Immuno 33 4 Linked

Journal of Blood Group Serology and Molecular Genetics
V o l u m e 3 3 , N u m b e r 4, 2017
This issue of Immunohematology
is supported by a contribution from
Grifols Diagnostics Solutions, Inc.
Dedicated to advancement and education in

molecular and serologic immunohematology
Immunohematology
Volume 33, Number 4, 2017
CO N TEN TS
147 Original Report

Assessment of common red blood cell pretreatments to yield an
accurate serologic antigen phenotype compared with genotype-
predicted phenotype
T. Horn, J. Hamilton, J. Kosanke, V.W. Hare, W. Kluver, W. Beres, S. Nance,
and M.A. Keller
152 Case Report

Anti-Vel alloimmunization and severe hemolytic disease of the
fetus and newborn
K.J. Moise Jr., Y. Morales, M.F. Bertholf, S.N. Rossmann, and Y. Bai
155 S ero lo g i c M e t h o d R e v i e w
Separation of multiple antibodies by adsorption with allogeneic
red blood cells
E.M. Ekema
159 Original Report

Hemovigilance and the Notify Library
B.I. Whitaker, D.M. Strong, M.J. Gandhi, and E. Petrisli
165 Original Report

Clinical and laboratory profile of anti-M
D. Basu, S. Basu, M. Reddy, K. Gupta, and M. Chandy
170 S ero lo g i c M e t h o d R e v i e w
Dithiothreitol treatment of red blood cells
C.B. Bub
173 C omm u n i c at i o n s
Thank You
S. Nance and C. Flickinger
174 175 183 187 191

E r r at u m Announcements A dv ertisements Instructions Subscription
for Authors I n f o r m at i o n
Editor-in- Chief Edi torial Board
Sandra Nance, MS, MT(ASCP)SBB
Philadelphia, Pennsylvania Patricia Arndt, MT(ASCP)SBB Geralyn M. Meny, MD
Pomona, California San Antonio, Texas
Managing Edi tor
Cynthia Flickinger, MT(ASCP)SBB Barbara J. Bryant, MD Paul M. Ness, MD
Wilmington, Delaware Galveston, Texas Baltimore, Maryland
Lilian M. Castilho, PhD Thierry Peyrard, PharmD, PhD
Te c h n i c a l E d i t o r s
Campinas, Brazil Paris, France
Janis R. Hamilton, MS, MT(ASCP)SBB
Detroit, Michigan Martha R. Combs, MT(ASCP)SBB S. Gerald Sandler, MD
Durham, North Carolina Washington, District of Columbia
Christine Lomas-Francis, MSc
New York City, New York Geoffrey Daniels, PhD Ira A. Shulman, MD
Bristol, United Kingdom Los Angeles, California
Joyce Poole, FIBMS
Bristol, United Kingdom Anne F. Eder, MD Jill R. Storry, PhD
Washington, District of Columbia Lund, Sweden
Dawn M. Rumsey, ART(CSMLT)
Norcross, Georgia Melissa R. George, DO, FCAP Nicole Thornton
Hershey, Pennsylvania Bristol, United Kingdom
Tiffany Walters, MT(ASCP)SBBCM
Julie K. Karp, MD
Charlotte, North Carolina
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A s s oc i at e M e d i c a l E d i t o r s New York City, New York Bristol, United Kingdom
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Philadelphia, Pennsylvania
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Durham, North Carolina
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O n O ur C ov er
Again the traffic lights that skim thy swift
Unfractioned idiom, immaculate sigh of stars,
Beading thy path—condense eternity:
And we have seen night lifted in thine arms.
The modernist American poet Hart Crane wrote these words in his proem To Brooklyn Bridge. Completed after 14
years in 1883 and considered a modern miracle, a fusing of creative epiphany with industrial power and precision,
the Brooklyn Bridge spans the East River in New York, joining the boroughs of Brooklyn and Manhattan. The
list of notable artists and writers inspired by the bridge also includes Joseph Stella, an Italian immigrant. For
modernists like Stella, the utilitarian and the aesthetic mingled in both art and public works, and the true rendition
of a painting’s subject addressed not only its form but its impact. Perhaps for Stella, the geometrically dazzling
cables and arches symbolized the tenuous and fantastical vault of immigration itself. He painted Brooklyn Bridge
in 1920 and would go on to make the bridge a subject of numerous later paintings. As a measure of his artistic
vision, it would span his life. An article in this issue of Immunohematology discusses the use of dithiothreitol
(DTT), a reducing reagent used to disrupt the bridging of the disulfide bonds between amino acid residues.
David Moolten, MD
ii I M M U N O H E M ATO LO GY, Vo l u m e 3 3 , N u m b e r 4, 2 017

Original Report
Assessment of common red blood cell

pretreatments to yield an accurate serologic
antigen phenotype compared with genotype-
predicted phenotype
T. Horn, J. Hamilton, J. Kosanke, V.W. Hare, W. Kluver, W. Beres, S. Nance, and M.A. Keller
For patients requiring multiple transfusions and patients with Key Words: antigen phenotype, microhematocrit separation,
positive direct antiglobulin tests (DATs), an extended red blood EDTA glycine acid (EGA), hypotonic saline wash, RBC
cell (RBC) phenotype can provide valuable information and
help to determine the risk of forming alloantibodies. In some
genotyping
instances, the phenotype may be used for prophylactic matching.
Phenotyping in this patient population is often hindered by the Red blood cell (RBC) phenotyping is valuable for trans-
presence of circulating donor cells and/or by a positive DAT. fusion management of multiply transfused patients, including
Several methods, such as EDTA glycine acid (EGA) treatment to
remove IgG, hypotonic saline wash to separate autologous RBCs, the determination of the risk of forming alloantibodies.
or reticulocyte separation, are often used in these situations to Extended phenotype matching is most commonly used in
isolate patient RBCs for serologic phenotyping. This study aimed situations where there is a need to avoid sensitization in a
to determine the accuracy of each RBC pretreatment method by
nontransfused patient1 or to avoid further alloimmunization in
comparing serologically determined antigen types with those
predicted by RBC genotyping. Forty-eight peripheral blood a patient who has already been sensitized.2–5 Phenotyping of
samples from recently transfused patients were phenotyped patients in these situations is often hindered by the presence of
for selected antigens in the Rh, Kell, MNS, Duffy, and Kidd circulating donor cells or by a positive direct antiglobulin test
systems. Treatment methods for the sample sets were reticulocyte
separation (N = 12), EGA (N = 16), and hypotonic saline wash
(DAT). Because a donor RBC can survive in circulation for up
(N = 20). DNA was extracted using standard methods, and to 120 days, it is not recommended to phenotype individuals
genotyping was performed using the HEA BeadChip panel. In who have been transfused in the past 3 months.6 In addition,
addition, 21 samples positive for RBC-bound IgG were EGA- phenotyping with certain types of antisera may be hindered
treated up to two times. These samples were analyzed pre- and
post-EGA treatment for RBC-bound IgG by tube DAT and by in patients with autoantibodies causing a positive DAT. In
flow cytometry with fluorescein isothiocyanate–labeled anti- these scenarios, methods—such as EDTA glycine acid (EGA)
human IgG. After reticulocyte separation, 3 of the 12 samples had treatment to remove IgG, hypotonic wash to separate autologous
discordant types with one antigen each: Fyb, N, and K; serologic
cells from patients with sickle cell disease, or microhematocrit
results were negative compared with genotype-predicted positive
phenotype results. The EGA-treated sample set showed one centrifugation to isolate reticulocytes—are often used in an
discordant type: Fyb; serologic results were negative compared attempt to obtain a phenotype.6 The effectiveness of removing
with genotype-predicted positive phenotype results. Four of the IgG from RBCs to obtain DAT-negative RBCs can vary between
20 samples had discordant types involving the following antigens:
Fyb, N, e, and M; serologic results were negative compared
methods.7 With the increasing availability of RBC genotyping,
with genotype-predicted positive phenotype results. After EGA more blood banks are using this testing to obtain a predicted
treatment of 21 samples, 14 (67%) were negative for RBC-bound RBC phenotype as an alternative to RBC pretreatments
IgG by tube DAT, and 7 remained positive. Using flow cytometry, followed by serologic antigen typing.8 A RBC genotyping
EGA treatment rendered only 4 samples negative, and 17 remained
positive. In the antigen testing sample set of 48 samples, 10 of panel such as the U.S. Food and Drug Administration (FDA)-
511 total antigen types tested were discordant. Discordant types approved PreciseType Molecular BeadChip9 (Immucor,
were most frequent in the hypotonic saline wash sample set (N = Norcross, GA) can predict antigen status for many of the major
6). In the flow cytometry sample set, 48 percent of the samples
clinically significant blood group systems (Table 1). Especially
negative by tube DAT after EGA elution had detectable RBC-
bound IgG by flow cytometry. These findings suggest that caution in patients with hemoglobinopathies, genotyping is a routine
should be taken when using phenotype results from all pretreated approach to obtaining an extended RBC phenotype.3,5,10 It has
RBCs and support the use of RBC genotyping to predict RBC been previously documented that genotyping can provide
antigen expression in samples from recently transfused patients.
Immunohematology 2017;33:147–151.
more accurate RBC phenotype results than routine serology.11
I M M U N O H E M ATO LO GY, Vo l u m e 3 3 , N u m b e r 4, 2 017 147

T. Horn et al.
Table 1. Blood group antigens predicted by the HEA BeadChip treatment for RBC-bound IgG by tube DAT (Immucor) and by
genotyping panel
flow cytometry (Becton Dickinson FACScalibur, San Jose, CA)
Blood group system Antigens with fluorescein isothiocyanate–labeled anti-human IgG (Life
RH C, c, E, e, V, VS Technologies, Carlsbad, CA).
FY Fy , Fyb, Fyb silenced
a
DO Doa, Dob, Hy, Joa Results

SC Sc1, Sc2
DI Dia, Dib Among the 147 antigen typing results in 12 samples
LW LW , LW
a b tested after reticulocyte separation, 3 (2.0%) were discordant
CO Coa, Cob
with 1 antigen each; 1 sample (R-1) phenotyped Fy(b–) and
was predicted to be Fy(b+w) by genotyping, 1 sample (R-4)
JK Jka, Jk b
phenotyped N– and was predicted to be N+ by genotyping,
KEL K, k, Kpa, Kpb, Jsa, Jsb
and 1 sample (R-7) phenotyped K– and was predicted to be
LU Lua, Lub
K+ by genotyping. Among the 116 antigen typing results in
MNS M, N, S, s, U, UVAR*
the 16 EGA-treated samples, 1 was discordant (0.8%); sample
*Includes UVAR (P2) and UVAR (NY).
(E-12) phenotyped Fy(b–) and was predicted to be Fy(b+w) by
genotyping. Among the 248 antigen typing results in the 20
A small study was performed to measure, by tube and flow hypotonic wash samples, 6 (2.4%) were discordant; 2 samples
cytometry, the effectiveness of EGA treatment in removing IgG (H-10, H-15) phenotyped Fy(b–) and were predicted to be
from RBCs. This study aimed to compare the accuracy of the Fy(b+) by genotyping, 2 samples (H-11, H-16) phenotyped N–
results obtained from the commonly used RBC pretreatment and were predicted to be N+ by genotyping, 1 sample (H-18)
methods with the results from a genotype-predicted phenotype phenotyped M– and was predicted to be M+ by genotyping,
using the BeadChip platform. and 1 sample (H-16) phenotyped e– and was predicted to be
e+ by genotyping (ruling out common e variants interrogated
Materials and Methods by the HEA BeadChip). The antigen typing results for all
samples are shown in Table 2. For comparison of effectiveness
This study was approved by the American Red Cross of EGA treatment by tube-DAT and flow cytometry, 21
institutional review board. A total of 48 peripheral blood samples were tested. Fourteen (67%) samples were negative
samples from recently transfused patients were phenotyped for RBC-bound IgG by tube DAT and 7 were positive. When
for RH, KEL, MNS, FY, and JK antigens (Table 2). RBC tested by flow cytometry, 4 (19%) samples were negative after
pretreatment methods included reticulocyte separation EGA treatment, and 17 remained positive. Interestingly, of
(N = 12), EGA treatment (N = 16), and hypotonic wash the 17 samples that were positive by flow cytometry, 10 were
(N = 20). Serologic antigen typing was performed by the tube negative by tube DAT after EGA treatment.
method with licensed antisera from various sources (American
Red Cross, Gaithersburg, MD; Immucor). Genomic DNA was Discussion
extracted from peripheral blood mononuclear cells using
DNA Blood Mini Kits (Qiagen, Carlsbad, CA), and genotyping This study aimed to compare antigen types obtained after
was performed per the manufacturer’s directions using the commonly used RBC pretreatments to RBC genotyping using
HEA BeadChip (Immucor) with a 96-well Veriti thermal an FDA-approved test. A total of 10 discordant results were
cycler (Applied BioSystems, Foster City, CA), InSlideOut discovered in 48 samples, with discordant types identified in
oven (Boekel Scientific, Feasterville, PA), and Array Imaging each of the three treatment sets and with each discordant result
System (Immucor). The blood group antigens predicted by the being a false negative by phenotyping after RBC treatment.
genotyping panel are listed in Table 1. For the samples studied Discordant types (N = 6) were most frequently identified in
by flow cytometry and tube-DAT testing, 21 additional samples the hypotonic wash sample set (N = 20), with 25 percent of
positive for RBC-bound IgG were EGA-treated (Gamma EGA samples being discordant with one or more antigens. The
kit, Immucor) until a negative tube DAT was obtained (up to total number of antigens that were tested on these samples
two times). The samples were analyzed before and after EGA was 248, 2.4 percent of which were found to be discordant.
148 I M M U N O H E M ATO LO GY, Vo l u m e 3 3 , N u m b e r 4, 2 017

Antigen phenotypes post RBC pretreatment
Table 2. Antigen typing results [positive (+), negative (0), or weak (+w)] for reticulocyte separation samples (R-1 through R-12), EDTA
glycine acid (EGA)-treated samples (E-1 through E-16), and hypotonic saline wash samples (H-1 through H-20).
Sample C E c e M N S s K Fya Fy b Jk a Jk b
R-1 + 0 + + 0 + 0 + 0 0 0/+w 0 +
R-2 + 0 0 + NT NT 0 + 0 + + + +
R-3 0 0 + + + + NT NT 0 NT NT 0 +
R-4 + 0 + + + 0/+ + + NT + 0 + +
R-5 0 0 + + + + 0 + 0 + 0 + +
R-6 0 0 + + + + 0 + NT 0 0 + 0
R-7 0 + + 0 + + + 0 0/+ 0 + + +
R-8 0 0 + + 0 + 0 + 0 0 0 + +
R-9 0 0 + + + + + + 0 0 0 + +
R-10 0 + + 0 0 + + + 0 + 0 + +
R-11 0 0 + + + 0 + + 0 0 0 + 0
R-12 0 + + + + + + + NT + + 0 +
E-1 NT NT NT NT NT NT + + NT 0 + + 0
E-2 NT NT NT NT NT NT 0 + NT + + + NT
E-3 NT NT NT NT NT NT 0 + NT 0 + + +
E-4 + 0 0 + 0 + 0 + NT + + 0 +
E-5 NT NT NT NT NT NT + 0 NT + + NT NT
E-6 NT NT NT NT NT NT 0 + NT 0 + NT NT
E-7 + + + + + + 0 + NT + + + +
E-8 0 0 + + + 0 + + NT + + + 0
E-9 NT NT NT NT NT NT + 0 NT + + NT NT
E-10 + 0 + + + 0 + + NT 0 + 0 +
E-11 + 0 + NT 0 + + 0 NT + 0 0 +
E-12 NT NT NT NT NT NT 0 + NT + 0/+w + +
E-13 NT NT NT NT NT NT + + NT + 0 + +
E-14 NT NT NT NT NT NT 0 + NT + + + 0
E-15 NT NT NT NT NT NT 0 + NT 0 + + 0
E-16 NT NT NT NT NT NT 0 + NT 0 + NT NT
H-1 + 0 + NT 0 + 0 + 0 0 + + +
H-2 + 0 + + 0 + 0 + 0 0 0 0 +
H-3 0 0 + NT NT NT 0 + 0 0 0 + 0
H-4 0 0 + NT + 0 + + 0 0 0 + 0
H-5 0 0 + + + NT 0 + NT 0 0 + +
H-6 + 0 0 NT + + 0 + 0 0 0 + 0
H-7 0 0 + NT + + + + 0 0 0 + 0
H-8 0 0 + + + + + + 0 0 0 + +
H-9 + 0 + + + + 0 + 0 0 0 + +
H-10 0 0 + + + 0 0 + 0 0 0/+ + +
H-11 0 0 + + NT 0/+ 0 + 0 0 0 + 0
H-12 + 0 + + + + 0 + 0 0 0 + +
H-13 0 + + + + + + 0 0 + 0 + 0
H-14 + 0 + + + + 0 + 0 0 + + +
H-15 0 0 + NT 0 + + + 0 0 0/+ + 0
H-16 0 + + 0/+ + 0/+ 0 + 0 + + + +
H-17 + 0 + + + + 0 + 0 0 0 + 0
H-18 0 0 + NT 0/+ + 0 + 0 0 0 + 0
H-19 0 0 + + + + 0 + 0 0 0 + 0
H-20 + 0 + + 0 + 0 + 0 0 0 0 +
Discordant antigens are highlighted in gray with serologic result presented first and genotype-predicted phenotype after the slash. Antigens not tested by
serology are indicated by NT.

T. Horn et al.
One discordant type was noted in the EGA-treated sample set may complicate provision of antigen-matched RBCs for
(N = 16), with 6 percent of samples being discordant with one transfusion-matching protocols requiring FY status. More
or more antigens and 0.8 percent of antigens tested (N = 116) recently, a similar variant was found that can cause weakening
being discordant. Three discordant types were noted in the of Fya.13
reticulocyte-separated sample set (N = 12), with 25 percent Furthermore, flow cytometry of EGA-treated RBCs
of samples being discordant with one or more antigens and suggests that samples negative by tube DAT after EGA elution
2 percent of total antigens tested (N = 147) being discordant may still have trace amounts of RBC-bound IgG detectable
(Figs. 1 and 2). only by flow cytometry.14 It is therefore critical that controls for
residual IgG coating be performed with each sample treated
with EGA (if the treatment is intended to remove RBC-bound
IgG for the purpose of antigen testing), since the effectiveness
of the treatment on removal of IgG may be different for
Number of samples
individual patient samples.

A recent survey of current practices for providing blood
to patients with warm-reactive autoantibodies (WAA) showed
that 75 percent of laboratories surveyed provided phenotype-
matched or genotype-matched RBCs for transfusion, with
80 percent of laboratories using RBC genotyping as part of
an antibody workup in patients with WAA.15 These findings
suggest that caution should be taken when using phenotype
Fig. 1. Concordance of samples tested by treatment. EGA = EDTA results from treated RBCs to confirm suspected antibody
glycine acid–treated sample set; Retic = reticulocyte-separated specificities or to provide extended matching for future
sample set; Hypo = hypotonic saline wash sample set.
transfusions. This study did not rule out the presence of
uncommon variants in the 10 samples with discordant antigen
types. The discrepancies described here were associated with
false-negative typings after cell treatment. False-negative
results may cause the laboratory to misidentify an antibody
specificity and distract the tester from identifying the true
Number of antigens
specificity, especially in patients with variant antigens when

the phenotype is being used to help rule in or out certain
antibodies.
For example, in our study, one sample was discrepant for
e (serologic E+e–, genotype predicted E+e+); if the patient
was receiving blood matched for RH antigens, e– blood would
have been sourced, increasing the complexity because of the
lower incidence of the e– phenotype. If this testing were to be
Fig. 2. Total discordant antigens per treatment. EGA = EDTA
glycine acid–treated sample set; Retic = reticulocyte-separated used to provide antigen-matched blood, it could potentially
sample set; Hypo = hypotonic saline wash sample set. cause delays in blood selection because of the perceived need
for more antigens to be negative than is needed.
This study shows the advantages of using genotyping
Two of the 10 total discordant samples (1 reticulocyte to predict RBC antigen expression and confirms that it is
separation, 1 EGA treatment) carried the missense variant preferable in difficult patient samples. Our results show that
c.265C>T in the ACKR1 gene, which encodes the FY antigens. RBC manipulation can result in serologic/genotypic antigen
This variant causes marked weakening of the Fyb antigen discrepancies and suggest that if extrapolated to general
(Fy x phenotype), which can be weakly agglutinated by some clinical use, additional antigen discrepancies—some with
commercial antisera but may be missed by others12 and is a significant clinical impact—could be recognized.
common cause of Fyb typing discrepancies. This scenario
may result in interpreting a patient with Fy x as Fy(b–) and

Antigen phenotypes post RBC pretreatment
Acknowledgments 11. da Costa DC, Pellegrino J Jr, Guelsin GA, Ribeiro KA, Gilli SC,
Castilho L. Molecular matching of red blood cells is superior to
serological matching in sickle cell disease patients. Rev Bras
The authors would like to thank the technical staff at Hematol Hemoter 2013;35:35–8.
the American Red Cross Immunohematology Reference 12. Tournamille C, Le Van Kim C, Gane P, et al. Arg89Cys
Laboratories and the National Molecular Laboratory who substitution results in very low membrane expression of the
Duffy antigen/receptor for chemokines in Fy(x) individuals.
tested the study samples.
Blood 1998;92:2147–56. Erratum in: Blood 2000;95:2753.
13. Arndt PA, Horn T, Keller JA, Heri SM, Keller MA. First example
References of an FY*01 allele associated with weakened expression of Fya
on red blood cells. Immunohematology 2015;31:103–7.
1. Shulman IA. Prophylactic phenotype matching of donors for 14. Beres W, Beauchamp CM, Nickle PA, Singh S, Nance
the transfusion of nonalloimmunized patients with sickle cell SJ. Comparing the removal of IgG from red blood cells
disease. Immunohematology 2006;22:101–2. (RBCs) by EDTA glycine acid (EGA) and chloroquine
2. Chou ST, Friedman DF. Transfusion practices for patients with diphosphate (CDP) measured by flow cytometry (FC), tube
sickle cell disease at the Children’s Hospital of Philadelphia. and gel direct antiglobulin test (DAT) methods. Transfusion
Immunohematology 2012;28:27–30. 2013;53:2S:181A.
3. Fasano RM, Chou ST. Red blood cell antigen genotyping 15. Ziman A, Cohn C, Carey PM, et al. Warm-reactive
for sickle cell disease, thalassemia, and other transfusion (immunoglobulin G) autoantibodies and laboratory testing
complications. Transfus Med Rev 2016;30:197–201. best practices: review of the literature and survey of current
4. Meny GM. Transfusion protocols for patients with sickle cell practice. Transfusion 2017;57:463–77.
disease: working toward consensus? Immunohematology
2012;28:1–2.
Trina Horn, MS, MLT(ASCP)SBBCM, Manager (corresponding author),
5. Matteocci A, Pierelli L. Red blood cell alloimmunization in
National Molecular Laboratory, American Red Cross Biomedical
sickle cell disease and in thalassaemia: current status, future
perspectives and potential role of molecular typing. Vox Sang Services, 700 Spring Garden Street, Philadelphia, PA 19123, Trina.
2014;106:197–208. Horn@redcross.org; Janis Hamilton, MS, MT(ASCP)SBB, Manager,
6. Fung MK, Eder AF, Spitalnik S, Westhoff CM, Eds. Technical Immunohematology Reference Laboratory, American Red Cross
manual. 19th ed. Bethesda, MD: AABB. Biomedical Services, Detroit, MI; Joanne Kosanke, MT(ASCP)
7. Burin des Roziers N, Squalli S. Removing IgG antibodies SBBCM, Director, Immunohematology Reference Laboratory,
from intact red cells: comparison of acid and EDTA, heat, and American Red Cross Biomedical Services, Columbus, OH; Virginia
chloroquine elution methods. Transfusion 1997;37:497–501. W. Hare, Supervisor, Immunohematology Reference Laboratory,
8. Sandler SG, Horn T, Keller J, Langeberg A, Keller MA. A model American Red Cross Biomedical Services, Douglasville, GA; Wendy
for integrating molecular-based testing in transfusion services. Kluver, MT(ASCP), Technologist III, Immunohematology Reference
Blood Transfus 2015;14:566–72. Laboratory, American Red Cross Biomedical Services, Madison, WI;
9. Hashmi G, Shariff T, Zhang Y, et al. Determination of 24 Wendy Beres, Immunohematology Assay Development II, American
minor red blood cell antigens for more high-throughput Red Cross Biomedical Services, Philadelphia, PA; Sandra Nance, MS,
DNA analysis. Transfusion 2007;47:736–47. Erratum in:
MT(ASCP)SBB, Senior Director, American Red Cross Biomedical
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Notice to Readers Attention:

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Case R ep ort
Anti-Vel alloimmunization and severe

hemolytic disease of the fetus and newborn
K.J. Moise Jr., Y. Morales, M.F. Bertholf, S.N. Rossmann, and Y. Bai
Only rare cases of anti-Vel–associated mild-to-moderate In the patient’s current pregnancy, her anti-Vel titer was
hemolytic disease of the fetus and newborn have been previously 32 at 11 weeks of gestation. A repeat at our regional blood
reported. No case of fetal anemia requiring prenatal therapy has
been noted to date. We report such a case recently encountered
center indicated a titer of 4. Consideration was given to begin
at our Fetal Center. Strategies are discussed for managing a plasmapheresis/intravenous immune globulin treatment;
pregnancy complicated with alloimmunization to an antibody given the patient’s low titer, however, this was not felt to be
to a high-prevalence antigen, including sources of red blood indicated. The patient returned to her local maternal-fetal
cells for intrauterine transfusions. Immunohematology
2017;33:152–154. medicine specialist and was followed with weekly middle
cerebral artery Doppler ultrasounds to measure the peak
Key Words: anti-Vel, Vel, red blood cell antibody, red blood systolic velocity (MCA-PSV). She was encouraged to donate
cell alloimmunization, hemolytic disease of the fetus and autologous RBC units in anticipation of a possible need for
newborn, intrauterine transfusion intrauterine transfusion later in gestation. The patient was able
to donate 4 units; these were kept refrigerated for 3–5 days and
The Vel red blood cell (RBC) antigen was first described in then frozen at the local blood center. At 24 weeks of gestation,
1952.1 The antigen is ubiquitous in the general population, with the MCA-PSV became elevated to 1.89 multiples of the median
only 0.04 percent of Caucasian individuals failing to exhibit (MoM), and the patient was referred back to our Fetal Center
expression.2 Only rare cases of anti-Vel–associated mild-to- (>1.5 MoM is indicative of severe fetal anemia8). A repeat
moderate hemolytic disease of the fetus and newborn (HDFN) MCA-PSV was 2.0 MoM, and the repeat maternal anti-Vel
have been previously reported.3–7 The neonatal manifestation titer was 16. A decision was made to proceed with intrauterine
of HDFN in these cases was limited to hyperbilirubinemia transfusion. Arrangements were made for transport of the
requiring only phototherapy. No case of fetal anemia requiring previously donated frozen maternal RBC units to our regional
prenatal therapy has been noted to date. We report such a case blood center. Initial sampling at the time of cordocentesis at 24
recently encountered at our Fetal Center. 1/7 weeks of gestation revealed a fetal hematocrit (Hct) of 11.8
percent (normal 36%) with a reticulocyte count of 14.7 percent
Case Report (normal <2%). RBC typing indicated the fetus to be Vel+.
One unit of maternal autologous RBCs was deglycerolized,
The patient was a 31-year-old woman (gravida/ irradiated, washed, and reconstituted with normal saline
para/abortus [GPA] = G2P1000) who was referred to our to achieve an Hct of 72 percent in the final blood product. A
Fetal Center from a neighboring state for evaluation of Vel 60-mL intravascular transfusion combined with a 40-mL
alloimmunization. Her past history was significant, with the intraperitoneal transfusion was performed successfully. A total
finding of a positive antibody detection test and identification of five intrauterine transfusions were subsequently undertaken
of anti-Vel during her previous pregnancy. Based on literature (Fig. 1). The patient donated 3 additional RBC units during
reports of only mild-to-moderate HDFN in association with the remainder of the pregnancy and maintained an Hct above
Vel alloimmunization, the patient’s obstetrician had elected 32 percent, with oral iron and folate supplementation. The
to follow the previous pregnancy conservatively. The patient additional RBC units for intrauterine transfusion came from
was delivered by vaginal birth at 39 6/7 weeks of gestation. a single local blood donor (2 frozen, 1 fresh) and another rare
The neonate was noted to exhibit severe anemia and was registry donor (1 fresh). The patient was delivered uneventfully
transferred to a tertiary level nursery. After a stormy neonatal by repeat caesarean section at 37 6/7 weeks of gestation of a
course that was complicated by pulmonary hypertension, the 4235-gram male infant with Apgar scores of 8 and 9. The infant
neonate died on day 13 of life. was noted to have an Hct of 46.2 percent (normal 53%) with a

Anti-Vel with severe HDFN
24w 1d 27w 2d 32w 2d

IUT (IVT/IPT) #1 IUT (IVT/IPT) #3 IUT (IVT/IPT) #5
11w 6d INITIAL Hct: 12% INITIAL Hct: 34% INITIAL Hct: 30% DELIVERED
Anti-Vel titer: 4 FINAL KB: N/A FINAL KB: 6.5% FINAL KB: 1.0% at 37w 2d
1/5/2017 1/9/2017 4/4/2017 4/5/2017 4/11/2017 4/27/2017 5/18/2017 6/1/2017 6/22/2017 7/10/2017 8/14/2017
11w 24w 25w 30w 2d 35w 2d TOP-UP

Anti-Vel titer: 32 Anti-Vel titer: 16 IUT (IVT/IPT) #2 IUT (IVT/IPT) #4 IUT (IVT/IPT) #6 Transfusion
MoM: 2.0 INITIAL Hct: 28% INITIAL Hct: 27% INITIAL Hct: 32.4% at 5w
FINAL KB: 20% FINAL KB: 1.8% FINAL KB: 1.9%
Fig. 1 Timeline of diagnostic testing and subsequent treatments. w = weeks; d = days; MoM = multiples of the median; IUT = intrauterine
transfusion; IVT = intravascular transfusion, IPT = intraperitoneal transfusion; Hct = hematocrit; KB = Kleihauer-Betke stain; N/A = not
applicable.
reticulocyte count of 2 percent (normal <7%) and a Kleihauer- essential element of our case. The high prevalence of Vel
Betke stain indicating 3.7 percent fetal cells remaining in the makes finding Vel– donors extremely difficult. The patient
infant’s circulation (normal 100%). The neonatal course was was evaluated before pregnancy, and no sibling or family
benign and required only treatment with bili light therapy for member was identified as a potential donor. A search by our
3 days; the maximum total bilirubin was 10.6 mg/dL (normal regional blood center found only one eligible donor locally.
6.5 mg/dL). He was discharged on day 7 of life. The infant was The patient was instructed to undergo autologous donations
followed weekly by a local pediatric hematologist. He required in the early part of pregnancy and was able to donate 4 units.
one top-up transfusion at 5 weeks of age and was released by Iron and folate supplementation was initiated in advance. We
the pediatric hematologist at 9 weeks of age. previously reported a series of 21 patients who donated 77
units of autologous blood as a source of RBCs for intrauterine
Discussion transfusion in the pre-HIV testing era.11 Three of the patients
in that series were able to donate 6 units with maintenance
Our case illustrates at least three important points. Vel of their baseline Hct levels. In our case, we were able to have
is expressed on fetal RBCs as early as 12 weeks of life; its our patient donate 7 units while maintaining her Hct with
strength appears to be equivalent to that on adult RBCs.9 hematopoietic pharmacologic supplementation. Because
Because alloimmunization to this antigen is so rare and only freshly donated RBCs are typically preferred as a source for
a few case reports of HDFN have been reported to date, it is intrauterine transfusion, we defined the specific time for the
difficult to determine a “critical titer” for this entity. Often, final 3 units of autologous donation and arranged allogeneic
anti-Vel is only IgM, which would not present a risk for donation times so that fresh blood was used for the scheduled
HDFN because transplacental passage cannot occur. Linz intrauterine transfusions. Our experience in timed blood
et al.10 recommended treatment with 2-mercaptoethanol preparation would be useful in managing similar patients.
or dithiothreitol to determine whether an IgG component is Finally, at delivery, we had multidisciplinary commu-
present. Both IgG and IgM had been detected in our case. A nication. To prepare for the urgent need for blood as well as to
predominance of IgG in our case may well explain the severe prevent wasting the rare blood product, we secured 2 frozen
degree of HDFN. One should assume that a paternal Vel autologous compatible RBC units in our regional blood center
antigen will be inherited by the fetus, given that Vel is a high- before the scheduled caesarean delivery. In the event of an
prevalence antigen. Thus, it would appear prudent to institute acute postpartum hemorrhage, we developed a plan with
serial MCA-PSV determinations in the early second trimester our blood bank for the emergency release of ABO-compatible
in the rare case that anti-Vel is detected during pregnancy. RBCs pending the deglycerolization of these frozen units.
The strategy in obtaining sufficient RBC units for
intrauterine transfusion to treat the HDFN is also an

K.J. Moise Jr. et al.
Acknowledgments 9. Toivanen P, Hirvonen T. Fetal development of red cell antigens

K, k, Lua, Lub, Fya, Fyb, Vel and Xga. Scand J Haematol
1969;6:49–55.
The authors would like to thank the blood bank staff at
10. Linz WJ, Fueger JT, Allen S, Johnson ST. Role for serial prenatal
the Memorial Hermann Hospital at the Texas Medical Center anti-Vel quantitative serologic monitoring with 2-ME serum
and the Gulf Coast Regional Blood Center for their assistance treatment during pregnancy: case report. Immunohematology
2010;26:8–10.
in diagnostic testing and blood procurement and processing.
11. Gonsoulin WJ, Moise KJ Jr, Milam JD, Sala JD, Weber
VW, Carpenter RJ Jr. Serial maternal blood donations for
References intrauterine transfusion. Obstet Gynecol 1990;75:158–62.
1. Sussman LN, Miller EB. [New blood factor: Vel.] Rev Hematol
1952;7:368–71. Kenneth J. Moise, Jr., MD, Professor (corresponding author),
2. Sussman LN, Pretshold H. The Vel blood group system. Department of Obstetrics, Gynecology, and Reproductive Sciences,
Haematologia (Budap) 1972;6:129–33. The McGovern School of Medicine at the University of Texas
3. Drachmann O, Lundsgaard A. Prenatal assessment of blood Health Science Center, and the Fetal Center at Children’s Memorial
group antibodies against “public” antigens: an example of anti- Hermann Hospital, Suite 700, 6410 Fannin Street, Houston, TX
Ve (Vel) in pregnancy. Scand J Haematol 1970;7:37–42. 77030, Kenneth.J.Moise@uth.tmc.edu; Yisel Morales, BSN, Clinical
4. Williams CK, Williams B, Pearson J, Steane SM, Steane EA. Research Coordinator, Department of Obstetrics, Gynecology, and
An example of anti-Vel causing mild haemolytic disease of the Reproductive Sciences, The McGovern School of Medicine at the
newborn. Transfusion 1985;25:462. University of Texas Health Science Center, and the Fetal Center at
5. Stiller RJ, Lardas O, Haynes de Regt R. Vel isoimmunization in Children’s Memorial Hermann Hospital, Houston, TX; Marsha F.
pregnancy. Am J Obstet Gynecol 1990;162:1071–2. Bertholf, MD, Associate Vice President, Medical Services, Gulf Coast
6. Le Masne A, Vachée A, Horbey C, et al. [Severe form of neonatal Regional Blood Center, Houston, TX; Susan N. Rossmann, MD, PhD,
hemolytic disease by anti-Vel allo-immunization.] Arch Fr
Chief Medical Officer, Gulf Coast Regional Blood Center, Houston, TX;
Pediatr 1992;49:899–901.
Yu Bai, MD, PhD, Associate Professor, The Department of Pathology
7. van Gammeren AJ, Overbeeke MA, Idema RN, van Beek
and Laboratory Medicine, The McGovern School of Medicine at the
RH, Ten Kate-Booij MJ, Ermens AA. Haemolytic disease
of the newborn because of rare anti-Vel. Transfus Med University of Texas Health Science Center, Houston, TX.
2008;18:197–8.
8. Mari G, Deter RL, Carpenter RL, et al. Noninvasive diagnosis
by Doppler ultrasonography of fetal anemia due to maternal
red-cell alloimmunization: Collaborative Group for Doppler
Assessment of the Blood Velocity in Anemic Fetuses. N Engl J
Med 2000;342:9–14.
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Separation of multiple antibodies by
adsorption with allogeneic red blood cells
E.M. Ekema
Antibody detection and identification are processes that are Reagents/Supplies

commonly performed in the transfusion service before transfusion
of allogeneic red blood cells (RBCs). Antibody identification
Reagents Supplies
usually follows the discovery of a positive antibody detection
test, or other factors such as ABO serum/cell discrepancy or an • Allogeneic red blood cells positive • 1 mL graduated,
for antigen to which antibody is disposable pipettes
incompatible crossmatch. Antibody identification is a necessary
directed against, and antigen
practice in blood banking to determine the suitability of blood • 37°C incubator
negative for other suspected
products for transfusion on an individual basis. When the presence antibody(ies) • Test tubes
of multiple antibodies is suspected, several methods, including • Phosphate-buffered saline • Centrifuge
neutralization of patient’s plasma, titration, elution, chemical
S E R O LO G I C M E T H O D R E V I E W
• Proteolytic enzyme
or enzyme treatment of reagent RBCs, and adsorption with
allogeneic RBCs, may be used to separate and properly identify
other atypical antibodies that are present in a single serum or Procedural Steps
plasma sample. This review will focus on the use of allogeneic
adsorption to identify antibody specificities in a patient’s sample.
• Select test RBCs to be used for allogeneic adsorption.
• Wash the test RBCs three to four times with phosphate-buffered
saline.
• Remove all supernatant saline from the washed RBCs.
Key Words: multiple antibodies, adsorption, allogeneic red
• If enzyme treatment is desired, follow manufacturer’s directions and
blood cells, antibody identification treat the RBCs.
• Wash the RBCs three to four times after treatment with enzymes.
• Add 1 volume serum or plasma to 1 volume washed, packed RBCs.
Principle
• Stopper, gently mix well, and incubate for 30–60 minutes at 37°C.
• Centrifuge for 5 minutes and remove the plasma/serum. Test
Antibody detection and identification are processes that against fresh sample of adsorbing cells to see if adsorption is
complete.
are commonly performed in the transfusion service before • Repeat the procedure with fresh adsorbing cells if adsorption is
the transfusion of allogeneic red blood cells (RBCs). Antibody incomplete.
identification usually follows the discovery of a positive RBCs = red blood cells.
antibody detection test, or other factors such as ABO serum/
cell discrepancy or incompatible crossmatch.1 Antibody
identification is a necessary practice in blood banking to
determine blood products that are suitable for transfusion to Performing pretransfusion testing with a plasma or
an individual. Routinely, antibody identification practices in a serum sample that contains multiple antibodies can present
blood bank comprise the testing of a patient’s plasma against serologic challenges. Differentiating and properly identifying
reagent RBCs using standard agglutination and indirect antibody specificities that are present in a sample containing
antiglobulin methods.1 There are, however, instances when multiple antibodies can be achieved by adsorption of patient
identification of multiple antibodies may be complicated and plasma or serum with allogeneic RBCs. Allogeneic adsorption
require additional serologic methods. When the presence of is used in blood banking to remove and/or separate antibody
multiple antibodies is suspected, several methods—including specificities from a plasma or serum sample with the use of
neutralization of patient’s plasma, titration, elution, chemical RBCs that express the corresponding antigen and that are
or enzyme treatment of reagent RBCs, and adsorption with antigen-negative for other antibodies.2
allogeneic RBCs—may be used to separate and properly If the patient’s phenotype is known, adsorptions with
identify antibodies that are present in a single serum or allogeneic RBCs that are phenotypically similar to the patient’s
plasma sample. RBCs may be used. This process must ensure that adsorption

E.M. Ekema
cannot remove any antibody(ies) that the patient may form antibodies with allogeneic RBCs can be performed, and the
based on his or her RBC phenotype. For example, if the patient adsorbed plasma can then be used for the identification of
types as C–, E–, K–, Fy(a–), Fy(b–), Jk(b–), and S–, the other antibodies.
adsorbing cells must be negative for these antigens so that, In the presence of multiple antibodies in a single plasma
if present, antibodies to these antigens will not be adsorbed or serum sample, allogeneic adsorptions are used to confirm or
onto the adsorbing cells. It is important to note, however, that exclude suspected antibodies against RBC antigens that may
the use of phenotypically similar cells for adsorption carries be masked by an antibody of known specificity. For example,
the inherent risk of adsorbing out alloantibodies to variant if the RBCs of a patient type as E– and the patient’s serum
antigens. This method should be used with extreme caution. contains anti-U, it will be statistically difficult to find sufficient
A preferred method is the use of differential adsorptions U–E+ RBC samples to confirm or exclude the presence of
using a set of three different adsorbing cells with known anti-E. In this scenario, the use of allogeneic adsorptions will
phenotypes. Generally, one cell should be R1R1, another should be necessary to remove anti-U from the test plasma or serum,
be R2R2, and the third cell should be rr. Treating the RBCs to leaving the adsorbed plasma with anti-E, if present. The
be used for allogeneic adsorption with proteolytic enzymes adsorbed plasma can then be tested against E+U+ and E–U+
can also be performed to enhance the adsorption process. cells to determine if the patient’s plasma contains anti-E.
Treatment with enzymes will destroy certain antigens that are If there is no known antibody specificity in the patient’s
sensitive to enzyme treatment such as antigens in the Duffy plasma, differential adsorptions may be necessary to exclude
and MNS blood group systems. or include all the common RBC antigens.
Application Procedure
Adsorption with allogeneic RBCs presents antibody(ies) Select group O allogeneic RBCs to be used for adsorption
to the corresponding antigen-positive RBCs under optimal that are positive for the antigen against which the antibody
conditions so that the antibody will attach to the antigen, to be removed is directed (for plasma with known antibody
thereby removing the antibody from the serum or plasma.3 specificity) and negative for the other antibodies that are
Allogeneic adsorption methods can be used to confirm an suspected to be present in a single plasma or serum sample.
antibody specificity, separate multiple antibodies present in When there is no known antibody specificity in the test plasma
a single plasma or serum, remove autoantibody to permit and the patient’s phenotype is known, select group O allogeneic
the detection of underlying alloantibodies, remove unwanted RBCs that are phenotypically similar to the patient’s RBCs.
antibody from serum that is suitable for the preparation of Note that the use of phenotypically similar cells for adsorption
typing reagents, and assist in detecting the presence of weak carries the inherent risk of adsorbing out alloantibodies to
antigens on RBCs.4 variant antigens and should be used with extreme caution.
When all or most cells of an antibody panel react at For patients with unknown phenotypes, select a set of three
different strengths or different phases in conjunction with a different RBCs with known phenotypes. The group O RBCs
negative autocontrol, the presence of multiple antibodies in the selected should be R1R1, R2R2, and rr. One of the cells must be
patient’s plasma or serum should be suspected, especially if negative for K, another negative for Jka, and the third negative
the reactivity does not fit a single antibody using inclusion and for Jkb to ensure that alloantibodies to common RBC antigens
exclusion methods.1,5 It is important to exclude the presence of a will be excluded.6 Adsorption can be performed with RBCs
possible single antibody to a high-prevalence antigen that may that are untreated or treated with proteolytic enzymes. If
be showing dosage on an RBC panel. If the patient’s phenotype adsorption is performed with untreated RBCs, one of the cells
is known or can be obtained, perform testing using a selected selected should be negative for S and Fya or Fyb.7
cell panel that is similar to the patient’s phenotype. Several Obtain an aliquot of the selected RBCs and wash at least
selected cell panels may be necessary to resolve the antibody three times with saline. After the last wash, centrifuge the
identification issue. There are instances where the separation RBCs for 5 minutes and remove the supernatant saline using
and identification of multiple antibodies cannot be attained disposable pipettes. Add 1 volume of washed, packed RBCs
using the methods described here because of insufficient to 1 volume of the patient’s plasma or serum and mix gently.
reagent RBCs to exclude or include all common alloantibodies. Incubate at 37°C for 30–60 minutes. Mix the serum or plasma/
In this instance, other methods such as adsorption of known

Antibody ID by allogeneic adsorption
cell mixture periodically throughout the incubation period to panel. Theoretically, the total adsorptions needed to completely
enhance adsorption.8 remove an unwanted antibody is the strength of reactivity on
After the incubation period has elapsed, centrifuge the the initial panel plus one. For example, if the initial panel’s
serum or plasma/cell mixture for 5 minutes to pack the RBCs. reactivity is 3+, perform four adsorptions.10 Repeat adsorptions
Remove the supernatant fluid (adsorbed plasma) with a should always be performed with a fresh volume of RBCs and
disposable pipette and dispense into a clean, properly marked not the RBCs already used for prior adsorptions.
test tube. Discard the RBCs if an eluate will not be prepared. Pretreating the RBCs used for allogeneic adsorptions with
If an elution study is indicated, save the RBCs. An elution proteolytic enzymes will reduce the number of adsorptions
study can be performed using the first adsorbing cell, or sets of needed for complete removal of unwanted antibodies because
adsorbing cells, to determine the antibody specificity that has enzyme treatment of cells enhances the uptake of antibodies
been removed by adsorption. Test an aliquot of the adsorbed against enzyme-resistant antigens. The use of a higher cell-to-
plasma or serum with an aliquot of fresh adsorbing cells to see serum ratio can help the adsorption process by increasing the
if adsorption is complete. This testing should be performed by amount of antigens able to adsorb the target antibody.6
the same method that will be used to test the reagent panel
cells with the adsorbed plasma. For example, if the adsorbed Limitations
plasma/reagent cell mixture will be enhanced by low-ionic-
strength saline (LISS) or polyethylene glycol (PEG), the same Adsorption with allogeneic RBCs does not only remove
method should be used for testing of the adsorbed plasma with targeted antibodies but also removes antibodies to high-
adsorbing cells. If adsorption is complete, no reactivity will be prevalence antigens when they are present in the test plasma
observed. If reactivity is observed, adsorption is incomplete. or serum. RBCs used for allogeneic adsorptions are presumed
For incomplete adsorption, repeat the process using a fresh to be positive for all high-prevalence antigens. If the test
volume of washed, packed RBCs until complete adsorption plasma or serum happens to contain an antibody to any of the
is attained. Once complete adsorption is observed, test the high-prevalence antigens, that antibody will be adsorbed onto
adsorbed plasma with a panel of reagent RBCs to exclude other the RBCs. In this case, the antibody will not be detected in the
underlying alloantibodies. adsorbed plasma but can be recovered in an eluate prepared
It is important to know which antibody specificities may be from the adsorbing cells.
present in each adsorbed plasma. R1R1 allogeneic RBCs adsorb Adsorption with allogeneic RBCs is a time-consuming
anti-D, -C, and -e and other antibodies to high-prevalence process that may take several hours to complete if multiple
antigens. Specificities left behind are anti-c and -E. R2R2 adsorptions are required, and this step may be contraindicated
allogeneic RBCs adsorb anti-D, -c, and -E and other antibodies for a patient who is in critical need of blood transfusion. When
to high-prevalence antigens. Specificities left behind are anti-C allogeneic adsorption is indicated for such a patient, transfusion
and -e. The rr cells adsorb anti-c and -e and antibodies to high- of phenotype-matched units may be necessary while testing is
prevalence antigens. Specificities left behind are anti-D, -C, and in process.
-E. In addition to these antibodies, alloantibodies detecting Adsorptions pose the risk of diluting the adsorbed plasma,
antigens that are absent from the adsorbing cells will also be thereby weakening the strength of antibody(ies) present.
left behind in the adsorbed plasma. The adsorbed plasma can Weak-reacting antibodies may be completely missed when
be tested with or without enhancements such as LISS or PEG. testing adsorbed plasma. To attenuate the possibility of missing
It is vital to note that adsorption is more effective if the area a weak-reacting antibody in an allogeneic adsorbed plasma,
of contact between the plasma or serum and RBCs is large. the number of adsorptions should be limited to a maximum of
This effect can be obtained by using large-bore test tubes (16 × six. Increasing the ratio of adsorbed plasma to reagent RBCs
100 mm) for adsorption.9 To completely remove an unwanted during the testing phase increases the chances of identifying
antibody, it may be necessary to perform multiple adsorptions, weak-reacting alloantibodies.
although there is an increased risk of diluting the serum with
each successive adsorption, which can lead to weak or false- Quality Control
negative reactions. The number of adsorptions necessary to
completely remove an unwanted antibody can be suggestively To ensure that allogeneic adsorption is complete, test the
determined by the strength of reactivity on the initial antibody adsorbed plasma with a freshly prepared, untreated, 3–5

E.M. Ekema
percent suspension of allogeneic RBCs used for adsorption 3. Harmening DM. Modern blood banking and transfusion
with or without enhancement. If reactivity persists, adsorption practices. 5th ed. Philadelphia, PA: F.A. Davis Company,
2005:519–32.
is not complete. A negative result proves that adsorption is
4. Judd WJ, Johnson ST, Storry JR. Judd’s methods in
complete, and the adsorbed plasma can be used for testing with immunohematology. 3rd ed. Bethesda, MD: AABB,
RBC panels. However, it is important to note the phenotype 2008:316–9.
of the adsorbing cells when testing for completeness. If anti- 5. Harmening DM. Modern blood banking and transfusion
practices. 5th ed. Philadelphia, PA: F.A. Davis Company,
Jka, for example, is present in the plasma, adsorption with 2005:257–8.
allogenic cells that are Jk(a–) will not remove this antibody 6. Issitt PD, Anstee D. Applied blood group serology. 4th ed.
from the plasma, and the adsorbed plasma will not react with Durham, NC: Montgomery Scientific Publications, 1998:891–3.
the untreated cells that are Jk(a–). 7. Judd WJ, Johnson ST, Storry JR. Judd’s Methods in
immunohematology. 3rd ed. Bethesda, MD: AABB,
2008:469–72.
Acknowledgments 8. Method 4–9: Adsorbing warm-reactive autoantibodies using
allogeneic red cells. In: Roback JD, Grossman BJ, Harris T,
The author thanks Nanette Johnson, MT(ASCP)SBB, et al., Eds. Technical manual. 17th ed. Bethesda, MD: AABB,
2011:925–6.
Director, Immunohematology Reference Laboratory, American
9. Tsimba-Chitsva F, Bishop S, Kezeor K. Warm autoadsorptions
Red Cross, Greater Chesapeake and Potomac Region, for with enzyme-treated red blood cells. Immunohematology
providing technical expertise and for her critical reading and 2012;28:88–90.
editing of the manuscript. 10. Barron C. Allogeneic red blood cell adsorption for removal of
warm autoantibody. Immunohematology 2014;30:153–5.
References
Ernest M. Ekema, BB(ASCP)CM, Technologist I, Immunohematology
1. Rudmann SV. Textbook of blood banking and transfusion Reference Laboratory, American Red Cross, Greater Chesapeake
medicine. 2nd ed. Philadelphia, PA: Elsevier Saunders, and Potomac Region, 4700 Mount Hope Drive, Baltimore, MD 21215,
2005:318–43. Ernest.Ekema@redcross.org.
2. Walker PS. Identification of antibodies to red cells antigens.
In: Roback JD, Grossman BJ, Harris T, et al., Eds. Technical
manual. 17th ed. Bethesda, MD: AABB, 2011:463–96.

Original Report

B.I. Whitaker, D.M. Strong, M.J. Gandhi, and E. Petrisli
Hemovigilance systems allow reporting of adverse occurrences The World Health Organization (WHO) promotes the
associated with blood transfusion to a central database where governance of MPHO in a manner that acknowledges their
events can be reviewed and analyzed for the benefit of patients and
donors. Hemolytic and serologic transfusion reactions are among
exceptional nature. From donation to the follow-up care of
the many types of reactions reported to these systems. The Notify the recipient, MPHO have a shared exposure to risks from
Library, a database of adverse events associated with medical breaches of ethical, legal, and safety standards—for example,
products of human origin, has incorporated hemovigilance into the risk of disease transmission and consequent morbidity or
its didactic resources. Students and practitioners are encouraged
to use the electronic library and to further enhance this resource mortality. Ensuring the protection of the donor, the recipient,
through review and recommendation of additional publications and society as a whole requires establishment of globally
in the area of immunohematology. Immunohematology consensual principles to govern the use of MPHO, such as the
2017;33:159–164.
noncommercial nature of the human body and its parts and
strict traceability associated with vigilance and surveillance.
Key Words: biovigilance, hemovigilance, transfusion The Notify Library1 is the first WHO initiative that covers
reactions, hemolytic transfusion reactions, serologic vigilance across the full MPHO scope.
transfusion reactions
Hemovigilance
Advances in science and health care technology have
led to the development of replacement medicine, with many Hemovigilance of blood transfusions encompasses a set
human body components being collected for the preparation of of surveillance procedures that cover the entire collection
medical products of human origin (MPHO). These collections to transfusion process: from the donor and donation, to the
encompass a wide range of medical products—from cells (e.g., processing of the donor’s blood and its components, to their
human stem cells from peripheral blood, bone marrow, or cord provision and transfusion to patients, to patient follow-
blood; gametes) and tissues (e.g., corneas, musculoskeletal up. Hemovigilance includes the monitoring, reporting,
tissues) to blood and blood components and organs, from investigation, and analysis of adverse events related to the
anatomical components to secretions (e.g., breast milk) and donation, processing, and transfusion of blood, as well as
excretions—all originating from the human body. Donated by the development and implementation of recommendations to
a human with the goal of benefitting others, these MPHO have prevent occurrence or recurrence.2
indeed saved and improved human lives through their clinical Hemovigilance systems arose as a response to the threat
application. to the blood supply from emerging infections, such as HIV
Vigilance is a powerful tool for improving safety and and the hepatitis viruses. Hemovigilance was first developed
quality. Vigilance includes monitoring and reporting adverse in Japan and then in France in 1993, featuring mandatory
events and outcomes associated with therapeutic treatments. reporting. The UK developed the first voluntary system in
Originating from hemovigilance for blood components, 1996, called Serious Hazards of Transfusion (SHOT).3 Over the
biovigilance is the term used for the monitoring of adverse years, systematic analyses from these systems and subsequent
outcomes associated with all MPHO. Sharing the lessons process improvements have led to enhanced patient safety.3–5
learned from adverse outcomes can allow for significant In 2002, the European Union (EU) Commission Directive
process improvements for the greater protection of donors 2002/98/EC set standards of quality and safety for the
and patients. These benefits apply not only to the institution collection, testing, processing, storage, and distribution of
where the incident occurred but also to other institutions human blood and blood components and amended Directive
where an identical or similar incident might occur. Detection, 2001/83/EC, thereby mandating hemovigilance in the
investigation, and communication of adverse events provide EU. Subsequently, other countries around the world have
the transparency and openness that these uniquely sourced established hemovigilance systems, adopting either active or
medical treatments demand. passive hemovigilance reporting. Systems engaging in active

B.I. Whitaker et al.
hemovigilance require a confirmation of a transfusion reaction or events that might have teaching value and assist in the
or the lack thereof after every transfusion. In 1998, the estimation of risk.
European Haemovigilance Network was established; in 2009, The core of the Notify Library Web site (www.notifylibrary.
it evolved into the International Haemovigilance Network to org) includes an online publicly accessible relational database
share common definitions, findings, and interventions and to of adverse occurrences collected and analyzed by dedicated
promote transfusion safety worldwide. editorial groups of international experts, regulators, and
The Haemovigilance Working Party of the International clinicians. The database is not a vigilance reporting program
Society of Blood Transfusion (ISBT) has developed transfusion but rather a collection of information identified primarily by
reaction definitions (Tables 1 and 2) and imputability criteria review of published articles in scientific journals and/or books.
(Table 3). Imputability is the strength of the relationship It also includes case reports from regulatory or professional
between the transfusion and the adverse transfusion reaction vigilance programs (gray literature). Sources include events
and is usually determined through a thorough investigation that may have occurred in procurement and processing, as
of the adverse reaction. Standard definitions for hemolytic well as in the clinical application, of blood, organs, tissues,
transfusion reactions include acute hemolytic transfusion and cells used in transfusion, transplantation, and assisted
reactions (AHTRs), delayed hemolytic transfusion reactions reproduction. For each adverse occurrence type, at least one
(DHTRs), and delayed serologic transfusion reactions (DSTRs) reference is cited. The project’s collaborating international
(Table 1).2 Non-hemolytic transfusion reactions are described experts provide a structured analysis, focused in particular on
in Table 2. how the adverse occurrence was recognized and how it was
shown to have been associated with the donation, processing,
Notify Library or clinical application of MPHO. Categories of occurrences that
are analyzed include transmitted infections, malignancies,
The Notify Library is a database developed through a donor reactions, clinical complications, and process-associated
collaboration of WHO and the Italian National Transplant incidents.
Center (CNT).6 It supports the sharing of published vigilance Currently, each database entry is given a title and a unique
information for teaching purposes and for greater public numeric identifier. Through the case analysis, the entry is
transparency on the use of MPHO. The library aims to support assigned an adverse occurrence type; an MPHO type; the
three key audiences: the general public (especially potential time to detection; alerting signs, symptoms, and evidence
donors or recipients), health professionals working in donor of occurrence; the estimated frequency; demonstration of
detection and selection or in the clinical application of MPHO, imputability and imputability grade; keywords; copy of the
and health authorities responsible for vigilance systems. It reference; and any expert comments about the reference or
aims to be comprehensive, describing all types of reactions occurrence. To facilitate a structured database search, all cases
have been classified according to a taxonomy of two main
Table 1. Hemolytic transfusion reaction definitions developed by the Haemovigilance Working Party of the International Society of Blood
Transfusion2
Adverse transfusion reaction Definition
Hemolytic transfusion A hemolytic transfusion reaction is one in which symptoms and clinical or laboratory signs of increased red cell
reactions (general) destruction are produced by transfusion. Hemolysis can occur intravascularly or extravascularly and can be immediate
(acute) or delayed.
Acute hemolytic transfusion AHTR has its onset within 24 hours of a transfusion. Clinical or laboratory features of hemolysis are present.
reaction (AHTR)
Not all clinical or laboratory features are present in cases of AHTR. Blood group serology usually shows abnormal results
but absence of immunological findings does not exclude AHTR. AHTR may also be due to erythrocyte auto-antibodies
in the recipient or to non-immunological factors like mechanical factors inducing hemolysis (malfunction of a pump, of a
blood warmer, use of hypotonic solutions, etc.).
Delayed hemolytic DHTR usually manifests between 24 hours and 28 days after a transfusion and clinical or laboratory features of hemolysis
transfusion reaction (DHTR) are present. Signs and symptoms are similar to AHTR but are usually less severe. DHTR may sometimes manifest as an
inadequate rise of post-transfusion hemoglobin level or unexplained fall in hemoglobin after a transfusion. Blood group
serology usually shows abnormal results.
Delayed serologic reaction There is a DSTR when, after a transfusion, there is demonstration of clinically significant antibodies against red blood
(DSTR) cells which were previously absent (as far as is known) and when there are no clinical or laboratory features of hemolysis.
This term is synonymous with alloimmunization.

Table 2. Non-hemolytic transfusion reaction definitions developed by the Haemovigilance Working Party of the International Society of
Blood Transfusion2
Febrile non hemolytic There is a FNHTR in the presence of one or more of the following: fever (≥38°C oral or equivalent and a change of ≥1°C
transfusion reaction from pretransfusion value), chills/rigors. These may be accompanied by headache and nausea. Symptoms occurring
(FNHTR) during or within four hours following transfusion without any other cause such as hemolytic transfusion reaction, bacterial
contamination or underlying condition. FNHTR could be present in absence of fever (if chills or rigors without fever).
Allergic reaction An allergic reaction may present only with mucocutaneous signs and symptoms: morbilliform rash with pruritus; urticaria
(hives); localized angioedema; edema of lips, tongue, and uvula; periorbital pruritus, erythema and edema; conjunctival
edema. Occurring during or within 4 hours of transfusion. In this form it usually presents no immediate risk to life of patient
and responds quickly to symptomatic treatment like antihistamine or steroid medications. This type of allergic reaction
is called ‘minor allergic reaction’ in many hemovigilance systems. For the purpose of classification this type of allergic
reaction would be graded as 1, i.e. non-severe.
An allergic reaction can also involve respiratory and/or cardiovascular systems and present like an anaphylactic reaction.
There is anaphylaxis when, in addition to mucocutaneous systems there is airway compromise or severe hypotension
requiring vasopressor treatment (or associated symptoms like hypotonia, syncope). The respiratory signs and symptoms
may be laryngeal (tightness in the throat, dysphagia, dysphonia, hoarseness, stridor) or pulmonary (dyspnea, cough,
wheezing/bronchospasm, hypoxemia). Such a reaction usually occurs during or very shortly after transfusion.
For the purpose of classification this type of allergic reaction would be graded as 2 (severe), 3 (life-threatening), or 4
(death) depending on the course and outcome of the reaction.
An allergic reaction classically results from the interaction of an allergen and preformed antibodies. A rise of mast cell
tryptase can support the diagnosis of an allergic reaction. IgA deficiency and/or anti-IgA in the recipient has been
associated with severe allergic reactions but is only one infrequent cause out of many others.
Transfusion associated TA-GVHD is a clinical syndrome characterized by symptoms of fever, rash, liver dysfunction, diarrhea, pancytopenia, and
graft-versus-host disease findings of characteristic histological appearances on biopsy occurring 1–6 weeks following transfusion with no other
(TA-GVHD) apparent cause. The diagnosis of TA-GVHD is further supported by the presence of chimerism.
Post-transfusion purpura PTP is characterized by thrombocytopenia arising 5–12 days following transfusion of cellular blood components with
(PTP) findings of antibodies in the patient directed against the human platelet antigen (HPA) system.
Transfusion-related acute In patients with no evidence of acute lung injury (ALI) prior to transfusion, TRALI is diagnosed if a new ALI is present (all
lung injury (TRALI) five criteria should be met):
1) Acute onset
2) Hypoxemia of PaO2/FiO2 <300 mmHg, or oxygen saturation of <90% on room air, or other clinical evidence
3) Bilateral infiltrates on frontal chest radiograph
4) No evidence of left atrial hypertension (i.e., circulatory overload)
5) No temporal relationship to an alternative risk factor for ALI, during or within 6 hours of completion of transfusion
Alternate risk factors for ALI include: direct lung injury (aspiration, pneumonia, toxic inhalation, lung contusion, near
drowning) or indirect lung injury (severe sepsis, shock, multiple trauma, burn injury, acute pancreatitis, cardiopulmonary
bypass, drug overdose).
Note: It has been suggested by the Toronto TRALI Consensus Panel to add a category of possible TRALI that would have
the same definition as TRALI except for the presence of a temporal relationship to an alternative risk factor for ALI (as
described earlier). In such a circumstance, TRALI should be indicated with a possible imputability to transfusion.
TRALI is therefore a clinical syndrome and neither presence of HLA or HNA antibodies in donor(s) nor confirmation of
cognate antigens in recipient is required for diagnosis.
Transfusion associated TACO is characterized by any 4 of the following occurring within 6 hours of completion of transfusion:
circulatory overload
1) Acute respiratory distress
(TACO)*
2) Tachycardia
3) Increased blood pressure
4) Acute or worsening pulmonary edema on frontal chest radiograph
5) Evidence of positive fluid balance.
An elevated brain natriuretic peptide (BNP) is supportive of TACO.
Transfusion associated TAD is characterized by respiratory distress within 24 hours of transfusion that does not meet the criteria of TRALI,
dyspnea (TAD) TACO, or allergic reaction. Respiratory distress should be the most prominent clinical feature and should not be explained
by the patient’s underlying condition or any other known cause.
Hypotensive transfusion This reaction is characterized by hypotension defined as a drop in systolic blood pressure of ≥ 30 mm Hg occurring
reaction during or within one hour of completing transfusion and a systolic blood pressure ≤ 80 mm Hg.
Haemosiderosis Transfusion-associated haemosiderosis is defined as a blood ferritin level of ≥ 1000 micrograms/L, with or without organ
dysfunction in the setting of repeated RBC transfusions.

Table 2. Non-hemolytic transfusion reaction definitions developed by the Haemovigilance Working Party of the International Society of
Blood Transfusion2 (continued)
Hyperkalemia Any abnormally high potassium level (> 5 mmol/L or ≥1.5 mmol/L net increase) within an hour of transfusion can be
classified as a transfusion- associated hyperkalemia.
Unclassifiable complication Occurrence of an adverse effect or reaction temporally related to transfusion, which cannot be classified according to an
of transfusion (UCT) already defined reaction type and with no risk factor other than transfusion and no other explaining cause.
*The definition of TACO is currently undergoing revision by the working party.
HLA = human leukocyte antigen; HNA = human neutrophil antigen; RBC = red blood cell.
Table 3. Imputability criteria developed by the Haemovigilance Working Party of the International Society of Blood Transfusion2
Imputability category* Definition
Definite (certain) There is conclusive evidence beyond reasonable doubt that the adverse event can be attributed to the transfusion.
Probable (likely) The evidence is clearly in favor of attributing the adverse event to the transfusion.
Possible The evidence is indeterminate for attributing the adverse event to the transfusion or an alternate cause.
Unlikely (doubtful) The evidence is clearly in favor of attributing the adverse event to causes other than the transfusion.
Excluded There is conclusive evidence beyond reasonable doubt that the adverse event can be attributed to causes other than the
transfusion.
*Only definite, probable, and possible cases should be used for international comparisons.
Infection
Harm to a
Immunological complications AHTR
recipient
Malignancy Allergic
Harm to a
Miscellaneous complications DHTR
donor
Adverse
Occurrence Non-infectious DSTR
Type Non-malignent transmissions
Harm to a fetus
Detr. Imm.
or offspring
GvHD
Risk of harm PTP
Rejection
TRALI
Fig. 1 Adverse occurrence type taxonomy (extract). AHTR = acute hemolytic transfusion reaction; DHTR = delayed hemolytic transfusion
reaction; DSTR = delayed serologic transfusion reaction; Detr. Imm. = detrimental immunization; GvHD = graft-versus-host disease; PTP =
post-transfusion purpura; TRALI = transfusion-related acute lung injury.

MPHO Type
MPHO-derived
Organs Tissues Cells Blood Other Reproductive
medicinal products
Heart Adipose tissue Adipocytes Cryoprecipate Fecal microbiota Embryo Cell-derived

medicinal products
Kidney Amniotic membrane Chondrocytes Granulocytes Milk Oocyte
Plasma derivates
Liver Cardiovascular Fibroblasts Plasma Topical products of Ovarian tissue

human origin Tissue-derived
medicinal products
Lung Dura mater Genetically modified Platelets Sperm
cells
Tissue- and cell-derived
Pancreas Larynx Red blood cells Testicular tissue medicinal products
Hematopoietic
progenitor cells
Small bowel Musculoskeletal Whole blood Combined
Hepatocytes
Composite tissue Nerve
grafts
Keratinocytes
Ocular
Combined
Leukocytes
Parathyroid glands
Type not specified
Limbal cells
Placenta
Mesenchymal stem
Skin cells
Trachea
Fig. 2 Medical products of human origin (MPHO) type taxonomy.
groups—adverse occurrence type (harm to a recipient, harm to risk of harm, and the remaining 1 percent are classified as
a donor, harm to a fetus or offspring, risk of harm) and MPHO harm to fetus or offspring. Not surprisingly, among entries
type (organs, blood, cells, tissues, etc.). Figure 1 provides relating to recipient harm, 52 percent are subsequently
an extract of the taxonomy for adverse occurrence type. For categorized as relating to infection and 17 percent as relating
example, immunological complications are further categorized to immunological complications. Of the 331 reports of adverse
into AHTRs, allergic reactions, DHTRs, DSTRs, detrimental occurrences associated with the MPHO of blood, 57 percent
immunization, graft-versus-host disease, post-transfusion are related to transfusion of red blood cells, 21 percent to
purpura, rejection, and transfusion-related acute lung injury platelets, 11 percent to plasma, 7 percent to whole blood, and
(TRALI). Figure 2 provides the taxonomy applied for MPHO 1 percent to granulocytes; the remaining are not specified. Of
type. This predefined classification enables searching by the 89 percent of blood reports of the occurrence type harm to
adverse occurrence type, by MPHO type, or both, as well as by recipient, 49 percent of reports are immunological in nature
using free text or key words. (Fig. 3), including DHTRs, AHTRs, DSTRs, TRALI, and
Of the current searchable entries in the Notify Library, allergic reactions.
68 percent are classified as harm to recipient, 18 percent
are classified as harm to donor, 13 percent are classified as

Other 6.4% Invitation to Join and Contribute

Allergic Reactions 9.7%
The Notify Library and the team of professionals

DHTR 45.33%
TRALI 18.13%
associated with its creation and maintenance are a global
resource in the area of biovigilance that encourages broad
global participation in the project. Access is open to the public.
Individual users are encouraged to search and review the
contents of the Library. Clinical and laboratory practitioners
and students are invited to identify information that might be
still missing, to refer new cases, propose new entries, involve
students and clinicians from domains outside transfusion
and transplantation, recommend references, and even join
an editorial group to review new cases. All organizations,
DSTR 21.15%
authorities, or professional societies that work on the safety
and quality of MPHO are invited to support and contribute to
AHTR 39.28%
this didactic initiative.
Fig. 3 Immunologic complications described in the Notify Library. References

TRALI = transfusion-related acute lung injury; DSTR = delayed
serologic transfusion reaction; AHTR = acute hemolytic transfusion 1. Notify Library. http://www.notifylibrary.org. Accessed 23
reaction; DHTR = delayed hemolytic transfusion reaction. April 2017.
2. De Vries RRP, Faber J-C, Eds. Appendix B: Proposed
standard definitions for surveillance of non infectious adverse
transfusion reactions. In: Hemovigilance: an effective tool for
Current Notify Library Activities improving transfusion safety. Oxford, UK: Wiley-Blackwell,
2012:351–9.
Competent authorities for blood, tissues, and cells in the 3. 2015 Annual SHOT Report. https://www.shotuk.org/wp-
28 countries of the EU have been approached through the content/uploads/SHOT-2015-Annual-Report-Web-Edition-
Final-bookmarked.pdf. Accessed 16 February 2017.
Vigilance and Inspection for the Safety of Transfusion, Assisted
4. FDA. Fatalities reported to the FDA following blood
Reproduction and Transplantation (VISTART) Joint Action collection. 2015. http://www.fda.gov/downloads/Biologics
supported by the European Commission and coordinated by Bl o o d Va c c i n e s/S a f e t yAv a i l a b i l i t y/ R e p o r t a P r o b l e m
TransfusionDonationFatalities/UCM518148.pdf. Accessed 23
CNT (since VISTART includes a specific Work Package on
April 2016.
Vigilance Communication dedicated to the Notify Library). 5. Agence nationale de sécurité du médicament et des produits
Moreover, a directory of vigilance is being created through the de santé. Rapport d’activité hémovigilance 2015. http://ansm.
WHO regional offices to associate all competent authorities for sante.fr/var/ansm_site/storage/original/application/5c7cbc28
90a1d5e436e11bd65e9ec512.pdf Accessed 16 February 2017.
MPHO at a global level to facilitate member states’ access and
6. Fehily D, Strong DM, Minutoli D, et al. Sharing vigilance
contribution to the Notify project, building a global network of experience and knowledge globally: a preliminary overview of
experts who are learning from each other. the Notify Library. Cells Tissues Organs 2013;16:117–25.
Since the beginning of the project, annual consultations
have been organized and, thanks to the collaboration of all Barbee I. Whitaker, PhD, Senior Director, Research (corresponding
editorial group members, the database is regularly updated author), AABB, 8101 Glenbrook Road, Bethesda, MD 2081,
bwhitaker@aabb.org; D. Michael Strong, PhD, Affiliate Professor,
and the Web site continues to improve. Further tools to support
Department of Orthopaedics and Sports Medicine, University of
vigilance have been developed over the years, including a
Washington, School of Medicine, Seattle, WA; Manish J. Gandhi,
comprehensive vigilance document available for download MD, Associate Professor of Laboratory Medicine and Pathology,
through the related section (Notify Booklet, currently in Consultant, Division of Transfusion Medicine, Mayo Clinic,
revision for the inclusion of hemovigilance data) and the ability Rochester, MN; Evangelia Petrisli, MD, Clinical/Scientific Database
to access a panel of experts providing support and advice on Content, NOTIFY operational team, Italian National Transplant
Centre (CNT), Rome, Italy.
request via the Notify Web site.

Original Report
Clinical and laboratory profile of anti-M

D. Basu, S. Basu, M. Reddy, K. Gupta, and M. Chandy
Anti-M is a frequently detected naturally occurring antibody that Materials and Methods
has been reported in various clinical settings and also in voluntary
donors. We describe here the clinical and laboratory findings
of 11 cases with anti-M detected at our center. This report is a
This report is from a tertiary care hemato-oncology
retrospective study in which we reviewed our immunohematology center in eastern India treating patients with hematologic
laboratory records for cases involving anti-M. Both donor malignancies and solid tumors (i.e., gynecologic,
and patient data from a 28-month period (September 2014 to gastrointestinal, head and neck, and soft tissue tumors).
December 2016) were reviewed. During this period, 11 examples
of anti-M were detected (8 patients, 1 voluntary whole blood donor, Pretransfusion antibody detection testing and crossmatch
and 1 hematopoietic stem cell donor. Anti-M was also detected in is performed for all RBC requests. Hematopoietic stem cell
one external quality assessment scheme sample received during transplantations are performed in patients with hematologic
this period. In conclusion, anti-M can be detected in various
as well as nonhematologic disorders. The pretransplant
clinical settings. This antibody can be clinically significant; in
the laboratory, it can present as a serologic problem such as an evaluation includes ABO blood grouping, extended RBC
ABO group discrepancy or an incompatible crossmatch. After phenotyping, antibody detection testing, direct antiglobulin
detection, management and course of action is determined test (DAT), and autocontrol in both donor and recipient. In
by both the antibody characteristics and the clinical setting.
addition, major and minor crossmatches are performed. ABO
grouping is performed by a conventional tube technique (CTT)
Key Words: anti-M, ABO group discrepancy, naturally with anti-A, anti-B, and anti-D reagents (Immucor India,
occurring, clinically significant New Delhi, India), and plasma ABO testing is performed
using in-house reagent RBCs (A, B, and O). A second anti-D
The MNS blood group system was identified by Landsteiner reagent (Tulip Diagnostics, Goa, India) is also tested. Antibody
and Levine in 1927.1 Many antibodies in this system may be detection testing and identification is performed by the
naturally occurring. The most common of these is anti-M, first column agglutination technique (CAT) (Biovue System; Ortho
described by Wolff and Johnson in 1933.2 Clinical Diagnostics, Raritan, NJ) using anti-IgG glass bead
The MNS blood group system consists of 49 antigens, cards and commercially available 3-cell panels (Surgiscreen;
of which two allelic pairs (M/N and S/s) are polymorphic in Ortho Clinical Diagnostics) and 11-cell panels (Resolve Panel
most populations.3 M and N are located on glycophorin A A; Ortho Clinical Diagnostics). Whenever necessary, the same
glycoprotein. Glycophorin A is expressed on the surface of is repeated in neutral or reverse diluent cards at 4°C. DAT,
mature as well as developing erythrocytes.4 Makroo et al.5 autocontrol, and crossmatch are performed by CAT. Extended
reported the following prevalence rates in a northern Indian phenotyping is performed by CTT using commercially available
blood donor population: 34.6 percent M+N–, 54.1 percent monoclonal antisera from Ortho Clinical Diagnostics. Titer is
M+N+, and 11.3 percent M–N+. Because the M antigen is performed by the double dilution method using double-dose
destroyed by routinely available proteolytic enzymes, anti-M M+ (M+N–) group O RBCs. Standard validated methods are
does not react with enzyme-treated red blood cells (RBCs).6 used.8
Anti-M is generally active below 37°C, with optimum activity
at 4°C.6 Hence, these antibodies are generally ignored in Results
transfusion practice, although they can interfere in ABO
plasma grouping and cause an ABO group discrepancy. If Over a period of 28 months (September 2014 to December
the antibody is active at 37°C, then M–, compatible RBCs 2016), 11 cases of anti-M were detected, comprising eight
must be transfused.7 We report here anti-M cases with varied patients, one donor, one hematopoietic stem cell transplant
presentations detected in our laboratory. donor, and one external quality assessment scheme (EQAS)
sample containing anti-M. The patient/donor ages ranged
from 2.5 to 82 years. Patient diagnoses and clinical profiles of
the cases are given in Table 1.

D. Basu et al.
Table 1. Clinical profile of the cases Specificity of the antibody was determined in seven
History of patients by antibody detection testing and identification at
previous 37°C. In one patient, anti-M was detected at 4°C. A dosage
Case Age (years)/ ABO group/ blood
number Donor/patient gender D type Diagnosis transfusion effect of anti-M, showing stronger agglutination strength with
1 Whole blood 24/F O+ NA No double-dose M+ reagent RBCs, was observed in four patients.
donor Antibody detection testing using serum treated with
2 Stem cell 3/F B+ NA No 0.01 M dithiothreitol (DTT) (Himedia Laboratories, Mumbai,
donor
India) was performed on samples from five patients. In all
3 Patient 3/F O+ Pre B ALL Yes
these patients, the agglutination strength decreased but did
4 Patient 2.5/F A+ Pre B ALL No
not disappear, suggesting the presence of both IgG and IgM
5 Patient 5/M O+ T ALL No components in the antibody. In two patients, DTT treatment of
6 Patient 63/M A+ Squamous cell No the serum could not be performed, and the antibody detection
carcinoma of
buccal mucosa testing was not repeated by a prewarmed test. Hence, whether
7 Patient 24/F O+ Carcinoma No this finding was a false reaction at 37°C due to high affinity
breast IgM anti-M or a reaction due to an IgG component of anti-M
8 Patient 66/M O+ Multiple No was not determined.
myeloma
Titration of the anti-M was performed in four cases and
9 Patient 82/F A+ Squamous cell No
carcinoma of was <1 (no reaction in neat plasma) at 37°C in all these cases.
cervix Because the test was performed by CTT, it is possible that
10 Patient 72/M A+ Seminoma No titration by CAT might have shown different results, since
testis
CAT is more sensitive.9 Phenotyping for M was performed on
NA = not applicable; Pre B ALL = Precursor B cell acute lymphoblastic
leukemia; T ALL = T cell acute lymphoblastic leukemia.
samples from all eight patients; seven were found to be M–
and one was M+. Of the seven M– patients, six had no known
history of blood transfusion; it thus appears that, in these
Immunohematologic Features of Anti-M in Patients patients, the anti-M was naturally occurring. In the seventh
An ABO blood group discrepancy was noted in six of the patient (who received RBC transfusion earlier [outside our
eight patients, where anti-M interfered in the reverse grouping hospital]), the anti-M could not be characterized as immune-
as detected by an extra reaction with O reagent RBCs. In five stimulated or naturally occurring because the donor of the
of these patients, this discrepancy was resolved by repeating transfused RBCs was unavailable for M antigen testing. One
the reverse group with a 15-minute incubation at 37°C. In one patient with anti-M (acute lymphoblastic leukemia [ALL] case)
patient, the discrepancy was resolved by using M– reagent was phenotyped as M+ with a positive DAT and autocontrol.
RBCs in reverse grouping. This was thus a case of autoanti-M. The immunohematologic
profile of these patients is summarized in Table 2.
Table 2. Serologic profile of anti-M in patients

Case
number Presentation DAT Class of antibody Thermal amplitude Titer Management
3 Antibody detection testing Positive IgM ± IgG 4°C, RT, and 37°C 4°C: 16 M– RBC transfusion
RT: 4
4 ABO group discrepancy Negative IgM + IgG 4°C, RT, and 37°C NP M– RBC transfusion
5 ABO group discrepancy Positive IgM + IgG 4°C, RT, and 37°C NP M– RBC transfusion
6 ABO group discrepancy Negative IgM + IgG 4°C, RT, and 37°C 4°C: 4 No transfusion
7 ABO group discrepancy Negative IgM ± IgG 4°C, RT, and 37°C NP M– RBC transfusion
8 Antibody detection testing Negative IgM + IgG 4°C, RT, and 37°C 4°C: 8 M– RBC transfusion
9 ABO group discrepancy Negative IgM + IgG 4°C, RT, and 37°C 4°C: 4 No transfusion
10 ABO group discrepancy Negative IgM 4°C, RT NP No transfusion
DAT = direct antiglobulin test; RT = room temperature; RBC = red blood cell; NP = not performed.

Profile of anti-M
Immunohematologic Features of Anti-M in Donors transfusion and responded well, confirmed by an increase in
Anti-M was found in one whole blood donor and one hemoglobin increment from 4.5 to 9.4 g/dL.
hematopoietic stem cell donor. In the whole blood donor, a The anti-M in the hematopoietic stem cell donor was
blood group discrepancy was noted and was resolved when detected during pretransplant evaluation. Incompatibility
the reverse group was repeated with prewarmed serum. The between donor’s plasma and patient’s RBCs was detected, and
anti-M was also detected at 4°C and not at 37°C, suggesting anti-M was then identified. Because the stem cell donor was 3
possibly only an IgM component. The donor’s RBCs tested as years of age, a marrow harvest was undertaken. RBC loss in
M–. the stem cell donor required transfusion of 1 RBC unit during
In the stem cell donor, anti-M was detected during the pre- the harvest. Because the donor had anti-M, group-specific,
transplant evaluation. Although both the donor and recipient M–, crossmatch-compatible RBCs were transfused. To further
were group B, D+, the minor crossmatch was incompatible. complicate the matter, the stem cell recipient was M+. Because
Antibody identification confirmed anti-M reactive at 37°C, the donor anti-M was clinically significant, the marrow
showing a dosage effect. Presence of IgG along with IgM harvest product was plasma-depleted before transfusion to
was confirmed by DTT treatment of the serum. The RBCs reduce chances of a hemolytic transfusion reaction. Hence,
were phenotyped as M–. Neither this donor nor the whole once anti-M is detected, further management of the patient
blood donor had history of blood transfusion, suggesting depends on the specific clinical setting.
that the anti-M in both donors was naturally occurring. The The donor unit, which had anti-M in the plasma, was
immunohematologic profile of these donors is summarized in separated into RBCs, platelets, and plasma components. The
Table 3. plasma was sent for fractionation, platelet concentrate was
transfused to an M– patient, and the RBC unit was transfused
EQAS Sample to an ABO group-specific patient.
During the study period, anti-M was identified in one
EQAS sample. This antibody was detected at 37°C and showed Discussion
dosage. The agglutination strength increased when the testing
was repeated at 4°C. DTT treatment of the plasma or antibody In patients, anti-M may be detected in various medical
titration studies could not be performed nor was a transfusion as well as surgical settings. Das et al.10 reported three cases
history supplied with the sample. of anti-M, all of which were detected during pre-transfusion
testing before surgery. In the present study, anti-M was
Clinical Outcome of Anti-M in Patients and Donors detected in our medical and surgical oncology patients at the
Among the patients with anti-M, five patients required time of blood grouping and antibody detection testing. Our
RBC transfusion. As in most of the cases, the IgG component population consisted of medical and surgical oncology patients.
was present along with IgM; hence, the anti-M was considered Generally, anti-M is IgM in nature, optimally reactive below
to be clinically significant, and these five patients received 22°C, and most often causes a blood grouping discrepancy.
M– RBC units. The patient with ALL who had autoanti-M It may also be detected incidentally during pretransfusion
had clinical as well as laboratory features of hemolysis. The evaluation. If there is no known stimulating event (i.e.,
unconjugated bilirubin was increased at 1.5 mg/dL (normal RBC transfusion, pregnancy, or other exposure to foreign
0–1.1 mg/dL), total bilirubin was 1.8 mg/dL (normal 0.2–1.3 RBCs), the antibodies are classified as naturally occurring.
mg/dL), reticulocyte count was 4% (normal 0.5–2.5%), and Autoanti-M has also been reported in the literature. Anti-M
lactate dehydrogenase was 4039 U/L (normal 313–618 U/L). is more commonly observed in children.6 In the present study,
This patient received injection dexamethasone along with RBC however, there were only four individuals younger than 10
Table 3. Serologic profile of anti-M in donors

Case
number Presentation DAT Class of antibody Thermal amplitude Titer Management
1 ABO group discrepancy NP IgM 4°C and RT NP NA
2 Minor crossmatch incompatible Negative IgM + IgG 4°C, RT, and 37°C NP M– RBC transfusion
DAT = direct antiglobulin test; NP = not performed; RT = room temperature; NA = not applicable; RBC = red blood cell.

D. Basu et al.
years of age (three patients and one stem cell donor). Mathur death20 to cases where the fetus is unaffected.21 Like anti-K,
et al.11 described one case of anti-M in a voluntary blood donor anti-M has been reported to cause suppression of erythroid
where the antibody had a thermal amplitude of 22°C to 37°C precursors, leading to RBC aplasia and prolonged anemia of
but showed no reaction at 4°C. This finding is in contrast to the newborn.22,23 Hence, anti-M in various clinical settings
our finding from the whole blood donor, where the anti-M was presents differently and demands different management.
reactive at room temperature with optimum activity at 4°C. In conclusion, anti-M can have varied clinical presentations
Sacher et al.12 described 17 cases of autoanti-M, of which four and can interfere during blood grouping and crossmatching. To
had symptoms of cold agglutinin disease, such as Raynaud’s determine the clinical significance of anti-M, elaborate methods
phenomenon, livedo reticularis, and acrocyanosis, and two such as antibody titration, the determination of antibody class,
had mild hemolysis; the rest were asymptomatic despite and thermal amplitude testing may be necessary. In the case
having a significant titer of the autoanti-M. of a transfusion requirement for a patient with anti-M, the
Patients with anti-M have been described by Tandon specific clinical setting along with the antibody characteristics
et al.13 and Kaur et al.14 These antibodies were identified as IgM, would determine the course of action to be taken. This report
causing ABO grouping discrepancy, with optimum reactivity also highlights the fact that, in all pretransplant evaluations, a
below 37°C. Anti-M with both IgM and IgG components and minor crossmatch or antibody screening of the donor must be
wide thermal amplitude (4°C–37°C) were also described. performed to ensure that a donor antibody is not missed. Lastly,
Hence, those authors suggest that when evaluating for the IgG in a stem cell donor, the harvest may require modification to
component of anti-M, strict prewarmed conditions must be avoid any adverse transfusion reaction in the recipient.
maintained during antibody identification to avoid interference
by high-affinity, high-titer IgM. References
Anti-M, which is commonly IgM in nature, can cause
1. Landsteiner K, Levine P. On individual differences in human
complement activation. In 1991, Combs et al.15 described blood. J Exp Med 1928;47:757–7.
an autoanti-M causing hemolysis in vitro by activating 2. Wolff E, Johnson B. Studien uber die untergruppen A1 and A2 mit
complement in CTT at low-ionic strength. besonderer berucksichtigung der paternitatsuntersuchungen.
Dtsch Ztschr Gerichtl Med 1933;22:65–85.
Naturally occurring anti-M with only an IgG component
3. Fung KM, Eder AF, Spitalnik S, Westhoff CM, Eds. Technical
has been reported very rarely.6 Anti-M shows a dosage effect, manual. 19th ed. Bethesda, MD: AABB, 2017:323–6.
that is, it shows a stronger reaction with RBCs with double- 4. Southcott MJ, Tanner MJ, Anstee DJ. The expression of
dose expression of M (M+N–) than with RBCs with single- human blood group antigens during erythropoiesis in a cell
culture system. Blood 1999;93:4425–35.
dose expression (M+N+). For this reason, the prevalence of
5. Makroo RN, Bhatia A, Gupta R, et al. Prevalence of Rh, Duffy,
naturally occurring anti-M in blood donors was shown to Kell, Kidd & MNSs blood group antigens in the Indian blood
be 1 in 2500 when testing with M+N– RBCs and 1 in 5000 donor population. Indian J Med Res 2013;137:521–6.
when using M+N+ RBCs.7 In our series, a dosage phenomenon 6. Klein HG, Anstee DJ. Other red cell antigens. In: Klein HG,
was noted in 6 of the 11 cases described. Anti-M has also Anstee DJ, Eds. Mollison’s blood transfusion in clinical
medicine. 11th ed. London: Blackwell Science, 2005;209–52.
demonstrated increased reactivity when tested with serum
7. Daniels G. Human blood groups. 3rd ed. Oxford: Blackwell
that has been adjusted to a pH of 6.5.3 Beattie and Zuelzer16 Scientific Publications, 2002:94–161.
described two anti-M examples that were identified after 8. Fung KM, Eder AF, Spitalnik S, Westhoff CM, Eds. Technical
acidification of the serum. manual. 19th ed. Bethesda, MD: AABB, 2017: Method 3-15.
Delayed hemolytic transfusion reaction is very rare 9. Bhangale A, Pathak A, Pawar S, Jeloka T. Comparison of
antibody titers using conventional tube technique versus column
because of alloanti-M. Sancho et al.17, Alperin et al.18, and agglutination technique in ABO blood group incompatible
Furlong and Monaghan19 reported cases of alloanti-M that renal transplant. Asian J Transfus Sci 2017;11:131–4.
were undetectable during pretransfusion evaluation but were 10. Das R, Dubey A, Agarwal P, Chaudhry RK. Spectrum of
anti-M: a report of three unusual cases. Blood Transfus
subsequently detected 5–15 days posttransfusion during 2014;12:99–102.
investigation of posttransfusion hemolysis. Anti-M has 11. Mathur A, Dontula S, Jagannathan L. An unusual case of
also been implicated in hemolytic disease of the fetus and potentially clinically significant anti-M antibody in a healthy
newborn (HDFN). The clinical effects in HDFN as described male blood donor without any history of blood transfusion.
Blood Transfus 2011;9:339.
in the literature range from severe HDFN with intrauterine

Profile of anti-M
12. Sacher RA, Abbondanzo SL, Miller DK, Womac B. Auto 21. Thompson DJ, Stults DZ, Daniel SJ, et al. Anti-M antibody in
anti-M: clinical and serological findings of seven patients from pregnancy. Obstet Gynecol Surv 1989;44:637–41.
one hospital and review of the literature. Am J Clin Pathol 22. Arora S, Doda V, Maria A, et al. Maternal anti-M induced
1979;32:154–7. hemolytic disease of newborn followed by prolonged anemia in
13. Tandon R, Kataria R, Chaudhry R. Anti-M: report of two cases newborn twins. Asian J Transfus Sci 2015;9:98–101.
and review of literature. Asian J Transfus Sci 2008;2:81–3. 23. Hinchliffe RF, Nolan B, Vora AJ, et al. Neonatal pure red
14. Kaur G, Basu S, Kaur P, et al. Clinically significant anti-M cell aplasia due to anti-M. Arch Dis Child Fetal Neonatal Ed
antibodies: a report of two cases. Transfus Apher Sci 2006;9:F467–8.
2012;47:259–61.
15. Combs MR, O’Rourke MM, Issitt PD, Telen MJ. An auto anti-M Debapriya Basu, MD, Fellow (corresponding author), Transfusion
causing haemolysis in vitro. Transfusion 1991;31:756–8.
Medicine, Tata Medical Center, 14 Middle Arterial Road (EW),
16. Beattie KM, Zuelzer WW. The frequency and properties of pH-
Rajarhat, New Town, Kolkata 700160, India, jhumpa.kumar11@
dependent anti-M. Transfusion 1965;4:322–6.
gmail.com; Sabita Basu, MD, Head of the Department, Transfusion
17. Sancho JM, Pujol M, Fernandez F, et al. Delayed haemolytic
Medicine; Mahua Reddy, PGDMLT, Technologist, Transfusion
transfusion reaction due to anti-M antibody. Br J Haematol
1998;103:268–9. Medicine; Kaushik Gupta, DMLT, Technologist, Transfusion
18. Alperin JB, Riglin H, Branch DR, et al. Anti-M causing delayed Medicine; and Mammen Chandy, MD, Head of the Department,
hemolytic transfusion reaction. Transfusion 1983;23:322–4. Clinical Haematology, Tata Medical Center, Kolkata, India.
19. Furlong MB, Monaghan WP. Delayed hemolytic episodes due
to anti-M. Transfusion 1981;21:45–9.
20. Wikman A, Edner A, Gryfelt G, et al. Fetal hemolytic anemia
and intrauterine death caused by anti-M immunization.
Transfusion 2007;47:911–7.
For information concerning the National Reference

Immunohematology is on the Web!
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Dithiothreitol treatment of red blood cells
C.B. Bub
Dithiothreitol (DTT), a reducing reagent, has multiple

applications in blood bank testing. DTT disrupts the bridging of Treatment to Destroy RBC Antigens
the disulfide bonds between amino acid residues necessary for
Reagents/Supplies
structural conformation of some proteins and the bonds holding
an IgM molecule in the pentameric formation. DTT treatment Reagents Supplies
of red blood cells (RBCs) can denature or modify certain blood
• PBS, pH 7.3 • Test tubes
group antigens—in particular, those in the Kell, Lutheran, YT,
JMH, LW, Cromer, Indian, Dombrock, and Knops systems—and • 0.2 M DTT • Pipettes
prevent recognition by the corresponding antibodies. It also • RBC test cell • Centrifuge
destroys RBC CD38, allowing DTT-treated RBCs to be used to • K+ RBCs (control cell) • pH meter
avoid testing interference by therapeutic anti-CD38 preparations.
• Anti-K (reagent or serum/plasma • Water bath or 37°C
DTT treatment can be used to disperse spontaneous agglutination specimen) incubator
S E R O LO G I C M E T H O D R E V I E W
of RBCs caused by heavy IgM autoantibody coating that

invalidates ABO/Rh cell grouping and direct antiglobulin tests. RBC = red blood cell; PBS = phosphate-buffered saline;
DTT = dithiothreitol.
Procedural Steps
Key Words: dithiothreitol (DTT), indirect antiglobulin test,
Kell system, daratumumab 0.2 M DTT Preparation
• Dissolve 1 g DTT in 32 mL PBS, pH 8.0. Adjust final pH of DTT to
8.0 with 0.1 M HCl/0.1 M NaOH as needed.
Principle • Aliquot and store at –20°C or colder for up to 12 months.
Procedure
Dithiothreitol (DTT) is a reducing agent capable of • Wash 1 volume of the test RBCs and control RBCs with PBS,
pH 7.3. Decant.
irreversibly cleaving accessible disulfide bonds when the • Add 4 volumes of 0.2 M DTT, pH 8.0.
solution pH is >7. DTT treatment of red blood cells (RBCs) • Incubate at 37°C for 30 minutes.
will modify the tertiary structure of protein-based erythrocyte • Wash three to four times with PBS.
• Resuspend the cells to a 2–5 percent suspension in PBS.
membrane antigens if their confirmation depends on disulfide
• Test DTT-treated cells with serum containing the antibody in
bonds.1 Antigens of the following blood group systems are question. Test K+ treated RBCs with anti-K.
destroyed or weakened by 0.2 M DTT treatment: KEL, IN, DTT = dithiothreitol; PBS = phosphate-buffered saline; RBCs = red
JMH, YT, LU, MER2, KN, DO, CROM, and LW.2,3 Antibodies blood cells.
directed at antigens in these systems will not react or will be

significantly weaker with the treated RBCs.
DTT will also cleave the disulfide bonds that connect at a DTT-sensitive antigen, treated RBCs may be used to detect
the monomeric subunits and the J chain of the IgM antibody or exclude underlying antibodies to DTT-resistant antigens.
pentameric form. When heavy coating of RBCs with IgM Treatment of autologous RBCs with 0.01 M DTT can resolve
autoantibody causes spontaneous agglutination, DTT spontaneous agglutination due to potent IgM autoantibodies
treatment will disrupt the IgM structure and disperse the that interfere with ABO/Rh and direct antiglobulin testing.4
agglutination. Monoclonal anti-CD38 (daratumumab), approved by the
U.S. Food and Drug Administration for treatment of multiple
Indications myeloma, targets the CD38 antigen on malignant plasma cells.
CD38 is also weakly expressed on all normal RBCs, including
DTT-treated reagent RBCs can be used in antibody those in RBC reagents used in pretransfusion testing.
identification to suggest the possible blood group specificity This expression complicates the identification of clinically
of an unidentified antibody based on whether reactivity is significant RBC antibodies because the plasma/serum of such
affected by the treatment. When a known antibody is directed patients will react with most or all RBCs in antibody detection

DTT-modified RBCs
suspension if the testing is performed in the tube or to the

Treatment to Disperse Spontaneous concentration required for non-tube test methods. Assess the
Agglutination batch for completeness of treatment using anti-K as described
Reagents/Supplies in the quality control section. If the reactivity of the test serum
Reagents Supplies is eliminated, appropriate RBC samples can be treated and
• PBS, pH 7.3 • Test tubes tested to exclude other clinically significant alloantibodies.
• 0.2 M DTT • Pipettes
• 6% albumin • Centrifuge Procedure to Disperse Spontaneous Agglutination
• Water bath or 37°C
incubator In a properly labeled tube, wash autologous RBCs three
PBS = phosphate-buffered saline; DTT = dithiothreitol. times in saline. Washing with warm saline can also aid in
removing cold-reacting IgM autoantibodies. Dilute washed
Procedural Steps RBCs to a 50 percent suspension in PBS. Add an equal volume
0.01 M DTT Preparation of 0.01 M DTT. Mix and incubate at 37°C for 15 minutes.
• Dilute 1 volume 0.2 M DTT with 19 volumes PBS, pH 7.3. Wash treated RBCs at least three times in PBS. Resuspend
Procedure an aliquot of the RBCs to a 2–5 percent suspension or to the
• Wash autologous RBCs at least three times with PBS and dilute to
a 50% concentration in PBS. concentration required by the test method being performed.
• Add an equal volume of 0.01 M DTT to the RBC suspension. Test the RBCs for removal of the spontaneous agglutination as
• Incubate at 37°C for 15 minutes. described in the quality control section.
• Wash RBCs three to four times in PBS.
• Resuspend the RBCs to a 2–5 percent suspension.
• Test the treated RBCs with 6% albumin (immediate-spin test) to Limitations
assess for dispersal of spontaneous agglutination.
DTT = dithiothreitol; PBS = phosphate-buffered saline; RBCs = red As in all serologic procedures, factors such as contaminated
blood cells.
materials or inadequate incubation time, temperature, or
centrifugation may produce false results. Preparation of the
0.2 M DTT at pH 8.0 is required for irreversible denaturation
and identification tests performed by the indirect antiglobulin of antigens.2 Reagent preparations at a lower pH may cause
test.5 Because CD38 configuration depends on disulfide bonds, inadequate and reversible reduction of disulfide bonds. Treated
DTT treatment of reagent RBCs is used to denature the red RBCs cannot be typed for any antigens destroyed by DTT.
cell CD38 and avoid this interference.6 A large volume of RBCs Antibodies to antigens destroyed or weakened by DTT
can be treated with DTT and stored in Alsever’s solution, since cannot be excluded on nonreactive treated RBCs. Because
the stability of common RBC antigens has been demonstrated anti-K is the most frequently encountered clinically important
for up to 14 days.7 Serologic investigations within this time antibody in this group, K– RBCs should be selected for
period are more efficiently performed when pretreated cells transfusion if the patient’s RBCs are K– or the K antigen status
are available. is unknown, unless anti-K has been excluded in other tests.
Procedure to Destroy RBC Antigens Quality Control
In a properly labeled tube, place 1 volume of the patient's When performing treatment to destroy RBC antigens,
packed RBCs (approximately 50 μL). Wash the RBCs three K+ RBCs should be DTT-treated with each test batch. The
times with phosphate-buffered saline (PBS), pH 7.3. Decant treated and untreated K+ RBCs are then tested with anti-K.
the supernatant completely after the last wash. Add 4 volumes The treated RBCs should be nonreactive; otherwise, the DTT
of 0.2 M DTT (approximately 200 μL) to 1 volume of RBCs treatment was not adequate. Other antigens of the Kell system
to be treated. Mix well, and incubate at 37°C for 30 minutes. can also serve as controls.1
Wash the RBCs four times with PBS. If marked hemolysis If dispersal of spontaneous agglutination is being
occurs, repeat the procedure with fresh RBCs and a smaller performed, test the treated RBCs using 6 percent albumin. No
volume of DTT. Resuspend the RBCs to a 2–5 percent agglutination should be present if the treatment was successful.

C.B. Bub
Summary 3. Reid ME, Lomas-Francis C, Olsson M. The blood group antigen

factsbook. 3rd ed. London:Elsevier Academic Press, 2012.
4. Fung MK, Grossman BJ, Hillyer CD, Westhoff CM. Eds.
DTT treatment of RBCs is an informative method to aid
Technical manual. 19th ed. Bethesda, MD: American
in antibody identification and to determine whether a serum Association of Blood Banks, 2017: Method 2–18.
contains additional alloantibodies when an antibody to a 5. Murphy MF, Dumont LJ, Greinacher A. Interference of new
DTT-sensitive antigen is present. It also destroys CD38 on the drugs with compatibility testing for blood transfusion. N Engl
J Med 2016;375:295–6.
RBC surface, thereby avoiding interference from anti-CD38
6. Chapuy CI, Aguad MD, Nicholson RT, Aubuchon JP, Cohn CS,
in pretransfusion testing. Donor units typed as K– must be Delaney M, et al. International validation of a dithiothreitol
transfused when antibody detection/identification has been (DTT)-based method to resolve the daratumumab
performed using DTT-treated RBCs, unless anti-K is excluded interference with blood compatibility testing. Transfusion
2016;56:2964–72.
by another method.
7. Disbro WL. Stability guidelines for dithiothreitol-treated red
Spontaneous agglutination of autologous RBCs causing blood cell reagents used for antibody detection methods in
false-positive results in ABO/Rh and direct antiglobulin patients treated with daratumumab. Immunohematology
2017;33:105–9.
testing can be resolved by using DTT to degrade the IgM
antibody coating the RBC.
Carolina Bonet Bub, MD, PhD, Hematology and Hemotherapy
Physician, Hematology and Cell Therapy Department, Hospital
References Israelita Albert Einstein, 627 – 3º andar, Bloco E, CEP: 05651-901
1. Fung MK, Grossman BJ, Hillyer CD, Westhoff CM. Technical Sao Paulo, SP, Brazil.
manual. 19th ed. Bethesda, MD: American Association of
Blood Banks, 2017: Method 3–18.
2. Branch DR, Muensch HA, Sy Siok Hian S, Petz LD. Disulfide
bonds are a requirement for Kell and Cartwright (Yta) blood
group antigen integrity. Br J Haematol 1983;54:573–8.
For information concerning Immunohematology or the

Notice to Readers
Immunohematology Methods and Procedures manual,
Immunohematology is printed on acid-free paper. contact us by e-mail at immuno@redcross.org.
Manuscripts
The editorial staff of Immunohematology welcomes manuscripts submitting scientific articles, case reports, and review articles,
pertaining to blood group serology and molecular genetics for see Instructions for Authors in every issue of Immunohematology
consideration for publication. We are especially interested in or e-mail a request to immuno@redcross.org. Include fax and
review articles, case reports, papers on platelet and white cell phone numbers and e-mail address with all manuscripts
serology, scientific articles covering original investigations or new and correspondence. E-mail all manuscripts to immuno@
blood group alleles, papers on molecular testing, and papers on redcross.org.
new methods for use in the blood bank. To obtain instructions for

C o m m u n i c at i o n s
To Contributors to the 2017 Issues
Immunohematology depends on many contributors to make each issue a success: authors who submit reviews, scientific
reports, case reports, blood group allele information, book reviews, and letters to the editors; peer reviewers who review each
submission for accuracy and completeness of content; medical editors who oversee each submission for medical content and
appropriateness for publication; technical editors who ensure that the technical aspects of each submission are correct; and the
copy editor, proof reader, and graphic designer who format each issue to ensure that it is aesthetically pleasing to the eye of
the reader. Each submission is reviewed and managed by an editorial staff who move the manuscripts through the review/
editing process and who determine the acceptability of each for publication, namely, the editor-in-chief, the managing editor,
and the medical and molecular editors. Behind the scenes are our administrative staff who process and manage subscriptions,
reply to emails in our mailbox, and various other duties that keep the journal on track. In addition, our prestigious editorial
board members submit manuscripts and provide peer reviews, guidance in policy, ideas for focused issues, and suggestions for
improvement of the journal. Without all of these contributors, Immunohematology would not exist. We appreciate the time and
effort each contributes to the journal.
The authors are showcased in their publications. The editorial staff, administrative staff, medical and technical editors, and
current members of the editorial board are listed in the front of each issue of the journal along with the copy editor, proof reader
and graphic designer: our sincerest appreciation to each.
Our peer reviewers did an excellent job this year. We take this opportunity to list each by name below with heartfelt gratitude
to all.
Roy A. Alther Jr., MT(ASCP)SBB Hein Hustinx

Patricia Arndt, MT(ASCP)SBB Catherine Hyland, PhD, MSc
Debra Bailey, MT(ASCP)SBB Susan T. Johnson, MSTM, MT(ASCP)SBB
Christina Barron, MT(ASCP)SBB Richard Kaufman, MD
Nancy Benitez, MHS(ASCP)SBB Jessica Keller, MS
Joan S. Boyd, MT(ASCP)SBB, CQA(ASQ) Paul M. Mansfield, MT(ASCP)SBB
Barbara J. Bryant, MD Jose Lima, MD
Lilian M. Castilho, PhD Ankit Mathur, MD
Laura Cooling, MD, MS Geralyn M. Meny, MD, MS
William S. Crews Jr., MD Jessica Poisson, MD
Geoffrey Daniels, PhD Glenn Ramsey, MD
Robertson Davenport, MD S. Gerald Sandler, MD
Meghan Delaney, DO, MPH Chelsea Sheppard, MD
Emmanuel A. Fadeyi, MD, FCAP Ira A. Shulman, MD
Dolores Figueroa, MT(ASCP)SBB Marilyn J. Telen, MD
Melissa R. George, DO, FCAP, FASCP Jeff Trimble, B.Sc., ART(CSMLS)
Gregory R. Halverson, MT(ASCP)SBB Carlos H. Villa, MD, PhD
Trina Horn, MS, MLT(ASCP)SBBCM F. Bernadette West, MD
Finally, we thank our readers, whose enthusiasm and interest in the journal make it all worthwhile.
Sandra Nance, MS, MT(ASCP)SBB

Editor-in-Chief
Cynthia Flickinger, MT(ASCP)SBB

Managing Editor

E r r at u m
Daniels G. The Augustine blood group system, 48 years in the making.

An error was detected in the review of the Augustine blood group system: Ata was reported as AUG1, when it should have
been AUG2; the antigen defined by the antibody produced by the woman with the null phenotype was reported as AUG 2, when
it should have been AUG1. Below is the corrected Table 1.
Table 1. Antigens of the Augustine system and their molecular backgrounds

Antigens Molecular basis (SLC29A1)*
Name ISBT number Nucleotides Exon/intron Amino acids
— AUG1 036001 c.589+1G (C) Intron 6 Splice site mutation
Ata AUG2 036002 c.1171G (A) Exon 12 Glu391 (Lys)
*Molecular basis of antigen-negative phenotype provided in parentheses.

Announcements

Announcements, cont.




Masters of Science (MSc) in Transfusion and Transplantation Sciences

at the University of Bristol, England
Applications are invited from medical or science graduates for the Master of Science (MSc) degree in
Transfusion and Transplantation Sciences at the University of Bristol. The course starts in October
2018 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
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Pathology and Microbiology. It includes:
• Scientific principles of transfusion and transplantation
• Clinical applications of these principles
• Practical techniques in transfusion and transplantation
• Principles of study design and biostatistics
• An original research project
Application can also be made for a Diploma in Transfusion and Transplantation Sciences or a Certificate
in Transfusion and Transplantation Sciences.
The course is accredited by the Institute of Biomedical Sciences.
Further information can be obtained from the Web site:

http://ibgrl.blood.co.uk/MSc/MscHome.htm
For further details and application forms, please contact:
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

The Johns Hopkins Hospital Specialist in Blood Bank Technology Program
The Johns Hopkins Hospital was founded in 1889. It is located in Baltimore, MD, on the original founding site, just 45
minutes from Washington, DC. There are approximately 1,000 inpatient beds and another 1,200 outpatient visits daily;
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The Johns Hopkins Hospital Transfusion Medicine Division is one of the busiest in the country and can provide
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The Johns Hopkins Hospital Specialist in Blood Bank Technology Program is an onsite work-study, graduate-level
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The variety of patients, the size, and the general intellectual environment of the hospital provide excellent opportunities
for training in Blood Banking. It is a challenging program that will prepare competent and knowledgeable graduates who
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transfusion/sbb.cfm for additional information.
Contact: Lorraine N. Blagg, MA, MLS(ASCP)CMSBB

Program Director
E-mail: lblagg1@jhmi.edu
Phone: (410) 502-9584
The Johns Hopkins Hospital

Department of Pathology
Division of Transfusion Medicine
Sheikh Zayed Tower, Room 3100
1800 Orleans Street
Baltimore, MD 21287
Phone (410) 955-6580

Fax (410) 955-0618
Web site: http://pathology.jhu.edu/department/divisions/transfusion/index.cfm

Online Specialist in Blood Bank (SBB)

Certificate and Masters in Clinical Laboratory
Management Program
Rush University
College of Health Sciences
Continue to work and earn graduate credit while the Rush University SBB/MS program prepares
you for the SBB exam and the Diplomat in Laboratory Management (DLM) exam given by ASCP
Board of Certification! (Please note acceptable clinical experience is required for these exams.)
Rush University offers online graduate level courses to help you achieve your career goals.
Several curricular options are available. The SBB/MS program at Rush University is currently accepting
applications for Fall 2018. For additional information and requirements, please visit our Web site
at: www.rushu.rush.edu/cls/
Rush University is fully accredited by the Higher Learning Commission (HLC) of the North Central
Association of Colleges and Schools, and the SBB Certificate Program is accredited by the
Commission on Accreditation of Allied Health Education Programs (CAAHEP).
Applications for the SBB/MS Program can be submitted online at the following Web site:
http://www.rushu.rush.edu/admiss/hlthadm.html
Contact: Yolanda Sanchez, MS, MLS(ASCP)CMSBB, Director, by e-mail at Yolanda_Sanchez@rush.edu

or by phone at 312-942-2402 or Denise Harmening, PhD, MT(ASCP), Director of Curriculum,
by e-mail at Denise_Harmening@rush.edu

A dv e rtise m e nts

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Becoming a Specialist in Blood Banking (SBB)

What is a certified Specialist in Blood Banking (SBB)?
• Someone with educational and work experience qualifications who successfully passes the American Society for Clinical Pathology
(ASCP) board of registry (BOR) examination for the Specialist in Blood Banking.
• This person will have advanced knowledge, skills, and abilities in the field of transfusion medicine and blood banking.
Individuals who have an SBB certification serve in many areas of transfusion medicine:
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Florida Academic Center at OneBlood St. Petersburg, FL
Illinois Rush University Chicago, IL
Indiana Indiana Blood Center Indianapolis, IN
Louisiana LifeShare Blood Center Shreveport, LA
University Medical Center New Orleans New Orleans, LA
Maryland National Institutes of Health Clinical Center Bethesda, MD
The Johns Hopkins Hospital Baltimore, MD
Walter Reed National Military Medical Center Bethesda, MD
Texas University Health System and Affiliates School of Blood Bank Technology San Antonio, TX
University of Texas Medical Branch Galveston, TX
Wisconsin BloodCenter of Wisconsin Milwaukee, WI
Revised October 2017

Reference and Consultation Services IgA Testing
Antibody identification and problem resolution IgA testing is available to do the following:
HLA-A, B, C, and DR typing • Identify IgA-deficient patients
HLA-disease association typing • Investigate anaphylactic reactions
Paternity testing/DNA • Confirm IgA-deficient donors

Our ELISA for IgA detects protein to 0.05 mg/dL.
F or infor mation , c ontac t :
F or additional infor mation c ontac t :
Mehdizadeh Kashi
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at (503) 280-0210
or e-mail:
or write to: Sandra.Nance@redcross.org
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ATTN: Sandra Nance
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National Reference Laboratory Donor IgA Screening

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Immunohematology Reference Laboratory
• Gel diffusion test that has a 15-year proven track record:
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24-hour phone number:
IgA deficient by this method are confirmed by the more
(215) 451-4901
Fax: (215) 451-2538 sensitive testing methods
American Rare Donor Program F or additional infor mation :

24-hour phone number:
(215) 451-4900 Kathy Kaherl
Fax: (215) 451-2538 at (860) 678-2764
ardp@redcross.org e-mail:
Immunohematology Katherine.Kaherl@redcross.org
Phone, business hours:
(215) 451-4902 or write to:
Fax: (215) 451-2538
Reference Laboratory
immuno@redcross.org
Quality Control of Cryoprecipitated–AHF Connecticut Region
Phone, business hours: 209 Farmington Avenue
(215) 451-4903 Farmington, CT 06032
Fax: (215) 451-2538

National Reference Laboratory National Neutrophil Serology Reference Laboratory

for Specialized Testing
Our laboratory specializes in granulocyte antibody detection
Diagnostic testing for: and granulocyte antigen typing.
• Neonatal alloimmune thrombocytopenia (NAIT) Indications for granulocyte serology testing
• Post-transfusion purpura (PTP)
include:
• Refractoriness to platelet transfusion
• Alloimmune neonatal neutropenia (ANN)
• Heparin-induced thrombocytopenia (HIT)
• 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:
• Molecular analysis for HPA-1a/1b • HLA (PRA) Class I and Class II antibody detection
For further information, contact: For further information, contact:
Platelet Serology Laboratory (215) 451-4205 Neutrophil Serology Laboratory (651) 291-6797
Sandra Nance (215) 451-4362 Randy Schuller (651) 291-6758
Sandra.Nance@redcross.org Randy.Schuller@redcross.org
American Red Cross Biomedical Services American Red Cross Biomedical Services
Musser Blood Center Neutrophil Serology Laboratory
700 Spring Garden Street 100 South Robert Street
Philadelphia, PA 19123-3594 CLIA licensed St. Paul, MN 55107 CLIA licensed

Immunohematology
Instructions for Authors
I. GENERAL INSTRUCTIONS b. Use short headings for each column needed and capitalize first letter of first
Before submitting a manuscript, consult current issues of Immunohematology for style. word. Omit vertical lines.
Number the pages consecutively, beginning with the title page. c. Place explanation in footnotes (sequence: *, †, ‡, §, ¶, **, ††).
8. Figures
II. SCIENTIFIC ARTICLE, REVIEW, OR CASE REPORT WITH
a. Figures can be submitted either by e-mail or as photographs (5 ×7″ 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 following ending with a period. If figure is submitted as a glossy, place first author’s
order: 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 possible:
2. Abstract l l s s n n.
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 code) for all
6. Author information authors. List country when applicable. Provide e-mail addresses of all authors.
7. Tables
8. Figures III. EDUCATIONAL FORUM
B. Preparation of manuscript A. All submitted manuscripts should be approximately 2000 to 2500 words with
1. Title page pertinent references. Submissions may include:
a. Full title of manuscript with only first letter of first word capitalized (bold 1. An immunohematologic case that illustrates a sound investigative approach with
title) clinical correlation, reflecting appropriate collaboration to sharpen problem-solving
b. Initials and last name of each author (no degrees; ALL CAPS), e.g., M.T. skills
JONES, J.H. BROWN, AND S.R. SMITH 2. Annotated conference proceedings
c. Running title of ≤40 characters, including spaces B. Preparation of manuscript
d. Three to ten key words 1. Title page
2. Abstract a. Capitalize first word of title.
a. One paragraph, no longer than 300 words b. Initials and last name of each author (no degrees; ALL CAPs)
b. Purpose, methods, findings, and conclusion of study 2. Text
3. Key words a. Case should be written as progressive disclosure and may include the
a. List under abstract following headings, as appropriate:
4. Text (serial pages): Most manuscripts can usually, but not necessarily, be divided i. Clinical Case Presentation: Clinical information and differential diagnosis
into sections (as described below). Survey results and review papers may need ii. Immunohematologic Evaluation and Results: Serology and molecular
individualized sections testing
a. Introduction — Purpose and rationale for study, including pertinent iii. Interpretation: Include interpretation of laboratory results, correlating
background references with clinical findings
b. Case Report (if indicated by study) — Clinical and/or hematologic data and iv. Recommended Therapy: Include both transfusion and nontransfusion-
background serology/molecular based therapies
c. Materials and Methods — Selection and number of subjects, samples, items, v. Discussion: Brief review of literature with unique features of this case
etc., studied and description of appropriate controls, procedures, methods, vi. Reference: Limited to those directly pertinent
equipment, reagents, etc. Equipment and reagents should be identified in vii. Author information (see II.B.9.)
parentheses by model or lot and manufacturer’s name, city, and state. Do not viii. Tables (see II.B.7.)
use patients’ names or hospital numbers.
d. Results — Presentation of concise and sequential results, referring to IV. LETTER TO THE EDITOR
pertinent tables and/or figures, if applicable A. Preparation
e. Discussion — 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 introduction 2. Title (first word capitalized)
5. Acknowledgments: Acknowledge those who have made substantial contributions 3. Text (written in letter [paragraph] format)
to the study, including secretarial assistance; list any grants. 4. Author(s) (type flush right; for first author: name, degree, institution, address
6. References [including city, state, ZIP code, and country]; for other authors: name, degree,
a. In text, use superscript, Arabic numbers. 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…) and use no punctuation at the end of the title. Send all manuscripts by e-mail to immuno@redcross.org

Immunohematology
Instructions for Authors | New Blood Group Allele Reports
A. For describing an allele that has not been described in a peer-reviewed publication and for which an allele name or provisional allele name has been assigned by the
ISBT Working Party on Blood Group Allele Terminology (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/blood-group-
terminology/blood-group-allele-terminology/)
B. Preparation
1. Title: Allele Name (Allele Detail)
ex. RHCE*01.01 (RHCE*ce48C)
2. Author Names (initials and last name of each [no degrees, ALL CAPS])
C. Text
1. Case Report
i. Clinical and immunohematologic data
ii. Race/ethnicity and country of origin of proband, if known
2. Materials and Methods
Description of appropriate controls, procedures, methods, equipment, reagents, etc. Equipment and reagents should be identified in parentheses by model or lot and
manufacturer’s name, city, and state. Do not use patient names or hospital numbers.
3. Results
Complete the Table Below:
Phenotype Allele Name Nucleotide(s) Exon(s) Amino Acid(s) Allele Detail References
e weak RHCE*01.01 48G>C 1 Trp16Cys RHCE*ce48C 1
Column 1: Describe the immunohematologic phenotype (ex. weak or negative for an antigen).
Column 2: List the allele name or provisional allele name.
Column 3: List the nucleotide number and the change, using the reference sequence (see ISBT Blood Group Allele Terminology Pages for reference sequence ID).
Column 4: List the exons where changes in nucleotide sequence were detected.
Column 5: List the amino acids that are predicted to be changed, using the three-letter amino acid code.
Column 6: List the non-consensus nucleotides after the gene name and asterisk.
Column 7: If this allele was described in a meeting abstract, please assign a reference number and list in the References section.
4. Additional Information
i. Indicate whether the variant is listed in the dbSNP database (http://www.ncbi.nlm.nih.gov/snp/); if so, provide rs number and any population frequency information, if available.
ii. Indicate whether the authors performed any population screening and, if so, what the allele and genotype frequencies were.
iii. Indicate whether the authors developed a genotyping assay to screen for this variant and, if so, describe in detail here.
iv. Indicate whether this variant was found associated with other variants already reported (ex. RHCE*ce48C,1025T is often linked to RHD*DIVa-2).
D. Acknowledgments
E. References
F. Author Information
List first name, middle initial, last name, highest degree, position held, institution and department, and complete address (including ZIP code) for all authors. List country when
applicable.

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*Make check payable in U.S. dollars to THE AMERICAN RED CROSS. Mail this form and check
in an envelope addressed to: Immunohematology, P.O. Box 40325, Philadelphia, PA 19106
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Card Number: Security Code: Exp. Date: /
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or given via phone to Marge Manigly at 215-451-4902
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Journal of Blood Group Serology and Molecular Genetics

Grifols Diagnostics Solutions, Inc.

Dedicated to advancement and education in

147 Original Report

152 Case Report

159 Original Report

165 Original Report

174 175 183 187 191

E d i t o r i a l A s s i s ta n t Immunohematology is indexed and included in Index Medicus and MEDLINE on the

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Assessment of common red blood cell

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DO Doa, Dob, Hy, Joa Results

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individual patient samples.

specificity, especially in patients with variant antigens when

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Notice to Readers Attention:

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Anti-Vel alloimmunization and severe

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24w 1d 27w 2d 32w 2d

11w 24w 25w 30w 2d 35w 2d TOP-UP

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Acknowledgments 9. Toivanen P, Hirvonen T. Fetal development of red cell antigens

Attention: SBB and BB Students

You are eligible for a free 1-year subscription to

Ask your education supervisor to submit the name and

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Antibody detection and identification are processes that are Reagents/Supplies

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Hemovigilance and the Notify Library

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Risk of harm PTP

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Heart Adipose tissue Adipocytes Cryoprecipate Fecal microbiota Embryo Cell-derived

Liver Cardiovascular Fibroblasts Plasma Topical products of Ovarian tissue

Fig. 2 Medical products of human origin (MPHO) type taxonomy.

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Other 6.4% Invitation to Join and Contribute

The Notify Library and the team of professionals

Fig. 3 Immunologic complications described in the Notify Library. References

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Clinical and laboratory profile of anti-M

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Table 2. Serologic profile of anti-M in patients

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Table 3. Serologic profile of anti-M in donors

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For information concerning the National Reference

Free Classified Ads and Announcements

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Contact: Lorraine N. Blagg, MA, MLS(ASCP)CMSBB

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Check if home address used Check enclosed*

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