values (Figs. 1–4). She was
discharged 10 days after her admission to the referral hos- Fig. 4 Alanine aminotransferase (ALT) and aspartate aminotrans-
pital. ferase (AST) values for day 0 to day 20 are shown.
Table 2 presents the patient’s laboratory data on ad-
mission to the referral hospital on Day 13.
Table 2. Laboratory data on admission at the referral hospital, Day reside on the C-terminal domain of Kell in the structural
13 sequence N-terminal to the zinc-binding catalytic motif,
Test Result which is the major site for endothelin-3–converting enzyme
activity.
Haptoglobin (reference range 1 mg/dL
34–200 mg/dL) Kell antigens are important in transfusion medicine
owing to their strong immunogenicity, and the correspond-
ABO, D (by Ortho gel) O, D positive
ing antibodies are clinically significant.9 Anti-Kpa has been
Antibody screen (0.8% Surgiscreen) Negative
implicated in mild to moderate HDFN and DHTR. Severe
Panel cells, A Negative DHTR as presented in this case has not been reported.
Panocell 16 Positive (anti-Kpa identified) Clinical signs and the laboratory values presenting on Day 7
Kp typing of patient’s RBCs
a
Negative with peak laboratory values at Days 10 and 11 after transfu-
DAT—using polyclonal, IgG, and Negative sion of a unit of RBCs positive for Kpa support a diagnosis
C3b/C3d AHG in this case of a severe DHTR with intravascular hemoly-
Panel with eluate Negative sis attributable to anti-Kpa. The cause of the primary Kpa
exposure could not be determined from the patient’s his-
Crossmatch with 1 of 5 units trans- Incompatible, Kp(a+)
fused at referring hospital tory. AHG crossmatch of all implicated RBC units despite
Kpa typing of incompatible unit Kp(a+): 1+ by tube and 2+ by gel
the negative DAT or antibody screen would have positively
techniques identified the incompatible Kp(a+) RBC as the implicated
unit for the DHTR.
Discussion The patient under discussion experienced unusually
Kpa was first described by Allen and Lewis in 1957.2 The severe hepatotoxicity, and the reasons for this remain un-
Kpa antigen is a member of the Kell blood group system and clear: there was no prior history of liver disease. Severe
is carried on the Kell glycoprotein. The prototypical gene hemolysis with resultant high levels of free heme can cause
for the Kell protein family was cloned and characterized in undesirable toxicity, leading to organ, tissue, and cellu-
the early 1990s.9 As discussed by Lee and colleagues,9 the lar injury. The mechanism of action is through free heme
antigens of the Kell blood group system result from single that catalyzes the oxidation, covalent crosslinking, and ag-
nucleotide changes in the Kell protein. Most Kell antigens gregate formation of protein and its degradation to small
peptides. It also catalyzes the formation of cytotoxic lipid 3. Smoleniec J, Anderson N, Poole G. Hydrops fetalis
peroxide via lipid peroxidation and damages DNA through caused by a blood group antibody usually undetected
oxidative stress. Heme, being a lipophilic molecule, inter- in routine screening. Arch Dis Child Fetal Neonatal Ed
calates in the membrane and impairs lipid bilayers and or- 1994;71:F216–7.
ganelles, such as mitochondria and nuclei, and destabilizes 4. Pinkerton PH, Coovadia AS, Goldstein J. Frequency of
the cytoskeleton. It also causes endothelial cell injury, lead- delayed hemolytic transfusion reactions following an-
ing to vascular inflammation, and stimulates the expression tibody screening and immediate-spin crossmatching.
of intracellular adhesion molecules. As a proinflammatory Transfusion 1992;32:814–17.
molecule, heme induces inflammation that results in toxic 5. Garratty G. How concerned should we be about miss-
effects on the kidney, liver, central nervous system, and car- ing antibodies to low incidence antigens? Transfusion
diac tissue.10 The severe heme toxicity may also be attribut- 2003;43:844–47.
able to the markedly compromised renal condition owing to 6. Reid ME, Lomas-Francis C. The blood group antigen
the severe stenosis of the right renal artery. factsbook. San Diego, CA: Academic Press, 1997:182–
The severity of the DHTR in this case may warrant con- 4.
sideration for inclusion of clinically significant low-frequency 7. Tuson M, Koyal K, Desai R, et al. Probable suppression
antigens that may be missed by current screening cells for of fetal erythropoiesis by anti-Kp (abstract). Transfu-
detection of clinically significant RBC antibodies. How- sion 2007;47(3 Suppl):166A.
ever, the risk of overt DHTRs to low-incidence antigens is 8. Shulman IA. The risk of an overt hemolytic transfusion
estimated at 1 per 650,000 crossmatches.5 The calculation reaction following the use of an immediate spin cross-
was based on report of probability of acute overt hemolytic match. Arch Pathol Lab Med 1990;114:412–14.
transfusion reaction at 1 per 260,000 with immediate-spin 9. Lee S, Zambas ED, Marsh WL, Redman CM. Molecular
crossmatches of 1.3 million negative antibody screens.8 As cloning and primary structure of Kell blood group pro-
the risk of overt DHTR is extremely small, the cost benefit tein. Proc Natl Acad Sci U S A 1991;88:6353–7.
should be considered for inclusion of low-frequency an- 10. Kumar S, Bandyopadhyay U. Free heme toxicity and
tigens such as Wra, Cw, and Kpa in the antibody screening its detoxification systems in human. Toxicol Lett
cells.5 We concur with the previous observation. AHG phase 2005;157:175–88.
crossmatch must be included in the investigation of any
suspected DHTR irrespective of a negative DAT or nega- Ranie Koshy, MD (corresponding author), Director, Blood
tive antibody screen. Appropriate and timely patient care to Bank and Transfusion Services, and Bhishma Patel, SBB,
avert further patient harm depends on a thorough investi- Blood Bank and Transfusion Safety Officer, Department
gation. of Pathology and Laboratory Medicine, NJMS/UMDNJ,
C101, University Hospital, Newark, NJ 07103; and Jona-
References than S. Harrison, MD, Department of Medicine, Hema-
1. Roback, JD, ed. Technical Manual. 16th ed. Bethesda, tology, Robert Wood Johnson Medical School—Pisc/New
MD: American Association of Blood Banks, 2008. Brunswick, New Brunswick, NJ.
2. Allen FH Jr, Lewis SJ. Kpa (Penney), a new antigen in
the Kell blood group system. Vox Sang 1957;2:81–7.
The antigens of the ABO system were the first to be recognized as Variation in A antigen expression was also recognized
blood groups and actually the first human genetic markers known. very early in the twentieth century (reviewed in Race and
Their presence and the realization of naturally occurring antibod- Sanger10), and the A blood group was divided into A1 and
ies to those antigens lacking from the cells made sense of the
A2.1,11 Other descriptions of weakened A antigen expression
erratic failure of blood transfusion hitherto and opened up the
followed, and the A blood group was subdivided further
possibility of a safe treatment practice in life-threatening blood
loss. Although initially apparently simple, the ABO system has based on characteristic reactivity with human polyclonal
come to grow in complexity over the years. The mass of knowl- antisera, i.e., strength of reactivity and presence of mixed
edge relating to carbohydrate chemistry, enzymology, molecular field agglutination; presence of anti-A1; and whether A or H
genetics, and structural and evolutionary biology is now enormous blood group substance was present in the saliva of secretor
thanks to more than a century of research using ABO as a principal subjects (Table 1). Weak forms of B antigen were also found
model. This has provided us with data to form a solid platform but are typically more difficult to define serologically into
of evidence-based transfusion and transplantation medicine used specific categories although some subgroups (e.g., Bel) are
every day in laboratories and clinics around the globe. This review
analogous to their A counterparts (Ael).
aims to summarize key findings and recent progress made toward
The frequency of the common ABO phenotypes (A1, A2,
further understanding of this surprisingly polymorphic system.
Immunohematology 2009;25:48–59. B, A1B, A2B, and O) varies greatly among different popula-
tions.12,13 Populations with a high frequency of A phenotype
Key Words: ABO, blood group, antigen, allele, genotype are found mainly in Northern and Central Europe, and the
T
Table 1. Subgroups of A—agglutination reaction patterns adapted
here have been many reviews of the ABO blood group
from textbooks
system written throughout the years, covering dif-
ferent aspects of this fascinating topic. A limited se- Sub- Reactions* with Sub- Anti-
group stances A1 in
lection of such reviews can be found in the reference list, Anti-A Anti-A,B Anti-A1 Anti-H
of A in saliva† serum
particularly for readers who want to focus more on the dis-
A1 ++++ ++++ ++++ 0 A, H No
covery,1 biochemistry,2,3 enzyme structure,4 and molecular
A2 ++++ ++++ 0 ++++ A, H Some-
genetics.5,6 Our intention is not to reproduce them but to times
follow the guidelines of this new series of blood group sys-
Aint ++++ ++++ ++(+) +++ A, H No
tematic reviews in Immunohematology to provide a brief
introduction to this amazingly complex blood group system. A3 ++(+) mf
++(+) mf
0 ++++ A, H Some-
times
A2 phenotype reaches its peak among the Lapps in North- dbRBC Web site (http://www.ncbi.nlm.nih.gov/projects/
ern Scandinavia but is very rare in Asia. The B phenotype gv/rbc20). In this review, alleles will be referred to in the
is most frequent in Central Asia and almost absent in Am- traditional way but with dbRBC terminology given in brack-
erindians. Blood group O is the most frequent phenotype ets, e.g., A1 [A101] and O1 [O01].
in a global perspective, with Native American Indians be- It can be specifically noted that it is important for clar-
ing almost exclusively blood group O. Parts of Africa and ity and consistency to use subscripts, superscripts, and
Australia also show high frequencies of blood group O. The italics appropriately. For example, A1, A1, and A1 mean the
reason for the differences observed among populations is antigen, the phenotype, and the allele, respectively.
not fully understood although several theories have arisen.
The concept of evolutionary selection based on pathogen- Inheritance and Molecular Genetics
driven blood group changes will be discussed later (see The A and B antigens are inherited as Mendelian char-
section on pathogen interactions). The early importance of acteristics in a codominant autosomal fashion. In 1908, Ep-
ABO diversity is supported by reports in which the group stein and Ottenberg21 were the first to suggest in a short case
O-defining single base pair deletion at nucleotide position report that ABO blood groups might be inherited. This was
261 (see section on molecular genetics) has been found in later proved by von Dungern and Hirszfeld in 1910 (trans-
both Neandertal people14 and ancient Egyptian mummies,15 lated by Pohlmann22). In fact, ABO inheritance was one of
suggesting that selection pressure and survival of the fittest the first genetic markers to be used in paternity testing and
were indeed early features during our co-evolution with in forensic medicine.23,24
pathogens like malaria parasites and many others. Unlike the majority of blood groups, the antigens of the
six currently known carbohydrate systems are not coded
Nomenclature by genes directly. Instead, these blood group genes encode
The ABO system was the first to be discovered and has glycosyltransferases that in turn synthesize the oligosac-
therefore been given the number 001 in the official Inter- charide epitopes (Fig. 1). Thus, the A and B antigens are
national Society of Blood Transfusion (ISBT) terminology. made by A and B glycosyltransferase, respectively, encoded
Nomenclature for the ABO antigens is actually straightfor- by the ABO gene carried on the long arm of chromosome 9
ward since the antigen status is determined by the pres- (9q34). As with many of the blood group genes, the position
ence or absence of specific carbohydrate molecules. This is of the ABO locus was known for many years before the gene
particularly true for the A and B antigens but may be less was cloned.25 The genes encoding the A-synthesizing 3-α-N-
clear for the two other antigens (A,B and A1) given official acetylgalactosaminyltransferase and the B-synthesizing
antigen status by the ISBT. The A,B antigen is thought to 3-α-N-galactosaminyltransferase were cloned by Yamamo-
be an epitope not involving the A versus B–differentiating to and colleagues in 199026,27 after purification and partial
surfaces, but consists of a common recognition motif16 amino acid sequencing of A transferase from lung tissue.28
found when either the A or B antigens are present. The de- Probing cDNA libraries obtained from human adenocar-
bate surrounding the real identity of the A1 antigen is still cinoma cell lines of different ABO types, the main alleles
ongoing,17,18 but the A1 phenotype reveals many differences were defined.26 They determined that the B-specific mRNA
compared with A2, both quantitative and qualitative, so the differed from the A-specific gene by only 7 of 1062 coding
question is quite complex (see section on biochemistry). nucleotides, of which 4 result in amino acid differences in
Table 2 shows both the numerical and traditional nomen- the enzyme product. The difference between the A1 [A101]
clature according to the ISBT Working Party on Terminol- and O1 [O01] genes was shown to be a single guanosine (G)
ogy for Red Cell Surface Antigens.19 deletion, which alters and severely truncates the open reading
frame (ORF) as shown in Figure 2. The A2 phenotype was
Table 2. ABO nomenclature shown to depend on a cytosine deletion in the 5′ end of the
System ABO antigen notation gene, resulting in elongation of the ORF.29 The organization
ISBT number 001 ABO1 ABO2 ABO3 ABO4 of the ABO locus30,31 is shown in Figure 3. The gene consists
(001001) (001002) (001003) (001004)
ISBT name ABO A B A,B A1 O H O H
Fig. 1 Principal C H 2O H
O
C H 2O H
O
O H
O H
structure of the A, B, HO C H 2O H HO C H 2O H
and H oligosaccha- O O
CH 3
O H
Table 7 in Chester and Olsson6). Typically, alleles are re- lighted by arrows.
A tr is a c c h a r id e B t r is a c c h a r id e
C H 2O H
O
O O H
H d is a c c h a r id e
of seven exons (plus an alternative exon 1a located upstream have been described that encode glycosyltransferases ca-
of exon 132), with the majority of the catalytic domain of the pable of synthesizing easily detectable levels of both A and
enzyme encoded by exon 7. B antigens. This group includes alleles conveying the two
Since the groundbreaking cloning paper, 215 ABO se- unusual phenotypes cisAB and B(A). Fifty-eight alleles are
quence entries have been submitted to the dbRBC Web predicted to give rise to proteins without enzymatic activity
site20 (accessed on April 15, 2009) and curated by the dbR- of which 45 contain 261delG, the mutation that alters the
BC staff and experts, but new alleles are constantly being translational ORF and predicts a shortened protein product
identified. Figure 4 shows a breakdown of the 181 alleles with no transferase activity. This large group of O alleles
in the dbRBC categorized by their association with normal includes four principally important evolutionary lineages:
or altered phenotypes: these include 65 different A alleles, O1 [O01], O1v [O02], Otlse09 or O1(467T;318T) [O09], and O1bantu
47 B alleles, 58 O alleles, and 11 “AB” alleles. The remain- [O54].33,34 Numerous minor variants of these main alleles35
ing 34 sequences listed in the current dbRBC consist of 20 as well as hybrid O alleles combining A2 or B with different
sequences encompassing the 5′ noncoding region including 261delG-containing O sequences36 have also been found,
the repetitive and polymorphic CCAAT box binding fac- most notably in individuals of African ancestry. Of the re-
tor/nuclear factor Y (CBF/NF-Y) motif and 14 intronic or maining 13 “nondeletional” alleles, 3 suffer similar fates to
overlap sequences. Of the A alleles, 6 and 11 encode nor- that caused by 261delG because they contain nonsense mu-
mal A1 or A2 phenotypes on RBCs, respectively, whereas 48 tations resulting in altered ORFs caused by nucleotide in-
describe mutated A alleles (Aweak) associated with different sertions. Another 3 have nonsense mutations introducing
variants of weak A antigen expression (Table 1); the B al- immediate stop codons, thereby truncating the ORF at the
leles include 9 normal and 38 weak alleles; and 11 alleles same codon where they occur. Finally, 7 recorded nonde-
261
261 467 467 526
526 703* *
703 1060
1060 letional O alleles are crippled by at least one missense mu-
A1
tation each, giving rise to critical amino acid substitutions,
the most famous example of which is 802G>A resulting in
268Arg. For a recent review about this latter group of inter-
A2
esting O alleles designated O2 [O03], see Yazer and Olsson.37
What is most intriguing about these nondeletional O alleles
B
is that they are all attributable to mutations in A allele back-
bone sequences. This has two main practical consequences:
O1 first, virtually all ABO genotyping methods will signal the
O2 70
60
Fig. 2 Schematic representation of predicted open reading frames
for some common ABO alleles. White rectangles represent trans- 13
50
lated A1 [A101] consensus; black rectangles represent translated
non-A1 consensus (nonsense). Vertical bars indicate nucleotide
alleles
40 48
(nt) positions (given above bars) of mutations leading to amino acid Unusual/weak
Unusual/weakphenotype
alleles
phenotype
phenotype
30
Number
38
O2 [O03] allele at nt 802. Number of alleles
45
20
10
17
9 11
0
A B O AB
Blood
B lo o d blood
g ro u pspecificity
s pe cificity
presence of A1 or A2 alleles unless specifically designed cal phenotypes can have more than one molecular genetic
to detect rare O alleles, which will result in the potentially background.
catastrophic prediction of group A in a group O person. The regulatory mechanism of the ABO gene has been
Second, the phenotype actually conveyed by inheritance of investigated extensively. An enhancer element located ap-
one of these so-called O alleles is not always O but often proximately 4 kbp upstream of exon 150 was found to con-
weak A or O-like but without anti-A present in plasma. The tain four 43-bp repeats in all alleles except A1 [A101] and the
reason for this is currently unclear, but there has recently infrequent O2 [O03], which have only one copy.51 This may
been a series of papers investigating the serologic pattern play a role in expression.52 The enhancer region contains
and mechanisms behind this interesting phenomenon.38–42 a CBF/NF-Y binding site; mutations in this site decrease
There is currently no O allele described that is based on a B enhancer activity in a gastric cancer cell line,50 and altera-
sequence, but we predict that when such an allele is found, tions in this region may even cause rare B subgroup pheno-
it will show the opposite serology, i.e., either weak B expres- types.53 However, it was recently shown that A1 [A101] or A2
sion or O-like without anti-B in plasma. [A201] transcripts are virtually undetectable in peripheral
It is the molecular genetics that make this system so fas- blood whereas B and O (including O2 [O03]) mRNA is read-
cinating because mutations ranging from single nucleotide ily found.54,55 This appears to speak against a critical role for
changes to more complex hybrid gene formations can alter CBF repeats in erythroid ABO regulation, but more work is
the specificity of the enzyme, the efficacy of the enzyme, or required. There is also an Sp1-binding site in the proximal
both; changes that, at the phenotypic level, are manifested promoter that may be important for erythroid ABO regula-
simply by altered antigen expression. Good examples are tion.56
the various molecular bases of the Ax phenotype, which can Although much is understood regarding the cause of
either result from a single missense mutation in exon 7 of weak A/B antigen expression on RBCs when it comes to in-
an A1 [A101] allele or be based on different hybrids, for in- herited weak ABO subgroups,5,6 less is known about altered
stance between the 5′ end of B and the 3′ end of O alleles.43,44 ABO expression in hematologic disorders. Erythrocytes los-
The B3 phenotype in the Taiwanese population has also ing A, B, or H antigen have been noted in patients with he-
been shown to result from different molecular backgrounds, matologic malignancy, especially in the myeloid lineage.57,58
one a missense mutation but more commonly, a principally Very recently it was suggested that methylation of the ABO
interesting mutation that was the first one shown to affect proximal promoter is the reason for such leukemia-associated
splicing of ABO mRNA and cause exon skipping.45 In ad- downregulation.59 Reduced A and B antigen expression in
dition to altered specificity or enzymatic efficacy, aberrant bladder and oral carcinomas is partially attributable to loss
intracellular trafficking of ABO transferase may cause weak of heterozygosity or hypermethylation.60,61 Furthermore,
subgroup phenotypes.46 urothelial tumor tissue contains decreased amounts of A/B
As alluded to previously, this added layer of complexity, antigens that correlated to decreased levels of ABO mRNA
in which mutations affect enzyme activity and not the anti- compared with cells from normal tissue.62 Transient depres-
gen directly, provides difficulties in designing genotyping sion of A antigens has also been observed in some pregnant
assays that will detect rare variants, which if misinterpreted women,44 but the reason for this is still unknown.
can have serious consequences in ABO group determina-
tion.47 Biochemistry
There are problems associated with all the more than The biochemistry of the A and B antigens was elucidat-
30 ABO genotype screening methods published or commer- ed by the astonishingly early and brilliant work from the
cially available so far (reviewed in Olsson and Chester48). groups of Morgan and Watkins, and Kabat (reviewed by
The vast majority is only designed to determine between Watkins2 and Kabat63). The A, B, and H determinants were
three and six of the common alleles, although additional hypothesized to reside on water-soluble glycoproteins able
alleles can often surface if their polymorphisms interfere to inhibit agglutination of RBCs by antibodies or lectins.
with the detection system. However, virtually all methods A precursor substance, H, was hypothesized as a building
will fail to predict the phenotype of a sample in the pres- structure for A and B, and the terms O- (or H-) substance
ence of most A/B subgroup alleles, rare nondeletional null and anti-H were introduced in 1948.64
alleles, cisAB and B(A) alleles, or hybrid alleles, which can Owing to difficulties in obtaining sufficient quantities of
lead to serious consequences. Because this is particularly blood group active material after extraction from RBCs, most
dangerous if blood or transplant recipients are typed (as of the early studies were performed on ovarian cyst or animal
suggested, e.g., by Procter et al.49) we recently developed mucin, rich in secreted blood group substances. Before isola-
and implemented an improved ABO genotype screening tion and chemical identification of these substances, inhibi-
method that addresses these problems and can be used in a tion with simple sugars indicated N-acetyl-d-galactosamine
clinical laboratory setting.47 In summary, the complex genet- (GalNAc), d-galactose (Gal), and l-fucose (Fuc) as the defin-
ics of ABO is a major challenge for ABO genotyping efforts, ing sugars in blood groups A, B, and O, respectively.65,66
not least because of the disturbing fact that a certain allele Independent of the blood group of the individual
can lead to more than one phenotype and seemingly identi- investigated, a surprisingly similar composition of carbo-
hydrate residues (mainly Fuc, Gal, GalNAc and N-acetyl- epithelial cells that line the lumen of the gastrointestinal,
d-glucosamine [GlcNAc]) was found. This was taken as a respiratory, and reproductive tracts as well as in salivary
sign of large similar precursor molecules carrying small glands and skin. This wide distribution is a common fea-
residues that differentiate blood groups.67 The minimal ture for many of the carbohydrate blood groups, which has
determinant structures were subsequently shown to be resulted in the term histo-blood group often being used to
trisaccharides as shown in Figure 1. The important concept reflect this wide distribution. A and B antigen synthesis
of precursor-product relationship between H and A/B was occurs during normal glycosylation of proteins and lipids
formulated,67,68 and the critical role of nucleotide-bound in the Golgi compartment.77 The precursor H substance is
sugars as substrates in oligosaccharide synthesis was appre- synthesized by one of two fucosyltransferases depending on
ciated.69 The biosynthetic pathway of the ABO blood group the acceptor substrate used. The FUT1 gene that encodes
structures is as outlined in Figure 5, and the presence of the 2-α-fucosyltransferase (α2FucT1) is responsible mainly
A and B glycosyltransferases was first predicted70 and then for the synthesis of the H antigen on type 2 (and type 4) car-
experimentally established.71–73 Thus, the A and B glycosyl- bohydrate precursors found on RBCs.78 The closely related
transferases use UDP-GalNAc and UDP-Gal, respectively, FUT2 gene encodes a very similar 2-α-fucosyltransferase
as substrates. Both require the H determinant as acceptor. (α2FucT2) that is expressed in epithelial cells and synthe-
The O protein is nonfunctional, leaving the H-defining sizes H antigen mainly on type 1 and type 3 chains.77 The
βα
terminal Fuc unaltered. major precursor types present in different tissues and
U D PUDP
- G a-lN
GaINAc
Ac UUDP
DP
secretions are shown in Table 3.
GaINAc α 3Galβ - R
G a lN A c α 3 G a l β - R
G D P- -Fuc
GDP Fu c GGDP
DP
A antigen α2
α2
Table 3. Peripheral core structures and their principal tissue
GGalβ
a l β--R
-R A a n tig e n
distribution (modified from Clausen and Hakomori3)
A transferase
A tr a n s fe r a s e Fuc
Fuc
H Htrtransferase
a n s fe r a s e α2
G aGalβ
l β --RR α2
Fu c
Fuc BB tr a n s fe r a s e
transferase
B antigen
B a n tig e n G Galα3Galβ
a l α 3 G a-l Rβ - R
Peripheral core type Structure Distribution
H antigen
P rPrecursor
e c u rs o r H a n tig e n α2
α2 Type 1 Galβ1→3GlcNAcβ1→R Endodermal, secretions,
F Fuc
UPD - Gal
U D P -G a l
UDP
UDP
uc
plasma
Type 2 Galβ1→4GlcNAcβ1→R Ecto- and mesodermal
Fig. 5. Schematic depiction of the biosynthetic pathways for con-
(e.g., erythrocytes)
version of H determinants to A or B determinants. R represents the
core structure. See text for enzyme abbreviations. Type 3 Galβ1→3GalNAcα1→R O-linked mucin-type,
repetitive A
Type 4 Galβ1→3GalNAcβ1→R Glycolipids in kidneys
(and erythrocytes)
Although the composition of the A and B antigens is R = inner core structure or linkage.
apparently straightforward, the biochemistry behind the
shared A,B antigen recognized by many group O plasmas In the Bombay phenotype (Oh), a silenced FUT1 gene is
has only recently been elucidated experimentally. Bovin present together with a silenced FUT2 gene. Because H an-
and his colleagues16 synthesized a deacetylated A trisaccha- tigen is the precursor substrate for both A and B antigens,
ride structure and bound it to the precursor in such a way neither antigen can be synthesized without α2FucT activity,
as to expose what they hypothesized would be the common independent of the ABO genotype. The para-Bombay phe-
A,B epitope. They were able to demonstrate binding of both notype results from either (1) a silenced FUT1 gene present
monoclonal and polyclonal anti-A,B to the synthetic struc- together with an active FUT2 gene, which permits the syn-
ture. thesis of H type 1 (and therefore A/B antigens) that may be
Although it is well known that there are approximately adsorbed onto the RBC from the plasma; or (2) a mutated
five times fewer A antigens on A2 than on A1 RBCs, the un- FUT1 gene in which the encoded enzyme activity is greatly
derlying basis for the qualitative differences between them diminished, so that very low amounts of H antigen (and
is less well defined. The A2 transferase is 10 times less ef- A/B antigen) are produced. It may be present with or with-
ficient than the A1 transferase and has a different pH opti- out an active FUT2 gene. In both cases, H antigen (and A/B
mum and pI.74,75 The A2 enzyme is also less able to use chains antigen) is very weakly expressed and is often only detected
other than type 1 or 2 carbohydrate precursors like the ex- by adsorption and elution tests with the appropriate blood
tended type 3 (repetitive A) and type 4 (globo-A) chains on group reagents. The H blood group system (ISBT 018) will
RBC glycolipids.17,76 More recently, Svensson et al.18 have be the subject of another review later in this Immunohematology
produced somewhat contradictory data indicating that al- series.
though the A2 transferase can readily use H type 3 chains to The A and B glycosyltransferases are type II membrane
synthesize A antigen, the low levels of type 4 chains remain proteins located in the Golgi compartment,79,80 although
unconverted. soluble forms are found in plasma and other body fluids.
The ABH sugars are found on glycolipids (approximate- The enzyme consists of a short transmembrane domain, a
ly 10%) and glycoproteins (approximately 90%) on the RBC stem region, and a catalytic domain that extends into the
as well as on many different tissues and cell types, including Golgi lumen (Fig. 6). The crystal structure elucidated by
ABO groups demonstrated specific neutralization with 59 percent. Surprisingly, however, many reports show that
anti-A of those isolates grown in group A PBMCs but not approximately 50 percent of patients who are inadvertently
those cultured with group B or group O cells.89 Measles vi- transfused with a major ABO-mismatched unit of blood tol-
rus, when cocultured in a system expressing ABH glycosyl- erate the blood without any apparent signs of a transfusion
transferases (to mimic an in vivo environment), expressed reaction.101 The mechanisms underlying the ability of some
A or B epitopes, or not, according to the enzymes expressed. individuals to tolerate ABO-mismatched blood are not well
Viral particles could then be neutralized by normal poly- understood but are very interesting as identification of
clonal anti-A or anti-B sera in the presence of complement resistance markers could be exploited in transplantation
if the corresponding glycans were present.90 Conversely, therapy. Despite our relative sophistication in providing ap-
the A and B (and H) antigens are also used as receptors for propriately ABO-matched blood for patients, there is recent
pathogen invasion by attachment to host cells as mentioned clinical evidence that even the concept of ABO-compatible
previously, so the equilibrium between invasion and eva- (as opposed to ABO-identical) products may not be as safe
sion is a fine balance on both sides. as it would appear. A study that followed the posttransfu-
Furthermore, growing evidence for a leading role of sion mortality among more than 86,000 patients receiving
Plasmodium falciparum as the major force shaping the hu- plasma showed that exposure to ABO-compatible but non-
man genome including the distribution of the blood groups ABO-identical plasma was associated with an increased risk
has emerged.91,92 The parasite-induced RBC surface protein of death.102 Whether this is associated with immune com-
pfEMP1 is known to bind to the A antigen trisaccharide,93 plexes between anti-A/B and soluble A/B antigen in AB
and it has been shown that the severity, including mortality, plasma, for instance, remains to be studied. Furthermore,
of malarial disease is significantly lower in group O children reports of the usage of platelet concentrates in cardiac
than in other groups, mainly through the mechanism of re- surgery has also demonstrated less favorable outcomes in
duced rosetting.94,95 It is the focus on pediatric disease that those patients receiving ABO-mismatched products.103 The
is thought to make this pathogen particularly effective in authors hypothesized that the formation of immune com-
exerting a selection pressure on our genome. plexes may trigger cellular and inflammatory changes that
The general theme here is the concept of herd immu- adversely affect patient outcome.
nity, i.e., the fact that differences in the population will keep Hemolytic disease of the fetus and newborn (HDFN) as
at least a fraction of all individuals protected from most if a result of ABO incompatibility between mother and baby
not all pathogens for humanity to survive. ABO differences is a relatively common event in group O mothers carrying a
as part of our innate immunity appear to be one of the bet- group A or group B fetus. However, the disease is generally
ter examples of this idea.96 mild and rarely requires treatment, most often by photo-
therapy,97 although inhibition of antibodies by administra-
Clinical Significance tion of soluble A or B trisaccharides has been used success-
Of all antibodies to RBC blood group antigens, anti-A fully.104 Mild HDFN is a consequence of the comparatively
and anti-B are arguably the most clinically important. On low levels of IgG ABO antibodies (which are often mainly
the other hand anti-A1 and anti-H are very seldom found IgG2 or IgG4) capable of crossing the placenta and also is
to correlate with hemolytic adverse events but often show related to the immaturity of the glycosylated structures on
lower thermal optima. Before the discovery of ABO, trans- fetal RBCs. In addition, ABH antigens are present on many
fusion between humans had been attempted with mixed other cell types so the antibody concentration on RBCs is
success. In some cases, the patient survived and got bet- limited and the DAT often only weakly positive.
ter. In others the patient died rapidly, which we now re- The clinical significance of anti-A and anti-B extends
alize was attributable to rapid intravascular hemolysis of beyond transfusion medicine and is important in both solid
ABO-incompatible RBCs (reviewed by Mollison et al.97). organ and hematopoietic transplantation. Although ABO-
It has been reported that transfusion of as little as 30 mL mismatched hematopoietic stem cell transplantation is
of ABO-mismatched blood can result in a rapid fatal in- standard practice with a favorable outcome in most cases,
travascular transfusion reaction (reviewed in Issitt and transplantation of ABO-incompatible solid organs has been
Anstee98). Together with transfusion-related acute lung established only relatively recently with moderately suc-
injury (TRALI) and bacterial contamination of blood com- cessful outcome.105 Children younger than 3 years of age
ponents, ABO incompatibility is still one of the main risks have been shown to tolerate mismatched organs better, and
for major morbidity and mortality associated with transfu- West and colleagues have demonstrated good posttrans-
sions, after erroneous transfusions.99,100 The latest FDA re- plant survival in infants undergoing ABO-incompatible
port on transfusion-associated fatalities (found at http:// heart transplants,106 showing that the relatively immature
www.fda.gov/cber/blood/fatal08.htm) shows that blood B-cell response in these patients can be exploited.107 In a
group incompatibility was judged as the cause of death in study of 46 patients undergoing ABO-mismatched renal
37 percent of all cases during 2008, with TRALI at 35 per- transplants, Tobian et al.108 demonstrated that therapeu-
cent. Among the hemolytic fatalities ABO was the cause in tic apheresis to reduce anti-A and anti-B titers to less than
16 together with standard immunosuppression protocols we recently introduced the first microarray-based ABO typ-
results in successful and stable engraftment. Progress ing system that takes the first steps toward this goal on a
continues in this field, driven by a shortage of appropriate higher throughput system.110 Efforts to renew methods for
organs for transplant. Yazer and Triulzi109 conclude that serologic typing are also ongoing. For instance, the young
immune hemolysis remains a major complication in ABO- field of microfluidics holds promise for alternative blood
mismatched transplantation of solid organs and to a lesser grouping technology being more rapid, using smaller vol-
extent hematopoietic progenitor cells. However, passenger umes of reactants, and being applicable to broader testing
lymphocyte syndrome attributable to ABO or other blood platforms.111
group antibodies can now often be ameliorated by mono- The temptation to create ABO-universal blood for
clonal antibody therapy, once clinicians realize the prob- transfusion purposes has kept scientists busy since the
lem. early 1980s when the first successful report of deliberate
transfusion with modified RBCs across the ABO barrier was
Summary and Future Perspectives published.112 Progress from those early days, using a poorly
Despite the relative simplicity of the A and B antigens, effective B-converting exoglycosidase from green coffee
perhaps especially considering the minor biochemical dif- beans, has continued with the aims of eliminating ABO-
ference between them, the ABO blood group system remains associated hemolytic transfusion reactions and simplify-
one of the most interesting, both clinically and scientifi- ing blood logistics and inventory management. Not only
cally, dividing the world’s population including patients has the process taken important strides toward becoming
and donors into four groups irrespective of origin or creed. a reality with both A- and B-degrading enzymes recombi-
Figure 7 summarizes the four principal levels at which the nantly available and necessary clinical trials in progress or
ABO system can be considered and shows how immunohe- being designed, but the project has also brought with it the
matologists have been able to exploit the steadily increasing scientifically exciting discovery of a large new class of bac-
knowledge of this system by devising tests taking advan- terially derived exoglycosidase enzymes unknown hitherto
tage of each of the different levels. It also shows some of the and without clear homology or resemblance to any other
natural consequences generated and their relation to the group of molecules.113,114 It is unknown at this point why so
microbes surrounding us. many different bacterial species have developed these blood
Analytic modalities Natural consequences
group–converting enzymes. Speculation includes that it
Analytic modal ities Natural conse quences
would simply be a means of digesting potential nutrient
Genetic level
Genotyping
Genotypin
Genotypingg
(DNA, mRNA )
Polymorphic population saccharides, but it also may be yet another way for microbes
to make sure they are able to attach to the host cell surface,
Counteracting enzymes
Enzymic activity
Enzym ic activity Enzyme level (exoglycosidases)
Counteracting developed
enzymes (exoglyco - even if the histo-blood group of the current host happens
(glycosyltransferase)
(glyco syltransferase) by bacteria
sidas es) developed by bacteria not to fit the bacterial lectins initially. It is actually quite
Forward
Forward typing
typing
Glycoconjugate level Cell surface receptor variation likely that yet other glycosidase specificities will be found
Adsorption/elution
Adsorption/ elution Cell surface receptor variation
Flow cytomery
Flow cytometry
(carbohydrate antigens) Neutralization by secreted antigens
Neutralization by secreted antigens in this extended arsenal of newly discovered enzymes. A
Saliva
Saliva inhibi tion
inhibition
recently exploited example is the Galα3Gal (also known as
Reverse typing Immunological level
Neutralizatiom
the straight B, fucose-less B, or Galili antigen) -degrading
Reverse typing Neutralizatio
Neutralizationn byby immunoglobulins
immunoglobu lins
(antibodies )
Titration
Titration enzyme subfamily of galactosidases that can, for instance,
Fig. 7 Principles of the ABO system at four molecular levels and be used to degrade xenograft antigenicity in pig tendons for
their modes of interaction are schematically represented by arrows use in knee surgery.115
in black. On the left side, the laboratory analytical modalities cur- Finally, it is easy to make the mistake of sitting back
rently exploited at each level are listed; the right side shows some of and looking at the ABO system as close to complete when
the innate mechanisms used between host and pathogen. it comes to knowledge and discovery. We predict that both
the antigenic diversity and the way we look at antibody
specificities in this and related systems will change dramat-
A whole array of ABO-related research and other proj- ically during the next few years based on recent and future
ects is currently in progress. Even if a surprising number findings made possible by new technologies. ABO has for a
of ABO alleles has been discovered already, no doubt there long time served as a great model for genetics, enzymology,
are more to come. ISBT is currently working to facilitate and biochemistry thanks to our deep understanding of the
communication and reporting by introducing an official al- interindividual variation in this system, and there is noth-
lele nomenclature. Although ABO genotyping cannot yet be ing to suggest that it will stop now when high-throughput
used as a stand-alone analysis to support clinical decisions, genetics and glycotope arrays are here to help us. On the
improvement of the current status is highly desirable for contrary, it should be expected that there are still more sur-
its use as an independent addition to serology in the refer- prises waiting around the corner.
ence laboratory. Together with colleagues across Europe,
31. Bennett EP, Steffensen R, Clausen H, Weghuis DO, van 45. Yu LC, Twu YC, Chou ML, Chang CY, Wu CY, Lin M.
Kessel AG. Genomic cloning of the human histo-blood Molecular genetic analysis for the B(3) allele. Blood
group ABO locus. Biochem Biophys Res Commun 2002;100:1490–2.
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32. Kominato Y, Hata Y, Takizawa H, et al. Alternative lar trafficking of a variant B glycosyltransferase. Trans-
promoter identified between a hypermethylated up- fusion 2008;48:1898–905.
stream region of repetitive elements and a CpG island 47. Hosseini-Maaf B, Hellberg A, Chester MA, Olsson ML.
in human ABO histo-blood group genes. J Biol Chem An extensive PCR-ASP strategy for clinical ABO blood
2002;277:37936–48. group genotyping that avoids potential errors caused
33. Hosseini-Maaf B, Smart E, Chester MA, Olsson ML. by null, subgroup and hybrid alleles. Transfusion
The Abantu phenotype in the ABO blood group system 2007;47:2110–25.
is due to a splice-site mutation in a hybrid between a 48. Olsson ML, Chester MA. Polymorphism and recombi-
new O1-like allelic lineage and the A2 allele. Vox Sang nation events at the ABO locus: a major challenge for
2005;88:256–64. genomic ABO blood grouping strategies. Transfus Med
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oujon JM, Blancher A. Molecular polymorphism of O secretor status and its potential for organ transplants.
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tology 2008;24:138–47. scription of the blood group ABO gene. Transfus Med
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A
nti-IT was originally described as a benign, naturally turned for a splenectomy, which was pathologically nega-
occurring, IgM cold agglutinin in Melanesians1 and tive for lymphoma. Sections showed lymphoid hyperplasia
later in Venezuela Indians.2 The anti-IT agglutinin with a benign phenotype consisting of T and B cells with no
did not demonstrate classical I or i specificity but reacted aberrant expression of CD5, CD23, or bcl-2. Serologic stud-
strongly with cord RBCs, weakly with normal adult I RBCs, ies at that time showed similar reactivity of anti-IT. Three
and most weakly with the rare adult i RBCs. It was thought months later, the patient exhibited mild thrombocytopenia
the agglutinin recognized a transition state of i to I, thus the with slightly elevated LDH. A repeat bone marrow showed
designation IT (T for transition).1 Subsequent data do not lymphocytosis suggestive of low-grade B-cell lymphopro-
support this hypothesis; IT is expressed nearly as well or as liferative disease. A year later, after rituximab and other
well on fetal RBCs ranging in age from 11 to 16 weeks as on chemotherapy, the bone marrow showed atypical lymphoid
cord RBCs.3 The biochemical structure of IT is still unclear. infiltrates. The infiltrates were composed primarily of small
The first four examples of IgG anti-IT were in sera of mature lymphocytes with occasional larger lymphocytes
patients with autoimmune hemolytic anemia (AIHA) and noted, most likely representing a lymphoproliferative dis-
Hodgkin’s lymphoma.3,4 Others reported a similar associa- order. Flow cytometry showed an entire B-cell population
tion.5 Hafleigh et al.6 found three examples of IgG autoanti- that did not express CD20. Less than 3 years after original
IT in patients who did not have Hodgkin’s disease or AIHA. presentation, the patient was transfusion-dependant and
One example of IgM warm anti-IT and one IgM cold plus diagnosed with aplastic anemia. She was discharged to hos-
IgG anti-IT in patients with AIHA were not associated with pice care with end-stage liver disease, lymphoproliferative
Hodgkin’s disease, but one of the cases was associated with disorder, and hemolytic anemia.
non-Hodgkin’s lymphoma.7,8 We report an unusual 37°C
reactive IgM agglutinin plus an IgG anti-IT9 and a 37°C re- Case 2
active IgM agglutinating anti-IT plus broadly reactive IgG A previously healthy 19-year-old Korean man presented
autoantibody in two patients with hemolytic anemia who to the emergency room with insidious onset of fatigue, short-
were subsequently evaluated for lymphoproliferative dis- ness of breath, dyspnea on exertion, and pallor. Laboratory
ease owing to the specificity of the antibodies reported. data revealed thrombocytopenia and hemolytic anemia:
The Duffy antigen receptor for chemokines (DARC or Fy glyco- RBC morphology.3 SAO is widespread in several popula-
protein) carries antigens that are important in blood transfusion tions of Papua New Guinea (PNG), where its prevalence
and is the main receptor used by Plasmodium vivax to invade correlates with malaria endemicity and altitude.4 In these
reticulocytes. Southeast Asian ovalocytosis (SAO) results from populations, homozygosity is apparently incompatible with
an alteration in RBC membrane protein band 3 and is thought to
full development of the fetus inasmuch as no persons ho-
mitigate susceptibility to falciparum malaria. Expression of some
RBC antigens is suppressed by SAO, and we hypothesized that mozygous for the band 3 mutation have yet been described,
SAO may also reduce Fy expression, potentially leading to reduced but heterozygosity confers strong protection against cere-
susceptibility to vivax malaria. Blood samples were collected from bral malaria.5,6 Although early studies had suggested that
individuals living in the Madang Province of Papua New Guinea. the SAO trait may afford some protection against vivax or
Samples were assayed using a flow cytometry assay for expres- falciparum parasitemia,7–9 other studies did not support
sion of Fy on the surface of RBC and reticulocytes by measuring this hypothesis.10 The mechanism of protection against
the attachment of a phycoerythrin-labeled Fy6 antibody. Reticu- cerebral malaria is not completely understood,11,12 but the
locytes were detected using thiazole orange. The presence of the specific protection against cerebral malaria suggests that it
SAO mutation was confirmed by PCR. There was a small (approxi-
may operate by means of alterations in sequestration of in-
mately 10%) but statistically significant (p=0.049, Mann-Whitney
U test) increase in Fy expression on SAO RBC compared with RBC fected RBC. However, SAO confers limited or no protection
from individuals without this polymorphism: mean Fy expres- against placental malaria, which is also linked to sequestra-
sion (mean fluorescence intensity [MFI]) was 10.12 ± 1.22 for SAO tion of infected RBCs.13,14
heterozygotes versus an MFI of 8.95 ± 1.1 for individuals without Individuals whose RBCs are negative for expression of
SAO. For reticulocytes the MFI values were 27.61 ± 19.12 for SAO Duffy antigens illustrate homozygote advantage for vivax
heterozygotes and 16.47 ± 3.81 for controls. SAO is associated with malaria in that their RBCs are completely refractory to in-
increased and not decreased Fy6 expression so that susceptibility vasion by Plasmodium vivax.15 Historic serologic data sug-
to P. vivax infection is unlikely to be affected. gest expression of some antigens on human RBCs was sup-
Immunohematology 2009;25:63–66.
pressed by the SAO trait, but expression of Duffy antigens
did not appear suppressed as determined by the less sensi-
Key Words: Duffy, Southeast Asian ovalocytosis, antigen
tive serologic instruments available at that time.16 In those
expression
studies, antibodies of varying specificities were placed in
I
tubes using serial dilution, and results were read macro-
n 1949 Haldane proposed that malaria had a dramatic
scopically with the aid of an eyepiece using a standard-
influence on the human genome, by selecting for ge-
ized scoring system. In this study, we used molecular and
netic polymorphisms that were known or suspected to
cytometric techniques to diagnose SAO and to reexamine
protect against malaria.1 Although some of these polymor-
whether there was a difference in expression of Duffy an-
phisms can be detrimental when individuals are homozy-
tigen receptor for chemokines (DARC) between SAO and
gous, e.g., sickle cell anemia, their apparent selection in hu-
control individuals in the context of possible differential
man populations living in malaria-endemic areas is evidence
susceptibility to infection with different Plasmodium spe-
that they offer some protection against death from malaria,
cies
an infectious disease that continues to kill more than 1 mil-
lion persons annually.2 Critical to this theory is the concept
Materials and Methods
of heterozygote advantage. That is, carriage of the mutant
Blood was collected by venipuncture in CPD anticoag-
gene on one member of a chromosomal pair is beneficial
ulant or by finger prick using a BD Microtainer (Becton
whereas homozygosity is deleterious. An extreme example
Dickinson, San Jose, CA) with EDTA anticoagulant from
of heterozygote advantage in Melanesian populations is
healthy volunteers from two villages, Sempi and Bemlon,
Southeast Asian ovalocytosis (SAO), a hereditary form of
on the north coast of the Madang Province, PNG. Oral in-
ovalocytosis that is caused by a 27-base pair deletion in the
formed consent was obtained after approval for the study
gene encoding the major integral RBC membrane protein
by the Medical Research Advisory Committee of the PNG
band 3 (AE1, SLC4A1), leading to a distinctive change in
Ministry of Health. After collection, samples were kept on Table 1. Mean fluorescence intensity (± standard deviation) of Fy6
ice or refrigerated at 4°C. Within 1 week, flow cytometric antibody binding to erythrocytes and reticulocytes
studies were undertaken with antibody to Fy6 (kindly pro- All Subjects All Subjects
vided by John Barnwell, CDC, Atlanta, GA). This is a mouse (Reticulocytes) (Erythrocytes)
monoclonal antibody that attaches to the first extracellular SAO heterozygotes (N = 19) 27.61 ± 19.12 10.12 ± 1.22
domain of the Fy glycoprotein, in an area thought to cor- Non-SAO individuals (N = 20) 16.47 ± 3.81 8.95 ± 1.1
respond to the binding site of P. vivax merozoites.17,18 The P value for difference (Mann- 0.65 0.0487
antibody was conjugated directly to a phycoerythrin (PE) Whitney U test)
label (Prozyme, San Leandro, CA) according to the manu-
facturer’s instructions. A 5-µL aliquot of blood was washed
three times in 50 µL of PBS and pelleted. RBCs were incu- Discussion
bated with 50 µL of a 1 in 50 dilution of Fy6 antibody for 15 The main finding of this study was that the SAO trait
minutes at 37°C. Cells were then washed twice in PBS and does not result in reduced expression of Fy antigen. In fact,
resuspended in thiazole orange (TO) solution according to we observed a 10 percent increase (p=0.049) in Fy antigen
the manufacturer’s specifications (Becton Dickinson). The expression by SAO RBCs when compared with RBCs from
samples were analyzed on a flow cytometer (Mo-Flo, Da- non-SAO individuals from the same area. This difference
koCytomation, Fort Collins, CO) equipped with a 70-mW was significant when expression by all RBC populations
air-cooled argon ion laser at 488 nm. TO fluorescence was was evaluated but not when only SAO reticulocytes were
read with a 525-nm bandpass optical filter and PE fluores- compared with non-SAO reticulocytes (although a sugges-
cence with a 570-nm bandpass filter. Compensation was tive trend existed). As anticipated, reticulocyte expression
applied, and negative fluorescence signals were adjusted of Fy6 antigen exceeded RBC expression.20 The reasons for
until they were orthogonal. Forward-scatter, side-scatter, the increased RBC Fy6 antigen expression in SAO are un-
and fluorescence data were analyzed. A count of 5000 TO- clear at present, but it may reflect either a decreased rate of
positive cells was taken per sample. This was approximately loss of Fy6 antigen from the surface of SAO RBC relative to
1 to 2 percent of the total number of cells assayed for each control RBC, perhaps in relation to the increased rigidity of
individual. Beads of known immunofluorescence (Immu- the SAO cell wall, or differences in the surface area of ovalo-
no-Brite, Coulter, Puerto Rico) were used to standardize cytes versus non-SAO RBC. Because there was only a small
experiments undertaken at different times. Detection of the increase in Fy6 antigen expression on SAO RBC and there
SAO mutation was undertaken using previously described was no statistically significant increase in the reticulocyte
PCR methods.19 Experienced microscopists from the PNG subpopulation, which is the only subpopulation susceptible
Institute of Medical Research performed light microscopic to infection by P. vivax, it is unlikely that this alteration af-
inspection of Giemsa-stained blood films for malaria para- fects susceptibility to this parasite, although there is other
sites. Statistical analysis was undertaken by using SPSS in vitro evidence that alterations in numbers of Fy antigens
(Statview 4.51, Abacus Concepts, Berkeley, CA). We have expressed may alter that susceptibility.21 It is not surprising
used nonparametric testing because we do not have evi- that the difference in Fy levels was not shown in the paper
dence for normal distribution of DARC abundance in the of Booth et al.16 because they were using less sensitive se-
surface of SAO erythrocytes. rologic methods or because Fy6 antigen is more available
or more expressed in SAO ovalocytosis over the antigens
Results tested in the previous study. However, their findings with
Samples from 19 SAO heterozygotes and 20 non-SAO respect to other antigens, including Rh polypeptides, have
donors were examined. Mean age of donors did not dif- subsequently been confirmed by molecular techniques.22
fer significantly between the two groups (mean age, 29.32 Fy antigen negativity caused by homozygosity for a pro-
± 13.70 years for SAO heterozygotes versus 26.91 ± 11.80 moter mutation that silences transcription of the FY gene23
years for non-SAO individuals). Four individuals had ma- is observed in many ethnic groups in sub-Saharan Africa,
laria infection based on inspection of blood smears. One and presumably accounts for the relative lack of vivax ma-
SAO and one non-SAO individual had Plasmodium malari- laria in this part of the world. The apparently independent
ae, and two non-SAO donors had Plasmodium falciparum. emergence of heterozygosity for this same promoter muta-
SAO RBC showed increased expression of Fy glycoprotein tion has also been described in the Wosera area of PNG,
relative to non-SAO RBC as measured by binding of the Fy6 where SAO is rare or nonexistent. There is a need for further
monoclonal antibody (Table 1). The level of Fy6 antigen evaluation of whether other common RBC polymorphisms
was also higher in SAO reticulocytes compared with control affect Fy antigen expression, especially those that are as-
reticulocytes, but the difference was not statistically sig- sociated with changes in RBC shape and surface area. For
nificant (Table 1). Subsequent analysis of the data did not example, α-thalassemia is common in PNG, and in many
suggest the differences were caused by a systemic bias at- areas, including the Madang Province, nearly all individuals
tributable to sex, village of origin, or method of obtaining have the typical α-globin mutation seen in Melanesians.24 It
blood (data not shown).
seems unlikely that this common polymorphism would act 9. Serjeantson S, Bryson K, Amato D, Babona D. Malaria
as a confounding factor in our study on the grounds of bio- and hereditary ovalocytosis. Hum Genet 1977;37:161–
logic plausibility, low likelihood of linkage disequilibrium, 7.
and very high prevalence in the region (assuming therefore 10. Foo LC, Rekhraj V, Chiang GL, Mak JW. Ovalocytosis
more or less equal distribution between SAO and control protects against severe malaria parasitemia in the Ma-
individuals within the population studied). Finally, it will layan aborigines. Am J Trop Med Hyg 1992;47:271–5.
be important to determine whether environmental vari- 11. Cortes A, Benet A, Cooke BM, Barnwell JW, Reeder JC.
ables extant in malaria-endemic populations might change Ability of Plasmodium falciparum to invade Southeast
Fy antigen expression. Another example would be iron defi- Asian ovalocytes varies between parasite lines. Blood
ciency, which is prevalent in PNG and many other malaria- 2004;104:2961–6.
endemic regions of the world. 12. Cortes A, Mellombo M, Mgone CS, Beck HP, Reeder JC,
There is evidence that the SAO trait affects several steps Cooke BM. Adhesion of Plasmodium falciparum–
of the biologic cycle of P. falciparum that may be relevant infected red blood cells to CD36 under flow is enhanced
for the selective advantage conferred by this trait against by the cerebral malaria-protective trait South-East Asian
malaria. In particular, SAO RBCs are partly resistant to in- ovalocytosis. Mol Biochem Parasitol 2005;142:252–7.
vasion in culture by merozoites of Plasmodium knowlesi 13. O’Donnell A, Raiko A, Clegg JB, Weatherall DJ, Allen
and several lines of P. falciparum,25,26 and the SAO trait SJ. Southeast Asian ovalocytosis and pregnancy in a
also results in altered adhesion of infected RBCs to CD36. malaria-endemic region of Papua New Guinea. Am J
As with ABO,27 it is likely that the evolutionary pressure ex- Trop Med Hyg 2007;76:631–3.
erted by survival with SAO is related to survival of children 14. Benet A, Khong TY, Ura A, et al. Placental malaria in
from cerebral malaria before reproductive age. Although Fy women with South-East Asian ovalocytosis. Am J Trop
expression probably affects P. vivax infection,28 this evolu- Med Hyg 2006;75:597–604.
tionary pressure is almost certainly caused by P. falciparum 15. Miller LH, Mason SJ, Clyde DF, et al. The resistance
through its relationship to RBC adhesion. factor to Plasmodium vivax in blacks. The Duffy blood
group genotype, FyFy. N Engl J Med 1976;295:302–4.
Acknowledgment 16. Booth PB, Serjeantson S, Woodfield DG, Amato D. Se-
This study was funded in part by a Covance Fellowship lective depression of blood group antigens associated
from the Royal Australasian College of Physicians (I.W.). with hereditary ovalocytosis among Melanesians. Vox
Sang 1977;32:99–110.
References 17. Nichols ME, Rubenstein P, Barnwell J, et al. A new hu-
1. Haldane JBS. Disease and evolution. Ric Sci Suppl A man Duffy blood group specificity defined by a murine
1949;19:68–76. monoclonal antibody. Immunogenetics and association
2. Maitland K, Bejon P, Newton CR. Malaria. Curr Opin with susceptibility to Plasmodium vivax. J Exp Med
Infect Dis 2003;16:389–95. 1987;166:776–85.
3. Jarolim P, Palek J, Amato D, et al. Deletion in erythro- 18. Wasniowksa K, Blanchard D, Janvier D, et al. Identifi-
cyte band 3 gene in malaria-resistant Southeast Asian cation of the Fy6 epitope recognised by two monoclonal
ovalocytosis. Proc Natl Acad Sci U S A 1991;88:11022– antibodies in the N-terminal extracellular portion of
6. the Duffy antigen receptor for chemokines. Mol Im-
4. Mgone CS, Koki G, Paniu MM, et al. Occurrence of the munol 1996;33:917–23.
erythrocyte band 3 (AE1) gene deletion in relation to 19. Jarolim P, Palek J, Amato D, et al. Deletion in erythro-
malaria endemicity in Papua New Guinea. Trans R Soc cyte band 3 gene in malaria-resistant Southeast Asian
Trop Med Hyg 1996;90:228–31. ovalocytosis. Proc Natl Acad Sci U S A 1991;88:11022–
5. Genton B, al-Yaman F, Mgone CS, et al. Ovalocytosis 6.
and cerebral malaria. Nature 1995;378:564–5. 20. Woolley IJ, Hotmire KA, Sramkoski RM, Zimmerman
6. Allen SJ, O’Donnell A, Alexander ND, et al. Prevention PA, Kazura JW. Differential expression of the Duffy
of cerebral malaria in children in Papua New Guinea by antigen receptor for chemokines according to RBC age
Southeast Asian ovalocytosis band 3. Am J Trop Med and FY genotype. Transfusion 2000;40:949–53.
Hyg 1999;60:1056–60. 21. Michon P, Woolley I, Wood EM, Kastens W, Zimmer-
7. Fowkes FJ, Michon P, Pilling L, et al. Host erythrocyte man PA, Adams JH. Duffy-null promoter heterozygosi-
polymorphisms and exposure to Plasmodium falcipar- ty reduces DARC expression and abrogates adhesion of
um in Papua New Guinea. Malar J 2008;7:1. the P. vivax ligand required for blood-stage infection.
8. Cattani JA, Gibson FD, Alpers MP, Crane GG. Heredi- FEBS Lett 2001;495:111–14.
tary ovalocytosis and reduced susceptibility to malar-
ia in Papua New Guinea. Trans R Soc Trop Med Hyg
1987;81:705–9.
22. Beckmann R, Smythe JS, Anstee DJ, Tanner MJ. Coex- 27. Cserti CM, Dzik WH. The ABO blood group sys-
pression of band 3 mutants and Rh polypeptides: dif- tem and Plasmodium falciparum malaria. Blood
ferential effects of band 3 on the expression of the Rh 2007;110:2250–8.
complex containing D polypeptide and the Rh complex 28. Kasehagen LJ, Mueller I, Kiniboro B, et al. Reduced
containing CcEe polypeptide. Blood 2001;97:2496– Plasmodium vivax erythrocyte infection in PNG Duffy-
505. negative heterozygotes. PLoS ONE 2007;2:e336.
23. Tournamille C, Colin Y, Cartron JP, Le Van Kim C. Dis-
ruption of a GATA motif in the Duffy gene promoter Ian J. Woolley (corresponding author), Department of
abolishes erythroid gene expression in Duffy-negative Infectious Diseases, Monash Medical Centre and Monash
individuals. Nat Genet 1995;10:224–8. University, Clayton, Victoria, Australia, Paul Hutchinson,
24. Cockburn IA, Mackinnon MJ, O’Donnell A, et al. A Centre for Inflammatory Diseases, Monash Medical Cen-
human complement receptor 1 polymorphism that re- tre, Clayton, Victoria, Australia, John C. Reeder, Papua
duces Plasmodium falciparum rosetting confers protec- New Guinea Institute of Medical Research, Goroka, Papua
tion against severe malaria. Proc Natl Acad Sci U S A New Guinea (current affiliation: Burnet Institute, Prah-
2004;101:272–7. ran, Victoria, Australia), James W. Kazura, Center for
25. Kidson C, Lamont G, Saul A, Nurse GT. Ovalocytic Global Health & Diseases, Case Western Reserve Univer-
erythrocytes from Melanesians are resistant to invasion sity, Cleveland, Ohio, USA, and Alfred Cortés, Papua New
by malaria parasites in culture. Proc Natl Acad Sci U S Guinea Institute of Medical Research, Madang, Papua
A 1981;78:5829–32. New Guinea (current affiliation: ICREA and Institute for
26. Hadley T, Saul A, Lamont G, Hudson DE, Miller LH, Research in Biomedicine, Barcelona, Spain).
Kidson C. Resistance of Melanesian elliptocytes (ovalo-
cytes) to invasion by Plasmodium knowlesi and Plas-
modium falciparum malaria parasites in vitro. J Clin
Invest 1983;71:780–2.
Manuscripts
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S
ickle cell disease (SCD) is described as the first identi- commonly found in western Africa. About one in every 400–
fied “molecular” disease since its manifestations stem 500 African Americans, or 80,000, has SCD. About 9000
from a substitution of valine for glutamic acid in the African Americans, or one in 12, have sickle cell trait.4
structure of the β chain hemoglobin molecule.1 As a result On the other hand, patients who have manifestations
of this change, RBCs form characteristic “sickle” shapes and of their sickle hemoglobin are considered to have SCD. This
the surface of these RBCs attract each other, polymerizing includes patients who are homozygous for Hgb SS, as de-
when in a low oxygen environment. This seemingly “small” scribed previously. In addition, some patients who inherit
variation in the structure of the RBC causing polymerization Hgb S from one parent and another abnormal hemoglobin
leads to manifestations such as chronic occlusion of blood from the other parent can also have SCD. Common exam-
vessels (vaso-occlusion), reduced blood flow to vital organs ples are designated as Hgb SC and Sβ-thalassemia. These
(ischemia), and alterations of the immune system. In ad- individuals can have a milder clinical course than that of
dition, the abnormal sickle cells are prematurely removed individuals who are homozygous for Hgb S.
from circulation, resulting in hemolytic anemia. Transfu- Since RBCs with Hgb S are abnormal, they are removed
sion is a vital component of the treatment of some of the from circulation in the spleen more rapidly than normal
complications of SCD. It is also a modality used to prevent RBCs. This leads to a reduced life in the circulation of 16–
some of these complications from occurring. Patients with 20 days in comparison to 120 days for normal RBCs.5 The
SCD are unique because those who are transfused usually premature destruction of RBCs, with the accompanying
require chronic transfusion, resulting in exposure to many decrease in hemoglobin, is classified as a hemolytic anemia.
different blood donors over the course of treatment. In
addition, most patients with SCD in the United States are Table 1. Composition of normal adult hemoglobin
African American, and most donors are Caucasian from Hemoglobin Composition Percent (%)
Western European descent. As a result of this difference,
Hgb A α2β2 96–98
patients with SCD are exposed to RBC antigens that they
Hgb A2 α2δ2 1.5–3.5
lack, putting them at risk for forming alloantibodies, de-
fined as RBC alloimmunization.2 Therefore, it is important Hgb F α2γ2 <1
to understand the issues involved in the safe and effective
transfusion of patients with SCD.
precursors, suggestive of reticulocytes, are released from to refer these patients to specialists and provide state-of-
the marrow prematurely. Therefore, the peripheral smear the-art patient care, identification of children with SCD at
will also show these immature RBCs, which are larger than birth is an important public health intervention. Screening
mature RBCs and have a slightly blue color since they are can be done in the neonatal period using fetal DNA during
not fully hemoglobinized.3 The term reticulocytes will be pregnancy, or more commonly, performing DNA analysis
used in this education activity, and refers to these RBCs. soon after birth. Prenatal testing can be done between 14–
16 weeks of gestation in pregnancies where there is a chance
that the baby will inherit Hgb S from both parents.1,3,4
More commonly, newborn infants are screened through
mandatory programs required by most states, soon after
birth.1,3,4 Several methods exist for detecting abnormal he-
moglobins. These include:
• High performance liquid chromatography (HPLC)—
Considered one of the best methods to be able to detect
abnormal hemoglobins and to be able to differentiate
them from normal hemoglobin. HPLC uses reactions in
columns of reagents. This technique also measures the
quantity of the various hemoglobins.
• Hemoglobin electrophoresis—Separates different he-
Fig. 1 The arrowed cell is a classic example of a sickle cell. Anoth-
moglobins according to their characteristic migration
er sickle cell is located in the upper left corner. There is a nucleated
red blood cell in the lower right corner. The large cell in the center across a gel of either cellulose acetate at an alkaline
is a polychromatophilic red cell or reticulocyte. (Source: Figure pH or citrate agar at an acid pH, based on charge. The
HE-42. In: Glassy EF, ed. Color Atlas of Hematology. Northfield, IL: different types of hemoglobin will migrate at different
College of American Pathologists, 1998:97.) rates, occupying a characteristic position on the gel.
• Isoelectric focusing—If further separation is necessary,
Another consequence of the high rate of hemolysis and then isoelectric focusing can be performed. An electric
erythropoiesis, or increased RBC turnover, is the accumula- current is passed through the gel that has areas of dif-
tion of unconjugated or indirect bilirubin. As a result, some ferent pH. The identity of the hemoglobin can be made
patients will have yellowing of the white of the eyes, scleral by its pattern of migration.
icterus, and skin, or jaundice. The increase in bilirubin also • Solubility test—Taking advantage of the fact that re-
puts patients at increased risk for gallstones, and the pa- duced Hgb S is insoluble in certain solutions, a simple
tient may have to have their gallbladder removed. Table 3 solubility test can be used to detect the presence of Hgb
lists the common laboratory findings in SCD and their S. After lysis, RBCs are reduced by sodium hydrosulfite.
etiologies. If Hgb S is present, the solution will be turbid; if not
Early diagnosis is essential in order to treat and even present, then the hemoglobin remains in solution and
prevent some of the complications of SCD. In the past, the suspension is clear. Since this test requires a certain
approximately 25% of children between the ages of four amount of whole blood, it is not routinely used for new-
months and five years with SCD died of pneumonia. The born screening. Note that this test will just detect the
use of prophylactic penicillin in children with SCD in the presence of Hgb S, so patients who have sickle cell trait
setting of comprehensive care reduced the risk of death in (heterozygous for Hgb S) will also be positive.
childhood to less than 3%.6–8 Therefore, in order to be able When performing patient testing, it is always important to
follow the package insert and ensure that proficiency test-
Table 3. Common laboratory findings in sickle cell disease ing is performed in compliance with the Clinical Laboratory
Laboratory value Etiology Improvement Amendments of 1988 (CLIA). CLIA regula-
Low hemoglobin and hematocrit Hemolysis tions require that laboratories enroll in a Centers for Medi-
High mean cellular volume (MCV) Reticulocytes care and Medicaid Services (CMS) approved proficiency
High white blood cell (WBC) Inflammation and increased marrow
testing (PT) program for all regulated tests that the labora-
count production tory performs.
High platelet count Increased marrow production
Clinical Manifestations of Sickle Cell Disease
High lactate dehydrogenase Hemolysis
(LDH) The function of a normal RBC is dependent upon its
ability to flow freely through blood vessels and to be flexible
Low haptoglobin Hemolysis
enough to move in and out of vessels and tissue where oxygen
High total and indirect bilirubin Hemolysis
delivery occurs. Due to the rigidity of the sickle RBC, these
High alkaline phosphatase until Elevated bone marrow activity properties are lost. Interestingly, when a patient with SCD
puberty
has a higher percentage of hemoglobin F, the course of their
disease is milder and their life expectancy is longer. This is blood flow through the internal carotid and middle cerebral
probably the result of the lower percentage of Hgb S in these arteries of the brain, a study found that one could predict
patients, and that RBCs with Hgb F do not form polymers.9 patients at risk for a first stroke.14 Chronic transfusion ther-
These chronic physiologic changes can lead to problems in apy has been used successfully to prevent primary stroke
all organ systems; heart, lungs, and kidneys. SCD can be and then subsequent stroke (see Treatment of Sickle Cell
seen as a disease based on vaso-occlusion (VOC), chronic Disease).15,16
hemolytic anemia, and immunologic impairment.
Acute Chest Syndrome
Occlusive Disease Acute chest syndrome (ACS) is the leading cause of
There are a variety of ways by which sickle cells cause death in adults with SCD and a major cause of morbidity
obstruction. One minor mechanism is whereby irreversibly in patients of all ages. It is characterized by the onset of
sickled cells obstruct small capillaries. More commonly, worsening respiratory status, fever, and new infiltrate on
sickle cells are trapped in small venules and capillaries.1,10 chest x-ray. The inciting event can be respiratory infection;
The process of sickling and occlusion involves a complex in- however, since a majority of cases of acute chest syndrome
teraction between chemical mediators, as well as elements in adults follow pain crisis, it is believed that an embolus
other than the RBC itself. There is increasing understanding of fatty tissue from the bone marrow (the site of the initial
that the walls of blood vessels are involved. There appears to vaso-occlusion) into the lungs is the culprit. Patients need
be increased adherence of sickle cells to the vascular walls, enhanced oxygenation, so supplemental oxygen is admin-
due to properties of the RBCs, as well as changes in the en- istered. Transfusion is an essential component in the treat-
dothelial cells of blood vessels. The lipid bilayer of RBCs is ment of acute chest syndrome, as will be discussed in more
disrupted in sickle cells, causing their early removal from detail later.17
circulation. Additionally, changes in their interactions with Of interest, there are many investigators looking at the
other cells within the blood stream, such as sticky white role of inflammatory chemicals in the pathophysiology of
blood cells (WBCs) and activated platelets, contribute to SCD, as well as their potential for developing new treat-
vaso-occlusion.10,11 These changes can also cause the acti- ments. For example, there is a reduction in levels of an ad-
vation of coagulation, leading to an increased risk of deep hesion molecule (VCAM-1) in patients with SCD on chronic
venous thrombosis.10,12 transfusion protocols.18 Therefore, chronic transfusion pro-
Vaso-occlusion manifests itself as one of the most com- tocols might prevent vaso-occlusion. Another recent study
mon findings in a patient with SCD, pain crisis. Prior to found that the increases in the inflammatory mediator,
routine screening of newborns, children were commonly secretory phospholipase A2, can predict acute chest syn-
diagnosed with SCD prior to the age of two, with occlu- drome. Transfusing when levels are elevated can prevent
sion in the hands and feet presenting as painful swelling, acute chest syndrome.19
or dactylitis.4,5 This would not occur until six months of age
because the higher level of fetal hemoglobin prior to six Hemolytic Anemia
months is protective. Most patients will often have severe The abnormal shape and surface of sickle cells result
pain due to occlusion of blood flow to bones, bone marrow, in their shortened life span in the circulation and lead to
muscles, or organs, and in their arms, legs, back, abdomen, the characteristic anemia. As RBCs are cycled through the
and chest. Treatment consists of analgesia (many patients spleen, the site of their ultimate destruction, they also injure
with SCD require chronic medication) and hydration, since the tissue of the spleen. Their rigidity impairs their ability to
dehydrated RBCs are more likely to sickle. The cause of cri- flow smoothly through the sinusoids, and their sharp edges
sis can vary between patients and between episodes in the cause them to be stuck, and to damage splenic tissue. In
same patient, but may be related to infection, extremes of children under the age of five, blood can pool in the spleen,
temperature, medication, or any other physiologic changes. which becomes a site of sequestration of RBCs. When this
Occlusion is also associated with bony infarcts leading to occurs, the child has a painful, enlarged spleen accompa-
osteonecrosis, skin ulcers, organ occlusion (including the nied by a drop in hemoglobin. Splenic sequestration can be
spleen, resulting in functional asplenia or autosplenecto- life-threatening since there is also a drop in blood volume in
my), acute chest syndrome, and cerebrovascular accidents the vasculature, or hypovolemia. Transfusion management
or strokes. is essential in these cases. Many of these patients eventu-
ally need to have their spleen removed. As a patient gets
Stroke older, the damage to the spleen is chronic, and the spleen
Stroke in any patient is a devastating event, with ap- actually becomes smaller, eventually shriveling and losing
proximately 11 percent of patients with Hgb SS having a function.1,4,5
stroke by the age of 20.13 As blood vessels become occluded, In order to keep up with this chronic destruction, the
their diameter gets smaller and the rate of blood flow in- body attempts to compensate by increasing RBC produc-
creases, thereby increasing the risk of stroke. Using a type tion. This chronic hemolysis may also create an inflamma-
of ultrasound, transcranial Doppler (TCD), to measure tory state.1,10,11 Also, the free hemoglobin that is released
from hemolyzed RBCs causes additional damage to the lin- The chemotherapeutic agent, hydroxyurea (HU), increases
ing of blood vessels.10,13 The marrow is very active, as evi- the amount of Hgb F that is made by the bone marrow.
denced by immature RBCs or reticulocytes in the peripheral RBCs with Hgb F lack the β chain, which is responsible for
blood. As the marrow increases production of RBCs, plate- the sickling seen in Hgb S RBCs. Therefore, these RBCs
let and WBC production also increases. As a consequence of do not sickle, nor can they polymerize. This is one of the
increased erythropoiesis, many patients do not suffer from mechanisms considered to be responsible for the improved
signs and symptoms of anemia, even though they may have outcomes. In addition, HU has been found to reduce WBC
a low hemoglobin. As long as the individual can continue production, thereby reducing the number of circulating in-
to compensate, the anemia does not present a problem. flammatory cells, capable of adhering to blood vessel walls.
When the marrow cannot keep up with production (i.e., a Platelet counts also drop and this may inhibit one of the
lack of folic acid), or has its production impaired by viral ill- factors responsible for vaso-occlusion. HU also influences
ness, such as parvovirus B-19, an aplastic crisis may occur. nitric oxide metabolism. Nitric oxide leads to the dilation
Parvovirus B-19 is a common childhood virus responsible of blood vessels and seems to reduce the adhesion between
for Fifth’s Disease. This disease causes a rash, the classic RBCs and blood vessel walls.22
“slapped-cheek” appearance, and high fevers. Parvovirus HU has been found to reduce mortality due to an induc-
can suppress bone marrow production by destroying the tion of Hgb F and a reduction in vaso-occlusive events. In
RBC precursor cells in the bone marrow. In these cases of addition, it reduces the incidence of acute chest syndrome,
severe anemia, transfusion therapy is necessary.5 transfusion requirements, episodes of hospitalization,
pain crisis, and reduces blood flow through intracranial
Immunologic and Infectious Manifestations blood vessels, as measured by transcranial Doppler in chil-
The spleen can be affected in SCD due to occlusive forc- dren.22–24 Currently, there is controversy about whether or
es, but it is also damaged by the pointed, inflexible sickle not HU can be used to prevent repeat strokes as effectively
cells that travel through it and are stuck. The spleen is an as chronic transfusion in children.13,24,25 Since HU is a che-
important immunologic organ, helping to fight infections motherapeutic agent, there is concern that it will have long
with encapsulated organisms (S. pneumoniae, H. influ- term side effects. It is not used in women who are preg-
enzae, and N. meningitidis). Therefore, it is important to nant or planning to become pregnant. In addition, some
recognize children with SCD and immunize them. As stated patients have to discontinue therapy because their WBC
earlier, the use of prophylactic penicillin has been found to count becomes too low. The effects of hydroxyurea are not
reduce death in children.9,20 All infections should be treated immediate, generally taking a few months to be effective.23
aggressively. Therefore, it does not provide rapid response during acute
illness.
Treatment of Sickle Cell Disease Bone marrow transplant, peripheral blood stem cell
Prevention of early complications, such as infection, is transplant, and umbilical cord blood transplant are all
important in the overall treatment plan for patients with strategies that can cure SCD. Due to the morbidity and
SCD. As more is being learned about the basic mechanisms mortality secondary to transplant, it is important to care-
of the disease, new treatments are being developed. Anal- fully choose patients with severe disease with a high risk
gesics, including opioids, are a mainstay of treatment for of morbidity and mortality, such as those with recurrent
people suffering with pain crisis. In addition, it is impor- strokes. Therefore, trials have centered on treating children
tant to give patients with SCD vitamins, such as folic acid, or young adults, and there are some encouraging results.
which are essential to the production of RBCs. Hydration is Unfortunately, one impediment is the low availability of
also key, since RBCs sickle when dehydrated. In addition, matched sibling donors.26–28
hydration improves the viscosity of the patient’s blood and
allows the RBCs to move more easily. As hemoglobin rises, Transfusion Management
the viscosity of the bloodstream increases and there is an Transfusion therapy should only be initiated in patients
increased risk of occlusive disorders, as well as an increased with signs and symptoms of anemia. As mentioned, most
risk of pain crisis.21 There is no data to show that transfu- patients with SCD, though anemic, do not generally have
sion should be part of the routine treatment for crisis.5,20 daily signs and symptoms of anemia. Therefore, RBC trans-
The role of nitric oxide is also being investigated as a thera- fusion should only be used for specific indications. RBC
peutic modality, since nitric oxide plays an important role transfusion, when necessary in patients with SCD, provides
in the physiology of RBCs.1,4 some additional benefits. While increasing the patient’s he-
Patients of all ages who have higher concentrations moglobin, transfusion dilutes the Hgb S with Hgb A. The
of Hgb F have milder disease and lower mortality than RBCs with Hgb A have longer survival than the RBCs with
patients with lower levels of Hgb F. Increasing the pro- Hgb S and neither sickle nor polymerize. In addition, trans-
duction of Hgb F seems like a reasonable therapeutic in- fusion will suppress the patient’s own erythropoiesis, or
tervention. In the laboratory, Hgb F has been shown to RBC production. As a consequence, they will produce less
interfere with the polymerization of deoxygenated Hgb S. of their own Hgb S RBCs.4,5,29
The decision to transfuse should take into account the Splenic Sequestration: When a child’s hemoglobin
known benefits in the face of risks. All blood products are drops and they become symptomatic, transfusion is neces-
capable of transmitting certain infectious diseases and sary, in this setting. Of interest, transfusion increases the
causing transfusion reactions. In addition, the recipient is hemoglobin beyond what’s expected, so it is important to
exposed to certain inflammatory mediators that accumu- transfuse slowly to avoid over-transfusion. Hepatic seques-
late in transfused blood and to foreign antigens from the tration can also occur.5,29
donor. Exposure to foreign RBC antigens has important im- Pregnancy: In uncomplicated pregnancies, there is no im-
plications for patients with sickle cell disease. provement in outcomes in women who are transfused. Pa-
RBCs can be transfused as a simple transfusion or via tients with other complications of SCD should be transfused
exchange transfusion (erythrocytopheresis).5,29 During accordingly.5,29,30
simple transfusion, one or two units of RBCs are transfused Priapism: Priapism is the painful engorgement of the
through a peripheral IV. Exchange transfusion is usually penis, as a result of vaso-occlusion. Supportive therapy
performed using an automated machine that is designed to consisting of hydration and analgesia is recommended, and
remove whole blood from the patient, separate into its vari- a urologist should be consulted. When priapism does not
ous components, and then discard the patient’s Hgb S RBCs. resolve, simple transfusion or exchange transfusion should
RBCs from the blood bank are then used as replacement. be considered. There are no studies to direct transfusion
Typically, an RBC exchange exchanges one to two RBC vol- therapy. In addition, exchange transfusion has been associ-
umes (total blood volume × hematocrit = 1 blood volume). ated with adverse neurologic sequelae, such as seizures and
A larger bore, stiff-walled central venous catheter is usually increases in intracranial pressure, so there is a reluctance
required due to the flow requirements of the automated in- to perform exchange until the patient is refractory to other
strument. In the absence of automated equipment, manual treatments.5,29,31
exchange transfusion can be done. This is not optimal, since Presurgical Prophylaxis: Patients with SCD are at high
this can create blood pressure and volume changes during risk for complications when undergoing major surgery.
the alternating removal of whole blood through a peripheral Currently, some practitioners recommend that patients
vein with subsequent reinfusion of banked RBCs. Each type be transfused to a hemoglobin of 10 g/dL prior to surgery.
of transfusion has its own risks and benefits, as outlined in A study done comparing exchange transfusion to simple
Table 4 and in Table 5. transfusion found that exchange transfusion is unneces-
Table 4. The benefits and risks of simple transfusion sary.5,29,32 There are investigations underway to determine
Benefits Risks whether or not a hemoglobin lower than 10 g/dL is safe for
certain surgeries.33
Technical ease—requires only peripheral Increases viscosity
IV access Risk of iron overload Acute Chest Syndrome (ACS): Transfusion, either sim-
ple or exchange, implemented early in the course, improves
Low donor exposure—only 1 or 2 units oxygenation and alleviates organ dysfunction. For patients
of RBCs necessary who are stable, simple transfusion should be performed. If
Dilution of Hgb S the patient deteriorates, does not improve, or has a rapidly
evolving course, exchange transfusion is recommended.17,34
Simple transfusion should only be performed until the he-
Table 5. The benefits and risks of exchange transfusion moglobin reaches about 10 g/dL. Beyond that, there is con-
Benefits Risks cern for vaso-occulsion.5,29
Produces rapid reduction in Hgb S Requires large gauge IV, usually Stroke: Due to the ease of simple transfusion in pediat-
No increase in viscosity central venous catheter ric patients, they usually undergo simple transfusion. For
No risk of iron overload—some Requires expertise and special
observe reductions in serum equipment—may require transfer of
patients who have an initial stroke, exchange transfusion
ferritin over time patient to another facility is used to rapidly reduce the amount of Hgb S that can be
Higher donor exposure—at least 4 recruited and extend the immediate damage to the brain.
units of RBCs for an adult, usually Once a patient has a stroke, they are at risk for additional
more
Higher expense
strokes. By performing a monthly transfusion, either sim-
ple or exchange, this risk of recurrence is reduced. A recent
study showed that stopping monthly transfusion after the
Indications for Transfusion in Sickle Cell Disease return of normal flow by transcranial Doppler results in
Indications for transfusion in SCD include: recurrent strokes and the return of abnormal TCDs.13,15,16,35
Aplastic Crisis: When RBC production in the bone marrow Of interest, during studies to reduce the risk of first stroke,
is interrupted, the delicate balance to maintain RBC pro- patients on a chronic transfusion protocol also had a reduc-
duction during chronic hemolysis is disrupted. Therefore, tion in the risk of acute chest syndrome and a reduction in
if the patient has a drop in their hemoglobin and becomes the number of pain crises.15,16
symptomatic, transfusion is required until the underlying
process abates.5,29
Adverse Consequences of Transfusion of Patients with vent this, medications are used to remove, or chelate, iron.
Sickle Cell Disease Intravenous chelators have been used, but their efficacy
Adverse consequences of transfusion of patients with is hindered by its half-life and poor patient compliance.
SCD include: A new generation of oral chelators is effective since there
Alloimmunization: Due to the disparity between do- is increased compliance, and mode of actions results in a
nors and patients with SCD in the United States, patients more continuous chelation. Serum ferritin is monitored in
with SCD are among those most frequently alloimmmu- patients on chronic transfusion protocols who have SCD.45
nized. Studies have shown that the most common antibod- The aim of treatment is to reduce serum ferritin and to
ies formed in this population are C, E, and K1.2,36 There are remove iron from organs.
also other antibodies that are formed due to donor–recipi-
ent disparity. Therefore, in order to prevent alloimmuniza- Transfusion Recommendations
tion, some centers routinely perform RBC phenotypes on When a patient develops complications from SCD, it
patients with SCD and only transfuse RBCs that lack C, E, also makes sense not to transfuse them with RBCs with ad-
and K1, if the patient is negative for the antigen. This strat- ditional Hgb S. Therefore, many laboratories will do a sim-
egy reduces the rate of antibody formation in these at-risk ple solubility test, as described earlier, and select units that
patients.36 Despite these findings, practice is variable. lack Hgb S. Realize that individuals with SCD are anemic
Analyzing the results of the 2003 J-C College of American and cannot serve as blood donors, however, there are active
Pathologists Proficiency Testing Survey, Osby and Shul- donors with sickle cell trait.41
man found that only 37 percent of North American hospi- Leukoreduction (LR) of blood products has been prov-
tals routinely perform antigen typing on the RBCs of non- en to reduce the risk of cytomegalovirus (CMV) transmis-
alloimmunized patients with SCD. In the 439 laboratories sion, reduce the risk of febrile non-hemolytic transfusion
that do perform RBC phenotyping, C, E, and K1 were the reactions, and reduce the risk of human leukocyte antigen
most frequent antigens that were matched.37 In a survey of (HLA) alloimmunization.41 There are also studies, though
50 academic medical centers in the US and Canada, 73 per- controversial, that show there are deleterious immunologic
cent of centers reported that they performed routine phe- sequelae of transfusion that are prevented by reducing the
notyping with 89% of those centers also matching for C, E, load of WBCs in transfused blood products. Since patients
and K1.38 There is controversy about providing RBCs that with SCD are chronically transfused, many believe that these
lack additional antigens. As you match for additional anti- patients should receive LR blood products. Of note, there is
gens, it becomes more difficult to find compatible units. It is also a study that shows reduced rates of RBC alloimmuniza-
argued that it is a better use of resources to save those rare tion in patients who receive LR blood products.46 Transfu-
units for patients with existing antibodies.39 Another strat- sion recommendations for patients with SCD are:
egy has been to use RBCs from donors who are ethnically • Blood products that are sickle hemoglobin negative
similar to the patient, thereby creating a better chance that • Blood products that are leukoreduced
the donor and patient will match more closely.40 Whenever • Blood products that are negative for C, E, and K1 anti-
a patient develops an antibody, they will also receive RBCs gens if the patient lacks these antigens
lacking the corresponding antigen.41 • Blood products that are negative for any additional an-
Hyperhemolytic Syndrome: A serious type of hemo- tigens against which the patient has antibody
lytic transfusion reaction, called the “hyperhemolytic syn- • Blood products that are from African American donors,
drome,” can occur in the setting of transfusion. During these if a program exists
episodes, the patients are typically being transfused, and
instead of rising, their hemoglobin falls with subsequent Summary
transfusion. It is felt that there is a “bystander” hemolysis of The substitution of one amino acid in the hemoglobin
the patient’s own RBCs, as well as destruction of transfused molecule results in sickle hemoglobin. As a result, RBCs
RBCs. Of interest, the units transfused are crossmatch com- sickle in low oxygen states causing occlusion of blood ves-
patible, and no new alloantibodies are identified at the time sels, increased viscosity, and inflammation. These RBCs
of transfusion. Further transfusion compounds the prob- are prematurely removed from the circulation, resulting in
lem. Therefore, it is important to recognize the syndrome a chronic hemolytic anemia. With newborn screening and
and, if possible, stop transfusing. If transfusion is neces- early treatment, the death rate among children with SCD
sary, intravenous immunoglobulin (IVIG) and intravenous has declined. In addition, a variety of treatments are being
steroids have been found to be effective. If transfusion is introduced to help manage the various manifestations of
required because of life-threatening anemia, it should be disease. Transfusion, simple or exchange, is a mainstay of
done cautiously, using IVIG and steroids concurrently.42–44 therapy, since it reduces the amount of Hgb S in circulation
Iron Overload: Each unit of transfused RBCs contains and suppresses erythropoiesis. Transfusion is indicated
about 200–250 mg of iron. With chronic transfusion, this for symptomatic anemia and specifically to prevent stroke
iron accumulates and can be deposited into organs such (first or recurrent), during acute stroke, and for acute chest
as the heart, liver, and endocrine glands. In order to pre- syndrome. Unfortunately, transfusion carries risks
for infectious disease transmission, as well as immunologic 14. Adams RJ, McKie VC, Nichols F, et al. The use of
and inflammatory sequelae. For patients with SCD who may transcranial ultrasonography to predict stroke in sick-
be chronically transfused, iron overload occurs frequently. le cell disease. N Engl J Med 1992;326:605–10.
In addition, due to differences in RBC antigens between do- 15. Adams RJ, McKie VC, Hsu L, et al. Prevention of a first
nors and recipients, these patients are at increased risk for stroke by transfusions in children with sickle cell ane-
development of RBC alloantibodies, which can complicate mia and abnormal results on transcranial Doppler ultra-
further transfusion. It is, therefore, important to prevent sonography. N Engl J Med 1998;339:5–11.
alloimmunization by transfusing leukoreduced RBCs that 16. Adams RJ, McKie VC, Brambilla D, et al. Stroke pre-
match the patient for the C, E, and K1 antigens. Human vention trial in sickle cell anemia. Control Clin Trials
progenitor cell (from bone marrow, peripheral blood stem 1998;19:110–29.
cells, or umbilical blood) transplant can cure the disease, 17. Vichinsky EP, Neumayr LD, Earles AN, et al. Causes and
and is used for patients with severe disease for whom con- outcomes of the acute chest syndrome in sickle cell dis-
ventional therapy may not be effective. ease. National Acute Chest Syndrome Study Group. N
Engl J Med 2000;342:1855–65.
References 18. Sakhalkar VS, Rao SP, Weedon J, Miller ST. Elevated
1. Frenette PS, Atweh GF. Sickle cell disease: old discov- plasma sVCAM-1 levels in children with sickle cell dis-
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2007;117:850–8. matol 2004;76:57–60.
2. Vichinsky EP, Earles A, Johnson RA, et al. Alloimmu- 19. Styles LA, Abboud M, Larkin S, Lo M, Kuypers FA.
nization in sickle cell anemia and transfusion of racially Transfusion prevents acute chest syndrome predicted
unmatched blood. N Engl J Med 1990;322:1617–21. by elevated secretory phospholipase A2. Br J Haematol
3. Elghetany MT, Banki K. Erythrocytic disorders. In: 2007;136:343–4.
McPherson RA, Pincus MR, eds. Henry’s clinical diag- 20. Claster S, Vichinsky EP. Managing sickle cell disease.
nosis and management by laboratory methods. 21st ed. BMJ 2003;327:1151–5.
Philadelphia, PA: Saunders Elsevier, 2007:504–44. 21. Platt O, Thorington B, Brambilla D, et al. Pain in sickle
4. Ogedegbe HO. Sickle cell disease: an overview. Lab Med cell disease. N Engl J Med 1991;325:11–16.
2002;33:515–43. 22. Steinberg MH, Barton F, Castro O, et al. Effect of hy-
5. Ohene-Frempong K. Indications for red cell transfusion droxyurea on mortality and morbidity in adult sickle cell
in sickle cell disease. Semin Hematol 2001;38:5–13. anemia. Risks and benefits up to 9 years of treatment.
6. Gaston MH, Verter JI, Woods G, et al. Prophylaxis with JAMA 2003;289:1645–51.
oral penicillin in children with sickle cell anemia. A ran- 23. Charache S, Terrin ML, Moore RD, et al. Effect of hy-
domized trial. N Engl J Med 1986;314:1593–9. droxyurea on the frequency of painful crises in sickle cell
7. Leikin SL, Gallagher D, Kinney TR, et al. Mortality in chil- anemia. N Engl J Med 1995;332:1317–22.
dren and adolescents with sickle cell disease. Cooperative 24. Ware RE, Zimmerman SA, Shultz WH. Hydroxyurea as
study of sickle cell disease. Pediatrics 1989;84:500–8. an alternative to blood transfusion for the prevention
8. Gill FM, Sleeper LA, Weiner SJ, et al. Clinical events in of recurrent stroke in children with sickle cell disease.
the first decade in a cohort of infants with sickle cell dis- Blood 1999;94:3022–6.
ease. Blood 1995;86:776–83. 25. Zimmerman SA, Schultz WH, Burgett S, et al. Hydroxyu-
9. Platt OS, Brambilla DJ, Rosse WF, et al. Mortality in rea therapy lowers transcranial Doppler flow velocities in
sickle cell disease—life expectancy and risk factors for children with sickle cell anemia. Blood 2007;110:1043–7.
early death. N Engl J Med 1994;330:1639–44. 26. Locatelli F, Rocha V, Reed W, et al. Related umbilical
10. Telen MJ. Role of adhesion molecules and vascular en- cord blood transplantation in patients with thalassemia
dothelium in the pathogenesis of sickle cell disease. He- and sickle cell disease. Blood 2003;101:2137–43.
matol Am Soc Hematol Educ Program 2007;84–90. 27. Bernaudin F, Socie G, Kuentz M, et al. Long-term results
11. Kupyers FA. Membrane lipid alterations in hemoglobin- of related myeloablative stem-cell transplantation to
opathies. Hematology Am Soc Hematol Educ Program cure sickle cell disease. Blood 2007;110:2749–56.
2007;68–73. 28. Woodard P, Jeng M, Handgretinger R, et al. Sum-
12. Key NS, Slungaard A, Dandelet L, et al. Whole blood tis- mary of symposium: the future of stem cell transplan-
sue factor procoagulant activity is elevated in patients tation for sickle cell disease. J Pediatr Hematol Oncol
with sickle cell disease. Blood 1998;91:4216–23. 2002;34:512–17.
13. Wang WC. The pathophysiology, prevention, and treat- 29. Josephson CD, Lu LL, Killyer KL, Hillyer CD. Transfu-
ment of stroke in sickle cell disease. Curr Opin Hematol sion in the patient with sickle cell disease: a critical review
2007;14:191–7. of the literature and transfusion guidelines. Transfus
Med Rev 2007;21:118–33.
30. Koshy M, Burd L, Wallace D, et al. Prophylactic red-cell 41. Brecher ME. Technical Manual. 15th ed. Bethesda, MD:
transfusions in pregnant patients with sickle cell disease. American Association of Blood Banks, 2005.
N Engl J Med 1988;319:1447–52. 42. King KE, Shirey RS, Lenkiewica MW, et al. Delayed
31. Rackoff WR, Ohene-Frempong K, Month S, et al. Neuro- hemolytic transfusion reactions in sickle cell disease:
logic events after partial exchange transfusion for pria- simultaneous destruction of recipients’ red cells. Trans-
pism in sickle cell disease. J Pediatr 1992;120:882–5. fusion 1997;37:376–81.
32. Vichinsky EP, Haberkern CM, Neumayr L, et al. A com- 43. Petz LD, Calhoun L, Shulman IA, et al. The sickle cell
parison of conservative and aggressive transfusion regi- hemolytic transfusion reaction syndrome. Transfusion
mens in the perioperative management of sickle cell dis- 1997;37:382–92.
ease. N Engl J Med 1995;333:206–13. 44. Win N, Yeghen T, Needs, et al. Use of intravenous immuno-
33. Buck J, Casbard A, Llewelyn C, Johnson T, Davies SC, globulin and intravenous methylprednisolone in hyper-
Williamson LM. Preoperative transfusion in sickle cell haemolysis syndrome in sickle cell disease. Hematology
disease: a survey of practice in England. Eur J Haematol 2004;9:433–6.
2005;75:14–21. 45. Porter JB. Concepts and goals in the management of trans-
34. Platt OS. The acute chest syndrome of sickle cell disease. fusional iron overload. Am J Hematol 2007;82:1136–9.
N Engl J Med 2000;342:1904–7. 46. Blumberg N, Heal JM, Gettings KF. Leukoreduction
35. Adams RJ, Brambilla D; Optimizing Primary Stroke of red cell transfusion is associated with a decreased
Prevention in Sickle Cell Anemia (STOP 2) Trial Inves- incidence of red cell alloimmunization. Transfusion
tigators. Discontinuing prophylactic transfusions used 2003;43:945–52.
to prevent stroke in sickle cell disease. N Engl J Med
2005;353:2769–78. Additional Resources
36. Vichinsky EP, Luban NL, Wright E, et al. Prospective Mayo Clinic. Sickle cell anemia. Available at: http://www.
RBC phenotype matching in a stroke-prevention trial in mayoclinic.com/health/sickle-cell-anemia/DS00324. Ac-
sickle cell anemia: a multicenter transfusion trial. Trans- cessed July 20, 2009.
fusion 2001;41:1086–92. Sickle Cell Disease Association of America, Inc. Available
37. Osby M, Shulman IA. Phenotype matching of donor red at: http://www.sicklecelldisease.org/. Accessed July 20,
blood cells units for non alloimmunized sickle cell dis- 2009.
ease patients: a survey of 1182 North American laborato- March of Dimes. Available at: http://www.marchofdimes.
ries. Arch Pathol Lab Med 2005;129:190–3. com/. Accessed July 20, 2009.
38. Afenyi-Annan A, Brecher ME. Pre-transfusion pheno- Lab Tests On Line. Available at: http://www.labtestsonline.
type matching for sickle cell disease patients. Transfu- org/. Accessed July 20, 2009.
sion 2005;44:619–20.
39. Ness PM. To match or not to match: the question for Susan D. Roseff, MD, Medical Director, Transfusion Medi-
chronically transfused patients with sickle cell anemia. cine, Professor, Virginia Commonwealth University, De-
Transfusion 1994;34:558–60. partment of Pathology, PO Box 980662, Richmond VA
40. Hillyer KL, Hare VW, Josephson CD, et al. Partners for 23298-0662.
life: the transfused program for patients with sickle cell
disease offered at the American Red Cross Blood Services,
Southern Region, Atlanta, Georgia. Immunohematology
2006;22:108–11.
You are eligible for a free 1-year subscription to All articles published, including communications and
Immunohematology. book reviews, reflect the opinions of the authors and do
not necessarily reflect the official policy of the American
Ask your education supervisor to submit the name and Red Cross.
complete address for each student and the inclusive
dates of the training period to immuno@usa.redcross.
org.
The Consortium for Blood Group Genes is a worldwide organiza- of appropriately worded reports of molecular analyses for
tion whose goal is to have a vehicle to interact, establish guide- both blood donors and transfusion recipients. Members
lines, operate a proficiency program, and provide education for continue to support the current recommendation that blood
laboratories involved in DNA and RNA testing for the prediction components should not be labeled with molecular test re-
of blood group, platelet, and neutrophil antigens.
sults as the sole means of antigen identification. Molecular
Immunohematology 2009;25:75–78.
data continue to be a source of information in the resolution
of complex serologic problems, and are not intended as the
Key Words: Blood group alleles, Consortium for Blood
sole means for patient transfusion management decisions.
Group Genes, proficiency, target alleles
T
Target Alleles
he Consortium for Blood Group Genes (CBGG) is a
The list of target alleles prepared by the CBGG after
nonprofit organization whose mission is “To estab-
meeting in 2007 was adopted. The preferred current termi-
lish guidelines, to provide education, and to provide
nology is listed in Table 1 of this report under the heading
a proficiency exchange for laboratories involved in DNA or
Target antigen (target allele). However, the final naming
RNA testing for the determination of blood group, platelet,
of alleles relies on the decision of the International Soci-
and neutrophil antigens.” The consortium was established
ety for Blood Transfusion. When referring to a particular
at the inaugural meeting on October 23, 2004, in Balti-
single-nucleotide polymorphism, nucleotide changes fol-
more, Maryland, by a group of people with scientific or in-
low the designated position, e.g., 125G>A; intronic nucle-
dustry experience and interest in the field. The consortium
otide changes represented in the lower case, e.g., –33t>c.
is coordinated by Marion Reid with assistance of country
The associated amino acid substitutions (not shown) flank
coordinators: Lilian Castilho for Brazil, Gregory Denomme
the designated position, e.g., Pro103Ser or P103S. Because
(with Maryse St. Louis as successor in 2009) for Canada,
gene numbering systems vary, and to avoid ambiguity in
and Connie Westhoff for the United States. All members are
the location of nucleotide changes, the CBGG has adopted
expected to interact and participate. New members are en-
GenBank gene reference sequences (RefSeqGene) and ref-
couraged to refer to the previously published information
erence SNP numbers (rs#) for blood group genes and nucle-
for background and progress information.1–4 The exchange
otides (Table 1). These numbers provide the set of common
of information is mainly accomplished through electronic
references used to communicate or report molecular testing
mailings, proficiency evaluation exercises, and a yearly
results.
meeting.
Guidelines for Molecular Testing
The CBGG Document
The CBGG has developed guidelines for free and gener-
The CBGG Document outlines the function of the CBGG.
al distribution. In 2007, the CBGG guidelines were shared
This document contains information on the structure, or-
with the AABB Molecular Testing Standards Program Unit
ganizational rules and bylaws, regulatory compliance plan,
(SPU). In addition, five CBGG members are committee
preferred terminology, and progress on the working parties
members of the AABB Molecular Testing SPU, and one
including proficiency exchange program, guidelines of prac-
member (MER) holds liaison status with the committee.
tice, DNA repository, funding, forms and disclaimers, and
The AABB Standards for Molecular Testing for Red Cell,
proposed Web site. It is highly recommended that CBGG
Platelet, and Neutrophil Antigens was published in 2008.5
members refer to this document for the aforementioned
The intent of the CBGG guidelines is to maintain an inde-
duties and activities. Meetings are held annually, in part,
pendent forum and voice for proposed molecular testing
for members to discuss outstanding issues and to provide
standards in ISO format for use by international laborato-
the opportunity to give input and accept amendments to
ries. The members will update, modify, or otherwise amend
the document. The CBGG Document is distributed to mem-
the CBGG guidelines by process of discussion and consen-
bers and is available to nonmembers on request.
sus. The CBGG guidelines will not become standards as
such to reflect the fact that the CBGG is not responsible for
Template Disclaimers
laboratory inspection or accreditation.
CBGG members continue to recognize the importance
* Full listing of the members of the CBGG can be found in the appendix.
Please e-mail all manuscripts for consideration to For more information, send an e-mail message
immuno@usa.redcross.org to immuno@usa.redcross.org
S (GPB*S) GYPB (NG_007483.1) nt 143T>C (rs7683365) Disclaimer required if null alleles not
s (GPB*s) tested.
Rh (RH) D RHD (NG_007494) Exon 4 & 7 Targets may vary as there are many
004 RHDΨ Ex 4 37 bp insert approaches
Cw nt 122A>G †
C x
nt 106G>A †
V & VS nt 733C>G (rs1053361) †
V (VS-) nt 1006G>T †
Lutheran (LU) Lua (LU*01) LU (NG_007480.1) nt 230A>G (rs28399653)
005 Lub(LU*02)
Kell (KEL) K (KEL*01) KEL (NG_007492.1) nt 578T>C (rs8176058)
006 k (KEL*02)
Kpa (KEL*03) nt 841T>C (rs8176059)
Kpb (KEL*04)
Jsa (KEL*06) nt 1790C>T (rs8176038)
Jsb (KEL*07)
Duffy (FY) Fya (FY*A) FY (NC_000001.9) nt 125G>A (rs12075)
008 Fyb (FY*B)
Fyx nt 265C>T (rs34599082) †
Fy null (RBC) nt -33t>c (rs2814778) Promoter GATA-1 box
Kidd (JK) Jk (JK*A)
a
JK (NC_000018.8) nt 838G>A (rs1058396) Testing for null alleles may be ap-
009 Jkb (JK*B) propriate.
Diego (DI) Dia DI (NG_007498.1) nt 2561T>C (rs2285644)
010 Dib
Yt (YT) Yta YT (NG_007474.1) nt 1057C>A (rs1799805)
011 Ytb
Scianna (SC) Sc1 SC (NG_008749.1) nt 169G>A (rs56025238) †
013 Sc2
Mark Yazer, MD
RBC Serology Reference Laboratory
Centralized Transfusion Service
University of Pittsburgh
3636 Boulevard of the Allies
Pittsburgh, PA 15213
Immunohematology will publish classified ads and announcements (SBB schools, meetings, symposia, etc.)
without charge. Deadlines for receipt of these items are as follows:
Deadlines
1st week in January for the March issue
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W
. John Judd, NYABB; and Suzanne Leiden Memorial Lecturer, CABB
FIBMS, MIBiol, Society. In North Carolina, the extra room over the garage
is being honored is called the FROG (furnished room over garage); however,
by Immunohematology, in John’s case, it is called a TROG (trophy room over ga-
Journal of Blood Group rage) for all of his well-deserved awards!
Serology and Education John received these awards for his many investigative
for his many contribu- studies, research reports, review papers, presentations, and
tions to the journal and to publications. In 2008, John coauthored the third edition of
the field of blood banking. Judd’s Methods in Immunohematology, a major reference
John served on the edito- methods manual for laboratory workers. This edition con-
rial board of Immunohe- tains more than 150 methods and procedures documents.
matology for 7 years, from During the span of his career, he has had more than 80
2002 through 2008. We scientific papers published in peer-reviewed journals. Ad-
thank him for giving gen- ditionally, John is the author of review articles on lectins,
erously of his time and polyagglutination, elution studies, pretransfusion studies,
ideas to improve the scope Rh blood groups, MNS-system antibodies, and investiga-
and concept of the journal. tion of the positive DAT. These are just some of the topics
This included contributing nine articles published between that he studied and investigated in depth.
1979 and 2005 that were significant to the interest of the A popular speaker, John gave numerous presenta-
readers of Immunohematology. He also was a dedicated, tions at local, state, national, and international meetings.
knowledgeable, and responsive peer reviewer of submitted One could always count on John to talk! In fact, he and the
articles from the beginning of the publication and during late John Case, of Gamma Biologicals, Inc., were famous
the length of his very important career. for their “chats” on the AABB SSCC forum and were well
John retired in 2008 after 34 years as director of a first- known for debating, in depth, many questions concerning
rate AABB-accredited reference laboratory at the Univer- blood group serology and its complexities.
sity of Michigan. His career is a tribute to his determination In addition to this impressive scientific career, John
to investigate the unusual and the undiscovered. In 1972, also managed to be active on many committees of the AABB,
he studied at the Sir John Cass College in London, England, MABB, and International Society of Blood Transfusion. He
where he was elected a Fellow of the Institute of Biomedical was also on the board of the AABB for 4 years and was on
Sciences (FIBMS). He came to the University of Michigan the board and president of the MABB.
in 1974 and was the director of the reference laboratory and The editors, authors, and reviewers of Immunohematology,
professor of immunohematology in the Department of Pa- Journal of Blood Group Serology and Education, would
thology until his retirement in 2008. He is now emeritus like to thank John for his contributions to the field of blood
professor of immunohematology, Department of Pathol- banking during his 34 years at the bench as an investigator,
ogy, at the University of Michigan. John was awarded the and as an author, educator, and mentor. We would particu-
usual blue and gold embossed chair at retirement to prove larly like to acknowledge his contributions during the past
it! John and his wife, Jane, then moved to North Carolina to several years as an editor, peer reviewer, and contributing
relax and watch the golfers go by. author to Immunohematology. John, you may be gone
John has received many awards, including the Ivor from the field, but you cannot be forgotten. Like the impor-
Dunsford Memorial Award and the John Elliott Memo- tant scientists before you, you have left a large and lasting
rial Award from the American Association of Blood Banks legacy of knowledge and respect. Thank you, and may you
(AABB); the Founders Award and Kay Beattie Lecture from and Jane have a long, healthy, and happy retirement.
the Michigan Association of Blood Banks (MABB); the Pet-
taway-Sheppard Award of the North Carolina Association Delores Mallory, MT(ASCP)SBB
of Blood Banks; L. Jean Stubbins Memorial Lecture of the Emeritus Editorial Board
UTMB Medical Branch; Ronald Dubin Memorial Lecturer, Immunohematology
T
he editors of Immunohematology present a new feature that is extremely exciting. Starting with this issue, Immunohe-
matology will publish a series of 28 “Blood Group Reviews” in upcoming issues. Experts in the field have agreed to put
their considerable knowledge and energy into writing a thorough review of each of the 28 selected areas. Readers will
then have comprehensive reviews of all the major blood group systems and other topics at their fingertips. Of additional value
will be the references for each article to which the reader can refer if interested in reading the original work. The authors will
present each blood group review in the following format: history, nomenclature, genetics, molecular basis, biochemistry, an-
tibodies in system, and clinical significance. The topics that will appear in sequential issues are detailed in the table below.
When all the topics have been published, Immunohematology will publish the entire collection as a stand-alone resource.
This series provides an excellent opportunity for experienced staff to obtain continuing education, and it will be an educa-
tional tool for new staff. The editors envision a copy of the collected reviews as a must-have for transfusion medicine facilities,
libraries, and personal bookshelves everywhere.
ABO FY H CR JMH
MNS JK DO KN P/Globoside
Rh and RHAG DI CO IN Lows
LU YT LW I GIL
KEL and Kx XG Ch/Rg OK
LE SC GE RAPH
Sandra Nance
Connie M. Westhoff
Editors-in-Chief
Immunohematology
Applications are invited from medical or science graduates for the Master of Science (MSc)
degree in Transfusion and Transplantation Sciences at the University of Bristol. The course
starts in October 2009 and will last for 1 year. A part-time option lasting 2 or 3 years is also
available. There may also be opportunities to continue studies for PhD or MD following the
MSc. The syllabus is organized jointly by The Bristol Institute for Transfusion Sciences and the
University of Bristol, Department of Pathology and Microbiology. It includes:
Application can also be made for Diploma in Transfusion and Transplantation Science or a
Certificate in Transfusion and Transplantation Science.
Dr Patricia Denning-Kendall
University of Bristol, Paul O’Gorman Lifeline Centre, Department of Pathology and
Microbiology, Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, England
Fax +44 1179 595 342, Telephone +44 1779 595 455, e-mail: p.a.denning-kendall@bristol.
ac.uk.
September 25
Illinois Association of Blood Banks (ILABB)
The Illinois Association of Blood Banks (ILABB) Fall Meeting will be held September 25, 2009, at the Lisle/Naperville Hil-
ton in Lisle, Illinois. For additional details please visit the Web site at www.ilabb.org or contact Kristi Williams at (309)
745-8999 or WilliaKr@usa.redcross.org.
For additional information, contact: Gregory Halverson, New York Blood Center, 310 East 67th Street, New York, NY
10021; e-mail: ghalverson@nybloodcenter.org; phone: (212) 570-3026; fax: (212) 737-4935; or visit the web site at http://
www.nybloodcenter.org >research >immunochemistry >current list of monoclonal antibodies available.
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National Platelet Serology Reference Laboratory National Neutrophil Serology Reference
Laboratory
Diagnostic testing for: Our laboratory specializes in granulocyte antibody detection
• Neonatal alloimmune thrombocytopenia (NAIT) and granulocyte antigen typing.
• Posttransfusion purpura (PTP)
• Refractoriness to platelet transfusion Indications for granulocyte serology testing include:
• Heparin-induced thrombocytopenia (HIT) • Alloimmune neonatal neutropenia (ANN)
• Alloimmune idiopathic thrombocytopenia purpura (AITP) • Autoimmune neutropenia (AIN)
• Transfusion related acute lung injury (TRALI)
Medical consultation available
Methodologies employed:
Test methods: • Granulocyte agglutination (GA)
• GTI systems tests • Granulocyte immunofluorescence by flow cytometry (GIF)
— detection of glycoprotein-specific platelet antibodies • Monoclonal antibody immobilization of neutrophil antigens
— detection of heparin-induced antibodies (PF4 ELISA) (MAINA)
• Platelet suspension immunofluorescence test (PSIFT)
• Solid phase red cell adherence (SPRCA) assay TRALI investigations also include:
• Monoclonal immobilization of platelet antigens (MAIPA) • HLA (PRA) Class I and Class II antibody detection
• Molecular analysis for HPA-1a/1b For further information contact:
For further information, contact Neutrophil Serology Laboratory (651) 291-6797
Platelet Serology Laboratory (215) 451-4205 Randy Schuller(651) 291-6758
schullerr@usa.redcross.org
Maryann Keashen-Schnell (215) 451-4041 office
mschnell@usa.redcross.org
Sandra Nance (215) 451-4362 American Red Cross Blood Services
snance@usa.redcross.org
Neutrophil Serology Laboratory
American Red Cross Blood Services 100 South Robert Street
Musser Blood Center St. Paul, MN 55107
700 Spring Garden Street
Philadelphia, PA 19123-3594
For information, contact Our ELISA for IgA detects protein to 0.05 mg/dL.
Mehdizadeh Kashi at (503) 280-0210, or write to:
For additional information contact Cindy Flickinger at
Pacific Northwest Regional Blood Services (215) 451-4909, or e-mail: flickingerc@usa.redcross.org,
ATTENTION: Tissue Typing Laboratory
or write to:
American Red Cross
3131 North Vancouver American Red Cross Blood Services
Musser Blood Center
Portland, OR 97227
700 Spring Garden Street
Philadelphia, PA 19123-3594
CLIA licensed, ASHI accredited ATTN: Cindy Flickinger
CLIA licensed
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 Ordering Information
to be found in testing 100 donors. Variations in different The book, which costs $25, can be
ethnic groups are given. ordered in two ways:
• Characteristics of the antibodies and optimal technique(s) • Order online from the publisher
for their detection. at: www.sbbpocketbook.com
• Technical tips to aid their identification. • Order from the authors, who
• Whether the antibody has been found as an autoantibody. will sign the book. Send a check,
made payable to “New York Blood
Center” and indicate “Pocket
Pocketbook Education Fund book” on the memo line, to:
The authors are using royalties generated from the sale of
this pocketbook for educational purposes to mentor people Marion Reid
in the joys of immunohematology as a career. They will Laboratory of Immunochemistry
accomplish this in the following ways: New York Blood Center
• Sponsor workshops, seminars, and lectures 310 East 67th Street
• Sponsor students to attend a meeting New York, NY 10065
• Provide copies of the pocketbook Please include the recipient’s
(See www.sbbpocketbook.com for details to apply for funds) complete mailing address.
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
Why be an SBB?
Professional growth Job placement Job satisfaction Career advancement
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
Community Blood Center/CTS Dayton, Ohio Nancy Lang 937-461-3293 nlang@cbccts.org http://www.cbccts.org/education/ On line
sbb.htm
Gulf Coast School of Blood Bank Technology Clare Wong 713-791-6201 cwong@giveblood.org www.giveblood.org/education/ On line
distance/htm
Hoxworth Blood Center, Univ. of Cincinnati Susan Wilkinson 513-558-1275 susan.wilkinson@uc.edu www.hoxworth.org On site
Indiana Blood Center Jayanna Slayten 317-916-5186 jslayten@indianablood.org www.indianablood.org On line
Univ. of Texas SW Medical Center Barbara Laird- 214-648-1785 barbara.fryer@UTSouthwestern.edu http://telecampus.utsystem.edu On line
Fryer
Additional Information can be found by visiting the following Web sites: www.ascp.org, www.caahep.org and www.aabb.org Revised August 2007
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 87
Immunohematology
Immunohematology
Journal
JOURNAL of
OF Blood
BLOOD Group SerologyAND
GROUP SEROLOGY andEDUCATION
Education
Instructions
Instructions tofor
theAuthors
Authors
I. GENERAL INSTRUCTIONS b. Use short headings for each column needed and capitalize first letter
Before submitting a manuscript, consult current issues of of first word. Omit vertical lines.
Immunohematology for style. Double-space throughout the manuscript. c. Place explanation in footnotes (sequence: *, †, ‡, §, ¶, **, ††).
Number the pages consecutively in the upper right-hand corner, beginning 8. Figures
with the title page. a. Figures can be submitted either by e-mail or as photographs (5″ × 7″
II. SCIENTIFIC ARTICLE, REVIEW, OR CASE REPORT WITH glossy).
LITERATURE REVIEW b. Place caption for a figure on a separate page (e.g. Fig. 1 Results of...),
A. Each component of the manuscript must start on a new page in the ending with a period. If figure is submitted as a glossy, place first
following order: author’s name and figure number on back of each glossy submitted.
1. Title page c. When plotting points on a figure, use the following symbols if
2. Abstract possible: � � � � � �.
3. Text 9. Author information
4. Acknowledgments a. List first name, middle initial, last name, highest degree, position held,
5. References institution and department, and complete address (including ZIP
6. Author information code) for all authors. List country when applicable.
7. Tables
8. Figures III. EDUCATIONAL FORUM
B. Preparation of manuscript A. All submitted manuscripts should be approximately 2000 to 2500
1. Title page words with pertinent references. Submissions may include:
a. Full title of manuscript with only first letter of first word capitalized 1. An immunohematologic case that illustrates a sound investigative
(bold title)
approach with clinical correlation, reflecting appropriate collaboration
b. Initials and last name of each author (no degrees; all CAPS), e.g., M.T.
JONES, J.H. BROWN, AND S.R. SMITH to sharpen problem solving skills
c. Running title of ≤40 characters, including spaces 2. Annotated conference proceedings
d. Three to ten key words B. Preparation of manuscript
2. Abstract 1. Title page
a. One paragraph, no longer than 300 words a. Capitalize first word of title.
b. Purpose, methods, findings, and conclusion of study b. Initials and last name of each author (no degrees; all CAPs)
3. Key words 2. Text
a. List under abstract a. Case should be written as progressive disclosure and may include the
4. Text (serial pages): Most manuscripts can usually, but not necessarily, following headings, as appropriate
be divided into sections (as described below). Survey results and i. Clinical Case Presentation: Clinical information and differential
review papers may need individualized sections
diagnosis
a. Introduction
ii. Immunohematologic Evaluation and Results: Serology and
Purpose and rationale for study, including pertinent background
references molecular testing
b. Case Report (if indicated by study) iii. Interpretation: Include interpretation of laboratory results,
Clinical and/or hematologic data and background serology/molecular correlating with clinical findings
c. Materials and Methods iv. Recommended Therapy: Include both transfusion and
Selection and number of subjects, samples, items, etc. studied and nontransfusion-based therapies
description of appropriate controls, procedures, methods, equipment, v. Discussion: Brief review of literature with unique features of this
reagents, etc. Equipment and reagents should be identified in case
parentheses by model or lot and manufacturer’s name, city, and state. vi. Reference: Limited to those directly pertinent
Do not use patient’s names or hospital numbers. vii. Author information (see II.B.9.)
d. Results viii. Tables (see II.B.7.)
Presentation of concise and sequential results, referring to pertinent
tables and/or figures, if applicable IV. LETTER TO THE EDITOR
e. Discussion A. Preparation
Implication and limitations of the study, links to other studies; if 1. Heading (To the Editor)
appropriate, link conclusions to purpose of study as stated in
2. Title (first word capitalized)
introduction
3. Text (written in letter [paragraph] format)
5. Acknowledgments: Acknowledge those who have made substantial
contributions to the study, including secretarial assistance; list any grants. 4. Author(s) (type flush right; for first author: name, degree, institution,
6. References address [including city, state, Zip code and country]; for other authors:
a. In text, use superscript, Arabic numbers. name, degree, institution, city and state)
b. Number references consecutively in the order they occur in the text. 5. References (limited to ten)
7. Tables 6. Table or figure (limited to one)
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. Send all manuscripts by e-mail to immuno@usa.redcross.org
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 6 215
88 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
FIRST CLASS
U.S. POSTAGE
PAID
SOUTHERN MD
Musser Blood Center PERMIT NO. 4144
700 Spring Garden Street
Philadelphia, PA 19123-3594