Exeley Inc.

Klejbor, Ilona

Mahmood, Saleh

Melka, Natalia

Ebertowska, Adriana

Morys, Janusz

Stachowiak, Ewa K.

Stachowiak, Michal K.

Patel, Mulchand S.

Phenylbutyrate administration reduces changes in the cerebellar Purkinje cells population in PDC-deficient mice

Tanpowpong, Natthaporn

Panichyawat, Sineenart

Comparison of sonographic hepatorenal ratio and the degree of hepatic steatosis in magnetic resonance imaging-proton density fat fraction

Drakonaki, Eleni E.

Symvoulakis, Emmanouil K.

Gliatis, John

High-resolution ultrasound of the ankles in Lofgren syndrome: attention to detail may be the key to diagnosis

EDD, SIERRA

Radical Traversals

WILDCAT, MATTHEW

Replacing Exclusive Sovereignty with a Relational Sovereignty

DUCHENEAUX, TALON BAZILLE “SHOOTS THE ENEMY”

The Deer with 3: Expressing Indigeneity Through Experimental Hip Hop

Dean-El, C.

Quraishy, N.

IMMUNOHEMATOLOGY, Volume 35, Number 2, 2019 45
Elution removes antibodies coating red blood cells and allows 
for serologic testing of the recovered antibodies. Heat elution 
may be used in the investigation of ABO hemolytic disease of the 
fetus and newborn, in the detection of weak A and B antigens in 
combination with adsorption-elution using polyclonal antibodies, 
and for the resolution of interfering IgM agglutinating antibodies. 
Immunohematology 2019;35:45–47.
Key Words: DAT, direct antiglobulin test, elution, heat 
elution, ABO, IgM antibodies, HDFN
Principle
Antibodies and antigens fit together in a “lock and key” 
mechanism, stabilized by van der Waals forces,1 hydrogen 
bonds, and electrostatic and hydrophobic interactions. The 
avidity of an antibody correlates with its binding strength—
for example, the IgM pentamer with its 10 antigen-binding 
sites has high antigen avidity in contrast to the monomeric 
IgG. The affinity of an antibody for a given antigen depends 
on the binding capacity of a single antigen-binding site. The 
interaction between the antibody and its antigen is reversible 
and is affected by the ionic strength, temperature, and pH of 
the testing environment. The process of removing an antibody 
attached to red blood cells (RBCs), either in vivo or in vitro, is 
termed elution, and the recovered, concentrated antibody in 
solution is called an eluate.
Heat elution, the first RBC elution procedure,2 was 
described by Landsteiner and Miller3 in their studies on 
chimpanzees. The original method involved a 5-minute 
incubation at 56°C of antibody-coated RBCs in normal saline, 
but resulted in eluates with low antibody levels.4 Currently, a 
modified Landsteiner-Miller heat elution method described by 
Judd5 and discussed in the AABB Technical Manual6 is used.
The exothermic reaction for formation of antigen (Ag)–
antibody (Ab) complexes is represented by the following 
equation:2
Ag + Ab = [AgAb] + calories
An increase in temperature on the Ag–Ab complex results 
in intensification of the thermal motion of atoms and molecules 
driving the reaction in reverse and causing dissociation of the 
Ag–Ab complex.
Additionally, heat causes a conformational change in 
proteins, resulting in a loss of the “lock and key” interaction 
between antigen and antibody.
Indications
Elution studies may be performed on patient samples 
having a positive direct antiglobulin test (DAT) due to auto- 
or alloantibodies, due to drug-induced antibodies, or in the 
investigation of hemolytic disease of the fetus and newborn 
(HDFN).7 They may also be used in combination with in vitro 
adsorption studies to identify weakly expressed blood group 
antigens and for the identification of multiple alloantibodies.
Heat elution: a modification of the 
Landsteiner-Miller method
C. Dean-El and N. Quraishy
S
E
R
O
L
O
G
IC
 M
E
T
H
O
D
 R
E
V
IE
W
Reagents/Supplies
Reagents Supplies
• Saline (ice-cold for IgM cold 
agglutinins) 
• 6% bovine albumin
• Reagents for testing, including 
group A, B, and O RBCs, if 
applicable
• 56°C water bath or heat block
• 12 × 75 mm test tubes
• Centrifuge
• Optical aid
• Timer
Procedural Steps
• Wash patient RBCs four to six times with saline. Use ice-cold 
saline washes for IgM cold agglutinins.
• Remove and place the final wash in a clean, labeled test tube.
• Place an equal volume of washed RBCs and bovine albumin 
in another clean, labeled test tube. Cap the tube, and mix the 
contents thoroughly by gently inverting the tube.
• Incubate the tube at 56°C for 10 minutes. Periodically, invert the 
tube, and mix the contents during incubation.
• After incubation, centrifuge the tube at 900–1000g for 2–3 
minutes (use a heated centrifuge if available).
• Transfer supernatant eluate into a clean, labeled test tube.
• Test the eluate and final wash in parallel, using the appropriate 
test phase(s) in a manner that is best for detection of the antibody, 
following the laboratory’s standard operating procedure.
RBCs = red blood cells.
46 IMMUNOHEMATOLOGY, Volume 35, Number 2, 2019
C. Dean-El and N. Quraishy
Indications for heat elution include:
• Investigation of ABO HDFN. Elution is rarely required, 
however, because the diagnosis is generally made from 
clinical findings consistent with HDFN in an ABO-
incompatible mother and newborn.2
• Identification of weak A and B antigens by a combination 
of adsorption and elution methods. Polyclonal anti-A 
or anti-B is adsorbed onto the RBCs, and heat elution 
is used to harvest the adsorbed-bound antibody to 
determine the presence of a weak A or B antigen.8
• Identification of IgM cold autoantibodies. Heat elution 
has been described as an effective method for the 
identification of IgM cold autoantibodies2,9 when 
compared with acid stroma (digitonin), organic solvent, 
and Lui freeze–thaw elution methods.
Procedure
Preparing the Eluate
Before performing the elution, the sensitized RBCs must 
be washed5,6 thoroughly four to six times with large volumes 
of saline. Use ice-cold saline to prevent dissociation of cold-
reactive IgM antibodies. The wash step is critical for removing 
cell-free antibody while retaining RBC-bound antibody. After 
the last wash is performed, harvest the supernatant wash into 
a clean, labeled test tube.
Place an equal volume of washed packed RBCs and 6 
percent bovine albumin in a labeled 12 × 75 mm test tube. A 
preferable starting volume is 1 mL or 20 drops of each. Cap 
the tube, and mix the contents well by gently inverting the 
tube. Immediately place the tube in a 56°C water bath or heat 
block, ensuring complete submersion of the tube in the heated 
water. Incubate for 10 minutes, with periodic agitation of the 
tube. Centrifuge the tube at 900–1000g for 2–3 minutes. If 
available, use a centrifuge that is heated to 56°C.
Transfer the supernatant eluate into a clean, labeled 
12 × 75 mm test tube. The eluate is now ready for testing using 
standard antibody identification methods. Perform testing 
immediately after elution, since eluates are not stable.5
Testing the Eluate
Test the eluate using the appropriate test phase(s) in a 
manner that is best for detection of the antibody, including a 
reading at room temperature if applicable. Include group A, B, 
and O cells if testing for ABO antibodies. Test the recovered 
last wash in parallel with, and in the exact same manner, as 
the eluate.
Use clean test tubes during the elution procedure. 
Contaminated test tubes carry the risk of antibody adhering 
to proteins on the contaminated test tube surface and being 
reintroduced to the last wash or eluate.2
Quality Control
The last wash must be tested in parallel with the eluate 
and must be negative for the eluate results to be valid. A 
positive last wash indicates the presence of unbound antibody 
due to inadequate washing of the sensitized RBCs. In that case, 
the elution procedure must be repeated after more thorough 
washing of the RBCs.
Another option for quality control is to perform a DAT on 
the RBCs after the heat treatment, and compare it with the 
pre–heat treatment DAT. A negative DAT post–heat treatment 
indicates successful elution.
Implementation of a new procedure requires performance 
of verification studies in accordance with Code of Federal 
Regulations standard 42CFR493.1253.
Limitations
The heat elution method is simple to perform but has 
limited applications, as described under Indications.
Drawbacks of heat elution include the following:
• There is the possibility of marked hemolysis of the 
eluate. The red color of the eluate can make agglutination 
difficult to read during testing.
• Heat can denature the RBC membrane and affect 
clinically significant blood group antigens, weakening 
or destroying them; thus, the RBCs used in the elution 
procedure should not be used for phenotyping.10
• Heat elution is not an effective method for removal of 
warm autoantibodies or alloantibodies, other than 
ABO.2,11
As with any elution procedure:
• Inadequate washing of the RBCs before the elution 
method will result in interference from unbound 
antibodies.
• There is a risk of the Matuhasi-Ogata phenomenon.2,5
IMMUNOHEMATOLOGY, Volume 35, Number 2, 2019 47
References
 1. Howard PL. Principles of antibody elution. Transfusion 
1981;21:477–82.
 2. Judd WJ. Elution: dissociation of antibody from red blood 
cells: theoretical and practical considerations. Transfus Med 
Rev 1999;13:297–310.
 3. Landsteiner K, Miller CP. Serological studies on the blood of 
primates. II. The blood groups in anthropoid apes. J Exp Med 
1925;42:853–62.
 4. Rubin H. Antibody elution from red blood cells. J Clin Path 
1963;16:70–3.
 5. Judd WJ. Section IV. Elution techniques. Pages 147–149.  In: 
Judd’s methods in immunohematology. 3rd ed. Bethesda, MD: 
AABB, 2008.
 6. Fung MK, Eder AF, Spitalnik SL, Westhoff CM. Method 
4.3. Heat elution procedure. In: Technical Manual. 19th ed. 
Bethesda, MD: AABB, 2017.
 7. Haberman S, Krafft CJ, Luecke PE, et al. ABO isoimmunization: 
the use of the specific Coombs and heat elution tests in the 
detection of hemolytic disease. J Pediatr 1960;56:471–7.
Heat elution
 8. Liu F, Li G, Mao X, Hu L. Detection of IgG anti-A/B is essential 
for safe transfusion support in patients undergoing ABO 
incompatible allogeneic HSCT. Transfus Apher Sci 2011; 
44:123–7.
 9. Ellis JP, Sokol RJ. Detection of IgM autoantibodies in eluates 
from red blood cells. Clin Lab Haematol 1990;12:9–15.
 10. Burin des Roziers N, Squalli S. Removing IgG antibodies 
from intact red cells: comparison of acid and EDTA, heat, and 
chloroquine elution methods. Transfusion 1997;37:497–501.
 11. Nathalang O, Sthabunsawasdigarn S, Bejrachandra S, et al. 
A comparative study of three techniques for eluting red cell 
antibodies. J Med Assoc Thai 1997;80:S5–8.
Celeste Dean-El, MS, SBB(ASCP)CM, Immunohematology Supervisor, 
Cleveland Clinic, Cleveland, OH, and NurJehan Quraishy, MD, 
Transfusion Medicine Staff (corresponding author), Q6-2, Clinical 
Pathology, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 
44195, quraisn@ccf.org.


Heat elution: a modification of the Landsteiner-Miller method

Erratum

Mougey, R.

An update on the Chido/Rodgers blood group system

George, M.R.

154 IMMUNOHEMATOLOGY, Volume 35, Number 4, 2019
An update on the Cartwright (Yt) blood 
group system
M.R. George
Blood Group revie w
This update of the Cartwright blood group system (George MR. 
Cartwright blood group system review. Immunohematology 
2012;28:49–54) reports the addition of three new antigens. From 
1956 until 2017, the Cartwright (Yt) blood group system consisted 
of two antigens, Yta and Ytb. Yta is a high-prevalence antigen, and 
its antithetical antigen, Ytb, shows much lower prevalence. In 
2017, YTEG was identified, and, in 2018, the International Society 
of Blood Transfusion added high-prevalence antigens YTLI and 
YTOT. Cartwright antigens result from point mutations in the 
acetylcholinesterase gene on chromosome 7q. Little is known about 
antibodies against YTEG, YTLI, and YTOT. Discovery of these 
new antigens required plasma inhibition studies and molecular 
analysis for further characterization. Based on experience with 
Yta and Ytb, Cartwright antibodies have rarely demonstrated 
clinical significance; nevertheless, cases of in vivo hemolysis 
have been reported, suggesting that clinical significance should 
be interpreted on a case-by-case basis. Immunohematology 
2019;35:154–155.
Key Words: Cartwright, Yt, YTEG, YTLI, YTOT
Molecular Update
Historically, the majority of molecular methods used 
in red blood cell (RBC) antigen testing used specific single 
nucleotide polymorphisms or single nucleotide variants. These 
methods can only detect a limited selection of known alleles. 
This limitation can result in missing new or uncommon 
alleles and can subsequently lead to incorrect interpretation of 
phenotypes. Next generation sequencing (NGS) methods do 
not have this limitation and appear to be better suited to detect 
novel polymorphisms.1 Scharberg et al.2 demonstrated the 
use of genotyped RBCs in antibody identification in a study of 
nearly 17,000 blood samples from close to 8500 patients tested 
in their reference lab. In this 3-year study, 21 antibodies from 
10 blood group systems were identified in 126 patients by using 
the genotype information of these reagent RBCs. Antibodies 
to Cartwright antigens were among the most frequent, 
accounting for antibodies identified in 31 patients (25%). 
This study suggests that genotyping may extend the range 
of detectable specificities to expedite antibody identification. 
Indeed, the use of NGS sequencing was instrumental in the 
identification of YTEG as being a homozygous mutation 
c.266G>A in exon 2, which resulted in an amino acid change 
p.Gly89Glu in acetylcholinesterase (AChE).3,4 YT4 (or YTLI) 
was found through a complex antibody workup in a pregnant 
woman with anti-Jka. The patient’s RBCs typed Yt(a+b–), 
YTEG+, and molecular ACHE sequencing analysis confirmed 
a novel homozygous mutation c.169G>A in exon 2, encoding 
p.Gly57Arg change in AChE. This sequence was found in 
both the patient and her crossmatch-compatible brother.5 The 
most recent addition to the YT system, YT5 (or YTOT), was 
discovered in the workup of a pregnant woman with sickle cell 
disease. She had multiple RBC antibodies and an unknown 
specificity inhibited by soluble recombinant Yt antigen (srYt). 
Antibodies to other Yt antigens (Yta, YTEG, and YTLI) were 
excluded, although ACHE sequence analysis revealed a 
novel homozygous mutation c.101G>A in exon 2, encoding 
p.Arg34Gln change in AChE.5 Given the recent discovery of 
the three new YT antigens (YTEG, YTLI, and YTOT), too 
little is known about their genetics/inheritance, other than 
to note that these three antigens are all of high prevalence.3–5 
The molecular basis of the Yt antigens can be summarized in 
Table 1.3–8
Biochemistry Update
Of note, Cartwright antigens are sensitive to dithiothreitol 
(DTT) treatment used to negate interference of daratumumab 
(DARA), an IgG1k monoclonal antibody against CD38 on 
plasma cells in plasma cell myeloma. This finding lends further 
support for RBC antigen phenotyping or genotyping before the 
patient is treated with DARA, because Cartwright antibodies, 
along with several others, could be missed in the DTT test 
environment.9 Inhibition studies using srYt were instrumental 
in further characterizing the three new Yt antigens discovered 
in recent years—YTEG, YTLI, and YTOT.
Nomenclature
The first two antigens, Yta and Ytb, were named after the 
patient, Mrs. Cartwright, in whom the antibody was first 
IMMUNOHEMATOLOGY, Volume 35, Number 4, 2019 155
Update on Yt
identified; the third antigen, YTEG, was named after the 
proband; and the fourth and fifth, YTLI and YTOT, were 
named after the referring laboratories. The nomenclature 
across various systems is denoted in Table 2.3–6
References
 1. Jakobsen MA, Dellgren C, Sheppard C, Yazer M, Sprogøe U. 
The use of next-generation sequencing for the determination of 
rare blood group genotypes. Transfus Med 2019;29:162–8.
 2. Scharberg E, Rink G, Portegys J, et al. The impact of using 
genotyped reagent red blood cells in antibody identification. 
Transfus Med Hemother 2018;45:218–24.
 3. Storry JR, Clausen FB, Castilho L, et al. International 
Society of Blood Transfusion Working Party on Red Cell 
Immunogenetics and Blood Group Terminology: report of 
the Dubai, Copenhagen and Toronto meetings. Vox Sang 
2019;114:95–102.
 4. Laundy R, Karamatic Crew V, Davies H, et al. A novel high 
frequency antigen in the Yt blood group system. Transfus Med 
2017;27(Suppl 2):42–3.
 5. Karamatic Crew V, Laundy R, Walser P, et al. Serological and 
molecular characterisation of two novel high frequency antigens 
in the Yt blood group system. Vox Sang 2018;113(Suppl 1): 
5–347.
Table 1. Molecular basis of the Yt antigens
Yta Ytb YTEG YTLI YTOT
Amino acid His 353 Asn 353 p.Gly89Glu p.Gly57Arg p.Arg34Gln
Nucleotide C at bp 1057 in exon 2 A at bp 1057 in exon 2 Novel 
homozygous 
mutation 
c.266G>A 
in exon 2
Novel 
homozygous 
mutation 
c.169G>A in 
exon 2
Novel 
homozygous 
mutation 
c.101G>A in 
exon 2
Additional 
features
Nucleotide at position 1431C>T 
in exon 3 also differentiates Yta 
from Ytb but does not alter the 
encoded amino acid. A second 
silent mutation in exon 5 does not 
correlate with the Yt polymorphism.
Nucleotide at position 1431C>T in 
exon 3 also differentiates Yta from 
Ytb but does not alter the amino 
acid. A second silent mutation in 
exon 5 does not correlate with the 
Yt polymorphism.
Table 2. Nomenclature for the Yt blood group system
System name System symbol Gene symbol
Traditional/ISBT ISBT number Traditional ISBT ISBT number Traditional ISBT ISGN
Yt or Cartwright 011 Yt YT Yt YT ACHE
Yta YT1 011.001
Ytb YT2 011.002
YTEG YT3 011.003
YTLI YT4 011.004
YTOT YT5 011.005
ISBT = International Society of Blood Transfusion; ISGN = International System for Gene Nomenclature.
 6. International Society of Blood Transfusion. Table of blood 
group antigens v.8.1_181111. Available from www.isbtweb.
org/fileadmin/user_upload/Working_parties/WP_on_
Red_Cell_Immunogenetics_and/Table_of_blood_group_
antigens_within_systems_v8.1_181111.pdf.
 7. Garratty G, Dzik W, Issitt PD, Lublin DM, Reid ME, Zelinski 
T. Terminology for blood group antigens and genes: historical 
origins and guidelines in the new millennium. Transfusion 
2000;40:477–89.
 8. Reid ME, Lomas-Francis C. The blood group antigen factsbook. 
2nd ed. New York: Elsevier Academic Press, 2004:332–7.
 9. Branch DR, Meunsch HA. Disulfide bonds are a requirement 
for Kell and Cartwright (Yta) blood group antigen integrity. Brit 
J Haem 1983;54:573–8.
Melissa R. George, DO, FCAP, FASCP, Medical Director, Transfusion 
Medicine; Program Director, Pathology Residency, and Associate 
Director, Transfusion Medicine Fellowship, Penn State Health 
Milton S. Hershey Medical Center, 500 University Drive, Hershey, 
PA 17033, mgeorge5@pennstatehealth.psu.edu.


An update on the Cartwright (Yt) blood group system

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