Exeley Inc.

MACIOSZEK, Elżbieta

KUREK, Agata

ROAD TRAFFIC DISTRIBUTION ON PUBLIC HOLIDAYS AND WORKDAYS ON SELECTED ROAD TRANSPORT NETWORK ELEMENTS

MYTROFANOV, Oleksandr

PROSKURIN, Arkadii

POZNANSKYI, Andrii

RESEARCH OF ROTARY PISTON ENGINE USE IN TRANSPORT POWER PLANTS

Covin, Randal B.

Evans, Karen S.

Olshock, Richard

Thompson, Hannis W.

I M M U N O H E M A T O L O G Y ,   V O L U M E  1 7,  N U M B E R  2,  2 0 0 1 45
Acute hemolytic transfusion
reaction caused by anti-Coa
R.B. COVIN, K.S. EVANS, R. OLSHOCK, AND H.W. THOMPSON
Coa is a high-frequency blood group antigen in the Colton blood
group system expressed on red blood cells (RBCs) of
approximately 99.8 percent of random persons. Anti-Coa has been
reported to cause delayed hemolytic transfusion reactions,
hemolytic disease of the newborn, and accelerated clearance of
RBCs in vivo. Acute hemolytic transfusion reactions (AHTRs) have
not previously been reported. A 58-year-old man was hospitalized
for vascular surgery. Initial blood bank evaluation revealed anti-Fya.
The patient received six units of RBCs during his initial
hospitalization and developed anti-E. A subsequent sample was sent
to the reference laboratory when all units of RBCs appeared
incompatible. Additional studies, including alloadsorptions, revealed
the presence of anti-E, anti-Fya, and an apparent warm autoantibody.
One unit of least-incompatible RBCs was transfused during surgery.
The patient had an increase in temperature. Hemoglobinuria and a
decrease in hematocrit were also noted. Due to the clinical
impression of an AHTR, the pre- and postreaction samples were re-
evaluated in the reference laboratory and demonstrated the
presence of anti-Coa in both. Based on clinical and laboratory
evaluation this patient appears to have had an AHTR due to anti-Coa.
This is the first known reported case of an AHTR caused by anti-Coa.
Immunohematology 2001;17:45–49.
Key Words: blood groups, transfusion reaction,
hemolysis, anti-Coa, Colton A (Coa) antigen
Coa is a high-frequency blood group antigen
expressed on red blood cells (RBCs) of 99.8 percent of
Caucasians and may be more frequent in other ethnic-
racial groups.1 The Colton protein has been identified
as aquaporin 1, a red cell water-selective transport
protein that regulates water homeostasis.2 The first
examples of anti-Coa were described by Heisto et al. in
1967.3 Anti-Coa has been reported to cause delayed
hemolytic transfusion reactions,4 hemolytic disease of
the newborn,5 and accelerated clearance of RBCs in an
in vivo survival study.6 Acute hemolytic transfusion
reactions (AHTRs) due to anti-Coa have not previously
been reported. The case presented here concerns a
patient who developed an apparent AHTR due to anti-
Coa. We believe that this case is the first reported case
of an AHTR caused by anti-Coa.
Case Report
A 58-year-old group A, D+ man with longstanding
venous insufficiency was admitted to the hospital on
February 18, 2000 for left leg valve transplantation. His
medical history was significant for a 38-year history of
pain, edema, and discomfort in his left lower extremity
due to venous insufficiency, ulcers, and thromboses.
He had received multiple red cell transfusions during
surgery for ulcer excisions followed by skin grafts,
multiple nerve blocks, and a sympathectomy. He had
a history of moderate-to-severe aortic stenosis and of
gastrointestinal bleeding in 1993. At that time he was
also noted to have anti-Fya. Medications on admission
included oxycodone, amitriptyline, zolpidem, and
warfarin. Admission hemoglobin was 11.9 g/dL.
Initial blood bank evaluation confirmed anti-Fya. The
patient’s RBC phenotype was reported as Fy (a–), Jk (a),
and S–. The patient underwent a left lower extremity
venous valve transplant on February 18, 2000. A
left deep venous thrombosis necessitating heparin
therapy complicated the patient’s postoperative course.
Subsequently, the patient developed left thigh
and popliteal hematomas that were evacuated on
February 28, 2000. During this phase of the patient’s
hospitalization, and prior to this second operation,
the patient received 6 units of RBCs. An anti-E was
detected for the first time. A type and crossmatch just
prior to the patient’s second surgery revealed no
compatible units and the specimen was sent to the
reference laboratory. Additional studies including
alloadsorptions revealed the presence of anti-E, -Fya, a
cold autoantibody, and an apparent warm autoantibody.
Further phenotyping revealed that the patients RBCs
also were E– and K–. Eight units of least-incompatible
RBCs were sent to the patient’s hospital. During the
second surgery, the patient was transfused with one of
the units and the other seven were returned. The
transfused unit was E–, Fy (a–), K–, and Jk (a–). In
the postanesthesia recovery unit, within 60 minutes
from the start of the transfusion, the patient was noted
to develop rigors, increased temperature, and
hemoglobinuria. The hematocrit was also noted to have
decreased. There was no clinical evidence of bleeding
46 I M M U N O H E M A T O L O G Y ,   V O L U M E  1 7,  N U M B E R  2,  2 0 0 1
R.B. COVIN ET AL.
to account for the decline in hematocrit. Due to the
clinical impression of an AHTR, the pre- and immediate
postreaction samples were re-evaluated in the reference
laboratory. A panel of RBCs to test for the presence
of antibodies to high-frequency antigens demonstrated
anti-Coa. A posttransfusion sample revealed a new
anti-Jka. The patient’s RBCs were Co(a–) and Jk(a–).
Materials and Methods
Polyspecific anti-IgG, -C3d, murine monoclonal anti-
IgG (Gamma Biologicals, Houston, TX) and murine
monoclonal anti-C3b, C3d (Ortho-Clinical Diagnostics,
Raritan, NJ) were used for the direct antiglobulin
test (DAT). Testing was performed at immediate spin
and after a 10-minute incubation.
The following monoclonal reagents were used for
the initial reference laboratory RBC phenotyping:
anti-A, -B, -D, -C, -c, -E, and -e (Ortho); the following
indirect antiglobulin reagents were used: anti-K, -Fya,
-S (Ortho) and -Jka (Gamma). The patient’s ABO serum
grouping was performed using A
1
 and B cells (Ortho).
Initial panels used in the reference laboratory
investigation included select cells from Panocell 16
(Lot # 09280, Immucor, Norcross, Georgia) and Panel
One (Lot numbers 0222 and 0118, Gamma). Panels
were read at immediate spin, followed by the addition
of two drops of N-Hance (Gamma). Each tube was
incubated at 37°C for 10 minutes, followed by
centrifugation and reading. Each tube was washed x 4
with saline, two drops of anti-IgG (Gamma) were added,
followed by centrifugation and reading. Negative
antihuman globulin reactions were checked using
Coombs Control Cells (Ortho).
A cold autoantibody, one of the suspected antibodies,
interfered with the patient’s ABO serum grouping. The
test was repeated and resolved using two drops of the
patient’s serum plus A
1
 and B cells, and O cord cells. A
prewarmed panel was performed, using the same cells
as the original select cell panel. The following were
warmed separately at 37°C for 5 to 10 minutes: one
drop of each panel cell to be tested, enough N-Hance to
add two drops to each tube, and the patient’s serum.
Two drops of prewarmed serum and N-Hance were
added to each panel cell tube. All tubes were incu-
bated at 37°C for 10 minutes. All tubes were washed
x 4 with warm saline, followed by the addition of two
drops of anti-IgG (Gamma) to each tube.
An acid eluate was performed using Elu-Kit II
(Gamma). As a control, the last wash was tested against
two selectogen cells (Ortho). Polyethylene glycol (PEG)
adsorptions were performed to remove the apparent
warm autoantibody from the serum. Raw PeG (Sigma
Chemicals, St. Louis, MO) was obtained and prepared as
a reagent for use in our laboratory using the following
method: 20 grams of PEG dissolved into 100 mL of
phosphate-buffered saline. Three different donor’s cells
(Bonfils Blood Center, Denver, CO) lacking the following
antigens were used to perform the adsorptions:
• Donor 1 (O, D+) lacking E, c, Fya, Jkb, and K antigens
• Donor 2 (O, D+) lacking C, E, Fya, Fyb, Jkb, K, and
S antigens
• Donor 3 (O, D–) lacking E, Fya, Jka, K, and s antigens
The cells from the three donors were tightly packed,
with as much donor plasma as possible removed.
Equal volumes of packed adsorbing RBCs, PEG, and
patient’s serum were incubated at 37°C for 15 minutes.
Following centrifugation, the adsorbed serum was
harvested and tested against a select cell panel from the
initial panels used in the investigation.
Upon report of the suspected transfusion reaction,
the patient’s pretransfusion serum was tested with the
following cells: one Rh
null
 / Fy (a+), one Rh
null
 / Fy (a–),
three E– / Fy (a–), and one Co (a–). The pretransfusion
eluate was also tested against the Rh
null
/Fy (a–) cell.
The patient’s RBCs were further phenotyped for the
following antigens: Vel, Kpb, I, Coa, Lub, Yta, PP
1
Pk, Ge,
and LAN. A select cell panel was run using methods
already described, testing at least one cell that was
negative for each of the high-frequency antigens listed
above. Seven more Co(a–), E–, Fy (a–) RBCs were tested
with the patient’s serum. Six of these cells were Jk (a+).
All rare cells and antisera were obtained from either the
Serum, Cells, and Rare Fluids (SCARF) program or
Bonfils Blood Center donors.
Results
Pretransfusion antibody study
As the patient’s type and crossmatch prior to his
second surgery revealed no compatible units, the
specimen was sent to the reference laboratory for
further evaluation. This evaluation revealed an anti-E
reactive at 37°C with N-Hance and anti-Fya plus -E at the
antiglobulin test using anti-IgG. A cold autoantibody at
immediate spin (IS) and an apparent warm
autoantibody were also present. The serologic
evaluation is shown in Figure 1. All negative anti-
human globulin reactions showed agglutination with
check cells.
I M M U N O H E M A T O L O G Y ,   V O L U M E  1 7,  N U M B E R  2,  2 0 0 1 47
An eluate showed a panagglutinin at IS. At the
antiglobulin test, anti-E plus a panagglutin was present
and the adsorbed serum contained only anti-E plus -Fya
(Fig. 1).
antigen. The results of this evaluation are shown in
Figure 2. An antibody to Coa by LISS IgG was identified.
All negative anti-human globulin reactions showed
AHTR DUE TO ANTI-COA
Table 1. Vital signs, laboratory, and blood bank evaluation pre- and
post-AHTR*
Pre Post Unit
Vital signs
Temperature (C) 38.4 39.2
Blood pressure (mmHg) 155/95 150/95
Pulse 115 105
Respirations 16 16
Laboratory evaluation
Hematocrit (%) 22.3 18.3
Creatinine (mg/dL) 1.1 1.8
Bilirubin (mg/dL) — 3.9
Urinalysis — hemoglobinuria
Blood bank evaluation
ABO and Rh A+ A+ A+
Serum normal icteric normal
DAT (IgG) +/– 1+w
Clerical check OK OK
*Acute hemolytic transfusion reaction
Fig. 1.Pretransfusion evaluation performed by reference laboratory. Antibodies identified included anti-E, anti-Fya, and “cold” and “warm” autoantibodies.
Transfusion reaction clinical, laboratory, and
serologic evaluation
Following transfusion of one of the units of RBCs, the
patient developed signs and symptoms suggestive of
an AHTR. The results of the hospital’s transfusion
reaction evaluation are shown in Table 1. The sample
was drawn within 3 hours from the time of the
suspected AHTR. Most notable were the laboratory
values and an increase in the strength of the DAT.
Identification of anti-Coa
Due to the AHTR, the reference laboratory re-
evaluated the patient’s pretransfusion sample in an
attempt to identify an antibody to a high-frequency
48 I M M U N O H E M A T O L O G Y ,   V O L U M E  1 7,  N U M B E R  2,  2 0 0 1
Fig. 2.Re-evaluation performed by reference laboratory on the pretransfusion sample following the report of an AHTR. Anti-Coa was identified. Patient’s
phenotype was Co (a–).
agglutination with check cells. Phenotyping of the
patient’s RBCs revealed the patient was Co (a–).
In further testing, Rh
null
 / Fy(a+) cells tested 2+
positive, whereas Rh
null
 / Fy (a–) cells tested ± and
antibodies to Vel, Kpb, I, Lub, Yta, PP
1
Pk, Ge, and LAN were
ruled out. A posttransfusion sample, in addition to anti-
Coa, -E, and -Fya, also revealed the presence of an anti-Jka.
Discussion
An AHTR is defined as the rapid destruction of RBCs
that occurs during or shortly after a blood transfusion
(< 24 hours). The most common cause of AHTR is
transfusion of ABO-incompatible RBCs with formation
of antigen-antibody complexes that induce activation of
the complement cascade and lead to intravascular
hemolysis. Most often, these antibodies are IgM and are
naturally acquired; however, antibodies may be IgG
and can be acquired due to a previous pregnancy or
blood transfusion. The clinical features of an AHTR are
variable and depend on the quantity of incompatible
RBCs transfused, the type of antibody and its antigen
specificity, the thermal amplitude of the antibody,
complement activation, and the clinical condition of the
patient. The most frequent presentation is fever (greater
than a 1°C rise in temperature) with or without chills.
Other signs and symptoms include hemoglobinemia,
hemoglobinuria, hypotension, chest pain, dyspnea,
flushing, oliguria or anuria, and disseminated
intravascular coagulation. Laboratory diagnosis usually
includes a decline in hematocrit, hemoglobinemia,
hemoglobinuria, and a positive DAT that may
show a mixed-field appearance. An unconjugated
hyperbilirubinemia, abnormal coagulation studies,
and worsening renal function may also be identified.7
Our patient developed an increased temperature
(although not greater than 1°C), a decrease in
hematocrit, hemoglobinuria, and increased strength of
the DAT consistent with the clinical impression of an
AHTR. Re-evaluation of the patient’s serum samples
identified an antibody to the high-frequency antigen Coa
and a new anti-Jka. Phenotyping of the patients RBCs
revealed the patient to be Co (a–). Because Co (a–), Jk
(a+) cells did not show agglutination during repeat
testing of the pretransfusion sample, we feel that it is
highly likely that the transfusion reaction was caused by
anti-Coa. In addition, the reaction was most consistent
with a preformed antibody and the anti-Jka was
identified only after the transfusion reaction. The only
Jk (a+) units that were transfused were given 1 month
prior to the reaction and this length of time is not
consistent with a delayed transfusion reaction. Our
patient did not receive any further transfusions during
his hospital stay so we cannot document an increase
in hematocrit and a lack of reaction to a transfusion of
Co(a–) RBCs.
The differential diagnosis for an AHTR includes
thermal destruction of RBCs by heating and cooling
devices, concomitant administration of drugs or
R.B. COVIN ET AL.
I M M U N O H E M A T O L O G Y ,   V O L U M E  1 7,  N U M B E R  2,  2 0 0 1 49
nonisotonic fluids, bacterial contamination, or a
hemolytic condition within the recipient (e.g.,
autoimmune hemolytic anemia).7 In our patient, no
other causes for the AHTR were identified.
This case describes what we believe to be the first
reported case of an AHTR caused by anti-Coa. Previous
reports have implicated anti-Coa in delayed hemolytic
transfusion reactions4 and hemolytic disease of the
newborn.5 In addition, accelerated clearance of Co(a+)
RBCs has been identified in patients with anti-Coa
during in vivo survival studies.3,6 In their initial report,
Heisto et al.3 described a shortened T
50
 when Co(a+)
RBCs were injected into a patient with anti-Coa. These
findings were later confirmed by Kurtz et al.6 who
found a T
50
 of only 5 minutes in a 56-year-old G3P2
woman with a history of four previous transfusions. She
was identified as having anti-Coa and rapidly cleared
51Cr-labeled Co(a+) RBCs within 24 hours. The authors
suggested that Co(a–b+) RBCs be used for transfusion
in patients with anti-Coa. Although anti-Coa is most often
IgG, some of these antibodies have been shown to bind
complement and examples of IgM antibodies have been
reported.6 Even though Coa has not previously been
reported to cause an AHTR, Lee and Bennett8 reported
a case of a 74-year-old man who developed an AHTR
due to anti-Cob.
In our case, the anti-Coa was initially diagnosed as a
warm autoantibody (WAA) due to its reactivity with all
test cells. The differentiation between a WAA and a high-
frequency antigen can be difficult. Cash et al.9 reported
similar difficulty with a 60-year-old female who was
initially suspected to have a warm autoantibody.
After transfusion of two units of RBCs, she developed
a delayed hemolytic transfusion reaction. The antibody
was subsequently identified as anti-Ata.
We believe the clinical presentation and laboratory
evaluation is consistent with an AHTR due to anti-Coa.
AHTR DUE TO ANTI-COA
References
1. Issitt PD, Anstee DJ. Applied blood group serology.
4th ed. Montgomery Scientific Publishers, 1998.
2. Mudad R, Telen MJ. Biologic functions of blood
group antigens. Curr Opinion Hematol 1996; 3:
473–9.
3. Heisto H, Van Der Hart M, Grethe M, et al. Three
examples of a new red cell antibody, anti-Coa. Vox
Sang 1967; 12:18–24.
4. Montanaro-Kitzke H, Julius H, Delaney M, Studnicka
L, Landmark J. Anti-Coa implicated in delayed
hemolytic transfusion reaction. Transfusion 1982;
22:407.
5. Simpson WKH. Anti-Coa and severe haemolytic
disease of the newborn. S Afr Med J 1973; 47:
1302–4.
6. Kurtz SR, Kuszaj T, Ouellet R, Valeri CR. Survival of
homozygous Coa (Colton) red cells in a patient with
anti-Coa. Vox Sang 1982; 43:28–30.
7. Jeter EK, Spivey MA. Noninfectious complications of
blood transfusion. Hematol Oncol Clin North Am
1995; 9:187–204.
8. Lee EL, Bennett C. Anti-Cob causing acute hemolytic
transfusion reaction. Transfusion 1982; 22:159–60.
9. Cash KT, Brown T, Sausais L, Uehlinger J, Reed, LJ.
Severe delayed hemolytic transfusion reaction
secondary to anti-At(a). Transfusion 1999; 39:834–7.
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Randal B. Covin, MD, University of Colorado Health
Sciences Center, Department of Pathology, Denver,
Colorado; Karen S. Evans, MT(ASCP)SBB, Bonfils
Blood Center, Denver, Colorado; Richard Olshock, MD,
Lutheran Medical Center, Department of Pathology,
Wheat Ridge, Colorado; and Hannis W. Thompson, MD
(corresponding author), Director, Transfusion Service,
Department of Pathology, University of Colorado
Health Sciences Center, 4200 East Ninth Avenue,
Box A022, Denver, Colorado 80262.


Acute hemolytic transfusion reaction caused by anti-Coa

Touray, Mustapha

Cimen, Harun

Gulsen, Sebnem H.

Ulug, Derya

Erdogus, Dolunay

Shapiro-Ilan, David

Hazir, Selcuk

The impact of chemical nematicides on entomopathogenic nematode survival and infectivity

Arnoni, Carine Prisco

Latini, Flavia R.M.

Muniz, Janaína Guilhem

Person, Rosangela Duarte de Medeiros

Vendrame, Tatiane Aparecida de Paula

Gazito, Diana

Castilho, Lilian

20 IMMUNOHEMATOLOGY, Volume 31, Number 1, 2015
A simple approach to screen rare donors  
in Brazil
C.P. Arnoni, F.R.M. Latini, J.G. Muniz, R.D.M. Person, T.A.P. Vendrame, D. Gazito, and L. Castilho 
Original repOrt
Providing blood units for patients with an antibody to a high-
prevalence antigen or with multiple common antibodies is a 
constant challenge to the blood banks. Finding a compatible donor 
requires extensive screening, which incurs a large amount of 
investment. In this article, we share our experience of organizing 
a rare donor inventory with limited resources, we include the 
strategy used for finding rare donors, and we share the difficulties 
found during the implementation of the approach and the results 
obtained. Immunohematology 2015;31:20–23.
Key Words: rare donor, high prevalence antigen, 
phenotyping, genotyping
Rare donor identification began to get attention in 
the 1960s when the American Red Cross and American 
Association of Blood Banks began compiling a rare donor 
database.1 Both institutions realized that ensuring blood 
supply for patients with antibodies to high prevalence antigens 
or with multiple common antibodies was a big challenge.1,2 
Thereafter, rare donor programs were created in some 
countries and, in 1998, a national American program was 
formed (the American Rare Donor Program)1 to manage and 
supply rare blood, which currently provides approximately 
1800 units per year in the United States.1,3 Concomitantly, 
in 1965, the International Society of Blood Transfusion 
(ISBT) established the International Panel of Rare Blood 
Donors gathered by the International Blood Group Reference 
Laboratory in Bristol, U.K. Consequently, since 1984, when 
the ISBT Working Party on Rare Donors was formed, the 
provision of an effective exchange program has been discussed 
worldwide.4,5 In 2004, the ISBT Working Party on Rare 
Donors reported that the International Panel of Rare Blood 
Donors held 4000 rare donors registered from 24 countries.5,6 
Other national rare donor programs were also established—
for example, the French program conducted by the French 
National Reference Laboratory for Blood Groups7 and the 
Israeli program headquartered by the Magen David Adom 
National Blood Services.8 Unfortunately, however, there are 
still some countries without a national program.2
Currently, we are starting discussions in Brazil to establish 
a national panel of rare donors. Implementation steps include 
the screening of large numbers of donors, the education 
of blood center personnel, the development of a computer 
system, recruitment of donors, and maintenance of the rare 
units.2 Since 2011, Colsan, a blood bank in São Paulo, has 
implemented a process to screen for rare blood types, trying 
to set a reliable, useful, and efficient strategy to find rare blood 
donors. Taking into account the limitations we have related 
to financial support and the use of different methods, we 
developed an approach to converge resources and strengthen 
the efforts that aim to find the best combination of tests to 
successfully screen large numbers of donors, reduce costs, 
and increase the chances of encountering rare phenotypes and 
genotypes.
Different serology methods, as well as DNA typing, are 
being used to screen rare donors.2 As serology has some 
limitations attributable to the scarcity of commercial and 
potent antisera to type rare antigens, molecular protocols for 
large-scale genotyping have emerged as a tool to overcome 
these restrictions.9,10
Because the definition of what can be considered as rare 
blood differs between countries, the first step is to establish 
which antigens should be screened. It is very difficult to obtain 
a negative example of a high prevalence antigen when it only 
occurs in less than 1 in 1000 individuals.11 Then, finding donors 
whose red cells are negative for multiple common antigens can 
also be a challenge. Therefore, a donor whose red cells lack 
multiple common antigens is also considered a rare donor.
In a multi-ethnic population such as we find in Brazil, 
the prevalence of the different blood group antigens varies 
significantly, and it is necessary to have a range of rare 
blood types available. According to the prevalence of rare 
blood groups in the Brazilian population, we established an 
approach to search for S–s–, k–, Rhnull, r′r′, r″r″, Di(b–), 
Vel–, Wr(b–), Co(a–), Yt(a–), Js(b–), Kp(b–), Jo(a–), and 
Hy– donors. Additionally, we searched for R2R2 donors with 
a combination of negative clinical antigens. This approach 
includes serologic and molecular screening in repeated donors, 
in D– donors, and in donors with RhCE variants. We herein 
share our approach, the difficulties we experienced during 
implementation, and the results obtained after approximately 
3 years of the program.
IMMUNOHEMATOLOGY, Volume 31, Number 1, 2015 21
Screening of rare donors in Brazil
Materials and Methods
Rare Donor Screening Strategy
Screening for rare PhenotyPeS in rePeat DonorS
Repeat donors, including group O donors with at least two 
prior donations, are selected for screening by serology as well 
as by DNA typing.
1. Serologic Screening: We first perform a cross-match 
between the donor’s red blood cells (RBCs) and plasmas 
containing antibodies, such as anti-K, anti-Dia, anti-s, and 
anti-E by gel test in an automated instrument (Wadianna, 
Grifols, Barcelona, Spain). To optimize the searching and 
to reduce costs, we mix plasmas with anti-K and anti-Dia 
and perform the tests on the pool. If the test is positive, K 
and Dia are individually typed. All the positive results are 
confirmed with commercial sera.
  The aim of this serologic strategy is to find donors 
with k–, S–s–, and Di(b–) phenotypes and R2R2 donors 
with an interesting combination of antigen-negative 
results. Figure 1 shows the four scenarios obtained 
with this strategy and the further steps performed with 
serologic and molecular protocols.
2. Molecular Screening: Taking into account the lack 
of commercial antisera to type the majority of high-
prevalence antigens and the limitations of conventional 
polymerase chain reaction (PCR), we developed a 
SNaPshot protocol to identify rare donors, as previously 
described by Latini et al.12
Briefly, SNaPshot is a mini-sequencing assay that permits 
analysis of several single nucleotide polymorphisms (SNPs) 
from numerous donors in a short period of time. The protocol 
we developed identifies alleles of the Diego, Colton, Cartwright, 
KEL, Dombrock, and VEL blood group systems. All samples 
previously typed by serology, such as Di(a+) and/or K+, R1r, 
R1R1, R2R2, or R0r are selected for this molecular analysis.
Screening of rare PhenotyPeS in D-negative DonorS
All D– donors are typed for C, E, and K by hemagglu-
tination in microplates using an automated instrument (Neo, 
Immucor, Birkenfeld, Germany). This approach identifies rare 
Rh phenotypes such as r′r′, r″r″, and Rhnull and also the k– 
phenotype in the samples typed as K+. 
Molecular Screening for rhce variantS
Molecular screening for RhCE variants is performed in 
repeat donors of group O African descendants and in donors 
with altered expression of C, c, E, and e. We also perform 
molecular searching for CE variants in donors with altered 
expression of D because RhD variants have been shown to 
be associated with RhCE variants. In these cases, we select 
samples with weak D expression for immediate spin with 
two monoclonal anti-D reagents (IgM RUM-1 and blend 
D175+D415) on an automated instrument (Neo, Immucor).
Because the most important RhCE variants are those 
silencing the high prevalence antigens, hrS and hrB, our 
strategy includes screening the following alleles: RHCE*ceAR, 
RHCE*ceEK, RHCE*ceAG, RHCE*ceMO, RHCE*ceBI, 
RHCE*ceSM, RHCE*ceCF, and RHCE*ceS (RHCE*ce733G) 
that cause the hrS– and/or hrB– phenotypes. To identify 
these variants, we first use a PCR–restriction fragment–
length polymorphism (PCR-RFLP) for detection of the SNPs 
712A>G, 254C>G, 667G>T and 733C>G. Depending on the 
results obtained, we then use a strategy to identify the variant, 
as shown in Figure 2.
Fig. 1. This figure shows the four scenarios obtained by serologic screening. (A) All K+ RBCs are typed for k to identify k– phenotypes. (B) 
s– and K– RBCs are typed for S and all S–s– samples are genotyped for GYPB to identify U– and U+var phenotypes. (C) All Di(a+) and K– 
samples are typed for RhCE. R1r, R1R1, R0r, and R2R2 RBCs are molecular typed by SNaPshot. (D) All E+ K– RBCs are typed for e, and all 
R2R2 samples are typed for other clinically relevant antigens.
A
B
C
D
22 IMMUNOHEMATOLOGY, Volume 31, Number 1, 2015
C.P. Arnoni et al.
We also perform molecular tests to identify RhD variants 
in all samples with RhCE variants, based on previously 
published protocols.13 
Results 
Identified rare donors are included in our rare donor 
registry. Table 1 presents the results obtained in our 3 years of 
experience of building our rare donor inventory. To all donors 
included in this inventory, we send a folder with a simple 
explanation of their rare blood type and instructions regarding 
their next donation. We also ask that they keep their contact 
information updated. 
Additionally, we also phenotype and genotype all the 
clinical relevant antigens and freeze RBC aliquots in liquid 
nitrogen. Phenotype information on rare donors is submitted 
to the database housed in our blood center. The database 
allows the identification of the donors and facilitates searching 
for a requested phenotype. The steps of the process are 
schematically described in Figure 2.
The inventory of donors with 
RhCE variants in the homozygous 
state or in the heterozygous state with 
Ce or cE haplotypes allows for RH 
molecular matching to patients with 
sickle cell disease, preventing Rh 
alloimmunization and delayed hemolytic 
transfusion reactions. Six of our 11 
donors with RhCE variants were related 
to RhD variants; all hrS– linked to weak 
D type 4.2.2 and all hrB– linked to weak 
D type 4.0.
Discussion
The strategy presented herein 
has allowed us to search for rare 
donors and to create a rare donor 
inventory at our institution. After 
the complete implementation of the 
molecular laboratory, the investment 
is approximately $2.00 per sample for 
serologic screening and $13.00 per 
sample for SNaPshot screening for RHCE 
variants. The search for D– donors is part 
of our laboratory routine; the additional 
k typing is the equivalent of $0.70 per 
sample. The development of the SNaPshot method has helped 
increase throughput and reduce costs. Therefore, this strategy 
to find rare donors, based on a focused search, is cost-effective 
Fig. 2. Flowchart used to identify hrS– and hrB– donors. The investigation starts with the 
use of polymerase chain reaction–restriction fragment–length polymorphism to detect the 
single nucleotide polymorphisms 712A>G, 254C>G, 667G>T, and 733C>G. According to 
the results, we perform the analyses indicated in the flowchart to identify the RHCE variants.
Table 1. Rare donor inventory
Screening
Total number of 
donors screened
Phenotypes and predicted 
phenotypes from genotypes n
Serologic screening 4500 U+var*
U–*
k–
6
1
5
Molecular screening 1600 Yt(a-b+)
Js(a+b–)
Co(a–b+)
Kp(a+b–)
Di(a+b–)
4
1
1
1
2
RhD negative 2000 r˝r˝
r´r´
k–
2
2
2
RhCE variants 590 hrS–
hrB–
hrS–, hrB–
7
3
1
TOTAL 38
*U– and U+var were determined by polymerase chain reaction–allele specific 
and restriction fragment–length polymorphism.14
IMMUNOHEMATOLOGY, Volume 31, Number 1, 2015 23
Screening of rare donors in Brazil
as compared with other strategies using microarrays and may 
help other blood centers to build their rare donor inventory.
The benefit of this investment is documented when an 
alloimmunized patient with a rare phenotype requires a rare 
unit of blood and is successfully transfused with a compatible 
unit. In a period of 3 years, we received 18 requests for rare 
units, and 8 of these were fulfilled using our rare donor 
inventory. Applying this approach, we intend to meet the 
needs of the patients with a rare phenotype in our institution 
and help other blood centers.  Nevertheless, we still have some 
limitations to ensure that blood is made available to specific 
patients. Although we observed a great collaboration of the 
donors when recruited, some of these may be unable to donate 
when required. Freezing blood would be a good option, but 
this procedure is currently a challenge in Brazil, since the 
blood bag and the glycerol solution used for freezing are not 
licensed by our regulatory agency.
Limitations of the Approach
Our strategy for searching for rare donors does not include 
screening for D– –, Ge:-2, and Ko phenotypes, which have 
already been found in the Brazilian population, but our future 
screening activities will focus on the identification of such 
donors. Another unfavorable point of this strategy is the long 
duration of the whole process. In general, when we find a rare 
donor, the RBC unit has already been transfused and we need 
to wait until the next donation to freeze RBC aliquots and to 
perform complete phenotyping.
Conclusion
It is widely accepted that a rare donor program is 
important to ensure a safe transfusion. In our experience, 
we realized that the implementation of a rare donor program 
requires efforts from both technical and administrative areas. 
On the other hand, we show herein that a simple and focused 
strategy can help fulfill the requests for units of rare blood for 
patients with antibodies against high-prevalence antigens or 
patients with multiple antibodies. Exchanges of experience 
and collaboration between different centers, through a 
national program, will allow us to ensure that blood will be 
made available to patients with rare blood phenotypes.
Acknowledgments
We are grateful to Nichola Conran for English review and 
to Grifols, who sponsored part of the rare donor searching 
by serology and helped us to implement this protocol. We 
thank Silvia Iversson for her efforts to maintain, contact, and 
recruit the rare donors and Carla Renata Messias for technical 
support.
References
 1. Flickinger C, Petrone T, Church A. Review: American rare 
donor program. Immunohematology 2004;20:239–43.
 2. Nance ST. How to find, recruit and maintain rare blood donors. 
Curr Opin Hematol 2009;16:503–8.
 3. Meny GM, Flickinger C, Marcucci C. The American Rare Donor 
Program. J Crit Care 2013; 28:110.
 4. Mourant AE. The establishment of an international panel of 
blood donors of rare types. Vox Sang 1965;10:129–32.
 5. Anstee D, Levene C, Mallory D, et al. Rare blood. An ISBT 
Working Party report on rare blood donors. International 
Society of Blood Transfusion. Vox Sang 1999;77:58-62.
 6. Woodfield G, Poole J, Nance ST, Daniels G. A review of the 
ISBT rare blood donor program. Immunohematology 2004;20: 
244–8.
 7. Peyrard T, Pham BN, Le Pennec PY, Rouger P. [The rare 
blood groups: a public health challenge]. Transfus Clin Biol 
2008;15:109–19.
 8. Levene C, Asher O, Shinar E, Yahalom V. Rare blood donors: a 
personal approach. Immunohematology 2006;22:64–8.
 9. Avent ND. Large-scale blood group genotyping: clinical 
implications. Br J Haematol 2009;144:3–13.
 10. Daniels G. The molecular genetics of blood group 
polymorphism. Hum Genet 2009;126:729–42.
 11. Reesink HW, Engelfriet CP, Schennach H, et al. Donors with a 
rare pheno (geno) type. Vox Sang 2008;95:236–53.
 12. Latini FR, Gazito D, Arnoni CP, et al. A new strategy to identify 
rare blood donors: single polymerase chain reaction multiplex 
SNaPshot reaction for detection of 16 blood group alleles. Blood 
Transfus;12(Suppl 1):s256–63.
 13. Arnoni CP, Latini FR, Muniz JG, et al. How do we identify RHD 
variants using a practical molecular approach? Transfusion 
2014;54:962–9.
 14. Storry JR, Reid ME, Fetics S, Huang CH. Mutations in 
GYPB exon 5 drive the S-s-U+(var) phenotype in persons of 
African descent: implications for transfusion. Transfusion 
2003;43:1738–47.
Carine Prisco Arnoni, PhD (corresponding author), Adjunct 
Director; Flavia R.M. Latini, PhD, Director; Janaína Guilhem Muniz, 
Biologist; Rosangela Duarte de Medeiros Person, Biologist; Tatiane 
Aparecida de Paula Vendrame, Biologist; Diana Gazito, Biologist, 
Technical and Scientific Department, Colsan-Associação Beneficente 
de Coleta de Sangue, Avenida Jandira 1260, Indianópolis 04080-
006, São Paulo, SP, Brazil; and Lilian Castilho, PhD, Professor and 
Researcher, Hemocentro-Unicamp, Rua Carlos Chagas 480, Barão 
Geraldo 13083-878, Campinas, SP, Brazil.


A simple approach to screen rare donors in Brazil

Dinardo, Carla Luana

Bonifácio, Sílvia Leão

Mendrone Júnior, Alfredo

IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 1
Indirect antiglobulin test-crossmatch 
using low-ionic-strength saline–albumin 
enhancement medium and reduced incubation 
time: effectiveness in the detection of most 
clinically significant antibodies and impact on 
blood utilization
C.L. Dinardo, S.L. Bonifácio, and A. Mendrone, Jr.
Indirect antiglobulin test-crossmatch (IAT-XM) using 
enhancement media such as low-ionic-strength saline (LISS) 
and polyethylene glycol (PEG) usually requires 15 minutes of 
incubation. These methods are necessary when testing samples 
from blood recipients who have a higher risk of alloimmunization. 
In emergency situations, IAT-XM can be time-consuming and 
can influence presurgery routine, resulting in more red blood cell 
(RBC) units being tested and stored to avoid the transfusion of 
uncrossmatched ones. The objective of this study was to evaluate 
the performance of a LISS-albumin enhancer to intensify 
antigen-antibody reaction after 5 minutes of 37°C incubation and 
compare this performance with that of other enhancers, gel, and 
conventional tube testing. Second, the study evaluated the impact 
of this method’s implementation in the C:T ratio (crossmatched 
to transfused RBC units) of a transfusion laboratory. Ninety 
serum samples containing alloantibodies of potential clinical 
significance were tested against phenotyped RBCs using four 
different methods: (1) tube with LISS-albumin enhancer 
(5 minutes of incubation), (2) tube with LISS-albumin and PEG 
(15 minutes of incubation), (3) gel, and (4) conventional tube 
method (60 minutes of incubation). In parallel, the study compared 
the C:T ratio of a tertiary-hospital transfusion laboratory in 
two different periods: 3 months before and 3 months after the 
implementation of the 5-minute IAT-XM protocol. The use of 
LISS-albumin with 5 minutes of incubation exhibited the same 
performance as LISS-albumin, PEG, and gel with 15 minutes 
of incubation. Conventional tube method results were equally 
comparable, but reactions were significantly less intense, except 
for anti-c (p = 0.406). Accuracy was 100 percent for all selected 
methods. After the implementation of the 5-minute IAT-XM 
protocol, the C:T ratio fell from 2.74 to 1.29 (p < 0.001). IAT-XM 
can have its incubation time reduced to 5 minutes with the use 
of LISS-albumin enhancement. We suggest this strategy should 
be used to quickly prepare RBC units for surgical patients, 
keeping transfusion safety without compromising blood supplies. 
Immunohematology 2014;30:1–5.
Key Words: crossmatch, alloantibody, antibody screening, 
LISS, PEG, gel testing
Crossmatching of donor red blood cells (RBCs) and 
recipient’s serum is an important step required to complete 
pretransfusion tests. Crossmatch can be performed 
electronically or by an immediate-spin method, in which 
no incubation or anti-human globulin (AHG) steps are 
performed. Those modalities of crossmatch are applied to 
nonalloimmunized recipients, with the detection of ABO 
mismatches their main goal. Whenever there is a positive 
antibody screen (current or past) or a history of pregnancy, 
crossmatch demands 37°C incubation and AHG steps 
(indirect antiglobulin test-crossmatch; IAT-XM), increasing 
the time needed for completion of pretransfusion tests, which 
can become critical in emergency situations.
In Brazil, legislation requires IAT-XM for all recipients 
who have ever been transfused, pregnant, or alloimmunized, 
and as a consequence, the routine of performing a type and 
screen on samples from surgical patients is frequently not 
applied, as most transfusion laboratories perform IAT-XM 
before the surgery and segregate all the units that may be used 
during the surgical procedure until its end. This obviously 
increases the number of blood units in store and the costs 
of the pretransfusion tests. Also, the implementation of any 
automation may be compromised.
Classic saline indirect antibody test (SIAT) demands 60 
minutes of incubation (37°C) and three wash steps before 
the addition of AHG and interpretation of the results. This 
method allows 99 percent of antibody uptake onto RBCs for 
the detection of an immunoglobulin G (IgG) antibody present 
in the recipient’s serum.1 This length of incubation can be 
shortened with the help of enhancement media such as low-
ionic-strength saline (LISS), albumin, polyethylene glycol 
(PEG), and hexadimethrine bromide (Polybrene), whose 
RepoRt
2 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014
C.L. Dinardo et al.
major functions are to speed up the rate of antigen-antibody 
association, promoting a higher rate of antibody uptake by 
RBCs in a shorter time.
In emergency situations, even when data referring to 
recipients’ previous alloimmunization is not known, saline 
immediate-spin crossmatch (IS-XM) is frequently chosen by 
laboratory analysts, as it allows fast availability of RBC units. For 
recipients who present a higher risk of alloimmunization, such 
as those with sickle cell anemia, myelodysplastic syndrome, 
or multiparity, this approach may be risky. Enhancement 
medium containing a mixture of LISS and albumin typically 
requires 15 minutes of incubation before the AHG phase. Our 
hypothesis is that this time can be shortened to 5 minutes 
without losing sensitivity, making this method suitable for 
RBC and serum crossmatch in emergency situations.
The main objective of this study is to evaluate the 
performance of a LISS-albumin enhancer to  intensify the 
antigen-antibody reaction after 5 minutes of 37°C incubation, 
allowing the detection of clinically significant alloantibodies 
present in recipients’ serum. Second, the impact of this method’s 
implementation in the routine of a transfusion laboratory will 
also be evaluated. To meet this second objective, we chose to 
evaluate the C:T ratio (ratio of units crossmatched to units 
transfused) before and after the implementation of the type 
and screen associated with the 5-minute IAT-XM protocol in 
our presurgery routine. In most countries, a C:T ratio less than 
2:1 is an indicator of efficient preparation of blood units for 
elective surgery.
Materials and Methods
Ninety serum samples containing alloantibodies of 
potential clinical significance (anti-D, -c, -K, -Jka, -Jkb, -Fya) 
from our inventory were tested against phenotyped RBCs 
using two different methods: tube and column agglutination 
gel (DiaMed Latino América, Lagoa Santa, Brazil). Tube testing 
was performed conventionally (without enhancers) and with 
two different enhancement media: LISS-albumin (Dialiss, 
DiaMed, enhancer 1, and LISS ADD, Lorne Laboratories, 
Lower Earley, UK, enhancer 2) and PEG (BioPeg, Fresenius 
HemoCare, São Paulo, Brazil, enhancer 3). One of the LISS-
albumin enhancers (enhancer 1) was chosen to be tested with 
the proposed protocol of 5 minutes of incubation. All tests 
were performed in parallel by the same analyst, according 
to the specific method described in a later section. We chose 
RBCs from donors who were heterozygous for the allele 
encoding the corresponding antigen against the alloantibody 
being tested.
Gel Microcolumn Assay Method
RBCs were washed once in 0.9 percent saline and 
suspended in LISS (ID-Diluent 2, DiaMed) to achieve a final 
0.8 to 1 percent concentration. Then 50 µL of 0.8 to 1 percent 
RBC suspension and 25 µL of sera were added to LISS-Coombs 
gel cards (IgG/C3d, DiaMed), incubated for 15 minutes at 
37°C, and centrifuged (85g) for 10 minutes according to 
manufacturer’s instructions.
Tube Testing
RBCs were washed three times in 0.9 percent saline 
and then adjusted to a final concentration of 3 percent. After 
this step, 50 µL of each RBC suspension and 100 µL of each 
specific serum were added to each tube. For the conventional 
method, tubes were incubated for 1 hour at 37°C. For the 
methods involving enhancers 2 and 3, tubes were incubated 
for 15 minutes at 37°C. After addition of enhancer 1, the tubes 
were incubated for 5 minutes at 37°C. After the incubation 
step, all tubes were washed three times with 0.9 percent saline 
before AHG was added and final reading of the results was 
performed. All reactions were read macroscopically. Positive 
reactions were graded from 1+ to 4+ and expressed in scores.2
Statistical Analysis
We compared the scores obtained from the five studied 
methods (median value) for each specific antibody using 
the Friedman statistical test (nonparametric statistical test 
suitable for multiple comparisons of different methods or 
treatments, SPSS version 17, SPSS Inc., Chicago, IL). We 
considered as statistically significant a probability value less 
than 0.05. Scores were calculated based on AABB standards.
Analysis of Crossmatched-to-Transfused Ratio
From June 2011 to September 2011, after the implemen-
tation of our new legislation, only an ABO/Rh type and antibody 
screen was performed for all preoperative transfusion requests 
(type and screen strategy), except in cases of alloimmunization, 
in which phenotyped units were prepared. If during the surgery 
any RBC unit was requested, our laboratory analysts performed 
the IAT-XM using the LISS-albumin enhancer and 5 minutes 
of 37°C incubation. We calculated the C:T ratio at the end of 
this 3-month period and compared it with our retrospective 
C:T ratio (March 2011 to May 2011).
Results
Enhancer 1 (LISS-albumin, 5 minutes of incubation), 
enhancer 2 (LISS-albumin, 15 minutes of incubation), 
IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 3
Rapid technique for IAT-crossmatch
enhancer 3 (PEG, 15 minutes of incubation), and gel 
microcolumn agglutination methods exhibited similar 
performance, expressed in terms of score, for all selected 
antibodies. Conventional tube method results were equally 
comparable, but reactions were significantly less intense than 
those presented by the other methods, except in the case of 
anti-c (p = 0.406). Sensitivity and specificity were 100 percent 
for all selected methods. Enhancer 1 presented the highest 
scores in the presence of anti-Jkb, -c, -K, and -Fya. In the case 
of anti-D and -Jka, gel microcolumn agglutination method 
exhibited higher scores, but they were not statistically different 
from those presented by all other enhancers. Table 1 shows 
mean scores for the different antibodies using each selected 
method. Figures 1 and 2 summarize the data.
After the implementation of the type and screen routine 
associated with the 5-minute IAT-XM protocol using a LISS-
albumin enhancer, we had 2080 elective surgeries: 360 RBC 
units were crossmatched and only 81 were not used during 
the surgery. Three months before the implementation of this 
protocol, we had a C:T ratio of 2.74 (2040 elective surgeries, 
795 units crossmatched and 290 units transfused). Our C:T 
ratio fell from 2.74 to 1.29 (p < 0.001) in 3 months.
After the implementation of the 5-minute IAT-XM 
protocol, no transfusion reactions suggestive of hemolysis were 
reported to the blood bank. In one case, antibody screening 
was negative (gel microcolumn agglutination method) and 
IAT-XM was positive. The antibody identified after the 
performance of 16°C antibody screening (tube method) was 
an anti-M without activity at 37°C. In all other cases, IAT-XM 
was negative.
Discussion
Our results demonstrate that the required 37°C 
incubation time for detection of significant RBC alloantibodies 
can be decreased to 5 minutes when using a LISS-albumin 
enhancement medium, without loss of sensitivity or specificity. 
This strategy improved our C:T ratio, an important quality 
indicator of blood utilization, as unexpected needs for urgent 
transfusions directed to surgical patients could be met in less 
than 10 minutes.
Reducing time without losing sensitivity in RBC antigen-
antibody reactions was always a subject of discussion in 
the literature before the emergence of the gel microcolumn 
agglutination method. PEG and LISS, either isolated or 
combined with albumin, are the most widely used enhancement 
media and intensify RBC sensitization while decreasing the 
required incubation time of 60 to 90 minutes (conventional 
tube method) to 15 to 20 minutes.3 Even though extending 
the incubation time in the presence of enhancers may increase 
the detection of antibodies directed against antigens of clinical 
significance,4 extending this time to more than 40 minutes 
may result in a paradoxical loss of sensitivity.3
Performance of RBC crossmatch between donor and 
recipient before transfusion is suggested either when the 
antibody screen is positive or when the risk of previous 
alloimmunization is higher than usual (multiply transfused 
patients and multiparous). There are a considerable number 
of reports of hemolytic transfusion reactions caused by 
antibodies that were not detected by antibody screen or by 
IS-XM, but that would have been detected by IAT-XM.5–8 
Even though those antibodies mainly are cold-reactive or 
are against low-incidence antigens, the odds of encountering 
them increase in patients who are immunologic responders 
and consequently prone to RBC alloimmunization.9 Indeed, 
the decision to eliminate IAT-XM is associated with a risk of 
hemolytic transfusion reactions of 1:2000 transfused units,10 
which is a significant value, especially in large transfusion 
laboratories.
The IS-XM approach is suitable for detecting donor-
patient ABO incompatibilities and may be used in situations 
when antibody screening is negative. However, there is the 
Table 1. Performance of different IAT methods in detecting clinically significant antibodies*
Antibody  
(number of samples)
Enhancer 1 (LISS-albumin): 
5-min incubation
Enhancer 2 (LISS-albumin): 
15-min incubation
Enhancer 3 (PEG):  
15-min incubation Gel Tube: 60-min incubation
Anti-D (50) 9.32 ± 0.79 9.3 ± 1.18 9.18 ± 0.92 9.52 ± 0.93 5.64 ± 1.27
Anti-Jka (12) 3.34 ± 2.23 4 ± 2.89 3.92 ± 1.88 4.25 ± 3.17 1.67 ± 1.37
Anti-Fya (11) 6.82 ± 2.75 6 ± 2.24 6 ± 2.28 6 ± 2.24 2.91 ± 1.87
Anti-Jkb (4) 8.5 ± 1 8.5 ± 1 6.5 ± 1.73 8 ± 0 4 ± 0
Anti-c (11) 12 ± 0 12 ± 0 11.81 ± 0.6 12 ± 0 11.81 ± 0.6
Anti-K (48) 9.38 ± 1.61 9.15 ± 1.51 8.75 ± 1.64 7.48 ± 1.89 5.27 ± 1.97
IAT = indirect antiglobulin test; LISS = low-ionic-strength saline; PEG = polyethylene glycol.
*Mean ± standard deviation.
4 IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014
substantial risk of not detecting an A2B (donor)-B (recipient) 
mismatch or the presence of an alloantibody against a low-
incidence antigen.11 In countries where IAT-XM is required for 
all patients with a previous history of pregnancy or transfusion, 
as in ours (Brazil), or in areas where the incidence of some low-
incidence antigens is higher owing to ethnic characteristics, a 
5-minute IAT-XM may be a less time-consuming and more 
effective strategy than a 15-minute IAT-XM.
Our present results indicate that the LISS-albumin 
enhancement medium detects clinically significant antibodies 
after 5 minutes of incubation and that the intensity of reactions 
did not differ from that presented by this same enhancer or 
by PEG after 15 minutes of incubation. After implementation 
of routine type and screen testing on samples from surgical 
patients and of preparing RBC units only after intraoperation 
requests, we were able to reduce significantly the number of 
units that were prepared and not transfused. In our country 
this strategy is of great importance, as our legislation requires 
IAT-XM for all recipients who have ever been transfused, 
pregnant, or alloimmunized. As a referral hospital, we have 
plenty of previously transfused or pregnant patients, and 
preparing and reserving RBC units for all of them compromised 
our blood supplies.
In conclusion, crossmatch incubation time can be reduced 
to 5 minutes with the use of LISS-albumin enhancement 
medium. We suggest this strategy be used to quickly prepare 
RBC units for surgical patients, maintaining transfusion safety 
without compromising blood supplies.
C.L. Dinardo et al.
Fig. 1 Mean scores ± 95 percent confidence interval (CI) of antigen-antibody reactions using different indirect antiglobulin test methods 
for the detection of anti-D, -Jka, and -Fya. Enhancers 1, 2, and 3 had a performance similar to that of gel testing. Conventional tube testing 
expressed significantly lower reaction intensity. *p < 0.05; **p < 0.01, ***p < 0.001.
M
ea
n 
Sc
or
e
10
8
6
4
2
0
Enhancer 1 Enhancer 1 Enhancer 1Enhancer 2 Enhancer 2 Enhancer 2Enhancer 3 Enhancer 3 Enhancer 3
anti-D anti-Jka anti-Fya
GEL GEL GELConventional Conventional Conventional
x
x
x
x
x
x
x
Error bars: 95% Cl
anti-Jkb anti-c anti-K
Fig. 2 Mean scores ± 95 percent confidence interval (CI) of antigen-antibody reactions using different indirect antiglobulin test methods 
for the detection of anti-Jkb, -c, and -K. Enhancers 1, 2, and 3 had a performance similar to that of gel testing. Conventional tube testing 
expressed significantly lower reaction intensity, except for anti-c. *p < 0.05; **p < 0.01, ***p < 0.001.
M
ea
n 
Sc
or
e
10
8
6
4
2
0
Enhancer 1 Enhancer 1 Enhancer 1Enhancer 2 Enhancer 2 Enhancer 2Enhancer 3 Enhancer 3 Enhancer 3GEL GEL GELConventional Conventional Conventional
x
x
x
x
x
Error bars: 95% Cl
12
IMMUNOHEMATOLOGY, Volume 30, Number 1, 2014 5
References
 1. Rumsey DH, Ciesielski DJ. New protocols in serologic 
testing: a review of techniques to meet today’s challenges. 
Immunohematology 2000;16:131–7.
 2. Roback JD, Grossman BJ, Harris T, Hillyer CD. Technical 
manual. 17th ed. Bethesda, MD: American Association of 
Blood Banks, 2011.
 3. Jørgensen J, Nielsen M, Nielsen CB, Nørmark J. The influence 
of ionic strength, albumin and incubation time on the sensitivity 
of the indirect Coombs’ test. Vox Sang 1979;36:186–91.
 4. Skaik YA. Detection of Rh antibodies using two low ionic 
diluents: extension of the incubation time and the number of 
Rh antibodies detected. J Postgrad Med 2008;54:4–6.
 5. Reznicek MJ, Cordle DG, Strauss RG. A hemolytic reaction 
implicating Sda antibody missed by immediate spin 
crossmatch. Vox Sang 1992;62:173–5.
 6. Judd WJ, Steiner EA, Abruzzo LV, et al. Anti-i causing acute 
hemolysis following a negative immediate-spin crossmatch. 
Transfusion 1992;32:572–5.
 7. Wise SC, Larson PJ, Cook LO. Antibodies to low-incidence 
antigens and elimination of the antihuman globulin phase of 
the crossmatch-case report: anti-Wra. Immunohematology 
1997;13:20–2.
 8. Koshy R, Patel B, Harrison JS. Anti-Kpa-induced severe 
delayed hemolytic transfusion reaction. Immunohematology 
2009;25:44–7.
 9. Higgins JM, Sloan SR. Stochastic modeling of human RBC 
alloimmunization: evidence for a distinct population of 
immunologic responders. Blood 2008;112:2546–53.
 10. Judd WJ, Fullen DR, Steiner EA, Davenport RD, Knafl PC. 
Revisiting the issue: can the reading for serologic reactivity 
following 37 degrees C incubation be omitted? Transfusion 
1999;39:295–9.
 11. Chapman JF, Milkins C, Voak D. The computer crossmatch: 
a safe alternative to the serological crossmatch. Transfus Med 
2000;10:251–6.
Carla Luana Dinardo, MD, PhD, student (corresponding author), 
Chief of Immunohematology Division, Sílvia Leão Bonifácio, MSc 
student, Chief of Immunohematology Quality Control Department, 
and Alfredo Mendrone Júnior, MD, PhD, Director of Fundação Pró-
Sangue/Hemocentro de São Paulo, Avenida Dr. Enéas de Carvalho 
Aguiar, 155—1st floor, Cerqueira César, São Paulo—SP, Brazil 
05403-000.
Rapid technique for IAT-crossmatch


Indirect antiglobulin test-crossmatch using low-ionic-strength saline&ndash;albumin enhancement medium and reduced incubation time: effectiveness in the detection of most clinically significant antibodies and impact on blood utilization

Hayes, Michele

Raph blood group system

Reid, Marion E.

A review of the JR blood group system

Sloan, Steven R.

IMMUNOHEMATOLOGY, Volume 28, Number 1, 2012 17
Transfusions for patients with sickle cell 
disease at Children’s Hospital Boston
S.R. Sloan
RepoRt
Introduction
Children’s Hospital Boston is a large pediatric medical 
center with 390 inpatient beds and several outpatient clinics. 
All transfusions of hospitalized patients with sickle cell disease 
(SCD) occur at the main hospital site in Boston. Outpatient 
transfusions take place at the main site in Boston and at a 
satellite clinic in Waltham, Massachusetts, which is about a 
25-minute drive from the main Boston site. Approximately 
180 patients with SCD are treated by Children’s Hospital 
Boston annually. These patients are predominantly children. 
At age 18 to 21 years, patients are transitioned to an adjacent 
hospital that treats adult patients with SCD.
Red blood cell (RBC) units for transfusions at both sites 
are provided by one blood bank located at the main hospital 
in Boston. Children’s Hospital Boston blood bank includes 
a blood donor center, transfusion service laboratory, and 
therapeutic apheresis unit. The apheresis unit performs 
RBC exchange transfusions for emergencies and for chronic 
transfusion therapy. Currently, approximately two RBC 
exchange procedures are performed each week. The blood 
donor center collects blood in a fixed site blood donor center 
adjacent to the main hospital lobby and from a bloodmobile 
that collects blood throughout eastern Massachusetts. The 
blood donor center collects enough blood to supply most of the 
RBC unit needs for Children’s Hospital Boston. Although not a 
full immunohematology reference laboratory, the transfusion 
service laboratory performs many immunohematology 
laboratory tests and also has a molecular testing area that 
performs single-nucleotide polymorphism (SNP) analysis 
using the BioArray Solutions System of Immucor Corporation 
(Norcross, GA). Genotyping is performed on a large proportion 
of the blood donors.
Transfusion Protocol
In addition to ABO and D, SCD patients are prospectively 
matched for K, C, and E. The treating hematologist sends a 
sample of blood from each patient with SCD who is at least 1 
year old for the blood bank to determine the patient’s phenotype 
for these antigens using serologic methods. Patients lacking K, 
C, or E antigens will receive RBC units lacking those antigens. 
No attempt is made to match for k, c, or e. Having a large 
number of RBC units genotyped facilitates rapid identification 
of appropriate RBC units, but genotyping is used as a screening 
test with serology used to confirm that specific antigens are 
lacking on RBC units.
Genotyping and serologic information are entered into 
blood donor records, and donors who are particularly useful 
for frequently transfused patients with SCD are targeted for 
extra recruitment efforts. This is done regardless of donor race. 
Indeed, the blood donor center does not track the race of the 
blood donors.
The blood needs for patients with SCD have increased in 
recent years as more patients have been chronically transfused 
based on findings of the STOP and STOP II trials.1,2 This has 
led to more patients with iron overload and subsequently 
more patients transfused by RBC apheresis to reduce the iron 
burden.3 Although RBC apheresis reduces the iron burden, it 
also increases the number of RBC units used by the patients 
and hence has increased the demand on our blood bank. 
The Children’s Hospital Boston blood donor center has made 
initial attempts to have blood drives at locations with high 
proportions of African Americans, but these efforts have not 
met the increased demand and the hospital has had to import 
RBC units for some of these patients.
RBC Selection Protocol
Children’s Hospital Boston will use RBC units of any 
age for patients with SCD unless they also are in a category 
of patients who receive fresher RBC units, such as infants 
undergoing surgery. Except in emergencies, all RBC and 
platelet units at Children’s Hospital Boston are irradiated, 
and patients with SCD receive irradiated units just as all 
other patients do. Patients with SCD receive RBC units that 
are negative for hemoglobin S (HbS). Although the BioArray 
molecular system tests for the HbS gene, the Sickledex (Streck, 
Inc., Omaha, NE) test is used to confirm that an RBC unit is 
negative for HbS.
18 IMMUNOHEMATOLOGY, Volume 28, Number 1, 2012
S.R. Sloan
All RBC and platelet units at Children’s Hospital Boston 
are leukocyte reduced and considered cytomegalovirus (CMV) 
safe. In addition, some units are known to be serologically 
negative for CMV antibodies. Such units are preferentially 
provided to patients at higher risk for CMV infection such 
as hematopoietic progenitor cell transplant patients who are 
CMV negative. Patients with SCD are not considered at high 
risk for CMV disease and so do not preferentially receive CMV 
serologically negative units.
Children’s Hospital Boston plans to test its RBC units 
for babesiosis using investigational tests and will attempt to 
have its entire RBC inventory tested for this disease. This will 
likely not be possible at all times. When there is an inventory of 
Babesia-tested and -untested RBC units, Babesia-tested RBC 
units will be preferentially provided to patients with SCD.
Emergency Protocols
If blood is emergently needed, blood untested for HbS 
may be transfused, at the discretion of the blood bank 
physician. Additionally, matching for the C, E, or K antigens 
may be skipped at the discretion of the blood bank physician. 
If no compatible blood is available, then Children’s Hospital 
Boston will attempt to procure RBC units lacking the 
necessary antigens from other sources. In these situations, 
the blood bank physician will usually be in communication 
with the treating hematologist. If no crossmatch-compatible 
RBC units are available anywhere, or if the RBC units are 
needed before they arrive at the hospital, incompatible RBC 
units may be transfused. However, if the patient has clinically 
significant antibodies, for example to antigens in the Rh 
system, or if the patient has developed hyperhemolysis, then 
the recommendation may be to avoid the transfusion. Indeed, 
transfusions have successfully been avoided by providing 
supportive care for patients with SCD who have very low blood 
hemoglobin concentrations for extended periods of time.
If a patient has a warm autoantibody, underlying 
alloantibodies are identified with the use of adsorption 
techniques. In addition, the patient’s predicted extended 
phenotype is determined by DNA methods. Such a patient is 
preferentially transfused with genotyped RBC units matched 
for several antigens. With this protocol, the blood bank knows 
the foreign RBC antigens to which the patient has been exposed 
and hence the potential alloantibodies that may develop.
Genotyping may also be performed on some select patients 
who do not have autoantibodies. These are determined on an 
individual case basis. In general, genotyping may help the 
laboratory investigation in patients with complex serologic test 
results. Also, genotyping can be extremely helpful in patients 
suspected of having Rh variant alleles based on serologic 
results. In these cases, Children’s Hospital Boston will generally 
send out the specimen for further molecular characterization 
that cannot be provided by the standard BioArray chip.
Protocol Outcomes
Antibody development has generally been manageable 
with the protocols used by Children’s Hospital Boston. 
Although we have not directly compared the antibodies 
developed under this protocol with antibodies that developed 
without D, C, E, and K matching, very few antibodies to these 
antigens have developed. The few patients who have developed 
antibodies in the Rh system usually have Rh variants or were 
transfused elsewhere. Specifically, a few patients developed 
antibodies that appeared to have a relative or absolute 
specificity for e antigen, and these patients were subsequently 
found to have variant RHCE alleles. One patient with SCD 
exhibited anti-D. Subsequent testing suggested that this 
patient had a partial RHD gene that was not detected before 
his transfusion of D+ RBCs. No patients with SCD in the past 
several years have developed panreactive autoantibodies. 
At least two patients have developed hyperhemolysis 
syndrome. The patients who developed hyperhemolysis did 
not have detectable autoantibodies, and one of the patients 
had no detectable antibodies against minor RBC antigens 
(negative antibody screen). The blood bank almost always 
has sufficient RBC inventory to avoid incompatibilities with 
any alloantibodies that have developed in  patients with SCD, 
and few  of these patients have developed autoantibodies. The 
main difficulties have been with patients with unusual RH 
alleles, some of whom have developed antibodies reactive with 
the Rh proteins present on most donor units. In rare cases, 
Children’s Hospital Boston has needed to acquire RBC units 
from blood supplies for these patients. Although there are 
significant costs associated with genotyping, this testing has 
aided in the identification of valuable blood donors and greatly 
speeds the process of identifying antigen-negative RBC units. 
This improves the turnaround time, an improvement that is 
especially valuable for  patients with SCD requiring emergent 
transfusions.
Summary
During the past decade, Children’s Hospital Boston 
has had four changes affecting transfusion therapy for SCD 
patients:
IMMUNOHEMATOLOGY, Volume 28, Number 1, 2012 19
Transfusion of patients with SCD at Children’s Hospital Boston
1. A bloodmobile has helped increase blood collections. 
This could allow for more drives at locations with 
many African Americans, but attempts to date have 
not met the growing need.
2. Genotyping has allowed for predicting the extended 
phenotype of many RBC units and patients with 
autoantibodies.
3. Transfusion-transmitted babesiosis cases have 
increased, and Children’s Hospital Boston will soon be 
providing Babesia-tested RBC units.
4. The number of RBC units transfused to patients with 
SCD has increased as a result of studies finding the 
benefits of transfusion therapy in decreasing stroke 
risk. This has significantly impacted the inventory, 
making it difficult and sometimes impossible to 
provide RBC units matched for multiple antigens.
References
 1. Adams RJ, Brambilla D; Optimizing Primary Stroke 
Prevention in Sickle Cell Anemia (STOP 2) Trial Investigators. 
Discontinuing prophylactic transfusions used to prevent stroke 
in sickle cell disease. N Engl J Med 2005;353:2769–78.
 2. Adams RJ, McKie VC, Hsu L, et al. Prevention of a first 
stroke by transfusions in children with sickle cell anemia and 
abnormal results on transcranial Doppler ultrasonography. N 
Engl J Med 1998;339:5–11.
 3. Adams DM, Schultz WH, Ware RE, Kinney TR. 
Erythrocytapheresis can reduce iron overload and prevent the 
need for chelation therapy in chronically transfused pediatric 
patients. J Pediatr Hematol Oncol 1996;18:46–50.
Steven R. Sloan, MD, PhD, Medical Director, Children’s Hospital 
Boston, Department of Laboratory Medicine, 300 Longwood 
Avenue, Boston, MA 02115.


Transfusions for patients with sickle cell disease at Children&#39;s Hospital Boston

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